erglanhzfl 5 3 .. u. 1.3.: , I 3. . x 1 . .p .z . . ‘ , . V , éf Afiauhm... n .. «a n. .1? ..n.m......:;.xi..m.fl.u. . a .‘l. ‘ v. .1 4f. . .htz \ can. 2...: k. X. . d) .2. 4. .. 5x178. €33. ., E. .Ilnui. , 3,431.11. .V Hula. .54.: xzuurki «. ii". «3.1. .u Ax" \ 1... ,1 5|; h. .4 . 3.7% x w; 1v. .3: I}... 1!: . . r Juan . 9...}: v 6.34...) in! ! ”rum”: 4?. \z ‘(nll‘lyn V“ . 3-4155.» u 5:: O This is to certify that the dissertation entitled STUDIES OF INTERLEUKlN-1 RECEPTOR ANTAGONIST AS A POSITIONAL CANDIDATE GENE IN ALLERGIC ASTHMA presented by RAVISANKAR A. RAMADAS has been accepted towards fulfillment of the requirements for the PhD degree in Comparative Medicine and Integrative Biologl /\ ”W It \-- (WMLKTJC’ \L Major PrdQs}or’ 5 Signature 9)»: l A “241% -. 1 x. v— .5 Date MSU is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University .-.-.--—-—.—--.—-.—.-.--o—u—o—.—.—.-.—.--o-u-c-n-a—n—o—-—--—-n--._.—.--o—-o-.—.—o-._--—.-.-.-—o—-—o—---n-.---n—-— PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/ClRC/DateDue.indd-p.1 STUDIES OF INTERLEUKIN-l RECEPTOR ANTAGONIST AS A POSITIONAL CANDIDATE GENE IN ALLERGIC ASTHMA By Ravisankar A. Ramadas A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Comparative Medicine and Integrative Biology Graduate Program 2006 ABSTRACT STUDIES OF INTERLEUKIN-l RECEPTOR ANTAGONIST AS A POSITIONAL CANDIDATE GENE IN ALLERGIC ASTHMA By Ravisankar A. Ramadas Asthma is a chronic airway inflammatory disease due to inappropriate immune responses to common environmental factors. It is characterized by AHR, eosinophilic infiltration, airway obstruction, increased mucus production and increased serum IgE levels. Asthma is controlled by genetic and environmental factors, and their interactions. IL-1 receptor antagonist plays a protective role in asthma by inhibiting the pro- inflammatory cytokine IL-l. Our mouse model for allergic asthma is comprised of airway hyperreponsive (All) and hyporesponsive (C3H/HeJ) strains. Genetic linkage analyses performed in AU backcross mice ((A/J x C3H/HeJ) F1 x All) identified two quantitative trait loci for AHR (AbhrI and Abhr2) on mouse chromosome 2. The murine IL-1 receptor antagonist gene (Illrn) is located within Abhrl. We hypothesized that genetic polymorphisms in [HM were responsible for the difference in AHR manifestation between N] and C3H/Hel strains. Hence, based on the genetic evidences, we investigated 111m as a positional candidate gene for allergic asthma in our mouse model at DNA, mRNA and protein levels. We sequenced the 111m gene (~16 kb) in AU and C3H/HeJ mice, but did not find polymorphisms that could explain the differences in AHR manifestation between N] and C3H/HeJ strains. A time course of allergen induced mRNA and protein levels of IL-1 receptor antagonist was performed by real-time RT-PCR and ELISA. The mRNA expression of lL-l receptor antagonist was increased due to ovalbumin treatment, and this increase was significantly higher in A/J mice at the earlier timepoints. The protein production of IL-1 receptor antagonist was increased due to ovalbumin treatment only in the AU strain, and not in the C3H/HeJ strain. These results indicate that IL-1 receptor antagonist plays an important role in allergic asthma, but the absence of qualitative differences at the DNA level indicates that it might not be the quantitative trait gene for the QTL AbhrI . We also have access to a human birth cohort characterized for asthma phenotypes over 10 years. We tested for the association of the human IL-1 receptor antagonist gene polymorphisms with asthma phenotypes in our birth cohort, to comparatively investigate the effect of IL-1 receptor antagonist in humans. We hypothesized that polymorphisms in the human 1L1 RN was associated with asthma and related phenotypes. i We tested three 1L1 RN SNPs for associations with asthma, chest infections, BHR and FEVl/FVC ratios. At the single SNP level, we found the SNPs to be associated with asthma at age 2 and chest infections at age 2. Haplotype pair analysis confirmed that the haplotype pair containing the minor alleles at all loci (GCT/GCT) conferred increased risk of asthma and chest infection in the children tested. Then, we tested for the effect of environmental tobacco smoke exposure on this association. We also found that maternal smoking during pregnancy coupled with postnatal tobacco smoke exposure caused several-fold increase in the risk of getting asthma and chest infection in children possessing the GCT/GCT haplotype pair. Taken together, our results suggest a major role of IL] RN in asthma and chest infections in this population. Copyright by RAVISANKAR A. RAMADAS 2006 DEDICATION This dissertation is dedicated to my parents, sister, friends and Jorge Luis Borges. ACKNOWLDEGEMENTS I thank Dr. Susan Ewart for her undaunted support for my dissertation and invaluable professional guidance and personal attention. She has been a great mentor and friend, she taught me the importance of clarity of thought and communication in a scientific career, and most importantly, helped me to acclimate to the scientific atmosphere in the United States. I cannot thank her enough. I am grateful to the valuable technical help and conceptual input from my committee members, Dr. Venu Gangur, Dr. Jack Harkema, Dr. Wilfried Karmaus and Dr. Patrick Venta, which helped shape this dissertation to its final form. Dennis Shubitowski taught me all the molecular biology techniques I know now, and was always available for healthy scientific discussions. From culinary tips to geopolitics, from music to American pop culture — I learnt a lot from him. He is a very good friend, and he made our lab a very pleasant and fun place to work — my special thanks to him. I owe a lot to Xingnan Li and Jessica Eason-Butler for their support and guidance during my initial days in the Ewart Lab. I also thank Dr. Sridhar Samineni from the Food Allergy & Immunology Lab for his help with ELISA assays. I thank Dr. P.S. Mohankumar and Dr. Sheba Mohankumar for their support and guidance all these years at MSU. I thank Dr. Vilma Yuzbasiyan-Gurkan for her excellent professional and personal support through the CMIB graduate program. I thank my friends who have made my stay. at MSU very pleasant - especially Badri, Shaiju, Galeb, Madhu, Nick, Maria, Annerose and Camilo. My parents and sister - I cannot thank them enough for their understanding, support and love. vi TABLE OF CONTENTS LIST OF TABLES ................................................................................... ix LIST OF FIGURES .................................................................................. xi LIST OF ABBREVIATIONS ..................................................................... xii Chapter One: Background and significance A. Asthma .................................................................................... 2 B. Genetics of asthma ....................................................................... 7 C. Environmental influences on asthma ................................................ 12 D. Gene-environment and gene-gene interactions ..................................... 13 E. lL-l receptor antagonist and its role in asthma ..................................... 15 F. Summary ................................................................................. 25 Chapter Two: IL-1 receptor antagonist: Mouse studies A. Mouse model of allergic asthma ...................................................... 27 B. Sequencing of the mouse IL-1 receptor antagonist gene .......................... 30 C. Transcript and protein studies in IL-1 receptor antagonist and related genes...68 D. Summary ................................................................................. 86 Chapter Three: IL-1 receptor antagonist: Human studies A. Isle of Wight birth cohort .............................................................. 89 B. IL-1 receptor antagonist SNP association studies .................................. 95 C. IL-1 receptor antagonist haplotype pair association studies ..................... 101 D. Summary ............................................................................... 109 Chapter Four: Discussion of mouse and human IL-1 receptor antagonist studies A. Role of IL-1 receptor antagonist in NJ and C3H/HeJ mice ..................... 111 B. Role of IL-1 receptor antagonist in the Isle of Wight birth cohort ............. 116 Chapter Five: Materials and Methods A. Ovalbumin sensitization and challenge ............................................. 123 B. DNA sequencing ...................................................................... 123 C. TaqMan/SYBR Green real-time RT-PCR .......................................... 126 D. Statistical analysis of mRNA expression data .................................... 130 E. Protein collection from lung tissues ................................................ 130 F. Enzyme linked immunosorbant assay (ELISA) ................................... 131 G. Pyrosequencing ........................................................................ 133 vii Appendix ............................................................................................ 140 Table 13. IL-1 receptor antagonist mRNA expression in lungs .................... 140 Table 14. IL-1 beta mRNA expression in lungs ...................................... 145 Table 15. IL-l alpha mRNA expression in lungs .................................... 148 Table 16. IlIf9 mRNA expression in lungs ........................................... 150 Table 17. IL-1 receptor type I mRNA expression in lungs ......................... 152 Table 18. IL-1 receptor type II mRNA expression in lungs ........................ 154 Table 19. IL-1 receptor antagonist protein production in lungs .................... 156 Table 20. IL-1 beta protein production in lungs ...................................... 159 Table 21. IL-1 receptor antagonist mRNA expression in spleen .................. 162 Table 22. IL-1 beta mRNA expression in spleen .................................... 165 Table 23. Isle of Wight genotypes for the SNPs rs2234678, rs878972 and rs454078 ..................................................................... 168 Biblography ......................................................................................... 1 86 viii LIST OF TABLES Table 1. Asthma or atopy genes identified using genetic approaches ....................... 25 Table 2. Primers used to sequence 111 m in NJ and C3H/HeJ mice ......................... 34 Table 3. Population characteristics of Isle of Wight birth cohort ............................ 94 Table 4. IL—1 receptor antagonist single nucleotide polymorphisms tested ................ 95 Table 5. Pairwise comparison of linkage disequilibrium for [L] RN SNPs ................. 98 Table 6. Association of IL] RN SNPs, asthma and chest infections ........................ 100 Table 7. IL-1 receptor antagonist haplotypes and haplotype pairs ......................... 102 Table 8. Haplotype pair analysis for asthma and related phenotypes ...................... 106 Table 9. Effect of smoke exposure on asthma and recurrent chest infection - haplotype pair analyses .......................................................................... 107 Table 10. Effect of illm haplotype pairs on asthma (repeated measurements: ages 1, 2, 4, 10) stratified for recurrent chest infection ....................................... 108 Table 11. Primers and probes used for real-time RT-PCR assays .......................... 131 Table 12. Primers used for genotyping by Pyrosequencing .................................. 136 Table 13. IL-1 receptor antagonist mRNA expression in lungs ............................. 140 Table 14. IL-1 beta mRNA expression in lungs ............................................... 145 Table 15. IL-l alpha mRNA expression in lungs ............................................. 148 Table 16. [1pr mRNA expression in lungs .................................................... 150 Table 17. IL-1 receptor type I mRNA expression in lungs .................................. 152 Table 18. IL-1 receptor type II mRNA expression in lungs ................................. 154 Table 19. IL-1 receptor antagonist protein production in lungs ............................. 156 Table 20. IL-1 beta protein production in lungs ............................................... 159 I Table 21. IL-1 receptor antagonist mRN A expression in spleen ........................... 162 Table 22. IL—1 beta mRNA expression in spleen ............................................. 165 Table 23. Isle of Wight genotypes for the SNPs rs2234678, r3878972 and rs454078....168 LIST OF FIGURES Figure 1. Location of IL-1 complex genes on mouse and human chromosomes .......... 18 Figure 2. Sequencing of the murine IlIrn gene ................................................ 33 Figure 3. Experimental time line of in vivo allergen exposure .............................. 70 Figure 4. The threshold cycle (CT) of TaqMan real-time RT-PCR .......................... 73 Figure 5. Protein and mRNA production profiles of IL-1 receptor antagonist in 1ungs...81 Figure 6. Protein and mRNA production profiles of IL-1 beta in lungs ..................... 82 Figure 7. Messenger RNA profile of lL-l alpha and IL-1 family member 9 ............... 83 Figure 8. Messenger RNA profile of IL-1 receptor type I and IL-1 receptor type II. .....84 Figure 9. Messenger RNA profile of IL-1 receptor antagonist and IL-1 beta in spleen...85 Figure 10. Schematic representation of the progress of the enzyme reaction in liquid- phase pyrosequencing .............................................................................. 97 Figure 11. Pyrograms from pyrosequencing .................................................... 97 Figure 12. Percentage incidence of asthma and chest infection in children with specific haplotype pairs ........................................................................... 105 xi Abhr Ach AHR AP-l BAL BHR C.I CT ECRHS ELISA FceRl F EV1 FVC GM-CSF IgE lL IL] A Illa 1L1 B 11 l b IL-1 Ra IL- 1 ra IL 1 RN 11 lm IL- 1 (1 IL- 1 0 LD MCP MIT NF-kB OR OVA PBS QTG QTL SNP VNTR LIST OF ABBREVIATIONS allergen induced bronchial hyperresponsiveness acetylcholine airway hyperresponsiveness activating protein-1 bronchoalveolar lavage bronchial hyperresponsiveness confidence intervals cycle threshold European community respiratory health survey enzyme linked immunosorbant assay immunoglobulin crystallizable fraction epsilon receptor I forced expiratory volume in 1 second forced vital capacity granulocyte macrophage — colony stimulating factor immunoglobulin E interleukin interleukin-1 alpha (human gene) interleukin-1 alpha (mouse gene) interleukin-1 beta (human gene) interleukin-1 beta (mouse gene) interleukin-1 receptor antagonist (human protein) interleukin-1 receptor antagonist (mouse protein) interleukin-1 receptor antagonist (human gene) interleukin-1 receptor antagonist (mouse gene) interleukin-1 alpha (protein) interleukin-1 beta (protein) linkage disequilibrium macrophage chemoattractant protein Massachusetts Institute of Technology nuclear factor of kappa light chain gene enhancer in B-cells odds ratio ovalbumin phosphate buffered saline quantitative trait gene quantitative trait locus single nucleotide polymorphism T helper cell variable number of tandem repeat xii 517 F3 .O E“ Chapter One: Background and significance Asthma Genetics of asthma using mouse models Genetics of asthma in human populations IL-1 receptor antagonist in asthma Summary A. ASTHMA Phenotype description: Asthma is a multifactorial chronic respiratory disorder occurring in genetically susceptible individuals due to inappropriate immune responses. The cardinal pathophysiological features of asthma are airway hyperresponsiveness (AHR), airway inflammation and elevated serum immunoglobulin (Ig) E levels]. It manifests as a chronic syndrome of the airway with recurrent wheezing, coughing, chest tightness and shortness of breath. The expression of the asthma phenotype is a result of interplay between multiple genetic and environmental factors. Gene products that contribute to the asthmatic phenotype can be derived from a variety of physiological pathways“, and exert their effect based on variations in their sequence and/or expression. Environmental factors such as allergens and smoke from various sources, exercise and season also substantially contribute to the asthmatic phenotype. The impact of genes on asthma is modified by the layers of environmental influence over the individual in a complex disease like asthmas. The definition and classification of the phenotypes in complex diseases such as asthma can vary based on the etiology, manifestation and associated symptoms6. In a random mating population like humans with a high degree of genetic diversity, this interaction between genes and environment is a critical factor in explaining the disease idiosyncrasies observed among individuals or population subgroups. Not all the mechanisms and genetic reasons for asthma are clear and remain to be elucidated. Epidemiology The prevalence, morbidity and mortality of asthma have dramatically increased in recent years. Asthma ranked 25th in the worldwide list of Disability-Adjusted Life Years lost due to common disorders7. It affects about 300 million people worldwide, and the prevalence of asthma has been shown to be higher in industrialized countries with a modern lifestyle compared to countries with traditional lifestyless. This asthma incidence gradient has also been shown to be proportional to the level of urbanization. An additional 100 million people are estimated to be affected with asthma as urbanization in the world population is projected to increase from 45% to 59% by 20259. In the United States alone, about 31 million people have been diagnosed with asthma at least once in their lifetime10 . Non-Hispanic blacks and American Indians had current asthma prevalence 30% higher than the non-Hispanic whites. Females had a 30% higher prevalence compared to males, and this pattern was reversed among children"). It is the most common chronic condition of childhood in the United States affecting about 4.8 million children, most of whom are diagnosed with asthma by 6 years of age”. Another alarming trend in the United States is the increasing rate of asthma mortality after 1970, in contrast to the declining asthma mortality rate in other western countries”. Various forms of asthma: Asthma is a multifactorial chronic inflammatory disease of the lung characterized by symptoms of recurrent episodes of coughing, wheezing and breathlessness. The manifestation of the clinical signs of asthma depends on the subject’s genetic makeup, environmental conditions and biological statuses such as age and gender. The interplay of these factors results in a multitude of features characteristic of asthma. A subject can display a few but not necessarily all of these phenotypes, and yet be clinically diagnosed as an asthmatic. Overlapping phenotypes and the lack of consensus on a combination of objective and subjective parameters necessary to draw a clinically definitive border around asthma is a major problem in airway disease taxonomy". Based on the severity of the disease, asthma has been classified into four groups - mild intermittent, mild persistent, moderate persistent and severe persistent”. Asthma is also classified based on the pattern of clinical presentation or on the suggested etiology (extrinsic vs intrinsic, occupational, aspirin induced)”"5. It can also be classified as atopic and non-atopic asthma based on the levels of IgE-mediated response. Atopy is the hereditary predisposition to develop certain hypersensitivity reactions on exposure to specific antigens. Atopic allergic asthma, the most common form of asthma, is an inflammatory disorder arising as a result of inappropriate immune responses to common environmental antigens in genetically susceptible individuals”. It is characterized by the cardinal features of airway hyperresponsiveness (AHR) to a variety of stimuli, pulmonary eosinophilia, increased mucus production and elevated serum IgE levels. Diagnosis: Physician-based subjective observations and pulmonary function test-based objective parameters are used for asthma diagnosis. Forced expiratory volume in 1 second (FEVl), forced expiratory vital capacity (PVC) and expiratory peak flow (PEF) are the important indices measured using pulmonary function tests. The ratio between forced expiratory volume in one second to forced vital capacity (FEVl/FVC) is decreased in asthmatic patients. National Institutes of Health (NIH) expert panel report on Guidelines for the diagnosis and treatment of asthma classifies the severity of asthma based on a combination of symptoms, nighttime symptoms and pulmonary function test results”. Serum IgE levels measured by Enzyme-linked Immunosorbant Assay (ELISA) and number of eosinophils in the bronchoalveolar lavage (BAL) fluid and lung tissues are also used as indicators of asthma. Airway hyperresponsiveness, the ability of the airways to constrict when exposed to small concentrations of bronchoconstrictor agents”, is a key phenotype used to model human asthma in mice. Therapy: Therapy for asthma is based on the severity of symptoms. The drugs used against asthma function through two main mechanisms. Bronchodilators aim at relaxing the airways to ensure ease in breathing, and anti-inflammatory therapies aim at reducing the inflammation that is responsible for this airway constriction. The major bronchodilators used are 02 adrenergic receptor agonists (salbutamol, terbutaline, salmeterol and forrnoterol), inhaled anticholinergics (ipatropium bromide and tiotropium bromide) and slow-release preparations of the drugs theophylline and aminophylline. [32 adrenergic receptors exist in an active form and an inactive form in vivo, and are located in a wide variety of cells such as airway smooth muscle cells, epithelial and endothelial cells of the lung and mast cells. When the receptor is in the active form, [32 adrenergic agonists bind to the receptor, increase the production of cyclic AMP (CAMP), and result in airway smooth muscle relaxation through mechanisms not fully understood”. The efficacy and duration of action of these drugs depend on tissue stochastics such the number of receptors available, and the potential functional antagonism of other bronchoconstrictors acting simultaneously on the airways. Selectivity of the agonists that bind to [32 adrenergic receptors, compared to [31 and [33 receptors, and potential side effects due to their non-specific binding on cardiac B adrenergic receptors are important factors taken into consideration for devising and usage of this class of drugs"). There are a variety of drugs that target the anti-inflammatory mechanisms involved in asthma. The major classes of drugs that are currently used to treat the inflammatory component of asthma are inhaled corticosteroids (budesonide, fluticasone propionate, beclomethasone dipropionate and mometasone), antileukotrienes (monteleukast, pranleukast and zafirleukast), 5-lipoxygenase inhibitors (zileuton), cromones (sodium cromoglycate and nedocromil sodium) and anti-IgE (omalizumab). Corticosteroids have been shown to inhibit histone acetylation and promote histone deacetylation in the chromatin, resulting in the transcriptional suppression of many pro-inflammatory transcription factors such as AP-l and NF-KBZO'ZZ. Leukotriene inhibitors and 5-lipoxygenase inhibitors help to reduce the asthma-like pathophysiological responses resulting from leukotrienes and other products from the 5- lipoxygenase pathway. This class of drugs might have particular advantages over the other drugs in the treatment of exercise-induced asthma and aspirin-induced asthma”. The exact anti-allergic mechanisms of cromones are not known with certainty“. The recently developed anti-IgE drug omalizumab binds to the crystallizable fraction (Fc) of IgE and prevents its binding to the high affinity immunoglobulin crystallizable fraction epsilon receptor 1 (FcERl) present on the mast cells. This prevents the degranulation of the mast cells and the type I hypersensitivity reactions that results from the inflammatory mediators secreted from the degranulated mast cells25 . Only a few new asthma treatment drugs have reached the clinic in the past few decades. A combination of inhalable corticosteroids and long-acting [32 agonists seems to be the treatment of choice at present and in the near future, although several other molecules in other pathophysiological pathways are being investigated26. B. GENETICS OF ASTHMA It is well established that asthma is under the control of both genetic and environmental influences. The relative contribution of these two influences might vary between populations. In humans, family history, twin studies and segregation analyses are used for the focused investigation of the genetic component of asthmazms. Major genetic approaches used for asthma gene discovery: Candidate gene and genome-wide screening approaches have been used in animal models and human studies to determine the genes responsible for asthma (Table 1). Candidate genes are selected based on their functional relevance to asthma, and hence the process depends on selecting and characterizing one or several genes whose role has been implicated in a functional pathway leading to the asthma phenotype. Genome screens, unlike the candidate gene approach, do not need a priori knowledge about genes or their functional relevance to the disease under investigation. In humans, genetic markers throughout the genome are genotyped in family members to identify chromosomal regions that are co-inherited (‘linked’) with specific phenotypes such as asthma, bronchial hyperresponsiveness (BHR) or a positive skin prick test (SPT)4. In animal models of asthma, phenotypes such as airway hyperresponsiveness (AHR), the rodent corollary to BHR, can be treated as quantifiable continuous traits. Genome screens performed in segregating backcrosses of inbred strains of mice for a number of genetic markers equally spaced across the genome identifies chromosomal regions on the genome that are responsible for regulating such quantitative traits, and the loci thus identified are termed quantitative trait loci (QTLs). These QTLs, whose statistically defined boundaries cover several megabases of genomic DNA, contain tens to hundreds of genes. This is then followed up with fine-mapping of the linked regions, followed by positional cloning or positional candidate cloning to identify specific genetic variations in one or more genes within the fine-mapped region that are responsible for genetic susceptibility. Recent advances in asthma gene discovery approaches: While the discovery of broad genomic regions that control susceptibility to asthma and other complex diseases has been relatively easy, progression from those regions to specific gene(s) controlling the phenotype has been difficult. A variety of genetic, molecular and bioinformatics approaches have been suggested, and are being used to determine quantitative trait genes (QTGs) in animal models”. These approaches use the flexibility animal models offer in terms of breeding, availability of genetic information in the public domain and amenability to phenotype studies. Genetic studies in humans are accelerating faster than ever, and the current research largely makes use of the genetic variability available in humans in the form of single nucleotide polymorphisms (SNPs). Any two human genomes differ from each other by 0.1% of the nucleotide sequences (on an average of 1 variant per 1000 basepairs of DNA in the genome)30’32. The most common variation in the human and other mammalian genomes sequenced are SNPs, and gene-based analysis of SNPs and their assocrations with disease phenotypes, such as asthma, have been at the forefront of genetic studies in asthma33'37. Hapmap, a global effort to catalogue and classify these polymorphisms from populations around the world38'40, has opened the way for genome- wide association studies. Genome-wide association studies aim at genotyping millions of SNPs spread over the entire genome in individuals who have the disease and those who don’t have the disease4'43. This is a logical extension of single gene association studies, with the difference being that a majority of the genes in the genome are simultaneously investigated instead of a single gene or a few genes at a time. Though at present the costs are prohibitive, it is considered to be a strategy for the future, at least for the next decade. Types of association: Association studies begin with genetic analysis of samples from a population where incidence of the investigated disease is reasonably common. The individuals in the population are then genotyped for several polymorphisms in the candidate genes, and the frequency of the alleles, genotypes and haplotypes are determined. Association studies rely on the detection of such polymorphisms in candidate genes and on the demonstration that particular polymorphisms are associated with one or more phenotypic traits“. The first possibility with a positive association is that an allele might directly influence the phenotype by causing a functional change at the genomic, mRNA or protein level. The other possibility is that an allele associated with the phenotype in the study might have been co-inherited (linked) with another allele in the vicinity or in another locus, which is the real functional regulator of the disease phenotype. When two alleles are indirectly related in this manner, they are said to be in ‘linkage disequibrium’, which is a widely used approach to determine allelic associations. The success of an association study depends on the population investigated and also the statistical and genetic techniques used for analysis. The population tested should be a random-mating population, which can be tested by the conformity of the selected SNPs to Hardy-Weinberg equilibrium. Hardy-Weinberg equilibrium states that in a random-mating population, the gene and genotype frequencies remain constant from generation to generation in the absence of migration, mutation and selection. The frequencies of the disease causing alleles might be different in different populations, and hence replication of results in different populations is difficult. Moreover, the associations could also be confounded by population stratification, in which the investigated population consists of a mixture of two or more subpopulations that have different allele frequencies and disease risks44‘45. Presence of multiple disease-causing alleles in a gene (allele heterogeneity) and presence of multiple disease-causing genes or loci in a disease (genetic or locus heterogeneity) also influence the power of the reported 6’47. The major statistical constraint faced in genetic assocration studies are associations inflated type I error rates due to multiple hypothesis testing. Type I errors, which occur when a null hypothesis is rejected when it is true, usually result in false positive associations. Such false positive results are compounded by the problem of multiple hypotheses testing in association studies, which involve large number of subjects, SNPs 10 and phenotypes. To reduce false positives resulting from this problem, the resulting significance values are adjusted for Type I errors due to multiple testing using conventional tests such as Bonferroni correction or by Bayesian approaches44'48. Apart from these, errors in genotyping could result in another major constraint in determining accurate genotype data for association studies”. These problems can be circumvented by carefully selecting the population, stringent definition of phenotypes investigated, efficient genotyping methods and applying suitable statistical tests to reduce type I error rates. Types of populations used in genetic association studies: Genetic association studies in human populations are performed either as cross- sectional case-control studies or longitudinal cohort-based studies. In the former approach, allele, genotype or haplotype frequencies in a population are compared between a set of people affected with the disease (cases) and a set of people not affected with the disease (controls). This approach provides a snapshot of the gene-phenotype association at a particular age in which the disease was diagnosed in the population investigated. However, this approach has a marked disadvantage because, being a chronic disease, asthma manifests as a combination of phenotypes over the various stages of life of an individual. Therefore investigation of the effect of the candidate genes on the disease at various ages of an individual would provide valuable information about the trajectory of the disease and its severity, and shed light on the most suitable points of therapeutic intervention. Longitudinal cohort-based genetic studies satisfy this need by investigating if a gene is associated with a phenotype over a period of time or at a 11 specific period during the progression of the disease. This helps to develop preventive strategies by elucidating the earliest stage in which gene-specific therapeutic intervention is possible in relation to a specific asthma pathway. Results from the data accumulated in this manner will be of tremendous importance in asthma-specific gene therapies in the future. Associations reported so far: While candidate gene studies provide definitive evidence for the role of a particular gene in asthma, the genome screen approach offers the additional possibility that novel genes, whose roles haven’t been previously implicated in the asthmatic process can also be identified. Several asthma regulatory loci have been identified on the human and mouse genomes so far, especially on human chromosomes 5 (containing lL-4, IL-5, IL-13 and GM-CSF genes), 6 (the MHC gene cluster) and 11 (chRl-B, the 0 chain of the high affinity receptor for IgE)50'5 l , all of which are important in the pathophysiology of asthma. These genetic approaches have recently been used to identify several asthma- influencing genes (Table 1, reviewed in”). More than 60 candidate genes have been investigated using this method“, and this approach can be used to identify and confirm the validity of candidate genes directly in human populations, and also to confirm the results from animal studies. One such recent confirmation across species is a polymorphism in the myostatin genesz'54 , which is responsible for downregulating the muscle mass formation. A polymorphism in the myostatin gene resulted in a splice site disruption, preventing the formation of myostatin protein and resulting in increased muscle mass in mice and humans, and double-muscling in cattle. Such polymorphic asthma-influencing 12 genes, if identified in mice and confirmed in humans or directly identified in humans, may prove to be excellent therapeutic targets to counter the disease process of asthma. C. ENVIRONMENTAL INFLUENCES ON ASTHMA: Environmental influence is an important dimension in the conceptual scaffold of asthmass. While animal models can be investigated under controlled environmental conditions ranging from specific pathogen free environments to selective environmental exposures, the same is not possible in humans. Depending on personal and community lifestyle and geographical location, humans are influenced by a variety of indoor and outdoor environmental factors that lead to their susceptibility or resistance to asthma. Environmental influences on asthma are studied under the framework of several hypotheses“. Probably the most widely investigated of these is the hygiene hypothesis proposed by David Strachan in 1989”. It suggested that infection in early childhood, transmitted by unhygienic contact with older siblings, or acquired prenatally from a mother infected by contact with her older children could prevent the development of allergic symptoms. This view has been supported by studies that reported a decreased incidence of asthma and atopy in children living under farming conditionssg'60 , where chances of being exposed to such protective influences are high. Von Mutius et al., reported that children in former West Germany had more asthma and atopy prevalence than children from former East Gemanym, showing that level of industrialization is an important factor in asthma prevalence. 