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Immunopathological Diagnosis and Molecular Immunopathogenesis of Idiopathic Inflammatory Myopathies

Author: DaiTingJun
Tutor: YanChuanZhu
School: Shandong University
Course: Neurology
Keywords: Major histocompatibility antigen Ⅰ CD8/MHC- Ⅰ molecule complex Polymyositis Dermatomyositis Inclusion body myositis Muscular dystrophy Steroids reaction necrotizing myopathy Immune pathological diagnosis B-cell activating factor Steroid hormones
CLC: R746.9
Type: PhD thesis
Year: 2010
Downloads: 207
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Background idiopathic inflammatory myopathy (idiopathic inflammatory myopathies, IIMs) are a heterogeneous group of skeletal muscle autoimmune diseases, clinical progressive muscle weakness associated with skeletal muscle inflammatory cell infiltration characteristics. Depending on the clinical and pathological morphological characteristics, clinical will be divided into three subtypes of polymyositis (polymyositis, PM), dermatomyositis (dermatomyositis, DM) and inclusion body myositis (inclusion-body myositis IBM). PM and DM has a good effect of corticosteroids and other immunosuppressive agents, therefore both timely and accurate diagnosis is very important for the treatment of patients even prognosis. Weeks atrophy on the beam of the the characteristic rash DM or pathological characteristics can contribute to the early identification and diagnosis of PM has been timely and correct diagnosis is the problem of clinical neurology and rheumatology. Although a variety of different diagnostic criteria have been proposed so far, most doctors and research institutions still using the diagnostic criteria proposed by Bohan and Peter 1975 years, but for the DM and PM. This diagnostic criteria based on clinical manifestations and common pathological basis, including the infiltration of inflammatory cells to occupy an important place in the diagnosis of PM. Drawn limitations or prior to the biopsy has been taking hormones and other immunosuppressive therapy, and other reasons, however, the infiltration of inflammatory cells is not often exist, resulting in missed diagnosis often influence the PM. In addition, in some types of muscular dystrophy, such as facioscapulohumeral muscular dystrophy (facialscapulohumeral muscular dystrophy, FSHD) and dysferlinopathies in often secondary inflammatory cell infiltration, pathology very similar with the PM, as well as easily lead to misdiagnosis. In addition, you can not PM and drawn limitations Erzhi found no rimmed vacuoles (rimmed vacuole, RV) IBM to identify open to pathology. In recent years, with the in-depth studies of the pathogenesis of DM and PM come to realize that there are significant differences both in immunopathogenesis. Studies have shown that immune pathology and molecular pathology based on the diagnostic criteria proposed by Bohan and Peter obvious flaw. In view of this, the 2003 Dalakas and Hohlfeld summarize the IIMs immunology research progress in recent years, and the first time that the immune pathological features differences immunopathological new diagnostic standards for PM and DM. The diagnostic criteria for projecting the importance of the detection of the major histocompatibility antigen class I molecules (MHC-I), and was first introduced the term CD8/MHC-I molecular complexes (2 complex, CD8/MHC-I), especially CD8 T lymphocytes invade MHC-1 molecules expression of positive non-necrotic muscle fibers, and that such a characteristic damage skeletal muscle inflammation of primary inflammation PM characteristic immune pathological features. At home and abroad have multiple diagnostic value of detection in the IIMs for the expression of MHC-1 molecules and / or MHC-II molecules so far, the diagnostic value of the expression of MHC-I molecules in the IIMs still exists considerable The big controversy, and do not have a clear conclusion. Whether negative can exclude the IIMs the diagnosis or MHC-I expression was positive to establish the diagnosis? Also, as far as we know, at home and abroad no for CD8/MHC-I complex in the PM diagnosis based solely on the expression of MHC-I The value of the study. Often with previous research in IIMs as a whole different study, this study focuses on the MHC-I molecules and CD8/MHC-I of complex diagnostic value in the PM. We first investigate the MHC-I