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AIM: To explore the mechanism of the nitric oxide (NO) synthesis induced by human C5b-9 complex in glomerular mesangial cells(MC) of rats. METHODS: The MC of rats were cultured and stimulated with human complement C5b-9 complex to induce TNF? and IL-1?. At the same time, several parameters related to NO synthesis were measured at 3 h, 6 h and 24 h after C5b-9 stimulation. The effects of monoclonal antibodies against TNF? and IL-1? on NO synthesis were examined in this system. RESULTS: TNF? concentration in supernatant from MC in C5b-9 group was higher than that of control group at 6, 24 h after stimulation with C5b-9 complex and reversed by adding anti-TNF? McAb. C5b-9 complex didn't stimulate the release of IL-1? in same system. In addition, the expression of iNOS mRNA in MC was observed at 3 h after stimulation with C5b-9. Levels of iNOS mRNA expression and cGMP in MC and NO - 3/NO - 2 in supernatant from MC in C5b-9 group were higher than those in control group at 6, 24 h after C5b-9 stimulation, these changes were also reversed by adding monoclonal antibody against TNF?. CONCLUSION: C5b-9 complex could induce iNOS mRNA expression at 3 h after C5b-9 stimulation, and the synthesis of NO at 6, 24 h was related to TNF? released from cultured MC of rats by C5b-9 complex to a certain extent.
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The aim of the current study was to determine whether tumor-specific T cells can be primed in and obtained from sponge implants loaded with tumor associated peptides. Naive C57BL/6 mice as well as C57BL/6 mice previously primed with FBL-3 tumor cells were implanted with small polyurethane sponges containing FBL-3 gag peptides CCLCLTVFL(gPr80 gag 85-93)or RSPTNLAKV(Pr65 gag p30 131-139). Both FBL-3 gag peptides were shown could bind to H-2 Db molecules. Ten days later, cells that had accumulated in the sponges were harvested, stimulated in vitro with the immunizing peptide, and tested for cytolytic activity against FBL-3 tumor and FBL-3 gag peptides. The results demonstrated that peptide-specific CD8+ CTL could be elicited and obtained from the sponge implants of both naive and immune mice. The FBL-3 gag p85-93 peptide induced CTL could specifically lyse syngeneic targets pulsed with the FBL-3 gag p85-93 peptide as well as FBL-3 tumor. However, the FBL-3 gag p131-139 peptide induced CTL lysed only the FBL-3 gag p131- 139 peptide pulsed syngeneic targets but not the FBL- 3 tumor. Tumor-specific T cells obtained from peptide-loaded sponge implants could be induced to grow to large numbers in vitro by periodic restimulation with the immunizing peptide plus syngeneic APC and low concentrations of IL- 2. Adoptive transfer of the resultant expanded FBL-3 gag p85-93 peptide-induced CTL into mice with disseminated FBL-3 could mediate effective anti-tumor therapy. Thus,in vivo immunization with peptide-loaded sponges provides a potentially useful technique for procuring primed peptide- specific T cells for tumor therapy.
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T lymphocytes play a very important role in tumor immunity, which recognize tumor antigenic peptide presented by MHC molecules on the surface of tumor cells through T cell receptor ( TCR) . Cytotoxic T lymphocytes kill tumor cells after recognizing antigenic peptide in the cleft of MHC class I molecules. It is now generally accepted that peptides naturally presented by a given MHC class I molecule have a specific motif which is referred to as MHC class I allele-specific consensus motif and that synthetic peptide corresponding to the identified naturally presented antigens exhibit remarkable activity in inducing T-cell responses. FBL-3 is a transplantable Friend virus-induced leukemia of B6 (H-2b) origin,which can induce the CTL against FBL-3 by immunizing B6 mice with atenuated FBL-3.Seven peptides were predicted as candidates of FBL-3 antigenic peptides in the light of H-2Db specific peptide binding motif and gag gene sequence of Friend virus. The synthetic peptides, named gagl to gag7, were obtainted. The results showed that CTL specific to gag3 could be induced by in vivo immunizing B6 mice with gag3 in IFA, but a primary T cell response could not be induced by in vitro immuniztion with the peptides. gag3 and gag5 could be recognized by specific CTL to FBL-3,because the CTLs obviously killed target cells (EL-4) pulsed with gag3 or gag5. Immunization with gag3 and gag5 could not induce an immune response to protect the subsequent challenge of FBL-3 in B6 mice which could be induced by immunizing B6 mice with parental FBL-3 tumor cells. This indicates these 7 synthetic peptides may not be the same antigens of the FBL-3.