13 62-65 Environmental exposure to a variety of allergens , or pollutants like ozone, sulphur dioxide, nitrogen dioxide and diesel exhaust particles can strongly incite or accentuate asthma-like symptoms‘r’é"73 . Climate changes have also been shown to influence asthma symptoms, such as bronchoconstriction due to inhalation of cold air. Period of thunderstorms have also been shown to be associated with increased incidence of asthma attacks, possibly due to bursting of pollen and the release of paucimicronic allergenic particles in the atmosphere74’77. It has been shown that the prevalence of asthma is much higher during childhood in males and conversely higher post-puberty in females. The in utero and postnatal influences such as maternal smoking and breastfeeding are also being extensively investigated as important factors in asthma susceptibility78'82. D. GENE-ENVIRONMENT INTERACTIONS: While underlying genetic factors play an important role in asthma, they also interact with one or more environmental factors to influence the outcome of the final phenotype. While this gene-environment interaction plays a very important role in asthma studies in human populations, this effect of environment can be controlled to a major degree in animal models, such as rodent models of asthma. By housing the experimental and control animals under the same environmental conditions, the phenotypes observed in the animal models can be largely attributable to genetic factors. The concept of gene- environment interactions is used to explain the situations where a particular gene is associated with the disease in some populations, but not others. 14 The essence of such gene-environment interaction effects have been captured most successfully by the hygiene hypothesis”. The hygiene hypothesis is supported by the fact that asthma incidence has increased severalfold over the last few decades84 especially in urbanized industrial lifestyles. This poses some interesting questions and provides new perspectives about the etiology of asthma. While it is possible that misdiagnosis and underreporting of cases in the previous decades could be a factor in this surge, it is less likely that such factors would significantly change the observed increasing trends. Moreover. it is also less likely that this surge in asthma incidence over the last few decades could be solely by genetic factors, because the amount of causal genetic variation required to bring about such an increase in incidence could not have been introduced in 85'87. A paradigm such a short interval of time in a random mating population like humans that explains this temporal variation that integrates the effects of genes and environment is epigenetic variation. Heritable short term alterations not involving changes in the nucleotide sequence resulting in disease phenotypes are classified as ‘epigenetic’ changes”. Epigenetic changes can occur due to several factors such aging and diets that 89-92 supply methyl groups for metabolic enzyme activities . The most extensively investigated epigenetic changes are methylation of nucleotides in the DNA sequence”, and modification of histone proteins94 that surround the DNA sequence to form the chromatin structure. Both these modifications have been shown to influence asthma by modulating transcription factors like NF-kB, which play a major role in asthma pathophysiology95'99. As asthma is a complex disease driven by multiple genes, interaction between the genes influencing asthma is also gaining importance. In these lines, interactions between 15 interleukin-13 (1L13) and interleukin-4 receptor alpha chain (IL4RA) genes have been shown to be associated with asthma'oo’m' . Thus, asthma and associated phenotype manifestations result from genes, environment and epigenetic factors, which interact within and between themselves in multiple combinations. E. INTERLEUKIN-l RECEPTOR ANTAGONIST AND ITS ROLE IN ASTHMA: This dissertation research is based on the results obtained from an asthma linkage study performed by Ewart et al16 in a murine model of allergic asthma with A/J (asthma hyperresponsive) and C3H/HeJ (asthma hyporesponsive) mouse strains. This study identified two QTLs on mouse chromosome 2, which control allergen induced bronchial hyperresponsiveness (A bhrI and Abhr2). Positional candidate genes within each of these regions were chosen for further investigation to determine if those genes are responsible for the difference in airway hyperresponsiveness between these two strains. While complement factor 5 (C5) has been shown as the susceptibility gene for the locus AbhrZ'OZ, such a gene has not been identified for the locus Abhr]. The murine IL-1 receptor antagonist gene (111m) is located within the Abhr] QTL, and based on its functional relevance in asthma as explained in the subsequent sections, it was chosen as the positional candidate gene for investigation of the QTL Abhr] . The interleukin-1 (IL-1) gene complex consists of two agonists, IL-la and IL-1 [3. Both these agonists have similar functions, and the IL-IB gene (11 1 b) is hypothesized to be a reverse-transcriptase mediated duplication product of the gene for IL-la (111a)I03 . Apart from the agonists, the complex consists of genes for the functional receptor (IL-1 receptor type I — IL-lRI), a decoy receptor (IL-1 receptor type II —IL-1RII), and an 16 antagonist (IL-1 receptor antagonist — IL-lra). In mice, the genes for these products are indicated by the symbols [1 1 r1 , II 1 r2 and 111m respectively. Recently, several new members (I! 1f5 — IlIf10) have also been identified and added to the IL-1 gene complex. In the humans, all the genes encoding these proteins (ILIA, [L] B, [L1 RN, 1L1 R1, 1L1 R2 and IL] F5 — 1L1F10) are located in the long arm of chromosome 2. In the mice, III r1 and III r2 are located in chromosome 1, while all the other genes are located in chromosome 2 (Figure l). Interleukin-1 receptor antagonist is a major anti-inflammatory cytokine in the IL- ] cascade involved in a variety of chronic diseases like asthma, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease'm'los. The functional significance of IL-lra in asthma pathophysiology can be viewed better in the context of other members of the IL-1 complex, especially the agonists IL-la and IL-IB. IL-lra abrogates the pro- inflammatory effects of IL-1, and hence the mechanistic perspectives on IL-1 receptor antagonist function have always coexisted with those on IL-1. The mechanism of IL-1 receptor antagonist activity: Human ILIRN gives rise to two different isoforms, an intracellular (ic) and an extracellular (ec) isoform. They are created by the alternative splicing of different first exons; the first exon of the intracellular isoform is located ~9.4 kb upstream from the first exon for the extracellular isoformm. The intracellular isoform lacks a functional leader signal peptide, and remains in the cytoplasmm. Two additional intracellular isoforms have also been described'm‘mg. The longest transcript in the human IL-1 receptor 17 antagonist gene (NCBI Refseq mRNA: NM_173841) contains six exons, and the longest transcript in the murine gene contains five exons (NCBI Refseq mRNA: NM_031167). Mouse Chr.1 Mouse Chr.2 Human Chr.2 . . r111m 111 1 Abhr1{ «— ”U5 4— r [11f6 {IlIr2 Abh’Z { < Illj8 111/9 (ILIRN KIWI” IL1F5 — 10 . ILIA <—) ILIB ILIRI LILIRII Figure 1. Location of lL-l complex genes on mouse and human chromosomes. Abhr -— Allergen induced bronchial hyperresponsiveness. . — centromere. The IL-lRI binds with the IL-1 receptor accessory protein (IL-RAcP) to form a dimer on the cell surface, which acts as the functional receptor complex and transduces signals on agonist ligand (IL-la or IL-lB) binding. On the contrary, when IL-lra binds to ILl-RI, it does not elicit any downstream signal transduction. It has been shown that IL- lRAcP is also a critical factor for IL-1 mediated signal transductionlog'm. The interactions between these proteins and the signal transduction mechanisms have been 18 elucidated by studies of their crystal structures. lL-lRl consists of an intracellular Toll IL-1 receptor (TIR) domain and three extracellular immunoglobulin (lg) domains. Crystal structures of IL-lRI bound to IL-lB”2 and IL-lRaH3 have shown that the first two Ig domains in the receptor are tightly linked, whereas the third domain was separate and connected to the first two domains by a flexible linker. When IL-IB binds to IL-lRI, it binds to all the three lg domains. The receptor then wraps around IL-IB and this is thought to result in the dimerization of the receptor with IL-lRAcP, resulting in signal transduction. On the contrary, IL-1 receptor antagonist binds only to the first two lg domains and not to the flexible third domain, hence the receptor could not wrap around the ligand and this could be the reason for the lack of signal transduction in this situation (reviewed inl 14). Another mechanism of IL-1 antagonizing is mediated through the decoy receptor (IL-IRII), which has three extracellular Ig domains, but lacks the cytoplasmic TIR domain critical for signal transduction. IL-lRII is released from the cells, binds to IL-1 and limits the binding of IL-1 to the functional receptor IL-lle and limits the availability of IL-lRAch’. Moreover, IL-lRII has only a very weak affinity to IL-1 receptor antagonist'”, and hence it does not hinder the anti-inflammatory properties exerted by the actions of IL-1 receptor antagonist. Thus, both IL-1 receptor antagonist and IL-lRII act as independent anti-inflammatory mechanisms. IL-1 receptor antagonist was chosen for this study because it was located within the region of genetic linkage observed in our mouse study”, and the other major members of the IL-1 complex were not chosen because they were not located inside the region of genetic linkage. 19 The role of IL-1 receptor antagonist in asthma As mentioned previously, the role of IL-1 receptor antagonist in asthma can be most efficiently explained from the perspective of its counter-regulatory capacity on the pro-inflammatory effects of IL-1. Interleukin-1 is directly involved in both the major stages of disease progress —- airway hyperresponsiveness and inflammation. Although asthma typically involves reversible airway obstruction, in some cases it becomes irreversible due to airway remodeling”. Accumulation of inflammatory mediators and growth factors burden the airways with additional workload, and this might lead to these irreversible changes in the airways that hamper the normal breathing capacity. IL-1 receptor antagonist serves to endogenously counter the pro-inflammatory effects of IL-1, and is also a suitable molecule for the therapeutic management of asthma'lg’uo. Being a pleiotrophic cytokine, IL-l seems to exert its effect at various stages of asthma pathophysiology. Hence its role is not in essence restricted to one specific pathway that leads all the way from IL-l to asthma, and it has been shown to be involved in many pathways that lead to asthma. The anti-inflammatory role of IL-1 receptor antagonist also should be viewed from a similar perspective. 0 IL-1 receptor antagonist in asthma - Functional evidences: IL-l , on binding to the functional receptor, IL-lRI, stimulates the expression of a large number of proinflammatory proteinsl”. IL-1 is one of the first wave cytokines, along with TNF-a and IL-6, that may be released on exposure to inhaled allergens via FceRII receptorsz. Studies by Nakae et al. utilizing IL-1u'/[3' or IL-lra deficient mice in an ovalbumin (OVA) exposure model demonstrated that allergen-induced AHR, OVA- specific T-cell proliferative responses and the levels of Th2 cytokines IL-4 and IL-5 were 20 significantly decreased in IL-la/B'/' mice, but were significantly increased in IL-lra'/' mice compared to the wild-type mice12 '. This study showed that IL-1 and IL-lRa have a direct effect on AHR, the major phenotype tested in animal asthma models, and also on T helper 2 (Th2) cell cytokine expression. On similar lines, in a guinea pig model of pulmonary anaphylaxis, IL-lra has been shown to inhibit bronchoalveolar lavage fluid inflammatory leukocyte influx and antigen-induced airway hyperreactivity to intravenous substance P in a time dependent mannerm. IL-lra pretreatment reduced the generation of late asthmatic responses in terms of pulmonary resistance and reduced the presence of hypodense eosinophils in the bronchoalveolar lavage fluid in another guinea pig model, where Ascaris antigen was used for sensitization123 . In a toluene-diisocyanate model of murine allergic asthma, blocking IL-1 activity attenuated AHR and inflammationm. Similarly, IL-1 receptor antagonist has also been shown to attenuate AHR following 125. Thus, IL-lRa is able to decrease AHR induced by a variety 0f exposure to ozone antigens in both allergic and non allergic animal models of asthma. In humans, the levels of IL-IB in BAL fluid from patients with asthma were found to be increased compared with those of non-asthmatic volunteers126 and increased levels of both IL-IB and IL-lra have been identified in asthmatic bronchial epitheliumm. As IL-lB and IL-lRa are co- regulated, it has been suggested that the ratio between them could also be an important ‘28. These studies have either demonstrated the effect of IL-1 factor in inflammation receptor antagonist on AHR, or provided a snapshot of IL-1 receptor antagonist in asthmatic conditions. The molecular mechanisms behind these results have also been examined, mostly using in vitro studies. The allergic component of asthma is characterized by the IgE 21 dependent triggering of the mast cells and the subsequent release of inflammatory mediators. IL-1 has been shown to induce a variety of pro-inflammatory cytokines such 129. Activated mast cells have also been as IL-5, IL-6 and IL-9 from the murine mast cells shown to express IL-13'30, a central cytokine mediator of asthmam’m. IL-1 treatment increased the expression of IL-13 through 3 NF -KB dependent mechanism, and increased IL-13 promoter activity and mRNA stability in human mast cellsm. IL-l results in the production of a NF-KB'”, and NF-KB has been shown to be critical for the expression of the Th2 cell specific transcription factor GATA-3'35. GATA-3 binds to the promoter regions of the Th2 cytokines IL-4, IL-5 and IL-13, induces their expression and increases allergic inflammation'36'139. These results establish the functionally relevant role of IL-1 receptor antagonist in the inflammatory component of asthma. Changes due to asthma also include changes in airway smooth muscles, inflammatory mediators, airway epithelial and subepithelial damage. Contrary to the notion that airway obstruction in asthma is reversible, it is now beginning to be accepted that in certain asthmatic conditions the obstruction may be irreversiblemo. Airway smooth muscle cell hyperplasia, subepithelial fibrosis, bronchial neovascularization and pro- inflammatory exudates from the smooth muscles are some major factors that drive this airway remodeling“. In the smooth muscles, IL-1 has been shown to increase the expression of granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP) -1, MCP-2, MCP-3, RANTES and eotaxin'“. IL-IB has been shown to reduce the airway smooth muscle response to bronchodilator agonists 2 operating through the Bz-adrenergic receptorsl4 , and cause airway thickening, subepithelial fibrosis and mucus cell metaplasiam. Extracellular regulated kinase (ERK) 22 as well as p38 mitogen associated protein kinase (MAPK) and Jun n-terminal kinase (JN K) pathways have been identified as major regulators of lL-IB induced aiway smooth muscle constriction and proliferationm. IL-la has been shown to induce the activation of the p38 MAPK, and result in the inhibition of glucocorticoid receptor function. lL-l mediated contractile responses to acetylcholine was ablated on pretreatment with IL-1 receptor antagonist in human atopic asthmatic smooth muscle cellsm‘m. 0 IL-1 receptor antagonist in asthma - Genetic evidences: Only a few genetic studies have investigated the role of human ILIRN polymorphisms in asthma and related phenotypes. Gohlke et al., found significant association of ILIRN polymorphisms with asthma in a German population, and the results were also confirmed in an independent Italian populationm. This study was performed in collections of father-mother—affected child trios from Germany, Sweden and Italy where one or neither of the parents had confirmed clinical asthma. The association 8 consisting of adult individuals was later reconfirmed in another German populationl4 participating in the follow-up of the European Community Respiratory Health Survey (ECRHS). The second intron of ILIRN contains variable numbers of an 86-bp tandem repeat (VNTR)149, and five alleles (alleles 1-5) have been described for this polymorphism. ILIRN*2 allele has primarily been associated with diseases of epithelial cellsm. The ILIRN*2 allele is associated with non-atopic asthma, while asthmatics and non-asthmatics possessing the IL] RN*2 allele had significantly lower serum IL-lra levels in a Japanese population'zs. The genotype combination containing homozygotes of ILIA *1 (lLlA SNP+4845; GG genotype in a G/T polymorphism in exon 5), ILIB*1 (SNP +3954; CC genotype in a C/T polymorphism) and IL] RN*2 was associated with the 23 highest risk of skin prick test positivity'so. A homozygous genotype for the G allele in the ILlA +4845 (G/T) polymorphism was associated with nasal polyposis, a chronic inflammatory disease often found coexisting with asthmam. ILIA, 1L1 B and ILIRN are located on chromosome 2 in both mice and humans (Fig. 1). Due to the complex biology of the IL-1 signaling system, it is possible that individual or a distinct combination of alleles from ILIA, [L] B and IL] RN might determine the susceptibility or resistance of an individual to asthma directly or by regulation of other cytokines involved in the inflammatory process. 24 Table 1. Asthma or atopy genes identified using genetic approaches (adapted froms) Gene Location Name Genes identified by positional cloning followifl linkage studies ADAM33 20p13 A disintegrin and metalloproteinase-33 PHFII 13q14 Plant homeodomain zinc finger protein 1 l DPPIO 2ql4 Dipeptidyl peptidase 10 GPRA 7p15-pl4 G-protein-related receptor for asthma ' HLA-G 6p21 Human leukocyte antigen G C YF1P2 5q33 Cytoplasmic fragile X mental retardation protein interacting protein 2 Genes identified by candidate gene studies and replicated in 25 samples 1L4 5q3] lL-4 ILI3 5q31 lL-l3 ADRBZ 5q32-q34 Adrenergic receptor [3 2 TNF 6p21 TNF LTA 6p21 Lymphotoxin a HLA -DRBI 6p21 HLA-DR FCERBI 11q13 Beta chain of the high-affinity Fc receptor for lgE IL4RA l6p12-pl l lL-4 receptor a chain Genes identified by candidate gene studies and replicated in 2—4 samples [“0 lq31-q32 IL-lO CTLA 4 2q33 Cytotoxic T lymphocyte antigen 4 CCR5 3p21 CC chemokine receptor 5 C014 5q3] Cluster of differentiation antigen 14 LTC4S 5q35 Leukotriene C4 synthase NOS3 7q36 Nitric oxide synthetase 3 CC I 0 11q12-ql3 Clara cell secretory 10 kD protein STA T6 12q l 3 Signal transducer and activator of transcription 6 IFNG 12q14 lFN-y NOS] 12q24 Nitric oxide synthetase l CARD/5 16q12 Caspase-recruitment domain containing protein 15 RANTES l7q1 l-q12 Regulated on activation, normal T cell expressed and secreted SCYA I] 17321 Small inducible cytokine Al 1 Genes identified by candidate gene studies and replicated in 2—4 samples since 2003 TLRIO 4p14 TLRIO SPINK5 5q32 Serine protease inhibitor Kazal type 5 ILIZB 5q3 I-q33 lL-IZB TIMI 5q33 T cell immunoglobulin- and mucin-domain-containing molecule 1 TLR4 9q32-q33 TLR4 1Ll8 llq22 lL-18 C YSL TR2 13ql4 Cysteinyl-leukotriene receptor 2 PTGDR 14q22 Prostanoid DP receptor ITGB3 l7q21 lntegrin B3 TGFBI 19q13 TGFjil 25 SUMMARY Asthma is a chronic airway inflammatory disease due to inappropriate immune responses to common environmental factors. It is characterized by AHR, eosinophilic infiltration, airway obstruction, increased mucus production and increased serum IgE levels. Asthma is controlled by genetic and environmental factors, and their interactions. IL-1 receptor antagonist plays a protective role in asthma by inhibiting the pro- inflammatory cytokine IL-l. Our mouse model for allergic asthma is comprised of airway hyperreponsive (AU) and hyporesponsive (C3H/HeJ) strains. Genetic linkage analyses performed in A/J backcross mice ((A/J x C3H/HeJ) F 1 x A/J) identified two quantitative trait loci for AHR(Abhr1 and A bhr2) on mouse chromosome 2. The murine IL-1 receptor antagonist gene is located within Abhr] . We hypothesized that genetic polymorphisms in 111m were responsible for the difference in AHR manifestation between NJ and C3H/HeJ strains. Hence, based on the genetic and functional evidences, we investigated 111m as a positional candidate for allergic asthma in our mouse model at DNA, mRNA and protein levels. We also have access to a human birth cohort characterized for asthma phenotypes over 10 years. We tested for the association of the human IL-1 receptor antagonist gene polymorphisms with asthma phenotypes in our birth cohort, to comparatively investigate the effect of IL-1 receptor antagonist in humans. We hypothesized that polymorphisms in the human 1L1 RN was associated with asthma and related phenotypes. 26 Chapter 2. IL-1 receptor antagonist -— Mouse studies A. Mouse model of allergic asthma B. Sequencing of mouse IL-1 receptor antagonist gene C. Transcript and protein studies in IL-1 receptor antagonist and related genes D. Summary of the mouse study 27 A. MOUSE MODEL OF ALLERGIC ASTHMA 0 Animal models of asthma Animal models of asthma are primarily used to investigate the pathophysiologic mechanisms in human asthma. Investigation of complex genetic traits such as asthma in humans is difficult because of various factors like genetic heterogeneity, phenocopies and 152 incomplete penetrance . Mice, rats, guinea pigs, rabbits, ferrets, cats and horses are '53, of which the mouse has been the most some of the existing animal models of asthma widely used for investigation of asthma pathophysiology. The mouse model has several advantages, the most important of which are the availability of the complete sequence of the murine genome and the availability of several inbred strains. Inbred strains of mice differ markedly in their susceptibility to asthma, which can be effectively used to identify novel genes that contribute to the different susceptibilities. All the individuals in any single inbred strain are genetically identical (homozygous alleles at all loci), and this reduces the problem of genetic complexity in an outbred population like humans. Inbred mice have a short life span (1.5-2.5 years), early sexual maturity, short generation '54. Moreover, availability of knock-out mice and reagents intervals and good litter size required for immunological investigation facilitate the rapid investigation of asthmatic phenotypes. Ethical considerations limit the type of experiments that could be done in humans, and the techniques like bronchial biopsies and BAL fluid analyses are not free from risk for the patient. Moreover, the ease of taking samples from organs like bronchioles or pulmonary parenchyma and the ability to conduct temporal studies also make animal models better suited for investigation of asthma than humans153 . For example, the causal link between systemic IL-5 and eosinophil recruitment to airways'ss, 28 the central role of the pro-asthmatic cytokine lL-13'3l and the transcription factor T-bet'56 were first established in mice. Mice are also used to study the relationship between the inflammatory processes in the upper and lower airways, facilitating the investigation of the so-called ‘united airway disease’ concept (reviewed in'57 ). Despite all these advantages in the mouse models, it has been suggested that in vivo models can only be used to model one or more traits of human asthma, and the possibility of having an overall model of asthma should be treated with caution“. However, information obtained from inbred mouse models is invaluable to reveal the common pathways of human diseases, and identify novel therapeutic targets for the management of asthma. 0 AA] and C3H/HeJ inbred mouse model The animal model used in this study consists of two strains of mice.— AU and C3H/IleJ, modeled for the traits AHR, IgE levels and eosinophilic infiltration. The QTL controlling airway responsiveness to acetylcholine (Ach) without allergen sensitization or challenge was mapped to chromosome 6 in this model by Ewart et al15 9. The same model was subjected to the following allergen challenge protocol. Both the strains of mice were sensitized with an intraperitoneal injection of OVA or phosphate buffered saline (PBS) at day 0, and a tracheopharyngeal instillation of OVA or PBS was used to challenge the sensitized mice on day 14. AU mice showed increased levels of AHR, pulmonary eosinophilia and serum IgE levels compared to C3H/HeJ mice in the OVA treated group on day 17”. Of those phenotypes, AHR was pursued for the subsequent linkage analyses because it is an outcome that most closely mimics the clinical manifestations of asthma. The time-integrated rise in peak inspiratory pressure subsequent to intravenous 29 acetylcholine challenge was calculated and reported as airway pressure time index (APTI)"’°. Without OVA treatment, A/J mice had higher APTI than that of C3H/HeJ mice. Following OVA treatment, the APTI of NJ mice increased sharply relative to that of PBS treated A/J mice. Contrary to this observation, no significant changes occurred in the C3H/HeJ mice following OVA treatment. The APTI of the F1 mice (A/J x C3H/HeJ or C3H/HeJ x NJ) was intermediate to the parental strains and no changes occurred following OVA treatment”. The APTI distribution in AU backcross mice was broad, covering those of A/J and C3H/HeJ mice; therefore they were suitable for linkage analysis. To determine the chromosomal locations that control the allergen-induced AHR, a genome wide linkage analysis was performed in A/J backcross mice by using microsatellite markers spaced at approximately 10 cM intervals. Two QTLs were found on chromosome 2: Abhr] (between D2Mit359 and D2Mit416, Lod score = 4.2) and Abhr2 (between D2Mit238 and DZMit298, Lod score = 3.7). The QTL Abhr2 has been resolved to a quantitative trait gene (QTG), which is the complement factor 5 (C5) by Karp et all”. The gene encoding IL-1 receptor antagonist (111m) maps within the Abhr] region. It was chosen as the positional candidate gene for Abhr] based on the genetic evidence and its functional relevance in the asthmatic pathway. 30 B. SEQUENCING OF THE MOUSE IL-l RECEPTOR ANTAGONIST GENE: 0 Introduction According to National Center for Biotechnological Information (NCBI), the murine IL-1 receptor antagonist gene has been mapped to 10.0 cM on chromosome 2. Refined linkage mapping done in our laboratory (Li et al — unpublished results) showed that the sequence-tagged site (STS) marker D2Mit60, located in 111m, was mapped to the QTL Abhr]. The physical map available from Ensembl (http://www.ensembl.org/) showed that IIIrn is located from 24,269,046 bp to 24,283,646 bp on mouse chromosome 2. As 111m has been shown to have a significant role to play in the pathophysiology of asthma (Chapter 1, section B), it was chosen as a positional candidate for allergen- induced AHR in our mouse model of asthma. To investigate the role of 111m in allergen-induced AHR, the first step was to compare its gene sequences between NJ and C3H/HeJ mice to determine whether DNA polymorphisms were present. Polymorphisms could be of several kinds - repeats of a short stretch of nucleotides (microsatellites), repeats of a long stretch of nucleotides (minisatellites or VNTR), or single nucleotide polymorphisms (substitution, insertion and deletion). These polymorphisms could be present in the introns, exons or the regulatory regions of the gene. Murine 111m spans across a region of ~14.5 kb on chromosome 2. Prior studies in humans have demonstrated that alternative splicing of two different first exons of the 111m mRNA produces a secretory protein containing a leader sequence (sIL-lra) and an intracellular isoform (icIL-lra), which lacks the leader sequence and remains intracellular '04. The first exon of the intracellular isoform lies ~9.4 kb upstream from the first exon of 31 the extracellular isoform'o", and both the isoforms have been shown to be regulated by separate upstream regulatory elements'6"'62. The most proximal regulatory region in human ILIRN is the promoter for the intracellular isoform of ILIRN. A 1.8 kb region upstream of the first exon of intracellular ILIRN has been shown to be critical for its promoter activity and has binding sites for several transcription factorsm. Similarly in the mice, the most proximal regulatory sequences for the intracellular isoform of murine IlIrn have been shown to be located in the -598 and -288 bp region upstream of the transcription start site'63 . o Sequencing of 111m in NJ and C3H/HeJ mice Primers were designed to sequence introns, exons and regulatory regions of both the intracellular and extracellular isoforms, spanning a total length of ~16 kb (Figure 2). Genomic sequence of 111m (mCG4837) from Celera (www.celeradiscoverysystem.com) was used to design the primers (Table 2). The Celera IlIrn sequence was a consensus sequence from 129x1/SvJ, 129Sl/SvlmJ, DBA/2J and AH mouse strains. Primers designed covered the most proximal regulatory regions described in the literature so far, and the regulatory region for the secreted isoform was included in the intron between the first exons of the intracellular and the extracellular isoforms. Genomic DNA isolated from the kidneys of NJ and C3H/HeJ mice were used for sequencing. Sequencing was performed at the MSU Research Technology Support facility (http://genomics.msu.edu) using fluorescence-labeled dideoxy sequencing method. A total of 16 kb of genomic DNA encompassing film was sequenced in both the strains, and the sequences were submitted to NCBI. The Genbank accession numbers for the submitted sequences are DQ383807 (NJ) and DQ383808 (C3H/HeJ). 32 A< \f Il1f5, Il1f6, Il1f8, Il1f9, Il1f10 "10, "lb Abhr1 Abhr2 e ............. ‘l ........................... "1m ................................ ’3. ........................... ‘ 5' —[r m 3’ 1 i/c 1 e/c 2 3 4 Figure 2. Sequencing of the murine 111m gene. (A) Several IL-l family genes map to murine chromosome 2. (B) 111m contains 4 exons, including alternative exon 1 for intracellular (i/c) and extracellular (e/c) isoforms. [:1 Untranslated regions in exons; .1 coding regions in exons. * Dinucleotide repeat polymorphism. 33 35.53. 8:882 £5 9 8:22 5 583.3 :M M92056 3 8:50 5:82 05 8:0: do MEN: Q< - howmwmoe 20: wow: 8:33.». 8:0qu2 05 .Mo 9:ng ofiMo panama: E085 I 3538 SEEM EMM ~ ASE 35 memeD Eco M5950 .50 .5 women 5383 3 action _ vnmm othM :5 EMMM OHHUEOOOHU<HOOHOOH<<M.UOO.MIM>M.O.MIM>M. 5&8 EnofiMFm ”M v vaM Mm an EMMMoM OCIOHO14459 /gene="Il l m" mRNA join(<1812..1821,10349..10400,12295..l2383, 13345..13457,14244..>l4459) /gene="Il 1 m" /product="IL-1 receptor antagonist isoform" /note="transcript for possible intracellular isoform; alternatively spliced" CDS join(1812..1821,10349..l0400,12295..12383,13345..13457, 14244nl4459) /gene="Illrn" /note="possible intracellular isoform; similar to NM_031 167; alternatively spliced" /codon_start=1 /product="IL-1 receptor antagonist isoform" /protein_id="ABD43 197.1 " /db_xref="GI:88595943" 38 /translation="MASEAACRPSGKRPCKMQAFRIWDTNQKTFYLRNNQLIAGYLQG PNIKLEEKIDMVPIDLHSVFLGIHGGKLCLSCAKSGDDIKLQLEEVNITDLSKNKE EDKRFTFIRSEKGPTTSFESAACPGWFLCTTLEADRPVSLTNTPEEPLIVTKFYFQE DQ" mRNA join(<10282..10400,]2295..]2383,13345..13457, 14244..>14459) /gene="ll 1 m" /product="IL-1 receptor antagonist isoform" /note="transcript for possible extracellular isoform; alternatively spliced" CDS join(10282..10400,12295..12383,]3345.. 1 3457,14244..14459) /gene="lllrn" /note="possible extracellular isoform; similar to GenBank Accession Number M64404; alternatively spliced" /codon_start= 1 /product="IL-1 receptor antagonist isoform" /protein_id="ABD43 196. 