Objective To produce anti-thrombomodulin antibodies.Methods Using genetic immunization: Eukaryotic expression plasmid pcDNA3.1/TM(LEO),encoding all the extracellular domain of human thrombomodulin and signal peptides but lacking the transmembrance and cytoplasmic domains was constructed, which recombinant thrombomodulin was secreted soluble product. The plasmid was isolated from large-scale bacterial cultures by treatment with alkali and SDS, purified by precipitation with polyethylene Glycol (PEG). Recombinant plasmid was injected into tibial muscle of BALB/c mice. The productions of TM and anti-TM have been detected. Results The positive of RT-PCR and expressed TM identified the function of the recombinant plasmid. The pcDNA3.1/TM(LEO) induced higher titer of anti-TM. The antibody titer peaked between the 5th and 7th injection with a titer of 1∶8 000 detected by cell-ELISA coated with EVC-304. Specificity has been identified by western blot and immunohistochemistry.Conclusion The production of antibody through genetic immunization was a feasible method due to the difficulties in obtaining and purification of natural thrombomodulin.

Our previous study showed that transmembrane TNF-alpha (TM-TNF-alpha) had broader tumoricidal spectrum than secretory TNF-alpha (s-TNF-alpha). This study examined the difference between the two kinds of TNF-alpha in inducing cells and the relationship between the apoptosis induced by TM-TNF-alpha and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-alpha on cell lines L-929 and HepG2. TUNEL was used to detect apoptosis and the TdT and PI co-staining were used for determining the phase of apoptotic cells. Our results showed that TM-TNF-alpha could kill not only s-TNF-sensitive L929 cells but also s-TNF-tolerant HepG2 cells. TM-TNF-alpha predominantly induced apoptosis while s-TNF could induce both apoptosis and necrosis. The apoptosis of L-929 cells induced by TM-TNF-alpha mainly occurred in S phase and the apoptosis of HepG2 predominantly took place in G(1) phase. It is concluded that the cytotoxic effects of the two TNF differ substantially. Since TM-TNF-alpha works locally, mainly induces apoptosis and has broader anti-tumor spectrum, it may be more effective for the treatment of tumor than s-TNF.

To compare the difference in tumor immunity and autoimmunity elicited by adenovirus (Ad) encoding human or murine tyrosinase-related protein 2 (AdhTRP2 or AdmTRP2), and to find the most effective way to induce immunity by AdhTRP2 or AdmTRP2, C57BL/6 mice were immunized with AdhTRP2 or AdmTRP2 intramuscularly at different doses of 10(5), 10(6), 10(7) and 10(8) separately (10 mice for each dose). Two weeks after the immunization, in vivo CTL assay and intracellular staining (ICS) of IFN-gamma were carried out to analyze the dose-effect relationship. Tumor growth and vitiligo (as an sign of autoimmunity) were observed until 3 months after challenge with 10(5) B16F10 tumor cells. The results showed that Ad encoding AdmTrp2 induced weak tumor immune response. Similar immunization with AdhTrp-2 elicited stronger protective immunity. CTL activity and IFN-gamma-produced CD8+T cells were directly proportional to dose of AdhTrp2 or AdmTrp2. Moreover, AdhTrp2 group showed tumor rejection in 100% of challenged mice till the end of 3rd month while 60% of mice immunized with AdmTrp2 were protected against tumor. In the whole process of this experiment, no vitiligo was observed in mice immunized either with AdhTrp2 or AdmTrp2. It is concluded that anti-melanoma responses induced by genetic vaccination expressing xenoantigens breaks immune tolerance effectively and is able to elicit strong antigen-specific cytotoxic T cell response without vitiligo.
Adenoviridae/metabolism ; Antineoplastic Agents/*pharmacology ; Cell Line, Tumor ; Cytokines/metabolism ; Immune System ; Immune Tolerance ; Interferon-gamma/metabolism ; Intramolecular Oxidoreductases/*biosynthesis ; Intramolecular Oxidoreductases/*genetics ; Mice, Inbred C57BL ; T-Lymphocytes, Cytotoxic/*metabolism ; Vitiligo/*metabolism

AIM and METHODS: The purpose of the study was to characterize the time-related effect of Danazol therapy on endometriosis explant using the rat model. Endometriosis was induced in mature female rats. One group of treated animals as well as controls were sacrificed at 2,4,6 and 8 weeks after treatment at which time the explant was evaluated. RESULTS: Explant volume was significantly reduced in all treatment groups, and the effect was more significant in animals treated for 4 weeks than those treated for only 2 weeks. CONCLUSION: Danazol treatment can cause gradual regression of endometrial explant in a time-related manner.

Our previous study showed that transmembrane TNF-α (TM-TNF-α) had broader tumoricidal spectrum than secretory TNF-α (s-TNF-α). This study examined the difference between the two kinds of TNF-α in inducing cells and the relationship between the apoptosis induced by TM-TNF-α and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-α on cell lines L-929 and HepG2. TUNEL was used to detect apoptosis and the TdT and PI co-stainin gwere used for determining the phase of apoptotic cells. Our results showed that TM-TNF-α could kill not only s-TNF-sensitive L929 cells but also s-TNF-tolerant HepG2 cells. TM-TNF-α predominantly induced apoptosis while s-TNF could induce both apoptosis and necrosis. The apoptosis of L-929 cells induced by TM-TNF-α mainly occurred in S phase and the apoptosis of HepG2 predominantly took place in G1 phase. It is concluded that the cytotoxic effects of the two TNF differ substantially.Since TM-TNF-α works locally, mainly induces apoptosis and has broader anti-tumor spectrum, it may be more effective for the treatment of tumor than s-TNF.

To compare the difference in tumor immunity and autoimmunity elicited by adenovirus (Ad) encoding human or murine tyrosinase-related protein 2 (AdhTRP2 or AdmTRP2), and to find the most effective way to induce immunity by AdhTRP2 or AdmTRP2, C57BL/6 mice were im-munized with AdhTRP2 or AdmTRP2 intramuscularly at different doses of 105, 106, 107 and 108 separately (10 mice for each dose). Two weeks after the immunization, in vivo CTL assay and in- tracellular staining (ICS) of IFN-γ were carried out to analyze the dose-effect relationship. Tumor growth and vitiligo (as an sign of autoimmunity) were observed until 3 months after challenge with 105 B I6F10 tumor cells. The results showed that Ad encoding AdmTrp2 induced weak tumor im- mune response. Similar immunization with AdhTrp-2 elicited stronger protective immunity. CTL activity and IFN-γ-produced CD8+T cells were directly proportional to dose of AdhTrp2 or AdmTrp2. Moreover, AdhTrp2 group showed tumor rejection in 100% of challenged mice till the end of 3rd month while 60% of mice immunized with AdmTrp2 were protected against tumor. In the whole process of this experiment, no vitiligo was observed in mice immunized either with AdhTrp2 or AdmTrp2. It is concluded that anti-melanoma responses induced by genetic vaccina- tion expressing xenoantigens breaks immune tolerance effectively and is able to elicit strong anti-gen-specific cytotoxic T cell response without vitiligo.