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1

Establishment of a Vero cell line persistently infected with African swine fever virus.  

PubMed Central

A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-P cells were growing logarithmically and increased considerably in confluent cultures when lysis appeared in a fraction of the cell population. Images

Salas, J; Vinuela, E

1986-01-01

2

Optimization of Toxoplasma gondii cultivation in VERO cell line.  

PubMed

In vitro culture of Toxoplasma gondii can provide tachyzoites which are active, viable and with desirable purity. Thus the aim of this study was to optimize the cell culture method for T. gondii propagation to obtain a consistent source of parasites with maximum yield and viability, but minimum host cell contamination for use in production of excretory-secretory antigen. Tachyzoites with seed counts of 1x10(6), 1x10(7) and 1x10(8) harvested from infected mice were added to VERO cells of different degrees of confluence, namely 50%, 85% and 100%, and examined periodically using an inverted microscope. When the maximum release of the tachyzoites was observed from the host cells, the culture supernatant was removed and the tachyzoites harvested. Using a Neubauer chamber, the percentages of viable tachyzoites and host cell contamination were determined using trypan blue stain. Parameters that gave the best yield and purity of viable tachyzoites were found to be as follows: VERO cells at 85% confluence in DMEM medium and inoculum comprising 1x10(7) tachyzoites. After about 3 days post infection, the tachyzoites multiplied 78x, with a yield of ~7.8x10(8) per flask, 99% viability and 3% host cell contamination. This study has successfully optimized the method of propagation of T. gondii tachyzoites in VERO cells which produce parasites with high yield, purity and viability. PMID:20562822

Saadatnia, G; Haj Ghani, H; Khoo, B Y; Maimunah, A; Rahmah, N

2010-04-01

3

Live cold-adapted influenza A vaccine produced in Vero cell line  

Microsoft Academic Search

The African green monkey kidney (Vero) cell line was used as a substrate for the development of a live cold-adapted (ca) reassortant influenza vaccine. For that purpose, a new master strain was generated by an adaptation of the wild type (wt) A\\/Singapore\\/1\\/57 virus to growth at 25°C in a Vero cell line. The resulting cold-adapted (ca) muster strain A\\/Singapore\\/1\\/57ca showed

Julia Romanova; Dietmar Katinger; Boris Ferko; Brigitta Vcelar; Sabine Sereinig; Oleg Kuznetsov; Marina Stukova; Marjana Erofeeva; Oleg Kiselev; Hermann Katinger; Andrej Egorov

2004-01-01

4

Photoirradiation study of gold nanospheres and rods in Vero and Hela cell lines  

NASA Astrophysics Data System (ADS)

Photoirradiation effect of gold nanospheres in conjucation with green light and rods in conjugation with red light corresponds to their absorption wavelength range found to be appreciable. In this present work concentration of nanomaterial and light dose were optimized. Gold nanospheres were synthesized by reduction technique using Sodium Borohydrate as reducing agent and Trisodium Citrate as capping agent. Au nanorods having 680-900nm absorption were synthesized using reduction techniques with CTAB and BDAC polymers. From UV-Vis absorption and Transmission Electron Microscopy the size of nanoparticles were confirmed. 30nm Gold nanospheres and green light source of 530nm wavelength with power 30mW were applied to Vero and Hela cell lines shows higher toxicity for Hela cells. Nanorods were applied and irradiated with 680nm wavelength light source with light intensity 45mW. Post irradiation effect for 24hrs, 48hrs confirms cell proliferation in normal rate in viable cells. The morphological changes in irradiated spot leads to apoptotoic cell death was confirmed with microscopic imaging. The LD50 value was also calculated.

Gananathan, Poorani; Aruna, Prakasarao; Ganesan, Singaravelu; Elanchezhiyan, Manickan

2014-03-01

5

Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum.  

PubMed

In this study the tachyzoite yields of Neospora caninum were compared in two cell lines: Vero (African Green Monkey Kidney) and suspension culture of murine macrophage (J774) cell lines. Then, N. caninum were continuously passaged in these cell lines for 3 months and the effect of host cells on virulence of tachyzoites was assessed by broiler chicken embryonated eggs. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs with different dilutions (0.5 × 10(4), 1.0 × 10(4), 1.5 × 10(4)) of tachtzoites isolated from these cell cultures. The mortality pattern and pathological changes of the dead embryos and hatched chickens were noted. Tissue samples of brain, liver and heart were examined by histopathological and detection of DNA of parasite by polymerase chain reaction (PCR). Also, consecutive sections of the tissues examined histologically were used for immunohistochemical (IHC) examination. Embryos inoculated with tachyzoites derived from Vero cell line (group V) showed a higher mortality rate (100%) than the embryos that received tachyzoites derived from J774 cell line (group J) (10% mortality rate). The results of this study indicated that the culture of N. caninum in J774 cell led to a marked increase in the number of tachyzoite yields and rapid attenuation in comparison to Vero, so the results were confirmed by IHC and PCR. This study is the first report of the significant effect of host cell on the attenuation of virulence of N. caninum tachyzoites. These findings could potentially provide a practical approach in the mass production of N. caninum tachyzoites, and also in producing live attenuated vaccine. PMID:23684321

Khordadmehr, Monireh; Namavari, Mehdi; Khodakaram-Tafti, Azizollah; Mansourian, Maryam; Rahimian, Abdollah; Daneshbod, Yahya

2013-10-01

6

Phorbol Esters Isolated from Jatropha Meal Induced Apoptosis-Mediated Inhibition in Proliferation of Chang and Vero Cell Lines  

PubMed Central

The direct feeding of Jatropha meal containing phorbol esters (PEs) indicated mild to severe toxicity symptoms in various organs of different animals. However, limited information is available on cellular and molecular mechanism of toxicity caused by PEs present in Jatropha meal. Thus, the present study was conducted to determine the cytotoxic and mode of action of PEs isolated from Jatropha meal using human hepatocyte (Chang) and African green monkey kidney (Vero) cell lines. The results showed that isolated PEs inhibited cell proliferation in a dose-dependent manner in both cell lines with the CC50 of 125.9 and 110.3 ?g/mL, respectively. These values were compatible to that of phorbol 12-myristate 13-acetate (PMA) values as positive control i.e., 124.5 and 106.3 ?g/mL respectively. Microscopic examination, flow cytometry and DNA fragmentation results confirmed cell death due to apoptosis upon treatment with PEs and PMA at CC50 concentration for 24 h in both cell lines. The Western blot analysis revealed the overexpression of PKC-? and activation of caspase-3 proteins which could be involved in the mechanism of action of PEs and PMA. Consequently, the PEs isolated form Jatropha meal caused toxicity and induced apoptosis-mediated proliferation inhibition toward Chang and Vero cell lines involving over-expression of PKC-? and caspase-3 as their mode of actions.

Oskoueian, Ehsan; Abdullah, Norhani; Ahmad, Syahida

2012-01-01

7

Systematically experimental investigation on carcinogenesis or tumorigenicity of VERO cell lines of different karyotypes in nude mice in vivo used for viral vaccine manufacture.  

PubMed

Many cell lines used for vaccine production have a potentially strong tumorigenic character. Some of those routinely used need to be checked at different passage numbers for this characteristic. Using HeLa cell cultures as positive controls, and primary canine kidney cell (CKC) or feline kidney cell (FKC) cultures purified in vitro on passage three as negative controls, the tumorigenicity of VERO cell sublines was tested in 219 nude mice. The master cell stocks (MCS) and working cell banks (WCB) of eight strains of VERO African green monkey kidney cell (AGMKC) line used for canine, feline and mink vaccine preparation were established in China. The hypo-tetra-ploid JA or hyper-diploid KA strain of VERO line was highly tumorigenic. These data showed a variable chromosome karyotype of VERO line, and contraindicated the use of JA or KA strain of VERO line for the preparation of attenuated viral vaccines. JA or KA strain of VERO line could be a substitute for HeLa line as a positive-control malignant tumor (MT) cell model. The non-carcinogenic YB, JC, M and JB strains of VERO line were therefore selected for the preparation of modified live rabies viral vaccine in place of BHK-21. The cell sub-lines are comparatively stable in terms of their heritable characters, and show little significant changes between passages. In summary, we have found that: 1) the tumorigenicity of cell line is different among different-karyotypic cells; 2) it is the genetic characteristics of chromosomes of cell lines that determines their tumorigenicity, but with species-specific carcinogenicity; 3) the chromosome number variation of cell lines has positive relationship with their carcinogenesis; 4) highly variable strains of tumor cell line can be selected quickly and successfully in nude mice by alternate cultivation in vitro and in vivo. Malignant rhabdoid tumor (MRT) was evolved in nude mice inoculated with violently variable HeLa or VERO cells. The importance of assessing the tumorigenicity in cell sublines used for vaccine production is emphasised. PMID:15473315

Zhang, De-Li; Ji, Liang; Li, Liu-Jin; Huang, Gao-Sheng

2004-07-01

8

Isolation of measles virus from clinical specimens using B95a and Vero/hSLAM cell-lines.  

PubMed

The clinical presentation of acute measles is normally quite typical, especially in the presence of Koplik's spots, that laboratory test is seldom required to confirm the diagnosis. However, with wide measles vaccination coverage and the extensive use of immunosuppressive chemotherapy, the diagnosis of atypical manifestations of acute measles may require laboratory confirmation. When compared with B95a cell-line, this study shows that the Vero/hSLAM cell-line is sensitive and is recommended for use in the primary isolation of wild-type measles virus from clinical specimens. Throat swab and urine specimens are the clinical specimens of choice and both are recommended for optimal isolation of measles virus from patients suspected of acute measles virus infection. PMID:19852319

Keniscope, C; Juliana, R; Subri, H; Shangari, S R; Wan Nor Azlina, W A; Hamizah, A; Emmi, E E; Nor Azlina, M D; Norizah, I; Chua, K B

2009-03-01

9

[Vitaherpavac is the first Russian herpes simplex virus vaccine obtained on the Vero B continuous cell line].  

PubMed

Vitaherpavac, a dry inactivated herpes simplex virus (HSV) culture vaccine, has been obtained, by using the Vero B continuous cell line as a substrate for accumulation of herpes simplex virus types 1 (US strain) and 2 (VN strain). Vitaherpavac and the similar vaccine Herpovax made by the Research Institute of Vaccines and Sera, Saint Petersburg (for which preparation a primary trypsinized chick embryo cell culture used as a substrate for accumulation of HSV types 1 and 2), underwent comparative clinical trials. The tolerability and therapeutic effectiveness of the vaccine were tested in patients diagnosed as having chronic frequently recurring herpes. The trials have yielded positive results that suggest that it is expedient to introduce of the new vaccine Vitaherpavac into practice to treat chronic recurrent herpetic infection of various localizations. Vitaherpavac has been registered in the Russian Federation and permitted for medical application. PMID:19882901

Barkhaleva, O A; Ladyzhenskaia, I P; Vorob'eva, M S; Shalunova, N V; Podcherniaeva, R Ia; Mikha?lova, G R; Khorosheva, T V; Barinski?, I F

2009-01-01

10

Autophagic Cell Death Is Induced by Acetone and Ethyl Acetate Extracts from Eupatorium odoratum In Vitro: Effects on MCF-7 and Vero Cell Lines  

PubMed Central

Eupatorium odoratum (EO) contains many biologically active compounds, the anticancer effects of which are not well documented. This study evaluates the cytotoxic effects and mechanism of action of EO extracts on MCF-7 and Vero cell lines. Evaluation of the cytotoxic activity using MTT assay, morphological alterations, and apoptosis were carried out. Autophagy was evaluated by LC3-A protein expression. Cytotoxic activity, membrane blebbing and ballooning at 24 hours, replacement by mass vacuolation, and double membrane vesicles mimicking autophagy and cell death were observed in the cancer cells. No apoptosis was observed by DNA fragmentation assay. Overexpression of LC3-A protein indicated autophagic cell death. Cell cycle analysis showed G0 and G2/M arrest. The Vero cells did not show significant cell death at concentrations <100??g/mL. These results thus suggest that acetone and ethyl acetate extracts of EO induce cell death through induction of autophagy and hold potential for development as potential anticancer drugs.

Harun, Faizah Bt.; Syed Sahil Jamalullail, Syed Mohsin; Yin, Khoo Boon; Othman, Zulkhairi; Tilwari, Anita; Balaram, Prabha

2012-01-01

11

Isolation of bovine coronavirus (bcoV) in vero cell line and its confirmation by direct FAT and RT-PCR.  

PubMed

Bovine Coronavirus (BCoV) is widespread both in dairy and beef cattle throughout the world. The virus is one of the largest RNA virus and has specific tropism for intestinal and pulmonary epithelial cells. It is responsible for huge economic losses by causing winter dysentery in adult dairy cattle and respiratory and intestinal tract infections leading to pneumo-enteritis in young calves. Isolation of BCoV has been reported to be difficult. Studies regarding epidemiology, virus isolation and molecular detection from India are very few. In the present study Vero cell line was used for isolation of the BCoV from Enzyme Linked Immunosorbent Assay (ELISA) positive samples. Direct florescent antibody technique (dFAT) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to confirm the isolated virus strains at antigenic and genomic levels, respectively. Out of the 15 positive fecal samples, virus from only seven was able to infect vero cell line. Subsequently BCoV got adapted to the vero cell line upto three passages, which was confirmed both at genomic and antigenic levels by dFAT and RT-PCR testing. It can be concluded that vero cell line can be used for isolation of BCoV, however due to the enormous stain diversity of the virus it is possible that many stains can't grow and get adapt in this cell line. Further studies are required for isolation of different viral strains, finding the susceptible cell lines and also to confirm the variations among the BCoV isolates at antigenic/genomic levels. PMID:24511744

Hansa, A; Rai, R B; Dhama, K; Wani, M Y; Saminathan, M; Ranganath, G J

2013-11-01

12

Autophagic cell death is induced by acetone and ethyl acetate extracts from Eupatorium odoratum in vitro: effects on MCF-7 and vero cell lines.  

PubMed

Eupatorium odoratum (EO) contains many biologically active compounds, the anticancer effects of which are not well documented. This study evaluates the cytotoxic effects and mechanism of action of EO extracts on MCF-7 and Vero cell lines. Evaluation of the cytotoxic activity using MTT assay, morphological alterations, and apoptosis were carried out. Autophagy was evaluated by LC3-A protein expression. Cytotoxic activity, membrane blebbing and ballooning at 24 hours, replacement by mass vacuolation, and double membrane vesicles mimicking autophagy and cell death were observed in the cancer cells. No apoptosis was observed by DNA fragmentation assay. Overexpression of LC3-A protein indicated autophagic cell death. Cell cycle analysis showed G0 and G2/M arrest. The Vero cells did not show significant cell death at concentrations <100??g/mL. These results thus suggest that acetone and ethyl acetate extracts of EO induce cell death through induction of autophagy and hold potential for development as potential anticancer drugs. PMID:22666123

Harun, Faizah Bt; Syed Sahil Jamalullail, Syed Mohsin; Yin, Khoo Boon; Othman, Zulkhairi; Tilwari, Anita; Balaram, Prabha

2012-01-01

13

VERO cells (cercopithecus aethiops kidney) — growth characteristics and viral susceptibility for use in diagnostic virology  

Microsoft Academic Search

Summary An investigation of some of the characteristics of the VERO cell line (Cercopithecus aethiops kidney) is reported, in which the suitability of the cells for use in routine diagnostic virology was examined. VERO cells will:1.grow to monolayers as rapidly as other heteroploid cell lines, but will maintain as usable monolayers in conventional maintenance medium for a significantly longer time;2.grow

D. E. Macfarlane; R. G. Sommerville

1969-01-01

14

African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses.  

PubMed

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly alpha2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelia] cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic. PMID:8764064

Govorkova, E A; Murti, G; Meignier, B; de Taisne, C; Webster, R G

1996-08-01

15

Humoral and cell-mediated immunity to vero cell-derived influenza vaccine.  

PubMed

A candidate trivalent influenza whole virus vaccine produced in a continuous mammalian cell line (Vero), and analogous commercially available egg-derived vaccines, were compared for their ability to induce humoral and cell-mediated immunity in Balb/c mice. Substantial haemagglutination-inhibition titre and high levels of influenza virus-specific IgG were found in all groups of immunized mice, irrespective of the vaccine formulation. The IgG responses were predominantly of IgG1 and IgG2a/2b isotypes. Virus-specific secretory IgA antibodies were detected only in mice immunized intranasally with a live virus, derived either from Vero cells or eggs. T-cell proliferative responses and T-helper 1 type cytokine release was significantly higher in mice immunized with Vero cell-derived influenza vaccine compared to egg-derived vaccine formulations. We have demonstrated that the immunogenicity of the trivalent Vero cell-derived whole influenza virus vaccine was comparable to that of the equivalent egg-derived vaccine, with respect to humoral immune response and was superior with respect to cellular response. PMID:11137251

Brühl, P; Kerschbaum, A; Kistner, O; Barrett, N; Dorner, F; Gerencer, M

2000-12-01

16

Humoral and cell-mediated immunity to Vero cell-derived influenza vaccine  

Microsoft Academic Search

A candidate trivalent influenza whole virus vaccine produced in a continuous mammalian cell line (Vero), and analogous commercially available egg-derived vaccines, were compared for their ability to induce humoral and cell-mediated immunity in Balb\\/c mice. Substantial haemagglutination-inhibition titre and high levels of influenza virus-specific IgG were found in all groups of immunized mice, irrespective of the vaccine formulation. The IgG

Peter Brühl; Astrid Kerschbaum; Otfried Kistner; Noel Barrett; Friedrich Dorner; Marijan Geren?er

2000-01-01

17

Polyvinyl formal surface promotes continuous growth of Vero cells in protein-free medium.  

PubMed

The effect of polyvinyl formal (PVF) culture surface on the growth of 10 mammalian continuous cell lines, including Swiss 3T6, NCTC clone 929 L, BHK-21 clone 13, CHO-K1, PK 15, A 431, HeLa, MDCK, LLC-MK2 and Vero in protein-free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 supplemented with trace elements and L-ascorbic acid 2-phosphate, was investigated. Most of the cell lines showed only some initial proliferation on PVF similar to the polystyrene (PS) surface of commercially available culture flasks. In contrast, proliferation of monkey kidney cell line Vero was by far greater on PVF than on PS or poly-D-lysine treated culture surface. In addition, Vero cells on PVF could be subcultured in the protein-free medium without any significant decrease of growth rate in successive passages. These results showed that PVF provides a culture surface which selectively promotes continuous growth of Vero cells in protein-free, chemically defined medium. PMID:1610559

Cinatl, J; Cinatl, J; Rabenau, H; Doerr, H W

1992-03-01

18

Recycle of Cytodex-3 in Vero cell culture  

Microsoft Academic Search

It is commonly considered not desirable to use microcarriers more than once in the cultivation of anchorage-dependent animal cells. However, our experiment contradicts this belief. The collagen-coated microcarriers, Cytodex-3, from a batch culture of Vero cells, were collected, cooled to 4, agitated in basic phosphate-buffered solution to detach the cells, and then fully washed to remove the cell debris. The

Y. Wang; F. Ouyang

1999-01-01

19

Development of Vero cell-derived inactivated Japanese encephalitis vaccine.  

PubMed

We have established a manufacturing system for a Vero cell-derived inactivated Japanese encephalitis vaccine at a 500l scale. The production system involves expansion of Vero cells using microcarrier, followed by virus infection. Except for an additional purification step, the downstream purification processes are similar to those used for the current mouse brain-derived vaccine; cell removal, concentration and removal of low-molecular weight impurities by membrane filtration, formalin-inactivation, sucrose density gradient ultracentrifugation, and Sulfate-Cellulofine column chromatography are conducted. The antigen obtained from the manufacturing system was highly purified and its physico-chemical and immunological properties were comparable with those of antigen derived from mouse brains. Our system is very simple and could be easily scaled-up to allow vaccine production at a several thousand litre scale. PMID:12421588

Sugawara, Keishin; Nishiyama, Kiyoto; Ishikawa, Yuji; Abe, Motoharu; Sonoda, Kengo; Komatsu, Kazuhiro; Horikawa, Yoshikane; Takeda, Kengo; Honda, Tomitaka; Kuzuhara, Shoji; Kino, Yoichiro; Mizokami, Hiroshi; Mizuno, Kyosuke; Oka, Tetsuya; Honda, Kennosuke

2002-12-01

20

Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines.  

PubMed

The development of cell culture systems for virus propagation has led to major advances in virus vaccine development. Primary and diploid cell culture systems are now being replaced by the use of continuous cell lines (CCLs). These substrates are gaining increasing acceptance from regulatory authorities as improved screening technologies remove fears regarding their potential oncogenic properties. The Vero cell line is the most widely accepted CCL by regulatory authorities and has been used for over 30 years for the production of polio and rabies virus vaccines. The recent licensure of a Vero cell-derived live virus vaccine (ACAM2000, smallpox vaccine) has coincided with an explosion in the development of a range of new viral vaccines, ranging from live-attenuated pediatric vaccines against rotavirus infections to inactivated whole-virus vaccines against H5N1 pandemic influenza. These developments have illustrated the value of this cell culture platform in the rapid development of vaccines against a range of virus diseases. PMID:19397417

Barrett, P Noel; Mundt, Wolfgang; Kistner, Otfried; Howard, M Keith

2009-05-01

21

Decreased sensitivity to diphtheria toxin of Vero cells cultured in serum-free medium  

Microsoft Academic Search

Vero cell cultures are used in the quality control of Diphtheria vaccines: to estimate vaccine potency and to determine residual toxicity and reversion to toxicity. The impact of replacing foetal calf serum containing medium (SCM) by serum free media (SFM) on the sensitivity of Vero cells to Diphtheria Toxin was studied. Compared to SCM, SFM showed an eight-fold decrease in

S. J. Piersma; J. W. van der Gun; C. F. M. Hendriksen; M. Thalen

2005-01-01

22

Optimization of transfection methods for Huh-7 and Vero cells: comparative study.  

PubMed

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI and Lipofectamine 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 +/- 2.36 and 73.9 +/- 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI was respectively 14.2 +/- 0.69 and 28 +/- 1.11% for the same cells. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed the superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines. PMID:23285746

Hashemi, A; Roohvand, F; Ghahremani, M H; Aghasadeghi, M R; Vahabpour, R; Motevau, F; Memarnejadian, A

2012-01-01

23

Improved poliovirus d-antigen yields by application of different Vero cell cultivation methods.  

PubMed

Vero cells were grown adherent to microcarriers (Cytodex 1; 3gL(-1)) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1×10(6) cellsmL(-1) during batch cultivation to 1.8, 2.7 and 5.0×10(6) cellsmL(-1) during semi-batch, perfusion and recirculation, respectively. The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher d-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific d-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific d-antigen production. The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing. PMID:24583004

Thomassen, Yvonne E; Rubingh, Olaf; Wijffels, René H; van der Pol, Leo A; Bakker, Wilfried A M

2014-05-19

24

Adaptation of High-Growth Influenza H5N1 Vaccine Virus in Vero Cells: Implications for Pandemic Preparedness  

PubMed Central

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 108 TCID50/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.

Huang, Mei-Liang; Yeh, Wei-Zhou; Weng, Tsai-Chuan; Chen, Yu-Shuan; Chong, Pele; Lee, Min-Shi

2011-01-01

25

[Fluorescent derivatives of diphtheria toxin subunit B and their interaction with Vero cells].  

PubMed

Diphtheria toxin's B subunit provides toxin interaction with its receptor on the cell surface and translocation of toxin's A subunit from endosome to cytozole of sensitive cells. Functional analogues of B subunit with fluorescent label are considered as perspective tools for studying the above mentioned processes. The aim of the work was to obtain fluorescent B subunit analogues and to detect the specificity of their interaction with Vero line cells. B subunit fluorescent analogues were obtained in two different ways. The first one was B subunit chemical conjugation with fluorescein isothiocyanate and the second one was genetic fusion of recombinant B subunit chain with enhanced green fluorescent protein chain. Specific interaction of B subunit fluorescent derivatives with Vero cells was studied by flow cytometry and confocal microscopy. Using competitive analysis it was shown that B subunit fluorescent analogues possessed different affinity for cells. The affinity of EGFP-SbB was higher than FITC-SbB. Our results indicate the possibility to use the fluorescent derivatives of B subunit as tools for identification of diphtheria toxin's receptor (HB-EGF) expression on the cell surface as well as for studying the interaction and penetration of diphtheria toxin to the cell. PMID:19877418

Kaberniuk, A A; Labyntsev, A Iu; Kolybo, D V; Oli?nyk, O S; Redchuk, T A; Korotkevych, N V; Horchev, V F; Karakhim, S O; Komisarenko, S V

2009-01-01

26

Kinetics and metabolic specificities of Vero cells in bioreactor cultures with serum-free medium  

Microsoft Academic Search

The aim of this study was to understand the metabolism kinetics of Vero cells grown on microcarriers in bioreactors in serum-free\\u000a medium (SFM). We sought to determine what nutrients are essential for Vero cells and how they are consumed. Contrary to glucose\\u000a and to most of the amino acids, glutamine and serine were very quickly depleted in this medium and

Sébastien Quesney; Annie Marc; Catherine Gerdil; Cyrille Gimenez; Jacqueline Marvel; Yves Richard; Bernard Meignier

2003-01-01

27

Development of a Vero cell-derived influenza whole virus vaccine.  

PubMed

Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and the presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The WHO-approved Vero cell line was used in serum-free culture to grow many influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter. A purification scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation. PMID:10494963

Kistner, O; Barrett, P N; Mundt, W; Reiter, M; Schober-Bendixen, S; Eder, G; Dorner, F

1999-01-01

28

Vero cell culture-derived pandemic influenza vaccines: preclinical and clinical development.  

PubMed

Several subtypes of influenza A viruses with pandemic potential are endemic in bird populations throughout Asia, Africa and the Middle East, and evidence suggests that these viruses are adapting to the mammalian host. As emphasized by the high mortality rate of humans infected with H5N1 viruses, this situation presents a substantial risk to global human health. The Vero cell culture platform has been used to develop whole-virus influenza vaccines that provide broad cross-clade protection against viruses with pandemic potential, at low antigen doses, without the requirement for adjuvantation. The safety and immunogenicity of these vaccines has been demonstrated in studies with more than 10,000 individuals, including healthy adult and elderly subjects, children and various risk groups. These Vero cell-derived vaccines are licensed for prepandemic and pandemic use. The Vero platform is also being explored to develop next-generation live-attenuated and recombinant vaccines. PMID:23560920

Barrett, P Noel; Portsmouth, Daniel; Ehrlich, Hartmut J

2013-04-01

29

Preflucel®: a Vero-cell culture-derived trivalent influenza vaccine.  

PubMed

Vaccination is the principal means to reduce the impact of influenza infection. Effective vaccination programs require a reliable and safe production system. Traditionally, influenza vaccines are produced in embryonated chicken eggs. Over the last two decades, new cell culture-derived vaccines have been licensed and manufactured, and other vaccines are still in various phases of development. Vero cells have been used for the development of a wide variety of vaccines including influenza vaccines. Pandemic and avian influenza vaccines derived from Vero cells have been shown to be well tolerated and immunogenic in animal and Phase I-II clinical studies. A Phase III randomized, double-blind, placebo-controlled trial of a trivalent influenza vaccine produced in Vero-cell culture was conducted in 7250 adults aged 18-49 years. Overall protective efficacy for antigenically matched influenza vaccine was 78.5%. The vaccine was well tolerated with no treatment-related serious adverse events and compared favorably with egg-derived vaccines from previous trials. Vero-cell-derived influenza vaccines have the potential to be an important parts of the influenza vaccine strategy, especially if an avian-derived strain becomes predominant or the demand outstrips the capacity of egg-based production systems. PMID:22913252

Chan, Candice Yuen-Yue; Tambyah, Paul Anantharajah

2012-07-01

30

Development of a mammalian cell (Vero) derived candidate influenza virus vaccine  

Microsoft Academic Search

Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The World Health Organisation (WHO) approved Vero

O Kistner; P. N. Barrett; W. Mundt; M. Reiter; S. Schober-Bendixen; F. Dorner

1998-01-01

31

A purified inactivated Japanese encephalitis virus vaccine made in vero cells  

Microsoft Academic Search

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals.

Ashok K. Srivastava; J. Robert Putnak; Sung H. Lee; Sun P. Hong; Sang B. Moon; David A. Barvir; Bangti Zhao; Russell A. Olson; Soo-Ok Kim; Wang-Don Yoo; Andrew C. Towle; David W. Vaughn; Bruce L. Innis; Kenneth H. Eckels

2001-01-01

32

Japanese encephalitis virus production in Vero cells with serum-free medium using a novel oscillating bioreactor  

Microsoft Academic Search

A novel oscillating bioreactor, BelloCell, was successfully applied for the cultivation of Vero cells using serum-free medium, and the production of Japanese encephalitis virus. The BelloCell requires no air sparging, pumping, or agitation, and thus provides a low shear environment. Owing to its simple design, BelloCell is extremely easy to handle and operate. Using this BelloCell (500ml culture), Vero cells

Hiroko Toriniwa; Tomoyoshi Komiya

2007-01-01

33

Development of an animal-component free medium for vero cells culture.  

PubMed

This work describes the development of an animal-component free medium (IPT-AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum-free medium (SFM) referred as IPT-SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT-SFM was further improved to obtain an animal-component free medium named IPT-AFM. IPT-AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue-culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non-animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT-AFM were investigated in T-flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 +/- 0.18 and a specific growth rate micro 0.019 +/- 0.003 h(-1) were achieved in IPT-AFM. PMID:19768803

Rourou, Samia; van der Ark, Arno; van der Velden, Tiny; Kallel, Héla

2009-01-01

34

Enhanced Growth of Influenza Vaccine Seed Viruses in Vero Cells Mediated by Broadening the Optimal pH Range for Virus Membrane Fusion  

PubMed Central

Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines.

Murakami, Shin; Ito, Mutsumi; Takano, Ryo; Katsura, Hiroaki; Shimojima, Masayuki

2012-01-01

35

Differential activities of plant polyphenols on the binding and internalization of cholera toxin in vero cells.  

PubMed

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization. PMID:15814610

Morinaga, Naoko; Iwamaru, Yoshifumi; Yahiro, Kinnosuke; Tagashira, Motoyuki; Moss, Joel; Noda, Masatoshi

2005-06-17

36

Decreased sensitivity to diphtheria toxin of Vero cells cultured in serum-free medium.  

PubMed

Vero cell cultures are used in the quality control of Diphtheria vaccines: to estimate vaccine potency and to determine residual toxicity and reversion to toxicity. The impact of replacing foetal calf serum containing medium (SCM) by serum free media (SFM) on the sensitivity of Vero cells to Diphtheria Toxin was studied. Compared to SCM, SFM showed an eight-fold decrease in sensitivity to Diphtheria Toxin. This decrease was almost immediate, indicating that this phenomenon was not caused by a change in membrane structure or protein expression. We investigated the effect of SFM on Diphtheria Toxin in order to determine the cause of the decrease in sensitivity. Our results show that oligopeptides, which are often used in SFM as part of the replacement of foetal calf serum, are the most likely cause. PMID:15905099

Piersma, S J; van der Gun, J W; Hendriksen, C F M; Thalen, M

2005-06-01

37

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells  

Microsoft Academic Search

Rabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B- and 3CD-like as well as viral RNAs co-localize within membranous structures

Rosa Casais; Lorenzo González Molleda; Angeles Machín; Gloria del Barrio; Alberto García Manso; Kevin P. Dalton; Ana Coto; José Manuel Martín Alonso; Miguel Prieto; Francisco Parra

2008-01-01

38

Isolation of Mycoplasma genitalium from First-Void Urine Specimens by Coculture with Vero Cells?  

PubMed Central

Isolation of Mycoplasma genitalium from clinical specimens remains difficult. We describe an improvement of the Vero cell coculture method in which the growth of M. genitalium was monitored by quantitative real-time PCR. Four new M. genitalium strains were isolated from six first-void urine specimens of male Japanese patients with urethritis. In two of them, only M. genitalium was detected: one also contained Ureaplasma urealyticum, and one contained Chlamydia trachomatis, Neisseria gonorrhoeae, U. urealyticum, and Ureaplasma parvum. In the specimens yielding isolates of M. genitalium, growth was documented by quantitative PCR after two to five passages in Vero cells. The complete isolation procedure from the initial inoculation to completion of single-colony cloning took about 1 year. Isolation of M. genitalium from urine specimens proved to be more difficult than from swab specimens. Due to the cytotoxic effect of urine, a procedure involving washing of the urinary sediment was introduced. Furthermore, prolonged storage of the urine specimens before culture was shown to be detrimental to the success of isolation, as shown by the lack of success in attempts to isolate M. genitalium from mailed urine specimens as well as by simulation experiments. High concentrations of penicillin G and amphotericin B were surprisingly inhibitory to the growth of wild-type M. genitalium strains, but penicillin G at 200 IU/ml and polymyxin B at 500 ?g/ml could be used as selective antibiotics to avoid bacterial overgrowth in the Vero cell cultures.

Hamasuna, Ryoichi; Osada, Yukio; Jensen, J?rgen Skov

2007-01-01

39

Structure-Function Relationship of the Ion Channel Formed by Diphtheria Toxin in Vero Cell Membranes  

Microsoft Academic Search

.   Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed\\u000a by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane\\u000a of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming\\u000a transmembrane domain

M. Lanzrein; P. Ø. Falnes; O. Sand; S. Olsnes

1997-01-01

40

Prevention of lipid peroxidation induced by ochratoxin A in Vero cells in culture by several agents.  

PubMed

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. It is a structural analogue of phenylalanine and contains a chlorinated dihydroisocoumarinic moiety. Ochratoxin A inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction. Recently lipid peroxidation induced by OTA has been reported, indicating that the lesions induced by this toxin could also be related to oxidative damage. An attempt to prevent its toxic effect, mainly the lipid peroxidation, has been made using aspartame (L-aspartyl-L-phenylalanine methyl ester) a structural analogue of both OTA and phenylalanine, piroxicam, a non steroidal anti-inflammatory drug and superoxide dismutase+catalase (endogenous oxygen radical scavengers). Lipid peroxidation was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (5-50 microM). After 24 h incubation OTA induced lipid peroxidation in Vero cells in a concentration dependent manner, as measured by malonaldehyde (MDA) production. The MDA production, in Vero cells, was significantly increased by 50.5% from 694.1 +/- 21.0 to 1041.5 +/- 23.5 pmol/mg of protein. In the presence of superoxide dismutase (SOD)+catalase (25 micrograms/ml each) the MDA production induced by OTA was significantly decreased. At 50 microM of OTA concentration (optimal production of MDA) the MDA production decreased from 1041.5 +/- 23.5 to 827.5 +/- 21.3 pmol/mg of protein. SOD and catalase, when applied prior to the toxin, seemed to prevent lipid peroxidation more efficiently than piroxicam (at a ten-fold higher concentration than OTA) and aspartame (at equimolar concentration). These molecules also partially prevented the OTA-induced leakage of MDA in the culture medium. PMID:9158693

Baudrimont, I; Ahouandjivo, R; Creppy, E E

1997-04-18

41

Vero cells expressing porcine circovirus type 2-capsid protein and their diagnostic application.  

PubMed

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) in swine. Although the incidences of PCV2-related diseases are ubiquitous throughout the world, the serological tools are rather limited, mainly because the virus does not induce any cytopathic effects in cells. The purpose of this study was to develop a rapid, sensitive and easy quantitative immunofluorescence assay (QIFA) using the recombinant PCV2 nucleocapsid protein (NCP) for the detection of PCV2-specific antibodies in pig sera. The recombinant PCV2 NCP was expressed in Vero cells by a lentivirus system. The performance of QIFA using these Vero cells as a diagnostic antigen was compared with currently available C-ELISA and I-ELISA; the relative sensitivity turned out to range from 92.5% up to 99.3%. The relative specificity was 93.3% when compared to C-ELISA as the gold standard. The serological experiment also indicated the inverse relationship between QIFA and the viral load in serum, semen, feces samples from 7 PCV2-positive boars. In addition, the PCV2 sequence detected from bone marrow cells shows 99% of sequence identity with PCV2 genome, confirming the infectivity of PCV2. PMID:23954842

Kim, Yeon-Hee; Kweon, Chang-Hee; Kang, Seung-Won; Oh, Yoon I; Song, Jae Young; Lee, Kyoung Ki; Park, Se Chang

2013-12-01

42

Immunological equivalence between mouse brain-derived and Vero cell-derived Japanese encephalitis vaccines.  

PubMed

The persistent spread via animal reservoirs urges expanding vaccination programs against pathogens like the Japanese encephalitis virus, JEV. The JEV is spreads to new areas by domestic as well as by wild animals. Although there is a safe and efficient vaccine on the market, this is derived from infected mouse brains, why today's situation requires overcoming the potential risk caused by using animal tissues. To meet this demand we have developed a Vero cell-derived JEV vaccine, using the same virus strain as in the established one. A phase III clinical study of the new vaccine has recently been completed with positive outcome. Like the established mouse brain-derived vaccine, the Vero cell-derived one is a formalin inactivated whole virus vaccine. We here demonstrate the very good agreement in immunological tests between the two antigens. The study includes analyses with two neutralizing monoclonal antibodies that blocks cell entry at a late stage in infection, assumedly interfering with fusion-related refolding in the virus fusion protein. It is obvious that the formalin inactivation treatment, with both virus preparations, retains these essential vaccine epitopes. PMID:16815584

Abe, Motoharu; Shiosaki, Kouichi; Hammar, Lena; Sonoda, Kengo; Xing, Li; Kuzuhara, Syoji; Kino, Yoichiro; Holland Cheng, R

2006-11-01

43

A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.  

PubMed

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers. PMID:11483284

Srivastava, A K; Putnak, J R; Lee, S H; Hong, S P; Moon, S B; Barvir, D A; Zhao, B; Olson, R A; Kim, S O; Yoo, W D; Towle, A C; Vaughn, D W; Innis, B L; Eckels, K H

2001-08-14

44

Non-clinical and phase I clinical trials of a Vero cell-derived inactivated Japanese encephalitis vaccine.  

PubMed

The safety and effectiveness of a Vero cell-derived inactivated Japanese encephalitis (JE) vaccine were compared with those of a current JE vaccine in non-clinical studies and a phase I clinical trial. The single-dose toxicity study showed no toxicity of either the current JE vaccine or the investigational Vero cell-derived JE vaccine. In a local irritation study, the degree of irritation caused by both vaccines was determined to be the same as that induced by normal saline. To investigate genotoxicity, a chromosomal aberration test was conducted and the results were negative. Both JE vaccines were administered to a group of 30 subjects who were seronegative (neutralizing antibody titer <10(1)) for JEV virus (Beijing-1 Strain). Each subject was subcutaneously inoculated twice at an interval of 1-4 weeks, followed by an additional booster inoculation 4-8 weeks later, and clinical reactions and serological responses were subsequently investigated. Adverse drug reactions of local reaction, headache and malaise were mild, occurring at a rate of 6.7 and 20.0% after administration of the Vero cell-derived JE vaccine and the current JE vaccine, respectively. The seroconversion rate after three doses of both JE vaccines was 100%, while the geometric mean titer for the Vero cell-derived and current JE vaccines was 10(2.35) and 10(2.03), respectively. These results suggest that the safety and effectiveness of the Vero cell-derived inactivated JE vaccine are equal to those of the currently available conventional vaccine in humans, and that the Vero cell-derived vaccine could be a useful second-generation JE vaccine. PMID:14575762

Kuzuhara, Syoji; Nakamura, Hideki; Hayashida, Kenshi; Obata, Junko; Abe, Motoharu; Sonoda, Kengo; Nishiyama, Kiyoto; Sugawara, Keishin; Takeda, Kengo; Honda, Tomitaka; Matsui, Hajime; Shigaki, Takamichi; Kino, Yoichiro; Mizokami, Hiroshi; Tanaka, Masahiko; Mizuno, Kyosuke; Ueda, Kohji

2003-11-01

45

Purification, potency and immunogenicity analysis of Vero cell culture-derived rabies vaccine: a comparative study of single-step column chromatography and zonal centrifuge purification.  