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A recombinant eukaryotic expression vector containing HSA(Heat-stable Antigen) cDNA and PcDNA 3 plasmid was constructed and then transfected into mouse lymphoma cell line---EL-4 by electroporation. The transfected tumor cells were selected in RPM11640 containing G418 (400?g/ml).HSA expression was detected by FACS using indirect immunofluorescene technique with HSA mAb (Ml/69).To obtain high expression of HSA'EL-4 cells ,the transfected cells were recloned by limiting dilution. In animal experiments, we found that the tumorigenicity of HSA+ EL-4 is weaker than HSA- EL-4(EL-4 or EL-4-v). The size of HSA+ EL-4 tumors were significantly smaller than that of HSA- EL-4 tumors. The tumor growth speed and survival time of tumor-bearing mice are also different. A protective effect against the subsequently challenge with low dose (2 ? 1 03/mouse) wild type tumor cells was found in immunized mice with inactivated HSA+ EL-4 tumor cells but not in those animals immunized with HSA- EL-4 tumor cells. Moreover, HSA+ EL-4 could be used as tumor vaccine to cure the established tumor at the initial stage.
ABSTRACT
T lymphocytes, which play a very important role in tumor immunity, recognize the tumor antigenic peptide presented by MHC molecules on the surface of tumor cells. FBL-3 is a transplantable Friend virus-induced leukemia of B6 (H-2~(b) ) origin, which can induce the CTL against FBL-3 by immunizing B6 mice with attenuated FBL-3. The present paper was to elute immunogenic peptides ( CD8+ T cell epitopes) from class I complexes at the cell surface of viable FBL-3 cells by acid treatment. The acid treated cells remained viable but lost sensitivity to be lysed by FBL-3 specific cytotoxic T lymphocytes (CTLs). The sensitivity of the acid-treated cell was restored to the FBL-3 specific CTLs by supplement of the acid-eluted cell-free supematants. Paptides were subsequently fractionated by reverse-phase high performance liquid chromatography (RP-HPLC) . The fractionated peptides in HPLC fractions 5~6 and 23 ~ 60 (tubes) showed the capacity to sensitize RAM-S cells, which could be lysised by FBL-3 specific CTLs. The experiment indicates that this method may be useful in the definition of petide epitopes relevant to tumor cells.
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We have studied the effects of B7-1 on antitumor immunity induced in vivo by murine B7-1 gene transfected EL-4 lymphoma. At a lethal dose of wild - type(wt) or plasmid controlled (pc) B7-1 negative tumor cells. B7-1 gone transfected EL-4 cells inoculated in syngeneic mice were regressed. In contrast to the mice immunized with B7-1 tumor cells, immunization of mice with B7-l~EL-4 tumor cells showed significant protective effects against the sequence rechallenge of wt EL-4 tumor. Vaccination with X-irradiated B7-1 positive tumor cells at the early stage ( 7 days) after inoculation of wt EL-4 tumor cells showed some therapeutically effects but not at the late stage (14 days after inoculation). Vaccination with B7-l~+ EL-4 tumor cells delayed the occurrence of wt EL-4 tumors and prolonged the survival period of tumor-bearing mice. Our results indicate that the transfcction of B7-1 into tumor cells can increase the immunogenicity of EL-4 tumor and improve host response to wt EL-4 lymphoma. Immunization with B7-1 positive tumors can elicit effective antitumor immunity.
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Objective: To induce an immune response by intramuscular immunization with HER2/neu oncogene extracellular do- main (ECD) DNA and observe the effect in vivo by an abortion model in mice.Methods: Human and rat HEH2/neu ECD gene were cloned and inserted into expressive vector pCDNA3. The DNA immunization, expression of HER2/neu ECD, antibody de- tection and abortion in immunized mice were studied. Results: HER2/neu ECD protein expressed on muscle cells in the injected site, specific antibody to HER2/neu was induced by DNA immunization. The average of the offspring per delivery from immu- nized mice was significantly lower than that from control group, and the abortive embryos were found in uterus from immunized mice.Conclusion: The results show that both human and rat HER2/neu ECD gene can induced effective immune response by DNA immunization.
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Objective: To study the cellular immune response and the anti-tumor effects induced by intramuscular DNA immu- nization with HER2/neu extracellular domain gene. Methods: HEH2/neu oncogene extracelltilar domain gene was isolated and inserted to pCDNA3 expression vector. The cellular immune response and its anti-tumor effects were analyzed after intramuscular DNA immunization. Results: A specific cytotoxic activity by spleen cells from HER2/neu ECD gene immunized mice was found in vitro. Tumor growth was inhibited after tumor challenge in vivo in immunized mice. Conclusion: Specific cellular immune re- sponse and anti-tumor effect were elicited by HEB2/neu extracellular domain gene immunization.
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100 d) than those injected BMCs at the fifth day (
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To investigate the mechanism of inducing a prolongation of allograft survival by immunizing the syngeneic mouse with activated murine spleen cells,we used an anti—activated mouse Tlymphocyte antigen monoclonal antibody(2H_3)made in our laboratory to examine the effect of 2H3on allograft survival in vivo and on cell—mediated immune rection in vitro.The results showed that 2H3 could significantly prolong the allograft survival in vivo and obviously inhibit the proliferation in MLR and CTL generation in bulk MLR as well as the T lymphocyte prolifer-ation to ConA in vitro.These data indicate that one of the mechanisms of the prolongation of al-lograft survival by immunizing the syngeneic mouse with activated murine spleencells may be due to the generation of an anti—activated mouse lymphocyte antigen antibodies in immunized ani-mals which can block process of allorection.
