Cell cultures

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 plates, where pipetting and centrifugation are indispensable for washing cells and changing media . However, our microdevice requires no external centrifugation or pump . Utilizing this microdevice, we attained dramatically shorter total analytical time with a high-throughput screening system for proteomic analysis (1 min per 12 samples; one eightieth of the conventional time) . Protein expression of Jurkat cells during stress-shock induced apoptosis was readily analyzed using this system . We found that a seaweed extraction effectively induced apoptosis of Jurkat cells.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Oct 15, 810(1), 119 - 27
Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents; Shen CF et al.; Lipids (fatty acids) at a concentration range of 10-100 microg/L are essential components included in most serum-free cell culture medium formulations . A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed . The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined . The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10-230 microg/L . Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent . Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery . Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68 . Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.

Biochimie, 2004 Jun, 86(6), 373 - 8
Cell culture media are potent antioxidants that interfere during LDL oxidation experiments; Faure P et al.; In vitro cell-induced low-density lipoprotein (LDL) oxidation is a model frequently used for studies on antioxidant compounds which may be potentially antiatherogens . Using Cu2+ or the free radical generator 2,2'-azobis-{2-amidinopropane} dihydrochloride (AAPH) to oxidize human LDL, we showed that the cell culture media Ham's F10 and RPMI are potent antioxidants which reduce LDL-protective effect of various thyroid compounds . The culture media interfered with the compounds depending on their mechanism of action, and RPMI had the greatest antioxidant effect, completely hiding antioxidant efficiency of the compounds whatever the prooxidant agent was . We suggest some recommendations for study of antioxidant compounds using cell-induced LDL oxidation models.

J Microbiol, 2004 Mar, 42(1), 25 - 31
Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures; Kim H et al.; Sf9 cells have obvious advantages for the conventional production technology of vaccine . They are useful tools for high concentration and large-scale cultures . Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media . To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus . By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days . Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative . Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis . Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining . To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days . The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml . The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay . It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.

Virus Res, 2004 Oct, 105(2), 183 - 94
Protective efficacy of intranasal cold-adapted influenza A/New Caledonia/20/99 (H1N1) vaccines comprised of egg- or cell culture-derived reassortants; Palker T et al.; Live, cold-adapted, temperature-sensitive (ca/ts) Russian influenza A vaccines are prepared in eggs by a 6:2 gene reassortment of the ca/ts donor strain A/Leningrad/134/17/57 (H2N2) (Len/17) with a current wild-type (wt) influenza A strain contributing hemagglutinin (HA) and neuraminidase (NA) genes . However, egg-derived reassortant vaccines are potentially more problematic to manufacture in large quantities than vaccines from cell-based procedures . To compare egg- and cell culture-derived reassortant vaccines, we prepared in Madin Darby canine kidney (MDCK) cells two cloned, ca/ts reassortants (25M/1, 39E/2) derived from Len/17 and a wt reference strain A/New Caledonia/20/99 (H1N1) (NC/wt) . Both 25M/1 and 39E/2 reassortants preserved the ca/ts phenotype and mutations described for internal genes of the A/Len/17 parent . When compared to a commercial, egg-derived ca/ts Russian A/17/NC/99/145 (H1N1) New Caledonia vaccine (NC/145), the MDCK-derived reassortant 39E/2 vaccine conferred similar levels of protection in ferrets challenged i.n . with 7 x 10(10) pfu of NC/wt . In a dose-ranging study, the protective vaccine dose for 50% of ferrets (PD50) was less than 1.2 x 10(4) pfu for the 25M/1 vaccine derived by recombination and amplification in MDCK cells . Clonal isolates of ca/ts influenza A/New Caledonia/20/99 (H1N1) obtained by recombination and amplification entirely in MDCK cells can be highly protective i.n . vaccines.

Neuro Endocrinol Lett, 2004 Jun, 25(3), 223 - 8
Effect of bromocriptine on cell apoptosis and proliferation in GH3 cell culture; Wasko R et al.; OBJECTIVES: In our study, with the use of GH3 cells line we decided to examine 1) what is the relation between the dose of bromocriptine and the development of apoptosis in GH3 cells 2) whether the induction of apoptosis is accompanied by alterations in bcl-2 and p53 content and 3) whether dibutyryl-cAMP or phorbol esters affect the initiation of apoptosis in GH3 cells . RESULTS: The current study demonstrated the absence of alterations in GH3 cells incubated for 24 h with bromocriptine at the concentrations of up to 15 micromol/l . Apoptotic and necrotic changes were observed after 48 h incubation with bromocriptine at the concentrations of 25 micromol/l . The ratio of necrotic to apoptotic cells increased at 40 micromol/l of bromocriptine concentration . An inhibitory effect of bromocriptine on cell proliferation was also observed . Phorbol-12-myristate-13-acetate (PMA), at concentrations ranging between 25 ng to 200 ng/ml, reduced the amount of apoptotic cells . CONCLUSIONS: Application of dibutyryl-cAMP at the concentration of 1 to 8 mmol/l resulted in an inhibition of apoptosis, followed by an increase in the number of cultured cells . Ultrastructural studies showed evident apoptotic lesions in the cells.

J Mater Sci Mater Med, 1997, 8(7), 423 - 6
XPS characterization of surface films formed on surface-modified implant materials after cell culture; Leitao E et al.; Nitrogen ion-implanted Ti-6Al-4V, Ti-5Al-2.5Fe and 316 L stainless steel and nitrogen or carbon sputter-coated samples were inoculated with rat bone marrow . The interface between the cell layer and the substrata was studied by X-ray photo-electron spectrometry and observed by scanning electron microscopy (SEM) . Ca and P were detected on all materials after in vitro cell culture . Titanium appears to be present mainly in the form of TiO2.

J Mater Sci Mater Med, 2002 Apr, 13(4), 421 - 32
Surface topography modulates the osteogenesis in human bone marrow cell cultures grown on titanium samples prepared by a combination of mechanical and acid treatments; Diniz MG et al.; Titanium samples of different roughness R(a) and morphology were prepared using a combination of mechanical (grinding with a SiC paper or blasting with aluminum oxide particles with 65 or 250 microm) and chemical (attack with a sulphuric acid based solution or a hydrofluoric acid based solution) treatments . The biological performance of the prepared surfaces was evaluated using human bone marrow osteoblastic cell cultures . Mechanically treated samples presented different R(a) values and surface morphology . The hydrofluoric acid solution was more effective than the sulphuric acid solution in smoothing titanium surface and also in eliminating aluminum contamination resulting from the blasting process . Bone marrow cells seeded on the different titanium samples showed a similar pattern of behavior during cell attachment and spreading . Cells proliferated very well on all the titanium surfaces and cell growth was observed during approximately two to three weeks . The samples treated with the hydrofluoric acid solution presented higher alkaline phosphatase activity . Only the blasted samples treated with the acid solutions allowed seeded bone marrow cells to form a mineralized extracellular matrix . The best biological performance was found in the blasted samples treated with the hydrofluoric acid solution, which could be related to the characteristic microtopography of these samples that presented a homogeneous and smooth roughness.

J Mater Sci Mater Med, 2003 Aug, 14(8), 727 - 9
Gland cell cultures into 3D hyaluronan-based scaffolds; Zavan B et al.; In this study we report a preliminary investigation of the feasibility of non-woven/sponge fabrics of a hyaluronan derived biomaterials (benzyl ester of HA (HYAFF-11 trade mark FAB, Abano Terme, Italy) for the in vitro culture of rat hepatocytes and rat beta cells . Cell growth on hyaluronan derived biomaterials were tested in the presence of complete medium and in the presence of ECM (extracellular matrix) secreted by fibroblasts previously cultured into the scaffold . Hepatocytes and beta cells were extracted from rat liver/pancreas and seeded either on the HYAFF-11 trade mark scaffold alone, or on HYAFF-11 trade mark scaffold containing ECM . Direct assay of cell proliferation was performed with MTT test . For morphological observations samples were stained with hematoxylin and eosin . The results obtained by MTT test showed that hepatocytes cultivated in both the above described conditions were able to proliferate up to 14 days and Langerhans islet up to 21 days . After this time, cells started to undergo apoptosis . The morphological analyses showed cell aggregation in three-dimensional structures promoted by the fibers of the biomaterial . Our results confirmed that HYAFF-11 trade mark meshes represent a suitable scaffold for hepatocyte adhesion/Langerhans islet organization and proliferation . In particular, the presence of a fibroblast secreted extracellular matrix improves the biological property of the scaffold.

J Mater Sci Mater Med, 2001 Jun, 12(6), 523 - 8
Microstructuring ceramic scaffolds for hepatocyte cell culture; Petronis S et al.; Both extracorporeal liver support devices and tissue engineering of liver for transplantation require the maintenance of functionality of liver cells (hepatocytes) in cell culture for a long time . One approach to achieve this is to optimize hepatocyte in vitro environment by using a scaffold with topographic structure at sub-millimeter scale which controls cell distribution . Therefore, a set of new type of titania ceramic scaffolds, containing cavities of several sizes, has been produced for deducing the best choice of cavity dimensions for culturing hepatocytes . The aim of this paper is to describe in detail the production methods and characterization of such ceramic scaffolds . Experimental production of the scaffolds consists of microfabrication of silicon templates as well as preparation and molding of titania ceramics . The templates, containing arrays of conical protrusions arranged in close-packed hexagonal order, have been achieved using microfabrication methods of photolithography and anisotropic etching in KOH at 50 degrees C . Protrusion dimensions and overall quality of the templates has been evaluated by scanning electron microscopy . The microfabricated templates have resulted in well-defined and reproducible cavities of corresponding dimensions on the titania ceramic surface after injection-molding . Alternatively, simple embossing of the plastified green ceramics with the silicon templates attached to a metal plate also creates cavities on the ceramic surface . While both methods yield good results, they have different advantages: the injection-molding provides a higher quality of imprints while embossing is quicker and less complicated, and is not limited by dimensions of specific molding equipment .

J Mater Sci Mater Med, 2000 Mar, 11(3), 141 - 53
Proliferation/differentiation of osteoblastic human alveolar bone cell cultures in the presence of stainless steel corrosion products; Costa MA et al.; Human osteoblastic alveolar bone cells were cultured for 28 days in control conditions and in the presence of three non-lethal concentrations of AISI 316L stainless steel (SS) corrosion products . Cells were exposed to SS corrosion products in two experimental situations: (i) in selected stages of the incubation time (during the first, second, third and fourth week of culture); and (ii) during the 28 days incubation period . Cultures were characterized for cell proliferation, total protein content, alkaline phosphatase activity (ALP) and ability to form mineralized deposits; culture media was analyzed for ionized calcium (Ca) and phosphorus (P) concentrations throughout the incubation period . The presence of SS corrosion products during the different stages of the incubation period did not significantly affect the cell proliferation; however, a significant dose-dependent deleterious effect was observed on the levels and pattern of ALP activity, concentration of ionized Ca and P in the culture medium and, also, ability to form mineralized deposits, especially in cultures exposed during the first and second week of culture (respectively, lag phase and exponential cell growth phase) . Similar effects were observed in cultures exposed to the SS corrosion products during the 28 days incubation period . However, the presence of such products during the third week (when the mineralization process occurs) and, also, during the fourth week, resulted in little or no significant effects on the behavior of alveolar bone cells . Results suggested that SS corrosion products above certain non-lethal concentrations may disturb the proliferation/differentiation relationship of osteoblastic human alveolar bone cell cultures .

J Mater Sci Mater Med, 1999, 10(10/11), 589 - 94
Endothelial cell cultures as a tool in biomaterial research; Kirkpatrick CJ et al.; Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques . Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs . In the past endothelial cells (EC) have frequently been used in cytotoxicity testing of materials, especially polymers, used in blood-contacting implants, as well as for investigating seeding technologies for vascular prostheses . At present the exponential development both in theory and practice of cell and molecular biology of the endothelium offers great promise in the biomaterial field . Up until now this EC research field has mostly been non-biomaterial orientated . Nevertheless, the relevance for biomaterial research is apparent . Four aspects will be concisely reviewed under the headings inflammation, with special reference to cell adhesion molecules (CAMs) and cytokines, angiogenesis, focusing on the healing response, signal transduction, presenting examples from cytokine- and metal ion-induced up-regulation of genes coding for CAMs, and, finally, endothelial functionality, with emphasis on the principal characteristics of the physiological endothelial phenotype . Finally, the application of these fields to three foci of biomaterial research will be discussed, emphasizing the role of EC culture techniques in controlling the host response to biomaterials (microvascular EC), controlling EC functionality (promoting positive effects and down-regulating negative effects), and tissue engineering (integration of EC into hybrid organs/biosensors) . The need for more co-culture and three-dimensional models will be stressed and data from the authors' laboratory presented to illustrate these principles .

J Mater Sci Mater Med, 1999 Dec, 10(12), 747 - 54
Chemical micropatterning of polymeric cell culture substrates using low-pressure hydrogen gas discharge plasmas; Ohl A et al.; Micropatterned cell cultures will allow a new quality of bioartificial systems . Here, an approach to chemical micropatterning of polymer substrates is presented, which is completely based on low pressure gas discharge processes . Well expressed micropatterned cell cultures on polystyrene and poly (ether ether ketone) were obtained with many different cell types . No impairment of typical cell behavior was observed .

BMC Infect Dis . 2004 Sep 06;4(1):32.
Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003; Vega VB et al.; BACKGROUND: The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome . The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations . METHODS: We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate . These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates . Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated . Phylogenetic analysis was used to uncover molecular relationships between the isolates . RESULTS: Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission . Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible . The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 x 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014) . Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet . Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings . However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes . CONCLUSIONS: Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations . We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China . The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3'--most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.

FASEB J, 2004 Nov, 18(14), 1749 - 51 Epub 2004 Sep 02.
Amyloidogenesis recapitulated in cell culture: a peptide inhibitor provides direct evidence for the role of heparan sulfate and suggests a new treatment strategy; Elimova E et al.; To date 22 different polypeptides, including Abeta in Alzheimer's disease and PrP(Sc) in prion disorders, are known to re-fold and assemble into highly organized fibrils, which associate with heparan sulfate (HS) proteoglycans to form tissue deposits called amyloid . Mononuclear phagocytes have long been thought to be involved in this process, and we describe a monocytic cell culture system that can transform the acute-phase protein serum amyloid A (SAA1.1) into AA-amyloid and appears to recapitulate all the main features of amyloidogenesis observed in vivo . These features in common include nucleation-dependent kinetics, identical proteolytic processing of SAA1.1, and co-deposition of HS with the fibrils . Heparin and polyvinylsulfonate previously reported to block AA-amyloidogenesis in mice are also effective inhibitors in this cell culture model . Furthermore, a synthetic peptide (27-mer) corresponding to a HS binding site of SAA, blocks amyloid deposition at a concentration that is several-orders-of-magnitude lower than any other peptide-based inhibitor previously reported . The 27-mer's inhibitory activity may target the amyloidogenic pathway specifically as it does not interfere with the binding of SAA to monocytes . These data provide direct evidence that SAA1.1:HS interactions are a critical step in AA-amyloidogenesis and suggest a novel treatment strategy for other amyloidoses.

J Immunol Methods, 2004 Aug, 291(1-2), 27 - 38
Validation and comparative analysis of a multiplexed assay for the simultaneous quantitative measurement of Th1/Th2 cytokines in human serum and human peripheral blood mononuclear cell culture supernatants; Prabhakar U et al.; There is increasing evidence suggesting a relationship between cytokine levels and disease pathogenesis, which has led to interest in analyzing multiple cytokines in biological fluids and culture supernatants for various research and clinical studies . The introduction of methodologies allowing simultaneous measurement of interrelated biomarkers/cytokines has further revolutionized this process . In contrast to tissue culture supernatant, the measurement of cytokines in serum has proven to be difficult to characterize in multiplexed formats because of the presence of large dynamic concentration ranges of proteins and other interfering factors that are present in this matrix . In the present study, we have used the microsphere-based multiplex method to simultaneously quantitate and compare six analytes, encompassing a representation of the Th1/Th2 cytokine panel (interleukin (IL)-2, IL-4, IL-5, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and IL-10), in both serum and culture supernatants from peripheral blood mononuclear cells (PBMCs) . A detailed validation procedure for these determinations is described along with a comparative analysis of the performance of the multiplexed assay in serum and culture supernatant matrices . Our results indicate that precision of the multiplexed assay is comparable in both culture supernatant and serum . However, the accuracy of quantification of cytokines in the serum matrix but not in culture supernatant may be compromised depending upon the cytokine being analyzed . Therefore, one must use caution when interpreting data from such complex matrices . Nevertheless, this assay format is appropriate to profile cytokines in clinical trial samples.

Stem Cells Dev, 2004 Aug, 13(4), 344 - 9
Endothelial progenitor cell culture for vascular regeneration; Ishikawa M et al.; A certain population of mononuclear cells in the peripheral blood is capable of contributing to new vessel formation by differentiating into endothelial cells . The basis for native as well as therapeutic neovascularization is not restricted to angiogenesis but includes postnatal vasculogenesis . These cells were discovered by Asahara in 1997 and named endothelial progenitor cells (EPCs) . Clinical usefulness of EPCs from human peripheral blood is also suggested in the animal experiments . These results indicate that administering EPCs can be a new clinical strategy for treating ischemic disease, diabetic retinopathy, or neoplasm in which the promotion or inhibition of neovascular formation is critical . However, expansion of EPCs ex vivo is not currently suitable for the clinical setting because of the animal products that are necessary for EPC expansion . To approach the autologous cell-based clinical application of EPCs, we established EPC culture using auto serum . In this article, we will discuss the sources that can generate EPCs and show the usefulness and potential of EPC culture using auto serum in the basic and clinical setting of neovascular formation.

Cancer Res, 2004 Sep 1, 64(17), 6304 - 9
Direct assessment of drug penetration into tissue using a novel application of three-dimensional cell culture; Kyle AH et al.; The failure of many anticancer drugs to control growth of solid cancers may stem in part from inadequate delivery to tumor regions distant from vasculature . Although the identification of new anticancer drug targets has led to the development of many new drug candidates, there is a lack of methodology for identifying drugs that adequately penetrate tumor tissue . We have developed a novel multilayered cell culture-based assay, which detects the penetration of anticancer drugs based on their effect within tissue . Drug exposures are made over 1 hour to one side of a disk of tissue approximately 150-microm thick, with the other side temporarily closed off, and penetration is then assessed 1-3 days later via bromodeoxyuridine-based detection of S-phase cells . Using this assay, the tissue distribution of a selection of anthracycline analogues was assessed . At clinically relevant exposures, none of the agents were able to affect cells on the far side of the culture at levels approaching that seen on the near (exposed) side . Doxorubicin and epirubicin exhibited approximately 10-fold decreases in the drug exposure seen by the cells on the far side relative to those on the near side of the cultures, whereas for daunorubicin and mitoxantrone, approximately 30-fold and >30-fold decreases were observed respectively . Results were consistent with the observed gradients in drug-derived fluorescence of doxorubicin, epirubicin, and daunorubicin . This model could be applied as a simple anticancer drug development screen to discover drugs that exhibit desirable penetration properties.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2003 Sep, 17(3), 205 - 8
{Anti-SARS virus activities of different recombinant human interferons in cell culture system}; Duan ZJ et al.; BACKGROUND: To study the anti-SARS virus activities of different recombinant human interferons on the cell culture system . METHODS: Anti-SARS virus activities of interferons were determined by using CPE inhibition test in human skeletal muscle sarcoma (Rda) cell culture . RESULTS: The average minimum amount of interferon alpha 2b, alpha 1b, beta 1b or omega 1b to inhibit 50% CPE in Rda cell culture was (160.5+/-129.5) IU/ml, (149.0+/-71.7) IU/ml, (69.5+/-61.5) IU/ml, (87.3+/-47.1) IU/ml, respectively or (0.6+/-0.5) ng/ml, (10.6+/-5.1) ng/ml, (3.5+/-3.1) ng/ml, (0.9+/-0.5) ng/ml, respectively . CONCLUSION: All the tested recombinant interferons showed anti-SARS virus activities on the Rda cell culture with different sensitivities.

