Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 15 - 8 TSH control of PKA catalytic subunit activity in thyroid cell cultures; Ben Abdelkhalek M et al.; The protein expression and the enzyme activity of the catalytic subunit (C) of the cAMP-dependent protein kinases were studied in porcine thyroid cell primary cultures stimulated with two doses of TSH (0.1 mU/ml and 1 mU/ml) for 1 to 3 days . In TSH-stimulated cells the desensitization of the catalytic subunit activity was accompanied by a simultaneous and parallel decrease of its immunoreactivity . The loss of catalytic subunit was rapid and reached its maximum after 1 day of culture . It is similar in the two subcellular compartments: cytosol and particulate extracts . Contrary to the observed loss of the C subunit protein molecules in TSH-stimulated cells, the expression of the Cbeta subunit mRNA in these cells was increased fivefold compared to controls, while no significant change was observed on the Calpha subunit mRNA . These results suggest that TSH controls the Cbeta subunits of PKA at two levels: at the transcriptional level it increases Cbeta mRNA expression, and at the translational or posttranslational level TSH decreases the amount and the activity of the Cbeta protein molecules . Biotechnol Bioeng, 1999, 66(4), 231 - 7 Development of a bioartificial pancreas: II . Effects of oxygen on long-term entrapped betaTC3 cell cultures; Papas KK et al.; Tissue-engineered pancreatic constructs based on immunoisolated, insulin-secreting cells are promising in providing an effective, relatively inexpensive, long-term treatment for type I (insulin-dependent) diabetes . An in vitro characterization of construct function under conditions mimicking the in vivo environment is essential prior to any extensive animal experimentation . Encapsulated cells may experience hypoxic conditions postimplantation as a result of one or more of the following: the design of the construct; the environment at the implantation site; or the development of fibrosis around the construct . In this work, we studied the effects of 3- and 4-day-long hypoxic episodes on the metabolic and secretory activities and on the levels of intracellular metabolites detectable by phosphorus-31 nuclear magnetic resonance ((31)P NMR) of alginate/poly-L-lysine/alginate entrapped betaTC3 mouse insulinomas continuously perfused with culture medium . Results show that, upon decreasing the oxygen concentration in the surrounding medium, the encapsulated cell system reached a new, lower metabolic and secretory state . Hypoxia drove the cells to a more anaerobic glycolytic metabolism, increased the rates of glucose consumption (GCR) and lactate production (LPR), and reduced the rates of oxygen consumption (OCR) and insulin secretion (ISR) . Furthermore, hypoxia reduced the levels of intracellular nucleotide triphosphates (NTP) and phosphorylcholine (PC) and caused a rapid transient increase in inorganic phosphate (P(i)) . Upon restoration of the oxygen concentration in the perfusion medium, all parameters returned to their prehypoxic levels within 2 to 3 days following either gradual unidirectional changes (ISR, NTP, PC) or more complicated dynamic patterns (OCR, GCR, LPR) . A further increase in oxygen concentration in the perfusion medium drove OCR, ISR, NTP, PC, and P(i) to new, higher levels . It is concluded that (31)P NMR spectroscopy can be used for the prolonged noninvasive monitoring of the bioenergetic changes of encapsulated betaTC3 cells occurring with changes in oxygen tension . The data also indicate that the oxygen-dependent states might be related to the total number of viable, metabolically active cells supported by the particular oxygen level to which the system is exposed . These findings have significant implications in developing and non-invasively monitoring a tissue-engineered bioartificial pancreas based on transformed beta cells, as well as in understanding the biochemical events pertaining to insulin secretion from betaTC3 insulinomas . Biotechnol Bioeng, 1999, 66(4), 219 - 30 Development of a bioartificial pancreas: I . long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures; Papas KK et al.; Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes . Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations . We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells . Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium . Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels . Concurrently, a reorganization of cell distribution within the beads was observed . Following this growth period, the measured metabolic and secretory parameters remained constant with time . During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive . Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment . It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions . These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion . Biotechnol Bioeng, 1999, 66(4), 238 - 46 Improvement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction; Irani N et al.; Continuous mammalian cell lines are important hosts for the production of biological pharmaceuticals . However, these cell lines show some severe disorders in primary metabolism, which they have in common with many cancer cells . This leads to a high throughput of substrates giving a low energy yield and ample toxic side products such as lactate and ammonia . Because the enzymatic connection between glycolysis and the tricarboxylic acid cycle (TCA) is very poor, glucose is mainly degraded via oxidative glycolysis . It will be shown that introducing a pyruvate carboxylase gene expressed in the cytoplasma into a continuous BHK-21 cell line, and thus reconstituting the missing link between glycolysis and TCA, can reduce this problem . Thus, glucose consumption could be reduced by a factor of four and glutamine utilization up to a factor of two, compared with control . Moreover, a 1.4-fold-higher adenosine triphosphate (ATP) content was achieved . The flux of labeled {(14)C}-glucose into the TCA is shown to be enhanced, indicating a higher rate of oxidative glucose degradation . Host cell lines with an improved energy metabolism will therefore result in better exploitation of substrates, an increasing yield by the more efficient use of carbon source, and higher product integrity combined with lower production costs . J Leukoc Biol, 1999 Nov, 66(5), 822 - 8 Modulatory effects of human herpes virus-7 on cytokine synthesis and cell proliferation in human peripheral blood mononuclear cell cultures; Atedzoe BN et al.; Human herpes virus-7 (HHV-7) infects cells of the immune system and thus may modulate their function . To investigate the potential immunomodulatory effects of this virus, we performed an in vitro study in which we investigated effects of HHV-7 on the synthesis of several key immunomodulatory cytokines, i.e . tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, IL-6, and transforming growth factor beta (TGF-beta) . This was examined after in vitro infection of human peripheral blood mononuclear cells (PBMC) with HHV-7 . We found elevated levels of TNF-alpha, TGF-beta, and IFN-gamma in the supernatants of HHV-7-infected cells . By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, using cytokine-specific primers, we found that levels of TNF-alpha, TGF-beta, and IFN-gamma mRNA were increased in the infected cells . The HHV-7 infection also significantly (P < 0.05) decreased the production of IL-2 from activated, IL-2-producing PBMC . Furthermore, mitogen- and cytokine-induced cellular proliferative responses of human PBMC were found to be significantly (P < 0.05) down-regulated by this virus . On the other hand, HHV-7 did not affect IL-4 and IL-6 synthesis . Overall, our results indicate that HHV-7 infection causes significant immunomodulatory effects with regard to cytokine synthesis in these cells as well as inhibiting lymphocyte proliferation by various stimuli. Plant J, 1999 Oct, 20(1), 109 - 17 Millisecond UV-B irradiation evokes prolonged elevation of cytosolic-free Ca2+ and stimulates gene expression in transgenic parsley cell cultures Frohnmeyer H, Loyall L, Blatt MR, Grabov A. Chalcone synthase (CHS) is a key enzyme leading to the generation of protective flavonoids in plants under environmental stress . Expression of the CHS gene is strongly upregulated by exposures to UV light, a response also observed in heterotrophic parsley cell cultures . Although there are hints that the stimulus for CHS expression may be coupled to UV-B irradiation through a rise in cytosolic-free Ca2+ ({Ca2+}i), the temporal relationship of these events has never been investigated critically . To explore this question, we have used a CHS promoter/luciferase (CHS/LUC) reporter gene fusion and recorded its expression and {Ca2+}i elevation in a transgenic parsley cell culture following millisecond light pulses . Luciferase expression was enhanced maximally seven- (+/- 2) fold by 30 10 ms flashes of UV-B light . The response was specific to wavelengths of 300-330 nm and could be inhibited in the presence of the Ca2+ channel blocker nifedipine . In parallel measurements, using Fura-2 fluorescence ratio microphotometry, we found that 10 ms UV-B flashes also evoked a gradual and prolonged rise of {Ca2+}i in the parsley cells which was irreversible within the timescale of these experiments, but could be prevented by prior treatment with nifedipine . These, and additional results, indicate a remarkably high temporal sensitivity to, and specificity for, UV-B light in CHS gene expression independent of UV-mediated DNA damage by thymine dimerization . The ability of transient UV-B stimulation to evoke prolonged elevations of {Ca2+}i suggests a functional coupling between the initial light stimulus and subsequent gene expression that takes place many tens of minutes later. Acta Physiol Scand, 1999 Oct, 167(2), 161 - 6 Visualization of nitric oxide formation in cell cultures and living tissue; Wiklund NP et al.; We have visualized nitric oxide (NO) released from cell cultures and living tissue . NO was visualized by a reaction with luminol and hydrogen peroxide to yield photons which were counted using a microscope coupled to a photon counting camera . Murine macrophages were activated with interferon-gamma (IFN-gamma) and endotoxin (LPS) . Cultured endothelial cells were stimulated with bradykinin, and neurones in the guinea-pig myenteric plexus and the rabbit hypogastric nerve trunk were electrically stimulated . There was a marked increase in photons emitted from the cultured cells as well as from the living tissues during stimulation . The stimulation-induced photon emission was markedly reduced by inhibition of nitric oxide synthase (NOS); removal of L-arginine from the medium also decreased photon counts . The present method allowed integration times in the order of minutes to improve signal-to-noise ratio . However, the high sensitivity of this method also makes it possible to generate an image in seconds, allowing the production of real time films . Photon emission was enhanced under conditions known to increase NO production, and diminished in the presence of NO inhibitors . Thus, this method has demonstrated specificity for the L-arginine:NO pathway from a wide range of biological sources such as cultured cells and living tissues, and has the potential for real time imaging of NO formation, with high temporal and spatial resolution. Int J Tissue React, 1999, 21(2), 43 - 9 Plasma cytokine concentration and the cytokine producing ability of whole blood cell cultures from healthy females with pharmacologically induced hyperprolactinemia; Rovensky J et al.; We investigated the in vitro effect of domperidone-induced hyperprolactinemia on plasma cytokine concentration and blood leukocyte cytokine production in healthy female volunteers . No changes were found in the plasma concentration of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, IL-10, IL-6 and IL-13 during hyperprolactinemia when compared with control values . Using unseparated blood leukocytes, we found that the spontaneous production of IL-6 (4-8 h) and transforming growth factor (TGF)-beta 1 (2-4 h) was significantly decreased and that the in vitro stimulated production of IFN-gamma (2-8 h) and TNF (4 h) was significantly increased compared with control . Our data concerning the increased IFN-gamma and TNF producing capacity of unseparated leukocytes during pharmacologically induced hyperprolactinemia strongly support the possibility that the lymphocyte production of these cytokines can be rapidly amplified by prolactin via a priming mechanism. J Clin Microbiol, 1999 Dec, 37(12), 3971 - 4 Comparison of four clinical specimen types for detection of influenza A and B viruses by optical immunoassay (FLU OIA test) and cell culture methods; Covalciuc KA et al.; Although laboratory diagnosis of respiratory viruses has been widely studied, there is a relative insufficiency of literature examining the impact of specimen type on the laboratory diagnosis of influenza A and B . In a clinical study comparing the FLU OIA test with 14-day cell culture, clinical specimens from nasopharyngeal swabs, throat swabs, nasal aspirates, and sputum were obtained from patients experiencing influenza-like symptoms . A total of 404 clinical specimens were collected from 184 patients . Patients were defined as influenza positive if the viral culture of a specimen from any sample site was positive . Patients were defined as influenza negative if the viral cultures of specimens from all sample sites