1 " /db_xref="GI:88595942" /translation="MEICWGPYSHLISLLLILLFHSEAACRPSGKRPCKMQAFRIWDT NQKTFYLRNNQLIAGYLQGPNIKLEEKIDMVPIDLHSVFLGIHGGKLCLSCAKSG DDIKLQLEEVNITDLSKNKEEDKRFTF IRS EKGPTTSFESAACPGWFLCTTLEADR PVSLTNTPEEPLIVTKFYFQEDQ" 39 repeat_region 15542..15581 /note="microsatellite" /rpt_type=tandem /rpt_unit_seq="gt" ORIGIN 1 tcattcaaca gatgtttcca aagtcaccaa aatacagttt acaaagctga tatttgaagc 61 tctgaatgcc agaccttgca gtaccacatg acctcctctt gtcatatcag atatgcacac 121 tagacaggct cccccaacaa cttccagact tccctcccag agcagcttcc caggtttgca 181 aagctggagt ttgtagagtt tccaggtttg caaagctagt tcctgattct tcacaaacat 241 ttctctacaa aagctccttt ctaattccta gttaaggctg aaagacctag acagtctagc 301 ttggtaagcc taaactaaca agagcaggaa gttagggccc aatgggaagt ccctgcggtt 361 cagagtgcca ggcagggatc ctttctcacg tcagggcctc agtgcctccc acacacatgt 421 cagtcccaat agagagacct tgggaatgag tccagatgaa caaacagcta gaggaaaagc 481 aggggacttt ggccaggtca ctcaagtgag ggtgagctgt cctgtcccat ccgcagagac 541 agacttgcag gtggcaagat tacaggcagc attatgtctg cctgccttct ccttcatctt 601 tgtattaaga cattgcctgg agcaaggcct ggttttaagt gcacacaatt tataaaccat 661 tttatggtct gcatatcagt ctaaggctgg gcagggagtg tcaggttgtt tttgcttcca 721 ttaaagtaag gcctagaggg gaaaatgaca tgcccaggct ctctcaaaga gttactgtca 781 gcccaaggga tcacaggttg ctgttaccca cctatctgcc taatggttgt taagcacaca 841 agagttgtcc actccttggt gggagggcac agaggcagga agccagagca agttgtttct 901 gagctgcaga ggaagaggag tcagagaatt gagaacttcc agagaaagtt gagagagtgc 961 atctggcctt ctggagtcag tcttgacagt ggcgcattgg tgaaatgcaa gcatacttgg 40 uotfiar meansdn ‘g 1021 aaaatggggt aaacctctaa gtgatgctgt gatcacatag tagtagtagt agtagtagtt \ 1081 agcataatac tcagatcatt aaatatccca tcccaagtca tcttccttgc tgtttcctgt 1141 ggttgtaaca ttctttccct tccccagctc aaatgccacc attctcaaag aggcttccct 1201 ggacaccttc gaagatatct gcctgggtac ctcatagaca tttgatttct cctgtgacct 1261 ttacccccat agagaattag cttattttat tctttccctc tccactttct ctcattagaa 1321 cacaagtggc ctttgtttgg cttgcaaccg gtgtttgtga gagcacttgt aagtaccaca 1381 ggcaggacta agtcagaatg tgttttaaac aaatttatgc acacataatg gtctcataac 1441 agctgggttt ggggtgatac aaaggtccca cctcataaat ccagctcctt tcccacatgt 1501 gacctccagg tttacatata taagaaccag tttggcttct gctagactga gtcacgcctc 1561 tggaagctgg gcctggcttg gcttcagtca tcagcaacca acctcctagg gctgactggc 1621 ctgtgtagga gtatgggagt ggccatctcc ccccttttat tctgcttagg tagctgggaa 1681 ggaggggcag ttccaccctg ggaaggtctg tgccatagac actgcctggg tgctccttta 1741 tacacagcaa gtctctctgg agtgagacgt tggaaggcag tggaagacct tgtgtcctgt 1801 ttagctcacc c4atggcttca ggtaagatct ttgaggtcat atgaaaatta agggagtaat 1861 tgtccaccgc tagtgttgac tcaatgctca atgccaggtg catggagcac taggctatca 1921 ggaaaatctg gccaatggga agccacaaaa tcccaggaaa ctacctagat atgctggggc 1981 agagagggta tgcccgatat gagaaaagtc atcattgaat ggtgttggcc attctccact 2041 gtttgtgaaa acaagaaata gatgaaatgt ctctgtaaga tggtgaaaag aagtatgtgt 2101 gtaatgaaaa ctgttgatat aaagtcatct gtgtccaagg ttgaagtgtg atagagaaag 2161 atagagtctg cagaggagta acagattgac acttaggcaa catgtttgcc atagagaata 2221 taggctcagg tggacattct taaagggaaa gccaaatatt atctgtgcta ctacctggta 2281 cttccaaaac cagacagaat cagacacatt aatgttacat gctggtgagc atgtattggg 2341 ttgaaccagt gcaagcaagc acaatataga caccacagtc tctgaaagaa ggaatcagaa / 4 Start codon of the intracellular (i/c) lL-l receptor antagonist isoform 41 uotfiar meansdn 5 11110505; 0/1 1 uonul 10} arts 11mg 2401 acagcatcaa aatccaaaga gatgggagct tccaaatgtg accaagcaga aaccatggct \ 2461 ctaagaacgg agaagttaag catgtgactt gatggggggt ggggcttgca gagtaaggac 2521 aggagcaagg gtgtaccctg tgcttccctg ggactttact gtctctctcc atggagtctg 2581 gcttcttgcc tcacagatga tgcttgtaaa gaagggaggg agaaagagaa ggagaaagca 2641 atggtaatgg gaagtataaa tcctgtttag gacccgcatc tgaagatagt ctaagaatgc 2701 tagacactaa cattgtctta agataaatgg cttaacttgg aatgaatcaa tggtggatgt 2761 gaccattctt ttatttacta tggtctcttc ctgggtgaga agttcatttt tggcttttgt 2821 atatatttca gttatctatt tgtctgtctg tctgtcttat ttataatcta tgaatttgtc 2881 tatcatccaa actgcatacc atctatccaa atggatcgta cctttgccaa taataacaag 2941 ctaaattagc aagtgacagt cattaagtca ttgtgtttgc aatttgtggc accatccacc 3001 aggattcaca atttaggttt tcaaagactg aggaggtcac tgaagtatag aatgttcagt 3061 aatgaagcta aaatagtctt tgacaaacca aggcagtttg ccatcctagt ccataaccaa 3121 gtaaaatggc tttcattata gcctatacaa aggttttaaa ttattttcct tacattccac 3181 atgcttttcc atatttccaa gcagttatga actaaatagt tactccatag gataatgagc 3241 cagagccaaa catttaaatc ataagtaaag gtcttgctct tgttgacctt acaatgaagc 3301 aaagaaggta aacacctgta gcacaactgt atgttgtatg ggaaagaaca aaaaccagaa 3361 tttaaacctt gccttagaca gcgtctaaaa gaaatgtagt ctttctgggc cttgagtttt 3421 tttctgcaag ctatccaggt tcattcccat catgaacgtg tgttctatgc ccggttggtg 3481 ggctgtctgg ggaatttagg agatgcagcc ttgcttgagg agagcattac agtgagtagg 3541 accgtttaac acctcagtcc acttccagtt catatctgct ttgtgtctgt ggttgaaaat 3601 gtgaactccc aactgcctgc tccagccacc atgctttccc tgcaatgtag actcgtatcc 3661 cttcttgagc catataaact cttttttttt ctataagttt cttcaaatta tggtgtttta 3721 tcactgcagc ataaaagtag tgaatgtgaa aacagaggtg ggcacagcaa atgacagcac j 42 1 uonuI 3781 agctgtgccc tctctcagaa ttcctgccca tctccaaatt ctcttgcctt atgaagttaa \ 3841 ccatcagaga aaagagtcag catgtacctc tgaaaatgta ataactataa aaacaatgac 3901 gttcagacca atacatatca cccgcacacc atggaattaa aagccatcta ggaagactta 3961 acctcagaaa ggattcaggg aaggtttcct atctggatct gggaagaaat tgagcctctt 4021 gaaaagcact gatccacaat tgttggttta tgccctgtct cctgaatgac ctttgctagc 4081 catgagagta tacatgatca ccttgacatc agaaagctag agggctaaaa cttaggtaga 4141 aagagccagg agcacagatg tttccctggg gagcttccaa caaggtgtat tattaagatg 4201 agctcaaata cctgacatga ttaggaagga tcagatggac aaaaaaaaaa aaaaatcagg 4261 ctataccctc aggtgttctt ttttttcccc ctacttcctc tcatactagt atatttatat 4321 atgacttgga aggaaaaact ttgacccact gttttagtaa gaattagcaa agttaggata 4381 atattgtcac aactgtgtgg ttcataccac acagctaagc tattagagag ttggctagag 4441 ggctacgagg tgattttgtt acaacttagg gtgcatattt attcacccaa ggccaaatgc 4501 atgcatctca caaatggtgc ttagtggcac aagctcagct ccaacaggca ggggtaatgc 4561 tcacatcagt attcattaaa gttttctgga aatatatatt caaaagctca agtccaagtg 4621 aatcaaggac ctccacataa aaccagagac actgaaactt atagaggaga aagtggggaa 4681 gaaacttgaa catatgggca cagtggaaat tttcctgaac agaataccaa tggcttgtgc 4741 tgtaagatca agaatcgaca aatgggacct cataaaattg cccagcttct gaaagtcaaa 4801 ggacactgtc aataagacaa aaaggcaacc aacagattgt gaaaaaatct ttaccaaccc 4861 taaattcaat atagggctaa tatccaatat atacaaagaa ctcaagaagt tagactccag 4921 agagccaaat aacccttttt taaaatgggg taaagagcta aacaacaaat tctcaactga 4981 ggaataccaa atggttgaga agcacctaaa ataatgttca acatccttag ccatcaggga 5041 aatgcaaatc ataacaaccc tgagattcca cctcacacca gtctgaatgg ctaagataaa ) 5101 aaactcaggt gacagcagat gctggcgaag atgtggagaa agaggaacac tcctccattg 43 1 uonul 5161 ttggtgggat tgcaagctgg tacaaccact ctggaaatca gttttgaggt gcctcagaaa 5221 attggacata gtacgatcgg aggatccagc aatacctctc ctgggtatac acccaggaga 5281 tgctccaaca tgtaataagg acacatgttc cactatgttc atagtagtct tatttataat 5341 agccatacac tgggaagaac ccagttgtcc ctcaacagag gaatggatac agaaaatgtg 5401 gtacattttc acaatggagt actactcagc tattacaaac aatgaattta tgaaattctt 5461 aggcaaatgg atggatctgg aggatatcat cctgagtgag gtaacccaat cacaaaagaa 5521 cacacatgat atatactcac tgataagtgg atattagccc agaagctcaa aatacccaag 5581 atacaatttg taaaatacat gaagctcaag aagaaggaag atcaaagtgt gtaaacttct 5641 attcttctta gaaggggaag caaaacaccc atggaaagag ttacagagac aaagtgtgga 5701 gcagagactg accgtaaggc catccagaga ctgcccccac ctggggatcc atcccatata 5761 taatcaacca aacccagaca cttttgtgga tgccaacaac tgcttgctga caggagcctg 5821 atatagctgt ctcctgaaag gctctgccag gacctgacaa ataaagatgt ggatgctcac 5881 acctatccat ggaactgagc acagggtccc caatgaagga gctagagaaa gtacccaagg 5941 agttaaaggg gtttgcagcc ccataggagg aacaacaata tgaactaacc agtaccctca 6001 gagctcccag ggaataaacc accaaccaaa gagtacacat cgtgggactc atggatccag 6061 ctgcatatgt agcagaggat ggcctaattg acatcaatgg gagaagaggc ccttggtcct 6121 gtgaaggctc tataccccag tgtaggggaa tgctagggtc aggaattggg agtgggtaga 6181 ttggtgagca gggaggaggg ggaatgggat ggggagacag ggttttggag gggaaatgag 6241 caaaggggat aacattttaa atgtaaataa agaaaatatc caataaagtc aaagaaggag 6301 gaggagaagg attgtggatc agtagcaggg agaggaagag ttgtacaagc tgcacatgtg 6361 gaccaagact gtaggggtgg tagacactgt gactcatccc tcccattttg cctgaagtaa 6421 ggaactatgc acaatcacta ctgtttgagt aattcccacg tgccagtaca tcatctcaga 6481 gtcttactga attctcatag caacccacat catggtttcc agtattaaac ctggttaatg 44 1 uonuI 6541 ggtaatgaaa ctgagatcta gagagtctgg gcaccctgtc tgaggcagtg atgtttcaga 6601 atgatgtaag aggattgaaa cgtgaggtca gcccaagaac tcctttcctg cacactcgga 6661 aagaacatac tgctggcctt tgtggaggtg agagtggtca tgggaagaag gcacagacga 6721 ggggagtctg ggagcccaat ctgtccaacc tctaggaata gtgtcttccc atctgaattt 6781 gggctgagat cagagaaaca ttactcatca gctatcatgt gggggcaggg gggaaggatt 6841 tcacttggat agctgtctga gggcagctat ggagcctcgc cattcacctg ccaagtcaca 6901 tctcttccac agtcttcaag aatgtttgag atgatgatac tgatcagatg ttctagcaaa 6961 gtctcgccag tttgctaaag tttggctcct ttagacactt atcctcatta agaatctcta 7021 gttttcattc accgctatat cagtagccac caaaacttcg gttttcaaaa aaacaacatt 7081 ctataaaaat ctgaaaatgg aatgaaaaga ataacaaacc ctaatatggt aaattacata 7141 ttaagaaact gaaagaatgt ataacttaat gacattcttg tatcctgcat atagaaatca 7201 catcacttgt aaatatcata atcacaggac aatgtgatat aaataatttg tgcttcactt 7261 caactatatt gtcttgtttt tcttgtgtta atttttgtct tcctatgaat gcataattaa 7321 taagaagttg aatatattta aggcataggc ttaacatcgt gttataagtt gcagaatatt 7381 caccataatc aggctaatta acacatcttc ctcctcacat agatcccatt ttctttctcc 7441 ttcctttttc ctccctccct ttccccttct gtccctctca ttttccttct cctttccttt 7501 tagaggggag aggtagtgaa ggctactcag caaccactct tgcaagtttc agtgtttagt 7561 aaacagtact atcactgtca taagatcctc agataacgct caccttagca acttggcata 7621 cattagatct gtgaagtggc acagaggcag aagagagaca ggcgtctgcc cttcaaccga 7681 tggggtagcc tctctccctc agagaactgg atggtctacc aggaggcaaa gatactgcaa 7741 tcctttatag ggatggggtg gggagtttgg gaacgaggaa attgttcctg ctgtagtggc 7801 ctacacatct ctggttcctg atgttgttgg ctcaaagcca gacaggctgt cctttctggg 7861 gcaactggta accgttgagc aaagtcacct gcactctctt aggaatcctg ctacacatac 45 l [ uonul 7921 aactgaggct ccaatccttg catttaactc tatgcattcc ttgtcatctc ctgcctctgg \ 7981 caattatcaa cctattgtct acttctgaat tttaatgtga attttattga tatgcaaaat 8041 gtagcatttg tacaaataaa taagtaataa gagagtccag agtggcttca aaagcacatt 8101 aaacaagcat aagagcttct gggagcagga tggcagctgc ttctcctttc agcagctgtg 8161 atagcaacag tttgtacctg atgactaata aacctttcag aaacttgaaa gcttaattaa 8221 ttcaacagtt tctggatatt gtaaataact agacaagttt atgcacattc cctctttcag 8281 ccagcttcac aaaagatttt caaaacaaga aatgagcaaa tagaatagtc ccttgactgt 8341 cacaagtaga tatagcattt ttctacagtc actcaagaaa gatatggact tgccattttg 8401 actctcaaaa attatcatcc agcattgtag tagcagacat gtcccatttt gtggggtggg 8461 ggacagaaga acttcttaaa gacttgatag ttctttagct tttccatgaa aggatttaac 8521 tctttgaaga tactctggtt tcttgggttg aaataaggtc agatgcatcc attgagcagt 8581 ggtgggcaca cctttaatcc cagcatgagg gaagcagaga caggaggatc tctgagttca 8641 aagtcagcct ggtctacaaa gaatgttcca ggacacccag ggctatacag agcaaccctg 8701 tctcaaaaac aaaaagaaaa aagaaaatga ggtcaaattc atcaagatca atgagtgcca 8761 taagaactgg ggtttgatcc tctgccatta acaatctcta caacttttcc taatgtctct 8821 ttctacatct ctttaaagta gtgagaggtt gagataaggt gaccactatg ctccaaccca 8881 gttctgaaaa tcctctgcct gtgctcctga cagcactctc ccttgttgga acacacaagg 8941 actctcttca ccttttgata acataaacta ggaagagcct tacctttccc caactgtcca 9001 gaaaattgtg tgaagggaac ttaatgtttt ttaatgctca cttgggatca aaatttaact 9061 cccttctttc ctactacctc caagaaagcc atgatcctcc tcattctgag aataaagaag 9121 cagagacaca aataaaagat tttttcaagg tcacacagat gatagtgaca agcagcaaag 9181 accggttgct gcccacacac ttaatccact tttccatgat ttgggaatga agtcacctct 9241 aaaggactcc aacttcacag aggaccacca caataggctc ttgtctgcaa acataacaga j 46 1 uonul 9301 gtatcattaa tgatgtcagg gattgatggt tgcccatggg atggtctcaa gttgggccaa \ 9361 ttattggttg gccattcctt cagtctctgt tccatagttg ttctgcattt ttaggggaga 9421 caaattttga accaaaagtt ttatagtttt gtgtgcattt ggaatgtaaa taaataagat 9481 aattaatttt acaaaagaaa cttcaacttt ccagatgcag aattgggaaa agatggccaa 9541 ttttaacaca cctcttggga ggacttattt ctctagggca gaggtcagca aacttctaca 9601 ctacgggtga cctttcctgt tttgtaaaca aggcatgttg gagcagagtt gacttttctg 9661 gcttacaaat tgcttgtgag cgcttttgca ccatgacagc cgagctgagt attatgttag 9721 attggatagt tcacaaaata ccaaatattt accatccggt tctggacaga atgtttatgt 1 uonuI 9781 ctagccaact gtcccttctc caaagaacac aataacagtg acatgacact gtcctttgtt 9841 caccaggccc tattgcttgc cttcaaatga aaaggggaca tttctattca gatctggttt 9901 tttttggggg gggaggggga tcgggacagg gggatcagca aatagactcg gagtacctgt 9961 catgcaaatg agggagtctg gttttcattg tgctcttctt cccaggaaca ccatgaaggg 10021 gaaacagaga acttaatttt ggggaaatta cacagggtaa gggggaggag atcagttaca 10081 acacaccatt gcgacacttt cagggttgac agcgacagca gtaaaggttt ctctttttgg 10141 aaatatgagg gtttttccgc ttctgacagt ggaacggaat gacagcagca caggctggtg 10201 aatgactact ttctttataa gcaaccacct tgagcctgaa atggcagtcg ctagtctcta ‘5 10261 ttgccttgct gtggcctcgg gsatggaaatc tgctggggac cctacagtca cctggtctct ) 1 uoxg 10321 ctccttctca tccttcggtt tcattcaga6g gcagcctgcc gcccttctgg gaaaagaccc 10381 tgcaagatgc aagccttcag gtaagtcttc caaagacaca ggattgcata gaccaaggac 3, 10441 cagagacaca tgccatatgt ccagagcata tgcaggaata ggagatatat atacatgtat 10501 aatatatata atgcgtgtgt gtatgtgtgt atacacatat gtatgtatgt atgtgtatat z uonul 10561 atatatatat atatatatat atataatgtg tgtgtataca catatgtatg tatgtgtata J 5 Start codon of the extracellular (e/c) 1L-1 receptor antagonist isoform 6 Alternate splice site where the start codon and subsequent sequences of intracellular (i/c) isoform joins to form the first exon of the V0 IL-1 receptor antagonist isoform. 47 10621 tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtatc cttgattcaa 10681 gaacagcatg ctaaatgcag tctttaagtc ttatgtttta aaatattcca tgcatggaca 10741 acaagacagt taactgtgct cactttctca gacacctaga tgttcagtaa gtgatggaca 10801 ggcatccggg aataatgcta gctttgggat cgagcaaaga ggaatacttc agcaggacac 10861 agtcaaaggc tcagaccaac agtctacact ctgtatctgt gttgacttgg aagatatctc 10921 tcgttggagt ccccagtttc cttatctgta acatgatact gctctgatga taaccccttg 10981 tgtgccttac agggtgaaca ctaaatacat gagtgatact gtaaccatgt tctgagacct 1 1041 atgctctgag aactgtaaag tgcctgaaaa ataacctgag ttttaaaaat tggatcaaaa 11101 gccttgggag atgccatcaa ccttatagta aaaatggcag gcctcgattt tgattttaaa 11 161 atgaataaag agattgttgg tgcatatgat ctgttcttga tccttcctga gagtgaagtc 11221 tgtgttgagt cacttcccct ttgaccctgt ctgctttgga tccacagctg gaggctggga 11281 ctctaactgt gattctatac atctatccca aggcaagtct gtcccacaga tccagtaact 11341 gcttcgtgag atttaccatc atcacatcct cttagcagcc tcaagagagg tccctggagt 11401 cctgttagca agactattga gtcccttgag tttgaagctc accagagata tagacaccag 11461 tcacaaaggc acaaatactc tttcacgtgc agagtacttg gtttgtcctc cacccatccc 11521 tgagctccta ggctgctcca agctactcaa aaagtcctgt cagctctgct gaccaggtaa 11581 agagataagg gacagatcca aggtcatatc atcaggcctc ttaccacacc tcacaggtgc 11641 ctgcctctct ggaagccaga gggcctttca ccaagaagtc agagagtaac aaacaggccc 11701 tggctgagct agacaggaag ctgacttatt tccaaggaca gctgtccctg tcaggcccag 11761 agcagatggt cccacaagag gttttagttg tagacttgca ggtctaagta gagtagcttg 11821 aggtaggagt agtggagcca gactagcttg gctacaatac attctaaccc ttgaacctgt 1 1881 aacactatga tgtggtggcc acgagctaca agtggccatc taaatttaca cataaacgca 11941 tgaaagcaga agaaagtcct gtacctggca actctattta gtggagtgac tataggatgt 48 z uonul 12001 gcttgcatcg cctaagtttc tatcagatgc tgacgctcta tagaaaattc tgctaaagtc 12061 atggatgtcc atgctgggat tctgaggtga ggaacaagaa aaagaggttt tctgttcacc 12121 agatgtgaga gatgggctca tttcttacat ggtatttgct taaatcttcc catttgtgtt 12181 atgaacttgg taagtacgac acttccagca agtctagatg taaattaggt gactctgagg 12241 aagctggaaa gggctctgta ctgcctactc cagctaggcc attttgcttt tcagaatctg 12301 ggatactaac cagaagacct tttacctgag aaacaaccag ctcattgctg ggtacttaca 12361 gggaccaagt atcaaactag aaggtgagtg gataacaggg aagctggtgt aatatggaca 12421 tagagtcctt tgccctgctc ctctgcctgg aggtgggatg tcctcatttc tgttgagttg 12481 gaaatgagag atttgaccac caggggacat atgggagtgg cctcaagaga gcagaaaaga 12541 taaagactgg gtcacaatgc tccagggaca cagctgagag gaacagaggc cagaaggcac 12601 ctgggcacct ccttagtcct tctgtgctgg tagtccacta taccccagtg ttattcgaac 12661 tctacccttg ccctaggcta atataacatg tatgtgggct gggtagcatt tttactgtgg 12721 acaccaccct catcatgtac cctctaaact aggacaaagc cacatgaact tggaggagca 12781 ttacccacag attcttcagt ttttctttta gaaaaaatga gggcacttag ttgacagaat 12841 ctctgtttgt gagggaacga agcattactt gtatctcctc aggatccccc aagccttctg 12901 ctttcctgta tcactcagca gttatgcaac tggcttttcc tgtctttcta gtaattctcc 12961 catgaacaca ctcaagcata gaaggtgctg gctttctatt gctacccagt aacaggatgg 13021 aaaggtgaac tgtgtggaac ctattcatgg gccttgtgag cttttgtgcc tctgtctact 13081 aacagcaaat ctgttgactt ggaggtctgg ttcactgtag aaagtaaagg aaagttggga 13141 gcagtgtaga atctaggaag ctggtcctta catagagtgt gctcatttgg atcttttgct 13201 tggaggcaga ctagaaagat agagccttct tgaccttctt gaccttctag ttttataaaa 13261 aggaagacag aaaatacaca cagacgctcc cctacccttg cctcctcttc tctctttctg 13321 acaccatcct ctactcttct ccagaaaaga tagacatggt gcctattgac cttcatagtg 49 g uoxg z uonul z uoxg g uonul 13381 t ctt catccac caa ct t cct tctt t ccaa ct a at ata 13441 tcaagctcca gctggaggta agaatctggt ttagctatca aatccttcta aaacccaatg 13501 gttatgacaa cctcaggtgt ttctcataac cctgagcatg caaagatgag ggaggctttt 13561 ccttcttcac agagtactat tttgaggtca ctccttaagc agtttccaca atgttcttgg 13621 ttgatattgg gtgtccaagg tggtttctca ttctctcaac taccctttac gtaacttctt 13681 tgcattcagt caacactctg agcttcctta agcgtggtga ccaactttta tgagagattg 13741 ttccagaaag atgagcctca atgtgaaagt gcttattaag cttgggctta tgtaagtcta 13801 ttggcagaag cctgtgacgt ggttgatatg gactcattgt agaaaggtac tgcacaagga 13861 tctaaacttt aggaggagac atggtcatta gaggagcacg acctgaacca ccatgggtct 13921 tgtgcctcct aaaccagttg agcctacctt cttctagcaa ggtcaattct caagactata 13981 cactcccaag catcatctat gctatttatt atctacgctc ctaatttaca tcccacacag 14041 acctgtgtca cttactcctt tacctagtca gtagtaatgg gctgttcaaa cattatcttg 14101 agggattagc tggacaaact tttaatccaa ctgcaaatag ccacaagcat gagtttgttg 14161 ataactctta ccaatggaca ggaacacctt ttagaggact ttctcagccc tcggcaatta 14221 cctgaccatt tcttgacttc caggaagtta acatcactga tctgagcaag aacaaagaag ) 14281 aagacaagcg ctttaccttc atccgctctg agaaaggccc caccaccagc tttgagcag 14341 ctgcctgtcc aggatggttc ctctgcacaa cactagaggc tgaccgtcct gtgagcctca 14401 ccaacacacc ggaagagccc cttatagtca cgaagttcta cttccagaaa gaccaa 7tagt; 14461 actgccgagg cctgtaataa tcaccaactg cctgatcact ctggccatca ttggggcctg 14521 aggaacaact tttgcagggt gtatgtacag tagaaggaga cagaagagtt ctgatgatag 14581 atctctgcct cagtctgttg gctggcctaa tccccatgat gattccagaa taatcttgca 14641 aattggatca tggcaggtgc ttgttcaaag ccctttcttg ttgcctctgc catctgggtg 14701 aagtctagac cacttgcttg gcctaggtgt cttctgctct accacccacc ctacccctgc J 7 Stop codon for both the intracellular and extracellular lL-l receptor antagonist isoforms. 50 g uoxg 17 uonuI p uoxg HIT] .E 14761 cacaaacaca cacttttttt gtttttgttt tttccattgt tctgcacttc cacagtccag 14821 accaatcaag tcacttgaca atatgcccca agtgactccc ttaccctgtt ttataaacct \ 14881 gtgcctgtct atggagaagg ttttaattct ccttgttatt cattttgggc tttttgatga 14941 aaccaccagg gcatcacata tactaagcat gtgctctacc atcatgctat gcttccagct 15001 caggggggca cttttaagga tctagaaaac agaaattaag gatctcatag ttattttatt 15061 aggccagcct tattccatgt cggcaagagg tttcttgtgg aaattatgtc ctttctgaga 15121 ggagctgggg attagatgct 'cctgcatttg tgaaatggtt ataagcatag aaaaataggt 15181 ggtaagcttt ccttctttcc ttattttgtg tgatgcctta aactgaaaag ttaaaaattg 15241 atggattgta gcattcccat aatctccccc ttcttttttt ttcctttgga aatgtccaat 15301 agtctatatt cctctgtccc gcccaaacac catcttcact ccaagcctac cacagatgcc 15361 tgaagaagtt cctcactatc tgcaaatgtg gctctcaggc ccttcctgat gtgatgaatg 15421 aatctactaa tcatttcttg accattcatt ttatcacttc taaccttgaa acatgtggaa 15481 gtagctatgt tcctgactgt ttcctctgcc agacaatgaa ctctggagat cagggagctt ‘1 15541 cgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgcgtgcgcg cgcgcgtgcg _ 15601 cacgcacgtg catgcacatg ctatgtattg ggtccctcca aggatgaacc ctctctttgg 15661 cttagaaggc actcagagaa tatgtgttat tcgtgctcac ggaaagtttc ttactcatcc 15721 ctgtgacttt ggctttattt tacaataaaa cactgaaaat gtccactttg ttagttgtga 15781 acatgagccc aggcctaagg tgctgggaaa cagaaagggc gggagatttt tctttattct 15841 atggctagaa aatagttacc tcctctctga aagtcttctt cctcatttct gggtaacaga 15901 atatcaaaca ccttgcttat aagttataaa gtagtgttgt ccaccatgaa cccaccaagt / 15961 aaaaacaacc caaataccta tcatggatga ata 51 souanbas ureansumop + )1 1.11 .E “(1.9) 0 111m gene sequence of C3H/HeJ strain — NCBI Genbank entry LOCUS DQ383808 15997 bp DNA linear ROD 26-FEB-2006 DEFINITION Mus musculus strain C3H/HeJ IL-1 receptor antagonist (111m) gene, complete cds, alternatively spliced. ACCESSION DQ383808 VERSION DQ383808.1G1288595944 KEYWORDS. SOURCE Mus musculus (house mouse) ORGANISM Mus musculus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Mus. REFERENCE 1 (bases 1 to 15997) AUTHORS Ramadas,R.A., Li,X., Shubitowski,D.M. and Ewart,S.L. TITLE Direct Submission JOURNAL Submitted (30-JAN-2006) Large Animal Clinical Sciences, Michigan State University, 242 National Food Safety and Toxicology Center, East Lansing, MI 48824, USA FEATURES Location/Qualifiers source 1..15997 /organism="Mus musculus" /mol_type="genomic DNA" /strain="C3H/HeJ" 52 /db_xref="taxon: 10090" gene <1813..>14460 /gene="ll 1m" mRNA join(<1813..1822,10350..10401,12296..12384, 13346..13458,14245..>14460) /gene="111rn" /product="IL-1 receptor antagonist isoform" /notc="transcript for possible intracellular isoform; alternatively spliced" CDS join(1813..1822,10350..10401,12296..12384,13346..13458, 14245nl4460) /gene="ll 1 m" /notc="possible intracellular isoform; similar to NM_031 167; alternatively spliced" /codon_start=1 /product="IL-1 receptor antagonist isoform" /protein_id="ABD43 199.1 " /db_xref="G1: 88595 946" /translation="MASEAACRPSGKRPCKMQAFRIWDTNQKTFYLRNNQLIAGYLQG 53 PNIKLEEKIDMVPIDLHSVFLGIHGGKLCLSCAKSGDDIKLQLEEVNITDLSKNKE EDKRFTFIRSEKGPTTSFESAACPGWFLCTTLEADRPVSLTNTPEEPLIVTKFYFQE DQ" mRNA join(<10283..10401,12296..12384,1 3346.13458, 14245..>14460) /gene="111rn" /product="IL-1 receptor antagonist isoform" /note="transcript for possible extracellular isoform; alternatively spliced" CDS join(10283..10401,12296..12384,13346..13458,14245.. 14460) /gene="Il 1 m" /note="possible extracellular isoform; similar to GenBank Accession Number M64404; alternatively spliced" /codon_start=1 /product="IL-1 receptor antagonist isoform" /protein_id="ABD43 198.1 " /db_xref——~"GI:88604776" /translation="MEICWGPYSHLISLLLILLFHSEAACRPSGKRPCKMQAFRIWDT NQKTFYLRNNQLIAGYLQGPNIKLEEKIDMVPIDLHSVFLGIHGGKLCLSCAKSG DDIKLQLEEVNITDLSKNKEEDKRFTFIRSEKGP'ITSFESAACPGWFLCTTLEADR PVSLTNTPEEPLIVTKFYFQEDQ" 54 repeat_region 15543.. 155 84 /note="microsatellite" /rpt_type=tandcm /rpt_unit_seq="gt" ORIGIN 1 ttcattcaac agatgtttcc aaagtcacca aaatacagtt tacaaagctg atatttgaag \ 61 ctctgaatgc cagaccttgc agtaccacat gacctcctct tgtcatatca gatatgcaca 121 ctagacaggc tcccccaaca acttccagac ttccctccca gagcagcttc ccaggtttgc 181 aaagctggag tttgtagagt ttccaggttt gcaaagctag ttcctgattc ttcacaaaca 241 tttctctaca aaagctcctt tctaattcct agttaaggct gaaagaccta gacagtctag 301 cttggtaagc ctaaactaac aagagcagga agttagggcc caatgggaag tccctgcggt 361 tcagagtgcc aggcagggat cctttctcac gtcagggcct cagtgcctcc cacacacatg 421 tcagtcccaa tagagagacc ttgggaatga gtccagatga acaaacagct agaggaaaag 481 caggggactt tggccaggtc actcaagtga gggtgagctg tcctgtccca tccgcagaga 541 cagacttgca ggtggcaaga ttacaggcag cattatgtct gcctgccttc tccttcatct 601 ttgtattaag acattgcctg gagcaaggcc tggttttaag tgcacacaat ttataaacca 661 ttttatggtc tgcatatcag tctaaggctg ggcagggagt gtcaggttgt ttttgcttcc 721 attaaagtaa ggcctagagg ggaaaatgac atgcccaggc tctctcaaag agttactgtc 781 agcccaaggg atcacaggtt gctgttaccc acctatctgc ctaatggttg ttaagcacac 841 aagagttgtc cactccttgg tgggagggca cagaggcagg aagccagagc aagttgtttc 901 tgagctgcag aggaagagga gtcagagaat tgagaacttc cagagaaagt tgagagagtg 961 catctggcct tctggagtca gtcttgacag tggcgcattg gtgaaatgca agcatacttg ) 55 uotfiar umonsdn ‘g 1021 gaaaatgggg taaacctcta agtgatgctg tgatcacata gtagtagtag tagtagtagt \ 1081 tagcataata ctcagatcat taaatatccc atcccaagtc atcttccttg ctgtttcctg 1141 tggttgtaac attctttccc ttccccagct caaatgccac cattctcaaa gaggcttccc 1201 tggacacctt cgaagatatc tgcctgggta cctcatagac atttgatttc tcctgtgacc 1261 tttaccccca tagagaatta gcttatttta ttctttccct ctccactttc tctcattaga 1321 acacaagtgg cctttgtttg gcttgcaacc ggtgtttgtg agagcacttg taagtaccac 1381 aggcaggact aagtcagaat gtgttttaaa caaatttatg cacacataat ggtctcataa 1441 cagctgggtt tggggtgata caaaggtccc acctcataaa tccagctcct ttcccacatg 1501 tgacctccag gtttacatat ataagaacca gtttggcttc tgctagactg agtcacgcct 1561 ctggaagctg ggcctggctt ggcttcagtc atcagcaacc aacctcctag ggctgactgg 1621 cctgtgtagg agtatgggag tggccatctc ccccctttta ttctgcttag gtagctggga 1681 aggaggggca gttccaccct gggaaggtct gtgccataga cactgcctgg gtgctccttt 1741 atacacagca agtctctctg gagtgagacg ttggaaggca gtggaagacc ttgtgtcctg 1801 tttagctcac ccsatggcttc aggtaagatc tttgaggtca tatgaaaatt aagggagtaa fl” 1861 ttgtccaccg ctagtgttga ctcaatgctc aatgccaggt gcatggagca ctaggctatc 1921 aggaaaatct ggccaatggg aagccacaaa atcccaggaa actacctaga tatgctgggg 1981 cagagagggt atgcccgata tgagaaaagt catcattgaa tggtgttggc cattctccac 2041 tgtttgtgaa aacaagaaat agatgaaatg tctctgtaag atggtgaaaa gaagtatgtg 2101 tgtaatgaaa actgttgata taaagtcatc tgtgtccaag gttgaagtgt gatagagaaa 2161 gatagagtct gcagaggagt aacagattga cacttaggca acatgtttgc catagagaat 2221 ataggctcag gtggacattc ttaaagggaa agccaaatat tatctgtgct actacctggt 2281 acttccaaaa ccagacagaa tcagacacat taatgttaca tgctggtgag catgtattgg 2341 gttgaaccag tgcaagcaag cacaatatag acaccacagt ctctgaaaga aggaatcaga ) 8 Start codon for the intracellular (i/c) lL-l receptor antagonist isoform 56 uotfiar meansdn ‘g l uonul moms; on 10} ans 11mg 2401 aacagcatca aaatccaaag agatgggagc ttccaaatgt gaccaagcag aaaccatggc \ 2461 tctaagaacg gagaagttaa gcatgtgact tgatgggggg tggggcttgc agagtaagga 2521 caggagcaag ggtgtaccct gtgcttccct gggactttac tgtctctctc catggagtct 2581 ggcttcttgc ctcacagatg atgcttgtaa agaagggagg gagaaagaga aggagaaagc 2641 aatggtaatg ggaagtataa atcctgttta ggacccgcat ctgaagatag tctaagaatg 2701 ctagacacta acattgtctt aagataaatg gcttaacttg gaatgaatca atggtggatg 2761 tgaccattct tttatttact atggtctctt cctgggtgag aagttcattt ttggcttttg 2821 tatatatttc agttatctat ttgtctgtct gtctgtctta tttataatct atgaatttgt 2881 ctatcatcca aactgcatac catctatcca aatggatcgt acctttgcca ataataacaa 2941 gctaaattag caagtgacag tcattaagtc attgtgtttg caatttgtgg caccatccac 3001 caggattcac aatttaggtt ttcaaagact gaggaggtca ctgaagtata gaatgttcag 3061 taatgaagct aaaatagtct ttgacaaacc aaggcagttt gccatcctag tccataacca 3121 agtaaaatgg ctttcattat agcctataca aaggttttaa attattttcc ttacattcca 3181 catgcttttc catatttcca agcagttatg aactaaatag ttactccata ggataatgag 3241 ccagagccaa acatttaaat cataagtaaa ggtcttgctc ttgttgacct tacaatgaag 3301 caaagaaggt aaacacctgt agcacaactg tatgttgtat gggaaagaac aaaaaccaga 3361 atttaaacct tgccttagac agcgtctaaa agaaatgtag tctttctggg ccttgagttt 3421 ttttctgcaa gctatccagg ttcattccca tcatgaacgt gtgttctatg cccggttggt 3481 gggctgtctg gggaatttag gagatgcagc cttgcttgag gagagcatta cagtgagtag 3541 gaccgtttaa cacctcagtc cacttccagt tcatatctgc tttgtgtctg tggttgaaaa 3601 tgtgaactcc caactgcctg ctccagccac catgctttcc ctgcaatgta gactcgtatc 3661 ccttcttgag ccatataaac tctttttttt tctataagtt tcttcaaatt atggtgtttt 3721 atcactgcag cataaaagta gtgaatgtga aaacagaggt gggcacagca aatgacagca / 57 1 uonul 3781 cagctgtgcc ctctctcaga attcctgccc atctccaaat tctcttgcct tatgaagtta \ 3841 accatcagag aaaagagtca gcatgtacct ctgaaaatgt aataactata aaaacaatga 3901 cgttcagacc aatacatatc acccgcacac catggaatta aaagccatct aggaagactt 3961 aacctcagaa aggattcagg gaaggtttcc tatctggatc tgggaagaaa ttgagcctct 4021 tgaaaagcac tgatccacaa ttgttggttt atgccctgtc tcctgaatga cctttgctag 4081 ccatgagagt atacatgatc accttgacat cagaaagcta gagggctaaa acttaggtag 4141 aaagagccag gagcacagat gtttccctgg ggagcttcca acaaggtgta ttattaagat 4201 gagctcaaat acctgacatg attaggaagg atcagatgga caaaaaaaaa aaaaaatcag 4261 gctataccct caggtgttct tttttttccc cctacttcct ctcatactag tatatttata 4321 tatgacttgg aaggaaaaac tttgacccac tgttttagta agaattagca aagttaggat 4381 aatattgtca caactgtgtg gttcatacca cacagctaag ctattagaga gttggctaga 4441 gggctacgag gtgattttgt tacaacttag ggtgcatatt tattcaccca aggccaaatg 4501 catgcatctc acaaatggtg cttagtggca caagctcagc tccaacaggc aggggtaatg 4561 ctcacatcag tattcattaa agttttctgg aaatatatat tcaaaagctc aagtccaagt 4621 gaatcaagga cctccacata aaaccagaga cactgaaact tatagaggag aaagtgggga 4681 agaaacttga acatatgggc acagtggaaa ttttcctgaa cagaatacca atggcttgtg 4741 ctgtaagatc aagaatcgac aaatgggacc tcataaaatt gcccagcttc tgaaagtcaa 4801 aggacactgt caataagaca aaaaggcaac caacagattg tgaaaaaatc tttaccaacc 4861 ctaaattcaa tatagggcta atatccaata tatacaaaga actcaagaag ttagactcca 4921 gagagccaaa taaccctttt ttaaaatggg gtaaagagct aaacaacaaa ttctcaactg 4981 aggaatacca aatggttgag aagcacctaa aataatgttc aacatcctta gccatcaggg 5041 aaatgcaaat cataacaacc ctgagattcc acctcacacc agtctgaatg gctaagataa 5101 aaaactcagg tgacagcaga tgctggcgaa gatgtggaga aagaggaaca ctcctccatt ] 58 1 uonul 5161 gttggtggga ttgcaagctg gtacaaccac tctggaaatc agttttgagg tgcctcagaa 5221 aattggacat agtacgatcg gaggatccag caatacctct cctgggtata cacccaggag 5281 atgctccaac atgtaataag gacacatgtt ccactatgtt catagtagtc ttatttataa 5341 tagccataca ctgggaagaa cccagttgtc cctcaacaga ggaatggata cagaaaatgt 5401 ggtacatttt cacaatggag tactactcag ctattacaaa caatgaattt atgaaattct 5461 taggcaaatg gatggatctg gaggatatca tcctgagtga ggtaacccaa tcacaaaaga 5521 acacacatga tatatactca ctgataagtg gatattagcc cagaagctca aaatacccaa 5581 gatacaattt gtaaaataca tgaagctcaa gaagaaggaa gatcaaagtg tgtaaacttc 5641 tattcttctt agaaggggaa gcaaaacacc catggaaaga gttacagaga caaagtgtgg 5701 agcagagact gaccgtaagg ccatccagag actgccccca cctggggatc catcccatat 5761 ataatcaacc aaacccagac acttttgtgg atgccaacaa ctgcttgctg acaggagcct 5821 gatatagctg tctcctgaaa ggctctgcca ggacctgaca aataaagatg tggatgctca 5881 cacctatcca tggaactgag cacagggtcc ccaatgaagg agctagagaa agtacccaag 5941 gagttaaagg ggtttgcagc cccataggag gaacaacaat atgaactaac cagtaccctc 6001 agagctccca gggaataaac caccaaccaa agagtacaca tcgtgggact catggatcca 6061 gctgcatatg tagcagagga tggcctaatt gacatcaatg ggagaagagg cccttggtcc 6121 tgtgaaggct ctatacccca gtgtagggga atgctagggt caggaattgg gagtgggtag 6181 attggtgagc agggaggagg gggaatggga tggggagaca gggttttgga 8938333183 6241 gcaaagggga taacatttta aatgtaaata aagaaaatat ccaataaagt caaagaagga 6301 ggaggagaag gattgtggat cagtagcagg gagaggaaga gttgtacaag ctgcacatgt 6361 ggaccaagac tgtaggggtg gtagacactg tgactcatcc ctcccatttt gcctgaagta 6421 aggaactatg cacaatcact actgtttgag taattcccac gtgccagtac atcatctcag 6481 agtcttactg aattctcata gcaacccaca tcatggtttc cagtattaaa cctggttaat 59 l [ uonul 6541 gggtaatgaa actgagatct agagagtctg ggcaccctgt ctgaggcagt gatgtttcag \ 6601 aatgatgtaa gaggattgaa acgtgaggtc agcccaagaa ctcctttcct gcacactcgg 6661 aaagaacata ctgctggcct ttgtggaggt gagagtggtc atgggaagaa ggcacagacg 6721 aggggagtct gggagcccaa tctgtccaac ctctaggaat agtgtcttcc catctgaatt 6781 tgggctgaga tcagagaaac attactcatc agctatcatg tgggggcagg ggggaaggat 6841 ttcacttgga tagctgtctg agggcagcta tggagcctcg ccattcacct gccaagtcac 6901 atctcttcca cagtcttcaa gaatgtttga gatgatgata ctgatcagat gttctagcaa 6961 agtctcgcca gtttgctaaa gtttggctcc tttagacact tatcctcatt aagaatctct 7021 agttttcatt caccgctata tcagtagcca ccaaaacttc ggttttcaaa aaaacaacat 7081 tctataaaaa tctgaaaatg gaatgaaaag aataacaaac cctaatatgg taaattacat 7141 attaagaaac tgaaagaatg tataacttaa tgacattctt gtatcctgca tatagaaatc 7201 acatcacttg taaatatcat aatcacagga caatgtgata taaataattt gtgcttcact 7261 tcaactatat tgtcttgttt ttcttgtgtt aatttttgtc ttcctatgaa tgcataatta 7321 ataagaagtt gaatatattt aaggcatagg cttaacatcg tgttataagt tgcagaatat 7381 tcaccataat caggctaatt aacacatctt cctcctcaca tagatcccat tttctttctc 7441 cttccttttt cctccctccc tttccccttc tgtccctctc attttccttc tcctttcctt 7501 ttagagggga gaggtagtga aggctactca gcaaccactc ttgcaagttt cagtgtttag 7561 taaacagtac tatcactgtc ataagatcct cagataacgc tcaccttagc aacttggcat 7621 acattagatc tgtgaagtgg cacagaggca gaagagagac aggcgtctgc ccttcaaccg 7681 atggggtagc ctctctccct cagagaactg gatggtctac caggaggcaa agatactgca 7741 atcctttata gggatggggt ggggagtttg ggaacgagga aattgttcct gctgtagtgg 7801 cctacacatc tctggttcct gatgttgttg gctcaaagcc agacaggctg tcctttctgg 7861 ggcaactggt aaccgttgag caaagtcacc tgcactctct taggaatcct gctacacata ) 60 [ uonul 7921 caactgaggc tccaatcctt gcatttaact ctatgcattc cttgtcatct cctgcctctg 7981 gcaattatca acctattgtc tacttctgaa ttttaatgtg aattttattg atatgcaaaa 8041 tgtagcattt gtacaaataa ataagtaata agagagtcca gagtggcttc aaaagcacat 8101 taaacaagca taagagcttc tgggagcagg atggcagctg cttctccttt cagcagctgt 8161 gatagcaaca gtttgtacct gatgactaat aaacctttca gaaacttgaa agcttaatta 8221 attcaacagt ttctggatat tgtaaataac tagacaagtt tatgcacatt ccctctttca 8281 gccagcttca caaaagattt tcaaaacaag aaatgagcaa atagaatagt cccttgactg 8341 tcacaagtag atatagcatt tttctacagt cactcaagaa agatatggac ttgccatttt 8401 gactctcaaa aattatcatc cagcattgta gtagcagaca tgtcccattt tgtggggtgg 8461 gggacagaag aacttcttaa agacttgata gttctttagc ttttccatga aaggatttaa 8521 ctctttgaag atactctggt ttcttgggtt gaaataaggt cagatgcatc cattgagcag 8581 tggtgggcac acctttaatc ccagcatgag ggaagcagag acaggaggat ctctgagttc 8641 aaagtcagcc tggtctacaa agaatgttcc aggacaccca gggctataca gagcaaccct 8701 gtctcaaaaa caaaaagaaa aaagaaaatg aggtcaaatt catcaagatc aatgagtgcc 8761 ataagaactg gggtttgatc ctctgccatt aacaatctct acaacttttc ctaatgtctc 8821 tttctacatc tctttaaagt agtgagaggt tgagataagg tgaccactat gctccaaccc 8881 agttctgaaa atcctctgcc tgtgctcctg acagcactct cccttgttgg aacacacaag 8941 gactctcttc accttttgat aacataaact aggaagagcc ttacctttcc ccaactgtcc 9001 agaaaattgt gtgaagggaa cttaatgttt tttaatgctc acttgggatc aaaatttaac 9061 tcccttcttt cctactacct ccaagaaagc catgatcctc ctcattctga gaataaagaa 9121 gcagagacac aaataaaaga ttttttcaag gtcacacaga tgatagtgac aagcagcaaa 9181 gaccggttgc tgcccacaca cttaatccac ttttccatga tttgggaatg aagtcacctc 9241 taaaggactc caacttcaca gaggaccacc acaataggct cttgtctgca aacataacag 61 [ uonul 9301 agtatcatta atgatgtcag ggattgatgg ttgcccatgg gatggtctca agttgggcca \ 9361 attattggtt ggccattcct tcagtctctg ttccatagtt gttctgcatt tttaggggag 9421 acaaattttg aaccaaaagt tttatagttt tgtgtgcatt tggaatgtaa ataaataaga 9481 taattaattt tacaaaagaa acttcaactt tccagatgca gaattgggaa aagatggcca 9541 attttaacac acctcttggg aggacttatt tctctagggc agaggtcagc aaacttctac 9601 actacgggtg acctttcctg ttttgtaaac aaggcatgtt ggagcagagt tgacttttct 9661 ggcttacaaa ttgcttgtga gcgcttttgc accatgacag ccgagctgag tattatgtta 9721 gattggatag ttcacaaaat accaaatatt taccatccgg ttctggacag aatgtttatg I uonul 9781 tctagccaac tgtcccttct ccaaagaaca caataacagt gacatgacac tgtcctttgt 9841 tcaccaggcc ctattgcttg ccttcaaatg aaaaggggac atttctattc agatctggtt 9901 ttttttgggg ggggaggggg atcgggacag ggggatcagc aaatagactc ggagtacctg 9961 tcatgcaaat gagggagtct ggttttcatt gtgctcttct tcccaggaac accatgaagg 10021 ggaaacagag aacttaattt tggggaaatt acacagggta agggggagga gatcagttac 10081 aacacaccat tgcgacactt tcagggttga cagcgacagc agtaaaggtt tctctttttg 10141 gaaatatgag ggtttttccg cttctgacag tggaacggaa tgacagcagc acaggctggt 10201 gaatgactac tttctttata agcaaccacc ttgagcctga aatggcagtc gctagtctct 10261 attgccttgc tgtggcctcg gggatggaaat ctgctgggga ccctacagtc acctaatctc 1 uoxg 10321 tctccttctc gtccttctgt ttcattcaga 'Oggcagcctgc cgcccttctg ggaaaagacc \ J! 10381 ctgcaagatg caagccttca ggtaagtctt ccaaagacac aggattgcat agaccaagga 10441 ccagagacac atgccatatg tccagagcat atgcaggaat aggagatata tatacatgta z uonul 10501 taatatatat aatgcgtgtg tgtatgtgtg tatacacata tgtatgtatg tatgtgtata 10561 tatatatata tatatatata tatataatgt gtgtgtatac acatatgtat gtatgtgtat J 9 Start codon for the extracellular (e/c) lL-l receptor antagonist isoform '0 Alternate splice site where the start codon and subsequent sequences of intracellular (i/c) isoform joins to form the first exon of the i/c lL-l receptor antagonist isoform. 