Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hIL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10(6) cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10(6) cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-gamma (IFN-gamma) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.
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PMID:Construction and characterization of retroviral vectors expressing biologically active human interleukin-12. 771 Nov 42

We have expanded ovarian tumor-infiltrating lymphocytes (TIL) in low concentrations of recombinant interleukin-2 (rIL-2) to conduct intraperitoneal adoptive immunotherapy trials in patients with ovarian cancer. We have previously demonstrated that certain T cell lines and clones derived from ovarian TIL exhibit in vitro autologous tumor-specific cytotoxicity and/or cytokine production (interferon-gamma, tumor necrosis factor-alpha) preferentially in response to autologous tumor cells. Studies that utilize a marker gene introduced into the DNA of TIL can provide useful information on specific uptake or localization of TIL at tumor sites and on the survival of TIL in vivo. We have conducted a series of preclinical experiments in which we have successfully transfected TIL with G1Na, which encodes the gene for neomycin phosphotransferase (neoR). NeoR was detected in at least 10% of CD8+ cells (mean = 10.4%) and between 2.5 and 20% of CD4+ TIL (mean = 8.5%). Transduction of ovarian TIL with G1Na caused no substantial changes to the T cell phenotypes or in vitro cytotoxicities against ovarian and hematogenous tumor cell targets, or on the rIL-2 requirements of TIL for growth and proliferation. In addition, the intact G1Na provirus in transduced TIL cells was rescuable by replication-competent retrovirus and was transferred into the genome of NIH-3T3 fibroblasts, which were rendered resistant to G418. An enhanced polymerase chain reaction (PCR) procedure utilizing detection by ethidium bromide staining was developed. The enhanced PCR detected 1 in 100,000 neoR-labeled cells. Furthermore, detection of the G1Na genome in transduced TIL by in situ hybridization with an RNA probe provided evidence for expression of the neoR gene in transduced TIL. Results obtained from these studies suggest that ovarian TIL-derived T cell lines transduced with the neoR gene post infection with the G1Na retroviral vector can be utilized to examine the in vivo trafficking pattern of ovarian TIL-derived T cell lines expanded in low concentrations of rIL-2 and their survival.
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PMID:Transduction of rIL-2 expanded CD4+ and CD8+ ovarian TIL-derived T cell lines with the G1Na (neor) replication-deficient retroviral vector. 857 11

IL-12 is a heterodimeric cytokine that is known to induce tumor regression and long-term antitumor immunity. Recombinant adeno-associated virus (rAAV) vectors are advantageous for gene therapy in that they lack pathogenicity in humans, infect dividing as well as nondividing cells, and show a broad range of infectivity. We constructed an rAAV vector expressing interleukin-12 (IL-12) for cancer immunotherapy studies in a mouse model by inserting murine IL-12 (mIL-12) p35 and p40 cDNAs into the plasmid pRep4 and inserting the encephalomyocarditis virus internal ribosomal entry site between the p35 and p40 cDNAs. The mIL-12 expression cassette containing the Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal was subcloned into the AAV plasmid p008Sub/NeoR, which contains two AAV inverted terminal repeat sequences and the NeoR gene driven by the thymidine kinase promoter. rAAV virions (10(4) infectious particles/ml) were generated by cotransfection of rAAV-mIL-12 and a helper plasmid (pAAV/Ad) into 293 cells previously infected with adenovirus 5. After infection of D6 fibroblasts with rAAV-mIL-12, G418-resistant clones were isolated. Each of the 1D D6 clones isolated produced up to 5.2 ng/10(6) cells/48 hours of mIL-12 as determined by enzyme-linked immunosorbent assay. Induction of interferon-gamma, enhanced lymphocyte proliferation, and cytotoxicity assays confirmed biologically functional IL-12 production by the vector. This is the first report indicating that an rAAV vector expresses mIL-12, which can be used to model the effects of mIL-12 alone and/or in combination with other antitumor agents.
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PMID:Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12). 1077 Jun 41