were negative . By this gold standard, culture and FLU OIA test results for each sample type were compared . For each of the four specimen types, the viral culture and FLU OIA test demonstrated equal abilities to detect the presence of influenza A or B virus or viral antigen . Sputum and nasal aspirate samples were the most predictive of influenza virus infection . Throat swabs were the least predictive of influenza virus infection, with both tests failing to detect influenza virus in nearly 50% of the throat samples studied. Pharmacol Toxicol, 1999 Oct, 85(4), 162 - 8 Growth-modulating effects of dichloro myristic and dichloro stearic acid in cell cultures; Hostmark AT et al.; Chloro-containing fatty acids are a major fraction of extractable, organically bound chlorine in fish . It has been suggested that dichloro stearic acid (9,10-dichlorooctadecanoic acid) (C18) is metabolized to dichloro myristic acid (5,6-dichlorotetradecanoic acid) (C14) which accumulates in tissues . Hence, the biological effects of the C18 dichloro fatty acid could be due to formation of the C14 dichloro fatty acid . In this study we have compared the effects of dichloro stearic and dichloro myristic acid on growth of three widely differing cell lines . Both fatty acids inhibited cell growth; however, dichloro myristic acid had a weaker growth inhibitory effect than dichloro stearic acid . Dichloro myristic acid had a biphasic effect (i.e . growth was stimulated at low concentrations, followed by inhibition at higher concentrations) on the growth of human hepatoma cells and immortalized human kidney epithelial cells, but no such effect on human microvascular endothelial cells . The order of potency for growth inhibition by dichloro myristic acid was consistently human hepatoma cells>immortalized human kidney epithelial cells >human microvascular endothelial cells, whereas the relative potency of dichloro stearic acid was variable . Albumin alone stimulated cell growth and had a stronger protective effect against growth inhibition by dichloro myristic acid than against that of dichloro stearic acid . It seems unlikely that a major part of the effect of dichloro stearic acid on cell growth is caused by conversion to dichloro myristic acid. J Virol, 1999 Dec, 73(12), 9891 - 8 Ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon; Chinsangaram J et al.; A genetic variant of foot-and-mouth disease virus lacking the leader proteinase coding region (A12-LLV2) is attenuated in both cattle and swine and, in contrast to wild-type virus (A12-IC), does not spread from the initial site of infection after aerosol exposure of bovines . We have identified secondary cells from susceptible animals, i.e., bovine, ovine, and porcine animals, in which infection with A12-LLV2, in contrast to A12-IC infection, does not produce plaques; this result indicates that this virus cannot spread from the site of initial infection to neighboring cells . Nevertheless, A12-LLV2 can infect these cells, but cytopathic effects and virus yields are significantly reduced compared to those seen with A12-IC infection . Reverse transcription-PCR analysis demonstrates that both A12-LLV2 and A12-IC induce the production of alpha/beta interferon (IFN-alpha/beta) mRNA in host cells . However, only supernatants from A12-LLV2-infected cells have significant antiviral activity . The antiviral activity in supernatants from A12-LLV2-infected embryonic bovine kidney cells is IFN-alpha/beta specific, as assayed with mouse embryonic fibroblast cells with or without IFN-alpha/beta receptors . The results obtained with cell cultures demonstrate that the ability of A12-IC to form plaques is associated with the suppression of IFN-alpha/beta expression and suggest a role for this host factor in the inability of A12-LLV2 to spread and cause disease in susceptible animals. Plant Physiol, 1999 Nov, 121(3), 805 - 812 Okadaic Acid Mimics Nitrogen-Stimulated Transcription of the NADH-Glutamate Synthase Gene in Rice Cell Cultures; Hirose N et al.; Okadaic acid (OKA), a potent and specific inhibitor of protein serine/threonine phosphatases 1 and 2A, induced the accumulation of NADH-glutamate synthase (GOGAT) mRNA within 4 h in rice (Oryza sativa L.) cell cultures . In contrast to the transient accumulation of NADH-GOGAT mRNA by NH(4)(+), OKA caused a continuous accumulation for at least 24 h . The induction of NADH-GOGAT mRNA by OKA was not inhibited in the presence of methionine sulfoximine, which inhibited the NH(4)(+)-induced accumulation of mRNA . These results suggest that the OKA-sensitive protein phosphatase is involved in the regulation of NADH-GOGAT gene expression and probably plays a role in the signal transduction pathway downstream from NH(4)(+), although a signal transduction pathway other than that of nitrogen sensing could be responsible . Nuclear run-on assays demonstrated that the accumulation of NADH-GOGAT mRNA induced by the supply of either NH(4)(+) or OKA was mainly regulated at the transcription level . OKA effects were synergistic to the NH(4)(+)-induced expression of the NADH-GOGAT gene . In the presence of K-252a, a protein kinase inhibitor, the accumulation of NADH-GOGAT mRNA induced by either NH(4)(+) or OKA was reduced . The possible roles of protein phosphatases in the regulation of NADH-GOGAT gene expression are discussed. Hum Exp Toxicol, 1999 Oct, 18(10), 634 - 9 Oxygen reactive radicals production in cell culture by okadaic acid and their implication in protein synthesis inhibition; Matias WG et al.; Okadaic acid (OA), a diarrhetic shellfish toxin is a potent promoter of tumours in mouse skin and a specific inhibitor of protein phosphatases 1 and 2A . Recently it has been shown that OA inhibited protein synthesis in a cell-free system, with 50% inhibitory concentration of 6.3x10(-12) M but the mechanism whereby this inhibition is mediated was still unclear . In the present study, the effect of OA on protein synthesis in Vero cell cultures was investigated . Protein synthesis was inhibited by OA alone in Vero cells in a concentration-dependent manner (IC50=27 ng/ml i.e . 3 . 3x10(-8) M) . Since OA also induced lipid peroxidation and likely oxygen reactive radicals, it was interesting to know whether these radicals impair the protein synthesis process . Therefore, SOD+catalase known as scavenger of active oxygen radicals were added in the culture medium in the presence of OA and labelled leucine . These enzymes partially prevented the inhibition of protein synthesis induced by OA, indicating that the formation of high reactive oxygen free radicals could be one of the pathways this marine toxin induces its toxicity . Since the prevention by SOD+catalase was only partial (the IC50 increased from 27 ng/ml to 48 ng/ml i.e . 3.3x10(-8) M to 5.9x10(-8) M) it was speculated that the production of oxygen reactive radical scavengered by SOD+catalase is not the main mechanism whereby OA induces its cytotoxicity . Vitamins E and C completely prevent the lipid peroxidation induced by OA in cells, but failed to reduce the inhibition of protein synthesis to the same level, indicating that a more specific mechanism might be responsible for protein synthesis inhibition . That is the hyperphosphorylation of elongation factor EF-2 in the protein synthesis machinery . However our results pointed to lipid peroxidation being a precocious phenomenon following the OA exposure, since a concentration with enhanced MDA production was lower than that inducing significant cellular protein synthesis inhibition. Eur J Immunol, 1999 Nov, 29(11), 3538 - 48 Myasthenia gravis: selective enrichment of antiacetylcholine receptor antibody production in untransformed human B cell cultures; Padberg F et al.; B cells producing antibodies against the acetylcholine receptor (AchR) play a central role in the pathogenesis of myasthenia gravis (MG) . Although anti-AchR autoantibodies have been studied extensively, not much is known about autoimmune B cells and their antigen-driven activation . This has mainly been due to difficulties in establishing and maintaining untransformed antigen-specific B cells in vitro . In this study, we show that highly enriched B cells from peripheral blood and thymus of MG patients can be maintained in culture over a period of 4 weeks when grown on the AchR-expressing rhabdomyosarcoma cell line TE671 together with an anti-CD40 stimulus and lymphokines . Anti-AchR antibody secretion could be detected in the majority of B cell cultures on TE671 cells up to 4 weeks . In contrast, B cells cultured on CDw32-transfected L cells binding anti-CD40 antibodies (the CD40 system) produced only small amounts of anti-AchR antibodies at single time points, whereas the overall IgG production was higher than on TE671 cells . The expression of the relevant autoantigen on the adherent cell line in addition to other growth stimuli could account for this difference and may provide a useful tool for investigating antigen-dependent B cell activation in MG and other B cell-mediated autoimmune conditions. J Immunol Methods, 1999 Oct 29, 229(1-2), 81 - 95 Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay; Plasier B et al.; Apoptosis is a form of cell death in which the dying cell plays an active part in its demise . At the morphological level, it is characterised by cell shrinkage rather than the swelling seen in necrotic cell death . In cell culture, apoptosis limits the yield of economically and medically important products, and can result in synthesis of imperfect molecules . Therefore, this process must be identified, monitored and fully understood, so that a means to regulate it can be developed . We have developed a new automatic image analysis assay for detecting apoptosis in animal cell culture on the basis of the annexin-V affinity assay . The results of this assay were compared with data generated by flow cytometry and manual scoring . All three methods were found to correspond well but image analysis like flow cytometry offers operator-independent results, and can be used as a tool for rapid monitoring of viable cell number, apoptosis and necrosis in animal cell culture . Furthermore, reduction in cell size was measured and was found to precede the appearance of phosphatidylserine on the cell surface. Arch Environ Contam Toxicol, 2000 Jan, 38(1), 52 - 8 Biochemical responses of fish sac fry and a primary cell culture of fish hepatocytes exposed to polychlorinated naphthalenes; Pesonen M et al.; Chlorinated naphthalenes are planar halogenated aromatic compounds, which are widespread in the environment . Knowledge of their biochemical and toxicological actions in aquatic biota is, however, limited . The objective of this study was to assess the toxicity of highly chlorinated naphthalene congeners found in the aquatic environment on fish sac fry and to study their effects on xenobiotic metabolizing enzymes (CYP) using a short-term primary culture of fish hepatocytes and liver microsomes . A few days after hatching, rainbow trout sac fry were administered either Hallovax 1014, a mixture of 1,2,3,4,6,7-hexachloronaphthalene and 1,2,3,5,6, 7-hexachloronaphthalene (HxCN-mix), or 1,2,3,4,5,6, 7-heptachloronaphthalene (HpCN) (0.08, 0.8, and 4 microg/sac fry injected into the yolk sac) . The exposure was terminated 2 weeks later . The naphthalene preparations did not cause any clinical signs of toxicity or difference in mortality rates between the control and treated groups . Immunohistochemical analysis of CYP1A expression in the treated sac fry revealed that staining was most pronounced in the hepatocytes and thereafter in kidney tubular epithelial cells . Moderate CYP1A staining was also seen in the mucosal epithelium of pyloric caecae and mild staining in the epithelium of olfactory organ . Staining in control sac fry was weak or absent . Exposure of the primary cell culture of trout hepatocytes to a low doses (</=10 ng/ml) of the chlorinated naphthalenes increased significantly CYP1A-associated EROD activity and CYP1A mRNA content, HxCN-mix being the most potent and thereafter HpCN and Hallovax 1014 . The higher doses (50-100 ng/ml) of each naphthalene also inhibited EROD activity . However, the content of CYP1A mRNA or the intensity of the CYP1A protein band (58 kDa) recognized by anti-trout CYP1A peptide antibodies were not decreased with increasing polychlorinated naphthalene (PCN) concentration, indicating that the inhibition was not due to reduced protein synthesis . Furthermore, in vitro analyses of the inhibitory potential of PCNs on CYP1A activity with trout liver microsomes suggested that these naphthalene preparations may be CYP1A substrates and act as competitive inhibitors of CYP1A catalyst . Our results demonstrate that highly chlorinated naphthalenes are potent modulators of fish CYP1A enzyme and suggest that hepatocytes and tubular epithelial cells are the cell types that may be vulnerable to their metabolic products for cell injury in fish sac fry. Neurosci Lett, 1999 Nov 5, 275(1), 53 - 6 Early hypoxia modulates the phenotype of dopaminergic cells in rat di- and mesencephalic cell cultures and induces a higher vulnerability of non-dopaminergic neurons to a second hypoxic exposure; Husemann B et al.; To investigate long-term effects of hypoxia on a cellular level, di- and mesencephalic cell cultures were exposed to hypoxia on in vitro day 2 (incubation in culture medium, pO2 = 10-20 mmHg, 24 h) and on in vitro day 13 (incubation in an electrolyte solution, pO2 = 10-20 mmHg, 8 h) . The numbers of neuron-specific enolase immuno-reactive (NSE-IR) and tyrosine hydroxylase