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Sample records for vero cells expressing

  1. The expression of essential components for human influenza virus internalisation in Vero and MDCK cells.

    Science.gov (United States)

    Ugiyadi, Maharani; Tan, Marselina I; Giri-Rachman, Ernawati A; Zuhairi, Fawzi R; Sumarsono, Sony H

    2014-05-01

    MDCK and Vero cell lines have been used as substrates for influenza virus replication. However, Vero cells produced lower influenza virus titer yield compared to MDCK. Influenza virus needs molecules for internalisation of the virus into the host cell, such as influenza virus receptor and clathrin. Human influenza receptor is usually a membrane protein containing Sia(α2,6) Gal, which is added into the protein in the golgi apparatus by α2,6 sialyltransferase (SIAT1). Light clathrin A (LCA), light clathrin B (LCB) and heavy clathrin (HC) are the main components needed for virus endocytosis. Therefore, it is necessary to compare the expression of SIAT1 and clathrin in Vero and MDCK cells. This study is reporting the expression of SIAT1 and clathrin observed in both cells with respect to the levels of (1) RNA by using RT-PCR, (2) protein by using dot blot analysis and confocal microscope. The results showed that Vero and MDCK cells expressed both SIAT1 and clathrin proteins, and the expression of SIAT1 in MDCK was higher compared to Vero cells. On the other hand, the expressions of LCA, LCB and HC protein in MDCK cells were not significantly different to Vero cells. This result showed that the inability of Vero cells to internalize H1N1 influenza virus was possibly due to the lack of transmembrane protein receptor which contained Sia(α2,6) Gal.

  2. [The protein expression profiles induced by trimethyltin chloride in Vero cells].

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    Xiao, Yun; Zhu, Li-jin; Jv, Li; Qian, Ya-ling; Zhang, Xing

    2011-10-01

    To explore the biomarkers and mechanism of kidney toxicity induced by trimethyltin chloride (TMT-Cl) through analyzing the differences of protein expression profiles between vero cells and vero cells exposed to TMT-Cl. The differences of protein expression levels of three paired samples of vero cells and vero cells exposed to TMT-Cl were compared by two-dimensional gel electrophoresis (2-DE) and liquid chromatography-electrospray ionization-linear trap quadrupole (LC-ESI-LTQ). The differences of expression levels of Annexin A1 and α-Tubulin proteins were validated with western blot assay, and the differences of mRNA expression levels of Annexin A1 and α-Tubulin genes were detected with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Fifteen spots of differential expression in protein profiles between vero cells and vero cells exposed to TMT-Cl were found, and 9 of these spots were identified by LC-ESI-LTQ. The expression levels of 3 proteins (Annexin A1,similar to RAN protein and a hypothetical protein) increased and the expression levels of 6 proteins(growth factor receptor-bound protein 10, tubulin alpha 6, heterogeneous nuclear ribonucleoprotein, similar to elongation factor SIII p15 subunit, S-adenosylhomocysteine hydrolase and a hypothetical protein) reduced. The expression levels of α-Tubulin protein and mRNA significantly decreased in vero cells exposed to TMT-Cl, as compared with vero cells (P < 0.01). The expression of Annexin A1 protein in all exposure groups was significantly up-regulated, the expression of Annexin A1 mRNA in the groups exposed to 25 and 50 µmol/L TMT-Cl was significantly down-regulated, and The expression of Annexin A1 mRNA in the group exposed to 100 µmol/L TMT-Cl was significantly up-regulated (P < 0.01). The results of present study suggest that 9 proteins with differential expression detected by LC-ESI-LTQ may be related to the kidney toxicity induced by TMT-Cl, which can serve as the biomarkers of early

  3. Percent of Cytopathic Effect in Vero vs Vero-76 cells

    OpenAIRE

    Redding, Taylor; Neilson, Skot; Day, Craig; Westover, Jonna

    2017-01-01

    Vero cells and Vero-76 cells are both used to evaluate the antiviral effect of drugs in vitro. This study tested whether Vero and Vero-76 cells yield similar cytopathic effects when inoculated with yellow fever Virus (YFV) or Dengue Virus(DV). Each virus was plated on replicated 96 well plates of Vero and Vero-76 cells with different cell concentrations. The two cells lines did not give significantly different results for most cell and virus concentrations.

  4. Isolation and propagation of Dengue virus in Vero and BHK-21 cells expressing human DC-SIGN stably.

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    Phanthanawiboon, Supranee; A-nuegoonpipat, Atchareeya; Panngarm, Narawan; Limkittikul, Kriengsak; Ikuta, Kazuyoshi; Anantapreecha, Surapee; Kurosu, Takeshi

    2014-12-01

    The "standard" methods of isolating dengue virus (DENV) utilize the mosquito cell line C6/36, monkey kidney LLC-MK2 cells, Vero cells, or baby hamster kidney (BHK-21) cells. However, these cells lines lack a particular DENV receptor, known as dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which is expressed on immature dendritic cells and monocytes/macrophages. This may result in less efficient virus isolation and propagation. The present study used a lentivirus vector to establish Vero and BHK-21 cell lines (Vero-DC and BHK-DC) that express human DC-SIGN stably. Five DENV strains, each passaged several times in C6/36 cells, replicated more efficiently in Vero-DC and BHK-DC than in the parental Vero or BHK-21 cells. Vero/Vero-DC and BHK-21/BHK-DC were used to isolate virus from buffy coats and plasma samples derived from 13 patients infected with DENV. Most of the viruses showed increased production in cell lines expressing DC-SIGN. However, the isolation rate was lower (15.4-46.2%) than that from C6/36 cells (84.6%). Interestingly, when the viruses were isolated in C6/36 cells prior to infecting Vero/Vero-DC and BHK-21/BHK-DC, the rate of virus production increased markedly, reaching levels higher than those initially achieved in C6/36 cells. These data suggest that Vero-DC and BHK-DC could be useful tools for virus propagation, and that human specimens may contain a factor that interferes with virus growth in mammalian cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. [Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

    Science.gov (United States)

    Zhao, Jianjun; Yan, Ruxun; Zhang, Hailing; Zhang, Lei; Hu, Bo; Bai, Xue; Shao, Xiqun; Chai, Xiuli; Yan, Xijun; Wu, Wei

    2012-12-04

    The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

  6. Antisense downregulation of SARS-CoV gene expression in Vero E6 cells.

    Science.gov (United States)

    Shi, Yi; Luo, Haifeng; Jia, Jie; Xiong, Jie; Yang, Dehua; Huang, Bing; Jin, Youxin

    2005-01-01

    Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). It is an enveloped, single-stranded, plus-sense RNA virus with a genome of approximately 30 kb. The structural proteins E, M and N of SARS-CoV play important roles during host cell entry and viral morphogenesis and release. Therefore, we have studied whether expression of these structural proteins can be down-regulated using an antisense technique. Vero E6 cells were transfected with plasmid constructs containing exons of the SARS-CoV structural protein E, M or N genes or their exons in frame with the reporter protein EGFP. The transfected cell cultures were treated with antisense phosphorothioated oligonucleotides (antisense PS-ODN, 20mer) or a control oligonucleotide by addition to the culture medium. Among a total of 26 antisense PS-ODNs targeting E, M and N genes, we obtained six antisense PS-ODNs which could sequence-specifically reduce target genes expression by over 90% at the concentration of 50 microM in the cell culture medium tested by RT-PCR. The antisense effect was further proved by down-regulating the expression of the fusion proteins containing the structural proteins E, M or N in frame with the reporter protein EGFP. In Vero E6 cells, the antisense effect was dependent on the concentrations of the antisense PS-ODNs in a range of 0-10 microM or 0-30 microM. The antisense PS-ODNs are effective in downregulation of SARS. The findings indicate that antisense knockdown of SARS could be a useful strategy for treatment of SARS, and could also be suitable for studies of the pathological function of SARS genes in a cellular model system.

  7. Generation and characterization of a potentially applicable Vero cell line constitutively expressing the Schmallenberg virus nucleocapsid protein.

    Science.gov (United States)

    Zhang, Yongning; Wu, Shaoqiang; Song, Shanshan; Lv, Jizhou; Feng, Chunyan; Lin, Xiangmei

    2017-02-01

    Schmallenberg virus (SBV) is a Culicoides-transmitted orthobunyavirus that poses a threat to susceptible livestock species such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV is an ideal diagnostic antigen for the detection of viral infection. In this study, a stable Vero cell line, Vero-EGFP-SBV-N, constitutively expressing the SBV-N protein was established using a lentivirus system combined with puromycin selection. This cell line spontaneously emitted green fluorescent signals distributed throughout the cytoplasm, in which the expression of SBV-N fusion protein was confirmed by western blot analysis. The expression of SBV-N protein in Vero-EGFP-SBV-N cells was stable for more than fifty passages without puromycin pressure. The SBV-N fusion protein contained both an N-terminal enhanced green fluorescent protein (EGFP) tag and a C-terminal hexa-histidine (6 × His) tag, by which the N protein was successfully purified using Ni-NTA affinity chromatography. The cell line was further demonstrated to be reactive with SBV antisera and an anti-SBV monoclonal antibody in indirect immunofluorescence assays. Taken together, our results demonstrate that the Vero-EGFP-SBV-N cell line has potential for application in the serological diagnosis of SBV infection.

  8. Vero cells infected with the Lederle strain of canine distemper virus have increased Fas receptor signaling expression at 15 h post-infection.

    Science.gov (United States)

    Del Puerto, H L; Martins, A S; Braz, G F; Alves, F; Heinemann, M B; Rajão, D S; Araújo, F C; Martins, S F; Nascimento, D R; Leite, R C; Vasconcelos, A C

    2011-10-18

    We evaluated the expression of the Fas receptor gene in Vero cells infected with the Lederle vaccine strain of canine distemper virus using RT-PCR. Vero cells were plated, and after being grown for 24 h in MEM with 5% FBS, 80-90% confluent monolayer cultures were infected with the virus. The cells were harvested at 3, 6, 9, and 15 h post-infection. Uninfected Vero cells were used as a control. Total RNA was isolated from Vero cells using 1 mL Trizol(®) LS, and RT was performed using 2 μg total RNA. Primer pairs for RT-PCR amplification for the canine distemper virus nucleocapsid gene, the S26 reference gene, and the Vero rFas gene were used to analyze expression in Vero cells. RT-PCR results revealed virus activity at 3, 6, 9, and 15 h in the virus-infected Vero cells. The S26 housekeeping gene was amplified in virus infected and control samples. However, expression of the cell death receptor Fas was detected in Vero cells only at 15 h post-infection. We suggest that the Lederle vaccine induces apoptosis by Fas receptor signaling, possibly through caspase-8 signaling rather than through mitochondrial signaling in the infected cells.

  9. Short hairpin RNA-mediated inhibition of HSV-1 gene expression and function during HSV-1 infection in Vero cells.

    Science.gov (United States)

    Liu, Yuan-yuan; Deng, Hai-ying; Yang, Guang; Jiang, Wen-lin; Grossin, Laurent; Yang, Zhan-qiu

    2008-08-01

    To evaluate the efficiency of 3 short hairpin RNA (shRNA) interfering with the herpes simplex virus type 1 (HSV-1) gene coding glycoprotein D (gD) for inhibiting the gD expression and virus replication in vitro. Vero cells were selected for an in vitro model of infection. Three shRNA sequences (shRNAgD1, -gD2, and -gD3) targeting specifically the gD gene of HSV-1 were selected for evaluating the antiviral effects. The antiviral effects of shRNA in the cells infected with HSV-1 were evaluated by cytopathic effect (CPE) observations and plaque assays. The transcription level of viral RNA and the gD expression were studied by RT-PCR, Western blotting, and flow cytometry. With the 3 shRNA at a final concentration of 120 nmol/L, a significant inhibition of CPE in the HSV-1-infected cells was observed. The ED50 of shRNA-gD1, gD2, and gD3 were 48.74+/-2.57, 57.13+/-3.24, and 114.64+/-5.12 nmol/L, respectively. The gD gene decreased significantly after viral infection in the Vero cells pretreated with shRNA compared to the virus group. The expressions of the gD protein, determined by Western blotting and flow cytometry, were also drastically decreased in shRNA-transfected cells. Exogenous shRNA molecules can suppress the HSV-1 gD expression. They are inhibitors of HSV replication during infection in Vero cells.

  10. Growth and Maintenance of Vero Cell Lines

    OpenAIRE

    Ammerman, Nicole C.; Beier-Sexton, Magda; Azad, Abdu F.

    2008-01-01

    Vero cells are derived from the kidney of an African green monkey, and are one of the more commonly used mammalian continuous cell lines in microbiology, and molecular and cell biology research. This unit includes protocols for the growth and maintenance of Vero cell lines in a research laboratory setting.

  11. VERO stable cell lines expressing full-length human epidermal growth factor receptors 2 and 3: platforms for subtractive phage display.

    Science.gov (United States)

    Hedayatizadeh-Omran, Akbar; Valadan, Reza; Rafiei, Alireza; Tehrani, Mohsen; Alizadeh-Navaei, Reza

    2015-09-01

    Cross-talk between human epidermal growth factor receptor 2 and 3 (HER2 and HER3) may potentially contribute to therapeutic resistance in human breast cancer. Subtractive phage display allows highly specific selection for antibody fragments directed against cells surface HER2 and HER3. The strategies to select conformation- and activation-specific antibodies against HER2 and HER3 require tightly regulated HER2 and HER3 expressing cells that allow controlled activation/inactivation of these receptors during panning procedures. To achieve this, first, we found that the VERO cell line is an appropriate cell line for heterogeneous expression of HER2 and HER3, and then we established a panel of VERO stable cell lines expressing high levels of HER2 and HER3 alone and in combination. We also showed that HER2 and HER3 expressed in VERO cells were biologically active and could form heterodimer following neuregulin1 treatment. The cell line established here not only provided platforms for phage display-based methods but also could be used in any HER-related studies.

  12. Characterization of a shiga-toxin 1-resistant stock of vero cells.

    Science.gov (United States)

    Sekino, Takaomi; Kiyokawa, Nobutaka; Taguchi, Tomoko; Takenouchi, Hisami; Matsui, Jun; Tang, Wei-Ran; Suzuki, Toyo; Nakajima, Hideki; Saito, Masahiro; Ohmi, Kazuhiro; Katagiri, Yohko U; Okita, Hajime; Nakao, Hiroshi; Takeda, Tae; Fujimoto, Junichiro

    2004-01-01

    Shiga toxins (Stxs, also referred to as verotoxins) were first described as a novel cytotoxic activity against Vero cells. In this study, we report the characterization of an Stx1-resistant (R-) stock of Vero cells. (1) When the susceptibility of R-Vero cells to Stx1 cytotoxicity was compared to that of Stx1-sensitive (S-) Vero cells by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, cell viability after 48-hr exposure to 10 pg/ml of Stx1 was greater than 80% and less than 15%, respectively. (2) Although both a binding assay of fluorescence-labeled Stx1 and lipid analysis indicated considerable expression of Gb3Cer, a functional receptor for Stxs, in both Vero cells, anti-Gb3Cer monoclonal antibodies capable of binding to S-Vero cells failed to effectively label R-Vero cells, suggesting a conformational difference in the Gb3Cer expressed on R-Vero cells. (3) The lipid analysis also showed that the R-Vero cells contained significant amounts of Gb4Cer. In addition, introduction of exogenous Gb4Cer into S-Vero cells slightly inhibited Stx1 cytotoxicity, suggesting some correlation between glycosphingolipid composition and Stx1 resistance. (4) Both butyrate treatment and serum depression eliminated the Stx1 resistance of R-Vero cells. (5) The results of the analysis by confocal microscopy suggest a difference in intracellular transport of Stx1 between R-Vero and S-Vero cells. Further study of R-Vero cells may provide a model of Stx1 resistance via distinct intracellular transport of Stx1.

  13. MicroRNAs as potential biomarkers for VERO cell tumorigenicity.

    Science.gov (United States)

    Teferedegne, Belete; Macauley, Juliete; Foseh, Gideon; Dragunsky, Eugenia; Chumakov, Konstantin; Murata, Haruhiko; Peden, Keith; Lewis, Andrew M

    2014-08-20

    MicroRNA expression appears to capture the process of neoplastic development in vitro in the VERO line of African green monkey kidney (AGMK) cells (Teferedegne et al. PLoS One 2010;5(12):e14416). In that study, specific miRNA signatures were correlated with the transition, during serial tissue-culture passage, of low-density passaged 10-87 VERO cells from a non-tumorigenic phenotype at passage (p) 148 to a tumorigenic phenotype at p256. In the present study, six miRNAs (miR-376a, miR-654-3p, miR-543, miR-299-3p, miR-134 and miR-369-3p) were chosen from the identified signature miRNAs for evaluation of their use as potential biomarkers to track the progression of neoplastic development in VERO cells. Cells from the 10-87 VERO cell line at passage levels from p148 to p256 were inoculated into newborn and adult athymic nude mice. No tumors were observed in animals inoculated with cells from p148 to p186. In contrast, tumor incidences of 20% developed only in newborn mice that received 10-87 VERO cells at p194, p234 and p256. By qPCR profiling of the signature miRNAs of 10-87 VERO cells from these cell banks, we identified p194 as the level at which signature miRNAs elevated concurrently with the acquisition of tumorigenic phenotype with similar levels expressed beyond this passage. In wound-healing assays at 10-passage intervals between p150 to p250, the cells displayed a progressive increase in migration from p165 to p186; beginning at p194 and higher passages thereafter, the cells exhibited the highest rates of migration. By qPCR analysis, the same signature miRNAs were overexpressed with concomitant acquisition of the tumorigenic phenotype in another lineage of 10-87 VERO cells passaged independently at high density. Correlation between the passages at which the cells expressed a tumorigenic phenotype and the passages representing peaks in expression levels of signature miRNAs indicates that these miRNAs are potential biomarkers for the expression of the VERO cell

  14. Overexpression of α-2,6 sialyltransferase stimulates propagation of human influenza viruses in Vero cells.

    Science.gov (United States)

    Li, N; Qi, Y; Zhang, F Y; Yu, X H; Wu, Y G; Chen, Y; Jiang, C L; Kong, W

    2011-01-01

    Human influenza viruses are major concern as the leading cause of global pandemics. In infecting cells, they preferentially bind to sialyloligosaccharides containing terminal N-acetyl sialic acid linked to galactose by an α-2,6-linkage (NeuAcα2,6Gal). The amount of NeuAcα2,6Gal in Vero cells, which are predominantly used for production of influenza vaccines over the past 30 years, may not be as high as that in epithelial cells of human respiratory tract, what leads to the suboptimal virus growth in Vero cells. In this study, we stably transfected Vero cells with cDNA of human α-2,6-sialyltransferase (SIAT1), an enzyme catalyzing α-2,6-sialylation of galactose on glycoproteins. Overexpression of SIAT1 in the transfected Vero cells (Vero-SIAT1 cells) was confirmed by Western blot analysis and immunofluorescence microscopy. Vero-SIAT1 cells expressed 7 times higher amounts of NeuAcα2,6Gal, but 3 times lower amounts of NeuAcα2,3Gal as compared to parental Vero cells. Furthermore, the influenza viruses A (H1N1 and H3N2) and B grew in Vero-SIAT1 cells to the higher titers than in Vero cells. Taken together, these results imply that Vero-SIAT1 cells are useful not only for the propagation of human influenza viruses, but also for the preparation of influenza vaccines.

  15. Vero/BC-F: an efficient packaging cell line stably expressing F protein to generate single round-infectious human parainfluenza virus type 2 vector.

    Science.gov (United States)

    Ohtsuka, J; Fukumura, M; Tsurudome, M; Hara, K; Nishio, M; Kawano, M; Nosaka, T

    2014-08-01

    A stable packaging cell line (Vero/BC-F) constitutively expressing fusion (F) protein of the human parainfluenza virus type 2 (hPIV2) was established for production of the F-defective and single round-infectious hPIV2 vector in a strategy for recombinant vaccine development. The F gene expression has not evoked cytostatic or cytotoxic effects on the Vero/BC-F cells and the F protein was physiologically active to induce syncytial formation with giant polykaryocytes when transfected with a plasmid expressing hPIV2 hemagglutinin-neuraminidase (HN). Transduction of the F-defective replicon RNA into the Vero/BC-F cells led to the release of the infectious particles that packaged the replicon RNA (named as hPIV2ΔF) without detectable mutations, limiting the infectivity to a single round. The maximal titer of the hPIV2ΔF was 6.0 × 10(8) median tissue culture infections dose per ml. The influenza A virus M2 gene was inserted into hPIV2ΔF, and the M2 protein was found to be highly expressed in a human lung cancer cell line after transduction. Furthermore, in vivo airway infection experiments revealed that the hPIV2ΔF was capable of delivering transgenes to hamster tracheal cells. Thus, non-transmissible or single round-infectious hPIV2 vector will be potentially applicable to human gene therapy or recombinant vaccine development.

  16. Infectious bursal disease virus recovery from Vero cells transfected with RNA transcripts is enhanced by expression of the structural proteins in trans.

    Science.gov (United States)

    Peters, M A; Lin, T L; Wu, C C

    2005-11-01

    Positive sense RNA transcripts of infectious bursal disease (IBD) virus genome segments A and B have previously been shown to be infectious. In this study we demonstrate that recovery of IBD virus from the transfection of Vero cells with positive sense RNA transcripts of genome segments A and B was enhanced by expression of the viral structural proteins VP2 with VP3 or by expression of viral polyprotein VP243 from DNA plasmids in trans. Expression of individual viral proteins VP2, VP3, or VP4 alone from DNA plasmids did not enhance IBD virus recovery. Earliest virus recovery from transfection of positive sense RNA transcripts of genomic segments A and B was at 36 h and mean titers were 10(1.8) pfu/ml. IBD virus was recovered 6 hours after transfection in cells concurrently expressing either VP2 with VP3 or VP243 and mean titers were 10(8.5) pfu/ml or 10(9.2) pfu/ml, respectively. Likewise, expression of the viral polyprotein from DNA plasmid increased the permissiveness of Vero cells for infection with non-culture adapted IBD virus. The titer of recovered non-culture adapted virus from 10(3.3) pfu/ml to 10(10.3) pfu/ml with expression of the viral polyprotein. This report is the first to describe a reverse genetics model for IBD virus with high efficiency of virus recovery for non-culture adapted strains.

  17. Potential neoplastic evolution of Vero cells: in vivo and in vitro characterization.

    Science.gov (United States)

    Andreani, N A; Renzi, S; Piovani, G; Ajmone Marsan, P; Bomba, L; Villa, R; Ferrari, M; Dotti, S

    2017-10-01

    Vero cell lines are extensively employed in viral vaccine manufacturing. Similarly to all established cells, mutations can occur during Vero cells in vitro amplification which can result in adverse features compromising their biological safety. To evaluate the potential neoplastic evolution of these cells, in vitro transformation test, gene expression analysis and karyotyping were compared among low- (127 and 139 passages) and high-passage (passage 194) cell lines, as well as transformed colonies (TCs). In vivo tumorigenicity was also tested to confirm preliminary in vitro data obtained for low passage lines and TCs. Moreover, Vero cells cultivated in foetal bovine serum-free medium and derived from TCs were analysed to investigate the influence of cultivation methods on tumorigenic evolution. Low-passage Vero developed TCs in soft agar, without showing any tumorigenic evolution when inoculated in the animal model. Karyotyping showed a hypo-diploid modal chromosome number and rearrangements with no difference among Vero cell line passages and TCs. These abnormalities were reported also in serum-free cultivated Vero. Gene expression revealed that high-passage Vero cells had several under-expressed and a few over-expressed genes compared to low-passage ones. Gene ontology revealed no significant enrichment of pathways related to oncogenic risk. These findings suggest that in vitro high passage, and not culture conditions, induces Vero transformation correlated to karyotype and gene expression alterations. These data, together with previous investigations reporting tumour induction in high-passage Vero cells, suggest the use of low-passage Vero cells or cell lines other than Vero to increase the safety of vaccine manufacturing.

  18. Analysis of protein expression changes of the Vero E6 cells infected with classic PEDV strain CV777 by using quantitative proteomic technique.

    Science.gov (United States)

    Sun, Dongbo; Shi, Hongyan; Guo, Donghua; Chen, Jianfei; Shi, Da; Zhu, Qinghe; Zhang, Xin; Feng, Li

    2015-06-15

    Recent outbreaks of porcine epidemic diarrhea virus (PEDV) have caused widespread concern. The identification of proteins associated with PEDV infection might provide insight into PEDV pathogenesis and facilitate the development of novel antiviral strategies. We analyzed the differential protein profile of PEDV-infected Vero E6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. A total of 126 proteins were identified that were differentially expressed between the PEDV-infected and mock-infected groups (PVero E6 cells, involving in integrin β2/β3, cystatin-C. The Gene Ontology analysis indicated that the molecular function of the differentially expressed proteins (DEPs) was primarily related to binding and catalytic activity, and that the biological functions in which the DEPs are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. Among the disease-related functions, certain anti-viral pathways and proteins, such as the RIG-I-like receptor, Rap1, autophagy, mitogen-activated protein kinase, PI3K-Akt and Jak-STAT signaling pathways, and integrin β2/β3 and cystatin-C proteins, represented potential factors in PEDV infection. Our findings provide valuable insight into PEDV-Vero E6 cell interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney

    Directory of Open Access Journals (Sweden)

    Asako Murayama

    2016-06-01

    Full Text Available A hepatitis C virus (HCV cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122, an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE, a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells.

  20. Canine distemper virus isolated from a monkey efficiently replicates on Vero cells expressing non-human primate SLAM receptors but not human SLAM receptor.

    Science.gov (United States)

    Feng, Na; Liu, Yuxiu; Wang, Jianzhong; Xu, Weiwei; Li, Tiansong; Wang, Tiecheng; Wang, Lei; Yu, Yicong; Wang, Hualei; Zhao, Yongkun; Yang, Songtao; Gao, Yuwei; Hu, Guixue; Xia, Xianzhu

    2016-08-02

    In 2008, an outbreak of canine distemper virus (CDV) infection in monkeys was reported in China. We isolated CDV strain (subsequently named Monkey-BJ01-DV) from lung tissue obtained from a rhesus monkey that died in this outbreak. We evaluated the ability of this virus on Vero cells expressing SLAM receptors from dog, monkey and human origin, and analyzed the H gene of Monkey-BJ01-DV with other strains. The Monkey-BJ01-DV isolate replicated to the highest titer on Vero cells expressing dog-origin SLAM (10(5.2±0.2) TCID50/ml) and monkey-origin SLAM (10(5.4±0.1) TCID50/ml), but achieved markedly lower titers on human-origin SLAM cells (10(3.3±0.3) TCID50/ml). Phylogenetic analysis of the full-length H gene showed that Monkey-BJ01-DV was highly related to other CDV strains obtained during recent CDV epidemics among species of the Canidae family in China, and these Monkey strains CDV (Monkey-BJ01-DV, CYN07-dV, Monkey-KM-01) possessed a number of amino acid specific substitutions (E276V, Q392R, D435Y and I542F) compared to the H protein of CDV epidemic in other animals at the same period. Our results suggested that the monkey origin-CDV-H protein could possess specific substitutions to adapt to the new host. Monkey-BJ01-DV can efficiently use monkey- and dog-origin SLAM to infect and replicate in host cells, but further adaptation may be required for efficient replication in host cells expressing the human SLAM receptor.

  1. Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney.

    Science.gov (United States)

    Murayama, Asako; Sugiyama, Nao; Wakita, Takaji; Kato, Takanobu

    2016-06-14

    A hepatitis C virus (HCV) cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122), an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI) was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE), a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells. HCV is a major cause of chronic liver diseases worldwide, and an effective prophylactic HCV vaccine is needed. For safety reasons, the current HCV cell culture system using HuH-7 cells, which was established from a hepatocellular carcinoma, is not suitable for the production of a vaccine against HCV. A robust HCV production system using non-cancer-derived cells is indispensable for

  2. Comparative susceptibility of vero and baby hamster kidney cell ...

    African Journals Online (AJOL)

    This study was undertaken to assess the comparative susceptibility of the different cell lines to PPRV using virus isolation by Vero and BHK cell lines from field samples. The inoculated BHK and Vero cells supported the growth of the virus with syncytia formation more commonly observed in the BHK cells while vacuolation ...

  3. Limited uptake of the cyanobacterial toxin cylindrospermopsin by Vero cells.

    Science.gov (United States)

    Froscio, S M; Cannon, E; Lau, H M; Humpage, A R

    2009-11-01

    Cylindrospermopsin (CYN) is a cyanobacterial toxin increasingly found in drinking water sources worldwide. Toxicity studies have shown CYN can induce effects in a range of different cell types with primary hepatocytes consistently shown to be the most sensitive cellular model. How CYN enters the intracellular environment is not clear, although the size and hydrophilic nature of the toxin suggest it would not readily cross a lipid bilayer. In this study, a Vero cell line expressing green fluorescent protein (GFP) was used to monitor for CYN uptake based on the toxin's potent effects on protein synthesis. Effects on the GFP signal were compared with inhibitors cycloheximide (CHEX) and emetine. While CYN potency was demonstrated in a cell-free system (CYN>CHEX>emetine) it was considerably reduced in the Vero-GFP cell model (CHEX, emetine>CYN). In contrast to other inhibitors, CYN effects on GFP signal increased 6 fold over 4-24 h incubation indicating slow, progressive uptake of the toxin. Confirming that the uptake process is not energy dependent CYN entry also occurred at 4 degrees C, while competition experiments excluded the uracil nucleobase transporter system as potential mechanism for CYN uptake. Dilution of media enhanced CYN uptake by Vero-GFP cells although mechanism by which this occurred is unknown.

  4. Leucine affects the fibroblastic Vero cells stimulating the cell proliferation and modulating the proteolysis process.

    Science.gov (United States)

    Gonçalves, Estela Maria; Gomes-Marcondes, Maria Cristina Cintra

    2010-01-01

    Branched-chain amino acids, especially leucine, exert regulatory influences on protein and carbohydrate metabolism, ribosome biogenesis and gene expression. This study investigated the effects of leucine in fibroblastic cells analysing viability, proliferation, morphology, proteolysis enzymes activities and protein turnover. After exposure to culture medium enriched with 25 or 50 microM leucine for 24, 48 and 72 h, Vero cells have no alterations on viability and morphology. Leucine-treated cells showed increase on alkaline phosphatase activity and proliferation. The protein synthesis was slightly increased, whereas the protein degradation showed a deep reduction after leucine incubation. The chymotrypsin-like, cathepsin B and H and calpain activities were decreased in leucine-treated cells. In conclusion, the proteolytic pathways and the total protein degradation were modulated by leucine in Vero cells. Our observations support the concept that Vero cells may represent a new model for protein turnover study.

  5. A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.

    Science.gov (United States)

    Wang, Kening; Kappel, Justin D; Canders, Caleb; Davila, Wilmer F; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I

    2012-12-01

    We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

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

    OpenAIRE

    Salas, J; Viñuela, E

    1986-01-01

    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-...

  7. Adhesion and internalization differences of COM nanocrystals on Vero cells before and after cell damage

    Energy Technology Data Exchange (ETDEWEB)

    Gan, Qiong-Zhi; Sun, Xin-Yuan; Ouyang, Jian-Ming, E-mail: toyjm@jnu.edu.cn

    2016-02-01

    The adhesion and internalization between African green monkey kidney epithelial (Vero) cells (before and after oxidative damage by hydrogen peroxide) and calcium oxalate monohydrate (COM) nanocrystals (97 ± 35 nm) were investigated so as to discuss the molecular and cellular mechanism of kidney stone formation. Scanning electron microscope (SEM) was used to observe the Vero–COM nanocrystal adhesion; the nanocrystal-cell adhesion was evaluated by measuring the content of malonaldehyde (MDA), the activity of superoxide dismutase (SOD), the expression level of cell surface osteopontin (OPN) and the change of Zeta potential. Confocal microscopy and flow cytometry were used for the observation and quantitative analysis of crystal internalization. In the process of adhesion, the cell viability and the SOD activity declined, the MDA content, Zeta potential, and the OPN expression level increased. The adhesive capacity of injured Vero was obviously stronger than normal cells; in addition the injured cells promoted the aggregation of COM nanocrystals. The capacity of normal cells to internalize crystals was obviously stronger than that of injured cells. Cell injury increased adhesive sites on cell surface, thereby facilitating the aggregation of COM nanocrystals and their attachment, which results in enhanced risk of calcium oxalate stone formation. - Graphical abstract: The adhesion and internalization differences between Vero cells before and after oxidative damage and calcium oxalate monohydrate nanocrystals were comparatively studied. - Highlights: • Adhesion capacity of injured Vero cells was stronger than normal cells. • Internalization capacity of injured Vero cells was weaker than normal cells. • Injured cells promoted the aggregation of COM nanocrystals. • COM adhesion could aggravate cell injury in both normal and injured cells.

  8. Involvement of endoplasmic reticulum and autophagy in microcystin-LR toxicity in Vero-E6 and HepG2 cell lines.

    Science.gov (United States)

    Menezes, Carina; Alverca, Elsa; Dias, Elsa; Sam-Bento, Filomena; Pereira, Paulo

    2013-02-01

    This work investigates the involvement of the endoplasmic reticulum (ER) and autophagy in microcystin-LR (MCLR) toxicity in Vero-E6 and HepG2 cell lines. Additionally, morphological alterations induced by MCLR in lysosomes and mitochondria were studied. Cytotoxicity evaluation showed that pure MCLR and MCLR from LMECYA110 extract induce concentration dependent viability decays after 24h exposure. HepG2 cells showed an increased sensitivity to MCLR than Vero cells, with lower cytotoxic thresholds and EC(50) values. Conversely, LC3B immunofluorescence showed that autophagy is triggered in both cell lines as a survival response to low MCLR concentrations. Furthermore, MCLR induced a MCLR concentration-dependent decrease of GRP94 expression in HepG2 cells while in Vero cells no alteration was observed. This suggests the involvement of the ER in HepG2 apoptosis elicited by MCLR, while in Vero cells ER destructuration could be a consequence of cytoskeleton inflicted damages. Additionally, in both cell lines, lysosomal destabilization preceded mitochondrial impairment which occurred at high toxin concentrations. Although not an early cellular target of MCLR, mitochondria appears to serve as central mediators of different signaling pathways elicited by the organelles involved in MCLR toxicity. As a result, kidney and hepatic cell lines exhibit cell type and dose-dependent mechanisms to overcome MCLR toxicity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Morphological and growth alterations in Vero cells transformed by cisplatin.

    Science.gov (United States)

    Gonçalves, Estela Maria; Ventura, Cláudio Angelo; Yano, Tomomasa; Rodrigues Macedo, Maria Lígia; Genari, Selma Candelária

    2006-06-01

    Cisplatin is an antineoplastic agent used to treat solid tumours, such as ovarian, testicular and bladder tumours. However, studies in vitro and in vivo have shown that cisplatin is mutagenic, genotoxic and tumorigenic in other tissues and organs. In this work, we examined the effect of cisplatin on Vero cells, a fibroblast-like cell line. The morphological characteristics were investigated using phase contrast microscopy, scanning electron microscopy and the actin cytoskeleton was labelled with fluorescein isothiocyanate-phalloidin. Cell proliferation was assessed based on the growth curve. Cultured Vero cells treated with cisplatin showed behavioural and morphological alterations associated with cellular transformation. The transformed cells grew in multilayers and formed cellular aggregates. The proliferation and morphological characteristics of the transformed cells were very different from those of control ones. Since transformed Vero cells showed several characteristics related to neoplastic growth, these cells could be a useful model for studying tumour cells in vitro.

  10. Interaction of Leishmania (L. chagasi with the Vero cell line

    Directory of Open Access Journals (Sweden)

    Pessotti J.H.

    2004-03-01

    Full Text Available The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L. chagasi. This strain (MHOM/BR/501/MS00 was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20 % of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.. chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.

  11. Interaction of Leishmania (L.) chagasi with the Vero cell line.

    Science.gov (United States)

    Pessotti, J H; Zaverucha Do Valle, T; Corte-Real, S; Gonçalves Da Costa, S C

    2004-03-01

    The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20% of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.

  12. Replication-competent human adenovirus 11p vectors can propagate in Vero cells

    Energy Technology Data Exchange (ETDEWEB)

    Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

    2016-08-15

    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

  13. New World hantaviruses activate IFNlambda production in type I IFN-deficient vero E6 cells.

    Science.gov (United States)

    Prescott, Joseph; Hall, Pamela; Acuna-Retamar, Mariana; Ye, Chunyan; Wathelet, Marc G; Ebihara, Hideki; Feldmann, Heinz; Hjelle, Brian

    2010-06-17

    Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon lambda, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner. We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNlambda. Three New World hantaviruses were similarly able to induce IFNlambda expression in this cell line. The IFNlambda contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs. Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNlambda. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNlambda production in these cells might increase their utility for virus

  14. New World hantaviruses activate IFNlambda production in type I IFN-deficient vero E6 cells.

    Directory of Open Access Journals (Sweden)

    Joseph Prescott

    Full Text Available Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS. These viruses induce a strong interferon-stimulated gene (ISG response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN, rendering them unable to mount an efficient innate immune response to virus infection. Interferon lambda, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner.We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNlambda. Three New World hantaviruses were similarly able to induce IFNlambda expression in this cell line. The IFNlambda contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7 without inducing ISGs.Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNlambda. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNlambda production in these cells might increase their utility

  15. Adhesion and internalization differences of COM nanocrystals on Vero cells before and after cell damage.

    Science.gov (United States)

    Gan, Qiong-Zhi; Sun, Xin-Yuan; Ouyang, Jian-Ming

    2016-02-01

    The adhesion and internalization between African green monkey kidney epithelial (Vero) cells (before and after oxidative damage by hydrogen peroxide) and calcium oxalate monohydrate (COM) nanocrystals (97±35nm) were investigated so as to discuss the molecular and cellular mechanism of kidney stone formation. Scanning electron microscope (SEM) was used to observe the Vero-COM nanocrystal adhesion; the nanocrystal-cell adhesion was evaluated by measuring the content of malonaldehyde (MDA), the activity of superoxide dismutase (SOD), the expression level of cell surface osteopontin (OPN) and the change of Zeta potential. Confocal microscopy and flow cytometry were used for the observation and quantitative analysis of crystal internalization. In the process of adhesion, the cell viability and the SOD activity declined, the MDA content, Zeta potential, and the OPN expression level increased. The adhesive capacity of injured Vero was obviously stronger than normal cells; in addition the injured cells promoted the aggregation of COM nanocrystals. The capacity of normal cells to internalize crystals was obviously stronger than that of injured cells. Cell injury increased adhesive sites on cell surface, thereby facilitating the aggregation of COM nanocrystals and their attachment, which results in enhanced risk of calcium oxalate stone formation. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Carbohydrates-chitosan composite carrier for Vero cell culture.

    Science.gov (United States)

    Lin, Ya-Ching; Chen, Guan-Ting; Wu, Sheng-Chi

    2016-12-01

    In this study, carbohydrate-chitosan composite including glucose-chitosan, sucrose-chitosan and starch-chitosan with varied carbohydrate concentrations were prepared as carriers for Vero cell culture. Our results show that among these composites, 30 % starch-chitosan composite (STC) were the best carriers for the growth of Vero cells. The initial number of attached cells on the surface of composite carriers did not have any significant effect on subsequent cell production. A higher glucose level in the growth medium during the exponential phase of cell growth, however, played an important factor for cell production. Vero cells on the STC carriers were able to convert starch inside the composite carriers into glucose and further utilized the glucose for their growth. Moreover, by crosslink with serum the STC carriers supported an even better cell production in the normal medium without adding fetal bovine serum, as well as a good extracellular virus production. The STC composite is therefore a promising alternative carrier for Vero cell culture.

  17. Use of a SLAM transfected Vero cell line to isolate and characterize marine mammal morbilliviruses using an experimental ferret model.

    Science.gov (United States)

    Nielsen, Ole; Smith, Greg; Weingartl, Hana; Lair, Stéphane; Measures, Lena

    2008-07-01

    Two ferrets (Mustela putorius furo) were experimentally infected with phocine distemper virus (PDV), from the 1988 seal epizootic in Europe, in order to determine whether the stable transfected Vero cell line (Vero.DogSLAMtag) expressing canine "signaling lymphocyte activation molecules" (SLAM; CD150) receptors, was more suitable for isolating and characterizing PDV when compared with Vero (American Type Culture Collection # C1008) and primary seal kidney (PSK) cells. Both ferrets displayed characteristic clinical signs of distemper, including fever and rash, 10 days postinoculation (dpi) and, due to increased morbidity, they were euthanized 12 dpi. Histologic lesions, suggestive of infection with morbilliviruses, were observed in tissues from both ferrets, and the tissues stained positive using immunohistochemistry. Isolation of PDV from isolated peripheral blood lymphocytes (PBLs), taken at 5 and 10 dpi, was achieved by cocultivation with Vero and PSK cells, following several passages. Cytopathic effects (CPE) were observed in Vero cell cultures at 29 dpi and in PSK cell cultures at 22 dpi. Phocine distemper virus was isolated from frozen infected ferret lung tissue within 48 hr, when isolation was attempted using the Vero.DogSLAMtag cell line. In addition, a reverse transcriptase polymerase chain reaction (RT-PCR) test was developed to detect a 114 base pair (bp) portion of the nucleocapsid gene found only in PDV. This RT-PCR methodology was used to confirm the identity of the virus subsequently isolated from the ferrets. Viral isolates from the infected ferrets, as well as cultures of virus originally isolated from a dolphin and a porpoise and maintained in Vero cells, also replicated faster and produced higher titers of virus when propagated in Vero.DogSLAMtag cells. These results indicate that Vero.DogSLAMtag cells offer a substantial improvement (including faster viral replication resulting in primary viral isolation in a shorter period of time, and higher

  18. Antiproliferative efficacy of Tabernaemontana divaricata against HEP2 cell line and Vero cell line.

    Science.gov (United States)

    Kumar, Arvind; Selvakumar, S

    2015-05-01

    Laryngeal cancer may also be called cancer of the larynx or laryngeal carcinoma. Conventional plants are a precious source of novel anticancer agents and are still in performance better role in health concern. The study was intended to estimation of the anticancer activity of the chloroformic extract of Tabernaemontana divaricata on the human epidermoid larynx carcinoma cell line (Hep 2). The aerial parts (leaves, stem, and flowers) of T. divaricata were tested for its inhibitory effect in 96 microplate formats against Hep 2 cell line. The anticancer activity of samples on Hep 2 and Vero was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and various enzymatic parameters like catalase, reduced glutathione (GSH), GSH peroxidase, and superoxide anion scavenging activity. Viable cells were determined by the absorbance at 540 nm. Measurements were performed, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of Hep 2 and Vero cells was expressed as the % cell viability. The extract on Hep 2 cell line up to 7.8 μg/ml and that IC50 value on Hep 2 cell line was 112 μg whereas 94 μg for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line. Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line).

  19. Adherence and intracellular parasitism of Paracoccidioides brasiliensis in Vero cells.

    Science.gov (United States)

    Hanna, S A; Monteiro da Silva, J L; Giannini, M J

    2000-07-01

    Paracoccidioides brasiliensis is a dimorphic fungus known to produce invasive systemic disease in humans. The 43-kDa glycoprotein of P. brasiliensis is the major diagnostic antigen of paracoccidioidomycosis and may act as a virulence factor, since it is a receptor for laminin.Very little is known about early interactions between this fungus and the host cells, so we developed in vitro a model system employing cultured mammalian cells (Vero cells), in order to investigate the factors and virulence mechanisms of P.brasiliensis related to the adhesion and invasion process. We found that there is a permanent interaction after 30 min of contact between the fungus and the cells. The yeasts multiply in the cells for between 5 and 24 h. Different strains of P. brasiliensis were compared, and strain 18 (high virulence) was the most strongly adherent, followed by strain 113 (virulent), 265 (considered of low virulence) and 113M (mutant obtained by ultraviolet radiation, deficient in gp43). P. brasiliensis adhered to the epithelial cells by a narrow tube, while depressions were noticed in the cell surface, suggesting an active cavitation process. An inhibition assay was performed and it was verified that anti-gp43 serum and a pool of sera from individuals with paracoccidioidomycosis were able to inhibit the adhesion of P. brasiliensis to the Vero cells. Glycoprotein 43 (gp43) antiserum abolished 85% of the binding activity of P. brasiliensis. This fungus can also invade the Vero cells, and intraepithelial parasitism could be an escape mechanism in paracoccidioidomycosis.

  20. Proteome analysis of virus-host cell interaction: rabies virus replication in Vero cells in two different media.

    Science.gov (United States)

    Kluge, Sabine; Rourou, Samia; Vester, Diana; Majoul, Samy; Benndorf, Dirk; Genzel, Yvonne; Rapp, Erdmann; Kallel, Héla; Reichl, Udo

    2013-06-01

    The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8 days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection.

  1. Carbamazepine induces mitotic arrest in mammalian Vero cells

    Energy Technology Data Exchange (ETDEWEB)

    Perez Martin, J.M.; Fernandez Freire, P.; Labrador, V. [Departamento de Biologia, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Hazen, M.J. [Departamento de Biologia, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)], E-mail: mariajose.hazen@uam.es

    2008-01-01

    We reported recently that the anticonvulsant drug carbamazepine, at supratherapeutic concentrations, exerts antiproliferative effects in mammalian Vero cells, but the underlying mechanism has not been elucidated. This motivates us to examine rigorously whether growth arrest was associated with structural changes in cellular organization during mitosis. In the present work, we found that exposure of the cells to carbamazepine led to an increase in mitotic index, mainly due to the sustained block at the metaphase/anaphase boundary, with the consequent inhibition of cell proliferation. Indirect immunofluorescence, using antibodies directed against spindle apparatus proteins, revealed that mitotic arrest was associated with formation of monopolar spindles, caused by impairment of centrosome separation. The final consequence of the spindle defects induced by carbamazepine, depended on the duration of cell cycle arrest. Following the time course of accumulation of metaphase and apoptotic cells during carbamazepine treatments, we observed a causative relationship between mitotic arrest and induction of cell death. Conversely, cells released from the block of metaphase by removal of the drug, continued to progress through mitosis and resume normal proliferation. Our results show that carbamazepine shares a common antiproliferative mechanism with spindle-targeted drugs and contribute to a better understanding of the cytostatic activity previously described in Vero cells. Additional studies are in progress to extend these initial findings that define a novel mode of action of carbamazepine in cultured mammalian cells.

  2. Influence of culture conditions on Vero cell propagation on non-porous microcarriers

    Directory of Open Access Journals (Sweden)

    Marta Cristina de Oliveira Souza

    2005-06-01

    Full Text Available Animal cell cultures are widely employed for the production of viral vaccines and for recombinant protein expression. The cell line Vero is a continuous, adherent cell line, which has been recommended by the World Health Organization for the production of human vaccines. For the large-scale production of vaccines, microcarriers, which are microspheres that serve as support for the cells, are being increasingly used. The use of microcarriers in stirred bioreactors allows high cell densities and, consequently, high virus titres to be achieved. With the aim of selecting appropriate culture conditions for the cultivation of Vero cells at high cell densities, in this work the influence of several variables (agitation rate, ratio of inoculated cells to microcarrier mass and fetal bovine serum concentration on cell growth on Cytodex 1 microcarriers was studied. Under the best conditions determined, a comparison with Vero cell cultivation on Cytodex 3 microcarriers was carried out.Cultivos de células animais são amplamente utilizados para a produção de vacinas virais e para a expressão de proteínas recombinantes. A linhagem celular Vero é uma linhagem contínua, dependente de ancoragem, recomendada pela Organização Mundial de Saúde para a produção de vacinas de uso humano. Para a produção de vacinas virais em larga escala, vêm sendo cada vez mais empregados microcarregadores, que são microesferas que servem de suporte para as células. O emprego de microcarregadores em biorreatores agitados permite a obtenção de altas densidades celulares e, conseqüentemente, de altos títulos de antígenos virais. Com o objetivo de selecionar condições de cultivo adequadas, estudou-se, neste trabalho, o efeito das variáveis agitação, razão de células inoculadas por microcarregador e concentração de soro fetal bovino sobre o crescimento de células Vero em microcarregadores Cytodex 1. Nas melhores condições selecionadas, o desempenho dos

  3. Replication-competent human adenovirus 11p vectors can propagate in Vero cells.

    Science.gov (United States)

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    2016-08-01

    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Hepatitis C virus infection of a Vero cell clone displaying efficient virus-cell binding.

    Science.gov (United States)

    Valli, M B; Carloni, G; Manzin, A; Nasorri, F; Ponzetto, A; Clementi, M

    1997-01-01

    The susceptibility of Vero cells and derivative cell clones to hepatitis C virus (HCV) infection was assayed by qualitative and quantitative polymerase chain reaction (PCR)-based methods. Cell extracts from Vero cells inoculated with HCV were tested for the presence of both positive and negative strands of HCV RNA; in parallel, cell-free HCV genomes were assayed in culture supernatant fluids. Quantitation of genomic HCV RNA molecules in infected cells by competitive reverse transcription PCR (cRT-PCR) indicated that HCV replication was more efficient in a derivative clone (named clone 10) than in parental Vero cells or other clones under study. Analysis of HCV-binding to cell receptors, performed by cRT-PCR quantitation of viral particles adsorbed to the cell surface, demonstrated a 10-fold higher virus-binding level of clone 10 than that of parental Vero cells. The results shown here indicate that the Vero clone 10 may constitute an efficient model system for analysing early events in HCV infection as well as a source of virus for diagnostic and biotechnological applications.

  5. Assessment of potential miRNA biomarkers of VERO-cell tumorigenicity in a new line (AGMK1-9T7) of African green monkey kidney cells.

    Science.gov (United States)

    Teferedegne, Belete; Rotroff, Daniel M; Macauley, Juliete; Foseh, Gideon; Lewis, Gladys; Motsinger-Rief, Alison; Lewis, Andrew M

    2017-10-04

    Patterns of microRNA expression appear to delineate the process of spontaneous neoplastic development-transformation (SPNDT) occurring in the African green monkey kidney (AGMK) VERO cell line (Teferedegne et al., 2010). Analysis of microarray data identified 6 microRNAs whose high-level of expression peaked when the World Health Organization 10-87 VERO cells became tumorigenic at passage (p) 190. Six miRNAs were identified as potential biomarkers for the expression of the VERO-cell tumorigenic phenotype (Teferedegne et al., 2014). However, the question remained whether these miRNA biomarkers are specific for VERO cells or can be generalizable to other cells originating from African green monkey kidneys. To examine miRNA expression patterns in AGMK cells at lower passage levels and to re-examine the identified miRNAs as biomarkers associated with tumorigenic phenotype of VERO cells in another independently-derived line, we established a new line of African green monkey kidney cells (AGMK1-9T7) by serially passaging kidney cells from another AGM. The AGMK1-9T7 cells became tumorigenic in nude mice at p40. Evaluation of miRNA expression at intervals from p1 to p40 revealed similarities between the evolution of miRNA expression during SPNDT in the AGMK1-9T7 cells and the 10-87 VERO cells. Four of the 6 potential biomarker miRNAs (miR-376a, miR-654-3p, miR-543, miR-134) in our earlier reports were detected by microarray in the AGMK1-9T7 cells; RT-qPCR analysis detected all 6 miRNAs. All 6 of these miRNAs have been associated with human tumors. Detection of the same miRNAs associated with the tumorigenic p40 AGMK1-9T7 cells and tumorigenic 10-87 VERO cells confirmed our proposal that these miRNA represent biomarkers for the tumor-forming ability of AGMK/VERO cells. The similarities of expression of miRNAs in different AGMK cell lines that were established 50years apart suggest that the process of SPNDT in these non-human primate cells in tissue culture is based upon

  6. TMPRSS2 and MSPL Facilitate Trypsin-Independent Porcine Epidemic Diarrhea Virus Replication in Vero Cells.

    Science.gov (United States)

    Shi, Wen; Fan, Wenlu; Bai, Jing; Tang, Yandong; Wang, Li; Jiang, Yanping; Tang, Lijie; Liu, Min; Cui, Wen; Xu, Yigang; Li, Yijing

    2017-05-18

    Type II transmembrane serine proteases (TTSPs) facilitate the spread and replication of viruses such as influenza and human coronaviruses, although it remains unclear whether TTSPs play a role in the progression of animal coronavirus infections, such as that by porcine epidemic diarrhea virus (PEDV). In this study, TTSPs including TMPRSS2, HAT, DESC1, and MSPL were tested for their ability to facilitate PEDV replication in Vero cells. Our results showed that TMPRSS2 and MSPL played significant roles in the stages of cell-cell fusion and virus-cell fusion, whereas HAT and DESC1 exhibited weaker effects. This activation may be involved in the interaction between TTSPs and the PEDV S protein, as the S protein extensively co-localized with TMPRSS2 and MSPL and could be cleaved by co-expression with TMPRSS2 or MSPL. Moreover, the use of Vero cells expressing TMPRSS2 and MSPL facilitated PEDV replication in the absence of exogenous trypsin. In sum, we identified two host proteases, TMPRSS2 and MSPL, which may provide insights and a novel method for enhancing viral titers, expanding virus production, and improving the adaptability of PEDV isolates in vitro.