PubMed

Continuous Vero cell lines are more suitable for large-scale production of rabies vaccine. The purification of Vero cell-derived rabies vaccine is critical because of the residual cellular DNA and serum proteins. The perfection of techniques using column chromatography with different matrix material, gel filtration and zonal centrifugation is of paramount importance for the optimal purification of rabies vaccine, leaving minimal residual cellular DNA, below the permissible level of 100 pg per dose and serum protein content of 1 ppm. In this study the potency, immunogenicity and safety of Vero cell-derived rabies vaccines were compared following purification by densely or loosely packed DEAE-sepharose CL-6B columns with different bed heights or by zonal centrifugation. The optimal virus recovery and maximum removal of substrate DNA and serum proteins were achieved only when the sepharose CL-6B column bed height was maintained at a thickness of 2-2.5 cm. The rabies virus material was purified by layering over the matrix without applying pressure. DEAE-sepharose CL-6B column purification using a simplified, cost effective technique as described in this study enhances the antigen yield by 50% in comparison with zonal purification. PMID:16046167

Prem Kumar, Ananda Arone; Mani, Kavaratty Raju; Palaniappan, Chitrambalam; Bhau, Lakshman Narasingha Rao; Swaminathan, Krishnaswami

2005-07-01

46

A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions  

Microsoft Academic Search

Rabies virus strain production in Vero cells grown on Cytodex 1 in a 2L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO+0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific

Samia Rourou; Arno van der Ark; Tiny van der Velden; Héla Kallel

2007-01-01

47

Characterization of Vero cell growth and death in bioreactor with serum-containing and serum-free media  

Microsoft Academic Search

The density of viable cells in a culture results from a balance between cell proliferation and cell death. The aim of this\\u000a study was to characterize and compare these two phenomena in Vero cell cultures in one serum containing medium (ScA) and one\\u000a serum free medium (SfB) in bioreactors. Cell growth was evaluated by cell counting(after crystal violet staining) and

Sébastien Quesney; Jacqueline Marvel; Annie Marc; Catherine Gerdil; Bernard Meignier

2001-01-01

48

Genetic stability of oral polio vaccine prepared on primary monkey kidney cells or Vero cells--effects of passage in cell culture and the human gastrointestinal tract.  

PubMed

The genetic stability of the three Sabin oral poliovaccine (OPV) strains produced on either primary monkey kidney (VK) or Vero cell substrates was compared in vivo and in vitro by measuring the rate at which the bases most strongly associated with attenuation and reversion to neurovirulence (positions 480, 481, and 472 in the 5' non-coding region of Sabin 1, 2 and 3 respectively, and 2034 in VP3 of Sabin 3) reverted during passage of the vaccine strains in the gastrointestinal tract of primary vaccinees and in cell culture. For the in vivo study, the poliovirus excretion patterns of 21 infants receiving OPV produced on either VK or Vero cells were followed for 21 days. No significant differences in either the frequency of excretion or the rate of reversion were observed between the two vaccine groups. The rate of accumulation of revertants during passage in vitro was compared for the three Sabin strains passaged 10 times in either VK or Vero cells. For types 1 and 3, revertants accumulated faster upon passage through VK cells compared with passage through Vero cells. Type 2 appeared to be stable as no revertants were detected in either cell type. Results of this study suggest that the use of Vero as opposed to VK cells as substrate for the manufacture of OPV does not negatively influence the genetic stability of the three Sabin OPV strains in vivo or in vitro. PMID:9796061

Chezzi, C; Dommann, C J; Blackburn, N K; Maselesele, E; McAnerney, J; Schoub, B D

1998-12-01

49

Immunogenicity of single-dose Vero cell-derived Japanese encephalitis vaccine in Japanese adults.  

PubMed

In Japan, intensive immunization against Japanese encephalitis (JE) was performed from 1967 to 1976, and regular JE immunization was performed thereafter. However, for Japanese adults facing JE risk, dates of vaccination with new inactivated Vero cell-derived JE vaccine are unavailable. This study investigated how a single dose of Vero cell-derived JE vaccine affects Japanese adults. Neutralizing antibodies were measured pre- and post-JE vaccination in 79 participants (age 40.7 ± 9.4 years), enrolled between October 2009 and March 2011, whose JE-vaccination data were gathered from vaccination records and history taking. Before vaccination, the participants' seroprotection rate (SPR) was 51.9%, whereas SPR after vaccination was 93.7%. The seroconversion rate (SCR), which measures seronegative cases that turn seropositive after vaccination, was 86.8%. The geometric mean titer (GMT) was 14.7 before vaccination and 70.1 after vaccination. Age was a significant difference between seroprotected (42.8 years) and non-seroprotected (38.7 years) groups before vaccination. Then the difference of age, SCR, pre-vaccination GMT, post-vaccination GMT and sex ratio were also significant in participants aged 25-39 years and ?40 years, who represent generations born when Japan's JE-vaccination policy changed. SCR was 100% in participants aged 25-39 years with a vaccination recorded 55.6% in participants aged 25-39 without a vaccination record, and 96.0% in participants aged ?40 years. Thus, more participants aged 25-39 years were seroprotected before vaccination, but SCR was higher in those aged ?40 years. Most Japanese adults can be protected after one-dose vaccination, but this may be insufficient for people aged 25-39 years without recorded JE vaccination. PMID:24485326

Takeshita, Nozomi; Lim, Chang-Kweng; Mizuno, Yasutaka; Shimbo, Takuro; Kotaki, Akira; Ujiie, Mugen; Hayakawa, Kayoko; Kato, Yasuyuki; Kanagawa, Shuzo; Kaku, Mitsuo; Takasaki, Tomohiko

2014-04-01

50

The effect of serial passage on Sabin oral poliomyelitis vaccine virus in secondary monkey kidney versus Vero cells as measured by the monkey neurovirulence test.  

PubMed

The three Sabin strains of poliomyelitis seed virus were serially passaged in either secondary monkey kidney or Vero cell cultures and the tenth passage of each virus harvest compared to non-passaged Sabin reference virus of the same type using the monkey neurovirulence test. All three types were further attenuated by passage in Vero cells, whereas only type 2 became further attenuated after passage in secondary monkey kidney cells. After passage in Vero cells, type 3 poliomyelitis virus became more heat stable, as measured by its replicative capacity at 40 degrees C. PMID:7731028

Dommann, C J

1994-09-01

51

Canine distemper virus induces apoptosis through caspase-3 and -8 activation in vero cells.  

PubMed

We investigated the signal-transduction pathway of canine distemper virus-Onderstepoort (CDV-Ond) vaccine strain-mediated apoptosis in Vero cells. Canine distemper virus-Onderstepoort at a multiplicity of infection (MOI) of 0.1 induced DNA fragmentation 48 h after infection. Immunofluorescence staining revealed that 57% +/- 4% of the CDV-N-protein-positive cells were terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive, and all TUNEL-positive cells were CDV-N-protein-positive, indicating that CDV-Ond induced apoptosis only in the infected cells. We also found that CDV-Ond infection induced activation of caspase-3 and caspase-8. In the semi-quantitative reverse transcription-polymerase chain reaction assay for apoptosis-related genes, the expression of mRNA of the death receptor, Fas, was also increased in CDV-Ond-infected cells. In contrast, the expressions of Bcl-2 and Bax, regulators for intrinsic apoptotic signaling through the mitochondria, did not change. These results suggest that CDV-Ond induced apoptosis by activating caspase-3, possibly through caspase-8 signaling rather than through p53/Bax-mediated, mitochondrial signaling in the infected cells. PMID:16907958

Kajita, M; Katayama, H; Murata, T; Kai, C; Hori, M; Ozaki, H

2006-08-01

52

Label free detection of pseudorabies virus infection in Vero cells using laser force analysis.  

PubMed

The rapid and robust identification of viral infections has broad implications for a number of fields, including medicine, biotechnology and biodefense. Most detection systems rely on specific molecules, such as nucleic acids or proteins, to identify the target(s) of interest. These molecules afford great specificity, but are often expensive, labor-intensive, labile and limited in scope. Label free detection methods seek to overcome these limitations by instead using detection methods that rely on intrinsic properties as a basis for identifying and separating species of interest and thus do not rely on specific prior knowledge of the target. Optical chromatography, one such technique, uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we present the application of individual optical force measurements as a means of investigating pseudorabies virus infection in Vero cells. Optical force differences are seen between cells from uninfected and infected populations at a multiplicity of infection as low as 0.001 and as soon as 2 hours post infection, demonstrating the potential of this technique as a means of detecting viral infection. Through the application of a pattern recognition neural network, individual cell size data are combined with optical force as a means of classifying cell populations. Potential applications include the early detection of bloodborne pathogens for the prevention of sepsis and other diseases as well as the detection of biological threat agents. PMID:24492491

Hebert, Colin G; Hart, Sean J; Terray, Alex

2014-03-21

53

Role of kinesin-1 and cytoplasmic dynein in endoplasmic reticulum movement in VERO cells  

PubMed Central

Summary Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that ?50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought.

Wozniak, Marcin J.; Bola, Becky; Brownhill, Kim; Yang, Yen-Ching; Levakova, Vesselina; Allan, Victoria J.

2009-01-01

54

Lipophilic organic pollutants induce changes in phospholipid and membrane protein composition leading to Vero cell morphological change.  

PubMed

Membrane damage related to morphological change in Vero cells is a sensitive index of the composite biotoxicity of trace lipophilic chemicals. However, judging whether the morphological change in Vero cells happens and its ratio are difficult because it is not a quantitative characteristic. To find biomarkers of cell morphological change for quantitatively representing the ratio of morphological changed cell, the mechanism of cell membrane damage driven by typical lipophilic chemicals, such as trichlorophenol (TCP) and perfluorooctanesulphonate (PFOS), was explored. The ratio of morphologically changed cells generally increased with increased TCP or PFOS concentrations, and the level of four major components of phospholipids varied with concentrations of TCP or PFOS, but only the ratio of phosphatidylcholine (PC)/phosphatidylethanolamine (PE) decreased regularly as TCP or PFOS concentrations increased. Analysis of membrane proteins showed that the level of vimentin in normal cell membranes is high, while it decreases or vanishes after TCP exposure. These variations in phospholipid and membrane protein components may result in membrane leakage and variation in rigid structure, which leads to changes in cell morphology. Therefore, the ratio of PC/PE and amount of vimentin may be potential biomarkers for representing the ratio of morphological changed Vero cell introduced by trace lipophilic compounds, thus their composite bio-toxicity. PMID:25065828

Liao, Ting T; Wang, Lei; Jia, Ru W; Fu, Xiao H; Chua, Hong

2014-10-01

55

Aggravation of cold-induced injury in Vero-B4 cells by RPMI 1640 medium - Identification of the responsible medium components  

PubMed Central

Background In modern biotechnology, there is a need for pausing cell lines by cold storage to adapt large-scale cell cultures to the variable demand for their products. We compared various cell culture media/solutions for cold storage of Vero-B4 kidney cells, a cell line widely used in biotechnology. Results Cold storage in RPMI 1640 medium, a recommended cell culture medium for Vero-B4 cells, surprisingly, strongly enhanced cold-induced cell injury in these cells in comparison to cold storage in Krebs-Henseleit buffer or other cell culture media (DMEM, L-15 and M199). Manufacturer, batch, medium supplements and the most likely components with concentrations outside the range of the other media/solutions (vitamin B12, inositol, biotin, p-aminobenzoic acid) did not cause this aggravation of cold-induced injury in RPMI 1640. However, a modified Krebs-Henseleit buffer with a low calcium concentration (0.42 mM), a high concentration of inorganic phosphate (5.6 mM), and glucose (11.1 mM; i.e. concentrations as in RPMI 1640) evoked a cell injury and loss of metabolic function corresponding to that observed in RPMI 1640. Deferoxamine improved cell survival and preserved metabolic function in modified Krebs-Henseleit buffer as well as in RPMI 1640. Similar Ca2+ and phosphate concentrations did not increase cold-induced cell injury in the kidney cell line LLC-PK1, porcine aortic endothelial cells or rat hepatocytes. However, more extreme conditions (Ca2+ was nominally absent and phosphate concentration raised to 25 mM as in the organ preservation solution University of Wisconsin solution) also increased cold-induced injury in rat hepatocytes and porcine aortic endothelial cells. Conclusion These data suggest that the combination of low calcium and high phosphate concentrations in the presence of glucose enhances cold-induced, iron-dependent injury drastically in Vero-B4 cells, and that a tendency for this pathomechanism also exists in other cell types.

2012-01-01

56

Comparison of viral glycosylation using lectin blotting with Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines.  

PubMed

We produced a Vero cell-derived inactivated Japanese encephalitis vaccine using a serum-free medium, as a substitute for the conventional mouse brain-derived Japanese encephalitis vaccine. The immunogenicity of this cell-derived vaccine was higher than that of the conventional mouse brain-derived vaccine. The results of a clinical study in humans also demonstrated higher immunogenicity of this cell-derived vaccine. No gene mutation was found in the viral structural proteins derived from Vero cells and mouse brain. So, we conducted a lectin blot analysis, assuming differing glycosylation as a cause of the higher immunogenicity in humans. The results demonstrated that vaccine reactivity varied with lectins, particularly with WGA, DBA, MAM, SSA, SBA, and GS-II. Thus, glycosylation differed with the vaccines, suggesting a possible cause of the differing immunogenicity between mice and humans. PMID:21195800

Toriniwa, Hiroko; Komiya, Tomoyoshi

2011-02-24

57

Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane  

PubMed Central

Two substances possessing the ability to bind to diphtheria toxin (DT) were found to be present in a membrane fraction from DT-sensitive Vero cells. One of these substances was found on the basis of its ability to bind DT and inhibit its cytotoxic effect. This inhibitory substance competitively inhibited the binding of DT to Vero cells. However this inhibitor could not bind to CRM197, the product of a missense mutation in the DT gene, and did not inhibit the binding of CRM197 to Vero cells. Moreover, similar levels of the inhibitory activity were observed in membrane fractions from DT-insensitive mouse cells, suggesting the inhibitor is not the DT receptor which is specifically present in DT-sensitive cells. The second DT-binding substance was found in the same Vero cell membrane preparation by assaying the binding of 125I-labeled CRM197. Such DT-binding activity could not be observed in membrane preparation from mouse L cells. From competition studies using labeled DT and CRM proteins, we conclude that this binding activity is due to the surface receptor for DT. Treatment of these substances with several enzymes revealed that the inhibitor was sensitive to certain RNases but resistant to proteases, whereas the DT receptor was resistant to RNase but sensitive to proteases. The receptor was solubilized and partially purified by chromatography on CM- Sepharose column. Immunoprecipitation and Western blotting analysis of the partially purified receptor revealed that a 14.5-kD protein is the DT receptor, or at least a component of it.

1988-01-01

58

Long-term stability of Vero cell-derived inactivated Japanese encephalitis vaccine prepared using serum-free medium.  

PubMed

We established a method of producing a Vero cell-derived Japanese encephalitis vaccine using serum-free medium, and tested its stability using various stabilizers during the inactivation process and storage at 4 degrees C and 28 degrees C. Similar to previously reported results of cell culture in serum-containing medium, Vero cells were cultured in a serum-free medium multiplied well, and the viral yield was successfully increased to about 10(9)PFU/ml. Following formalin-inactivation and purification via ethanol precipitation and sucrose density ultracentrifugation of the virus solution, the vaccine had the same quality as, and higher immunogenicity, the mouse brain-derived vaccine in current use. Testing of several stabilizers showed that the addition of 0.5% glycine during the virus inactivation process facilitated the maintenance of immunogenicity for a long period of time. Furthermore, the addition of 0.5% glycine and 1.0% sorbitol as vaccine stabilizers after purification led to the maintenance of immunogenicity for 1 year, not dependent on the storage temperature (4 degrees C or 28 degrees C). These results indicate that, in contrast to the current mouse brain-derived vaccine, the Vero cell-derived vaccine can be prepared using serum-free medium containing no animal-derived components, and that the vaccine can be stored at room temperature by adding stabilizers, suggesting the possibility of producing room temperature-stable vaccines. PMID:18534722

Toriniwa, Hiroko; Komiya, Tomoyoshi

2008-07-01

59

Safety profile of the Vero cell-derived Japanese encephalitis virus (JEV) vaccine IXIARO(®).  

PubMed

Japanese encephalitis (JE) is the most common cause for viral encephalitis in Asia and can be effectively prevented by vaccination. IXIARO(®) is a Vero cell-derived, inactivated JE virus vaccine which has been licensed and distributed in the US, Europe, Canada, Hongkong, Israel, and distributed in Australia under the trade name JESPECT(®). This paper reviews the safety profile of IXIARO(®) in the first 12months after licensure and discusses the observed profile in the context of clinical trial results for IXIARO(®) and post-marketing safety data for JE-VAX(®). The clinical safety profile is derived from a pooled analysis including safety data from 10 phase III trials in 4043 subjects who received at least one IXIARO(®) vaccination and were followed-up for up to 3years after the primary immunization. Local and systemic tolerability of IXIARO(®) was similar to an earlier safety analysis at the time of licensure of the vaccine. In post-marketing AE reports, the system organ classes affected following vaccination with IXIARO(®) were similar to the previously observed clinical trial profile. No serious allergic reactions were observed in the 12-month post-marketing period. This comprehensive safety review confirms the good safety profile of IXIARO(®) in clinical and post-marketing use. PMID:21907747

Schuller, Elisabeth; Klingler, Anton; Dubischar-Kastner, Katrin; Dewasthaly, Shailesh; Müller, Zsuzsanna

2011-11-01

60

In-vitro maturation of round spermatids using co-culture on Vero cells.  

PubMed

In an attempt to determine whether co-culture could promote sperm maturation, three patients with non-obstructive azoospermia, two with maturation arrest at the level of primary spermatocytes and one patient with <1% tubules showing complete spermatogenesis, and one patient with total globozoospermia, gave consent to experimentally co-culture round spermatids retrieved from the testicle on Vero cell monolayers. In all azoospermic patients elongating spermatids could be obtained from round spermatids. In one case of maturation arrest, of 37 round spermatids co-cultured for up to 5 days, 30% developed flagella, 46% matured to elongating and 19% to elongated spermatids, with one mature spermatozoon also obtained (3%). In the same patient, primary cultures of three round spermatids with flagella enabled development of one further mature spermatozoon. In the case with total globozoospermia, of six round spermatids co-cultured for up to 5 days, one mature spermatozoon was obtained, with a flagellum and normal head morphology. These preliminary findings suggest that it may be possible to overcome the round spermatid block, and even the triggering of morphological abnormalities arising at the spermiogenic level, by in-vitro maturation under special environmental conditions. PMID:10325279

Cremades, N; Bernabeu, R; Barros, A; Sousa, M

1999-05-01

61

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells.  

PubMed

Rabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B- and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated. PMID:18625274

Casais, Rosa; Molleda, Lorenzo González; Machín, Angeles; del Barrio, Gloria; Manso, Alberto García; Dalton, Kevin P; Coto, Ana; Alonso, José Manuel Martín; Prieto, Miguel; Parra, Francisco

2008-10-01

62

Cell lines.  

PubMed

We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals. PMID:24434506

Cherbas, Lucy; Gong, Lei

2014-06-15

63

Human Respiratory Syncytial Virus Memphis 37 Grown in HEp-2 Cells Causes more Severe Disease in Lambs than Virus Grown in Vero Cells  

PubMed Central

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and young children. A small percentage of these individuals develop severe and even fatal disease. To better understand the pathogenesis of severe disease and develop therapies unique to the less-developed infant immune system, a model of infant disease is needed. The neonatal lamb pulmonary development and physiology is similar to that of infants, and sheep are susceptible to ovine, bovine, or human strains of RSV. RSV grown in Vero (African green monkey) cells has a truncated attachment G glycoprotein as compared to that grown in HEp-2 cells. We hypothesized that the virus grown in HEp-2 cells would cause more severe clinical symptoms and cause more severe pathology. To confirm the hypothesis, lambs were inoculated simultaneously by two different delivery methods (intranasal and nebulized inoculation) with either Vero-grown or HEp-2-grown RSV Memphis 37 (M37) strain of virus to compare viral infection and disease symptoms. Lambs infected with HEp-2 cell-derived virus by either intranasal or nebulization inoculation had significantly higher levels of viral RNA in lungs as well as greater clinical disease including both gross and histopathologic lesions compared to lambs similarly inoculated with Vero-grown virus. Thus, our results provide convincing in vivo evidence for differences in viral infectivity that corroborate previous in vitro mechanistic studies demonstrating differences in the G glycoprotein expression by RSV grown in Vero cells.

Derscheid, Rachel J.; van Geelen, Albert; McGill, Jodi L.; Gallup, Jack M.; Cihlar, Tomas; Sacco, Randy E.; Ackermann, Mark R.

2013-01-01

64

Human respiratory syncytial virus Memphis 37 grown in HEp-2 cells causes more severe disease in lambs than virus grown in Vero cells.  

PubMed

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and young children. A small percentage of these individuals develop severe and even fatal disease. To better understand the pathogenesis of severe disease and develop therapies unique to the less-developed infant immune system, a model of infant disease is needed. The neonatal lamb pulmonary development and physiology is similar to that of infants, and sheep are susceptible to ovine, bovine, or human strains of RSV. RSV grown in Vero (African green monkey) cells has a truncated attachment G glycoprotein as compared to that grown in HEp-2 cells. We hypothesized that the virus grown in HEp-2 cells would cause more severe clinical symptoms and cause more severe pathology. To confirm the hypothesis, lambs were inoculated simultaneously by two different delivery methods (intranasal and nebulized inoculation) with either Vero-grown or HEp-2-grown RSV Memphis 37 (M37) strain of virus to compare viral infection and disease symptoms. Lambs infected with HEp-2 cell-derived virus by either intranasal or nebulization inoculation had significantly higher levels of viral RNA in lungs as well as greater clinical disease including both gross and histopathologic lesions compared to lambs similarly inoculated with Vero-grown virus. Thus, our results provide convincing in vivo evidence for differences in viral infectivity that corroborate previous in vitro mechanistic studies demonstrating differences in the G glycoprotein expression by RSV grown in Vero cells. PMID:24284879

Derscheid, Rachel J; van Geelen, Albert; McGill, Jodi L; Gallup, Jack M; Cihlar, Tomas; Sacco, Randy E; Ackermann, Mark R

2013-11-01

65

Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors  

Microsoft Academic Search

The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus\\u000a (Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120\\/h and 0.0106\\/h,\\u000a respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of 106.75 and 106.67 TCID50 per 50 µl; signifying

O.-W. Merten; R. Wu; E. Couvé; R. Crainic

1997-01-01

66

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor  

Microsoft Academic Search

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work.\\u000a Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent\\u000a experiments, Vero cell growth in IPT-AF medium, on 2 g\\/l Cytodex 1 was consistent. An average Cd (cell division number) of\\u000a 3.3?±?0.4 and

Samia Rourou; Arno van der Ark; Samy Majoul; Khaled Trabelsi; Tiny van der Velden; Héla Kallel

2009-01-01

67

Preclinical evaluation of Vaxfectin-adjuvanted Vero cell-derived seasonal split and pandemic whole virus influenza vaccines.  

PubMed

Increasing the potency and supply of seasonal and pandemic influenza vaccines remains an important unmet medical need which may be effectively accomplished with adjuvanted egg- or cell culture-derived vaccines. Vaxfectin, a cationic lipid-based adjuvant with a favorable safety profile in phase 1 plasmid DNA vaccines trials, was tested in combination with seasonal split, trivalent and pandemic whole virus, monovalent influenza vaccines produced in Vero cell cultures. Comparison of hemagglutination inhibition (HI) antibody titers in Vaxfectin-adjuvanted to nonadjuvanted vaccinated mice and guinea pigs revealed 3- to 20-fold increases in antibody titers against each of the trivalent influenza virus vaccine strains and 2- to 8-fold increases in antibody titers against the monovalent H5N1 influenza virus vaccine strain. With the vaccine doses tested, comparable antibody responses were induced with formulations that were freshly prepared or refrigerated at conventional 2-8°C storage conditions for up to 6 mo. Comparison of T-cell frequencies measured by interferon-gamma ELISPOT assay between groups revealed increases of between 2- to 10-fold for each of the adjuvanted trivalent strains and up to 22-fold higher with monovalent H5N1 strain. Both trivalent and monovalent vaccines were easy to formulate with Vaxfectin by simple mixing. These preclinical data support further testing of Vaxfectin-adjuvanted Vero cell culture vaccines toward clinical studies designed to assess safety and immunogenicity of these vaccines in humans. PMID:23857272

Smith, Larry R; Wodal, Walter; Crowe, Brian A; Kerschbaum, Astrid; Bruehl, Peter; Schwendinger, Michael G; Savidis-Dacho, Helga; Sullivan, Sean M; Shlapobersky, Mark; Hartikka, Jukka; Rolland, Alain; Barrett, P Noel; Kistner, Otfried

2013-06-01

68

Preclinical evaluation of Vaxfectin®-adjuvanted Vero cell-derived seasonal split and pandemic whole virus influenza vaccines  

PubMed Central

Increasing the potency and supply of seasonal and pandemic influenza vaccines remains an important unmet medical need which may be effectively accomplished with adjuvanted egg- or cell culture-derived vaccines. Vaxfectin®, a cationic lipid-based adjuvant with a favorable safety profile in phase 1 plasmid DNA vaccines trials, was tested in combination with seasonal split, trivalent and pandemic whole virus, monovalent influenza vaccines produced in Vero cell cultures. Comparison of hemagglutination inhibition (HI) antibody titers in Vaxfectin®-adjuvanted to nonadjuvanted vaccinated mice and guinea pigs revealed 3- to 20-fold increases in antibody titers against each of the trivalent influenza virus vaccine strains and 2- to 8-fold increases in antibody titers against the monovalent H5N1 influenza virus vaccine strain. With the vaccine doses tested, comparable antibody responses were induced with formulations that were freshly prepared or refrigerated at conventional 2–8°C storage conditions for up to 6 mo. Comparison of T-cell frequencies measured by interferon-gamma ELISPOT assay between groups revealed increases of between 2- to 10-fold for each of the adjuvanted trivalent strains and up to 22-fold higher with monovalent H5N1 strain. Both trivalent and monovalent vaccines were easy to formulate with Vaxfectin® by simple mixing. These preclinical data support further testing of Vaxfectin®-adjuvanted Vero cell culture vaccines toward clinical studies designed to assess safety and immunogenicity of these vaccines in humans.

Smith, Larry R.; Wodal, Walter; Crowe, Brian A.; Kerschbaum, Astrid; Bruehl, Peter; Schwendinger, Michael G.; Savidis-Dacho, Helga; Sullivan, Sean M.; Shlapobersky, Mark; Hartikka, Jukka; Rolland, Alain; Barrett, P. Noel; Kistner, Otfried

2013-01-01

69

Characterization and detection of Vero cells infected with Herpes Simplex Virus type 1 using Raman spectroscopy and advanced statistical methods.  

PubMed

Herpes viruses are involved in a variety of human disorders. Herpes Simplex Virus type 1 (HSV-1) is the most common among the herpes viruses and is primarily involved in human cutaneous disorders. Although the symptoms of infection by this virus are usually minimal, in some cases HSV-1 might cause serious infections in the eyes and the brain leading to blindness and even death. A drug, acyclovir, is available to counter this virus. The drug is most effective when used during the early stages of the infection, which makes early detection and identification of these viral infections highly important for successful treatment. In the present study we evaluated the potential of Raman spectroscopy as a sensitive, rapid, and reliable method for the detection and identification of HSV-1 viral infections in cell cultures. Using Raman spectroscopy followed by advanced statistical methods enabled us, with sensitivity approaching 100%, to differentiate between a control group of Vero cells and another group of Vero cells that had been infected with HSV-1. Cell sites that were "rich in membrane" gave the best results in the differentiation between the two categories. The major changes were observed in the 1195-1726cm(-1) range of the Raman spectrum. The features in this range are attributed mainly to proteins, lipids, and nucleic acids. PMID:24582780

Salman, A; Shufan, E; Zeiri, L; Huleihel, M

2014-07-01

70

Expression of the newcastle disease virus fusion glycoprotein in vero cells using attenuated Salmonella typhimurium as transgenic carrier.  

PubMed

The full-length cDNA of fusion protein (F) gene of newcastle disease virus (NDV)strain F48E9 was amplified by RT-PCR and inserted into pcDNA3 under the control of human cytomegalovirus (hCMV) immediate early enhancer and promoter. The resulting recombinant plasmid pcDNA3-F was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam(-) and phoP(-)), which was then used to transfect the Vero cells. The DNA and RNA dot blotting revealed that the F gene was transcribed into mRNA in the Vero cells. There was expression of the F protein as shown by indirect immunofluorescent assay. The expression began at 48 h post-infection and increased thereafter, as indicated by ELISA. A 55 kD band of the F protein was identified by SDS-PAGE and Western blotting. These results clearly show that the expressed fusion protein was immuno-reactive with chicken anti-NDV serum. PMID:12098773

Fang, Wei-Huan; Liang, Xue-Ya

2002-07-01

71

Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells.  

PubMed

The aim of the present study was to analyze the influence of virus origin, mammalian or mosquito cell-derived, on antiviral susceptibility of DENV-2 to entry inhibitors and the association of this effect with any alteration in the mode of entry into the cell. To this end, ten serial passages of DENV-2 were performed in mosquito C6/36 cells or monkey Vero cells and the antiviral susceptibility of each virus passage to sulfated polysaccharides (SPs), like heparin and carrageenans, was evaluated by a virus plaque reduction assay. After serial passaging in Vero cells, DENV-2 became increasingly resistant to SP inhibition whereas the antiviral susceptibility was not altered in virus propagated in C6/36 cells. The change in antiviral susceptibility was associated to a differential mode of entry into the host cell. The route of endocytic entry for productive Vero cell infection was altered from a non-classical clathrin independent pathway for C6/36-grown virus to a clathrin-mediated endocytosis when the virus was serially propagated in Vero cells. Our results show the impact of the cellular system used for successive propagation of DENV on the initial interaction between the host cell and the virion in the next round of infection and the relevant consequences it might have during the in vitro evaluation of entry inhibitors. PMID:24583230

Acosta, Eliana G; Piccini, Luana E; Talarico, Laura B; Castilla, Viviana; Damonte, Elsa B

2014-05-12

72

Growth and poliovirus production of Vero cells on a novel microcarrier with artificial cell adhesive protein under serum-free conditions.  

PubMed

A microcarrier is used for the three-dimensional (3D) culture of adhesion-dependent mammalian cells. We developed a novel microcarrier by binding ProNectin F, an artificial cell adhesive protein synthesized by genetically engineered Escherichia coli to a polyacrylic superabsorbent polymer. The microcarrier is characterized by containing no animal-derived components. The serum-free culture of Vero cells for vaccine production using the microcarrier increased the number of Vero cells by approximately 30% compared with the existing dextran beads coated with porcine Type I collagen, which resulted in approximately a 30% to 40% increase in the infectivity titer of the Sabin 2 strain of poliovirus. These results suggested that the developed microcarrier should be unprecedented in permitting high-yield vaccine production by means of a serum-free culture. PMID:21262586

Kurokawa, Masato; Sato, Shigehiro

2011-05-01

73

Differentiation of a Vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3.  

PubMed

A porcine epidemic diarrhea virus (PEDV) designated DR13 was isolated in Vero cells and serially passaged by level 100. The virus was titrated at regular intervals of the passage level. Open reading frame (ORF) 3 sequences of the virus at passage levels 20, 40, 60, 80, and 100 were aligned and compared using a computer software program. Suitability of the restriction fragment length polymorphism (RFLP) analysis for differentiating the virus from other Korean field strains was investigated. The DR13 field isolate was successively adapted in Vero cells as observed through polymerase chain reaction (PCR) and titration of the virus. RFLP analysis identified change in cleavage sites of HindIII and Xho II from passage levels 75 and 90, respectively; these RFLP patterns of ORF 3 differentiated the Vero cell-adapted virus from its parent strain, DR13, and 12 other strains of PEDV studied. The cell adapted DR13 was tested for its pathogenicity and immunogenicity in piglets and pregnant sows. The results indicated that cell adapted DR13 revealed reduced pathogenicity and induced protective immune response in pigs. Differentiation between highly Vero cell-adapted virus and wild-type virus could be the marker of adaptation to cell culture and a valuable tool for epidemiologic studies of PEDV infections. The results of this study supported that the cell attenuated virus could be applied as a marker vaccine candidate against PEDV infection. PMID:12706667

Song, D S; Yang, J S; Oh, J S; Han, J H; Park, B K

2003-05-16

74

Apoptosis induced in an early step of African swine fever virus entry into vero cells does not require virus replication.  

PubMed

Permissive Vero cells develop apoptosis, as characterized by DNA fragmentation, caspases activation, cytosolic release of mitochondrial cytochrome c, and flow cytometric analysis of DNA content, upon infection with African swine fever virus (ASFV). To determine the step in virus replication that triggers apoptosis, we used UV-inactivated virus, inhibitors of protein and nucleic acid synthesis, and lysosomotropic drugs that block virus uncoating. ASFV-induced apoptosis was accompanied by caspase-3 activation, which was detected even in the presence of either cytosine arabinoside or cycloheximide, indicating that viral DNA replication and protein synthesis were not required to activate the apoptotic process. The activation of caspase-3 was released from chloroquine inhibition 2 h after virus absorption, while the infection with UV-inactivated ASFV did not induce the activation of the caspase cascade. We conclude that ASFV induces apoptosis in the infected cell by an intracellular pathway probably triggered during the process of virus uncoating. PMID:12009879

Carrascosa, Angel L; Bustos, María J; Nogal, María L; González de Buitrago, Gonzalo; Revilla, Yolanda

2002-03-15

75

Non-Linear Relationships between Aflatoxin B1 Levels and the Biological Response of Monkey Kidney Vero Cells  

PubMed Central

Aflatoxin-producing fungi contaminate food and feed during pre-harvest, storage and processing periods. Once consumed, aflatoxins (AFs) accumulate in tissues, causing illnesses in animals and humans. Most human exposure to AF seems to be a result of consumption of contaminated plant and animal products. The policy of blending and dilution of grain containing higher levels of aflatoxins with uncontaminated grains for use in animal feed implicitly assumes that the deleterious effects of low levels of the toxins are linearly correlated to concentration. This assumption may not be justified, since it involves extrapolation of these nontoxic levels in feed, which are not of further concern. To develop a better understanding of the significance of low dose effects, in the present study, we developed quantitative methods for the detection of biologically active aflatoxin B1 (AFB1) in Vero cells by two independent assays: the green fluorescent protein (GFP) assay, as a measure of protein synthesis by the cells, and the microculture tetrazolium (MTT) assay, as a measure of cell viability. The results demonstrate a non-linear dose-response relationship at the cellular level. AFB1 at low concentrations has an opposite biological effect to higher doses that inhibit protein synthesis. Additional studies showed that heat does not affect the stability of AFB1 in milk and that the Vero cell model can be used to determine the presence of bioactive AFB1 in spiked beef, lamb and turkey meat. The implication of the results for the cumulative effects of low amounts of AFB1 in numerous foods is discussed.

Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

2013-01-01

76

Non-linear relationships between aflatoxin B? levels and the biological response of monkey kidney vero cells.  

PubMed

Aflatoxin-producing fungi contaminate food and feed during pre-harvest, storage and processing periods. Once consumed, aflatoxins (AFs) accumulate in tissues, causing illnesses in animals and humans. Most human exposure to AF seems to be a result of consumption of contaminated plant and animal products. The policy of blending and dilution of grain containing higher levels of aflatoxins with uncontaminated grains for use in animal feed implicitly assumes that the deleterious effects of low levels of the toxins are linearly correlated to concentration. This assumption may not be justified, since it involves extrapolation of these nontoxic levels in feed, which are not of further concern. To develop a better understanding of the significance of low dose effects, in the present study, we developed quantitative methods for the detection of biologically active aflatoxin B? (AFB1) in Vero cells by two independent assays: the green fluorescent protein (GFP) assay, as a measure of protein synthesis by the cells, and the microculture tetrazolium (MTT) assay, as a measure of cell viability. The results demonstrate a non-linear dose-response relationship at the cellular level. AFB1 at low concentrations has an opposite biological effect to higher doses that inhibit protein synthesis. Additional studies showed that heat does not affect the stability of AFB1 in milk and that the Vero cell model can be used to determine the presence of bioactive AFB1 in spiked beef, lamb and turkey meat. The implication of the results for the cumulative effects of low amounts of AFB1 in numerous foods is discussed. PMID:23949006

Rasooly, Reuven; Hernlem, Bradley; He, Xiaohua; Friedman, Mendel

2013-08-01

77

Comparison of N-Glycan Pattern of Recombinant Human Coagulation Factors II and IX Expressed in Chinese Hamster Ovary (CHO) and African Green Monkey (Vero) Cells.  

PubMed

The N-glycan patterns of recombinant human coagulation factors II (rF-II) and IX (rF-IX), derived from both transfected Chinese hamster ovary (CHO) cells and African green monkay (Vero) cells produced at industrial scale, were analyzed by binding to carbohydrate-specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. Human plasma-derived coagulation factors II (hpF-II) and IX (hpF-IX) exhibited complex-type glycan structures with carbohydrate chains capped with alpha(2-6)-sialic acid. Terminal galactose-beta(1-4)-N-acetylglucosamine units were detected in hpF-IX. Both CHO cell-derived rF-II and rF-IX exhibited complex-type glycosylation and contained alpha(2-3)-sialic acid in addition to terminal galactose-beta(1-4)-N-acetylglucosamine. Vero cell-derived rF-IX exhibited a complex-type glycan structure similar to that of CHO cell-derived rF-IX. In contrast, rF-II produced by Vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "high-mannose" structures and complex-type glycosylation containing alpha(2-3)-sialic acid. Galactose-beta(1-4)-N-acetylglucosamine structures and a low concentration of alpha(2-6)-sialic acid were detected in both microheterogeneity fractions of Vero cell-derived rF-II. Although different in their carbohydrate structures, coagulation factors II and IX obtained recombinantly from both transformed CHO cells and Vero cells exhibited coagulation activities comparable with the plasma-derived proteins. PMID:10608038

Fischer; Mitterer; Dorner; Eibl

1996-01-01

78

A/H5N1 prepandemic influenza vaccine (whole virion, vero cell-derived, inactivated) [Vepacel®].  

PubMed

The influenza A subtype H5N1 virus is a likely causative agent for the next human influenza pandemic. Pandemic influenza vaccine production can begin only after a novel pandemic virus emerges. Cell-based vaccine production has advantages over conventional egg-based methods, allowing more rapid large-scale vaccine production. A reliable Vero cell culture system is available for pandemic and prepandemic influenza vaccine production. Prepandemic influenza vaccines are an important component of influenza pandemic preparedness plans, as their targeted use in the pandemic alert period or early in a pandemic is likely to mitigate the consequences of an influenza outbreak. Vepacel® is a prepandemic influenza vaccine (whole virion, Vero cell-derived, inactivated) containing antigen of H5N1 strain A/Vietnam/1203/2004 and is approved for use in the EU. Clinical immunogenicity studies with the vaccine have demonstrated good rates of functional neutralizing antibody responses against the vaccine strain (A/Vietnam/1203/2004), meeting established immunogenicity criteria for seasonal influenza vaccines, and cross-reactivity against H5N1 strains from other clades. In phase I/II and III studies, a heterologous (A/Indonesia/05/2005) booster vaccine administered to healthy adult and elderly volunteers 6-24 months after the two-dose priming vaccine (A/Vietnam/1203/2004) regimen induced good immunogenic responses against both H5N1 strains, demonstrating strong immunological memory. Broadly similar, albeit less robust, responses were observed in two special risk cohorts of immunocompromised and chronically ill patients. In general, adverse events observed in clinical immunogenicity studies with H5N1 vaccine (A/Vietnam/1203/2004) were similar to those reported with non-adjuvanted, inactivated, seasonal influenza vaccines. PMID:22788239

Plosker, Greg L

2012-07-30

79

Inhibitory effect of oryzacystatins and a truncation mutant on the replication of poliovirus in infected Vero cells.  

PubMed

Poliovirus, a picornavirus family member, requires the processing of its poly-protein by its own cysteine proteinase for replication. Oryzacystatin-I and oryzacystatin-II, proteinaceous cysteine proteinase inhibitors (cystatins) of rice seed origin, were found to inhibit the replication of poliovirus effectively in infected Vero cells in vitro. Truncated oryzacystatin-I, which lacks the NH2-terminal 25 amino acid residues of the intact protein, is an even more effective inhibitor, eliciting its effect at concentrations of less than 0.25 nmol/ml. The low molecular weight cysteine proteinase inhibitors, E-64, E-64C and loxistatin, showed no anti-viral effect at any concentration investigated. PMID:1312033

Kondo, H; Ijiri, S; Abe, K; Maeda, H; Arai, S

1992-03-24

80

A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions.  

PubMed

Rabies virus strain production in Vero cells grown on Cytodex 1 in a 2 L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO+0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific growth rate and cell division number) similar to that obtained in serum containing medium. To design a scalable process that is totally free of animal-derived substances, two proteases: TrypLE Select and Accutase, were assessed as an alternative to trypsin which is routinely used for cell passage. Growth performance of Vero cells grown in VP-SFM and MEM+10% fetal calf serum (FCS) over four passages and subcultivated with either TrypLE Select or Accutase was evaluated. TrypLE Select showed the best performance in terms of specific growth rate and cell division number. Kinetics of cell growth and rabies virus production (LP2061/Vero strain) were investigated in spinner flask and in a 2 L bioreactor. In spinner flask, a maximal cell density level of 1.85x10(6) cells/mL was achieved when the cells were grown in VP-SFM on 2 g/L Cytodex 1. Cell infection experiments conducted at an MOI of 0.3 and without the medium exchange step, typically needed for serum containing rabies virus production, resulted in a maximal virus titer equal to 2x10(7) (Fluorescent Focus Unit) FFU/mL. In stirred tank bioreactor, Vero cell growth in VP-SFM on 3 g/L Cytodex 1 was shown to be sensitive to the aeration mode. Sparging the culture was detrimental for cell growth, whereas cell density level was greatly enhanced when only headspace aeration was used. A cell density level of 2.6x10(6) cells/mL was obtained when the cells were grown on 3g/L Cytodex 1 and in batch culture mode. Cell infection at an MOI of 0.1 without any medium exchange, yielded a maximal rabies virus titer of 2.4x10(7) FFU/mL. Furthermore, Vero cell growth in a 2 L bioreactor using recirculation culture mode during cell proliferation step and perfusion for virus multiplication phase was investigated. In comparison to batch culture, a higher cell density level that was equal to 5x10(6) cells/mL was reached. Cell infection under conditions similar to batch culture, resulted in a maximal virus titer equal to 1.38x10(8) FFU/mL. The potency of the pooled inactivated virus harvests showed an activity of 2.58 IU/mL which was comparable to that obtained in serum supplemented medium. PMID:17307281

Rourou, Samia; van der Ark, Arno; van der Velden, Tiny; Kallel, Héla

2007-05-10

81

Purification of Vero cell derived live replication deficient influenza A and B virus by ion exchange monolith chromatography.  

