The Study Of Nasal Immunological Tolerance On Experimental Autoimmune Myasthenia Gravis In Young Mice

PARTⅠINDUCE A MODEL OF EXPERIMENTAL AUTOIMMUNE MYASTHENIA GRAVIS ON YOUNG MICEObjective: To induce an animal model of Experimental autoimmune myasthenia gravis (EAMG) on young C57BL/6 mice with 1.0mg/Kg of nicotinic acetylcholine receptor monoclonal antibody-mAb35 through intraperitoneal injection, identify the model whether does succeed,and to provide an animal model for further study of the pathogenesis and immunotherapy of human MG.Methods: The young C57BL/6 mice in the experimental groups were divided into two subgroups-Model group(M) and Control group(C), injected intraperitonealy (i.p.) with 0.2 ml of Ringer's buffer solution containing 0.5mg/kg of capital monoclonal antibody mAb35 of acetylcholine receptor . The control group (C) were i.p. injected with 0.2 ml of Ringer's buffer solution with no mAb35. In order to decide whether the model was a success, the clinical symptoms of the mice were observed, evaluation experiments on pharmacology were done with neostigmine methlsulfate, repetitive nerve stimulation (RNS) was carried out, the serum level of acetylcholine receptor antibody (AChR–Ab) was detected by ELISA.Results: All mice of the experiment groups had myasthenia symptoms: loss of weight, decrease of action and depress of muscle power. The action and ingestion of control group were normal and myasthenia symptoms were not observed.All mice were induced myasthenia gravis symptoms (myasthenia and fatigability), and the changes in electrophysiology (decrement of RNS), characteristics of pharmacology (prostigmin test positive), serum level of AChR–Ab (increased) and ultrastructure change were in accordance with those of MG patients.Conclution: B6 mice is a susceptible breed for inducing an animal model of EAMG. The model of EAMG can be successfully induced on young B6 mice with mAb35, with 1.0mg/kg as It's effective dose. The method is convenient and reliable. A fine animal model is, hence, produced for experimental study of myasthenia gravis in children. PARTⅡTHE STUDY OF NASAL IMMUNOLOGICAL TOLERANCE ON EXPERIMENTAL AUTOIMMUNE MYASTHENIA GRAVIS IN YOUNG MICEObjective: To establish a new therapy for myasthenia gravis in childhood and provide a reliable theory on it, the experimental autoimmune myasthenia gravis (EAMG) young C57BL/6 (B6) mice were treated with a specific toleragen-dual analogue (Lys262–Ala207) nasally in different time points,The effect of Lys262–Ala207 on the invasion process of mice model and the clinical symptoms were observed, It's clinical effects were assessed, and the underlying mechanisms and feasibility of nasal tolerance were explored.Methods: The model of experimental autoimmune myasthenia gravis was induced by mAb35 on C57BL/6 (B6) mice. The experimental animals were divided into four groups: Tolerance group (A and B),and control group (CA and CB). Dual analogues (Lys262–Ala207) or control PBS were given nasally before(GroupA or CA) or on the day(Group B and CB) of immunization with mAb35 for 10 consecutive days. The body weight and clinical scores were evaluated. The serum levels of AChR–Ab and the main cytokines (IL–4,IFN–γ,IL–10) were detected with ELISA.The number of mononuclear cells expressing CD4+ and CD4~+CD25~+ regulation T cells from spleen were enumerated with flow cytometry. Results: Comparaed with the corresponding control groups.the body weight increase, severity reduced, D5 positive rate of CAMP was decremental in C57BL/6 mice in group A or B developed EAMG. The levels of anti-AChR IgG(0.162±0.068)and(0.226±0.069)were also decreased. The number of cells secreting IL–4,IFN–γor IL–10 was decreased. the number of mononuclear cells expressing CD4+ and CD4~+CD25~+ regulation T cells from spleen were higher in group A(4.516±0.598)and(3.671±0.300)than in those corresponding control groups.Conclution:1. Nasal administration with a dual analogue Lys262–Ala207 could ameliorated the muscular weakness in EAMG young mice, with the decreased levels of AChRAb,IL–4,IFN–γand IL–10 in serum,and the increased the number of mononuclear cells expressing CD4+ and CD4~+CD25~+ regulation T cells from spleen.2. The therapy effect is possibly correlated with the function of immune system.3. Nasal administration with a dual analogue Lys262–Ala207 at two different time points has different effects:there is more effective results before the day of immunization than on the day of immunization.4. Our experiment verified that nasal administration with a dual analogue Lys262–Ala207 is an effect therapy for clinical EAMG. this might provide potential therapy for human MG and other autoimmune diseases by mucosal administration of dual analogue. It is possible to utilize the immunological nasal tolerance to treat MG in childhood and need further investigation for improment .