Int J Cardiol, 2004 Jun, 95 Suppl 1, S29 - 33
Autologous human serum for cell culture avoids the implantation of cardioverter-defibrillators in cellular cardiomyoplasty; Chachques JC et al.; BACKGROUND: Current clinical experience with cellular cardiomyoplasty (using serum bovine-cultivated myoblasts) has demonstrated significant malignant ventricular arrhythmias and sudden deaths in patients . In some ongoing clinical trials the implantation of cardioverter-defibrillator is mandatory . We have hypothesized that contact of human cells with fetal bovine serum results after 3-week fixation of animal proteins on the cell surface, representing an antigenic substrate for immunological and inflammatory adverse events . METHODS AND RESULTS: Autologous myoblasts were transplanted into infarcted LV in 20 patients (90% males, mean age 62+/-8 years) . Cells were cultivated in a complete human medium during 3 weeks, using the patients' own serum obtained from a blood sample or from plasmapheresis . Injections were performed during CABG (2.1 grafts/pt) . All patients had an uneventful recovery . At a mean follow-up of 14 +/- 5 months without mortality, no malignant cardiac arrhythmias are reported . LV ejection fraction improved from 28 +/- 3% to 52 +/- 4.7% (p = 0.03), and regional wall motion score index (WMSI) from 3.1 to 1.4 (p = 0.04) in the cell-treated segments . Myocardial viability tests showed areas of regeneration . Patients moved from mean NYHA class 2.5 to class 1.2 . CONCLUSIONS: A total autologous cell culture procedure was used in cellular cardiomyoplasty reducing the risk of arrhythmia . Human-autologous-serum cell expansion avoids the risk of prion, viral or zoonoses contamination . Since patients treated with noncultivated bone marrow cells are free of arrhythmia, the bovine-culture medium seems to be responsible for this complication . Cellular cardiomyoplasty may be efficient to avoid progression of ventricular remodeling and subsequent heart failure in ischemic heart disease.

J Cell Physiol, 2004 Nov, 201(2), 181 - 9
IGF-I mRNA levels in bovine satellite cell cultures: effects of fusion and anabolic steroid treatment; Kamanga-Sollo E et al.; Androgenic and estrogenic steroids enhance muscle growth in a number of species; however, the mechanism by which anabolic steroids enhance muscle growth is not known . Castrated male cattle (steers) provide a particularly good model system in which to study the effects of anabolic steroids on muscle growth because they respond dramatically to treatment with both estrogens and androgens . The goal of this study was to determine if treatment of bovine satellite cell (BSC) cultures with 17beta-estradiol (E(2)) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)-alpha, androgen receptor, and growth factors that have been shown to affect muscle growth (insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3, and myostatin) . BSC cultures were established from the semimembranosus muscles of steers and then treated for 48 h with various concentrations of E(2) or trenbolone ranging from 0.001 to 10 nM . IGF-I mRNA levels in proliferating BSC cultures were significantly increased at 0.01 (1.9-times control values, P < 0.02) and at 0.1, 1, and 10 nM E(2) (2.9-, 3.5-, and 3.5-times control values, respectively, P < 0.0001) . Additionally both 1 and 10 nM trenbolone increased IGF-I mRNA levels to 1.7-times control values (P < 0.02) . ER-alpha mRNA was detectable in BSC cultures, and levels were increased (2.3-times control levels, P < 0.001) in cultures treated with 0.001 nM E(2) but not in cultures treated with higher concentrations of E(2) . Androgen receptor mRNA levels also were increased (1.5-times control levels, P < 0.02) in cultures treated with 0.001 nM trenbolone but not by treatment with higher concentrations of trenbolone . Levels of IGFBP-3 were increased (1.4-times control values, P < 0.02) by treatment with 0.001 nM E(2) but not by treatment with high concentrations of E(2) . Myostatin mRNA levels were not affected by any concentration of either of the steroids . Although, levels of IGF-I mRNA were 10-times greater (P < 0.02) in fused BSC cultures than in proliferating cultures, treatment of fused cultures for 48 h with 10 nM E(2) increased IGF-I mRNA levels (2.5-times control levels, P < 0.02) . Both E(2) and trenbolone increased (3)H-thymidine incorporation rate (1.5-times control levels, P < 0.001) in BSC cultures in media containing serum from which IGFBP-3 had been removed by anti-IGFBP-3 affinity chromatography . In summary, treatment of BSC cultures with either E(2) or trenbolone increased IGF-I mRNA level and proliferation rate, thus, establishing that these steroids have direct anabolic effects on cells present in the BSC culture .

Magn Reson Med, 2004 Sep, 52(3), 495 - 505
Simultaneous 1H PFG-NMR and confocal microscopy of monolayer cell cultures: effects of apoptosis and necrosis on water diffusion and compartmentalization; Minard KR et al.; We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed-field-gradient (PFG) 1H-NMR and simultaneous confocal microscopy . In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b-values (0-27000 s/mm2), 1H-NMR data collected with untreated cells exhibited multiexponential behavior . Analysis with a slow-exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 x 10(-3) mm2/s) and volume fractions (0.96 and 0.04) . During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred . Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident . These results indicate that PFG 1H-NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis . Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions .

ALTEX, 2004, 21(3), 129 - 34
Comparison of human corneal cell cultures in cytotoxicity testing; Zorn-Kruppa M et al.; The cytotoxic pattern of cosmetic or pharmaceutical compounds within different layers of the human cornea is of special interest with respect to ocular safety testing . The aim of this study was to evaluate the ability of a newly developed human corneal keratocyte (HCK) cell line as an in vitro model to predict toxicity towards keratocytes in the corneal stroma . The cytotoxic response of immortalised HCK cultures towards different surfactants was compared to that of primary cultures of human corneal keratocytes . Our studies revealed comparable results for immortalised and primary keratocytes . Furthermore, we quantified surfactant-induced cytotoxic effects on immortalised cultures of corneal epithelium and endothelium . In conclusion, the HCK cell line represents an appropriate model to test keratocyte-specific toxicity and may serve as a useful building block in the construction of three-dimensional human cornea equivalent models.

J Mol Histol, 2004 Feb, 35(2), 133 - 9
Optimization of an Acridine Orange-bisbenzimide procedure for the detection of apoptosis-associated fluorescence colour changes in etoposide-treated cell cultures; Landex NL et al.; This study was initiated in order to investigate the possibility of improving fluorescence microscopy as a method for evaluating apoptosis in cells by combining two fluorescent dyes with different staining characteristics . Cells were vitally stained with bisbenzimide (1.3 microM) and Acridine Orange (6.6 microM) and observed using the following filter configuration: excitation 380 nm, beamsplitter 395 nm and longpass filter 397 nm . Control cells exhibited clear blue fluorescent nuclei and red fluorescing lysosomes . In cells treated with etoposide to induce apoptosis, two distinct occurrences were observed: a change in the spectrum of emitted light from bisbenzimide bound to the nuclear region and an increase in lysosomal Acridine Orange fluorescence . The two occurrences together permit a more unbiased detection of apoptosis than most assays . Only one filter set is required for evaluation and the resulting images can be easily evaluated visually or processed further by image analysis.

Arch Biochem Biophys, 2004 Oct 1, 430(1), 77 - 88
Intestinal absorption and metabolism of carotenoids: insights from cell culture; During A et al.; Cell culture models are useful for studying intestinal absorption and metabolism of carotenoids . The human intestinal cell line, Caco-2, has been the most widely used model for these studies . The PF11 and TC7 clones of Caco-2 exhibit beta-carotene-15,15'-oxygenase activity, a key enzyme in the conversion of carotenoids to vitamin A . Studies on the recent cloning of this enzyme are discussed . An in vitro cell culture system used to study intestinal absorption of carotenoids is presented . Under conditions mimicking the postprandial state, Caco-2 cells on membranes take up carotenoids and secrete them incorporated into chylomicrons . Both the cellular uptake and secretion of beta-carotene are saturable, concentration-dependent processes . The selective absorption of all-trans beta-carotene versus its cis isomers, the differential absorption of individual carotenoids, and the specific interactions between carotenoids during their absorption are discussed . The participation of a specific epithelial transporter in the intestinal absorption of carotenoids is proposed.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2004 Jul, 18(4), 327 - 30
{Study on biological behavior of osteoblast and vascular endothelial cell culture}; Rui G et al.; OBJECTIVE: To study the biological behavior of osteoblast and vascular endothelial cell culture . METHODS: The osteoblasts and vascular endothelial cells were obtained from calvarial bone and renal cortex of 2-week rabbits respectively . The experiment were divided into group A (osteoblasts), group B (vascular endothelial cells) and group C (co-cultured osteoblasts and vascular endothelial cells) . The cells were identified with cytoimmunochemical staining . The cellular biological behavior and compatibility were observed under inverted phase contrast microscope and with histological staining . The cells viability and alkaline phosphatase (ALP) activity were measured . RESULTS: The cytoimmunochemical staining showed that the cultured cells were osteoblasts and vascular endothelial cells . The cellular compatibility of osteoblasts and vascular endothelial cells was good . The ALP activity was higher in group C than in group A and group B (P<0.01), and it was higher in group A than in group B (P<0.05) . In group C, the cell proliferation were increased slowly early, but fast later . CONCLUSION: The cellular compatibility of osteoblasts and vascular endothelial cells were good . The vascular endothelial cells can significantly increased the osteoblast viability and ALP activity, and the combined cultured cells have greater proliferation ability.

J Biol Chem, 2004 Oct 15, 279(42), 43371 - 3 Epub 2004 Aug 18.
The innate antiretroviral factor APOBEC3G does not affect human LINE-1 retrotransposition in a cell culture assay; Turelli P et al.; APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G) is an innate intracellular antiretroviral factor that can inhibit viral retroelements such as retroviruses and hepadnaviruses . However, it is unknown whether it can act on non-viral substrates . Retrotransposons are transposable elements that cumulatively account for about one third of the human genome . They are commonly classified in long terminal repeat (LTR) retrotransposons, which are strongly homologous to retroviruses, and non-LTR retrotransposons also known as L1 elements or LINE-1 (long interspersed nucleotide element-1) elements . Most of the L1 elements are defective and only a small number are very active in vivo, but they are responsible for nearby all of the retrotransposition in the human population . The cloning of active human L1 elements has allowed the development of tissue culture-based assays for measuring their retrotransposition potential . We used such an assay to demonstrate that APOBEC3G, which impairs the replication of exogenous retroelements, does not affect the replication of endogenous L1 retrotransposons.

J Biomater Sci Polym Ed, 2004, 15(7), 851 - 64
Fibroin hydrogels for biomedical applications: preparation, characterization and in vitro cell culture studies; Motta A et al.; Silk fibroin hydrogels prepared either by treating a 2% (w/v) silk fibroin aqeuous solution at 4 degrees C (thermgel) or by adding 30% (v/v) of glycerol (glygel), were characterized by using Environmental Scanning Electron Microscopy (ESEM), Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Thermogravimetrical Analysis (TGA) and molecular weight determination . The preparation procedure affected morphology and molecular weight of hydrogels, with no or negligible differences being displayed by FT-IR and DSC analyses . While thermgel presented a well uniform porous structure, the morphology of glygel appeared to be non-porous and heterogeneous . Glygel presented lower water content and lower degradation temperatures, associated with the presence of glycerol but likely also to less-organized protein structures . Cytoxicity tests with human osteoblast-like cells indicated that both gels were not cytoxic, while cell cultures pointed out a faster cell proliferation on glygel and a higher cell activation and differentiation on thermgel . These gels could be used as scaffolds able to promote in situ bone regeneration.

J Mol Neurosci, 2004, 24(1), 15 - 21
Cell cultures from animal models of Alzheimer's disease as a tool for faster screening and testing of drug efficacy; Trinchese F et al.; Approximately 2 million people in the United States suffer from Alzheimer's disease (AD), which is the most common cause of chronic dementia among the aging population . During the last 7 yr, excellent opportunities to screen drugs against AD have been provided by animal models of the disease . Because even in the fastest model, AD pathology does not start before the end of the second month, it has been necessary to wait at least until that age to inject drugs into the animal to assess whether they prevent, reduce, or revert synaptic impairment, plaque formation, and increase of beta-amyloid (Abeta) levels, the main features of the disease . A solution to the problems mentioned above is achieved by the present fast, efficient, and reproducible cultured cell system from animal models of AD or Abeta-associated diseases, for the screening and testing of compounds for the treatment and therapy of AD or Abeta-associated diseases .

Arch Biochem Biophys, 2004 Sep 15, 429(2), 154 - 63
Antioxidant activity of sugar-lysine Maillard reaction products in cell free and cell culture systems; Jing H et al.; Model Maillard reaction (MR) products (MRPs) employing lysine with an aldohexose (e.g., glucose), ketohexose (e.g., fructose), and aldopentose (e.g., ribose) sugars were generated (e.g., pH 9.0; over 2h heating at 120 degrees ) and fractionated with ethanol into low (LMW) and high (HMW) molecular weight fractions . Characteristically different temporal patterns of fluorescence and ultraviolet/visible absorption spectra were obtained from the three distinct sugar-lysine MRPs, and corresponded to different yields of total and dialyzable carbon, indicating that relative reaction rates and degree of polymerization favored the Rib-Lys MRP, compared to Glu-Lys and Fru-Lys MRPs, respectively (p<0.05) . Further characterization of antioxidant activity of the sugar specific-lysine MRPs in chemical (e.g., hydrophobic (1,1,-diphenyl-2-picryl-hydrazyl radical (DPPH) and hydrophilic (Fenton reaction-induced hydroxyl radical) in vitro scavenging assays showed that Rib-Lys HMW MRPs had the highest (p<0.05) affinity to scavenge free radicals . All sugar-Lys MRPs, however, displayed similar protection of cultured Caco-2 cells from exposure to H(2)O(2)-, 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH)-, ferrous (Fe(2+))-, and cupric (Cu(2+))-induced cytotoxicity, evaluated both from redox (e.g., MTT response) and cell membrane integrity (e.g., LDH secretion) . HMW-MRPs exhibited stronger (p<0.05) antioxidant activity to scavenge hydroxyl and DPPH radicals, and a greater (p<0.05) protective effect against both Fe(2+)- and Cu(2+)-induced cytotoxicity in Caco-2 cells than corresponding LMW-MRPs . We conclude that HMW MRPs possess affective antioxidant protection against oxidizable substrates; however, the degree of polymerization of this product, characteristic to the source of monosaccharide used in the reaction, is not a distinguishable factor for this bioactivity.

In Vitro Cell Dev Biol Anim, 2004 Mar-Apr, 40(3-4), 102 - 7
Identification of cells responsible for urokinase-type plasminogen activator synthesis and secretion in human diploid kidney cell cultures; Beqaj S et al.; Human urokinase-type plasminogen activator (uPA) is a serine protease that converts plasminogen to plasmin . It is produced and secreted by a variety of different human cells in vivo and in vitro . We have studied human diploid kidney cell (HKC) cultures prepared from neonatal kidney tissue and cultures of purified populations of HKC to determine which cells synthesize and secrete uPA into the culture medium . Antibodies against cell specific antigens and uPA were used to correlate specific kidney cell types with uPA synthesis . In addition, secretion of uPA activity into growth and uPA production media was determined for each cell type and cultures containing a mixture of cell types . The results of these studies demonstrated that glomerular visceral epithelial and kidney tubular epithelial cells synthesize and secrete uPA into the culture medium.

Vaccine, 2004 Sep 3, 22(25-26), 3329 - 33
Live vaccinia-rabies virus recombinants, but not an inactivated rabies virus cell culture vaccine, protect B-lymphocyte-deficient A/WySnJ mice against rabies: considerations of recombinant defective poxviruses for rabies immunization of immunocompromised individuals; Lodmell DL et al.; Presently, commercially available cell culture rabies vaccines for humans and animals consist of the five inactivated rabies virus proteins . The vaccines elicit a CD4+ helper T-cell response and a humoral B-cell response against the viral glycoprotein (G) resulting in the production of virus neutralizing antibody . Antibody against the viral nucleoprotein (N) is also present, but the mechanism(s) of its protection is unclear . HIV-infected individuals with low CD4+ T-lymphocyte counts and individuals undergoing treatment with immunosuppressive drugs have an impaired neutralizing antibody response after pre- and post-exposure immunization with rabies cell culture vaccines . Here we show the efficacy of live vaccinia-rabies virus recombinants, but not a cell culture vaccine consisting of inactivated rabies virus, to elicit elevated levels of neutralizing antibody in B-lymphocyte deficient A/WySnJ mice . The cell culture vaccine also failed to protect the mice, whereas a single immunization of a vaccinia recombinant expressing the rabies virus G or co-expressing G and N equally protected the mice up to 18 months after vaccination . The data suggest that recombinant poxviruses expressing the rabies virus G, in particular replication defective poxviruses such as canarypox or MVA vaccinia virus that undergo abortive replication in non-avian cells, or the attenuated vaccinia virus NYVAC, should be evaluated as rabies vaccines in immunocompromised individuals.

Vaccine, 2004 Sep 3, 22(25-26), 3237 - 9
Rabies cell culture vaccines reconstituted and stored at 4 degrees C for 1 year prior to use protect mice against rabies virus; Lodmell DL et al.; Human exposure to rabid dogs in developing countries is an ongoing problem that continues to demand effective, safe, and affordable post-exposure rabies vaccinations . Sheep and suckling mouse brain rabies vaccines used in developing countries are being replaced by expensive inactivated-virus cell culture vaccines . Human studies using cell culture vaccines have determined that cost is reduced and protection is maintained by injecting the unused portion of vaccines that have been reconstituted and stored refrigerated for 1 week . Here we determined whether reconstituted purified chick embryo cell and human diploid cell vaccine that had been stored at 4 degrees C for intervals up to 1 year elicit neutralizing antibody, and protect mice against rabies virus . Undiluted, or 1:5 and 1:25 dilutions of both vaccines injected immediately after reconstitution, or after reconstitution and storage at 4 degrees C for 1 week, 1 month, 3 months, 6 months or 1 year elicited high levels of neutralizing antibody and protected 100% of the mice injected with rabies virus.

Horm Metab Res, 2004 Jul, 36(7), 445 - 52
Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures; Kawase T et al.; In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation . In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture . Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers . Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase . Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective . No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3) . Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures . Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.