  7. Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells.

    Science.gov (United States)

    Gaudin, Raphaël; Kirchhausen, Tomas

    2015-11-09

    Many viruses have evolved strategies of so-called "superinfection exclusion" to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.

  8. Intracellular localization of Saffold virus Leader (L) protein differs in Vero and HEp-2 cells.

    Science.gov (United States)

    Xu, Yishi; Victorio, Carla Bianca Luena; Ng, Qimei; Prabakaran, Mookkan; Tan, Yee-Joo; Chua, Kaw Bing

    2016-10-12

    The Saffold virus (SAFV) genome is translated as a single long polyprotein precursor and co-translationally cleaved to yield 12 separate viral proteins. Little is known about the activities of SAFV proteins although their homologs in other picornaviruses have already been described. To further support research on functions and activities of respective viral proteins, we investigated the spatio-temporal distribution of SAFV proteins in Vero and HEp-2 cells that had been either transfected with plasmids that express individual viral proteins or infected with live SAFV. Our results revealed that, with the exception of the Leader (L) protein, all viral proteins were localized in the cytoplasm at all the time points assayed. The L protein was found in the cytoplasm at an early time point but was subsequently translocated to the nucleus of HEp-2, but not Vero, cells. This was observed in both transfected and infected cells. Further mutational analysis of L protein revealed that Threonine 58 of the Ser/Thr-rich domain of L protein is crucial for protein trafficking between the cytoplasm and nucleus in HEp-2 cells. These findings contribute to a deeper understanding and stimulate investigation of the differetial cellular responses of HEp-2 cells in comparison to other mammalian cell lines during SAFV infection.

  9. Novel endogenous simian retroviral integrations in Vero cells: implications for quality control of a human vaccine cell substrate.

    Science.gov (United States)

    Sakuma, Chisato; Sekizuka, Tsuyoshi; Kuroda, Makoto; Kasai, Fumio; Saito, Kyoko; Ikeda, Masaki; Yamaji, Toshiyuki; Osada, Naoki; Hanada, Kentaro

    2018-01-12

    African green monkey (AGM)-derived Vero cells have been utilized to produce various human vaccines. The Vero cell genome harbors a variety of simian endogenous type D retrovirus (SERV) sequences. In this study, a transcriptome analysis showed that DNA hypomethylation released the epigenetic repression of SERVs in Vero cells. Moreover, comparative genomic analysis of three Vero cell sublines and an AGM reference revealed that the genomes of the sublines have ~80 SERV integrations. Among them, ~60 integrations are present within all three cell sublines and absent from the reference sequence. At least several of these integrations consist of complete SERV proviruses. These results strongly suggest that SERVs integrated in the genome of Vero cells did not retrotranspose after the establishment of the cell lineage as far as cells were maintained under standard culture and passage conditions, providing a scientific basis for controlling the quality of pharmaceutical cell substrates and their derived biologics.

  10. Proteomic analysis of membrane proteins of vero cells: exploration of potential proteins responsible for virus entry.

    Science.gov (United States)

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong; Sun, Dongbo

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells.

  11. Severe acute respiratory syndrome coronavirus persistence in Vero cells.

    Science.gov (United States)

    Palacios, Gustavo; Jabado, Omar; Renwick, Neil; Briese, Thomas; Lipkin, W Ian

    2005-03-20

    Several coronaviruses establish persistent infections in vitro and in vivo, however it is unknown whether persistence is a feature of the severe acute respiratory syndrome coronavirus (SARS-CoV) life cycle. This study was conducted to investigate viral persistence. We inoculated confluent monolayers of Vero cells with SARS-CoV at a multiplicity of infection of 0.1 TCID50 and passaged the remaining cells every 4 to 8 days for a total of 11 passages. Virus was titrated at each passage by limited dilution assay and nucleocapsid antigen was detected by Western blot and immunofluoresence assays. The presence of viral particles in passage 11 cells was assessed by electron microscopy. Changes in viral genomic sequences during persistent infection were examined by DNA sequencing. Cytopathic effect was extensive after initial inoculation but diminished with serial passages. Infectious virus was detected after each passage and viral growth curves were identical for parental virus stock and virus obtained from passage 11 cells. Nucleocapsid antigen was detected in the majority of cells after initial inoculation but in only 10%-40% of cells at passages 2-11. Electron microscopy confirmed the presence of viral particles in passage 11 cells. Sequence analysis at passage 11 revealed fixed mutations in the spike (S) gene and ORFs 7a-8b but not in the nucleocapsid (N) gene. SARS-CoV can establish a persistent infection in vitro. The mechanism for viral persistence is consistent with the formation of a carrier culture whereby a limited number of cells are infected with each round of virus replication and release. Persistence is associated with selected mutations in the SARS-CoV genome. This model may provide insight into SARS-related lung pathology and mechanisms by which humans and animals can serve as reservoirs for infection.

  12. The genome landscape of the african green monkey kidney-derived vero cell line.

    Science.gov (United States)

    Osada, Naoki; Kohara, Arihiro; Yamaji, Toshiyuki; Hirayama, Noriko; Kasai, Fumio; Sekizuka, Tsuyoshi; Kuroda, Makoto; Hanada, Kentaro

    2014-12-01

    Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover, a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of Vero cells. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  13. Development of a Vero cell DNA reference standard for residual DNA measurement in China.

    Science.gov (United States)

    Cao, Shouchun; Dong, Guanmu; Tang, Jianrong; Li, Jia; Liu, Jinghua; Shi, Leitai; Li, Changgui; Wang, Junzhi

    2013-02-01

    This collaborative study developed a Vero cell DNA reference for standardizing dot blot hybridization, an assay widely employed to measure residual DNA contents of viral vaccines prepared with Vero cells. High purity of Vero cell DNA was extracted and characterized by Hind III enzyme digestion and DNA sequencing. Then, with a cooperative calibration, the concentration of Vero cell DNA reference bulk solution was determined (64.0 ± 1.9 μg/mL, OD 260/OD 280 = 1.87) and diluted (40 ng/mL) with Tris-EDTA buffer containing bovine serum albumin as freeze-dried excipients. With industrial filling apparatus, the diluted bulk was loaded into ampoules (0.5 mL each) which were heat sealed after nitrogen filling. Finally, a collaborative study showed that the Vero cell DNA reference could reach a sensitivity of 1 to 5 pg/dot and maintained good stability after accelerated destruction test. The successful establishment of the Vero cell DNA quantitative reference will facilitate the standardization of dot blot hybridization for testing residual host cell DNA.

  14. [Mitochondrial injury in enterovirus 71-infected Vero cells and its mechanism].

    Science.gov (United States)

    Lin, Peixin; Shen, Hong; Gao, Lulu; Huang, Yeen; Zhang, Yaozhong; Chen, Qing

    2015-06-01

    To investigate the mitochondrial injury in enterovirus 71 (EV71)-infected Vero cells and explore the possible mechanism. A clinical isolate of EV71 was inoculated to Vero cells and the EV71 antigen was detected by immunofluorescence assay. The morphological changes of Vero cells were observed using optical microscopy and transmission electron microscopy. The diameter and area density of the viral particles and the ratio and area density of vacuolated mitochondria in the cells were measured on the ultrastructural images. EV71-infected Vero cells underwent obvious changes and to a spherical morphology followed by cell death EV71 particles were detected in the cytoplasm by immunofluorescence. Ultrastructurally, the infected cells contained a large number of viral particles in the cytoplasm, with a clustered distribution and lattice-like arrangement. The diameter of the particles were 16.3 nm and the mean area density was 38.3%. Most of the mitochondria presented with swelling, vacuoles and degeneration. The ratio of the vacuolated mitochondria was 90.9% with a mean area density of 89.2%. Viral particles were also found in some mitochondria. EV71 proliferates in the cytoplasm and invades the mitochondria of infected Vero cells leading to mitochondrial injury and cell death, suggesting that mitochondria are the targets for EV71 infection.

  15. [Efficiency of in vitro culture of Toxoplasma gondii in THP1 and Vero cell lines].

    Science.gov (United States)

    Cuéllar, Jorge Andrés; Hernández, Alejandro; Villegas, Enrique; Gómez, Jorge Enrique

    2012-09-01

    Cell culture is an important method for isolating Toxoplasma gondii to make clinical diagnosis or for biotechnological purposes. The percentage of invasion and production levels of T. gondii was determined for THP1 and Vero cell lines. The growth conditions for T. gondii in Vero and THP1 cell lines were determined by counting in hemocytometer chamber. The percentage of invasion of T. gondii in THP1 and Vero cells was determined by flow cytometry in different cell/tachyzoite ratios: 1/5, 1/20, 1/50. The growth performance index of the T. gondii RH strain and the CIBM1 isolate was calculated for THP1 cells. Vero cells multiplied faster than the THP1 cells, showing an exponential and a sigmoidal growth curve respectively, within a period of 7 days. The CIBM1 isolate infected the THP1 cells in three different parasite concentrations: 1/5, 1/20 and 1/50, with invasion percentages in THP1 cells of 57.1%, 15.5% and 12.2% and for the Vero cells 25.3%, 17.8% and 8.8% respectively. The RH strain of T. gondii had the lowest invasion percentage with 32.6%, 14.8% and 8.1% in THP1 cells and 22.3%, 14.1% and 3.4% in Vero cells. The CIBM1 isolate had a higher yield than the RH strain of T. gondii in THP1 cells. THP1 cells were indicated to be a good model for the study of invasion and for the assays of new pharmacological candidates against T. gondii.

  16. ADAPTATION OF AN INDIGENOUS VERY VIRULENT INFECTIOUS BURSAL DISEASE VIRUS ON VERO CELL LINE

    Directory of Open Access Journals (Sweden)

    I. Hussain and M. H. Rasool

    2005-07-01

    Full Text Available In the present study, Vero cell line was tested for its ability to support the replication of indigenous very virulent infectious bursal disease virus (vvIBDV. The frozen cells were resuscitated to prepare monolayer, which was further sub-cultured to prepare semi-confluent monolayers using M199 growth medium supplemented with 5% foetal calf serum. The semi confluent monolayers were then infected with 0.25 ml of indigenous vvIBDV. The passage 1 virus was harvested and used for the next passage. In this way virus was given three serial passages on Vero cell line, where characteristic cytopathic effects (CPEs were observed. During the first passage, no CPEs were found. The Vero cell monolayers remained normal in first passage upto 144 hours post-infection. During second passage, rounding of cells was observed after 72 hours of infection. However, clear and consistent CPEs were not observed in 2nd passage. Typical aggregation, rounding and granulation of Vero cells was noticed in passage 3 (P3 from 72 hours upto 144 hours post-infection. The positive results of agar gel precipitation test (AGPT confirmed that the adapted (P3 virus was IBDV. The infectivity titer of adapted vvIBDV was found to be log10 7.60 TCID50/ ml at 72 hours post-infection. The indigenous vvIBDV was well adapted to Vero cell line after three successive passages.

  17. A Vero-cell-adapted vaccine donor strain of influenza A virus generated by serial passages.

    Science.gov (United States)

    Hu, Weibin; Zhang, Hong; Han, Qinglin; Li, Li; Chen, Yixin; Xia, Ningshao; Chen, Ze; Shu, Yuelong; Xu, Ke; Sun, Bing

    2015-01-03

    A cell culture-based vaccine production system is preferred for the large-scale production of influenza vaccines and has advantages for generating vaccines against highly pathogenic influenza A viruses. Vero cells have been widely used in human vaccine manufacturing, and the safety of these cells has been well demonstrated. However, the most commonly used influenza-vaccine donor virus, A/Puerto Rico/8/1934 (PR8) virus, does not grow efficiently in Vero cells. Therefore, we adapted the PR8 virus to Vero cells by continuous passaging, and a high-growth strain was obtained after 20 passages. Sequence analysis and virological assays of the adapted strain revealed that mutations in four viral internal genes (NP, PB1, PA and NS1) were sufficient for adaptation. The recombinant virus harboring these mutations (PR8-4mut) displayed accelerated viral transport into the nucleus and increased RNP activity. Importantly, the PR8-4mut could serve as a backbone donor virus to support the growth of the H7N1, H9N2 and H5N1 avian viruses and the H1N1 and H3N2 human viruses in Vero cells without changing its pathogenicity in either chicken embryos or mice. Thus, our work describes the generation of a Vero-adapted, high-yield PR8-4mut virus that may serve as a promising candidate for an influenza-vaccine donor virus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Cell culture-derived influenza vaccines from Vero cells: a new horizon for vaccine production.

    Science.gov (United States)

    Montomoli, Emanuele; Khadang, Baharak; Piccirella, Simona; Trombetta, Claudia; Mennitto, Elisa; Manini, Ilaria; Stanzani, Valerio; Lapini, Giulia

    2012-05-01

    In the 20th century, three influenza pandemics killed approximately 100 million people. The traditional method of influenza vaccine manufacturing is based on using chicken eggs. However, the necessity of the availability of millions of fertile eggs in the event of a pandemic has led research to focus on the development of cell culture-derived vaccines, which offer shorter lead-in times and greater flexibility of production. So far, the cell substrates being evaluated and in use include Vero, Madin-Darby canine kidney, PER.C6 and insect cells. However, Vero cells are the most widely accepted among others. This review introduces briefly the concepts of advanced cell culture-derived influenza vaccine production and highlights the advantages of these vaccines in terms of efficiency, speed and immunogenicity based on the clinical data obtained from different studies.

  19. [Effect of autoinducer 2 on Riemerell antatipestifer adherence and invasion to Vero cells].

    Science.gov (United States)

    Liu, Lei; Han, Xiangan; Liu, Rui; Bai, Hao; Dong, Hongliang; Ding, Chan; Liu, Haiwen; Yu, Shengqing

    2013-03-04

    Autoinducer 2 (AI-2), used to communicate among bacterial species, regulates numerous physiological functions of bacteria. In this study, we studied the effect of AI-2 on adherence and invasion of Riemerella antatipestifer (RA) strain CH3 to Vero cells and transcriptional levels of virulence-related and metabolism-related genes were investigated. To verify whether the adherence and invasion of CH3 was affected by AI-2, we added different concentrations of AI-2 to the cocultures of Vero cells and CH3 and then calculated adherence percentages and invasion percentages of tested groups. We further added AI-2 (184.0 micromol/L) to the tryptone soya broth culture of CH3 and then detected the effect of transcriptional levels of related genes of CH3 using real-time PCR. The adherence of CH3 to Vero cells was decreased most to 62% with 18.4 micromol/L AI-2 and the invasion of CH3 to Vero cells was increased most to 194% with 184.0 micromol/L AI-2. The result of real-time PCR shows that AI-2 increased transcriptional levels of some virulence-related genes and decreased transcriptional levels of some metabolism-related genes. These results suggest that AI-2 affected adherence and invasion of CH3 to Vero cells. Moreover, AI-2 could regulate some genes of CH3 to modulate particular physiological behaviors.

  20. Construction high-yield candidate influenza vaccine viruses in Vero cells by reassortment.

    Science.gov (United States)

    Yu, Wei; Yang, Fan; Yang, Jinghui; Ma, Lei; Cun, Yina; Song, Shaohui; Liao, Guoyang

    2016-11-01

    Usage of influenza vaccine is the best choice measure for preventing and conclusion of influenza virus infection. Although it has been used of chicken embryo to produce influenza vaccine, following with WHO recommended vaccine strain, there were uncontrollable factors and its deficiencies, specially, during an influenza pandemic in the world. The Vero cells are used for vaccine production of a few strains including influenza virus, because of its homology with human, recommended by WHO. However, as known most of the influenza viruses strains could not culture by Vero cells. It was used two high-yield influenza viruses adapted in Vero cells as donor viruses, such as A/Yunnan/1/2005Va (H3N2) and B/Yunnan/2/2005Va (B), to construct high-yield wild influenza virus in Vero cells under antibody selection pressure. After reassortment and passages, it obtained the new Vaccine strains with A/Tianjin/15/2009Va (H1N1), A/Fujian/196/2009Va (H3N2) and B/Chongqing/1384/2010Va (B), which was not only completely keeping their original antigenic (HA and NA), but also grown well in Vero cells with high-yield. All results of gene analysis and HA, HI shown that this reassortment method could be used to find new direction to product the influenza vaccine. J. Med. Virol. 88:1914-1921, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Citotoxicity evaluation of three dental adhesives on vero cells in vitro

    OpenAIRE

    Catunda, Raisa-Queiroz; Vieira, Jeymesson-Raphael-Cardoso; de Oliveira, Erwelly-Barros; da Silva, Eliete-Cavalcanti; Brasil, Veruska-Lima-Moura; Perez, Danyel-Elias-da Cruz

    2017-01-01

    Background To evaluate, in vitro, the potential cytotoxicity of three different dental adhesives systems (Adper Single Bond 2 -SB, Silorane System Adhesive Bond -SSAB and Single Bond Universal -SBU) on cultivated Vero cells after different contact times. Material and Methods The cells were cultured in a concentration of 2 x 105 cells/mL for 24h and grown to sub-confluent monolayers. VERO cells were exposed to 25?l of conditioned extracts obtained from 24h, 48h and 72h immersion of adhesive sa...

  2. High genetic stability of dengue virus propagated in MRC-5 cells as compared to the virus propagated in vero cells.

    Directory of Open Access Journals (Sweden)

    Chia-Chyi Liu

    Full Text Available This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4, including dengue virus type 4 (DEN-4 recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-free and serum-containing cultures were found to yield comparable titers of these viruses. The cloned DNA-derived DEN-4 started genetically more homogeneous was used to investigate the genetic stability of the virus propagated in Vero cells and MRC-5 cells. Sequence analysis revealed that the DEN-4 propagated in MRC-5 cells maintained a high genetic stability, compared to the virus propagated in Vero cells. Amino acid substitutions of Gly(104Cys and Phe(108Ile were detected at 70%, 60%, respectively, in the envelope (E protein of DEN-4 propagated in Vero cells, whereas a single mutation of Glu(345Lys was detected at 50% in E of the virus propagated in MRC-5 cells. Sequencing of multiple clones of three separate DNA fragments spanning 40% of the genome also indicated that DEN-4 propagated in Vero cells contained a higher number of mutations than the virus growing in MRC-5 cells. Although Vero cells yielded a peak virus titer approximately 1 to 17 folds higher than MRC-5 cells, cloned DEN-4 from MRC-5 cells maintained a greater stability than the virus from Vero cells. Serum-free microcarrier cultures of MRC-5 cells offer a potentially valuable system for the large-scale production of live-attenuated DEN vaccines.

  3. Chemical Induction of Endogenous Retrovirus Particles from the Vero Cell Line of African Green Monkeys▿

    Science.gov (United States)

    Ma, Hailun; Ma, Yunkun; Ma, Wenbin; Williams, Dhanya K.; Galvin, Teresa A.; Khan, Arifa S.

    2011-01-01

    Endogenous retroviral sequences are present in high copy numbers in the genomes of all species and may be expressed as RNAs; however, the majority are defective for virus production. Although virus has been isolated from various Old World monkey and New World monkey species, there has been no report of endogenous retroviruses produced from African green monkey (AGM) tissues or cell lines. We have recently developed a stepwise approach for evaluating the presence of latent viruses by chemical induction (Khan et al., Biologicals 37:196–201, 2009). Based upon this strategy, optimum conditions were determined for investigating the presence of inducible, endogenous retroviruses in the AGM-derived Vero cell line. Low-level reverse transcriptase activity was produced with 5-azacytidine (AzaC) and with 5′-iodo-2′-deoxyuridine (IUdR); none was detected with sodium butyrate. Nucleotide sequence analysis of PCR-amplified fragments from the gag, pol, and env regions of RNAs, prepared from ultracentrifuged pellets of filtered supernatants, indicated that endogenous retrovirus particles related to simian endogenous type D betaretrovirus (SERV) sequences and baboon endogenous virus type C gammaretrovirus (BaEV) sequences were induced by AzaC, whereas SERV sequences were also induced by IUdR. Additionally, sequence heterogeneity was seen in the RNAs of SERV- and BaEV-related particles. Infectivity analysis of drug-treated AGM Vero cells showed no virus replication in cell lines known to be susceptible to type D simian retroviruses (SRVs) and to BaEV. The results indicated that multiple, inducible endogenous retrovirus loci are present in the AGM genome that can encode noninfectious, viruslike particles. PMID:21543506

  4. Morphological alterations of Vero cell exposed to coplanar PCB 126 and noncoplanar PCB 153.

    Science.gov (United States)

    Shen, Kaili; Shen, Chaofeng; Chen, Lei; Chen, Xincai; Chen, Yingxu

    2012-01-01

    Polychlorinated biphenyls (PCBs) are widespread, persistent environmental contaminants that display a complex spectrum of toxicological properties. Exposure to PCBs has been associated with morphological anomalies in cell cultures. However, most mechanistic studies of PCBs' toxic activity have been focused on coplanar congeners. It is of importance to determine whether PCB treatment would influence cell configuration and whether these changes would depend on the structural characteristics of PCBs. In this study, we investigated cell morphological alteration in Vero cell cultures after exposure to coplanar PCB 126 and noncoplanar PCB 153. The survival of Vero cells was measured through the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. Cytotoxicity results suggested that PCB congeners had a toxic, antiproliferative effect on Vero cells. Morphological studies described structural modifications and provided evidence that apoptosis might be the main cell death pathway in PCB 153-treated cells. The comparison between PCB 126 and PCB 153 indicated that the cell death mechanisms involved in coplanar or noncoplanar PCB congener exposure were different in Vero cells. Copyright © 2010 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2013-11-22

    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.

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

    Directory of Open Access Journals (Sweden)

    Rachel J. Derscheid

    2013-11-01

    Full Text Available 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.

  7. Citotoxicity evaluation of three dental adhesives on vero cells in vitro.

    Science.gov (United States)

    Catunda, Raisa-Queiroz; Vieira, Jeymesson-Raphael-Cardoso; de Oliveira, Erwelly-Barros; da Silva, Eliete-Cavalcanti; Brasil, Veruska-Lima-Moura; Perez, Danyel-Elias-da Cruz

    2017-01-01

    To evaluate, in vitro, the potential cytotoxicity of three different dental adhesives systems (Adper Single Bond 2 -SB, Silorane System Adhesive Bond -SSAB and Single Bond Universal -SBU) on cultivated Vero cells after different contact times. The cells were cultured in a concentration of 2 x 105 cells/mL for 24h and grown to sub-confluent monolayers. VERO cells were exposed to 25µl of conditioned extracts obtained from 24h, 48h and 72h immersion of adhesive samples in culture medium (DMEM), immediately after polymerization. Fresh DMEM was used as negative control. Cell metabolism was evaluated by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyl-tetrazolium bromide). The data were analyzed statistically by ANOVA, considering a significance of 5%. The values of cell viability ranged from 94.2% at 72h (SBU) to 109.6% at 48h (SB). The mean percentage of viability after exposure to the extracts of SB, SSAB and SBU were 103.2%, 100.63% and 97.43%, respectively. There was no statistically significant difference (p= 0.342) between the experimental and negative control groups. At all exposure times, all adhesives tested in this study presented no cytotoxicity to Vero cells in vitro. Key words:Biocompatibility, cytotoxicity, dental adhesives, Vero cells.

  8. Cytotoxic effects of etephon and maleic hydrazide in Vero, Hep2, HepG2 cells.

    Science.gov (United States)

    Yurdakok, Begum; Baydan, Emine; Okur, Hamza; Gurcan, Ismayil Safa

    2014-10-01

    The toxicity of etephon and maleic hydrazide, used as plant growth regulators in agriculture, were reported as low in mammals in previous studies. However, in vitro cytotoxicity studies in mammalian cells are currently missing to understand their toxicity at molecular level. In the current study, the cytotoxicity of these compounds, were studied in Vero (African green monkey kidney epithelium), HepG2 (human hepatocellular carcinoma), Hep2 (human epidermoid cancer) cells by MTT ((3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromure) and LDH (lactate dehydrogenase) assays. Maleic hydrazide had lower IC50 values for all cell lines compared to ethephon. Least cytotoxic effect treated by ethephon were observed in Vero, followed by HepG2 and Hep2. Similarly maleic hydrazide also showed least cytotoxicity on Vero cells, followed by Hep2 and HepG2 cells (p Vero cells, followed by HepG2 and Hep2 cells (p 0.868 (p cells to be supplemented by further studies.

  9. Citotoxicity evaluation of three dental adhesives on vero cells in vitro

    Science.gov (United States)

    Catunda, Raisa-Queiroz; Vieira, Jeymesson-Raphael-Cardoso; de Oliveira, Erwelly-Barros; da Silva, Eliete-Cavalcanti; Brasil, Veruska-Lima-Moura

    2017-01-01

    Background To evaluate, in vitro, the potential cytotoxicity of three different dental adhesives systems (Adper Single Bond 2 -SB, Silorane System Adhesive Bond -SSAB and Single Bond Universal -SBU) on cultivated Vero cells after different contact times. Material and Methods The cells were cultured in a concentration of 2 x 105 cells/mL for 24h and grown to sub-confluent monolayers. VERO cells were exposed to 25µl of conditioned extracts obtained from 24h, 48h and 72h immersion of adhesive samples in culture medium (DMEM), immediately after polymerization. Fresh DMEM was used as negative control. Cell metabolism was evaluated by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyl-tetrazolium bromide). The data were analyzed statistically by ANOVA, considering a significance of 5%. Results The values of cell viability ranged from 94.2% at 72h (SBU) to 109.6% at 48h (SB). The mean percentage of viability after exposure to the extracts of SB, SSAB and SBU were 103.2%, 100.63% and 97.43%, respectively. There was no statistically significant difference (p= 0.342) between the experimental and negative control groups. Conclusions At all exposure times, all adhesives tested in this study presented no cytotoxicity to Vero cells in vitro. Key words:Biocompatibility, cytotoxicity, dental adhesives, Vero cells. PMID:28149465

  10. In vitro anticancer and cytotoxic activities of some plant extracts on HeLa and Vero cell lines.

    Science.gov (United States)

    Tugba Artun, Fulya; Karagoz, Ali; Ozcan, Gul; Melikoglu, Gulay; Anil, Sezin; Kultur, Sukran; Sutlupinar, Nurhayat

    2016-01-01

    The aim of our study was to evaluate the effect of in vitro anticancer and cytotoxic activity of the methanolic extracts of 14 medicinal plants, 8 of which are endemic species in Anatolia, against the human HeLa cervical cancer cell line and to compare to the normal African green monkey kidney epithelial cell line (Vero) using the MTT colorimetric assay. Values for cytotoxicity measured by MTT assay were expressed as the concentration that causes 50% decrease in cell viability (IC50, μg/mL). The degree of selectivity of the compounds can be expressed by its selectivity index (SI) value. High SI value (>2) of a compound gives the selective toxicity against cancer cells (SI = IC50 normal cell/IC50 cancer cell). Dose-dependent studies revealed IC50 of 293 mg/mL and >1000 mg/mL for Cotinus coggygria Scop., IC50 of 265 μg/mL and >1000 mg/mL for Rosa damascena Miller, IC50 of 2 μg/mL and 454 mg/mL for Colchicum sanguicolle K.M. Perss, IC50 of 427 μg/mL and >1000 μg/mL for Centaurea antiochia Boiss. var. praealta (Boiss & Bal) Wagenitz on the HeLa cells and the Vero cells, respectively. Four plants showed significant SI values which were 227 for Colchicum sanguicolle K.M. Perss (endemic species), >3.8 for Rosa damascena Miller, >3.4 for Cotinus coggygria Scop. and >2.3 for Centaurea antiochia Boiss. var. praealta (Boiss & Bal)Wagenitz (endemic species). According to our study, 4 methanolic extracts of 14 tested plants exhibit greater activity on the HeLa cell line and little activity on the Vero cell line, meaning that these plants can be evaluated for potential promising anticancer activity.

  11. Flavone Enhances Dengue Virus Type-2 (NGC Strain Infectivity and Replication in Vero Cells

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    Keivan Zandi

    2012-02-01

    Full Text Available This study investigates the effects of 2-phenyl-1-benzopyran-4-one (flavone on DENV-2 infectivity in Vero cells. Virus adsorption and attachment and intracellular virus replication were investigated using a foci forming unit assay (FFUA and quantitative RT-PCR, respectively. Addition of flavone (100 μg/mL significantly increased the number of DENV-2 foci by 35.66% ± 1.52 and 49.66% ± 2.51 when added during and after virus adsorption to the Vero cells, respectively. The average foci size after 4 days of infection increased by 33% ± 2.11 and 89% ± 2.13. The DENV-2 specific RNA copy number in the flavone-treated infected cells increased by 6.41- and 23.1-fold when compared to the mock-treated infected cells. Flavone (100 μg/mL did not promote or inhibit Vero cell proliferation. The CC50 value of flavone against Vero cells was 446 µg/mL. These results suggest that flavone might enhance dengue virus replication by acting antagonistically towards flavonoids known to inhibit dengue virus replication.

  12. Heat shock protein 90β in the Vero cell membrane binds Japanese encephalitis virus.

    Science.gov (United States)

    Wang, Yuan; Li, Yan; Ding, Tianbing

    2017-08-01

    The pathogenesis of Japanese encephalitis virus (JEV) is complex and unclearly defined, and in particular, the effects of the JEV receptor (JEVR) on diverse susceptible cells are elusive. In contrast to previous studies investigating JEVR in rodent or mosquito cells, in this study, we used primate Vero cells instead. We noted that few novel proteins co‑immunoprecipitated with JEV, and discovered that one of these was heat shock protein 90β (HSP90β), which was probed by mass spectrometry with the highest score of 60.3 after questing the monkey and human protein databases. The specific HSP90β‑JEV binding was confirmed by western blot analysis under non‑reducing conditions, and this was significantly inhibited by an anti‑human HSP90β monoclonal antibody in a dose‑dependent manner, as shown by immunofluorescence assay and flow cytometry. In addition, the results of confocal laser scanning microscopic examination demonstrated that the HSP90β‑JEV binding occurred on the Vero cell surface. Finally, JEV progeny yields determined by plaque assay were also markedly decreased in siRNA‑treated Vero cells, particularly at 24 and 36 h post‑infection. Thus, our data indicate that HSP90β is a binding receptor for JEV in Vero cells.

  13. Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium.

    Science.gov (United States)

    Mattos, Diogo A; Silva, Marlon V; Gaspar, Luciane P; Castilho, Leda R

    2015-08-20

    In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Identification of Putative Vero Cell Protein(s) that Bind Specifically to ...

    African Journals Online (AJOL)

    Results: The 45 KDa, 43 KDa and 30 KDa plasma membrane proteins were identified as viral envelope targets. Competitive binding assay showed these proteins competing with dengue virus binding. MTT assay indicate that viability of vero cells increases in cultures pretreated with 45 KDa, 43 KDa and 30 KDa proteins ...

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

    NARCIS (Netherlands)

    Thomassen, Y.E.; Rubingh, O.; Wijffels, R.H.; Pol, van der L.A.

    2014-01-01

    Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L-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

  16. The Effect of Cumin Seed Extracts against Herpes Simplex Virus Type 1 in Vero Cell Culture

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    Mohammad Motamedifar

    2010-12-01

    Full Text Available Background: Cumin (Cuminum cyminum L. [family Apiaceae]seed essential oil is reported to have antiseptic activity.Until now the antiviral properties of cumin seed extracts onviruses such as herpes simplex virus-1 (HSV-1 have not beenstudied. The objective of this study was to investigate the invitro effects of aqueous, methanolic and hydroalcoholic extractsof cumin seed on HSV-1 growth in Vero cell line.Methods: Antiviral activity of various concentrations aqueous,hydroalcoholic and methanolic extracts of cumin seed in Verocells were studied using plaque reduction assays. The 50%cytotoxic concentration (CC50, 50% inhibitory concentration(IC50, and therapeutic index of the effective extracts were calculated.Results: Methanolic extract of cumin seed showed a significantantiviral activity on HSV-1 in Vero cell line. Its CC50 forVero cells, IC50 and the therapeutic index for HSV-1 were0.45, 0.18 mg/mL and 2.5, respectively. Aqueous and hydroalcoholicextracts of cumin seeds showed no inhibitory effecton HSV-1.Conclusion: The methanolic extract of cumin seed producesanti-HSV-1 effect. Probable interference of phenolic compoundswith fusion of Vero cell membrane and HSV-1 envelopemight be the mechanism of such inhibitory effect. Furtherstudies are required to ascertain its in vivo antiviral propertiesand potential toxicity.Iran J Med Sci 2010; 35(4: 304-309.

  17. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

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    Laura Lafon-Hughes

    2014-10-01

    Full Text Available Poly-ADP-ribose (PAR is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs and degraded by poly-ADP-ribose-glycohydrolase (PARG. Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair. Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt. In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO. PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

  18. Clinical development of a Vero cell culture-derived seasonal influenza vaccine.

    Science.gov (United States)

    Ehrlich, Hartmut J; Berezuk, Gregory; Fritsch, Sandor; Aichinger, Gerald; Singer, Julia; Portsmouth, Daniel; Hart, Mary Kate; El-Amin, Wael; Kistner, Otfried; Barrett, P Noel

    2012-06-19

    Cell culture technologies have the potential to improve the robustness and flexibility of influenza vaccine supply and to substantially shorten manufacturing timelines. We investigated the safety, immunogenicity and efficacy of a Vero cell culture-derived seasonal influenza vaccine and utilized these studies to establish a serological correlate of vaccine protection. Two multicenter, randomized, double-blind phase III trials were undertaken in the US during the 2008-2009 Northern hemisphere influenza season, in young (18-49 years) and older (50-64 years and ≥ 65 years) adult subjects. 7250 young adults were randomized 1:1 to receive either Vero-derived vaccine or placebo. 3210 older adult subjects were randomized 8:1 to receive either Vero-derived vaccine or a licensed egg-derived vaccine. Serum hemagglutination inhibition antibody titers were assessed 21 days post-vaccination. Vaccine efficacy in preventing cell culture-confirmed influenza infection was determined for the young adult population. Local and systemic adverse events were recorded in both studies. The Vero-derived vaccine was safe and well tolerated in both young and older adults. All US and European immunological licensing thresholds were comfortably met in both populations. Vaccine efficacy in young adults was 79% against A/H1N1 viruses antigenically matching the corresponding vaccine strain and 78.5% for all antigenically matched influenza viruses. A hemagglutination inhibition antibody titer of ≥ 1:15 provided a reliable correlate of protection for the Vero-derived influenza vaccine, with no additional benefit at titers >1:30. Bridging of the correlate of protection established in the young adult population to the older adult immunogenicity data demonstrated the likely effectiveness of the Vero-derived vaccine in the older adult population. A Vero cell culture-derived seasonal influenza vaccine is safe, immunogenic and protects against infection with influenza virus. The novel vaccine

  19. Establishment of an analyzing method for a Japanese encephalitis virus neutralization test in Vero cells.

    Science.gov (United States)

    Abe, Motoharu; Kuzuhara, Syoji; Kino, Yoichiro

    2003-05-16

    We established a 50% plaque reduction analyzing method of neutralizing antibody for human serum to Japanese encephalitis virus (JEV) in Vero cells, called the '3 points least-squares regression method' (3LSRM). Our method shows a high correlation with the chick embryo cell method (the current standard method for human serum), using the chart method established by the National Institute of Infectious Diseases of Japan, which is an equation made with retrospective data obtained with the 50% plaque reduction method as a standard measurement method for neutralizing antibody titers to JEV. Our new method is much simpler and more reliable than the current method in that it uses an established cell line, Vero cells, and its results are computed by the 3LSRM.

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

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    Syahida Ahmad

    2012-10-01

    Full Text Available 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 μg/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 μg/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-δ 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-δ and caspase-3 as their mode of actions.

  1. High Genetic Stability of Dengue Virus Propagated in MRC-5 Cells as Compared to the Virus Propagated in Vero Cells

    OpenAIRE

    Chia-Chyi Liu; Shiang-Chi Lee; Michael Butler; Suh-Chin Wu

    2008-01-01

    This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4), including dengue virus type 4 (DEN-4) recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-fr...

  2. Effect of interferon on the development of Trypanosoma cruzi in tissue culture "Vero" cells

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    R. R. Golgher

    1980-01-01

    Full Text Available Results are presented on the effects of interferon on the intracellular stages of T. cruzi in tissue culture "Vero" cells. Interferon was obtained by infecting monolayers of human amniotic cells with inactivated Newcastle disease virus. Interferon has not affected the cell infection by T. cruzi culture infective stages and neither has it prevented the transformation of amastigote into trypomastigote stages.Interferon obtido através da infecção de células amnióticas humanas por vírus inativado da doença de Newcastle foi incapaz de influir sobre a infectividade de formas de cultura do T. cruzi para células "Vero" de cultura de tecido. A transformação amastigota-tripomastigota também não foi afetada pelo interferon.

  3. Intracellular African swine fever virus DNA remains unmethylated in infected Vero cells.

    Science.gov (United States)

    Weber, Stefanie; Hakobyan, Astghik; Zakaryan, Hovakim; Doerfler, Walter

    2018-01-12

    Sequence-specific CpG methylation of eukaryotic promoters is an important epigenetic signal for long-term gene silencing. We have now studied the methylation status of African swine fever virus (ASFV) DNA at various times after infection of Vero cells in culture. ASFV DNA was detectable throughout the infection cycle and was found unmethylated in productively infected Vero cells as documented by bisulfite sequencing of 13 viral DNA segments. ASFV DNA does not become de novo methylated in the course of infection in selected segments spread across the entire genome. Thus DNA methylation does not interfere with ASFV genome transcription. Lack of de novo methylation has previously been observed for free intracellular viral DNA in cells permissively infected with human adenoviruses, with human papillomaviruses and others.

  4. Estimation of the Cultured Cells' Volume and Surface Area: Application of Stereological Methods on Vero Cells Infected by Rubella Virus.

    Science.gov (United States)

    Noorafshan, Ali; Motamedifar, Mohammad; Karbalay-Doust, Saied

    2016-01-01

    Morphological changes of the cells infected with rubella virus cannot be observed easily. Estimation of the size of the cultured cells can be a valuable parameter in this condition. This study was conducted to find answers to the following questions: How much time after infection with rubella virus, the volume and surface area of the Vero cells and their nuclei get started to change?How is it possible to apply stereological methods to estimate the volume and surface area of the cultured cells using the invariator, nucleator, and surfactor techniques? The cultured Vero cells were infected with rubella virus. The cells of the control and experimental groups were harvested at 2, 4, 8, 24, and 48 hours following the incubation period. The cells were processed and embedded in paraffin. Invariator, nucleator, and surfactor were applied to estimate the size of the Vero cells and their nuclei. The cell volume was decreased by 15-24%, 48 hours after the infection in comparison to the non-infected cells. Besides, the cell surface area was decreased by 13%, 48 hours after the infection. However, no changes were detected in the nuclei. The values of the standard deviation and coefficient of variation of the cells, estimated by invariator, were lower compared to those measured by the nucleator or surfactor. In this study, the volume and surface area of the Vero cells were reduced by rubella virus 48 hours after infection. Invariator is a more precise method compared to nucleator or surfactor.

  5. Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium.

    Science.gov (United States)

    da Costa-Silva, Thaís Alves; da Silva Meira, Cristina; Frazzatti-Gallina, Neuza; Pereira-Chioccola, Vera Lucia

    2012-04-01

    Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different

  6. Size-dependent cellular uptake mechanism and cytotoxicity toward calcium oxalate on Vero cells

    Science.gov (United States)

    Sun, Xin-Yuan; Gan, Qiong-Zhi; Ouyang, Jian-Ming

    2017-02-01

    Urinary crystals with various sizes are present in healthy individuals and patients with kidney stone; however, the cellular uptake mechanism of calcium oxalate of various sizes has not been elucidated. This study aims to compare the internalization of nano-/micron-sized (50 nm, 100 nm, and 1 μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The internalization and adhesion of COM and COD crystals to Vero cells were enhanced with decreasing crystal size. Cell death rate was positively related to the amount of adhered and internalized crystals and exhibited higher correlation with internalization than that with adhesion. Vero cells mainly internalized nano-sized COM and COD crystals through clathrin-mediated pathways as well as micron-sized crystals through macropinocytosis. The internalized COM and COD crystals were distributed in the lysosomes and destroyed lysosomal integrity to some extent. The results of this study indicated that the size of crystal affected cellular uptake mechanism, and may provide an enlightenment for finding potential inhibitors of crystal uptake, thereby decreasing cell injury and the occurrence of kidney stones.

  7. Probiotic inhibits the cytopathic effect induced by Escherichia coli O157:H7 in Vero cell line model.

    Science.gov (United States)

    Tahamtan, Y; Kargar, M; Namdar, N; Rahimian, A; Hayati, M; Namavari, M M

    2011-05-01

    The effectiveness of four strains of Bifidobacteria against enterohemorrhagic Escherichia coli O157:H7 infection was studied using a Vero cell model. E. coli O157 was inoculated on the Vero cell line before and after treatment with probiotic. The cytopathic effect (CPE) was evaluated during 24 h of incubation. The results indicated that Shiga toxin activity was inhibited by the probiotic. To prevent a Stx2 CPE, the probiotic needs one log more than the Stx1. The Vero cell assay, in particular, is a good model to evaluate the effect of Bifidobacteria inhibiting bacterial attachment because of soluble substances and the competitive aspect and could be used in a variety of foods like milk and yoghurt to protect pathogen bacteria. Probiotics could control pathogenic bacteria and Vero cell introduce as a model for evaluation of probiotics against pathogen bacteria. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  8. Experimental infection of BHK21 and Vero cell lines with different Mycoplasma spp.

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    Cristiane Netto

    2014-12-01

    Full Text Available Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts generally require two essential inputs, bovine serum and cells. The study herein aims to evaluate the mycoplasma concentrations that affect the growing of BHK21 and Vero cells. The species used were: Mycoplasma orale, M. salivarium, M. arginini and M. hyorhinis, cultivated in a SP4 media. Two contamination tests were performed with BHK21 and Vero cells and one of them applied different concentrations of mycoplasma. In the first one, mycoplasma was applied at the day zero and, in the second one, the contamination was performed after the monolayer establishment. The both cellular cultures presented cytopathic effects with mycoplasma contamination, but the Vero cells suffered more damages than the BHK21 ones. It was also observed that the severity of the cytopathic effect depended on the mycoplasma specie, on the concentration and on the time of contact with the cellular culture, which evidences the importance of controlling the presence of mycoplasma in biotechnological industries.

  9. Amino Acid substitutions in matrix, fusion and hemagglutinin proteins of wild measles virus for adaptation to vero cells.

    Science.gov (United States)

    Xin, Ji Yi; Ihara, Toshiaki; Komase, Katsuhiro; Nakayama, Tetsuo

    2011-01-01

    Wild-type measles virus (MV) is isolated in B95a but not in Vero cells. Through an adaptation process of wild-type MV to Vero cells, several amino acid substitutions were reported. Six strains were adapted to Vero cells and membrane (M), fusion (F) and hemagglutinin (H) genes were sequenced. Cell fusion was assessed and recombinant MVs were constructed, having wild-type H or M gene with or without mutations. No F gene substitution was noted. Amino-acid substitutions at positions 481 from Asn to Tyr (N481Y) and 546 from Ser to Gly (S546G) were observed in the H protein. Glu at position 89 of the M protein was substituted for Gly (E89G) and two mutations were noted at positions 62 (S62R) and 83 (S83P) in M protein. Recombinant viruses with mutation(s) detected in Vero-adapted strains induced a cytopathic effect and grew well in Vero cells, but those with the wild type did not. Recombinant viruses with mutation(s) demonstrated lower viral growth in B95a cells. Substitutions of E89G, S62R and S83P of the M protein were newly observed through adaptation to Vero cells, besides the mutations described in previous reports, with varying adaptation for each strain. Copyright © 2011 S. Karger AG, Basel.

  10. Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence.

    Science.gov (United States)

    Šantak, Maja; Markušić, Maja; Balija, Maja Lang; Kopač, Sandra Keć; Jug, Renata; Örvell, Claes; Tomac, Jelena; Forčić, Dubravko

    2015-03-01

    Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5' untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence. These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  11. The 3a Protein of SARS-coronavirus Induces Apoptosis in Vero E6 Cells.

    Science.gov (United States)

    Y Waye, Mary; W Law, Patrick; Wong, Chi-Hang; C Au, Thomas; Chuck, Chi-Pang; Kong, Siu-Kai; S Chan, Paul; To, Ka-Fai; I Lo, Anthony; W Chan, Judy; Suen, Yick-Keung; Edwin Chan, H Y; Fung, Kwok-Pui; Y Sung, Joseph; Lo, Y M; W Tsui, Stephen

    2005-01-01

    An outbreak of severe acute respiratory syndrome (SARS) occurred in China and the first case emerged in mid November 2002. The etiologic agent of this disease was found to be a previously unknown coronavirus, SARS-CoV. The detailed pathology of SARS-CoV infection and the host response to the viral infection are still not known. The 3a gene encodes a non-structural viral protein which is predicted to be a transmembrane protein. In this study, we showed that the 3a protein was localized to the endoplasmic reticulum (ER) in 3a-transfected monkey kidney Vero E6 cells. In vitro experiments of chromatin condensation and DNA fragmentation suggest that the 3a protein may trigger apoptosis. Our data show that over-expression of a single SARS-CoV protein can induce apoptosis in vitro. Thus GFP-3a fusion protein could also be used as a biosensor for monitoring the cytopathic features of SARS infection, e.g. lymphopenia, in animal model systems, similar to nucleocapsid and 7a proteins.

  12. Reassortment of high-yield influenza viruses in vero cells and safety assessment as candidate vaccine strains.

    Science.gov (United States)

    Zhou, Jian; Yang, Fan; Yang, Jinghui; Ma, Lei; Cun, Yina; Song, Shaohui; Liao, Guoyang

    2017-01-02

    Vaccination is the practiced and accessible measure for preventing influenza infection. Because chicken embryos used for vaccine production have various insufficiencies, more efficient methods are needed. African green monkey kidney (Vero) cells are recommended by the World Health Organization (WHO) as a safe substitute for influenza vaccine production for humans. However, the influenza virus usually had low-yield in Vero cells, which limits the usage of Vero cellular vaccines. This study used 2 high-yield influenza viruses in Vero cells: A/Yunnan/1/2005Va (H3N2) and B/Yunnan/2/2005Va (B) as donor viruses. It used 3 wild strain viruses to reassort new adaptation viruses, including: A/Tianjin/15/2009(H1N1), A/Fujian/196/2009(H3N2), and B/Chongqing/1384/2010(B). These three new viruses could maintain the characteristic of high-yield in Vero cells. Furthermore, they could keep the immunogenic characteristics of the original wild influenza viruses. Importantly, these viruses were shown as safe in chicken embryo and guinea pigs assessment systems. These results provide an alternative method to produce influenza vaccine based on Vero cells.

  13. Respiratory syncytial virus grown in Vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans.

    Science.gov (United States)

    Kwilas, Steven; Liesman, Rachael M; Zhang, Liqun; Walsh, Edward; Pickles, Raymond J; Peeples, Mark E

    2009-10-01

    Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.

  14. [A study of the antiherpetic activity of the chaga mushroom (Inonotus obliquus) extracts in the Vero cells infected with the herpes simplex virus].

    Science.gov (United States)

    Polkovnikova, M V; Nosik, N N; Garaev, T M; Kondrashina, N G; Finogenova, M P; Shibnev, V A

    2014-01-01

    The chaga mushroom (Inonotus obliquus) contains a wide range of excellent bioactive compounds. However, limited information exists on the antiviral activity of the compounds extracted from chaga. A number of subfractions of chaga were obtained using different solvents and different procedures. The subfractions of chaga extracted with water, alcohol, alkali were tested for their toxicity for the Vero cell culture and antiviral effect in the Vero cells infected with the Herpes simplex virus (HSV), Type 1. It was shown that most of the subfractions were not toxic for the Vero cells and had protective effect on the Vero cells infected with HSV. The subfraction IV in the concentration 5 microg/ml protected the Vero cells from cytodestructive action of HSV and no viral DNA was detected in infected cells treated with chaga extracts. Best protective effect was observed when compound was added before or within one hour after the Vero cells were infected with HSV.

  15. Suscetibilidade da linhagem de células Vero a cepas vacinais do vírus do sarampo Susceptibility of Vero cell line to vaccine strains of the measles virus

    Directory of Open Access Journals (Sweden)

    Célia Sayoko Takata

    1994-06-01

    Full Text Available A suscetibilidade da linhagem de células Vero ao vírus do sarampo é bem conhecida e sua utilização no controle da potência da vacina contra o sarampo é amplamente difundida. Com o objetivo de comparar a suscetibilidade de células Vero empregadas em titulações, amostras provenientes de dois laboratórios controladores (Vero IB e Vero INCQS, foram testadas frente a três cepas vacinais: Moraten, Schwarz e Biken CAM-70. Foram titulados 72 lotes de vacinas contra o sarampo, sendo 25 produzidos com a cepa Moraten, 24 com a cepa Schwarz e 23 com a cepa Biken CAM-70. A análise estatística dos resultados obtidos nas titulações, feita através dos testes Limites para uma Média e "t" de Student, mostrou que para as cepas Moraten e Biken CAM-70, as diferenças de títulos não foram estatisticamente significantes, o mesmo não ocorrendo com a cepa Schwarz, para a qual as células Vero IB se mostraram mais sensíveis.Vero cells used by distinct measles vaccine control laboratories had their susceptibility to Moraten, Schwarz and Biken CAM-70 vaccine strains assayed. Of a total of 72 lots of measles vaccine whose potency was titrated by microtechnique in two Vero cell samples (Vero IB and Vero INCQS, 25 had been produced with Moraten strain, 24 with Schwarz and 23 with Biken CAM-70. The statistical analysis of the results demonstrated that both Vero cells assayed presented comparable susceptibility to Moraten and Biken CAM-70 strains. As to the Schwarz strain, Vero IB cells were more susceptible than the other cell sample tested, thus confirming the existence of different sensitivities of Vero cells to some measles vaccine strains, or even to viruses derived from the same strain but with different passage histories. An altered cell susceptibility to virus replication may significantly alter the results in potency testing. Such alteration may be caused not only by the adoption of distinct protocols for the maintenance of cell cultures by

  16. Cytotoxicity of methanol extracts of Elaeis guineensis on MCF-7 and Vero cell lines.

    Science.gov (United States)

    Vijayarathna, Soundararajan; Sasidharan, Sreenivasan

    2012-10-01

    To investigate the cytotoxic effect of Elaeis guineensis methanol extract on MCF-7 and Vero cell. In vitro cytotoxicity was evaluated in by MTT assay. Cell morphological changes were observed by using light microscope. The MTT assay indicated that methanol extract of the plant exhibited significant cytotoxic effects on MCF-7. Morphological alteration of the cell lines after exposure with Elaeis guineensis extract were observed under phase contrast microscope in the dose dependent manner. The results suggest the probable use of the Elaeis guineensis methanol extract in preparing recipes for cancer-related ailments. Further studies on isolation of metabolites and their in vivo cytotoxicity are under investigation.

