PS Cell Line Research Articles

Highly Efficient Derivation of Pluripotent Stem Cells from Mouse Preimplantation and Postimplantation Embryos in Serum-Free Conditions.

Pluripotency refers to the potential of cells to generate all cell types of the embryo proper. Pluripotency spans a spectrum of cellular states. At one polar extreme is naïve pluripotency, which is identified based on the potential to form germline chimeras. At the other polar extreme is primed pluripotency, in which pluripotent cells are primed to differentiate. Mouse naïve PS cells can be derived from preimplantation embryos. Primed epiblast stem (EpiS) cells are typically isolated from epiblasts of early postimplantation mouse embryos. In this chapter, we describe protocols for highly efficient derivation and propagation of murine naïve and primed PS cell lines in serum-free conditions from preimplantation and postimplantation embryos. We describe generation of mouse naïve PS cells using LIF and inhibitors of MEK and GSK3 kinases and of mouse primed PS cells using FGF2 and IWR1 compound which induces the stabilization of Axin proteins.

Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis.

Mastitis remains a major disease of cattle with a strong impact on the dairy industry. There is a growing interest in understanding how cell mediated immunity contributes to the defence of the mammary gland against invading mastitis causing bacteria. Cytokines belonging to the IL-17 family, and the cells that produce them, have been described as important modulators of the innate immunity, in particular that of epithelial cells. We report here that expression of IL-17A and IL-17F genes, encoding two members of the IL-17 family, are induced in udder tissues of cows experimentally infected with Escherichia coli. The impact of IL-17A on the innate response of bovine mammary epithelial cells was investigated using a newly isolated cell line, the PS cell line. We first showed that PS cells, similar to primary bovine mammary epithelial cells, were able to respond to agonists of TLR2 and to LPS, provided CD14 was added to the culture medium. We then showed that secretion of CXCL8 and transcription of innate immunity related-genes by PS cells were increased by IL-17A, in particular when these cells were stimulated with live E. coli bacteria. Together with data from the literature, these results support the hypothesis that IL-17A and IL-17 F could play an important role in mediating of host-pathogen interactions during mastitis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0201-4) contains supplementary material, which is available to authorized users.

Adaptation and propagation of classical swine fever virus in different cell lines

Adaptation and propagation of 10 classical swine fever virus (CSFV) field isolates and the lapinized vaccine strain of CSFV were made in PK-15 and PS cell lines. None of the field isolates and the vaccine strain could be adapted in MDBK cell line. Four field isolates of CSFV produced cytopathic effect in both the cell lines. All the field isolates and the vaccine strain were propagated in both the cell lines up to fifth passage level after the initial adaptation. A gradual increase of the viral antigen based on the OD value in sandwich ELISA test was observed from the first to the fifth passages in the cell lines. The OD value of the virus isolates in the PK-15 cell line ranges from 0.612 to 1.538 and in the PS cell line ranges from 0.703 to 1.742 at fifth passage level. The log TCID50 of the virus isolates in the PK-15 cell line varies from 2.75 to 6.25 and in the PS cell line ranges from 2.25 to 6.50 at the fifth passage level. Although no statistically significant differences in the OD values and the logTCID50 values of the virus isolates and the vaccine virus strain was observed in the PK-15 and PS cell lines, but the use of PS cell line for adaptation and propagation of CSFV is the first report and this cell line appears to be one of the best alternatives of PK-15 cell line for propagation of CSFV.

Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

BackgroundChandipura virus (CHPV), a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.MethodsPrimers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice) in ovo (eggs), in vitro (Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.ResultsReal-time one step RT-PCR was optimized using in vitro transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml). Vero and PS cell-lines (1.2 × 103 PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.ConclusionOn account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.

Distribution of E and NS1 proteins of TBE virus in mammalian and tick cells.

Four monoclonal antibodies recognizing TBEV proteins were prepared. Three of them (2/H6, 8/A10, 5/F9) specifically recognize the structural E protein and the fourth binds to the nonstructural NS1 protein. These antibodies were used for an immunofluorescence study of TBEV protein distribution in infected mammalian host and tick vector tissue culture cells. Any differences in the distribution of the E and NS1 proteins were revealed. In porcine PS cell line the proteins were localized in the cytoplasm with a distinct increase of the signal in the perinuclear area 16 h post-infection (p.i.). The area gradually expanded and at 24 h p.i. covered the major part of the cytoplasm. The localization of the proteins in tick RA-257 cells revealed a uniform spread of the proteins in whole cytoplasm of the infected cells. The signal was very weak at 16 h p.i. and gradually increased during progressing time p.i., although, the distribution of the proteins did not exhibit any changes. Difference in viral protein distribution in the two cell types points to the possibility that the maturation process of TBEV exhibits different features in mammalian and tick cells.

