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THE NUMBER OF NEUTROPHIL IN SPUTUM INDUCTION OF ASYMPTOMATIC SMOKERS AND SMOKERS WITH PULMONARY EMPHYSEMA BASED ON RADIOLOGIC FINDINGS

FINAL ASSIGNMENT
To fulfill the requirements for Degree of Bachelor of Medicine

By : Uthaya Kumar Nallayan NIM : 0810714039

MEDICAL PROGRAMME FACULTY OF MEDICINE UNIVERSITY OF BRAWIJAYA MALANG 2011

CERTIFICATION PAGE FINAL PROJECT

THE NUMBER OF NEUTROPHIL IN SPUTUM INDUCTION OF ASYMPTOMATIC SMOKERS AND SMOKERS WITH PULMONARY EMPHYSEMA BASED ON RADIOLOGIC FINDINGS
By: UTHAYA KUMAR NALLAYAN SRN : 0810714039 Has been examined on: Day: Friday Date: 20 January 2012 and declared to pass by:

Examiner I,

Dr.dr.Retty Ratnawati,M.Sc NIP: 19550201 198503 2 001

Examiner II / Supervisor I,

Examiner III / Supervisor II,

dr.Triwahju Astuti Sp,P,MKes NIP: 19632210 199601 2001

dr.Maimun ZulhaidahA,Mkes,SpPK

NIP: 19700526 199702 2005

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ACKNOWLEDGEMENT
First of all, I would like to thank God for blessing me to finish up my Final Assignment to fulfill the precondition to achieve Medical Degree in Medical Faculty, Brawijaya University. My title of final assignment is THE NUMBER OF NEUTROPHIL IN SPUTUM INDUCTION OF ASYMPTOMATIC SMOKERS AND SMOKERS WITH PULMONARY EMPHYSEMA BASED ON RADIOLOGIC FINDINGS. Taking this opportunity, I would like to thank everyone whom always gives me support and encouragement throughout my Final Assignment. I would like to thank : 1. Dr.dr Karyono Mintaroem, SpPA, as the Dean of Medical Faculty,

Brawijaya University for providing the facilities in Medical Faculty, Brawijaya University. 2. dr.Triwahju Astuti Sp,P,MKes as my first facilitator who spent her

precious time despite of her busy schedule helping and always supporting, advising and correcting to make my research better. 3. dr.Maimun Zulhaidah A,Mkes,SpPK as my second facilitator who is full

of graciousness and willing to spend precious time for my final assignment and providing necessary corrections. 4. My examiner, Dr.dr.Retty Ratnawati,M.Sc. who made me think out of the box with her interesting questions and ideas relating to my thesis, and examining my research with a smile. 5. My research advisor, dr Andreas Infianto, for helping and correcting my mistakes despite of his busy schedule. 6. All the staffs in Respiratory Department and Pathology Clinic Laboratory of Saiful Anwar who really helped a lot.

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7.

My wonderful group mate, Kaviprathaa for always being there for me and lending her help in completion of this thesis.

8. My family for their support, undying love and sacrifices. My heartfelt gratitude goes to my parents Nallayan and Kaliammal as well as my three siblings, Suresh Kumar, Balasubramaniam, Thunesh Kumar. 9. My dearest brother, Thunesh for the constant support and encouragement through the thick and thin of my research. 10. All my friends whom never failed to lend a helping hand when I needed them the most. 11. Final Assignment Team. 12. Those whom helped me directly and indirectly in completing this study.

Last but not least, I hope that my research will provide a great beneficial contribution to society in the future. To accomplish that, I need critics and comment from everyone who read my final assignment. Thank you very much.

Malang, February, 2012

Uthaya Kumar Nallayan

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ABSTRAK Nallayan, Uthaya Kumar. 2011. Perbedaan jumlah neutrofil dalam induksi sputum perokok non symptomatis dan perokok emfisema berdasarkan gambaran radiologis.Tugas akhir Fakultas Kedokteran Universitas Brawijaya. Pembimbing: (1) dr.Triwahyu Astuti Sp,P.MKes (2) dr.Maimun Zulhaidah A,Mkes,SpPK Rokok dengan kandungan radikal bebasnya dapat menyebabkan berbagai kerusakan di paru dan saluran nafas. Keadaan inflamasi yang terus menerus dapat berpengaruh pada keseimbangan neutrofil di alveoli pada perokok dan seterusnya dapat melandasi patogenesis terjadinya emfisema paru. Penelitian ini bertujuan untuk menentukan jumlah neutrophil dalam sputum pada perokok non simptomatis dan perokok dengan emfisema paru yang dipilih dengan menggunakan gambaran radiologis.Desain penelitian adalah Observational Cross Sectional dilakukan secara in vivo pada manusia. Terdapat 4 kelompok yang masing-masing terdiri dari 10 subyek yang dibahagi menjadi perokok ringan, perokok sedang, perokok berat dan untuk perokok simptomatis peserta yang dipilih adalah perokok berat dengan emfisema paru berdasarkan gambaran radiologis. Pada setiap subyek dicatat data klinisnya (darah lengkap, EKG, foto thoraks, spirometri) dan diambil sputum dan sampel darahnya sebanyak 5 ml untuk mengetahui kondisi badan peserta. Hasil penelitian menunjukkan bahawa hanya kelompok perokok simptomatis dengan emfisema paru yang memberi hasil signifikan (p<0.05) dan rerata jumlah neutrofil dalam kelompok lain tidak memberikan perbedaan yang bermakna. Di samping itu, berdasarkan Pearson test menunjukkan bahawa derajat merokok tidak mempengaruhi jumlah neutrofil pada sputum induksi perokok. Oleh yang demikian, kesimpulan daripada penelitian ini adalah, merokok akan meningkatkan jumlah neutrophil di alveolar tetapi derajat merokok tidak mempengaruhi jumlah neutrophil di alveolar yang ada di dalam sputum seseorang perokok. Kata kunci : Rokok, neutrofil di alveolar, emfisema paru

ABSTRACT Nallayan, Uthaya Kumar. 2011.The Number of Neutrophil in Sputum induction of Asymptomatic Smokers And Smokers With Pulmonary Emphysema Based on Radiologic Findings Final assignment Fakultas Kedokteran Universitas Brawijaya. Pembimbing: (1) dr.Triwahyu Astuti Sp,P.MKes (2) dr.Maimun Zulhaidah A,Mkes,SpPK. Cigarette contains various substances and free radicals that may be harmful to the smoker. Continous and progressive state of inflammation cause the recruitment of Neutrophil in alveolar in the smoker and this induces the pathogenesis of the pulmonary emphysema in the smoker.The study was aimed to determine the correlation of smoking with the number of neutrophil in alveolar that are recruited in the smokers. This study was a Cross Sectional Observational study which was carried out in vivo in humans. There were four groups each consisting of 10 patients where the asymptomatic smokers are grouped into three different groups which were classified as mild, moderate and severe smoker and the fourth group was the smoker with pulmonary emphysema based on radiologic findings. In each patient recorded the clinical data (complete blood count, ECG, chest X-ray, spirometry), sputum and 5ml of blood samples were taken. Based on the study, only the smoker with pulmonary emphysema group gave a significant result (p<0.05) whereas the other groups have an average distribution of neutrophil approximately the same. Despite of that, based on the Pearson test that was done to identify the correlation of smoking with the number of neutrophil, it revealed that, the stages of smoking are not proportional to the number of neurophil in the sputum induction of the smoker.Therefore, based on this study, it can be concluded that, smoking will increase the number of neutrophil in alveolar in the smoker but the stages of smoking has no effect on the number of neutrophil in alveolar from the sputum induction of the smoker.

Keywords: Cigarette, neutrophil in alveolar, pulmonary emphysema

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TABLE OF CONTENTS Pages Title ...................................................................................................................... i Certification page................................................................................................................ii Preface...............................................................................................................................iii Abstract ......................................................................................................................v Table of Contents..............................................................................................................vii List of Figures.....................................................................................................................x List of Tables......................................................................................................................xi List of Appendixes.............................................................................................................xii List of Abbreviation.xiii CHAPTER I INTRODUCTION 1.1 Background.............................................................................................................1 1.2 Statement of Study Problem...................................................................................4 1.3 Objectives of the Study...........................................................................................4 1.3.1 General Purpose............................................................................................4 1.3.2 Specific Purpose............................................................................................4 1.4 Significance of the Study........................................................................................5 CHAPTER II REVIEW OF RELATED LITERATURE 2.1 Smoking.................................................................................................................6 2.1.1 Smoker Classification...................................................................................6 2.1.2 The Composition of Cigarette......................................................................7 2.1.3 Factors that influence a person to smoke......................................................7 2.1.4 Complication of Smoking...............................................................................8 2.1.4.1 Chronic Diseases...............................................................................8 2.1.4.2 Chronic Obstructive Pulmonary Disease.........................................10 2.1.4.2.1 Emphysema......................................................................12 2.1.4.2.2 Chronic Bronchitis............................................................14

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2.1.5 Management of Smokers. .15 2.1.5.1 Drug Therapy...15 2.1.5.2 Non Drug Therapy ..18 2.1.5.3 Combination Therapy.....18 2.2 Neutrophils.19 2.2.1 Introduction..19 2.2.2 Classification20 2.2.3 Mechanism of Production and Regulation.21 2.2.4 Biological Function of Neutrophil24 2.2.5 The Role of Neutrophil In Pulmonary Emphysema..28 CHAPTER III CONCEPTUAL FRAMEWORK AND HYPOTHESIS 3.1 Conceptual Framework........................................................................................31 3.2 Hypothesis of the Study.......................................................................................32 CHAPTER IV METHODOLOGY 4.1 Study Design........................................................................................................33 4.2 Population and Sample of Study..........................................................................33 4.2.1 Population....................................................................................................33 4.2.2 Sample Size.................................................................................................33 4.2.3 Sample Size Estimation...............................................................................34 4.2.4 Sample Statistic...........................................................................................35 4.3 Location and time of study...................................................................................35 4.3.1 Place of Study..............................................................................................35 4.3.2 Time of Study...............................................................................................35 4.4 Variable................................................................................................................35 4.4.1 Dependent Variable....................................................................................35 4.4.2 Independent Variable.................................................................................36 4.5 Inclusion and Exclusion criteria of the Study.......................................................36 4.6 Operational Definition...........................................................................................37 4.7 Instruments...........................................................................................................38 4.7.1 Studies Tools and Substances..................................................................38 4.8 Study Work Plan...................................................................................................39 4.8.1 Sputum Induction.......................................................................................39 viii

4.8.2 Sputum Decontamination and Centrifugation.............................................40 4.8.3 Neutrophil Counting....................................................................................40 4.9 Study Framework.................................................................................................42 CHAPTER V RESEARCH RESULT 5.1 Subject and Study Location.................................................................................43 5.2 Characteristic of the Subject................................................................................43 5.3 Characteristic Of Supportive Examination Data of Subject.................................45 5.4 Neutrophil Counting in the sputum induction of the Subjects...............................46 5.5 Data Analysis........................................................................................................47 5.5.1 Normality Test..............................................................................................47 5.5.2 Neutrophils Comparative hypothesis with using Mann Whitney U Test.......47 5.5.3 One way Anova Test....................................................................................48 5.5.4 Correlation Test of Different Stages of Smoking..........................................48 CHAPTER VI DISCUSSION 6.1 Characteristic of Study Subjects...........................................................................50 6.2 Neutrophils Counting in the Sputum Induction......................................................51 6.3 The Correlation between smoking stages and number of Neutrophils.................52 6.4 The Weakness of The Study.................................................................................53