molecules PM, DM, IBM, following different expression characteristics of muscular dystrophy, inflammatory cell infiltration, and these types of steroids reaction necrotizing myopathy myopathy, and then further assess CD8/MHC-I molecular complex diagnostic value in the diagnosis of PM. Methods This study was a retrospective case - control study. Cases of sources for the period from September 2004 to December 2008 in Neuromuscular Disease Institute of Shandong University Qilu Hospital muscle biopsy patients. We selected 65 patients were divided into five groups. The first group of 20 patients diagnosed with PM patients meet the diagnostic criteria of Bohan and Peter, all cases muscle biopsy specimens were seen significant inflammatory cell infiltration. The second group of 20 cases of patients with DM Bohan and Peter diagnostic criteria, which are visible characteristic rash and / or beam Week atrophy pathological features. The third group consists of the five cases of patients with IBM, Griggs diagnostic criteria muscle pathology seen significant inflammatory cell infiltration and characteristic rimmed vacuoles pathological features. The fourth group of 10 cases of muscle biopsy specimens associated with muscular dystrophy, inflammatory cell infiltration, including six cases of facioscapulohumeral muscular dystrophy, and four cases dysferlinopathies. The fifth group of 10 cases of clinically suspected diagnosis of PM, but no inflammatory cell infiltration and serum autoantibody-negative cases on the pathological. According to the good response to corticosteroids, the exclude other common necrotizing myopathy etiology, including some types of muscular dystrophy, the application of muscle toxicity drugs, contact-related toxins and rhabdomyolysis diagnosis of steroid response necrotizing myopathy (steroid-responsive necrotizing myopathy, SRNM). Another 10 patients with myasthenia gravis symptoms but normal muscle biopsy ultimately confirmed for other diseases rather than cases of neuromuscular disease served as normal controls. Immunoenzyme staining method to detect the expression of MHC-1 molecules in different types of myopathy, application of double immunofluorescence staining method to detect CD8/MHC-I molecular complex expression in the IIMs. Results 1.MHC-I molecule expression in each group of patients in the 10 cases of normal muscle biopsy specimens only vascular endothelial cells express MHC-I molecules, and muscle fibers expressing MHC-I molecules. (1) PM.80% (16/20) visible in the presence of MHC-I-positive non-necrotic muscle fibers, the positive fibers were scattered in the distribution, or in small groups in the PM muscle biopsy specimens showed focal expression patterns. The number of non-necrotic muscle fibers of the MHC-I-positive between 10-60%, the degree of staining as weakly positive (1) or positive (2). (2) DM: 20 cases of DM muscle biopsy specimens in the presence of visible expression of MHC-I-positive non-necrotic muscle fibers, the positive rate was 100%. MHC-I in DM muscle biopsy specimens showed diffuse expression of almost all muscle fibers showed a strongly positive sarcolemma at the MHC-I expression (3). Part of the muscle fibers is still visible cytoplasmic expression of MHC-I and the sarcolemma only at the MHC-I expression in muscle fibers and white distribution, showing a mosaic-like expression pattern. Another in the the DM cases of a beam Week atrophy pathological features, is still visible beam Week to atrophic fibers degree of positive expression of MHC-I enhanced. (3) IBM: 5 cases of IBM muscle biopsy specimens in the presence of visible expression of MHC-I-positive non-necrotic muscle fibers, the positive rate was 100%. MHC-I was in the three cases IBM patients diffuse positive expression, and the other two cases similar PM, showed focal expression. The number of the positive fibers in two different expression pattern of the large differences between cases, at 10% to 100%. (4) muscular dystrophy: accompanied by inflammatory cell infiltration in 10 patients with muscular dystrophy cases, the visible expression of MHC-I-positive non-necrotic muscle fibers in the muscle biopsy specimens of 6 patients (60%), including four cases of facioscapulohumeral muscular dystrophy and two cases dysferlinopathies. MHC-I-positive non-necrotic muscle fibers in muscular dystrophy was scattered, usually less than 5% the