Melanoma Res, 2004 Aug, 14(4), 257 - 62
Expression of Melan-A/MART-1 in primary melanoma cell cultures has prognostic implication in metastatic melanoma patients; Murer K et al.; The lack of melanoma-associated antigen (MAA) expression has been associated with the reduced overall survival in melanoma patients . In order to investigate whether the MAA expression detected on cell cultures established from melanoma patients might relate to the overall survival in these patients, we screened primary cell cultures derived from 37 melanoma metastases for the expression of five known MAA: Melan-A, tyrosinase, gp-100, MAGE-1 and MAGE-3 by polymerase chain reaction (PCR) and fluorescence-activated cell sorting (FACS) . MAA expression detected by PCR was found at a high percentage in evaluated melanoma cell lines: 25 of 28 (89%) were positive for Melan-A, 22 of 28 (79%) were positive for tyrosinase, 26 of 28 (93%) were positive for gp-100, and 18 of 28 (64%) were positive for MAGE-3 expression . Using the FACS method the percentage of MAA-positive cell lines was much lower: 14 of 31 (45%) cell lines were positive for Melan-A, eight of 31 (26%) were positive for tyrosinase, 13 of 31 (42%) were positive for gp-100, six of 31 (19%) were positive for MAGE-1, and 14 of 31 (45%) were positive for MAGE-3 expression . Kaplan-Meier survival analysis demonstrated that the patients whose cell lines were positive for Melan-A expression by PCR had significantly longer overall survival time as Melan-A PCR-negative cases (P=0.0038) . This could not be shown for any of the markers tested by FACS . Our results suggest that the expression of Melan-A/MART-1 in patient-derived cell cultures may help to identify a group of melanoma patients with prolonged survival .

Appl Biochem Biotechnol, 1999 Oct, 82(1), 17 - 26
Assessment of Various Carbon Sources and Nutrient Feeding Strategies for Panax ginseng Cell Culture; Wu J et al.; Ginseng (root of Panax ginseng C . A . Meyer) cells were cultivated on medium supplemented with various carbohydrates including sucrose, glucose, and fructose, at initial concentrations ranging from 10 to 110 g/L . Sucrose was shown to be the superior carbon source to the monosaccharides for ginseng cell growth and the optimal concentration was between 30 and 50 g/L . An increase in the initial concentration within this range increased the maximum cell density and growth index significantly, whereas much higher concentrations inhibited cell growth . Feeding of sucrose and some other medium components during the growth (fed-batch mode) was more effective in enhancing the cell growth and biomass productivity, increasing the growth index by more than 60-70% and biomass productivity by more than 50%.

Brain Res Brain Res Protoc, 2004 Aug, 13(3), 144 - 50
Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining; Daniel B et al.; The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) stain is in wide use for measuring apoptosis in neurons, as well as in other cell types . TUNEL may give false positive results due to variations in labeling technique as well as staining of cells that have undergone non-apoptotic DNA strand breaks . Therefore, in isolation, TUNEL is not a certain indicator of apoptosis . Recently, we have demonstrated the potent apoptotic effect of secreted phospholipase A2 from group III (sPLA2-III) on primary cortical neurons from rat . Here we describe a computer-assisted method for quantifying TUNEL-positive neurons after sPLA2-III induced apoptosis . Extent of TUNEL is normalized to total nuclear content using 4',6-diamidino-2-phenylindole (DAPI) staining . Furthermore, DAPI counterstaining allows for determination of a nuclear morphology indicator, based on nuclear size and roundness, which we call the nuclear area factor . We found that the nuclear area factor is an early indicator of cell death (significant after 4 h post treatment), while TUNEL staining is significant at later times (26 h) . Thus, the independent staining techniques using TUNEL and DAPI complement each other, and with commercially available image analysis software, may be used to indicate early as well as delayed cell injury processes.

Free Radic Biol Med, 2004 Sep 1, 37(5), 682 - 94
Effect on endothelial cell gene expression of shear stress, oxygen concentration, and low-density lipoprotein as studied by a novel flow cell culture system; Warabi E et al.; A new cell culture system has been developed that reflects the vascular microenvironment . By means of this system the cultured cells are exposed not only to shear stress by the circulating culture medium, but also to an oxygen concentration gradient and certain critical blood components such as low-density lipoprotein (LDL) and monocytes . DNA microarray analysis was performed for human umbilical vein endothelial cells cultured in this system in the absence and presence of laminar flow at a low shear stress, 0.2 dyn/cm(2) . In addition to shear stress, either an oxygen concentration gradient, or LDL (1 mg/ml), or both were applied . Many Nrf-2-regulating genes, such as heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, solute carrier family 7 No . 11, and glutamate-cysteine ligase modifier subunit, were induced by laminar flow at very low shear stress regardless of the additional conditions . Certain genes were specifically affected by exposure to the oxygen gradient and/or LDL under shear stress, but the degree was very low . These results suggest that shear stress is the most critical factor affecting gene expression in endothelial cells and that Nrf-2-regulating proteins may contribute to protecting endothelial cells against other vascular stress . This system should provide highly relevant and useful information about both vascular physiology and pathology, in the latter on such urgent matters as the specific steps involved in atherogenesis.

Cancer Biother Radiopharm, 2004 Jun, 19(3), 343 - 9
Effect of simultaneous administration of Avemar and cytostatic drugs on viability of cell cultures, growth of experimental tumors, and survival tumor-bearing mice; Szende B et al.; Avemar (Biromedicina Co., Budapest, Hungary), a wheat germ preparation with immunomodulant and antimetastatic activity, was applied simultaneously with cytostatic drugs of different modes of action, in vitro and in vivo, in order to find out whether this simultaneous administration exerts an antagonistic or a synergistic effect on the viability of cell cultures, tumor growth, and survival of animals, inoculated with a transplantable mouse tumor (3LL-HH) . In vitro, Avemar did not influence the effect on the viability of MCF-7, HepG2, or Vero cells, exerted by Dacarbazine, 5-fluorouracyl, or Adriblastina . In vivo, Avemar, combined with Endoxan, Navelbine, and doxorubicin, did not prevent the tumor growth inhibitory effect of the cytostatic drugs . The combination of Avemar with the cytostatic drugs did not increase the toxicity of the cytostatic compounds, and did not exert any toxic effect . Avemar may be administered together with cytostatic drugs, without the risk of increasing toxicity or decreasing antiproliferative activity.

Hum Mol Genet, 2004 Oct 1, 13(19), 2207 - 20 Epub 2004 Jul 28.
Phenotypic analysis of neurofilament light gene mutations linked to Charcot-Marie-Tooth disease in cell culture models; Perez-Olle R et al.; Mutations in the neurofilament light (NFL) gene cause Charcot-Marie-Tooth (CMT) disease . There is a wide range of clinical presentations in CMT patients harboring NFL mutations, with patients classified as CMT2E or CMT1F . In this study, we analyzed the effects of five NFL mutations on the assembly and intracellular distribution of intermediate filaments (IFs), and compared the results with those obtained previously for other NFL mutations . Although all NFL mutants affected the formation of IF networks, our data show differential effects on the assembly of IFs depending on the exact nature of the mutation . Defective transport of the mutant NFL subunits was observed for all the CMT-linked NFL mutations, but the characteristics of this defect also depended on the specific mutation . These results show that defects in the assembly and transport of NFs are common to all NFL mutants studied thus far, but the exact nature of the defect appears to be correlated with each mutant genotype.

J Biol Chem, 2004 Sep 24, 279(39), 40629 - 33 Epub 2004 Jul 26.
Evidence against the rescue of defective DeltaF508-CFTR cellular processing by curcumin in cell culture and mouse models; Song Y et al.; Curcumin, the yellow colored component of the spice turmeric, has been reported to rescue defective DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) cellular processing in homozygous mutant mice, restoring nasal potential differences and improving survival (Egan, M . E., Pearson, M., Weiner, S . A., Rajendran, V., Rubin, D., Glockner-Pagel, J., Canny, S., Du, K., Lukacs, G . L., and Caplan, M . J . (2004) Science 304, 600-602) . Because of the implied potential use of curcumin or similar compounds in the therapy of cystic fibrosis caused by the DeltaF508 mutation, we tried to reproduce and extend the pre-clinical data of Egan et al . Fluorometric measurements of iodide influx in Fischer rat thyroid cells expressing DeltaF508-CFTR showed no effect of curcumin (1-40 microm) when added for up to 24 h prior to assay in cells grown at 37 degrees C . Controls, including 27 degrees C rescue and 4 mm phenylbutyrate at 37 degrees C, were strongly positive . Also, curcumin did not increase short circuit current in primary cultures of a human airway epithelium homozygous for DeltaF508-CFTR with a 27 degrees C rescue-positive control . Nasal potential differences in mice were measured in response to topical perfusion with serial solutions containing amiloride, low Cl-, and forskolin . Robust low Cl- and forskolin-induced hyperpolarization of 22 +/- 3 mV was found in wild type mice, with 2.1 +/- 0.4 mV hyperpolarization in DeltaF508 homozygous mutant mice . No significant increase in Cl-/forskolin hyperpolarization was seen in any of the 22 DeltaF508 mice studied using different curcumin preparations and administration regimens, including that used by Egan et al . Assay of serum curcumin by ethyl acetate extraction followed by liquid chromatography/mass spectrometry indicated a maximum serum concentration of 60 nm, well below that of 5-15 microm, where cellular effects by sarcoplasmic/endoplasmic reticulum calcium pump inhibition are proposed to occur . Our results do not support further evaluation of curcumin for cystic fibrosis therapy .

Invest Ophthalmol Vis Sci, 2004 Aug, 45(8), 2778 - 85
Safety testing of indocyanine green and trypan blue using retinal pigment epithelium and glial cell cultures; Jackson TL et al.; PURPOSE: Indocyanine green (ICG) and trypan blue have been advocated as vital stains for use during macular surgery . The safety of these agents was tested using a cell culture model . METHODS: Human retinal pigment epithelium (RPE) and Muller cell lines were exposed to ICG over a range of concentrations up to 0.5%, and trypan blue up to 0.2% . Cells were exposed to each dye for 5, 15, or 30 minutes, rinsed, and incubated 24 hours . Cell viability was measured using a mitochondrial dehydrogenase-assay and fluorescent live-dead probe . Experiments were repeated using 0.5% and 1% ICG and 0.06% and 0.12% trypan blue, with follow-up at 0, 1, 5, and 15 days . ICG experiments were repeated in the presence of illumination from a xenon light-source channeled through a surgical endolight, and using reduced osmolarity solutions of 0.1%, 0.5%, and 1% (185 vs . 275 mOsM) . RESULTS: There was no clear relationship between cell viability and the concentration of the agent or duration of follow-up, except in RPE cells exposed to 1% ICG . These showed a linear (R(2) 0.9952) decline in viability with time, with a significant reduction by day 15 (P = 0.016) . RPE cells exposed to ICG and illumination were not significantly different from the negative control, but when illumination was combined with low osmolarity, viability was reduced (P = 0.0016) . ICG and illumination reduced Muller cell viability (P < 0.0001 for both 185 and 275 mOsM) . Muller cells incubated with 185 mOsM 1% ICG showed a significant reduction in viability (P < 0.0001) not seen with the 185 mOsM 0.5% or 0.1% solutions or in the low-osmolarity RPE groups . CONCLUSIONS: The combination of exposure to 0.5% ICG and the newer endoillumination light-sources can damage cultured Muller cells . Although the preparations of ICG most commonly used clinically did not produce significant damage, relatively small changes in ICG osmolarity and concentration did . This suggests that safety margins are not large . Trypan blue is safe in a cell culture model.

Immunol Lett, 2004 Jul 15, 94(3), 209 - 14
Role of interaction between Ly49 inhibitory receptors and cognate MHC I molecules in IL2-induced development of NK cells in murine bone marrow cell cultures; Das A et al.; Murine bone marrow (BM) cell preparations lack mature cytotoxic natural killer (NK) cells, but NK cells may be induced in these cell preparations by culturing with interleukin-2 (IL2) . Present study was aimed at studying the role of interactions between Ly49 molecules and major histocompatibility complex (MHC) class I molecules during IL2-induced development of mature NK cells in BM cell cultures . Addition of monoclonal antibodies (mabs) specific to class I MHC molecules of H-2b haplotype, to block any interaction of MHC I molecules with their receptors, was found to inhibit NK cell development . Mouse NK cells express several types of Ly49 molecules including Ly49C, which is an inhibitory receptor specific to MHC I molecules of H-2b haplotype . Blocking Ly49-MHC I interaction by using anti-Ly49C mab inhibited the development of cytotoxic NK cells . Addition of anti-Ly49A (no specificity for H-2b MHC I molecules) or anti-Ly49D (activating receptor specific for MHC I molecules of many H-2 haplotypes including H-2b) mabs, however, had no effect on IL2-induced NK cell development in BM cells . Mabs specific to Ly49C molecule and MHC I molecules of H-2b haplotype inhibited the development of mature NK cells from highly purified NK precursor cell population . These results indicate that specific interaction between inhibitory self-reactive Ly49 molecules and MHC I molecules may be crucial for NK cell development . We propose a model in which Ly49-MHC I interaction may have a permissive role in allowing development of only such NK cell clones that expresses at least one self-reactive inhibitory Ly49 molecule so that lysis of autologous healthy cells by mature NK cells may be avoided.

J Neuroendocrinol, 2004 Aug, 16(8), 695 - 703
Melanocortin peptides stimulate prolactin gene expression and prolactin accumulation in rat pituitary aggregate cell cultures; Langouche L et al.; Treatment for 40 h of reaggregate pituitary cell cultures from 14-day-old female rats with nanomolar concentrations of gamma3-melanocyte-stimulating hormone (MSH) increased prolactin mRNA but not growth hormone (GH) mRNA expression levels as measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) . During the 40 h incubation, gamma3-MSH stimulated prolactin accumulation in the culture medium . alpha-MSH, a potent agonist of the rat melanocortin-3 receptor (MC3R) and Ala(8)-gamma2-MSH, a very weak agonist of the MC3R, increased prolactin mRNA expression at a similar concentration range as gamma3-MSH . The effect of gamma3-MSH on prolactin mRNA expression was abolished when aggregates were cultured in the presence of thyroid or glucocorticoid hormones, but not of oestradiol . By contrast, oestradiol abolished the stimulatory effect of Ala(8)-gamma2-MSH on prolactin mRNA expression . In GH3 cells stably transfected with the enhanced green fluorescent protein (eGFP) gene under control of a 3-kb prolactin promoter fragment, a dose as low as 1 nMgamma3-MSH, added for 24 h, significantly increased eGFP fluorescence . Agouti-related protein (AgRP(83-132)), a known endogenous MC3R and MC4R antagonist, did not reduce the stimulation of prolactin mRNA expression by gamma3-MSH or Ala(8)-gamma2-MSH . On its own, AgRP(83-132) significantly increased prolactin mRNA expression level and prolactin accumulation . Both gamma2-MSH and Ala(8)-gamma2-MSH increased {S(35)}GTPgammaS binding in membrane preparations of 14-day-old rat pituitaries and of GH3 cells . Whereas MC3R and MC5R mRNA were detectable by RT-PCR in normal pituitary, these receptor mRNAs were undetectable in GH3 cells using various oligonucleotide primer sets . The present findings indicate that melanocortin peptides stimulate prolactin gene expression and production and that, at least in part, a receptor different from the classic MCR is involved . AgRP appears to have other actions than its known antagonistic activity on the MC3R and MC4R.

J Gen Virol, 2004 Aug, 85(Pt 8), 2479 - 83
Evaluation of new cell culture inhibitors of protease-resistant prion protein against scrapie infection in mice; Kocisko DA et al.; In vitro inhibitors of the accumulation of abnormal (protease-resistant) prion protein (PrP-res) can sometimes prolong the lives of scrapie-infected rodents . Here, transgenic mice were used to test the in vivo anti-scrapie activities of new PrP-res inhibitors, which, because they are approved drugs or edible natural products, might be considered for clinical trials in humans or livestock with transmissible spongiform encephalopathies (TSEs) . These inhibitors were amodiaquine, thioridazine, thiothixene, trifluoperazine, tetrandrine, tannic acid and polyphenolic extracts of tea, grape seed and pine bark . Test compounds were administered for several weeks beginning 1-2 weeks prior to, or 2 weeks after, intracerebral or intraperitoneal 263K scrapie challenge . Tannic acid was also tested by direct preincubation with inoculum . None of the compounds significantly prolonged the scrapie incubation periods . These results highlight the need to assess TSE inhibitors active in cell culture against TSE infections in vivo prior to testing these compounds in humans and livestock.

Nat Biotechnol, 2004 Aug, 22(8), 985 - 92 Epub 2004 Jul 18.
Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture; Durr E et al.; Endothelial cells can function differently in vitro and in vivo; however, the degree of microenvironmental modulation in vivo remains unknown at the molecular level largely because of analytical limitations . We use multidimensional protein identification technology (MudPIT) to identify 450 proteins (with three or more spectra) in luminal endothelial cell plasma membranes isolated from rat lungs and from cultured rat lung microvascular endothelial cells . Forty-one percent of proteins expressed in vivo are not detected in vitro . Statistical analysis measuring reproducibility reveals that seven to ten MudPIT measurements are necessary to achieve > or =95% confidence of analytical completeness with current ion trap equipment . Large-scale mapping of the proteome of vascular endothelial cell surface in vivo, as demonstrated here, is advisable because distinct protein expression is apparently regulated by the tissue microenvironment that cannot yet be duplicated in standard cell culture.

J Altern Complement Med, 2004 Jun, 10(3), 535 - 9
Immunomodulatory effects of Premna tomentosa (L . Verbenaceae) extract in J 779 macrophage cell cultures under chromate (VI)-induced immunosuppression; Devi KP et al.; OBJECTIVE: In the present study, the immunomodulatory effects of Premna tomentosa extract against chromate (VI)-induced toxicity was assessed in J 779 macrophage cell line . DESIGN: The cells were analyzed for cytotoxicity, phagocytosis, oxidant burst, antioxidant status, and cell proliferation . RESULT: Chromate treatment resulted in a significant increase in cytotoxicity and free radical production . Furthermore, there is a significant decrease in reduced glutathione (GSH) levels and glutathione peroxidase activity (GPx) . There was an appreciable decrease in cell proliferation and phagocytosis by macrophages in the presence of chromate . However, pretreatment of the cells with P . tomentosa extract (500 microg concentration), 30 minutes prior to chromate (VI) treatment resulted in a significant inhibition of chromate-induced cytotoxicity and reactive oxygen species production . The extract also restored the antioxidant status, cell proliferation, and phagocytosis similar to that of control cells . CONCLUSION: The results confirm the cytoprotective and immunomodulatory effects of the leaves of P . tomentosa and its possible usage in immunosuppressed conditions . Copyright Mary Ann Liebert, Inc.

Indian J Exp Biol, 2003 Aug, 41(8), 821 - 6
Isolation of etiological agent of hydropericardium syndrome in chicken embryo liver cell culture and its serological characterization; Kumar R et al.; The virus causing hydropericardium syndrome was isolated in chicken embryo liver (CEL) cell culture from livers obtained from naturally infected broilers . The cytopathic effects characterized by rounding and degeneration of cells were visible 36 hr post infection in first passage . At 4th passage level, the infectivity titre was 5.24 log10 TCID50/ml . In May-Grunwald and Giemsa stained cells, basophilic intranuclear inclusions ('bird eye' inclusion), typical of aviadenovirus infection, were observed . The specificity of inclusion was confirmed by indirect immunofluorescence . Various serological tests, such as agar gel precipitation test, counter immuno electrophoresis, micro serum neutralization test and enzyme linked immunosorbent assay were also standardized to confirm the isolation of etiological agent of hydropericardium syndrome in CEL cell culture and to diagnose the disease in poultry.