CHAPTER VII CONCLUSION AND SUGGESTION 7.1 Conclusion............................................................................................................54 7.2 Suggestion............................................................................................................55 LIST OF REFERENCES..............................................................................................56 APPENDIXES...............59 STATEMENT OF ORIGINALITY..................................66

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LIST OF FIGURES Pages Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 3.1 The common adverse effect of smoking....10 A thin section of lung tissue stained with hematoxylin and eosin.. 13 Histopathology Sample of chronic bronchitis15 Bacterial phagocytosis and destruction by a neutrophil 28 Pathogenesis of emphysema and smokers .........................................30 Conceptual Framework of Pathogenesis of emphysema and association of Neutrophil31

LIST OF TABLES

Pages Table 5.1 Characteristics of study subjects in each group...........................................44 Table 5.2 Characteristics Of Supportive Examination Data of Subject........................46

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LIST OF APPENDIXES Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Test of Normality...59 Test of Homogeneity of Variances60 One Way Anova Analysis60 Mann-Whitney Test...61 t-Test62 Correlations63 Raw Data.64

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List of Abberviation

SGOT SGPT

: :

Serum Glutamic Oxaloacetic Transaminase Serum Glutamate Pyruvate Transaminase Analysis Of Variance

ANOVA :

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CHAPTER 1 INTRODUCTION 1.1. Background Tobacco use is responsible for more than 5 million deaths per year and is the leading preventable cause of premature death worldwide. Tobacco companies have gradually shifted their market from high-income to low-income countries, where many people are poorly informed about the health risks of tobacco use and anti-smoking policy is relatively weak.Few examples of such countries are Indonesia,India,and Vietnam (Charlesworth et al., 2010). The smoking exacerbates the effects of poverty, as expenditures for tobacco may divert household income from food, clothing, housing, health and education. The amount of money spent on tobacco is especially problematic in low-income countries. For example, in Vietnam in 1996, smokers spent an average of $US 49.05 on cigarettes per year, which was 1.5 times that spent on education, ve times that spent on health care and about one-third that spent on food per capita in the household each year. In the poorest households in Indonesia, more money was spent on tobacco than on education and health care combined. Indonesia is the fth largest market for tobacco in the world, with 182 billion sticks consumed per year. The absolute domestic consumption of tobacco increased by 159% between 1970 and 1980, coincident with the mechanisation of the cigarette industry in Indonesia in the early 1970s (Semba et al., 2006).

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According to the World Health Organization (WHO) Tobacco Atlas of the year 2004,, smoking can be classified into few lucid types such as manufactured cigarettes consist of shredded or reconstituted tobacco processed with hundreds of chemical, Bidis consist of a small amount of tobacco, hand-wrapped in dried temburni leaf and tied with string ,Cigars are made of air-cured and fermented tobaccos with a tobacco wrapper, and come in many shapes and sizes, from cigarettesized cigarillos, double coronas, cheroots, stumpen, chuttas and dhumtis. Kreteks are clove-flavoured cigarettes. Pipes are made of briar, slate, clay or other substance and tobacco is placed in the bowl and inhaled through the cigarettes, sometimes through water and lastly sticks are made from sun-cured tobacco known as brus and wrapped in cigarette paper. Smoking is known to have a major impact on human health, adversely affecting almost every organ. Exposure to cigarette smoke increases the risk of many diseases, including a wide range of cancers (from lung to pancreatic cancer), cardiovascular diseases (including atherosclerosis and coronary heart disease), a range of respiratory diseases (including chronic obstructive pulmonary disease and pneumonia), as well as various other adverse health effects such as increased risk of cataracts, infection and poor wound healing, and is generally detrimental to the overall health of individuals who smoke (Semba et al., 2006). Emphysema is defined pathologically as an abnormal permanent enlargement of air spaces distal to the terminal bronchioles, accompanied by the destruction of alveolar walls and without obvious fibrosis. Inflammatory response is normally amplified in emphysema The inflammation is further amplified by oxidative stress and protease production and this suggest the increase of macrophages in smokers. Oxidants are produced from cigarette smoke whereas proteases are produced by macrophages. This xv

leads to a protease-antiprotease imbalance that leads to destruction of elastin and other structural elements. This is believed to be central in the development of emphysema (Demirjian, 2011). Neutrophils play a major role in defense mechanism in a persons immune response. Neutrophils are considered to be central to the pathogenesis of most forms of acute lung injury (ALI). For the sake of clarity, neutrophil involvement in ALI can be conceptualized as consisting of sequential stages, beginning with their sequestration in the pulmonary microvasculature, followed by adhesion and activation, and culminating in the production of a microbicidal or effector response, such as the generation of reactive oxygen species or release of proteolytic enzymes. Great strides have been made in elucidating these various stages of neutrophil involvement. Recent studies have focused on the intracellular signaling pathways that govern neutrophil activation and have elucidated complex cascades of kinases and other intracellular signaling molecules that allow for amplication of the neutrophil response, yet simultaneously confer specificity of a response. Inflammatory response in ALI may initially be adaptive, such as the pivotal role played by neutrophils in a bacterial or fungal infection. Ultimately, it is the persistence or the dysregulation of neutrophil activation that may lead to ALI (Lee and Downey, 2001). Smoking tobacco has done a vast majority of harmful effect to the mankind. Research about smoking and adverse effect of smoking should be encouraged and done more frequently from now on because it may save millions of lives. I am very intrested to be a part of the research which might help to find the corelation of smoking and adverse effect which will be useful in curing and decreasing the death toll because of the smoking.

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1.2.1

Statement of Study Problem The problems to be solved in this study are,

1) Does smoking increase the number of neutrophil in alveolar of the smokers? 2) Are there any differences in the number of neutrophil in alveolar according to different stages of smokers? 3) Are there any differences in the number neutrophil in alveolar of smoker with pulmonary emphysema and smoker without pulmonary emphysema? 1.3.1 1.3.2 Objective of the Study General purpose Generally, this study is conducted to determine the influence of smoking towards the number of neutrophil in alveolar of the smokers. 1.3.3 Specific purpose 1. To determine the number of neutrophils in mild, moderate and severe smokers. 2. To find out the difference in neutrophils number in pulmonary emphysema smoker and non-pulmonary emphysema smoker. xvii

3. To determine the correlation of stages of smoking to the body mass index (BMI) of the smokers. 4. To determine the correlation of stages of smoking to the age of the smokers. 1.4 Significance of the Study Benefits of this study are: 1. Can be used to give information to the smokers about the risk factor of smoking to the development of emphysema and eventually can promote smoking cessation to the smokers. 2. Can be used to study the long term effect of smoking in chronic obstructive pulmonary disease such as, emphysema and chronic bronchitis. 3. Can be used to compare the difference in neutrophil count in smoker with pulmonary emphysema and smoker without pulmonary emphysema.

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CHAPTER 2 LITERATURE REVIEW 2.1 Smoking

2.1.1 Smoker Classification Smokers can be classified according to the Brinkman index (BI), which is defined as numbers of cigarette smoked per day times smoking years which can be categorized into mild,moderate and severe smokers. A mild smoker has Brinkman Index value of 1 to 199, moderate smokers with a value of 200 to 399 and severe smokers with a value of 400-599 (Kume et al., 2009). Besides that, smoker also can be classified according to Indrayans smoking index where it is measured according to the number of cigarretes per day and duration

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of smoking. A persons amount of smoking may also vary from time to time. A measure could be the total number of cigarettes smoked so far in life. This number is given by S1 = n1x1 + n2x2 + ... +nK xK, where nk (k = 1, 2, , K) cigarettes per day (intensity) are smoked for xk years (duration). This is more exact than pack-years generally used for smoking. For an example,S1 suffers from the same demerit as the pack-years, namely that smoking 10 cigarettes a day for 25 years is the same as smoking 25 cigarettes per day for 10 years (Indrayan, 2008).

2.1.2

The Composition of Cigarette The tar, nicotine, and carbon monoxide content of cigarettes varies markedly

according to the different brands and types of cigarettes. For example, carbon monoxide content in cigarettes can vary from less than 0.05 to 3.0 mg per cigarette. This difference would affect the intravascular levels of carbon monoxide and carboxyhemoglobin and would therefore affect the degree of any presumed pathophysiologic effect on the arterial walls but, this effect is difficult to quantify, since tar, carbon monoxide, and nicotine levels determined by the amount of smoke that the smoker takes in. Therefore it depends on how they smoke their cigarettes. The amount of tar and nicotine a smoker actually gets can also increase if the smoker blocks tiny ventilation holes in cigarette filters that are designed to dilute smoke with air. In addition, many smokers of low tar or light cigarettes compensate by taking deeper,

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longer, or more frequent puffs from their cigarettes and causes more harm to the smokers eventually (Schillinger et al., 2004). 2.1.3 Factors that influence the person to smoke There are many reason for a person to smoke, but the main reason a person tend to smoke throughout his life is because of dependency or in other word, addiction. The cigarette-dependence process, like other pathogenically induced diseases, is influenced by host or individual factors, environmental factors, and the level of exposure to the pathogen. Initiation is often mediated by a variety of social and cultural factors. However, over time the reinforcing effects of the drug strengthen and the individual's control over use weakens. Although other factors continue to operate, cigarette dependence is powerfully and critically driven by the positively and negatively reinforcing effects of nicotine .Like other drug dependencies, nicotine dependence is a "progressive," "chronic," "relapsing" disorder. Mean age of cigarette smoking onset is 13-14 years. The level of nicotine dependence in adults is inversely related to the age of smoking initiation according to the diagnostic criteria of the American Psychiatric Association (Henningfield et al., 1999). According to the WHO Tobacco Atlas year 2002, the teenage smoker, in contrast to the middle aged chronic smoker,experiences primarily the pleasant effects of smoking. In fact,impairment of pulmonary function can be demonstrated in teenage smokers but only by rather subtle tests . By middle age and older these differences are more pronounced .The pathological changes induced by smoking in various parts of the body slowly accumulate over time measured in years and decades and if death occurs in middle age or later these can be fairly easily identified by the pathologist. Every child

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should know that smokers lungs are darker than non-smokers because smoke residues accumulate there. 2.1.4 Complication of smoking

2.1.4.1 Chronic Diseases Several components in tobacco smoke contribute to its cardiovascular harm. Substances such as carbon monoxide reduce the oxygen carrying capacity of red blood cells, thus forcing the circulatory system to increase its efforts to deliver needed oxygen to all cells of the body while also predisposing the heart to rhythm disturbances. Oxidizing chemicals, such as polycyclic aromatic hydrocarbons, cause inflammation and can lead to atherosclerosis. These same oxidizing chemicalscan cause endothelial dysfunction and promote vascular damage. Other toxins in tobacco smoke are thrombogenic and can increase platelet adhesiveness, predisposing to clot formation within the vessel. Nicotine itself has some modest physiologic effects on pulse, blood pressure, and vascular tone. However, these are mild in comparison with the other cardiovascular effects of the numerous other toxins . However, despite the degree to which smoking leads to coronary artery disease and cerebrovascular disease, cessation treatment is still not widely implemented (Burton et al., 2007). Besides that,smoking also has high relation with kidney disease. Cigarette smoking has been reported to exacerbate existing diabetic and non-diabetic kidney disease and may also be an etiologic factor triggering the onset of proteinuria and reduced renal function. Despite the recognized relationship between cigarette smoking and kidney disease, it is unclear whether smoking cessation or reduction can attenuate the progression of renal injury in CKD (Burton et al., 2007).

