Alpha-1-antitrypsin Deficiency: Biology, Diagnosis, Clinical Significance, and Emerging Therapies

Alpha-1-antitrypsin Deficiency

Biology, Diagnosis, Clinical Significance, and Emerging Therapies

Noor Kalsheker

University of Nottingham, Nottingham, United Kingdom

Robert Stockley

Birmingham University, Birmingham, United Kingdom

Table of Contents

Cover

Title page

Copyright

List of Contributors

Preface

Acknowledgments

Chapter 1: Historical Perspective

Abstract

Introduction

The discovery of alpha-1-antitrypsin deficiency (AATD)

AATD and association with pulmonary emphysema

AATD and association with liver disease

Other disease associations

Chapter 2: The Swedish Alpha-1-Antitrypsin Screening Study: What We Have Learnt From Birth to Adult Life

Abstract

Introduction

Fetal life

The liver

Chapter 3: Alpha-1-Antitrypsin Gene Regulation

Abstract

Introduction

AAT, acute-phase, and tissue expression

The serpin cluster on 14q31-31.2, the AAT gene, and transcripts

AAT hepatocyte promoter and enhancers

Control of AAT expression in nonhepatic tissue

AAT locus control region and interaction with transcription factors

Chapter 4: Alpha-1-Antitrypsin Variants

Abstract

Introduction

Alpha-1-antitrypsin nomenclature and isoelectric focusing

Noncoding DNA variants

Chapter 5: Alpha1-Antitrypsin: Structure and Dynamics in Health, Disease and Drug Development

Abstract

Introduction

Conformational states associated with function and dysfunction

Importance of dynamics

In vitro studies of Alpha1-Antitrypsin polymerization: lessons and limitations

Translation

Studying polymerization in cells, organisms, and clinical samples

Conclusions

Chapter 6: Novel Biological Functions of A1AT

Abstract

Introduction

A1AT is a broad-spectrum inhibitor of proteases

A1AT as an antiinflammatory and immunomodulatory protein

Modified molecular forms of A1AT and their biological functions

Plasma purified A1AT as an antiinflammatory drug

Chapter 7: AATD and Lung Disease

Abstract

Introduction

Range of severity

Emphysema

Evaluation and progression

Airways disease

Bronchiectasis

Exacerbations

Heterozygotes

Pathophysiology

Other proteinases

Noninhibitory functions of AAT

Treatment

Registries

Conclusions

Chapter 8: Alpha-1-Antitrypsin Deficiency Liver Disease

Abstract

Introduction

Clinical presentation

Diagnosis and management

Heterozygotes and other genotypes

Molecular and cellular pathophysiology of AAT ZZ liver disease

New therapeutic technologies

Conclusions

Chapter 9: Alpha-1-Antitrypsin Deficiency: Epidemiological Studies and Other AATD Associated Diseases

Abstract

The prevalence of alpha-1-antitrypsin deficiency

Evidence that alpha-1–antitrypsin deficiency is under-recognized

Clinical features

Detection and diagnosis of AATD-associated liver disease

Other proposed disease associations

Chapter 10: Thoracic CT Imaging in AATD

Abstract

Introduction

Emphysema: the pathological basis for defining emphysema

The role of computed tomography in phenotyping AATD-associated lung disease

CT assessment of airways disease

Incidental pulmonary nodules

CT lung densitometry

The validation and acceptance of CT densitometry as an outcome measure

Chapter 11: Animal Models of Alpha-1-Antitrypsin Deficiency

Abstract

Role of animal models in understanding lung disease

Proteinase–antiproteinase hypothesis

Naturally occurring animal model of genetic alpha-1 antitrypsin deficiency

Other naturally occurring animal models of emphysema

Cigarette smoking models

Genetically modified animal models

Protease inhibitor knockout mouse models

Transgenic mouse model

Chapter 12: Establishing European Registries for AATD and Clinical Trials for Lung Disease

Abstract

History of choice of cross-sectional data collection for the AIR database

Initial choices of items to collect and definition of a minimal data set for entry of patients

Subsequent additions: gas transfer and health status questionnaire results

Improvements of quality control on data entry

Description of the cross-sectional data collected over 15 years

History of prospective data collection for the AIR database

Spin-off: a database for children with alpha-1-antitrypsin deficiency

Description of prospective data collected over 5 years

Impact of replacement therapy for lung disease

Chapter 13: Establishing the USA Registry: Logistics, Impact, and Early Clinical Trials

