IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HEART-REACTIVE AUTOANTIBODY IN SYSTEMIC LUPUS ERYTHEMATOSUS: POSSIBLE SEROLOGICAL MARKER FOR EARLY MYOCARDIAL DYSFUNCTION XU QIAN NATIONAL UNIVERSITY OF SINGAPORE 2007 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HEART-REACTIVE AUTOANTIBODY IN SYSTEMIC LUPUS ERYTHEMATOSUS: POSSIBLE SEROLOGICAL MARKER FOR EARLY MYOCARDIAL DYSFUNCTION XU QIAN (M. Med., Shanghai Second Medical University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MEDICINE YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENT First of all, I would like to express my deepest gratitude and appreciation to my supervisor Associate Professor Fong Kok Yong, Chairman of Division of Medicine, senior consultant, Department of Rheumatology and Immunology in Singapore General Hospital, for his invaluable supervision, support and inspiration. His open-mindedness, critical comments, provoking discussions as well as continuous encouragement and patience have enlightened me and inspired my independent thinking. Apart from these, I have also benefited from his integrity, preciseness and explorative philosophy to research. I am also thankful to my co-supervisor Vice-Dean and Associate Professor Koh Dow Rhoon, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, for his guidance. I am also very grateful to Dr Tin Soe Kyaw for his help in my research. His kindness, patience, and intelligence in research work are very impressive to me. Thanks to Prof Ling Lieng Hsi and his staff: Ms Yang Hong, Ms Gong lingli for doing the echocardiography for the patients. Thanks to Ms Connie Tse, for helping to collect the blood samples and the clinical data. Thanks to Dr Sivalingam SP for his humor, kindness, and help. Thanks to all staff of Deparment of Rheumatology and Immunology, Singapore General Hospital for their help and friendship. ii Thanks to all staff of Department of Clinical Research, Singapore General Hospital, Dr Aw, Ms Cindy Goh, and the other staff. Thanks for their kind help and encouragement. I greatly acknowledge National University of Singapore for offering me the Research Scholarship and Singapore General Hospital for providing me technical and research support. Finally, I would like to give the greatest gratitude to my family for their infinite love and support. Thanks to my father Mr Xu Houcai, my mother Mrs Xu Wenying, and my sister Xu xu, for without them, I could not have come so far. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………….ii TABLE OF CONTENTS…………………………………………………………iv SUMMARY………………………………………………………………………ix LIST OF TABLES……………………………………………………………… .xi LIST OF FIGURES…………………………………………………………… .xiii LIST OF ABBREVIATIONS………………………………………………… xv LIST OF PUBLICATIONS……………………………………………………xxi CHAPTER INTRODUCTION 1.1 Systemic lupus erythematosus (SLE) .……………………………………….2 1.1.1 Epidemiology .…………………………………………………………….2 1.1.2 Pathogenesis of SLE……………………………………………………….5 Genetics……………………………………………………………………5 Zero Enviromental factors…………………………………………………… 10 Hormones……………………………………………………………… .13 Infection and inflammation……………………………………………….14 Immunology……………………………………………………… .……17 Immune clearance deficiency…………………………………………….20 Autoantibodies in SLE……………………………………………………24 Pathogenesis of cardiovascular involvements in SLE………………… 26 iv 1.1.2.8.1 General cardiovascular manifestations in SLE ………………………27 1.1.2.8.2 Factors contributing to cardiovascular disease in SLE……………… 30 1.1.2.8.3 Heart reactive autoantibodies in SLE…………………………………31 1.1.2.8.4 Interleukin-18 and cardiovascular involvements in SLE Therapies for SLE… .………………………………………………………………37 1.2 Diagnostic approaches of cardiovascular involvements in SLE…………….39 1.2.1 Echocardiography……………………………………………………… 40 1.2.1.1 Transthoracic echocardiography (TTE) and Transesophageal echocardiography (TEE)…………………………………………………………40 1.2.1.2 Myocardial contrast echocardiography………………………………… .42 1.2.1.3 Doppler Tissue Imaging (TDI)………………………………………… .43 1.2.1.4 Strain and Strain Rate…………………………………………………….45 1.2.2Cardiac troponin I.………………………………………………………… 48 1.3 Gaps………………………………………………………………………… 52 1.4 Objectives of the study…………………………………………………… .52 CHAPTER MATERIALS AND METHODS 2.1 Heart reactive autoantibodies study………………………….……………….55 2.1.1 Recruitment of patients and controls and collection of clinical data……….55 2.1.2 Sources of organ samples .