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a1-Antitrypsin Deficiency What Has It Ever Done for Us? Robert A. Stockley, MD, DSc

The first five cases of a1-antitrypsin deficiency were originally published in 1963. This changed our whole concept about the pathophysiology of emphysema, including the role of inflammation and, in particular, the role of proteolytic enzymes. However, the observation also had a significant 50-year impact on many aspects of protein biochemistry, genetics, cell biology, and disease concepts outside the lung as well as the study of COPD in general. CHEST 2013; 144(6):1923–1929 Abbreviations: AAT 5 a1-antitrypsin; AATD 5 a1-antitrypsin deficiency; NE 5 neutrophilic elastase

two significant and linked events. ThisFirst,yearthehas50thseenanniversary of the publication of the

first description of a1-antitrypsin deficiency (AATD) and its clinical features,1 and second, the unfortunate death of Gordon Snider, who contributed extensively to our understanding of emphysema. Indeed, his Amberson lecture in 19922 remains an excellent background to the original descriptions of emphysema dating back to the late 1600, the epidemiologic link to cigarette smoking and the surrogate markers, including lung function and CT scanning. His subsequent work with animal models and human studies based on the AATD observation helped establish current thinking on the pathophysiology of this destructive lung process. Background It has not always been an enthusiastic path. In 1972, as an introduction to a book on emphysema and proteolysis, William Briscoe wrote “It is sometimes suggested that testing for AATD should be included in mass screening programmes designed to protect the health of normal population. There are others, experienced physicians with whom I agree, who consider this fruitless meddling and who say that if they or any of their relatives had AATD they would rather not

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know, since nothing can be done about it.”3 This view still persists in some countries and physicians’ minds. So, with hindsight, what has this original observation done for our thinking since 1963? Emphysema is one of the pathologic features of the COPD syndrome. It was first recognized as part of the normal aging process by Lyn Reid4 in 1967, leading to the possibility also expressed by William Briscoe3 in his introduction that the disease should be regarded as “an accelerated ageing process,” although the mechanism was obscure. The fact that COPD ran in families5,6 suggested a clear genetic component, and the fact that the majority of cigarette smokers retained relatively normal lungs indicated a susceptibility that reflected possible gene/environmental interactions. However, despite extensive genetic screening studies, little has emerged (perhaps reflecting the heterogeneity of the COPD syndrome) that outranks the chance discovery of AATD. a1-Antitrypsin (AAT) is a classic proteinase inhibitor, and the relationship of deficiency suggests that the destructive process leading to emphysema was driven by an enzyme(s) normally controlled by AAT. By the mid-1970s, the concept that it was destruction of lung elastin central to the process led to the demonstration that elastase from the neutrophil was the CHEST / 144 / 6 / DECEMBER 2013

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likely culprit.7 Indeed, AAT has the greatest affinity for this enzyme, rapidly inactivating it, and this led to substantiation of the proteinase/antiproteinase theory of emphysema. Release of elastase by migrating neutrophils was assumed to cause the damage to elastin and the development of emphysema. Since these cells represent a key component of the lung defenses, this concept is consistent with both the age-related changes in “healthy individuals” and the accelerated changes in AATD. The next logical step, therefore, was to enhance the protection of the lung by augmentation of the low levels of AAT toward a normal or “protective” level. In the 1980s, purification of AAT from human plasma was shown to be feasible, and weekly infusions boosted the levels of AAT to those believed to be protective,8 leading to the belief that the problem/disease was resolved. Thereafter, US Food and Drug Administration approval resulted in the acceptance of augmentation therapy based on a biochemical replenishment outcome, and it became widely administered in many countries. Classic clinical trials using the gold standard of FEV1 as an outcome were deemed impractical in part because of the time and expense required but mainly because of the unlikely ability to power such a study due to the rarity of AATD.9 This path from discovery to treatment took approximately 20 years and led to a general lull in research into the deficiency. However, studies carried on with the identification of the gene and its chromosomal location and an understanding of the genetic defect resulting in the common severe deficiency gene (the Z phenotype). The point mutation resulted in a single animo acid change at position 342 (glu to lys). However, multiple, though less frequent, defects became recognized, including other point mutations, insertions, and deletions resulting in premature stop codons and even gene deletion.10 At this time, lung disease led genetic disease monitoring. The mechanism for the accumulation of the Z protein in the hepatocyte was finally elucidated by Lomas and colleagues,11 who demonstrated that the single Z point mutation altered the tertiary structure of the AAT molecule such that the mobile reactive loop of one molecule became inserted into a second molecule Manuscript received August 14, 2013; revision accepted August 16, 2013. Affiliations: From the Lung Function and Sleep Department, ADAPT Project, Queen Elizabeth Hospital Birmingham, Edgbaston, Birmingham, England. Correspondence to: Robert A. Stockley, MD, DSc, Lung Function and Sleep Department, ADAPT Project, Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB, England; e-mail: [email protected] © 2013 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.13-1901 1924

