Allergy Frontiers - Volume 4: Diagnosis and Health Economics

Allergy Frontiers: Diagnosis and Health Economics Volume 4

Ruby Pawankar • Stephen T. Holgate Lanny J. Rosenwasser Editors

Allergy Frontiers: Diagnosis and Health Economics Volume 4

Ruby Pawankar, M.D., Ph.D. Nippon Medical School 1-1-5 Sendagi, Bunkyo-ku Tokyo Japan

Lanny J. Rosenwasser, M.D. Children’s Mercy Hospitals and Clinics UMKC School of Medicine 2401 Gillham Road Kansas City, MO 64108 USA

Stephen T. Holgate, M.D., Ph.D. University of Southampton Southampton General Hospital Tremona Road Southampton UK

ISBN: 978-4-431-98293-7 e-ISBN: 978-4-431-98349-1 DOI: 10.1007/978-4-431-98349-1 Springer Tokyo Berlin Heidelberg New York Library of Congress Control Number: 2009928622 © Springer 2009 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Foreword

When I entered the field of allergy in the early 1970s, the standard textbook was a few hundred pages, and the specialty was so compact that texts were often authored entirely by a single individual and were never larger than one volume. Compare this with Allergy Frontiers: Epigenetics, Allergens, and Risk Factors, the present sixvolume text with well over 150 contributors from throughout the world. This book captures the explosive growth of our specialty since the single-author textbooks referred to above. The unprecedented format of this work lies in its meticulous attention to detail yet comprehensive scope. For example, great detail is seen in manuscripts dealing with topics such as “Exosomes, naturally occurring minimal antigen presenting units” and “Neuropeptide S receptor 1 (NPSR1), an asthma susceptibility gene.” The scope is exemplified by the unique approach to disease entities normally dealt with in a single chapter in most texts. For example, anaphylaxis, a topic usually confined to one chapter in most textbooks, is given five chapters in Allergy Frontiers. This approach allows the text to employ multiple contributors for a single topic, giving the reader the advantage of being introduced to more than one viewpoint regarding a single disease. This broad scope is further illustrated in the way this text deals with the more frequently encountered disorder, asthma. There are no fewer than 26 chapters dealing with various aspects of this disease. Previously, to obtain such a comprehensive approach to a single condition, one would have had to purchase a text devoted solely to that disease state. In addition, the volume includes titles which to my knowledge have never been presented in an allergy text before. These include topics such as “NKT ligand conjugated immunotherapy,” “Hypersensitivity reactions to nano medicines: causative factors and optimization,” and “An environmental systems biology approach to the study of asthma.” It is not hard to see that this textbook is unique, offering the reader a means of obtaining a detailed review of a single highly focused subject, such as the neuropeptide S receptor, while also providing the ability to access a panoramic and remarkably in-depth view of a broader subject, such as asthma. Clearly it is intended primarily for the serious student of allergy and immunology, but can also serve as a resource text for those with an interest in medicine in general. v

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I find it most reassuring that even though we have surpassed the stage of the one-volume, single-author texts, because of the wonderful complexity of our specialty and its broadening scope that has evolved over the years, the reader can still obtain an all-inclusive and comprehensive review of allergy in a single source. It should become part of the canon of our specialty. Phil Lieberman, M.D.

Foreword

When I started immunology under Professor Kimishige Ishizaka in the early 1950s, allergy was a mere group of odd syndromes of almost unknown etiology. An immunological origin was only suspected but not proven. The term “atopy,” originally from the Greek word à-topòs, represents the oddness of allergic diseases. I would call this era “stage 1,” or the primitive era of allergology. Even in the 1950s, there was some doubt as to whether the antibody that causes an allergic reaction was really an antibody, and was thus called a “reagin,” and allergens were known as peculiar substances that caused allergy, differentiating them from other known antigens. It was only in 1965 that reagin was proven to be an antibody having a light chain and a unique heavy chain, which was designated as IgE in 1967 with international consensus. The discovery of IgE opened up an entirely new era in the field of allergology, and the mechanisms of the immediate type of allergic reaction was soon evaluated and described. At that point in time we believed that the nature of allergic diseases was a mere IgE-mediated inflammation, and that these could soon be cured by studying the IgE and the various mediators that induced the inflammation. This era I would like to call “stage 2,” or the classic era. The classic belief that allergic diseases would be explained by a mere allergenIgE antibody reaction did not last long. People were dismayed by the complexity and diversity of allergic diseases that could not be explained by mere IgE-mediated inflammation. Scientists soon realized that the mechanisms involved in allergic diseases were far more complex and that they extended beyond the conventional idea of a pure IgE-mediated inflammation. A variety of cells and their products (cytokines/chemokines and other inflammatory molecules) have been found to interact in a more complex manner; they create a network of reactions via their receptors to produce various forms of inflammatory changes that could never be categorized as a single entity of inflammation. This opened a new era, which I would like to call the modern age of allergology or “stage 3.” The modern era stage 3 coincided with the discovery that similar kinds of cytokines and cells are involved in the regulation of IgE production. When immunologists investigated the cell types and cytokines that regulate IgE production, they found that two types of helper T cells, distinguishable by the profile of

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cytokines they produce, play important regulatory roles in not only IgE production but also in regulating allergic inflammation. The advancement of modern molecular technologies has enabled detailed analyses of molecules and genes involved in this extremely complex regulatory mechanism. Hence, there are a number of important discoveries in this area, which are still of major interest to allergologists, as can be seen in the six volumes of this book. We realize that allergology has rapidly progressed during the last century, but mechanisms of allergic diseases are far more complex than we had expected. New discoveries have created new questions, and new facts have reminded us of old concepts. For example, the genetic disposition of allergic diseases was suspected even in the earlier, primitive era but is still only partially proven on a molecular basis. Even the molecular mechanisms of allergic inflammation continue to be a matter of debate and there is no single answer to explain the phenomenon. There is little doubt that the etiology of allergic diseases is far more varied and complex than we had expected. An immunological origin is not the only mechanism, and there are more unknown origins of similar reactions. Although therapeutic means have also progressed, we remain far from our goal to cure and prevent allergic diseases. We have to admit that while we have more knowledge of the many intricate mechanisms that are involved in the various forms of allergic disease, we are still at the primitive stage of allergology in this respect. We are undoubtedly proceeding into a new stage, stage 4, that may be called the postmodern age of allergology and hope this era will bring us closer to finding a true solution for the enigma of allergy and allergic diseases. We are happy that at this turning point the editors, Ruby Pawankar, Stephen Holgate, and Lanny Rosenwasser, are able to bring out such a comprehensive book which summarizes the most current knowledge on allergic diseases, from epidemiology to mechanisms, the impact of environmental and genetic factors on allergy and asthma, clinical aspects, recent therapeutic and preventive strategies, as well as future perspectives. This comprehensive knowledge is a valuable resource and will give young investigators and clinicians new insights into modern allergology which is an ever-growing field. Tomio Tada, M.D., Ph.D., D.Med.Sci.

Foreword

Allergic diseases represent one of the major health problems in most modern societies. The increase in prevalence over the last decades is dramatic. The reasons for this increase are only partly known. While in former times allergy was regarded as a disease of the rich industrialized countries only, it has become clear that all over the world, even in marginal societies and in all geographic areas—north and south of the equator—allergy is a major global health problem. The complexity and the interdisciplinary character of allergology, being the science of allergic diseases, needs a concert of clinical disciplines (internal medicine, dermatology, pediatrics, pulmonology, otolaryngology, occupational medicine, etc.), basic sciences (immunology, molecular biology, botany, zoology, ecology), epidemiology, economics and social sciences, and psychology and psychosomatics, just to name a few. It is obvious that an undertaking like this book series must involve a multitude of authors; indeed, the wide spectrum of disciplines relevant to allergy is reflected by the excellent group of experts serving as authors who come from all over the world and from various fields of medicine and other sciences in a pooling of geographic, scientific, theoretical, and practical clinical diversity. The first volume concentrates on the basics of etiology, namely, the causes of the many allergic diseases with epigenetics, allergens and risk factors. Here, the reader will find up-to-date information on the nature, distribution, and chemical structure of allergenic molecules, the genetic and epigenetic phenomena underlying the susceptibility of certain individuals to develop allergic diseases, and the manifold risk factors from the environment playing the role of modulators, both in enhancing and preventing the development of allergic reactions. In times when economics plays an increasing role in medicine, it is important to reflect on this aspect and gather the available data which—as I modestly assume— may be yet rather scarce. The big effort needed to undertake well-controlled studies to establish the socio-economic burden of the various allergic diseases is still mainly ahead of us. The Global Allergy and Asthma European Network (GA2LEN), a group of centers of excellence in the European Union, will start an initiative regarding this topic this year.

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In volume 2, the pathomechanisms of various allergic diseases and their classification are given, including such important special aspects as allergy and the bone marrow, allergy and the nervous system, and allergy and mucosal immunology. Volume 3 deals with manifold clinical manifestations, from allergic rhinitis to drug allergy and allergic bronchopulmonary aspergillosis, as well as including other allergic reactions such as lactose and fructose intolerances. Volume 4 deals with the practical aspects of diagnosis and differential diagnosis of allergic diseases and also reflects educational programs on asthma. Volume 5 deals with therapy and prevention of allergies, including pharmacotherapy, as well as allergen-specific immunotherapy with novel aspects and special considerations for different groups such as children, the elderly, and pregnant women. Volume 6 concludes the series with future perspectives, presenting a whole spectrum of exciting new approaches in allergy research possibly leading to new strategies in diagnosis, therapy, and prevention of allergic diseases. The editors have accomplished an enormous task to first select and then motivate the many prominent authors. They and the authors have to be congratulated. The editors are masters in the field and come from different disciplines. Ruby Pawankar, from Asia, is one of the leaders in allergy who has contributed to the understanding of the cellular and immune mechanisms of allergic airway disease, in particular upper airway disease. Stephen Holgate, from the United Kingdom, has contributed enormously to the understanding of the pathophysiology of allergic airway reactions beyond the mere immune deviation, and focuses on the function of the epithelial barrier. He and Lanny Rosenwasser, who is from the United States, have contributed immensely to the elucidation of genetic factors in the susceptibility to allergy. All three editors are members of the Collegium Internationale Allergologicum (CIA) and serve on the Board of Directors of the World Allergy Organization (WAO). I have had the pleasure of knowing them for many years and have cooperated with them at various levels in the endeavor to promote and advance clinical care, research, and education in allergy. Together with Lanny Rosenwasser as co-editor-in-chief, we have just started the new WAO Journal (electronic only), where the global representation in allergy research and education will be reflected on a continuous basis. Finally, Springer, the publisher, has to be congratulated on their courage and enthusiasm with which they have launched this endeavor. Springer has a lot of experience in allergy—I think back to the series New Trends in Allergy, started in 1985, as well as to my own book Allergy in Practice, to the Handbook of Atopic Eczema and many other excellent publications. I wish this book and the whole series of Allergy Frontiers complete success! It should be on the shelves of every physician or researcher who is interested in allergy, clinical immunology, or related fields. Johannes Ring, M.D., Ph.D.

Preface

Allergic diseases are increasing in prevalence worldwide, in industrialized as well as industrializing countries, affecting from 10%–50% of the global population with a marked impact on the quality of life of patients and with substantial costs. Thus, allergy can be rightfully considered an epidemic of the twenty-first century, a global public health problem, and a socioeconomic burden. With the projected increase in the world’s population, especially in the rapidly growing economies, it is predicted to worsen as this century moves forward. Allergies are also becoming more complex. Patients frequently have multiple allergic disorders that involve multiple allergens and a combination of organs through which allergic diseases manifest. Thus exposure to aeroallergens or ingested allergens frequently gives rise to a combination of upper and lower airways disease, whereas direct contact or ingestion leads to atopic dermatitis with or without food allergy. Food allergy, allergic drug responses and anaphylaxis are often severe and can be life-threatening. However, even the less severe allergic diseases can have a major adverse effect on the health of hundreds of millions of patients and diminish quality of life and work productivity. The need of the hour to combat these issues is to promote a better understanding of the science of allergy and clinical immunology through research, training and dissemination of information and evidence-based better practice parameters. Allergy Frontiers is a comprehensive series comprising six volumes, with each volume dedicated to a specific aspect of allergic disease to reflect the multidisciplinary character of the field and to capture the explosive growth of this specialty. The series summarizes the latest information about allergic diseases, ranging from epidemiology to the mechanisms and environmental and genetic factors that influence the development of allergy; clinical aspects of allergic diseases; recent therapeutic and preventive strategies; and future perspectives. The chapters of individual volumes in the series highlight the roles of eosinophils, mast cells, lymphocytes, dendritic cells, epithelial cells, neutrophils and T cells, adhesion molecules, and cytokines/chemokines in the pathomechanisms of allergic diseases. Some specific new features are the impact of infection and innate immunity on allergy, and mucosal immunology of the various target organs and allergies, and the impact of the nervous system on allergies. The most recent, emerging therapeutic strategies are discussed, including allergen-specific immunotherapy and anti-IgE treatment, xi

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while also covering future perspectives from immunostimulatory DNA-based therapies to probiotics and nanomedicine. A unique feature of the series is that a single topic is addressed by multiple contributors from various fields and regions of the world, giving the reader the advantage of being introduced to more than one point of view and being provided with comprehensive knowledge about a single disease. The reader thus obtains a detailed review of a single, highly focused topic and at the same time has access to a panoramic, in-depth view of a broader subject such as asthma. The chapters attest to the multidisciplinary character of component parts of the series: environmental, genetics, molecular, and cellular biology; allergy; otolaryngology; pulmonology; dermatology; and others. Representing a collection of stateof-the-art reviews by world-renowned scientists from the United Kingdom and other parts of Europe, North America, South America, Australia, Japan, and South Africa, the volumes in this comprehensive, up-to-date series contain more than 150 chapters covering virtually all aspects of basic and clinical allergy. The publication of this extensive collection of reviews is being brought out within a span of two years and with the greatest precision to keep it as updated as possible. This sixvolume series will be followed up by yearly updates on the cutting-edge advances in any specific aspect of allergy. The editors would like to sincerely thank all the authors for having agreed to contribute and who, despite their busy schedules, contributed to this monumental work. We also thank the editorial staff of Springer Japan for their assistance in the preparation of this series. We hope that the series will serve as a valuable information tool for scientists and as a practical guide for clinicians and residents working and/or interested in the field of allergy, asthma, and immunology. Ruby Pawankar, Stephen Holgate, and Lanny Rosenwasser

Contents

Part I Diagnosis and DD of Allergic Diseases History of Allergy ........................................................................................... Alan Edwards

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Allergy Diagnosis ........................................................................................... Pascal Demoly, Francesco Gaeta, Jean Bousquet, and Antonino Romano

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Nasal and Bronchial Provocation Tests ........................................................ Donald W. Cockcroft, Beth E. Davis, and John K. Reid

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Nasal and Bronchial Nonallergic Provocation Tests ................................... John K. Reid, Donald W. Cockcroft, and Beth E. Davis

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Sputum Tests and Exhaled NO in the Diagnosis and Monitoring of Asthma ................................................................................... Myron Zitt Lung Function and Bronchial Challenge Testing for the Allergist ........... Klaus F. Rabe, Adrian Gillissen, and Zuzana Diamant Investigative Bronchoprovocation and Bronchoscopy in Patients with Bronchial Asthma ............................................................... Hironori Sagara and Ruby Pawankar

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Exhaled NO in Asthma .................................................................................. D. Robin Taylor

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Basophil Activation Tests for Allergy Diagnosis ......................................... Alain L. de Weck and Maria Luisa Sanz

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CD203c for Allergy Diagnosis ....................................................................... Vanessa G. Brown and Madeleine Ennis

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Flow-Assisted Analysis of Basophils: A Valuable Instrument for In Vitro Allergy Diagnosis............................... Didier G. Ebo and Chris H. Bridts

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Recombinant Allergens for the Diagnosis and Treatment of House Dust Mite Allergy................................................. Martin D. Chapman and L. Karla Arruda

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Diagnosis of Occupational Rhinitis .............................................................. Roy Gerth van Wijk

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Asthma: Clinical Descriptions and Definitions ........................................... William K. Dolen

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Occupational Asthma .................................................................................... Joaquin Sastre

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Hypersensitivity Pneumonitis ....................................................................... Moisés Selman and Andrew Churg

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Clinical Aspects and Diagnosis of Atopic Eczema ...................................... Matthias Möhrenschlager, Stephan Weidinger, and Johannes Ring

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Clinical Aspects and Diagnosis of Anaphylaxis........................................... Phil Lieberman

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Angioedema in the Emergency Department................................................ Malcolm W. Greaves and Allen P. Kaplan

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Diagnosis of Aspirin Sensitivity in Aspirin Exacerbated Respiratory Disease ................................................................. Marek L. Kowalski

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Food Allergy: Diagnosis of Food Allergy ..................................................... Scott H. Sicherer

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Evaluation of the Patient with Photosensitivity .......................................... Ann K. Haylett and Lesley E. Rhodes

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Clinical Manifestations of Allergic Diseases: Drug Hypersensitivity .................................................................................... Benno Schnyder and Werner J. Pichler

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State of the Art: Multiple Chemical Sensitivity .......................................... Michael Lacour, Klaus Schmidtke, Peter Vaith, and Carl Scheidt

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Monitoring the Allergic Inflammation ......................................................... Per Venge

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Part II Allergy and Asthma Education Asthma Patient Education ............................................................................ Vanessa M. McDonald and Peter G. Gibson The Costs of Allergy and Asthma and the Potential Benefit of Prevention Strategies .................................................................... Jay M. Portnoy and Mercedes C. Amado

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Outcome Measures in Asthma Management............................................... Michael Schatz

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Index ................................................................................................................

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Contributors

Mercedes C. Amado Children’s Mercy Hospitals and Clinics, 2401 Gillham Road, Kansas City, MO 64108, USA L. Karla Arruda Department of Medicine, School of Medicine of Ribeirão Preto – University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil Jean Bousquet Exploration des Allergies – Maladies Respiratoires et INSERM, Hôpital Arnaud de Villeneuve, University Hospital of Montpellier, 34295 Montpellier, France Chris H. Bridts University of Antwerp, Faculty of Medicine, Immunology – Allergology – Rheumatology, Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerpen, Belgium Vanessa G. Brown Respiratory Research Group, Centre for Infection and Immunity, School of Medicine and Dentistry, Queen’s University Belfast, Belfast BT12 6BN, UK Martin D. Chapman Indoor Biotechnologies, Inc., Charlottesville, VA, USA Andrew Churg Department of Pathology, University of British Columbia, Vancouver, BC, Canada Donald W. Cockcroft Division of Respirology, Critical Care and Sleep Medicine, Royal University Hospital/University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada

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Beth E. Davis Department of Medicine, Division of Respirology, Critical Care and Sleep Medicine, Royal University Hospital/University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada Pascal Demoly Exploration des Allergies -– Maladies Respiratoires et INSERM, Hôpital Arnaud de Villeneuve, University Hospital of Montpellier, 34295 Montpellier, France Zuzana Diamant Centre for Human Drug Research, Leiden, The Netherlands William K. Dolen Allergy-Immunology Section, Departments of Pediatrics and Medicine, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA Didier G. Ebo University of Antwerp, Faculty of Medicine, Immunology – Allergology – Rheumatology, Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerpen, Belgium Alan Edwards The David Hide Asthma and Allergy Research Centre, St. Mary’s Hospital, Newport, Isle of Wight, PO30 5TG, UK Madeleine Ennis Respiratory Research Group, Centre for Infection and Immunity, School of Medicine and Dentistry, Queen’s University Belfast, Belfast BT12 6BN, UK Francesco Gaeta Unità di Allergologia, Complesso Integrato Columbus, via G. Moscati, 31, I-00168 Rome, Italy Roy Gerth van Wijk Section of Allergology, Department of Internal Medicine, Erasmus Medical Centre, Rotterdam, The Netherlands Peter G. Gibson Department of Respiratory and Sleep Medicine, John Hunter Hospital, Lookout Rd, New Lambton, NSW 2305, Australia; Hunter Medical Research Institute, Lookout Rd, New Lambton, NSW 2305, Australia; University of Newcastle, University Drive, Callaghan, NSW 2308, Australia Adrian Gillissen Robert-Koch-Hospital, St. George Medical Center, Leipzig, Germany

Contributors

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Malcolm W. Greaves London Allergy Clinic, 66, New Cavendish St London W1 and Institute of Dermatology, St Thomas’ Hospital, London SE1, UK; Guy’s Kings and St Thomas’ Schools of Medicine and Dentistry, University of London, London, UK Ann K. Haylett Photobiology Unit, Dermatological Sciences, University of Manchester, Salford Royal Foundation Hospital, Stott Lane, Manchester, M6 8HD, UK Allen P. Kaplan National Allergy and Urticaria Centres of Charleston, Charleston, South Carolina, USA Marek L. Kowalski Department of Immunology, Rheumatology and Allergy, Chair of Immunology, Faculty of Medicine, Medical University of Łód , Łód , Poland Michael Lacour Department of Psychotherapy and Psychosomatic Medicine, Freiburg University Hospital, Hauptstraße 8, D-79104 Freiburg, Germany Phil Lieberman Division of Allergy and Immunology, Departments of Medicine and Pediatrics, University of Tennessee, Memphis, TN, USA Vanessa M. McDonald Department of Respiratory and Sleep Medicine, John Hunter Hospital, Lookout Rd, New Lambton, NSW 2305, Australia; Hunter Medical Research Institute, Lookout Rd, New Lambton, NSW 2305, Australia; University of Newcastle, University Drive, Callaghan, NSW 2308, Australia Matthias Möhrenschlager Division of Environmental Dermatology and Allergology GSF/TUM, Department of Dermatology and Allergy Biederstein, Technical University of Munich, Biedersteiner Street 29, D-80802 Munich, Germany Ruby Pawankar Nippon Medical School, Tokyo, Japan Werner J. Pichler Division of Allergology, Clinic for Rheumatology and Clinical Immunology/ Allergology, Inselspital, University of Bern, 3010-Bern, Switzerland Jay M. Portnoy Children’s Mercy Hospitals and Clinics, 2401 Gillham Road, Kansas City, MO 64108, USA

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Klaus F. Rabe Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands John K. Reid Department of Medicine, Division of Respirology, Critical Care and Sleep Medicine, Royal University Hospital/University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada Lesley E. Rhodes Photobiology Unit, Dermatological Sciences, University of Manchester, Salford Royal Foundation Hospital, Stott Lane, Manchester, M6 8HD, UK Johannes Ring Division of Environmental Dermatology and Allergology GSF/TUM, Department of Dermatology and Allergy Biederstein, Technical University of Munich, Biedersteiner Street 29, D-80802 Munich, Germany Antonino Romano Unità di Allergologia, Complesso Integrato Columbus, via G. Moscati, 31, I-00168 Rome, Italy; IRCCS Oasi Maria S.S., Troina, Italy Hironori Sagara Department of Respiratory Medicine, Dokkyo Medical University Koshigaya Hospital, Japan Maria Luisa Sanz Department of Allergology and Clinical Immunology, Clinica Universitaria, University of Navarra, 31080 Pamplona, Spain Joaquin Sastre Fundacion Jiménez Diaz, Servicio de Alergia, Av. Reyes Catolicos 2, 28040 Madrid, Spain Michael Schatz Department of Allergy, Kaiser Permanente Medical Center, San Diego, CA, USA Carl Scheidt Department of Psychotherapy and Psychosomatic Medicine, Freiburg University Hospital, Hauptstraße 8, D-79104 Freiburg, Germany Klaus Schmidtke Center for Geriatric Medicine and Gerontology, Freiburg University Hospital, Lehener Straße 88, D-79106 Freiburg, Germany Benno Schnyder Division of Allergology, Clinic for Rheumatology and Clinical Immunology/ Allergology, Inselspital, University of Bern, 3010-Bern, Switzerland

Contributors

Moisés Selman Instituto Nacional de Enfermedades Respiratorias, México Scott H. Sicherer The Elliot and Roslyn Jaffe Food Allergy Institute, Division of Allergy and Immunology, Department of Pediatrics, Mount Sinai School of Medicine, New York, NY, USA D. Robin Taylor Dunedin School of Medicine, University of Otago, P.O. Box 913, Dunedin, New Zealand Peter Vaith Department of Rheumatology and Clinical Immunology, Freiburg University Hospital, Hugstetter Straße 55, D-79106 Freiburg, Germany Per Venge Department of Medical Sciences, Clinical Chemistry, and Asthma and Allergy Research Centre, Uppsala University, Uppsala, Sweden Alain L. de Weck Department of Allergology and Clinical Immunology, Clinica Universitaria, University of Navarra, 31080 Pamplona, Spain Stephan Weidinger Division of Environmental Dermatology and Allergology GSF/TUM, Department of Dermatology and Allergy Biederstein, Technical University of Munich, Biedersteiner Street 29, D-80802 Munich, Germany Myron Zitt Medicine, State University of New York, Stony Brook Medical Center, 9 Cypress Drive, Woodbury, NY 11797, USA

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History of Allergy Alan Edwards

The term ‘Allergy’ was first used by the Austrian paediatrician, Clemens von Pirquet (1874–1929) (Fig. 1), in a paper in the journal Münchener Medizinische Wochenschrift in 1906 [1]. In the latter years of the nineteenth century, great progress had been made in the treatment of diseases such as tetanus and diphtheria using antitoxins raised in animals by the injection of a toxin. Emil von Behring (1854–1917) carried out the first successful treatment of a diphtheria patient using diphtheria antitoxin raised in dogs in 1891. However, it had also been recognised that giving an animal repeated injections of a toxic substance did not always provide protection against that toxin, immunity; sometimes it resulted in hypersensitivity or supersensitivity, the latter term used by Carl Praunitz [2] in his English translation of von Pirquet’s paper. von Behring described the sudden death of animals hyperimmunised against tetanus that occurred when they were subsequently injected with a small dose of the same toxin as a ‘paradoxical reaction’. Charles R. Richet (1850–1935) was among the first to recognise the significance of supersensitivity. He was also the first to use another important word, anaphylaxis. In a joint paper with Paul J. Portier (1866–1962) in the Bulletin of the French Biological Society in 1902 [3], he used it to describe the attribute which certain poisons possess of increasing instead of diminishing the sensitivity of an organism to their action. He described anaphylaxis as the condition opposite to protection (phylaxis). To return to the paper by von Pirquet, he debated whether immunity and supersensitivity were separate entities or were related. He concluded that they were closely interrelated. He went on to write: What we need is a new generalised term, which prejudices nothing but expresses the change in condition which an animal experiences after contact with any organic poison, be it animate or inanimate. The vaccinated person behaves towards vaccine lymph, the syphilitic towards the virus of syphilis, the tuberculous patient towards tuberculin, the person injected with serum A. Edwards () The David Hide Asthma and Allergy Research Centre, St. Mary’s Hospital, Newport, Isle of Wight, PO30 5TG, UK e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_1, © Springer 2009

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Fig. 1 Clemens Freiherr von Pirquet (From [31])

towards the serum, in a different manner from him who has not previously been in contact with such an agent. Yet he is not insensitive to it. We can only say of him that his power to react has undergone a change.

For this general concept of a changed reactivity I propose the term Allergy. ‘Allos’ implies deviation from the original state, from the behaviour of the normal individual, as it is used in the words Allorhythmia, Allotropism.

Further on in the paper, he distinguishes between Allergen and Antigen. The word antigen implies a substance capable of giving rise to an antibody. The term Allergen is more far reaching. The allergens comprise, besides the antigen proper the many protein substances which lead to no production of antibodies but to supersensitivity.

The paper was written before the identification of IgE antibodies and the mechanisms of supersensitivity were unknown. Finally von Pirquet wrote: The term immunity must be restricted to those processes in which the introduction of the foreign substance into the organism causes no clinically evident reaction, where, therefore, complete insensitivity exists

Over the years the term allergy has lost its original definition as provided by von Pirquet whereby it just implied a changed reactivity and is now used synonymously with hypersensitivity or supersensitivity. In a lecture on the hypersensitivity reactions given in 1991 [4], R.R.A. Coombs (1921–2006) argued for a return to von Pirquet’s original concept which he illustrated as shown in Fig. 2.

History of Allergy

5 Clinical Outcome

Biological Response

Allergic resp. (prejudices nothing)

IMMUNITY (protective) HYPERSENSITIVITY (Harmful)

Immune resp. (prejudiced)

IMMUNITY (protective) ALLERGY (harmful)

Fig. 2 Biological response and clinical outcomes (From [4])

Previous History In the years before von Pirquet coined the word allergy, conditions and diseases that probably resulted from allergy had been described and documented. Although there are many examples it is perhaps worth recording a few of the more interesting cases.

Hippocrates (460–375 BC) Amongst the writings of Hippocrates is the first mention of the diseases asthma and eczema. Asthma is a word of Greek origin meaning ‘panting’. Eczema similarly comes from a Greek root meaning ‘to bubble, boil, burst forth’. Hippocrates was one of the first to describe food allergy: [C]heese does not harm all men alike; some can eat their fill of it without the slightest hurt, nay, those it agrees with are wonderfully strengthened thereby. Others come off badly. So the constitutions of these men differ, and the difference lies in the constituent of the body which is hostile to cheese, and is roused and stirred to action under its influence.

Mithridates VI Eupator (132 BC) Mithridates the Great was king of Pontus in Asia Minor. He experimented with poisons and antidotes to poisons. When he was unable to find a good antidote, in order to guard himself against certain poisons he deliberately took very small doses of the poisons which he gradually increased until he became ‘immune’ to them. He gave us the word ‘mithridate’ defined by Webster’s dictionary as ‘an antidote against poison’.

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Leonardo Botallo (1519–1588) Leonardo Botallo, an Italian surgeon, was the first to describe allergic rhinitis. He had a patient who hated roses, since they caused his nose to itch intolerably, made him sneeze and gave him nasty headaches. This condition he called ‘rose fever’. Rose fever has been described by other physicians and has been used as a synonym of hay fever. A Roman Cardinal, Oliveri Caraffa, is said to have posted guards outside his palace with orders to send away any visitor who came with a bunch of roses.

Pietro Andrea Mattioli (1501–1577) Pietro Andrea Mattioli was an Italian doctor who described the first case of cat allergy. His patient was so sensitive to cats that if he was sent into a room with a cat he reacted with agitation, sweating and pallor.

Bernardo Ramazzini (1633–1714) Bernardo Ramazzini was a Professor of Theory of Medicine at the University of Modena, Italy. In his treatise, Diseases of Tradesmen, he described how Bakers, Millers, and Sifters and Measurers of grain often suffered as a result of inhaling the flour, what is now called Baker’s Asthma: [T]he throat is choked and dried up with dust, the pulmonary passages become coated with a crust formed by the dust, and results in a dry and obstinate cough; the eyes are much inflamed and watery; and almost all are short of breath and cachectic and rarely reach old age.

John Bostock (1773–1846) In 1819 John Bostock, a medical graduate of the University of Edinburgh, Scotland, in a talk to the Royal Medical and Chirurgical Society of London, described a condition he termed Catarrhus aestivus: About the beginning or middle of June in every year the following symptoms make their appearance, with a greater or less degree of violence. A sensation of heat and fullness is experiences in the eyes. The eyes become extremely inflamed and discharge very copiously a thick mucous fluid. To this succeeds irritation of the nose, producing sneezing, which occurs in fits of extreme violence, coming on at uncertain intervals. To the sneezings are added a further sensation of tightness of the chest, and a difficulty in breathing, with a general irritation of the fauces and trachea.

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Pollen and Hay Fever Two physicians, Morrill Wyman in the USA and Charles Blackley in England, connected the symptoms of seasonal allergies with pollens from plants.

Morrill Wyman (1812–1903) Morrill Wyman was a graduate of Harvard Medical School, Massachusetts, USA. He had noted that hay fever did not occur in the White Mountains of New Hampshire. In 1872 he described how he gathered some Roman wormwood flowers in full bloom, covered with pollen, carried them to the White Mountain Glen about 1200 feet above tide. The parcel [that] was containing it was then opened and freely sniffed by myself and son. We were both seized with sneezing and itching of nose, eyes, and throat with a limpid discharge. My nostrils were stuffed and my uvula swollen, without cough, but with the other usual symptoms of Autumnal Catarrh. These troubles continued through the night and did not disappear till the afternoon following.

Roman wormwood is a member of the same botanical genus as ragweed, perhaps the commonest cause of allergic rhino-conjunctivitis in the USA.

Charles H. Blackley (1820–1900) Charles H. Blackley (Fig. 3) was a qualified M.R.C.S. from the Pine Street Medical School, affiliated with Manchester Royal Infirmary in England in 1858. He studied the causation of hay fever and hay asthma in himself. He published his findings in two books, Experimental Researches on the Causes and Nature of Catarrhus Aestivus (1873) and Hay Fever: Its Causes, Treatment and Effective Prevention, Experimental Researches (1880). Blackley started his experiments with grass and other pollens which were applied to the mucous membranes of the nose producing the symptoms of hay fever. Application to the conjunctiva or inhalation produced conjunctivitis and asthma, respectively. His other major experiment was to carry out pollen counts in the atmosphere and relate this to the prevalence of hay fever and to the intensity of the symptoms. For this purpose he devised an instrument that arranged for the exposure of microscope slides covered in glycerine under a little roof and these were exposed for 24 h before they were removed and the number of pollen grains counted under the microscope (Fig. 4). In 1866, for example, pollen counts were found to increase in early June, reach a peak in late June and fall off during July. The principles for pollen counting have not changed much to this day. Blackley also sent microscope slides up into the atmosphere on kites. He found the pollen counts to be greater at higher rather than at lower levels and that pollen could be blown long distances in the wind.

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Fig. 3 Dr Charles Blackley (From [32])

Fig. 4 Pollen trap built by Dr Charles Blackley (From [32])

Blackley’s conclusions that pollen was the cause of allergic rhino-conjunctivitis and asthma were not fully accepted by his contemporaries as they were published at a time when germs in the form of bacteria were thought to be the likely cause of all diseases.

William P. Dunbar (1863–1922) Although born in St. Paul, Minnesota, USA, William P. Dunbar studied medicine at Giessen, Germany, and remained there for his working life. In 1893 he became Director of the State Hygiene Institute at Hamburg. In 1901 he was joined by Carl Prausnitz, who became his assistant. The main work of the Institute at that time was dealing with cholera epidemics and also with outbreaks of plague, typhoid, diphtheria and tuberculosis. Both Dunbar and Prausnitz suffered from hay fever and despite their busy life were able to carry out experiments in this disease. Dunbar was sure that the activity of pollen was derived from a proteid which he extracted using salt solutions and then purified. He reported: Normal persons were unaffected even by the instillation of concentrated proteid solutions into the eye or nose. The majority of hay fever patients were affected by one drop of a solution of 1:20,000 to 1:30,000. But some patients were found susceptible to one drop of a million fold dilution. This corresponds to the contents of two or three pollen grains [5].

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In his account of the life of Carl Prausnitz, David Hide [6] tells of some of the experiments carried out by Dunbar and Prausnitz on themselves. Prausnitz injected himself subcutaneously in the forearm with a solution of the grass pollen proteid. Within the next half hour very severe symptoms developed in the mucous membranes of the eye, nose and mouth. He suffered from pain in the chest, expectorated tough mucous sputum and perspired profusely. Respiration was accelerated and difficult, the pulse rate quickened and the voice became hoarse. Prausnitz described how he and Dunbar were working together on a pollen antitoxin, Pollantin, a preparation obtained by immunising horses with pollen proteid. On one occasion Prausnitz administered an injection of the horse serum extract to Dunbar, who fell unconscious to the floor. Hypersensitivity to the horse serum among a number of recipients of Pollantin caused Dunbar and Prausnitz to abandon their pioneering work on desensitising for hay fever; otherwise they might have been credited with starting this advance in medical practice. There are many other great scientists who have contributed to our understanding and knowledge of allergy. Mainly these were reports on the clinical manifestations of allergy, particularly asthma, urticaria, hay fever and food allergy.

Desensitisation It was fortuitous that at the same time that Dunbar, Prausnitz and Blackley were investigating the causes of hay fever much work was going on in the treatment of bacterial diseases such as diphtheria, tetanus, cholera and rabies by using antibodies to the toxins or the bacteria raised in animals. Dunbar’s view that hay fever was caused by a ‘pollen toxin’ and their attempts to develop an antitoxin failed. At the same time as diseases were being treated with passive immunisation, active immunisation with altered cultures of bacteria was also beginning to be used. One of the centres was the Department of Therapeutic Inoculation at St. Mary’s Hospital, London, England, established by Sir Almroth Edward Wright (1861–1947). He was responsible for the development of a vaccine for typhoid fever given successfully to thousands of British soldiers during the First World War and he also developed vaccines for tuberculosis, cholera and staphylococcal infections. The department at St. Mary’s Hospital soon gained an international reputation and Wright was joined in the early 1900s by two research fellows, Leonard Noon (1877–1913) and John Freeman (1877–1962). Working together and using extracts of Timothy grass pollen (Phleum pratense) collected by Noon’s sister Dorothy, they established a protocol for the diagnosis and treatment of hay fever using active immunisation. Treatment consisted of giving gradually increasing doses of pollen extract. The diagnosis was confirmed by dropping a small amount of pollen extract into the eye and observing the reaction. This technique was also used to monitor the effect of treatment. The importance of their work was the care they employed in preparing, quantifying and administering the pollen extracts. For quantifying the strength of the extracts they used the Noon unit, which is the amount of extract obtained from 1 µm of P. pratense. This unit has been used since

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that time although other units of allergen activity have been used in other countries. Leonard Noon died from tuberculosis in 1913, shortly after publishing the first article on desensitisation in The Lancet in 1911 entitled ‘Prophylactic inoculation against hay fever’ [7]. The work on establishing the dosages and protocols needed in the treatment of allergies using subcutaneous injections of increasing doses of allergen extracts was continued by Freeman until his death in 1962. Initially injections were given at the clinic at intervals of once a week to once a month. In later years Freeman established a special hay fever clinic where the patients were instructed how to give themselves the pollen injections. They were taught to inject themselves daily with the correct dose of the pollen extract from the correct bottle of ten provided. Patients were also taught how to deal with a reaction should it occur by giving themselves an injection of adrenaline, which was also provided. Daily injections were given for 54 days and great care was taken to ensure that all patients, including children, knew exactly what dose to give each day. Freeman also invented ‘rush’ inoculation for patients who came to the clinic just before the season started [8]. Eight injections were given at the hospital every 2–3 h starting at 5 am and ending at 11 pm over the course of 7 days. The allergy clinic at St. Mary’s Hospital became one of the busiest in the world. Supplies of pollens became a critical factor and in 1936 the Pollenarium was built on land in Surrey, England, where the plants were grown and the pollen collected. Freeman was succeeded as Director of the Department of Allergy Department by Dr A.W. (Bill) Frankland who together with Rosa Augustin carried out and published the first controlled trial of desensitisation [9]. Desensitisation using increasing doses of allergen extracts delivered by subcutaneous injections became the established treatment for allergic disease throughout the world, and has remained so to this day. Standardisation of allergen dose and the production of allergen extracts were taken over by commercial companies. Other routes of delivering the allergen have been tried over the years. The use of a tincture or syrup of allergens as oral drops was first reported by H. Holbrook Curtis in 1900 [10], and by J. Harvey Black in 1927 [11]. Double-blind placebo-controlled trials using the oral route for the treatment of inhalant allergy were published in the 1980s but in more recent years the administration of allergen extracts delivered sublingually either as drops or tablets has become more common.

Immunoglobulin E Von Pirquet had recognised that the supersensitive reactions he described resulted from antigen–antibody reactions and attempts were made to identify the antibodies in the sensitised individuals using the techniques of precipitation, complement binding and neutralisation prevalent at the time. The first published work that identified the presence of specific allergy antibodies in the serum of sensitised individuals was the paper entitled ‘Studies on Supersensitivity’ by Carl Prausnitz (1876–1963) and Heinz Küstner (1897–1963) in 1921 [12]. [The English quotations from the

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original German paper are Carl Prausnitz’s own translations published in the first three editions of Clinical Aspects of Immunology [13]. This paper contains so much detail relevant to our understanding of allergy today that it should perhaps be required reading for every aspiring allergist. Both suffered from supersensitivity: Prausnitz suffered from grass pollen hay fever and Küstner was very sensitive to heated but not raw white fish. They investigated the nature of the antigen: A standard solution was prepared by mincing fresh marine fish (usually haddock), boiling it in ten times its weight of distilled water, filtering through paper and sterilizing the filtrate for half an hour in the steam sterilizer. The clear, non-opalescent fluid this obtained proved inactive on the patient’s conjunctiva, but highly active on intradermal injection. After intradermal injection of 0.1 cc. of the standard solution, there developed at the site of the injection within 10 minutes a very itching wheal which rapidly, under our eyes, grew to about 4 cm in diameter. The fully developed wheal was raised high above the surrounding skin, white, with an indented margin; it was surrounded by a deep red flare about 10 cm wide. After 20 minutes there developed the syndrome of severe generalised intoxication previously described (urticaria of the entire body, intense congestion of the conjunctivae and upper air passages, irritating cough, dyspnoea).

They showed that a distinct local reaction could be obtained by 0.1 cc. of a 1,000-fold dilution of the standard solution but not by 0.1cc. of a 10,000-fold dilution. After more work to characterise the nature of the antigen and to compare the effects of fish antigen in Küstner and pollen antigen in Prausnitz they tried to produce passive anaphylaxis in guinea pigs by intraperitoneal injection of the fish-supersensitive patient’s serum followed 24 h later by intravenous injection of standard fish solution. When this failed to produce a result they attempted to transfer the supersensitive state passively to non-susceptible human beings. They did this by injecting both reactants (serum and antigen) intradermally into the same spot of skin. The critical experiment which is a lesson in controlled trials is described as follows: On July 19th 1920, a person, not sensitive to fish solution received into abdominal skin intradermal injections of 0.1cc. of the following substances: (1) Serum of the fish-sensitive patient, undiluted; (2) Serum of the fish-sensitive patient, diluted 1 in 10; (3) Serum of the fish-sensitive patient, diluted 1 in 100; (4) Serum of a healthy person, free from any idiosyncrasy; (5) Normal saline solution On July 20th 1920, 0.1 cc of standard fish solution was injected into each of these spots and (6) into an untreated spot of skin. The arrangement of the wheals was as follows:

Table 1 Arrangement of injections into the abdominal skin. Experiment carried out by Carl Prausnitz and Heinz Küstner [12] 4 1 Right 3 Navel 5 Left 6 2

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A. Edwards After 15 minutes a marked subjective and objective reaction was present only in the spots 1, 2 and 3 pretreated with the specific serum. It was strongest where the undiluted serum had been administered. The control wheals 4, 5 and 6 showed only a trifling traumatic reaction.”

More details are provided and they concluded: The specific transfer of fish supersensitivity to normal, not fish-sensitive human beings, was successfully accomplished.

This technique of demonstrating and identifying skin-sensitising antibodies by transfer in the serum of sensitising individuals known as the Prausnitz–Küstner reaction or P-K test remained a standard test in the identification of allergy antibodies and diagnosis of allergy for many years until it was stopped because of the risk of transferring virus hepatitis. Over the next few years a number of investigators confirmed the work of Prausnitz and Küstner. The sensitising bodies transferred in the serum and responsible for the specific skin reaction were called ‘atopic reagins’ by Coca and Grove [14]. Over subsequent years attempts were made to characterise it. Initially, it was thought to be immunoglobulin A until it was shown that reaginic activity occurred in a patient with IgA deficiency. In the 1960s, working at the Children’s Asthma Research Institute and Hospital in Denver, Colorado, USA, the husband and wife team of Kimishige and Teruko Ishizaka, using the reaginrich fraction of the serum of a patient supersensitive to ragweed, raised antibodies to the reagin fraction in rabbits. They showed that the reaginic activity was due to a new and distinct class of immunoglobulins to which they gave the name of γE-globulin [15]. Essentially all the reaginic activity in the sera of ten patients was lost upon absorption of the sera with an anti-γE serum which did not contain any antibody against γG-, γA, γM, or γD-globulins.

At about the same time, Hans Bennich and S. Gunnar Johansson, working at the Institute of Biochemistry, University of Uppsala, Uppsala, Sweden, discovered an atypical protein obtained from a myeloma patient. They called the protein IgND after the initials of the patient. The abstract of a joint paper they published with the Ishizakas reads as follows [16]: IgND, represented by a myeloma protein ND and γE-globulin, detected in and isolated from atopic patients’ sera with reaginic activity, belong to the same immunoglobulin class, which is distinct from IgG, IgA, IgM and IgD. The antigenic determinants characteristic of the fifth immunoglobulin class, which should be designated IgE, are localized in the Fc portion of the molecule. The association of reaginic activity with IgE was confirmed by the fact that anti-γE globulin as well as an antiserum specific for the Fc portion of myeloma-IgND precipitated the reaginic factor in atopic patients’ sera.

At a conference in 1968 in Lausanne, Switzerland, of the WHO Reference Centre for Immunoglobulins, the meeting concluded that reaginic antibodies were a new, fifth class of human immunoglobulins to be called IgE.

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Food Allergy and Food Intolerance It is interesting that one of the first recorded cases of allergy was by Hippocrates about the constitutions of some men differing with respect to their ability to tolerate cheese and the identification by Prausnitz and Küstner of a transferable fraction in the blood of a patient allergic to fish. Over the centuries the exact mechanisms involved in food allergy have probably been the least investigated. By a series of detailed analyses, Prausnitz and Küstner had identified that the likely causative factor in the fish to which Küstner was sensitive was a ‘fairly high molecular decomposition product of fish protein’. Using the P-K reaction as the end point, Matthew Walzer, in a series of experiments in the 1920s, showed that both normal and sensitised individuals absorbed undigested protein that reacted with sensitised skin to produce positive reactions [17]. In a series of challenge experiments with egg albumin in patients sensitised to egg and control subjects to egg, Claudio Carini, Jonathan Brostoff and Deryck Wraith showed that egg-sensitised patients absorbed greater quantities of egg albumin compared to control subjects [18].

Chemical Mediators of Allergy and the Mast Cell The range of inflammatory mediators, cytokines and other chemicals derived from the cells involved in allergy is now very large. It all started, however, with the recognition that the release of histamine is the main mediator giving rise to the symptoms of allergy. In 1909, William H. Schultz (1873–1953), working at the Hygienic Laboratory of the Public Health and Marine Hospital Service in the USA, published his experiments on the effects of anaphylaxis on smooth muscle [19]. Using an isolated strip of guinea pig intestine suspended in a bath of physiologic solution he showed that intestine from guinea pigs that had been sensitised with horse serum contracted more vigorously than intestine from non-sensitised animals, when treated with dilute horse serum. His position in the laboratory was as Associate Pharmacologist and he concluded the following from his experimental results: These experiments furnish the first direct proof of cytological sensitization and if the serum acts upon the receptive substance of the cell, a good pharmacological explanation is possible for the action of such substances as atropine, chloral, etc. in alleviating the symptoms of acute anaphylaxis.

In 1911, Henry H. Dale (1875–1968), working as a pharmacologist at the Wellcome Physiological Research Laboratories in London, England, and using uterine muscle strip of the guinea pig, showed a similar contractile response to that demonstrated by Schultz but challenged the muscle strip with β-iminazolylethamine (histamine) [20]. He concluded that histamine was the chemical mediator that

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caused anaphylaxis. The results of the experimental procedure became known as the Schultz–Dale phenomenon and became the basis for the identification and assay of anti-allergic and antihistamine drugs. The existence of another chemical in addition to histamine that caused spasm of isolated muscle strips was first identified by Joseph Harkavy (1890–1980) in the sputum of asthmatics in 1925. In the late 1930s, a group of workers under the direction of Charles H. Kellaway (1899–1952) showed that the contraction of the smooth muscle caused by snake venoms was neither due to the direct effect of the venom nor brought about by the release of histamine. They showed that a chemical substance they called the ‘slow-reacting smooth muscle stimulating substance’ was released from tissue constituents by enzymatic action of the venoms. They then carried out a series of experiments to see if the same thing occurred in the antigen–antibody reaction of anaphylaxis. The injection of antigen into the perfused lung of sensitised guinea pigs caused the liberation of histamine and in addition a substance that appears in the perfusate after the anaphylactic reaction. They called this substance S.R.S. [21]. This became known in later years as S.R.S.-A or the Slow Reacting Substance of Anaphylaxis. It was another 25 years before S.R.S.-A was characterised as the leukotrienes and their role in asthma, allergy and inflammation described by Bengt I. Samuelsson (1934–) [22]. The main source of histamine was identified as the mast cell by James F. Riley (1912–1985) and Geoffrey B. West (1916–) in 1953 [23]. This focused work on the mast cell as the central cell involved in allergic inflammation has remained valid to this day (Fig. 5).

Allergen Challenge Tests Exposing the body surface to allergens has long been used as a means of confirming the diagnosis of hypersensitivity to the allergen concerned. In the past years food was the allergen most often used. Both Morrill Wyman and Charles Blackley sniffed pollen into their noses to demonstrate that it was the pollen that caused their symptoms of hay fever. Inhalation of the relevant allergen into the bronchial tree results in an asthmatic reaction in sensitised subjects and, as the degree of bronchoconstriction produced can be measured accurately, this has become the most widely used method of increasing our understanding of allergic reactions. It has also been used as a screening test for drugs used in the treatment of allergic disease. Inhalation of house dust extract to produce an asthmatic reaction was started in the 1930s [24], but the effects were recorded by auscultation. It was Irving W. Schiller and Francis C. Lowell in the 1940s who began to use the reduction in vital capacity as the measurement of the effects of inhalation [25].

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Fig. 5 Mast cell (From Fisons Pharmaceutical Division, Research and Development Laboratories, Loughborough, UK)

In the 1963 edition of their textbook Clinical Aspects of Immunology [13], Gell and Coombs first presented the classification, Types I–IV, of the various hypersensitivity reactions. Recognition that the different types of allergic reactions could occur following a single exposure to an antigen was examined in detail in bronchial antigen challenge experiments by allergy departments in London, England [26, 27], and in Groningen, the Netherlands [28]. These experiments showed very clearly that in addition to immediate reactions occurring 10–20 min after allergen exposure there were also late reactions that occurred 4–8 h after exposure. Such reactions occurred either one after the other or were isolated immediate or late reactions.

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These observations and the effects of different drugs on the reactions were the important steps forward in the understanding of allergy. Standardisation of the bronchial challenge and of the dose of antigen used became an important part of the work of R.E.C. Altounyan when he used bronchial challenge on himself to screen and evaluate inhaled drugs for asthma. His experiments, carried out between 1957 and 1982, resulted in two drugs used in the treatment of allergy: sodium cromoglicate (cromolyn sodium) and nedocromil sodium [29]. The chromones provided a new approach to the treatment of allergies. Although developed originally for the treatment of asthma, administered by inhalation, the first chromone, sodium cromoglicate, was soon used intranasally for allergic rhinitis, as eyedrops for vernal keratoconjunctivitis and allergic conjunctivitis, as oral capsules and sachets for food allergy and more recently as a skin lotion for allergic eczema. It is believed that the primary mode of action of the chromones is to prevent the release of the chemical mediators that cause allergic symptoms from sensitised mast cells. This is supported by its use in the treatment of systemic mastocytosis. Otherwise the principle treatments for allergic disease have not changed markedly in recent years. Adrenaline (epinephrine) administered by injection was an early treatment for the bronchoconstriction of asthma, which subsequently changed to isoproteronol (isoprenaline) and more recently to both short-acting bronchodilators salbutamol and terbutaline, and long-acting bronchodilators salmeterol and formoterol. These drugs administered by inhalation have almost replaced oral treatment with ephedrine and theophyllines. The introduction of extracts of the adrenal cortex, the corticosteroids, was a major advance in the treatment of allergic disease. The corticosteroids were first discovered by Tadeus Reichstein (1897–1996) in the 1930s. They were first used in humans in the treatment of rheumatoid arthritis, and Reichstein together with his co-workers Edward Kendall and Philip Hench were awarded the Nobel Prize for Medicine and Physiology in 1950 for their work. Modifications of the original cortisone and hydrocortisone molecules have become standard treatment for all allergic diseases. The antihistamines remain the first-line treatment for most allergic diseases other than asthma. The first compound shown to have antihistaminic effect was thymo-ethyl-diethylamine known as F929. Its actions in guinea pigs were described by Daniel Bovet (1907–1992) working at the Pasteur Institute in Paris in 1937. For his work he was awarded the Nobel Prize for Medicine and Physiology in 1957. F929 proved too toxic to be used in humans and the first antihistamine suitable for humans was developed in 1942 by Bovet and Bernard Halpern (1904–1978), a pharmacologist working for the Rhône-Poulenc company. It was called Antergan (phenbenzamine). A later product first produced in 1944, Neo-Antergan (mepyramine), is still in use today, as is the first antihistamine produced in the USA, diphenhydramine, discovered by George Rievschl (1916–2007) in 1946. Since 1946 many antihistamines have been discovered and introduced, the main advance in recent years being the non-sedating forms.

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The latest class of drugs introduced for the treatment of allergic disease has been the leukotriene antagonists. These either inhibit the production of leukotrienes or block the pharmacological effects.

Recent History Perhaps the most striking happening in the history of allergy over the past 30 years has been the increase in the prevalence of atopy as a marker of allergic disease. In a study measuring the amount of serum IgE to a mixture of 12 common inhalant allergens, Law et al. reported that the proportion of men aged 40–64 years with positive results increased from 30% in 1975–1976 to 42% in 1996–1998 [30]. All other studies measuring the prevalence of allergic diseases have shown similar increases. So far no definitive cause for this increase has been identified. On the scientific front over the same time period, the major advances have been in the molecular biology of allergens, the recognition that cross-linking of IgE molecules attached to the surface of mast cells by antigens is the primary event in allergic reactions and the identification of the molecular and cellular pathways that result from this event. Identification standardisation of allergens together with the availability of diagnostic tests to identify the primary allergens causing allergic reactions in an individual has led to improvements in introducing antigen tolerance as an aim of treatment using improved methods of immunotherapy. In the future, the identification and control of the factors that lead to intolerance in the first instance must be a major aim of future research.

Conclusions This has been a very brief overview of some of the main happenings and publications in the history of allergy. There is much more and many more workers, both scientists and clinicians, who have contributed greatly to our understanding of this disease that is increasing in prevalence. Space has not allowed consideration of the important work that has been done in the identification and standardisation of allergens, the standardisation of diagnostic skin tests, the standardisation of allergen challenge tests given orally, and by inhalation and the measurement of total and specific IgE antibodies in the serum. Apart from original papers my thanks for background sources are to Sheldon G. Cohen and Max Samter for their book Excerpts from Classics in Allergy published by Symposia Foundation, Carlsbad, CA, USA, and to Allergy: The History of a Modern Malady by Mark Jackson published by Reaktion Books Ltd. London, UK.

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References 1. von Pirquet C (1906) Allergie. Munch Med Wochenschr 53:1457–158 2. C v Pirquet (1963) Appendix A. Allergy, (Translated from the German original by Carl Prausnitz) In Gell PGH, Coombs RRA (eds). Clinical aspects of immunology. Oxford: Blackwell Scientific 3. Portier P, Richet C (1902) De l’action anaphylactique de certains venins. C R Soc Biol (Paris) 54:170 4. Coombs RRA (1992) The Jack Pepys lecture. The hypersensitivity reactions – some personal reflections. Clin Exp Allergy 22:673–680 5. Dunbar WP (1913) The present state of our knowledge of hay fever. J Hygiene 13:105 6. Hide DW (1992) Carl Prausnitz – Father of clinical allergy. Southampton Med J 8(2) 7. Noon L (1911) Prophylactic inoculation against hay fever. Lancet 1:1572 8. Freeman J (1930) “Rush” inoculation. Lancet 1:744 9. Frankland AW, Augustin R (1954) Prophylaxis of summer hay fever and asthma: a controlled trial comparing crude grass-pollen extracts with the isolated main protein component. Lancet 1: 1055–1057 10. Curtis HH (1900) The immunizing cure of hay fever. Med News (NY) 77:16–18 11. Black JH (1927) The oral administration of pollen. J Lab Clin Med 12:1156 12. Prausnitz C, Kustner H (1921) Studien uber die uberempfindlichkeith. Zentralblatt fur Bakteriologie 86:160–169 13. Gell PGH, Coombs RRA (eds) (1963) Clinical aspects of immunology. Oxford: Blackwell Scientific 14. Coca AF, Grove EF (1925) Studies in hypersensitivness. J Immunol 10:445–464 15. Ishizaka K, Ishazaki T (1967) Identification of gamma-E antibodies as a carrier of reaginic activity. J Immunol 99:1187 16. Bennich H, Ishizaka K, Ishizaka T, Gunnar Johansson S (1969) A comparative antigenic study of λE-globulin and myeloma-IgND. J Immunol 102:826–831 17. Walzer M (1927) Studies in absorption of undigested proteins in human beings. J Immunol 14:143–174 18. Carini C, Brostoff J, Wraith DG (1987) IgE complexes in food allergy. Ann Allergy 59:110–117 19. Schultz WH (1909) Physiological studies in anaphylaxis. I. The reaction of smooth muscle of the guinea-pig sensitised with horse serum. J Pharmacol Exp Ther 1:566 20. Dale HH, Laidlaw PP (1911) The physiological action of β-iminazolylethylamine. J Physiol 41:318 21. Kellaway C, Trethwaite E (1940) The liberation of a slow reacting smooth muscle-stimulating substance in anaphylaxis. Q J Exp Physiol 30:121 22. Samuelsson, B (1983) Leukotrienes: mediators of immediate hypersensitivity reactions and inflammations. Science 220:568–575 23. Riley J, West GB (1953) Histamine and tissue mast cells. J Physiol 120:528 24. Peipers A (1931) Ueber die Frage der Identität des hausstauballergens. Zeitschrift Immunitätsforch 71:359–364. 25. Lowell FC, Schiller IW (1947) Reduction in the vital capacity of asthmatic subjects following exposure to aerolised pollen extracts. Science 105:317 26. Hargreave FE, Pepys J, Longbottom JL, Wraith DG (1966) Bird breeder’s (Fancier’s) lung. Lancet 1:445–449 27. Pepys J, Hargreave FE, Chan M, McCarthy DS (1968) Inhibitory effects of disodium cromoglycate on allergen-inhalation tests. Lancet 2:134–137 28. Booij-Noord H, Orie NGM, de vries K (1971) Immediate and late bronchial obstructive reactions to inhalation of house dust and protective effects of disodium cromoglycate and prednisolone. J Allergy Clin Immunol 48(6):344–354

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29. Edwards AM, Howell JBL (2000) The chromones: history, chemistry and clinical development: a tribute to the work of REC. Altounyan Clin Exp Allergy 30:756–774 30. Law M, Morris JK, Wald N, Luczynska C, Burney P (2005) Changes in atopy over a quarter of a century, based on cross sectional data at three time periods. BMJ 330:1187–1188.

31. Silverstein AM (2000) Clemens Freiherr von Pirquet: Explaining immune complex disease in 1906. Nature Immunol 1: 453–455 32. Blackley CH (1873) Experimental Researches on the Nature and Causes of Catarrhus Aestivus. London: Balliere Tindall & Cox

Allergy Diagnosis Pascal Demoly, Francesco Gaeta, Jean Bousquet, and Antonino Romano

The diagnosis of IgE-mediated allergy is based on the confirmation of a typical history of allergic symptoms by diagnostic tests. IgE is the major isotype of anaphylactic antibodies, and although, theoretically, IgG4 can also act as a reagin, its clinical importance is not significant. Thus in vivo and in vitro tests are used in the diagnosis to detect free or cell-bound IgE (Fig. 1). The diagnosis of allergy has been improved by allergen standardisation, which provides satisfactory extracts for both in vivo and in vitro tests for most inhalant allergens, and the introduction of recombinant allergens. In the present chapter, neither non-specific challenges nor food and drugs will be considered, since they are presented in detail in other chapters.

Skin Tests Immediate-reading skin tests are widely used to demonstrate an IgE-mediated allergic reaction and represent a major diagnostic tool. If properly performed, they yield useful confirmatory evidence for a diagnosis of specific allergy. As they are complex to perform and interpret, it is recommended that they be carried out by experienced personnel [1].

P. Demoly () and J. Bousquet Exploration des Allergies – Maladies Respiratoires et INSERM, Hôpital Arnaud de Villeneuve, University Hospital of Montpellier, 34295 Montpellier, France e-mail: [email protected] F. Gaeta and A. Romano Unità di Allergologia, Complesso Integrato Columbus, via G. Moscati, 31, I-00168 Rome, Italy A. Romano IRCCS Oasi Maria S.S., Troina, Italy

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_2, © Springer 2009

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Fig. 1 Diagnosis of IgE-mediated allergy

Methods Skin Testing Methods Several methods of skin testing are available. • Prick and puncture tests (SPT) There is a high degree of correlation between symptoms and the results of both prick tests and provocative challenges. The modified prick test introduced by Pepys [2] is the current reference method, although the variability of this test has been shown to be greater than that of the intradermal test. Puncture tests with various devices [3–7] were introduced to decrease the variability of prick tests. Opinions concerning these so-called standardised methods vary according to the skill, experience and aims of the investigator. They are highly reproducible. Prick tests should be 2 cm apart. • In some instances (e.g. weak allergen solution), intradermal skin tests may be employed. Although they are more sensitive than prick tests, they may induce some false-positive reactions and correlate less well with symptoms [8]. Intradermal tests are less safe than prick tests, since, although rarely, systemic reactions may occur [9]. Particular care should be taken in patients treated with β-blocking agents, which may increase the risk of systemic reactions. Intradermal tests are not considered useful for the diagnosis of inhalant allergy when standardised extracts are available [1, 9–11]. As a general rule, the starting dose of intracutaneous extract solutions in patients with a preceding negative prick test should

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range between 100- and 1,000-fold dilutions of those used for prick-puncture tests [12]. The European Academy of Allergology and Clinical Immunology [13] and the US Joint Council of Allergy Asthma and Immunology [12, 14] therefore recommend skin prick-puncture tests for the diagnosis of IgE-mediated allergic diseases and for research purposes. • Prick–prick tests: Prick plus prick tests with fresh foods, mainly fruits and vegetables, were introduced [15, 16] because the commercial extracts of some foods are not sufficiently sensitive or may not even be available. • Atopy patch tests: The atopy patch test is a procedure involving epicutaneous patch tests with allergens known to elicit IgE-mediated reactions and the evaluation of eczematous skin lesions after 24–72 h in patients with aeroallergen- and food-triggered atopic dermatitis [17]. It has been standardised with regard to the use of vehicle and dose response relationships [18, 19]. Although there is increasing evidence that a small subset of patients with atopic dermatitis show atopy patch test positivity and serum-specific IgE negativity to the same allergen, as far as food allergy is concerned, the atopy patch test still requires standardisation [20, 21]. It may also be difficult to differentiate between irritative and allergic reactions [20, 21]. Wheat gluten in particular has been suspected of causing false-positive (irritant) reactions [22]. The diagnostic performance of this test with food allergens can be evaluated by comparing the results with double-blind, placebo-controlled food challenges (DBPCFC). There is a task force to study standardisation of the challenge procedures for delayed reactions [21]. Instead, the problem with aeroallergens is that a ‘gold standard’ provocation test in atopic eczema does not exist [23].

Negative and Positive Control Solutions Because of inter-patient variability in cutaneous reactivity, it is necessary to include negative and positive controls in every skin test study. The negative control solutions are the diluents concerned. Rare dermographic patients will have wheal-and-erythema reactions to the negative control. The latter will also detect traumatic reactivity induced by the skin test device (with a wheal which may approach a diameter of 3 mm with some devices) and/or the technique of the tester [12]. Any reaction at the negative control test sites will hinder interpretation of the allergen sites [12]. Positive control solutions are used to detect possible suppression by medication or disease, exceptional patients who are poorly reactive to histamine or variations in technician performance. The usual positive control for prick-puncture testing is histamine dihydrochloride, used at a concentration of 5.43 mmol/l (or 2.7 mg/ml, equivalent to 1 mg/ml of histamine base) [1]. Wheal diameters with this preparation range from 2 to 7 mm. However, a tenfold greater concentration is more appropriate [24], with a mean wheal size ranging between 5 and 8 mm. For the intradermal test, the concentration routinely used is 0.0543 mmol/l. The mean wheal size elicited ranges from 10 to 12 mm. Mast cell secretagogues such as codeine phosphate 2.5% [5] or 9% [25] may

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also be used. Histamine-induced skin reactions reflect vascular reactivity and are useful to determine whether histamine antagonists are present, while codeine skin reactions are a function of both mast cell reactivity and vascular responsiveness. In this, the reaction pathway to codeine is comparable to that of allergens, which bind to specific IgE on high-affinity receptors on the mast cell surface and stimulate mediator release, including histamine. The use of both substances therefore may provide a more accurate impression of the reactivity of the individual than one substance alone [26].

Skin Tests with Recombinant Allergens Current diagnosis of allergy relies on natural extracts, which may lack standardisation and/or be degraded rapidly in solution. Recombinant DNA technology allows production of pure biochemically characterised proteins. Skin tests with recombinant allergens have been available since the 1990s for pollens [27], moulds such as Aspergillus [28], mites [29], venoms [30], and latex [31]. Skin tests with recombinant and natural allergens have a similar value [32–34] if the recombinant allergens have been well selected and represent all or most epitopes of the natural allergen [35, 36]. Allergy diagnosis based on allergenic molecules is important in patients with multiple pollen sensitisation [37], since this condition appears to be determined by sensitisation to defined allergenic components (panallergens) or by pollen of multiple species [38]. Detection of IgE to non-panallergenic molecules using skin tests and/or serum IgE allows more important allergenic sources to be identified. Food allergens are usually non-standardised and unstable in solution. Recombinant allergens should be useful for the diagnosis of food allergy such as apple [39], celery [40], peanut [41], and cherry [42]. Skin tests with recombinant food allergens can be an alternative to prick–prick tests with foods.

Grading of Skin Tests and Criteria of Positivity Skin tests should be read at the peak of their reaction by measuring (in mm) the wheal and the flare approximately 15 min after the performance of the tests. Late-phase reactions are not recorded because their exact significance is not known [12, 13]. Some scoring systems have been proposed and may be used in daily practice. In the USA, for example, for skin-prick tests: neg = 0 reaction, 1 + = 1 mm wheal above saline control; 2 + = 1–3 mm wheal above saline control; 3 + (the first point we consider a positive reaction) = 3–5 mm wheal above saline control plus an accompanying flare; 4 + = > 5 mm wheal above saline control, plus an accompanying flare. For prick tests, when the control site is completely negative, small wheals of a mean diameter of at least 3 mm of the negative control represent a positive immunological response [1, 2], but these reactions do not necessarily imply the presence of a clinical allergy [1].

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Factors Affecting Skin Testing Skin reaction is dependent on a number of variables that may alter the performance of skin tests: • The quality of the allergen extract is important. When possible, allergens that are standardised by using biological methods and that are labelled in biological units should be used [12, 13]. Recombinant allergens can also be used effectively (see above). • Age is known to affect the size of the reaction [1], but positive skin-prick tests can be found early in infancy [43, 44]. In old age, the size of skin tests decreases [45]. • Seasonal variations related to specific IgE antibody synthesis have been demonstrated in pollen allergy [46]. Skin sensitivity increases after the pollen season and then declines. This effect has some importance in patients with low sensitivity [47] and/or in patients sensitised to allergens such as cypress pollen [48]. • Drugs affect skin tests and it is always necessary to ask patients about the drugs they have taken. Some drugs such as astemizole (no longer available in many countries) can depress or abolish responses to skin tests for a period of up to 6 weeks (for review see [1]). Discontinuing other H1-antihistamines 1 week before is required [1]. Montelukast does not appear to reduce skin test reactivity [49] and does not need to be discontinued before skin testing. Butterbur Petasites hybridus, a herbal remedy for the treatment of allergic rhinitis, does not produce any significant effects on the histamine and allergen cutaneous response [49]. • Patients with dermographism (urticaria) or widespread skin lesions should not be tested by prick puncture tests. Intradermal tests with the proper negative control may sometimes be feasible.

Interpretation of Skin Tests Carefully performed and correctly interpreted skin tests with high-quality allergen extracts and a battery that includes all relevant allergens of the patient’s geographic area are a simple, painless and highly effective method. Therefore, skin testing represents one of the primary tools for allergy diagnosis. Both false-positive and false-negative skin tests may occur because of improper technique or material. False-positive skin tests may result from dermographism or may be caused by ‘irritant’ reactions or a non-specific boost from a nearby strong reaction [1]. False-negative skin tests can be caused by: extracts of poor initial potency or subsequent loss of potency [8], drugs inhibiting the allergic reaction, diseases attenuating the skin response, decreased skin reactivity in infants and elderly patients, improper technique (no or weak puncture). The use of positive control solutions may overcome some of the false-negative results because reactions will be either decreased or inhibited in patients with poorly reactive skin.

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The occurrence of positive responses to skin tests does not necessarily imply that the patient’s symptoms are due to an IgE-mediated allergy, since skin-prick tests are positive in 15–35% of symptom-free individuals depending on the allergen and the area (for review see [1]). The presence of positive skin tests in asymptomatic subjects may precede the onset of allergic symptoms [50, 51], especially if the allergen load is high.

Clinical Value of Skin Tests Even after false-positive and false-negative tests have been eliminated, the proper interpretation of results requires a thorough knowledge of the history and the physical findings. A positive skin test alone does not necessarily confirm a definite clinical reactivity to an allergen. With inhalant allergens, skin test responses represent one of the first-line diagnostic methods and when they correlate with the clinical history, in vitro tests may not be required [12, 13, 52]. For foods, particular caution should be used, since very few extracts are standardised and some may be insufficiently sensitive. Extracts made from fruits and vegetables are usually of poor quality, since the allergens are rapidly destroyed. Skin tests with fresh foods are more sensitive [1]. Recombinant allergens will certainly improve the diagnosis of food allergy. For occupational rhinitis, skin tests are often unreliable, except in the case of high-molecular-weight compounds such as latex or grain dust.

Nasal Challenge Tests Nasal challenge tests are used in research and to a lesser extent in clinical practice, but they are particularly important in the diagnosis of occupational rhinitis. Recommendations on and critical analysis of nasal provocations and methods to measure the effects of such tests have already been published by a subcommittee of the ‘International Committee on Objective Assessment of the Nasal Airways’ [53–55] (Table 1), which has devised guidelines for nasal provocation tests concerning indications, techniques and evaluation of the tests.

Methods Different methods for the provocation and measurement of nasal responses have been used. Each technique has its own advantages and disadvantages. For clinical purposes, techniques for qualitative measurements may be appropriate, but for experimental research, quantitative measurements with high reproducibility are essential [56] (Table 2).

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Table 1 Indications for nasal challenge tests [54] 1. Allergen provocations: • When discrepancies between history of allergic rhinitis and tests or between tests are present (e.g. in cases of diagnostic doubt). • For diagnosis of occupational allergic rhinitis. • Before immunotherapy for allergic rhinitis. Although it is still not very common to use nasal provocation before starting immunotherapy, a laborious long-lasting therapy is justified by a proper diagnosis. This holds true particularly in the case of perennial allergic rhinitis. • For research. 2. Lysine-aspirin: Nasal provocation is recommended as a substitute for oral provocation in aspirin hypersensitivity. Whenever such nasal provocation is negative, an oral test is still required. 3. To test non-specific hyperreactivity: • Nasal provocation with non-specific stimuli (histamine, methacholine, cold dry air, etc.) is not relevant for daily clinical practice and diagnosis, but can be used in research.

Table 2 Recommendations for the performance of nasal challenge tests [54] 1. Provoking agent • Use solutions at room temperature • Standardised extracts • Isotonic solutions buffered to a pH of about 7 • Use control solutions 2. Administration into the nose • Metre-dose pump spray • Paper disks 3. Assessment of the nasal response: symptom scores are combined with objective measures • Counting sneezes or attacks of sneezing • Measuring volume or weight of nasal secretion • Changes of nasal patency, airflow or airflow resistance 4. Methods to evaluate nasal patency, airflow and airflow resistance • Rhinomanometry • Acoustic rhinometry • Rhinostereometry • Nasal inspiratory or expiratory peak flow Less common methods are: • Head-out body plethysmography • Oscillometry

Provoking Agents Allergens are usually administered in an aqueous solution, but although the solution is easy to administer into the nostrils, this form of challenge has many limits: • Allergen extracts are not always standardised. Only standardised ones should be used when available. • Allergen extracts may not represent the native allergen and the amount of allergen administered is far greater than that entering the nose during natural allergen

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exposure. This is also true for recombinant allergens. As an example, although rBet v 1 alone is sufficient for a reliable diagnosis of birch pollen allergy in most patients and induces skin test reactivity comparable to that of birch pollen extract, it provokes fewer allergic reactions in nasal provocations [57]. • The potency of an aqueous extract often decreases rapidly and it is advisable, at least for research projects, to use standardised and lyophilised extracts of the same batch freshly reconstituted on the day of the test. • Preservatives such as glycerol, benzalkonium chloride and phenol can induce non-specific nasal reactions. • Temperature, pH and osmolarity of the solution should be checked carefully. Allergens can also be administered in the form of a powder [58], as a solution adsorbed on a paper disk, or in the form of pollen grains mixed with lactose in capsules.

Administration in the Nose Aqueous allergen extracts can be delivered from atomisers and an exact dose can be applied. Other investigators use a pipette and allergens are deposited during rhinoscopy. When using any of these methods, care should be taken to avoid non-specific responses, and for all experiments, the diluent of the allergen extract must be administered before the allergen, to test for the non-specific response. Small paper disks can be directly applied to the nostrils and allergen powders or pollen grains can be insufflated easily with Spinhalers or derived devices. Other methods are of interest. In the Vienna Challenge Chamber [59] or the environmental exposure unit [60], patients are challenged under controlled conditions with purified airborne grass pollen. However, these conditions are only used for large clinical trials and are not useful in the diagnosis of allergic rhinitis.

Assessment of the Response Different methods have been used to assess the response to allergens. None of these are fully accepted by all investigators. Symptoms produced after a challenge can be recorded. Sneezing, rhinorrhea and nasal blockage are easy to assess and yield valuable information. However, patients can react with different symptoms on different test days and it is preferable to record more than one symptom [61]. Nasal obstruction is one of the cardinal symptoms in allergic rhinitis and the major symptom of the late-phase reaction following allergen challenges. The objective measurement of this symptom is therefore of greatest importance. However, physiological fluctuations in nasal resistance may be a problem in monitoring the nasal provocation test [62]. So far, rhinomanometry is the best evaluated and standardised technique [53, 63]. Active anterior rhinomanometry was recommended by an international committee

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in 1984 [53]. Unlike active posterior rhinomanometry, with active anterior rhinomanometry, unilateral measurements can be made. Acoustic rhinometry [64], characterised by a low coefficient of variation, has been used in nasal challenge tests with bradykinin, histamine and allergens. It appears to be a safe, non-invasive, objective and validated measure of nasal obstruction. However, acoustic rhinometry has limitations and pitfalls. The value of acoustic rhinometry in evaluating nasal responses after provocation in routine clinical work is not yet established although it is gaining more importance [65]. Rhinostereometry [66] can be used to record changes in the thickness of the nasal mucosa. With a microscope, 0.2 mm changes can be recorded in test subjects fixed to the apparatus using an individually made plastic splint adapted to the teeth. Rhinostereometry is, however, a time-consuming method. It seems useful for comparisons between well-defined groups of subjects and between the same subjects on different occasions. It may be combined with laser Doppler flowmetry [67]. Nasal peak flow appears to correlate very well with rhinomanometry [68]. Other methods, such as rhinostereometry and whole body plethysmography, have been proposed [69], but they have not yet been fully assessed. Comparisons between methods are also available. In order to find the most sensitive method, assessments were made by means of symptom score, acoustic rhinometry, nasal peak expiratory and inspiratory flow, and rhinomanometry during histamine challenges [70]. There was no difference in the mucosal reactivity between patients and controls, regardless of the method used, but nasal peak flows were more sensitive to mucosal changes than the other methods studied.

Measurement of Mediators and Cells during Challenges Allergen-specific nasal challenges are a valid and reliable tool for studying the pathophysiological mechanisms involved in allergic inflammation. Nasal challenges induce an immediate and late response in allergic subjects with the release of pro-inflammatory mediators. Nasal biopsies may also be obtained and have been used in many drug trials [71]. Measurement of mediators in the nose may increase the sensitivity of nasal challenges [72], but more data are needed.

Factors Affecting Nasal Challenges As in any other in vivo tests, the major factors affecting nasal challenges are the quality of the allergens used and the drugs taken by the patient. Other factors are more specific to nasal challenges, including technical problems already discussed and inflammation of the nasal mucosa. Sodium cromoglycate should be withdrawn 48 h before the test, the second generation H1-antihistamines and nasal steroids 5–6 days before, ketotifen and

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imipramines 2 weeks before, and astemizole at least 1 month before. Nasal vasoconstrictors may not modify nasal challenges. Specific immunotherapy decreases the sensitivity of the nose to allergens. The nasal mucosa may be altered by several factors and the response to allergens may be greatly affected. Indeed, it has been shown that an allergic reaction significantly increases the reactivity of the nose following a subsequent stimulation because of the priming effect initially described by Connell [73]. Viral infections, pollutants induce the release of pro-inflammatory mediators and cytokines in nasal secretions. Nasal challenges should thus be performed at least 2–4 weeks after any allergic or infectious episode. Finally, the nasal cycle [62] should be taken into consideration when rhinomanometry is used.

Challenges with Occupational Agents The diagnosis of occupational rhinitis is often complex and requires nasal provocation tests with the relevant occupational agent [74–76]. The challenge can be carried out in the form of natural exposure, especially if the relevant allergen is unavailable. For example, this has been done for laboratory animal allergy in a vivarium during cage cleaning [76].

Bronchial Challenge Tests Asthma is a multifactorial disease in which the non-specific bronchial hyperreactivity is a key factor, and so it is sometimes difficult to determine the exact importance of allergy. However, bronchial challenge tests with allergens are mainly used in research and to a lesser extent in clinical practice maybe with the exception of occupational asthma. Although easier to carry out, nasal or conjunctival provocation tests cannot replace bronchial challenge tests [77].

Technique The traditional method for performing bronchial challenges is to use allergens in a solution form administered by nebulisers or in chambers, the patient handling the suspected allergen. Usually, the response is measured using FEV1 and a provocative dose inducing a 20% reduction from the baseline is required to classify a bronchial challenge as positive (PD20FEV1) [78, 79]. Provoking Agents The vast majority of inhaled allergens are in the form of aqueous solutions similar to those used for nasal challenges. Because most asthmatics have increased bronchial hyperreactivity, inhaled solutions must be isotonic, iso-osmolar and close to

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body temperature and without irritant preservatives [80]. In research trials, it is recommended to use standardised lyophilised extracts of the same batch reconstituted on each of the test days. However, administration of nebulised extracts bears little resemblance to natural allergen exposure in terms of allergen form, size, dose and site of deposition in the airways. In the bronchial provocation test, enormous doses of 1–4 µm droplets inhaled through the mouth reach all parts of the airways [80]. During natural exposure, the 20–60 µm pollen grains, animal allergens, or mite faeces are trapped mostly in the nose. Daily exposure consists of thousands of microprovocations of very small amounts of allergen. In case of animal dander or exposure to mould, the size of the particles is smaller (1–5 µm). However, aerosol challenges are still widely used and attempts have been made to standardise the methods. Since severe asthmatic reactions may occur during allergen challenges, increasing doses of allergens are administered until a positive immediate reaction demonstrates that the maximum tolerable dose has been administered. Natural allergen exposures are made in environment challenge exposure chambers, which are discussed below.