…………………………………………………56 2.1.3 Membrane protein extraction……………………………………………….57 2.1.4 Immunoblotting…………………………………………………………….57 v 2.1.5 Detection of heart reactive autoantibody (HRAA)…………………………59 2.1.6 Two-dimensional (2-D) gel electrophoresis……………………………… 59 2.1.7 Peptide mass fingerprinting……………………………………………… .60 2.1.8 Calculation of molecular weights……………… …………………………61 2.1.9 Characterisation of the HRAA protein…………… ………… ………… .61 2.1.9.1 Western blot comparations between lupus serum and troponin antibodies.61 2.1.9.2 Immunoprecipitation…………………………………………………… 62 2.1.9.3 Competitive binding by human cardiac troponin I proteins…………… .62 2.1.10 Statistics………………………………………………………………… .63 2.2 Echocardiography…………………………………………………………….63 2.2.1 Patients…………………………………………………………………… 63 2.2.2 Echocardiography……………… …………………………………………64 2.2.3 Doppler tissue imaging and strain rates measurements…………………….65 2.2.4 Statistics…………………………………………………………………….65 2.3 Interleukin 18 and cardiac involvements in SLE…………………………… 65 2.3.1 Patients recruitment and sample preparation ………………………………65 2.3.2 Genomic DNA extraction………………………………………………… 66 2.3.3 Specific Sequence Primer PCR…………………………………………… 66 2.3.4 Restriction Fragment Length Polymorphisms (RFLP) analysis………… 67 2.3.5 Enzyme-linked immunosorbent assay (ELISA) for circulating interleukin 18 levels in SLE patients……………………………………………………….67 2.3.6 Statistics…………………………………………………………………….68 vi CHAPTER RESULTS 3.1 Patient demographics… …………………………………………………… 70 3.2 Prevalence of HRAA in lupus patients, non-lupus patients and healthy controls………………………………………………………………… … .72 3.3 Tissue and species specificity of HRAA…………………………………… .76 3.4 Peptide mass fingerprinting of heart antigens reactive to HRAA…………….78 3.5 Confirmation of cardiac troponin I……………………………………………79 3.5.1 Comparision of the western blotting images……………………………… 79 3.5.2 Immunoprecipitation……………………………………………………….80 3.5.3 Competitive western blotting……………………………………………….82 3.6 Echocardiography…………………………………………………………….83 3.7 Interleukin 18 results………………………………………………………….89 3.7.1 Clinical data……………………………………………………………… .89 3.7.2 Allelic frequencies of Interleukin 18 promoter gene polymophisms at position -607 and -137 in SLE patients and controls……………………………… 89 3.7.3 Genotypic frequencies of Interleukin 18 promoter gene polymorphisms at position -607 and -137 in SLE patients and controls……………………….90 3.7.4 Correlation of Interleukin 18 promoter gene polymorphism and circulating Interleukin 18 protein levels……………………………………………… 93 3.7.5 Patients with HRAA have higher circulating Interleukin 18 levels.……… 96 CHAPTER DISCUSSION………… …………………………………………97 vii CHAPTER CONCLUSION… .…………………………………………… .112 REFERENCES… …………………………………………………………… .114 APPENDICES………………………………………………………………… 158 viii SUMMARY Early cardiac involvement is difficult to diagnose in SLE. Autoantibodies have been associated with lupus cardiac involvement. A novel 29-kDa heart reactive autoantibody was identified in lupus sera by immunoblots, and characterized as anti-cTnI (cardiac troponin I) antibody by 2-D electrophoresis and mass spectrophotometry of the mouse heart antigen. This was further confirmed by competitive inhibition assays. The prevalence of this antibody was found to be 12% in a cohort of 109 lupus patients. Immunoblotting results using human heart lysates showed concordant results with those obtained by mouse heart lysates. No anti-cTnI was detected in the sera of 118 non-lupus rheumatic patients (primary antiphospholipid syndrome, rheumatoid arthritis, osteoarthritis, polymyositis) and 50 patients with acute myocardial infarction. Polymyositis patients and lupus patients with myositis showed a positive protein band of 27 kDa, which was demonstrated to be similar to skeletal TnI. Clinical data were obtained by chart review. 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Arthritis Rheum 44: 361-7 157 APPENDICES 158 RECIPE OF SOLUTIONS 1X PBS (pH 7.4) NaCl 9.00g/L Na2HPO4·7H2O 0.795g/L KH2PO4 0.144g/L ddH2O 5X sample buffer for immunoblotting Tris-HCl (pH 6.8) 0.625M SDS 10% Glycerol 50% β-mercapthoethanol 5% bromophenol blue pinch ddH2O 1X running buffer for immunoblotting Tris 6g/L Appendices 179 Glycine 28.8g/L SDS 1g/L ddH2O 1X transferring buffer for immunoblotting Glycine 14.41g/L Tris base 3.03g/L Methanol 10% (v/v) ddH2O 1X wash buffer for immunoblotting (TBST) Tris pH 7.5 10mM NaCl 100mM TWEEN 20 0.1% ddH2O Striping buffer for immunoblotting Tris-HCl (pH 6.8) 62.5mM SDS 2% β-mercaptoethanol 0.8% ddH2O 159 1X TBE buffer (pH 8.35) Tris 90M Boric acid 50M EDTA 2mM ddH2O 160 [...]... 