PubMed

We explored the possibilities for purification of various ?NS1 live, replication deficient influenza viruses on ion exchange methacrylate monoliths. Influenza A ?NS1-H1N1, ?NS1-H3N2, ?NS1-H5N1 and ?NS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. ?NS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the same pH, while ?NS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A ?NS1-H1N1 virus were 1.9E+10 TCID50/ml, 1.0E+10 TCID50/ml and 8.9E+08 TCID50/ml, respectively. Purification of ?NS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious virus on CIM QA were between 70.8±32.3% and 87±30.8%. Total protein removal varied from 93.3±0.4% to 98.6±0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease. PMID:24631091

Banjac, Marko; Roethl, Elisabeth; Gelhart, Franz; Kramberger, Petra; Jarc, Barbara Lah; Jarc, Marko; Strancar, Aleš; Muster, Thomas; Peterka, Matjaž

2014-05-01

82

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System  

Microsoft Academic Search

BackgroundEnterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.Principal FindingIn this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g\\/L Cytodex 1 microcarrier. The viral titer was >106 TCID50\\/mL

Chia-Chyi Liu; Meng-Shin Guo; Fion Hsiao-Yu Lin; Kuang-Nan Hsiao; Kate Hsuen-Wen Chang; Ai-Hsiang Chou; Yu-Chao Wang; Yu-Ching Chen; Chung-Shi Yang; Pele Choi-Sing Chong

2011-01-01

83

The major outer membrane protein rOmpB of spotted fever group rickettsiae functions in the rickettsial adherence to and invasion of Vero cells  

Microsoft Academic Search

The role of one of the major outer membrane proteins, rOmpB, of spotted fever group rickettsiae was examined. Antibodies generated against native rOmpB inhibited plaque formation by Rickettsia japonica in Vero cells when applied at the time of inoculation of the rickettsiae. However, antibodies to heat-denatured rOmpB did not. Moreover, the soluble recombinant rOmpB also inhibited plaque formation to some

Tsuneo Uchiyama; Hiroaki Kawano; Yoshito Kusuhara

2006-01-01

84

A vero cell-derived whole-virus H5N1 vaccine effectively induces neuraminidase-inhibiting antibodies.  

PubMed

A Vero cell-derived whole-virus H5N1 influenza vaccine has been shown to induce neutralizing antibodies directed against the hemagglutinin (HA) protein of diverse H5N1 strains in animal studies and clinical trials. However, neuraminidase-inhibiting (NAi) antibodies can reduce viral spread and may be of particular importance in the event of an H5N1 pandemic, where immunity due to HA antibodies is likely absent in the general population. Here we demonstrate the effective induction of NAi antibody titers after H5N1 vaccination in humans. In contrast to the immune response directed toward HA, a single vaccine dose induced a strong NAi response that was not significantly boosted by a second dose, most probably due to priming by previous vaccination or infection with seasonal influenza viruses. After 2 immunizations, seroconversion rates based on antibody titers against HA and NA were similar, indicating the induction of equally strong immune responses against both proteins by this H5N1 vaccine. PMID:22090447

Fritz, Richard; Sabarth, Nicolas; Kiermayr, Stefan; Hohenadl, Christine; Howard, M Keith; Ilk, Reinhard; Kistner, Otfried; Ehrlich, Hartmut J; Barrett, P Noel; Kreil, Thomas R

2012-01-01

85

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine.  

PubMed

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (?5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm(2) for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations. PMID:24560672

Lackner, Cornelia; Leydold, Sandra M; Modrof, Jens; Farcet, Maria R; Grillberger, Leopold; Schäfer, Birgit; Anderle, Heinz; Kreil, Thomas R

2014-04-11

86

Bovine colostrum ultrafiltrate supplemented with adult bovine serum and transferrin: An effective fbs substitute for cultivation of vero and CHO-K1 cells  

Microsoft Academic Search

Summary  A mixture containing an ultrafiltrate fraction (UF) of bovine colostrum (6.7%), adult bovine serum (BS) (1%), and human holo-transferrin\\u000a (hTF) (5 mg\\/liter) was developed for cultivation of Chinese hamster ovary cells (CHO-K1) and African green monkey kidney cells\\u000a (Vero). The growth-supporting activity of the mixture (UF\\/BS\\/hTF) was comparable to that of 1 to 10% fetal bovine serum (FBS)\\u000a and considerably

Raimo Pakkanen

1994-01-01

87

Induction of indistinguishable gene expression patterns in rats by Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines.  

PubMed

Transcriptomics is an objective index that reflects the overall condition of cells or tissues, and transcriptome technology, such as DNA microarray analysis, is now being introduced for the quality control of medical products. In this study, we applied DNA microarray analysis to evaluate the character of Japanese encephalitis (JE) vaccines. When administered into rat peritoneum, Vero cell-derived and mouse brain-derived JE vaccines induced similar gene expression patterns in liver and brain. Body weights and blood biochemical findings were also similar after administration of the two vaccines. Our results suggest that the two JE vaccines are likely to have equivalent characteristics with regard to reactivity in rats. PMID:20093758

Momose, Haruka; Imai, Jun-ichi; Hamaguchi, Isao; Kawamura, Mika; Mizukami, Takuo; Naito, Seishiro; Masumi, Atsuko; Maeyama, Jun-ichi; Takizawa, Kazuya; Kuramitsu, Madoka; Nomura, Nobuo; Watanabe, Shinya; Yamaguchi, Kazunari

2010-01-01

88

Establishment of cell lines with increased susceptibility to EV71/CA16 by stable overexpression of SCARB2  

PubMed Central

Background Human enterovirus type 71 (EV71) and Coxsackievirus A group type 16 (CA16) belong to human Enterovirus species A of the family Picornaviridae. These viruses are recognized as the major pathogens responsible for epidemics of hand-foot-mouth disease (HFMD), which presents with fever and vesicular eruptions of palms, soles of the feet or mouth. Human scavenger receptor class B, member 2 (SCARB2) has been identified as the receptor for both EV71 and CA16, as overexpression of SCARB2 in cells can enhance virus replication significantly. Methods In this study, we used a lentivirus packaging vector to transduce the SCARB2 gene into human embryonic kidney cells (293), human rhabdomyosarcoma cells (RD) and African green monkey kidney cells (Vero) to create stable expression lines. Expression of SCARB2 in the resulting three transgenic cell lines was confirmed by real-time RT-PCR, immunofluorescence and flow cytometry. Results Levels of SCARB2 mRNA determined by real-time RT-PCR in 293-SCARB2 (293S) or RD-SCARB2 (RDS) transgenic cell lines were approximately 2?×?102 times higher than those in 293 and RD cells, respectively, and three times higher in Vero-SCARB2 (VeroS) than in Vero cells. Furthermore, EV71 and CA16 virus titers in 293S and RDS cells were 102–103-fold higher (detected in RD cell) than those in the parental cells, and a 10-fold higher titer of EV71 was achieved in VeroS cells compared with that in Vero cells. Conclusions We established for the first time three cell lines stably overexpressing SCARB2, which showed drastic increases in susceptibility to EV71/CA16 infection. These optimal cell lines may be utilized to develop inactivated vaccines for EV71/CA16 and facilitate rapid detection and isolation of HFMD pathogens or other Enterovirus serotypes. Furthermore, these stable cell lines also can serve as tools to facilitate drug screenings as well as molecular studies on virus-host interactions and pathogenesis of causative agents for HFMD.

2013-01-01

89

Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E.  

PubMed

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin mainly produced by Fusarium graminaerum, found as a world-wide contaminant mainly of corn and wheat. Previous studies have demonstrated that among several other effects on animals and humans, ZEN also displays hepatotoxicity, immunotoxicity and nephrotoxicity. ZEN is mainly known as a hormonal disrupter due to its estrogenic activities and consequent toxicity for reproduction. Furthermore, mutagenic and genotoxic proprieties of ZEN were disclosed recently, the molecular mechanisms of which are not yet well understood. In the present study, the genotoxic potential of ZEN was evaluated using genotoxicity tests: the 'cytokinesis block micronucleus assay' in Vero monkey kidney cells and the 'in vivo mouse bone marrow micronucleus assay'. In cultured cells treated with 5, 10 and 20 microM ZEN, the frequency of binucleated micronucleated cells (BNMN) was assessed in 1000 binucleated cells and in mice given oral doses of 10, 20 and 40 mg/kg bw, the frequency of polychromatic erythrocytes micronucleated (PCEMN) in bone marrow cells was assessed in 2000 polychromatic erythrocytes (PCE). The potential prevention of ZEN-induced effects by 25 microM Vitamin E (Vit E) was also evaluated. In vivo, doses of 10, 20 and 40 mg/kg bw ZEN representing, respectively 2, 4 and 8% of the LD50 (LD50 of ZEN in mice is 500 mg/kg bw), were administered to animals either with or without pre-treatment with Vit E (216.6 mg/kg bw) in order to evaluate its preventive potential.ZEN was found to induce micronuclei (MN) in a dose-dependent manner in cultured Vero cells as well as in mouse bone marrow cells. The present data emphasise the likely clastogenic pathway among the molecular mechanisms that underlay the ZEN-induced genotoxicity. Vit E was found to prevent partially-from 30 to 50%-these toxic effects, most likely acting either as a structural analogue of ZEN or as an antioxidant. PMID:12834755

Ouanes, Zouhour; Abid, Salwa; Ayed, Imen; Anane, Rachid; Mobio, Théophile; Creppy, Edmond E; Bacha, Hassen

2003-07-01

90

In vitro cytotoxic activity of seed oil of fenugreek against various cancer cell lines.  

PubMed

In the present study, investigations were carried out to screen the anticancer activities of fenugreek seed oil against cancer cell lines (HEp-2, MCF-7, WISH cells), and a normal cell line (Vero cells). Cytotoxicity was assessed with MTT and NRU assays, and cellular morphological alterations were studied using phase contrast light microscopy. All cells were exposed toi 10-1000 ?g/ml of fenugreek seed oil for 24 h. The results show that fenugreek seed oil significantly reduced the cell viability, and altered the cellular morphology in a dose dependent manner. Among the cell lines, HEp-2 cells showed the highest decrease in cell viability, followed by MCF-7, WISH, and Vero cells by MTT and NRU assays. Cell viability at 1000 ?g/ml was recorded as 55% in HEp-2 cells, 67% in MCF-7 cells, 75% in WISH cells, and 86% in Vero cells. The present study provides preliminary screening data for fenugreek seed oil pointing to potent cytotoxicity against cancer cells. PMID:23679282

Al-Oqail, Mai Mohammad; Farshori, Nida Nayyar; Al-Sheddi, Ebtesam Saad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2013-01-01

91

[Use of continuous human and animal cell lines for the production of viral vaccines].  

PubMed

History of development of safety criteria for continuous human and animal cell lines approved for manufacture of immunobiologic preparations. It was noted that current WHO documents recommend mandatory use of respective WHO's reference cell cultures (Vero-10-87 for continuous cell lines, and Wi-38 or MRC-5 for diploid cell lines) during attestation of new cell cultures proposed for the manufacturing of immunobiologic preparations. Examples of practical use of continuous cell lines (CCLs) for production of viral vaccines on industrial scale are described. On the basis of modern data most important principles were formulated which should be considered to provide safety and efficacy of vaccines produced on the CCLs. PMID:18368760

Grachev, V P; Khapchaev, Iu Kh

2008-01-01

92

Comparison of human immune responses to purified Vero cell and human diploid cell rabies vaccines by using two different antibody titration methods.  

PubMed Central

Antibody responses to a conventional rabies preexposure regimen of a new purified Vero cell rabies vaccine (PVRV) and a human diploid cell vaccine (HDCV) were compared in 80 healthy Kenyan veterinary students. Forty-three of the students received the PVRV and 37 received the HDCV on days 0, 7, and 28. Antibody responses were monitored by using the rapid fluorescent-focus inhibition test (RFFIT) and an inhibition enzyme immunoassay (INH EIA) on days 0, 7, 28, and 49. Both vaccines elicited a rapid antibody response. A good correlation between the RFFIT titers and the INH EIA titers was obtained (r = 0.90). Our results also showed that the INH EIA was more reproducible and might therefore be a suitable substitute for the more expensive and less reproducible RFFIT. The geometric mean titers determined by both tests in the two groups of students were statistically similar during the test period. The RFFIT and the INH EIA gave comparable geometric mean titers, which differed significantly only on day 28 in the PVRV group. The effect of the new PVRV is comparable to that of the more expensive HDCV, as determined by the present test systems. The PVRV could therefore be the vaccine of choice, especially in tropical rabies-endemic areas, where the high cost of the HDCV has confined its use to a privileged few.

Kitala, P M; Lindqvist, K J; Koimett, E; Johnson, B K; Chunge, C N; Perrin, P; Olsvik, O

1990-01-01

93

Tick Cell Lines.  

National Technical Information Service (NTIS)

This invention relates to new continuous cell lines from embryonic tissues of ticks (Acari Ixodidae); the use of such cell lines for replicating selected microorganisms; and the use of the replicated microorganisms for diagnosis, prophylaxis and control o...

C. E. Yunker J. C. Cory H. R. Meibos

1981-01-01

94

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture  

PubMed Central

Objective This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. Methods A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. Results The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P < 0.05). The rates of embryonic development after 48 hours (morula?) and 96 hours (blastocyst?) were significantly higher in 20% SSS and 10% SPS than in 20% hFF supplementation (P < 0.05). And the rates of embryonic development after 96 hours (hatching blastocyst?) were significantly higher in 10% SPS (94.5%) than in 20% SSS (82.6%) and 20% hFF supplementation (68.5%). The rates of embryonic development according to storage period of the SF-VCM supplemented with 10% SPS showed no significant difference between control, 2 weeks and 4 weeks group. However developmental rate in 6 weeks storage group was significantly lower than other groups. Conclusion The rate of embryonic development after 96 hours (hatching blastocyst?) was significantly higher in SF-VCM supplemented with 10% SPS. And storage period of media up to 4 weeks did not affect on embryonic development.

Lee, Jong-Seon; Kim, Ju-Hwan; Seo, Young-Seok; Yang, Jung-Bo; Kim, Yong-Il; Kim, Hye-Jin

2013-01-01

95

An animal component free medium that promotes the growth of various animal cell lines for the production of viral vaccines.  

PubMed

IPT-AFM is a proprietary animal component free medium that was developed for rabies virus (strain LP 2061) production in Vero cells. In the present work, we demonstrated the versatility of this medium and its ability to sustain the growth of other cell lines and different virus strains. Here, three models were presented: Vero cells/rabies virus (strain LP 2061), MRC-5 cells/measles virus (strain AIK-C) and BHK-21 cells/rabies virus (strain PV-BHK21). The cell lines were first adapted to grow in IPT-AFM, by progressive reduction of the amount of serum in the culture medium. After their adaptation, BHK-21 cells grew in suspension by forming clumps, whereas MRC-5 cells remained adherent. Then, kinetics of cell growth were studied in agitated cultures for both cell lines. In addition, kinetics of virus replication were investigated. PMID:24583007

Rourou, Samia; Ben Ayed, Yousr; Trabelsi, Khaled; Majoul, Samy; Kallel, Héla

2014-05-19

96

Morphological analysis of the transfer of VSV ts-045 G glycoprotein from the endoplasmic reticulum to the intermediate compartment in vero cells.  

PubMed

Vero cells were infected with the ts-045 strain of vesicular stomatitis virus, and the cells were incubated at 39 degrees C to accumulate the mutant G glycoprotein in the ER as a misfolded aggregate. Cycloheximide was added to the culture medium 3.5 h after infection to prevent further protein synthesis, and the temperature was lowered to 10, 15, or 31 degrees C. At these temperatures, the mutant G glycoprotein correctly folds and oligomerizes. Immunofluorescence light microscopy showed that the G glycoprotein was exported to the Golgi complex at 31 degrees C and to the intermediate compartment (IC) at 15 degrees C, but no export was observed at 10 degrees C. However, incubations at 10 degrees C followed by shift to 15 or 31 degrees C resulted in the normal transfer of the glycoprotein to the IC and the Golgi, respectively. Immunoelectron microscopical analysis confirmed all these results, but showed also that the glycoprotein was frequently clustered in the ER at 10 degrees C. Conventional electron microscopy showed that the morphology of the ER, IC, and Golgi complex remained essentially unchanged at all temperatures. The only significant difference detectable in cells incubated at 10 degrees C was the increased number of partially coated ER protrusions, longer than those detected at higher temperatures. These results demonstrate that the transport toward the Golgi complex of G glycoprotein can be arrested at a step preceding the entry into the IC, thus suggesting that ER and IC are separate stations in the exocytic pathway. PMID:8831570

Lotti, L V; Torrisi, M R; Erra, M C; Bonatti, S

1996-09-15

97

Inhibition of cytotoxicity of Shiga toxin of Escherichia coli O157:H7 on vero cells by Prosopis alba Griseb (Fabaceae) and Ziziphus mistol Griseb (Rhamnaceae) extracts.  

PubMed

The capacity of Prosopis alba Griseb. and Ziziphus mistol Griseb. fruit extracts to inhibit the toxic action of Shiga toxin (Stx) was investigated. Purification of Stx from Escherichia coli O157:H7 was performed by saline precipitation and affinity chromatography using a column with globotriaosylceramide, while the fruits were subjected to ethanolic or aqueous extractions. The protective action of both fruits was determined by pre-, co-, and postincubation of one 50% cytotoxic dose per ml of Stx with different concentrations of ethanolic and aqueous extracts in confluent monolayers of Vero cells for 72 h at 37°C (5% CO2). The inhibition of the cytotoxic effect of Stx by fruit extracts was determined by the neutral red vital staining technique. The extraction of the polyphenols and flavonoids was effective, and more polyphenols per milligram of dissolved solids were obtained from P. alba than from Z. mistol. However, there were more flavonoids in Z. mistol than in P. alba. Components of both fruits increased the viability of cells treated with Stx when the extracts were preincubated with Stx for 1 h before being applied to the cell cultures, with the ethanolic extract of P. alba showing 95% cell viability at a concentration of 2.45 mg/ml. The extracts were less effective in protecting cells when Stx, extracts, and cells were coincubated together without a previous incubation of Stx; only the concentrations of 19.46 mg/ml for the P. alba aqueous extract and 3.75 mg/ml for the Z. mistol ethanolic extract resulted in the inhibition of cytotoxicity, with 52 and 56% cell viability occurring, respectively. Investigation into this difference in the protection of cells indicated that the protein molecule of Stx suffered degradation to advanced oxidative protein products during preincubation with extracts, principally with P. alba, which exhibited a greater amount of nonflavonoid polyphenols than Z. mistol. The prooxidant action on Stx favored the cells and enhanced the protective action of both fruits. PMID:24112573

Pellarín, M G; Albrecht, C; Rojas, M J; Aguilar, J J; Konigheim, B S; Paraje, M G; Albesa, I; Eraso, A J

2013-10-01

98

Safety analysis of a Vero-cell culture derived Japanese encephalitis vaccine, IXIARO (IC51), in 6 months of follow-up.  

PubMed

Japanese encephalitis (JE) is the most common viral encephalitis in Asia. IXIARO is a Vero cell-derived, inactivated JE virus vaccine which has recently been approved in the US, Europe, Canada and Australia (trade name JESPECT). This overview of the safety and tolerability of IXIARO, for 6 months after the first vaccination in 7 Phase III trials, includes: 3558 subjects with at least one IXIARO vaccination, 435 subjects with a JE-VAX (manufactured by BIKEN, distributed by Sanofi Pasteur) vaccination, and 657 with phosphate-buffered saline solution with 0.1% Al(OH)(3) (PBS+Alum) control vaccination. The percentage of subjects reporting solicited local adverse events (AEs) with IXIARO (54%) was similar to PBS+Alum vaccination (56%) as were solicited systemic adverse events (40% IXIARO; 40% PBS+Alum vaccination). JE-VAX showed a higher frequency of subjects with solicited local adverse events (61%) but a slightly lower frequency of subjects with solicited systemic adverse events (36%). The frequency of subjects with any solicited and unsolicited AE with IXIARO (64%) was also similar to PBS+Alum vaccination (61%) and JE-VAX (64%); as for subjects with serious AEs (1% IXIARO; 2% PBS+Alum vaccination, 1% JE-VAX). No serious allergic reactions were observed in any group. This safety analysis indicates that IXIARO has a favorable safety profile, comparable to PBS+Alum control vaccination and appears to have a better local tolerability profile than JE-VAX. PMID:20673824

Dubischar-Kastner, Katrin; Kaltenboeck, Astrid; Klingler, Anton; Jilma, Bernd; Schuller, Elisabeth

2010-09-01

99

The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells.  

PubMed Central

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIII were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4 degrees C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIII is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances. Images

Flynn, S J; Burgett, B L; Stein, D S; Wilkinson, K S; Ryan, P

1993-01-01

100

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System  

PubMed Central

Background Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. Principal Finding In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10?5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). Conclusion These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.

Liu, Chia-Chyi; Guo, Meng-Shin; Lin, Fion Hsiao-Yu; Hsiao, Kuang-Nan; Chang, Kate Hsuen-Wen; Chou, Ai-Hsiang; Wang, Yu-Chao; Chen, Yu-Ching; Yang, Chung-Shi; Chong, Pele Choi-Sing

2011-01-01

101

Impact of Host Cell Line Adaptation on Quasispecies Composition and Glycosylation of Influenza A Virus Hemagglutinin  

PubMed Central

The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried “rescue” mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for “rescue” variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit.

Roedig, Jana Verena; Rapp, Erdmann; Hoper, Dirk; Genzel, Yvonne; Reichl, Udo

2011-01-01

102

Process standardization for optimal virus recovery and removal of substrate DNA and bovine serum proteins in Vero cell-derived rabies vaccine.  

PubMed

Purification of a rabies vaccine by a single zonal centrifugation run was replaced by two runs with optimal standardization of the sucrose density gradient. As a result, significant reductions in the levels of substrate DNA and bovine serum protein in the Vero cell-derived human rabies vaccine were achieved. Following many trials, for the first run, loading of the 3.2-l capacity K-3 rotor with 1800 ml of 60% sucrose solution and 1400 ml of vaccine PBS buffer solution gave a satisfactory linear gradient. However, after the first run, the substrate DNA and bovine serum contents exceeded the required levels. After protamine sulphate and Tween-80 treatment of the concentrated inactivated material, a second run using the same procedure as in the first run was tried. However, these purification procedures resulted in low virus recovery. To achieve optimal virus recovery, and removal of substrate DNA and bovine serum protein, the peak fractions from the first run as indicated by the haemagglutination, sucrose concentration, and optical density values were pooled and the sucrose concentration of the pooled fractions was increased to 60%. A second (flotation) run was then carried out. Using this method, the virus recovery rate was more than 95% that of the first run, and the levels of cellular DNA and bovine serum protein were well within the acceptable limits of less than 100 pg/dose and one part per million, respectively. The substrate DNA was quantified by both radioactive labeling and non-radioactive biotin labeling methods. For the quantification of calf serum protein, a counter-immunoelectrophoresis method was developed and effectively applied. A potency assay was performed using the National Institutes of Health (NIH) and well-standardized in vitro single radial immuno diffusion (SRD) methods. Finally, an immunogenicity study was conducted with human volunteers and the results were confirmed by a rapid fluorescent focus inhibition test (RFFIT). PMID:16233321

Kumar, Ananda Arone Prem; Rao, Yarlagadda Udaya Bhaskara; Joseph, Arokiaswami Leo William; Mani, Kavaratty Raju; Swaminathan, Krishnaswami

2002-01-01

103

Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells.  

PubMed

The cDNA encoding the E and NS1 proteins of the yellow fever virus (YFV) was expressed in Spodoptera frugiperda cells via the recombinant baculovirus Ac-E. NS1 as a gp100 precursor which was cleaved to generate the recombinant proteins E and NS1 similar in size, folding and antigenicity to the authentic ones. Recombinant protein E exhibited immunodominant epitopes as judged by its reactivity with YFV-neutralizing MAbs. Using the Triton X-114 phase separation system, authentic and recombinant E proteins as well as the gp100 precursor exhibited hydrophobic properties similar to those of integral membrane proteins. Recombinant protein E was found neither in the extracellular medium nor on the cell surface, suggesting that it did not migrate within the secretory pathway of insect cells. Analysis of protein NS1 expressed in primate and insect cells revealed that the newly synthesized 48K NS1 glycoprotein was converted to a heat-labile gp72 homo-oligomeric form. This phenomenon did not require the presence of carbohydrate groups. Using the Triton X-114 phase separation system, the oligomeric form of NS1 was shown to be associated with cellular membranes although it appeared less hydrophobic than protein E and gp100. A small fraction of YFV NS1 oligomers were transported throughout the secretory pathway to be shed into the extracellular medium of primate cells. YFV NS1 oligomers migrated from the endoplasmic reticulum to the Golgi complex, whereas their N-oligosaccharides of the high-mannose type are processed to the complex-mannose type. Protein NS1 expressed by recombinant baculovirus-infected insect cells was not found in the extracellular medium but associated with the plasma membrane of the cells. Two recombinant NS1 forms were detected in insect cells: a major one with an apparent Mr of 48K and a minor one of 47K in which N-linked glycans were probably processed to a trimannosyl core without further elongation. Thus, it appears that the transport strategy as well as the N-glycosylation of NS1 in insect cells infected with recombinant baculovirus were different from those of the NS1 in primate cells infected with YFV. PMID:1710649

Desprès, P; Girard, M; Bouloy, M

1991-06-01

104

Cytopathic effects of toxogenic strains of Helicobacter pylori on different cell lines.  

PubMed

Purpose: Many virulence factors are involved in the pathomechanism of infection caused by Helicobacter pylori. Toxins such as vacuolating cytotoxin, encoded by the vacA gene and the immunogenic protein cagA, encoded by the cagA gene (cytotoxin-associated gene) are major factors conferring the property of virulence. The current study is aimed at isolation of H. pylori and separation of its toxin from antral biopsies of patients. Materials and Methods: The following cell lines were used to demonstrate the cytopathic effect (CPE) of the separated toxin: African green monkey kidney (Vero), baby hamster kidney, human lung carcinoma (LLC-MK2), and human epithelial. Results: H. pylori was isolated from 27 out of 45 patients (60%) selected for the study. CPE of H. pylori toxin was highly significant on Vero cells than other cell lines used as it reached a high dilution titer of toxin (1/16) in 13 isolated strains (48.15%). No significant difference in CPE of toxin in different dilutions was detected among other cell lines used in different groups. H. pylori toxin could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis as a distinct band with a molecular weight ranging between 66 and 97 kDa and closely related to 87 kDa. Conclusion: H. pylori vacuolating cytotoxin plays a vital role in the pathogenesis of gastroduodenal diseases (gastritis, gastric ulcer, duodenal ulcer, and gastric cancer). The Vero cell lines were found to be the most suitable form of tissue culture when compared with other cell lines used in our study for demonstrating the activity of H. pylori toxin. PMID:24943747

Gowda, K Lakshmana; Marie, Mohammed Ali M; John, James; Pradeep, C S; Dabwan, Khaled Homoud M; Sangeetha, G

2014-01-01

105

Comparative study on the cytotoxicity of different Myrtaceae essential oils on cultured vero and RC-37 cells.  

PubMed

Medicinally and commercially important essential oils from the family Myrtaceae, i.e. cajuput, clove, kanuka and manuka were phytochemically analysed by GC-MS. Cytotoxicity of these essential oils was evaluated in a standard neutral red assay. Maximum noncytotoxic concentrations for cajuput oil and clove oil were determined at 0.006%, kanuka oil and manuka oil were more cytotoxic with a maximum noncytotoxic concentration of 0.001%. The compounds alpha-pinene, eugenol and leptospermone demonstrated maximum noncytotoxic concentrations at dilutions of 0.001%, 0.003% and 0.001%, respectively. However, the terpene 1,8-cineole was about 100 times less toxic to cultured cells with a maximum noncytotoxic concentration of 0.1% and a TC50 value of 0.44%. Manuka essential oil exhibited high levels of virucidal activity against HSV-1 as well against drug-resistant HSV-1 isolates in viral suspension tests. Determination of cytotoxicity of natural products is an important prerequisite for application in cosmetic and health care products and in antiviral tests. PMID:19069246

Schnitzler, P; Wiesenhofer, K; Reichling, J

2008-11-01

106

The specific activities of Shiga-like toxin type II (SLT-II) and SLT-II-related toxins of enterohemorrhagic Escherichia coli differ when measured by Vero cell cytotoxicity but not by mouse lethality.  

PubMed Central

Characteristically, enterohemorrhagic Escherichia coli (EHEC) strains produce Shiga-like toxin type I (SLT-I), SLT-II, or both of these immunologically distinct cytotoxins. No antigenic or receptor-binding variants of SLT-I have been identified, but a number of SLT-II-related toxins have been described. Because EHEC O91:H21 strain B2F1, which produces two SLT-II-related toxins, is exquisitely virulent in an orally infected, streptomycin-treated mouse model (oral 50% lethal dose [LD50], < 10 organisms), we asked whether the pathogenicity of strain B2F1 was a consequence of SLT-II-related toxin production. For this purpose, we compared the lethality of orally administered E. coli DH5 alpha (Strr) strains that produced different cytotoxic levels of SLT-II, SLT-IIvha (cloned from B2F1), SLT-IIvhb (also cloned from B2F1), or SLT-IIc (cloned from EHEC O157:H7 strain E32511) on Vero cells. We also calculated the specific activities of purified SLT-IIvhb and SLT-II in intraperitoneally injected mice and on Vero cells. The two purified toxins were equally toxic for mice, but SLT-IIvhb was approximately 100-fold less active than SLT-II on Vero cells and bound to the glycolipid receptor Gb3 with lower affinity than did SLT-II. In addition, characterization of SLT-II-related toxin-binding (B) subunit mutants generated in this study revealed that the reduced in vitro cytotoxic levels of the SLT-II-related toxins were due to Asn-16 in the B subunit. Taken together, these findings do not support the idea that B2F1 is uniquely virulent because of the in vivo toxicity of SLT-II-related toxins but do demonstrate differences in in vitro cytotoxic activity among the SLT-II group produced by human EHEC isolates. Images

Lindgren, S W; Samuel, J E; Schmitt, C K; O'Brien, A D

1994-01-01

107

Cell line provenance  

Microsoft Academic Search

Cultured cell lines have become an extremely valuable resource, both in academic research and in industrial biotechnology.\\u000a However, their value is frequently compromised by misidentification and undetected microbial contamination. As detailed elsewhere\\u000a in this volume, the technology, both simple and sophisticated, is available to remedy the problems of misidentification and\\u000a contamination, given the will to apply it. Combined with proper

R. Ian Freshney

2002-01-01

108

A Single Dose of Vero Cell-Derived Japanese Encephalitis (JE) Vaccine (Ixiaro) Effectively Boosts Immunity in Travelers Primed With Mouse Brain-Derived JE Vaccines  

PubMed Central

Background.?A significant part of the world population lives in areas with endemic Japanese encephalitis (JE). For travelers from nonendemic countries, Vero cell–derived vaccine (JE-VC; Ixiaro) has replaced traditional mouse brain–derived vaccines (JE-MB) associated with safety concerns. The 2 vaccines are derived from different viral strains: JE-VC from the SA14-14-2 strain and JE-MB from the Nakayama strain. No data exist regarding whether JE-VC can be used to boost immunity after a primary series of JE-MB; therefore, a primary series of JE-VC has been recommended to all travelers regardless of previous vaccination history. Methods.?One hundred twenty travelers were divided into 4 groups: Volunteers with no prior JE vaccination received primary immunization with (group 1) JE-MB or (group 2) JE-VC, and those primed with JE-MB received a single booster dose of (group 3) JE-MB or (group 4) JE-VC. Immune responses were tested before and 4–8 weeks after vaccination using plaque reduction neutralization test (PRNT) against both vaccine strains. Results.?In vaccine-naive travelers, the vaccination response rate for test strains Nakayama and SA14-14-2 was 100% and 87% after primary vaccination with JE-MB and 87% and 94% after JE-VC, respectively. Antibody levels depended on the target virus, with higher titers against homologous than heterologous PRNT50 target strain (P < .001). In travelers primed with JE-MB, vaccination response rates were 91% and 91%, and 98% and 95% after a booster dose of JE-MB or JE-VC, respectively. Subgroup analysis revealed that a higher proportion of primed (98%/95%) than nonprimed (39%/42%) volunteers responded to a single dose of JE-VC (P < .001). Conclusions.?A single dose of JE-VC effectively boosted immunity in JE-MB–primed travelers. Current recommendations should be reevaluated. Clinical Trials Registration.?NCT01386827.

Erra, Elina O.; Askling, Helena Hervius; Rombo, Lars; Riutta, Jukka; Vene, Sirkka; Yoksan, Sutee; Lindquist, Lars; Pakkanen, Sari H.; Huhtamo, Eili; Vapalahti, Olli; Kantele, Anu

2012-01-01

109

Establishment and characterization of a new Aedes aegypti (L.) (Diptera: Culicidae) cell line with special emphasis on virus susceptibility.  

PubMed

A new cell line from the neonate larvae of Aedes aegypti (L) mosquito was established and characterized. The cell line at the 50th passage (P) level consisted of three prominent cell types, i.e., epithelial-like cells (92%), fibroblast-like cells (7%), and giant cells ( approximately 1%). Karyological analysis showed diploid (2n = 6) number of chromosomes in >75% cells at P-50. The growth kinetics studied at 52nd passage level showed approximately tenfold increase in cell number over a 10-d study period. The species specificity studies using DNA amplification fingerprinting profile analysis using RAPD primers demonstrated 100% homology with the host profile showing the integrity of the cell line. Electron microscopy revealed the absence of mycoplasma or other adventitious agents. The cell line supported the multiplication of seven arboviruses, i.e., Chikungunya (CHIK), Japanese encephalitis, West Nile, dengue 2 (DEN-2), Chandipura, vesicular stomatitis, and Chittoor viruses. The cell line did not replicate Ganjam and Kaisodi viruses. CHIK virus yield in the new cell line was approximately 3log and 0.5log 50% tissue culture infective dose (TCID(50))/mL higher than Vero E6 and C6/36 cell lines, respectively. In the case of DEN-2 virus, it yielded 1log TCID(50)/mL higher than Vero E6, but lesser than C6/36 cell line. Due to its high susceptibility to a broad spectrum of viruses, the new cell line may find application in virus isolation during epidemics and in antigen production. PMID:19533252

Sudeep, A B; Parashar, Deepti; Jadi, Ramesh S; Basu, Atanu; Mokashi, Chetan; Arankalle, Vidya A; Mishra, Akhilesh C

2009-10-01

110

Plaque Assay of Rickettsiae in a Mammalian Cell Line  

PubMed Central

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N?-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells. Images

Cory, J.; Yunker, C. E.; Ormsbee, R. A.; Peacock, M.; Meibos, H.; Tallent, G.

1974-01-01

111

Plaque assay of rickettsiae in a mammalian cell line.  

PubMed

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells. PMID:4208640

Cory, J; Yunker, C E; Ormsbee, R A; Peacock, M; Meibos, H; Tallent, G

1974-06-01

112

Safety and Immunogenicity of Inactivated, Vero Cell Culture-Derived Whole Virus Influenza A/H5N1 Vaccine Given Alone or with Aluminum Hydroxide Adjuvant in Healthy Adults  

PubMed Central

Dosage-sparing strategies, adjuvants and alternative substrates for vaccine production are being explored for influenza vaccine development. We assessed the safety and immunogenicity of a Vero cell culture-grown inactivated whole virus influenza A/H5N1 vaccine with or without aluminum hydroxide adjuvant [Al(OH)3] in healthy young adults. Vaccines were well tolerated, but injection site discomfort was more frequent in groups receiving Al(OH)3. Dose-related increases in serum antibody levels were observed. Neutralizing antibody titers varied significantly when tested by two different laboratories. Al(OH)3 did not enhance HAI or neutralizing antibody responses, and contributed to increased injection site pain. Because influenza antibody titers vary significantly between different laboratories, international standardization of assays is warranted.

Keitel, Wendy A.; Dekker, Cornelia L.; Mink, ChrisAnna; Campbell, James D.; Edwards, Kathryn M.; Patel, Shital M.; Ho, Dora Y.; Talbot, Helen K.; Guo, Kuo; Noah, Diana L.; Hill, Heather

2011-01-01

113

Vero cell-derived inactivated West Nile (WN) vaccine induces protective immunity against lethal WN virus infection in mice and shows a facilitated neutralizing antibody response in mice previously immunized with Japanese encephalitis vaccine.  

PubMed

A novel Vero cell-derived inactivated WN vaccine (WN-VAX) was prepared from virus strain NY99-35262. Two immunizations with WN-VAX induced high levels of neutralizing antibody to WN virus. All immunized mice were protected against challenge with a lethal dose of WN virus. No WN viremia was detected, and the level of WN virus-neutralizing antibody increased rapidly. WN-VAX was then examined for immunogenicity in mice previously immunized with Japanese encephalitis vaccine (JE-VAX). Immunization with WN-VAX induced WN virus-neutralizing antibody in all mice previously immunized with JE-VAX but in only half of the control mice at 10 weeks. These results indicate that WN-VAX induced complete protective immunity against lethal WN infection and that the WN-VAX-induced antibody response is facilitated in JE-VAX-immunized mice. This WN-VAX is thus a candidate WN vaccine for humans. PMID:18221765

Lim, Chang-Kweng; Takasaki, Tomohiko; Kotaki, Akira; Kurane, Ichiro

2008-04-25

114

Long-term immunogenicity of the new Vero cell-derived, inactivated Japanese encephalitis virus vaccine IC51 Six and 12 month results of a multicenter follow-up phase 3 study.  

PubMed

Japanese encephalitis (JE) is the most common viral encephalitis in Asia. IC51 is a new Vero cell-derived, inactivated JE virus vaccine with non-inferior immunogenicity (after 2 months) compared to the US-licensed vaccine JE-VAX (mouse brain-derived, inactivated) and with a more convenient (two injections instead of three) intramuscular dose schedule. Adult subjects from two studies were followed-up for comparative immunogenicity (JE-VAX) at 6 months and long-term immunogenicity of IC51 alone at 12 months. At 6 months, immunogenicity was higher with IC51 (seroconversion rate [SCR] 95%; geometric mean titer [GMT] 84) than with JE-VAX (SCR 74%; GMT 34). At 12 months, the SCR was 83% and the GMT (41) remained above the protective titer of 1:10. Most people immunized with IC51 will have protective neutralizing antibody levels for at least a year. PMID:18599165

Schuller, E; Jilma, B; Voicu, V; Golor, G; Kollaritsch, H; Kaltenböck, A; Klade, C; Tauber, E

2008-08-12

115

Quantitative Proteomics Using Stable Isotope Labeling with Amino Acids in Cell Culture Reveals Changes in the Cytoplasmic, Nuclear, and Nucleolar Proteomes in Vero Cells Infected with the Coronavirus Infectious Bronchitis Virus*  

PubMed Central

Virus-host interactions involve complex interplay between viral and host factors, rendering them an ideal target for proteomic analysis. Here we detail a high throughput quantitative proteomics analysis of Vero cells infected with the coronavirus infectious bronchitis virus (IBV), a positive strand RNA virus that replicates in the cytoplasm. Stable isotope labeling with amino acids in cell culture (SILAC) was used in conjunction with LC-MS/MS to identify and quantify 1830 cellular and two viral proteins from IBV-infected cells. Fractionation of cells into cytoplasmic, nuclear, and nucleolar extracts was used to reduce sample complexity and provide information on the trafficking of proteins between the different compartments. Each fraction showed a proportion of proteins exhibiting ?2-fold changes in abundance. Ingenuity Pathway Analysis revealed that proteins that changed in response to infection could be grouped into different functional categories. These included proteins regulated by NF-?B- and AP-1-dependent pathways and proteins involved in the cytoskeleton and molecular motors. A luciferase-based reporter gene assay was used to validate the up-regulation of AP-1- and NF-?B-dependent transcription in IBV-infected cells and confirmed using immunofluorescence. Immunofluorescence was used to validate changes in the subcellular localization of vimentin and myosin VI in IBV-infected cells. The proteomics analysis also confirmed the presence of the viral nucleocapsid protein as localizing in the cytoplasm, nucleus, and nucleolus and the viral membrane protein in the cytoplasmic fraction. This research is the first application of SILAC to study total host cell proteome changes in response to positive sense RNA virus infection and illustrates the versatility of this technique as applied to infectious disease research.

