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Intravenous Augmentation Therapy Does Not Decrease Airway Epithelial Cell Inflammation in Individuals with Alpha-One Ant...

Permanent Link: http://ufdc.ufl.edu/UFE0022787/00001

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Title: Intravenous Augmentation Therapy Does Not Decrease Airway Epithelial Cell Inflammation in Individuals with Alpha-One Antitrypsin Deficiency
Physical Description: 1 online resource (28 p.)
Language: english
Creator: Olson, Eric
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antitrypsin, deficiency
Clinical Investigation (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Alpha-1-Antitrypsin (AAT) deficiency is an autosomal recessive genetic disease associated with the development of early onset emphysema, bronchiectasis and chronic bronchitis. Homozygous individuals have deficient production of alpha-1 antitrypsin, an anti-protease protein with known additional anti-inflammatory properties. Individuals with two severe deficiency alleles maintain a markedly increased genetic risk for developing progressive emphysema. However, a significant variability in the development of lung disease is consistently observed. Exposure to environmental toxicities such as cigarette smoke clearly contributes to an accelerated decline in lung function in deficient patients. Other factors such as asthma, history of previous pneumonias or other childhood respiratory illnesses, chronic bronchitis and sex have all been associated with a more rapid development of severe lung disease. The effect of these factors suggests that both acute and chronic airway inflammation contributes to the development of airflow obstruction and progressive emphysema. Intravenous (IV) augmentation therapy with human blood-derived AAT has been demonstrated to restore blood and epithelial lining fluid concentrations of AAT to protective levels and to decrease inflammatory markers in both expectorated sputum and in bronchoalveolar lavage fluid, presumably by limiting neutrophil chemoattraction. The effect of IV augmentation therapy on limiting airway epithelial cell inflammation remains undetermined. Therefore, this study was designed to investigate the potential reduction in bronchial airway epithelial cell inflammation following IV augmentation therapy. This study was a prospective, observational pilot study in 11 AAT deficient individuals to evaluate the effect of IV AAT augmentation therapy on epithelial cell inflammation as measured by epithelial cell gene expression of the inflammatory markers TNF-?, IL-8, IL-15, HSP-1 and OSM-receptor. BIP and IRE-1, genes associated with the unfolded protein response, were also evaluated, as was AAT gene expression. After 12 weeks of therapy, no significant changes in gene expression were noted for any of the selected genes. AAT gene expression was demonstrated in the airway epithelial cells and also remained constant after 12 weeks of therapy. The results of this pilot study suggest that AAT IV augmentation therapy does not significantly affect airway epithelial cell inflammation and that additional therapeutic strategies are warranted in order to reduce the progression of airways disease in AAT deficient individuals.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Eric Olson.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Brantly, Mark L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022787:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022787/00001

Material Information

Title: Intravenous Augmentation Therapy Does Not Decrease Airway Epithelial Cell Inflammation in Individuals with Alpha-One Antitrypsin Deficiency
Physical Description: 1 online resource (28 p.)
Language: english
Creator: Olson, Eric
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antitrypsin, deficiency
Clinical Investigation (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Alpha-1-Antitrypsin (AAT) deficiency is an autosomal recessive genetic disease associated with the development of early onset emphysema, bronchiectasis and chronic bronchitis. Homozygous individuals have deficient production of alpha-1 antitrypsin, an anti-protease protein with known additional anti-inflammatory properties. Individuals with two severe deficiency alleles maintain a markedly increased genetic risk for developing progressive emphysema. However, a significant variability in the development of lung disease is consistently observed. Exposure to environmental toxicities such as cigarette smoke clearly contributes to an accelerated decline in lung function in deficient patients. Other factors such as asthma, history of previous pneumonias or other childhood respiratory illnesses, chronic bronchitis and sex have all been associated with a more rapid development of severe lung disease. The effect of these factors suggests that both acute and chronic airway inflammation contributes to the development of airflow obstruction and progressive emphysema. Intravenous (IV) augmentation therapy with human blood-derived AAT has been demonstrated to restore blood and epithelial lining fluid concentrations of AAT to protective levels and to decrease inflammatory markers in both expectorated sputum and in bronchoalveolar lavage fluid, presumably by limiting neutrophil chemoattraction. The effect of IV augmentation therapy on limiting airway epithelial cell inflammation remains undetermined. Therefore, this study was designed to investigate the potential reduction in bronchial airway epithelial cell inflammation following IV augmentation therapy. This study was a prospective, observational pilot study in 11 AAT deficient individuals to evaluate the effect of IV AAT augmentation therapy on epithelial cell inflammation as measured by epithelial cell gene expression of the inflammatory markers TNF-?, IL-8, IL-15, HSP-1 and OSM-receptor. BIP and IRE-1, genes associated with the unfolded protein response, were also evaluated, as was AAT gene expression. After 12 weeks of therapy, no significant changes in gene expression were noted for any of the selected genes. AAT gene expression was demonstrated in the airway epithelial cells and also remained constant after 12 weeks of therapy. The results of this pilot study suggest that AAT IV augmentation therapy does not significantly affect airway epithelial cell inflammation and that additional therapeutic strategies are warranted in order to reduce the progression of airways disease in AAT deficient individuals.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Eric Olson.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Brantly, Mark L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022787:00001