Administration into the Airways The administration of allergens into the airways depends on many factors, including the aerodynamic size of the particles [80]. Two methods have gained more acceptance than others and provide remarkably similar results: intermittent generation of aerosols during full deep inspiration or continuous aerosol generation during tidal volume [81]. However, there are more sophisticated methods available, such as computerised equipment for the delivery of inhaled doses of solid particles in specific bronchial challenges [82]. Assessment of the Response Many different techniques are also available. It is now accepted that the test should be stopped when an immediate response is observed with at least a 20% fall in FEV1 (PD20FEV1), a 25% fall in the maximum mid-expiratory flow rate, or a 35% increase in specific airway resistances [78, 80]. However, the most used is PD20FEV1 since FEV1 is the most reproducible lung function test [83]. In some cases, PD15FEV1 is used, making it advisable to measure the pulmonary function serially to monitor late-phase reactions, if they occur. Early- and Late-Phase Reactions The typical reaction to inhaled allergens is characterised by an early (immediate) airway response that fully develops within 10–20 min and is rapidly reversible after inhalation of a short-acting β2 agonist. This reaction is sometimes followed by a

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late-phase reaction that develops within 3–5 h after the challenge and usually peaks at 6–10 h. In some patients, these reactions are followed for several days, usually in the morning, by episodes of bronchoconstriction. The early-phase reaction is typically due to bronchospasm, whereas the latephase reaction is associated with airway inflammation. Reproducibility of the Response Many studies have confirmed that asthmatic responses induced by allergen challenges have a good reproducibility [84]. This is the reason for the widespread use of bronchial challenges in the development of drugs.

Factors Affecting Bronchial Challenges The response to inhaled allergens is determined by the level of allergenic sensitivity as well as by the level of non-specific airway hyper-responsiveness [85, 86]. As in all in vivo tests, the major factors affecting nasal challenges are the quality of both the allergens used and the drugs taken by the patient. There are also other factors more specific to bronchial challenges, including baseline airway calibre, viral infections, recent asthma exacerbations and pollutants (in particular tobacco smoke). It is usually recommended to avoid allergen challenges if the baseline FEV1 is under 70% of predicted values, because the reaction induced by the inhaled allergen may lead to a severe asthmatic reaction [79]. Use of short-acting inhaled β2-agonists, aspirin, theophylline or anticholinergics should be stopped for at least 12 h prior to the test. Long-acting β2 agonists should be stopped for 24 h. Cromoglycate and inhaled corticosteroids should be stopped for a week.

Environmental Exposure Units Pollen exposure in the environmental exposure unit is an effective, reproducible, safe and suitable method for single-centre clinical studies [59, 60]. These exposure units are mostly used to assess the efficacy of anti-allergic treatments. However, the priming effect [73] on the nasal mucosa is not considered in many studies and the results of the challenges do not usually accord with clinical data. In cat allergy, exposure to cats in environmental exposure units has been widely used [87], but there is a high variability of cat allergen during these studies. There are also environmental exposure units that are used for the diagnosis of occupational allergy. These are of great value and have been used for latex sensitisation [88].

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Conjunctival Challenge Tests The conjunctival challenge test is useful in clinical research. When indicated in clinical routine, it is a safe and easy test with good precision [89–91]. Nasal and conjunctival challenge tests give similar results in many, but not all patients [92]. Occupational allergy can also be determined using conjunctival challenges [93].

Technique Provoking Agents Allergens are administered in an aqueous solution. Administration in the Eye Conjunctival provocation tests are usually performed by applying 20 µl of the diluent and then 20 µl of the allergen solution in the conjunctival cul-de-sac. Assessment of the Response The positivity of conjunctival provocation tests can be assessed by symptom score or by the release of mast cell-derived mediators. Abelson proposed an easy scoring system [91] (Table 3). Conjunctival provocation tests is positive when the cumulative score is ≥ 5. For clinical purposes, only the early-phase reaction is recorded [94]. For research or pharmacologic purposes, both the early- and late-phase responses may be examined. Cells and inflammatory mediators can be assessed [95], but they are only useful in research and for assessing drug mechanisms [96]. Many studies have been performed using conjunctival challenges to assess the efficacy of oral [96, 97] or ocular drugs [98, 99] and specific immunotherapy [100]. More sophisticated methods can be used, such as digital imaging [101], scanning and imaging technology [102] or using a fractional millimetre reticule in the eyepiece of a slit lamp microscope for quantifying eyelid swelling [103], spectroradiometer or colorimeter for measuring erythema [103].

Factors Affecting Conjunctival Challenges As for all in vivo tests, the major factors affecting conjunctival provocation tests are the quality of both the allergens used and the drugs taken by the patient. There are also other factors more specific to nasal challenges, including technical problems already discussed and inflammation of the nasal mucosa.

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Table 3 Scoring system to measure the signs and symptoms of allergic conjunctivitis [91] Redness, eyelid swelling 0: None 1: Mild 2: Moderate 3: Severe Chemosis 0: None 1: Mild, detectable with slit lamp, conjunctiva separated from sclera 2: Moderate (visually evident, raised conjunctiva, especially in the limbal area) 3: Severe (ballooning of conjunctiva) Tearing 0: None 1: Mild (eyes feel slightly watery) 2: Moderate (blows nose occasionally) 3: Severe (tears rolling down cheeks) Itching (to be graded by subject) 0: None 1: Mild (intermittent tickling sensation) 2: Moderate (continual awareness but without the desire to rub) 3: Severe (continual awareness with the desire to rub the eyes) 4: Incapaciting itching (subject insists on rubbing eyes)

Clinical Value and Indication for Conjunctival Challenges The results of conjunctival provocation tests appear to be more reproducible than those of nasal challenges, but vary more than the results of skin tests or IgE measurement. The challenge is time-consuming and may be unpleasant, and so it is not routinely used. Conjunctival provocation tests, however, may be used in clinical practice to confirm the allergy of a patient, especially when there is a discrepancy between the results of skin and in vitro tests or when immunotherapy is indicated. They can also be used to assess the results of specific immunotherapy or drug trials.

Laboratory Tests The diagnosis of IgE-mediated hypersensitivity is based on positive histories, skin tests and/or specific-IgE assays. Sometimes identification of the causal factors is not easy because of the high number of allergens and the fact that not all allergies are IgE-dependent. In vitro tests are of great interest to allergists in order to establish the culprit agent and to avoid provocations, especially for patients exposed to several allergens simultaneously or with histories of life-threatening reactions.

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Serum-Specific IgE Assays The discovery of IgE in 1967 was a major advance in the understanding and diagnosis of allergic diseases [104, 105]. In normal subjects, levels of IgE increase from birth (0–1 KU/l) to adolescence and then decrease slowly and reach a plateau after the age of 20–30 years. In adults, levels of over 100–150 KU/l are considered to be above normal. Allergic and parasitic diseases as well as many other conditions increase the levels of total IgE in serum. Thus, the measurement of total serum IgE is barely predictive for allergy screening and should no longer be used as a diagnostic tool. In contrast to the low predictive value of total serum IgE measurements in the diagnosis of immediate type allergy, the measurement of allergen-specific IgE in serum is of importance. Methods The first technique used to accurately measure serum specific IgE was the RAST (radioallergosorbent test) [106]. New techniques are now available using either radio- or enzyme-labelled anti-IgE [107, 108]. The different reagents are critical for an appropriate assay. Results are expressed in terms of total radioactive counts bound (cpm), arbitrary units (RAST class, PRU/ml) or units of IgE (IU/ml, KU/l). Factors Affecting the Measurement of Serum Specific IgE Specific IgE measurements are not influenced by drugs or skin diseases. Many technical factors can affect the measurement of IgE. The quality of reagents used (allergens, anti-IgE antibodies) is of importance. IgE antibody assays need to be sensitive and specific to make quantitative measurements over as wide a range as possible [109]. A high-capacity solid phase provides a large excess of allergen that maximises the binding of IgE antibody. The anti-IgE preparations applied must be Fcε-specific and preferably combinations of monoclonal antibodies with specificities against more than one epitope on the Fc fragment and with complementary dose-response characteristics. Calibrators should be traceable to the WHO International Reference Preparation for human IgE, 75/502 [110]. As for skin tests, the quality of allergens is of critical importance and, when possible, only standardised extracts should be used. Standardisation of the allergen source material in combination with adequate reagent design provides precise and reproducible data increasing the accuracy and efficiency of allergy diagnostic testing. However, using molecular biology, it is possible to obtain large quantities of major allergens for many species. Recombinant Bet v I produced in bacterial expression systems allows accurate in vitro diagnosis of birch pollen allergy in over

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95% of birch pollen allergic patients [111]. Other studies have found similar values for recombinant allergens. Thus, single recombinant allergen or a combination of a few major recombinant allergens or the addition of some relevant recombinant allergens to an allergen extract can substitute the crude extract for in vitro diagnostic purposes [112]. It also seems that in vitro diagnostics for pollen allergy can be simplified using cross-reactivities. Current diagnostic extracts for grass pollen allergy are usually composed of mixtures of pollen from different grass species. Their complex composition hampers accurate standardisation. It was recently shown that the use of one grass species is sufficient for the in vitro diagnosis of grass pollen allergy. Purified natural Lol p 1 and Lol p 5 detect over 90% of grasspositive patients. Around 80% of the IgE response to grass pollen is directed to these major allergens [113]. The same is true for purified natural Bet v 1, Bet v 2 and profiling to diagnose Fagales pollen allergy [114]. Significance of Measurement of Serum Allergen Specific IgE Several studies have shown that serum-specific IgE results correlate closely to those of skin tests and nasal challenges. As in skin tests, the presence or absence of specific IgE in the serum does not preclude symptoms, and some symptom-free subjects have serum-specific IgE. Although a low specific IgE titre may not be clinically relevant, the titre of serum-specific IgE is usually unrelated with symptoms. This is because the severity of symptoms depends not only on IgE antibodies but also on the releasability of mediators, the response of the target organ to mediators and non-specific hypersensitivity. When using single allergen tests, the cost of serum-specific IgE measurement is high and only a selected list of allergens can usually be tested. Screening Tests Using Serum Specific IgE Some methods use either a mixture of several allergens in a single assay [115] or test several different allergens during a single assay. These tests can therefore be used by specialised doctors and non-allergists as screening tests for the diagnosis of allergic diseases. The clinical relevance of these tests has been extensively studied and it has been shown that their efficiency (specificity and sensitivity) in allergy diagnosis is often over 85% [115]. However, using these tests, the patient is defined only as allergic or nonallergic and more extensive investigations for rhinitis are needed if the test is positive.

Basophil Activation Tests Basophils are major effector cells of allergic disease; they express the highaffinity IgE receptor (FcεRI), which is the main activating receptor for these cells. Flow cytometry has been used to investigate activation of human

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basophils, both in vitro and in vivo [116], and it gives information on anaphylactic reactions. The main advantages of the latest-generation flow cytometry analysers are the capability of identifying cells even in very small percentages of the total population examined (below 1%). In patients with immediateallergic reactions, a flow cytometric basophil activation test to detect specific surface markers with monoclonal antibodies can also be performed. At present, the most commonly used antigens in basophil activation tests are CD63 and CD203c. However, a new basophil identification antigen, CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper 2 cells) and the recently described activation antigens CD13, CD164 (behaving as CD203c), and CD107a (paralleling CD63 expression) could be applied in order to improve it [117]. However, the assessment of basophil activation as a clinical tool for the diagnosis of allergic disease is still in its infancy. Cytometry-assisted investigation of human basophils has been used to evaluate patients sensitised to inhalant allergens [118, 119], food [120, 121], latex [122, 123], drugs [124–127], and insect venom [128, 129], and is also helpful to study patients with autoimmune chronic urticaria [130, 131].

Microarrays The microarray is an ordered array of microscopic elements on a planar substrate that allows the specific binding of genes or proteins [132, 133]. Protein microarrays are suitable to study protein–ligand interactions in which the ligand can be a protein, peptide, DNA, RNA, oligosaccharide or chemical compound. Any microarray assay provides a precise measure of the number, amount or concentration of the molecules present in a sample. The feasibility of using allergen-chips or arrayed allergens for multiallergen testing has been reported [134, 135]. In contrast to conventional allergy diagnosis, allergen microarray permits the simultaneous investigation of several hundred allergens and the measurement of different classes of immunoglobulin (IgE/IgG4) in a single step, and requires only a small amount of serum. Microarrays were first used in the field of allergic diseases to analyse recombinant allergens to determine the individual sensitisation profile of patients [136]. DNA and protein microarrays have then been assessed for the diagnosis of respiratory allergy, and particularly bronchial asthma and rhinitis, atopic dermatitis, food allergy, and hypersensitivity to aspirin [137–141]. Microarray technologies have already proven to be invaluable in the field of genomics and proteomics and solutions to these challenges will undoubtedly facilitate the development of clinically useful diagnostic chips in the foreseeable future.

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Clinical Value of the Tests and Correlation Between Tests The diagnosis of allergy is based on the correlation between the clinical history and diagnostic tests for allergy. It is not possible to diagnose allergy based solely on responses to skin tests, in vitro tests, or even challenges. Factors affecting tests should always be checked before investigations, since drug therapy may modify the results of in vivo tests for days or even weeks. The importance of allergen challenges depends on the disease and the quality of the allergen extract considered. The different tests used in the diagnosis of allergy do not have the same biological and clinical significance.

Diagnosis of Inhalant Allergy Skin tests represent the primary diagnostic tools used for immediate-type hypersensitivity. Comparisons between the measurement of specific IgE and skin tests depend on the quality and standardisation of the allergens used in both types of tests and, to a lesser extent, on the method of skin testing used. The weakest correlations have been obtained with mould and unstandardised dander extracts. For standardised allergens, challenges are usually not necessary to confirm the diagnosis of allergy. There are good correlations between a strongly positive response to a skin test and the detection of serum-specific IgE and between a negative response to a prick test and the lack of detection of serum-specific IgE, whereas small wheals induced by prick tests and positive results of intradermal tests with concentrated extracts are less frequently associated with the detection of serum-specific IgE. Positive responses to skin tests and serum-specific IgE can be found in totally symptom-free subjects with a similar prevalence. Correlations between responses to skin tests and to the measurement of allergen-specific IgE with inhalation challenges are less consistent because of the non-specific hyper-reactivity. Poor correlations are observed with un-standardised extracts, weakly positive responses to skin testing, and the RAST, or when there is a discrepancy between the clinical history and skin tests. When there are both a suggestive history and strongly positive responses to skin testing and the RAST, inhalation challenges are often positive. The prevalence of positive responses to inhalation challenges in symptom-free patients is lower than that of positive responses to skin testing or the RAST, but pollen-allergic patients who suffer only from rhinitis often manifest a positive response to bronchial challenge with pollen extracts. Correlations between responses to skin tests and serum-specific IgE with nasal challenges are less consistent because of the non-specific nasal hyper-reactivity. Poor correlations are observed with un-standardised extracts, weak positive responses to skin testing, and the RAST, or when there is a discrepancy between the clinical history and skin tests.

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Before the discovery of IgE and when poorly characterised allergens were used, false-negative results were frequent and the bronchial challenge was useful to diagnose allergic asthma. It is now mostly restricted to research protocols, assessment of drugs in development, and occupational medicine. Correlations have not been performed with environmental exposure chambers or conjunctival challenges.

Diagnosis of Food Allergy While allergic reactions to foods are usually due to IgE-mediated hypersensitivity mechanisms, there are a number of immune mechanisms that may contribute to adverse reactions to foods. Tests for allergic reactions include both skin-prick tests and the measurement of serum allergen-specific IgE antibodies. The diagnosis of food allergy is complicated, however, because allergen extracts and the test reagents currently available are not standardised and their stability is poorly determined. The presence of food-specific IgE in serum or a positive skin test to a foodstuff does not always correlate with a food allergy since some patients outgrow their allergy with age and not all patients with food-specific IgE have a clinical sensitivity. In many instances, the diagnosis has to be confirmed by a double-blind food challenge, which should be carried out under precisely specified conditions and by staff who have the competence to manage anaphylactic reactions. As for other forms of allergy, unproven and controversial techniques such as cytotoxic tests, VEGA testing or sublingual provocation tests have no proven value.

Diagnosis of Occupational Allergy Occupational rhinitis must be more precisely confirmed than allergic rhinitis of other aetiology. In practice, interviews concerning the causal relation, frequency, latent period and atopic disposition often provide suggestions, but sometimes give unreliable evidence for diagnosing occupational nasal allergy. Therefore, examinations such as skin tests, nasal provocation tests and determination of the IgE antibody level are necessary to confirm the causality between the disease and the work exposure.

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102. Abelson MB, Pratt S, Mussoline JE, Townsend D (2003) One-visit, randomized, placebocontrolled, conjunctival allergen challenge study of scanning and imaging technology for objective quantification of eyelid swelling in the allergic reaction with contralateral use of olopatadine and artificial tears. Clin Ther 25:2070–2084 103. Friedlaender MH (2004) Objective measurement of allergic reactions in the eye. Curr Opin Allergy Clin Immunol 4:447–453 104. Ishizaka K, Ishizaka T (1967) Identification of gamma-E-antibodies as a carrier of reaginic activity. J Immunol 99:1187–1198 105. Johansson SG (1967) Raised levels of a new immunoglobulin class (IgND) in asthma. Lancet 2:951–953 106. Wide L, Bennich H, Johansson SG (1967) Diagnosis of allergy by an in-vitro test for allergen antibodies. Lancet 2:1105–1107 107. Bousquet J, Chanez P, Chanal I, Michel FB (1990) Comparison between RAST and Pharmacia CAP system: a new automated specific IgE assay. J Allergy Clin Immunol 85:1039–1043 108. van Houte AJ, Bartels PC (1992) Comparative evaluation of the pharmacia CAP system and the DPC AlaSTAT system for in vitro detection of allergen-specific IgE with the skin prick test. Eur J Clin Chem Clin Biochem 30:101–105 109. Bernstein I (1988) Proceedings of the Task Force on Guidelines for standardizing old and new technologies used for the diagnosis and treatment of allergic diseases. J Allergy Clin Immunol 82:487–526 110. Yman L (1991) Standardization of IgE antibody assays. J Int Fed Clin Chem 3:198–203 111. Menz G, Dolecek C, Schonheit-Kenn U, Ferreira F, Moser M, Schneider T, Suter M, BoltzNitulescu G, Ebner C, Kraft D, Valenta R (1996) Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen. Clin Exp Allergy 26:50–60 112. Olsen E, Mohapatra SS (1994) Recombinant allergens and diagnosis of grass pollen allergy. Ann Allergy 72:499–506 113. van Ree R, van Leeuwen WA, Aalberse RC (1998) How far can we simplify in vitro diagnostics for grass pollen allergy?: A study with 17 whole pollen extracts and purified natural and recombinant major allergens. J Allergy Clin Immunol 102:184–190 114. Van Ree R, Van Leeuwen WA, Akkerdaas JH, Aalberse RC (1999) How far can we simplify in vitro diagnostics for Fagales tree pollen allergy? A study with three whole pollen extracts and purified natural and recombinant allergens. Clin Exp Allergy 29:848–855 115. Eriksson NE (1990) Allergy screening with Phadiatop and CAP Phadiatop in combination with a questionnaire in adults with asthma and rhinitis. Allergy 45:285–292 116. Bochner BS (2000) Systemic activation of basophils and eosinophils: markers and consequences. J Allergy Clin Immunol 106:S292–S302 117. Ebo DG, Sainte-Laudy J, Bridts CH, Mertens CH, Hagendorens MM, Schuerwegh AJ, De Clerck CK, Stevens WJ (2006) Flow-assisted allergy diagnosis: current applications and future perspectives. Allergy 61:1028–1039 118. Sanz ML, Sanchez G, Gamboa PM, Vila L, Uasuf C, Chazot M, Diéguez I, De Weck AL (2001) Allergen-induced basophil activation: CD63 cell expression detected by flow cytometry in patients allergic to Dermatophagoides pteronyssinus and Lolium perenne. Clin Exp Allergy 31:1007–1013 119. Saporta M, Kamei S, Persi L, Bousquet J, Arnoux B (2001) Basophil activation during pollen season in patients monosensitized to grass pollens. Allergy 56:442–445 120. Moneret-Vautrin DA, Sainte-Laudy J, Kanny G, Fremont S (1999) Human basophil activation measured by CD63 expression and LTC4 release in IgE-mediated food allergy. Ann Allergy Asthma Immunol 82:33–40 121. Shreffler WG (2006) Evaluation of basophil activation in food allergy: present and future applications. Curr Opin Allergy Clin Immunol 6:226–233

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139. Shreffler WG, Lencer DA, Bardina L, Sampson HA (2005) IgE and IgG4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2. J Allergy Clin Immunol 116:893–899 140. Izuhara K, Saito H (2006) Microarray-based identification of novel biomarkers in asthma. Allergol Int 55:361–367 141. Noh G, Ahn HS, Cho NY, Lee S, Oh JW (2007) The clinical significance of food specific IgE/IgG4 in food specific atopic dermatitis. Pediatr Allergy Immunol 18:63–70

Nasal and Bronchial Provocation Tests Donald W. Cockcroft, Beth E. Davis, and John K. Reid

Introduction Allergens are important in the pathogenesis of both asthma and rhinitis. Provocation tests with allergen are valuable in investigating the pathogenesis of allergic disease and also the pharmacotherapy of disease. This chapter deals with bronchial and nasal allergen provocation tests.

Bronchial Allergen Challenge History It is nearly 200 years since the first good clinical description of allergen-induced airway disease was published [1]. Charles Blackley in 1873 identified pollen as one cause of allergic rhinitis (AR) and allergic asthma using nasal and bronchial provocation with natural whole pollen grains [2]. His own accidental exposure to a heavy load of grass pollen produced the first clinical description of what we now recognize as the allergen-induced late asthmatic response (LAR). Throughout the mid-part of the last century, there were several published studies involving allergen challenge [3–11]. Most of these involved inhalations of aqueous allergen extracts and targeted only the early asthmatic response (EAR). Many of these studies were performed before, or shortly after the development of expiratory flow rates. The end points were monitored crudely by such things as symptoms, coarse breathing, and D.W. Cockcroft, B.E. Davis, and J.K. Reid Department of Medicine, Division of Respirology, Critical Care and Sleep Medicine, Royal University Hospital/University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada D.W. Cockcroft () Division of Respirology, Critical Care and Sleep Medicine, Royal University Hospital, 103 Hospital Drive, Ellis Hall, Rm 551, Saskatoon, SK S7N 0W8, Canada e-mail: [email protected]

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objectively by relatively insensitive lung function measurements such as maximum breathing capacity or vital capacity. The late asthmatic response was rediscovered by Herxheimer in the early 1950s primarily based on (re-)appearance of symptoms several hours after allergen challenge [6, 9]. He mistakenly thought that this might represent a continuous allergen-induced response with recurrence of symptoms and airflow obstruction after the effect of the inhaled bronchodilator prescribed to treat the EAR had worn off [9]. The classic biphasic nature of the allergen-induced response, the so-called dual asthmatic response (DAR) became evident over the next 10 or 15 years and the description and basic clinical pharmacology were well studied by Professor Orie’s group in the Netherlands [12] and Professor Pepys’ group in the UK [13]. Allergen-induced increase in airway hyperresponsiveness (AHR) to histamine or methacholine was first noted clinically during the grass pollen season by Altounyan [14] and first identified following allergen inhalation challenges in the laboratory by ourselves [15] and its association with the allergen-induced LAR was noted [16]. The allergen-induced increase in AHR has now become a component of many standardized allergen challenge protocols. Allergen-induced eosinophilic airway inflammation and its association with the LAR was first noted by the bronchoscopic studies of de Monchy et al. [17]; this can be studied much less invasively by examination and analysis of induced sputum cells [18].

Airway Response to Allergen The airway response to inhaled allergen is relatively complex particularly when compared to the responses to nonselective stimuli. Airway responses include the allergen-induced EAR, as well as the allergen-induced late sequelae, which include the LAR [6, 9, 12, 13], induced increases in AHR [15, 16], and induced eosinophilic inflammation [17, 18]. These will be outlined below. The allergen-induced EAR (Fig. 1) is an episode of airflow obstruction secondary to the release of mast-cell mediators, which results from the allergen–IgE interaction. The airflow obstruction generally develops within 10 min of the inhalation and is maximal at 20–30 min. The airflow obstruction resolves spontaneously between 90 min and 2–3 h. The EAR probably has limited clinical significance other than, when occurring upon natural exposure, to alert the individual and their physician to clinically relevant allergens. In many regards, the EAR behaves like the nonselective indirect mast-cell mediator releasing stimuli. This includes the clinical pharmacology which involves inhibition by functional antagonists [19], specific antagonists (anti-IgE) [20], and single-dose inhaled cromones but not single-dose inhaled corticosteroid [19]. The LAR (Fig. 1) is an episode of airflow obstruction, which develops after spontaneous resolution of the EAR [21]. The LAR generally commences between 3 and 4 h after allergen challenge and is somewhat variable in its duration; as with most induced airway responses, more severe responses tend to be longer lasting. The LAR lasts up to 7 or 8 h under most circumstances but may persist for 12 h or more. The

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Fig. 1 Early and late asthmatic response to inhalation of grass pollen extract. FEV1 on the vertical axis and time after inhalation of diluent (dotted line) or grass pollen extract (solid line) in hours on the horizontal axis (Reproduced with permission from Elsevier [31])

immunopathogenesis, once a topic of some controversy, has now clearly been shown to be IgE-mediated [22]. The physiology involves primarily bronchoconstriction as the LAR does respond reasonably well to bronchodilators [23]. More severe LARs are incompletely responsive to bronchodilators raising the likelihood, recognized early on, that the LAR was probably caused by and/or associated at least in part with cellular or noncellular aspects of airway inflammation [24]. The LAR is not inhibited by inhaled β2 agonists administered before the EAR [19, 25] but can be masked by large doses, repeated doses, or long-acting drugs of this category [26]. Pre-allergen administration of cromones significantly inhibits the LAR [19, 27]. Single doses of inhaled corticosteroids administered either before allergen [19] or up to 2 h after allergen challenge [28] provide marked inhibition of the LAR. Allergen-induced increases in AHR have been studied primarily using the nonselective direct stimuli histamine or methacholine [15, 16]. AHR has generally not increased by 2 h after allergen challenge [29], however, has been shown to increase 3 [30], 7 [15], and 24–30 h [15, 16, 29] after allergen challenge. Measurement of histamine or methacholine PC20/PD20 at 7 h is difficult to interpret since, in the presence of an LAR, the airway caliber may be significantly reduced. Assessment of allergen-induced AHR at 3 h is attractive since the post-allergen inhalation airway caliber in subjects with a DAR is usually at its best and this would minimize the amount of time required by the subjects. However, there may be difficulty in differentiating the airflow obstruction induced by histamine or methacholine from that of the progressing LAR. From a practical perspective, allergen-induced increase

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in histamine or methacholine responsiveness is generally measured from the day before to the day after allergen challenge either in the mornings 24 h before and 24 h after allergen challenge or in the afternoons 16–18 h prior and 30–32 h after allergen challenge. Increased responsiveness can persist for several days or longer (Fig. 2) and may be associated with increased asthma symptoms on exposure to nonallergic stimuli such as smoke, dust, and cold air [31]. Several studies have documented that increased histamine or methacholine responsiveness is seen primarily in conjunction with the LAR [15, 16] and correlates reasonably well with the magnitude of the LAR [16]. The increased responsiveness is not associated with reduced airway caliber or, more appropriately, persists after airway caliber has returned to baseline [16, 29]. The precise mechanism of the increased airway responsiveness is unclear. The association with the LAR [16] and the allergen-induced eosinophilic inflammation [18], and its inhibition by inhaled corticosteroids [19], makes it attractive to speculate that the increased airway responsiveness is somehow caused by the airway inflammation. There are, however, two studies which have cast doubt on this hypothesis. Anti-IL-5 satisfactorily prevented allergen-induced eosinophilia without inhibition of the LAR or the induced AHR to histamine [32].

Fig. 2 Allergen-induced increase in nonallergic bronchial responsiveness to inhaled histamine. A dual asthmatic response (DAR) is shown, with spontaneous recovery, to a single inhalation of ragweed pollen extract. Bronchial responsiveness to inhaled histamine (PC20) increased after allergen exposure, associated with asthma symptoms on exposure to nonallergic stimuli (Reproduced with permission from Elsevier [31])

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The same group investigated a 4-week course of IL-12 and demonstrated inhibition of allergen-induced eosinophilia with a nonsignificant reduction of the LAR but little inhibition of the allergen-induced fall in histamine PC20 [33]. Allergen-induced eosinophilia was first identified by de Monchy in 1985. Using bronchoscopic studies, this group demonstrated significant increases in bronchoalveolar lavage (BAL) eosinophils 6–7 h post-allergen inhalation in subjects with an LAR but not in subjects with an isolated EAR. They also demonstrated no significant BAL eosinophilia at 2–3 h in subjects with a DAR or an isolated EAR [17]. Standardized measurement of inflammatory cells in sputum induced by hypertonic saline is now well described and provides a much less invasive means to investigate allergen-induced airway eosinophilia [18].

Allergen Challenge Methods: DAR Model Detailed methods for performing controlled allergen challenges have been published [34–36] (see Figs. 1 and 2). The complete standardized allergen challenge requires 3 or 4 consecutive days. A control day is recommended at least once in an individual to ascertain stability of spirometry. This includes the need to differentiate any EAR from an irritant effect of inhaling the diluent or performing repeated spirometries and to differentiate an LAR from spontaneous afternoon fluctuations in FEV1. The allergen diluent is inhaled on three occasions at 10–15 min intervals either using tidal breathing or a dosimeter. The FEV1 is initially measured in triplicate prior to inhalations and is repeated on two occasions, 10 min after completion of each inhalation. Following the completion of inhalations, the FEV1 is measured in duplicate at 10-min intervals for the first hour, at 30-min intervals for the second hour, and then hourly up to 7 h. The best FEV1 at each time point is recorded and the percentage change in FEV1 is calculated using the highest pre-inhalation value and the highest post-inhalation value. Allergen challenge is done on a subsequent day using a protocol that mimics the diluent day. Doubling, tripling, or half log (3.2-fold) incremental concentrations are prepared from the best quality aqueous extracts available. A 1:8 dilution from the stock solution is made using an appropriate diluent; we frequently use 0.5% phenol with a small amount of human serum albumin as the stabilizer. From the 1:8 dilution, serial (doubling) dilutions are prepared up to 1:1,024, and further if required. The allergen concentration which causes a 20% fall in FEV1 (EAR) can be predicted with reasonable (±2–3 concentration range) accuracy from the skin test end point produced by the allergen concentrations to be used for inhalation and the methacholine PC20 [36]. Allergen challenges can safely be started three or four concentrations below this prediction. Allergen inhalations are continued until the 10-min FEV1 has fallen by 15–20%. The next concentration is administered when the 10-min FEV1 has fallen by less than 10%. Modifications are often used when there is an intermediate 10–15% FEV1 fall at 10 min. For example, the same dose of allergen may be repeated or the next dose may be inhaled for 1.5 rather

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than 2 min. Following completion of allergen challenges, spirometry is monitored for 7 h as per the control day. Allergen-induced increase in AHR is usually monitored by methacholine PC20 measured the day before and the day after allergen challenge. Allergen-induced airway inflammation can likewise be measured, generally after completion of the methacholine challenges, using induced sputum the day before and the day after. The allergen-induced EAR is determined as the maximum percentage fall in the first hour (or two) after allergen challenge and the allergen-induced LAR as the maximum percentage fall in FEV1 between 3 and 7 h after allergen challenge. Some investigations prefer to look at the area under the curve generally from 0 to 2 (or 3) h for the EAR and from 3 to 7 h for the LAR using a trapezoidal calculation. Recommendations include continuous personal attendance by a physician during the allergen challenge and the EAR and readily available physician for the following 24 h. Equipment and facility for reversal of induced bronchoconstriction and indeed for complete cardiopulmonary resuscitation should be at hand. Some authorities recommend that allergen challenge should be restricted to hospital settings. Repeat challenges within an individual should be done with at least a 7-day washout. Spirometry (±10%) and baseline methacholine PC20 (±1 doubling concentration) should be stable. Avoidance of respiratory tract infections and natural allergen exposure is essential. In order to interpret effects of interventions, it is important to achieve identical dosage of allergen for subsequent challenges; this is occasionally difficult because of the occurrence of a large EAR at a smaller allergen concentration. The DAR model is widely used for investigations of asthma pathophysiology and drug treatments. The focus for the majority of these studies is on the allergeninduced late sequelae (LAR, AHR, and eosinophilia) since these most closely mimic naturally occurring asthma.

Allergen Challenge Methods: EAR Model The allergen-induced EAR can be studied using a protocol similar to methacholine or other challenges with the EAR being expressed as the allergen PC15 or PC20 [20]. Repeat studies looking at interventions can be done to target changes in allergen PC20 [20, 37–40]. The allergen PC20 model has several attractive advantages and one major disadvantage. Perhaps the most important advantage is the improved ability to discriminate between the magnitude of the response for very effective treatments. For example, inhaled β2 agonists can shift the allergen PC20 to more than four doubling concentrations [37]. The magnitude of this protective effect would be lost looking at the percentage inhibition of the maximum FEV1 fall after the same dose of allergen. A one-doubling-dose improvement in allergen PC20 would equate to a 50% reduction in EAR (defined by percentage fall in FEV1). A two-dose improvement in PC20 would equate to a 75% reduction in EAR, and so on. It becomes difficult, therefore, to discriminate changes with the same-dose allergen challenge

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model for improvements greater than a two-doubling-dose improvement in PC20; it is only possible to say that the treatments were very effective. Other advantages of the allergen PC20 model include a larger number of available subjects since 50% or more allergen challenge patients do not have a late response; improved stability of patients since in our experience isolated early responders often have less volatile asthma; and the obviation of the need to administer the same dose of allergen at each setting. The major disadvantage of the allergen PC20 model is its failure to capture the more clinically relevant late sequelae. The PC20 model involves inhalation of doubling concentrations of allergen at 12-min intervals followed by measuring the percentage fall in FEV1 as noted above. The study is continued until there is a 20% or greater fall in FEV1 and the allergen PC20 is calculated using the same algebraic formula that is used to calculate the methacholine PC20. Following completion of the data acquisition, the airflow obstruction can be rapidly relieved with an inhaled beta agonist and a single dose of inhaled corticosteroid (e.g., fluticasone 500 µg) can be administered to prevent any LAR [20]. This is particularly important when measuring the PC20 after inhibition by a beta agonist since the larger dose of allergen administered after a beta agonist has the potential to produce large LARs [41]. The allergen PC20 model has been successfully used in studies addressing beta agonist tolerance [37], increased allergen response after regular beta agonist use [37–39], and in preliminary studies assessing the effect of asthma therapies such as anti-IgE [20] and combination treatment with a leukotriene receptor antagonist and an H1 blocker [40].

Allergen Challenge Methods: Repeated Low-Dose Challenge Allergen challenges using single brief relatively high-dose inhalations of already dissolved allergen do not mimic natural allergen exposure. Several investigators have developed an allergen protocol that involves repeated daily low-dose allergen exposure below the threshold of a detectable change in FEV1 [42, 43]. After 5 days of low-dose challenge, susceptible subjects demonstrate significant increases both in airway responsiveness and airway eosinophilia. This type of challenge more closely mimics natural exposure and may well be a safer allergen challenge alternative. However, the time required increases markedly, the washout between repeat allergen challenges may be longer, and there is a question of how practical this approach would be for routine clinical research.

Allergen Challenge Methods: Segmental Allergen Challenge The final method for allergen challenge which has been occasionally used for physiologic studies is that of bronchoscopic segmental allergen challenge [44].

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It is possible to administer a relatively high amount of allergen locally via a bronchoscope and make measurements via the same route. This is, of course, quite invasive and has been reserved for research applications.

Allergen Challenges: Uses At one time, allergen challenges were recommended as a clinical tool to determine which allergens might be relevant in a given individual [45]. Since the early asthmatic response can be reasonably accurately predicted from easier and safer measures (methacholine challenge, skin test end point) [36], there seems little to be gained by performing allergen challenges regarding clinical relevance. It is not likely that any more clinically relevant data will be obtained from using the same often less than adequately standardized allergens by inhalation as there is by skin test. Occasionally, it has been said that some patients may benefit from seeing the proof of a cause and effect regarding a specific allergen [45], generally a house pet. Most authorities therefore agree that allergen challenge studies are limited to research purposes. The most extensive use of the allergen challenge, generally the dual asthmatic response model, is for investigation of new asthma therapies. It is a reasonable hypothesis that therapeutic interventions which suppress the late sequelae should prove effective asthma therapies whereas those which do not inhibit the late sequelae are likely to prove ineffective. This hypothesis has not been completely validated. The late asthmatic response and associated sequelae are inhibited by inhaled corticosteroid used before [19, 26] or after [27] allergen challenge, by inhaled cromones, by leukotriene receptor antagonists [46, 47], and by anti-IgE [48]. These responses are not influenced by pre-allergen inhaled β2 agonist in a dose sufficient to completely inhibit the EAR [19, 25]. It is possible to administer a larger dose of allergen after a beta agonist and this will result in appearance or accentuation of late responses [41]. Investigations of longer-acting functional antagonists such as the longacting beta agonists [26, 49] have produced data which are difficult to interpret. These drugs have effects which last long enough to mask some of the late sequelae. The apparent separation of some of the airway physiology with the airway inflammation in the IL-12 [32] and anti-IL-5 [33] studies stress the need to measure all aspects of the allergen response. The other major research utility of the allergen challenge is investigation into the pathogenesis of asthma. This can be accomplished by the detailed evaluation of the airway responses, and either sputum or biopsy studies of cells, mediators, cytokines, etc. A final research use for allergen challenge is identification and definite confirmation of new allergens [45].

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Nasal Allergen Challenge History As previously noted, reports of upper airway allergic disease began in the early 1800s [1] and the first documented nasal challenge was described in 1862 [2]. Crude as it was, the sniffing of grass pollen was observed to induce hay fever, a term still used today but largely replaced by allergic rhinitis in the medical community. Nasal provocation testing (NPT) with specific allergic stimuli is in many ways similar to bronchial allergen challenge. A known or suspect naturally provoking stimulus is introduced to the nasal mucosa; symptoms (e.g., itchy, runny nose, sneezing) are scored and changes in nasal airflow and patency are measured. Investigations provide insight into the mechanisms of action and pathophysiology of allergic rhinitis (AR) and are useful in evaluating pharmacological efficacy but there has been limited clinical application in North America.

Nasal Response to Allergen The response of the upper airway to allergic stimuli is an IgE-mediated Type I hypersensitivity reaction. In sensitized individuals, antigen–IgE–FcεRI interactions cause mast cells to release mediators, which subsequently activate neural, vascular, glandular, and inflammatory responses leading to sneezing, itching, rhinorrhea, and congestion/obstruction. Response kinetics include both an early and a late phase. The early or acute phase is characterized by subjective symptoms (e.g., sneezing) resulting in large part from the physiological effects of histamine. The late phase is associated with congestion/obstruction and decreased nasal airflow that coincides with inflammation [50–53]. First-line therapies therefore include H1 antagonists and nasal corticosteroids; other therapeutic interventions which may be efficacious include environmental control, alpha adrenergic stimulants, mast-cell stabilizers, leukotriene receptor antagonists, and immunotherapy.

Methods Guidelines for objective measurements [54], methods for clinicians [55], and reviews of NPT [56, 57] have been published; however, an accepted standardized methodology has yet to be developed. Diagnostic tests require high sensitivity and specificity, and must generate reproducible data that correlates with clinical findings. The primary rationale for nonacceptance in North America is that

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objective measurements of nasal airway patency (e.g., rhinomanometry and acoustic rhinometry) provide little additional information to that obtained from physical examination, nasal endoscopy, CT imaging, or self-assessment. NPT is, however, clinically used in many European countries. Clinical indications include identification of an occupational sensitivity and confirmation of a suspect sensitivity where history and skin tests are inconclusive; NPT is contraindicated during acute respiratory infection, allergic exacerbation, and pregnancy, and should not be conducted if lung function is poor. Rhinoscopic examination of the nasal passages should precede NPT to assess baseline nasal mucosa and rule out differential nasal pathology (e.g., deviated septum, polyps) and should follow an ambient acclimatization period of about 20–30 min. Skin tests (prick or intradermal) should also be performed to identify the sensitizing agent to be used for the NPT and to assist in the preparation of allergen dilutions. Testing should occur in the morning to avoid confounding influences from other stimuli (e.g., fumes, food) and appropriate washout of relevant medications (e.g., anti-histamines, anti-cholinergics, anti-inflammatories) must be considered when interpreting a negative test. Baseline nasal function (airflow and resistance) and subjective measurements of symptoms (e.g., itching, sneezing, rhinorrhea) are recorded for post-NPT comparison. Techniques for measuring nasal airflow and resistance include nasal peak-flow rates (expiratory – NPEFR and inspiratory – NPIFR) and active anterior, passive anterior, or active posterior rhinomanometry. Measurements of nasal peak-flow rates are attractive due to their low cost and relatively simple use. Active anterior rhinomanometry is commonly used but complete obstruction in one nare limits its use. Active posterior rhinomanometry is a more sensitive test, which is impractical for clinical use but well suited for a research laboratory [58]. Acoustic rhinometry, which uses sound waves to measure changes in the cross-sectional area of a nare, and optical rhinometry, which measures the passage of light through nasal tissue, are emerging technologies [59–61]. The mean of three reproducible measurements (within 10–15% of each other) is used as the comparator for changes occurring post allergen. The mean should be within the normal range as false negatives may occur in the presence of baseline obstruction. The challenge begins with the administration of diluent. A breathhold during administration is important to prevent deposition of the stimuli into the lower airway. The wider nare receives the stimulus preferably via a metered dose device and the response is measured in both. Symptoms are recorded over the next 10 or 15 min and nasal resistant is repeated at 10 or 15 min. If the response is negative, the predetermined starting concentration of allergen is applied and the process continues, with approximately threefold increases (e.g., 1:10,000, 1:3,000, 1:1,000), until a positive response occurs or until the highest concentration is administered. Dilutions of 1:500 or less may lead to false positives due to nonspecific irritation. Interpretation of the response is somewhat ambiguous. Consideration should be given to both the objective and subjective measurements so that a “true” negative result would be no change in airflow and the absence of symptoms. Positive responses vary according to the method of measurement. For example, a decrease of 20% in NPEFR and the presence of symptoms (not scored);

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the occurrence of any two of the following: 50% decrease in NPEFR, ≥5 sneezes, nasal secretions; a 20% decrease in nasal airflow combined with a total symptom score (TSS) of 2 or greater; a TSS of 3 or greater (i.e., no objective measurement) or a decrease in nasal airflow of ≥40% (i.e., no subjective measurement) have all been considered positive tests. A standardized symptom scoring system has recently been proposed [62]. In summary, NPT has not evolved as a clinical tool in the management or diagnosis of allergic rhinitis in Canada and the USA. Given the ease and relative noninvasiveness of obtaining samples, the use of NPT in drug development and the pathophysiology of allergic upper (and lower) airway disease is attractive. However, general agreement on a standardized methodology would greatly improve the utility of NPT. Acknowledgments The authors would like to thank Jacquie Bramley for assisting in the preparation of this manuscript.

References 1. Bostock J (1819) Case of a periodical affection of the eyes and chest. Med Chir. Trans. 10:161–164 2. Blackley CH (1873) In: Experimental Researches on the Cause and Nature of Catarrhus Aestivus (Hay-Fever or Hay-Asthma). Balliere Tindall & Cox, London. Reprinted by Dawson’s of Pall Mall, 1959:80 3. Stevens FA (1934) A comparison of pulmonary and dermal sensitivity to inhaled substances. J Allergy 5:285–287 4. Schiller IW, Lowell FC (1947) The effect of drugs in modifying the response of asthmatic subjects to inhalation of pollen extracts as determined by vital capacity measurements. Ann Allergy 5:564–566 5. Lowell FC, Schiller IW (1948) Measurement of changes in vital capacity as a means of detecting pulmonary reactions to inhaled aerosolized allergenic extracts in asthmatic subjects. J Allergy 19:100–107 6. Herxheimer H (1951) Bronchial hypersensitization and hyposensitization in man. Int Arch Allergy Appl Immunol 2:40–59 7. Herxheimer H (1951) Bronchial obstruction induced by allergens, histamine and acetyl-betamethylcholinechloride. Int Arch Allergy 2:27–39 8. Herxheimer H (1951) Induced asthma in man. Lancet 260:1337–1341 9. Herxheimer H (1952) The late bronchial reaction in induced asthma. Int Arch Allergy Appl Immunol 3:323–333 10. Colldahl H (1952) A study of provocation tests on patients with bronchial asthma. I. The reliability of provocation tests performed under different conditions. Acta Allergol 5:133–142 11. Colldahl H (1952) A study of provocation tests on patients with bronchial asthma. II. The outcome of provocation tests with different antigens. Acta Allergol 5:143–153 12. Booij-Noord H, deVries K, Sluiter HJ, Orie NGM (1972) Late bronchial obstructive reaction to experimental inhalation of house dust extract. Clin Allergy 2:43–61 13. Pepys J, Chan M, Hargreave FE, McCarthy DS (1968) Inhibitory effects of disodium cromoglycate on allergen-inhalation tests. Lancet 2:134–137 14. Altounyan REC (1964) Variation of drug action on airway obstruction in man. Thorax 19:406–415

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15. Cockcroft DW, Ruffin RE, Dolovich J, Hargreave FE (1977) Allergen-induced increase in nonallergic bronchial reactivity. Clin Allergy 7:503–513 16. Cartier A, Thomson NC, Frith PA, Roberts R, Hargreave FE (1982) Allergen-induced increase in bronchial responsiveness to histamine: relationship to the late asthmatic response and change in airway caliber. J Allergy Clin Immunol 70:170–177 17. de Monchy JGR, Kauffman HF, Venge P, Koeter GH, Jansen HM, Sleuter HJ, de Vries K (1985) Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am Rev Respir Dis 131:373–376 18. Pin I, Freitag AP, O’Byrne PM, Girgis-Gabardo A, Watson RM, Dolovich J, Denburg JA, Hargreave FE (1992) Changes in the cellular profile of induced sputum after allergen-induced asthmatic responses. Am Rev Respir Dis 145:1265–1269 19. Cockcroft DW, Murdock KY (1987) Comparative effects of inhaled salbutamol, sodium cromoglycate and beclomethasone dipropionate on allergen-induced early asthmatic responses, late asthmatic responses and increased bronchial responsiveness to histamine. J Allergy Clin Immunol 79:734–740 20. Boulet LP, Chapman KR, Cote J, Kalra S, Bhagat R, Swystun VA, Laviolette M, Cleland LD, Deschesnes F, Su JO, De Vault A, Fick RB Jr, Cockcroft DW (1997). Inhibitory effects of an anti-IgE antibody E25 on allergen-induced early asthmatic response. Am J Respir Crit Care Med 155:1835–1840 21. O’Byrne PM, Dolovich J, Hargreave FE (1987) State of the art: late asthmatic responses. Am Rev Respir Dis 136:740–751 22. Kirby JG, Robertson DG, Hargreave FE, Dolovich J. Asthmatic responses to inhalation of anti-human IgE. Clin Allergy 1986;16:191–194. 23. Dorsch W, Baur X, Emslander HP, Fruhmann G (1980) Zu pathogenese und therapie der allergeninduzierten verzogerten bronchialobstruktion. Prax Pneumol 34:461–468 24. Pepys J, Hutchcroft BJ (1975) Bronchial provocation tests in etiologic diagnosis and analysis of asthma. Am Rev Respir Dis 112:829–859 25. Hegardt B, Pauwels R, Van Der Straeten M (1981) Inhibitory effect of KWD 2131, terbutaline, and DSCG on the immediate and late allergen-induced bronchoconstriction. Allergy 36:115–122 26. Twentyman OP, Finnerty JP, Harris A, Palmer J, Holgate ST (1990). Protection against allergen-induced asthma by salmeterol. Lancet 336:1338–1342 27. Booij-Noord H, Orie NGM, deVries K (1971) Immediate and late bronchial obstructive reactions to inhalation of house dust and protective effects of disodium cromoglycate and prednisolone. J Allergy Clin Immunol 48:344–354 28. Cockcroft DW, McParland CP, O’Byrne PM, Manning P, Friend JL, Rutherford BC, Swystun VA (1993) Beclomethasone given after the early asthmatic response inhibits the late response and the increased methacholine responsiveness and cromolyn does not. J Allergy Clin Immunol 91:1163–1168 29. Cockcroft DW, Murdock KY (1987) Changes in bronchial responsiveness to histamine at intervals after allergen challenge. Thorax 42:302–308 30. Durham SR, Craddock CF, Cookson WO, Benson MK (1988) Increases in airway responsiveness to histamine precede allergen-induced late asthmatic responses. J Allergy Clin Immunol 82:764–770 31. Cockcroft DW (1983) Mechanism of perennial allergic asthma. Lancet 2:253–256 32. Leckie MJ, ten Brinke A, Khan J, Diamant Z, O’Connor BJ, Walls CM, Mathur AK, Cowley HC, Chung KF, Djukanovic R, Hansel TT, Holgate ST, Sterks PJ, Barnes PF (2000) Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyperresponsiveness, and the late asthmatic response. Lancet 356:2144–2148 33. Bryan SA, O’Connor BJ, Matti S, Leckie MJ, Kanabar V, Khan J, Warrington SJ, Renzetti L, Rames A, Bock JA, Boyce MJ, Hansel TT, Holgate ST, Barnes PJ (2000) Effects of recombinant human interleukin-12 on eosinophils, airway hyperresponsiveness, and the late asthmatic response. Lancet 356:2149–2153 34. Sterk PJ, Fabbri LM, Quanjer PH, Cockcroft DW, O’Byrne P, Anderson SD, Juniper EF, Malo JL (1993) Airway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Eur Respir J Suppl 16:53–83

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35. Cockcroft DW with assistance from R. Hopp (2004) Allergen Bronchoprovocation. AAAAI teaching slide set. Posted on AAAAI website, www:aaaai.org 36. Cockcroft DW, Murdock KY, Kirby J, Hargreave FE (1987) Prediction of airway responsiveness to allergen from skin sensitivity to allergen and airway responsiveness to histamine. Am Rev Respir Dis 135:264–267 37. Cockcroft DW, McParland CP, Britto SA, Swystun VA, Rutherford BC (1993) Regular inhaled salbutamol and airway responsiveness to allergen. Lancet 342:833–837 38. Cockcroft DW, Swystun VA, Bhagat R (1995) Interaction of inhaled β2 agonist and inhaled corticosteroid on airway responsiveness to allergen and methacholine. Am J Respir Crit Care Med 152:1485–1489 39. Bhagat R, Swystun VA, Cockcroft DW (1996) Salbutamol-induced increased airway responsiveness to allergen and reduced protection vs. methacholine: dose-response. J Allergy Clin Immunol 97:47–52 40. Davis BE, Todd DC, Cockcroft DW (2005) Effect of combined montelukast and desloratadine on the early asthmatic response to inhaled allergen. J Allergy Clin Immunol 116:768–772 41. Lai CKW, Twentyman OP, Holgate ST (1989) The effect of an increase in inhaled allergen dose after rimiterol hydrobromide on the occurrence and magnitude of the late asthmatic response and the associated change in nonspecific bronchial responsiveness. Am Rev Respir Dis 140:917–923 42. Sulakvelidze I, Inman MD, Rerecich T, O’Byrne PM (1998) Increases in airway eosinophils and interleukin-5 with minimal bronchoconstriction during repeated low-dose allergen challenge in atopic asthmatics. Eur Respir J 11:821–827 43. Gauvreau GM, Sulakvelidze I, Watson RM, Inman MD, Rerecich TJ, O’Byrne PM (2000) Effects of once daily dosing with inhaled budesonide on airway hyperresponsiveness and airway inflammation following repeated low-dose allergen challenge in atopic asthmatics. Clin Exp Allergy 30:1235–1243 44. Metzger WJ, Zavala D, Richerson HB, Moseley P, Iwamota P, Monick M, Sjoerdsma K, Hunninghake GW (1987) Local allergen challenge and bronchoalveolar lavage of allergic asthmatic lungs. Description of the model and local airway inflammation. Am Rev Respir Dis 135:433–440 45. Spector S, Farr R (1979) Bronchial inhalation challenge with antigens. J Allergy Clin Immunol 64:580–586 46. Diamant Z, Grootendorst DC, Veselic-Charvat M, Timmers MC, De Smet M, Leff JA, Seidenberg BC, Zwinderman AH, Peszek I, Sterk PJ (1999) The effect of montelukast (MK-0476), a cysteinyl leukotriene receptor antagonist, on allergen-induced airway responses and sputum cell counts in asthma. Clin Exp Allergy 29:42–51 47. Leigh R, Vethanayagam D, Yoshida M, Watson RM, Rerecich T, Inman MD, O’Byrne PM (2002). Effects of montelukast and budesonide on airway responses and airway inflammation in asthma. Am J Respir Crit Care Med 166:1212–1217 48. Fahy JV, Fleming HE, Wong HH, Liu JT, Su JQ, Reimann J, Fick RB Jr, Boushey HA (1997) The effect of an anti-IgE monoclonal antibody on the early- and late-phase responses to allergen inhalation in asthmatic subjects. Am J Respir Crit Care Med 155:1828–1834 49. Wong BJ, Dolovich J, Ramsdale EH, O’Byrne P, Gontovnick L, Denburg JA, Hargreave FE (1992) Formoterol compared with beclomethasone and placebo on allergen-induced asthmatic responses. Am Rev Respir Dis 146:1158–1160 50. Howarth PH, Salagean M, Dokic D (2000) Allergic rhinitis: not purely a histamine-related disease. Allergy 64:7–16 51. Hansen I, Klimek L, Mosges R, Hormann K (2004) Mediators of inflammation in the early and the late phase of allergic rhinitis. Curr Opin Allergy Clin Immunol 4(3):159–163 52. Gelfand EW (2004) Inflammatory mediators in allergic rhinitis. J Allergy Clin Immunol 114:S135–138 53. Jeffrey PK, Haahtela T (2006) Allergic rhinitis and asthma: inflammation in a one-airway condition. BMC Pulm Med 6:S5 54. Malm L, Gerth van Wijk R, Bachert C (2000) Guidelines for nasal provocations with aspects on nasal patency, airflow, and airflow resistance. International Committee on Objective Assessment of the Nasal Airways, International Rhinologic Society. Rhinology 38(1):1–6

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55. Litvyakova LI, Baraniuk JN (2002) Human nasal allergen provocation for determination of true allergic rhinitis: methods for clinicians. Curr Allergy Asthma Rep 2(3):194–202 56. Druce HM, Schumacher MJ (1990) Nasal provocation challenge. The Committee on Upper Airway Allergy. J Allergy Clin Immunol 86(2):261–264 57. Gosepath J, Amedee RG, Mann WJ (2005) Nasal provocation testing as an international standard for evaluation of allergic and nonallergic rhinitis. Laryngoscope 115(3):512–516 58. Nathan RA, Eccles R, Howarth PH, Steinsvag SK, Togias A (2005) Objective monitoring of nasal patency and nasal physiology in rhinitis. J Allergy Clin Immunol 115:S442–459 59. Clement PAR, Gordts F (2005) Consensus report on acoustic rhinometry and rhinomanometry. Rhinology 43:169–179 60. Uzzaman A, Metcalfe DD, Komarow HD (2006) Acoustic rhinometry in the practice of allergy. Ann Allergy Asthma Immunol 97(6):745–751 61. Wustenberg EG, Zahnert T, Huttenbrink KB, Hummel T (2007) Comparison of optical rhinometry and active anterior rhinomanometry using nasal provocation testing. Arch Otolaryngol Head Neck Surg 133(4):344–349 62. Riechelman H, Bachert C, Goldschmidt O, Hauswald B, Klimek L, Schlenter WW, Tasman AJ, Wagenmann M (2003) Application of the nasal provocation test on diseases of the upper airways. Position paper of the German Society for Allergology and Clinical Immunolgy (ENT Section) in cooperation with the Working Team for Clinical Immunology, Laryngorhinootologie 82(3), pp. 183–188

Nasal and Bronchial Nonallergic Provocation Tests John K. Reid, Donald W. Cockcroft, and Beth E. Davis

Introduction Provocation challenges, both bronchial and nasal, can be performed with nonselective or selective stimuli. These are often referred to as nonallergic and allergic stimuli, respectively. This chapter deals with nonselective (nonallergic) bronchial and nasal provocation tests.

Nonallergic Bronchial Provocation Bronchoprovocation tests are widely used both diagnostically and in the investigation of the pathogenesis of asthma [1]. The nonselective or nonallergic provocation tests which are or might be relevant to all subjects with asthma can be divided into direct-acting and indirect-acting [2]. Direct bronchoconstrictors are those which act directly on airway smooth muscle receptors such as cholinergic agonists acting on muscarinic receptors and histamine acting on H1 receptors. Indirect bronchoconstrictors act through intermediate pathways to lead to bronchoconstriction. Many (but not all) of these involve mast cell mediator release, either osmotically (exercise, hyperventilation, nonisotonic aerosols, mannitol) or non-osmotically (AMP).

J.K. Reid, D.W. Cockcroft, and B.E. Davis Department of Medicine, Division of Respirology, Critical Care and Sleep Medicine, Royal University Hospital/University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada D.W. Cockcroft () Royal University Hospital, Division of Respirology, Critical Care and Sleep Medicine, 103 Hospital Drive, Ellis Hall, Rm 551, Saskatoon, SK S7N 0W8, Canada e-mail: [email protected]

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Direct Bronchial Challenges: Methacholine Background Airway hyperresponsiveness (AHR) is regarded as a characteristic and defining feature of the syndrome we call asthma [3]. Airway hyperresponsiveness has been most often measured clinically using the direct nonselective stimuli histamine or methacholine; both stimuli give similar results [4] and methacholine is now most widely used because it causes less systemic side effects.

History There are reports from the first half of the twentieth century of bronchoconstriction induced by parenteral administration of muscarinic agonists or histamine [5, 6]. The era of bronchoprovocation, however, began with Robert Tiffeneau in the 1940s. Tiffeneau was the first to use spirometry to measure expiratory flow rates as opposed to simple volumes [7]. Indeed, the forced expired volume in one second (FEV1 or VEMS in French) was referred to as Tiffeneau’s Test. Tiffeneau used inhaled isoproterenol as a bronchodilator and inhaled acetylcholine as a bronchoconstrictor and suggested that these tests might be useful in evaluating asthma and other lung diseases [8]. Both types of tests targeted a standardized end point, namely a 20% change in FEV1. While it was safe to administer a high concentration of bronchodilator, it was not safe to start with a high concentration of a bronchoconstrictor and, consequently, progressive dosing regimens were developed for safety reasons. Little attention was paid to dose other than an arbitrary designation of a maximal dose. We appreciate now that airway responsiveness to histamine and methacholine is unimodally distributed in the population [9], that the test is relatively imprecise (provocation concentration causing a 20% fall in FEV1 or PC20 repeatability is at best ±1 doubling concentration) [10], and that airway responsiveness can be measured into the normal range. For all these reasons, it is important to standardize methacholine inhalation tests so as to administer as reproducible a dose as possible. This is both relevant for repeatability within a laboratory and within an individual method and for comparative data between laboratories and between methods.

Methacholine Inhalation Methods Several years ago, the American Thoracic Society (ATS) published detailed methods for two of the more commonly used methacholine inhalation protocols [11]. The published methods are identical except for the method of aerosol generation and inhalation.

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The 2-min tidal breathing method was developed by ourselves [12] by modification of a Dutch method [13]. Aerosols are generated by a durable and reproducible jet nebulizer calibrated to deliver a mass loss of 0.13 g/min of an aerosol with a particle size between 1.5 and 5 µm. This mass loss is interpreted as an output of 0.13 ml/min, however, it must be appreciated that determination of nebulizer output by weighing it before and after a period of nebulization overestimates the true output because the mass loss includes a significant proportion (25% or more) due to evaporation rather than nebulization [14]. Using a noseclip and either a mask or mouthpiece, aerosols are inhaled through the mouth for 2 min of tidal breathing exposing the individual at each concentration to approximately 90 µl of aerosol (0.13 ml/min × 2 min × a duty cycle of approximately 0.35). The dosimeter method was modified [15] from that described by the American Academy of Allergy [16]. Aerosols are inhaled from a breath-activated dosimeter via a jet nebulizer. Inhalations are recommended to go from functional residual capacity (FRC) to total lung capacity (TLC) with a slow 5 s inhalation and a 5 s breathhold. The nebulizer is calibrated to deliver 9 µl per inhalation and each concentration is inhaled with five TLC inspirations. This method exposes individuals to 45 µl (9 µl × 5 breaths) for each concentration stepup. The remainder of the methods are identical for both protocols and include initial full spirometry in triplicate, inhalation of the diluent, followed by inhalation of doubling concentrations of methacholine from 0.03 up to 16 mg/ml or higher with 5-min intervals between the commencement of one inhalation and the next. Single determinations of FEV1 (some use a truncated spirogram of 1.5–2 s to avoid fatigue) are made at 30 and 90 s. Percent change in FEV1 is determined either from the lowest post-saline to the lowest post-methacholine or the highest post-saline to the highest post-methacholine; similar results are obtained by either method [17]. The test is continued until the FEV1 has fallen by ≥20% or until the highest concentration has been administered. The PC20 is calculated either graphically or with an algebraic equation [18] from the log concentration versus response curve. The provocation dose causing a 20% FEV1 fall or PD20 can be approximated by multiplying the PC20 by a constant. At the time these guidelines were written, there were few comparative data between methods. Despite the twofold larger aerosol exposure for the tidal breathing method, equivalent results had been seen in a small study [15]; this was attributed to more efficient retention and deposition of aerosol with the dosimeter method [11]. We have participated in several comparative studies [19–21] and agree that this is generally true, however, some subjects with asthma and mild AHR demonstrate TLC-induced bronchoprotection and have, consequently, negative methacholine challenges when the dosimeter method is performed by the ATS recommendation (see Fig. 1). While the prevalence of this is unknown, it occurs in precisely those individuals who might be undergoing a diagnostic methacholine challenge; i.e., those with asthma symptoms but normal lung function. This issue can be overcome by using submaximal inhalations with the dosimeter, or by the 2-min tidal breathing method and we would recommend that methacholine challenges by any method be done without TLC inhalations [22].

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Fig. 1 Tidal breathing PC20 (left) and dosimeter PC20 (right) values. PC20 values are expressed in a log scale on the vertical axis. The geometric mean tidal breathing PC20 is 1.8 mg/ml, and the dosimeter PC20 is 4.1 mg/ml (P < 0.00001). The nine subjects shown in green have dosimeter PC20 values of between 32 and 128 mg/ml, and the five subjects depicted in red have dosimeter PC20 values of between 16 and 32 mg/ml (Reproduced from [22]. With permission form Elsevier.)

Interpretation Methacholine challenge tests are primarily used as a diagnostic aid for asthma. It seems likely that methacholine challenges may occasionally be overinterpreted or misinterpreted, as many believe that a positive methacholine challenge is synonymous with asthma. The arbitrary cutpoints from the ATS guidelines are as follows [11]. A PC20 > 16 mg/ml is normal, PC20 4–16 mg/ml (i.e., 8 mg/ml ± 1 concentration)

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borderline, PC20 1–4 mild, 0.25–1.0 mg/ml moderate, and PC20 < 0.25 mg/ml marked airway hyperresponsiveness. Using these cutpoints, the methacholine challenge is highly sensitive for symptoms of current asthma [23, 24]. The sensitivity and negative predictive value approach 100%, that is, a methacholine PC20 > 16 mg/ml excludes current asthma with reasonable certainty. Positive methacholine challenges are often overinterpreted. It is difficult to evaluate the performance of the test since there is no easy confirmatory diagnostic test for asthma (i.e., variable airflow obstruction) in subjects with symptoms and repeatedly normal spirometry. The positive predictive value, however, improves with an increased pretest likelihood (i.e., application of the test to a group with a high prevalence of disease) and, although not validated, we believe that the diagnostic value increases if the methacholine-induced symptoms mimic the naturally occurring symptoms. Since the methacholine challenge is highly sensitive, false negative results should be relatively uncommon. Perhaps the most common reason for a false negative test would be the TLC bronchoprotection noted above. Other considerations include elite athletes, some of whom have negative methacholine challenges despite positive exercise challenges [25]. This issue has to be interpreted with caution since strenuous exercise can also induce non-bronchoconstrictive airway dysfunction [26]. Other reasons for false negative challenges include evaluation for symptoms which are not clinically current and the accidental failure to withhold either specific antagonists (anti-muscarinic bronchodilators or other medications with anticholinergic side effects) or functional antagonists (primarily inhaled β2 agonists). There are several important caveats that must be recalled for accurate interpretation of methacholine challenges. These are outlined in Table 1 and will be reviewed briefly below. It is important that symptoms are clinically current, since airway responsiveness can normalize with allergen [24] and occupational sensitizer avoidance, sometimes within a few days [27]. This is one reason for confusion regarding methacholine test interpretation since epidemiologic studies, which define current as occurring within the past year, have often shown a poor negative predictive value [28]. Controller asthma medications can also result in a negative methacholine challenge [29] but only if the patients have been completely asymptomatic for a significant period of time.

Table 1 Methacholine challenge Interpretation caveats 1. 2. 3. 4. 5. 6. 7.

Symptoms must be current (important reinterpretation of a negative test) Flow rates (FEV1) must be normal (important reinterpretation of a positive test) PC20 is imprecise (±1–2 dose repeatability) leading to a borderline range May be of limited value in therapeutic decisions in isolated cough Severity of methacholine AHR does not equate with severity of asthma Recall medications can be a cause of false negative results (especially anticholinergics) Inhalation of methacholine by maximal inhalationscan cause false negative tests by bronchoprotection in subjects with mild AHR

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The second important caveat is that flow rates (FEV1) should be normal. Patients with airflow obstruction due to reasons other than asthma (i.e., COPD) exhibit airway hyperresponsiveness to the direct stimuli histamine and methacholine which correlates highly with the magnitude of the FEV1 reduction [30, 31]. Accordingly, methacholine airway responsiveness cannot be used to differentiate asthma from COPD. The requirement for normal or near normal baseline FEV1 is more an issue of interpretation than an issue of safety. The addition of a borderline range is relatively new and often not appreciated. Methacholine PC20’s in the borderline range (4 and 16 mg/ml) must be interpreted with caution, since many subjects with no asthma or no other lung disease may have PC20’s in this range [24]. Methacholine challenges are often performed in subjects with isolated cough looking for cough-variant asthma. Although valuable in assessing underlying physiology, the methacholine challenge is of limited value therapeutically. Positive methacholine challenges have been shown not to correlate well with response to asthma therapy [32]. Eosinophilic bronchitis (eosinophilic airway inflammation with normal methacholine challenge and no variable airflow obstruction) is a significant cause of cough which is closely related to asthma [33]. The cough associated with eosinophilic bronchitis responds well to inhaled corticosteroids and, consequently, a negative methacholine challenge does not correlate with nonresponse to asthma therapy. Accordingly, we have suggested that from a practical perspective, a diagnostic trial of inhaled corticosteroids might be preferred over a methacholine challenge. The severity of airway hyperresponsiveness as outlined above does not correlate with the severity of asthma. This is a common misconception that has arisen particularly from various regulatory agencies (Armed Forces, Police Forces, etc.). For example, mild airway hyperresponsiveness does not equate with mild asthma; the majority of subjects with mild airway hyperresponsiveness in the population do not have asthma at all, whereas the majority of subjects with moderate airway hyperresponsiveness will have mild asthma clinically [24]. Methacholine challenges are usually not required diagnostically in subjects with moderate or severe asthma. It is important to recall that unrecognized use of either respiratory medications or non-respiratory medications (particularly those with anticholinergic effects) can occasionally result in false negative challenges. The medication history in subjects whose methacholine challenges are not consistent with the clinical picture must be carefully reviewed. Finally, it is important to recall that methods-related bronchoprotection can cause false negative challenges and that if indicated, methacholine challenges should be repeated with a submaximal inhalation method.

Summary Methacholine challenges, the most widely performed inhalation bronchoprovocation tests, are valuable diagnostic aids for asthma. The test functions best to exclude current asthma with reasonable certainty when it is negative. Positive challenges

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must be interpreted with caution based on pretest probability and whether or not methacholine mimics the naturally occurring symptoms. There are several important caveats that must be borne in mind when interpreting methacholine challenges. The most important of these are the clinical currency of the symptoms under investigation and the normality of the baseline FEV1.

Indirect Bronchial Challenges Overview Several years ago, Pauwels et al. proposed the differentiation of (nonselective) bronchoconstrictors into direct and indirect [2]. They suggested that, since naturally occurring bronchoconstriction in subjects with asthma occurred via indirect pathways that indirect airway responsiveness should correlate better with the clinical syndrome we call asthma. This topic has recently been extensively reviewed [34, 35]. Subsequent investigations have confirmed this. For example, the osmotic stimuli exercise and cold air differentiate asthma from COPD [36, 37] whereas the direct stimuli histamine and methacholine do not. Similarly, AMP responsiveness has been shown to correlate better than methacholine responsiveness with respect to eosinophilic airway inflammation in asthma [38, 39]. There are data to suggest that indirect responsiveness improves to a greater extent than direct airway responsiveness with anti-inflammatory (or controller) therapies such as environmental control [40] or inhaled corticosteroids [41]. There is also little doubt that the indirect challenges are much more specific for asthma. In fact, a technically well-performed exercise challenge, when positive, has virtually a 100% specificity for exercise-induced bronchoconstriction (EIB). Unfortunately, with the increased specificity, these tests tend to have relatively low sensitivity. We have found that many if not the majority of patients with mild and/ or well-controlled current asthma fail to respond to indirect stimuli such as exercise or AMP. Many of the indirect stimuli are dose limited; i.e., there is a maximum dose of stimulus which cannot be exceeded. This is true for the physical stimuli (exercise, EVH, cold air, etc.) and for AMP which is administered by inhalation in the most concentrated solution that can be achieved. The inability to exceed these maximum doses may add to the increased diagnostic specificity and decreased diagnostic sensitivity. Most indirect stimuli act either osmotically (exercise, cold air, hyperventilation, nonisotonic aerosols, mannitol) or non-osmotically (AMP) to cause mast cell degranulation and mediator release. Since this includes all of the commonly used indirect challenges, this review will focus exclusively on challenges involving this mechanism. There are, however, other indirect mechanisms. Beta blockers, for example, produce bronchoconstriction by blocking beta receptors and unmasking cholinergic tone. Propranolol inhalation tests have been used as an indirect stimulus and have been shown, like exercise and cold air, to better differentiate asthma from COPD [42]. Other indirect mechanisms may also be involved in responses to other stimuli.

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Exercise Exercise is the most commonly performed indirect challenge. Exercise challenge methodology has recently been reviewed by the ATS [11]. The standard exercise protocol involves a single near maximal challenge with exercise either using a treadmill or a cycle ergometer. Approximately, 6 min of exercise targeting the heart rate to 80–90% of predicted maximum (220-age) or about 50% of predicted maximum voluntary ventilation (FEV1 × 35) is required. The patient should be inspiring dry (relative humidity less than 50%), cool (less than 25°C) air through the mouth (noseclip in place). The response is measured by FEV1 using the best baseline FEV1 and the best FEV1 measured at intervals between 5 and 30 min after exercise. A positive response has variably been defined as a 10–15% fall in FEV1 from baseline. Exercise is a single relatively high-dose challenge compared with methacholine and many others which employ a dose–response curve. As such, although it is a natural stimulus, there is the potential for very large falls in FEV1 in some subjects. Care must be taken to guard against and treat such episodes of severe obstruction.

EVH EVH (eucapnic voluntary hyperpnea) is a surrogate for exercise challenge. A standard EVH challenge [25, 43] involves a 6-min period of hyperpnea with dry air containing 5% CO2 to maintain eucapnea targeting 30 times the FEV1 (85% of maximum voluntary ventilation or MVV). FEV1 is measured before and at time intervals (1, 3, 5, 7, 10 min) following EVH. The EVH test is considered positive if there is a greater than 10% fall in FEV1 [43]. The EVH challenge produces similar results to exercise and requires less expensive and less sophisticated equipment. In addition, the EVH test can be performed in subjects who for one reason or another are not able to participate in an exercise challenge.

Cold Air Standardized bronchoprovocation challenges with dry, cold air, like EVH, involve eucapnic hyperpnea in this instance of cold (−18°C), dry (0% humidity) air. One method allowed construction of a dose–response curve by serial inhalations at minute ventilation rates of 7.5, 15, 30, and 60 l/min followed by MVV, each dose lasting for 3 min [44]. FEV1 was measured before and 30, 90, 180 s (and more often if necessary) after each inhalation targeting a 20% decline in FEV1. This is in effect a modified EVH challenge which requires more expensive and sophisticated equipment. Cold air challenges are not widely performed. One can extrapolate from this study [44] that it should also be possible to perform the EVH challenge in a dose– response fashion. This might avoid the occasional large decrease in FEV1 seen with both exercise and EVH when they are performed as single large dose challenges.

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Non-isotonic Aerosols Both hypotonic aerosols (distilled water or fog [45]) and hypertonic saline [46, 47] have been used as indirect stimuli. Inhaled distilled water does provoke bronchoconstriction and correlates to some extent with EIB but is not very sensitive. Hypertonic saline challenges have been widely used particularly in Australia. A standardized hypertonic saline challenge involves doubling doses of aerosol inhaled from a high output (1.2 ml/min) ultrasonic nebulizer. After a control inhalation, 4.5% saline is inhaled for doubling times (0.5, 1, 2, 4, and 8 min) [47]. FEV1 is measured before and at 30, 90, and 180 s after each inhalation with a targeted decline in FEV1 of 20%. Hyperresponsiveness to hypertonic saline correlates with both EVH and exercise [46, 47]. The hypertonic saline challenge is attractive in that it allows a dose–response curve to be constructed as opposed to exercise and the current EVH protocols. A second advantage is that hypertonic saline has been used as a tool to obtain sputum for investigation of inflammatory cells which is a useful (some might argue more useful) diagnostic tool in asthma [48]. Hypertonic saline sputum induction is generally preceded by the prophylactic administration of an inhaled β2 agonist to prevent bronchoconstriction. However, the use of an un-premedicated hypertonic saline inhalation can serve a dual purpose, namely determination of indirect airway responsiveness and induction of sputum for cellular analysis [49].

AMP Adenosine monophosphate (AMP), when inhaled by asthmatics, results in mast cell mediator release [50]. Doubling dose AMP inhalation challenges are performed analogous to methacholine challenges using either the dosimeter or tidal breathing method. The concentrations of AMP required are up to 400 mg/ml, namely 20–50 times greater than methacholine concentrations. The AMP challenge has been widely used particularly in Europe as an indirect challenge correlating better (than methacholine) with eosinophilic inflammation and demonstrating greater changes with both pro-inflammatory and anti-inflammatory asthma stimuli/treatments [38–41]. Even using the more sensitive tidal breathing method for AMP inhalation, we found AMP to be insensitive in mild well-controlled asthmatics.

Mannitol Mannitol is a naturally occurring alcohol sugar, an isomer of sorbitol. Anderson et al. have recently developed a simple and portable dose–response mannitol inhalation challenge and propose this as a useful alternative as an indirect osmotic challenge. Increasing doses of dry powder mannitol are inhaled from a simple portable dry powder inhaler. The doses are 0 (placebo), 5, 10, 20, 40, 80, 160, 160, and 160 mg for cumulative doses ranging from 0 to 635 mg. The FEV1 is measured before

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and 60 s after each dose with the target being a 15% or greater reduction in FEV1, the results being expressed as a cumulative PD15 in milligram of mannitol administered [51]. The challenge has been shown to correlate well with other indirect osmotic challenges [52, 53]. Unlike the other indirect challenges described above, the mannitol challenge is not so dose limited. The ability to administer a relatively large dose of stimulus may result in increased diagnostic sensitivity [34]. This may be of value, particularly if it is not accompanied by a reduction in diagnostic specificity. Investigations are currently underway to assess this.

Clinical Utility of Nonselective Broncial Challenges Diagnostic Test for Asthma The nonselective challenges are most widely used as diagnostic tests for asthma. The most widely performed challenge, the methacholine challenge, is sensitive enough so that it excludes current asthma with reasonable certainty when the challenge is negative. In a high prevalence subgroup, i.e., individuals with asthma-like symptoms and normal lung function, a positive methacholine challenge is supportive of a diagnosis of asthma, particularly if methacholine-induced symptoms mimic the naturally occurring symptoms. Methacholine challenges are not always positive in subjects with EIB, particularly elite athletes [25]. For investigation of exercise-induced symptoms, or for regulatory agencies where the issue is EIB (e.g., Armed Services, police forces, scuba diving, international Olympic Committee requirements), an exercise challenge or reasonable surrogate such as EVH is recommended. Methacholine challenges in particular, although often used in these circumstances are overly sensitive, and, at least for elite athletes, not specific enough.

Occupational Asthma Nonselective challenges are valuable in several areas in the investigation of occupational asthma. While methacholine challenges have most commonly been used, it seems likely that indirect challenges might perform better. This remains to be evaluated. First, in occupational asthma, it is important to objectively confirm that the patient does indeed have asthma, i.e., variable airway obstruction and/or airway hyperresponsiveness. Since airway responsiveness can rapidly return to normal with sensitizer avoidance, even within a few days [27], it is important to perform the test within close proximity to recent exposures and symptoms. Serial methacholine challenges can also be used diagnostically. Exposure to a sensitizing agent (or allergen) can increase airway responsiveness sometimes

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markedly [54, 55]. In fact, there is little else that will have such an effect on airway responsiveness other than the acute effect of some medications. Consequently, significant work-related increases in airway responsiveness, and improvements when away from work, can be used to infer exposure to a sensitizer at work. These changes must occur without changes in baseline FEV1 and without changes in medication. A significant change in methacholine PC20 must be at least two and preferably three doubling concentrations or more and ideally should be reproducible on a couple of occasions. While being strongly suggestive of exposure to a sensitizer, this approach fails to identify the specific sensitizer. It is in this area that one would anticipate that an indirect challenge might show greater changes. Finally, airway responsiveness is valuable in follow up of occupational asthma both to help in determining eventual issues surrounding disability and impairment, and to help assess adequacy of environmental control.

Monitoring Asthma Control Asthma is traditionally monitored clinically by symptoms and spirometry. It has been suggested that more detailed measurements of inflammation either directly by sputum inflammatory cell analysis [56] or indirectly by exhaled NO [57] or AHR [58] might be helpful. Sont et al. have shown that a therapeutic strategy designed to maximize improvement in methacholine PC20 improved asthma control and reduced airway remodeling at the expense of doubling the inhaled corticosteroid requirement [58]. Again, this is an area in which the indirect challenges should perform better [34, 38–41]. At this point, since the majority of patients with asthma do not even have simple clinical control criteria routinely checked [59], it would seem that additional tests are not to be recommended on a regular basis but remain a research tool. Nevertheless, in some subjects, particularly those who might be complicated, i.e., with more than one cause for breathlessness (e.g., anxiety hyperventilation combined with asthma), bronchial challenges might prove helpful in monitoring adequacy of treatment.