Abstract

Introduction

The NHLBI Registry

AAT augmentation therapy in the NHLBI Registry

The Alpha-1 Foundation Research Registry

The Alpha Coded Testing (ACT) Study for home diagnostic testing

Future designs

Conclusions

Acknowledgments

Chapter 14: Gene Therapy

Abstract

Introduction

General principles of gene therapy

Animal models of gene therapy

Features of vectors used in gene therapy studies of AATD

Human trials of gene therapy

Future developments

Conclusions

Chapter 15: The Future of Alpha-1-Antitrypsin Research

Abstract

Summary of progress to date

What are some of the important questions for future research?

Conclusions

Index

Copyright

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List of Contributors

Samuel Alam PhD,     University of Cambridge, Cambridge, United Kingdom

Bibek Gooptu BSc (Hons.), MBBChir, PhD, AFHEA, MRCP

Institute of Structural & Molecular Biology, University College London

London Alpha-1 Antitrypsin Deficiency Service, The Royal Free Hospital

Kings College London, Guy’s Hospital, London

Leicester Institute for Structural & Chemical Biology and NIHR Leicester BRC Respiratory, Leicester, United Kingdom

Marian Hill PhD,     Nottingham University Hospitals National Health Service Trust, Nottingham, United Kingdom

James A. Irving PhD

University College London, Respiratory

Institute of Structural and Molecular Biology, University College London, London, United Kingdom

Alistair Jagger BSc

University College London, Respiratory

Institute of Structural and Molecular Biology, University College London, London, United Kingdom

Sabina Janciauskiene PharmD, PhD, PhD,     Hannover Medical School, Hannover, Germany

Noor Kalsheker MD, MSc, FRCPath,     University of Nottingham, Nottingham, United Kingdom

David A. Lomas PhD, ScD, FHEA, FRCP, FMedSci,     University College London School of Life and Medical Sciences, University College London, London, United Kingdom

Ravi Mahadeva MD,     University of Cambridge, Cambridge, United Kingdom

David Parr MD

University Hospitals Coventry and Warwickshire NHS Trust, Coventry

Warwick University, Warwick, Warwickshire, United Kingdom

S. Tamir Rashid MBBS, PhD,     Centre for Stem Cells & Regenerative Medicine and Institute for Liver Sciences, Kings College London, Guy’s Hospital, London, United Kingdom

Robert A. Sandhaus MD, PhD, FCCP

National Jewish Health, Denver, CO

AlphaNet, Alpha-1 Foundation, Coral Gables, FL, United States

Robert Stockley MD, DSc,     Birmingham University, Birmingham, United Kingdom

Jan Stolk MD, PhD,     Leiden University Medical Center, Leiden, The Netherlands

James K. Stoller MD, MS,     Education Institute Cleveland Clinic, Cleveland, OH, United States

Charlie Strange MD,     Medical University of South Carolina, Charleston, SC, United States

Tomas Sveger MD, PhD,     University Hospital, Malmo, Sweden

Jeffrey H. Teckman MD,     Saint Louis University School of Medicine, Saint Louis, MO, United States

Alice Turner MBChB (Hons), MRCP, PhD,     University of Birmingham, Birmingham, United Kingdom

Preface

The last edition of a textbook entitled Alpha-1-antitrypsin deficiency (AATD) was published in 1996, 33 years after the original discovery of the condition in 1963. We have made enormous progress since then, and this has been the stimulus for writing this book. Much of the earlier and more recent work is described in this book. The advent of powerful biomedical research methods and a collective will to establish patient registries has enabled us to make progress in a number of key areas and should ensure further progress in the years ahead. The idea behind the book was to collate and update the information in the field of AATD and to provide a resource to the AAT community, both researchers and patients.

The editors of this book have contributed to the field of AAT research over several decades and this provided the stimulus to the conception of this textbook. Many other investigators, not all of whom are included in the authorship of this book, have made important contributions to the field. The editors acknowledge the important cumulative effort of all involved that has resulted in the progress made in the field.