62 10621 atatatatat atgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat ccttgattca 10681 agaacagcat gctaaatgca gtctttaagt cttatgtttt aaaatattcc atgcatggac 10741 aacaagacag ttaactgtgc tcactttctc agacacctag atgttcagta agtgatggac 10801 aggcatccgg gaataatgct agctttggga tcgagcaaag aggaatactt cagcaggaca 10861 cagtcaaagg ctcagaccaa cagtctacac tctgtatctg tgttgacttg gaagatatct 1092 1. ctcgttggag tccccagttt ccttatctgt aacatgatac tgctctgatg ataacccctt 10981 gtgtgcctta cagggtgaac actaaataca tgagtgatac tgtaaccatg ttctgagacc 11041 tatgctctga gaactgtaaa gtgcctgaaa aataacctga gttttaaaaa ttggatcaaa 11 101 agccttggga gatgccatca accttatagt aaaaatggca ggcctcgatt ttgattttaa 11 161 aatgaataaa gagattgttg gtgcatatga tctgttcttg atccttcctg agagtgaagt 1 1221 ctgtgttgag tcacttcccc tttgaccctg tctgctttgg atccacagct ggaggctggg 1 1281 actctaactg tgattctata catctatccc aaggcaagtc tgtcccacag atccagtaac 11341 tgcttcgtga gatttaccat catcacatcc tcttagcagc ctcaagagag gtccctggag 11401 tcctgttagc aagactattg agtcccttga gtttgaagct caccagagat atagacacca 11461 gtcacaaagg cacaaatact ctttcacgtg cagagtactt ggtttgtcct ccacccatcc 11521 ctgagctcct aggctgctcc aagctactca aaaagtcctg tcagctctgc tgaccaggta 11581 aagagataag ggacagatcc aaggtcatat catcaggcct cttaccacac ctcacaggtg 11641 cctgcctctc tggaagccag agggcctttc accaagaagt cagagagtaa caaacaggcc 11701 ctggctgagc tagacaggaa gctgacttat ttccaaggac agctgtccct gtcaggccca 11761 gagcagatgg tcccacaaga ggttttagtt gtagacttgc aggtctaagt agagtagctt 11821 gaggtaggag tagtggagcc agactagctt ggctacaata cattctaacc cttgaacctg 11881 taacactatg atgtggtggc cacgagctac aagtggccat ctaaatttac acataaacgc 1 1941 atgaaagcag aagaaagtcc tgtacctggc aactctattt agtggagtga ctataggatg 63 z uonul 12001 tgcttgcatc gcctaagttt ctatcagatg ctgacgctct atagaaaatt ctgctaaagt 12061 catggatgtc catgctggga ttctgaggtg aggaacaaga aaaagaggtt ttctgttcac 12121 cagatgtgag agatgggctc atttcttaca tggtatttgc ttaaatcttc ccatttgtgt 12181 tatgaacttg gtaagtacga cacttccagc aagtctagat gtaaattagg tgactctgag 12241 gaagctggaa agggctctgt actgcctact ccagctaggc cattttgctt ttcagaatct 12301 gggatactaa ccagaagacc ttttacctga gaaacaacca gctcattgct gggacttac 12361 aaggaccaaa tatcaaacta gaaggtgagt ggataacagg gaagctggtg taatatggac 12421 atagagtcct ttgccctgct cctctgcctg gaggtgggat gtcctcattt ctgttgagtt 12481 ggaaatgaga gatttgacca ccaggggaca tatgggagtg gcctcaagag agcagaaaag 12541 ataaagactg ggtcacaatg ctccagggac acagctgaga ggaacagagg ccagaaggca 12601 cctgggcacc tccttagtcc ttctgtgctg gtagtccact ataccccagt gttattcgaa 12661 ctctaccctt gccctaggct aatataacat gtatgtgggc tgggtagcat ttttactgtg 12721 gacaccaccc tcatcatgta ccctctaaac taggacaaag ccacatgaac ttggaggagc 12781 attacccaca gattcttcag tttttctttt agaaaaaatg agggcactta gttgacagaa 12841 tctctgtttg tgagggaacg aagcattact tgtatctcct caggatcccc caagccttct 12901 gctttcctgt atcactcagc agttatgcaa ctggcttttc ctgtctttct agtaattctc 12961 ccatgaacac actcaagcat agaaggtgct ggctttctat tgctacccag taacaggatg 13021 gaaaggtgaa ctgtgtggaa cctattcatg ggccttgtga gcttttgtgc ctctgtctac 13081 taacagcaaa tctgttgact tggaggtctg gttcactgta gaaagtaaag gaaagttggg 13141 agcagtgtag aatctaggaa gctggtcctt acatagagtg tgctcatttg gatcttttgc 13201 ttggaggcag actagaaaga tagagccttc ttgaccttct tgaccttcta gttttataaa 13261 aaggaagaca gaaaatacac acagacgctc ccctaccctt gcctcctctt ctctctttct 13321 gacaccatcc tctactcttc tccagaaaag atagacatgg tgcctattga ccttcatagt 64 \ J J z uonul z uoxg g uoxg g uonul 13381 mflcflgg gcatccacgg gggcaagctg tgcctflctt gtgccaagtc tggagatgat 13441 atcaagctcc agctggaggt aagaatctgg tttagctatc aaatccttct aaaacccaat E 13501 ggttatgaca acctcaggtg tttctcataa ccctgagcat gcaaagatga gggaggcttt 13561 tccttcttca cagagtacta ttttgaggtc actccttaag cagtttccac aatgttcttg 13621 gttgatattg ggtgtccaag gtggtttctc attctctcaa ctacccttta cgtaacttct 13681 ttgcattcag tcaacactct gagcttcctt aagcgtggtg accaactttt atgagagatt 13741 gttccagaaa gatgagcctc aatgtgaaag tgcttattaa gcttgggctt atgtaagtct 13801 attggcagaa gcctgtgacg tggttgatat ggactcattg tagaaaggta ctgcacaagg 13861 atctaaactt taggaggaga catggtcatt agaggagcac gacctgaacc accatgggtc 13921 ttgtgcctcc taaaccagtt gagcctacct tcttctagca aggtcaattc tcaagactat 13981 acactcccaa gcatcatcta tgctatttat tatctacgct cctaatttac atcccacaca 14041 gacctgtgtc acttactcct ttacctagtc agtagtaatg ggctgttcaa acattatctt 14101 gagggattag ctggacaaac ttttaatcca actgcaaata gccacaagca tgagtttgtt 14161 gataactctt accaatggac aggaacacct tttagaggac tttctcagcc ctcggcaatt 14221 acctgaccat ttcttgactt ccaggaagtt aacatcactg atctgagcaa gaacaaagaa j 14281 gaagacaagc gctttacctt catccgctct gagaaaggcc ccaccaccag ctttgagtca 14341 gctgcctgtc caggatggtt cctctgcaca acactagagg ctgaccflcc tgtgagcctc 14401 accaacacac aggaagagcc ccttatagta acgaagtgct acttccagga agaccaal Itag 14461 tactgccgag gcctgtaata atcaccaact gcctgatcac tctggccatc attggggcct \ 14521 gaggaacaac ttttgcaggg tgtatgtaca gtagaaggag acagaagagt tctgatgata 14581 gatctctgcc tcagtctgtt ggctggccta atccccatga tgattccaga ataatcttgc 14641 aaattggatc atggcaggtg cttgttcaaa gccctttctt gttgcctctg ccatctgggt 14701 gaagtctaga ccacttgctt ggcctaggtg tcttctgctc taccacccac cctacccctg J ” Stop codon for both intracellular and extracellular 1L-1 receptor antagonist isoforms 65 g uoxg J HID ‘5 17 uoxa 14761 ccacaaacac acactttttt tgtttttgtt ttttccattg ttctgcactt ccacagtcca 14821 gaccaatcaa gtcacttgac aatatgcccc aagtgactcc cttaccctgt tttataaacc \ 14881 tgtgcctgtc tatggagaag gttttaattc tccttgttat tcattttggg ctttttgatg 14941 aaaccaccag ggcatcacat atactaagca tgtgctctac catcatgcta tgcttccagc 15001 tcaggggggc acttttaagg atctagaaaa cagaaattaa ggatctcata gttattttat 15061 taggccagcc ttattccatg tcggcaagag gtttcttgtg gaaattatgt cctttctgag 15121 aggagctggg gattagatgc tcctgcattt gtgaaatggt tataagcata gaaaaatagg 15181 tggtaagctt tccttctttc cttattttgt gtgatgcctt aaactgaaaa gttaaaaatt 15241 gatggattgt agcattccca taatctcccc cttctttttt tttcctttgg aaatgtccaa 15301 tagtctatat tcctctgtcc cgcccaaaca ccatcttcac tccaagccta ccacagatgc 15361 ctgaagaagt tcctcactat ctgcaaatgt ggctctcagg cccttcctga tgtgatgaat 15421 gaatctacta atcatttctt gaccattcat tttatcactt ctaaccttga aacatgtgga 15481 agtagctatg ttcctgactg tttcctctgc cagacaatga actctggaga tcagggagct 15541 tcgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgcgtgc gcgcgcgcgt . 15601 gcgcacgcac gtgcatgcac atgctatgta ttgggtccct ccaaggatga accctctctt 4 15661 tggcttagaa ggcactcaga gaatatgtgt tattcgtgct cacggaaagt ttcttactca 15721 tccctgtgac tttggcttta ttttacaata aaacactgaa aatgtccact ttgttagttg 15781 tgaacatgag cccaggccta aggtgctggg aaacagaaag ggcgggagat ttttctttat 15841 tctatggcta gaaaatagtt acctcctctc tgaaagtctt cttcctcatt tctgggtaac 15901 agaatatcaa acaccttgct tataagttat aaagtagtgt tgtccaccat gaacccacca 15961 agtaaaaaca acccaaatac ctatcatgga tgaataa j 66 lino) BOUQUDGS LUBQJISUAAOP + film ‘E 0 Results from sequencing We optimized PCR conditions for all sequencing primer sets, and performed bidirectional sequencing on all the regions to ensure sequence accuracy, hence we are confident that our results are accurate and the sequences are of high quality. We aligned the NJ and C3H/HeJ 111m sequences with the Celera 111m sequence (mCG4837) and the sequence from the NCBI contig AL732528, which contains the murine 111m gene to check our sequence results. A previously identified microsatellite was confirmed in the 3’ untranslated region (UTR) in the last exon of 111m gene. The microsatellite is a GT dinucleotide repeat, with 21 copies in the C3H/HeJ mice [(GT)21] and 20 copies in the AH mice [(GT)20]. As this polymorphism exists in the non-coding region, it is less likely to contribute to a major functional difference. Apart from this, no other polymorphisms were observed between N] and C3H/HeJ strains. We found only one other difference between the four 111m sequences we thus compared. A guanine (G) nucleotide was deleted in the Celera 111m sequence at position 15578 (position number based on Genbank Accession No. DQ3 83807). The missing G nucleotide was present in the NCBI contig AL732528, and was also present in both AH and C3H/HeJ strains in our sequence. We conclude that the deletion in the Celera 111m sequence is probably due to a sequencing error. We have sequenced all the exons and introns of Illrn, and have sequenced all the regulatory regions reported in the literature that are important for transcriptional regulation. This low level of genetic variation in ~16 kb of examined sequence extends a greater distance than the commonly reported single nucleotide polymorphism rate of 1/ 1,000 bp as a theoretical possibility across the genome“. However, the mosaic structure of the mouse genome, as recently described by Wade and 67 colleagues, results in long segments of DNA with extremely high (~40 SNPs/ 10 kb) or extremely low (~0.5 SNPs/ 10 kb) polymorphisms rates'é’5 . Thus, the genomic region containing 111m appears to reside in a low SNP block. The lack of genetic variation in coding sequence may further indicate strong conservation pressure on this gene and underscores the importance of Illrn. C. TRANSCRIPT AND PROTEIN STUDIES IN IL-l RECEPTOR ANTAGONIST AND RELATED GENES 0 Introduction: Along with thesequencing of 111m described previously (Chapter 2, Section B), we also tested for the mRNA expression and protein production levels in our mouse model of allergic asthma. IL-1 receptor antagonist is a major component of the IL-1 complex of genes consisting of IL-1 agonists, antagonists, receptors and accessory genes required for signal transduction. With the IL-1 receptor antagonist operating from within such a complex, its effects on asthma can be better interpreted in conjunction with the other genes. The IL-l family genes present within the QTL Abhr1 include Illrn, IlIf5, 111 f6, 11] f8, IlIf9 and 111f10. Of these, 111m has been shown to be involved in a variety of inflammatory disordersm’los , while all the other genes have been discovered and mapped fairly recently'“. The roles of the genes III/3 — IlIflO in asthma haven’t been clearly elucidated. Previous studies in the same mouse model have shown that there is a clear temporal pattern in Th1 and Th2 cytokines in both the strains of mice (Li et a1 — manuscript in review). In a similar manner, we mapped the expression profiles of the 68 genes in the lL-l complex, with an emphasis on our positional candidate gene 111m. This will help to expand our understanding temporality of the cytokine patterns in this model, and most importantly, elucidate the role of 111m and related genes in the pathophysiological mechanisms. IL-1 complex is being actively investigated as a therapeutic target to devise asthma intervention strategies'lg’lzo. Elucidation of the temporal patterns of 111m and related genes would shed light on their role in airway inflammation and airway obstruction and assist such processes. The maximal AHR in our mouse model was observed at 72 h after allergenic challenge”, hence we decided to measure 111m mRNA and protein levels at various timepoints (6, 12, 24, 48 and 72 h) after allergen challenge. 0 Experimental time line Age-matched, virus-free, AU and C3H/HeJ male mice obtained from the Jackson Laboratory (Bar Harbor, ME) at 4 wk of age were allowed to acclimatize for 1-2 wk before experimentation. They were housed under HEPA filtered laminar flow hoods in an environmentally controlled facility and allowed free access to ovalbumin-free rodent chow and water. All animals were maintained and treated in accordance with the specific guidelines provided by the All University Committee on Animal Use and Care of Michigan State University. NJ and C3H/HeJ mice (n = 6/group) were sensitized with 10 pg chicken egg ovalbumin (crude grade IV; Sigma, St. Louis, MO) in 0.2 ml calcium and magnesium- free phosphate-buffered saline (PBS) or an equivalent amount of PBS alone on day 0. On day 14 mice were anesthetized (ketamine, 45 mg/kg, intraperitoneally and xylazine, 8 69 mg/kg, intraperitoneally), challenged by pharyngeo-tracheal instillation of 1.5% ovalbumin in 45 ul PBS, or PBS alone. The mice were sacrificed and the lungs, tracheobronchial lymph nodes and spleens were collected 6, 12, 24, 48 and 72 h post challenge (Figure 3). Lungs were collected because they are the pertinent foci of inflammation in the asthmatic process. Spleens were collected because of their importance as a peripheral component of the immune system containing T and B cells which play a major role in inflammation. . . . Challenge 3611511123110“ OVA (or saline) 45 ul 1.5% OVA (or 3311113) 10 118 IP tracheopharyngeal instillation 1 day 0 day 14 6, 12, 24, 48, 72 h Tissue harvest Figure 3. Experimental time line of in vivo allergen exposure - Real-time RT-PCR A variety of techniques are available to detect mRNA, such as the classical Northern blot hybridization and the recent quantitative real-time reverse transcription- polymerase chain reaction (qRT-PCR). Northern blot hybridization is only semi- quantitative, and requires radioactive labeled DNA or RNA probes, while quantitative real-time PCR is much more sensitive than Northern blots. There are two major types of real-time PCR chemistries — SYBR Green based assays and 5’ nuclease assays (commercially known as TaqMan® assays). These are the most widely used assays for 70 detecting mRNA expression at present, and possess numerous advantages over the conventional RNA detection methods'67. They are very sensitive in detecting RNA transcripts in very low copy numbers, have very high sequence-specificity, require very minimal post-amplification processing and are amenable to high-throughput analyses. Moreover, availability of efficient primer design software, experimental design and data analysis protocols developed for these assays also helps to minimize the causes of intra- and inter-assay variations and enhance the accuracy of quantitative results. SYBR Green real-time PCR technique uses a PCR buffer containing the fluorescent dye SYBR Green, which binds to double stranded DNA products that are produced during the PCR process. As the PCR proceeds, fluorescence is produced proportional to the amount of double stranded products formed, and is quantified by a laser-assisted fluorescence detection system. It is a powerful technique, but the only downside is that it detects all double-stranded products, and hence careful primer design is required for accurate quantification. Non-specific amplifications can be easily detected by performing a dissociation curve run subsequent to the real-time PCR run. The non- specific products with smaller sizes dissociate at lower temperatures while the products of the correct size (usually between 80-150 bp) dissociate at higher temperatures. Compared with SYBR Green techniques, TaqMan real-time RT-PCR assays are more specific, and these involve a forward primer, a reverse primer and a probe that is located between the two primers. The primers and probe are sequence specific, and they span a gene-specific location of about 80-100 bp. The probe is labeled with 5’ reporter dye (FAM or VIC) and 3’ quencher (TAMRA or non fluorescent) dyes. During the PCR process, the 5’-3’ nucleolytic activity of the AmpliTaq Gold polymerase enzyme cleaves 71 the probe when it is bound to the target. The reporter dye is released from the cleaved probe and emits the fluorescent signal, which is then detected and quantified. The PCR reaction proceeds usually for 40 cycles, during which there is an exponential amplification of the PCR product. The results obtained are in terms of cycle threshold (CT) values, which is the cycle number at which a statistically significant signal over the background signal is detected. The CT value is used as the index of the original copy number of target mRNA (Figure 4). The higher the original copy number of target mRNA, the lower the CT value obtained. One CT unit difference is equal to a two-fold difference in target mRNA difference. Since we were more interested in the relative gene expression between A/J and C3H/HeJ mice and between PBS- and OVA-treated mice, rather than the absolute gene copy numbers, a relative quantification of gene expression method was used. We used 18S rRNA as an endogenous reference (internal control). Equal quantities of total RNA from lung tissues were reverse transcribed and diluted five fold with nuclease-free water. This product was used as the template for real-time PCR reactions (more details available in ‘Materials and Methods’ section). Real-time PCR was performed on all the samples in duplicate and the average of the two duplicates was used for further data analysis. If the difference in CT value between the duplicates of a sample was more than 1.0, data fi'om that particular sample was excluded fi'om the analysis. When the real-time PCR amplification efficiency is 100%, a CT difference of 1.0 translated to two-fold differences in mRNA expression. When one of the duplicates did not work, data from such samples were also excluded from the analyses. 72 A standard series was used with each run and data were obtained using the standard curve-separate tubes method“. A series dilution of 20, 10, 5, 2, 1 and 0.5 ng of total RNA of a single sample was used as the standard curve. 1" .. ::.-,' lilr 7 31-2'31" géf‘a' 1 11111 1 i§:j :;.r;'? 1 |:111 l u- .— .1 s. t,_., ' 11..-1-- I ‘t 1'; 5 f} f' 1 Rn+ 1? ??§'-f 1 Sample ' ‘ Rn qUJW 1‘?” f 5'? := " l . I1,'I1 : AR” ‘ Threshold ."j ; - ...,, ,‘ ,I.L.zq., I . -. T’ 81154’ i I 4+ 5: 112111: 1.:-§a‘1.::i;11 .. ‘ I .. ..' -4 .1. 1; -..}--5.:_.‘\_. . 11 Ctafisjggfliji’bRn- . ....%.1111‘2.:.1111=;11111Hs;!1 I II T] [I II II It’rl II II II II II II II 1: ll ri*r1' 1 02468101214 16 18 20 22 24 26 28 30 32 34 36 38 40 Cu ole Figure 4. The threshold cycle (CT) of TaqMan real-time RT-PCR. Rn is the ratio of the emission intensity of the reporter dye to the passive reference (ROX). Rn— is the Rn value of the unreacted sample or early cycles without signal increases (baseline). Rn+ is the Rn value of a reaction sample. ARn is the difference between Rn+ and Rn-, which shows the signal increases due to PCR amplification. The threshold cycle (CT) is the cycle at which a statistically significant signal increase in ARn is first detected. All the samples in a single timepoint were assayed in a single plate with standard curves. The same standard curve was used for samples assayed from all time points for any single target gene, facilitating comparison across time points. The amplification efficiency of the standard curve was used to quantify the expression levels in the samples. 73 The expression levels in each strain/treatment/time group were averaged, and the averages and standard deviations were used to calculate the relative expression of target genes after normalizing the values to 188'“. Normalized gene/18S value of the 6 h— PBS—C3H/HeJ group was used as the calibrator, and the expression of strain/treatment groups within all the timepoints were calculated in relation to this value. 0 Data analysis The ratio of the RNA value of the target gene to the RNA value of 18S rRNA for each sample (ratio) was used as the input data set for statistical analysis. The data was analyzed using two or three-way ANOVA tests using PROC MIXED model in SAS v 9.1. Model significance levels were tested for the factors time, strain, treatment and the interactions time*strain, time*treatment, strain*treatment and time*strain*treatrnents. Differences of least-square means were then calculated for these factors and interactions. All the p values obtained from the differences in least square means were adjusted for multiple comparisons using Tukey (for balanced datasets) and Tukey-Kramer tests (for unbalanced datasets). A residual plot of predicted values vs residuals was analyzed, along with normal probability plots and stem and leaf plots, and if trends are observed in residuals due to lack of normality or unequal variances, the input data (ratio) was log transformed and the analysis was repeated using the transformed data. All the significance values indicated in figures 4-7 are Tukey-adjusted significance values (p < 0.05). These values were obtained by comparing the least square means between time*strain*treatment interaction groups in the three-way ANOVA approach. In certain situations when the entire dataset was used for analysis, adjustment for multiple 74 comparisions led to the inflation of Type 11 error rates due to the presence of too many unplanned comparisons. In such situations, we used a two-way ANOVA model instead of a three—way ANOVA model, and investigated the strain*treatment interactions within each time. We followed this approach because the most important effects that we wanted to identify from these analyses were effects due to treatment, strain and time, in that order. Using the three-way ANOVA, we were able to calculate significance values for the effects of treatment and strain, and the effect of time on treatment*strain combinations across all timepoints. The two-way ANOVA approach was able to calculate the significance values for the effects of treatment and strain, but could not capture the information about the effect of time on treatment*strain combinations across all timepoints. This was because the third factor ‘time’ was used to physically slice the dataset so that maximum information about the most important factors — treatment and strain, could be obtained from the model without inflating type II error rates and losing actual significance values. Two-way ANOVA approach was used for analysis of 111m mRNA and protein data, and three-way ANOVA approach was used for the analysis of mRNA and protein data of all other genes. 0 Transcript expression profiles for film and genes from the IL-1 complex IL-1 receptor antagonist We observed both treatment-induced and strain-specific increases in [11m mRNA expression in the lungs of NJ mice at 6, 12 and 24 h subsequent to ovalbumin challenge (Figure 5A). The level of 111m mRNA in NJ mice returned to baseline at the 72 h time point. Expression of [11m was also increased in C3H/HeJ mice at 6 and 12 h, but it was to a significantly lesser extent than in the NJ strain and returned to baseline by 24 h. At 75 48 h, there was only a treatment induced increase in expression in both AU and C3H/HeJ mice, but at much lesser levels compared to the earlier time points. In the spleens, no statistically significant changes in film mRN A expression were observed due to the effects of either treatment or strain (Figure 9A). IL-l agonists The expression of IL-la and IL-lb was examined in an effort to get a more complete assessment of the IL-1 related role in our model. While little is known about the mechanisms of the newly-identified IL-l family members, 111 f5, 11] f6, 11] f8, [11 f9 and IlIf10, they were also examined as they map near 111m within the Abhr1 locus. The active agonist, 1!] b, showed a similar expression pattern as film with significant treatment-related increases in expression in the lungs of ovalbumin-treated A/J mice at 6, 12 and 24 h time points (Figure 6A). Strain-related increase in expression in NJ mice compared to C3H/HeJ mice was observed only at 12 h time point (Figure 6A). In the C3H/I-IeJ mice, there was a treatment-specific increase in expression of 111 b at 6 and 12 h time points, but not at 24 h timepoint. In the spleens, no statistically significant changes in II 1 b mRN A expression were observed due to the effects of either treatment or strain (Figure 9B). Transcript levels of 111a also showed significant treatment-induced increases at the 6 h time point in both the strains, but no strain-specific differences were observed (Figure 7A). Both treatment— and strain-specific increases were observed in the new IL-l family member, 111 f9, at 6 h (Figure 7B), with the expression levels in the AU mice being higher than in C3H/HeJ mice. The levels of all IL-l agonists detected returned to (or 76 below) baseline levels by 72 h. We were not able to quantify the expression of 111f3, 111 f6, Ill/8 or 111f10 in treated or control lung tissues using the same template concentrations and real-time PCR conditions that were used to detect and quantify the other RNAs. These RNAs were not quantifiable despite using three different SYBR Green primer pairs. IL-l receptors The active receptor, 1]] r1 , was not induced by allergen exposure in either strain (Figure 8A), but its expression was decreased in both the strains at 72 h. In contrast, the decoy receptor, IlIr2, showed an increased expression in the lungs of NJ mice due to ovalbumin treatment at 6 h (Figure 8B), but there were no significant strain-specific differences. The expression levels of these two receptors returned to the baseline level at 72 h after ovalbumin challenge. 0 ELISA assays for IL-lra and IL-18 Of the cellular products, proteins are the most important drivers of biological functions. So, we tested for differences in protein production between N] and C3H/HeJ strains under allergen challenge. Although we found no major genetic polymorphisms between N] and C3H/HeJ strains, mRNA studies showed that there was a difference in the expression of 111m and IL-1 complex genes between the two mouse strains due to OVA treatment. To confirm these findings, we decided to measure the protein production ' levels of the positional candidate gene 111m, and its major agonist II] b in a subset of time points. 77 Protein quantification can be performed by two major methods — western blot and enzyme-linked immunosorbant assay (ELISA). Based on the principle of antigen- antibody interaction, ELISA is a quantitative approach that is suited to our aims of measuring treatment-dependent and strain-dependent changes in protein production with greater accuracy. The ELISA process involves the following steps. A primary antibody to the analyte (IL-Ira or IL-IB) tested for is coated on an ELISA plate. Then, the samples are added to the plate, and the analytes are bound by the primary antibody. To this, a secondary antibody (linked with an enzyme) is added, and it binds to a different epitope on the analyte. A substrate is added to this analyte-antibody complex, which is enzymatically converted by the enzyme linked to the secondary antibody to produce a color reaction or light proportional to the amount of analyte bound in the complex. A dilution series is prepared from purified recombinant protein (IL-Ira or IL-lfi), and is used as a standard with each assay for the corresponding protein. This colorimetric reaction can be measured in an ELISA plate reader to quantify the protein present in the samples based on the colorimetric quantification obtained from the standard series. ELISA kits are commercially available, but only a limited number of assays could be performed with each kit, and the kits are expensive. So we tried to develop our own ELISA assays, and succeeded in developing an assay for lL-lB, which was used to measure IL—lB protein levels in our experimental samples. As we were unable to develop a similar assay for IL-lra, therefore we purchased commercial IL-lra ELISA kits and used them to measure the protein levels in our experimental samples. Mouse lungs from the experiments (Figure 3) were homogenized in leBS- Tween20 buffer, centrifuged, and the supematants containing the proteins were aliquoted 78 and used for analysis using ELISA (more details available in ‘Materials and methods’). The time points used for the protein study were 6, 24 and 48 h post allergen challenge. 0 Protein production profiles of IL-lra and IL-lB IL-lra lL-lra protein was significantly induced by ovalbumin treatment in All mice at all time points examined (Figure 5B). While some increase in IL-lra protein levels were observed in the C3H/HeJ strain these changes were not significant. No strain-dependent increases in IL-lra expression were observed in any of the time points examined. IL-lB IL-lB protein production was significantly increased due to OVA in both strains of mice at the 6 h time point, and declined to baseline at 24 and 48 h (Figure 6B). The protein production at 6 h was not significantly different between the strains. There are some minor differences between the transcript and the protein levels in these genes at individual time points, but the overall trend remains the same. In the mRNA studies, we have shown that all the genes in the IL-1 complex, including [11m show an increased expression at the earlier time points and decline with time. This trend was also observed in the protein studies, but to a lesser degree in IL-lra protein. This is probably due to the fact that its stable expression over time is required to counteract the pro-inflammatory properties of IL-1. Maximal IL-lra protein production was observed at 24 h, contrary to 6 h in mRNA studies. This is also understandable because the protein production follows mRNA production, and the mRNA and protein production could peak 79 at different timepoints. No other studies have measured the in vivo temporal pattern of IL-1 receptor antagonist mRNA and protein productions in an allergic asthma model so far. The studies that have measured IL-1 receptor antagonist production in allergic asthma models have only done so at a single timepoint, usually when AHR measurements were donem. In other cases, temporality has been measured in cell cultures from lung or '46. Our measurements show the in vivo temporal variation in our mouse airway tissues model, and we believe this is a more accurate representation of the biological levels of the gene products. Thus, our results have provided a profile of the entire temporal variation pattern in mRNA and protein expression preceding the AHR measurement. 80 A N l A 6hr 12hr I 24hr 48 hr 72 hr g 10. = = o E 5 . a 5 [was '8 8 1 5 . E : 1 w .1! ‘ 5 + I :E i: iIOVA’ -' s = s= =5 + *1 E 6‘ .2 i ‘ ’ 2 c: 4- g * I . .g 1 * g + 1; m : t . :‘e 21E. * l m . E 1. E o. . 5 134:. CI,C.L:ICI,C.1 C3H AJ C3H AJ C3H AJ C3H AJ C3H AJ 5000 1 3 0 B 4500 1 6hr ii 24 hr 48 hr , 4000 . g ‘ 1 , 1 3500 1 . 10"“1 1 15' 3000 ., 1: .IOVA. a 2500 1 * . .‘E’ 2000 3 = j. * 1 :1. 1500 1 * 1 ‘ 1 1000 : , 1 5°“ I I . I I 1 0 ; I : -. . C3H AJ C3H AJ C3H AJ Figure 5. Lung homogenates were assayed for 111m (A) mRNA (n = 6/ group) using TaqMan assay and (B) protein (n = 5/ group) using ELISA. IL-1 receptor antagonist message and protein were increased in OVA-treated All mice. Samples were assayed in duplicate and values reported as mean iSEM. Significance differences (p < 0.05) due to: * treatment; + strain. 81 A 3°: ' 6h 5 12h ' 24h 25. g i ,L 1 1 1DPBS1 . 20. # E # 1 1 1 1 a E 1 1.0VA 1 1 E + . ' 15 . 5 a.................. Iltb expression normalized to 18$ =— ,_i 1 C3H AJ C3H AJ C3H AJ 10000 ; . B 90001 6hr . 24hr 1 48 hr 8000 1 C E 1 . 7000 1 E * 1 :1 ‘DPBSI E 6000 1 11 1I0VA1 1 E 50001 1 ' * {3 40001 1 1 =‘ 30001 ‘ 1 2000 ' 1 a 1:- :i . o i . J, ,E , 1::- 1 C3H AJ C3H AJ C3H AJ Figure 6. Lung homogenates were assayed for [11b (A) mRNA (n = 6/group) using assays-on demand, and (B) protein (n = 5/group) using ELISA. IL-1 beta message and protein were increased in OVA-treated A/J mice. Samples were assayed in duplicates and values are reported as mean iSEM. Significant differences (p < 0.05) due to: * treatment; + strain; # different from 24 h time point; and C different from all other time points. 82 A ‘2 . 1 ’ 1 6h 1 72h § 10 '1 3 g. 1 3 8 _ # # :5 DPBS' 1 -o 1 a» at 1 g 1 .IOVA, 1 7.: 6; E 1 1 8 41 : c i .2 . 1 8 2 1 I § 1 0 '. ti.— .' m 0 1 [=h—. . E C3H AJ B 12 1 1 1 1 6h 72h 1 a 10 1 # ' 1 B 1 1DPBS 1 .0 8 1 + 1 1 . £1: 9 * ' IOVA1 1 E 6 2 . 2 1 1 1: 4 _ 1 .2 1 :3. 2 1 I Q ' : 55 1 1 0 1 A a . o . E AJ C3H AJ Figure 7. Lung homogenate (n = 6/ group) transcript expression profiles of (A) 111a showed treatment- and time-specific differences, but no strain difference, and (B) IIIf9 was increased in OVA-treated A/J mice at the 6 hr time point. SYBR Green assays were used to measure mRNA levels. Samples were assayed in duplicate and values are reported as mean :tSEM. Significant differences (p < 0.05) due to: * treatment; + strain; and # time. 83 A112 ' 11 1 1 6h " 72h , 2 10 1 . 1 :3 8 1 1 10 P351 1 (D ‘ 1 £1 1 ' 1IOVA1 m 1 1 - v . E 6 -1 1 8 . c 4 1 1 .2 2 2 # ’1‘ i 1 r:- :I * ’ m 1 * ‘1: 0 w} 2. 2 :12 1:1— :h 1 E C3H AJ C3H AJ B 12 -1 1 ’ 1 53 1o . 6h 1 72h ‘- 1 , ._ 1 1 .9 8 .. 1DP8$1 1 E 1 # 1IOVA1 1 “ 6 “* . E 1 * 1 o , C 4 1 C 1 .2 ‘ 1 8 2- 1 E} 1 ‘3: 0 1:21— L I C3H AJ C3H AJ Figure 8. Lung homogenates (n = 6/group) were assayed for (A) II 1 r] and (B) 111 r2 message expression. Ill r2 was upregulated in OVA-treated A/J mice at the 6 h time point, in contrast no change was detected in II 1 r] in any group. SYBR Green assays were used to measure mRNA levels. All the samples were assayed in duplicate and values reported as mean iSEM. Significant differences (p < 0.05) due to: * treatment; and # time. 84 > :3 . 6 hr 12 hr 24 hr ‘é 1° 1 1 . 1 2 8 . 1UPBS1 ; B 1 IOVA. 1 .r: 1 : 1m . 76 6 1 E 1 E E 1 g 4 .1 g 1 C 1 1 : 2 1 E g 2 : I 5 a. . E x . 3 ‘1’ 0 1 4L : E ; C3H AJ C3H AJ B 12 . W 7 1: 1 1 s a: :1 1:. pigs? 1 g 3 1 as a ; 1 1 '5‘ ‘ ” 1IOVA_ 1 E 6 ’ 1‘ ‘ 1 2 4 1 1 , 1 C 1 ' 1' ‘ .2 : 1 E} 2 1 fl 1 g- .: 1 Q‘ 1 ‘ z '2 0 *1 — «17" #1 4-1 :ifl ii 1 = C3H AJ C3H AJ Figure 9. Spleen homogenates (n = 6/group) were assayed for (A) 111m and (B) 111 b message expression. No significant treatment- or strain-specific differences were observed in 111m and 111 b expression. Taqman and assay-on demand were used to measure mRNA levels of Illrn and 111 b respectively. All the samples were assayed in duplicate and values reported as mean iSEM. 85 D. SUMMARY OF THE ROLE OF IL-l RECEPTOR ANTAGONIST IN THE MOUSE MODEL We investigated the role of IL-1 receptor antagonist as a positional candidate gene for the QTL Abhr1 in our murine model of allergic asthma. We hypothesized that genetic polymorphisms in IL-1 receptor antagonist were responsible for the difference in AHR manifestation between A/J and C3H/He] strains. We sequenced the murine IL-1 receptor antagonist gene (111m) in A/J and C3H/He] strains of mice to identify polymorphisms in IL-1 receptor antagonist that might make A/J an airway hyperresponsive strain and C3H/He] an airway hyporesponsive strain. In the same model, we simultaneously examined the mRNA and protein production profiles of IL-1 receptor antagonist and its major agonist IL-lbeta. We also tested the mRNA expression profiles of genes in the IL- 1 complex, to determine the role of the IL-1 complex in this model of allergic asthma, with the focus on IL-1 receptor antagonist as a positional candidate gene. We sequenced a region of ~16 kb of genomic DNA from Al] and C3H/He] strains containing Him, but found no functional or regulatory polymorphisms. The only polymorphism we found was a previously established microsatellite in the 3’UTR. Being a microsatellite, it is less likely to contribute to the differences in transcript expression or protein production. There are some evidences to suggest that it could influence mRNA 1 - 7] . . . . . 