Objective To provide experimental evidence for the development of multi-epitope-baseded marker vaccines through investigating the humoral and cellular immune responses in BALB/c mice induced by the multiple antigen peptides (MAPs) with the mimic epitope.Methods Three types of MAPs in eight branched forms containing the mimic epitope of Mycoplasma genitalium adhesion protein (MgPa) were prepared using poly-lysine as the core matrix.The purity of MAPs was analyzed by reverse phase high performance liquid chromatography (RP-HPLC).The molecular weights of MAPs were characterized by Mass Spectrometry.The BALB/c mice were immunized intramuscularly for four times with single or mixed MAPs.The specific IgG antibody and the subtype of IgG antibody in serum of the immunized mice were detected by indirect ELISA.The proliferative responses of the spleen lymphocytes were detected using MTT assay.The ELISA were used to detect IFN-γ and IL-4 levels in the cultured supematant of spleen lymphocytes.Results The three types of MAPs containing the mimic epitopes were successfully prepared with high purity.They,could stimulate mice to produce specific IgG antibodies,of which,the major antibody isotype was Th1 immune response-associated IgG2a.Compared with the single MAP immunization group,the mixed-MAPs immunized mice produced more IgG,IgG1 and IgG2a antibody (P＜0.05).Furthermore,these MAPs could enhance the specific proliferation of spleen lymphocytes in immunized mice and induce the production of IFN-γ and IL-4.The levels of IFN-γand IL-4 in mixed-MAPs group were significantly higher than those of the single MAPs group (P＜0.01).Conclusion The three types of MAPs could induce strong specific cellular and humoral immune responses.The immunological competence of the mixed-MAPs was stronger than those of the single MAP.

Objective To explore the effects of CD4+CD25+Treg cell on the allograft after infusion of dendritic cells (DCs) with low expression of CD40 from donor in mouse heart transplantation.Methods In vitro,mouse bone marrow-derived DCs were infected by CD40-RNAi lentiviral vector,and tolerogenic DCs (Tol-DCs) with low expression of CD40 were prepared.A heterotopic abdominal heart transplantation model was established in mice,and the other three groups that were control group,noninfected DC group and lentivirus infected DC group were designed correspondingly.Cardiac allograft survival time was recorded and pathological grading for acute rejection was assessed on the 7 d after heterotopic abdominal heart transplantation.Concentrations of CD4+CD25+Treg cells in peripheral blood were analyzed before and after transplantation by flow cytometry.Results After 48 h infection of DCs by CD40-RNAi lentiviral vector in vitro,the expression of CD40 mRNA was down-regulated significantly,whose inhibition rate was 80.9％.The expression of CD40 was decreased from 74.37％ ±4.08％ to 40.07％ ± 4.03％ (P＜0.05) after 48 h infection.Compared with the control group and the noninfected DC group,the cardiac allograft survival time was significantly prolonged in the CD40-RNAi lentivirus infected DC group,which was (14 ± 4) d(P＜0.01) ; concentrations of CD4+CD25+Treg cells in peripheral blood were increased both on the 3 d and the 7 d after transplantation (P＜0.05) ; the pathological grading for acute rejection was decreased on the 7 d after transplantation (P＜0.05).Conclusion The CD4+CD25+Treg cell in peripheral blood was protective to cardiac allograft in prolonging its survival time in mouse heart transplantation.

Objective:In order to elucidate the derivation and differentiation mechanisms for derivation of myofibroblasts in oral submucous fibrosis.Methods:Oral keratinocytes and fibroblasts were isolated and cultured,and a co-cultur system composed of the keratinocytes and fibroblasts was established in vitro.Expressions of ?-SMA protein and mRNA in fibroblasts were detected by immunohistochemistry and RT-PCR. The level of TGF-?1 in conditioned medium were evaluated by ELISA.Results:The expressions of ?-SMA mRNA and protein in fibroblasts co-cultured with keratinocytes preprocessed by arecoline was higher than that in fibroblasts co-cultured with keratinocytes and without preprocessed by arecoline. The level of TGF-?1 in conditioned medium of fibroblasts-keratinocytes preprocessed by arecoline co-cultures was higher than those of fibroblasts cultured alone.Conclusion:The derivation of myofibroblast from FB can be promoted by keratinocytes preprocessed by arecoline and TGF-?1 may play a role in the process.

Objective:To construct pEGFP-C3-B7.2-MAGE-1 eukaryotic expression plasmids and to study the immunological effects of esophageal cancer cell vaccine modified by human B7.2/MAGE-1 gene.Methods:The B7.2 and MAGE-1 cDNA,which was amplified by RT-PCR, and the eukaryotic expression plasmid pEGFP-C3-B7.2-MAGE-1 was constructed. Human esophageal cancer cell EC9706 was transfected with the vector of pEGFP-C3-B7.2-MAGE-1 using the technique of lipofectamine transfection.The dendritic cells(DCs)from peripheral blood mononuclear cells(PBMC)were loaded with the tumor antigen,and co-cultured with congeneric T cells derived from PBMCs for 3 days to obtain the tumor specific cytotoxicity T lymphocytes(CTL). Methy1 thiazoly1 tetrazolium (MTT) assay was used to detect inhibition effect of CTL on transfected and untransfected EC9706 cells.Results:The CTL had stronger inhibition response against the cancer cells transfected with pEGFP-C3-B7.2-MAGE-1 than to the cancer cells transfected with pEGFP-C3 and the untransfected cancer cells(P

