INTRODUÇÃO: A Miastenia gravis (MG) é uma desordem imunológica com antígenos alvos conhecidos, com produção de anticorpos contra o receptor nicotínico de acetilcolina, AAChR e Musk, na junção neuromuscular, dificultando a transmissão do impulso nervoso e provocando fadiga e fraqueza flutuantes na musculatura ocular, facial, dos membros e respiratória. A terapêutica sintomática com inibidores de acetilcolinesterase e a etiopatogênica como a timectomia, corticosteróides, agentes citostásticos e imunoglobulinas são utilizadas e indicadas em acordo com a incapacidade e gravidade clínicas. A qualidade de vida (QV) é uma ferramenta utilizada para quantificar a eficácia e a resposta às terapêuticas adotadas, avaliar a efetividade e custos econômicos de novas estratégias terapêuticas, contribuir para planejar e aplicar os recursos para a saúde na comunidade. OBJETIVO: Avaliar a influência da terapêutica na QV e evolução clínica dos pacientes com MG. CASUÍSTICA: Foram avaliados 51 pacientes com MG, 38 submetidos a tratamento conservador e 13 timectomizados. MÉTODOS: Os instrumentos genéricos quantitativos de QV como WHOQOL, qualidade de vida relacionada à saúde (QVRS) SF-36, e depressão (BDI) foram utilizados. ESTATÍSTICA: Empregou-se análise univariada com os testes de Wilcoxon, U-Mann-Whitney, Fisher e razão de chance para avaliar a evolução clínica, a QV e QVRS e depressão; análise multivariada para a caracterização dos dois grupos de terapêutica considerando a interação de múltiplos fatores. RESULTADOS: O grupo submetido à terapêutica conservadora foi constituído por 16 pacientes do sexo masculino e 22 do feminino, com média de idade de 33,71±2,76 anos; o grupo timectomizado foi constituído por 6 homens e 7 mulheres, média de idade de 32,23±4,16 anos. Os pacientes do grupo conservador apresentaram melhora clínica estatisticamente significante (p <0,05) dos aspectos clínicos, da QV, da QVRS e depressão. Os timectomizados apresentaram melhora estatisticamente significante dos aspectos físicos e psicológicos da QV e QVRS e depressão, e melhora clínica evidente e não significante (p = 0,06). A análise multivariável mostrou r=0,65 para comparação das áreas; Wilks? Lambda para analisar as distâncias, X2 = 22,67; gl = 7; p = 0,05; as medidas centrais mostraram média = -0,42 dp = 1,0 para o grupo conservador; e média = 1,22, dp = 1,0 para o grupo timectomizado. CONCLUSÕES: Nesta amostra e no período avaliado constatamos que: 1) Os pacientes submetidos ao tratamento conservador apresentaram diferenças significantes com melhora clínica acentuada, de QV, de QVRS e depressão; 2) os timectomizados apresentaram melhora clínica, da QV e QVRS em seus aspectos físicos e psicológicos, e efetiva e significante melhora dos índices de depressão; 3) a análise multivariada revelou que a timectomia produziu efeito benéfico e significativo na recuperação da saúde e bem estar nos pacientes. / INTRODUCTION: Myasthenia gravis (MG) is an immune mediated disease with production of antibodies against post-synaptic acetylcholine receptor of neuromuscular junctions (AAChR,Musk) and orders in nervous impulse transmission. The disease´s clinical characteristics include fatigability and fluctuating weakness of voluntary muscles. Acetylcholinesterase inhibitors, thymectomy, corticosteroids, cytostatic agents and immunoglobulin are widely used and are indicated according to the patient´s disability and severity. Clinical manifestations, Quality of life (Qol), health-related (HRQol) analysis are used to evaluate response to therapy. Nowadays, Qol index is an important tool to evaluate the medical outcome, treatment efficacy, cost effectiveness and net benefit of new therapeutic strategies to determine whether their cost can be justified in the planning and application of health policies. OBJECTIVE: To evaluate the influence of conservative treatment and thymectomy on Qol and clinical response of myasthenic patients. SUBJECTS: Fifty-one myasthenic patients were chosen; 38 were submitted to conservative therapy and 13 to thymectomy. METHODS: Quantitative Qol tools such as WHOQOL, SF-36 and BDI were employed to evaluate Qol, HRQol and depression. STATISTICS: Univariate analysis by means of the Wilcoxon, U-Mann Whitney and Fisher tests, Chi-Square, odd ratio were used to follow the patient?s clinical status, evolution of Qol, HRQol and depression. Discriminant analysis was used to analyze the interation of multiple factors in the characterization of conservative and thymectomized groups. RESULTS: The conservative group of patients was constituted of 16 males and 22 females average age 33.71±2.76 years; the thymectomized group was composed of 6 males, 7 females, average age 32.23±4.16 years. Patients submitted to conservative therapy improved significantly in clinical progress, Qol, HRQol and depression. The follow-up of thymectomized patients showed a strong trend for clinical progress and significant improvement in physical and psychological Qol domains as well as in depression index. Discriminant analysis showed r = 0.65, p <0.05; Wilk?s Lambda X2 = 22.67, gl = 7; mean = -0.42, SD = 1.0 for conservative group; and mean = 1.22, SD = 1.0 for timectomized group. CONCLUSIONS: A prospective evaluation of a myasthenic patients sample revealed: 1) conservative treatment was found to have a strong and significant impact on clinical progress, Qol, HRQol and depression; 2) Thymectomy partly influenced Qol, specially physical and psychological aspects. There was also improvement in depression and clinical progress; 3) the evaluation of multiple parameters pointed to a strong and positive influence of thymectomy in the recovery of the patients.