69 I , investigation of a Similar role was beyond stability and play a role in intron splicing the scope of this study. Moreover, the positional candidate gene for Abhr2 (C5) had a deletion mutation in the NJ strain, resulting in a premature stop codon that abrogated the production of C5 protein in that strain. The lack of C5 protein in NJ strain has been shown to be responsible for insufficient IL-12 production, resulting in decreased 86 interferon-gamma (IFN 7) production, and increased airway inflammation and AHRIOZ. Our investigation at this level involved the conductance of a set of studies aimed at detecting a gene with similar effects, an approach widely followed in refining QTLs to QTGs based on positional candidate gene candidate gene approachesm. We found transcript and protein level increases in IL-1 receptor antagonist production in the airway hyperresponsive A/J strain compared to the hyporesponsive C3H/HeJ strain. This increase was co-regulated with the increase in the major agonist (IL-1(3) levels and that of other genes in the IL-1 complex. While we cannot totally exclude the role of the microsatellite repeat difference between these strains, investigation of its role at this point is beyond the scope of this study, and will have to be pursued in the future. In summary, we found limited 111m genetic polymorphisms between A/J and C3H/He] strains, but found that mRNA and protein levels were increased in the NJ strain. A similar pattern was observed with the mRNA expression of other IL-l complex genes. While we conclude that only these changes are not sufficient to produce increased AHR in Al] mice compared to C3H/HeJ mice, it is nevertheless plays an important role in preparing the milieu for the other gene products to take over the inflammatory process. Other potentital candidate genes within Abhr1 should also be investigated, that might have a major effect on AHR in our mouse model. 87 Chapter Three: IL-1 receptor antagonist — Human studies A. Isle of Wight birth cohort B. IL-1 receptor antagonist SNP association studies C. IL-1 receptor antagonist haplotype pair association studies D. Summary of the human study 88 A. ISLE OF WIGHT BIRTH COHORT 0 Introduction Asthma is a multifactorial disorder resulting from multiple genes, environmental factors and their interactions (Chapter 1, Sections B & C). In a multifactorial disease like asthma, patients manifest a dynamic combination of multiple phenotypes at various stages of disease during the course of their lifetimeé. Genetic association studies test the association of polymorphisms in a candidate gene (or locus) with various disease phenotypes. Of the approaches used in genetic association studies, the case-control study design tests for such associations at a single point of time, in a set of people affected (cases) and not affected (controls) with the disease. On the other hand, population-based longitudinal cohort studies investigate such associations in a recruited cohort of people over various points of life. These are usually prospective studies, which follow the recruits at various stages of their lives to record and investigate the appearance, morbidity patterns and the trajectory of the disease over time. In a disease like asthma, which is driven by both genetic and environmental factors and exhibits age and gender specific trajectories, longitudinal studies are probably the best tools available at present to dissect the pathophysiology of asthma. This study is an extension of our linkage studies in mice”, and the investigation of murine 111m, as described in Chapter 2. Mouse and human IL-1 receptor antagonist genes are syntenic (present in the same chromosome in both the species — Chromosome 2). Moreover, human ILIRN is located (human chromosome 2q14) very close to the recent positionally cloned novel asthma gene DPP10'73. Based on these evidences, we 89 hypothesized that human ILIRN gene is associated with asthma and related phenotypes, and polymorphisms in IL 1 RN have an important role to play in asthma pathophysiology. Most of the studies that have examined the relationship between ILIRN polymorphisms and asthma or related phenotypesm’wo’l5"174 have been performed on cross-sectional adult case-control populations. Longitudinal genetic association studies that examine the dynamics of both objective and subjective asthma phenotypes over different ages provide additional valuable information for preventive and age—specific asthma management strategiesm’m. A few existing and concluded longitudinal birth cohort studies have effectively investigated asthma and relative phenotypes using this approach'77'18'. The primary objective of our study was to establish the effect of the ILIRN gene on asthma in a longitudinal cohort of children who were evaluated for asthma and related phenotypes at ages 1, 2, 4 and 10 years. Apart from asthma, the additional phenotypes we tested for association with [L] RN polymorphisms were recurrent chest infections, bronchial hyperresponsiveness (BHR) and F BVl/F VC ratios. 0 Isle of Wight birth cohort — population characteristics Between January 1989 and February 1990 children born on the Isle of Wight, UK. were recruited to participate in a longitudinal study (n = 1,456). The study was approved by the Local Research Ethics Committee and informed written parental consent was obtained for all the participants. The population is largely Caucasian (99%), living in a semi-rural environment with no heavy industry. At birth, data from birth records and extensive questionnaires were collected, including information on asthma and allergy family history, as well as maternal smoking 90 habits. Maternal and cord sera were collected and assayed for IgE. At ages 1 and 2 years, the questionnaire-based data collection was repeated, physical examinations were performed on the children by a study physician and symptoms of asthma and allergic diseases were recorded. At age 4, questionnaires and physical examinations were repeated and skin prick tests to common aeroallergens and food allergens were performed182 . At age 10, a subset of the population underwent pulmonary function testing (Table 1). Along with the physical examinations and questionnaire information update at age 10, anticoagulated blood samples were collected and stored frozen for subsequent DNA analysis (n = 92]). Additionally, International Study of Asthma and Allergy in Childhood (ISAAC) written questionnaires were used to assess respiratory, nasal and 183 dermatological symptoms . The characteristics of the study population are shown in Table 3. 0 Outcomes tested for genetic association in the Isle of Wight birth cohort We investigated the following four outcomes, which were assessed by the study physician based on questionnaire data, clinical diagnosis and pulmonary function tests as applicable to the various ages in which the phenotypes were measured. The analyses were carried out on a representative unselected subset of 921 individuals from the Isle of Wight birth cohort, whose DNA was available for genetic studies. 02° Asthma: At ages 1, 2 and 4, asthma was defined as having three or more episodes of wheezing, each lasting for more than three days in the past twelve months based on questionnaire data. At age 10 asthma was defined as ever having a physician diagnosis of 91 asthma in addition to wheeze in the past twelve months. 02° Recurrent chest infections: Recurrent chest infection was defined as parental report of two or more episodes of productive cough lasting for five or more days in the past year. The presence of wheeze and antibiotic usage were not prerequisites for the diagnosis of chest infection. This phenotype was measured at ages 1 and 2. '3' Bronchial Hyperresponsiveness (BHR): BHR was defined as being present when the PC20 was < 4.0 mg/ml of methacholine during pulmonary function tests. This phenotype was measured at age 10. '3' FEV1/FVC1 ratio: The ratio of forced expiratory volume in one second (F EVI) and forced vital capacity (F VC) was calculated from the forced expiratory maneuver during spirometry. This phenotype was measured at age 10. 0 Risk factors evaluated for the outcomes tested: 03° Genetic risk factors: Genotypes in the single SNP analyses and haplotype pairs in the multilocus analyses were evaluated as the risk factors that could explain the effect of IL] RN gene on the outcomes investigated. Maternal smoking during pregnancy, environmental tobacco smoke exposure, low birth weight (< 2,500 g), male gender and breastfeeding status at least until 3 months were used as confounders in these analyses. 02' Environmental risk factors: 92 The modifying effect of the various degrees of exposure to environmental tobacco smoke (ETS) on the genetic risk that the children would have for asthma, chest infections, BHR and reduced FEVl/FVC ratios was evaluated Other relevant environmental factors, such as low birth weight (< 2,500 g), male gender and breastfeeding for at least 3 months, were used as confounders in these analyses. The levels of ETS exposure on children were classified into the following three groups. When mothers did not smoke during pregnancy and there was no exposure to household ETS in children up to the age of 10, children were categorized under the group “ETS-0” (n = 431). When mothers did not smoke during pregnancy, but household members (including mothers) smoked within the home at some point up to the children’s age of 10 years, the exposure status was categorized as “ETS-1” (n =194). When mothers smoked during pregnancy and the children were also exposed to household ETS at some point up to the age of 10, the exposure was categorized as “ETS-2” (n = 293). No children had mothers who smoked during pregnancy but no exposure to household tobacco smoke afier birth. 93 TABLE 3. POPULATION CHARACTERISTICS OF ISLE OF WIGHT BIRTH COHORT Initial sample (%) No. used in analysis Variable n = 1,491 (%) n = 921 Asthma at age 1 Yes 133 (8.9) 95 (10.3) No 1,241 (83.2) 776 (84.3) Missing 1 17 (7.9) 50 (5.4) Asthma at age 2 Yes 132 (8.9) 105 (l 1.4) No 1,099 (73.7) 707 (76.8) Missing 260 (17.4) 109 (l 1.8) Asthma at age 4 Yes 181 (12.1) 133 (14.4) No 1,033 (69.3) 698 (75.8) Missing 277 (18.6) 90 (9.8) Asthma at age 10 Yes 178 (11.9) 134 (14.6) No 1,192 (80.0) 786 (85.3) Missing 12] (8.1) l (0.1) Chest infections at age 1 Yes 10] (6.8) 7] (7.7) No 1,273 (85.4) 800 (86.9) Missing 1 17 (7.8) 50 (5.4) Chest infections at age 2 Yes 157 (10.5) 118 (12.8) No 1,074 (72.0) 694 (75.4) Missing 260 (17.4) 109 (11.8) BHR Yes 169 (11.3) 157 (17.0) No 614 (41.2) 542 (58.8) Missing 708 (47.5) 222 (24.1) FEV l/FVC 5"‘-95‘h percentile 1033 (69.3) 912 (99) Missing 458 (30.7) 9 (1.0) Smoke exposure ETS-0 647 (43.4) 431 (46.8) ETS-l 464 (31.1) 194 (21.1) ETS-2 370 (24.8) 293 (31.8) Missing 10 (0.7) 3 L03) Definition of abbreviations: ETS-0, mothers did not smoke during pregnancy and children not exposed to environmental tobacco smoke in the household; ETS-1, mothers did not smoke during pregnancy, but children were exposed to environmental tobacco smoke in the household; ETS-2, mothers smoked during pregnancy and children were exposed to environmental tobacco smoke in the household. 94 B. IL-l RECEPTOR ANTAGONIST SNP ASSOCIATION STUDIES 0 Polymorphism selection and genotyping: We checked SNPper (http://snpper.chip.org) and dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNPl) databases for SNPs in the ILIRN gene. None of the reported SNPs found in the databases resulted in an amino acid change. We analyzed the ILIRN SNP information available from Hapmap (http://hapmap.org/) and found that majority of the SNPs within the gene were in strong linkage disequilibrium (LD). LD is a statistical measure of the strength of association between two alleles at different markers”. Previous reports from Gohlke et al also reported that the SNPs they tested covering the IL] RN gene were also in strong LDW. As our population is primarily Caucasian, similar to the population used by Gohlke et a1, and as the SNPs within the genes are in strong LD, we chose to investigate the three SNPs that were associated with asthma in the Golke study (r32234678, rs878972 and rs454078). All these SNPs had minor allele frequencies greater than 10 per cent (Table 4). TABLE 4. ILIRN SINGLE NUCLEOTIDE POLYMORPHISMS TESTED SNP Alleles Allele frequency Gengtype Genotype frequency (n) r52234678 A/G 0.75/0.25 AA/GA/GG 0.56/0.37/0.07 (921) rs878972 A/C 0.75/0.25 AA/AC/CC 0.56/0.38/0.06 (921) rs454078 A/T 0.74/0.26 AA/AT/TT 0.55/O.38/0.07 (918) Definition of abbreviations: n = Number of individuals for whom genotype information is available. (Total number of individuals genotyped = 921) 95 Genomic DNA was isolated from blood samples using QIAamp DNA Blood Kits (Qiagen, Valencia CA) or the ABI PRISM 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA). DNA yields were quantified by spectrophotometry (NanoDrop technologies, Wilmington DE) or by measuring the quantity of the single copy gene, RNAseP, using 5’nuclease fluorescent chemistry PCR normalized to known genomic DNA standards by cycle threshold. Genotyping was performed by . 4 Pyrosequencrng®18 . To avoid background signals, a blocking primer was used as required for individual SNPsI85 . Primers were designed using pyrosequencing primer design resources (http://primerdesign.pyrosequencing.com/j sp/TemplateInput.j sp, http://biodev.hgen.pitt.edu/sop3/index.php). The SNPs selected were genotyped in all children with available DNA (n = 918 - 921). - Pyrosequencing: Pyrosequencing is a non-electrophoretic method for DNA sequencing, and the technique works in the following way. Primers are designed for a region of ~150 bp around the SNP to be genotyped. One of the primers is biotinylated at one end. The PCR product formed contains two strands of DNA -— a non-biotinylated strand and a biotinylated strand. The biotinylated strand is immobilized on a Pyrosequencing reaction plate, and 10 — 15 nucleotides around the SNP are sequenced by addition of each of A, G, T and C nucleotides in a predetermined order, as inferred from the sequence around the SNP. With the incorporation of each nucleotide, a pyrophosphate is released, and it undergoes an enzymatic reaction to give light (Figure 10), which is captured by a laser- assisted camera. The resulting sequences, called pyrograms (Figure 11), are used to determine the genotype of an individual. 96 Polymerase (NA)n + Nucleotide > (NA)n+1 + PPi ATP sulfurylase PPi + APS % ATP 4» 8042' _ . Luciferase ATP + Lucrferm + 02 g AMP + PPi + Oxyluciferin + C02 4» Light Figure 10. Schematic representation of the progress of the enzyme reaction in pyrosequencing. PPi indicates pyrophosphate. (Figure and legend adapted fromm). AT 267 24 .- _ 24 20 __ 20 14 ....._.. ..--.. ‘ 14 s 12.4.-.-” .. 12". 10 ‘ 1o ‘ 8 _ 8 , 6 l ' 5 . I 2 ' d4 ,,...- "' 2 0 Tr 1 r r r . ' 0- ram. ”w q . ; ‘ . T O y ATG G GTAT rA ATGAGATGATA AT AGATGATA Figure 11. Pyrograms of the investigated sequence (A/T)GAAGATGGGA The SNP is in parentheses; A and T are the alleles. AA and TT are homozygote genotypes and AT is a heterozygote genotype (Figure adapted from186 ), as visualized in the pyrograms. AA genotype has a single peak at the A position, and no peak at the G position. GG genotype has a single peak at the G position, and no peak at the A position. AG genotype has one peak at the A position and one peak at the G position, and both these peaks are half the height of either the A or G peaks in AA or GG homozygotes. 97 0 Statistical analysis: The SNPs were tested for Hardy-Weinberg equilibrium and linkage disequilibrium using Haploview 3.2 (http://www.broad.mit.edu/mpg/haploview/) software. Using the genotype data from SNPs r32234678, rs878972 and rs454078, chi- square tests were used to test for associations of IL] RN SNPs with asthma, recurrent chest infection, BHR and FEV/FVCI ratio. Permutation tests were performed using R software v2.1.1 (http://cran.r-project.org) to confirm that the associations between [L] RN SNPs and the outcomes tested were not spurious. The statistical significance threshold was p < 0.05. 0 Results All SNPs were in Hardy-Weinberg equilibrium, and were in high LD with each other (Table 5) based on D’ and pairwise r2 values calculated by the default algorithm in Haploview software187 . Of the children genotyped, 54.25% of children were homozygous for the major allele in all three loci (n = 498), 35.51% were heterozygous at all three loci (n = 326), and 5.88% were homozygous for the minor allele at all three loci (n = 54). Table 5. Pairwise comparison of linkage disequilibrium for [L] RN SNPs SNP ID rs2234678 rs878972 rs454078 rs2234678 1 0.988 (0.968) 0.970 (0.899) r5878972 1 0.976 (0.902) rs454078 1 Values indicated in the cells: pairwise LD estimates - D’(r2) The SNPs r32234678 and rs878972 were significantly associated with asthma at age 2, as well as with recurrent chest infection at age 2 (Table 6). Similarly, permutation 98 tests based on 50,000 Monte Carlo simulations identified significant associations of the SNPs r32234678 and r3878972 with asthma and recurrent chest infection (p < 0.05), limited to age 2. We did not find any associations with BHR or the FEVl/FVC ratio. The significance values for the associations of the individual SNPs (Table 6) were adjusted for false discovery rates using the Benjamini-Hochberg algorithm'gs. 99 .ooflEon E @0339: mos—g a 33$:me .Eficowfi maniac: - EmEmEom mam: moose @9686 02mm Sm 335.5% 823 a 0 Banana 3va 28 ©9686 02mm 38880 .850 =m 8m 823 a 2850 9 wow: 82$ 3mg 0833.50 .32? oogowmcwmm 05 How 9 com: 83 EB c9823? .oBmtg 080350 msosfifioo m £ 038 U>nS >mE m< ... and mmd wvd vmd Nod 36 N5 _.. U>nS>mE mod mad cod mod 36 3d 3% 52 12m 36 mad 3:... N36 3?: mac... vac m: N own “a mcotoomfi 30:0 ovd m _ .o 56 _.c ovd m _.o cow K _ own as mcozoflfi 36:0 36 wed wnd mod 3d wd of. 3: 3 own “a mEEflQ mwd wed 8d omd 56 $6 wow m2 v own an «Enumxx wmd 36 ”no... 25.: we... 3.: 2:. m3 N own an «EEQQ .86 mod and wed 36 Ed 0: 3 fl own an «853‘ 353cm ya..— o=_m> m toms?“ Maw 02? a @8392“ Man 02"; m .538...“ 32 6398.3. 2.38:0 wgvmvfl «Saga.— wvevmnnfi 5.6.29 we Loan—=2 mZOEmeZH HmmIU Q72 HOZmZm DmEiA:0 .006:0w 0.03 0.36:0 0:. 5 60:: 80650600 8...... 8.”... - 00.. 8.0 :8... .08 - 8... 2...: 8... 8.8 . 8... 8.: <<<..<<< 8 000.000 .8... 8.: - 8... .0... 8... 8.. - .m... 80... m0... 8.: - 8... 8.. < .. .0 2.8 :0 0:...> .. .0 2.8 :0 2...; .. .0 2.8 :0 029... v.0: a... u 5 0.000 .8: n 5 .08. .08 u e. 0.8.. — own 50300:: «mesa 25.2590“ 82...... 0...... - 00.. 2... 2.... 2.: - 8... S... 2.... 8... - 8... N2. <<<.<<< 8 000.000 8... 8.: - 8... 8. .. 8... 2... - mm... 2.... .2... 2... - 8... .8... 5225. e, <<.0m:0 mum. @8033: 3m .m:0_.0>:0m:0 8% 300.33 .mm .m:0_.0>..0m:0 sac—fl Ntmhm— 7um otmkm 3 6:0 6 .N .— 00w: .0 0.5—.00 :0 305000000... 60.0023— mmm>q<2< ~:HOiE:0 ..06:0w 0.03 0.00—0:0 0:. 0. 600.. 0.065.020“. .0. 0m... 00.: .o.m N0... 2.. <<<\<<< m> Pompom. 0.0.. .m... 6m... 0: m0... m0... 3% m> «103000 - - . - - . 3% 0> <<<\<<< .0 ..\..m0 m0 .0 {ema mo 000.00.. :03 0.00.3.5 an. .m..0..0>..00:0 009 0.00.30... 03 .0..0..0>..00:0 Sin At 00.0000... 200:0 2.00.503. 3 00.0000... 200:0 2.00.503. 20.0.0907... ram—IO HZmdMDUmM MO... DmEEEHm 8. .0 .N .. mm0< . mHZmEmmDmxme DmhHqu 4 kb. If the product size was 500 bp — 4 kb, addition of isopropanol has no effect on DNA yield. Then, QIAquick spin columns were placed in the provided 2 m1 collection tube. The solution in the tube was transferred to the spin columns, and centrifuged at 14,000 rpm for 1 min. The flow-through was discarded and the spin columns were placed again in the collection tube. 0.5 ml of Buffer QG was added to the QIAquick columns, and centrifuged again at 14,000 rpm for 1 min. The flow-through was discarded again, the QIAquick columns were placed again in the collection tubes. 0.75 ml of Buffer PE was added to the column, allowed to stand for 5 minutes and centrifuged at 14,000 rpm for 1 min. The flow-through was discarded, the QIAquick column placed again inside the collection tube and centrifuged under the same conditions one more time. Then, The QIAquick column was removed and placed inside a clean 1.5 ml microcentrifuge tube. 30 pl of Buffer EB was added to the center of the QIAquick membrane in the column, and allowed to stand for 1 minute. The fresh collection tube with the column was centrifuged again at 14,000 rpm for 1 minute. About 28 pl of eluate containing the amplified DNA fragments was obtained, and was stored at 4°C for further use. A mixture of 9 pl eluate 127 and 3 pl 10 pM forward primer, and another mixture containing 9 pl eluate and 3 pl 10 pM reverse primer was submitted for fluorescent automated sequencing in Research Technology Support Facility (http://genomics.msu.edu) in Michigan State University. Sequencing was done using BigDyeTM terminator Cycle Sequencing Ready reaction DNA sequencing kits (Applied Biosystems, Foster City, CA) in an ABI Prism 377 DNA sequencer. Sequences were analyzed and aligned using Chromas v1.45 (Technelysium Pty Ltd, Australia) and DNAssist v1.02 (Bellville, S. A.) software packages. Multiple alignments of Genbank, Celera, A/J and C3H/He] 111 m sequences were performed with ClustalW option in BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). C. TaqMan/SYBR Green real-time RT-PCR Total RNA was extracted from lungs using TRIzol reagent (Invitrogen, Carlsbad, CA). Tissues were homogenized in 2 m1 of TRIzol reagent in 10 ml tissue dounces at room temperature using about 10 strokes. The pestle was rinsed with 1 ml TRIzol reagent. This 1.5 ml mixture was poured into a sterile microfuge tube. The sample was incubated at room temperature for 5 min to allow complete dissociation of cells. Subsequently, 200 pl chloroform were added, tubes were capped securely and shaken vigorously by hand for 15 sec, then incubated at room temperature for an additional 3 min. Samples were centrifuged at 12,000 rpm at 4°C for 15 min. The upper, aqueous phase containing the RNA was transferred to a new, sterile 1.5 ml microfuge tube and RNA precipitated by adding 500 pl isopropyl alcohol. This mixture was incubated at room temperature for 10 min. Samples were centrifuged at 12,000 rpm at 4°C for 15 min. The RNA precipitate formed a gel-like paste on the bottom of the tube. The supematants were carefully 128 removed and the pellets washed with 1 ml of 75% ethanol. The samples were vortexed and centrifuged at 9,500 rpm for 5 min at 4°C. The supematants were carefully removed and air-dried for 15-30 min being careful not to contaminate the samples by aerosol or over dry. RNA pellets were dissolved in 300 pl 0.2% DEPC-treated water and stored at -70°C. Genomic DNA was removed by DNAseI treatment. Total RNA (300 pl) was combined with 20 pl 1M Tris (pH = 7.5), 4 pl 1M MgClz, 2 pl 10 mg/ml BSA, 4 pl DNAseI (RNAse free), and 70 pl DEPC-HzO to make 400 p1 total volume. This was incubated at 37°C for 30 min and then precipitated with 40 pl 3M sodium acetate (pH = 5.2). The tubes were filled with ice-cold 95-100% ethanol and incubated at -20°C for 30 min, then centrifuged at 12,000 rpm for 5 min at room temperature. The supematants were carefully removed and 1 ml 75% ethanol was added, the samples were vortexed, and re-spun under the same conditions. The pellets were air dried for 5-10 min and resuspended in 300 pl DEPC-HzO. Complementary DNA (cDNA) was reverse transcribed from total RNA using TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA). According to the manufacturer’s instructions, 2 pl 10X RT buffer was combined with 4.4 pl 25 mM MgC12, 4 pl deoxy NTPs mixture, 1 pl random hexamers, 0.4 pl RNAse inhibitor, 1.25 pl Multiscribe reverse transcriptase, 400 ng total RNA and a suitable amount of DEPC-HZO to make 20 pl total volume. The reagents were capped and centrifuged at 2000 rpm for 2 minutes. Thermal cycling was conducted as follows: 25°C (10 min), 37°C (60 min), 95°C (5 min), followed by an indefinite hold period at 4°C. Samples were diluted to 4 ng cDNA/ p] in double distilled H20. 129 Three different types of mRNA expression assays were used. 111m was assayed using a self-designed primer set and a TaqMan probe. 111 b mRNA levels were measured using assay-on demand gene expression assays (Applied Biosystems, Foster City, CA). The other genes were measured using SYBR Green assays using self-designed primers. For the Taqman assays, 12.5 pl 2X TaqMan mastermix, 1.25 pl 20X Illrn probe, 2.25 pl 10 pM forward primer, 2.25 pl 10 pM reverse primer, 5.75 pl RNase free H20 and 1 pl of cDNA from the corresponding samples were added to the Applied Biosystems optical plates and sealed with optical caps. The plate was centrifuged at 2,000 rpm for 2 min in an Eppendorf 5810R centrifuge (Eppendorf, Westbury, NY). For assay-on demand, 12,5 pl 2X TaqMan Universal PCR mastermix, 1.25 pl 20X assay-on demand Gene expression assay mix, 10.25 pl RNAse free water and 1.0 pl cDNA from corresponding samples were added to the Applied Biosystems optical plates and sealed with optical caps. The plate was centrifuged at 2,000 rpm for 2 min in an Eppendorf 5810R centrifuge (Eppendorf, Westbury, NY). Real-time PCR was performed for these TaqMan or assay- on demand assay plates in an ABI7700 sequence detection system (Applied Biosystems, Foster City, CA) with the following conditions: 50°C (2 min), 95°C (10 min), followed by 40 cycles of 95°C (15 sec) and 60°C (1 min). Data were collected during all the steps in the PCR reaction. For SYBR Green assays, 2.5 p] 10X SYBR Green buffer, 0.25 pl of 10 pM forward primer, 0.25 p1 of 10 pM reverse primer, 3.0 pl 25 mM MgC12, 2.0 pl 12.5 mM dNTPs, 0.15 pl 5U/p1 AmpliTaq Gold, 0.25 pl Uracil N-glycosylase, 15.6 pl double distilled H20 and 1 pl of cDNA from corresponding samples were added to the Applied Biosystems optical plate, sealed with optical caps and centrifuged at 2,000 rpm for 2 min. 130 in an Eppendorf 5810R centrifuge (Eppendorf, Westbury, NY). Real-time PCR was performed for these TaqMan or assay-on demand assay plates in an ABI7700 sequence detection system (Applied Biosystems, Foster City, CA) with the following conditions: 50°C (2 min), 95°C (10 min), followed by 40 cycles of 95°C (15 sec), 60°C (1 min) and 72°C (15 sec). Data were collected at 72°C. Primers and probes used for real-time RT- PCR are listed in Table 11. Table 1 1. PRIMERS AND PROBES USED FOR REAL-TIME RT-PCR ASSAYS *Primers & probe Sequence (5’ — 3’) Assay based on Illrn GenBank M64404 Forward primer AGTACTGCCGAGGCCTGTAATAA Reverse primer TTGTTCCTCAGGCCCCAAT Probe ACCAACTGCCTGATCACTCTGGCCAT ' Illa GenBank NM_O l 05 54 Forward primer CAGGGCAGAGAGGGAGTCAAC Reverse primer CAGGAACTTTGGCCATCTTGAT III/9 GenBank NM_15351 1 Forward primer CCCTTGTGACAGTTCCACGAA Reverse primer GGGTACTTGCATGGGAGGATAG IlIrI GenBank NM_0083 62 Forward primer CGGCGCATGTGCAGTTAATA Reverse primer TGTAGCCGTGAGGATGATAAAGC 111r2 GenBank NM_010555 Forward primer AGTGCAGCAAGACTCTGGTACCTA Reverse primer AGTTCCACAGACATTTGCTCACA 18S rRNA Forward primer CGGCTACCACATCCAAGGAA Reverse primer GCTGGAATTACCGCGGCT * Primers & probe designed using Primer Express® software D. Statistical analysis of mRNA expression data Complementary DNA from a single time point (n=24, 6 samples/group) as well as a standard curve were assayed in 96 well optical plates in duplicates. The CT values of duplicates were averaged, and the relative amounts of target RNA were calculated by referring to the standard curve. The averages and standard deviations of target RNA and 188 RNA from each strain/treatment/time group (n = 6 samples) were calculated. These 131 averages were compared to show the effects of strain, treatment and time using the manufacturer’s protocol“. The data of the relative ratio of target gene/188 rRNA were analyzed using SAS v 9.1 (SAS Institute, Cary, NC) sofiware. The data was analyzed by two- or three—factor ANOVA mixed model, and the P values were adjusted for multiple comparisons using Tukey or Tukey-Kramer tests. The results were considered statistically significant when P values were < 0.05. When the residual plots in SAS v 9.1 showed trends indicating unequal variances within the groups, the data was analyzed after subjecting it to a logarithmic transformation. E. PROTEIN COLLECTION FROM LUNG TISSUES Al] and C3H/HeJ mice were sensitized and challenged as described above (Materials and methods — Section A). 1X PBS-Tween 20 buffer required for protein collection was prepared as follows. A 10X PBS stock buffer was prepared by adding 1.25 g NaHzPO4.HzO, 7.1 g NazHPO4 and 43.85 g NaCl to a suitable quantity of nanopure double distilled HzO to make a volume of 500 ml. 1X PBS-Tween 20 buffer was prepared as follows: To 100 ml of 10X PBS, 900 ml of nanopure ddeO was added, and the pH was adjusted to 7.2. To this, 1 ml of Tween-20 was added, mixed well with a stirrer, autoclaved and stored at 4°C. Mice were sacrificed and lungs and spleens were collected at 6, 24 and 48 hrs post-challenge. Right lungs and spleens were collected and placed in sterile 5 ml tubes (USA Scientific, Ocala, FL) on ice. After all the right lung. lobes and spleens were collected in a single time point, the tissues were weighed using a Mettler Toledo AX504 132 balance (Mettler Toledo, Columbus, OH), and 20 pl 1X PBS-Tween 20 buffer was added to each mg of tissue. The tissues in the buffer were then homogenized using a Pro 200 homogenizer with a 5mm x 75mm generator (Pro Scientific Inc, Oxford, CT). The homogenized tissues were centrifuged at 5000 rpm for 5 min at 4°C. The supematants containing the proteins were aliquoted and stored at -80°C. F. ENZYME LINKED IMMUNOSORBANT ASSAY (ELISA) o ELISA for IL-1 ra ' IL-lra protein levels in the lungs were determined by an IL-lra/IL-1F3 Quantikine ELISA kit (R&D Systems, Minneapolis, MN). All the reagents were reconstituted and the recombinant standard for the IL-lra assay was prepared as per the manufacturer’s protocol (http://www.mdsystems.com/pdf/mra00.pdf). A microplate coated with a polyclonal antibody specific for mouse IL-lra is available with the kit. The plate layout was recorded prior to the assay. 50 pl of assay diluent RDlW was added to each well, followed by the addition of 50 p1 of standard, control or sample per well. The wells were covered with the adhesive strip provided. They were incubated for 2 h at room temperature on a horizontal orbital microplate shaker set at 500 i: 50 rpm. This was followed by washing the plate five times with 400 pl wash buffer. After the last wash, the plate was inverted and blotted against clean paper towels. 100 pl of mouse IL-lra conjugate was added to each well, the plate was covered with new adhesive cover strips and incubated for 2 h at room temperature on the shaker. After incubation, the plate was washed five times using 400 pl washing buffer, and blotted on clean paper towels after the last wash. 100 pl of Substrate Soultion was added to each well. The plate was 133 incubated for 30 minutes at room temperature at the benchtop, protected from light. After the incubation, 100 p1 stop solution was added to the wells and gently tapped to ensure even mixing of the solutions. The optical densities of the samples were read in an ELISA reader (Microplate ELISA Reader, SoftMax program, Molecular Devices, Sunnyvale, CA) at 450 nm, with the correction set to 570 nm. The concentrations of IL- 1ra in the samples (pg/ml) were calculated based on the standard curve. The sensitivity of the assay was 4-13 pg/ml. o ELISA for IL-lB Anti-mouse IL-lB antibody, biotinylated anti mouse IL-1B antibody and recombinant mouse lL-IB were purchased from R&D Systems (Minneapolis, MN). The following materials were purchased from sources as indicated in parenthesis. p-nitro-phenyl phosphate (PNPP) (Sigma, St Louis, MO, USA); Streptavidin alkaline hosphatase (Jackson ImmunoResearch, West Grove, PA); ELISA plates (Costar, Corning Inc., Corning, NY). ELISA plates (96-well EIA/RIA plate, 96-well easy washTM, high binding, Corning, NY) were coated with anti-mouse IL-lB antibody (purified and unlabeled; lpg/ml) diluted in carbonate buffer (0.005 M, pH 9.6) and incubated overnight at 4°C. Unbound antibody was discarded and the plates were blocked (0.17% BSA/PBS) at 37°C for 3 h. After washing (0.05% Tween 20 in PBS) recombinant IL-IB protein (standard) in two-fold dilutions and samples at appropriate dilutions (1 in 5 dilutions for 6 h and 1 in 2.5 dilutions for 24 and 48 h) in dilution buffer (0.085% BSA, 0.05% Tween 20 in PBS), were added to the plates and incubated overnight at 4°C. Following incubation, plates were washed four times and biotin-labeled IL-lB was added (0.1 pg/ml) and incubated at 134 37°C for 90 min. After incubation, plates were washed four times and streptavidin alkaline phosphatase (SAP) conjugate was added at 1:4000 (in dilution buffer). Subsequently, plates were washed again and p-nitro phenyl phosphate (PNPP) substrate added (1 tablet per 5 ml substrate buffer, according to manufacturer’s instructions). Reactions were allowed to develop at room temperature in the dark and absorbance was measured in a microplate reader with dual mode of wavelength at 405 nm (peak) minus 690 nm (background) using KC4 software program (Synergy HT Multifunction Reader, Bio-Tek, Winooski, VT). According to the manufacturer’s instructions, dual mode provides relatively better measurements since it adjusts the reading for background interference. All reagents were used at a final volume of 50 pl/well except for blocking buffer that was used at 75 pl/well. The sensitivity of the assay was 13 pg/ml. G. PYROSEQUENCING - Polymerase chain reaction for Pyrosequencing The sequences surrounding the SNPs tested were obtained from dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNPO. Primers were designed using pyrosequencing resource (http://primerdesign.pyrosequencing.com/jsp/Templatelnput.jsp, http://biodev.hgen.pitt.edu/sop3/index.php) available on internet. The primers used for pyrosequencing are listed in Table 12. Forward and reverse primers were obtained first, and PCR reactions were performed with the primer pairs in a subset of samples as follows. 