This book is divided into 15 chapters beginning with a historical perspective, through to the basic biology of alpha-1-antitrypsin (AAT), the regulation of the gene, the structural biology of the AAT protein, animal models of lung and liver disease, rare variants causing AATD through to the epidemiology, natural history, and clinical presentation of AATD. Patient registries has been established and replacement therapy has been trialled for the lung disease. The three-dimensional structure of the AAT protein molecule has been determined and facilitated our understanding of the elegant mechanism for proteinase inhibition. The propensity for some of the more common molecular variants causing deficiency, particularly the Z variant to polymerize and consequently accumulate in hepatocytes leading to liver disease in a proportion of proteinase inhibitor (Pi) homozygous Z individuals. This has also facilitated the search for synthetic polymer blockers which could be exploited therapeutically to alleviate the liver cell damage that occurs. An increasing array of biological functions in addition to the classical role as a proteinase inhibitor have been described, highlighting the broad-spectrum role of this molecule in the inflammatory state and in regulating aspects of the immune system.

We have learnt about how the gene is regulated and the complex interactions that controls expression of the gene. The use of high throughput screening methods has allowed us to characterize an increasing number of rare variants causing deficiency, providing mechanistic insights into the basis of the deficiency, and in identifying other genetic variants of unknown significance, with some potentially modulating AAT expression.

Importantly, our understanding of the natural progression of the disease, has improved as a result of establishing Registries in Europe and North America in addition to the long-term follow-up of patients from the Swedish Registry. The use of computerized tomography (CT) to better quantify the extent of lung damage that occurs in the emphysema related to AATD will allow for a more objective assessment of responses to AAT replacement therapy which has been trialled in several centers. We are now at the cusp of correction of genetic defects through gene editing, both in the germ line and somatically and it is only a matter of time before human trials of some of these technologies will be tested. Clinical trials and long-term studies of AATD patients should help in the evaluation of replacement therapy in these patients who desperately need ways to delay the onset of progressive disease.

There are many hurdles to cross but the future holds much promise in this area. The AATD patients deserve our urgent attention in making future developments work their way into clinical practice.

Noor Kalsheker

Robert Stockley

Acknowledgments

The editors would like to thank the staff of Elsevier for their help and patience in bringing this book to fruition. In particular, the enormous help of Sam Young who was always there to provide help and advice. NK would like to thank Caroline Kalsheker for the support and encouragement she has given him toward editing this book. Finally, I would like to thank my fellow editor, Rob Stockley, without whom this task would have been truly daunting.

Chapter 1

Historical Perspective

Robert A. Sandhaus, MD, PhD, FCCP

Abstract

The association between alpha-1-antitrypsin deficiency (AATD) and an increased risk of lung disease was described in 1963. In 1969, childhood liver disease and AATD were linked. These descriptions set in motion a cascade of experimental and observational discoveries that have informed our understanding of proteinase-mediated tissue injury, protein misfolding and the cellular responses to such misfolding, and the roles of alpha-1-antitrypsin (AAT) in both health and disease. In addition, the insights gained in studying AATD have contributed to our understanding of much more common diseases of the lung and liver, including chronic obstructive pulmonary disease, liver cirrhosis, and diseases related to protein misfolding. We have learned that AATD is not so rare as initially thought and that some suspected disease associations were likely based on an underappreciation of the prevalence of this genetic condition. This chapter presents one view of the historical arc that led to our current understanding of AATD.

Keywords

historical perspective

pulmonary emphysema

alpha-1-antitrypsin discovery 1963

liver disease 1969

proteinases and animal models

human neutrophil elastase

other disease associations

Introduction

In 1698, Sir John Floyer published A Treatise of the Asthma [1] in which the anatomy of the chest of a broken-winded mare was described. The thorax was said to appear puffed-up or swelled and much larger than that found in the normal horse, although the animal appeared otherwise emaciated. When the chest cavity was entered, the lungs did not collapse as they would in a healthy animal and air blown into the lungs did not appear to come out again and remained … in the over-distended bladders. This, concluded Floyer, was the cause of the dyspnea noted in animals suffering from this condition: external air can’t pass freely thro’ the trachea and its branches in inspiration or expiration; and this difficulty occasions the labor and nisus of the respiratory muscles. Although Bonet correlated dyspnea and orthopnea with autopsy findings of distended lungs in 1679 [2] and Ruysch used the title Vesiculas Pulmonales Obtouctas for an article in 1691 illustrating distended air vesicles in the lung [3], Floyer’s correlation of signs, symptoms, and pathology is considered the first conclusive report of emphysematous anatomy. Margagni reported two cases which would likely be called bullous emphysema today, in 1761 [4].