Emmott, Edward; Rodgers, Mark A.; Macdonald, Andrew; McCrory, Sarah; Ajuh, Paul; Hiscox, Julian A.

2010-01-01

116

Cytotoxicity of municipal solid waste incinerator ash wastes toward mammalian kidney cell lines.  

PubMed

In this study, three municipal solid waste incinerator (MSWI) ash wastes-bottom ash, scrubber residue, and baghouse ash-were extracted using a toxicity characteristic leaching procedure (TCLP) extractant. These so-called final TCLP extracts were applied to African green monkey kidney cells (Vero), baby hamster kidney cells (BHK-21), and pig kidney cells (PK-15), multi-well absorption reader analysis was performed to test how the cytotoxicity of the incineration ashes would affect the digestive systems of animals. Ion-coupled plasma analyses indicated that the baghouse ash extract possessed the highest pH and heavy metal concentration, its cytotoxicity was also the highest. In contrast, the bottom ash and the scrubber residue exhibited very low cytotoxicities. The cytotoxicities of mixtures of baghouse ash and scrubber residue toward the three tested cell lines increased as the relative ratio of the baghouse ash increased, especially for the Vero cells. The slight cytotoxicity of the scrubber residue arose mainly from the presence of Cr species, whereas the high cytotoxicity of the baghouse ash resulted from its high content of heavy metals and alkali ions. In addition, it appears that the dissolved total organic carbon content of these ash wastes can reduce the cytotoxicity of ash wastes that collect in animal cells. PMID:18329068

Huang, Wu-Jang; Tsai, Jia-Lin; Liao, Ming-Huei

2008-05-01

117

DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E  

Microsoft Academic Search

Zearalenone (ZEN) is a non-steroidal oestrogenic mycotoxin produced by several Fusarium species growing on cereals. ZEN and its metabolites bind to human oestrogen receptors and hence display oestrogenic and anabolic properties. Several lines of investigation suggest that ZEN may be genotoxic in vivo. ZEN damages DNA in Bacillus subtilis recombination tests, and it induces sister chromatid exchange and chromosomal aberration

Salwa Abid-Essefi; Isabelle Baudrimont; Wafa Hassen; Zouhour Ouanes; Théophile A. Mobio; Rachid Anane; Edmond E. Creppy; Hassen Bacha

2003-01-01

118

Plant cell lines in cell morphogenesis research.  

PubMed

Plant organs and tissues consist of many various cell types, often in different phases of their development. Such complex structures do not allow direct studies on behavior of individual cells. In contrast, populations of in vitro-cultured plant cells represent valuable tool for studying processes on a single-cell level, including cell morphogenesis. Here we describe characteristics of well-established model tobacco and Arabidopsis cell lines and provide detailed protocol on their cultivation, characterization, and genetic transformation. PMID:24132432

Seifertová, Daniela; Klíma, Petr; Pa?ezová, Markéta; Petrášek, Jan; Zažímalová, Eva; Opatrný, Zden?k

2014-01-01

119

[Development of a novel influenza vaccine derived from a continuous cell line].  

PubMed

Influenza viruses for production are presently produced in embryonated hen"s eggs. This conventional standard methodology is extremely cumbersome; it requires millions of eggs and an extensive purification to reduce the amount of contaminating egg proteins and to minimise the risk of allergies against egg albumin. The shortage of eggs in a pandemic situation, the selection of egg-adapted variants and the presence of adventitious viruses has emphasised the necessity for production of Influenza vaccines on a well characterised stable cell line. Our established serum and protein free Vero cell technology has been successfully adapted to large scale production of a huge variety of Influenza virus strains. The production in 1200 liter fermenter cultures under serum free conditions gave antigen yields comparable to the conventional embryonated egg technology. The development of a rapid and efficient purification scheme resulted in a safe high purity vaccine which was at least as immunogenic as conventional egg-derived vaccines in a mouse model. Clinical trials in the UK, Poland and Austria demonstrated that the Vero cell derived influenza vaccine is well tolerated, safe and highly immunogenic in humans. PMID:11248852

Kistner, O; Barrett, N; Mundt, W; Reiter, M; Schober-Bendixen, S; Eder, G; Dorner, F

2001-01-01

120

Pandemic influenza A H1N1 vaccine in recipients of solid organ transplants: immunogenicity and tolerability outcomes after vero cell derived, non-adjuvanted, whole-virion vaccination.  

PubMed

During the 2009/10 pandemic of influenza A (H1N1), the American Society of Transplantation and other health organizations recommended that immunocompromised patients should be vaccinated as the key preventive measure. Since there are no data available for the immunogenicity of the unadjuvanted pandemic influenza vaccine in immunocompromised patients - as opposed to the adjuvanted preparation - the objective of this study was to evaluate the immunogenicity of an adjuvant-free H1N1 vaccine in recipients of solid organ transplants. Patients were recruited at the Vienna General Hospital, Austria. The vaccination schedule consisted of 2 doses of a whole-virion, vero cell derived, inactivated, non-adjuvanted influenza A/California/07/2009 (H1N1) vaccine given with an interval of 3 weeks. A hemagglutination inhibition (HI) assay on blood samples obtained prior to the first and after each vaccination was used for serologic analysis. The primary immunologic endpoint was the seroconversion rate, defined as the proportion of subjects with an individual 4-fold increase in HI titer of at least 1:40. In addition, virus-specific IgG antibodies to the pandemic H1N1 strain were measured using a commercially available ELISA. Twenty-five organ transplant patients (16 males, 9 females) aged 25-79 years were vaccinated and provided blood samples for serologic analysis. The time elapsed since transplantation was 10 months to 25 years (mean: 9 years; 95% CI 6-13 years). The vaccine was well tolerated and no local adverse events were noticed. After two vaccinations 37% of the patients demonstrated seroconversion in the HI assay as defined above and 70% had virus-specific IgG antibodies. Among the HI vaccine responders were 6 of 14 heart transplant recipients and 1 of 4 liver transplant recipients. The number and type of immunosuppressive agents did not significantly differ in their effect on the immune response. Our results show that the novel vero cell derived and adjuvant-free pandemic A/California/07/2009 (H1N1) influenza vaccine induced limited but measurable immune responses in adult recipients of solid organ transplants. PMID:21803100

Lagler, Heimo; Wenisch, Judith M; Tobudic, Selma; Gualdoni, Guido A; Rödler, Susanne; Rasoul-Rockenschaub, Susanne; Jaksch, Peter; Redlberger-Fritz, Monika; Popow-Kraupp, Theresia; Burgmann, Heinz

2011-09-16

121

Effects of sulfated fucan, ascophyllan, from the brown Alga Ascophyllum nodosum on various cell lines: a comparative study on ascophyllan and fucoidan.  

PubMed

The effects of fucose-containing sulfated polysaccharides, ascophyllan and fucoidan, isolated from the brown alga Ascophyllum nodosum, on the growth of various cell lines (MDCK, Vero, PtK(1), CHO, HeLa, and XC) were investigated. In a colony formation assay, ascophyllan and fucoidan showed potent cytotoxic effects on Vero and XC cells, while other cell lines were relatively resistant to these polysaccharides. Almost no significant effects of these polysaccharides were observed in the cell lines tested using the Alamar blue cytotoxicity assay over 48 h with varying initial cell densities (2500-20,000 cells/well) in growth medium. Interestingly, a significant growth promoting effect of ascophyllan on MDCK cells was observed, whereas treatment with fucoidan showed growth suppressive effects on this cell line under the same experimental conditions. These results suggest that ascophyllan is distinguishable from fucoidan in terms of their bioactivities. This is the first report of the growth promoting effects of a sulfated fucan on a mammalian cell line under normal growth conditions. PMID:20541128

Jiang, Zedong; Okimura, Takasi; Yokose, Takeshi; Yamasaki, Yasuhiro; Yamaguchi, Kenichi; Oda, Tatsuya

2010-07-01

122

Comparison of procedures for the extraction of supernatants and cytotoxicity tests in Vero cells, applied to assess the toxigenic potential of Bacillus spp. and Lactobacillus spp., intended for use as probiotic strains.  

PubMed

Interest in using Bacillus strains as probiotic components of animal feeds has grown in recent years. However, some of these strains, especially those taxonomically related to the Bacillus cereus group, may have enterotoxigenic activity. Assessment of their toxigenic potential by well-established and robust protocols is required before authorizing their use in animal nutrition. Three methods of extraction and concentration of supernatants of Bacillus and Lactobacillus strains (methanol extraction, ammonium sulphate and ultrafiltration concentration) and three cytotoxic tests in Vero cells (WST-1, LDH and protein synthesis inhibition assays) for the assessment of the cytotoxicity activity of Lactobacillus strains (as probiotic strains in human and animal nutrition) and Bacillus toyonensis BCT-7112(T) (as animal probiotic strain in animal nutrition-Toyocerin®-) were evaluated in this study. Methanol extraction was not useful under any circumstances. The other two concentration methods (ammonium sulphate and ultrafiltration) were feasible, with slightly greater sensitivity achieved by ultrafiltration. The probiotic strain B. toyonensis BCT-7112(T) proved to be a non-cytotoxic strain in all the protocols tested. However, some Lactobacillus strains showed cytotoxicity activity, regardless of the protocols applied. PMID:24938520

Blanch, Anicet R; Méndez, Javier; Castel, Susana; Reina, Manuel

2014-08-01

123

Comparison of vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes.  

PubMed

Mosquitoes collected during the epidemic of West Nile virus (WN) in Staten Island, NY, during 2000 were identified to species, grouped into pools of up to 50 individuals, and tested for the presence of WN by using TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) to detect West Nile viral RNA, Vero cell plaque assay to detect infectious virus, and VecTest WNV/SLE Antigen Panel Assay. A total of 10,866 specimens was tested in 801 pools. Analysis of results indicated that TaqMan RT-PCR detected 34 WN-positive pools, more than either of the other techniques. The plaque assay detected 74% of the pools positive by TaqMan, and VecTest detected 60% of the pools positive by TaqMan. The VecTest assay detected evidence of West Nile viral antigen in 67% of the pools that contained live virus detected by plaque assay. A WN enzyme immunoassay performed similarly to the VecTest WN assay. Differences in performance were related to relative sensitivity of the tests. Infection rates of WN in Culex pipiens and Cx. salinarius calculated by the 3 techniques varied, but each estimate indicated a high infection rate in the population. Positive and negative attributes of each procedure, which may influence how and where they are used in surveillance programs, are discussed. PMID:12542186

Nasci, Roger S; Gottfried, Kristy L; Burkhalter, Kristen L; Kulasekera, Varuni L; Lambert, Amy J; Lanciotti, Robert S; Hunt, Ann R; Ryan, Jeffrey R

2002-12-01

124

Morphology and infectivity of virus that persistently caused infection in an AGS cell line.  

PubMed

A recent report has indicated that proteins and genes of simian virus 5 (SV5) are detected in a human gastric adenocarcinoma (AGS) cell line, which is widely provided for oncology, immunology, and microbiology research. However, the production of infective virions has not been determined in this cell line. In this study, the morphology and infectivity of the virus particles of the AGS cell line were studied by light and electron microscopy and virus transmission assay. The virus particles were approximately 176.0 ± 41.1 nm in diameter. The particles possessed projections 8-12 nm long on the surface and contained a nucleocapsid determined to be 13-18 nm in width and less than 1,000 nm in length. The virus was transmissible to the Vero cell line, induced multinuclear giant cell formation, and reproduced the same shape of antigenic virions. In this study, the persistently infected virus in the AGS cell line was determined to be infective and form reproducible virions, and a new morphological feature of SV5 was determined. PMID:22179184

Ooi, Yukimasa; Daikoku, Eriko; Wu, Hong; Aoki, Hiroaki; Morita, Chizuko; Nakano, Takashi; Kohno, Takehiro; Takasaki, Tomohiko; Sano, Kouichi

2011-12-01

125

A Three-Dimensional Comparison of Tick-Borne Flavivirus Infection in Mammalian and Tick Cell Lines  

PubMed Central

Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection.

Offerdahl, Danielle K.; Dorward, David W.; Hansen, Bryan T.; Bloom, Marshall E.

2012-01-01

126

Investigations of porcine circovirus type 1 (PCV1) in vaccine-related and other cell lines.  

PubMed

Porcine circovirus type 1 (PCV1) is highly prevalent in swine and was recently reported in some rotavirus vaccines. Since animal-derived raw materials, such as cells, trypsin, and serum, can be a major source of introducing virus contamination in biological products, we have investigated PCV1 in several cell lines obtained from ATCC that have broad use in research, diagnostics, or vaccine development. It is expected that these cell lines have been exposed to bovine and porcine viruses during their establishment and passage history due to the use of serum and trypsin that was not qualified according to current testing guidances or processed using new virus-inactivation methods. This study showed that Vero, MRC-5, and CEFs, which represent cell substrates used in some U.S. licensed vaccines, and other cell lines used in investigational vaccines, such as MDCK, HEK-293, HeLa, and A549, were negative for PCV1 using a nested PCR assay; some were also confirmed negative by infectivity analysis. However, MDBK cells, which are used for some animal vaccines, contained PCV1 sequences, although no virus was isolated. Although the results showed that PCV infection may not have occurred under previous culture conditions, the recent cases of vaccine contamination emphasizes the need for continued efforts to reduce the likelihood of introducing viruses from animal-derived materials used in product manufacture. PMID:21835219

Ma, Hailun; Shaheduzzaman, Syed; Willliams, Dhanya K; Gao, Yamei; Khan, Arifa S

2011-10-26

127

Safety and Immunogenicity of a Vero Cell Culture-Derived Whole-Virus H5N1 Influenza Vaccine in Chronically Ill and Immunocompromised Patients.  

PubMed

The development of vaccines against H5N1 influenza A viruses is a cornerstone of pandemic preparedness. Clinical trials of H5N1 vaccines have been undertaken in healthy subjects, but studies in risk groups have been lacking. In this study, the immunogenicity and safety of a nonadjuvanted cell culture-derived whole-virus H5N1 vaccine were assessed in chronically ill and immunocompromised adults. Subjects received two priming immunizations with a clade 1 A/Vietnam H5N1 influenza vaccine, and a subset also received a booster immunization with a clade 2.1 A/Indonesia H5N1 vaccine 12 to 24 months later. The antibody responses in the two populations were assessed by virus neutralization and single radial hemolysis assays. The T-cell responses in a subset of immunocompromised patients were assessed by enzyme-linked immunosorbent spot assay (ELISPOT). The priming and the booster vaccinations were safe and well tolerated in the two risk populations, and adverse reactions were predominantly mild and transient. The priming immunizations induced neutralizing antibody titers of ?1:20 against the A/Vietnam strain in 64.2% of the chronically ill and 41.5% of the immunocompromised subjects. After the booster vaccination, neutralizing antibody titers of ?1:20 against the A/Vietnam and A/Indonesia strains were achieved in 77.5% and 70.8%, respectively, of chronically ill subjects and in 71.6% and 67.5%, respectively, of immunocompromised subjects. The T-cell responses against the two H5N1 strains increased significantly over the baseline values. Substantial heterosubtypic T-cell responses were elicited against the 2009 pandemic H1N1 virus and seasonal A(H1N1), A(H3N2), and B subtypes. There was a significant correlation between T-cell responses and neutralizing antibody titers. These data indicate that nonadjuvanted whole-virus cell culture-derived H5N1 influenza vaccines are suitable for immunizing chronically ill and immunocompromised populations. (This study is registered at ClinicalTrials.gov under registration no. NCT00711295.). PMID:24739978

van der Velden, Maikel V W; Geisberger, Alexander; Dvorak, Thomas; Portsmouth, Daniel; Fritz, Richard; Crowe, Brian A; Herr, Wolfgang; Distler, Eva; Wagner, Eva M; Zeitlinger, Markus; Sauermann, Robert; Stephan, Christoph; Ehrlich, Hartmut J; Barrett, P Noel; Aichinger, Gerald

2014-06-01

128

Molluscan cells in culture: primary cell cultures and cell lines  

PubMed Central

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

Yoshino, T. P.; Bickham, U.; Bayne, C. J.

2013-01-01

129

Differential cytotoxic effects of gold nanoparticles in different mammalian cell lines.  

PubMed

Gold nanoparticles (AuNPs) possess unique properties that have been exploited in several medical applications. However, a more comprehensive understanding of the environmental safety of AuNPs is imperative for use of these nanomaterials. Here, we describe the impacts of AuNPs in various mammalian cell models using an automatic and dye-free method for continuous monitoring of cell growth based on the measurement of cell impedance. Several well-established cytotoxicity assays were also used for comparison. AuNPs induced a concentration-dependent decrease in cell growth. This inhibitory effect was associated with apoptosis induction in Vero cells but not in MRC-5 or NIH3T3 cells. Interestingly, cDNA microarray analyses in MRC-5 cells supported the involvement of DNA damage and repair responses, cell-cycle regulation, and oxidative stress in AuNP-induced cytotoxicity and genotoxicity. Moreover, autophagy appeared to play a role in AuNPs-induced attenuation of cell growth in NIH3T3 cells. In this study, we present a comprehensive overview of AuNP-induced cytotoxicity in a variety of mammalian cell lines, comparing several cytotoxicity assays. Collectively, these assays offer convincing evidence of the cytotoxicity of AuNPs and support the value of a systematic approach for analyzing the toxicology of nanoparticles. PMID:24316248

Chueh, Pin Ju; Liang, Ruei-Yue; Lee, Yi-Hui; Zeng, Zih-Ming; Chuang, Show-Mei

2014-01-15

130

Generating Mammalian Stable Cell Lines by Electroporation  

PubMed Central

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr– Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method.

Longo, Patti A.; Kavran, Jennifer M.; Kim, Min-Sung; Leahy, Daniel J.

2014-01-01

131

Leea indica Ethyl Acetate Fraction Induces Growth-Inhibitory Effect in Various Cancer Cell Lines and Apoptosis in Ca Ski Human Cervical Epidermoid Carcinoma Cells  

PubMed Central

The anticancer potential of Leea indica, a Chinese medicinal plant was investigated for the first time. The crude ethanol extract and fractions (ethyl acetate, hexane, and water) of Leea indica were evaluated their cytotoxicity on various cell lines (Ca Ski, MCF 7, MDA-MB-435, KB, HEP G2, WRL 68, and Vero) by MTT assay. Leea indica ethyl acetate fraction (LIEAF) was found showing the greatest cytotoxic effect against Ca Ski cervical cancer cells. Typical apoptotic morphological changes such as DNA fragmentation and chromatin condensation were observed in LIEAF-treated cells. Early signs of apoptosis such as externalization of phosphatidylserine and disruption of mitochondrial membrane potential indicated apoptosis induction. This was further substantiated by dose- and time-dependent accumulation of sub-G1 cells, depletion of intracellular glutathione, and activation of caspase-3. In conclusion, these results suggested that LIEAF inhibited cervical cancer cells growth by inducing apoptosis and could be developed as potential anticancer drugs.

Yau Hsiung, Wong; Abdul Kadir, Habsah

2011-01-01

132

Leea indica Ethyl Acetate Fraction Induces Growth-Inhibitory Effect in Various Cancer Cell Lines and Apoptosis in Ca Ski Human Cervical Epidermoid Carcinoma Cells.  

PubMed

The anticancer potential of Leea indica, a Chinese medicinal plant was investigated for the first time. The crude ethanol extract and fractions (ethyl acetate, hexane, and water) of Leea indica were evaluated their cytotoxicity on various cell lines (Ca Ski, MCF 7, MDA-MB-435, KB, HEP G2, WRL 68, and Vero) by MTT assay. Leea indica ethyl acetate fraction (LIEAF) was found showing the greatest cytotoxic effect against Ca Ski cervical cancer cells. Typical apoptotic morphological changes such as DNA fragmentation and chromatin condensation were observed in LIEAF-treated cells. Early signs of apoptosis such as externalization of phosphatidylserine and disruption of mitochondrial membrane potential indicated apoptosis induction. This was further substantiated by dose- and time-dependent accumulation of sub-G(1) cells, depletion of intracellular glutathione, and activation of caspase-3. In conclusion, these results suggested that LIEAF inhibited cervical cancer cells growth by inducing apoptosis and could be developed as potential anticancer drugs. PMID:21423690

Yau Hsiung, Wong; Abdul Kadir, Habsah

2011-01-01

133

Safety and immunogenicity of two different doses of a Vero cell-derived, whole virus clade 2 H5N1 (A/Indonesia/05/2005) influenza vaccine.  

PubMed

A successful vaccine development strategy for areas with clustered H5N1 events requires conduct of vaccine trials in potentially non-naïve subjects and evaluation of post-vaccination responsiveness. An open-label, randomized, phase I/II study therefore assessed the immunogenicity and safety of two different dose levels of an inactivated, non-adjuvanted, whole virus clade 2.1 (A/Indonesia/05/2005) H5N1 Vero cell-derived influenza vaccine in healthy adults (21-45 years) from a region where the virus has been circulating (Hong Kong) as well as Singapore. Subjects (N=110) were randomized 1:1 to receive two vaccinations with either 3.75 ?g or 7.5 ?g H5N1 haemagglutinin antigen 21 days apart. Safety, immunogenicity (microneutralization [MN] and single radial haemolysis [SRH] at baseline and post-vaccination) and cross-reactivity against a heterologous clade 1 strain (A/Vietnam/1203/2004) of the vaccine were assessed. Pre-existing immunity to the vaccine strain was 14% which is higher than previously reported for these regions. Two vaccinations with either vaccine formulation induced high seroprotection rates (MN titre ? 1:20) against the vaccine strain A/Indonesia/05/2005: 82.7% and 86.5% in the 3.75 ?g and 7.5 ?g dose groups. Seroconversion rates and fold increase exceeded the CPMP criterion of >40% and >2.5 for MN and SRH in both dose groups after the second vaccination, while the seroprotection rate in the 7.5 ?g dose group determined by SRH was only marginally lower (69.2%) than the CPMP criterion of >70%. Thus, 11 of 12 CHMP criteria were fulfilled. A cross-reactive antibody response against the heterologous A/Vietnam/1203/2004 strain was demonstrated after the second vaccination (>21% by MN and ? 25% by SRH). Persistence of antibodies against the vaccine strain was also demonstrated 6 months after the first vaccination, indicating that a booster vaccination would be effective in those who have received two priming doses. No serious adverse events were reported. The H5N1 influenza vaccine against clade 2.1 strain A/Indonesia/05/2005 was well tolerated and immunogenic after two vaccinations, and induced a cross-neutralizing antibody response, with no dose effect. PMID:22080174

Tambyah, Paul A; Wilder-Smith, Annelies; Pavlova, Borislava G; Barrett, P Noel; Oh, Helen M L; Hui, David S; Yuen, Kwok-yung; Fritsch, Sandor; Aichinger, Gerald; Loew-Baselli, Alexandra; van der Velden, Maikel; Maritsch, Friedrich; Kistner, Otfried; Ehrlich, Hartmut J

2012-01-01

134

Anticancer Activity of Certain Herbs and Spices on the Cervical Epithelial Carcinoma (HeLa) Cell Line.  

PubMed

Acetone extracts of selected plant species were evaluated for their in vitro cytotoxicity against a noncancerous African green monkey kidney (Vero) cell line and an adenocarcinoma cervical cancer (HeLa) cell line. The plants studied were Origanum vulgare L. (Oregano), Rosmarinus officinalis L. (Upright and ground cove rosemary), Lavandula spica L. (Lavender), Laurus nobilis L. (Bay leaf), Thymus vulgaris L. (Thyme), Lavandula x intermedia L. (Margaret Roberts Lavender), Petroselinum crispum Mill. (Curly leaved parsley), Foeniculum vulgare Mill. (Fennel), and Capsicum annuum L. (Paprika). Antioxidant activity was determined using a quantitative DPPH (1,1-diphenyl-2-picryl hydrazyl) assay. The rosemary species exhibited effective radical scavenging capacity with 50% inhibitory concentration (IC(50)) of 3.48 ± 0.218??g/mL and 10.84 ± 0.125??g/mL and vitamin C equivalents of 0.351?g and 1.09?g for McConnell's Blue and Tuscan Blue, respectively. Cytotoxicity was measured using XTT (Sodium 3'-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate) colorimetric assay. Only L. nobilis and O. vulgare exhibited pronounced effects on the HeLa cell line. Dose-dependent studies revealed IC(50) of 34.46 ± 0.48??g/mL and 126.3 ± 1.00??g/mL on the HeLa cells and on the Vero cells 124.1??g/mL ± 18.26 and 163.8??g/mL ± 2.95 for L. nobilis and O. vulgare, respectively. Light (eosin and haematoxylin staining) and confocal microscopy (Hoechst 33342, acridine orange, and propidium iodide staining) were used to evaluate the cytotoxic mechanism of action for L. nobilis and O. vulgare. PMID:22649474

Berrington, Danielle; Lall, Namrita

2012-01-01

135

How Embryonic Stem Cell Lines are Made  

NSDL National Science Digital Library

Use of embryonic stem cells in research has been hotly debated for several years. This animation presents the basics on how stem cell lines are established. This animation from Cold Spring Harbor Laboratory's Dolan DNA Learning Center presents how embryonic stem cell lines are made through a series of illustrations of the processes involved.

2012-03-23

136

Differential Response of the Human Renal Proximal Tubular Epithelial Cell Line HK-2 to Shiga Toxin Types 1 and 2 ?  

PubMed Central

Shiga toxins (Stxs) are expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Stx-producing bacteria cause bloody diarrhea with the potential to progress to acute renal failure. Stxs are potent protein synthesis inhibitors and are the primary virulence factors responsible for renal damage that may follow diarrheal disease. We explored the use of the immortalized human proximal tubule epithelial cell line HK-2 as an in vitro model of Stx-induced renal damage. We showed that these cells express abundant membrane Gb3 and are differentially susceptible to the cytotoxic action of Stxs, being more sensitive to Shiga toxin type 1 (Stx1) than to Stx2. At early time points (24 h), HK-2 cells were significantly more sensitive to Stxs than Vero cells; however, by 72 h, Vero cell monolayers were completely destroyed while some HK-2 cells survived toxin challenge, suggesting that a subpopulation of HK-2 cells are relatively toxin resistant. Fluorescently labeled Stx1 B subunits localized to both lysosomal and endoplasmic reticulum (ER) compartments in HK-2 cells, suggesting that differences in intracellular trafficking may play a role in susceptibility to Stx-mediated cytotoxicity. Although proinflammatory cytokines were not upregulated by toxin challenge, Stx2 selectively induced the expression of two chemokines, macrophage inflammatory protein-1? (MIP-1?) and MIP-1?. Stx1 and Stx2 differentially activated components of the ER stress response in HK-2 cells. Finally, we demonstrated significant poly(ADP-ribose) polymerase (PARP) cleavage after exposure to Stx1 or Stx2. However, procaspase 3 cleavage was undetectable, suggesting that HK-2 cells may undergo apoptosis in response to Stxs in a caspase 3-independent manner.

Lentz, Erin K.; Leyva-Illades, Dinorah; Lee, Moo-Seung; Cherla, Rama P.; Tesh, Vernon L.

2011-01-01

137

Molecular Characterization of Putative Chordoma Cell Lines  

PubMed Central

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.

Bruderlein, Silke; Sommer, Joshua B.; Meltzer, Paul S.; Li, Sufeng; Osada, Takuya; Ng, David; Moller, Peter; Alcorta, David A.; Kelley, Michael J.

2010-01-01

138

CellLineMiner: a knowledge portal for human cell lines  

PubMed Central

Experimental models of human tissues and disease phenotypes frequently rely upon immortalized cell lines, which are easily accessible and simple to use due to their infinite capability of cell division. For decades, cell lines have been used to investigate cellular mechanisms of disease and the efficacy of drugs, most prominently for human cancers. However, the large body of knowledge with respect to human cell lines exists primarily in an unstructured fashion, that is, as free text in the scientific literature. Here we present CellLineMiner, a novel text mining-based web database that provides a comprehensive view of human cell line knowledge. The application offers a simple search in all indexed cell lines, accompanied by a rapid display of all identified literature associations. The CellLineMiner is intended to serve as a knowledge resource companion to the cellular model systems used in biomedical research. Availability CellLineMiner is accessible at http://dev.pubgene.com/cellmine

Nakken, Sigve; Johansen, Morten; Fillebeen, Julien; Berge, Ole Petter; Kirker?d, Harald; Jenssen, Tor-Kristian; Hovig, Eivind

2012-01-01

139

Investigation of the uptake of drugs by individual cells using a scanning proton microprobe (SPM).  

PubMed

In this paper we demonstrate the use of micro-PIXE (proton induced X-ray emission) for measuring the quantitative uptake of anti-AIDS drugs, containing metal atoms, by individual Vero cells (African green monkey kidney cell line). Hetero-polytungstates, which are assessed to present an activity against the HIV virus, were studied using Vero cells. It was found that unlike other techniques, SPM offers both the sensitivity and the spatial resolution to carry out these programs of investigations. The use of elemental analysis in single cells of cultured cell lines has shown to have distinct advantages over peripheral blood lymphocytes. PMID:8833668

Cholewa, M; Turnbull, I F; Legge, G J; Weigold, H; Marcuccio, S M; Holan, G; Tomlinson, E; Wright, P J

1996-02-01

140

Human Esophageal Epithelial Cell Lines.  

National Technical Information Service (NTIS)

Human esophageal epithelial cells having replicative capacity in cell culture that is enhanced compared to normal cells and are unable to produce tumors is disclosed. Normal human esophagus tissue from two autopsy specimens was explanted in serum-free med...

G. D. Stoner R. R. Reddel C. C. Harris R. Roger

1989-01-01

141

Renal Cell Carcinoma (ccRCC) Human Cell Line  

Cancer.gov

A renal cell carcinoma (RCC) cell line designated UOK171 has been developed from the resected tumor of a patient diagnosed with stage IV high nuclear grade clear cell type renal cell carcinoma (ccRCC). The UOK171 cell line was immortalized spontaneously by mincing the resected tumor into pieces followed by propagation of the cells over more than twenty generations. One of the most prominent characteristics of this cell line is its intact, nonmutated von Hippel-Lindau (VHL) tumor suppressor gene.

142

Thyroid cancer cell lines: an overview.  

PubMed

Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution. PMID:23162534

Saiselet, Manuel; Floor, Sébastien; Tarabichi, Maxime; Dom, Geneviève; Hébrant, Aline; van Staveren, Wilma C G; Maenhaut, Carine

2012-01-01

143

Thyroid cancer cell lines: an overview  

PubMed Central

Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution.

Saiselet, Manuel; Floor, Sebastien; Tarabichi, Maxime; Dom, Genevieve; Hebrant, Aline; van Staveren, Wilma C. G.; Maenhaut, Carine

2012-01-01

144

Antibacterial effect of theaflavin, polyphenon 60 ( Camellia sinensis) and Euphorbia hirta on Shigella spp. — a cell culture study  

Microsoft Academic Search

Antibacterial effect of compounds extracted from Camellia sinensis L. and the methanol extract of Euphorbia hirta L. were studied against dysentery causing Shigella spp. using the Vero cell line. Cytotoxicity studies of the extracts were performed using the cell line and the non-cytotoxic concentration of the extract was tested for antibacterial activity against the cytopathic dose of the pathogen. These

K. Vijaya; S. Ananthan; R. Nalini

1995-01-01

145

Chromosomes of human hepatoma cell lines  

Microsoft Academic Search

The karyotypes of three human hepatoma cell lines Hep G2, Hep 3B and PLC\\/PRF\\/5 were investigated by G- and C-banding techniques. In addition to ploidy changes, typical for most carcinoma cell lines, certain markers were found that remained stable throughout passage of these cultures. Chromosome I is involved in multiple translocations, resulting in at least three copies of the chromosome

Daniela Simon; David P. Aden; Barbara B. Knowles

1982-01-01

146

Cell-host, LINE and environment  

PubMed Central

Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship.

Del Re, Brunella; Giorgi, Gianfranco

2013-01-01

147

Formation and accumulation of protoporphyrin IX in tumor and nontumor cell lines induced by 5-aminolevulinic acid  

NASA Astrophysics Data System (ADS)

The endogenous photosensitizer 5-aminolevulinic acid (ALA) is a haem precursor and induces the synthesis of protoporphyrin IX (PpIX) in mitochondria-containing cells. Due to the slow conversion of porphyrins to haem, high levels of PPIX are found in the tissues, sufficient to produce a photodynamic effect following exposure to light. Since PpIX accumulates effectively in tumor cells, the use of ALA leads to a better photoselectivity than Photofrin. However, this selectivity has not been sufficiently studied. As far as we know there is just one study comparing the amount of accumulated PpIX in non-tumor and tumor cell lines. In this work we attempt to compare not just the production but also the accumulation and cytotoxicity of PpIX in non-tumor (VERO) versus tumor (Hep-2) cells induced by the use of ALA. The results have shown that both non-tumor and tumor cell lines produce the same amount of PpIX but just the tumor cells can accumulate PpIX. So, under illumination, only the tumor cells will be killed.

Fernandez, Sandra R.; Milanetto, Marilia; Bagnato, Vanderlei S.; Imasato, Hidetake; Perussi, Janice R.

2005-04-01

148

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines  

Microsoft Academic Search

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http:\\/\\/bioinformatics.istge.it\\/hypercldb\\/) is a hyper- text version of CLDB that improves data accessibil- ity by also allowing information retrieval

Paolo Romano; Maria Assunta Manniello; Ottavia Aresu; Massimiliano Armento; Michela Cesaro; Barbara Parodi

2009-01-01

149

Generation of rabbit pluripotent stem cell lines.  

PubMed

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology. PMID:22925641

Tancos, Z; Nemes, C; Polgar, Z; Gocza, E; Daniel, N; Stout, T A E; Maraghechi, P; Pirity, M K; Osteil, P; Tapponnier, Y; Markossian, S; Godet, M; Afanassieff, M; Bosze, Z; Duranthon, V; Savatier, P; Dinnyes, A

2012-11-01

150

Rickettsia prowazekii requires host cell serine and glycine for growth.  

PubMed Central

The growth requirement of Rickettsia prowazekii for the amino acids serine and glycine was assessed in both wild-type cell lines and a mutant cell line. X-irradiated L929 cells supported the growth of R. prowazekii when the cells were incubated in Eagle minimal essential medium supplemented with serum. In contrast, in this medium, X-irradiated Vero cells did not support the growth of rickettsiae unless cycloheximide, serine, or glycine was added. Other nonessential amino acids, additional glucose, and potential products of host cell metabolism of serine and glycine were nonstimulatory. The concentration of serine or glycine required to support rickettsial growth had no effect on the doubling time of uninfected, unirradiated Vero cells. A comparison of intracellular amino acid pools indicated that the serine and glycine concentrations in mock-infected Vero cells were approximately 31 and 14% of the respective concentrations in mock-infected L929 cells. The pools of both amino acids in Vero cells increased markedly upon treatment of the cells with cycloheximide. Interconversion of serine and glycine catalyzed by serine hydroxymethyltransferase was detected in cell-free extracts of purified rickettsiae. However, this enzymatic activity did not permit rickettsial growth in a glycine-requiring clone (772-56d) of the Chinese hamster ovary cell CHO-K1 in the absence of glycine supplementation. These data indicate that R. prowazekii depends on the host cell for serine or glycine.

Austin, F E; Turco, J; Winkler, H H

1987-01-01

151

Molecular cloning of membrane cofactor protein (MCP; CD46) on B95a cell, an Epstein-Barr virus-transformed marmoset B cell line: B95a-MCP is susceptible to infection by the CAM, but not the Nagahata strain of the measles virus.  

PubMed Central

Measles virus (MV) infects not only human beings but also some simian species. The MV receptor on Vero cells (a cell line established from African Green monkey kidney cells) and human cells has been shown to be the membrane cofactor protein MCP/CD46, which is an inhibitor of autologous complement (C) activation. B95a, an Epstein-Barr virus (EBV)-transformed marmoset B cell line, is a simian cell line used for MV selection and is much more susceptible to MV than Vero cells. In the present study, we isolated cDNAs encoding MCP homologues from B95a cDNA library and assessed whether B95a-MCP is responsible for the high susceptibility of B95a to MV. The deduced amino acid sequence of the cDNA of B95a-MCP was 76% identical to that of human-MCP, and the recombinant B95a-MCP exerts C inhibitor activity. Although CAM, a vaccine strain of MV, infected Chinese hamster ovary (CHO) cells expressing B95a-MCP, Nagahata strain, a wild type of MV, failed to infect the CHO transfectants, suggesting that additional membrane molecules of B95a are responsible for the high susceptibility of B95a to the Nagahata strain.

Murakami, Y; Seya, T; Kurita, M; Fukui, A; Ueda, S; Nagasawa, S

1998-01-01

152

Human cell line authentication: the critical first step in any project using human cell lines.  

PubMed

Short tandem repeat (STR) typing is a standard procedure used in many laboratories for the authentication of human cell lines. This technology, which is based on the informativeness of known polymorphism of numerous loci to uniquely identify a human cell line, has allowed for direct-amplification of human DNA stored on FTA(®) paper. We describe an application of this technology to create a unique STR profile by direct amplification of HCT 116 (ATCC(®) CCL-247™) cell line DNA, a cell line commonly used in colon research. The ability to perform direct-amplification of DNA opens up the possibility of using FTA(®) paper as a way to maintain long-term storage of DNA samples from a cell line and other human tissues, such as buccal cells. PMID:23296621

McLaren, Robert S; Reid, Yvonne; Storts, Douglas R

2013-01-01

153

Isolation and identification of a novel chlorophenol from a cell suspension culture of Helichrysum aureonitens.  

PubMed

A novel chlorophenol, 4-chloro-2-(hepta-1,3,5-triyn-1-yl)-phenol (1), was isolated as the major phenolic compound from the cells of Helichrysum aureonitens suspension cultures. Compound 1 has been proposed to be an intermediate in the acetylene biosynthetic pathway of other acetylenic compounds in Helichrysum spp. The ethanol extract of cell suspension cultures and compound 1 were evaluated for their cytotoxicity against monkey kidney Vero (Vero cells) and human prostate epithelial carcinoma (DU145) cell lines, also, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Mycobacterium tuberculosis H37Rv were determined as well. PMID:19881282

Ziaratnia, Seyed Mahdi; Ohyama, Kiyoshi; Hussein, Ahmed Abdel-Fattah; Muranaka, Toshiya; Lall, Namrita; Kunert, Karl Josef; Meyer, Jacobus Johannes Marion

2009-11-01

154

Umbelliprenin Induces Apoptosis in CLL Cell Lines.  

PubMed

Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V-FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

2012-01-01

155

Umbelliprenin Induces Apoptosis in CLL Cell Lines  

PubMed Central

Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate.

Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

2012-01-01

156

CHARACTERIZATION OF A UNIQUE MUSCLE CELL LINE  

PubMed Central

A clonal cell line derived from a mouse neoplasm is described which shares many properties with smooth muscle. The cells have electrically excitable membranes capable of generating overshooting action potentials, and they contract both spontaneously and with electrical stimulation. They respond to the iontophoretic application of acetylcholine with a depolarizing response, and to norepinephrine with a hyperpolarizing response. Electron microscopy reveals that the cells have a morphology similar in many, but not all, respects to that of smooth muscle cells in vivo. The cells secrete soluble collagen-like molecules in addition to several proteins of undefined function. Finally, there is an increase in the specific activities of creatine phosphokinase and myokinase associated with increased cell density and the cessation of cell division.

Schubert, David; Harris, A. John; Devine, Carrick E.; Heinemann, Stephen

1974-01-01

157

Catecholamines in a macrophage cell line  

Microsoft Academic Search

This study provides the first evidence for catecholamine synthesis and release in the RAW264.7 cell line, an important macrophage model. Although catecholamines were low in unstimulated cells, activation with lipopolysaccharide (LPS) induced tyrosine hydroxylase (TH) mRNA and increased extracellular norepinephrine and intracellular dopamine within 48 h. The catecholamine synthesis inhibitor ?-methyl-para-tyrosine (?-mpt) decreased extracellular norepinephrine levels, suggesting release and rapid

Scott W Brown; Randall T Meyers; Karen M Brennan; Julie M Rumble; Nedathur Narasimhachari; Edmund F Perozzi; John J Ryan; Jennifer K Stewart; Krista Fischer-Stenger

2003-01-01

158

Radiation sensitivity of Merkel cell carcinoma cell lines  

SciTech Connect

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others] [Queensland Institute of Medical Research (Australia); and others

1995-07-30

159

Granulocyte growth modulators elaborated by human cell lines  

Microsoft Academic Search

Two human monocyte-like cell lines have been established which produce colony stimulating activity (CSA) and colony inhibitory activity (CIA) for man and other species. Cell line CSA has been partially characterized. Cell line CIA, a low molecular weight hydrophobic molecule has been partially purified and shown to inhibit not only CFU-C growth but the growth of various lymphoid cell lines.