  17. Adaptation and growth kinetics study of an Indian isolate of virulent duck enteritis virus in Vero cells.

    Science.gov (United States)

    Aravind, S; Kamble, Nitin M; Gaikwad, Satish S; Shukla, Sanjeev Kumar; Dey, Sohini; Mohan, C Madhan

    2015-01-01

    Duck virus enteritis, also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with chicken embryo adapted live virus that is known to be poorly immunogenic and elicits only partial protection. Further, the embryo propagated vaccine virus pose a threat of harboring other infectious agents. Seeing these limitations, the present study reports for the first time regarding propagation and adaptation of a virulent Indian isolate of duck enteritis virus in Vero cell line. In this study isolation of an outbreak virus from Kerala state was done in chicken embryo fibroblast cell culture (CEF). Then adapted the DEV isolate in the Vero cell line. The characteristic cytopathic effects (CPE) of clumping and fusion of Vero cells were observed starting from the 7th passage onwards. The presence of the virus and its multiplication in Vero cells was confirmed by detection of viral specific DNA and antigen by using polymerase chain reaction (PCR) and indirect immuno fluorescent assay (IIFA), respectively. PCR detection of DEV using self designed primers for US4 (gD) and UL30 (DNA Polymerase) gene has been reported for the in the present study. The kinetics of DEV in Vero cells revealed a maximum infectivity titer of 10(5.6) TCID 50/ml after 48hr of viral infection. Compared to chicken embryo adapted DVE vaccine virus, the Vero cell culture system is free from other infectious agents. So it will be a good candidate for cultivation and propagation of duck enteritis virus vaccine strain. Further research studies are suggested to explore the feasibility of utilizing this Vero cell culture adapted DEV isolate for developing an attenuated vaccine virus against duck virus enteritis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. HSP70 induced by Hantavirus infection interacts with viral nucleocapsid protein and its overexpression suppresses virus infection in Vero E6 cells.

    Science.gov (United States)

    Yu, Lu; Ye, Ling; Zhao, Rong; Liu, Yan Fang; Yang, Shou Jing

    2009-07-15

    Hantavirus (HTV) infection is known to induce innate cellular response, a more specified cellular response in the host cells. However, whether it stimulates synthesis of stress proteins, particularly associations of viral proteins, is entirely unknown. The primary focus of this research is using Vero E6 cells infected with Hantaan 76-118 (HTNV) as an in vitro infection model to examine the individual contribution of HTV infection to heat shock response. This study shows that HTNV infection rapidly induced HSP70 expression in Vero E6 cells, which underwent a nucleo-cytoplasmic shuttle that lasted for more than 3 d. The increased HSP70 was preceded by induction of HSP70 mRNA. The physical association of HSP70 with viral nucleocapsid protein (NP) in infected cells was demonstrated by co-localization and immunoprecipation. Vero E6 cells that constitutively overexpress HSP70 after stable transfection with HSP70 gene, when infected with HTNV, showed selectively reduced NP synthesis. These findings suggest HSP70 is actively involved in the control of the expression level of viral structural proteins and possibly involved in virus assembly by binding of NP to HSP70. Overexpression of HSP70 does not favor viral propagation.

  19. Vero cell growth and differentiation on poly(L-lactic acid) membranes of different pore diameters.

    Science.gov (United States)

    Santos, A R; Barbanti, S H; Duek, E A; Dolder, H; Wada, R S; Wada, M L

    2001-01-01

    In the last few years, the demand has increased for research on polymeric materials, which can be used as substitutes for injured tissues and organs or to improve their regeneration. In this work, we studied poly(L-lactic acid) (PLLA) membranes, a resorbable biomaterial, which were either dense or had different pore diameters (less than 45 microm, between 180 and 250 microm, and between 250 and 350 microm), in relation to stimulation of cell adhesion, growth, and differentiation in vitro. We used Vero cells, a fibroblastic cell line, as the biological model of investigation. We found that cells attached slowly to all PLLA membranes studied. On the other hand, once the adhesion occurs, the cells are able to grow and differentiate on the different polymers. The cells grew to form a confluent monolayer and were capable of producing collagen Type IV and fibronectin on different PLLA membranes. This behavior indicates that cells try to create a better environment to stimulate their growth. This also indicates that Vero cells alter their differentiation pattern once they are producing extracellular matrix molecules related to epithelial differentiation.

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

    Science.gov (United States)

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

    2014-05-19

    Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L(-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) cells mL(-1) during batch cultivation to 1.8, 2.7 and 5.0×10(6) cells mL(-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. Copyright © 2014. Published by Elsevier Ltd.

  1. Comparative growth of spotted fever group Rickettsia spp. strains in Vero cells

    Science.gov (United States)

    Silva, Arannadia Barbosa; Duarte, Myrian Morato; Vizzoni, Vinicius Figueiredo; Duré, Ana Íris de Lima; Lopéz, Diego Montenegro; Nogueira, Rita de Maria Seabra; Soares, Carlos Augusto Gomes; Machado-Ferreira, Erik; Gazêta, Gilberto Salles

    2016-01-01

    In Brazil, the spotted fever group (SFG) Rickettsia rickettsii and Rickettsia parkeri related species are the etiological agents of spotted fever rickettsiosis. However, the SFG, Rickettsia rhipicephali, that infects humans, has never been reported. The study of growth dynamics can be useful for understanding the infective and invasive capacity of these pathogens. Here, the growth rates of the Brazilian isolates R. rickettsii str. Taiaçu, R. parkeri str. At#24, and R. rhipicephali HJ#5, were evaluated in Vero cells by quantitative polymerase chain reaction. R. rhipicephali showed different kinetic growth compared to R. rickettsii and R. parkeri. PMID:27508322

  2. Dual ligand insertion in gB and in gD of oncolytic HSVs for the retargeting to a producer Vero cell line and to cancer cells.

    Science.gov (United States)

    Petrovic, Biljana; Leoni, Valerio; Gatta, Valentina; Zaghini, Anna; Vannini, Andrea; Campadelli-Fiume, Gabriella

    2017-12-20

    Oncolytic viruses gain cancer specificity in several ways. Like the majority of viruses, they grow better in cancer cells which are defective in mounting the host response to viruses. Often they are attenuated by deletion or mutation of virulence genes which counteract the host response, or are naturally occurring oncolytic mutants. In contrast, retargeted viruses are not attenuated or deleted; their cancer-specificity rests on a modified, specific tropism for cancer receptors. For herpes simplex virus (HSV)-based oncolytics, the detargeting-retargeting strategies employed so far were based on genetic modifications of gD. Recently, we showed that even gH or gB can serve as retargeting tools. To enable the growth of retargeted HSVs in cells that can be used for clinical grade virus production, a double retargeting strategy has been developed. Here we show that several sites in the N-terminus of gB are suitable to harbour the 20 aa long GCN4 peptide, which readdresses HSV tropism to Vero cells expressing the artificial GCN4 receptor, and thus enables virus cultivation in the producer non-cancer Vero-GCN4R cell line. The gB modifications can be combined with a minimal detargeting modification in gD, consisting in the deletion of two residues, aa 30 and 38, and replacement of aa 38 with the scFv to HER2, for retargeting to the cancer receptor. The panel of recombinants was analysed comparatively in terms of virus growth, cell-to-cell spread, cytotoxicity, in vivo anti-tumor efficacy to define the best double retargeting strategy. IMPORTANCE There is increasing interest in oncolytic viruses, following FDA and EMA approval of HSV Oncovex GM-CSF , and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly checkpoint inhibitors. A strategy to gain cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. Cultivation of fully retargeted

  3. Mathematical model of adherent Vero cell growth and poliovirus production in animal component free medium.

    Science.gov (United States)

    Ursache, Ramona V; Thomassen, Yvonne E; van Eikenhorst, Gerco; Verheijen, Peter J T; Bakker, Wilfried A M

    2015-03-01

    Sabin-IPV (or sIPV, inactivated polio vaccine based on attenuated Sabin strains) is anticipated to replace the oral polio vaccine for the endgame in polio eradication. Optimization of sIPV production will lead to a better economically feasible vaccine. To assist process optimization, we studied Sabin type 1 poliovirus (PV) infection kinetics on Vero cells in controlled bioreactor vessels. The aim of our study was to develop a descriptive mathematical model able to capture the dynamics of adherent Vero cell growth and PV infection kinetics in animal component free medium. The model predicts the cell density, metabolites profiles, and viral yields in time. We found that the multiplicity of infection (MOI) and the time of infection (TOI) within the investigated range did not affect maximal PV yields, but they did affect the process time. The latter may be reduced by selecting a low TOI and a high MOI. Additionally, we present a correlation between viral titers and D-antigen, a measure for immunogenicity, of Sabin type 1 PV. The developed model is adequate for further studies of the cell metabolism and infection kinetics and may be used to identify control strategies to increase viral productivity. Increased viral yields reduce costs of polio vaccines with large implications on public health.

  4. Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease.

    Science.gov (United States)

    Li, Si-Ming; Bai, Fu-Liang; Xu, Wen-Juan; Yang, Yong-Bi; An, Ying; Li, Tian-He; Yu, Yin-Hang; Li, De-Shan; Wang, Wen-Fei

    2014-09-01

    The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  5. Generation of a Vero-Based Packaging Cell Line to Produce SV40 Gene Delivery Vectors for Use in Clinical Gene Therapy Studies

    Directory of Open Access Journals (Sweden)

    Miguel G. Toscano

    2017-09-01

    Full Text Available Replication-defective (RD recombinant simian virus 40 (SV40-based gene delivery vectors hold a great potential for clinical applications because of their presumed non-immunogenicity and capacity to induce immune tolerance to the transgene products in humans. However, the clinical use of SV40 vectors has been hampered by the lack of a packaging cell line that produces replication-competent (RC free SV40 particles in the vector production process. To solve this problem, we have adapted the current SV40 vector genome used for the production of vector particles and generated a novel Vero-based packaging cell line named SuperVero that exclusively expresses the SV40 large T antigen. SuperVero cells produce similar numbers of SV40 vector particles compared to the currently used packaging cell lines, albeit in the absence of contaminating RC SV40 particles. Our unique SV40 vector platform named SVac paves the way to clinically test a whole new generation of SV40-based therapeutics for a broad range of important diseases.

  6. Real-time Imaging of Rabies Virus Entry into Living Vero cells

    Science.gov (United States)

    Xu, Haijiao; Hao, Xian; Wang, Shaowen; Wang, Zhiyong; Cai, Mingjun; Jiang, Junguang; Qin, Qiwei; Zhang, Maolin; Wang, Hongda

    2015-01-01

    Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets. PMID:26148807

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

    OpenAIRE

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

    1996-01-01

    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. K...

  8. Colistin inhibits E. coli O157:H7 Shiga-like toxin release, binds endotoxins and protects Vero cells.

    Science.gov (United States)

    Percivalle, Elena; Monzillo, Vincenzina; Pauletto, Alessandro; Marone, Piero; Imberti, Roberto

    2016-04-01

    The role of antibiotics in the treatment of Shiga-like toxin (Stx)-producing E. coli infection is still controversial. This study investigated the effects of colistin on Vero cell cytotoxicity caused by the enterohemorrhagic EC O157:H7, and the effects of colistin on Stx and endotoxin release by EC O157:H7. Vero cells were incubated with supernatant collected from EC O157:H7 cultured for 18 h without (control) or with various concentrations of colistin. In the absence of colistin, Vero cell viability after 48 h was 29.1±6.5%. Under the same conditions, the overnight presence of colistin reduced cytotoxicity to Vero cells (viability: 97±3.5 to 56.5±14.4% for colistin concentrations ≥MIC). Sub-MIC concentrations of colistin also provided partial protection (viability: 38.8±12.5 to 36.6±14% for 0.125 and 0.06 mcg/ml colistin, respectively). Endotoxins contributed to the cytotoxic effects on Vero cells since lower but still significant protection was observed when colistin was added directly to the supernatant collected from cultures of untreated EC O157:H7. Colistin reduced Stx release in a concentration-dependent manner, also at sub-MIC concentrations. Coincubation of the supernatant from EC O157:H7 cultures with colistin markedly reduced the endotoxin concentration at all doses investigated. In conclusion, colistin protects Vero cells from EC O157:H7 at supra- and sub-MIC concentrations by inhibiting Stx release and binding endotoxins. Colistin might be a valuable treatment for clinically severe forms of EC O157:H7 infection.

  9. Apoptotic activity and anti-Toxoplasma effects of artemether on the tachyzoites and experimental infected Vero and J774 cell lines by Toxoplasma gondii.

    Science.gov (United States)

    Mikaeiloo, Hajar; Ghaffarifar, Fatemeh; Dalimi, Abdolhossein; Sharifi, Zohreh; Hassan, Zuhair Mohammad

    2016-01-01

    Drugs used for toxoplasmosis have limited efficacy and also severe side effects. A new drug with good efficacy and limited side effects is need of the hour. We studied the effects of artemether on Toxoplasma gondii in vitro conditions. Artemether (methyl-ether-qinghaosu) was tested for tachyzoites, J774, and Vero cell lines infected by T. gondii. For evaluating the effect of drugs on Vero cells infected with T. gondii, we designed two separate experiments; in the first experiment, the Vero cells were infected with tachyzoites and then treated with artemether; while in the second one, the tachyzoites were exposed to artemether and then Vero cells were infected with treated tachyzoites. For evaluating the apoptotic effect of artemether on tachyzoites and infected J774 macrophages cell line with T. gondii, we used flow cytometry method. Inhibitory concentration (IC50) was evaluated by intracellular replication of tachyzoites in Vero cells. IC50 for infected Vero cells with tachyzoites was determined as 49.13 μg/ml. In pretreated tachyzoites with artemether before entering into Vero cells, IC50 was calculated as 13.15 μg/ml. In both experiments, artemether showed a higher inhibitory effect than sulfadiazine (positive control). Artemether even at the highest concentrations only showed low cytotoxicity on Vero and J774 cell lines. Apoptosis in tachyzoites rise with an increasing concentration of artemether. Our findings indicate that artemether is effective to control the tachyzoites of T. gondii in vitro and maybe a good alternative drug for toxoplasmosis.

  10. Diphtheria toxin-induced channels in Vero cells selective for monovalent cations

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    Sandvig, K.; Olsnes, S.

    1988-09-05

    Ion fluxes associated with translocation of diphtheria toxin across the surface membrane of Vero cells were studied. When cells with surface-bound toxin were exposed to low pH to induce toxin entry, the cells became permeable to Na+, K+, H+, choline+, and glucosamine+. There was no increased permeability to Cl-, SO4(-2), glucose, or sucrose, whereas the uptake of /sup 45/Ca2+ was slightly increased. The influx of Ca2+, which appears to be different from that of monovalent cations, was reduced by several inhibitors of anion transport and by verapamil, Mn2+, Co2+, and Ca2+, but not by Mg2+. The toxin-induced fluxes of N+, K+, and protons were inhibited by Cd2+. Cd2+ also protected the cells against intoxication by diphtheria toxin, suggesting that the open cation-selective channel is required for toxin translocation. The involvement of the toxin receptor is discussed.

  11. Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line.

    Science.gov (United States)

    Azizi, Ali; Aidoo, Francisca; Gisonni-Lex, Lucy; McNeil, Bryan

    2013-12-01

    The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. The evaluation of gene copy numbers by a qPCR method, is one of the most common approaches used to assess the consistency of transgenes in a constructed cell line. The cell line AV529-19 is a Vero-based cell line specifically engineered to express the HSV-1 UL5 and UL29 open reading frames. AV529-19 is used to support the replication of a defective HSV-2 viral candidate vaccine called HSV529. To assess the genetic stability of the UL5 and UL29 transgenes in AV529-19 cells, a digital PCR-based approach was developed. During characterization of the test method, the specificity, accuracy, and intermediate precision of the assay was investigated based on regulatory guidelines. The developed assay was used to monitor the stability of the transgenes in the manufactured AV529-19 cell lines by comparison of transgene copy numbers in the master cell bank (MCB) with their copy numbers in the extended cell bank (ECB). Results showed that the UL29 and UL5 transgenes are stable in that there are one and three copies of the UL29 and UL5 genes, respectively, per cell in both the AV529-19 MCB and ECB. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2011-01-01

    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. PMID:22022351

  13. Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness.

    Directory of Open Access Journals (Sweden)

    Yu-Fen Tseng

    Full Text Available 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 10(8 TCID(50/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.

  14. The aqueous extract of cinnamon bark ameliorated cisplatin-induced cytotoxicity in vero cells without compromising the anticancer efficiency of cisplatin.

    Science.gov (United States)

    ElKady, Ayman I; Ramadan, Wafaa S

    2016-09-01

    Cis-diammine dichloroplatinum (CDDP) is one of the most important chemotherapeutic agents for cancer treatment. Nonetheless, its notable side effect, nephrotoxicity, undermines its clinical use. The current study was undertaken to evaluate the protective potential of the aqueous extract (AEC) of Cinnamomum cassia (cinnamon) against the cytotoxic effect of CDDP in vitro and to elaborate the molecular mechanism underlying protection. MTT assay was performed to assess viability of the normal kidney Vero cells treated with CDDP and/or AEC. Cells were stained with Coomassie blue, acridine orange and ethidium bromide to highlight morphological features of apoptosis. Caspase-3 activity, DNA fragmentation and reactive oxygen species (ROS) level were monitored to assess biochemical hallmarks of apoptosis. Quantitative RT-PCR and Western blot analyses were performed to elucidate expression of cellular molecules underlying the protective potential of AEC. CDDP-treated Vero cells exhibited hallmarks of apoptosis; these hallmarks were significantly suppressed in the presence of AEC. AEC did not alter activity of CDDP-induced cytotoxicity of breast and liver cancer cells. AEC treatment of Vero cells prevented CDDP-induced increased expression of mitochondrial Bax protein, release of mitochondrial cytochrome c, caspase-3 activation, DNA fragmentation and generation of ROS. AEC up-regulated expression of the cytoprotective gene (heme oxygenase (HO)-1). These findings suggest AEC has protective effects against CDDP-induced toxicity via preventing the activation of various cellular mechanisms mediating apoptotic cell death, without compromising the anticancer efficiency of CDDP. Thus, cinnamon may represent one of the most feasible ways to reduce the risk of CDDP-induced toxicity.

  15. Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells.

    Science.gov (United States)

    Nikolay, Alexander; Castilho, Leda R; Reichl, Udo; Genzel, Yvonne

    2017-03-23

    The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging. Here we present a production platform based on BHK-21 suspension (BHK-21 SUS ) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1-48PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV PE ) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21 SUS cells with ZIKV PE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9×10 3 PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2×10 7 cells/mL, and virus titers of 3.9×10 7 PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards

  16. Production of Newcastle disease virus by Vero cells grown on cytodex 1 microcarriers in a 2-litre stirred tank bioreactor.

    Science.gov (United States)

    Arifin, Mohd Azmir; Mel, Maizirwan; Abdul Karim, Mohamed Ismail; Ideris, Aini

    2010-01-01

    The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was 1.93 x 10(6) cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95 x 10(5) cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3( * *)(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.

  17. Production of Newcastle Disease Virus by Vero Cells Grown on Cytodex 1 Microcarriers in a 2-Litre Stirred Tank Bioreactor

    Directory of Open Access Journals (Sweden)

    Mohd Azmir Arifin

    2010-01-01

    Full Text Available The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM which was 1.93×106 cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95×105 cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3∗∗(3-1 Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v, centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.

  18. Intracellular ATP and total adenylate concentrations are critical predictors of reovirus productivity from Vero cells.

    Science.gov (United States)

    Burgener, A; Coombs, K; Butler, M

    2006-07-05

    The productivity of reovirus type-3 Dearing was studied in cultures of Vero cells in serum-free media. Viral productivity was dependent upon the metabolic state of the cells rather than the phase of growth at which the cells were infected. Cells at different energy states were established by 24-h incubation in nutrient-depleted media. This resulted in variable intracellular nucleotide concentrations but high cellular viability was maintained. Of the nucleotides analyzed at the time of infection only the intracellular [ATP] and total adenylate nucleotides were positively correlated with viral productivity. The correlated data followed a sigmoidal plot with an equation defined by polynomial regression analysis. Apparent threshold values of 3.2 fmol/cell and 3.3 fmol/cell were established for ATP and total adenylate, respectively, at which the viral production was 50% the maximal value. Cultures with lower ATP and total adenylate levels at the time of infection resulted in as much as a 95% reduction in overall viral titer compared to the control. The adenylate energy charge (AEC) showed a negative correlation with viral production with an AEC value >0.97 resulting in low virus productivity. Intracellular ATP or total adenylate concentration at the point of infection may be used as a predictor of viral yield in bioprocesses designed for virus/vaccine production. (c) 2006 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2014-01-01

    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.

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

    Science.gov (United States)

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

    2014-03-01

    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.

  1. Inhibition of the PI3K/Akt pathway by Ly294002 does not prevent establishment of persistent Junín virus infection in Vero cells.

    Science.gov (United States)

    Linero, Florencia N; Fernández Bell-Fano, Pablo M; Cuervo, Eugenia; Castilla, Viviana; Scolaro, Luis A

    2015-02-01

    In previous work, we demonstrated that the arenavirus Junín virus (JUNV) is able to activate Akt by means of the phosphatidylinositol-3-kinase (PI3K) survival pathway during virus entry. This work extends our study, emphasizing the relevance of this pathway in the establishment and maintenance of persistent infection in vitro. During the course of infection, JUNV-infected Vero cells showed a typical cytopathic effect that may be ascribed to apoptotic cell death. Treatment of infected cultures with Ly294002, an inhibitor of the PI3K/Akt pathway, produced an apoptotic response similar to that observed for uninfected cells treated with the drug. This result suggests that virus-induced activation of the PI3K/Akt pathway does not deliver a strong enough anti-apoptotic signal to explain the low proportion of apoptotic cells observed during infection. Also, inhibition of the PI3K/Akt pathway during the acute stage of infection did not prevent the establishment of persistence. Furthermore, treatment of persistently JUNV-infected cells with Ly294002 did not alter viral protein expression. These findings indicate that despite the positive modulation of the PI3/Akt pathway during Junín virus entry, this would not play a critical role in the establishment and maintenance of JUNV persistence in Vero cells.

  2. Non-hemolytic enterotoxin of Bacillus cereus induces apoptosis in Vero cells.

    Science.gov (United States)

    Liu, Xiaoye; Ding, Shuangyang; Shi, Peijie; Dietrich, Richard; Märtlbauer, Erwin; Zhu, Kui

    2017-04-01

    Bacillus cereus is an opportunistic pathogen that often causes foodborne infectious diseases and food poisoning. Non-hemolytic enterotoxin (Nhe) is the major toxin found in almost all enteropathogenic B. cereus and B. thuringiensis isolates. However, little is known about the cellular response after Nhe triggered pore formation on cell membrane. Here, we demonstrate that Nhe induced cell cycle arrest at G 0 /G 1 phase and provoked apoptosis in Vero cells, most likely associated with mitogen-activated protein kinase (MAPK) and death receptor pathways. The influx of extracellular calcium ions and increased level of reactive oxygen species in cytoplasm were sensed by apoptosis signal-regulating kinase 1 (ASK1) and p38 MAPK. Extrinsic death receptor Fas could also promote the activation of p38 MAPK. Subsequently, ASK1 and p38 MAPK triggered downstream caspase-8 and 3 to initiate apoptosis. Our results clearly demonstrate that ASK1, and Fas-p38 MAPK-mediated caspase-8 dependent pathways are involved in apoptotic cell death provoked by the pore-forming enterotoxin Nhe. © 2016 John Wiley & Sons Ltd.

  3. Development of a new purified vero cell rabies vaccine (Rabivax-S) at the serum institute of India Pvt Ltd.

    Science.gov (United States)

    Kulkarni, Prasad S; Sahai, Ashish; Gunale, Bhagwat; Dhere, Rajeev M

    2017-04-01

    Rabies is a 100% fatal disease with significant disease burden in Asia and Africa but preventable with vaccines and immunoglobulins. There are very few WHO prequalified cell culture derived rabies vaccines available globally for use in humans. We have developed a new purified vero cell rabies vaccine (Rabivax-S) to meet this demand. Areas covered: In this review, we have described the detailed manufacturing process of Rabivax-S and summary of preclinical and clinical development based on the data generated in-house. Expert commentary: Rabivax-S has been developed on Vero ATCC CCL81 cells using Pitman Moore (PM3218) strain. Following all the GMP requirements the vaccine was tested in GLP toxicology studies. Further it underwent clinical trials in preexposure and postexposure settings and was found safe and immunogenic.

  4. Enhancement of cytotoxicity against Vero E6 cells persistently infected with SARS-CoV by Mycoplasma fermentans.

    Science.gov (United States)

    Mizutani, T; Fukushi, S; Kenri, T; Sasaki, Y; Ishii, K; Endoh, D; Zamoto, A; Saijo, M; Kurane, I; Morikawa, S

    2007-01-01

    We previously reported that cells with persistent severe acute respiratory syndrome coronavirus (SARS-CoV) infection were established after apoptotic events. In the present study, we investigated the cytopathic effects of dual infection with SARS-CoV and Mycoplasma fermentans on Vero E6 cells. Dual infection completely killed cells and prevented the establishment of persistent SARS-CoV infection. M. fermentans induced inhibition of cell proliferation, but the cells remained alive. Apoptosis was induced easily in M. fermentans-infected cells, indicating that they were primed for apoptosis. These results indicated that M. fermentans enhances apoptosis in surviving cells that have escaped from SARS-CoV-induced apoptosis.

  5. A single NS2 mutation of K86R promotes PR8 vaccine donor virus growth in Vero cells.

    Science.gov (United States)

    Zhang, Hong; Han, Qinglin; Ping, Xianqiang; Li, Li; Chang, Chong; Chen, Ze; Shu, Yuelong; Xu, Ke; Sun, Bing

    2015-08-01

    Vaccination is the most effective way to prevent and control infection by influenza viruses, and a cell-culture-based vaccine production system is preferred as the future choice for the large-scale production of influenza vaccines. As one of the WHO-recommended cell lines for producing influenza vaccines, Vero cells do not efficiently support the growth of the current influenza A virus vaccine donor strain, the A/Puerto Rico/8/1934 (PR8) virus. In this study, a single mutation of K86R in the NS2 protein can sufficiently render the high-yielding property to the PR8 virus in Vero cells. Further analysis showed that the later steps in the virus replication cycle were accelerated by NS2(K86R) mutation, which may relate to an enhanced interaction between NS2(K86R) and the components of host factor F1Fo-ATPase, FoB and F1β. Because the NS2(K86R) mutation does not increase PR8 virulence in either mice or embryonated eggs, the PR8-NS2(K86R) virus could serve as a promising vaccine donor strain in Vero cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Pre-clinical development of cell culture (Vero)-derived H5N1 pandemic vaccines.

    Science.gov (United States)

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

    2008-05-01

    The rapid spread of avian influenza (H5N1) and its transmission to humans has raised the possibility of an imminent pandemic and concerns over the ability of standard influenza vaccine production methods to supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains, produced at a variety of manufacturing scales, were demonstrated to be highly immunogenic in animal models without the need for adjuvant. The vaccines induce cross-neutralising antibodies and are protective in a mouse challenge model not only against the homologous virus but against other H5N1 strains, including those from other clades. These data indicate that cell culture-grown, whole virus vaccines, based on the wild-type virus, allow the rapid high-yield production of a candidate pandemic vaccine.

  7. Toxigenicity testing of clinical isolates of non-typhoidal salmonellae in Vero cell culture & Caenorhabditis elegans model.

    Science.gov (United States)

    Jesudason, Mary V; V Balaji, V; Densibai, Shoba

    2006-06-01

    The non-typhoidal salmonellae (NTS) are recognized agents of gastroenteritis worldwide. Some of the NTS do not produces cytotoxic changes in tissue culture and not much is known about the endotoxicity of the clinical isolates of NTS (mostly Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Enteritidis). We examined the exotoxic (cytotoxin) and endotoxic activity of clinical isolates of NTS in two assay models namely Vero cell culture and the nematode, Caenorhabditis elegans. Bacteria-free culture supernatants of 40 isolates NTS were tested in 96 well microtitre plate containing confluent monolayers of Vero cells. For the effects on C. elegans, the worms were exposed to bacteria free culture supernatants in 24 well microtitre plate for 24 h and then transferred to OP50 Escherichia coli lawn culture. The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of NTS separately. No cytopathic effect was observed with NTS tested in Vero cell culture assay. Likewise, the worms exposed to the bacteria-free culture supernatants were found active up to 7 days. In the co-culture killing assay, worms were found dead with characteristic stiff and straight appearance by 16(th) day. The worms were alive up to 21 days in OP50 E. coli. Bacteria-free culture supernatants did not have any deleterious effect on worms or in Vero cell culture, suggesting that there is no soluble toxic factor (diffusible toxin) in the culture supernatants. However, live NTS were found to be lethal to the worms; indicating that direct interaction between viable NTS and C. elegans is necessary for killing.

  8. Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly.

    Science.gov (United States)

    Sandekian, Véronique; Lemay, Guy

    2015-01-22

    In a recent study, the serotype 3 Dearing strain of mammalian orthoreovirus was adapted to Vero cells; cells that exhibit a limited ability to support the early steps of reovirus uncoating and are unable to produce interferon as an antiviral response upon infection. The Vero cell-adapted virus (VeroAV) exhibits amino acids substitutions in both the σ1 and μ1 outer capsid proteins but no changes in the σ3 protein. Accordingly, the virus was shown not to behave as a classical uncoating mutant. In the present study, an increased ability of the virus to bind at the Vero cell surface was observed and is likely associated with an increased ability to bind onto cell-surface sialic acid residues. In addition, the kinetics of μ1 disassembly from the virions appears to be altered. The plasmid-based reverse genetics approach confirmed the importance of σ1 amino acids substitutions in VeroAV's ability to efficiently infect Vero cells, although μ1 co-adaptation appears necessary to optimize viral infection. This approach of combining in vitro selection of reoviruses with reverse genetics to identify pertinent amino acids substitutions appears promising in the context of eventual reovirus modification to increase its potential as an oncolytic virus. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. An inactivated yellow fever 17DD vaccine cultivated in Vero cell cultures.

    Science.gov (United States)

    Pereira, Renata C; Silva, Andrea N M R; Souza, Marta Cristina O; Silva, Marlon V; Neves, Patrícia P C C; Silva, Andrea A M V; Matos, Denise D C S; Herrera, Miguel A O; Yamamura, Anna M Y; Freire, Marcos S; Gaspar, Luciane P; Caride, Elena

    2015-08-20

    Yellow fever is an acute infectious disease caused by prototype virus of the genus Flavivirus. It is endemic in Africa and South America where it represents a serious public health problem causing epidemics of hemorrhagic fever with mortality rates ranging from 20% to 50%. There is no available antiviral therapy and vaccination is the primary method of disease control. Although the attenuated vaccines for yellow fever show safety and efficacy it became necessary to develop a new yellow fever vaccine due to the occurrence of rare serious adverse events, which include visceral and neurotropic diseases. The new inactivated vaccine should be safer and effective as the existing attenuated one. In the present study, the immunogenicity of an inactivated 17DD vaccine in C57BL/6 mice was evaluated. The yellow fever virus was produced by cultivation of Vero cells in bioreactors, inactivated with β-propiolactone, and adsorbed to aluminum hydroxide (alum). Mice were inoculated with inactivated 17DD vaccine containing alum adjuvant and followed by intracerebral challenge with 17DD virus. The results showed that animals receiving 3 doses of the inactivated vaccine (2 μg/dose) with alum adjuvant had neutralizing antibody titers above the cut-off of PRNT50 (Plaque Reduction Neutralization Test). In addition, animals immunized with inactivated vaccine showed survival rate of 100% after the challenge as well as animals immunized with commercial attenuated 17DD vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Comparison of herpes simplex (HSV) proteins synthesized in Vero, HEP-2 and human megakaryocyte-like cell lines

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    Soslau, G.; Pastorino, M.B.; Morgan, D.A.; Brodsky, I.; Howett, M.K.

    1986-05-01

    The natural human host blood cell capable of supporting herpes virus replication has yet to be defined. They found that a recently cultured human megakaryocyte-like (Meg) cell line can support HSV 1 and 2 replication as demonstrated by growth inhibition, CPE, virus production and HSV DNA synthesis. The HSV proteins synthesized and post-translationally modified in Vero and HEp-2 infected cells were compared to the protein species produced in the infected Meg cell since differences may influence antigenic properties and host range. Host cell protein synthesis was greatly reduced in all three cell lines within hours post infection (pi). However, maximum viral protein synthesis occurs between 4 and 24 hrs pi with the Meg cells as compared to 24-48 hrs pi with the other cell lines. The immunoprecipitated /sup 35/S-methionine labeled HSV protein gel patterns for each infected cell line are qualitatively and quantitatively very different from each other. Dramatic differences were also observed when infected cells were labeled with /sup 32/P-ATP (in vitro method) or /sup 32/Pi (in vivo method). Finally, analysis of /sup 3/H-mannose labeled HSV glycoproteins demonstrates that the post-translational modifications of these proteins are significantly altered in the Meg cell as compared to the Vero and HEp-2 cells.

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

    Science.gov (United States)

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

    2013-10-01

    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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. MDCK and Vero cells for influenza virus vaccine production: a one-to-one comparison up to lab-scale bioreactor cultivation.

    Science.gov (United States)

    Genzel, Yvonne; Dietzsch, Christian; Rapp, Erdmann; Schwarzer, Jana; Reichl, Udo

    2010-09-01

    Over the last decade, adherent MDCK (Madin Darby canine kidney) and Vero cells have attracted considerable attention for production of cell culture-derived influenza vaccines. While numerous publications deal with the design and the optimization of corresponding upstream processes, one-to-one comparisons of these cell lines under comparable cultivation conditions have largely been neglected. Therefore, a direct comparison of influenza virus production with adherent MDCK and Vero cells in T-flasks, roller bottles, and lab-scale bioreactors was performed in this study. First, virus seeds had to be adapted to Vero cells by multiple passages. Glycan analysis of the hemagglutinin (HA) protein showed that for influenza A/PR/8/34 H1N1, three passages were sufficient to achieve a stable new N-glycan fingerprint, higher yields, and a faster increase to maximum HA titers. Compared to MDCK cells, virus production in serum-free medium with Vero cells was highly sensitive to trypsin concentration. Virus stability at 37 degrees C for different virus strains showed differences depending on medium, virus strain, and cell line. After careful adjustment of corresponding parameters, comparable productivity was obtained with both host cell lines in small-scale cultivation systems. However, using these cultivation conditions in lab-scale bioreactors (stirred tank, wave bioreactor) resulted in lower productivities for Vero cells.

  13. Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

    Directory of Open Access Journals (Sweden)

    Yan Mylene L

    2011-08-01

    Full Text Available Abstract Background Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1 gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. Results Five commercially available animal-component free, serum-free media (SFM were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. Conclusions We describe for the

  14. Serum-free microcarrier based production of replication deficient influenza vaccine candidate virus lacking NS1 using Vero cells.

    Science.gov (United States)

    Chen, Allen; Poh, Swan Li; Dietzsch, Christian; Roethl, Elisabeth; Yan, Mylene L; Ng, Say Kong

    2011-08-11

    Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1) gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. Five commercially available animal-component free, serum-free media (SFM) were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. We describe for the first time the production of Influenza viruses using Vero

  15. Characterization of temperature-sensitive HVJ (Sendai virus) infection in Vero cells: inhibitory mechanism of viral production at 41 degrees.

    Science.gov (United States)

    Hirayama, Etsuko; Ishida, Yo-ichi; Sugimoto, Masao; Hiraki, Akihiro; Kim, Jeman

    2003-01-01

    In a previous study, it was found that the synthesis of hemagglutinating virus of Japan (HVJ; Sendai virus)-specific proteins was inhibited at the transcriptional level at 41 degrees in LLC-MK2 cells. During an investigation of the temperature sensitivity of HVJ production in other host cells, the synthesis of HVJ-specific proteins was recognized even at 41 degrees in Vero cells. Viral production, however, was not detected, indicating the inhibition of steps after the synthesis of viral proteins. Hemadsorption activity was not detected at 41 degrees, suggesting problems with the envelope proteins, especially hemagglutinin-neuraminidase (HN) protein, at the cell membrane. Immunofluorescent staining and surface immunoprecipitation showed that HN protein was not present on the surface in spite of its localization in the cytoplasm. Further, analysis of the cell membrane fraction suggested that fusion (F) protein was integrated into the cell membrane but HN protein was not at 41 degrees. Electron microscopic observation showed that budding sites with spike structures formed and nucleocapsids assembled under the sites at 41 degrees without HN protein, although budded HVJ virions were not detected. At this time, F protein was exposed to the cell membrane and interacted with matrix and nucleocapsid proteins. The results suggested that the suppression of HVJ production at 41 degrees was due to the absence of HN protein in the membrane of Vero cells. Copyright 2003 S. Karger AG, Basel

  16. Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures.

    Science.gov (United States)

    Corry, Jacqueline; Johnson, Sara M; Cornwell, Jessica; Peeples, Mark E

    2015-11-18

    All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines. Worldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein are less infectious

  17. Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

    Science.gov (United States)

    Corry, Jacqueline; Johnson, Sara M.; Cornwell, Jessica

    2015-01-01

    ABSTRACT All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines. IMPORTANCE Worldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein

  18. Effective primary isolation of wild-type canine distemper virus in MDCK, MV1 Lu and Vero cells without nucleotide sequence changes within the entire haemagglutinin protein gene and in subgenomic sections of the fusion and phospho protein genes.

    Science.gov (United States)

    Lednicky, John A; Meehan, Thomas P; Kinsel, Michael J; Dubach, Jean; Hungerford, Laura L; Sarich, Nicolene A; Witecki, Kelley E; Braid, Michael D; Pedrak, Casandra; Houde, Christiane M

    2004-06-15

    Canine distemper virus (CDV) is an important pathogen of many carnivores. We are developing a field-based model of morbillivirus virulence and pathogenesis through a study of distemper in naturally infected free-ranging raccoons. The isolation of CDV from raccoon tissues is essential for this work. CDV has often been isolated from animals only after co-cultivation of infected tissues with peripheral blood mononuclear cells derived from specific pathogen-free dogs or similar methods. We explored the utility and consequences of a simpler and cheaper alternative: CDV isolation in Vero, MDCK, and MV1 Lu cells. Virus growth was detected first in MDCK cells, whereas viral cytopathic effects were most obvious in Vero cells. CDV growth in MV1 Lu cells was relatively protracted and occurred without the formation of cytopathic effects. In primary CDV isolates, the entire nucleotide sequence of the receptor binding haemagglutinin (H) gene, and subgenomic fusion (F) and phospho (P) protein gene sequences corresponding to nt 5399-5733 and 2132-2563 of CDV reference strain Onderstepoort, respectively, were identical to those in matched infected tissues. Virus isolation confirmed the presence of CDV in instances where RT-PCR failed to detect CDV in infected tissues. Different viral phenotypes and genotypes were detected. The conservation of H gene sequences in primary CDV isolates suggests that MDCK, MV1 Lu, and Vero cells express proper receptors for wild-type CDV.

  19. Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus.

    Directory of Open Access Journals (Sweden)

    Debora Ferreira Barreto-Vieira

    Full Text Available Zika virus (ZIKV is a member of the flavivirus genus, and its genome is approximately 10.8 kilobases of positive-strand RNA enclosed in a capsid and surrounded by a membrane. Studies on the replication dynamics of ZIKV are scarce, which limits the development of antiviral agents and vaccines directed against ZIKV. In this study, Aedes albopictus mosquito lineage cells (C6/36 cells and African green monkey kidney epithelial cells (Vero cells were inoculated with a ZIKV sample isolated from a Brazilian patient, and the infection was characterized by immunofluorescence staining, phase contrast light microscopy, transmission electron microscopy and real-time RT-PCR. The infection was observed in both cell lineages, and ZIKV particles were observed inside lysosomes, the rough endoplasmic reticulum and viroplasm-like structures. The susceptibility of C6/36 and Vero cells to ZIKV infection was demonstrated. Moreover, this study showed that part of the replicative cycle may occur within viroplasm-like structures, which has not been previously demonstrated in other flaviviruses.

  20. Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus.

    Science.gov (United States)

    Barreto-Vieira, Debora Ferreira; Jácome, Fernanda Cunha; da Silva, Marcos Alexandre Nunes; Caldas, Gabriela Cardoso; de Filippis, Ana Maria Bispo; de Sequeira, Patrícia Carvalho; de Souza, Elen Mello; Andrade, Audrien Alves; Manso, Pedro Paulo de Abreu; Trindade, Gisela Freitas; Lima, Sheila Maria Barbosa; Barth, Ortrud Monika

    2017-01-01

    Zika virus (ZIKV) is a member of the flavivirus genus, and its genome is approximately 10.8 kilobases of positive-strand RNA enclosed in a capsid and surrounded by a membrane. Studies on the replication dynamics of ZIKV are scarce, which limits the development of antiviral agents and vaccines directed against ZIKV. In this study, Aedes albopictus mosquito lineage cells (C6/36 cells) and African green monkey kidney epithelial cells (Vero cells) were inoculated with a ZIKV sample isolated from a Brazilian patient, and the infection was characterized by immunofluorescence staining, phase contrast light microscopy, transmission electron microscopy and real-time RT-PCR. The infection was observed in both cell lineages, and ZIKV particles were observed inside lysosomes, the rough endoplasmic reticulum and viroplasm-like structures. The susceptibility of C6/36 and Vero cells to ZIKV infection was demonstrated. Moreover, this study showed that part of the replicative cycle may occur within viroplasm-like structures, which has not been previously demonstrated in other flaviviruses.

  1. The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets.

    Science.gov (United States)

    Wang, Yanhui; Gao, Xiaojing; Yao, Yali; Zhang, Yunjing; Lv, Chaochao; Sun, Zhe; Wang, Yuzhou; Jia, Xiangrui; Zhuang, Jinshan; Xiao, Yan; Li, Xiangdong; Tian, Kegong

    2015-10-02

    A severe porcine epidemic diarrhea (PED) epizootic has been affecting pigs of all ages that are characterized by high mortality among suckling piglets in China since late 2010, causing significant economic losses. Obtaining a current-circulating PEDV variant isolate that can grow efficiently in cell culture is prerequisite for the development of efficient vaccines. In this study, PEDV strain HN1303 was isolated successfully on Vero cells with supplemental trypsin, and the isolate has been serially propagated in cell culture for over 95 passages. The infectious titers of the virus during the first 10 passages ranged from 10(2.6) to 10(5.8) 50% tissue culture infective doses (TCID50)/ml, and the titers of 20-95 passages ranged from 10(6.2) to 10(8.0)TCID50/ml. The growth curve of Vero cell-adapted HN1303 in cell culture was determined, and dynamics of virus production was confirmed by immunoperoxidase monolayer assay (IPMA). Sequence and phylogenetic analysis based on spike gene indicate that the HN1303 strain belongs to genotype IIa. In addition, the fourth passage cell-culture HN1303 was subjected to 2-day old piglets. All piglets orally inoculated developed severe watery diarrhea and vomiting within 24 hours post-inoculation (hpi) and died within 72 hpi. The results of animal experiments reveal that this strain is highly pathogenic to 2-day old piglets. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection.

    Science.gov (United States)

    Piccini, Luana E; Castilla, Viviana; Damonte, Elsa B

    2015-01-01

    The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis.

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

    Science.gov (United States)

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

    2004-07-01

    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

  4. [Comparative evaluation of stx-PCR, Vero cell assay and Verotoxin enzyme imminoassay for detection of Shiga toxin-producing Escherichia coli].

    Science.gov (United States)

    Bai, Li; Hu, Yujie; Wang, Jiahui; Wu, Qing; He, Yingying; Wang, Wei; Gan, Xin; Han, Chunhui; Li, Fengqin; Xu, Jin

    2014-09-01

    To evaluate the suitability of stx-PCR, Vero cell assay and commercial enzyme immunoassay for detection of Shiga toxin Escherichia coli and to compare sensitivity and specificity of three different methods for detection of Shiga toxin-producing Escherichia coli. Using stx-PCR, Vero cell assay and commercial enzyme immunoassay to detect 35 Escherichia coli reference strains and 45 strains isolated from food. The three methods all had good specificity. 31 strains gave positive reaction in the Vero cell assay and in the stx-PCR. The consistency between the Vero cell assay and stx-PCR was 100%. Only 38 strains can be detected by commercial enzyme immunoassay. stx-PCR method can serve as a routine rapid detection method in the laboratory. Vero cell assay is recommended to be the gold standard to determine whether the bacteria had the functionally active toxin. Commercial kit was suitable for preliminary rapid detection during clinical testing and outbreaks of food-borne disease.

  5. Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment1[S

    Science.gov (United States)

    Steil, Daniel; Schepers, Catherine-Louise; Pohlentz, Gottfried; Legros, Nadine; Runde, Jana; Humpf, Hans-Ulrich; Karch, Helge; Müthing, Johannes

    2015-01-01

    Shiga toxins (Stxs) are produced by enterohemorrhagic Escherichia coli (EHEC), which cause human infections with an often fatal outcome. Vero cell lines, derived from African green monkey kidney, represent the gold standard for determining the cytotoxic effects of Stxs. Despite their global use, knowledge about the exact structures of the Stx receptor glycosphingolipids (GSLs) and their assembly in lipid rafts is poor. Here we present a comprehensive structural analysis of Stx receptor GSLs and their distribution to detergent-resistant membranes (DRMs), which were prepared from Vero-B4 cells and used as lipid raft equivalents. We identified globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) as the GSL receptors for Stx1a, Stx2a, and Stx2e subtypes using TLC overlay detection combined with MS. The uncommon Stx receptor, globopentaosylceramide (Gb5Cer, Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), which was specifically recognized (in addition to Gb3Cer and Gb4Cer) by Stx2e, was fully structurally characterized. Lipoforms of Stx receptor GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:0/C24:1 or C16:0 fatty acid. Moreover, co-occurrence with lipid raft markers, SM and cholesterol, in DRMs suggested GSL association with membrane microdomains. This study provides the basis for further exploring the functional impact of lipid raft-associated Stx receptors for toxin-mediated injury of Vero-B4 cells. PMID:26464281

  6. Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment.

    Science.gov (United States)

    Steil, Daniel; Schepers, Catherine-Louise; Pohlentz, Gottfried; Legros, Nadine; Runde, Jana; Humpf, Hans-Ulrich; Karch, Helge; Müthing, Johannes

    2015-12-01

    Shiga toxins (Stxs) are produced by enterohemorrhagic Escherichia coli (EHEC), which cause human infections with an often fatal outcome. Vero cell lines, derived from African green monkey kidney, represent the gold standard for determining the cytotoxic effects of Stxs. Despite their global use, knowledge about the exact structures of the Stx receptor glycosphingolipids (GSLs) and their assembly in lipid rafts is poor. Here we present a comprehensive structural analysis of Stx receptor GSLs and their distribution to detergent-resistant membranes (DRMs), which were prepared from Vero-B4 cells and used as lipid raft equivalents. We identified globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) as the GSL receptors for Stx1a, Stx2a, and Stx2e subtypes using TLC overlay detection combined with MS. The uncommon Stx receptor, globopentaosylceramide (Gb5Cer, Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), which was specifically recognized (in addition to Gb3Cer and Gb4Cer) by Stx2e, was fully structurally characterized. Lipoforms of Stx receptor GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:0/C24:1 or C16:0 fatty acid. Moreover, co-occurrence with lipid raft markers, SM and cholesterol, in DRMs suggested GSL association with membrane microdomains. This study provides the basis for further exploring the functional impact of lipid raft-associated Stx receptors for toxin-mediated injury of Vero-B4 cells. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  7. Evaluation of Vero-cell-derived simian endogenous retrovirus infection in humans by detection of viral genome in clinicopathological samples and commercialized vaccines and by serology of Japanese general population.

    Science.gov (United States)

    Fukumoto, Hitomi; Hishima, Tsunekazu; Hasegawa, Hideki; Saeki, Hidehisa; Kuroda, Makoto; Katano, Harutaka

    2016-05-23

    Vero cells are used in laboratories for the isolation of viruses and the production of vaccines. Recently, the sequence of simian endogenous retrovirus (SERV) was identified in Vero cells (SERVagm-Vero), with homology to exogenously transmitted, pathogenic simian retroviruses (SRVs). Although SERVagm-Vero was shown to be noninfectious to human cells in vitro, SERV infection in humans is controversial. In this study, we evaluated the status of SERV infection in humans by detecting the viral genome in clinicopathological samples and commercialized vaccines, and with a serological survey of the Japanese general population. Real-time polymerase chain reaction (PCR) and reverse transcription-PCR were used to detect the SERVagm-Vero genome. We also examined the seroprevalence of SERV in 1000 individuals in the Japanese population with an enzyme-linked immunoabsorbent assay (ELISA) using mixed SERVagm-Vero gag, pol, and env proteins as antigens. Real-time PCR failed to detect SERVagm-Vero genomic fragments in 783 human clinicopathological samples, and all 13 human cell lines tested were negative for the SERVagm-Vero genome. Thirteen commercialized vaccines, including five Vero-based vaccines, were also negative for the SERVagm-Vero genome on real-time PCR and reverse transcription-PCR. Eight (0.8%) were seropositive on ELISA, and western blotting showed that all eight sera contained anti-pol antibodies. All SERV-seropositive individuals were born before 1965, suggesting that SERV infection in Japan might not be associated with vaccine, because more than 90% of Japanese children born from 1964 to 2012 have received live poliovirus vaccines containing virus produced in Vero cells since the 1980s. We have confirmed that the vaccines we use today are free of SERVagm-Vero. Moreover, SERV infection in humans is very rare and unlikely to be associated with vaccines in the Japanese general population. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. In Vitro Studies with Modoc Virus in Vero Cells: Plaque Assay and Kinetics of Growth, Neutralization, and Thermal Inactivation

    Science.gov (United States)

    Davis, James W.; Hardy, James L.

    1973-01-01

    A sensitive and quantitative assay system is described for plaquing Modoc virus in Vero cells. Neutralizing antibodies to Modoc virus could be detected by using this in vitro system by their interference with viral plaque formation. Virus was readily neutralized within 30 min at 37 C by a 1:10 dilution of hyperimmune hamster serum. The rate of neutralization and the total amount of virus neutralized was not altered significantly by the addition of 20 U of guinea pig complement to the hyperimmune hamster serum. A study of the growth of Modoc virus in Vero cells is also presented. After an initial latent period of 20 h, viral titer increased exponentially for 20 h. By 83 h after infection, 8,000 plaque-forming units of virus were detected per cell. The stability of viral infectivity in phosphate-buffered saline at pH 7.4 was evaluated. No reduction in viral titer was detected after 3 days at 7 or 22 C. A continuous decrease in infectivity at 37 C was observed, however, throughout the observation period. PMID:4201641

  9. Adaptation of yellow fever virus 17D to Vero cells is associated with mutations in structural and non-structural protein genes.

    Science.gov (United States)

    Beasley, David W C; Morin, Merribeth; Lamb, Ashley R; Hayman, Edward; Watts, Douglas M; Lee, Cynthia K; Trent, Dennis W; Monath, Thomas P

    2013-09-01

    Serial passaging of yellow fever virus 17D in Vero cells was employed to derive seed material for a novel inactivated vaccine, XRX-001. Two independent passaging series identified a novel lysine to arginine mutation at amino acid 160 of the envelope protein, a surface-exposed residue in structural domain I. A third passage series resulted in an isoleucine to methionine mutation at residue 113 of the NS4B protein, a central membrane spanning region of the protein which has previously been associated with Vero cell adaptation of other mosquito-borne flaviviruses. These studies confirm that flavivirus adaptation to growth in Vero cells can be mediated by structural or non-structural protein mutations. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Establishment of Vero cell RNA polymerase I-driven reverse genetics for Influenza A virus and its application for pandemic (H1N1) 2009 influenza virus vaccine production.

