Mutation of LoopAB in HuIFN 1c/86D and enhancement of antiviral activity / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Based on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity.</p><p><b>METHODS</b>Two unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB. Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project.</p><p><b>RESULTS</b>The mutated residues M31?D,D32?P of LoopAB in parent IFN were produced. The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed. The recombinant protein has been expressed in E.coli JM103. The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT. 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference.</p><p><b>CONCLUSIONS</b>The authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.</p>

Experimental study on the effect and mechanism of action of active immunization with elemene combo-tumor cell vaccine / 中国病理生理杂志

Elemene is a new anticancer drug isolated from a Chinese traditional medicine Curcuma aromatica. In previous work, we discovered that tumor cell vaccine (TCV) treated with oleum Curcuma aromatica or elemene could induce significant immunoprophylactic effect against a variety of aminal tumor strains and the method of preparation of elemene combo-TCV(EC-TCV) already got China's inventive patent. In this paper we further studied the active immunotherapeutic effect and the possible cellular/molecular mechanisms of EC-TCV immunization. The results were as follows:(1) EC-TCV immunization showed significant therapeutic effects (P＜0.05) against murine Ca761 syngeneic mammary carcinoma (H-2k) and HCa-F allogeneic hepatic carcinoma (H-2-) models; (2) The spleen cells of Hca-F EC-TCV immunized mice displayed higher cytotoxicity and IL-12 level while the secretion of IL-10 was decreased (P＜0.05); (3) Similar to heat shock, elemene(E), mitomycin C(MMC) and glutaraldehyde (G) could act alone as stressor, and induce significant changes of the expression of membrane heat shock proteins(HSP70 or/and HSP90) on L615 leukemia and HCa-F hepatoma cells and the EC-TCVs (E+MMC+G treated in combination) showed the highest level of membrane HSPs expression (P＜0.05 or P＜0.01 );(4) The HSP70-peptide complex isolated from HCa-F EC-TCV through ADP-agarose affinity chromatographic system could induce active immunoprotection against lethal dose challenge of HCa-F hepatic cancer cell but could not protect against the cross challenge of lethal dose of L615 leukemia. The results indicated that the immunoprotective effect of EC-TCV was in some extent tumor-specific, MHC-nonrestricted, and HSPs might play an important role in its molecular mechanisms.

The role of DCs in the immunoprotective effects of active immunization of elemene combo-tumor cell vaccine / 中国病理生理杂志

In this experiment, Hca-F or L615 elemene combo-TCV (H-TCV and L-TCV), H-TCV lysates, corynebacterium parvum (CP) were used to immunize 615 and Balb/c inbred mice, and their splenic DCs were prepared and pulsed in vitro with tumor cell lysates (H or L) and TCV lysates (TH or TL). The capacities of DCs to stimulate the proliferation of syngeneic nonadherent spleenic cells were tested with MTT assay. The results showed that the splenic DCs from normal mice in vitro pulsed with TH or TL could induced syngeneic noradherent splenic cells to proliferate (P＜0.01), while the H or L pulsed DCs could not. The splenic DCs from H-TCV or L-TCV or TH immunized mice re-pulsed in vitro with TH or TL exhibited stronger stimulating effects than the DCs from normal mice pulsed in vitro for the firth time pulsed with TH or TL (P＜0.01 or P＜0.05); The capacities of DCs to induce proliferation of syngeneic nonadherent splenic cells could be further enhanced by CP immunization, especially when were pulsed with TH (P＜0.01). Normal inbred 615 mice were transferred with DCs pulsed with lysates of elemene TCV (TDCs) or pulsed with lysates of Hca-F tumor cells (HDCs) on day 7 before challenged with lethal dose of live Hca-F cells, significant adoptive immunoprotective effects were seen, with 61.6% tumor inhibition rate and 25% survival in TDC adoptive transfer group. This study indicated that DCs might play a role in the mechanisms of active immunization and pulsing DCs with lysate of elemene combo TCV and isolating DCs from elemene combo TCV immunized mice were useful methods for DCs vaccine preparation.