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1 INTRAVENOUS AUGMENTATION THER APY DOES NOT DECREASE AIRWAY EPITHELIAL CELL INFLAMMATION IN INDIVIDUALS WITH ALPHA-ONE ANTITRYPSIN DEFICIENCY By ERIC OLSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Eric Olson

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3 To Sam: You are the reason Im doing what Im doing.

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4 ACKNOWLEDGMENTS I thank the members of my supervisory committee for their mentoring, support, and guidance throughout this project. I acknowle dge the Alpha-1 Foundation/CHEST Foundation Clinical Research Award in Alpha-1 Antitrypsin (AAT) Deficiency and Kamada, Ltd. for the financial support of this pr oject and I deeply thank the participants in this research study. Most importantly, I thank my wife Jenny for her unwav ering love and support; without her, none of this would have been possible.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........6 LIST OF FIGURES................................................................................................................ .........7 ABSTRACT....................................................................................................................... ..............8 CHAPTER 1 INTRODUCTION..................................................................................................................10 Alpha-1-Antitrypsin Deficiency.............................................................................................10 AAT and Airway Inflammation..............................................................................................10 2 MATERIALS AND METHODS...........................................................................................12 Study Design................................................................................................................... ........12 Study Overview...............................................................................................................12 Subjects....................................................................................................................... .....12 Baseline Procedures.........................................................................................................13 Study Procedures.............................................................................................................13 Bronchoscopy..................................................................................................................14 Quantification of Gene Expression.................................................................................14 Sample Size.................................................................................................................... .16 Statistical Analysis........................................................................................................... .......16 3 RESULTS........................................................................................................................ .......17 Treatment...................................................................................................................... ..........17 Gene Expression Analysis......................................................................................................18 Adverse Outcomes............................................................................................................... ...18 4 DISCUSSION AND CONCLUSION....................................................................................22 Discussion..................................................................................................................... ..........22 Conclusion..................................................................................................................... .........24 REFERENCES..................................................................................................................... .........26 BIOGRAPHICAL SKETCH.........................................................................................................28

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6 LIST OF TABLES Table page 3-1 Patient characteristics................................................................................................... ..........18 3-2 Quantitative PCR relative gene expression units at baseline and af ter 12 weeks of AAT augmentation therapy.........................................................................................................19

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7 LIST OF FIGURES Figure page 3-1 AAT quantitative PCR relative gene expression..............................................................19 3-2 HSP-A1A quantitative PCR relative gene expression.......................................................19 3-3 TNFquantitative PCR relative gene expression............................................................20 3-4 IRE-1 quantitative PCR relative gene expression..............................................................20 3-5 BiP quantitative PCR relative gene expression.................................................................20 3-6 IL-8 quantitative PCR relative gene expression................................................................21 3-7 IL-15 quantitative PCR relative gene expression..............................................................21 3-8 OSM-receptor quantitative PCR relative gene expression................................................21

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INTRAVENOUS AUGMENTATION THER APY DOES NOT DECREASE AIRWAY EPITHELIAL CELL INFLAMMATION IN INDIVIDUALS WITH ALPHA-ONE ANTITRYPSIN DEFICIENCY By Eric Olson December 2008 Chair: Mark Brantly Major: Medical Sciences Clinical Investigation Alpha-1-Antitrypsin (AAT) deficiency is an autosomal recessive genetic disease associated with the development of early onset emphysema, bronchiectasis and chronic bronchitis. Homozygous i ndividuals have deficient production of alpha-1 antitrypsin, an antiprotease protein with known additional anti-inf lammatory properties. Individuals with two severe deficiency alleles maintain a markedly increased genetic risk fo r developing progressive emphysema. However, a significant variability in the development of lung disease is consistently observed. Exposure to environmenta l toxicities such as cigarette smoke clearly contributes to an accelerated declin e in lung function in deficient pa tients. Other factors such as asthma, history of previous pneumonias or other childhood respirator y illnesses, chronic bronchitis and sex have all been associated with a more rapi d development of severe lung disease. The effect of these factors suggests that both acute and chronic airway inflammation contributes to the development of airflow obstruction and progre ssive emphysema. Intravenous (IV) augmentation therapy with human blood-deri ved AAT has been demonstrated to restore blood and epithelial lining fluid concentrations of AAT to protective le vels and to decrease inflammatory markers in both expectorated sp utum and in bronchoalveolar lavage fluid,