    Science.gov (United States)

    Song, Min-Suk; Baek, Yun Hee; Pascua, Philippe Noriel Q; Kwon, Hyeok-Il; Park, Su-Jin; Kim, Eun-Ha; Lim, Gyo-Jin; Choi, Young-Ki

    2013-06-01

    The constant threat of newly emerging influenza viruses with pandemic potential requires the need for prompt vaccine production. Here, we utilized the Vero cell polymerase I (PolI) promoter, rather than the commonly used human PolI promoter, in an established reverse-genetics system to rescue viable influenza viruses in Vero cells, an approved cell line for human vaccine production. The Vero PolI promoter was more efficient in Vero cells and demonstrated enhanced transcription levels and virus rescue rates commensurate with that of the human RNA PolI promoter in 293T cells. These results appeared to be associated with more efficient generation of A(H1N1)pdm09- and H5N1-derived vaccine seed viruses in Vero cells, whilst the rescue rates in 293T cells were comparable. Our study provides an alternative means for improving vaccine preparation by using a novel reverse-genetics system for generating influenza A viruses.

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

    Science.gov (United States)

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

    2014-05-12

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Chemical synthesis, characterisation, and biocompatibility of nanometre scale porous anodic aluminium oxide membranes for use as a cell culture substrate for the vero cell line: a preliminary study.

    Science.gov (United States)

    Poinern, Gérrard Eddy Jai; Le, Xuan Thi; O'Dea, Mark; Becker, Thomas; Fawcett, Derek

    2014-01-01

    In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72 h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells.

  13. Evaluation of apoptotic activity of Withania coagulans methanolic extract against human breast cancer and Vero cell lines.

    Science.gov (United States)

    Ahmad, Rumana; Fatima, Afreen; Srivastava, A N; Khan, Mohsin Ali

    The genus Withania (Family: Solanaceae) holds an important position in Ayurveda, the Indian traditional system of medicine. Withania somnifera Dunal and Withania coagulans Dunal have been documented in folklore as panaceas for various ailments since time immemorial. W. coagulans (WC), commonly called as Indian cheese maker is used for fermenting milk for cheese production in various parts of India. In the study, in vitro cytotoxicity of methanolic extract of dried fruits (berries) of WC was evaluated in a dose dependent manner using trypan blue dye exclusion method against human breast cancer cell line MDA-MB-231 and normal kidney epithelial cell line Vero in the range 20-200 μg/ml. The percentage viability of the cell lines was determined by using MTT assay and cytometry. Methanolic extract of WC showed significant anticancer activity against MDA-MB-231 cell line. Cell viability was reduced to about 50% at 40 μg/ml of methanolic extract in 50% DMSO. Cytotoxicity of the extract was lower in 10% and 1% DMSO. On the other hand, methanolic extract of WC did not exhibit any significant in vitro activity against Vero cells at 170 and 200 μg/ml. AGE of isolated DNA from treated cancer cells revealed characteristic ladder like fragmentation, a hallmark of apoptosis. HPLC profiling was carried out for identification of the active components, which demonstrated the presence of Withaferin A in the methanolic extract. Methanolic extract of WC possesses apoptotic activity against human breast cancer cells in vitro albeit lower in comparison to W. somnifera and warrants further investigation. Copyright © 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Published by Elsevier B.V. All rights reserved.

  14. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion.

    Science.gov (United States)

    Roehrig, John T; Butrapet, Siritorn; Liss, Nathan M; Bennett, Susan L; Luy, Betty E; Childers, Thomas; Boroughs, Karen L; Stovall, Janae L; Calvert, Amanda E; Blair, Carol D; Huang, Claire Y-H

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. Published by Elsevier Inc.

  15. Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers

    NARCIS (Netherlands)

    Martens, D E; Nollen, E A; Hardeveld, M; Velden-de Groot, C A; Gooijer, C D; Beuvery, E C; Tramper, J

    The death rate of Vero cells grown on Cytodex-3 microcarrierswas studied as a function of the gas flow rate in a smallair-lift loop reactor. The death rate may be described byfirst-order death-rate kinetics. The first-order death-rateconstant as calculated from the decrease in viable cells,

  16. Dengue type 4 live-attenuated vaccine viruses passaged in vero cells affect genetic stability and dengue-induced hemorrhaging in mice.

    Science.gov (United States)

    Lee, Hsiang-Chi; Yen, Yu-Ting; Chen, Wen-Yu; Wu-Hsieh, Betty A; Wu, Suh-Chin

    2011-01-01

    Most live-attenuated tetravalent dengue virus vaccines in current clinical trials are produced from Vero cells. In a previous study we demonstrated that an infectious cDNA clone-derived dengue type 4 (DEN-4) virus retains higher genetic stability in MRC-5 cells than in Vero cells. For this study we investigated two DEN-4 viruses: the infectious cDNA clone-derived DEN-4 2A and its derived 3' NCR 30-nucleotide deletion mutant DEN-4 2AΔ30, a vaccine candidate. Mutations in the C-prM-E, NS2B-NS3, and NS4B-NS5 regions of the DEN genome were sequenced and compared following cell passages in Vero and MRC-5 cells. Our results indicate stronger genetic stability in both viruses following MRC-5 cell passages, leading to significantly lower RNA polymerase error rates when the DEN-4 virus is used for genome replication. Although no significant increases in virus titers were observed following cell passages, DEN-4 2A and DEN-4 2AΔ30 virus titers following Vero cell passages were 17-fold to 25-fold higher than titers following MRC-5 cell passages. Neurovirulence for DEN-4 2A and DEN-4 2AΔ30 viruses increased significantly following passages in Vero cells compared to passages in MRC-5 cells. In addition, more severe DEN-induced hemorrhaging in mice was noted following DEN-4 2A and DEN-4 2AΔ30 passages in Vero cells compared to passages in MRC-5 cells. Target mutagenesis performed on the DEN-4 2A infectious clone indicated that single point mutation of E-Q(438)H, E-V(463)L, NS2B-Q(78)H, and NS2B-A(113)T imperatively increased mouse hemorrhaging severity. The relationship between amino acid mutations acquired during Vero cell passage and enhanced DEN-induced hemorrhages in mice may be important for understanding DHF pathogenesis, as well as for the development of live-attenuated dengue vaccines. Taken together, the genetic stability, virus yield, and DEN-induced hemorrhaging all require further investigation in the context of live-attenuated DEN vaccine development.

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

    Science.gov (United States)

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

    2012-01-01

    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. PMID:22090129

  18. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: jtr1@cdc.gov [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  19. iTRAQ-based comparative proteomic analysis of Vero cells infected with virulent and CV777 vaccine strain-like strains of porcine epidemic diarrhea virus.

    Science.gov (United States)

    Guo, Xiaozhen; Hu, Han; Chen, Fangzhou; Li, Zhonghua; Ye, Shiyi; Cheng, Shuang; Zhang, Mengjia; He, Qigai

    2016-01-01

    The re-emerging porcine epidemic diarrhea virus (PEDV) variant related diarrhea has been documented in China since late 2010 and now with global distribution. Currently, a virulent PEDV CH/YNKM-8/2013 and a CV777 vaccine strain-like AH-M have been successfully isolated from the clinical samples. To dissect out the underlying pathogenic mechanism of virulent PEDV and clarify the differences between virulent and CV777 vaccine strain-like PEDV infections, we performed an iTRAQ-based comparative quantitative proteomic study of Vero cells infected with both PEDV strains. A total of 661 and 474 differentially expressed proteins were identified upon virulent and CV777 vaccine strain-like isolates infection, respectively. Ingenuity Pathway Analysis was employed to investigate the canonical pathways and functional networks involved in both PEDV infections. Comprehensive studies have revealed that the PEDV virulent strain suppressed protein synthesis of Vero cells through down-regulating mTOR as well as its downstream targets 4EBP1 and p70S6K activities, which were validated by immunoblotting. In addition, the virulent strain could activate NF-κB pathway more intensively than the CV777 vaccine strain-like isolate, and elicit stronger inflammatory cascades as well. These data might provide new insights for elucidating the specific pathogenesis of PEDV infection, and pave the way for the development of effective therapeutic strategies. Porcine epidemic diarrhea is now worldwide distributed and causing huge economic losses to swine industry. The immunomodulation and pathogenesis between PEDV and host, as well as the difference between virulent and attenuated strains of PEDV infections are still largely unknown. In this study, we presented for the first application of proteomic analysis to compare whole cellular protein alterations induced by virulent and CV777 vaccine strain-like PEDV infections, which might contribute to understand the pathogenesis of PEDV and anti

  20. Non-linear relationships between aflatoxin B₁ levels and the biological response of monkey kidney vero cells.

    Science.gov (United States)

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

    2013-08-14

    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.

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

    Directory of Open Access Journals (Sweden)

    Mendel Friedman

    2013-08-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2007-05-10

    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

  3. High-yield production of a stable Vero cell-based vaccine candidate against the highly pathogenic avian influenza virus H5N1

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Fangye; Zhou, Jian; Ma, Lei; Song, Shaohui; Zhang, Xinwen; Li, Weidong; Jiang, Shude [No. 5, Department of Bioproducts, Institute of Medical Biology, Chinese Academy of Medical Science and Pecking Union Medical College, Jiaoling Avenue 935, Kunming, Yunnan Province 650102, People' s Republic of China (China); Wang, Yue, E-mail: euy-tokyo@umin.ac.jp [National Institute for Viral Disease Control and Prevention, China Center for Disease Control and Prevention, Yingxin Lane 100, Xicheng District, Beijing 100052, People' s Republic of China (China); Liao, Guoyang, E-mail: liaogy@21cn.com [No. 5, Department of Bioproducts, Institute of Medical Biology, Chinese Academy of Medical Science and Pecking Union Medical College, Jiaoling Avenue 935, Kunming, Yunnan Province 650102, People' s Republic of China (China)

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Vero cell-based HPAI H5N1 vaccine with stable high yield. Black-Right-Pointing-Pointer Stable high yield derived from the YNVa H3N2 backbone. Black-Right-Pointing-Pointer H5N1/YNVa has a similar safety and immunogenicity to H5N1delta. -- Abstract: Highly pathogenic avian influenza (HPAI) viruses pose a global pandemic threat, for which rapid large-scale vaccine production technology is critical for prevention and control. Because chickens are highly susceptible to HPAI viruses, the supply of chicken embryos for vaccine production might be depleted during a virus outbreak. Therefore, developing HPAI virus vaccines using other technologies is critical. Meeting vaccine demand using the Vero cell-based fermentation process has been hindered by low stability and yield. In this study, a Vero cell-based HPAI H5N1 vaccine candidate (H5N1/YNVa) with stable high yield was achieved by reassortment of the Vero-adapted (Va) high growth A/Yunnan/1/2005(H3N2) (YNVa) virus with the A/Anhui/1/2005(H5N1) attenuated influenza vaccine strain (H5N1delta) using the 6/2 method. The reassorted H5N1/YNVa vaccine maintained a high hemagglutination (HA) titer of 1024. Furthermore, H5N1/YNVa displayed low pathogenicity and uniform immunogenicity compared to that of the parent virus.

  4. Safety and immunogenicity of an inactivated whole virus Vero cell-derived Ross River virus vaccine: a randomized trial.

    Science.gov (United States)

    Aichinger, Gerald; Ehrlich, Hartmut J; Aaskov, John G; Fritsch, Sandor; Thomasser, Christiane; Draxler, Wolfgang; Wolzt, Michael; Müller, Markus; Pinl, Fritz; Van Damme, Pierre; Hens, Annick; Levy, Jack; Portsmouth, Daniel; Holzer, Georg; Kistner, Otfried; Kreil, Thomas R; Barrett, P Noel

    2011-11-21

    Ross River virus (RRV) is endemic in Australia and several South Pacific Islands. Approximately 5000 cases of RRV disease, which is characterized by debilitating polyarthritis, are recorded each year in Australia. This study describes the first clinical trial of a candidate RRV vaccine. An inactivated whole-virus Vero cell-derived RRV vaccine was tested in 382 healthy, RRV-naïve adults in a phase 1/2 dose-escalation study at ten sites in Austria, Belgium and The Netherlands. Subjects were equally randomized to receive 1.25 μg, 2.5 μg, 5 μg, or 10 μg aluminum hydroxide-adjuvanted or non-adjuvanted RRV vaccine, with a second dose after three weeks and a booster at six months. Vaccine immunogenicity was determined by measurements of serum IgG and neutralizing antibody titers. Vaccine tolerability and safety were monitored over the entire study period. The optimal vaccine formulation was the adjuvanted 2.5 μg dose, as calculated using a repeated mixed model analysis of covariance comparing log-transformed RRV-specific IgG titers between different dose groups. Geometric means of RRV-specific serum antibodies measured 21 days after the third vaccination with the 2.5 μg adjuvanted formulation were 520.9 (90% CI 377.2-719.4) as determined by IgG ELISA and 119.9 (82.6-173.9) as determined by virus neutralization assay, resulting in seropositivity rates of 92.9% (82.6-98.0) and 92.7% (82.2-98.0), respectively. All vaccine formulations and doses were well tolerated after the first, second and third vaccination. The adjuvanted, inactivated whole-virus Vero cell-derived Ross River virus vaccine is highly immunogenic in RRV-naïve adults and well tolerated at all dose levels. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Amino acid substitutions in σ1 and μ1 outer capsid proteins are selected during mammalian reovirus adaptation to Vero cells.

    Science.gov (United States)

    Jabre, Roland; Sandekian, Véronique; Lemay, Guy

    2013-09-01

    Establishment of viral persistence in cell culture has previously led to the selection of mammalian reovirus mutants, although very few of those have been characterized in details. In the present study, reovirus was adapted to Vero cells that, in contrast to classically-used L929 cells, are inefficient in supporting the early steps of reovirus uncoating and are also unable to produce interferon as an antiviral response once infection occurs. The Vero cell-adapted reovirus exhibits amino acids substitutions in both the σ1 and μ1 proteins. This contrasts with uncoating mutants from persistently infected L929 cells, and various other cell types, that generally harbor amino acids substitutions in the σ3 outer capsid protein. The Vero cell-adapted virus remained sensitive to an inhibitor of lysosomal proteases; furthermore, in the absence of selective pressure for its maintenance, the virus has partially lost its ability to resist interferon. The positions of the amino acids substitutions on the known protein structures suggest an effect on binding of the viral σ1 protein to the cell surface and on μ1 disassembly from the outer capsid. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Sensitivity of different cytotoxic responses of Vero cells exposed to organic chemical pollutants and their reliability in the bio-toxicity test of trace chemical pollutants.

    Science.gov (United States)

    Liao, Ting-Ting; Shi, Yan-Ling; Jia, Jian-Wei; Wang, Lei

    2010-06-01

    To find a sensitive cytotoxic response to reflect the bio-toxicity of trace organic pollutants, the sensitivity and reliability of morphological change and proliferation inhibition of Vero cells exposed to 2, 4, 6-trichlorophenol (TCP) and the leachate from products related to drinking water (PRDW) were compared, and the mechanism of the morphological change in Vero cells exposed to chemical pollutants was studied. Vero cells were treated by different concentration of TCP and the leachate from PRDW. Methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay was carried out for proliferation inhibition. Bioluminescence method was carried out as another method to test the toxicity of TCP. Flow Cytometry assay was used to test cell Apoptosis and damage of cell-membrane. 0.25 mg/L TCP had an effect on cell morphology, and the proportion of morphologically changed cells increased with increasing TCP concentration. At low TCP concentrations, inhibition of cell proliferation did not seem to correlate to TCP concentration, and was negative when TCP concentration was cells increased with extracting temperature, but the inhibition of cell proliferation failed to reflect the correlation between extracting temperature and proliferation inhibition of Vero cells. Although the Sensitivity of bioluminescence method seems to be similar to morphological change in Vero cells, the bacterial in this method is not homologous enough with human body cells to reflect the toxicity to human body. These imply cell morphological change is a more sensitive and reliable method to reflect bio-toxicity of organic pollutants than proliferation inhibition. Flow cytometry analysis and cell rejuvenation experiments indicated cell membrane damage, which results in cell morphological change, was an early and sensitive cytotoxic response comparing with necrosis. These results indicated that the cell membrane toxicity represented by morphological changes is a more sensitive and reliable method to

  7. Photodynamic efficiency of hypericin compared with chlorin and hematoporphyrin derivatives in HEp-2 and Vero epithelial cell lines.

    Science.gov (United States)

    Bernal, Claudia; Ribeiro, Anderson O; Andrade, Gislaine P; Perussi, Janice R

    2015-06-01

    Hypericin (HY) is a photoactive aromatic dianthraquinone that is considered a potent photodynamic agent. In this study, hypericin and two other photosensitizers, a hematoporphyrin derivative (Photogem(®); PG) and a chlorin derivative (Photodithazine(®); PZ), were compared in terms of their phototoxicity toward two cell lines, HEp-2 and Vero. The median inhibitory concentration (IC(50)) of each of the photosensitizers was obtained after a 16.2J cm(-2) dose of irradiation at 630 ± 10 nm. The IC(50) values were 0.07 ± 0.01 (HY), 1.0 ± 0.2 (PZ), and 9 ± 1 μgmL(-1) (PG) in HEp-2 cells and 0.3 ± 0.1 (HY), 1.6 ± 0.2 (PZ) and 11 ± 1 μgmL(-1) (PG) in Vero cells, showing that HY is more phototoxic than the others when irradiated at 630 nm. If these results are analyzed, simultaneously, with the first-order constant for BSA tryptophan photooxidation, obtained by fluorescence decay (λ(excitation)=280 nm), which are 11×10(-3) min(-1)±1. 10(-3) min(-1) (HY), 10 × 10(-3) min(-1)±1 × 10(-3) min(-1) (PZ), and 6 × 10(-3)min(-1) ± 1×10(-3)min(-1) (PG), it is possible to infer that the photodynamic efficiency alone is not sufficient to explain the higher HY phototoxicity. The lipophilicity is also an important factor for an efficient target cell accumulation and was assessed for all sensitizers through the octanol-water partition coefficient (log P): 1.20 ± 0.02 (HY), -0.62 ± 0.03 (PZ), and -0.9 ± 0.2 (PG). The higher value for HY correlates well with its observed superior efficiency to promote damage at low concentrations and doses. As HY is used for the long-term treatment of mild depression, it is considered safe for humans. This fact and the present results reinforce the great potential of this photosensitizer to replace porphyrin derivatives, with the advantages that mean it could be used as photosensitizer in clinical photodynamic therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Vero cells as a model to study the effects of adenoviral gene delivery vectors on the RNAi system in context of viral infection.

    Science.gov (United States)

    Matskevich, Alexey A; Jung, Jiun-Shan; Schümann, Michael; Cascallo, Manel; Moelling, Karin

    2009-01-01

    Technology based on RNA interference (RNAi) is a promising source for new antiviral therapies. Although the application of RNAi has been studied extensively, significant problems with using RNAi remain. Very few studies have specifically assessed model systems for testing the effects of viruses or gene delivery vectors on the RNAi system. Since viruses have developed efficient strategies to circumvent the interferon (IFN) response, an IFN-deficient model system should be considered. Here we show that in Vero cells, which lack IFN-alpha and IFN-beta genes, knockdown of Dicer, a key RNAi component, led to accelerated death of cells infected with other evolutionary distinct viruses: influenza A virus, vesicular stomatitis virus and poliovirus. We also demonstrate that transduction of Vero cells with adenoviral vector with subsequent infection with influenza A virus also resulted in increased mortality of infected cells. These effects were much weaker in IFN-producing A549 and Hela cell lines. Thus, the Vero cell line could serve as an interesting model for studying the effects of gene delivery vectors on the RNAi system in the context of virus-related disorders. Copyright 2009 S. Karger AG, Basel.

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

    Science.gov (United States)

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

    2013-11-01

    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.

  10. Cytotoxicity effects induced by Zearalenone metabolites, alpha Zearalenol and beta Zearalenol, on cultured Vero cells.

    Science.gov (United States)

    Othmen, Zouhour Ouanes-Ben; Golli, Emna El; Abid-Essefi, Salwa; Bacha, Hassen

    2008-10-30

    Zearalenone (Zen) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium. It has been implicated in several mycotoxicosis in farm animals and in humans. The major metabolites of this mycotoxin in various species are alpha and beta Zearalenol. In vivo, Zen is mainly reduced to these alcoholic metabolites which cause reproductive tract disorders and impaired fertility due to their estrogenic activities. In this study, we examined the cytotoxicity of alpha and beta Zearalenol in cultured cells. For this purpose, the MTT assay was carried out and the influence of alpha and beta Zearalenol on protein and DNA syntheses was assessed. To evaluate the cell stress caused by these two metabolites, oxidative stress measured by MDA induction and stress protein induction (Hsp 70, Hsp 27) were tested. Results showed that alpha and beta Zearalenol were metabolites that caused cytotoxicity by inhibiting cell viability, protein and DNA syntheses and inducing oxidative damage and over-expression of stress proteins. However, the Zen metabolites exhibited lower toxicity than Zen, with beta zearalenol being the more active of the two metabolites.

  11. Anti-Proliferative Activity and Apoptosis Induction of an Ethanolic Extract of Boesenbergia pandurata (Roxb.) Schlecht. against HeLa and Vero Cell Lines.

    Science.gov (United States)

    Listyawati, Shanti; Sismindari; Mubarika, Sofia; Murti, Yosi Bayu; Ikawati, Muthi

    2016-01-01

    Rhizomes of Boesenbergia pandurata (Roxb.) Schlecht have been reported to contain active compounds with anticancer properties. This research was carried out to examine anti-proliferative and apoptotic induction against HeLa and Vero cells-line. Dried powder of B. pandurata rhizomes was extracted by a maceration method using 90% ethanol. Cytotoxic assays to determine IC50 and anti-proliferative effects were carried out by MTT methods. Observation of apoptosis was achieved with double staining using acridine orange and ethidium bromide. The results showed that ethanolic extract of B. pandurata was more cytotoxic against HeLa cells (IC50 of 60 μg/ mL) than Vero cells (IC50 of 125 μg/mL). The extract had higher anti-proliferative activity as well as apoptotic induction in HeLa than Vero cells. Therefore, it was concluded that the ethanolic extract of B. pandurata had anti-proliferative as well as apoptosis induction activity dependent on the cell type.

  12. Detection of Vero Cells Infected with Herpes Simplex Types 1 and 2 and Varicella Zoster Viruses Using Raman Spectroscopy and Advanced Statistical Methods.

    Science.gov (United States)

    Huleihel, Mahmoud; Shufan, Elad; Zeiri, Leila; Salman, Ahmad

    2016-01-01

    Of the eight members of the herpes family of viruses, HSV1, HSV2, and varicella zoster are the most common and are mainly involved in cutaneous disorders. These viruses usually are not life-threatening, but in some cases they might cause serious infections to the eyes and the brain that can lead to blindness and possibly death. An effective drug (acyclovir and its derivatives) is available against these viruses. Therefore, early detection and identification of these viral infections is highly important for an effective treatment. Raman spectroscopy, which has been widely used in the past years in medicine and biology, was used as a powerful spectroscopic tool for the detection and identification of these viral infections in cell culture, due to its sensitivity, rapidity and reliability. Our results showed that it was possible to differentiate, with a 97% identification success rate, the uninfected Vero cells that served as a control, from the Vero cells that were infected with HSV-1, HSV-2, and VZV. For that, linear discriminant analysis (LDA) was performed on the Raman spectra after principal component analysis (PCA) with a leave one out (LOO) approach. Raman spectroscopy in tandem with PCA and LDA enable to differentiate among the different herpes viral infections of Vero cells in time span of few minutes with high accuracy rate. Understanding cell molecular changes due to herpes viral infections using Raman spectroscopy may help in early detection and effective treatment.

  13. Modulation of the captopril interference with the activity of some enzymatic biomolecules in monkey kidney vero cells by drug delivery mesoporous silica system

    Directory of Open Access Journals (Sweden)

    Roxana Popovici

    2011-12-01

    Full Text Available The in vitro effect of different formulations of captopril on some cellular enzymatic equipments activities of monkey kidney Vero cells was investigated in the present research. The preparation of the samples of the mesoporous silica nanocomposites, loaded or not with captopril, was described and their effect on membranary Na+-K+-ATP-ase, cell Mg2+-ATP-ase, LDH, Px, GSH-Px, SOD, CAT, ACP, ALP activities were studied. The Vero cells were incubated, for a period of 144 hours, with growing medium renewed twice. When the cells reached confluence in the monolayer stage, the cultures were divided into control cell cultures and other 4 treated groups. To the 12 hours treated cells were added: Cap H2, SBA–15, unfunctionalized SBA-15_CapH2_RT and functionalized SBA-15_APTES_CapH2_80°C nanocomposites, each of them in a dose of 0.4μg./flask. As compared with the control Vero cells, which are characterized by a specific level of the enzymatic activities, the cultures treated with SBA-15 have not presented significant alterations of them. The comparative study of captopril interactions with some membrane bound and intracellular enzymatic biomolecules of monkey kidney Vero cells has revealed either an enhancement of membranary Na+-K+-ATP-ase, intracell total ATP- ase , LDH, ACP , and GSH-Px activities or a repression of cellular CAT, Px and SOD activities. These variations of the enzymatic activities – which induce modifications of the membranary and metabolic processes – could be due to a direct or indirect interaction of captopril with cellular (plasmalemma or subcellular (organelles structures and with intracellular biomolecules (enzymes, DNA, RNA etc.. The association of captoptil with SBA – 15 or SBA – 15 _ APTES mesoporous silica matrices and treatment of Vero cells with these nanocomposites were correlated with modulation of the captopril interference with the activity of investigated enzymatic biomolecules, its sense (stimulation or

  14. Evaluation of physicochemical and biological properties of chitosan/poly (vinyl alcohol) polymer blend membranes and their correlation for Vero cell growth.

    Science.gov (United States)

    Sharma, Parul; Mathur, Garima; Dhakate, Sanjay R; Chand, Subhash; Goswami, Navendu; Sharma, Sanjeev K; Mathur, Ashwani

    2016-02-10

    The blend membranes with varying weight ratios of chitosan/poly (vinyl alcohol) (CS/PVA) (1:0, 1:1, 1:2.5, 1.5:1, 1.5: 2.5) were prepared using solvent casting method and were evaluated for their potential application in single-use membrane bioreactors (MBRs). The physicochemical properties of the prepared membranes were investigated for chemical interactions (FTIR), surface morphology (SEM), water uptake, protein sorption (qe), ammonia sorption and growth kinetics of Vero cells. CS/PVA blend membrane having weight ratio of 1.5:1 had shown enhanced membrane flexibility, reduced water uptake, less protein sorption and no ammonium sorption compared to CS membrane. This blend membrane also showed comparatively enhanced higher specific growth rate (0.82/day) of Vero cells. Improved physicochemical properties and growth kinetics obtrude CS/PVA (1.5:1) as a potential surface for adhesion and proliferation with possible application in single use membrane bioreactors. Additionally, new insight explaining correlation between water holding (%) of CS/PVA (1.5:1) blend membrane and doubling time (td) of Vero cells is proposed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Safety and immunogenicity of chromatographically purified Vero cell rabies vaccine for intradermal pre- and post-exposure rabies prophylaxis.

    Science.gov (United States)

    Tantawichien, Terapong; Sibunruang, Suda; Tantawichien, Thanphet; Angsanakul, Jaruboot; Benjavongkulchai, Maneerat; Limsuwan, Kornvika; Udomchaisakul, Piyada; Khomvilai, Sumana; Sitprija, Visith

    2014-12-01

    Improved rabies pre- and post-exposure prophylaxis (PrEP and PEP) in developing countries uses an economic multi-site intradermal vaccination. To evaluate immunogenicity of chromatographically purified Vero cell vaccine (CPRV) for intradermal PrEP and PEP. The subjects received conventional PrEP with CPRV or PVRV in PrEP study or received intradermal PEP with CPRV or PVRV and rabies immunoglobulin in PEP study. All subjects who received PrEP with CPRV had protective neutralizing antibody (Nab) titers (≥0.5 IU/ml) 14 days after completing vaccination. In PEP study, Nab titers in the CPRV groups reached ≥ 0.5 IU/ml in all subjects by day 14 through day 90 after vaccination. The geometric mean titers of Nab in the CPRV groups had significantly higher titers than the PVRV group on day 14 through day 365 (p < 0.05). No serious adverse reactions were observed. CPRV is safe and immunogenic when given for intradermal PrEP and PEP.

  16. Development of an in situ detachment protocol of Vero cells grown on Cytodex1 microcarriers under animal component-free conditions in stirred bioreactor.

    Science.gov (United States)

    Rourou, Samia; Riahi, Nesrine; Majoul, Samy; Trabelsi, Khaled; Kallel, Héla

    2013-08-01

    Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in six-well plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L(-1). Cells were also grown in spinner flask on 2 g L(-1) Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78 ± 8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70 ± 19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L(-1) Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another.

  17. types sat 1 and sat 2 in bhk, bk, vero and lk cell

    African Journals Online (AJOL)

    BSN

    vcro cells (established cell lines) and Bovine kidne) (BK) and Lamb Kidney (LK) cells (Primary. Cells) were grown I Ocells were gro\\ ...

  18. Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

    Directory of Open Access Journals (Sweden)

    Sydow Farid FO von

    2000-01-01

    Full Text Available Fluorescent activated cell sorter (FACS analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus and Vero cells (green monkey kidney. Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML. FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+ are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

  19. Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen.

    Science.gov (United States)

    Sydow, F F; Santiago, M A; Neves-Souza, P C; Cerqueira, D I; Gouvea, A S; Lavatori, M F; Bertho, A L; Kubelka, C F

    2000-01-01

    Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

  20. An Electron Microscope Study of VERO Cells Infected with Homogeneous and Heterogeneous Virus of VEE

    Science.gov (United States)

    1974-12-10

    formation of giant viricns with distinctly defined nucleoids (Illustration, B and C). It should be noted tat a najority CC the gient cells observed in these...infection), giant virions with distinctly defined nucleoides were only rarely observed. Instead, on the cell surfaces and also in the intercellular spaces...containing two or more nuclcoids within the sam~e membrane, and particles containinj, i :n.terial of the sam.o density as nucleoids . In this connection, it

  1. types sat 1 and sat 2 in bhk, bk, vero and lk cell

    African Journals Online (AJOL)

    BSN

    highest virus titres (6.85 log10 TCID 50/ml) in BK cells follo\\\\ed ti: BHK eel' (5 6lo; TUD,. ml) while the lowest titres v.ere obtained in LK cells (3 5 log10 TCID,o mll lhe <;\\- ~ '1rat:is ~relded lc-•.1er 11r11s tilres in BK lells ..... TABLE V: ANTISERA - Fl\\ID SPECIFIC ANTIGE 1 REACTION BY CIE AND CF. TESTS. --. NNO. 017.

  2. [Comparison of the indirect immunofluorescence assay performance of Bartonella henselae antigens obtained by co-cultivation in Vero and HeLa cells].

    Science.gov (United States)

    Ergin, Cağrı; Akkaya, Yüksel; Kiriş Satılmış, Ozgün; Yılmaz, Cansev

    2011-07-01

    The laboratory diagnosis of Bartonella henselae infection is mainly based on serological testing by indirect immunofluorescence assay (IFA). Cell line co-cultivation with B.henselae and agar derivated antigens are the two major procedures used for evaluation of anti-Bartonella antibodies. Vero and Hep-2 cell lines are the most commonly used media for co-cultivation both in-house and commercial diagnostic kits production. However, HeLa cells which are easily supplied and grown, also can easily be infected by B.henselae. The aim of this study was to compare the performances of antigens obtained by co-cultivation of B.henselae ATCC 49882 (Houston-1) in Vero and HeLa Cells in IFA serology. Out of 381 sera samples, 127 (33.3%) were found positive and 195 (51.2%) were found negative by IFA performed by both cell line co-cultivations. The total agreement between the methods were found as 84.5% (322/381), and Cohen kappa value was calculated as 0.68 (strong, coherent). As a result, He-La cells were found to be useful for the preparation of B.henselae antigens to be used in IFA for the serologic diagnosis of B.henselae infections. However different genotype strains and cross reactions with other infectious agents should be investigated by further studies before routine applications of HeLa cell co-cultivations procedure is established.

  3. Co-cultura de embriões humanos em células vero e transferência em fase de blastocisto Human embryo coculture in vero cells and blastocyst stage transfer

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    Carlos Gilberto Almodin

    1999-08-01

    Full Text Available Objetivos: determinar se o dia da transferência ou o estágio em que o embrião é transferido interferem nas taxas de gravidez e implantação. Métodos: cento e sete pacientes tiveram seus oócitos aspirados e submetidos à fertilização in vitro. Os embriões foram co-cultivados em células Vero e transferidos no dia 3 ou dia 5 pós-fertilização, após avaliação morfológica. Resultados: a taxa de implantação dos embriões transferidos no dia 5 foi significativamente maior que quando os embriões eram transferidos no dia 3, mas as taxas de gravidez não variaram. Observou-se uma diferença significativa nas taxas de gravidez quando se comparou o estágio em que o embrião era transferido, obtendo-se 70,6% de gravidez quando se transferiam blastocistos expandidos e 20,0% e 10,5% quando eram transferidos blastocistos iniciais ou mórulas, respectivamente. Conclusões: as taxas de implantação e gravidez são significativamente aumentadas quando se transferem embriões em estágio de blastocisto expandido, mas os meios e condições de cultura de que dispomos no momento ainda são insuficientes para nos fornecer uma taxa satisfatória de embriões neste estágio.Purpose: to determine whether the transfer day or the stage that the embryo is transferred interferes in pregnancy and implantation rates. Methods: oocytes we recovered from 107 patients and submitted to in vitro fertilization. The embryos were cocultured on Vero cells and transferred on day 3 or day 5 post-fertilization, after morphological assessment. Results: the implantation rate of the transferred embryos on day 5 was significantly higher than when the embryos were transferred on day 3, but the pregnacy rates did not change. However, a significant difference was observed in the pregnancy rates for embryos transferred at the expanded blastocyst stage (70.6% of pregnancy when compared to 20.0% and 10.5% at the earlier blastocyst and morula stages, respectively. Conclusions

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

    Science.gov (United States)

    2012-01-01

    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. PMID:23046946

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

    Science.gov (United States)

    Pless-Petig, Gesine; Metzenmacher, Martin; Türk, Tobias R; Rauen, Ursula

    2012-10-10

    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. 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. 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.

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

    Directory of Open Access Journals (Sweden)

    Pless-Petig Gesine

    2012-10-01

    Full Text Available Abstract 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.

  7. Human papillomavirus type 16 and 18 L1 protein peptide binding to VERO and HeLa cells inhibits their VLPs binding.

    Science.gov (United States)

    Vera-Bravo, Ricardo; Ocampo, Marisol; Urquiza, Mauricio; García, Javier E; Rodríguez, Luis E; Puentes, Alvaro; López, Ramses; Curtidor, Hernando; Suárez, Jorge E; Torres, Elizabeth; Guzmán, Fanny; Díaz, Diana; Cortes, Jimena; Bravo, María M; Cómbita, Alba L; Orozco, Oscar; Patarroyo, Manuel E

    2003-11-10

    Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus-host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20-mer peptides, covering the entire protein, HPLC-purified, (125)I-radiolabeled and tested in VERO and HeLa cell-binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54-77) and 18294 (274-308) from HPV16 L1, as well as 18312 (59-78) and 18322 (259-278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface-exposed protein. There was a 60% reduction in peptide 18283 binding to heparin lyase-treated cells. Cross-linking assays showed that these proteins molecular weights were around 69 and 54 kDa. Peptides 18283 and 18294 specifically inhibited HPV-16 VLP binding to HeLa cells. According to the L1- and VLP-reported structure, both peptides are close on the VLP-surface, belonging to the outer surface broad pockets suggested as being potential receptor sites. Furthermore, it has been reported that a conserved motif from peptide 18294 is the target for neutralizing antibodies. These results suggest that such binding sequences are used by the virus as cell-binding regions. Copyright 2003 Wiley-Liss, Inc.

  8. Genetic and phenotypic properties of vero cell-adapted Japanese encephalitis virus SA14-14-2 vaccine strain variants and a recombinant clone, which demonstrates attenuation and immunogenicity in mice.

    Science.gov (United States)

    Gromowski, Gregory D; Firestone, Cai-Yen; Bustos-Arriaga, José; Whitehead, Stephen S

    2015-01-01

    The live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine, produced in primary hamster kidney cells, is safe and effective. Past attempts to adapt this virus to replicate in cells that are more favorable for vaccine production resulted in mutations that significantly reduced immunogenicity. In this study, 10 genetically distinct Vero cell-adapted JEV SA14-14-2 variants were isolated and a recombinant wild-type JEV clone, modified to contain the JEV SA14-14-2 polyprotein amino acid sequence, was recovered in Vero cells. A single capsid protein mutation (S66L) was important for Vero cell-adaptation. Mutations were also identified that modulated virus sensitivity to type I interferon-stimulation in Vero cells. A subset of JEV SA14-14-2 variants and the recombinant clone were evaluated in vivo and exhibited levels of attenuation that varied significantly in suckling mice, but were avirulent and highly immunogenic in weanling mice and are promising candidates for the development of a second-generation, recombinant vaccine. © The American Society of Tropical Medicine and Hygiene.

  9. Immunogenicity and protective efficacy of a Vero cell culture-derived whole-virus H7N9 vaccine in mice and guinea pigs.

    Science.gov (United States)

    Wodal, Walter; Schwendinger, Michael G; Savidis-Dacho, Helga; Crowe, Brian A; Hohenadl, Christine; Fritz, Richard; Brühl, Peter; Portsmouth, Daniel; Karner-Pichl, Anita; Balta, Dalida; Grillberger, Leopold; Kistner, Otfried; Barrett, P Noel; Howard, M Keith

    2015-01-01

    A novel avian H7N9 virus with a high case fatality rate in humans emerged in China in 2013. We evaluated the immunogenicity and protective efficacy of a candidate Vero cell culture-derived whole-virus H7N9 vaccine in small animal models. Antibody responses induced in immunized DBA/2J mice and guinea pigs were evaluated by hemagglutination inhibition (HI), microneutralization (MN), and neuraminidase inhibition (NAi) assays. T-helper cell responses and IgG subclass responses in mice were analyzed by ELISPOT and ELISA, respectively. Vaccine efficacy against lethal challenge with wild-type H7N9 virus was evaluated in immunized mice. H7N9-specific antibody responses induced in mice and guinea pigs were compared to those induced by a licensed whole-virus pandemic H1N1 (H1N1pdm09) vaccine. The whole-virus H7N9 vaccine induced dose-dependent H7N9-specific HI, MN and NAi antibodies in mice and guinea pigs. Evaluation of T-helper cell responses and IgG subclasses indicated the induction of a balanced Th1/Th2 response. Immunized mice were protected against lethal H7N9 challenge in a dose-dependent manner. H7N9 and H1N1pdm09 vaccines were similarly immunogenic. The induction of H7N9-specific antibody and T cell responses and protection against lethal challenge suggest that the Vero cell culture-derived whole-virus vaccine would provide an effective intervention against the H7N9 virus.

  10. Immunogenicity and protective efficacy of a Vero cell culture-derived whole-virus H7N9 vaccine in mice and guinea pigs.

    Directory of Open Access Journals (Sweden)

    Walter Wodal

    Full Text Available A novel avian H7N9 virus with a high case fatality rate in humans emerged in China in 2013. We evaluated the immunogenicity and protective efficacy of a candidate Vero cell culture-derived whole-virus H7N9 vaccine in small animal models.Antibody responses induced in immunized DBA/2J mice and guinea pigs were evaluated by hemagglutination inhibition (HI, microneutralization (MN, and neuraminidase inhibition (NAi assays. T-helper cell responses and IgG subclass responses in mice were analyzed by ELISPOT and ELISA, respectively. Vaccine efficacy against lethal challenge with wild-type H7N9 virus was evaluated in immunized mice. H7N9-specific antibody responses induced in mice and guinea pigs were compared to those induced by a licensed whole-virus pandemic H1N1 (H1N1pdm09 vaccine.The whole-virus H7N9 vaccine induced dose-dependent H7N9-specific HI, MN and NAi antibodies in mice and guinea pigs. Evaluation of T-helper cell responses and IgG subclasses indicated the induction of a balanced Th1/Th2 response. Immunized mice were protected against lethal H7N9 challenge in a dose-dependent manner. H7N9 and H1N1pdm09 vaccines were similarly immunogenic.The induction of H7N9-specific antibody and T cell responses and protection against lethal challenge suggest that the Vero cell culture-derived whole-virus vaccine would provide an effective intervention against the H7N9 virus.

  11. A Comparative Study on the Adverse Reactions of Purified Chick Embryo Cell Vaccine (PCECV) and Purified Vero Cell Rabies Vaccine (PVRV).

    Science.gov (United States)

    Ramezankhani, Roghieh; Shirzadi, Mohammad Reza; Ramezankhani, Azra; Poor Mozafary, Jamshid

    2016-07-01

    Human rabies is preventable by prompt application of post-exposure prophylaxis (PEP). The aim of this study was to compare the adverse reactions of purified vero cell rabies vaccine (PVRV) with purified chick embryo cell vaccine (PCECV) vaccination for the PEP. In this double blind clinical trial study, 1449 people bitten by animals (279 females), were recruited from 9 different cities of Iran, and randomly assigned to receive intramuscular injections of the PVRV (n = 702) and PCECV (n = 747) vaccines in 5-dose regimen. The local and systemic adverse reactions were compared between two groups. The mean age was 26.8 years (SD, ± 13.1 years) and 27.4 years (SD, ±13.9 years) in PVRV and PCECV group, respectively. Bites were most often located on the lower extremities in both groups. The most common local adverse reaction in both groups was pain at the injection site (4%). Most of the reported systemic adverse reactions were headache (2.5%) and fever (1.9%) in PCECV and PVRV group, respectively. The incidence of itching was higher in the PVRV group compared to the PCECV group (1% vs. 0.1%) (P vaccination was associated with fewer itching at the injection site. There was no significant difference between PCECV and PVRV vaccine regarding local and systemic adverse reactions. Therefore, the PCECV vaccine can be administered instead of PVRV, when our country encounters serious challenges in PVRV vaccine supply.

  12. Patterns of microRNA expression in non-human primate cells correlate with neoplastic development in vitro.

    Directory of Open Access Journals (Sweden)

    Belete Teferedegne

    Full Text Available MicroRNAs (miRNAs are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines, progenitor primary African green monkey kidney (pAGMK cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP; tumorigenic, high-passage VERO cells (10-87 HP; and a cell line (10-87 T derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

  13. Patterns of microRNA expression in non-human primate cells correlate with neoplastic development in vitro.

    Science.gov (United States)

    Teferedegne, Belete; Murata, Haruhiko; Quiñones, Mariam; Peden, Keith; Lewis, Andrew M

    2010-12-22

    MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines), progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

  14. Characterization of a Mutant Diphtheria Toxin that is Defective in Binding to Cell Membrane Receptors on Vero Cells

    Science.gov (United States)

    1982-08-13

    the primary injury produced by diphtheria toxin is the inhibition of protein synthesis In eucaryotic cells . Because early studies of diphtheria...different animal species were reported (58, 181), yet the protein-synthesizing systems in cell - free extracts from all eucaryotic cells are equally...the translocation of toxin to the cell cytosol. Macromolecules which possess a hydrophobic domain can penetrate the lipid bilayer of eucaryotic

  15. The Vero cell-derived, inactivated, SA14-14-2 strain-based vaccine (Ixiaro) for prevention of Japanese encephalitis.

    Science.gov (United States)

    Erra, Elina O; Kantele, Anu

    2015-01-01

    With an estimated 68,000 cases each year, Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia. Vaccination against the disease is recommended for endemic populations and also for travelers at risk. Recently, a Vero cell-derived, inactivated, SA14-14-2 strain-based JE vaccine (JE-VC) became available for travelers from non-endemic regions, replacing the traditional mouse brain-derived vaccines. First licensed in 2009, JE-VC is currently available in Europe, the USA, Canada, Australia and several other countries. In 2013, the vaccine was approved by the European Medicines Agency and the US Food and Drug Administration for use in children. This review summarizes current data on the immunogenicity, safety and clinical use of JE-VC.

  16. Use of blends of bioabsorbable poly(L-lactic acid/poly(hydroxybutyrate- co-hydroxyvalerate as surfaces for Vero cell culture

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    A.R. Santos Jr.

    2005-11-01

    Full Text Available Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops, were cultured in F-10 Ham medium supplemented with 10% fetal calf serum at 37°C on membranes of poly(L-lactic acid (PLLA, poly(hydroxybutyrate-co-hydroxyvalerate (PHBV and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100. The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50 slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60 blends presented flattened cells on smooth areas. PLLA/PHBV (0/100, which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.

  17. 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.

    Science.gov (United States)

    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

    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

  18. Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses.

    Science.gov (United States)

    Kistner, Otfried; Howard, M Keith; Spruth, Martin; Wodal, Walter; Brühl, Peter; Gerencer, Marijan; Crowe, Brian A; Savidis-Dacho, Helga; Livey, Ian; Reiter, Manfred; Mayerhofer, Ines; Tauer, Christa; Grillberger, Leopold; Mundt, Wolfgang; Falkner, Falko G; Barrett, P Noel

    2007-08-10

    The rapid spread and the transmission to humans of avian influenza virus (H5N1) have induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but also against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine.

  19. Feasibility of using the Vero SBRT system for intracranial SRS.

    Science.gov (United States)

    Burghelea, Manuela; Verellen, Dirk; Gevaert, Thierry; Depuydt, Tom; Poels, Kenneth; Simon, Viorica; De Ridder, Mark

    2014-01-06

    The Vero SBRT system was benchmarked in a planning study against the Novalis SRS system for quality of delivered dose distributions to intracranial lesions and assessing the Vero system's capacity for SRS. A total of 27 patients with one brain lesion treated on the Novalis system, with 3 mm leaf width MLC and C-arm gantry, were replanned for Vero, with a 5 mm leaf width MLC mounted on an O-ring gantry allowing rotations around both the horizontal and vertical axis. The Novalis dynamic conformal arc (DCA) planning included vertex arcs, using 90° couch rotation. These vertex arcs cannot be reproduced with Vero due to the mechanical limitations of the O-ring gantry. Alternative class solutions were investigated for the Vero. Additionally, to distinguish between the effect of MLC leaf width and different beam arrangements on dose distributions, the Vero class solutions were also applied for Novalis. In addition, the added value of noncoplanar IMRT was investigated in this study. Quality of the achieved dose distributions was expressed in the conformity index (CI) and gradient index (GI), and compared using a paired Student's t-test with statistical significance for p-values ≤ 0.05. For lesions larger than 5 cm3, no statistical significant difference in conformity was observed between Vero and Novalis, but for smaller lesions, the dose distributions showed a significantly better conformity for the Novalis (ΔCI = 13.74%, p = 0.0002) mainly due to the smaller MLC leaf width. Using IMRT on Vero reduces this conformity difference to nonsignificant levels. The cutoff for achieving a GI around 3, characterizing a sharp dose falloff outside the target volume was 4 cm3 for Novalis and 7 cm3 for Vero using DCA technique. Using noncoplanar IMRT, this threshold was reduced to 3 cm3 for the Vero system. The smaller MLC and the presence of the vertex fields allow the Novalis system to better conform the dose around the lesion and to obtain steeper dose falloff outside the lesion

  20. Immunogenicity, safety and antibody persistence of a purified vero cell cultured rabies vaccine (Speeda) administered by the Zagreb regimen or Essen regimen in post-exposure subjects.

    Science.gov (United States)

    Shi, Nianmin; Zhang, Yibin; Zheng, Huizhen; Zhu, Zhenggang; Wang, Dingming; Li, Sihai; Li, Yuhua; Yang, Liqing; Zhang, Junnan; Bai, Yunhua; Lu, Qiang; Zhang, Zheng; Luo, Fengji; Yu, Chun; Li, Li

    2017-06-03

    To compare the safety, immunogenicity and long-term effect of a purified vero cell cultured rabies vaccine in post-exposure subjects following 2 intramuscular regimens, Zagreb or Essen regimen. Serum samples were collected before vaccination and on days 7, 14, 42, 180 and 365 post vaccination. Solicited adverse events were recorded for 7 d following each vaccine dose, and unsolicited adverse events throughout the entire study period. This study was registered with ClinicalTrials.gov (NCT01821911 and NCT01827917). No serious adverse events were reported. Although Zagreb regimen had a higher incidence of adverse reactions than Essen regimen at the first and second injection, the incidence was similar at the third and fourth injection between these 2 groups as well. At day 42, 100% subjects developed adequate rabies virus neutralizing antibody concentrations (≥ 0.5IU/ml) for both regimens. At days 180 and 365, the antibody level decreased dramatically, however, the percentage of subjects with adequate antibody concentrations still remained high (above 75% and 50% respectively). None of confirmed rabies virus exposured subjects had rabies one year later, and percentage of subjects with adequate antibody concentrations reached 100% at days 14 and 42. Rabies post-exposure prophylaxis vaccination with PVRV following a Zagreb regimen had a similar safety, immunogenicity and long-term effect to the Essen regimen in China.

  1. Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system.

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    Chia-Chyi Liu

    Full Text Available 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 >10(6 TCID(50/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.

  2. Analysis of the growth pattern of Vero cells cultured on dense and porous poly (L-Lactic Acid scaffolds

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    Arnaldo Rodrigues Santos Jr

    2009-09-01

    Full Text Available Poly (L-lactic acid (PLLA polymers are the most frequently used substrates for cell culture, tissue regeneration and orthopedic prostheses, mainly because of their atoxic characteristics and good biocompatibility. The objective of this study was to evaluate whether a higher density or different pore diameters (less than 45, 180-250, and 250-350 µm would change the growth pattern of cultured cells. The cells were found to adhere to and spread over all PLLA scaffolds studied. The cells also showed similar proliferation on dense and porous PLLA scaffolds, except for PLLA scaffolds with a smaller pore diameter. The cytochemical data showed high metabolic cellular activity on the various substrates. Overall, the results indicated satisfactory cell growth and proliferation on the different PLLA scaffolds studied, especially for those with pore diameters of 180-250 µm and 250-350 µm.

  3. Immunogenicity of one dose of Vero cell culture-derived Japanese encephalitis (JE) vaccine in adults previously vaccinated with mouse brain-derived JE vaccine.

    Science.gov (United States)

    Woolpert, Tabitha; Staples, J Erin; Faix, Dennis J; Nett, Randall J; Kosoy, Olga I; Biggerstaff, Brad J; Johnson, Barbara W; Sracic, Michael; Fischer, Marc

    2012-04-26

    There are no data on the use of inactivated Vero cell culture-derived Japanese encephalitis (JE) vaccine (JE-VC) as a booster among individuals who previously received inactivated mouse brain-derived JE vaccine (JE-MB). Military personnel who received ≥3 doses of JE-MB or were JE vaccine-naïve were vaccinated with 2 doses of JE-VC on days 0 and 28. Serum neutralizing antibodies were measured pre-vaccination and 28 days after each dose. Non-inferiority was evaluated for seroprotection rate and geometric mean titer (GMT) between previously vaccinated participants post-dose 1 and vaccine-naïve participants post-dose 2. Fifty-three previously vaccinated and 70 JE vaccine-naïve participants were enrolled. Previously vaccinated participants had significantly higher GMTs pre-vaccination, post-dose 1, and post-dose 2. Seroprotection rates among previously vaccinated participants post-dose 1 (44/44, 100%) were noninferior to those achieved in previously naïve participants post-dose 2 (53/57, 93%). The GMT was significantly higher in previously vaccinated participants post-dose 1 (GMT 315; 95% CI 191-520) compared to previously naïve participants post-dose 2 (GMT 79; 95% CI 54-114). Among military personnel previously vaccinated with ≥3 doses of JE-MB, a single dose of JE-VC adequately boosts neutralizing antibody levels and provides at least short-term protection. Additional studies are needed to confirm these findings in other populations and determine the duration of protection following a single dose of JE-VC in prior recipients of JE-MB. Published by Elsevier Ltd.