INTRODUÇÃO: A Miastenia gravis (MG) é uma desordem imunológica com antígenos alvos conhecidos, com produção de anticorpos contra o receptor nicotínico de acetilcolina, AAChR e Musk, na junção neuromuscular, dificultando a transmissão do impulso nervoso e provocando fadiga e fraqueza flutuantes na musculatura ocular, facial, dos membros e respiratória. A terapêutica sintomática com inibidores de acetilcolinesterase e a etiopatogênica como a timectomia, corticosteróides, agentes citostásticos e imunoglobulinas são utilizadas e indicadas em acordo com a incapacidade e gravidade clínicas. A qualidade de vida (QV) é uma ferramenta utilizada para quantificar a eficácia e a resposta às terapêuticas adotadas, avaliar a efetividade e custos econômicos de novas estratégias terapêuticas, contribuir para planejar e aplicar os recursos para a saúde na comunidade. OBJETIVO: Avaliar a influência da terapêutica na QV e evolução clínica dos pacientes com MG. CASUÍSTICA: Foram avaliados 51 pacientes com MG, 38 submetidos a tratamento conservador e 13 timectomizados. MÉTODOS: Os instrumentos genéricos quantitativos de QV como WHOQOL, qualidade de vida relacionada à saúde (QVRS) SF-36, e depressão (BDI) foram utilizados. ESTATÍSTICA: Empregou-se análise univariada com os testes de Wilcoxon, U-Mann-Whitney, Fisher e razão de chance para avaliar a evolução clínica, a QV e QVRS e depressão; análise multivariada para a caracterização dos dois grupos de terapêutica considerando a interação de múltiplos fatores. RESULTADOS: O grupo submetido à terapêutica conservadora foi constituído por 16 pacientes do sexo masculino e 22 do feminino, com média de idade de 33,71±2,76 anos; o grupo timectomizado foi constituído por 6 homens e 7 mulheres, média de idade de 32,23±4,16 anos. Os pacientes do grupo conservador apresentaram melhora clínica estatisticamente significante (p <0,05) dos aspectos clínicos, da QV, da QVRS e depressão. Os timectomizados apresentaram melhora estatisticamente significante dos aspectos físicos e psicológicos da QV e QVRS e depressão, e melhora clínica evidente e não significante (p = 0,06). A análise multivariável mostrou r=0,65 para comparação das áreas; Wilks? Lambda para analisar as distâncias, X2 = 22,67; gl = 7; p = 0,05; as medidas centrais mostraram média = -0,42 dp = 1,0 para o grupo conservador; e média = 1,22, dp = 1,0 para o grupo timectomizado. CONCLUSÕES: Nesta amostra e no período avaliado constatamos que: 1) Os pacientes submetidos ao tratamento conservador apresentaram diferenças significantes com melhora clínica acentuada, de QV, de QVRS e depressão; 2) os timectomizados apresentaram melhora clínica, da QV e QVRS em seus aspectos físicos e psicológicos, e efetiva e significante melhora dos índices de depressão; 3) a análise multivariada revelou que a timectomia produziu efeito benéfico e significativo na recuperação da saúde e bem estar nos pacientes. / INTRODUCTION: Myasthenia gravis (MG) is an immune mediated disease with production of antibodies against post-synaptic acetylcholine receptor of neuromuscular junctions (AAChR,Musk) and orders in nervous impulse transmission. The disease´s clinical characteristics include fatigability and fluctuating weakness of voluntary muscles. Acetylcholinesterase inhibitors, thymectomy, corticosteroids, cytostatic agents and immunoglobulin are widely used and are indicated according to the patient´s disability and severity. Clinical manifestations, Quality of life (Qol), health-related (HRQol) analysis are used to evaluate response to therapy. Nowadays, Qol index is an important tool to evaluate the medical outcome, treatment efficacy, cost effectiveness and net benefit of new therapeutic strategies to determine whether their cost can be justified in the planning and application of health policies. OBJECTIVE: To evaluate the influence of conservative treatment and thymectomy on Qol and clinical response of myasthenic patients. SUBJECTS: Fifty-one myasthenic patients were chosen; 38 were submitted to conservative therapy and 13 to thymectomy. METHODS: Quantitative Qol tools such as WHOQOL, SF-36 and BDI were employed to evaluate Qol, HRQol and depression. STATISTICS: Univariate analysis by means of the Wilcoxon, U-Mann Whitney and Fisher tests, Chi-Square, odd ratio were used to follow the patient?s clinical status, evolution of Qol, HRQol and depression. Discriminant analysis was used to analyze the interation of multiple factors in the characterization of conservative and thymectomized groups. RESULTS: The conservative group of patients was constituted of 16 males and 22 females average age 33.71±2.76 years; the thymectomized group was composed of 6 males, 7 females, average age 32.23±4.16 years. Patients submitted to conservative therapy improved significantly in clinical progress, Qol, HRQol and depression. The follow-up of thymectomized patients showed a strong trend for clinical progress and significant improvement in physical and psychological Qol domains as well as in depression index. Discriminant analysis showed r = 0.65, p <0.05; Wilk?s Lambda X2 = 22.67, gl = 7; mean = -0.42, SD = 1.0 for conservative group; and mean = 1.22, SD = 1.0 for timectomized group. CONCLUSIONS: A prospective evaluation of a myasthenic patients sample revealed: 1) conservative treatment was found to have a strong and significant impact on clinical progress, Qol, HRQol and depression; 2) Thymectomy partly influenced Qol, specially physical and psychological aspects. There was also improvement in depression and clinical progress; 3) the evaluation of multiple parameters pointed to a strong and positive influence of thymectomy in the recovery of the patients.