Nonallergic Nasal Provocation Background Although allergic nasal provocation was first performed in the 1800s, NPT with nonspecific agents was not described until much later. One of the first challenges was conducted by van Lier in 1959 [60], when he challenged patients with veratrine (a mixture of plant alkaloids) noting an increase in response during the pollen season. This demonstrated the concept of nasal hyperresponsiveness, the most common

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symptoms being sneezes, rhinorrhea, and nasal blockage. Common nonspecific environmental stimuli include dust particles, cold dry air (CDA), tobacco smoke, perfumes, and paint fumes. Allergen exposure has been shown to increase responsiveness not only to the allergen in question, but also to nonspecific stimuli such as histamine and methacholine [61–64] in a way analogous to the lower airway hyperresponsiveness of asthma. Proposed mechanisms of increased responsiveness include increased epithelial permeability, neural reactivity, changes at the neuronal level, receptor sensitivity, and non-IgE-mediated mast cell stimulation [61]. As with bronchial challenge, both direct and indirect acting agents have been used for NPT. The direct challenge agents methacholine and histamine have been most widely used. Histamine acts on the nasal mucosa via a number of mechanisms including stimulation of type C neurons bearing the H1 receptor that mediate itch and recruit cholinergic parasympathetic reflexes causing glandular exocytosis; and plasma extravasation by stimulation of endothelial H1-receptor and subsequent hydrostatically driven exudation across the epithelium, which results in edema, rhinorrhea, and nasal obstruction. These mechanisms all occur in the ipsilateral nasal cavity after unilateral stimulation with histamine. Only the reflex-induced glandular secretion occurs in the contralateral side. Methacholine induces glandular secretion only, via cholinergic mechanisms. Both histamine and methacholine have been extensively used to assess nonspecific nasal hyperrespnonsiveness. Other agents which have been used, but not to the same extent include: capsaicin, bradykinin, nicotine, and serotonin. Bradykinin induces vasodilation and permeablility due to stimulation of vascular receptors [61, 65, 66]. Nicotine and serotonin also induce sensory nerve stimulation. Interestingly, as capsaicin actually damages type C fibers, it has been proposed as a treatment of nonallergic rhinitis [61, 67]. Other irritant (indirect) agents which have been used include ozone, sulfur dioxide, formaldehyde, cigarette smoke, CDA, rewarming after cold air exposure, hypertonic saline, and mannitol. Indirect acting agents, particularly CDA, may be more clinically relevant as they more closely match environmental exposure. In fact, CDA has been shown to evoke a more predictable response than histamine [68]. Acetylsalicylic acid has also been used as an assessment of aspirin-sensitive asthma, but is reported to be less sensitive than the oral challenge [61, 69]

Testing Method The testing procedure for nonselective agents is the same as for selective NPT. Nasal secretions and symptoms such as itching, sneezing and nasal obstruction are monitored. Symptom scores may be augmented by assessment of nasal resistance and flow, utilizing rhinometry, acoustic rhinometry [61, 70, 71] and nasal expiratory peak flow [72]. Although the methodology varies between studies, standardized assessment criteria have been published [70]. NPT is contraindicated in the following circumstances: acute bacterial or viral rhinosinusitis, exacerbation of allergic disease, anaphylaxis to specific agent,

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severe general disease specifically cardiac or pulmonary, and pregnancy. Relative contraindications include allergic rhinitis within 2–4 weeks, and recent nasal surgery within 8 weeks. H1 blockers should be withheld for 3 days (ketotifen 2 weeks, astemizole >4 weeks), nasal corticosteroids, oral corticosteroids and sodium cromoglycate for several weeks, nonsteroidal anti-inflammatory drugs for 1 week, central-acting antihypertensives (reserpine, clonidine, etc.), oral contraceptives (which cause congestion and other nalas mucosal histologic changes), and tricyclic antidepressants. False positive tests mostly occur because of the effects of the nasal cycle. Nasal priming due to allergen or irritant exposure other than the substance being tested is an additional reason. False negative tests are due to medication effects, nonstandardized or obsolete testing substances, or very low nasal airflow before provocation. Previous nasal surgery can decrease the sensitivity if there is lack of reconstitution of reactive nasal mucosa.

Clinical Utility of Nonselective Nasal Challenge Although not widely used in North America for clinical application, NPT is a safe and potentially useful tool the diagnosis of allergic and nonallergic rhinitis. NPT may also offer insights into the lower airways because the reactions of the upper and lower airways often are the same. Certainly nasal allergic reactions are thought to be predictive of bronchial reactions, and may be useful in assessing the reactions to specific allergens. Nonselective NPT has less clinical utility and therefore is less widely used. However, nonselective NPT can be employed to assess nasal hyperresponsiveness and thereby assist in therapeutic follow up [73]. Nonselective NPT may also provide insights into the pathophysiology of nonallergic rhinitis, however, its role in this regard has yet to be established. Acknowledgments The author would like to thank Jacquie Bramley for assisting in the preparation of this manuscript.

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Sputum Tests and Exhaled NO in the Diagnosis and Monitoring of Asthma Myron Zitt

Background Since 1987, with the identification of nitric oxide (NO) as the previously uncharacterised endothelial-derived relaxing factor, it has become apparent that this endogenous multifunctional regulatory molecule, which is widely distributed throughout the body, plays a significant role in human physiology [1, 2]. In 1998, Furchgott, Ignarro, and Murad were awarded the Nobel Prize for their work with NO which had, several years earlier, been recognised by Science as 1992s “Molecule of the Year” [3]. In the respiratory system, NO is a neurotransmitter for bronchial non-adrenergic, non-cholinergic neurons and promotes dilation of blood vessels and relaxation of bronchial smooth muscle, thus regulating vascular and bronchial tone [4–6]. Its cytotoxic and bacteriostatic properties play a role in host defense [4–6]. NO also promotes mucus secretion and is instrumental in coordinating the ciliary beat frequency of airway epithelial cells, thus affecting mucociliary clearance [4–6]. The discovery by Gustafsson et al. in 1991 that NO can be detected in the exhaled breath of animals and humans, and the subsequent observation by Alving and coworkers that NO is elevated in the exhaled air of patients with asthma has led to evolving research evaluating the significance of varying concentrations of this molecule in exhaled air in inflammatory airway disease [7, 8].

Nitric Oxide Formation The production of NO is mediated by a family of haem-containing enzymes, the NO synthases, which convert the amino acid l-arginine to NO and l-citrulline in the presence of oxygen and NADPH [9]. Three NO synthase isoforms exist, all of which have been identified in the airways of humans [6, 10, 11]. Type I or neuronal NO synthase (nNOS, NOS 1), and Type III or endothelial NO synthase (eNOS, NOS 3) M. Zitt () Clinical Associate Professor, Medicine, State University of New York, Stony Brook Medical Center, 9 Cypress Drive, Woodbury, NY 11797, USA e-mail: [email protected]

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are constitutively expressed and produce relatively constant low quantities of NO, with amounts varying with changes in intracellular calcium concentration. Type II or inducible NO synthase (iNOS, NOS 2), is found largely in airway epithelium. It is not constitutively active and is little influenced by calcium fluxes [6, 10, 11]. Nonetheless, it has the capacity to generate large amounts of NO when transcriptionally upregulated by pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF α), interferon gamma (IFN γ), interleukin 1-beta (IL-1β) and endotoxin [12]. In patients with asthma, as part of the inflammatory process, the expression of airways iNOS messenger RNA is upregulated and has been shown to correlate with levels of exhaled NO [13]. Of interest is that nitric oxide formed by constitutively expressed nNOS and eNOS may lead to cGMP-dependent relaxation of airway smooth muscle, while high amounts of NO released by iNOS appear to lead to proinflammatory effects [14, 15]. While exhaled NO is elevated in patients with asthma, those receiving corticosteroid therapy exhibit a decreased expression of iNOS protein and messenger RNA compared to those not receiving treatment [13]. Similarly, the oral administration of a selective iNOS inhibitor has been shown to reduce exhaled NO significantly in both asthma patients and healthy volunteers [16].

Exhaled Nitric Oxide Measurement Reproducibility, Yet Inter and Intra-Individual Variability Presently, the most widely used method for online determination of fractional exhaled nitric oxide (FENO) is chemiluminescence after reaction with ozone, which allows measurement down to approximately 1 part per billion (ppb) [17]. While work is ongoing to develop smaller, and increasingly more cost-effective technologies to measure FENO, in the USA several devices have currently been approved by the Food and Drug Administration (FDA) for clinical use in patients with asthma, with an indication to monitor the response to anti-inflammatory medications, in adults up to 65 years of age and children older than 4 years [18]. NO is formed both in the upper and lower respiratory tract and diffuses in a flow-dependent fashion into the lumen by gaseous diffusion down a concentration gradient. FENO levels are inversely proportional to expiratory flow rates. Therefore, the more rapid the flow rates, the shorter the time for alveolar gas to diffuse into the airway and the lower the FENO [19, 20]. To avoid variations due to flow, the European Respiratory Society and the American Thoracic Society have recommended a standardised single-breath online technique for measuring FENO in adults and children [21]. Subjects are instructed to inhale NO-free gas to total lung capacity and to then exhale at a constant flow rate of 50 ml/s until a steady plateau is reached. During exhalation, sufficient pressure is created in the oral cavity to ensure closure of the velum and prevent nasal airflow from significantly contributing to the FENO measurement [22]. This is important as significant amounts of NO emanate from the paranasal sinuses. While measurement of FENO has been shown to be highly reproducible [23], levels can vary over time in patients with

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stable asthma [24]. In addition, it must be noted that there are multiple factors that determine individual variability. FENO levels appear to relate to airway size, as they are lower in healthy women than healthy men, and are lower in children than adults, but tend to increase with age [25, 26]. Low values have been reported with cystic fibrosis [27] and ciliary dysmotility syndromes [28] and may be depressed by cigarette smoking [29]. On the other hand, certain foods and drinks that are high in nitrates may increase levels [30]. Patients with allergic rhinitis, have been shown to have increased levels of FENO [31] and it has been suggested that those with the highest levels might be at risk for the development of asthma. Regardless, individuals with allergic rhinitis and asthma have higher FENO levels than those with asthma alone [32, 33]. In patients with allergic asthma, FENO levels increase after allergen challenge [34] or during the allergy season [35] and decrease with reduction in allergen exposure [36]. Viral infections may increase FENO in asthma patients [37], while anti-inflammatory therapies including corticosteroids [38], leukotriene modifying agents (LTM) [39] and omalizumab [40] will decrease levels. Genetic polymorphisms regarding NOS 1 and NOS 3 have been associated with varying levels of FENO and serum IgE [41]. Regarding the NOS 1 gene, asthma patients with >12 AAT repeats in intron 20 have lower exhaled NO values than those with fewer repeats. Spirometry, sputum induction or airway hyperresponsiveness studies can also decrease FENO levels [42–44]. Thus, the measurement of FENO should precede these studies if they are to be done during the same patient visit. In addition to patients with asthma, those with a wide variety of diseases including mycobacterial infection [45], liver disease [46], lung cancer [47], bronchiectasis [48] lung transplant rejections [49] and chronic obstructive pulmonary disease [50, 51] have all been reported to have increased FENO levels. Therefore, care must be taken when interpreting FENO values in the diagnosis and treatment of patients with asthma to ensure that increased levels due to factors other than asthmatic airway inflammation are considered.

Asthma Heterogeneity Although asthma has been defined as a chronic inflammatory disorder of the airways characterised by bronchial hyperreactivity and reversible airways obstruction, it has become evident that airways obstruction is not necessarily entirely reversible, despite management as recommended by current guidelines [52, 53]. Because of the heterogeneity of this disease, some patients can experience impaired lung growth during childhood and/or a progressive decline in pulmonary function in adulthood [54–56]. In some, chronic inflammation may lead to permanent structural changes in the airways termed ‘remodelling’ which, though poorly defined, has been characterised by extracellular matrix deposition with subepithelial fibrosis, angiogenesis, transformation of fibroblasts to myofibroblasts, increased smooth muscle mass, mucus gland hyperplasia and epithelial damage [57, 58]. To date, studies aimed at early intervention, including the Children’s Asthma Management Program (CAMP) [59], the Prevention of Early Asthma in Kids (PEAK) [60] and Inhaled Fluticasone in Wheezy Infants (IFWIN) [61]

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have been unable to demonstrate that early administration of inhaled corticosteroids (ICS) is disease modifying or can alter the natural course of asthma. Heterogeneity is also evident in patient response to medication. A bell-shaped response has been demonstrated to ICS as well as to leukotriene modifying agents (LTM) indicating varying degrees of response or non-response to both drug categories [62]. The CLIC study (Characterizing the response to a Leukotriene receptor antagonist and an Inhaled Corticosteroid study) comparing FEV1 responses to 8 weeks of LTM or ICS therapy in children with moderate persistent asthma, determined the percentage in each group that would achieve improvement of >7.5%. While 17% responded to both therapeutic agents, 23% to ICS alone and 5% to LTM alone, surprisingly 55% responded to neither drug [63]. Heterogeneity in response to ICS was also evidenced in the GOAL (Gaining Optimal Asthma Control) study where it was demonstrated that 30% of moderate persistent asthma patients could not achieve optimal asthma control despite maximal recommended doses of inhaled fluticasone/salmeterol combination therapy [64].

The Unmet Need To date, asthma continues to be under-diagnosed and under-treated, resulting in an unacceptably high mortality rate and a less than optimal quality of life for those with the disease [52, 53]. Guideline measures of asthma severity and control are based on assessments of symptoms, the frequency with which rescue medication is employed and pulmonary function but have failed to address airways inflammation which is part and parcel of the disease. In fact, these conventional measures correlate poorly with histological evidence of inflammation and reputed surrogate markers such as airway hyperresponsiveness (AHR), sputum eosinophilia and FENO [65, 66]. Included among our unmet needs in the management of asthma would be to heighten diagnostic capabilities, to better understand and determine the significance of the association between inflammation and disease control or impairment or the risk of experiencing exacerbations or airway remodelling. In addition, there is a need to better account for asthma heterogeneity by predicting and monitoring individual responsiveness to various therapeutic agents, in hopes that assessing their effects and titrating their doses through the use of non-invasive surrogate markers of inflammation could improve outcomes [67].

Markers of Inflammation Airway Hyperreactivity The AMPUL Study Group conducted a randomised, prospective parallel group 2-year trial involving 75 adults with mild to moderate asthma to determine whether a treatment strategy aimed at reducing AHR in addition to utilising the recommendations

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of existing guidelines would lead to more effective control of asthma and greater improvement in chronic airway inflammation than a conventional therapeutic strategy based on guideline recommendations alone [68]. While the patients treated with a strategy aimed at reducing AHR required higher ICS dosing, they experienced a 1.8-fold lower incidence of mild exacerbations compared to those in the conventional group. Lung function characterised to be FEV1 was also significantly higher in those whose ICS dose was titrated based on AHR (p ≤ 0.05), while bronchial biopsy revealed a decrease in the thickness of the subepithelial reticular layer, which correlated with a decrease in tissue eosinophil counts. Although a therapeutic strategy based on the monitoring of AHR with methacholine bronchoprovocation appeared to improve asthma outcomes, this non-invasive technique is generally impractical in a clinical setting and cannot be performed in severe or poorly controlled asthma patients.

Induced Sputum Eosinophil Analysis Eosinophils are a predominant cell in the airways of asthmatic patients [69], have been shown to increase in sputum prior to the onset of asthma exacerbations [70] and are very sensitive to corticosteroid therapy with their suppression associated with an amelioration of symptoms and an improvement in lung function [71]. In addition, a significant correlation has been observed between methacholine-induced airway responsiveness and eosinophilic inflammation [72]. Therefore, a study was designed, utilising induced sputum analysis, to determine if titrating ICS dosing to normalise sputum eosinophil counts at or below 3% could result in improved outcomes as compared to therapy based on the assessment of symptoms, peak expiratory flow and the use of inhaled beta agonists consistent with British Thoracic Society (BTS) guidelines [73]. For this 12-month study, 74 patients with moderate to severe asthma were randomised to the BTS, or sputum management group. For those patients unable to produce sputum, FENO was used as a surrogate marker of inflammation. Patients in the sputum management maintained a 63% lower sputum eosinophil count while experiencing significantly fewer severe exacerbations hospitalisations than the BTS group. In contrast to the AMPUL study, the average daily dose of corticosteroids did not differ between the groups. While this technique reduced asthma exacerbations and hospital admissions in patients with moderate to severe asthma, it is unclear whether this success could be applied to patients with less severe asthma. In addition, the technique might be impractical to perform in an out-patient setting as it requires albuterol inhalation before the procedure in an effort to prevent potential bronchospasm, which could result from the inhalation of hypertonic saline for a recommended 12–30 min followed by expectoration every 2 min for up to 30 min. Not all patients can successfully produce sputum as was evident in the 77% success rate observed during CAMP [74]. In addition, technical expertise is necessary to prepare and analyse the sputum. Therefore, the authors recommend the use of FENO as a simpler and more practical surrogate marker of inflammation [73].

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FENO Determination The determination of FENO is a non-invasive procedure which requires little technical expertise, and is quick, reproducible and easy to perform [75]. As a positive correlation has been observed between FENO and sputum eosinophils [76, 77] bronchoalveolar lavage fluid eosinophils [78, 79], tissue eosinophils [80] and major basic protein on bronchial biopsy [81], it would appear that FENO is an accurate indicator of eosinophilic airway inflammation and has potential adjunctive practical clinical applicability in asthma diagnosis and management.

Practical Applications of FENO Measurement Evaluating the Effect of Allergen Exposure or Avoidance on Airway Inflammation In atopics, FENO will change rapidly with allergen exposure or avoidance, as was demonstrated in a study of asthmatic children residing in a sea level community with high airborne dust mite concentrations [82]. Within 2 weeks of transporting the children to a high-altitude low-mite environment, initially elevated FENO levels decreased significantly and remained low, despite inhaled steroid withdrawal. Within 2 weeks of re-exposing the children to a mite-rich, sea level atmosphere, FENO values again increased to elevated levels. Throughout the study, FEV 1 levels changed very little. Repeated low-dose allergen challenge mimics natural allergen exposure, providing a model for early mechanisms in the triggering of asthma. In a recent study, asymptomatic patients with mild asthma completed two 7-day repeated low-dose challenge periods with either diluent or allergen. Neither diluent nor allergen induced either asthma symptoms or a change in FEV1. Nonetheless, repeated allergen exposure did induce low-grade inflammation characterised by increases in histamine responsiveness (2.3 doubling doses), a small increase in the mast cell marker 9α11β prostaglandin F2 and a significant increase in FENO compared to baseline which was detected within a week of allergen challenge [83]. Thus, serial measurements of FENO have the potential to provide a very sensitive strategy for early detection of emerging airway inflammation and/or improvement not reflected by measurements of lung function.

Asthma Diagnosis The diagnosis of asthma is currently based on symptoms, measures of airflow limitation, assessment of bronchodilator response and, in some circumstances,

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the somewhat arduous measurement of airway hyperresponsiveness. However, symptoms are non-specific and not closely related to the presence and severity of airway inflammation [66]. Because the disease continues to be under-diagnosed, a study was undertaken to determine the validity and accuracy of FENO to improve diagnostic capabilities by evaluating 240 non-smoking, steroid-naive adults with symptoms suggestive of asthma [84]. By demonstrating reversibility of airways obstruction and/or histamine hyperresponsiveness, 160 were diagnosed with asthma. While baseline lung function, including FVC, FEV1 and FEV1/FVC were similar in asthmatic and non-asthmatic cohorts, FENO measured at a flow rate of 200 ml/s (equivalent to 33 ppb at a standardised flow rate of 50 ml/s) was significantly higher in those with asthma (25 ppb) compared to those without (11 ppb). Using a receiver operator characteristic (ROC) curve analysis, to determine a level that would reflect optimal sensitivity and specificity for the diagnosis of asthma, a cut point or threshold value of 13 ppb was chosen. An FENO above this value showed a sensitivity of 85%, a specificity of 80% and a positive and negative predictive value of 90%. The authors concede that there was overlap between the two groups and that specificity may have been limited by the potential influence of various other factors such as smoking or concurrent viral infection on FENO values as was noted above. They point out that the most accurate method of establishing a diagnosis of asthma is bronchoprovocation testing with a specificity of 100% and a sensitivity of 85% [85]. However, by utilising FENO, in the population studied, relatively comparable results were observed with greater simplicity. It was concluded that FENO should be considered as a complementary test for asthma and that its use for diagnostic screening could theoretically reduce the number of tests needed to diagnose asthma resulting in decreased health care costs. When FENO values are below the cut point or threshold value, the likelihood of asthma is low, and this simple non-invasive measurement may obviate the need for a challenge study. This hypothesis was tested and confirmed at a military setting where screening for asthma can be quite expensive. In a study of 172 basic trainees with symptoms of asthma, a diagnosis was established in 80% by using physical examination, spirometry and histamine bronchoprovocation [86]. When the same group was evaluated using FENO measurements, a diagnostic cut point for asthma of 10.5 ppb when measured at a standard exhalation rate of 50 ml/s showed a sensitivity of 86% for a positive histamine challenge. To determine applicability in younger patients, 143 children between 3.8 and 7.5 years were evaluated for symptoms suspicious for asthma [87]. As young children often cannot adequately perform spirometry, diagnosing them with asthma is often difficult and is largely based on symptoms and clinical response to short acting β agonists. After the measurement of FENO at a flow rate of 50 ml/s, an ROC analysis determined a threshold for asthma, which correlated with a clinical diagnosis, at 9.7 ppb. An FENO above this value revealed a sensitivity of 86%, a specificity of 92% and positive and negative predicted values of 78% and 95% respectively, confirming the diagnostic value of FENO in children. Deykin et al. utilised online and offline measurement techniques to determine that FENO can serve as a robust discriminator between 34 subjects with asthma

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diagnosed through reversibility in airways obstruction or hyperresponsiveness to methacholine and 28 non-asthmatic subjects (70% sensitivity and specificity) [88]. Similarly, Smith and colleagues demonstrated greater sensitivity of FENO (88%) and the measurement of sputum eosinophils (86%) than conventional parameters, including history and pulmonary function and peak flow variability in diagnosing asthma (0–47%) [66]. FENO was preferred because of its speed and ease of use. When FENO values are below 30 ppb, the likelihood of asthma is low, and this simple non-invasive measurement may obviate the need for a challenge study. A diagnosis of asthma in patients presenting with cough is entertained, but difficult to confirm without evaluating airway hyperresponsiveness. Patients with gastro-oesophageal reflux disease (GERD) often present with symptoms of cough. FENO levels have been found to be lower in allergic children with coexisting asthma-like symptoms and GERD as compared to asthmatic children [89]. The correlations between FENO levels and measurements of 24-h oesophageal pH suggest that inhalation of acid gastric content may interfere with NO production in the airways. Thus, elevated levels of FENO in a coughing patient would favour a diagnosis of asthma rather than GERD. Although current guidelines do not define specific cut-point values for the diagnosis of asthma, several recent studies have attempted to provide normal reference ranges for children and adults. A study of healthy adults determined that the inter-quartile normal range for FENO was 11.9–22.4 ppb [91] while a second study of healthy non-atopic adults determined an upper limit of normal of 33.1 ppb [92]. A study of 405 children determined that while FENO was age-dependent, the upper limit of the 95% confidence interval ranged from 15.7 ppb at 4 years of age to 25.2 ppb for adolescents [90]. Another study of nonatopic children concluded that height is the best determinant of FENO in this group, but cautioned that because of the strong effect of atopy, that FENO should not be interpreted without knowing the atopic status of the child. The authors stressed that “reliance on the purely statistical concept of reference range as equivalent to abnormality may be misleading, and results should always be interpreted in their clinical context” [93]. The speed and simplicity with which FENO can be obtained make it an excellent asthma screening tool and a valuable adjunct to our current diagnostic armamentarium. While the lack of specific cut points, individual variability and the influence of comorbidities place limitations on its diagnostic value, the following algorithm can be applied. Levels within the normal range indicate that eosinophilic airway inflammation is unlikely. In the absence of symptoms, no further asthma evaluation would appear indicated. However, in the presence of respiratory symptoms, diagnoses such as GERD, bacterial rhinosinusitis, vocal cord dysfunction, cystic fibrosis, cardiac disease or neutrophilic asthma should be entertained. Values between 25 and 50 ppb are equivocal and diagnosis must be based on clinical presentation and conventional diagnostic studies. On the other hand, FENO values >50 ppb, particularly when combined with any evidence of airway obstruction, strongly suggest a diagnosis of asthma and predict that a positive response to a trial of inhaled or oral steroids is likely [94].

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Predicting a Response to Asthma Therapy In a single blind crossover trial, patients with undiagnosed cough, without demonstrable reversibility in airflow limitation, were treated with either inhaled Fluticasone Propionate (FP) or placebo after the determination of spirometry, adenosine PC20 AMP and FENO [95]. Patients with FENO values >47 ppb demonstrated significantly greater changes in symptom scores, FEV 1, morning peak flow rates (PEFR) and PC20 AMP following FP compared with patients with lower FENO values. The authors concluded that FENO may help to identify patients with respiratory symptoms without a firm diagnosis who could benefit from ICS therapy. Recognising asthma heterogeneity, Zeiger et al. sought to determine inter-individual and intra-individual response profiles and predictors of response to 8 weeks of therapy with ICS or LTM in 144 children, ages 6–17, with mild to moderate persistent asthma treated with only as-needed bronchodilators [96]. While both controller therapies were effective, patients on ICS achieved more favourable outcomes including greater asthma control days (ACDs), and improved lung function and FENO values. The inflammatory marker FENO was shown to be both a predictor of ACDs (P = 0.011) and a response indicator (P = 0.003) in discriminating the difference in ACD response between fluticasone and montelukast. The authors, and those of the previously mentioned CLIC study [63] concluded that FENO, as a predictor of response, might help to identify individual children not receiving controller medication who would achieve greater improvement in asthma control with an ICS compared with leukotriene receptor antagonist (LTRA).

Comparing Clinical Potency of ICS Formulations The introduction of new ICS formulations has necessitated the development of a clinically relevant model to compare their clinical potency. A recent study sought to determine whether a dose response to an ICS could be demonstrated by measuring surrogate markers of inflammation [97]. Sputum eosinophils and FENO were extremely sensitive to the effects of an ICS, decreasing with increasing doses of medication and correlating with decreasing airway responsiveness to methacholine. With this in mind, a study comparing inhaled ciclesonide with FP indicated that the former had stronger anti-inflammatory activity in patients with mild asthma, because a once-daily dose of ciclesonide induced a faster and greater decrease in FENO than a comparable twice-daily dose of FP [98].

Determining Response to ICS Therapy FENO levels in patients with asthma are very sensitive to ICS and will begin to decrease within 6 h of treatment and plateau within 3–4 weeks [99]. In patients with

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steroid-naive mild persistent asthma a dose-dependent FENO response was observed after treatment with 100 and 400 ug/day of inhaled budesonide. FENO and FEV1 were measured at baseline and after a 2-week course of ICS treatment in 65 adults and children with uncontrolled asthma. Symptomatic improvement correlated with a significant decrease in FENO, but not with spirometry results. In patients whose asthma was exacerbated after their Beclomethasone Dipropionate (BDP) dose was reduced, a linear inverse relation was demonstrated between FENO and the log of the reduced ICS dose [100]. One can conclude that FENO, which correlates poorly with spirometry, is a quick and sensitive indicator of the anti-inflammatory response to ICS, decreasing in a dose-dependent fashion with ICS treatment and increasing if inflammation increases as ICS is withdrawn.

Predicting Asthma Relapse During ‘Remission’ As asthma severity is variable [101], it is not uncommon for patients who are symptom-free, particularly during the summer months, to be weaned off ICS therapy (steroid holiday). Though not approved by current national guidelines, justification for this controversial practice is found in the CAMP and PEAK studies indicating that chronic ICS use can control symptoms but cannot affect the natural course of the illness [59, 60]. Further support is found in the IMPACT study, which demonstrated that patients with mild persistent asthma would not experience an increase in exacerbations using as-needed versus daily ICS [102]. A reliable tool to predict an asthma relapse in patients weaned off ICS would be extremely useful in preventing hospitalisations and adverse outcomes. A recent study evaluated symptom-free children with asthma, who had been withdrawn from a mean daily dose of budesonide of 400 µg/day, to determine whether the measurement of FENO at regular intervals could predict an asthma relapse defined as more than 1 exacerbation per month, the need for β agonist use on 4 days per week for at least 2 weeks or peak flow variability of >20% [103]. With the use of ROC curves to determine optimal specificity and sensitivity, it was determined that an FENO > 49 ppb after ICS withdrawal was highly predictive of a relapse in asthma.

Predicting Loss of Asthma Contol/Impending Exacerbation Clinical improvement in asthma and a reduction in inflammation have been shown to correlate with a decrease in FENO indicating that this measurement could be used to complement existing tools to monitor asthma and confirm control. In contrast, FENO will increase prior to the onset of an asthma exacerbation, suggesting that it could have utility as a marker of impending loss of asthma control. Jones and colleagues evaluated 78 patients with mild to moderate asthma who had stopped their ICS treatment and were followed weekly for 6 weeks [104]. Loss of control (LOC)

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was experienced by 60 patients. FENO increased 2.16-fold in the LOC patients versus 1.44-fold in those who did not experience LOC (P = 0.004). FENO > 15 ppb (33 ppb at 50 ml/s) or an FENO increase >60% since the last visit was highly indicative of LOC at the next visit (positive predictive value = 83%). Harkins et al. evaluated 22 patients with moderate to severe persistent asthma to determine if monitoring FENO could indicate when additional treatment would be necessary to avoid excessive morbidity and hospitalisation [105]. FENO was evaluated during a routine clinic visit and patients were questioned on their next visit as to whether they experienced an exacerbation in the interim. While there was no significant difference in lung function (FEV1 or PEF) between the groups, those who exacerbated had a significantly higher FENO (29.67 ppb) versus those who did not experience an asthma flare (12.92 ppb, P = 0.0002).

ICS Titration to Optimise Asthma Control A study monitoring asthma patients with FENO was designed to duplicate the decreased exacerbations observed when the ICS dose required to achieve optimal asthma control was titrated using sputum eosinophils and/or methacholine sensitivity. Often physicians look at symptoms to guide inhaled corticosteroid dosing in asthma. However, symptoms are non-specific and are not closely related to the presence and severity of airway inflammation. In a parallel study, 46 patients had their ICS dose titrated using Global Initiative for Asthma (GINA) guidelines while 48 patients had their ICS dose determined by maintaining FENO at a cut point of 15 ppb, (equivalent to 33 ppb at 50 ml/s) [106]. The mean dose of ICS (FP) was significantly lower in the FENO adjusted group (370 vs. 641 ug/day) while no significant difference in exacerbation rates or symptom-free days was observed between the groups. A similar study evaluating children with asthma determined that 1 year of titrating inhaled steroid doses based on FENO and symptoms rather than symptoms alone resulted in improved airway hyperresponsiveness and fewer exacerbations without the need for higher steroid doses [107]. The authors of both studies concluded that titrating ICS doses against FENO values would be preferable to symptom-based treatment that could lead to excessive ICS doses for fear of losing asthma control or to under-dosing, for fear, particularly in children, of potential steroid side effects. More recent studies have not been as favorable in their assessment of the value of monitoring FENO to titrate ICS dosing in patients with persistent asthma. Shaw and coworkers [108], in evaluating 118 adult patients with a diagnosis of asthma over a 1 year period concluded that a treatment strategy employing FENO to adjust treatment “did not result in a large reduction in asthma exacerbations (0.33 per patient per year in the FENO group vs. 0.42 in the control group) or in the total amount of inhaled corticosteroid therapy” when compared to traditional guideline driven management. While the overall use of ICS was 11% greater in the FENO group, the final

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daily ICS dose, was nonetheless lower (557 µg) than in the traditionally treated (895 µg) group. Szefler and colleagues [109] conducted a multi-center study in which 780 inner city patients, aged 12–20 years, with persistent asthma, were evaluated over 46 weeks after a 3 week run-in period during which non-adherent patients were excluded. The remaining patients, who, as a group, demonstrated significant improvement during the run in period and throughout the study, were randomized to a National Asthma Education and Prevention Program (NAEPP) driven “conventional” strategy or a management program where standard NAEPP treatment was modified based on measurements of FENO. The authors concluded that the addition of FENO as an indicator asthma control provided “no clinically important improvement” in symptomatic asthma control, while resulting in higher overall ICS doses. Nonetheless, the risk of at least one prednisone course for asthma exacerbations was lower in the FENO monitored group than in the controls while a sub-group of patients with obesity, high blood eosinophil counts, and atopy showed a larger decrease in days with asthma symptoms. Most recently, de Jongste and associates [110] evaluated 30 weeks of daily telemonitoring of children with atopic asthma, utilizing recently FDA approved portable FENO analyzers. The study revealed that ICS adjustment using symptom monitoring alone or in conjunction with FENO was associated with a significant improvement of symptoms and a reduction in ICS doses. However, the investigators found “no added value of daily FENO monitoring compared with daily symptom monitoring only”. Evaluating and comparing ICS titration studies is difficult and confounded by varying FENO cutpoints and by varying thresholds for symptoms and measures of lung function that would prompt treatment change, each of which can effect overall ICS dosing. Similarly the maintenance use in some studies of long acting beta agonists can influence ICS dosing. Exacerbations must be clearly and uniformly defined when their frequency is evaluated as a study endpoint. Perhaps a study design weakness is, that in the presence of symptoms and low FENO levels, no changes were made in ICS dosing. Yet, low values are highly predictive of the absence of eosinophilic inflammation, the absence of long-term steroid requirements in children and a low risk of deterioration of asthma control in adults. Lowering ICS doses in these patients might have altered study outcomes. From a practical standpoint, patient symptoms in the presence of low FENO levels should prompt an investigation for comorbid conditions such as GERD rhinosinusitis or vocal cord dysfunction [111,112]. Finally, FENO levels can vary depending on age, height and atopic status. Elevated levels are not specific for asthma and, as was mentioned earlier, can be observed in numerous other conditions. An inexplicably high “normal range” may be found in some patients which is not significantly affected by ICS and which correlates poorly with sputum eosinophils. Not accounting for these variables can also influence study outcomes. The conclusions of a recent meta-analysis indicate that the role of utilizing FENO to tailor the dose of ICS is currently uncertain and that this approach needs further investigation before it can be advocated in clinical practice [113].

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Determining Adherence/Compliance with Asthma Therapy As estimates suggest that only about 50% of patients adhere to therapeutic recommendations from health care providers, it is likely that poor patient compliance will contribute to suboptimal asthma outcomes [114]. Monitoring patient adherence and providing feedback has been shown to improve asthma outcomes [115, 116] and reduce health care costs [117]. It would be desirable to have a compliance marker in asthma management comparable to glycosylated haemoglobin A1C for diabetes. Delgado-Corcoran et al. monitored ICS compliance in 30 children, aged 7–17 years, with varying degrees of asthma, evaluating control based on symptoms, pulmonary function, β agonist use and FENO [118]. The latter proved to be a sensitive marker of compliance, even when FEV1 was not significantly decreased. Beck-Ripp and colleagues, in studying 54 children with persistent asthma, similarly showed a positive correlation between a reduction in FENO and compliance with an ICS which was not detected using conventional lung function [119]. Similar observations, showing a failure of FENO to fall with ICS treatment in non-compliant asthma patients, lends further support that FENO measurements could be of value in identifying patients not adhering to health care provider treatment recommendations.

Potential Measurement of Distal Airway Inflammation There is accumulating evidence to indicate that airway inflammation in asthma occurs not only in the large airways but in the distal airways (≤2 mm in diameter) and alveoli as well [120, 121]. A study examining surgically resected lungs reported inflammatory changes and greater numbers of activated eosinophils in the distal than the central airways [122]. Another, using trans-bronchial biopsies, demonstrated significant eosinophilic inflammation in the alveoli of patients with nocturnal asthma which increased in the early morning hours to the point where eosinophils accumulated to a greater extent in alveolar compared with proximal lung tissue [123]. Other studies confirm that the peripheral airways are a predominant site of asthmatic airways obstruction [124–126], with one of the patients with mild asthma and normal spirometry, indicating that peripheral airway resistance was increased up to sevenfold when compared with control individuals and correlated with methacholine responsiveness [127]. It has been postulated that airway-wall remodelling, potentially leading to a component of fixed airflow obstruction, can occur in small airways. The inability of currently available larger particle-sized ICS formulations, delivered by chloroflurocarbon or dry powdered inhalers, to reach the small airways may at least in part, be responsible for their inability to alter the natural course of asthma and prevent disease progression and/or remodelling [128]. More information is needed regarding small airways disease and its response to therapeutic interventions. Nonetheless, this has been difficult because of the absence of a reliable, noninvasive measure of peripheral airways inflammation. Observations of the flow

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dependence of FENO have led to the development of a mathematical model theorising that at very low flow rates, FENO would largely reflect alveolar inflammation, whereas at very high flows, FENO would reflect bronchial inflammation [129]. A study employing this model determined that alveolar FENO was elevated in subjects with asthma with nocturnal symptoms but not in subjects with asthma without nocturnal symptoms or in non-asthmatic controls [130]. While no invasive studies were performed for this trial, as nocturnal asthma appears to involve the distal airway, it is plausible that FENO performed at slow flow rates would reflect alveolar inflammation. At this point, further studies are necessary to evaluate alveolar FENO and correlate the results with tissue pathology, prior to and after therapeutic intervention targeted at the distal airways.

Conclusions Asthma remains a prevalent, yet under-diagnosed and under-treated disease with significant morbidity and mortality and the potential for disease sufferers to develop airway remodelling and/or progressive loss of lung function. While airway inflammation represents the underlying pathophysiologic feature of asthma, current guideline recommendations base diagnosis and treatment largely on symptoms and pulmonary function, neither of which accurately reflects airway inflammation. There is a growing body of evidence to suggest that the measurement of FENO, a non-invasive, standardised procedure, which requires little technical expertise, and is quick, reproducible and easy to perform might be a useful clinical ‘inflammometer’ or surrogate marker of eosinophilic airway inflammation that could complement conventional methods of asthma diagnosis and management with hopes of improving outcomes [131]. Because of its sensitivity, FENO can detect subclinical airway inflammation which may exist in the absence of symptoms or pulmonary function abnormalities. This can be of potential value in the diagnosis of asthma or in evaluating the effect of allergen exposure or therapeutic intervention on airway inflammation. Determining cut points for diagnosis is difficult, in view of inter-patient variability and numerous factors including atopy, smoking, concurrent infection or the use of corticosteroids which can affect FENO levels. While an algorithm for diagnosis based on FENO reference ranges is offered, it is important to stress that results must always be interpreted, in the context of prevailing clinical information. Additional potential practical applications for the measurement of FENO include comparing the clinical potency of various ICS formulations and predicting patient response to various therapeutic agents including ICS. In patients already being treated with ICS, FENO can be of value in determining an ICS response, predicting an asthma relapse during ‘remission’, predicting loss of control or an impending exacerbation, determining adherence and/or compliance with therapy. The use of FENO to titrate ICS doses to optimize therapy appears to have promise, but is currently uncertain and requires further investigation before this application can be advocated for routine clinical practice. While cut-point values are cited for loss of control, impending exacerbations or poor compliance, it seems

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equally practical to establish personal best values for patients and to monitor FENO at regular intervals to detect changes in inflammation and the need to increase or decrease anti-inflammatory therapy accordingly. As measurement devices become more portable and cost-effective, it is conceivable that patients could monitor FENO at home as easily as they measure peak expiratory flow rates. Finally, the potential to measure alveolar FENO could provide more information regarding the deposition and the effect of medication in the distal airways where irreversible lung damage may occur. With increasing studies to support its value, using the measurement of FENO to complement guideline recommended methods of asthma diagnosis and management appears to have significant merit. Further studies will be necessary to determine the long-term effect on asthma outcomes, including airway remodelling, quality of life and health care costs.

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Lung Function and Bronchial Challenge Testing for the Allergist Klaus F. Rabe, Adrian Gillissen, and Zuzana Diamant

General Introduction The diagnosis and management of respiratory allergies in general, bronchial asthma, and rhinitis inevitably requires objective measurement of respiratory function. A wide variety of lung function equipment is commercially available, but varying expertise in its use and general reservations towards the complexity of some measurements likely have hampered the widespread and optimal use of state-of-the-art technology in general allergy practice. Curiously, there are almost no specific guidelines for the standards, scope or interpretation, or the quality assurance of lung function testing issued from the large allergy societies. Bronchoprovocation testing with direct bronchoconstrictor stimuli, with allergen, or with clinical models is relevant for the diagnosis of allergies and asthma, in particular when baseline lung function is normal. These methods need a standardized approach and involve reliable functional assessment of lung function to be safe, reproducible, and of adequate quality. Lately the functional assessment of airway function has been complemented by noninvasive techniques to measure components in exhaled breath, breath condensate, and in sputum. These techniques have in part been introduced into clinical algorithms, they are increasingly used in respiratory research, and they complement the classical techniques of physiological lung function measurements. This chapter, therefore, strives to give a concise overview of relevant techniques for lung function testing, spanning from simple peak flow measurements to classical physiological techniques to novel developments for airway “inflammometry.”

K.F. Rabe () Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands e-mail: [email protected] A. Gillissen Robert-Koch-Hospital, St. George Medical Center, Leipzig, Germany Z. Diamant Centre for Human Drug Research, Leiden, The Netherlands

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Measurement of Lung Function Pulmonary function tests provide measures of lung volumes, flow rates, gas exchange, and respiratory muscle function. Since Hutchinson first developed the spirometer in 1846, measurements of the dynamic lung volumes and of maximal flow rates have been used in the detection and quantification of diseases affecting the airways and the lung parenchyma. Basic pulmonary function tests that are available in the ambulatory setting include peak flow and spirometry [1, 2]. These tests provide physiologic measures of pulmonary function and can be used to quickly narrow a differential diagnosis and suggest a subsequent strategy of additional testing or therapy. Additional lung function tests include body plethysmography to measure the amount of thoracic gas volume, which is important to quantify hyperinflation and the airway resistance, esophageal pressure measurements to determine pressure–volume relationships, blood gas analysis, and exercise testing [3, 4]. These provide a more detailed description of physiologic abnormalities and the underlying pathology. The choice and sequence of testing are guided by information from the history, the physical information, and after all by the technical availability.

Peak Flow The maximal peak expiratory flow is measured with a peak-flow meter, which is a very user-friendly device, portable, made from plastic, and therefore inexpensive, although electronic versions are available [5]. Peak-flow measurements can be performed by the patients themselves, and can therefore be used for self-management purposes, diagnosis, or to detect environmental exposure situations causing airway obstruction. Peak-flow measurements provide a simple, quantitative measure of limitation of exhaled airflow typically seen in obstructive airway diseases. Daily monitoring can be used to detect worsening of lung function in the absence for symptoms, to assess variations in lung function throughout the day, to identify triggers, to make appropriate medication decisions, and to monitor the patient’s response to therapy. Peak-flow measurements can be done in adults and also in children over 5 years of age. Optimal peak-flow assessment requires some degree of patient education: • The patients should know that it is a key measure to monitor asthma at home [6]. • The device and the breathing technique must be explained. It is important that the patient perform the peak-flow measurement always in the same body position, preferably when standing straight. After taking a deep breath, the patient must blow as hard as possible without bending over. • Most commonly, peak flow is measured first thing in the morning prior to the treatment. • The number on the indicator scale is the maximal exhaled airflow. • These steps should be repeated two or three times, and the largest number measured should be recorded in a peak-flow meter protocol.

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Budesonide 200µg bid + Formoterol 9µg bid 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

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Fig. 1 Before therapy high daily variability of peak flows indicate poorly controlled asthma

During the day peak-flow measurements can be repeated as often as necessary. The best peak-flow number is important for the patient because it is the comparator for the best achievable value, which serves as a reverence value for monitoring the effects of changes over time [7]. A decrease of ≥20% of the personal-best number (or ≥60 ml/min) or a diurnal variation of >20% (2×/day reading: >10%) may indicate (a) an asthma attack or (b) it shows an inadequately controlled disease [8] (Fig. 1). A fall in peak flow, especially when accompanied by symptoms such as increased cough, shortness of breath, or wheezing may signal the onset of an exacerbation, requiring immediate treatment to prevent complications. High diurnal variability is an important feature of poorly controlled asthma. There are two ways to calculate the peak-flow variability: • The difference between the maximum and the minimum value for the day, expressed as a percentage of the mean daily peak-flow value and averaged over 1–2 weeks, or • The minimum morning pre-bronchodilator peak flow over 1 week, expressed as a percentage of the recent best min/max (%). This has been suggested to be the best index of airway lability for clinical practice because it requires only a 1×/ day reading, it is easy to calculate, and it correlates best with airway hyperresponsiveness (AHR). However, peak-flow measurements have also the following disadvantages [9]: • The degree of airflow limitation can be underestimated, particularly as airflow limitation and gas trapping worsens. • Values vary considerably according to a person’s age, sex, body size, and between different peak-flow meters. • Peak-flow meters cannot be calibrated [10].

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Fig. 2 An example of a peak-flow meter protocol. The “best of the three” is the reading to record. Mark this with a cross on the chart

Figure 2 shows an example of a chart the patient can use for recording his peakflow values on a day-to-day basis.

Spirometry The simplest form of pulmonary function testing is spirometry, which in principle measures how quickly air can be expelled from the lungs [11]. Spirometry is performed by blowing into a spirometer, which measures timed expired and inspired volumes. A spirogram is thus either a volume–time curve or a flow–volume curve, e.g., expressed as a peak expiratory flow or as a function of volume (Fig. 3). Although highly dependent on individual factors, these curves are reproducible for any individual, but vary considerably between different lung diseases and disease stages. Furthermore, the spirographic values are dependent on the factors age, height, and gender (see below). The following parameters are usually measured (Fig. 4): • Vital capacity (VC): It is the maximum volume of air which can be exhaled or inspired during either a forced (FVC) or a slow (VC) maneuver. • Force expired volume in one second (FEV1): It is the volume expired in the first second of maximal expiration after a maximal inspiration and is a useful measure of how quickly full lungs can be emptied. FEV1 is the most widely used parameter in asthma and chronic obstructive pulmonary disease (COPD). Besides the symptoms, FEV1 and FEV1/FVC determine the severity of both diseases. The extent of FEV1 decline is a strong predictor of poor disease outcome and indicates poor asthma management and vice versa [2, 3]. • FEV1/FVC or FEV1/VC: The ratio of FEV1 is expressed as a percentage of FVC or VC and gives a clinically useful index of airflow limitation, even if airway

Fig. 3 A typical flow–volume curve (a) and a volume–time curve (b). VC = vital capacity, FEV1 = forced expired volume in one second

obstruction is very moderate. In contrast, interstitial pulmonary diseases have normal FEV1/FVC and FEV1/VC volumes due to limitation of both lung function parameters. • FEF25–75%: It is the average expired flow over the middle half of the FVC maneuver and is regarded as a more sensitive measure of small airways narrowing than FEV1. Unfortunately, FEF25–75% has a wide range of normality, is less reproducible than FEV1, and is difficult to interpret if the VC or FVC is reduced or increased.

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Fig. 4 Normal spirogram showing the spirographic parameters: FEV1 =forced expired volume in one second; FVC = forced vital capacity; VT = tidal volume or tidal breathing; ERV = expired residual volume; IRV = inspired residual volume; FRC = functional residual capacity; RV = residual volume; VC = vital capacity; TLC = total lung capacity

Spirometry should be performed only when the spirometer is heated up to body temperature and pressure saturated with water vapor. Temperature differences between, for example, the warm exhaled air within a cold spirometer will cause an underestimation of the true lung volumes. Further, spirometers need to be calibrated prior to use.

Technique – How to Use It: Pitfalls and Problems To ensure an acceptable result, the spirometry maneuver must be performed with greatest effort starting with maximum expiration followed by a deep inspiration and an expiration phase lasting at least 6 s. The spirogram should be a smooth continuous curve. To achieve good results the procedure should be done only in a patient sitting erect with feet firmly on the ground (though standing gives a similar result) with a nose clip applied. The lips must seal firmly the mouthpiece and the patient must be urged to exhale forcibly. At least three technically acceptable blows should be obtained. Only the measurement with the largest VC or FVC is recorded. A good reproducibility means a difference of less than 200 ml between the highest and the second highest FVC [2, 3, 12]. Many patient-related problems may occur and result in inefficient flow–volume curves (Fig. 5):

Fig. 5 Common pitfalls with the breathing technique of the patients

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Submaximal effort Leaks between the lips and the mouthpiece Incomplete inspiration or expiration prior to or during the forced maneuver Hesitation at the start of expiration, cough, and/or vocalization during the forced maneuver • Obstruction of the mouthpiece by the tongue • Poor posture

Hazards and Quality Control Spirometry is a safe procedure and hazards have only been anecdotally reported. The quality of the spirometry can be just as good as the operator understands the principles underlying measurement and equipment operation. Various components

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of the spirometry system, including mouthpieces, nose clips, pneumotachs, valves, and tubing are potential vehicles for transmission of infection to subjects and staff. A tight infection control is therefore recommended.

Interpretation of the Results Spirometry allows the distinction between obstructive and restrictive pulmonary diseases [13]. Further, reversibility of an airway obstruction can be tested easily (see bronchodilator [BD] test). In order to compare the individual results to healthy persons, reference tables or reference equations have been established and abnormalities are evaluated against predicted results. Predicted values are calculated based on age, height, and gender. Office spirometers are typically preprogrammed with prediction equations derived from the study of Caucasians, such as the European Community for Steel and Coal (ECSC) [14]. An obstructive ventilatory abnormality is defined as a disproportionate reduction in maximal airflow from the lung with respect to the maximal volume that can be displaced from the lung. Typically, reduction of FEV1/FVC (or FEV1/VC) FEV1 is seen in asthma and COPD. The extent of lung function decline defines the disease stage of both diseases [8, 15]. The diagnosis of an obstruction should be followed up with a bronchodilator test (see below). Severity or airway obstruction is graded according to percentage of predicted FEV1 [8]. A restrictive ventilatory defect is characterized physiologically by a reduction in total lung capacity (TLC) which, however, cannot be determined by spirometry. Regardless of this limitation, reduced FVC or VC together with reduced FEV1 resulting in normal or high FEV1/FVC or FEV1/VC indicate a restrictive pattern. A range of conditions can reduce FEV1 and FVC (or VC) such as diseases impeding the movement of the chest wall including pain, neuromuscular weakness, lung parenchymal diseases (interstitial lung diseases), or conditions causing reduced lung volume (lung resection).

Bronchodilator Test The purpose of the bronchodilator (BD) test is to determine whether an airway obstruction, as measured by low FEV1% predicted and/or low FEV1/VC is reversible with an inhaled rapid acting β2-agonist. The test can be standardized as follows: • • • •

Two puffs (100 µg) of salbutamol or equivalent are administered. A waiting period of at least 10 min is introduced. Two reproducible flow–volume curves (FEV1 and/or FVC) are again obtained. The best post-bronchodilator FEV1 is evaluated.

A significant improvement of at least 12% (or 15% depending on the society) and 200 ml from the best pre-bronchodilator FEV1 is regarded as a positive reversibility,

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a strong indication for asthma. Typically, COPD patients lack a positive test result due to fixed airway obstruction [3, 16, 17]. Percentage improvement in FEV1 can be calculated using the following formula: [FEV1 pre-BD 2 FEV1 post-BD/FEV1 pre-BD] × 100 For an accurate interpretation of a negative response, patients must have been weaned from bronchodilators for at least 12 h, if medically possible.

Limitations Spirometry is an effort-dependent test that requires careful instructions and the cooperation of the test subject. Inability to perform acceptable maneuvers may be due to poor subject motivation or failure to understand instructions. Physical impairment and young age (e.g., children over 5 years of age) may also limit the subject’s ability to perform spirometric tests. These limitations do not preclude attempting spirometry, but should be noted and taken into consideration when the results are interpreted.

Bodyplethysmography The measurement of absolute lung volumes, residual volume (RV), functional residual capacity (FRC), and total lung capacity (TLC) are technically more challenging procedures, which has limited the use of bodyplethysmography in clinical practice. Also, the role of lung volume measurements in the assessment of disease severity, functional disability, course of disease, and response to treatment remains to be determined for all age groups, although sometimes measurements of lung volume are necessary for a correct physiological diagnosis [3].

Definitions of Lung Volumes The term “lung volume” usually refers to the volume of gas within the lungs, as measured by body plethysmography and the measurements obtained are graphically illustrated in Fig. 6.

Measurement of FRC Using Body Plethysmography Plethysmographic measurements are based on Boyle’s law, which states that, under isothermal conditions, when a constant mass of gas is compressed or decompressed, the gas volume decreases or increases and gas pressure changes such that the product of volume and pressure at any given moment is constant [18, 19].

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Fig. 6 The functional residual capacity (FRC) is the volume of gas present in the lung at endexpiration during tidal breathing. The expiratory reserve volume (ERV) is the volume of gas that can be maximally exhaled from the end-expiratory level during tidal breathing (i.e., from the FRC). The maximum volume of gas that can be inspired from FRC is referred to as the inspiratory capacity (IC). The inspiratory reserve volume is the maximum volume of gas that can be inhaled from the end-inspiratory level during tidal breathing. The residual volume (RV) refers to the volume of gas remaining in the lung after maximal exhalation. The volume of gas inhaled or exhaled during the respiratory cycle is called the tidal volume (TV or VT). The thoracic gas volume (TGV or VTG) is the absolute volume of gas in the thorax at any point in time and any level of alveolar pressure. The total lung capacity (TLC) refers to the volume of gas in the lungs after maximal inspiration, or the sum of all volume compartments

The changes in thoracic volume that accompany a compression or decompression of the gas in the lungs during respiratory maneuvers can be obtained using a body plethysmograph by measuring the changes either as: (1) pressure within a constant-volume chamber (variable-pressure plethysmograph); (2) volume within a constant-pressure chamber (volume-displacement plethysmograph); or (3) airflow in and out of a constant-pressure chamber (flow plethysmograph). Regardless of the type, a transducer capable of measuring mouth pressure ≥ ±5 kPa (≥ ±50 cm H2O), with a flat frequency response in excess of 8 Hz, is essential for measurements (Fig. 7). The transducer measuring changes in the chamber pressure must be capable of accurately measuring a range of ±0.02 kPa (±0.2 cm H2O) [20]. All components of plethysmographs that are used for the measurement of lung volumes should meet published standards for the accuracy and frequency response of spirometric devices [20, 21].

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Fig. 7 Body plethysmorgraphy (MasterScreen®, CardinaHealth) consists of a body box (right) and an analyzing unit (left)

Measurement Technique Measurements should practically adhere to the following steps: 1. The equipment should be turned on and allowed an adequate warm-up time. 2. The equipment is set up for testing, including calibration, according to manufacturer’s instructions. 3. The equipment is adjusted so that the patient can sit comfortably in the chamber and reach the mouthpiece without having to flex or extend the neck. 4. The patient is seated comfortably, with no need to remove dentures. The procedure is explained in detail, including that the door will be closed, the patient’s cheeks are to be supported by both hands, and a nose clip is to be used.

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5. The plethysmograph door is closed, and time is allowed for the thermal transients to stabilize and the patient to relax. 6. The patient is instructed to attach to the mouthpiece and breathe quietly until a stable end-expiratory level is achieved (usually three to ten tidal breaths). 7. When the patient is at or near FRC, the shutter is shortly closed at end-expiration and the patient is instructed to perform a series of gentle pants (~±1 kPa [~±10 cm H2O]) at a frequency between 0.5 and 1.0 Hz [22, 23]. 8. A series of three to five technically satisfactory panting maneuvers should be recorded after which the shutter is opened and the patient performs an ERV maneuver, followed by a slow IVC maneuver. If needed, the patient can come off the mouthpiece and rest between TGV/VC maneuvers. 9. For those unable to perform appropriate panting maneuvers (e.g., young children), an alternative is to perform a rapid inspiratory maneuver against the closed shutter. In this situation, it is essential that the complete rather than the simplified version of the TGV computation equation [18] be used in the calculation of TGV. 10. With regard to repeatability, at least three FRCpleth values that agree within 5% should be obtained and the mean value reported.

Quality Control The accuracy of the flow and volume output of the mouth flow-measuring device should comply with published recommendations [2]. The mouth pressure transducer should be physically calibrated daily. The plethysmograph signal should also be calibrated daily, using a volume signal of similar magnitude and frequency as the respiratory maneuvers during testing. A validation of accuracy using a known volume should be performed periodically using a container of known volume [18, 24]. Care should be taken to adjust the calculated volumes to ambient temperature and saturated conditions (BTPS) during the calculations. The accuracy of adult plethysmographs in measuring the gas volume of the container should be ±50 ml or 3%, whichever is greater, based on a mean of five determinations [18]. Monthly, or when errors are suspected, two reference subjects should have their FRCpleth and related RV and TLC measured. Values that differ significantly (e.g., 0.10% for FRC and TLC, or 0.20% for RV) from the previously established measurements suggest errors that need to be followed up [25].

Reference Values Interpretation of lung volumes critically depends on reference values which are related to body size, with standing height being the most important factor. In children and adolescents, lung growth appears to lag behind the increase in standing

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height during accelerated growth, shifting the relationship between lung volume and height during this period [26, 27].

Pitfalls and Problems A number of factors need to be considered when selecting predictive values for absolute lung volumes. The reference populations need to match the patient populations and this information is not always easily available. Furthermore, the appropriate extrapolation of regression equations needs to be exerted considering the size and age range of subjects actually studied. Finally, the differences in testing methodology between clinical laboratories and studies from which predicted reference values are derived need to be considered before clinical interpretation of plethysmographic measurements.

Bronchoprovocation Tests Direct and Indirect Bronchial Challenges Airway hyperresponsiveness (AHR) is a key characteristic of asthma and can be subdivided into a transient (relatively reversible) and more persistent (relatively irreversible) component [28]. Measurements of airway responsiveness are particularly helpful when lung function tests are normal despite symptoms or history of respiratory allergies. In general, the pathophysiology of AHR is complex and not entirely clarified. Transient AHR can be induced by indirect stimuli (e.g., exercise, cold dry air, hypertonic saline, adenosine monophosphate (AMP), and mannitol) and is generally well-responsive to inhaled corticosteroids. Hence, there is evidence that this component of AHR is linked to airway inflammation [28]. Indeed, some indirect stimuli are applied as exacerbation models of airway inflammation [29]. Alternatively, persistent AHR is more marked to direct stimuli, including cholinergic agents and histamine, appears relatively refractory to inhaled corticosteroids, and reflects the structural changes within the airways, referred to as “airway remodeling” (Table 1) [28].

Direct Challenges Direct stimuli or agonists, exemplified by methacholine and histamine, induce airway narrowing through interaction with specific receptors on airway smooth muscle cells [28]. Bronchial challenge tests with these agonists are standardized, usually performed by the 2-min tidal breathing method, and amply used for the assessment and quantification of the AHR in clinical and research settings [28, 30]. The severity

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Table 1 Tests identifying pathophysiologic characteristics of asthma eNO EBC Sputum BAL Biopsy

Airway inflammation

Airway remodeling

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Airway hyperresponsiveness

Transbronchial biopsy Imaging Methacholine BPT (?)

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Asthma symptoms

Key features of asthma and possible sampling methods. eNO = exhaled NO, EBC = exhaled breath condensate, BAL = bronchoalveolar lavage, AMP = Adenosine 5' monophosphate, BPT = bronchoprovocation test.

(mild to severe) of AHR is expressed by the provocative dose or concentration causing a 20% fall in FEV1 from baseline (PD20 and PC20, respectively) [30]. Direct challenges are highly sensitive, although not specific for asthma, and can be used as diagnostic tools in subjects with a history of asthma with normal spirometry and no reversibility to inhaled β2-agonists [28, 30].

Indirect Challenges Indirect stimuli contract airway smooth muscle causing subsequent bronchoconstriction through pathways mediated by the release of mediators from inflammatory cells and sensory nerves [31]. Therefore, when performed serially, an adequate washout period should be allowed between consecutive challenges to correct for these pro-inflammatory “carry-over” effects [31, 32]. Similarly to direct provocation tests, most indirect challenges are standardized procedures [30, 33]. In 2003, an ERS Task Force document has been published on the pathophysiology, methodology, and applicability of indirect challenges with physical or pharmacological stimuli [33]. Physical stimuli with exercise challenge and the related isocapnic hyperventilation of cold, dry air are validated exacerbation models of exercise-induced bronchoconstriction (EIB) [31]. Bronchoprovocation with the pharmacological stimulus hypertonic saline (mostly NaCl 4.5%), aerosolized by an ultrasonic nebulizer, shares similar pathophysiological mechanisms with EIB and cold, dry air hyperventilation [33]. This simple, inexpensive method is an attractive tool for both diagnostic (EIB, risk assessment for scuba divers with asthma) and research

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purposes (EIB-modeling, airway sampling by sputum induction) [31, 34]. Mannitol is another hyperosmolar pharmacological stimulus, comprising of a dry powder inhaler and capsules [35]. Sharing a similar pathophysiological mechanism, i.e., increasing airway osmolarity through dehydration, both hypertonic saline and mannitol have been shown to be well-related to exercise and cold, dry air in steroid-naive asthmatics [35, 36]. Despite mechanistic similarities, there are marked differences in the methodology, equipment, and costs [30, 33, 35]. Making mannitol possesses superior properties: being a simple, reproducible, and relatively inexpensive method [35, 37]. Apart from its diagnostic potential, mannitol appeared as a sensitive predictor of a drug’s anti-inflammatory efficacy and thus may be applicable for monitoring of asthma control both in clinical practice and in clinical trials [33, 37]. Adenosine monophosphate (AMP) is another related, pharmacological stimulus that could act as a marker of disease activity [31, 33].

Exacerbation Models Asthma exacerbations can be modeled by indirect stimuli that can trigger more or less specific immunological pathways and, consequently, mimic various pathophysiological features of asthma (Table 2) [30, 38–45]. In early clinical trials, validated exacerbation models can be applied for proof of mechanism (POM), as they only require short-term pretreatment in a (relatively) small number of patients [38, 39]. For obvious reasons, the choice of an adequate exacerbation model should

Table 2 Most commonly known direct stimuli and indirect stimuli/exacerbation models Direct stimuli Indirect stimuli Cholinergic agonists: Acetylcholine Carbachol Methacholine Histamine

Physical stimuli: Exercise Cold, dry air

Pharmacological stimuli: Hypertonic saline Mannitol Adenosine monophosphate (AMP) Exacerbation models/cellular mechanism: Exercise (mast cells) Endotoxin (LPS) (neutrophils) Ozone (neutrophils) Virus (eosinophils/neutrophils) Aspirin (eosinophils) Allergen (mast cells/eosinophils) Steroid tapering (eosinophils)

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fit the asthma phenotype and the drug’s target. In this chapter, we will address the most commonly applied exacerbation models.

Mast Cell-Triggered Challenges Exercise and Related Challenges Exercise challenge and isocapnic hyperventilation of cold, dry air have been shown to induce bronchoconstriction in over 70% of the asthmatics [32]. Although the underlying mechanisms mediated by these stimuli are not fully understood, there is convincing evidence that airway cooling and drying cause the release of bronchoactive mediators from mast cells and stimulation of sensory nerves [31, 33]. Other indirect challenges acting through similar pathways, comprise adenosine monophosphate (AMP), hypertonic saline, and mannitol [33, 35]. When performed serially in a clinical trial, these indirect challenges require washout periods ranging from hours up to several days to correct for refractoriness and carry-over effects [31, 33, 46].

Models of Eosinophilic Airway Inflammation Allergen Challenge Allergy is a key mechanism inducing the development of persisting inflammation and structural changes within the airways that may result in symptomatic asthma. Several novel treatment modalities are presently being developed targeting the allergic mechanisms. Therefore, methods and tools that can rapidly predict a new drug’s clinical efficacy are pivotal for the development of novel therapies. Allergen challenge meets most of these criteria and is a useful model for POM studies with anti-allergic therapies [44]. Presently, there are three allergen challenge techniques. First, nasal allergen challenge is a validated and reproducible method in which relevant allergens are administered intranasally to sensitized subjects [47, 48]. The subsequent upper airway response mimics symptoms and signs of allergic rhinitis and upper airway inflammation. Consequently, this model enables to study the (relationship between) kinetics of the clinical symptoms and the allergic upper airway response [49]. The allergen-induced nasal early response occurs within 10 min of the provocative allergen concentration and is mainly characterized by nasal congestion, itching, sneezing, and rhinorrhea, caused by mast cell-derived pro-inflammatory mediators. These symptoms can be scored and quantified by a validated questionnaire according to Lebel and colleagues [50]. Alternatively, the nasal late response is dominated by a prolonged nasal congestion and airway eosinophilia [49]. Rhinomanometry can be used for an objective assessment of the changes in nasal patency and several sampling techniques can be used to quantify

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the inflammatory nasal response [51]. Although nasal biopsies are the golden standard to study the cellular inflammatory response, serial samplings are limited due to its invasive nature [52]. Being less invasive techniques, nasal brushings, nasal lavages, and possibly nasal NO seem to be alternatives for repetitive nasal samplings of airway inflammation [53–55]. In patients with combined allergic rhinitis and asthma syndrome (CARAS), nasal allergen challenge may induce airway inflammation and hyperresponsiveness within the lower airways, thus allowing studying the link between both airway compartments [56–58]. The second technique comprises segmental allergen challenge, an invasive method requiring bronchoscopy for direct airway sampling (biopsy, bronchoalveolar lavage) following locally instilled allergen [59]. A safe and effective allergen dose is determined by prior inhaled allergen bronchoprovocation [60]. However, serial applicability of this method is limited due to its invasive nature, providing histopathological/cytological information on a restricted part of the lower airways. The third method includes inhaled allergen challenge, a highly reproducible exacerbation model mimicking several acute and some chronic features of (allergic) asthma [29, 30, 61]. Traditionally, the allergen dose is individually determined by a diluted skin prick test and/or methacholine PC20 by Cockcroft’s formula or a derived method [62, 63]. Within 10–30 min of inhalation, this “provocative allergen dose” induces an early asthmatic response (EAR) in sensitized asthmatics that usually subsides at 3 h post-allergen. The EAR is an acute inflammatory airway narrowing, characterized by a self-limiting 15–20% fall in FEV1 from baseline, in approximately 50% followed by a late asthmatic response (LAR) [29, 32]. In the pathophysiology of the EAR, IgE-triggered mast cells have been shown to play the key role and convincing evidence points to their involvement in the LAR and the subsequent AHR [64]. The LAR represents an episode of progressive and longer-lasting airway narrowing, characterized by a fall in FEV1 of at least 15% from baseline, usually occurring between 3 and 7 h post-allergen [30, 62]. In parallel, there is a more persistent airway inflammation in which activated eosinophils and their mediators play a key effector role causing tissue damage with subsequent AHR [65]. Consequently, to avoid carry-over effects in clinical trials, a washout period of at least 3 weeks is recommended between two consecutive allergen challenges [66]. The allergen-induced airway responses are defined as maximal percentage fall from baseline FEV1, or, more precisely, as the area under the time–response curve post-allergen (AUC), during the EAR (0–3 h), and the LAR (3–≥7 h), respectively [30]. Especially when combined with noninvasive sampling techniques enabling to study several aspects of the allergen-induced airway responses and their relation to the inflammatory events within the airways, the allergen challenge is a useful tool in POM studies with novel drugs targeting allergen-induced mechanisms [29, 44, 67]. And if properly conducted, allergen challenge can predict a drug’s clinical efficacy [44]. Indeed, agents inhibiting the sequelae of the LAR have generally shown efficacy in clinical asthma (e.g., inhaled corticosteroids, leukotriene modifiers, and anti-IgE), while those that did not affect the LAR have not. Based on previous studies and although not all drugs that did not show efficacy against the LAR have been tested in a clinical setting, this model seems to have a moderate positive predictive value, but an excellent negative predictive value [44].

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Aspirin Challenge Aspirin challenge is a more or less specific provocation test that can be used to confirm the diagnosis of aspirin-exacerbated respiratory disease better known as aspirin-induced asthma (AIA). The incidence of this asthma phenotype ranges from 3% to 21% among adult asthmatics and usually starts after the age of 30 years [68]. Upper respiratory tract symptoms are usually the first manifestations of AIA (rhinorrhea, nasal congestion, and polyps), while asthma and aspirin hypersensitivity develop some years later, persisting throughout life. Despite avoidance of aspirin and related nonsteroidal anti-inflammatory drugs (NSAIDs), approximately 50% of patients have severe persistent asthma, characterized by profound blood and airway eosinophilia [68]. Aspirin challenge is a specific and sensitive tool for both diagnostic and research purposes. This challenge can be performed with oral aspirin (ASA) or local (intranasal, endobronchial, or inhaled) solutions of lysine-aspirin (L-ASA) [43, 69]. In sensitized patients, aspirin challenge provokes an acute bronchoconstrictor response, often accompanied by extra-bronchial symptoms (conjunctivitis, rhinorrhea, nasal congestion, gastro-intestinal symptoms, and flushing of head and neck). The aspirin-induced airway inflammatory response is characterized by profound eosinophilia releasing large amounts of cysteinyl leukotrienes: bronchoactive mediators that can be quantified by urinary leukotriene E4 [43, 69]. Despite a timeconsuming procedure requiring close monitoring for potential anaphylaxis, aspirin challenge is a useful tool for intervention studies with drugs targeting the aspirininduced airway responses, e.g., anti-leukotriene therapy [70].

Noninvasive Airway Sampling Methods and Biomarkers In contrast with the classical concepts of asthma, symptoms and lung function adequately reflect neither the activity of underlying airway inflammation nor the severity of AHR [71]. Hence, the efficacy of novel drugs should be evaluated by multiple (surrogate) biomarkers instead of symptom scores and lung function measurements only [38]. Consequently, an increasing number of noninvasive sampling methods have been developed to study the components of the asthmatic airway inflammation in search of responsive and reproducible biomarkers [32].

Exhaled Nitric Oxide Measurement of the fraction of nitric oxide in exhaled breath (FENO) is a simple and versatile method of noninvasive inflammometry. Nitric oxide (NO) is implicated in several biological processes, including the regulation of vascular and bronchial tone, neurotransmission, and inflammation. This volatile molecule

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is nonspecific for asthma and affected by several exogenous and endogenous factors including food, sex, atopic status, and smoking [72]. In patients with (allergic) asthma uncontrolled by ICS, FENO has been found to correlate with eosinophils, key components of (allergic) airway inflammation, and thus may be indicative of asthma control [73–76]. In agreement with this finding, ICS and other anti-eosinophil therapies, including leukotriene modifiers and anti-IgE, dose-dependently reduced FENO [77–80]. Similarly, several tapering studies have shown that loss of asthma control is associated with an increase in FENO [81–83]. Hence, FENO has been proposed as a biomarker for the diagnosis of asthma and treatment monitoring. To this end, the American Thoracic Society (ATS) and the European Respiratory Society (ERS) issued recommendations for standardized exhaled NO measurements from upper and lower respiratory tracts in adults [84]. A separate document for children was approved by ATS and ERS [85]. The currently recommended FENO sampling technique is performed during a singlebreath exhalation against a fixed resistance allowing online measurements by validated chemoluminescence analyzers [84]. More recently, a two-compartment model has been proposed to discriminate between alveolar and conducting airways NO fractions [86, 87]. Using this model in patients with severe refractory asthma, alveolar NO appeared as a potential biomarker for distal airway inflammation that could not be reached by ICS [88]. The introduction of the less expensive, handheld NIOX MINO®, yielding comparable measurements to the stationary, costly chemoluminescence NO-analyzers, will enable further implementation of NO measurements into daily practice [89].

Sputum Induction Induced sputum is another validated sampling method of the airways. This relatively simple, though demanding, method requires specific equipment and expertise including a dedicated lab and a certified (cyto)pathologist. The introduction of this reproducible technique helped to identify several components of the airway inflammation and their response to treatment, resulting in the definition of inflammatory asthma phenotypes [90]. In 2002, the ERS Task Force formulated guidelines for standardization of the different induction and processing techniques [34]. The commonly recommended method comprises inhalations of hypertonic saline (NaCl 4.5%) for three to four times 5–7 min through a mouthpiece – the aerosols being generated by an ultrasonic nebulizer [34]. Subsequently, the sputum is processed within 2 h according to guidelines and divided into a cell pellet and supernatant [84, 91]. The pellet is resuspended, cytospined, stained, and the viability of the cells is assessed. Subsequently, cells are counted and the differentials are expressed as percentage of 500 nucleated, non-squamous cells. In analyzable sputum samples the percentage of squamous cells should not exceed 80% [84]. Depending on the availability of validated bioassays, several soluble biomarkers can be quantified in the sputum supernatant [91].

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Sputum eosinophil and neutrophil counts are reproducible biomarkers of airway inflammation and well-correlated with asthma severity [81, 90, 92, 93]. Sputum eosinophils are increased in models of eosinophilic exacerbations (e.g., allergen-induced LAR) and in eosinophil-dominated asthma phenotypes with an overall good response to anti-inflammatory interventions, especially inhaled corticosteroids [94–96]. In line with phenotype-directed treatment, Green and colleagues demonstrated that sputum eosinophils are superior guides of asthma control in patients with moderate to severe persistent asthma than symptom scores and lung function [90, 97]. Alternatively, the neutrophilic asthma phenotype, reflecting more severe asthma, is relatively refractory to inhaled corticosteroids and hence requires different targeted therapy [90]. In general, sputum eosinophil and neutrophil counts can serve as biomarkers for diagnostic purposes and early drug development [38]. Presently, several novel detection techniques are being tested to optimize quantification of soluble markers in the sputum supernatant, including peptidomics and metabolomics [98, 99].

Exhaled Breath Condensate Exhaled breath condensate (EBC) is another, relatively novel, noninvasive technology, allowing collection and measurement of various volatile compounds potentially implicated in disease processes [91]. An ATS/ERS Task Force document has been published on EBC addressing methodology and yet unresolved issues [91]. Exhaled breath consists of two components: the gaseous phase, containing volatile organic compounds (VOCs) such as NO and carbon dioxide (CO2), and a liquid phase containing nonvolatile components including water-soluble inflammatory markers (Fig. 8).

Fig. 8 CO-diffusion capacity, rebreathing functional residual capacity (FRC) but also NO-CO-diffusion capacity can easily be measured in daily routine (MasterScreen®, PFT series, Cardinal Health)

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So far, there has been no standardization of EBC sample collection or analysis and this needs to be resolved before results can be interpreted and compared. Overall, analysis of exhaled breath seems a promising tool for both research and future clinical monitoring, enabling disease (severity) recognition by smellprints, metabolomics, and proteonomics [100, 101]. In addition, measurement of drug concentrations in EBC may allow studying the link between pharmacokinetic properties and pharmacodynamic effects in the future.

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Investigative Bronchoprovocation and Bronchoscopy in Patients with Bronchial Asthma Hironori Sagara and Ruby Pawankar

Introduction Information directly obtained from inflamed airways provides important clues to the pathogenesis, diagnosis, and treatment of respiratory diseases. To study the pathogenesis of airway inflammation, bronchoscopy with bronchoalveolar lavage (BAL) and endobronchial biopsy have been found to be useful for evaluating changes in histologic features as compared with healthy subjects and for measuring cell activation markers, chemical mediators, and cytokines. BAL has been used for the diagnosis of various diseases, including diffuse pulmonary disease, granulomatous pulmonary disease, neoplastic pulmonary disease, and pulmonary infections. Histopathological, immunologic, and genetic studies of endobronchial-biopsy specimens allow tumorous, inflammatory, and immunologic lesions to be evaluated. Recently, these techniques have been used clinically for the diagnosis of inflammatory diseases of the airways, including bronchial asthma and chronic obstructive pulmonary disease (COPD). In this chapter, we explain techniques for bronchoscopy, BAL, and endobronchial biopsy and briefly describe the characteristics of asthma and COPD. We then focus on bronchial asthma and explain the roles of various types of inflammatory cells in airway inflammation and finally summarize associated histologic changes.

Techniques for Bronchoscopy Bronchoscopy is performed in the following order: 1. Patients abstain from all food and drinks for 4 h before examination, and vascular access is secured. H. Sagara () Department of Respiratory Medicine, Dokkyo Medical University Koshigaya Hospital, Japan e-mail: [email protected] R. Pawankar Nippon Medical School, Tokyo, Japan

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2. Premedication: Atropine sulfate (0.5 mg) is given intramuscularly 15 min before examination. Atropine sulfate is contraindicated in patients with glaucoma and relatively contraindicated in patients with heart disease. Pentazocine (15–30 mg) or hydroxyzine (Atarax P®) (25 mg) is given intramuscularly to induce sedation. Midazolam (Dormicam®) (10 mg) is given intravenously in patients with severe anxiety. Patients with asthma receive two puffs of an inhaled β2 agonist (Saltanol®) to prevent asthma attacks. 3. Anesthesia: The pharynx and larynx are anesthetized with 4% xylocaine spray (5 ml). 4. Bronchoscope insertion: A fiberoptic bronchoscope is inserted orally through a mouthpiece. During examination, 2% xylocaine (1–2 ml) is used as needed for anesthesia. Necessary examinations are performed after examination of the bronchial lumen. During examination, the partial pressure of arterial oxygen, blood pressure, electrocardiogram, and pulse rates are monitored. 5. Patients abstain from all food and drinks for 2 h after examination. Patients with asthma are confirmed to be free of asthma attacks on auscultation and monitoring of peak expiratory flow.

Bronchoalveolar Lavage As recommended by previous studies, the lateral segmental bronchus (B4) and medial segmental bronchus (B5) are selectively washed three to four times with 50-ml aliquots of physiologic saline solution. B4 and B5 are considered to represent nearly the entire lung. The resulting lavage fluid is filtered with a sterile gauze to remove mucous substances and is centrifuged for 10 min at 240–250 × g at room temperature to separate cellular and humoral constituents. For the cellular constituents, cell counts, differential counts, and lymphocyte subpopulations are assessed. For the humoral constituents, protein, albumin, immunoglobulins, lipids, enzymes, and cytokines are assessed. BAL is useful for identifying inflammatory cells infiltrating the airways and soluble factors associated with inflammation. In patients with asthma, many eosinophils, lymphocytes, macrophages, and mast cells are detected as cellular constituents in BAL fluid from patients with asthma. As for disease type, patients with atopic asthma have higher levels of eosinophil expression than do those with nonatopic asthma. As for age, neutrophil counts are higher in patients who are middle-aged or older at disease onset than in those who are younger. This trend is stronger in patients with severe, refractory asthma. In BAL fluid, the levels of mediators such as the eosinophil-derived granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) are increased. The levels of chemokines (eotaxin, regulated on activation normal T cell expressed and secreted [RANTES] and the eosinophilic chemotactic factors monocyte chemoattractant protein-1 [MCP-1] and macrophage inflammatory protein-1α [MIP-1α]) are also increased. The levels of mast cell-derived mediators including histamine, leukotriene C4, tryptase, prostaglandin D2, and chymase are also elevated. The main type of lymphocyte in BAL fluid is CD4 + T cells. A type 2 helper T (Th2) cell-like pattern has been confirmed on the basis of cytokine production.

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The main inflammatory cells in BAL fluid from patients with COPD are macrophages, neutrophils, and CD8 + T cells. As for mediators, CD8 + T cells secrete a number of cytokines, including tumor necrosis factor-α, interferon-γ, and interleukin-12, as well as neutrophilic chemotactic factors such as interleukin-8 and leukotriene B4.

Endobronchial Biopsy A forceps is inserted under bronchoscopic guidance. Biopsy is performed at the bifurcation of the second- and fourth-order bronchi. Three to six biopsy specimens can be obtained, unless bleeding is severe. Endobronchial biopsy is useful for evaluating histopathological changes in the airways.

Histopathological Features of Airways in Patients with Bronchial Asthma Asthma is characterized by infiltration of inflammatory cells such as eosinophils, T lymphocytes, mast cells, and basophils and by structural changes, including airway-epithelium desquamation, epithelial goblet cells, submucosal-gland hyperplasia, mucosal and submucosal edema, basement-membrane thickening, smooth-muscle hypertrophy, and neovascularization.