Establishment of highly metastatic variants of murine colon adenocarcinoma 26 under serum‐free conditions

Tumor cell variants were established in vitro by treating a metastatic variant of murine colon adenocarcinoma 26 (P-26-select) in serum-free RPMI 1640 medium for 6 to 10 weeks. Five tumor cell lines were established from independent cultures and designated PS-1 to PS-5. The PS cells showed higher growth potential in vitro under serum-free or low-serum conditions than the parental P-26-select. In comparison to P-26-select, the PS cell lines possessed enhanced lung-colonizing ability after i.v. inoculation and metastasized spontaneously to the lung following s.c. inoculation into the flank or the right fore-footpads of BALB/c mice. Metastatic nodules were also observed in the liver. Spleens and livers of the tumor-bearing mice were enlarged mainly as a result of hematopoiesis, suggesting the production of CSF-like substance(s) by the tumor cells. The PS cells secreted increased amounts of putative autocrine growth factor(s) and CSF-like substance(s) in vitro. These characteristics might be related to the in vivo metastatic ability of the PS cells.

Antineoplastic Agents, 107. Isolation of Acteoside and Isoacteoside from Castilleja linariaefolia

The southwestern Indian paintbrush, Castilleja linariaefolia, yielded extracts that displayed in vivo activity against murine P-388 (PS) lymphocytic leukemia. Separation guided by PS cell line inhibition led to isolation of cytotoxic compounds that were identified as the known glycosides acteoside [1] (ED50 2.6 micrograms/ml) and isoacteoside [2] (ED50 10 micrograms/ml). The identifications were established by spectral measurements and degradation studies. Mannitol was also found in this plant.

Isolation, structure, synthesis, and antimitotic properties of combretastatins B-3 and B-4 from Combretum caffrum.

Further investigation of a CH2Cl2 fraction prepared from the South African tree Combretum caffrum for substances inhibitory to the murine P-388 lymphocytic leukemia (PS system) cell line has led to the isolation of two new bibenzyls, designated combretastatins B-3 and B-4, accompanied by the previously known bibenzyls 7, 8, and 9. The structure of each substance was ascertained by results of mass and nmr spectral analyses and confirmed by crystal structure determination (for 7) or synthesis. Combretastatins B-3 and B-4 gave PS ED50 values of 0.4 and 1.7 micrograms/ml, respectively, and bibenzyls 7, 8, and 9 were comparably cell growth inhibitory against the PS cell line with ED50 results of 1.7, 2.5, and 0.25 ug/ml, respectively. All the bibenzyls caused leukemia cells to accumulate in mitosis at cytotoxic drug concentrations; however, a wide range of in vitro activity against the protein tubulin (the major component of the mitotic spindle) was observed.

An experimental inactivated virus vaccine against bovine ephemeral fever 1. Studies of the virus

Studies were carried out to find methods for obtaining optimum yields of bovine ephemeral fever (BEF) virus and for concentrating the virus in order to develop inactivated virus vaccines. Cells from the SVP cell line, which was derived from the pig kidney PS cell line, were most satisfactory for growing and assaying BEF virus. BHK 21 and Vero cells also gave similar yields of virus but were not as useful for virus assay. A plaque assay in SVP cells, in which there was 0.1 μg of actinomycin D per ml of overlay, produced reproducible clear plaques and was slightly more sensitive than assays in BHK 21 cell roller tubes. High multiplicities of infection (MOI), around 1 PFU/cell, produced low yields of infectious virus, whereas decreasing the MOI approximately 100-fold led to an increase in virus yield of up to four logs. BEF virus could be concentrated using zinc acetate or ammonium sulphate but not polyethylene glycol 6000. Ammonium sulphate proved most suitable and produced an easily handled precipitate with up to 100% recovery of virus infectively, and 100-fold concentration was possible. This concentrated virus could be rapidly desalted by gel filtration through Sephadex G-75. The virus could be further purified by sucrose density gradient ultracentrifugation provided the gradient contained a protein stabilizer of 0.1% bovine serum albumin. Inactivation kinetics with 0.025% β-propiolactone was similar to that reported for other rhabdoviruses.