OBJECTIVETo study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes.

METHODSPlasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group.

RESULTSIn vivo, the mtHSP70/HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10(9) CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly.

CONCLUSIONResults of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth .

Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; pharmacology ; Genetic Therapy ; methods ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Melanoma, Experimental ; microbiology ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis ; genetics ; Salmonella typhimurium ; genetics ; immunology ; Simplexvirus ; enzymology ; genetics ; Skin Neoplasms ; therapy ; Thymidine Kinase ; genetics ; immunology ; Vaccines, Attenuated ; genetics ; immunology ; pharmacology ; Vaccines, DNA ; genetics ; immunology ; pharmacology

OBJECTIVETo investigate the therapeutic effect of cationic liposome-mediated interleukin-12 gene delivery on established murine melanoma in vivo.

METHODSThe lipofectin encapsulated pCmIL-12 plasmid was given to C57BL/6 mice on the day 3,5,7,9 after inoculation of B16 melanoma cells. The tumor size, the survival time of mice and the NK cell activity were observed.

RESULTSThe pCmIL-12 plasmid coupled with cationic liposome inhibited the tumor growth and improved the survival of mice bearing established melanoma. The activity of NK cells was also enhanced after interleukin-12 gene delivery in vivo.

CONCLUSIONCationic liposome-mediated interleukin-12 gene delivery has significantly therapeutic effects on mice melanoma in vivo.

Animals ; Cations ; DNA ; therapeutic use ; Female ; Interleukin-12 ; genetics ; therapeutic use ; Killer Cells, Natural ; immunology ; Liposomes ; Melanoma, Experimental ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Tumor Cells, Cultured

OBJECTIVETo observe the inhibitory effect of dendritic cells (DCs) activated cytotoxicity T lymphocyte (CTL) combined with MAGE-1 nonapeptide on transplanted human hepatocyte carcinoma (HCC) in nude mice.

METHODSA model of HCC transplanted tumor was established by injecting BEL-7402 cell line HCC cells subcutaneously on the back of nude mice. Successful transplantation rate was 73%. Specific CTLs (1 x 10(6)), which were activated by DCs combined with MAGE-1 nonapeptide, were injected into the site of transplanted tumor (group A, n = 5). Another group of 17 mice were treated with same amounts of different kinds of cells, and they were divided into groups B, C, D, E, and F. The growth of tumor was observed, and pathological examination was also done.

RESULTS(1) The activated lymphocytes induced by DCs combined with MAGE-1 nonapeptide could suppress the growth of tumor and reduce the tumor size. In group A, 5/5 mice survived for at least two weeks, while the tumors grew rapidly and the majority of the mice died within two weeks in other groups (groups B, C, D, E, F) (P < 0.01). (2) Extensive necrosis and apoptosis were found in transplanted tumors in group A.

CONCLUSIONSThe DCs combined with MAGE-1 nonapeptide could not only inhibit the growth of HCC, but also result in produce death and apoptosis of HCC, hence preventing tumor metastasis and recurrence. The mechanism underlying tumor immunization resulted from DCs might be enhanced in apoptosis of tumor cells. MAGE-1 nonapeptide combined with DCs might be a potential novel tumor vaccine for the treatment of HCC.

Animals ; Antigens, Neoplasm ; Apoptosis ; Cancer Vaccines ; immunology ; Dendritic Cells ; immunology ; Humans ; Liver Neoplasms, Experimental ; immunology ; pathology ; therapy ; Melanoma-Specific Antigens ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Proteins ; administration & dosage ; genetics ; Neoplasm Transplantation ; T-Lymphocytes, Cytotoxic ; immunology ; Transplantation, Heterologous

OBJECTIVETo investigate the enhanced effect of bone marrow-derived dendritic cells (DC) pulsed with SVYDFFVWL, a MHC class I peptide located in 180-188 amino acid residues of human melanoma-associated antigen tyrosinase- related protein 2 ( hTRP2) on the immunity against melanomas elicited by adenovirus encoding hTRP2 (Ad hTRP2).

METHODSThe mice were intradermally immunized with Ad hTRP2, and three weeks later with Ad hTRP2 or DC/SVYDFFVWL once more. Analysis of CTL killing activity and IFN-gamma-producing CD8 + T cells in the total CD8 + T cells of spleen were made using in vivo CTL and intracellular staining of IFN-gamma, respectively. Additionally, the survival of mice was checked after the subcutaneous inoculation with mouse melanoma B16. F10 cells.