Indian J Exp Biol, 2003 May, 41(5), 457 - 72
Registration of spontaneous photon emission from virus-infected cell cultures: development of experimental system; Lipkind M; Detection of spontaneous photon emission from virus-infected cells was attempted using cell monolayer cultures prepared from the established cell lines differing by origin and sensitivity to viruses . The experimental system was elaborated permitting maintenance of the cell monolayer cultures grown upon quartz slides placed inside quartz cuvettes within the photomultiplier chamber during prolonged time periods (till 24-36 hr) covering the whole virus multiplication cycle . Rich nutritive medium was employed, providing undisturbed cell viability and virus-induced cytopathic effect (CPE) development during such prolonged experiment, each ingredient of the medium being checked as potential parasitic emitter or extinguisher of the cell-specific emission . As presupposed 'positive control', the in vivo cultivated chorio-allantoic membranes (CAM) of 10-days-old chick-embryonated eggs were used . The virus-infected CAMs showed specific peculiarities of the emission dynamics as compared to monotonous dynamics shown by non-infected CAMs . Similar dynamic regularities were observed in cell monolayer cultures containing much lesser (by order) number of cells per exposed sample . Using the elaborated system, some specific changes in the virus-infected cells were found, being correlated with two stages of virus replication cycle: the initial stage, synchronous penetration of the pre-adsorbed virus inside the cell, and a later stage, characterized by intensive CPE manifestations.

Plant Physiol, 2004 Jul, 135(3), 1346 - 66 Epub 2004 Jul 02.
Binding of arabinogalactan proteins by Yariv phenylglycoside triggers wound-like responses in Arabidopsis cell cultures; Guan Y et al.; Arabinogalactan-proteins (AGPs) are cell wall proteoglycans and are widely distributed in the plant kingdom . Classical AGPs and some nonclassical AGPs are predicted to have a glycosylphosphatidylinositol lipid anchor and have been suggested to be involved in cell-cell signaling . Yariv phenylglycoside is a synthetic probe that specifically binds to plant AGPs and has been used to study AGP functions . We treated Arabidopsis suspension cell cultures with Yariv phenylglycoside and observed decreased cell viability, increased cell wall apposition and cytoplasmic vesiculation, and induction of callose deposition . The induction of cell wall apposition and callose synthesis led us to hypothesize that Yariv binding of plant surface AGPs triggers wound-like responses . To study the effect of Yariv binding to plant surface AGPs and to further understand AGP functions, an Arabidopsis whole genome array was used to monitor the transcriptional modifications after Yariv treatment . By comparing the genes that are induced by Yariv treatment with genes whose expressions have been previously shown to be induced by other conditions, we conclude that the gene expression profile induced by Yariv phenylglycoside treatment is most similar to that of wound induction . It remains uncertain whether the Yariv phenylglycoside cross-linking of cell surface AGPs induces these genes through a specific AGP-based signaling mechanism or through a general mechanical perturbation of the cell surface.

Am J Ophthalmol, 2004 Jul, 138(1), 64 - 9
Comparison of the in vitro toxicity of indocyanine green to that of trypan blue in human retinal pigment epithelium cell cultures; Gale JS et al.; PURPOSE: To compare the in vitro toxicity of indocyanine green (ICG) to that of trypan blue (TB) in human retinal pigment epithelium cell cultures . The use of ICG and TB in macular hole surgery is discussed . DESIGN: In vitro cell biology experimental study . METHODS: The ICG dye and TB were applied to ARPE-19, a commercially available human retinal pigment epithelium cell line . Cultures were established and maintained according to supplier protocols . The ICG dye, TB or Hank's balanced salt solution (controls) were then applied to the cells at varying concentrations and over various exposure periods . Fiberoptic light was also applied to cells to assess for the possibility of a potentiating phototoxic effect . Cell viability fractions were determined using a well-studied mitochondrial dehydrogenase assay . RESULTS: The TB was not toxic to the retinal pigment epithelium cell cultures at any concentration or over any period of exposure, whereas ICG dye demonstrated dose-dependent and exposure-dependent toxicity . The ICG dye was found to be toxic to the cells at all tested concentrations between 5.0 mg/ml (stock concentration, 26.1% cell survival) and 0.5 mg/ml (92.8% cell survival) over a 3-minute exposure . No toxicity to TB was seen at the stock concentration of 1.5 mg/mL . Addition of light to the cultures did not significantly alter cell viability with either dye . Long periods of exposure, 2 hours, 24 hours, and 72 hours, to minute concentrations of either dye did not produce significant cell death . CONCLUSIONS: Indocyanine green demonstrates more toxicity than TB to human retinal pigment epithelium cell cultures . This is independent of any phototoxic potentiating effect of fiberoptic light or solvent toxicity . A clinically useful concentration of 0.5-mg/ml ICG causes low cytotoxicity at 3 minutes' exposure (cell survival 92.8%) and shows no detectable toxicity at 1-minute exposure (cell survival 102%).

FASEB J, 2004 Sep, 18(12), 1404 - 6 Epub 2004 Jul 01.
Purinoceptor expression in regenerating skeletal muscle in the mdx mouse model of muscular dystrophy and in satellite cell cultures; Ryten M et al.; ATP is an important extracellular signaling molecule mediating its effects by activation of P2X and P2Y receptors . P2 receptors are expressed during muscle development, and recent findings demonstrate that ATP can regulate myoblast proliferation and differentiation in vitro . However, the role of purinergic signaling during regeneration of injured skeletal muscle has not been investigated . To examine this process in a clinically relevant system, we used the mouse model of muscular dystrophy (mdx), in which muscle degeneration is rapidly followed by regeneration . The latter process, in vivo muscle regeneration, was the focus of this study, and to study the cellular mechanisms involved in it, a parallel study on normal rat skeletal myoblast cultures was conducted . Using immunohistochemistry, RT-PCR, and electrophysiology, we investigated the expression of the P2X1-7 receptor subtypes and the P2Y1,2,4,6 receptors . Experiments in vitro and in vivo demonstrated the sequential expression of the P2X5, P2Y1, and P2X2 receptors during the process of muscle regeneration . The P2X5 and P2Y1 receptors were expressed first on activated satellite cells, and the P2Y1 receptor was also expressed on infiltrating immune cells . Subsequent P2X2 receptor expression on newly formed myotubes showed significant colocalization with AChRs, suggesting a role in regulation of muscle innervation . Thus, this study provides the first evidence for a role for purinergic signaling in muscle regeneration and raises the possibility of new therapeutic strategies in the treatment of muscle disease.

Pol J Vet Sci, 2004, 7(2), 103 - 8
Effect of KLP-602 on virus replication in cell cultures; Malaczewska J et al.; The effect of KLP-602 (active substance: lysozyme dimer) on the replication of two animal viruses: the TK900 strain of Aujeszky's disease virus and the Roakin strain of the Newcastle disease virus were investigated . The maximal tolerable dose of the drug was determined for two cell cultures (CECC and GMK) and the effect of the medicine on the titre range of infectious viruses and their adsorption was assayed . The direct impact of KLP-602 on the viral strains used was also determined . And finally the replication dynamics of viruses in the presence of KLP-602 preparation was estimated . KLP-602 showed no direct effect on either the viruses applied in the study or their adsorption . The drug, introduced into the culture 24 hours before its infection, did not affect the replication of the pseudorabies virus, but decreased the titre of the Newcastle disease virus . KLP-602 introduced simultaneously with the infection considerably lowered the final titres of both viruses . The medicine had the greatest inhibitory effect on the replication dynamics of both types of viruses in the CECC and of the pseudorabies virus in the GMK culture upon the maximal tolerable concentrations of drug and low infectious doses of viruses applied.

Pol J Vet Sci, 2004, 7(2), 97 - 102
Effect of methisoprinol on virus replication in cell cultures; Malaczewska J et al.; The effect of Methisoprinol (active substance: isoprinozine) on the replication of two animal viruses, the TK900 strain of Aujeszky's disease virus and the Roakin strain of the Newcastle disease virus was investigated . When the maximal tolerable doses of the drug were added to two cell cultures (CECC and GMK), its effect on the level of infectious titres of theviruses and their adsorption were assayed . Investigations were also performed to assess the direct effect of Methisoprinol on the viral strains used . The final stage of the experiment aimed at analysing of the replication dynamics of the viruses in the presence of Methisoprinol . Methisoprinol showed no direct effect on the viruses used in the study . Nor did it affect their adsorption . The preparation applied to the culture 24 hours before infection did not influence the replication of viruses, but administered simultaneously with the infection significantly lowered the final titres of viruses . The highest inhibitory effect of the drug was observed during the analysis of the replication dynamics of both viruses in CECC and of pseudorabies virus in GMK cell culture upon the application of the maximal tolerable doses of Methisoprinol and low infectious doses of the viruses.

Lab Anim (NY), 2004 Jul-Aug, 33(7), 37 - 46
Cell culture and animal models of viral hepatitis . Part I: hepatitis B; Guha C et al.; Despite the existence of a preventative vaccine, HBV represents a substantial threat to public health, suggesting the need for research to develop new treatments to combat the disease . The authors review the available in vitro and in vivo models, including recently developed transgenic and chimeric mouse models.

Hum Fertil (Camb), 2004 Jun, 7(2), 113 - 8
Validation of cell culture media components; Stacey G; It is difficult to predict the influence that a particular combination of processes and culture media may have on cells and tissues manipulated in vitro . Accordingly, it is important that the procedures, media and reagents used in the manipulation of cells for clinical application are specified and have been appropriately assessed for safe and reliable use . This presents an approach to the validation of cell culture media and reagents which is a vital process in the overall quality assurance that is increasingly demanded for cells and tissues used in humans.

Mult Scler, 2004 Jun, 10(3), 290 - 7
Interferon-beta inhibits the expression of metalloproteinases in rat glial cell cultures: implications for multiple sclerosis pathogenesis and treatment; Liuzzi GM et al.; Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in multiple sclerosis (MS) and it has recently been reported that treatment of MS patients with interferon-beta (IFN-beta) reduces MMP-9 serum levels and in vitro release from monocytes . We investigated whether IFN-beta is able to modulate the expression of MMPs in glial cell cultures . Rat microglial and astrocyte cultures were treated with different doses of IFN-beta, then activated by exposure to LPS . In another set of experiments cells were simultaneously activated with LPS and treated with IFN-beta . Culture supernatants collected from astrocytes and microglia were subjected to zymography for the assessment of MMP-2 and MMP-9 . Increased amounts of MMP-9 and MMP-2 were observed in supernatants from LPS-treated astrocytes in comparison with supernatants from nontreated control cells . MMP-9 also increased in LPS-treated microglia . The treatment of astrocytes and microglia with IFN-beta inhibited dose-dependently the expression of both MMP-2 and MMP-9 in LPS-treated astrocytes and of MMP-9 in LPS-treated microglia . These results demonstrate a modulating effect of IFN-beta on the release of MMPs from CNS cells . This effect represents an additional mechanism by which IFN-beta, may decrease the development of new CNS lesions in the course of MS.

Proteomics, 2004 Jul, 4(7), 1883 - 96
Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts; Imin N et al.; Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells . The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population . We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage . Over 3000 proteins could reproducibly be resolved over a pI range of 4-11 . Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing . This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine . These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M . truncatula . The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Cell Tissue Res, 2004 Aug, 317(2), 173 - 85 Epub 2004 Jun 22.
Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture; Kikuchi H et al.; In an organ culture system under a three-dimensional microenvironment that provides the conditions needed for odontoblast differentiation, a row of odontoblasts can be induced (Kikuchi et al . 1996, 2001) . Therefore, in a newly designed three-dimensional cell culture system that fulfils the conditions necessary for odontoblast differentiation (Kikuchi et al . 2002), we examined whether dental papilla cells in rat mandibular incisors could differentiate into tubular dentine-forming cells . In our previously established organ culture system, CM-Dil-labeled cells that were microinjected into isolated dental papillae were replaced by a row of odontoblasts . In a three-dimensional cell culture system, which consists of two kinds of type I collagen in the upper layer over multi-layered cells seeded onto collagen containing Matrigel in the lower layer and which acts as a structural meshwork, dental papilla cells were incubated as multi-layered cells in an artificial extracellular matrix (ECM) . The cells aggregated to form a cell mass and invaginated as a cell mass into the ECM . The cells also extended fine fibrillar processes into the ECM . With regard to invagination, the proteolytic activities of matrix metalloproteinase-2 (MMP-2)/membrane type 1-matrix metalloproteinase (MT 1-MMP) were observed on the outer multi-layers of cells within a cell mass adjacent to the ECM . The cell mass progressively shrank to about one-half to one-third of its original diameter and was organized as a tissue surrounded by a newly secreted ECM, like dental pulp-dentine . The cells adjacent to the secreted ECM were constructed as a row of polarized columnar cells . They extended slender processes into the new ECM, which is characteristic of tubular matrix . Dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP 1) genes, which are specific for odontoblast differentiation, were expressed in an aggregated cell mass where tubular matrix-forming cells were induced . Furthermore, the tubular matrix became mineralized under prolonged culture . These results imply that the putative progenitor cells/stem cells residing in dental papillae can differentiate into odontoblasts under appropriate conditions in vitro .

J Nat Prod, 2004 Jun, 67(6), 973 - 7
Hyperjovinols A and B: Two new phloroglucinol derivatives from Hypericum jovis with antioxidant activity in cell cultures; Athanasas K et al.; Two new and four known phloroglucinol derivatives were isolated from the dichloromethane extract of the Greek endemic plant Hypericum jovis . Their antioxidant activity was evaluated in vitro with the DPPH assay and in cell cultures using the DCFH-DA assay . All six compounds demonstrated significant antioxidant activity, while two of them possessed activity at a cellular level comparable to Trolox, protecting against ROS.

Tsitologiia, 2004, 46(3), 185 - 90
{Development and morphofunctional characterization of the osteoblastic phenotype in cell culture in vitro}; Deev RV et al.; The objective of this research was to study osteogenic properties of cultured rabbit bone marrow stromal cells, newborn rat cranium bone cells and rat osteocarcoma ROS 17-2/8 cells . For this purpose cytochemical reaction for alkaline phosphatase was performed by the Lowry method, mineral deposition was assessed by staining of the cultures after von Kossa . Cranium bone cells were shown to synthesize alkaline phosphatase (34 +/- 7 nmol/min/10(6) cells), the matrix mineralization being found . Bone marrow stromal cells displayed a lower activity alkaline phosphatase level than did cranium bone cells (4 +/- 0.6 nmol/min/10(6) cells) . However, cell cultivation in the presence of dexamethasone in the medium (10(-8) M) induced a higher activity of alkaline phosphatase (9 +/- 1 nmol/min/10(6) cells), mineralization of the extracellular matrix being the case . The highest level of alkaline phosphatase activity was found for ROS 17-2/8 cells (60 +/- 12 nmol/min/10(6) cells) but no matrix mineralization was determined . According to these data, matrix calcification and formation of bone-like nodules are the most important properties of osteoblastic differentiation in vitro.

Can J Microbiol, 2004 Apr, 50(4), 269 - 78
Development of an astrovirus RT-PCR detection assay for use with conventional, real-time, and integrated cell culture/RT-PCR; Grimm AC et al.; Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries . Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water . We have developed and optimized a reverse transcription - polymerase chain reaction (RT-PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction . In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected . The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared . The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT-PCR (ICC/RT-PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less . Also, the sensitivity of the ICC/RT-PCR assay was compared with RT-PCR alone . The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.

J Agric Food Chem, 2004 Jun 30, 52(13), 4330 - 7
Assessment of carotenoid bioavailability of whole foods using a Caco-2 cell culture model coupled with an in vitro digestion; Liu CS et al.; Epidemiological studies have shown that consumption of carotenoid-rich fruits and vegetables is associated with a reduced risk of developing chronic diseases . beta-Carotene, alpha-carotene, and beta-cryptoxanthin are precursors of vitamin A, a nutrient essential for human health . However, little is known about the bioavailability of carotenoids from whole foods . This study characterized the intestinal uptake performance of carotenoids using monolayers of differentiated Caco-2 human intestinal cells and mimicked human digestion to assess carotenoid absorption from carrots and corn . Results showed that Caco-2 cellular uptake of beta-carotene and zeaxanthin was higher than that of lutein . Uptake performances of pure carotenoids and carotenoids from whole foods by Caco-2 cells were both curvilinear, reaching saturated levels after 4 h of incubation . The time kinetics and dose response of carotenoid uptake presented a similar pattern in Caco-2 cells after plating for 2 and 14 days . Furthermore, the applicability of this new model was verified with whole grain corn, showing that cooked corn grain significantly enhanced carotenoid bioavailability . These results support the feasibility of the in vitro digestion cell model for assessing carotenoid absorption from whole foods as a suitable and cost-effective physiological alternative to current methodologies.

J Agric Food Chem, 2004 Jun 30, 52(13), 4097 - 100
Gluten of spelt wheat (Triticum aestivum subspecies spelta) as a source of peptides promoting viability and product yield of mouse hybridoma cell cultures; Franek F; The enzymic hydrolysate of gluten from spelt wheat (Triticum aestivum subsp . spelta), an ancient protein-rich wheat subspecies, was subjected to repeated chromatography runs on the small pore size exclusion chromatography matrix, Biogel P-2 . Two small peptide fractions were purified by rechromatography . The amino acid analyses carried out upon total hydrolysis of these fractions have shown a very high proportion of glutamic acid/glutamine, leucine, and methionine . The biological activity of the peptide fractions was tested on a model hybridoma at a concentration range from 0.02 to 0.2% . The most striking effect of peptide fractions, apparent even at the lowest concentrations tested, was a significantly higher persistence of viable cells on day 6, i.e., at the decline phase of the cultures . Culture viability values in the presence of peptide fractions were 64-74%, in comparison with 56% in the control culture . The results of this work are consistent with the concept that peptide molecules may act as antiapoptotic agents, survival factors, rather than serving as metabolic substrates.

Bone, 2004 Jul, 35(1), 83 - 95
Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell cultures by increased donor age and glucocorticoid treatment; Chen TL; Primary cultures of bone marrow stromal cells (BMSC) from long bones of young (4-5 months) and old (22-25 months) C57BL/6 male mice were used to study how donor age affects growth and differentiation of osteoblasts and their sensitivity to dexamethasone (DEX) . We assessed changes in the number and area of alkaline phosphatase-positive bone-forming osteolastic colonies (CFU-ALP) and in the total number of colonies (CFU-F) that include ALP negative colonies . Cell proliferation and apoptosis, specific activity of ALP, were also measured for growth and differentiation . We found that the number of nucleated cells harvested from old mice was significantly higher (approximately 20% more) than that from young mice . However, the number of colonies formed by old cells was fewer and the total area less than those formed by young cells plated at the same density . Young and old cells responded similarly to DEX showing a dose-dependent decrease in colony number and area with more inhibition for area than number . DEX affected CFU-ALP more than CFU-F indicating a greater inhibition for osteoprogenitor cells than other cell types . Inhibition of cell attachment at early culture was the major cause for the DEX reduction of colony number and the major cause of area reduction was inhibition of cell proliferation . This was demonstrated by a severe dose-dependent lowering of bromodeoxyuridine (BrdU) incorporation to less than 40% of the control . Although the number of apoptotic cells in the DEX-treated cultures was higher, apoptosis was not a major factor since the number of apoptotic cells was less than 5% even with DEX treatment . Despite these negative effects on colony number and size, DEX-enhanced osteoblastic differentiation activity by stimulating ALP activity of the colonies up to 25-fold in the young and 5-fold in the old . Our data suggest that increased age lowered the number of osteoprogenitor cells and their growth in BMSC cultures . DEX decreased the attachment and proliferation of BMSC in culture . These changes reflect age-related and glucocorticoid-induced osteopenia . Mouse BMSC cultures therefore may serve as a useful in vitro model to study the mechanisms of type II osteoporosis.