2.5 pl 10X PCR buffer, 2.0 pl 25 mM MgC12, 0.3125 pl 10mM dNTPs, 0.15 pl 5U/pl AmpliTaq Gold DNA polymerase (all from Applied Biosystems, Foster City, CA) 0.5 pl 10 pM forward primer, 135 0.5 pl 10 pM reverse primer (both from IDT, Coralville, IA), 14.0375 pl double distilled H20 and 5.0 pl genomic DNA (2 ng/pl) were added to PCR reaction tubes or plates, capped and centrifuged at 2,000 rpm for 2 minutes. TABLE 12. PRIMERS USED FOR GENOTYPING BY PYROSEQUENCING Primers Primer sequences (5’ — 3’) Forward Primer Reverse Primer“ Sequencing Primer Blocking primer Forward Primer Reverse Primer* Sequencing Primer Blocking primer Forward Primer Reverse Primer* Sequencing Primer Blocking primer dbSNP ID: r82234678 TGCTACTTTATGGGCAGCAG /5 ’ Bio/TGAGAGTGGAAGGAGCTTACC TTGAGTTAGAGTCTGGAAGA TGCTAC l l l ATGGGCAGddC dbSNP ID: r8878972 TCCCACCACTTCCCTTACAG /5 ’ Bio/GCCTAAAATTGTTTTCAAACTTGG TGCTGACTCAAAGGGTA TGGAGGAGGAGGAGAAGGTGAAGAddC dbSNP ID: rs454078 CAGTGGCTTGAAACAACCAA l5 ’Bio/TGAATGCAGCTTCCAAAGTG TTGAAACAACCAA None * Biotinylated Primer PCR reactions were performed in MJ Research Peltier FTC-200 or PTC-225 thermal cyclers (MJ Research, Watertown, MA) with the following conditions: 95°C (5 min), followed by 45 cycles of 95°C ( 15 sec), 60°C (30 sec), 72°C (15 sec), and a final extension and hold temperatures of 72C° (5 min) and 4°C (forever), respectively. The PCR products were separated on 2% agarose gels, and if the PCR reaction produced a clean, robust product, biotinylated primers were obtained. PCR products obtained from the reactions using one ordinary and one biotinylated primer were used for 136 pyrosequencing. All PCR reactions were performed on DNase/RNase free non-skirted 96 well PCR plates (Dot Scientific Inc, Burton, MI). 0 Sample preparation using the vacuum prep tool Four reagents were required for sample preparation using the vacuum prep tool for pyrosequencing — binding buffer, annealing buffer, denaturing reagent and washing buffer. In the same PCR plate, 25 pl of each biotinylated PCR product was mixed with 3 pl Streptavidin-Sepharose high performance beads (Amersham Biosciences, Uppsala, Sweden), 12 pl of nanopure H20 and 40 pl of binding buffer (10 mM Tris, 2 M NaCl, 1 mM ethylene diamine tetraacetic acid (EDTA) and 1 ml Tween-20/1iter of buffer; pH 7.6). The plate containing the biotinylated PCR product and the binding buffer was shaken at 1400 rpm for 10 minutes at room temperature. While the plate was shaking, 0.2 pl 100 pM sequencing primer and 40 pl 1X annealing buffer (20 mM Tris, 2mM Magnesium acetate tetrahydrate; pH 7.6) were added to all the wells in a PSQTM plate (Biotage, Uppsala, Sweden). A master mix was prepared for the number of reactions for every assay, and was added to the plate using a multi-channel pipette. Four troughs supplied with the Pyrosequencing vacuum prep tool were filled with approximately 180 ml of high purity water, 70% ethanol, denaturing solution (0.2 M Sodium hydroxide) and washing buffer (10 mM Tris; pH 7.6). These troughs were refilled whenever needed. The probes in the vacuum tool were primed by applying vacuum and lowering the tool into the trough with high purity water for approximately 30 137 seconds to wash the filter probes. The PCR plate containing the biotinylated PCR product and the binding reaction was removed from the shaker, and the sepharose beads containing the immobilized biotinylated DNA strand were immediately captured by slowly lowering the vacuum prep tool with the probes into the PCR plate. The probes captured the streptavidin beads containing the biotinylated DNA strand. The PCR plate and the vacuum prep tool were carefully lifted together to check if all the beads had been captured on the probes. Without touching the sides of the wells in the PCR plates, the vacuum prep tool was lifted from the PCR plate and washed for 5 seconds in the troughs with 70% ethanol, denaturing solution and the washing buffer. Then, the vacuum connection was removed from the vacuum prep tool, and the beads in the probes were released into the PSQTM plate containing the sequencing primer and the annealing buffer. Release into the PSQTM plate was facilitated by gently rubbing the filter probes in small circles against the bottom of the wells. After the beads were released, the vacuum prep tool with the filter probe was placed in nanopure water to clean the probes for subsequent use. The PSQTM plate containing the beads with biotinylated DNA strand, sequencing primer and the annealing buffer was heated at 80°C for 2 min using the PSQ 96 HS Sample Prep Thermoplate Kit. The plates were removed and cooled at room temperature for approximately 10 min, and the sequencing reaction was done in a PSQTM 96MA pyrosequencer in the following way. The PSQTM program was started on the computer connected to the PSQTM 96MA pyrosequencer, and later PSQTM 96MA pyrosequencer was turned on, and allowed to warm up for 15 minutes. Information about the SNPs and the plates assayed was filled in 138 the necessary places in the program, and the program calculated the amount of reagents required for pyrosequencing each 96 well plate depending on the sequence composition. The cooled PSQTM plate was then placed in the assigned slot in the PSQTM 96MA pyrosequencer. The slots in the cartridge that dispensed the A, G, C, T nucleotides, the enzyme and substrate were filled with appropriate amounts of respective reagents (Pyrosequencing PSQTM 96MA reagent kit, Biotage, Uppsala, Sweden) and placed in the cartridge slot. Pyrosequencing reactions were initiated, and the results were exported for data analyses and pyrograms were printed and saved for lab records. The exported results were later imported into the Isle of Wight SNP genotyping database in Dr. Susan Ewart’s laboratory. 139 o; v $5 I :0“ mo «:85 comma—2: 05 “one no: Ev swab: xosbm mo=_a> .mufiosme v.8 N6 98 CD * 83 be.“ 33. t: o.iw NS 33 8: £18 v8.0 80% Scam 3.<>o-_zo 2: 8m.“ 8; 83m 819 Seem SEN 3.430....6 09 8E exam v28 v8.0- 5va team 3359.10 8 83 Eon BEN 23 «8.8 SEN 3r<>oexm 8 5: $3. $08 $3- 83m 808 3.<>oe:m 3 so... 83 a: can o-io 8 $3 0me $er 48.0 SEN v2.8 18-/$0-50 mm 83 :3 80% 05.0 80% 50mm Imo-<>o-im 3 mil.» v13 83m $3 Sta :mmm I8-<>o-io 8 $2 53 5% 24.0- SNR E8 18-/$9.10 mm 82 93 ENE 83 8on nova Imo-<>o-.:o 5 53 2:.” $3 $3 <2: 92 mm: we: 59mm 83 80.2 3mg 3-84.10 8 mm; #3 O88 9.0.0- v38 89% _<<.mmn_-fo mm :3 we: mama #3 55 BEN Sewmaazo 8 mama SE 3.8 80.0 8va 84.8 _.\<.mmn_-.zm S 23. Son «SR S3 858 80R gumaero 8 $2.. .83 ~88 and «83 02.8 3-mma.iw mm 83 3°..." 83 $3 < 89m 23 8va $00 N58 83w Imoémaexw g Sea 53 mag 23. Roam .8va :8-wma-£w mm Boa 83 80% 80.0- 8th 80.8 18-84.10 S 0me 23 02.8 20.0 50.2 8va Imoamaezm 5 33” 5? 83m mono- 08.3 3.8 Imowmaexo on #3. 83 ENE Ema- «.8.me 83m Imoémaaxo 2 am at... < 05.0 08.9 08.8 08.0 000.8 80.8 3-050.018 08 8:. 000.: 000.8 000.0- 000.8 000.8 02-<>o.__._~0 08 08.0 000.0 000.8 08.0- 3.58 80.8 3-050-018 08 000.0 000.0 02.8 000.0- 000.8 03.8 2<-<>o.f~e 80 000.0 08.0 0-.0~ 88.0 000.8 8.8 3-050-018 80 ~80 000.0 80.8 03.0 000.8 08.8 3.050.018 8 E; 000.0 08.0 000.0 <20 0>< 000.0 000.0 03.8 000.0 8.8 000.8 I0o-<>o-__._~0 08 ~80 $8. 808 E00 :88 -08 I0o-<>o-i~r 0: 000.8 00:: 8.8 05.0 08.8 88.8 I0o-<>o-_:~F 0: «0&0 003 08.8 08.0 0 00.8 80.8 :00-<>o-i~r E 000.0 v8~ 808 30.0 0008 80. 8 :00-<>o-i~0 0: 08.0 ~00.~ 088 :00 0008 :08 100050-018 0: 003 80.0 80.. :08 <20 92 000.0 08.0 08.8 08.0- 000.00 000.00 3000.58 .0: 0.0.0 5.0.? 000.8 80.0 8.8 08.8 3.000018 0: 000.0 50.0 08.8 83 ~88 E08 02.00%:8 ~: 000.0 08.0 000.8 03.0- 80.8 80.8 02-000-.I~F E 000.0 E; 0008 0:0 ~88 000.8 3-004.728 0: 000.0 000; 08.8 80.0 88.8 000.8 02-00%18 08 08.0 000.0 02.0 80.0 < 000.0 000.0 000.8 50 ~88 000.8 100-00%:8 02 000.0 0000 0:8 08.0 000.8 :08 100-00%18 Be 80.0 08.0 000.8 000.0- 000.8 ~88 80.00858 00? 804 3%.... 808 8:? 000.8 08.8 80-00%:8 000 08.0. 0004 :08 80.8- 000.00 :08 80.00858 09 000.0 80.0 08.00 3.00. 000.8 000.8 100000.018 000 00 as 083:9: 000 N00 0:0 :0 0 000.. 0.0.0 000.. 000.~ < 80.0 000.0 0:81 000.0- 000.8 80.8 02.<>o-i0~ 0~ 000.0 000.0 00 0.8 000.0- 2 0.8 00 F .8 02-<>o-f0~ 8 000.0 000.0 00 0.8 80.0- 5.8 000.8 02-<>oa:0~ - 000.0 000.0 000.8 000.0- 08.8 000.8 0Z-<>o-.:0~ 0~ 000.0 000.0 000.8 000.0- 000.8 R08 02-<>o-_:0~ 8 000.0 000.0 000.8 000.0- 000.8 80.8 02-<>o-.:0~ 00 08.0 08.0 000.0 000.0 <20 0>< ~00.0 ~00.0 $0.00 000.0 ~00.F0 000.00 _-_0o-<>o-£0~ 00 000.0 0:0 ~88 000.0- 000.00 000.00 z0o-<>o-i0~ D 000.0 00: 000.8 000.0 08.8.1 000.00 :0o-<>o-f0~ 0F :00 000.0 000.8 000.0 80.8 000.8 :0o-<>o-._-_0~ 00 08.0 000.0 000.00 000.0 08.00 08.00 :0o-<>o-.:0~ 0F F~0.~ 000.0 000.00 80.0- 028 0050 :0o-<>o-._-_0~ 00 08.0 000.0 08.0 000.0 <20 0>< 000.~ 000.0 08.8 08.0- 08.8 0008 30000.58 8 000.0 R00 08.8 00~.0- 000.00 000.00 02000.58 3 000.0 000.0 000. 00 8 0 .0- 80. 00 ~00. 00 3000-£8 00 000.~ 000.0 000. 00 80.0 00 F. 00 00 08 02-000018 0 0000 000:0 ~28 B~. 0- 0:8 000. 00 2.0-000-58 0 000.~ 08.0 80.8 08.0. 000.8 08.8 3000-i8 ~ 000.~ 000.0 000.0 08.0 <20 0>< 000.~ 000.0 08.8 000.0- 08.8 08.8 100000-008 0 000.0 000.0 000.00 08.0- 0008 80.00 100000.18 0 x x 000.00 000.0 000.00 000.00 0.00-000-58 0 000.0 000.0 808 0:0- 80.00 088 :00-0ma-i0~ 0 08.0 000.0 000. 00 08.0- 088 0020 0.00-000-018 ~ 80.0 000.0 08.00 000.0- .008 000.00 0.00-000-008 0 00 as 080230.. 020 30 was :0 0 0.0.. 00~.0 000.0 000.. 22102 000.0 . .0. . 0 .08 .000- .008 000.00 :< -<>o .0100 00 000.0 .00.. 000.00 00 . .0- 80.00 000.00 2< <5 .0200 .0 .0..0 0.0.. 80.00 000.0- 000.00 0.0.00 2< .050 0:00 00 ~.0.~ 000. . 08.00 000.0- 000.00 .-.00 2< -<>o .0100 00 000.0 000.0 0.0.8 000.0- 00~.8 0... .0 2< -<>o .0100 00 000.0 00..~ 80.8 00.0 80.8 0 .08 2< -<>o .0200 00 000.. 000.0 000.0 000.. o -0100 .0 0000 000.0 00.00 8... 08.00 000.00 I0o-<>o-_I00 00 000.~ 80.. 000.00 .000 00.8 80.00 :00 <5 2:00 00 80.0 8... 000.8 .-.0- .008 000.8 :00 <5 0:00 00 00.0 0...~ 000.8 000.0- .008 000.8 :00 -<>o -.100 .0 00.0 000.0 8.0 0.... <20 0%< 00.0 08.. 000.00 00~.0 80.00 .0000 :< -000 .0200 00 0.0.0 08.. 000.00 8. .0- 000.00 808 :< -000 2:00 00 ~.0.0 000.0 08. .0 000.0 .00. .0 . .0. .0 3. -000 -0200 00 2.0 80.. 08. .0 80.0- .00. .0 000. .0 2< -000 -0100 00 .80 00... -..00 000.0 00.8 00.00 2< -000 .0100 8 00.0 000. . 000.00 00.0- ~00. .0 000.00 :< -000 -0100 .0 .8. 000.0 00~.0 000.. <20 0% 0.0.0 00... 0~..00 00.0 ~008 000.00 :00 -000 0:00 00 00~.0 0.0.. 000. .0 000.0- .8. .0 ..08 :00 -000 2:00 8 000.0 000.. 000. .0 .000 ~.0. .0 000. .0 :00 -000 0:00 8 80.0 000.. 000.00 00~.0- 000. .0 000.00 :00 -000 0:00 8 .000 000.. 0.000 000.0- .008 .88 :00 -000 .0200 8 00.0 000.0 . .0. .0 000.0- 000. .0 8.. .0 :00 -000 2:00 8 00 as 2002 00 302205... 00205 89.8 ~.o .5 xx .0 02:- 0.0800 00:00. 9:... 20560-0 E w: 0.0.0080 0. 2000 143 o; v 3.0 I :8 .0 0.52.5 .5622: on. .00.: .0: 20 .3052. x250 00=_0> .0800.th 000 N00 «0:0 :0 0 nub-o Von... maMd Fond <21 u>< vmwd MMvd omméM Med- omo.NM NMMéM 2< -<>O -0INN N.- Mewé 00m... MMNdM 90.0 MNNdM wmmdM 2< -<>O .0INN E Mmmé nmmé vmfoM Mde- MMMdM ommdm 2< .<>O -0INN on mmMé tmd VMM. FM Mrmd EMA-M omwdm 0?. -<>O LINN mm ovr.N mwmd vow. 0M mmmd voméM McINM 2< -<>O -0INN. Mw vmmd 00~.0 _-:.OM 000.0- erdM RodM 2< -<>O -0INN nw ave... Mmhé «mud awn-o <2”. m>< vmvd Bad NoodM $0.0- wNMdM onmdm IMO -<>O --_INN mm M. I. 900.. andM Mood- vomdM .vvdM IMO -<>O -0INN mm Nome mmnd MOFeM novd- MOMéM romdM IMo -<>O -0INN v0 0.04-Fw- Nwwd thM 3M0 MNmNM VMMMM IMO -<>O -0INN Mm MFVN .omd omw. 0M 5M6 ONE—M KoNM IMO -<>O -0INN. Nw 03M mm... .3de M56 MNNdM FmNdM IMO -<>O -0INN 5 2nd war-N mun-o mam-c < MR: MMMd EYNM god www.mM oomNM 2< -mmn. .0INN ow vowN Nomd rmwéM mmvd mtg-M MOINM _.<< -wmn. -0IN.. on CNN MMmd wwodM mid thM 80.5 02 -wmn. -._IN\. mm MMMN vwa... ~6de oMod- owvdM $10.8 2< -wmm -0INN mm mom; ommd 2.4-M 9.0.0 NMMéM MNONM a)... -mma .0INN mm mme pond mnoéM Food- mnoéM vnoem 2< -wmn. -0IN. mm vad mmM-r {#6 wood < vwme owwé 90de M _- r .o omwdm MOVMN IMO -wmm Jim... Vm M09. mmnd nmo. .M med- MNVéM oRdM IMO -wma -0IN. Mm CNNN vmwd MMM. .M vad- 3m. 0M 50. _-M IMO -wmm -0INN Nm KNEN coo..- vadM MMIO- vmmdM MMMdM IMO -wmn. LINN rm omM.. $0.0 3M. 0M MMFd mmN. .M MMV. _.M IMO -mmm -0INN om BM;- o.m.o deM word- 30. 0M .MMdM IMO -mmn. -0INN 9. am 32mm? ow fizzzms v: lommho>< N00. 50 N00 50 xx a 0E:- 0&an cmE ah ..I Nb :3.— FF: 76.0.53 2 050.- 144 09.00 95.030 8 0:0 000288 0.9000 00 ”Q— v 35 I :8 mo 0:00.00 020305 0.0 .008 .0: 20 5:85 0.0050 0020> 0802.00. 0.0 flu 0:0 :0 a 00.0 0.0.0 000.. 000.0 000.22 000.0 0...0 000.00 000.0 000.00 0...00 3.050010 00. 000.0 0.0.0 000.8 0.00 000.8 000.8 30.050010 .0. 000.0 000.0 .0000 0.0.0- .0000 000.00 02-050010 00. 00.0 000.. .0000 000.0- 00.00 0.0.00 3.050010 00 000.. ..0.. 000mm] 000.0- 08.8 000.00 0205-000 00 00.00 00.00 80.00 00.0 80.00 8000 0205-010 .0 000.0 000.0 00.0 000.. 000.030 .000 000.. 00.00 0...0 0.0.00 000.00 0.00-050-010 00 000.0 00... 000.00 00.0 000.00 .0000 .000-«$0-010 00 000.. 0...0 0. ..00 000.0 000.0 00000 100.050.0000 00 0...0 .000 0.000 000.0 0. .00 80.00 :0o-<>o-i0 00 00.0 .8. ..000 0.00. 0.000 08.00 I0o-<>o-i0 00 000.0 00... 000.00 .000- 000.00 000.00 0.00-050-010 .0 .0..0 00.0 00.0 00.0 000.030 00.0 00.0 ..000 .000- 000.8 .0000 02.00000 00 00.0 00.0 .0000 000.0 00mm 000.8 3.000000 00 ..00 000.0 000.00 000.0 00.00 000.0 3.000010 00 000.0 000.0 000.8 000.0- 0.0.00 0.0.8 30.000000 .0 0.0.0 0000 .0..0 00.0 000.0 08.0 2.0-000-010 00 00.0 000.0 08.00 0.00 0.0.8 000.00 3.000010 00 0.0.0 000.0 000.0 00.0 002030 000.0 .000 000.0 000.0 .00..0 «0...0 .000-000.010 00 .000 .. ..0 ....00 000.0- 008W .0000 .000-000-010 00 .000 00.0 000.00 000.0- .0000 000.8 .000-000.010 00 000.0 .0..0 .0000 0.0.0 00.001 0.0.8 000000-010 .0 000.0 00.0 ..000 000.0 000.00 000.00 0.00-000.010 00 .. ..0 00.0 0.000 00.0- ..000 000.8 0.00-000-010 0. 00 as 002000. 00 05,020 0... 0 0.22 0.9.6 0.0 so 00 .0 as: 20500 .1 w. :3..— UCNEQD :0 NNIMW< 308:— 0w:3 5 00000098 <73... .2: .3 «Eu-_- 145 0.. v EU I :8 .0 0:00.00 .8330... 05 .00.: 8: E0 zwsofi. x0050 00:_0> 00.00.30. 000 m5 000 :0 ... 000.0 000.0 .0..0 000.0 00003. 0.0.0 000.0 00.00 ..00 00.0% 000.00 3.0.5.010. 00. 00.0 00.0 ..000 0.0.0 ..0001 000.00 3305.010. 00. 0.00 0.0.. 000.00 ..00 .0..00 0.0.00 3.050010. 00. 0.0.0 .00.. 00.00 .000- 00.00 00.00 30-050010. 00. 000.0 000.. 000.00 000.0 0.0.00 0.0.00 02.050010. 00. 000.0 000.0 000.00 00.0 000.00 000.00 02-05008. .0. 0.0.. 0.0.0 000.0 00.0 00mg... 000.0 ..00 .00..0 000.0 000.0 80.0 :0o-<>o-._._0. 00. .0..0 000.0 0.0.00 0000 .0000 000.00 100050-010. 0.. 000.. 000.0 0.0.00 .000 00.801 000.00 :0o-<>o-i0. 0.. .0..0 000.0 000.0 .000 000.0 00.00 :0o-<>o-i0. ... 000.0 000.0 000.00 00.0- 000.00 000.00 I0o-<>o-i0. 0.. 000.0 000.0 .....0 000.0 00...0 000.0 I0o-<>o.f0. 0.. 000.. .000 000.0 00.0 000.03. 000.. 000.0 0.0.00 000.0 .0000 000.00 3-000-010. 0.. 000.0 000.0 0...00 00.0- 000.00 ..000 02-000010. 0.. 000.0 000.0 00000 000.0 000.00 000.00 02-00008. 0.. 000.0 .000 00.00 000.0 00.00 00.00 02-00008. ... 000.0 ..00 000.0 000.0- 000.0 000.0 02-00008. 0.. 00.0 ..00 000.00 000.0 000.00 .0000 02000-50. 00. 0.0.. 0.0.0 0.0.0 000.0 M00003. 000.0 ..00 .0000 0.0.0 000.00 000.00 100-000010. 00. 000.0 .000 000.00 000.0- 0.0.00 0.0.00 100000-010. .0. .000 000.0 000.00 000.0 0...00 0.0.00 18-000010. 00. 000.. 000.0 0.0.00 000.0- 00.00 000.00 :0o-000-i0. 00. .000 0000 .0000 00.0 0.0.00 00.00 100000-010. 00. 000.0 000.0 000.00 000.0 0.0.00 000.00 08-000010. 00. 00 00.5.0208. 00 002200.: 1.00.1222 0.0-50 0.0 :0 00.005: 0.00.00 ..I Nrnmczl- tam—:60 :0 Nuwm< mafia:— A...0.Eoov 3 030.0 146 o._ v 3.0 I :0. ..o ...-.0..00 02020.: 05 .008 .0: 0.0 .3385 x030 0030> 00:00:00.. 0.0 N5 0:0 :0 ... 000.. 0.0.0 0.0.0 .000 002020 0.0.0 000.0 0.0.00 0.0.0 000.00 000.00 02-<>o.._x00 00 000.0 000.0 0.000 00.0- 000.00 000.00 2.0-050-0100 00 0...0 0.0.0 00.8 00.0 0.0.00 000.00 :<.<>o-£00 00 00.0 00.0 0.0.00 .000- 000.00 00000 02.050.00.00 .0 0.0.0 0000 000.00 000.0- .0000 00000 2<.<>o..x00 00 .000 00.0 .0..00 0000 000.00 0.0.00 :<.<>o.._-_00 0. 000.0 .0..0 000.0 0.0.0 08.030 000.0 0000 000.00 000.0 .0000 0.0.00 10005-0100 0. 000.0 000.0 00000 00... .0000 .00..0 0.00-0500100 .. 000.0 0.0.0 000.0 .0..0 0.000 0....0 I00-<>o...-_00 0. ..00 0...0 000.00 00.0- 000.00 .0..00 I00-<>o._:00 0. 0000 000.0 000.0 .000 000.0 000.0 0.00-0.5.0.00 0. 0000 0000 000.00 000..- 00000 000.00 100-050.0100 0. 000.0 000.0 000.0 0.0.0 M0803. 000.0 0.0.0 000.00 000.0 000.00 000.00 02.000-00.00 0. 000.0 000.0 000.00 0000- 00000 0.0.00 02.000000 .. 0000 0.0.0 0.0.00 00.0 000.00 0.0.00 0.<.000..I00 0. 000.0 0000 0.000 0...0 0.000 00.00 02.00.0018 0 000.0 0.0.0 000.00 000.0 00.00 .0000 3.000.018 0 000.0 000.0 00000 0.0.0- 00000 0.0.00 02-000.00.00 . 0.0.0 000.0 000.0 000.0 000.020 0.0.0 0.0.0 .0000 000.0 00.00 000.0 100-000.0000 0 000.0 000.0 000.0 000.0 .00..0 000.0 100-000.0100 0 x x 000.00 000.0 00000 000.00 0.00-000.000 0 00.0 000.0 .00..0 0.0.0- 0.0..0 000.0 I00-000-.I00 0 000.0 .000 0.000 0000- 00000 00.00 0.00-000.0100 0 .000 000.0 000.0 .000- 000.0 .0..0 0.00-00.2100 . 00 30:20.00. 00 .5m 000.00 00:05.0..00 .oom .:0.0_0:00 0. 0:0 000..—00m . 5.. v NO I :8 .0 3.00.0 8530:. 0:. .00:. .0: E0 ..w:o.:. 0.0..-..0- 0030> 00:00:00. 0.0 N.O 0:0 :0 0 Mud and 36 Mad claw—03‘ Mo.M nmN MIMN ONMd- vaN NMNN <>O 3.. w... M No. mwfi N06 BEN orNd- 5....N vNN <>O 0< 0.: M ..0.. _.M.M omM omNN CNNO .0.-...le MNMN <>O 0< 0.: o 00. MM mvN MYMN 03.0 :MN mme. <>O _..< 0.: .0. mm 5N NON MNMN ommd .mMN NoMN <>O 0< 0.: M Mm 00.0.. Nod-_- OMMM cord MNMM MMMM <>O 0< m... o 5 05.0 MMé VIN 0.9-M Olnl6.o>< Nwhw wad nodM ONON MOMN ...rM <>O IMO 2.. m cm om. 5N NNMN omrd NMMN .MMN <>O IMO 0E o mm mod.- NNLO. mmdM 000.0 8.8 NMdM (>0 IMO 0.; o vm Nm.m NNM MMMN omrd MMMN NoNN <>O IMO 0E M Mm No.m 9.0 OMEN OMIO MmNN MMNN <>O IMO 0E M Na MIM no.0 MIMN omod M..MN NNMN <>O IMO 0.: M 0m QNN NM-M #06 MM... omflo>< wmhm 3.4 MMMN 0N0. NmMN .0..0M mmn. 0< 2.. 0 cm NIM Mvd MM..M OMIO MN._,M NveM mmn. 0< 0.: M MM .36 NNI... woMN OM... vaN NMMN mmn. _..< m... M MM MMM 30.0 5.5 ONIO mN._.M NMeM mmm 0< 0.: M ..M No.N NN.o woNM oo..o MMNM MMNM mm... 0< w... M MM wwhm mod 5.3” 0M0. mMMM MDVM wma 0< 0.: M MM ..mé QM...” hNd mvd 0ImI03>< MMM omd ONdM 000.0 NMMN MmdM mmn. IMO m... 0 VM an... mmd $0.0M 0.0.0 mo._.M moeM wmn. IMO 00.. o MM SM No.0 NFOM oomd NvdM No._-M wma IMO 0E 0 NM MMN OMd QINM OM50- mVNM .55 mm... IMO w... m M NAN mrd NMNM oer MVNM NNMM mma IMO m... 0 0M VN.M MMd 2.0M 000.0 00.5 VMéM mma IMO 0E M an om Ila-m5 <2mmMr am 3:22”.— uE. 0m20>< _ $0-20 Nam. ...O .9.“ 59.5 0.5.. 2man 0mg. E 5.03.9.0 <73... 02~ .m— 030,—- 148 0.. v 3.0 I SOB ..o 0...0...0 02030... 05 .00... .0.. 0.0 $00.... x028 0020> 00.00.30. 0.0 NO 0:0 CO ... 00.0 8.. 00.0 00.0 00202 80 - 80 8.8 0.0.0 8.8 00.8 <>o 2 2.. 00 00 00.4 - $0 0.8 8... 8.8 8.00 <>o 2 2.. 00 .0 8... - $0 8.8 80.0 0.8 00.8 <>o 2 2.. 00 00 8.. x 00.8 000.0 8.8 8.8 <>o 2 2.. 00 8 0...0 30 8.8 08.. 00.8 8.8 <>o 2 2.. 00 8 8.0 8.0 8.8 08.0 0.8 8.8 <>o 2 2.. 00 00 8.0 0... 00.0 8.0 00202 0.2.0 .00 8.8 000.0 8.8 8.8 <>o .80 2.. 00 8 0... 8.0 00.8 0.00 .08 8.8 <>o .80 2.. 00 8 00.. 8.0 8.8 000.0- 8.8 8.8 <>o .80 2.. 00 8 .0.. .00 8.8 8.0 8.8 .08 <>o .80 2.. 00 8 8.0 8.0 0.8 80.0- 8.8 8.8 <>o .80 2.. 00 00 ...8 8.0 8.8 08.0 8.8 8.8 <>o .80 2.. 00 .0 00.0 00.0 .0..0 0.0 0 202 00.. 8.0 8.8 08.0 .08 8.8 000 2 2.. 0. 8 0.0 8.0 00.8 000.0 00.8 8.8 000 2 2.. 0. 8 00.0 8.0 00.8 0.0.0- 8.8 00.00 000 2 2.. 00 8 8.0 ...0 8.8 08.0 00.08 8.8 000 2 2.. 00 00 RE. 090 8.8 08..- 8.8 00.8 000 2 2.. 00 8 8.0 8.0 00.8 08.0- 00.8 00.8 00.. 2 2.. 00 8 00.0 00.. ...0 0.0 08.02 8.. 00.0 0...0 0.0.0 .08 00.8 80 :00 2.. 00 8 8.1. 88 8.8 8...- 00.8 .08 000 .80 2.. 00 8 8.0 8.0 8.8 000.0- 8.8 88 00a. .80 2.. 00 00 8.0 090 8.8 08..- 8.8 00.8 000 :00 2.. 00 .0 8.. 8.0 008 0.0.0. 8.8 .08 000 .80 2.. 00 8 00.. 8.0 00.8 08.0- 8.8 .08 80 .80 2.. 00 8 00 a... <28... 00 3:54.20 .0... 00.29... 0.0.8 0.0 ..0 xx 0 0.2.0 0...... 20.5.0 620.2800 2 0E0... 149 003:.00 wc... 000.620.88.05. .0 00.0.. .0... :. 0.0080 50.0 Mm>m 08.00 00:05.0..00 .00.. 0.0.0.050 0. 000 00020me5.. V 3.0 I ..O. ..o 0..0...0 5.00.0... 0... .00... .0: 0.0 00.8.... .00..0 00...0> 00.00.30. 0.0 N.O 0:0 .6 ... 0..... 8.8 00.. 8.. 000.102 8.0 8.0 00.8 00.0 0...0 8.8 <>o 2 2.. 0 00. 8... 8.8 8.00 8.0- 8.00 8.00 <>o 2 2.. 0 .0. 8.0 0... 8.8 8.0 8.8 00.00 <>o 2 2.. 0 00. 8.0 0... 8.00 .00 8.00 8.00 <>o 2 2.. 0 00 .00 8.0 8.00 8.0 00.00 8.00 <>o 2 2.. 0 00 00.0. 000. 8.8 00.0- 8.8 .08 <>o 2 2.. 0 8 0... 8.0 8.0 0...0 00.1205. 8.0 8.0 8.00 .00- 800 8.00 <>o 80 2.. 0 00 00.. 8.0 00.8 00.0- 8.8 8.8 <>o .80 2.. 0 00 000. 09.0. 8.8 0.0- 0.8 8.8 <>o 80 2.. 0 8 00.0 00.. 8.00 .00 00.00 8.00 <>o .80 2.. 0 8 00.0 8.0 .000 8.0 00.00 8.00 <>o .80 2.. 0 00 0.0 8.0 8.8 00.0- 00.8 00.8 <>o .80 2.. 0 .0 8.0 00.0 8.0 00.0 «0.1202 8.0 00.0 8.00 0.0- 8.00 .000 000 2 2.. 0 8 0.8 8.0 8.8 00.0- 02E 00.8 000 2 2.. 0 8 .00 8.0 00.00 0.0- 8.00 8.00 00a. 2 2.. 0 8 8.0 8.0 8.8 0.0- 8.8 8.8 000 2 2.. 0 8 00.0 8.0 .000 00.0 0.00 8.00 00.. 2 2.. 0 8 8.8 8.0 00.8 00.0- .0..0 00.8 000 2 2.. 0 8 8.. 8... ...... 8.0 00202 8.0 8.0 8.00 8.0- 8.00 8.00 000 .80 2.. 0 8 0.... 8.0 8.8 8.0- 8.8 8.8 000 80 2.. 0 8 .00 8.0 8.8 00.0 8.8 8.8 000 .80 2.. 0 00 8.0 0.0 00.8 8.0 8.00 00.00 00.. 80 2.. 0 .0 00.0 00.0 8.8 8.0 8.8 .0..0 00.. .80 2.. 0 8 8.0 .00 8.8 0.0 8.8 8.8 000 80 2.. 0 0. 00 .0... 2208. loml .0032”. 0..: 00:02 0.0.6 0.0 ..0 5.0 0.2.0 0..... 2 E8 0. .5. ... 00.80.96 <72... ab: .0. 0...0-_- 150 0.. V HN.O I ..0. ..0 0..0...0 00.020... 0... .00... .0.. 0.0 0000.... .00..0 00:.0> 00.00....0. 0.0 N.O 0:0 :0 * ....0 .00 0..... ...... 000.02 000 $0 ...8 8.. 8.8 8.8 <>0 2 2.. 00 0. 00+ «00 00.8 8.. 8.8 00.8 <>0 2 2.. 0. K 00.. .00 00.8 00.0 00.8 8.8 <>o 2 2.. 0. 00 8.. .00 00.8 8.0 8.8 00.8 <>o 2 2.. 0. 8 ....0 .00 8.8 8.0- 8.8 8.8 <>0 2 2.. 0. 8 00.0 .00 8.8 00.0 8.8 8.8 <>o 2 2.. 0. 8 00.0 8.0 ...... 0..... 80.02 0Y0 000 0.8 00.. 8.8 .08 <>o .80 2.. 0. 00 0.8 $0 0000 .00 8.8 8.8 <>o .80 2.. 0. 8 .8... $0 ...8 8.0- 8.8 008 <>o 80 2.. 0. 8 .0.. 00.0 8.8 8.0 00.8 8.8 <>0 80 2.. 0. 8 ....0 8.0 8.8 0.0 00.8 ...8 <>0 .80 2.. 0. 00 ...0 00.0 00.8 00.0 8.8 ...8 <>o .80 2.. 0. .0 0...0 8.0 0..... 00.0 000.02 008 $0 .08 8.0- 8.8 00.8 000 2 2.. 00 8 0.0 .00 8.8 8.0- 8.8 008 000 2 2.. 0. 00 00.0 0.0 00.8 .00 00.8 8.8 000 2 2.. 00 00 8.0 8.0 8.8 00.0- 00.8 8.8 000 2 2.. 0. 8 .0.. 00.0 8.8 00.0- 00.8 00.8 8.0 2 2.. 0. 8 00.0 - 00.0 8.8 8.0- 8.8 00.8 000 2 2.. 0. 00 8... 8.0 ...... 8... ..0.-202 8.4 $0 8.8 00.0- 00.8 00.8 000 80 2.. 0. 8 88 $0 008 .00- 00.8 8.8 000 .80 2.. 0. . 8 00.0 00.0 ...8 0.0 8.8 0.8 00.. .80 2.. 0. 00 8.0 8.0 8.8 8.0- 0.8 00.8 000 80 2.. 0. .0 00.4 80 00.8 8.. .08 00.8 000 .80 2.. 0. 8 004 000 00.8 ....- ...8 00.8 000 .80 2.. 0. 8 00 as <28... 00 .0022”. 0.... ..0.-202 0.0- ..0 0.0 ..0 xx ... 0.2.0 0..... 0.0500 A. . .0..000. 0. 0...0-.- 151 020.000 was. 000.000.005-853. ..o 00.0.. 00.0 0. 0.00000 0020 Mm>m 000.00 00:05:88.. .80 0:80.050 9 0:0 000..—ova. .. 6.. v .60 I .5. ..o 0..—8.00 00.020... 20 .00.: .0: 0.0 swab: 0.00.50 0020> 0200.32 000 N00 000 :0 ... 0.0 000 00.0 00... “000.02 000 0..... 00.00 0.0.0 00.00 00.00 <>o 2 20 0 00. 00... 000 00.0 0000- 0...0 00.00 <>o 2 02 0 .0. .00 00.0 .000 0000 00.00 00.00 <>o 2 0:. 0 00. 000 00... .000 00...0- 00.00 00.00 <>o 2 20 0 00 .00 ..0 00.00 0000 0.00 00.0 <>o 2 02 0 00 000. 00.00 00.0 0000- 0.00 00.0 <>o 2 02 0 .0 0... 00... 00.. 00... 0000.22 .00 0.0 .000 0.00- 0.00 00.00 <>o :00 e; 0 00 00.. ..0 00.0 00.0 ....0 00.0 <>o I00 20 0 00 090. 0...0. .000 000..- 0000 0000 <>o I00 02 0 00 00.0 00.0 00.00 0...0- 0..00 00.00 <>o I00 0:. 0 00 00.0 0.0 00.00 000.0 00.00 00.00 <>o I00 0.0 0 00 0.0 .00 00.00 0.0.0- 00.00 00.00 <>o :00 9; 0 .0 00.0 000 00.0 000 000.02 000, .00 00.0 0.... ...00 00.0 000 2 05 0 00 0.0 00.0 00.0 000.0- 00.0 00.0 000 2 20 0 00 .00 0... ...00 0000- 00.00 0...0 000 2 02 0 00 00.0 00.0 00.0 0000- .000 00.0 000 2 2; 0 .0 00.0 000 00.00 0000 .000 .0..0 000 2 20 0 00 000 00.. .000 0.0.0 ...00 00.00 000 2 02 0 00 .0.. 000 00.0 00.0 000.02 00.0 .0... 00.00 0.00 ...00 .000 000 :00 2; 0 .0 0.... .00 0...0 0000 .000 .0..0 000 :00 20 0 00 .00 .0.. 00.0 00.0 .0..0 00.00 000 :00 m: 0 00 00.0 0... 0.00 000.0 00.0 00.00 000 :00 20 0 .0 0.0 00.0 00.0 000.0 ....0 00.0 000 :8 20 0 00 ..00 000 00.0 0.0.0- 0...0 00.00 000 :00 e; 0 0. 00 as «.2000. owl! .0022”. ...: 000402 06.6 05 .8 5.0 52.0 25.. 200.00 0 .5. :. 00.000098 0800.32 000 N00 000 ..U ... 0.0 0... 0.0 00.0 00.202 000 0.0 .000 0000- .000 00.00 <>o 2 02 0. 0. 00.. 0.0 00.00 0000- 00.00 0.00 <>o 2 02 0. .. 00.. ...0 0.00 00.0- 0000 ...00 <>o 2 e; 0. 0. 00.. 000 00.0 0000- 0.0 00.0 <>o 2 e; 0. 00 2.0 .00 0...0 0000 00.0 0.0 <>o 2 02 0. 00 00.0 000 0000 00.0 .000 .000 <>o 2 20 0. .0 00.0 0... ...0 0.0 00202 000 0.0 .000 0000- 0000 .000 <>o :00 e; 0. 00 0... 00.0 0...0 00.0- 00.00 0...0 <>o I00 9.. 0. 00 .0.. 0.0 00.00 0000- ...00 .000 <>o I00 0.0 0. 00 .0.. 0.0 .000 0000 0.00 300 <>o I00 2.. 0. 00 .00 000 .0..0 000.0 .000 ....0 <>o :00 2.. 0. 00 ..0 000 0.00 0000 0.00 000 <>o I00 02 0. .0 00.0 0.0 00.0 00.0 00.202 .0.. 000 0000 0000- 0000 0000 000 2 02 0. 00 0.0 .00 00.0 0000- ....0 0.0 000 2 2.. 0. 00 0.0 00.. 0.00 0...0- 00.00 0.00 000 2 0: 0. 00 00.0 .00 00.00 00.0- 00.00 00.00 000 2 20 0. .0 .... 000 00.00 00.0- 0000 00.00 000 2 05 0. 00 00.0 0.0 .000 0000- 0000 00.00 000 2 02 0. 00 00.0 00.. 00.0 00.0 100202 00.. 00.0 0000 0000 00 00 000 :8 90 0. 00 00.. 000 0000 000.0- 0000 .000 000 :8 a; 0. 00 00.0 00.. 00.00 0000- 00.00 ...00 000 :00 02 0. 00 0.0 000 00.00 0...0- 00.00 00.00 000 :00 02 0. .0 00.. ...0 0000 0000. .000 00.00 000 :00 02 0. 00 00.. 000 0000 0000- 0000 0000 000 :00 0... 0. 00 00 a... 0. .0 00.0.. .0... 0. 0.00000 000.0 Mm>m 000.00 0000.05.00 .000 0080.008 0. 000 000.00%. . 5.. v 3.0 I ..0. ..o 0..0...0 00.00.00. 0... .00.: .00 0.0 09.0.... 0.00.00 000_0> 00.00.30. 0.0 N5 000 ..U .. 0.0 000 .0.. 00.0 00mm... 000 00.0 0.00 0...0 00.00 0.00 <>o 2 0:. 0 00. 00... 0.0 0.00 00.0 .000 ...00 <>o 2 2: 0 .0. .00 .00 0000 0.00 0000 .. .0 <>o 2 0:. 0 00. .00 00.. 0...0 0.0.0 ....0 0...0 <>o 2 05 0 00 .00 0... 00.0 0.0.0- 00.0 0...0 <>o 2 05 0 00 000. 000. 0.00 0...0- 0000 ...00 <>o 2 02 0 .0 0... 00... 0.0 .0.. 00.032 .00 000 0.00 0. .0 0000 ..00 <>o :00 0.0 0 00 00.. 000 00.00 0...0- .000 0.00 <>o :00 0:. 0 00 000k 090. ...00 0000- .000 0.00 <>o I00 05 0 00 00.0 .0.. 00.0 000.0 .0..0 00.0 <>o I00 20 0 00 000 000 .0..0 000.0- 0.. .0..0 <>o :00 2.. 0 00 0.0 00.4 0...0 000.. .00 00.00 <>o :00 05 0 .0 00.0 000 00.0 0.0 00.052 000 00.. 0.00 0..0- 0.00 .000 00.. 2 2; 0 00 0.0 .00 0000 00.0- ...00 0000 00. 2 0.; 0 00 .00 00.. .0..0 0000- 0.00 0...0 00. 2 20 0 00 00.0 .00 ..00 0.00 0000 00 00.. 2 02 0 .0 00.0 000 00.00 00.0- 00.00 0.00 00.. 2 02 0 00 000 ..00 .000 00.0 00.00 00.00 000 2 2; 0 00 .0.. ...00 0...0 0...0 $0.02 00.0 00.. 00.00 0.00- 0.00 .0..0 000 I00 0.0 0 .0 0.... 000 00.0 0000 00.00 .0..0 000 :00 02 0 00 .00 000 00.00 0.00 0.00 00.00 00. :00 05 0 00 00.0 000 0.00 000.0- ...00 0.00 000 I00 0:. 0 .0 0.0 0.0 00.0 000.0 00.0 0...0 000 :00 0:. 0 00 000 000 0.00 000.0 00.00 00.00 00.. :00 02 0 0. 00 .0... <2000. 00 .0022”. 0.... 1000.02 0.0-.00 0.0 :0 000 52.0 0.5. 0.0.50 0mg. 0. 00.000.00.00 <72... TE. .2 0.00,.- 154 0.. v 3.0 I ..0. ..o 0..0...0 00.0200. 0... .00... .00 0.0 ..w:0.... 0.02.0 000_0> 00.00.10. 0.0 N5 000 ..U ... 00.0 00.. .00 00.0 00202 00.0 000 00.0 0000- 0.00 ....0 <>o 2 0:. 0. 0. 00.. 000 0.00 0000 00.00 .000 <>o 2 0.0 0. .. 004 000 .000 000..- 0.. .000 <>o 2 0:. 0. 0. 0.... .00 .0..0 000.0- 00.0 00.0 <>0 2 0... 0. 00 ...0 000 .0..0 0.00- 0.00 00.0 <>o 2 02 0. 00 00.0 30 00.00 02.. ...00 0...0 <>o 2 02 0. .0 .0.. .00 00.0 .00 00.202 0.00 .0..0 00.0 000.. ....0 00.00 <>o I00 0.; 0. 00 0... 00.0 00.0 0400- 0.0 00.0 <>o I00 02 0. 00 .04 .00 00.0 000.0 ...00 .000 <>o :00 02 0. 00 00.4 30 .0..0 0.0..- ...00 00.0 <>o :00 a; 0. 00 .00 000 00.00 0000 .000 .000 <>o :00 0:. 0. 00 ..0 .00 00.00 0000 0.00 .000 <>o :00 0:. 0. .0 .00 00.0 0.0 .00 00202 00.4 20 0...0 000.. 0000 00.0 000 2 0.0 0. 00 0.0 000 00.0 00.0- .000 .0..0 000 2 0.0 0. 00 0.0 0...0 00.0 00.0- 00.0 0.0 000 2 02 0. 00 .00 000 00.0 030- .000 0.0 000 2 02 0. .0 .... 000 00.00 000.0 00.00 00.00 000 2 0.0 0. 00 000 00.0 00.00 0.0..- 00.00 00.00 000 2 2; 0. 00 00.0 00.0 00.0 ...0 000.02 x x 000.. 0000 0000 0.. 000 I00 0:. 0. v0 00.. 000 0.00 0000- 00.00 00.00 000 :00 0... 0. 00 00.0 ...0 .000 00...0- 0000 0000 000 :00 0: 0. 00 0.0 .00 .000 0.0.0 .000 00.00 000 :00 02 0. .0 00.. 0.0 00.0 000.0- .0.... ...00 000 :00 0.0 0. 00 004 000 00.00 000.0 ...00 00.00 000 ...00 02 0. 0.. 00 .05 22000. 00 0.220 0.... 0022;. 0.0...0 0.0 ..0 .20 52.0 25. 20.5.0 0.0.0.8.. 0. 200.- 155 33 «do». as»; $8. 83. v.32 <>o 2 E m 8. «SE 3. .N 3% <>o 2 E m m... 33. 3.8. Rom <>o 2 E o 3. $8. ... .m. Zoom 25 2 E o 9. 38. «on: 8.0. <>o 2 E o N... ...... .38 8&3. New. 38. 3.3. mm: 2 ... o .v. 83 33.3 8.3.. mm: 2 E o o... 02.» 2.8.. 9.3 mm“. 2 ... m 8. .38 38 3.3 mm: 2 E o 8. 3% a...» 8.8 m8 2 E o 5. 3...” ...ma. 8292 ..mmwm $8 ..ommm <>o :8 E m on. 88. 33. 2.... <>o :8 z o 8. 33... 3%. 82m <>o :8 E o 3. .82 ..mom. 2&8 <>o :8 E o mm. v.8: 3.... ~28. <>o :8 z e mm. «.3 28.. «922... «3.. .2. 05. mm: :8 E m .m. 3%.. 2%.. 3.5. mm: :8 E m on. .9... 8... 38. m8 :8 E m 8. ...mm. E8. 3%. mm: :8 E o mm. QNN: 3m... «.8. 8a :8 ... m R. a.” 0wgo>u 50“th 87.: N .. EOuOun- 0..—WI: F u Equum 27.: X1 5 £65m 05:. ”EB—am 2. o - «9.3 * 00.0 0.0000 mama: 0.030 0.0000 0000 <>o 2 E v. 000 0.0000 0.0..0 0.000 <>o 2 E v. 00. 00000 0...0 .0000 <>o 2 E ..N 000 ...00. .00.. .0.: <>o 2 E ... .00 0.0000 000.~ 00000 <>o 2 E ... 000 0.000 ..0.0 00202 0.0.. 00.00 0.000 00. 2 E v. 000 ...00. 0.0.. 00...0 00. 2 E v. 00. 0.0... 00.00 .0000 000 2 E ..N 000 0.80 0.00.. 00.8 000. 2 E .0 N0. 0000. ....0. 0.000. 00. 2 E v. .00 0.300 0.000. #00032 000... 0.2000 000.0 <>o :8 E ..N 00. 0.0.0. 0.0.0. ...00. <>o :8 E ..N 0.0 0.000. 0.000. 0.0.0. <>o :8 E ... 0.0 0.0... ...N. .0000 <>o :8 E .0 ... 000. 0.00. 0.00. <>o :8 E ... 0.0 0..... ..0.. $on 0.000 .000. 00.000 000 :8 E «N 0.0 0.00 .0000 0...000 000. :8 E v. ...N 30.0 0:00.00 $0.0 84:8 0.20 0.. 00.0 0000 .00..0 00. :8 E .0 0.. 0.000 .0..00 00000 00: :8 E ... ..N D.“ om20>u Eouoha 0..—.-.: N u Sung—l a.— ruu= r u Eon—OLE G.— r-|= XM— “ Eunuw GEE. macaw E ... . 0 5.. <0_._m 2.-.: 0.0.0.8 2 200. 157 ..00. 0.03. 00.203. 0.0.0. 0.0.0. 000. <>o 2 E 00 0.0 0.000. 0.000. 0.00. <>o 2 E 00 000 000.. ...... 0.00.. <>o 2 E 00 000 0.0.0. 0000. 0.0.0. <>o 2 E 00 .00 ...00. 0.000. 0.000. <>o 2 E 00 000 ..0.. «..0.. 00222 0.000 0.000 ...000 000 2 E 00 000 0.0.0 0.0... 00000 000 2 E 00 000 0.30 00.0.0 00.0.0 000 2 E 0.. 000 0.000 00.000 00.000 000 2 E 00 000 003 00.00.. 0.03. 000 2 E 00 .00 ...00 0.0.0. 09202 .000. .000. 0.0.0. <>o :8 E 00 000 ..0.: 0.000. 0.000. <>o :8 E 00 000 0.000. 0.00... 0..00. <>o :8 E 0.. 000 .0..0. 0.000. ..000. <>o :8 E 00 .00 0.0000 0.0.00 0...00 <>o :8 E 00 000 0.00. 0.00.. 80.02 0.000 0.0.0 00000 000 :8 E 0.. 000 «..0.. 0.000. 0.000. 000 :8 E 00 .00 ..0.00 0.0000 0.000. 000 :8 E 0.. 000 0....0 00...0 0.000 000 :8 E 0.. 000 0.000. 0.000. 000. 000 :8 E 0.. .00 D.w \ou90>u EOuOLQ «hr-n: N . Ecucsm «LP-d —. u Swap—n— u.— Fun= xx d figum 05:. carp—am E 0.. - 00.5.. .0030 0..-... A. . 0008.0 0. 200. 158 00.0000 00.000 0000.02 000.00 00.0000 0.000.. 00.0.0 <>o 2 0E0 00. 0000.0 00.0000 0000.0 00.0.0 <>o 2 0E0 0... 000.00 0000.0 0.0000 00.0000 <>o 2 0E0 3.. 00.....0 00.0000 00.0000 00.000 <>o 2 0E0 0... 00.00... 00.000. 00.0.0. 00.000. <>o 2 0E0 00. 000.. 00.00 0020:. .0..0 00.0 30> 0: 0.00 000 2 0E0 .0. 00.00 00.00 00.00 00.... 000 2 0E0 00. 00.0. ...0. 0...00. 00.00. 000 2 0E0 00. 0...0 00.00 00.0 00.00 000 2 0E0 00. 00.00. 00.00. 0.00. 0..00. 000 2 0E0 .0. 00.0.0. .0..0.0 3% 00.0000 00.0000 00....0 00000. <>o :8 0E0 00. 00.0000 0000.0 000.00 0..0.00 <>o :8 0E0 00. 00.000 0.0000 00.0000 00.0000 <>o :8 0E0 ..0. 00.000 0.0... 00.000 00.000 <>o :8 0E0 00. 00.0.00 00.0000 00.0.00 00.0.00 <>o :8 0E0 00. ....00 00.80 00.202 00.00. 0.0.. 00.00. 00.0.0 000 :8 0E0 .0. 0.0.. 0_ 500 o: 0.00. 00.00. 000 :8 0E0 00. 00.0.0 ....000 00.000 3000 000 :8 0E0 00. 00.00. 00.0.. 0..000 00.00. 000 :8 0E0 00. 0.00. 00.0. 00.0. 0.00. 000 :8 0E0 .0. 00 5320 0.-.: 0: £220 0...: 0.529.. 0.-.: 0.532. 0.-: 7:32.. 0.-.: 5... .0 52.0 2:. 0.0% E0 . 00.5.. <0_._m 0.-.: 0%.:— 05 E 05.80605 588.. 9k: .3 030,—. 159 .00..—8w 06 mo 03.00.800.002 on. Ho: 2:8 85: 9.0 .0590 2:: 2: E 020800 3 .080 05 BE cows—cm .020 0 .o: 003 mum .02 29:00 So... 3.00:0". 0500050050 05 00 40200806 0.03 finch: x250 003.5 0. .0..0 00. .00. «000402 00.000. 00.000. 00.000. <>o 2 E 00 000 0..000. 00.000. 00.000. <>o 2 z 00 000 00.000. 00.0.0. 00.000. <>o 2 0: 00 000 00.00.. 00.0.0. 00.00.. <>o 2 E 00 .00 00.00. 00.000. 00.000. <>o 2 E 00 0001 .0000 00.000 000.02 0..000 00.000 00.000 000 2 E 00 000 00.000 0..000 00.00 000 2 E 00 000 00.000 .000. 0..000 000 2 E 00 000 0..000 00.000 .0000 000 2 E 00 000 00.0.. 00.00.. 0..0.0. 000 2 E 00 .001 00.00. 00.000. «0.1202 00.0000 00.0.00 00.0000 <>o :8 E 00 000 00.000 00.000. 00.0.0 <>o :8 E 00 0.0 00.000. 00.000. 00.000. <>o :8 z 00 0.0 .000. 00.00. 00.00. <>o :8 E 00 ..0 00.00 00.00 00.000 <>o :8 0: 00 0.0 0..000 .0000 000.02 00.0. .. 0..0.0. .0000 000 :8 0: 00 0.0 .0..00 ...0.0 00.000 000 :8 E 00 0.0 00.0.. 00.0.. 00.0.. 000 :8 E 00 0.0 .0000 00.000 00.000 000 :8 E 00 ..0 am 5323.-.: 02. £220 0 ...: 0-5220 0.-.: 7522.. 0 .-.: x: .0 52.0 0:... lad-cam! E 00 - 0000.. <0_._m 0...: A. . 6.0.88 om 0500- 160 00.00 00.0.0 Mamas. 00.000 0...00 ...000 <>o 2 E 00 0.0 0...00 00.000 00.0.0 <>o 2 E 00 000 00.000 00.000 00. .00 <>o 2 E 00 000 0.000 .0.000 00.00 <>o 2 E 00 .00 00.000 00.000 0...00 <>o 2 E 00 000 0...0. 00.000 00MB... 00.000 .0.0.0 00000 000 2 E 00 000 00.00 00.00 000 2 E 00 000 0...00 00.000 0..000 000 2 E 00 000 00.000 00.000 0...00 000 2 0: 00 000 0.0.. 00.0. 0.0.. 00.0 2 E 00 .00 00.000. 0..000. 00.0.02 .0.0.0 .0.000 00.000 <>o :8 0: 00 000 00.00. 00.0.. 00.00. <>o :8 0: 00 000 00.000. 00.0.0. 00.00. <>o :8 0: 00 000 00.0.0. 00.000. 0...... <>o :8 E 00 .00 00...00 0...000 0..0.00 <>o :8 E 00 000 00.000 .0.000 mam}. 00.0.0 .0.0.0 0..000 000 :8 E 00 000 00.000 00.000 00.00 000 :8 z 00 000 0..0000 00.0000 000.00 000 :8 E 00 000 00.000 00.00 00.00 000 :8 E 00 0mm1 00.0.0. 00.0.0. 00.000. 000 :8 0.. 00 .00 00 £820 0.-.: 0>0 £0.20 0 ..: 0.532.. 0.-.: 2:32: 0.-.: xx .0 5.2.0 as: 0.0.000 E 00 . 0000-. <0_._m 0 .-.: A. . .0008. 00 200. 161 o; v ~.0.-00 I :00 ..0 0:00.00 c2022: 2: .30: 0o: Ev swab: x250 0020> 088:3. 80 ~00 00:00 :0 ... 000. . 000.0 000.0 000.0 00mg... 000.. 000.0. 000.00 .0..0- 00.00 000.00 02-<>o-.:0 00. 000.0 000.0 000.00 .0..0 000.00 000. .0 02-<>o.0:0 .0. 000.0 000.0 000. .0 000.0- 0.0.00 .0..0 02.<>o-.:0 00. - 000.0 - 8000 000.00 0.0. .- 000.0 000.00 02.<>o-0:0 00 000.0 0...0 0.0.0 0000 000.0 000. .0 02-<>o._:0 00 00.0 000.. 000.00 000.0 0.0.00 .0000 02-<>o-.:0 .0 .0... 0.0.0 00.... 00.... £000.02 000.0 .000 000.00 ..00 .0000 000. .0 :0o-<>o-_:0 00 .000 000.0. ..000 000.0- 000.00 00.00 :8-<>o-.:0 00 ....0 00.0 0.0.00 000.0- 000.00 .0000 :0o-<>o-0:0 00 .000 000.. .0000 .000 000.00 .0000 :0o-<>o._:0 00 000.0 000.. 0.0.00 0.0.0 .0000 000.00 :8-<>o-_:0 00 000.. 000.00 .00..0 0.0.0- 00...0 0.0..0 :8-<>o-.:0 .0 00.0 000.0 000.0 000.0 000.02 00.0 .000 000.00 000.0- 0.0.00 0.0.00 22-00000 00 000.0 000.0 ..000 0.0.0 00000 00.00 0.2-000-.:0 00 000.0 000.. 000.00 ..00 0.0.00 000.00 02-00%:0 00 000.. 00.0 .0000 00.0- 00.00 0.0.00 0.2-000..:0 .0 000.0 00... 000.00 00.0 000.00 00.00 0.2-000-.:0 00 .000 .00.. .0000 000.0- 000.00 000.00 0.2-000..:0 00 0.0.0 0.0.. 00 ..0 .000 0 .202 000.0. 000. .. 