Die Myasthenia Gravis (MG) ist ein Prototyp einer Antikörper-vermittelte Autoimmunerkrankung. Die Autoantikörper richten sich hauptsächlich gegen den Acetylcholinrezeptor (AChR). Welche Bedeutung TH17-Zellen für die Pathogenese der MG haben, konnte bisher noch nie direkt gezeigt werden. Mithilfe des Tiermodells Experimentelle Autoimmune Myasthenia Gravis (EAMG) sollte die Rolle der TH17-Zellen im Rahmen dieser Arbeit analysiert werden. Eine signifikante Anzahl tAChR-spezifischer CD4+ T-Zellen, die IL17 exprimieren, konnte nach der Immunisierung mit torpedo AChR in CFA in Wildtyp-Mäusen (WT) beobachtet werden. Die IL17ko Mäuse entwickelten weniger oder keine EAMG Symptome, obwohl weder die Frequenz tAChR-spezifischer CD4+ T-Zellen, die IL2, IFNgamma oder IL21 sezernierten noch der prozentuale Anteil der FoxP3+ Treg-Zellen einen Unterschied aufwiesen. Im Gegensatz dazu waren die Level pathogener anti-muriner AChR Antikörper statistisch geringer in IL17ko Mäusen, während bei anti-tAChR Antikörpertitern kein Unterschied festzustellen war. Ähnliche Resultate erbrachten TCRbeta/delta ko Mäuse rekonstituiert mit entweder WT oder IL17ko CD4+ T-Zellen. Die Depletion von Treg-Zellen mithilfe von DEREG Mäusen in der frühen Erkrankungsphase zeigte keine signifikanten Unterschiede bezüglich der analysierten Parameter. Zusammenfassend lässt sich hier festhalten, dass die Frequenz und Differenzierung Antigen-spezifischer CD4+ T-Zellen sowie der Antikörpertiter gegen den tAChR nicht durch die IL17-Defizienz im EAMG Modell beeinflusst wird. Auch hat eine frühe Treg-Zell-Depletion keinen Einfluß auf die Erkrankungsstärke. Allerdings scheint das Durchbrechen der B-Zell Toleranz, das zur Produktion von pathogenen Anti-mAChR-spezifischen Antikörpern und somit zu einer Induktion der Erkrankung führt, abhängig von IL17-produzierenden CD4+ T-Zellen zu sein. Der Einsatz von Anti-IL17-Antikörpern könnte insofern auch für die MG eine Therapieoption darstellen. / Myasthenia gravis (MG) is an antibody-mediated autoimmune disease. The autoantibodies are directed against the acetylcholine receptor (AChR). The importance TH17 cells have for MG pathogenesis has never been directly demonstrated. Therefore, the analysis of TH17 cells in the Experimental Autoimmune Myasthenia Gravis (EAMG) animal model was the aim of this work. Here, it is shown that in wildtype mice (WT) significant numbers of IL17-producing tAChR-specific CD4+ T cells could be observed after immunization with torpedo AChR in CFA. IL17ko mice developed less or no EAMG symptoms, although frequencies of tAChR-specific CD4+T cells secreting IL2, IFNgamma or IL21 as well as percentage of FoxP3+ Treg cells were similar. In contrast, pathogenic anti-murine AChR antibody levels were significantly lower in IL17ko mice, while anti-tAChR antibody levels were equal. Similar results were obtained by the reconstitution of TCR beta/delta ko mice with CD4+ T cells of either WT or IL17ko origin. For the depletion of Treg cells using DEREG mice in the initial phase of the disease no significant differences could be detected in terms of the analyzed parameters. In summary, this thesis demonstrates, that frequencies and differentiation of antigen specific CD4+ T cells as well as the level of anti-tAChR specific antibody titers are not affected by IL17-deficiency in the EAMG model. Likewise, an early Treg cell depletion seems to have no impact on disease severity. However, breaking of B cell tolerance resulting in pathogenic anti-murine AChR specific antibodies and subsequent disease induction, seems to be dependent on IL17 producing CD4+ T cells. In this respect, the application of anti-IL17 antibodies could also become a MG therapy option.

Die vorliegende Arbeit dient der Entwicklung und Evaluierung eines neuen operativen Zugangsweges zur Durchführung einer kompletten Thymektomie (Thx). Die erste klinische Umsetzung erfolgte 1994. Der thorakoskopische Zugang für die Thx wurde an der Chirurgischen Klinik der Charité nach den Prinzipien der "Good clinical practice" zur Einführung eines neuen operativen Verfahrens entwickelt. Eine retrospektive Kohortenstudie bestimmte den internen Standard der erreichbaren Ergebnisse einer Thx in konventioneller Operationstechnik bei MG. Die Daten des eigenen Krankengutes an der Charité waren mit den Literaturangaben übereinstimmend und somit repräsentativ für die Zielstellung eines neuen Operationsverfahrens. Die komplexe Evaluation des neuen operativen Zuganges der thorakoskopischen Thx (tThx) umfaßte neben dem Nachweis der Durchführbarkeit der tThx eine experimentelle anatomische Demonstration der adäquaten Radikalität. Es konnte gezeigt werden, daß eine unilaterale linksseitige thorakoskopische Operationstechnik mit 3 Trokaren im Vergleich zu einer rechtsseitigen 3-Trokar-Technik besser eine komplette Thx realisieren kann. Es folgte die Ausarbeitung einer detaillierten operativen Technik der tThx für den klinischen Einsatz, die den individuell verschiedenen anatomischen Gegebenheiten Rechnung trägt und beschrieben wird. Nach ermutigenden ersten klinischen Ergebnissen wurden dann die Resultate der prospektiven klinischen Untersuchung des Verfahrens an 60 konsekutiven Patienten erstmals nach den Empfehlungen für klinische Forschung der Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America standardisiert dargestellt. Die Hypothese der geringeren Beeinträchtigung und schnelleren Erholung der Atemfunktion nach tThx wurde in einem prospektiven Vergleich der tThx mit der am meisten akzeptierten und weitesten verbreiteten Operationstechnik der Thx untersucht und bewiesen. Schließlich wurden die Ergebnisse der funktionellen Besserung der MG nach tThx, Thx durch mediane Sternotomie oder Thx durch anterolaterale Thorakotomie in einer Matched-pair-Studie verglichen. Obwohl technisch anspruchsvoll, ließen sich vergleichsweise adäquate Resultate bei objektiven und subjektiven patientenbezogenen Vorteilen für die tThx nachweisen. Ein Studiendesign mit höherem Evidenzgrad erscheint unizentrisch aus ethischen und epidemiologischen Gründen sowie bezogen auf die Pathogenese der MG gegenwärtig für diese Fragestellung schwer erreichbar. In der Zusammenfassung kann die tThx das in der Einleitung beschriebene Dilemma der Suche nach dem optimalen operativen Verfahren zur Thx lösen, indem eine minimale Invasivität, die nicht weiter reduziert werden kann, durch einen optimalen Zugang über 3 Trokare erreicht wird. Dies führt zur Möglichkeit einer radikalen Thx durch die weite Exposition des vorderen Mediastinums. Die tThx sollte bevorzugt werden, da sie adäquate Raten der Verbesserung der MG mit einer minimal-invasiven Operationstechnik erreicht. Die umfassende Untersuchung dieser Technik sollte fortgesetzt werden. / The aim of the present work was to develop and evaluate a novel operation technique to perform a complete thymectomy (Thx). This new approach was first used in a clinical setting in 1994. The thoracoscopic approach for Thx was developed at the Humboldt University Medical School (Charité), Clinic of Surgery, Campus Mitte according to the principles of "good clinical practice" for the introduction of a new operation technique. The success of conventional Thx for MG was determined by a retrospective cohort study as an internal standard. The results of our own clinical series at the Charité corresponded with the data of large published series worldwide and, were thus representative for the aim of developing a new operation technique. Apart from the approval of feasibility, the complex evaluation of the new surgical approach comprised an experimental anatomical study to demonstrate adequate radicality. It could be shown that a unilateral left-sided thoracoscopic operation technique with only 3 trocars can better accomplish a complete Thx as compared to a right-sided 3-trocar-technique. The next step was the development of the detailed and standardized operation technique for the clinical application of thoracoscopic Thx (tThx) which considers the individually different anatomical circumstances and is described. After encouraging first results, the findings of the prospective clinical investigation of the procedure in the first 60 patients were presented, standardized according to the suggestions for clinical research of the Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America. The hypothesis of smaller impairment and faster recovery of pulmonary function after tThx was investigated and could be proved in a prospective comparison of tThx versus the most accepted and most common approach for Thx.Consequently, the results of functional improvement of MG after tThx, median sternotomy for Thx, and anterolateral thoracotomy for Thx were compared in a matched-pair study. Though technically demanding, tThx showed adequate results combined with objective and subjective patient-related advantages. For ethical and epidemiological reasons and due to the pathogenesis of MG a single-center study design with higher level of evidence seems difficult to achieve. In conclusion, tThx may solve the dilemma of the search for an optimal operation technique for Thx because minimal invasion is obtained by an optimal approach with only 3 trocars. This creates the possibility of radical Thx by wide exposure of the anterior mediastinum. The technique of tThx should be prefered because an adequate rate of improvement of MG is achieved by a minimally-invasive operation technique. The comprehensive investigation of this technique should be continued.

