The cytokine inducible SH2‐domain protein (CISH) is a well‐known STAT5 target gene, but its role in the immune system remains uncertain. In this study, we found that CISH is predominantly induced ...during dendritic cell (DC) development from mouse bone marrow (BM) cells and plays a crucial role in type 1 DC development and DC‐mediated CTL activation. CISH knockdown reduced the expression of MHC class I, co‐stimulatory molecules and pro‐inflammatory cytokines in BMDCs. Meanwhile, the DC yield was markedly enhanced by CISH knockdown via cell‐cycle activation and reduction of cell apoptosis. Down‐regulation of cell proliferation at the later stage of DC development was found to be associated with CISH‐mediated negative feedback regulation of STAT5 activation. In T‐cell immunity, OT‐1 T‐cell proliferation was significantly reduced by CISH knockdown in DCs, whereas OT‐2 T‐cell proliferation was not affected by CISH knockdown. CTLs generated by DC vaccination were also markedly reduced by CISH knockdown, followed by significant impairment of DC‐based tumor immunotherapy. Taken together, our data suggest that CISH expression at the later stage of DC development triggers the shutdown of DC progenitor cell proliferation and facilitates DC differentiation into a potent stimulator of CTLs.
We have previously demonstrated that semimature dendritic cell- (smDC-) based immunotherapy is effective for the treatment of collagen-induced arthritis (CIA) prior to disease onset. In the present ...study, we examined the efficacy of combination therapy with smDCs and methotrexate (MTX) in advanced CIA with a score of 2-3. Combination therapy with low-dose MTX and type II collagen- (CII-) pulsed smDCs (CII-smDCs) was more effective in inhibiting disease progression than high or low-dose MTX alone or a combination of high dose MTX and CII-smDCs. The effect of CII-smDCs alone was also comparable to the combination therapy. CD4+Foxp3+ Treg populations and IL-10 secretion markedly increased, and CII-specific autoreactive T cells decreased in mice treated with CII-smDCs alone or in combination with MTX. Combination therapy reduced the secretion of interferon-γ (IFN-γ) and IL-17 with little influence on the IL-4 secretion in the mixed leukocyte reaction. These results imply that the combination therapy with low-dose MTX and smDCs is effective in controlling advanced CIA by enhancing Treg population and suppresses antigen-specific Th1/Th17 immunity, rather than initiating Th1 to Th2 immune deviation. Our findings provide a better understanding of the DC therapy in combination with MTX for the treatment of patients with rheumatoid arthritis (RA).
We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by ...caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5'-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32-), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32- produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32- compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32- contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18.
We found that JBP1, known as a human homolog (Skb1Hs) of Skb1 of fission yeast, interacts with NS3 of the hepatitis C virus in a yeast two-hybrid screen. Amino acid sequence analysis revealed that ...Skb1Hs/JBP1 contains conserved motifs ofS-adenosyl-l-methionine-dependent protein-arginine methyltransferases (PRMTs). Here, we demonstrate that Skb1Hs/JBP1, named PRMT5, is a distinct member of the PRMT family. Recombinant PRMT5 protein purified from human cells methylated myelin basic protein, histone, and the amino terminus of fibrillarin fused to glutathione S-transferase. Myelin basic protein methylated by PRMT5 contained monomethylated and dimethylated arginine residues. Recombinant glutathione S-transferase-PRMT5 protein expressed in Escherichia coli also contained the catalytic activity. Sedimentation analysis of purified PRMT5 on a sucrose density gradient indicated that PRMT5 formed distinct homo-oligomeric complexes, including a dimer and tetramer, that comigrated with the enzyme activity. The PRMT5 homo-oligomers were dissociated into a monomer in the presence of a reducing agent, whereas a monomer, dimer, and multimer were detected in the absence or at low concentrations of a reducing agent. The results indicate that both covalent linkage by a disulfide bond and noncovalent association are involved in the formation of PRMT5 homo-oligomers. Western blot analysis of sedimentation fractions suggests that endogenous PRMT5 is present as a homo-oligomer in a 293T cell extract. PRMT5 appears to have lower specific enzyme activity than PRMT1. Although PRMT1 is known to be mainly located in the nucleus, human PRMT5 is predominantly localized in the cytoplasm.
Solid tumors contain normoxic and hypoxic regions depending on the distance from the capillary. Normal cells may also be exposed to hypoxia under certain physiological conditions. Tumor hypoxia has ...been shown to associate strongly with tumor propagation and malignant progression. Hypoxia-inducible factor (HIF)-1alpha is stable under hypoxia and induces transcription of target genes by binding to the hypoxia-response element (HRE). Here we investigated the oncolytic effects of a novel adenovirus mutant with a deleted E1B55 gene (Ad.Delta55.HRE), in which the expression of E1A, which is essential for adenoviral replication, is regulated under the control of an HRE-expression system. Ad.Delta55.HRE expressed E1A under normoxia and more E1A under hypoxia and exhibited oncolytic effects on various cultured tumor cells, but its cytotoxic effect is relatively attenuated in normal fibroblast cells under normoxic and hypoxic conditions. Ad.Delta55.HRE lysed Huh-7 hepatoma cells stably expressing HIF-1alpha more effectively compared to parental cells. Ad.Delta55.HRE treatment exhibited significant antitumor activity in PC-3 prostate- and MDA-MB-435 breast tumor-bearing nude mice in which HIF-1alpha protein was immunohistochemically detected. The E1A and hexon proteins of adenovirus were immunostained in MDA-MB-435 xenografts after Ad.Delta55.HRE treatment, suggestive of viral replication. Our results suggest that Ad.Delta55.HRE may be useful for the treatment of solid tumors.
TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, selectively induce apoptosis in various transformed cell lines but not in almost-normal tissues. It is regulated by 2 death ...receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2 and 2 decoy receptors, TRAIL-R3 and TRAIL-R4. However, the determining factors of the sensitivity to TRAIL-induced apoptosis are not clearly understood. Herein, we investigated the expression of TRAIL-R, c-FLIP, FADD-like interleukin-1beta-converting enzyme inhibitory protein, and TRAIL-induced apoptosis in human hepatocellular carcinoma (HCC) cell lines. Seven of ten HCC cell lines showed resistance to TRAIL-induced apoptosis and five of seven TRAIL-resistant cell lines became sensitive to TRAIL by co-treatment with cycloheximide. In HCC cell lines, their TRAIL resistance did not correlate with the basal expression level of TRAIL receptors or c-FLIP, however, in human tissues, TRAIL-R1 and TRAIL-R2 expressions were notably decreased compared to normal counterpart. Cisplatin showed synergistic effect on TRAIL-induced apoptosis in most HCC cell lines regardless of their p53 status and TRAIL-R1 was induced by cisplatin treatment in certain cell lines. Inhibition of nuclear factor K B (NF-kappaB) by SN50, a peptide inhibitor of NF-KB activity, had no effect on TRAIL-induced apoptosis in HCC cells. These results suggest that (a) the majority of human HCC cell lines are resistant to TRAIL-induced apoptosis and cycloheximide-sensitive short-lived antiapoptotic molecule(s) is responsible for this resistance, (b) the expression of TRAIL-R1 and TRAIL-R2 is reduced in HCC tissues, and the increased expression of TRAIL-R1 may be a mechanism of cisplatininduced sensitization to TRAIL-induced apoptosis in some HCC cells, and (c) the activation of NF-kappaB may not be involved in the TRAIL resistance of HCC cells
Hypoxia-inducible factor-1 (HIF-1) is one of the key mammalian transcription factors and shows increased levels in both protein stability and intrinsic transcriptional activity during low oxygen ...tension. Hypoxia-activated functional HIF-1 protein binds to hypoxia-responsive elements (HRE) in the enhancers of several genes including VEGF, the major player in angiogenesis, and initiates their mRNA expression. The molecular mechanisms regulating the gene expression under hypoxic conditions could increase the therapeutic window of tumor-specific delivery systems. In this study, to examine hypoxia-specific production of anti-angiogenic therapeutic gene, we constructed 5 copies of HRE (5xHRE) of human VEGF linked to soluble Tie2 (sTie2) driven by minimal SV40 promoter (5xHRE/SV40/sTie2). Our data showed that under hypoxia the secreted sTie2 selectively inhibited tube formation and migration capacities of endothelial cells in vitro. Hence, we propose that the vector system, 5xHRE/SV40/sTie2, might be a useful tool for down-regulating tumor angiogenesis under hypoxic condition.
The E1 and E2 proteins of hepatitis C virus (HCV) are believed to be the viral envelope glycoproteins that are major candidate antigens for HCV vaccine development. We reported previously that the ...replication-competent recombinant adenovirus encoding core-E1-E2 genes of HCV (Ad/HCV) produces serologically reactive E1 and E2 proteins forming a heterodimer in substantial amounts. Here, we examined immunogenicity of the E1E2 proteins copurified from HeLa cells infected with Ad/HCV virus in mice. Furthermore, we constructed a replication-defective recombinant adenovirus encoding the core-E1-E2 genes of HCV (Ad.CMV.HCV) and examined immunogenicity of the virus in mice. The mice immunized intraperitoneally with the copurified E1E2 proteins induced mainly antibodies to E2, but not to E1 by Western blot analysis. The sera of mice immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81. E2-specific cytotoxic T cells (CTLs), but not antibodies to the E1E2 antigens were induced in the mice intramuscularly immunized with Ad.CMV.HCV virus. When immunized with both Ad.CMV.HCV virus and the E1E2, mice elicited E2-specific CTLs and antibodies to the E1E2 antigens. The results suggest that immunization of Ad.CMV.HCV virus combined with E2 protein is an effective modality to induce humoral as well as cellular immune response to E2 antigen.
The cation channel of sperm （CatSper） protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are ...localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca2＊ influx, real.time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca2＊ levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression.
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