000.00 00.0- .0000 000.00 :0o-000-.:0 00 0.0.0 000.0 0.0..0 0. ..0 000.0 000.0 00-000-0:0 00 0...0 00.0 00000 000.0- 000.00 0.0.00 00-000-:0 00 00.0 0.0.0 000. .0 000.0- 000. .0 000. .0 00-000-0:0 .0 000.0 000.. .0000 00.0 000.00 .0000 :8-000-.:0 00 0.0.. 00.0 000.00 00.0- 0.0.00 000.00 :0o-000¢:0 0. 00 0 <28... 00 00.0.0205... 030.2 0.0-.6 06 .6 xx .0 2:: 0.00.00 ..Iw ...00—mm . :NEMNP c0 :— 52000 E nonmemxo O-0IN. mm. .0..0 NNmNN 000.00 80.0- 3.00 000.00 2<-<>O¢IN. 00. 000.0. 80.0 «00...0 o.o.o- .Nv..m ...0.0 02-<>O--_IN. mm. .0000 0.0..0 000.00 0.0.0 00.00 ..000 02.<>o-.:0. 00. 000.0 0.... 0.0.00 momd- .NN.mm 0.000 0.2-030.010. .N. ....n .0 .... $0.3 30.3 o a00>< 50.0 .090 80.00 900.0 0.0on 000.00 Imo-<>O.:IN. om. 50.0. 000.00 000.00 mm . .0. 000.00 3me ImO-«SO-LIN. 0.. 08.0 80.0.. 000.0 o. ..0. 0.0.0 mm. ..0 IMHO-«$0-010. m .. m .06. 08.00 80.0 $06. 80.0 .00..0 Inc-($0-010. ... om..o. mom... 00...0 3.6. 0.0.0 000..... Inc-«$0-010. 0.. 0...0. 8.00 ...000 80.0. 00000 8.00 ...00-«$0.010. 0.. 03.0 $0.. 0.0... .00... a Eo>< 8.0 80.0 000.00 ovod 0.0.00 0...00 02-wmm-._IN. 0.. 0.0.0 ...0.0 8.00 mm..o 00.00 000.00 2<-wmn_-:._w. 0.. .8. .. .000 000.00 000.0 000.00 08.8 0.2-$0.010. 0.. 0.0.0 900.0 000.00 05.0 80.00 0.0.00 02.wmn_.0_._~. ... 0.0.. 000.00 0.000 000.0. ..o.m~ 0.000 02.mmn_¢Im. 9. 0.0.0 ..w.m 000.00 .mmd- . .060 000.00 20rwma-fm. mo. 0.0.0 000..... 000.00 «no.3 Mala-$3. 50.00.. 900.0. «.0..0 000.0. 000.0 00...0 Inc-wma-i~. mo. 000.00 000.0. 0.0.00 mood 0.0.00 www.mw Inc-wmm-fm. .o. 000. .N no . . .. 50.00 00..? 0.00.8 0.0.00 ImOfimg-fm. mo. 000.0 .00.. one...” 08.0- 0.0.0 000..... Inc-wmm-EN. mo. 00...0 08.8 000.00 .00.? 0.0.00 000.5 Inc-mma-fm. 0.0. 000.~. .86. 000.8 03.0. ommdm .ovdm Inc-wmnEIN. mo. ow = < 000.50 0.0 :0 xx a 0E. 0_ .53 .01 Nr :02 m . an»: «F E :— 0 . 0.0.8 .0 200. 163 o._ v .90 I :8 0o 0.08:0 0.20205 2: 088 .0: En 5:85 03.50 0030> 028:0». 20 N00 .50 :0 ... .3...» o.o.n_. wand o3.» u 20>< wood. mmo. ova-«om N.o.o- vmmom Nwmom :2-<>O..IVN vm momo. no... ovwom o .vd- wmoom ovmom 2<-<>O-:Ivm mm mmmd mood Nwoom .mmo omoom .oNdm w?.<>O-..I¢N mm .mm. .N .wwé. ommdw mood .mm.mm mmoom :2.<>O-.IVN .N . .m.m Nwmd mov. .m omoo- wmv. .m 03. .m 0.2-«$0.0va. ow no.0. .. 9006 Von. .m wood .mm. .m .3... .m 0.2-($0...va o. {No mood. ova-v. v....—. 01mm;>< momfiw owed mmmom Nvoo- vwmom Nvmom Im0-<>O¢IVN 9. wood no.6 mvmom Novo 30. do ovmom ImO-(>O..IVN .. Slum. m.m.o. mmmdw mood vmmow www.mm ImO-«SO-ivw 0. «mod mNN..v mom..N ammo- Nooow mwm..w InO-<>O-..Iv~ m. mooém mood. mvvom 00...0 www.mw ommom ImO.<>O-._IVN v. omnéw Nvmo. oNoom ...0.0- mmoom NNodm ImO-<>O¢Ivm m. 93.... wow-or mood woo-v umM.c>< - 000..]. - wow...- mNmNm .N.. .- mo..mm vooNo 0.2.wmn7fvm N. mo... moo. moodm o...o- N.v.mm mmmNm ..Z-mma¢1vw .. mono me... woman vmmo- NN..Nm movdm ...(émmiIvm o. ommd. ~.0. v...Nm NNmo- mmon m.o.wm 0.2-wmnIva o ommd. mood ®.N.om Nmoo oowdm waom ...(émmufvm m 09.40 m .m... .modw ommo- mooom mm..oN ...(émqfvw . .mud armor ..o.o .nod. 00 go>< med. vmoh .oo..m vvmo m.m.om ow...” Zoo-wmm-._Ivm o v.0.ow wv..NN Noodm wm..o- ..o.m~ m .m.wN IMO-wmm-LIVN m - 000.0 - 30.0 oooov oooo ooo.ov oooo.0 loo-wmm-EVN v .Nv... mood o.o.om mmoo- mooom mmoom Im0-wmm¢I.0N m mm..v. mvoo oomom No..o- oovom w.N.om IMO-wmn.-._Ivm N m.m... .mo. vmmom voNo- m.m.om Nmmom ImOéQu-fvm . Dm C < N00. 30 «.0 50 09— w 25... Ems—am ..IVN :62 m - cm... a... E :— A. . 6.0.50 5 0300. 164 $3 2......” .8." Sad magi 3,: £3 SQR 80.0 85R Rafi 3.<>o-io N2 83 83. :3“ «Ed {EN 82% 3-<>o-.zo SF 3% 33 ~38 83 8:8 80.8 ..2.<>o-fm 09 93 ~93 «8.2 53 «8.8 80% a2.<>o.._._m 8 33. 8: 80% fine Seam 23m 3r<>o-im 8 85, 23 ENS 83- $08 50.8 ..2.<>o-fm B #5 23. as.» 83.. a .223 m8.~ 269 SEN mid 3mg EQR Imo-<>o.io 8 53. 53 SN? 95.0 Smmm BN3 Imo-<>o-io 8 :5. RI: SEN $3 9.2m mama Imo-<>o-im E 53. $1: www.mm mmod- 308 83a :8-<>o-fm 8 98m, 33 «ER Bod 83m 808 Imo-<>o..:o 8 83 «3.8 v2.8 83- 5mg 85% Imo-<>o.fo a a: 8% 33 53 o 22E 8; 83 «3.8 23 83K $0.8 2<.mmn_-fo 8 33 «SN. 03.2. $3. «8.8 59% Sammie 8 $3 9: 23m 83 RE v3.3 2<¢mntxo 8 83 83 898 and 325 N? F . F, _,\<-mmn_-im S 83 $3 $08 2.3 2.6.8 83m n2.mmn.-im 8 593 8g EN; 800. 205 80.8 _<<-wmn_-fo 3 2o." 23. 53.. 89: awaé $59 08.8 893. 03.0 80% 8mg Imowmméo vm 20m :3 SEN Eco :05 «8.8 Imo-wma-__._m 8 2:. Bow 8me «and. N88 3me Imo-mmn_-_:m mm v2.0 mm: 98.5 :3. v8.5 SEN Imo-mmn_-_zw 5 $3 85 www.mm Bro mmgm g2 :8-mmn_-=._o om was 808 SEN 89o- SE 505 Imoémaéo 2'1 am a om 3:35.52. Imamai «6-50 «6 :0 Edge: 295m 020232 20 ~00 0:00 :0 * 00~.0 000.2 2.0.2 02.00 mama: - 030 - 0000 000.00 000.0 000.00 000.00 3.<>o.i§ 0N? - no? - 030 000.00 000.0 000.00 000.00 02-050018 00? 53 000.00 000.5 000.0- 0F 000 SEN 32>?er 09 000.00 00:; 000.00 02.0 000.00 02.00 3.050023 00? 80.00 000.00 0005 000.0- 30.00 «00.00 3-050%? NS 20.0 000.00 05.00 20.0 80.00 000.00 325019 E :3 5.: 000.3. 2.0.00 00202 000.0 $0.: 3000 000.0 000.00 03.00 I8.<>o-.:§ 09 00.00 000.00 000.00 140.000- 30.00 «00.00 18-05005 0: 000.0 000.00 08.00 00 F .0- 000.00 000.00 18-/$0019 0: 05.2 05.000 000.00 E0- 9000 000.00 18005-58 E 00 F .00 000.8 03.00 000.0- 000.00 000.00 :8-<>o-i§ 0: 0:9 500.3. 000.5 3.0.0 00000 SEN 18-050019 0: 000.~ 00~.0 aha 05.3 mafia: 000.0 000.00 000.00 30.0 000.00 20.00 02-02-53 «2 000.0 000.0 000.00 02.0 000.00 :000 3.000.019 0: 80.: 000.00 000.00 080 $0.00 30.00 3-000013 0: «~00 80.? E00 0000 000.00 000.00 3-000-500 5 ES 000.00 000.00 00~.0- R000 20.00 02-00%sz 0: 000.0 000.0 000.00 R00- #000 000.00 3.0000100 02 80.0 000.2 ~00.? 000.00 a 22$ 50.00 20.000 30.00 000.0- 0:00 000.00 $9004.19 02 000.00 03.00 000.3 000.0. 000.5 000.00 50-000019 SF 000. E 000.00 000.00 02.0- 02.00 000.5 18.00.0015 000 000.0 30.00 000.00 0000. $0.00 000.00 18.00.0019 08 02.0 00:00 0030 00~.0- $0.3 $0.8 18-000013 «00 000.~? $0. 00 000.00 000.0- 000.00 50.00 18-000018 02 00 0 <2”. 02 00 0 020332 20 N00 “0:0 :0 ... 20.0 000.0. 000.0 000.0 E00902 000.00 000.00 000.00 000.0 0:00 80.00 02050100 00 000.00 000. 2 00:0 0:0 SEN 03R $05-$00 00 000.0 000.0 00:0 000.0- 000.00 000.00 30005-0100 00 50. a 000.0 000.00 300 E00 000.00 0205-0100 E 20.0 000.0 000.00 000.0 000.00 000.00 3-0500100 00 000. 3 000.0 000.00 000.0. 000.00 000.00 3350-500 2 2.0.0 000.00 000.0 0.00.0 mama: 000.00 50.0 000.00 000.0- 000.00 000.-0 100-050-0100 00 000.0 000.0 000.00 000.0 000.00 000.00 100-050-0100 C F0000 000.0 000.00 000.0 000.00 20.00 I0o-<>o-._-_0N 00 000.0 20.: 000.00 000.0- 000.00 80.00 :0o-<>o-i00 00 000.00 000.0 000.00 000.0- 000.3. 000.00 100-050-0100 00 000.00 000.9 000.00 050 000.00 0050 I0o-<>o-i00 00 000.0 $0.0 000.~ 0E0 £000.05 000.0 000.0 05.00 000.0 08.00 00000 020000100 3 00: 000.0 000.00 000.0- 00 r .00 000.00 3-0000100 : 000.0 000.0 000.00 000.0- 000.00! 000.00 3000-£00 00 000.00 000.0 F0000 200 30.5 000.00 02.000.00.000 0 000.00 000.0 BYE 000.0- 000.00 000.00 02.000-00.00 0 000.0 000.0 000.00 000.0 000.00 50.00 02-0mm-i00 0 00~.0 20.0. 000.0 000.0 00.2030 02.00 R00 000.00 0000 $0.00 000.00 100.000.5000 0 000.00 000.00 000.00 R00- 000.00 000.00 100-000.0100 0 - 800.0 - 0000 000.00 000.0 000.00 000.00 100-000.0100 0 R05 000.: 00 EN 00:0- 0050 000.00 100-000.0100 0 000.00 20.0 000.00 000.0- 000.00 E000 100-000-0000 0 000.: 000.0 000.00 000.0- 80.00 000.00 0.00-000-.:00 F 00 c <20. 00. 00 83.02”. E:_ o 222 06.50 06 :o E .0 as: 0.0500 i 00 :00. 0 - 00:. a» 0:. A. . 0.0080 00 2000 167 < -_. < O < 0 oo < < < < < < oo 4. < < < < < on < ._. < 0 < 0 mo < < < < < < on < < < < < < om < < < < < < oo - - .. - - - om .. - - - - - ow - - - - - - no .. - - - - - mm - .. - - - - mm < < < < < < 0o .4. h < 0 < 0 mm - - - - - - 0N < ... < 0 < 0 no .. - - - .. - mm < ._. < 0 < 0 mm < .r < O < 0 vo < .r < 0 < 0 3 - .. - - u - vm < < < < < .4. mo < < < < < .0. mm < .r < 0 < 0 mm .r ._. 0 O .0 0 No < -_. < 0 < 0 mm - - - - - - NN - - - - - - 0o - - - - .. - rm 4. < < < < < ..N < < < < < < oo - - - - - - on < < < < < < ow - - - - - - on - - - - - - ov - - - - - - or < < < < < < on - - - - - - ov - - - - - - or < -_- < 0 < 0 NA .. - - .. - - Du - - - - - .- t .- - - - - - 05 < ..- < 0 < 0 ow < H < 0 < O or < h < 0 < 0 mm - - - - - - mv - - - - - .. m.- - - - - - - .K < -_- < 0 < 0 30 < -_- < 0 < 0 3. - - - - - - on < .r < 0 < 0 mv - - - - - - 9. < < < < < < Nu - - - - - - NV < < < < < < NF < -_- < O < O K - - - .. - - :0 < -_- < O < 0 _. _. < -_- < O < 0 on < < < < < mu ov < < < < < < o r < < < < < < mo < P < O < 0 on < .r < 0 < 0 m < < < < < < oo - - - - - .. on < < < < < < o H H 0 0 0 0 no < -_- < o < 0 no - - - - - - n < < < < < < mo 4. < < < < < mm .. - - - - - o < < < < < < mo - -. - - - - mm < .r < O < 0 m H .r 0 O 0 0 v0 < -_- < 0 < 0 X” .r H 0 0 0 0 v < < < < < < mo - - - - - - mm 4. -_. < 0 < O m < < < < < < No < < < < < < mm < < < < < < N r - - - - - - Fm - - - - - - ..m < -_. < 0 < 0 —. ohovmvmg «momma».— ohofinNNE a. whovmvm. «mambo». ohogflue D. ohovmvfl «nacho».— ohmvnNNE D. $903000 28 $330.0 £33.88 0mZm 2: 8m mumbocow Emma mo 03 .MN 030,—- 168 < < < < < < oow < < < < < < om.- < ... < O < O oNF - - - - - - at. < < < < < < o3 < < < < < < o.- F < < < < < < o: - - - - - - o3- - - - - - - o.- _- < .r < 0 < 0 N: - .. - - - - BI. - - - - - - t _- - - - - - .. o: - - - - - - o: < < < < < < o— r - .. - - - .. mt. < < < < < 4. 9: < < < < < < or F - - - - - - v: < -_. < 0 < 0 S: < -_- < < < < 3 _- < -_. < 0 < 0 m: - - - - - - m3 - - - - - - m0 _. - - - - - - N: < ._- < O < 0 N3 - .. - - - - N _- w < ... < 0 < 0 Ft - - - - - - El < -_. < 0 < 6 —. _- _- - - - - .. - o: - - - - - - o: < .r < 0 < 0 o_. F < -_. < O < 0 mo? < < < < < < on? - - - - - - oop- - .. - .- - - o9. < < < .< < < mm? 4. .r < 0 < 0 wow < < < < < < 500 < .r < O < 0 59- < < < < < < Now - - .. - - .. mo.- < < < < < < 09. < -_- < 0 < 0 09. < < < < < < nor < H < 0 < 0 mm? - - - - - - mo? < < < < < < .00—- < < < < < < vow < ..- < 0 < 0 vor < < < < < .4. now .0. -_- < 0 < 0 mm? - - - - - - nor < -_. < 0 < 0 No.- - - - - - - Nor - .. - - - - No v < < < < < < For < < < < < < :3 - - - - - - For < < < < < < om? < ._. < 0 < 0 om? - - -. - - - oor < < < < < < omw - - - - - - on < < < < < < mm .- - - - - - on? < < < < < < oNv - - - - - - mo - - - - - - nmw .. - - - - - 5N0 - - - - - - no .- - - - - - om? - - - - - - on < < < < < < co < < < < < < mm? - - - - .. - mNF -. - - - - - mo < ._. < 0 < 0 VB. - - -. - - - VN _. - - - - .. - vo - - - - - - on? < < < < < < mNF - - - - - - mm - - - - - - wa - - - - -. - NN_- - - - - - - No < .r < 0 < 0 wmw - - - - - .. 0N0 - - - - - - 0o ohovmvw. Nboohofl ohovnNNE o. onofimwfi Nhoohoe ohovnNNm. o. ohovmvm. thohofl ohovnNNm. D. A. . 0.0080 00 2000 169 < < < < < < CNN - - - - - - OVN - - - - - - 0 —N < < < < < < moN - - - - - - mmN < < < < < < QON < ._. < 0 < 0 woN < ._. < 0 < 0 RN < < < < < < wON < < < < < < moN < < < < < < NMN - - - - - - NON - - - - - - omN < < < < < < omN - - - - - .. ®ON - - - - - - mON < < < < < .4. RN < .r < O < 0 mON < < < < < < va < < < < < < VMN < h < 0 < O VON < < < < < < mmN < ._. < 0 < 0 mmN < < < < < < MON - - - - - - NoN .. - - - .. - NMN - - - - - - NoN < .r < < < < EN 4. .r < 0 < 0 EN < ... < 0 < 0 SN < < < < < < omN < < < < < < OMN < ... < 0 < 0 CON < ... < 0 < O mmN < < < < < < mNN < < < < < < mm? .. - - - - .. mmN .r ._. < O < 0 wNN - - - - - - we < ._. < O < 0 no N < < < < < < NNN - - - - - - 59‘ < ... < 0 < 0 on N < ._. < 0 < 0 oNN < ._. < 0 < 0 02 < ._. < 0 < O mmN .. - - - .. - mNN - - - - .. - mar < < < < < < va - - - - - .. VNN - - .. - - - var < ... < O < 0 mmN - - - - - - mNN - - - - - - mar - - - - - - NmN - - - - - - NNN < ._. < O < 0 N? < < < < < < 3N - - - - - - _.NN < < < < < < 5? < ._. < 0 < 0 omN < < < < < < ONN < ... < O < 0 09. < < < < < < mVN < < < < < < m N - - - - - - am? < ._. < 0 < 0 QVN < ._. < 0 < 0 m ..N - - - - - . mm? < < < < < < NVN - - - - - - 5 N < ._. < 0 < 0 53 < < < < < < ®VN < < < < < < o N < < < < < < 03 < < < < < 4. EN < < < < < < m N < < < < < < m9. < ._. < 0 < 0 SN < ._. < U < 0 EN - - - - - ; v? < < < < < < MVN - - - - - - m N < < < < < < mm r ... ._. 0 0 O 0 NVN - - - - - - N N < < < < < < Nor ._. .r 0 0 0 0 SN < < < < < < r N < ... < O < 0 For @5933». thwhmfl mhwvnNNE o. ”5:332 thahwm. whoVnNNE D. mhcvmvm. thwhufl whmgNNE o. _ Azfiaé MN 03$ 170 - - - - - - com < < < < < < 0mm - - - - - - oom < < < < < < mmm < < < < < < mNm < .r < 0 < O mmN - - - - .. - mmm < ._. < 0 < 0 wNm - - - - - - me - - .. - .. - 5mm 4. < < < < < NNM < < < O < < mmN < < < < < < 0mm .. - - - - - on < .r < 0 < 0 omN < < < < < < mom - - - - - - mNm < < < < < < mmN < < < < < < vmm ._. ._. O 0 O O VNm - - - - - - va < < < < < < mmm - - - - - - mNm - - - - - - mmN .. .. - - - - Nmm - - - - - - NNm < < < < < < NmN < < < < < < rmm < ._. < 0 < 0 NM < P < O < 0 SN - - - - - - 0mm - - - - - - ONm - - - - - - omN < < < < < < mvm - - - - - - a PM - - - - - - mwN .. - - - - .. 9am .. - - - - - m 5 - - - - - - wwN < .r < 0 < 0 NS.” - - - - .. - N 5 - - - - - - an < .r < < < 0 mvm - - - .. - - m _‘m - - - - - - owN - .. .. - - .. mvm < ._. < O < O m Pm - - - - - - mwN < < < < < 4. 3m < < < < < < v Fm - - - .. - - va - - .. - - - mvm - - - - - - m Fm - - - - - - mmN - - - - - - Nvm < ._. < 0 < 0 N 5 - - - - .. - NwN - - - u - - Sum ._. H O O 0 0 _‘ Fm - - - - - - 5N - - - - - - ovm < .r < 0 < 0 o Fm - - - - - - omN < < < < < < mmm < < < < < < mom < < < < < < mNN < < < < < < mmm - - - - - - mom < < < < < < mNN - - - - - - 5mm < < < < < < mom < ._. < 0 < 0 RN < h 0 0 0 O mmm .. - - - - - mom. < ._. < O < 0 mKN - - - - - - mmm - - - - - .. mom - .. - - - - mNN < < < < < < me - - - .. .. - vow < < < < < < VNN - - - - - - mmm - - - - - - mom - - - - - - MKN < .r < O < 0 Nmm < .r < 0 < 0 Non .. - - - .. - NNN < < < O < < Sum < h < O < 0 won - - - .. - - KN 2.63ku thohwfi ahngNE n: ”2.3me mewhow. whovnNNE D. whovmvfl thmhmfl mhmvnNNE a. A. . .Paoe mm 03$ 171 - - - - - - omv < ._. < 0 < O ONv < < < < < < 0mm < ._. < 0 < O mvv < < < < < < m _‘v .. - .. - - - mwm - - u - - - wvv < < < < < < w v - - - u - - wwm < ._. < O < 0 va - - .. - - - N _.v < < < < < < Nam - - - - - - ovv ._. ._. 0 O O O w v .. .. .. - - - mwm .. .. .. - - - mvv < < < < < < m _‘v - - - - - - mwm < < < < < < vvv < ._. < O < 0 v _.v - - - - - - vmm - .. - u .. - mvv - - - - .. - n v - - - - - - mom - - - - - - va < .r < O < 0 N v - - - - - - Nwm < h < 0 < 0 rvv - - - - - .. r v < .r < 0 < O ..wm < < < < < < ovv < < < < < < o v < ._. < 0 < 0 own < < < < < < mmv - - - - - - mov - - - - - - mum. < < < < < < wmv < .r < 0 < 0 wov - - - - - - mum - - - - - .. nmv - - - - - - nov < .r < O < 0 55m .4. < < < < < on v < ._. < O < .0 oov < < < < < < mum < ._. < 0 < O mmv - - - - - - mov < < < < < < mum - - .. - .. .. vmv < H < o < 0 vov < < < < < < vnm < < < < < < mmv < ._. < O < O mov < < < < < < mum < < < < < < va < < < < < < Nov < < < < < < Nun < < < < < < Fmv - - .. - - - Fov - - - - - - Km - - - .. - - omv < ._. < 0 < 0 oov < H < 0 < O cum - - - - - - va < < < < < < mam - - < O < 0 mom < < < < < < wNv < ._. < O < 0 com .. - .. - - - mom < < < < < < NNv < < < < < < mom .. - - - - - non - - - - - - oNv < < < < < < 0mm - - - - - - 00m < ._. < O < 0 va - - - - - .. mam < < < < < < mmm . - - .. - - va < < < < < < vmm < H < 0 < 0 vow < ._. < O < 0 MNv < < < 0 < 0 man h .r < < 0 0 non .. - - - - - NNv < < < < < < Nmm < < < < < < Nmm < ._. < O < O ..Nv < ._. < 0 < 0 5m < < < < < < _‘mm whcvmvw. thwhwfi uhcvnNNw. o. whovmvm. thmhww— 3.ngth D. _ mhcvmvw. thmhmm. whovNNNE o. A. . .Paoov mm 0:3 172 - .. u - - - ovm < ._. < O < 0 o 5 < < < < < < omv - - - - - - mmm < < < < < < mom < ._. < 0 < O va < < < < < < wmm < < < < < < mom < ._. < 0 < O wNv < ._. < 0 < 0 Nmm < < < < < .4. Non < < < < < < NNv - - - - - - 0mm - - - .. - - mom < < < < < < va < < < < < < mmm < < < < < < mom < ._. < 0 < 0 va - - .. - - - vmm < < < < < < vow < ._. < 0 < 0 va < < < < < < mmm < P < 0 < 0 non - - - - - - MNv < < < < < < Nmm < < < < < < Nom < < < < < < NNv < < < < < < ..mm - - .. - - - ..om < < < < < 0 ..Nv < < < < < < 0mm < ._. < 0 < 0 com - - - - - .. ONv < < < < < < mNm < < < < < < mmv - - - - - - mmv < < < 0 < 0 wNm - - - - - - wmv - - - - - - mmv < < < < < < NNm - - - - - - va - - - - - - va < < < < < < on - - - - - - omv < ._. < 0 < 0 00v < < < < < < mNm - - - - - - mmv - - - - - - mmv ._. ._. O 0 0 0 va - - - - - - vmv < ._. < 0 < 0 vov .. - .. - - - MNm - - .. - - - mmv < .r < 0 < 0 mov < < < < < < NNm - .. - - - .. Nov < ._. < O < 0 Nov < < < < < < wNm - .. - - - - 5v < N < 0 < 0 Nov .. - - .. - - ONm ._. ._. 0 0 O 0 omv - - - - u .. oov - - - - - - m ..m - - - - - - mwv - - - - - - mmv - - - - - - m E - .. - - - - mwv < < < < < < mmv - - - - - - N E ._. ._. O O 0 O va < < < < < < va ._. ._. 0 0 0 0 o E - - - - - - omv - - - - - .. mmv < < < < < < m E - - - - - - mmv < ._. < O < 0 mmv - - - - - - v rm < < < < < < vwv < < 4. < < < vmv - - u - - - m rm < ._. < 0 < O mwv < < < < < < mmv - - - - - - N B < N < 0 < < va < ._. < O < 0 va < < < < < < F E H .N 0 O 0 0 5v < ._. < O < 0 wmv ”Novmvm. NNmmem. «NovnNNE a. «Novmve NNmme2 wvanNNE n= «chmvm. NNmmeE mchnNNm. n: A. . 383 mm 22¢ 173 - - - - - - omo < < < < < < ooo ._. ._. 0 U 0 O ONm < ._. < 0 < O oNo < < < < < < mom . .. - - - - mom 4. < < < < < oNo - - u u - - mom < .r < 0 < 0 mom - - - - - - NNo ._. .F O 0 0 0 Now - u - - .. - Nom - - - .. - - oNo - - - - u - mom < .r < O < 0 00m < ._. < 0 < 0 mNo - .. - - - - mom - .. - - - - mom < < < < < < vNo ._. ._. < O < 0 vow . - - - - - vow - - - - - .. oNo < ._. < 0 < 0 mom < .F < 0 < 0 mom . - - - .. - NNo < < < < < < Non < < < < < .4. Now < ._. < O < 0 FNo < ._. < O < 0 Foo < .r < O < 0 Foo 4. .F < 0 < O ONo .F ._. 0 O 0 0 oom < ._. < 0 < 0 oom - - - - - - o Fm - - - - - - mom < < < < < < mom - - - - - - o Fo - - - - - - mom - - - - - - omm < < < < < < N Fo < < < < < < Non ._. .r O O 0 0 Now - - - - .. - o Fo - - - - - - mom - - .. - - - 0mm .N N. < O < 0 m Fo - - - - - - mom ._. ._. 0 O 0 0 non < < < < < < v F o - - - - - .. vow - - .. - - - vmm < < < < < < m Fo < ._. < 0 < 0 mom .4. < < < < < mom < < < < < < N F0 4. < < < < < Nom - - - - - - Nmm < < < < < < F Fm - - - - - .. Fom < < < < < < 50 .F .F U 0 0 0 o Fo < .F < 0 < 0 oom < < < < < < omm - - - - - - moo .F ._. O 0 O 0 on < .r < 0 < 0 mvm - - - - - - woo < ._. O O O 0 on - - - - - - ovm < < < < < < Now - - - - - - NNm < < < < < < Nvm < < < < < < moo - - .. - - - on - - - - - .. ovm < ._. < 0 < 0 moo - - .. - - - mNm - u - - - - mvm - - - - - - vow < < < < < < va < ._. < 0 < 0 vvm < ._. < 0 < 0 moo - - - .. .. - MNm < < < < < < mvm < < < < < < Noo - - - - - - NNm - - - - - - Nvm - - - - - - Foo < N < 0 < 0 FNm < < < < < < va oNovmvE NNooNow. oNovNNNE a. oNovmvE NNmoNom.h oNovnNNE n: oNovmvE NNooNoE oNovnNNm. o. A. . .Paoa 8 29¢ 174 - - - - - - oNN < < < < < < ooo < < < < < < ooo < < < < < < o FN < ._. < 0 < 0 moo < < < < < < ooo ._. ._. O 0 0 0 o FN < .F < O < O ooo ._. .F O 0 0 0 ooo - - - - - - N FN .. u - - - - Noo < < < < < < Noo - - - - - - oFN < < < < < < ooo - - .. - - - ooo - - - - - - o FN < < < < < < ooo < < < < < < ooo - - - - - - vFN - - u - - - voo < < < < < < voo - - - - - - m FN < ._. < O < 0 moo - - - - - - moo < < < < < < N FN ... ._. 0 0 0 O Noo < < < < < < Noo < < < < < 4. F FN < ._. < 0 < 0 Foo .. - - - - - Foo - - - - - - 0 FN < < < < < < ooo - - - - - - ooo - - - .. - - ooN < < < < < < oNo - - - - - - ovo - - - - - - ooN < < < < < < oNo - - - - - - ovo - - - .. - - NON - - - - - - NNo - - - - - - Nvo - - - .. - - ooN < < < < < < oNo < ._. < 0 < O ovo < < < < < < ooN - - - - - - oNo < .F < 0 < 0 ovo < < < < < < voN < .N < < < < vNo < < < < < < vvo < < < < < < moN < .F < O < O «No < < < < < < ovo - - - - .. - NON H ._. O O 0 0 NNo < ._. < 0 < 0 Nvo < < < < < < FON < < < < < < FNo - - - - - - Fvo - - - - - - OON < < < < < < oNo < H < 0 < O ovo - - - - - - moo < ._. < 0 < 0 ooo < ._. < 0 < 0 moo < < < < < < ooo < < < < < < ooo < < < < < < omo < ._. < < < < Noo < ... < 0 < O Noo - - - - - - Noo < < < < < < ooo .. - - - - - ooo < < < < < < omo - .. - - - - moo - - - - - - ooo ._. ._. O O 0 0 omo < ._. < 0 < 0 voo < .F < O < 0 voo < < < < < < vmo < .F < 0 < 0 moo - - - .. .. - moo - - - - - - moo < < < < < < Noo < < < < < < Noo < < < < < < Noo < . < < < < < Foo < ._. < O < 0 Foo < < < < < < Foo oNovm NNooNoE oNovnNNE o. oNovovm. NNooNoE oNovoNNE D. oNovovE NNooNoE oNovnNNE o. A. . .Peooo MN 2A5 175 - - - - - - o Fo < ._. < 0 < 0 ooN < < < < < < ooN < ._. < 0 < O ooo - - - .. - - oNN < < < < < < ovN - - - - - - ooo .. - - - .. - oNN < < < < < < ovN < < < < < < Noo - - - - .. - NNN < ._. < O < 0 NvN ._. ._. O 0 0 0 ooo - - - - - - oNN < < < < < < ovN - - - - - - ooo - - - - - - oNN < < < < < < ovN - - - - - - voo < P < 0 < 0 vNN < < < < < < va - - - - - - moo < < < < < < mNN < ._. < < < < mvN < < < < < < Noo - - - - - .. NNN < < < < < < NvN < < < < < 4. Foo - - - - - - FNN < ._. < O < O FvN - u - - - - ooo < < < < < < oNN - - - - - - ovN < < < < < < ooN .. - - - - - ooN - - - - - - omN ._. ._. 0 0 0 0 ooN - - - - - - ooN < ._. < O < 0 omN - - - .. - - NoN < ._. < < < < NoN < < < < < < NoN < < < < < < ooN < P < O < O ooN < < < < < < omN - u - - - .. ooN - - - - - - ooN < ._. < O < 0 omN - - < < < < voN - - - - - - voN - .. - - - - va < ._. < 0 < 0 ooN - - - - - - moN < < < < < < moN - - .. - - - NoN < ._. < 0 < 0 NoN - - - - - - NmN - - - - - - FoN < < < < < < FoN - - - - - - FmN < ._. < 0 < 0 ooN - - - - - - ooN - - - .. - - omN < < < < < < ooN < .F < 0 < 0 ooN < < < < < < oNN .F ._. < 0 < 0 ooN < ._. < 0 < O ooN < < < < < < oNN < < < < < < NoN < < < < < < NoN < ._. < < < < NNN .. - .. - - - ooN < < < < < < ooN - - - - - - oNN - - - - - - ooN - - - - - - ooN - - - - n - oNN < < < < < < voN - - - - - - voN < < < < < < vNN - - - - - - moN - - - - - - moN < < < < < < oNN < < < < < < NoN - - - - - - NoN < < < < < < NNN < ._. < 0 < 0 FoN < h < 0 < O FoN < .r < 0 < 0 FNN oNovmvE NNooNom. oNovaNE D. oNovmvE NNooNoE oNovaNm. D. oNovmvE NNooNoE oNovaNE D. A. . .Paoe mm 2me 176 < < < < < < ooo < ._. < 0 < 0 ONo < < < < < < ovo < < < < < < omo < < < < < < ooo ._. ._. 0 0 O 0 ooo - - - - - - ooo < ._. < 0 < 0 ooo < < < < < < omo - - - - - - Noo - - - - - - Noo < < < < < < Nmo < < < < < < ooo < ._. < 0 < 0 ooo < < < < < < ooo < .r < 0 0 O ooo < .F < 0 < 0 ooo < ._. < O < 0 omo - - - - - - voo - - .. - - .. voo < ._. < O < O vmo < ._. < 0 < O ooo - - - .. .. - moo < < < < < < omo ._. ._. < O < 0 Noo < .F < 0 < 0 Noo - - - - - - Noo - - - - - - Foo < < < < < < Foo ._. .r 0 O 0 0 Foo < .F < 0 < O ooo < < < < < < ooo - - - - - - omo - - - - - - ooo < < < < < < ooo < ._. < 0 < O oNo < < < < < < ooo - - - .. - - ooo 4. .F < O < O oNo - - - - - - Noo ._. ._. 0 0 0 0 Noo - - - - - - NNo - .. - - - - ooo < ._. < O < 0 ooo - - - - - - oNo < < < < < < ooo - - - - - - ooo - - - - - - oNo ._. ._. 0 0 0 0 voo ._. ._. < 0 < 0 voo .. - - u - - vNo < < < < < < moo .. - - - - - moo - - - - - - oNo < .F < 0 < O Noo < < < < < < Noo < < < < < < NNo - - - - - .. Foo < < < < < < Foo < < < < < < FNo - - - - - - ooo < .F < 0 < O ooo - - - - - - oNo - - - - - - oNo < ._. < 0 < O mvo - - - - - - m Fo < < < < < < oNo < < < < < < ovo < < < < < < o Fo < .F < 0 < 0 NNo < < < < < < Nvo - - - - - - N Fo - - - - - - oNo < ._. < 0 < 0 ovo - - - - .. - oFo < < < < < < oNo < ._. < O < 0 ovo < ._. < 0 < O o Fo - - - - - - vNo - - - - - - vvo - - - - - - vFo - - - - - - mNo - .. - - - - mvo ._. .F O 0 0 0 m Fo - - .. - - - NNo < < < < < < Nvo .r ._. 0 O 0 0 N Fo < ._. < 0 < O FNo - - - . - - - Fvo < ._. < O < 0 F Fo oNovovmh NNooNomb oNovoNNm. o. oNovovE NNooNoE oNovaNw. D. oNovovE NNooNomg oNovang o. L A. . 38a 8 2me 177 < ._. < 0 < 0 ooo < ._. < 0 < 0 ooo - - - - - - ooo < ._. < O < 0 ooo < < < < < < ooo - - - - - - oNo < < < < < < ooo < < < < < < ooo < .F < 0 < 0 oNo < ._. < < < < Noo < ._. < O < 0 Noo - - - - - - NNo < ._. < 0 < 6 ooo < < < < < < ooo - .. - - - - oNo - - - - - - ooo < < < < < < ooo - - - - - - oNo < < < < < < voo < < < < < < voo < < < < < < vNo < < < < < < moo < < < < < < moo ._. ._. 0 0 O 0 m No < ._. < O < 0 Noo - .. - - - - Noo - - - - - - NNo < < < < < < Foo - - - - - - Foo - - - - - - FNo < ._. < 0 < 0 ooo - - - - - - ooo < ._. < 0 < 6 oNo - - - - - - oNo - - - - - - mvo < < < < < < o Fo - - - - - - oNo < < < < < < ovo < < < < < < o Fo < ._. < 0 < 0 NNo < < < < < < Nvo < < < < < < N Fo - .. - - .. - oNo < < < < < < ovo - - - .. - - o Fo < ._. < 0 < 0 oNo - - - - - - ovo < ._. < 0 < O o Fo - - - - - - vNo < < < < < < vvo < ._. < 0 < 0 v Fm < o. < 0 < 0 mNo - - - - - - mvo - - - - - .. m Fo - - - - - - NNo - - - - - - Nvo < < < < < < N Fo < .r < 0 < O FNo < < < < < < Fvo .. - - - - - F Fo < ._. < 0 < O oNo < ._. < 0 < 0 ovo - - - - - - 0 Fa - - - - - - ooo - - - - - - ooo < ._. < O < 0 ooo < < < < < < ooo - - - - - .. ooo - - - - - - ooo < < < < < < Noo < < < < < < Noo - - - - - - Noo - - - - - - ooo - - - - - - omo - - - - - - ooo - - - - - - ooo < ._. < < < < ooo - - - - - - ooo < < < < < < voo < < < < < < vmo - - - - - - voo - .. - - - - moo < < < < < < mmo .F ._. 0 0 0 0 moo < .r < 0 < 0 Noo < < < < < < Noo < < < < < < Noo < ._. < O < 0 Foo < ._. < O < O Fmo - - - - - - Foo oNovovfl NNooNom.h oNovaNE o. oNovmvE NNooNog oNovaNE o. oNovovmg NNooNom. oNovoNNE D. A. . .PESV MN 2me 178 < ._. < O < 0 ooo F < < < < < < ooo F < < < < < < ooo F - - - - - - oNo F < < < < < < ovo F < < < < < < ovo F - - - u - - oNo F < .—. < 0 < 0 ovo F < ._. < 0 < 0 ovo F < < < < < < NNo F < < < < < < Nvo F < < < < < < Nvo F < ._. < O < 0 oNo F < < < < < < ovo F < < < < < < ovo F - - - - - - oNo F < < < < < < ovo F < < < < < < ovo F < < < < < < vNo F < < < < < < vvo F < < < < < < vvo F < < < < < < mNo F < < < < < < ovo F < < < < < < ovo F - - - - - .. NNo F < < < < < < Nvo F < < < < < < Nvo F - - - - - - FNo F < < < < < < Fvo F < < < < < < Fvo F < < < < < < oNo F < < < < < < ovo F < < < < < < ovo F < ._. < 0 < 0 ooo F < ._. < 0 < 0 omo F < ._. < O < 0 omo F ._. ._. 0 0 O 0 ooo F - - - - - - omo F - - .. - - - omo F < ._. < 0 < 0 Noo F - - - - - - Noo F - .. - .. - - Nmo F - - - - - - ooo F < .r < 0 < O ooo F < ._. < 0 < 6 omo F .. - .. - - - ooo F < ._. < O < 0 omo F < ._. < O < 0 omo F - - - - - - voo F - - - - - - vmo F - - - - .. - vmo F - - - - - - ooo F < < < < < < mmo F < < < < < < mmo F - - .. - - - Noo F - - - - - - Noo F - - - - - - Nmo F - - - - - - Foo F < < < < < < Fmo F < < < < < < Fmo F - - .. - - - ooo F < ._. < 0 < 0 omo F < ._. < O < 0 omo F < < < < < < ooo F < < < < < < oNo F < < < < < < oNo F .. - .. - .. - ooo F < < < < < < oNo F < < < < < < oNo F < .r < 0 < 0 Noo F < ._. < O < 0 NNo F < ._. < 0 < O NNo F < < < < < < ooo F - - - - - - oNo F - - - - - - oNo F < .F < < < < ooo F < ._. < O < 0 oNo F < ._. < 0 < 0 oNo F - - - - - - voo F - - - - .. - vNo F - - - - - - vNo F - - - - - - moo F < < < < < < mNo F < < < < < < oNo F - - - - .. - Noo F < ._. < O < O NNo F < ._. < D < O NNo F - - - - - - Foo F - - - - - - FNo F - - - - - - FNo F oNovovE NNooNoE oNovaNE o. oNovmvE NNooNoE oNovaNm. D. oNovmvE NNooNog oNovaNE a. A. . 38s MN 2.3 179 - - - - - - oNFF - - - - - .. ovFF - - - - - - oFFF - - - .. - - ooFF < < < < < < mmFF < .r < 0 < O ooFF < < < < < < ooFF < < < < < < ooFF < < < < < < ooFF - - - - - - NoFF < < < < < < NoFF - - - - - - NoFF < < < < < < ooFF - - .. - - - omFF - - - - - - ooFF < < < < < < ooFF ._. ._. O O 0 0 omFF - - - - - - ooFF < < < < < < voFF < < < < < < voFF < < < < < < voFF < ._. < O < 0 moFF < .F < 0 < 0 mmFF .. - - - - . moFF < ._. < 0 < 0 NoFF < < < < < < NmFF < ._. < 0 < 0 NoFF - - - - - - FoFF < < < < < < FoFF - - - - - - FoFF < < < < < < ooFF - - - - - - omFF < .r < 0 < 0 ooFF - - - - - .. ooFF < ._. < 0 < 0 oNFF - - - - - - oooF < < < < < < ooFF < ._. < O < 0 oNFF < < < < < < oooF < < < < < < NoFF ._. .r 0 0 0 0 NNFF - - - - - - NooF - - - - - - ooFF < < < < < < oNFF < ._. < < < < oooF < < < < < < ooFF < ._. < O < 0 oNFF < < < < < < oooF - u - - - - voFF - - - - - - vNFF - - - - - - vooF - - - - - - moFF - - - - - .. mNFF < < < < < < mooF < < < < < < NoFF < < < < < < NNFF < < < O < 0 NooF - - - - - - FoFF < < < < < < FNFF < ._. < 0 < 0 FooF < ._. < 0 < 0 ooFF .r .F 0 U 0 0 ONFF - - - - - - oooF - - - - - - ovFF .. - - - - - oFFF - - - - - - oooF < < < < < < ovFF - - - - - - oFFF < ._. < 0 < O oooF - - - .. - - NvFF < < < < < < NFFF < ._. < 0 < 0 NooF - - - - - - ovFF < < < < < < oFFF - - - - - - oooF - - - - - - ovFF < ._. < 0 < O oFFF - - - - - - oooF - - .. - - - vvFF - - - - - - vFFF - - - - - - vooF < < < < < < ovFF < ._. < O < 0 oFFF - - - .. - - mooF - - - - - - NvFF < < < < < < NFFF < ._. < 0 < O NooF < ._. < O < O FvFF < < < < < < FFFF - .. - - - - FooF IoNovovm. NNooNoE oNovaNmL n: oNovome NNooNo2 oNovoNNB n: oNovovw. NNooNoE oNovaNP. n: A. . .383 MN 2me 180 - - - - - - ooNF < < < < < < omNF < ._. < O < O OONF < .F < < < < ooNF < .r < 0 < 0 oNNF < < < < < < ooF F < ._. < 0 < 0 ooNF < < < < < < oNNF - - - - - - ooF F - - - - - - NoNF < ._. < 0 < 0 NNNF - - - - - - NoF F .r ._. 0 0 0 0 ooNF - - - - - - oNNF < ._. < 0 < 0 ooF F < < < < < < ooNF - - - - - - oNNF - - - - - - ooF F < ._. < O < 0 voNF < .... < 0 < 0 vNNF ._. ._. O 0 0 0 voF F < ._. < 0 < O moNF < .r < 0 < < mNNF - - - - - - ooF F - - - - - - NoNF < < < < < < NNNF - - - - .. .. NoF F < < < < < < FoNF - - - - - - FNNF < < < < < < FoF F < < < < < < ooNF < < < < < < oNNF .. - - - - - ooF F u - - .. - - ovNF < P < 0 < 0 oFNF - - - - - - ooFF < ._. < O < 0 ovNF - .. .. - - - oFNF - - - - - - ooFF ._. .r 0 0 0 0 NvNF < < < < < < NFNF < ._. < 0 < O NoFF .. - - - - - ovNF . - - - - .. oFNF < < < < < < ooF F - - - - - - ovNF - - - - - - oFNF < < < < < < ooFF - - - .. - - vaF < < < < < < vFNF < ._. < 0 < 0 voF F - - - - - - ovNF - - - - - - mFNF < < < < < < moFF < < < < < < NvNF < < < < < < NFNF - - - - u - NoF F - - - - - - FvNF < ._. < 0 < 0 F FNF < < < < < < FoF F - - - - - .. ovNF - - - - - - oFNF ._. .—. 0 0 0 0 ooFF - - - - - - ooNF - - .. - - - ooNF < < < < < < oNF F - u - - - - omNF - - u - - - ooNF - - - - - - oNF F < < < < < < NmNF - .. - - - - NoNF - - .. - - - NNF F - - - - - - omNF < .r < O < 0 ooNF - - - - - - oNF F - - .. - - - omNF < ._. < 0 < 0 ooNF - - - - - - oNF F < < < < < < vaF < ._. < 0 < 0 voNF < < < < < < vNF F - - - - - - omNF < ._. < 0 < 0 moNF < ._. < 0 < 0 oNF F - - - - - - NmNF - - - - - - NoNF - - - - - - NNF F - - - - - - FmNF < < < < < < FoNF - - - - - - FNF F oNovovE NNooNoE oNovaNE D. oNovmvm. NNooNo2 oNovaNg a. oNovovE TNNooNoE oNovoNNE o. A. . .383 MN 23. 181 < .r < 0 < 0 oom F < < < < < < ONm F < < < < < < ooN F < < < < < < mvo F ._. ._. 0 0 0 0 o Fo F < < < < < < ooN F - - - - - - ovm F - - - - - - o Fm F - - - - - - ooN F < < < < < < Nvm F < < < < < < N Fm F .. - - - - - NoN F - - - - - - ovm F .. - - - - - oFm F < < < < < < ooN F - .. .. - - - ovm F < < < < < < o Fm F - - - - - - ooNF < < < < < < vvm F < < < < < < v Fm F ._. ._. O 0 0 O voN F < < < < < < mvo F < < < < < < m Fm F < < < < < < moN F - - - - - - Nvm F < ._. < O < 0 N Fm F < ._. < 0 < 0 NoN F .. - .. - .. .. Fvo F < < < < < < F Fm F - .. - .. - - FoNF < < < < < < ovm F - - - - - - o Fo F < < < < < < ooN F < .r < 0 < 0 omm F - - - .. - - oom F < .r < O < 0 oNNF - - - - - - omo F < < < < < < ooo F - .. .. - - - oNN F - - - - - - Nom F - - - - - - Noo F < < < < < < NNN F < ._. < 0 < 0 omm F < < < < < < ooo F < < < < < < oNN F - - - - - - omm F < < < < < < oom F < < < < < < oNN F < < < < < < vmm F < < < < < < vom F < < < < < < vNN F .. - - - - - mmo F < < < < < < moo F - - .. - - - mNN F - - .. - - - Noo F - - - - - - Noo F < < < < < < NNNF < < < < < < me F < .r < 0 < 0 Foo F - - .. - - - FNN F < < < < < < oom F < < < < < < ooo F < ._. < O < 0 ONN F - - - - - - on F < .r < 0 < 0 ooN F < < < < < < ooN F < < < < < < oNo F < .r < O 0 0 ooN F < < < < < < ooN F < < < < < < NNm F < < < < < < NoN F ._. .r O 0 0 O NoN F - - - - - - oNo F < < < < < < ooN F < ._. < 0 < 0 ooN F < < < < < < oNo F .. - - - - - ooN F < ._. < O < 0 ooN F < < < 0 < 0 va F - - - - .. - voN F < < < < < < voN F - .. - - .. - mNo F < < < < < < ooN F < < < < < < moN F < ._. < 0 < 0 NNm F < < < < < < NoN F - - .. - - - NoN F < .r < < < < FNm F < < < < < < FoN F < < < < < < FoN F oNovovg NNooNoE oNovaN2 a. oNovovm. NNooNoE oNovaNm. D. oNovmvfl NNooNoE oNovaNg a. A. . .Paooo MN 2me 182 - - - - - - ovv F < < < < < < o Fv F < < < < < < ooo F - .. - - - - omv F < ._. < 0 < 0 oov F < H < 0 < 0 oNo F .. - - - - - ova < ._. < O < 0 oovF < ._. < 0 < 0 onF - - - - - - va F < ._. < 0 < 0 Nov F < < < < < < NNm F .. - .. - - - omv F - - - - - - oov F < < < < < < oNo F - - - - - - omv F < < < < < < oov F < < < < < < oNo F < ._. < 0 < 0 vmv F < ._. < O < 0 vov F < < < < < < vNo F - - - - - - mmv F - - - - - - oov F < ._. < 0 < 0 mNm F - - - - - - va F < < < < < < Nov F - - .. - - - NNm F < ._. < 0 < 0 Fmv F < ._. < 0 < 0 Fov F - - - - - - FNm F < < < < < < omv F < .F < 0 < 0 oov F < ._. < < < < ONm F < < < < < < oNvF < < < < < < mom F < < < < < < mom F - - - - - - oNv F - - - - - - oom F < < 4. < < < oom F < ._. < O < 0 NNv F < < < < < < Nom F < < < < < < Noo F < < < < < < oNv F < ._. < O < 0 ooo F < < < < < < ooo F ._. ._. 0 0 O 0 oNv F < ._. < 0 < 0 ooo F < < < < < < ooo F < < < < < < vaF < ._. < O < 0 vom F < ._. < 0 < 0 vom F - - - - - - va F < ._. < O < 0 ooo F ._. ._. < 0 < 0 mom F - - - - .. - NNvF - - - - - - Nom F < < < < < < Nom F < < < < < < FNvF < < < < < < Fom F < < < < < < FomF < < < < < < oNvF < < < < < < oom F < ._. < 0 < 0 oom F - - - - - - o Fv F < < < < < < mom F - .. - - - - mom F 4. .F < 0 < O o Fv F < < < < < < ooo F < < < < < < oom F < < < < < < N Fv F ._. ._. O 0 0 0 Nom F - - - - - - Nom F - - - - - - o F v F < 4. < < < < oom F < ._. < 0 < 0 oom F - .. - - - - o Fv F < < < < < < oom F < ._. < O < 0 mom F - - - - - - v F v F - - .. - - - vom F < < < < < < vom F < < < < < < o Fv F ._. ._. 0 0 0 0 ooo F < < < < < < moo F - - - - - - N FvF - - - - - - Nom F - - - - - - Nom F < ._. < 0 < 0 F FvF < < < < < 4. Foo F - - .. - - - Fom F oNovmvE NNooNoB oNovaNE D. oNovovm. NNooNoe oNovaNE o. oNovovm. NNooNofl oNovaNm. a. A. . 383 MN 0:5 183 < ._. < O < O omo F < < < < < < ooo F - - - - - - ONvF < ._. < O < 0 omo F < ._. < O < O oovF ._. H O 0 0 0 oovF - - - - - - voo F < ._. < O < O oovF < < < < < < oov F - - - - - - moo F < ._. < 0 < O NovF < ._. < 0 < 0 NovF < < < < < < Nmo F - .. - - - - oov F - - - - - - oov F - - - - - - Foo F - .. - - - - oovF - u - - - - oovF < ._. < O < 0 omo F < < < < < < vovF - - - - - - vovF < ._. < O < 0 omo F < < < < < < movF - - - - - - movF - - - - - - vmo F .. - - - .. - NovF - - - - - - NovF - - - - - - mmo F < .r < 0 < 0 FovF < ._. < 0 < O FovF < < < < < < Nmo F < < < < < < oovF ._. ._. < O < O oovF - - - - .. - Fmo F < < < < < < oovF < ._. < O < 0 oovF < ._. < 0 < 0 omo F - - - - .. - oovF < < < < < < oovF < ._. < O < 0 omo F - - - - - .. NovF < .r < 0 < 0 NovF - - - - - - vmo F < ._. < O < O oovF .. - .. - - - oovF - - - - - - omo F - - - - - - oovF - - - - - - oovF < < < < < < Nmo F < ._. < O < 0 vovF - u - - - - vovF - - .. - - - Foo F < ._. < 0 < 0 movF < < < < < < movF < .r < 0 < 0 omo F - - - - - - NovF - - - - - - NovF < .r < O < 0 omo F - - - - - - FovF - - - - - - FovF - - - - - - voo F < < < < < < oovF < .r < 0 < 0 oovF - - - - - - mmo F - - - - - - oNvF - - - - - - ovvF < < < < < < Nmo F - - - - - - oNvF - - - .. - - ovvF - - - - - - Fmo F - - - - - - NNv F .r ._. < 0 < 0 vaF < H < 0 < 0 omo F - - - - - - oNvF < ._. < O < O ovvF < ._. < 0 < 0 omo F < < < < < < oNvF < < < < < < ovvF .. - u - - - vmo F - u - - - - vaF < < < < < < vvvF - - - - - - omo F < ._. < 0 < O vaF < .r < 0 < 0 mvv F < < < < < < Noo F < < < < < < NNvF < < < < < < vaF - - - - - - Fmo F - - - - .. - FNvF - - - - - - FvvF oNovovE NNooNoE oNovaNfi D. oNovovw. NNooNomL oNovang o. oNovovm. NNooNo2 oNovMNNm. n: A. . 