and so on, forming polymers. These were retained in the endoplasmic reticulum, markedly reducing secretion of AAT from hepatocytes. This understanding of the molecular mechanism lead to the recognition of the same pathologic process involving other serine proteinase inhibitors in other organ systems, the serpinopathies.12 Once the mechanism was understood, processes to interfere with polymerization became one small step,13 and although such a process inactivates AAT, it would prevent liver accumulation and damage. Research continues to design peptides that can prevent polymerization while retaining function, extending our knowledge of protein structure/function relationships. The 1990s saw a resurgence of interest in AATD following its recognition as a problem by the World Health Organization,14 the establishment of the Alpha1-Foundation, and the insistence of many countries that evidence of clinical efficacy was required to support initiation or continuation of augmentation therapy. An Alpha-1 International Registry (AIR) was formed and quickly established an extensive database, currently in excess of 5,000 patients,15 to facilitate collaborative research and establish a list of patients not receiving augmentation therapy who could and were involved in subsequent clinical trials.16,17 Closer clinical observation established the variations between clinical features/impact in individual patients even within families18 and highlighted the limitations of spirometry in the assessment of severity and monitoring of progression.19,20 Factors other than smoking that influenced the disease progression emerged.21-23 Studies revealed that the emphysema process progressed most physiologically when the FEV1 impairment was worse and had stabilized22 and thus probably reflected a shift of the emphysema process from the bases to involve the apical regions. CT scan technology emerged as the most sensitive monitoring tool20 and was shown to reflect a relatively constant progression from early to late disease.24 Patients with “atypical” apical-dominant emphysema were identified25 even before spirometry deteriorated26 and, together with gas transfer, may reflect the earliest changes before spirometry becomes impaired,27 providing a strategy for early monitoring. Registry and observational studies started to provide indirect evidence that augmentation therapy did more than correct the low AAT level. The National Institutes of Health registry suggested that mortality was higher in individuals who did not receive augmentation therapy.28 Furthermore, the decline in spirometry was less in patients on augmentation therapy if the FEV1 was between 35% and 60% predicted. The interpretation of these data was partly muted by an imbalance of social characteristics and health-care exposure of the treated and untreated groups. However, comparisons of spirometry between European Special Features

countries where augmentation was or was not available supported the concept of benefit.29 Furthermore, sequential studies in Germany also provided support for benefit,30 as did a recent meta-analysis comparing the data from treated and untreated patients from multiple sources.31 These observational studies do have their drawbacks, as they are based on spirometry, which is a poor surrogate of the emphysema process. Also, the National Institutes of Health observation has led to the belief that augmentation is only effective in a limited FEV1 range. However, this likely reflects the fact that FEV1 decline is greatest in this range,22 and it is becoming increasingly recognized in clinical trials that enriching populations for the proposed outcome increases the statistical power. This is an important point, as emphysema assessed by lung densitometry24 and gas transfer22 shows progression even when the FEV1 is most stable. Indeed, the specificity and sensitivity of CT densitometry to emphysema progression20 has resulted in this being accepted as a relevant outcome in emphysema trials. The initial use of CT scan in the study of augmentation therapy was published in 1999 as part of a secondary outcome measure of a placebo-controlled augmentation study32 suggesting efficacy. The methodology was improved and further validated by Parr and colleagues33,34 and used as an exploratory outcome in the Exacerbations and CT Scan as Lung Endpoints (EXACTLE) trial,16 again suggesting (though not proving) efficacy. The data were further analyzed on a regional basis, suggesting the potential benefit was most evident at the lung bases,35 where the classic AATD emphysema occurs. Combining data from the two studies increased the power and provided further support for the benefit of augmentation,36 although all the data represented secondary outcomes or post hoc analyses. More recently, a larger study has been completed with CT densitometry as the primary outcome, suggesting statistically significant benefit of augmentation therapy.37 Nevertheless, the data do not mean that all deficient patients require augmentation, as the progression is variable both in never smokers and after smoking cessation. The strategy, therefore, requires optimization of usual therapy (including smoking cessation) and then either direct observation of subsequent decline or the use of predictive biomarkers to guide therapy. Concepts, and studies that urgently need exploring. The Mechanism(s) of Deficiency Study of the AAT protein and gene has led to a clear understanding of the mechanisms that result in low circulating (and, hence, lung) AAT. Of most importance was the demonstration of the loop sheet polymerization process that causes the most common journal.publications.chestnet.org

