Biologicals, 2004 Dec, 32(4), 183 - 93 Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins; Eldering JA et al.; Chinese hamster ovary (CHO) cell cultures used to produce biopharmaceuticals are tested for mycoplasma contamination as part of the ensurance of a safe and pure product . The current U.S . Food and Drug Administration (FDA) regulatory guideline recommends using two procedures: broth/agar cultures and DNA staining of indicator cell cultures . Although these culture methods are relatively sensitive to most species, theoretically capable of detecting as few as 1-10 cfu/ml of most species, the overall procedure is lengthy (28 d), costly and less sensitive to noncultivable species . The detection of mycoplasma using the polymerase chain reaction (PCR) method has been considered an alternative method because it is relatively fast (1-2 d), inexpensive, and independent of culture conditions, however, limitations in sensitivity (limit of detection >/=1000 cfu/ml) and the risk of false positive and false negative results have prevented PCR from replacing the traditional culture methods in the industrial setting . In this report, we describe a new PCR assay for mycoplasma detection that appears to resolve these issues while being sufficiently simple and inexpensive for routine use . This assay applies readily available techniques in DNA extraction together with a modified single-step PCR using a previously characterized primer pair that is homologous to a broad spectrum of mycoplasma species known to infect mammalian cell cultures . Analysis is made easy by the detection of only a single amplification product within a narrow size range, 438-470 bp . A high sensitivity and specificity for mycoplasma detection in CHO cell production cultures is made possible through the combination of three key techniques: 8-methoxypsoralen and UV light treatment to decontaminate PCR reagents of DNA; hot-start Taq DNA polymerase to reduce nonspecific priming events; and touchdown- (TD-) PCR to increase sensitivity while also reducing nonspecific priming events . In extracts of mycoplasma DNA, the limit of detection for eight different mycoplasma species is 10 genomic copies . In CHO cell production cultures containing gentamicin, the limit of detection for a model organism, gentamicin-resistant M . hyorhinis, is 1 cfu/ml . The sensitivity and specificity of this PCR assay for mycoplasma detection in CHO cell production cultures appear similar to the currently used culture methods and thus should be considered as an alternative method by the biopharmaceutical industry. Neurourol Urodyn, 2005, 24(1), 81 - 8 Intracellular Ca2+ regulation in a human prostate stromal cell culture; Wu C et al.; AIMS: Prostate stromal cell cultures are used in vitro to study the cellular pathophysiology of benign prostatic hyperplasia (BPH), but their functional properties are poorly understood . This study characterized intracellular Ca2+ ({Ca2+}i) regulation in a cultured cell line in comparison to freshly isolated cells, as a background to understanding contractile regulation and cellular proliferation in this tissue . METHODS: Prostate stromal cells were isolated from either PrS6 cell cultures, with an extended life span by transfection with the SV40 T-antigen, tsA58-U19, or freshly obtained transition zone prostate samples, primary cells . {Ca2+}i was measured in vitro with the indicator Fura-2 by epifluorescence microscopy . RESULTS: Phenylephrine, high-K+, and caffeine induced Ca2+-transients in primary cells (resting {Ca2+}i 94 +/- 8 nM, n = 29; peak 193 +/- 26 nM, n = 19) . In PrS6 cells resting {Ca2+}i was 96 +/- 8 nM (n = 78) and in 34 of these 78 cells, 30 microM phenylephrine increased {Ca2+}i to 296 +/- 28 nM . 5-methyl-urapidil (10-30 microM) inhibited this response in 10 of 16 cells . Spontaneous Ca2+-transients were also observed in 91% of phenylephrine-responsive cells, but in only 20% of non-responsive cells (P < 0.01) . Ca2+-transients were also induced by high-K+ solution, and 20 mM caffeine . The latter abolished the response to subsequent phenylephrine application . Depletion of intracellular Ca2+ stores by caffeine or restoration from a Ca2+-free superfusate caused a substantial rise of {Ca2+}i . CONCLUSIONS: PrS6 prostate stromal cells express functional alpha1-adrenoceptors associated with spontaneous intracellular Ca2+-transients . They exhibit functional Ca2+ channels, intracellular Ca2+ stores, and Ca2+ entry induced by store depletion . Stromal cultures can therefore be used to characterize the cellular physiology of prostate stromal cell contraction and proliferation. Biochemistry, 2004 Dec 7, 43(48), 15296 - 302 High-level expression of rabbit 15-lipoxygenase induces collapse of the mitochondrial Ph gradient in cell culture; Vijayvergiya C et al.; A critical step in the development of mammalian erythroblasts into mature red blood cells is the extrusion of the nucleus, followed by intracellular degradation of the remaining organelles . It has been hypothesized that the breakdown of cellular organelles in rabbit reticulocytes is initiated by 15-lipoxygenase . In vitro, the purified rabbit reticulocyte 15-lipoxygenase binds and permeabilizes organellar membranes, thereby releasing the lumenal contents of the organelle . Here, we demonstrate that ectopic expression of 15-lipoxygenase leads to the collapse of the mitochondrial pH gradient in nonerythroid cells, using a novel reporter of mitochondrial pH, mito-pHluorin . No change in mitochondrial pH was observed with a mutant of 15-lipoxygenase that lacks enzymatic activity . These data demonstrate that 15-lipoxygenase is capable of disrupting the pH gradient maintained by mitochondria in living cells without additional factors specific for red blood cell development. Clin Diagn Virol, 1995 Aug, 4(2), 195 - 205 Specific enzyme immunoassays for the rapid detection of Ross River virus in cell cultures inoculated with infected mosquito homogenates; Oliveira NM et al.; Background: Ross River (RR) virus is a mosquito-borne alphavirus and one of the aetiological agents of epidemic polyarthritis in humans . Early detection of increased virus activity in mosquito populations enables public health authorities to implement measures to reduce the number of human infections during epidemics . However, current surveillance techniques require a minimum of four weeks for viruses to be isolated and identified . Objectives: This study was carried out to assess the use of enzyme immunoassays (EIA) as rapid alternatives to traditional cell culture techniques for detection of RR virus in mosquitoes . Study design: Enzyme immunoassays and immunoperoxidase assays were developed using RR-specific monoclonal antibodies and compared to traditional methods for detection of RR virus in field-caught mosquito samples . Results: By inoculation of {Formula: see text} cell cultures with mosquito homogenates and testing monolayers and culture supernatant by EIA, RR virus was detected and identified in all infected samples within 6 days . Conclusions: The use of EIA provides a rapid, sensitive and specific alternative to traditional methods for the detection of RR virus in mosquito vectors. Clin Diagn Virol, 1994 Apr, 2(2), 105 - 12 Comparison of the rapid second generation directigen((R)) EIA with cell culture and immunofluorescence for the detection of respiratory syncytial virus in nasopharyngeal aspirates; Lipson SM et al.; Background: A second generation-RSV enzyme immunoassay (EIA) was compared with cell culture and immunofluorescence to determine the improved efficacy of this reformulated system . Objectives: This study was performed to identify whether the EIA might serve an ancillary function during non-operational virology hours, or whether the EIA may serve as a replacement of the commonly used direct fluorescent assay (DFA) . Study design: During the 1992-1993 (September-April) RSV season, 124 freshly collected nasopharyngeal (NP) aspirates were tested by the EIA and the DFA for the presence of antigen to the infectious agent . Infectivity was performed by tube culture cytopathic effect (TC-CPE) in parallel with the two antigen detection methodologies . Results: Thirteen of the 48 confirmed positive specimens (27%) failed to yield infectious virus by TC-CPE . The sensitivity, specificity, positive and negative predictive values of the EIA were 90, 95, 91 and 94% respectively . This observed sensitivity of 90% using freshly collected NP aspirates, represents a marked improvement over an earlier generation EIA kit . Conclusions: Considering the simplicity and speed of this EIA (10 min), the test is recommended for use by medical personnel when facilities for DFA and traditional virus culture are not readily available. Clin Diagn Virol, 1993 Jul, 1(2), 123 - 7 Detection of influenza A in clinical specimens and cell culture fluid by a commercial EIA; Gleaves CA et al.; A MoAb-based capture EIA for the direct detection of influenza A from clinical samples was compared with cell culture isolation . A total of 330 respiratory specimens were submitted for detection of influenza A and/or other respiratory viruses . Influenza A was detected in 39 of 330 (12%) respiratory samples by culture or EIA . There were 33 concordant (EIA+/Culture-) samples (82%), and 6 discordant samples (3 EIA +/Culture-; 3 EIA-/Culture+) . Compared to viral isolation, the EIA had a sensitivity of 92%, a specificity of 98%, with positive and negative predictive values of 92% and 99%, respectively. Rapid Commun Mass Spectrom, 2004, 18(24), 3099 - 104 Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry; Tian Q et al.; The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS) . During positive ion LC/ESI-MS, LG formed an abundant sodiated species ({M+Na}+) while the protonated molecule was barely observable . {M+Na}+ further fragmented into the less abundant {LARL+H}+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation . The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water . In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, {aglycone+Na}+, while LARL fragmented into {M+H}+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures . The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively . PLoS Biol . 2004 Dec;2(12):e432 . Epub 2004 Nov 30. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages; Wobus CE et al.; Noroviruses are understudied because these important enteric pathogens have not been cultured to date . We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages . MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes . An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo . This is the first report of replication of a norovirus in cell culture . The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology. Neuroscience, 2004, 129(4), 935 - 45 The role of aquaporin-4 in the blood-brain barrier development and integrity: studies in animal and cell culture models; Nicchia GP et al.; Aquaporin-4 (AQP4) is the major water channel expressed in brain perivascular astrocyte processes . Although the role of AQP4 in brain edema has been extensively investigated, little information exists regarding its functional role at the blood-brain barrier (BBB) . The purpose of this work is to integrate previous and recent data regarding AQP4 expression during BBB formation and depending on BBB integrity, using several experimental models . Results from studies on the chick optic tectum, a well-established model of BBB development, and the effect of lipopolysaccharide on the BBB integrity and on perivascular AQP4 expression have been analyzed and discussed . Moreover, data on the BBB structure and AQP4 expression in murine models of Duchenne muscular dystrophy are reviewed . In particular, published results obtained from mdx(3cv) mice have been analyzed together with new data obtained from mdx mice in which all the dystrophin isoforms including DP71 are strongly reduced . Finally, the role of the endothelial component on AQP4 cellular expression and distribution has been investigated using rat primary astrocytes and brain capillary endothelial cell co-cultures as an in vitro model of BBB. Anim Reprod Sci, 2005 Jan, 85(1-2), 41 - 52 Effects of leptin on gonadotropin-releasing hormone release from hypothalamic-infundibular explants and gonadotropin release from adenohypophyseal primary cell cultures: further evidence that fully nourished cattle are resistant to leptin; Amstalden M et al.; In rodents and pigs, leptin stimulates the release of gonadotropin-releasing hormone (GnRH) from hypothalamus, gonadotropins from adenohypophyseal (AP) explants and cells, and luteinizing hormone (LH) from full-fed animals . In the current studies, we investigated whether leptin could stimulate the release of GnRH from bovine hypothalamic-infundibular (HYP) explants and gonadotropins from bovine adenohypophyseal cells . In Experiment 1A, HYP explants collected from 17 bulls and seven steers were incubated with Krebs-Ringer bicarbonate buffer (KRB) containing 0, 10, 100, or 1000 ng/ml recombinant ovine leptin (oleptin) for 30 min after a 3-h period of equilibration . None of the doses of leptin affected (P > 0.05) GnRH release into the media . In Experiment 1B, HYP explants collected from six steers were incubated with KRB containing 0 or 1000 ng/ml oleptin for two consecutive 30-min periods and challenged with 60 mM K(+) afterwards . Leptin did not affect (P > 0.05) basal or K(+)-stimulated release of GnRH . In Experiment 2, adenohypophyses from steers were collected at slaughter and cells dispersed and cultured for 4 days . On day 5, cells were treated with media alone (control) or media containing 10(-11), 10(-10), 10(-9), and 10(-8)M oleptin . Three independent replications were performed . None of the doses of leptin stimulated (P > 0.05) the release of LH . Although leptin at 10(-11), 10(-10), and 10(-9)M increased (P < 0.03) slightly the release of FSH compared to control-treated cells in one replicate, this effect was not confirmed in the other two replicates . Results support the hypothesis that leptin has limited effects on the release of GnRH and gonadotropins in full-fed cattle and reiterate important species differences in responsiveness to leptin. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Nov, 20(6), 741 - 3 {IL-18 expression in whole blood cell cultures from active lupus nephritis and the inhibitory effects of FK506, cyclosporin A and Dexamethasone.}; Pan ZX et al.; AIM: To explore the expression of IL-18 in patients with active lupus nephritis(LN) and the inhibitory effects of immunesupressive agents FK506, cyclosporin A (CsA) and dexamethasone(DEX) . METHODS: Peripheral blood samples were collected from 16 LN patients and 10 health volunteers and cultured with or without PHA/LPS, PHA/LPS+FK506, PHA/LPS+CsA and PHA/LPS+DEX for 24 hours . The IL-18 level in cultured supernatants and the IL-18 mRNA expression in cultured whole blood cells were detected by ELISA and semi-quantitative RT-PCR respectively, and the inhibitory effects of FK506, CsA and DEX on IL-18 expression were investigated . RESULTS: The expression levels of IL-18 mRNA and its protein in whole blood cell cultures from LN patients were higher than those in normal control group (P<0.01) either spontaneously or after stimulation with LPS/PHA . FK506, CsA and DEX suppressed significantly the expressions of IL-18 mRNA and its protein (P<0.001) in LPS/PHA-stimulated whole blood cell from LN patients.The inhibitory effects of FK506, CsA and DEX on the IL-18 protein expression in LN patients were stronger than that in normal contral group (P<0.01 or P<0.05) . CONCLUSION: IL-18 may play an important role in the pathogenesis of LN and inhibition of IL-18 production may be an useful way to treat LN. J Biomed Mater Res A, 2005 Feb 1, 72A(2), 180 - 9 Human skin cell cultures onto PLA(50) (PDLLA) bioresorbable polymers: Influence of chemical and morphological surface modifications; Garric X et al.; Poly(alpha-hydroxy acid)s derived from lactic and glycolic acid are bioresorbable polymers which can cover a large range of thermal, physical, mechanical, and biological properties . Human keratinocytes have been shown as able to grow on a poly(DL-lactic acid) film . However the keratinocyte growth was delayed with respect to culture on standard tissue culture polystyrene, even though the same plateau level was observed after 2 weeks . In order to improve the performance of poly(DL-lactic acid) films as skin culture support, their surface was modified by creating tiny cavities using a method based on the leaching out of poly(ethylene oxide) from poly(lactic acid)-poly(ethylene oxide) heterogeneous blends . The surface of the films was also chemically modified by alkaline attack with sodium hydroxide and by type-I collagen coating . Murine fibroblast cell line and primary cultures of human fibroblasts and of two types of keratinocytes were allowed to adhere and to grow comparatively on the different films . The presence of cavities affected neither the adhesion of dermal fibroblasts nor that of keratinocytes . Only keratinocyte proliferation was significantly reduced by the presence of cavities . Collagen coating improved skin cell adhesion and proliferation as well, except in the case of murine fibroblasts . In the case of the NaOH treatments, similar trends were observed but their extent depended on the treatment time . In the case of chemical modifications, fluorescence microscopy bore out adhesion and proliferation tendencies deduced from MTT tests . (c) 2004 Wiley Periodicals, Inc . J Biomed Mater Res 72A: 180-189, 2005. Biochem Pharmacol, 2004 Dec 15, 68(12), 2347 - 58 A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides; Lemaire G et al.; A stable hepatoma cell line expressing the human pregnane X receptor (hPXR) and the cytochrome P4503A4 (CYP3A4) distal and proximal promoters plus the luciferase reporter gene was developed to assess the ability of several xenobiotic agents to induce CYP3A4 and CYP2B6 . After selection for neomycin resistance, one clone, displaying high luciferase activity in response to rifampicin (RIF), was isolated and the stable expression of hPXR was confirmed by reverse transcription polymerase chain reaction (RT-PCR) . Dose-response curves were generated by treating these cells with increasing concentrations of RIF, phenobarbital (PB), clotrimazole (CLOT) or 5beta-pregnane-3,20-dione (5beta-PREGN) . The effective concentrations for half maximal response (EC50) were determined for each of these compounds . RIF was the most effective compound, with maximal luciferase activity induced at 10 microM . The agonist activities of PXR-specific inducers measured using our stable model were consistent with those measured in transient transfectants . The abilities of organochlorine (OC), organophosphate (OP) and pyrethroid pesticides (PY) to activate hPXR were also assessed and found to be consistent with the abilities of these compounds to induce CYP3A4 and CYP2B6 in primary culture of human hepatocytes . These results suggest that CYP3A4 and CYP2B6 regulation through PXR activation by persistent pesticides may have an impact on the metabolism of xenobiotic agents and endogenous steroid hormones . Our model provides a useful tool for studying hPXR activation and for identifying agents capable of inducing CYP3A4 and CYP2B6. Radiat Res, 2004 Dec, 162(6), 660 - 6 Changes in micronucleus frequency resulting from preirradiation of cell culture surfaces; Medvedeva N et al.; We have initiated a series of experiments to quantify the impact of environmental variables on the observed frequency of micronuclei in monolayer cultures . In this paper the influence of preirradiation of cell culture vessels on micronucleus formation in Chinese hamster ovary cells was examined . Dry cell culture vessels were preirradiated with 2 Gy of either alpha particles or X rays and immediately plated with nonirradiated cells . About 48 h later a group of randomly chosen containers was set aside, and the rest of the containers were exposed to a range of doses of X rays or alpha-particle radiation . Nonirradiated cells plated on previously irradiated cell culture surfaces manifested nearly as many micronuclei as the irradiated cells . In all experiments, preirradiation of the cell substrate (the culture dish) led to a significantly increased micronucleus frequency relative to unirradiated substrate . These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be a confounding factor, particularly in low-dose experiments. J Virol, 2004 Dec, 78(23), 13306 - 14 Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture; Neddermann P et al.; Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations . In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations . The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication . Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture . Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds . The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3 . This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors. Mol Cell Endocrinol, 2004 Dec 30, 228(1-2), 79 - 102 Cell lines and primary cell cultures in the study of bone cell biology; Kartsogiannis V et al.; Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite and amorphous calcium phosphate crystals deposited in an organic matrix . Bone has two main functions . It forms a rigid skeleton and has a central role in calcium and phosphate homeostasis . The major cell types of bone are osteoblasts, osteoclasts and chondrocytes . In the laboratory, primary cultures or cell lines established from each of these different cell types provide valuable information about the processes of skeletal development, bone formation and bone resorption, leading ultimately, to the formulation of new forms of treatment for common bone diseases such as osteoporosis. J Immunol Methods, 2004 Oct, 293(1-2), 127 - 42 Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function; Reddy M et al.; Activation of lymphocytes is a complex, yet finely regulated cascade of events that results in the expression of cytokine receptors, production and secretion of cytokines and expression of several cell surface molecules that eventually lead to divergent immune responses . Assessing the qualitative and quantitative nature of lymphocyte function following immunotherapy provides valuable information about the immune responses mediated by a therapeutic agent . To facilitate evaluation of the immunomodulatory activity of therapeutic agents, we have established a platform of in vitro immunoassays with normal human peripheral blood mononuclear cells (PBMCs) treated with several polyclonal activators that are known to exhibit different modes of action . We evaluated the kinetics of cell surface marker expression and cytokine release from PBMCs stimulated in parallel with various activating agents over a time course . These stimulating agents induced early (CD69 and CD71) and late (CD25 and HLA-DR) activation markers to varying antigen densities, indicated different cytokine profiles, and showed differential inhibition with dexamethasone (DEX), an inhibitor of early signaling events . Based on the association or correlation of the kinetics of activation marker expression and secreted cytokines, the results of our study indicate the appropriate time points for the simultaneous measurement of both these activation products . This study defines the kinetics for both measures of T cell activation and provides a comprehensive review with various polyclonal activators that can serve as a reference for monitoring lymphocyte function in clinical study samples. J Biomed Mater Res A, 2005 Jan 1, 72A(1), 10 - 8 Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidic devices: cell culture and flow studies with glial cells; Peterson SL et al.; Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies . Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids . However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells . To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies . For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive . However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40% . Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion. Vet Microbiol, 2004 Nov 30, 104(1-2), 119 - 24 Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture; La Ragione RM et al.; Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only non-O157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals . However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement . In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E . coli O157:H7 . To investigate this, and prior to animal studies, E . coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to HEp-2 tissue culture alone and in competition one with another . Applied together, both strains adhered in similar numbers but lower than when either was applied separately . Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second . It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E . coli O157:H7 . The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. Appl Environ Microbiol, 2004 Nov, 70(11), 6695 - 705 Use of cell culture-PCR assay based on combination of A549 and BGMK cell lines and molecular identification as a tool to monitor infectious adenoviruses and enteroviruses in river water; Lee C et al.; Viral contamination in environmental samples can be underestimated because a single cell line might reproduce only some enteric viruses and some enteric viruses do not exhibit apparent cytopathic effects in cell culture . To overcome this problem, we evaluated a cell culture-PCR assay based on a combination of A549 and Buffalo green monkey kidney (BGMK) cell lines as a tool to monitor infectious adenoviruses and enteroviruses in river water . Water samples were collected 10 times at each of four rivers located in Gyeonggi Province, South Korea, and then cultured on group 1 cells (BGMK cells alone) and group 2 cells (BGMK and A549 cells) . Reverse transcription and multiplex PCR were performed, followed by phylogenetic analysis of the amplicons . Thirty (75.0%) of the 40 samples were positive for viruses based on cell culture, and the frequency of positive samples grown on group 2 cells (65.0%) was higher than the frequency of positive samples grown on group 1 cells (50.0%) . The number of samples positive for adenoviruses was higher with A549 cells (13 samples) than with BGMK cells (one sample); the numbers of samples positive for enteroviruses were similar with both types of cells . By using phylogenetic analysis, adenoviral amplicons were grouped into subgenera A, C, D, and F, and enteroviral amplicons were grouped into coxsackieviruses B3 and B4 and echoviruses 6, 7, and 30, indicating that A549 and BGMK cells were suitable for recovering a wide range of adenoviral and enteroviral types . The cell culture-PCR assay with a combination of A549 and BGMK cells and molecular identification could be a useful tool for monitoring infectious adenoviruses and enteroviruses in aquatic environments. Cell Mol Life Sci, 2004 Oct, 61(19-20), 2523 - 34 Immortalization protocols used in cell culture models of human breast morphogenesis; Gudjonsson T et al.; Defining the key players in normal breast differentiation is instrumental to understanding how morphogenesis becomes defective during breast cancer progression . During the past 2 decades much effort has been devoted to the development of technologies for purification and expansion of primary human breast cells in culture and optimizing a relevant microenvironment, which may help to define the niche that regulates breast differentiation and morphogenesis . In contrast to the general property of cancer, normal human cells have a finite lifespan . After a defined number of population doublings, normal cells enter an irreversible proliferation-arrested state referred to as replicative senescence . To overcome this obstacle for continuous long-term studies, replicative senescence can be bypassed by treatment of cells with chemical agents such as benzopyrene, by radiation or by transfection with viral oncogenes or the gene for human telomerase (human telomerase reverse transcriptase, hTERT) . A drawback of some of these protocols is a concurrent introduction of chromosomal changes, which sometimes leads to a transformed phenotype and selection of a subpopulation, which may not be representative of the tissue of origin . In recent years, we have sought to establish immortalized primary breast cells, which retain crucial characteristics of their original in situ tissue pattern . This review discusses various approaches to immortalization of breast-derived epithelial and stromal cells and the application of such cell lines for studies on human breast morphogenesis. Eur J Neurosci, 2004 Nov, 20(9), 2267 - 75 Cannabinoid-mediated neuroprotection following interferon-gamma treatment in a three-dimensional mouse brain aggregate cell culture; Jackson SJ et al.; Multiple sclerosis is increasingly recognized as a neurodegenerative disease which is triggered by inflammation in the central nervous system (CNS) . Demyelination-associated axonal or neuronal damage is a primary cause of disability and has thus far not been successfully targeted by available drug therapies . The neuroprotective properties of both endogenous and administered cannabinoids have been shown in in vivo and in vitro models of CNS damage following excitotoxic, oxidative, traumatic and ischaemic insults, with a predominantly apoptotic effector mechanism . In this study a foetal mouse telencephalon aggregate cell culture system was developed to compare tissue from cannabinoid receptor 1 knockout mice with wildtype counterparts . Aggregate formation and neurofilament/myelin basic protein accumulation were dependent on the age of foetal dissection and species used . Following treatment with interferon-gamma, levels of myelin basic protein, neurofilament, neuronal dephosphorylation and caspase 3 activation were assessed in telencephalon tissue in vitro . Cytokine treatment resulted in significant loss of the neuronal marker neurofilament-H in cannabinoid receptor 1 knockout cultures but not in wildtypes, indicating that presence of the cannabinoid receptor 1 gene can be neuroprotective . Caspase 3 activation was higher in cultures from knockout animals, indicating an apoptotic mechanism of cell death . Dephosphorylated neurofilament levels were significantly elevated in knockout mice, lending support to the premise that neurofilament dephosphorylation is a marker for neuronal damage . Taken together, these results indicate that neuroprotection could be elicited through the cannabinoid receptor 1, and point towards a potential therapeutic role for cannabinoid compounds in demyelinating conditions such as multiple sclerosis. Chem Biol Interact, 2004 Nov 1, 150(1), 129 - 36 A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells; Li AP et al.; In vitro assays involving primary cells are used routinely to evaluate organ-specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity . A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism . A novel cell culture system, the integrated discrete multiorgan cell culture system (IdMOC), is described here . The IdMOC is based on the "wells within a well" concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells . Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium) . On the day of toxicity testing, a volume of drug-containing medium is added to the containing well to flood all inner wells, thereby interconnecting all the small wells . After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate endpoints . We report here the application of IdMOC in the evaluation of the cytotoxicity of tamoxifen, an anticancer agent with known human toxicity, on primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells) as well as the MCF-7 human breast adenocarcinoma cells . IdMOC produced results that can be used for the quantitative evaluation of its anticancer effects (i.e., cytotoxicity towards MCF-7 cells) versus its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels). Cancer Res, 2004 Nov 1, 64(21), 7702 - 5 Tumor cells fail to trans-induce telomerase in human umbilical vein endothelial cell cultures; Pascale E et al.; The shortening of the telomeres that occurs in most somatic cells and untransformed cell cultures is considered a hallmark of cellular senescence . Re-activation of telomerase, which is usually present in immortal cells, avoids telomere shortening and considerably extends the culture life span . Normal human endothelial cells are characterized by an accelerated rate of telomere shortening and reach replicative senescence after a limited number of cell divisions . It has recently been reported that human telomerase reverse transcriptase expression may be strongly up-regulated in human endothelial cells cocultivated with tumor cells . Due to the important implications of this finding on tumor progression, we have extensively analyzed for the presence of telomerase in primary human endothelial cells either cocultivated with tumor cells or grown with tumor-conditioned medium . We found modest, but readily detectable, amounts of telomerase in all human endothelial cell cultures analyzed that disappeared as the cultures approached senescence . Quantitative reverse transcription-PCR also showed a direct correlation between human telomerase reverse transcriptase expression and the proliferative index of the cultures . Nevertheless, we did not find any evidence of induction of telomerase activity by tumor cells in any of the tested conditions . All data indicate that telomerase in human endothelial cells follows an activation program that is strictly associated to the culture growth rate. Anal Biochem, 2004 Dec 1, 335(1), 119 - 25 Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection; Genzel Y et al.; The direct separation detection of amino acids by anion exchange chromatography with integrated pulsed amperometric detection was optimized for the analysis of typical mammalian cell culture broth samples . Existing gradient elution conditions were adapted, considering the additions of peptone (2 g/L) and 10 vol% fetal calf serum to the medium as well as changing concentrations of glucose from 5.5 g/L up to complete consumption . Samples had to be analyzed in two dilutions with water (1:33.3 and 1:200) due to the strongly varying amino acid concentrations in the samples as a result of the medium composition and cell metabolism . The method was validated in a linear working range for the most common amino acids (2.5-7.5 and 1.25-3.75 microM for cystine/cysteine with 15 microl injection volume) . The relative standard deviation of the method for all amino acids was less than 5%, with detection limits of less than 0.6 microM and quantitation limits of less than 1.6 microM . As an example, data for the amino acid composition of different media used for the production of inactivated influenza vaccines in cell culture are shown. Virology, 2004 Nov 24, 329(2), 261 - 9 Markedly reduced severity of Dengue virus infection in mosquito cell cultures persistently infected with Aedes albopictus densovirus (AalDNV); Burivong P et al.; AalDNV-infected C6/36 cells serially passaged for over 10 weeks showed a decline in percentage of anti-AalDNV-positive cells (APC) from an initial 92% to approximately 20% . Cultures of persistent APC were indistinguishable from uninfected cultures by direct microscopy but most stained cells from early APC passages had enlarged nuclei with eosinophilic inclusions, while late APC passages had few and naive cells none . Super challenge of persistent APC cultures did not increase percentage APC and supernatants from persistent APC cultures gave low APC (40%) in naive C6/36 cell cultures . When challenged with dengue virus serotype 2 (DEN-2), naive C6/36 cells showed severe cytopathic effects (CPE) and high mortality within 4 days, as did early passage APC cultures . Remarkably, DEN-2 infections in persistent APC cultures were much less severe, being characterized by reduced DEN-2 infection percentage, retarded DEN-2 virion production, no CPE and no significant mortality . Reasons for rapid reduction in APC and resistance to superinfection upon serial passage remain unproven but may relate to production of AalDNV-defective interfering particles (DIP) by molecular mechanisms still open to speculation . More difficult to explain is cross-protection against DEN-2-induced mortality seen in persistent APC cultures . However, by comparison to work on shrimp viruses, we speculate that this may involve blockage of viral-triggered apoptosis . The phenomena described raise questions regarding the potential for persistent infections by unknown viruses to confound experimental results with insect cell lines. Hum Genet, 2005 Jan, 116(1-2), 51 - 61 Epub 2004 Oct 23. RGD-containing fibrillin-1 fragments upregulate matrix metalloproteinase expression in cell culture: A potential factor in the pathogenesis of the Marfan syndrome; Booms P et al.; The Marfan syndrome (MFS), a relatively common autosomal dominant disorder of connective tissue, is caused by mutations in the gene for fibrillin-1 (FBN1) . Fibrillin-1 is the main component of the 10- to 12-nm microfibrils that together with elastin form elastic fibers found in tissues such as the aortic media . Recently, FBN1 mutations have been shown to increase the susceptibility of fibrillin-1 to proteolysis in vitro, and other findings suggest that up-regulation of matrix metalloproteinases (MMP), as well as fragmentation of microfibrils, could play a role in the pathogenesis of MFS . In the present work, we have investigated the influence of fibrillin-1 fragments on the expression of MMP-1, MMP-2, and MMP-3 in a cell culture system . Cultured human dermal fibroblasts were incubated with several different recombinant fibrillin-1 fragments . The expression level of MMP-1, MMP-2, and MMP-3, was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and the concentration of the