Histopathological Features of Airways in Patients with COPD Histopathologically, COPD is characterized by epithelial mucosal goblet cells, squamous metaplasia, inflammatory-cell infiltration, hypertrophy and hyperplasia of bronchial glands, smooth-muscle hypertrophy, vascular-wall thickening, and airway-wall thickening. Infiltration of macrophages as well as CD8 + T lymphocytes and neutrophils is seen in the bronchial mucosa of patients with COPD.

Indications for Bronchoscopy Bronchoscopy can be performed safely in patients who meet the following criteria: 1. Lung function is stable and unchanged during the 3 weeks before examination. 2. Forced expiratory volume in one second (FEV1) is higher than 60% of the predicted value (patients with asthma).

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3. Airway obstruction is confirmed to be reversible (patients with asthma). 4. No evidence of cardiovascular diseases. However, bronchoscopic examination may lead to a transient reduction in lung function, causing coughing, wheezing, and dyspnea. These signs and symptoms promptly respond to treatment with bronchodilators or steroids and resolve. As for the indications and safety of bronchoscopy, please refer to a recent report summarizing a National Institutes of Health workshop [1]. In a study of 159 patients with asthma, both BAL and endobronchial biopsy were performed 228 times and endobronchial biopsy alone was performed 45 times. Side effects such as airway constriction, worsening of asthmatic symptoms, fever, and chest pain occurred in 32 patients who underwent both BAL and bronchoscopy and in 2 who underwent bronchoscopy alone. These results show that bronchoscopic examination is safe [2].

Inflammatory Cells in Airways of Patients with Asthma Mast Cells The number of mast cells in the bronchial lamina propria is small in healthy subjects and large in patients with asthma. Some studies have demonstrated increased numbers of mast cells in the airway epithelium of patients with asthma [3, 4], whereas others have not [5]. In association with the aggregation of mast cells on the surface of the bronchial lumen, the percentage of mast cells collected by BAL is increased in patients with asthma [6]. This finding suggests the migration of mast cells from deeper layers to the epithelium and bronchial lumen. Histopathological examination of bronchial-biopsy specimens in patients with asthma shows increased numbers of mast cells, with degranulation in some regions [4, 7]. In addition to evidence of mast-cell activation on electron microscopy, in vitro analysis of mast cells obtained from the BAL fluid of patients with asthma shows spontaneous or IgE-dependent increased release of histamine. We compared the numbers of mast cells infiltrating biopsy specimens of the airways between patients with atopic or nonatopic asthma and healthy controls. The number of mast cells was higher in patients with asthma than in healthy controls. In patients with atopic asthma, the number of mast cells in the bronchial mucosa, particularly the epithelium, was significantly higher than that in patients with nonatopic asthma [8]. Electron microscopy showed many degranulated mast cells in patients with atopic asthma. These results suggest that mast cells are not only increased, but also functionally activated in patients with asthma. Mediators such as histamine, leukotriene C4, tryptase, prostaglandin D2, and chymase are released into BAL fluid through mast-cell degranulation, inducing immediate asthmatic responses such as bronchial constriction, mucosal edema, and mucus secretion. Inflammatory cytokines such as interleukin-4, 5, 6, 8, and 13, granulocyte-macrophage colonystimulating factor (GM-CSF)-α, tumor necrosis factor-α, and chemokines are also

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released and participate in late asthmatic responses. Clinical studies have reported that increased numbers of mast cells correlate with airway hypersensitivity [9, 10].

Eosinophils Eosinophils are recognized as important effector cells among airway inflammatory cells in asthma. Increased numbers of eosinophils have been confirmed in the lamina propria and epithelium of the bronchi [3, 5, 7, 11–13]. The number of eosinophils in BAL fluid and the bronchial mucosa and the levels of major basic protein (MBP) and eosinophil cationic protein, derived from degranulated eosinophils, strongly correlate with FEV1 and other indices of the severity of asthma [12, 14]. Larger numbers of eosinophils and higher levels of MBP in BAL fluid are associated with increased numbers of desquamated airway epithelial cells and correlate with the degree of airway hypersensitivity [7, 10]. Histopathological studies by electron microscopy show that eosinophils infiltrating the mucosa are activated and secrete granules and that eosinophilic infiltration is associated with expanded airway epithelial cellular spaces as well as with increased airway hypersensitivity [15]. Eosinophils produce platelet-activating factor, lipid mediators such as leukotriene C4, a potent smooth-muscle constrictor, cytokines such as interleukin-3, interleukin-5, and GM-CSF, and chemokines (RANTES and eotaxin). In a clinical study of patients with asthma who received antihuman anti-interleukin-5 antibody, the activation of eosinophils was blocked, but airway hypersensitivity and biphasic asthmatic responses were not inhibited. The role of eosinophils in the pathogenesis of asthma has been thus questioned. Another cytokine that is produced by eosinophils is transforming growth factor (TGF)-β. TGF-β acts as a mitogen of fibroblasts and participates in the proliferation of myofibroblasts and collagen deposition in the basement membrane during the repair of epithelial damage. Matrix metalloproteinase-9 is released by eosinophils, fibroblasts, and macrophages and plays an important role in the degradation of excessive extracellular matrix of connective tissue.

T Cells Many studies have shown slightly increased numbers of T lymphocytes in bronchial-biopsy specimens from patients with asthma. Patients with atopic [13] or nonatopic asthma [14–16] have increased levels of CD25 and HLA-DR, indices of T lymphocyte activation, and a morphologic study [17] has shown high indices of T lymphocyte activation in asthmatic airways. Patients with atopic asthma have increased numbers of Th2 cells in BAL fluid, which secrete interleukin-2, 4, and 5, and GM-CSF [18, 19]. The number of activated T cells in BAL fluid positively correlates with the number of eosinophils. These variables correlate with

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FEV1, airway hyperresponsiveness, and asthmatic symptoms [10]. The number of interleukin-5-producing cells in BAL fluid also correlates with FEV1, airway hyperresponsiveness, and asthmatic symptoms. Infiltration of activated Th2 cells is marked in the airway tissue and correlates with the number of eosinophils, the severity of asthma, and the degree of airway hyperresponsiveness [11, 19]. This is because CD4 + Th2 cells present in the airway mucosa of patients with asthma up-regulate the messenger RNA expression of interleukin-3, GM-CSF, and interleukin-5. The production of these cytokines may promote the activation of eosinophils.

Neutrophils Activated neutrophils produce and release various mediators (platelet-activating factors, leukotriene B4, interleukin-8, TNF-α, elastase, collagenase, protease, superoxide, and nitric oxide) that participate in mechanisms underlying acute exacerbations of asthma, airway inflammation, airway remodeling, and airway hyperresponsiveness. Neutrophil elastase has a particularly important role as a potent destroyer of tissues. Autopsy studies have examined inflammatory cells infiltrating airway tissue in patients who died of asthma. Neutrophils were the most common cells in patients who died within 1 h after the onset of an asthma attack, whereas eosinophils were most common in those who died 2 h or more after an attack. These findings suggest that neutrophils are involved in acute exacerbations of asthma. Neutrophils may be also related to respiratory syncytial virus, causing one type of airway viral infection associated with the onset and exacerbation of asthma. Wenzel et al. found increased numbers of neutrophils in the airway mucosa of patients with severe steroid-dependent asthma [20]. Lamblin et al. reported increased levels of interleukin-8, neutrophils, and elastase in BAL fluid during the initial phase of repeated asthma attacks in patients with mild asthma [21]. Infants who had prolonged wheezing without a diagnosis of asthma were found to have increased numbers of nonspecific cells and neutrophils in BAL fluid, as compared with the number of eosinophils. These findings suggest that the presence of neutrophilic airway inflammation may be involved in the pathogenesis of asthma. However, many questions remain unanswered. Further studies are therefore required.

Peripheral Airway Inflammation Recently, attention has focused on evidence that peripheral airway inflammation is associated with the pathogenesis of asthma. Hamid et al. compared the numbers of inflammatory cells between the central and the peripheral airways in resected lung tissue from patients with asthma and lung cancer. They found increased numbers of inflammatory cells such as T lymphocytes, MBP, EG2-positive eosinophils, and mast cells at both sites. At least the number of activated eosinophils (EG2 cells) was

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significantly higher in the peripheral airways than in the central airways. However, the distribution of eosinophils differed considerably between these sites. Higher accumulations of cells were found between the basement membrane and the smooth muscle in the central airways and between the smooth muscle and the outer wall in the peripheral airways [22]. This distribution of cells suggests that inflammation originates from the lumen and involves the airway, extending to the lateral side of the smooth muscle. The expression of Th2 cytokines (interleukin-4 mRNA and interleukin-5 mRNA) is up-regulated not only in the central airways, but also in the peripheral airways. In particular, the expression of interleukin-5 mRNA is significantly up-regulated in the peripheral airways, as compared with the central airways [23]. Kraft et al. performed endobronchial biopsy of the central airways and transbronchial lung biopsy of the alveoli at 4 a.m. and 4 p.m. in patients with nocturnal asthma to analyze the numbers of inflammatory cells in tissues. The number of eosinophils in the alveoli obtained at 4 a.m. was significantly higher than that at 4 p.m. [24]. In patients with nocturnal asthma, the numbers of eosinophils and CD4 + T lymphocytes in the alveoli obtained at 4 a.m. correlated with decreased lung function [25]. Balzar et al. performed endobronchial biopsy of the central airways and transbronchial lung biopsy of the peripheral airways in patients with severe asthma and found that alveolar inflammation in the peripheral airways was severer than that in the central airways [26].

Airway Remodeling We will describe histologic changes, such as desquamation of the airway epithelial cells, epithelial goblet cells, basement-membrane thickening, smooth-muscle hypertrophy, and neovascularization, which are signs of asthmatic airway remodeling in response to allergic inflammation. These findings are useful for the assessment of airway inflammation. Recent studies have reported that airway remodeling occurs early after the onset of asthma [27].

Airway Epithelium The airways of patients with asthma are generally characterized by desquamation of bronchial epithelial cells and expansion of epithelial cellular spaces. In fact, epithelial fragility is a feature of asthma [7, 17] and correlates with the degree of airway hyperresponsiveness [28]. Bronchial epithelial cells secrete various types of cytokines, growth factors, and chemokines and may actively contribute to airway inflammation, instead of acting only as a barrier. Desquamated epithelial cells are mainly ciliated columnar epithelial cells. The epithelium is constantly repaired by the proliferation and migration of basal cells

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existing on the surface of the basement membrane. In patients with moderate to severe asthma, airway secretions are increased. In healthy individuals, mucin-type glycoproteins are thought to be produced mainly by the submucosal glands. In the presence of chronic airway inflammation, goblet cells are also believed to participate in the production of mucin-type glycoproteins. Asthma is characterized by marked goblet-cell hyperplasia in the lung. The major function of goblet cells, a type of mucous cell, is the secretion of mucin. Even in patients with mild to moderate asthma, abnormal mucin-gene expression and goblet-cell hyperplasia are found in airway tissues [29]. Expression of mucin genes (particularly MUC5AC) in airway secretions is induced by growth factors, leukotrienes, platelet-activating factors, oxidative stress, and elastase. In particular, Th2 cytokines such as interleukin-4, 9, and 13 cause goblet-cell hyperplasia in airway epithelium. The airways of patients with asthma show up-regulated expression of epidermal growth factor receptors [30] and calcium-activated chloride channels [31], the distribution of which is consistent with that of goblet cells. Therefore, epidermal growth factor receptors and calcium-activated chloride channels have been reported to have important roles in mucin production. Recently, amphiregulin, produced by human mast cells in response to FcεRI cross-linking stimulation, has been found to up-regulate the expression of mucin mRNA. In patients with asthma, 30–50% of mast cells in the airway mucosa express amphiregulin. In healthy subjects, no amphiregulin-positive cells are present in the airway mucosa. The number of amphiregulin-positive mast cells correlates with the degree of goblet-cell hyperplasia. Mast cells participate in mucin production [32].

Thickening of the Basement Membrane (Subepithelial Fibrosis) The basement membrane of the human bronchial mucosa consists of the basal lamina in the shallow layer and the lamina reticularis in the deep layer. Thickening of the basement membrane on light microscopy has been confirmed to be caused by thickening of the deeper lamina reticularis on electron microscopy. Deposition of collagens (type III or V), fibronectin, proteoglycans, and tenacin is found at sites of basement-membrane thickening, a phenomenon better termed “subepithelial basement-membrane fibrosis.” The thickness of collagen is directly proportional to the number of fibroblasts in the mucosa [33]. Fibroblasts may thus participate in the synthesis of connective tissue. Metalloproteinases are degrading enzymes of extracellular matrix containing excessive deposits of collagens and elastin. Lack of a balance between metalloproteinases and tissue inhibitors of metalloproteinases may lead to fibrosis. TGF-β is a fibroblast activator that is mainly produced by eosinophils. The number of TGFβ-positive cells correlates with the number of fibroblasts [34]. Recent studies have shown that the expression of Smad2, a transcription factor involved in TGF-β signaling, is up-regulated in the airway mucosa of patients with asthma. The degree of Smad2 expression correlates with thickening of the basement

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membrane, airway hyperresponsiveness, and clinical severity of disease. Conversely, the expression of Smad7, an inhibitory signal transmitter, is down-regulated in the airway mucosa of patients with asthma [35, 36]. As for the relations of basementmembrane thickening to lung function and clinical symptoms, studies contrasting the concentration of histamine that lowers the FEV1 by 20% (provocative concentration, PC20), base-line FEV1, and the duration of disease reported no significant correlations among these variables [37]. Another study divided patients with asthma into three groups according to disease severity (mild, moderate, and severe) and assessed the deposition rate of collagen type III in the tissue below the basement membrane as an index of airway remodeling. No significant differences were found among these three groups [38]. The deposition rate of collagen type III also did not correlate significantly with the duration of disease. Jeffery et al. [17] examined bronchial tissue by light microscopy and investigated the relation between basement-membrane thickness and the PC20 of methacholine. Hypersensitivity to methacholine was associated with a thicker basement membrane, and the degree of airway epithelial desquamation significantly correlated with hypersensitivity. Chetta et al. [39] reported that the thickness of subepithelial basement membrane negatively correlated with the base-line FEV1, positively correlated with the diurnal variation of peak expiratory flow, and negatively correlated with the P20 of methacholine. They also found that the thickness of the basement membrane increased in parallel with the severity of asthma. However, the duration of asthma was unrelated to the degree of basementmembrane thickening. In our previous study [34], the thickness of the subepithelial basement membrane positively correlated with asthmatic symptoms and negatively correlated with airway hyperresponsiveness. However, there were no correlations among the duration of disease, FEV1 at base line, or the diurnal variation of peak expiratory flow. These findings suggest that thickening of the basement membrane due to airway inflammation may occur even in the early phase of asthma.

Hypertrophy and Hyperplasia of Airway Smooth Muscle As compared with healthy subjects, the amount of bronchial smooth muscle is significantly increased in patients with asthma. Smooth-muscle spasms can cause severe airway obstruction. Increased smooth muscle can be caused by hypertrophy and hyperplasia. Studies examining preferential sites in the airways for the development of hypertrophy or hyperplasia of bronchial smooth muscle showed two types of thickening: thickening of smooth muscle only in large-caliber central airways and thickening of the entire airway, including both the central and peripheral airways. The former type is associated with an increased number of smooth-muscle cells, unaccompanied by hypertrophy. The latter type is characterized mainly by hypertrophy, particularly marked in the bronchioles [40]. Proliferation of bronchial smooth muscle in patients with asthma is thought to involve growth factors such as epidermal growth factor and its receptors [41], platelet-derived growth factor, fibroblast growth factor, and insulin-like growth factors, as well as leukotriene D4 (LTD4), a chemical mediator.

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It is noteworthy that in a recent study by Brightling et al. mast cells, not present in the bronchial smooth muscle of healthy subjects, diffusely infiltrated the smooth muscle of patients with asthma [42]. Tryptase, a mediator derived from mast cells, may activate protein-activated receptor 2, leading to the proliferation of bronchial smooth-muscle cells. Smooth-muscle cells are constantly in contact with mast cells, inducing fibrosis and smooth-muscle remodeling. Bronchial smooth-muscle cells from patients with asthma have higher proliferative activity in vivo than those from healthy subjects. A recent study by Benayoun et al. [43] showed that bronchial smooth muscle obtained by biopsy is significantly thicker in patients with asthma than in healthy subjects.

Neovascularization As for vascular remodeling in asthma, some studies reported no increase in airway blood vessels in asthma. However, Kuwano et al. [44] reported that neovascularization occurs in the airways of patients with asthma, with marked dilation and increased numbers of blood vessels in autopsy specimens of the lung obtained from patients with fatal asthma attacks. A recent study reported an increased number and caliber of submucosal vessels in bronchial-biopsy specimens obtained from patients with asthma [45]. The number of vessels in the airways of patients with asthma correlates with the number of mast cells. Mast cells secrete various factors that promote neovascularization, suggesting that these cells contribute to neovascularization in the airway wall of patients with asthma. The relative area of the airway mucosa occupied by blood vessels inversely correlates with airway hyperresponsiveness and FEV1 [46]. Increased numbers of blood vessels may be related to the severity of submucosal edema associated with inflammation and contribute to airway obstruction. Factors participating in neovascularization in the airways of patients with asthma include vascular endothelial growth factor (VEGF), fibroblast growth factor, angiogenin, and platelet-derived growth factor [47]. In particular, the expression of VEGF mRNA and VEGF receptors (flt-1 and flk-1) is up-regulated in the airways of patients with asthma. The number of VEGF-positive cells significantly correlates with the relative area of the airway mucosa occupied by blood vessels.

Conclusions The underlying pathogenesis of asthma and COPD is chronic airway inflammation, associated with various types of inflammatory cells. Mediators and cytokines form complex networks, acting to promote and sustain airway inflammation and leading to irreversible changes of airway tissue. Bronchoscopic examination with BAL and endobronchial biopsy are useful procedures that are essential for a better

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understanding of the local inflammatory status of the airways. Limitations of these procedures include their invasive nature, problems associated with the dilution of BAL fluid, inability to obtain bronchial-biopsy specimens from deep layers of the airways, and the provision of only snapshots reflecting changes at the time of examination.

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17. Jeffery PK, Wardlaw AJ, Nelson FC, et al.: Bron-chial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 140: 1745–1753, 1989 18. Robinson DS, Hamid Q, Ying S, et al.: Predominant Th2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 326: 298–304, 1992 19. Robinson DS, Bentley AM, Hartnell A, et al.: Activated memory T helper cells in bronchoalveolar lavage fiuid from patients with atopic asthma: relation to asthma symptoms, lung function, and bronchial responsiveness. Thorax 48: 26–32, 1993 20. Wenzel SE, Szefler S, Leung DYM, et al.: Bronchoscopic evaluation of severe asthma. Am J Respir Crit Care Med 156: 737–743, 1997 21. Lamblin A, Gosset P, Tillie-Leblond I, et al.: Bronchial neutrophilia in patients with noninfectious status asthmatics. Am J Respir Crit Care Med 157: 394–402, 1998 22. Hamid Q, Song Y, Kotsimbos T, et al.: Inflammation of small airways in asthma. J Allergy Clin Immunol 100: 44–51, 1997 23. Minshall EM, Hogg JC, Hamid QA: Cytokine mRNA expression in asthma is not restricted to the large airways. J Allergy Clin Immunol 101: 386–390, 1998 24. Kraft M, Djukanovic R, Wilson S, et al.: Alveolar tissue inflammation in asthma. Am J Respir Crit Care Med 154: 1505–1510, 1996 25. Kraft M, Martin RJ, Wilson S, et al.: Lymphocyte and eosinophil influx into alveolar tissue in noctur-nal asthma. Am J Respir Crit Care Med 159: 228–234, 1999 26. Balzar S, Wenzel SE, Chu HW: Transbronchial biopsy as a tool to evaluate small airways in asthma. Eur Respir J 20: 254–259, 2002 27. Cokugras H, Akcakaya N, Seckin I, et al.: Ultra-structural examination of bronchial biopsy specimens from children with moderate asthma. Thorax 56: 25–29, 2001 28. Laitinen LA, Heino M, Kava T, et al.: Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 131: 599–606, 1985 29. Ordonez CL, Khashayar R, Wong HH, et al.: Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am J Respir Crit Care Med 163: 517–523, 2001 30. Takeyama K, Fahy JV, Nadel JA, et al.: Relationship of epidermal growth factor receptors to goblet cell production in human bronchi. Am J Respir Crit Care Med 163: 511–516, 2001 31. Hoshino M, Morita S, Iwashita H, et al.: Increased expression of the human Ca’+-activated CI- channel I (CaCC1) gene in the asthmatic airway. Am J Respir Crit Care Med 165: 1132– 1136, 2002 32. Okumura S, Sagara H, Fukuda T, et al.: FceRI-mediated amphiregulin production by human mast cells increases mucin gene expression in epithelial cells. J Allergy Clin Immunol 115: 272–279, 2005 33. Brewster CEP, Howarth PH, Djukanovic R, et al.: Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 3: 507–511, 1990 34. Hoshino M, Nakamura Y, Sim JJ, et al.: Expression of growth factors and remodelling of the airway wall in bronchial asthma. Thorax 53: 21–27, 1998 35. Sagara H, Okada T, Okumura K, et al.: Activation of TGF-fi/Smad 2 signaling is associated with airway remodeling in asthma. J Allergy Clin Immunol 110: 249–254, 2002 36. Nakao A, Sagara H, Setoguchi Y, et al.: Expression of Smad 7 in bronchial epithelial cells is inversely correlated to basement membrane thickness and airway hyperresponsiveness in patients with asthma. J Allergy Clin Immunol 110: 873–878, 2002 37. Roche WR, Beasley R, Williams JH, et al.: Sub-epithelial fibrosis in the bronchi of asthmatics. Lancet I: 520–524, 1989 38. Chu HW, Halliday JL, Martin RJ, et al.: Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease. Am J Respir Crit Care Med 158: 1936–1944, 1998 39. Chetta A, Foresi A, Del Donno M, et al.: Bronchial responsiveness to distilled water and methacholine and its relationship to inflammation and remodeling of the airways in asthma. Am J Respir Crit Care Med 153: 910–917, 1996

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Exhaled NO in Asthma D. Robin Taylor

Introduction Until recently, the diagnosis of asthma and ongoing assessment of asthma control have been based largely on clinical parameters. Objective evidence to support clinical decision making has been obtained by measuring either airflow obstruction and its variability, or airway hyperresponsiveness (AHR). Measurements such as serial peak flows and spirometry, and, in some centres, bronchial challenges with methacholine or histamine are employed. However, these tests provide only a limited perspective on the asthma phenotype and its current control status. They focus largely on the functional consequences of the underlying inflammatory process, which characterizes asthma, rather than the pathology itself. Perhaps this emphasis has arisen because of the ease of availability of physiological tests rather than because they are necessarily the most helpful. It is now clear that correlations between airway inflammation and symptoms, airflow obstruction and airway hyperresponsiveness are weak [1]. Each comprises an independent domain of the ‘asthma syndrome’ [2] and there is a need for each to be considered if a thorough assessment of asthma is to be undertaken. Further, it is increasingly recognized that the ‘asthma syndrome’ comprises not just one but several different inflammatory subtypes [3], with differing phenotypic features including differing responsiveness to anti-inflammatory treatment [4]. Against this background, the ability to assess airways pathology, either directly or indirectly, would be a major advance. To date, however, this has not been possible except in research centres. The cost–benefit ratio for bronchial biopsy procedures may be acceptable in the diagnosis of endobronchial tumour, but clearly this is not the case for the common airways diseases such as asthma! There is a need for a simple, inexpensive and easily available alternative. On the face of it, induced sputum analysis ought to fit the bill. This technique, pioneered by Hargreave, has been standardized [5] and provides very useful information about the pathology

D.R. Taylor () Dunedin School of Medicine, University of Otago, P.O. Box 913, Dunedin, New Zealand e-mail: [email protected]

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of inflammatory airways disease in patients who present with non-specific cough, wheeze and/or shortness of breath. Although it may be regarded as the gold standard for such assessment, sputum induction and analysis requires considerable expertise in order to obtain reproducible results, is relatively time-consuming, and the results are not immediately available. For this reason the hunt for a suitable biomarker has continued. Candidates include exhaled breath condensate, serum eosinophilic cationic protein (ECP), and – the topic of this chapter – exhaled nitric oxide (NO).

Exhaled Nitric Oxide: What Are We Measuring? The production of NO in the airways is complex and its pathological significance in asthma is unclear [6]. Both constitutive and inducible NO synthases (cNOS and iNOS) are found in airway and lung tissues, although there is evidence that in disease states such as asthma it is the inducible isoform (iNOS) that is upregulated [7] resulting in increased exhaled levels. However, NO production in the airways occurs in health as well as disease, i.e. NO levels are not zero in healthy subjects. Both genetic and environmental factors influence NO production, and a recent twin study indicates that if anything, genetic factors are predominant [8]. A number of important demographic and anthropometric factors affect the fraction of nitric oxide in exhaled air (FENO) [9]. Levels increase with increasing age throughout childhood and even possibly in adults [10, 11]. Perhaps most importantly, the majority of studies indicate that females have lower levels than males [9, 12–14], with the magnitude of the difference being in the range of 20–40%. This is likely to be clinically relevant. After adjusting for sex, height and weight appear not to be significant factors. Other common variables, which require to be taken into account include atopy [15–17], a history of rhinitis [18] and current cigarette smoking [19, 20]. The magnitude of effect for each of these and other factors is shown in Table 1. Even after taking these factors into account, it is by no means clear that the increase in NO production seen in asthma results directly from airway inflammation [6]. It may be that NO production is an epiphenomenon. However, the association between NO levels in exhaled air and specifically eosinophilic airway inflammation

Table 1 Effect of different demographic and other factors on FENO in a population of 895 members of the Dunedin Multidisciplinary Health and Development Cohort (unpublished data from the author) Factor Percentage difference in FENO compared to controls Gender Smoking Atopy Current rhinitis Current asthma

30% less in females 35% less in current smokers 62% greater in atopic subjects 31% greater 43% greater

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is a consistent and reproducible finding [21, 22]. In contrast, there appears to be no relationship between FENO and other inflammatory cell types, e.g. neutrophils. This distinction is of key importance in the interpretation and application of FENO in the clinical setting. Correlations between induced sputum eosinophil counts and FENO are relatively high, averaging between 70% and 80% [21–23]. However, this implies that the positive and negative predictive values for FENO as a marker for eosinophilic airway inflammation are significantly less than 100%. In the study by Berry et al. [23], the optimum cut-point for FENO as a predictor of a clinically significant increase in sputum eosinophils (>3%) was 19 ppb (corrected for an expiratory flow rate of 50 ml/s), yielding a sensitivity and specificity of 71% and 72%, respectively. An important corollary of these findings is that in the 15% or so of patients with FENO lying above the so-called normal range (up to 33 ppb [24]), the sputum eosinophil count will be normal despite having an elevated FENO. Correlations between FENO and AHR are also high [22, 25–27]. Based on these findings, it has been suggested that FENO measurements might be used as a substitute marker for AHR. However, this concept is misleading because AHR is itself nonspecific. In studies where the majority of subjects have AHR secondary to eosinophilic airway inflammation, then the relationship with FENO will be strong. However, in more heterogeneous populations comprising both eosinophilic and non-eosinophilic asthma phenotypes, the relationship between FENO and AHR is much weaker. This is illustrated in the results of a recent study, which reported that the performance characteristics of FENO as a predictor of AHR to methacholine were much weaker than as a predictor for doctor-diagnosed asthma with negative predictive values of 71% and 85%, respectively [28].

FENO in the Diagnosis of Asthma Early studies reported that FENO levels are increased in asthma but not in other respiratory conditions, at that time drawing attention to the potential role of FENO in differentiating asthma from other pathologies [29–31]. But has this potential been realized? In cross-sectional studies comparing asthma patients with healthy controls, the discriminant properties for FENO were found to be good but by no means perfect. Deykin et al. [32] reported that FENO distinguished 34 steroid-naive asthmatics from 28 controls with a sensitivity of 72.2% and a specificity of 70.6%. In another study comprising 96 pre-school children with asthma symptoms and 62 age-matched controls, FENO was superior to lung function testing for predicting steroid-naive asthma (n = 21), with a sensitivity of 86% and specificity of 92% [33]. In the steroid-treated asthmatics (n = 29), FENO levels were similar to those of healthy controls emphasizing the importance of obtaining FENO measurements prior to any treatment with inhaled corticosteroids. FENO measurements provide similar diagnostic accuracy whether the ‘gold standard’ for asthma is based on bronchodilator reversibility or AHR, or is exclusively ‘doctor-diagnosed’.

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Similar performance characteristics have been reported where the pre-test probabilities were somewhat higher, i.e. the populations being studied all had active respiratory symptoms. In the study by Dupont et al., 240 non-smoking steroidnaive patients with current symptoms were enrolled, of whom 160 (67%) were diagnosed with asthma using conventional criteria. FENO was highly predictive of asthma with a sensitivity and a specificity of 85% and 90%, respectively [34]. Smith et al. [35] evaluated 47 patients presenting with nonspecific respiratory symptoms of whom 17 had asthma. In that study, comparisons were made between FENO and a comprehensive range of conventional pulmonary function tests, including diurnal peak-flow variation, spirometry, the response to oral corticosteroid challenge and induced sputum cell analysis. Sensitivities for the ‘physiological’ tests were exceedingly low, ranging from 0% to 47% – much lower than for FENO (88%) and sputum eosinophils (86%). The overall diagnostic accuracy was greatest for FENO and sputum eosinophils. Interestingly the combination of FENO (cut-point 33 ppb) and spirometry (cut-point for FEV1 of 80% predicted) yielded a sensitivity of 94% and specificity of 93%. It is important to understand that the optimum cut-point for distinguishing asthma from non-asthma derived from each of these published studies depends on pretest probabilities, and that comparisons between studies can only be made where the methods for FENO measurement complies with recommended guidelines [36]. Taking these considerations into account, there is a broad consensus that a diagnostic cut-point of 20–25 ppb in adults appears to be optimum [34, 35]. However, because the ‘normal range’ for FENO is markedly skewed to the right [11], the interpretation of FENO values in the range of 20–35 ppb ought to be cautious and made with strict regard to the history of respiratory symptoms. Conversely, a patient may indeed have asthma as defined by the GINA guidelines [37], but have a low FENO of less than 25 ppb, i.e. the test is falsely negative. This is entirely possible, given that not all asthma is eosinophilic. Indeed, patients with non-eosinophilic asthma (between 15% and 30% in most populations of patients with asthma [38]) may be inappropriately diagnosed if FENO is relied upon as a diagnostic test. There is one other catch: sometimes patients have transiently elevated FENO levels in association with a viral respiratory tract infection [39, 40]. Therefore, the test is best used in the work-up of patients with persistent symptoms of 6 weeks duration or longer. Taken together, it is important to assert that an FENO is not a diagnostic test for asthma as such, even although that role has been actively pursued (and claimed) in many clinical studies. Finally, in older patients presenting with symptoms of airways disease, particularly if they have a prior history of smoking, clinicians may seek to distinguish asthma and chronic obstructive pulmonary disease (COPD), although the relevance of making the distinction is still debated! Here, FENO may have a role but it remains to be more fully established. In the study by Fabbri et al. [41], 46 patients, all with fixed airflow obstruction due to either COPD (n = 27) or asthma (n = 19) were investigated. The two groups had similar degrees of airflow limitation, and there was considerable overlap for most of the other physiological tests, making them non-discriminatory. However, both FENO and sputum eosinophils were the most reliable measures for

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distinguishing asthma from COPD. Thus, in this setting, FENO measurements offer the opportunity to obtain insights into the underlying airway pathology, i.e. the presence of an eosinophilic component to the airways inflammation, which might otherwise not be possible.

FENO in the Diagnosis of Steroid Responsiveness Arguably, establishing whether a patient has the potential to respond to corticosteroid therapy is more important than applying a diagnostic label. The latter is only helpful if indeed it provides the basis for anticipating either the natural history of the disease or the response to treatment. There is consistent and compelling evidence that eosinophilic airway inflammation is steroid-responsive – in contrast to other inflammatory subtypes. The relationship between blood eosinophilia and steroid-responsiveness has been established for many years [42]. In airways disease, an increased sputum eosinophil count indicates a beneficial short- and long-term response to corticosteroid therapy irrespective of the clinical context in which it is present [4, 43, 44]. More specifically, Meijer et al. have demonstrated in 120 patients with unstable asthma that, following a trial of inhaled or oral corticosteroid, the improvement in FEV1 was highly correlated with baseline sputum eosinophils [45]. Conversely, in patients who do not demonstrate sputum eosinophilia, any improvement in symptoms or lung function with inhaled steroid therapy is limited [46]. Against this background, it is plausible that as a marker for airway eosinophilia, FENO might also serve as a marker for potential steroid-responsiveness in patients with airways symptoms. Identifying such patients is a frequent challenge particularly in primary care practice, where clinicians often prescribe inhaled corticosteroids empirically. Information to guide the appropriateness of using inhaled corticosteroids would be very useful. A number of studies have addressed this issue. In an open-label, parallel group study by Szefler et al. [47], dose–response relationships for inhaled fluticasone and beclomethasone were evaluated in 30 patients with ‘asthma’. ‘Good responders’ (characterized by an improvement of 15% or greater in FEV1) were found to be associated with a high FENO (median of 17.6 ppb), and this contrasted with the ‘poor responders’ (<5% improvement in FEV1) whose FENO was 11.1 ppb (p = 0.002). Little et al. have also shown that the benefits of steroid therapy in poorly controlled asthma are most likely to be achieved in patients with the highest FENO levels [48]. In a more detailed study, Smith et al. compared the predictive accuracy of FENO measurements with other baseline tests: lung function (FEV1 percentage predicted), FEV1 bronchodilator response, peak-flow variation (amplitude percentage mean) and bronchial challenge testing (methacholine PD20) as predictors of steroidresponsiveness in a placebo-controlled crossover study of inhaled fluticasone in 52 patients with nonspecific respiratory symptoms [49]. Irrespective of how steroid response was measured (symptoms, lung function [peak flows and FEV1],

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or AHR to adenosine monophosphate), the performance characteristics for FENO were better than for any of the other predictors. More importantly, the study also demonstrated that steroid response was most likely to occur in subjects whose FENO at baseline was greater than 47 ppb (see Fig. 1). Further analysis of the outcomes of this study also shed interesting light on the relationship between the clinical diagnosis (of asthma) and the response to steroid: it is very weak. Patients were stratified into two groups based on the diagnosis of ‘asthma’ versus ‘not asthma’. This showed that even in patients who did not fulfil criteria for a diagnosis of asthma (typical symptoms and either a positive bronchodilator response OR a positive methacholine challenge test) a positive response to steroid might still occur, and again this was best predicted by the baseline FENO (see Fig. 2). This finding confirms that identifying the characteristics of underlying airway inflammation is more informative than a diagnostic label, at least in terms of predicting the response to anti-inflammatory therapy in patients with respiratory symptoms. In this regard FENO is a useful biomarker, and a once-off measurement prior to commencing inhaled drug therapy is very informative. In patients whose FENO was high, there was a negative predictive value for steroid response of nearly 90%, i.e. the absence of an FENO indicates that a response to steroid is unlikely.

Composite symptom score

Peak flow (percent change) 25 20 15 10 5 0 –5

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Fig. 1 Steroid responsiveness in relation to baseline FENO measurements in 52 patients with nonspecific respiratory symptoms. Mean (SEM) changes from baseline in (a) forced expiratory volume in one second (FEV1), (b) morning peak flow over last 7 days of treatment, (c) composite symptom score, and (d) provocative concentration of adenosine monophosphate causing a 20% fall in FEV1 (PC20AMP) following treatment with inhaled fluticasone 500 µg/day stratified by tertiles (Reproduced from Smith et al. [49]. With permission of the publishers)

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D FEV1

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0 All subjects (n = 52)

“Asthma” (n = 19)

“Not asthma” (n = 33)

Fig. 2 Percentage change in FEV1 (mean, SEM) in relation to baseline FENO stratified by tertiles following a trial of inhaled fluticasone 500 µg/day in 52 patients presenting with nonspecific respiratory symptoms. The population was then further stratified by post hoc diagnosis: ‘asthma’ versus ‘non-asthma’. The results show that a positive response to inhaled steroid was obtained in patients with and without ‘asthma’, and that this was strongly related to baseline FENO (Data are taken from Smith et al. [49] but have not been published previously)

Monitoring Asthma: Can FENO Be Used to Predict Loss of Control or Exacerbations? Persistent asthma is characterized by variability in the frequency and severity of symptoms, and optimizing treatment involves, among other things, adjusting antiinflammatory therapy in order to achieve good current control as well as to minimize future risk of poor control. The question arises as to whether FENO may be used to assist in this strategy. On the face of it, such an objective would seem to be very plausible. FENO levels increase during asthma exacerbations and are lower in stable asthma [50–52]. However, despite this, evidence to support the use of inflammatory biomarkers for predicting future asthma control is somewhat contradictory. For sputum eosinophil counts, studies show that the intensity of eosinophilic airway inflammation is dissociated from symptoms/disordered airway function in patients with asthma [2, 53]. On the other hand, in steroid reduction studies, a raised sputum eosinophil count is predictive of the future development of an exacerbation [54, 55], and when a management strategy aimed at normalizing the sputum eosinophil count is used, there is a reduction of up to 60% in the number of severe asthma exacerbations requiring treatment with oral corticosteroids [56, 57]. For FENO, the same dissociation between FENO levels and asthma severity has been observed [58, 59], although this may be because patients with non-eosinophilic asthma have been included in such analyses. In a study designed to address the

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prognostic value of both sputum eosinophils and FENO, Jatakanon et al. reduced the dose of inhaled budesonide to 200 µg/day in 15 patients and reviewed them twice weekly for 8 weeks [54]. In those who went on to develop an exacerbation, the mean FENO (measured at a flow rate of 100 ml/s) increased from 11.3 ppb (5.6, 17.1) to 34.2.1 ppb (20.7, 47.8), whereas in those who did not experience an exacerbation FENO increased minimally from 9.1 ppb (6.0, 12.2) to 13.1 ppb (6.1, 20.1). Correlations between the changes in sputum eosinophils and changes in FENO against changes in morning peak flows and FEV1 indicated that sputum eosinophils were more reliable than FENO. Predictive values were not calculated. In a larger study by Jones et al. inhaled steroid was withdrawn in 78 patients, and of these 60 went on to develop loss of control [60]. The optimum positive predictive values for absolute as well as changes in FENO levels were similar ranging from 80% to 85% depending on the cut-point used. For sputum eosinophils a value of greater than 4% provided a similar positive predictive value of 84% for future loss of control. The authors concluded that an absolute value of greater than 35 ppb (at a flow rate of 50 ml/s) or a change of greater than 60% from baseline identified patients with ongoing airway inflammation and the potential for breakthrough symptoms. More recently, Gelb et al. prospectively evaluated the predictive values of FENO for an asthma exacerbation over a 3-year interval [61]. Each patient was taking regular salmeterol/fluticasone combination therapy. The positive and negative predictive values at a cut-point for FENO of 28 ppb were 77% and 87%, respectively, with a relative risk of 3.4 (95% CI: 1.3–9.1; p = 0.007). This outcome was independent of baseline FEV1. In patients with a low FENO (<28 ppb) and a normal/high FEV1 (defined as >76% predicted), there was a 0% probability of exacerbation on regular treatment during follow-up. More detailed analysis of data from the study by Jones et al. [60] reveals that the confidence intervals for change in FENO after withdrawal of inhaled steroid and the development of loss of control are very wide. The mean increase in FENO was +25 ppb, but with a range of −10 to +141 ppb. In the study by Beck-Ripp et al. [62] the mean change in FENO between stability and loss of control was +16 ppb. Thus, the use of group mean data to predict individual outcomes may be inappropriate. Anecdotally, it is more important to measure serial FENO levels in individual patients, and then establish the relationship between changes in FENO and the level of asthma control. The technology that will permit this to be done inexpensively and reliably is still being developed. Until it is more readily available, the scope for using FENO to predict future loss of control will be limited to selected patients with difficult asthma, usually attending secondary care facilities, in whom trends in serial FENO measurements are matched against treatment levels and control status.

Monitoring Asthma: Can FENO Be Used to Guide Inhaled Corticosteroid Requirements? There are a number of cogent reasons why the use of a biomarker such as FENO to guide anti-inflammatory treatment in asthma might be helpful. Firstly, the aim of anti-inflammatory treatment is to minimize inflammation, it is therefore

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appropriate that the primary end point should be an objective measurement of airway inflammation! To date this simple logic has not been applied in the management of asthma because it has not been possible to do so. Secondly, given that FENO levels fall when inhaled corticosteroid treatment is commenced [50], and the reduction occurs in a dose-dependent manner [63, 64], the arguments for using FENO as a biomarker in this setting are even more substantial. Thirdly, conventional guidelines advocate that inhaled corticosteroid therapy should be adjusted in response to patients’ reports of their asthma symptoms. But this assumes that the symptoms are indeed due to asthma, and in particular, that they are due to uncontrolled airway inflammation. This may not always be the case. Studies show that, in general, correlations between symptoms and airway inflammation are very weak [2]. Furthermore, other clinical problems such as anxiety-hyperventilation or gastro-oesophageal reflux may complicate asthma and give rise to overlapping symptoms, which may be difficult to distinguish from the asthma itself. Clearly, they will not respond to increasing doses of ICS. Finally, it is now clear that a significant proportion of patients have non-eosinophilic asthma, and as such it is less likely to be steroid-responsive. The use of a biomarker, which indirectly distinguishes the inflammatory phenotype would enable the clinician to avoid inappropriate increases in the dose of ICS. Two proof of concept studies preceded those which were specifically designed to investigate the role of FENO to guide ICS therapy. The first, by Sont et al. (the so-called AMPUL study [65]), showed that by titrating the dose of ICS against symptoms and AHR, a net reduction of 1.8-fold in asthma exacerbation rates could be achieved, although at the cost of increased ICS dose (median increase in dose of inhaled steroids of 400 µg/day). The second, more relevant study by Green et al. reported that by using sputum eosinophil counts to guide ICS dose adjustments, the frequency of severe asthma exacerbations was reduced from 2.95 per patient per annum in the group whose ICS dose adjustments were based on a conventional symptoms-based algorithm, to 0.95 per patient per annum in the sputum strategy group (p < 0.01) [57]. At least five studies in which FENO has been used to guide ICS treatment have now been conducted, of which three have been reported at the time of writing [66–68]. The results provide inconclusive support for using FENO in this setting than for using sputum eosinophils. In the study by Pijnenburg et al. [66], comprising a total of 85 children (39 in the FENO group), the use of a three-tier FENO strategy resulted in a significant reduction in AHR in the strategy group (2.5 doubling doses) compared to the control group (1.1 doubling doses, p = 0.04). There was a reduction in exacerbations from 8 among 39 patients in the FENO group to 18 among 46 patients in the conventional group (NS). However, the study was not powered to assess the impact of the FENO strategy on exacerbations rates. In the study by Shaw et al. [67] there were no significant differences in any of the study outcomes using the FENO strategy, notably the primary end point, exacerbations. However, again, the study was underpowered for this particular end point. For asthma control, as measured by ACQ, [69] there were no significant differences suggesting that even if the study had been larger, the impact of using FENO on exacerbations would not necessarily have been significant. In the study by Smith et al. [68],

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the difference in exacerbation rates achieved using the FENO strategy also failed to reach significance, this time in a study which was powered for this end point. Interestingly, the dose of ICS required was nearly 40% less in the FENO group, and it may be that this is the most clinically important, rather than the most statistically significant study outcome (see Fig. 3). A number of issues need to be considered in the interpretation of these results [70]. Firstly, there is whether the algorithm for ICS adjustment using FENO has two or three tiers. The latter will have two cut-points for FENO, the higher being the level above which the ICS dose is increased (e.g. 35 ppb), the lower being the level below which the ICS dose is reduced (e.g. 25 ppb). For a FENO that lies between these cut-points the dose will remain unchanged. Secondly, there is the choice of cut-points. In the study by Pijnenburg et al. [66] the cut-point for FENO were based on the upper limit of ‘normal’ values for FENO [10], whereas in the study by Smith et al. [68], they were based on values obtained in patients with stable asthma [60]. In the study by Shaw et al. [67], cut-points were chosen in relation to a clinically relevant sputum eosinophil count: 26 ppb corresponded to a sputum eosinophil count of 2% [71]. The issue of cut-points is also important in relation to the algorithm used to adjust ICS dose in the control groups (see Table 2). Thirdly, there is the confounding influence of long-acting agonists (LABAs): intervention with LABA treatment was permitted in the study by Shaw et al. [67] and has the potential to confound the study outcomes when treatment adjustments are based on symptoms rather than those based on FENO, by reducing the exacerbation rates preferentially in the conventionally managed group. In a later study investigating the role of sputum eosinophil counts as a guide to ICS dose adjustment, Jayaram et al. reported that the benefits of using the ‘sputum strategy’ were found in the subgroup of patients with more severe asthma, characterized by a

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Fig. 3 Distribution of final dose of inhaled fluticasone in two groups of patients after back titration of dose using either FENO (FENO group) or symptoms and peak flows (conventional group) as the basis for dose adjustment (Data derived from but not published in [68])

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Table 2 Details from treatment algorithms from three studies in which FENO results were used to guide inhaled corticosteroid adjustment in patients with chronic persistent asthma Author and reference Pijnenburg et al. [66] Smith et al. [68] Shaw et al. [67] Cut point(s) and their derivation/ origin

Treatment decision in relation to cut-points

30 ppb – 2SD above mean for healthy children From [10]

>30 ppb – increase ICS dose <30 ppb and symptomatic – no change <30 ppb and asymptomatic – decrease ICS dose Algorithm for ‘control’ Cumulative symptoms groups and their using validated cut-points if ‘good symptom score control’ criteria cut-point: <14 were met then ICS points = good dose was decreased control

35 ppb – upper limit of 95% CI for stable asthma From [60]

>35 ppb – increase ICS dose <35 ppb – decrease ICS dose

26 and 16 ppb – equivalent to sputum eosinophils counts of 3% and 1%, respectively From [23] >26 ppb – increase ICS dose <16 ppb or <26 ppb on two visits – decrease ICS dose

GINA-based scoring: Juniper ACQ [84] cut-point: all of cut-point: <1.57 five criteria for for good control asthma control had to be met for good control

high ICS maintenance dose requirement [56]. It may be that for FENO the same is true, i.e. the use of the biomarker achieves significant outcomes only in patients whose steroid requirements are high or allegedly high. The studies to date do not address this issue, and the question will only be answered in further studies, which focus on this targeted subgroup of patients. In an interesting but unpublished analysis of original data [68], the use of an FENO-based management strategy permitted a reduction in ICS dose to zero in four out of five patients with a high Nijmegen score. This score identifies patients with symptoms of hyperventilation by means of a questionnaire [72]. Clearly, such symptoms may occur with asthma, which is associated with anxiety overlay, or alternatively, they may occur with anxiety presenting as pseudo-asthma and which, in the absence of knowing the patient’s inflammatory phenotype, is treated as such. In patients with a high Nijmegen score who were treated according to a symptoms-based algorithm the ICS dose requirements increased (see Fig. 4).

How to Interpret High FENO Levels in Patients with Diagnosed Asthma In the clinical setting, the measurement of FENO with or without spirometry can be very helpful as a guide to interpreting a patient’s current asthma status. The principles are well established, but the numeric definition of ‘high’ remains

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Nijmegen score 50

Nijmegen score 50

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Fig. 4 Individual Nijmegen scores (y-axis) versus optimized dose of inhaled fluticasone (x-axis) in two groups of patients. The first group (FENO group) had their inhaled fluticasone dose optimized by stepwise back titration of ICS dose at monthly intervals based on FENO levels. The second group (conventional group) had their inhaled fluticasone dose optimized by stepwise backtitration based on symptoms and peak flows. A full account of the methods is contained in Smith et al. (68) The trend in the conventional group was significant (p = 0.04), but was non-significant in the FENO group (p = 0.31). A Nijmegen score of 23 or above is considered to be clinically significant for hyperventilation [72]

exceedingly problematic. If ‘high’ is defined as greater than the upper limit of the confidence interval for FENO in patients with stable asthma, then 35–40 ppb appears to be an appropriate cut-point [60]. If ‘high’ is defined as a level above which clinically meaningful events occur, i.e. steroid-responsiveness [49] or relapse following steroid withdrawal [73], then 45–50 ppb appears to be more relevant. As stated previously, the importance of these figures may be superseded if serial measurements in an individual patient demonstrate a significant increase or decrease (60% or greater [60]). It is critical to interpret FENO result in the light of the patient’s symptoms (see Table 3). In an asymptomatic patient with a high FENO, the dose of ICS ought not to be changed as there is a strong likelihood of relapse. Pijnenburg et al. have shown that in children with stable well-controlled asthma, relapse is likely following withdrawal of inhaled steroids in patients with high baseline FENO levels prior to withdrawal, or in those who demonstrate a rise in FENO within 4 weeks of ICS withdrawal. The cut-point predicting relapse was 49 ppb [73]. High FENO levels may persist despite apparently optimum ICS treatment [74]. The most frequent cause for this is poor compliance with ICS therapy, or perhaps poor inhaler technique. In some patients, the inflammatory process is predominantly alveolar rather than in the airways, as judged by increased alveolar NO [71, 75]. This is reflected in a positive response to systemic rather than inhaled corticosteroid therapy [75]. Alternatively, persistently increased FENO levels may be the result of high constitutive NO production, rather than due to ongoing eosinophilic airway

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Table 3 Reasons for high FENO levels (usually greater than 45 ppb) in a patient with diagnosed asthma Asymptomatic Patient has endogenously high FENO, which is outside the ‘normal range’ but is ‘normal’ for them Symptomatic patient has Poor compliance with anti-inflammatory treatment. Dose of ICS uncontrolled does not need to be changed, but steps to improve compliance eosinophilic airway need to be addressed inflammation Poorly controlled asthma. Dose of ICS needs to be increased, or additional therapy needs to be added if very unstable (prednisone, anti-IgE) Truly steroid dependent asthma (rare)

inflammation. Indeed sputum eosinophilia may be absent despite persistently high FENO levels [67]. Often serial measurements of FENO are helpful in determining how to interpret high levels in individual patients, as they can be matched against particular therapeutic interventions.

How to Interpret Low FENO Levels in Patients with Diagnosed Asthma A low FENO is generally accepted to be one of less than 25 ppb in adults. This is based not only on the results of studies in which the optimum cut-point separating asthma from non-asthma has been determined (see earlier section of this chapter), but also on data from the large study by Berry et al. which showed that a sputum eosinophil count of 2% is comparable to a FENO level of 26 ppb [23]. However, again it needs to be emphasized that these data are population-based and need not apply to individual patients. A falsely low FENO result is often obtained in current smokers [19, 20], but the individual effect of cigarette smoking cannot be estimated and for this reason it is doubtful whether any attempt to use FENO measurements in current smokers is worthwhile. Establishing that a patient has low FENO levels is very helpful in the management of (non-smoking) patients with chronic asthma (see Table 4). There are a number of possible interpretations. Firstly, the results may prompt a review of the working diagnosis. The differential diagnosis includes vocal cord dysfunction, anxiety-hyperventilation (primary), chronic obstructive pulmonary disease, cardiac disease, bronchiectasis, and, in children, primary ciliary dyskinesia. However, as stated previously, low levels do not exclude a diagnosis of asthma. Rather they imply that the asthma is not eosinophilic. Low FENO levels may also occur in symptomatic patients who do indeed have atopic (eosinophilic) asthma, but whose symptoms are due to an alternative aetiology. This information is helpful in the management of difficult or severe asthma, which is often multifactorial. Uncontrolled symptoms may be due to the effects of obesity (perhaps made worse by frequent courses of steroids), anxiety-hyperventilation (secondary), gastro-esophageal reflux disease,

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Table 4 Reasons for low FENO levels (usually greater than 25 ppb) in a patient with diagnosed asthma Asymptomatic If the patient has previously had high FENO, then this implies either good control as a result of current treatment OR good control in spite of current treatment. Attempts to reduce ICS dose should be considered depending on the history Symptomatic Non-eosinophilic asthma: in such cases a test for AHR is likely to be positive. A response to ICS therapy is unlikely Eosinophilic asthma, which in itself is well controlled but with complications giving rise to asthma-like symptoms • Rhinosinusitis • Obesity • Gastro-esophageal reflux Alternative diagnosis • Vocal cord dysfunction • Hyperventilation • Chronic obstructive pulmonary disease • Cardiac disease • Bronchiectasis • Primary ciliary dyskinesia (children)

or rhinosinusitis. None of these is associated with elevated FENO, and none will respond to an increase in anti-inflammatory therapy! Thus, a low FENO level may direct the clinician away from managing the asthma per se towards the management of one of its complications. In stable asymptomatic asthmatic patients, low FENO levels raise the question as to whether the dose of ICS steroid may be reduced or even withdrawn altogether. Zacharasiewicz et al. explored this question in a group of 40 children with stable asthma [76]. The dose of ICS was progressively halved at 8-weekly intervals. During the study, 30% of children were able to be weaned off ICS therapy without relapsing, while 38% experienced loss of control according to a priori criteria. The sensitivity and specificity for loss of control were 78.6% and 68.6%, respectively. A FENO of 22 ppb yielded the optimum values for positive and negative predictive values, i.e. low FENO (less than 22 ppb) predicted successful reduction/withdrawal of ICS.

FENO in the Diagnosis of Occupational Asthma The diagnosis of occupational asthma is often difficult, and is dependent on demonstrating differences in symptoms, peak flows, and AHR at work compared to periods of elective absence from work, e.g. holidays or weekends. Not all occupational sensitizers give rise to IgE-mediated eosinophilic airway inflammation, but for those that do, it would be useful to obtain complementary evidence to support a diagnosis of asthma using an appropriate biomarker such as induced sputum or FENO. Identifying changes in induced sputum in relation to occupational exposures is reported to be informative in this regard [77, 78].

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Fig. 5 Percentage change in FEV1, with baseline = 100%, in a sensitized patient after challenged by exposure to a laboratory rat. An early asthmatic reaction occurred with changes in symptoms and lung function at 30–60 min. The patient went on to demonstrate an increase in FENO over the next 24 h indicating the advent of eosinophilic airway inflammation. This was not accompanied by significant changes in lung function

The exact role of FENO in the diagnosis of occupational asthma has yet to be fully explored. There are data to suggest that FENO may have a role. Specific challenge tests resulting in an increase in sputum eosinophils are simultaneously associated with an increase in FENO consistent with the time-course for the late asthmatic reaction [79]. In a controlled study comparing aspirin-sensitive and aspirin-tolerant asthmatic patients, a single dose of aspirin elicited a rise in sputum eosinophils in the sensitized patients, and this was highly correlated with changes in FENO [80]. Cross-sectional studies have explored the role of FENO in the diagnosis of occupational asthma in at-risk populations. [81, 82] However, the potential use of FENO as a sensitive marker of response either to a specific allergen challenge (in which changes in symptoms and lung function may be entirely absent) [83] or with workplace exposure. We have observed a rise in FENO over 24 h in a patient with asthma secondary to laboratory animal allergy following exposure to rat antigen (see Fig. 5). Anecdotally, we have also noted a progressive day-on-day increase in FENO in another patient who was sensitized to guinea pig, rat and mouse antigens during 5 consecutive working days of animal laboratory exposure. These case studies indicate that serial FENO measurements may provide supportive evidence for a diagnosis of occupational asthma where the sensitizing agent of interest is known to evoke an eosinophilic inflammatory response. Further studies are awaited.

Summary Exhaled nitric oxide is by no means a perfect test, but at the present time is the easiest to use biomarker for the assessment of airways inflammation. Its role is as an indirect marker for eosinophilic airway inflammation, although importantly,

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increased eosinophilic inflammation is not the only reason for elevated FENO levels. It is certainly not a diagnostic test for asthma; nor is it a substitute measurement for AHR. In a patient presenting with nonspecific respiratory symptoms who has a high FENO (>45 ppb in an adult), this points helpfully towards the likelihood of a positive response to steroid. Arguably this information is more clinically relevant than merely confirming a diagnostic label. In patients with established asthma, monitoring FENO yields important information, which informs the adjustment or may even allow for safe withdrawal of inhaled steroid therapy. Reference to whether or not the patient is symptomatic is critical to the interpretation of an FENO result. High (>45 ppb in an adult) or progressively increasing FENO levels are associated with uncontrolled eosinophilic airway inflammation, often due to noncompliance with ICS therapy. Low levels (<25 ppb in an adult) suggest that symptoms are due to a cause other than eosinophilic airway inflammation and therefore increasing ICS therapy is inappropriate. Using FENO measurements in individual patients with asthma requires fine-tuning, and interpreting results against those obtained from large population studies ought to be cautious. Serial rather than one-off measurements are recommended.

References 1. Sont JK, Han J, van Krieken JM, et al. Relationship between the inflammatory infiltrate in bronchial biopsy specimens and clinical severity of asthma in patients treated with inhaled steroids. Thorax 1996; 51:496–502. 2. Rosi E, Ronchi MC, Grazzini M, Duranti R, Scano G. Sputum analysis, bronchial hyperresponsiveness, and airway function in asthma: results of a factor analysis. J Allergy Clin Immunol 1999; 103:232–237. 3. Simpson JL, Scott R, Boyle MJ, Gibson PG. Inflammatory subtypes in asthma: assessment and identification using induced sputum. Respirology 2006; 11:54–61. 4. Pavord ID, Brightling CE, Woltmann G, Wardlaw AJ. Non-eosinophilic corticosteroid unresponsive asthma. Lancet 1999; 353:2213–2214. 5. Djukanovic R, Sterk PJ, Fahy JV, Hargreave FE. Standardised methodology of sputum induction and processing. Eur Respir J Suppl 2002; 37:1s–2s. 6. Ricciardolo FL. Multiple roles of nitric oxide in the airways. Thorax. 2003; 58:175–182. 7. Lane C, Knight D, Burgess S, et al. Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath. Thorax 2004; 59:757–760. 8. Lund MB, Kongerud J, Nystad W, Boe J, Harris JR. Genetic and environmental effects on exhaled nitric oxide and airway responsiveness in a population-based sample of twins. Eur Respir J 2006; 1:1. 9. Travers J. MS, Aldington S., Williams M., Shirtcliffe P., Pritchard A., Weatherall M., Beasley R. Reference ranges for exhaled nitric oxide derived from a random community survey of adults. Am J Respir Crit Care Med 2007; 176:238–242. 10. Buchvald F, Baraldi E, Carraro S, et al. Measurements of exhaled nitric oxide in healthy subjects age 4 to 17 years. J Allergy Clin Immunol 2005; 115:1130–1136. 11. Olin AC, Rosengren A, Thelle DS, Lissner L, Bake B, Toren K. Height, age, and atopy are associated with fraction of exhaled nitric oxide in a large adult general population sample. Chest 2006; 130:1319–1325.

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12. Jilma B, Kastner J, Mensik C, et al. Sex differences in concentrations of exhaled nitric oxide and plasma nitrate. Life Sci 1996; 58:469–476. 13. Tsang KW, Ip SK, Leung R, et al. Exhaled nitric oxide: the effects of age, gender and body size. Lung 2001; 179:83–91. 14. Olivieri M, Talamini G, Corradi M, et al. Reference values for exhaled nitric oxide (reveno) study. Respir Res 2006; 7:94. 15. van Amsterdam JG, Janssen NA, de Meer G, et al. The relationship between exhaled nitric oxide and allergic sensitization in a random sample of school children. Clin Exp Allergy 2003; 33:187–191. 16. Jouaville LF, Annesi-Maesano I, Nguyen LT, Bocage AS, Bedu M, Caillaud D. Interrelationships among asthma, atopy, rhinitis and exhaled nitric oxide in a population-based sample of children. Clin Exp Allergy 2003; 33:1506–1511. 17. Gratziou C, Lignos M, Dassiou M, Roussos C. Influence of atopy on exhaled nitric oxide in patients with stable asthma and rhinitis. Eur Respir J 1999; 14:897–901. 18. Henriksen AH, Sue-Chu M, Lingaas Holmen T, Langhammer A, Bjermer L. Exhaled and nasal NO levels in allergic rhinitis: relation to sensitization, pollen season and bronchial hyperresponsiveness. Eur Respir J 1999; 13:301–306. 19. Kharitonov SA, Robbins RA, Yates D, Keatings V, Barnes PJ. Acute and chronic effects of cigarette smoking on exhaled nitric oxide. Am J Respir Crit Care Med 1995; 152:609–612. 20. Persson MG, Zetterstrom O, Agrenius V, Ihre E, Gustafsson LE. Single-breath nitric oxide measurements in asthmatic patients and smokers. Lancet 1994; 343:146–147. 21. Payne DN, Adcock IM, Wilson NM, Oates T, Scallan M, Bush A. Relationship between exhaled nitric oxide and mucosal eosinophilic inflammation in children with difficult asthma, after treatment with oral prednisolone. Am J Respir Crit Care Med 2001; 164:1376–1381. 22. Jatakanon A, Lim S, Kharitonov SA, Chung KF, Barnes PJ. Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma. Thorax 1998; 53:91–95. 23. Berry MA, Shaw DE, Green RH, Brightling CE, Wardlaw AJ, Pavord ID. The use of exhaled nitric oxide concentration to identify eosinophilic airway inflammation: an observational study in adults with asthma. Clin Exp Allergy 2005; 35:1175–1179. 24. Kharitonov SA, Gonio F, Kelly C, Meah S, Barnes PJ. Reproducibility of exhaled nitric oxide measurements in healthy and asthmatic adults and children. Eur Respir J 2003; 21:433–438. 25. Dupont LJ, Rochette F, Demedts MG, Verleden GM. Exhaled nitric oxide correlates with airway hyperresponsiveness in steroid-naive patients with mild asthma. Am J Respir Crit Care Med 1998; 157:894–898. 26. Franklin PJ, Stick SM, Le Souef PN, Ayres JG, Turner SW. Measuring exhaled nitric oxide levels in adults: the importance of atopy and airway responsiveness. Chest 2004; 126:1540–1545. 27. Steerenberg PA, Janssen NA, de Meer G, et al. Relationship between exhaled NO, respiratory symptoms, lung function, bronchial hyperresponsiveness, and blood eosinophilia in school children. Thorax 2003; 58:242–245. 28. Berkman N, Avital A, Breuer R, Bardach E, Springer C, Godfrey S. Exhaled nitric oxide in the diagnosis of asthma: comparison with bronchial provocation tests. Thorax 2005; 60:383–388. 29. Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6:1368–1370. 30. Berlyne GS, Parameswaran K, Kamada D, Efthimiadis A, Hargreave FE. A comparison of exhaled nitric oxide and induced sputum as markers of airway inflammation. J Allergy Clin Immunol 2000; 106:638–644. 31. Kharitonov SA, Yates D, Springall DR, et al. Exhaled nitric oxide is increased in asthma. Chest 1995; 107:156S–157S. 32. Deykin A, Massaro AF, Drazen JM, Israel E. Exhaled nitric oxide as a diagnostic test for asthma: online versus offline techniques and effect of flow rate. Am J Respir Crit Care Med 2002; 165:1597–1601.

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33. Malmberg LP, Pelkonen AS, Haahtela T, Turpeinen M. Exhaled nitric oxide rather than lung function distinguishes preschool children with probable asthma. Thorax 2003; 58:494–499. 34. Dupont LJ, Demedts MG, Verleden GM. Prospective evaluation of the validity of exhaled nitric oxide for the diagnosis of asthma. Chest 2003; 123:751–756. 35. Smith AD, Cowan JO, Filsell S, et al. Diagnosing asthma: comparisons between exhaled nitric oxide measurements and conventional tests. Am J Respir Crit Care Med 2004; 169:473–478. Epub 2003 Nov 2025. 36. ATS/ERS Recommendations for Standardized Procedures for the Online and Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide. Am J Respir Crit Care Med 2005; 171:912–930. 37. GINA. GINA guidelines Update 2006. http://www.ginasthma.org 2006. 38. Douwes J, Gibson P, Pekkanen J, Pearce N. Non-eosinophilic asthma: importance and possible mechanisms. Thorax 2002; 57:643–648. 39. Kharitonov SA, Yates D, Barnes PJ. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. Eur Respir J 1995; 8:295–297. 40. de Gouw HW, Grunberg K, Schot R, Kroes AC, Dick EC, Sterk PJ. Relationship between exhaled nitric oxide and airway hyperresponsiveness following experimental rhinovirus infection in asthmatic subjects. Eur Respir J 1998; 11:126–132. 41. Fabbri LM, Romagnoli M, Corbetta L, et al. Differences in airway inflammation in patients with fixed airflow obstruction due to asthma or chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003; 167:418–424. Epub 2002 Nov 2008. 42. Brown HM. Treatment of chronic asthma with prednisolone; significance of eosinophils in the sputum. Lancet. 1958; 2:1245–1247. 43. Brightling CE, Ward R, Wardlaw AJ, Pavord ID. Airway inflammation, airway responsiveness and cough before and after inhaled budesonide in patients with eosinophilic bronchitis. Eur Respir J. 2000; 15:682–686. 44. Brightling CE, Monteiro W, Ward R, et al. Sputum eosinophilia and short-term response to prednisolone in chronic obstructive pulmonary disease: a randomised controlled trial. Lancet 2000; 356:1480–1485. 45. Meijer RJ, Postma DS, Kauffman HF, Arends LR, Koeter GH, Kerstjens HA. Accuracy of eosinophils and eosinophil cationic protein to predict steroid improvement in asthma. Clin Exp Allergy 2002; 32:1096–1103. 46. Green RH, Brightling CE, Woltmann G, Parker D, Wardlaw AJ, Pavord ID. Analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids. Thorax 2002; 57:875–879. 47. Szefler SJ, Martin RJ, King TS, et al. Significant variability in response to inhaled corticosteroids for persistent asthma. J Allergy Clin Immunol 2002; 109:410–418. 48. Little SA, Chalmers GW, MacLeod KJ, McSharry C, Thomson NC. Non-invasive markers of airway inflammation as predictors of oral steroid responsiveness in asthma. Thorax 2000; 55:232–234. 49. Smith AD, Cowan JO, Brassett KP, et al. Exhaled Nitric Oxide: A Predictor of Steroid Response. Am J Respir Crit Care Med 2005; 18:18. 50. Massaro AF, Gaston B, Kita D, Fanta C, Stamler JS, Drazen JM. Expired nitric oxide levels during treatment of acute asthma. Am J Respir Crit Care Med 1995; 152:800–803. 51. Baraldi E, Azzolin NM, Zanconato S, Dario C, Zacchello F. Corticosteroids decrease exhaled nitric oxide in children with acute asthma. J Pediatr 1997; 131:381–385. 52. Baraldi E, Dario C, Ongaro R, et al. Exhaled nitric oxide concentrations during treatment of wheezing exacerbation in infants and young children. Am J Respir Crit Care Med 1999; 159:1284–1288. 53. Crimi E, Spanevello A, Neri M, Ind PW, Rossi GA, Brusasco V. Dissociation between airway inflammation and airway hyperresponsiveness in allergic asthma. Am J Respir Crit Care Med. 1998; 157:4–9. 54. Jatakanon A, Lim S, Barnes PJ. Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med 2000; 161:64–72.

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55. Pizzichini MM, Pizzichini E, Clelland L, et al. Prednisone-dependent asthma: inflammatory indices in induced sputum. Eur Respir J. 1999; 13:15–21. 56. Jayaram L, Pizzichini MM, Cook RJ, et al. Determining asthma treatment by monitoring sputum cell counts: effect on exacerbations. Eur Respir J 2006; 27:483–494. 57. Green RH, Brightling CE, McKenna S, et al. Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet 2002; 360:1715–1721. 58. Franklin PJ, Turner SW, Le Souef PN, Stick SM. Exhaled nitric oxide and asthma: complex interactions between atopy, airway responsiveness, and symptoms in a community population of children. Thorax 2003; 58:1048–1052. 59. Strunk RC, Szefler SJ, Phillips BR, et al. Relationship of exhaled nitric oxide to clinical and inflammatory markers of persistent asthma in children. J Allergy Clin Immunol 2003; 112:883–892. 60. Jones SL, Kittelson J, Cowan JO, et al. The predictive value of exhaled nitric oxide measurements in assessing changes in asthma control. Am J Respir Crit Care Med 2001; 164:738–743. 61. Gelb AF, Flynn Taylor C, Shinar CM, Gutierrez C, Zamel N. Role of spirometry and exhaled nitric oxide to predict exacerbations in treated asthmatics. Chest 2006; 129:1492–1499. 62. Beck-Ripp J, Griese M, Arenz S, Koring C, Pasqualoni B, Bufler P. Changes of exhaled nitric oxide during steroid treatment of childhood asthma. Eur Respir J 2002; 19:1015–1019. 63. Jones SL, Herbison P, Cowan JO, et al. Exhaled NO and assessment of anti-inflammatory effects of inhaled steroid: dose-response relationship. Eur Respir J 2002; 20:601–608. 64. Silkoff PE, McClean P, Spino M, Erlich L, Slutsky AS, Zamel N. Dose-response relationship and reproducibility of the fall in exhaled nitric oxide after inhaled beclomethasone dipropionate therapy in asthma patients. Chest 2001; 119:1322–1328. 65. Sont JK, Willems LN, Bel EH, van Krieken JH, Vandenbroucke JP, Sterk PJ. Clinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment. The AMPUL Study Group. Am J Respir Crit Care Med 1999; 159:1043–1051. 66. Pijnenburg MW, Bakker EM, Hop WC, De Jongste JC. Titrating steroids on exhaled nitric oxide in asthmatic children: a randomized controlled trial. Am J Respir Crit Care Med 2005; 23:23. 67. Shaw DE, Berry MA, Green RH, Brightling CE, Wardlaw AJ, Pavord ID. The use of exhaled nitric oxide to guide asthma management: a randomsied controlled trial. Am J Respir Crit Care Med 2007; 176;231–237. 68. Smith AD, Cowan JO, Brassett KP, Herbison GP, Taylor DR. Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med 2005; 352:2163–2173. Epub 2005 May 2124. 69. Juniper EF, O’Byrne PM, Guyatt GH, Ferrie PJ, King DR. Development and validation of a questionnaire to measure asthma control. Eur Respir J 1999; 14:902–907. 70. Taylor DR. Exhaled nitric oxide: forwards, backwards or sideways? Am J Respir Crit Care Med 2007; 176:221–222. 71. Berry M, Hargadon B, Morgan A, et al. Alveolar nitric oxide in adults with asthma: evidence of distal lung inflammation in refractory asthma. Eur Respir J 2005; 25:986–991. 72. van Dixhoorn J, Duivenvoorden HJ. Efficacy of Nijmegen questionnaire in recognition of the hyperventilation syndrome. J Psychosom Res 1985; 29:199–206. 73. Pijnenburg MW, Hofhuis W, Hop WC, De Jongste JC. Exhaled nitric oxide predicts asthma relapse in children with clinical asthma remission. Thorax 2005; 60:215–218. 74. Pijnenburg MW, Bakker EM, Lever S, Hop WC, De Jongste JC. High fractional concentration of nitric oxide in exhaled air despite steroid treatment in asthmatic children. Clin Exp Allergy 2005; 35:920–925. 75. Gelb AF, Taylor CF, Nussbaum E, et al. Alveolar and Airway Sites of Nitric Oxide Inflammation in Treated Asthmatics. Am J Respir Crit Care Med 2004; 30:30. 76. Zacharasiewicz A, Wilson N, Lex C, et al. Clinical use of noninvasive measurements of airway inflammation in steroid reduction in children. Am J Respir Crit Care Med 2005; 171:1077–1082. Epub 2005 Feb 1011.

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77. Lemiere C. The use of sputum eosinophils in the evaluation of occupational asthma. Curr Opin Allergy Clin Immunol 2004; 4:81–85. 78. Girard F, Chaboillez S, Cartier A, et al. An effective strategy for diagnosing occupational asthma: use of induced sputum. Am J Respir Crit Care Med 2004; 170:845–850. Epub 2004 July 2021. 79. Kharitonov SA, O’Connor BJ, Evans DJ, Barnes PJ. Allergen-induced late asthmatic reactions are associated with elevation of exhaled nitric oxide. Am J Respir Crit Care Med 1995; 151:1894–1899. 80. Rolla G, Di Emanuele A, Dutto L, et al. Effect of inhalation aspirin challenge on exhaled nitric oxide in patients with aspirin-inducible asthma. Allergy 2004; 59:827–832. 81. Lund MB, Oksne PI, Hamre R, Kongerud J. Increased nitric oxide in exhaled air: an early marker of asthma in non-smoking aluminium potroom workers? Occup Environ Med 2000; 57:274–278. 82. Olin AC, Andersson E, Andersson M, Granung G, Hagberg S, Toren K. Prevalence of asthma and exhaled nitric oxide are increased in bleachery workers exposed to ozone. Eur Respir J 2004; 23:87–92. 83. Ihre E, Gyllfors P, Gustafsson LE, Kumlin M, Dahlen B. Early rise in exhaled nitric oxide and mast cell activation in repeated low-dose allergen challenge. Eur Respir J 2006; 27:1152–1159. Epub 2006 Mar 1151. 84. Juniper EF, Bousquet J, Abetz L, Bateman ED. Identifying ‘well-controlled’ and ‘not wellcontrolled’ asthma using the asthma control questionnaire. Respir Med 2006; 100:616–621. Epub 2005 Oct 2013.

Basophil Activation Tests for Allergy Diagnosis Alain L. de Weck and Maria Luisa Sanz

Introduction Peripheral blood basophils and tissue mast cells are primary effector cells in IgEmediated immediate allergic reactions such as rhinitis, asthma, and anaphylaxis. They may also be involved in other kinds of allergic or pseudo-allergic reactions in which other activation mechanisms such as complement activation, non-IgE-mediated stimulation, or non-immunological mechanisms are implicated [1]. Basophils represent less than 0.5% of the total leukocytes in peripheral blood, which makes their purification difficult. Since these cells play an important role in immediate allergic reactions, some functional in vitro tests have been developed that detect their activation. Following microscopic evaluation of basophil degranulation [2], one of the first was the histamine release test, a technique much used in allergy research but which did not find extensive clinical application due to its insufficient sensitivity and specificity [3]. Release of other mediators such as sulfidoleukotrienes (CAST) has also found some clinical diagnostic applications [4]. But in the past decade, an increasing number of groups has taken advantage of flow cytometry and developed new tools to monitor basophil activation after antigen-specific stimulation, using the expression of various membrane surface markers [5–11].

Mechanisms of Basophil Activation The complex mechanisms of IgE-receptor-mediated activation and the subsequent cascade of intracellular signals leading to the release of various mediators such as histamine, LTC4 (sLT), and lymphokines such as interleukin (IL)-4 and IL-13, have A.L. de Weck () Beaumont 18, CH-1700, Fribourg, Switzerland e-mail: [email protected] A.L. de Weck and M.L. Sanz Department of Allergology and Clinical Immunology, Clinica Universitaria, University of Navarra, 31080 Pamplona, Spain

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been extensively investigated [12]. However, the mechanisms of expression of various membrane proteins, which are the basis of diagnostic basophil activation tests (BAT), as well as the various mechanisms of non-IgE-mediated activation have not yet been entirely differentiated. The contention that the same quantitative and qualitative rules may apply to the various outcomes of basophil activation (e.g., CD63/CD203c expression, histamine release, LTC4, and lymphokines formation) [13] does not appear to be correct. In fact, the various test readouts do not follow strictly and in all respects the rules established for histamine release: a number of differences in the various activation cascades and regulatory factors have been well documented [14–16]. These differences probably explain why in clinical practice, although often correlated, the various outcomes of basophil activation may occur independently from each other in some individuals [17]. Even the expression of two different membrane markers, CD63 and CD203c, obeys different rules and regulations [18, 19]. One must therefore be careful to extrapolate general conclusions drawn from experience gained with histamine release to other basophil activation assays.

Historical Background of Flowcytometric BAT The background of the flowcytometric basophil activation test goes back to early studies performed by Nakagawa et al. [20] in the early 1980s, followed by Knol et al. [21] and Gane et al. [22], among others, in the early 1990s, and the application of BAT to clinical allergy diagnosis by Sainte-Laudy et al. [23, 30].

Basis of the Flowcytometric Basophil Activation Test Flow cytometry is a useful tool for the analysis of different cellular types and can be used to identify specific cell populations, even those that are present in low amounts [24]. The basis of the BAT assays is in a first step the identification of basophils by specific fluorescent antibodies against more or less specific basophil markers, such as anti-IgE, anti-CD 203c, anti-CD123 (IL-3 receptor), anti-CRTH2 (DP2 receptor), or anti-CCR3 all associated with anti-HLA-DR in order to exclude other cell types (e.g., T cells) carrying the same marker. In a second step, basophil stimulation is detected by the appearance or increase of certain membrane phenotypes that are upregulated after exposition to allergen. Upregulation has been described for expression of various molecules such as CD45, CD11b, and CD11c. However, most studies make reference to the expression of CD63 [6–9] or of CD203c [10, 25] on basophils after in vitro activation. Whether in the future, some novel basophil activation markers such as CD13, CD164, and CD107a can be applied in flow-assisted allergy diagnosis remains to be established [19]. In addition, a decrease in the mean fluorescent intensity of IgE-carrying basophils after in vitro stimulus with allergen has been described as a possible activation parameter [26, 27]. Bridging of IgE receptors by the action of di- or plurivalent allergens provokes the intracellular fusion of the granules and the fusion of their membrane with

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the plasmatic cell membrane, followed by exocytosis (degranulation). Molecules present on the granule membrane, such as the CD63 molecule, and are then expressed on the basophil membrane upon activation. CD63 is a tetraspan 53 Kda granular protein that is expressed not only on basophil granules but also on monocytes, macrophages, and platelets. The expression of this marker correlates well with degranulation and histamine release, which makes it an ideal marker of basophil activation [19]. An alternative form of activation, detected by the basophil-specific CD203c marker, results in mediator release without granular exocytosis (so-called piecemeal degranulation) [28]. Other, non-IgE-mediated forms of basophil activation, such as complement activation (through the C5a receptor) or stimulating peptides (e.g., fMLP) also manifest themselves by CD63 or CD203c expression, but their regulation may be different from that of anti-IgE. The first step in performing the test consists of in vitro stimulation with allergen. Peripheral blood cells either as whole blood or as isolated leukocytes are incubated with the suspected allergen for 15–40 min at 37°C. After stopping the reaction, the cells are labeled with anti-CD63-PE and anti-IgE-FITC monoclonal antibodies. The cells are then analyzed according to successive gating parameters in a flowcytometer (Fig. 1). Two controls are usually used: (a) a negative control in which the cells are incubated with the stimulation buffer alone used in the assay and that often contains IL3; (b) a positive control of stimulation for which an anti-IgE, an anti-IgE receptor antibody, or an activating peptide (fMLP) can be used.

Fig.1 Flowcytometric analysis of basophils activated by allergen (Ag). The upper panels show the successive gating of lymphocytes, IgE-bearing basophils and Ige-bearing basophils expressing CD63 (from left to right). The lower panels show the mean fluorescence intensity of cells bearing IgE and expressing CD63

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In order to evaluate the results, the percentage of activated basophils is assessed and the determination of a positivity cut-off must be made for each allergen with the help of receiver operator characteristic (ROC) curves (optimal sensitivity vs specificity) [29]. The BAT technique has been increasingly considered useful for in vitro allergologic diagnosis [6–11] and has by now been validated clinically for various allergens such as inhalants, hymenoptera venoms, latex, muscle relaxants, betalactam antibiotics, and pyrazolones. In addition, BAT has been reported useful in diagnosing hypersensitivity to nonsteroidal anti-inflammatory drugs (NSAID) and to many other compounds. More recently, its diagnostic reliability has also been studied using recombinant allergens, in the monitoring of antigen-specific immunotherapy, in food allergy diagnosis, and in chronic urticaria.