Bone, 2004 Jul, 35(1), 47 - 56
Cyclosporine A and FK506 induce osteoclast apoptosis in mouse bone marrow cell cultures; Igarashi K et al.; Studies were carried out to characterize the effects of cyclosporines and FK506 on the formation and survival of osteoclasts deriving from mouse bone marrow cultures . Cyclosporin A (CsA), cyclosporin B (CsB), cyclosporin H (CsH), and FK506 all inhibited receptor activator of NFkappaB ligand (RANKL)-stimulated tartrate-resistant acid phosphatase (TRAP) activity and generation of TRAP+ multinucleated cells in the cultures . CsA and CsG were approximately equipotent, CsH was approximately one order of magnitude less potent than the other cyclosporines, and FK506 was approximately two orders of magnitude more potent than CsA and CsG . All of the inhibitors demonstrated greater potency and efficacy on decreasing the number of TRAP+ multinucleated cells than on decreasing total TRAP activity . Further evidence that late stages were more sensitive to inhibition was obtained in experiments in which CsA was present for different segments of the RANKL-stimulated culture period . CsA was as efficacious when added for the final 2 days of a 4-day culture as when added for the entire culture period, whereas it was less effective if added for only the first 2 days of the culture . When CsA or FK506 were added for 1 day to cultures in which osteoclasts had already formed, the numbers of TRAP+ osteoclasts decreased . Treatment with CsA or FK506 produced nuclear fragmentation and disruption of the multinucleated osteoclasts and an increase in caspase-3 activity . The apoptosis inhibitor z-VAD partially prevented the inhibitory effects of CsA and FK506 on the survival of TRAP+ multinucleated cells in the cultures and also preserved the normal osteoclast morphology . The data indicate that an important component of the inhibitory effects of CsA and FK506 on marrow-derived osteoclasts is the induction of apoptosis.

Br J Ophthalmol, 2004 Jul, 88(7), 957 - 61
Conditioned medium from mixed retinal pigmented epithelium and Müller cell cultures reduces in vitro permeability of retinal vascular endothelial cells; Tretiach M et al.; AIM: To investigate the in vitro effect of laser photocoagulation on blood-retinal barrier permeability . METHODS: Retinal capillary endothelial cells were exposed to supernatants from long term co-cultured cells that were argon laser treated . Endothelial cell permeability was analysed by (1) measurement of transendothelial electrical resistance and (2) equilibration of {(3)H} inulin and {(14)C} albumin across the cell monolayer . RESULTS: Laser photocoagulation of various retinal cells and control ECV304 cells in the lower chamber did not appreciably improve permeability of the endothelial cell monolayer compared with that of unlasered cells . However, medium that was conditioned by mixed retinal pigmented epithelium and Muller cells significantly reduced both inulin (43.2% (SD 6.5%) equilibration in mixed cultures v 59.8% (SD 7.0%) control cells, p<0.05) and albumin (15.1% (SD 3.8%) v 31.1% (SD 6.7%), p<0.05) permeability of the endothelial cell monolayers . A fourfold increase in transendothelial electrical resistance was also seen . CONCLUSIONS: These results are consistent with the hypothesis that interaction of Muller cells with retinal pigmented epithelium induced by laser treatment results in secretion of soluble factor(s), which reduces permeability of retinal vascular endothelium . Identification of these factor(s) may have implications for the clinical treatment of macular oedema secondary to diabetic retinopathy and other diseases.

Hematology, 2004 Jun, 9(3), 199 - 205
Generation of dendritic cells using cell culture bags--description of a method and review of literature; Buchler T et al.; Anticancer immunotherapy using dendritic cell-based vaccines is a strategy aimed at the induction and maintenance of immune responses against cancer cells . Clinical applications of dendritic cells (DCs) require stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization of DC-based vaccine preparation . Recently, closed systems for DC culture have been developed with a goal to minimize the risk of contamination . Here, we compare the yield, immunophenotype, and functional properties of DCs generated in Lifecell X-Fold culture bags and in plastic wells, both from adherence-selected monocytes, and review the current literature on closed systems for DC generation . We found that both the overall yield and the yield of CD83+ cells in cell culture bags was lower than in the standard culture method . No statistically significant differences were observed in the expression of DC immunophenotypic markers . The capability of DCs cultured in bags and in wells to induce the proliferation of allogeneic mononuclear cells were equivalent . The performance of DCs in mixed lymphocyte reaction correlated significantly (p = 0.005) with the CD83 expression but not with the CD80, CD86, HLA-DR, CD1a, and CD1c expression . We conclude that the immunophenotype and stimulatory properties of DCs cultured in closed cell culture bags are similar to those generated by conventional method using cell culture wells .

Methods Mol Biol, 2004, 283, 21 - 36
Characterization of endothelial internalization and targeting of antibody-enzyme conjugates in cell cultures and in laboratory animals; Muro S et al.; Streptavidin-biotin conjugates of enzymes with carrier antibodies provide a versatile means for targeting selected cellular populations in cell cultures and in vivo . Both specific delivery to cells and proper subcellular addressing of enzyme cargoes are important parameters of targeting . This chapter describes methodologies for evaluating the binding and internalization of labeled conjugates directed to endothelial surface adhesion molecules in cell cultures using anti-intercellular adhesion molecule/catalase or antiplatelet endothelial cell adhesion molecule/catalase conjugates as examples . It also describes protocols for characterization of biodistribution and pulmonary targeting of radiolabeled conjugates in rats using anti-intercellular adhesion molecule/tPA conjugates as an example . The experimental procedures, results, and notes provided may help in investigations of vascular immunotargeting of reporter, experimental, diagnostic, or therapeutic enzymes to endothelial and, perhaps, other cell types, both in vitro and in vivo.

Biotechnol Lett, 2004 May, 26(9), 763 - 7
On-line oxygen uptake rate and culture viability measurement of animal cell culture using microplates with integrated oxygen sensors; Deshpande RR et al.; O2 uptake rates of animal cells (Chinese hamster ovary-CHO) were measured in 96-well microtiter plates by integrating with fluorescent sensors thereby measuring fluorescence intensity ratios of an O2-sensitive and an insensitive fluorophor . O2 consumption rate was estimated from measured dissolved O2 and from O2 mass transfer coefficient determined in advance . Specific uptake decreased with time from 3.2 x 10(-13) mol O2 cell(-1) h(-1) at 15 h cultivation to 1.8 x 10(-13) mol O2 cell(-1) h(-1) at 48 h . Specific O2 uptake was also determined by sampling from a spinner-flask culture giving identical values . A cell viability assay for cultures based on O2 measurements is described in which cells are incubated outside the fluorescence reader and then the dissolved O2 is measured only once at a fixed time after the start of incubation . This protocol can be directly applied for high-throughput measurements.

ALTEX, 2004, 21(2), 61 - 6
Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing . Part 2: toxicological evaluation; Jennings P et al.; The goal of replacement, refinement and reduction of animal testing is critically dependent on the development and assessment of novel in vitro methodologies and the further development of existing methodologies . Here, we evaluated the use of a modified perfusion cell culture apparatus for application to chronic in vitro nephrotoxicity testing using DMSO, SDS, paracetamol and cyclosporine A as test compounds . Renal epithelial monolayers were cultured on microporous growth supports and exposed to test compounds under static or perfusion conditions . Alamar Blue reduction, gamma-glutamyl transpeptidase activity (GGT), lactate dehydrogenase activity (LDH) and remnant protein were used to assay cell toxicity . There was no significant difference in IC(50) values between static and perfusion cultures up to 72 hours exposure . However, the perfusion system allowed continuous real-time monitoring of plasma membrane damage, which gives important information of time, duration and scale of toxicity . The complexity of the system restrains its use to low-throughput analysis . However, the real and theoretical advantages of this and similar systems merit further investigations.

ALTEX, 2004, 21(2), 51 - 60
Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing . Part 1: technical description; Koppelstaetter C et al.; In vitro models for chronic toxicity, defined as a recurring exposure to compounds over a prolonged period of time, are still underrepresented in drug evaluation processes . The classical approach to cell culture is not readily suitable to long term repetitive applications . Therefore, we assessed the use of a commercially available perfusion cell culture apparatus in its applicability to chronic renal toxicity testing and describe the technical aspects of adopting the perfusion cell culture system to our purposes . It was apparent that there is a subtle dynamic difference between human renal proximal tubular cells cultured under perfusion and static conditions as illustrated by the accumulation of lactate dehydrogenase (LDH) and the secondary metabolism of resazurin to hydroresorufin, which occurred only under static conditions . The major achievement was the standardisation of the handling of this system with regard to cell cultivation, pH regulation, temperature regulation, and reproducibility of common toxicity endpoints.

Mol Biol Cell, 2004 Aug, 15(8), 3863 - 75 Epub 2004 Jun 11.
The bone morphogenetic protein type ib receptor is a major mediator of glial differentiation and cell survival in adult hippocampal progenitor cell culture; Brederlau A et al.; Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation . They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb . Little is known about functional differences between the three type I receptors . Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs) . The dn receptors have a nonfunctional intracellular but functional extracellular domain . They thus trap BMPs that are endogenously produced by AHPs . We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors . Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased . No effect on neuronal differentiation was seen . Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling . After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling . We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture.

Am J Physiol Lung Cell Mol Physiol, 2004 Oct, 287(4), L824 - 34 Epub 2004 Jun 11.
Mucins and their O-Glycans from human bronchial epithelial cell cultures; Holmen JM et al.; A longstanding question in obstructive airway disease is whether observed changes in mucin composition and/or posttranslational glycosylation are due to genetic or to environmental factors . We tested whether the mucins secreted by second-passage primary human bronchial epithelial cell cultures derived from noncystic fibrosis (CF) or CF patients have intrinsically different specific mucin compositions, and whether these mucins are glycosylated differently . Both CF and non-CF cultures produced MUC5B, predominantly, as judged by quantitative agarose gel Western blots with mucin-specific antibodies: MUC5B was present at approximately 10-fold higher levels than MUC5AC, consistent with our previous mRNA studies (Bernacki SH, Nelson AL, Abdullah L, Sheehan JK, Harris A, William DC, and Randell SH . Am J Respir Cell Mol Biol 20: 595-604, 1999) . O-linked oligosaccharides released from purified non-CF and CF mucins and studied by HPLC mass spectrometry had highly variable glycan structures, and there were no observable differences between the two groups . Hence, there were no differences in either the specific mucins or their O-glycans that correlated with the CF phenotype under the noninfected/noninflammatory conditions of cell culture . We conclude that the differences observed in the mucins sampled directly from patients are most likely due to environmental factors relating to infection and/or inflammation.

Glia, 2004 Aug 1, 47(2), 130 - 7
Ensheathing cell cultures from the olfactory bulb and mucosa; Jani HR et al.; Transplantation of cells cultured from the nerve layers of the adult rat olfactory bulb has been shown to repair CNS tract injuries . The precise cellular composition of the culture appears to be important for this effect . Comparison was made of tissue cultured from the adult rat olfactory mucosa with that from the olfactory bulb . Both yielded mixtures of p75 immunoreactive cells and fibronectin immunoreactive cells . In sequential observations over 21 days in culture, the population of p75-expressing cells was maintained and continued to proliferate for longer in the samples from the olfactory mucosa . For derivation of cells for transplantation, the mucosa can be accessed without the need for intracranial surgery .

Lab Invest, 2004 Aug, 84(8), 1060 - 70
Inducible short-term and stable long-term cell culture systems reveal that the PAX3-FKHR fusion oncoprotein regulates CXCR4, PAX3, and PAX7 expression; Tomescu O et al.; In the pediatric cancer alveolar rhabdomyosarcoma (ARMS), the 2;13 chromosomal translocation juxtaposes the PAX3 and FKHR genes to generate a chimeric transcription factor . To explore molecular pathways altered by this oncoprotein, we generated an inducible form by fusing PAX3-FKHR to a modified estrogen receptor ligand-binding domain and expressed this construct in the RD embryonal rhabdomyosarcoma cell line . This inducible system permits short-term evaluation of downstream expression targets of PAX3-FKHR and complements a panel of stable long-term RD subclones constitutively expressing PAX3-FKHR . Using these two sets of resources, we investigated several candidate PAX3-FKHR target genes . First, we demonstrated in both short-term and long-term systems that PAX3-FKHR upregulates expression of the gene encoding the chemokine receptor CXCR4 . In addition, we found that expression of wild-type PAX3 is upregulated, whereas expression of wild-type PAX7 is downregulated by PAX3-FKHR . In the presence of cycloheximide, CXCR4 and PAX3 are still inducible, supporting the hypothesis that these genes are direct transcriptional targets of PAX3-FKHR . Finally, studies of ARMS tumors revealed CXCR4, PAX3, and PAX7 expression levels consistent with our cell culture results . These findings of genes regulated by PAX3-FKHR will direct future biological and clinical investigation to important pathways contributing to ARMS tumorigenesis and progression.

Dev Comp Immunol, 2004 Jul 1, 28(9), 863 - 9
Cell culture of clonal ginbuna crucian carp hematopoietic cells: differentiation of cultured cells into erythrocytes in vivo; Moritomo T et al.; We cultivated kidney hematopoietic cells from clonal triploid ginbuna crucian carp, Carassius auratus langsdorfii . Proliferating cells from hematopoietic cell cultures were harvested and injected into tetraploid hybrids (clonal triploid ginbuna and goldfish hybrid) which possess three sets of chromosomes from a triploid clone and a haploid set of chromosomes from goldfish (Carassius auratus) . After injection of cultured triploid cells (donor cells), blood samples were obtained from tetraploid hybrids (recipients) every other week . Blood cells stained with acridine orange were measured by flow cytometry to trace the injected donor cells by means of differences in DNA content . Although erythrocytes were not produced in donor cell cultures, such cultures maintained precursor cells capable of differentiation into erythrocytes in vivo . After 4-12 weeks of transplantation, mature erythrocytes derived from the donors were observed in the blood circulation of the recipient fish . These results indicated that the ginbuna hematopoietic cell culture system is an in vivo situation suitable for the study of hematopoietic control mechanisms.

Eur J Neurosci, 2004 Jun, 19(11), 2915 - 22
Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice; Slemmer JE et al.; The homeostasis of intracellular Cl(-) concentration ({Cl(-)}(i)) is critical for neuronal function, including gamma-aminobutyric acid (GABA)ergic synaptic transmission . Here, we investigated activity-dependent changes in {Cl(-)}(i) using a transgenetically expressed Cl(-)-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons . Application of glutamate (100 microm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence . The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV . Glutamate application resulted in a slight neuronal acidification . Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl(-)-free solution, demonstrating that the EYFP signal represented an increase in {Cl(-)}(i) . Similar to glutamate, a rise in {Cl(-)}(i) was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular {K(+)}, indicating that an increase in driving force for Cl(-) suffices to increase {Cl(-)}(i) . To elucidate the membrane mechanisms mediating the Cl(-) influx, a series of blockers of ion channels and transporters were tested . The glutamate-induced increase in {Cl(-)}(i) was resistant to furosemide, bumetanide and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) . We conclude that membrane depolarization increases {Cl(-)}(i) via several pathways involving NFA- and NPPB-sensitive anion channels and GABA(A) receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl(-) transporters . The present study highlights the vulnerability of {Cl(-)}(i) homeostasis after membrane depolarization in neurons.

Toxicol Lett, 2004 Jun 15, 151(1), 163 - 70
Bovine chromaffin cell cultures as model to study organophosporus neurotoxicity; Quesada E et al.; Based on the high level of phenyl valerate esterase activities, and in particular of neuropathy target esterase (NTE) found in bovine adrenal medulla, chromaffin cells culture have been proposed as an alternative model for the study of organophosphorus neurotoxicity . Organophosphorus-induced polyneuropathy is a syndrome related to the inhibition and further modification by organophosphorus compounds of NTE (a protein that displays phenyl valerate esterase activity resistant to mipafox and sensitive to paraoxon) . Total phenyl valerate esterase activities found in homogenate, particulate and soluble fractions of bovine adrenal medulla were 5200+/-35, 5000+/-280 and 1700+/-260 mU/g tissue, respectively . Cultured chromaffin cells displayed a total hydrolysing activity of 41+/-5 mU/10(6) cells . Homogenates of bovine adrenal medulla displayed only about 6% of activity sensitive to paraoxon . Most of the phenyl valerate esterase activity inhibited by mipafox (a neuropathy inducing compound) was found in particulate fraction . Cultured chromaffin cells displayed kinetics of inhibition by mipafox similar to the kinetics displayed by homogenates of bovine adrenal medulla . We conclude that NTE could be assayed in this system by only using one inhibitor (mipafox) instead of two (paraoxon and mipafox) . Also, the proposal is supported of using chromaffin cells as in vitro model for the study of the role of NTE and related esterases in organophosphorus-induced polyneuropathy.

Synapse, 2004 Aug, 53(2), 122 - 30
Differential properties of GABAergic synaptic connections in rat hippocampal cell cultures; Ivanova SY et al.; Based on the effect of prolonged tetanic stimulation (30 Hz, 4 sec), we divided GABAergic synaptic connections in hippocampal cell cultures into two groups: connections facilitated ( approximately 45%) and connections depressed ( approximately 55%) by the tetanic stimulation . In order to reveal possible reasons for the differential effect of the tetanization, we compared several properties of the connections belonging to both groups . We found that, on average, evoked IPSCs in the connections facilitated by the tetanization have a smaller amplitude and larger coefficient of variation (CV) of IPSC amplitude compared to connections depressed by the tetanization . We also estimated quantal parameters for both groups of connections assuming that transmitter release is reasonably described by a binomial distribution . We found that a background release probability (P) is substantially lower in the connections facilitated by the tetanization (P approximately 0.5) than in the connections depressed by the tetanization (P approximately 0.9) and suggest that this difference may underlie the differential effect of the tetanization . We also found that the tetanization induces the opposite effect on connections made by distinct presynaptic neurons with the same postsynaptic cell (convergent connections) in a fraction of postsynaptic neurons studied (3 out of 9) . These results support the idea that properties of the presynaptic neuron are of primary importance for the observed differential effect of the tetanization, but they do not exclude a role of the postsynaptic neuron in this effect .

