Protection Of Recombined Sap2 Protein Vaccination On Immunosuppressed Mouse Systematically Infected By Candida Albicans

BackgroundWith such developments of novel treatment technologies as bone marrow transplantation, organ transplantation and intensive treatment and the wide use of broad spectrum antibiotics and immunosuppressors, the phenomenon of clinical infection of candida becomes more and more widespread, meanwhile the limitation of antifungal agents derived from the side effects and drug resistance makes prevalence of candida albicans high in the patients of low immunity. Thus, in order to explore the preventive measures against the infection of candida albicans in the low immune patients, it's necessary to establish certain animal model of the infection of candida albicans after immunosuppression, which mimicking the conditions of the low immune patients, and followed by further investigation of the protection of vaccination for the systemic candida albicans infection in the low immune patients.Part oneObjectivesTo establish the immunosuppressive mouse model of systemic infection of candida albicans and observe the main influence factors.Methods1)6-week old BALB/c mice were injected intraperitoneally with two different dosages of cyclophosphamide, 100mg/kg and 200mg/kg respectively, then 48 hours later candida albicans were injected into the caudal veins at the following concentrations as 1×105,106,107/ml.the median lethal dose was defined by the mortality of each group.2)The inhibitory degrees of the white blood cell count,lymphocytes count in peripheral blood were observed after single intraperitoneal injection of cyclophosphamide with a dosage of 50 mg/kg orl00mg/kg.3)4- and 8-week old mice were used to observe the influence of week age on the mortality.4)The mouse model was validated by histopathological examination and fungal culture of renal tissues. Results1)The mortality of 6-week old balb/c mice was close to 50%,which were injected intraperitoneally with a single dose of 100mg/kg cyclophosphamide,followed by injection of candida albicans into caudal vein 48 hours later.2)The decreases of the white blood cells and lymphocytes in peripheral blood were more than 30% after single intraperitoneal injection of 100mg/kg cyclophosphamide.3)The mortality of 4-week old mice was high because of bad tolerability to all the conditions required for the mouse model.4)The results of histopathological examinations and cultures of fungi in renal tissue indicated that there were hypha growth and focal abcesse formation in the kidney of the mouse model.Conclusions1) The mouse model of systemic infection of candida albicans after immunosuppression was successfully established.2) High affinity of candida albicans to renal tissues was observed in the conditions of the mouse model.Part twoObjectivesTo observe the protection profile of recombinant Sap2 protein, with which the model mice were inoculated for 3 times (every 2 weeks), from systemic infection of candida albicans.Methods6-week old balb/c mice were randomized into three groups:1)recombinant Sap2 combined with complete Freund's adjuvant(CFA) were injected intramuscularly for 3 times at one week interval in the tibial anterior area, then single dose of 100mg/kg cyclophosphamide was injected intraperitoneally according to body weight one week after the third inoculation,48 hours after which 1×105/ml candida albicans were injected into caudal vein at a dosage of 0.01ml/g.2) CFA were injected intramuscularly for 3 times at one week interval in the tibial anterior area at the same other interventions as group one.3)PBS were injected intramuscularly for 3 times at one week interval in the tibial anterior area at the same other interventions as group one. Mice were raised by the above three grouping ways and were determined at different time point the following indexes:1)mortality of each group within 21 days after infection of candida albicans.2)levels of specific IgG antibody in the sera of each group of mice determined by indirect ELISA.3)serous levels of IL-2,IL-4,IFN-γ, TNF-αdetermined by ELISA.4)the ratio of CD4+/CD8+T cells in the spleen by flow cytometry.5)fungal quantity in the kidney of each group of mice.6)comparison of splenetic weight of each group of mice.7)histopathological examination of the kidney, liver and lung of each group of mice.Results1)Mortality of the inoculated mice group was significantly lower than that of control.2)The specific serous IgG antibody was significantly higher than that of control.3)At the test time point of day1/4/7 after candida albicans challenge, the serous levels of IL-2,IL-4,IFN-γand TNF-αat day4 were higher than that at dayl and day7.of note, the levels of TNF-αkept lower than that of normal control throughout the experiment.4) At the test time point of day1/4/7 after candida albicans challenge, the ration of CD4+/CD8+T cells in all groups presented with similar changes, of which the measured values at day4 were lower than that at dayl and day7. 5)The fungal quantities of inoculated mice were significantly lower that of control. 6)The splenic weight of the inoculated mice were significantly higher than that of control.7) The prevalence of hypha and focal abcesses in the kidney of inoculated mice was lower than that of control.Conclusions1)Three-time inoculation of these model mice (every 2 weeks) with recombinant Sap2 protein had certain protection against systemic infection of candida albicans.2) Three-time inoculation of the model mice (every 2 weeks) with recombinant Sap2 protein led to splenomegaly.