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9 presumably by limiting neutrophil chemoattraction. The effect of IV augmentation therapy on limiting airway epithelial cell in flammation remains undetermined. Therefore, this study was designed to investigate the poten tial reduction in bronc hial airway epithelial cell inflammation following IV augmentation therapy. This study was a prospective, obs ervational pilot study in 11 AAT deficient individuals to evaluate the effect of IV AAT augmentation therapy on epithelial cell inflammation as measured by epithelial cell gene expression of the inflammatory markers TNF, IL-8, IL-15, HSP-1 and OSM-receptor. BIP and IRE-1, genes associated with the unfolded protein response, were also eval uated, as was AAT gene expression. After 12 weeks of therapy, no significant ch anges in gene expression were noted for any of the selected genes. AAT gene expression was demonstrated in the airway epith elial cells and also remained constant after 12 weeks of therapy. The resu lts of this pilot study suggest that AAT IV augmentation therapy does not significantly aff ect airway epithelial cell inflammation and that additional therapeutic strategies are warranted in order to reduce the progression of airways disease in AAT deficient individuals.

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10 CHAPTER 1 INTRODUCTION Alpha-1-Antitrypsin Deficiency Alpha-1-Antitrypsin (AAT) deficiency is an in herited disorder characterized by low serum concentrations of the AAT protein and is associated with the early onset of severe lung disease. AAT deficiency, the most common inherited cause of COPD, can lead to the development of severe emphysema even with minimal exposure to tobacco smoke. Individuals with the most common AAT deficiency phenotype, Pi ZZ, have an 18-fold greater risk of developing severe emphysema compared to individual s with adequate AAT levels. AAT deficiency occurs with a prevalence rate of 1 in 1,600 to 1 in 4,000. Up to 10% of individuals with COPD have at least one copy of the Z gene mutation. Currently, no cure exists for AAT deficiency [1]. AAT deficiency is associated with the premature onset of progressive COPD, characterized by basilar emphysema, chronic bronchitis and bronchi ectasis, especially in smokers [2]. AAT is a serum acute-phase glycoprotein that inhibits several types of proteolytic enzymes, notably neutrophil elastase (NE). The increased activity of NE in AAT deficient individuals has been long believed to be the primary cause of lung damage. However, a growing body of clinical evidence shows that the absence of sufficient AAT in the lower respiratory tract is associated with an increased burden of pro-inflammatory factors such as TNF-al pha, IL-6, IL-8 and Creactive protein (CRP) and that AAT may have broa d anti-inflammatory prope rties [3] [4] [5]. AAT and Airway Inflammation Asthma-like symptoms and airway hyperresponsiveness are common and important findings in many patients with AAT deficiency. One la rge cohort study revealed 82% of AAT deficient patients reported wheezing at th eir well baseline and almost 90% reported wheezing with cold symptoms [6]. Almost 50% of the cohort had a significant response to bronchodilator therapy.

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11 Longitudinal study in patients with AAT deficiency demonstrates th at the most rapid decline in airway function, measured by the forced expiratory volume in one second (FEV1), occurs in the third and fourth decades of life, which is also when the onset of self -reported wheezing occurs [7]. Additionally, the diagnosis of asthma and significant airway hyper-responsiveness have both been associated with a more rapid decline in lung function in AAT deficiency [7, 8]. These results suggest that the absence of sufficient AAT in the respiratory tract is associated with increased airway inflammation. Increased ai rway inflammation likel y results in airway remodeling, worsened airflow obstructi on, and accelerated decline in FEV1. Airway disease, including bronchiectasis and changes in the ai rway walls, has been identified in subjects with AAT deficien cy. Approximately 40% of patie nts with AAT deficiency have chronic cough and sputum expectoration [9]. Clinically significant bronchiectasis (radiologic bronchiectasis in 4 or more bronchopulmonary se gments together with symptoms of regular sputum production) has been demonstrated in 27 % of patients with AAT deficiency [10]. These findings demonstrate that while emphysema is the predominant component of AAT deficiency related lung injury, airway inflammation has an import impact on disease progression. To date, studies evaluating airway inflammation in AAT de ficiency have utilized either sputum or bronchoalveolar lavage (BAL) flui d, neither of which specifically targets the inflammatory changes in bronchial airway epithelial cells. An improved understanding of the airway epithelial cell inflammatory response to AAT deficien cy and augmentation therapy has important implications for patient outcomes and also fo r drug development, specifically aerosolized therapeutics, and is therefore a crucial area of investigation.