Clinical Applications of BAT Beyond a large number of anecdotal studies at the beginning of the BAT era in the 1990s [23, 39], during the past 5 years, a large number of clinically validated studies have been published. In this review, we consider as validated clinical studies those encompassing at least 15–20 confirmed allergic patients, as demonstrated by compelling history and/or provocation tests and other positive tests such as skin tests and/or allergen-specific IgE. In addition, at least ten non-allergic controls, negative to other tests and with no history of aeroallergen allergy, should be included. For some allergens, e.g., foods, it is needed to include atopic patients, that is, IgEsensitized to aeroallergens but negative to challenge with the food allergen tested. Many of these studies have been reviewed recently [9–11, 17]. Herewith, various tables summarize the results of validated clinical studies performed with various allergens such as inhalant allergens (Table 1), hymenoptera venoms (Table 2), latex (Table 3), foods (Table 4), and drugs (Tables 5–7). These studies are also classified according to the applied techniques. In our opinion, these studies demonstrate the diagnostic interest of BAT in a number of clinical indications.

Inhalant Allergens The results of BAT in patients sensitized to aeroallergens such as grass pollens or house dust mites have been extensively compared with skin tests, specific IgE determinations (CAP, RAST), or mediator release assays (histamine release, CAST) [17, 31]. They usually show high diagnostic efficiency (Table 1) but are, in this indication only of minor clinical interest since the other diagnostic tests (skin tests, specific IgE) are easier and cheaper to perform. It might be interesting, however, to make some additional studies in children, particularly in terms of follow-up of immunotherapy.

Nb 10 13 34 58 51 20 56

Controls HC HC HC HC HC HC HC

33 38 38 10 14

Nb

Sensitivity 78% 100% 91.2% 91.3% 91.3% 100% 85% 72%

Specificity 91% 100% 100% 98.4% 98.4% 100% 100% 92%

Cells Wb Wb Wb IL IL Wb Wb Wb

CD 63 63 63 63 63 203c 63 63

IL-3 +/− +/− + + + − + +

Test

Basotest BD For references, see the reference list and [17]. In addition to the listed studies, several other studies have been presented and published in abstract form [17]. Abbreviations: SPT: skin prick test; HC: healthy controls; Wb: whole blood; IL: isolated leukocytes – Baso: BASOTEST; BD: FastImmune Beckton Dickinson a All references cited in this table are to be found in Ref. [17].

SPT+ SPT+ SPT+ SPT+ SPT+ SPT+ SPT+

Cozon, 1999 Cozon, 1999 Paris-Kohler, 2000 Sanz, 2001 Sanz, 2001 Hauswirth, 2002 Michova, 2006

Mites d1 Grass pollens Cypress pollen Mites d1 Lol perenne Bet v1,2 Phlp 1,3,5 Grass pollens

Table 1 Clinically validated studies for aeroallergens Authorsa Allergens Patients

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HC

HC HC HC HC HC HC HC HC

Controls

Ebo, 2007 Wasp AS 80 HC In addition to the listed studies, some other reports [17] have been published. Abbreviations: AS: anaphylactic shock a All references cited in this table are to be found in Ref. [17].

67 43

44 45 20 22 28 50 57 14 150

Eberlein, 2006 Eberlein, 2006

Nb

AS AS AS AS AS AS AS AS AS AS AS AS

Sabbah, 1999 Sainte Laudy, 2000 Freitag, 2001 Platz, 2002 Lambert, 2003 Erdmann, 2003, 2004 Sturm, 2004 Eberlein, 2004 Scherrer, 2005, 2007

Hymenoptera Hymenoptera Wasp Hymenoptera Hymenoptera Wasp Hymenoptera Hymenoptera Wasp Bee Wasp,bee Wasp, bee

Table 2 Clinically validated studies for insect venoms Authorsa Allergens Patients

14

25

32 12 13 8 20 30 5 40

Nb 100% 93% 70–100% 91% 91% 92% 88% 78% 93% 94% 97% 97% 89% 86%

Sensitivity

90% 80% 87% 100% 85% 95% 100% 89% 100% 100%

100% 100% 100%

Specificity

Cells Wb IL Wb Wb Wb Wb Wb Wb IL IL Wb Wb Wb Wb

CD 63 63 63 203c 63 63 63 63 63 63 63 203c 63 63

IL-3 − − + − − − − + + + + − + +

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Latex Latex Latex

Sanz, 2002 Erdmannn, 2003 Boumiza, 2003

Hx+ ST+ sIgE+ Hx+ ST− sIgE− Hx− ST− sIgE+ Hx+ ST+ Hx+ ST+ Hx+ ST+

29 13 24 43 20 16

Nb HC− HC− sIgE+ 17% HC, Prov− HC5, AC5 HC

Controls

Hemery, 2005 Latex Hx+ ST+ sIgE+ 46 HC Sanz, 2006 Hev b5,6 Hx+ ST+ sIgE+ 23 HC Nettis, 2006 Hev b5, 6 Hx+ ST+ IgE+ 24 HC In addition to the listed studies, some other reports [17] have been published. Abbreviations: Hx: history; ST: skin test; sIgE: specific IgE; Prov: provocation a All references cited in this table are to be found in Ref. [17].

Latex

Ebo, 2002

Table 3 Clinically validated studies for latex allergens Authorsa Allergens Patients

33 10 19

12 24 83% 20 10 12

Nb

93% 90% 50% 75% 85% 95% 96%

91.7% 100%

Sensitivity

100= 80% 100% 100% 97% 100% 100%

100% 87%

Specificity

IL Wb Wb Wb Wb IL

Wb

Cells

63 63 63 203c 63 63

63

CD

+ + − − + +

+

IL-3

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Various Various Peach, rPrup3 Carrot, ceéery, hazelnut, Apple

Nb

Controls

Nb

Sensitivity

Prov+ 31 HC 80% 100% Hx+ 74 AC 64 82.3% AS 17 HC 5 90% OAS+ 29 HC 20 85–90% OAS+ 29 HC 10 100% OAS− 23 86% Erdmann, 2005 rMald1, rApig1, rDauc1 OAS+ 32 HC 10 65-75% OAS− 12 Caceres, 2005 rMald1,3,5; rPrup3 AS 24 HC 8 67-92% Gamboa rMald1,3,4, rPrup3 Prov+ 30 HC 29 ~100% In addition to the listed studies, a number of reports on individual food-related allergens have been published [17]. Abbreviations: OAS: Oral Allergy Syndrome; AC: atopic controls a All references cited in this table are to be found in Ref. [17].

Monneret-Vautrin, 1999 Garcia-Aviles, 2000 Diaz-Perales, 2003 Erdmann, 2003 Ebo, 2005

Table 4 Clinically validated studies for food allergens Authorsa Allergens Patients

63

Wb IL IL

83–100% ~100%

63 63

+ 63 63 63 63

CD 63 IL IL Wb Wb

Cells

IL 62.5% 100% 80–90% 100% 75% 68–100%

Specificity

+ +

+

+ + + +

IL-3

168 A.L. de Weck and M.L. Sanz

Nb 15 41 39 21

HC Prov− HC Prov− HC Prov− 10, HC 7 HC

Controls

Kvedariene, 2006 Myorelacants AS ST? 47 AC 40 HC 5 Ebo, 2006 Rocuronium AS ST+ 14 HC ST− Prov− * In patients investigated within 3 years after the clinical reaction a All references cited in this table are to be found in Ref. [17].

AS AS ST+ AS ST+ AS ST+/−

Leynadier, 1999 Abuaf, 1999 Monneret, 2002 Sudheer, 2005

Myorelaxants Myorelaxants Myorelaxants Myorelaxants

Table 5 Clinically validated studies for myorelaxants Authorsa Allergens Patients Nb

45 8

9 21 17 10

Sensitivity 66% 64% 54% 79% 36% 36% (86%*) 91%

Specificity 87% 100% 100% 100% 100% 93% 100%

Cells Wb Wb Wb Wb Wb Wb Wb

CD 63 63 63 63 203c 63 63

IL-3

− + +

− − −

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Nb

Controls

Nb

Sanz, 2002 Betalactams Imm, ST+ 58 HC 30 Torres, 2004 Betalactams Imm, ST+ 70 HC 40 Arnoux, 2004 Betalactams Imm 35 AC 40 Gamboa, 2004 Betalactams Imm, ST− Prov+ 23 HC 30 Kvedariene, 2005 Betalactams Imm 22 HC 29 Erdmann, 2005 Betalactams Imm 20 De Weck ENDA, 2006 Betalactams Imm ST+/− 181 HC 80 Some additional reports have been published [17]. Abbreviations: Imm: Immediate type reactions (anaphylactic shock, urticaria, angioedema) a All references cited in this table are to be found in Ref. [17].

Table 6 Clinically validated studies for betalactams Authorsa Allergens Patients 50% 48% 9% 39% 34% 35% 46%

Sensitivity

93%

93% 91% 100% 93% 83%

Specificity

IL Wb Wb IL Wb Wb IL

Cells

63 63 63 63 63 63 63

CD

+ + + + + + +

IL-3

170 A.L. de Weck and M.L. Sanz

Sanz, 2004 NSAID NSAID synd Kvedariene, 2005 NSAID NSAID synd Erddmann, 2005 NSAID NSAID synd De Weck ENDA, 2005 NSAID NSAID synd a All references cited in this table are to be found in Ref. [17]. * With a particular method of basophil isolation (buffy coat)

60 15 20 140

Table 7 Clinically validated studies for hypersensitivity to NSAIDs Authorsa Allergens Patients Nb Controls HC Prov− HC − HC Prov−

Nb 30 10 − 163

Sensitivity 74% 25% 30% 76%

Specificity 92% 80% − 92%*

Cells IL Wb Wb IL

CD 63 63 63 63

IL-3 + + + +

Basophil Activation Tests 171

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Hymenoptera Allergy The interest of BAT in hymenoptera venom hypersensitivity has been confirmed hitherto by several studies (Table 2). The main interest of BAT in that indication is that it may confirm clinical history in the absence of positive skin tests or positive allergen-specific IgE. In addition, it often allows detecting specifically the insect responsible for clinical sensitivity and to confirm the indication for immunotherapy [32–34]. In the largest series reported so far [35], correlation of BAT with skin tests and specific IgE oscillated between 0.71 and 0.82. In a recent large series of 118 patients, a definite diagnosis of venom anaphylaxis (history + unequivocal single positive sIgE and skin test) was observed in only about 55% of the patients. In 40%, diagnosis was hindered by equivocal or discrepant test results (mostly double positive sIgE results for both venoms). In seven patients (5%), there was no proof of sensitization (sIgE and skin test negative). Considering only the BAT responders (86% of all patients), the BAT confirmed a final diagnosis in almost two thirds of the patients with equivocal results and, most importantly, in all seven patients with negative sIgE and skin test results [36]. A recent paper [37] has documented that BAT may also be used to monitor venom immunotherapy. When BAT tests are performed in whole blood, the question remains whether the decrease in reactivity observed after immunotherapy is due to competing venom-specific serum IgG antibodies, as shown to be the case for histamine release [38], or whether a true decrease of cellular reactivity also occurs in parallel.

Latex Allergy In several validated studies [39–42] (Table 3), the BAT was shown to demonstrate a sensitivity and a specificity for CD63 expression of over 90%, with one exception in which sensitivity was reported to be 75% for CD203c and 50% for CD63 assay [43]. BAT is the test that reflects the most closely to clinical provocation. In particular, it enables to sort out the clinically “false positive” cases, namely those with negative latex challenge but positive latex-specific IgE determinations due to cross-reactive carbohydrate determinants (CCD) [40]. With other allergens also, IgE to CCD determinants seem to have little functional activity [44] and BAT should permit sorting out of such cases.

Food Allergy Food allergy will probably become one of the areas of allergy diagnosis in which BAT will give most useful services. Although double-blind placebo-controlled food challenge (DBPCFC) is constantly advocated as the gold standard, it is not flawless

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and is much lessoften used in practice than publicly advocated. In numerous instances, it is not practicable or even not ethical (e.g., history of anaphylactic shock). There are several validated studies in the literature (Table 4), some including recombinant food allergens [45–49]. In birch-allergic patients with or without oral allergy syndrome (OAS) demonstrated by DBPCFC, cellular tests such as CAST [50] and BAT [48] have been found to be better predictive of provocation outcome than quantitative food-specific IgE. BAT may help to discriminate between relevant and irrelevant IgE to apple [48]. BAT has also shown its diagnostic usefulness in allergies to various foods (for review, see [17]).

Chronic “Idiopahtic” Urticaria Numerous cases of chronic “idiopathic” urticaria seem due to autoimmune phenomena, in particular, to autoantibodies directed against IgE or IgeR1 receptors. This leads not only to immediate skin test reactivity against autologous serum but also to basophil hyperreactivity. An increasing number of studies confirm the usefulness of BAT with donor basophils in the diagnosis and pathogenetic elucidation of chronic urticaria cases [50–57].

Drug Hypersensitivity It is probably in the field of immediate-type allergic reactions to drugs that BAT has encountered the most interest in recent years, due to the fact that the performance of drug- specific IgE tests has been rather poor, especially in non-betalactam drugs, and that they are not available for many drugs. BAT can also detect immediate drug hypersensitivities due to non-IgE mechanisms.

Myorelaxants Several validated studies [26, 58–61] (Table 5) show the interest of BAT in diagnosis of immediate reactions to myorelaxants, with a sensitivity oscillating between 54% and 91% for CD63 expression and 36% for CD203c expression [59]. One may argue that sensitivity is too low to be useful in routine diagnosis [58], but one must recognize that it is at least equal if not superior to that of skin tests and drug-specific IgE. On the other hand, BAT is highly specific. Accordingly, like for many other tests in drug allergy, a positive test in an individual case is probably meaningful, while a negative test does not permit to exclude clinical drug allergy. Sensitivity apparently increases up to 80% when BAT is performed shortly after the clinical reaction [60]. BAT has proven to be complementary to skin tests in the evaluation of cross-reactivity between myorelaxants [61].

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Betalactams The percentage of BAT-positive tests is only about 50% in patients with a history of betalactam immediate-type allergy and positive skin tests [62–64]. But BAT are also positive in about 40% of patients with entirely negative skin tests and positive clinical history of immediate systemic reaction confirmed by provocation [65, 66], who constitute about 25% of all betalactam allergic patients. (Table 6). In all studies where sensitivity and specificity of BAT have been compared on the same patients [62–66] to those of betalactam-specific IgE (sIgE), BAT has appeared to be superior by 5–10% or more. Sensitivity of betalactam sIgE oscillates between 24% and 74% [67, 68] depending on the group of patients selected and the time elapsed since the clinical reaction. These results have been recently confirmed in a large multicentric study implicating 10 European groups, 181 betalactam allergic patients, and 80 control individuals [65]. In that study, it was also shown that the combination with a CAST assay increases sensitivity to 62%. In terms of diagnostic performance, the combined use of skin tests, specific IgE, and BAT increases sensitivity to 85% and the addition of CAST to 95%.

Pyrazolones In IgE-mediated allergies to single analgesics, such as pyrazolones, the specific case of metamizol has been investigated [69]. Such patients have nothing to do with the NSAID hypersensitivity syndrome: they react specifically to a single drug or to a group of drugs of similar immunochemical structure. In 26 patients reacting to metamizol, BAT was found positive in 42%. BAT was also highly specific (100%). In combination with skin tests, sensitivity rose to 69% and with the addition of CAST, reached 77%.

NSAIDs Since the description by several authors that blood leukocytes from patients suffering from the NSAID hypersensitivity syndrome may produce sLTs, as detected by CAST [4], the dogma that no in vitro tests may be associated with that condition has been crumbling. The first studies on BAT and NSAIDs by Sanz and Gamboa et al. [70, 71] have included 60 NSAID-hypersensitive patients, among which 18 have been positively challenged while the remaining 42 had a documented history of at least two or more immediate reactions to at least two different NSAID drugs, a rather strict clinical inclusion criterion. In these patients, BAT performed with five NSAIDs at three concentrations was found positive in 55% of the cases. These findings have been recently confirmed in a large multicentric study including 12 European groups, 150 NSAID-hypersensitive patients, and 163 controls,

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showing a sensitivity of 75% [72] In that study, when two similar populations, positively provoked or not (but with at least two clinical events) have been analyzed separately, an identical percentage of positive BAT (about 75%) was found. A major new finding is that some NSAID-tolerant individuals also react with BAT to higher concentrations of NSAIDs: the clinical NSAID hypersensitivity syndrome appears therefore to be reflected in a shift of the BAT dose–response curve to NSAIDs [72–74]. It has also been shown that an index based on BAT reactions to two concentrations of aspirin, diclofenac, and naproxen (ADN index) enables us to distinguish NSAID-hypersensitive from NSAID-tolerant patients with 80% accuracy [72]. Indeed, provocation remains the ultimate proof needed for validation but the BAT tests and the ADN index seems by now to be a practical alternative in many cases, when provocation cannot be done (Table 7). It has been questioned [13] why a BAT test to some NSAID could be considered positive when the culprit drug itself could be negative. One should realize that the mechanism of BAT in the NSAID hypersensitivity syndrome has nothing to do with an immunological reaction and that the classical notions of specificity for culprit drug and cross-reactivity do not apply. In fact, BAT reactions in NSAID hypersensitivity are closely related to the pharmacological COX-1 inhibiting activity of the NSAIDs and are strictly dose-dependent [72–74]. The most likely and main mechanism of basophil activation is the pharmacological abolition of PGE2 synthesis, a natural inhibitor of basophil activation, partly responsible for homeostasis [75–77]. In vitro, diclofenac and naproxen have a much stronger pharmacological activity than aspirin and are more likely to cause a BAT reaction in vitro, even if aspirin had been responsible for the clinical reaction in vivo. In some cases of anaphylactic shock to aspirin, however, an IgE-related mechanism has been claimed to be the cause of a positive BAT [77].

Others Individual cases of immediate-type allergy to a number of other drugs or materials used in diagnostic or therapeutic procedures have been reported in recent years: iodinated povidone, cyclosporine, heparins, chlorhexidine, hydroxyethyl starch, patent blue, omeprazol, hyaluronidase, Viscum album, gelofusine, dexchlopheniramine, or bovine serum albumin (for references, see [17]). The main interest of BAT in such cases is to confirm the diagnosis, particularly when skin testing and/or determination of drug-specific IgE are not available.

Indications, Performance, and Interpretation of BAT Tests BAT tests are not primary diagnostic measures; they are essentially complementary to skin tests and allergen-specific IgE determinations, particularly when these cannot be performed or have given equivocal/doubtful results in comparison with

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clinical history. They are particularly indicated in insect venom allergy, food allergy, latex allergy, and other major immediate-type drug allergies. BAT tests appear to be useful for the study of cross-reactivity among various protein allergens and drugs and to monitor immunotherapy with some allergens, particularly with anti-IgE antibodies [12, 34]. The proper performance of the test requires some precise attention to detail, particularly in the conservation of the blood samples, the preparation and quality of the reagents used, and the flowcytometric gating processes established. Most important is the quality and standardization of the allergens used, which must be submitted to special quality control measures for use in flowcytometry. If these flowcytometric quality control measures are not provided by the allergen commercial source, the laboratory will have to perform them by itself. In fact, experience with allergens is probably more important for quality BAT tests than experience with basophil handling, which is easily acquired by any flowcytometry laboratory. Among the technical aspects to be considered that may influence the results are the use of whole blood or isolated leukocytes, various factors possibly affecting the negative control (e.g., exposure to allergen in vivo), the efficiency of the positive control (anti-IgE, anti-IgE receptor, or fMLP) and the incidence of non-responders, the effects of blood storage and of the time of incubation with allergen, the role of allergen concentration and the need to establish dose–response curves, as well as the possible beneficial effect of addition of IL-3. These technical details have been extensively reviewed [6, 9, 10]; some points require additional comparative studies. Intra-assay reproducibility assessed by duplicate determinations for different allergens is usually very high, which often allows performance of a single test for each allergen concentration [6]. The reproducibility of BAT was found in drug allergy quite remarkably with an intra-assay variation coefficient of 0.89 (p < 0.0001) for betalactams, 0.99 (p < 0.0001) for metamizol, and 0.92 (p < 0.0001) for NSAIDs. For appropriate evaluation of the results, two values should be taken into account: (a) The absolute number of basophils evaluated, which should be at least over 150. (b) The percentage of activated basophils. In the negative (non-stimulated) control, the percentage of activated basophils is usually below 5%. The positive control after activation of the cells with anti-IgE or anti-IgE antireceptor should be above 10% and the stimulation index * above 2. Some authors describe also a decreased mean fluorescence emitted by the cells activated in vitro by allergens [26, 27], a phenomenon which does not seem to affect the percentage of basophils expressing CD63. It is necessary to establish validated cut-off points for each allergen. For this, it is recommended to have proper negative and positive controls and to set up receiver operator characteristic (ROC) curves establishing optimal sensitivity versus specificity [29]. Some allergens, particularly foods, can provoke unspecific stimulations. Other allergens, such as drugs, usually cause lower responses than those obtained with protein allergens.

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In our experience, the cut-off points offering the highest specificity and sensitivity values determined by ROC curves are the following: For inhalant allergens >15%; food allergens >15%; latex >10%; hymenoptera venoms >10%; *betalactam antibiotics >5% and SI >2; *metamizol >5% and SI >5; *aspirin and NSAIDs >5%; and SI >2. *SI: Stimulation index = stimulation by allergen/basal stimulation or negative control. For most other groups [9, 10], cut-off points have been very similar but usually not adding a stimulation index as additional requirement. For some special questions, such as NSAID, special criteria (e.g., ADN index) [72] have to be used. It is still open whether the quantitative degree of basophil activation, measured by sLT production or BAT, has some clinical diagnostic value. Some authors have found a correlation between the degree of clinical sensitivity and the percentage of activated basophils [78] but other authors did not confirm this observation. It has been claimed [37] that patients at risk of anaphylaxis upon insect sting challenge may show higher BAT baselines but this study used different technologies and mode of evaluation than most BAT studies reported hitherto. For muscle relaxants, a correlation between the degree of skin sensitivity and of CD63 expression has been reported [26]. In hymenoptera venom allergy, the degree of BAT reactivity has been reported to be predictive of side effects during venom immunotherapy [79] but this is not agreed upon by all [79]. The occurrence of high CAST and/or BAT levels to betalactams has been correlated to systemic anaphylaxis [63]. Finally, it has been demonstrated that sLT levels (CAST) [50] and BAT [48] are highly predictive of double-blind placebocontrolled food challenge and of oral allergy syndrome in food allergy.

Comparison with Other Cellular In Vitro Diagnostic Techniques Comparisons among various in vitro tests have been made for several allergens, BAT usually shows a good correlation with histamine release and/or sulfidoleukotriene determinations (CAST) [6, 31, 45, 62, 80], but BAT seems more sensitive and specific than the other tests. For diagnosis of immediate reactions to drugs in general [63, 65], and for immediate reactions to Betalactams in particular, the joint use of BAT and CAST offers better results and allows detection of up to 80% of the cases. The same is true for pyrazolones [69].

Conclusions There should be little doubt for those following the literature that BAT tests are rapidly becoming an important addition to our allergy-diagnostic capacities. However, the number of European groups possessing varied and sustained practical

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BAT experience in routine diagnosis is still rather limited. Furthermore, some defiance has been entertained since years by position papers often authored by scientists not yet using the method in their own laboratory and always requiring still “more evaluation” [3, 81]. Some opinion papers [13] give the wrong impression of an “extraordinary variability of BAT responses,” of an avalanche of pitfalls and of a requirement to possess many skills before engaging in clinical BAT diagnosis, “which should be restricted to selected cases and to experienced laboratories.” It would be a pity if these warnings would deprive many patients from the benefits of BAT and of flowassisted allergy diagnosis any longer. In fact, the common positive scientific and clinical experience of several groups with BAT over a number of years is much more significant than the few technical issues still debated. In addition to a number of validated and published “home protocols” [5, 6, 9], we dispose at present of at least three widely clinically validated and commercially available BAT technologies. These may be considered as BAT tests of first generation. Modifications have been proposed [10] and additional tests of second generation still await an equivalent clinical validation.

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50. Ballmer B.K., Weber J.M., Vieths S., Wühtrich B. (2008) Predictive value of the sulfidoleukotriene release assay (CAST-ELISA) in oral allergy syndrome to celery, hazelnut and carrot. J Investig Allergol Clin Immunol. 18: 93–99 51. De Swerdt A., Van Den K.C., Ksran A., Cadot P., Neyens K., Coorevits L., Kochuyt A.M., Degreef H., Ceuppens J.L. (2005) Detection of basophil-activating IgG autoantibodies in chronic idiopathic urticaria by induction of CD63. J Allergy Clin Immunol, 116: 662–667 52. Frezzolini A., Provini A., Teofoli P., Pomponi D., De Pita O. (2006) Serum-induced basophil CD63 expression by means of a tricolour flowvytometric method for the in vitro diagnosis of chronic urticaria. Allergy, 61: 1071–1077 53. Grattan C.E.H., Walpole D., Francis D.M., Niimi D.M., Dooston G., Edler S., Corbett M.F., Barr R.M. (1997) Flow cytometric analysis of nasophil numbers in chronic urticaria: basopenia is related to serum histamine releasing activity. Clin Exp Allergy, 27: 1417–1424 54. Gyimesi E., Sipka S., Danko K., Kiss E., Hidvegi B., Gal M., Hunyadi J., Irinyi B., Szegedi A. (2004) Basophil CD63 expression assay on highly sensitized atopic donor leukocytes – a useful method inn chronic autoimmune urticaria. Br J Dermatol, 151: 388–396 55. Szegedi A., Irinyi B., Gal M., Hunyadi J., Danko K., Kiss E., Sipka S., Szegedi G., Gyomesi E. (2006) Significant correlation between the CD63 assay and the histamine release assay in chronic urticaria. Br J Dermatol, 155: 67–75 56. Vasagar K., Vonakis B.M., Gober L.M., Viksman A., Gibbons S.P. Jr., Saini S.S. (2006) Evidence of in vivo basophil activation in chronic urticaria. Clin Exp Allergy, 36: 770–776 57. Wedi B., Novacovic V., Koerner M., Kapp A. (2000) Chronic urticaria serum induces histamine release, leukotriene production and basophil CD63 surface expression- inhibitory effects of anti-inflammatory drugs. J Allergy Clin Immunol 105: 552–560 58. Monneret G., Benoit Y., Debard A.L., Gutowski M.C., Topenot I., Bienvenu J. (2002) Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relaxant drugs. Clin Immunol, 102: 192–199 59. Sudheer P.S., Hall J.E., Read G.F., Rowbottom A.W., Williams P.E. (2005) Flowcytometric investigation of peri-anaesthetic anaphylaxis using CD63 and CD203c. Anaestzhesia, 60: 251–256 60. Kvedariene V., Kamey S., Ryckwaert Y., Rongier M., Bousquet J., Demoly P., Arnoux B. (2006) Diagnosis of neuromuscular blocking agent hypersensitivity reactions using cytofluorimteric anapysis of basophils. Allergy, 61: 311–315 61. Ebo D.G., Bridts C.H., Hagendorens M.M., Mertens C.H., De Clerck L.S., Stevens W.J. (2006) Flow-assisted diagnostic management of anaphylaxis from rocuronium bromide. Allergy, 61: 935–939 62. Sanz M.L., Gamboa P.M., Uasuf C., Vila L., Garcia-Aviles C., Chazot M., De Weck A.L. (2002) Flow-cytometric basophil activation test by detection of CD63 expression in patients with immedate type reactions to betalactam antibiotics. Clin Exp Allergy, 32: 277–286 63. De Weck A.L., Gamboa P.M., Sanz M.L. (2002) Clinical evaluation of in vitro tests in diagnosis of immediate allergic reactions to Betalactam antibiotics. ACI Int, 14: 185–192 64. Torres M.J., Padial A., Mayorga C., Fernandez T., Sanchez-Sabate E., Cornejo-Garcia A., Antunez C., Blanca M. (2004) The diagnostic interpretation of basophil activation test in immedaite allergic reactions to betalactams. Clin Exp Allergy, 34: 1768–1775 65. De Weck A.L., Sanz M.L., Gampoa PM, Aberer W., Bilo B., Blanca M., Torres M., Mayorga L., Campi P., Drouet M., Sainte-Laudy J., Romano A., Jermann T. Weber J.M and the ENDA group. (2006) Diagnosis of immediate-type Betalactam allergy in vitro by flowcytometry (Flow-CAST) and sulphidoleikotriene production. A multicentric study. EAACI Vienna, p 686 66. Gamboa P.M., Garcia-Aviles M.C., Urrutia I., Antepara I., Esparza R., Sanz M.L. (2004) Basophil activation and sulfidoleukotriene production in patients with immediate allergy to betalactam antibiotics and negative skin tests. J Invest Allergol Clin Immunol, 14: 278–283 67. Blanca M., Mayorga C., Torres M.J., Reche M., Moya M.C., Rodriguez J.L., Romano A., Juarez C. (2001) Clinical evaluation of Pharmacia CAP system RAST FEIA amoxicilloyl and benzylpenicilloyl in patients with penicillin allergy. Allergy, 56: 862–870

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68. Fontaine C., Mayorga C., Bousquet P.J., Arnoux B., Torres M.J., Blanca M., Demoly P. (2007) Relevance of the determination of serum specific IgE antibodies in the diagnosis of immediate Beta-lactam allergy. Allergy, 62: 47–52 69. Gamboa P.M., Sanz M.L., Caballero M.R., Antepara I., Urrutia I., Jauregui I., Gonzales G., Dieguez I., de Weck A.L. (2003) Use of CD63 expression as a marker of in vitro basophil activation and leukotriene determination in metamizol allergic patients. Allergy, 58:312–317 70. Sanz M.L., Gamboa P., de Weck A.L. (2005) A new combined test with flowcytometric basophil activation anddetermination of sulfidoleukotrienes Is useful for in vitro diagnosis of hypersensitivity to aspirin and other nosteroidal anti-imnflammatory drugs. Int Arch Allergy, 36: 58–72 71. Gamboa P., Sanz M.L., Caballero M.R., Urrutia I., Antepara I., Esparza I., de Weck A.L. (2004) The flow-cytometric determination of basophil activation induced by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is useful for in vitro diagnosis of the NSAID hypersensitivity syndrome. Clin Exp Allergy, 34: 1448–1457 72. De Weck A.L., Sanz ML, Gamboa PM, Aberer W, Sturm G., Blanca M, Torres M, Mayorga L, Correia S, Kowalski M, Medrala W, Sczeklik A, and ENDA group. (2006) Diagnosis of hypersensitivity to non-steroidal anti-inflammatory drugs (NSAIDs) in vitro by flowvytometry (Flow-CAST) and sulfidoleukotriene assay. A multicentric study. EAACI Vienna, p 653 73. De Weck A.L., Sanz M.L., Gamboa P.M. (2007) Further considerations on the mechanism of NSAID hypersensitivity. CIA Symposium (Ring, J., Holgate, S., Kay, B. Eds) Hogrefe & Huber, Göttingen, J World Allergy Org, Suppl. 2: 297–300 74. De Weck A.L., Gamboa P.M., Esparza R., Sanz M.L. (2006) Hypersensitivity to aspirin and other nonsteroidal ati-inflammarory drugs (NSAIDs) Curr Pharm Des, 2006; 12: 3347–3458 75. Schäfer D., Lindenthal U., Wagner M., Bölcksei P.L., Baenkler H.W. (1996) Effect of prostaglandin E2 on eicosanoid release by human bronchial biopsy specimens from normal and inflammed mucosa. Thorax, 51: 919–929 76. Szczeklik A., Stevenson D. (2003) Aspirin-induced asthma: advances in pathogenesis, diagnosis and management. J Allergy Clin Immunol, 111: 913–921 77. Sainte-Laudy J. (2002) Nine cases of suspected IgE-mediated anaphylaxis induced by aspirin. ACI Int, 14: 220–222 78. Kosnik M., Silar M., Bajrovic N., Music E., Korosec P. (2005) High sensitivity of basophils predicts side effects in venom immunotherapy. Allergy, 60: 1401–1406 79. Eberlein-König B., Schmodt-Leidescher C., Begrendt H, Ring J. (2006) Predicting sideeffects in venom immunotherapy by basophil activation ? Allergy, 61:897 80. Sainte-Laudy J., Le Provost A., André C., Vallon C. (1995) Comparison of four methods for human basophil activation measurement: alcian blue staining, histamine and LTC4 release and flow cytometry. In Proceedings of the 16th EAACI Congress (Basomba A., Hernadez M.D., De Rojas F. Eds), 257–260 81. Renz H., Becker W.M., Bufe A., Kleine-Tebbe J., Raulf-Heimsoth M., Saloga J., Werfel T., Worm M. (2002) In vitro Allergie-Diagnostik. Allergo J, 11: 492–506

CD203c for Allergy Diagnosis Vanessa G. Brown and Madeleine Ennis

Introduction Diagnosis of allergy is usually based on taking a careful clinical history supplemented with measurement of total and specific IgE, skin tests and in some cases organspecific challenges. Unfortunately, these tests do not always elicit the causative agent. Total IgE may be normal even in cases of severe allergy. The sensitivity and specificity of specific IgE measurements depends very much on the allergen being tested. Skin test reactivity may be reduced in the young or the elderly and adverse events may be induced in some susceptible individuals. Organ-specific challenges can be difficult to perform. Turning to other laboratory-based assays, measurement of plasma histamine or tryptase levels can indicate that a reaction has taken place. However, often more than one agent is given at the same time. These assays are also unable to be used when investigating cross-reactivity to other agents. For a laboratory-based assay to be accepted for clinical diagnosis ideally it should offer good sensitivity, specificity and positive and negative predictive values which have been established in well-conducted clinical trials; it must be reproducible and technically straightforward and it must be cost-effective. Since the initial description in 1994 assessing basophil activation using CD63 upregulation, flow cytometric methods have proven interesting to allergists. This review will compare and contrast the use of CD63 and CD203c as markers of basophil activation in the allergy diagnosis.

Basophil Activation Basophil activation induced by IgE-dependent (e.g. specific allergen, anti-IgE antibody) involves cross-linking of high-affinity IgE receptors which causes structural modification of the plasma membrane IgE receptor site. This causes V.G. Brown () and M. Ennis Respiratory Research Group, Centre for Infection and Immunity, Microbiology Building, Queen’s University Belfast, Grosvenor Road, Belfast BT12 6BN, Northern Ireland, UK e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_10, © Springer 2009

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activation of adenyl-cyclase which catalyses the transformation of ATP to cAMP. This in turn leads to an increase in the concentration of Ca + + in the cytosol causing release of granule associated basic molecules such as histamine (Fig. 1). These events cause the basophil to lose their staining affinity for basic dyes. If the reaction is intense, exocytosis of the granule occurs with the release of preformed (e.g. histamine and /or newly synthesised (e.g. LTC4) mediators, depending on the agonist used to activate the cells [1]). Microscopy has revealed two different pathways for basophil activation; anaphylactic degranulation, which is characterised by the fast morphological changes and exocytosis of intracellular granules or else by what is known as piecemeal degranulation, which involves slow morphological changes of the cytoplasmic granules that are packed into vesicles and transported to the plasma membrane with no intergranule or plasma membrane fusion [2].

Methods to Assess Basophil Activation Studies in the late 1980s/early 1990s implicating basophils in inflammation [3–6] and their ability to produce IL-4 and IL-13 [7–9], led to the development of methods for analysis of basophil activity based on membrane surface markers, such as IgE, and therefore redirected attention to this important cell. Originally, basophil activation was quantified by measurement of secreted histamine [10] and by microscopic examination of the percentage of degranulated cells [11]. However, small numbers of circulating basophils are a major limitation of the histamine release

Anti-IgE

Y

YY Adenylcyclase ATP

Preformed mediators -histamine -proteases -IL-3, IL-4, IL-5, IL-6 -GM-CSF, TNF

Ca++

cAMP

Newly formed mediators -leukotrienes -prostaglandins -thromboxanes -platelet-activating factor -free radicals

Fig. 1 Simplified scheme of the intracellular events in anti-IgE induced basophil degranulation and the exocytosis of granules leading to release of various mediators

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assay and more recently flow cytometry has been used to measure basophil activation by monitoring the upregulation of cell surface markers. Upon basophil activation, expression of CD45 [12], CD11b and CD11c [13] is upregulated, and CD62L expression is downregulated [13]. Two markers, CD63 and CD203c, have been mainly identified for use in an in vitro basophil stimulation assay, where activated basophils can be identified via flow cytometry. Changes in expression of basophilic surface markers can be quantified by multicolour flow cytometry using specific monoclonal antibodies.

CD63 As a Marker of Basophil Activation CD63 is a 53 kDa lysosomal associated membrane protein (LAMP) and belongs to the tetraspanin transmembrane-4 super family (TM4SF) subfamily 1. It is a type 1 integral membrane glycoprotein of late endosomes and lysosomes. It is expressed primarily on the cytoplasmic granules of various resting cells and only weakly expressed on the cell membrane. It has been shown that activation of human basophils in vitro by allergen or anti-IgE antibody results in degranulation and high-density surface expression of CD63 [14–16] after the fusion of cytoplasmic granules with the cell surface membrane. The kinetics of the increased CD63 expression and of the accompanying histamine release are very similar, and there is a strong positive correlation between these two events [16, 17]. Microscopic fluorimetry showed that the CD63 epitope was present intracellularly in resting basophils, and that CD63 expression was an all-or-nothing response with the level of response after stimulation varying from donor to donor [18]. Apart from expression on activated basophils, CD63 is present on platelets [19], endothelial cells [20], monocytes [21] and neutrophils [22]. CD63 has been shown to be predominantly expressed on basophils with little or no expression on B cells, T cells or monocytes [15] and using anti-IgE (early activation marker) to firstly identify basophils followed by analysis of the upregulation of CD63 expression (later activation marker) has been recommended [23]. A large number of studies using CD63 have been carried out across a wide range of allergens, unfortunately also with a number of different experimental protocols. Flow cytometry has been used to investigate food allergy [24–26], venom allergy [24, 27–31], house dust mite (HDM) [24, 32–34], pollen [17, 32–35], latex [36–39], and allergy to various drugs including muscle relaxants (MR) [40–42] and betalactam antibiotics [23, 43–45] and non-steroidal anti-inflammatory drugs [46, 47] with varying degrees of success. The mechanism by which basophils are activated appears to have a bearing on whether an increase in CD63 expression occurs [46]. A significant correlation has been reported between the flow cytometric method using CD63 and anti-IgE and histamine release assay after basophil activation by allergens or anti-IgE [17, 33, 48, 49] and the high level of sensitivity and specificity of the basophil activation test indicates that it can be a very reliable diagnostic tool.

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In subjects with food allergies, both the basophil activation test (CD63/IgE) and the LTC4 release test (LRT) were shown to be more reliable than histamine release tests [25]. The sensitivity of the CD63/IgE test has been shown to be increased in house dust mite-allergic and grass pollen-allergic patients following preincubation with IL-3, however the specificity of the assay decreased with increasing concentrations of lL-3 [32]. The CD63 basophil activation assay has also been used to diagnose venom allergy, with a high sensitivity and high specificity compared to other diagnostic tests such as skin tests, specific IgE, histamine and leukotriene release tests [27]. Similarly, a significant correlation has been observed between the CD63 assay and specific IgE test, histamine release and Ag-specific sulphidoleukotriene production (CAST) in subjects sensitised to Der p (house dust mite) and Lol P (timothy pollen) [33]. Venom allergy was investigated using flow cytometry along with specific IgE, histamine release, and Cys-LT release, with flow cytometry sensitivity and specificity reaching 100% with CD63 as the activation marker. Sensitivities for specific IgE, histamine release, and Cys-LT release were 88%, 89% and 100% respectively [27]. In contrast, MR allergy has been investigated using a typical IgE FITC/ CD63 PE protocol, and the observed sensitivity was 54%, with a specificity 100%. Sensitivity rose to 80% when flow cytometry was used in conjunction with specific IgE testing [41]. Another study, utilising the commercial Basotest® whole blood test kit, showed that Basotest® provided a characteristic dose–response curve for suxamethonium while indicating no response to other MR such as pancuronium, vecuronium and rocuronium [50]. In betalactam allergy, flow cytometry identified 50% of patients, giving better specificity and sensitivity than RAST, although the differences were not statistically significant. Again, sensitivity was raised when flow cytometry was used in conjunction with RAST, with 65.5% of patients identified [45]. A number of initial studies used CD45, the leukocyte common antigen, to assess basophil activation and one report suggests that 80% of basophils are identified using IgE and CD45 together [51]. CD45 is only overexpressed on activated basophils and therefore presents difficulties interpreting the percentage of activated cells. Monneret et al. published work using a tricolour flow cytometric method that incorporated using anti-CD45 and-CD63 together with anti-IgE, and showed using a small group of subjects a specificity of 100% [24]. Although the tricolour system is thought to be more efficient since it screens out interference from platelets, a number of studies using the tricolour assay for basophil activation showed variations in sensitivities and specificities [28, 40, 42, 52, 53] and that the contribution of platelets is negligible [14] (see Table 1). One drawback to the IgE/CD63 and/or CD45 basophil activation assay could be the potential of the IgE antibody to cause activation of the basophils and therefore generate false-positive results. Nevertheless, low concentrations of anti-IgE antibodies do not induce detectable basophil activation [23]. CD123 (IL-3R α chain), a marker for resting basophils [54], has been used with HLA-DR as an alternative to IgE [31, 55, 56], and the expression of CD123 has been reported as being less variable than surface IgE [57]. However, in subjects with grass pollen allergy,

Wasp venom

Latex Latex Bee/wasp/paper wasp venom Wasp/Bee venom

93.1% 50% 75% 93% 79.3% 91% Wasp 0.45 µg/ml–78.6% 0.045 µg/ml–25.6% 0.0045 µg/ml–7.1 % Bee 0.45 µg/ml–50% 0.045 µg/ml–35.7% 0.0045 µg/ml–0% 92%

85% 72%

80%

91.7% 100% 100% 100% 96.7% 90% 100%

100% 92%

IgE/CD63

17% (ROC curves) At least 2 sequential dilutions = >10% positive basophils 10% (ROC curves) 22% (ROC curves) 5% (no ROC curves) >15% (according to manufacturers instructions)

Latex Latex

15% (ROC curves)

[58]

Basotest® FastImmune (CD123+/ HLA-DR-/CD63+) IgE/CD63 IgE/CD45/CD63 IgE/CD45/CD203c FAST Basotest® IgE/CD45/CD63 Basotest®

Grass pollen

[30] (continued)

[38] [39] [28] [29]

[36] [37]

[35]

Basotest®

Cypress pollen

[32] [33] [34]

91–100% 98.4% 96% 100% 100%

IgE/CD63 IgE/CD63 CD123+/HLA-DR-/CD63+

56–70% 93.3% 96% 82% 91.2%

10% (no ROC curves) 15% (ROC curves) 18%(16 µg/ml) 8% (1.6 µg/ml) (ROC curves) >15% (according to manufacturers instructions) > 9.5% (ROC curves)

HDM HDM, timothy pollen HDM

Reference

Test

Table 1 ROC analysis to determine best cut-off with regard to highest sensitivity and specificity Allergen Cut-off Sensitivity Specificity

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Celery Carrot Neuromuscular blocking agents Neuromuscular blocking agents Neuromuscular blocking agents Rocuronium bromide Metamizol

Apple

Food allergies Carrot Celery Hazelnut Apple

Anisakis

>10% for ≥ 2 successive concentrations >10% (according to manufacturers instructions) 4% (ROC curves) 5% SI ≥ 5 (for any two concentrations)

15% (no ROC curves)

15% (CD63) 14% (CD203c) (ROC curves) 33% (15 µg) 21% (1.5 µg) 8% (0.12 µg) (ROC curves) ≥5% for ≥ 2 successive concentrations 8.9% 6.3% 6.7% (ROC curves) 10%b 17%c (ROC curves) 10% (mean basophil activation + 4SD)

25% (2.5-fold increase in number activated cells)

Bee venom

Wasp venom Bee/wasp venom

Cut-off

Allergen

Table 1 (continued)

100% 100% 100% 100% 100%

79% 36% 91.7% 42.3%

86% 82% 93%

83.3% 100% 89% 78% 96% 89% 97% 85% 80% 80% 100% 75% 64%

90%

Specificity

54%

65% 75% 64%

85.3% 89a, 88, 83% 97a, 73, 66% 97% 100% 91% 58% 85% 85% 85% 100% 88% 75%

91.3%

Sensitivity

IgE/CD45/CD63 OR CCR3/CD24/CD63 IgE/CD45/CD63 IgE/CD45/CD203c CD123+/HLA-DR-/CD63+ FAST

IgE/CD45/CD63

[56] [82]

[42]

[41]

[40]

[81]

[80]

IgE/CD63 IgE/CD63

[25] [26]

[55]

[65]

[31]

Reference

IgE/CD63 IgE/CD63

Basotest® IgE/CD203c CD123+/HLA-DR-/CD63+

CD123+/HLA-DR-/CD63+

Test

188 V.G. Brown and M. Ennis

5% (ROC curves) plus SI >2 SI ≥ 2 5% (ROC) SI ≥ 2 @ any 2 concentrations, plus activation ≥ 5% ≥ 5% above basal level plus SI >2 SI ≥ 2 @ at least 1 concentration >2 SD above controls 8.1% (mean +2SD control) 25% 49% 20% 60% 100%

15–55% 42.85% 50% 39.1% 100% 91% 79% 100% 95% 95%

74–100% 100% 93.3% 93.3% IgE/CD63 Basotest® IgE/CD45/CD63 IgE/CD45/CD203c IgE/CD45/CD63 IgE/CD45/CD203c

FAST Basotest® FAST FAST

[53]

[23] [45] [66]

[83] [47] [43] [44]

1.38 SI (mean + 2SD control) 95% HDM: house dust mite, NSAIDs: non-steroidal anti-inflammatory drugs; FAST: flow cytometry stimulation test; SI: stimulation index; SD: standard deviation a With regard to history, skin test and radioallergosorbent test (RAST) respectively b OAS subjects (birch pollen allergy and apple-mediated oral allergy syndrome) versus healthy controls c OAS subjects (birch pollen allergy and apple-mediated oral allergy syndrome) versus subjects with birch pollen allergy

Betalactams Betalactams Betalactams (amoxicillin anaphylaxis) Cat

Aspirin & other NSAIDs NSAIDs Betalactams Betalactams

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a lower percentage of CD63 + basophils were obtained with the FastImmune assay (CD63/CD123/HLA-DR) compared to the Basotest®, possibly due to the different stimulation and activation protocols [58]. More recently a study has identified analysis of p38 phosphorylation in combination with CD63 as a potential new marker for allergy (birch) [59], and will require further studies to investigate its use in other allergies or in situations where CD63 is not reliable.

CD203c As a Marker of Basophil Activation In 1999, Bühring et al. showed that another marker, CD203c, is upregulated in response to IgE-dependent basophil activation [60] and in recent years this has been suggested as a potential marker for basophil activation in allergy diagnosis. CD203c is also known as ectonucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) [61]. It is a type II transmembrane protein, and cleaves a variety of phosphodiester and phosphosulphate bonds, and may be involved in the clearance of extracellular nucleotides. Compared to CD63, CD203c has been described as being selectively expressed by basophils, mast cells and by their CD34 + progenitors [60, 61]. It seems that the upregulation of CD63 and CD203c follow different kinetics, with rapid CD203c upregulation (reaching maximum levels at 5–15 min of stimulation) compared to CD63 (20–40 min) [62]. In addition to their differing kinetics, CD63 expression is more similar to anaphylactic degradation [62]. However, upregulation of CD203c is always accompanied by upregulation of CD63, and as is the case with CD63, upregulation of CD203c and degranulation occur simultaneously [63].

CD63 Compared to CD203c The first study to directly compare allergen-induced upregulation of CD63 and CD203c, revealed a strong correlation between both markers of basophil activation and concluded that due to the specificity of CD203c for blood basophils, such a method may have several advantages over CD63 when used in allergy diagnosis [63]. Following this, a number of studies showed that CD203c is a sensitive marker for grass allergens [64] and may be more sensitive than CD63 for the diagnosis of latex allergy [37], venom allergy [65] and amoxicillin allergy [66] (see Table 1). Initially, CD203c expression was shown to increase significantly on allergen challenge, which indicates that CD203c plays a role in, and may be functionally involved in, IgE-dependent activation of basophils [67]. Some authors are critical of this upregulation and the increase in sensitivity using CD203c compared to CD63, suggesting that although CD203c is upregulated, it is not as obvious as CD63 upregulation [68, 69]. However, as mentioned earlier, there are a number of differences in the activation and regulation of both CD63 and CD203c, and more

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recently it has been shown that CD203c expression can be upregulated by a nonIgE mediated mechanism (IL-3) with no effect on CD63 [70]. A comparative study of CD63 and CD203c in subjects with allergies to drugs used in the perioperative period showed that the sensitivities and specificities for CD63 and CD203c were 79% and 100%, and 36% and 100% respectively [42]. In cat allergy sensitivities of 100% and 95% and specificities of 95% and 95% for CD63 and CD203c positive basophils were reported. [53]. The only other study directly comparing CD63 and CD203c staining, found an improvement in the sensitivity of basophil activation analysis using the CD203c assay to detect latex allergy (75% compared to 50%) [37]. A number of functional and technical issues may explain discrepancies between studies.

Methodological Aspects Although CD203c is a specific basophil marker, staining with anti-IgE may be critical for the precise gating of basophils with low levels or basal CD203c expression due to the low spontaneous expression of the marker [37, 53]. Also, in patients with high spontaneous levels of CD203c, false-negative results can be observed where upregulation may be completely absent and it has been recommended to exclude patients with severe inflammation [71]. Interleukin 3 activates human blood basophils via high-affinity binding sites and perhaps similar to CD63, the inclusion of an IL-3 preincubation step may also increase the sensitivity of the CD203c assay [31]. The majority of published studies using CD63 have stimulated the basophils with IL-3 with concentrations varying from 2 to 60 ng/ml (Fig. 2). On average between 10% and 20% of donors have little or no response to stimulation by anti-IgE [72, 73] and therefore IL-3 has been recommended for use in IgE nonresponders to increase the CD203c response [74, 75]. Studies using CD203c as the activation marker are not as numerous as those using CD63 and therefore it is very difficult to compare directly the efficacy of preincubation with IL-3 with CD63 compared to CD203c (Fig. 3). Overall it has been suggested that IL-3 preincubation is not necessary but may be useful in low sensitivity drug allergen analysis [76].

31%

Fig. 2 Percentage of CD63 studies incubating basophils with (light grey, n = 28) [14, 23, 25–27, 29, 30, 32, 33, 35, 36, 38, 39, 44, 45, 47, 53, 55, 56, 58, 65, 80–87] and without (dark grey, n = 13) IL-3 stimulation [24, 28, 31, 34, 37, 40–42, 53, 63, 66, 67, 74]

69%

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Fig. 3 Percentage of CD203c studies incubating basophils with (light grey, n = 2) [53, 74] and without (dark grey, n = 9) [37, 42, 53, 63–67, 74] IL-3 stimulation 18 %

82 %

Fig. 4 Percentage of studies using whole blood and isolated basophils for the assessment of CD63 upregulation (light grey; whole blood n = 25 [24, 26–30, 32, 34–37, 39, 41, 42, 45, 47, 53, 55, 56, 58, 63, 65, 67, 80, 81, 86] (dark grey; separated leukocytes by either dextran sedimentation, histopaque separation or centrifugation n = 13 [14, 23, 25, 33, 38, 40, 43, 44, 66, 82, 83, 86, 88, 89]

33 % 67 %

Protocols for the CD63- and CD203c-based assays of basophil activation have employed whole blood, leukocyte preparations or purified basophil samples. Using whole blood is easier and faster to prepare than separated cells, although it is possible that thrombocytes, which also express CD63, can adhere to basophils in whole blood samples [77], potentially resulting in false positive results. Since CD203c is selectively expressed by basophils, mast cells and their progenitors, the potential to use whole blood in this particular assay may seem greater than in the CD63 assay (Fig. 4). However, there are still several disadvantages: a reduction in the overall numbers of basophils per sample, interference with serum components will reduce the sensitivity of the assay and there may be interference by aggregated platelets. Indeed a recent review on some of the technical issues surrounding the basophil activation test, outlines that the sensitivity of using either whole blood or separated cells varies with allergen [76].

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Importance Issues Regarding the Allergen

Number of studies

The importance of developing standard dose response curves for each allergen has recently been emphasised [78], and it has been suggested that for protein allergies, two allergen concentrations are sufficient, while for drug allergies three concentrations have been recommended [76]. Several studies have shown that there is a dose-dependent increase in CD63 and/or CD203c in response to pollen [35, 63, 64], food [26], drugs [40, 43], latex [38] and venom [67]. Similarly, it is important to establish the optimal incubation times for the relevant assay, since both CD63 and CD203c have differing activation kinetics. CD63 is maximum at around 25–30 min and CD203c around 10–20 min, which declines after 15–20 min [62]. Despite this, incubation times vary considerably from study to study (Fig. 5). For CD203c, time course experiments have shown that for birch and grass pollen and bee and wasp venom allergies the optimal time for allergen incubation is 15 min [63, 67]. It is possible that reducing the incubation time by combining the allergen with the antibody to CD203c, could considerably shorten the overall time of the assay with minimal affect on assay performance [74]. Platz et al. investigating CD203c in sensitised individuals found that recombinant allergens were the preferred reagents in response to hymenoptera venom allergy [67]. Natural allergens have numerous disadvantages over recombinant allergens, in particular the heterogeneity of the extract by contamination with unwanted materials or allergens from other sources and therefore variations between batches may occur and jeopardise concluding data. Indeed the use of recombinant allergens has increased over the years with increasing availability and have been recommended for use in allergy diagnosis [74, 79] 13 12 11 10 9 8 7 6 5 4 3 2 1 0 5

10

15

20

30

40

45

Incubation time (mins)

Fig. 5 Variation in length of time for allergen incubation in studies using CD63 (black bars [23, 25–30, 32–45, 47, 53, 56, 66, 67, 80–83, 86, 88]) and CD203c (white bars [37, 42, 53, 64, 66, 67, 74]). Only those studies that specifically state an incubation time have been included

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Analysis of Results Activation of basophils is calculated as a percentage of the total basophil population or as change in mean fluorescence intensity (MFI). When expressing data as percentage positive basophils, it is necessary to establish a negative cut-off level. When mean fluorescence intensity is used, the increase in CD63 or CD203c expression is given as a relative value (stimulation index, SI), which is a ratio of MFI of stimulated cells to MFI of control cells. It is generally accepted that a stimulation index greater than 2 is positive basophil activation. Receiver Operator Characteristic (ROC) curves have not been produced for all allergens in order to select an appropriate cut off point. ROC curves have become widely accepted for describing and comparing the accuracy of tests and are important to achieve the highest sensitivity by an optimal specificity. Published cut-off values for various stimulants are outlined in Table 1 and discrepancies between studies may be due to several technical factors, including the method used to determine the threshold of positivity. One CD63 study into latex allergy used a ROC curve to set a 17% increase in activation as the cut off point to achieve sensitivity and specificity exceeding 90% [36], whereas similar studies into latex allergies set a 10% or 22% increase in activation as the cut off using ROC curves [38, 39]. A shortage of studies using CD203c means that comparisons of cut-off values for CD203c with the same allergen are limited. Two studies investigating bee and wasp venom allergies with the CD203c assay used different criteria for setting the threshold of positivity – Binder et al. used a SI of greater or equal to 1.4 [74], while Eberlein-König et al. used ROC curves to establish a cut-off of 14% [65].

Conclusion The specificity of CD203c on the surface of basophils led to the development of a novel basophil activation assay. Current evidence regarding the use of CD203c as a diagnostic test in allergic diseases suggests that it may offer more quantitative and reliable results compared to other tests based on mediator release and may be just as useful as or perhaps more useful than the CD63 assay. However, the functional differences between CD63 and CD203c and the suggestion that these markers examine two different mechanisms of basophil activation [62] means that each method will have its own advantages and disadvantages, which has led to the recommendation of using both CD63 and CD203c for the precise analysis of allergen reactivity [75]. In order for flow cytometric-based assays of basophil activation to become part of the routine tests for allergy, the methodology needs to be optimised and standardised, and specifically in the case of CD203c where there is limited data available, still requires further validation before it becomes a widely accepted clinical test. This will include careful investigation of the type of sample required (whole blood etc.); the role of IL-3 preincubation; the optimum time and

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allergen concentration(s) for stimulation; the calculation of allergen specific cut-off points etc. This must be performed in a large enough sample size and using appropriate controls and comparator tests.

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Flow-Assisted Analysis of Basophils: A Valuable Instrument for In Vitro Allergy Diagnosis Didier G. Ebo and Chris H. Bridts

Summary It is clear that the basophil activation test (BAT) that rests upon flow-assisted quantification of in vitro-activated basophils provides the physician with a novel diagnostic tool that could rapidly spread because of the numerous flow cytometers already set in clinical and experimental laboratories. Nowadays, the technique has been adopted for the diagnosis of inhalant allergy, natural rubber latex allergy, primary and secondary food allergies, hymenoptera venoms allergy and allergy for certain drugs. Applying donor basophils, the technique has proven to be useful in the detection of auto-antibodies responsible for chronic autoimmune urticaria. As the technique closely resembles the in vivo pathway ultimately leading to symptoms, it has already been successfully adopted for other objectives such as differentiation between clinically relevant and irrelevant IgE antibody results, quantitative evaluation of (residual) allergenicity of recombinant proteins and allergoids, component resolved diagnosis, to assess efficacy of anti-IgE treatment, to select and monitor specific immunotherapy and to study signalling in basophils. Finally, because basophils can be stimulated ex vivo, they provide the theoretical potential of measuring more than specific IgE, but rather a biological response from cells that were, until shortly before the assay, subject to the host’s milieu.

The Basophil Basophils are granulocytes that develop from CD34 + pluripotent progenitor stem cells, differentiate and mature in the bone marrow, and circulate in the periphery where they represent less than 1% of the leukocytes. Basophils exhibit a segmented nucleus and are identified by means of metachromatic staining with basic dyes, D.G. Ebo () and C.H. Bridts University of Antwerp, Faculty of Medicine, Immunology, Allergology, Rheumatology, Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerpen, Belgium e-mail: [email protected]

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Table 1 Mechanisms of basophil activation [65] FcεRI-dependent FcεRI-independent Allergens (Glyco)proteins Chemicals Auto-antibodies (>IgG) To IgE To FcεRI

Receptors for endogenous substances Chemokines Complement (anaphylatoxins) Cytokines IgG (FcγR) Neuropeptides β2-adrenergics Glucocorticoids Histamine These receptors can also be triggered by pharmacological interaction and auto-antibodies Receptors for exogenous substances fMLP (FPRL receptors) Formyl peptides Bacterial peptideglycans (via TLR) FPRL: formyl peptide receptor-like (also known as FPRH: formyl peptide receptor-homologue) TLR: toll-like receptors (e.g. TLR2 and TRL4)

such as toluidine blue. Human basophils express a variety of cytokine receptors: interleukin (IL)-3R [α-chain (CD123), β-chain (CD131)], IL-5R [α-chain (CD125), β-chain (CD131)], IL-18R [α-chain (CDw218a), β-chain (CDw218b)], GM-CSFR (CD116), tumour necrosis factor (TNF)-related apoptosis-inducing ligand receptor (TRAIL)-R1 (CD261) and TRAIL-R2 (CD262), chemokines (CCR1 [CD191], CCR2 [CD192], CCR3 [CD193], CCR7 [CD197], CXCR1 [CD181]), complement (CD11b, CD11c, CD35 and CD88), prostaglandin D2 (CRTH2 or PD2) (CD294) and immunoglobulin Fc receptors (FcεRI and FcγRII) [1–4]. Basophils express also transmembrane activator and calcium-modulator, and cyclophilin ligand interactor (TACI) (CD267), toll-like receptor-4 (CD284), leukocyte-associated immunoglobulinlike receptor (LAIR)-1 (CD305), EMR-2 (CD312) a member of the adhesionG-protein-coupled receptors (GPCR) family, junctional adhesion molecule-1 (JAM1 or CD321) and JAM2 (CD322) [4]. Human basophils, in contrast to their murine counterparts, do not express FcγRIII receptors [5]. The basophil can be activated by a number of pathways, which may be differentiated whether or not the high-affinity IgE receptor (FcεRI) is involved (see Table 1). The requirements for effective FcεRI cross-linking and subsequent degranulation are explained elsewhere [6]. It is clear that both cellular (FcεRI-IgE) and allergen characteristics determine the final outcome.

Flow-Assisted Allergy Diagnosis: Principles The basis of flow-assisted allergy diagnosis is analysis and quantification of alterations in expression of basophilic surface membrane markers. Electron microscopic studies have revealed that basophil activation may follow two distinct

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pathways: (i) ‘anaphylactic’ degranulation (AND), which is characterised by quick morphological changes, exocytosis of the intracellular granules and release of presynthesised mediators; and (ii) piecemeal degranulation (PMD) characterised by slow morphological changes without exocytosis of the granules [7, 8]. Basophils, upon encounter with allergen that is recognised by FcεRI-bound specific IgE, not only secrete and generate quantifiable mediators (e.g. histamine, leukotrienes, IL-4, IL-13), but also up-regulate the expression of different activation markers. These alterations can rapidly be detected on a single-cell basis by (multicolour) flow cytometry using specific fluorochrome-conjugated monoclonal antibodies. At present, the most commonly used markers in flow-assisted analysis of in vitro basophil activation experiments are CD63 and CD203c.

Sampling and Preservation It is obvious that correct sampling and preservation of the blood is of critical importance in order to obtain optimal cellular viability and functionality. At the University of Antwerp a whole-blood technique is applied and analysis is performed within 3 h after sampling in preservative and endotoxin-free heparinised tubes that are kept at room temperature. When cell isolation techniques are used, blood is normally anticoagulated with ethylenediaminetetraacetic acid (EDTA) or acid citrate dextrose (ACD).

Whole Blood or Isolated Basophils Flow-assisted analysis and quantification of in vitro-activated basophils can be applied either upon whole heparinised blood or upon isolated cells. White bloods cells can easily be isolated using centrifugation techniques or density gradient techniques, whereas when pure basophils are needed more advanced techniques like magnetic bead isolation or cell sorting will be needed. The use of whole blood has the main advantage of being very practical with simple manipulation (less centrifugation steps) mirroring physiological conditions. However, interference with serum components such as IgG-blocking antibodies, anti-IgE and anti-IgE receptor antibodies, C5a, cytokines, drugs, as well as basophil inhibition by co-aggregation of FcεRI and FcγRII (principally the immunoreceptor tyrosine-based inhibitory motifs [ITIM]-containing FcγRIIB), cannot entirely be excluded. Alternatively, cell separation is generally accompanied by loss of basophils and in vitro activation of cells can occur. Currently, there is insufficient evidence to state one of the techniques to be superior for allergy diagnosis. For proteinaceous allergens such as natural rubber latex, pollen, house dust mite, foods and hymenoptera venom both techniques give rather similar results in terms of sensitivity and specificity. For smaller chemical allergens such as drugs a technique based upon isolated cells might be more sensitive. However, recently a sensitivity of 91.7% was attained for rocuronium in a whole

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blood-based BAT in which basophils were gated out upon SSC/CD123/HLADR and activation was quantified by CD63 [9].

Stimulation Conditions Basophils react immediately upon activation and are highly dependent on stimulation conditions requiring optimal temperature and optimal incubation times, as well as appropriate composition of buffer in which reagents have to be diluted. Even prewarming of reagents can be necessary to reach optimal stimulation conditions (personal observation).

Pre-activation or Priming with Interleukin-3 IgE-mediated basophil activation can either be inhibited or enhanced by several compounds. For example, histamine can act as an autocrine inhibitor via binding the basophilic H2-receptor leading to sustained cAMP production and decreased calcium response [10]. Alternatively, interleukin-3 (IL-3) can considerably enhance basophil activation, though the precise mechanisms remain to be established [11]. Recent data indicate that stimulation of human endothelium with IL-3 induces selective basophil accumulation in vitro [12]. There is no general consensus on the need of priming with IL-3 to improve sensitivity of the BAT. Although some authors have demonstrated that short preincubation with IL-3 might increase sensitivity of CD63-based assays [13, 14], this seems not be critical for proteinaceous allergens [15] and stimulation with serum of patients with autoimmune urticaria [16]. On the other hand, when investigating allergens that elicit relative little basophil activation such as drugs, increase of assay sensitivity might be relevant. Priming with IL-3, by shifting dose–response to the left, might allow widening the sometimes narrow gap between specific allergen stimulation and unspecific activation or cytotoxicity as IL-3-primed basophils may react to allergen concentrations a 10- to 100-fold lower than those for non-primed cells [17]. For CD203c-based assays, priming with IL-3 might be detrimental [14]. The differential influence of IL-3 priming on CD63 and CD203c might (to some extent) explain contradictory results and contribute to the controversy in the literature regarding which is the ‘best’ marker for measuring basophil activation [18].

Characterisation of Basophils (Whole-Blood Technique) When the intended application is the diagnosis of IgE-mediated allergy, most studies have currently applied an anti-IgE antibody to identify basophils in peripheral blood. The amount of IgE on the basophils varies greatly between different donors,

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ranging from 6,000 to 600,000 molecules per cell, and is related to the concentration of IgE in the serum [19]. The number of FcεRI per basophil ranges from approximately 30,000 to 700,000 between different donors and is also related to the concentration of IgE in the serum [20]. It has emerged that it is IgE itself that regulates expression of its high-affinity receptor [21, 22]. Ligand-mediated upregulation of FcεRI by IgE is biphasic. First, IgE stabilises receptor complexes at the surface and protects them from degradation. Subsequently, once all FcεRI are at the surface, continued expression is maintained by synthesis of new complexes from pre-existing transcripts [23]. It has been argued that other cells (e.g. monocytes) that also express FcεRI and CD63 might interfere with the test and that exposure of basophils to anti-IgE prior to allergen stimulation may alter their responsiveness [24, 25]. However, compared with basophils these cells demonstrate distinct side-scatter characteristics and the average cell-surface FcεRI expression by monocytes is low [26]. In difficult cases an additional staining with anti-HLADR can help to discriminate between HLA-DR− basophils and HLA-DR+ monocytes (see Fig. 1). Alternatively, anti-IgE antibodies applied for basophil characterisation do not induce cell activation. Furthermore, in classical basophil activation assays, cells are exposed to an anti-IgE for flow cytometric identification only after the stimulation phase and presumed additional activation by anti-IgE is avoided by fixing and/or cooling the cells between allergen stimulation and staining with a fluorochrome-conjugated anti-IgE. CD203c (PD-Iβ, B10, gp130RB 13-6) is a glycosylated type II transmembrane molecule that belongs to the family of ecto-nucleotide pyrophosphatase/ phosphodiesterase (E-NPP) enzymes that catalyse the hydrolysis of oligonucleotides, nucleoside phosphates and NAD (for review see [27]). Among haematopoietic cells, expression of CD203c is restricted to basophils, mast cells and their precursors, and has been described as specific for this lineage [28]. As in peripheral blood, CD203c expression is exclusively and constitutionally expressed on the surface of resting basophils, it can be applied for basophil characterisation. However,

Fig. 1 (a) Determination of basophils with a combination of CD123 and side scatter. CD123 + cells are gated out and shown in (b), where dendritic cells are HLA-Dr-positive and basophils are HLA-Dr-negative. Activated basophil is CD63+. (c) Shows a histogram of the activated CD63positive basophils (red) in combination with a negative blank (open)

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spontaneous expression of CD203c by resting basophils can be weak hampering clear characterisation of the cells [14]. Alternatively, as CD203c expression quickly up-regulates upon encounter of the basophil with specific allergen (see below), it offers the advantage of studying the basophil activation without additional staining in a ‘single-colour BAT’. Apart from the usefulness of analysing basophil activation for allergy diagnosis, anti-CD203c is also a suitable reagent to define and purify viable basophils, including their precursor cells, using paramagnetic separation procedures [29]. Prostaglandin D2 (PGD2), a product of the arachidonic acid/cyclo-oxygenase pathway, is generated at sites of inflammation and is believed to exert a key regulatory function in inflammatory and immune responses. Being predominantly released by resident mast cells [30], PGD2 has a prominent role in allergic reactions [31, 32], due to its ability to induce extravasation of leukocytes and by acting as a chemoattractant for Th2 cells, eosinophils and basophils [33–36]. To date, two distinct G-protein-coupled receptors for PGD2 have been described; the D prostanoid receptor (DP or PD1) [37] and the chemoattractant receptor-homologous molecule expressed by Th2 lymphocytes (CRTH2, PD2, CD294) [38, 39]. Actually, PGD2 favours Th2 functions via CRTH2 while restraining Th1 functions via DP [40, 41]. Basophils can readily be discriminated from CRTH2 carrying eosinophils and TH2 cells on the basis of light scattering and an additional staining with anti-CD3, respectively [42]. Finally, some studies [43–47] have applied the combination of anti-CD123 and anti-HLADR to characterise basophils in peripheral blood. CD123 is a glycosylated type I transmembrane molecule that belongs to the cytokine receptor superfamily. CD123 is the primary low-affinity subunit of the IL-3 receptor that associates with CD131, the common β-chain of the IL-3, IL-5 and GM-CSF receptor, to form the high-affinity IL-3 receptor. The IL-3 receptor is involved in cell signalling for cell growth and differentiation. In peripheral human blood, the CD123 antigen is expressed at high levels only on plasmacytoid dendritic cells and basophilic granulocytes but at low levels also on monocytes, eosinophilic granulocytes, myeloid dendritic cells and subsets of haematopoietic progenitor cells. However, in contrast to basophils these cells stain positive with an anti-HLADR antibody. CD123 expression appears to be less variable than expression of IgE and is independent of the allergy status of the donor. CD123 is not present on mast cells [48]. For a review on CD123 the reader is referred elsewhere [49].

Assessment of Basophil Activation At present, the most commonly used markers in basophil activation experiments are CD63 and CD203c. The major characteristics of CD63 and CD203c are summarised in Table 2. CD63 (gp 53 or lysosyme-associated membrane protein [LAMP]-3) is a member of the transmembrane-4 superfamily and is expressed by different cell types

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Table 2 Characteristics of CD63 and CD203c [102] Synonyms

Gp53 or lysosyme-associated membrane protein (LAMP)-3

(Super)family

Transmembrane-4 superfamily (tetraspanins) Barely detectable (need for additional marker to identify basophils, e.g. IgE or CD123/HLADR) Up-regulation within 10 min (RT)

Resting basophils

IgE-activated basophils

IL-3 priming Parallel expression

Neural cell-surface differentiation antigen E-NPP-3 (also: PD-Iβ, B10, gp130RB 13–6) Pyrophosphatase/phospodiesterases (E-NPPs) Constitutively expressed (no need for additional marker to identify basophils)

Up-regulation stars within minutes and peaks within 5–10 min Expressed with a high density Up-regulation less prominent (at least 1 log scale) on a when compared with CD63, subpopulation of the basophils but present on all basophils No effect on expression Up-regulated expression (takes +90 min) CD107a (LAMP-1), CD107b CD13, CD164 (LAMP-2)

E-NPP: ecto-nucleotide pyrophosphatase/phosphodiesterase RT: room temperature (up-regulation maximal after 5 min for stimulation at 37°C)

such as basophils, tissue mast cells, macrophages and platelets [50–52]. In resting basophils, CD63 is anchored at the intracytoplasmatic granules and is barely expressed on the outer cell membrane, both in healthy individuals and allergic patients. In contrast, as a result of the fusion of the granule and the outer cell membrane, CD63 is expressed with a high density on IgE-activated cells [53]. It has been argued that a fraction of CD63 might come from contaminating activated platelets that adhere to basophils. However, platelets from patients do not up-regulate CD63 expression in response to stimulation with allergen or anti-IgE [54, 55] and a vast majority of anti-IgE+/anti-CD63+ cells have been phenotyped as CD45+, CD14−, CD16− and CD41− [56], CD41 being the gpIIB antigen expressed by platelets and megakaryocytes. Basophil activation can also been assessed by quantification of the up-regulation of CD203c (see section on Characterisation of Basophils in Whole Blood). Recently, Hennersdorf et al. [57] screened over 260 monoclonal antibodies for reactivity with resting and anti-IgE-activated CD203-positive basophils. They identified four additional markers of basophil activation (CD13, CD107a, CD107b and CD164) and helped to elucidate upon the observation that the pathways of CD63 and CD203c up-regulation are clearly distinct [58, 59]. Actually, Hennersdorf et al. [57] demonstrated that the lysosyme-associated membrane proteins CD107a (LAMP-1), CD107b (LAMP-2) and CD63 (LAMP-3) are up-regulated concomitantly, according to their localisation on the membrane of intracellular granules. These granules contain the pre-synthesised bioactive mediators that are released upon FcεRI cross-linking and are distinct from the small vesicles associated

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with piecemeal degranulation. It is this second pathway to be associated with up-regulation of the transmembrane glycoprotein sialomucin endolyn (CD164) and the ecto-enzymes CD13 (gp150, N-aminopeptidase or human myeloid plasma membrane glycoprotein) and CD203c. These two pathways can be distinguished by kinetics, pharmacological inhibitors and differing sensitivities to various stimulants. For example, both IL-3 and prostaglandin D2 rapidly induce up-regulation of CD203c, but not CD63 [14, 58–61]. The observation that CD203c up-regulation might also result from IL-3 stimulation might adversely affect the CD203c assay by decreasing sensitivity of the assay [14]. Comparative studies have shown that CD63 and CD203c are clearly different in their up-regulation profile. Up-regulation of CD63 is generally bimodal with a subpopulation cells that express CD63 with a high density versus a population with almost no expression. Up-regulation of CD203c is generally less prominent but often occurs in almost all cells. By consequence results for CD63 expression can be expressed as percentage activated CD63+ basophils, whereas results for CD203c are expressed as stimulation indexes of mean fluorescence [14, 57, 59] (see Fig. 2).

Allergen Selection Proper selection of allergen is of paramount importance that cannot be overemphasised. Currently, flow-assisted analysis and quantification of in vitro-activated basophils generally relies upon the use of natural allergen extracts that have proven to be quite satisfactory. Nevertheless, it should be kept in mind that extracts prepared from natural source material might be heterogeneous with varying composition, presence of non-specific stimulatory and/or inhibitory stimulatory components such as preservatives, endotoxins and lectins. Potency of extracts might significantly decrease over time, due to spontaneous degradation of the constituent proteins. Therefore, extreme care must be taken in preparing and conserving allergen extracts. It is absolutely mandatory that these extracts should be properly and repeatedly characterised. To some extent, these issues can be circumvented by the use of isolated and/or recombinant allergens that are becoming available for an increasing number of allergens. Moreover, flow-assisted allergy diagnosis using isolated and/or recombinant allergens might be a highly reliable assay for component-resolved allergy diagnosis [25, 62, 63]. Commercially available skin test extracts are rarely suitable for basophil activation experiments, as they contain cytotoxic and/or inhibitory preservatives. To some extent, dialysing the extract might solve this problem. For drugs, the injectable (intravenous and/or intramuscular) formulation is recommended. Although some companies provide numerous allergen extracts for basophil activation experiments, they are rarely clinically validated.

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Fig. 2 Triple-stain (IgE/CD203c/CD63) dot plots of a patient allergic to tree pollen and grass pollen (above) and a negative control. Basophils (red) were selected on high-positive IgE, while dendritic cells (green) have a lower IgE density on their membrane. Plots show a different upregulation in CD63- and CD203c-positive basophils

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Positive and Negative Control Stimulation It has to be emphasised that for correct interpretation in each assay, relevant negative and positive control stimulation has to be included. In most publications the positive control is a polyclonal or monoclonal anti-IgE antibody. An attractive alternative positive stimulant is monoclonal anti-FcεRI antibody [14, 64]. In the absence of positive control stimulation, patients with unresponsive basophils would simply be recorded as (potentially false) negatives. As formyl-methionylleucylphenylalanine (fMLP) acts via a G-protein-coupled receptor (FPR-1) that activates MAP kinase pathways and phospholipase C and does not mimic an IgE-mediated activation of the basophil, it cannot be recommended as an appropriate positive control. However, fMLP can be applied as a tool to assess viability of the cells. Negative control stimulation, to assess spontaneous expression of the activation markers, generally implies incubation of the cells with stimulation buffer at the same volume of the allergens.

Dose-Finding It has been argued that basophil responses tend to be highly heterogeneous and demonstrate a considerable inter- and intra-individual variability [65]. This manifests itself in terms of ‘cellular reactivity’ (i.e. maximal secretory response after optimal IgE-mediated stimulation) that is determined by the intracellular signal transduction and ‘cellular sensitivity’ (i.e. threshold for distinct cellular response). The latter is governed by the total FcεRI cell-surface density, ratio of membranebound allergen sIgE antibodies over total IgE and the so-called intrinsic sensitivity (i.e. number of IgE molecules required for half-maximal response). In clinical practice, however, this does not appear to be a major issue. In most individuals, basophil responses demonstrate a clear up-regulation of CD63 and/or CD203c spanning several log scales offering the opportunity to restrict activation experiments to one or two ‘optimal’ stimulation concentrations that discriminate between patients and controls [66–70]. There is generally no need to expand analysis over more stimulation concentrations, irrespective of the nature of the allergen (proteinaceous, chemical). On the other hand, the evaluation of diagnostic tests cannot be considered as appropriate when it failed to address the possibility of conditions that could give rise to values outside the normal range. Immunochemical cross-reactivity between allergens has clearly been demonstrated to be of major importance in allergy testing, mainly affecting the specificity of the assay. In accordance with others [71], we demonstrated that basophils from patients with birch pollen allergy would degranulate when stimulated with high concentrations of apple extract, irrespective the presence or absence of a birch pollen-associated apple allergy [68]. Nevertheless, using an optimum allergen stimulation concentration and ROC analysis to define

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the best CD63 percentage threshold between birch pollen-allergic patients with and without apple allergy, sensitivity and specificity of the BAT was 88% and 75%, respectively. Another important reason to perform dose-finding experiments is to establish the allergen stimulation concentrations that evoke maximal and sub-maximal cell activation, respectively. Recently, in contrast to others [72], we demonstrated that the BAT can help to monitor successful venom immunotherapy, provided the cells are challenged sub-maximally [70]. It is also of paramount importance to identify stimulation concentrations that are cytotoxic or result in unspecific stimulation of the cells, as this might result in false-negative and false-positive results, respectively. Finally, optimal stimulation concentrations may vary from one protocol to another (whole blood versus purified basophils, priming with IL-3, etc.).

Expression of Results Results can be expressed in two ways: first, as percentages of activated basophils that are corrected by subtracting spontaneous expression (negative control) from the value obtained with allergen stimulation; and second, as a mean fluorescence intensity (MFI) stimulation index (response allergen/basal). The latter being particularly applied for CD203c-based assays, as up-regulation of CD203c by activated cells, is less prominent and often occurs on almost all basophils hampering correct discrimination between negative (non-activated CD203c+low) and positive (activated CD203c+bright) cells [14, 73].

