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Objective To construct a novel M.tb DNA vaccine (p846) co-expressing mycobacterial triple antigens including Rv3615c, Mtb10.4 and Rv2660c, and evaluate its cellular immune response and protective efficacy against tuberculosis infection in BALB/c mice. Methods We constructed the p846 by using the cloning technology. The 6- to -8-week old female BALB/c mice were randomly divided into 4 groups: p846, pcDNA3.1, PBS and the BCG group. All mice were administrated intramuscularly with 50 μg recombinant plasmids at 0, 2, 4, 6 week. A single dose of BCG was injected subcutaneously in the BCG group. Two weeks after the final immunization, 10 mice in each group were used for cell proliferation, ELISPOT and FCM assay, BCG challenge experiment and HE staining of lung were performed at 4, 6 weeks later, respectively. Results The p846 vaccine could effectively induce the specific T cell proliferation(P < 0.001) and increase the numbers of IFN-γ+T cells(P <0.001), compared with those in the PBS group and the vector conreol group. The mouse lung tissue presented very mild lung inflammation in the p846 group, compared with other groups. Conclusion Vaccine p846 could not only induce strong cellular immune response, but also efficiently protect BALB/c mice against M.tb infection.
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C-reactive protein (CRP), an acute-phase protein with an ability to bind to nuclear antigen, has been reported to regulate cytokine secretion and modulate immune responses. We previously reported that activated syngeneic lymphocyte-derived apoptotic DNA (apopDNA) could induce macrophage activation and contribute to the initiation and progression of lupus nephritis. It is reasonable to hypothesize that CRP might regulate apopDNA-induced macrophage activation. Herein, CRP was shown to promote macrophage-mediated apopDNA uptake by binding to apopDNA (CRP/apopDNA complex). Notably, CRP/apopDNA treatment inhibited the production of inflammatory cytokines and chemokines by macrophages which could be induced by apopDNA alone. Further coculture and transwell studies revealed that CRP/apopDNA-induced macrophages prohibited apopDNA-induced macrophage activation in an IL-10 dependent manner. These results provide insight into the potential mechanism of CRP regulatory activity in macrophage activation induced by apopDNA in the context of lupus nephritis and other autoimmune diseases.
Subject(s)
Animals , Female , Mice , C-Reactive Protein , Metabolism , Pharmacology , Cell Line, Tumor , DNA , Metabolism , Pharmacology , Enzyme-Linked Immunosorbent Assay , Lupus Erythematosus, Systemic , Metabolism , Lupus Nephritis , Metabolism , Macrophage Activation , Physiology , Mice, Inbred BALB C , Protein Binding , Real-Time Polymerase Chain ReactionABSTRACT
Objective To explore the mechanism of lymphotactin(LTN) to exert mucosal adjuvant activity. Methods Complexes of chitosan-pVP1 and chitosan-pLTN were seperately prepared by co-cojugation method, then 50μg(DNA) of each complex was administered intranasally to male BALB/c mice 4times biweekly. Two weeks after the final immunization, mice were challenged with 3LD50 CVB3 to cause viral myocarditis, heart histopathological changes were examined 7 days later. Meanwhile, T cell immune responses, DC percentage and its membrane CD86 expression were monitored in spleen, mesenteric lymph node(MLN) and cervical lymph node(CLN). Results Co-immunizaiton with LTN remarkbly alleviated CVB3-induced cardial injury. This improvement was accompanied with enhanced T cell proliferation and IFN-γ-secreting ability, increased DC frequency and membrane CD86 expression both in spleen and mucosal draining lymph nodes( MLN, CLN). Conclusion LTN exerts its mucosal adjuvant function in augmenting specific T cell immune responses systemically and mucosally via DC enrichment in spleen, MLN and CLN and up-regulation of DC maturation.