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12 CHAPTER 2 MATERIALS AND METHODS Study Design This study was a prospective, observationa l pilot study approved by the Institutional Review Boards at the University of Florid a and the University of Texas at Tyler. The specific aim of this pilot study was to determine the effect of intravenous AAT augmentation therapy on the gene expression of in flammatory markers in airway epithelial cells. Markers of inflammation tested were TNF, IL-8, IL-15, HSP-1 and OSM-receptor. BIP and IRE-1 were tested to evaluate markers of the unfolded protein response. Additionally, AAT gene expression was determined in all samples. Th e primary hypothesis tested was that intravenous AAT augmentation therapy would decrease bronc hial airway epithe lial cell inflammation. Study Overview After providing voluntary informed consent, subjects receiving exogenous AAT therapy underwent a 5 week washout period. Prior to receiving study related AAT augmentation therapy, all subjects underwent baseline bronchoscopy with br onchial brushings to obtain bronchial epithelial cells. Subjects then receiv ed functional human AAT (either Kamada API or Prolastin) via IV drip at a dos e of 60mg/kg body weight and weekly for 12 weeks. Subjects then were evaluated with a second bronchoscopy and bronc hial brushing to inves tigate the effect of AAT augmentation therapy on bronchial airway epithelial cell inflammation. Subjects Inclusion criteria included age 18 years, presence of at-risk alleles associated with serum AAT levels 11 M, and evidence of AAT deficiency related lung disease identified by either FEV1 < 80% predicted post bronchodilator, loss of lung function over a one year period of greater than 35mL in FEV1 or high resolution CT (HRCT) evidence of pulmonary emphysema.

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13 Subjects who were actively treated with augmentati on therapy prior to enrollment had to agree to not receive any exogenous AAT product (i.e. washout period) for five weeks prior to the first study infusion. All subjects had to be on a stable dose of inhaled cortic osteroids for two weeks prior to first bronchoscopy and th rough the remainder of the study. Exclusion criteria involved safety and effi cacy parameters for delivering intravenous AAT therapy and for undergoing bronchoscopy. Cu rrent or recent (within the past 3 months) tobacco use, known allergy to plasma proteins uncontrolled hypertension and/or tachycardia excluded participation. Factor s potentially affect ing immune function including laboratory evidence of severe immunoglobulin A deficiency, pregnancy or lactation, current malignancy, previous organ transplantation, hi story of infection with HCV, HB V, and or HIV, and any acute respiratory tract infections within the prio r 6 weeks requiring antib iotics or systemic corticosteroid treatment exclude d participation. Finally, FEV1 < 45%, allergy to lidocaine, or any other inability to undergo bronc hoscopy also excluded participation. Baseline Procedures Patients were enrolled at the University of Florida and the University of Texas at Tyler. Patients meeting inclusion criter ia and agreeing to provide in form consent underwent further screening with a routine medical assessment, a complete physical examination and blood tests for hematologic, biochemical, and virology scre ening. Evidence of lung disease was confirmed by an HRCT and complete pulmonary function testing (PFTs) including spirometry, lung volumes, and diffusing capacity. Study Procedures After baseline procedures were complete d, all subjects underwen t bronchoscopy with bronchial brushing within 10 days of, but no less than 2 days prior to, the fi rst infusion of AAT. Blood samples for the measurements of functi onal and antigenic AAT levels were drawn

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14 immediately prior to the infusion. Subjects rece ived an infusion of f unctional human AAT via IV drip at a dose of 60 mg/kg body weight. Initial week one infusions were administered at the bronchoscopy center at either research site. Subs equent weekly doses (211) were administered IV via each individuals home health agency. Between weeks 10 and 12, all subjects undergoing repeat bronchoscopy received complete PFTs. Bronchoscopy Prior to undergoing bronchoscopy, subjects coul d not have a history of adverse reactions to the local anesthetic, sedatives or pre-me dications employed. Intravenous morphine and midazolam were used for conscious sedation in all subjects. Subj ects were monitored by electrocardiogram and pulse-oxim etry throughout the procedures. All subjects received inhaled bronchodilator therapy prior to re ceiving local airway anesthesia. Bronchial airway brushings were obtaine d from the fourth through sixth segmental airways utilizing a sheathed cytologic brus h system (Olympus Endo Therapy disposable cytology brush). Brushing samples were proces sed immediately after bronchoscopy. Brushing specimens were initially collect ed in sterile RPMI-1640 media a nd centrifuged at 800X g force for 15 minutes at 4 C. Cell pellets were resuspended in 1mL RTL buffer/ -mercaptoethanol mixture and stored at -80 C until future processing. Quantification of Gene Expression Total RNA was isolated and cDNA synthesis pe rformed according to commonly standardized protocols. Quantitative PCR was then performe d to measure gene expression of human TNF, IL-8, IL-15, HSP-1A, OSM-receptor, BIP, IRE1 and AAT. Quantitative PCR was performed as follows. Primers and FAM/VIC labe led probes were purchased as ei ther pre-developed assays or custom designed assays using the Applied Bi osystemss Assay-on-demand and Assay-by-design