Objective:To investigate the clinical significance of Th1/Th2 balance of peripheral blood Th cells and of serum high-sensitivity C-Reactive Protein(hs-CRP) in type 2 diabetic patients.Methods:Th1/Th2 cells were determined by using the tri-colored fluorescence labeled flow cytometry serum hs-CRP concentrations were determined by ELISA in type 2 diabetic patients (n=60) and the controls (n=25).Results:(1)Th1 cells and Th1/Th2 ratios were significantly reduced in type 2 diabetic patients than the controls (P 0.05 ). Th1 cells negatively correlated with HbA1c (r=-0.688 P

Objective:To explore the expression of intercellular adhesion molecule-1(ICAM-1 )induced by cigarette smoking extract(CSE) as well as the influence of the adenovirus E1A gene in pulmonary epithelial cells.Methods:A549 cells were transfected with a plasmid carrying the adenovirus E1A gene and stable trasfectants expressing E1A protein were selected. The cells were tested for the expression of ICAM-1 after stimulation with different concentrations of CSE.Results:ICAM-1expression was increased in A549 markedly with CSE stimulation,and the higher CSE concentration were,the more expression of ICMA-1 was observed. As compared with parental cells or cells transfected with control plasmid, ICAM-1 expression was markedly increased with stimulation of different concentrations of CSE in E1A-positive A549 cells.Conclusion:CSE exposure induces the surface expression of ICAM-1 and adenovirus E1A gene can markedly increase ICAM-1 expression induced by CSE in pulmonary epithelial cells, which suggest that latent adenovirus infection may amplify the inflammation process present in airways of smokers.

Objective:To investigate the changes of lung lymphocytes in OVA induced murine asthma model,and study the involvement of NK cells in this process.Methods:C57BL/6J(B6) mice were induced to develop asthma by intrapenitoneal injection of OVA with alum as adjuvant, and then inhalation of nebulized OVA. After collecting serum and Bronchoalveolar Lavage Fluid(BALF),IL-4 level was determined by ELISA. The kinetics of pulmonary lymphocyte recruitment and cytokine release were detected by flow cytometry.Results:IL-4 expression increased in BALF after OVA nebulization, while there was no significant difference in serum. IFN-?,IL-4+NK cells accumulation in lung parenchymal tissues,and exhibited an evident NK2 shift in mice with asthma.Conclusion:NK cell involved in OVA-induced mouse asthma and NK2 shift accompany with Th2, indicating that NK2 played an important role in this process.

Objective:To search for the efficient immune adjuvant for rotavirus DNA vaccine by evaluating the efficacy in modifying immune responses to rotavirus.Methods:BALB/c mice were immunized by intramuscular and intranasal routes with 3 doses of naked DNA and liposome-coated DNA vaccine at two-week intervals. Titers of serum IgG and IgA were measured by ELISA.Results:Serum titers of rotavirus specific IgG in naked DNA and liposome-coated DNA vaccine-immunized mice were significantly higher than that of the unimmunized controls. Interestingly, only those mice administrated with liposome-coated DNA vaccine by intramuscular route generated serum rotavirus IgA,and neither by naked plasmid nor by intranasal immunization with DNA-liposome complex.Conclusion:These results indicate that liposome can act as both the vector and adjuvant for DNA vaccine against rotavirus by intramuscular injection,which induces rotavirus-specific IgA in the sera,and may provide partial protection against rotavirus infection.

Objective:To clone human anti-ricin antibodies from large phage antibody library.Methods:Panning for a large phage library against ricin toxin was conducted to select specific antibodies against ricin. The binding activities and specificities were tested by ELISA method. Soluble ScFvs were prepared through infecting E coli. HB2151 with the selected phage antibodies and induction with IPTG. Results:Forty positive clones were obtained after 5 rounds of panning, and 12 clones had specific binding ability to ricin toxin. DNA fingerprinting showed 7 different band patterns indicating 7 different positive clones. DNA sequencing showed that variable regions of these ScFvs belonged to different subgroups.Conclusion:Human anti-ricin antibodies were successfully obtained from large phage antibody library.

Objective:To investigate the effect of intrathymic injection of allogenic antigen to sciatic nerve transplantation.Methods:C57BL/6(H-2b) mice were used as donors and BALB/c(H-2d) as recipients. The recipients were divided into four groups: auto-transplantation group, allogenic transplantation group, allogenic transplantation and using immunosuppressive drugs,intrathymic injection group: 3 mol/L KCl MHC antigen extractions were injected into the recipients’ thymus before two weeks before the sciatic nerves were transplanted. In the third week all the recipients underwent immunological detections for IL-2R,TNF-?,mixed lymphocytes culture and apoptosis.Results:All the detections indicated that it was of significant difference between intrathymic injection group and those in allogenic transplantation group.Conclusion:The immunological rejection of allogenic peripheral nerve transplantation can be somewhat inhibited by intrathymic injection of allogenic MHC antigen.