Non-responders Any use of BAT must contend with the issue of ‘non-responders’; that is, individuals whose cells are unresponsive to FcεRI cross-linking. This phenomenon was already observed in early studies quantifying histamine release, and the prevalence has been estimated at 10–20%. These non-responders also fail to up-regulate expression of CD63 and CD203c [14, 73]. Several investigations have identified defects in early intracellular signal transduction involving the tyrosine kinases lyn and syk [74–76]. For some ‘non-responders’ IL-3 can reverse the defect in vitro after several days of exposure [75, 77]. Although these non-responders represent 5–10% of patients and control individuals tested, they are (too) often not reported. Obviously, this is not correct. Non-responders not only influence sensitivity and specificity of the assay, but most importantly non-responsiveness can result in false-negative results with potentially dangerous implications for the individual patient. In fact, in analogy with skin tests, if positive control stimulation results in non-responsiveness of the cells, it is impossible to correctly interpret negative allergen stimulation. For such individual

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patients the tests are lost as a diagnostic instrument, whereas for study purposes such results should be considered as ‘false negatives’ and be reported. False-negative results might also be explained by recent exposure to the allergen, resulting in a temporary refractoriness of the cells. Alternatively, as a result of consumption during the acute event, patients might have transient decreased levels off allergen-specific circulating and surface-bound IgE antibodies. On the other hand, sensitivity of the assay might significantly decrease over time [78]. Therefore, it is recommended to perform analysis between 6 weeks and 12 months after the acute event. As already reviewed elsewhere [79], intake and/or administration of drugs, particularly glucocorticosteroids and antihistamines can influence basophil activation.

Decision Thresholds It cannot be over-reemphasised that one should always establish optimal allergenspecific thresholds and abandon predefined and usually arbitrarily chosen decision thresholds relative to spontaneous expression for determination of sensitivity, specificity and predictive value of a test. All possible combinations of sensitivity and specificity that can be achieved by changing the test’s cut-off can be summarised using a single parameter, i.e. the area under the receiver operating characteristic (ROC) curve. The recommendation to apply ROC analysis to calculate discriminative thresholds has now been followed by the majority of groups publishing on the BAT.

Clinical and Research Applications Analysis of Patient Basophils Currently, flow-assisted analysis and quantification of in vitro-activated basophils proved reliable for the diagnosis of several IgE-mediated allergies including classical inhalant allergens (house dust mite, cat epithelium, pollen), hymenoptera venom allergy, natural rubber latex allergy, primary and secondary food allergies and drug allergies (see Table 3). In individual patients the BAT confirmed diagnosis of different food [80] and drug [81–85] allergies. Moreover, in some of the patients with drug allergy the BAT contributed to establish the individual therapeutic alternative and/or allowed identification of potentially cross-reactive structures. In venom allergy, it was recently demonstrated the BAT to constitute an additional diagnostic tool for patients that yield equivocal or negative sIgE and/or skin test results. In addition, the technique allowed component resolved diagnosis of pollen allergy [25], venom allergy [62] and natural rubber latex allergy [63]. The technique was also repeatedly applied to assess and to compare the (residual) allergenicity of

Table 3 Clinical applications [99] Stimulus Reference test Inhalant allergens House dust mite Dactylis glomerata Cypress pollen HDM and Lolium perenne Cat epithelium (Fel d 1) Latex Latex Latex Latex Latex (Hev b5, Hev b 6.01, Hev b 6.02) Hymenoptera venom Wasp Wasp Bee Bee Wasp Wasp Drugs NMBA NMBA NMBA NMBA Rocuronium (NMBA) β-Lactam β-Lactam Metamizol Aspirin and NSAID Food Carrot, celery, hazelnut Apple Apple Apple (Mal d 1) Celery (Api g 1) Carrot (Dau c 1) Autoimmune urticaria Serum of patients with AIU

Sens

Spec

N

Reference

H + IgE and/or ST H + IgE and/or ST H + ST + PT H + IgE + ST H + IgE and/or ST

56–78a 73–100 91 93 100a

91–100 100 100 98 95

20 20 75 128 39

[13] [13] [103] [104] [14]

H + IgE + ST H + ST H + IgE and/or ST H

93 93 80 96

92 100 97 100

102 73 79 33

[105] [64] [106] [63]

H H H H H H

92 85 91 100 97 99b

80 83 90 100 100 100

70 87 87 12 39 94

[72] [44] [44] [106] [106] [70]

H H ± ST H H + ST H + ST H + ST H ± ST ± IgE ± PT H± PT H ± PT

64 54 79 36–86c 92 50 49 42 15–55

93 100 100 93 100 93 91 100 74–100

50 60 24 92 22 88 110 55 90

[55] [107] [108] [78] [46] [109] [110] [111] [112]

H (OAS) H (OAS) H (OAS) H (OAS) H (OAS) H (OAS)

85–90 100d 88 75 75 65

80–90 100 75e 68 77 100

20 59 59 54 54 54

[66] [68] [68] [113] [113] [113]

H + ASST 20 70 65 [96] H + ASST 96 91 64 [47] Fel d 1: major allergen from cat (Felis domesticus); Sens: sensitivity; Spec: specificity; N: total number of patients and controls; NMBA: neuromuscular blocking agent; OAS: oral allergy syndrome; AIU: autoimmune urticaria; H: history; ST: skin test; PT: provocation tests; ASST: autologous serum skin test. a In the studies by Cozon et al. [13] and Ocmant et al. [14] there is some evidence for IL-3 to increase sensitivity of the CD63-based BAT. b Taken into account the non-responders sensitivity is 84%. c In the study by Kvedariene et al. [78] it is demonstrated sensitivity of the BAT to decrease over time. d Taken into account the non-responders sensitivity is 90%. e In an additional comparison between birch pollen-allergic patients with and without appleinduced OAS.

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natural allergen extracts, chemically modified allergoids and recombinant allergens [25, 67, 86–90]. Finally, the technique was applied to evaluate passive IgE sensitisation through blood transfusion [91].

Analysis of Non-atopic/atopic Donor Basophils Flow cytometric analysis and quantification of in vitro-activated basophils can also be applied on cells obtained from non-atopic and/or atopic donors. In a first application, generally referred as the passive sensitisation procedure, (stripped) donor basophils, prior to allergen challenge, are pre-incubated with serum of the patient. These passive sensitisation protocols to detect allergen-specific IgE in the patients’ serum, however, are quite laborious, difficult to standardise and highly dependent on the reactivity of the donor cells. In addition, passive sensitisation assays have been proven to be less sensitive than the direct basophil activation assays [56]. Apart from the diagnostic applications, passive sensitisation protocols have also been applied to assess functional differences between peanut sIgE antibodies [92], to functionally analyse cross-reactivity of sIgE antibodies [93, 94] and to characterise allergens [95]. A second application comprises demonstration of functional auto-antibodies against the α-chain of the high-affinity IgE receptor (FcεRI), or less commonly, against IgE itself in patients with auto-immune chronic urticaria [47, 96–98]. These auto-antibodies are able to induce histamine from basophils and mast cells via a direct cross-linking of adjacent IgE or IgE receptors [99].

Future Perspectives To date, flow cytometric basophil activation techniques have focused on changes in membrane morphology. With the advent of multi-parameter flow cytometric analysis, subsets of cells can also be defined on the different kinase activation states. It is understood that signalling cascades play an important role in nearly every cell activation step [100]. In mast cells and basophils cross-linking of FcεRI induces a cascade of signalling events that eventually results in degranulation and release of mediators. As more and more monoclonal antibodies against phosphoproteins and kinases becomes available, flow cytometric techniques can be used to correlate subpopulations with the activation state of kinases in signalling pathways. To achieve this, many technical considerations must be addressed including accessibility of the phospho-epitope, surface phenotype integrity and antibody suitability for staining inside fixed and permeabilised cells [101]. As a concept of proof, phosphorylated MAPK p38 was shown to be correlated with allergen-specific basophil activation [114,115] (Fig. 3).

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Fig. 3 Basophils are gated out on an IgE+ and side-scatter gate (left). Two scatter plots demonstrate that phosphorylated MAPK p38 is maximally unregulated in basophils after 3 min activation with latex antigen (centre) and is diminished after 20 min while CD63 is maximally unregulated (right). Quadrants are chosen on cells without activation at the time points used

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Recombinant Allergens for the Diagnosis and Treatment of House Dust Mite Allergy Martin D. Chapman and L. Karla Arruda

Introduction Allergen-specific immunotherapy has been extensively used to treat patients with allergic diseases. Controlled studies provided evidence of the efficacy of immunotherapy as part of the treatment of patients with asthma, allergic rhinitis, and those with severe reactions to hymenoptera venoms [1–3]. In some cases, long-term effects of immunotherapy have been demonstrated, several years following discontinuation of treatment, and preventive effects on development of additional sensitizations or asthma have become evident [1, 4]. At present, immunotherapy is carried out using extracts from natural sources. Although some extracts are currently standardized based on biological potency, many extracts remain nonstandardized. The absolute and relative concentration of individual allergens in a given extract may differ, reflecting the composition of the raw material. Although major allergens are defined by the ability to be recognized by the majority of allergic patients, individual patients may present different reactivity profiles. In addition, natural extracts contain antigenic proteins, other antigens, and macromolecules which are not relevant to the treatment. It is possible that efficacy and safety of allergen immunotherapy could be improved by the use of well-defined and purified allergen preparations. Over the past 30 years, allergens from most relevant sources have been identified, characterized, and produced as recombinant proteins with biological activity comparable to that of their natural counterparts. Therefore, recombinant allergens may be used as part of novel strategies to improve immunotherapy [5, 6]. Recombinant allergens: (i) provide standardized and known doses of allergen; (ii) provide balanced doses of allergens; (iii) correct for allergens not well represented in natural extracts; (iv) allow hypoallergenic variants with less IgE reactivity to be generated M.D. Chapman Indoor Biotechnologies, Inc., Charlottesville, VA, USA L.K. Arruda () Department of Medicine, School of Medicine of Ribeirão Preto – University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil e-mail: [email protected]

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(these variants retain T-cell reactivity and theoretically would be effective and safer, allowing higher doses to be administered); and (v) formulate vaccines to include the most relevant allergens in defined concentrations and to match them to sensitizations of individual patients [7]. In this regard, in vitro methods such as CAP FEIA, streptavidin CAP assay, multiplex flow cytometric assay, and microarrays have been successfully used to determine IgE reactivity profiles in individual patients [7–10]. These assays may differentiate true IgE-mediated sensitization from allergenic cross-reactivity. In order to use recombinant allergens for immunotherapy, approval for use in humans requires preparation of allergens under good manufacturing practice (GMP) conditions. This requires extensive characterization of the allergen preparation, including stability of the construct, fermentation conditions, purity, structure, potency, and stability of the final product, as well as preclinical animal studies to ensure safety of the product. Demonstration of safety and efficacy in controlled studies is necessary. Finally, an important issue is to define whether all allergens are necessary or just the major specificities. The first immunotherapy trials have been conducted with recombinant grass pollen allergens and hypoallergenic variants of the major birch pollen allergen Bet v 1, and showed that recombinant allergen-based immunotherapy has effects that are similar to those induced by natural allergen vaccines [11–13]. The results indicated that recombinant allergen-based immunotherapy will improve current immunotherapy practice and may open possibilities for new treatment strategies. In this chapter, we will review the current perspectives for using recombinant mite allergens for immunotherapy to treat patients with asthma and allergic rhinitis.

Mite Allergens House dust mites are among the most common sources of indoor allergens worldwide. It is well established that sensitization to house dust mite allergens is a major cause of asthma and allergic rhinitis in many areas of the world [14–16]. The principal domiciliary species worldwide are Dermatophagoides pteronyssinus and D. farinae; Blomia tropicalis is also abundant in tropical and subtropical areas [16]. Analysis of house dust mite extracts has indicated that over 20 different proteins can induce IgE antibody in patients allergic to the house dust mite. However, the group 1 and group 2 allergens account for much of the allergenicity of extracts. Up to 85% of mite allergic patients have positive skin test and/or serum IgE to group 1 and group 2 allergens, and in some patients, IgE antibodies to these allergens may account for most of the IgE to Dermatophagoides, and contribute to a large portion of the total IgE [15–17]. A complete list of mite allergens which have been characterized to date is presented in Table 1. Sequence analysis revealed that several Dermatophagoides allergens have homologues in other domiciliary species such as B. tropicalis and Euroglyphus maynei, as well as in storage mite species. Mite tropomyosin (group 10) allergens show extensive sequence identity to tropomyosins from other invertebrates including cockroach,

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Table 1 Official list of mite allergens by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) allergen nomenclature subcommitteea Group MW (SDS-PAGE) (kDa) Function Species 1 2

24, 25, 27, 39 15, 16

Cysteine protease Dp, Df, Dm, Em, Bt Nieman-Pick disease Dp, Df, Em, Ld, Tp, type C2 family Gd, Bt 3 29, 31 Trypsin Dp, Df, Em, Bt 4 56, 60 α-Amylase Dp, Bt, Em 5 14 Unknown Dp, Bt, Ld 6 25 Chymotrypsin Dp, Df, Bt 7 26, 30, 31 Unknown Dp, Df, Ld 8 27 Glutathione-S-transferase Dp 9 29 Collagenolytic serine protease Dp 10 33, 36, 37 Tropomyosin Dp, Df, Bt, Ld, Tp 11 98, 103, 110 Paramyosin Dp, Df, Bt 12 14 Unknown Bt 13 15 Fatty acid-binding protein Df, Bt, Ld, Tp, As 14 177 Apolipophorin Dp, Df, Em 15 98/109 Chitinaseb Df 16 53 Gelsolin/villin Df 17 53 Ca-binding EF protein Df 18 60 Chitinaseb Df 19 7 Antimicrobial peptide Bt 20 n.a. Arginine kinase Dp Dp: Dermatophagoides pteronyssinus; Df: D. farinae; Dm: D. microceras; Ds: D. sibonei; Em: Euroglyphus maynei; Bt: Blomia tropicalis; Ld: Lepidoglyphus destructor; Tp: Tyrophagus putrescentiae; Gd: Glycyphagus domesticus; As: Acarus siro a Last updated on March 3, 2009 (www.allergen.org). Limited information available for Der p 21, Blo t 21, Der f 22, Der p 23 and Tyr p 24. b The cDNA for Dermatophagoides pteronyssinus chitinase Der p 15 encodes mature proteins of 58.8 and 61.4 kDa, showing 90% identity to Der f 15; cDNA for D. pteronyssinus chitinase Der p 18 codes for a mature protein of 49.2 kDa, with 88% sequence identity to Der f 18. Both Der p 15 and Der p 18 bind to IgE in allergic human sera, and are important allergens for humans as well as dogs.

shrimp, and Ascaris lumbricoides, and may be involved in clinically relevant IgE cross-reactivity [18]. The tertiary structure of the group 1 and group 2 allergens has been determined from recombinant proteins and it is an excellent model for the investigation of modified allergens. An important property of the group 1, group 2, and group 3 allergens is the high degree of polymorphism found by cDNA analysis.

Immunotherapy with Mite Extracts Specific immunotherapy with mite extracts administered by the subcutaneous route has provided clinical benefits to patients with asthma and/or allergic rhinitis, demon strated in several double-blind, placebo-controlled trials and systematic reviews of

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the literature [1–3, 19, 20]. More recently, the use of house dust mite extracts in sublingual immunotherapy (SLIT) [21, 22] has been shown to be effective for treatment of pediatric and adult patients with rhinoconjunctivitis and/or asthma, and selected patients with atopic dermatitis [23, 24]. Sublingual immunotherapy (SLIT) with mite extracts has been used with increasing frequency in Europe. Most successful studies of mite immunotherapy have used native allergen extracts adsorbed onto aluminum hydroxide, or chemically modified mite-allergen extracts.

Recombinant Mite Allergens for Diagnosis The efficacy of using recombinant allergens for diagnosis has been investigated by both in vitro and in vivo methods. Skin testing data with recombinant wild-type allergens has been available for a large panel of allergens derived from mites, cockroach, grass, birch and olive pollens, molds, latex, venom allergens, and food allergens. Schmid-Grendelmeier and Crameri compiled the available studies (25 published studies, 1,600 allergic individuals tested with 21 different recombinant allergens), and reported that no increased side effects have occurred with recombinant allergens, using concentrations ranging from 1 to 100 µg/ml, and occasionally up to 1,000 µg/ml for skin prick testing, and doses between 10−2 and 10 µg/ml for intradermal testing [25]. A study was conducted in Brazil to investigate the diagnostic efficacy of recombinant mite allergens in a large, well-defined group of Brazilian patients with asthma and/or rhinitis. A group of 120 patients was selected based on the presence of positive skin tests to D. pteronyssinus extract. Recombinant allergens, rDer p 1, rDer f 1, rDer p 2, rDer p 5, and rBlo t 5, produced in Escherichia coli or Pichia pastoris, were used separately in skin testing or formulated as cocktails, designated DM1 and DM2, that included both the group 1 and Der p 2 allergens. Serum IgE to Der p 1 and Der p 2 were quantitated by chimeric enzyme-linked immunosorbent assay (ELISA). Recombinant allergens were tested at 5 and 50 µg/ml concentrations. The results showed that the prevalence of positive skin tests to group 1, Der p 2, and group 5 allergens was 78%, 85%, and 48–55%, respectively; and to DM1 and DM2 was 95% and 98%, respectively. Wheal sizes of skin reaction induced by DM1 and DM2 and to D. pteronyssinus extract showed significant correlation. There was also a strong correlation of the presence of positive skin tests and detectable serum IgE antibodies to Der p 1 and Der p 2. In addition, prick tests to natural and recombinant Der p 1 and Der f 1 were compared. There was a significant correlation between wheal diameters to nDer p 1 and rDer p 1 (r = 0.73, p < 0.0001) and to nDer f 1 and rDer f 1 (r = 0.87, p < 0.0001). Skin tests using recombinant allergens were well tolerated and did not cause adverse reactions. The conclusion of the study was that cocktails of recombinant allergens can be safely and effectively used for diagnosis of mite allergy among patients with asthma and/or rhinitis [26]. Previous small-scale studies using rDer p 2, rDer p 5, and rDer p 7 at concentrations up to 100 µg/ml, to skin test children with asthma and/or rhinitis in Venezuela, also revealed a high prevalence of positive skin tests to these allergens [27].

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In that parasite-endemic region, testing with purified Der p 2 was better for discriminating clinically relevant allergy from a “nonsymptomatic atopic.” In addition, a hypoallergenic variant of the mite allergen Der p 2, generated by site-directed mutagenesis, showed marked reduced IgE reactivity on skin testing, while maintaining T-cell epitopes [28]. Similar studies were conducted with the storage mite allergen Lep d 2 and a variant in which all six cysteine residues were replaced with serine. The variant induced smaller and fewer skin prick test reactions in the 17 patients tested, and dermal infiltrates and the number of activated eosinophils were also reduced [29]. Skin test reactivity to recombinant Blo t 5, a major B. tropicalis allergen, has also been studied among patients from Taiwan and Singapore, and shown to be comparable to the natural allergen [30, 31]. A panel of seven purified natural or recombinant D. pteronyssinus allergens (nDer p 1, nDer p 4, rDer p 2, rDer p 5, rDer p 7, rDer p 8, rDer p 10) has been used to establish IgE reactivity profiles in an European mite-allergic population. A combination of IgE immunoblotting, CAP FEIA measurements, and IgE competition studies was carried out. The results revealed that more than 95% of the patients could be diagnosed with a combination of nDer p 1 and rDer p 2, and that miteallergic patients who were mainly sensitized to the major D. pteronyssinus allergens (Der p 1, Der p 2) could be discriminated from patients with a broad sensitization profile, including highly cross-reactive allergens (e.g., Der p 10, tropomyosin) as well as reactivity to storage mites. The conclusion was that testing with individual allergens may improve the diagnostic selection of patients for immunotherapy with D. pteronyssinus extracts, and could be useful for monitoring of patients undergoing immunotherapy [7].

Immunotherapy with Recombinant Allergens: Clinical Trials with Tree and Grass Pollen Allergens The first study of allergen-specific immunotherapy with recombinant preparations investigated the clinical effects and immunological responses in patients with rhinoconjunctivitis allergic to birch pollen, using two hypoallergenic preparations of the major birch pollen allergen Bet v 1: a mixture of two Bet v 1 fragments and a Bet v 1 trimer, in comparison to placebo. Immunotherapy was carried out with a course of eight pre-seasonal injections of increasing concentrations from 1 to 80 µg total protein, with further injections up until the beginning of the pollen season [12]. Results showed that both active preparations induced Bet v 1-specific IgG1, IgG2, and IgG4 antibody responses as well as an IgA response. Correlations of IgG1 antibody titers and both improvement in clinical symptoms, as judged by a ten-point interval scale, and reduction in skin test reactivity to Bet v 1 were demonstrated. Measurement of Bet v 1-specific IgE responses showed a threefold increase in the placebo group as a consequence of seasonal pollen exposure, whereas the responses in the two treatment groups were blunted. A double-blind placebo-controlled clinical trial with a mixture of recombinant grass pollen allergens was conducted with 62 grass pollen allergic patients suffering

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from rhinoconjunctivitis with or without asthma [13]. The mixture of recombinant allergens was administered in subcutaneous injections of increasing concentrations at 7-day intervals, with doses up to 10 µg Phlp1, 5 µg Phlp2, 10 µg Phlp5a, 10 µg Phlp5b, and 5 µg Phlp6 (40 µg total protein in 0.8 ml). Maintenance injections were continued until after the subsequent pollen season with a 50% reduction during each pollen season. A combined symptom–medication score showed a 39% improvement in the active treatment group relative to placebo (p = 0.041). Symptoms alone improved by 37% (p = 0.015) and the use of symptomatic medication decreased by 36.5% relative to placebo. Data from a validated rhinitis quality of life questionnaire, administered following the maximum pollen count, demonstrated benefits for those subjects on active treatment during the first pollen season, but even greater differences were seen between active and placebo treatment during the second season with an overall significant benefit (p = 0.024). Active treatment induced highly significant increases in both IgG1 and IgG4 grass pollen-specific antibody concentrations together with a significant decrease in IgE. IgG4 achieved an approximately 4,000-fold increase by the end of treatment. Adverse events related to treatment were seen in association with 78 injections (10.7%) in the active treatment group and 44 injections (5.9%) in the placebo group. Local reactions accounted for nearly all these reactions; single systemic reactions including urticaria, dyspnea, rhinoconjunctivitis, and asthma were seen in seven active treatment and two placebo subjects. Conclusions from clinical trials with recombinant allergens revealed that the use of cocktails of grass pollen allergens, or hypoallergenic variants of Bet v 1, showed safety of the use of recombinant preparations, as well as immunologic effects and clinical improvement.

Future Perspectives for the Use of Recombinant Mite Allergens for Immunotherapy Up to now, no clinical trials conducted with recombinant mite allergens for immunotherapy have been reported. It is clear that allergen-specific immunotherapy using crude mite extracts to treat mite-allergic patients with asthma and/or rhinitis, and perhaps selected children with atopic dermatitis, is an effective adjuvant treatment in conjunction with environmental control measures and pharmacological therapy. Most currently available commercial mite extracts are standardized based on biological activity, and contain sufficient amounts of major group 1 and group 2 allergens to ensure development of an immunological response which underlies improvement of clinical outcomes. However, these extracts also contain several non-allergenic components which may be difficult to measure and/or standardize, including mite DNA and endotoxin, which may or may not be relevant for a successful treatment [32]. Using recombinant mite allergens for immunotherapy would provide an opportunity to perform a “proof-of-concept” investigation, to determine whether the beneficial effect is actually allergen-specific. One attractive strategy

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would be to use a panel of recombinant mite allergens to establish the reactivity profile of individual patients, and use the information to prepare a customized vaccine. However, the feasibility of this strategy needs to be evaluated. Alternatively, a limited number of allergens could be chosen, and in this regard, the major allergens Der p 1 and Der p 2 could be good candidates for the following reasons: (1) most mite-allergic patients show strong IgE reactivity to group 1 and group 2 allergens; (2) high-level expression systems for production of biologically active group 1 and group 2 allergens have recently become available; and (3) it is possible that vaccines with group 1 and group 2 allergens would be sufficient to induce a beneficial effect, through a bystander effect towards reactivity to other mite allergens the patient might be eventually sensitized to. In fact, it is thought that the clinical and immunological effects of the currently used commercial extracts are due mainly to high contents of group 1 and group 2 allergens in the extracts, with other allergens being usually underrepresented. Another interesting possibility would be to investigate the use of recombinant mite allergens for sublingual immunotherapy; preliminary data suggest that wild-type allergens, rather than modified allergens, should be used for this treatment modality [11]. Once the efficacy and safety of immunotherapy with recombinant mite allergens are established in controlled trials, the use of modified, hypoallergenic allergens could be investigated, with the aim of reducing undesired side effects of immunotherapy and allowing higher maintenance doses to be achieved. The efficacy of this strategy could be therefore compared with that of the use of wild-type recombinant allergens or crude commercial allergen extracts for immunotherapy.

Conclusions Over the past 30 years there has been great progress in identification of allergens and production of these molecules as recombinant proteins with biological activity similar to their natural counterparts. Recent studies reporting the use of recombinant pollen allergens in clinical trials revealed that these highly purified, well-characterized proteins could be safely and effectively used in immunotherapy, to treat pollen allergic patients. At preclinical level, constructs of recombinant peanut and bee venom allergens have caused decreasing of symptoms and reduction of IgE responses. A series of studies using recombinant mite allergens for skin testing and in vitro testing demonstrated that recombinant allergens could be used safely for diagnostic purposes and may be helpful in establishing IgE reactivity profiles of individual patients, increasing specificity over currently available allergenic extracts. Although, as yet, clinical trials with recombinant mite allergens have not been conducted, the major allergens are well defined and these trials should proceed once pharmaceutical grade recombinant allergens are available. These trials will allow different formulations of recombinant mite allergens to be compared for safety and efficacy and the immunologic mechanisms of the response to be evaluated. Ultimately, this should improve current treatment modalities for mite-allergic patients.

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Acknowledgments Dr. L. Karla Arruda’s research on recombinant mite allergens in Brazil is supported by CNPq – Instituto de Investigação em Imunologia – iii. She is a recipient of a CNPq scholarship.

References 1. Joint Task Force on Practice Parameters; American Academy of Allergy, Asthma and Immunology; American College of Allergy, Asthma and Immunology; Joint Council of Allergy, Asthma and Immunology. Allergen immunotherapy: a practice parameter second update. J Allergy Clin Immunol 2007; 120(3 Suppl.):S25–85. 2. Abramson MJ, Puy RM, Weiner JM. Allergen immunotherapy for asthma (Cochrane review). In: The Cochrane Library, Issue 1, 2006. Oxford: Update Software. 3. Calderon MA, Alves B, Jacobson M, Hurwitz B, Sheikh A, Durham S. Allergen injection immuno therapy for seasonal allergic rhinitis. Cochrane Database Syst Rev 2007; 1:CD001936. 4. Larché M, Akdis CA, Valenta R. Immunological mechanisms of allergen-specific immunotherapy. Nat Rev Immunol. 2006; 6:761–771. 5. Chapman MD, Smith AM, Vailes LD, Arruda LK, Dhanaraj V, Pomés A. Recombinant allergens for diagnosis and therapy of allergic disease. J Allergy Clin Immunol 2000; 106:409–418. 6. Valenta R, Niederberger V. Recombinant allergens for immunotherapy. J Allergy Clin Immunol. 2007; 119:826–830. 7. Pittner G, Vrtala S, Thomas WR, Weghofer M, Kundi M, Horak F, Kraft D, Valenta R. Component-resolved diagnosis of house-dust mite allergy with purified natural and recombinant mite allergens. Clin Exp Allergy 2004; 34:597–603. 8. Satinover SM, Reefer AJ, Pomes A, Chapman MD, Platts-Mills TAE, Woodfolk JA. Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens. J Allergy Clin Immunol 2005; 115:803–809. 9. King EM, Vailes LD, Tsay A, Satinover SM, Chapman MD. Simultaneous detection of total and allergen-specific IgE by using purified allergens in a fluorescent multiplex array. J Allergy Clin Immunol 2007; 120:1126–1131. 10. Hiller R, Laffer S, Harwanegg C, Huber M, Schmid W, Twardosz A, Becker WM, Blaser K, Brelteneder H, Chapman MD, Crameri R, Duchene M, Ferreira F, Fiebig H, HoffmannSommergruber K, King TP, Kleber-Janke T, Kurup VP, Lehrer SB, Lidholm J, Müller U, Pini C, Reese G, Scheiner O, Scheynius A, Shen H, Spitzauer S, Suck R, Swoboda I, Thomas W, Tinghino R, van Hage-Hamsten M, Virtanen T, Kraft D, Müller MW, Valenta R. Microarrayed allergen molecules: Diagnostic gatekeepers for refined allergy treatment. FASEB J 2002; 16:414–416. 11. Cromwell O, Fiebig H, Suck R, Kahlert H, Nandy A, Kettner J, Narkus A. Strategies for recombinant allergen vaccines and fruitful results from first clinical studies. Immunol Allergy Clin N Am 2006; 26:261–281, vii. 12. Niederberger V, Horak F, Vrtala S, Spitzauer S, Krauth M-T, Valent P et al. Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc Nat Acad Sci USA 2004; 101(Suppl. 2):14677–14682. 13. Jutel M, Jaeger L, Suck R, Meyer H, Fiebig H, Cromwell O. Allergen-specific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol 2005; 116:608–613. 14. Platts-Mills TA. The role of indoor allergens in chronic allergic disease. J Allergy Clin Immunol 2007; 119:297–302. 15. Erwin EA, Rönmark E, Wickens K, Perzanowski MS, Barry D, Lundbäck B, Crane J, PlattsMills TA. Contribution of dust mite and cat specific IgE to total IgE: relevance to asthma prevalence. J Allergy Clin Immunol 2007; 119:359–365. 16. Thomas WR, Smith WA, Hales BJ, Mills KL, O’Brien RM. Characterization and immunobiology of house dust mite allergens. Int Arch Allergy Immunol 2002; 129:1–18.

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17. Trombone AP, Tobias KR, Ferriani VP, Schuurman J, Aalberse RC, Smith AM, Chapman MD, Arruda LK. Use of a chimeric ELISA to investigate immunoglobulin E antibody responses to Der p 1 and Der p 2 in mite-allergic patients with asthma, wheezing and/or rhinitis. Clin Exp Allergy 2002; 32:1323–1328. 18. Santos AB, Chapman MD, Aalberse RC, Vailes LD, Ferriani VP, Oliver C, Rizzo MC, Naspitz CK, Arruda LK. Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens. J Allergy Clin Immunol 1999; 104:329–337. 19. Fernández-Caldas E, Iraola V, Boquete M, Nieto A, Casanovas M. Mite immunotherapy. Curr Allergy Asthma Rep 2006; 6:413–419. 20. Cohon A, Arruda LK, Martins MA, Guilherme L, Kalil J. Evaluation of BCG administration as an adjuvant to specific immunotherapy in asthmatic children with mite allergy. J Allergy Clin Immunol 2007; 120:210–213. 21. Cox LS, Linnemann DL, Nolte H, Weldon D, Finegold I, Nelson HS. Sublingual immunotherapy: a comprehensive review. J Allergy Clin Immunol 2006; 117:1021–1035. 22. Calamita Z, Saconato H, Pelá AB, Atallah AN. Efficacy of sublingual immunotherapy in asthma: systematic review of randomized-clinical trials using the Cochrane Collaboration method. Allergy 2006; 61:1162–1172. 23. Ozdemir C, Yazi D, Gocmen I, Yesil O, Aydogan M, Semic-Jusufagic A, Bahceciler NN, Barlan IB. Efficacy of long-term sublingual immunotherapy as an adjunct to pharmacotherapy in house dust mite-allergic children with asthma. Pediatr Allergy Immunol 2007; 18: 508–515. 24. Pajno GB, Caminiti L, Vita D, Barberio G, Salzano G, Lombardo F, Canonica GW, Passalacqua G. Sublingual immunotherapy in mite-sensitized children with atopic dermatitis: a randomized, double-blind, placebo-controlled study. J Allergy Clin Immunol 2007; 120:164–170. 25. Schmid-Grendelmeier P, Crameri R. Recombinant allergens for skin testing. Int Arch Allergy Immunol 2001; 125:96–111. 26. Genov IR, Jorge PPO, Trombone APF, Tobias KRC, Ferriani VPL, Smith AM, Chapman MD, Arruda LK. Use of cocktails of recombinant allergens for diagnosis of mite allergy in patients with asthma and/or rhinitis. J Allergy Clin Immunol 2002; 109:S163 [Abstract]. 27. Lynch NR, Puccio FS, Di Prisco MC, Escudero JE, Nozzolino M, Hazel LA, Smith WA, Thomas WR. Association between allergic disease and reactivity to recombinant Der p 2 allergen of house dust mites in a tropical situation. J Allergy Clin Immunol 1998; 101:562–564. 28. Smith AM, Chapman MD, Taketomi EA, Platts-Mills TA, Sung SS. Recombinant allergens for immunotherapy: a Der p 2 variant with reduced IgE reactivity retains T-cell epitopes. J Allergy Clin Immunol 1998; 101:423–425. 29. Kronqvist M, Johansson E, Whitley P, Olsson S, Gafvelin G, Scheynius A, van Hage-Hamsten M. A hypoallergenic derivative of the major allergen of the dust mite Lepidoglyphus destructor, Lep d 2.6Cys, induces less IgE reactivity and cellular response in the skin than recombinant Lep d 2. Int Arch Allergy Immunol 2001; 126:41–49. 30. Yi FC, Chua KY, Cheong N, Shek LP, Lee BW. Immunoglobulin E reactivity of native Blo t 5, a major allergen of Blomia tropicalis. Clin Exp Allergy 2004; 34:1762–1767. 31. Tsai JJ, Yi FC, Chua KY, Liu YH, Lee BW, Cheong N. Identification of the major allergenic components in Blomia tropicalis and the relevance of the specific IgE in asthmatic patients. Ann Allergy Asthma Immunol 2003; 91:485–489. 32. Trivedi B, Valerio C, Slater JE. Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 2003; 111:777–783.

Diagnosis of Occupational Rhinitis Roy Gerth van Wijk

Introduction Occupational rhinitis refers to rhinitis caused by stimuli from the occupational environment. It can be either allergic or non-allergic. According to the ARIA classification both allergic and non-allergic rhinitis can be subdivided into intermittent or persistent rhinitis [1]. Allergic rhinitis to common inhalant allergens and occupational allergic rhinitis may share identical clinical features such as sneezing, itch and watery discharge. However, at an immunological level occupational rhinitis is not always characterized by an IgE-mediated mechanism. As a consequence, immunological tests to establish the diagnosis are not always available even if an immunologic mechanism is suspected. Whereas a diagnosis of allergic rhinitis to house dust mites, pollen and animal epithelia can be easily established with skin tests and determination of allergenspecific IgE nasal challenge tests may be required to confirm the diagnosis of occupational allergic rhinitis. Non-allergic occupational rhinitis is a less well-defined entity. The pathophysiological background of non-allergic rhinitis may comprise increased permeability of the nasal epithelium, increased sensitivity of sensory nerve fibres or autonomic dysregulation [2]. Non-allergic occupational rhinitis has been subdivided into irritative or irritant rhinitis [3] and corrosive rhinitis [4, 5]. In irritant rhinitis, exposure to irritating chemicals may lead to nasal symptoms. It has been postulated that these agents induce neurogenic inflammation accompanied by secretion and nasal blockage [3]. Corrosive rhinitis is the consequence of exposure to high concentrations of corrosive chemicals such as strong acids and alkali, oxidizing and dehydrating

R. Gerth van Wijk () Section of Allergology, Department of Internal Medicine, Erasmus Medical Centre, Rotterdam, The Netherlands e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_13, © Springer 2009

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agents. Irreversible tissue damage resulting in nasal obstruction, dryness, bleeding and even septal perforation is a feature of this type of occupational rhinitis [5]. The reactive upper-airways dysfunction syndrome (RUDS) has been put forward as an analogue of reactive airway dysfunction syndrome (RADS). These types of chronic rhinitis or asthma originate from a single exposure to strong and highly concentrated irritants [6]. Next to the classification based on immunological or non-immunological mechanisms, rhinitis can also be classified as occupational rhinitis, work-aggravated rhinitis and rhinitis-like conditions [4]. It will be clear that the different types of occupational rhinitis require various diagnostic approaches. The characteristics of the provoking agents may determine the diagnostic possibilities. High molecular weight (HMW) agents, generally proteins may lead to allergic sensitization, which can be identified with skin tests or radioallergosorbent test (RAST). Also low molecular weight (LMW) agents may form a hapten–protein complex and thus sensitize subjects. Many LMW agents exert their effect on the nasal mucosa by irritation or unknown way mechanisms, which may hamper identification of provoking agents and establishment of the diagnosis. However, the diagnosis needs to be made on firm grounds, as the consequences of either diagnosing OR when absent or missing the diagnosis when present are substantial.

Clinical History Occupational History To establish the diagnosis of occupational rhinitis not only is a good medical history necessary, but also a detailed history of current and previous jobs and exposure. Questionnaires can be used to obtain the relevant information. As with occupational asthma, [7] the history should substantiate the temporal association between symptoms and working environment. The occupational history comprises questions to job duties, work processes, work environment and recent changes in processes and environment. Data on compounds to which subjects are directly exposed are needed, but also information on other compounds may be relevant. Sometimes, a visit of the working place is required to get insight in potential harmful exposures.

Medical History Occupational allergic rhinitis is characterized by the same clinical features as common allergic rhinitis. Symptoms comprise sneezing, nasal itch, watery discharge

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and nasal blockage. However, other symptoms may be important. In a small study among bakers, 75% reported crusts in the nose [8]. In another study among workers exposed to paper dust nasal symptoms were dominated by obstruction and crusting, but not by nasal discharge, itch or sneezing [9]. Also, epistaxis should be looked for [10]. Conjunctivitis may be present as well. Against the background of the similarities and tight interactions between rhinitis and asthma [1, 11], also occupational rhinitis may be accompanied by occupational asthma [12]. Thus, questions focusing on lower airway pathology should be included.

Patterns In occupational allergy exacerbations and recovery of symptoms may be linked to exposure. Improvement of symptoms when not at work during holidays and weekends and progression of symptoms during the week are suggestive for occupational allergy. In contrast to controls a gradual decrement of peak nasal flow and a corresponding significant gradual increase in nasal blockage in woodwork teachers during the week has been observed [13]. In a study in bakery workers, 57 out of 160 subjects reported improvement during holidays [14]. Workers in bell pepper green houses, sensitized to bell pepper pollen, experienced less problems outside the bell pepper flowering period as measured with a rhinitis-related quality of life (QoL) questionnaire [15]. A prospective study to the natural course of occupational allergy in animalhealth apprentices demonstrated that nasal obstruction seems to develop later than other symptoms such as sneezing, rhinorrhea and itchy eyes [16]. However, in many cases this pattern is absent, as symptoms are also usually present outside the work place, being triggered by exposure to irritants. Symptoms may even be more severe at home, and weekends may not be long enough to allow for recuperation. An early onset of symptoms after the start of exposure suggests the presence of irritant-induced rhinitis, whereas a latency time may reflect the development of sensitization in occupational allergic rhinitis. Latency time may vary. Potent sensitizers such as laboratory animals may sensitize workers within a few months [17], whereas sensitization to wheat and α-amylase in bakers may appear after several years [18]. All associations between exposure, interruption of exposure and fluctuation in nasal symptoms have been studied at a group level. Some questions are useful to identify subjects with concomitant occupational asthma. Nasal itching at work has been clearly associated with the presence of asthma [19]. However, a history suggestive of an occupational allergy, even in an employee exposed to a known occupational agent, is not sufficient to make the diagnosis. A questionnaire is a sensitive, but non-specific tool. Even in the hands of experienced physicians, the predictive value of a positive asthma questionnaire was only 63%, while the

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predictive value of a negative questionnaire was 83% [7]. It is expected that also in rhinitis questionnaires are not sufficiently specific to diagnose occupational rhinitis. Instead, the history should support the diagnosis.

Health-Related Quality of Life According to the ARIA guidelines the impact of rhinitis on work, school, leisure and sport should be estimated [1]. It has been recognized that rhinitis has a major impact on daily life [20]. Therefore, assessment of health-related quality of life (HRQL) is not only important in the case of perennial or seasonal allergies, but also when an occupational allergy is involved. Quality of life can be categorized into four domains namely; physical status and functional abilities, psychological status and well-being, social functioning and economic and/or vocational status. As the true quality of life value cannot be measured directly, one has to resort to questionnaires to measure these four domains indirectly. For this purpose, HRQL instruments have generally been constructed [21]. Several instruments (questionnaires) are currently available, which can be broadly classified as either generic or disease-specific. The generic instruments are general questionnaires and can be applied to all medical conditions, measuring physical, psychological and social domains, irrespective of the underlying disease. Disease-specific questionnaires are used to evaluate quality of life in a particular disease state such as asthma and rhinoconjunctivitis. Juniper and colleagues have developed several questionnaires for measuring HRQL in adults with rhinitis, which can easily be used in allergy research in health care practice as well as in research. These provide a method for quality of life assessment, which has been profoundly tested and accepted in terms of reliability, responsiveness and validity [22]. However, little is known about the relative impact of an occupational allergy on daily life and until now, only a few studies in asthma have been published on this topic [23, 24], whereas in one study only the relationship between occupational allergy and rhinitis-related quality of life has been assessed [15]. In this study bell pepper greenhouse employees appeared to be impaired in QoL because of their sensitization to bell pepper pollen, suggesting that bell pepper pollen is the most important occupational allergen in greenhouse workers with allergic symptoms. A common allergy did not have more impact on a person’s day-to-day life than an occupational allergy; however, there was a clear difference in the way in which an occupational group is hampered compared with a nonoccupational group. The available instruments to evaluate health-related quality of life such as the rhinitis-related quality of life questionnaire (RQLQ) [22] are designed for the use in clinical trials and are not appropriate for assessment of quality of life at an individual level. Nevertheless, the physician should be aware of the impact of occupational allergy on day-to-day life. Therefore, questions to the effects of nasal disease on leasure, sport, school and work productivity should be asked.

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Physical Examination An ENT examination using anterior rhinoscopy and nasal endoscopy may reveal the typical signs of allergic rhinitis (i.e. mucosal swelling, nasal discharge), and also crusts, bleeding lesions and ulceration may be seen in case of corrosive rhinitis. There are no clear characteristic features, however. An ENT investigation is important to rule out other pathologies. Particularly, with unilateral symptoms and complaints one should be aware of septal deviations, polyps or malignancies. In addition, the close interaction between rhinitis and asthma, also in occupational allergy warrants a general physical examination.

Diagnostic Tests Skin Tests Skin tests are commonly used to determine sensitization to inhalant allergens in patients with allergic rhinitis. Similarly, skin prick tests with suspected agents may be used to support the diagnosis of occupational rhinitis. However, a few major reasons may hamper the use of both skin tests and determination of specific IgE. Firstly, extracts of the appropriate agents are not always available. Commercially produced extracts, whenever available, are not standardized. Often home-made crude extracts have to be prepared. Material for extraction needs to be sampled from the working place. Samples may vary in allergen content, purity and therefore allergenicity. The source of allergen is important. Stamen and pollen from bell pepper plants are more allergenic than leaf and stem [25]. Variation in environmental conditions and working activities may also be responsible for fluctuation in samples. Before applying tests on patients, healthy subjects should be tested to rule out non-specific reactions. Furthermore, the characteristics of the agent determine the availability of test extracts. High molecular weight (HMW) agents are easier to produce than low molecular weight (LMW) allergens. Few LMW substances such as platinum salts are soluble and easily used in skin tests. Secondly, the use of skin tests assumes the presence of an IgE-mediated background, whereas in many cases the pathophysiology of occupational rhinitis is not clearly understood or based on nasal irritation. Non-allergic occupational rhinitis does not fit in a diagnostic algorithm using skin tests and specific IgE. Thirdly, as in common inhalant allergy positive skin tests indicate the presence of sensitization. Positive tests do not necessarily point at allergic disease. The chance that sensitization develops into clinical disease depends on level and intensity of exposure and on environmental factors. In a Swedish study six out of seven subjects sensitized to laboratory animals developed allergic symptoms in less than 18 months [26]. In a Canadian study 53% and 37% of employees sensitized to laboratory animals developed symptoms of rhinoconjunctivitis and asthma respectively in a timeframe of 44 months after the start of exposure [27, 28].

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In Polish sensitized baker apprentices 16.6% developed rhinitis, whereas 37.5% developed asthma [29].

Specific IgE to Occupational Allergens Specific IgE to occupational allergens may be determined with RAST or enzymelinked immunosorbent assay (ELISA) techniques. Basically, these tests share the limitations of skin tests. Allergens need to be obtained, interpretation is restricted to IgE-mediated allergy and a positive test indicates sensitization without necessarily the presence of allergic symptoms. Skin tests are more sensitive than RASTs in estimating sensitization. In greenhouse workers with work-related symptoms, 69% of subjects with positive skin tests to bell pepper pollen showed specific IgE to bell pepper pollen [25]. Fifty-eight percent of subjects with skin test to predatory mites revealed specific IgE antibodies [30], whereas 52% of workers sensitized to chrysanthemum pollen according to skin test results demonstrated IgE to these pollen [31].

Nasal Challenge Tests in the Laboratory Challenge tests are still considered to be the gold standard in the confirmation of the diagnosis of occupational asthma [7, 32], and also rhinitis [33]. By gradually exposing the nose to increasing doses of the suspected allergen, work exposure is mimicked and reproduction of clinical disease can be achieved. Challenge tests are obviously more conclusive when performed in a controlled, laboratory setting, with known and available substances, and specially equipped facilities. They are more difficult when dealing with an unknown agent, multiple agents, dangerous agents or when specific testing facilities are not available. In these instances, a work place challenge is recommended. Challenges should always be carried out under close supervision of an expert physician. In most subjects, the tests can be carried out on an outpatient basis or in the work environment, restricting hospitalization to subjects who have severe late reactions.

Methodology Water-Soluble HMW Allergens Nasal challenge with HMW agents is relatively easy. The technique does not differ from challenges with common allergens as grass pollen or house dust mites. From position papers the following recommendations can be extracted [34].

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Solutions should have room temperature before application. Control solutions should be sprayed first and the reactions should be monitored. Deep inspiration should be performed before spraying in order to avoid deposition in the lower airways. Metre-dose pump sprays or discs can be used with a reproducible delivery of allergen. One or both nostrils may be challenged. Ideally, purified and standardized allergenic extracts should be used when such reagents are available. Alternatively, extracts may be freshly prepared into saline (phenolated) solutions [30, 33]. Dilutions from freeze-dried stock solutions should be used where available. Standardized allergen extracts and units are recommended. To avoid non-specific stimulation with solutions, these should be isotonic and buffered to a pH close to 7.

Non-Water-Soluble Agents Agents may be applied as dry powder, vapour, aerosol or gas. In particular, the characteristics of LMW agents require such ways of application. In these cases, a challenge room with adequate facilities should be available. Ideally, conditions at the work place should be reproduced as much as possible. Also, exposure should be controlled, if possible. Such challenge tests can only be performed in specialized centres.

Assessment of the Nasal Response For the assessment of the nasal response it is recommended to combine symptom scores with objective measurements (counting sneezes or attacks of sneezes, measuring volume or weight of nasal secretion, and results of changes of nasal patency, airflow or airflow resistance). Several methods to assess nasal patency, airflow or airflow resistance are available.

Rhinomanometry Anterior rhinomanometry measures one-sided nasal airway, whereas posterior rhinomanometry assesses the total resistance of nasal airways. The latter technique requires more collaboration of the patients [35]. Active anterior rhinomanometry has been recommended for evaluation of rhinomanometric responses after nasal provocations in a simple way assessing changes in percent of values obtained after application of solutions without allergen. Allergen may be applied unilaterally. The drawback with active anterior rhinomanometry is that the nasal cycle may affect the results. It is possible that this drawback is of minor importance when application is done in the most open side.

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Acoustic Rhinometry Acoustic rhinometry is a technique that assesses nasal patency by determining the cross-sectional area and volume of nasal cavities using the reflection of sound waves [35]. In nasal provocations with histamine and bradykinin acoustic rhinometry appeared to be more sensitive than posterior rhinomanometry [36]. Pirila also found acoustic rhinometry to be more sensitive than rhinomanometry to monitor nasal provocations [37]. Acoustic rhinometry, however, has limitations and pitfalls. The value of acoustic rhinometry to evaluate nasal responses after provocation in routine clinical work is not established yet.

Nasal Peak Airflow The inspiratory flow meter is more suitable than the expiratory one because it avoids contamination with secretions. The performance of nasal peak flow has been demonstrated in a long-term pollen immunotherapy trial testing the nasal reactivity with provocations once a year using different methods and symptoms among them nasal peak flow. The reactivity measured with the peak flow method decreased significantly between the four occasions the 31 patients were tested [38]. The authors advocated, however, a total nasal provocation score to be used, also including scores for sneezes and secretion. Peak inspiratory nasal flow and peak expiratory nasal flow (rate), especially the former, can be recommended for long-time control of pharmacologic or immunologic treatment of different types of rhinitis. For detecting nasal changes after provocations they are less accurate than active anterior rhinomanometry according to all publications found.

Symptom Scores In addition to the objective measurements of nasal patency and airflow a variety of methods to assess the response to allergen or non-specific stimuli are available. These methods comprise measuring nasal secretion, counting sneezes, assessment of severity of symptoms and visual analogue scores (VAS). The composite symptom score of Lebel [39] has been validated in terms of responsiveness to treatment, correlation with daily nasal symptoms and discrimination between patients and healthy subjects [30, 40, 41].

Markers of Inflammation in Nasal Lavage Nasal lavage before and after nasal provocation can be used to estimate the involvement of nasal inflammation. Tryptase, leucotrienes and eosinophilic cationic protein derived from mast cells and eosinophils can be found during the

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early- and late-phase nasal reaction [41–43]. Also in the diagnosis of occupational rhinitis nasal lavage has been used [44, 45]. However, although this technique may be of help in clarifying the background of nasal disease, nasal lavage has been predominantly used in research.

Nitric Oxide A recent study found that nasal provocation with common allergens induced a decrease in the level of nasal NO, which was followed by an increase at 7 and 24 h post challenge [46]. The role of nasal NO as a biomarker of airway inflammation during nasal provocation with occupational agents requires further investigation.

Exposure at the Work Place Exposure at the work place and monitoring of the nasal response may be warranted if responsible agents cannot be identified, multiple agents may be involved or a nasal challenge test at the laboratory is not feasible because of its complexity (i.e. if chemical products are involved) [47]. A comparison should be made between days at work and control days. Also, irritant-induced rhinitis and work-aggravated rhinitis can be studied with monitoring at the work place [48]. Theoretically, methods of nasal assessment used to monitor the results of nasal challenges might be used at the work place. Rhinomanometry and acoustic rhinometry have never been used to monitor individual patients with suspected occupational rhinitis at the work place. In contrast, it is possible to identify workers with occupational rhinitis with both rhinomanometry [49] and acoustic rhinometry [9, 47] at a group level. Also, nasal peak flow has been used to monitor workers exposed to soft paper dust [9] and to study bakers [8]. Again, these studies have been performed at a group level. In a study among woodwork teachers serial peak flow measurements are recommended for studies of groups with nasal obstructive symptoms. But because of the variability in peak flow the authors considered it important to study a large number of patients with several measurements a day [48]. One study only used serial nasal peak-expiratory-flow-rate recordings to detect changes in nasal patency. A hospital nurse with allergy to latex showed a marked decrease in nasal flow when exposed to latex gloves in a challenge test and in her work environment. The authors proposed using the method as a diagnostic tool for allergic rhinitis in occupational and other exposure situations in which nasal blockage is a prominent symptom [50]. However, more research is needed to validate nasal peak flow measurements in the diagnosis of individual subjects with suspected occupational rhinitis. Nasal lavage has been used in waste handlers exposed to bioaerosols [47], in workers exposed to isocyanates [51] and other populations [8, 9, 52] but again this technique is not validated for the use in individuals.

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Symptom scores have been used to monitor work-related nasal obstruction in woodwork teachers. On a group level, rated nasal blockage was higher in the woodwork teachers compared to controls. Nasal blockage also gradually increased during the working week [48]. Also in flour-exposed bakers subjects had more nasal symptoms than controls [8]. In conclusion, if nasal challenge is not feasible, exposure in the working place is the best option to diagnose occupational rhinitis. An exacerbation of symptoms underwrites the diagnosis. However, methods of assessing changes in nasal function or symptoms have mainly been validated in groups of patients. Thus, the findings of peak flow measurements, acoustic rhinometry and other nasal function tests, and also of symptom scores, should be interpreted with caution.

Nasal Hyperreactivity Non-specific nasal hyperreactivity is an important feature of allergic and non-allergic rhinitis and can be defined as an increased nasal response to a normal stimulus resulting in sneezing, nasal congestion, and/or nasal secretion [53, 54]. Nasal challenge tests with histamine or methacholine [53] have been proposed as a method to quantify non-specific upper airway hyperreactivity. Assessing non-specific nasal hyperreactivity might be relevant, since work-related rhinitis symptoms might result, at least in part, from nasal reactions to irritants [55]. In addition, nasal hyperreactivity may increase after nasal challenges with allergens [41]. There is, however, controversy as to whether nasal reactivity to histamine can accurately distinguish patients with allergic rhinitis from healthy subjects [53, 54].

Diagnostic Considerations In conclusion, due to the absence of standardized and validated tools to diagnose occupational rhinitis the diagnosis may be established with varying degrees of certainty. A stepwise approach is required. The first step comprises a thorough and extensive occupational and medical history. The second step includes immunological tests if available. Such tests can be performed in case of a suspected allergy to HMW allergens and some LMW allergens [56–58]. The third step should include evidence for a causal relationship between rhinitis and work environments in an objective way. In spite of all limitations nasal challenge is still the gold standard to obtain a definite diagnosis. If the nasal challenge is negative, challenge (i.e. exposure) in the work environment may be considered. Also, if a nasal challenge in the laboratory is not feasible challenge in the work place is the best next step. With this stepwise approach a probable diagnosis of occupational rhinitis will be based on the combination of a suggestive history and positive immunological tests with standardized agents. A definite diagnosis of occupational rhinitis will be

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established by either nasal challenge in the laboratory (recommended option) or environmental exposure in the working place [59].

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43. van Wijk RG, Zijlstra FJ, van Toorenenbergen AW, Vermeulen A, Dieges PH. Isolated early response after nasal allergen challenge is sufficient to induce nasal hyperreactivity. Ann Allergy 1992;69(1):43–47. 44. Krakowiak A, Ruta U, Gorski P, Kowalska S, Palczynski C. Nasal lavage fluid examination and rhinomanometry in the diagnostics of occupational airway allergy to laboratory animals. Int J Occup Med Environ Health 2003;16(2):125–132. 45. Palczynski C, Walusiak J, Krakowiak A, Szymczak W, Wittczak T, Ruta U, et al. Nasal lavage fluid examination in diagnostics of occupational allergy to chloramine. Int J Occup Med Environ Health 2003;16(3):231–240. 46. Boot JD, de Kam ML, Mascelli MA, Miller B, van Wijk RG, de Groot H, et al. Nasal nitric oxide: longitudinal reproducibility and the effects of a nasal allergen challenge in patients with allergic rhinitis. Allergy 2007;62(4):378–384. 47. Heldal KK, Halstensen AS, Thorn J, Djupesland P, Wouters I, Eduard W, et al. Upper airway inflammation in waste handlers exposed to bioaerosols. Occup Environ Med 2003;60(6):444–450. 48. Ahman M. Nasal peak flow rate records in work related nasal blockage. Acta Otolaryngol 1992;112(5):839–844. 49. Chloros D, Sichletidis L, Kyriazis G, Vlachogianni E, Kottakis I, Kakoura M. Respiratory effects in workers processing dried tobacco leaves. Allergol Immunopathol (Madr) 2004;32(6):344–351. 50. Ahman M, Wrangsjo K. Nasal peak-flow-rate recording is useful in detecting allergic nasal reactions – a case report. Allergy 1994;49(9):785–787. 51. Littorin M, Welinder H, Skarping G, Dalene M, Skerfving S. Exposure and nasal inflammation in workers heating polyurethane. Int Arch Occup Environ Health 2002;75(7):468–474. 52. Douwes J, Wouters I, Dubbeld H, van Zwieten L, Steerenberg P, Doekes G, et al. Upper airway inflammation assessed by nasal lavage in compost workers: a relation with bio-aerosol exposure. Am J Ind Med 2000;37(5):459–468. 53. Gerth van Wijk R, Dieges PH. Nasal reactivity to histamine and methacholine: two different forms of upper airway responsiveness. Rhinology 1994;32(3):119–122. 54. Gerth van Wijk RG, de Graaf-in ‘t Veld C, Garrelds IM. Nasal hyperreactivity. Rhinology 1999;37(2):50–55. 55. Plavec D, Somogyi-Zalud E, Godnic-Cvar J. Nonspecific nasal responsiveness in workers occupationally exposed to respiratory irritants. Am J Ind Med 1993;24(5):525–532. 56. Yokota K, Johyama Y, Yamaguchi K. A cross-sectional survey of 32 workers exposed to hexahydrophthalic and methylhexahydrophthalic anhydrides. Ind Health 2002;40(1):36–41. 57. Nielsen J, Welinder H, Ottosson H, Bensryd I, Venge P, Skerfving S. Nasal challenge shows pathogenetic relevance of specific IgE serum antibodies for nasal symptoms caused by hexahydrophthalic anhydride. Clin Exp Allergy 1994;24(5):440–449. 58. Santucci B, Valenzano C, de Rocco M, Cristaudo A. Platinum in the environment: frequency of reactions to platinum-group elements in patients with dermatitis and urticaria. Contact Dermatitis 2000;43(6):333–338.

59. Moscato G, Vandenplas O, Gerth Van Wijk R, Malo JL, Quirce S, Walusiak J et al. Occupational rhinitis. EAACI Position paper. Allergy 2008;63:969–980.

Asthma: Clinical Descriptions and Definitions William K. Dolen

Asthma is like love: everyone knows what it is but no two people can agree the terms for its definition. (Quoted by Carswell) [1]

Asthma: Symptom or Disease? The English word “asthma” is a direct transliteration of the Greek άσθµα, a noun used in Homer’s Iliad to denote breathlessness or “panting” [2]. Hippocrates brought the term into the field of medicine, in addition to his use of the terms orthopnea, tachypnea, and dyspnea. He reported a seasonal pattern of symptoms and an association with middle age [3], although it is not clear whether he was describing a symptom or attempting to define a disease entity. Some 500 years later, Aretaeus of Cappadocia wrote of “asthma” as a disease characterized by episodic paroxysmal dyspnea, and distinguished between acute and chronic forms of the condition. In describing exercise-induced asthma, he writes: “If from running, gymnastic exercises, or from any other work, the breathing becomes difficult it is called asthma” [2]. Galen also recognized exercise-induced asthma and the more chronic form, describing the latter as a “lack of room in the cavities of the lungs” due to bronchiolar obstruction from “thick secretions” [2]. In the seventeenth century, English medical education still drew heavily on the writings of Hippocrates and Galen. In A Treatise of the Asthma (1698), Sir John Floyer (himself a patient with asthma) clearly defined asthma as a condition separate from other forms of breathlessness, and attributed the cause to bronchoconstriction. He made many clinical observations based on his own experience with the disease, noting an inherited form, exacerbation by environmental factors, and lack of disease progression: I cannot remember the first occasion of my asthma, but I have been told that it was a cold when I first went to school. As my asthma was not hereditary from my ancestors, so I thank God neither of my two sons are inclined to it, who are now past the age when it seized me. … W.K. Dolen () Allergy-Immunology Section, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_14, © Springer 2009

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I never had any considerable fit in Oxford for twelve years that I lived there. But, as oft as I came into Staffordshire, into my native air, I was usually visited with a severe fit or two. … I have met with some asthmatics who have been so for fifty years, as they informed me, and yet in tolerable health without any considerable decay of their lungs … which I oft reflect on to encourage my patients and myself, who yet can study, walk, ride and follow my employment, eat, drink and sleep, as well as ever I could. [4]

Henry Hyde Salter and the Definition of Asthma By the nineteenth century, the invention and clinical use of the stethoscope permitted Laennec to define asthma as “paroxysmal dyspnea between two intervals of normal respiration” with “sibilant and sonorous râles” [5]. Salter (Fig. 1) considered asthma as “a morbid proclivity of the musculo-nervous system of [the] bronchial tubes to be thrown into a state of activity” [6]. He described the clinical course of acute asthma in great detail (Table 1), and noted clinical heterogeneity as

Fig. 1 Henry Hyde Salter (1823–1871) (Image courtesy of Dr. Sheldon Cohen, National Library of Medicine)

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Table 1 Salter’s clinical description of asthma [6] Premonitory and initiatory symptoms Drowsiness (many patients): “On the night previous to the attack; but in some cases for a longer time.” Alertness (some patients): “Extreme wakefulness and unusual mental activity and buoyancy of spirits.” Onset of symptoms in the early morning: “Almost invariably in the early morning, from two to six o’clock.” Position of the body: “An extra pillow may prevent [the attack].” Relation to meals: “If [a certain patient] goes to sleep [after taking a supper], his asthma will come on immediately; but by thus sitting up till his supper is fairly digested, his stomach empty, and the source of irritation thus removed, he may go to sleep fearlessly and have a good night’s rest.” Diuresis: “The patient will fill half a chamber-pot with pale, limpid water … generally lasts for the first three of four hours, and then ceases altogether.” Arthralgias and myalgias: “A deep-seated aching in the limbs and joints; the testicles, too, are very apt to be affected with it.” The attack Wheezing: “The patient … dreams, perhaps, that he is under some circumstances that make his respiration difficult; while yet asleep the characteristic wheezing commences.” Awakening: “The increasing difficulty quite wakes him … he sits up in bed … falls back [to sleep] to be again awoke … sleep is no longer possible … throws himself forward, plants his elbows on his knees, and with fixed head and elevated shoulders labors for his breath like a dying man.” In distress: “He sits fixed in a chair, immovable, unable to speak … his whole figure is deformed … at every breath his head is thrown back.” Pulse: “The pulse during severe asthma is always small … so feeble sometimes that it can hardly be felt.… I have never known the small pulse absent in severe asthma.” Itching under the chin: “The itching is incessant … often accompanied with the same itching sensation over the sternum and between the shoulders … it appears the moment the first tightness of breathing is felt, and goes off when the paroxysm has been confirmed.” Use of accessory muscles: “All the muscles passing from the head to the shoulders, clavicles, and ribs are rigid … all the muscles that increase the capacity of the chest are straining their utmost … the chest is almost motionless.” Breathing pattern: “The breathing … is not short; on the contrary, it is even sometimes longer than natural … [implying] stricture of some part of the air passages … the relative length of the inspiration and expiration is reversed … the expiration is longer than the inspiration.… I believe it is peculiar to asthma.” Chest auscultation: “Sonorous and sibilant rhonchi of every variety … a very orchestra … high and shrill … the points of stricture are constantly changing their place.” No breath sounds heard “if the spasm is severe.” Time course: Maximal intensity reached “within a quarter of an hour” in some patients, and “in others the dyspnea creeps slowly on, getting deeper and deeper for hours.” “In an hour or two the severity of the paroxysm gives way, and, even if it does not completely disappear, the patient experiences a sense of inexpressible relief … in others the attack comes quickly on and as quickly and completely subsides … in others it lasts the entire day … in others … the second night is worse than the first.” Resolution Expectoration: “When the spasm finally subsides it generally does so coincidently with the first appearance of expectoration. Up to that time the wheezing has been dry.” Salter described an inflammatory response (“cellular elements”) in the sputum of asthma patients.

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Table 2 Salter’s “varieties of asthma” [6] I. Spasmodic (idiopathic, uncomplicated) asthma (A) Intrinsic asthma, with six “varieties.” “The lungs are alone concerned; the source of irritation is applied to the very part that is the seat of the diseased phenomena.” 1. Caused by fog, smoke, or fumes of various kinds. Described as “very common.” Patients do not have symptoms at other times. 2. Ipecacuanha powder. Considered “very rare.” Symptoms do not occur under any other circumstances. All patients were medical students. 3. Hay fever. “Distinct asthmatic dyspnea” associated with allergic rhinitis. 4. Exposure to animals. Similar to the hay fever variety (including presence of rhinoconjunctivitis), but caused by exposure to cat or rabbit fur. 5. Change in locale. “Asthma induced by the air of certain localities.” 6. Toxemic asthma. “Asthma produced by blood poisoning … [from] beer, wine, sweets.” (B) Reflex (excito-motory) asthma, with three varieties. “The source of irritation has a distant seat, far removed from the lungs.” 1. Peptic asthma. “An error in diet … supervenes on a full meal.” 2. Organic nervous irritation. “Asthma from a loaded rectum or uterine irritation.” 3. Peripheral cerebrospinal irritation. Caused by a stimulus to the “external surface” of the body. (C) Central asthma. “The perverted innervation starts from the brain or spinal cord,” as in epileptic or emotional causes. (D) Periodic asthma. “No exciting cause of the attacks can be detected” in contradistinction to the above three varieties. II. Organic (symptomatic, complicated) asthma. More frequent paroxysms, symptoms easy to induce, imperfect remission between attacks. Considered to be of vascular or neurogenic cause.

“varieties of asthma” (Table 2). Some of these “varieties” seem to reflect different irritant triggers of asthma, but others suggest different mechanisms leading to episodic bronchoconstriction and form the basis of the modern study of asthma. In defining asthma clinically, Salter recognized the conceptual difference between “the cause of the disease” of asthma and “the cause of the paroxysms” of asthma. Despite the popularity of Salter’s work, physicians in the early twentieth century indiscriminately used the term “asthma” to refer to dyspnea (or breathlessness) of any cause, including pulmonary (bronchial asthma), cardiac (cardiac asthma), or renal (renal asthma) etiologies [7]. Salter’s (and earlier) clinical observations remain the basis of modern clinical definitions of asthma, although subsequent writers emphasized the inflammatory nature of the disease, as well as diagnostic criteria of reversible episodic wheezing, dyspnea, and a prolonged expiratory phase of respiration [5].

Clinical Definitions of Asthma The 2007 Guidelines for the Diagnosis and Management of Asthma of the National Heart, Lung, and Blood Institute of the United States states: “Asthma is a common chronic disorder of the airways that is complex and characterized by variable and recurring symptoms, airflow obstruction, bronchial hyperresponsiveness, and

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an underlying inflammation” [8]. The 2006 guidelines published by the Global Initiative for Asthma state: Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. These episodes are usually associated with widespread, but variable, airflow obstruction within the lung that is often reversible either spontaneously or with treatment. [9]

When the guideline definitions are put into clinical practice, the diagnosis of asthma rests on eliciting symptoms and defining their severity (a subjective exercise), and when possible, objectively demonstrating reversible obstruction. The demonstration of bronchial hyperresponsiveness and underlying inflammation is not a routine aspect of patient care.

Asthma Subtypes Cooke taught that asthma is a disease entity with dyspnea as a symptom [10]. Likewise, Salter’s description of “varieties of asthma” assumed that asthma is one disease entity with different clinical features in different individuals. His “varieties” are now termed “clinical phenotypes,” and there is increasing evidence that asthma is not a single disease, but several diseases of the lung causing episodic symptoms. In the twentieth century, various authors attempted to simplify or elaborate on Salter’s asthma “varieties” in proposing asthma classifications. Rackemann (Fig. 2) noted some “patients whose asthma depends upon some cause outside of the body,” and called this “extrinsic asthma” [11]. Cooke termed this “non-infective asthma” [10]. Rackemann reported other patients in whom “the cause of the trouble depends on something wrong inside their body,” calling this “intrinsic asthma,” synonymous with Cooke’s “infective asthma” [10]. This simple classification, based on “the cause of the paroxysms” rather than “the cause of the disease” has limited use because Rackemann’s original definitions are no longer employed. In modern times, “extrinsic asthma” has for some clinicians become synonymous with “allergic asthma” (i.e., asthma paroxysms triggered by IgE-dependent mechanisms), and “intrinsic asthma” with “nonallergic asthma.” Rackemann also developed a more elaborate clinical classification of asthma based on his knowledge of the literature and his clinical observations of the natural history of the disease (Table 3). This classification warrants careful study.

Discovering the Cause of the Disease In our modern era, clinical research as well as basic science research have defined multiple causes for diabetes mellitus, hypertension, and many other diseases. It now seems clear that asthma is not a single disease entity, but many diseases

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Fig. 2 Francis M. Rackemann (1887–1973) (Image courtesy of Dr. Sheldon Cohen, National Library of Medicine)

with a common pathway that leads to breathlessness and wheezing. Genotyping offers the promise to help define the diseases of asthma at a basic level, but early genetic studies seem to have assumed that asthma is one disease with various clinical presentations. Furthermore, they seem not to have distinguished between the asthmatic diathesis, the atopic diathesis, and the allergic diathesis. Unfortunately, the terms “atopy” [12] and “allergy” [13] have multiple definitions on which clinicians and scientists cannot agree. These conflicting and overlapping definitions are not particularly useful as the basis for detailed molecular studies. As a result, research to date has identified a vastly complex panoply of genes that seem to play some role in some aspect of asthma pathogenesis (often, different genes found in different patient populations), as well as various gene–gene interactions and gene– environment interactions. The complexity of the genomics of asthma currently limits the application of principles of genomic medicine to diagnosis and management of the disease.

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Table 3 Rackemann’s “clinical types of asthma patients” [11] Type I. The normal-looking child subject to asthmatic attacks About one third of children with asthma Isolated attacks, frequently following a cold, appears normal between episodes Eczema in the present or past Positive family history of allergy Positive skin tests in 46/79 (58%) of patients Good prognosis; 29/79 (37%) reported as “cured” within 2 years of presentation; all others were improved Type II. The asthmatic child About two thirds of children with asthma More obvious symptoms, with cough and sleep disturbance “Frail, tired looking”; “thin delicate skin of rather pale color, with blue lips” Prominent upper sternum, “a thick anteroposterior diameter, with dilated veins over the chest wall” Scattered wheezy râles; variable quantities of sputum, perhaps thick and mucopurulent May show areas of chronic eczema Positive skin tests (pollens, animals, foods) in 78/153 (51%) patients Recovery possible “if they can be maintained free from acute attacks” Type III. Normal adults with clear cut attacks separated by considerable intervals Almost entirely composed of extrinsic cases, with infrequent exposure to triggering agent Triggered by allergy or by colds Demonstrable focus of infection (usually in sinuses) in “about a third,” “but evidently these foci are unimportant” Type IV. Normal appearing adults with more frequent attacks, but who are still free of symptoms between them Various etiological factors: 1. Patients with persistent symptoms due to persistent exposure to an extrinsic cause. “If they enter the hospital, they recover promptly” 2. Patients with persistent symptoms due to “secondary infections on top of the primary cause” 3. Patients with recurrent respiratory infections, including patients “who harbor a definite focus of infection” Subtypes: 1. Young adults (under age 30) with frequent attacks. “About half of these have extrinsic factors which are usually complicated by secondary infections” 2. Well-nourished, frequently obese, men and women with asthma of moderate but variable severity, occurring in attacks with no symptoms between them. A third of the Type IV group, with more women than men and twice as many intrinsic cases than extrinsic. Patients “with a shorter duration of asthma were older at the time of onset,” and intrinsic asthma is more common. “Asthma frequently dates from some acute infection.” “Fatal asthma belongs in this group” (continued)

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Table 3 (continued) Type V. Asthmatic adults with essentially constant symptoms but without chronic bronchitis and emphysema Characteristics No great evidence of chronic changes in the bronchi and lungs Cough relatively unimportant, sputum thin and small in amount Chest examination shows soft, high-pitched sounds, with “breathing of various qualities” About 10% of patients of both sexes, and age beyond childhood Subtypes: 1. Young adults with asthma and eczema together and both severe. Considered uncommon, but “very characteristic.” Positive skin tests, “but the importance of them is not clear.” Poor response to immunotherapy 2. “School teacher type.” “Middle aged, thin, tired men and women … with long standing asthma which is severe and persistent and with poor general health.” “If positive skin tests occur, they are not clinically important. Fatigue and anxiety play important roles. Indigestion is common … cough is unimportant” 3. “Gall-bladder type.” “Occurs chiefly in women.” Difficult to separate from the Type IV group, but symptoms are more persistent. “They are always short of breath; they rarely sleep through the entire night.” Common associated symptoms are “indigestion, with gas and distress after meals and frequently persistent pain in the epigastrum.” Not usually associated with actual gall bladder disease 4. “Chronic severe asthma”. About 2% of asthma patients, “seen not infrequently in the wards of the hospital.” Most patients are men, and “the asthma in these men is of maximum severity” Type VI. Chronic bronchitis and emphysema

Can Clinical Phenotypes Assist in Defining Genotypes? When a patient reports characteristic symptoms of asthma, has a substantial objective response to an inhaled bronchodilator, and achieves appropriate asthma “control” with simple therapy, the diagnosis of asthma is straightforward, and there is no need for complex molecular diagnostic techniques. When a patient with apparent asthma does not respond to therapy, the diagnosis of asthma warrants reconsideration, and in such cases it would be useful to have a reliable diagnostic test for the disease. Furthermore, the emerging discipline of pharmacogenomics ought to assist clinicians in prescribing therapy with optimal efficacy and minimal side effects and knowledge of mechanisms should assist clinicians in monitoring the outcome of therapy. The classic clinical descriptions of asthma by Salter, Rackemann, and others remain a useful basis for the study of asthma, not only in the clinic but also in the laboratory. Additional clinical subtypes may be defined by a differential response to modern pharmacotherapy (implying different underlying disease mechanisms), and by the detailed study of inflammatory mechanisms. When these are listed individually (Table 4), there is obvious overlap, for example, the patient with adult-onset asthma who has chronic sinusitis, nasal polyps, and aspirin sensitivity. Computer analysis of clinical and genomic data might well identify genes that are directly responsible for clinically important disease phenotypes. Thus, renewed

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Table 4 Modern definitions of asthma phenotypes (Adapted from [14–19]) Clinical patterns Age of onset Onset following respiratory infection (particularly respiratory syncytial virus and parainfluenza virus) in early childhood Remission in adolescence, recurrence in adulthood Onset following respiratory infection in adulthood, including Chlamydia pneumoniae and Mycoplasma pneumoniae Occupational, chemical, toxic exposures; tobacco smoke exposure Presence of associated rhinitis, atopic dermatitis Presence of associated immediate “Type 1” or other patterns of hypersensitivity Associated chronic sinusitis, nasal polyps, gastroesophageal reflux Exercise-induced asthma Family history of asthma (whether or not associated with immediate “Type 1” hypersensitivity) Family history of other atopic diseases (rhinitis, atopic dermatitis) Family history of immediate “Type 1” or other patterns of hypersensitivity Clinical severity Progression to allergic bronchopulmonary aspergillosis Progression to Churg–Strauss syndrome Pharmacologic patterns Aspirin sensitivity Response to challenge with methacholine, histamine, other agents Response to β2-adrenergic receptor agonists, cromolyn, antileukotrienes, antimuscarinics β2-adrenergic receptor subsensitivity Corticosteroid insensitivity Types of inflammation Eosinophilic, neutrophilic, mixed granulocytic, paucigranulocytic, lymphocytic Patterns of T-cell activation, determined by analysis of bronchoalveolar lavage or peripheral blood CD4 + CD3 + NK cells Expression of leukotrienes, cytokines, chemokines, other mediators, and their receptors Various signaling proteins involved in inflammatory mechanisms Tissue repair mechanisms (“airway remodeling”) Other HLA genotyping

interest in asthma “subtypes” or phenotypes will focus further genomic research, define genetic subtypes of asthma, and simplify the study of the various individual diseases that produce the symptom of άσθµα.

References 1. Carswell F (2001) Poetry tells us that our souls have a shadow: can science respond? Nature 411:885 2. Marketos SG, Ballas CN (1982) Bronchial asthma in the medical literature of Greek antiquity. Journal of Asthma 19:263–269

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3. Netuveli G, Hurwitz B, Sheikh A (2007) Lineages of language and the diagnosis of asthma. Journal of the Royal Society of Medicine 100:19–24 4. Gibbs DD (1969) Sir John Floyer, M.D. (1649–1734). British Medical Journal I:242–245 5. Guidotti TL (1994) Consistency of diagnostic criteria for asthma from Laënnec (1819) to the National Asthma Education Program (1991). Journal of Asthma 31:329–338 6. Salter HH (1882) Asthma: Its Pathology and Treatment. New York: William Wood & Company 7. Vaughan WT, Black JH (1954) Practice of Allergy. Third ed. St. Louis, MO: C. V. Mosby 8. National Heart, Lung, and Blood Institute (2007) Expert Panel Report 3: Guidelines for the Diagnosis and Management of Asthma. Washington, DC: National Institutes of Health 9. Global strategy for asthma management and prevention. Global Initiative for Asthma, 2006. Available from http://www.ginasthma.com/Guidelineitem.asp?l1=2&l2=1&intId=1388 10. Cooke RA (1947) Allergy in Theory and Practice. Philadelphia/London: W. B. Saunders 11. Rackemann FM (1931) Clinical Allergy, Particularly Asthma and Hay Fever: Mechanism and Treatment. New York: Macmillan 12. Nelson HS (1985) The atopic diseases. Annals of Allergy and Asthma Immunology 55:441–447 13. Johansson SGO, Hourihane J, Bousquet J, et al. (2001) A revised nomenclature for allergy: an EAACI position statement from the EAACI nomenclature task force. Allergy 56:813–824 14. Kiley J, Smith R, Noel P (2007) Asthma phenotypes. Current Opinion in Pulmonary Medicine 13:19–23 15. Diamant Z, Boot JD, Virchow JC (2007) Summing up 100 years of asthma. Respiratory Medicine 101:378–388 16. Haldar P, Pavord ID (2007) Noneosinophilic asthma: a distinct clinical and pathologic phenotype. Journal of Allergy and Clinical Immunology 119:1043–1052 17. Balaci L, Spada MC, Olla N, et al. (2007) IRAK-M Is involved in the pathogenesis of early-onset persistent asthma. American Journal of Human Genetics 80:1103–1114 18. Wenzel SE (2006) Asthma: defining of the persistent adult phenotypes. Lancet 368:804–813 19. Simpson JL, Scott R, Boyle MJ, et al. (2006) Inflammatory subtypes in asthma: assessment and identification using induced sputum. Respirology 11:54–61

Occupational Asthma Joaquin Sastre

Definition For clinical diagnosis occupational asthma (OA) is a disease characterized by variable airflow limitation and/or nonspecific bronchial hyperresponsiveness and/or inflammation due to causes and conditions attributable to a particular occupational environment and not to stimuli encountered outside the workplace [1]. The definition may vary considering other purposes such as epidemiologic studies, workplace surveillance programs, or medicolegal assessment.

Classification Two types of OA are distinguished by whether they appear after a latency period [1]: (1) Immunologic – OA appears after a latency period of exposure necessary for the worker to acquire immunologically mediated sensitization to the causal agent. This type encompasses OA that is induced by an IgE mechanism (most high- and some low-molecular-weight agents), and OA in which an IgE mechanism has not been demonstrated consistently (low-molecular-weight agents, such as diisocyanates, western red cedar, acrylates,…). (2) Nonimmunologic – OA is characterized by the absence of a latency period. It occurs after accidental exposure to high concentrations of a workplace irritant. This clinical entity has been defined as irritant-induced asthma [2]. The most definitive form of irritant-induced asthma is “reactive airway dysfunction syndrome” (RADS) occurring after a single exposure to high levels of an irritating vapor, fume, or smoke [3]. OA caused by low doses of irritants occurs after repeated contact with low doses of the causative agent. It is a condition of particular current relevance but that is still under discussion.

J. Sastre () Fundacion Jiménez Diaz, Servicio de Alergia. Av. Reyes Catolicos 2, 28040 Madrid, Spain e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_15, © Springer 2009

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Variant forms of OA include eosinophilic bronchitis, asthma-like disorders caused by exposure to organic dusts and potroom asthma. The term work-aggravated asthma refers to the situation in which there is evidence of worsening of preexisting asthma as a consequence of environmental exposure in the workplace, but is not considered as OA.

