Jiro Imanishi

Interleukin (IL)-21 and IL-15 genetic transfer synergistically augments therapeutic antitumor immunity and promotes regression of metastatic lymphoma

IL-21 supports proliferation of mature T and B cells and facilitates expansion and maturation of natural killer (NK) cells in synergy with IL-15. However, the biological implications of IL-21 in vivo have not been fully elucidated. IL-21 and IL-15 expression plasmids were intravenously injected under high pressure into the tail veins of mice, which were subsequently challenged by an intravenous injection of RLmale1 lymphoma cells. The IL15 gene transfection significantly reduced the numbers of metastatic tumor foci in the liver. In contrast, when IL21 and IL15 genes were cotransfected, complete regression was achieved in 80% of the mice. The cytokine gene therapy was also performed in mice that had been intravenously inoculated with the tumor cells. Forty percent of mice that received a single injection of a mixture of cytokine genes successfully rejected the preestablished metastatic lymphoma and showed tumor-free survival for more than 300 days. IL-21 significantly elevated the cytotoxic T lymphocyte activity in the spleens of tumor-inoculated mice, while the two cytokines augmented NK killing activity in a synergistic manner. These results strongly suggest that the codelivery of IL-21 and IL-15 elicits powerful antitumor immune responses, resulting in marked therapeutic efficacy against metastatic tumors.

Relation between Cytokines and Helicobacter pylori in Gastric Cancer

Helicobacter pylori is etiologically involved in the development of gastric cancer and infected gastric mucosa has been shown to possess elevated levels of cytokines [for example interleukin (IL)‐1β, IL‐6 and IL‐8]. Because specific cytokines have also been shown to enhance the development of certain cancers, we examined the relationship between the levels of cytokines, the type and stage of gastric cancers, and the H. pylori infection.

Cytokine Expression and Production by Purified Helicobacter pylori Urease in Human Gastric Epithelial Cells

ABSTRACT Cytokines have been proposed to play an important role inHelicobacter pylori-associated gastroduodenal diseases, but the exact mechanism of the cytokine induction remains unclear. H. pylori urease, a major component of the soluble proteins extracted from bacterial cells, is considered to be one of the virulence factors for the inflammation in the gastric mucosa that is produced in H. pylori infection. However, the response of human gastric epithelial cells to the stimulation of urease has not been investigated. In the present study, we used human gastric epithelial cells in a primary culture system and examined whetherH. pylori urease stimulates the gastric epithelial cells to induce proinflammatory cytokines by reverse transcription-PCR and enzyme-linked immunosorbent assay. First, by using peripheral blood mononuclear cells (PBMC) and a gastric cancer cell line (MKN-45 cells), we confirmed the ability of purified H. pylori urease to induce the production of proinflammatory cytokines. Furthermore, we demonstrated that the human gastric epithelial cells produced interleukin-6 (IL-6) and tumor necrosis factor alpha, but not IL-8, following stimulation with purified urease. The patterns of cytokine induction differed among human PBMC, MKN-45 cells, and human gastric epithelial cells. These results suggest that the human gastric epithelial cells contribute to the induction of proinflammatory cytokines by the stimulation of H. pylori urease, indicating that the epithelial cells were involved in the mucosal inflammation that accompanied H. pylori infection.

Highly efficient suicide gene expression in hepatocellular carcinoma cells by Epstein-Barr virus-based plasmid vectors combined with polyamidoamine dendrimer

The present study was aimed at devising an efficient nonviral strategy for suicide gene therapy of hepatocellular carcinoma (HCC). To improve the efficiency of DNA delivery and expression, we applied Epstein-Barr virus (EBV)-based plasmid vectors instead of conventional plasmid vectors and combined them with cationic liposome (EBV/lipoplex) or polyamidoamine dendrimer (PAAD) (EBV/polyplex). When the β-galactosidase gene was transferred to HuH7, PLC/PRF/5, or HLE cells, ≤50-fold higher β-galactosidase activities were demonstrated in the cells transfected with EBV vector compared with those transfected with conventional plasmid vectors. PAAD-mediated transfection of HCC with pSES.Tk (an EBV-based vector carrying the herpes simplex virus-1 thymidine kinase gene) resulted in a marked reduction in viable cell number by the addition of ganciclovir (GCV). The HCC cells transfected with pSES.Tk/PAAD showed 100- to 1000-fold higher susceptibilities to GCV than those transfected with pS.Tk (a conventional plasmid vector carrying herpes simplex virus-1 thymidine kinase gene)/PAAD. The pSES.Tk-transfected HCC cells were effectively killed by day 9 in culture with a clinically feasible concentration of GCV (25 μM), whereas the pS.Tk-transfected cells survived the culture. These results demonstrate highly efficient suicide gene transfer into various HCC cells by EBV-based plasmid vectors in vitro, suggesting the possible application of this nonviral vector system to gene therapy of HCC.