immuno-reactive (TH-IR) neurons and the levels of dopamine, its main metabolites and the spontaneous and potassium-stimulated DA release were determined on DIV 15 . Hypoxia on DIV 2 did not affect the numbers of NSE-IR and TH-IR neurons, but increased the dopamine content and dopamine release by about 100% in both di-and mesencephalic cultures . In addition, this hypoxia increased the vulnerability of non-TH-IR neurons to the second hypoxic episode applied during more advanced stages of the culture development on DIV 13 . On the contrary, hypoxia exposure did not affect the vulnerability of TH-IR cells. Clin Oral Implants Res, 1999 Oct, 10(5), 379 - 93 Cell culture tests for assessing the tolerance of soft tissue to variously modified titanium surfaces; Sauberlich S et al.; The aim of our research project was to achieve an improvement in the integration of enossal dental implants in the region of peri-implantary soft tissue . Improvement in the adhesion of the gingiva of the surface of enossal implants was to be achieved by modification of the titanium surface . The effect of different modifications on the biocompatibility of the modified titanium surfaces was tested: sulfur dioxide plasma treatment of titanium; acetylene plasma treatment of titanium followed by sulfur dioxide plasma etching; plasma nitration of titanium; replacement of titanium by glycidoxypropyltrimethoxy silane; coating titanium with poly{(ethene-co-vinyl acetate)-graft-vinyl chloride} and coating titanium with fibronectin . Determination of the chemical composition of the surface was carried out using X-ray photospectroscopy . The adsorption of fibronectin at the surface of the titanium was tested using an Enzyme Linked Immunosorbent Assay . In selected in vitro tests with human gingival fibroblasts, cell morphology was assessed using scanning electron microscopy and light microscopy . Cell proliferation and protein synthesis, as well as the activity of mitochondrial dehydrogenases were evaluated . By means of centrifugation and by determining initial cell adhesion, the adhesion of gingival fibroblasts was investigated . According to the kind of modification made to the titanium surfaces, it was possible to observe differences in the cellular behavior of gingiva fibroblasts on the differently modified surfaces of the implants . Coating the titanium using fibronectin produced optimization of cell growth and improvement in the adhesion of gingiva fibroblasts to the implant surface . In contrast, modification of the titanium with poly{(ethene-co-vinyl acetate)-graft-vinyl chloride} generally resulted in a deterioration of the biocompatibility of the surface . A marked correlation between the cellular compatibility of the modified titanium and the surface modification made did not become apparent . One reason for this is the large number of parameters determining the interaction between implant and tissue. Arch Virol, 1999, 144(10), 1961 - 75 Antigenicity and pathogenicity characteristics of molecularly cloned chicken anaemia virus isolates obtained after multiple cell culture passages; Scott AN et al.; The Cux-1 isolate of chicken anaemia virus (CAV), which had received 310 (P310) cell culture passages, was substantially less pathogenic than virus that had been passaged 13 times (P13) . Molecularly cloned virus isolates, selected from the P310 and P13 virus populations using recombinant DNA cloning and transfection procedures, reacted differently with 4 CAV-specific monoclonal antibodies (MAbs), which had been raised to low-passage Cux-1 virus . In contrast to the strong immunofluorescence (IF) reactivities exhibited by all P13 cloned isolates tested, 80% and 57% of the P310 cloned isolates reacted weakly with MAbs 2A9 and 4H4, which are directed against conformational epitopes on the capsid protein, VP1 . Sequence analysis of the VP1 coding regions possessed by ten P310 and two P13 cloned isolates showed that 6 amino acid changes within VP1 had been selected by multiple-cell culture passage . One of these at position 89 in VP1 appeared to be crucial for determining reactivity with MAb 2A9 . Of nine P310 cloned isolates evaluated, 8 were substantially attenuated compared to the low-passage Cux-1 virus pool . It is concluded that the individual virus variants comprising the P310 virus pool differ with regards to their antigenicity and pathogenicity. Mol Cell Biol Res Commun, 1999 Aug, 2(2), 131 - 7 Development of human and rabbit vaginal smooth muscle cell cultures: effects of vasoactive agents on intracellular levels of cyclic nucleotides; Traish A et al.; In this study, we subcultured and characterized human and rabbit vaginal smooth muscle cells and investigated the synthesis of second messenger cyclic nucleotides in response to vasodilators and determined the activity and kinetics of phosphodiesterase (PDE) type 5 (EC 3.1.4.35 3',5'-cyclic GMP phosphodiesterase) . Cultured vaginal cells exhibited growth characteristics typical of smooth muscle cells and immunostained with antibodies against alpha smooth muscle actin . The cells retained functional prostaglandin E and beta-adrenergic receptors as demonstrated by increased intracellular cAMP synthesis in response to PGE1, or isoproterenol . The response to these vasoactive substances was augmented with forskolin, suggesting stabilization of G-protein-activated adenylyl cyclases . Treatment with the nitric oxide donor, sodium nitroprusside, in the presence of sildenafil, a PDE type 5 inhibitor, enhanced intracellular cGMP synthesis and accumulation . Incubation of rabbit vaginal tissue with sildenafil, sodium nitroprusside, and PGE1 or forskolin produced a marked increase in intracellular cGMP . These observations were similar to findings with cultured cells and suggest that subcultured cells retain functional characteristics exhibited in intact tissue . The cells retained phosphodiesterase type 5 expression as shown by specific cGMP hydrolytic activity . Sildenafil and zaprinast inhibited cGMP hydrolysis competitively and bound with high affinity (Ki = 7 and 250 nM, respectively) . These observations suggest that cultured human and rabbit vaginal smooth muscle cells retained their metabolic functional integrity and this experimental system should prove useful in investigating the pathway of nitric oxide and PDE type 5 inhibitors in modulating vaginal smooth muscle tone. Invest Ophthalmol Vis Sci, 1999 Nov, 40(12), 3012 - 6 Antioxidant pattern in uveal melanocytes and melanoma cell cultures; Blasi MA et al.; PURPOSE: To investigate the antioxidant status of cultured uveal melanocytes from patients with uveal melanoma and uveal melanoma cells to characterize some of the biochemical properties of these cells in respect to the normal cutaneous melanocytes . METHODS: The fatty acid pattern of membrane phospholipids, intracellular vitamin E level, and superoxide dismutase (SOD) and catalase activities were studied in uveal melanocytes (n = 10) and uveal melanoma cell (n = 10) cultures, by gas chromatography mass spectrometry or by spectrophotometer . RESULTS: Among the uveal melanocyte cultures, two groups were differentiated, according to catalase activity: group A with catalase values comparable to those of cutaneous ones and higher SOD activity and group B with catalase values 2 SD lower (P<0.001) and lower SOD activity . Vitamin E concentration was not significantly different between melanoma cells and melanocytes, whereas a significantly higher percentage of polyunsaturated fatty acids was found in melanoma cells and the B group of melanocytes (P = 0.022) . In uveal melanoma cells SOD activity was significantly lower than that detected in uveal melanocytes (P< 0.005) . CONCLUSIONS: These results show a different pattern of antioxidants in uveal melanocytes with respect to cutaneous ones, possibly related to the anatomic distribution . However, as in cutaneous melanocytes, two subgroups were identified on the basis of the antioxidant pattern that could be the expression of a constitutional increased susceptibility to oxidative stress in some subjects . Moreover, an imbalance of the antioxidants was observed in melanoma cells, possibly related to the disease status and progression. Biochem Biophys Res Commun, 1999 Nov, 265(1), 233 - 9 TGF-beta enhances osteoclast differentiation in hematopoietic cell cultures stimulated with RANKL and M-CSF; Sells Galvin RJ et al.; TGF-beta has been shown to inhibit and stimulate osteoclastogenesis . The purpose of this study was to evaluate the effects of TGF-beta in hematopoietic cell cultures stimulated with RANKL and M-CSF . In cocultures of hematopoietic cells and BALC cells (a calvarial-derived cell line), TGF-beta inhibited tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell formation . In contrast, TGF-beta enhanced TRAP-positive multinucleated cell formation up to 10-fold in hematopoietic cell cultures containing few osteoblastic/stromal cells . Likewise, TGF-beta increased the number of calcitonin receptor (CTR)-positive multinucleated and mononucleated cells in a concentration-dependent manner . An increase in cell size and multinuclearity was also observed in the presence of TGF-beta . The stimulatory effects of TGF-beta were dependent on the presence of M-CSF and RANKL . When differentiated on bovine cortical bone slices, these cells formed resorption lacunae . These results suggest that TGF-beta has a direct stimulatory effect on osteoclastogenesis in hematopoietic cells treated with RANKL and M-CSF . Ann Urol (Paris), 1999, 33(5), 382 - 8 {Urothelium cell culture: what is the future? An experimental study in rabbits}; Bertschy C; Since the first human bladder reconstruction in 1989 using an ileal segment, many alternatives have been proposed to recreate a bladder reservoir as adapted as possible to physiological conditions . Since the development of urothelial cell culture alone and then in combination with matricial supports, various experimental trials have studied the possibility of using this neurothelium for surgical purposes . This experimental study in rabbits tested the compatibility of two different biosynthetic supports in an enterocystoplasty and the survival in in vitro urothelial cells grafted onto this support. Can J Physiol Pharmacol, 1999 Aug, 77(8), 598 - 605 Slow wave and spike action potentials recorded in cell cultures from the muscularis externa of the guinea pig small intestine; Espinosa-Luna R et al.; Intracellular recordings were obtained to investigate whether slow wave and spike type action potentials are present in cell cultures of the muscularis externa from the guinea pig small intestine . The muscularis externa of the small intestine was dissociated by using specific purified enzymes and gentle mechanical dissociation . Cells were plated on cover slips and maintained in culture for up to 4 weeks . Dissociated cells obtained in this way reorganized themselves in a few days to form small cell clumps showing spontaneous movements . Intracellular recordings of these clumps displayed both spike and slow wave type action potentials . Spikes were observed on top of some slow waves and were abolished by the addition of nifedipine or the removal of extracellular calcium . Slow waves, however, were nifedipine insensitive and temperature sensitive, and were abolished by octanol (a gap junction blocker) and forskolin (an adenyl cyclase activator) . Slow waves were never observed in small clumps (<50 microm), suggesting that a critical mass of cells might be required for their generation . These observations demonstrated for the first time the presence of nifedipine-insensitive slow waves in cell cultures of the muscularis externa from the guinea pig small intestine . Cell cultures allow rigorous control of the immediate environment for the cells and this should facilitate future studies on the molecular and cellular mechanisms responsible for the slow waves in the gastrointestinal tract. J Neurochem, 1999 Nov, 73(5), 1894 - 900 Serotonin N-acetyltransferase mRNA levels in photoreceptor-enriched chicken retinal cell cultures: elevation by cyclic AMP; Greve P et al.; Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is a key regulatory enzyme in the biosynthesis of melatonin . Previous studies have shown that the activity of this enzyme in the chicken retina is regulated by a cyclic AMP-dependent mechanism . In the present report, we investigated whether cyclic AMP can regulate the levels of AA-NAT mRNA in photoreceptor-enriched chick retinal cell cultures . AA-NAT mRNA levels were elevated by acute treatment with cyclic AMP protagonists, including forskolin; this response was blocked by H-89, a selective inhibitor of cyclic AMP-dependent protein kinase . Forskolin did not alter the rate of disappearance of AA-NAT mRNA in actinomycin D-treated cells, suggesting that cyclic AMP enhances transcription of the AA-NAT gene . Forskolin-induced elevation of AA-NAT mRNA levels was enhanced by cycloheximide, which decreased the degradation of the transcript in cells treated with actinomycin D . These studies indicate that the abundance of AA-NAT mRNA is regulated in part through a cyclic AMP-dependent mechanism. Glia, 1999 Nov, 28(2), 114 - 27 Neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha; Downen M et al.; We examined cytokine-mediated neuronal death in neuron-astrocyte cultures from second trimester human fetal cerebrum . In these cultures, high-output inducible nitric oxide synthase (NOS) and tumor necrosis factor-alpha (TNFalpha) are expressed in astrocytes after exposure to IL-1beta/IFNgamma . Neuronal