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15 service, respectively (Applied Biosystems, Foster City CA). Primer sequences for each gene probed are included: 1. AAT (primer ID HS00165475_m1) gcaaatggga gagacccttt gaagtcaagg acaccgagga agaggacttc cacgtggacc aggtgacca c cgtgaaggtg cctatgatga agcg 2. HSP-A1A (HS00359163_s1) tgcagagat gaatttatac tgccatctta cgactatttc ttctttttaa tacacttaac tcaggccatt ttttaagttg gttacttcaa ag taaataaa ctttaaaatt caaaaaaaaa aaaaaa 3. TNF alpha (HS00174128_m1) c aaaccctcaa gctgaggggc agctccagtg gctgaaccgc cgggccaatg ccctcctggc caatggcgtg gagctgaga 4. IRE-1 (HS00176385_m1) aact tccttttacc atcccagaat tggtgcaggc atccccatgc cgaagttcag atggaatcct ctacatgggt aaaaagca 5. BiP (HS00607129_gh) c ggcgtgttca agaacggccg cgtggagatc atcgccaacg atcagggcaa ccgcatcacg ccgtcctatg tcgccttcac tcctgaaggg gaacgtctga ttggcgatgc cgccaagaac cagctcacct ccaaccccga gaaca 6. IL-8 (HS00174103_m1) gtgca gttttgccaa ggagtgct aa agaacttaga tgtcagtgca taaagacata ctccaaacct ttccacccca aa tttatcaa agaactgaga gtgattg 7. IL-15 (HS99999039_m1) ccagttg caaagtaaca gcaatg aagt gctttctctt ggagttacaa gttatttcac ttgagtccgg agatgcaagt attcatgata cagtagaaaa tctgatcatc ctagcaaaca acagtttgtc ttctaat 8. OSM-receptor (HS01051640_m1) gg gtggggaatt acagcaccac tgtgaagtgg aaccaggttc tgcattggag ctgggaatct gagctccc tt tggaatgtgc cacacacttt gtaa gaataa agagtttggt ggacgatgcc aagttccctg agcca. One hundred ng of cDNA (i.e. cDNA equivalent of 100 ng total RNA) of epithelial cell and human universal reference (18s) RNA-derived cDNAs were used as templates in hot-start PCR with gene-specific primers and Taqman probe using a universal PCR master mix (Applied Biosystems, Foster City CA) in a 25 l total reaction volume, using the protocol supplied by the manufacturer. Replicates were used so that stat istical analysis could be performed on the data generated. Data analysis was perf ormed using Ct values determined and normalized to the 18s housekeeping gene and analyzed using the deltadelta Ct method (Analysis of Relative Gene Expression data Using Real-Time Quantitative PCR and the 2Ct Method) to obtain fold change

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16 in gene expression between experimental sample s and human universal reference RNA control. Expression differences were e xpressed as fold-change ratio from baseline to 12 weeks. Sample Size This pilot study had no preliminary data to provide power calculations. A previously published study evaluating the effect of intr avenous AAT augmentation therapy on sputum neutrophilic inflammatory ma rkers found significant changes in sputum leukotriene B4 concentrations with 12 subjects[4] This previously published study suggests that a sample size of 12 or more subjects, although small, would be an adequate target enrollment for our pilot study. Statistical Analysis Changes in gene e xpression of human TNF, IL-8, IL-15, HSP-1A, OSM-receptor, BIP, IRE-1 and AAT were all independent primary anal ysis variables. Statistical analysis was performed for each probe using the Wilcoxon matche d pairs t-test for non-parametric paired samples. Statistical significan ce was accepted at a level of p < 0.05.

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17 CHAPTER 3 RESULTS Fifteen individuals were initially enrolled, 11 at the University of Florida and 4 at the University of Texas at Tyler. One subject elec ted to withdraw from the study and two subjects were withheld from repeat bronc hoscopy at the investig ators discretion due to the development of adverse events (pneumothorax, pulmonary embo lism) subsequent to the first bronchoscopy. Inadequate cellular return and resulting inadequate RNA concen tration prevented the final study of a fourth individuals samples. Therefore, paired baseline and after therapy bronchial epithelial cell brushings were available for analysis from 11 subjects. However, do to limited concentrations of RNA recovered from the bronchoscopies, gene expression analyses was performed on only eight samples for OSM-receptor and IL-15. Demographics The 11 subjects completing the study had an average age of 54.8 years of age. There were 7 males and 4 females in the sample group. Seven of the 11 were previous smokers with a pack year history ranging from 5 to 70 pack years. The average FEV1% predicted for all subjects was 61% predicted. All but one of the study su bjects had an obstructive ventilatory defect on pulmonary function testing determined by FEV1 % predicted. That individual met study inclusion criteria by having evidence of pulmonary emphysem a on high resolution CT (HRCT) of the chest (Table 3-1). Treatment All 11 subjects received AAT augmentation th erapy throughout the co urse of the study. Serum AAT levels were drawn prio r to initiation of therapy to demonstrate AAT deficiency and again prior to follow-up bronchoscopy to ensure appropriate response to therapy (4.3 1.4 M at