重症肌無力是由自身抗體在神經肌肉接頭特異性的結合乙酰膽鹼受體和肌肉特異性激酶引起的一種獲得性免疫性疾病。疾病的主要症狀是骨骼肌的軟弱無力和易疲勞性。這一症狀在運動後尤為顯著，休息後會有所緩解。重症肌無力在世界範圍的發病率是百萬分之三到三十。由於近年來患者的數量在不斷增加，重症肌無力引起了醫學界的廣泛關注。但是，目前的診斷和治療措施還不能完全滿足臨床病人的需要。在本課題研究中，我們希望運用代謝組學的手段建立一種新的更加有效可靠的方法用於重症肌無力的診斷。同時，我們希望在代謝物的水平上來闡釋強肌健力方（一種中藥復方）對重症肌無力的治療作用。 / 本研究所用樣本來自42個重症肌無力病人和16個健康志願者。樣本由廣州中醫藥大學第一附屬醫院於二零零七年到二零零八年收集所得。診斷後，病人每日口服一定劑量的強肌健力方接受治療，連續服藥兩個月。分別在服藥前和治療後對病人抽血採樣。進一步分離血清後，樣品進行質譜分析。多元統計學方法如主成分分析，正交偏最小二乘和正交偏最小二乘判別分析等用於質譜數據的分析。 / 通過和健康者比較分析，我們在重症肌無力病人的血液中找到142個顯著改變的離子。其中，14個離子得到鑒定，包括：γ-氨基丁酸，2-哌啶酸，鳥氨酸，5,8-十四碳二羧酸，精胺，己酰肉毒鹼，N-油酰基甘氨酸，鞘氨醇-1-磷酸，聯原膽酸，糞甾烷酸，植物鞘氨醇-1-磷酸，鵝去氧膽酸甘氨酸結合物，輔酶Q4和甘氨酸膽。基於以上142個離子建立的數學診斷模型在診斷重症肌無力時表現出很高的靈敏度和特異性，分別高達92.8%和83.3%。強肌健力方能夠逆轉由重症肌無力引起的特異性代謝變化，將病人體內被改變的代謝網絡恢復正常，特別是大部分的代謝標誌物在治療後都恢復到了相對正常水平，包括：γ-氨基丁酸，哌啶酸，鳥氨酸，5,8-十四碳二羧酸，精胺，己酰肉毒鹼，N-油酰甘氨酸，鞘氨醇-1-磷酸，聯原膽酸，輔酶Q4和甘氨酸膽。 / 本研究揭示了基於液質聯用的代謝組學方法適用於探索重症肌無力的代謝標誌物，並提供了一種可用於診斷重症肌無力的新方法。同時，本研究證實強肌健力方適用於重症肌無力的治療，且無明顯副作用。 / Myasthenia gravis (MG) is an acquired autoimmune disease caused by specific autoantibodies against acetylcholine receptors (AChRs) and muscle-specific kinase (MuSK) proteins at the neuromuscular junctions. The disease is characterized by weakness and fatigability of the voluntary muscles that gets worse with exertion and improves with rest. The global incidence rate of MG is about 3-30 cases per million per year. In recent years, the worldwide prevalence rate of MG is increasing as a result of increased awareness. However, current diagnostic measures and treatments are not conclusive and satisfactory for MG. In this study, a mass spectrometry-based metabolomic strategy was applied to develop a novel and reliable diagnostic measure for MG on the basis of metabolic analysis, and to explore the therapeutic effect of Qiangji Jianli Fang (QJF, a newly developed Chinese medicine formula) on MG at the metabolite level. / Total 42 MG patients (13 males and 29 females) and 16 volunteers (5 males and 11 females) were recruited at the First Affiliated Hospital of Guangzhou University of Chinese Medicine between March 2007 and March 2008. The patients took QJF once per day for 2 months. Peripheral blood from patients was collected at diagnosis and after 2-month treatment, respectively. Sera prepared from the blood samples were monitored by the liquid chromatography Fourier transform mass spectrometry (LC-FTMS). Mass spectral data were analyzed by multivariate statistical analyses, including principal component analysis (PCA), orthogonal partial least squares (OPLS), and orthogonal partial least squares discriminant analysis (OPLS-DA). / By comparing analysis with the healthy volunteers, 142 significantly changed ions from serum metabolic profile of MG patients were picked out as the potential biomarkers of MG. Among of them, 14 ions were temporarily identified. They were gamma-aminobutyric acid (GABA), pipecolic acid, ornithine, 5,8-tetradecadienoic acid, spermine, hexanoylcarnitine, N-oleoyl glycine, sphingosine-1-phosphate (S1P), bisnorcholic acid, coprocholic acid, phytosphingosine-1-P, chenodeoxycholylglycine, coenzyme Q4, and cholylglycine. The developed OPLS-DA diagnostic model based on the 142 special ions showed a high sensitivity (92.8%) and specificity (83.3%) in detecting MG. QJF showed a powerful action on MG by recovering the holistic serum metabolic profile from the disease level to the normal level. Especially, the levels of GABA, pipecolic acid, ornithine, 5,8-tetradecadienoic acid, spermine, hexanoylcarnitine, N-oleoyl glycine, S1P, bisnorcholic acid, coenzyme Q4, and cholylglycine in MG patients were regulated to a relatively normal level after QJF treatment. / My results first indicated that the LC-FTMS-based metabolomics was a useful tool in biomarkers exploration of MG, and it was potentially applicable as a new diagnostic approach for MG. Also, my results demonstrated that QJF was a good optional choice for the treatment of MG, with no reported side effects. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Yonghai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 113-129). / Abstract also in Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Abstract (in English) --- p.iii / Abstract (in Chinese) --- p.vi / Acknowledgements --- p.viii / Table of contents --- p.ix / Abbreviations --- p.xiv / List of Tables --- p.xviii / List of Figures --- p.xix / Chapter 1: Introduction --- p.1 / Chapter 1.1 --- Myasthenia gravis --- p.1 / Chapter 1.1.1 --- History --- p.1 / Chapter 1.1.2 --- Epidemiology --- p.2 / Chapter 1.1.3 --- Clinical features --- p.2 / Chapter 1.1.4 --- Clinical classification --- p.4 / Chapter 1.1.5 --- Pathophysiology --- p.5 / Chapter 1.1.6 --- Diagnosis --- p.9 / Chapter 1.1.6.1 --- Physical examination --- p.9 / Chapter 1.1.6.2 --- Blood test --- p.10 / Chapter 1.1.6.3 --- Electrodiagnostic test --- p.10 / Chapter 1.1.6.4 --- Edrophonium test --- p.11 / Chapter 1.1.6.5 --- Imaging --- p.11 / Chapter 1.1.6.6 --- Pulmonary function test --- p.11 / Chapter 1.1.7 --- Treatment --- p.12 / Chapter 1.1.7.1 --- Medication --- p.12 / Chapter 1.1.7.2 --- Thymectomy --- p.12 / Chapter 1.1.7.3 --- Plasmapheresis and intravenous immunoglobulin --- p.13 / Chapter 1.2 --- Qiangji Jianli Fang --- p.14 / Chapter 1.2.1 --- Huang qi --- p.15 / Chapter 1.2.2 --- Dang shen --- p.16 / Chapter 1.2.3 --- Bai shu --- p.16 / Chapter 1.2.4 --- Dang gui --- p.17 / Chapter 1.2.5 --- Sheng ma --- p.17 / Chapter 1.2.6 --- Chai hu --- p.18 / Chapter 1.2.7 --- Chen pi --- p.18 / Chapter 1.2.8 --- Gan cao --- p.19 / Chapter 1.3 --- Metabolomics --- p.19 / Chapter 1.3.1 --- What’s metabolomics? --- p.20 / Chapter 1.3.1.1 --- Metabolites --- p.20 / Chapter 1.3.1.2 --- Metabolome --- p.21 / Chapter 1.3.1.3 --- Two terms: metabolomics and metabonomics --- p.21 / Chapter 1.3.2 --- How metabolomics works? --- p.22 / Chapter 1.3.2.1 --- Sample preparation --- p.22 / Chapter 1.3.2.1.1 --- Quenching --- p.23 / Chapter 1.3.2.1.2 --- Separating metabolites --- p.24 / Chapter 1.3.2.1.3 --- Sample concentration --- p.24 / Chapter 1.3.2.2 --- Analytical technologies (Sample analysis) --- p.25 / Chapter 1.3.2.3 --- Data analysis --- p.26 / Chapter 1.3.2.4 --- Database --- p.28 / Chapter 1.3.3 --- Why metabolomics? --- p.29 / Chapter 1.3.4 --- Metabolomics for human diseases --- p.30 / Chapter 1.3.5 --- Metabolomics for Traditional Chinese Medicine --- p.32 / Chapter 1.4 --- Objectives and significances of the present study --- p.34 / Chapter Chapter 2 --- Metabolic biomarkers of myasthenia gravis --- p.36 / Chapter 2.1 --- Introduction --- p.36 / Chapter 2.2 --- Materials and methods --- p.40 / Chapter 2.2.1 --- Chemicals --- p.40 / Chapter 2.2.2 --- Patients --- p.40 / Chapter 2.2.3 --- Volunteers --- p.42 / Chapter 2.2.4 --- Blood collection --- p.43 / Chapter 2.2.5 --- QC samples --- p.43 / Chapter 2.2.6 --- Sample processing --- p.43 / Chapter 2.2.7 --- Liquid chromatography-mass spectrometry --- p.44 / Chapter 2.2.8 --- Data analysis --- p.45 / Chapter 2.2.9 --- Metabolite identification --- p.45 / Chapter 2.3 --- Results --- p.46 / Chapter 2.3.1 --- Method validation --- p.46 / Chapter 2.3.2 --- An overall comparative analysis between 28 patients and 10 volunteers --- p.48 / Chapter 2.3.3 --- Classification of MG --- p.53 / Chapter 2.3.4 --- Comparative analysis of the metabolic changes in early- and late-stage MG patients respectively --- p.54 / Chapter 2.3.5 --- Biomarker identification --- p.56 / Chapter 2.4 --- Discussion --- p.58 / Chapter 2.5 --- Conclusion --- p.63 / Chapter Chapter 3 --- A novel diagnostic approach for myasthenia gravis --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.2 --- Materials and methods --- p.68 / Chapter 3.2.1 --- Chemicals --- p.68 / Chapter 3.2.2 --- Patients and Volunteers --- p.69 / Chapter 3.2.2.1 --- Training set for establishment of diagnostic model --- p.69 / Chapter 3.2.2.2 --- Test set for evaluation of diagnostic model --- p.69 / Chapter 3.2.3 --- QC samples --- p.70 / Chapter 3.2.4 --- Sample processing --- p.71 / Chapter 3.2.5 --- Chromatography --- p.71 / Chapter 3.2.6 --- Mass spectrometry --- p.72 / Chapter 3.2.7 --- Data analysis --- p.72 / Chapter 3.3 --- Results --- p.72 / Chapter 3.3.1 --- Method validation --- p.73 / Chapter 3.3.2 --- Alterations in serum metabolic profile under MG --- p.74 / Chapter 3.3.3 --- Prediction of MG based on biomarkers --- p.74 / Chapter 3.3.4 --- Establishment of diagnostic model on the basis of metabolic profile --- p.77 / Chapter 3.3.5 --- Prediction of MG with diagnostic model --- p.79 / Chapter 3.4 --- Discussion --- p.80 / Chapter 3.5 --- Conclusion --- p.83 / Chapter Chapter 4 --- Qiangji Jianli Fang treatment for myasthenia gravis --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Chemicals --- p.88 / Chapter 4.2.2 --- Herbs --- p.88 / Chapter 4.2.3 --- Participants --- p.88 / Chapter 4.2.4 --- QC