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Gene Transfer Method (gene + transfer_method)
Selected AbstractsAntitumor effect of simultaneous transfer of interleukin-12 and interleukin-18 genes and its mechanism in a mouse bladder cancer modelINTERNATIONAL JOURNAL OF UROLOGY, Issue 8 2004SATOKO HIKOSAKA Abstract Background:, The objectives of this study were to evaluate the antitumor effects of the simultaneous introduction of interleukin 12 (IL-12) and IL-18 genes into a mouse bladder cancer cell line (MBT2). We intended to compare these with those of either gene alone and to investigate the mechanism of the effects induced by the transfer of IL-12 and/or IL-18 genes in this model system. Methods:, We transfected the IL-12 and/or IL-18 genes into MBT2 cells by the liposome-mediated gene transfer method. We confirmed the secretion of IL-12 and/or IL-18 by enzyme-linked immunosorbent assay. Parental (MBT2/P), IL-12-transfected (MBT2/IL-12), IL-18-transfected (MBT2/IL-18) or both IL-12- and IL-18-transfected (MBT2/Both) cells were subcutaneously or intravenously injected into syngeneic C3H mice. To analyze the mechanism of tumor rejection, these clones were subcutaneously injected into naive nude mice and those depleted with natural killer (NK) cells by antibody. Results:, MBT2/IL-12, MBT2/IL-18 and MBT2/Both were completely rejected when they were injected subcutaneously or intravenously into syngeneic mice. However, MBT2/IL-12, but not MBT2/IL-18, could grow in nude mice. Moreover, the antitumor effect of MBT2/IL-18 was partially abrogated when injected into nude mice of which NK cells were depleted by antibody treatment. MBT2/Both was completely rejected in both nude mice with and without NK cells. Conclusion:, The results of the present study indicate that T cells and NK cells seem to play important roles in the antitumor effects by the secretion of IL-12 and IL-18, respectively, and MBT2/Both possesses both mechanisms. [source] Ex vivo hypothermic recirculatory adenoviral gene transfer to the transplanted pig heartTHE JOURNAL OF GENE MEDICINE, Issue 7 2006Keiji Oi Abstract Background To facilitate the application of adenoviral gene therapy in clinical heart transplantation, we developed an ex vivo hypothermic recirculatory adenoviral gene transfer method to the transplanted pig heart. Methods Experimental animals were assigned into three groups; controls, 1 × 108 plaque-forming units (pfu)/ml group and 1 × 109 pfu/ml group. During the 30 min gene transfer perfusion, 200 ml of University of Wisconsin solution containing the adenoviral vector was recirculated through the coronary vessels. The myocardial temperature was maintained below 4 °C and the perfusion pressure was adjusted at 50 mmHg. Results Cardiac myocyte transduction efficiencies in the 1 × 108 pfu/ml group were 0.04% and 0.07%, whereas transduction efficiencies in the 1 × 109 pfu/ml group were widely distributed from 0.45% to 22.62%. The gene transduction efficiency increased with the virus titer. Additionally, no difference in the transduction efficiency was observed between different segments of the left ventricle. The current gene transfer method at 1 × 109 pfu/ml of adenovirus titer enabled homogeneous gene transduction into the transplanted pig heart up to a maximum of 22.62%. Conclusions This model can be applied to a large isolated heart and will greatly facilitate the investigation of gene therapy in large animal models of heart transplantation. Copyright © 2006 John Wiley & Sons, Ltd. [source] An efficient gene transfer method mediated by ultrasound and microbubbles into the kidneyTHE JOURNAL OF GENE MEDICINE, Issue 1 2005Hiromi Koike Abstract Background Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non-viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression. Methods In this study, we examined the potential of ultrasound for gene transfer into the kidney. First, we transfected rat left kidney with luciferase plasmid mixed with microbubbles, Optison, to optimize the conditions (duration of ultrasound and concentration of Optison). Then, 4, 7, 14 and 21 days after gene transfer, luciferase activity was measured. Next, localization of gene expression was assessed by measuring luciferase activity and green fluorescent protein (GFP) expression. Expression of GFP plasmid was examined under a fluorescence microscope at 4 and 14 days after gene transfer. Finally, to examine the side effects of this gene transfer method, biochemical assays for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cre) were