Causal Agents More than 350 agents have been implicated in the development of OA. A complete list of those agents can be found in articles, reviews [4, 5], and web pages (e.g., www.worldallergy.org/professional/allergic_diseases_center/occupational_allergens; www.asmanet.com, www.asthme.csst.qc.ca). These agents are categorized into high-molecular-weight and low-molecular-weight agents. High-molecular-weight agents are proteins of animal or vegetable origin acting through an IgE-mediated mechanism. Low-molecular-weight agents include organic and inorganic compounds that, with a few exceptions, are not associated with an IgE mechanism. Chlorine, sulfur dioxide, combustion products, and ammonia are the most common agents that can induce irritant-induced asthma. Some workers may be exposed to both sensitizers and irritants and both diagnosis can be present in the same worker.

Epidemiology and Risk Factors A recent review of the literature estimated a median value of 15% the magnitude of the attributable risk of asthma due to occupation [6]. But it varied in different studies from 4% to 58% depending on the definition used, study population, and design of study. These include cross-sectional studies, population-based studies, randomized control trials, prospective or retrospective cohorts, case-referent studies, and case series. Each type of design has its own bias and varies regarding advantages, limitations, and information obtained. When objective testing was included in the studies, prevalence rates of about 5% for high-molecularweight agents and greater than 5% for low-molecular-weight agents were found. OA caused by high-molecular-weight substances is more frequent than that caused by low-molecular-weight agents [7]. RADS is estimated to occur in 36% of cases referred to hospital for assessment of OA [8]. In addition, 11–15% of all work-related asthma is reported to be caused by irritants [9]. In OA with latency period, it was observed that the higher the degree of exposure to an occupational agent, the higher the prevalence of asthma, showing that the intensity of exposure is an important determinant of sensitization and asthma caused by respiratory sensitizers [10–12]. Once a subject is sensitized, the main factor that influences the onset of symptoms is the degree of exposure. However, there is still a lack of information regarding the risk of sensitization at low concentrations or the existence of a “no-effect level.” It is still not known whether peak rather than

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mean or continuous exposures are more important in causing sensitization and OA. An important concept to keep in mind is that the concentration of an allergen that sensitizes is quite different from the one that provokes symptoms in workers already sensitized [13]. The minimum concentration that induces sensitization is at least one order, and probably two orders, of magnitude greater than the minimum concentration that elicits symptoms. What is known is that, even if the level of exposure is a critical factor for the development of OA, given the same level of exposure, only a small proportion of workers will develop sensitization and/or OA, suggesting that host susceptibility may also be an important factor. Atopy (skin reactivity to common inhalants) is a predisposing factor in workers exposed to high-molecular-weight agents, but it is a weak predictor of sensitization and development of OA [14]. An important question concerns whether sensitization to common aeroallergens precedes or follows sensitization to occupational allergens. For exposure to high-molecular-weight work-related allergens, subjects with new occupational sensitization are at greater risk of developing sensitization to common aeroallergens than are subjects without sensitization. In addition, new sensitization to common aeroallergens often occurs at about the same time as sensitization to work-related agents. Other factors besides atopy, such as rhinoconjunctivitis symptoms and having a measurable methacholine PC 20 (provocative concentration of methacholine producing a 20% decrease in FEV1-forced expiratory volumen in the first second), could be important in the development of respiratory symptoms and in the development and/or worsening of asthma. Cigarette smoking has been reported to be associated with the development of OA only in workers exposed to platinum salts and anhydride compounds, which are chemicals that cause asthma through an IgE mechanism. Sex plays a role in the distribution of occupational lung diseases, because there are sex differences in specific jobs and therefore differences in exposure to agents causing these diseases.

Genetics The phenotype of individuals with OA appears to be generated through the involvement of genes of the major histocompatibility complex on chromosome 6p coding for class II human leukocyte antigen (HLA) molecules. These molecules are required for presentation of an antigen to a T-cell receptor to initiate the cascade of events that lead to an antibody response. Associations of HLA molecules has been described in laboratory animal workers sensitized to rat lipocalins, which showed that about 40% of OA in the population examined could be attributed to an HLA-DRβ1*07 phenotype. Associations have been also described of some HLA molecules in sensitization to complex platinum salts, natural rubber latex, acid anhydrides, and in the case of asthma caused by red cedar, in which an increase in the HLA-DQBI*0603 and HLA-DQBI*0302 alleles and a decrease in the DQBI*050134 allele has been observed [15].

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In the case of isocyanates, an association has been described between this disease and the HLA-DQBQ0503 allele and protection in the presence of the HLADBQ0501 allele. The marker for susceptibility is the substitution of the aspartate residue at position 57 of HLA-DBQ [16]. But this HLA association has not been confirmed for diisocyanates by other investigations. Genes of the glutathione S-transferase and N-acetyltransferase superfamilies, that is critical for protecting cells from oxidative stress products, including lipid peroxides, also appear to be involved in OA, among subjects who were exposed to toluene diisocyanate (TDI) for 10 years or more. The frequency of the GSTP1 Val/Val genotype was lower in those who had asthma and in those with moderate to severe airway hyperresponsiveness to methacholine [17]. Even though HLA class II molecules are involved in immune recognition of some occupational agents, HLA associations are not strong enough to be used for generating preventative recommendations. Moreover, any reported association between a genetic marker and risk for disease cannot be considered definitive until the findings of the study have been replicated.

Pathogenesis IgE-dependent mechanisms. Most high-molecular-weight substances that cause OA are animal- or vegetable-derived proteins or glycoproteins that act via a mechanism involving IgE. OA induced by IgE-dependent agents is similar to allergic asthma that is unrelated to work. These proteins behave as complete antigens that stimulate the production of IgE. Nevertheless, some low-molecular-weight substances (e.g., acid anhydrides and platinum salts) are known to be highly reactive and capable of binding certain specific sites on proteins in the airway [18, 19] that will also stimulate IgE production. When these substances are inhaled they bind the specific IgE found on the surface of mast cells and basophils, triggering a sequence of cellular events that leads to the release of preformed or de novo synthesized mediators and the recruitment and activation of inflammatory cells that ultimately provoke an inflammatory reaction in the airways characteristic of asthma. IgE-independent mechanisms. Most low-molecular-weight substances that cause OA act via a mechanism that, while probably immunologic, does not involve IgE. Specific IgG and IgG4 antibodies appear to be associated more with the level of exposure than with the disease itself [20]. It is possible that cellular or delayed hypersensitivity is involved in these cases. Increased numbers of activated T lymphocytes (which express the receptor for IL-2), activated eosinophils, and mast cells have been observed in bronchial biopsies from patients with OA caused by low-molecular-weight substances [21]. Other low-molecular-weight agents, such as diisocyanates and plicatic acid, share the clinical and pathologic features of immunologic asthma, but do not consistently induce specific IgE antibodies. In addition, some substances can have nonimmunologic proinflammatory effects. If they bind glutathione, they cause intracellular glutathione deficiency, which can reduce

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defense against oxidizing agents [22]. In fact, it has been reported that exposure to isocyanates is associated with elevated intracellular concentrations of peroxide. Damage to cells of the bronchial mucosa caused by such process could amplify or initiate an immune response to low-molecular-weight substances. Irritation or toxicity. Many reports indicate that unintentional high-level respiratory irritant exposures can induce the new onset of asthma [8]. The massive initial epithelial lesion probably would not only induce changes in vascular permeability but would also provoke an increase in mucosal secretion that would contribute to the chronic inflammation seen in biopsy material [19]. Consequences of the damage in the bronchial epithelium are the loss of relaxing factors derived from epithelium, the exposure of nerve endings leading to neurogenic inflammation, and the release of inflammatory mediators and cytokines after the nonspecific activation of mast cells. Pathologic changes consist of marked fibrosis of the bronchial wall and denudation of the mucosa with fibrinohemorrhagic exudates in the submucosa [23]. During the process of recovery the inflammation would be resolved, leading to recovery of the epithelium, inhibition of neuronal activity, and improvement of vascular integrity. However, complete recovery would not always be achieved and sequelae of the inflammatory response would persist in the form of hyperreactivity and bronchial obstruction.

Bronchial Inflammation The airway inflammation process is similar in IgE-dependent and IgE-independent asthma and is characterized by the presence of eosinophils, lymphocytes, mast cells, and thickening of the reticular basement membrane (Fig. 1) [24]. In the bronchial airways, inflammatory cells are not only increased in number but are also activated, resulting in the secretion of a wide range of proinflammatory mediators and proteins; these mediators and proteins have a variety of harmful effects, such as toxic damage to epithelial cells. In the airway inflammatory process of OA, eosinophilia is associated with an increased number of T cells, especially CD4+ cells, which exhibit signs of activation [24]. Along with the increased expression of lymphocyte activation markers, in asthma induced by low-molecular-weight agents (e.g., diisocyanates) an increased number of cells producing proinflammatory cytokines have been reported. These proinflammatory cytokines, produced primarily by mononuclear phagocytes, may contribute to airway inflammation by several mechanisms, including increased expression of adhesion molecules, chemotaxis, and stimulation of inflammatory leukocytes. Together with these findings, antigen stimulation of monocyte chemoattractant protein 1 and tumor necrosis factor-α has been shown in isocyanate-induced asthma, suggesting that isocyanate-specific cellular immune reactions result in activation of macrophages, which may be important in the pathogenesis of this type of OA [25]. It has been suggested that CD8+ cells are also key cells in OA with an IgEindependent mechanism. A controversial issue is the role of neutrophils in OA.

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Fig. 1 Characteristics of asthma in the workplace

What is unclear in OA is whether neutrophilia can also be considered a marker of severe disease due, perhaps, to higher levels of exposure, or whether, for unknown reasons, the same level of exposure may induce an inflammatory airway infiltrate in which eosinophils or neutrophils are the predominant cells [26]. In grain dustinduced asthma-like disorder, the number of neutrophils in the bronchial mucosa is higher than that in allergic asthma, and the levels of IL-8 in induced sputum are significantly higher after inhalation challenge with grain dust extract than at baseline [27]. Potroom asthma has been recognized for many years in workers employed in the production of aluminum smelting, where the alumina is partially dissolved in an electrolyte of molten cryolite at about 960°C. But is still unknown whether fluorides are causative agents, coagents, or simply markers for the causative agent(s) of potroom asthma. Only one study has documented a dual asthmatic reaction with an associated increase in nonspecific airway responsiveness after exposure to the potroom workplace [28]. Bronchial biopsies obtained in workers with potroom asthma have shown that pathologic alterations are similar to those described in other types of asthma [29].

Diagnosis of Immunologic Occupational Asthma Diagnosis of OA should be confirmed by objective testing for asthma and then by establishing the relation between asthma and work [30]. Physicians should consider the possibility of OA in all adults with asthma. Figure 2 shows the algorithm used for diagnosis OA.

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Skin Prick Tests and /or specific IgE determination (if possible)

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Fig. 2 Diagnostic algorithm for immunologic occupational asthma. * May require measurement of exposure

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Clinical History The patient should be questioned not only about the existence of bronchial symptoms but also about eyes, nasal, and skin symptoms. In general, improvements of respiratory symptoms are observed at the weekend or during holidays, but this is not always the case. Clinical history provides high sensitivity (87–92%) but low specificity (14–22%) [31]. Physical examination, chest radiography, lung function testing, and standard workup do not differ in OA from those performed in any other asthmatic patient. A test to reveal nonspecific bronchial hyperreactivity, such as the methacholine or histamine test, is necessary when the bronchodilator test is negative due to the absence of bronchial obstruction at that time. In addition, if the methacholine or histamine test is negative, the existence of OA can be ruled out in practice, so long as the test is performed when the patient is working, since airway hyperresponsiveness can normalize following a variable period without exposure to the causative substance [32].

Immunologic Tests The results of immunologic tests can indicate exposure and sensitization but by themselves are unable to confirm a diagnosis of OA. A positive test does not always imply the existence of clinical signs. To prevent erroneous interpretations, the sensitivity and specificity of each of the antigens used must be known when any such tests are performed, since various substances can give rise to false positive or negative reactions. Either in vivo (prick test) or in vitro (analysis of specific IgE antibodies) techniques can be used. Sometimes allergen extracts have to be prepared in the laboratory due to a lack of commercial availability. In general, high-molecular-weight substances display a high sensitivity. Most low-molecular-weight substances are irritants and, therefore, prick tests are not appropriate. Only some low-molecularweight substances, such as isocyanates, appear to display a good specificity in in vitro studies. However, even though an IgE antibody to diisocyanates, which has strong positive response levels (an RAST score of 3 or greater) is present and exhibits high specificity [33], it has no sensitivity in detecting OA. The sensitivity increases when a blood sample is taken less than 30 days from the last exposure, which is consistent with the observed approximate 6 months of half-life of specific IgE.

Assessment of Inflammatory Markers Induced Sputum Various authors have described the usefulness of induced sputum in the diagnosis and monitoring of OA. Some studies have demonstrated that an increase in the

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number of eosinophils in sputum when the patient is working compared with out of working days, as well as a significant increase in the number of eosinophils and neutrophils following specific bronchial challenge in patients with OA caused by both high- and low-molecular-weight substances can aid the diagnosis of the disease [34]. It has been reported that additional analysis of cells in induced sputum increases the specificity of PEF (peak expiratory flow) monitoring [35]. It has also been demonstrated to be useful during specific challenge tests.

Exhaled Nitric Oxide Various studies have reported abnormalities in the concentrations of NO in respiratory diseases characterized by inflammatory processes. This marker has been extensively studied in asthma and it has been observed to be correlated with the number of eosinophils and the concentration of eosinophil cationic protein in sputum. Although the measurement of NO has been demonstrated to be useful for the diagnosis and follow-up of patients with asthma [36], its usefulness in the case of OA is less clear. Elevated concentrations of NO have been observed in asthma mediated by immunologic mechanisms involving IgE; this association is less clear in patients whose asthma is nonimmunologic or mediated by irritants [34].

Exhaled Breath Condensate Exhaled breath contains aerosols and water vapor that can be condensed by freezing. It is noninvasive, simple, and safe. However, analysis of this type of sample must be subjected to extensive standardization to allow future comparison of data obtained in different laboratories and assessment of its possible usefulness in patients with OA.

Bronchial Provocation in the Workplace: Peak Expiratory Flow Monitoring Bronchial provocation can confirm clinical suspicion of bronchial asthma caused by an agent that is present in the workplace or produced by work activities. The measurement relates the occupation to the disease but does not indicate which specific substance or agent is involved. However, if it is known that in that particular occupation a product is used that is commonly linked to OA, and if evidence of sensitization of the patient to a particular agent can be obtained through immunologic tests, diagnosis of OA caused by that agent is highly likely. The test must be performed during or after a period of time in which the patient is working and

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during or after another period in which the individual is not. Those periods must generally be at least 2 weeks long and interference in the test due to factors such as use of bronchodilators, presence of exacerbations, etc., should be prevented. In some cases, such as when it is suspected that irritant concentrations of particular substances are reached in the workplace, it may be necessary to measure the concentrations of the agent under suspicion. Measurement of the changes between two periods can be performed in various ways. The method that is most widely used and probably possesses the greatest diagnostic efficiency is serial monitoring of peak expiratory flow (PEF) during periods of exposure and lack of exposure, although serial monitoring of forced expiratory volume in 1 s (FEV1) during both periods or periodic monitoring of FEV1 or nonspecific bronchial hyperreactivity at the end of each period can also be useful [37]. In any case, they are not incompatible with each other and sometimes a method such as testing of nonspecific bronchial hyperreactivity and changes in cellularity of induced sputum can reinforce the diagnosis obtained using serial monitoring of PEF. The sensitivity and specificity of serial PEFs were found to be 73% and 100%, respectively. Although there is some lack of consensus regarding what represents a significant change, a difference of more than 20% in PEF or FEV1, or a reduction of at least threefold in the concentration of methacholine PC20 between the two periods would be considered definitively positive [5, 38, 39]. It is noteworthy that qualitative visual analysis of serial PEF recordings by an expert has a very high sensitivity and specificity, the highest among the different systems mentioned. Measurement four times per day is usually acceptable for most patients. Using that method, four types of response have been identified: (a) deterioration during the working day, such that on returning the following day the patient has completely recovered; (b) progressive deterioration over the course of the week with recovery at the weekend; (c) week-by-week deterioration with recovery only after at least 3 days away from work; and (d) maximal deterioration on Monday with recovery over the course of the week. Sometimes different patterns can also be observed, such as periodic reductions when the worker is exposed to a specific substance only occasionally over the course of the day or only on particular days. However, as with other respiratory function tests, experience and correct interpretation of the data can draw attention to manipulations of some individuals seeking work or financial advantages. Nowadays, however, electronic devices are available in which the use of a computer program allows the information to be stored and prevents it from being manipulated.

Specific Bronchial Provocation Test Although specific bronchial provocation tests are considered the gold standard for diagnosis of OA, in most cases they cannot be considered for routine diagnosis. They may be indicated in the following situations: (a) when there is a new agent that may be a possible cause of asthma; (b) to identify the causative agent from among various substances to which a worker is exposed; (c) when severe asthmatic reactions may

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occur when the individual returns to work; (d) when diagnosis is still doubtful after other tests have been performed, and (e) in assessment of protective devices. Exposure to the agent can be performed in several ways, always in specialized centers [40, 41]. When the agents are soluble, antigen solutions are administered as aerosols at increasing concentrations. The concentration at which the technique is initiated is calculated using a formula based on the PC20 (mg/ml) for methacholine and the lowest concentration that generates a positive response in skin prick tests. Forced spirometry is performed 10 min after each nebulization. The test result is positive if there is a fall in FEV1 of at least 20% from baseline. The results are expressed as the PC20 of the allergen, or as the PD20 of the allergen if a dosimeter is used. If the result is negative a higher concentration is administered. During the 24 h following inhalation it is important to monitor FEV1 every hour until bedtime to identify delayed responses. When the agents are insoluble different types of challenges can be used. Any test used must assure exposure of the patient to a nonirritant concentration of the suspected causative agent. For this reason, means of measuring the concentration of those agents should be available if possible. The length of exposure varies according to the agent and the characteristics of the patient. If the test is negative, exposure is repeated for a longer period of time or with a higher concentration of the product on successive days. When non-water-soluble dust is used, it can be passed from one tray to another mixed with lactose to produce a cloud of dust. The use of lactose alone allows a placebo test to be performed. Drug-inhalation devices that employ capsules containing a specific amount of dust have also been used. Some hospitals have developed equipment for closed-circuit exposure to dust, which in theory offers greater control over exposure and makes it safer for health care personnel. When gases or fumes are tested, the methods used to generate a given concentration can be classified as static or dynamic (continuous flow). In the static systems, a known quantity of gas is mixed with another of air to produce a given concentration. In dynamic systems, the airflow and the addition of gas is controlled to produce a specific dilution. These systems offer a continuous flow and allow rapid and predictable changes in the concentration to be made, favoring good mixture and minimizing loss through adsorption to the walls of the chamber.

Diagnosis and Treatment of Nonimmunologic Occupational Asthma Reactive Airways Dysfunction Syndrome The diagnostic criteria for RADS are [3]: (1) Absence of prior respiratory symptoms, (2) exposure to a gas, smoke, or vapor present at high concentrations and with irritant qualities, (3) onset of symptoms within the first 24 h of exposure and

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persistence for at least 3 months, (4) symptoms similar to asthma with cough, wheezing, and dyspnea, (5) objective evidence of bronchial asthma, (6) other types of lung disease ruled out. Reports of experience with a small number of cases have indicated that early treatment with high doses of corticosteroids can improve prognosis [42]. However, many patients with RADS continue to present symptoms of bronchial irritation and hyperreactivity years after exposure. Asthma caused by exposure to grain dust. Asthma caused by exposure to dust from cereal grain occurs mainly in workers involved with grain silos, mills, or bakeries but is also seen in agricultural workers [43]. The specific cause is unknown but could be a component of the cereal, parasitic fungi such as smut or rust or saprophytes such as Aspergillus species, organisms such as weevils, mites, or gramnegative bacteria. In close to 50% of cases the symptoms improve or disappear spontaneously, suggesting a process of desensitization in some cases. Asthma in livestock workers. A higher rate of nonatopic asthma has been demonstrated in farm workers who are exposed to livestock, particularly birds, cattle, and pigs. This type of asthma is associated with exposure to endotoxins, fungal spores, and ammonia [44].

Eosinophilic Bronchitis Eosinophilic bronchitis causes chronic cough, expectoration, dyspnea, and on rare occasions, wheezing. Its main characteristic is the presence of a large number of eosinophils in sputum and the absence of variable airflow obstruction and/or bronchial hyperresponsiveness. It should be noted that cases of eosinophilic bronchitis have been described associated with exposure to certain workplace-related substances (Tetrahydrophtalic anhydride, Cyanoacrylate, Methacrylate, Latex Mushroom spores, Lysozyme, Welding fumes, Formaldehyde, Chloramine T, Isocyanate (MDI), Cereal flour) [45]. In such cases, and in the absence of recognizable bronchial hyperresponsiveness, diagnosis is provided when significant reproducible changes in the number of eosinophils in sputum are seen to be associated with workplace exposure.

Work-Aggravated Asthma The term work-aggravated asthma refers to the situation in which there is evidence of worsening of preexisting asthma as a consequence of environmental exposure in the workplace. Although it manifests as an increase in the frequency and/or severity of asthma symptoms and/or an increase in the medication required to control the disease during working days, diagnosis should be performed on the basis of

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changes in bronchial diameter, the degree of bronchial hyperresponsiveness, or the extent of inflammation of the airway in relation to workplace exposure. In these cases specific bronchial challenge test are negative.

Environmental Monitoring of Allergens and Chemical Agents Quantification of environmental allergens has various applications that can also be useful in the diagnosis of OA and it is sometimes necessary to confirm a diagnosis of OA in the laboratory or workplace and to monitor the agent following introduction of safety measures or workplace changes [13]. Inventory and identification of substances that may be present in the working environment is necessary. It is very useful in reviewing a manufacturer’s safety data sheets, which nearly always provide the necessary information on the substances used. Assessment of the state of the agent as a dust, aerosol, gas, or vapor must be investigated, since this can affect its interaction with the body and the way in which it must be analyzed. Prior to sampling and analysis it is usually indispensable to first focus suspicion on a specific causative agent. Otherwise, it is difficult, and sometimes impossible, to identify the agent. It must be remembered that a specific agent normally requires a particular type of sampling in order to then use the appropriate analytic technique. The web pages of various organizations publish sampling methods and analytic techniques for a variety of chemicals (www.c.cdc.gov/niosh/nmam/nmammenu. html; www.o.osha.gov/dts/sltc/methods/index.html).

Management When asthma is induced by a workplace sensitizer, strict exposure control is needed. Early removal of the employee from exposure to the offending agent, although associated with a better medical outcome, has the worst socioeconomic outcome in many cases [46]. Although respirators have not usually been considered safe for sensitizer-induced asthma, there is evidence that the use of respirators and other environmental controls to lower exposures may be helpful in OA [47, 48]. The use of respirators requires worker adherence, professional guidance to assure correct device selection, and user training. For patients with OA induced by an acute exposure to an irritant at work, steps should be taken to prevent further exposure to high concentrations of the irritant. Patients with preexisting asthma that is aggravated at work should limit exposure to irritants, tobacco smoke, and relevant environmental allergens.

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Pharmacologic treatment for patients with OA caused by a respiratory sensitizer and/or irritant should be the same as that for patients with nonoccupational asthma. The beneficial effects of inhaled glucocorticoids are more evident when treatment starts soon after diagnosis [49]. Immunotherapy with extracts of high-molecular-weight occupational allergens has been studied only for natural rubber latex. Clinicians should support the patient in the pursuit of appropriate compensation. The regulations affecting compensation policies vary according to the country or region.

Natural History and Long-Term Consequences To summarize the natural history of OA and the role of specific agents, the risk of OA is highest soon after the first exposure, because most subjects develop asthma within 1–2 years of exposure. Nevertheless, the latency period can vary from months to years [50]. The rate of acquiring both sensitization and asthmatic symptoms may differ according to the nature of the agent, and the intensity of exposure. Discontinuation of exposure to the causative agent is associated with an improvement in symptoms and lung function that does not normally exceed 50% in affected individuals. Lung function and nonspecific bronchial hyperreactivity is only normalized in around 25% of individuals. In general, the prognosis of a given patient in whom contact with the causative agent is removed depends on the severity of the condition when diagnosis was established. On the other hand, if exposure to the causative agent continues, it almost always leads to clinical and functional deterioration of the patient [7].

Prevention and Surveillance To prevent OA, removal of the offending agent and substitution of a nontoxic agent are the best approach because they eliminate the asthma hazard. If substitution is not possible, ongoing maintenance of engineering controls, such as enclosure of the industrial process and improving work area ventilation, are useful, particularly when the employee works at a constant location and does the same tasks [50]. For primary prevention, host and environmental factors are taken into consideration; for secondary prevention, preclinical changes in the disease need to be identified; and for tertiary prevention, workers should be diagnosed in an early phase of the disease and appropriate management of the disease should be offered [51]. The reduction of respiratory exposure has been achieved in some workplaces, such as in the manufacture of detergent enzymes by improved dust control and the encapsulation of the enzymes and for natural rubber latex by the use of powderfree low-protein gloves. Secondary prevention addresses preclinical changes, namely the immunologic sensitization that generally precedes the development

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of OA. Although the positive predictive value of skin reactivity as an indicator of immunologic sensitization is low, those with positive skin tests to high-molecularweight agents should be monitored closely. For these agents, rhinoconjunctivitis can be considered a predictor of the later development of OA. Early detection of disease could be accomplished by periodic examination of workers employed in high-risk industries, but this procedure is costly. Medical surveillance programs for sensitization and OA consist of a questionnaire given before employment and repeated periodically; in addition, immunologic tests (skin tests/or specific IgE) and pulmonary function tests may be considered. Tertiary prevention aims at the prevention of permanent asthma. Examples of tertiary prevention include therapy with inhaled glucocorticoids, substitution of non-isocyanate-containing spray paints in the workplace of an employee with isocyanate-induced asthma, and strict avoidance of exposure to mice in an animal handler’s asthma that resulted from an allergy to mice. Early recognition of the disease and early removal of the patient from exposure make it more likely that the patient will avoid permanent asthma.

References 1. Bernstein IL, Bernstein DI, Chan-Yeung M, Malo JL. Definition and classification of asthma in the workplace. In: Bernstein IL, Chan-Yeung M, Malo JL, Bernstein DI, editors. Asthma in the workplace. New York: Taylor & Francis; 2006. pp. 1–8. 2. Tarlo SM, Broder I. Irritant induced asthma. Chest 1989; 96:297–300. 3. Brooks SM, Weiss MA, Bernstein IL. Reactive airways dysfunction syndrome (RADS): persistent asthma syndrome after high level irritant exposures. Chest 1985; 88:376–384. 4. Mapp CE. Agents, old and new, causing occupational asthma. Occup Environ Med 2001; 58:354–360. 5. Mapp CE, Boschetto P, Maestrelli P, Fabri LM. Occupational asthma. Am J Respir Crit Care Med 2005; 172:280–305. 6. Balmes J, Becklake M, Blanc P, Henneberger P, Kreiss K, Mapp CE, et al. American thoracic society statement: occupational contribution to the burden of airway disease. Am J Respir Crit Care Med 2003; 167:787–797. 7. Becklake MR, Malo JL, Chan-Yeung M. Epidemiological approaches in occupational asthma. In: Bernstein IL, Chan-Yeung M, Malo JL, Bernstein DI, editors. Asthma in the workplace. New York: Taylor & Francis; 2006. pp. 37–86. 8. Tarlo SM. Workplace respiratory irritants and asthma. Occup Med. 2000. 9. McDonald JC, Keynes HL, Meredith SK. Reported incidence of occupational asthma in the United Kingdom 1989–1997. Occup Environ Med 2000; 57:823–829. 10. Venables KM, Newman Taylor AJ. Exposure-response relationships in asthma caused by tetrachlorophthalic anhydride. J Allergy Clin Immunol 1990; 85:55–58. 11. Nieuwenhuijsen MJ, Putcha V, Gordon S, Heederik D. Venables KM, Cullinan P, Newman Taylor AJ. Exposure-response relations among laboratory animal workers exposed to rats. Occup Environ Med 2003; 60:104–108. 12. Heederik D, Houba R. An exploratory quantitative risk assessment for high molecular weight sensitizers: wheat Hour. Ann Occup Hyg 2001; 45:175–185. 13. Swanson MC. Immunochemical measurement of occupational aeroallergenic bioaerosols: determination of permissible exposure limits? Proceedings of the first Jack Pepys Occupational Asthma Symposium. Am J Respir Crit Care Med 2003; 167:456–458.

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14. Malo JL. Prevention of occupational asthma. Proceedings of the first Jack Pepys Occupational Asthma Symposium. Am J Respir Crit Care Med 2003; 167:463–464. 15. Newman Taylor AJ, Yucesoy B. Genetics and occupational asthma. In: Bernstein IL, ChanYeung M, Malo JL, Bernstein DI, editors. Asthma in the workplace. New York: Taylor & Francis; 2006. pp. 87–108. 16. Mapp CE, Beghe B, Balboni A, Zamorani G, Padoan M, Jovine L, et al. Association between HLA genes and susceptibility to toluene diisocyanate-induced asthma. Clin Exp Allergy 2000; 30:651–656. 17. Mapp CE, Fryer AA, de Marzo N, Pozzato V, Padoan M, Boschetto P. Strange RC, Hemmingsen A, Spiteri MA. Glutathione S-transferase GSTP1 is a susceptibility gene for occupational asthma induced by isocyanates. J Allergy Clin Immunol 2002; 109:867–872. 18. Sastre J, Vandesplas O, Park H-S. Pathogenesis of occupational asthma. Eur Respir J. 2003; 22:364–367. 19. Gautrin D, Bernstein IL, Brooks S, Henneberger PK. Reactive airways dysfunction syndrome or irritant induced asthma. In: Bernstein IL, Chan-Yeung M, Malo JL, Bernstein DI, editors. Asthma in the workplace. New York: Taylor & Francis; 2006. pp. 581–630. 20. Park HS, Hong CS. The significance of specific IgG and IgG4 antibodies to a reactive dye in exposed workers. Clin Exp Allergy 1991; 21:357–362. 21. Saetta M, Di Stefano A, Maestrelli P, De Marzo N, Milani GF, Pivirotto F, et al. Airway mucosal inflammation in occupational asthma induced by toluene diisocyanate. Am Rev Respir Dis 1992; 145:160–168. 22. Day BW, Jin R, Basalyga DM, Kramarik JA, Karol MH, et al. Formation, solvolysis and transcarbamoylation reactions of bis(s-glutathionyl) adducts of 2,4- and 2,6-diisocyanotoluene. Chem Res Toxicol 1997; 10:424–431. 23. Lemière C, Malo JL, Boulet M. Reactive airways dysfunction syndrome due to chlorine: sequential bronchial biopsies and functional assessment. Eur Respir J 1997; 10:241–244. 24. Maestrelli P, Occari P. Turato G, Papiris SA, Di Stefano A, Mapp CE, Milani GF, Fabbri LM, Saetta M. Expression of interleukin (IL)-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI). Clin Exp Allergy 1997; 27:1291–1298. 25. Lummus ZL, Alam R, Bernstein JA, Bernsetin DI. Diisocyanate antigen-enhanced production of monocyte chemoattractant protein-1, IL-8, and tumor necrosis factor-α by peripheral mononuclear cells of workers with occupational asthma. J Allergy Clin Immunol 1998; 102:265–274. 26. Anees W, Huggins V, Pavord ID, Robertson AS, Burge PS. Occupational asthma due to low molecular weight agents: eosinophilic and non-eosinophilic variant. Thorax 2002; 57:231–236. 27. Park HS, Jung KS, Hwang SC, Nahm DH, Yim HE. Neutrophil infiltration and release of IL-8 in airway mucosa from subjects with grain dust-induced occupational asthma. Clin Exp Allergy 1998; 28:724–730. 28. Desjardins A, Bergeron JP, Ghezzo H, Cartier A, Malo JL. Aluminium potroom asthma confirmed by monitoring of forced expiratory volume in one second. Am J Respir Crit Care Med 1994; 150:1714–1717. 29. Sjaheim T, Halstensen TS, Lund MB, Bjortuft O, Drablos PA, Malterud D, Kongerud J. Airway inflammation in aluminium potroom asthma. Occup Environ Med 2004; 61:779–785. 30. Moscato G, Malo JL, Bernstein D. Diagnosing occupational asthma: how, how much, how far? Eur Respir J 2003; 21:879–885. 31. Baur X, Huber H, Degens PO, Allmers H, Ammon J. Relation between occupational asthma case history, bronchial melhacholine challenge, and specific challenge test in patients with suspected occupational asthma. Am J Ind Med 1998; 33:114–122. 32. Sastre J, Fernandez-Nieto M, Novalbos A, de Las Heras M, Cuesta J, Quirce S. Need for monitoring nonspecific bronchial hyperresponsiveness before and after isocyanate inhalation challenge. Chest 2003; 123:1276–1279. 33. Tee RD, Cullinan P, Welch J, Burge PS, Newman Taylor AJ. Specific IgE to isocyanates: a useful diagnostic role in occupational asthma. J Allergy Clin Immunol 1998; 101:709–715.

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34. Lemiere C. Induced sputum and exhaled nitric oxide as noninvasive markers of airway inflammation from work exposures. Curr Opin Allergy Clin Immunol 2007; 7:133–137. 35. Girard F, Chaboilliez S, Cartier A, Coté J, Hargreave F, Labrecque M, et al. An effective strategy for diagnosing occupational asthma. Am J Respir Crit Care Med 2004; 170:845–850. 36. Taylor DR, Pijnenburg MW, Smith AD, Jongste JCD. Exhaled nitric oxide measurements: clinical application and interpretation. Thorax 2006; 61:817–827. 37. Leroyer C, Perfetti L, Trudeau C, L’Archevêque, Chang-Yeung M, Malo JL. Comparison of serial monitoring of peak expiratory flow and FEV1 in the diagnosis of occupational asthma. Am J Respir Crit Care Med 1998; 158:827–832. 38. Perrin B, Lagier F, L’Archeveque J, Cartier A, Boulet LP, Cote J, et al. Occupational asthma: validity of monitoring of peak expiratory flow rates and non-allergic bronchial responsiveness as compared to specific inhalation challenge. Eur Respir J 1992; 5:40–48. 39. Gannon PFG, Newton DT, Belcher J, Pantin CF, Burge PS. Development of OASYS-2: a system for the analysis of serial measurement of peak expiratory flow in workers with suspected occupational asthma. Thorax 1996; 51:484–489. 40. Vandenplas O, Malo JL. Inhalation challenges with agents causing occupational asthma. Eur Respir J 1997; 10:2612–2629. 41. Quirce S, Sastre J. Occupational asthma. Allergy 1998; 53:633–641. 42. Chester E, Kaimal J, Payne CB Jr, Kohn PM. Pulmonary injury following exposure to chlorine gas. Possible beneficial effects of steroid treatment. Chest 1977; 72:247–250. 43. Chan-Yeung M, Emerson DA, Kennedy SM. The impact of grain dust on respiratory health. Am Rev Respir Dis 1992; 145:476–487. 44. Eduard W, Douwes J, Omenaas E, Heederick D. Do farming exposures cause or prevent asthma? Results from a study of adult Norwegian farmers. Thorax 2004; 59:381–386. 45. Quirce S. Eosinophilic bronchitis in the workplace. Curr Opin Allergy Clin Immunol 2004; 4:87–91. 46. Ameille J, Parion JC, Bayeux MC, Brochard P, Choudat D, Conso F, Devienne A, Gamier R, Iwatsubo Y. Consequences of occupational asthma on employment and financial status: a follow-up study. Eur Respir J 1997; 10:55–58. 47. Taivainen AI, Tukiainen HO, Terho EO, Husman KR. Powered dust respirator helmets in the prevention of occupational asthma among farmers. Scand J Work Environ Health 1998; 24:503–507. 48. American Thoracic Society. Respiratory protection guidelines. Am J Respir Crit Care Med 1996; 154:1153–1165. 49. Malo JL, Cartier A, Coté J, Milot J, Lablanc C, Paquette L, Ghezzo H, Boulet LP. Influence of inhaled steroids on the recovery of occupational asthma after cessation of exposure: an 18-month double-blind cross-over study. Am J Respir Crit Care Med 1996; 153:953–960. 50. American Thoracic Society. Guidelines for assessing and managing asthma risk at work, school, and recreation. Am J Respir Crit Care Med 2004; 169:873–881. 51. Cullinan P, Tarlo S, Nemery B. The prevention of occupational asthma. Eur Respir J 2003; 22:853–860.

Hypersensitivity Pneumonitis Moisés Selman and Andrew Churg

Introduction Hypersensitivity pneumonitis (HP), also known as extrinsic allergic alveolitis, is a syndrome that results from repeated inhalation of a wide spectrum of organic particles, including mammalian and avian proteins, fungi, thermophilic bacteria, nontuberculous mycobacteria, and certain small-molecular weight chemical compounds (Table 1). Exposure to these antigens provokes, in a susceptible individual, a diffuse and predominantly mononuclear cell inflammation of the small airways and pulmonary parenchyma [1].

Epidemiology The prevalence of HP is difficult to estimate accurately because it represents a diverse group of syndromes with different causative agents and because epidemiologic studies are scanty and lack uniform diagnostic criteria. In addition, it is important to consider that the prevalence and incidence of HP vary considerably around the world, depending upon disease definitions and diagnosis, intensity of exposure to offending antigens, geographical and local conditions, cultural practices, agricultural activities, and the presence of industrial manufacturing plants and host risk factors. The two most frequently reported types of HP are the farmer’s lung and the pigeon breeder’s lung. Among them, most epidemiological studies have been related to farmer’s lung, indicating that its prevalence in exposed farmers to be estimated at between 0.5% and 3% [2]. Old studies indicate that the prevalence of HP among bird fanciers ranges from 20 to 20,000 per 100,000 persons at risk [3, 4]. However, the prevalence of pigeon breeder’s lung among people with only a few birds at home, a type of exposure that usually provokes a more chronic lung disease, is largely unknown. M. Selman Instituto Nacional de Enfermedades Respiratorias, México A. Churg () Department of Pathology, University of British Columbia, Vancouver, BC, Canada e-mail: [email protected]

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Table 1 Antigens involved in hypersensitivity pneumonitis Disease Antigen

Source

Fungal and bacterial Farmer’s lung

Moldy hay, grain, silage

Ventilation/humidifier lung Metal-working fluid-associated HP Mushroom worker’s lung Woodworker’s lung Sauna taker’s lung Maple bark strippers’ lung Cheese washers’ lung Sequiosis Stipatosisb Suberosis Hot-tub lung Summer-type pneumonitis Animal proteins Pigeon breeder’s disease Furrier’s lung Animal handler’s lung; laboratory worker’s lung Chemical compounds Pauli’s reagent alveolitis Chemical worker’s lung Pyrethrum pneumonitis

Saccharopolyspora rectivirgula, Thermoactinomyces vulgaris T. vulgaris, T. sacchari Mycobacterium immunogenum

Contaminated forced-air systems; water reservoirs Metal-working fluids

Aureobasidium sp, Pullularia Cryptostroma corticale Penicillium caseii Pullularia Aspergillus fumigatus P. frequentans, A. fumigatus M. avium complex Trichosporon cutaneum

Moldy mushroom compost Oak, cedar, mahogany dust, pine, and spruce pulp Contaminated sauna water Mouldy maple bark Mouldy cheese Mouldy sawdust Esparto fibers Cork dust Hot tubs; swimming pools Contaminated old houses

Avian droppings, feathers, serum Animal-fur dust Rats, gerbils

Parakeets, budgerigars, pigeons, chickens, turkeys Animal pelts Urine, serum, pelts proteins

Sodium diazobenzene sulfate Isocyanates; trimellitic anhydride Pyrethrum

Laboratory reagent Polyurethane foams, spray paints, special glues Insecticide

T. sacchari Alternaria sp., wood dust

HP occurs in many different settings (Table 1) but there are no epidemiological approaches to evaluate the magnitude of the problem. Small outbreaks are often reported in occupational settings. Thus, for example, several outbreaks, putatively attributed to Mycobacterium immunogenum, or some bacteria such as Pseudomonas sp, have been detected in workers exposed to metal working fluids [5, 6]. It is important to consider that in the USA alone over 1.2 million workers involved in machine finishing, machine tooling, and other metal-working and metal-forming operations are potentially exposed [7].

Risk Factors Only a few exposed individuals develop HP, indicating that genetic factors and other host or environmental factors influence an individual’s risk of disease. It is likely that the immunopathological response in the lung microenvironment is the

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final manifestation of the interaction of multiple genes involved in the immune response. However, studies dealing with promoting (or protecting factors) are scanty. Genetic susceptibility associated with the major histocompatibility complex (MHC), primarily class II genes, has been described both in the human disease and in an experimental model of HP [8–11]. The class II genes code the molecules HLA-DR, -DQ, and -DP, expressed in a subgroup of antigen-presenting immune cells, including B cells, activated T cells, macrophages, dendritic cells, and thymic epithelial cells, and are crucial regulators of the immune response. Also, the frequency for the tumor necrosis factor (TNF)-α A2 allele, a genotype associated with high TNF-α production in vitro, was found to be significantly higher in farmer’s lung patients [12]. However, a more recent study did not confirm this association or other cytokine gene polymorphisms, including interleukin (IL)-10 (−592C/A, −819C/T, −1082G/A), transforming growth factor (TGF)-beta 1 (−509C/T, +869T/C), and IL-6 (−634C/G) in patients with summer-type HP and pigeon breeder’s disease [13]. Some environmental factors (a second non-antigen exposure) may also trigger the disease. Thus, in experimental HP it has been demonstrated that mice previously infected with respiratory syncytial virus or Sendai virus display an exaggerated inflammatory response compared with the virus or the antigen alone [14, 15]. Likewise, common respiratory viruses are usually present in the lower airways of patients during acute HP [16]. Viral infections may increase the antigenpresenting capacity of alveolar macrophages and decrease antigen clearance, which among other effects favors the expansion of T lymphocytes in the lung parenchyma. Along the same line of thought, it has been suggested that exposure to pesticides, mainly organochlorine and carbamate pesticides, may be a potential risk factor for farmer’s lung [17]. Some host situations may also contribute as a risk factor. A recent work provided evidence for increased frequency of circulating microchimerism including the presence in the lung of fetal microchimeric cells in patients with HP [18]. However, the putative role of these microchimeric fetal cells in the HP lungs is presently unknown, although they seem to increase the severity of the disease.

The Effect of Smoking It has been known for a long time that HP occurs more frequently in nonsmokers, than in smokers, under the same risk exposure [19, 20]. Similar findings have also been reported in patients with HP provoked by the inhalation of aerosols of contaminated air conditioners and in the summer-type HP [21, 22]. The mechanisms involved in the protective effect of cigarette smoking on the development of hypersensitivity pneumonitis remain unclear. Cigarette smoking can suppress specific antibody formation, reduce the proliferative response of T-helper (CD4+) lymphocytes in bronchoalveolar lavage (BAL) fluid and impair

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macrophage effector function thus interfering with the exaggerated reaction necessary to develop HP [23]. More recently, it was found that nicotine reduces the alveolar inflammatory response to Saccharopolyspora rectivirgula antigen and affects some functions of alveolar macrophages in vitro [24]. Nicotine may affect the expression of key cytokines (e.g., interleukin-10, TNF-α, and interferon gamma) both in vivo and in vitro [24]. However, it is important to emphasize that when HP occurs in smokers, the clinical course of the disease may change to a more insidious and chronic form [1]. Thus, smoker patients with farmer’s lung had more recurrences than nonsmokers after the initial diagnosis and exhibited higher mortality at 10-year follow-up [25]. Also, in an experimental model in guinea pigs, cigarette smoking decreased the initial inflammatory response, but retarded the eventual recovery during maintenance of antigen exposure [26].

Clinical Presentation The clinical features of the disease are usually similar, regardless of the type or nature of the inhaled dust. The disease presents in three overlapping clinical forms: acute, subacute, or chronic [1, 27]. HP is a complex dynamic clinical syndrome where clinical expression depends upon several factors including the nature of the inhaled dust, intensity and frequency of exposure to the antigens, and the host susceptibility.

Acute Presentation Episodes of the acute form often present after intermittent and high-level exposure to the offending antigen over a short period of time. Typically symptoms occur a few hours after exposure to the offending antigen and consist of the abrupt onset of a flu-like syndrome characterized by fever, chills, and malaise. Pulmonary symptoms include dyspnea and nonproductive cough. These symptoms and signs gradually clear over the next few days, but often recur after the next (intense) inhalation of the causative antigen. Between acute attacks the individual is usually completely normal. Differential diagnosis of acute HP includes acute viral respiratory infection and atypical pneumonia caused by viral or mycoplasmal agents. Here, a history of illness occurring within hours of exposure to an identifiable antigen is fundamental to suspect acute HP. In farmers, the differential diagnosis must include the organic dust toxic syndrome (ODTS), which is usually associated to the exposure of bacterial endotoxins and fungal toxins of moldy hay in unloading silos [31]. ODTS is a self-limited syndrome characterized by a fever with chills, malaise, myalgia, headache, and dyspnea. In contrast to patients with acute HP, ODTS patients generally

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have no precipitins to antigens of molds, and usually present with normal clinical findings upon respiratory examination and chest radiographs.

Subacute Presentation The subacute form appears gradually over several days to weeks, and is characterized by cough and dyspnea. Patients often present with fever at the onset of the illness. Occasionally, the disease evolves rapidly and patients need to be hospitalized for severe respiratory failure. Differential diagnosis includes some granulomatous lung infections such as tuberculosis, noninfectious granulomatous lung disorders like sarcoidosis, and some of the idiopathic interstitial pneumonias, i.e., nonspecific interstitial pneumonia and cryptogenic organizing pneumonia.

Chronic Presentation The chronic form results from continual low-level exposure to inhaled antigens, usually in the domestic environment. Chronic disease may occur in two clinical settings, i.e., in patients following recurrent (unrecognized and untreated) acute/ subacute episodes or appearing insidiously without any history of acute episodes [28, 29]. In the insidious group, patients have usually few (if any) acute symptoms and the disease is characterized by slowly progressive dyspnea, anorexia, weight loss, fatigue, malaise, and cough. Tachypnea and bibasilar crackles are the usual clinical findings in all forms of HP. In chronic HP, overt manifestations of right-sided heart involvement and digital clubbing may be found [30]. Chronic HP may mimic any chronic interstitial lung disease, including idiopathic pulmonary fibrosis, and precise diagnosis often needs lung biopsy [32]. It has been proposed that acute HP is provoked by immune complexes while subacute and chronic hyperreactivity is orchestrated by T lymphocytes [1].

Imaging Chest Radiographs In general, chest x-ray is useful to approach the diagnosis of interstitial lung disease, but lacks sensitivity and specificity for a precise diagnosis. Thus, 20–30% of patients with acute and even subacute forms of HP may have normal chest radiographs [33, 34].

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In the acute presentation, a transient diffuse ground-glass or ill-defined airspace consolidation is usually seen. In the subacute presentation the chest radiograph reveals a fine nodular or reticulonodular shadowing with some degree of groundglass attenuation. The chronic stages are characterized by a predominantly reticular pattern superimposed on some subacute infiltrates.

Computed Tomography In acute HP, high-resolution computed tomography (HRCT) features may include diffuse ground-glass attenuation (predominating in the lower lobes) and patchy or widespread air space opacification (Fig. 1). Subacute HP is characterized by a combination of patchy or diffuse bilateral ground-glass opacities, poorly defined small centrilobular nodules (usually less than 5 mm in diameter), and lobular areas of decreased attenuation on inspiration and of air trapping on expiration [27] (Fig. 2). A confident diagnosis of subacute HP can be done with these abnormalities [27]. HP patients with organizing pneumonia exhibit areas of consolidation. Also, thin-walled cysts are occasionally found in patients with subacute HP [27, 35]. The cysts vary in number (they are typically few), and have a random distribution [35]. Patients with chronic disease show fine and coarse reticular opacities superimposed on subacute changes such as micronodules and ground-glass attenuation. Reticulation

Fig. 1 High-resolution computed tomography (HRCT) scan of the chest of a patient with acute HP who consulted 2 days after the beginning of severe dyspnea. Large areas of ground-glass attenuation and consolidation can be observed

Fig. 2 High-resolution computed tomography (HRCT) of a patient with subacute HP showing small, poorly defined centrilobular nodules, areas of ground-glass opacities and focal areas of decreased attenuation (mosaic pattern)

Fig. 3 High-resolution computed tomography (HRCT) image of a chronic case of HP showing bilateral reticulation superimposed to patchy areas of ground-glass attenuation

may eventually evolve to honeycombing (Fig. 3). Patients with chronic farmer’s lung may develop emphysema, and this seems to occur more frequently than interstitial fibrosis [36].

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Pulmonary Function Tests Lung function abnormalities are neither specific nor diagnostic for HP and similar changes are found in most interstitial lung diseases. Independent of the clinical presentation, HP patients show a restrictive functional pattern with loss of lung volumes and capacities [1]. The decrease is usually more significant in vital capacity than in total lung capacity, whereas the residual volume is relatively well preserved. Patients with subacute and chronic HP display a decreased compliance over the entire range of the reduced inspiratory capacity [37]. On gas exchange patients present with hypoxemia (or normoxemia) at rest, which usually worsens with exercise, normal or slightly decreased arterial carbon dioxide tension (PaCO2), and elevated alveolar-arterial oxygen gradient [P(A-a O2]. An early and sensitive functional abnormality is a reduction in the diffusing lung capacity for carbon monoxide (DLCO), which appears to be a good predictor of arterial oxygen desaturation during exercise. Following exposure to moldy hay, patients with a history of farmer’s lung present with a decreased breathing reserve and a greater [P(A-a)O2] gradient at baseline and show an increased [P(A-a)O2] gradient and dead space to tidal volume (VD/VT) ratio during exercise. In addition, a ventilation-perfusion mismatch develops [38]. Some degree of obstruction of the peripheral airways, as suggested by a decrease in the maximum to mid-flow rates and in the ratio of dynamic to static lung compliance, may be present due to bronchiolitis [39]. Furthermore, patients with chronic farmer’s lung may show functional defects reflecting bronchiolitis and emphysematous changes [40]. In these cases, the pattern of lung function impairment is characterized by airway obstruction, increased lung compliance, and reduced elastic recoil.

Routine Laboratory Tests With the exception of the detection of specific antibodies against the offending antigen, there are neither clinically useful diagnostic laboratory tests nor serologic markers to monitor disease activity or progression. The presence of specific antibodies is an important piece for diagnosis. However, it should be remembered that a number of exposed but asymptomatic individuals or exposed individuals with other diseases may present circulating specific precipitins (false positives). Also, false negatives might occur, mostly in chronic patients. Therefore, HP cannot be ruled in solely on the basis of positive antibodies or excluded on the basis of negative antibodies [2]. In the acute and subacute forms, a moderate neutrophilic leukocytosis with lymphopenia may occur. Increases in C-reactive protein, rheumatoid factor, immune complexes, and a mild increase in immunoglobulin G and M (IgG and

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IgM) have also been described as nonspecific findings [1]. It has been suggested that measurement of plasma lactate dehydrogenase (LDH) may be useful in assessing the disease activity, but this finding has not been corroborated in another cohort [41].

Bronchoalveolar Lavage A remarkable increase in the percentage of lymphocytes, primarily T cells, often over 40%, is usually found [42–44]. Smokers and chronic cases may have a moderate increase (>25%). The ratio of T cell CD4+/CD8+ subpopulations is quite variable. Patients with acute and subacute disease usually show an increase of CD8 + T cells with a decrease in the CD4+/CD8+ ratio [45, 46]. However, a number of patients with subacute HP and most patients with chronic HP have an increase of both subsets without changes in its ratio, or a predominance of CD4+ on the surface phenotypes of BAL T cells and a consequent increased CD4+/CD8+ ratio [47, 48]. In general terms, CD4+/CD8+ ratios seem to be related to the clinical form (acute versus chronic), smoking addiction, type/dose of inhaled antigen, and even the time elapsed between last antigen exposure and the obtaining of BAL [47, 49, 50]. However, increased BAL lymphocytes and CD8 + T cells have also been found in some asymptomatic pigeon breeders and dairy farmers [51–53]. Moreover, bronchoalveolar lymphocytosis appears to be a persistent phenomenon in a large number of asymptomatic dairy farmers who do not develop the disease [51]. Whether this finding represents an appropriate, normal inflammatory response or whether these individuals develop a subclinical, low-intensity alveolitis is presently unknown. Increased natural killer cells, nonmajor histocompatibility complex (MHC)restricted cytotoxic lymphocytes, and lymphokine-activated killer cells are usually detected in BAL from patients with HP [54, 55]. Small numbers of plasma cells are found in about half of the patients, which may be a feature of recent antigen exposure and of active alveolitis [56]. Also, a moderate but significant increase in the percentage of neutrophils has been found after inhalation challenge (acute transient neutrophil alveolitis), and in half of the patients with chronic pigeon breeder’s disease [57, 58]. Several studies have documented a small but significant increase of mast cells in HP [59, 60]. Electron microscopic examination suggests that these cells are activated and resemble bronchial subepithelial tissue mast cells rather than those from alveolar interstitial tissue [58, 60]. Nevertheless, as in the case of BAL lymphocytosis, an increase in the number of mast cells has been found in exposed farmers without disease [61]. Thus, the significance of these cells in the pathogenesis of this disorder or in the disease activity is unclear. A number of proteins are also elevated in BAL fluids of HP patients [1]. These molecules include immunoglobulins, immune complexes, leukotriene C4,

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β2-microglobulin, hyaluronic acid, procollagen 3 N-terminal peptide, fibronectin, vitronectin, KL-6 (a mucin-like glycoprotein), and SP-A.

Pathologic Features of HP Acute HP: Descriptions of the pathologic features of HP in the literature are somewhat confused by differing uses of the terms “acute,” “subacute,” and “chronic.” As used here, acute HP refers to the acute, self-limited, systemic illness caused by exposures to very high levels of antigen. Such cases are rarely biopsied because the clinical story is usually obvious, and pathologic descriptions are limited to a few case reports which illustrate neutrophilic or eosinophilic inflammation and sometimes diffuse alveolar damage [62, 63]. Subacute HP: Subacute HP is by far the most common pattern encountered in biopsy material and fundamentally consists of a small airway-centered chronic interstitial inflammatory infiltrate, which may be accompanied by individual giant cells and/or granulomas, and sometimes Schaumann bodies [64]. This description is very general because there is a wide range of appearances in subacute HP. Bronchiolitis, manifest as a peribronchiolar chronic inflammatory infiltrate, which sometimes invades the wall of the bronchiole, is frequently seen, and giant cells or granulomas have a tendency to be found in the peribronchiolar infiltrate (Figs. 4a, b). In the classic case the chronic interstitial infiltrate appears to spread away from the bronchiole, often leaving the more distal alveolar walls uninvolved (Figs. 4a, b). Some cases show only bronchiolar involvement with virtually no interstitial infiltrate. Other cases of subacute HP demonstrate a much more even distribution within the lobule and can be indistinguishable, apart from giant cells/granulomas, from cellular nonspecific interstitial pneumonia [65]. Ordinarily the lymphoid infiltrates in the alveolar walls are only a few cells thick, but the infiltrate can be marked, raising a question of lymphocytic interstitial pneumonia or lymphoma. Giant cells and granulomas are said to be seen in about two thirds of cases [66, 67], but even in their absence, the bronchiolocentric distribution of the infiltrates should suggest a diagnosis of HP. A history of known exposure is also very helpful in arriving at a correct diagnosis in such cases. Small amounts of organizing pneumonia, typically involving the respiratory bronchioles, are common, and occasionally organizing pneumonia is the predominant pattern. Chronic HP: Chronic HP by definition shows interstitial fibrosis and the distinction is important, since these patients tend to have a considerably worse prognosis than patients with subacute HP [68, 69]. There are relatively few pathologic descriptions of chronic HP, and some of these [70] are too dated to be incorporated into the current formulations of interstitial pneumonias. Recent studies [71, 72] suggest that chronic HP shows two basic patterns: [1] similar to fibrotic nonspecific interstitial pneumonia (NSIP); and [2] more or less resembling usual interstitial pneumonia (UIP). The latter is characterized by peripheral fibrosis in a patchy pattern with associated fibroblast foci (Fig. 5a). In our experience the fibrosis is often

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a

2375

b

Fig. 4a Subacute HP: note the interstitial infiltrate with a more intense infiltrate around and in the bronchiolar wall. A poorly formed granuloma is present at the arrow. Fig. 4b Higher power view of Fig. 4a to show the poorly formed granuloma (arrow)

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a

b

Fig. 5a Chronic HP showing a UIP-like pattern in a patient with exposure to molds. Note the fibroblast focus (open arrow) and areas of interstitial chronic inflammatory infiltrate (closed arrows). The chronic inflammatory infiltrate would not be typical of UIP but is seen in chronic HP (Reproduced from [71]. With permission). Fig. 5b Another area of the same case as shown in Fig. 5a. Note the small peribronchial granuloma and the peribronchial lymphoid infiltrate. This area is typical of subacute HP, and areas with a subacute pattern may be found in cases of chronic HP. (Reproduced from [71]. With permission)

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less marked and more delicate than in typical cases of UIP, but this difference can be very subtle. Areas of subacute HP may be present. An additional helpful finding is the presence of interstitial fibrosis in a peribronchiolar pattern, typically unconnected to the peripheral UIP-like fibrosis (Fig. 6). As in subacute HP, the finding of individual giant cells, granulomas, or Schaumann bodies is an important clue to the diagnosis (Fig. 5b), but older descriptions of chronic HP [70] suggest that giant cells/granulomas tend to disappear over time, particularly if the patient is removed from exposure. Some cases of chronic HP are pathologically indistinguishable from fibrotic NSIP [65] or from UIP [72], and only clinical or radiologic features indicate the correct diagnosis.

Treatment The most important interventions in managing HP are early diagnosis and avoidance of antigen exposure. In occupationally exposed individuals the risk of HP can be reduced by adapting modern practices and conditions that reduce the content of causative antigens. Likewise, elimination of the antigen source is key in patients with home-related HP. Thus, for example, when the colonization by Trichosporon cutaneum, the causative agent of summer-type HP in Japan, was eliminated from the domestic environment no recurrence was observed. By contrast, recurrence was

Fig. 6 Another pattern of chronic HP in a patient with long-standing bird exposure. Note both the peripheral and the peribronchiolar fibrosis, a common combination in chronic HP (Reproduced from [71]. With permission)

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observed in all patients who resided in homes that were not cleaned or in homes where cleaning was not adequate [73]. Nonetheless, there are reports indicating that some patients may stabilize or even may display complete remission of the disease, despite subsequent exposure to the offending antigen [74, 75]. Despite these occasional reports, the primary focus in the treatment of HP is prevention of additional exposure to the causative antigens. In this context, a change in patient’s habits, occupation, or environment to avoid future contact with the offending antigen should be considered. Nevertheless, in chronic HP patients with fibrotic changes subsequent antigen avoidance may not reverse the disease and some of them show progressive worsening and eventually die from the disease. Prednisone is recommended in subacute/chronic presentations, although longterm efficacy of these agents has yet to be determined. An optional approach consists of 0.5 mg per kg per day of prednisone for a month, followed by a gradual reduction until a maintenance dose of 10–15 mg per day is reached. Prednisone is discontinued when the patient is considered to be healed or when there is no clinical and/or functional response. If the patient worsens despite prednisone treatment, the addition of azathioprine can be considered.

Prognosis Data on survival rates in cohorts with HP are limited and often contradictory. In general, acute and subacute patients show a favorable prognosis, and most patients heal or display a significant improvement with some residual respiratory functional abnormalities remaining. A different picture emerged from studies with chronic HP, primarily pigeon breeder’s disease. In two different cohorts, chronic HP, usually associated with the development of fibrosis, was accompanied by a high rate of mortality with median survivals of 5 and 7 years, respectively [68, 69]. By contrast, patients with farmer’s lung, mainly those that experience recurrent acute attacks, develop more often airflow obstruction and emphysema although survival data is usually not provided [75]. Erkinjuntti-Pekkanen et al. evaluated the long-term outcome of 88 farmer’s lung patients and 83 matched control farmers [76]. At average of 14 years after the diagnosis, the HRCT examination revealed that 23% of the patients with farmer’s lung had emphysema compared with only 7% of the control farmers. The presence of emphysema was most frequent in smokers.

Conclusions Hypersensitivity pneumonitis is a complex syndrome that develops after exposure to a large variety of environmental antigens, causing variable clinical symptoms often making diagnosis uncertain. Diagnosis of HP relies on a constellation of

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findings that include a suggestive environmental history, symptoms, physical findings, BAL lymphocytosis, imaging abnormalities, pulmonary function changes, and serum-specific antibodies. HRCT has improved diagnostic confidence. In the acute and subacute forms, removing the patient from the suspected environment often results in spontaneous amelioration of symptoms and this finding is a useful clue to the diagnosis. Inhalation challenge may be performed in selected patients in whom the causal relationship between antigen and lung disease has not been established. Finally, lung biopsy is recommended when the diagnosis is not apparent. The pathogenesis of HP is intricate, and it is likely that both immune complexes and T-cell abnormalities participate in the development of the alveolitis, but in different phases and clinical forms of the disease. Importantly, HP occurs only in a few exposed individuals suggesting that some additional promoting factors (either genetic or environmental) play a role.

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52. Johnson MA, Nemeth A, Condez A, Clarke SW, Poulter LW (1989). Cell-mediated immunity in pigeon breeder’s lung: the effect of removal from antigen exposure. Eur Respir J 2:444–450. 53. Cormier Y, Létourneau L, Racine G (2004). Significance of precipitins and asymptomatic lymphocytic alveolitis: a 20-yr follow-up. Eur Respir J 23:523–525. 54. Semenzato G, Trentin L, Zambello R, Agostini C, Cipriani A, Marcer G (1988). Different types of cytotoxic lymphocytes recovered from the lungs of patients with hypersensitivity pneumonitis. Am Rev Respir Dis 137:70–74. 55. Ratjen F, Costabel U, Griese M, Paul K (2003). Bronchoalveolar lavage fluid findings in children with hypersensitivity pneumonitis. Eur Respir J 21:144–148. 56. Drent M, Wagenaar SjSc, Velzen-Blad H, Mulder PGH, Hoogsteden HC, van des Bosch JMM (1993). Relationship between plasma cell levels and profile of bronchoalveolar lavage fluid in patients with subacute extrinsic allergic alveolitis. Thorax 48:835–839. 57. Fournier E, Tonnel AB, Gosset PH, Wallaert B, Ameisen JC, Voisin C (1985). Early neutrophil alveolitis after antigen inhalation in hypersensitivity pneumonitis. Chest 88:563–566. 58. Haslam PL, Dewar A, Butchers P, Primett ZS, Newman-Taylor A, Turner-Warwick M (1987). Mast cells, atypical lymphocytes, neutrophils in bronchoalveolar lavage in extrinsic allergic alveolitis: comparison with other interstitial lung diseases. Am Rev Respir Dis 135:35–47. 59. Schildge J, Klar B, Hardung-Backes M (2003). Mast cells in bronchoalveolar lavage fluid of patients with interstitial lung diseases. Pneumologie 57:202–207. 60. Ishida T, Matsui Y, Matsumura Y, Fujimori N, Furutani M (1995). Bronchoalveolar lavage mast cells in summer-type hypersensitivity pneumonitis: increase in numbers and ultrastructural evidence of degranulation. Intern Med 34:357–363. 61. Laviolette M, Cormier Y, Loiseau A, Soler P, Leblanc P, Hance AJ (1991). Bronchoalveolar mast cells in normal farmers and subjects with farmer’s lung. Am Rev Respir Dis 144:855–860. 62. Travis WD, Colby TV, Koss MN, Rosado-de-Christenson ML, Müller NL, King TE (2002). Non-neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology, pp. 115–123. 63. Myers JL (2005). Idiopathic interstitial pneumonias. In: Churg AM, Myers JL, Tazelaar HD, Wright JL, editors: Thurlbeck’s Pathology of the Lung, 3rd Edition. New York: Thieme Medical Publishers, pp. 563–600. 64. Coleman A, Colby TV (1988). Histologic diagnosis of extrinsic allergic alveolitis. Am J Surg Pathol 12:514–518. 65. Vourlekis JS, Schwarz MI, Cool CD, Tuder RM, King TE, Brown KK (2002). Nonspecific interstitial pneumonitis as the sole histologic expression of hypersensitivity pneumonitis. Am J Med 112:490–493. 66. Kawanami O, Basset F, Barrios R, Lacronique JG, Ferrans VJ, Crystal RG (198). Hypersensitivity pneumonitis in man. Light- and electron-microscopic studies of 18 lung biopsies. Am J Pathol 110:275–289. 67. Reyes CN, Wenzel FJ, Lawton BR Emanuel DA (1982). The pulmonary pathology of farmer’s lung disease. Chest 81:142–146. 68. Perez-Padilla R, Salas J, Chapela R, Sanchez M, Carrillo G, Perez R, Sansores R, Gaxiola M, Selman M (1993). Mortality in Mexican patients with chronic pigeon breeder’s lung compared with those with usual interstitial pneumonia. Am Rev Respir Dis 148: 49–53. 69. Vourlekis JS, Schwarz MI, Cherniack RM, Curran-Everett D, Cool CD, Tuder RM, King TE Jr, Brown KK (2004). The effect of pulmonary fibrosis on survival in patients with hypersensitivity pneumonitis. Am J Med 116:662–668. 70. Seal RM, Hapke EJ, Thomas GO, Meek JC, Hayes M (1968). The pathology of the acute and chronic stages of farmer’s lung. Thorax 23:469–489. 71. Churg A, Muller NL, Flint J, Wright JL (2006). Chronic hypersensitivity pneumonitis. Am J Surg Pathol 30:201–208. 72. Ohtani Y, Saiki S, Kitaichi M, Usui Y, Inase N, Costabel U, Yoshizawa Y (2005). Chronic bird fancier’s lung: histopathological and clinical correlation. Thorax 60:665–671.

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Clinical Aspects and Diagnosis of Atopic Eczema Matthias Möhrenschlager, Stephan Weidinger, and Johannes Ring

Introduction Diagnosing atopic eczema (AE) may be sometimes challenging even for the experienced dermatologist. Several causes are responsible: the combinations of polygenic factors and modifications by individual exposures elicit a wide spectrum of signs and symptoms from minimal manifestations via mild eczematous lesions to severe and chronic AE. The morphology of skin lesions as well as affected body regions may change according to age and environmental influences. In addition, there is a strong variability in the inter-individual course of the disease. Furthermore, problems in diagnosing AE may arise from the impreciseness of nomenclature, especially of the terms “eczema/dermatitis” and “atopy” [1]. Eczema is a non-contagious inflammation of the epidermis and dermis with characteristic clinical (itch, erythema, papule, seropapule, vesicle, scales, crusts, lichenification, in the sense of a synchronous or metachronous polymorphy) and histopathological (spongiosis, acanthosis, parakeratosis, lymphocytic infiltrates, exocytosis) signs [2]. Since the introduction of the term “atopy” by Coca and Cooke in 1923 [3], its definition has been a matter of controversy. In the 1980s it has been designated as “familial tendency to develop certain diseases (asthma, rhinoconjunctivitis, eczema) on the basis of a hypersensitivity of skin and mucous membranes against environmental substances, associated with increased IgE production and/or altered non-specific reactivity” [4]. Some years ago, the European Academy of Allergy and Clinical Immunology (EAACI) proposed the following definition: “Atopy is a personal or familial tendency to produce IgE antibodies in response to low doses of allergens, usually proteins, and to develop typical symptoms such as asthma, rhinoconjunctivitis, or eczema/dermatitis” [5]. EAACI also coined the new term

M. Möhrenschlager (), S. Weidinger, and J. Ring Division of Environmental Dermatology and Allergology GSF/TUM, Department of Dermatology and Allergy Biederstein, Technical University of Munich, Biedersteiner Street 29, D-80802 Munich, Germany. e-mail: [email protected]

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“atopic eczema/dermatitis syndrome” (AEDS) [5]. This new syndrome was further divided into non-allergic and allergic AEDS, the latter further subdivided into “IgEassociated AEDS” and “non-IgE-associated allergic AEDS” [5]. On a global level, the World Allergy Organization has recently published a revised nomenclature where the problem of combining laboratory and clinical definitions of “atopy” and AE has been solved in a way that only the IgE-associated forms of the disease will be called AE [6]. In the future, the term “eczema” will replace the current term AE or atopic dermatitis. Other forms of dermatitis (e.g., contact dermatitis) will remain unaffected. It remains open whether this consensus will be accepted in daily life [1].

Morphology of Skin Lesions Since there does not exist any specific laboratory test or any pathognomonic histopathological finding, the diagnosis of AE is essentially clinical. In general, diagnosis is made on the basis of dermatologic examination of skin lesions under consideration of their age-specific morphology and distribution. Further cardinal symptoms leading to the diagnosis of AE are the chronicity of the disorder and its associated pruritus [7]. Depending on the severity of inflammation and different stages of healing, chronic scratching, and secondary infections, the morphology of skin lesions in AE may vary. Acute lesions often consist of papules and vesicles on erythematous skin (Fig. 1). Subacute lesions may develop scales and lichenification (Fig. 2).

Fig. 1 Childhood atopic eczema, acute flexural lesions

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Fig. 2 Subacute atopic eczematous lesions with scales and lichenification

Chronically involved areas appear dry, thick, and fibrotic and sometimes show nodules. Resolvement of lesions often leaves postinflammatory hypopigmentation or hyperpigmentation [8]. The distribution of skin lesions can be highly variable but is generally age-related [9].

Infantile Phase (0–2 years) The earliest clinical features are dryness and roughness of the skin. Distinct eczematous lesions appear from birth to 6 months of age. In infants, the dermatitis commonly affects face and scalp and spreads to involve the neck and trunk (Fig. 3). Typically, lesions are erythematous and have highly pruritic, moist, oozing papulovesicles that may crust and scale. Secondary impetiginization can occur. Nasolabial, groin, and napkin area is often spared. In children 1–2 years of age, the distribution of lesions moves from the face to the antecubital and popliteal fossae, neck, wrists, ankles, and retroauricular folds. Due to the developing ability to scratch, the primary lesions can be altered and a more variable clinical picture may develop with papules, poorly demarcated scaly patches, excoriations, and hemorrhagic crusts. While the truncal lesions are often diffuse, on the extremities localized patches prevail that tend to involve both extensor and flexor aspects and commonly the wrists and ankles [10, 11].

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Fig. 3 Infantile atopic eczema affecting face and trunk

Childhood Phase (2–12 years) During childhood, polymorphous manifestations with various types of skin lesions at different localizations are common. At sites of chronic involvement, thickened plaques with excoriation and mild lichenifications develop. During phases of exacerbation, acute erythema, plaque-like infiltrations and weeping or erosive skin lesions may occur. Other morphological variants of the childhood phase are nummular, papulovesicular, or lichenoid lesions. Flexures and buttocks become the predominant predilection sites (Fig. 4). The nails may become shiny and buffed from constant rubbing and long-lasting eczema of the periungual skin (“eczema nails”) [7, 10, 11].

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Fig. 4 Atopic eczema in childhood with involvement of buttocks

Adolescent and Adult Phase The main clinical picture of AE in adolescents is flexural lichenified and often excoriated skin lesions [12]. In addition, neck, wrists, ankles, and eyelids are frequently affected [13]. In more widespread disease, the upper trunk, shoulders, and scalp may show lesions. AE spontaneously clears in about 40% of children before or during adolescence but may remain quiescent in others until adulthood. A subgroup of patients exhibits first symptoms not before adulthood [7]. In patients who do not outgrow AE by adolescence, the disease typically worsens with the skin becoming more thick and dry with lichenified eczema being the predominant lesion type [4].

Morphological Variants Follicular Variant The follicular type of AE, which is common in Asian and Afro-American patients, is characterized by skin-colored, whitish, or red-brown, densely aggregated follicular papules (Fig. 5). Predilection sites are the lateral parts of trunk, neck, and extensor surfaces. The course is usually cyclic with exacerbations in winter and improvements during summer [11].

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Fig. 5 Atopic eczema: follicular variant

Papular Lichenoid Variant Skin lesions typical for the lichenoid variant of AE are skin-colored, flat, polygonal, or round papules symmetrically affecting the extensor surfaces (Fig. 6). The papules may be disseminated or aggregated, sometimes show desquamation, and tend to appear in spring or summer [11].

Prurigo Type This variant is rare in children, but may sometimes be seen in adolescents. Erythematous, often excoriated papules and hyper- or hypopigmented residual maculopapular lesions and sometimes indurated nodules are seen primarily on the extensor surfaces of the extremities and trunk (Fig. 7) [10, 11].

Nummular or Discoid Variant This type of AE is characterized by sharply demarcated, coin-sized patches of eczematous skin. Lesions are reddish in color, dry, often infiltrated, and may ooze and become crusty (Fig. 8). The legs are most commonly affected, but trunk and arms, especially the backs of the hands can also be affected. In adults, it commonly occurs as distinct entity without association with atopy. Exacerbation often occurs during winter [11, 14].

Fig. 6 Atopic eczema: papular lichenoid variant

Fig. 7 Prurigo type of atopic eczema

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Fig. 8 Nummular type of atopic eczema

Manifestations of Atopic Eczema at Special Body Areas Fingertip Eczema and Atopic Hand Eczema In patients with chronic hand eczema, including dyshidrosis (or pompholyx), an increased prevalence of atopy between 47% and 64% has been reported [4]. Palmar/plantar dermatitis has been reported to occur in 70% of children with AE [15, 16]. Fingertip eczema and/or palmar dermatitis may be characterized as “minimal variants” of AE. Clinically, the palmar surface of the fingertips shows dry, nonpruritic plaques, recurrent hyperkeratosis, and fissuring (pulpitis sicca). The palmar skin may appear slightly erythematous and scaly or appear thickened, dry, and leathery (Fig. 9). The dorsum of the hand can be similarly involved. In this context, nonspecific irritants (e.g., cleansers, solvents, wet work) may play a major role. Allergic and irritant contact dermatitis have to be delineated [4].

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Fig. 9 Dyshidrotic and hyperkeratotic rhagadiform atopic hand eczema

Atopic Winter Feet There is no consensus regarding the relation between the “atopic winter feet” and juvenile plantar dermatosis. The clinical picture is similar. A glittering erythema appearing like lacquered skin with scaling and fissuring of the plantar forefeet and toes is typical. The non-weight-bearing areas of the sole are spared. The condition usually starts at school age, shows a chronically relapsing course with worsening in winter, and heals about onset of puberty in the majority of patients [17–19]. Misdiagnosis and treatment as athlete’s foot or allergic contact dermatitis is frequent [19].

Eyelid Eczema Involvement of the eyelids is encountered often in patients with AE. In some atopic individuals, eyelid eczema represents the predominant dermatologic finding. The clinical picture reaches from soft scaly erythema to hyperpigmented lichenifications with excoriations (Fig. 10) [20]. Relapses of atopic eyelid eczema are found often due to the vulnerability of the thin skin of the eyelids, which is constantly exposed to contact irritants and allergens. Furthermore, pruritic eyelids can be easily rubbed by hands. Irritant and allergic contact dermatitis should be ruled out [20].

Nipple Eczema Nipple eczema occurs in 12–23% of the patients [21–23]. If present, it is highly susceptible for AE [4]. In the areolar area, a symmetric, oozing, papulovesicular erythema is seen that may extend onto the surrounding breast skin.

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Fig. 10 Bilateral atopic eyelid eczema

Fig. 11 Exfoliative cheilitis with perlèche

Cheilitis Cheilitis often starts in childhood as dry and scaly upper and lower lips. Eczematization of the lips may proceed to fissuring, angular cheilitis, and perioral eczema (Fig. 11). Besides the habitual lip-licking to ease the dryness and nibbling of adherent scales, the lips are constantly exposed to irritant fluids from foods and drinks [4].

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Pityriasis Alba In areas of previous eczema, especially in the face, neck, and upper trunk, finely scaling and diffusely demarcated hypopigmented patches sometimes resembling tinea corporis or vitiligo may develop. The condition is most prominent after prolonged sun exposure and represents postinflammatory hypopigmentation. Pityriasis alba has been reported to occur in 35–44% of AE patients [22, 23].

Stigmata of Atopy Stigmata of atopy are minor skin signs not representing actual “disease” that are characteristic but not specific for atopic individuals. They are significantly more common in patients with AE than in healthy individuals. They appear to be constitutional markers of the atopic state, since most of them are also found in atopic respiratory diseases [24]. Stigmata may be valuable clues to the diagnosis of AE.

Dry Skin (Xerosis) Xerosis (Figs. 1–11) is the most common skin finding in patients suffering from AE. It is characterized by slightly scaling and noninflamed skin affecting large areas of the body. Usually it persists throughout the patient’s life, but may show seasonal variations [25, 26]. Atopic skin appears rough and dry, which is the result of the atopic keratinocytes’ decreased ability to bind water, and an increased transepidermal water loss [27, 28]. Xerosis is one of the triggers of pruritus and contributes to an abnormal protective barrier layer in AE. It sometimes causes fissures that may serve as a portal of entry for infectious agents.

Hyperlinearity of the Palms/Soles Hyperlinearity of the palms (Fig. 9) or the soles is noted more often in atopic patients than in non-atopics and has been found in up to 88% of AE patients. An increased number of fine lines and accentuated markings is encountered and has to be distinguished from palmoplantar keratoderma of ichthyosis vulgaris [29].

Infraorbital Fold (Dennie–Morgan Fold) Dennie–Morgan folds are symmetric, striking single or double folds beneath the lower eyelids (Fig. 10) first reported by Dennie as mentioned by Morgan [30]. It may be seen in 50–60% of atopic patients with possible ethnic variations [24, 31].

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White Dermatographism In non-atopic individuals, firm stroking of the skin causes a red line with a reflex erythema. In contrast, the majority of AE patients show a delayed white line, which replaces the initial erythematous reaction after ~1 min [32]. It has been noted to be age-dependent and to develop during the first year of life [32, 33].

Facial Pallor Atopic persons frequently have paleness of the face (Fig. 10). Like white dermatographism it is believed to be caused by the AE patient’s altered vascular reactivity [4].

Periorbital Darkening Many AE patients exhibit a blue-gray hue around the eyes (Fig. 10) with accentuation of the suborbital area. This condition is more frequent in the young and often seen in other atopic family members [21].

Herthoge’s Sign This sign refers to the thinning or absence of the lateral portion of the eyebrows. A prevalence of 39% has been reported in AE patients compared to 1% in controls [23].

Keratosis Pilaris This disorder of keratinization of the xerotic hair follicles is characeterized by tiny rough bumps on the cutaneous surface (like “chicken skin”). Primarily, it appears on the back and outer sides of the upper arms, but can also occur on thighs and buttocks or any body part except palms or soles. It is frequently associated with AE but can also be seen in other inflammatory dermatoses or occurs in individuals without other skin lesions. It most often appears in childhood, reaches its peak in adolescence, and becomes less apparent during adulthood [34].

Long Eyelashes Luxurious, long eyelashes have been characterized as part of the AE patient’s phenotype [35–37]. Nevertheless, a recent cross-sectional study among 5- to 6-year-old

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preschoolers from Germany failed to show any significant difference in the length of eyelashes with or without AE [38].