1995; Ward 2004) makes this difference less obvious The lowest overall incidence was found in Iceland and Japan and highest in USA and France The overall prevalence was the lowest in Northern Ireland, UK and Finland, and the highest in Italy, Spain and Martinique The burden of SLE was consistently increased in non-white population of the USA, Europe, Canada and Australia (Danchenko et al 200 6a) 4 Introduction... presentation at singhealth scientific meeting 2004 October 2004, Singapore Manuscript: Xu Q, Ling LH, Thumboo J, Tin SK, Chua T, Tse C, Yang H, Fong KY Identification and characterization of a novel heart- reactive autoantibody in systemic lupus eryhematosus and correlation with Doppler myocardial strain rates: possible serological marker for early myocardial dysfunction xxii CHAPTER 1 INTRODUCTION 1 Introduction... those of incidence, but no clear North–South or East–West pattern emerged Studies of aboriginal populations in Australia and New Zealand report higher prevalence and incidence in this population than in Australian Caucasians Both racial and genetic differences, and more complex social factors related to poverty and access to care are likely associated with increased risk of the disease A higher prevalence... Plasma IL-18 protein levels in SLE patients 96 xiv LIST OF ABBREVIATIONS ACA anticardiolipin antibodies ACR American College of Rheumatology ACHA anticholesterol antibody aCL anticardiolipin antibodies ANA antinuclear autoantibody ANP atrial natriuretic peptide APS anti-phospholipid syndrome aPL anti-phospholipids antibodies ARA American Rheumatology Association ATP adenosine triphosphate BCIP 5-Bromo-4-Chloro-3'-Indolyphosphate... echocardiographic variables in SLE patients with and without anti-cTnI 84 Table 10 Longitudinal (Longit) and radial myocardial tissue velocity, strain rate and strain indexes in SLE patients with and without anti-cTnI 86 Table 11 Correlation of longitudinal strain rates with Anti-cTnI .87 Table 12 Valvular thickening and regurgitation in SLE patients according to anti-cTnI status... "Clinical applications of strain rate imaging." (Yip et al 2003) 46 Figure 4 Example of strain rate (SR) image Adapted from "Clinical applications of strain rate imaging." (Yip et al 2003) .48 Figure 5 Identification of Heart Reactive Autoantibody (HRAA) 73 Figure 6 The HRAA is heart- specific 76 Figure 7 Immunoblotting results of HRAA against heart membrane protein extracted from different... marker for lupus cardiac involvements x LIST OF TABLES Table 1 Environmental factors and SLE Adapted from "Environment and systemic lupus erythematosus: an overview."(Sarzi-Puttini et al 2005) 12 Table 2 Possible Factors contributing to cardiovascular disease in SLE 31 Table 3 Demographic data of controls, lupus and non -lupus patient cohort 71 Table 4 ACR criteria presentation of lupus patients at diagnosis... 72 Table 5 HRAA in controls, lupus and non -lupus groups 73 Table 6 Demographic data of HRAA -positive and -negative groups 74 Table 7 Autoantibody profiles of SLE patients positive and negative for HRAA 75 Table 8: Distribution of heart- related disease in SLE patients positive and negative for HRAA 75 Table 9 M-mode, two-dimensional, conventional Doppler and annular... (Zuniga et al 2001), Caucasian (Koene et al 1998), Korean (Salmon et al 1999) and an ethnically diverse population (Wu et al 1997), but not in African American (Oh et al 1999) or Caucasian SLE patients (Dijstelbloem et al 2000) Association of F158 to lupus nephritis was found in a Caucasian, Korean and a multiethnic population (Salmon et al 1999) but not in Germans or in Hispanics, Asian/Pacific Islanders... virus increases antibody titers, but these may be the result of polyclonal B cell activation (Arbuckle et al 200 3a) Other data suggest that there may be a link between infections early in life and an increased prevalence of antinuclear antibodies and SLE in adults Early exposure to microbial antigens or vaccines may predispose to lupus- like autoimmune disease 10 Introduction (Baxter et al 1994) The amount . IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HEART -REACTIVE AUTOANTIBODY IN SYSTEMIC LUPUS ERYTHEMATOSUS: POSSIBLE SEROLOGICAL MARKER FOR EARLY MYOCARDIAL DYSFUNCTION XU QIAN. NATIONAL UNIVERSITY OF SINGAPORE 2007 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HEART -REACTIVE AUTOANTIBODY IN SYSTEMIC LUPUS ERYTHEMATOSUS: POSSIBLE SEROLOGICAL MARKER FOR EARLY. samples and the clinical data. Thanks to Dr Sivalingam SP for his humor, kindness, and help. Thanks to all staff of Deparment of Rheumatology and Immunology, Singapore General Hospital for