Z-deficient protein to accumulate in the hepatocyte, reducing secretion and serum levels.11 Indeed, this common process is a recognized feature of other mutants of the serpin family resulting in other systemic diseases and referred to as the serpinopathies.12 Understanding the molecular process has provided a further understanding of the lung pathophysiology38 as well as the liver pathophysiology.39 Furthermore, this has resulted in the development of several potential treatment or curative strategies that include treatments to facilitate secretion from the liver, including the use of chemical chaperones40 and peptides to block the polymerization, gene therapy, and gene repair.13 Impact on Usual COPD The link between AATD and emphysema established the proteinase/antiproteinase balance as the important mechanism of lung tissue destruction. This led to studies of AAT and its phenotypes to determine whether this was a more general feature. However, most patients with COPD had normal serum levels of AAT, and so alternative mechanisms resulting in uncontrolled proteinase activity in the lung were sought. As the structure/function knowledge of AAT grew, it became recognized that the amino acid at position 358 was critical for its function. In normal AAT this is methionine, which gives the protein its specificity for interaction with the catalytic triad of serine proteinases, especially neutrophilic elastase (NE).41 The only other naturally occurring active site variant identified had arginine at this site changing the protein into an antithrombin.42 No such active site variants were found in COPD, but experiments showed that cigarette smoke oxidized the normal methionine, reducing the inhibition of NE 2,000-fold.43 Initial lung lavage studies demonstrated a reduction in AAT function in healthy smokers44 and evidence of oxidized methionine residues,45 adding credence to the theory that usual COPD emphysema was also due to a proteinase/antiproteinase imbalance due to cigarette smoke induction of functional AATD. This concept remains entrenched even though subsequent studies failed to confirm these initial findings,46-48 and indeed no or little evidence of oxidized AAT was found in airway secretions of patients with COPD.49 Furthermore, this simple biochemical inactivation failed to explain why most smokers do not develop emphysema/COPD. Indeed, other mechanisms of inactivation (cleavage of the reactive loop and complexing with enzyme) were also characterized50 as well as the presence of other, locally produced elastase inhibitors.51 This does not negate the role of oxidants, as there is undoubtedly evidence of oxidant stress in COPD,52 although the effect may largely be to amplify the CHEST / 144 / 6 / DECEMBER 2013

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inflammatory process,53 which may play a key role (see later discussion). Thus, inactivation of lung AAT by inhaled oxidants may be minimal or a bystander process. Intriguingly, however, migrating neutrophils pulse with sequential oxidant and protease bursts.54 It is therefore possible that such oxidant release in the microenvironment around the neutrophil inactivates the local AAT, facilitating matrix degradation as the cell migrates. This process is worthy of further study. Animal Models The first recognized animal model of emphysema was reported in 1964 as a surprising outcome following instillation of the metalloproteinase papain in the lungs. The concept that a proteolytic enzyme could induce emphysema fitted well with the prevalence of emphysema in AATD. The demonstration that human neutrophil elastase7 and proteinase 3,55 both serine proteinases, are capable of inducing an “emphysematous” phenotype in experimental animals seemed to close the loop. Lung elastin is a stable long-lived tissue in healthy humans,56 and the link between cutis laxa and emphysema57 supports elastin as being central to the emphysematous process. The proteolytic target was reasonably deemed to be lung elastin, and this was supported by studies showing that loss of lung elastin followed by rapid accumulation of nonfibrillary elastin58 loss due to starvation,59 prevention of elastin cross-linking,60 and other classes of enzymes capable of digesting elastin61 produced the same pathologic changes. Indeed, the demonstration that the interstitial NE signal related to the amount of emphysema in humans62 provided further supportive evidence of the NE pathway. Transgenic models overexpressing inflammatory cytokines63 and several knock-out mouse models64,65 added to the knowledge implicating inflammation, neutrophils, macrophages, metalloproteinases, autoimmunity, cell death, and tissue damage/repair in a complex cascade of events, all of which have received some credence from human studies and have been the subject of many reviews.66,67 However, the implication of many individual components reflects such complex interactions that it remains difficult to identify any one key step or process in human disease. It may be that there are many pathways leading to one central end point, but dissecting physiologic from pathologic response remains a major challenge while studying the inflammatory processes involved. Part of the problem has been that COPD has always been a generic term used to describe several pathologic sites and processes that result in a common physiologic marker. This linked to sampling of different sites in the lung (BAL, sputum, sputum induction, and biopsies of major or small airways or pathologic 1926