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Figure 2.1 Common adverse effect of smoking (Morrow, 2007). 2.1.4.2 Chronic Obstructive Pulmonary Disease ( COPD ) Chronic obstructive pulmonary disease (COPD) is a leading cause of disability and death worldwide. Many research and studies have indicated that long term use of smoking is highly related to serious respiratory complications such as chronic obstructive pulmonary disease ( COPD ). Chronic obstructive pulmonary disease (COPD) is estimated to affect 32 million persons in the United States and is the fourth leading cause of death in this country (Vogelmeier et al., 2011). Patients typically have symptoms of chronic bronchitis and emphysema, but the classic triad also includes asthma which reversible compared to the first two diseases mentioned earlier which cannot be reversed. Exacerbations of COPD indicate

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instability or worsening of the patients clinical status and progression of the disease and have been associated with the development of complications, an increased risk of subsequent exacerbations, a worsening of coexisting conditions, reduced health status and physical activity, deterioration of lung function, and an increased risk of death.The prevention of exacerbations therefore constitutes a major goal of treatment (Mosenifar, 2011). Pathophysiology of the development of COPD occurs in the large (central) airways, the small (peripheral) bronchioles, and the lung parenchyma. The normal inflammatory response is amplified in persons prone to COPD development. The pathogenic mechanisms are not clear but are most likely diverse. Increased numbers of activated polymorphonuclear leukocytes and macrophages release elastases in a manner that cannot be counteracted effectively by antiproteases, resulting in lung destruction. Increased oxidative stress caused by free radicals in cigarette smoke, the oxidants released by phagocytes, and polymorphonuclear leukocytes all may lead to apoptosis or necrosis of exposed cells. Accelerated aging and autoimmune mechanisms have also been proposed as having roles in the pathogenesis of COPD. Cigarette smoke causes neutrophil influx, which is required for the secretion of MMPs; this suggests, therefore, that neutrophils and macrophages are required for the development of emphysema (Mosenifar, 2011). Most patients with chronic obstructive pulmonary disease (COPD) seek medical attention late in the course of their disease. Patients often ignore the symptoms because they start gradually and progress over the course of years. Patients often modify their lifestyle to minimize dyspnea and ignore cough and sputum production. With retroactive questioning, a multiyear history can be elicited.Patients typically present with a combination of signs and symptoms of chronic bronchitis, emphysema, xxiv

and reactive airway disease. These include cough, worsening dyspnea, progressive exercise intolerance, sputum production, and alteration in mental status. Symptoms of COPD are such as productive cough or acute chest illness, breathlessness, wheezing. Systemic manifestations (decreased fat-free mass, impaired systemic muscle function, osteoporosis, anemia, depression, pulmonary hypertension, cor pulmonale, left-sided heart failure. A productive cough or an acute chest illness is common. The cough usually is worse in the mornings and produces a small amount of colorless sputum (Mosenifar, 2011). 2.1.4.2.1 Emphysema Emphyesema is defined as a chronic disease characterized by destruction of the alveolar walls, with subsequent abnormal permanent enlargement of the respiratory air spaces. Progressive breakdown of elastin in the lung parenchyma is a key feature in the pathogenesis of emphysema. The major known cause of emphysema is cigarette smoking, but the initiating elastinolytic factor in smoking-induced emphysema is still not clear. The other known cause of emphysema is a genetic deciency of a1-proteinase inhibitor, an inhibitor of neutrophil elastase. In a1-proteinase inhibitor deciencyassociated emphysema, lung elastin breakdown is undoubtedly triggered by the unopposed action of neutrophil elastase due to the insufficient levels of its inhibitor in the bronchoalveolar epithelial lining . In cigarette smoking-associated emphysema, there is debate whether increased macrophage and/or neutrophil elastinolytic activity within the alveolar matrix, resulting from a smoking-induced accumulation of macrophages and neutrophils in the lung, may be responsible for the elastinolytic damage (Foronjy et al., 2010).

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The 3 described morphological types of emphysema are centriacinar, panacinar, and paraseptal. Centriacinar emphysema begins in the respiratory bronchioles and spreads peripherally. Also termed centrilobular emphysema, this form is associated with long-standing cigarette smoking and predominantly involves the upper half of the lungs. Panacinar emphysema destroys the entire alveolus uniformly and is predominant in the lower half of the lungs. Panacinar emphysema generally is observed in patients with homozygous alpha1-antitrypsin (AAT) deficiency. In people who smoke, focal panacinar emphysema at the lung bases may accompany centriacinar emphysema. Paraseptal emphysema, also known as distal acinar emphysema, preferentially involves the distal airway structures, alveolar ducts, and alveolar sacs. The process is localized around the septae of the lungs or pleura. Although airflow frequently is preserved, the apical bullae may lead to spontaneous pneumothorax. Giant bullae occasionally cause severe compression of adjacent lung tissue (Demirjian, 2011).

Figure 2.2 A thin section of lung tissue stained with hematoxylin and eosin The individual suffers from emphysema (Demirjian, 2011).

2.1.4.2.2 Chronic Bronchitis

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Chronic bronchitis is defined clinically as cough with sputum expectoration for at least 3 months a year during a period of 2 consecutive years. Chronic bronchitis is associated with hypertrophy of the mucus-producing glands found in the mucosa of large cartilaginous airways. As the disease advances, progressive airflow limitation occurs, usually in association with pathologic changes of emphysema. This condition is called chronic obstructive pulmonary disease. Chronic bronchitis is a clinical syndrome defined by chronic sputum production,and it is associated with periodic exacerbations in which the patient experiences a worsening of respiratory symptoms (Saetta et al., 1994). Chronic bronchitis is associated with excessive tracheobronchial mucus production sufficient to cause cough with expectoration for 3 or more months a year for at least 2 consecutive years. The alveolar epithelium is both the target and the initiator of inflammation in chronic bronchitis. A predominance of neutrophils and the peribronchial distribution of fibrotic changes result from the action of interleukin 8, colony-stimulating factors, and other chemotactic and proinflammatory cytokines. Airway epithelial cells release these inflammatory mediators in response to toxic, infectious, and inflammatory stimuli, in addition to decreased release of regulatory products such as angiotensin-converting enzyme or neutral endopeptidase. Chronic bronchitis can be categorized as simple chronic bronchitis, chronic mucopurulent bronchitis, or chronic bronchitis with obstruction. Mucoid sputum production characterizes simple chronic bronchitis. Persistent or recurrent purulent sputum production in the absence of localized suppurative disease, such as bronchiectasis, characterizes chronic mucopurulent bronchitis (Fayyaz, 2011).

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Figure

2.3

Histopathology sample taken from a chronic bronchitis patient showing goblet cell hyperplasia (Demirjian, 2011).

2.1.5 Management of smokers Smokers move through stages in relation to quitting: of precontemplation, contemplation, readiness then action, followed by maintenance or relapse. Many move through this cycle several times before they finally quit, while others report they found it easier to quit than they expected. These stages are influenced by increased costs from tax increases or reduction of smuggling, illness in the smoker, family or friends dying from tobacco, the media, health profession, bans on promotion, creation of smokefree areas and, while most smokers still quit on their own, availability of support and treatment. There are now techniques to assist those who want to quit smoking, although these are not available in all parts of the world: social support, clinics, quitlines, internet sites; skills training; nicotine replacement therapy (NRT) and other pharmaceutical treatments (Crapo et al., 2004). 2.1.5.1 Drug Therapy (Pharmacotherapy) Drug therapy can be further categorized into pharmacotherapy, which comprises four nicotinic group such as, nicotinic gum, inhaler, nasal spray and patch, whereas,

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non-nicotinic agent is Sustained- Release Bupropion( bupropion SR) (Crapo et al., 2004). Nicotinic gum, it is given in a flexible dosage according to specific cravings of the smokers. An individual who smokes 1 pack per day should use 4-mg pieces. The 2mg pieces are to be used by individuals who smoke less than 1 pack per day. Instruct the patient to chew hourly and also to chew when needed for their initial cravings for 2 weeks. Gradually reduce the amount chewed over the next 3 months. Proper information should be given about the usage of the gum such as avoid drinking liquids while chewing the gum and using gum within 15 minutes of drinking acidic beverages(e.g., cola, coffee, citrus juice) may reduce the effect by decreasing the absorption of the nicotine (Crapo et al., 2004). The nicotine inhaler, also nicknamed "the puffer" is a thin, plastic cartridge that contains a porous nicotine plug in its base. By puffing on the cartridge, nicotine vapor is extracted and absorbed through the lining of the mouth. Each cartridge delivers up to 400 puffs of nicotine vapor. It takes at least 80 puffs to obtain the equivalent amount of nicotine delivered by one cigarette. The inhaler mimics the habitual hand-to-mouth action but without combustion, the smoker is not exposed to carbon monoxide, tar or other carcinogens that can be found in tobacco smoke. Few reported side effects from the inhaler are,coughing,rhinitis,and local irritation of the mouth and throat (Crapo et al., 2004). The patch is a nicotine delivery system that was developed in part because of the difficulty in the patients to optimize the usage of nicotine gum. It is applied on the non hairy surface of the skin and absorbed readily through the skin and distributed throughout the body, reducing withdrawal symptoms and the craving for tobacco.

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Transdermal nicotine patches are available readily for replacement therapy. Long-term success rates are 22-42%, compared with 2-25% with a placebo. These agents are well tolerated, and the adverse effects are limited to localized skin reaction. Nicotine replacement therapy patches are sold under the following trade names such as NicoDerm, Nicotrol, and Habitrol. Each of these products is dosed with a scheduled graduated decrease in nicotine over 6-10 weeks (Crapo et al., 2004). The nicotine nasal spray is the strongest form of nicotine replacement therapy, which is particularly useful and effective for highly dependent heavy smokers who cannot give up by any other means. The reason that the nasal spray is so much more effective is because of its fast action. Once the nicotine has been administered, it enters the bloodstream and reaches the brain within 10 minutes. Other methods take much longer. This method also most realistically mimics the fast "hit" obtained when smoking a cigarette. This makes it much easier to control and satisfy cravings if they suddenly arise.However it has highest level of side effect compared to other nicotine therapies which about 94% of the users reporting nasal irritation of moderate to severe intensity during initial use (Crapo et al., 2004). Bupropion SR is the only nonnicotine pharmacotherapy to be approved by Food and Drug Administration (FDA) to date. This atypical antidepressant will block the reuptake of dopamine or norepinephrine in the brain.This therapy is initiated 1 to 2 weeks before the the quiting date and started with a dosage of 150mg in the morning for 3 days and then increased into 150mg twice a day for up to 3 months following the quit date.Bupropion is contraindicated to patients with seizure or eating disorder,who took monoamine oxidase inhibitor within the previous 14days.Commonly reported side effect are insomnia and dry mouth.It is also sometimes be beneficial in treating patients with depression history (Crapo et al., 2004). xxx