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18 baseline, 18.9 5.2 M after treatment) (Table 3-1) At the end of the study, all subjects had serum AAT levels equal to or greater than 11 M the clinically accepted protective target [11]. Gene Expression Analysis Quantitative PCR relative gene expression units for AAT, HSP-A1A, TNF, IRE-1, BiP, IL-8, IL-15 and OSM-receptor are summarized in Table 3-2. No significant changes from baseline and following AAT augmentation therapy we re observed in any of the selected genes. Adverse Outcomes As noted, two individuals initially enrolled in the study did not undergo follow-up bronchoscopy due to investigators discretion after development of a pneumothorax in one individual and a catheter-related pulmonary embolism in another. Neither of these events was study drug related. No study drug adverse events were observed. Table 3-1. Patient characteristics Subjec t Age Sex AAT Genotype Smoking history Pack years FEV1 actual FEV1 % predicted AAT M baseline AAT M 12 weeks 1.55 M ZZ Former 702.74L68%3.8317.80 2.71 M ZZ Former 103.00L101%3.8517.90 3.58 M ZZ Former 252.16L57%6.1117.80 4.59 F ZZ N eve r 01.73L58%3.7318.70 5.44 F ZZ Former 121.60L41%3.2214.80 6.55 M MaltonMZ Former 402.55L63%6.5119.60 7.52 F ZZ Former 301.73L48%2.1526.60 8.56 F ZZ N eve r 01.94L76%3.2829.40 9.53 M ZZ N eve r 02.19L55%5.8118.70 10.54 M ZZ N eve r 01.61L48%5.716.20 11.46 M ZZ Former 52.18L51%3.1410.50

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19 Table 3-2. Quantitative PCR relative gene expres sion units at baseline and after 12 weeks of AAT augmentation therapy Gene Baseline 12 weeks P value (comparing baseline to 12 weeks) AAT 0.22 0.160.22 HSP-A1A 2.18 1.470.17 TNF6.16 6.660.86 IRE-1 0.32 0.240.31 BiP 0.59 0.380.26 IL-8 3.84 5.260.55 IL-15 0.75 0.660.65 OSM-receptor 0.13 0.110.22 AAT Baseline 12 Weeks 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7Relative Gene Expression Figure 3-1. AAT quantitative PCR re lative gene expression. p=0.22 HSP-A1A Baseline 12 Weeks 0 1 2 3 4 5 6 7 8Relative Gene Expression Figure 3-2. HSP-A1A quantitative P CR relative gene expression. p=0.17

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20 TNF-alpha Baseline 12 weeks 0 5 10 15 20 25 30 35 40 45Relative Gene Expression Figure 3-3. TNFquantitative PCR relative gene expression. p=0.86 IRE-1 Baseline 12 Weeks 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3Relative Gene Expression Figure 3-4. IRE-1 quantitative PCR relative gene expression. p=0.31 BIP Baseline 12Weeks 0 1 2 3Relative Gene Expression Figure 3-5. BiP quantitative PCR relative gene expression. p=0.26

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21 IL-8 Baseline 12 Weeks 0 10 20 30 40Relative Gene Expression Figure 3-6. IL-8 quantitative PCR relative gene expression. p=0.55 IL-15 Baseline 12 weeks 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3Relative Gene Expression Figure 3-7. IL-15 quantitative PCR re lative gene expression. p=0.65 OSM-receptor Baseline 12 weeks 0.00 0.05 0.10 0.15 0.20Relative Gene Expression Figure 3-8. OSM-receptor quantitative P CR relative gene expression. p=0.22