specimens) has endorsed the presence of inflammation but not the biologic process. Studies of healthy smokers, never smokers, and those with established disease need to be studied in parallel. Since only a proportion of smokers develop the COPD syndrome, only a proportion of never smokers should have the susceptibility factor(s); none of the healthy smokers (who should show the physiologic response) and all of the patients (enriched for the pathologic response) should have the susceptibility factor(s). The latter group consists of several clinical phenotypes (emphysema, small airways disease, chronic bronchitis, colonized airways, bronchiectasis, reversible/nonreversible), which may have different pathologic processes or pathways but still influence the local and systemic inflammatory signal. In addition, smoking cessation and treatments may also influence the data. Here, AATD may play a key role as a further control group, since the degree of physiologic abnormality, treatment, and even emphysema can be matched to usual COPD, seeking differences specific to the latter. Indeed, such an approach has been used to identify abnormal neutrophil behavior specific to patients with COPD. For some years it has been recognized that neutrophils from patients with COPD have increased chemotactic response and destructive capability,68 increased adhesion and spontaneous migration under flow conditions,69 and a chaotic chemotactic migration pathway.70 The latter two studies were consistently different from matched patients with AATD (overcoming any effects secondary to the presence and severity of airflow obstruction and treatment) and probably reflect abnormal signaling through PI3K.70 Studies of early emphysema, preceding the development of airflow obstruction, highlighted local neutrophilic inflammation present in BAL.71 More recently, PET CT scanning highlighted an enhanced neutrophilic signal in usual COPD localized to where the emphysema occurs (the apices) compared with subjects with AATD in whom the signal was normal.72 Since the release of proteinases by an activated neutrophil exceeds the inhibitory capacity of even normal concentrations of active AAT,73 some tissue destruction will be a consequence of any neutrophil migration. In AATD, this will be enhanced compared with normal because of the deficiency. In COPD it is also enhanced because of neutrophil overactivity, the same process but a different paradigm (Fig 1). It should, however, be emphasized that this conceptual line from AATD to usual COPD may only apply to the emphysema process. The more widespread use of CT scanning has opened up a new approach to COPD. Separation of the emphysematous and small airways phenotypes provides at least one step in the study of more specific pathophysiologic processes. With this Special Features

Figure 1. Neutrophils are recruited to the airway in response to chemoattractants released locally (1). In AATD, neutrophil adhesion and migration (2) lead to release of elastase, and degradation of lung connective tissue is greater than usual (3) because of the reduced level of a1-antitrypsin. In usual COPD, the same process occurs (4), but because of the rapid and less-directed migration, a greater area of damage occurs (5) despite normal levels of AATD. Tissue-derived chemotactic degradation products (6) together with proteinase- and oxidant-driven inflammation (7) amplify the processes. AATD 5 a1-antitrypsin deficiency.

in mind, more recent animal studies have suggested a different proteolytic path, in which metalloproteinases play a more central role in small airways disease rather than emphysema.74 However, the implications of emphysema on physiologic decline75 and long-term outcomes76 are beginning to re-emerge as clinical and research issues. This and recognition of the other clinical phenotypes lie central to the interpretation of the enormous amount of data being generated by genomic, proteomic, and metabolomic research that is likely to become more interpretable as clinical, radiologic, and pathologic phenotypes are characterized better and secondary events in the lung (colonization) and systemic effects (cardiovascular, metabolic, osteoporotic) are recognized as variants that may affect measurements. Indeed, even the variations in clinical phenotype in AATD itself suggest that the clinical condition is influenced by epigenetic factors, and this is being confirmed by more recent studies of gene screening, especially in well-characterized patients.77-80 The lessons learned have clear implications for our concepts of the proteinase/antiproteinase and the genetic complexities of AATD and COPD in general. Summary So What Has AATD Ever Done for Us? Well, • The identification of the most common genetic susceptibility factor for emphysema; • The development of protein structural biology to understand and resolve the defect; journal.publications.chestnet.org

