Epidemiology of Asthma

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INTRODUCTION

According to the Global Asthma Report 2014, asthma is a highly prevalent chronic airway disease that affects 334 million people of all ages worldwide and is expected to increase by another 100 million by 2025 due to urbanization. Asthma therefore imposes a substantial burden on healthcare system throughout the world and also on society where individuals are affected through loss of quality of life. Although asthma is common among children, the burden of asthma impact increases with age, especially in elderly women. Despite of a variety of treatment regimens, the prevalence of asthma is on the increase. Thus, there is a need to better understand the pathophysiology associated with asthma to translate scientific knowledge into improved treatment and patient management. Microscopy is an emerging technique that allows for the investigation of the ultrastructural properties of components of blood.
It can unfold a category of information regarding structural and mechanical properties of the cells, aiding in the understanding of the functional and physiological properties of the components associated with asthma. Asthma is a complex heterogeneous disease affecting the respiratory airways, characterised by airway inflammation, bronchoconstriction, airway remodelling and bronchial hyper reactivity. One of the major risk factor for asthma is allergy or atopy [a predisposition for immunoglobulin E (IgE) to allergens]. Both environmental and genetic factors play an important role in the pathogenesis of allergic asthma. A central feature of asthma pathology is airway inflammation. Similar to a chronic wound, a plethora of biochemical mechanisms initiate and propagate allergic inflammatory responses in asthma due to a wide range of environmental insults such as allergens, pathogens, pollutants, certain drugs and chemicals. Inflammation is also a multifaceted and complex process, which is well controlled and critical for the normal physiology of the body to maintain homeostasis.
Uncontrolled inflammation, however, can have detrimental consequences. A complex network of mediators and pathways orchestrate allergic inflammatory cascade in allergic asthma. These mechanisms cause the attraction and accumulation of immune cells and also the extravasation of plasma from the small blood vessels into the interstitial space in inflamed tissue. Altered coagulation cascade is one of the mechanisms that are potentiated due to inflammatory conditions. Evidence suggests a relationship exists between disturbed haemostasis and asthma pathogenesis. Haemostasis is a normal physiological process that maintains blood-fluidity and has the potential to induce the formation of a clot to prevent bleeding. Blood is a fluid, composed of erythrocytes (red blood cells), leukocytes (white blood cells), platelets and plasma proteins such as fibrinogen suspended in plasma. The functions of blood include: transporting oxygen and carbon dioxide, nutrients, signalling molecules such as hormones and waste materials. Coagulation, another function of blood during injury is to prevent excessive bleeding and reduce blood loss by forming a clot at the site of injury. Asthma is a chronic inflammatory disease in which abnormalities in coagulation and fibrinolysis feature prominently among multiple mechanisms that have been implicated in the pathogenesis of asthma.
Coagulation occurs through a cascade, involving the interactions of various blood components leading to the conversion of individual molecules of the soluble protein fibrinogen into insoluble fibrin fibres in a complex network of fibrin, erythrocytes and platelets. Blood components such as platelets, erythrocytes and fibrin networks are sensitive to inflammation, and changes in these components can influence the coagulation process. Therefore the coagulation profile and the resultant clot as well as assessment of the blood components can be used as indicators of disease status of an individual as well as progression and severity of disease. Changes in the structure and function of blood components manifest in many inflammatory related diseases such as alzheimer‟s disease, parkinson‟s disease, lupus, thrombotic diseases, rheumatoid arthritis and human immunodeficiency virus infection and acquired immunodeficiency syndrome (HIV/AIDS). Pathological changes in erythrocytes, platelets and fibrin have been studied extensively, however, little is known about the altered coagulation system leading to morphological change of blood components in asthma.
The effect of inflammation is not isolated to platelets and erythrocytes but also influences non-cellular components such as fibrin formation. Fibrin is a key component of blood clots and several factors influences its formation through the mode of polymerisation and cross-linking, consequently influencing the architecture of the resulting fibrin network and stability and persistence of the blood clot. Coagulation factor XIII (FXIII) is a unique coagulation protein that stabilizes the clot by determining its mechanical strength or stiffness, thus affecting fibrinolysis and ultimately, thrombolysis. The role of FXIII has recently come under the spotlight drawing much interest among researchers who study haemostasis. Therefore this study also investigated the messenger ribonucleic acid (mRNA) levels of FXIII subunit A (FXIII-A) in circulation to assess the potential for de novo synthesis and influence on clot properties. Ultrastructural and mechanical properties of cellular blood components involved in the formation of clots have yet to be explored in asthma. The overarching aim of this study was therefore to study plasma, erythrocytes and platelets in allergic asthma, as little is known about how inflammation affects these formed elements in atopic asthma. To the best of our knowledge this is the first study to evaluate the ultrastructural properties of blood, platelets and fibrin in allergic asthma patients not only in a South African cohort, but globally. Asthma is an incurable disease but its symptoms can be controlled with proper treatment. Therefore, this research can provide insight on the pathophysiology of the disease, which can shed light towards additional treatment options to support the available therapies.