performed. Results Optison and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 70,80% of total glomeruli could be transfected. Also, a significant dose-dependent effect of Optison was observed as assessed by luciferase assay (Optison 25%: 12.5 × 105 relative light units (RLU)/g tissue; 50%: 31.3 × 105 RLU/g tissue; 100%: 57.9 × 105 RLU/g tissue). GFP expression could be observed in glomeruli, tubules and interstitial area. Results of blood tests did not change significantly after gene transfer. Conclusions Overall, an ultrasound-mediated gene transfer method with Optison enhanced the efficiency of gene transfer and expression in the rat kidney. This novel non-viral method may be useful for gene therapy for renal disease. Copyright © 2004 John Wiley & Sons, Ltd. [source] Suppressive role of leukocyte cell,derived chemotaxin 2 in mouse anti,type II collagen antibody,induced arthritisARTHRITIS & RHEUMATISM, Issue 2 2008Akinori Okumura Objective We previously reported that the Val58Ile polymorphism of the leukocyte cell,derived chemotaxin 2 gene (LECT2) is associated with the severity of rheumatoid arthritis (RA). To define the role of LECT2 in inflammatory arthritides, we investigated the development of collagen antibody,induced arthritis (CAIA) in LECT2-deficient (LECT2,/,) mice. Methods CAIA was induced in mice by administering anti,type II collagen antibodies followed by lipopolysaccharide. Daily assessment of hind paw swelling was used to monitor the development of arthritis. The histopathologic features and expression of inflammatory cytokines were also analyzed. We confirmed the role of LECT2 by introducing a LECT2 expression vector into LECT2,/, mice, using a hydrodynamic gene transfer method. Results Arthritis in LECT2,/, mice was significantly exacerbated compared with that in wild-type (WT) controls. Histopathologic assessment of the tarsal joints showed that inflammation and erosion of cartilage and bone in LECT2,/, mice were more severe than that in controls. Interleukin-1, (IL-1,), IL-6, and certain chemokines were present at significantly higher levels in the arthritic hind paws of LECT2,/, mice. In contrast, the amount of LECT2 in the serum and locally in the hind paws was higher in arthritic WT mice. Finally, hydrodynamic gene transfer experiments revealed that the severity of arthritis was reduced by the systemic expression of exogenous mouse LECT2 protein in LECT2,/, mice. Conclusion These results strongly suggest that LECT2 directly suppresses the development of CAIA. Manipulation of LECT2 might provide a rationale for novel therapeutic approaches to the treatment of inflammatory arthritides such as RA. [source] Non-viral strategies of intra-ocular gene deliveryACTA OPHTHALMOLOGICA, Issue 2009F BEHAR-COHEN Purpose Systemic anti TNF strategies are efficient to treat intraocular inflammation but require repeated injections and are associated to severe systemic side effects. Our aim was to develop a non viral gene transfer method to produce locally anti-inflammatory proteins in a sustained and minimally invasive manner in the ocular media. For this purpose, we have transformed the ciliary muscle into a bioreactor, using an electrically assisted gene transfer technique. Methods Electrotransfer (ET) of plasmids, encoding for different variants of TNF alpha soluble receptors, was performed in the ciliary muscle cells. Using toptimized conditions, soluble receptors were dosed in the ocular media up to 8 months after a single treatment. The technique has been applied in two models of intraocular inflammation: Endotoxin-Induced Uveitis (EIU) and auto immune experimental uveitis (EAU) in rats. Results When performed 8 days or 3 months before the LPS challenge, ET significantly reduced both clinical and histological signs of EIU. Particularly, iNOS, IL6 and TNF were down regulated while IL10 was upregulated. Importantly, systemic TNF alpha was not decreased demonstrating a local effect of the treatment. In EAU, ET significantly delayed the onset of EAU and deceased its severity. Similarly, a switch towards a Th2 cytokines profile was observed in the ocular media without any effect on systemic TNF alpha. Conclusion - ET is a safe and efficient non viral method to produce locally TNF alpha soluble receptors. - Local anti TNF allows for a local intraocular immunomodulation, without affecting systemic TNF. ET could therefore be used to reduce systemic side effects of anti TNF and prevent repeated injections. [source] | ||||||||||