  4. Safety and immunogenicity of a new purified vero cell rabies vaccine (PVRV) administered by intramuscular and intradermal routes in healthy volunteers.

    Science.gov (United States)

    Kulkarni, Prasad S; Sapru, Amita; D'costa, Pradeep M; Pandit, Anand; Madhusudana, Shampur N; Yajaman, Ashwin Belludi; Mangrule, Somnath; Gunale, Bhagwat; Bavdekar, Ashish R

    2013-05-31

    Rabies is 100% fatal but preventable with modern vaccines and immunoglobulins. There is a huge demand for rabies vaccines in developing countries of Asia and Africa. We have developed a new purified vero cell rabies vaccine (PVRV) and evaluated its safety and immunogenicity in healthy volunteers by intramuscular (IM) and intradermal (ID) routes of vaccination. Sixty adults aged between 18 and 50 years were recruited in this actively controlled Phase I clinical study and were randomized to receive three 1 ml or 0.1 ml doses of new PVRV intramuscularly or intradermally on days 0, 7 and 21. The control group received commercially available PVRV (Verorab) by intramuscular route. Adverse events (AEs) were recorded with diary cards till day 28 post-vaccination. Immunogenicity was assessed on day 0, 7, 21 and 42 by rapid fluorescence focus inhibition test (RFFIT). In all, 116 solicited local and systemic events were reported across the three groups. Most were mild and resolved without sequelae. Also the few unsolicited events, deemed unrelated to the study vaccines, caused no problems. No significant changes in the routine laboratory parameters were found. Two doses of a vaccine elicited protective titres (≥ 0.5 IU/ml) in all subjects, the GMTs varying between 0.57 and 0.69 IU/ml on day 7, 3.07 and 3.97 IU/ml on day 21, and 6.12 and 8.52 IU/ml on day 42 post-vaccination. PVRV was well tolerated and showed good immunogenicity regardless of whether administered intramuscularly or, using a tenth of that volume, intradermally. Further studies with this new vaccine are warranted. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Adverse events following vaccination with an inactivated, Vero cell culture-derived Japanese encephalitis vaccine in the United States, 2009-2012.

    Science.gov (United States)

    Rabe, Ingrid B; Miller, Elaine R; Fischer, Marc; Hills, Susan L

    2015-01-29

    In March 2009, the U.S. Food and Drug Administration licensed an inactivated, Vero cell culture-derived Japanese encephalitis vaccine (JE-VC [Ixiaro]) for use in adults. The vaccine was licensed based on clinical trial safety data in 3558 JE-VC recipients. It is essential to monitor post-licensure surveillance data to evaluate the safety of JE-VC because rare adverse events may not be detected until the vaccine is administered to a larger population. We reviewed adverse events reported to the U.S. Vaccine Adverse Event Reporting System (VAERS) for adults (≥17 years) who received JE-VC from May 2009 through April 2012. Adverse event reporting rates were calculated using 275,848 JE-VC doses distributed. Over the 3 year period, 42 adverse events following vaccination with JE-VC were reported to VAERS for an overall reporting rate of 15.2 adverse events per 100,000 doses distributed. Of the 42 total reports, 5 (12%) were classified as serious for a reporting rate of 1.8 per 100,000 doses distributed; there were no deaths. Hypersensitivity reactions (N=12) were the most commonly reported type of adverse event, with a rate of 4.4 per 100,000 doses distributed; no cases of anaphylaxis were reported. Three adverse events of the central nervous system were reported (one case of encephalitis and two seizures) for a rate of 1.1 per 100,000; all occurred after receipt of JE-VC with other vaccines. These post-marketing surveillance data suggest a good safety profile for JE-VC consistent with findings from pre-licensure clinical trials. Post-licensure safety data should continue to be monitored for any evidence of rare serious or neurologic adverse events. Published by Elsevier Ltd.

  6. The progressive adaptation of a georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome.

    Science.gov (United States)

    Krug, Peter W; Holinka, Lauren G; O'Donnell, Vivian; Reese, Bo; Sanford, Brenton; Fernandez-Sainz, Ignacio; Gladue, Douglas P; Arzt, Jonathan; Rodriguez, Luis; Risatti, Guillermo R; Borca, Manuel V

    2015-02-01

    African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed. The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been

  7. Generation of High-Yielding Influenza A Viruses in African Green Monkey Kidney (Vero) Cells by Reverse Genetics

    OpenAIRE

    Ozaki, Hiroichi; Govorkova, Elena A.; Li, Chenghong; Xiong, Xiaoping; Webster, Robert G.; Webby, Richard J.

    2004-01-01

    Influenza A viruses are the cause of annual epidemics of human disease with occasional outbreaks of pandemic proportions. The zoonotic nature of the disease and the vast viral reservoirs in the aquatic birds of the world mean that influenza will not easily be eradicated and that vaccines will continue to be needed. Recent technological advances in reverse genetics methods and limitations of the conventional production of vaccines by using eggs have led to a push to develop cell-based strategi...

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

    Science.gov (United States)

    Schnitzler, P; Wiesenhofer, K; Reichling, J

    2008-11-01

    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.

  9. DETECTION OF HUMAN ANTI-ZIKA VIRUS IgG BY ELISA USING AN ANTIGEN FROM in vitro INFECTED VERO CELLS: PRELIMINARY RESULTS

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    Laura Masami SUMITA

    Full Text Available SUMMARY Zika virus (ZKV infection is a huge public health problem in Brazil because of the increased incidence of microcephaly in neonates from infected mothers. Detection of specific IgG antibodies in maternal serum samples constitutes an important approach for diagnosing ZKV infection and evaluating its relationship with neonatal microcephaly. However, as there is no serological test produced in Brazil to detect IgM and IgG antibodies against ZKV, we sought to examine specific IgG in serum samples from patients or suspected mothers to detect previous infection and to test for specificity with regard to flaviviral infections occurring in the same area. Brazilian Zika virus native antigens were obtained from infected Vero cell layers or free virions in the culture medium and then used in ELISA. We tested sera from eight ZKV RNA-diagnosed infected patients (ZKVR, seven neonates with microcephaly and their mothers after delivery (MM, 140 dengue virus IgM-positive (DM and IgG (DG-positive patients, and 100 yellow fever (YF-vaccinated patients. According to the ELISA, ZKVR samples were mostly positive (7/8, and all the MM serum samples were positive for ZKV IgG (7/7. In contrast, cross-reactions for dengue or yellow fever-vaccinated patients were observed, including DM (48/95, DG (10/45 or YF (3/100 serum samples; however, these cross-reactions exhibited low antigen avidity so that 6 M urea largely removed this cross-reactivity, with only a few cross-reacting samples remaining (8/140. ELISA based on extracted virions was much more specific, with all ZKVR (8/8 and MM sera being positive for ZKV IgG (7/7 and only borderline cross-reactivity found for DM (6/95, DG (3/45 or YF (4/100-vaccinated serum samples. This technique (ELISA can identify specific IgG in ZKV-infected patients and may be helpful in diagnosing congenital infetions after maternal RNA virus clearance or in epidemiological studies.

  10. [Expression of herpes simplex virus type 2 latency associated transcript ORF1 and its anti-apoptotic function].

    Science.gov (United States)

    Lv, Fangbiao; Yang, Huilan; Zhong, Feifei; Fan, Jianyong; Liu, Yanhua; Gao, Ruidi

    2013-12-01

    To study the expression of herpes simplex virus type 2 latency-associated transcript (LAT) open reading frame 1 (ORF1) and its anti-apoptosis function induced by actinomycin D in Vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, and the expression of ORF1 was identified by RT-PCR. The changes of Vero cells morphology induced by actinomycin D were observed by fluorescence microscopy, Hochest33258 fluorescence staining. Cells viability was evaluated by MTT assay and cells apoptosis rate was detected by flow cytometry. Double digestion and sequencing confirmed the pEGFP-ORF1 was constructed successfully, RT-PCR showed that the target gene was highly expressed in Vero cells. Hochest33258 staining reaveals that Vero cells transfected with pEGFP-ORF1 and induced apoptosis by actinomycin D had no changes in morphology. MTT assay showed that the viabilities of Vero cells transfected with recombinant plasmid pEGFP-ORF1 and induced apoptosis by actinomycin D has no statistically significant difference compared with the untreated normal control group (P > 0.05), but remarkable higher than Vero cells transfected with empty plasmid pEGFP-C2 and induced apoptosis by actinomycin D, the difference was statistically significant (P rate had no significant difference between pEGFP-ORF1 group and the normal group, but the cells apoptosis rate ofpEGFP-ORF1 was lower than the pEGFP-C2 group. HSV-2 LAT ORF1 gene can be expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.

  11. Modulatory activities of Zingiber offiinale Roscoe methanol extract on the expression and activity of MMPs and TIMPs on dengue virus infected cells

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    Binita Koirala Sharma

    2015-06-01

    Full Text Available Objective: To evaluate the effect of methanolic extract of Zingiber officinale (ZOM rhizome on the activity and expression profile of matrix metalloproteinase (MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP-1, TIMP-2 at the mRNA level in dengue virus infected Vero cells. Methods: Total phenolic content and [6]-gingerol content in ZOM were determined by utilizing Folin-Ciocalteu reagent and high performance liquid chromatography. IC50 value of ZOM for Vero cells was determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. Vero cells were infected with dengue virus to induce MMPs production. Modulatory effect of ZOM on the activity and expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were demonstrated by using gelatin zymography and real time RT-PCR respectively. Results: Amount of total phenolics in ZOM in terms of mg gallic acid equivalents/g was (252.89 ± 0.56 and it possessed (137.32 ± 2.47 mg [6]-gingerol content per gram of extract. The IC 50 value of ZOM was 221.5 µg/mL for Vero cells. The activities of MMP-2 and to a lesser extent MMP-9 were significantly enhanced in the conditioned media collected from the dengue virus infected Vero cells compared to conditioned media from non-infected cells and their activities were significantly inhibited by ZOM in dose-dependent manner. ZOM significantly downregulated the mRNA expression of MMP-2 and MMP-9 and upregulated the mRNA expression of TIMP-1 and TIMP-2 in dengue virus infected Vero cells in concentrationdependent manner. Conclusions: The results of this study suggest that ZOM may be effective in the control of dengue-virus-induced permeability through the reduction of activities and expression of proteases which degrade the adhesion molecules between cells. This may provide the basis for developing new and effective methods in controlling severe dengue complications.

  12. 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.

    Science.gov (United States)

    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

    2009-11-05

    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.

  13. Animal Cell Expression Systems.

    Science.gov (United States)

    Butler, M; Reichl, Ing U

    2017-10-03

    The glycan profile of therapeutic recombinant proteins such as monoclonal antibodies is a critical quality attribute, which affects the efficacy of the final product. The cellular glycosylation process during protein expression is dependent upon a number of factors such as the availability of substrates in the media, the intracellular content of nucleotide sugars, and the enzyme repertoire of the host cells. In order to control the variability of glycosylation it is important to understand the critical process parameters and their acceptable range of values to enable reproducible production of proteins with a predetermined glycan profile providing the desired biological function or therapeutic effect. The depletion of critical nutrients such as glucose or galactose, which may occur toward the end of a culture process, can lead to truncated glycans. Terminal galactosylation and sialyation are particularly variable but may be controlled by the presence of some key media components. Ammonia accumulation, pH, and dissolved oxygen levels are also known to be key bioprocess parameters that affect the glycosylation of recombinant proteins. Specific enzyme inhibitors can be added to the media to drive the formation of selected and predetermined glycan profiles. Various attempts have been made to predict the glycan profiles of cellular expressed proteins and have led to metabolic models based upon knowledge of metabolic flux and the kinetics of individual glycosylation reactions.In contrast to single recombinant proteins, the glycan profiles of viral vaccines are far more complex and difficult to predict. The example of influenza A virus shows that hemagglutinin, the major antigenic determinant, has three to nine N-glycans, which may influence the antigenicity and efficacy of the vaccine. Glycosylation of the influenza A virus has been largely unmonitored in the past as production has been from eggs, where glycan profiles of antigens are difficult if not impossible to

  14. Interaction between submicron COD crystals and renal epithelial cells.

    Science.gov (United States)

    Peng, Hua; Ouyang, Jian-Ming; Yao, Xiu-Qiong; Yang, Ru-E

    2012-01-01

    This study aims to investigate the adhesion characteristics between submicron calcium oxalate dihydrate (COD) with a size of 150 ± 50 nm and African green monkey kidney epithelial cells (Vero cells) before and after damage, and to discuss the mechanism of kidney stone formation. Vero cells were oxidatively injured by hydrogen peroxide to establish a model of injured cells. Scanning electron microscopy was used to observe Vero-COD adhesion. Inductively coupled plasma emission spectrometry was used to quantitatively measure the amount of adhered COD microcrystals. Nanoparticle size analyzer and laser scanning confocal microscopy were performed to measure the change in the zeta potential on the Vero cell surface and the change in osteopontin expression during the adhesion process, respectively. The level of cell injury was evaluated by measuring the changes in malonaldehyde content, and cell viability during the adhesion process. The adhesion capacity of Vero cells in the injury group to COD microcrystals was obviously stronger than that of Vero cells in the control group. After adhesion to COD, cell viability dropped, both malonaldehyde content and cell surface zeta potential increased, and the fluorescence intensity of osteopontin decreased because the osteopontin molecules were successfully covered by COD. Submicron COD further damaged the cells during the adhesion process, especially for Vero cells in the control group, leading to an elevated amount of attached microcrystals. Submicron COD can further damage injured Vero cells during the adhesion process. The amount of attached microcrystals is proportional to the degree of cell damage. The increased amount of microcrystals that adhered to the injured epithelial cells plays an important role in the formation of early-stage kidney stones.

  15. A spiroketal-enol ether derivative from Tanacetum vulgare selectively inhibits HSV-1 and HSV-2 glycoprotein accumulation in Vero cells.

    Science.gov (United States)

    Álvarez, Ángel L; Habtemariam, Solomon; Abdel Moneim, Ahmed E; Melón, Santiago; Dalton, Kevin P; Parra, Francisco

    2015-07-01

    The inhibitory effects of Tanacetum vulgare rhizome extracts on HSV-1 and HSV-2 in vitro replication were assessed. Unlike extracts obtained from the aerial parts, adsorption inhibition and virucidal activities seemed not to be relevant for the observed antiviral action of tansy rhizome extracts. Instead, the most significant effects were the inhibition of virus penetration and a novel mechanism consisting of the specific arrest of viral gene expression and consequently the decrease of viral protein accumulation within infected cells. Through a bioactivity-guided fractionation protocol we isolated and identified the spiroketal-enol ether derivative (E)-2-(2,4-hexadiynyliden)-1,6-dioxaspiro[4.5]dec-3-ene as the active compound responsible for this inhibitory effect. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. A randomized non-inferiority clinical study to assess post-exposure prophylaxis by a new purified vero cell rabies vaccine (Rabivax-S) administered by intramuscular and intradermal routes.

    Science.gov (United States)

    Bose, Anuradha; Munshi, Renuka; Tripathy, Radha Madhab; Madhusudana, Shampur N; Harish, B R; Thaker, Saket; Mahendra, B J; Gunale, Bhagwat; Gogtay, Nithya J; Thatte, Urmila M; Mani, Reeta Subramaniam; Manjunath, K; George, Kuryan; Yajaman, Ashwin Belludi; Sahai, Ashish; Dhere, Rajeev M; Alex, Reginald G; Adhikari, Debasis Das; Abhilash; Raghava, Venkata; Kumbhar, Dipti; Behera, Tapas Ranjan; Kulkarni, Prasad S

    2016-09-14

    Rabies is a 100% fatal disease but preventable with vaccines and immunoglobulins. We have developed a new purified vero cell rabies vaccine (Rabivax-S) and evaluated its safety and immunogenicity in post-exposure prophylaxis by intramuscular (IM) and intradermal (ID) routes. This was a randomized active-controlled non-inferiority study in 180 individuals (age 5years and above) with suspected rabies exposure (90 each with WHO Category II and Category III exposures). The participants received either Rabivax-S (1mL IM; five doses), Rabivax-S (0.1mL ID; eight doses) or purified chick embryo cell vaccine (PCEC, Rabipur®) (1mL IM; five doses). The IM doses were given on Day 0, 3, 7, 14 and 28 while the ID doses were given on days 0, 3, 7 and 28. Category III patients also received a human rabies immunoglobulin (HRIG) on Day 0. Adverse events (AEs) were recorded with diary cards till day 42. Rabies neutralizing antibody levels were measured on day 0, 7, 14, 28 and 42. In both the category II and III patients, the geometric mean concentration (GMC) ratios of Rabivax-S IM and Rabivax-S ID groups to PCEC IM were more than 1, thus proving the non-inferiority. GMCs were similar or higher in Rabivax-S groups at all the time points. Seroresponse against rabies (RFFIT titre⩾0.5IU/mL) was achieved in all participants. Mostly mild local and systemic adverse events were reported across the three groups and all resolved without sequelae. Rabivax-S was well tolerated and showed immunogenicity comparable to a licensed rabies vaccine by both IM and ID routes in post-exposure prophylaxis. Registry No.: CTRI/2012/11/003135. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. A randomised controlled study with whole-cell or acellular pertussis vaccines in combination with regular DT-IPV vaccine and a new poliomyelitis (IPV vero) component in children 4 years of age in the Netherlands

    NARCIS (Netherlands)

    Berbers GAM; Lafeber AB; Labadie J; Vermeer-de Bondt PE; Bolscher DJA; Plantinga AD; LVO; Stichting Thuiszorg Oost-Veluwe

    1999-01-01

    In deze veldproef is de immunogeniteit en de reactogeniteit van 3 verschillende ACVAs en WCV van het RIVM onderzocht, gecombineerd met DTP toegediend als booster bij 4-jarige kinderen. Bij deze kinderen is tevens de immuunrespons op IPVvero (geproduceerd op Vero cellen) vergeleken met het reguliere

  18. Refined equation for description of the influence of intracellular Mg2+ on the activity of single Ca2+-activated K+ channels in Vero kidney cells.

    Science.gov (United States)

    Kazachenko, V N; Kochetkov, K V

    2000-01-01

    A refined empirical equation relating the mean value of the logarithm of the apparent dissociation constant (K0.5) to intracellular Mg2+ concentration is given. The equation is derived based on a more precise adjustment of analytical expressions to experimental data.

  19. Localization of O-glycan initiation, sphingomyelin synthesis, and glucosylceramide synthesis in Vero cells with respect to the endoplasmic reticulum-Golgi intermediate compartment

    NARCIS (Netherlands)

    Schweizer, A..; Clausen, H.; van Meer, G.|info:eu-repo/dai/nl/068570368; Hauri, H.P.

    1994-01-01

    The identification of an endoplasmic reticulum-Golgi intermediate compartment (ERGIC), defined by the 53-kDa transmembrane marker protein ERGIC-53, has added to the complexity of the exocytic pathway of higher eukaryotic cells. Recently, a subcellular fractionation procedure was established for the

  20. Lactobacillus plantarum LB95 impairs the virulence potential of Gram-positive and Gram-negative food-borne pathogens in HT-29 and Vero cell cultures.

    Science.gov (United States)

    Dutra, Virna; Silva, Ana Carla; Cabrita, Paula; Peres, Cidália; Malcata, Xavier; Brito, Luisa

    2016-01-01

    Listeria monocytogenes, Salmonella enterica and verocytotoxigenic Escherichia coli (VTEC) are amongst the most important agents responsible for food outbreaks occurring worldwide. In this work, two Lactobacillus spp. strains (LABs), Lactobacillus plantarum (LB95) and Lactobacillus paraplantarum (LB13), previously isolated from spontaneously fermenting olive brines, and two reference probiotic strains, Lactobacillus casei Shirota and Lactobacillus rhamnosus GG, were investigated for their ability to attenuate the virulence of the aforementioned pathogens using animal cell culture assays. In competitive exclusion assays, the relative percentages of adhesion and invasion of S. enterica subsp. enterica serovar Enteritidis were significantly reduced when the human HT-29 cell line was previously exposed to LB95. The relative percentage of invasion by Listeria monocytogenes was significantly reduced when HT-29 cells were previously exposed to LB95. In the cytotoxicity assays, the cell-free supernatant of the co-culture (CFSC)of VTEC with LB95 accounted for the lowest value obtained amongst the co-cultures of VTEC with LABs, and was significantly lower than the value obtained with the co-culture of VTEC with the two probiotic reference strains. The cytotoxicity of CFSC of VTEC with both LB95 and LB13 exhibited values not significantly different from the cell-free supernatant of the nonpathogenic E. coli B strain. Our results suggested that LB95 may be able to attenuate the virulence of Gram-positive and Gram-negative food-borne pathogens; together with other reported features of these strains, our data reveal their possible use in probiotic foods due to their interesting potential in preventing enteric infections in humans.

  1. Alteration of cell cycle progression by Sindbis virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

    2015-07-10

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  2. A serum-free, purified vero cell rabies vaccine is safe and as immunogenic as the reference vaccine Verorab for pre-exposure use in healthy adults: results from a randomized controlled phase-II trial.

    Science.gov (United States)

    Pichon, Sylvie; Guinet-Morlot, Françoise; Minutello, Maria; Donazzolo, Yves; Rouzier, Regine; Chassard, Didier; Fitoussi, Serge; Hou, Victor

    2013-04-26

    Verorab was licensed in 1985 for both pre- and post-exposure prophylaxis of rabies. The next generation purified Vero cell rabies vaccine (PVRV-NG) is a highly purified vaccine. We performed a phase II clinical study in adults in France to assess its immunological non-inferiority and clinical safety for pre-exposure prophylaxis. In a randomized phase-II trial, 384 healthy adult subjects were randomized (2:1) to receive a three-dose primary series of PVRV-NG or Verorab. One year later, the PVRV-NG group received a PVRV-NG booster while the Verorab group participants were randomized to receive a booster of PVRV-NG or Verorab for. Rabies virus neutralizing antibodies (RVNA) were evaluated on days 0, 28 (subgroup), 42, months 6, 12 and 12+14 days. Safety was evaluated for seven days after each dose. Adverse event between doses, until 28 days after the final dose was recorded. Serious adverse events were recorded up to 6 months after the last dose. The criterion for non-inferiority was met in the per-protocol analysis set and confirmed in the full analysis set (FAS). In the FAS, 99.6% and 100% of subjects had RVNA titers ≥0.5 IU/mL in PVRV-NG and Verorab groups, respectively. While RVNA levels gradually decreased over the 12-month period, at 6 and 12 months after vaccination >89% and >77%, respectively, in both groups had RVNA titers ≥0.5 IU/mL. The PVRV-NG booster induced a strong response, irrespective of the vaccine given for the primary series. PVRV-NG was safe and well tolerated and its safety profile was similar to Verorab for unsolicited adverse events and solicited systemic reactions. The incidence of solicited injection-site reactions was lower with PVRV-NG than with Verorab after the primary series and the booster dose. PVRV-NG was shown to be at least as immunogenic as Verorab and to present a similar safety profile. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Ebola virus infection inversely correlates with the overall expression levels of promyelocytic leukaemia (PML protein in cultured cells

    Directory of Open Access Journals (Sweden)

    Szekely Laszlo

    2003-04-01

    Full Text Available Abstract Background Ebola virus causes severe, often fatal hemorrhagic fever in humans. The mechanism of escape from cellular anti-viral mechanisms is not yet fully understood. The promyelocytic leukaemia (PML associated nuclear body is part of the interferon inducible cellular defense system. Several RNA viruses have been found to interfere with the anti-viral function of the PML body. The possible interaction between Ebola virus and the PML bodies has not yet been explored. Results We found that two cell lines, Vero E6 and MCF7, support virus production at high and low levels respectively. The expression of viral proteins was visualized and quantified using high resolution immunofluorescence microscopy. Ebola encoded NP and VP35 accumulated in cytoplasmic inclusion bodies whereas VP40 was mainly membrane associated but it was also present diffusely in the cytoplasm as well as in the euchromatic areas of the nucleus. The anti-VP40 antibody also allowed the detection of extracellular virions. Interferon-alpha treatment decreased the production of all three viral proteins and delayed the development of cytopathic effects in both cell lines. Virus infection and interferon-alpha treatment induced high levels of PML protein expression in MCF7 but much less in Vero E6 cells. No disruption of PML bodies, a common phenomenon induced by a variety of different viruses, was observed. Conclusion We have established a simple fixation and immunofluorescence staining procedure that allows specific co-detection and precise sub-cellular localization of the PML nuclear bodies and the Ebola virus encoded proteins NP, VP35 and VP40 in formaldehyde treated cells. Interferon-alpha treatment delays virus production in vitro. Intact PML bodies may play an anti-viral role in Ebola infected cells.

  4. Reinjury risk of nano-calcium oxalate monohydrate and calcium oxalate dihydrate crystals on injured renal epithelial cells: aggravation of crystal adhesion and aggregation.

    Science.gov (United States)

    Gan, Qiong-Zhi; Sun, Xin-Yuan; Bhadja, Poonam; Yao, Xiu-Qiong; Ouyang, Jian-Ming

    2016-01-01

    Renal epithelial cell injury facilitates crystal adhesion to cell surface and serves as a key step in renal stone formation. However, the effects of cell injury on the adhesion of nano-calcium oxalate crystals and the nano-crystal-induced reinjury risk of injured cells remain unclear. African green monkey renal epithelial (Vero) cells were injured with H2O2 to establish a cell injury model. Cell viability, superoxide dismutase (SOD) activity, malonaldehyde (MDA) content, propidium iodide staining, hematoxylin-eosin staining, reactive oxygen species production, and mitochondrial membrane potential (Δψm) were determined to examine cell injury during adhesion. Changes in the surface structure of H2O2-injured cells were assessed through atomic force microscopy. The altered expression of hyaluronan during adhesion was examined through laser scanning confocal microscopy. The adhesion of nano-calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals to Vero cells was observed through scanning electron microscopy. Nano-COM and COD binding was quantitatively determined through inductively coupled plasma emission spectrometry. The expression of hyaluronan on the cell surface was increased during wound healing because of Vero cell injury. The structure and function of the cell membrane were also altered by cell injury; thus, nano-crystal adhesion occurred. The ability of nano-COM to adhere to the injured Vero cells was higher than that of nano-COD crystals. The cell viability, SOD activity, and Δψm decreased when nano-crystals attached to the cell surface. By contrast, the MDA content, reactive oxygen species production, and cell death rate increased. Cell injury contributes to crystal adhesion to Vero cell surface. The attached nano-COM and COD crystals can aggravate Vero cell injury. As a consequence, crystal adhesion and aggregation are enhanced. These findings provide further insights into kidney stone formation.

  5. [Expression of the cathepsin L1 gene of Fasciola hepatica eucaryotic cells].

    Science.gov (United States)

    Kuk, Salih; Kaplan, Mustafa; Kalkan, Ahmet; Ozdarendeli, Aykut

    2006-01-01

    The parasitic trematode Fasciola hepatica is the causative agent of fasciolosis that is common in ruminants especially sheep and cattle and is occasionally found in humans. Fasciolosis has a worldwide distribution including Turkey and causes major economic losses in agricultural industry. Cathepsin L1 is one of the major molecules in the excretory-secretory products of F. hepatica and is involved in tissue penetration, immune evasion and feeding and therefore may be used in vaccination and serological diagnosis. The aim of this study was to evaluate cloning and expression of the cathepsin L1 gene of F. hepatica eucaryotic cells. For this purpose, total RNA was extracted from adult F. hepatica. Cathepsin L1 DNA amplicons were obtained with the reverse transcription polymerase chain reaction (RT-PCR). The 981 base-coding gene region of cathepsin L1 was amplified using specific primers to the cathepsin L1 gene. Then, the cathepsin L1 gene was cloned into the pCI-neo mammalian expression vector. The presence of the cathepsin L1 gene was confirmed by PCR screening and enzyme digestion assays. So, the resulting recombinant plasmid was named pFhCL1. Afterwards, the pFhCL1 vector was transiently transfected into Vero cells. The presence of the cathepsin L1 proteins was shown by Western immunoblotting.

  6. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore...... to examine whether human bladder tumor cells express VDR. Tumor biopsies were obtained from 26 patients with TCC. Expression of VDR was examined by immunohistochemical experiments. All tumors expressed VDR. Biopsies from advanced disease contained more VDR positive cells than low stage disease (p ....05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...

  7. Expression of periostin in breast cancer cells.

    Science.gov (United States)

    Ratajczak-Wielgomas, Katarzyna; Grzegrzolka, Jedrzej; Piotrowska, Aleksandra; Matkowski, Rafal; Wojnar, Andrzej; Rys, Janusz; Ugorski, Maciej; Dziegiel, Piotr

    2017-10-01

    Periostin (POSTN) is a protein involved in multiple processes important for cancer development, both at the stage of cancer initiation and progression, as well as metastasis. The aim of this study was to determine the expression of POSTN in the cells of non-invasive ductal breast carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) and to correlate it with clinicopathological data. Immunohistochemical studies (IHC) were conducted on 21 cases of fibrocystic breast change (FC), 44 cases of DCIS and 92 cases of IDC. POSTN expression at mRNA (real-time PCR) and protein level (western blot analysis) was also confirmed in selected breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231 and BO2). Statistically significant higher level of POSTN expression in IDC and DCIS cancer cells compared to FC was noted. Also, the level of POSTN expression in the cytoplasm of IDC cells was shown to increase with the increasing degree of tumour malignancy (G) and significantly higher expression of POSTN was observed in each degree of tumour malignancy (G) relative to FC. Statistically significant higher POSTN expression was observed in tumours with estrogen receptor-negative (ER-) and progesterone receptor-negative (PR-) phenotypes in comparison to estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+) cases, as well as significant negative correlation between POSTN expression in cancer cells and expression of ER and PR (p<0.05). Additionally, statistically significant differences in POSTN expression were shown between particular breast cancer cell lines, both at mRNA and protein level. Observed POSTN expression was the lowest in the case of MCF-7, and the highest in MDA-MB-231 and BO2 of the most aggressive potential clinically corresponding to G3 tumours. POSTN expression in the cytoplasm of IDC cancer cells may play an important role in cancer transformation mechanism.

  8. Molecular expression in transfected corneal endothelial cells

    Science.gov (United States)

    Wang, Fan; Miao, Zhuang; Lu, Chengwei; Hao, Jilong

    2017-10-01

    To investigate the capability of human corneal endothelial cells serving as immunological cells. Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells which were pre-treated with and without -IFN respectively, activation of lymphocytes was determined by FACS analysis. In coculture system, T lymphocyte was activated by corneal endothelial cells, HLA-DP, -DQ, -DR and CD40 expression were increased by - IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. Human corneal endothelial cells were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells.

  9. Adaptation of a Madin-Darby canine kidney cell line to suspension growth in serum-free media and comparison of its ability to produce avian influenza virus to Vero and BHK21 cell lines

    NARCIS (Netherlands)

    Wielink, van R.; Kant-Eenbergen, H.C.M.; Harmsen, M.M.; Martens, D.E.; Wijffels, R.H.; Coco-Martin, J.M.

    2011-01-01

    Madin–Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in

  10. RISET SITOTOKSIK CAMPURAN EKSTRAK DAUN SIRSAK (Annona muricata L DAN KULIT BUAH MANGGIS (Garcinia mangostana L PADA SEL VERO DAN AML12

    Directory of Open Access Journals (Sweden)

    Tuty Erlina Mardja

    2016-12-01

    Full Text Available Campuran Daun sirsak dan Kulit buah manggis sekarang ini banyak digunakan sebagai fito-terapi penyakit kanker dan antioksidan. Tetapi penggunaan bahan ini belum diketahui data keamanannya. Tujuan dilakukan penelitian adalah untuk mengetahui efek sitotoksik campuran ekstrak daun sirsak dan kulit buah manggis terhadap sel vero dan sel AML12. Uji sitotoksik dapat memprediksi keberadaan senyawa yang bersifat toksik secara in vitro menggunakan sel normal atau sel yang telah mengalami transformasi. Uji ini menggunakan 2 jenis cell line, yaitu sel vero dan sel AML12. Sampel berupa simplisia daun sirsak dan kulit buah manggis yang dibuat ekstrak dengan menggunakan etanol 96%. Konsentrasi uji yang digunakan adalah 100; 50; 25; 12,5; 6,25 dan 3,125 mg/mL untuk sel vero dan 500; 250; 125; 62,5; 31,25 dan 15,625 mg/mL untuk sel AML12. Kultur sel dilakukan di wellplate 96 diinkubasi didalam inkubator CO2 pada suhu 37°C selama 24 jam kemudian ditambahkan sampel uji dan selanjutnya diinkubasi kembali didalam inkubator CO2 pada suhu 37°C selama 24 jam. Langkah selanjutnya uji MTT dan kemudian dibaca dengan ELISA reader pada panjang gelombang 570nm. Diperoleh hasil sebagai berikut: IC50 55,97 mg/mL pada sel vero, dan 43,292 mg/mL pada sel AML12. Kesimpulannya sampel campuran ekstrak daun sirsak dan kulit buah manggis ini mempunyai efek toksik terhadap sel vero dan sel AML12 (IC50<100 mg/mL. Kata kunci: Sitotoksik, Daun sirsak, Kulit buah manggis, sel vero, sel AML12, IC50 Abstract Mix of soursop leaves and mangosteen pericarp use as cancer phytotherapy and antioxidant, but it is not yet known its toxicities data. The aim of this study was evaluate cytotoxicity effect of mix of soursop leaves and mangosteen pericarps extract on vero cells and AML12 cells. Toxicity study is one ways to predict the presence of toxic compounds using normal cells or cells that have undergone a transformation. That study was using vero cell and AML12. Samples was ethanolic extract

  11. IAA8 expression during vascular cell differentiation

    Science.gov (United States)

    Andrew T. Groover; Amy Pattishall; Alan M. Jones

    2003-01-01

    We report the characterization of a member of the auxin-induced IAA gene family from zinnia, designated zIAA8, which is expressed by mesophyll cells differentiating as tracheary elements in vitro. Transcription of zIAA8 is upregulated within 3 h after cell isolation in inductive medium,...

  12. CNPase Expression in Olfactory Ensheathing Cells

    Directory of Open Access Journals (Sweden)

    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  13. Avaliação do possível efeito tóxico de um alcano semifluorinado de uso oftalmológico sobre cultura de células Vero Assessment of the possible toxic effect of a semifluorinated alkane on Vero cell culture

    Directory of Open Access Journals (Sweden)

    Paulo Estacia

    2004-12-01

    controlled cell culture conditions. METHODS: We analyzed indirect cytotoxicity, where the cells only come into contact with soluble elements that can be eliminated by perfluorohexiloctano. We therefore analyzed direct toxicity (contact toxicity of perfluorohexiloctano by means of scanning electronic microscopy and immunocytochemistry reagents for actin. Cells embedded in a treatment-free culture medium were used as control, a positive control for toxicity with an undeniably toxic effect on cells, and a weight control that produced a mechanical compression similar to the amount of perfluorohexiloctano used in the experiment. RESULTS: The indirect cytotoxicity test showed that F6H8 did not affect cell growth. Our direct toxicity tests showed that cellular alterations caused by perfluorohexiloctano were similar to those produced by the weight control and different from toxicity control. CONCLUSION: Perfluorohexiloctano does not present indirect toxicity and this product has a compressive rather than a toxic effect on cultured cells.

  14. Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells.

    Science.gov (United States)

    Heldring, Nina; Joseph, Bertrand; Hermanson, Ola; Kioussi, Chrissa

    2012-11-01

    Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

  15. Anticancer and Cytotoxic Activities of [Cu(C6H16N2O2)2][Ni(CN)4] and [Cu(C6H16N2O2)Pd(CN)4] Cyanidometallate Compounds on HT29, HeLa, C6 and Vero Cell Lines.

    Science.gov (United States)

    Aydın, Ali; Korkmaz, Sengul Aslan; Demir, Veysel; Tekin, Saban

    2017-01-01

    In cancer, apoptosis relevant proteins-such as CaM kinase, Bcl-2 or P53, topoisomerase I, cell migration feature and DNA/BSA-macromolecules represent significant targets for current chemotherapeutics. We recently reported two coordination compounds-[Cu(C6H16N2O2)2][Ni(CN)4] (1) and [Cu(C6H16 N2O2)Pd(CN)4] (2)-together with their IR spectra, magnetic properties, thermal analyses and crystal structures. Herein, we describe the ability of these complexes to induce apoptosis in relevant proteins and stimulate topoisomerase I activity, cell migration velocity and DNA/BSA binding properties. The in vitro antiproliferative effects and cell toxicity of both compounds were investigated through pharmacological measurement techniques, and interactions between both compounds and CT-DNA/BSA were studied with UV-Vis spectroscopy and fluorescence spectroscopy. Results & Conclusion: Studies on cells revealed that 2 (i) demonstrated a high antiproliferative effect, which was higher toward HeLa and C6 cancer cells than toward healthy Vero cells; (ii) impaired the migration of HeLa cells; (iii) altered the P53-Bcl-2 ratio in favor of apoptosis; (iv) strongly bound to DNA/BSA macromolecules; and (v) inhibited human topoisomerase I and KpnI or BamHI restriction endonucleases. In conclusion, this preliminary information demonstrates that 2 may represent a promising antiproliferative agent and a potential candidate for a therapeutic approach against HeLa. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Oncomirs Expression Profiling in Uterine Leiomyosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Bruna Cristine de Almeida

    2017-12-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. They play a key role in several biological processes. Their abnormal expression may lead to malignant cell transformation. This study aimed to evaluate the expression profile of 84 miRNAs involved in tumorigenesis in immortalized cells of myometrium (MM, uterine leiomyoma (ULM, and uterine leiomyosarcoma (ULMS. Specific cell lines were cultured and qRT-PCR was performed. Thirteen miRNAs presented different expression profiles in ULM and the same thirteen in ULMS compared to MM. Eight miRNAs were overexpressed, and five were underexpressed in ULM. In ULMS cells, five miRNAs exhibited an overexpression and eight were down-regulated. Six miRNAs (miR-1-3p, miR-130b-3p, miR-140-5p, miR-202-3p, miR-205-5p, and miR-7-5p presented a similar expression pattern in cell lines compared to patient samples. Of these, only three miRNAs showed significant expression in ULM (miR-1-3p, miR-140-5p, and miR-7-5p and ULMS (miR-1-3p, miR-202-3p, and miR-7-5p. Our preliminary approach identified 24 oncomirs with an altered expression profile in ULM and ULMS cells. We identified four differentially expressed miRNAs with the same profile when compared with patients’ samples, which strongly interacted with relevant genes, including apoptosis regulator (BCL2, epidermal growth factor receptor (EGFR, vascular endothelial growth factor A (VEGFA, insulin like growth factor 1 receptor (IGF1R,serine/threonine kinase (RAF1, receptor tyrosine kinase (MET, and bHLH transcription factor (MYCN. This led to alterations in their mRNA-target.

  17. Oncomirs Expression Profiling in Uterine Leiomyosarcoma Cells

    Science.gov (United States)

    Garcia, Natalia; Maffazioli, Giovana; Gonzalez dos Anjos, Laura; Chada Baracat, Edmund; Candido Carvalho, Katia

    2017-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. They play a key role in several biological processes. Their abnormal expression may lead to malignant cell transformation. This study aimed to evaluate the expression profile of 84 miRNAs involved in tumorigenesis in immortalized cells of myometrium (MM), uterine leiomyoma (ULM), and uterine leiomyosarcoma (ULMS). Specific cell lines were cultured and qRT-PCR was performed. Thirteen miRNAs presented different expression profiles in ULM and the same thirteen in ULMS compared to MM. Eight miRNAs were overexpressed, and five were underexpressed in ULM. In ULMS cells, five miRNAs exhibited an overexpression and eight were down-regulated. Six miRNAs (miR-1-3p, miR-130b-3p, miR-140-5p, miR-202-3p, miR-205-5p, and miR-7-5p) presented a similar expression pattern in cell lines compared to patient samples. Of these, only three miRNAs showed significant expression in ULM (miR-1-3p, miR-140-5p, and miR-7-5p) and ULMS (miR-1-3p, miR-202-3p, and miR-7-5p). Our preliminary approach identified 24 oncomirs with an altered expression profile in ULM and ULMS cells. We identified four differentially expressed miRNAs with the same profile when compared with patients’ samples, which strongly interacted with relevant genes, including apoptosis regulator (BCL2), epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), insulin like growth factor 1 receptor (IGF1R),serine/threonine kinase (RAF1), receptor tyrosine kinase (MET), and bHLH transcription factor (MYCN). This led to alterations in their mRNA-target. PMID:29295562

  18. GOLPH2 expression in renal cell cancer

    Directory of Open Access Journals (Sweden)

    Jung Klaus

    2008-11-01

    Full Text Available Abstract Background Renal cell carcinomas (RCC are among the most common and most lethal genitourinary malignancies. GOLPH2 (golgi phosphoprotein 2, GOLM1 has recently been proposed as a biomarker for hepatocellular and prostate cancer. In this study we analysed the expression patterns and the prognostic and diagnostic value of GOLPH2 in RCC. Methods GOLPH2 protein expression was analysed by immunohistochemistry in 104 clinically well characterized RCC cases in comparison with matched normal kidney tissue and in association with clinico-pathological parameters. Statistical analyses including Kaplan Meier analyses were performed with SPSS version 15.0. Results GOLPH2 was highly expressed in normal renal tubules and in almost half of RCC with a statistically significant predominance in the papillary and chromophobe histological subtypes. No other associations with clinico-pathological parameters were detectable. The Kaplan-Meier curves showed a weak trend for unfavourable prognosis of tumours with high GOLPH2 expression, but failed significance. Conclusion GOLPH2 protein is expressed in normal renal tissue (especially in distal tubular epithelia and is down-regulated in the majority of clear cell RCC. In papillary and chromophobe RCC GOLPH2 expression is consistently present. In contrast to its diagnostic value in hepatocellular and prostatic carcinomas, a prognostic or diagnostic value of GOLPH2 in RCC appears to be unlikely.

  19. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies that comp......It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies...... that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... survival might, therefore, act through such a matrix-to-cell suppression of apoptosis. Indeed, correlative mining of gene expression and patient survival databases suggests that poor survival in patients with metastatic cancer correlates highly with tumor expression of a common theme: the genes involved...

  20. Nanomaterials Enhanced Gene Expression in Yeast Cells

    Directory of Open Access Journals (Sweden)

    Su-Fang Chien

    2008-01-01

    Full Text Available Metal nanomaterials are shown to enhance gene expression for rice -galactosidase gene (-Gal in yeast cells. Au and Ag nanoparticles and their nanocomposites, silica-Au and silica-Ag, were prepared and characterized by UV-vis spectroscopy and TEM technique. The rice -galactosidase gene was cloned into the yeast chromosome, where the cloned cells were precultured and induced into a medium containing each of the testing nanomaterials. The nanomaterials were observed to incorporate inside the cells, and no cell death has been detected during the course of gene expression. The enzyme activity was determined by a synthetic substrate, p-nitrophenyl--D-galctopyranoside, and the yellow product yield was recorded in a spectrophotometer at 400 nm. When Au and Ag nanoparticles were incorporated with the culture, a 3–5 fold enhancement in -galactosidase was observed for intracellular activity as well as the secreted activity into the medium. The secreted protein was analyzed to have a pure form and displayed as a single protein band in the SDS-gel electrophoresis. The effects of size and chemical nature of nanomaterials on gene expression for the rice -galactosidase gene in yeast cells are discussed.

  1. MEMBRANE LEc EXPRESSION IN BREAST CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Ya. A. Udalova

    2009-01-01

    Full Text Available Affine chromatography was used to isolate Lec antibodies from the sera of a healthy female donor with the high titers of these anti- bodies, which were labeled with biotin. The study enrolled 51 patients with primary breast cancer (BC. Antigen expression was found by immunohistochemistry and flow cytometry. With these two techniques being used, the detection rate of Lec expression in BC cells was 65% (33/51; the antigen was most frequently found by flow cytometry as compared with immunohistochemistry: 72 and 58% of cases, respectively.

  2. One-year immunogenicity kinetics and safety of a purified chick embryo cell rabies vaccine and an inactivated Vero cell-derived Japanese encephalitis vaccine administered concomitantly according to a new, 1-week, accelerated primary series.

    Science.gov (United States)

    Cramer, Jakob P; Jelinek, Tomas; Paulke-Korinek, Maria; Reisinger, Emil C; Dieckmann, Sebastian; Alberer, Martin; Bühler, Silja; Bosse, Dietrich; Meyer, Seetha; Fragapane, Elena; Costantini, Marco; Pellegrini, Michele; Lattanzi, Maria; Dovali, Claudia

    2016-03-01

    Conventional rabies pre-exposure prophylaxis (PrEP) and Japanese encephalitis (JE) primary series vaccination regimens each require up to 4 weeks to complete and, thus, may not be feasible for individuals who need these immunizations on short notice. This Phase 3b, randomized, controlled, observer-blind study evaluated the immunogenicity and safety of concomitant administration of a purified chick embryo cell culture rabies vaccine and an inactivated, adsorbed JE vaccine according to an accelerated (1 week) regimen when compared with the conventional regimens (4 weeks). This report describes the kinetics of immune responses up to 1 year after vaccination. A total of 661 healthy adults (18 to ≤65 years) were randomized into the following accelerated or conventional vaccine regimens: Rabies + JE-Conventional, Rabies + JE-Accelerated, Rabies-Conventional and JE-Conventional. Immunogenicity was assessed by virus neutralization tests. Safety and tolerability were also evaluated. Irrespective of rabies vaccination regimen, ≥97% of subjects had adequate levels of rabies virus neutralizing antibody (RVNA) concentrations (≥0.5 IU/ml) up to Day 57, with percentages of subjects with RVNA concentrations ≥0.5 IU/ml at Day 366 ranging between 68% in the Rabies + JE-Accelerated group and 80% of subjects in the Rabies-Conventional group. The Rabies + JE-Accelerated group revealed high JE neutralizing antibody titers at all-time points. At Day 366, the percentage of subjects with antibody titers indicative of seroprotection (PRNT50 titers ≥1:10) remained high across JE vaccine groups (86-94%). The accelerated PrEP rabies and JE vaccination regimens, once licensed, could represent a valid alternative in the short-term to currently recommended conventional regimens. The concomitant administration of these two vaccines does not compromise immune responses to any of the vaccine antigens particularly when aiming for short-term protection. Further evidence

  3. Konsentrasi Aman Kurkumin dan PGV-0 terhadap Sel Vero Berdasarkan Hasil Uji Sitotoksik

    Directory of Open Access Journals (Sweden)

    Dewi Marbawati

    2015-08-01

    Full Text Available Curcumin (1,7-bis(3-methoxyphenyl 4'hidroksi -1.6 heptadien, 3,5-dione is the yellow pigment of Curcuma longa. Curcumin has various biological activities such as antioxidant, antibacterial, antifungal, antiprotozoal and antivirus. Various benefits of curcumin can not be separated from the weakness which is not stable to pH and light. Pentagamavunon-0 (PGV-0 were made by changing the β diketone group on cluster analog of curcumin into monoketon while eliminating active methylene group so it is more stable to pH and light. PGV-0 also has potential as a stronger antioxidant and antiinflammatory agent than other curcumin analogues. The objective of this research was to determine the safe concentrations of curcumin and PGV-0 on vero cells due to the increased use of the two compounds through the cytotoxic test. This research includes experimental research. Cytotoxic test performed with microculture tetrazolium technique (MTT method. Use of MTT to evaluate the cytotoxic is based on changes of tetrazolium salt into formazan crystals by mitochondrial enzyme succinate dehydrogenase with the help of cellular NADH. The results showed that the safe concentrations of curcumin and PGV-0 on vero cells respectively are 6.25 and 1.5625 ppm. Based on the cytotoxic test the secure concentration of curcumin was higher than PGV-0.

  4. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  5. Direct cell lysis for single-cell gene expression profiling

    Directory of Open Access Journals (Sweden)

    David eSvec

    2013-11-01

    Full Text Available The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA to be the best lysis agent, resulting in efficient cell lysis, high RNA stability and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single cells as well as samples composed of small numbers of cells.

  6. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    OpenAIRE

    Kimitoshi Kohno; Noriaki Kitamura; Akihiro Kuma; Yoshihiro Yasuniwa; Takahiro Yamaguchi; Masaki Akiyama; Hiroto Izumi

    2011-01-01

    We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-li...

  7. UJI SITOTOKSITAS DAN ANTIPROLIFERATIF SEL KANKER PAYUDARA T47D DAN SEL VERO BIJI Nigella sativa, L.

    Directory of Open Access Journals (Sweden)

    Laela Hayu Nurani

    2012-05-01

    Full Text Available Black cumin seeds contain oil classes atisiri, terpenes, and alkaloids that can be used for traditional medicine as anticancer. The purpose of this study was to assess the cytotoxic effects of ether extract, ethanol, and infusa seed Nigella sativa, L. (Black cumin to inhibit the growth of T47D and normal (Vero cells and its effect on the kinetics of T47D breast cancer cells proliferation. This study were used extracts of ether, ethanol, and black cumin seeds infusa that obtained by maceration method and infundasi. Cytotoxicity test was performed by incubating T47D breast cancer cells at a 2 x 104 density with concentrations series of 2000; 1000; 500: 250: 125; 62.5; 31.25 and 15.625 microg/ml for 24 hours. Vero cell with a density of 2 x 104 with concentrations series of 4000; 2000; 1000; 500; 250; 125 and 62.5 microg/ml. The number of cells was calculated by direct counting method and the calculated the death percentage. The LC50 values calculated using probit analysis. Observations on the nature of the growth inhibition were done by observing kinetics of cell proliferation with the addition of trypan blue at-24, 48 and 72 to determine its doubling time. The results showed that the ether extract, ethanol, and black cumin seeds infusa are cytotoxic to T47D breast cancer cells with successive LC50 of 32.63: 10.02, and 23.82 mg mL. Vero cell cytotoxicity test to produce successive LC50 of 300.6; 328.41, and 778.64 g/ml. Antiproliferative test results showed that in 62.5 ug/ml and 31.625 microg/ml prolong the doubling time. Ethanol extract of cumin seeds have a higher potential due to the highest security index compared to ether extract and infusa.

  8. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    Energy Technology Data Exchange (ETDEWEB)

    Izumi, Hiroto, E-mail: h-izumi@med.uoeh-u.ac.jp; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro; Kuma, Akihiro; Kitamura, Noriaki; Kohno, Kimitoshi [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 (Japan)

    2011-10-19

    We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1), aurora kinase B (AURKB) and some minichromosome maintenance complex components (MCM). However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells) and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  9. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    Directory of Open Access Journals (Sweden)

    Kimitoshi Kohno

    2011-10-01

    Full Text Available We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143 regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1, aurora kinase B (AURKB and some minichromosome maintenance complex components (MCM. However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  10. Forced Expression of ZNF143 Restrains Cancer Cell Growth.

    Science.gov (United States)

    Izumi, Hiroto; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro; Kuma, Akihiro; Kitamura, Noriaki; Kohno, Kimitoshi

    2011-10-19

    We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1), aurora kinase B (AURKB) and some minichromosome maintenance complex components (MCM). However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells) and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  11. High-dose hydrocortisone reduces expression of the pro-inflammatory chemokines CXCL8 and CXCL10 in SARS coronavirus-infected intestinal cells.