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Objective To observe the correlation of histologicalactivity(HAI) of chronic hepatitis B (CHB) with CCL20 expression, and to investigate the impact of CCL20 expression in CHB infection. Methods On the basis of established competitive quantitative RT-PCR with an internal standard, the expression of the CCL20 in the hepatocytes in different infected patterns of HBV infected cells and liver biopsies were quantified and at the same time its correlation to HAI were explored. Results In the cell levels, the expression quantity of CCL20 in control cells (HepG2), persistent HBV infected hepatocytes( HepG2. 2. 15) are (2. 65 ± 0. 02) pg/106 cells, ( 1.22± 0. 04) pg/106 cells, respectively. There were significantly differences between them ( t = 39. 66, P < 0. 01 ). The expression of CCL20 was enhanced in hepatocytes stimulated by PMA but their expression pauern was not changed. Moreover, CCL20 expression in liver biopsies with CHB was (3.54 ± 0. 65 ) pg/20 mg and CCL20 expression in control groups was ( 8. 74±0. 56) pg/20 mg. The expression of CCL20 between two groups was different (t =30. 09,P <0. 01 ) and correlation lied in between HAI and CCL20 expression in liver biopsies of CHB patients ( r = 0. 675, P =0. 023 ). Conclusion CCL20 expression was down-regulated and it was correlated to HAI of liver biopsies in CHB patients.
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Objective To investigate the expression of Th1/Th2 cytoki nes in early symptomatic syphilitic lesions and its relationship with the sero-c onversion of RPR test in patients with syphilis. Methods The expression of Th1/Th2 cytokines in lesions from 30 patients with early symptomatic syphilis was i mmunohistochemically detected with ABC method. The serum titers of RPR test in t hese patients were measured in 1, 3, 6, 9 and 12 months after routine benzathine penicillin treatment. Results Among 10 cases of primary syphilis, the express ion of both Th1 and Th2 cytokines in the lesions was found in 9 cases, while onl y Th1 cytokine expression was observed in the remainder case; and the sero-conve rsion of RPR test occurred in all 10 cases during the follow-up period. Among 20 cases of secondary syphilis, Th1/Th2 cytokines expressed in the lesions in 16 c ases, and only Th2 cytokines expressed in 4 cases; and the sero-conversion of RP R test was found in 12 cases during the follow-up period. The expression of Th1 cytokines in early syphilitic lesions was positively correlated with the sero-co nversion of RPR test. The higher the expression of IFN- the more likely the s ero-conversion of RPR test. Conclusion The early activation and persistence of the expression of Th1 cytokines may play an important role in the clearance of pathogens.
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95%,WT= 1 271 6 .Lymphoproliferation reaction of the splenocytes derived from BALB/C mice was inhibited by stimulating with the antisense peptide pre treatment of allogeneic splenic cells and cardiac cells of C57 mice.The inhibition rates up to 38 4% and 36 6%.Pre treatment of allogeneic cardiac grafts resulted in prolonging the survival of cardiac transplantation 5 4 days more than the control group(n=6,P
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Ectopic human chorionic gonadotrophin (hCG) is an autocrine hormone expressed by most malignant tumor cells. Increasing data recently showed that the expression pattern and biological properties of ectopic hCG was significantly different from that of the normal hCG secreted by trophoblastic cells. Evidence from different research groups strongly indicated that there was a direct relationship between the expression of ectopic hCG and the development of malignant tumor. Ectopic hCG may play a key role in regulation of tumor growth,metastasis and immune tolerance versus malignant tumor. The advances in the research of the molecular biologic characteristics and functions of ectopic hCG are reviewed and evaluated. [
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Purpose To oberve the dynamics of liver granuloma and the relative changes of Thl/Th2cytokine levels in mice infected with Schistosoma japonicum, and investigate the role of Th1/Th2 in S.japontcum granuloma formation and regulation. Methods Liver granuloma measurement were performedby histological examination and the ELISA were used for the quantitative determination of IL-2, IFNr andIL-4 in murine serum and spleen lymphocyte culture medium at 0,4,6,8,10 and 12 wk after infection.Results At 6 wk liver granuloma formation appeared and at 8 wk liver granuloma peaked. After 12 wk livergranuhoma diminished obviously. Meanwhile, at 4 - 6 wk IL-2, IFNr and IL-4 began to rise, at 8 wk thelevels of Th1 cytokines IL-2 and IFNγ peaked and then declined, and at 8 wk the levels of Th2 cytokines IL-4 were rapidly enhanced and increased obviously with a prolongation of the infection duration.Conclusions The Th1cytokines IL-2 and IFNγ were correlated well with S. japonicum granulomaformation and vigour, and the Th2 cytokines IL-4 might play an important role in down-regulating egggranuloma reaction at chronic schistosomiasis by inhibiting the Th1cytokines.