samples --- p.90 / Chapter 4.2.5 --- Sample processing --- p.90 / Chapter 4.2.6 --- Liquid chromatography-mass spectrometry --- p.90 / Chapter 4.2.7 --- Data analysis --- p.91 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Method validation --- p.91 / Chapter 4.3.2 --- Symptomatic examination after QJF treatment --- p.92 / Chapter 4.3.3 --- Holistic metabolic responses to QJF treatment --- p.93 / Chapter 4.3.4 --- MG biomarkers changes after QJF treatment --- p.95 / Chapter 4.3.5 --- Drug-related biomarkers of QJF --- p.97 / Chapter 4.4 --- Discussion --- p.100 / Chapter 4.5 --- Conclusion --- p.103 / Chapter Chapter 5 --- Conclusions --- p.104 / Chapter Chapter 6 --- Perspectives --- p.107 / Chapter 6.1 --- Experimental autoimmune myasthenia gravis model --- p.107 / Chapter 6.2 --- Chemical composition of Qiangji Jianli Fang --- p.111 / References --- p.113 / Appendices --- p.130

Myasthenia gravis (MG) is an autoimmune disease whose name means "grave muscular weakness". MG is a rare disease affecting only 200-400 persons per million and the characteristic symptoms include muscle weakness, particularly in highly active voluntary muscles. MG affects the neuromuscular junction in an antibody-mediated manner, resulting in impaired nerve-muscle cell communication in affected individuals. Specifically, two main proteins are targeted: nicotinic acetylcholine receptors (ACh receptors) and a muscle-specific tyrosine kinase (MuSK). Previous studies have discovered the mechanism of MG pathogenesis but the exact mechanisms which cause the failure to maintain self-tolerance have not been discovered. Based on current knowledge of MG, this paper will explore potential causes of the disease and provide numerous hypotheses directed at future research opportunities.

Η βαριά μυασθένεια (Myasthenia gravis-MG) είναι μία επίκτητη αυτοάνοση δυσλειτουργία στη νευρομυϊκή σύναψη που προκαλείται από αντισώματα που κατευθύνονται ειδικά έναντι του μυϊκού τύπου νικοτινικού υποδοχέας της ακετυλοχολίνης (AChR) που βρίσκεται στα μυϊκά κύτταρα. Τα αντισώματα μειώνουν τον αριθμό των λειτουργικών υποδοχέων με αποτέλεσμα οι μυς να μην μπορούν να ανταποκριθούν στα μηνύματα που λαμβάνουν από τα νευρικά κύτταρα. Η κλινική εικόνα χαρακτηρίζεται από χρόνια μυϊκή αδυναμία. Η σωστή διάγνωση από τον θεράποντα ιατρό και ο σχεδιασμός της κατάλληλης θεραπείας, αποτελούν τη βάση για την αντιμετώπιση μιας νόσου και την ενίσχυση της ποιότητας ζωής. Η μη συμμόρφωση όμως του ασθενούς, στη θεραπεία που του προτείνεται, αποτελεί αντικείμενο έρευνας τις τελευταίες δεκαετίες. Η μη συμμόρφωση έχει ως αποτέλεσμα την αύξηση του κόστους περίθαλψης, προκαλώντας μεγάλες κοινωνικοοικονομικές συνέπειες. Στην παρούσα έρευνα δημιουργήθηκε ένα ερωτηματολόγιο και διατέθηκε σε δείγμα 30 μυασθενών. Μέσω της συλλογής των απαντήσεων έγινε μια προσπάθεια να προσδιοριστούν οι παράγοντες που οδηγούν σε μη συμμόρφωση. Διερευνάται η στάση ζωής που κρατά ο ασθενής, η εικόνα που έχει ο ίδιος για τον εαυτό του και ο ρόλος που έχουν το στενό περιβάλλον, ο ιατρός και ο φαρμακοποιός του, ούτως ώστε, ο ασθενής να είναι συνεπής στην αγωγή που του έχει προταθεί. / Myasthenia Gravis (MG) is an acquired autoimmune neuromuscular junction disorder caused by antibodies directed specifically toward the skeletal muscle nicotinic acetylcholine receptor (AChR). The antibodies decrease the number of functional AChRs which compromises neuromuscular transmission. The clinical course of MG is characterized by muscular weakness and fatigability. The right diagnosis of the attending physician and the design of appropriate therapy are determinant for the treatment of a disease and enhance quality of life. Non-compliance of the patient (also adherence or capacitance) to the treatment that his doctor suggested, however, is being investigated in the last decades. Non-compliance increase health care costs and evoke considerable socioeconomic consequences. Worldwide, non-compliance is a major obstacle to the effective delivery of health care. In the present dissertation a questionnaire is created and distributed to a sample of 30 MG patients. Through the collection of responses we attempted to analyze the factors that lead to non-compliance. The objective was to identify how the patient sees himself. It was also made an effort to clarify the roles of doctor, pharmacist and family in patient compliance when long term medication is needed.