2.1.5.2 Non Drug Therapy There are a wide variety of non drug therapy can be given such as affect management, diet programs, hypnosis, acupuncture, social support, and sensory deprivation. Self-help materials such as pamphlets, manual , and audiotapes and videotapes were found to be a marginal effective method. Individual counseling from a smoking cessation specialist may help smokers to make a successful attempt to stop smoking by doing intra treatment such as communicating encouragement and concern to persons quitting while allowing them to speak openly about their experience and extra treatment social support, where, the clinician identify friends or family members who may be sources of support (Crapo et al., 2004). 2.1.5.3 Combination therapy The best therapy to those who motivated to make a serious quit attempt would be a combination of drug therapy and basic counseling which may give favorable result by increasing long term cessation to three to four times from what they would without a treatment (Crapo et al., 2004). 2.2 2.2.1 Neutrophils Introduction Neutrophil is a large numbers of polymorphonuclear neutrophil (PMN) granulocytes are rapidly recruited from the bloodstream to the site of infection or injury via transmigration through the vascular endothelium. Neutrophils constitute the rst line of defense and are considered as primary effector cells in infectioninduced acute inammatory reactions where they serve to destroy invading pathogens. Neutrophils are inherently short-lived cells with a half life of only z6-10 h in the circulation and

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rapidly undergo spontaneous apoptosis . In infected tissues their apoptosis can be delayed both by microbial constituents and by proin- ammatory stimuli . Finally, however, tissue neutrophils die in large numbers. Because uncontrolled release of toxic substances from dead neutrophils can propagate the inammatory response leading to tissue destruction, recognition of dying inammatory neutrophils has a critical function for the resolution of the in- ammatory response. It leads not only to the removal of the inammatory cells themselves, along with anything they have ingested, but also to the generation of anti-inammatory mediators that shut down the on going inammation (Esmann et al., 2010). Though neutrophils are short lived, with a half-life of four to ten hours when not activated and immediate death upon ingesting a pathogen, they are plentiful and responsible for the bulk of an immune response. They are the main component of pus and responsible for its whitish color. Neutrophils are present in the bloodstream until signaled to a site of infection by chemical cues in the body. They are fast acting, arriving at the site of infection within an hour (Esmann et al., 2010). Before ingesting invasive bacteria, neutrophils can release a net of fibers called a neutrophil extracellular trap (NET), which serves to trap and kill microbes outside of the cell. When neutrophils ingest microbes, they release a number of proteins in primary, secondary, and tertiary granules that help kill the bacteria. They also release superoxide, which becomes converted into hypochlorous acid, or chlorine bleach, which is theorized to play a part in killing microbes as well (Esmann et al., 2010). 2.2.2 Classification Neutrophil granulocytes are subdivided into segmented neutrophil(segs) and banded neutrophil (band). The segmented neutrophil term is derived from the xxxii

multilobed nature of the cells nucleus. Generally, neutrophil nuclei exhibit 3-5 segmented lobes (Witko et al., 2000). A second characteristic of functional importance is the large number of cytoplasmic granules. Three types of granules are present in the cytoplasm of neutrophils : Small, specific granules (0.1 m in diameter) Larger azurophilic granules (0.5 m in diameter) The newly discovered tertiary granules

Specific granules contain various enzyme and pharmacological enzymes and pharmacological agents tahat aid the neutrophil in performing its antimicrobial functions. Azurophilic granules are lysosomes, containing acid hydrolases, myeloperoxidase, the antibacterial agent lysozyme, bactericidal permeability increasing protein, cathepsin G, elastase, and nonspecific collagenase. Tertiary granules contain gelatinase and cathepsins as well as glycoproteins that are inserted into plasmalemma (Esmann et al., 2010). Besides that, like segmented neutrophil, the term banded neutrophil is derived from the cells characteristic nuclear staining. The nuclear material is in simple Ushaped pattern. The banded is an immature cell on the way to becoming a mature segmented neutrophil. When there is demand for neutrophils because of an infection or chronic cell damage, the bone marrow is stimulated to release its supply of mature(segmented) and some immature(banded) cells. Generally, in the presence of a long term bacterial infection or chronic tissue necrosis. The level of bands reported in CBC increases (Anderson, 1999). xxxiii

2.2.3

Mechanism of production and regulation Neutrophils are produced in the marrow, where they arise from progenitor and

precursor cells by a process of cellular proliferation and maturation. They differentiate from the pluripotential stem cell through a series of progressively more committed progenitor or colony forming units, including the granulocyte-monocyte colony forming unit and the granulocyte colony forming unit, which give rise to neutrophils. The early progenitor cells cannot be recognized under the microscope but can be identified by marrow culture. The earliest microscopically recognizable neutrophil precursor is the myeloblast. From there, the formal sequence of precursor development is myeloblast promyelocyte myelocyte metamyelocyte band neutrophil segmented neutrophil. The term granulocyte often is loosely used to refer to neutrophils but strictly speaking includes eosinophils and basophils. Eosinophilic and basophilic granulocytes develop from progenitors in a manner analogous to the neutrophils, although commitment to neutrophilic, eosinophilic, or basophilic development probably is established at an early progenitor stage (Williams, 2007). The normal human neutrophil production rate is 0.85 to 1.6 x 109 cells/kg/day. Mature neutrophils are stored in the marrow before they are released into the blood. They leave the circulation randomly, with a half-disappearance time of approximately 7 hours. The cells then enter the tissues and probably function for 1 or 2 days before their death or loss into the gastrointestinal tract through mucosal surfaces (Williams, 2007). The humoral regulators involved in granulopoiesis have been defined by in vitro culture systems. Originally identified by their ability to stimulate colony formation from marrow progenitor cells, the hemopoietins (cytokines) came to be called colony stimulating factors (CSF). With regard to neutrophil production, at least four human

xxxiv

CSFs have been defined. Granulocyte-monocyte colony stimulating factor (GM-CSF) is a 22,000 relative molecular mass (Mr) glycoprotein that stimulates the production of neutrophils, monocytes, and eosinophils. Granulocyte colony stimulating factor (G-CSF) has an Mr of 20,000 and stimulates only the production of neutrophils. Interleukin-3 (IL3), or multi-CSF, also has an Mr of 20,000 and acts relatively early in hematopoiesis, affecting pluripotential stem cells. Finally, stem cell factor (also known as c-kit ligand or steel factor), with an Mr of 28,000, acts in combination with IL-3 and/or GM-CSF to stimulate the proliferation of the early hematopoietic progenitor cells. In addition to their effects on neutrophil precursors, G-CSF and GM-CSF act directly on the neutrophil, enhancing its function. These cytokines regulate the production, survival, and functional activity of neutrophils. The mature neutrophil lacks IL-3 receptors and thus is not affected by IL-3. However, IL-3 receptors are present on mature eosinophils and monocytes. IL-3 is produced by activated T lymphocytes and thus is expected to have a physiologic role in circumstances of cell-mediated immunity. GM-CSF also is produced by activated lymphocytes. However, like G-CSF, it also is elaborated by mononuclear phagocytes and endothelial and mesenchymal cells when these cell types are stimulated by certain cytokines, including IL-1 and tumor necrosis factor, or bacterial products, such as endotoxin. Stem cell factor is secreted by a variety of cells, including marrow stromal cells, and affects the development of several kinds of tissues (Williams, 2007). The activities of exogenously administered biosynthetic (recombinant) human GCSF and GM-CSF in humans are well documented. G-CSF administration rapidly induces neutrophilia, whereas GM-CSF causes an increase in neutrophils, eosinophils, and monocytes. GM-CSF cannot be detected easily in normal plasma; thus, its role as a day-to-day, long-range modulator of neutrophil production is uncertain. Mice in which

xxxv

the GM-CSF gene is "knocked out" have generally normal hematopoiesis but show macrophage abnormalities, pulmonary alveolar proteinosis, and decreased resistance to microbial challenge. However, G-CSF appears to be a critical regulator of neutrophil development, as giving an animal an antibody to G-CSF leads to profound neutropenia.The G-CSF knockout mouse shows severe neutropenia.Neutropenia that results from a production disturbance, such as exposure to cytotoxic drugs, is associated with high circulating serum concentrations of G-CSF (Williams, 2007). 2.2.4 Biological Function of Neutrophils Neutrophils phagocytose and destroy the bacteria by the help of the their

various granules. Neutrophil interact with chemotactic agents to migrate to site of infection. They accomplish this by entering postcapillary venules in the region of inflammation and adhering to the various selectin molecules of endothelial cells of this vessels by use of their selectin receptors. The interaction between the neutrophils selectin receptor and the selectin of the endothelial cells cause neutrophils to roll slowly along the vessels endothelial lining. As the neutrophil are slowing their migrations, Interleukin-1 (IL-1) and tumor necrosis factor (TNF) induce the endothelial cells to express intercellular adhension molecule type 1 (ICAM-1), to which the integrin molecules of neutrophils avidly bind (Kerrigan et al., 2009). When binding occurs, the neutrophils stop migrating in prepartion fot their passage through the endothelium of postcapillary venule to enter connective tissue compartment. Once there, they destroy the infected cells by phagocytosis and by release of hydrolytis enzymes (and respiratory burst). In addition, by manufacturing and releasing leukotrienes, neutrophil assist in the initiation of the inflemmatory process (Doan et al., 2008).

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The sequence of event as follows: 1. The binding of neutrophil chemotactic agents to the neutrophils

plasmalemma facilitates the release of the content of tertiary granules into extracellular matrix 2. Gelatinase degrades the basal lamina, facilitating neutrophil migration.

Glycoproteins that become inserted in the cell membrane aid the process of phagocytosis. 3. The content of the specific granules are also released into the

extracellular matrix, where they attack the invading matrix and aid neutrophil migration. 4. Microorganisms, phagocytosed by neutrophils, become enclosed in

phagosomes. Enzymes and pharmacological agents of the azurophilic granules are released into the lumina of these intracellular vacuoles, where they destroy the ingested microorganisms. Because of their phagocytic functions, neutrophils are also known as microphages to distinguish them from the larger phagocytic cells, the macrophages. 5. Bacteria are killed not only by the actions of enzymes but also by

formation of reactive oxygen compounds within the phagosomes of neutrophils. These are superoxide, formed by the action of NADPH oxidase on oxygen in a respiratory burst; hydrogen peroxide, formed by action of superoxide; and hydrochlorus acid(HOCL), formed by the interaction of myeloperoxidase(MPO) and chloride ions with hydrogen peroxide.

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6.

Occasionally, the contents of the azurophillic granules are released into

extracellular matrix, causing tissue damage, but usually catalase and gluthione peroxidase degrade hydrogen peroxide. 7. Once neutrophils perform their function of killing microorganisms, they

also die, resulting in formation of pus, the accumulation of dead leukocytes, bacteria, and extracellular fluid. 8. Not only do neutrophils destroy bacteria, they also synthesize

leukotrienes from arachidonic acids in thier cell membranes. These newly formed leukotrienes aid the initiation of the inflammatory process (Doan et al., 2008). Phagocytosis of neutrophils involve cell surface receptors associated with specialized region called clathrin coated pits. The mechanism of phagocytosis involves : a) Recognition and attachment of microbes by phagocytes. Phagocytosis is initiated when a phagocyte binds a cell or molecule that has penetrated the bodys barrier. The binding occurs at various receptors on phagocyte surface. These include PRRs(including TLRs) that recognize microbe related molecule, complement receptors(CR) that recognize certain fragments of complements (especially C3b) that adhere to microbial surfaces, Fc receptors that recognize immunoglobins that have bound to microbial surfaces or other particles, scavenger receptors, and others. b) Ingestion of microbes and other materials :

xxxviii

Following attachment to the cell membrane, a microorganism or foreign particle is engulfed by extensions of the cytoplasm and cell membrane called pseudopodia and is drawn into the cell internalization or endocytosis. In addition to phagocytosis, dendritic cells can extend plasma membrane projections and encircle large amounts of extracellular fluids to form cytoplasmic vesicles independent to cell surface attachment. Once internalized, the bacteria are trapped within phagocytic vacuoles or cytoplasmic vesicles within the cytoplasm. The attachment and ingestion of microbes trigger changes within the phagocyte. It increases in size, becomes more aggressive in seeking additional microbes to bind and ingest, and elevates production of certain molecules. Some of these molecules contribute to destruction of the ingested microbes; others act as chemotactic agents and activators for other leukocytes. c) Destruction of ingested microbes and other materials: Phagosomes, the membrane bound organells containing the ingested microbes/materials, fuse with lysosomes to form phagolysosomes. Lysosomes employ multiple mechanisms for killing and degrading ingested matter. These include Lysosomal acid hydrolase, including protease and nucleases. Several oxygen radicals, including superoxide radicals,

hypochlorite, hydrogen peroxide, and hydroxyl radicals, that are highly toxic to microbes. The combined action of these molecules involves a period of heightened oxygen uptake known as the oxidative burst.

