[Changes of cytokine secretion in mice by immunization with leaf protein extracted from Echinococcus granulosus Eg95-EgA31 transgenic alfalfa].

OBJECTIVE: To investigate the weight reduction of hydatid cyst and the changes of cytokine secretion in mice immunized by leaf protein extracted from Echinococcus granulosus (Eg) Eg95-EgA31 transgenic alfalfa and challenged by Eg protoscoleces. METHODS: Leaf protein was extracted from E. granulosus Eg95-EgA31 transgenic alfalfa by heat coagulation method. Meanwhile, leaf protein extracted from transgenic alfalfa containing pBI121 and normal alfalfa served as control. 32 female BALB/c mice were randomly divided into 4 groups. Groups A and B were immunized intragastrically (100 p1) and intranasally (10 microl) respectively by leaf protein containing Eg95-EgA31 fusion antigen, group C was vaccinated intranasally by 10 microl. leaf protein with pBI121, group D was given intragastrically 100 microl normal leaf protein. All mice were immunized once per 3 days for 2 months. Mice in all groups were challenged with 50 Eg protoscoleces on the 8th week after vaccination and sacrificed on the 24th week after infection. The weight of hydatid cysts was measured and weight-reduction rate was calculated. Spleens were collected to prepare splenocytes which were cultured under stimulation with EgAg, concanavalin A (ConA) or lipopolysaccharide (LPS). The supernatant was collected to measure Objective To investigate the weight reduction of hydatid cyst and the changes of cytokine secretion in mice immunized by leaf protein extracted from Echinococcus granulosus (Eg) Eg95-EgA31 transgenic alfalfa and challenged by Eg protoscoleces. METHODS: Leaf protein was extracted from E. granulosus Eg95-EgA31 transgenic alfalfa by heat coagulation method. Meanwhile, leaf protein extracted from transgenic alfalfa containing pBI121 and normal alfalfa served as control. 32 female BALB/c mice were randomly divided into 4 groups. Groups A and B were immunized intragastrically (100 microl) and intranasally (10 microl) respectively by leaf protein containing Eg95-EgA31 fusion antigen, group C was vaccinated intranasally by 10 microl. leaf protein with pBI121, group D was given intragastrically 100 microl normal leaf protein. All mice were immunized once per 3 days for 2 months. Mice in all groups were challenged with 50 Eg protoscoleces on the 8th week after vaccination and sacrificed on the 24th week after infection. The weight of hydatid cysts was measured and weight-reduction rate was calculated. Spleens were collected to prepare splenocytes which were cultured under stimulation with EgAg, concanavalin A (ConA) or lipopolysaccharide (LPS). The supernatant was collected to measure the level of IL-12, IL-10, IFN-gamma and TNF-alpha by ELISA. RESULTS: The average weight of hydatid cysts in groups A, B, C, and D was (28.0 +/- 36.0), (41.0 +/- 33.0), (72.0 +/- 36.0) and (78.0 +/- 57.0) mg, respectively, the cyst weight of group A was lower than that of group D (P < 0.05), decreased by 64.1%. The levels of IFN-gamma, IL-12 and TNF-alpha in group A [(925.0 +/- 88.6), (22.5 +/- 2.7) and (82.5 +/- 11.7) pg/ml] were higher than those of group D (P < 0.01), while the IL-10 level in group A [(125.0 +/- 26.7) pg/ml] was significantly lower than that of group D (P < 0.01). The level of IFN-gamma [(750.0 +/- 100.0) pg/ml] and TNF-alpha [(80.0 +/- 13.1) pg/ml] in group B was significantly higher than those of group D (P < 0.01); but there was no significant difference in the level of IL-12 and IL-10 between the two groups (P > 0.05). No considerable difference in the cytokines was found between group C and group D (P > 0.05). The levels of the 4 cytokines in groups stimulated by EgAg, ConA or LPS were higher than those without stimulation (P < 0.05 or < 0.01), and those in groups stimulated by ConA or LPS were higher than groups stimulated by EgAg (P < 0.05 or < 0.01). CONCLUSION: Th1 response may be induced in mice by immunization with the leaf protein extracted from Echinococcus granulosus Eg95-EgA31 transgenic alfalfa to resist the challenge of Eg protoscoleces.

[The study on immune responses in BALB/c mice by immunization with the transgenic Alfalfa(Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus].

AIM: To investigate the immune responses and the protection induced by the transgenic Alfalfa (Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus against Eg protoscoleces. METHODS: The leaf protein was extracted from the transgenic alfalfa by heat-coagulation method, its concentration was prepared for 20 g/L. BALB/c mice were immunized intranasally or orally with the leaf protein once per 3 days for 2 months.At the same time, the leaf protein transfected with pBI-121 blank vector and the normal leaf protein without foreign antigen was served as control. The mice were then challenged intraperitoneally with Eg protoscoleces (50 protoscoleces per mouce)on week 8 after the last vaccination and sacrified on week 24 postinfection to count the rate of reduced hydatid cyst. The specific antibody (IgE, IgG and its subclasses)in the sera collected from the eyeballs was evaluated by ELISA. Splenocytes were separated and cultured in vitro with EgAg, ConA or LPS stimulus.The substes of CD4(+); and CD8(+); T cells were measured by FCM. The splenocytes' proliferation was determined by MTT method.Then the cells were collected, stained by PI and Annexin V-FITC, and analyzed by FCM to get the splenocytes' apoptotic rate.The supernatant was collected to measure the level of IL-12, IL-10, IFN-gamma and TNF-alpha by ELISA. RESULTS: Compared with the control group, The hydatid cyst weight in the oral immunization group decreased by 64.1%; the splenocytes' apoptotic rate got obviously lower than that in the control group; the splenocytes' proliferation increased significantly, the CD4(+); subsets and the ratio of the CD4(+);/CD8(+); did so, and the similar trend about the specific antibody titer and the level of cytokines could be seen in this group. CONCLUSION: Apoptosis of splenocytes may be inhibited in mice by immunization with the transgenic alfalfa, splenocytes' proliferation and Th1 response can be induced in the mice against the challenge of Eg protoscoleces. CD4(+); T cell and the specific antibody(IgG, IgG2b and IgE) may play important roles in the protection induced by the transgenic alfalfa vaccine.