RESULTSThe 6 h CTL killing and IFN-gamma producing CD8 +T cells in the total CD8 ' T cells of spleens were 68. 40%+/-5. 50% and 0. 67%+/-0.16% in Ad hTRP2 (priming)-Ad hTRP2 (boosting) group,28. 50%+/-6.40% and 0.22%+/-0.07% in DC/SVYDFFVWL (priming)-DC/ SVYDFFVWL (boosting) group,and 98. 90%+/-0.90% and 1.05%+/-0.21% in Ad hTRP2 (priming)-DC/ SVYDFFVWI, (boosting) group, respectively. In the tumor-bearing model, none of mice survived in DC/SVYDFFVWL (priming)-DC/SVYDFFVWL (boosting) group, and just only 40% of mice were tumor-free in Ad hTRP2 (priming) -Ad hTRP2 (boosting) group, whereas 100% of mice survived in Ad hTRP2 (priming)-DC/SVYDFFVWL (boosting) group.

CONCLUSIONBoosting with DC/ SVYDFFVWL can significantly enhance the immunity against melanomas elicited by priming with Ad hTRP2, indicating that first priming with Ad hTRP2 and then boosting with DC/SVYDFFVWL is a potentially effective regimen for overcoming the disadvantage that anti-tumor immune response can not be significantly increased by readministration of adenovirus.

Adenoviridae ; genetics ; Animals ; CD8-Positive T-Lymphocytes ; immunology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cells, Cultured ; Cytotoxicity Tests, Immunologic ; Dendritic Cells ; cytology ; immunology ; Female ; Genetic Vectors ; genetics ; Humans ; Immunization, Secondary ; methods ; Intramolecular Oxidoreductases ; immunology ; Melanoma, Experimental ; immunology ; pathology ; therapy ; Membrane Proteins ; immunology ; Mice ; Mice, Inbred C57BL ; Peptide Fragments ; immunology ; Survival Analysis ; T-Lymphocytes, Cytotoxic ; immunology

Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1-CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA-Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-alpha, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-gamma cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine.
Animals ; Cancer Vaccines/genetics/immunology ; Cell Line, Tumor ; Cell Proliferation ; Dendritic Cells/immunology ; Exosomes/genetics/*metabolism ; Gene Expression Regulation ; Gene Transfer Techniques ; Immunity, Cellular/immunology ; Immunity, Humoral/immunology ; Immunotherapy ; Lymphocyte Activation/immunology ; Melanoma, Experimental/mortality/pathology/*physiopathology ; Mice ; Mice, Inbred C57BL ; Nuclear Proteins/*genetics/*metabolism ; Survival Analysis ; T-Lymphocytes/immunology/metabolism ; Trans-Activators/*genetics/*metabolism ; Transduction, Genetic

We examined the effect of class II transactivator (CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenecity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.
Transplantation, Homologous ; Transfection ; Trans-Activators/genetics/*immunology/metabolism ; Trans-Activation (Genetics)/genetics/immunology ; Skin Transplantation ; Nuclear Proteins/genetics/*immunology/metabolism ; Mutation ; Mice, Transgenic ; Mice, Knockout ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Melanoma, Experimental/genetics/immunology/pathology ; Male ; Interferon Type II/pharmacology ; Humans ; Histocompatibility Antigens Class II/genetics/*immunology/metabolism ; Graft Survival/genetics/immunology ; Graft Rejection/genetics/*immunology ; Genes, MHC Class II/genetics/immunology ; Flow Cytometry ; DNA, Complementary/genetics ; Cell Proliferation/drug effects ; Cell Line, Tumor ; Animals

OBJECTIVETo improve the anti-tumor effect of dendritic cytoma vaccine (DCV) for finding an effective anti-tumor biotherapy.

METHODSDC vaccine prepared by transfection of adenovirus mediated melanoma-associated antigen gene (gp100) into bone marrow-derived dendritic cell (DC) was used to study the immuno-therapeutic effect and the mechanism of lentinan (LNT) in different dosages, used alone or combined with gp100-DC for treatment of B16 melanoma bearing mice.

RESULTSAfter being treated with LNT combining gp100-DC, the growth of malignant melanoma was inhibited with the tumor-free survival in the experimental animals being 66.7%. The treatment could also significantly enhance the activity of cytotoxicity T lymphocyte (CTL) and natural killer (NK) cells, elevate the levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in splenocytes, and histological examination showed that a large amount of inflammatory cells infiltrated inside and around the tumor, and obvious necrosis of tumor cells was found.

