| Objective1. Anti-Mi-2antibodies and anti-SRP antibodies are classical myositis specific antibodies(MSAs). The clinical phenotypes and the positive rate of these two kinds of MSAs in Chinese idiopathic inflammatory myopathies patients are unknown. In this research, we analyzed the prevalence of anti-Mi-2/SRP antibodies and their association with clinical and muscle pathological characteristics in a large dermatomyositis(DM) and polymyositis(PM) cohort, and compared our results with the published studies, for giving meaningful evidence of anti-Mi-2/SRP antibodies associated myositis subtypes for the clinical work.2. It is a common and important method for the researchers to study the pathogenesis of idiopathic inflammatory myopathies by using animal models of autoimmune myositis. Recently, new kinds of autoimmune myositis animal models containing advanced mature technology were established in America and Japan, but in our country, studies of constructing new kinds of animal models were very rare. The aim of this research was to prepare recombinant human myosin-binding protein C, fast type (MYBPC2) by optimizing prokaryotic expression and affinity chromatograph, and to establish a new mouse model of experimental autoimmune myositis by immunizing mice with MYBPC2. We wished to provide a new kind of autoimmune myositis animal model for studying the pathogenesis of myositis.Methods1. Serum samples from DM and PM patients were detected for anti-Mi-2and anti-SRP antibodies by immunoblotting tests. Clinical and muscle pathological characteristics were collected by review of medical records and follow-up study was also completed. Through systemic literature review, the characteristics of anti-Mi-2/SRP antibodies associated myositis were analyzed.2. According to the mRNA sequence of MYBPC2, a pair of primers was designed. The nucleotide sequence corresponding to284th and579th amino acid residue of the full length protein was sub-cloned into pMALc-5e plasmid, then the recombinant plasmid was transformed into Escherichia coli. MYBPC2protein was expressed and purified by affinity chromatography. The validity of MYBPC2was confirmed by Western blot with anti-MYBPC2antibodies. Animals were immunized with purified protein to obtain antiserum. The immunogenicity of the protein was confirmed by using the antiserum.The purified MYBPC2was emulsified with complete Freund’s adjuvant, then C57BL/6mice were immunized by subcutaneous multi-point injection (0days,7days), and given intraperitoneal injection of pertussis toxin2ug simultaneously.(1) To explore the pathological changes of mice with different immunizing dose at the preconceived time.(2) To immunize mice with600ug each time, muscle endurance was tested on the21th day. Expression of MHC class-I and the surface biomarkers of the inflammatory cells in muscle tissues were observed.Results1. Serum samples from5DM patients in125PM and DM patients (4%) were positive for anti-Mi-2antibodies, and the positive rate in DM was5.6%. The rates of Gottron’s papules/sign, heliotrope rash, facial erythema, limbs muscle involvement were higher in anti-Mi-2positive group than in negative group, but without statistical significance. Combined with the literature analysis, patients with anti-Mi-2antibodies had low frequency of Interstitial lung diseases (ILD) and cardiac damage, and cancers were rarely found. Patients with anti-Mi-2antibodies showed a good response to immunosuppressive treatment. Muscle pathology findings were mainly peri-vascular inflammatory cells infiltration with T lymphocytes as the dominating infiltrating cells, and up-regulated expression of MHC-I in the surface of muscle fibers.2. Serum samples from9patients with PM in148PM and DM patients (6.08%) were positive for anti-SRP antibodies, while the positive rate in PM patients was21.43%. Compared with antibody negative patients, high incidence rate of dysphagia (P=0.005) and low incidence rate of C reactive protein elevation (P=0.04) in the positive group were the main findings. Compared with the anti-Jo-1antibodies positive patients, significant differences between the two groups were focused on fever, dysphagia, ILD, the level of erythrocyte sedimentation rate and C-reactive protein (P<0.05). Combined with the literature analysis, patients with anti-SRP antibodies had low incidence of serious cardiac damage and no obvious correlation with ILD. Muscle pathological features were scattered muscle fiber degeneration, atrophy and necrosis, positive expression of MHC class-I molecule in muscle fibers with scattered or focal infiltration of T lymphocytes, but with no infiltration of B lymphocytes. Patients responded well to immunosuppressive therapy. The mean follow-up period was20.6months, while complete remission rate was37.5%and partial remission rate was50%.3. The recombinant plasmid inserted MYBPC2gene was constructed. Each liter of culture medium could produce30mg purified fusion protein. This recombinant protein could combine with its antibodies and antiserum specifically.4.(1) With the increase of immunizing dose, muscle damage and inflammation tend to be more serious. On the21th and28th day, muscle lesions were the most significant. Muscle fiber degeneration and necrosis and inflammatory cells infiltration could be seen in the experimental group.(2) Compared with the control group, muscle endurance of mice in the experimental group decreased significantly (P=0.017). MHC class-I positive on the muscle fiber surface of the experimental group, scattered infiltration of CD4+, CD8+T lymphocytes and CD68+macrophages between muscle fibers and around the vascular areas, CD20+B lymphocytes mainly distributed in the area around the blood vessels, nevertheless rarely seen between muscle fibers. Conclusion1. The positive rate of anti-Mi-2antibodies in the Chinese PM/DM patients of our centre was4%, and the rate in DM was5.6%. Anti-Mi-2antibodies were relatively specific for the diagnosis of DM. They had a strong association with skin rash and muscle lesions, but no obvious association with cancer. The muscular pathology feature was not so specific. Patients with this antibodies responded well to immunosuppressive treatment with prognoses rather different among individuals.2. The positive rate of anti-SRP antibodies in the Chinese PM/DM patients of our centre was6.08%, and the rate in PM was21.43%. Anti-SRP antibodies were relative specific antibodies of PM. They had a remarkable corelation with dysphagia, but low frequency of serious cardiac damage. The infiltration of T lymphocytes existed in muscle tissues of the myositis subtype.3. The MYBPC2fusion protein could be efficiently synthesized and purified by the optimizing methods, that showed favorable antigen specificity and immunogenicity.4. Experimental autoimmune myositis models of mice were successfully induced by immunization with MYBPC2in China for the first time, and showed similar clinical and pathological features of human polymyositis. This new model can be used for studying the pathogenesis of autoimmune myositis. |
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