Pulsing effects on bone marrow dendritic cells of H_(TA)-HSP70_( BCG) / 中国免疫学杂志

Objective:Normal murine DCs were pulsed with complex of tumor antigen from elemene-combo tumor cell vaccine-heat shock protein 70 of BCG (H TA-HSP70 BCG).Their proliferation and antigen presenting function were evaluated.Methods:The dendritic cells(DCs)were cultured in complete media containing GM-CSF and IL-4 and pulsed with H TA-HSP70 BCG,H TA or HSP70 BCG.Their proliferation and stimulating effects on spleen nonadherent cells were evaluated with MTT assay.Their capability of endocytosing FITC labeled dextran was assayed with FACscan,morphological changes of DC were observed in electron microscope.Results:Proliferation index of DCs pulsed with H TA-HSP70 BCG was 2.107?0.013,proliferation index of DCs pulsed with H TA-HSP70 BCG mixed with normal nonadherent spleen cell was 1.927?0.073.The percent of DCs endocytosed FITC labeled dextran was 58.61%.Above changes were more significant than those of DCs pulsed with H TA or HPS79 BCG.Conclusion:H TA-HPS70 BCG had more potent activity to pulse DC and strengthen antigen presenting of DC.

Isolation and Sequencing the Differential Gene Fragments Expressed in Human Stomach Cancer Tissue / 中国肿瘤生物治疗杂志

Objective: Identification of the genes specially expressed in tumor cell but not in normal cell is important for understanding the molecular mechanisms of carcinogencsis. This study will focus on identification of differentially expressed gene fragments in human stomach cancer. Methods: By using the new developing mRNA differential display (DD) technique, genes fragments differentially expressed in stomach cancer tissues from a patient and the adjacent normal tissues beyond the tumor mass were studied. Results: Two differentially displayed complementary DNA fragments from stomach cancer tissues, scgl and scg2 (stomach cancer-associated gene, scg), cofirmed by Northern Blot, were cloned and sequenced. The nucleotide length of scgl is 194 base pairs and that of scg2 is 343 base pairs. After searching against GenBank databases by BLASTN, neither scgl nor scg2 had significant homological gene sequences with the known genes. Conclusion: These results suggested scgl and scg2 might be complementary DNA fragments of novel genes expressed in stomach cancer tissues, but not in normal tissues and may play a role in the occurrence and development of stomach cancer. Further characterization of full-length of these two complementary DNA fragments will be continued.

The Role of JNK/SAPK Signaling-Transduction Pathway in the Effect of El-emene Against Hepatocarcinoma / 中国肿瘤生物治疗杂志

Objective:To find out the role of JNK/SAPK signaling-transduction pathway in the effect of elemene against hepatocarcinoma, offering the clue to ilustrate the molecular mechanisms of antitumor effects of elemene. Methods: The detection of the distribution of elemene in Hca-F cells was detected by gas chomatography and apoptotic changes in elemene treated. SMMC7721 cells were examined by TEM. After elemene treatment, the activation of JNK/SAPK in HepG2 cellls and the DAXX gene expression in SMMC7721 cells were detected by Western blotting and RT-PCR respectively. Results: Gas chomatography showed that elemene was detected at 8. 42 minute. The SMMMC7721 cells treated by elemene for 3 hours began to show typical apoptotic changes . The JNK/SAPK activity in HepG2 cell treated with heat shock was the highest of all groups and the group treated with elemene was the next and the control group is the lowest one. There was no DAXX gene expression in SMMC7721 cells treated with elemene. Conclusion: Elemene can diffuse into cells. Tumor cell apoptosis treated with elemene may be induced by JNK/SAPK activating and DAXX signal pathway may not play key role in JNK/SAPK activation induced by elemene.

Experimental Study on TNF-a Gene Expression and Anti-Pulmonary Metastases of Macrophages Activated by New Castle Disease Virus / 中国肿瘤生物治疗杂志

The activating effect of NDV-L on murine PEM? in vitro and the anti-metastatic effect of NDV-L-PEM? in vitro against B16 melonoma were investigated. The results showed that NDV-L could not only activate murine PEM? morphologically , but also enhance the TNF-? gene expression. If the activated NDV-L PEM? were adoptively transferred in the B16 melanoma bearing mice, significant inhibiting effect against pulmonary metastases could be exhibited.

