[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).


CONCLUSIONS
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.
Ye
Yan-ju
YJ
Institute of Infectious and Parasitic Diseases, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Li
Wen-gui
WG
D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology*; D004443:Echinococcosis / Q000276:immunology* / Q000517:prevention & control; D048209:Echinococcus granulosus / Q000235:genetics*; D005260:Female; D050939:Gene Fusion; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology*; D016753:Interleukin-10 / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D000455:Medicago sativa / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D018417:Th1 Cells / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000947:Antigens, Helminth; C446034:EG95 protein, Echinococcus granulosus; D015801:Helminth Proteins; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D018664:Interleukin-12