TABLE OF CONTENTS :

• Description Page
• DECLARATION
• DEDICATION
• ACKNOWLEDGEMENTS
• PUBLICATIONS
• PRESENTATIONS
• ABSTRACT
• LIST OF ABBREVIATIONS
• LIST OF FIGURES
• LIST OF TABLES
• CHAPTER 1 INTRODUCTION
• CHAPTER 2 LITERATURE REVIEW
  • 2.1 Overview of Asthma
  • 2.2 Epidemiology of Asthma
  • 2.3 Classification of Asthma
  • 2.4 Genetic/Hereditary factors
  • 2.5 Non-genetic (environmental and behavioural) risk factors
  • 2.6 Inflammation
  • 2.7 Specific pro-inflammatory mediators in asthma
  • 2.8 Haemostasis
  • 2.9 Endothelium
  • 2.10 Coagulation proteins
  • 2.11 Coagulation
    • 2.11.1 Phase 1: Initiation
    • 2.11.2 Phase 2: Amplification
    • 2.11.3 Phase 3: Propagation
    • 2.11.4 Phase 4: Stabilization
  • 2.12 Fibrinolysis
  • 2.13 Altered haemostasis in allergic asthma
  • 2.14 Role of platelets in Asthma
  • 2.15 Role of fibrin and factor XIII in asthma
  • 2.16 Role of erythrocytes in asthma
• CHAPTER 3 PATIENTS DEMOGRAPHICS AND DESCRIPTIVE ANALYSIS OF HAEMATOLOGY AND MATERIALS AND METHODS
  • 3 Introduction
  • 3.1 Study Design
    • 3.1.1 Patient recruitment
    • 3.1.2 Blood Sample Collection
  • 3.2 Patient demographics and biochemical parameters
  • 3.3 Haematology analysis
  • 3.4 Red blood cell indices
    • 3.4.1 Haemoglobin
    • 3.4.2 Erythrocyte count
    • 3.4.3 Haematocrit
    • 3.3.4 White blood cell count
    • 3.3.5 Platelet Count
  • 3.4 Experiments
    • 3.4.1 Preparation of erythrocytes sample for Light Microscopy
      • 3.4.2.1 Preparation of red blood cells for Atomic Force Microscopy
      • 3.4.2.2 AFM measurements for membrane deformability of cells
      • 3.4.2.3 Statistical Analysis of AFM data
• 3.5 Thromboelastography
• 3.6 Preparation of fibrin clot for scanning electron microscopy
• 3.7 Preparation of platelets for Transmission Electron Microscopy
• 3.8 Preparation of platelets for Confocal Microscopy
• 3.9 Quantitative Polymerase Chain Reaction
  • 3.9.1 Extraction of RNA
  • 3.9.2 cDNA synthesis and amplification of FXIII-A mRNA
• 3.10 Statistical Analysis
• CHAPTER 4 ANALYSIS OF THE FORMED ELEMENTS IN BLOOD: ERYTHROCYTES AND PLATELETS
  • 4. Introduction
  • 4.1 Erythrocytes
    • 4.1.1 Light microscopy and axial ratio
    • 4.1.2 Atomic force microscopy on erythrocyte membrane elasticity
    • 4.1.3 Scanning electron microscopy analysis of erythrocytes
  • 4.2 Platelets
    • 4.2.1 Scanning electron
    • 4.2.2 Transmission electron microscopy
    • 4.2.3 Confocal microscopy
  • 4.3 Discussion
    • 4.3.1 Erythrocyte morphological changes observed
    • 4.3.2 Platelet morphology and activation
    • CHAPTER 5 KINETICS OF COAGULATION, ULTRASTRUCTURE OF FIBRIN AND FACTOR XIII-A MRNA ANALYSIS
      • 5. Introduction
      • 5.1 Thromboelastography assessment of coagulation profile
      • 5.2 Ultrastructural changes of fibrin fibres and factor XIII mRNA levels
      • 5.2.1 Scanning electron microscopy
      • 5.3 Quantitative polymerase chain reaction
      • 5.4 Discussion
        • 5.4.1 Coagulation profiles
        • 5.4.2 Fibrin and factor XIII
      • 6. CONCLUDING DISCUSSION
      • REFERENCES
      • APPENDIX 1 Ethical approval
      • APPENDIX 2 Statistical approval
      • APPENDIX 3 PhD approval
      • APPENDIX 4 Accepted abstract to International Conference
      • APPENDIX 5 Proof of submission of Research Article
      • APPENDIX 6 Informed consent template for controls
      • APPENDIX 7 Informed consent template for asthma patients

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