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Nitrous oxide (NO) Decrease pH Other microbial molecules

d) Secretion of cytokines and chemokines : Once activated, phagocytes secrete cytokines and chemokines that attract and activate other cells involved in innate immune responses. Cytokines or chemical messengers such as interleukin-1(IL-1) and interleukin-6(IL-6) induce the production of proteins that lead to elevation of body temperature. Other cytokines, such as tumour necrosis factor-(TNF- ), increase the permiability of local vascular epithelial to increase its permiability and enhance the movement of cells and soluble molecules from the vasculature into tissues. Still others, such as interleukin-8(IL-8) and interleukin-12(IL-12) attract and activate leukocytes such as neutrophils and NK cells (Doan et al., 2008).

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Figure 2.4 Bacterial phagocytosis and destruction by a neutrophil (Doan et al., 2008). 2.2.5 The Role Of Neutrophil in Pulmonary Emphysema When oxidant from cigarrete smoke is exposed to the lungs, macrophage will be activated causing, histone deacetylase-2 to be inactivated, shifting the balance toward acetylated or loose chromatin, exposing nuclear factor B sites and resulting in transcription of matrix metalloproteinase-9, proinflammatory cytokines interleukin 8 (IL8), and tumor necrosis factor(TNF). This will lead to neutrophil recruitment (Longo et al., 2011). CD8+ T-cells are also recruited in response to cigarette smoke and release interferon inducible protein-10 (IP-10, CXCL-7) that in turn leads to macrophage production of macrophage elastase [matrix metalloproteinase-12 (MMP-12)]. Matrix metalloproteinases and serine proteinases, most notably neutrophil elastase, work together by degrading the inhibitor of the other, leading to lung destruction. Proteolytic cleavage products of elastin also serve as a macrophage chemokine, fueling this destructive positive feedback loop that lead to emphysema (Longo et al., 2011). Collagen turnover in emphysema is complex. The three collagenases (MMP-1, MMP-8, and MMP-13) that initiate the cleavage of interstitial collagens are also induced in both inflammatory cells and structural cells in emphysema. While collagen is disrupted as alveolar units are obliterated, overall there is a net increase in collagen

xli

content in the emphysema , with prominent accumulation in the airway submucos (Longo et al., 2011).

Figure 2.5 Pathogenesis of emphysema and association of neutrophil (Longo et al, 2011).

xlii

CHAPTER 3 CONCEPTUAL FRAMEWORK AND HYPOTHESIS SMOKIN G OXIDANTS

ALVEOLAR MACROPHAGE

CD 8+ Cells DEACYTELASE-2 INACTIVETED TRANSCRIPTION OF MM9 INTERLEUKIN-8 TUMOR NECROSIS FACTOR- NEUTROPHI L

PROTEASE

PARENCYMAL LUNG DAMAGE

1-AT deficiency

COP D xliii

Related To Macrophage Profile EMPHYSEMA Indirectly related to Study

Figure 3.1

Conceptual Framework of Pathogenesis of emphysema and association

of macrophage

3.1

Conceptual Framework of the Study Long term smoking is directly related to emphysema. Under normal

condition,alveolar macrophage will patrol the lower airspace but when chronic exposure to smoking occurs, it will activate the alveolar macrophages and

subcequenly,macrophages will accumulate in the alveolar area.This is due to the oxidants produced by cigarettes. Activated macrophages will release several chemical substances such as tumor necrosis factor-alpha1 (TNF-1), interleukin-8 ( IL-8), and protease.This will lead to the neutrophil recruitment. Accumulated neutrophils will play its role by further activating macrophages to produce excessive protease.This will cause protease and anti protease imbalance. that leads to destruction of elastin and other structural elements which cause recurrent inflammation.This mechanism will lead to irreversible enlargement of the air spaces distal to the terminal bronchioles. This parenchymal lung damage will cause the breakdown of elasticity and loss of fibrous and muscle tissue, making the lungs less compliant or in other word,emphysema is occured in the person. 3.2 Hypothesis of the Study

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Based on the summary of problem and study purpose, the hypothesis is, 1) Smoking will increase the number of neutrophil in alveolar in the smokers. 2) The number of neutrophil in alveolar will be increased by the three degree of smokers, that are mild, moderate and severe smokers. 3) There is a difference in neutrophil count in the alveolar between asymptomatic smoker and smoker with pulmonary emphysema. CHAPTER 4 STUDY METHOD 4.1 Study design This study was a laboratory observational study with a study design of cross sectional of the groups posttest only on smokers without any other systemic complication (mild, moderate, severe) and smokers with pulmonary emphysema without any other systemic complication. The purpose of the study is to observe the effect of smoking on the level of neutrophil among different groups of smokers. 4.2 4.2.1 Population and Sample of Study Population

The study populations were smokers from 4 groups which composed of 3 types of smokers and have the following requirements: Mild smokers ( Brinkman Index : 1 to199 )

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4.2.2

Moderate smokers ( Brinkman Index : 200 to 399 ) Severe smokers ( Brinkman Index : 400 to 599 ) Smokers with pulmonary emphysema

Sample size The sample used was sputum collected from the four groups. The groups were asymptomatic mild, moderate and severe smokers according to the Brinkman Index and the fourth group is smokers with pulmonary emphysema. The criteria for the fourth group were chest x-ray with a posteroanterior (PA) position.

4.2.3

Sample size estimation

( z S ) n = 2 d
Explanation = = = = Accordingly, sample size standard deviation

absolute accuracy of the different levels mean value (0.5) z value at alpha 5% is 1.96

(1.96 0.5) n = 2 0.5


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n = 7.6
Rounded to the nearest value it will be 10.

(Hamilton, 2009).

The total samples were taken from ten persons in each group and we had 4 groups as a sample. So, the total sample for the study would be forty. 4.2.4 Sample statistic The statistic that was used for this observational study is Anova, because it uses logical extension of T test. Since we have data from four independent groups. Anova would be the best method to calculate the statistic. The observations was independent which means the value of one group is not correlated with the other group, therefore, the observation in each group was normally distributed and the variance of each group was equal to the that of any other group (homogeneity of variances). 4.3 4.3.1 Location and time of study Place of study The study was carried out from the smokers around the RSSA and also smokers with emphysematous lung disease who came to RSSA as outpatient. The research was done in Respiratory Department and Central Laboratory of RSSA. 4.3.2 Time of study

Research was done at the estimated time of thirty days.

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4.4 4.4.1

Variable Dependent variable Dependent variable in this study was the level of neutrophil in the sputum that was collected from four different types of comparison groups.

4.4.2

Independent variable The independent variables of this study consist of four groups that were the following: Group 1 : Mild smoker without any other systemic complication. Group 2 : Moderate smoker without any other systemic complication. Group 3 : Severe smoker without any other systemic complication. Group 4 : Smoker with pulmonary emphysema without any other systemic complication.

4.5 Inclusion and exclusion criteria of the study Inclusion Criteria : Male Smoker 45- 75 years old -Smoker with pulmonary emphysema is chosen based on a radiologic findings ECG in a normal range

xlviii

Exclusion Criteria : COPD patient with acute exacerbation Female With a sign of cor pulmonale Heart attack, tuberculosis, Asthme bronchitis, Hypertension

4.6 Operational Definition i. Neutrophil is a a polymorphonuclear granular leukocyte having a nucleus with three to five lobes connected by threads of chromatin, and cytoplasm containing very fine granules which involves in phagocytic process. ii. Smoker is defined as the person who knowingly smoke the cigarette by inhaling and exhaling the cigarette smoke with minimal cigarette smoking one per day. iii. Mild smoker is categorized according to the Brinkman Index by multiplying number of cigarettes smoked per day with years of smoking and the

score should be 0 199. iv. Moderate smoker is categorized according to the Brinkman Index by multiplying number of cigarettes smoked per day with years of smoking and the score should be 200 599.

xlix

v.

Severe smoker is categorized according to the Brinkman Index by multiplying number of cigarettes smoked per day with years of smoking and the score should be more than 599.

vi.

Brinkman Index is multiplying number of cigarettes smoked per day with years of smoking.

vii.

Non symptomatic smoker is a person that inhales and exhales cigarette smoke purposely without any clinical findings in the respiratory system.

viii.

Non smoker is a person who does not smoke and neither family member nor working colleague smokes and exposed to cigarette smoke less than eight hours per week.

ix.

Positive control smoker is a person that inhales and exhales cigarette smoke purposely and has x-ray findings of pulmonary emphysema.

x.

Passive smoker is a person that exposed to cigarettes smoke emitted from cigarettes smoke by other person for more than four hours per day.

xi.

Pulmonary emphysema is described radiologically as an abnormal permanent enlargement of air spaces distal to the terminal bronchioles, that the x-ray image shows an appearance of hyperlucent and hyperinflation at both the lungs.

xii. 4.7 4.7.1 Instruments Studies Tools and Substances

40 pieces of sterile disposable plastic pots for sputum collection Sticker labels 1 unit spirometry equipment (nebulizer ultrasonic) Salbutamol

2 fl 3% Nacl NaOH 4% Water bottles Laboratory request form

Gloves Mask 10 ml syringe 15ml centrifuge tube Tube rack

Pasteur pipette Biosafety Cabinet class II Vortex mixer

Bacti-cinerator

li

4.8 4.8.1

Bio contained centrifuge 3000g Sysmex XT-2000i Hemoanalyzer Computer with monitor Printer Tissue paper

Study Work Plan Sputum Induction i. Detailed information and instruction to the subject were given and consent to the procedure was taken. ii. The safety of the equipment was checked and the ultrasonic nebulizer was prepared (output approximately 1ml/ min) iii. Subjects were asked to rinse their mouth using hypertonic saline solution (NaCl 3%) and asked to breathe through the nose in order to avoid contamination of saliva. Saliva removed prior to begin the sputum induction procedure. iv. Subject was asked to cough and throw up the sputum, firstly in the fifth minute and the secondly at tenth minute. v. Sputum collected in container with subjects name label on it.

4.8.2

Sputum Decontamination and Centrifugation

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i.

Collected sputum was mixed with sodium hydroxide (NAOH) 4% in a ratio of (1: 2) and fastened firmly.

ii.