• The recognition of the serpinopathies in disease pathophysiology affecting other organs; • The understanding of neutrophilic tissue damage, inflammation, migration, and the function of proteinases and oxidants relevant to the pathophysiology of lung and other inflammatory diseases; • The variation and implication of clinical phenotypes and structure-function relationships; • The role and validation of CT scanning and acceptance as a primary outcome for clinical trials; • A specific therapy that may have added value beyond AATD81; • The emergence of a strong patient organization leading to the COPD Foundation with political influence; • International collaboration and the emergence of multiple leaders in the field; • A realization that even a single gene defect does not explain variations in clinical phenotype and impact. Well, Granted, but Apart From That What Has AATD Ever Done for Us? Fifty years has certainly extended our methodologies and knowledge of COPD with major impact into other fields of clinical medicine. Although there is still much to do, thank you Sten Eriksson and CarlBertil Laurell! Acknowledgments Financial/nonfinancial disclosures: The author has reported to CHEST the following conflicts of interest: Dr Stockley has lectured widely as part of pharmaceutical sponsored symposia, sat on numerous advisory boards for drug design and trail implementation, and received noncommercial grant funding from Grifols and AstraZeneca.

References 1. Laurell C, Eriksson S. The electrophorectic alpha 1-globulin pattern of serum in alpha 1-antitrypsin deficiency. Scand J Clin Lab Invest. 1963;15:132-140. 2. Snider GL. Emphysema: the first two centuries—and beyond. A historical overview, with suggestions for future research: part 1. Am Rev Respir Dis. 1992;146(5 pt 1):1334-1344. 3. Briscoe WA. Introductory remarks. In: C. Mittman, ed. Pulmonary Emphysema and Proteolysis. New York, NY: Academic Press; 1972:3-5. 4. Reid L. The Pathology of Emphysema. Philadelphia, PA: Lippincott; 1967. 5. Larson RK, Barman ML, Kueppers F, Fudenberg HH. Genetic and environmental determinants of chronic obstructive pulmonary disease. Ann Intern Med. 1970;72(5):627-632. 6. Kueppers F, Miller RD, Gordon H, Hepper NG, Offord K. Familial prevalence of chronic obstructive pulmonary disease in a matched pair study. Am J Med. 1977;63(3):336-342. 7. Senior RM, Tegner H, Kuhn C, Ohlsson K, Starcher BC, Pierce JA. The induction of pulmonary emphysema with human leukocyte elastase. Am Rev Respir Dis. 1977;116(3):469-475. 8. Wewers MD, Casolaro MA, Sellers SE, et al. Replacement therapy for alpha 1-antitrypsin deficiency associated with emphysema. N Engl J Med. 1987;316(17):1055-1062. CHEST / 144 / 6 / DECEMBER 2013

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9. Schluchter MD, Stoller JK, Barker AF, et al. Feasibility of a clinical trial of augmentation therapy for alpha(1)-antitrypsin deficiency. The Alpha 1-Antitrypsin Deficiency Registry Study Group. Am J Respir Crit Care Med. 2000;161(3 pt 1):796-801. 10. Brantly ML, Nukiwa T, Crystal RG. Molecular basis of alpha1-antitrypsin deficiency. Am J Med. 1988;84(6A):13-31. 11. Lomas DA, Evans DL, Finch JT, Carrell RW. The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature. 1992;357(6379):605-607. 12. Lomas DA, Belorgey D, Mallya M, et al. Polymerisation underlies alpha1-antitrypsin deficiency, dementia and other serpinopathies. Front Biosci. 2004;9:2873-2891. 13. Mallya M, Phillips RL, Saldanha SA, et al. Small molecules block the polymerization of Z alpha1-antitrypsin and increase the clearance of intracellular aggregates. J Med Chem. 2007; 50(22):5357-5363. 14. Alpha 1-antitrypsin deficiency: memorandum from a WHO meeting. Bull World Health Organ. 1997;75(5):397-415. 15. Stockley RA, Dirksen A, Stolk J. Alpha-1 antitrypsin deficiency: the European experience. COPD. 2013;10(suppl 1):50-53. 16. Dirksen A, Piitulainen E, Parr DG, et al. Exploring the role of CT densitometry: a randomised study of augmentation therapy in alpha1-antitrypsin deficiency. Eur Respir J. 2009;33(6): 1345-1353. 17. Stolk J, Stockley RA, Stoel BC, et al. Randomised controlled trial for emphysema with a selective agonist of the g-type retinoic acid receptor. Eur Respir J. 2012;40(2):306-312. 18. Wood AM, Needham M, Simmonds MJ, Newby PR, Gough SC, Stockley RA. Phenotypic differences in alpha 1 antitrypsindeficient sibling pairs may relate to genetic variation. COPD. 2008;5(6):353-359. 19. Dowson LJ, Guest PJ, Hill SL, Holder RL, Stockley RA. Highresolution computed tomography scanning in a1-antitrypsin deficiency: relationship to lung function and health status. Eur Respir J. 2001;17(6):1097-1104. 20. Stolk J, Cooper BG, Stoel B, et al. Retinoid treatment of Emphysema in Patients on the Alpha-1 International Registry. The REPAIR study: study design, methodology and quality control of study assessments. Ther Adv Respir Dis. 2010;4(6): 319-332. 21. Dowson LJ, Guest PJ, Stockley RA. Longitudinal changes in physiological, radiological, and health status measurements in alpha(1)-antitrypsin deficiency and factors associated with decline. Am J Respir Crit Care Med. 2001;164(10 pt 1): 1805-1809. 22. Dawkins PA, Dawkins CL, Wood AM, Nightingale PG, Stockley JA, Stockley RA. Rate of progression of lung function impairment in alpha1-antitrypsin deficiency. Eur Respir J. 2009;33(6):1338-1344. 23. Wood AM, Harrison RM, Semple S, Ayres JG, Stockley RA. Outdoor air pollution is associated with rapid decline of lung function in alpha-1-antitrypsin deficiency. Occup Environ Med. 2010;67(8):556-561. 24. Parr DG, Stoel BC, Stolk J, Stockley RA. Validation of computed tomographic lung densitometry for monitoring emphysema in a1-antitrypsin deficiency. Thorax. 2006;61(6):485-490. 25. Parr DG, Stoel BC, Stolk J, Stockley RA. Pattern of emphysema distribution in a1-antitrypsin deficiency influences lung function impairment. Am J Respir Crit Care Med. 2004; 170(11):1172-1178. 26. Holme J, Stockley RA. Radiologic and clinical features of COPD patients with discordant pulmonary physiology: lessons from alpha1-antitrypsin deficiency. Chest. 2007;132(3): 909-915. 27. Holme J, Stockley JA, Stockley RA. Age related development of respiratory abnormalities in non-index a-1 antitrypsin deficient studies. Respir Med. 2013;107(3):387-393. 1928