cell death was evident at >/=48 h following cytokine stimulation . Neutralizing anti-TNFalpha antiserum inhibited ( approximately 48%) neurotoxicity in IL-1beta/IFNgamma-treated cultures, demonstrating a role for endogenously produced TNFalpha . Interestingly, the degree of neuroprotection conferred by superoxide dismutase or N-methyl D-aspartate (NMDA) receptor antagonists in these cultures was smaller and variable . Similarly, the effect of the NOS inhibitor, N(G)-monomethyl L-arginine (NMMA) on IL-1beta/IFNgamma-induced neuronal death was variable, showing no statistically significant effect when results from more than 30 independent cultures were averaged . Neurons die by apoptosis in cytokine-treated human fetal CNS cultures as shown by the characteristic nuclear morphology as well as positive labeling for TUNEL . Our results demonstrate a potent neurotoxicity mediated by the cytokine combination IL-1beta/IFNgamma in primary human neuron-astrocyte cultures and a crucial role for endogenous TNFalpha in mediating neurotoxicity in this system . These results firmly establish the neurotoxic potential of the inflammatory cytokines IL-1beta and TNFalpha in the human CNS . Life Sci, 1999, 65(14), 1455 - 61 Paradoxical effect of neuroleptic drugs on prolactin secretion by rat pituitary cell cultures; Braghiroli L et al.; Several antipsychotic drugs reverse the dopamine-induced inhibition of prolactin release by rat pituitary cell cultures . Paradoxically, at high doses and without dopamine, antipsychotic drugs can also inhibit prolactin secretion . The mechanism underlying this phenomenon is unclear . Some evidence suggests that these drugs have an agonistic action . We sought to verify whether clozapine and fluphenazine, at doses higher than those reversing dopamine-induced inhibition of prolactin secretion in vitro, show this paradoxical effect and eventually a partial agonistic action . Both antipsychotics inhibited prolactin secretion, clozapine at doses starting from 10(-6) M and fluphenazine from 10(-7) M . Haloperidol reversed clozapine-induced prolactin inhibition but left fluphenazine-induced inhibition unchanged . These in vitro findings suggest that clozapine has a partial agonistic action on dopaminergic receptors but fluphenazine does not. Brain Res, 1999 Oct 2, 843(1-2), 95 - 104 Cellular localization of dopamine-releasing protein (DARP) in rat C6 glioma and primary mesencephalic cell cultures; Smith S et al.; Dopamine-releasing protein (DARP) is a multisubunit protein shown to have dramatic effects on development, recovery, and function of the rat catecholaminergic (CA) system . This study details efforts to determine if glial cells are responsible for the production of DARP in the central nervous system (CNS) . Enzyme-linked immunosorbent assays (ELISA), Western blotting, and immunocytochemical techniques were employed to measure DARP levels and identify DARP immunoreactive proteins in rat C6 glioma cells and medium, respectively . ELISA analysis of serum-free C6 culture media revealed a maximal concentration of DARP by culture day 1 . However, ELISA analysis of C6 cultures grown in F-12K/serum medium revealed that maximal levels of DARP were detected on culture day 6 with a 108% increase in DARP immunoreactivity from culture day 1 . These values were determined using a polyclonal antibody generated against DARP-36aa (anti-DARP-36aa), a synthetic peptide with dopamine (DA) releasing activity, and anti-DARP B9-B10, a monoclonal antibody generated against partially purified DARP . Western blot analysis revealed that anti-DARP B9-B10 recognized proteins of approximately 60, 50, and 45 kDa in C6 cell homogenates while anti-DARP-36aa had immunoreactivity with the 60-kDa protein alone . Immunocytochemical studies demonstrated that anti-DARP-36aa and anti-DARP B9-B10 had strong immunoreactivity with proteins throughout the cytosol and in several processes of C6 cells . These results reveal that DARP is detected in glioma cells and secreted in a time-dependent fashion during culture . Primary rat mesencephalic cultures were also examined using immunocytochemistry . Incubation with DARP antibodies and antisera against glial fibrillary acidic protein (GFAP) revealed that DARP and GFAP immunoreactivity co-localized in primary mesencephalic cultures . However, the majority of DARP immunoreactivity was localized to cells without GFAP staining . These findings reveal that DARP is detected in astrocytes although the majority of DARP immunoreactivity is found in non-astrocyte type cells. Neurobiol Dis, 1999 Oct, 6(5), 347 - 63 Neurons undergo apoptosis in animal and cell culture models of diabetes; Russell JW et al.; Recent clinical trials indicate that the severity of diabetic neuropathy is correlated with the level of patient glycemic control . In the current study, hyperglycemia induces apoptotic changes in dorsal root ganglion neurons and Schwann cells in vivo both in streptozotocin-treated diabetic rats and in rats made acutely hyperglycemic with infused glucose . Typical apoptotic nuclear and cytoplasmic changes are observed . In addition mitochondrial changes recently reported to occur as part of the apoptotic cascade, such as ballooning of mitochondria and disruption of the internal cristae, are seen in diabetic dorsal root ganglion neurons and Schwann cells . Similar changes have been reported in neurons in the presence of oxidative stress . In order to study the neurotoxic effects of high glucose we developed an in vitro model using rat dorsal root ganglion neurons . In dorsal root ganglion cultured in defined medium, addition of moderate glucose levels results in neurite degeneration and apoptosis . These changes are coupled with activation of caspase-3, dependent on the concentration of glucose . The apoptotic changes observed in vitro are similar to those observed in vivo . In contrast, addition of IGF-I, even at physiological concentrations, prevents activation of caspase-3 and neuronal apoptosis in vitro . We suggest that oxidative stress may promote the mitochondrial changes in diabetic animals and lead to activation of programmed cell death caspase pathways . These results imply a new pathogenetic mechanism for diabetic sensory neuropathy . J Clin Microbiol, 1999 Nov, 37(11), 3722 - 4 Isolation of Rickettsia prowazekii from blood by shell vial cell culture; Birg ML et al.; A blood sample from a patient who returned from Algeria with a fever inoculated on human embryonic lung fibroblasts by the shell vial cell culture technique led to the recovery of Rickettsia prowazekii . The last clinical strain was isolated 30 years ago . Shell vial cell culture is a versatile method that could replace the classic animal and/or embryonated egg inoculation. Brain Res Dev Brain Res, 1999 Sep 6, 116(2), 159 - 67 Brain endogenous insulin effects on neurite growth within fetal rat neuron cell cultures; Schechter R et al.; We have previously described insulin to be synthesized "de novo" within the fetal rat brain and that brain endogenous insulin {I(n)} promoted neurofilament distribution within fetal neurons . In this study, we investigated the role of I(n) in neuron axonal growth . Rat fetal brain stem cells from 16-day gestational age were cultured in an IFDM and treated with an insulin antibody . In addition, the cell cultures were also treated in defined medium with the addition of: 5 ng, 20 ng or 100 ng/ml of insulin or 100 ng/ml insulin-like growth factor 1 (IGF-1) . The neuron cell cultures were studied at 1 and 3 days of incubation . The presence of preproinsulin mRNA and insulin immunoreaction confirmed the "de novo" synthesis of insulin by the fetal neuron cell cultures . Axonal growth was similar by day 1 of the study in all the media, but in insulin medium containing 100 ng/ml of insulin the axonal length was significantly longer . By day 3 of incubation I(n) promoted axonal growth . Treating the neurons with an insulin antibody confirmed these findings, with a significant decrease in axonal length (p<0.05) . The treatment with different concentrations of exogenous insulin did not promote axonal growth beyond I(n) by day 3 of incubation . IGF-1 did not promote axonal growth by day 3 of incubation . In summary, I(n) may promote axonal growth during brain development. Vet Microbiol, 1999 Sep 1, 69(1-2), 67 - 8 Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and persistently infected cats; Radford AD et al.; The study determines the sequence evolution of feline calicivirus both in cell culture and in persistently infected cats and relates this to changes in virus neutralisation. J Med Chem, 1999 Oct 7, 42(20), 4122 - 8 Design and synthesis of lipophilic phosphoramidate d4T-MP prodrugs expressing high potency against HIV in cell culture: structural determinants for in vitro activity and QSAR; Siddiqui AQ et al.; A series of new substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized as membrane-soluble nucleotide prodrugs, to extend and quantify the SAR observed for an earlier series of related derivatives . All of the compounds were found to be significantly more potent against HIV in cell culture than the nucleoside analogue d4T, and most were also found to be significantly more potent than the parent phosphoramidate . A Hansch type QSAR analysis was applied to the combined series of 21 compounds . The results of this analysis revealed anti-HIV activity to be principally dependent on lipophilicity in a quadratic manner, with terms representing substituent steric bulk and electronic effects having a minimal significance. Biomaterials, 1999 Oct, 20(19), 1773 - 82 Biological evaluation of RGD peptidomimetics, designed for the covalent derivatization of cell culture substrata, as potential promotors of cellular adhesion; Marchand-Brynaert J et al.; Our aim was to replace the proteins and peptides, generally used for the biocompatibilization of polymer substrata, with synthetic molecules mimicking the RGD (Arg-Gly-Asp) active sequence . Based on the (L)-tyrosine template, RGD peptidomimetics were constructed; one molecule 3 was equipped with an anchorage arm that allowed its covalent grafting on a culture substratum made from poly(ethylene terephthalate) (PET) microporous membrane . The amount of fixed molecules was readily determined by XPS, using a fluorine tag incorporated in the peptidomimetic structure . The binding of peptidomimetics 1-3 to the vitronectin (VN) and fibronectin (FN) receptors could not be revealed in a test of inhibition of MSC 80 cells adhesion, by the synthetic compounds in solution placed in competition with the adhesive proteins (VN and FN) coating polystyrene plates . However, the cell-attachment activity of peptidomimetic 3 was shown by culturing CaCo2 cells, in the absence of serum, on the PET substratum grafted with 3 . The performance of this support was similar to that of PET grafted with the reference peptide RGDS (Arg-Gly-Asp-Ser), and only reduced by half comparatively to the PET grafted with FN. Histol Histopathol, 1999 Oct, 14(4), 1231 - 40 Quantitative in situ hybridization for the evaluation of gene expression in asynchronous and synchronized cell cultures and in tissue sections; Barlati S et al.; We describe an image analysis (IA) system that has been applied for the quantitative evaluation of mRNAs evidenced by in situ hybridization (ISH) with radiolabelled probes in cultured cells and in tissue sections . The ISH-IA method was used for the evaluation of cultured cell morphological parameters such as cell and nucleous area (CA and NA, respectively) in parallel with the levels of mRNAs detected as hybridization grains areas (GA) . The evaluation of these parameters, together with the analysis of the levels of mRNAs (c-jun, cyclin A) specific for given cell cycle phases (i.e . G1 and S/G2), allowed the identification, in asynchronous cultures of human skin fibroblasts, of cells in G1 and S/G2 phases . The mRNA levels measured by ISH-AI were comparable with those detected by RT-PCR . This method was also applied for the analysis of fibronectin (FN) gene expression in control skin fibroblasts in relationship with the different phases of the cell cycle and in comparison with a tumor cell line (Sk-Hep1), heterogeneous either for morphometric parameters or for the levels of this transcript . Finally, the ISH-AI was applied for the semiquantitative evaluation of the expression, localization and alternative splicing pattern of FN mRNA in normal liver and in hepatocellular carcinoma (HCC) tissue sections. AIDS Res Hum Retroviruses, 1999 Sep 20, 15(14), 1265 - 77 Effect of extracellular human immunodeficiency virus type 1 glycoprotein 120 on primary human vascular endothelial cell cultures; Huang MB et al.; During the course of an HIV-1 infection, free infectious and noninfectious virus particles, and free HIV-1 proteins, circulate within the host, exposing the host endothelium to these viral factors, even if the endothelium is not infected . This suggests that extracellular HIV-1 proteins could influence endothelial cell function, leading to pathogenesis . In light of this, we have used primary cultured human vascular endothelial cells (HUVECs) to screen for effects of the HIV-1 protein gp120 on endothelial cell function . The results of this study show that short exposure of HUVEC cultures to this protein causes significant levels of cytotoxicity . Further, using several different assays, we have shown that this cytotoxic effect on HUVECs appears to be due to induction of an apoptotic program . The biphasic nature of gp120 titration curves suggests that