Successful genetic transduction in vivo into synovium by means of electroporation

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.

Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo

To accomplish efficient nonviral gene therapy against prostate cancer (PC), Epstein–Barr virus (EBV)-based plasmid vectors containing EBNA1 gene and oriP were employed and combined with a cationic polymer or cationic lipid. When EBV-plasmid/poly-amidoamine dendrimer complex was injected into PC-3-derived tumors established in severe combined immunodeficiency mice, a considerable expression of marker gene was obtained in the tumors, and the expression level was more than eight-fold higher than that achieved by conventional plasmid vector/dendrimer. Since most PC cells express the apoptotic signal molecule Fas (Apo-1/CD95) on their surface, Fas ligand (FasL) gene was transferred into PC cells to kill the tumor cells. In vitro transfection with pGEG.FasL (an EBV-plasmid with the FasL gene) significantly reduced the viability of PC cells, which subsequently underwent apoptosis. Intratumoral injections of pGEG.FasL into PC induced significant growth suppression of the xenograft tumors, in which typical characteristics of apoptosis were demonstrated by TUNEL staining and electron microscopic observations. When pGEG.FasL transfer was accompanied by systemic administrations of cisplatin, the tumors were inhibited even more remarkably, leading to prolonged survival of the animals. FasL gene transfection by means of EBV-based plasmid/cationic macromolecule complexes may provide a practical therapeutic strategy against PC.

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors.

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein–Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 μg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 μg of DNA. More than 70% of liver cells stained with X-gal when β-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver. Gene Therapy (2001) 8, 1508–1513.

Role of apoptosis induction in both peripheral lymph nodes and thymus in progressive loss of CD4+ cells in SHIV-infected macaques.

To investigate the role of apoptosis in the progressive loss of CD4+ lymphocytes in HIV infection, we have used macaques infected with SHIV, a hybrid virus of HIV and simian immunodeficiency virus (SIV). In the present study, we sequentially analyzed apoptosis induction in the acute phase of SHIV infection. Four macaques infected with a pathogenic SHIV, SHIV89.6P, and four macaques infected with a nonpathogenic SHIV, NM-3rN, were analyzed during the first 2 or 4 weeks postinfection. In the 89.6P-infected macaques the number of peripheral CD4+ cells sharply decreased at 2 weeks postinfection and was maintained below 50/microl until 4 weeks postinfection, while in the NM-3rN-infected macques the number of the CD4+ cells did not change significantly. Plasma viral loads peaked at 2 and 2-3 weeks postinfection, and the peak values were about 1 x 10(9) and 10(6)-10(7) copies/ml in the 89.6P- and the NM-3rN-infected macaques, respectively. In the 89.6P-infected macaques, Fas antigen expression and the extent of apoptosis in PBMCs and peripheral lymph nodes increased at 1-2 weeks postinfection. A high number of apoptotic cells was also observed in thymus sections 2 and 4 weeks postinfection. On the other hand, apoptosis was scarcely induced in the NM-3rN-infected macaques. These results suggest that the extent of apoptosis induction is closely correlated with the pathogenicity of SHIV and that the apoptosis induction in peripheral lymphoid tissues and thymus, where T cell maturation occurs, may play an important role in the depletion of CD4+ lymphocytes in 89.6P infection.

Interleukin-2 abolishes myeloid cell accumulation induced by Lewis lung carcinoma.

Immune aberration in cancer patients can be at least partly ascribed to an accumulation of immature myeloid cells and monocytes/macrophages with immunosuppressive functions. Mice implanted with Lewis lung carcinoma 2 (LL/2) cells show marked splenomegaly as the tumors progress, and this condition is accompanied by impaired T cell activities. We characterized the cells that accumulated in the spleens of LL/2 tumor-bearing mice and attempted to restore the normal cell population by employing interleukin-2 (IL-2). Flow cytometric analysis revealed that the cells expressing Mac1, B7, NK-K1, Gra-1, and MHC class II antigens on their surfaces drastically decreased in number when LL/2 had been engineered to produce IL-2. IL-2 also restored the concanavalin A (ConA)-mediated proliferative response and IL-2 production of the spleen cells. The in vivo growth of IL-2-producing tumors was significantly slower than that of parental LL/2 cells. Therefore, local IL-2 production may reverse systemic immune abnormality by stopping myeloid cell accumulation.