J. F. DiPersio; J. K. Brennan; M. A. Lichtman

1978-01-01

160

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines  

SciTech Connect

Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany) [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany)] [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany)] [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany)] [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)] [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)

2011-04-01

161

MHC class I bound peptides of a colon carcinoma cell line, a Ki-ras gene-targeted progeny cell line and a B cell line  

Microsoft Academic Search

MHC class I associated peptides on cancer cells represent potential targets for CD8+ cytotoxic T cell activity against tumor cells. We eluted the naturally bound MHC class I peptides of a colon carcinoma cell line and compared them to peptides isolated from a B cell line and a slow-growing activated Ki-ras-disrupted colon cancer cell line. While we failed to detect

Christopher J Savoie; Nobuhiro Kamikawaji; Tohru Sudo; Masanori Furuse; Senji Shirasawa; Takeshi Tana; Takehiko Sasazuki

1998-01-01

162

Investigating citrullinated proteins in tumour cell lines  

PubMed Central

Background The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. Studies have found PADI4, an enzyme performing citrullination, to be highly expressed in a variety of malignant tumours and have shown that PADI4 participates in the process of tumorigenesis. However, as citrullinated proteins have not been systematically investigated in tumours, the present study aimed to identify novel citrullinated proteins in tumours by 2-D western blotting (2-D WB). Methods Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 tumour cell lines. The expression profiles on a 2-DE gel were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in tumour cell lines. Results 2-D WB and mass spectrometry identified citrullinated ?-enolase (ENO1), heat shock protein 60 (HSP60), keratin 8 (KRT8), tubulin beta (TUBB), T cell receptor chain and vimentin in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumour cell lines. Conclusions The citrullination of these proteins suggests a new mechanism in the tumorigenic process.

2013-01-01

163

Anticancer effect of the extracts from Polyalthia evecta against human hepatoma cell line (HepG2)  

PubMed Central

Objective To investigate the anticancer activity of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep against human hepatoma cell line (HepG2). Methods The anticancer activity was based on (a) the cytotoxicity against human hepatoma cells (HepG2) assessed using a neutral red assay and (b) apoptosis induction determined by evaluation of nuclei morphological changes after DAPI staining. Preliminary phytochemical analysis of the crude extract was assessed by HPLC analysis. Results The 50% ethanol-water crude leaf extract of P. evecta (EW-L) showed greater potential anticancer activity with high cytotoxicity [IC50 = (62.8 ± 7.3)µg/mL] and higher selectivity in HepG2 cells than normal Vero cells [selective index (SI) = 7.9]. The SI of EW-L was higher than the positive control, melphalan (SI = 1.6) and the apoptotic cells (46.4 ± 2.6) % induced by EW-L was higher than the melphalan (41.6 ± 2.1)% (P<0.05). The HPLC chromatogram of the EW-L revealed the presence of various kinds of polyphenolics and flavonoids in it. Conclusions P. evecta is a potential plant with anticancer activity. The isolation of pure compounds and determination of the bioactivity of individual compounds will be further performed.

Machana, Sasipawan; Weerapreeyakul, Natthida; Barusrux, Sahapat

2012-01-01

164

In vitro anticancer effect of venom from Cuban scorpion Rhopalurus junceus against a panel of human cancer cell lines.  

PubMed

In Cuba the endemic species of scorpion Rhopalurus junceus has been used in traditional medicine for cancer treatment. However, there is little scientific evidence about its potential in cancer therapy. The effect of a range of scorpion venom concentrations (0.1, 0.25, 0.5, 0.75 and 1mg/ml) against a panel of human tumor cell lines from epithelial (Hela, SiHa, Hep-2, NCI-H292, A549, MDA-MB-231, MDA-MB-468, HT-29), hematopoietic origins (U937, K562, Raji) and normal cells (MRC-5, MDCK, Vero) was determined by the MTT assay. Additionally, the effect of venom on tumor cell death was assayed by Fluorescence microscopy, RT-PCR and western blot. Only the epithelial cancer cells showed significant cell viability reduction, with medium cytotoxic concentration (IC50) ranging from 0.6-1mg/ml, in a concentration-dependent manner. There was no effect on either normal or hematopoietic tumor cells. Scorpion venom demonstrated to induce apoptosis in less sensitive tumor cells (Hela) as evidenced by chromatin condensation, over expression of p53 and bax mRNA, down expression of bcl-2 mRNA and increase of activated caspases 3, 8, 9. In most sensitive tumor cells (A549), scorpion venom induced necrosis evidenced by acridine orange/ethidium bromide fluorescent dyes and down-expression of apoptosis-related genes. We concluded the scorpion venom from R. junceus possessed a selective and differential toxicity against epithelial cancer cells. This is the first report related to biological effect of R. junceus venom against a panel of tumor cells lines. All these results make R. junceus venom as a promise natural product for cancer treatment. PMID:23946884

Díaz-García, Alexis; Morier-Díaz, Luis; Frión-Herrera, Yahima; Rodríguez-Sánchez, Hermis; Caballero-Lorenzo, Yamira; Mendoza-Llanes, Dianeya; Riquenes-Garlobo, Yanelis; Fraga-Castro, José A

2013-01-01

165

Establishment of a follicular lymphoma cell line (FLK-1) dependent on follicular dendritic cell-like cell line HK  

Microsoft Academic Search

A novel cell line, FLK-1, was established from bone marrow cells of a patient with follicular lymphoma by means of co-culture with follicular dendritic cell (FDC)-like cell line HK. Immunophenotypic analysis showed that FLK-1 expressed CD10, CD19, CD20, CD38, IgG and HLA-DR, which is a typical feature of germinal center B cells. Cytogenetic analysis of FLK-1 demonstrated t(14;18)(q32;q21) translocation involving

Y Kagami; J Jung; YS Choi; K Osumi; S Nakamura; Y Morishima; M Seto

2001-01-01

166

Colonic myofibroblast cell line stimulates colonoid formation.  

PubMed

A stable and efficient system for the culture of murine colon epithelial cells or crypts is required to facilitate studies of the dynamics and factors affecting colon stem cell niche and crypt formation. Survival of colonic epithelial cells or crypts in vitro was not established until recently, when it was found that exogenous Wnt3A and R-spondin could promote cell survival and formation of spheroids (colonospheres) or some advanced organoids with well-developed crypts (colonoids). However, after 6-8 days in these culture conditions, only small numbers of colonospheres form organoids with crypt-like structures (colonoids). This study describes the use of a myofibroblast cell line and a coculture system that increases the efficiency of colonoid formation from isolated crypts. The enhanced coculture system has significantly improved colonoid-forming efficiency compared with results from previous systems. Crypt formation can be detected as early as day 2. The coculture system will facilitate the characterization of the colon stem cell niche and the changes that occur as a result of perturbations or mutations in colon stem or epithelial cells, such as those that favor precancerous adenoma or cancer. PMID:24481605

Hirokawa, Yumiko; Yip, Kelvin Hon Yan; Tan, Chin Wee; Burgess, Antony W

2014-04-01

167

Cell lines that support replication of a novel herpes simplex virus 1 U{sub L}31 deletion mutant can properly target U{sub L}34 protein to the nuclear rim in the absence of U{sub L}31  

SciTech Connect

Previous results indicated that the herpes simplex virus 1 (HSV-1) U{sub L}31 gene is necessary and sufficient for localization of the U{sub L}34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U{sub L}31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U{sub L}31 gene. The replication of the U{sub L}31 deletion virus was restored on U{sub L}31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U{sub L}34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U{sub L}34 protein localized at the nuclear membrane in rabbit skin cells, and U{sub L}31 complementing CV1 cells infected with the U{sub L}31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U{sub L}34 protein to the nuclear membrane. We speculate that this function partially complements that of U{sub L}31 and may explain why U{sub L}31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.

Liang Li [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (United States); Tanaka, Michiko [Department of Virology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Kawaguchi, Yasushi [Department of Virology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Baines, Joel D. [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (United States)]. E-mail: jdb11@cornell.edu

2004-11-10

168

Establishment of stable cell lines of Drosophila germ-line stem cells  

PubMed Central

Each Drosophila ovariole has three independent sets of stem cells: germ-line stem cells (GSCs) and escort stem cells, located at the anterior tip of the germarium, and somatic stem cells (SSCs), located adjacent to the newly formed 16-cell cysts. Decapentaplegic (Dpp) is required to maintain the anterior stem cells, whereas Hedgehog is required for maintenance and cell division of the SCCs. In an effort to establish a new in vitro system to analyze intrinsic and extrinsic factors regulating the division and differentiation of GSCs of Drosophila, we tested various culture conditions for growing GSCs, derived from bag of marbles (bam) mutant ovaries. We have shown that bam? GSCs can be maintained and promoted to divide in vitro in media containing Dpp. These cells retain the morphological features of GSCs, i.e., expression of Vasa and Nanos and spectrosomes, even after several months of culture. Somatic cells are induced to grow in culture by the presence of sonic Hedgehog. The somatic cells produce Dpp. GSCs associate with the somatic cells via DE-cadherin, features that are also prominent at the niche of a normal germarium. Finally, we have established stable cell cultures consisting of GSCs and sheets of somatic cells, which are dependent on the addition of fly extract. A somatic cell line, lacking GSCs, has also been established. These cells are thought to be descendants of SCCs. Our in vitro system may provide the opportunity to manipulate GSCs genetically and to analyze the interaction of germ-line stem cells and soma.

Niki, Yuzo; Yamaguchi, Takafumi; Mahowald, Anthony P.

2006-01-01

169

Establishment of stable cell lines of Drosophila germ-line stem cells.  

PubMed

Each Drosophila ovariole has three independent sets of stem cells: germ-line stem cells (GSCs) and escort stem cells, located at the anterior tip of the germarium, and somatic stem cells (SSCs), located adjacent to the newly formed 16-cell cysts. Decapentaplegic (Dpp) is required to maintain the anterior stem cells, whereas Hedgehog is required for maintenance and cell division of the SCCs. In an effort to establish a new in vitro system to analyze intrinsic and extrinsic factors regulating the division and differentiation of GSCs of Drosophila, we tested various culture conditions for growing GSCs, derived from bag of marbles (bam) mutant ovaries. We have shown that bam(-) GSCs can be maintained and promoted to divide in vitro in media containing Dpp. These cells retain the morphological features of GSCs, i.e., expression of Vasa and Nanos and spectrosomes, even after several months of culture. Somatic cells are induced to grow in culture by the presence of sonic Hedgehog. The somatic cells produce Dpp. GSCs associate with the somatic cells via DE-cadherin, features that are also prominent at the niche of a normal germarium. Finally, we have established stable cell cultures consisting of GSCs and sheets of somatic cells, which are dependent on the addition of fly extract. A somatic cell line, lacking GSCs, has also been established. These cells are thought to be descendants of SCCs. Our in vitro system may provide the opportunity to manipulate GSCs genetically and to analyze the interaction of germ-line stem cells and soma. PMID:17056713

Niki, Yuzo; Yamaguchi, Takafumi; Mahowald, Anthony P

2006-10-31

170

Cytosine methylation profiling of cancer cell lines.  

PubMed

DNA-methylation changes in human cancer are complex and vary between the different types of cancer. Capturing this epigenetic variability in an atlas of DNA-methylation changes will be beneficial for basic research as well as translational medicine. Hypothesis-free approaches that interrogate methylation patterns genome-wide have already generated promising results. However, these methods are still limited by their quantitative accuracy and the number of CpG sites that can be assessed individually. Here, we use a unique approach to measure quantitative methylation patterns in a set of >400 candidate genes. In this high-resolution study, we employed a cell-line model consisting of 59 cancer cell lines provided by the National Cancer Institute and six healthy control tissues for discovery of methylation differences in cancer-related genes. To assess the effect of cell culturing, we validated the results from colon cancer cell lines by using clinical colon cancer specimens. Our results show that a large proportion of genes (78 of 400 genes) are epigenetically altered in cancer. Although most genes show methylation changes in only one tumor type (35 genes), we also found a set of genes that changed in many different forms of cancer (seven genes). This dataset can easily be expanded to develop a more comprehensive and ultimately complete map of quantitative methylation changes. Our methylation data also provide an ideal starting point for further translational research where the results can be combined with existing large-scale datasets to develop an approach that integrates epigenetic, transcriptional, and mutational findings. PMID:18353987

Ehrich, Mathias; Turner, Julia; Gibbs, Peter; Lipton, Lara; Giovanneti, Mara; Cantor, Charles; van den Boom, Dirk

2008-03-25

171

Cytosine methylation profiling of cancer cell lines  

PubMed Central

DNA-methylation changes in human cancer are complex and vary between the different types of cancer. Capturing this epigenetic variability in an atlas of DNA-methylation changes will be beneficial for basic research as well as translational medicine. Hypothesis-free approaches that interrogate methylation patterns genome-wide have already generated promising results. However, these methods are still limited by their quantitative accuracy and the number of CpG sites that can be assessed individually. Here, we use a unique approach to measure quantitative methylation patterns in a set of >400 candidate genes. In this high-resolution study, we employed a cell-line model consisting of 59 cancer cell lines provided by the National Cancer Institute and six healthy control tissues for discovery of methylation differences in cancer-related genes. To assess the effect of cell culturing, we validated the results from colon cancer cell lines by using clinical colon cancer specimens. Our results show that a large proportion of genes (78 of 400 genes) are epigenetically altered in cancer. Although most genes show methylation changes in only one tumor type (35 genes), we also found a set of genes that changed in many different forms of cancer (seven genes). This dataset can easily be expanded to develop a more comprehensive and ultimately complete map of quantitative methylation changes. Our methylation data also provide an ideal starting point for further translational research where the results can be combined with existing large-scale datasets to develop an approach that integrates epigenetic, transcriptional, and mutational findings.

Ehrich, Mathias; Turner, Julia; Gibbs, Peter; Lipton, Lara; Giovanneti, Mara; Cantor, Charles; van den Boom, Dirk

2008-01-01

172

Commissioning and initial stereotactic ablative radiotherapy experience with Vero.  

PubMed

The purpose of this study is to describe the comprehensive commissioning process and initial clinical performance of the Vero linear accelerator, a new radiotherapy device recently installed at UT Southwestern Medical Center specifically developed for delivery of image-guided stereotactic ablative radiotherapy (SABR). The Vero system utilizes a ring gantry to integrate a beam delivery platform with image guidance systems. The ring is capable of rotating ± 60° about the vertical axis to facilitate noncoplanar beam arrangements ideal for SABR delivery. The beam delivery platform consists of a 6 MV C-band linac with a 60 leaf MLC projecting a maximum field size of 15 × 15 cm² at isocenter. The Vero planning and delivery systems support a range of treatment techniques, including fixed beam conformal, dynamic conformal arcs, fixed gantry IMRT in either SMLC (step-and-shoot) or DMLC (dynamic) delivery, and hybrid arcs, which combines dynamic conformal arcs and fixed beam IMRT delivery. The accelerator and treatment head are mounted on a gimbal mechanism that allows the linac and MLC to pivot in two dimensions for tumor tracking. Two orthogonal kV imaging subsystems built into the ring facilitate both stereoscopic and volumetric (CBCT) image guidance. The system is also equipped with an always-active electronic portal imaging device (EPID). We present our commissioning process and initial clinical experience focusing on SABR applications with the Vero, including: (1) beam data acquisition; (2) dosimetric commissioning of the treatment planning system, including evaluation of a Monte Carlo algorithm in a specially-designed anthropomorphic thorax phantom; (3) validation using the Radiological Physics Center thorax, head and neck (IMRT), and spine credentialing phantoms; (4) end-to-end evaluation of IGRT localization accuracy; (5) ongoing system performance, including isocenter stability; and (6) clinical SABR applications. PMID:24710458

Solberg, Timothy D; Medin, Paul M; Ramirez, Ezequiel; Ding, Chuxiong; Foster, Ryan D; Yordy, John

2014-01-01

173

CellLineNavigator: a workbench for cancer cell line analysis  

PubMed Central

The CellLineNavigator database, freely available at http://www.medicalgenomics.org/celllinenavigator, is a web-based workbench for large scale comparisons of a large collection of diverse cell lines. It aims to support experimental design in the fields of genomics, systems biology and translational biomedical research. Currently, this compendium holds genome wide expression profiles of 317 different cancer cell lines, categorized into 57 different pathological states and 28 individual tissues. To enlarge the scope of CellLineNavigator, the database was furthermore closely linked to commonly used bioinformatics databases and knowledge repositories. To ensure easy data access and search ability, a simple data and an intuitive querying interface were implemented. It allows the user to explore and filter gene expression, focusing on pathological or physiological conditions. For a more complex search, the advanced query interface may be used to query for (i) differentially expressed genes; (ii) pathological or physiological conditions; or (iii) gene names or functional attributes, such as Kyoto Encyclopaedia of Genes and Genomes pathway maps. These queries may also be combined. Finally, CellLineNavigator allows additional advanced analysis of differentially regulated genes by a direct link to the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources.

Krupp, Markus; Itzel, Timo; Maass, Thorsten; Hildebrandt, Andreas; Galle, Peter R.; Teufel, Andreas

2013-01-01

174

GPVI oligomerisation in cell lines and platelets  

PubMed Central

Summary Background Glycoprotein VI (GPVI) is a physiological receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerisation of GPVI has been proposed as necessary for the interaction with collagen, although direct evidence of dimerisation has not been reported in cell lines or platelets. Objectives To investigate oligomerisation of GPVI in transfected cell lines and in platelets under nonstimulated conditions. Methods and Results By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extra-cellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerisation of GPVI under these conditions. A chemical cross-linker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the FcR ?-chain. Conclusions The present results directly demonstrate GPVI-FcR ?-chain oligomerisation at the surface of the platelet, and thereby add to the growing evidence that oligomerisation of GPVI may be a pre-requisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.

2007-01-01

175

On the Ontology Based Representation of Cell Lines  

PubMed Central

Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT) integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed.

Ganzinger, Matthias; He, Shan; Breuhahn, Kai; Knaup, Petra

2012-01-01

176

CALCIUM OXALATE CRYSTAL ATTACHMENT TO CULTURED KIDNEY EPITHELIAL CELL LINES  

Microsoft Academic Search

PurposeCultured kidney epithelial cell lines have frequently been used in urolithiasis research, and in particular in studies related to the interactions between stone crystals and cell membranes. There is evidence that when epithelial cell lines are transformed or serially passed to immortalize them, they experience changes in both cell physiology and morphology. Stone research utilizing cell cultures is frequently necessary

MICHAEL W. BIGELOW; JOHN H. WIESSNER; JACK G. KLEINMAN; NEIL S. MANDEL

1998-01-01

177

Catecholamines in a macrophage cell line.  

PubMed

This study provides the first evidence for catecholamine synthesis and release in the RAW264.7 cell line, an important macrophage model. Although catecholamines were low in unstimulated cells, activation with lipopolysaccharide (LPS) induced tyrosine hydroxylase (TH) mRNA and increased extracellular norepinephrine and intracellular dopamine within 48 h. The catecholamine synthesis inhibitor alpha-methyl-para-tyrosine (alpha-mpt) decreased extracellular norepinephrine levels, suggesting release and rapid turnover of newly synthesized norepinephrine. High concentrations of dopamine or norepinephrine (>/=100 microM) decreased proliferation and increased apoptosis of macrophages. These anti-proliferative effects were prevented by simultaneous treatment with the anti-oxidant ascorbic acid. Pre-incubation with a glutathione synthesis inhibitor (L-buthionine-[S,R]-sulfoximine [L-BSO]) increased sensitivity to catecholamine-stimulated apoptosis, suggesting that glutathione protects macrophages from both endogenous and exogenous catecholamines. PMID:12576223

Brown, Scott W; Meyers, Randall T; Brennan, Karen M; Rumble, Julie M; Narasimhachari, Nedathur; Perozzi, Edmund F; Ryan, John J; Stewart, Jennifer K; Fischer-Stenger, Krista

2003-02-01

178

Effect of black tea extract on herpes simplex virus-1 infection of cultured cells  

PubMed Central

Background The purpose of this investigation was to determine if black tea extract (BTE), consisting primarily of flavanol compounds called theaflavins, could inhibit herpes simplex virus type-1 (HSV-1) infection in cultured A549 (human epithelial) and Vero cells. Methods The effect of BTE both on A549 and Vero cultured cells and on HSV-1 was assessed by using phase contrast and fluorescent microscopy, and cell viability and proliferation assays. After establishing the maximum non-cytotoxic concentration of BTE, A549 and Vero cells and HSV-1 virions were treated with varying concentrations of BTE, respectively. A549 and Vero cells were infected with HSV-1 with green fluorescent protein (GFP) insert at the UL46 gene. The effect of infectivity was determined by viral DNA extraction followed by PCR, plaque assays, adsorption assays, and electrophoresis of PCR products. Results BTE was not cytotoxic to A549 and Vero cells, as confirmed by cell viability and proliferation assays, in which BTE treated groups paralleled the positive control group. For both cell lines, plaque assays and fluorescent microscopy indicated an inverse relationship between BTE concentration (from 0.14 ?M – 1.4 mM) and HSV-1 infectivity. Specifically, PCR and electrophoresis showed a reduction in the viral genome following treatment with BTE. In addition, there was a noticeable decrease in the amount of viral plaques for BTE treated samples in the adsorption assays. Conclusions BTE consisting primarily of theaflavins is not cytotoxic and can reduce or block the production of infectious HSV-1 virions in cultured A549 and Vero cells, thus inhibiting the infectivity of the virus by interfering in the attachment, penetration and viral DNA replication of HSV-1 particles. These findings indicate that BTE enriched with theaflavins has the potential to be developed as a safe, therapeutic antiviral agent to prevent the spread of HSV-1.

2013-01-01

179

Tetanus toxin as a marker for small-cell lung cancer cell lines  

Microsoft Academic Search

Tetanus toxin labeling of human lung cancer cell lines was investigated using direct and indirect immunofluorescence and immunohistochemical staining. Cells of characterized permanent cell lines, eight small-cell lung cancer (SCLC) cell lines of classic subtype, six SCLC cell lines of variant subtype and seven non-small-cell lung cancer (NSCLC) cell lines, were incubated with a saturating concentration of tetanus toxin. For

Jochen Heymanns; Kurt Neumann; Klaus Havemann

1989-01-01

180

Detection algorithm for the validation of human cell lines.  

PubMed

Cell lines are an important tool in understanding all aspects of cancer growth, development, metastasis and tumor cell death. There has been a dramatic increase in the number of cell lines and diversity of the cancers they represent; however, misidentification and cross-contamination of cell lines can lead to erroneous conclusions. One method that has gained favor for authenticating cell lines is the use of short tandem repeats (STR) to generate a unique DNA profile. The challenge in validating cell lines is the requirement to compare the large number of existing STR profiles against cell lines of interest, particularly when considering that the profiles of many cell lines have drifted over time and original samples are not available. We report here methods that analyze the variations and the proportional changes extracted from tetra-nucleotide repeat regions in the STR analysis. This technique allows a paired match between a target cell line and a reference database of cell lines to find cell lines that match within a user designated percentage cut-off quality matrix. Our method accounts for DNA instability and can suggest whether the target cell lines are misidentified or unstable. PMID:22419365

Eltonsy, Névine; Gabisi, Vivian; Li, Xuesong; Russe, K Blair; Mills, Gordon B; Stemke-Hale, Katherine

2012-09-15

181

Personalized chemotherapy profiling using cancer cell lines from selectable mice  

PubMed Central

Purpose High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult, because contaminating stromal cells overgrow the malignant cells. Experimental Design We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Results Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified seventy-seven FDA approved drugs with activity, including two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Conclusion Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice.

Kamiyama, Hirohiko; Rauenzahn, Sherri; Shim, Joong Sup; Karikari, Collins A.; Feldmann, Georg; Hua, Li; Kamiyama, Mihoko; Schuler, F. William; Lin, Ming-Tseh; Beaty, Robert M.; Karanam, Balasubramanyam; Liang, Hong; Mullendore, Michael E.; Mo, Guanglan; Hidalgo, Manuel; Jaffee, Elizabeth; Hruban, Ralph H.; Jinnah, H. A.; Roden, Richard B. S.; Jimeno, Antonio; Liu, Jun O.; Maitra, Anirban; Eshleman, James R.

2013-01-01

182

DNA profiling and characterization of animal cell lines.  

PubMed

The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines. PMID:24297409

Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

2014-01-01

183

Match criteria for human cell line authentication: where do we draw the line?  

PubMed

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ?80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines. PMID:23136038

Capes-Davis, Amanda; Reid, Yvonne A; Kline, Margaret C; Storts, Douglas R; Strauss, Ethan; Dirks, Wilhelm G; Drexler, Hans G; MacLeod, Roderick A F; Sykes, Gregory; Kohara, Arihiro; Nakamura, Yukio; Elmore, Eugene; Nims, Raymond W; Alston-Roberts, Christine; Barallon, Rita; Los, Georgyi V; Nardone, Roland M; Price, Paul J; Steuer, Anton; Thomson, Jim; Masters, John R W; Kerrigan, Liz

2013-06-01

184

Functional Analysis of an Established Mouse Vascular Endothelial Cell Line  

Microsoft Academic Search

Background: In vitrostudies using cell lines are useful for the understanding of cellular mechanisms. The purpose of our study is to develop a new immortalized aortic vascular endothelial cell (EC) line that retains endothelial characteristics and can facilitate the study of ECs. Methods: A mouse aortic vascular EC line (MAEC) was established from p53-deficient mouse aorta and cultured for over

Tatsuaki Nishiyama; Kenji Mishima; Fumio Ide; Koichi Yamada; Kumi Obara; Aki Sato; Noriko Hitosugi; Hiroko Inoue; Kazuo Tsubota; Ichiro Saito

2007-01-01

185

Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3)-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv).  

PubMed

Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro. PMID:24671184

Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Chow, Vincent T K; Chua, Kaw Bing

2014-01-01

186

Phenotypic and Genotypic Characteristics of Novel Mouse Cell Line (NIH/3T3)-Adapted Human Enterovirus 71 Strains (EV71:TLLm and EV71:TLLmv)  

PubMed Central

Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus’s inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136–150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Chow, Vincent T. K.; Chua, Kaw Bing

2014-01-01

187

Polar release of pathogenic Old World hantaviruses from renal tubular epithelial cells  

PubMed Central

Background Epithelio- and endotheliotropic viruses often exert polarized entry and release that may be responsible for viral spread and dissemination. Hantaviruses, mostly rodent-borne members of the Bunyaviridae family infect epithelial and endothelial cells of different organs leading to organ dysfunction or even failure. Endothelial and renal epithelial cells belong to the target cells of Old World hantavirus. Therefore, we examined the release of hantaviruses in several renal epithelial cell culture models. We used Vero cells that are commonly used in hantavirus studies and primary human renal epithelial cells (HREpC). In addition, we analyzed MDCKII cells, an epithelial cell line of a dog kidney, which represents a widely accepted in vitro model of polarized monolayers for their permissiveness for hantavirus infection. Results Vero C1008 and primary HREpCs were grown on porous-support filter inserts for polarization. Monolayers were infected with hantavirus Hantaan (HTNV) and Puumala (PUUV) virus. Supernatants from the apical and basolateral chamber of infected cells were analyzed for the presence of infectious particles by re-infection of Vero cells. Viral antigen and infectious particles of HTNV and PUUV were exclusively detected in supernatants collected from the apical chamber of infected Vero C1008 cells and HREpCs. MDCKII cells were permissive for hantavirus infection and polarized MDCKII cells released infectious hantaviral particles from the apical surface corresponding to the results of Vero and primary human epithelial cells. Conclusions Pathogenic Old World hantaviruses are released from the apical surface of different polarized renal epithelial cells. We characterized MDCKII cells as a suitable polarized cell culture model for hantavirus infection studies.

2012-01-01

188

An activity specified by the osteosarcoma line U2OS can substitute functionally for ICP0, a major regulatory protein of herpes simplex virus type 1.  

PubMed Central

Among the five immediate-early regulatory proteins of herpes simplex virus (HSV) type 1, only ICP0 is capable of activating all kinetic classes of viral genes. Consistent with its broad transactivating activity, ICP0 plays a major role in enhancing the reactivation of HSV from latency both in vivo and in vitro. Although not essential for viral replication, ICP0 confers a significant growth advantage on the virus, especially at low multiplicities of infection. In this report we describe the expression of a novel activity by the osteosarcoma cell line U2OS that can substitute functionally for ICP0. Compared with Vero cells, both U2OS cells and cells of the ICP0-expressing line 0-28 significantly enhanced the plating efficiency of an ICP0 null mutant, 7134. In contrast, the plating efficiencies of the wild-type virus in all three cell types were similar. Single-step growth experiments demonstrated that the yield of 7134 in U2OS cells was severalfold higher than that in 0-28 cells and about 100-fold higher than that in Vero cells. In order to identify the viral genes whose expression is enhanced by the activity in U2OS cells, levels of expression of selected viral proteins in extracts of Vero and U2OS cells were compared by Western blot (immunoblot) analysis following low-multiplicity infection. At a multiplicity of 0.1 PFU per cell, the levels of expression of the immediate-early protein ICP4 and the early protein gD in 7134-infected U2OS cells were significantly higher than those in 7134-infected Vero cells. When infections were carried out at a multiplicity of 1 PFU per cell, however, no major differences in the levels of expression of these proteins in U2OS and Vero cells were observed. Cycloheximide reversal experiments demonstrated that the cellular activity expressed in U2OS cells that promotes high-level expression of ICP4 is not synthesized de novo but appears to exist as a preformed protein(s). To confirm this observation and to determine whether, like immediate-early genes, early, delayed-early, and late viral genes are also responsive to the cellular activity, transient-expression assays were performed. The results of these tests demonstrated that basal levels of expression from immediate-early, early, and delayed-early promoters, but not that from a late promoter, were significantly higher in U2OS cells than in Vero cells and that this enhancement occurred in the absence of viral proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Yao, F; Schaffer, P A

1995-01-01

189

Cell line misidentification: the beginning of the end  

Microsoft Academic Search

Cell lines are used extensively in research and drug development as models of normal and cancer tissues. However, a substantial proportion of cell lines is mislabelled or replaced by cells derived from a different individual, tissue or species. The scientific community has failed to tackle this problem and consequently thousands of misleading and potentially erroneous papers have been published using

2010-01-01

190

Differential signaling of the GnRH receptor in pituitary gonadotrope cell lines and prostate cancer cell lines  

PubMed Central

The GnRH receptor (GnRHR) mediates the pituitary functions of GnRH, as well as its anti-proliferative effects in sex hormone-dependent cancer cells. Here we compare the signaling of GnRHR in pituitary gonadotrope cell lines vs. prostate cancer cell lines. We first noticed that the expression level of PKC?, PKC?II and PKC? is much higher in ?T3-1 and L?T2 gonadotrope cell lines vs. LNCaP and DU-145 cell lines, while the opposite is seen for PKC?. Activation of PKC?, PKC?II and PKC? by GnRH is relatively transient in ?T3-1 and L?T2 gonadotrope cell lines and more prolonged in LNCaP and DU-145 cell lines. On the otherhand, the activation and re-distribution of the above PKCs by PMA was similar for both gonadotrope cell lines and prostate cancer cell lines. Activation of ERK1/2 by GnRH and PMA was robust in the gonadotrope cell lines, with a smaller effect observed in the prostate cancer cell lines. The Ca2+ ionophore A23187 stimulated ERK1/2 in gonadotrope cell lines but not in prostate cancer cell lines. GnRH, PMA and A23187 stimulated JNK activity in gonadotrope cell lines, with a more sustained effect in prostate cancer cell lines. Sustained activation of p38 was observed for PMA and A23187 in Du-145 cells, while p38 activation by GnRH, PMA and A23187 in L?T2 cells was transient. Thus, differential expression and re-distribution of PKCs by GnRH and the transient vs. the more sustained nature of the activation of the PKC-MAPK cascade by GnRH in gonadotrope cell lines vs. prostate cancer cell lines respectively, may provide the mechanistic basis for the cell context-dependent differential biological responses observed in GnRH interaction with pituitary gonadotropes vs. prostate cancer cells.

Sviridonov, Ludmila; Dobkin-Bekman, Masha; Shterntal, Boris; Przedecki, Fiorenza; Formishell, Linor; Kravchook, Shani; Navi, Liat Rahamim-Ben; Bar-Lev, Tali Hana; Kazanietz, Marcelo G.; Yao, Zhong; Seger, Rony; Naor, Zvi

2014-01-01

191

Effect of melatonin on cytotoxicity of doxorubicin toward selected cell lines (human keratinocytes, lung cancer cell line A-549, laryngeal cancer cell line Hep-2).  

PubMed

The pineal hormone melatonin (MLT) has been recognised as a substance capable of alleviating in vivo nephro-, cardio- and myelotoxicity of doxorubicin (DOX) and of other anthracyclines in animal models. However, few data are available on the effects of MLT on cytotoxicity of antineoplastic drugs toward tumor cells in vitro. The present study aimed at the evaluation of effects of MLT and of DOX on selected cell lines. The experiments were conducted on human keratinocytes (primary culture), non-small cell lung cancer (A-549) and laryngeal cancer cell lines (HEp-2). In keratinocytes and in A-549 cells, MLT used at pharmacological concentrations (0.1 and 1.0 mM) was observed to intensify apoptotic lesions. MLT exerted no clear-cut effects on the HEp-2 cell line. In contrast, DOX at concentrations of 0.1 and 1.0 microg/ml intensified apoptosis and augmented the frequency of necrotic lesions in cell nuclei in all the examined cell lines. MLT intensified cytotoxicity of DOX in all cell lines, significantly decreasing cell numbers and promoting apoptosis. The effect was MLT concentration-dependent. MLT decreased the proportion of cells with necrotic lesions. PMID:17591362

Fic, Magdalena; Podhorska-Okolow, Marzena; Dziegiel, Piotr; Gebarowska, Elzbieta; Wysocka, Teresa; Drag-Zalesinska, Malgorzata; Zabel, Maciej

2007-01-01

192

Biological behaviors and proteomics analysis of hybrid cell line EAhy926 and its parent cell line A549  

Microsoft Academic Search

BACKGROUND: It is well established that cancer cells can fuse with endothelial cells to form hybrid cells spontaneously, which facilitates cancer cells traversing the endothelial barrier to form metastases. However, up to now, little is known about the biologic characteristics of hybrid cells. Therefore, we investigate the malignant biologic behaviors and proteins expression of the hybrid cell line EAhy926 with

Ze Jun Lu; Ya Qiong Ren; Guo Ping Wang; Qi Song; Mei Li; Sa Sa Jiang; Tao Ning; Yong Song Guan; Jin Yang; Feng Luo

2009-01-01

193

Human Neuron-Committed Teratocarcinoma NT2 Cell Line Has Abnormal ND10 Structures and Is Poorly Infected by Herpes Simplex Virus Type 1  

PubMed Central

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stimulates the initiation of lytic infection and reactivation from quiescence in human fibroblast cells. These functions correlate with its ability to localize to and disrupt centromeres and specific subnuclear structures known as ND10, PML nuclear bodies, or promyelocytic oncogenic domains. Since the natural site of herpesvirus latency is in neurons, we investigated the status of ND10 and centromeres in uninfected and infected human cells with neuronal characteristics. We found that NT2 cells, a neuronally committed human teratocarcinoma cell line, have abnormal ND10 characterized by low expression of the major ND10 component PML and no detectable expression of another major ND10 antigen, Sp100. In addition, PML is less extensively modified by the ubiquitin-like protein SUMO-1 in NT2 cells compared to fibroblasts. After treatment with retinoic acid, NT2 cells differentiate into neuron-like hNT cells which express very high levels of both PML and Sp100. Infection of both NT2 and hNT cells by HSV-1 was poor compared to human fibroblasts, and after low-multiplicity infection yields of virus were reduced by 2 to 3 orders of magnitude. ICP0-deficient mutants were also disabled in the neuron-related cell lines, and cells quiescently infected with an ICP0-null virus could be established. These results correlated with less-efficient disruption of ND10 and centromeres induced by ICP0 in NT2 and hNT cells. Furthermore, the ability of ICP0 to activate gene expression in transfection assays in NT2 cells was poor compared to Vero cells. These results suggest that a contributory factor in the reduced HSV-1 replication in the neuron-related cells is inefficient ICP0 function; it is possible that this is pertinent to the establishment of latent infection in neurons in vivo.

Hsu, Wei-Li; Everett, Roger D.

2001-01-01

194

Polyamine Synthesis in Maize Cell Lines 1  

PubMed Central

Uptake of [14C]putrescine, [14C]arginine, and [14C]ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average, 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions.

Hiatt, Andrew

1989-01-01

195

Investigation of Radiosensitivity Gene Signatures in Cancer Cell Lines  

PubMed Central

Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n?=?16] and head and neck [n?=?11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

Hall, John S.; Iype, Rohan; Senra, Joana; Taylor, Janet; Armenoult, Lucile; Oguejiofor, Kenneth; Li, Yaoyong; Stratford, Ian; Stern, Peter L.; O'Connor, Mark J.; Miller, Crispin J.; West, Catharine M. L.

2014-01-01

196

Continuous human cell lines and method of making same  

DOEpatents

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

Stampfer, Martha R. (Oakland, CA)

1989-01-01

197

Continuous human cell lines and method of making same  

DOEpatents

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

Stampfer, M.R.

1985-07-01

198

Shortest path based splitting line finding for touching cells  

NASA Astrophysics Data System (ADS)

A shortest path based algorithm is proposed in this paper to find splitting lines for touching cells. Firstly, an initial splitting line is obtained through the distance transform of a marker image and the watershed algorithm. Then, the initial splitting line is separated into different line segments if necessary, and the start and end points of these line segments act as the start and end points of shortest path. Finally, the shortest path algorithm is used to find the splitting line between the start and end points, and the final result of touching cells splitting can be formed by the contour of the touching cells and the splitting lines. Experimental results show that the proposed algorithm is efficient for different types of touching cells.

Bai, Xiangzhi; Sun, Changming; Wang, Peng; Zhou, Fugen

2013-10-01

199

The cell line data base and the new catalogue: Detailed information on 2650 human and animal cell lines  

Microsoft Academic Search

The Cell Line Data Base (CLDB), set up within the Interlab Project, is a relational database containing data on 2650 Human and animal cell lines which are available in labs and cell banks all over Europe. The second edition of the catalogue, directly generated from the database, has been produced, and will be published in the first months of 1993.

A. Manniello; O. Aresu; B. Parodi; P. Romano; B. Iannotta; G. Rondanina; L. Viegi; T. Ruzzon

1993-01-01

200

Differentiation of Embryonic Stem Cell Lines Generated from Adult Somatic Cells by Nuclear Transfer  

Microsoft Academic Search

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and

Teruhiko Wakayama; Viviane Tabar; Ivan Rodriguez; Anthony C. F. Perry; Lorenz Studer; Peter Mombaerts

2001-01-01

201

Complement-Fixing Antigens in Burkitt Lymphoma Cell Lines  

PubMed Central

The complement-fixing (CF) activity of antigens from cultured Burkitt lymphoma cells was determined by using normal American sera as the source of antibody. Approximately 75% of the sera fixed complement with the positive cell lines. These lines contained the herpes-like virus detectable by electron microscopy. The content of CF antigen depended on the cell line used but appeared to be independent of the number of cells which produced Henle's immunofluorescence (IF) antigen. Only sera that reacted in the IF test also contained CF antibodies to the crude cell extracts.

McCormick, Kenneth J.; Stenback, Wayne A.; Mumford, David M.; Trentin, John J.

1971-01-01

202

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls  

PubMed Central

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

2013-01-01

203

Regulated expression of erythropoietin by two human hepatoma cell lines  

SciTech Connect

The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

1987-11-01

204

Replicative Capacity of MERS Coronavirus in Livestock Cell Lines.  

PubMed

Replicative capacity of Middle East respiratory syndrome coronavirus (MERS-CoV) was assessed in cell lines derived from livestock and peridomestic small mammals on the Arabian Peninsula. Only cell lines originating from goats and camels showed efficient replication of MERS-CoV. These results provide direction in the search for the intermediate host of MERS-CoV. PMID:24457147

Eckerle, Isabella; Corman, Victor M; Müller, Marcel A; Lenk, Matthias; Ulrich, Rainer G; Drosten, Christian

2014-02-01

205

GREG cells, a dysferlin-deficient myogenic mouse cell line  

SciTech Connect

The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S. [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Morree, Antoine de [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands)] [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands); Pekkurnaz, Gulcin [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Nagaraju, Kanneboyina [Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC 20010 (United States)] [Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC 20010 (United States); Zimmerberg, Joshua, E-mail: zimmerbj@mail.nih.gov [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)

2012-01-15

206

Phase III clinical trials comparing the immunogenicity and safety of the vero cell-derived Japanese encephalitis vaccine Encevac with those of mouse brain-derived vaccine by using the Beijing-1 strain.  