Biochemistry (Mosc), 2004 Apr, 69(4), 413 - 9
Inhibition of gap junction intercellular communications in cell culture by polycyclic aromatic hydrocarbons (PAH) in the absence of PAH metabolism; Sharovskaja JJ et al.; We have studied the effect of polycyclic aromatic hydrocarbons (PAH) on gap junction intercellular communications (GJIC) in culture of hepatoma cells Hep G2 and G27 . Carcinogenic PAH inhibited GJIC in both cultures in contrast to non-carcinogenic PAH . We showed that both constitutive and inducible expressions of mRNAs of Ah receptor and cytochrome P4501A1 (the main isoform involved in PAH metabolism) were absent in hepatoma G27 cells . We concluded that the initial, non-metabolized molecules of carcinogenic PAH are responsible for changes in GJIC through interaction with an unknown factor in the cellular membrane.

Phys Rev Lett . 2004 May 14;92(19):198103 . Epub 2004 May 13.
Regular and alternant spiral waves of contractile motion on rat ventricle cell cultures; Hwang SM et al.; We demonstrate that meandering as well as regular spiral waves can form in a well-controlled culture layer of rat ventricle cells and that the meandering spiral wave, in particular, can generate an alternant rhythm . These observations are made possible by a newly developed, noninvasive phase contrast macro-optics that is simple but highly effective in visualizing the contractile motion of the populations of cardiac cells.

J Gen Virol, 2004 Jun, 85(Pt 6), 1791 - 9
Cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie; Solassol J et al.; Over the last 30 years, many drugs have been tested both in cell culture and in vivo for their ability to prevent the generation of prions and the development of transmissible spongiform encephalopathies . Among the compounds tested, dendrimers are defined by their branched and repeating molecular structure . The anti-prion activity of new cationic phosphorus-containing dendrimers (P-dendrimers) with tertiary amine end-groups was tested . These molecules had a strong anti-prion activity, decreasing both PrP(Sc) and infectivity in scrapie-infected cells at non-cytotoxic doses . They can bind PrP and decrease the amount of pre-existing PrP(Sc) from several prion strains, including the BSE strain . More importantly, when tested in a murine scrapie model, the dendrimers were able to decrease PrP(Sc) accumulation in the spleen by more than 80 % . These molecules have a high bio-availability and therefore exhibit relevant potential for prion therapeutics for at least post-exposure prophylaxis.

Tissue Eng, 2004 Mar-Apr, 10(3-4), 465 - 74
Novel starch-based scaffolds for bone tissue engineering: cytotoxicity, cell culture, and protein expression; Salgado AJ et al.; Starch-based biomaterials and scaffolds have been proposed for several biomedical applications . In the present work new scaffolds based on a 50/50 (wt%) blend of corn starch/ethylene-vinyl alcohol (SEVA-C) were studied . These scaffolds were processed by a melt-based technology, which has been used before with other starch-based materials but never with SEVA-C . Scanning electron microscopy (SEM) observation showed that the developed porous structures were 60% porous with pore size between 200 and 900 microm and a reasonable degree of interconnectivity . Moreover, scaffolds presented a compressive modulus of 117.50 +/- 3.7 MPa and a compressive strength of 20.8 +/- 2.4 MPa . Cytotoxicity evaluation was performed according to ISO/EN 10993 part 5 guidelines, and revealed that the developed scaffolds were nontoxic and did not inhibit cell growth . Direct contact assays were also carried out by use of a cell line of human osteoblast-like cells (SaOS-2) . Cells were seeded (3 x 10(5) per scaffold) and allowed to grow for 4 weeks at 37 degrees C, in a humidified atmosphere containing 5% CO(2) . Total protein assay showed that the cells were able to grow for the 4 weeks of the experiment . These data were further confirmed by SEM . Moreover, a cell viability assay (MTS test) demonstrated that cells were perfectly viable after the 4 weeks of culture, showing the adequacy of the developed structure in supporting them . Finally, Western blot analysis revealed that osteopontin was being actively expressed by the cells, which, in association with collagen deposition observed by SEM, seems to indicate that bone extracellular matrix was being deposited . Consequently it is believed that starch-based scaffolds should be considered as an alternative for bone tissue-engineering applications in the near future.

Calcif Tissue Int, 2004 Sep, 75(3), 231 - 42
Mineralization of decalcified bone occurs under cell culture conditions and requires bovine serum but not cells; Hamlin NJ et al.; The purpose of this study was to develop an in vitro model system for bone matrix mineralization in the absence of cells . For this model, we utilized EDTA-decalcified new-born rat tibias with the cartilaginous ends intact, allowing us to visually determine the specificity of mineralization within the bone . Our results show that supplementation of DMEM culture medium with 10mM beta-glycerophosphate and 15% fetal bovine serum (FBS) results in non-physiological mineral percipitation in the tibia because of the generation of supraphysiological (5mM) levels of inorganic phosphate in the medium . The same medium supplemented only with inorganic phosphate to a final concentration of 2mM failed to mineralize a decalcified tibia matrix . However, additional supplementation of this medium with as little as 5% FBS resulted in mineralization of those regions of the type I collagen where mineral was found prior to decalcification, with no evidence for mineralization in the cartilage at the bone ends or in the periosteum . Analysis of the mineral by Fourier-transform infrared spectroscopy and powder X-ray diffraction shows that tibias that have been decalcified and then remineralized contain an apatitic mineral that is strikingly similar to the mineral in normal bone . Tendon, a type I collagen matrix not normally mineralized in vivo, also mineralizes when incubated in DMEM containing 2mM Pi and as little as 1.5% FBS, but not when incubated in DMEM without serum . These data indicate that serum contains a nucleator of type I collagen matrix mineralization, and that mineralization of type I collagen under cell culture conditions requires serum but not living cells.

Proc Natl Acad Sci U S A, 2004 Jun 8, 101(23), 8768 - 73 Epub 2004 May 25.
Two Creutzfeldt-Jakob disease agents reproduce prion protein-independent identities in cell cultures; Arjona A et al.; Human Creutzfeldt-Jakob disease (CJD) and similar neurodegenerative diseases such as sheep scrapie are caused by a variety of related infectious agents . They are associated with abnormal host prion protein (PrP), which is assessed by limited proteolysis to yield resistant PrP bands (PrP-res) . Although PrP-res has been posited as the infectious agent, purified PrP-res itself is not infectious . To establish the independence of CJD agent characteristics from those of PrP-res, two different mouse-passaged CJD strains were propagated in neuronal cell lines whose PrP-res patterns differ markedly from each other and from those found in infected brain . In mouse brain, the fast CJD strain, FU, elicits many PrP-res deposits, whereas the slow SY strain elicits few . Both strains evoked PrP-res in cultured murine cells, although SY induced PrP-res only transiently . PrP-res patterns in FU- and SY-infected GT1 cells were identical, and were significantly different from those in brain and in N2a cells . Nevertheless, all FU-infected cell lines reproduced their original fast disease in mice, even after extensive subculture, whereas SY-infected cells produced only slow disease . These data indicate PrP-res neither encodes nor alters agent-specific characteristics . PrP-res was also a poor predictor of infectivity because SY cells that had lost PrP-res were approximately 10-fold more infectious than PrP-res-positive cultures . Furthermore, FU titers increased 650-fold, whereas PrP-res remained constant . Passaged FU-infected cells had titers comparable to brain, and >30% of cells displayed abundant cytoplasmic PrP-res aggregates that may trap agent . The continuous substantial replication of CJD in monotypic cells will further the discrimination of agent-specific molecules from pathological host responses to infection.

J Cell Sci, 2004 Jun 15, 117(Pt 14), 2917 - 24 Epub 2004 May 25.
Non-genomic regulation of transmitter release by retinoic acid at developing motoneurons in Xenopus cell culture; Liao YP et al.; Although the long-term effects of all-trans retinoic acid (RA) on neuronal growth and differentiation have been intensively studied, nothing is known about its effect on synaptic transmission . Here we show that RA rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch-clamp recording . Acute addition of RA dose-dependently and reversibly enhances the frequency of spontaneous synaptic currents (SSCs) . Application of the lipophilic RA analogue all-trans retinol or RA metabolites produced by light-induced decomposition failed to provoke similar changes in SSC frequency, indicating the specificity of RA-induced facilitation of spontaneous transmitter release . Protein synthesis inhibitors anisomycin or cycloheximide had no effect on RA-induced SSC frequency facilitation . Treating cells with pan RA receptor (RAR) selective agonist or RARbeta-selective agonist, but not RARalpha-, RARgamma- or retinoid X receptor (RXR)-selective agonists, mimicked the action of RA . These results suggest that RA acts through the activation of RARbeta, to induce a rapid, non-genomic increase in the frequency of spontaneous transmitter release at developing neuromuscular synapses.

Fitoterapia, 2004 Jun, 75(3-4), 296 - 301
Production of two volatile glucosinolate hydrolysis compounds in Nasturtium montanum and Cleome chelidonii plant cell cultures; Songsak T et al.; Callus and suspension cultures established from Nasturtium montanum and Cleome chelidonii were shown to produce glucosinolates by analysis of their hydrolysis products . Large increases in two glucosinolate hydrolysis products were noted when cultures were supplemented with L-cysteine and L-methionine, and further increases were produced in N . montanum with l-tryptophan supplementation .

Eur J Pharm Sci, 2004 Jun, 22(2-3), 181 - 9
Reversible, temperature-dependent, and AM404-inhibitable adsorption of anandamide to cell culture wells as a confounding factor in release experiments; Karlsson M et al.; Relatively little is known about the process whereby the endocannabinoid anandamide (AEA) is released from cells . A simple way of studying this process is to sample the appearance in the medium of tritium following preloading of cells with {(3)H}AEA under conditions where its metabolism is prevented . However, this approach may be complicated by the ability of AEA to be adsorbed reversibly to the cell culture wells . In the present study, it is found that cell culture wells adsorb almost half of the added AEA in a manner prevented by fatty acid-free bovine serum albumin, and by the prototypical uptake inhibitors AM404 and VDM11 with IC(50) values of 3 and 1 microM, respectively . After incubation followed by washing of the plates, AEA is released into the medium from the wells by a first order process (K approximately 0.1 min(-1)) that is temperature-dependent and increased by AM404 and fatty acid-free bovine serum albumin . When assays were run with 0.15% fatty acid-free bovine serum albumin during the loading, washing and release phases of the assay, the release from the well was greatly reduced and a first order, temperature-sensitive release from C6 glioma cells could be unmasked . It is concluded that the reversible adsorption of AEA by cell culture wells can be a confounding factor in release experiments.

Vet J, 2004 Jul, 168(1), 74 - 80
Growth characteristics of porcine chlamydial strains in different cell culture systems and comparison with ovine and avian chlamydial strains; Schiller I et al.; Porcine Chlamydiaceae were cultivated under various culture conditions and we compared their growth characteristics with those of ruminant and avian strains . The combination of centrifugation assisted cell culture infection and cycloheximide treatment of Vero cell coverslip cultures provided the highest inclusion numbers with all chlamydial strains . Interestingly, the use of Iscove's modified Dulbecco's medium instead of Eagle's minimal essential medium significantly increased Chlamydia suis inclusion counts . C . suis and Chlamydophila pecorum inclusion numbers were markedly increased in CaCo cells, compared with Vero cells . This accelerated growth of porcine Chlamydiaceae under certain cultivation conditions may be helpful for the propagation of low chlamydial numbers or for their isolation from field samples . The intracellular distribution of porcine Chlamydiaceae in polarised CaCo cells clearly demonstrated differences between the chlamydial strains: C . pecorum 1710S inclusions were predominantly localised in the apical cytoplasm, C . suis S45 inclusions, however, were mostly situated in lower cytoplasmatic compartments . These findings might reflect biological differences in vivo.

J Nutr Biochem, 2004 Jun, 15(6), 342 - 9
Caffeine suppresses the expression of the Bcl-2 mRNA in BeWo cell culture and rat placenta; Nomura K et al.; Chronic caffeine exposure during pregnancy has an effect on fetal growth; however, the adverse effects of caffeine on embryogenesis are not well understood and controversial . We used cDNA microarray technology to determine whether caffeine alters gene expressions in a human cytotrophoblast-like cell line, BeWo . We found that the expression of the B-cell CLL/lymphoma 2 (Bcl-2) gene in BeWo cells was down-regulated by caffeine, suggesting that chronic exposure during the gestational period could exert an influence on embryogenesis . We then focused on the Bcl-2- and Bcl-2-associated X protein gene, Bax, to study the responsive gene expression in BeWo cells as well as placentas of pregnant rats fed a diet supplemented with caffeine (2 mg/100 g body weight) during gestation, and analyzed the gene expressions using LightCycler-based quantitative real-time polymerase chain reaction assays . We found a significantly decreased level of Bcl-2 mRNA expression, which demonstrated the influence of caffeine on placental function.

Br J Pharmacol, 2004 May, 142(2), 231 - 55
Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?
Halliwell B, Whiteman M.
Free radicals and other reactive species (RS) are thought to play an important role in many human diseases . Establishing their precise role requires the ability to measure them and the oxidative damage that they cause . This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress . It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies . Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent "probes" of RS, are also critically reviewed . The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.

Expert Opin Biol Ther, 2004 May, 4(5), 709 - 17
The role of cell culture vaccines in the control of the next influenza pandemic; Audsley JM et al.; Pandemic influenza A viruses of avian origin are of particular concern and have crossed the species barrier several times in recent years, giving rise to illness and occasionally death in humans . This situation could become dramatically worse if the infectivity of avian viruses for humans were increased by reassortment between the genes of human and avian viruses . Co-infection of humans or an intermediate host with an avian strain and an existing human strain could produce new viruses of unknown pathogenicity to which the entire population would be susceptible . Inactivated vaccines against influenza have been prepared for many years using viruses grown in embryonated chicken eggs . However, the use of eggs presents difficulties when vaccine supplies need to be expanded at short notice . It seems likely that future vaccines will be prepared in high-yielding cell cultures from continuous lines that are preferably anchorage-independent . At present, only certain preparations of the Vero and Madin-Darby canine kidney cell lines, grown and maintained in serum-free medium, are acceptable to all regulatory authorities . However, this situation is likely to change with increasing need for non-pandemic and pandemic vaccines.

Neurosci Res, 2004 Mar, 48(3), 315 - 23
Effects of beta-AP peptides on activation of the transcription factor NF-kappaB and in cell proliferation in glial cell cultures; Casal C et al.; The effect of two beta amyloid peptides (Abeta 25/35 and Abeta 1/42) on the activation of the transcription factor kappaB (NF-kappaB) in pure astroglial, pure microglial and mixed glial cell cultures was compared by means of single or double immunofluorescence and Western blot techniques . We also studied the effect of both peptides in cell proliferation in mixed glial cultures and pure astrocytes . The Abeta 1/42 peptide induced the activation of NF-kappaB in all studied cell cultures and its effect was potentiated by interferon-gamma (IFN-gamma) . Abeta 25/35 alone did not induce NF-kappaB activation, but Abeta 25/35 plus IFN-gamma induced the activation of the transcription factor in the mixed and pure microglial cultures, although not in pure astroglia . The Abeta 1/42 peptide, but not Abeta 25/35, induced proliferation in pure astroglial and mixed glial cell cultures . The results suggest that the state of peptide aggregation is related to their ability to activate glial cells.

Ann N Y Acad Sci, 2004 Apr, 1014, 137 - 9
Ca(2+) channel properties in neuroendocrine tumor cell cultures investigated by whole-cell patch-clamp technique; Mergler S et al.; Neuroendocrine tumor (NET) cells of the gastroenteropancreatic system express a variety of voltage-operated Ca(2+) channels (VOCCs) . In addition, NET cells release hormones and biogenic amines in distinct patterns, leading to typical hypersecretion syndromes . Interestingly, these patterns depend on the primary location of the NET . The aim of this study was to investigate a possible link between clinically distinct entities of NETs and specific properties of VOCCs . Using whole-cell patch-clamp technique, electrophysiological data were obtained from permanent NET cell cultures as well as from primary cultivated NET cells from foregut and midgut tumors . Definite Ca(2+) channel characteristics were analyzed by a color-coding method . As a result, we could demonstrate specific Ca(2+) channel characteristics in NET cells from midgut tumors that were not found in NET cells from a foregut location . This may be important functionally with respect to different cell biological functions of NET cells . In summary, clear differences exist in the VOCC pattern in NET cells from foregut versus midgut . This may be functionally relevant in the hypersecretion syndrome predominantly found in foregut NETs . Therefore, these data on the characteristics of VOCCs could be useful in the development of a novel therapy for NET disease; for example, specific VOCC subtype modulators may help.

Br J Dermatol, 2004 May, 150(5), 860 - 8
Human follicular stem cells: their presence in plucked hair and follicular cell culture; Gho CG et al.; BACKGROUND AND OBJECTIVES: A considerable portion of the hair follicle remains attached to plucked hair and can be used for follicle cell culture . In this study we have phenotyped these cells in an attempt to identify the stem cell fraction . Reports in the literature have indicated that this cell population may be positive for cytokeratin (CK) 19 . Because stem cells in general need to be protected from apoptosis, the presence of the apoptosis-suppressing Bcl-2 protein, together with the absence of the apoptosis-promoting Bax and the CK profile may be used as an indicator of the stem cell population in the hair follicle, and in cultures of hair follicle cells . METHODS: Hair follicles from skin biopsies and plucked hair were derived from the scalps of healthy volunteers . Follicular cells were cultured from the plucked hairs . These hair follicles, plucked hairs and cultured cells were examined for their CK profiles, which are indicative of the type of cell (basal/stem cells) and for their status with respect to the proliferation marker Ki-67, Bax and Bcl-2 . RESULTS: We found coexpression for CK19 and Bcl-2, but not Bax in two distinct areas, localized in the upper and lower third of the follicle from both skin biopsies and plucked hairs, while proliferation markers were negative in these areas . CK19 and Bcl-2 were also coexpressed in combination in a fraction of the follicular cell culture . The skin basal cell marker CK14 could be found throughout the outer root sheath of the hair follicle from both skin biopsies and plucked hairs, as well as in the follicular cell culture . CONCLUSIONS: Thus, CK19/Bcl-2-positive and Bax-negative cells can be obtained from cells derived from plucked hair and are retained in cultures made from these cells . If this phenotype represents follicular stem cells, our finding endorses the assumption that stem cells are located in the bulge area of the hair follicle, as we did not find them in or near the dermal papilla.

Arch Toxicol, 2004 Oct, 78(10), 599 - 608 Epub 2004 May 18.
The effects of enrofloxacin on decorin and glycosaminoglycans in avian tendon cell cultures; Yoon JH et al.; Tendonitis and tendon rupture have been reported to occur during or following therapy with fluoroquinolone antibiotics . Though the pathogenesis is unknown, several studies suggest that fluoroquinolone antibiotics alter proteoglycan content in soft tissues, including tendons, and thereby alter collagen fibrillogenesis . To better understand the mechanism of action of fluoroquinolones, we studied the effects of enrofloxacin, a widely used fluoroquinolone in veterinary medicine, on avian tendon cell cultures established from gastrocnemius tendons from 18-day-old chicken embryos . We found that cell proliferation was progressively inhibited with increasing concentrations of enrofloxacin . This was accompanied by changes in morphology, extracellular matrix content and collagen fibril formation as detected by electron microscopy . We also observed a 35% decrease in the content of total monosaccharides in enrofloxacin-treated cells . The ratio of individual monosaccharides was also altered in enrofloxacin-treated cells . Enrofloxacin also induced the synthesis of small amounts of keratan sulfate in tendon cells . Moreover we observed enrofloxacin-induced changes in glycosylation of decorin, the most abundant tendon proteoglycan, resulting in the emergence of multiple lower molecular bands that were identifiable as decorin after chondroitinase ABC and N-glycanase treatment of extracts from enrofloxacin-treated cells . Medium conditioned by enrofloxacin-treated cells contained less decorin than did medium conditioned by control cells . We hypothesize that enrofloxacin induces either changes in the number of N-linked oligosaccharides attached to the core protein of decorin or changes in decorin degradation process . In conclusion, our data suggest that enrofloxacin affects cell proliferation and extracellular matrix through changes in glycosylation.