28. Survival and FEV1 decline in individuals with severe deficiency of alpha1-antitrypsin. The Alpha-1-Antitrypsin Deficiency Registry Study Group. Am J Respir Crit Care Med. 1998;158(1): 49-59. 29. Seersholm N, Wencker M, Banik N, et al. Does alpha1antitrypsin augmentation therapy slow the annual decline in FEV1 in patients with severe hereditary alpha1-antitrypsin deficiency? Wissenschaftliche Arbeitsgemeinschaft zur Therapie von Lungenerkrankungen (WATL) alpha1-AT study group. Eur Respir J. 1997;10(10):2260-2263. 30. Wencker M, Fuhrmann B, Banik N, Konietzko N; Wissenschaftliche Arbeitsgemeinschaft zur Therapie von Lungenerkrankungen . Longitudinal follow-up of patients with alpha(1)-protease inhibitor deficiency before and during therapy with IV alpha(1)-protease inhibitor. Chest. 2001;119(3): 737-744. 31. Chapman KR, Stockley RA, Dawkins C, Wilkes MM, Navickis RJ. Augmentation therapy for alpha1 antitrypsin deficiency: a meta-analysis. COPD. 2009;6(3):177-184. 32. Dirksen A, Dijkman JH, Madsen F, et al. A randomized clinical trial of alpha(1)-antitrypsin augmentation therapy. Am J Respir Crit Care Med. 1999;160(5 pt 1):1468-1472. 33. Parr DG, Sevenoaks M, Deng C, Stoel BC, Stockley RA. Detection of emphysema progression in alpha 1-antitrypsin deficiency using CT densitometry; methodological advances. Respir Res. 2008;9(1):21. 34. Parr DG, Stoel BC, Stolk J, Nightingale PG, Stockley RA. Influence of calibration on densitometric studies of emphysema progression using computed tomography. Am J Respir Crit Care Med. 2004;170(8):883-890. 35. Parr DG, Dirksen A, Piitulainen E, Deng C, Wencker M, Stockley RA. Exploring the optimum approach to the use of CT densitometry in a randomised placebo-controlled study of augmentation therapy in alpha 1-antitrypsin deficiency. Respir Res. 2009;10(75):75. 36. Stockley RA, Parr DG, Piitulainen E, Stolk J, Stoel BC, Dirksen A. Therapeutic efficacy of a-1 antitrypsin augmentation therapy on the loss of lung tissue: an integrated analysis of 2 randomised clinical trials using computed tomography densitometry. Respir Res. 2010;11:136. 37. Chapman KR, Burdon JGW, Piitulainen E et al. IV alpha antitrypsin (A1AT) preserves lung density in homozygous alpha-1-antitrypsin deficiency (A1ATD): a randomized placebocontrolled trial. Am J Resp Crit Care Med. 2013;187:A6069. 38. Gooptu B, Lomas DA. Polymers and inflammation: disease mechanisms of the serpinopathies. J Exp Med. 2008;205(7): 1529-1534. 39. Teckman JH. Liver disease in alpha-1 antitrypsin deficiency: current understanding and future therapy. COPD. 2013; 10(10 suppl 1):35-43. 40. Burrows JA, Willis LK, Perlmutter DH. Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc Natl Acad Sci U S A. 2000;97(4):1796-1801. 41. Travis J, Matheson NR. Properties of mutant forms of alphaproteinase inhibitor prepared by recombinant DNA technology. In: Taylor JC, Mittman C, eds. Pulmonary Emphysema and Proteolysis. Orlando, FL: Academic Press; 1986:133-141. 42. Owen MC, Brennan SO, Lewis JH, Carrell RW. Mutation of antitrypsin to antithrombin. alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder. N Engl J Med. 1983;309(12):694-698. 43. Johnson D, Travis J. The oxidative inactivation of human alpha-1-proteinase inhibitor. Further evidence for methionine at the reactive center. J Biol Chem. 1979;254(10): 4022-4026. Special Features