corresponding proteins was estimated by quantitative Western blotting . Our results establish that treatment of cultured human dermal fibroblasts with recombinant fibrillin-1 fragments containing the arginine-glycine-aspartic acid (RGD) integrin-binding motif of fibrillin-1 induces up-regulation of MMP-1 and MMP-3 . A similar effect was seen upon stimulation with a synthetic RGD peptide . The expression of MMP-2 was not influenced by treatment . Our results suggest the possibility that fibrillin fragments could themselves have pathogenic effects by leading to up-regulation of MMPs, which in turn may be involved in the progressive breakdown of microfibrils thought to play a role in MFS. Clin Exp Rheumatol, 2004 Jul-Aug, 22(4 Suppl 34), S45 - 9 The level of soluble Granzyme A is elevated in the plasma and in the Vgamma9/Vdelta2 T cell culture supernatants of patients with active Behçet's disease; Accardo-Palumbo A et al.; OBJECTIVE: Gramzyme A (GrA) is a serine proteinase with trypsin-like activity that is released extracellularly during the degranulation of cytotoxic cells . Among the cytotoxic cells, gamma/delta T cells participate in the early phases of the immune response and are known to express perforin and granzymes constitutively in agreement with their cytolytic pontential . METHODS: GrA activity was detected using the synthetic substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester in the plasma and supernatants of peripheral blood mononuclear cell cultured in the presence of Dimethylallyl pyrophosphate to obtain Vgamma9/Vdelta2 T cell expansion . RESULTS: Significantly high levels of GrA were found in the serum and supernatants of lymphocytes from patients with active Behcet's disease cultured in the presence of DMAPP . Levels were found to be significantly lower after remission . A positive correlation was observed between GrA levels in the supernatants and the Vgamma9/Vdelta2 T cell expansion factor . CONCLUSION: These results strongly suggest that Vgamma9/Vdelta2 T cells are active participants in the pathogenesis of the disease through their degranulation and granzyme release. Proc Natl Acad Sci U S A, 2004 Nov 9, 101(45), 15861 - 6 Epub 2004 Oct 28. Computerized microfluidic cell culture using elastomeric channels and Braille displays; Gu W et al.; Computer-controlled microfluidics would advance many types of cellular assays and microscale tissue engineering studies wherever spatiotemporal changes in fluidics need to be defined . However, this goal has been elusive because of the limited availability of integrated, programmable pumps and valves . This paper demonstrates how a refreshable Braille display, with its grid of 320 vertically moving pins, can power integrated pumps and valves through localized deformations of channel networks within elastic silicone rubber . The resulting computerized fluidic control is able to switch among: (i) rapid and efficient mixing between streams, (ii) multiple laminar flows with minimal mixing between streams, and (iii) segmented plug-flow of immiscible fluids within the same channel architecture . The same control method is used to precisely seed cells, compartmentalize them into distinct subpopulations through channel reconfiguration, and culture each cell subpopulation for up to 3 weeks under perfusion . These reliable microscale cell cultures showed gradients of cellular behavior from C2C12 myoblasts along channel lengths, as well as differences in cell density of undifferentiated myoblasts and differentiation patterns, both programmable through different flow rates of serum-containing media . This technology will allow future microscale tissue or cell studies to be more accessible, especially for high-throughput, complex, and long-term experiments . The microfluidic actuation method described is versatile and computer programmable, yet simple, well packaged, and portable enough for personal use. Acta Otolaryngol, 2004 Oct, 124(8), 890 - 5 Effect of 17 beta-estradiol on diastrophic dysplasia sulfate transporter activity in otosclerotic bone cell cultures and SaOS-2 cells; Imauchi Y et al.; OBJECTIVE: Diastrophic dysplasia sulfate transporter (DTDST) is involved in the regulation of bone turnover, and its activity in otosclerosis has been shown to be abnormally high . Taking into account the role of estrogens in the progression of otosclerosis, the possible effect of estrogens on DTDST was investigated in otosclerotic bone cell cultures and in SaOS-2, a human osteoblastic cell line . MATERIAL AND METHODS: Primary bone cell cultures of stapes and external auditory canal (EAC) bone were obtained from 33 patients with otosclerosis and 18 control patients undergoing cerebellopontine angle tumor surgery . These cultures were assessed in parallel with SaOS-2 cells . Estrogen receptors (ERs) were detected using reverse transcriptase polymerase chain reaction . DTDST activity was assessed by sulfate uptake at baseline and after 24 h of incubation with 17 beta-estradiol at concentrations ranging from 10(-12) to 10(-6) M . RESULTS: Stapes and EAC cultures predominantly expressed mRNA of ER alpha, while ER beta expression was predominant in SaOS-2 cells . In stapes and EAC cultures no modification of DTDST activity was observed with 10(-8) M 17 beta-estradiol . In SaOS-2 cells, DTDST activity was inhibited by 17 beta-estradiol (93.5+/-9.21 vs 83.6+/-8.83 pmol/mg protein/5 min, n=29; mean of differences=10.0+/-3.22, paired t-test, p<0.01) . CONCLUSION: DTDST activity is regulated by estrogens in SaOS-2 cells, but not in primary cell cultures from stapes and EAC . This difference in the regulation mechanisms may be related to the type of estrogen receptor expressed. Analyst, 2004 Nov, 129(11), 1026 - 31 Epub 2004 Aug 11. Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation; Zhu X et al.; This paper describes the use of arrays of horizontally-oriented reservoirs to deliver liquids through microchannels at a constant flow rate over extended periods of time (hours to days) . The horizontal orientation maintains a constant hydraulic pressure drop across microfluidic channels even as the volumes of liquids within the reservoirs change over time . For a given channel-reservoir system, the magnitude of the flow velocity depends linearly on the height difference between reservoirs . The simple structure and operation mechanism make this pumping system versatile . A one-inlet-one-outlet system was used to continuously deliver media for perfusion cell culture, and an array of inlet reservoirs coupled to an outlet reservoir via microchannels was used to drive flows of multiple laminar streams . The parallel pumping scheme conveniently generated various smooth and step concentration gradients, and allowed evaluation of the effect of colchicine on myoblasts . Since the reservoir arrays are configured to be compatible with commercialized multichannel pipettors designed for 96 well plate handling, this simple pumping scheme is envisioned to be broadly useful for medium to high throughput microfluidic perfusion cell culture assays, cell migration assays, multiple laminar flow drug tests, and any other applications needing multiple microfluidic streams. J Virol, 2004 Nov, 78(22), 12613 - 24 Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers; Pegtel DM et al.; Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo . Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo . In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo . Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation . Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia . A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus . We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection . We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant . Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo . This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection. Arch Virol . 2004 Oct 20; {Epub ahead of print} Site directed mutagenesis of the carboxyl terminus of human cytomegalovirus glycoprotein B leads to attenuation of viral growth in cell culture; Strive T et al.; A viable human cytomegalovirus (HCMV) mutant was generated harbouring a glycoprotein B (gB) in which the carboxyl-terminal amino acids DRLRHR (aa 885-900) were changed to AALREE . Characterization of the phenotype of the recombinant virus revealed significant reduction of infectious progeny release and only moderate reduction of viral DNA replication indicating its diminished specific infectivity . This observation was in line with immunogold labeling of extracellular virions demonstrating that the amount of gB protein was markedly reduced in the envelope of the mutant virus . Our results suggest that the conserved carboxyl-terminus of the gB molecule is critical for HCMV maturation. ASAIO J, 2004 Sep-Oct, 50(5), 464 - 7 Use of omentum as an in vivo cell culture system in tissue engineering; Suh S et al.; Many modifications of in vitro culture techniques have been applied to promote tissue formation, resulting in limitations . Because the omentum is composed of lobes of adipose tissue with abundant blood vessels and has been used for organ reconstruction, we used the omentum as an in vivo culture system to promote cellular proliferation upon the scaffold . Two kinds of autogenous cells, oral epithelial cells and rib chondrocytes, obtained from canine were isolated and then seeded on porous poly-lactic-glycolic acid scaffolds of a pre-determined shape and size . Comparison was performed in two groups . In Group 1, cell-polymer constructs were cultured in vitro for 2 weeks, and in group 2, cell-polymer constructs were cultured in vitro for 1 week following the same protocol as group 1 but were then implanted into the omentum of same canines for the next week . We performed histologic analysis of tissue formation between the two groups . In group 1, seeded cells were presented spatially along the porous polymer surface only . However, in group 2, the cell-polymer constructs maintained their original dimensions and showed formation of a multicell layered structure with abundant blood vessels . We concluded that the use of the omentum as an in vivo culture medium offers possibilities as an efficient and effective method for tissue engineering with greater vascularization and more consistent cell spacing throughout the construct. Sci China C Life Sci, 2004 Aug, 47(4), 303 - 12 Mitogen-activated protein kinases mediate the oxidative burst and saponin synthesis induced by chitosan in cell cultures of Panax ginseng; Hu X et al.; Chitosan (CHN) specially induced the activities of 39 kD and 42 kD protein kinases in ginseng cells, which could be suppressed by an inhibitor of mitogen-activated protein kinase (MAPK) pathway, PD98059 . The immunoprecipitation (IP) using MAPK antibody or kinase assay in vitro also showed that CHN-induced 42 kD and 39 kD protein kinases belonged to the MAPK family . PD98059 suppressed CHN-induced transcriptions of ginseng squalene synthase and ginseng squalene epoxidase genes (gss and gse), CHN-induced accumulation of beta-Amyrin synthase (beta-AS) and synthesis of saponin . These results showed that CHN-induced activities of MAPKs were necessary for the CHN-induced saponin synthesis . EGTA and LaCl3 suppressed CHN-induced 39 kD and 42 kD MAPK activities . Ruthenium red (RR) could suppress CHN-induced 39 kD activity . All of them suppressed CHN-induced saponin synthesis . These results indicated that CHN-induced increment of cytosolic calcium was necessary for CHN-induced saponin synthesis . PD98059 also suppressed CHN-induced oxidative burst (including the increment of activity of plasma membrane NADPH oxidase and production of H2O2), but diphenylene iodonium (DPI), dimethylthiourea (DMTU) and 2,5-dihydroxycinnamic acid methyl ester (DHC) could not suppress CHN-induced MAPK activities, which indicated that MAPK was possibly function upstream of CHN-induced oxidative burst. Methods Mol Med, 2004, 107, 325 - 42 Conjunctiva organ and cell culture; Berry M et al.; Conjunctiva, the mucous membrane of the eye, covers its surface from the limbus, the junction with the cornea, to the edges of the eyelids where it meets the skin, thus forming a blind sac that permits free movement of the eye . Topologically, the conjunctiva is divided into bulbar (covering the sclera, adherent at the limbus), tarsal (lining the lids), and fornical (from the medial "corner" of the eye) . At the limbus, where putative corneal stem cells reside, a barrier whose nature has yet to be determined stops conjunctival epithelium from migrating over the cornea . When this barrier is damaged, the migration of conjunctiva over the cornea is accompanied by invasion of blood vessels