The pace of examination of obstructive lung disease picked up in the 19th century. In 1808, Badham reported findings similar to Floyer but in human autopsies of people dying with severe dyspnea [5]. Ten years later, Bailie described additional autopsy results of a large number of individuals who died with chronic pulmonary symptoms [5]. He found the same hyperexpanded lungs but noted, in addition, the presence of distended air-filled vesicles on the lung surface and a preponderance of enlarged air spaces enclosed by thin membranes of pulmonary tissue. He suggested that this picture might be due to trapping of inspired air with rupture of contiguous septa.

The earliest accurate clinical observations and anatomic correlations of pulmonary emphysema in humans have been attributed to Rene Laennec. In 1819, Laennec described a dilatation of the air-cells to which the bronchus leads. [6] Many of the pathological descriptions in the past did not have in-life findings on physical examination. Laennec felt this was due to the subtlety of the clinical signs and that his invention of the stethoscope would allow more to be diagnosed during life. After extensive evaluation of the clinical and pathological course of many individuals with obstructive lung disease, Laennec went so far as to propose a pathogenetic mechanism for emphysema which points toward modern disease concepts. He reasoned that since the airways were often partially obstructed by mucus or by the tumefactions of the lining membrane of the bronchi, and since the muscles of inspiration are strong and numerous, whereas expiration is effected by the elasticity of the parts and feeble contractions of the intercostal muscles, inspired air is likely to be trapped, in ever increasing volume, distal to the site of impaired flow. In addition, he suggested that air, inspired at room temperature, would be heated to that of the body in the air-cells and thus further expand the volume of entrapped gas. This entrapment, he reasoned, leads to dilation of the air-cells and must become a fixed and permanent condition. In 1830s, William Stokes adopted Laennec’s stethoscope to evaluate a large number of patients with airway obstruction [7]. He noted the association of long-standing obstructive lung disease with enlargement of the right side of the heart. On the treatment of these patients, Stokes is clear, It seems scarcely possible that any effort of medical skill can restore the lung to its original condition, and all that we can hope for is to palliate the symptoms.

Later in that same decade, George Budd reviewed the emphysema literature of the previous 20 years [8]. His conclusions included a description of the increased antero-posterior diameter of the chest, the loss of rib obliquity, and the use of accessory muscles for inspiration. He went on to suggest that in the early stages of emphysema, small portions of the lung might be affected by this loss of elasticity and these small areas would not empty as completely as the rest of the lung during expiration, thus the air also which these portions contain will be very imperfectly renewed: in fact, they will contribute little to the act of breathing, which will be performed almost entirely by the rest of the lungs. Budd also thought that the loss of alveolar capillary blood supply accounted for the dilation of the right heart and peripheral edema seen in advanced cases of emphysema. Budd felt that it was important to distinguish between emphysema and bronchitis. He pointed out that emphysema often develops itself without the occurrence of pulmonary catarrh.

Of special note, Budd cites a Mr. Jackson who examined a series of 28 patients with emphysema and found that 18 were the offspring of parents with the disease. Budd’s conclusion? Emphysema is very frequently a hereditary disease.

The discovery of alpha-1-antitrypsin deficiency (AATD)

The diagnosis of pulmonary emphysema was difficult in the mid-20th century. Chest radiography gave suggestions of radiolucency in parallel with pruning of the vascular tree on plane films of the thorax. The flattened diaphragms and increased antero-posterior diameter suggested lung destruction but could be simple air-trapping. Pulmonary function testing beyond spirometry was somewhat brutal, asking short-of-breath individuals to swallow balloons on the end of stiff tubing so that lung compliance could be calculated. Many young physicians were taught that while chronic bronchitis could be diagnosed based on symptoms, emphysema was a disease diagnosed most accurately at autopsy, or by lung biopsy during life.