    Science.gov (United States)

    Cinatl, Jindrich; Michaelis, Martin; Morgenstern, Birgit; Doerr, Hans Wilhelm

    2005-02-01

    Clinical observations and our high-density oligonucleotide microarray results demonstrated increased expression of proinflammatory chemokines after SARS-CoV infection. Here, we investigated the influence of SARS-CoV infection on CXCL8 (interleukin 8) and CXCL10 (interferon-gamma-inducible protein 10) in human intestinal epithelial (Caco2) cells. RT-PCR and ELISA showed time-dependent up-regulation of both chemokines after SARS-CoV infection. Electric mobility shift assay revealed increased DNA binding activity of the cellular transcription factors activator protein 1 (AP-1) and nuclear factor (B (NF-kappaB) in SARS-CoV infected cells. High hydrocortisone concentrations (> or =50 microg/ml) completely prevented increased DNA binding activity of AP-1 and NF-kappaB and inhibited up-regulation of CXCL8 and CXCL10, but did not reduce chemokine expression to basal levels. Ribavirin that does not inhibit SARS-CoV replication in Vero cells inhibited SARS-CoV replication in Caco2 cells at therapeutical concentrations. Hydrocortisone neither influenced SARS-CoV titres alone nor in combination with ribavirin. Our results show that corticosteroids may be of limited benefit in the suppression of chemokine production by SARS-CoV-infected cells.

  12. Recombinant Wild-Type and Edmonston Strain Measles Viruses Bearing Heterologous H Proteins: Role of H Protein in Cell Fusion and Host Cell Specificity

    OpenAIRE

    Takeuchi, Kaoru; Takeda, Makoto; Miyajima, Naoko; Kobune, Fumio; Tanabayashi, Kiyoshi; Tashiro, Masato

    2002-01-01

    Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but...

  13. CD25+ B-1a Cells Express Aicda

    Directory of Open Access Journals (Sweden)

    Hiroaki Kaku

    2017-06-01

    Full Text Available B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID, a product of the Aicda gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for Aicda in B-1a cells has been suggested on the basis of experiments with knock out (KO mice, whether B-1a cells express Aicda, and if so, which B-1a cell subpopulation expresses Aicda, remains unknown. Here, we demonstrate that B-1 cells express Aicda, but at a level below that expressed by germinal center (GC B cells. We previously reported that B-1a cells can be subdivided based on CD25 expression. We show here that B-1a cell Aicda expression is concentrated in the CD25+ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of Aicda expression may have inadvertently included an unknown number of CD25+ B-1a cells. Although B-1a cells develop normally in the absence of Aicda, a competitive reconstitution assay reveals enhanced vigor for AID KO B-1a cell bone marrow (BM progenitors, as compared with wild-type BM B-1 cell progenitors. These results suggest that AID inhibits the development of B-1a cells from BM B-1 cell progenitors in a competitive environment.

  14. Progressive adaptation of a Georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major changes of the viral genome

    Science.gov (United States)

    African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified or cell culture-adapted ASFV have been evaluated but no commercial vaccine is available to control African Swine Fev...

  15. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Carolyn A. de Graaf

    2016-09-01

    Full Text Available Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

  16. Canine Signaling Lymphocyte Activation Molecule発現Vero細胞における3タイプのイヌジステンパーウイルス株の増殖特徴

    OpenAIRE

    LAN, Nguyen Thi; Yamaguchi, Ryoji; Kai, Kazushige; Uchida, Kazuyuki

    2005-01-01

    To know growth profiles of canine distemper virus (CDV) on Vero cells stably expressing canine signaling lymphocyte activation molecule (Vero-DogSLAMtag; Vero-DST cells), the propagation of three strains of CDV was tested in Vero-DST cells in comparison with parental Vero cells. Strain MD77 could grow well in both cell lines, but demonstrated no syncytium formation or indistinguishable rounding cytopathic effects (CPE) in Vero cells. Strains Onderstepoort and KDK-1 also grew well in Vero-DST ...

  17. Gene expression profiles in adenosine-treated human mast cells ...

    African Journals Online (AJOL)

    The role of mast cells in allergic diseases and innate immunity has been widely researched and much is known about the expression profiles of immune-related genes in mast cells after bacterial challenges. However, little is known about the gene expression profiles of mast cells in response to adenosine. Herein, we ...

  18. Identifying gene expression modules that define human cell fates

    OpenAIRE

    Germanguz, I; Listgarten, J; Cinkornpumin, J.; Solomon, A; Gaeta, X.; Lowry, W. E.

    2016-01-01

    Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in f...

  19. 33 CFR 110.73b - Indian River at Vero Beach, Fla.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Indian River at Vero Beach, Fla. 110.73b Section 110.73b Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Special Anchorage Areas § 110.73b Indian River at Vero Beach, Fla. (a) Area...

  20. Physical disruption of cell-cell contact induces VEGF expression in RPE cells.

    Science.gov (United States)

    Farjood, Farhad; Vargis, Elizabeth

    2017-01-01

    To investigate the role of RPE cell-cell contact in vascular endothelial growth factor (VEGF) protein expression in cultures of primary human RPE (hRPE) cells and a human RPE cell line (ARPE-19). Two in vitro methods, scratching and micropatterning, were used to control the physical dissociation of RPE cell-cell junctions. Scratching was performed by scoring monolayers of RPE cells with a cell scraper. Micropatterning was achieved by using a stencil patterning method. Extracellular VEGF expression was assessed by using an enzyme-linked immunosorbent assay (ELISA) kit. Immunocytochemistry (ICC) was performed to visualize the expression and localization of VEGF and intercellular proteins zonula occludens-1 (ZO-1), N-cadherin, β-catenin, and claudin-1 in RPE cultures. Higher expression of VEGF protein by cells on the edges of the scratched RPE layers was confirmed with ICC in short-term (1 day after confluency) and long-term (4 weeks after confluency) cultures. According to the ICC results, ZO-1, N-cadherin, β-catenin, and claudin-1 successfully localized to cell-cell junctions in long-term cultures of ARPE-19 and hRPE cells. However, unlike N-cadherin, β-catenin, and claudin-1, only ZO-1 localized junctionally in short-term cultures of both cell types. Moreover, removing cell-cell junctions by scratching resulted in the delocalization of ZO-1 from tight junctions to the cytoplasm. The loss of tight junction formation and the accumulation of ZO-1 in the cytoplasm correlated with increased VEGF expression. Micropatterning RPE cells on different sized circular patterns produced varying concentrations of cells with lost cell-cell junctions. When fewer cells formed intercellular junctions, increased extracellular VEGF secretion was observed from the ARPE-19 and hRPE cells. VEGF expression increases after physical disruption of RPE cell-cell connections. This increase in VEGF expression correlates with the loss of intercellular junctions and the localization of ZO-1 in

  1. Human thymic epithelial cells express functional HLA-DP molecules

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

    T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA-DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA-DP molecules. The expression of HLA-DP molecules was comparable to or higher than the expression...... of HLA-DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN-gamma treatment strongly upregulated the HLA-DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels...

  2. Modeling bi-modality improves characterization of cell cycle on gene expression in single cells.

    Science.gov (United States)

    McDavid, Andrew; Dennis, Lucas; Danaher, Patrick; Finak, Greg; Krouse, Michael; Wang, Alice; Webster, Philippa; Beechem, Joseph; Gottardo, Raphael

    2014-07-01

    Advances in high-throughput, single cell gene expression are allowing interrogation of cell heterogeneity. However, there is concern that the cell cycle phase of a cell might bias characterizations of gene expression at the single-cell level. We assess the effect of cell cycle phase on gene expression in single cells by measuring 333 genes in 930 cells across three phases and three cell lines. We determine each cell's phase non-invasively without chemical arrest and use it as a covariate in tests of differential expression. We observe bi-modal gene expression, a previously-described phenomenon, wherein the expression of otherwise abundant genes is either strongly positive, or undetectable within individual cells. This bi-modality is likely both biologically and technically driven. Irrespective of its source, we show that it should be modeled to draw accurate inferences from single cell expression experiments. To this end, we propose a semi-continuous modeling framework based on the generalized linear model, and use it to characterize genes with consistent cell cycle effects across three cell lines. Our new computational framework improves the detection of previously characterized cell-cycle genes compared to approaches that do not account for the bi-modality of single-cell data. We use our semi-continuous modelling framework to estimate single cell gene co-expression networks. These networks suggest that in addition to having phase-dependent shifts in expression (when averaged over many cells), some, but not all, canonical cell cycle genes tend to be co-expressed in groups in single cells. We estimate the amount of single cell expression variability attributable to the cell cycle. We find that the cell cycle explains only 5%-17% of expression variability, suggesting that the cell cycle will not tend to be a large nuisance factor in analysis of the single cell transcriptome.

  3. Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

    Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

  4. Safety and immunogenicity of a freeze-dried, Vero cell culture-derived, inactivated Japanese encephalitis vaccine (KD-287, ENCEVAC®) versus a mouse brain-derived inactivated Japanese encephalitis vaccine in children: a phase III, multicenter, double-blinded, randomized trial.

    Science.gov (United States)

    Yun, Ki Wook; Lee, Hoan Jong; Kang, Jin Han; Eun, Byung Wook; Kim, Yae-Jean; Kim, Kyung-Hyo; Kim, Nam Hee; Hong, Young Jin; Kim, Dong Ho; Kim, Hwang Min; Cha, Sung-Ho

    2015-01-08

    Although mouse brain-derived, inactivated Japanese encephalitis vaccines (JE-MBs) have been successfully used for a long time, potential rare neurological complications have prompted the development of a Vero cell culture-derived inactivated vaccine (JE-VC). In a phase III clinical study, we aimed to compare the safety and immunogenicity of a JE-VC, KD-287 with a JE-MB, JEV-GCC, in children. In this multicenter, double-blinded, randomized controlled trial, the study population consisted of 205 healthy Korean children aged 12-23 months. Each subject was subcutaneously vaccinated with either KD-287 or JEV-GCC twice at an interval of 2 weeks and then vaccinated once 12 months after the second vaccination. Neutralizing antibodies were measured by the plaque reduction neutralization test using the homologous and heterologous, as a post hoc analysis, challenge virus strains. The three-dose regimen of KD-287 showed a comparable safety profile with JEV-GCC except higher incidence of fever after the first dose (30.4% and 14.7%, respectively). Most of the fever was mild degree (61.3% and 66.7%, respectively). KD-287 fulfilled the non-inferiority criteria for seroconversion rate (SCR) and geometric mean titer (GMT) of the neutralizing antibody, which were the primary endpoints, at 4 weeks after the third vaccination (95% CI: -1.00, 3.10 for the SCR difference and 10.8, 17.6 for the GMT ratio). The SCRs of KD-287 were all 100% and the GMTs were higher in the KD-287 group than in the JEV-GCC group after the second vaccination and before and after the third vaccination (GMT ratio: 5.59, 20.13, and 13.79, respectively, p < 0.001 in all). GMTs were higher in the KD-287 group in the heterologous analysis also (GMT ratio: 4.05, 5.15, and 4.19, respectively, p < 0.001 in all). This study suggests that the KD-287, a JE-VC is as safe as and may be more effective than the licensed MB-derived vaccine. KD-287 could thus be useful as a second-generation vaccine and substitute

  5. Regulation of Mu Opioid Receptor Expression in Developing T Cells

    OpenAIRE

    Zhang, Lily; Belkowski, Judith Sliker; Briscoe, Tammi; Rogers, Thomas J.

    2012-01-01

    We have previously reported that functionally active μ-opioid receptors (MOR) are constitutively expressed at relatively low levels by developing T cells in the thymus. However, very little is known about the regulation of MOR expression by immature T cells. In this report, we first attempted to determine the effect of T cell receptor-induced T cell activation on the expression of MOR. We activated T cells with either the combination of anti-CD3 and CD28, or with superantigen, and observed a ...

  6. Advantages and Applications of CAR-Expressing Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Wolfgang eGlienke

    2015-02-01

    Full Text Available In contrast to donor T cells, natural killer (NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD. In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/ on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

  7. Increased expression of T-helper cell activation markers in ...

    African Journals Online (AJOL)

    Ehab

    part, by the actions of eosinophil-active cytokines derived predominantly from activated CD4 T-cells, of which interleukin-5 (IL-5) is uniquely eosinophil- specific 3,4. T-helper cells are a heterogeneous group of cells that can be divided into subpopulations on the basis of the expression of the cell surface markers. The.

  8. Investigation of factors responsible for cell line cytoplasmic expression differences

    Directory of Open Access Journals (Sweden)

    Finn Jonathan D

    2005-05-01

    Full Text Available Abstract Background Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line and COS-7 (a low expressing cell line. Results The main findings of this work are that the individual elements of the cytoplasmic expression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence are functioning similarly in both cell types. Both cell types were found to have the same amount of cytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellular processing of DNA -cationic lipid complexes. Conclusion After exploring many factors, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in cytoplasmic gene expression.

  9. Gene expression signatures of human cell and tissue longevity.

    Science.gov (United States)

    Seim, Inge; Ma, Siming; Gladyshev, Vadim N

    2016-01-01

    Different cell types within the body exhibit substantial variation in the average time they live, ranging from days to the lifetime of the organism. The underlying mechanisms governing the diverse lifespan of different cell types are not well understood. To examine gene expression strategies that support the lifespan of different cell types within the human body, we obtained publicly available RNA-seq data sets and interrogated transcriptomes of 21 somatic cell types and tissues with reported cellular turnover, a bona fide estimate of lifespan, ranging from 2 days (monocytes) to a lifetime (neurons). Exceptionally long-lived neurons presented a gene expression profile of reduced protein metabolism, consistent with neuronal survival and similar to expression patterns induced by longevity interventions such as dietary restriction. Across different cell lineages, we identified a gene expression signature of human cell and tissue turnover. In particular, turnover showed a negative correlation with the energetically costly cell cycle and factors supporting genome stability, concomitant risk factors for aging-associated pathologies. In addition, the expression of p53 was negatively correlated with cellular turnover, suggesting that low p53 activity supports the longevity of post-mitotic cells with inherently low risk of developing cancer. Our results demonstrate the utility of comparative approaches in unveiling gene expression differences among cell lineages with diverse cell turnover within the same organism, providing insights into mechanisms that could regulate cell longevity.

  10. Gene expression profiling of Drosophila tracheal fusion cells.

    Science.gov (United States)

    Chandran, Rachana R; Iordanou, Ekaterini; Ajja, Crystal; Wille, Michael; Jiang, Lan

    2014-07-01

    The Drosophila trachea is a premier genetic system to investigate the fundamental mechanisms of tubular organ formation. Tracheal fusion cells lead the branch fusion process to form an interconnected tubular network. Therefore, fusion cells in the Drosophila trachea will be an excellent model to study branch fusion in mammalian tubular organs, such as kidneys and blood vessels. The fusion process is a dynamic cellular process involving cell migration, adhesion, vesicle trafficking, cytoskeleton rearrangement, and membrane fusion. To understand how these cellular events are coordinated, we initiated the critical step to assemble a gene expression profile of fusion cells. For this study, we analyzed the expression of 234 potential tracheal-expressed genes in fusion cells during fusion cell development. 143 Tracheal genes were found to encode transcription factors, signal proteins, cytoskeleton and matrix proteins, transporters, and proteins with unknown function. These genes were divided into four subgroups based on their levels of expression in fusion cells compared to neighboring non-fusion cells revealed by in situ hybridization: (1) genes that have relative high abundance in fusion cells, (2) genes that are dynamically expressed in fusion cells, (3) genes that have relative low abundance in fusion cells, and (4) genes that are expressed at similar levels in fusion cells and non-fusion tracheal cells. This study identifies the expression profile of fusion cells and hypothetically suggests genes which are necessary for the fusion process and which play roles in distinct stages of fusion, as indicated by the location and timing of expression. These data will provide the basis for a comprehensive understanding of the molecular and cellular mechanisms of branch fusion. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Identifying gene expression modules that define human cell fates.

    Science.gov (United States)

    Germanguz, I; Listgarten, J; Cinkornpumin, J; Solomon, A; Gaeta, X; Lowry, W E

    2016-05-01

    Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in fact define cell fate. Lastly, we introduce a web-based database to disseminate the results. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Science.gov (United States)

    Pilbrough, Warren; Munro, Trent P; Gray, Peter

    2009-12-23

    Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise

  13. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Directory of Open Access Journals (Sweden)

    Warren Pilbrough

    Full Text Available Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean, approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations. Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50

  14. CD133 expression in chemo-resistant Ewing sarcoma cells

    Directory of Open Access Journals (Sweden)

    Kovar Heinrich

    2010-03-01

    Full Text Available Abstract Background Some human cancers demonstrate cellular hierarchies in which tumor-initiating cancer stem cells generate progeny cells with reduced tumorigenic potential. This cancer stem cell population is proposed to be a source of therapy-resistant and recurrent disease. Ewing sarcoma family tumors (ESFT are highly aggressive cancers in which drug-resistant, relapsed disease remains a significant clinical problem. Recently, the cell surface protein CD133 was identified as a putative marker of tumor-initiating cells in ESFT. We evaluated ESFT tumors and cell lines to determine if high levels of CD133 are associated with drug resistance. Methods Expression of the CD133-encoding PROM1 gene was determined by RT-PCR in ESFT tumors and cell lines. CD133 protein expression was assessed by western blot, FACS and/or immunostaining. Cell lines were FACS-sorted into CD133+ and CD133- fractions and proliferation, colony formation in soft agar, and in vivo tumorigenicity compared. Chemosensitivity was measured using MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxy-methoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium assays. Results PROM1 expression was either absent or extremely low in most tumors. However, PROM1 was highly over-expressed in 4 of 48 cases. Two of the 4 patients with PROM1 over-expressing tumors rapidly succumbed to primary drug-resistant disease and two are long-term, event-free survivors. The expression of PROM1 in ESFT cell lines was similarly heterogeneous. The frequency of CD133+ cells ranged from 2-99% and, with one exception, no differences in the chemoresistance or tumorigenicity of CD133+ and CD133- cell fractions were detected. Importantly, however, the STA-ET-8.2 cell line was found to retain a cellular hierarchy in which relatively chemo-resistant, tumorigenic CD133+ cells gave rise to relatively chemo-sensitive, less tumorigenic, CD133- progeny. Conclusions Up to 10% of ESFT express high levels of PROM1. In some tumors and cell

  15. TRPM5-expressing microvillous cells in the main olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Liman Emily R

    2008-11-01

    Full Text Available Abstract Background The main olfactory epithelium (MOE in the nasal cavity detects a variety of air borne molecules that provide information regarding the presence of food, predators and other relevant social and environmental factors. Within the epithelium are ciliated sensory neurons, supporting cells, basal cells and microvillous cells, each of which is distinct in morphology and function. Arguably, the least understood, are the microvillous cells, a population of cells that are small in number and whose function is not known. We previously found that in a mouse strain in which the TRPM5 promoter drives expression of the green fluorescent protein (GFP, a population of ciliated olfactory sensory neurons (OSNs, as well as a population of cells displaying microvilli-like structures is labeled. Here we examined the morphology and immunocytochemical properties of these microvillous-like cells using immunocytochemical methods. Results We show that the GFP-positive microvillous cells were morphologically diversified and scattered throughout the entire MOE. These cells immunoreacted to an antibody against TRPM5, confirming the expression of this ion channel in these cells. In addition, they showed a Ca2+-activated non-selective cation current in electrophysiological recordings. They did not immunoreact to antibodies that label cell markers and elements of the transduction pathways from olfactory sensory neurons and solitary chemosensory cells of the nasal cavity. Further, the TRPM5-expressing cells did not display axon-like processes and were not labeled with a neuronal marker nor did trigeminal peptidergic nerve fibers innervate these cells. Conclusion We provide morphological and immunocytochemical characterization of the TRPM5-expressing microvillous cells in the main olfactory epithelium. Our data demonstrate that these cells are non-neuronal and in terms of chemosensory transduction do not resemble the TRPM5-expressing olfactory sensory neurons

  16. High expression of markers of apoptosis in Langerhans cell histiocytosis

    DEFF Research Database (Denmark)

    Petersen, Bodil Laub; Lundegaard, Pia Rengtved; Bank, M I

    2003-01-01

    AIMS: Langerhans cell histiocytosis is a rare disease with clonal proliferation of dendritic histiocytes, occurring most frequently in infancy and early childhood. In the localized form (single system), the disease is self-limiting, but in the cases of multisystem disease a third of the patients...... develop organ dysfunction. In these cases the prognosis is poor. Our objective has been to study the immunohistochemical expression of Fas and Fas-ligand (Fas-L) in order to determine whether the level of expression of these proteins could predict the outcome of the disease. We also wanted to determine......53 and the number of cells in apoptosis detected with TUNEL. Langerhans cell histiocytosis cells showed strong expression of p53 and in some cases co-expression of Fas and Fas-L. The expression of Fas-L was significantly higher in infiltrates from patients with single-system disease. The actual...

  17. Oct-4 expression maintained stem cell properties in prostate cancer ...

    African Journals Online (AJOL)

    The purpose of the present study is to isolate cancerous stem-like cells from normal healthy volunteers and prostate cancer patients (CD133+) which also express MDR1 and to ascertain the influence of Oct-4 on 'stem-ness' and differentiation of these CD133+ cells towards epithelium. Methods: CD133+ cells were isolated ...

  18. Regulatory natural killer cell expression in atopic childhood asthma ...

    African Journals Online (AJOL)

    Introduction: Different subsets of natural killer (NK) cells were found to play a role in pathogenesis of allergy. We sought to investigate the expression of regulatory NK cells (CD56+CD16+CD158+) in atopic children with bronchial asthma in order to outline the value of these cells as biomarkers of disease severity and/or ...

  19. Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells.

    Science.gov (United States)

    Peng, Weidan; Bao, Yunhua; Sawicki, Janet A

    2011-10-01

    To establish a method for efficient and relatively easy isolation of a cell population containing epithelial prostate stem cells, we developed two transgenic mouse models, K5/CFP and K18/RFP. In these models, promoters of the cytokeratin 5 (Krt5) and the cytokeratin 18 (Krt18) genes regulate cyan and red fluorescent proteins (CFP and RFP), respectively. CFP and RFP reporter protein fluorescence allows for visualization of K5(+) and K18(+) epithelial cells within the cellular spatial context of the prostate gland and for their direct isolation by FACS. Using these models, it is possible to test directly the stem cell properties of prostate epithelial cell populations that are positively selected based on expression of cytoplasmic proteins, K5 and K18. After validating appropriate expression of the K5/CFP and K18/RFP transgenes in the developing and adult prostate, we demonstrate that a subset of CFP-expressing prostate cells exhibits stem cell proliferation potential and differentiation capabilities. Then, using prostate cells sorted from double transgenic mice (K5/CFP + K18/RFP), we compare RNA microarrays of sorted K5(+)K18(+) basal and K5(-)K18(+) luminal epithelial cells, and identify genes that are differentially expressed. Several genes that are over-expressed in K5(+) cells have previously been identified as potential stem cell markers. These results suggest that FACS isolation of prostate cells from these mice based on combining reporter gene fluorescence with expression of potential stem cell surface marker proteins will yield populations of cells enriched for stem cells to a degree that has not been attained by using cell surface markers alone.

  20. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  1. [EGFR-expression in pulmonary neuroendocrine cell hyperplasia].

    Science.gov (United States)

    Kuhnen, C; Winter, B U

    2006-03-01

    15 cases of pulmonary neuroendocrine cell hyperplasia (carcinoid-tumorlets, diffuse idiopathic pulmonary neuroendocrine cell hyperplasia/DIPNECH) and 20 neuroendocrine pulmonary tumors (10 carcinoid tumors, 5 large cell neuroendocrine, and 5 small cell neuroendocrine lung carcinomas) were immunohistochemically analyzed for the expression of epidermal growth factor receptor (EGFR, = HER-1). All cases of neuroendocrine cell hyperplasia exhibited a maximum EGFR expression (score 3 in 100% of cells) showing predominantly membranous, partly cytoplasmic staining. 4 ot the 10 carcinoid tumors were strongly positive for EGFR, whereas the other 6 were EGFR-negative. A total of 90% of large cell neuroendocrine and small cell neuroendocrine carcinomas were negative for EGFR. Overexpression of EGFR in pulmonary neuroendocrine cell hyperplasia might be significant for the pathogenesis of these lesions. As DIPNECH is characterized by clinical signs and symptoms including mild cough and obstructive functional impairment, a specific antagonistic therapeutic trial could aim at blocking EGFR/HER-1 or its subsequent signal transduction pathway.

  2. Differentiation of chronic lymphocytic leukemia B cells into immunoglobulin secreting cells decreases LEF-1 expression.

    Directory of Open Access Journals (Sweden)

    Albert Gutierrez

    Full Text Available Lymphocyte enhancer binding factor 1 (LEF-1 plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig secreting cells (ISC using the TLR9 agonist, CpG, together with cytokines (CpG/c. CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation.

  3. [Expression of a human FL eukaryotic expressing plasmid mediated by lipofectamine in HFCL cells].

    Science.gov (United States)

    Ma, L J; Wang, G J; Li, L; Hong, X; Pei, X T

    2001-05-01

    Bone marrow stromal cell line-HFCL were transfected with the recombinant eukaryotic expressing vector-pIRESlneo/hFL by using liposome-mediated gene transfer method and get a stable expression. HFCL cells were transfected with the recombinant eukaryotic expressing vector-pIRESlneo/hFL by using liposome lipofectamine. Integration of hFL in the genome, transcription of hFL mRNA and expression of hFL protein in the transfected HFCL cells were assayed by Southern blot, Northern blot, Western blot and ELISA, the experiment of the human umbilical blood CD34+ cell multiplication. hFL cDNA was integrated into HFCL genome successfully, hFL mRNA was transcripted, hFL protein was expressed with (60.3 +/- 0.1) ng. 10(6) cell(-1) x d(-1) and the experiment of the human umbilical blood CD34+ cell multiplication shows that hFL has obvious biological activity in the supernatant. The recombinant plasmid is proved to be stably expressed in HFCL cells and obvious biological activity of hFL was detectable in the supernatant of the transfected cells.

  4. Engineering cells to improve protein expression.

    Science.gov (United States)

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J

    2014-06-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines.

    Directory of Open Access Journals (Sweden)

    Bisrat G Debeb

    Full Text Available BACKGROUND: The extraembryonic endoderm (ExEn defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines", which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. CONCLUSIONS/SIGNIFICANCE: Our findings (i suggest strongly that the ExEn precursor is a self-renewable entity, (ii indicate that active Oct4 gene expression (transcription plus translation is part of its molecular identity, and (iii provide an in vitro model of early ExEn differentiation.

  6. Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells.

    Science.gov (United States)

    Manokawinchoke, J; Sumrejkanchanakij, P; Subbalekha, K; Pavasant, P; Osathanon, T

    2016-12-01

    Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Matrix metalloproteinase expression in keratocystic odontogenic tumors and primary cells.

    Science.gov (United States)

    Amm, Hope M; Casimir, Monee D; Clark, Dakota B; Sohn, Phillip; MacDougall, Mary

    2014-08-01

    Keratocystic odontogenic tumors (KCOTs) are locally invasive, rapidly proliferating cystic lesions of the jaw. The bone-invasive nature of these tumors has been previously associated with the expression of matrix metalloproteinases (MMPs), which degrade the extracellular matrix. The purpose of this study was to assess the expression and activity of MMPs in primary KCOT cells and tumor tissue. Four independently established KCOT primary cell populations were grown in Dulbecco's modified Eagle medium supplemented with 10% FBS and antibiotics. Primary cells were analyzed by qRT-PCR and immunohistochemistry (IHC), and for secretion of active MMPs. Primary tumor sections were analyzed by IHC. Of the 18 human MMPs examined, 9 were consistently expressed in primary KCOT cells. MMP-2 and MMP-14 were highly expressed in all KCOT populations, while MMP-1, 3, 11, 12, 16, 17, and 19 were moderately expressed. MMP-3, 11, 12, 16, 17 and 19 were shown to be expressed in KCOTs for the first time. No significant differences in MMPS profiles were found between syndromic (KCOT-3) and non-syndromic cell populations (KCOT-1/2/4). Protein expression of MMP-1, 11, 12, 14 and 16 was confirmed in each KCOT cell populations by IHC. KCOT-3 cells secreted active MMP-2 as determined by a gel zymography assay. Expression of MMP-1, 2, 3, 11, 12, 14, and 16 was confirmed in matching primary KCOT tumor sections representing syndromic and non-syndromic KCOTs. KCOT primary cell populations and tumors express a wide range of MMPs, which likely play a role in the bone-invasive nature of these tumors.

  8. Respiratory Syncytial Virus Infection Upregulates NLRC5 and Major Histocompatibility Complex Class I Expression through RIG-I Induction in Airway Epithelial Cells.

    Science.gov (United States)

    Guo, Xuancheng; Liu, Taixiang; Shi, Hengfei; Wang, Jingjing; Ji, Ping; Wang, Hongwei; Hou, Yayi; Tan, Ren Xiang; Li, Erguang

    2015-08-01

    Respiratory syncytial virus (RSV) is the leading cause of acute respiratory tract viral infection in infants, causing bronchiolitis and pneumonia. The host antiviral response to RSV acts via retinoic acid-inducible gene I (RIG-I). We show here that RSV infection upregulates major histocompatibility complex class I (MHC-I) expression through the induction of NLRC5, a NOD-like, CARD domain-containing intracellular protein that has recently been identified as a class I MHC transactivator (CITA). RSV infection of A549 cells promotes upregulation of NLRC5 via beta interferon (IFN-β) production, since the NLRC5-inducing activity in a conditioned medium from RSV-infected A549 cells was removed by antibody to IFN-β, but not by antibody to IFN-γ. RSV infection resulted in RIG-I upregulation and induction of NLRC5 and MHC-I. Suppression of RIG-I induction significantly blocked NLRC5, as well as MHC-I, upregulation and diminished IRF3 activation. Importantly, Vero cells deficient in interferon production still upregulated MHC-I following introduction of the RSV genome by infection or transfection, further supporting a key role for RIG-I. A model is therefore proposed in which the host upregulates MHC-I expression during RSV infection directly via the induction of RIG-I and NLRC5 expression. Since elevated expression of MHC-I molecules can sensitize host cells to T lymphocyte-mediated cytotoxicity or immunopathologic damage, the results have significant implications for the modification of immunity in RSV disease. Human respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants and young children worldwide. Infection early in life is linked to persistent wheezing and allergic asthma in later life, possibly related to upregulation of major histocompatibility class I (MHC-I) on the cell surface, which facilitates cytotoxic T cell activation and antiviral immunity. Here, we show that RSV infection of lung epithelial cells induces

  9. Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells.

    Science.gov (United States)

    Boraas, Liana C; Guidry, Julia B; Pineda, Emma T; Ahsan, Tabassum

    2016-01-01

    Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this

  10. Impact of cell culture process changes on endogenous retrovirus expression.

    Science.gov (United States)

    Brorson, Kurt; De Wit, Christina; Hamilton, Elizabeth; Mustafa, Mehnaz; Swann, Patrick G; Kiss, Robert; Taticek, Ron; Polastri, Gian; Stein, Kathryn E; Xu, Yuan

    2002-11-05

    Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other

  11. Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144 during endothelial differentiation

    Directory of Open Access Journals (Sweden)

    Libermann Towia

    2008-05-01

    Full Text Available Abstract Background Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods Mouse embryonic stem (ES cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that

  12. Expression of heparanase in basal cell carcinoma and squamous cell carcinoma.

    Science.gov (United States)

    Pinhal, Maria Aparecida Silva; Almeida, Maria Carolina Leal; Costa, Alessandra Scorse; Theodoro, Thérèse Rachell; Serrano, Rodrigo Lorenzetti; Machado, Carlos D'Apparecida Santos

    2016-01-01

    Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

  13. Noise in Gene Expression Determines Cell Fate in Bacillus subtilis

    National Research Council Canada - National Science Library

    Hédia Maamar; Arjun Raj; David Dubnau

    2007-01-01

    .... In Bacillus subtilis, noise in ComK, the protein that regulates competence for DNA uptake, is thought to cause cells to transition to the competent state in which genes encoding DNA uptake proteins are expressed...

  14. Intraclonal protein expression heterogeneity in recombinant CHO cells

    National Research Council Canada - National Science Library

    Pilbrough, Warren; Munro, Trent P; Gray, Peter

    2009-01-01

    .... Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers...

  15. Analysis of allelic differential expression in human white blood cells

    National Research Council Canada - National Science Library

    Pant, P V Krishna; Tao, Heng; Beilharz, Erica J; Ballinger, Dennis G; Cox, David R; Frazer, Kelly A

    2006-01-01

    .... To identify human genes with allelic expression differences, we genotype DNA and examine mRNA isolated from the white blood cells of 12 unrelated individuals using oligonucleotide arrays containing 8406 exonic SNPs...

  16. [Construction of Trim6 eukaryotic expression vector and its expression in HEK293 cells].

    Science.gov (United States)

    Sun, Da-Kang; An, Xin-Ye; Hu, Feng-Ai; Li, Cai-Yu; Zheng, Jing

    2011-09-01

    To construct the recombinant eukaryotic expression vector pcDNA3.1 (+)-Trim6, and observe its expression in HEK293T cells in vitro. The total RNA was isolated from HeLa cells. After amplification with reverse transcription polymerase chain reaction (RT-PCR), the target sequences were cloned into the pcDNA3.1(+). The recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing. Then it was transfected into HEK293T cells.After 24 hours, the Trim6 expression was detected by Western blot. The results of the restriction enzyme digestion, PCR and sequencing confirmed the vector was constructed successfully, and it can express Trim6 protein in HEK293T cells. The vector is constructed successfully, which establishes the foundation for future research on the effect of Trim6.

  17. Cardiomyocyte expression and cell-specific processing of procholecystokinin

    DEFF Research Database (Denmark)

    Gøtze, Jens P.; Johnsen, Anders H.; Kistorp, Caroline

    2015-01-01

    Heart muscle cells produce peptide hormones such as natriuretic peptides. Developing hearts also express the gene for the classic intestinal hormone cholecystokinin (CCK) in amounts similar to those in the intestine and brain. However, cardiac expression of peptides other than natriuretic peptide...

  18. Expression of Podoplanin in Different Grades of Oral Squamous Cell ...

    African Journals Online (AJOL)

    Background: The expression of podoplanin is up‑regulated in a number of different human cancers, including squamous cell carcinoma of the oral cavity and its relationship with tumor invasion raises the possibility that podoplanin expression could be used as a biomarker for diagnosis and prognosis. Aim: The aim of the ...

  19. Increased expression of T-helper cell activation markers in ...

    African Journals Online (AJOL)

    Objective: To study the dynamic changes in peripheral blood CD4 cells expressing the activation markers naïve/memory (CD45RA/CD45RO) and interleukin–2 light chain receptor (CD25) in asthmatic children during and after resolution of acute asthma attacks and to determine whether the expression of these activation ...

  20. Differential expressed genes in ECV304 Endothelial-like Cells ...

    African Journals Online (AJOL)

    Background: Human cytomegalovirus (HCMV) is a virus which has the potential to alter cellular gene expression through multiple mechanisms. Objective: With the application of DNA microarrays, we could monitor the effects of pathogens on host-cell gene expression programmes in great depth and on a broad scale.

  1. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstr...

  2. Mechanosensory hair cells express two molecularly distinct mechanotransduction channels.

    Science.gov (United States)

    Wu, Zizhen; Grillet, Nicolas; Zhao, Bo; Cunningham, Christopher; Harkins-Perry, Sarah; Coste, Bertrand; Ranade, Sanjeev; Zebarjadi, Navid; Beurg, Maryline; Fettiplace, Robert; Patapoutian, Ardem; Mueller, Ulrich

    2017-01-01

    Auditory hair cells contain mechanotransduction channels that rapidly open in response to sound-induced vibrations. We report here that auditory hair cells contain two molecularly distinct mechanotransduction channels. One ion channel is activated by sound and is responsible for sensory transduction. This sensory transduction channel is expressed in hair cell stereocilia, and previous studies show that its activity is affected by mutations in the genes encoding the transmembrane proteins TMHS, TMIE, TMC1 and TMC2. We show here that the second ion channel is expressed at the apical surface of hair cells and that it contains the Piezo2 protein. The activity of the Piezo2-dependent channel is controlled by the intracellular Ca(2+) concentration and can be recorded following disruption of the sensory transduction machinery or more generally by disruption of the sensory epithelium. We thus conclude that hair cells express two molecularly and functionally distinct mechanotransduction channels with different subcellular distributions.

  3. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  4. CD34 expression in glioblastoma and giant cell glioblastoma.

    Science.gov (United States)

    Galloway, M

    2010-01-01

    This study aimed to determine whether CD34 is expressed in glioblastomas and giant cell glioblastomas, as this information may be of value when attempting to differentiate between giant cell glioblastomas and other relevant differential diagnoses such as pleomorphic xanthoastrocytomas with anaplastic features and anaplastic gangliogliomas. 11 giant cell glioblastomas and 16 non-giant cell glioblastomas were assessed with immunocytochemical staining for CD34. Standard immunocytochemical techniques were used, to reflect the staining patterns likely to be seen in routine diagnostic practice. Positive staining refers to staining of neoplastic cells. 73% of giant cell glioblastomas showed some degree of staining for CD34, and 55% showed strong widespread staining. 56% of non-giant cell glioblastomas showed some degree of CD34 staining, and 25% showed strong widespread staining. Both giant cell and non-giant cell glioblastomas frequently show CD34 expression by neoplastic cells, which may in some cases be strong and diffuse. Strong widespread staining of neoplastic cells for CD34 was more frequent in giant cell than non-giant cell glioblastomas, however this difference was not statistically significant. CD34 staining in isolation is unlikely to be of assistance in differentiating between giant cell glioblastoma and pleomorphic xanthoastrocytomas with anaplastic features or anaplastic gangliogliomas.

  5. Expression of stanniocalcin 1 in thyroid side population cells and thyroid cancer cells.

    Science.gov (United States)

    Hayase, Suguru; Sasaki, Yoshihito; Matsubara, Tsutomu; Seo, Daekwan; Miyakoshi, Masaaki; Murata, Tsubasa; Ozaki, Takashi; Kakudo, Kennichi; Kumamoto, Kensuke; Ylaya, Kris; Cheng, Sheue-yann; Thorgeirsson, Snorri S; Hewitt, Stephen M; Ward, Jerrold M; Kimura, Shioko

    2015-04-01

    Mouse thyroid side population (SP) cells consist of a minor population of mouse thyroid cells that may have multipotent thyroid stem cell characteristics. However the nature of thyroid SP cells remains elusive, particularly in relation to thyroid cancer. Stanniocalcin (STC) 1 and 2 are secreted glycoproteins known to regulate serum calcium and phosphate homeostasis. In recent years, the relationship of STC1/2 expression to cancer has been described in various tissues. Microarray analysis was carried out to determine genes up- and down-regulated in thyroid SP cells as compared with non-SP cells. Among genes up-regulated, stanniocalcin 1 (STC1) was chosen for study because of its expression in various thyroid cells by Western blotting and immunohistochemistry. Gene expression analysis revealed that genes known to be highly expressed in cancer cells and/or involved in cancer invasion/metastasis were markedly up-regulated in SP cells from both intact as well as partial thyroidectomized thyroids. Among these genes, expression of STC1 was found in five human thyroid carcinoma-derived cell lines as revealed by analysis of mRNA and protein, and its expression was inversely correlated with the differentiation status of the cells. Immunohistochemical analysis demonstrated higher expression of STC1 in the thyroid tumor cell line and thyroid tumor tissues from humans and mice. These results suggest that SP cells contain a population of cells that express genes also highly expressed in cancer cells including Stc1, which warrants further study on the role of SP cells and/or STC1 expression in thyroid cancer.

  6. HIV-1 induces DCIR expression in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Alexandra A Lambert

    2010-11-01

    Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

  7. HIV-1 Induces DCIR Expression in CD4+ T Cells

    Science.gov (United States)

    Lambert, Alexandra A.; Imbeault, Michaël; Gilbert, Caroline; Tremblay, Michel J.

    2010-01-01

    The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4+ T cells found in the synovial tissue from rheumatoid arthritis (RA) patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons) and cells acutely infected in vitro (seen in both virus-infected and uninfected cells). Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals) and -independent intrinsic apoptotic pathways (involving the death effector AIF). Finally, we demonstrate that the higher surface expression of DCIR in CD4+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4+ T cells, a process that might promote virus dissemination throughout the infected organism. PMID:21085612

  8. Selective expression of erg isoforms in human endothelial cells.

    Science.gov (United States)

    Hewett, P W; Nishi, K; Daft, E L; Clifford Murray, J

    2001-04-01

    Erg and Fli-1 are closely related members of the ets family of transcription factors. There are at least five human Erg isoforms (Erg-1, Erg-2, Erg-3/p55(Erg), p49(Erg) and p38(Erg)) produced through differential mRNA splicing and alternative use of translational start codons. However, relatively little is known about the expression or function of these isoforms in vitro or their distribution in vivo. We used RT-PCR to screen a panel of primary and established human cell lines for erg and fli-1 consensus sequences. Whilst fli-1 was expressed in several human cell types, erg was detected mainly in endothelial cells. To identify which erg isoforms are expressed in endothelial cells we used RT-PCR, Northern blotting and 5'-RACE. Erg-3/p55(Erg) and p38(Erg)/p38(Erg)-like transcripts were detected in both microvascular and large vessel endothelial cells affinity-purified from different vascular beds. Moreover, these erg isoforms were present in both freshly isolated, and confluent endothelial cells following several passages in culture, indicating that endothelial erg expression in vitro may be broadly representative of that in vivo. The selective expression of the Erg-3/p55(Erg) and p38(Erg)/p38(Erg)-like isoforms in endothelial cells indicates their involvement in the regulation of endothelial-restricted genes.

  9. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Sidjanin, D. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences; Grdina, D. [Argonne National Lab., IL (United States); Woloschak, G.E. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  10. Gene expression dynamics during cell differentiation: Cell fates as attractors and cell fate decisions as bifurcations

    Science.gov (United States)

    Huang, Sui

    2006-03-01

    During development of multicellular organisms, multipotent stem and progenitor cells undergo a series of hierarchically organized ``somatic speciation'' processes consisting of binary branching events to achieve the diversity of discretely distinct differentiated cell types in the body. Current paradigms of genetic regulation of development do not explain this discreteness, nor the time-irreversibility of differentiation. Each cell contains the same genome with the same N (˜ 25,000) genes and each cell type k is characterized by a distinct stable gene activation pattern, expressed as the cell state vector Sk(t) = xk1(t) ,.. xki(t),.. xkN(t), where xki is the activation state of gene i in cell type k. Because genes are engaged in a network of mutual regulatory interactions, the movement of Sk(t) in the N-dimensional state space is highly constrained and the organism can only realize a tiny fraction of all possible configurations Sk. Then, the trajectories of Sk reflect the diversifying developmental paths and the mature cell types are high-dimensional attractor states. Experimental results based on gene expression profile measurements during blood cell differentiation using DNA microarrays are presented that support the old idea that cell types are attractors. This basic notion is extended to treat binary fate decisions as bifurcations in the dynamics of networks circuits. Specifically, during cell fate decision, the metastable progenitor attractor is destabilized, poising the cell on a `watershed state' so that it can stochastically or in response to deterministic perturbations enter either one of two alternative fates. Overall, the model and supporting experimental data provide an overarching conceptual framework that helps explain how the specifics of gene network architecture produces discreteness and robustness of cell types, allows for both stochastic and deterministic cell fate decision and ensures directionality of organismal development.

  11. The expression of periostin in dental pulp cells.

    Science.gov (United States)

    Wiesen, Robert M; Padial-Molina, Miguel; Volk, Sarah L; McDonald, Neville; Chiego, Daniel; Botero, Tatiana; Rios, Hector F

    2015-05-01

    Dental pulp repair is a common process triggered by microbial and mechanical challenges. Matricellular modulators, such as periostin, are key for extracellular matrix stability and tissue healing. In the scope of the dental pulp, periostin expression has been reported during development and active dentinogenesis. However, the specific dental pulp cell population capable of expressing periostin in response to known regulators has not been clearly defined. Among the different relevant cell populations (i.e., stem cells, fibroblasts and pre-odontoblasts) potentially responsible for periostin expression in the dental pulp, this study aimed to determine which is the primary responder to periostin regulators. Human dental pulp stem cells (DPSCs), human dental pulp fibroblasts (DPFs), and rat odontoblast-like cells (MDPC-23) were treated with different concentrations of TGF-β1 or different regimens of biomechanical stimulation to evaluate periostin expression by qRT-PCR, Western blot and ELISA. Statistical analyses were performed by Student's t-test and ANOVA with Fisher's LSD post hoc tests (p ≤ 0.05). DPSC and MDPC-23 showed a statistically significant increase in periostin mRNA expression after exposure to TGF-β1 for 48 h. TGF-β1 also up-regulated periostin protein levels in DPSC. However, periostin significantly down-regulated protein expression in DPF. Different regimens of biomechanical stimulation showed different patterns in protein and mRNA periostin expression. Expression of periostin was identified in each of the analysed dental pulp cell lines, which can be regulated by TGF-β1 and biomechanical stimulation. Overall, DPSCs are the most responsive cells to stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. [Construction of eucaryotic expression plasmid carrying the BMP7 gene and expression in mesenchymal stem cells].

    Science.gov (United States)

    Hou, Shu-xun; Sun, Da-ming; Du, Gui-xin; Tong, Yi-gang; Fu, Xiao-bing

    2003-06-01

    To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs. The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs. PCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs. Construction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.

  13. Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.

    Science.gov (United States)

    Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan

    2007-10-01

    A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.

  14. Expression of GABAergic receptors in mouse taste receptor cells.

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    Margaret R Starostik

    Full Text Available BACKGROUND: Multiple excitatory neurotransmitters have been identified in the mammalian taste transduction, with few studies focused on inhibitory neurotransmitters. Since the synthetic enzyme glutamate decarboxylase (GAD for gamma-aminobutyric acid (GABA is expressed in a subset of mouse taste cells, we hypothesized that other components of the GABA signaling pathway are likely expressed in this system. GABA signaling is initiated by the activation of either ionotropic receptors (GABA(A and GABA(C or metabotropic receptors (GABA(B while it is terminated by the re-uptake of GABA through transporters (GATs. METHODOLOGY/PRINCIPAL FINDINGS: Using reverse transcriptase-PCR (RT-PCR analysis, we investigated the expression of different GABA signaling molecules in the mouse taste system. Taste receptor cells (TRCs in the circumvallate papillae express multiple subunits of the GABA(A and GABA(B receptors as well as multiple GATs. Immunocytochemical analyses examined the distribution of the GABA machinery in the circumvallate papillae. Both GABA(A-and GABA(B- immunoreactivity were detected in the peripheral taste receptor cells. We also used transgenic mice that express green fluorescent protein (GFP in either the Type II taste cells, which can respond to bitter, sweet or umami taste stimuli, or in the Type III GAD67 expressing taste cells. Thus, we were able to identify that GABAergic receptors are expressed in some Type II and Type III taste cells. Mouse GAT4 labeling was concentrated in the cells surrounding the taste buds with a few positively labeled TRCs at the margins of the taste buds. CONCLUSIONS/SIGNIFICANCE: The presence of GABAergic receptors localized on Type II and Type III taste cells suggests that GABA is likely modulating evoked taste responses in the mouse taste bud.

  15. Melanopsin expressing human retinal ganglion cells

    DEFF Research Database (Denmark)

    Hannibal, Jens; Christensen, Anders Tolstrup; Heegaard, Steffen

    2017-01-01

    microscopy and 3D reconstruction of melanopsin immunoreactive (-ir) RGCs, we applied the criteria used in mouse on human melanopsin-ir RGCs. We identified M1, displaced M1, M2 and M4 cells. We found two other subtypes of melanopsin-ir RGCs, which were named "gigantic M1 (GM1)" and "gigantic displaced M1 (GDM...

  16. Microarray gene expression profiling and analysis in renal cell carcinoma

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    Sadhukhan Provash

    2004-06-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  17. PAX8 expression in ovarian surface epithelial cells.

    Science.gov (United States)

    Adler, Emily; Mhawech-Fauceglia, Paulette; Gayther, Simon A; Lawrenson, Kate

    2015-07-01

    High-grade serous ovarian carcinoma (HGSOC) is usually diagnosed at a late stage and is associated with poor prognosis. Understanding early stage disease biology is essential in developing clinical biomarkers to detect HGSOC earlier. While recent studies indicate that HGSOCs arise from fallopian tube secretory epithelial cells, a considerable body of evidence suggests that HGSOC can also arise from ovarian surface epithelial cells (OSECs). PAX8 is overexpressed in HGSOCs and expressed in fallopian tube secretory epithelial cells, but there are conflicting reports about PAX8 expression in OSECs. The purposes of this study were to comprehensively characterize PAX8 expression in a large series of OSECs and to investigate the role of PAX8 in early HGSOC development. PAX8 protein expression was analyzed in the OSECs of 27 normal ovaries and 7 primary OSEC cultures using immunohistochemistry and immunofluorescent cytochemistry. PAX8 messenger RNA expression was quantified in 66 primary OSEC cultures. Cellular transformation was evaluated in OSECs expressing a PAX8 construct. PAX8 was expressed by 44% to 71% of OSECs. Calretinin and E-cadherin were frequently coexpressed with PAX8. Expression of PAX8 in OSECs decreased cellular migration (P = .028), but had no other effects on cellular transformation. In addition, PAX8 expression was significantly increased (P = .003) in an in vitro stepwise model of neoplastic transformation. In conclusion, PAX8 is frequently expressed by OSECs, and endogenous levels of PAX8 expression are non-transforming. These data indicate that in OSECs, PAX8 expression may represent a normal state and that OSECs may represent an origin of HGSOCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Suppression of MHC gene expression in cancer cells

    NARCIS (Netherlands)

    Bernards, R.A.

    1987-01-01

    The class I antigens of the major histocompatibility complex play a crucial part in the recognition of foreign antigens by cytotoxic T lymphocytes. Some cancer cells exhibit a reduced expression of these antigens and this may help these cells to escape immune surveillance.

  19. Differential expression of tetraspanin superfamily members in dendritic cell subsets

    NARCIS (Netherlands)

    M. Zuidscherwoude (Malou); K. Worah (Kuntal); A. Van Der Schaaf (Alie); S.I. Buschow (Sonja I.); A.B. Spriel (Annemiek )

    2017-01-01

    textabstractDendritic cells (DCs), which are essential for initiating immune responses, are comprised of different subsets. Tetraspanins organize dendritic cell membranes by facilitating protein-protein interactions within the so called tetraspanin web. In this study we analyzed expression of the

  20. Differential expression of tetraspanin superfamily members in dendritic cell subsets

    NARCIS (Netherlands)

    Zuidscherwoude, M.C.; Worah, K.; Schaaf, A. van der; Buschow, S.I.; Spriel, A.B. van

    2017-01-01

    Dendritic cells (DCs), which are essential for initiating immune responses, are comprised of different subsets. Tetraspanins organize dendritic cell membranes by facilitating protein-protein interactions within the so called tetraspanin web. In this study we analyzed expression of the complete

  1. Tff3 is Expressed in Neurons and Microglial Cells

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    Ting Fu

    2014-11-01

    Full Text Available Background/Aims: The trefoil factor family (TFF peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. Methods: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. Results: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. Conclusion: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

  2. Expression of ZNF396 in basal cell carcinoma.

    Science.gov (United States)

    Bai, Juncheng; Kito, Yusuke; Okubo, Hiroshi; Nagayama, Tomoko; Takeuchi, Tamotsu

    2014-05-01

    Zfp191 represses differentiation and keeps various cells in the stem/progenitor stage. Here, we report that a Zfp191 homolog protein, ZNF396, is expressed in basal cell carcinoma (BCC) and possibly represses the expression of a Notch system effector molecule, Hes1 (hairy and enhancer of split-1), and prevents BCC cells from undergoing Notch-mediated squamous cell differentiation. ZNF396 immunoreactivity was found in the nucleus of 35 of 38 cutaneous BCC and 4 of 74 squamous cell carcinoma tissue specimens. In non-tumorous epidermal tissues, ZNF396 immunoreactivity was restricted in basal cells. siRNA-mediated silencing of ZNF396 induced the expression of Notch2, Hes1, and involucrin in cultured BCC cells. Finally, we found that siRNA-mediated silencing of ZNF396 gene inhibited the proliferation of TE354.T basal cell carcinoma cells. ZNF396 might repress Notch-Hes1 signaling axis and prevent tumor cells from undergoing squamous differentiation in BCC.

