Detection of Staphylococcal Enterotoxins in Patients with Rheumatoid Arthritis or Closed Fractures PhD Medicine A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to LANCASTER UNIVERSITY July 2016 By LAURA ELIZABETH GRACE BSc (Hons) TABLE OF CONTENTS i ABSTRACT .................................................................................................................................. xi ACKNOWLEDGEMENTS .......................................................................................................... xiii DECLARATION .......................................................................................................................... xiv LIST OF FIGURES ...................................................................................................................... xv LIST OF TABLES ........................................................................................................................ xxx LIST OF ABBREVIATIONS ....................................................................................................... xxxvi 1. INTRODUCTION ............................................................................................................ 1 1.1 Motivation ................................................................................................................ 1 1.2 Aims ......................................................................................................................... 1 1.3 The Immune System ................................................................................................ 2 1.4 Rheumatoid Arthritis ............................................................................................... 4 1.5 Epidemiology ........................................................................................................... 7 1.5.1 Prevalence ...................................................................................................... 7 1.5.2 Morbidity ....................................................................................................... 8 1.5.3 Current Treatment .......................................................................................... 8 1.5.4 Cancer ............................................................................................................ 10 1.5.5 Mental Health ................................................................................................. 11 1.5.6 Infection as a Result of RA ............................................................................ 11 1.5.7 Mortality ........................................................................................................ 12 1.6 Hygiene Hypothesis .................................................................................................. 13 1.6.1 Background .................................................................................................... 13 1.6.2 Molecular Mimicry ........................................................................................ 15 1.6.3 Bystander Activation ..................................................................................... 18 ii 1.6.4 Infection as a Cause of RA ............................................................................ 19 1.7 Staphylococcus Aureus ............................................................................................ 21 1.7.1 Staphylococcus Aureus .................................................................................. 21 1.7.2 S.aureus Toxins .............................................................................................. 24 1.7.2.1 Alpha Haemolysin ............................................................................. 24 1.7.2.2 Staphylococcal Enterotoxin B ........................................................... 25 1.7.2.3 Staphylococcal Enterotoxin C ........................................................... 27 1.7.2.4 Toxic Shock Syndrome Toxin 1 ....................................................... 28 1.8 Aetiology .................................................................................................................. 29 1.8.1 Age ................................................................................................................. 29 1.8.2 Gender ............................................................................................................ 30 1.8.3 Birth Order ..................................................................................................... 31 1.8.4 Sharing a Bedroom ........................................................................................ 32 1.8.5 Exposure to Animals ...................................................................................... 32 1.8.6 Socioeconomic Status .................................................................................... 33 1.8.7 Smoking ......................................................................................................... 35 1.8.8 Urbanisation ................................................................................................... 36 1.8.9 Race & Culture .............................................................................................. 37 1.8.10 Seasonal Variation ......................................................................................... 38 1.8.11 Genetic Influence ........................................................................................... 39 1.9 Conclusion ............................................................................................................... 41 2. MATERIALS & METHODS ........................................................................................... 43 2.1 Chemicals & Reagents ............................................................................................. 43 2.1.1 Toxins .......................................................................................................... 45 iii 2.1.2 Antibodies .................................................................................................... 46 2.1.3 Enzymes ....................................................................................................... 46 2.1.4 Buffers .......................................................................................................... 47 2.1.5 Gels .............................................................................................................. 48 2.1.6 Reduction & Alkylation ............................................................................... 49 2.1.7 Mass Spectrometry Sample Preparation ...................................................... 50 2.1.8 Mass Spectrometry ....................................................................................... 50 2.2 Sample Population ................................................................................................... 52 2.2.1 Sample Size Calculation .............................................................................. 52 2.2.2 Inclusion/Exclusion Criteria ........................................................................ 54 2.2.3 Risks/Benefits .............................................................................................. 54 2.2.4 Questionnaire Design ................................................................................... 55 2.2.5 Study Design ................................................................................................ 56 2.3 Anonymisation ........................................................................................................ 57 2.4 Sample Collection and Storage ............................................................................... 57 2.5 Enzyme Digestion ................................................................................................... 57 2.5.1 Trypsin ......................................................................................................... 58 2.5.1.1 Trypsin Singles .............................................................................. 58 2.5.1.1 Trypsin ........................................................................................... 59 2.5.2 Pepsin ........................................................................................................... 59 2.5.3 Papain ........................................................................................................... 60 2.5.4 Chymotrypsin ............................................................................................... 60 2.5.5 Lysine C &Trypsin ...................................................................................... 61 2.6 Western Blot for the Detection of Staphylococcal Toxins ...................................... 61 iv 2.7 ELISA for the Detection of Staphylococcal Toxins ................................................ 63 2.8 Mass Spectrometry Sample Preparation .................................................................. 64 2.9 mMass ...................................................................................................................... 67 2.10 Mass Spectrometry ................................................................................................... 68 2.10.1 HPLC-ESI-MS ........................................................................................... 68 2.10.2 LCMS-IT-TOF ........................................................................................... 69 2.11 Statistical Analysis ..................................................................................................