Experimental Study on the Immunoprotective Effect and Mechanism of Action of Specific Active Immunization with Tumor Cell Vaccine / 中国肿瘤生物治疗杂志

Through several murine transplantable tumor models, we studied the immunoprotective effect and mechanism of action of specific active immunization with various tumor cell vaccines (TCV) treated by: (1) Oleum Curcuma Aromatica (OCA, a Chinese anticancer medicament) and its effective monomer ?-elemene; (2) MMC and glutaraldehyde; (3) ~(60)Co and X-ray irradiation; and (4) vaccinia virus (VV) and newcastle disease virus (NDV), including virus infected TCV membrane fraction (Mfr). The reproducible results of immunoprotective effect indicated that the specific active immunotherapy with such TCVs might be of practical value in clinical immunological approaches to cancer therapeutics.

The Effect of Antitumor Immunity Elicited by Heat Shock Protein70-Associated Peptide Complexes on Hepatoma in Mice / 中国肿瘤生物治疗杂志

Objective: To observe effective of antitumor immunity elicited by heat shock protein70(HSP70) associated peptide complexes isolated from hepatoma (HCaF), a tumor model of no MHC Ⅰmolecule expression in mice. Methods: Specific active immunization and adoptive cellular immunization assay was adopted to observe the immunoprotective effect elicited by HSP70 associated peptide complexes purfied from the HCaF. Results: Mice immunized with HSP70 associated peptide complexes were protected from HCaF challenge. The immunoprotective effect in female mice was better than the male groups. Adoptively transferred immune spleen cells of mice which had been immunized with HSP70 associated peptide complexes could be elicited immunity against HCaF challenge, and the mice free of tumor can resist repeated challenge, the maximal challenge quantities exceeded 1?10 7 HCaF cells. Mice immunized once with spleen cells pulsed by HSP70 associated peptide complexes could also be elicited certain immunity against HCaF challenge. Conclusions: HSP70 associated peptide complexes derived from the HCaF can elicit no MHC Ⅰ molecule restrictive immunoprotective effect against HCaF and could be continuously enhanced by repeated challenge with alive HCaF cells. Adoptively transferred immune spleen cells of mice which had been immunized with HSP70 associated peptide complexes could provide protection against HCaF cells challenge.

Immunotherapeutic Effects of Elemene Combo Tumor Cell Vaccine on Hca-F and Changes of IL-10 and IL-12 Secretion by Splenocytes of Treated Mice / 中国肿瘤生物治疗杂志

Objective: To investigate the immunotherapeutic effects of elemene combo tumor cell vaccine(EC TCV) on Hca F murine hepatic carcinoma and changes of IL 10 and IL 12 secretion by splenocytes of treated mice. Methods: BALB/c mice inoculated with Hca F were treated with EC TCV, CY, EC TCV+CY or PBS control, tumor growth was measured, concentration of IL 10 and IL 12 in supernatants of mixed splenocytes and Hca F culture(MSTC) was assayed with ELISA. Results: After EC TCV immunotherapy and CY chemotherapy, the latent period of tumor growth prolonged, mean tumor weight (MTW) decreased, even no nodule was observed in EC TCV+CY chemoimmunotherapy group, the concentration of IL 10 in supernatants of MSTC of EC TCV immunotherapy group decreased significantly(1.82?0.29 pg/ml, P

Studies on the Mechanisms of the Apoptotic Cell Death of Target Cells Mediated by Human Monocytes / 中国肿瘤生物治疗杂志

The mechanisms of cytotoxicity mediated by pokeweed-mitogen (PWM)-activated human peripheral blood monocytes was investigated in this study. By DNA electrophoresis and propidium iodide ( Pl) -DNA staining flow cytometry, the study demonstrated that apoptotic cell death of target U937 cells and Raji cells was induced in lectin-depen-dent monocyte-mediated cytotoxicity ( LDMC) . The LDMC-mediated DNA fragmentation in U937 cells could be completly inhibited by anti-TNF-?monoclonal antibodies McAbs, instead of the addition of monosaccharide (N-acetyl glucosamine, Glc NAC) . In contrast, Glc NAC inhibited the DNA fragmentation in Raji cells induced by PWM-monocytes while the anti-TNF-a McAbs had no effect. By flow cytometry, we also demonstrated that PWM could bind with tumor cells as well as with monocytes, at the stimulation of the production of nitric oxide ( NO-) from monocytes. This NO-production was enhanced in the presence of target cells, but the enhacement was abolished by the treatment of GlcNAc.The presence of Lectin-like receptors on the surface of monocytes and tumor cells may bring the effector target cells together, thus facilitating the induction of apoptosis in target cells by triggering the production of cytolytic factors and the modification of target cell surface antigens.