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22 CHAPTER 4 DISCUSSION AND CONCLUSION Discussion In this prospective, observati onal pilot study we te sted the hypothesis that treatment of AAT deficient individuals with intravenous AAT augmentation therapy to clinically accepted serum concentrations would resu lt in a decrease in airway in flammation measured by epithelial cell inflammatory gene expressi on. Our findings in 11 patients did not demonstrate significant changes in the inflammatory genes TNF, IL-8, IL-15, HSP-1 and OSM-receptor. BIP and IRE-1, genes associated with the unfolded protei n response, were also not affected. We did demonstrate via cells obtained in vivo from br onchoscopy that bronchial epithelial cells express AAT, which to our knowledge has previously only b een reported from cell cultures. No changes in AAT gene expression were observed followi ng augmentation therapy in our study subjects. These data have important implications. Th e link between AAT deficiency and increased airway inflammation resulting in clinically evid ent airways disease, such as bronchospasm and bronchiectasis, is well document ed [2] [8]. This airway remode ling contributes to diminished airflow and further progression of lung disease [8]. Furthe r supporting that a peripheral inflammatory process contribute s to airflow obstruction in patie nts with AAT deficiency, the addition of extra-fine inhaled corticosteroids to long acting beta-agonists has been shown to decrease airway narrowing, mostly in the small airways, further reduci ng dynamic hyperinflation and resulting in a marked improvement in exer cise tolerance and dyspnea [12]. While IV AAT augmentation therapy has been shown to decrease ne utrophilic inflammation in the airways [4], a direct effect of therapy on diminishing airway epithelial cell inflammation has not been demonstrated.

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23 There are several possibilities as to why we did not observe decreased bronchial epithelial cell inflammation in this pilot therapeutic study. Firs t, AAT concentrations obtained in the upper airway epithelial cells via IV augmen tation therapy may be insufficient to downregulate inflammation. While serum and lower ai rway/alveolar epithelial lining fluid (ELF) concentrations correlate well due to the close proximity of th e alveolar-capillary membranes [11], the upper airways do not shar e this same type of vascular perfusion. Thus, it is unlikely that increased serum concentratio ns of AAT affect th e epithelial cells in the upper airways the same as in the lower airways. Second, the increased sputum found in the upper airways also likely contributes to a lessened e ffect of IV augmentation. Not only does airway mucous create an additional barrier to serum AAT diffusion, but this mucous also contains many PMNs and other inflammatory cells, thus increasing AAT c onsumption. While we demonstrated that upper airway bronchial epithelial cells express AAT intrinsically and thus have some low level intracellular production, this producti on is unlikely to be adequate to ameliorate the observed airway inflammation, especially in AAT deficien t individuals. Aerosolized AAT, an alternative therapeutic delivery mechanism currently being studied, may have a greater impact on airway inflammation by achieving much highe r concentrations of AAT to the airway epithelial cells. Third, the duration of the study intervals may ha ve been inadequate. A washout period of 5 weeks for the therapeutic AAT augmentation therapy our subjects were receiving prior to beginning the study may not have been long enou gh for a complete return to a non-treatment baseline inflammation in their airway epithelial cells. Also, du e to the factors affecting upper airway inflammation discussed previously, a ther apeutic course longer than 12 weeks of therapy may be necessary to observe any significant dow n-regulation in airway inflammation. However, decreased neutrophilic inflammation in the sputum of AAT deficient patient s and cystic fibrosis

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24 patients receiving IV and aerosoli zed treatment have been observed in as little as 4 weeks of therapy [13] [4]. Finally, the absence of any significant dow n-regulation of bronchial epithelial cell inflammation following IV augmentation therapy observed in our study could also be due to Type II error because of our small sample size. No preliminary data existed for this pilot study, so adequate power calculations were not available. Previous studies investigating sputum neutrophilic inflammation in AAT deficient individuals sugges ted that a sample size of approximately 10 would be sufficient to observe a significant therapeutic effect [5] [4] [14]. However, the effect of increased serum AAT c oncentrations on sputum neutrophil inflammation may be much more profound than it is for upper airway epithelial cells, necessitating a larger sample size to detect a significant difference. Mo st studies investigating airway inflammation in AAT deficiency utilize expectorat ed sputum due to the high cost difficulty, and invasiveness of bronchoscopy. Repeating this study on a larger scale with a larger sample size would be difficult due to the cost and invasiveness of the necessary sampling. Although no data exist for power calculations, another possible study would enail the use of aerosoli zed AAT rather than IV AAT augmentation therapy, thus increasing the therap eutic delivery to the ta rget cells, and providing another means to test whether such treatment could modulate the expression of inflammatory markers. Conclusion Upper airway bronchial epithe lial cells obtained from indivi duals with AAT deficiency express AAT in vivo. To our knowledge, this is the first report dem onstrating that human bronchial epithelial cells express AAT outside of a cell cu lture model. Although increased airway inflammation is clearly as sociated with AAT deficiency, we were unable to demonstrate that intravenous AAT augmentation therapy de creases upper airway br onchial epithelial cell

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25 inflammation. Further study regard ing the role of AAT deficien cy on bronchial epithelial cell inflammation is warranted, specifically utilizi ng aerosolized AAT as a therapeutic modality.