multiple cellular factors are mediating these gp120-induced effects . Competition studies appear to confirm this by showing that the apoptotic effect is mediated through two cell surface receptors on HUVECs, CCR5 and CXCR4 . Alternatively, competition studies examining CD4 receptors suggests that CD4 played no role in gp12O-induced effects on HUVECs. Neurosci Lett, 1999 Sep 24, 273(1), 57 - 60 Astrocytes rather than neurones mediate interleukin-1beta dependent nitric oxide and superoxide radical release in primary hypothalamic rat cell cultures; Tolias CM et al.; The cellular sources of nitric oxide in the hypothalamus are thought to be 'NOergic' neurones . Using free radical electrochemical sensors we investigated nitric oxide and superoxide radical release in primary hypothalamic cell cultures . We present evidence that under interleukin-1beta (IL-1beta) stimulation hypothalamic astrocytes rather than neurones release nitric oxide . Under L-arginine deprivation and IL-1beta stimulation a concentration-dependent release of superoxide was also observed, which was inhibited in the presence of nitric oxide synthase inhibitor nitro-L-argininemethyl-ester . These findings support the hypothesis that the balance between nitric oxide and superoxide may be of vital importance in hypothalamic pathophysiology. Life Sci, 1999, 65(13), PL167 - 70 Effect of estradiol metabolites on prostacyclin synthesis in human endothelial cell cultures; Seeger H et al.; Estradiol can stimulate prostacyclin production in the vessel wall, thereby eliciting vasodilatation . In the present work the effect of the estradiol metabolites estrone, 2-methoxyestrone, 2-methoxyestradiol, and 16alpha-hydroxyestrone were investigated to find out if they are also able to stimulate prostacyclin synthesis . All metabolites triggered an increase of prostacyclin synthesis in human endothelial cells starting at a concentration of 10(-9) M . The parent substance, 17beta-estradiol, accomplished this effect only starting at a concentration of 10(-8) M . These results indicate that estradiol metabolites may take part in the estradiol-induced vasodilatation in vivo. Gynecol Oncol, 1999 Oct, 75(1), 72 - 7 Ovarian carcinoma cell cultures are resistant to TGF-beta1-mediated growth inhibition despite expression of functional receptors; Yamada SD et al.; OBJECTIVE: The purpose of this study was to determine the response of ovarian carcinoma cells to TGF-beta1 and to examine components of the TGF-beta signaling pathway . METHODS: Twenty-three primary ovarian cancer cell (CSOC) cultures established from solid ovarian carcinomas were treated with TGF-beta1 and assayed for growth response by MTT assay . Expression of TGF-beta receptor I (TbetaR-I) and receptor II (TbetaR-II), essential for effective signaling, was determined by Western analysis of CSOC cultures . TGF-beta1 ligand-induced phosphorylation of TbetaR-I was determined by immunoprecipitation of TbetaR-I followed by a protein kinase assay to assess TbetaR-I phosphorylation, an essential first step in TGF-beta signal transduction . Gelatin zymography performed on 5 CSOC cultures incubated with TGF-beta1 was used to determine TGF-beta's effect on matrix metalloproteinase production . Normal ovarian surface epithelial cells were used for comparison . RESULTS: Eighteen of twenty-three (78%) CSOC cultures demonstrated no significant growth inhibition in response to TGF-beta1 treatment . All cell cultures expressed TbetaR-I and TbetaR-II and exhibited TbetaR-I phosphorylation following TGF-beta1 treatment . CSOC cultures produced significantly higher levels of matrix metalloproteinase-2 (MMP-2) than normal ovarian surface epithelial cells; however, the level of MMP-2 expression was not regulated by TGF-beta1 . CONCLUSION: These results indicate that TGF-beta1 resistance and higher levels of MMP-2 production may be inherent properties of the ovarian cancer phenotype . The initial steps in the TGF-beta signaling pathway, receptor expression, ligand binding, and TbetaR-I phosphorylation, appear to be functional in primary ovarian cancer cell cultures . Therefore, the mechanism of growth resistance is downstream of TbetaR-I phosphorylation . J Neurosci Res, 1999 Oct 15, 58(2), 308 - 17 Aging in a dish: age-dependent changes of neuronal survival, protein oxidation, and creatine kinase BB expression in long-term hippocampal cell culture; Aksenova MV et al.; Results from different experimental systems demonstrate that increased oxidative damage plays a role in normal aging and age-associated pathology . In the current study, long-term cultures of hippocampal neurons were examined as a model system . It was established that neuronal survival in long-term culture decreases according to the Gompertz law and that neuronal "aging in the dish" is associated with increased oxidative damage of cell proteins . The increase of protein carbonyl formation in aged neurons was demonstrated both by Western blot analysis for oxidized proteins and by in situ immunocytochemical method, which was developed to analyze protein oxidation in fixed cells . In aging neuronal cultures, a gradual increase in creatine kinase (CK) content but decreased activity of enzyme per immunoreactive protein was found, suggesting the accumulation of inactive CK molecules . The increase in CK content was not a result of generalized protein elevation, since analysis of beta-actin content showed a time-dependent loss, probably reflecting decreased number of cellular processes with aging . These findings, showing "aging in a dish," consistent with the notion that aging is associated with increased protein oxidation, provide a system for study of age-related neurodegenerative disorders associated with oxidative stress . Adv Exp Med Biol, 1999, 457, 415 - 21 Cellular drug resistance in childhood acute myeloid leukemia . A mini-review with emphasis on cell culture assays; Kaspers GJ et al.; Cellular drug resistance is an important limiting factor in the success of chemotherapy in childhood acute myeloid leukemia (AML) . We summarize the results of the studies published sofar that have focussed on drug resistance in childhood AML, using cell culture assays . We also briefly report our own results of an ongoing study . Finally, potential applications of cellular drug resistance testing are discussed . It appears that cellular drug resistance differs between AML and acute lymphoblastic leukemia and between subgroups of AML patients, that AML cells of relapsed patients are more resistance to cytarabine than those of untreated patients, and that in vitro resistance to cytarabine and daunorubicin is related to a worse prognosis . However, more and larger studies are required to determine the exact role of cellular drug resistance testing in the treatment of childhood AML. Toxicology, 1999 Aug 13, 136(1), 27 - 39 Extracellular calcium is required for the polychlorinated biphenyl-induced increase of intracellular free calcium levels in cerebellar granule cell culture; Mundy WR et al.; Recent studies from the laboratory indicate that polychlorinated biphenyl (PCB) congeners can alter signal transduction and calcium homeostasis in neuronal preparations . These effects were more pronounced for the ortho-substituted, non-coplanar congeners, although the mechanisms underlying these effects are not clear . In the present study the time-course and concentration-dependent effects of coplanar and non-coplanar PCBs on intracellular free calcium concentration ({Ca2+}i) in cerebellar granule cell cultures were compared using the fluorescent probe fura-2 . The ortho-substituted congeners 2,2'-dichlorobiphenyl (DCB) and 2,2',4,6,6'-pentachlorobiphenyl (PeCB) caused a gradual increase of {Ca2+}i while the non-ortho-substituted congeners 4,4'-DCB and 3,3',4,4',5-PeCB had no effect . The increase of {Ca2+}i produced by 2,2'-DCB was time- and concentration-dependent . Further studies examined possible mechanisms for this rise in {Ca2+}i . In contrast to the muscarinic agonist carbachol, the effects of 2,2'-DCB on {Ca2+}i were not blocked by thapsigargin and required the presence of extracellular calcium . The effects of ortho-substituted PCBs may depend on their ability to inhibit calcium sequestration as 2,2'-DCB significantly inhibited 45Ca2+-uptake by microsomes and mitochondria while 3,3',4,4',5-PeCB had no effect . In addition, 2,2'-DCB significantly increased the binding of {3H}inositol 1,4,5-trisphosphate to receptors on cerebellar microsomes, suggesting another possible mechanism by which ortho-substituted PCBs can mobilize {Ca2+}i . These results show that PCBs increase {Ca2+}i in vitro via a mechanism that requires extracelluar calcium, and support previous structure-activity studies indicating that ortho-substituted PCBs are more potent than non-ortho-substituted PCBs. Bioorg Med Chem Lett, 1999 Sep 6, 9(17), 2555 - 60 Simple mono-derivatisation of the aryl moiety of D4A and DDA-based phosphoramidate prodrugs significantly enhances their anti-HIV potency in cell culture; Siddiqui AQ et al.; Simple mono-derivatisation of the aryl moiety of some phosphoramidate pronucleotide derivatives of d4A and ddA served to increase the lipophilicity of these membrane-soluble prodrugs . A concomitant and significant enhancement of potency against HIV-1 and HIV-2 in vitro was observed for the ddA- and d4A-based prodrugs compared to the original underivatised prodrugs. Pharm Res, 1999 Sep, 16(9), 1380 - 5 Effects of pharmaceutical compounds on ciliary beating in human nasal epithelial cells: a comparative study of cell culture models; Agu RU et al.; PURPOSE: To test two in vitro human nasal epithelial cell culture systems for their ability to screen the effects of pharmaceutical compounds on ciliary beating . METHODS: Human nasal epithelial cells were cultured as monolayer and in a sequential monolayer-suspension culture with in vitro ciliogenesis . The influence of reference cilio-stimulatory compounds (glycocholate, isoprenaline), reference cilio-inhibitory compounds (chlorocresol, diphenhydramine) and pH on ciliary beating was investigated using computerized microscope photometry . RESULTS: Sodium glycocholate (0.5% w/v) maximally and reversibly increased CBF of the cells in both culture systems by 26 +/- 4% (monolayer) and 18 +/- 6% (suspension) . Similarly, isoprenaline (10(-3) M) maximally, but irreversibly increased CBF of the cells by 14 +/-3% (monolayer) and 17 +/- 4% (suspension) . Chlorocresol (0.005% w/ v) reversibly reduced the CBF of the cells by 50 +/- 6% (monolayer) and 34 +/- 4% (suspension); at a higher concentration (0.1% w/v) it resulted in instantaneous and irreversible ciliostasis . Diphenhydramine (0.1% w/v) reversibly reduced CBF in both culture systems by 45 +/- 13% (monolayer) and 69 +/- 5% (suspension); irreversible cilio-stasis occurred in less than 2 minutes in both culture systems upon cell exposure to diphenhydramine (1.0% w/v) . In the monolayer culture system, CBF was stable only within the physiological pH range of 6.5-8.0; ciliary beating in the suspension culture remained stable within a pH range of 4.0-10.0 . CONCLUSIONS: Both cell culture systems are suitable for screening the effects of pharmaceutical compounds on ciliary beating . Especially the sequential monolayer-suspension culture appears to be very promising as ciliary activity can be preserved for as long as 6 months. Cell Physiol Biochem, 1999, 9(3), 150 - 72 Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures . Are renal cells in tissue culture hypoxic? Gstraunthaler G, Seppi T, Pfaller W. When renal proximal tubular cells are brought into tissue culture, they revert from oxidative metabolism and gluconeogenesis to high rates of glycolysis . Among the factors possibly responsible for this metabolic conversion, limited oxygen availability and/or substrate supply are discussed . In order to study the role of these factors on long-term cultures, the impact of growth conditions, culture media volume, and glucose content on carbohydrate metabolism of the continuous renal cell lines LLC-PK(1) (porcine kidney) and OK (opossum kidney) was investigated . The impact of culture media volumes and glucose content, respectively, was determined by overlaying confluent monolayer cultures of LLC-PK(1) and OK cells (i) with increasing volumes of culture medium and thus increasing amounts of glucose, and (ii) with increasing culture medium volumes at constant absolute amounts of glucose by adding glucose-free medium, in order to increase volume at a constant glucose supply . Alternatively, and in order to improve cell oxygenation, LLC-PK(1) cells were also cultured in roller bottles . Cell carbohydrate metabolism was assessed by measuring rates of glucose consumption and lactate production, respectively, and by determination of specific activities of the key glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) . Mitochondrial phosphate-dependent glutaminase (PDG) was assayed as marker enzyme of oxidative metabolism of glutamine . In LLC-PK(1) and OK cells, rates of glucose consumption were independent of the initial glucose concentrations and/or the culture media volumes used . Glucose was quantitatively converted to lactate, which accumulated in a 1:2 molar ratio . Lactate in culture media reached a maximum content after 24 h, and was reutilized by the cell lines thereafter . Interestingly, the rates of lactate reuptake strictly depended on culture medium volume, indicating a volume-induced stimulation of oxidative lactate metabolism . Marked changes were found for the specific activities of