positive degree weakly positive (1). (5) steroids a reactive necrotizing myopathy: five cases of muscle biopsy specimens visible presence of positive expression of MHC-I non-necrotic muscle fibers, the positive rate was 50% in the 10 cases of steroid response necrotizing myopathy. And PM similar, MHC-I in the group of patients also showed focal expression, expressed as scattered or small group of distribution of the MHC-I-positive muscle fibers, the number of positive fibers between 5-50% positive degree as weakly positive or positive. Expression of 2. CD8/MHC-I molecule complex in each group of cases we apply double immunofluorescence labeled CD8 and MHC-I molecules to detect the CD8/MHC-I molecule complex exists. Visible in only 4 patients (20%) of 20 patients diagnosed with PM Case typical CD8/MHC-I molecular complexes. IBM patients in five cases, three cases (60%) can be seen Typical CD8/MHC-I molecular complexes. DM, while in the other group of patients were not seen in the presence of the CD8/MHC-I molecule complex accompanied by inflammatory cell infiltration of the muscular dystrophy and steroid reaction necrotizing myopathy. Conclusion 1.MHC-I molecules in IIMs (PM) in the expression showing heterogeneity exclude having a specificity to merely rely on the detection of MHC-I molecules is not possible to reliably PM and accompanied by inflammatory cells infiltration muscular dystrophy or reactive steroids the necrotizing myopathy identify off. In contrast, more consistent with its expression in DM detection of MHC-I molecules diagnosis of DM may have a certain value. CD8/MHC-I molecule complex with high specificity in PM / IBM, but due to its low sensitivity in the PM detection method more complex, therefore not an ideal PM diagnostic markers. Background idiopathic inflammatory myopathy (idiopathic inflammatory myopathies, IIMs) are a heterogeneous group of skeletal muscle autoimmune diseases, clinical progressive muscle weakness associated with skeletal muscle inflammatory cell infiltration characteristics. Depending on the clinical and pathological morphological characteristics, clinical will be divided into three major subtypes of dermatomyositis (dermatomyositis, DM), polymyositis (polymyositis, PM) and inclusion body myositis (inclusion-body myositis , IBM). DM is generally considered to be a humoral immune-mediated adaptive immunity (adaptive immune) based autoimmune diseases, inflammatory cell infiltration mainly to the large number of B lymphocytes in the skeletal muscle and CD4 T cells, antibodies and complement mediated mediated capillary damage and pathological bundle weeks of muscle fibers atrophy as the main feature. PM and IBM by autoreactive CD8 T cell-mediated cellular immunity mainly CD8 cytotoxic T cells invade the expression of MHC-I-positive non-necrotic muscle fibers. CD8 T cell-mediated cellular immune response based PM and IBM, the B lymphocyte infiltration is relatively rare, but recent study found that the gene chip (microarray) technology-based PM and IBM in the muscle tissue of patients with a large number of immunoglobulin (immunoglobulin, Ig) gene transcription, and the application of the method of immunohistochemistry PM and IBM muscle biopsy specimens of patients found the presence of a large number of plasma cells. These results suggest that the infiltration and activation of B lymphocytes may not only in DM PM and IBM pathogenesis occupies an important position. B lymphocytes to further clarify the role in the pathogenesis of IIMs, Bradshaw by the BCR lgH chain gene transcription analysis found that the three IIMs 1g experienced affinity maturation, confirming local antigen-driven B-lymphocyte activation , so that the local antigen-driven humoral immune response may be a co-existence of the three IIMs immune mechanisms. The higher incidence of autoantibodies often clinical patients with DM and PM and IBM patients with monoclonal gammopathy disease (monoclonal gammopathy) suggest that abnormal activation of B cells produce immunoglobulin may IIMs pathogenesis occupy a certain position. In addition, the clinical application of anti-CD20 chimeric monoclonal antibody rituximab rituximab (rituximab) the Clear B cell treatment of DM, PM and IBM also further confirmed the role of B lymphocytes in the pathogenesis of IIMs. B cell activating factor (B cell activating factor, BAFF) is a newly discovered in