The sample was mixed gently with vortex mixer and placed in

temperature of 15 C room temperature to stimulate homogenization. iii. iv. v. vi. vii. viii. ix. Centrifugation was done at 3000 x g for 15 minutes. The aerosol was left to become sediment for 10 minutes. Supernatant was removed. Distilled water was added up to the 15ml. The solution was centrifuged again at 3000 x for 15 minutes The aerosol was left to become sediment for 10 minutes. Supernatant was removed and the homogenized sample kept to do neutrophil counting through sysmex-XT hemoanalyzer. 4.8.3 Neutrophil Counting i. ii. iii. Sysmex XT-2000i hemoanalyzer was turned on. Computer CPU and printer were turned on too. QC *QC analysis click * select a QC file to be executed, press OK * Enter the e - check that has been in homogenization into the sample probe. * Press Start. * Make sure the QC results in target and click Accept. * To see the QC chart, graph and select the desired type. iv. The sample was ran according to the instruction below:

liii

v. vi.

Perform first order on the work list Enter Number and type of test sample Enter the patient ID and patient data (if any) Click Save Click Manual Type of sample Number (adjust to the work list) Press OK Enter been homogenizing samples into the sample probe Press START Results can be viewed by clicking on the Explorer or the Data Browser Switch off the appliance: Click shut down on-screen menu Enter Cell clean the sample probe into Press the START 1 (one) time Wait until the process is complete, then turn off the equipment

and programs Click START on the Windows program Click shut down liv

Turn off the monitor and printer

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4.8

Study Framework

Mild Smokers (10 Subjects)

Moderate Smokers (10 Subjects)

Severe Smokers (10 Subjects)

Smokers with pulmonary emphysema based on radiologic findings (10 Subjects)

Sputum samples were collected from each group using sputum pot

Collected sputum Mixed with NAOH 4% in a ratio of (1 :2) for decontamination Centrifugation is done at 3000 x g for 15 minutes

Supernatant is removed, add distilled water up to the15ml

Centrifugation is done at 3000 x g for 15 minutes

Sediment Sysmex XT-2000i instrument Result been recorded according to the group lvi

(A) Smokers with Pulmonary Emphysema

(B)Mild Smokers Neutrophil Counting (C) Moderate Smokers

(D) Severe Smokers Figure 4.1 Study Framework

CHAPTER 5 STUDY RESULTS 5.1. Subject and Study Location There were 42 subjects been given informed consent about the purpose, procedures and the approval letters to be signed. Two subjects were excluded, one due to pulmonary tuberculosis lesion from radiological study and the other due to lysis of blood plasma obtained. So, we used 40 subjects to study and they have been divided into 4 groups. Where 10 subjects of light smoker, 10 subjects of moderate smokers, 10 subjects of severe smokers and lastly 10 severe smokers with pulmonary emphysema. 5.2. Characteristic of the subjects The numbers of subjects of this study are 40 people and all are male. The mean age of the subjects of the study was 60.78 + 7.82 years with the youngest age range 46 years old and the oldest is 77 years of age as shown in the table.

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Table 5.1 Characteristics of study subjects in each group


Characteristic Mild Smokers (n = 10) Moderate Smokers (n = 10) Heavy Smokers (n = 10) Smokers with Pulmonary Emphysema (n = 10) 67,40 + 4,29

Age (Years)

53,60 +6,68

58,30 +5,90

63,80 + 6,61

Occupation 1. 2. 3. 4. 5. 6. 7. 8.

Retired PNS Retired TNI/Polri Private Lecturer Farmers Enterpreneur Salesman BUMN (PDAM/PLN) Security Guard

1 1 1 5 2 -

3 2 3 1 1

7 2 1

6 1 1 1 1 -

Jenis Rokok 1. Clove Cigarettes 2. Filter and Filter Clove Cigarettes 3. Hand-rolled Cigarettes 4. Clove Cigarettes and Filters

2 6 2

2 8

2 6 2

7 1 1 1

Brinkman Index (Cigarettes/year)

145,8 +63,3

366,8 +87,9

819,8 + 224,97

845,40 + 213,18

Body Mass Index 1. Underweight (< 18,5 kg/m2)

2. 3.

Normal (18,5-24,9 kg/m ) Overweight (> 25-29,9 kg/m2)

7 3 23,50 + 3,86

1 3 6 24,70 + 3,38

1 7 2 22,70 + 3,84

4 5 1 20,20 + 4,07

BMI (kg/m2) Data on the total and the mean (SD)

From the characteristic of the subjects of the study, the average age of the mild smoker is between 46 to 63 and moderate ranges from 48 to 67, followed by severe at lviii

57 to 77 and pulmonary emphysema group which has a age range of 61 to 73 years old. Most of the candidates taken for the research are retired PNS. Most usual type of cigarette smoked by study subjects are a mixture of clove cigarettes and

filters (alternate) as much as 80% (as shown in the figure) the mean of the Brinkman index, in the smokers with pulmonary emphysema group is at a number of 845.40+ 213.18 cigarettes / year, in about 145.8+ 63.3 cigarettes /year, in 366.8 + 87.9 cigarettes/year, 819.8 224.97 + cigarettes/year. in The the group of group of the mild smokers is

the group the

moderate

smokers is

about is

of severe smokers

mean Brinkman index in all study

subjects

are 544.45 + 344.90 cigarettes/year. From the subjects of the study Brinkman index from the mild smokers

range from 10-192 cigarettes / year (p <0.05), moderate smokers in the group with a range of 230-468 cigarettes / year (p <0.05), a group of severe smokers with a range of 630-1152cigarettes / year (p <0.05) and in the fourth group with a range of 6401120 cigarettes /year (p <0.05) as shown below. Throughout the period of the samples were taken, subjects did not show any adverse effect based on the anamnesis, physical examination and additional examination that was done here. Similarly, most of the vital signs such as blood pressure, pulse rate and respiratory rate was normal during the time samples were taken. The mean body mass index or Body Mass Index (BMI) of study subjects from each group is a norm weight and its distribution can be seen in the picture. 5.3. Characteristics Of Supportive Examination Data of Subject

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Routine blood test, SGOT, and SGPT that was done among all the subjects from different types of group, showed normal value. The results of

electrocardiographic examination showed normal value for 12 respondents and only one respondent showed right axis deviation. PA position of radiographic

examinations obtained from 13 study subjects had normal radiological picture, and 17 study, subjects had an increased bronchovascular pattern, while radiographical picture in the fourth group gained from 10 subjects with radiographical studies with pulmonary emphysema. Tabel 5.2 Characteristics Of Supportive Examination Data of Subject

Characteristics

Mild Smoker (n = 10)

Moderate Smoker (n = 10)

SevereSmoker (n = 10)

Smokers with Pulmonary Emphysema (n = 10)

Routine Blood Test 1. Haemoglobin (gr/dl) 2. Leukocytes(gr/dl)

14,03+ 0,91 6656+ 2368,6

14,26+ 1,59 7882+ 2915,1

13,94+ 1,63 7294+ 2602,4

13,8+ 1,28 8453+ 3475,5

ECG 1. 2.

Normal RAD

10 -

10 -

9 1

10 -

Chest X-Ray (PA) 1. Normal 2. Increase Bronchovascular pattern 3. Emphysema Neutrophil in Sputum (/mm3)

7 3 617.8 + 600.7

4 6 1369.4 +1362.2

2 8 953.9 + 749.9

10 789.44 + 571.5

Data on the total and the mean (+ SD) (source: study)

5.4

Neutrophil counting in the sputum induction of the subjects

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The average counting of neutrophils from the sputum induction of the subjects has the highest value in moderate group at a number of 1369.4 + 1362.2/mm3 (p<0,05), and severe smokers about 953.9 + 749.9/mm3 (p>0,05) and then pulmonary emphysema group at about 789.44 + 571.5/mm3 (p>0,05) and finally mild smokers at a number 617.8 + 600.7/mm3 (p<0,05). Only severe smokers pulmonary emphysema group and another group which is the severe smokers gave the significant result and normally distributed compared to the other group which are the mild and moderate smokers. When comparing the non pulmonary emphysema group and pulmonary emphysema group, it can be shown that the numbers are 981.0 + 376.3 /mm3 and 789.4 + 571.6 /mm3 that are shown in the picture. 5.5 5.5.1 Data Analysis Normality Test Normality test was done to identify whether neutrophils are distributed normally in the different stages of smoking such as mild,moderate and severe and also the pulmonary emphysema group.From the Shapiro Wilik with Liliefors correlation test, only pulmonary emphysema group and asymptomatic severe smokers group are normally distributed with p > 0.05. Because the other stages do not give normal distribution, so further test that is Mann Whitney test was done. 5.5.2 Neutrophils Comparative Hypotesis Test with Using Mann Whitney U Test Based on the Mann- Whitney test that was done on the neutrophils counting by taking pulmonary emphysema group as one group and the combination group of mild, moderate and severe smokers as another group, the value obtained was p

lxi

>0.05.Therefore it can be concluded that there is no any differences between the groups or in other word, there value is not significant.

5.5.3

One Way Anova Test This observation used variable that is the neutrophils counting in the sputum

induction from all the groups of smokers based on the different stages of smokers ( > 2 unpaired groups ) without involving the fourth group which is the severe smokers with pulmonary emphysema. With considering the result of the test of homogeneity of neutrophils or on the other word the Levene test, with an arrangement of p >0.05, it can be concluded that there is no variable differences of the data of the groups that are compared and can be further continued with the one way Anova test. Based on the Levene variant test it can be concluded there is no differences in the neutrophils variable from the different groups according to the stages of smoking with a distribution of p> 0.05. Since the data of the variants are the same, one way Anova test is valid with a value of p >0.05 from the neutrophils variable and therefore can be concluded there is no significant differences between the variable of all the three groups. 5.5.4 Correlation Test Of Different Stages Of Smoking

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Correlation test was done to identify the influence of the different stages of smoking with the neutrophils variable by using normally distributed Product Momen Pearson numerical correlative hypothesis test. It can be shown clearly that only BMI and age give significant result with the value p < 0.05. The correlation of the stages of smoking with the BMI value has negatively correlation value, therefore it can be said that the bigger the stage of smoking, the lower the value of the BMI of the person. Contrarily for this statement, the age of the smoker has positive correlation with the stages of smoking. So, the bigger the stage of smoking, the higher the age of the smoker. It was found out that, the stages of smoking with the counting of the neutrophils from the sputum induction has positive correlation with p > 0.05, so therefore it is not significant also.