glycolytic enzymes . In LLC-PK(1) cells, increased glucose supply caused increases in HK, PFK, PK and LDH activities, which were superimposed to the stimulatory effects of increased media volumes . Enzyme activity showed a biphasic response, indicating that both glucose supply and culture media volumes covering the cell monolayer are factors determining glycolytic rates of LLC-PK(1) renal cells . Conversely, in OK cells glycolytic enzyme activities decreased with increasing culture media volumes at constant glucose levels . As expected, under conditions of enhanced oxygenation of LLC-PK(1) cells in roller bottle culture, glycolytic enzyme activities decreased, whereas PDG activity increased, which was paralleled by increased rates of ammonia generation . Thus, changes in nutrient supply and oxygenation of renal epithelial cell cultures by altered culture media volumes dramatically influence metabolic rates and levels of enzyme activities, respectively. Cell Physiol Biochem, 1999, 9(3), 139 - 49 Cell culture conditions determine apolipoprotein CIII secretion and regulation by fibrates in human hepatoma HepG2 cells; Clavey V et al.; Fibrates are widely used drugs which lower triglycerides and increase HDL concentrations in serum . Recent findings from our laboratory have shown that fibrates repress apolipoprotein (apo) CIII gene expression, an effect that explains partially the triglyceride-lowering activity of these drugs . The goal of the present study was to compare the effect of various fibrates on apo CIII gene expression in the human hepatoblastoma cell line HepG2 . First, we demonstrate that the level of apo CIII secretion by HepG2 cells is controlled by serum factors whereas apo CIII mRNA levels are not and even increase under conditions when apo CIII secretion dramatically decreases . Twelve different fetal calf serum batches were tested during this study and apo CIII secretion in cell medium could only be detected with three of them . The effect of serum on apolipoprotein secretion was more pronounced for apo CIII whereas other apolipoproteins (apo E, apo B, apo AII and apo AI) were affected to a lesser extent . Under serum conditions allowing apo CIII secretion, treatment with the peroxisome-proliferator activated receptor (PPAR)alpha activators fenofibrate, gemfibrozil and Wy-14643 result in a marked lowering of apo CIII secretion and gene expression, this effect being most pronounced with Wy-14643 . Comparison of the activity of a PPARgamma-specific ligand, the antidiabetic thiazolidinedione, BRL-49653 and a PPARalpha ligand Wy-14643 showed a marked decrease of apo CIII secretion and gene expression after activation of PPARalpha but not PPARgamma . In conclusion, fibrates down-regulate apo CIII gene expression in human HepG2 cells, most likely via PPARalpha but not via PPARgamma . However, these effects are only observed in HepG2 cells cultured under appropriate conditions. Dev Biol Stand, 1999, 98, 177 - 81; discussion 197 Cell culture influenza vaccine: an Australian perspective; Chang L et al.; The Australia Influenza Vaccine Committee makes independent decisions concerning the influenza vaccine formulation for Australia . Large-scale cell culture using MDCK cells would improve response time for vaccine production in the face of a new pandemic . There must be a consensus with respect to the use of MDCK or other cells before the next pandemic . It is unrealistic to expect any national regulatory authority to determine what safety requirements should be met before approving a cell substrate for vaccine production during an influenza pandemic . Regulatory issues seen as obstacles to the approval of MDCK cells as an accepted cell substrate for influenza vaccine production are identified. Dev Biol Stand, 1999, 98, 171 - 5; discussion 197 Regulatory perspective in the United States on cell cultures for production of inactivated influenza virus vaccines; Levandowski RA; The United States Code of Federal Regulations requires that all influenza virus vaccines produced for use in the United States adhere to specific regulatory standards including the demonstration of safety and efficacy . For vaccines produced in cell lines, rigorous characterization for manufacturing is particularly important . Influenza vaccines produced by the passage of viruses in mammalian cell lines will require careful evaluation to ensure the removal or inactivation of potential adventitious agents. Dev Biol Stand, 1999, 98, 93 - 100; discussion 111 Influvac: a safe Madin Darby Canine Kidney (MDCK) cell culture-based influenza vaccine; Brands R et al.; Influenza vaccine production technology based on large scale cell culture technology has been developed . From the characterization of the continuous cell line MDCK as well as drug safety studies we conclude that this cell line and the cell culture system are suitable for biological production . The Down Stream Process (DSP) of the virus-containing harvest fluids guarantees sufficient inactivation of influenza viruses and adequate removal or inactivation of putative adventitious or endogenous viruses, mycoplasma or bacteria . Our data indicate that the tissue culture-based production technology is feasible. Biol Reprod, 1999 Oct, 61(4), 1090 - 8 Expression and regulation of relaxin-like factor gene transcripts in the bovine ovary: differentiation-dependent expression in theca cell cultures; Bathgate R et al.; The relaxin-like factor (RLF) was recently discovered as a new member of the insulin-insulin-like growth factor-relaxin family of growth factors and hormones predominantly in the Leydig cells of the testis . In cattle, in contrast to other species, the RLF gene is also expressed to a high level in the ovary, where its expression pattern in the corpus luteum of the late cycle and pregnancy is similar to that of relaxin in the pig . The RLF gene was also transcribed to a high level in the theca cells of estrogen-rich, large antral follicles . Long-term primary cultures of bovine theca cells showed that expression was insulin dependent . After an initial decline in specific mRNA concentrations, there was a switch to a transcript with a longer poly(A) tail at about Day 6 of culture, which continued to increase to very high levels by Day 15 of culture . Addition of fetal calf serum to cultures caused a rapid and irreversible down-regulation of the RLF gene . Also, LH caused a decline in specific gene expression in long-term primary theca cell cultures . As in the Leydig cells of the testis, the pattern of RLF gene expression appears to reflect the differentiation state of the ovarian theca-luteal cell lineage, and should prove useful for mapping the fate of these cells under differing stimulation regimes. Artif Organs, 1999 Sep, 23(9), 881 - 4 Partially degradable film/fabric composites: textile scaffolds for liver cell culture; Karamuk E et al.; In this study, a composite scaffold combining textile superstructures and biomimetic glycopolymers is introduced, which may allow engineering of organotypic liver tissue in vitro . Woven poly(ethylene therephtalat) (PET) fabrics were coated on one side with a thin biodegradable polymer film (poly{D-L-lactic-co-glycolic acid} PLGA), in order to obtain a polar structure . The composite structure ensured the stability of the membrane during in vitro degradation, independently of mesh size . Matrix porosity increased when a polymer blend matrix was used . For hepatocyte culturing studies, the scaffolds were additionally coated with an artificial glycopolymer (poly{N-p-vinylbenzyl-D-lactoamide}, PVLA) in order to improve cell attachment . It was observed that formation of aggregates depends on the scaffold geometry as well as on the pretreatment and medium conditions . After 4 days in culture, the pores of the fabric were filled with aggregates illustrating the possibility of immobilizing hepatocyte aggregates in well-defined spatial configurations on textile structures. Artif Organs, 1999 Sep, 23(9), 809 - 16 Recombinant human erythropoietin stimulates production of interleukin 2 by whole blood cell cultures of hemodialysis patients; Bryl E et al.; The impairment of function of human T lymphocytes, leading to an inappropriate cytokine production, is partially responsible for defective immunological response in hemodialysis patients (HD) . Recent data suggest that recombinant human erythropoietin (rhEPO) may exert immunological effects . The aim of this study was to find out whether rhEPO treatment of the HD patients may have an effect on the interleukin 2 (IL-2) production by their whole blood cell cultures . The study was carried out in 10 HD patients receiving rhEPO for 6 months . Compared with the levels seen before the treatment, the concentration of IL-2 increased in the phytohemagglutinin-stimulated whole blood cell cultures of 7 of 10 patients under study . Addition of rhEPO in vitro to the whole blood cell cultures of the HD patients before implementation of erythropoietin confirmed that rhEPO is able to directly stimulate IL-2 production . Our studies show that the therapy with rhEPO affects IL-2 secretion. J Biomed Mater Res, 1999 Dec 5, 47(3), 424 - 33 Osteoclastic responses to various calcium phosphates in cell cultures; Doi Y et al.; Disks made of hydroxyapatite, beta-tricalcium phosphate, carbonate apatite, tetracalcium phosphate, alpha-tricalcium phosphate, dicalcium phosphate dihydrate, and octacalcium phosphate were incubated in osteoclastic cell cultures for 2 days . The first five salts were sintered and the last two were compressed before incubation . Osteoclasts resorbed only the sintered carbonate apatite disks . However, osteoclasts were able to resorb octacalcium phosphate disks that were preincubated for 1 day in medium without cells, indicating that surface conditioning was important for osteoclastic resorption of this calcium phosphate . Although resorption did not occur, medium calcium and phosphorus changed to an appreciable extent after a 2-day incubation of beta-tricalcium phosphate, tetracalcium phosphate, alpha-tricalcium phosphate, and dicalcium phosphate dihydrate . These changes in the medium calcium and phosphate concentrations could explain why osteoclasts appeared to have lost their activity on these calcium phosphate disks and were not capable of resorbing them . With hydroxyapatite disks no changes were observed in the medium calcium and phosphorus before and after incubation . Moreover, the osteoclasts appeared to be essentially the same as with the sintered carbonate apatite disks and with bone slices used as a control . Nevertheless, no pits or lacunae were observed on the hydroxyapatite disks, indicating that sintered carbonate apatite should be superior to sintered hydroxyapatite as a bioresorbable bone substitute . Hum Mol Genet, 1999 Oct, 8(11), 1975 - 84 Cis and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model; Amack JD et al.; The mutation causing myotonic dystrophy (DM) has been identified as a CTG expansion in the 3'-untranslated region (3'-UTR) of the DM protein kinase gene ( DMPK ), but the mechanism(s) of pathogenesis remain unknown . Studies using DM patient materials have often produced confusing results . Therefore, to study the effects of the DM mutation in a controlled environment, we have established a cell culture model system using C2C12 mouse myoblasts . By expressing chimeric reporter constructs containing a reporter gene fused to a human DMPK 3'-UTR, we identified both cis and trans effects that are mediated by the DM mutation . Our data show that a mutant DMPK 3'-UTR, with as few as 57 CTGs, had a negative cis effect on protein expression and resulted in the aggregation of reporter transcripts into discrete nuclear foci . We determined by deletion analysis that an expanded (CTG) (n) tract alone was sufficient to mediate these cis effects . Furthermore, in contrast to the normal DMPK 3'-UTR mRNA, a mutant DMPK 3'-UTR mRNA with (CUG)(200)selectively inhibited myogenic differentiation of C2C12 myoblasts . Genetic analysis and the Cre- loxP system were used to clearly demonstrate that the myoblast fusion defect could be rescued by eliminating the expression of the mutant DMPK 3'-UTR transcript . Characterization of spontaneous deletion events mapped the inhibitory effect to the (CTG) (n) expansion and/or the 3' end of the DMPK 3'-UTR . These results provide evidence that the DM mutation acts in cis to reduce protein production (consistent with DMPK haploinsufficiency) and in trans as a 'riboregulator' to inhibit myogenesis. J Immunol Methods, 1999 Jul 30, 227(1-2), 53 - 63 The use of Teflon cell culture bags to expand functionally active CD8+ cytotoxic T lymphocytes; Garland RJ et al.; We have investigated the ability of Teflon cell culture (TCC) bags, compared to conventional tissue culture flasks and plates, to support the expansion of human CD8+ T cells in response to an allogeneic stimulus . TCC bags, which are compatible with good manufacturing practice (GMP), facilitated CD8+ T cell growth as well as conventional culture vessels and resulted in cytotoxic T cells which were able to kill allogeneic targets . Growth characteristics were compared by investigating the number, immunophenotype and cell cycle properties of the cells generated . The kinetics of cell growth were not significantly different over the first 14 days of culture in each vessel type, with the cell counts being highest at day 10 in all cases . However, the TCC bags resulted in a significantly higher proportion of cells with the morphology of typical lymphocytes than tissue culture flasks after 