Objective To investigate the feasibility of inducing neonatal immunotolerance against Graves'disease by gene TSH receptor (TSHR) 289 and its possible mechanism.Methods Neonatal (0-24 h) female BALB/c mice were divided into intraperitoneal injection group,intramuscular injection group,model group,and normal control group.The intraperitoneal group and the intramuscular group were further divided into low-dosage,middle-dosage,high-dosage tolerance groups,and the coresponding control groups.The tolerance groups and the controls were intraperitoneally or intramuscularly pretreated with low-dosage( 1×106 particles),middle-dosage( 1 × 108particles),high-dosage( 1 × 1010 particles)of Ad-TSHR 289 or Ad-lacz respectively.6 to 7 weeks later,the normal control group received intramuscular injection with Ad-lacz; the other groups were immunized with Ad-TSHR289,three times at 3 weeks interval.10 days after the first immunization,serum TRAb was detected.4 weeks after the last immunization,serum TRAb,TT4,splenic CD4 + CD25 + Foxp3/CD4 + were tested,and the thyroid tissues were examinated histologically.Results Ten days after the first immunization,no antibody response against TSHR was detected in the two high-dose tolerance groups,but the TRAb titer in respective controls was significantly higher( P＜0.05 ).4 weeks after the last injection,in high-dose tolerance groups,only 1/10 of mice immunized by intraperitoneal or intramuscular injection elicited anti-TSHR antibody,and no mice immunized intraperitoneally had elevated serum TT4.Two of ten mice challenged intramuscularly showed slightly increased TT4 levels,but the respective controls displayed a strong antibody response( P＜0.01 ) and elevated TT4 level ( P＜0.05 ).The similar percentages of high TT4 and thyroid hyperplasia were found in all groups.Additionally,the frequencies of CD4+CD25 +Foxp3/CD4+in two high-dose tolerance groups were significantly increased as compared to those in controls( P＜0.05 ).The incidence of Graves' disease in the other groups by intraperitoneal or intranuscular injections was not statistically different from those in the corresponding control groups and the model group.Conclusions The immune tolerance against Graves'disease is induced in neonatal mice by either intraperitoneal or intramuscular pathway with specific antigen of TSHR 289,carried by adenovirus vector,and then inhibits Graves' disease in adults. Stimulation with the high-dosage antigen is liable to induce immune unresponsiveness.CD4 + CD25 + Foxp3 +T cells may play an important role in the induction and maintenance of tolerance.

Objective To compare the efficacy of Graves disease animal models induced by thyroid stimulating hormone receptor (TSHR) plasmid DNA (pcDNA3.1-TSHR) and by TSHR A subunit recombinant adenovirus(Ad-TSHR289),and to investigate the influence of duration for preparing animal model induced by Ad-TSHR289 on Graves hyperthyroidism and its related indices.Methods The plasmid group and the adenovirus group were set up respectively.The plasmid group:21 female BALB/c mice were randomly divided into model group (n =12) and control group (n =9).The model group were injected intradermally with pcDNA3.1-TSHR 50 μg,once every 3 weeks,totally 3 times.Then 4 weeks after the last immunization,the mice were euthanized to obtain blood for testing TSHR antibody (TRAb),total T4,and thyroid tissue for histological examination.The controls were injected with the same dose of pcDNA3.1 in the same way.The adenovirus group:52 female BALB/c mice were divided into 10-week model group (n =8),14-week model group (n =10) and 18-week model group (n =8),and the respective controls (n =8,n =10,n =8) were set up.All model groups were injected intramuscularly with Ad-TSHR289,three times at three weekly intervals.Then the mice were euthanized at 4,8 and 12 weeks to test TRAb,total T4 level and to observe the change of thyroid histology.The controls were treated with the same dose of Ad-lacz in the same way.Another 8 mice were scheduled to test the dynamic variation of TRAb before and after the 3 times immunization.Results In the plasmid model group,only two of 12 mice developed weak antibody responses against TSHR,and no elevated total T4 level and no hyperplasia changes of thyroid were observed.In the 10-week model group,all mice had high level TRAb [(807.65 ± 136.33)U/L,Six-eighths mice had hyperthyroidism exhibited hyperplasia changes.In the 14-week model group,the TRAb level [(650.12 ± 192.88) U/L]and the incidence of hyperthyroidism (3/10) were lower than those in 10-week group.Histologically,the degree of thyroid hyperplasia lightened to a small extent,but its positive rate did not decline.In the 18-week model group,only 2 of 8 mice displayed slightly elevated TRAb level,and no mice showed increased total T4 level.Additionally,thyroid tissues of 2 mice were mildly abnormal.Compared with the model groups at different time,the change of antibody levels of the mice for TRAb dynamic observation exhibited the similar trend.Conclusions Being good at repeatability and high incidence of hyperthyroidism,the animal model of Graves disease induced by Ad-TSHR289 is still an ideal research tool presently.The duration of model ean be maintained 18 weeks,and 10 weeks is the best period to snstain characteristic of Graves disease.

Objective To investigate the effect of mice gender on the TSH receptor antibody(TRAb)titers, the levels of TT4,and the degree of thyroid hyperplasia by establishing an animal model of Graves′ disease in male and female BALB/c mice. Methods Male and female BALB/c mice were immunized with recombinant adenovirus expressing TSHRA subunit(Ad-TSHR289)to induce Graves′ disease. Animals were injected 3 times at intervals of 3 weeks. All mice were sacrificed 4 weeks after the last injection to obtain blood for measurement of TSHR antibody titers and TT4evels, and thyroid glands for histological examination. Results TRAb positive rates were 100% both in female or male mice. No significant difference was observed in titers of TRAb between them. The incidence of hyperthyroidism in female mice was higher than that in male mice, being 75.0% and 41.7% respectively. There was statistical difference in levels of TT4between females and males(P＜0.01). Mice with high TT4exihibited marked thyroid hyperplasia. Conclusion Despite TSHR antibodies were similar between female and male mice, the incidence and degree of hyperthyroidism showed sex bias in Graves′s animal model. The results indicated that it was easier to induce model in females than in males by immunizing BALB/c mice with Ad-TSHR289.