To modulate alloreactivity after hematopoietic stem cell transplantation, suicide gene-expressing donor T cells can be administered with an allogeneic T-cell-depleted bone marrow graft. Immune competence of such cells is a critical issue. The impact of the ex vivo gene transfer protocol (12-day culture period including CD3/interleukin-2 [IL-2] activation, retroviral-mediated gene transfer, and G418-based selection) on the anti-Epstein-Barr virus (EBV) potential of gene-modified cells has been examined. Cytotoxic (pCTL) and helper (pTh) cell precursor limiting dilution assays, interferon-gamma enzyme-linked immunospot, or fluorescence-activated cell sorter analysis after tetrameric HLA-A2/EBV peptide complexes revealed that the frequency of anti-EBV T cells was lower in gene-modified cells (GMCs) than in similarly cultured but untransduced T cells and was even lower than in fresh peripheral blood mononuclear cells, demonstrating both an effect of the culture and of the transduction or selection. The culture-dependent loss of EBV-reactive cells resulted from the preferential induction of activation-induced cell death in tetramer(+) cells. Replacing the initial CD3/IL-2 activation by CD3/CD28/IL-2 partially restored the anti-EBV response of GMCs by reducing the initial activation-induced cell death and enhancing the proliferation of EBV-tetramer(+) cells. Moreover, the G418 selection, and not the transduction, was directly toxic to transduced tetramer(+) cells. Replacing the G418 selection by an immunomagnetic selection significantly prevented the selection-dependent loss of EBV-specific cells. Overall, ex vivo gene modification of primary T cells can result in a significant reduction in EBV-reactive T cells through both culture-dependent and selection-dependent mechanisms. Improving immune functions of GMCs through modifications of the cell culture conditions and transduction/selection processes is critical for further clinical studies.
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PMID:Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein-Barr virus potential through both culture-dependent and selection process-dependent mechanisms. 1183 Apr 62

The porcine interferon-gamma (PoIFN-gamma) gene, in which the sequence encoding signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-PoIFN-gamma was then transformed into Pichia pastoris GS115 cells by electroporation and stable multicopy recombinant Pichia pastoris strains were selected by G418 resistance. Two recombinants of multiple inserts were obtained. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant PoIFN-gamma, 17kD and 23kD proteins, were secreted into the culture medium. Target proteins, 60% of total proteins, were obtained in the culture medium at the concentration of 108 mg/L. This is the first secreted expression of porcine interferon-gamma gene in Pichia pastoris.
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PMID:[Secreted expression of porcine interferon-gamma gene in Pichia pastoris]. 1628 13

Porcine interferon-gamma (PoIFN-gamma) of Chinese local brand, Meishan porcine, was cloned and inserted into retroviral vector pLXSN (neo r) . Using Lipofectamine, this recombinant plasmid was transfected into retroviral packing cell line, PA317 cells. These transfected cells were selected by DMEM containing 400microg/mL G418 for one week. RNA was extracted from the supernatant of these selected PA317 cells and the PoIFN-gamma gene could be amplified by RT-PCR. Pocine kidney cells and PK-15 cells were infected by the supernatant and were selected by 400 microg/mL, 600 microg/mL and 800 microg/mL G418, respectively. Those PK-15 cells were detected by indirect immunofluorescence assay and it was found that PoIFN-gamma mainly anchored in cellular membrane. The supernatant of the selected PK-15 was tested for the antiviral bioactivity after 48 hours of passage. The anti-VSV (vesicular stomatitis virus) activity in MDBK (bovine kidney cell) was 1200IU/10(6) cells. In addition, the effect of rPoIFNgamma-anti-FMDV was determined using cytopathic effect inhibition. The results indicate that PoIFN-gamma has been inserted into retroviral vector and recombinant retrovirus has been successfully packaged in PA317 cells. Furthermore, this retrovirus can infect PK-15 cells and express PoIFN-gamma with natural antiviral bioactivity and can inhibit VSV and FMDV.
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PMID:[Construction of recombinant retroviral vector carrying porcine interferon-gamma and its expression in porcine kidney cells (PK-15)]. 1743 41

This study was designed to establish an interleukin-12 (IL-12)-expressing murine Lewis lung carcinoma (LLC) cell vaccine (LLC/murine IL-12 [mIL-12]) and assess its antitumor efficacy and mechanism in vivo. The recombinant IL-12 plasmid was transfected into LLC cells and screened by G418, and positive clones were obtained. C57BL/6 tumor-bearing mouse model was established and tumor-bearing mice were randomly divided into three groups (n = 20), that is, treated with an intratumoral injection of phosphate-buffered solution, blank plasmid, or LLC/mIL-12 vaccine, respectively, at days 0, 7, and 14. Tumor size was measured before and after treatment. Tumor growth curve was plotted, cytolytic T lymphocyte (CTL) activity assay and natural killer (NK) cell activity assay were performed, CD4(+) and CD8(+) T lymphocyte were quantitated using flow cytometry, and the expression of interferon-gamma (IFN-gamma), IL-12, and interferon-inducible protein-10 (IP-10) in serum was detected by ELISA. Microvessel density was determined by immunohistochemistry after all mice were euthanized at day 21. The study revealed suppressed tumor growth, elevated levels of IFN-gamma, IP-10, and IL-12, augmented NK and CTL cell activities, and decreased microvessel density of tumor tissues. There were abundant CD4(+) and CD8(+) T lymphocyte infiltration in the vaccine group. This study demonstrated that the antitumor mechanism of LLC/mIL-12 vaccine was to promote IFN-gamma and IL-12 secretion, augment the NK and CTL cell activities, and decrease the microvessel density of tumors.
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PMID:Antitumor mechanism of recombinant murine interleukin-12 vaccine. 2057 31