14 and 18 days in culture . There were no significant differences in the percentage of typical lymphocytes expanded in TCC bags compared to those expanded in plates . Expanded CD8+ cells maintained their initial level of expression of CD3, CD11a, CD18 and T cell receptor (alphabeta heterodimer, TCR (alphabeta)) but increased expression of CD45RO, CD95 and of activation markers HLA-DR and CD25 in each culture vessel . Studies of cell cycle parameters showed that each vessel supported CD8+ T cell stimulation, as demonstrated by significantly higher levels of S phase than fresh PBMN cells . The cells generated in TCC bags were able to kill allogeneic targets and also possessed natural killer (NK) cell activity . Thus, TCC bags are able to support the expansion of CD8+ T lymphocytes as well as flasks or tissue culture plates and are applicable to lymphocyte expansion for use in immunotherapy. Mikrobiol Z, 1999 May-Jun, 61(3), 37 - 45 {Virus reproduction in cell cultures synchronized by macromolecular platinum complexes}; Hyrin VM et al.; A new method of application of platinum macromolecular complex poly inverted question markhexacis{chloroamine aquaplatinum (II)} inverted question mark-mu-deoxyribonucleate (MCP) as a cell cycle synchronization inductor has been developed . The process of some RNA-genome viruses reproduction in cell cultures synchronized by MCP was studied . It was shown that the amount of virus protection in such cultures under special conditions can be increased 100-350 times without increasing the cell substrate amount . The obtained results may be used in biotechnology to increase production of virus antigens and vaccines. Plant Cell Physiol, 1999 Jun, 40(6), 651 - 5 One type of chalcone synthase gene expressed during embryogenesis regulates the flavonoid accumulation in citrus cell cultures; Moriguchi T et al.; To elucidate the relationship between the expression of chalcone synthase (CHS) genes and the production of flavonoid in citrus cell cultures, two cDNA clones encoding CHS were isolated (CitCHS1 and CitCHS2) from the citrus . The accumulation of CitCHS2 mRNA was notably induced by embryogenesis but CitCHS1 mRNA was not . There was no detectable accumulation of flavonoid in the undifferentiated calli, but flavonoid accumulated after the morphological changes to embryoids . These results indicate that two CHS genes differentially expressed during citrus somatic embryogenesis and CitCHS2 may regulate the accumulation of flavonoid in citrus cell cultures. J Infect Dis, 1999 Oct, 180(4), 976 - 86 Hepatocytes are permissive for human cytomegalovirus infection in human liver cell culture and In vivo; Sinzger C et al.; The cytopathic potential of human cytomegalovirus (HCMV) in human liver cells was analyzed in cell culture and in tissue sections from patients with HCMV hepatitis . Liver cell cultures, consisting of hepatocytes, bile duct epithelial cells, and stromal cells were infected by various HCMV strains . Cytopathic effects, viral gene expression, and virus production were detected . Infected cell types were identified by immunocytochemical double labeling . Hepatocytes were the predominant target cells of HCMV infection in liver tissues and in cell culture . Late-stage infected cultured hepatocytes produced infectious progeny virus, and infectious virus was propagated from liver tissue specimens . HCMV infection in cultured liver cells closely resembled in vivo infection of the liver with regard to the target cell spectrum and the permissive course of infection . It is concluded that HCMV can cause direct liver parenchyma damage by efficient cytolytic infection of hepatocytes. Mutat Res, 1999 Jul 21, 444(1), 217 - 25 Evaluation of the genotoxicity of 2,4-dichlorophenoxyacetic acid and its derivatives in mammalian cell cultures; Gollapudi BB et al.; 2,4-dichlorophenoxyacetic acid and its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants . The genetic toxicity of an ester (2,4-D 2-butoxyethylester) and two salts (2,4-D isopropylamine and 2,4-D triisopropanolamine) was investigated in cultured mammalian cells . The end points used were the induction of chromosomal aberrations in primary cultures of rat lymphocytes and forward mutations at the HGPRT locus of Chinese hamster ovary cells . There was no evidence of genotoxicity for the test materials in the experimental systems used . These results were consistent with the general lack of genotoxic potential for 2,4-D in a number of other test systems. Anticancer Drugs, 1999 Jun, 10(5), 445 - 52 Selective cell kill of the combination of gemcitabine and cisplatin in multilayered postconfluent tumor cell cultures; Padron JM et al.; Both gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cisplatin (cis-diammine-dichloroplatinum) have significant anticancer activity against ovarian, head and neck, and non-small cell lung cancer (NSCLC) . dFdC can be incorporated into DNA and RNA, and inhibit DNA repair, while cisplatin can form Pt-DNA adducts . We previously observed schedule-dependent synergism of the combination of dFdC and cisplatin in monolayer cell cultures . We now evaluated whether the combination would also enable selective cell kill in multilayered postconfluent cell cultures, since each compound showed variable activity in multilayered cells . The combination was tested in multilayered cultures from cell lines with a different histological origin: the human head and neck squamous cell carcinoma cell line UMSCC-22B (22B), the human NSCLC cell line H322, and ADDP, a cisplatin-resistant variant of the human ovarian cancer cell line A2780 . Sensitivity of the multilayered cells was dependent on exposure duration and sequence of the drug combinations, which were added in a constant molar ratio (dFdC:cisplatin 1:100), with a total exposure time of 96 h . The type of interaction was related to the degree of resistance of the cell lines to either dFdC or cisplatin . Thus, the very sensitive 22B cells only showed an additive effect when cells were preincubated for 24 h with dFdC prior to exposure to the combination . In contrast, in the resistant ADDP and H322 cells, synergism was the most common profile (three out of four schedules tested) . This is of special relevance when we take into account that these drugs only show cytostatic effects when administered alone, whereas the combination produced cytotoxic cell killing . In conclusion, combining dFdC with cisplatin can be at least additive, but synergistic in multilayered postconfluent cells resistant to dFdC and cisplatin. Clin Diagn Lab Immunol, 1999 Sep, 6(5), 729 - 33 Cell culture of sporadic hepatitis E virus in China; Huang R et al.; The isolation and identification of the 87A strain of epidemic hepatitis E virus (HEV) by means of cell culturing have been described previously . This paper reports the successful isolation of a sporadic HEV strain (G93-2) in human lung carcinoma cell (A549) cultures . The etiology, molecular and biological properties, and serological relationship of this new strain to other, epidemic HEV strains are described . The propagation of both sporadic and epidemic HEV strains in a cell culture system will facilitate vaccine research. FEBS Lett, 1999 Aug 27, 457(2), 200 - 4 Induction of myelin gene expression in Schwann cell cultures by an interleukin-6 receptor-interleukin-6 chimera; Haggiag S et al.; Expression of myelin basic protein (MBP) and Po gene products is induced during the final postnatal maturation of Schwann cells and reinduced during nerve regeneration . We show that a chimeric protein containing interleukin-6 fused to its soluble receptor (IL6RIL6 chimera) induces MBP and Po RNAs and proteins in cultures of dorsal root ganglia (DRG) from 14 day old mouse embryos . Activation of gp130 signaling by IL6RIL6 appears comparable to cyclic AMP elevating agents to induce the myelin gene products in DRG and in pure Schwann cell cultures. J Neurovirol, 1999 Aug, 5(4), 384 - 91 Restricted replication of herpes simplex virus in satellite glial cell cultures clonally derived from adult mice; Wilkinson R et al.; To determine the possible influence of satellite glial cells on restricting the spread of herpes simplex virus in the peripheral nervous system, HSV replication was studied in clonally derived cultures of satellite glial cells from adult animals . Satellite cells were purified by exploiting their close anatomical association with primary sensory neurons . Dissociated neurons from dorsal root ganglia were micro-manipulated to remove all but one of the attached satellite cells and cultured in the presence of the mitogenic stimulators bovine pituitary extract and cholera toxin . Following a lag phase of 20-30 days some of the individual satellite cells began to proliferate . Initial cultures demonstrated bipolar morphology similar to cultured Schwann cells, some of which differentiated into large astrocytic whorl-like cells on subsequent passage . Immunocytochemical and molecular studies demonstrated that these cells, designated Sat.1, express glial fibrillary acidic protein, confirming their glial origin and by electron microscopy they were shown to be phagocytic . Under single step viral growth conditions Sat.1 cells were restrictive for HSV replication, producing in the order of 1000 times less infectious virus than Vero cells, a standard permissive cell line . These results suggest that satellite cells, which tightly encase sensory neurons, play a role in restricting interneural spread of HSV within the peripheral nervous system. Folia Med (Plovdiv), 1999, 41(1), 43 - 5 In vitro study on cytotoxic effect of some chemicals on serum McCoy and serum-free McCoy-Plovdiv cell culture systems; Docheva D et al.; The cytotoxicity of test agents on serum-free McCoy cultures has not been studied at all . The cytotoxic effect of EDTA, methanol, DMSO, and cycloheximide on serum-free McCoy-Plovdiv cell culture (SF) was detected visually on inverted microscope and quantitatively by tests for viability (NR) and total protein (KBP) . The IC50 values for the tested chemicals were calculated . SF showed the lowest IC50 values for cycloheximide, DMSO and EDTA and the highest for methanol according to both tests . EDTA, methanol, DMSO and cycloheximide had dose-effect relationship in the cell test systems after treatment . The data indicate that McCoy-Plovdiv cell line is a suitable serum-free cell system for in vitro cytotoxic studies. Histochem Cell Biol, 1999 Jul, 112(1), 41 - 9 Characterisation of caveolins from cartilage: expression of caveolin-1, -2 and -3 in chondrocytes and in alginate cell culture of the rat tibia; Schwab W et al.; This study was performed to determine if rat articular chondrocytes express caveolin, the structural protein of caveolae, and to determine differences in the distribution of the caveolin subtypes 1, 2 and 3 in knee joints of newborn and adult rats . All three subtypes of caveolin were detected in adult cartilage by immunocytochemical staining . In newborn rats, only caveolin-1 was found in the hyaline cartilage . Caveolin-1, -2 and -3 messenger RNA and protein were also detected in chondrocyte cell cultures . Ultrastructural investigations of cell culture and cartilage tissue revealed the presence of caveolae at the plasma membrane of chondrocytes . These findings represent the first report on the different expression of caveolin isoforms, in particular the expression of the muscle cell-specific caveolin-3 in chondrocytes . There is evidence that caveolin-2 and -3 are upregulated during growth and development of articular cartilage, suggesting a role for caveolins in chondrocyte differentiation. Proc Soc Exp Biol Med, 1999 Sep, 221(4), 386 - 90 5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures; McDuffie JE et al.; Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells . The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures . Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated {3H}L-citrulline ({3H}L-Cit) formation in BAEC cultures . We found that 5-HT stimulated the conversion of {3H}L-arginine ({3H}L-Arg) to {3H}L-Cit, indicating eNOS activation . The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated {3H}L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent . Maximal responses were observed within 10 min following agonist exposures . These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO) . Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins . These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT . Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity. J Med Virol, 1999 Oct, 59(2), 215 - 20 Comparison of two nested PCR, cell culture, and antigen detection for the diagnosis of upper respiratory tract infections due to influenza viruses; Magnard C et al.; Influenza surveillance requires sensitive and rapid diagnostic methods . Different diagnostic procedures have been evaluated on a selected set of nasal swabs sample collected from patients presenting with acute respiratory infection . One hundred fifty-four samples collected during the peak of the influenza epidemic recorded during winter of 1997-1998 in the south of France were processed for influenza detection using antigen detection (ELISA-immunocapture assay), two different nested RT-PCR assays (targeting M and HA genes), and cell culture . Among 154 samples, 93 (60.4%) were positive for influenza detection . Forty specimens (26%) were positive by ELISA, 77 (50%) by culture, 88 (57.1%) using the multiplex HA-PCR and 76 (49.4%) using the M-PCR . Multiplex HA-PCR was thus the most sensitive test . The PCR assay offers an alternative to culture for influenza detection . Nevertheless, culture is efficient for influenza diagnosis and is the only technique that allows the reference centres to collect viral strains and characterise fully new variants . Acta Anat (Basel), 1992, 145(4), 307 - 20 Experiments on the induction of antibody dependent macrophage-mediated cellular cytotoxicity in mixed brain cell cultures; Korn J; These examinations were based on the discussion whether in demyelinating diseases anti-lipid antibody associated with brain macrophages could have a cytotoxic effect on oligodendrocytes . We used mixed brain cell cultures of newborn rats where, among others, both oligodendrocytes and vacuolated macrophage-like cells were found . On these macrophage-like cells, the presence of Fc-receptors was proven . Besides Fc-receptor-dependent phagocytosis, these cells showed an Fc-receptor-independent type of phagocytosis . The Fc-receptor-bearing cells moved within the culture and adhered to glass fibers . In the cytoplasm of these cells, unspecific esterase, acid phosphatase and peroxidase could be visualized . The vacuolated cells showed strong autofluorescence, expressed a surface marker found on all types of rat leukocytes and were marked by Griffonia simplicifolia lectin . These results definitely characterized the vacuolised cells as macrophages . We saw globular and pleomorphic macrophages . After incubation of anti-GC serum in a highly diluted solution, significantly more macrophages bound to oligodendrocytes than in the controls . In these cases, we found target cell lysis . It could be shown in vitro that anti-GC serum together with macrophages of neonatal brains can induce a cytotoxic effect on oligodendrocytes. Bone Marrow Transplant, 1999 Jul, 24(2), 179 - 89 Natural killer (NK) cells prevent virus production in cell culture; Baraz L et al.; Natural killer (NK) cells (CD3-/CD16+/CD56+ lymphocytes) play an important role in early immune defense against viral infection, a fact which is of prime significance for heavily immunosuppressed patients after bone marrow transplantation . In this study we demonstrate that NK cells preferentially lyse human colon adenocarcinoma (Colo-205) tumor cells infected with herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV) and autologous T cells infected with VV . This phenomenon was assessed by the viral infectious center (IC) method and compared with the results obtained by means of the standard 51Cr-release assay . Using the IC assay, we found that NK cells lyse virus infected cells at an early stage of infection, thereby preventing viral dissemination to neighboring cells . 51Cr-release assay verified by propidium iodide (PI) penetration showed that the early effects of NK mediated anti-viral activity are not the result of membrane damage . The effect of NK cells on HSV-1 infected Colo-205 cells appears to be independent of the level of expression of major histocompatibility complex (MHC) class I molecules while the killing of autologous VV-infected T cells correlates with a reduction in MHC class I expression . Our results suggest that additional factors besides MHC play a role in the regulation of NK cell-mediated lysis of virus infected cells . This may be of clinical importance in patients who are heavily immunosuppressed after bone marrow transplantation. Clin Infect Dis, 1999 May, 28(5), 1100 - 3 Antibody response after a four-site intradermal booster vaccination with cell-culture rabies vaccine; Tantawichien T et al.; The current World Health Organization recommendation for booster vaccination of previously immunized individuals with potential exposure to rabies is two doses of vaccine intramuscularly or intradermally on days 0 and 3 . We report responses to two types of postexposure treatment of healthy individuals who had received preexposure rabies vaccination 1 year previously . Group A individuals received four intradermal doses (one-fifth of the diluent volume of vaccine per dose) on day 0, and group B individuals received two intramuscular doses on days 0 and 3 . Immunogenicity of the two booster regimens was assessed by titrating the amount of neutralizing antibody (Nab) . We found that the booster doses of vaccine produced remarkable responses in all subjects . Nab titers of > or = 0.5 IU/mL (acceptable antibody level for protection against rabies) were detected in all subjects on day 14, and they were shown to be consistently high 1 year after the booster vaccination . We also found that the Nab titers for group A were significantly higher (two- to eightfold) than those for group B on days 5, 14, 150, and 360 after the initial booster vaccination (P < .05) . Our study shows that the four-site intradermal booster regimen with use of one-fifth of the diluent volume of cell-culture rabies vaccine on day 0 is associated with a significantly higher antibody response than is the conventional booster regimen for subsequent postexposure rabies treatment of individuals who have received preexposure rabies vaccination with cell-culture rabies vaccine 1 year previously. FEBS Lett, 1999 Jul 30, 456(1), 41 - 4 Short-term block of Na+/K+-ATPase in neuro-glial cell cultures of cerebellum induces glutamate dependent damage of granule cells; Stelmashook EV et al.; Granule cells in a dissociated neuro-glial cell culture of cerebellum when exposed to ouabain (10(-3) M) for 25 min apparently swell, increase their {Ca2+}i with obvious depolarization of the mitochondrial membrane . In 3 h after ouabain was omitted from the solution, 62 +/- 3% of granule cells had pycnotic nuclei . The supplement of a solution with competitive specific antagonist of NMDA receptors, L-2-amino-7-phosphonoheptanoate (10(-4) M, APH) together with ouabain prevented cells from swelling, mitochondrial deenergization, neuronal death and increase of {Ca2+}i . These data suggest that cellular Na+/K+-ATPase inactivation in neuro-glial cell cultures of cerebellum leads to glutamate (Glu) accumulation, hyperstimulation of glutamate receptors, higher Ca2+ and Na+ influxes into the cells through the channels activated by Glu . This process leads to cell swelling, mitochondrial deenergization and death of granule cells . Possibly, the decrease of Na+/K+-ATPase activity in brain cells can lead to the onset of at least some chronic neurological disorders. Zhonghua Yan Ke Za Zhi, 1997 Sep, 33(5), 357 - 9 {Cell culture of human lamina cribrosa in vitro}; Luo X et al.; OBJECTIVE: To investigate the physiology and pathology of the biosynthesis of extracellular matrix (ECM) by lamina cribrosa cells (LCC) and to establish a model of cultured LCC . METHODS: The LCC of 10 human dead fetal eyes were cultured with enzyme digestion procedures in vitro . The cultured cells were identified by immunohistochemistry assays and electron microscopy . RESULTS: The cultured cells were flat, polygonal in shape, and their cytoplasm contained abundant perinuclear granules . The nuclei were relatively transparent and nucleoli were clear, there were basement membrane-like material and ECM surrounding the cell membrane . The immunofluorescent staining was positive for collagen type I, III, IV, fibronectin and laminin, negative for glial fibrillar acid protein and factor-VIII . The characteristics mentioned above were coincident with the morphology of LCC . This cell line has been cultured to its fourth generation . CONCLUSION: We have successfully cultured a cell line of LCC, and it may be utilized in the study of pathogenesis of glaucoma. J Nutr Sci Vitaminol (Tokyo), 1999 Apr, 45(2), 203 - 12 Formation of 8,11,14-octadecatrienoic acid (18:3 n-4) from naturally occurring unique fatty acid, 9,12-hexadecadienoic acid (16:2 n-4), in animal cell cultures; Nakano N et al.; 9,12-Hexadecadienoic acid (16:2 n-4), present in small amounts in fish oils as a naturally occurring unique fatty acid, was incorporated into the phospholipids in rat liver BRL-3A cells to a similar extent as linoleic acid (18:2 n-6) . 11,14-Octadecadienoic acid (18:2 n-4) and 8,11,14-octadecatrienoic acid (18:3 n-4) were detected in the cellular lipids of BRL-3A cells when incubated in a medium supplemented with 16:2 n-4 methyl ester . The cellular levels of these acids increased in parallel with 16:2 n-4 methyl ester added to the medium . These compounds were probably formed through conversion from 16:2 n-4 to 16:3 n-4 by delta 6 desaturation, and then 18:3 n-4 was produced by elongation, and part of the surplus 16:2 n-4, not desaturated to 16:3 n-4, elongated to 18:2 n-4 . These results suggested that 16:2 n-4 was desaturated by delta 6 desaturase in vitro . It was also shown that 16:2 n-4 inhibited arachidonic acid synthesis from exogenous linoleic acid in BRL-3A cells as efficiently as alpha-linolenic acid (18:3 n-3). Int J Cancer, 1999 Sep 24, 83(1), 135 - 40 Inverse regulation of neuronal cellular adhesion molecule (NCAM) by IFN-gamma in melanoma cell cultures established from CNS lesions; Geertsen R et al.; In advanced stages of malignant melanoma (MM), metastases to the CNS are frequently observed . Few results are available on trophic factors and immunological features involved in the process of invasion and adhesion of circulating metastatic cells into the CNS . A direct comparison of remote metastases found in different locations of the same patient might help to identify such properties . For this purpose, we screened a panel of MM cell cultures, which had been established from patients with surgically removed MM lesions of the CNS, for expression and regulation of immunorelevant molecules . The results were compared with standard controls and cultures established from non-CNS metastatic lesions of the same patients . No significant differences were observed for expression of HLA-I, HLA-II, ICAM-1 and the melanoma-associated antigens Mage-3, MelanA and tyrosinase . Constitutive expression of the neuronal cell adhesion molecule (NCAM) was found in all CNS-derived samples and in fewer than 50% of non-CNS derived cultures . IFN-gamma was found to have a weak up-regulating effect in all non-CNS-derived cultures, except normal melanocytes . However, in 6/7 CNS-derived cultures, pre-treatment with IFN-gamma reduced expression of NCAM to 28% to 77% of the level in untreated cultures . The presence and regulation of NCAM differs between MM cells derived from CNS metastases and non-CNS-derived melanocytic cells . Thus, NCAM might be a candidate immunoregulating molecule involved in the formation of CNS metastases of MM . Brain Res, 1999 Jul 17, 835(1), 74 - 9 Instability of the CAG repeat in immortalized fibroblast cell cultures from Huntington's disease transgenic mice; Manley K et al.; Huntington's Disease transgenic mice were used for an exploration into the stability of a trinucleotide repeat . The brain shows heterogeneous somatic instability that increases quantitatively with age . To test somatic CAG-repeat alterations during long-term culture, DNA was extracted from transgenic tissue, primary fibroblasts, and SV40-immortalized fibroblasts at intervals of approximately 100 cell doublings . In fibroblasts derived from an adult mouse, there was an initial short truncation of the repeat, followed by an emerging population of cells showing continuous slow expansion . After 15 months in continuous culture (approximately 600 cell doublings following transformation) the major CAG peak has increased from 155 to approximately 170 triplets . This in vitro system can now be used to assay factors that affect instability . J Cell Sci, 1999 Sep, 112 ( Pt 17), 2823 - 32 Matrix expression and proliferation of primary gingival fibroblasts in a three-dimensional cell culture model; Hillmann G et al.; The growth of cultured primary human gingival fibroblasts and the three-dimensional arrangement of the extracellular matrix in a polyester carrier system was investigated using various histological techniques . The results were compared with monolayer cultures . Collagen types I, III, V, and VI were investigated by conventional and fluorescence microscopy, scanning and transmission electron microscopy, and confocal laser scanning microscopy . Human gingival fibroblasts were obtained from tissue biopsies of five donors and were cultivated up to 5 weeks under three-dimensional culture conditions . The cells displayed an elongated, spindle-like or stellate morphology resembling the in vivo situation . Collagen type I revealed thick fiber bundles, and collagens type III and V were distributed as fine fibrils or small bundles throughout the culture system . Frequently, the fibers were oriented parallel to the long axis of the cells . Type VI collagen formed thin fibers and revealed a reticular pattern . In histological sections the cultured cells exhibited a morphology clearly different from that of cells cultured in monolayers . Their shape and spatial distribution resembled that of cells in tissue biopsies more closely . The culture system presented here promotes a dynamic model for performing studies for instance on the interactions of cultured cells with extracellular matrix molecules, on the pathogenesis of inflammatory processes or on the interactions with biomaterials, thus providing qualitative and quantitative information. J Neuroendocrinol, 1999 Jul, 11(7), 491 - 502 Expression of functional growth hormone secretagogue receptors in human pituitary adenomas: polymerase chain reaction, triple in-situ hybridization and cell culture studies; Barlier A et al.; We examined the expression of functional growth hormone secretagogue receptors (GHS-R) in a series of 3