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26 REFERENCES 1. American Thoracic Society/European Respir atory Society Statement: Standards for the diagnosis and management of individuals with alpha-1 an titrypsin deficiency. Am J Respir Crit Care Med 2003;168:818-900. 2. Brantly ML, Paul LD, Miller BH, Falk RT, Wu M, Crystal RG. Clinical features and history of the destructive lung disease associated with alpha -1-antitrypsin deficiency of adults with pulmonary symptoms Am Rev Respir Dis 1988;138:327-366. 3. Brantly, M, Alpha 1-Antitrypsin: Not just an antiprotease: Extendi ng the half-life of a natural anti-inflammatory molecule by c onjugation with polyethylene glycol. Am J Respir Cell Mol Bi ol 2002;27(6):652-654. 4. Stockley RA, Bayley DL, Unsal I, Dowson LJ. The effect of augmentation therapy on bronchial inflammation in alpha 1-antitrypsin deficiency. Am J Respir Crit Care Med 2002;165;1494-1498. 5. Gompertz S, Hill AT, Bayley DL, Stockley RA. Effect of expectoration on inflammation in induced sputum in Alpha 1-Antitryps in Deficiency. Respiratory Medicine 2006;100:1094-1099. 6. The Alpha-1 Antitrypsin Deficiency Registry Study Group. Survival and FEV1 decline in individuals with severe defici ency of alpha 1-antitrypsin. Am J Respir Crit Care Med 1998;158:49-59. 7. Eden E, Hammel J, Rouhani FN, Brantly ML, Barker AF, Buist AS, Fallat RJ, Stoller JK, Crystal RG, Turino GM. Asthma features in severe alpha1-antitrypsin deficiency: Experience of the National Heart, L ung, and Blood Institute Registry. Chest 2003;123:765-771. 8. Demeo DL, Sandhaus RA, Barker AF, Bran tly ML, Eden E, McElvaney NG, et al. Determinants of airflow obstruction in seve re alpha-1-antitrypsi n deficiency. Thorax 2007;62:806-813. 9. Tobin M., Cook P, Hutchinson D. Alpha-1-A ntitrypsin Deficiency: The clinical and physiological features of pulmonary emphysema in subjects homozygous for Pi Type Z: A survey by the British Thoracic As sociation. Br J Dis Chest 1983;77:14-27. 10. Parr DG, Guest PG, Reynolds JH, Dowson LJ, Stockley RA. Prevalence and impact of bronchiectasis in alpha 1antitrypsin deficiency. Am J Respir Crit Care Med 2007;176:1215-1221. 11. Wewers MD, Casolaro MA, Sellers SE, Swayze SC, McPhaul KM, Wittes JT, Crystal RG. Replacement therapy for alpha 1-antitrypsi n deficiency associated with emphysema. N Engl J Med 1987;316:1055-1062.

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27 12. Corda L, Bertella E, La Piana GE, Boni E, Redolfi S, Tantucci C. Inhaled corticosteroids as additional treatment in alpha-1-antitr ypsin-deficiency-relate d COPD. Respiration 2008;76:61-68. 13. Griese M, Latzin P, Kappler M, Weckerle K, Heinzlmaier T, Bernhardt T, Hartl D. alpha1-antitrypsin inhalation redu ces airway inflammation in cy stic fibrosis patients. Eur Respir J 2007;29:240-250. 14. Malerba M, Ricciardolo F, Radaeli A, To rregiani C, Ceriani L, Mori E, et al. Neutrophilic inflammation and IL-8 levels in induced sputum of alpha-1-antitrypsin PiMZ subjects. Thorax 2006;61:129-133.

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28 BIOGRAPHICAL SKETCH Eric Lee Olson, M.D., was born on November 26, 1970 in Fort Collins, Colorado. He graduated from Otis High School in Otis, Colora do and then pursued his undergraduate degree at the University of Colorado at Boulder. After completing his B.A. in molecular, cellular and developmental (MCD) biology at CU in 1993, Eric wo rked as a laboratory research technician in the Pulmonary and Critical Care Division at the University of Colorado H ealth Sciences Center and at the National Jewish Center for Immunolo gy and Respiratory Medicine in Denver for two years. In the fall of 1995, Eric began his medical school training at the University of Vermont (UVM) College of Medicine in Burlington, VT. Upon graduation from UVM in 1999, Eric moved to the University of No rth Carolina at Chapel Hill, where he completed his internal medicine residency and then fellowship traini ng in Pulmonary and Critical Care Medicine, concluding in 2005. He then accepted a position as an assistant professor of medicine at the University of Florida and has held that posi tion since that time. In December, 2008, Eric received his M.S. in medical scie nces with a concentration in clinical investigation from UF. Erics research focuses on the pa tho-physiology of the airways and how genetic diseases such as Alpha-1 Antitrypsin deficiency and cystic fi brosis and environmental stimuli alter airway function. Eric is married to Jennifer A. C. Olson and has one child, Samuel Olson.