PubMed

The immunogenicity and safety of an inactivated cell culture Japanese encephalitis vaccine (CC-JEV) were compared with those of an inactivated mouse brain-derived Japanese encephalitis vaccine (MB-JEV) in phase III clinical multicenter trials conducted in children. The vaccines contain the same Japanese encephalitis virus strain, the Beijing-1 strain. Two independent clinical trials (trials 1 and 2) were conducted. Trial 1 was conducted in 468 healthy children. Each subject was injected with 17 ?g per dose of either CC-JEV or MB-JEV, and the immunogenicity and safety of the vaccines were investigated. Trial 1 showed that CC-JEV was more immunogenic and reactive than MB-JEV at the same dose. Therefore, to adjust the immunogenicity of CC-JEV to that of MB-JEV, a vaccine that has had a good track record regarding its efficacy for a long time, trial 2 was conducted in 484 healthy children. To improve the stability, CC-JEV was converted from a liquid type to a freeze-dried type of vaccine. Each subject was injected subcutaneously with either 4 ?g per dose of CC-JEV, 8 ?g per dose of CC-JEV, or 17 ?g per dose of MB-JEV twice, at an interval of 2 to 4 weeks, followed by an additional booster immunization 1 to 15 months after the primary immunization. Based on the results of trial 2, 4 ?g per dose of the freeze-dried CC-JEV (under the label Encevac) was selected as a substitute for the MB-JEV. Encevac was approved and launched in 2011 and has since been in use as a 2nd-generation Japanese encephalitis vaccine in Japan. (These studies have been registered at the JapicCTI under registration no. JapicCTI-132063 and JapicCTI-080586 for trials 1 and 2, respectively). PMID:24334689

Miyazaki, Chiaki; Okada, Kenji; Ozaki, Takao; Hirose, Mizuo; Iribe, Kaneshige; Yokote, Hiroyuki; Ishikawa, Yuji; Togashi, Takehiro; Ueda, Kohji

2014-02-01

207

HLA Homozygous Stem Cell Lines Derived from Human Parthenogenetic Blastocysts  

Microsoft Academic Search

Individual HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines have the potential for cell-based therapy in a significant number of individuals, provided the HLA haplotype is prevalent. We report the successful derivation of four stable hpSC-Hhom lines from both HLA homozygous and HLA heterozygous donors. Of these, the hpSC-Hhom-4 line carries the HLA haplotype found most commonly within the U.S.

E. S. Revazova; N. A. Turovets; O. D. Kochetkova; L. S. Agapova; J. L. Sebastian; M. V. Pryzhkova; V. Iu. Smolnikova; L. N. Kuzmichev; J. D. Janus

2008-01-01

208

Phenotype and Genotype of Pancreatic Cancer Cell Lines  

PubMed Central

The dismal prognosis of pancreatic adenocarcinoma (PA) is due in part due to a lack of molecular information regarding disease development. Established cell lines remain a useful tool for investigating these molecular events. Here we present a review of available information on commonly used PA cell lines as a resource to help investigators select the cell lines most appropriate for their particular research needs. Information on clinical history, in vitro and in vivo growth characteristics, phenotypic characteristics, such as adhesion, invasion, migration and tumorigenesis, and genotypic status of commonly altered genes (KRAS, p53, p16, and SMAD4) was evaluated. Identification of both consensus and discrepant information in the literature suggests careful evaluation before selection of cell lines and attention be given to cell line authentication.

Deer, Emily L.; Gonzalez-Hernandez, Jessica; Coursen, Jill D.; Shea, Jill E.; Ngatia, Josephat; Scaife, Courtney L.; Firpo, Matthew A.; Mulvihill, Sean J.

2009-01-01

209

Undermethylation of specific LINE-1 sequences in human cells producing a LINE-1-encoded protein.  

PubMed

Nucleotide sequences near the 5' ends of some long interspersed elements-1 (LINE-1) from Homo sapiens (L1Hs) are undermethylated in cell lines which produce a L1Hs-encoded protein. In contrast, these sequences are methylated in cell lines with little or no detectable L1Hs expression. The fact that the 5' end of L1Hs is differentially methylated in cells exhibiting different levels of L1Hs expression suggests that the methylation state of this region plays a role in L1Hs expression. PMID:7693554

Thayer, R E; Singer, M F; Fanning, T G

1993-11-15

210

Regulatory networks define phenotypic classes of human stem cell lines  

PubMed Central

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine1, 2 have highlighted the need for a general, reproducible method for classification of these cells3. We report here the creation and analysis of a database of global gene expression profiles (“Stem Cell Matrix”) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent, and differentiated cell types. Using an unsupervised clustering method4, 5 to categorize a collection of ~150 cell samples, we discovered that pluripotent stem cell lines group together, while other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis6 we uncovered a protein-protein network (“PluriNet”) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas, and induced pluripotent cells). Analysis of published data showed that the PluriNet appears to be a common characteristic of pluripotent cells, including mouse ES and iPS cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotence and self-renewal are under tight control by specific molecular networks.

Muller, Franz-Josef; Laurent, Louise C.; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Schwartz, Philip H.; Schmidt, Nils O.; Loring, Jeanne F.

2008-01-01

211

Further characterization of the first seminoma cell line TCam-2.  

PubMed

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs. PMID:18050305

de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; Hersmus, Remko; van Gurp, Ruud J H L M; van de Geijn, Gert-Jan M; van Drunen, Ellen; Beverloo, H Berna; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Kitazawa, Sohei; van Zoelen, E Joop; van Roozendaal, Kees; Oosterhuis, J Wolter; Looijenga, Leendert H J

2008-03-01

212

Characterization of Liposarcoma Cell Lines for Preclinical and Biological Studies  

PubMed Central

Liposarcoma cell lines represent in vitro models for studying disease mechanisms at the cellular level and for preclinical evaluation of novel drugs. To date there are a limited number of well-characterized models available. In this study, nine immortal liposarcoma cell lines were evaluated for tumor-forming ability, stem cell- and differentiation potential, and metastatic potential, with the aim to generate a well-characterized liposarcoma cell line panel. Detailed stem cell and differentiation marker analyses were also performed. Five of the liposarcoma cell lines were tumorigenic, forming tumors in mice. Interestingly, tumor-forming ability correlated with high proliferative capacity in vitro. All the cell lines underwent adipocytic differentiation, but the degree varied. Surprisingly, the expression of stem cell and differentiation markers did not correlate well with function. Overall, the panel contains cell lines suited for in vivo analyses (LPS141, SA-4, T778, SW872, and LISA-2), for testing novel drugs targeting cancer stem cells (LPS141) and for studying tumor progression and metastasis (T449 and T778).

Stratford, Eva W.; Castro, Russell; Daffinrud, Jeanette; Skarn, Magne; Lauvrak, Silje; Munthe, Else; Myklebost, Ola

2012-01-01

213

Development and characterization of immortalized ovine endometrial cell lines.  

PubMed

The objective of this study was to generate immortalized endometrial epithelial and stromal cell lines from the ovine uterus. Luminal (LE) and glandular epithelial (GE) cells and stromal (ST) cells were enzymatically isolated from the uterus of a Day 5 cyclic ewe (estrus on Day 0), and primary cultures were immortalized by transduction with a retroviral vector (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analogue G418 (0.6-0.8 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for more than 40 passages. Phase-contrast microscopy revealed that LE and GE cells exhibited a cobblestone morphology whereas immortalized ST cells were spindle shaped. The epithelial origin of LE and GE was confirmed by positive cytokeratin immunostaining, and ST cells were vimentin positive. All cell lines were negative for smooth muscle alpha-actin staining. Western blot analyses of cell extracts revealed the presence of signal transducers and activators of transcription (STAT) proteins 1, 2, and 3. In the LE cells, interferon tau (IFNtau) induced nuclear translocation of STAT proteins 1 and 2 and up-regulated several IFN-inducible genes, including STATs 1, 2, and 3 and ubiquitin cross-reactive protein (UCRP/ISG17). In the LE cell line, IFN regulatory factor one was transiently up-regulated and then down-regulated by IFNtau. Immunostaining revealed the presence of nuclear estrogen receptor and progesterone receptor in all cell lines. These ovine endometrial cell lines provide useful in vitro model systems for the study of hormone and cytokine action, signal transduction pathways, cell-cell interactions, and gene expression in specific cell types of the ovine endometrium. PMID:10529281

Johnson, G A; Burghardt, R C; Newton, G R; Bazer, F W; Spencer, T E

1999-11-01

214

Murine bone marrow cell line producing colony-stimulating factor  

SciTech Connect

A cell line (H-1) derived from the adherent layer of a 14-wk-old Dexter bone marrow culture has been maintained as cloned and uncloned lines through 21 passages at the time of these studies. These cell lines develop many fat droplets as they age and become confluent. The uncloned line produces increasing amounts of colony-stimulating activity as the cells become confluent. Feeder-layers or supernatants from the nonconfluent or confluent fat-laden cells stimulate the formation of greater numbers of colonies derived from cultures of colony-forming units (CFU) than does medium from L cell culture containing colony-stimulating factor (CSF). Antibody to the CSF-containing medium from L cell culture neutralizes the colony-stimulating activity, thus showing immunologic similarity to a known molecular species that stimulates colony production in a CFU culture that produces granulocyte or macrophage populations, or both.

Harigaya, K. (Brookhaven National Lab., Upton, NY); Cronkite, E.P.; Miller, M.E.; Shadduck, R.K.

1981-11-01

215

Generation of mesenchymal stem cell lines from murine bone marrow.  

PubMed

Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages. PMID:22836234

Sreejit, P; Dilip, K B; Verma, R S

2012-10-01

216

Quantitative methods to characterize morphological properties of cell lines.  

PubMed

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages. PMID:22619183

Mancia, Annalaura; Elliott, John T; Halter, Michael; Bhadriraju, Kiran; Tona, Alessandro; Spurlin, Tighe A; Middlebrooks, Bobby L; Baatz, John E; Warr, Gregory W; Plant, Anne L

2012-07-01

217

Small RNAs: Keeping Stem Cells in Line  

PubMed Central

Stem cells and RNA silencing have emerged as areas of intense interest for both basic and clinical research. Recently these fields have converged with reports implicating small regulatory RNAs in the maintenance and pluripotency of stem cells.

Stadler, Bradford M.; Ruohola-Baker, Hannele

2010-01-01

218

Eternity and functionality - rational access to physiologically relevant cell lines.  

PubMed

In the first 50 years of cell culture, the development of new cell lines was mainly based on trial and error. Due to the understanding of the molecular networks of aging, senescence, proliferation, and adaption by mutation, the generation of new cell lines with physiologic properties has become more systematic. This endeavor has been supported by the availability of new technological achievements and increasing knowledge about the biology of cell differentiation and cell-cell communication. Here, we review some promising developments that are contributing toward this goal. These include molecular tools frequently used for the immortalization process. In addition to these broadly acting immortalization regimens, we focus on the developments of cell type-specific immortalization and on the methodologies of how to control the growth of newly established cell lines. PMID:23863696

Lipps, Christoph; May, Tobias; Hauser, Hansjörg; Wirth, Dagmar

2013-12-01

219

Apoptosis as a Cause of Death in Measles Virus-Infected Cells  

Microsoft Academic Search

To determine the mechanism of measles virus-induced cell death, we studied the infection of Vero cells and monocytic cell lines with wild-type (Chicago-1) and vaccine (Edmonston) strains of measles virus. DNA fragmentationindicativeofapoptosiswasapparentbyflowcytometry,agarosegelelectrophoresis,andelectron microscopy. Within syncytia, DNA strand breaks were demonstrated by end labeling with terminal transferase and then by visualization. A number of viruses have recently been shown to cause

LISA M. ESOLEN; SUK W. PARK; J. MARIE HARDWICK; ANDDIANE E. GRIFFIN

1995-01-01

220

Development of a conditionally immortalized human pancreatic ? cell line  

PubMed Central

Diabetic patients exhibit a reduction in ? cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human ? cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human ? cell line (EndoC-?H1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-?H1 cells display many functional properties of adult ? cells, including expression of ? cell markers and insulin secretion following glucose stimulation; however, unlike primary ? cells, EndoC-?H1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human ? cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-?H2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of ? cell–specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-?H2 cells are highly representative of human ? cells and should be a valuable tool for further analysis of human ? cells.

Scharfmann, Raphael; Pechberty, Severine; Hazhouz, Yasmine; von Bulow, Manon; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Guez, Fanny; Rachdi, Latif; Lohmann, Matthias; Czernichow, Paul; Ravassard, Philippe

2014-01-01

221

Antineoplastic activity of rinvanil and phenylacetylrinvanil in leukaemia cell lines  

PubMed Central

In the search for novel chemotherapeutic agents for cancer treatment, capsaicin has been shown to inhibit proliferation and induce apoptosis in various types of cancer cell line, including leukaemia cell lines. The capsaicin analogues, rinvanil and phenylacetylrinvanil (PhAR), share a binding affinity for vanilloid receptors and may have biological activities similar to capsaicin; however, their anticancer potential has not yet been reported. This study analyses the antineoplastic activities of rinvanil and PhAR in leukaemia versus normal cells. P388, J774 and WEHI-3 leukaemia cell lines, as well as mouse bone marrow mononuclear cells, were cultured with varying concentrations of rinvanil and PhAR. Following this, proliferation and apoptosis were determined by the sulforhodamine B (SRB) assay and DNA ladder. Cultured leukaemia cell lines and mouse bone marrow mononuclear cells demonstrated a dose-dependent inhibition of proliferation, while non-diseased cells were less sensitive to the cytotoxic effect of capsaicin, rinvanil and PhAR. Rinvanil and PhAR also induced apoptosis in leukaemia cell lines but not in bone marrow. Given the lower IC50 values for apoptosis induction in leukaemia cells compared with that of normal cells, PhAR is a promising selective anticancer agent.

LUVIANO, AXEL; AGUINIGA-SANCHEZ, ITZEN; DEMARE, PATRICIA; TIBURCIO, REYNALDO; LEDESMA-MARTINEZ, EDGAR; SANTIAGO-OSORIO, EDELMIRO; REGLA, IGNACIO

2014-01-01

222

UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma  

Cancer.gov

This technology describes the UOK 268 cell line, a spontaneously immortalized renal tumor cell line that may be of great interest to industry for studying HLRCC, drug screening, and searching for tumor markers related to diagnosis, prognosis, and drug resistance.

223

Search for the critical characteristics of phenotypically different B cell lines, Burkitt lymphoma cells and lymphoblastoid cell lines, which determine differences in their functional interaction with allogeneic lymphocytes  

Microsoft Academic Search

Summary Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines

Javier Avila-Carifio; Sigurbjörg Torsteinsdottir; Barbro Ehlin-Henriksson; Maria G. Masucci; Eva Klein

1991-01-01

224

[DNA fingerprinting analysis of silkworm embryo cell lines].  

PubMed

DNA extraction and the polymerase chain reaction (PCR) were used on DNA genomes study of cell lines of Bombyx mori. DNA polymorphic marker analysis was conducted and DNA fingerprint of cell lines of Bombyx mori. was carried out using ISSR and RAPD. Primers that can reliably find polymorphic bands were screened out. 26 ISSR primers were selected from them any available, and 797 polymorphic bands were abtained through PCR amplification in 9 samples, including 3 embryo cell lines of Bombyx mori (BmE-SWU1, BmE-SWU2, BmE-SWU3), 5 passage cell lines (BmE, BmN, Sf9, Sf21, Hi5) and the embryos from which BmE-SWU1 originated. The ration of polymorphic bands was 89.9%. 43 RAPD primers were selected out through PCR amplification, and 1205 polymorphic bands were obtained in 9 samples. The ration of polymorphic bands was 76.6%. There were many DNA polymorphic bands differences in the cell lines of Bombyx mori. The special DNA markers of the 3 embryo cell lines were found respectively. The similarity index Nei and genetic distance of the 9 samples were calculated and the phylogeny tree of 9 samples was constructed by UPGMA. Results showed that 2 groups were divided,one group including the 3 embryo cell lines and the embryo of XQ has close relative. Another group constructed by five insect cell lines came from different species, their genetic distance was closer than the 3 embryo cell lines. PMID:17348206

Pan, Min Hui; Feng, Zhen Yue; Tian, Zhi Qiang; Liu, Min; Lu, Cheng

2006-12-01

225

Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines  

Microsoft Academic Search

The recent availability in culture of embryo-derived pluripotential cells which exhibit both a normal karyotype and a high differentiative ability1-3 has encouraged us to assess the potential of these cells to form functional germ cells following their incorporation into chimaeric mice. We report here the results of blastocyst injection studies using three independently isolated XY embryo-derived cell lines (EK.CP1, EK.CC1.1

Allan Bradley; Martin Evans; Matthew H. Kaufman; Elizabeth Robertson

1984-01-01

226

Vaccine production: upstream processing with adherent or suspension cell lines.  

PubMed

The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered. PMID:24297427

Genzel, Yvonne; Rödig, Jana; Rapp, Erdmann; Reichl, Udo

2014-01-01

227

Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed to 12C6+ ions  

NASA Astrophysics Data System (ADS)

AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

Jing, X.; Yang, J.; Li, W.; Guo, C.; Dang, B.; Wang, J.; Zhou, L.; Wei, W.; Gao, Q.

228

Engineering cultured insulin-secreting pancreatic B-cell lines  

Microsoft Academic Search

Despite many triumphs, a significant limitation of the usefulness of many of the available B-cell lines for the study of\\u000a insulin secretion are either inappropriate or lack of responsiveness to glucose. Commonly employed cell lines generated prior\\u000a to the 1990s following X-ray irradiation (RINm5F cells) or simian virus 40 B-cell transformation (HIT-T15 cells and BTC) fall\\u000a into this category. More

Neville H. McClenaghan; P. R. Flatt

1999-01-01

229

Characterization of a hormone-producing ovarian carcinoma cell line.  

PubMed

An ovarian carcinoma cell line (OTN 11) was produced from the ascitic fluid of a patient with a moderately to well differentiated papilliferous cystadenocarcinoma of the ovary. The cell line was characterized using electron microscopy karyotyping, immunohistochemical techniques with monoclonal antibodies against keratins as epithelial markers, and the monoclonal antibodies OV-TL 3 and OC 125 as ovarian carcinoma markers. These techniques revealed the epithelial and adenocarcinomatous nature of the cell line and the presence of ovarian carcinoma-related surface markers. The adenocarcinomatous nature of the cell line also became apparent after heterotransplantation of cell suspensions into nude mice and nude rats, in which adenomatous tumor structures were formed. These xenografts had the same ultrastructural and immunohistochemical properties as the cell line. Despite the adenocarcinomatous character of the tumor the cultured cells release estradiol into the culture medium. We may conclude that OTN 11 is an ovarian carcinoma cell line which has retained highly differentiated functions, such as the production of an ovarian hormone. PMID:2463217

Poels, L G; Jap, P H; Ramaekers, F F; Scheres, J M; Thomas, C M; Vooijs, P G; Croes, H J; Mungyer, G

1989-02-01

230

Vascular mimicry in cultured head and neck tumour cell lines  

PubMed Central

Introduction Vascuologenesis is the de novo establishment of blood vessels and vascular networks from mesoderm-derived endothelial cell precursors (angioblasts). Recently a novel mechanism, by which some genetically deregulated and aggressive tumour cells generate "micro-vascular" channels without the participation of endothelial cells and independent of angiogenesis, has been proposed. This has been termed "vasculogenic mimicry" and has implications beyond angiogenesis and adds another layer of complexity to the current concept for the generation of tumour micro-circulation. We suggest this is common phenomenon in head and neck squamous cell carcinoma (HNSCC) cell lines and other aggressive tumour cell lines. We present experimental evidence of vasculogenic mimicry in HNSCC cell lines and compare them with other tumours and a positive control vascular cell line. Materials and methods The cell lines used were HUVEC, HN 2a, 2b (primary and metastatic tongue base squamous carcinoma cell line), HCT116 (colonic carcinoma cell line) and DU145 (prostate carcinoma cell line). Pilot experiments were undertaken to assess growth of a bank of tumour cell lines on (growth factor reduced) matrigel (Sigma) with standard media (DMEM with 10% Fetal Calf Serum). A functional growth assay was performed by preparing the appropriate cell suspension in serum free medium plated onto either bare plastic or a well pre-coated with growth factor reduced type 4 collagen analogues. Phase contrast photomicrographs were taken at 4 hours and 24 hours. Image analysis was performed; particular features of interest were two dimensional area (surrogate of growth and migration), branch points and end point measurements (surrogate of intercellular complexity). Results There were observable differences in growth of the cells on laboratory plastic and collagen matrix. Tumour cells formed capillary like networks similar to HUVEC cells. Metastatic HNSCC cells lines were found to have vasculogenic properties similar to HUVEC cell lines when compared to cell lines from their corresponding primary tumour. The endothelial growth factor antibodies used did not inhibit or stimulate cell growth when compared to control but did discourage vascular mimicry. Other tumour cell lines also displayed this property. Discussion Tumour "vasculogenic mimicry" must still be regarded as a controversial issue whose existence is not proven. The clinical importance of this phenomenon however, is that it does explain the lack of complete efficacy of current anti-angiogenic treatments due to the added layer of complexity. It provides a feasible mechanism of early tumour vascular supply which can co-exist and incorporate with later angiogenic mechanisms. We suggest that "vasculogenic mimicry" maybe a common neoplastic phenomena which appears to also be dictated by the cells micro-environment. Its existence also suggests a further process that of the development of tumour mosaic vessels as the neo-vasculature integrates with the existing endothelial lined systems.

2011-01-01

231

Characteristics of nine newly derived mesothelioma cell lines.  

PubMed

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 microns, and the doubling times varied from 31 to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte-monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and interleukin-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies. PMID:7695406

Pass, H I; Stevens, E J; Oie, H; Tsokos, M G; Abati, A D; Fetsch, P A; Mew, D J; Pogrebniak, H W; Matthews, W J

1995-04-01

232

[The effects of actovegin on cell proliferation of permanent lines].  

PubMed

The influence of Actovegin on proliferation activity and mitotic regimen of cells of permanent lines PK-15-IEKVM and BHK-21 clone 13/04 was investigated. Addition of Actovegin into growth media containing bovine serums of different components and concentrations stimulates cell proliferation. Conclusion has been made that Actovegin can be used in cell culture biotechnology. PMID:18411759

Gulevski?, A K; Trifonova, A V; Lavrik, A A

2008-01-01

233

Development of multidrug resistance in a canine lymphoma cell line.  

PubMed

New multidrug resistant cell lines developed from the canine B cell lymphoma cell line (GL-1) were characterized in terms of chemosensitivity to some antineoplastics and P-glycoprotein (Pgp) expression. GL-1 was continuously exposed to a culture medium containing gradually increasing levels of doxorubicin and the cells that could grow in the presence of doxorubicin were obtained. Chemosensitivity of these cells to various antineoplastics were investigated with or without verapamil, which reversed Pgp-mediated drug resistance. The expression of Pgp on the cells was also examined by Western blot analysis. As a result, three kinds of resistant cell lines, designated as GL-DOX60, 300, and 4000 were obtained. These cell lines showed stable proliferation in the medium containing 60, 300, and 4000 ng/ml, respectively. These cells were much more resistant to vincristine than doxorubicin. This resistance was strongly reversed by the presence of verapamil. On the other hand, cisplatin was effective enough in killing these derived cells. In the Western Blot analysis, some bands that reacted to the anti-human Pgp monoclonal antibodies were observed in GL-DOX4000. The cells derived from GL-1 have multidrug resistance potential mediated by canine Pgp. The cells produced in this experimental trial are considered to be useful models for various investigations on canine multidrug resistance. PMID:15766940

Uozurmi, K; Nakaichi, M; Yamamoto, Y; Une, S; Taura, Y

2005-06-01

234

Establishment and characterization of an oral melanoma cell line (ME)  

Microsoft Academic Search

A new cell line, ME, has been established from a melanoma of the palatal mucosa. The cultured monolayer of cells was fusiform and melanin-producing. The cells were highly tumorigenic and metastatic in nude mice. The xenographic tumors resembled the original tumor in morphology, melanin production, and the expression of S-100 and HMB-45 antigens. The metaphase karyotype of ME indicated multiple

Kuo-Wei Chang; Shu-Chun Lin; Shou-Yee Chao; Po-Cheung Kwan; Chun-Po Chiu; Yong-Kie Wong

2001-01-01

235

Evolution and ultrastructure of the bovine spermatogonia precursor cell line  

Microsoft Academic Search

The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in

Karl-Heinz Wrobel; Daniela Bickel; Richard Kujat; Margit Schimmel

1995-01-01

236

Clonal cell lines from the rat central nervous system  

Microsoft Academic Search

Five neuronal and a large collection of putative glial cell lines from the rat central nervous system have been established in clonal cell culture and partially characterised. These cells shed new light on the distribution of neurotransmitter synthesis and brain-specific antigens among nerve and glia.

D. Schubert; S. Heinemann; W. Carlisle; H. Tarikas; B. Kimes; J. Patrick; J. H. Steinbach; W. Culp; B. L. Brandt

1974-01-01

237

Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines  

Microsoft Academic Search

Purpose: Doxorubicin (DOX) is a commonly used anticancer drug which causes DNA damage and kills cancer cells mainly by apoptosis. However, the process leading to killing of cancer cells and the molecular basis of resistance to DOX are not well understood. To evaluate the role of p53 and the cellular effects of DOX on hepatoma cell lines, we examined three

Terence Lee; Tracy Lau; Irene Ng

2002-01-01

238

Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.  

PubMed

L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time. PMID:24240092

Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

2014-01-01

239

MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES  

SciTech Connect

A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

2009-05-08

240

Effects of ethanol on an intestinal epithelial cell line  

SciTech Connect

The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

Nano, J.L.; Cefai, D.; Rampal, P. (Laboratoire de Gastroenterologie et de Nutrition, U.E.R. de Medecine, Nice (France))

1990-02-01

241

DNA Fingerprinting of the NCI-60 Cell Line Panel  

PubMed Central

The National Cancer Institute’s NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Since many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also demonstrate that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

Lorenzi, Philip L.; Reinhold, William C.; Varma, Sudhir; Hutchinson, Amy A.; Pommier, Yves; Chanock, Stephen J.; Weinstein, John N.

2009-01-01

242

Cancer Stem Cell-Like Side Population Cells in Clear Cell Renal Cell Carcinoma Cell Line 769P  

PubMed Central

Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells as compared with other four cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

Yao, Zhi Jun; Chen, Xu; Guo, Sheng Jie; Mao, Xiao Peng; Wang, Dao Hu; Chen, Jun Xing; Qiu, Shao Peng

2013-01-01

243

Human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50 are admixtures of the human colon carcinoma cell line HCT 116  

Microsoft Academic Search

In two recently described human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50, derived from one tumour, we detected identical E-cadherin and ?-catenin gene mutations as in colon carcinoma cell line HCT 116. We demonstrate by HLA-typing, mutation analysis and microsatellite analysis that cell lines JROECL 47 and JROECL 50 are admixtures of the human colon adenocarcinoma cell line

B P L Wijnhoven; M G J Tilanus; A G Morris; S J Darnton; H W Tilanus; W N M Dinjens

2000-01-01

244

Sensing cell line to dioxin-type chemicals  

NASA Astrophysics Data System (ADS)

Detection dioxins-type chemicals by a sensing cell line containing luciferase reporter gene under the control of dioxin responsive elements was compared with the traditional ethoxyresorufin O-deethylase (EROD) induction. The result suggested that the luciferase induction was 30-fold more sensitive than EROD induction, the detection time was shorter for 68-hour and the detection procedure was also simpler. The data showed the sensing cell line can screen lots of samples and quickly semi-quantitate.

Zhang, Zhiren; Xu, Shunqing; Zhou, Yikai; Cai, Xiaokun; Liu, Zhiwei

2001-09-01

245

Screening Services – NCI-60 DTP Human Tumor Cell Line Screen  

Cancer.gov

The In Vitro Cell Line Screening Project (IVCLSP) is a dedicated service providing direct support to the DTP anticancer drug discovery program. The in vitro cell line screen was implemented in fully operational form in April of 1990. It required approximately five years (1985 - 1990) to develop, and persistence in the effort reflected dissatisfaction with the performance of prior in vivo primary screens. This project is designed to screen up to 3,000 compounds per year for potential anticancer activity.

246

Isoenzyme patterns in soybean- Nicotiana somatic hybrid cell lines  

Microsoft Academic Search

Zymograms obtained by polyacrylamide gel electrophoresis of alcohol dehydrogenase (EC 1.1.1.1.) and aspartate aminotransferase (EC 2.6.1.1.) from 5 different soybean-Nicotiana hybrid cell lines showed enzymatic characteristics derived from both parents. Variations in the zymogram of the cell lines were observed during a culture period of 8 months (more than 100 generations). These variations may be related to chromosomal loss from

L. R. Wetter

1977-01-01

247

Reliable in vitro studies require appropriate ovarian cancer cell lines  

PubMed Central

Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future.

2014-01-01

248

Modulation of melphalan cytotoxic activity in human melanoma cell lines.  

PubMed

The aim of the present study was to potentiate the cytotoxic effects of melphalan through pharmacological and physical modulators. The combination of the cytotoxic agent with ethacrynic acid, a glutathione-S-transferase pi (GST pi) inhibitor, or topotecan, a topoisomerase I inhibitor, or mild hyperthermia was investigated. The selected cell lines exhibited variable levels of expression of GST pi, DNA topoisomerase I and heat-shock proteins. Mild hyperthermia (42 degrees C) alone potentiated melphalan cytotoxicity, especially in the two cell lines exhibiting low basal levels of HSP70 expression. The combination of the GST inhibitor with melphalan resulted in a potentiation of drug cytotoxicity only in JR8 cells, one of the two cell lines which expressed high levels of GST pi mRNA and which were the less responsive to ethacrinic acid alone. A synergistic interaction between topotecan and melphalan was observed only in the cell lines expressing low levels of topoisomerase I even if all cell lines exhibited a comparable sensitivity to this agent. The results support an involvement of GST and DNA topoisomerase in cell defense and response to the alkylating agent. However, the variable potentiation of the cytotoxic effects of melphalan achieved in different cell systems suggests that factors other than the level of expression of the modulation target are responsible of such potentiation. PMID:8862730

Supino, R; Caserini, C; Orlandi, L; Zaffaroni, N; Silvestrini, R; Vaglini, M; Zunino, F

1996-07-01

249

Mammalian cell line developments in speed and efficiency.  

PubMed

Mammalian cell expression systems are the dominant tool today for producing complex biotherapeutic proteins. In this chapter, we discuss the basis for this dominance, and further explore why the Chinese hamster ovary (CHO) cell line has become the prevalent choice of hosts to produce most recombinant biologics. Furthermore, we explore some of the innovations that are currently in development to improve the CHO cell platform, from cell line specific technologies to overarching technologies that are designed to improve the overall workflow of bioprocess development. PMID:24196317

Estes, Scott; Melville, Mark

2014-01-01

250

Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines  

PubMed Central

Solanum nigrum is used in various traditional medical systems for antiproliferative, antiinflammatory, antiseizure and hepatoprotective activities. We have evaluated organic solvent and aqueous extracts obtained from berries of Solanum nigrum for antiproliferative activity on leukemic cell lines, Jurkat and HL-60 (Human promyelocytic leukemia cells). The cell viability after the treatment with Solanum nigrum extract was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Results indicated increased cytotoxicity with increasing extract concentrations. Comparative analysis indicated that 50% inhibitory concentration value of methanol extract is the lowest on both cell lines.

Gabrani, Reema; Jain, Ramya; Sharma, Anjali; Sarethy, Indira P.; Dang, Shweta; Gupta, S.

2012-01-01

251

Dengue2 virus infection of human mononuclear cell lines and establishment of persistent infections  

Microsoft Academic Search

Summary Twenty three human mononuclear cell lines including ten myelomonocytic cell lines, eight B cell lines and five T cell lines, were examined to determine whether they could be infected with dengue-2 virus. All the cell lines were infected with dengue-2 virus as determined by immunofluorescent staining and by virus titration of culture supernatant fluids. K 562, Jiyoye and Jurkat,

I. Kurane; U. Kontny; J. Janus; F. A. Ennis

1990-01-01

252

Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines  

PubMed Central

Background Taurolidine (TRD) represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines. Materials and methods Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas) were incubated with increasing concentrations of TRD (100 ?M, 250 ?M and 1000 ?M) for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining). Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC) and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death. Results All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines. Conclusion This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.

2010-01-01

253

BHD Tumor Cell Line and UOK257-2 wild type FLCN-restored Renal Cell Line  

Cancer.gov

Center for Cancer Research, Urologic Oncology Branch is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize kidney cancer tumor cell lines.

254

Lack of functional erythropoietin receptors of cancer cell lines.  

PubMed

Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation. PMID:17990315

Laugsch, Magdalena; Metzen, Eric; Svensson, Tanja; Depping, Reinhard; Jelkmann, Wolfgang

2008-03-01

255

Replication of Borna disease virus in cell cultures.  

PubMed

Borna disease (BD) virus from infected brain tissue of horses or rabbits could be grown in embryonic brain cells from rabbits or rats with high virus yields. The cells became persistently infected and could be subcultivated without loss of infectivity. Cocultivation of infected rabbit brain (ERB) cells with GMK-, Vero-, or MDCK-cells led to persistently infected cell lines. BD virus grown in MDCK cells after cocultivation became adapted to this cell type and could be used directly for further infection of MDCK cells. PMID:6772932

Herzog, S; Rott, R

1980-01-01

256

p53 is frequently mutated in Burkitt's lymphoma cell lines.  

PubMed Central

A panel of 12 Burkitt's lymphoma cell lines and four other B cell lines were tested for the presence of mutations in p53. Protein analysis using a mutant-specific antibody and sequencing of both cDNA and genomic DNA revealed changes relative to the standard p53 protein sequence in 12 of the 16 lines studied, including 10 of the BL lines. Mutation of p53 in the BL lines was usually accompanied by loss of the other allele of p53. Testing of the mutated p53 cDNAs for gain of transforming activity or loss of growth suppression activity showed that several of the BL mutants were functionally altered from wild-type p53. Images

Farrell, P J; Allan, G J; Shanahan, F; Vousden, K H; Crook, T

1991-01-01

257

Stem-like Cells in Bladder Cancer Cell Lines with Differential Sensitivity to Cisplatin  

PubMed Central

Background Recurrence is a common problem in bladder cancer; this has been attributed to cancer stem cells. In this study, we characterized potential cancer stem cell populations isolated from three cell lines that demonstrate different responses to cisplatin. Materials and Methods The ALDEFLUOR® assay was used to isolate cells from TCCSUP, T24, and 5637 cell lines, and these cells were evaluated for their ability to form colonies, differentiate, migrate and invade. Results The cell lines demonstrate a spectrum of aldehyde dehydrogenase high (ALDHHigh)populations that correlate with resistance to cisplatin. In the two resistant cell lines, T24 and 5637, the ALDHHigh cells demonstrate increased colony formation, migration, invasion, and ability to differentiate. The resistant T24 and 5637 cell lines may serve as models to investigate alternative therapies for bladder cancer.

Sarachine Falso, Miranda J.; Buchholz, Bruce A.; deVere White, Ralph W.

2013-01-01

258

Generation of stable Drosophila cell lines using multicistronic vectors  

PubMed Central

Insect cell culture is becoming increasingly important for applications including recombinant protein production and cell-based screening with chemical or RNAi libraries. While stable mammalian cell lines expressing a protein of interest can be efficiently prepared using IRES-based vectors or viral-based approaches, options for stable insect cell lines are more limited. Here, we describe pAc5-STABLEs, new vectors for use in Drosophila cell culture to facilitate stable transformation. We show that viral-derived 2A-like (or "CHYSEL") peptides function in Drosophila cells and can mediate the multicistronic expression of two or three proteins of interest under control of the Actin5C constitutive promoter. The current vectors allow mCherry and/or GFP fusions to be generated for positive selection by G418 resistance in cells and should serve as a flexible platform for future applications.

Gonzalez, Monika; Martin-Ruiz, Itziar; Jimenez, Silvia; Pirone, Lucia; Barrio, Rosa; Sutherland, James D.

2011-01-01

259

[Effect of NKG2D in eliminating hematological malignant cell lines by natural killer cells].  

PubMed

The aim of this study was to clarify whether NKG2D plays an activating role in eliminating hematological malignant cells lines by natural killer (NK) cells. Several hematological malignant cell lines (K562, NB4, Kasumi-1 THP-1, MV-4-11, MOLT-4, Jurkat, RS4; 11, Raji) were used as target cells. The expression levels of major histocompatibility complex class I (MHC I)-related molecules A/B (MICA, MICB), whose corresponding ligand was NKG2D, were detected in target cells by flow cytometry. Firstly, the target cell lines were co-incubated with carboxyfluorescein succinimidyl ester (CFSE) for 30 min. In the meanwhile, NK92MI, a kind of NK cell line, was co-incubated respectively with isotype control antibody or blocking antibody, the latter could block NKG2D specifically. Then, NK92MI cells were co-cultured with different target cell lines. After incubation for 2 h, the apoptotic ratio of each target cell line was detected by flow cytometry. The results demonstrated that there was a significant reduction of the apoptotic ratio in Kasumi-1, an acute myeloid leukemia cell line, when NK92MI cells were incubated with NKG2D blocking antibody previously. In contrast, the apoptotic ratio of other cell lines varied minimally. It is concluded that NKG2D can activate NK cells through inducing cytotoxicity to certain target cells. PMID:22541085

Wang, Wei; Gao, Li; Ma, Yi-Gai

2012-04-01

260

Novel cell lines established from pediatric brain tumors.  

PubMed

The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat "fingerprints" matching that of the patient's tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors. PMID:22120608

Xu, Jingying; Erdreich-Epstein, Anat; Gonzalez-Gomez, Ignacio; Melendez, Elizabeth Y; Smbatyan, Goar; Moats, Rex A; Rosol, Michael; Biegel, Jaclyn A; Reynolds, C Patrick

2012-04-01

261

Novel cell lines established from pediatric brain tumors  

PubMed Central

The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat “fingerprints” matching that of the patient’s tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors.

Xu, Jingying; Erdreich-Epstein, Anat; Gonzalez-Gomez, Ignacio; Melendez, Elizabeth Y.; Smbatyan, Goar; Moats, Rex A.; Rosol, Michael; Biegel, Jaclyn A.

2012-01-01

262

Three-dimensional cultured glioma cell lines  

NASA Technical Reports Server (NTRS)

Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

Gonda, Steve R. (inventor); Marley, Garry M. (inventor)

1991-01-01

263

Human embryonic stem cell lines derived from single blastomeres.  

PubMed

The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. In this proof-of-principle study, multiple biopsies were taken from each embryo using micromanipulation techniques and none of the biopsied embryos were allowed to develop in culture. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos. PMID:16929302

Klimanskaya, Irina; Chung, Young; Becker, Sandy; Lu, Shi-Jiang; Lanza, Robert

2006-11-23

264

Isolation and characterization of cancer stem-like cells from MHCC97H Cell Lines  

Microsoft Academic Search

ObjectiveTo identify and isolate CD133 positive cancer stem-like cells (CD133+ cells) from the highly invasive human hepatocellular carcinoma cell line(MHCC97H), and examine their potential for clonogenicity and tumorigenicity.

Shanyong Yi; Kejun Nan; Aihua Yuan; Chuangxin Lu

2009-01-01

265

Establishment and characterization of human B cell precursor-leukemia cell lines  

Microsoft Academic Search

A large number of continuous human leukemia cell lines have been established over the last three decades. Clearly, leukemia cell lines have become important research tools. Here, we have summarized the immunological, molecular and standard cytogenetic features of a panel of well characterized B cell precursor (BCP)-leukemia cell lines which were derived from patients with acute lymphoblastic\\/undifferentiated leukemia (ALL\\/AUL) or

Yoshinobu Matsuo; Hans G Drexler

1998-01-01

266

Growth inhibition of newly established human glioma cell lines by leukemia inhibitory factor  

Microsoft Academic Search

We have established three new cell lines deriving from malignant human gliomas. The cell lines were described in terms of both morphology and growth characteristics. Most cells in all three cell lines expressed the neuroepithelial marker protein GFAP. In terms of growth characteristics, the cells showed only slight differences. The cell lines showed no expression of the neural form of

Hartmut Halfter; Joachim Kremerskothen; Jürgen Weber; Ursula Hacker-Klom; Angelika Barnekow; E. Bernd Ringelstein; Florian Stögbauer

1998-01-01

267

HEL Cells: A New Human Erythroleukemia Cell Line with Spontaneous and Induced Globin Expression  

Microsoft Academic Search

A new human erythroleukemia cell line has been established. This line, designated HEL, is capable of spontaneous and induced globin synthesis, producing mainly Ggamma and Agamma chains. Embryonic chains (? , zeta ) and alpha chains are detectable in very small amounts; beta chains are undetectable. This line provides a new model system for studying aspects of erythroid cell differentiation

Paul Martin; Thalia Papayannopoulou

1982-01-01

268

Membrane Nanotubes in Urothelial Cell Line T24  

Microsoft Academic Search

Membrane nanotubes (also referred as tunnelling nanotubes—TNTs, nanotu- bules, cytonemes), that directly connect separated neighboring cells, may offer a very specific and effective way of intercellular transport and communication. Our experiments on T24 cell line show that TNTs can be divided into two types with respect to their biochemical and biophysical characteristics and the nature of their formation. As type

CHAPTER T HREE; Marusa Lokar; Sarka Perutkova; Veronika Kralj-Iglic; Peter Veranic

269

Chapter 3 Membrane Nanotubes in Urothelial Cell Line T24  

Microsoft Academic Search

Membrane nanotubes (also referred as tunnelling nanotubes—TNTs, nanotubules, cytonemes), that directly connect separated neighboring cells, may offer a very specific and effective way of intercellular transport and communication. Our experiments on T24 cell line show that TNTs can be divided into two types with respect to their biochemical and biophysical characteristics and the nature of their formation. As type I

Maruša Lokar; Šárka Perutková; Veronika Kralj-Igli?; Aleš Igli?; Peter Verani?