Pharm Res, 2004 Apr, 21(4), 675 - 82
Effective transfection of rabies DNA vaccine in cell culture using an artificial lipoprotein carrier system; Alanazi F et al.; PURPOSE: To evaluate the transfection efficiency in cell culture of rabies plasmid DNA vaccine carried by a novel artificial lipoprotein system . METHODS: Phospholipid nanoemulsions resembling the lipid core of natural lipoproteins were prepared . The artificial lipoprotein carrier system for DNA was constructed by assembling of the nanoemulsion (NE)-palmitoyl-poly-L-lysine (p-PLL)-rabies DNA complex . Agarose gel electrophoresis, zeta potential, and mobility measurement were conducted to determine the surface charge balance in various complex compositions . Transfection and transfection efficiency were examined by fluorescence microscopy and flow cytometry, respectively . RESULTS: The artificial lipoprotein system was successfully constructed and the rabies DNA vaccine was effectively transfected in glioma cell line SF-767 . The amount of p-PLL incorporated into the artificial lipoprotein formulations had a significant effect on transfection efficiency . The new system also proved to be more efficient in cellular transfection of rabies DNA vaccine than the commercial lipofectamine formulation . CONCLUSIONS: Effective transfection of rabies DNA vaccine in cell culture can be achieved using the novel artificial lipoprotein carrier system, and the charge balance of the NE-p-PLL-DNA complex appears an important factor.

J Agric Food Chem, 2004 May 19, 52(10), 2915 - 23
Chemical and biological characterization of cinnamic acid derivatives from cell cultures of lavender (Lavandula officinalis) induced by stress and jasmonic acid; Nitzsche A et al.; Cell cultures of lavender (Lavandula officinalis) were analyzed for the metabolite profile under normal growth conditions and under stress as well as after jasmonic acid treatment . The main compound synthesized was rosmarinic acid, which was also secreted into the culture medium . Different solvent extraction methods at different pH values altered the profile slightly . Anoxic stress induced the synthesis of a cinnamic acid derivative, which was identified as caffeic acid by gas chromatography-mass spectrometry . Caffeic acid was also induced after treatment of the cell cultures with jasmonic acid . Although the antioxidative activity of both compounds, rosmarinic acid and caffeic acid, was confirmed in an assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was demonstrated that both substances have a low cytotoxic potential in vitro using acute myeloid leukemia (HL-60) cells . The potential of the system for finding new bioactive compounds is discussed.

Neurosci Lett, 2004 May 6, 361(1-3), 86 - 9
Longterm stability and developmental changes in spontaneous network burst firing patterns in dissociated rat cerebral cortex cell cultures on multielectrode arrays; Van Pelt J et al.; Spontaneous action potentials were recorded longitudinally for 4-7 weeks from dissociated rat occipital cortex cells cultured on planar multi-electrode plates, during their development from isolated neurons into synaptically connected neuronal networks . Activity typically consisted of generalized bursts lasting up to several seconds, separated by variable epochs of sporadic firing at some of the active sites . These network bursts displayed discharge patterns with age-dependent firing rate profiles, and durations significantly increasing in the 3rd week in vitro and decreasing after about 1 month in vitro, when they evolved into short events with prompt onsets . These findings indicate that after about a month in vitro these cultured neuronal networks have developed a degree of excitability that allows almost instantaneous triggering of generalized discharges . Individual neurons tend to fire in specific and persistent temporal relationships to one another within these network bursts, suggesting that network connectivity maintains a core topology during its development.

Biotechnol Adv, 2004 Jul, 22(6), 433 - 44
Acoustic cell filter: a proven cell retention technology for perfusion of animal cell cultures; Shirgaonkar IZ et al.; This article is a review highlighting the application of the acoustic filter as a reliable cell retention device during the long-term perfusion of animal cell cultures . Critical operating parameters such as duty cycle, perfusion and re-circulation flow rates, acoustic power and backflush frequency are discussed with regard to influence on the separation efficiency and optimal operating ranges have been identified . Perfusion data gathered from the literature have been complemented with original data from a series of perfusion experiments carried out in the context of industrial projects for industrially relevant cell lines including NS0, HEK-293, SP2-derived hybridoma and insect cells in different serum-supplemented and serum-free media at different perfusion rates and acoustic chamber volumes . Finally, scale-up potential of the acoustic filter for large-scale industrial applications is discussed.

Shanghai Kou Qiang Yi Xue, 2004 Apr, 13(2), 103 - 5
{Investigation of primary cell culture for dental pulp: comparing of coronal pulp and root pulp}; Liu SH et al.; PURPOSE: To compare and establish an ideal method for the culture of human primary dental coronal and root pulp cells . METHODS: The whole dental pulp tissues were obtained from healthy and young human teeth extracted for orthodontic purposes . The cells in coronal and root pulp were separately cultured by the tissue explant and tissue explant-collagenase digestion(0.1% type I collagenase) methods . By evaluating the attachment efficiency,viability and the successfulness of the cultured cells, the optimal primary cell culture process of human dental coronal and root pulp was determined . RESULTS: The tissue explant culture had more successfulness,more viability and less attachment efficiency than that of tissue explant-collagenase digestion method in the culture of human coronal and root dental pulp cells . CONCLUSIONS: Tissue explant method was superior to tissue explant-collagenase digestion method for the culturing of human dental coronal and root pulp cells . The ideal method for dental coronal and root pulp cells was established in this study.

J Biomed Mater Res, 2004 Jun 1, 69A(3), 417 - 27
Degradation and cell culture studies on block copolymers prepared by ring opening polymerization of epsilon-caprolactone in the presence of poly(ethylene glycol); Huang MH et al.; Poly(epsilon-caprolactone) (PCL) and its block copolymers with poly(ethylene glycol) (PEG) were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of ethylene glycol or PEG, using zinc metal as catalyst . The resulting polymers were characterized by various analytical techniques such as (1)H NMR, SEC, DSC, IR, X-ray, ESEM, and CZE . PCL/PEG copolymers with long PCL chains presented the same crystalline structure as PCL homopolymer, whereas PEG-bearing short PCL blocks retained the crystalline structure of PEG and exhibited an amphiphilic behavior in aqueous solutions . Degradation of PCL and PCL/PEG diblock and triblock copolymers was realized in a 0.13 M, pH 7.4 phosphate buffer at 37 degrees C . The results indicated that the copolymers exhibited higher hydrophilicity and degradability compared with the PCL homopolymer . Large amounts of PEG were released from the bulk after 60 weeks' degradation . In vitro cell culture studies were conducted on scaffolds manufactured via solid free form fabrication by using primary human and rat bone marrow derived stromal cells (hMSC, rMSC) . Light, scanning electron, and confocal laser microscopy, as well as immunocytochemistry, showed cell attachment, proliferation, and extracellular matrix production on the surface, as well as inside the scaffold architecture . Copolymers showed better performance in the cell culture studies than the PCL homopolymer .

Chromosome Res, 2004, 12(3), 275 - 83
Expression of XIST sense and antisense in bovine fetal organs and cell cultures; Farazmand A et al.; Untranslated RNAs transcribed from sense and antisense strands of a gene referred to as X-inactive specific transcript (XIST) play crucial roles in the genetic inactivation and condensation of one of the two X chromosomes in the somatic cells of female mammals . X inactivation is also thought to occur in mammalian male germ cells mainly based on the formation of a condensed structure referred to as a sex body or XY-body, during spermatogenesis . Molecular identity of the sex body, the roles of sense and antisense XIST RNAs in its formation, and the relevance of the sex body to spermatogenesis are not known . Here we report the results of our strand-specific RT-PCR approach to identify the amplicon detected in fetal bovine testes previously referred to as XIST and to test for sense/antisense expression in male and female organs and cell cultures of different sex chromosome constitution . Our results showed that the transcript detected consistently in male gonads and variably in somatic organs represents XIST antisense RNA and that XIST sense and antisense RNAs are co-expressed in female somatic tissues and cultured cells including cells of sex chromosome aneuploids (XXY and XXX) . Our results, which differ from those of other investigators in this area, are discussed in the light of the recently reported differences in the expression pattern of murine Xist/Tsix loci and their structural and functional differences in different mammalian species.

Prikl Biokhim Mikrobiol, 2004 Mar-Apr, 40(2), 159 - 64
{Ecdysone 20-monooxygenase activity of cytochrome P450 in plants and cell culture of Ajuga reptans L.}; Alekseeva LI; The concentration of cytochrome P450 and ecdysone 20-monooxygenase activity in plants and callus cell culture of the bugleweed Ajuga reptans L . were determined . The maximal ecdysone 20-monooxygenase activity of cytochrome P450 was found in vegetative rosettes of intact plants . During the stage of flowering, the ecdysone 20-monooxygenase activity of cytochrome P450 in plant leaves was higher than in other organs . It was demonstrated that the content of ecdysteroids in callus cell culture is higher than in the intact plant with concurrent retention of a high ecdysone-20-monooxygenase activity.

J Chromatogr A, 2004 Apr 30, 1035(1), 63 - 73
Simultaneous separation and quantitation of amino acids and polyamines of forest tree tissues and cell cultures within a single high-performance liquid chromatography run using dansyl derivatization; Minocha R et al.; The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures . The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl amino acids include: use of a 10 cm column to reduce the total run time by approximately 15 min; modification of the dansyl derivatization process and gradient profile to elute amino acids and common polyamines within the same run; addition of steps for column cleaning within each run; shorter re-equilibration time; and finally, column cleaning and physically reversing the column at the end of a loop of samples . These changes improved peak resolution and increased column longevity by several-fold . Over 1000 foliar samples from mature forest trees could be analyzed with the same column as compared to only 200-250 samples before the incorporation of these changes . This method eluted 22 amino acids within 40 min plus all three common polyamines between 44 and 47 min . The total run time is 53.6 min for amino acids only and 55.6 min for both amino acids and polyamines.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Jun 5, 805(1), 33 - 9
Determination of malondialdehyde by liquid chromatography as the 2,4-dinitrophenylhydrazone derivative: a marker for oxidative stress in cell cultures of human hepatoma HepG2; Mateos R et al.; Malondialdehyde (MDA) is considered a presumptive biomarker for lipid peroxidation in live organisms and cultured cells . The present study adapts an accurate and reproducible method to measure MDA by high-performance liquid chromatography (HPLC) as its 2,4-dinitrophenylhydrazone derivative in human hepatoma HepG2 cells in culture . Since MDA is assumed to increase in conditions of cellular oxidative stress, two compounds that induce pharmacological oxidative stress in cell cultures, hydrogen peroxide (H(2)O(2)) and tert-butyl hydroperoxide (t-BOOH), have been used in HepG2 cells . The results report a significant increase in the content of MDA derivative after treatment with 200 and 500microM t-BOOH for 3h, while H(2)O(2) in doses up to 500microM failed to evoke a similar response, indicating a stronger lipid peroxidation of t-BOOH to HepG2 cells than H(2)O(2) . Thus, MDA can be used as a reliable biomarker for cellular oxidative stress in human hepatoma HepG2.

Invest Ophthalmol Vis Sci, 2004 May, 45(5), 1499 - 506
Effect of somatostatin on nitric oxide production in human retinal pigment epithelium cell cultures; Vasilaki A et al.; PURPOSE: To investigate the presence of somatostatin and its receptors (sst(1-5) receptors) and their possible involvement in the regulation of nitric oxide (NO) production in human RPE cell cultures . METHODS: Human RPE cells (D407) were used for all studies performed . Somatostatin levels were detected by radioimmunoassay, and RT-PCR and immunocytochemistry studies were performed to identify the somatostatin receptors (sst1-sst5) . Radioligand binding assays were also performed examining the ability of certain somatostatin ligands (sst1, sst2, sst5) to compete for {125I}Tyr11 somatostatin binding . The presence of NO synthase in the cultures was assayed with NADPH-diaphorase cytochemistry, and RT-PCR, and NO levels were assessed by examining the production of its stable metabolites NO2- and NO3- (NOx-) . RESULTS: SRIF was detected in a concentration of 0.56 +/- 0.13 picomoles/mg protein . sst1, sst2, and sst5 mRNAs were detected, yet only sst2B and sst5 immunoreactivity was observed in human RPE cell cultures . sst1- and sst5- but not sst2-selective ligands displaced the specific {125I}Tyr11 somatostatin binding to RPE cell membranes . NADPH-diaphorase stain and iNOS mRNA were detected . SRIF and the sst2-selective analogue MK678 increased the levels of NOx- in a concentration-dependent manner . This increase was blocked by the sst2 antagonist CYN-154806 (Ac-4NO2-Phe-c(dCys-Tyr-dTrp-Lys-Thr-Cys)-dTyr-NH2) . CONCLUSIONS: These results demonstrate the presence of somatostatin, and its receptors sst1, sst2B, and sst5 in human RPE cells and suggest an autocrine or paracrine role for somatostatin . Somatostatin's ability to regulate NO production, by activating sst2 receptors, provides a functional role of somatostatin in the RPE.

Dev Biol, 2004 May 15, 269(2), 459 - 78
A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses; Kuppers-Munther B et al.; Neurons in primary cell cultures provide important experimental possibilities complementing or substituting those in the nervous system . However, Drosophila primary cell cultures have unfortunate limitations: they lack either a range of naturally occurring cell types, or of mature physiological properties . Here, we demonstrate a strategy which supports both aspects integrated in one culture: Initial culturing in conventional serum-supplemented Schneider's medium (SM(20K)) guarantees acquisition of all properties known from 30 years of work on cell type-specific differentiation in this medium . Through subsequent shift to newly developed active Schneider's medium (SM(active)), neurons adopt additional mature properties like the ability to carry out plastic morphological changes, neurotransmitter expression and electrical activity . We introduce long-term FM-dye measurements as a tool for Drosophila primary cell cultures demonstrating the presence of increased, action potential-dependent synaptic activity in SM(active) . This is confirmed by patch-clamp recordings, which in addition show that SM(active)-cultured neurons display different spiking patterns . Furthermore, we demonstrate that transmission can be evoked in SM(active) cultures, revealing the existence of synaptic plasticity . Thus, these culture conditions support developmental, structural and physiological properties known or expected from the nervous system, enhancing possibilities for future experiments complementing or substituting those in nervous systems of Drosophila.

Bioelectrochemistry, 2004 Jun, 63(1-2), 347 - 51
Electrochemical determination of lead and glutathione in a plant cell culture; Vacek J et al.; In this work we established differential pulse anodic stripping voltammetry (DPASV) as the tool for analysis of lead in the plant cell culture . For the cultivation procedure, lead in Pb(II)-ethylenediaminetetraacetic acid (Pb-EDTA) chelate has been used . The detection limit of lead was found at 500 pM in phosphate buffer (pH 5.5), and 100 nM in prepared cells intracellular extract (20 pg Pb(II)/mg cells) . For determination of cysteine-rich peptides, voltammetry in differential mode (DPV) in cobalt(III)-containing ammonia buffer (Brdicka reaction) was used . In this short communication, we present suitable voltammetric techniques for the physiological study of lead and thiols in plant cell culture.

J Hosp Infect, 2004 Apr, 56 Suppl 2, S58 - 63
Assessment of new chemical disinfectants for HBV virucidal activity in a cell culture model; Payan C et al.; Several new chemical disinfectants were processed for Hepatitis B virus (HBV) virucidal activity in a cell culture model . A pooled HBV infected human plasma with 10(10.4) HBV DNA copies/mL was treated with the tested disinfectant . It was then subjected, for three days at several dilutions, to cell culture using the human hepatoma cell line, HepG2, with 4% polyethyleneglycol and 3 mM sodium butyrate . Thirty-seven assays were performed on 12 products, with up to 3 concentrations and 3 time exposures for each product tested . The mean viral titre without disinfectant was 10(5.18) infectious units per mL . Our results showed that products all four hand rubs examined, two of the three surface disinfectants and two of the three instrument disinfectants were highly active whatever concentrations and time exposures, reducing viral times by factors of 10(3)-10(4) . However, other products such as one of the surface disinfectants was only active at concentrations above 0.5% for 15 min . Similarly the skin disinfectant, one of the instrument disinfectants and the hand wash agent (diluted to 50%) were less or not active (of <10(3) fold reduction) . This is the first study using a cell culture model to assess virucidal activity against HBV of new disinfectants . It showed that most 9/12 products were active by either HBs antigen alteration (8/9) or probable envelope disruption (1/9) . Further studies are in progress using this model to assess the activity of other chemical disinfectants such as peracetic acid against HBV.

Plant Cell Rep, 2004 Sep, 23(3), 174 - 9 Epub 2004 Apr 24.
Enhancement of vitamin E production in sunflower cell cultures; Caretto S et al.; The most biologically active component of vitamin E, alpha-tocopherol, is synthesized in its most effective stereoisomeric form only by photosynthetic organisms . Using sunflower cell cultures, a suitable in vitro production system of natural alpha-tocopherol was established . The most efficient medium was found to be MS basal medium with naphthaleneacetic acid and 6-benzylaminopurine with the addition of casaminoacids and myo-inositol . Culture feeding experiments using biosynthetic precursors showed that alpha-tocopherol production improved by 30% when homogentisic acid was used . Interestingly, time-course experiments with sunflower suspension cultures showed a possible increase of 78% in alpha-tocopherol production when using cultures of longer subculture intervals . Compared to the starting plant tissue, an overall 100% increase of alpha-tocopherol was reached by these sunflower cell cultures.

Crit Rev Eukaryot Gene Expr, 2004, 14(1-2), 43 - 51
Development of cell cultures with competency for contributing to the zebrafish germ line; Fan L et al.; The zebrafish is an established model for the genetic analysis of vertebrate development . Forward-genetic screens have generated thousands of mutations, and antisense-based methods have been used to transiently knockdown gene expression during embryogenesis . Although these methods have made the zebrafish a valuable system for the identification and functional characterization of developmentally important genes, one deficiency of the zebrafish model is the absence of methods to introduce targeted mutations to generate knockout lines of fish . Application of gene-targeting methods has been limited in nonmurine species due to the absence of germ-line competent embryonic stem (ES) cell lines . Recently, progress was made in addressing this problem by the derivation of zebrafish embryo cell lines that remain pluripotent and germ-line competent for multiple passages in culture . Zebrafish germ-line chimeras were generated using cultures derived from embryos at two different developmental stages, and targeted insertion of vector DNA by homologous recombination was demonstrated in both cultures . Several strategies are being used to optimize the production and identification of germ-line chimeras . The zebrafish embryo cell culture system should provide the basis of a gene-targeting approach that will complement other genetic strategies and improve the utility of the zebrafish model for studies of development and disease.