Orientador: Luiz Antônio de Lima Resende / Banca: Cláudia Ferreira da Rosa Sobreira / Banca: José Luiz Pedro / Resumo: No estado de São Paulo é comum aplicação de pesticidas na agricultura sem equipamentos de proteção, com grande exposição dos trabalhadores aos agentes tóxicos. O uso doméstico de inseticidas também é muito difundido. Foram demonstrados defeitos da transmissão neuromuscular, com miastenia, relacionados a pesticidas organofosforados. Inseticidas do grupo químico das piretrinas e piretróides, de uso doméstico, atuam sobre o sistema nervoso dos insetos e dos seres humanos. O objetivo deste trabalho foi procurar correlações entre Miastenia Grave e exposição crônica a pesticidas em nosso meio. Foi aplicado questionário sobre exposição a pesticidas em 217 pacientes com diagnóstico clínico e ENMG de Miastenia Grave, sendo 163 mulheres e 54 homens, com idades de 14 a 84 anos, provenientes dos serviços médicos da UNESP e UNIFESP, e em 227 controles, sendo 179 mulheres e 48 homens, com idades de 17 a 78 anos. Os dados foram analisados estatisticamente pelo Teste de Associação do Qui-quadrado. A análise estatística do total de pacientes expostos e não expostos sugeriu possível associação de exposição a pesticidas com miastenia grave (p < 0.0001) / Abstract: In the São Paulo state is very common application of pesticides in agriculture without protective equipment, with great exposure of workers to the toxic agents, and domestic use of insecticides is also widespread. Neuromuscular transmission defects with myasthenia related to the organophosphate pesticides were demonstrated in human and animals. The chemical group of pyrethrins and pyrethroids has action on the peripheral nervous system of insects and humans. The aim of this study was to search correlations between Myasthenia Gravis and chronic exposure to pesticides in our geographic region. Questionnaires were applied on exposure to pesticides in 217 patients with clinical and EMG diagnosis of Myasthenia Gravis, 163 women and 54 men, aged from 14-84 years, seen in two medical services of UNESP and UNIFESP, and in 227 controls, 179 women and 48 men, aged from 17-78 years. Data were statistically analyzed by the Association Chi-square test. The statistical analysis of all patients exposed or not exposed suggested possible association of myasthenia gravis with chronic exposure to pesticides (p <0.0001) / Mestre

Orientador: Antonio José Maria Cataneo / Coorientador: Daniele Cristina Cataneo / Coorientador: Paulo Eduardo de Oliveira Carvalho / Banca: Vânia dos Santos Nunes / Banca: Olavo Ribeiro Rodrigues / Resumo: Introdução: A miastenia gravis é uma doença que afeta a junção neuromuscular e leva a fraqueza da musculatura esquelética. O tratamento atual pode ser clínico ou cirúrgico através da ressecção completa do timo. Objetivo: Comparar, através de uma revisão sistemática da literatura, os resultados do tratamento cirúrgico e clínico da miastenia gravis. Métodos: Foi realizada uma busca nos principais bancos de dados a fim de encontrar ensaios clínicos randomizados que comparassem as duas modalidades terapêuticas. Como não existem estudos dessa categoria, foram selecionados aqueles com menor nível de evidência e que continham as duas intervenções, com pelo menos 10 pacientes em cada braço do estudo. A análise estatística foi feita com software StatsDirect, versão 3.0.121. Resultados: A busca encontrou 592 artigos na base Medline, 1925 artigos na base Embase e 204 artigos na base Lilacs. Após a exclusão dos estudos duplicados, 51 artigos foram analisados integralmente e nove foram selecionados para esta revisão. O número total de pacientes avaliados foi de 3.211. A mortalidade nos grupos cirúrgico e clínico foram respectivamente de 7 e 19%, com diferença estatística significante. A taxa de remissão nos grupos foi de 17% para o grupo cirúrgico e de 13% para o clínico, sem significância estatística. Para o desfecho melhora, o grupo cirúrgico apresentou uma taxa de 23% e o clínico de 29%, também sem diferença estatística. Conclusões: Apesar da baixa evidência disponível, a timectomia pode ser considerada uma opção terapêutica na miastenia gravis, com menores índices de mortalidade e taxas de remissão e controle semelhantes ao tratamento clínico / Abstract: Introduction: Myasthenia gravis is a disease that affects the neuromuscular junction and leads to weakness of the skeletal muscles. Current treatment is guided by the clinical and surgical conduction through the complete resection of the thymus. Objective: Compare, through a systematic review, the results of surgical and medical treatment of myasthenia gravis. Methods: A search was conducted in major databases to find randomized controlled trials that compared the two treatment modalities. As there are no studies that category, were selected studies with a lower level of evidence and that contained both interventions and at least 10 patients in each arm of the study. Statistical analysis was performed with StatsDirect version 3.0.121 software. Results: The search found 592 articles in Medline, 1925 articles in the Embase and 204 articles in the Lilacs. After exclusion of duplicate studies, 51 articles were fully analyzed and nine were selected for this review. The total number of patients was 3,211. The mortality in the surgical and medical groups were respectively 7 and 19%, with significant differences. The remission rate in both groups was 17% for the surgical group and 13% for clinical, without statistical significance. To improve the outcome, the surgical group had a rate of 23% and 29% of the clinical, also no statistical difference. Conclusion: Despite the low available evidence, thymectomy may be considered a therapeutic option in myasthenia gravis, with lower rates of mortality and rates of remission and control similar to clinical treatment Keywords: myasthenia gravis, thymectomy, clinical treatment / Mestre