and, consequently, loss of sight . There is a change in morphology from the fornix, where cylindrical cells give rise to a columnar stratified conjunctival epithelium, to the limbus and lid margins where flattened cells result in a squamous stratified tissue . In rodents and rabbits, goblet cells appear in clusters . Their apical openings are decorated with actin collars . These are not seen in human tissue, where goblet cells may appear either singly or in clusters . Although these cells are larger than the surrounding squames, they do not necessarily span the entire epithelial thickness.The equal efficiency of expanding primary epithelial cultures from every region of the human conjunctiva, i.e., limbal, bulbar, tarsal, and fornical, suggested that this epithelium does not have stem cells . On the other hand, phorbol ester treatment and pulse 3H-thymidine labeling of conjunctival epithelium in vivo indicated the presence of slow cycling cells, consistent with a stem cell population, in the fornix . Clonal expansion of cells from every part of the conjunctiva yielded cultures in which goblet cells differentiated after a specific number of cell divisions . Expansion from explants also resulted in epithelial cultures where goblet cells were observed again after a period of absence . Electron microscope images have been interpreted as refilling of goblet cells. Methods Mol Med, 2004, 107, 269 - 82 Glomerular epithelial and mesangial cell culture and characterization; Wilson HM et al.; The advent of in vitro culture techniques has enabled the culture of homogeneous populations of glomerular mesangial and epithelial cells to aid our understanding of the development of glomerular disease at the cellular level . Advances in our knowledge of the pathogenic mechanisms have made it clear that the response of intrinsic glomerular cells to external stimuli plays an important role in glomerular injury . Glomerular cells from several mammalian species have been isolated and propagated, and, in some instances, cell lines have been generated by viral or nonviral oncogenic transformation.The renal glomerulus is a complex anatomical structure that contains many different cell types, including visceral and parietal epithelial cells, mesangial cells, and endothelial cells . These cells have long been recognized as distinct entities, because they occupy defined anatomical locations in vivo and have distinguishable morphological and cytochemical features . This compartmentalization is, however, lost in culture, as are several of the anatomical characteristics such as endothelial fenestrations and epithelial pedicels; in addition, cultured cells may undergo dedifferentiation . Despite these limitations, the study of glomerular cells in culture has proven useful, and valuable information has been obtained about their physiology and pathophysiology . Human glomeruli are usually obtained from the normal pole of kidneys surgically removed from patients with renal carcinoma or from donor kidneys that cannot be used for transplantation for technical reasons . Glomeruli are isolated using sieves such that they can be rendered virtually free of tubule contaminatio . Thereafter, glomeruli can be seeded into culture flasks, and after a week, cells can be seen growing out of the glomerular core . Alternatively, glomeruli can be dissociated by incubation with an enzyme, such as collagenase before culture. Methods Mol Med, 2004, 107, 163 - 72 Human fetal brain cell culture; Mattson MP; The human brain is a highly evolved and complex organ system, consisting of more than 10 billion nerve cells and at least three times as many glial cells . Because of its cellular complexity, it is important to develop technical approaches that allow isolation and study of nerve and glial cells under conditions in which their environment can be precisely manipulated . Such methods have proven valuable in discovering the cellular and molecular mechanisms of brain development, function, and disease in invertebrates and rodents . However, several factors have contributed to the relative dearth of information concerning the mechanisms responsible for the development and proper function of the human brain compared to the knowledge base in lower species . A major impediment has been the ethical considerations surrounding the use of fetal human tissue obtained from elective abortions and the lack of an alternative source of viable normal human brain cells . Although research that employs human fetal tissue has been limited, what has been done has made a major impact in the development of prophylactics and therapeutics for several important human diseases . For example, the use of fetal tissue from elective abortions was key to development of the polio vaccine. Methods Mol Med, 2004, 107, 111 - 24 Cell cultures of autopsy-derived fibroblasts; Meske V et al.; Until now, for most of the neurodegenerative diseases, such as Alzheimer's disease (AD), ideal animal systems do not exist . Hence, cell-biological experiments, which would help to elucidate the degenerative processes, cannot be performed with affected tissue . On the other hand, biopsy-derived human brain tissue from patients, as an alternative source for living cell material, is rare and, in autopsy-derived tissue, neuronal cells are generally already dead, before any experiments can be performed . There is evidence that peripheral tissue also expresses pathophysiological mechanisms relevant for brain dysfunction, and there are many reports dealing with disease-related abnormalities in the physiology of fibroblasts of AD patients . The advantage of using autopsy-derived fibroblasts from deceased patients is that both the validity of the clinical diagnoses and the severity of neuropathological changes can be assessed reliably by subsequent histological investigations of the brain.Fibroblasts are robust cells that tolerate hypo-oxygen conditions . For that reason, living cells can be isolated from autopsy-derived tissue specimen gained from individuals even with long postmortem delays (<48 h tested) . We used connective tissue to isolate fibroblasts . There are several sources for connective tissue that can be used, for example, dermis of the skin, capsules and stroma of various organs, and mucous and serous membranes . We prefer skin specimens as a source, which are easy to obtain . The dissection of the tissue can be accomplished in several ways . The most noninvasive method is using explants (small parts of the tissue) and allow cells to migrate from the tissue samples . The second method is the mechanical disaggregation of the tissue, using the shear forces that occur during vigorous pipetting or pressing tissue into a mesh/sieve . The third method is based on the enzymatic digestion of the tissue using proteases (trypsin, collagenase, or elastase), which disrupt cell-cell and cell-matrix connections . We used both the explant method and the disaggregation method to obtain living fibroblasts from autopsy-derived skin specimens. Int J Infect Dis, 2004 Oct, 8 Suppl 2, S31 - 44 ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense; Monath TP et al.; The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness . In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture . The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox . A variety of clones were evaluated, and many were unacceptably virulent in animal models . One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells . ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates . To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions . The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes . ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials . The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax. Planta Med, 2004 Oct, 70(10), 936 - 41 Anatalline and other methyl jasmonate-inducible nicotine alkaloids from Nicotiana tabacum cv . By-2 cell cultures; Hakkinen ST et al.; Anatalline {2,4-di(3-pyridyl)piperidine} accumulation was shown to be induced by methyl jasmonate in Nicotiana tabacum cv . BY-2 cell cultures . Beside anatabine, anatalline represented the most abundant alkaloid, moreover, it was always present in two isomeric forms occurring always in similar concentrations . Both isomers could be completely separated by GC-MS . For structural analysis, the isolation of both isomers was performed using a semi-preparative HPLC system . The structures of anatalline {cis-2,4-di(3-pyridyl)piperidine} and its stereoisomer trans-2,4-di(3-pyridyl)piperidine were confirmed by MS and 1D and 2D NMR spectral data . The biosynthetic origin of anatalline was studied by feeding alkaloid precursors to BY-2 cell cultures. J Clin Invest, 2004 Oct, 114(8), 1037 - 40 Endothelial cell culture: beginnings of modern vascular biology; Nachman RL et al.; Endothelial cells derived from human umbilical veins were first successfully cultured in vitro in 1973 . Weibel-Palade bodies and the von Willebrand factor antigen were used as morphological, immunohistochemical, and functional markers to unequivocally identify the cells . These landmark studies helped initiate the growth of modern vascular biology. Mycopathologia, 2004 Jul, 158(1), 17 - 24 Cytokine quantification in the supernatant of mononuclear cell cultures and in blood serum from patients with Jorge Lobo's disease; Vilani-Moreno FR et al.; Few studies are available about the participation of the immune response in the control or the development of Jorge Lobo's disease . Thus, the objective of the present study was to quantify macrophage and lymphocyte cytokines in the supernatant of cell cultures and in blood serum from patients with this disease . The study was conducted on 15 patients with the mycosis and on 15 healthy adult individuals (control group) . Blood samples were collected in order to obtain serum and mononuclear cells . Monocytes were cultured for 24 h in the presence or absence of LPS and L . loboi, and lymphocytes were cultured for 48 h in the presence or absence of PHA and L . loboi . Cytokines IL-1beta, TNF-alpha and IL-6 were quantified by ELISA in the supernatants of monocyte cultures and in serum . Cytokines IL-2, IFN-gamma, IL-4 and IL-10 were quantified by FLISA in the supernatants of lymphocyte cultures and in serum . The quantification of the cytokines in the culture supernatant revealed a greater IL-4 and IL-6 production and lower IL-2 levels in patients compared to control . The production of IL-1beta, TNF-alpha, IL-10 and INF-gamma was similar in patients and controls . The mononuclear cells from patients with the non-localized form of the disease produced higher INF-gamma levels than those of patients with the localized form . The results suggest that patients with Jorge Lobo's disease show altered cytokine profiles represented by a predominance of the Th2 profile . However, further studies are needed to assess the participation of cytokines in the cell-fungus interaction in situ. BMC Biotechnol . 2004 Oct 15;4(1):23. Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer; Vancha AR et al.; BACKGROUND: Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates . Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent . A less known use of this cationic polymer as an attachment factor was explored with several cell lines . RESULTS: Polyethyleneimine compared favorably to traditional attachment factors such as collagen and polylysine . PC-12 and HEK-293 cells plated on dishes coated with polyethyleneimine showed a homogeneous distribution of cells in the plate, demonstrating strong cell adhesion that survived washing procedures . The polymer could also be used to enhance the adherence and allow axonal outgrowth from zebrafish retinal explants . The effects of this coating agent on the transfection of loosely attaching cell lines were studied . Pre-coating with polyethyleneimine had the effect of enhancing the transfection yield in procedures using lipofection reagents . CONCLUSION: Polyethyleneimine is an effective attachment factor for weakly anchoring cell lines and primary cells . Its use in lipofection protocols makes the procedures more reliable and increases the yield of expressed products with commonly used cell lines such as PC-12 and HEK-293 cells. J Virol, 2004 Nov, 78(21), 11591 - 604 Viral evolution and interferon resistance of hepatitis C virus RNA replication in a cell culture model; Sumpter R Jr et al.; Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy . We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region . A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure . In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-beta production and low-level IFN-stimulated gene (ISG) expression . When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication . The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence . Replication of the HP RNA resisted suppression by IFN-alpha treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation . Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly . These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype. J Appl Microbiol, 2004, 97(5), 1105 - 12 Detection of infectious hepatitis A virus by integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction; Jiang YJ et al.; AIMS: A novel integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay was established for detection of infectious hepatitis A virus (HAV) . METHODS AND RESULTS: The specificity of tagged RT-PCR was assessed using HAV genomic positive-strand RNA extracted from HAV virions as reference . Water samples artificially contaminated with infectious or formalin-inactivated HAV were subjected to integrated cell culture (ICC)/RT-PCR and ICC/strand-specific RT-PCR assays respectively . The tagged RT-PCR had high specificity for HAV negative-strand RNA . By demonstrating the formation of negative-strand RNA replicative intermediate, ICC/strand-specific RT-PCR can distinguish between infectious and non-infectious HAV . The described method detected infectious HAV at inoculation level of 10(0) TCID50 per flask within 4 days . CONCLUSIONS: The ICC/strand-specific RT-PCR is a novel, rapid, sensitive and reliable method for detection of infectious HAV . SIGNIFICANCE AND IMPACT OF THE STUDY: Coupled with a suitable virus concentration and purification system, ICC/strand-specific RT-PCR will provide a novel and rapid method for detection of infectious HAV in clinical, environmental and food samples . This assay may be used as an alternative method to test the effective inactivation of inactivated virus vaccines . It may also be adapted to assess the efficacy of disinfection of HAV and enteric viruses in foods and water. In Vitro Cell Dev Biol Anim, 2004 May-Jun, 40(5-6), 150 - 8 Spontaneous cell transformation: karyoplasts derived from multinucleated cells produce new cell growth in senescent human epithelial cell cultures; Walen KH; Previously, it was shown that SV40-induced cell transformation of human diploid (2N), epithelial cells was a dynamic process of nuclear and cellular events . In this process, nuclei of polyploid (above 2N) cells broke down into multinucleated cells (MNCs) by amitotic division . An induced mass karyoplast (i.e., small cell with reduced amount of cytoplasm) budding process from the MNCs produced transformed cells with extended life span (EL) and altered morphology . In this study, without the use of SV40 and no induction of karyoplast budding, the same sequence of cellular events was found to occur spontaneously for the same type of cells at replicative senescence (no mitosis) . These cell transformation events were followed by phase-contrast photography of living cell cultures . Primary, diploid, epithelial cell cultures grew for two to three passages and then entered senescence . Cells remaining in the cultures after widespread cell death (mortality stage 1; M1) developed the typical large, flat-cell morphology of senescence with increased cytoplasmic volume . Some of these cells were MNCs, mostly with two to four nuclei . Cytokinesis in MNCs and spontaneous karyoplast budding from MNCs were observed, and new, limited EL cell growth was present either in foci of cells or as prolonged cell growth over one to two passages . At the end of their replicative phase, the EL cells entered another death crisis (M2) from which no cells survived . In M2-crisis, rarely transformed cells appear with immortal cell growth characteristics (i.e., cell lines) . Numerous examples of fragmentation or amitosis of polyploid nuclei in the production of multinucleated cells (MNCs) are presented . Such nuclear divisions produced nuclei with unequal sizes, which suggest unbalanced chromosomal segregations . The nuclear and cellular events in cell transformation are compared with a natural (no induction) occurrence of MNC-offspring cells in mammalian placentas . The possibility of a connection between these two processes is discussed . And finally the difference in the duration of EL cell growth from SV40-MNCs versus from senescent-MNCs is ascribed to increased mutational load in SV40-induced MNCs as compared with that in senescence MNCs. Drug Chem Toxicol, 2004 Aug, 27(3), 269 - 80 Effects of BSO and L-cysteine on drug-induced cytotoxicity in primary cell cultures: drug-, cell type-, and species-specific difference; Lu Y et al.; The effects of DL-buthionine-(S,R)-sulfoximine(BSO) and L-cysteine(CYS) on cytotoxicity induced by cisplatin(CP) and diclofenac(DIC) in primary cell cultures of hepatocytes and renal tubular epithelial cells(RTEC) isolated from rats or monkeys were studied . Hepatocytes and RTEC were inoculated into collagen-coated 96-well culture plates . After preincubation, a series of concentrations of CP or DIC were added, and 16 h and 4 h prior to CP and DIC, 40 microM BSO and 5 mM CYS were added, respectively . MTT assays were performed to evaluate cytotoxicity(concentrations of drug that inhibited 50% cell growth, IC50) . CYS made IC50s of CP in rat and monkey RTEC increase up to more than 5 mM, but BSO made IC50s of CP in rat RTEC lower down with bigger magnitude than that in monkey RTEC; similarly, CYS made IC50s of CP in rat hepatocytes increase up to more than 5 mM, but BSO made IC50s lower down with bigger magnitude than that in rat RTEC . However, neither CYS nor BSO had significant effects on all IC50s of DIC in all examined cells . These results suggested that during CP-induced stress state, rat hepatocytes were more susceptible to changes of GSH level than rat RTEC, and rat RTEC were more dependent on intracellular GSH status than monkey RTEC . DIC-induced cytotoxicity in RTEC and hepatocytes is independent of GSH level. J Mater Sci Mater Med, 2004 Aug, 15(8), 915 - 23 A comparative investigation of biodegradable polyhydroxyalkanoate films as matrices for in vitro cell cultures; Shishatskaya EI et al.; The paper describes the production and investigation of flexible films made of high-purity polyhydroxyalkanoates (PHAs)--polyhydroxybutyrate {poly-(3HB)} and poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate {poly(3HB-co-3HV)}, containing 4-30 mol % hydroxyvalerate . Poly(3HB-co-3HV) films have a more porous structure than poly-(3HB) films, which are more compact, but their surface properties, such as wettability and surface and interface energies, are the same . Sterilisation of the PHA films by conventional methods (heat treatment and gamma-irradiation) did not impair their strength . Cells cultured on PHA films exhibited high levels of cell adhesion . Cell morphology, protein synthesis and DNA synthesis were estimated by extent of 3H-thymidine incorporation into the animal cell cultures of various origins (fibroblasts, endothelium cells, and isolated hepatocytes) in direct contact with PHAs . The investigation showed that this material can be used to make matrices for in vitro proliferous cells . The investigated properties of poly-(3HB) and poly(3HB-co-3HV) films proved to be fundamentally similar. J Nutr, 2004 Oct, 134(10), 2717 - 21 An in vitro digestion/Caco-2 cell culture system accurately predicts the effects of ascorbic acid and polyphenolic compounds on iron bioavailability in humans; Yun S et al.; We developed a rapid in vitro digestion/Caco-2 cell culture model for assessing relative bioavailabilities of iron in foods and meals . The objective of the present study was to determine how closely our Caco-2 model reflects the human response . Meals described in published reports of studies on effects of varying levels of ascorbic acid (AA) and tannic acid (TA) on iron absorption by human subjects were carefully replicated . Iron absorption ratios (iron absorption from meals containing AA or TA divided by iron absorption from identical meals without these enhancers or inhibitors) were determined using the Caco-2 model . Ferritin formation by the Caco-2 cells was used as an indicator of iron absorption . Response patterns of effects of AA and TA on absorption ratios (AR) calculated from Caco-2 and human data were very similar: AA increased ARs in a dose-response manner and TAs decreased AR . The natural logs of the ARs determined in Caco-2 and human studies were correlated: R = 0.935 (P = 0.012) and 0.927 (P = 0.007) for AA and TA, respectively . When results from meals with AA and TA were pooled, a linear relation between the natural logs of ARs from Caco-2 and human studies was observed (R = 0.968, P < 0.001) . We conclude that our Caco-2 model accurately predicts the human response to AA and TA in the meals we tested . If future work reproduces the precision and accuracy shown in this paper for predicting iron bioavailability to humans, then the implications for saving time and resources in iron bioavailability measurements are considerable. J Cyst Fibros, 2004 Aug, 3 Suppl 2, 55 - 7 Three-dimensional human airway epithelial cell cultures; Ulrich M et al.; An epithelial airway-derived 3-D cell culture model is described . The long lifetime of this model, compared to monolayer cultures of primary cells, allows many experiments with material from one single patient to be performed. J Cyst Fibros, 2004 Aug, 3 Suppl 2, 37 - 41 Immunohistochemistry of CFTR in native tissues and primary epithelial cell cultures; Mendes F et al.; Studies on CFTR protein expression and localization in native tissues or in primary cultures of human epithelial cells are scarce due to the intrinsic instability of this protein, its low expression in most tissues and also to technical difficulties . However, such data are of the highest importance to understand the pathophysiology of CF . The purpose of this article is to outline several assays for the characterization of primary epithelial cultures and to review different CFTR immunostaining protocols. Ann Nucl Med, 2004 Jul, 18(5), 363 - 8 Antisense targeting in cell culture with radiolabeled DNAs--a brief review of recent progress; Hnatowich DJ et al.; The promise of antisense targeting that any tissue with a unique genetic expression can be specifically localized with radioactivity in the living subject is the holy grail that drives this research today . If antisense targeting were to achieve even a fraction of its promise, the results could well lead a revolution in diagnostic nuclear medicine . Despite its obvious complexities, antisense targeting with radiolabeled oligomers such as DNA is making considerable progress in cell culture . As is documented in this brief review, evidence is becoming overwhelming that an antisense mechanism is probably responsible for the accumulation in tumor cells in culture of radiolabeled DNAs with base sequences antisense to target messenger RNAs (mRNAs) . That an increased accumulations of these DNAs compared to control DNAs has now been seen in a substantial number of tumor cell types and mRNA targets largely eliminates any possibility of an aptameric effect being responsible for these specific accumulations . In addition, the number of antisense DNAs accumulating specifically in cells in culture has been shown to be orders of magnitude larger than that expected on the basis of steady state mRNA levels . Thus, two of the main concerns regarding antisense targeted, namely that the mechanism of localization may not be attributed to antisense and that the degree of accumulation will be impractically low for imaging, have been addressed in recent research . The remaining obstacle to successful targeting may be delivery . This review will provide a brief review of recent results, primarily from the laboratory of one of the authors (DJH), obtained in tissue culture in studies of antisense targeting and will conclude with several suggestions for future approaches. Acta Virol, 2004, 48(2), 115 - 21 Genetic stability of the attachment glycoprotein of human respiratory syncytial viruses during serial passages in cell cultures; de Sierra M et al.; Thirteen isolates of human respiratory syncytial viruses (HRSV) of groups A and B were isolated in HEp-2 cells from nasopharyngeal aspirates (NPA) from the children with acute respiratory infections . Three isolates of HRSV of group A were propagated in HEp-2 cells in 20 serial passages . Nucleotide sequences of the products obtained by RT-PCR from the glycoprotein (G) hypervariable region of the original virus isolates in NPA and those after one or several passages were compared . All the isolates analyzed showed no changes during passaging in HEp-2 cells. Acta Virol, 2004, 48(2), 85 - 9 Genetic diversity of chicken anemia virus following cell culture passaging in MSB-1 cells; Hasmah MS et al.; It has been shown that a chicken anemia virus (CAV) isolates which had undergone 60 passages in MSB-1 cells (SMSC-1/P60, 3-1/P60) acquired 33-66 nucleotide substitutions at the coding region resulting in 13-16 amino acid changes as compared to the CAV isolates passaged only 5 times in MSB-1 cells (SMSC-1 and 3-1) (Chowdhury et al., Arch . Virol . 