CONCLUSIONBy combined use with LNT the anti-tumor immuno-reaction of DCV vaccine could be enhanced effectively.

Adjuvants, Immunologic ; pharmacology ; therapeutic use ; Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Cancer Vaccines ; administration & dosage ; immunology ; Cell Line ; Cell Line, Tumor ; Combined Modality Therapy ; Dendritic Cells ; cytology ; immunology ; Female ; Humans ; Lentinan ; pharmacology ; therapeutic use ; Melanoma, Experimental ; immunology ; pathology ; therapy ; Membrane Glycoproteins ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Shiitake Mushrooms ; chemistry ; Survival Analysis ; Transfection ; gp100 Melanoma Antigen

This study is to clone the gene of staphylococcal enterotoxins O, obtain recombinant protein (rSEO) and investigate its activity on mice lymphocyte. Staphylococcus aureus O gene is cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEO was used to transform E. coLi BL21, where the GST-SEO fusion protein was expressed efficiently. Then SEO was purified by Glutathione Sepharose 4B affinity column and digested with thrombin. The bioactivity of SEO was analyzed by MTT assay on mice lymphocyte and tumor cells. The nucleotide sequence was confirmed to code for the protein correctly, and soluble SEO was expressed efficiently in E. coli BL21 with pGEX-4T-SEO. The protein purified by affinity chromatography resulted to be one single band by SDS-PAGE detection. The MTT assay of the purified rSEO demonstrated that its abilities of stimulating T cells and inhibiting the proliferation of K562, K562-ADM and B16 cells were equivalent to that of SEC in vitro. The expression plasmid pGEX-4T-SEO was constructed and the recombinant superantigen was expressed successfully, which may provide a foundation for the further research of the anticancer activity of SEO.
Animals ; Cell Proliferation ; Cloning, Molecular ; Enterotoxins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Vectors ; Humans ; K562 Cells ; Lymphocytes ; cytology ; Male ; Melanoma, Experimental ; pathology ; Mice ; Mice, Inbred ICR ; Plasmids ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Spleen ; cytology ; Staphylococcus aureus ; genetics ; Superantigens ; genetics ; immunology ; metabolism

OBJECTIVETo establish a syngeneic mouse model of liver tumor stably expressing hepatitis B virus (HBV) antigens.

METHODSMelanoma cell line B16 cells were transfected with pLXSN-2HBV. Cells (named B16/HBV) stably and persistently expressing HBV surface (HBsAg) and core (HBcAg) antigens were identified. The cells were injected into the hepatic subcapsular space of fifteen C57BL/6J mice. The mice were divided into 3 groups, receiving 100, 1000 or 5000 cells in a total volume of 5 µl per mouse, respectively, five mice in each group. Two weeks after the tumor cell inoculation, serum samples from the mice were collected weekly and the serum concentration of HBsAg and anti-HBs was quantified by ELISA. The tumor growth in the mouse liver was monitored by a high-resolution ultrasound system. Expression of HBsAg and HBcAg in the tumor tissues was determined by immunohistochemistry.

RESULTSLiver tumors were formed in all the mice receiving 1000 and 5000 B16/HBV cells per mouse, and in 80% of the mice receiving 100 B16/HBV cells. HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation. The mice receiving 100 cells per mouse began to die 4 weeks, those receiving 1000 cells per mouse began to die 3 - 4 weeks and those receiving 5000 cells began to die 2 - 3 weeks after the cell inoculation. All the tumor cells expressed HBsAg and HBcAg.

CONCLUSIONSThe B16/HBV cells stably and persistently express HBV antigens both in vitro and in vivo. A mouse model of transplanted liver tumor stably expressing HBV antigens has been successfully established by inoculation of those cells into the hepatic subcapsular space.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Liver Neoplasms, Experimental ; immunology ; virology ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVEThe aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF.

METHODSoHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded.

RESULTSBoth oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P < 0.01). The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree.

CONCLUSIONThe findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vive to the transplanted B16R tumor models.

Animals ; Cell Line, Tumor ; Female ; Gene Deletion ; Genetic Engineering ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Herpesvirus 2, Human ; genetics ; immunology ; Humans ; Immediate-Early Proteins ; genetics ; metabolism ; Melanoma, Experimental ; pathology ; therapy ; virology ; Mice ; Mice, Inbred C57BL ; Oncolytic Virotherapy ; methods ; Oncolytic Viruses ; genetics ; physiology ; Random Allocation ; Tumor Burden ; Viral Proteins ; genetics ; metabolism ; Xenograft Model Antitumor Assays