Noninvasive biomarkers for early smoking related lung disease

9 INTRODUCTION.11 REVIEW OF THE LITERATURE.14 1. Definitions and classification.14 2. Epidemiology and aetiology of COPD.16 3. Pathogenesis of COPD.18 3.1. Inflammation in the lung in COPD.18 3.2. Inflammatory cells in COPD.19 3.2.1. Neutrophils.19 3.2.2. Macrophages.20 3.2.3. Lymphocytes.21 3.2.4. Eosinophils.22 3.3. Biomarkers/mediators in COPD.22 3.3.1. Oxidative and nitrosative stress markers in COPD.22 3.3.2. Matrix metalloproteinases in COPD.28 3.3.3. Surfactant protein-A and -D in COPD.35 4. Diagnosis of COPD.36 5.Treatment strategies for COPD.37 6. Prognosis of COPD.38 AIMS OF THE STUDY.40 MATERIALS AND METHODS.41 1. Subjects.41 2. Samples and data collection.44 5 2.1. Pulmonary function tests.44 2.2. Sputum induction and processing.44 2.3. Plasma specimens.45 2.4. NO measurements (Study I) . 45 3. Studies conducted on the sputum cells, supernatants and plasma samples.46 3.1. Enzyme-Linked Immunosorbent Assay (ELISA)/Enzyme Immunoassay (EIA) (Studies I, II, III, IV, V) . 46 3.2. Immunocytochemistry for sputum cells (Studies I, III) . 46 3.3. Western blot analysis (Study III) . 47 3.4. Gelatinase assay (Study III) . 48 3.5. Measurement of serine proteinase activity (Study IV) . 48 3.6. MMP-8 immunofluorometric assay (Study IV) . 49 4. Statistical analysis.49 RESULTS.51 1. Characteristics of subjects.51 2. Cell profile.54 3. Oxidative/nitrosative stress in smokers.56 3.1. Markers of increased oxidative stress in smokers (Study I) . 56 3.1.1. The expression of oxidative stress markers in induced sputum of non-smokers and smokers.56 3.1.2. ECP, lactoferrin and FENO in smokers.57 3.1. 3. Correlations between oxidative stress markers, inflammatory cell profile and lung function values.58 3.2. 8-Isoprostane as a marker of oxidative stress (Study II) . 58 4. Matrix metalloproteinases as non-invasive markers for chronic obstructive pulmonary disease.59 4.1. Matrix metalloproteinases in smokers and early stages of COPD (GOLD Stage 0) (Study III) . 59 4.1.1. Increased sputum and plasma levels of matrix metalloproteinases in nonsymptomatic and symptomatic chronic smokers (Stage 0 COPD) . 60 4.1.2. Immunocytochemistry of MMPs and TIMP-1 in induced sputum cytospins.61 4.1. 3. MMP-9 activity by zymography and Western blotting.61 4.1. 4. Correlations of MMPs and TIMP-1.62 6 4.2. Neutrophil proteinases in COPD exacerbation (Study IV) . 63 4.2.1. Neutrophil elastase, MMP-8 and -9 in sputum samples.63 4.2.2. Neutrophil proteinases correlated with sputum cells and lung function parameters.65 5. Age and smoking affect plasma biomarkers (Study V). 65 5.1. Surfactant protein A.65 5.2. Surfactant protein D . 66 5.3. Matrix metalloproteinase -9 . 66 5.4. TIMP-1 and MMP-9/TIMP-1.66 5.5. SP-A may be a promising marker for COPD.67 DISCUSSION.68 1. The potential markers of COPD and their significance in the early diagnosis of this disease (Studies I, II, and III). 69 1.1. The expression of oxidative stress markers in symptomatic and non-symptomatic smokers.69 1.2. Matrix metalloproteinases as specific markers of early COPD.73 2. Changes in inflammatory profile and levels of proteases during COPD exacerbation and its recovery period (Study IV). 76 3. Age related alterations and value of plasma surfactant proteins (SP) and MMP-9 in healthy non-smokers and smokers (Study V). 77 CONCLUSIONS.80 ACKNOWLEDGEMENTS.82 REFERENCES.85