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Chapter 6 Discussion 6.1 Characteristic of the Research Subjects Based on the characteristic of the respondent from all the groups, it can be found out that all the respondents are male with an average age of 60,775 + 7,816 years old and most of them are retired PNS who have smoking habit currently. T.B. Grydeland et al, (2009) concluded that emphysema is more frequently occurs in male compared to female. The higher emphysema score in males could have several explanations. Emphysema quantification is very sensitive to the level of inspiration, but even though males have larger lung, there are no indications that males inspired deeper. Environmental cause is another possible explanation, as males are more exposed to occupational airborne agents, which are potentially harmful to the lungs. The prevalence of smoking is more prominent in developing countries such as China and India compared to developed countries such as United States and United

Kingdom. This is due to most of the people who practice routine smoking hailed from low social-economical status society who are low in literacy and do not have proper job and eventually have low per capital income.

lxiv

The type of cigarettes smoked by the smokers are clove, filtered, hand rolled and most of the respondent has the habit of smoking a combination of kretek and filtered cigarettes. Smoking filtered cigarettes has many beneficial such as reduced lavel of nicotine and tar compared to the non-filtered cigarettes, however the level of carbon monoxide is stil the same in both filtered and non-filtered, so the amount of hazardous toxic effect that are produced by both the cigarettes are the same. Hence neither type will protect the airway of the person. The weight of the respondents were taken to identify the level of Body Mass Index (BMI).BMI is the index used to categorize the person into underweight, normal, overweight, and obese category. From the studies it is identified that most of the respondents have higher BMI level compared to the controlled group which is the smoker with emphysematous lung. Similarly, Verbeken et al, (1992) also found out that BMI level of the normal healthy smoker is higher compared to the level of emphysematous patients. Hence, emphysema together with smoking increase the workload of the lung, and the airway muscles, so much energy will be used to compensate this process and this causes the smoker with pulmonary emphysema to be thinner. 6.2 Neutrophil Counting In Sputum Induction The counting of neutrophils of the sputum induction is higher in the fourth group which is the severe smokers with pulmonary emphysema. Apparently Morrison et al, (1998) also found out that neutrophils are normally higher in emphysematous patient compared to healthy smoker. This is because the underlying mechanisms of emphysema include inflammatory processes in the lung and airways. Cigarette smoke and other irritants activate macrophages and airway epithelial cells in the respiratory

lxv

tract, which release neutrophil chemotactic factors, including IL-8 and leukotriene (LT) B4. Neutrophils and macrophages then release proteases that break down the connective tissue in the lung parenchyma, resulting in emphysema. Therefore the higher number of neutrophil in emphysematous patient is because of increased activity of inflammation in the lung compared to healthy smoker. Based on Mann Whitney test that was conducted on the neutrophils counts of the two groups which are the pulmonary emphysema group and the non pulmonary emphysema group which is the combination group of mild, moderate and severe smokers, it can be concluded that there is no any significant differences between the both groups. Apparently, this result is quite parallel to the research done by Domagala et al, (2003) where they counted the neutrophils level in smokers with emphysematous lung and healthy ex-smokers and they showed that there are no significant differences in the cellular profile of induced sputum of the samples between patients with COPD who are active smokers and those who have ceased smoking. Based on the one way Anova test, which was done among the mild, moderate and severe smokers, there is no any difference between the groups and it can be concluded that the neutrophils level is not significantly variable among the groups, so the values are approximately the same. It also can be clearly identified that smoking stages do not directly affect the variability of the observation subjects, unless subjects that are studied are non-smoker or not smoking currently. 6.3 The correlation between smoking stages and number of neutrophil in alveolar Pearson test was used to identify the correlation of stages of smoking to the level of neutrophils and it was found that there is no any correlation of the stages of lxvi

smoking that was characterized according to Brinkman Index as mild, moderate and severe smokers to the quantity of neutrophils in the person. It is because the value of p > 0.05, therefore, it is not significance. From the research, it can be concluded that, the number of neutrophil increases significantly even in the mild stage and therefore, the severity of smoking is the same regardless the number of cigarette smoked per day.

6.4

The Weakness of the Study The study was done in a population of 40 candidates and this can be referred as

a small population to do this study. Therefore, the result would have been more accurate and reliable if the study population was bigger. Apart from that, the smokers with pulmonary emphysema were chosen based on the solitary confirmation from x-ray radiologic findings. This would have created lesser accuracy because not any further diagnosis was done to confirm the candidates are having pulmonary emphysema. Hence, if more examinations were done, then it would have increase the accuracy of the study.

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CHAPTER 7 CONCLUSION 7.1 Conclusion From the study result, it can be concluded that: 1. Smoking does influence the level of macrophages in sputum induction of a smoker compared to the healthy person 2. The numbers of neutrophils do not increase proportionally according to Brinkman Index from mild, moderate and severe smoker, hence, there is a significant increase of macrophage even in mild smoker 3. There neutrophil counts in sputum induction of smoker with pulmonary emphysema or non-pulmonary emphysema smoker (mild, moderate, severe) are approximately the same. 4. Different stages of smoking are inversely proportional to the level of BMI of the smoker

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5. Different stages of smoking are directly proportional to the age of the smoker.

7.2

Suggestions From this study, suggestion or opinions in future study are: 1. Further study should be done to identify how actually stages of smoking influence the recruitment of neutrophils in the sputum induction of the smoker. 2. Further study should be done to identify the correlation of smoking to the level of neutrophils in the sputum induction of the smoker. 3. The number of the candidates used should be increased to decrease the unwanted error in the study. 4. Another research should be done to identify how the mechanism of recruitment of neutrophil in the sputum induction from the alveolar of a smoker.

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REFERENCE

Anderson, W. 1999. Immunology, Integrated Medical Sciences, 1st Ed, p. 24-30. Burton, J.C., Vessal, G., Brown, J., Dowlinan, T.C., Fink, J.C.. 2007. Urinary cotinine as an objective measure of cigarette smoking in chronic kidney disease, Clinical Nephorology, 2007, 22 (7): 1950-1954. Charlesworth, J.C., Curran, J.E., Johnson, M.P., Goring,H.H.H., Dyer, T.D., Diego, V.P., Kent, J.W., Mahaney, M.C., Almasy, L., Jean, W.M., Moses, E.K., Blangero, J.. 2010. Transcriptomic epidemiology of smoking: the effect of smoking on gene expression in lymphocyte, BMC Medical Genomics, 2010, 29 (3): 1-11. Crapo, J.D., Glassroth, J., Karlinsky, J., King, T.K.. 2004. Management of The Smokers, Baums Textbook of Pulmonary Disease, 7th Ed, p. 500-534. Demirjian, B.G.. 2011. Pathophysiology of Emphysema. http://emedicine.medscape.com/article/298283-overview#a0104 Doan, T., Melvold, R., Viselli, S., Waltenbaugh, C.. 2008. Granular Leukocytes, Lippincotts Illustrated Reviews: Immunology, 1st Ed, p. 37-53. Domagaa, J.K., Warzchowska, M.M., Kraszewska, I., Chazan, R.. 2003. The Cellular Composition and Macrophage Phenotype in Induced Sputum in Smokers and Ex-Smokers With COPD, Chest, 2003, 123 (4): 10541059. Esmann, L., Idel, C., Sarkar, A., Helberg, L., Bejen, M.. 2010. Phagocytosis of Apoptytic Cells By Neutrophil Granulocytes: Diminished Proinflammatory Neutrophil Functions in the Presence of Apoptotic Cells, The Journal Of Immunology, 2010, 184: 391-400. lxx

Fayyaz, J.. 2011. Pathophysiology of Chronic Bronchitis. http://emedicine.medscape.com/article/297108-overview Foronjy R., Imai K., Shiomi T., Mercer B., Sklepkiewicz P., Thankachen J., Bodine P., D'Armiento J.. 2010. The divergent roles of secreted frizzled related protein-1 (SFRP1) in lung morphogenesis and emphysema, 2010, 177 (2): 598607. Global Strategy for Diagnosis, Management, and Prevention of COPD, Global Initiative For Chronic Obstructive Lung Disease (GOLD). 2010. http://www.goldcopd.org/uploads/users/files/GOLDReport_April112011.pdf Grydeland, T.B., Dirksen, A., Coxson, H.O., Pillai, S.G., Sharma, S., Eide, G.E., Gulsvik, A., Bakke P.S.. 2009. Quantitative computed tomography: emphysema and airway wall thickness by sex, age and smoking, Eur Respir J. 2009, 34 (4): 858-865. Guyton AC, Hall JE. 2006.Textbook of Medical Physiology, 11th Ed. P. 471-533. Hamilton AC. 2009. Statistics With Stata, Updated For Version 10, Brooks& Cole Publishing, p. 155-168. Henningfield, J.E., Jude, N.R.. 1999. Prevention of nicotine addiction: Neuropsychopharmacological issues, Nicotine And Tobacco Research, 1999, 1 (1): 41-48 Indrayan, A.. 2008. A comprehensive index of smoking (Indrayans smoking index). http://indrayan.weebly.com/smoking-index1.html Kerrigan, A.M., Dennehy, K.M., Trindade, D.M.F., Willment, J.A., Taylor, P.R., Eble, J.A., Sousa, C.R., Brown, G.D. 2009. CLEC-2 Is a Phagocytic Activation Receptor Expressed on Murine Peripheral Blood Neutrophils, J Immunol, 2009, 182: 4150-4157. Kumar V, Abbas AK, Fausto N. 2005. Robbins And Cotran Pathologic Basic of Disease, 7th Ed. p. 711-730. Kume, A., Kume, T., Masuda, K., Shibuya, F., Yamazaki, H.. 2009. Dose Dependents Effect of Cigarette Smoke in Healthy Japanese Volunteers, Observation of Smoking and Non Smoking,Journal of Health Science, 2009, 55 (2): 259-264. Lee, W.L., Downey, G.P.. 2001. Neutrophil activation and acute lung injury, 2001, 7 (1): 1-7. Longo, D.L., Fauci, A.S., Kasper, D.L., Hauser, S.L., Jameson, J.L., Loscalzo, J. 2011. Harrisons Principles of Internal Medicine, 18th Ed, p. 2151-2159

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Morrison, D., Strieter, R.M., Donnelly, S.C., Burdick, M.D., Kunkel, S.L., MacNee, W. 1998. Neutrophil chemokines in bronchoalveolar lavage fluid and leukocyte-conditioned medium from nonsmokers and smokers, Eur Respir J. 1998, 12: 10671072. Morrow, J. 2007. Common Adverse Effect of Tobacco Smoking. http://www.jarretmorrow.com/how-does-smoking-cigaretts-affect-your-jointhealth/ Mosenifar, Z.. 2011. Chronic Obstructive Pulmonary Presentation. http://emedicine.medscape.com/article/297664-clinical Disease Clinical

Saetta, M., Stefano, A.D., Maestrelli, P., Turato,G., Ruggieri, M.P., Roggeri, A., P Calcagni, P., Mapp,C.E., Ciaccia, A., Fabbri, L.M.. 1994. Airway eosinophilia in chronic bronchitis during exacerbations, Respiratory and Critical Care Medicine, 1994, 150 (6): 1646-1652. Schillinger, M., Exner, M., Mlekusch, W., Haumer, M., Sabeti, S., Ahmadi, R., Wagner, O., Minar, E..2004. Effect of smoking on restenosis during the 1st year after lower-limb endovascular interventions, Radiology, 2004, 31 (2): 831-838. Schillinger, M., Exner, M., Mlekusch, W., Haumer, M., Sabeti, S., Ahmadi, R., Wagner, O., Minar, E.. 2004. Effect of smoking on restenosis during the 1st year after lower-limb endovascular interventions, Radiology, 2004, 231: 831 838. Semba, R.D., Kalm, L.M.,Pee, S., Ricks, M.O., Sari, M., Bloem, M.W.. 2006. Paternal smoking is associated with increased risk of child malnutrition among poor urban families in Indonesia. Public Health Nutrition, 2006, 98 (10): 1824 1826. The Tobacco Atlas, World Health Organization (WHO), 1st Ed, 2002. http://www.who.int/tobacco/media/en/title.pdf Verbeken EK, Cauberghs M, Mertens I, Clement J, Lauweryns JM, Van de Woestijne KP. The senile lung: comparison with normal and emphysematous lungs: 1: structural aspects, Chest. 1992, 10 (1): 793799. Vogelmeier, C., Hederer, B., Glaab, T., Schmidt, H., Beeh, K.M., Rabe, K.F., Fabbri, L.M.. 2011. Tiotropium versus Salmeterol for the Prevention of Exacerbations of COPD, 2011, 64 (3):1093-1103. Wallace, W.A.H., Ramage, E.A., Lamb, B., Howie, S.E.M.. 1995. A type 2 (Th2like) pattern of immune response predominates in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis (CFA), Clin Exp Immunol 1995, 101: 436-441. Williams, L.J. Fazilleau, N.R. (2007) Local development of effector and memory T helper cells. Curr. Opin. Immunol. 19 (3) 259-267. lxxii

Witko, S.V., Rieu, P., Descamps, L.B., Lesavre, P., Halbwachs, M.L.. 2000. Neutrophils: Molecules, Functions And Pathophysiological Aspects, 2000, 80 (5): 617-53.