Οι υποδοχείς της ακετυλοχολίνης (AChRs) είναι διαμεμβρανικές πρωτεΐνες ενεργοποιούμενες με τη δέσμευση της ακετυλοχολίνης (ACh). Με κριτήρια, όπως η χημική συγγένεια που εμφανίζουν για σηματοδότικά μόρια και οι φαρμακολογικές τους ιδιότητες, ταξινομούνται στην ομάδα των νικοτινικών AChRs και στην ομάδα των μουσκαρινικών AChRs. Οι νικοτινικού τύπου υποδοχείς δημιουργούνται από τη συναρμογή πέντε ομόλογων υπομονάδων και υποδιαιρούνται σε μυϊκού τύπου, ευρισκόμενους κυρίως στους σκελετικούς μύες των σπονδυλωτών και σε νευρικού τύπου, απαντώμενους κατά κύριο λόγο στο κεντρικό και περιφερικό νευρικό σύστημα. Οι AChRs σχετίζονται με σειρά παθολογικών καταστάσεων, μεταξύ των οποίων και η βαρειά μυασθένεια (Myasthenia Gravis-MG). Η μυασθένεια χαρακτηρίζεται από χρόνια μυϊκή αδυναμία, προκαλούμενη από τη δράση αντισωμάτων υψηλής συγγένειας έναντι του μυϊκού τύπου AChR. Με απώτερο σκοπό τη διερεύνηση της παθογονικότητας των αυτοαντισωμάτων έναντι μεμονωμένων υπομονάδων του AChR, προχωρήσαμε στην παραγωγή ανασυνδυασμένων πολυπεπτιδικών τμημάτων των υπομονάδων στο ζυμομύκητα Pichia pastoris. Τα πολυπεπτίδια χρησιμοποιήθηκαν στην παρασκευή χρωματογραφικών-ανοσοπροσροφητικών στηλών, που εφαρμόστηκαν ακολούθως για την απομόνωση αυτοαντισωμάτων από επιλεγμένους ορούς μυασθενικών ατόμων. Η παθογόνος δράση των απομονωμένων αυτοαντισωμάτων ελέχθηκε μέσω της προκαλούμενης απώλειας υποδοχέων (αντιγονική τροποποίηση-antigenic modulation) σε κυτταρική σειρά (ΤΕ671) που εκφράζει τον AChR και μέσω της χορήγησή τους σε πειραματόζωα και τον έλεγχο της εμφάνισης χαρακτηριστικών συμπτωμάτων της νόσου. Εκτενής συγκριτική μελέτη μεταξύ τεσσάρων επιλεγμένων ορών και αντισωμάτων έναντι της α1 και της β υπομονάδας του υποδοχέα, που απομονώθηκαν από τους συγκεκριμένους ορούς, έδειξαν πως τα αυτοαντισώματα ευθύνονται για δράση των ορών στους υποδοχείς των κυττάρων. Τόσο οι ολικοί οροί όσο και τα απομονωμένα-καθαρά αυτοαντισώματα έναντι των υπομονάδων α1 και β, προκάλεσαν δοσοεξαρτώμενη απώλεια υποδοχέων στα κύτταρα και μάλιστα τα αντι-α1 αντισώματα εμφανίστηκαν περίπου τέσσερις φορές δραστικότερα από τα αντι-β. Η ικανότητα των μερικώς απαλλαγμένων από αυτοαντισώματα έναντι του υποδοχέα ορών να προκαλούν απώλεια υποδοχέων στα κύτταρα, φάνηκε να ποικίλλει και να συσχετίζεται άμεσα με το είδος των αντισωμάτων που έχουν παραμείνει στον ορό, υποστηρίζοντας μια διαφορετικότητα στην παθογονικότητα των επιμέρους αντισωμικών κλασμάτων. Με σκοπό την επιβεβαίωση και ενίσχυση των αποτελεσμάτων που προκύπτουν από τα in vitro πειράματα, ακολούθησαν προσπάθειες για την πρόκληση πειραματικής μυασθένειας σε πειραματόζωα, με τη χορήγηση ορών μυασθενικών και καθαρών αυτοαντισωμάτων έναντι διαφόρων υπομονάδων του υποδοχέα. Η χορήγηση σε ζώα τόσο του ολικού ορού, όσο και καθαρών αντισωμάτων έναντι της α1-υπομονάδας του υποδοχέα, προκάλεσαν σημαντική απώλεια βάρους και εμφάνιση έντονων συμπτωμάτων μυϊκής αδυναμίας, μέχρι και το θάνατο. Πειραματόζωα που ενέθηκαν με το κλάσμα του ορού από το οποίο έχουν απομακρυνθεί τα συγκεκριμένα αντισώματα εμφάνισαν πολύ ηπιότερα ή και καθόλου συμπτώματα, ενώ απουσία συμπτωμάτων καταγράφηκε και κατά τη χορήγηση ορού που περιείχε αποκλειστικά αντισώματα έναντι της β υπομονάδας, αλλά και απομονωμένων αντι-β αντισωμάτων. Η παρούσα μελέτη υπέδειξε τα αυτοαντισώματα έναντι του AChR ως τον μοναδικό παθογόνο παράγοντα στον ορό μυασθενικών ατόμων, συμβάλλοντας στην κατανόηση της παθοφυσιολογίας της νόσου. Επιβεβαίωσε την υπεροχή των αντι-α1 αντισωμάτων έναντι των αντι-β, ως πρός την παθογονικότητά τους, τόσο in vitro όσο και in vivo, με την επιφύλαξη βέβαια που επιβάλλει ο μικρός αριθμός δειγμάτων που μελετήθηκαν. Η δυνατότητα λήψης αντισωμάτων που στοχεύουν σε συγκεκριμένη υπομονάδα μπορεί να συμβάλλει στη λεπτομερή μελέτη της δραστικότητας του κλάσματος και να οδηγήσει στη συσχέτισή του με την εμφάνιση συγκεκριμένων συμπτωμάτων της νόσου. / Acetylcholine receptors (AChRs) are integral membrane proteins that respond to the binding of acetylcholine (ACh), which is synthesized, stored and finally released by cholinergic neurons. Like other transmembrane receptors, AChRs have been classified according to either their pharmacological properties or their relative affinities for various molecules, and can therefore be further divided into: i) nicotinic AChRs, which are particularly responsive to nicotine and ii) muscarinic AChRs, which are particularly responsive to muscarine. AChRs are involved in myasthenia gravis (MG) and many other physiological disorders, mainly affecting the central and peripheral nervous system. In MG, autoantibodies are directed against the nicotinic AChR at the neuromuscular junction. The disease is characterized by various symptoms, including muscle weakness and fatigability, due to defective neuromuscular transmission. To obtain an insight into the role of the various anti-AChR antibody specificities in MG, we isolated and studied the in vitro and in vivo activity of autoantibodies targeting individual AChR subunits. Using recombinant proteins corresponding to extracellular domains (ECDs) of individual AChR subunits as immunoadsorbents; we isolated autoantibodies which specifically bind to these subunits. We then used the well established TE671 human muscle cell line to examine the in vitro functions of subunit-specific autoantibody populations through their ability to induce nAChR antigenic modulation. Isolated subunit-specific autoantibodies were also used to determine their capacity to passively transfer experimental MG into lab animals. Our results clearly demonstrated that autoantibodies against the α1 or β subunit can cause AChR loss via antigenic modulation in a dose-dependent manner, the anti-α1 autoantibodies being much more effective than the anti-β autoantibodies. Furthermore, we showed that the autoantibody-depleted sera were much less effective, or were completely inactive, at causing AChR loss. In in vivo experiments, the administration of MG sera derivatives to lab animals showed that sera enriched in anti-α1 autoantibodies, as well as the corresponding pure anti-α1 autoantibodies from two individuals, are efficient in inducing MG like symptoms to the animals. A single serum contained almost 100% anti-β antibodies and the corresponding purified antibodies did not cause any clinical MG symptoms. The depleted fraction of MG sera tested, induced mild symptoms or no symptoms were observed, and this is in agreement with the in vitro results, strongly suggesting that the anti-AChR autoantibodies in MG sera and mainly the anti-α1 specificities are the sole pathogenic factor in anti-AChR antibody-seropositive MG.