| Schistosoma japonicum (S. japonicum) is an amphixenosis that could result in severe morbidity and mortality. Current schistosomiasis control in Asia is based primarily on snail eradication and large scale chemotherapy using the effective drug praziquantel. Although active drug praziquantel is available, evidence is now accumulating that if it can reduce the overall incidence of severe forms of the disease. Moreover, the parasite resistance to the drug has appeared. Thus, safe and efficacious vaccine development is an important measure for long-term integrated control of schistosomiasis.The vaccines against S. japonicum, including dead vaccine, attenuated live vaccine, genetically engineering and nucleic acid vaccine. The advantages of DNA vaccines over the traditional vaccine are the low cost of production, thermal stability and their ability to induce a wide variety of cellular and humoral immune response. Currently, DNA vaccine has become the highest priority of vaccine against schistosome. But to this day, there is still not yet a commercial DNA vaccine to the world. These results are perhaps explained by the fact that the schistosome parasite is such an antigenically complex organism with very complicated genome. Furthermore, they have achieved many mechanisms of immunized escape generated during long-term evolutional process together with host.More recently, there is convincing evidence that CD4+CD25+ Tregs are a variety of T cell subsets with suppressive properties, which play an important role in the progress of parasites escaping from the host immune assault. Evidence suggests that regulatory T cells help to protect the host from the hepatocyte damage induced by S. mansoni eggs and to prevent death from the infection through immune-mediated pathology. Thus, there is a need to explore the level of regulatory T cells in the host infected of S. japonicum, and to adopt related method for immunization of S. japonicum vaccine.Cimetidine (CIM) is a histamine-2-receptor antagonist that with highly successful use in gastric acid-mediated gastrointestinal disorders. Its use in clinical medicine is reported not only reduces or inhibits some infectious diseases induced impairment of the immune response such as chronic Herpes virus infection and HIV, but suppresses angiogenesis and tumor growth with few adverse biologic effects. Evidence suggests that CIM enhances a variety of immunologic functions because of its inhibitory effects on suppressor T cell function. There was convincing evidence that simultaneous administration of praziquantel and CIM could improve further the efficacy of the single therapy with praziquantel for cysticercosis and other arasitic diseases, such as schistosomiasis. Moreover, they had also achieved successful results to simultaneous administration cimetidine and variable vaccine.So in the present study, we evaluated the feasibility of using CIM to co-immunization with a S. japonicum DNA vaccine, the gene encoding 26 kDa glutathione S-transferase of S. japonicum (Sj26), which is one of promising vaccine candidated selected by WHO. Explore the adjuvant effect and mechanism of CIM in the protective efficacy against S. japonicum generated by pEGFP-Sj26GST DNA vaccine.The main contents and results of this thesis include three parts:Part 1:Studies on the effect of CIM on the immune responds of mice infected with S. japonicumObjective:Explore the effect of different schedule of CIM treatment on the S. japonicum infected mice.Methods:BALB/c mice were randomly divided into three groups.50 mg/kg CIM group (CIM50),25 mg/kg CIM group (CIM25) and infected control group (control).Animals in CIM50 treated with 50 mg/kg CIM subcutaneously (s.c) injection three times; Animals in CIM25 treated with 25 mg/kg CIM subcutaneously (s.c) injection three times; Six weeks of the treatment, mice in each group were challenged percutaneously with cercariae for 20 min. After 6 weeks post infection, all mice were succumbed. The percent of CD4+CD25+ Tregs in spleen was measured with flow cytometer. The expression of gamma interferon (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-5 (IL-5) in cultural suspension of splenic cells was detected by sandwich-ELISA after stimulation with ConA.Results:Compare with the Control group, the percentage of CD4+CD25+ regulatory T cells was significiantly decreased in the spleens of CIM25 Group mice (P<0.01), the cytokine production of splenocytes showed that animals in CIM25 group promoted an elevated level of IFN-γand IL-2 (P<0.05). But compare with the Control group, neither the percentage of CD4+CD25+ regulatory T cells nor the cytokine production of splenocytes in the spleens of CIM50 Group mice showed significiantly different (P>0.05).Conclusion:CIM can be used as an immunomodulator drug and could be useful in protective immunity against schistosome infection of mice in a dose-response manner.Part 2:Studies on the effcet of co-immunization with CIM and pEGFP-Sj26GST DNA vaccine on the protective efficacy in miceObjective:To study the protective efficacy of co-immunization with pEGFP-Sj26GST DNA vaccine and CIM against S.japonicum in mice.Methods:The plasmid pEGFP-Sj26GST and pEGFP vector were amplified as DNA vaccine to immunize BALB/c mice.50 female BALB/c mice were randomly divided into five groups, each with 10 animals. CIM with pEGFP-Sj26GST DNA vaccine immunization was used as co-treated group. The pEGFP or pEGFP-Sj26GST alone was used as the immune challenge group. CIM alone was used as adjuvant groups. Sixteen non-treated mice were used as infected control group. Animals treated with CIM received a daily subcutaneously (s.c) injection of 25 mg/kg from day-2 through the entire 6-week. After 1, 14 and 28 days of treatment mice were immunized intramuscularly with 100μg pEGFP-Sj26GST DNA vaccine or pEGFP vector. Control animals received an equivalent volume of Sodium Chloride via the same route. After two weeks of the final boost, mice were challenged percutaneously with 40 of cercariae for 20 min by the cover glass method. On day 42 following the challenge, adult worms were perfused from hepatic portal system and also manually removed from mesenteric veins after mice were sacrificed. Percent of worm reduction and percent reduction in liver eggs in co-immunization group were compared with that in pEGFP-Sj26GST alone group and infected control group. Liver tissue sections were stained by histopathological examination to detect S. japonicum egg granuloma. The histopathological examination showed a significantly (P<0.01) greater number of egg granulomas in the control group (16.25±2.87) than in the CIM plus pEGFP-Sj26GST group (4.5±1.76). Sections of groups of mice immunized with pEGFP-Sj26GST DNA vaccine plus CIM showed fewer and smaller egg granuloma. In contrast, sections of the control groups showed a greater number of portal egg granulomas.Results:The recombinant plasmid pEGFP-Sj26GST was successfully abstracted, and could be used as DNA vaccine. Compared with the infected control group, mice inoculated intramuscularly with pEGFP-Sj26GST showed worm reduction of 27%(P<0.05), and co-injection of CIM and pEGFP-Sj26GST dramatically enhanced worm reduction to 79% (P<0.01), there was significant difference between the two groups. In addition, there was substantial reduction in the number of eggs present in the livers and feces of the pEGFP-Sj26GST alone and plus CIM immunized groups (P<0.05) when compared to the control groups.Conclusion:Compared with the pEGFP-Sj26GST DNA vaccine alone group, the mice co-immunization with pEGFP-Sj26GST and CIM could significiant enhance the protective efficacy against S. japonicum. The immunization with pEGFP-Sj26GST plus CIM could impair the development of schistosome egg granulomas.Part 3:Studies on the mechanism of CIM to enhance the protective efficacy induced by pEGFP-Sj26GST DNA vaccineObjective:To explore the effect of pEGFP-Sj26GST DNA vaccine and cimetidie on the percentages of CD4+CD25+ Tregs; To explore the mechanism of CIM in the protective efficacy against S. japonicum generated by pEGFP-Sj26GST DNA vaccine. Methods:80 female BALB/c mice were randomly divided into five groups, each with 16 animals. CIM with pEGFP-Sj26GST DNA vaccine immunization