148, 2437-2448, 2003) . In this study we found that a low CAV (BL-5) and a high CAV passage (BL-5/P90) differed by only 15 nucleotide substitutions resulting in 11 amino acid changes . Phylogenetic analysis based on VP1 also revealed that both isolates were close to each other but not to other CAV isolates from Malaysia, namely SMSC-1 and 3-1. J Med Chem, 2004 Oct 7, 47(21), 5126 - 39 Effects of altering the electronics of 2-methoxyestradiol on cell proliferation, on cytotoxicity in human cancer cell cultures, and on tubulin polymerization; Edsall AB et al.; A series of new analogues of 2-methoxyestradiol (1) were synthesized to further elucidate the relationships between structure and activity . The compounds were designed to diminish the potential for metabolic deactivation at positions 2 and 17 and were analyzed as inhibitors of tubulin polymerization and for cytotoxicity . 17alpha-methyl-beta-estradiol (30), 2-propynyl-17alpha-methylestradiol (39), 2-ethoxy-17-(1'-methylene)estra-1,3,5(10)-triene-3-ol (50) and 2-ethoxy-17alpha-methylestradiol (51) showed similar or greater tubulin polymerization inhibition than 2-methoxyestradiol (1) and contained moieties that are expected to inhibit deactivating metabolic processes . All of the compounds tested were cytotoxic in the panel of 55 human cancer cell cultures, and generally, the derivatives that displayed the most activity against tubulin were also the most cytotoxic. J Insect Sci . 2002;2:9 . Epub 2002 May 20. Methods for maintaining insect cell cultures; Lynn DE; Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology . As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique . This paper describes methods that are effective for maintaining various insect cell lines . The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains. Toxicol Lett, 2004 Nov 28, 153(3), 311 - 26 Polychlorinated biphenyl mixtures (Aroclors) induce apoptosis via Bcl-2, Bax and caspase-3 proteins in neuronal cell cultures; Sanchez-Alonso JA et al.; Polychlorinated biphenyls (PCBs) are a group of persistent and widely dispersed environmental pollutants, some of which may be neurotoxic . In the present study, we have investigated the effect of PCB commercial mixtures (Aroclors) on neuronal cell cultures by assessing cell viability and apoptotic cell death . We have combined morphological and biochemical techniques to establish the relevance of apoptosis in neuronal cell death induced by Aroclors . Treatment with both Aroclor 1248 and Aroclor 1260 caused the loss of cell viability and accelerated apoptosis both in a concentration- and time-dependent manner . However, the extent of apoptosis resulted greater for Aroclor 1248 than for Aroclor 1260 . This is correlated with the loss of cell viability since Aroclor 1248 is more cytotoxic . The apoptosis induced by Aroclors involves the increase of caspase-3 activity . To correlate the caspase-3 activity with respect to changes in protein processing, caspase-3 precursor protein (procaspase-3) was evaluated by Western blot analysis . Also, Bcl-2 and Bax protein were assessed in order to elucidate the cell death machinery induced in cortical neuronal cell cultures by Aroclor 1248 . The results indicate that the increase in Aroclor-induced apoptosis correlates with a reduction in the expression of antiapoptotic Bcl-2 and an increase in the expression of proapoptotic Bax . These results suggest that, with our experimental conditions, Aroclors induce apoptosis in primary cultures of cortical neurons via proteins of the Bcl-2 and caspase families. Bone, 2004 Oct, 35(4), 859 - 69 Long-term effects of neridronate on human osteoblastic cell cultures; Frediani B et al.; Bisphosphonates (BPs) are widely used in the treatment of a variety of bone-related diseases, particularly where the bone turnover is skewed in favor of osteolysis . The mechanisms by which BPs reduce bone resorption directly acting on osteoclasts are now largely clarified even at molecular level . Researches concerning the BP's effects on osteoblast have instead shown variable results . Many in vitro studies have reported positive effects on osteoblasts proliferation and mineralization for several BPs; however, the observed effects differ, depending on the variety of different model system that has been used . OBJECTIVES: We have investigated if neridronate, an aminobisphosphonate suitable for pulsatory parenteral administration, could have an effect on human osteoblastic proliferation and differentiation in vitro . METHODS: We have investigated whether prolonged addition of neridronate (from 10(-3) to 10(-11) M) to different human osteoblasts cultures, obtained from 14 different bone specimens, could affect the cells number, the endogenous cellular alkaline phosphatase (ALKP) activity, and the formation of mineralized nodules . RESULTS: Our results show that neridronate does not negatively affect in vitro the viability, proliferation, and cellular activity of normal human osteoblasts even after a long period addition of the drug (20 days) at concentrations equal or lower than 10(-5) mol/l (therapeutic dose) . In addition, neridronate seems to enhance the differentiation of cultured osteoblasts in mature bone-forming cells . A maximum increase of alkaline phosphatase activity (+50% after 10 days; P < 0.01) and mineralized nodules (+48% after 20 days; P < 0.05) was observed in cultures treated with neridronate 10(-8) M . CONCLUSIONS: These results encourage the use of neridronate in long-term therapy of demineralizing metabolic bone disorders. Invest Ophthalmol Vis Sci, 2004 Oct, 45(10), 3697 - 703 Safety testing of infracyanine green using retinal pigment epithelium and glial cell cultures; Jackson TL et al.; PURPOSE: To undertake safety testing of infracyanine green (IFCG) in a cell culture model . METHODS: Experiments were undertaken in a cell culture model used previously to perform safety testing of indocyanine green (ICG) . Human retinal pigment epithelium (RPE) and Muller cells were exposed to IFCG for 5 minutes, over a range of concentrations up to 0.5% . Experiments were repeated, using double-staining with trypan blue . Cell viability was measured at days 1, 5, and 15 using a mitochondrial dehydrogenase assay and a fluorescent live-dead probe containing calcein and ethidium homodimer-1 . Viability was measured after exposure to 0.5% IFCG and 5 minutes of illumination with a vitrectomy endolight powered by a xenon light source . RESULTS: RPE viability was not reduced over the range of concentrations and follow-up intervals . RPE cells exposed to IFCG and illumination had reduced viability relative to the negative control (cells exposed to saline), but not relative to those exposed to saline and illumination . Glial cells showed reduced viability at days 1 and 5, but not day 15 . Illumination did not further reduce viability . CONCLUSIONS: IFCG has been advocated as a safer macular vital stain than ICG . These results suggest that it is less likely to produce phototoxicity, but despite being nearly iso-osmolar, IFCG also produces damage in cultured glial cells. Toxicol Appl Pharmacol, 2004 Oct 1, 200(1), 7 - 15 Mechanism of differential inhibition of hepatic and pancreatic fatty acid ethyl ester synthase by inhibitors of serine-esterases: in vitro and cell culture studies; Kaphalia BS et al.; Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively . We have also reported that only hepatic FAEE synthase is inhibited by tri-o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells . The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity . To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di-o-tolyl-o-( proportional, variant -hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP) . The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines . Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible . However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP . Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation . Studies with HepG2 cells also showed a significant inhibition of FAEE synthase-esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells . 3-BCP did not inhibit FAEE synthase-esterase activity either in HepG2 or AR42J cells . Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase-esterase is supported by our earlier in vivo and in vitro studies . Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase-esterase activities. J Neurobiol, 2005 Jan, 62(1), 121 - 33 Distinct temporal genetic signatures of neurogenic and gliogenic cues in cortical stem cell cultures; Sauvageot C et al.; Cortical progenitor cells from rat embryos give rise to neurons or glia following exposure to platelet derived growth factor (PDGF) or ciliary neurotrophic factor (CNTF), respectively . Both growth factors impart their developmental cues quickly through a transcription-dependent mechanism . Do the alternate developmental responses to PDGF and CNTF reflect induction of qualitatively distinct genes? Alternatively, do the same genes respond to each growth factor, but with quantitatively distinct kinetics? Using differential library screening and custom cDNA microarrays we show that a common set of genes responds to either growth factor . However, quantitative differences in the onset and duration of gene induction equate to the expression of factor-specific gene signatures . Multitissue cluster analysis also reveals tissue-specific gene signatures that may play important roles in the developing brain . (c) 2004 Wiley Periodicals, Inc. J Mater Sci Mater Med, 2004 May, 15(5), 575 - 82 Thermally-reversible gel for 3-D cell culture of chondrocytes; Jasionowski M et al.; Regeneration of destroyed articular cartilage can be induced by transplantation of cartilage cells into a defect . The best results are obtained by the use of autologus cells . However, obtaining large amounts of autologus cartilage cells causes a problem of creating a large cartilage defect in a donor site . Techniques are currently being developed to harvest a small number of cells and propagate them in vitro . It is a challenging task, however, due to the fact that ordinarily, in a cell culture on flat surfaces, chondrocytes do not maintain their in vivo phenotype and irreversibly diminish or cease the synthesis of the phenotypic markers for articular chondrocytes . Therefore, the research is continuing to develop culture conditions for chondrocytes with the preserved phenotype . We have investigated the use of thermoreversible gelling polymer based on N-isopropylacrylamide for the in vitro cell culture of chondrocytes. Tissue Cell, 2004 Oct, 36(5), 323 - 32 Cytokeratin expression in primary epithelial cell culture from bovine conjunctiva; Paladino G et al.; Aim of the present study is to extend our previous observations on a model of primary epithelial cell culture obtained from bovine conjunctiva, and analyse the maintenance of the conjunctival phenotype, relative to cytokeratin (CK) expression, through extended periods of cultivation under different conditions . Conjunctival epithelial cells were grown in transwell filters, and cultured either under liquid covered (LC), or air-interface (AI) conditions . The physiological state of the cells was monitored daily by measurement of the trans-epithelial electrical resistance (TEER) . Analysis of cytokeratin expression was then carried out at different time points (up until 14 days), and compared to the original profile of the conjunctival tissue in order to assess deviations from the primitive phenotype . Immunodetection studies, carried out by both western immunoblot and immunofluorescence analyses, revealed constant expression of the pan-epithelial marker AE3 (recognizing basic type cytokeratins), confirming the epithelial nature of the culture . Other cytokeratins characteristic of non-keratinized stratified epithelia (CK4 and CK13) were absent in corneal tissue, while in conjunctival epithelial cells were more expressed under AI than under LC culture conditions . Expression of CK12, a specific marker of corneal tissue, revealed by the antibody AE5, was never observed in conjunctival epithelial cells . These results indicate that the conjunctival phenotype is conserved during extended periods of culturing, making this system a reliable substitute of conjunctival tissue for pharmaceutical analyses. Zhong Yao Cai, 2004 May, 27(5), 313 - 4 {Production of shikonin by cell cultures of Lithospermum erythrorhizon}; Hu L; OBJECTIVE: To explore cultural conditions of shikonin production by cell cultures of Lithospermum erythrorhizon . METHOD: Orthogonal design was applied in determination of shikonin within the medium . Flask test was applied in the study of shikonin production by the amount of ventilation . RESULT: The best medium consisted of 100 mg/L L-phenylalanine, 2 mg/L IAA and 800 mg/L Ca(NO3)2 4H2O . The best amount of ventilation was get by shaken at 150 r/min . CONCLUSION: This test provided data for producing shikonin by cell cultures of Lithospermum erythrorhizon. Biomaterials, 2005 Mar, 26(9), 979 - 86 Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen; Dhiman HK et al.; Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo . Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals . A biodegradable, biocompatible and non-toxic polymer chitosan was employed for 3D culture of MCF-7 cell lines . Cells grown on chitosan scaffold produce more lactate from glucose in comparison to that secreted by cells grown on tissue culture plate, thus indicating the suitability of chitosan scaffold as an in vitro model resembling cancer tissue growth in vivo . Cytotoxic effect of tamoxifen at different concentrations was evaluated for MCF-7 breast cancer cell lines grown on tissue culture plate as well as on 3D chitosan scaffold . At a tamoxifen concentration of 10(-6) M, 50% reduction in cell growth was observed in tissue culture plate-grown cells where 15% reduction in cell growth was observed when cells were grown in chitosan scaffold . Higher tamoxifen concentrations were required to achieve comparable cytostatic action in 3D culture, supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro . Carbohydrate metabolism of MCF-7 cells in terms of glucose utilization and lactate production in 3D and monolayer culture were unaffected by tamoxifen treatment . Cathepsin D activity, an autocrine growth factor in breast cancer cells was monitored in all experiments . In 3D culture, addition of tamoxifen promoted cathepsin D secretion but inhibited its uptake by cells . Growth of cells in 3D chitosan scaffold indicated that action of tamoxifen on estrogen positive cancer cells is also mediated through inhibition of cathepsin D uptake from the culture medium. Pancreas, 2004 Oct, 29(3), e77 - 83 HPAF-II, a cell culture model to study pancreatic epithelial cell structure and function; Rajasekaran SA et al.; OBJECTIVES: Epithelial cells have distinct apical and basolateral plasma membrane domains separated by tight junctions . This phenotype is essential for the directional transport functions of epithelial cells . Here we characterized a well-differentiated pancreatic epithelial cell line to establish a useful model for understanding the mechanisms involved in the regulation of junctional complexes, polarity, and disease processes in the pancreas . METHODS: Immunofluorescence of cell junction marker proteins and electron microscopy were used to determine the presence of tight junctions, adherens junctions, and desmosomes . The functionality of tight junctions was tested by transepithelial resistance measurements and transepithelial permeability studies of nonionic molecules . Tight junction function in polarity was determined by laser scanning confocal microscopy . RESULTS: Immunofluorescence analysis in HPAF-II cells revealed tight junction localization of ZO-1, occludin, and claudin-4; adherens junction localization of E-cadherin and beta-catenin; and desmosomal localization of desmocollin . Transmission electron microscopy showed the presence of tight junctions, adherens junctions, and des-mosomes, and freeze-fracture electron microscopy revealed the presence of distinct anastomosing tight junction strands . Transepithelial electrical resistance and permeability measurements revealed functional tight junctions . In addition, 3-dimensional images of the monolayer generated by laser scanning confocal microscopy revealed that HPAF-II cells show polarity . Immunoblotting and RT-PCR analyses revealed high expression levels of E-cadherin and Na,K-ATPase beta-subunit but low levels of the transcription factor Snail in HPAF-II cells compared with MiaPaCa-2 cells . CONCLUSION: The HPAF-II cell line is a well-differentiated human pancreatic carcinoma cell line that should be useful as a model for studies aimed at understanding epithelial polarity, regulation of junctional complexes, and disease processes in pancreas. J Pharm Pharm Sci, 2004 Jun 29, 7(2), 186 - 99 A new topological descriptors based model for predicting intestinal epithelial transport of drugs in Caco-2 cell culture; Marrero Ponce Y et al.; PURPOSE: Quantitative Structure-Permeability Relationships (QSPerR) of the intestinal permeability across the (Caco-2) cells monolayer could be obtained by the application of new molecular descriptors . METHOD: A novel topologic-molecular approach to computer molecular design ( TOMOCOMD-CARDD ) has been used to estimate the intestinal-epithelial transport of drug in Caco-2 cell culture . RESULTS: The Permeability Coefficients in Caco-2 cells (P) for 33 structurally diverse drugs were well described using quadratic indices of the molecular pseudograph's atom adjacency matrix as molecular descriptors . A quantitative model that discriminates the high-absorption compounds from those with moderate-poor absorption was obtained for the training data set, showing a global classification of 87.87% . In addition, two QSPerR models, through a multiple linear regression, were obtained to predict the P {apical to basolateral (AP-->BL) and basolateral to apical (BL-->AP)} . A leave- n -out and leave- one -out cross-validation procedure revealed that the discriminant and regression models respectively, had a good predictability . Furthermore, others 18 drugs were selected as a test set in order to assess the predictive power of the models and the accuracy of the final prediction was similar to achieve for the data set . Besides, the use of both regression models, in a combinative way, is possible to predict the Permeability Directional Ratio (PDR, BL-->AP/AP-->BL) value . The found models were used in virtual screening of drug intestinal permeability and a relationship between calculated P and percentage of human intestinal absorption for several compounds was established . Furthermore, this approximation permits us to obtain a good explanation of the experiment based on the molecular structural features . CONCLUSIONS: These results suggest that the proposed method is able to predict the P values and it proved to be a good tool for studying the oral absorption of drug candidates during the drug development process. Eur J Orthod, 2004 Aug, 26(4), 421 - 6 In vitro cytotoxicity of orthodontic archwires in cortical cell cultures; David A et al.; There have been a number of studies regarding the toxicity of orthodontic archwires, but little is known concerning the mechanism of their toxicity . This investigation used murine cortical cell cultures to examine the in vitro neurotoxicity of commonly used orthodontic metallic archwire alloys . The materials examined included 0.016 inch nickel-titanium (NiTi), copper-nickel-titanium, titanium-molybdenum, Elgiloy, and stainless steel archwire alloys . Standard sized samples of each material were placed on tissue culture inserts suspended above the cell cultures . Neuronal death was determined using the lactate dehydrogenase release assay 24 hours after exposure to the archwires . The results indicated that NiTi, copper-nickel-titanium and titanium-molybdenum alloys were not neurotoxic, while stainless steel and Elgiloy were significantly toxic . Washing the archwires for 7 days in a saline solution did not alter the toxicity . However, the free radical scavenger, trolox, blocked the toxicity of both stainless steel and Elgiloy, indicating that the death was free radical mediated . The caspase inhibitor, Z-VAl-Ala-Asp-fluoromethylketone (zVAD-FMK), blocked the toxicity of stainless steel, but not Elgiloy, suggesting that stainless steel induced apoptosis . Further evidence that stainless steel induced apoptosis was provided by propidium staining which showed nuclear chromatin condensation and fragmentation into discrete spherical or irregular shapes, characteristic of apoptosis . The specific metal responsible for the toxicity was not determined; the metals common to each of the toxic archwires were nickel, iron, and chromium. Eur Urol, 2004 Oct, 46(4), 531 - 7 Bladder cell culture on small intestinal submucosa as bioscaffold: experimental study on engineered urothelial grafts; Campodonico F et al.; OBJECTIVES: To investigate the feasibility to perform primary urothelial cell culture using porcine small intestinal submucosa as a delivery scaffold both in vitro and after in vivo implantation in a rabbit model . MATERIALS AND METHODS: Bladder mucosa samples were aseptically obtained from a group of eight male rabbits . The mucosa was cut into fragments and placed on small intestinal submucosa matrices for selective urothelial cell culture . After complete in vitro epithelization the matrices were shaped into tubes and placed in the subcutaneous tissue and subdartos of donor rabbits . The pattern of cell growth and delivery was evaluated on retrieved grafts using histology and immunostaining at the end of the in vitro phase; then 5, 10 and 20 days after implantation . RESULTS: Histological and immunohistochemical analysis of the in vitro primary culture showed the acellular matrices covered with a thin uninterrupted monolayer of urothelial cells . The implants examined on the day 5 maintained the epithelial configuration of the cultured grafts in all samples retrieved . On the day 10 the urothelium showed increased thickness taking on a bilayer configuration . On day 20, all grafts presented the transitional cells arranged in a double layer closely resembling the natural urothelium . The immunostaining pattern displayed the maintaining of urothelial cell phenotype . No differences in epithelium growth and delivery were noted between the two sites of implantation . Five days after implantation, the histological analysis of small intestinal submucosa showed a medium degree tissue reaction with the presence of acute inflammatory cells . Angiogenesis was demonstrated by the development of several new vessels inside the matrix . After twenty days, small intestinal submucosa was gradually replaced with host tissue . CONCLUSION: The small intestinal submucosa proved to function as a means of delivering of autologous urothelial cells cultured in vitro . After ectopic in vivo implantation the bioscaffold maintained viability and growth of the surrounding cells until its degradation. Anal Chem, 2004 Sep 15, 76(18), 5273 - 81 A three-dimensional flow control concept for single-cell experiments on a microchip . 1 . Cell selection, cell retention, cell culture, cell balancing, and cell scanning; Peng XY et al.; An ideal microchip for single-cell experiments should be able to allow us to culture cells, to select any desired single cell from a group, to retain the cell for convenient cellular signal detection, and to deliver any buffer or reagent directly to the cell at any time during continual detection and observation . Most importantly, any negative impact on the live cell should be minimized . To accomplish all these functions, we developed a three-dimensional liquid flow control concept and employed special liquid flow fields to manipulate and retain a single yeast cell freely in the chip . A zero-speed point was controlled to retain the cell for three-dimensional cell balancing and cell scanning . A dispersive flow delivered reagents at a high speed to very near the cell and provided them to the cell at a low speed . No force stronger than its gravitational force was exerted on the cell, which could be balanced on different positions on an arc-sloping wall, thus minimizing any negative impact on the cell due to strong liquid flows . Specifically, we demonstrate on-chip single-cell culture, cell wall removal, and reagent delivery . Subsequently, single-cell fluorescence detection was performed, and noise filtering and background correction were applied for data processing. J Proteome Res, 2004 Jul-Aug, 3(4), 871 - 7 Self-contained on-chip cell culture and pretreatment system; Tabuchi M et al.; In this study, we describe a simple on-chip cell culture and pretreatment system that requires no external machines . Conventional cell culture utilizes culture dishes or microtiter