2009-01-01

270

In-line load cell for flexible strength member materials  

Microsoft Academic Search

An in-line load cell for flexible strength member materials is presented. An aluminum cylindrical body section houses two steel pins that perpendicularly pass through the longitudinal axis of the body section. Both steel pins extend from either side of the body section to provide four wrapping points for a flexible strength member to be tested. The load cell is placed

Joseph Liguore

1992-01-01

271

Epithelial Cell Line Expressing a Cystic Fibrosis Phenotype.  

National Technical Information Service (NTIS)

An airway epithelial cell line (CF/T43) was developed by infecting cultured cystic fibrosis (CF) airway epithelial cells with the pZIPneoSV(X)1/SV40T retrovirus and selecting for Genetian (G418) resistance and ion transport properties. The distinctive chl...

A. M. Jetten J. R. Yankaskas

1989-01-01

272

Differences in Arachidonic Acid Metabolism by Human Myelomonocytic Cell Lines.  

National Technical Information Service (NTIS)

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-gamma (IFN-gamma, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, ...

M. C. Madden S. Becker H. S. Koren M. Friedman

1992-01-01

273

Elimination of mycoplasma from human B-lymphoblastoid cell lines.  

PubMed

Intraperitoneal passage of human B-lymphoblastoid cell lines in nude mice was examined as a means of mycoplasma eradication. Recovery of viable cells from the mice was facilitated by immediate plating on feeder layers of human foreskin fibroblasts. In all cases, nude mouse passage for as little as 5 days was totally effective in removing all contaminating mycoplasma. PMID:6983518

Howell, D N; Machamer, C E; Cresswell, P

1982-11-01

274

Generation and characterization of a mouse lymphatic endothelial cell line  

Microsoft Academic Search

Lymphatic vessels, by channeling fluid and leukocytes from the periphery into lymph nodes, play a central role in the development of the immune response. Despite their importance in homeostasis and disease, the difficulties in enriching and culturing lymphatic endothelial cells limit studies of their biology. Here, we report the isolation, stabilization, and characterization of a mouse lymphatic endothelial cell line

Marina Sironi; Annarita Conti; Sergio Bernasconi; Anna M. Fra; Fabio Pasqualini; Manuela Nebuloni; Eleonora Lauri; Maida De Bortoli; Alberto Mantovani; Elisabetta Dejana; Annunciata Vecchi

2006-01-01

275

MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES  

EPA Science Inventory

We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

276

Taurine chloramine induces apoptosis in human osteosarcoma cell lines.  

PubMed

Although combination of surgery with chemotherapy has noticeably improved the survival rate of osteosarcoma patients, the application of anticancer drugs is still associated with significant adverse reactions, for instance acquisition of drug-resistant phenotypes, necessitating the development of new chemotherapeutical agents. Therefore, the aim of this study was to research, if taurine chloramine (NCT) induces apoptosis in the osteosarcoma cell lines HOS, MG-63, and SAOS-2. Proliferation of osteosarcoma cells was detected with the "EZ4U Cell Proliferation and Cyotoxicity Assay" showing a time- and dose-dependent cytotoxic effect of NCT on these cell lines. After 3 h of incubation all cell lines showed significantly less cells at 5.5 mM NCT solutions, after 6 h at concentrations of 1.1 and 2.2 mM. Acridine-orange fluorescence nuclear staining showed characteristic features of apoptosis. DNA fragmentation was detected via ELISA, showing significant results for HOS and MG-63 after 6 h at an NCT concentration of 3.3 mM. Results of JC-1 mitochondrial FACS analysis presented a significant increase in apoptotic cells after 6 h at 3.3 mM for the tested cell lines. Summarized, the results of this study indicate that NCT is a promising agent in osteosarcoma therapy. PMID:22674504

Pilz, Magdalena; Holinka, Johannes; Vavken, Patrick; Marian, Brigitte; Krepler, Petra

2012-12-01

277

Pluripotency network in porcine embryos and derived cell lines.  

PubMed

Huge amounts of work have been dedicated to the establishment of embryonic stem cell lines from farm animal species since the successful isolation of embryonic stem cells from the mouse and from the human. However, no conclusive results have been obtained so far, and validated lines have yet to be established in domestic animals. Many limiting factors have been suggested and need to be studied further to isolate truly pluripotent cell lines from livestock. In this review, we will discuss the difficulties in deriving and maintaining embryonic stem cell lines from farm animal embryos and how can this lack of success be explained. We will summarize results obtained in our laboratory regarding derivation of pluripotent cells in the pigs. Problems related to the identification of standard methods for derivation, maintenance and characterization of cell lines will also be examined. We will focus our attention on the need for appropriate stemness-related marker molecules that can be used to reliably investigate pluripotency in domestic species. Finally, we will review data presently available on functional key pluripotency-maintaining pathways in farm animals. PMID:22827355

Brevini, Tal; Pennarossa, G; Maffei, S; Gandolfi, F

2012-08-01

278

Differential effects of monastrol in two human cell lines.  

PubMed

The kinesin-related protein HsEg5 plays essential roles in mitotic spindle dynamics. Although inhibition of HsEg5 has been suggested as an aid in cancer treatment, the effects of such inhibition on human cells have not been characterized. Here we studied the effects of monastrol, an allosteric HsEg5 inhibitor, on AGS and HT29 cell lines and compared them to those of taxol. While both cell lines were similarly sensitive to taxol, AGS cells were more sensitive to monastrol. The differences in sensitivity were determined by the degree of inhibitory effect on cell proliferation, reversibility of monastrol-induced G2/M arrest, intracellular phenotypes and induction of apoptosis. In both cell lines, monastrol-induced apoptosis was accompanied by mitochondrial membrane depolarization and poly-ADP-ribose polymerase 1 cleavage. In AGS, but not HT29 cells, monastrol-induced apoptosis involved a prominent cleavage of procaspases 8 and 3. While in AGS cells, monastrol induced the formation of symmetric microtubule asters only, in HT29 cells, asymmetric asters were also formed, which may be related to specific HsEg5 functions in HT29 cells. PMID:15316655

Leizerman, I; Avunie-Masala, R; Elkabets, M; Fich, A; Gheber, L

2004-08-01

279

Metabolic characteristics of different malignant cancer cell lines.  

PubMed

Extracellular AMP inhibits cell proliferation of MCF-7 and MDA-MB-453 whereas cell proliferation of the highly malignant Novikoff cell line is not affected. In medium with low glucose supply MDA-MB-453 cells grow well, Novikoff cells are slightly inhibited and MCF-7 cells are totally unable to grow. Isoelectric focusing revealed that a glyclytic enzyme complex exists in all three cell lines. In addition to the glycolytic enzymes, c-Raf-kinase, adenylate kinase, and nucleoside diphosphate kinase are also found within the complex. The differences in glucose in dependence of the three cell lines can be explained by the different constitutions of shuttle enzymes. MDA-MB-453 and Novikoff cells contain cytsolic glycerol 3-phosphate dehydrogenase which is associated with glyceraldehyde 3-phosphate dehydrogenase within the glycolytic enzyme complex and which is responsible for the transport of cytoslic hydrogen in the mitochondria. MCF-7 and Novikoff cells contain the pI 7.8 form of malate dehydrogenase which couples glycolysis with glutaminolysis. PMID:9858895

Mazurek, S; Grimm, H; Wilker, S; Leib, S; Eigenbrodt, E

1998-01-01

280

Cystoisospora belli: In vitro multiplication in mammalian cells  

Microsoft Academic Search

Intracellular development of Cystoisospora belli was demonstrated in 4 different mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12h

M. B. Oliveira-Silva; E. Lages-Silva; D. V. Resende; A. Prata; L. E. Ramirez; J. K. Frenkel

2006-01-01

281

Basic protocols for zebrafish cell lines: maintenance and transfection.  

PubMed

Cell lines derived from zebrafish embryos show great potential as cell culture tools to study the regulation and function of the vertebrate circadian clock. They exhibit directly light-entrainable rhythms of clock gene expression that can be established by simply exposing cultures to light-dark cycles. Mammalian cell lines require treatments with serum or activators of signaling pathways to initiate transient, rapidly dampening clock rhythms. Furthermore, zebrafish cells grow at room temperature, are viable for long periods at confluence, and do not require a CO2-enriched atmosphere, greatly simplifying culture conditions. Here we describe detailed methods for establishing zebrafish cell cultures as well as optimizing transient and stable transfections. These protocols have been successfully used to introduce luciferase reporter constructs into the cells and thereby monitor clock gene expression in vivo. The bioluminescence assay described here lends itself particularly well to high-throughput analysis. PMID:17417032

Vallone, Daniela; Santoriello, Cristina; Gondi, Srinivas Babu; Foulkes, Nicholas S

2007-01-01

282

Reconstruction of endometrium from human endometrial side population cell lines.  

PubMed

Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1-7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45?) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ER?) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis. PMID:21712999

Cervelló, Irene; Mas, Aymara; Gil-Sanchis, Claudia; Peris, Laura; Faus, Amparo; Saunders, Philippa T K; Critchley, Hilary O D; Simón, Carlos

2011-01-01

283

Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines  

PubMed Central

Background Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Methodology/Findings Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. Conclusions/Significance The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

Crameri, Gary; Todd, Shawn; Grimley, Samantha; McEachern, Jennifer A.; Marsh, Glenn A.; Smith, Craig; Tachedjian, Mary; De Jong, Carol; Virtue, Elena R.; Yu, Meng; Bulach, Dieter; Liu, Jun-Ping; Michalski, Wojtek P.; Middleton, Deborah; Field, Hume E.; Wang, Lin-Fa

2009-01-01

284

Non-targeted radiation effects in vertebrate cell lines  

NASA Astrophysics Data System (ADS)

Radiation effects, such as bystander effects, hyper radiosensitivity/induced radioresistance (HRS/IRR) and adaptive response that are not related to direct DNA damage are now accepted. However the inter-relationship between them and the possible impact on the scientific basis for radiation protection are highly controversial. This thesis attempts to elucidate the mechanisms of some of these well known but little understood effects. Each paper examines some aspect of bystander effects, adaptive responses and HRS/IRR in an effort to understand how they vary with cell type, dose and time of exposure to single or multiple doses. All the effects involve non-linear dose effect curves and are mainly evident following low doses. Overall findings of the thesis include (1) A clear difference was observed between radioresistant, tumorigenic cell lines with mutant p53 gene expression, and radiosensitive, more normal, cell lines with wild type p53. In general death inducing bystander responses are induced in normal cell populations exposed to low doses of radiation while survival inducing IRR and adaptive responses are seen in the radioresistant tumorigenic cell lines. (2) A cohort of fish cell lines which demonstrated survival promoting bystander effects, also did not show a protective adaptive responses. (3) Adaptive responses traditionally occur when a large challenge dose is given 4--6hrs following low (10--100mGy) priming doses but this thesis shows that for the epithelial cell lines tested, the size of the priming dose (range 0.1--2Gy) does not appear to alter the size of the recovery response. Additionally increased survival could be detected in some cases when the challenge dose was given within one hour of the priming dose. The overall conclusion is that cell lines induce either a bystander response or a protective/adaptive response depending on genetic background and other factors. Care is needed in the interpretation of data generated from only one or two cell lines and in the extrapolation of mechanistic ideas based on one or two cell lines to other cell types or to the in vivo situation.

Ryan, Lorna

285

Artificial islets from hybrid spheroids of three pancreatic cell lines.  

PubMed

Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely ? cells, ? cells, ? cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a ?-cell line), ? TC1 clone 6 (an ?-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development. PMID:24815150

Jo, Y H; Jang, I J; Nemeno, J G; Lee, S; Kim, B Y; Nam, B M; Yang, W; Lee, K M; Kim, H; Takebe, T; Kim, Y S; Lee, J I

2014-05-01

286

Nitric Oxide-Dependent Apoptosis in Ovarian Carcinoma Cell Lines  

Microsoft Academic Search

Objective. In a recent study, we found different profiles of inducible nitric oxide synthase (iNOS) gene expression in the ovarian carcinoma cell lines OVCAR-3, HOC-7, and 2774 following stimulation by proinflammatory cytokines. The present study was performed to determine whether nitric oxide (NO) synthesis correlates with programmed cell death in these cells.Methods. NO-Dependent apoptosis was detected by DNA fragmentation analysis

Josef Rieder; Rolf Jahnke; Michaela Schloesser; Maja Seibel; Monika Czechowski; Christian Marth; Georg Hoffmann

2001-01-01

287

Beta-cell gene expression and functional characterisation of the human insulinoma cell line CM  

Microsoft Academic Search

Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on ‚-cells. In contrast, human insulinoma cell lines are rarely used because of diYculties in obtaining and culturing them for long periods. The aim of our study was to investigate, under diVerent experimental conditions, the

M G Baroni; M G Cavallo; M Mark; L Monetini; B Stoehrer; P Pozzilli

1999-01-01

288

Adaptation of an insect cell line ( Agallia constricta ) in a mammalian cell culture medium  

Microsoft Academic Search

Summary  An established insect cell line (AC20) from the leafhopperAgallia constricta has been adapted to a mammalian cell culture medium based on the formulation of two commercially available media. The cell\\u000a population doubling time of the adapted line in this medium is approximately 45 hr at 30C.

Arthur H. Mc Intosh; K. Maramorosch; C. Rechtoris

1973-01-01

289

Second-line treatment outcomes after first-line sunitinib therapy in metastatic renal cell carcinoma.  

PubMed

This study was conducted to evaluate the treatment outcomes associated with common second-line targeted therapies given after first-line sunitinib for metastatic renal cell carcinoma (mRCC). The sample comprised patients with mRCC (n = 257) who were receiving second-line everolimus, sorafenib, or temsirolimus between April 1, 2008, and February 29, 2011, after first-line sunitinib treatment. The patients were followed-up from the start of second-line treatment until treatment failure (defined as advancement to a third-line therapy or to mortality) or the last observation in the medical and pharmacy databases. Treatment failure was observed in 38.5% (n = 99) of cases: 20.2% of patients (n = 52) advanced a line of treatment; and 18.3% of patients (n = 47) died. Kaplan-Meier analysis indicated a statistical difference in time to treatment failure among the 3 second-line targeted therapies (log-rank test, P = .045). The estimated 1-year cumulative probabilities of treatment failure were 49.9% for everolimus, 68.4% for sorafenib, and 71.4% for temsirolimus. In a multivariate Cox proportional hazards model, a higher adjusted risk of treatment failure vs. everolimus was observed for both temsirolimus (hazard ratio [HR] 2.05 [95% CI, 1.26-3.35]; P = .004) and sorafenib (HR 1.77 [95% CI, 1.02-3.07]; P = .043). The results of this real-world data analysis suggest that the risk of second-line treatment failure after first-line sunitinib was significantly higher with temsirolimus and sorafenib compared with everolimus. PMID:22682982

Chen, Chi-Chang; Hess, Gregory P; Liu, Zhimei; Gesme, Dean H; Agarwala, Sanjiv S; Garay, Carlos C; Hill, Jerrold W; Guo, Amy

2012-12-01

290

Cytopathogenesis of Naegleria fowleri Thai strains for cultured human neuroblastoma cells.  

PubMed

The aim of this study is to evaluate cellular interaction between free-living amoebae Naegleria fowleri strains and mammalian target cells in vitro. Two Thai strains of N. fowleri; Khon Kaen strain from the environment and Siriraj strain from the patient's cerebrospinal fluid and the Center of Disease Control VO 3081 strain from Atlanta (US) were studied. Human neuroblastoma (SK-N-MC) and African Green monkey Kidney (Vero) cells were used as target cells. Each cell line was inoculated with each strain of N. fowleri at a ratio of 1:1 and observed for 7 days. The uninoculated target cells and each strain of N. fowleri were used as control. The numbers of the challenged and unchallenged cells as well as the free-living amoebae were counted three times by trypan blue exclusion method. The inoculation began when the amoebae attached to the cell membrane and ingested the target cells. In this study, extensive cytopathogenesis with many floating inoculated cells and abundant number of amoebae were observed. The destruction pattern of both inoculated SK-N-MC and Vero target cells were similar. Interestingly, SK-N-MC was more susceptible to N. fowleri strains than the Vero cell. In addition, N. fowleri Siriraj strain showed the highest destruction pattern for each target cell. Our findings suggest that the SK-N-MC should be used as a base model for studying the neuropathogenesis in primary amoebic meningoencephalitis patients. PMID:18214541

Tiewcharoen, Supathra; Malainual, Nat; Junnu, Virach; Chetanachan, Pruksawan; Rabablert, Jundee

2008-04-01

291

Establishment and characterization of five human small cell lung cancer cell lines from early tumor xenografts.  

PubMed

Five small-cell lung carcinoma (SCLC) cell lines were established from xenografted tumor lines. These tumor lines were established after transplantation into nude mice of primary tumors or metastatic foci obtained surgically, from untreated (IRSC-2M, IRSC6M, IRSC-10M and IRSC-61M) or treated patients (IRSC-74M). They were then set-up in culture as parallel cell lines. Histologically, these tumor lines were classified as being of the classic (IRSC-2M, IRSC-10M and IRSC-61M) or intermediate type (IRSC-6M and IRSC74M). Four of these 5 SCLC cell lines grew as floating cell aggregates, while one (IRSC6M) grew as an adherent cell monolayer. Growth rates were slow (doubling times ranged between 120 and 194 h) but could be accelerated (67 to 144 h) by cultivating cells in medium mixed (v/v) with self-conditioned medium. Electron microscopical examination revealed that all SCLC cell lines contained dense core granules, characteristic of their neuroendocrine origin. These cell lines formed colonies in agarose with colony forming efficiencies ranging from 0.02-0.36%. The classic-type cell lines retained their tumorigenic capacity when re-injected intracranially into naive nude mice, whereas the intermediatetype cells did not. Cytogenetic analysis confirmed the human origin of SCLC xenografts and cultured cell lines. Various numerical and structural chromosome abnormalities were found, with deletion in the short arm of chromosome 3 being the most common (4 of the 5 cell lines). Deletions in or loss of the chromosome 10 were also observed. Oncogene expression was studied in 3 representative cell lines (IRSC-10M, IRSC-2M and IRSC-74M). L-myc was overexpressed only in IRSC-74M, while the GRP gene was overexpressed in the classic (IRSC-2M and IRSC-10M) but not in the intermediate-type cells (IRSC-74M). The Ki-ras oncogene was overexpressed in the 3 cell lines, while c-myc, N-myc, Ha-ras, N-ras, erb B2 and sis were not detected in any of them. The 3 cell lines weakly expressed the MDR1 gene, while the GST-pi gene was not expressed. These cell lines constitute a multifaceted well-characterized in vitro model for studying the biology of these phenotypically diverse cancer cells. PMID:7847823

Arvelo, F; Poupon, M F; Le Chevalier, T

1994-01-01

292

Identity of some human bladder cancer cell lines.  

PubMed

Recent reports on transfection of mouse cells with DNA from the established human urinary bladder cancer cell lines T24, J82 and EJ (MGH-U1), and the presence of an identical genetic modification in T24 and EJ cells have led us to examine the identity of these and other cultures of urothelial origin. By the criteria of HLA-A-B-C typing 7 and isozyme analysis, we conclude that EJ (MGH-U1) and some cultures of J82 are in fact T24 cells. However, five other bladder cancer cell lines, J82 (CO'T), RT4, RT112, TCCSuP and SCaBER, are clearly distinct from T24 by HLA typing (ref. 7) and/or isozyme patterns. PMID:6823318

O'Toole, C M; Povey, S; Hepburn, P; Franks, L M

1983-02-01

293

A cell line from an anaplastic transitional cell carcinoma of human urinary bladder.  

PubMed

A cell line, TCCSUP, derived from an undifferentiated, Grade IV transitional cell carcinoma is described. The karyotype showed an abnormal distribution of chromosomes, with no obvious modal number. Distinct marker chromosomes were observed in both early and late in vitro passages. These cells have been subcultured over 50 times during a 20-month period. TCCSUP differs in certain morphological and immunological features from other cell lines from transitional cell carcinomas. PMID:836756

Nayak, S K; O'Toole, C; Price, Z H

1977-02-01

294

The ‘monocytic’ cell line I937 displays typical B cell characteristics  

Microsoft Academic Search

The monocytic cell line I937 was derived from U937 by sorting for cells with high expression of MHC class II molecules. Further analysis of these class II molecules revealed the presence of the HLA-DR3 subtype suggesting that the cell line was a potential candidate for testing antigen presentation to T cells restricted by HLA-DR3. We found that the T cell

I. G. Reischl; E.-M. Pöllabauer; S. Peiritsch; S. Schlager; P. Gladstone; W. C. Vooijs; M. Woisetschläger; G. C. Mudde

1996-01-01

295

Cell surfactomes of two endometrial epithelial cell lines that differ in their adhesiveness to embryonic cells.  

PubMed

Adhesiveness of the endometrial epithelium to an embryo plays a critical role in the initiation of pregnancy. Loss or gain of adhesiveness also dictates the potential of endometrial epithelial cells to metastasize, an event that can result from certain genetic insults. A proteomics-based exploration of the "adhesiveness" these epithelial cells was employed that could identify targets that could disrupt embryo-endometrium interactions and/or metastasis of endometrial cancer cells. The present study defined the surfactomes of two human endometrial epithelial cell lines known for their differential adhesiveness to embryonic cells. Comparative, two-dimensional electrophoretic analysis of the surfactomes of RL95-2 (exhibiting higher adhesiveness to the embryonic cell line JAr) and HEC-1A (exhibiting reduced adhesiveness to JAr cells) revealed 55 differentially enriched proteins. Of these, 10 proteins were identified by MALDI-TOF/TOF or LC-MS/MS. TUBB2C, ADAMTS3, and elongation factor beta were more abundant on the HEC-1A cell surface whereas HSP27, HSPA9, GP96, CRT, Tapasin-ERP57, PDI, and ?-actin were more abundant on the RL95-2 cell surface. Nano LC-MS/MS was also employed to generate a more comprehensive surfactomes of RL95-2 and HEC-1A. The study also demonstrated a pro-adhesive role of CRT and HSPA9 and an anti-adhesive role of TUBB2C populations found on the cell surface. In brief, this study identifies the cell-surface protein complements of two human endometrial epithelial cell lines, and reveals the role of three proteins in heterotypic cell adhesion. PMID:24415223

Bhagwat, Sonali R; Redij, Tejashree; Phalnikar, Kruttika; Nayak, Sumeet; Iyer, Swati; Gadkar, Sushama; Chaudhari, Uddhav; Kholkute, Sanjeeva D; Sachdeva, Geetanjali

2014-04-01

296

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line.  

PubMed

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF). PMID:4006009

Crespi, C L; Altman, J D; Marletta, M A

1985-05-01

297

Expression of the somatostatin gene in human astrocytoma cell lines.  

PubMed Central

Somatostatin (somatotropin release-inhibiting hormone; SRIH) has been demonstrated in neurons of the central nervous system (CNS) as well as in endocrine cells of the pancreas and gastrointestinal tract and can suppress various immune functions including lymphocyte proliferation, immunoglobulin synthesis, and cytokine production. Since astrocytes possess antigen-presenting activity and can secrete a wide array of immunoregulatory and inflammatory cytokines, we studied SRIH gene expression in both astrocyte cell lines and mitogen-stimulated peripheral blood mononuclear leukocytes from healthy donors. We now report by means of a complementary DNA-based reverse transcription PCR that differential levels of SRIH mRNA were expressed in 9 of 11 human astrocytoma cell lines tested but were undetectable in activated peripheral blood mononuclear leukocytes as well as in a variety of human lymphocyte and monocyte cell lines. The synthesis and secretion of SRIH protein by astrocytoma cells that expressed SRIH transcripts were confirmed by specific radioimmunoassay of cell culture fluids. These findings support the notion that SRIH gene expression occurs in human astrocytoma cells but not in mature lymphoid cells of the immune system.

Mercure, L; Tannenbaum, G S; Schipper, H M; Phaneuf, D; Wainberg, M A

1996-01-01

298

Neuroectoderm-associated antigens on Ewing's sarcoma cell lines.  

PubMed

The histogenesis of Ewing's sarcoma, the second most frequent primary bone tumor in humans, remains controversial. Ten Ewing cell lines were analyzed by immunological methods. Surface antigens recognized on Ewing cells were found to be related to the neuroectoderm lineage. They included ganglioside GD2, a marker of neuroectodermal tissues and tumors, and an acidic glycolipid detected by monoclonal antibody HNK-1 in the nervous system. The P61 rat monoclonal antibody that reacts with a peptide moiety of neural cell adhesion molecule (N-CAM) and a rabbit antiserum raised to purified mouse N-CAM also stained Ewing cells. Flow cytometry analysis performed using these reagents allowed the definition of four distinct Ewing phenotypes: all reagents equally stained group 1 lines; group 2 lines were strongly reactive with anti-N-CAM reagents, by contrast with a fainter staining with HNK-1 and anti-GD2 antibodies; all reagents but P61 were strongly reactive with group 3 lines; in group 4, Ewing lines were stained by P61 but only poorly by the anti-N-CAM antiserum. Several antibodies to melanoma and neuroblastoma associated antigens including two monoclonal antibodies to the nerve growth factor receptor were also found to react with Ewing cells. By contrast, all antibodies detecting antigens specifically expressed in hematopoietic cell lineages were totally unreactive. HLA class II antigens were never detected while the level of expression of class I antigens varied to a large extent. Ewing cells are characterized by a specific t(11;22)(q23-24;q12) translocation also observed in neuroepithelioma, a neuroectodermal tumor. Thus, Ewing's sarcoma cells share antigenic and karyotypic features with derivatives of the neuroectoderm possibly indicating a related histogenesis. PMID:3024814

Lipinski, M; Braham, K; Philip, I; Wiels, J; Philip, T; Goridis, C; Lenoir, G M; Tursz, T

1987-01-01

299

Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies  

PubMed Central

Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

Krampe, Britta

2010-01-01

300

Molecular cytogenetic analysis of breast cancer cell lines  

PubMed Central

The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at http://www.path.cam.ac.uk/~pawefish. © 2000 Cancer Research Campaign

Davidson, J M; Gorringe, K L; Chin, S-F; Orsetti, B; Besret, C; Courtay-Cahen, C; Roberts, I; Theillet, C; Caldas, C; Edwards, P A W

2000-01-01

301

[Cytogenetic studies on 3 esophageal cancer cell lines].  

PubMed

The cytogenetic studies on three esophageal cancer cell lines established in China were done. Thirty metaphases showing suitable chromosome length and good G-banding pattern from each of the cell lines were chosen and subjected to karyological analysis. The typical karyotypes from these cell lines were listed, and the variation of chromosome number as well as the morphological characteristics, possible sources and the incidence of the marker chromosomes were analysed. Eca 109 is the cell line first established and used extensively in China. A strictly regular karyotype pattern was found in it: the modal number of chromosomes being 63-64; the number of each type of chromosome varying between 1-5; a distal deletion of short arm of chromosome No. 1 being discernible in all metaphases, with break sites located within 1p22-1p33. Also a distal deletion of long arm of chromosome No. 4 was usually visible. There were seven marker chromosomes with high incidence. Among them, M1 marker chromosome was a large subacrocentric chromosome which was observed in the early passages of this cell line. The chromosome number of Ec 17 cell line was usually subtetraploid. In addition to the numerical variation in some of the chromosomes, six marker chromosomes were usually observed. Among them, M1 involved reciprocal translocation between chromosome No. 1 and No. 4. M3 of Ec 17 was in correspondence with M5 of Eca 109. Both were Rob (13q;14q). The chromosome number of Ec 56 was usually subtetraploid, and in addition to the numerical variation in some of the chromosomes, seven marker chromosomes were usually observed. PMID:3477417

Li, S D; Zhang, L R; Luo, X; Ao, P

1987-03-01

302

Generation of cell lines for monoclonal antibody production.  

PubMed

Monoclonal antibodies (mAbs) represent the largest group of therapeutic proteins with 30 products approved in the USA and hundreds of therapies currently undergoing clinical trials. The complex nature of mAbs makes their development as therapeutic agents constrained by numerous criteria such as quality, safety, regulation, and quantity. Identification of a clonal cell line expressing high levels of mAb with adequate quality attributes and generated in compliance with regulatory standards is a necessary step prior to a program moving to large-scale production for clinical material. This chapter outlines the stable transfection technology that generates clonal cell lines for commercial manufacturing processes. PMID:24515472

Alvin, Krista; Ye, Jianxin

2014-01-01

303

Isolation of three stem cell lines from human sacrococcygeal teratomas.  

PubMed

Sacrococcygeal teratomas (SCTs) are benign tumours of the newborn with absolute indication for surgery directly after birth. We recently described the presence of stem cells positive for the stem cell markers nanog and Oct4 in SCTs. Here we report the isolation of three stem cell lines from three different SCTs. Cells were propagated in mesenchymal or in embryonic stem cell medium. Non-clonal homogeneous stem cell lines were obtained after two to three passages and characterized in vitro by immunocytochemistry, RT-PCR, western blot, FACS analysis, and metaphase spreads. The differentiation potential was tested in vitro and in vivo. The isolated cell lines, which we refer to as human sacrococcygeal teratoma stem cells (hSctSCs), express nanog, Oct4 and stella, and are negative for malignancy markers alpha-fetoprotein and carcinoembryonic antigen. They can be induced in vitro to express neuronal, osteogenic, and chondrogenic traits. After grafting in vivo, spontaneous integration into the neural crest of the chick embryo and teratoma formation in the nude mouse were obtained. Our results indicate that SCTs are derived from remnants of the epiblast-derived primitive streak, which in the human embryo normally regresses but forms teratomas in children affected with SCT. The hSctSCs therefore may be comparable to mouse epiblast-derived stem cells (EpiSCs) and share characteristic features with human embryonic stem (hES) cells. Thus, SCT tissue obtained after surgery appears to be a novel source for the generation of human stem cells without the ethical implications associated with hES cells. PMID:19142973

Busch, Christian; Bareiss, Petra M; Sinnberg, Tobias; Just, Lothar; Wehrmann, Manfred; Fuchs, Jörg; Garbe, Claus; Drews, Ulrich

2009-03-01

304

In Vitro Expression of the Endothelial Phenotype: Comparative Study of Primary Isolated Cells and Cell Lines, Including the Novel Cell Line HPMEC-ST1.6R  

Microsoft Academic Search

Endothelial cell lines are commonly used in in vitro studies to avoid problems associated with the use of primary endothelial cells such as the presence of contaminating cells, the difficulty in obtaining larger numbers of cells, as well as the progressive loss of cell viability and expression of endothelial markers in the course of in vitro propagation. We have analyzed

Ronald E. Unger; Vera Krump-Konvalinkova; Kirsten Peters; C. James Kirkpatrick

2002-01-01

305

Extremely low-frequency electromagnetic fields cause DNA strand breaks in normal cells  

PubMed Central

Background Extremely low frequency electromagnetic fields aren’t considered as a real carcinogenic agent despite the fact that some studies have showed impairment of the DNA integrity in different cells lines. The aim of this study was evaluation of the late effects of a 100 Hz and 5.6 mT electromagnetic field, applied continuously or discontinuously, on the DNA integrity of Vero cells assessed by alkaline Comet assay and by cell cycle analysis. Normal Vero cells were exposed to extremely low frequency electromagnetic fields (100 Hz, 5.6 mT) for 45 minutes. The Comet assay and cell cycle analysis were performed 48 hours after the treatment. Results Exposed samples presented an increase of the number of cells with high damaged DNA as compared with non-exposed cells. Quantitative evaluation of the comet assay showed a significantly (<0.001) increase of the tail lengths, of the quantity of DNA in tail and of Olive tail moments, respectively. Cell cycle analysis showed an increase of the frequency of the cells in S phase, proving the occurrence of single strand breaks. The most probable mechanism of induction of the registered effects is the production of different types of reactive oxygen species. Conclusions The analysis of the registered comet indices and of cell cycle showed that extremely low frequency electromagnetic field of 100 Hz and 5.6 mT had a genotoxic impact on Vero cells.

2014-01-01

306

Establishment of new multidrug-resistant human osteosarcoma cell lines.  

PubMed

Multidrug-resistant clones of human osteosarcoma MNNG/HOS and MG63 cells were isolated by stepwise selection on exposure to increasing doses of doxorubicin (DXR). The final clones MNNG/HOS/DXR1000 and MG63/DXR1000, established after ethylmethane sulfonate mutagenesis, showed 96-fold and 121-fold higer resistance to DXR than their parental cell lines. They were also cross-resistant to vincristine, but not to cisplatinum or methotrexate. The levels of multidrug-resistance-1 (MDR1) mRNA expression increased gradually according to the concentration of DXR in both cell lines. Although the parental MNNG/HOS cells expressed a low level of MDR1 mRNA, the parental MG63 cells showed no MDR1 expression. The IC50 values of MNNG/HOS and its resistant variant to DXR were higher than those of MG63 and its resistant clone. Multidrug-resistant associated protein (MRP) mRNA expression was detected in MNNG/HOS or MG63 parental cell lines, and in their resistant variants. MG63 and its resistant variants revealed stable expression of MRP, whereas the resistant phenotype of MNNG/HOS showed decreased MRP expression, compared to its parental cell line. No alteration in the levels of hepatocyte growth factor (HGF) or its receptor c-MET was recognized between parental lines and their resistant variants. The results indicate that our DXR-resistant variants of MNNG/HOS and MG63 reveal a classical MDR phenotype and can offer a model with which to investigate the mechanisms of multidrug resistance in osteosarcoma. PMID:10854558

Oda, Y; Matsumoto, Y; Harimaya, K; Iwamoto, Y; Tsuneyoshi, M

2000-01-01

307

Mechanisms of resistance to azacitidine in human leukemia cell lines.  

PubMed

The DNA methylation inhibitor azacitidine (5-azacytidine) is used against myelodysplastic syndrome and acute myeloid leukemia, but drug resistance is an ongoing, intractable problem. To investigate resistance mechanisms, we generated two azacitidine-resistant cell lines, THP-1/AR and HL60/AR, and studied genetic disparities between them and their corresponding parental lines. In cells treated with azacitidine, significant mitotic variations were noted in parental cells which were absent in resistant cells, suggesting that resistance arises from negating azacitidine-mediated activation of apoptosis signaling and reestablishing G2/M checkpoint. Importantly, both resistant cell lines have common point mutations in the uridine-cytidine kinase 2 (UCK2) gene, which encodes the rate-limiting enzyme of the azacitidine activation pathway. Forced expression of mutated UCK2 in parental THP-1 cells abrogated azacitidine-induced apoptosis, whereas overexpression of wild type UCK2 in resistant THP-1/AR cells restored sensitivity to azacitidine, implying that UCK2 gene mutations perturb azacitidine activation and advance azacitidine resistance. Our study provides new insights into azacitidine resistance and establishes models useful in developing effective strategies to overcome it. PMID:24368162

Sripayap, Piyanuch; Nagai, Tadashi; Uesawa, Mitsuyo; Kobayashi, Hiroyuki; Tsukahara, Tomonori; Ohmine, Ken; Muroi, Kazuo; Ozawa, Keiya

2014-04-01

308

Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines  

PubMed Central

Canine and human osteosarcoma (OSA) have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

Legare, Marie E.; Bush, Jamie; Ashley, Amanda K.; Kato, Taka; Hanneman, William H.

2011-01-01

309

Evaluating cell lines as tumour models by comparison of genomic profiles  

PubMed Central

Cancer cell lines are frequently used as in vitro tumour models. Recent molecular profiles of hundreds of cell lines from The Cancer Cell Line Encyclopedia and thousands of tumour samples from the Cancer Genome Atlas now allow a systematic genomic comparison of cell lines and tumours. Here we analyse a panel of 47 ovarian cancer cell lines and identify those that have the highest genetic similarity to ovarian tumours. Our comparison of copy-number changes, mutations and mRNA expression profiles reveals pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and high-grade serous ovarian cancer tumour samples. We identify several rarely used cell lines that more closely resemble cognate tumour profiles than commonly used cell lines, and we propose these lines as the most suitable models of ovarian cancer. Our results indicate that the gap between cell lines and tumours can be bridged by genomically informed choices of cell line models for all tumour types.

Domcke, Silvia; Sinha, Rileen; Levine, Douglas A.; Sander, Chris; Schultz, Nikolaus

2013-01-01

310

Decorin Suppresses Bone Metastasis in a Breast Cancer Cell Line  

Microsoft Academic Search

Decorin, the prototype of an expanding family of small leucine-rich proteoglycans, is involved in a number of cellular processes including matrix assembly, fibrillogenesis and the control of cell proliferation. In this study, we investigated the role of decorin in suppressing tumor aggressiveness and bone metastases. We used a metastatic breast cancer cell line, MDA-MB-231, to show that decorin causes marked

Kentaro Araki; Hiroki Wakabayashi; Ken Shintani; Joji Morikawa; Akihiko Matsumine; Katsuyuki Kusuzaki; Akihiro Sudo; Atsumasa Uchida

2009-01-01

311

Celecoxib increases retinoid sensitivity in human colon cancer cell lines  

Microsoft Academic Search

Retinoid resistance has limited the clinical application of retinoids as differentiation-inducing and apoptosis-inducing drugs.\\u000a This study was designed to investigate whether celecoxib, a selective COX-2 inhibitor, has effects on retinoid sensitivity\\u000a in human colon cancer cell lines, and to determine the possible mechanism of said effects. Cell viability was measured using\\u000a the MTT assay. Apoptosis was detected via Annexin-V\\/PI staining

Jian-Pei Liu; Hong-Bo Wei; Zong-Heng Zheng; Wei-Ping Guo; Jia-Feng Fang

2010-01-01

312

Characterization of Butyrate Uptake by Nontransformed Intestinal Epithelial Cell Lines  

Microsoft Academic Search

Butyrate (BT) is one of the main end products of anaerobic bacterial fermentation of dietary fiber within the human colon.\\u000a Among its recognized effects, BT inhibits colon carcinogenesis. Our aim was to characterize uptake of BT by two nontransformed\\u000a intestinal epithelial cell lines: rat small intestinal epithelial (IEC-6) and fetal human colonic epithelial (FHC) cells.\\u000a Uptake of 14C-BT by IEC-6

Pedro GoncalvesJoao; João R. Araújo; Fátima Martel

2011-01-01

313

The NCI60 human tumour cell line anticancer drug screen  

Microsoft Academic Search

The US National Cancer Institute (NCI) 60 human tumour cell line anticancer drug screen (NCI60) was developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumours in anticancer drug screening. This screening model was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumour-cell

Robert H. Shoemaker

2006-01-01

314

Conditionally Immortalized Neural Cell Lines: Potential Models for the Study of Neural Cell Function  

PubMed

Studies on primary cell cultures have contributed significantly to our understanding of neural cell function. Nevertheless, for many studies the value of these primary cell cultures has been limited by the time the cultures survive in vitro, the quantity of cellular material available for analysis, and the need to prepare the cells on a regular basis from fresh tissue. Techniques for immortalizing cells have existed for some time, but the repertoire of immortalizing genes has grown significantly. This has expanded our ability to generate useful cell lines of specific neural types that are better models of the in vivo phenotype than previously. The constitutive expression of oncogenes keeps cells in a proliferative state that could lead to the loss of differentiated gene expression and function. An appealing improvement of immortalization methodology is the use of temperature-sensitive oncogenes that generate cell lines that can proliferate at a permissive temperature and "differentiate" at a nonpermissive temperature. The proliferation of such conditionally immortalized cell lines can be suppressed simply by increasing the temperature. Cell lines maintained at the nonpermissive temperature can enter into a stage in which they express differentiated properties of the cell. The potential ability of conditionally immortalized neural cell lines to accurately reflect their in vivo function has now been demonstrated on several occasions through transplantation experiments. In this report, the generation of these cell lines is described along with a discussion of their potential applications in neurobiology. PMID:8954859

Bongarzone; Foster; Byravan; Verity; Landry; Schonmann; Amur-Umarjee; Campagnoni

1996-12-01

315

Optimized protocol for derivation of human embryonic stem cell lines.  

PubMed

For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure. PMID:22614996

Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel

2012-09-01

316

[Establishment and biological characterization of human medulloblastoma cell lines].  