There was certainly a growing appreciation of the role played by cigarette smoking in emphysema and the spectrum of fixed airflow obstruction included in the umbrella term, chronic obstructive pulmonary disease or COPD. But there were always those patients who seemed to develop COPD in spite of minimal cigarette smoke exposure. More than that, some had appreciated a family predilection to the development of COPD, often with symptom onset relatively early in adult life.

It is on this background that alpha-1-antitrypsin deficiency (AATD) was first described by Laurell and Eriksson in 1963. In the mid-20th century, Malmö, Sweden was home to a number of scientists interested in identifying and quantifying plasma proteins. Carl-Bertil Laurell noted the absence of the α1 band in the serum protein electrophoresis (SPEP) of several patients (Fig. 1.1) [9]. He assigned a young clinician, Sten Eriksson, to investigate the potential clinical implications of this finding. Dr. Eriksson found that the missing band appeared in several families and that these families had an unexpectedly high incidence of precocious emphysema. Previous work had described that the α1 band on SPEP was composed predominantly of a protein that had been found to inhibit the pancreatic proteinase, trypsin. In a pair of publications, Laurell and Eriksson postulated an association between familial emphysema and a condition they referred to as AATD [9,10]. It was unclear at the time of the initial descriptions of AATD whether the circulating protein deficiency was due to a primary defect in the alpha-1-antitrypsin (AAT) gene or the processing and clearance of AAT protein within the body. A wonderful recounting by Dr. Eriksson of the initial work done in Malmö was published in 1991 [11] and republished in full in 2013 [12].

Figure 1.1   One of the first paper electrophoresis figures prepared by Sten Eriksson demonstrating the lack of α1 band in serum protein electrophoresis for normal and AATD individuals.

(Gift from Dr. Eriksson to the author).

In their initial reports of five individuals with AATD in 1963, three had evidence of COPD, two were entirely healthy. Laurell and Eriksson took the leap of suggesting a relationship between the deficiency of AAT and the development of pulmonary emphysema. Over the subsequent 2 years, Eriksson had collected a series of 33 individuals with severe AATD and confirmed the hereditary nature of this condition [13]. He noted the basilar predominance and panlobular nature of the emphysema. He also noted the highly variable penetrance of this clinical picture with some homozygous deficient individuals having precocious onset of emphysema while others remained healthy late into life.

As the identification of families continued, it became apparent that AATD was not the result of one particular mutation of the AAT gene. Fagerhol developed a potato starch gel electrophoresis method that could distinguish between the normal protein and the aberrant protein [14]. However, additional abnormal AAT proteins began appearing, many in individuals with similar clinical presentations to the initially described patients. A nomenclature system was developed called the Pi system (for Proteinase inhibitor) and AAT proteins were named by letters of the alphabet designating their speed of migration within the gel. The normal AAT protein, by design, migrated to the middle of the gel and was designate PiM. The initially identified abnormal/deficient protein barely migrated at all from the origin and was designated PiZ. Other letter designations were applied as new AAT variants were discovered. Individuals homozygous for the Z mutation (PiZZ) produce low amounts of circulating AAT protein, usually at a level about 10%–15% of the normal level. If an individual is heterozygotic for two different alleles, both proteins are found in the circulation (e.g., PiMZ). The presence of both proteins in the circulation led to the understanding that AAT is expressed in an autosomal-codominant fashion. A growing list of null mutations has been described ([15] and see Chapter 4). These mutations, designated Pi prefix Q0, lead to the production of no circulating AAT protein. Individuals with two null genes for AAT develop very early onset and rapidly progressive emphysema.

As subsequent chapters will describe, we long ago ran out of letters in the alphabet to describe the hundreds of AAT mutations we know of today. Later work described that AAT protein was coded on the long arm of chromosome 14 by the SERPINA1 gene [16].

By 1969, approximately 6 years after the initial description of AATD, the first descriptions of liver disease associated with this genetic condition began to appear. The serendipitous discovery of a hereditary plasma protein deficiency coupled with the research and intuition to link it to devastating lung and liver disease stands as an almost unique series of events in medicine. While there is a general appreciation that there are likely multiple genetic influences on the risk and severity of a large variety of pulmonary and hepatic conditions, and multiple candidate genes have been identified through modern methods of genetic linkage and genome-wide association studies, it is the personal scientific adventure of two Swedish scientists that has provided the most direct link between an inherited trait and destructive lung disease.