Neurochem Res, 2004 Apr, 29(4), 827 - 33
Inhibitory effects of extracellular matrix modulator(s) on lipopolysaccharide (LPS)- and acidosis-damaged glia containing cerebellar granule cell cultures; Fukui H et al.; The inhibitory effects of a novel chondroitin sulfate compound on lipopolysaccharide (LPS)- and acidosis-induced neuronal dysfunctions were examined . Cell viabilities in cultured neurons and/or astrocyte-rich cerebellar granule cells were measured by the calcein-AM method . Ten and 20 microg, as a final dosage, of LPS damaged less than 20% cells during a-2 h incubation . More than 5000 ng/ml of chondroitin sulphate-dipalmitoylphosphatidylethanolamine (CS-PE), but not chondroitin sulfate (CS) treatment, significantly inhibited such damage . Twenty microg of LPS damaged more than 40% cells during 24 h incubation, and these cell damages were significantly inhibited by less than 1000 ng/ml of CS-PE . Moreover, treatments with between 5 and 500 ng/ml CS-PE, but not CS, significantly reduced the number of acidosis-damaged cells in a dose-dependent manner . The current results indicate that modulator(s) of ECM and its derivative containing covalently linked dipalmitoylphosphatidylethanolamine show neuroprotective effects under conditions of brain inflammation.

Biochem Biophys Res Commun, 2004 May 14, 317(4), 1023 - 9
Cell culture propagation and biochemical analysis of the Ljungan virus prototype strain; Johansson ES et al.; Ljungan virus (LV) is proposed as a potentially important rodent harbored viral human pathogen . Little is known about the biophysical nature of the virus and despite being molecularly characterized, progress in epidemiological and basic biological studies of LV has been hampered by the lack of a robust and reliable cell culture propagation system . Here we report the first description of an efficient lytic multi-cycle cell culture propagation of the LV prototype strain (87-012) . Biophysical analysis of gradient purified LV virions generated by this system identified mature infectious virions to possess a sedimentation coefficient of 160S and in agreement with previous molecular prediction, polyprotein analysis suggests that the native virion is composed of only three major structural proteins . The nucleotide composition of the complete genome of the LV cell culture adapted virus was determined and compared to that of the parental prototype LV . Numerous mutations were observed scattered throughout the viral genome and particularly in VP1 . The development of this cell culture system for LV should open new avenues in the study of LV biology, structure, pathogenesis, and prevalence of natural infection in the wider community.

Biotechniques, 2004 Apr, 36(4), 650 - 4, 656, 658 passim
Combining optical and electrical impedance techniques for quantitative measurement of confluence in MDCK-I cell cultures; De Blasio BF et al.; A new method combining optical and electrical impedance measurements is described that enables submicroscopic cell movements to be monitored . The cells are grown on small gold electrodes that are transparent to light . This modified electrical cell-substrate impedance sensor (ECIS) allows simultaneous microscopic recording of both growth and motility, thus enabling cell confluence on the electrodes to be systematically correlated to the impedance in regular time intervals of seconds and for extended periods of time . Furthermore, the technique provides an independent measure of monolayer cell densities that we compare to calculated values from a theoretical model . We have followed the attachment and spreading behavior of epithelial Madin-Darby canine kidney strain I (MDCK-I) cell cultures on microelectrodes for up to 40 h . The studies reveal a high degree of correlation between the measured resistance at 4 kHz and the corresponding cell confluence in 4- to 6-h intervals with typical linear cross-correlation factors of r equaling approximately 0.9 . In summary, the impedance measured with the ECIS technique provides a good quantitative measure of cell confluence.

Cancer Res, 2004 Apr 15, 64(8), 2643 - 8
Nitrosative stress in rotated three-dimensional colorectal carcinoma cell cultures induces microtubule depolymerization and apoptosis; Laguinge LM et al.; Malignant cells undergo anoikis as they encounter fluid shear stress during transit to a metastatic site . We postulated that intracellular nitric oxide (NO) contributes to this cell death by comparing the growth of human colorectal carcinoma cells in low fluid shear stress rotated three-dimensional (Rotated 3-D) cultures with growth in static three-dimensional (Static 3-D) cultures on nonadherent surfaces and with two-dimensional monolayer (Monolayer 2-D) cultures . NO, loss of microtubules, and apoptosis increased significantly in Rotated 3-D cultures within 10 min and persisted at 24 h, whereas inhibition of NO synthase decreased apoptosis and intracellular NO and prevented tubulin degradation . Thus, fluid shear stress and three-dimensional growth increases NO synthase and NO to cause tubulin breakdown and induce anoikis . Intracellular NO in malignant cells entering the circulation may be a novel target for metastasis by colorectal carcinoma.

Nutr Cancer, 2003, 47(2), 195 - 209
Oral cancer cells differ from normal oral epithelial cells in tissue like organization and in response to lycopene treatment: an organotypic cell culture study; Livny O et al.; We established distinctive monolayer and organotypic cell culture techniques to assess possible differences in cross-talk and spatial and structural organization of oral cancer cells compared with normal oral cells and also to evaluate possible differential responses of the cells to carotenoids . In monolayers, we investigated the effect of lycopene on the proliferation of an established oral cancer cell line, KB-1, and compared it with a primary cell line obtained from normal oral mucosa . Lycopene exerted a significant inhibitory effect on KB-1 cell proliferation inducing a dose-dependent downregulation of proliferating cell nuclear antigen (PCNA) associated with upregulation of connexin-43 (Cx-43) expression, whereas in the normal oral mucosal cells lycopene did not affect either PCNA expression, which was very low, or the expression of Cx-43, which was basically very high . Lycopene significantly inhibited the formation of colonies induced by the carcinogen 3-methylcholanthrene (MCA) on normal oral cells and almost completely abrogated the hyperplastic effect induced by MCA . KB-1 cells and normal oral epithelial cells in the organotypic cell culture method differed in their stratification and intercellular adhesion patterns as well as in the expression profile of cytokeratins, vimentin, and Cx-43 . Lycopene induced Cx-43 expression in KB-1 cells grown by the organotypic raft method, similar to KB-1 cells grown as monolayers . We conclude that lycopene is a promising chemopreventive, pro-differentiating, and anticarcinogenic agent . No adverse effects of lycopene were detected in normal cells cultured in either monolayer or organotypic systems.

Zhonghua Nan Ke Xue, 2004 Mar, 10(3), 198 - 201
{Biological characteristics of female rabbit clitoral smooth muscle cell culture in vitro}; Liu B et al.; OBJECTIVE: To investigate the method of culturing rabbit clitoral smooth muscle cells(SMC) in vitro and their biological characteristics . METHODS: Rabbit clitoral SMCs were cultured in vitro by enzymatically dispersed method, and morphological observation, cell counting and immunohistochemical staining were employed to study their biological characteristics . RESULTS: Rabbit clitoral SMCs were spindle-shaped and parallelled along their longitudinal axis, and the attachment and proliferation in vitro were rapid . CONCLUSION: Rabbit clitoral SMCs cultured in vitro can grow and maintain their steady characteristics when provided with appropriate culture condition . Clitoral SMCs cultured in vitro can serve as a cell material for researches of clitoris.

Antivir Chem Chemother, 2004 Jan, 15(1), 43 - 55
Synthesis of beta-enantiomers of N4-hydroxy-3'-deoxypyrimidine nucleosides and their evaluation against bovine viral diarrhoea virus and hepatitis C virus in cell culture; Hollecker L et al.; N4-Hydroxycytidine (NHC) was recently reported to have anti-pestivirus and anti-hepacivirus activity . It is thought that this nucleoside acts as a weak alternative substrate for the hepatitis C virus (HCV) polymerase . In addition to NHC, 3'-deoxyuridine (3'-dU) was found to inhibit bovine diarrhoea virus (BVDV) production by 1 log10 at 37.2 microM . These initial findings prompted the synthesis of beta-D and beta-L analogues of (i) base-modified 3'-deoxy-NHC; (ii) 3'-deoxyuridine; and 3'-deoxycytidine . The antiviral activity of these 42 nucleosides was evaluated against BVDV and HCV bicistronic replicon in cell culture . Among the NHC analogues, the antiviral activity observed for the beta-L-3'-deoxy-5-fluoro-derivative 1-(3-deoxy-beta-L-erythro-pentofuranosyl)-5-fluoro-4-hydroxyaminopyrimidin-2(1H)-one and the beta-D-3'-deoxy-5-iodo-derivative 1-(3-deoxy-beta-D-erythro-pentofuranosyl)-5-iodocytosine in the replicon system (1 log10 reduction at 100 microM) was due to the concomitant toxicity towards intracellular ribosomal RNA levels (CC90 equal or lower than the EC90) . In conclusion, none of the newly synthesized derivatives exhibited enhanced antiviral activity compared to the parent nucleoside NHC.

Mol Vis, 2004 Mar 31, 10, 248 - 53
Expression of wild type and mutant ELOVL4 in cell culture: subcellular localization and cell viability; Karan G et al.; PURPOSE: ELOVL4 is a member of the fatty acid elongase (ELO) family of genes . Mutations of this gene are responsible for autosomal dominant Stargardt-like macular degeneration . However, the specific role of ELOVL4 in photoreceptor cells and the mechanism by which mutations in ELOVL4 causes macular degeneration are not known . In this study we examined the subcellular localization of wild type (wt) and mutant (mt) ELOVL4 EGFP fusion protein and the potential functional consequence of mtELOVL4 expression on cell viability . METHODS: Wt and mt ELOVL4 were expressed as EGFP fusion proteins in NIH 3T3 and HEK293 cells . Subcellular localizations of the fusion proteins were determined with a series of organelle-specific markers for endoplasmic reticulum (pDsRed2-ER), mitochondria (pDsRed2-Mito), peroxisomes (pDsRed2-Peroxi), and Golgi (BODIPY TR) . Transfected cells were viewed using confocal and episcopic-fluorescence microscopy . Western blot analysis was performed to assess protein expression using an anti-GFP antibody . TUNEL staining was used to quantify apoptotic cell death . RESULTS: In cell transfection studies, wtELOVL4/EGFP fusion protein localized preferentially to the endoplasmic reticulum (ER) and was not found to be discernibly present in mitochondria, peroxisomes, or Golgi . In contrast, the truncated mutant fusion protein (which has no ER retention signal) appeared to be mislocalized to other compartments within transfected cells . Transfected cells expressing mtELOVL4/EGFP fusion protein exhibited induction of apoptotic cell death . CONCLUSIONS: Unlike wtELOVL4/EGFP fusion protein, the mtELOVL4/EGFP fusion protein did not localize to the ER but rather appeared to be sequestered elsewhere in an aggregated pattern in the cytoplasm . The apoptosis induced by the mutant ELOVL4 fusion protein may be the mechanism whereby photoreceptor cells degenerate in Stargardt-like macular degeneration . Our study has provided an important in vitro model system for further assessment of ELOVL4 biochemical functions.

Biol Trace Elem Res, 2004 May, 98(2), 181 - 91
Transmembrane partitioning of boron and other elements in RAW 264.7 and HL60 cell cultures; Ralston NV et al.; The trace element boron is essential for all higher plants and is beneficial or has been established as essential for several animal models of human nutrition . To help identify the biomolecules that require boron for function in humans, we determined whether intracellular boron is retained against a concentration gradient . Cells (Abelson leukemia virus BALB murine monocyte-macrophage RAW 264.7 {RAW} and HL60) and supplemented media (Dulbecco's modified essential media {+ 10% fetal calf serum} and Iscove's modified Dulbecco's medium {+ 5% fetal calf serum}, respectively) were analyzed for mineral concentrations after culture and subculture . Special corrections were made for trapped extracellular media in cell pellets and endocytosed media . For RAW cells, the partitioning coefficients (PC; intracellular/extracellular ratios) were, in rank order, as follows: Mn, 110; Fe, 67; P, 65; Zn, 32; K, 15; Cu, 7.1; Mg, 4.3; B, 1.7; Ca, 0.4; Na, 0.3 . For HL60 cells, the partitioning coefficients were, in rank order, as follows: Mn, 212; Zn, 211; P, 123; K, 21; Fe, 16; Mg, 11; B, 1.7; Ca, 0.8; Na, 0.3 . Trapped extracellular media was estimated to be 6.7 +/- 0.8%; trapped extracellular and endocytosed media together was 24.8 +/- 0.3% of the mass within the isolated cell pellets . The partitioning coefficients indicate a positive gradient for intracellular accumulation of boron, zinc, phosphorus, manganese, magnesium, potassium, iron, and copper in RAW264.7 and HL60 cells . Specifically, the data indicate the existence of a selective boron-binding molecular species within the cell or the existence of a boron-specific membrane transporter.

Phytomedicine, 2004 Feb, 11(2-3), 157 - 64
Mechanisms underlying antigonadotropic effects of some traditional plant extracts in pituitary cell culture; Benie T et al.; Previous works have demonstrated that stem bark extracts of Cola nitida (Sterculiaceae), Afrormosia laxiflora and Pterocarpus erinaceus (Fabaceae) provoked a blockade of female rat ovulation and estrous cycle by inhibiting pituitary LH release in vivo . In addition, these plant extracts exerted an inhibitory effect on LH release of rat pituitary cells . Therefore, these data could explain inhibitory effects of plant extracts on LH release in vivo . The present study was undertaken to examine the mechanisms by which these plants exert their antigonadotropic activities . So, we studied the biological activities of these plant extracts on pituitary cells in culture . Data show that C . nitida, A . laxiflora and P . erinaceus extracts only inhibit LH release and have no effect on FSH release . In fact, A . laxiflora, P . erinaceus and C . nitida extracts diminish LH release in culture medium without acting on rat pituitary cell content . Plant extracts form complexes with basic glycoproteins (but not with acid glycoproteins) and prevent them from entering the cells.

Prostate Cancer Prostatic Dis, 2004, 7(2), 138 - 43
Proteasome inhibitors and their combination with antiandrogens: effects on apoptosis, cellular proliferation and viability of prostatic adenocarcinoma cell cultures; Zwergel T et al.; The 26S proteasome is a ubiquitin-dependent proteolytic system that has been implicated in the regulation of cell cycle progression and apoptosis . We investigated the effects of the proteasome inhibitors MG115 and PSI alone or in combination with different concentrations of the antiandrogen hydroxyflutamide on the cellular proliferation, apoptosis and viability of 10 prostatic adenocarcinoma cell cultures . Treatment with both proteasome inhibitors resulted in apoptosis induction, whereas the combinations with hydroxyflutamide generally did not, with the exception of MG115 combined with 10(-7) M hydroxyflutamide . MG115 caused a significant decrease in cellular proliferation, as did the combinations of both proteasome inhibitors with hydroxyflutamide, whereas hydroxyflutamide alone was only effective at a concentration of 10(-5) M . Cellular viability was significantly reduced when both proteasome inhibitors were combined with 10(-5) M hydroxyflutamide . Although the results varied among different cell lines, we conclude that proteasome inhibitors are able to induce apoptosis and reduce cellular proliferation . They might prove effective as antineoplastic substances in prostatic adenocarcinoma alone or in combination with antiandrogens.

Eur J Neurosci, 2004 Mar, 19(6), 1459 - 68
Neurogenic and intact or apoptotic non-neurogenic areas of adult brain release diffusible molecules that differentially modulate the development of subventricular zone cell cultures; Agasse F et al.; Abstract In the adult mammalian brain, neurogenic activity is maintained in the subventricular zone (SVZ) . Damage to non-neurogenic areas can stimulate SVZ cell proliferation and trigger addition of new neurons in the affected areas . We therefore examined the possible control exerted by specific microenvironment cues on SVZ neurogenic activity . To this end, neonatal SVZ neurospheres were maintained in the presence of diffusible signals derived from the adult neurogenic SVZ or from the non-neurogenic cerebral cortex either previously treated (apoptotic cortex) or not (untreated cortex) with staurosporine, a known apoptosis inducer . To restrict interactions to soluble signals, the explants were separated from the SVZ neurospheres by a microporous membrane . The results indicated that molecules released by the SVZ itself promoted the expansion of SVZ cell population through increased proliferation and reduced apoptosis . In contrast, untreated cortex factors reduced the expansion of SVZ cell population by decreasing proliferation . In addition, SVZ or untreated cortex factors, respectively, promoted or inhibited neuronal differentiation . Following apoptotic damage, cortex factors no longer inhibited and instead promoted the expansion of the SVZ cell population by increasing proliferation . These effects on cell numbers were replicated following use of culture media conditioned with the different explants but were no longer present following heat inactivation, which indicates that proteins were involved . These findings indicate that the neurogenic SVZ delivers autocrine/paracrine signals that promote neurogenesis whereas the non-neurogenic cerebral cortex releases signals that inhibit proliferation and neuronal differentiation . Interestingly, this constitutive growth inhibitory effect of the cerebral cortex is inverted following apoptotic lesion.

Biochemistry (Mosc), 2004 Mar, 69(3), 306 - 10
Cytochrome P-450 family 1 in rat embryo cell culture immortalized by Rausher leukemia virus; Vaiman AV et al.; We studied comparative expression and activity of cytochrome P450 family 1 (CYP1) isoforms in rat embryo cells, both primary and immortalized by Rausher leukemia virus (RLV) . In RLV-infected embryonal cells compared with the initial ones the expression levels of CYP1A1 and 1B1 mRNAs and benzo{a}pyrene (BP) hydroxylase activity were higher, regardless of their treatment with the CYP1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin . The sensitivity to BP and 7,12-dimethylbenzo{a}anthracene was higher in the cells immortalized with RLV . The expression level of mRNAs of induction-mediating proteins aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator was the same in both cell cultures tested . Higher sensitivity of cells immortalized with RLV compared with the initial embryo cells to transforming effect of BP, which was described previously, is possibly associated with elevated expression of CYP1 isoforms.

Biotechnol Prog, 2004 Mar-Apr, 20(2), 590 - 7
Incorporation of 3T3-L1 cells to mimic bioaccumulation in a microscale cell culture analog device for toxicity studies; Viravaidya K et al.; Deficiencies in the early ADMET (absorption, distribution, metabolism, elimination, and toxicity) information on drug candidates extract a significant economic penalty on pharmaceutical firms . We have developed a microscale cell culture analog (microCCA) device that can potentially provide better, faster, and more efficient prediction of human and animal responses to a wide range of chemicals . The system described in this paper is a simple four-chamber microCCA ("lung"-"liver"-"fat"-"other tissue") designed on the basis of a physiologically based pharmacokinetics (PBPK) model of a rat . Cultures of L2, HepG2/C3A, and differentiated 3T3-L1 adipocytes were selected to mimic the key functions of the lung, liver, and fat compartments, respectively . Here, we have demonstrated the application of the microCCA system to study bioaccumulation, distribution, and toxicity of selected compounds . Results from the bioaccumulation study reveal that hydrophobic compounds such as fluoranthene preferentially accumulated in the fat chamber . Only a small amount of fluoranthene was observed in the liver and lung chambers . In addition, the presence of the differentiated 3T3-L1 adipocytes in the microCCA device significantly reduced naphthalene and naphthoquinone-induced glutathione (GSH) depletion . These findings suggest the potential utilization of the microCCA system to assess ADMET characteristics of the compound of interest prior to animal or human trials.

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