Preliminary Study Of The High Protective Immunological Mechanisms Of Schistosomulum Cells

Chapter One Comparison of protective immunity induced by schistosomulum cells with that by UV-attenuated cercariae[Objective] To compare the resistances against challenge infection in two susceptible strains of the mice, which were both immunized with schistosomula cells and UV-attenuated cercariae.[Methods] Schistosomulum were obtained from rabbits12days after infected with freshly released s.j cercariae and cells prepared. Afterwards, The cells and cercaiae attenuated with UV light were both used to immunize Kunning strain and Balb/c strain of mice.40Kunning strain of mice and40Balb/c strain of mice were divided into tatal eight groups (D1group:Kunning strain of mice immunized with schistosomulum cells; E1group:Kunning strain of mice immunized with UV-attenuated cercariae; F1group: normal control group of Kunning strain mice injected with PBS; F1group:normal control group of Kunning strain mice stuck by using a cover glass with NS on it through plucked abdomen; D2group:Balb/c strain of mice immunized with schistosomulum cells; E2group:Balb/c strain of mice immunized with UV-attenuated cercariae; F2group: normal control group of Balb/c strain mice injected with PBS; F2’group: normal control group of Balb/c strain mice stuck by using a cover glass with NS on it through plucked abdomen. The mice of D1and D2groups were subcutaneously injected with cells prepared from12-day old juvenile worms from both sides of inguinal groove for three times at2wks interval. Correspondingly, as control groups, F1and F2groups were injected with PBS. At the day of injection of cells or NS, the mice in E1and E2groups were stuck through plucked abdomen with a cover glass with400UV-attenuated cercaiae on it. Similarly, both F1’ and F2’groups were stuck with a cover glass only with NS on it.2wks post final immunization of cells or6wks post immunization of attenuated cercariae, the mice were challenged with30±1freshly released cercariea. On the42th day after challenge infection, mice were sacrificed and perfused, the worm burdens and liver eggs per gram counted and the sizes of liver granulomas compared.[Results] D1exhibited45.9%reduction in worm burden,62.9%reduction of liver egg burden,39.6%reduction in average diameter of granulomas and63.7%reduction in average area of granulomas. E1group showed a reduction of53.3%in worm burden,62.4%in liver egg burden,42.7%in average diameter of granulomas and67.2%in average area of granulomas. Interestingly, as compared two immunized groups, D1with E1, there wasn’t significant difference on the above parasitological indexes excepted on worm reduction rate with that of E1significantly higher than that of D1(P<0.05). For the experiment by using Balb/c strain mice as model, D2exhibited a reduction of56.3%in worm burden,57.1%in liver egg burden,42.4%in average diameter of granulomas and66.9%in area of granulomas.E2showed49.5%reduction in worm burden,42.7%reduction in liver egg burden,31.4%reduction in average diameter of liver granunomas and52.9%reduction in area of liver granuloma. Unexpectedly, all indicators of D2group was significantly higher than that of E2(P<0.05).[Conclusion] By employing two susceptible strains of mice as models, the experiment results indicated that immunization with schistosomulum cells and UV-attenuated cercariae were both capable of inducing high level of protective immunity, and, unexpectedly, the level of protective immunity induced by schistosomulum cells was matched with and even higher than that by UV-attenuated cercariae. Chapter Two Analysis of protective mechanism induced by the schistosomulum cells and UV-attenuated cercariae[Objective] To analyze and compare the high protective mechanism through the discussion of immune response produced by schistosomulum cells and UV-attenuated cercariae.[Methods] the experimental group was the same with chapter one. The levels of IgG, IgG2a, IgG1against soluble juvenile worm antigen (SJWA) in the sera of the progenies collected at different time points were detected by ELISA.[Results] Both D1group and E1group were produced significantly higher IgG level than that of F1in Kunming strain mice.the IgG level of D1group was significantly higher than that of E1group between the first immune to infection (P<0.05), after the attack, the IgG level of E1group increased faster and significantly greater than D2Group until12Wks (P<0.05). Both D1and E1groups produced higher IgG1and IgG2a level and its IgG2a/IgG1are greater than1, IgG2a/IgG1of D1group decreased from2.03to1.81after the attack. IgG2a/IgG1of E1group increased from1.73to2.23; In the experiment of Balb/c strain mice, D2group and E2Group were also produced higher anti-SJWA-IgG level than that of the normal control group, the IgG level of the two group had no significantly differences before the attack (P>0.05), but the IgG level of D2group gradually higher than that of E2Group after the attack(P>0.05), IgG2a/IgG1of E2group had a certain amount of rise after the attack, however, there was no significant change in IgG2a/IgG1in D2group.[Conclusion] By employing two susceptible strains of mice as models, the experiment results indicated that immunization with schistosomulum cells and UV-attenuated cercariae were both capable of inducing the immune response against challenge infection, and the higher level of IgG1, IgG2a and IgG2a/IgG1showed that the two immune methods could both be produced hybrid immune response which based on cellular immunization. Chapter Three Protection against challenge infection in the offspring mice born to immunized mother mice with schistosomulum cells[Objective] To explore the resistance against challenge infection in the two susceptible strains of the offspring mice born to mother mice immunized with schistosomulum cells [Methods] Both susceptible female Kunming strain and Balb/c strain of mice to schiostosome were immunized with S.j cells for3time, respectively. After mating with the corresponding strain of male mice, the offspring mice either born to immunized mothers or to untreated mothers were divided into4groups, A1:experiment offspring mice born to immunized Kunming strain of mother mice, B1:control offspring mice born to untreated Kunming strain of mothers, A2:experiment offspring mice born to immunized Balb/c strain of mother mice, B2:control offspring mice born to untreated Balb/c strain of mothers. About6-8weeks post neutralization rearing (both strains of mice in weight of16-20g), the two strains of offspring mice either born to immunized mothers (Al and A2group) or born to untreated normal mothers (B1and B2group) were challenged with S.j cercariae and the protection efficiency was evaluated with parasitological and immunological indicators. The worm burdens and liver egg loads collected from above4groups were counted and compared; The liver sections were prepared and stained with HE, and the diameters and areas of liver egg granulomas were estimated; The levels of IgG against SJWA in the sera of the progenies collected at different time points were detected by ELISA.[Results]Compared with two strains of control groups B1and B2, both strains of experiment offspring mice possessed certain level of protection against challenge infection with either Kunming strain of offspring mice exhibiting35.6%reduction in worm burden and59.4%reduction of liver egg burden or Balb/c strain of offspring mice showing29.8%reduction in worm burden and47.4%reduction in liver egg burden. The liver appearances of Al and A2mice were relatively normal as compared with that of control mice. At the same time, the mean diamaters and areas of granulomas in the sections of livers from A1and A2groups were significantly smaller than that in the sections of livers from B1and B2groups;. Furthermore, ELISA results showed that the levels of specific IgG in the sera from A1and A2group of mice were higher than the IgG levels in the sera from corresponding strain of control groups.[Conclusion] By employing two susceptible strains of mice to S.j infection as models, the experiment results indicated that the mother mice immunized with live schistosomulum cells could be able to deliver the protective immunity to their embryo through certain intrauterine rout and stimulated the immunity related stem cells of the embryo or early immature immune cells to form specific immunological memory and even make the next generation to inherit partial immune protection against initial acquired challenge of S.j.