AATD and association with pulmonary emphysema

The ignition of interest in the subtleties of clinical diagnosis of the chronic obstructive lung diseases in the mid-20th century stimulated an equivalent burst of basic research into the pathogenesis of these diseases. The natural occurrence of disease in animals was known for centuries [1]. Horses, cows, whales, and rats had been shown to have spontaneous emphysema-like disease in life. As in man, however, the occurrence of this spontaneous disease was unpredictable and usually complicated in its clinical picture.

Investigators turned their attention to the development of an induced animal model of pulmonary emphysema. Techniques employed reflected, to a large extent, an investigator’s personal prejudice as to the etiology of the disease. Since airway obstruction was thought by some to be the primary lesion, obstructing devices were implanted in airways, often with valve-like mechanisms allowing free inspiration but restriction of expiration. These devices usually produced alveolar dilatation without parenchymal destruction, thus failing to meet current definitions of pulmonary emphysema which require alveolar destruction.

When industrial workers received an accidental exposure to a high concentration of cadmium fumes in 1938 [17] and it was discovered that survivors developed pulmonary emphysema many years later, [18] some studies were published attempting to mimic this mishap using laboratory animals [19–21]. The latter two groups were successful in producing disease in the rat, although both groups found inflammation and scarring to be a prominent component of the reaction to cadmium in solution or aerosol. This complication kept this method from wide use as a disease model. It is interesting to note that there may be some relevance of this model for the human disease, since cigarette smoke contains a significant amount of cadmium salts and elevated cadmium concentrations have been reported in the lungs of persons with severe emphysema [22].

Other methods have been employed with limited success, including cigarette smoke [23], nitrogen oxides ([24], nitrogen dioxide adsorbed to carbon particles [25], airway obstruction plus infection [26], X-irradiation [27], micro embolization into the pulmonary vasculature [28], injection of a sclerosing agent (chlorpromazine) into the bronchial vasculature [29], and inhalation of phosgene [30]. The reasons why these methods did not gain widespread attention in laboratories other than those of the initial investigators is difficult to explain. In some cases, the use of the horse as an experimental animal made use difficult, in others, the etiologic agent was dangerous to handle, and, in still others, the disease was equivocal or complicated in its presentation.

The proposed association between AATD and emphysema was an observational one and was based on a retrospective evaluation of clinical data in a small number of patients. The initial description of AATD found several affected family members with emphysema but there were also AAT deficient family members with no discernable lung disease. One had liver disease, although the clinical description of this patient was recalled only later by Eriksson [11].

A serum protein that was known to inhibit trypsin was missing or low and its transmission appeared to be inherited. The possibility that trypsin played a role in the development of emphysema was almost immediately dismissed. Instilling trypsin into the lungs of laboratory animals seemed to have no effect. But in the United States in 1965, in work concurrent but independent of the work moving forward in Sweden, Gross and associates described an animal model of emphysema produced by the direct intratracheal administration of the proteinase papain, derived from the latex of the papaya fruit [31]. This was followed, in 1968, by Goldring and associates who produced emphysema with aerosolized papain into Syrian hamsters [32]. Both groups reported rapid induction of alveolar destruction with little or no inflammatory reaction. The characteristic lesions developed in a matter of weeks after papain administration. This yielded a convenient, inexpensive method for studying a possible model of the human disease in small laboratory animals. The biggest question to be answered was the relevance of this model to the human disease. Papain appeared unlikely to have any direct etiologic relationship to pulmonary emphysema in man. On the other hand, morphologic and mechanical studies on animal lungs treated with papain showed a remarkable similarity to the changes in function and morphology of the lung in human emphysema [33–35]. Papain induced emphysema became an early, standard laboratory model of the disease.

It was rapidly appreciated that the ability of papain to induce emphysema in rodents and other animals was related to its ability to degrade one particular connective tissue protein in the lung: elastin. In fact, many elastolytic proteinases had been found by this time and evaluations of these various elastases began [36–38]. All were found to be capable of producing the anatomic, histologic, and physiologic changes associated with pulmonary emphysema and in a variety of different animals including rats, mice, guinea pigs, hamsters, and dogs. There was a problem with these models, however, none of the elastases evaluated, whether from a papaya tree, a pig pancreas, or bacteria, seemed