Bone loss in osteoporosis and rheumatoid arthritis diseases: The effects of disease mechanisms, age, gender and ethnic origin on responsiveness to treatment.

4 Table of contents . 5-8 List of figures . 9-12 List of tables . 13 Abbreviations . 14-16 Chapter One 17-59 1.1. Bone . 18 1.2. Structure and organisation of bone . 18 1.3. Bone homeostasis . 19-21 1.4. Factors that influence changes in bone metabolism . 22-23 1.4. 1. Changes in bone metabolism with age . 24 1.5. Methods used for the assessment of changes in bone density . 25 1.5.1. Radiographic protocols . 25 a) Dual energy X-ray absorptiometry (DEXA .25-26 b) Peripheral dual energy X-ray absorptiometry (PDEXA) . 27 c) Single X-ray absorptiometry (SXA) . 27 d) Quantitative ultrasound (QUS) . 27 e) Quantitative computed tomography (QCT) . 28 f) Radiographic absorptiometry . 28 1.5.2. Laboratory biomarkers of bone metabolism . 29 1.6. Pathological conditions associated with changes in bone metabolism . 29 1.6. 1. Osteoporosis . 30 1.6. 1.1. Primary osteoporosis . 31 a) Postmenopausal Osteoporosis . 31 b) Age-related osteoporosis . 31 1.6. 1.2. Secondary osteoporosis . 32 a) Bone loss induced by cancer . 32 b) Bone loss induced by Glucocorticoid . 33 c) Bone loss induced by chronic inflammatory diseases . 33-34 I. Rheumatoid arthritis (RA) . 34-36 II. Early diagnosis of patients with RA . 36 a) Anti-CCP autoantibodies . 36-37 b) Radiographic procedures for the detection and assessment of bone erosions in RA.37 c) Ultrasound (US) techniques . 38 III. Bone loss in RA: The role of the TNFα . 38 a) Osteoclastogenic effects of TNFα . 39-40 b) The effects of TNFα on osteoblastogenesis . 40-41 1.6. 2. Osteopetrosis . 42 1.6. 3. Paget’s disease . 43 1.7. Treatment of patients with osteoporosis . 43 6 a) Hormone replacement therapy (HRT) . 43-44 b) Bisphosphonates . 44-45 c) Selective oestrogen receptor modulators (SERMs) . 45-46 d) Calcitonin (CT) . 46-47 e) Teriparatide (Forsteo) . 47 f) New anti-osteoporosis therapies . 47-48 1.8. Biologic anti-inflammatory therapies and their effect on bone loss in RA . 48 a) Etanercept . 48 b) Infliximab . 48 c) Adalimumab . 49 d) Rituximab . 49 e) Abatacept . 50 f) Tocilizumab . 51 g) Anakinra . 51-52 1.9. The relationship between Osteoporosis and RA . 52-54 1.10. Reduced bone loss in patients treated with biologic anti-TNFα agents . 54-57 Hypothesis . 58 Aims and objectives . 58-59 Chapter Two 60-82 2.1. Patients . 61-62 2.2. Methods . 62 2.2.1. Dual energy X-ray absorptiometry (DEXA) . 62-63 a) Precision and accuracy . 63-64 b) In vitro short-term precision . 64-65 c) In vitro long-term precision . 65 d) Subject positioning and site of measurements . 66-67 e) BMD scan reports . 68-70 2.2.2 Digital X-ray radiogrammetry (DXR) . 71 a) Precision of DXR . 72 2.2. 3. Separation and stimulation of T and B lymphocytes and monocytes . 76 2.2. 4. Enrichment of peripheral blood mononuclear cells (PBMNCs) for evaluating the effect of treatment with biologic anti-TNFα agents on osteoclastogenesis . 76 2.2. 5. Enzyme-linked immunosorbent assay (ELISA) . 77 2.2. 5.1. ELISA of protein involved in regulating bone resorption . 76-77 2.2. 5.2. ELISA of bone turnover biomarkers . 78-79 2.2. 5.3. Reproducibility and accuracy of ELISA . 79 a) The effect of rheumatoid factor (RF) autoantibody interference on the reproducibility of ELISA for bone biomarkers . 81 b) ELISA for the measurement of RF . 82 2.2. 6. Measurement of cytokine levels produced by immune cells from RA patients treated with biologic anti-TNFα agents in relation to changes in BMD . 83 a) Cytokine analysis . 83 2.2. 7. Meso scale discovery (MSD) protocol for the measurement of cytokines . 83 2.2. 8. Inhibition of osteoclastogenesis by OPG and anti-TNFα . 84 2.2. 9. Fluorescence-activated cell sorting (FACS) analysis for measuring the frequency of OCPs before and after treatment of RA patients with biologic anti-TNFα agents . 84 2.2.10. Disease activity score 28 (DAS28) measurements . 84 2.2.11. RA patient selection . 85 2.2.12. Statistical Methods . 85 7 Chapter Three 86-112 3.1. Assessment of the DEXA protocol used in the study . 87 3.1.1. In-vitro short-term precision . 87-89 3.1.2. In vitro long-term precision assessment . 90-91 3.1.3. Outcome of precision assessments . 91-93 3.2. Assessment of accuracy and reproducibility of ELISA protocols . 93 3.2.1. The potential interference of RFs on the ELISA protocols of bone biomarkers . 9