  3. [Expression of HLA-G protein in trophoblast cells].

    Science.gov (United States)

    Wang, Yan-qiu; Chen, Shi-ling; Xing, Fu-qi

    2005-12-01

    To investigate the expression of human leucocyte antigen protein G (HLA-G) in different trophoblast cells and different stages of pregnancy. The expression of HLA-G protein in normal placenta and trophoblasts of different trimesters was detected using immunohistochemical method (SP). HLA-G protein expression exhibited spatio-temporal changes, which located in the extravillous trophoblast (EVT) and was higher in the placenta of the first and second trimesters while lower in the third trimester (PHLA-G protein expression in different stages of pregnancy and different trophoblasts may be related to the controlled invasion of the trophoblast.

  4. HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell

    Science.gov (United States)

    Boullé, Mikaël; Müller, Thorsten G.; Dähling, Sabrina; Jackson, Laurelle; Mahamed, Deeqa; Oom, Lance; Lustig, Gila

    2016-01-01

    Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses. PMID:27812216

  5. HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

    Science.gov (United States)

    Boullé, Mikaël; Müller, Thorsten G; Dähling, Sabrina; Ganga, Yashica; Jackson, Laurelle; Mahamed, Deeqa; Oom, Lance; Lustig, Gila; Neher, Richard A; Sigal, Alex

    2016-11-01

    Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

  6. Methylene blue modulates adhesion molecule expression on microvascular endothelial cells.

    Science.gov (United States)

    Werner, Isabella; Guo, Fengwei; Stock, Ulrich A; Lupinski, Michèle; Meybohm, Patrick; Moritz, Anton; Beiras-Fernandez, Andres

    2014-08-01

    As methylene blue (MB) has been recently proposed to preserve blood pressure in case of vasoplegic syndrome and shock, an entity directly related to systemic inflammation, we aimed to elucidate the effect of MB on the expression of adhesion-molecules in endothelial-cells. Human microvascular endothelial-cells (HuMEC-1) were treated with 10, 30 or 60 µM MB for 30 min and 2 h each. Additionally, the treated HuMEC-1 were co-cultured with either human peripheral blood mononuclear cells (PBMCs) or Jurkat cells (human T-lymphocytes) for 2 h. HuMEC-1 were analyzed after MB treatment and after co-culture experiments for expression of different adhesion-molecules (ICAM-1, VCAM-1, L-selectin, E-selectin) via FACS measurement and western blot analysis. The supernatants of the experiments were analyzed with regard to the soluble forms of the adhesion molecules. We found that MB is able to modulate the expression of adhesion-molecules on EC. Administration of MB increases the expression of E-selectin and VCAM-1 depending on the dosage and time of exposure. ICAM-1 measurements provide evidence that different circulating blood cells can differently alter the adhesion-molecule expression on EC after MB exposure. Our results provide evidence regarding the immunomodulatory effect of MB upon endothelial-cells after inflammation.

  7. Hypoxia-inhibited dual-specificity phosphatase-2 expression in endometriotic cells regulates cyclooxygenase-2 expression.

    Science.gov (United States)

    Wu, Meng-Hsing; Lin, Shih-Chieh; Hsiao, Kuei-Yang; Tsai, Shaw-Jenq

    2011-11-01

    Endometriosis is one of the most common gynaecological diseases that significantly reduces the life qualify of affected women and their families. Aberrant expression of cyclooxygenase-2 (COX-2), and thus over-production of prostaglandin E(2) (PGE(2) ) has been shown to play critical roles in the development of this disease. However, the mechanism responsible for COX-2 over-expression remains obscure. Here, we provide evidence for what we believe is a novel mechanism in regulating COX-2 expression in endometriotic stromal cells. Dual-specificity phosphatase-2 (DUSP2), a nuclear phosphatase that inactivates mitogen-activated protein kinase (MAPK), is markedly down-regulated in stromal cells of ectopic endometriotic tissues, which results in prolonged activation of extracellular signal-regulated kinase (ERK) and p38 MAPK and increased COX-2 expression. Expression of DUSP2 is inhibited by hypoxia inducible factor-1α (HIF-1α) at the transcriptional level. Treatment of normal endometrial stromal cells with hypoxia, or chemicals that cause HIF-1α accumulation, results in DUSP2 down-regulation, prolonged ERK phosphorylation and COX-2 over-expression. In contrast, forced expression of DUSP2 under hypoxia abolishes HIF-1α-induced ERK phosphorylation and COX-2 expression. Furthermore, suppression of DUSP2 by HIF-1α in eutopic endometrial stromal cells increases sensitivity of cox-2 gene to interleukin-1β stimulation, a phenomenon resembling endometriotic stromal cell characteristics. Taken together, these data suggest that DUSP2 is an important molecule in endometrial physiology and that hypoxia-inhibited DUSP2 expression is a critical factor for the development of endometriosis. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  8. The regulation of CD5 expression in murine T cells

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    Herzenberg Leonard A

    2001-05-01

    Full Text Available Abstract Background CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells.Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression. Results We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA. This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid. We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y and demonstrate the respective roles of the each region in the regulation of CD5 transcription. Conclusion Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.

  9. Expression of parafibromin in major renal cell tumors

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    C. Cui

    2012-10-01

    Full Text Available Parafibromin, encoded by HRPT2 gene, is a recently identified tumor suppressor. Complete and partial loss of its expression have been observed in hyperparathyroidism-jaw tumor (HPT-JT, parathyroid carcinoma, breast carcinoma, lung carcinoma, gastric and colorectal carcinoma. However, little has been known about its expression in renal tumors. In order to study the expression of parafibromin in a series of the 4 major renal cell tumors - clear cell renal cell carcinoma (ccRCC, papillary renal cell carcinoma (pRCC, chromophobe renal cell carcinoma (chRCC and oncocytoma. One hundred thirty nine renal tumors including 61 ccRCCs, 37 pRCCs, 22 chRCCs and 19 oncocytomas were retrieved and used for the construction of renal tissue microarrays (TMAs. The expression of parafibromin was detected by immunohistochemical method on the constructed TMAs. Positive parafibromin stains are seen in 4 out of 61 ccRCCs (7%, 7 out of 37 pRCCs (19%, 12 out of 23 chRCCs (52% and all 19 oncocytomas (100%. Parafibromin expression varies significantly (P< 8.8 x10-16 among the four major renal cell tumors and were correlated closely with tumor types. No correlation of parafibromin expression with tumor staging in ccRCCs, pRCCs and chRCCs, and Fuhrman nuclear grading in ccRCCs and pRCCs. In summary, parafibromin expression was strongly correlated with tumor types, which may suggest that it plays a role in the tumorigenesis in renal cell tumors.

  10. Differential ectonucleotidase expression in human bladder cancer cell lines.

    Science.gov (United States)

    Stella, Joséli; Bavaresco, Luci; Braganhol, Elizandra; Rockenbach, Liliana; Farias, Patrícia Fernandes; Wink, Márcia R; Azambuja, Alan A; Barrios, Carlos Henrique; Morrone, Fernanda Bueno; Oliveira Battastini, Ana Maria

    2010-01-01

    Bladder cancer is the most prevalent tumor in the genitourinary tract. Nucleotides are important molecules that regulate many pathophysiological functions in the extracellular space. Studies have revealed evidence of a relationship between purinergic signaling and urothelial malignancies. Nucleotide-mediated signaling is controlled by a highly efficient enzymatic cascade, which includes the members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDases), ectonucleotide pyrophosphatase/phosphodiesterase (E-NPPs), ecto-alkaline phosphatases, and ecto-5'-nucleotidase/CD73. In an attempt to identify possible differential expression of ectonucleotidases during bladder cancer progression, a comparative analysis between RT4 (grade 1) and T24 (grade 3) bladder cancer cell lines was performed. In RT4 cells, the hydrolysis of tri- and diphosphate nucleosides was higher than monophosphonucleosides. T24 cells, however, presented the opposite profile, a low level of hydrolysis of tri- and diphosphate nucleosides and a high level of hydrolysis of monophosphates. Phosphodiesterase activity was negligible in both cell lines at physiological pH, indicating that these enzymes are not active under our assay conditions, although they are expressed in both cell lines. The T24 cells expressed NTPDase5 mRNA, while the RT4 cells expressed NTPDase3 and NTPDase5 mRNA. Both cell lines expressed ecto-5'-nucleotidase/CD73 mRNA. The present work describes, for the first time, the differential pattern of ectonucleotidases in the more malignant bladder cancer cells compared with cells derived from an early stage of bladder cancer. Our results open new avenues for research into the physiological roles of this family of enzymes and their possible therapeutic potential in bladder cancer. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  11. Characterization, isolation, and differentiation of murine skin cells expressing hematopoietic stem cell markers.

    Science.gov (United States)

    Meindl, Simone; Schmidt, Uwe; Vaculik, Christine; Elbe-Bürger, Adelheid

    2006-10-01

    As the phenotype of adult dermal stem cells is still elusive, and the hematopoietic stem cell is one of the best-characterized stem cells in the body, we tested dermal cell suspensions, sections, and wholemounts in newborn and adult mice for hematopoietic stem cell marker expression. Phenotypic analysis revealed that a small population of CD45(+) cells and a large population of CD45(-) cells expressed CD34, CD117, and stem cell antigen-1 molecules. When cultivated in selected media supplemented with hematopoietic cytokines, total dermal cells, lineage(-), and/or highly enriched phenotypically defined cell subsets produced hematopoietic and nonhematopoietic colonies. When injected into lethally irradiated recipient mice, a small percentage of newborn dermal cells was able to migrate into hematopoietic tissues and the skin and survived through the 11-month monitoring period. Our ability to isolate a candidate autologous stem cell pool will make these cells ideal vehicles for genetic manipulation and gene therapy.

  12. Differential expression of tetraspanin superfamily members in dendritic cell subsets.

    Science.gov (United States)

    Zuidscherwoude, Malou; Worah, Kuntal; van der Schaaf, Alie; Buschow, Sonja I; van Spriel, Annemiek B

    2017-01-01

    Dendritic cells (DCs), which are essential for initiating immune responses, are comprised of different subsets. Tetraspanins organize dendritic cell membranes by facilitating protein-protein interactions within the so called tetraspanin web. In this study we analyzed expression of the complete tetraspanin superfamily in primary murine (CD4+, CD8+, pDC) and human DC subsets (CD1c+, CD141+, pDC) at the transcriptome and proteome level. Different RNA and protein expression profiles for the tetraspanin genes across human and murine DC subsets were identified. Although RNA expression levels of CD37 and CD82 were not significantly different between human DC subsets, CD9 RNA was highly expressed in pDCs, while CD9 protein expression was lower. This indicates that relative RNA and protein expression levels are not always in agreement. Both murine CD8α+ DCs and its regarded human counterpart, CD141+ DCs, displayed relatively high protein levels of CD81. CD53 protein was highly expressed on human pDCs in contrast to the relatively low protein expression of most other tetraspanins. This study demonstrates that tetraspanins are differentially expressed by human and murine DC subsets which provides a valuable resource that will aid the understanding of tetraspanin function in DC biology.

  13. Differential expression of tetraspanin superfamily members in dendritic cell subsets.

    Directory of Open Access Journals (Sweden)

    Malou Zuidscherwoude

    Full Text Available Dendritic cells (DCs, which are essential for initiating immune responses, are comprised of different subsets. Tetraspanins organize dendritic cell membranes by facilitating protein-protein interactions within the so called tetraspanin web. In this study we analyzed expression of the complete tetraspanin superfamily in primary murine (CD4+, CD8+, pDC and human DC subsets (CD1c+, CD141+, pDC at the transcriptome and proteome level. Different RNA and protein expression profiles for the tetraspanin genes across human and murine DC subsets were identified. Although RNA expression levels of CD37 and CD82 were not significantly different between human DC subsets, CD9 RNA was highly expressed in pDCs, while CD9 protein expression was lower. This indicates that relative RNA and protein expression levels are not always in agreement. Both murine CD8α+ DCs and its regarded human counterpart, CD141+ DCs, displayed relatively high protein levels of CD81. CD53 protein was highly expressed on human pDCs in contrast to the relatively low protein expression of most other tetraspanins. This study demonstrates that tetraspanins are differentially expressed by human and murine DC subsets which provides a valuable resource that will aid the understanding of tetraspanin function in DC biology.

  14. Gene expression profiling of granule cells and Purkinje cells in the zebrafish cerebellum.

    Science.gov (United States)

    Takeuchi, Miki; Yamaguchi, Shingo; Sakakibara, Yoshimasa; Hayashi, Takuto; Matsuda, Koji; Hara, Yuichiro; Tanegashima, Chiharu; Shimizu, Takashi; Kuraku, Shigehiro; Hibi, Masahiko

    2017-05-01

    The structure of the neural circuitry of the cerebellum, which functions in some types of motor learning and coordination, is generally conserved among vertebrates. However, some cerebellar features are species specific. It is not clear which genes are involved in forming these conserved and species-specific structures and functions. This study uses zebrafish transgenic larvae expressing fluorescent proteins in granule cells, Purkinje cells, or other cerebellar neurons and glial cells to isolate each type of cerebellar cells by fluorescence-activated cell sorting and to profile their gene expressions by RNA sequencing and in situ hybridization. We identify genes that are upregulated in granule cells or Purkinje cells, including many genes that are also expressed in mammalian cerebella. Comparison of the transcriptomes in granule cells and Purkinje cells in zebrafish larvae reveals that more developmental genes are expressed in granule cells, whereas more neuronal-function genes are expressed in Purkinje cells. We show that some genes that are upregulated in granule cells or Purkinje cells are also expressed in the cerebellum-like structures. Our data provide a platform for understanding the development and function of the cerebellar neural circuits in zebrafish and the evolution of cerebellar circuits in vertebrates. J. Comp. Neurol. 525:1558-1585, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Can dead bacterial cells be defined and are genes expressed after cell death?

    Science.gov (United States)

    Trevors, J T

    2012-07-01

    There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. TLR7-expressing cells comprise an interfollicular epidermal stem cell population in murine epidermis.

    Science.gov (United States)

    Yin, Chaoran; Zhang, Ting; Qiao, Liangjun; Du, Jia; Li, Shuang; Zhao, Hengguang; Wang, Fangfang; Huang, Qiaorong; Meng, Wentong; Zhu, Hongyan; Bu, Hong; Li, Hui; Xu, Hong; Mo, Xianming

    2014-07-25

    Normal interfollicular epidermis (IFE) homeostasis is maintained throughout the entire life by its own stem cells that self-renew and generate progeny that undergo terminal differentiation. However, the fine markers of the stem cells in interfollicular epidermis are not well defined yet. Here we found that TLR7 identified the existence of progenitors and interfollicular epidermal stem cells in murine skin. In vitro, TLR7-expressing cells comprised of two subpopulations that were competent to proliferate and exhibited distinct differentiation potentials. Three-dimensional (3D) organotypic culture and skin reconstitution assays showed that TLR7-expressing cells were able to reconstruct the interfollicular epidermis. Finally, TLR7-expressing cells maintained the intact interfollicular epidermal structures revealed in serial transplantation assays in vivo in mice. Taken together, our results suggest that TLR7-expressing cells comprise an interfollicular epidermal stem cell population.

  17. High epitope expression levels increase competition between T cells.

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    Almut Scherer

    2006-08-01

    Full Text Available Both theoretical predictions and experimental findings suggest that T cell populations can compete with each other. There is some debate on whether T cells compete for aspecific stimuli, such as access to the surface on antigen-presenting cells (APCs or for specific stimuli, such as their cognate epitope ligand. We have developed an individual-based computer simulation model to study T cell competition. Our model shows that the expression level of foreign epitopes per APC determines whether T cell competition is mainly for specific or aspecific stimuli. Under low epitope expression, competition is mainly for the specific epitope stimuli, and, hence, different epitope-specific T cell populations coexist readily. However, if epitope expression levels are high, aspecific competition becomes more important. Such between-specificity competition can lead to competitive exclusion between different epitope-specific T cell populations. Our model allows us to delineate the circumstances that facilitate coexistence of T cells of different epitope specificity. Understanding mechanisms of T cell coexistence has important practical implications for immune therapies that require a broad immune response.

  18. A Novel Protein Is Lower Expressed in Renal Cell Carcinoma

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    Ruili Guan

    2014-04-01

    Full Text Available Engrailed-2 (EN2 has been identified as a candidate oncogene in breast cancer and prostate cancer. It is usually recognized as a mainly nuclear staining in the cells. However, recent studies showed a cytoplasmic staining occurred in prostate cancer, bladder cancer and clear cell renal cell carcinoma. The inconsistency makes us confused. To clarify the localization and expression of EN2 in renal cell carcinoma, anti-EN2 antibody (ab28731 and anti-EN2 antibody (MAB2600 were used for immunohistochemistry (IHC respectively. Interestingly, we found that EN2 detected by ab28731 was mainly presented in cytoplasm while EN2 detected by MAB2600 was mainly presented in nucleus. To further investigate the different patterns observed above, lysates from full-length EN2 over expression in HEK293T cells were used to identify which antibody the EN2 molecule bound by western blot. Results showed ab28731 did not react with the lysates. For this reason, the novel specific protein detected by ab28731 was not the EN2 molecule and was named nonEN2. Then using the renal carcinoma tissue microarray and renal tissues, we found that the protein expression levels of nonEN2 in kidney tumor tissues was significantly lower than that in kidney normal tissues (p < 0.05, so was in renal cell lines. Taken together, nonEN2 is lower expressed and may play an important role in renal cell carcinoma.

  19. Foxd3 suppresses interleukin-10 expression in B cells.

    Science.gov (United States)

    Zhang, Yu; Wang, Zhiding; Xiao, He; Liu, Xiaoling; Zhu, Gaizhi; Yu, Dandan; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Ma, Ning; Shen, Beifen; Li, Yan; Wang, Tianxiao; Wang, Renxi

    2017-04-01

    Interleukin-10-positive (IL-10 + ) regulatory B (Breg) cells play an important role in restraining excessive inflammatory responses by secreting IL-10. However, it is still unclear what key transcription factors determine Breg cell differentiation. Hence, we explore what transcription factor plays a key role in the expression of IL-10, a pivotal cytokine in Breg cells. We used two types of web-based prediction software to predict transcription factors binding the IL-10 promoter and found that IL-10 promoter had many binding sites for Foxd3. Chromatin immunoprecipitation PCR assay demonstrated that Foxd3 directly binds the predicted binding sites around the start codon upstream by -1400 bp. Further, we found that Foxd3 suppressed the activation of IL-10 promoter by using an IL-10 promoter report system. Finally, knocking out Foxd3 effectively promotes Breg cell production by up-regulating IL-10 expression. Conversely, up-regulated Foxd3 expression was negatively associated with IL-10 + Breg cells in lupus-prone MRL/lpr mice. Hence, our data suggest that Foxd3 suppresses the production of IL-10 + Breg cells by directly binding the IL-10 promoter. This study demonstrates the mechanism for Breg cell production and its application to the treatment of autoimmune diseases by regulating Foxd3 expression. © 2016 John Wiley & Sons Ltd.

  20. Expression of functional recombinant antibody molecules in insect cell expression systems.

    Science.gov (United States)

    Reavy, B; Ziegler, A; Diplexcito, J; Macintosh, S M; Torrance, L; Mayo, M

    2000-03-01

    Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain. Copyright 2000 Academic Press.

  1. Regulation of stem cell factor expression in inflammation and asthma

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    Carla A Da Silva

    2005-03-01

    Full Text Available Stem cell factor (SCF is a major mast cell growth factor, which could be involved in the local increase of mast cell number in the asthmatic airways. In vivo, SCF expression increases in asthmatic patients and this is reversed after treatment with glucocorticoids. In vitro in human lung fibroblasts in culture, IL-1beta, a pro-inflammatory cytokine, confirms this increased SCF mRNA and protein expression implying the MAP kinases p38 and ERK1/2 very early post-treatment, and glucocorticoids confirm this decrease. Surprisingly, glucocorticoids potentiate the IL-1beta-enhanced SCF expression at short term treatment, implying increased SCF mRNA stability and SCF gene transcription rate. This potentiation involves p38 and ERK1/2. Transfection experiments with the SCF promoter including intron1 also confirm this increase and decrease of SCF expression by IL-1beta and glucocorticoids, and the potentiation by glucocorticoids of the IL-1beta-induced SCF expression. Deletion of the GRE or kappaB sites abolishes this potentiation, and the effect of IL-1beta or glucocorticoids alone. DNA binding of GR and NF-kappaB are also demonstrated for these effects. In conclusion, this review concerns new mechanisms of regulation of SCF expression in inflammation that could lead to potential therapeutic strategy allowing to control mast cell number in the asthmatic airways.

  2. Cd1d is expressed on dermal dendritic cells and monocyte-derived dendritic cells.

    Science.gov (United States)

    Gerlini, G; Hefti, H P; Kleinhans, M; Nickoloff, B J; Burg, G; Nestle, F O

    2001-09-01

    CD1 proteins are a family of cell surface molecules that present lipid antigens to T cells. We investigated skin dendritic cells and monocyte-derived dendritic cells for expression of CD1 molecules using a panel of 10 different monoclonal antibodies focusing on the recently described CD1d molecule. By immunohistochemical analysis, CD1d expression in normal human skin was restricted to dendritic appearing cells in the papillary dermis mainly located in a perivascular localization. Langerhans cells did not show detectable CD1d expression in situ. Epidermal/dermal cell suspensions analyzed by flow cytometry demonstrated distinct subpopulations of HLA-DR positive dermal dendritic cells expressing CD1a, CD1b, and CD1c. CD1d was expressed on HLA-DRbright dermal antigen-presenting cells in dermal suspensions (16% +/- 3.6%), as well as on highly enriched dermal dendritic cells migrating out of skin explants (60.5% +/- 8.0%). Migrated mature dermal dendritic cells coexpressed CD83 and CD1d. Western blot analysis on microdissected skin sections revealed the presence of a 50-55 kDa CD1d molecule in dermis, suggesting that CD1d is highly glycosylated in skin. Both immature and mature monocyte-derived dendritic cells cultured in autologous plasma expressed CD1d molecules. In contrast, culture in fetal bovine serum downregulated CD1d expression. In conclusion, antigen-presenting cells in skin express different sets of CD1 molecules including CD1d and might play a role in lipid antigen presentation in various skin diseases. Differential expression of CD1 molecules depending on culture conditions might have an impact on clinical applications of dendritic cells for immunotherapy.

  3. NFATc1 regulation of TRAIL expression in human intestinal cells.

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    Qingding Wang

    Full Text Available TNF-related apoptosis-inducing ligand (TRAIL; Apo2 has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA plus the calcium ionophore A23187 (Io increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA, an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.

  4. Single-Cell Expression Profiling and Proteomics of Primordial Germ Cells, Spermatogonial Stem Cells, Adult Germ Stem Cells, and Oocytes.

    Science.gov (United States)

    Conrad, Sabine; Azizi, Hossein; Skutella, Thomas

    2018-01-04

    The mammalian germ cells, cell assemblies, tissues, and organs during development and maturation have been extensively studied at the tissue level. However, to investigate and understand the fundamental insights at the molecular basis of germ and stem cells, their cell fate plasticity, and determination, it is of most importance to analyze at the large scale on the single-cell level through different biological windows. Here, modern molecular techniques optimized for single-cell analysis, including single fluorescence-activated cell sorting (FACS) and single-cell RNA sequencing (scRNA-seq) or microfluidic high-throughput quantitative real-time polymerase chain reaction (qRT-PCR) for single-cell gene expression and liquid chromatography coupled to tandem mass spectrometry (LC-MSMS) for protein profiling, have been established and are still getting optimized.This review aims on describing and discussing recent single-cell expression profiling and proteomics of different types of human germ cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), human adult germ stem cells (haGSCs), and oocytes.

  5. Tim-3 expression defines regulatory T cells in human tumors.

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    Jing Yan

    Full Text Available Tim-3, a member of the novel Tim (T cell immunoglobulin and mucin domain family, has been reported to negatively regulate the immune responses against viral infection and had implications for autoimmune disease. However, the nature and role of Tim-3(+ CD4 T cells in human tumors remain largely unknown. In the present study, we characterized Tim-3(+ CD4 T cells in 100 specimens from human hepatocellular, cervical, colorectal and ovarian carcinoma patients. Compared with peripheral blood and nontumor-infiltrating lymphocytes, the lymphocytes isolated from the corresponding tumor tissues of hepatocellular, cervical, colorectal and ovarian carcinoma patients contained significantly greater proportion of Tim-3(+ CD4 T cells. The majority of tumor-derived Tim-3(+ CD4 T cells exhibited an impaired capacity to produce IFN-γ and IL-2, but expressed higher levels of CD25, Foxp3, CTLA-4 and GITR than their Tim-3(- CD4 T cell counterparts. In contrast, most Tim-3(+ CD4 T cells isolated from the paired nontumor tissues and peripheral blood did not express these molecules. Moreover, tumor-derived Tim-3(+ CD4 T cells, but not tumor-derived Tim-3(- CD4 T cells, significantly suppressed the proliferation of autologous CD8(+ T cells in vitro. Notably, multi-color immunofluorescence and confocal microscopy demonstrated that Tim-3(+Foxp3(+CD4(+ cells were preferentially distributed in the tumor nest rather than the peritumoral stroma of hepatocellular carcinoma. Together, our data indicate that Tim-3-expressing CD4 T cells in human tumors could represent the functional regulatory T cells which contribute to the formation of the immune-suppressive tumor micromilieu.

  6. Migrating glioma cells express stem cell markers and give rise to new tumors upon xenografting

    DEFF Research Database (Denmark)

    Munthe, Sune; Sørensen, Mia D; Thomassen, Mads

    2016-01-01

    -related genes and the HOX-gene list in migrating cells compared to spheroids. Determination of GBM molecular subtypes revealed that subtypes of spheroids and migrating cells were identical. In conclusion, migrating tumor cells preserve expression of stem cell markers and functional CSC characteristics. Since...

  7. Pax4 Expression does not Transduce Pancreatic Alpha Cells to Beta Cells

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    Ling Chen

    2015-07-01

    Full Text Available Background/Aims: The lack of available beta cells greatly limits the use of beta cell transplantation as a therapy for diabetes. Thus, generation of beta cells from other sources is substantially required. Pax4 has been shown to induce reprograming of alpha cells into beta cells during embryogenesis. Nevertheless, whether expression of Pax4 in adult alpha cells could trigger this alpha-to-beta cell reprogramming is unknown. Methods: Here we generated an adeno-associated virus carrying Pax4 and GFP under a CMV promoter (AAV-Pax4. We used AAV-Pax4 to transduce a mouse alpha cell line in vitro, and to transduce primary alpha cells in diabetic mice. Reprogramming was examined by double immunostaining and by changes in beta cell number. The effects on blood glucose were evaluated by fasting blood glucose and glucose response. Results: In vitro, Pax4 overexpression neither induced insulin expression, nor suppressed glucagon expression in alpha cells. In vivo, Pax4 overexpression failed to increase beta cell number, and did not alter hyperglycemia and glucose response in diabetic mice. Conclusion: Pax4 expression is not sufficient to transduce pancreatic alpha cells into beta cells. Overexpression of Pax4 in alpha cells may not increase functional beta cell number in diabetic patients.

  8. Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

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    Dilyana Todorova

    Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

  9. Immunohistochemical expression of MYB in salivary gland basal cell adenocarcinoma and basal cell adenoma.

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    Rooney, Sydney L; Robinson, Robert A

    2017-10-01

    Basal cell predominant salivary gland neoplasms can be difficult to separate histologically. One of the most aggressive of basaloid salivary gland neoplasms is adenoid cystic carcinoma. MYB expression by immunohistochemistry has been documented in adenoid cystic carcinoma. Some investigators have suggested that using this expression can help in establishing the diagnosis of adenoid cystic carcinoma. Utilizing tissue microarrays, we studied a group of basal cell adenocarcinomas and basal cell adenomas to determine: (i) whether either tumor expressed MYB and (ii) the frequency of any expression in either tumors. Seventeen salivary gland basal cell adenocarcinomas and 30 salivary gland basal cell adenomas were used to construct microarrays. These tissue microarrays were used to assess for immunohistochemical MYB expression. Fifty-three percent (nine of 17) of salivary gland basal cell adenocarcinomas and 57% (17 of 30) of salivary gland basal cell adenomas showed MYB overexpression. For comparison, we studied 11 adenoid cystic carcinomas for MYB expression and found that 64% (seven of 11) overexpressed MYB. We found no relation to clinical course for basal adenomas or basal cell adenocarcinomas that overexpressed MYB vs those that did not. MYB expression does not help separate basal cell adenocarcinomas from basal cell adenomas, and our data suggest it does not differentiate between either of these neoplasms and adenoid cystic carcinoma. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. The effect of the colostral cells on gene expression of cytokines in cord blood cells.

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    Hrdý, Jiří; Novotná, Olga; Kocourková, Ingrid; Prokešová, Ludmila

    2017-11-01

    Beneficial effect of maternal milk is acknowledged, but there is still question whether maternal milk from allergic mother is as good as from healthy one. In our study, we have assayed the effect of cells from colostrum of healthy and allergic mothers on gene expression of cytokines in cord blood cells of newborns of healthy and allergic mothers. Cytokines typical for Th1 (IL-2, IFN-gamma), Th2 (IL-4, IL-13), Tregs (IL-10, TGF-beta), and IL-8 were followed. We were not able to detect significant influence of colostral cells on gene expression of cytokines in cord blood after 2-day coculture using Transwell system. There was no difference in gene expression of cytokines in nonstimulated cord blood cells of newborns of healthy and allergic mothers, but generally increased gene expression of cytokines except IL-10 and TGF-beta after polyclonal stimulation was detected in cord blood cells of children of allergic mothers. There was no difference in IL-10 expression in stimulated cord blood cells of children of healthy and allergic mothers. Gene expression of TGF-beta was even decreased in stimulated cord blood cells of children of allergic mothers in comparison to healthy ones. We have not observed difference in the capacity of colostral cells of healthy and allergic mothers to influence gene expression of cytokines in cord blood cells, but we have described difference in the reactivity of cord blood cells between children of allergic and healthy mothers.

  11. A functional profile of gene expression in ARPE-19 cells

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    Johnson Dianna A

    2005-11-01

    Full Text Available Abstract Background Retinal pigment epithelium cells play an important role in the pathogenesis of age related macular degeneration. Their morphological, molecular and functional phenotype changes in response to various stresses. Functional profiling of genes can provide useful information about the physiological state of cells and how this state changes in response to disease or treatment. In this study, we have constructed a functional profile of the genes expressed by the ARPE-19 cell line of retinal pigment epithelium. Methods Using Affymetrix MAS 5.0 microarray analysis, genes expressed by ARPE-19 cells were identified. Using GeneChip® annotations, these genes were classified according to their known functions to generate a functional gene expression profile. Results We have determined that of approximately 19,044 unique gene sequences represented on the HG-U133A GeneChip® , 6,438 were expressed in ARPE-19 cells irrespective of the substrate on which they were grown (plastic, fibronectin, collagen, or Matrigel. Rather than focus our subsequent analysis on the identity or level of expression of each individual gene in this large data set, we examined the number of genes expressed within 130 functional categories. These categories were selected from a library of HG-U133A GeneChip® annotations linked to the Affymetrix MAS 5.0 data sets. Using this functional classification scheme, we were able to categorize about 70% of the expressed genes and condense the original data set of over 6,000 data points into a format with 130 data points. The resulting ARPE-19 Functional Gene Expression Profile is displayed as a percentage of ARPE-19-expressed genes. Conclusion The Profile can readily be compared with equivalent microarray data from other appropriate samples in order to highlight cell-specific attributes or treatment-induced changes in gene expression. The usefulness of these analyses is based on the assumption that the numbers of genes

  12. Stem cell factor expression in B cell malignancies is influenced by the niche.

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    Fox, Matthew F; Pontier, Andrea; Gurbuxani, Sandeep; Sipkins, Dorothy A

    2013-10-01

    Our group has previously demonstrated that expression of the cytokine stem cell factor (SCF) by leukemic blasts is a frequent finding in pre-B acute lymphoblastic leukemia (ALL). Whether SCF expression is a feature of other B cell malignancies and whether cross-talk from the local microenvironment modulates malignant cell SCF production are, however, unknown. Here we show using immunohistochemistry that SCF is expressed by a wide variety of indolent and aggressive B cell malignancies involving the bone marrow (BM) or lymph nodes (LNs). In diseases such as follicular lymphoma (FL), however, where lymphoma cells uniquely associate with the BM endosteal niche, BM lymphoma does not express SCF, while LN involvement is SCF positive. In contrast, cases of FL with high-grade transformation in the BM are SCF positive. These data suggest that lymphoma cell interaction with the endosteal niche inhibits SCF production, and that FL cells become independent of this microenvironment effect following transformation.

  13. NOS2 expression in glioma cell lines and glioma primary cell cultures: correlation with neurosphere generation and SOX-2 expression.

    Science.gov (United States)

    Palumbo, Paola; Miconi, Gianfranca; Cinque, Benedetta; Lombardi, Francesca; La Torre, Cristina; Dehcordi, Soheila Raysi; Galzio, Renato; Cimini, Annamaria; Giordano, Antonio; Cifone, Maria Grazia

    2017-04-11

    Nitric oxide has been implicated in biology and progression of glioblastoma (GBM) being able to influence the cellular signal depending on the concentration and duration of cell exposure. NOS2 (inducible nitric oxide synthase) have been proposed as a component of molecular profile of several tumors, including glioma, one of the most aggressive primary brain tumor featuring local cancer stem cells responsible for enhanced resistance to therapies and for tumor recurrence. Here, we investigated the NOS2 mRNA expression by reverse transcription-PCR in human glioma primary cultures at several grade of malignancy and glioma stem cell (GSC) derived neurospheres. Glioma cell lines were used as positive controls both in terms of stemness marker expression that of capacity of generating neurospheres. NOS2 expression was detected at basal levels in cell lines and primary cultures and appeared significantly up-regulated in cultures kept in the specific medium for neurospheres. The immunofluorescence analysis of all cell cultures to evaluate the levels of SOX-2, a stemness marker aberrantly up-regulated in GBM, was also performed. The potential correlation between NOS2 expression and ability to generate neurospheres and between NOS2 and SOX-2 levels was also verified. The results show that the higher NOS2 expression is detected in all primary cultures able to arise neurosphere. A high and significant correlation between NOS2 expression and SOX-2 positive cells (%) in all cell cultures maintained in standard conditions has been observed. The results shed light on the potential relevance of NOS2 as a prognostic factor for glioma malignancy and recurrence.

  14. Patterns of expression of cell wall related genes in sugarcane

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    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  15. Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas

    Science.gov (United States)

    Azuma, Sakiko; Yokoyama, Yukihiro; Yamamoto, Akiko; Kyokane, Kazuhiro; Niida, Shumpei; Ishiguro, Hiroshi; Ko, Minoru S. H.

    2013-01-01

    We have recently identified the zinc finger and SCAN domain containing 4 (Zscan4), which is transiently expressed and regulates telomere elongation and genome stability in mouse embryonic stem (ES) cells. The aim of this study was to examine the expression of ZSCAN4 in the adult pancreas and elucidate the role of ZSCAN4 in tissue inflammation and subsequent regeneration. The expression of ZSCAN4 and other progenitor or differentiated cell markers in the human pancreas was immunohistochemically examined. Pancreas sections of alcoholic or autoimmune pancreatitis patients before and under maintenance corticosteroid treatment were used in this study. In the adult human pancreas a small number of ZSCAN4-positive (ZSCAN4+) cells are present among cells located in the islets of Langerhans, acini, ducts, and oval-shaped cells. These cells not only express differentiated cell markers for each compartment of the pancreas but also express other tissue stem/progenitor cell markers. Furthermore, the number of ZSCAN4+ cells dramatically increased in patients with chronic pancreatitis, especially in the pancreatic tissues of autoimmune pancreatitis actively regenerating under corticosteroid treatment. Interestingly, a number of ZSCAN4+ cells in the pancreas of autoimmune pancreatitis returned to the basal level after 1 yr of maintenance corticosteroid treatment. In conclusion, coexpression of progenitor cell markers and differentiated cell markers with ZSCAN4 in each compartment of the pancreas may indicate the presence of facultative progenitors for both exocrine and endocrine cells in the adult pancreas. PMID:23599043

  16. Connexin expression and gap-junctional intercellular communication in ES cells and iPS cells.

    Science.gov (United States)

    Oyamada, Masahito; Takebe, Kumiko; Endo, Aya; Hara, Sachiko; Oyamada, Yumiko

    2013-01-01

    Pluripotent stem cells, i.e., embryonic stem (ES) and induced pluripotent stem (iPS) cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC) during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming.

  17. Protein Expression Analyses at the Single Cell Level

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    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  18. Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Vindeløv, L L; Spang-Thomsen, M

    1993-01-01

    A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse....

  19. Regulation of cell-to-cell variability in divergent gene expression.

    Science.gov (United States)

    Yan, Chao; Wu, Shuyang; Pocetti, Christopher; Bai, Lu

    2016-03-24

    Cell-to-cell variability (noise) is an important feature of gene expression that impacts cell fitness and development. The regulatory mechanism of this variability is not fully understood. Here we investigate the effect on gene expression noise in divergent gene pairs (DGPs). We generated reporters driven by divergent promoters, rearranged their gene order, and probed their expressions using time-lapse fluorescence microscopy and single-molecule fluorescence in situ hybridization (smFISH). We show that two genes in a co-regulated DGP have higher expression covariance compared with the separate, tandem and convergent configurations, and this higher covariance is caused by more synchronized firing of the divergent transcriptions. For differentially regulated DGPs, the regulatory signal of one gene can stochastically 'leak' to the other, causing increased gene expression noise. We propose that the DGPs' function in limiting or promoting gene expression noise may enhance or compromise cell fitness, providing an explanation for the conservation pattern of DGPs.

  20. Stable expression of Anthozoa fluorescent proteins in mammalian cells.

    Science.gov (United States)

    Richards, Burt; Zharkikh, Ludmilla; Hsu, Forrest; Dunn, Christine; Kamb, Alexander; Teng, David H-F

    2002-06-01

    Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP. The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection. Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader. We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters

  1. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures.

    Science.gov (United States)

    Kisselbach, Lynn; Merges, Michael; Bossie, Alexis; Boyd, Ann

    2009-01-01

    Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.

  2. Male germ cells express abundant endogenous siRNAs

    Science.gov (United States)

    Song, Rui; Hennig, Grant W.; Wu, Qiuxia; Jose, Charlie; Zheng, Huili; Yan, Wei

    2011-01-01

    In mammals, endogenous siRNAs (endo-siRNAs) have only been reported in murine oocytes and embryonic stem cells. Here, we show that murine spermatogenic cells express numerous endo-siRNAs, which are likely to be derived from naturally occurring double-stranded RNA (dsRNA) precursors. The biogenesis of these testicular endo-siRNAs is DROSHA independent, but DICER dependent. These male germ cell endo-siRNAs can potentially target hundreds of transcripts or thousands of DNA regions in the genome. Overall, our work has unveiled another hidden layer of regulation imposed by small noncoding RNAs during male germ cell development. PMID:21788498

  3. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob

    2003-01-01

    Studies have suggested that in ulcerative colitis (UC), intercellular adhesion molecule-1 (ICAM-1) is involved in migration of leukocytes toward the colonic epithelium. A suitable in vitro model of chronic colonic inflammation does not exist, and the role of the epithelium is based on monolayers...... of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium....

  4. Modulation of GLO1 Expression Affects Malignant Properties of Cells

    Directory of Open Access Journals (Sweden)

    Antje Hutschenreuther

    2016-12-01

    Full Text Available The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO. Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1 that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

  5. Amiodarone inhibits tissue factor expression in monocytic THP-1 cells.

    Science.gov (United States)

    Yamamoto, Yumiko; Morita, Toshihiro; Tanaka, Tomofumi; Ikeda, Kenichi; Kikuchi, Hironobu; Oguri, Gaku; Nakamura, Fumitaka; Nakajima, Toshiaki; Nagai, Ryozo

    2013-02-15

    There is a possibility thrombus formation is closely involved in sudden cardiac death. Amiodarone, a potassium channel inhibitor, is known to reduce mortality in patients with coronary artery disease or low ejection fraction, having antithrombotic actions. Using human monocytic THP-1 cells, we investigated the effects of amiodarone on tissue factor mRNA and protein expression. The involvement of the two main potassium channels existing in THP-1 cells was also investigated. Amiodarone (10μM) significantly and almost completely inhibited the increase of tissue factor mRNA and protein expression induced by tumor necrosis factor-α (100ng/ml). The inhibitory effects of amiodarone on tissue factor mRNA expression showed dose-dependency. Margatoxin (1nM), a selective blocker of voltage-dependent potassium channel Kv1.3, also inhibited tissue factor protein expression, but didn't significantly inhibit mRNA expression. Ba(2+), a blocker of inwardly rectifying potassium channel Kir2.1, partly inhibited the increase of tissue factor mRNA and protein expression. This is the first study that shows amiodarone inhibits tissue factor expression in monocytic cells, by inhibiting mRNA transcription. The result may correlate with the facts amiodarone has antithrombotic actions in patients under extraordinary conditions where thrombus formation is enhanced. The inhibitory effects of amiodarone on tissue factor expression are drastic, different from those of margatoxin and Ba(2+). The result suggests amiodarone has an underlying mechanism to intensely inhibit tissue factor expression other than blocking Kv1.3 and Kir2.1. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Loss of c-KIT expression in thyroid cancer cells.

    Science.gov (United States)

    Franceschi, Sara; Lessi, Francesca; Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

    2017-01-01

    Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

  7. Chemokine receptor expression by inflammatory T cells in EAE

    DEFF Research Database (Denmark)

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6(+) and CXCR3(+) CD4(+) T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8(+) T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double......Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20...... receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed...

  8. EXPRESSION PROFILES AND METHYLATION GENES IN CLEAR CELL RENAL CARCINOMA

    Directory of Open Access Journals (Sweden)

    E. A. Braga

    2016-01-01

    Full Text Available Renal cancer (RC is a common malignancy of the genitourinary system. Clear cell renal cell carcinoma is the most common histological type of RC. In most cases diagnosis and prognosis of clear cell renal cell carcinoma are based on the results of instrumental tests, while search for novel molecular RC markers and their characterization remain relevant. Molecular genetic abnormalities accompanied with changes in gene expression underly the RC carcinogenesis; however, diagnostic panels of the expression markers of RC are still not widely used. This review represents the results of recent research in the area of gene expression markers of RC aimed to elaborate prognostic test systems. Application of the NotI-microarray methodology allowed for identification of many novel genes associated with RC pathogenesis. The relationship of alterations of expression level and methylation of chromosome 3 genes with RC progression and metastasis has been shown. Based on this data, a  diagnostic marker system for RC have been proposed with identification of expression and methylation profiles and novel markers, that is an urgent problem in modern urologic oncology.

  9. Nucleophosmin expression in renal cell carcinoma and oncocytoma.

    Science.gov (United States)

    Sari, Aysegul; Calli, Aylin; Altinboga, Aysegul Aksoy; Pehlivan, Fatma Seher; Gorgel, Sacit Nuri; Balci, Ugur; Ermete, Murat; Dincel, Cetin; Cakalagaoglu, Fulya

    2012-03-01

    The objective of this study was to investigate nucleophosmin/B23 (NPM) expression in renal cell carcinomas (RCC) and renal oncocytomas. The expression of NPM was studied by immunohistochemical methods on 59 RCCs, 9 oncocytomas, and 19 tumour-negative renal tissues. The expression was assessed relative to various clinicopathological variables and histological subtypes, to determine its potential role as a prognostic and diagnostic marker. All tumours showed nuclear staining, and a minority also exhibited cytoplasmic immunoreactivity. Two patterns of nuclear staining were observed: nuclear staining with a prominent nucleolus (nucleolar staining) and nuclear staining without a prominent nucleolus. There were significant differences, in both nuclear staining and cytoplasmic NPM expression, between oncocytomas and chromophobe RCCs (p oncocytomas. A statistically significant correlation was discovered between nucleolar staining and nuclear grade (p oncocytoma and chromophobe RCC. In addition, increased nucleolar NPM expression in RCCs appears to be associated with tumour progression. © 2011 The Authors. APMIS © 2011 APMIS.

  10. Phenotypic expression of human hepatoma cells in culture.

    Science.gov (United States)

    Cutroneo, Kenneth R; White, Sheryl L; Buttolph, Thomas R; Allison, Gretchen; Ehrlich, H Paul

    2007-04-01

    Hepatomas thrive in a hypoxic environment resulting in the induction of a cluster of hypoxia related genes. The protein phenotypic expression include hypoxia inducible factor-alpha, prolyl-4-hydroxylase, vascular endothelear growth factor and erythropoietin. The present study was undertaken to determine if human hepatoma cells when cultured for 72 h in the presence of serum under normoxia would maintain their cancerous phenotypic expression of certain hypoxia inducible genes. Our positive results affords an in vitro model system to test hypoxia inhibitors on the expression and the intracellular compartmentalization or the secretion of these hypoxia-inducible proteins. c 2007 Wiley-Liss, Inc.

  11. [6]-Gingerol Induces Cell Cycle Arrest and Cell Death of Mutant p53-expressing Pancreatic Cancer Cells

    Science.gov (United States)

    Park, Yon Jung; Wen, Jing; Bang, Seungmin; Park, Seung Woo

    2006-01-01

    [6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild-type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21cip1, was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53-expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53-expressing cells by inducing temporal growth arrest. PMID:17066513

  12. Euphorbia tirucalli modulates gene expression in larynx squamous cell carcinoma.

    Science.gov (United States)

    Franco-Salla, Gabriela Bueno; Prates, Janesly; Cardin, Laila Toniol; Dos Santos, Anemari Ramos Dinarte; Silva, Wilson Araújo da; da Cunha, Bianca Rodrigues; Tajara, Eloiza Helena; Oliani, Sonia Maria; Rodrigues-Lisoni, Flávia Cristina

    2016-05-21

    Some plants had been used in the treatment of cancer and one of these has attracted scientific interest, the Euphorbia tirucalli (E. tirucalli), used in the treatment of asthma, ulcers, warts has active components with activities scientifically proven as antimutagenic, anti-inflammatory and anticancer. We evaluate the influence of the antitumoral fraction of the E. tirucalli latex in the larynx squamous cell carcinoma (Hep-2), on the morphology, cell proliferation and gene expression. The Hep-2 cells were cultivated in complete medium (MEM 10 %) and treated with E. tirucalli latex for 1, 3, 5 and 7 days. After statistically analyzing the proliferation of the tested cells, the cells were cultivated again for RNA extraction and the Rapid Subtractive Hybridization (RaSH) technique was used to identify genes with altered expression. The genes found using the RaSH technique were analyzed by Gene Ontology (GO) using Ingenuity Systems. The five genes found to have differential expression were validated by real-time quantitative PCR. Though treatment with E. tirucalli latex did not change the cell morphology in comparison to control samples, but the cell growth was significantly decreased. The RaSH showed change in the expression of some genes, including ANXA1, TCEA1, NGFRAP1, ITPR1 and CD55, which are associated with inflammatory response, transcriptional regulation, apoptosis, calcium ion transport regulation and complement system, respectively. The E. tirucalli latex treatment down-regulated ITPR1 and up-regulated ANXA1 and CD55 genes, and was validated by real-time quantitative PCR. The data indicate the involvement of E. tirucalli latex in the altered expression of genes involved in tumorigenic processes, which could potentially be applied as a therapeutic indicator of larynx cancer.

  13. Gene expression profiling of chicken primordial germ cell ESTs

    Directory of Open Access Journals (Sweden)

    Lim Dajeong

    2006-08-01

    Full Text Available Abstract Background Germ cells are the only cell type that can penetrate from one generation to next generation. At the early embryonic developmental stages, germ cells originally stem from primordial germ cells, and finally differentiate into functional gametes, sperm in male or oocyte in female, after sexual maturity. This study was conducted to investigate a large-scale expressed sequence tag (EST analysis in chicken PGCs and compare the expression of the PGC ESTs with that of embryonic gonad. Results We constructed 10,851 ESTs from a chicken cDNA library of a collection of highly separated embryonic PGCs. After chimeric and problematic sequences were filtered out using the chicken genomic sequences, there were 5,093 resulting unique sequences consisting of 156 contigs and 4,937 singlets. Pearson chi-square tests of gene ontology terms in the 2nd level between PGC and embryonic gonad set showed no significance. However, digital gene expression profiling using the Audic's test showed that there were 2 genes expressed significantly with higher number of transcripts in PGCs compared with the embryonic gonads set. On the other hand, 17 genes in embryonic gonads were up-regulated higher than those in the PGC set. Conclusion Our results in this study contribute to knowledge of mining novel transcripts and genes involved in germline cell proliferation and differentiation at the early embryonic stages.

  14. Quantifying HER-2 expression on circulating tumor cells by ACCEPT.

    Science.gov (United States)

    Zeune, Leonie; van Dalum, Guus; Decraene, Charles; Proudhon, Charlotte; Fehm, Tanja; Neubauer, Hans; Rack, Brigitte; Alunni-Fabbroni, Marianna; Terstappen, Leon W M M; van Gils, Stephan A; Brune, Christoph

    2017-01-01

    Circulating tumor cells (CTCs) isolated from blood can be probed for the expression of treatment targets. Immunofluorescence is often used for both the enumeration of CTC and the determination of protein expression levels related to treatment targets. Accurate and reproducible assessment of such treatment target expression levels is essential for their use in the clinic. To enable this, an open source image analysis program named ACCEPT was developed in the EU-FP7 CTCTrap and CANCER-ID programs. Here its application is shown on a retrospective cohort of 132 metastatic breast cancer patients from which blood samples were processed by CellSearch® and stained for HER-2 expression as additional marker. Images were digitally stored and reviewers identified a total of 4084 CTCs. CTC's HER-2 expression was determined in the thumbnail images by ACCEPT. 150 of these images were selected and sent to six independent investigators to score the HER-2 expression with and without ACCEPT. Concordance rate of the operators' scoring results for HER-2 on CTCs was 30% and could be increased using the ACCEPT tool to 51%. Automated assessment of HER-2 expression by ACCEPT on 4084 CTCs of 132 patients showed 8 (6.1%) patients with all CTCs expressing HER-2, 14 (10.6%) patients with no CTC expressing HER-2 and 110 (83.3%) patients with CTCs showing a varying HER-2 expression level. In total 1576 CTCs were determined HER-2 positive. We conclude that the use of image analysis enables a more reproducible quantification of treatment targets on CTCs and leads the way to fully automated and reproducible approaches.

  15. Polyclonal T-cells express CD1a in Langerhans cell histiocytosis (LCH lesions.

    Directory of Open Access Journals (Sweden)

    Jennifer A West

    Full Text Available Langerhans cell histiocytosis (LCH is a complex and poorly understood disorder that has characteristics of both inflammatory and neoplastic disease. By using eight-colour flow cytometry, we have identified a previously unreported population of CD1a(+/CD3(+ T-cells in LCH lesions. The expression of CD1a is regarded as a hallmark of this disease; however, it has always been presumed that it was only expressed by pathogenic Langerhans cells (LCs. We have now detected CD1a expression by a range of T-cell subsets within all of the LCH lesions that were examined, establishing that CD1a expression in these lesions is no longer restricted to pathogenic LCs. The presence of CD1a(+ T-cells in all of the LCH lesions that we have studied to date warrants further investigation into their biological function to determine whether these cells are important in the pathogenesis of LCH.