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AIM:To study the effect of human complement C 5b～9 complex on nitric oxide(NO) synthesis of glomerular mesangial cells (MC). METHODS: First, the human complement C 5b～9 complexes were isolated and glomerular MC of rats were cultured. Second, the MC were stimulated with C 5b～9 complex and changes of metabolism products of NO(NO 3 and NO 2) in MC culture supernatant at 6,24 and 48 hours after C 5b～9 stimulating were detected. Moreover, cGMP levels in cultured MC were also measured. RESULTS: NO 3/NO 2 contents from culture supernatant and cGMP levels in MC were increased parallelly after C 5b～9 complex stimulation. Further, NO synthesis was inhibited by L-NG-nitro-arginine-methylester(L-NAME). CONCLUSION: NO synthesis of rat glomerular MC was incerased by human complement C 5b～9 stimulation.
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Objective:To explore the possibly mechanisms of inducing allogeneic chimerism and prolonging allografts survival in recipients by immature dendritic cells (imDCs) Methods:Bone marrow cells (BMCs) derived from donor (C57BL/6) were used to generate imDCs The splenocytes of recipients were pretreated by inactivated imDCs in vitro, or the recipients (Balb/C) were injected of inactivated imDCs via vein in vivo Then collected the splenocytes mixed with the inactivated splenocytes from donor to detect the responsiveness Mixed lymphocyte reaction was also used to evaluate the reactivity of the chimerism mice to the donor splenocytes At the same time the diversion of Th1/Th2 paradigm was studied by semi quantitative RT PCR Results:The splenocytes conditioned with imDCs pretreatment expressed hypo responsiveness to the donor stimulation, and the immunized mice also proliferated less degree compared with the naive mice The hyporeactivity was evidently seen within 72 hours after stimulation by donor splenocytes There was significant difference between them The chimerism mice showed unresponsiveness to donor antigens, while reactivity to the third party antigens was retained The result of RT PCR suggested, to some extent, there was a diversion of Th1/Th2 paradigm in the establishment of chimerism in the model Conclusion:The putative mechanism of immature dendritic cells inducing the generation of allogeneic chimerism may based on the hypo responsiveness produced by imDCs, and there may also exist some kinds of diversion of Th1/Th2 paradigm
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The McAb 2H3 to different target cells was investigated by using an indirect ELISA meth-ods. The result showed that the 2H3 could bind to the ConA actiuated mouse T lymphoblastsand CTLL-2 cells (an IL-2 dependent T cell line)but not to the resting mouse splenocytes,lymph node cells and thymocytes. The binding of FITC-anti-IL-2R to the activated mouse spleencells and CTLL-2 cells was seen as tested by immunofluorescence analysis. This binding reactionwas blocked when the target cells were previously treated with 2H3, but the binding reaction ofFITC-anti-Ia McAb to these target cells couldn't be blocded by 2H3. Further analysis of the tar-get antigen by using Western-Blotting technigue showed the molecular weight of this antihenrecognized by 2H3 was of 50～60KD in reducing condition. According to these data, target anti-gen recognized by the McAb 2H3 may be the IL-2R molecule(P_(55) mole cule)on the cell surface.
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Objective:To prolong the allograft survival time in immunized mice with immature dendritic cell, and to analyze the mechanism of hypo allo immuno responsiveness induced by immature dendritic cells Methods:Based on mouse cardiac allograft model, the survival of cardiac recipients immunized with mature or immature DC were observed Meanwhile the CTL activity of splenocytes from immature DC immunized recipients was detected Results:The survival time of cardiac allograft was substantially prolonged and the mean survival time extended from 9 1?0 73 days to 25 4?4 27 days It became more effective if those immunized mice were treated in combination with adriamycin application, the mean survival time of allograft was extended to 30 5?3 98 days It was proved that the CTL activity in spleen cells from the mice immunized with immature DCs was much lower (specific release was 16 32%) than that from the immunized mice with mature DCs (specific release was 39 58%) Conclusion:Immature DCs could induce prolongation of allograft survival time It may be possible that low CTL activity in recipients immunized with immature DCs promised this prolongation
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Objective:To study anti tumor immunity against Lewis lung cancer of dendritic cells derived from proliferated bone marrow cells Methods:After immunization with MUT 1 peptide pulsed DCs (DC MUT 1) intravenously, C57BL/6 mice were challenged with tumor cells subcutaneously to test the immune protection effect Meanwhile, CTL assay was analyzed Results:DC MUT 1 could induce durable and specific antitumor immunity in mice, and the T cells from immunized mice with pulsed DCs showed strong CTL activity Conclusion: An antitumor cellular immunity and a specific immune protection could be induced by immunization with DC MUT 1