Kolmogorov-Smirnov Group Statistic .348 .283 .241 .422 .263 .231 .147 .166 .180 .176 .135 .145 .289 .305 .137 .327 .406 .410 .482 .410 .175 .247 .299 .199 df 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Sig .001 .023 .103 .000 .049 .140 .200* .200* .200* .200* .200* .200* .017 .009 .200* .003 .000 .000 .000 .000 .200* .085 .012 .200*

Shapiro-Wilk Statistic .721 .906 .871 .655 .877 .867 .975 .888 .948 .925 .961 .969 .775 .778 .961 .779 .623 .708 .509 .708 .908 .791 .763 .870 df 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Sig .010** .315 .111 .010** .147 .096 .922 .212 .615 .423 .767 .863 .010** .010** .770 .010** .010** .010** .010** .010** .323 .014 .010** .0104

Type Of Occupation

Smoker with P.E Mild Smokers Moderate Smokers Heavy Smokers

Age

Smoker with P.E Mild Smokers Moderate Smokers Heavy Smokers

BMI

Smoker with P.E Mild Smokers Moderate Smokers Heavy Smokers

Brinkman Index

Smoker with P.E Mild Smokers Moderate Smokers Heavy Smokers

Type Of Cigarettes

Smoker with P.E Mild Smokers Moderate Smokers Heavy Smokers

Neutrophil

Smoker with P.E Mild Smokers Moderate Smokers Heavy Smokers

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APPENDIXES
Kolmogorov-Smirnov Group Macrophage Smokers With P.E Asymptomatic Smokers Statistic .175 .201 10 30 df Sig .200* .003 Shapiro-Wilk Statistic .908 .774 df 10 30 Sig .323 .010**

Table 1 : Test of Normality

Tests of Normality

**. This is an upper bound of the true significance *. This is a lower bound of true significance a. Lilliefors Significance Correction

Test of Normality

**. This is an upper bound of the true significance *. This is a lower bound of true significance a. Lilliefors Significance Correction

Table 2 : Test of Homogeneity of Variances

Test of Homogeneity of Variances NEUTROPHIL Levene Statistic 2.370 df1 2 df2 27 Sig. .113

Table 3 : One Way Anova Analysis Descriptives lxxiv

N Mild Smokers Moderate Smokers Heavy Smoker Total 10 10 10 30

Mean 617.8300 1369.1400 955.9300 980.9667

Std. Deviation 600.9134 1362.1994 749.9378 178.8969

Std. Error 190.0255 430.7653 237.1512 178.8969

95 % Confidence Interval for Mean Lower Upper Bound Bound 187.9625 394.6813 419.4568 615.0815 1047.6975 2343.5987 1492.4032 1346.8518

Minimum 126.70 228.00 236.40 126.70

Maximum 2061.70 4225. 80 2344.00 4225.80

ANO VA NEUTROPHIL Sum of Squares Between Groups 2831736 Within Groups 25011816 Total 27843552 df 2 27 29 Mean Square 1415868.050 926363.565 F 1.528 Sig. .235

Table 4: Mann-Whitney Test


Ranks Group NEUTROPHIL Smoker with P.E Asymptomatic Smokers Total N 10 30 40 Mean Rank 20.50 20.50 Sum of Ranks 205.00 615.00

Test Statisticsb Mann-Whitney U Wilcoxon W Z Asymp. Sig. (2-tailed) Exact Sig. [2*(1-tailed Sig.)] a. Not corrected for ties. Grouping Variable: Group b. NEUTROPHIL 150.000 615.000 .000 1.000 1.000
a

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Table 5: t-Test
Group Statistics Group Neutrophil Symptomatic Smokers Asymptomatic Smokers N 10 30 Mean 789.5000 980.9667 Std. Deviation 571.5877 979.8584 Std. Error Mean 180.7519 178.8969

Independent Samples Test


Levene's Test for quality of Variance t-test for Equality of Means 95% Confidence Interval of the Difference F Neutrophil Equal Variance assumed Equal Variance not assumed 1.469 Sig .233 t -.583 38 df Sig. (2tailed) .564 Mean Difference 191.4667 Std. Error Difference 328.6547 Lower 56.7933 Upper 73.8600

-.753

27.176

.458

191.4667

254.3135

13.1170

30.1836

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Table 6: Correlations
Correlations Brinkman Index -.057 .726 40 -.289 0.71 40 1.000 40 .667** .000 40 -.099 .542 40 -.516** .001 40 .099 .545 40 -352* .026 40 .667** .000 40 1.000 40 -.012 .943 40 -.357* .024 40 Type of Cigarettes -.015 .927 40 -.113 .489 40 -.099 .542 40 -.012 .943 40 1.000 40 .323* .042 40 Type of Occupation .255 .113 40 .034 .834 40 -.516** .001 40 -.357* .024 40 .323* .042 40 1.000 40

Macrophage Neutrophil Pearson Correlation Sig. (2-tailed) N Pearson Correlation Sig. (2-tailed) N Pearson Correlation Sig. (2-tailed) Brinkman Index N Pearson Correlation Sig. (2-tailed) Type of Cigarettes N Pearson Correlation Sig. (2-tailed) Type of Occupation N Pearson Correlation Sig. (2-tailed) N 1.000 40 -.030 .855 40 -.057 .726 40 .099 .545 40 -.015 .927 40 .255 .113 40

BMI -.030 .855 40 1.000 40 -.289 .071 40 -.352* .026 40 -.113 .489 40 .034 .834 40

AGE

BMI

AGE

*. Correlation is significant at the 0.05 level (2-tailed) **. Correlation is significant at the 0.01 level (2-tailed)

lxxvii

Table 7 : Raw Data


Type Pekerj aan A1 A2 A3 A4 A5 A6 A7 A8 A9 A11 mea n: B1 B2 B3 B4 B5 B6 B7 B8 5 5 7 7 5 2 5 5 1 1 1 2 4 1 1 5 1 6 Usi a BMI kg / m2 23 23 16. 87 18 21. 8 21. 4 21. 4 14. 3 28. 75 17. 7 20. 622 20. 2 23. 05 22. 6 19. 8 22. 4 24. 65 25 30. Indeks Brinkm ann 680 740 1120 1100 640 720 670 644 1080 1060 845.4 190 190 192 112 192 180 180 68 5 2 2 2 2 1 1 2 Bronkovaskular meningkat Bronkovaskular meningkat Normal Normal Normal Bronkovaskular meningkat Normal Normal Type Roko k 1 4 1 1 1 2 1 1 1 5 C R PA WB C 106 /ml 900 .0 200 .0 450 0.0 800 .0 240 0.0 280 0.0 540 0.0 460 0.0 240 0.0 230 0.0 263 0.0 0 160 0.0 800 .0 230 0.0 530 0.0 210 0.0 700 .0 110 0.0 120 MACR OPHA GE 106 /ml 61.2 11.8 423.0 95.2 249.6 114.8 540.0 501.4 223.2 250.7 247.09 184.0 79.2 188.6 519.4 216.3 67.2 107.8 120.0 NEUTRO PHIL 106 /ml 319.5 59.6 2079.0 333.6 513.6 596.4 901.8 1131.6 964.0 995.9 789.50 547.2 260.8 1143.1 2061.7 854.7 126.7 364.1 278.4

68 61 73 71 68 68 67 59 69 70 67. 4 63 60 53 46 48 46 60 47

Emphysema Emphysema Emphysema Emphysema Emphysema Emphysema Emphysema Emphysema Emphysema Emphysema

lxxviii

96 B9 B10 mea n: C1 C3 C4 C5 C6 C7 C8 C9 C10 C11 mea n: D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 mea n: A B C 9 1 53 60 53. 6 61 64 53 63 59 57 59 67 52 48 58. 3 65 59 68 57 57 68 77 68 57 62 63. 8 67. 4 67. 4 53. 6 58. 23. 3 27. 4 23. 936 27. 4 27. 6 22. 5 25. 7 24 25 18 30. 4 22. 7 27. 23 25. 053 19. 6 22. 4 24. 3 26 21. 8 23. 9 16. 6 23. 62 29. 36 24. 69 23. 227 20. 622 20. 622 23. 936 25. 144 10 145.8 310 230 390 258 490 400 468 450 336 336 366.8 1082 1056 1152 648 684 658 660 1020 608 630 819.8 845.4 845.4 145.8 366.8 5 2 1 2 2 2 1 2 2 5 Bronkovaskular meningkat Bronkovaskular meningkat Normal Bronkovaskular meningkat Bronkovaskular meningkat Normal Bronkovaskular meningkat Bronkovaskular meningkat Bronkovaskular meningkat Bronkovaskular meningkat 2 2 2 1 2 2 2 1 2 2 Normal Bronkovaskular meningkat Bronkovaskular meningkat Bronkovaskular meningkat Bronkovaskular meningkat Bronkovaskular meningkat Bronkovaskular meningkat Normal Normal Normal 5 2 Normal Normal

0.0 190 0.0 800 .0 178 0 820 0.0 100 0.0 240 0.0 220 0.0 800 0.0 420 0.0 100 0.0 230 0.0 280 0.0 120 0.0 333 0 310 0.0 460 0.0 110 0.0 800 0.0 270 0.0 200 0.0 120 0.0 220 0.0 170 0.0 150 0.0 281 0 263 0.0 0 178 0 333 159.6 66.4 170.85 787.2 87.0 242.4 202.4 704.0 399.0 92.0 230.0 218.0 110.4 307.24 229.4 432.4 69.3 768.0 288.9 174.0 121.2 173.8 212.5 156.0 262.55 410.4 131.2 617.83 4225.8 335.0 943.2 930.6 3456.0 1419.6 228.0 662.4 1027.6 463.2 1369.14 1667.8 1826.2 317.9 2344.0 820.8 564.0 236.4 1097.8 360.4 324.0 955.93

7 2 5 1 5 5 2 1 1 8

5 5 1 1 1 1 1 1 8 1

247.09 170.85 307.24

789.50 617.83 1369.14

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3 D TOTAL MEAN 63. 8 60. 775

053 23. 227 23. 209 5 819.8 544.45

0 281 0 263 7.5 262.55 246.93 25 955.93 933.1

STATEMENT OF ORIGINALITY The undersigned, Name NIM : Uthaya Kumar Nallayan : 0810714039

Study Program : General Medicine, Faculty of Medicine, Brawijaya University

Declares that this Final Project is an original research, not an acquisition of others writing and thoughts which I claim as my own. If later it is revealed that this Final Project contains partly or wholly plagiarized of others intellectual work of any kind, I will readily accept the sanction established by the university on this matter.

Malang, January 2012 Sincerely,

(Uthaya Kumar Nallayan) lxxx

0810714039

lxxxi