recent years, the tumor necrosis factor superfamily member, capable of specifically activated B lymphocytes, and to promote B-lymphocyte maturation and survival, induction of B lymphocyte proliferation, and differentiation into plasma mother cell, secrete autoantibodies, mediated antigen isotype switching (isotype switch), as well as a costimulatory signal to activated T lymphocytes, the regulation of T lymphocyte responses. Abnormal expression of BAFF can stimulate the proliferation and activation of peripheral B lymphocytes, to extend the survival time of autoreactive B cells, inhibition of apoptosis and prolong the survival time of the plasma cells in the tissue, disrupting the body's state of immune tolerance, causing autoimmune diseases. Much evidence that BAFF plays an important role in the pathogenesis of autoimmune diseases. The study found that overexpression the BAFF transgenic mouse model showed similar rheumatoid arthritis (rheumatoid arthritis, RA), systemic lupus erythematosus (systemic lupus erythematosus, SLE) and primary Sjogren's syndrome (primary Sjogren's syndrome, pSS) symptoms. In SLE, RA, pSS and idiopathic thrombocytopenic purpura (idiopathic thrombocytopenic purpera, ITP) serum were also found abnormally elevated levels of BAFF, and found pSS serum BAFF level with serum immunoglobulins and their own antibody titers. However, so far, at home and abroad still rarely BAFF molecule role in the the IIMs pathogenesis of research reports. Clinical treatment against IIMs mainly applied for the long course of the large doses (prednisone), wherein the majority of DM and PM patients showed good efficacy for IBM invalid, the current differences for this efficacy is no good explanation. Recent studies have found that high-dose steroids (dexamethasone) could down-regulate the expression of BAFF in the peripheral blood of patients with autoimmune disease (such as RA, ITP), however, is unclear steroid hormones for BAFF in vivo expression of the IIMs patients with or without impact. In this study, one hand is that clear BAFF role in three different subtypes IIMs, there is no difference, and to explore whether the expression in the peripheral blood and muscle tissue. The BAFF expression changes in peripheral blood the other hand, by comparing before and after treatment IIMs patients to to clear large doses of hormones (prednisone) Can cut IIMs in peripheral blood of patients with the expression of BAFF new intervention targets for IIMs treatment. Materials and Methods. Cases and controls in this study were collected prospectively IIMs patients with 21 cases, including 16 women and 5 men, aged 12 years old to 67 years old, with a median age of 46 years old. Cases of sources for all selected the IIMs cases, including DM, PM and IBM are consistent with the clinical diagnosis of patients attending clinics and wards of Neurology, Qilu Hospital of Shandong University, during October 2008 to May 2009, and confirmed by muscle biopsy in the cases of active disease, including 10 cases of DM 9 patients with PM, as well as two cases of IBM. All selected cases in the beginning of the enrollment, clinical and laboratory inspection be excluded overlap syndrome, collagen vascular disease, malignancy, and concomitant have HIV, lymphoma, immune dysfunction, hepatitis C and other diseases, and into the group before the application of any immune agents were not applied before treatment or immunosuppressive therapy but have been disabled for at least 6 months. Select another 10 healthy adult volunteers served as controls, including 6 females, 4 males between the age of 21 years old to 65 years old, with a median age of 45 years old. 2 treatment options in addition to two cases of IBM patients, all selected for DM and PM patients enrolled in the beginning herein are empirical prednisone monotherapy and follow-up of the appropriate treatment effect. Reference to the literature and combined with our own experience to develop the following specific treatment programs: 1mg/kg/d prednisone starting dose reduction begins when patients spend four to six weeks after the muscle strength to improve and CK levels, dose per Zhou 5mg decreasing until the daily dose is 25-30mg, then every other day regimen. In order to assess the patient the prednisone large doses (1mg/kg/d), the response to treatment in patients treated for 4-6 weeks after the initial reduction