44. Gadek JE, Fells GA, Crystal RG. Cigarette smoking induces functional antiprotease deficiency in the lower respiratory tract of humans. Science. 1979;206(4424):1315-1316. 45. Carp H, Miller F, Hoidal JR, Janoff A. Potential mechanism of emphysema: a 1-proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity. Proc Natl Acad Sci U S A. 1982;79(6):2041-2045. 46. Stone PJ, Calore JD, McGowan SE, Bernardo J, Snider GL, Franzblau C. Functional alpha 1-protease inhibitor in the lower respiratory tract of cigarette smokers is not decreased. Science. 1983;221(4616):1187-1189. 47. Boudier C, Pelletier A, Gast A, Tournier JM, Pauli G, Bieth JG. The elastase inhibitory capacity and the alpha 1-proteinase inhibitor and bronchial inhibitor content of bronchoalveolar lavage fluids from healthy subjects. Biol Chem Hoppe Seyler. 1987;368(8):981-990. 48. Afford SC, Burnett D, Campbell EJ, Cury JD, Stockley RA. The assessment of a 1 proteinase inhibitor form and function in lung lavage fluid from healthy subjects. Biol Chem Hoppe Seyler. 1988;369(9):1065-1074. 49. Morrison HM, Burnett D, Stockley RA. The effect of catalase and methionine-S-oxide reductase on oxidised alpha 1-proteinase inhibitor. Biol Chem Hoppe Seyler. 1986;367(5): 371-378. 50. Stockley RA, Afford SC. Qualitative studies of lung lavage alpha 1-proteinase inhibitor. Hoppe Seylers Z Physiol Chem. 1984;365(4):503-510. 51. Morrison HM, Kramps JA, Burnett D, Stockley RA. Lung lavage fluid from patients with alpha1-proteinase inhibitor deficiency or chronic obstructive bronchitis: anti-elastase function and cell profile. Clin Sci (Lond). 1987;72(3):373-381. 52. MacNee W. Oxidants and COPD. Curr Drug Targets Inflamm Allergy. 2005;4(6):627-641. 53. Bowler RP, Barnes PJ, Crapo JD. The role of oxidative stress in chronic obstructive pulmonary disease. COPD. 2004;1(2): 255-277. 54. Kindzelskii AL, Zhou MJ, Haugland RP, Boxer LA, Petty HR. Oscillatory pericellular proteolysis and oxidant deposition during neutrophil locomotion. Biophys J. 1998;74(1):90-97. 55. Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal JR. Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest. 1988;82(6):1963-1973. 56. Shapiro SD, Endicott SK, Province MA, Pierce JA, Campbell EJ. Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weaponsrelated radiocarbon. J Clin Invest. 1991;87(5):1828-1834. 57. Hu TH, Tai DI, Changchien CS, Chen TY, Chang WC. Double pylorus: report of a longitudinal follow-up in two refractory cases with underlying diseases. Am J Gastroenterol. 1995;90(5):815-818. 58. Kuhn C, Yu SY, Chraplyvy M, Linder HE, Senior RM. The induction of emphysema with elastase. II. Changes in connective tissue. Lab Invest. 1976;34(4):372-380. 59. Sahebjami H, MacGee J. Changes in connective tissue composition of the lung in starvation and refeeding. Am Rev Respir Dis. 1983;128(4):644-647. 60. Kuhn C III, Starcher BC. The effect of lathyrogens on the evolution of elastase-induced emphysema. Am Rev Respir Dis. 1980;122(3):453-460. 61. Lesser M, Padilla ML, Cardozo C. Induction of emphysema in hamsters by intratracheal instillation of cathepsin B. Am Rev Respir Dis. 1992;145(3):661-668. 62. Damiano VV, Tsang A, Kucich U, et al. Immunolocalization of elastase in human emphysematous lungs. J Clin Invest. 1986; 78(2):482-493. journal.publications.chestnet.org