  16. RBM4 promotes pancreas cell differentiation and insulin expression.

    Science.gov (United States)

    Lin, Jung-Chun; Yan, Yu-Ting; Hsieh, Wen-Kou; Peng, Pey-Jey; Su, Chun-Hao; Tarn, Woan-Yuh

    2013-01-01

    The RNA-binding protein RNA-binding motif protein 4 (RBM4) modulates alternative splicing of muscle-specific mRNA isoforms during muscle cell differentiation. To better understand the physiological function of RBM4, we exploited a gene knockout strategy in the present study. Mice with targeted disruption of one of the two Rbm4 genes exhibited hyperglycemia coincident with reduced levels of serum insulin and reduced size of pancreatic islets. The embryonic pancreases of Rbm4-deficient mice showed reduced expression or aberrant splicing of many transcripts encoding factors required for pancreas cell differentiation and function. Using pancreatic acinar AR42J cells, we demonstrated that RBM4 promoted insulin gene expression by altering the isoform balance of the transcription factors Isl1 and Pax4 via alternative splicing control. RBM4 overexpression was sufficient to convert AR42J cells into insulin-producing cells. Moreover, RBM4 may mediate glucose-induced insulin expression and insulin receptor isoform switches. These results suggest that RBM4 may have role in promoting pancreas cell differentiation and endocrine function, essentially via alternative splicing regulation.

  17. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Carlos Medina

    Full Text Available In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i the nasF transcriptional attenuator, which reduces basal expression levels, (ii a strong ribosome binding site, and (iii the Type III Secretion System (TTSS signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

  18. Global expression profile of tumor stem-like cells isolated from MMQ rat prolactinoma cell

    OpenAIRE

    Su, Zhipeng; Cai, Lin; Lu, Jianglong; Li, Chuzhong; Gui, Songbai; Liu, Chunhui; Wang, Chengde; Li, Qun; Zhuge, Qichuan; Zhang, Yazhuo

    2017-01-01

    Background Cancer stem cells (CSCs), which have been isolated from various malignancies, were closely correlated with the occurrence, progression, metastasis and recurrence of the malignant cancer. Little is known about the tumor stem-like cells (TSLCs) isolated from benign tumors. Here we want to explore the global expression profile of RNA of tumor stem-like cells isolated from MMQ rat prolactinoma cells. Methods In this study, total RNA was extracted from MMQ cells and MMQ tumor stem-like ...

  19. AIRE expressing marginal zone dendritic cells balances adaptive immunity and T-follicular helper cell recruitment.

    Science.gov (United States)

    Lindmark, Evelina; Chen, Yunying; Georgoudaki, Anna-Maria; Dudziak, Diana; Lindh, Emma; Adams, William C; Loré, Karin; Winqvist, Ola; Chambers, Benedict J; Karlsson, Mikael C I

    2013-05-01

    Autoimmune polyendocrine syndrome Type I (APS I) results in multiple endocrine organ destruction and is caused by mutations in the Autoimmune regulator gene (AIRE). In the thymic stroma, cells expressing the AIRE gene dictate T cell education and central tolerance. Although this function is the most studied, AIRE is also expressed in the periphery in DCs and stromal cells. Still, how AIRE regulated transcription modifies cell behaviour in the periphery is largely unknown. Here we show that AIRE is specifically expressed by 33D1(+) DCs and dictates the fate of antibody secreting cell movement within the spleen. We also found that AIRE expressing 33D1(+) DCs expresses self-antigens as exemplified by the hallmark gene insulin. Also, as evidence for a regulatory function, absence of Aire in 33D1(+) DCs led to reduced levels of the chemokine CXCL12 and increased co-stimulatory properties. This resulted in altered activation and recruitment of T-follicular helper cells and germinal centre B cells. The altered balance leads to a change of the early response to a T cell-dependent antigen in Aire(-/-) mice. These findings add to the understanding of how specific DC subtypes regulate the early responses during T cell-dependent antibody responses within the spleen and further define the role of AIRE in the periphery as regulator of self-antigen expression and lymphocyte migration. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Human dental follicle cells express embryonic, mesenchymal and neural stem cells markers.

    Science.gov (United States)

    Lima, Rodrigo Lopes; Holanda-Afonso, Rosenilde Carvalho; Moura-Neto, Vivaldo; Bolognese, Ana Maria; DosSantos, Marcos Fabio; Souza, Margareth Maria

    2017-01-01

    This study was conducted to identify and characterize dental follicle stem cells (DFSCs) by analyzing expression of embryonic, mesenchymal and neural stem cells surface markers. Design Dental follicle cells (DFCs) were evaluated by immunocytochemistry using embryonic stem cells markers (OCT4 and SOX2), mesenchmal stem cells (MSCs) markers (Notch1, active Notch1, STRO, CD44, HLA-ABC, CD90), neural stem cells markers (Nestin and β-III-tubulin), neural crest stem cells (NCSCs) markers (p75 and HNK1) and a glial cells marker (GFAP). RT-PCR was performed to identify the expression of OCT4 and NANOG in DFCs and dental follicle tissue. Immunocytochemistry and RT-PCR analysis revealed that a significant proportion of the DFCs evaluated expressed human embryonic stem cells marker OCT4 (75%) whereas NANOG was weakly expressed. A considerable amount of MSCs (90%) expressed Notch1, STRO, CD44 and HLA-ABC. However, they were weakly positive for CD90. Moreover, it was possible to demonstrate that dental follicle contains a significant proportion of neural stem/progenitors cells, expressing β-III-tubulin (90%) and nestin (70%). Interestingly, immunocytochemistry showed DFCs positive for p75 (50%), HNK1 (cells. This is the first study reporting the presence of NCSCs and glial-like cells in the dental follicle. The results of the present study suggest the occurrence of heterogeneous populations of stem cells, particularly neural stem/progenitor cells, in the dental follicle, Therefore, the human dental follicle might be a promising source of adult stem cells for regenerative purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Heterogeneity of premetastatic niches gene expression in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Tashireva L. A.

    2015-12-01

    Full Text Available Aim. To investigate the expression of the genes TGFB1, TNF, CSF1, CSF2, VEGFA and HIF1A in the patients with invasive breast carcinoma of no special type considering the intratumoral morphological heterogeneity. Methods. The technology of laser capture microdissection PALM was used to isolate five types of morphological tumor structures from three patients with invasive carcinoma of no special type (IC NST, luminal A subtype, T1-2NxMx. The level of expression of the cytokine (TNF, growth factor genes (TGFB1, CSF1, CSF2, VEGFA and the HIF1A gene was assessed in the samples obtained using real-time PCR, TaqMan-probes and specific oligonucleotides. Results. The study demonstrated the absence of the expression of the growth factor gene CSF2 in tumor cells of IC NST, and the expression of the gene CSF1, independent from the metastasis status and tumor structure type. The prevalence of the expression of the genes VEGFA and TGFB1 was revealed in the alveolar and solid structures along with the rare expression of the gene TNF. Conclusions. The expression of pre-metastatic niche genes in the tumors of patients with IC NST is heterogeneous. The hypoxia-mediated change in the cytokine gene expression may be expected in the alveolar and solid structures, which ultimately results in the formation of microenvironment, facilitating tumor growth and the formation of tumor metastatic potential.

  2. GLUT-1 Expression in Cutaneous Basal and Squamous Cell Carcinomas.

    Science.gov (United States)

    Abdou, Asmaa Gaber; Eldien, Marwa Mohammad Serag; Elsakka, Daliah

    2015-09-01

    Glucose uptake is a key regulating step in glucose metabolism and is mediated by facilitative glucose transporters (GLUTs), and GLUT-1 is the predominant glucose transporter in many types of human cells. Cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) represent the most common skin cancer in Egypt. The present study aimed at evaluation of the pattern and distribution of GLUT-1 in cutaneous BCC (16 cases) and SCC (16 cases) by means of immunohistochemistry. GLUT-1 was expressed in all SCC (100%) and in 62.5% of BCC. Membranous pattern of GLUT-1 was seen in 62.5% of SCC and 31.25% of BCC. Positivity (P = .02) and percentage (P = .000) of GLUT-1 expression were in favor of SCC in comparison to BCC. The high percentage of GLUT-1 expression was associated with high grade in SCC (P = .03). The immunoreactivity for GLUT-1 was more in the periphery of malignant nests of SCC while it was more in the center of BCC nests. GLUT-1 is overexpressed in cutaneous non-melanoma skin cancer. Its expression in SCC is related to differentiation status, and its expression in BCC is intimately associated with squamous metaplastic areas. © The Author(s) 2015.

  3. Versatile epitope tagging vector for gene expression in mammalian cells.

    Science.gov (United States)

    Hosfield, T; Lu, Q

    1998-08-01

    We have constructed an epitope-tagging vector, pCMV-Tag1, for gene expression in mammalian cells. This vector, which allows for N-terminal, C-terminal and internal tagging of the gene product of interest with the FLAG and/or c-myc epitopes, enables researchers to rapidly and efficiently characterize gene products in vivo.

  4. Regulation of HIV receptor expression in cervical epithelial cells by ...

    African Journals Online (AJOL)

    by real-time polymerase chain reaction (RT-PCR) analysis. HIV receptor .... PCR product. Results were calculated using the comparative cycle threshold ... data were expressed relative to an endogenous control of HeLa cell. cDNA included in ... Statistical analysis was performed using one-way analysis of variance and the ...

  5. Regulation of HIV receptor expression in cervical epithelial cells by ...

    African Journals Online (AJOL)

    Treatment of HeLa cells with LPS increased expression of the primary HIV chemokine ... and several alternative HIV receptors including CCR2b (p<0.01), CXCR6 (p<0.05) and GPR1 ... Cervical cancer tissue specimens were obtained at the time of surgery ..... body, including the gastrointestinal tract, prostate and cervix, has.

  6. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  7. Regulation of endothelial cell adhesion molecule expression by mast cells, macrophages, and neutrophils

    National Research Council Canada - National Science Library

    Zhang, Jie; Alcaide, Pilar; Liu, Li; Sun, Jiusong; He, Aina; Luscinskas, Francis W; Shi, Guo-Ping

    2011-01-01

    .... Using bone marrow-derived mast cells from wild-type, Tnf(-/-), Ifng(-/-), Il6(-/-) mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1...

  8. The expression and regulation of glucose transporters in tumor cells

    Directory of Open Access Journals (Sweden)

    Pengfei Zhao

    2016-12-01

    Full Text Available Glucose transporter proteins are involved in many physiological and biochemical processes. In particular, the high expressions of sodium-glucose cotransporter and glucose transporter proteins in tumor cells show that these two transporters play a key role in tumor cell metabolism. Studying the crystal structure and conformation of human glucose transporter proteins has enabled the development of drugs based on specific binding sites, opening up a new path towards more effective cancer treatments. This mini review serves to summarize our existing understanding of the metabolic pathways of tumor cells, focusing on the roles of glucose transporter proteins.

  9. COX-2 expression positively correlates with PD-L1 expression in human melanoma cells.

    Science.gov (United States)

    Botti, Gerardo; Fratangelo, Federica; Cerrone, Margherita; Liguori, Giuseppina; Cantile, Monica; Anniciello, Anna Maria; Scala, Stefania; D'Alterio, Crescenzo; Trimarco, Chiara; Ianaro, Angela; Cirino, Giuseppe; Caracò, Corrado; Colombino, Maria; Palmieri, Giuseppe; Pepe, Stefano; Ascierto, Paolo Antonio; Sabbatino, Francesco; Scognamiglio, Giosuè

    2017-02-23

    The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma. Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro. BRAFV600E/V600K and NRASQ61R/Q61L were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines. COX-2 expression correlates with and

  10. PROFILES OF GENE EXPRESSION ASSOCIATED WITH TETRACYCLINE OVER EXPRESSION OF HSP70 IN MCF-7 BREAST CANCER CELLS

    Science.gov (United States)

    Profiles of gene expression associated with tetracycline over expression of HSP70 in MCF-7 breast cancer cells. Heat shock proteins (HSPs) protect cells from damage through their function as molecular chaperones. Some cancers reveal high levels of HSP70 expression in asso...

  11. Profiling helper T cell subset gene expression in deer mice

    Directory of Open Access Journals (Sweden)

    Hjelle Brian

    2006-08-01

    Full Text Available Abstract Background Deer mice (Peromyscus maniculatus are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV, the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT, Th2 cells (GATA-3, STAT6, IL-4, IL-5 and regulatory T cells (Fox-p3, IL-10, TGFβ1. These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

  12. Higher expression of SIRT1 induced resistance of esophageal squamous cell carcinoma cells to cisplatin.

    Science.gov (United States)

    Cao, Bin; Shi, Qintong; Wang, Wengong

    2015-04-01

    High expression of Sirtuin type 1 (SIRT1) exists in some cancer cells. However, it is still unclear whether SIRT1 affects the sensitivity of esophageal cancer cells to cisplatin. This study was designed to explore the relationship between SIRT1 expression and resistance of esophageal squamous cell carcinoma (ESCC) cells to cisplatin and reveal the underlying mechanism. The tissue samples of 68 ESCC patients were collected from Nanjing Drum Tower Hospital, China. All the patients had undergone cisplatin based combination chemotherapy. The expression of SIRT1and Noxa in tissue samples were analyzed by quantitative real-time reverse PCR (qRT-PCR) and Western blot. Human ESCC cell line (ECa9706 cells) was cultured and a cisplatin-resistant subline (ECa9706-CisR cells) was established by continuous exposure to cisplatin at different concentrations. The expression of SIRT1 and Noxa in both cell lines was analyzed by qRT-PCR and Western blot. siRNA technology was utilized to down-regulate the SIRT1 expression in ECa9706-CisR cells. The influence of SIRT1 silence on sensitivity of ECa9706-CisR cells to cisplatin was confirmed using CCK-8 assay and flow cytometry. Furthermore, the level change of Noxa after SIRT1 silence in ECa9706-CisR cells was determined by qRT-PCR and Western blot. SIRT1 and Noxa expression in chemo-resistant patients was significantly increased and decreased respectively, compared with chemo-sensitive patients. SIRT1 expression in ECa9706-CisR cells was significantly increased with a lower Noxa level, compared with normal ECa9706 cells. Cisplatin 5 µM could cause proliferation inhibition, G2/M phase arrest and apoptosis in ECa9706-CisR cells and these effects could be enhanced dramatically by SIRT1 silencing. Moreover, Noxa expression was increased after treated with SIRT1 siRNA. Over-expression of SIRT1 may cause resistance of ESCC cells to cisplatin through the mechanism involved with Noxa expression.

  13. EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures.

    OpenAIRE

    Ford, M G; Valle, J D; Soroka, C J; Merchant, J L

    1997-01-01

    Gastrin release from the antral gastrin-expressing cell (G cell) is regulated by bombesin and luminal factors. Yet, these same extracellular regulators do not stimulate expression of the gene. Since the gastric mucosa expresses large quantities of EGF receptor ligands such as TGFalpha, we examined whether EGF receptor ligands stimulate gastrin gene expression in gastrin-expressing cell cultures. EGF receptor activation of primary cultures stimulated gastrin gene expression about twofold; wher...

  14. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T

    2013-01-01

    expressing increased amounts of human endogenous retrovirus antigens. MS patients also have increased antibody levels to these antigens. The target cells are spontaneously growing peripheral blood mononuclear cells (PBMCs) of B cell lineage, expressing human endogenous retrovirus HERV epitopes...

  15. Global expression profile of tumor stem-like cells isolated from MMQ rat prolactinoma cell.

    Science.gov (United States)

    Su, Zhipeng; Cai, Lin; Lu, Jianglong; Li, Chuzhong; Gui, Songbai; Liu, Chunhui; Wang, Chengde; Li, Qun; Zhuge, Qichuan; Zhang, Yazhuo

    2017-01-01

    Cancer stem cells (CSCs), which have been isolated from various malignancies, were closely correlated with the occurrence, progression, metastasis and recurrence of the malignant cancer. Little is known about the tumor stem-like cells (TSLCs) isolated from benign tumors. Here we want to explore the global expression profile of RNA of tumor stem-like cells isolated from MMQ rat prolactinoma cells. In this study, total RNA was extracted from MMQ cells and MMQ tumor stem-like cells. RNA expression profiles were determined by Agilent Rat 8 × 60 K Microarray. Then we used the qRT-PCR to test the result of Microarray, and found VEGFA had a distinct pattern of expression in MMQ tumor stem-like cells. Then WB and ELISA were used to confirm the VEGFA protein level of tumor sphere cultured from both MMQ cell and human prolactinoma cell. Finally, CCK-8 was used to evaluate the reaction of MMQ tumor stem-like cells to small interfering RNAs intervention and bevacizumab treatment. The results of Microarray showed that 566 known RNA were over-expressed and 532 known RNA were low-expressed in the MMQ tumor stem-like cells. These genes were mainly involved in 15 different signaling pathways. In pathway in cancer and cell cycle, Bcl2, VEGFA, PTEN, Jun, Fos, APC2 were up-regulated and Ccna2, Cdc25a, Mcm3, Mcm6, Ccnb2, Mcm5, Cdk1, Gadd45a, Myc were down-regulated in the MMQ tumor stem-like cells. The expression of VEGFA were high in tumor spheres cultured from both MMQ cell and human prolactinomas. Down-regulation of VEGFA by small interfering RNAs partially decreased cell viability of MMQ tumor stem-like cells in vitro. Bevacizumab partially suppressed the proliferation of MMQ tumor stem-like cells. Our findings characterize the pattern of RNA expression of tumor stem-like cells isolated from MMQ cells. VEGFA may act as a potential therapeutic target for tumor stem-like cells of prolactinomas.

  16. Nephroblastoma overexpressed gene (NOV) expression in rat hepatic stellate cells.

    Science.gov (United States)

    Lee, Sung Hee; Seo, Geom Seog; Park, Young Nyun; Sohn, Dong Hwan

    2004-10-01

    Using the expression-profiling method, we identified nephroblastoma overexpressed gene (NOV) mRNA as one member of the mRNA population that was upregulated in cultured activated hepatic stellate cell (HSC). Northern analysis showed that NOV mRNA was increasingly expressed during progressive activation of cultured rat HSCs, and a significant increase was observed in both the carbon tetrachloride-induced and bile duct ligation/scission rat models of liver fibrosis. RT-PCR showed human NOV mRNA was increased in most fibrotic livers compared with normal livers. The expression of NOV protein in fibrotic rat and human livers was predominantly located in areas of ductular proliferation and HSC of the fibrous septa. HSCs stimulated with transforming growth factor beta1 showed increased expression of NOV protein without changing its mRNA levels. Dexamethasone stimulated the expression of NOV mRNA and protein. Furthermore, we demonstrated that bile acids have a modulating effect on the induction of NOV mRNA expression. In conclusion, this study suggests that NOV is expressed during liver fibrogenesis and HSCs may be an important source of hepatic NOV.

  17. Hypertonic saline impedes tumor cell-endothelial cell interaction by reducing adhesion molecule and laminin expression.

    LENUS (Irish Health Repository)

    Shields, Conor J

    2012-02-03

    BACKGROUND: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism. METHODS: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours. Confluent human umbilical vein endothelial cells were similarly exposed. Cellular apoptosis and expression of adhesion molecules and laminin were measured by flow cytometry. Tumor cell adhesion to endothelium and laminin was assessed with fluorescence microscopy. Data are represented as mean +\\/- standard error of mean, and an ANOVA test was performed to gauge statistical significance, with P <.05 considered significant. RESULTS: Hypertonic exposure significantly reduced tumor cell adhesion despite the presence of the perioperative cell stressors (42 +\\/- 2.9 vs 172.5 +\\/- 12.4, P <.05), attenuated tumor cell beta-1 integrin (14.43 vs 23.84, P <.05), and endothelial cell laminin expression (22.78 +\\/- 2.2 vs 33.74 +\\/- 2.4, P <.05), but did not significantly alter cell viability. CONCLUSION: Hypertonic saline significantly attenuates tumor cell adhesion to endothelium by inhibiting adhesion molecule and laminin expression. This may halt the metastatic behavior of tumor cells shed at surgery.

  18. Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells.

    Science.gov (United States)

    Shirzad, Hedayatollah; Esmaeili, Fariba; Bakhshalizadeh, Shabnam; Ebrahimie, Marzieh; Ebrahimie, Esmaeil

    2017-04-01

    Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Targeted overexpression of cyclic AMP-dependent protein kinase subunit in Toxoplasma gondii promotes replication and virulence in host cells.

    Science.gov (United States)

    Sun, Hongchao; Wang, Suhua; Zhao, Xianfeng; Yao, Chaoqun; Zhuang, Haohan; Huang, Yechuan; Chen, Xueqiu; Yang, Yi; Du, Aifang

    2017-08-30

    Toxoplasma gondii (T. gondii) is one of the most common parasite that can infect almost any warm-blooded animals including humans. The cyclic nucleotide-dependent protein kinase (PKA) regulates a spectrum of intracellular signal pathways in many organisms. Protein kinase catalytic subunit (PKAC) is the core of the whole protein, and plays an important role in the life cycle of T.gondii. Here, T.gondii PKAC (TgPKAC) overexpression strain (TgPKAC-OE) was constructed. The growth of the TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains (TgPKAC-H89) were analysed by SYBR-green real-time PCR, and the ultrastructure was observed by transmission electron microscopy. The survival rate in mice was also recorded to analyse the virulence of the parasites. We also investigated the subcellular localization of TgPKAC in Vero cells by laser scanning microscope. We found that TgPKAC-OE strain exhibited obviously increased growth rate in Vero cells in vitro, and infected mice survived for a shorter time compared to wild type strain. Ultrastructural analysis found more autophagosomes-like structures in TgPKAC-H89 parasite compared to RH△Ku80 strain, and the relative expression level of Toxoplasma gondii autophagy-related protein (ATG8) in TgPKAC-H89 parasite was higher than wild type parasite. Laser confocal results showed that TgPKAC was mainly expressed in the cytoplasm of Vero cells. In conclusion, we hypothesized that inhibition of TgPKAC could cause autophagy of Toxoplasma gondii and then influence the replication of the parasite. TgPKAC plays an important role in parasite virulence in vivo, and the subcellular localization was successfully detected in Vero cells. Our data will provide a basis for further study of TgPKAC function and help screen drug targets of T. gondii. Copyright © 2017. Published by Elsevier B.V.

  20. Stage-specific embryonic antigen: determining expression in canine glioblastoma, melanoma, and mammary cancer cells.

    Science.gov (United States)

    Lin, Weiming; Modiano, Jaime F; Ito, Daisuke

    2017-03-30

    The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1(-) and SSEA-1(+) cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

  1. Behavioral experience induces zif268 expression in mature granule cells but suppresses its expression in immature granule cells

    Directory of Open Access Journals (Sweden)

    Kylie A. Huckleberry

    2015-08-01

    Full Text Available Thousands of neurons are born each day in the dentate gyrus (DG, but many of these cells die before reaching maturity. Both death and survival of adult-born neurons are regulated by neuronal activity in DG. The immediate-early gene (IEG zif268 is an important mediator of these effects, as its expression is induced by neural activity and knockout of zif268 impairs survival of adult-born neurons (Veyrac et al., 2013. Despite the apparent importance of zif268 for adult neurogenesis, its behavior-induced expression has not been fully characterized in adult-born neurons. Here we characterize behavior-evoked expression of zif268 in mature and newborn dentate granule cells (DGCs. In the general granule cell population, zif268 expression peaked 1 hour after novel environment exposure and returned to baseline by 8 hours post-exposure. However, in the doublecortin-positive (DCX+ immature neurons, zif268 expression was suppressed relative to home cage for at least 8 hours post-exposure. We next determined that exposure to water maze training, an enriched environment, or a novel environment caused approximately equal suppression of zif268 expression in DCX+ cells and approximately equal activation of zif268 in the general DGC population and in 6-week-old adult-born neurons. Finally, we asked whether zif268 suppression varied as a function of age within the DCX+ population, which ranges in age from 0 to approximately 4 weeks. Novel environment exposure had no significant effect on zif268 expression in 2- or 4-week-old BrdU-labeled neurons, but it significantly suppressed zif268 expression in 3-week-old neurons. In summary, behavioral experience transiently activated expression of zif268 in mature DGCs but caused a more long-lasting suppression of zif268 expression in immature, adult-born DGCs. We hypothesize that zif268 suppression inhibits memory-related synaptic plasticity in immature DGCs or mediates learning-induced apoptosis of immature adult

  2. Tangeretin induces cell cycle arrest and apoptosis through upregulation of PTEN expression in glioma cells.

    Science.gov (United States)

    Ma, Li-Li; Wang, Da-Wei; Yu, Xu-Dong; Zhou, Yan-Ling

    2016-07-01

    Tangeretin (TANG), present in peel of citrus fruits, has been shown to various medicinal properties such as chemopreventive and neuroprotective. However, the chemopreventive effect of TANG on glioblastoma cells has not been examined. The present study was designed to explore the anticancer potential of TANG in glioblastoma cells and to investigate the related mechanism. Human glioblastoma U-87MG and LN-18 cells were treated with 45μM concentration of TANG and cell growth was measured by MTT assay. The cell cycle distribution and cell death were measured by flow cytometry. The expression of cell cycle and apoptosis related genes were analyzed by quantitative RT-PCR and western blot. The cells treated with TANG were significantly increased cell growth suppression and cell death effects than vehicle treated cells. Further, TANG treatment increases G2/M arrest and apoptosis by modulating PTEN and cell-cycle regulated genes such as cyclin-D and cdc-2 mRNA and protein expressions. Moreover, the ability of TANG to decrease cell growth and to induce cell death was compromised when PTEN was knockdown by siRNA. Taken together, the chemopreventive effect of TANG is associated with regulation of cell-cycle and apoptosis in glioblastoma, thereby attenuating glioblastoma cell growth. Hence, the present findings suggest that TANG may be a therapeutic agent for glioblastoma treatment. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  3. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  4. Changes in cell adhesion molecule expression on T cells associated with systemic virus infection

    DEFF Research Database (Denmark)

    Andersson, E C; Christensen, Jan Pravsgaard; Marker, O

    1994-01-01

    Virus-induced changes in adhesion molecule expression on T cells were investigated to understand how antiviral effector cells migrate into infectious foci. FACS analysis revealed that after systemic infection with lymphocytic choriomeningitis virus a number of cell adhesion molecules, including VLA......, it was found that up-regulation of VLA-4 expression on splenic T cells correlated with influx of inflammatory cells into the cerebrospinal fluid of intracerebrally infected animals, and that the number of CD8+VLA-4hi cells increased from lymph nodes and spleen to blood and cerebrospinal fluid. These results......-4, LFA-1, and ICAM-1, are up-regulated on CD8+ cells, whereas the lymph node homing receptor MEL-14 is down-regulated during the infection; only marginal changes were observed for CD4+ cells. Basically similar but less marked results were obtained in mice infected with Pichinde virus. Further...

  5. Resveratrol Represses Pokemon Expression in Human Glioma Cells.

    Science.gov (United States)

    Yang, Yutao; Cui, Jiajun; Xue, Feng; Overstreet, Anne-Marie; Zhan, Yiping; Shan, Dapeng; Li, Hui; Li, Hui; Wang, Yongjun; Zhang, Mengmeng; Yu, Chunjiang; Xu, Zhi-Qing David

    2016-03-01

    POK erythroid myeloid ontogenic factor (Pokemon), an important proto-oncoprotein, is a transcriptional repressor that regulates the expression of many genes and plays an important role in tumorigenesis. Resveratrol (RSV), a natural polyphenolic compound, has many beneficial biological effects on health. In this study, we investigated the role of Pokemon in RSV-induced biological effects and the effect of RSV on the expression of Pokemon in glioma cells. We found that overexpression of Pokemon decreased RSV-induced cell apoptosis, senescence, and anti-proliferative effects. Moreover, we showed that RSV could efficiently decrease the activity of the Pokemon promoter and the expression of Pokemon. Meanwhile, RSV also inhibited Sp1 DNA binding activity to the Pokemon promoter; whereas, it did not influence the expression and nuclear translocation of Sp1. In addition, we found that RSV could increase the recruitment of HDAC1, but decreased p300 to the Pokemon promoter. Taken together, all these results extended our understanding on the anti-cancer mechanism of RSV in glioma cells.

  6. Differential expression of the klf6 tumor suppressor gene upon cell damaging treatments in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Gehrau, Ricardo C.; D' Astolfo, Diego S.; Andreoli, Veronica [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Bocco, Jose L., E-mail: jbocco@fcq.unc.edu.ar [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Koritschoner, Nicolas P. [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina)

    2011-02-10

    The mammalian Krueppel-like factor 6 (KLF6) is involved in critical roles such as growth-related signal transduction, cell proliferation and differentiation, development, apoptosis and angiogenesis. Also, KLF6 appears to be an emerging key factor during cancer development and progression. Its expression is thoroughly regulated by several cell-damaging stimuli. DNA damaging agents at lethal concentrations induce a p53-independent down-regulation of the klf6 gene. To investigate the impact of external stimuli on human klf6 gene expression, its mRNA level was analyzed using a cancer cell line profiling array system, consisting in an assortment of immobilized cDNAs from multiple cell lines treated with several cell-damaging agents at growth inhibitory concentrations (IC{sub 50}). Cell-damaging agents affected the klf6 expression in 62% of the cDNA samples, though the expression pattern was not dependent on the cell origin type. Interestingly, significant differences (p < 0.0001) in KLF6 mRNA levels were observed depending on the cellular p53 status upon cell damage. KLF6 expression was significantly increased in 63% of p53-deficient cells (122/195). Conversely, KLF6 mRNA level decreased nearly 4 fold in more than 70% of p53+/+ cells. In addition, klf6 gene promoter activity was down-regulated by DNA damaging agents in cells expressing the functional p53 protein whereas it was moderately increased in the absence of functional p53. Consistent results were obtained for the endogenous KLF6 protein level. Results indicate that human klf6 gene expression is responsive to external cell damage mediated by IC{sub 50} concentrations of physical and chemical stimuli in a p53-dependent manner. Most of these agents are frequently used in cancer therapy. Induction of klf6 expression in the absence of functional p53 directly correlates with cell death triggered by these compounds, whereas it is down-regulated in p53+/+ cells. Hence, klf6 expression level could represent a valuable

  7. Clock upregulates intercellular adhesion molecule-1 expression and promotes mononuclear cells adhesion to endothelial cells.

    Science.gov (United States)

    Gao, Yinghua; Meng, Dan; Sun, Ning; Zhu, Zhu; Zhao, Ran; Lu, Chao; Chen, Sifeng; Hua, Luchun; Qian, Ruizhe

    2014-01-10

    Clock is a basic helix-loop-helix (bHLH) transcription factor that plays important role in circadian rhythms of various physiological functions. Previous study showed that the expression of intercellular adhesion molecule-1 (ICAM-1) was reduced in the liver tissues of Clock mutant mice. However, how Clock regulates ICAM-1 expression and whether Clock affects cell adhesion function remain unknown. In the present study, we found that exogenous expression of Clock upregulated the gene expressions of ICAM-1 and other adhesion-related genes including VCAM1 and CCL-2, and increased the transcriptional activity of ICAM-1 in mouse brain microvascular endothelial cell lines. In contrast, loss of Clock decreased these gene expressions and ICAM-1 transcriptional activity. Chromatin immunoprecipitation (ChIP) assay revealed that Clock binds to the E-box-like enhancer of ICAM-1 gene. ICAM-1 gene showed rhythmic expression in endothelial cells after serum shock in vitro, suggesting ICAM-1 may be a Clock-controlled gene. Clock regulates the adhesion of mononuclear cells to endothelial cells via ICAM-1. Together, our findings show that Clock is a positive regulator of ICAM-1, and promotes the adhesion of mononuclear cells to endothelial cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Multiple melanocortin receptors are expressed in bone cells

    Science.gov (United States)

    Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.

    2005-01-01

    Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

  9. Heterogeneous expression of Drosophila gustatory receptors in enteroendocrine cells.

    Science.gov (United States)

    Park, Jeong-Ho; Kwon, Jae Young

    2011-01-01

    The gastrointestinal tract is emerging as a major site of chemosensation in mammalian studies. Enteroendocrine cells are chemosensory cells in the gut which produce regulatory peptides in response to luminal contents to regulate gut physiology, food intake, and glucose homeostasis, among other possible functions. Increasing evidence shows that mammalian taste receptors and taste signaling molecules are expressed in enteroendocrine cells in the gut. Invertebrate models such as Drosophila can provide a simple and genetically tractable system to study the chemosensory functions of enteroendocrine cells in vivo. To establish Drosophila enteroendocrine cells as a model for studying gut chemosensation, we used the GAL4/UAS system to examine the expression of all 68 Gustatory receptors (Grs) in the intestine. We find that 12 Gr-GAL4 drivers label subsets of enteroendocrine cells in the midgut, and examine colocalization of these drivers with the regulatory peptides neuropeptide F (NPF), locustatachykinin (LTK), and diuretic hormone 31 (DH31). RT-PCR analysis provides additional evidence for the presence of Gr transcripts in the gut. Our results suggest that the Drosophila Grs have chemosensory roles in the intestine to regulate physiological functions such as food uptake, nutrient absorption, or sugar homeostasis.

  10. Differential nephron HO-1 expression following glomerular epithelial cell injury.

    Science.gov (United States)

    Datta, Prasun K; Reddy, Sreenivas; Sharma, Mukut; Lianos, Elias A

    2006-01-01

    In proteinuria of glomerular origin there is upregulation of heme-oxygenase (HO), the rate-limiting enzyme of heme degradation, in the nephron in a segment-specific manner. To better characterize this phenomenon, we employed a model of proteinuria resulting from disruption of the glomerular capillary permeability barrier to protein by administration of the glomerular epithelial cell toxin puromycin aminonucleoside (PAN) to rats. In this model, we assessed nephron distribution of the expression of the inducible HO isoform, HO-1, and the role of free radicals in modulating HO-1 expression. Rats were injected with either vehicle (dimethyl sulfoxide) or PAN or the spin trap free radical stabilizer alpha-phenyl-N-tert butyl nitrone (PBN), or with both PAN and PBN. Ten days following the PAN injection, urine protein, creatinine, nitric oxide (NO) and malonyldialdehyde (MDA) were measured. Kidney sections and protein lysates were assessed for changes in HO-1 expression by immunohistochemistry and Western blot analysis. In control animals (DMSO or PBN alone) there was no proteinuria and very weak or absent HO-1 staining in nephron segments. PAN treatment induced proteinuria and increased urine MDA excretion. In these animals, there was a robust HO-1 expression mainly in tubules and in glomerular parietal but not visceral epithelial cells. Unilateral ureteral obstruction to interrupt glomerular filtration in animals treated with PAN abrogated tubular HO-1 expression in the kidney ipsilateral to the obstruction. Administration of PBN to PAN-treated animals reduced proteinuria and MDA excretion while it markedly augmented tubular HO-1 expression. This augmentation was prominent in tubular cells of the inner cortex/outer medulla. These observations indicate that upregulation of nephron HO-1 following disruption of the glomerular permeability barrier occurs at sites downstream of this barrier and is mediated by a filtered HO-1 inducer(s). Scavenging of free radicals potentiates

  11. CD117 expression on blast cells in acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Goryainova N.V.

    2015-09-01

    Full Text Available The aim of the present work was to analyze the frequency of CD117 (c-KIT antigen expression on the blast cells in acute myeloid leukemia (AML, evaluation of the presence of the relationship between the expression of the c-KIT and leukemia according to the FAB classification and definition of co-expression of the antigen CD117, antigens CD33 and CD34. The data of 47 patients with AML were diagnosed. M0 AML variant was established in 3 (6% patients, M1 – in 2 (4%, M2 – in 9 (20%, M4 – in 22 (47% and M5 – in 11 (23%. For immunophenotypic stu¬dies monoclonal antibodies (mAb that detect antigens of anti-CD34, anti-CD33 and anti-CD117 (Becton Dickinson, USA were used. The presence of the antigen CD117 was detected in 39 people, accounting for 83% of all surveyed. Antigen c-KIT was present in 48.117.0% cells on average: in all 3 cases – AML M0, in2 cases of AML M1, in 6 cases – AML M2, 20 of 22 cases – AML M4 and in 8 of 11 AML M5 cases. Average levels of CD117 in investigated leukemia cases statistically differed significantly (p=0.0067. Among 39 CD117- positive patients in 25 (53% co-expression of CD117+/CD34+ was revealed. Expression of CD117+/CD34- was observed in 14 cases (30%, CD117-/CD34+ – in 4 cases (8,5%, CD117-/CD34- – in 4 cases (8.5%. CD34 had of 64% of cells of myeloid origin. A high positive cor¬relation between expression of CD117 and CD34 (r=+0,5169 was determined, being statistically significant (p0,0067.

  12. Reelin expression in brain endothelial cells: an electron microscopy study.

    Science.gov (United States)

    Perez-Costas, Emma; Fenton, Erin Y; Caruncho, Hector J

    2015-03-24

    Reelin expression and function have been extensively studied in the brain, although its expression has been also reported in other tissues including blood. This raises the possibility that reelin might be able to cross the blood-brain barrier, which could be functionally relevant. Up-to-date no studies have been conducted to assess if reelin is present in the blood-brain barrier, which is mainly constituted by tightly packed endothelial cells. In this report we assessed the expression of reelin in brain capillaries using immunocytochemistry and electron microscopy. At the light microscope, reelin immunolabeling appeared in specific endothelial cells in brain areas that presented abundant diffuse labeling for this protein (e.g., layer I of the cortex, or the stratum lacunosum moleculare of the hippocampus), while it was mostly absent from capillaries in other brain areas (e.g., deeper cortical layers, or the CA1 layer of the hippocampus). As expected, at the electron microscope reelin labeling was observed in neurons of the cortex, where most of the labeling was associated with the rough endoplasmic reticulum. Importantly, reelin was also observed in some endothelial cells located in small capillaries, which confirmed the findings obtained at the light microscope. In these cells, reelin labeling was located primarily in caveolae (i.e., vesicles of transcytosis), and associated with the plasma membrane of the luminal side of endothelial cells. In addition, some scarce labeling was observed in the nuclear membrane. The presence of reelin immunolabeling in brain endothelial cells, and particularly in caveolar vesicles within these cells, suggests that reelin and/or reelin peptides may be able to cross the blood-brain barrier, which could have important physiological, pathological, and therapeutic implications.

  13. ADAM17 deletion in thymic epithelial cells alters aire expression without affecting T cell developmental progression.

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    David M Gravano

    2010-10-01

    Full Text Available Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach.We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted.In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and the mechanisms by which it regulates intrathymic T cell development

  14. Gene Expression Profiling Predicts Survival in Conventional Renal Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available BACKGROUND: Conventional renal cell carcinoma (cRCC accounts for most of the deaths due to kidney cancer. Tumor stage, grade, and patient performance status are used currently to predict survival after surgery. Our goal was to identify gene expression features, using comprehensive gene expression profiling, that correlate with survival. METHODS AND FINDINGS: Gene expression profiles were determined in 177 primary cRCCs using DNA microarrays. Unsupervised hierarchical clustering analysis segregated cRCC into five gene expression subgroups. Expression subgroup was correlated with survival in long-term follow-up and was independent of grade, stage, and performance status. The tumors were then divided evenly into training and test sets that were balanced for grade, stage, performance status, and length of follow-up. A semisupervised learning algorithm (supervised principal components analysis was applied to identify transcripts whose expression was associated with survival in the training set, and the performance of this gene expression-based survival predictor was assessed using the test set. With this method, we identified 259 genes that accurately predicted disease-specific survival among patients in the independent validation group (p < 0.001. In multivariate analysis, the gene expression predictor was a strong predictor of survival independent of tumor stage, grade, and performance status (p < 0.001. CONCLUSIONS: cRCC displays molecular heterogeneity and can be separated into gene expression subgroups that correlate with survival after surgery. We have identified a set of 259 genes that predict survival after surgery independent of clinical prognostic factors.

  15. Gene expression profiling predicts survival in conventional renal cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Hongjuan Zhao

    2006-01-01

    Full Text Available BACKGROUND: Conventional renal cell carcinoma (cRCC accounts for most of the deaths due to kidney cancer. Tumor stage, grade, and patient performance status are used currently to predict survival after surgery. Our goal was to identify gene expression features, using comprehensive gene expression profiling, that correlate with survival. METHODS AND FINDINGS: Gene expression profiles were determined in 177 primary cRCCs using DNA microarrays. Unsupervised hierarchical clustering analysis segregated cRCC into five gene expression subgroups. Expression subgroup was correlated with survival in long-term follow-up and was independent of grade, stage, and performance status. The tumors were then divided evenly into training and test sets that were balanced for grade, stage, performance status, and length of follow-up. A semisupervised learning algorithm (supervised principal components analysis was applied to identify transcripts whose expression was associated with survival in the training set, and the performance of this gene expression-based survival predictor was assessed using the test set. With this method, we identified 259 genes that accurately predicted disease-specific survival among patients in the independent validation group (p < 0.001. In multivariate analysis, the gene expression predictor was a strong predictor of survival independent of tumor stage, grade, and performance status (p < 0.001. CONCLUSIONS: cRCC displays molecular heterogeneity and can be separated into gene expression subgroups that correlate with survival after surgery. We have identified a set of 259 genes that predict survival after surgery independent of clinical prognostic factors.

  16. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    Science.gov (United States)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  17. Expression of Plant Receptor Kinases in Tobacco BY-2 Cells.

    Science.gov (United States)

    Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

    2017-01-01

    Although more than 600 single-transmembrane receptor kinase genes have been found in the Arabidopsis genome, only a few of them have known physiological functions, and even fewer plant receptor kinases have known specific ligands. Ligand-binding analysis must be operated using the functionally expressed receptor form. However, the relative abundance of native receptor kinase molecules in the plasma membrane is often quite low. Here, we present a method for stable and functional expression of plant receptor kinases in tobacco BY-2 cells that allows preparation of microsomal fractions containing the receptor. This procedure provides a sufficient amount of receptor proteins while maintaining its ligand-binding activities.

  18. Expression of BTG1 in hepatocellular carcinoma and its correlation with cell cycles, cell apoptosis, and cell metastasis.

    Science.gov (United States)

    Sun, G G; Lu, Y F; Cheng, Y J; Yang, C R; Liu, Q; Jing, S W; Han, X C

    2014-12-01

    This study aimed to analyze the expression, clinical significance of B cell translocation gene 1 (BTG1) in hepatocellular carcinoma, and the biological effect in its cell line by BTG1 overexpression. Immunohistochemistry and Western blot were used to analyze BTG1 protein expression in 70 cases of hepatocellular cancer and 32 cases of normal tissues to study the relationship between BTG1 expression and clinical factors. Recombinant lentiviral vector was constructed to overexpress BTG1 and then infect hepatocellular cancer HepG2 cell line. The level of BTG1 protein expression was found to be significantly lower in hepatocellular cancer tissue than normal tissues (P expression of BTG1 was significantly correlated with tumor invasion, lymph node metastasis, clinic stage, and histological grade of patients with hepatocellular cancer (P expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P protein expression compared with HepG2 cell-untransfected BTG1 (P expression decreased in hepatocellular cancer and correlated significantly with lymph node metastasis, clinic stage, histological grade, poor overall survival, proliferation, and metastasis in hepatocellular cancer cell by regulating CND1, Bcl-2, and MMP-9 protein expression, suggesting that BTG1 may play important roles as a negative regulator to hepatocellular cancer cell.

  19. Osteopontin upregulates the expression of glucose transporters in osteosarcoma cells.

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    I-Shan Hsieh

    Full Text Available Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients.

  20. Expression of P-glycoprotein in adult T-cell leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuwazuru, Y.; Hanada, S.; Furukawa, T.; Yoshimura, A.; Sumizawa, T.; Utsunomiya, A.; Ishibashi, K.; Saito, T.; Uozumi, K.; Maruyama, M. (Kagoshima Univ. (Japan))

    1990-11-15

    We have examined the expression of P-glycoprotein (P-gp) in adult T-cell leukemia (ATL) samples from 25 patients. Based on immunoblotting with a monoclonal antibody against P-gp, C219, 8 of 20 ATL patients were P-gp positive at the initial presentation. All 6 patients at the relapsed stage were P-gp positive, and refractory to chemotherapy. The expression of MDR1 mRNA in P-gp-positive ATL cells was increased at the relapsed stage of one patient. P-gp of this patient was photolabeled with (3H)azidopine and the labeling was inhibited with nimodipine, vinblastine and progesterone. These results suggest that P-gp expressed in ATL cells from patients at relapsed stage has the same binding site(s) for the drugs as that in multidrug resistant cells, and is correlated with the refractory nature of the cells to chemotherapy.

  1. Malignant T cells express lymphotoxin alpha and drive endothelial activation in cutaneous T cell lymphoma

    DEFF Research Database (Denmark)

    Lauenborg, Britt; Christensen, Louise; Ralfkiaer, Ulrik

    2015-01-01

    Lymphotoxin α (LTα) plays a key role in the formation of lymphatic vasculature and secondary lymphoid structures. Cutaneous T cell lymphoma (CTCL) is the most common primary lymphoma of the skin and in advanced stages, malignant T cells spreads through the lymphatic to regional lymph nodes...... to internal organs and blood. Yet, little is known about the mechanism of the CTCL dissemination. Here, we show that CTCL cells express LTα in situ and that LTα expression is driven by aberrantly activated JAK3/STAT5 pathway. Importantly, via TNF receptor 2, LTα functions as an autocrine factor by stimulating...... expression of IL-6 in the malignant cells. LTα and IL-6, together with VEGF promote angiogenesis by inducing endothelial cell sprouting and tube formation. Thus, we propose that LTα plays a role in malignant angiogenesis and disease progression in CTCL and may serve as a therapeutic target in this disease....

  2. Differential expression of cell-cycle regulators in human beta-cells derived from insulinoma tissue.

    Science.gov (United States)

    Ueberberg, Sandra; Tannapfel, Andrea; Schenker, Peter; Viebahn, Richard; Uhl, Waldemar; Schneider, Stephan; Meier, Juris J

    2016-05-01

    The low frequency of beta-cell replication in the adult human pancreas limits beta-cell regeneration. A better understanding of the regulation of human beta-cell proliferation is crucial to develop therapeutic strategies aiming to enhance beta-cell mass. To identify factors that control beta-cell proliferation, cell-cycle regulation was examined in human insulinomas as a model of increased beta-cell proliferation (n=11) and healthy pancreatic tissue from patients with benign pancreatic tumors (n=9). Tissue sections were co-stained for insulin and cell-cycle proteins. Transcript levels of selected cell-cycle factors in beta-cells were determined by qRT-PCR after performing laser-capture microdissection. The frequency of beta-cell replication was 3.74±0.92% in the insulinomas and 0.11±0.04% in controls (p=0.0016). p21 expression was higher in insulinomas (p=0.0058), and Rb expression was higher by trend (p=0.085), whereas p16 (pcell-cycle factors in beta-cells derived from insulinomas and healthy adults differs markedly. Targeting such differentially regulated cell-cycle proteins may evolve as a future strategy to enhance beta-cell regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Inhibition of dengue virus 3 in mammalian cell culture by synthetic ...

    African Journals Online (AJOL)

    Purpose: To evaluate the inhibition of Dengue virus 3 by synthetic siRNAs targeting the untranslated regions UTR and structural regions of DENV3 genome in Vero-81 cell line. Methods: Vero-81 cells transfected with synthetic siRNAs were challenged by DENV3. The effectiveness of siRNAs was confirmed by four ...

  4. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  5. CD100 on NK cells enhance IFNgamma secretion and killing of target cells expressing CD72.

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    Sa'ar Mizrahi

    2007-09-01

    Full Text Available NK cells are able to kill tumor and virus-infected cells without the need of prior antigen stimulation. The killing of these target cells is regulated by inhibitory, lysis and co-stimulatory receptors that are expressed on the surface of NK cells.CD100 (Semaphorin 4D, a 150kD transmembrane protein, is expressed on the surface of activated NK cells as a homodimer, mediates the killing of target cells by binding to CD72. CD100 is not involved directly in the killing process but is rather increases NK cytotoxicity by enhancing the adhesion between NK cells and their targets. This increased adhesion leads to a more efficient killing and enhanced IFNgamma secretion.Since CD72 is expressed on antigen presenting cells (APC and the CD100-CD72 interaction lead to the shading of CD100, we suggest that NK interacting with APC cells could be the early source of soluble CD100 which is crucial for the formation of antigen specific immune response. CD100-CD72 interaction can be the mechanism by which NK cell communicate with B cells.

  6. PAX2 regulates ADAM10 expression and mediates anchorage-independent cell growth of melanoma cells.

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    Sophia Boyoung Lee

    Full Text Available PAX transcription factors play an important role during development and carcinogenesis. In this study, we investigated PAX2 protein levels in melanocytes and melanoma cells by Western Blot and immunofluorescence analysis and characterized the role of PAX2 in the pathogenesis of melanoma. In vitro we found weak PAX2 protein expression in keratinocytes and melanocytes. Compared to melanocytes increased PAX2 protein levels were detectable in melanoma cell lines. Interestingly, in tissue sections of melanoma patients nuclear PAX2 expression strongly correlated with nuclear atypia and the degree of prominent nucleoli, indicating an association of PAX2 with a more atypical cellular phenotype. In addition, with chromatin immunoprecipitation assay, PAX2 overexpression and PAX2 siRNA we present compelling evidence that PAX2 can regulate ADAM10 expression, a metalloproteinase known to play important roles in melanoma metastasis. In human tissue samples we found co-expression of PAX2 and ADAM10 in melanocytes of benign nevi and in melanoma cells of patients with malignant melanoma. Importantly, the downregulation of PAX2 by specific siRNA inhibited the anchorage independent cell growth and decreased the migratory and invasive capacity of melanoma cells. Furthermore, the downregulation of PAX2 abrogated the chemoresistance of melanoma cells against cisplatin, indicating that PAX2 expression mediates cell survival and plays important roles during melanoma progression.

  7. In vitro expression of native H5 and N1 genes of avian influenza virus by using Green Fluorescent Protein as reporter

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    Risza Hartawan

    2011-10-01

    Full Text Available The hemagglutinin and neuraminidase are important immunogen of avian influenza virus that are suitable for recombinant experimentation. However, both genes have been experienced rapid mutation resulting in diverse variety of genotypes. Hence, gene expression in recombinant systems will be difficult to predict. The objective of the study was to examine expression level of H5 and N1 genes from a field isolate by cloning the genes into expression vector pEGFP-C1. Two clones respresenting fulllength of H5 and N1 gene in plasmid pEGFP-C1 were transfected into chicken embryo fibroblasts (CEF, rabbit kidney (RK13 and African green monkey kidney (VERO cells using Lipofectamine ‘Plus’ reagent. The experiment showed level of gene expression in the VERO cell was higher than in the RK13 and CEF cells. Observations using fluorescent microscopy and Western blotting revealed that the N1 gene was expressed better in all cells compared to the H5 gene.

  8. Mural granulosa cell gene expression associated with oocyte developmental competence

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    Jiang Jin-Yi

    2010-03-01

    Full Text Available Abstract Background Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. Methods Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC. Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. Results The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox and nerve growth factor receptor associated protein 1 (Ngfrap1, which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2, which is involved in the regulation of extracellular matrix organization and biogenesis. Conclusions The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and

  9. Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells.

    Science.gov (United States)

    Khan, Mohammed I; Czarnecka, Anna M; Lewicki, Sławomir; Helbrecht, Igor; Brodaczewska, Klaudia; Koch, Irena; Zdanowski, Robert; Król, Magdalena; Szczylik, Cezary

    2016-01-01

    Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs)-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have

  10. Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Mohammed I Khan

    Full Text Available Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin.Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2 and metastatic (ACHN renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis.Metastatic RCC cell lines (ACHN and Caki-1 demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations

  11. Alterations in integrin expression modulates invasion of pancreatic cancer cells.

    LENUS (Irish Health Repository)

    Walsh, Naomi

    2009-01-01

    BACKGROUND: Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood. METHODS: In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression. RESULTS: Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin beta1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion. CONCLUSION: Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma.

  12. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    Science.gov (United States)

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but al