PubMed

Two cell lines of human medulloblastoma (ONS-76 and ONS-81) were established, and their biological characteristics were investigated. The cell line, ONS-76, was established from a tumor specimens obtained from a large cerebellar tumor of a 2-year-old girl. The pathological diagnosis was a typical medulloblastoma. The other cell line, ONS-81, was derived from a metastatic tumor in right frontal lobe of a 9-year-old girl. The tumor specimens were minced into fragments approximately 1 mm in diameter and cultured in plastic culture flasks in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 50% patients serum. The cells growing as a monolayer were subcultured in RPMI 1640 supplemented with 10% FCS and initially with L-glutamine, sodium pyruvate, and nonessential amino acid. Microscopically, both cultured cells exhibited various morphological appearances, and this morphological heterogeneity seemed to be specific for medulloblastoma cells. The in vitro population doubling time of ONS-76 and ONS-81 were 18.6 and 19.2 hr, respectively. The ONS-76 and ONS-81 cells formed subcutaneous tumors in nude mice as serial transplantable xenograft, and these tumors had a microscopic appearance similar to that of the original medulloblastoma. Ultrastructurally++, the cultured cells showed primitive, undifferentiated appearance, and no neuronal or glial structures were not seen. Immunohistochemical studies showed that both cells expressed neuron-specific enolase (NSE) and neurofilament protein (NFP 200 K, 145 K), but glial fibrillary acidic protein (GFAP) and S-100 protein were not detected. The NFP immunoreactivities of both cultured cells were demonstrated as abnormal perinuclear deposits.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2818910

Yamada, M; Shimizu, K; Tamura, K; Okamoto, Y; Matsui, Y; Moriuchi, S; Park, K; Mabuchi, E; Yamamoto, K; Hayakawa, T

1989-07-01

317

Gene expression profiling of cell lines derived from T-cell malignancies  

Microsoft Academic Search

The expression profiles of eight cell lines derived from T-cell malignancies were compared to CD4-positive T-cells using cDNA microarray technology. Unsupervised hierarchical clustering of 4364 genes demonstrated substantial heterogeneity resulting in four distinct groups. While no genes were found to be uniformly up- or down-regulated across all cell lines, we observed 111 over-expressed genes (greater than two-fold) and 1118 down-regulated

G. Chris Fillmore; Zhaosheng Lin; Sandra D Bohling; Ryan S Robetorye; Chan-Hwan Kim; Stephen D Jenson; Kojo S. J Elenitoba-Johnson; Megan S Lim

2002-01-01

318

Susceptibility of MHC Class I Expressing Extravillous Trophoblast Cell Lines to Killing by Natural Killer Cells  

Microsoft Academic Search

Purified human first trimester extravillous trophoblast (EVT) cell lines HTR-8 and HT-116 were examined for susceptibility to natural killer (NK) cell-mediated lysis. Based upon nucleic acid sequencing of an amplified fragment of cDNA, Western blot analysis and immunostaining of fixed and live cells, it was shown that both EVT cell lines expressed HLA-G mRNA and protein within the cytoplasm when

M. Zdravkovic; G. Aboagye-Mathiesen; M.-J. Guimond; H. Hager; P. Ebbesen; P. K. Lala

1999-01-01

319

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines  

PubMed Central

Background Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

2011-01-01

320

Analysis of p53 in human cutaneous melanoma cell lines.  

PubMed

Mutations in the p53 tumour suppressor gene have been detected in a variety of human malignancies. Mutations have been found predominantly in conserved regions two to five. Our aim was to analyse p53 at the protein and DNA level in seven melanoma cell lines of cutaneous origin (HMB-2, DX3, LT5.1, MJM, SK23, A375P and A375M), including two parental/metastatic derivatives (A375P and A375M; DX3 and LT5.1). By immunohistochemical staining with three mouse monoclonal antibodies and a rabbit polyclonal serum, it was possible to observe differential nuclear expression of p53. The quantitation of p53 protein levels by ELISA correlated with the nuclear staining pattern. Western blotting showed an intact p53 protein in all cell lines; p53 was polymorphic in three cell lines (MJM, A375P and A375M). DNA sequencing studies showed that all cell lines had wild type p53. These results suggest that p53 is unlikely to play a significant role in the genesis of cutaneous melanoma. PMID:8152807

Montano, X; Shamsher, M; Whitehead, P; Dawson, K; Newton, J

1994-05-01

321

Mammalian Cell Lines Specifically Deficient in O-Linked Glycosylation.  

National Technical Information Service (NTIS)

Genetically modified cell lines that express a UDP-galactose 4-epimerase (GALE) capable of interconverting UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc), but essentially incapable of interconverting UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-ace...

H. M. Holden J. B. Thoden J. L. Fridovich-Kell J. M. Schulz M. Krieger

2004-01-01

322

Use of Cell Lines in the Investigation of Pharmacogenetic Loci  

PubMed Central

Drug response and toxicity, complex traits that are often highly varied among individuals, likely involve multiple genetic and non-genetic factors. Pharmacogenomic research aims to individualize therapy in an effort to maximize efficacy and minimize toxicity for each patient. Cell lines can be used as a model system for cellular pharmacologic effects, which include, but are not limited to, drug-induced cytotoxicity or apoptosis, biochemical effects and enzymatic reactions. Because severe toxicities may be associated with drugs such as chemotherapeutics, cell lines derived from healthy individuals or patients provide a convenient model to study how human genetic variation alters response to these drugs that would be unsafe or unethical to administer to human volunteers. In addition to the traditional candidate gene approaches that focus on well-understood candidate genes and pathways, the availability of extensive genotypic and phenotypic data on some cell line models has begun to allow genome-wide association (GWA) studies to simultaneously test the entire human genome for associations with drug response and toxicity. Though with some important limitations, the use of these cell lines in pharmacogenomic discovery demonstrates the promise of constructing a more comprehensive model that may ultimately integrate both genetic and non-genetic factors to predict individual response and toxicity to anticancer drugs.

Zhang, Wei; Dolan, M. Eileen

2009-01-01

323

Molecular cytogenetic analysis of the monoblastic cell line U937  

Microsoft Academic Search

Previous reports on the analysis of the human monoblastic cell line U937 had described several sublines containing unidentified rearrangements and marker chromosomes. In order to determine the true nature of the rearrangements, conventional banding analysis was carried out with various combinations of molecular cytogenetic techniques: comparative genomic hybridization, fluorescence in situ hybridization (FISH) with whole chromosome painting probes, and microdissection

Ji-Yun Lee; Chul-Hoon Lee; Sung-Han Shim; Han-Kyu Seo; Jee-Hong Kyhm; Sechin Cho; Youl-Hee Cho

2002-01-01

324

Curbing rampant cross-contamination and misidentification of cell lines.  

PubMed

A son's challenge started an emeritus professor of biology on a three-year odyssey to get biological researchers to correct a decades-long problem with cross-contaminated and misidentified cell lines. These errors may account for more than 15% of mammalian cultures, wasting resources and undermining the integrity of research. PMID:18816888

Nardone, Roland M

2008-09-01

325

Telomere stability genes are not mutated in osteosarcoma cell lines  

Microsoft Academic Search

Osteosarcoma (OS), the most common primary bone tumor in adolescents and young adults, is characterized by a high degree of chromosomal abnormalities. Because telomeres are important for maintaining chromosomal integrity, it is plausible that germ-line or somatic mutations in the genes responsible for stabilizing the telomere complex could contribute to OS. We performed bi-directional sequence analysis in five OS cell

Sharon A. Savage; Brian J. Stewart; Jason S. Liao; Lee J. Helman; Stephen J. Chanock

2005-01-01

326

DIFFERENCES IN ARACHIDONIC ACID METABOLISM BY HUMAN MYELOMONCYTIC CELL LINES  

EPA Science Inventory

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines were determined after incubation with interferongamma (IFNg; 500 U/ml) or vehicle for 4 days. ells were prelabeled with tritiated arachidonic acid for 4 hours, and media supernata...

327

USING NEUROBLASTOMA CELL LINES TO EXAMINE ORGANOPHOSPHATE NEUROTOXICITY  

EPA Science Inventory

The need to deploy IN VITRO models to test neurotoxic scribes the use of by industry and government regulatory agencies. his research describes the neuroblastoma cell lines to address the relationship between esterase inhibition and neurotoxic outcome following exposure to organo...

328

Ultra-wideband time-delay line inspired by composite right\\/left-handed transmission line unit cell  

Microsoft Academic Search

This paper presents a design of ultra-wideband time-delay line inspired by the composite right\\/left-handed transmission line (CRLH TL) unit cell. A rotated version of the conventional CRLH TL unit cell is used to increase the operating bandwidth. The time-delay line is optimized using computer simulation and then fabricated on a PCB for measurement. For comparison, the time-delay lines using the

J. Zhang; S. W. Cheung; T. I. Yuk

2010-01-01

329

A human gall-bladder signet ring cell carcinoma cell line.  

PubMed

To date, very few reports of the establishment of gall-bladder cancer cell lines have appeared, although many cancer cell lines of various kinds have been established. On the other hand, no reports could be found on signet ring cell carcinoma cell lines derived from the gall-bladder and only five cell lines from the stomach. A human gall-bladder cancer cell line (FU-GBC-2) was established in tissue culture from the ascitic fluid of a 69-year-old Japanese female patient. The tumor cells growing in tissue culture exhibited the morphological characteristics of signet ring cells in phase contrast and electron microscopy. The population doubling time was 43 hours. Heterotransplantation was succeeded by inoculation into the dermis of BALB/c nude mice. An immunocytochemical study showed that most of the cultured cells were positive for carcinoembryonic antigen, CA19-9 and epithelial membrane antigen, but negative for vimentin. The modal chromosome number was 120 with a range of 100-124. Flow cytometry showed an aneuploidy pattern in the cultured cells at passage 30. Markedly amplified c-myc oncogene was observed by Southern blot analysis. This cell line may be useful in the study of the morphological and biological characteristics of signet ring cell carcinoma and gall-bladder adenocarcinoma. PMID:9211524

Nishida, T; Iwasaki, H; Johzaki, H; Tanaka, S; Watanabe, R; Kikuchi, M

1997-06-01

330

Zebrafish kidney stromal cell lines support multilineage hematopoiesis  

PubMed Central

Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish.

Stachura, David L.; Reyes, Jason R.; Bartunek, Petr; Paw, Barry H.; Zon, Leonard I.

2009-01-01

331

Deoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification  

PubMed Central

Context: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. Objective: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. Results: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. Conclusions: Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

Schweppe, Rebecca E.; Klopper, Joshua P.; Korch, Christopher; Pugazhenthi, Umarani; Benezra, Miriam; Knauf, Jeffrey A.; Fagin, James A.; Marlow, Laura A.; Copland, John A.; Smallridge, Robert C.; Haugen, Bryan R.

2008-01-01

332

Effects of hypoxia on human cancer cell line chemosensitivity  

PubMed Central

Background Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia. Methods A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA). Results Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs. Conclusions A panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia.

2013-01-01

333

Endosialin expression in side populations in human sarcoma cell lines.  

PubMed

The Hoechst 33342 exclusion side population (SP) assay is a validated method used to identify cells with stem cell-like properties. When isolated from tumors, SP cells have been shown to have high malignant potential. SPs have been found in both carcinomas and sarcomas. The molecular profile of sarcoma SP is poorly understood. The purpose of the present study was to determine whether endosialin is a suitable therapeutic target for sarcomas. Six cell lines (HT-1080 fibrosarcoma, SJSA-1 and HOS osteosarcoma, A-673 and SK-ES-1 Ewing sarcoma) were used for the SP analysis. Flow cytometry was used to count and examine the cells. Results showed for the first time that endosialin (CD248), which was previously identified as a sarcoma marker, is expressed in sarcoma SP cells. This observation supports the hypothesis that endosialin is a promising therapeutic target for sarcomas. PMID:22740905

Rouleau, Cecile; Sancho, Jose; Campos-Rivera, Juanita; Teicher, Beverly A

2012-02-01

334

Metal mutagenesis in transgenic Chinese hamster cell lines.  

PubMed Central

Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium.

Klein, C B; Kargacin, B; Su, L; Cosentino, S; Snow, E T; Costa, M

1994-01-01

335

Cytotoxicity and genotoxicity of phenazine in two human cell lines.  

PubMed

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9-123?M) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2'-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24h IC50: 11?M; 48h IC50: 7.8?M) observed as low as 1.9?M. T24 cells experienced a minor antiproliferative effect (BrdU 24h IC50: 47?M; 48h IC50: 17?M). IC50 values for HepG2 proliferation and viability were 54-77% lower compared to T24 cells. In both cell lines, IC50 values for proliferation were 66-90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9-30.8?M) experienced no significant genotoxic effects, while T24 cells (7.7-123?M) experienced significant genotoxicity at ?61.5?M. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study. PMID:24380821

McGuigan, Claire F; Li, Xing-Fang

2014-06-01

336

Cytotoxic effects of Euterpe oleracea Mart. in malignant cell lines  

PubMed Central

Background Euterpe oleracea Mart., a plant from the Amazon region, is commonly known as açaí or juçara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Açaí is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines. Methods Cell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 ?g/mL of bark, seed, and total açaí fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate. Results We observed that of all the cell lines tested, MCF-7 was the only line that responded to açaí treatment. The extracts caused significant reduction (p?cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo™ assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events. Conclusions The present study demonstrated that açaí possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the anticancer potential of açaí may help in the development of chemopreventive drugs and may have therapeutic effects in the treatment of breast cancer.

2014-01-01

337

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines  

Microsoft Academic Search

Background  Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of\\u000a CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the\\u000a stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the

Lu Cao; Yanming Zhou; Beibei Zhai; Jian Liao; Wen Xu; Ruixiu Zhang; Jing Li; Yu Zhang; Lei Chen; Haihua Qian; Mengchao Wu; Zhengfeng Yin

2011-01-01

338

Singlet oxygen toxicity is cell line-dependent: A study of lipid peroxidation in leukemia cell lines  

Microsoft Academic Search

Singlet oxygen (1O2) can be quenched by water, lipids, proteins, nucleic acids and other small molecules. Poly- unsaturated fatty acids (PUFA) of cells principally quench 1O 2 by chemical mechanisms, producing lipid hy- droperoxides, while proteins physically and chemically quench 1O 2. Because cell lines can have different PUFA and protein levels, we hypothesized that 1O2 toxicity will vary between

Freya Q. Schafer; Garry R. Buettner

1998-01-01

339

Characterization of side population cells isolated from the colon cancer cell line SW480.  

PubMed

Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many types of cell lines and tissues have demonstrated the presence of SP cells, including colon cancer cell lines. This study aimed to identify cancer stem cells (CSCs) in the SP of the colon cancer cell line SW480. SP cells were isolated by fluorescence-activated cell sorting (FACS), followed by serum-free medium (SFM) culture. The self-renewal, differentiated progeny, clone formation, proliferation, invasion ability, cell cycle, chemosensitivity and tumorigenic properties in SP and non-SP (NSP) cells were investigated through in vitro culture and in vivo serial transplantation. The expression profiles of ATP-binding cassette (ABC) protein transporters and stem cell?related genes were examined by RT-PCR and western blot analysis. The human colon cancer cell lines SW480, Lovo and HCT116 contain 1.1±0.10, 0.93±0.11 and 1.33±0.05% SP cells, respectively. Flow cytometry analysis revealed that SP cells could differentiate into SP and NSP cells. SP cells had a higher proliferation potency and CFE than NSP cells. Compared to NSP cells, SP cells were also more resistant to CDDP and 5-FU, and were more invasive and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNA and protein expression of ABCG2, MDR1, OCT-4, NANOG, SOX-2, CD44 and CD133. SP cells isolated from human colon cancer cell lines harbor CSC properties that may be related to the invasive potential and therapeutic resistance of colon cancer. PMID:24926880

Xiong, Binghong; Ma, Li; Hu, Xiang; Zhang, Caiquan; Cheng, Yong

2014-09-01

340

Trichothecene-induced cytotoxicity on human cell lines  

Microsoft Academic Search

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type\\u000a D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2,\\u000a HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent\\u000a cell proliferation assay assessing

Carina Nielsen; Maximilian Casteel; Andrea Didier; Richard Dietrich; Erwin Märtlbauer

2009-01-01

341

Morphogenetic behavior of simian virus 40-transformed human mammary epithelial stem cell lines on collagen gels  

Microsoft Academic Search

Summary  Transformation of primary cultures of human breast cells with simian virus 40 and clonal selection has yielded single-cell-cloned,\\u000a epithelial cell lines, as well as myoepithelial-related cell lines. When grown on floating collagen gels, the epithelial cell\\u000a lines give rise to branching rays of cells, thick fingerlike protrusions, saclike structures, and degenerating areas. The\\u000a myoepithelial-related cell lines give rise only to

Philip S. Rudland; Gillian E. Ollerhead; Angela M. Platt-Higgins

1991-01-01

342

Targeted genetic modification of cell lines for recombinant protein production  

PubMed Central

Considerable increases in productivity have been achieved in biopharmaceutical production processes over the last two decades. Much of this has been a result of improvements in media formulation and process development. Though advances have been made in cell line development, there remains considerable opportunity for improvement in this area. The wealth of transcriptional and proteomic data being generated currently hold the promise of specific molecular interventions to improve the performance of production cell lines in the bioreactor. Achieving this—particularly for multi-gene modification—will require specific, targeted and controlled genetic manipulation of these cells. This review considers some of the current and potential future techniques that might be employed to realise this goal.

Piskareva, Olga; Muniyappa, Mohan

2007-01-01

343

Androglobin knockdown inhibits growth of glioma cell lines  

PubMed Central

Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of glioma cell lines determined by MTT assay and colony formation assay. ADGB knockdown also increased the apoptosis of glioma cell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro.

Huang, Bo; Lu, Yi-Sheng; Li, Xia; Zhu, Zhi-Chuan; Li, Kui; Liu, Ji-Wei; Zheng, Jing; Hu, Ze-Lan

2014-01-01

344

Over-expression of secreted proteins from mammalian cell lines  

PubMed Central

Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats.

Dalton, Annamarie C; Barton, William A

2014-01-01

345

The interaction of normal lymphocytes and cells from lymphoid cell lines  

PubMed Central

Cells from thirty-three human lymphoid cell lines and sublines have been typed for HL-A antigens by a microcytotoxicity test. Similar patterns of HL-A antigens were found for the cell line cells and for the fresh lymphocytes of the donor of the line (eleven cases). However, certain typing sera gave positive reactions with the cell line cells which were not found with the fresh lymphocytes. No correlation was noted between the pattern of these `extra' reactions and the HL-A typing of the cells. These same typing sera often gave positive reactions with blood lymphocytes cultured for several days in conventional media. These positive reactions were quantitatively more pronounced and sometimes quantitatively different if the cells had been stimulated. A range of normal sera failed to react with cell line cells suggesting that the HL-A typing sera giving `extra' reactions are detecting antigens in some way related to a histocompatibility system. Absorption studies performed with two of the lines confirmed the HL-A typing by the direct cytotoxicity test. Two sera giving `extra' reactions were also tested in the absorption experiments. The results indicated that antibodies other than those of the HL-A specificity designated for these sera were responsible for the `extra' reactions. It is suggested that `extra' reactions indicate a change in the apparent antigenic expression of lymphoid cells reflecting altered membrane characteristics as they adapt to a culture environment.

Mackintosh, Pauline; Wallin, Josephine; Hardy, D. A.; Ling, N. R.; Steel, C. M.

1973-01-01

346

Spontaneous transformation of human granulosa cell tumours into an aggressive phenotype: a metastasis model cell line  

Microsoft Academic Search

BACKGROUND: Granulosa cell tumours (GCTs) are frequently seen in menopausal women and are relatively indolent. Although the physiological properties of normal granulosa cells have been studied extensively, little is known about the molecular mechanism of GCT progression. Here, we characterise the unique behavioural properties of a granulosa tumour cell line, KGN cells, for the molecular analysis of GCT progression. METHODS:

Misa Imai; Miho Muraki; Kiyoshi Takamatsu; Hidekazu Saito; Motoharu Seiki; Yuji Takahashi

2008-01-01

347

Beta-cell differentiation from a human pancreatic cell line in vitro and in vivo.  

PubMed

Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human beta-cells, inducibly transformed pancreatic beta-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of beta-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional beta-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human beta-cells is a major step toward developing a cell transplantation therapy for diabetes. PMID:11222748

de la Tour, D; Halvorsen, T; Demeterco, C; Tyrberg, B; Itkin-Ansari, P; Loy, M; Yoo, S J; Hao, E; Bossie, S; Levine, F

2001-03-01

348

Heterogeneity of a human T-lymphoblastoid cell line  

SciTech Connect

A human T-lymphoblastoid cell line (Jurkat) was cloned, and four resulting sublines were characterized in a variety of ways with the objective of gaining information on heterogeneity in cell lines. Within a few weeks of cloning, distinct cellular morphologies and growth patterns became apparent in the four sublines. Growth rate measurements made over 3 months did not show any significant differences between the sublines. Surface protein profiles obtained by radioimmunoprecipitation using antisera in conjunction with extracts from (/sup 35/S)Met and /sup 125/I-labeled cells revealed differences between the sublines. Analysis of total cell DNA showed that one of the sublines possessed only half the chromosome complement of the other sublines and the parental line. Karyotyping confirmed this result and, in addition, demonstrated that chromosome numbers fluctuated around a mean value for each subline. Karyotypic variability became apparent within 2 months of cloning and tended to increase with time in culture. G-banding analysis showed that the analyzed cell populations contained distinctive cytogenetic aberrations. Properties of the cloned sublines were monitored over a 9-month period. One of the sublines that had shown heterogeneous morphology even after 6 weeks maintained the heterogeneity throughout this time. Another subline underwent a marked change in morphology (round to irregular) and growth habit (single cells to large clumps) with increasing time in culture. Interestingly, several alterations to surface proteins accompanied these growth changes. A third subline had relatively stable morphology and chromosome number throughout the 9-month period. The modal chromosome number was hypotetraploid for three sublines and the parent line, but was diploid for another subline.

Snow, K.; Judd, W.

1987-08-01

349

Carbon nanoparticles for gene transfection in eukaryotic cell lines.  

PubMed

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed ?-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests. PMID:24863237

Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

2014-06-01

350

Cytotoxicity evaluation of silica nanoparticles using fish cell lines.  

PubMed

Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO2 NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO2 NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO2 NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish, goldfish, haddock, and American eel) were used to comparatively evaluate viability of cells by measuring metabolic impairment using Alamar Blue. Toxicity of SiO2 NPs appeared to be size-, time-, temperature-, and dose-dependent as well as tissue-specific. However, dosages greater than 100 ?g/mL were needed to achieve 24 h EC50 values (effective concentrations needed to reduce cell viability by 50%). Smaller SiO2 NPs (16 nm) were relatively more toxic than larger sized ones (24 and 44 nm) and external lining epithelial tissue (skin, gills)-derived cells were more sensitive than cells derived from internal tissues (liver, brain, intestine, gonads) or embryos. Higher EC50 values were achieved when toxicity assessment was performed at higher incubation temperatures. These findings are in overall agreement with similar human and mouse cell studies reported to date. Thus, fish cell lines could be valuable for screening emerging contaminants in aquatic environments including NPs through rapid high-throughput cytotoxicity bioassays. PMID:24357037

Vo, Nguyen T K; Bufalino, Mary R; Hartlen, Kurtis D; Kitaev, Vladimir; Lee, Lucy E J

2014-05-01

351

Production and Origination of Cell Lines and Isolation of Histocompatibility Antigens from Lymphoid Cell Lines.  

National Technical Information Service (NTIS)

A method for culturing the L1210 cells so as to obtain large amounts of antigen with high levels of glycoprotein was developed. The feasibility of extraction of large quantities of H2-d antigen from cells frozen in dimethyl sulfoxide (DMSO) and liquid nit...

P. M. Lincoln J. L. Glick B. A. Maurer

1973-01-01

352

Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines  

PubMed Central

The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. However, we previously documented apoptosis in a B non-Hodgkin lymphoma cell line involving diacylglycerol analogue stimulation of Ras guanyl-releasing protein, a Ras activator, and Bim, a proapoptotic Bcl-2 family protein. To further explore the role of Bim, we examined several Bim-deficient B non-Hodgkin lymphoma cells for their responses to pico, a synthetic bryostatin-1-like compound. The Bim? mantle cell lymphoma cell lines Jeko-1, Mino, Sp53, UPN1, and Z138 and the Bim+ cell line Rec-1, as well as the Burkitt lymphoma cells lines BL2 (Bim?) and Daudi (Bim+), were examined for their response to pico using assays for proliferation and apoptosis as well as biochemical methods for Ras guanyl-releasing proteins and Bcl-2 family members. With the exception of UPN1, mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases.

Lopez-Campistrous, Ana; Song, Xiaohua; Schrier, Adam J.; Wender, Paul A.; Dower, Nancy A.; Stone, James C.

2014-01-01

353

Bioenergetic Analysis of Ovarian Cancer Cell Lines: Profiling of Histological Subtypes and Identification of a Mitochondria-Defective Cell Line  

PubMed Central

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1? in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs.

Dier, Usawadee; Shin, Dong-Hui; Hemachandra, L. P. Madhubhani P.; Uusitalo, Larissa M.; Hempel, Nadine

2014-01-01

354

Glycoprotein profiles of human breast cells demonstrate a clear clustering of normal/benign versus malignant cell lines and basal versus luminal cell lines.  

PubMed

Gene expression profiling has defined molecular subtypes of breast cancer including those identified as luminal and basal. To determine if glycoproteins distinguish various subtypes of breast cancer, we obtained glycoprotein profiles from 14 breast cell lines. Unsupervised hierarchical cluster analysis demonstrated that the glycoprotein profiles obtained can serve as molecular signatures to classify subtypes of breast cancer, as well as to distinguish normal and benign breast cells from breast cancer cells. Statistical analyses were used to identify glycoproteins that are overexpressed in normal versus cancer breast cells, and those that are overexpressed in luminal versus basal breast cancer. Among the glycoproteins distinguishing normal breast cells from cancer cells are several proteins known to be involved in cell adhesion, including proteins previously identified as being altered in breast cancer. Basal breast cancer cell lines overexpressed a number of CD antigens, including several integrin subunits, relative to luminal breast cancer cell lines, whereas luminal breast cancer cells overexpressed carbonic anhydrase 12, clusterin, and cell adhesion molecule 1. The differential expression of glycoproteins in these breast cancer cell lines readily allows the classification of the lines into normal, benign, malignant, basal, and luminal groups. PMID:22106898

Yen, Ten-Yang; Macher, Bruce A; McDonald, Claudia A; Alleyne-Chin, Chris; Timpe, Leslie C

2012-02-01

355

Internal arsenite bioassay calibration using multiple bioreporter cell lines  

PubMed Central

Summary Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error?prone. A new approach to control assay variations and improve application ease could be an internal calibration based on the use of multiple bioreporter cell lines with drastically different reporter protein outputs at a given analyte concentration. To test this concept, different Escherichia coli?based bioreporter strains expressing either cytochrome c peroxidase (CCP, or CCP mutants) or ??galactosidase upon induction with arsenite were constructed. The reporter strains differed either in the catalytic activity of the reporter protein (for CCP) or in the rates of reporter protein synthesis (for ??galactosidase), which, indeed, resulted in output signals with different intensities at the same arsenite concentration. Hence, it was possible to use combinations of these cell lines to define arsenite concentration ranges at which none, one or more cell lines gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of arsenite in drinking water (10?µg?l?1).

Wackwitz, Anke; Harms, Hauke; Chatzinotas, Antonis; Breuer, Uta; Vogne, Christelle; Van Der Meer, Jan Roelof

2008-01-01

356

Processing of proSAAS in neuroendocrine cell lines.  

PubMed Central

ProSAAS, a recently discovered granin-like protein, potently inhibits prohormone convertase (PC)1, and might also perform additional functions. In the present study, the processing of proSAAS was compared in two neuroendocrine cell lines overexpressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line. The processing of proSAAS was examined by pulse-chase analysis using [(3)H]leucine, by MS, and by chromatography and radioimmunoassay. Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN and big LEN. Because the PC-12 cells used in the present study do not express either PC1 or PC2, the finding that these cells efficiently cleave proSAAS indicates that these cleavages do not require either enzyme. Two of the peptides identified in AtT-20 media represent novel C-terminally truncated forms of PEN. In both cell lines, the secretion of the small proSAAS-derived peptides is stimulated by secretagogues. However, long-term treatment of wild-type AtT-20 cells with two different secretagogues (8-bromo-cAMP and a phorbol ester) does not affect levels of proSAAS mRNA; this treatment significantly increases PC1 mRNA by approx. 60-80%. The lack of co-regulation of proSAAS and PC1 mRNA implies that enzyme activity can be induced without an accompanying increase in the inhibitor. In addition, the finding that the peptides are secreted via the regulated pathway is consistent with the proposal that they may function as neuropeptides.

Mzhavia, Nino; Qian, Yimei; Feng, Yun; Che, Fa-Yun; Devi, Lakshmi A; Fricker, Lloyd D

2002-01-01

357

Epitope tagging of endogenous genes in diverse human cell lines  

PubMed Central

Epitope tagging is a powerful and commonly used approach for studying the physical properties of proteins and their functions and localization in eukaryotic cells. In the case of Saccharomyces cerevisiae, it has been possible to exploit the high efficiency of homologous recombination to tag proteins by modifying their endogenous genes, making it possible to tag virtually every endogenous gene and perform genome-wide proteomics experiments. However, due to the relative inefficiency of homologous recombination in cultured human cells, epitope-tagging approaches have been limited to ectopically expressed transgenes, with the attendant limitations of their nonphysiological transcriptional regulation and levels of expression. To overcome this limitation, a modification and extension of adeno-associated virus-mediated human somatic cell gene targeting technology is described that makes it possible to simply and easily create an endogenous epitope tag in the same way that it is possible to knock out a gene. Using this approach, we have created and validated human cell lines with epitope-tagged alleles of two cancer-related genes in a variety of untransformed and transformed human cell lines. This straightforward approach makes it possible to study the physical and biological properties of endogenous proteins in human cells without the need for specialized antibodies for individual proteins of interest.

Kim, Jung-Sik; Bonifant, Challice; Bunz, Fred; Lane, William S.; Waldman, Todd

2008-01-01

358

Serial analysis of gene expression in a microglial cell line.  

PubMed

We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte-activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN-7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695-696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT-PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology. PMID:10559785

Inoue, H; Sawada, M; Ryo, A; Tanahashi, H; Wakatsuki, T; Hada, A; Kondoh, N; Nakagaki, K; Takahashi, K; Suzumura, A; Yamamoto, M; Tabira, T

1999-12-01

359

First continuous human pheochromocytoma cell line: KNA Biological, cytogenetic and molecular characterization of KNA cells  

Microsoft Academic Search

Pheochromocytomas are rare tumours, with an incidence of 1–2 per million which arise from chromaffin cells of the adrenal medulla. They occur sporadically or as part of dominantly inherited cancer syndromes like multiple endocrine neoplasia 2 (MEN2A and 2B) and others. Continuous cell lines, not available so far, are essential tools for studies in these tumours. A continuous cell line

R. Pfragner; A. Behmel; D. P. Smith; B. A. J. Ponder; G. Wirnsberger; I. Rinner; S. Porta; T. Henn; B. Niederle

1998-01-01

360

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative  

Microsoft Academic Search

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the

Oluseun Adewumi; Behrouz Aflatoonian; Lars Ahrlund-Richter; Michal Amit; Gemma Beighton; Paul A Bello; Nissim Benvenisty; Lorraine S Berry; Simon Bevan; Barak Blum; Justin Brooking; Kevin G Chen; Andre B H Choo; Gary A Churchill; Marie Corbel; Ivan Damjanov; Jon S Draper; Petr Dvorak; Katarina Emanuelsson; Roland A Fleck; Angela Ford; Karin Gertow; Marina Gertsenstein; Paul J Gokhale; Rebecca S Hamilton; Ales Hampl; Lyn E Healy; Outi Hovatta; Johan Hyllner; Marta P Imreh; Joseph Itskovitz-Eldor; Jamie Jackson; Jacqueline L Johnson; Mark Jones; Kehkooi Kee; Benjamin L King; Barbara B Knowles; Majlinda Lako; Franck Lebrin; Barbara S Mallon; Daisy Manning; Yoav Mayshar; Ronald D G Mckay; Anna E Michalska; Milla Mikkola; Masha Mileikovsky; Stephen L Minger; Harry D Moore; Christine L Mummery; Andras Nagy; Norio Nakatsuji; Carmel M O'Brien; Steve K W Oh; Cia Olsson; Timo Otonkoski; Kye-Yoon Park; Robert Passier; Hema Patel; Minal Patel; Roger Pedersen; Martin F Pera; Marian S Piekarczyk; Renee A Reijo Pera; Benjamin E Reubinoff; Allan J Robins; Janet Rossant; Peter Rugg-Gunn; Thomas C Schulz; Henrik Semb; Eric S Sherrer; Henrike Siemen; Glyn N Stacey; Miodrag Stojkovic; Hirofumi Suemori; Jin Szatkiewicz; Tikva Turetsky; Timo Tuuri; Steineke van den Brink; Kristina Vintersten; Sanna Vuoristo; Dorien Ward; Thomas A Weaver; Lesley A Young; Weidong Zhang; Peter W Andrews

2007-01-01

361

Establishment and characterization of a new murine cell line (SR4987) derived from marrow stromal cells  

Microsoft Academic Search

A new murine cell line designated as SR-4987 was established by treating a long-term bone marrow culture with the supernatant\\u000a from Y-1 cells which actively produce viral C-particles (MuLV). The line showed a fibrolbast-like morphology and its mesodermal\\u000a origin was confirmed by immunocytochemical staining. Flow cytometric analysis of DNA index evidenced a tetraploid number of\\u000a chromosomes whereas cell cycle analysis

Augusto Pessina; Elisabetta Mineo; Maria Grazia Neri; Laura Gribaldo; Robert Colombi; Paolo Brambilla; Gintaras Zaleskis

1992-01-01

362

Establishment and characterization of renal cell carcinoma cell lines with multidrug resistance  

Microsoft Academic Search

Many of the discoveries of multidrug resistance (MDR) have resulted from studies using drug-resistant cultured tumor cell\\u000a lines as experimental models. To date, there has been no report on the detailed characterization of such a cell line from\\u000a renal cell carcinoma (RCC). By long-term exposure of an established RCC (RCC8701) to increasing concentrations of adriamycin,\\u000a we established a series of

Dah-Shyong Yu; Cheng-Ping Ma; Sun-Yran Chang

2000-01-01

363

Bovine viral diarrhea virus (BVDV) in cell lines used for somatic cell cloning.  

PubMed

Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus. PMID:15710188

Stringfellow, David A; Riddell, Kay P; Givens, M Daniel; Galik, Patricia K; Sullivan, Eddie; Dykstra, Christine C; Robl, James; Kasinathan, Poothapillai

2005-03-01

364

Human small cell lung cancer cell lines expressing the proopiomelanocortin gene have aberrant glucocorticoid receptor function.  

PubMed Central

Some human small cell lung carcinomas (SCLC) secrete proopiomelanocortin (POMC) derived peptides, but in contrast to the pituitary, glucocorticoids fail to inhibit this hormone production. We have previously described an in vitro model using human SCLC cell lines that express POMC and are resistant to glucocorticoids. We have now identified the glucocorticoid receptor (GR) in the SCLC cell line COR L24 using a whole cell ligand binding assay (Kd = 5.7 nM; Bmax = 11 fmol/million cells), while another cell line, DMS 79, lacked significant glucocorticoid binding. To analyze GR function both positive (GMCO) and negative (TRE)3-tkCAT), glucocorticoid-regulated reporter gene constructs were transfected into COR L24 cells. In the SCLC cell line, neither hydrocortisone nor dexamethasone (500-2,000 nM) significantly induced chloramphenicol acetyltransferase expression from GMCO; in addition, they did not suppress chloramphenicol acetyltransferase expression from (TRE)3-tkCAT. Similar results were obtained with two other POMC-expressing SCLC cell lines. Expression of wild type GR in COR L24 cells restored glucocorticoid signaling, with marked induction of GMCO reporter gene expression by dexamethasone (9,100 +/- 910%; n = 3), and an estimated EC50 of 10 nM. This failure of the GR explains the resistance of the POMC gene to glucocorticoid inhibition and may have implications for cell growth in SCLC. Images

Ray, D W; Littlewood, A C; Clark, A J; Davis, J R; White, A

1994-01-01

365

Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.  

PubMed

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages. PMID:23782837

Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

2013-08-01

366

75 FR 65581 - Proposed Amendment and Revocation of Class E Airspace, Vero Beach, FL  

Federal Register 2010, 2011, 2012, 2013

...designated as an extension to Class D surface area at Vero Beach Municipal Airport...Class E airspace designated as surface area to remove any reference to the...designated as an extension to Class D surface area to eliminate controlled...

2010-10-26

367

33 CFR 110.73b - Indian River at Vero Beach, Fla.  

Code of Federal Regulations, 2010 CFR

...Areas § 110.73b Indian River at Vero Beach, Fla. (a) Area A. Beginning at a point located on the eastern shore of Fritz Is. at latitude 27°39â²32.5â³ N., longitude 80°22â²20.6â³ W. following the shoreline northward to the northwest...

2010-07-01

368

33 CFR 110.73b - Indian River at Vero Beach, Fla.  

Code of Federal Regulations, 2010 CFR

...Areas § 110.73b Indian River at Vero Beach, Fla. (a) Area A. Beginning at a point located on the eastern shore of Fritz Is. at latitude 27°39â²32.5â³ N., longitude 80°22â²20.6â³ W. following the shoreline northward to the northwest...

2009-07-01

369

Red Cell Apheresis with Automated In-Line Filtration  

PubMed Central

Summary Background The aim of this study was to provide data on concurrent red blood cell (RBC) and platelet (PLT) apheresis with RBC in-line leukoreduction and automated addition of saline-adenine-glucose-mannitol (SAGM) using the new version (V6.0) of Trima Accel®. Methods In this two-center paired study, each subject completed a test and a control procedure with an interval of 9 weeks between procedures. In the test arm, single RBC and PLT units were collected on the Trima Accel V6.0 (in-line leukofiltration and automated addition of SAGM). In the control arm, they were collected on Trima Accel V5.1/V5.2 (post-collection leukoreduction, manual SAGM addition). RBC percent hemolysis, potassium concentration and adenosine triphosphate over storage, hemoglobin (Hb) yield, and residual white blood cells (WBC) were determined. Results 34 subjects successfully completed both test and control procedures. Post-storage hemolysis was similar in both groups, and all values were less than 0.8% for both arms. Residual WBC counts in all RBC units were less than 1 × 106/unit. In-line processed RBC units (V6.0) have a significantly higher volume and more Hb/unit due to filtration recovery improvements. All procedures were well tolerated by the subjects. Conclusion In-line filtration and automated addition of storage solution on Trima Accel V6.0 allows collection of ready-to-use RBC units that meet EU requirements.

Matthes, Gert; Ingilizov, Marin; Dobao, Maria Luz; Marques, Susana; Callaert, Martine

2014-01-01

370

Choosing the right cell line for breast cancer research.  

PubMed

Breast cancer is a complex and heterogeneous disease. Gene expression profiling has contributed significantly to our understanding of this heterogeneity at a molecular level, refining taxonomy based on simple measures such as histological type, tumour grade, lymph node status and the presence of predictive markers like oestrogen receptor and human epidermal growth factor receptor 2 (HER2) to a more sophisticated classification comprising luminal A, luminal B, basal-like, HER2-positive and normal subgroups. In the laboratory, breast cancer is often modelled using established cell lines. In the present review we discuss some of the issues surrounding the use of breast cancer cell lines as experimental models, in light of these revised clinical classifications, and put forward suggestions for improving their use in translational breast cancer research. PMID:21884641

Holliday, Deborah L; Speirs, Valerie

2011-01-01

371

Designing of promiscuous inhibitors against pancreatic cancer cell lines  

PubMed Central

Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

2014-01-01

372

Designing of promiscuous inhibitors against pancreatic cancer cell lines  

NASA Astrophysics Data System (ADS)

Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

2014-04-01

373

New cell lines from mouse epiblast share defining features with human embryonic stem cells.  

PubMed

The application of human embryonic stem (ES) cells in medicine and biology has an inherent reliance on understanding the starting cell population. Human ES cells differ from mouse ES cells and the specific embryonic origin of both cell types is unclear. Previous work suggested that mouse ES cells could only be obtained from the embryo before implantation in the uterus. Here we show that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper. These cells, which we refer to as EpiSCs (post-implantation epiblast-derived stem cells), express transcription factors known to regulate pluripotency, maintain their genomic integrity, and robustly differentiate into the major somatic cell types as well as primordial germ cells. The EpiSC lines are distinct from mouse ES cells in their epigenetic state and the signals controlling their differentiation. Furthermore, EpiSC and human ES cells share patterns of gene expression and signalling responses that normally function in the epiblast. These results show that epiblast cells can be maintained as stable cell lines and interrogated to understand how pluripotent cells generate distinct fates during early development. PMID:17597760

Tesar, Paul J; Chenoweth, Josh G; Brook, Frances A; Davies, Timothy J; Evans, Edward P; Mack, David L; Gardner, Richard L; McKay, Ronald D G

2007-07-12

374

Can we develop ethically universal embryonic stem-cell lines?  

Microsoft Academic Search

Human embryonic stem-cell (hESC) research faces opposition from those who object to the destruction of human embryos. Over the past few years, a series of new approaches have been proposed for deriving hESC lines without injuring a living embryo. Each of these presents scientific challenges and raises ethical and political questions. Do any of these methods have the potential to