of efficacy in patients by assessing changes in muscle strength and CK levels assessment. Separation of plasma and peripheral blood mononuclear cells were 4-6 weeks after the beginning of the enrollment, and hormone therapy various specimens the EDTA anticoagulant 15ml. At room temperature, the plasma was separated by centrifugation and peripheral blood mononuclear cells, and then separated by density gradient centrifugation method. Plasma BAFF levels detected by RD the company people ELISA kit detected plasma BAFF levels, the lower limit of detection 62.5pg/ml. BAFF mRNA expression of peripheral blood mononuclear cells using TRIzol extraction of peripheral blood mononuclear cell total RNA, the application of first-strand cDNA synthesis kit (Quantscript RT Kit) mRNA reverse transcribed into cDNA. SYBR Green real-time quantitative PCR, with reference to the ratio of BAFF and β-actin cycle threshold (threshold cycle, Ct) and by the formula 2 - △ △ Ct indicates the relative levels of mRNA. Muscle biopsy and immune staining applications open muscle biopsy drawn biceps routine HE BAFF molecule immunohistochemical staining, specimens were quick frozen processing, while application of CD3, CD4, CD8, CD20 and CD68, etc. Monoclonal antibody detection of inflammatory cells in the muscle tissue classification and composition. Results 1. Before treatment plasma BAFF levels increased in the DM and PM, but no increase in IBM patients the ELISA experimental results show that, compared with the normal control (767 ± 171 pg / ml), DM and PM plasma BAFF levels (3262 ± 896 pg / ml and 1713 ± 463 pg / ml, P <0.001), DM patients with elevated plasma levels than PM for the difference with statistical significance (P lt; 0.001) . The two cases IBM plasma BAFF levels in patients with no significant increase, within the normal range. Treatment before the DM and PM patients in PBMCs BAFF mRNA levels were up-regulated quantitative real-time PCR results showed that the treatment of patients with DM before BAFF mRNA relative expression level between 1.87 times to 4.89 times the normal controls, and treatment former PM patients 1.02 times 2.45-fold between. Mean relative expression levels of BAFF mRNA in the treatment of DM and PM were 3.26 times and 1.82 times the normal controls. The expression levels of BAFF in DM than PM (P <0.001). The treatment of patients with DM and PM plasma BAFF levels with PBMCs BAFF mRNA expression Pearson correlation analysis, we found that treatment of the former DM and PM plasma BAFF levels in peripheral blood PBMCs BAFF mRNA expression significantly correlated (r = 0.74 and 0.91, P lt; 0.001). 4 high-dose prednisone can inhibit the expression of BAFF in DM and PM after 4-6 weeks of high-dose prednisone in patients with DM and PM plasma BAFF levels than before treatment decreased (932 422 pg / ml vs 3262 ± 896 pg / ml, 658 ± 220 pg / ml vs 1713 ± 463 pg / ml, P lt; 0.001). Corresponding DM and PM patients after treatment, peripheral blood mononuclear cells BAFF mRNA expression also significantly down-regulated, respectively, 0.61 times and 0.57 times the pre-treatment (P lt; 0.001). 5.BAFF horizontal or content change has some correlation with prednisone efficacy after a sufficient amount of prednisone treatment, the average of all DM and PM plasma BAFF water decreased in 15 cases (78.9%) patients responded well to hormone showed partial remission or complete remission, while four patients (two cases each) PM and DM insensitive response to steroid therapy. 6.DM and PM muscle tissue in patients with BAFF negative expression using immunohistochemistry method to detect the expression of BAFF in patients with DM and PM muscle biopsy specimens. DM and PM muscle biopsy specimens in patients with the muscle fibers themselves, showed no expression of BAFF in inflammatory cell infiltration in the vascular endothelial cells, and muscle tissue. Conclusion upregulation 1.BAFF activities of DM and PM peripheral blood, and for the high-dose prednisone inhibited, suggesting that DM and PM hormone response mechanism may be related to hormones can inhibit BAFF in both peripheral blood upregulation. 2.IIMs peripheral blood of patients with skeletal muscle target organ BAFF expression differences, blocking the expression of BAFF in peripheral blood may be a new DM and PM therapeutic effect targets.

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