Affirmation On Cell-type Immunogen From Schistosomulum Japonicum And Observation Of The Effects Of Schistosomulum Cells Combined With Immunomodulators On Anti-infection

Chapter One Protective immunity induced by the cell-type antigen and non-cell type antigen of Schistosomulum japonicum[Objective] Previous researches have proved that immunization with intact live cells resourced from Schistosomulum japonicum (Sj) was capable of inducing production of immunoprotection against challenge infection by Sj cercaria in Kunming strain murine model. In the present study, the aim is directed to validate whether the protection induced by Sj cells was associated with the physical characters of the immunogens and compare the protective immunity induced by the particulate antigen of Sj cells with that induced by soluble antigen of Sj cells. [Methods] Schistosomulum were obtained from rabbits 18 days after infected with Sj cercaria. Afterwards, cells from the worms used for immunization were prepared, and PCR was performed to identify the genus and evaluate whether containing contamination from host's tissue. 50 Kunming strain mice were divided into PBS group,schistosomulum cell-type antigen (SCA) group, schistosomulum non-cell type antigen (SNCA) group, schistosomulum worms fraction antigen(SWFA) group and schistosomulum worms soluble antigen(SWSA) group. Immunization process in the experiment was subcutaneous and intramuscular injection in inguinal groove for three times with 2 wks interval. 4 wks of final immunization, the mice were challenged with 30±1 cercarea. On the 45^th day after the challenge infection, mice were sacrificed and perfused, the worm burden and liver egg load counted and the sizes of liver granulomaes compared. Synchronously, primary dynamic levels of specific total antibody classes were observed by ELISAs. [Results] No contaminant from host tissue was found in the prepared cells by PCR. By parallel comparison with control group, the results from experimental groups revealed that the reduction rate of worm burden and liver egg burden (LEPG) were 46.8% and 56.2% for SCA group, 35.3% and 38.2% for SNCA group, 31.4% and 30.6% for SWFA group and 8.0% and 20.2% for SWSA group, respectively. Compared to PBS control group, SWSA immunized mice showed no significant reductions in worm burden, LEPG, liver eggs per female worm and granuloma areas (P>0.05). SCA immunization revealed significant reductions in granuloma areas compared with other four groups (P<0.05). High levels of IgG were detected in all immunized groups, correlating positively with immunization times, but without significant difference between each group was found at the same time point. [Conclusions] The research showed in advance that schistosomulum cells were capable of inducing significant immunoprotection against schistosomiasis. One of its mechanisms might be the physical character of Immunogen.Chapter Two Dynamic observation of the mice immunized with either cell-type antigen or non-cell type antigen from Schistosomulum japonicum[Objective] To prove whether there is a connection between the mechanism of the protective immunity induced by cell-type antigen and the slow-release immunogen in vivo and observe the pathological changes induced by Sj cell antigen. [Methods] Kunming strain mice were divided into A and B groups. The mice of group A and group B were subcutaneously and intramuscularly injected with either 107 schistosomulum cells or non-cell type antigen prepared from equal amount of cells at quadriceps. Both groups of mice were then treated 24hrs,3d,6d,9d and 12d after injection. The materials were taken from the immune locus of the mice to identify and detect the immunogen by using immunohistochemistry and western blot techniques, respectively. Also, histopathology technique was used to observe the pathological changes. [ Results ] Analysis of SDS-PAGE showed that there was no significantly different band between extracts of quadriceps from group A and B. Western blot suggested that a 75kDa antigen of Sj could be detected in the muscular tissue from group A of mice at 12 day after immunization but disappeared in group B at 3 day after immunization. The result of immunohistochemistry matched the western-blot. Histopathological study demonstrated that there was no pathological change in both group A and group B after 1 day of injection. Inflammatory cell infiltration could be found in muscular tissue from both groups of mice and the phenomenon sustained in group A until 12th after injection. However, for group B, it could be barely found at this time. The inflammatory cells in group A and B are mainly neutrophils and lymphocytes. No pathological change was found in myocytes of both groups. [Conclusions] The schistosomulum cells released antigen ingredients slower than soluble antigen and induced more significant inflammatory responses. The characteristic of the schistosomulum cell antigen may be one of the important mechanisms in inducing protective immunity against schistosomiasis.Chapter Three Protective immunity induced by Schistosomulum cells combined with different immunomodulators[Objective] To observe whether the protective immunity was further enhanced by the schistosomulum cells by combining with different immunomodulators. [Methods] Schistosomulum cells were obtained from rabbits 18 days after infected with Sj cercaria, which were identified of genus and without contamination from host's tissue by PCR . Animal were divided into 8 groups: PBS group (A), schistosomulum cell (SC) group (B), L-2 group(C), L-2 + SC group(D), IFN-γgroup (E), IFN-γ+ SC group (F), Lentinan group (G) and Lentinan + SC group(H). Immunization rout in the experiment was subcutaneous and intramuscular injection in inguinal groove for three times with 2 wks interval. 4 wks of final immunization, the mice were challenged with 30±1 cercarea. On the 35^th day after the challenge infection, mice were sacrificed and perfused, and the worm burden and liver egg load were counted. Primary dynamic levels of specific total antibody classes were observed by ELISAs. [Results] No contaminant from host tissue was found in the prepared cells by PCR. By parallel comparison with control group, the results from experimental groups revealed that the reduction rate of worm burden and liver egg burden were 38.4% and 59.8% for group B; 13.5% and 26.6% for group C; 36.1% and 52.1% for group D; 32.7% and 47.7% for group E; 46.2% and 57.7% for group F; 18.3% and 34% for group G; 33.7% and 53.7% for group H. The reduction of female worm and eggs per female were 42.1% and 48.6% for group B; -2.1% and 25.2% for group C; 21.1% and 44.4% for group D; 38.9% and 34.7% for group E; 40% and 50% for group F; 30.5% and 28.5% for group G; 40% and 42.3% for group H. High levels of IgG were detected in all SC groups combined with different immunomodulators (P<0.05). However, no significant difference between each group combined with different immunomodulators and single SC group was found (P>0.05). [Conclusions] The protective immunity induced by Sj cells was not further enhanced by immunomodulators, though the single IFN- y group showed some protective immunity against Sj.