was used as co-treated group. The pEGFP or pEGFP-Sj26GST alone was used as the immune challenge group. CIM alone was used as adjuvant groups. Sixteen non-treated mice were used as infected control group. Animals treated with CIM received a daily subcutaneously (s.c) injection of 25 mg/kg from day-2 through the entire 6-week. After 1,14 and 28 days of treatment mice were immunized intramuscularly with 100μg pEGFP-Sj26GST DNA vaccine or pEGFP vector. That means the animals were inoculated three times with two weeks interval. Control animals received an equivalent volume of Sodium Chloride via the same route. One week after last immunization,6 mice from each group were sacrificed to determine the anti-Sj26GST antibodies in the individual serum samples (diluted in PBS) by GST-ELISA. Two weeks after the last immunization, mice were challenged percutaneously with 40 of cercariae for 20 min by the cover glass method. On day 42 following the challenge, spleens were obtained sterile, preparation single cell suspension. Flow cytometry was performed to detect the percentage of CD4+CD25+ Tregs. Single suspensions of splenocyte were harvested as describe previously and 2×105 cells/well were cultured in 96-well plates, in triplicate, in RPMI-1640 medium containing 5% FCS at 37℃in a 5% CO2 incubator for 48 h. Then we used MTT to express the splenocyte proliferation responses to ConA in vitro. The concentrations of IFN-γ, IL-12, IL-4, IL-5 and IL-10 in cell culture supernatants were determined as described by the manufacturer of the ELISA Kits.Results:1) The mean optical density values (mean±standard deviation) of anti-Sj26GST antibody in the mice immunized with pEGFP-Sj26GST was higher than that in the control group injected with CIM or saline alone (P<0.05). And at the same time, co-injection of CIM and pEGFP-Sj26GST did produce a significantly higher level of anti-Sj26GST antibody (P<0.05), compared with vaccination with pEGFP-Sj26GST alone.2) The percentage of CD4+CD25+Foxp3+ T cells in spleens of mice treated with pEGFP-Sj26GST was significantly decreased from infected control mice (P<0.05). Meanwhile the percentage of CD4+CD25+Foxp3+ T cells in groups with pEGFP-Sj26GST plus CIM was dramatically dropped down and was significantly lower than those in the infected control groups (P<0.01).3) Mice immunized with CIM and pEGFP-Sj26GST was seen to produce vigorous lymphocyte responses, compared with the control group (P<0.05).4) Co-injection of CIM and pEGFP-Sj26GST significantly increased the production of IFN-y and IL-12 compared with pEGFP-Sj26GST alone (P<0.05). However, spleen cells showed no difference in IL-4 and IL-5 production among all five of the groups tested and the expression of the anti-inflammatory cytokine (IL-10) were significantly decreased (P<0.05).Conclusion:1) The recombinant plasmid pEGFP-Sj26GST can be used as DNA vaccine. The co-immunization CIM with pEGFP-Sj26GST DNA vaccine could produce a significantly higher level of anti-Sj26GST antibody in BALB/c mice.2) The percentage of CD4+CD25+Foxp3- T cells in groups with pEGFP-Sj26GST plus CIM was dramatically dropped down and was significantly lower than those in the pEGFP-Sj26GST alone group.3) Mice immunized with CIM and pEGFP-Sj26GST could produce an enhanced cell-mediated response in vivo.4) CIM could alter the Th1-Th2 balance in murine hosts even during a Th2-promoting S. japonicum infection. The immunization with pEGFP-Sj26GST plus CIM could significantly enhance the protection of DNA vaccine through enhancing Th1 type immune responses. hi conclusion, these data demonstrated:1. In our study we confirmed the finding that CIM could exerts immunomodulating properties through enhancing Thl type immune responses during S. japonicum infection.2. It was firstly reported that the co-immunization CIM with pEGFP-Sj26GST DNA vaccine could significantly enhanced the protection of DNA vaccine. 3. Our data showed for the first time that vaccination of mice with pEGFP-Sj26GST DNA vaccine plus CIM could significantly impair the suppressive function of CD4+CD25+ Treg cells.
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