63. Wang Z, Zheng T, Zhu Z, et al. Interferon gamma induction of pulmonary emphysema in the adult murine lung. J Exp Med. 2000;192(11):1587-1600. 64. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Requirement for macrophage elastase for cigarette smokeinduced emphysema in mice . Science . 1997 ; 277 ( 5334 ): 2002-2004. 65. Shapiro SD, Goldstein NM, Houghton AM, Kobayashi DK, Kelley D, Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. Am J Pathol. 2003;163(6):2329-2335. 66. Churg A, Wright JL. Proteases and emphysema. Curr Opin Pulm Med. 2005;11(2):153-159. 67. Owen CA. Roles for proteinases in the pathogenesis of chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2008;3(2):253-268. 68. Burnett D, Chamba A, Hill SL, Stockley RA. Neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis. Lancet. 1987;2(8567):1043-1046. 69. Woolhouse IS, Bayley DL, Lalor P, Adams DH, Stockley RA. Endothelial interactions of neutrophils under flow in chronic obstructive pulmonary disease. Eur Respir J. 2005;25(4): 612-617. 70. Sapey E, Stockley JA, Greenwood H, et al. Behavioral and structural differences in migrating peripheral neutrophils from patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2011;183(9):1176-1186. 71. Yoshioka A, Betsuyaku T, Nishimura M, Miyamoto K, Kondo T, Kawakami Y. Excessive neutrophil elastase in bronchoalveolar lavage fluid in subclinical emphysema. Am J Respir Crit Care Med. 1995;152(6 pt 1):2127-2132. 72. Subramanian DR, Jenkins L, Edgar R, Quraishi N, Stockley RA, Parr DG. Assessment of pulmonary neutrophilic inflammation in emphysema by quantitative positron emission tomography. Am J Respir Crit Care Med. 2012;186(11):1125-1132. 73. Campbell EJ, Campbell MA, Boukedes SS, Owen CA. Quantum proteolysis by neutrophils: implications for pulmonary emphysema in alpha 1-antitrypsin deficiency. J Clin Invest. 1999;104(3):337-344. 74. Churg A, Zhou S, Wright JL. Series “matrix metalloproteinases in lung health and disease”: matrix metalloproteinases in COPD. Eur Respir J. 2012;39(1):197-209. 75. Cerveri I, Corsico AG, Grosso A, et al. The rapid FEV(1) decline in chronic obstructive pulmonary disease is associated with predominant emphysema: a longitudinal study. COPD. 2013;10(1):55-61. 76. Haruna A, Muro S, Nakano Y, et al. CT scan findings of emphysema predict mortality in COPD. Chest. 2010;138(3): 635-640. 77. Kim WJ, Wood AM, Barker AF, et al. Association of IREB2 and CHRNA3 polymorphisms with airflow obstruction in severe alpha-1 antitrypsin deficiency. Respir Res. 2012;13:16. 78. Sapey E, Wood AM, Ahmad A, Stockley RA. Tumor necrosis factor alpha rs361525 polymorphism is associated with increased local production and downstream inflammation in COPD. Am J Respir Crit Care Med, 2010;15:182(2):192-199. 79. McAloon CJ, Wood AM, Gough SC, Stockley RA. Matrix metalloprotease polymorphisms are associated with gas transfer in alpha 1 antitrypsin deficiency. Ther Adv Respir Dis. 2009; 3(1):23-30. 80. Demeo DL, Campbell EJ, Barker AF, et al. IL10 polymorphisms are associated with airflow obstruction in severe alpha1-antitrypsin deficiency. Am J Respir Cell Mol Biol. 2008;38(1):114-120. 81. Lewis EC. Expanding the clinical indications for a(1)-antitrypsin therapy. Mol Med. 2012;18:957-970. CHEST / 144 / 6 / DECEMBER 2013

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