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Memory T cells play an important role in the pathogenesis of tumors as a result of the long term of tumor immunity and the heterogeneity of T cells. Great progress in the studies on the generation, classification, mechanism of maintenance and function of memory T cells in tumor immunity have been made in recent years, which may facilitate our understanding of immunological memory and the clinical therapy of tumor.
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Objective: To study the anti-tumor efficacy induced by antibodized tumor epitope PDTRP gene immunization. Methods: Three copies of tumor associate gene PDTRP from MUCI tandem repeats were designed and mimicked the conformation of MUCI by Insight Ⅱ . The ?lneo-PDTRP plasmid was further constructed, in which the PDTRP target gene was inserted into CDR3 of the ?1 -neo vector. The specific humoral and cellular immune responses towards to PDTRP were detected after intraspleen immunized Balb/c mice with "ylneo-PDTRP. And the immune protection assay was also done to observe whether the mice immunized with ?lneo-PDTRP could prolong the survival after tumor challenge. Results: The conformation of three copies of PDTRP mimicked the conformation of MUCI tandem repeats. The expression of ?lneo-PDTRP could be detected after in vitro transfect. The specific antibody against PDTRP epitope could be induced and increase to a higher titer after intraspleen injection with a ?lneo-PDTRP plasmid. And the specific proliferation and cytotox-ic function of lymphocyte were also increased. There is a significant survival from mice immunized with ?lneo-PDTRP a-gainst the 4T1-PDTRP tumor challenge. Conclusions: Gene immunization with ?lneo-PDTRP could elicit both humoral and cellular tumor specific immune response and had the protective effect.
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Objective: To investigate whether the plasmid ?1neo-hgp100 could be expressed and presented in vitro and could protect the immunized mice from B16F10 challenge in vivo. Methods: ?1neo-hgp100 plasmid was constructed in which the DNA sequence encoding hgp100 CTL epitope inserted into CDR3 of ?1-neo vector. The expression of anti-bodized antigen and IFN-? in supernatant was measured by ELISA respectively after transfection J558L with ?1neo-hgp100 and further co-culture of J588L transfacted with ?1 neo-hgp100 and pmel TCR transgenic T cell. After introspleenic inoculation of ?1neo-hgp100, the protective efficacy of the gene vaccine was observed by means of measuring the tumor area every two days. Results: ?1neo-hgp100 could be expressed and presented in vitro, the immunogenecity of CTL epitope of hgp100 was strong enough and could activate gp100 specific T cell, the mice immunized with the gene vaccine could resist the tumor challenging in vivo. The mean survival time was prolonged to 36 days, compared to control group (P
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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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Objective:To investigate the effects of IP10(IFN-? inducible protein 10,CXCL10) on anti-tumor immune response and to explore its mechanisms involved in.Methods:A mammary carcinoma cell line 4T1 was transfected with pcDNA3-IP10(IP10-4T1) by electrophoration and positive clones were screened in the presence of G418.Growth kinetics of IP10-4T1 cells was observed in vitro and in vivo.Survival rate among the animals was determined by daily assessment.Proliferation activity of lymphocytes was analyzed with()~3H-TdR incorporation.The phenotypes of lymphocytes isolated from tumors by ficoll density gradient centrifugation were assayed by flow cytometry.Results:The growth rate of IP10-4T1 cells was similar with that of parental 4T1 cells and neo-vector transfected 4T1 cells in vitro.Growth of the tumors formed by IP10-4T1 cells was inhibited in vivo.Compared to those of controls,the size and the weight of the tumors formed by IP10-4T1 cells decreased significantly 35 days post tumor transplantation(P
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Objective:To study the relationship between the dose of active DNA and the induction of SLE-like syndrome.Methods: DNA from extracted ConA-activated spleen lymphocytes and immunized syngenic mice with different quantities of active DNA, the anti-dsDNA antibodies and anti-histone antibodies as well as the antibody subclass were detected by ELISA.The patterns of antinuclear antibodies and immune complexes in glomeruli were observed by immunofluorescent-stain. Results: 10 pig active DNA could induce all the animal to produce anti-dsDNA and anti-histone antibodies,and the induced autoann'bodies were mainly IgGl type.Only 25%animals produced autoantibodies immunized by 5 fjig active DNA. Conclusion:The minimum dose of active DNA to induce SLE-like syndrome was 10