383 MN 03:. 184 < ._. < 0 < 0 omo F < .r < 0 < 0 ooo F - - - - - - vmo F - - - - - - ooo F < < < < < < Nmo F - - - - - - Fmo F oNovome NNooNo2 oNovaNE a. AEPEOoV MN Bank 185 10. 11. 12. 13. BIBLIOGRAPHY Wills-Karp, M. & Ewart, S. L. Time to draw breath: asthma-susceptibility genes are identified. Nat Rev Genet 5, 376-87 (2004). Chung, K. F. & Barnes, P. J. Cytokines in asthma. Thorax 54, 825-57 (1999). Wills-Karp, M. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu Rev Immunol 17, 255-81 (1999). Hoftjan, S., Nicolae, D. & Ober, C. Association studies for asthma and atopic diseases: a comprehensive review of the literature. Respir Res 4, 14 (2003). Ober, C. & Thompson, E. E. Rethinking genetic models of asthma: the role of environmental modifiers. Curr Opin Immunol 17, 670-8 (2005). Wardlaw, A. 1., Silverman, M., Siva, R., Pavord, I. D. & Green, R. Multi- dimensional phenotyping: towards a new taxonomy for airway disease. Clin Exp Allergy 35, 1254-62 (2005). Masoli, M., Fabian, D., Holt, S. & Beasley, R. The global burden of asthma: executive summary of the GINA Dissemination Committee report. Allergy 59, 469-78 (2004). Von Hertzen, L. C. & Haahtela, T. Asthma and atopy - the price of affluence? Allergy 59, 124-37 (2004). GINA. httgflwwwginasthma.com/Reportltem.asp?l1=2&l2=2&intld=94. Global burden of asthma (2004, accessed 23rd March 2005). NIHS. http://www.cdggov/nchs/products/pubs/pubd/hestats/asthma/asthma.htm. Asthma Prevalence, Health Care Use and Mortality, 2002 (2005, accessed 23rd March 2005). O'Connell, E. J. The burden of atopy and asthma in children. Allergy 59 Suppl 78, 7-11 (2004). Beasley, R. The burden of asthma with specific reference to the United States. J Allergy Clin Immunol 109, 8482-9 (2002). Expert Panel Report 2: Guidelines for the Diagnosis and Management of Asthma. National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD. NIH Publication No. 97-4053. (1997). 186 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Bel, E. H. Clinical phenotypes of asthma. Curr Opin Pulm Med 10, 44-50 (2004). Fuhlbrigge, A. L. Asthma severity and asthma control: symptoms, pulmonary function, and inflammatory markers. Curr Opin Pulm Med 10, 1-6 (2004). Ewart, S. L. et a]. Quantitative trait loci controlling allergen-induced airway hyperresponsiveness in inbred mice. Am J Respir Cell Mol Biol 23, 537-45 (2000). Hargreave, F. E. et a1. Bronchial responsiveness to histamine or methacholine in asthma: measurement and clinical significance. J Allergy Clin Immunol 68, 347- 55 (1981). Johnson, M. The beta-adrenoceptor. Am J Respir Crit Care Med 158, 8146-53 (1998). Moore, R. H., Khan, A. & Dickey, B. F. Long-acting inhaled beta2-agonists in asthma therapy. Chest 113, 1095-108 (1998). Schacke, H. et a1. Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proc Natl Acad Sci U S A 101, 227-32 (2004). Heck, S. et al. A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-l. Embo J 13, 4087-95 (1994) Adcock, I. M., Nasuhara, Y., Stevens, D. A. & Barnes, P. J. Ligand-induced differentiation of glucocorticoid receptor (GR) trans-repression and transactivation: preferential targetting of NF-kappaB and lack of I-kappaB involvement. Br J Pharmacol 127, 1003-11 (1999). Drazen, J. M., Israel, E. & O'Byrne, P. M. Treatment of asthma with drugs modifying the leukotriene pathway. N Engl J Med 340, 197-206 (1999). Chu, E. K. & Drazen, J. M. Asthma: one hundred years of treatment and onward. Am J Respir Crit Care Med 171, 1202-8 (2005). Corry, D. B. & Kheradmand, F. 7. Control of allergic airway inflammation through immunomodulation. J Allergy Clin Immunol 117, 8461-4 (2006). Barnes, P. J. New drugs for asthma. Nat Rev Drug Discov 3, 831-44 (2004). Burke, W., Fesinmeyer, M., Reed, K., Hampson, L. & Carlsten, C. Family history as a predictor of asthma risk. Am J Prev Med 24, 160-9 (2003). 187 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. Sandford, A., Weir, T. & Pare, P. The genetics of asthma. Am J Respir Crit Care Med 153, 1749-65 (1996). DiPetn'llo, K., Wang, X., Stylianou, I. M. & Paigen, B. Bioinformatics toolbox for narrowing rodent quantitative trait loci. Trends Genet 21, 683-92 (2005). Li, W. H. & Sadler, L. A. Low nucleotide diversity in man. Genetics 129, 513-23 (1991) Wang, D. G. et al. Large-scale identification, mapping, and genotyping of single- nucleotide polymorphisms in the human genome. Science 280, 1077-82 (1998). Cargill, M. et a1. Characterization of single-nucleotide polymorphisms in coding regions of human genes. Nat Genet 22, 231-8 (1999). Neale, B. M. & Sham, P. C. The future of association studies: gene-based analysis and replication. Am J Hum Genet 75, 353-62 (2004). Palmer, L. J. & Cookson, W. 0. Using single nucleotide polymorphisms as a means to understanding the pathophysiology of asthma. Respir Res 2, 102-12 (2001) Brookes, A. J. The essence of SNPs. Gene 234, 177-86 (1999). Chakravarti, A. Population genetics--making sense out of sequence. Nat Genet 21, 56—60 (1999). Botstein, D. & Risch, N. Discovering genotypes underlying human phenotypes: past successes for mendelian disease, future approaches for complex disease. Nat Genet 33 Suppl, 228-37 (2003). Wall, J. D. & Pritchard, J. K. Haplotype blocks and linkage disequilibrium in the human genome. Nat Rev Genet 4, 587-97 (2003). The International HapMap Project. Nature 426, 789-96 (2003). Altshuler, D. et al. A haplotype map of the human genome. Nature 437, 1299-3 20 (2005) Syvanen, A. C. Toward genome-wide SNP genotyping. Nat Genet 37 Suppl, SS- 10 (2005). Hirschhom, J. N. & Daly, M. J. Genome-wide association studies for common diseases and complex traits. Nat Rev Genet 6, 95-108 (2005). 188 43. 44. 45. 46. 47. 48.. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. Wang, W. Y., Barratt, B. J ., Clayton, D. G. & Todd, J. A. Genome-wide association studies: theoretical and practical concerns. Nat Rev Genet 6, 109-18 (2005) Colhoun, H. M., McKeigue, P. M. & Davey Smith, G. Problems of reporting genetic associations with complex outcomes. Lancet 361, 865-72 (2003). Ardlie, K. G., Kruglyak, L. & Seielstad, M. Patterns of linkage disequilibrium in the human genome. Nat Rev Genet 3, 299-309 (2002). Terwilliger, J. D. & Weiss, K. M. Linkage disequilibrium mapping of complex disease: fantasy or reality? Curr Opin Biotechnol 9, 578-94 (1998). Schork, N. J ., Cardon, L. R. & Xu, X. The future of genetic epidemiology. Trends Genet 14, 266-72 (1998). Cardon, L. R. & Bell, J. 1. Association study designs for complex diseases. Nat Rev Genet 2, 91-9 (2001). Pompanon, F ., Bonin, A., Bellemain, E. & Taberlet, P. Genotyping errors: causes, consequences and solutions. Nat Rev Genet 6, 847-59 (2005). Hoftjan, S. & Ober, C. Present status on the genetic studies of asthma. Curr Opin Immunol 14, 709-17 (2002). Cookson, W. O. & Moffatt, M. F. Genetics of asthma and allergic disease. Hum Mol Genet 9, 2359-64 (2000). Schuelke, M. et a1. Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 350, 2682-8 (2004). McPherron, A. C. & Lee, S. J. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci U S A 94, 12457-61 (1997). McPherron, A. C. & Lee, S. J. Suppression of body fat accumulation in myostatin-deficient mice. J Clin Invest 109, 595-601 (2002). von Mutius, E. Influences in allergy: epidemiology and the environment. J Allergy Clin Immunol 113, 373-9; quiz 380 (2004). Marks, G. B. Environmental factors and gene-environment interactions in the aetiology of asthma. Clin Exp Pharmacol Physiol 33, 285-9 (2006). Strachan, D. P. Hay fever, hygiene, and household size. ij 299, 1259-60 (1989). Von Ehrenstein, O. S. et a1. Reduced risk of hay fever and asthma among children of farmers. Clin Exp Allergy 30, 187-93 (2000). 189 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. Riedler, J ., Eder, W., Oberfeld, G. & Schreuer, M. Austrian children living on a farm have less hay fever, asthma and allergic sensitization. Clin Exp Allergy 30, 194-200 (2000). Braun-Fahrlander, C. et al. Prevalence of hay fever and allergic sensitization in farmer's children and their peers living in the same rural community. SCARPOL team. Swiss Study on Childhood Allergy and Respiratory Symptoms with Respect to Air Pollution. Clin Exp Allergy 29, 28-34 (1999). von Mutius, E. et al. Prevalence of asthma and atopy in two areas of West and East Germany. Am J Respir Crit Care Med 149, 358-64 (1994). Britton, W. J. et al. Prevalence of bronchial hyperresponsiveness in children: the relationship between asthma and skin reactivity to allergens in two communities. Int J Epidemiol 15, 202-9 (1986). Turner, S. W. et al. Determinants of airway responsiveness to histamine in children. Eur Respir J 25, 462-7 (2005). Kuehr, J. et al. Sensitization to mite allergens is a risk factor for early and late onset of asthma and for persistence of asthmatic signs in children. J Allergy Clin Immunol 95, 655-62 (1995). Perzanowski, M. S. et a1. Association of sensitization to Altemaria allergens with asthma among school-age children. J Allergy Clin Immunol 101, 626-32 (1998). Balmes, J. R. The role of ozone exposure in the epidemiology of asthma. Environ Health Perspect 101 Suppl 4, 219-24 (1993). White, M. C., Etzel, R. A., Wilcox, W. D. & Lloyd, C. Exacerbations of childhood asthma and ozone pollution in Atlanta. Environ Res 65, 56-68 (1994). Whittemore, A. S. & Korn, E. L. Asthma and air pollution in the Los Angeles area. Am J Public Health 70, 687-96 (1980). Pandya, R. J ., Solomon, G., Kinner, A. & Balmes, J. R. Diesel exhaust and asthma: hypotheses and molecular mechanisms of action. Environ Health Perspect 110 Suppl 1, 103-12 (2002). Takenaka, H., Zhang, K., Dial-Sanchez, D., Tsien, A. & Saxon, A. Enhanced human IgE production results from exposure to the aromatic hydrocarbons from diesel exhaust: direct effects on B-cell IgE production. J Allergy Clin Immunol 95, 103-15 (1995). 190 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. Riedl, M. & Diaz-Sanchez, D. Biology of diesel exhaust effects on respiratory function. J Allergy Clin Immunol 115, 221-8; quiz 229 (2005). Roger, L. J. et al. Pulmonary function, airway responsiveness, and respiratory symptoms in asthmatics following exercise in N02. T oxicol Ind Health 6, 155-71 (1990) Sheppard, D., Wong, W. S., Uehara, C. F., Nadel, J. A. & Boushey, H. A. Lower threshold and greater bronchomotor responsiveness of asthmatic subjects to sulfur dioxide. Am Rev Respir Dis 122, 873-8 (1980). Knox, R. B. Grass pollen, thunderstorms and asthma. Clin Exp Allergy 23, 354-9 (1993) Spieksma, F. T., Nikkels, B. H. & Dijkman, J. H. Seasonal appearance of grass pollen allergen in natural, pauci-micronic aerosol of various size fractions. Relationship with airborne grass pollen concentration. Clin Exp Allergy 25, 234-9 (1995) Davidson, A. C., Emberlin, J ., Cook, A. D. & Venables, K. M. A major outbreak of asthma associated with a thunderstorm: experience of accident and emergency departments and patients' characteristics. Thames Regions Accident and Emergency Trainees Association. ij 312, 601-4 (1996). D'Amato, G., Liccardi, G., D'Amato, M. & Holgate, S. Environmental risk factors and allergic bronchial asthma. Clin Exp Allergy 35, 1113-24 (2005). Friedman, N. J. & Zeiger, R. S. The role of breast-feeding in the development of allergies and asthma. J Allergy Clin Immunol 115, 1238-48 (2005). Gdalevich, M., Mimouni, D. & Mimouni, M. Breast-feeding and the risk of bronchial asthma in childhood: a systematic review with meta-analysis of prospective studies. J Pediatr 139, 261-6 (2001). Strachan, D. P., Butland, B. K. & Anderson, H. R. Incidence and prognosis of asthma and wheezing illness from early childhood to age 33 in a national British cohort. ij 312, 1195-9 (1996). J anson, C. et al. Effect of passive smoking on respiratory symptoms, bronchial responsiveness, lung function, and total serum IgE in the European Community Respiratory Health Survey: a cross-sectional study. Lancet 358, 2103-9 (2001). Strachan, D. P. & Cook, D. G. Health effects of passive smoking. 6. Parental smoking and childhood asthma: longitudinal and case-control studies. Thorax 53, 204-12 (1998). 191 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. Vercelli, D. Genetics, epigenetics, and the environment: switching, buffering, releasing. J Allergy Clin Immunol 113, 381-6; quiz 387 (2004). Bach, J. F. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med 347, 911-20 (2002). Bromham, L. & Penny, D. The modern molecular clock. Nat Rev Genet 4, 216-24 (2003) Crow, J. F. The origins, patterns and implications of human spontaneous mutation. Nat Rev Genet 1, 40-7 (2000). Reich, D. E. et al. Human genome sequence variation and the influence of gene history, mutation and recombination. Nat Genet 32, 135-42 (2002). Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33 Suppl, 245- 54 (2003). Mays-Hoopes, L., Chao, W., Butcher, H. C. & Huang, R. C. Decreased methylation of the major mouse long interspersed repeated DNA during aging and in myeloma cells. Dev Genet 7, 65-73 (1986). Wilson, V. L., Smith, R. A., Ma, S. & Cutler, R. G. Genomic 5- methyldeoxycytidine decreases with age. J Biol Chem 262, 9948-51 (1987). Michaud, E. J. et al. Differential expression of a new dominant agouti allele (Aiapy) is correlated with methylation state and is influenced by parental lineage. Genes Dev 8, 1463-72 (1994). Van den Veyver, I. B. Genetic effects of methylation diets. Annu Rev Nutr 22, 255-82 (2002). Jones, P. A. & Takai, D. The role of DNA methylation in mammalian epigenetics. Science 293, 1068-70 (2001). Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074-80 (2001 ). Barnes, P. J ., Adcock, I. M. & Ito, K. Histone acetylation and deacetylation: importance in inflammatory lung diseases. Eur Respir J25, 552-63 (2005). Cosio, B. G. et a1. Histone acetylase and deacetylase activity in alveolar macrophages and blood mononocytes in asthma. Am J Respir Crit Care Med 170, 141-7 (2004). 192 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. Barnes, P. J. Corticosteroid effects on cell signalling. Eur Respir J 27, 413-26 (2006) Shin, H. J. et al. STAT4 expression in human T cells is regulated by DNA methylation but not by promoter polymorphism. J Immunol 175, 7143-50 (2005). Santangelo, S., Cousins, D. J ., Winkelmann, N. E. & Staynov, D. Z. DNA methylation changes at human Th2 cytokine genes coincide with DNase I hypersensitive site formation during CD4(+) T cell differentiation. J Immunol 169, 1893-903 (2002). Howard, T. D. et al. Gene-gene interaction in asthma: IL4RA and IL1 3 in a Dutch population with asthma. Am J Hum Genet 70, 230-6 (2002). Chan, 1. H. et al. Gene-gene interactions for asthma and plasma total IgE concentration in Chinese children. J Allergy Clin Immunol 117, 127-33 (2006). Karp, C. L. et al. Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma. Nat Immunol 1, 221-6 (2000). Clark, B. D., Collins, K. L., Gandy, M. S., Webb, A. C. & Auron, P. E. Genomic sequence for human prointerleukin 1 beta: possible evolution from a reverse transcribed prointerleukin 1 alpha gene. Nucleic Acids Res 14, 7897-914 (1986). Arend, W. P., Malyak, M., Guthridge, C. J. & Gabay, C. Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol 16, 27-55 (1998). Arend, W. P. & Guthridge, C. J. Biological role of interleukin 1 receptor antagonist isoforms. Ann Rheum Dis 59 Suppl 1, i60-4 (2000). Butcher, C., Steinkasserer, A., Tejura, S. & Lennard, A. C. Comparison of two promoters controlling expression of secreted or intracellular IL-1 receptor antagonist. J Immunol 153, 701-11 (1994). Muzio, M. et al. Cloning and characterization of a new isoform of the interleukin 1 receptor antagonist. J Exp Med 182, 623-8 (1995). Malyak, M. et a1. Characterization of a low molecular weight isoform of IL-1 receptor antagonist. J Immunol 161, 1997-2003 (1998). Guo, C., Dower, S. K., Holowka, D. & Baird, B. Fluorescence resonance energy transfer reveals interleukin (IL)-1-dependent aggregation of IL-1 type I receptors that correlates with receptor activation. J Biol Chem 270, 27562-8 (1995). 193 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. Wesche, H., Neumann, D., Resch, K. & Martin, M. U. Co-expression of mRNA for type I and type II interleukin-l receptors and the IL-1 receptor accessory protein correlates to IL-1 responsiveness. FEBS Lett 391, 104-8 (1996). Wesche, H. et al. The interleukin-1 receptor accessory protein (IL-lRAcP) is essential for IL-1 -induced activation of interleukin-1 receptor-associated kinase (IRAK) and stress-activated protein kinases (SAP kinases). J Biol Chem 272, 7727-31 (1997). Vigers, G. R, Anderson, L. J ., Caffes, P. & Brandhuber, B. J. Crystal structure of the type-I interleukin-1 receptor complexed with interleukin-lbeta. Nature 386, 190-4 (1997). Schreuder, H. et al. A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist. Nature 386, 194-200 (1997). Dunne, A. & O'Neill, L. A. The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci STKE 2003, re3 (2003). Colotta, F. et al. Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science 261, 472-5 (1993). Lang, D. et al. The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness. J Immunol 161, 6871-7 (1998). Symons, J. A., Young, P. R. & Duff, G. W. Soluble type II interleukin 1 (IL-1) receptor binds and blocks processing of lL-l beta precursor and loses affinity for IL-1 receptor antagonist. Proc Natl Acad Sci U S A 92, 1714-8 (1995). Lazaar, A. L. & Panettieri, R. A., Jr. Airway smooth muscle: a modulator of airway remodeling in asthma. J Allergy Clin Immunol 1 16, 488-95; quiz 496 (2005) Braddock, M. & Quinn, A. Targeting IL-1 in inflammatory disease: new opportunities for therapeutic intervention. Nat Rev Drug Discov 3, 330-9 (2004). Braddock, M., Quinn, A. & Canvin, J. Therapeutic potential of targeting IL-1 and IL-18 in inflammation. Expert Opin Biol T her 4, 847-60 (2004). Nakae, S. et al. IL-1 is required for allergen-specific Th2 cell activation and the development of airway hypersensitivity response. Int Immunol 15, 483-90 (2003). Selig, W. & Tocker, J. Effect of interleukin-1 receptor antagonist on antigen- induced pulmonary responses in guinea pigs. Eur J Pharmacol 213, 331-6 (1992) 194 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. Okada, S. et al. Potential role of interleukin-1 in allergen-induced late asthmatic reactions in guinea pigs: suppressive effect of interleukin-1 receptor antagonist on late asthmatic reaction. J Allergy Clin Immunol 95, 1236-45 (1995). Johnson, V. J ., Yucesoy, B. & Luster, M. 1. Prevention of IL-1 signaling attenuates airway hyperresponsiveness and inflammation in a murine model of toluene diisocyanate-induced asthma. J Allergy Clin Immunol 116, 851-8 (2005). Park, J. W. et al. Interleukin-1 receptor antagonist attenuates airway hyperresponsiveness following exposure to ozone. Am J Respir Cell Mol Biol 30, 830-6 (2004). Borish, L. et al. Detection of alveolar macrophage-derived IL-1 beta in asthma. Inhibition with corticosteroids. J Immunol 149, 3078-82 (1992). ' Sousa, A. R., Lane, S. J ., Nakhosteen, J. A., Lee, T. H. & Poston, R. N. Expression of interleukin-1 beta (IL-1beta) and interleukin-1 receptor antagonist (IL-Ira) on asthmatic bronchial epithelium. Am J Respir Crit Care Med 154, 1061-6 (1996). Mao, X. Q. et a1. Imbalance production between interleukin-1beta(IL-1beta) and IL-1 receptor antagonist (IL-lRa) in bronchial asthma. Biochem Biophys Res Commun 276, 607-12 (2000). Hultner, L. et al. In activated mast cells, IL-l up-regulates the production of several Th2-related cytokines including IL-9.J1mmunol 164, 5556-63 (2000). Burd, P. R., Thompson, W. C., Max, E. E. & Mills, F. C. Activated mast cells produce interleukin 13. J Exp Med 181, 1373-80 (1995). Wills-Karp, M. et al. Interleukin-13: central mediator of allergic asthma. Science 282, 2258-61 (1998). ' Grunig, G. et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282, 2261-3 (1998). Lee, S. A. et al. Molecular regulation of interleukin-13 and monocyte chemoattractant protein-1 expression in human mast cells by interleukin-lbeta. Am J Respir Cell Mol Biol 31, 283-91 (2004). Li, Q. & Verma, I. M. NF-kappaB regulation in the immune system. Nat Rev Immunol 2, 725-34 (2002). Das, J. et al. A critical role for NF-kappa B in GATA3 expression and TH2 differentiation in allergic airway inflammation. Nat Immunol 2, 45-50 (2001). 195 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. Ferber, I. A. et al. GATA-3 significantly downregulates IFN-gamma production from developing Th1 cells in addition to inducing IL-4 and IL-5 levels. Clin Immunol 91, 134-44 (1999). Zhang, D. H., Yang, L. & Ray, A. Differential responsiveness of the IL-5 and IL- 4 genes to transcription factor GATA-3. J Immunol 161, 3817-21 (1998). Yamashita, M. et al. Identification of a conserved GATA3 response element upstream proximal from the interleukin-13 gene locus. J Biol Chem 277, 42399- 408 (2002). Zhang, D. H. et al. Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3. Immunity 11, 473-82 (1999). Bleecker, E. R. Similarities and differences in asthma and COPD. The Dutch hypothesis. Chest 126, 93S-95S; discussion 1598-1618 (2004). Howarth, P. H. et al. Synthetic responses in airway smooth muscle. J Allergy Clin Immunol 114, 832-50 (2004). Shore, S. A., Laporte, J ., Hall, I. P., Hardy, E. & Panettieri, R. A., Jr. Effect of IL- 1 beta on responses of cultured human airway smooth muscle cells to bronchodilator agonists. Am J Respir Cell Mol Biol 16, 702-12 (1997). Lappalainen, U., Whitsett, J. A., Wert, S. E., Tichelaar, J. W. & Bry, K. Interleukin-lbeta causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung. Am J Respir Cell Mol Biol 32, 311-8 (2005). Zhai, W., Eynott, P. R., Oltmanns, U., Leung, S. Y. & Chung, K. F. Mitogen- activated protein kinase signalling pathways in IL-1 beta-dependent rat airway smooth muscle proliferation. Br J Pharmacol 143, 1042-9 (2004). Hakonarson, H., Maskeri, N., Carter, C., Chuang, S. & Grunstein, M. M. Autocrine interaction between IL-5 and IL-lbeta mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle. J Clin Invest 104, 657-67 (1999) Whelan, R. et al. Role and regulation of interleukin-1 molecules in pro-asthmatic sensitised airway smooth muscle. Eur Respir J 24, 559-67 (2004). Gohlke, H. et a1. Association of the interleukin-1 receptor antagonist gene with asthma. Am J Respir Crit Care Med 169, 1217-23 (2004). 196 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. Pattaro, C., Heinrich, J ., Werner, M., de Marco, R. & stt, M. Association between interleukin-1 receptor antagonist gene and asthma-related traits in a German adult population. Allergy 61, 239-44 (2006). Tarlow, J. K. et al. Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat. Hum Genet 91, 403-4 (1993) Pessi, T., Karjalainen, J ., Hulkkonen, J ., Nieminen, M. M. & Hurme, M. A common IL-l complex haplotype is associated with an increased risk of atopy. J Med Genet 40, e66 (2003). Karjalainen, J. et al. The ILlA genotype is associated with nasal polyposis in asthmatic adults. Allergy 58, 393-6 (2003). Lander, E. S. & Schork, N. J. Genetic dissection of complex traits. Science 265, 2037-48 (1994). Torres, R., Picado, C. & de Mora, F. [Use of the mouse to unravel allergic asthma: a review of the pathogenesis of allergic asthma in mouse models and its similarity to the condition in humans]. Arch Bronconeumol 41, 141-52 (2005). Silver, L. M. Mouse Genetics: Concepts and Applications (Oxford University Press, New York, 1995). Wang, J. et al. Circulating, but not local lung, IL-5 is required for the development of antigen-induced airways eosinophilia. J Clin Invest 102, 1132-41 (1998) Finotto, S. et al. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 295, 336-8 (2002). Hellings, P. W. & Ceuppens, J. L. Mouse models of global airway allergy: what have we learned and what should we do next? Allergy 59, 914-9 (2004). Kips, J. C. et al. Murine models of asthma. Eur Respir J 22, 374-82 (2003). Ewart, S. L., Mitzner, W., DiSilvestre, D. A., Meyers, D. A. & Levitt, R. C. Airway hyperresponsiveness to acetylcholine: segregation analysis and evidence for linkage to murine chromosome 6. Am J Respir Cell Mol Biol 14, 487-95 (1996) Ewart, S., Levitt, R. & Mitzner, W. Respiratory system mechanics in mice measured by end-inflation occlusion. J Appl Physiol 79, 560-6 (1995). 197 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. Jenkins, J. K. et al. Intracellular IL-1 receptor antagonist promoter: cell type- specific and inducible regulatory regions. J Immunol 158, 748-55 (1997). Smith, M. F., Jr. et al. Human IL-1 receptor antagonist promoter. Cell type- specific activity and identification of regulatory regions. J Immunol 149, 2000-7 (1992) La, E. & Fischer, S. M. Transcriptional regulation of intracellular IL-1 receptor antagonist gene by IL-1 alpha in primary mouse keratinocytes. J Immunol 166, 6149-55 (2001). Wang, D. G. et al. Large-scale identification, mapping, and genotyping of single- nucleotide polymorphisms in the human genome. Science 280, 1077-82 (1998). Wade, C. M. et al. The mosaic structure of variation in the laboratory mouse genome. Nature 420, 574-8 (2002). Nicklin, M. J. et al. A sequence-based map of the nine genes of the human interleukin-1 cluster. Genomics 79, 718-25 (2002). Wong, M. L. & Medrano, J. F. Real-time PCR for mRNA quantitation. Biotechniques 39, 75-85 (2005). AppliedBiosystems. in User bulletin # 2, AB] 7700 Sequence Detection System (www.appliedbiosystemscom, 2001 ). Bilbao, D. & Valcarcel, J. Getting to the heart of a splicing enhancer. Nat Struct Biol 10, 6-7 (2003). Hui, J ., Stangl, K., Lane, W. S. & Bindereif, A. HnRNP L stimulates splicing of the eNOS gene by binding to variable-length CA repeats. Nat Struct Biol 10, 33-7 (2003) Hui, J ., Reither, G. & Bindereif, A. Novel functional role of CA repeats and hnRNP L in RNA stability. Rna 9, 931-6 (2003). Abiola, O. et al. The nature and identification of quantitative trait loci: a community's view. Nat Rev Genet 4, 911-6 (2003). Allen, M. et al. Positional cloning of a novel gene influencing asthma from chromosome 2q14. Nat Genet 35, 258-63 (2003). Pattaro, C., Heinrich, J ., Werner, M., Marco, R. & stt, M. Association between interleukin-1 receptor antagonist gene and asthma-related traits in a German adult population. Allergy 61, 239-44 (2006). 198 175. 176. 177. 178.. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. Martinez, F. D. Links between pediatric and adult asthma. J Allergy Clin Immunol 107, 8449-55 (2001). Vonk, J. M. & Boezen, H. M. Predicting adult asthma in childhood. Curr Opin Pulm Med 12, 42-7 (2006). Taussig, L. M. et al. Tucson Children's Respiratory Study: 1980 to present. J Allergy Clin Immunol 111, 661-75; quiz 676 (2003). Lau, S. et al. The development of childhood asthma: lessons from the German Multicentre Allergy Study (MAS). Paediatr Respir Rev 3, 265-72 (2002). Oddy, W. H., de Klerk, N. H., Sly, P. D. & Holt, P. G. The effects of respiratory infections, atopy, and breastfeeding on childhood asthma. Eur Respir J 19, 899- 905 (2002). Oddy, W. H., Peat, J. K. & de Klerk, N. H. Maternal asthma, infant feeding, and the risk of asthma in childhood. J Allergy Clin Immunol 110, 65-7 (2002). Sears, M. R. et al. A longitudinal, population-based, cohort study of childhood asthma followed to adulthood. N Engl J Med 349, 1414-22 (2003). Arshad, S. H., Tariq, S. M., Matthews, S. & Hakim, E. Sensitization to common allergens and its association with allergic disorders at age 4 years: a whole population birth cohort study. Pediatrics 108, E33 (2001). Kurukulaaratchy, R. J ., Fenn, M., Twiselton, R., Matthews, S. & Arshad, S. H. The prevalence of asthma and wheezing illnesses amongst 10-year-old schoolchildren. Respir Med 96, 163-9 (2002). Ronaghi, M. Pyrosequencing sheds light on DNA sequencing. Genome Res 11, 3- 11(2001) Utting, M., Hampe, J ., Platzer, M. & Huse, K. Locking of 3' ends of single- stranded DNA templates for improved Pyrosequencing performance. Biotechniques 37, 66-7, 70-3 (2004). Alderbom, A., Kristofferson, A. & Hammerling, U. Determination of single- nucleotide polymorphisms by real-time pyrophosphate DNA sequencing. Genome Res 10, 1249-58 (2000). Gabriel, S. B. et al. The structure of haplotype blocks in the human genome. Science 296, 2225-9 (2002). Benj amini, Y. & Hochberg, M. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Royal Stat Soc B 57, 289-300 (1995). 199 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. Pritchard, J. K. & Przeworski, M. Linkage disequilibrium in humans: models and data. Am J Hum Genet 69, 1-14 (2001). Reich, D. E. et al. Linkage disequilibrium in the human genome. Nature 411, 199- 204 (2001). F risse, L. et al. Gene conversion and different population histories may explain the contrast between polymorphism and linkage disequilibrium levels. Am J Hum Genet 69, 831-43 (2001). Tishkoff, S. A. et al. Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of alleles that confer malarial resistance. Science 293, 455-62 (2001) Nordborg, M. & Tavare, S. Linkage disequilibrium: what history has to tell us. Trends Genet 18, 83-90 (2002). Zeggini, E. et al. An evaluation of HapMap sample size and tagging SNP performance in large-scale empirical and simulated data sets. Nat Genet 37 , 1320- 2 (2005). Nothnagel, M. & Rohde, K. The effect of single-nucleotide polymorphism marker selection on patterns of haplotype blocks and haplotype frequency estimates. Am J Hum Genet 77, 988-98 (2005). Gu, H. F. et al. Quantitative trait loci near the insulin-degrading enzyme (IDE) gene contribute to variation in plasma insulin levels. Diabetes 53, 2137-42 (2004). Oguma, T. et al. Role of prostanoid DP receptor variants in susceptibility to asthma. N Engl J Med 351, 1752-63 (2004). Zeger, S. L. & Liang, K. Y. Longitudinal data analysis for discrete and continuous outcomes. Biometrics 42, 121-30 (1986). Haskill, S. et al. cDNA cloning of an intracellular form of the human interleukin 1 receptor antagonist associated with epithelium. Proc Natl Acad Sci U S A 88, 3681-5 (1991). Wilmott, R. W., Kitzmiller, J. A., Fiedler, M. A. & Stark, J. M. Generation of a transgenic mouse with lung-specific overexpression of the human interleukin-1 receptor antagonist protein. Am J Respir Cell Mol Biol 18, 429-34 (1998). Tillie-Leblond, I. et al. Balance between proinflammatory cytokines and their inhibitors in bronchial lavage from patients with status asthmaticus. Am J Respir Crit Care Med 159, 487-94 (1999). 200 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. Fischer, E. et al. Interleukin-1 receptor antagonist circulates in experimental inflammation and in human disease. Blood 79, 2196-200 (1992). Smith, D. E. et a1. Four new members expand the interleukin-1 superfamily. J Biol Chem 275, 1169-75 (2000). Debets, R. et al. Two novel IL-I family members, IL-l delta and IL-1 epsilon, function as an antagonist and agonist of NF -kappa B activation through the orphan IL-l receptor-related protein 2. J Immunol 167, 1440-6 (2001). Towne, J. E., Garka, K. E., Renshaw, B. R., Virca, G. D. & Sims, J. E. Interleukin (IL)-1F6, IL-1F8, and IL-1F9 signal through IL-erp2 and IL-lRAcP to activate the pathway leading to NF -kappaB and MAPKs. J Biol Chem 279, 13677-88 (2004) Arshad, S. H. & Hide, D. W. Effect of environmental factors on the development of allergic disorders in infancy. J Allergy Clin Immunol 90, 235-41 (1992). Arshad, S. H., Stevens, M. & Hide, D. W. The effect of genetic and environmental factors on the prevalence of allergic disorders at the age of two years. Clin Exp Allergy 23, 504-11 (1993). Stein, R. T. et al. Influence of parental smoking on respiratory symptoms during the first decade of life: the Tucson Children's Respiratory Study. Am J Epidemiol 149, 1030-7 (1999). F loreani, A. A. & Rennard, S. I. The role of cigarette smoke in the pathogenesis of asthma and as a trigger for acute symptoms. Curr Opin Pulm Med 5, 38-46 (1999) Jaakkola, J. J. & Gissler, M. Maternal smoking in pregnancy, fetal development, and childhood asthma. Am J Public Health 94, 136-40 (2004). Weiss, S. T., Tager, I. B., Munoz, A. & Speizer, F. E. The relationship of respiratory infections in early childhood to the occurrence of increased levels of bronchial responsiveness and atopy. Am Rev Respir Dis 131, 573-8 (1985). Ehrlich, R. I. et al. Risk factors for childhood asthma and wheezing. Importance of maternal and household smoking. Am J Respir Crit Care Med 154, 681-8 (1996) Gilliland, F. D., Li, Y. F. & Peters, J. M. Effects of maternal smoking during pregnancy and environmental tobacco smoke on asthma and wheezing in children. Am J Respir Crit Care Med 163, 429-36 (2001). 201 214. 215. 216. 217. 218. 219. 220. 221. Chan-Young, M. & Dimich-Ward, H. Respiratory health effects of exposure to environmental tobacco smoke. Respirologz 8, 131-9 (2003). Dunne, A., Ejdeback, M., Ludidi, P. L., O'Neill, L. A. & Gay, N. J. Structural complementarity of Toll/interleukin-l receptor domains in Toll-like receptors and the adaptors Mal and MyD88. J Biol Chem 278, 41443-51 (2003). Ewart, S. L. & Ramadas, R. A. in The Mouse in Animal Genetics and Breeding Research (ed. Eisen, E. J.) 205-236 (Imperial College Press, London, 2005). Arshad, S. H., Kurukulaaratchy, R. J ., Fenn, M. & Matthews, S. Early life risk factors for current wheeze, asthma, and bronchial hyperresponsiveness at 10 years of age. Chest 127, 502-8 (2005). Kurukulaaratchy, R. J ., Matthews, 8., Holgate, S. T. & Arshad, S. H. Predicting persistent disease among children who wheeze during early life. Eur Respir J 22, 767-71 (2003). Martin, A. J ., McLennan, L. A., Landau, L. I. & Phelan, P. D. The natural history of childhood asthma to adult life. Br Med J 280, 1397-1400 (1980). Martinez, F. D., Morgan, W. J ., Wright, A. L., Holberg, C. J. & Taussig, L. M. Diminished lung function as a predisposing factor for wheezing respiratory illness in infants. N Engl J Med 319, 1112-7 (1988). Young, S., Amott, J ., O'Keeffe, P. T., Le Souef, P. N. & Landau, L. I. The association between early life lung function and wheezing during the first 2 yrs of life. Eur Respir J 15, 151-7 (2000). 202 111111111311111