Q127599704
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B210 cells are murine (BALB/c) cells transformed by bcr-abl fusion gene. After intravenous administration they are capable of inducing leukaemia-like disease in syngeneic mice. From these cells a thymidine-kinase less subline was derived. It was significantly less pathogenic than the parental cells. However, a highly pathogenic clone denoted B210cTK-/cl-2 was isolated from its population. As determined by Western blotting, these cells produced more p210 protein than the parental B210 cells. To successfully transfect these cells a modified electroporation method was introduced. Bicistronic plasmids carrying gene for herpes simplex thymidine kinase (HSV TK) and the gene for either granulocyte-monocyte colony stimulation factor (GM-CSF), interleukin-2 (IL-2) or interleukin 12 (IL-12) were used for the transfection experiments. Gradually, cell lines producing these cytokines were isolated in media supplemented with hypoxantin, aminopterin and thymidine (HAT). All of them were highly sensitive to ganciclovir in vitro confirming that the cells produced HSV TK. The genetic modification of B210cTK-/cl-2 was associated neither with the alteration of p210 bcr-abl production nor with any changes in expression of MHC class I molecules. From populations of each of the three lines several cell clones were isolated and tested for the production of the respective cytokines. The original uncloned population and several clones differing in the cytokine production were administered intravenously into mice. All animals survived without symptoms of the disease suggesting that the gene-modification was associated with the loss of pathogenicity. Keywords: CML, Bcr-Abl, HSV TK, cytokines, gene-modified tumour cells, pathogenicity.
- MeSH
- adjuvancia imunologická genetika MeSH
- bcr-abl fúzní proteiny analýza MeSH
- cytokiny biosyntéza genetika MeSH
- ganciklovir terapeutické užití MeSH
- geny abl MeSH
- MHC antigeny I. třídy analýza MeSH
- MHC antigeny II. třídy analýza MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nádorová transformace buněk MeSH
- thymidinkináza genetika MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
One of the gene therapy strategies in oncology is immunization with cancer cells that express various cytokines. We used a thymidine-kinase deficient (cTK-) cell line designated 123IA, which had been derived from HPV16-transformed mouse (C57BL/6) cells MK16/I/III/ABC (MK16). To obtain genetically modified cells, 123IA cells were transfected with bicistronic plasmid vectors carrying the herpes simplex type 1 thymidine kinase (HSV TK) gene and either the gene for the mouse B7.1 (CD80) co-stimulatory molecule or the gene for the monocyte-chemoattractant protein 1 (MCP-1). For control purposes, a plasmid vector carrying only the HSV TK gene was used. The transfected cells were cultivated in medium supplemented with hypoxanthine, aminopterin and thymidine. For comparative purposes we also used B9 cells, which express the granulocyte-macrophage colony stimulation factor (GM-CSF) and had been derived from 123A cells by transduction with the recombinant adeno-associated virus carrying the HSV TK gene and the mouse GM-CSF gene. All of the cell lines isolated were found to be sensitive to minute amounts of ganciclovir, revealing the production of HSV TK, and to express the respective transgenes. When inoculated into 5-week-old female syngeneic mice, cells expressing either GM-CSF or B7.1 were non-oncogenic. On the other hand, nearly all mice inoculated with MCP-1-producing cells developed tumours, though considerably later than animals inoculated with the same dose of the parental MK16 cells. Animals injected with GM-CSF- or B7.1-producing cells were protected against challenge with the parental MK16 cells. When another mouse (C57BL/6) HPV16-transformed oncogenic cell line, TC-1, which differs from the MK16 cells in a number of properties such as MHC class I and B7.1 expression, was used for the challenge, the protective effect was much less pronounced.
- MeSH
- antigeny CD80 genetika MeSH
- buněčné linie MeSH
- chemokin CCL2 genetika MeSH
- faktor stimulující granulocyto-makrofágové kolonie genetika MeSH
- financování organizované MeSH
- ganciklovir farmakologie MeSH
- geny MHC třídy I MeSH
- imunizace MeSH
- lidský papilomavirus 16 genetika MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- onkogenní proteiny virové genetika MeSH
- protinádorové vakcíny imunologie MeSH
- represorové proteiny genetika MeSH
- transfekce MeSH
- transformované buněčné linie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
This paper describes the direct label-free detection of antibodies against the Epstein-Barr virus (anti-EBNA) using a surface plasmon resonance (SPR) biosensor. The antibody detection was performed using the immunoreaction between anti-EBNA and a respective synthetic peptide (EBNA-1), which was conjugated with bovine serum albumin (BSA-EBNA) and immobilized on the sensor surface. Three immobilization chemistries for the attachment of BSA-EBNA were investigated to optimize ligand density and minimize loss of EBNA-1 immunoreactivity. The developed SPR biosensor functionalized with the optimal immobilization method was calibrated and characterized in terms of detection limit, reproducibility, regenerability and storability. It was demonstrated that the sensor is capable of detecting concentrations of anti-EBNA as low as 0.2 ng/ml (approximately 1 pM) both in buffer and 1% human serum and can be stored and regenerated for repeated use.
- MeSH
- analýza selhání vybavení MeSH
- biosenzitivní techniky metody přístrojové vybavení MeSH
- design vybavení MeSH
- financování organizované MeSH
- imunoanalýza metody přístrojové vybavení MeSH
- protilátky analýza imunologie MeSH
- virus Epsteinův-Barrové - jaderné antigeny analýza imunologie MeSH
- virus Epsteinův-Barrové imunologie MeSH
- MeSH
- DNA vakcíny terapeutické užití MeSH
- faktor stimulující granulocyto-makrofágové kolonie genetika MeSH
- finanční podpora výzkumu jako téma MeSH
- interleukin-2 genetika MeSH
- myši MeSH
- nádory terapie MeSH
- protinádorové vakcíny aplikace a dávkování terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