Diagnostic Criteria of Atopic Eczema Numerous lists of diagnostic criteria have been developed in order to establish a definition for AE with known validity and reproducibility by using reliable discriminators. Most widely accepted are the criteria of Hanifin and Rajka [39]. Other criteria include those of Diepgen et al. [40], the United Kingdom Working Party’s Diagnostic Criteria for Atopic Dermatitis [41], the Millennium Criteria for the Diagnosis of Atopic Dermatitis [42], and the criteria of Ring [4].

Diagnostic Criteria According to Hanifin and Rajka In 1980, Hanifin and Rajka [39] proposed criteria for diagnosis of AE based on the presence of at least three major and three additional minor criteria (Table 1). These criteria represented an important milestone in describing the clinical aspects of AE and established some degree of comparability in subsequent hospital studies. They have been evaluated by a number of investigators [21]. However, due to their partly unknown validity, their complexity and heterogeneity, and since some of the criteria are not precisely defined, very rare, or unspecific, their use in population-based epidemiological studies is limited [43–45].

Diagnostic Criteria According to Diepgen et al. Diepgen et al. [40] established a scoring system based on criteria derived from 110 AE patients and 527 controls. Their top five criteria turned out to be “itch when sweating,” “intolerance to wool,” xerosis, white dermographism, and Hertoghe’s sign [40].

UK Working Party’s Diagnostic Criteria for Atopic Dermatitis In 1994, a minimum list of discriminators for the diagnosis of AE was established and validated in a hospital setting with sensitivity and specificity of about 90% [41, 46]. The criteria have also been validated in other countries (e.g., Germany [47]).

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Table 1 Diagnostic criteria according to Hanifin and Rajka (according to [39]) At least three major criteria Plus three or more minor features • Pruritus • Typical morphology and distribution: Flexural lichenification or linearity in adults Facial and extensor involvement in infants and children • Chronic or chronically relapsing dermatitis • Personal or family history of atopy (asthma, allergic rhinitis, atopic dermatitis)

• Xerosis • Ichthyosis/palmar hyperlinearity/keratosis pilaris

• • • •

• • • • • • • • • • • • • • • • •

Immediate (type I) skin test reactivity Elevated serum IgE Early age at onset Tendency toward cutaneous infections (especially Staphylococcus aureus and Herpes simplex)/impaired cell-mediated immunity Tendency toward nonspecific hand or foot dermatitis Nipple eczema Cheilitis Recurrent conjunctivitis Denny–Morgan infraorbital fold Keratoconus Anterior subcapsular cataracts Orbital darkening Facial pallor/facial erythema Pityriasis alba Anterior neck folds Itch when sweating Intolerance to wool and lipid solvents Perifollicular accentuation Food intolerance Course influenced by environment/ emotional factors White dermatographism/delayed blanch

The diagnosis of AE can be made if an itchy skin condition plus three or more of the following criteria are present: • History of involvement of the skin creases (the folds of the elbows, the fronts of the ankles, around the neck, or the cheeks in children less than 4 years old) • History of asthma or hay fever in the patient, or of atopic disease in a firstdegree relative (i.e., mother, father, brother, or sister) if the child is less than 4 years old • History of general dry skin in the past year • Visible flexural eczema (or eczema involving the cheeks/forehead and outer limbs in children under 4 years) • Onset during the first 2 years of life (not used for children less than 4 years old)

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Criteria of Ring In 1982, Ring established a list of diagnostic criteria for AE [4]. The diagnosis of AE can be made if at least four of the following six criteria are present: • • • • • •

Age-specific morphology Pruritus Age-specific distribution of skin lesions Stigmata of AE (“typus neurodermiticus”) Personal or family history of atopy IgE-mediated sensitization

Assessment of Disease Severity: The SCORAD Index The heterogeneity in expression, severity, and extent of AE has led to the establishment of parameters or criteria for assessing the severity of the disease. The most common and universally accepted system used for assessment of disease severity was developed by the European Task Force on Atopic Dermatitis as Scoring Index of Atopic Dermatitis (SCORAD) [48]. SCORAD consists of an objective score that quantifies extent and intensity of skin lesions and a subjective score that quantifies daytime pruritus and sleep loss. The extent is measured as the percentage of affected skin surface area, and the intensity reflects different qualities of skin morphology and is scored in terms of erythema, edema, papulation, oozing, crusting, excoriation, lichenification, and xerosis. Subjective complaints are scored on a visual scale of 0–10. This scoring index is particularly useful in clinical trials.

Differential Diagnosis of Atopic Eczema Several disorders may mimic AE. These include: • Seborrhoeic dermatitis: In infants, seborrhoeic dermatitis is the most common differential diagnosis of AE. A clear-cut differentiation may be difficult at first presentation, as important clues to the diagnosis (e.g., course) are not available. Pruritus, age of onset and family history of atopy can not reliably discriminate the two entities. Lesions on forearms and shins as well as specific IgE to egg white and milk point towards AE. Smooth, nonscaling, sharply marginated, brightly erythermatous dermatitis in the axillae or initially only affecting the napkin area, and heavy yellow scales on the cheeks, trunk, and limbs favor the diagnosis seborrhoeic dermatitis [49, 50]. • Scabies: Scabies may be very difficult to distinguish from AE if secondary eczematization has occurred. A history of acute itchy conditions within the family, the presence of relatively large papules on the upper back and in the genital and

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axillary areas, vesicles on the palms and soles in infants, and sparing of the face in older individuals are signs in favor of scabies. Mite or ova can be demonstrated in scrapings of the vesicles [51]. • Psoriasis: Early-onset psoriasis with extensive involvement may also be confused with AE. It is rare in infancy. Usually, pruritus is not present. Sharply demarcated fawn-colored scaly lesions and psoriasis in one or more family members are helpful in distinguishing this disease from AE [4]. • Immunodeficiency syndromes: Several types of immunodeficiency syndromes are frequently associated with AE, for example, Wiskott–Aldrich syndrome [52], selective IgA deficiency [53], and hyperimmunoglobulin E syndrome [54, 55]. In a variety of other primary immunodeficiencies, occasional associations have been described [52]. They can be distinguished by the presence of additional symptoms like recurrent infections, generalized lymphadenopathies, chronic diarrhoea, hematological abnormalities or failure to thrive [56]. Metabolic disorders may also predispose to AE [4]. • Other disorders: In older children, contact dermatitis (irritant or allergic), tinea, eczematous pityriasis rosea, and rarely drug eruption have to be differentiated from AE [4]. Table 2 provides an overview on the most common differential diagnoses of AE.

Allergologic Tests in Atopic Eczema Skin Prick Test Skin prick testing (SPT) to common allergens will identify specific IgE-mediated sensitizations. However, positive test results do not necessarily indicate clinical relevance. In AE, one has to keep in mind that AE patients may also show an uneventful SPT, representing the intrinsic type of AE [57]. SPT only indicate that the patient has been sensitized to the particular antigens. To be informative, the SPT must be related to the clinical context of the patient’s history and the physical examination. The selection of the antigens and the administration of tests require experience and knowledge [58]. A SPT may be positive both before the allergy is clinically apparent and years after cessation of symptoms.

Total IgE Concentration in Serum Among atopic diseases, highest serum IgE levels are found in AE [59], and total serum IgE levels are elevated in the majority of AE patients. Although total serum IgE is one of the parameters used to discriminate intrinsic and extrinsic forms of AE [4, 57], the clinical applications and interpretation of total serum IgE concentrations

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Table 2 Differential diagnoses of atopic eczema Congenital disorders Netherton syndrome Familial keratosis pilaris Erythrokeratodermia variabilis Ichthyoses Inflammatory dermatoses Seborrhoeic dermatitis Contact dermatitis (allergic or irritant) Nummular dermatitis Psoriasis Lichen simplex chronicus Infectious diseases Scabies HIV infection Dermatophytosis (tinea corporis) Malignant neoplasias Cutaneous T-cell lymphoma (mycosis fungoides/Sézary syndrome) Langerhans cell histiocytosis Immunodeficiencies Wiskott–Aldrich syndrome Severe combined immunodeficiency Hyper-IgE syndrome Ataxia-teleangiectasia Selective IgA deficiency Metabolic disorders Acrodermatitis enteropathica Pyridoxine (vitamin B6) and niacin deficiency Multiple carboxylase deficiency Phenylketonuria Autoimmune diseases Dermatitis herpetiformis Pemphigus foliaceus Dermatomyositis Graft versus host disease

are of modest value. An elevated IgE concentration cannot be considered as a pathognonomic sign of atopy or allergy. Furthermore, a normal IgE level does not exclude allergy or AE, while highly elevated levels may be seen in non-atopic people. In addition, total IgE is elevated in a variety of disorders and syndromes like allergic bronchopulmonary aspergillosis, hyper-IgE syndrome, certain stages of HIV infection, lymphoproliferative diseases, drug-induced interstitial nephritis, graft versus host disease, parasitic diseases, and several immune deficiency diseases as well as idiopathically [55]. Total IgE is not of predictive value for the course of the disease or long-term prognosis [4].

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Specific IgE Concentration in Serum Specific IgE antibodies are most commonly detected by radioallergosorbent test (RAST) or related techniques measuring the amount of IgE directed to a specific allergen [60–63]. SPT are generally considered to be more sensitive than IgE assays, but AE patients may be SPT negative and RAST positive or vice versa. Thus, RAST may be particularly useful in patients with severe skin disease who cannot be skin tested. Quantitative specific IgE tests have a high reliability and sensitivity (about 90%) [4].

Atopy Patch Test The role of allergy has been debated rigorously in the past, partly due to the limited specificity of SPT and RAST with regard to the clinical course. The flare of eczematous lesions after contact with aeroallergens, a predictive lesional pattern affecting air-exposed skin, and a seasonal fluctuating course of the disease are wellknown clinical features in many patients with AE. In 1982 it could be demonstrated that epicutaneous application of several allergens on the uninvolved, abraded skin of patients with severe AE could induce eczematous lesions only in patients who also showed a positive immediate skin reaction to the same allergen [64]. Based on these observations, an epicutaneous patch test with allergens known to elicit IgE-mediated reactions used to evaluate the occurrence of eczematous skin lesions was named “atopy patch test” (APT) [65] and further developed. Several groups have used the APT as a model to study the role of aeroallergens in AE [66–74]. The APT reaction has been shown to be specific for eczema patients, and does not occur in healthy volunteers or in patients suffering from asthma or rhinitis [74–76]. The APT seems to act as a marker of exposure and may be viewed as a kind of provocation test for the dermo-epidermal unit in patients with AE [69]. However, due to differences in patient selection and in methodology, the results of APT may show large variations [76–78]. In this context, an international multicenter trial studied the correlation of positive APT reactions with history, SPT, and specific IgE in serum [76]. As many as 314 patients with AE were tested with house dust mite (Dermatophagoides pteronyssinus), cat dander, grass pollen, birch pollen, hen’s egg, celery, and wheat flour. APT, SPT, and specific IgE results showed significant agreement with history of AE-aggravation by contact with grass pollen and ingestion of hen’s egg [76]. Further significant correlations of APT results and patient’s history were shown in a previous study [74]. With regard to history, the APT had a much higher specificity (60–90%) than SPT and specific IgE, while sensitivity was higher for specific IgE and SPT [76]. Thus, the APT with aeroallergens may provide an important diagnostic tool in patients with an air-exposed AE distribution pattern. Open questions concern the

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clinical relevance of positive APT reactions in patients with a negative history and negative SPT or specific IgE, as there is no gold standard for the provocation of eczematous skin lesions in aeroallergen-triggered AE. Thus, the APT is widely accepted as an useful model for studying inflammatory reactions and the effect of topical treatment in AE [77]. In summary, although the diagnostic value of the APT in clinical practice is still a matter of discussion [79], it is a helpful tool for identification of the patient group suffering from aeroallergen-induced AE flares [80].

Outlook: Will Genetic Analysis Boost Atopic Eczema Diagnosis? Among the causal factors of AE, the relevance of genetics is highlighted in twin studies [81]. Several genetic analyses have identified different chromosome regions with a linkage to AE features: Th2 cell cytokine genes on 5q31–33, as well as regions located on 1q21, 3q21,17q25, 20p, which are closely coincident with some major psoriasis loci [82–85]. Additionally, further genetic regions associated with AE features include gene polymorphisms in the signal transducer and activator of transcription (STAT)-6, the proximal promoter of regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin (IL)-4, IL-4Rα, and transforming growth factor (TGF)-β [86–89]. Very recent findings of Weidinger et al. [90] showed prominent associations of the loss-of-function mutations R501X and 2282del4 within the filaggrin gene and AE. Furthermore, associations of the filaggrin mutations with the extrinsic subtype of AE, characterized by high total IgE concentration in serum and concomitant allergic sensitization were revealed [90]. Impairment of epidermal barrier function in AE due to a primary defect in epidermal differentiation by fillaggrin mutations may represent a strong genetic factor which can be useful for diagnostic purposes in the future. Nevertheless, diagnosis of AE exclusively based on genetic grounds seems to be a goal to be achieved in the future.

References 1. Möhrenschlager M, Darsow U, Schnopp C, Ring J (2006) What is new in atopic eczema? JEADV 20:503–511 2. Rudikoff D, Akhavan A, Cohen SR (2003) Color atlas: eczema. Clin Dermatol 21:101–108 3. Coca AF, Cooke RA (1923) On the classification of the phenomena of hypersensitiveness. J Immunol 8:163–182 4. Ring J (2005) Allergy in practice. Springer, Berlin, pp. 86–105 5. Johansson SG, Hourihane JO, Bousquet J, Bruijnzeel-Koomen C, Dreborg S, Haahtela T, Kowalski ML, Mygind N, Ring J, van Cauwenberge P, van Hage-Hamsten M, Wüthrich B; EAACI (European Academy of Allergology and Cinical Immunology) Nomenclature Task Force (2001) A revised nomenclature for allergy. Allergy 56:813–824

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6. Johansson SG, Bieber T, Dahl R, Friedmann PS, Lanier BQ, Lockey RF, Motala C, Ortega Martell JA, Platts-Mills TA, Ring J, Thien F, van Cauwenberge P, Williams HC (2004) Revised nomenclature for allergy for global use: report of the Nomenclature Review Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol 113:832–836 7. Rudikoff D, Lebwohl M (1998) Atopic dermatitis. Lancet 351:1715–1721 8. Thestrup-Pedersen K (2000) Clinical aspects of atopic dermatitis. Clin Exp Dermatol 25:535–543 9. Hanifin JM (1991) Atopic dermatitis in infants and children. Pediatr Clin North Am 38:763–789 10. Abeck D, Burgdorf W, Cremer H (2003) Common skin diseases in children. Steinkopff, Darmstadt, pp. 19–30 11. Kunz BIF, Ring J (2006) Clinical features and diagnostic criteria of atopic dermatitis. In: Harper J, Oranje A, Prose N (eds.), Textbook of pediatric dermatology (2nd edition). Blackwell, Oxford, pp. 227–244 12. Dotterud LK, Kvammen B, Lund E, Falk ES (1995) Prevalence and some clinical aspects of atopic dermatitis in the community of Sør-Varanger. Acta Derm Venereol (Stockh) 75:50–53 13. Schudel P, Wüthrich B (1985) Klinische Verlaufsbeobachtungen bei Neurodermitis atopica nach dem Kleinkindesalter. Z Hautkr 60:479–486 14. Cowan MA (1961) Nummular eczema: a review, follow up and analysis of a series of 325 cases. Act Derm Venereol (Stockh) 41:453 15. Breit R (1997) Neurodermatitis (Dermatitis atopica) im Kindesalter. Z Hautkr 2(Suppl):72–82 16. Holden CA, Berth-Jones J (2004) Eczema, lichenification, prurigo and erythroderma. In: Burns T, Breathnach S, Cox N, Griffiths C (eds.), Rook’s textbook of dermatology (vol. 1) (7th edition). Blackwell, Oxford, pp. 17.1–17.55 17. Neering H, van Dijk E (1978) Juvenile plantar dermatosis. Acta Derm Venereol (Stockh) 58:531–534 18. Kint A, van Hecke E, Leys G (1982) Dermatitis plantaris sicca. Dermatologica 165:500–509 19. Jones SK, English JS, Forsyth A, Mackie RM (1987) Juvenile plantar dermatosis – an 8-year follow-up of 102 patients. Clin Exp Dermatol 12:5–7 20. Svensson A, Moiler H (1986) Eyelid dermatitis: the role of atopy and contact allergy. Contact Dermatitis 15:178–182 21. Rudzki E, Samochocki Z, Rebandel P, Saciuk E, Galecki W, Raczka A, Szmurlo A (1994) Frequency and significance of the major and minor features of Hanifin and Rajka among patients with atopic dermatitis. Dermatology 189:41–46 22. Mevorah B, Frenk E, Wietlisbach V, Carrel CF (1988) Minor clinical features of atopic dermatitis. Dermatologica 177:360–364 23. Diepgen TL, Fartasch M (1992) Recent epidemiological and genetic studies in atopic dermatitis. Acta Derm Venereol (Stockh) 176(suppl):13–18 24. Przybilla B, Ring J, Enders F, Winkelmann H (1991) Stigmata of atopic constitution in patients with atopic eczema or atopic respiratory disease. Acta Derm Venereol (Stockh) 71:407–410 25. Svensson A (1989) Xerosis and a history of dry skin. In: Svensson A (ed.), Diagnosis of atopic skin disease based on clinical criteria. Lund University Press, Kristianstad, pp. 41–43 26. Linde YW (1992) Dry skin in atopic dermatitis. Acta Derm Venereol Suppl (Stockh) 177:9–13 27. Werner Y, Lindberg M (1982) The water-binding capacity of stratum corneum in non-eczematous skin of atopic eczema. Acta Derm Venereol (Stockh) 62:334–337 28. Eberlein-König B, Schäfer T, Huss-Marp J, Darsow U, Möhrenschlager M, Herbert O, Abeck D, Krämer U, Behrendt H, Ring J (2000) Skin surface pH, stratum corneum hydration, transepidermal water loss and skin roughness related to atopic eczema and skin dryness in a population of primary school children. Acta Derm Venereol (Stockh) 80:188–191 29. Przybilla B, Bauer C (2005) Stigmata of the atopic constitution. In: Ring J, Przybilla B, Ruzicka T (eds.), Handbook of atopic eczema (2nd edition). Springer, Berlin, pp. 61–73

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30. Morgan DB (1948) A suggestive sign of allergy. Arch Dermatol Syphol 57:1050–1053 31. Williams HC, Pembroke AC (1996) Infraorbital crease, ethnic group, and atopic dermatitis. Arch Dermatol 132:51–54 32. Whitfield A (1938) On the white reaction in dermatology. Br J Dermatol 50:71–76 33. Aizawa H, Tagami H (1989) Inability to produce white dermographism in the early stage of infantile eczema. Pediatr Dermatol 6:6–9 34. Judge MR, McLean WHI, Munro CS (2004) Disorders of keratinization. In: Burns T, Breathnach S, Cox N, Griffiths C (eds.), Rook’s textbook of dermatology (vol. 2) (7th edition). Blackwell, Oxford, pp. 34.1–34.111 35. Marks MB (1977) Recognizing the allergic person. Am Fam Physician 16:72–79 36. Hurwitz S (2006) Clinical pediatric dermatology (3rd edition). Elsevier/W.B. Saunders, Philadelphia, pp. 49–64 37. Levy Y, Segal N, Ben-Amitai D, Danon YL (2004) Eyelash length in children and adolescents with allergic disease. Pediatr Dermatol 21:534–537 38. Möhrenschlager M, Weidinger S, Huss-Marp J, Krämer U, Behrendt H, Ring J (2005) Do gender-specific differences in eyelash length in 5- to 6-year-old preschool children with and without atopic eczema exist? Results from the MIRIAM study conducted in Augsburg, Germany. Pediatr Dermatol 22:576–577 39. Hanifin JM, Rajka G (1980) Diagnostic features of atopic dermatitis. Acta Derm Venereol (Stockh) 92(Suppl):44–47 40. Diepgen TL, Sauerbrei W, Fartasch M (1996) Development and validation of diagnostic scores for atopic dermatitis incorporating criteria of data quality and practical usefulness. J Clin Epidemiol 49:1031–1038 41. Williams HC, Burney PG, Hay RJ, Archer CB, Shipley MJ, Hunter JJ, Bingham EA, Finlay AY, Pembroke AC, Graham-Brown RA (1994) The U.K. working party’s diagnostic criteria for atopic dermatitis. I. Derivation of a minimum set of discriminators for atopic dermatitis. Br J Dermatol 131:383–396 42. Bos JD, van Leent EJ, Sillevis Smitt JH (1998) The millennium criteria for the diagnosis of atopic dermatitis. Exp Dermatol 7:132–138 43. Schultz-Larsen F, Hanifin JM (1992) Secular change in the occurrence of atopic dermatitis. Acta Derm Venereol (Stockh) 176(suppl):7–12 44. Svensson A, Edman B, Möller H (1985) A diagnostic tool for atopic dermatitis based on clinical criteria. Acta Derm Venereol (Stockh) 114(suppl):33–40 45. Visscher MO, Hanifin JM, Bowman WJ (1989) Atopic dermatitis and atopy in non-clinical populations. Acta Derm Venereol (Stockh) 144(suppl):34–40 46. Williams HC, Burney PGJ, Strachan D, Hay RJ (1994) The UK working party’s diagnostic criteria for atopic dermatitis. II: observer variation of clinical diagnosis and signs of atopic dermatitis. Br J Dermatol 131:397–405 47. Möhrenschlager M, Schäfer T, Williams HC, von der Werth J, Krämer U, Behrendt H, Ring J (1998) Übersetzung und Validierung der britischen Diagnosekriterien des atopischen Ekzems bei 8- und 9- jährigen Schulkindern in Augsburg. Allergo J 7:373–377 48. European Task Force on Atopic Dermatitis (1993) Severity scoring of atopic dermatitis: the SCORAD index. Dermatology 186:23–31 49. Yates VM, Kerr REI, MacKie RM (1983) Early diagnosis of infantile seborrhoeic dermatitis and atopic dermatitis-clinical features. Br J Dermatol 108:633–638 50. Yates VM, Kerr REI, MacKie RM (1983) Early diagnosis of infantile seborrhoeic dermatitis and atopic dermatitis-total and specific IgE levels. Br J Dermatol 108:639–645 51. Prendiville JS (2006) Scabies and lice. In: Harper J, Oranje A, Prose N (eds.), Textbook of pediatric dermatology (2nd edition). Blackwell, Oxford, pp. 659–673 52. Saurat JH (1985) Eczema in primary immune deficiencies. Clues to the pathogenesis of atopic dermatitis with special reference of the Wiskott-Aldrich syndrome. Acta Derm Venereol (Stockh) 114:125–128 53. Plebani A, Ugazio AG, Monafo V (1989) Selective IgA deficiency: an update. Curr Probl Dermatol 18:66–78

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54. Buckley RH, Fiscus SA (1975) Serum IgD and IgE concentrations in immunodeficiency diseases. J Clin Invest 55:157–165 55. Ring J, Landthaler M (1989) Hyper IgE syndromes. Curr Probl Dermatol 18:79–88 56. Amman AJ (1987) Cutaneous manifestations of immunodeficiency disorders. In: Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austen KF (eds.), Dermatology in general medicine (3rd edition). McGraw-Hill, New York, pp. 2507–2522 57. Möhrenschlager M, Schäfer T, Huss-Marp J, Eberlein-König B, Weidinger S, Ring J, Behrendt H, Krämer U (2006) The course of eczema in children aged 5–7 years and its relation to atopy: differences between boys and girls. Br J Dermatol 154:505–513 58. Nelson HS (1995) Clinical application of immediate skin testing. In: Spector SL (ed.), Provocation testing in clinical practice. Marcel Dekker, New York, pp. 703–727 59. Wüthrich B, Fuchs W (1979) Serum IgE levels in atopic diseases in childhood. Helv Paediatr Acta 34:537–544 60. Ahlstedt S (2002) Understanding the usefulness of specific IgE tests in allergy. Clin Exp Allergy 32:11–16 61. Yman L (2001) Standardization of in vitro methods. Allergy 56(suppl 67):70–74 62. Dolen WK (1999) Skin testing and immunoassays for allergen-specific IgE: a workshop report. Ann Allergy Asthma Immunol 82:407–412 63. Sanz ML, Prieto I, Garcia BE, Oehling A (1996) Diagnostic reliability considerations of specific IgE determination. J Investig Allergol Clin Immunol 6:152–161 64. Mitchell EB, Crow J, Chapman MD, Jouhal SS, Pope FM, Platts-Mills TAE (1982) Basophils in allergen-induced patch test sites in atopic dermatitis. Lancet 1:127–130 65. Ring J, Darsow U, Gfesser M, Vieluf D (1997) The ‘atopy patch test’ in evaluating the role of aeroallergens in atopic eczema. Int Arch Allergy Immunol 113:379–383 66. Gondo A, Saeki N, Tokuda Y (1986) Challenge reactions in atopic dermatitis after percutaneous entry of mite antigen. Br J Dermatol 115:485–493 67. Bruijnzeel PL, Kuijper PH, Kapp A, Warringa RA, Betz S, Bruijnzeel-Koomen CA (1993) The involvement of eosinophils in the patch test reaction to aeroallergens in atopic dermatitis: its relevance for the pathogenesis of atopic dermatitis. Clin Exp Allergy 23:97–109 68. Adinoff AD, Tellez P, Clark RA (1988) Atopic dermatitis and aeroallergen contact sensitivity. J Allergy Clin Immunol 135:182–186 69. Clark RA, Adinoff AB (1989) Aeroallergen contact can exacerbate atopic dermatitis: patch tests as a diagnostic tool. J Am Acad Dermatol 21:863–869 70. Imayama S, Hashizume T, Miyahara H, Tanahashi T, Takeishi M, Kubota Y, Koga T, Hori Y, Fukuda H (1992) Combination of patch test and IgE for dust mite antigens differentiates 130 patients with atopic dermatitis into four groups. J Am Acad Dermatol 27:531–538 71. Darsow U, Vieluf U, Ring J (1996) The atopy patch test: an increased rate of reactivity in patients who have an air-exposed pattern of atopic eczema. Br J Dermatol 135:182–186 72. Darsow U, Vieluf U, Ring J (1999) Evaluating the relevance of aeroallergen sensitization in atopic eczema with the atopy patch test: a randomized, double-blind multicenter study. J Am Acad Dermatol 40:187–193 73. Darsow U, Vieluf D, Ring J (1995) Atopy patch test with different vehicles and allergen concentrations: an approach to standardization. J Allergy Clin Immunol 95:677–684 74. Darsow U, Ring J (2000) Airborne and dietary allergens in atopic eczema: a comprehensive review of diagnostic tests. Clin Exp Dermatol 25:544–551 75. Bruijnzeel PL, Kuijper PH, Kapp A, Warringa RA, Betz S, Bruijnzeel-Koomen CA (1993) The involvement of eosinophils in the patch test reaction to aeroallergens in atopic dermatitis: its relevance for the pathogenesis of atopic dermatitis. Clin Exp Allergy 23:97–109 76. Darsow U, Laifaoui J, Kerschenlohr K, Wollenberg A, Przybilla B, Wüthrich B, Borelli S, Giusti F, Seidenari S, Drzimalla K, Simon D, Disch R, Borelli S, Devillers AC, Oranje AP, de Raeve L, Hachem JP, Dangoisse C, Blondeel A, Song M, Breuer K, Wulf A, Werfel T, Roul S, Taieb A, Bolhaar S, Bruijnzeel-Koomen C, Brönnimann M, Braathen LR, Didierlaurent A, André C, Ring J (2004) The prevalence of positive reactions in the atopy patch test with aeroallergens and food allergens in subjects with atopic eczema: a European multicenter study. Allergy 59:1318–1325

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77. Langeveld-Wildschut EG, van Marion AM, Thepen T, Mudde GC, Bruijnzeel PL, BruijnzeelKoomen CA (1995) Evaluation of variables influencing the outcome of the atopy patch test. J Allergy Clin Immunol 96:66–73 78. Langeveld-Wildschut EG, Riedl H, Thepen T, Bihari IC, Bruijnzeel PL, Bruijnzeel-Koomen CA (2000) Modulation of the atopy patch test reaction by topical corticosteroids and tar. J Allergy Clin Immunol 106:737–743 79. Høst A, Andrae S, Charkin S, Diaz-Vázquez C, Dreborg S, Eigenmann PA, Friedrichs F, Grinsted P, Lack G, Meylan G, Miglioranzi P, Muraro A, Nieto A, Niggemann B, Pascual C, Pouech MG, Rancé F, Rietschel E, Wickman M (2003) Allergy testing in children: why, who, when and how? Allergy 58:559–569 80. Darsow U, Ring J (2002) Allergy diagnosis in atopic eczema with the atopy patch test. In: Bieber T, Leung DY (eds.), Atopic dermatitis. Marcel Dekker, New York, pp. 86–105 81. Schultz-Larsen F (1993) Atopic dermatitis: a genetic-epidemiologic study in a populationbased twin sample. J Am Acad Dermatol 28:719–723 82. Cookson WO, Ubhi B, Lawrence R, Abecasis GR, Walley AJ, Cox HE, Coleman R, Leaves NI, Trembath RC, Mofatt MF, Harper IJ (2001) Genetic linkage of childhood atopic dermatitis to psoriasis susceptibility loci. Nat Genet 27:372–373 83. Cookson WO, Moffatt MF (2002) The genetics of atopic dermatitis. Curr Opin Allergy Immunol 2:383–387 84. Leung DYM, Jain N, Leo HL (2003) New concepts in the pathogenesis of atopic dermatitis. Curr Opin Immunol 15:634–638 85. Lee YA, Wahn U, Kehrt R, Tarani L, Businco L, Gustafsson D, Andersson F, Oranje AP, Wolkertstorfer A, von Berg A, Hoffmann U, Küster W, Wienker T, Rüschendorf F, Reis A (2001) A major susceptibility locus for atopic dermatitis maps to chromosome 3q21. Nat Genet 26:470–473 86. Tamura K, Suzuki M, Arakawa H, Tokuyama K, Morikawa A (2003) Linkage and association studies of STAT6 gene polymorphisms and allergic diseases. Int Arch Allergy Immunol 131:33–38 87. Novak N, Kruse S, Kraft S, Geiger E, Klüken H, Fimmers R, Deichmann KA, Bieber T (2002) Dichotomic nature of atopic dermatitis reflected by combined analysis of monocyte immunophenotyping and single nucleotide polymorphisms of the interleukin-4/interleukin-13 receptor gene: the dichotomy of extrinsic and intrinsic atopic dermatitis. J Invest Dermatol 119:870–875 88. Arkwright PD, Chase JM, Babbage S, Pravica V, David TJ, Hutchinson IV (2001) Atopic dermatitis is associated with a low-producer transforming growth factor beta(1) cytokine genotype. J Allergy Clin Immunol 108:281–284 89. Yagi R, Nagai H, Iigo Y, Akimoto T, Arai T, Kubo M (2002) Development of atopic dermatitis-skin lesions in STAT6-deficient NC/Nga mice. J Immunol 168:2020–2027 90. Weidinger S, O’Sullivan M, Illig T, Baurecht H, Depner M, Rodriguez E, Ruether A, Klopp N, Vogelberg C, Weiland SK, McLean WH, von Mutius E, Irvine AD, Kabesch M (2008) Filaggrin mutations, atopic eczema, hay fever, and asthma in children. J Allergy Clin Immunol 121:1203–1209

Clinical Aspects and Diagnosis of Anaphylaxis Phil Lieberman

Definition of Anaphylaxis The difficulty in determining the clinical manifestations that define an anaphylactic event was highlighted in a symposium sponsored by the National Institute of Health and the Food Allergy and Asthma Network [1, 2]. The symposium was attended by representatives selected from 16 different medical organizations or government bodies from North America, Europe, and Australia. It was convened to define the clinical manifestations of anaphylaxis required to establish a diagnosis. No true definition, in the classic sense of the term, resulted from the deliberations of this group, but they did define a clear-cut constellation of signs and symptoms requiring the necessity for treatment with epinephrine. They formulated three clinical scenarios during which anaphylaxis was highly likely as a cause of the event and thus epinephrine therapy mandated. These scenarios can be summarized briefly as follows: 1. The acute onset of skin or subcutaneous tissue manifestations accompanied by either respiratory, cardiovascular, or both, signs and symptoms 2. An event occurring after exposure to a likely antigen, with two or more of the following symptoms: (a) Cutaneous manifestations (b) Respiratory symptoms (c) Cardiovascular symptoms (d) Gastrointestinal symptoms 3. After exposure to a known allergen for that patient, a fall in blood pressure or manifestations of hypotension with or without any other signs or symptoms.

P. Lieberman () Division of Allergy and Immunology, Departments of Medicine and Pediatrics, University of Tennessee, Memphis, 7205 Wolf River Boulevard, Suite 200, Germantown, TN 38138, USA e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_18, © Springer 2009

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Table 1 Terminology used to define anaphylaxis Traditional World allergy organization suggested change Anaphylaxis: event mediated by IgE Anaphylactoid: clinically similar event not mediated via IgE

Anaphylaxis Immunologic IgE-mediated Non-IgE-mediated Anaphylaxis non-immunologically mediated

As can be seen, these criteria do not establish a true “definition” of an anaphylactic event, but rather define the clinical manifestations that would make a diagnosis likely and therefore indicate treatment with epinephrine. The traditional definition of anaphylaxis is “a systemic, immediate hypersensitivity reaction caused by immunoglobulin E (IgE)-mediated immunologic release of mediators from mast cells and basophils.” The term “anaphylactoid” reaction has been traditionally defined as a clinically similar event not mediated by IgE [3, 4]. A change in this terminology was suggested by the World Allergy Organization [5] via a proposal that the term anaphylaxis refer to a “severe, life-threatening, generalized or systemic hypersensitivity reaction.” They further suggested the term “allergic anaphylaxis” be reserved for such events “mediated by an immunologic mechanism, e.g., IgE, IgG, and immune-complex complement-related,” and that anaphylaxis from a non-immunologic reaction be called “nonallergic anaphylaxis.” This suggested change in terminology would eliminate the term “anaphylactoid.” Table 1 compares the “traditional terminology” to that of the suggested revision proposed by the World Allergy Organization.

Epidemiology, Incidence, and Causative Agents The exact incidence of anaphylactic episodes is unknown. This issue was recently evaluated by a panel convened to review publications studying the incidence [6]. The panel concluded the incidence is approximately 50–2,000 episodes per 100,000 person-years. They also felt that there was a possible “lifetime” prevalence of about 0.05–2%, and that the best estimate of incidence might be from studies looking at “real time data” obtained from prescriptions dispensed for the outpatient use of injectable epinephrine [7]. In one study of prescription data it was found that approximately 1% of the population of Manitoba, Canada, filled a prescription for the outpatient use of epinephrine [7]. The panel also concluded that the incidence is increasing, a conclusion shared by others [8–10], and that the estimate of incidence is probably low due to under-reporting as has been noted previously [11]. Drugs and foods are clearly the most common causes of anaphylactic episodes. Drug reactions account for perhaps as many as 230,000 hospital admissions in the USA each year [12]. As many as 6% of children and 3–4% of adults suffer from food allergy [13]. The most common drugs to cause reactions in most series are antibiotics and nonsteroidal antiinflammatory drugs [4]. The most common foods

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Table 2 Factors affecting the incidence of anaphylaxis Factor Effect

References

Geographic location

[14, 15]

Socioeconomic status

Gender

Constancy of administration of antigen

Chronobiology Age

Race Atopy

In most instances no known effect has been documented. However, in one study geographical location as well as a rural environment affected the incidence. Two investigations have now shown a clear north–south gradient in the northern hemisphere with reactions appearing to be more frequent in northern climes Higher socioeconomic status has been associated with an increased frequency of anaphylaxis based upon outpatient epinephrine dispensing rates Reportedly more frequent in females for latex, aspirin, and muscle relaxants. Appears to be more frequent overall for females in random surveys. May be more frequent in males for Hymenoptera, perhaps a function of exposure. An age-related effect has been shown with males being affected under age 15 more frequently, and females affected more frequently after age 15 Gaps in administration may predispose to reactions; this has been shown for insulin allergy, for example, where reactions occur more frequently in situations where insulin therapy has been interrupted and then reinitiated (e.g., women with gestational diabetes and multiple pregnancies) No known effect of time of day or lunar cycle More frequent in adults than children for some agents: radiocontrast, plasma expanders, anesthetics – may be function of exposure frequency No known effect Risk factor for anaphylaxis as a result of ingested antigens, exercise anaphylaxis, idiopathic anaphylaxis, radiocontrast reactions, latex reactions; probably not risk factor for insulin, penicillin, and hymenoptera reactions

[16]

[4, 16, 11]

[4]

[17] [4, 17, 18]

[17, 18]

are probably shellfish, peanuts, and tree nuts [4]. Other causes include latex, insect stings and bites, and events occurring in special situations such as those during surgery and hemodialysis. The factors which affect incidence [14–18] are summarized in Table 2.

Clinical Manifestations The clinical manifestations of anaphylaxis are of course due to the mediators formed and released at the time of the event. The relationships between these mediators and the manifestations of anaphylaxis are summarized in Table 3. The signs and symptoms of anaphylaxis can be ascertained by a review of published series [17–25]. Such a review reveals striking similarities and demonstrates

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Table 3 Mediators of anaphylaxis and their roles in anaphylactic events Mediators Pathophysiologic activity Clinical correlates Smooth muscle spasm, Histamine and products mucus secretion, of arachidonic acid vasodilatation, increased metabolism (leukotrienes, vascular permeability, thromboxane, activation of nociceptive prostaglandins, and neurons, platelet adherence, platelet-activating factor) eosinophil activation, eosinophil chemotaxis Nitric oxide Smooth muscle relaxation causing vasodilatation of peripheral vascular bed, bronchodilatation, and coronary artery vasodilatation. In addition, nitric oxide causes increased vascular permeability Neutral proteases: Cleavage of complement tryptase, chymase, components, chemoattractcarboxypeptidase, ants for eosinophils and cathepsin G neutrophils, further activation and degranulation of mast cells, cleavage of neuropeptides, conversion of angiotensin I to angiotensin II Proteoglycans: Heparin, Anticoagulation, inhibition chondroitin sulphate of complement, binding phospholipase A2, chemoattractant for eosinophils, inhibition of cytokine function, activation of kinin pathway Chemoattractants: Calls forth cells to the site Chemokines, eosinophil chemotactic factors

Tumor necrosis factor alpha activates NKF-beta

Production of plateletactivating factor

Wheeze, urticaria, angioedema, flush, itch, diarrhea and abdominal pain, hypotension, rhinorrhea, and bronchorrhea

Perhaps relief of bronchospasm, but most important effect appears to be the production of hypotension and shock

May recruit complement by cleaving C3, may ameliorate symptoms by invoking a hypertensive response through the conversion of angiotensin I to angiotensin II and by inactivating neuropeptides. Also can magnify response due to further mast cell activation Can prevent intravascular coagulation and the recruitment of complement. Also can recruit kinins increasing the severity of the reaction

May be partly responsible for recrudescence of symptoms in late phase reaction or extension and protraction of reaction Vascular permeability and vasodilatation. Also since it is synthesized and released “late,” has been incriminated in production of late phase reactions

that the most common manifestations are cutaneous followed by respiratory and cardiovascular, and finally gastrointestinal manifestations (Table 4). However, there are exceptions to these classical manifestations. Cardiovascular collapse with shock can occur immediately and may not be accompanied by any other

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Table 4 Signs and symptoms of anaphylaxisa Signs and symptoms

Percentage of casesb

Cutaneous, subcutaneous Urticaria and angioedema Flush Pruritus without rash Respiratory Dyspnea, wheeze Upper airway angioedema Rhinitis Cardiovascular Dizziness, syncope, hypotension, arrhythmia, angina, myocardial infarction Gastrointestinal Nausea, vomiting, diarrhea, cramping abdominal pain Miscellaneous Headache Substernal pain Seizure Disseminated intravascular coagulation

> 90 85–90 45–55 2–5 40–60 45–50 50–60 15–20

a b

30–35

25–30

5–8 4–6 1–2 <1

Obtained from a review of references [4, 17–25]. Percentages are approximations (see text).

symptoms or signs [4, 19]. This may be related to the severity of the event. In a series of severe episodes [19], only 70% of patients with circulatory and/or cardiovascular collapse demonstrated cutaneous manifestations. Five percent had neurological symptoms including seizures, impaired consciousness, and muscle spasms. A relative paucity of subcutaneous and cutaneous manifestations in this group of patients could be due to the fact that the series included severe episodes reported in patients rescued by helicopter, and signs and symptoms were only obtained after the arrival of emergency personnel. Another possibility is that the lack of subcutaneous and cutaneous manifestations might be due to profound hypotension with loss of blood volume into the third space, and therefore lack of sufficient cutaneous and subcutaneous blood flow to manifest signs such as urticaria and angioedema. In addition, cutaneous symptoms may be more common in adults than children. For example, Sampson and associates [25] in evaluating the manifestations of oral food challenge in children with food sensitivity, although finding skin symptoms the most common manifestation, recorded such symptoms only in 84% of episodes. A similar finding was noted in a study involving 57 children presenting to an emergency room with anaphylaxis [21]. In this study, pruritus occurred in 40%, generalized erythema in 26% by history and 25% on examination, urticaria in 24%, and angioedema in 32%. The overall percent of children with cutaneous features was 82% – a finding echoing Sampson’s evaluation. However, the incidence of cutaneous features may have been reduced because of the time between the onset

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of symptoms and presentation to the emergency department. In contrast, in a larger series of anaphylaxis in children, cutaneous symptoms were more frequent [20]. Perioperative anaphylaxis may be different from those events occurring outside the operating room. Cutaneous symptoms are seen less frequently, and cardiovascular collapse more frequently. In addition there may be significant differences between perioperative IgE-mediated events and those which are nonIgE-mediated [4]. The incidence of cutaneous manifestations is approximately 75% for IgE-mediated events, and slightly higher for non-IgE-mediated events. Cardiovascular collapse, however, occurs significantly more frequently during IgE-mediated events than in those which are not IgE-mediated. Thus, overall, IgEmediated events during surgery are in general more severe than those which occur through non-IgE-mediated mechanisms. Symptoms usually begin 5–30 min after antigen injection. When antigen has been ingested, symptoms usually occur within the first 2 h. However, there can be a delay of several hours after ingestion of an antigen. In some instances, after ingestion symptoms can occur almost immediately. Such rapidly occurring events seem to be more severe. There is thought to be a direct correlation between the immediacy of onset of symptoms and the severity of a given attack. That is, the more rapid the onset, the more severe is the event. Biphasic reactions can occur as well. A biphasic event is characterized by a recrudescence of symptoms occurring after either a spontaneous remission or a remission due to treatment. Such biphasic events usually appear within 8 h after resolution. But such a return of symptoms has occurred even later in some individuals, and has been recorded in some instances 24 h or longer after resolution of the initial manifestations. In addition, attacks can be protracted, persisting for several days, during which multiple recurrences interrupted by asymptomatic periods are characteristic. The exact incidence of protracted and biphasic events is unknown. However, biphasic reactions have been recorded in as high as 20% of episodes [26]. The severity of the second response is highly variable, ranging from mild to severe. Fatalities have occurred during the biphasic response. There are no definitive features which predict a biphasic reaction. However, biphasic responses have been associated with the failure to administer epinephrine in a timely manner and in an adequate dose, when antigen is ingested, and related to failure to administer corticosteroids during the event. Cardiovascular manifestations of anaphylaxis are quite varied and can be profound. Characteristically anaphylaxis has been associated with compensatory tachycardia that occurs in response to a decreased effective vascular volume. This has been used as a sign to differentiate anaphylactic episodes from vasodepressor reactions. However, bradycardia, presumably caused by increased vagal activity, can also occur. Such bradycardia appears to be due to the Bezold-Jarisch reflex, a cardioinhibitory reflex that arises in sensory receptors in the inferoposterior wall of the left ventricle. The reflexes are carried by unmyelinated vagal C fibers. In a series of anaphylactic events conducted by Brown et al. [22], patients (8 of 21) exhibiting hypotension had an initial tachycardia followed by a relative bradycardia. The bradycardia was severe enough to require atropine therapy in two patients.

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Myocardial depression with decreased cardiac output as a result of contractile depression can occur and can persist for several days. Coronary artery vasospasm has been documented with coronary angiography and can be severe enough to result in myocardial infarction (Kounis syndrome) [23]. Numerous electrocardiographic abnormalities have been recorded. It is important to remember that anaphylaxis can present with unusual manifestations that make diagnosis difficult. Syncope without any other manifestation can occur. It has been reported after fire ant sting, exercise anaphylaxis, and in patients with mastocytosis. Such patients may simply lose consciousness without any other manifestation or can experience accompanying seizure activity. Unless anaphylaxis is suspected this presentation can result in unnecessary cardiovascular and neurological evaluations before a diagnosis of anaphylaxis is established. Anaphylactic events have mimicked foreign body aspiration in children and have resulted in adrenal hemorrhage and hypotensive episodes due to adrenal failure [4]. Severe episodes with hypotension can result in fibrinolysis and disseminated intravascular coagulation. Four cases experiencing hypotensive reactions (three with elevated tryptase) exhibited abnormal thromboelastograms and clear-cut evidence of disseminated intravascular coagulation. These events have occurred via both IgE-mediated and non-IgE-mediated mechanisms. It is interesting to note that tranexamic acid rapidly reversed coagulopathy in all cases [24].

Differential Diagnosis The differential diagnosis of anaphylaxis is seen in Table 5. The most common condition mimicking anaphylaxis is probably the vasodepressor reaction (vasovagal syncope). The characteristic features of vasodepressor reactions are hypotension, pallor, weakness, nausea, vomiting, and diaphoresis. There may be loss of consciousness in severe reactions. Vasodepressor reactions are the result of a threatening event or emotional trauma. The characteristic bradycardia has been used as a differential diagnostic feature to distinguish them from anaphylaxis. However, as noted, hypotensive anaphylactic episodes can be associated with bradycardia as well. Thus perhaps the most important distinguishing feature between anaphylaxis and vasodepressor reactions is the absence of urticaria, flush, angioedema, and pruritus in the vasodepressor response. Flushing reactions should be considered in the differential diagnosis. Flush occurs in a “wet” and “dry” form. In the wet form there is associated sweating. This form is mediated by sympathetic cholinergic nerves that supply sweat glands in the skin, and is characteristic of postmenopausal flushing. The postmenopausal flush lasts about 3–5 min and occurs several times a day. It can be aggravated by alcohol and stress. There are no known respiratory tract symptoms, gastrointestinal manifestations, and no hypotension. “Wet” flushing also occurs after the ingestion of spicy foods containing capsaicin. Direct vasodilatation without stimulation of the sweat glands produces a dry flush as is seen in the carcinoid syndrome. Other

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P. Lieberman Table 5 Differential dragnosis of anaphylaxis Anaphylaxis due to exogenously administered agents (e.g., drugs, foods) Anaphylaxis due to physical factors Exercise Cold Heat Sunlight Idiopathic anaphylaxis Vasodepressor reactions Flush syndromes Carcinoid Postmenopausal Alcohol Drugs Niacin Vasointestinal polypeptide secreting tumors Medullary carcinoma thyroid Gastrointestinal tumors Other forms of shock Hemorrhagic Cardiogenic Endotoxic “Restaurant syndromes” Monosodium glutamate (MSG) Sulfites Scrombroidosis Excess endogenous production of histamine syndromes Systemic mastocytosis Urticaria pigmentosa Basophilic leukemia Acute promyelocytic leukemia (tretinoin treatment) Hydbatid cyst Nonorganic disease Panic attacks Münchhausen stridor Vocal cord dysfunction syndrome Globus hystericus Undifferentiated somatoform anaphylaxis Miscellaneous Hereditary angioedema “Progesterone” anaphylaxis Urticarial vasculitis Phbeochromocytoma Hyperimmunoglobulin E, urticaria syndrome Neurologic (seizure, stroke) Pseudoanaphylaxis Red man syndrome (vancomycin) Capillary leak syndrome

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agents can induce a dry flush. These include niacin, nicotine, catecholamines, and angiotensin converting enzyme inhibitors. A dry flush is also seen in pancreatic and intestinal tumors, medullary carcinoma of the thyroid, hypoglycemia, pheochromocytoma, rosacea, and alcohol administration (both with and without associated drug administration). Flush also is characteristic of mastocytosis and can occur in panic attacks as well as autonomic epilepsy. It is not surprising that the carcinoid syndrome, pancreatic tumors, medullary carcinoma of the thyroid, and some gastrointestinal tumors produce flushing and other symptoms similar to anaphylaxis. This is because these tumors can secrete histamine, kallikrein, neuropeptides, prostaglandins, serotonin, pancreastatin, and other molecules producing vasodilatation and other pathophysiologic events similar to those occurring in anaphylaxis. In an alcohol-induced flush, a non-elevated, intense erythema located mainly over the trunk occurs within minutes after the ingestion of alcohol. Symptoms usually peak 30–40 min after ingestion, and usually subside within 2 h. There are two forms of alcohol-induced flushing. In one form the event is associated with the intake of drugs, and in the other, alcohol alone is sufficient to produce the episode. The latter form is most common in Asians, but also occurs in non-Asians as well. In both the drug-related and non-drug-related types, lightheadedness, dyspnea, anxiety, headache, and vomiting can occur. A number of different drugs have been incriminated in alcohol-induced flushing. These include disulfiram, griseofulvin, and cephalosporins. Alcohol-induced flushing has also been reported in patients with malignancies (including Hodgkin’s disease), hypereosinophilic syndrome, mastocytosis, and in patients who have undergone splenectomy. The alcohol-related, drug-independent form of alcohol-induced flush is due to a deficiency in aldehyde dehydrogenase-2. This enzyme metabolizes aldehyde, a metabolite of alcohol. In patients without this enzyme, the increased levels of aldehyde result in mast cell degranulation. A group of postprandial symptoms can produce manifestations similar to those occurring during anaphylactic episodes. Postprandial episodes occur after the ingestion of monosodium glutamate, sulfites, or histamine. Monosodium glutamate ingestion can cause chest pain, facial burning, flushing, paresthesias, sweating, dizziness, headaches, palpitations, nausea, and vomiting. In children there can be shivering and chills, irritability, screaming, and delirium. It is believed that monosodium glutamate causes a “transient acetylcholinosis,” which is responsible for these symptoms. Symptoms usually begin no later than 1 h after ingestion, but they can be delayed in onset up to 14 h. There may be a familial tendency to develop these reactions. Histamine poisoning, caused by the ingestion of histamine contained in spoiled fish, appears to be increasing in frequency. Histamine is the major chemical involved in the production of symptoms, but all symptoms are not caused by the uncomplicated ingestion of histamine alone. The ingestion of histaminecontaminated spoiled fish is more toxic than the ingestion of equal amounts of pure histamine administered by mouth. cis-Urocanic acid, an imidazole compound similar to histamine also found in spoiled fish, could account for this phenomenon. cis-Urocanic acid can degranulate mast cells, thus perhaps augmenting the response

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to endogenous histamine. Histamine itself is produced by histidine-decarboxylating bacteria that cleave histamine from histidine in the spoiled fish. The increase in histamine occurs early after death of the fish. It can therefore occur on the fishing vessel, at the processing plant, in the distribution system, or in the restaurant or home. Fish with elevated histamine levels usually do not have any look or smell that distinguishes them from unspoiled fish. Cooking does not destroy the histamine or the activity of cis-Urocanic acid. Histamine poisoning, known as Scombroidosis, is the most prevalent form of seafood-induced disease in the USA [4]. It is probably underreported because most episodes are mild. Some episodes are probably attributed to allergic reactions. It is most often due to the ingestion of scrombroid fish which belong to the family Scombroidae (e.g., tuna and mackerel) or Scomberesocidae (e.g., saury), but nonscombroid species, such as Mahi-Mahi, anchovies, and herring, can also produce reactions. Bacteria such as Morganella morganii, Klebsiella pneumoniae, and Hafniae alvei are most often responsible for the production of histamine and cis-Urocanic acid. This reaction usually occurs at room temperature, but once the bacterial population is enlarged, ongoing histamine production can occur even with refrigeration. The onset of symptoms in scombroidosis can occur a few minutes to several hours after the ingestion of fish. The episodes usually last a few hours but can persist for days. The signs and symptoms are similar to histamine toxicity and include urticaria, flush, angioedema, nausea, vomiting, diarrhea, and hypotension. Neurologic findings also can occur. Rarely, wheezing is a manifestation. The most common symptom is flushing of the face and neck, accompanied by a sensation of heat and discomfort. The rash can be similar in appearance to sunburn. Gastrointestinal symptoms occur second in frequency to cutaneous manifestations. Histamine fish poisoning can be distinguished from anaphylaxis, in that with scombroidosis: (1) several people at the dining table are often affected, (2) the cutaneous symptoms, as noted above, are usually somewhat different, (3) and measurement of tryptase, plasma histamine, and 24-h urinary histamine metabolites reveal elevated levels of histamine and its byproducts with normal levels of tryptase. Anaphylactic symptoms can occur due to the excessive endogenous production of histamine in conditions such as acute promyelocytic leukemia, basophil leukemia, and mastocytosis. Nonorganic conditions have been known to mimic anaphylaxis. These include panic attacks, undifferentiated somatoform anaphylaxis, and the vocal cord dysfunction syndrome. Panic attacks are usually accompanied by tachycardia, gastrointestinal symptoms, shortness of breath, and occasionally by flushing as well. However, there is no pruritus or airway obstruction. Undifferentiated somatoform anaphylaxis is a term used to describe patients who present with manifestations that mimic idiopathic anaphylaxis but who lack objective confirmatory findings, do not respond to therapy, and exhibit psychologic signs of an undifferentiated somatoform disorder. Other entities are traditionally listed in the differential diagnosis of anaphylaxis. They include hereditary angioedema, pheochromocytoma, tracheal foreign bodies, the “red man syndrome” due to the administration of vancomycin,

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pseudoanaphylaxis occurring after the administration of procaine penicillin, and the capillary leak syndrome. Some patients with hereditary angioedema exhibit an erythematous, serpiginous rash which can be distinguished from urticaria by the absence of pruritus. This rash when associated with airway obstruction due to angioedema can produce an event that mimics anaphylaxis. Capillary leak syndrome can also present in a similar way. This rare, often fatal disorder can be characterized by angioedema, gastrointestinal symptoms, and shock with hemoconcentration. It is associated with elevation in leukotriene levels. Recurrent episodes can mimic idiopathic anaphylaxis.

The Laboratory as an Aid in the Differential Diagnosis Several tests can be employed to confirm a diagnosis of anaphylaxis or a condition mimicking anaphylaxis. These are noted in Table 6. Serum tryptase, plasma histamine, and 24-h urinary histamine metabolites are traditionally ordered to confirm a diagnosis of anaphylaxis.

Table 6 The laboratory in the differential diagnosis and diagnosis of anaphylaxis Test Comment Serum tryptase

Plasma histamine

Serum tryptase levels usually peak 60–90 min after the onset of symptoms and persist 6 h. However, tryptase levels can be elevated for as long as 24 h. Nonetheless, ideally, measurement should be obtained between 1 and 2 h after onset of symptoms Plasma histamine levels rise earlier than tryptase levels, the onset of symptoms, increasing within 5–10 min after levels are elevated only evanescently and usually return to normal after 60 min. Thus they are of little help if the patient is seen more than an hour after the event began Urinary histamine metabolites can be elevated for up to 24 h after the onset of the event Used to rule out carcinoid syndrome

24-h urinary histamine metabolites (N-methylhistamine) Serum serotonin and urinary 5-hydroxyindoleacetic acid Gastrointestinal vasopeptides Useful to rule out the presence of a vasoactive polypeptide including pancreastatin, which can be associated with pancreatic or vaso-intestinal polypeptide, substance P, small bowel tumors and medullary carcinoma neurokinin, and others of the thyroid Plasma-free metanephrine and urinary Useful to rule out a paradoxical vanilmandelic acid pheochromocytoma Octreotide-enhanced CT of helpful If elevated levels of vasopeptides are noted, may be abdomen bowel in locating site of synthesis (e.g., pancreatic or small tumor)

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Tryptases are a subgroup of trypsin-family serine peptidases. Almost all human mast cells contain tryptases, and small amounts can be found in the human basophil. Serum tryptase is specific to these cells and is therefore a useful marker in the diagnosis of anaphylaxis. Tryptase is secreted constitutively in small amounts. Originally it was thought that alpha tryptase comprised the majority of constitutively secreted tryptase. Recently, however, subjects with null alleles for alpha tryptase have been found to exhibit “normal” levels of constitutively secreted tryptase. It is now known that constitutively secreted tryptase is, for the most part, beta protryptase (immature beta tryptase) with alpha tryptase contributing a small amount. With mast cell degranulation a marked increase in tryptase occurs. This increase is due to the secretion of mature beta tryptase. Thus constitutively secreted tryptase is a mixture of alpha and beta tryptase (primarily beta), with increases occurring in tryptase levels seen during anaphylaxis being due to the release of mature beta tryptase [27]. Serum tryptase levels peak 60–90 min after the onset of symptoms and usually persist for up to 6 h. However, elevated levels of tryptase can be present for several hours longer, perhaps even for 24 h after the onset of symptoms. Plasma histamine, in contrast to serum tryptase, begins to rise much more quickly. Plasma histamine levels are noted 5–10 min after the onset of the event, but are evanescent, usually returning to normal within 60 min after the onset of symptoms. Therefore, they are of little help if the patient is seen as long as an hour after the onset of the event. However, urinary histamine metabolites can be elevated for 24 h. Disparities between histamine and tryptase levels can occur [28]. In a study of allergic reactions presenting to an emergency room, elevated concentrations of plasma histamine were observed in 42 of 97 adult patients, whereas only 20 such patients had elevations of serum tryptase. Histamine levels also correlated better with clinical signs than did tryptase. Patients with elevated histamine were more likely to have urticaria, more extensive erythema, abnormal abdominal findings, and wheezing. Other markers of mast cell and basophil degranulation have also been evaluated. These include CD63 on basophils detected by flow cytometry, prostaglandin D2, nitric oxide, and carboxypeptidase. CD63 is a surface maker on basophils. The expression of CD63 is up-regulated by basophil degranulation and can be detected by flow cytometry. Flow-cytometry-assisted allergy diagnosis has been shown to be reliable for reactions induced by food, latex, hymenoptera venom, and drugs [29]. Prostaglandin D2 levels have been useful to confirm the diagnosis of anaphylaxis in patients with mastocytosis. Carboxypeptidase levels in serum or plasma collected within 8 h of the onset of allergic reactions in mastocytosis patients were significantly greater than those found in control groups. In 83% of cases that had an elevated tryptase, concentrations of carboxypeptidase were elevated. Out of 110 cases suspected of anaphylaxis that were tryptase negative, elevated concentrations of carboxypeptidase were found in 77 (70%) [30]. Exhaled levels of nitric oxide have also been reported to be elevated in a patient with an anaphylactic episode due to insect sting [31]. However, in a study of 77 patients with “acute allergic reactions,” nitric oxide levels did not correlate with

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plasma histamine or serum tryptase levels. In addition, nitric oxide levels were not correlated with urticaria or erythema, and were no higher in patients with hypotension and/or bronchospasm [32]. Finally, it should be noted that tryptase levels can be used to distinguish anaphylactic events occurring in patients with mastocytosis versus those unrelated to mastocytosis. The basis of this distinction rests in the fact that laboratory measurements of serum tryptase exist that can distinguish total tryptase from the mature form. As noted, total tryptase consists of both the mature and immature forms of beta tryptase and alpha tryptase. Most monoclonal antibodies used to measure tryptase recognize all forms. However, there is one monoclonal antibody which recognizes a linear epitope only on the mature form of tryptase [33]. The utilization of both antibodies can distinguish mastocytosis-related events from nonmastocytosis-related episodes. For example, at rest (not during an event) systemic mastocytosis patients will have increased levels of total tryptase (usually greater than 20 ng/ml), consisting mainly of immature tryptase and related to the total mast cell burden. On the other hand, patients with non-mastocytosis-related events will have normal levels of resting (asymptomatic phase) tryptase. During an event, the majority of the increase in serum tryptase is due to the release of mature tryptase. The ratio of total tryptase to mature tryptase can therefore be used to distinguish events related to mastocytosis from those occurring in non-mastocytosis patients. In patients with systemic mastocytosis, who have elevated baseline levels of immature tryptase, the ratio of total to mature tryptase is usually greater than 20. In nonmastocytosis-related events this ratio is usually less than 10. It is important to remember that there are other causes of elevated total serum tryptase level. These include myeloproliferative disorders, the hypereosinophilic syndrome associated with FIP1L1/PDGFRA mutations, and patients with end-stage kidney disease [33].

References 1. Sampson HA, Munoz-Furlong A, Bock SA, et al. (2005) Symposium on the definition and management of anaphylaxis: summary report. J Allergy Clin Immunol 115(3):584–592 2. Sampson HA, Munoz-Furlong A, Campbell RL, et al. (2006) Second symposium on the definition and management of anaphylaxis: summary report – Second National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network Symposium. J Allergy Clin Immunol 117(2):391–397 3. Lieberman P, Kemp S, Oppenheimer J, et al. (2005) The diagnosis and management of anaphylaxis: an updated practice parameter. J Allergy Clin Immunol 115:S483–S523 (supplement) 4. Lieberman P (1998) Anaphylaxis and anaphylactoid reactions. In: Middleton E Jr, Reed CE, Ellis EF, Adkinson NF Jr, Yunginger JW, Busse WW (eds.), Allergy: Principles and Practice, Vol. 2, 5th edition. St. Louis, MO: Mosby-Yeark, Section E Chapter 77, pp. 1079–1092 5. Johansson SGO, Bieber T, Dahl R, et al. (2004) Revised nomenclature for allergy for global use: report of the nomenclature review committee of the World Allergy Organization October 2003. J Allergy Clin Immunol 113:832–836 6. Lieberman PL, Camargo C, Simons FER, Jick H, Sheikh A, Bohlke K, Miller R (2006) Epidemiology of anaphylaxis: findings of the ACAAI (American College of Asthma, Allergy,

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30. Zhou X, Buckley MG, Lau LC, et al. (2006) Mast cell carboxypeptidase as a new clinical marker for anaphylaxis. J Allergy Clin Immunol 117(2):S85 31. Rolla G, Nebiolo F, Guida G, et al. (2006) Level of exhaled nitric oxide during anaphylaxis. Ann Allergy Asthma Immunol 97:264–265 32. Lin RY, Gupta A (2003) Nitric oxide levels in patients with acute allergic reactions. J Allergy Clin Immunol 111:S100 (abstract) 33. Schwartz LB (2006) Diagnostic value of tryptase in anaphylaxis and mastocytosis. Immunol Allergy Clin N Am 26:451–463

Angioedema in the Emergency Department Malcolm W. Greaves and Allen P. Kaplan

Introduction: Definition and Classification Angioedema is defined as the occurrence of one or more acute short-lived (almost invariably less than 72 h duration) erythematous or skin-coloured skin and/or mucosal non-pitting swellings. It is due to sudden onset of localized increase in vasopermeability affecting the deep mucosal or cutaneous dermis and submucosal or subcutaneous tissue. Pain may occur; itch is common in those cases associated with urticaria but frequently the swellings are asymptomatic. They subside without staining or desquamation of the skin or mucosae. Scratching or rubbing may cause bruising. The patient presenting in the Accident and Emergency department with a tentative diagnosis of angioedema must be subjected to careful history-taking (from the patient if possible, or alternatively from any first-hand witnesses) and meticulous clinical examinations. There is a long list of causes of swellings – mainly on the face – which can mimic angioedema. The more important are listed in Table 1. Angioedema, especially when it affects the upper respiratory or gastrointestinal tract is a common and highly alarming occurrence for patient, his or her family and friends and Emergency Room staff alike. This is because of the sudden onset of swelling, the angor animi and sense of impending doom due to feared or actual onset of asphyxia often experienced by the patient, and involvement of skin and mucocutaneous junctions bringing about a grotesque appearance. Not uncommonly the tongue becomes too big for the mouth and protrudes and speech may be impossible. Intestinal angioedema, which frequently mimics an acute abdomen with or without accompanying shock is also a challenging event for all concerned. Angioedema with any of these presentations can be due to a variety of causes, with important implications for treatment. Although elements of emergency treatM.W. Greaves () London Allergy Clinic, 66, New Cavendish St London W1 and Institute of Dermatology, St Thomas’ Hospital, London SE1, UK; Guy’s Kings and St Thomas’ Schools of Medicine and Dentisty, University of London, London, UK e-mail: [email protected] A.P. Kaplan National Allergy and Urticaria Centres of Charleston, Charleston, South Carolina, USA

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_19, © Springer 2009

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Table 1 Conditions causing facial swelling, which mimic angioedema (‘pseudoangioedema’ [1]) Facial condition Distinguishing featuresa ‘Ordinary’ urticaria Cellulitis Acute allergic contact dermatitis Acute photodermatitis Dermatomyositis Tumid lupus erythematosus Lymphoedema

Crohns disease Superior vena cava syndrome

No mucosal lesions, superficial (‘hives, nettle rash’), invariably red, pruritic Pain, fever, several days’ duration, then desquamation Intense itching, blistering, several days’ duration, desquamation Predominantly light-exposed areas, several days’ duration; desquamation Periorbital swelling, persistent Persistent erythema and oedema, ANF usually negative, mucin deposition on skin biopsy Persistent, classically in Melkersson–Rosenthal syndrome, which is complicated by recurrent cellulitis; scrotal tongue Persistent, complicated by lymphoedema, cobblestone thickening of oral mucosa Persistent oedema, associated with venous engorgement

a

In contrast to pseudoangioedema, ‘true’ angioedema has an abrupt onset, transient course (usually less than 3 days) and fades without desquamation. Table 2 Classification of angioedema With urticaria Acute Allergic (foods, drugs) Virus infections Idiosyncratic (NSAIDS; radiocontrast media) Contact urticaria, especially latex Idiopathic Chronic Idiopathic chronic urticaria Autoimmune Non-autoimmune Physical urticarias Urticarial vasculitis

No urticaria Hereditary angioedema With C'1 inhibitor deficiency Type1, type 2 With normal complement levels Type 3 Acquired C'1 esterase inhibitor deficiency

ACE inhibitor angioedema Idiopathic chronic angioedema NSAIDS: nonsteroidal anti-inflammatory drugs; ACE: angiotensin convertase inhibitor

ment are common to angioedema whatever the aetiology, awareness of the subtypes of angioedema and their diagnosis is essential for effective management. The principle subtypes of angioedema are listed in Table 2. It is convenient to classify angioedema according to the presence or absence of concurrent urticaria. According to one large UK study 49% of 554 patients with urticaria also had angioedema [2]. However, urticaria is not associated with angioedema in patients with hereditary forms of angioedema, those associated

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with C'1 esterase inhibitor (C'1 INH) deficiency or in angioedema due to angiotensin convertase enzyme (ACE) inhibitors. A chronic relapsing form of angioedema, in the absence of urticaria, which is not familial and in which there is no complement abnormality, termed idiopathic angioedema, is also seen. The clinical and laboratory features of the different subtypes of angioedema have been reviewed in detail elsewhere [1, 3] and will only be outlined here.

Angioedema with Urticaria Angioedema with Acute Urticaria Acute urticaria is accompanied by angioedema in about 5% of cases [3]. By currently accepted definition [4] acute angioedema with urticaria runs a course of less than 6 weeks, usually only a few days at the most. Common causes include food allergies, especially shellfish, nuts; drug allergies, especially penicillin and its derivatives; virus infections, especially in children; and idiosyncratic reactions to nonsteroidal anti-inflammatory drugs (NSAIDS) or radiocontrast media, insect stings in hypersensitive patients. In the severest cases, for example, those associated with peanut allergy, anaphylactic shock may supervene. In such patients abdominal pain and vomiting, dyspnoea due to bronchospasm or stridor due to laryngeal oedema may be prominent. Angioedema due to NSAIDS seems to be mainly an attribute of the COX 1 inhibitors, COX 2 inhibitors appearing to be relatively safe in this respect, although more data are required before arriving at a final conclusion [5]. Acute contact urticaria is defined as an acute local skin or mucosal reaction occurring within minutes of contact with the eliciting substance. It is an IgEmediated type 1 hypersensitivity reaction, which is frequently associated with urticaria, angioedema and even anaphylaxis, especially when associated with latex allergy [6].

Angioedema with Chronic Urticaria Chronic urticaria, with or without angioedema is defined as the daily or almost daily occurrence of wheals for at least 6 weeks [5]. Published figures for the frequency of angioedema in patients with chronic urticaria range from 59% to 87% [7, 8]. Chronic ‘idiopathic’ urticaria is now recognized to be due to an autoimmune process in at least 30–50% of patients [9, 10]. The autoantibodies are predominantly subclass IgG1 or IgG3 and are directed against the alpha component of FceR1 (the high affinity IgE receptor) (or less commonly IgE itself) expressed on

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dermal mast cells and basophils [11]. About 40% of patients with autoimmune urticaria also have angioedema [7]. This diagnosis cannot be made on clinical or histopathological grounds since there is too much overlap between the clinical findings and skin biopsy histology in autoimmune and non-autoimmune cases. Although poor response to antihistamines and a past history of thyroid disease may raise suspicions, the diagnosis can be supported by a positive autologous serum test [11] and confirmed by demonstrating that the patient’s serum releases histamine form basophil leucocytes of a healthy donor [12] or causes up-regulation of an activation marker, CD63, in the donor basophils [13]. The cause of the urticaria and angioedema in 50–60% of patients in whom no autoantibodies can be demonstrated (‘ordinary’ chronic urticaria) is unclear, although an abnormality of signal transduction in mast cells involving the ras-dependent signalling pathway has been proposed [14]. Physical urticarias comprise about 40% of all chronic urticarias [11, 15]. Some but not all physical urticarias cause angioedema (Table 3). In cold urticaria, immersion of forearm in cold water causes localized angioedema of the hand and wrist. An iced drink may cause glossal angioedema. Exertion, heat and emotion in patients with severe cholinergic urticaria may cause facial angioedema in addition to the characteristic pruritic monomorphic rash. Urticaria does not occur in association with the angioedema of vibratory angioedema. Angioedema rarely or never occurs in other physical urticarias. Urticarial vasculitis may be associated with angioedema, especially in hypocomplementaemic cases [16]. Urticarial vasculitis is rare, comprising around 1–2% of all chronic urticarias. Characteristically, individual urticarial wheals last longer than 24 h and may be associated with staining of the involved skin. Underlying systemic disease including autoimmune connective tissue disease, hepatitis and inflammatory bowel disease need to be sought. The diagnosis must be confirmed by skin biopsy. The angioedema of urticarial vasculitis, which may affect skin and/ or mucous membranes is unremarkable in appearance, resembling that occurring in other types of chronic urticaria.

Table 3 Physical urticarias with or without angioedema Physical urticaria Angioedema Comment Symptomatic dermographism (syn. factitious urticaria) Delayed pressure urticaria Cholinergic urticaria

No

Cold urticaria

Yes

Solar urticaria Heat urticaria Aquagenic urticaria Vibratory angioedema

Rare Rare No Yes

No Yes

Typical pruritic monomorphic rash provoked by exercise, heat and emotion may be associated with facial angioedema Lingual and glossal angioedema may be provoked by iced drinks, iced lollipop, ice cream

Angioedema is invariable; no urticaria

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Angioedema Without Urticaria Hereditary Angioedema Due to C'1 INH Deficiency This genetic disorder affects 1:50,000 persons. It was first recognized as an entity by William Osler [17] who established the dominantly inherited genealogy of this relapsing angioedema. Although most patients have a family history of the disorder, de novo mutations are common. It exists in two forms. Type 1 (about 85% of patients) manifests a reduction in synthesis of C'1 INH with corresponding loss of function, whereas the less common type 2 shows normal or increased synthesis of non-functional C'1 INH protein. In both types values of functioning C'1 INH of less than 50% of normal are likely to be associated with bouts of angioedema. This occurs because C'1 INH is the primary inhibitor of activated factor X11 (Hageman factor), which converts prekallikrein to kallikrein and it inhibits kallikrein as well. Kallikrein is a potent and ubiquitous bradykinin-forming enzyme, which is activated by local trauma, bradykinin being a potent vasopermeability factor. Thus, in patients with hereditary angioedema (HAE), minor trauma, dental procedures, infections, oestrogens, strenuous exercise, alcohol consumption or emotional stress trigger attacks of angioedema associated with elevated tissue and plasma bradykinin levels [18]. Oestrogen also increases the risk of relapses of HAE, for example, in early pregnancy [19], and following use of oral contraceptives [20], since they increase synthesis of factor X11. Since impaired C'1 inhibition also leads to increased turnover of C'1, C'2 and C'4, plasma levels of C'2 and C'4 are low – consistently so in the case of C'4, which is reduced even in between attacks, due to continual activation of C'1. Therefore, a normal C'4 even in an asymptomatic patient excludes HAE in 95% of cases and is often used as a screening test [21]. However in a case where the history and clinical picture of the patient is strongly suggestive of hereditary angioedema measurement of functional C′1 esterase inhibitor should be performed, even though the C′4 level is normal. The C'1 INH gene (SERPING1) maps to chromosome 11. For type 1 HAE gene defects include deletions, insertions, stop codons or frame-shift mutations [22], whereas type 2 is typically caused by a missense mutation. HAE is dominantly inherited and on that basis it would be expected that there would be a reduction of 50% in C'1 INH activity for either type. However, actual levels during and even in between attacks are usually lower than this, a finding probably explained by repression of synthesis or increased consumption of the gene product [23]. Clinical features HAE can occur at any age including the perinatal period and infancy, but frequently appears for the first time at school age [24]. There is no urticaria, but in children and adults a non-pruritic erythematous rash may occur before (prodromal) or during attack of HAE in up to 28% of patients [25, 26]. Oedema usually develops at a single site, is non-pitting, well-defined and skin-coloured. Face, extremities, genitals and mucosal surfaces are common sites, but it may occur anywhere, and swellings regress within 2–5 days. Most patients experience recurrent abdominal pain due to localized bowel oedema, which may even cause volvulus or intussusception but most often presents

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with ascites. Repeated abdominal episodes frequently result in a series of surgical interventions. Thus, HAE should be considered in any patient presenting to the emergency room with abdominal pain and multiple abdominal scars [27] and should prompt abdominal ultrasound imaging [28]. The white blood cell count, erythrocyte sedimentation rate and C-reactive protein are usually normal in attacks of abdominal HAE. Attacks subside after 72 h even without treatment; thus, surgery is rarely indicated and only in the exceptional circumstances outlined above or for unrelated acute abdomen. Laryngeal oedema is the most feared manifestation of HAE and may be fatal. In one series of 58 patients from 46 affected families, 23 patients (40%) aged 9–78 years of age died from asphyxia due to this cause [29]. Autoimmune diseases, especially lupus erythematosus, show an increased frequency in patients with HAE [30] but are usually mild. Laboratory diagnosis (Fig. 1) In a patient with angioedema the diagnosis of HAE is effectively excluded if the serum C'4 level is normal and the quantitative assay of C'1 INH protein is also normal in between or during attacks. However, C'4 and C'1 INH protein can very rarely (<1% of patients) be normal in between attacks [31, 32]. Therefore, if the clinical suspicion is high, a C'1 INH functional assay should be carried out. If the C'4 serum level is low this may be due to a variety of other conditions including autoimmune connective tissue diseases. Therefore, ideally a quantitative C'1 INH

Measure C`4

C`4, C`1 INH protein normal

Effectively excludes HAE types

Strong clinical suspicion despite normal C`4 between

C`4 low, normal / raised C`1 INH protein – consider C`4 low, C`1 INH protein low

Measure functional C`1 INH

Measure functional C`1 Functional C`1 INH low = HAE type 2

Normal functional C`1 INH – consider type 3 HAE in the right clinical

Fig. 1 HAE diagnostic algorithm

Low functional C`1

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assay should be performed alongside the C'4, and if these are low then the diagnosis of HAE type 1 is confirmed. If the C'4 is low, but the C'1 INH protein serum level is normal or raised, then a C'1 INH functional assay needs to be carried out to exclude type 2 HAE. Note that serological tests may be misleading in infants of 1 year of age or less and when in doubt, genetic typing should be performed [33].

Hereditary Angioedema Without C'1 INH Deficiency: HAE Type 3 In 1986, Warin et al. [34] reported two sisters who suffered attacks of angioedema only during pregnancy or when receiving the ethinyl oestradiol contraceptive pill. The C'1 INH and other complement components were consistently normal in these patients, both during and in between relapses. Subsequently, Bork [35] described ten women with HAE from ten families. Study of the families of these women revealed another 26 affected women. In all these patients the C'4, C'1 INH protein and C'1 INH functional assay were normal. Similar families have been reported elsewhere [36, 37]. The age of onset tends to be later than for classical (type 1 or 2) HAE, but otherwise the clinical presentation is similar including episodes of abdominal pain and laryngeal oedema. However, unlike types 1 and 2 HAE, attacks occur mainly during pregnancy, or when oestrogen-containing contraceptives are administered, and are unresponsive to intravenous C'1 INH concentrate (vide infra). The molecular basis and genetics of type 3 HAE have recently been elucidated. Dewald et al. [38] identified the F12 gene encoding the coagulation factor XII (F12 Hageman factor) as the HAE type III gene and reported two heterozygous missense gain-of-function mutations leading to increased bradykinin production by kallikrein. In practice, in the absence of available genetic studies, the diagnosis of type III hereditary angioedema is essentially clinical and by exclusion (Fig. 1).

Acquired C'1 INH Deficiency This rare entity has been reported to occur in patients with lymphosarcoma and lymphoma, especially B-cell lymphoma, and autoimmune co nnective tissue disorders such as systemic lupus erythematosus. The clinical presentation is similar to that seen in HAE, including abdominal and laryngeal symptoms. However, the onset is usually in middle age or later. In patients with lymphoma, angioedema with low serum C'1 INH is associated with low serum C'1q, which distinguishes this condition from HAE [39]. The cause of the low levels of these complement components is probably increased consumption due to circulating immune complexes, vicarious C'1 activation by tumour cell surface antigen or to C'1 fixation and C'1 INH depletion by an anti-idiotypic antibody bound to immunoglobulin on the surface of the B cells [40]. The clinical presentation of C'1 INH deficiency in lupus erythematosus [41] is similar to that of

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HAE and responds to androgen therapy, which increases hepatic synthesis of C'1 INH (vide infra). These patients are grouped together as type 1 acquired C'1 INH deficiency. In other cases, termed type 2-acquired C'1 INH deficiency, C'1 INH is depleted by IgG antibodies directed against the C'1 INH itself [42, 43]. This causes reduced serum levels of C'4, C'1q and C'1 INH, and a C'1 INH cleavage product, 95 kD C'1 INH, is also found in type 2-acquired but not type 1-acquired angioedma. In one rare clinical presentation of this type of acquired C'1 INH deficiency the paraprotein in a monoclonal gammopathy is an antibody against C'1 INH [44]. The abnormalities in the hereditary and acquired forms of angioedema are listed in Table 4.

Angioedema Due to Angiotensin Convertase Enzyme Inhibitors and Angiotensin Receptor Type 2 Blockers Angioedema due to these classes of drugs is still rare but as their usage increases the frequency of this serious adverse effect is rising and is the commonest (17–30%) [45] cause of angioedema seen in an Accident and Emergency setting. The incidence of ACE inhibitor angioedema is estimated at around 1–7 per 1,000 patients [46] and accounts for the rising hospitalization rate for angioedema in the USA (4.2 per 100,000) [47]. It is higher in Afro-Americans and Afro-Caribbeans and is life-threatening in up to 20% [48]. Although commonly occurring within the first few weeks of treatment, its onset may be delayed many months into the course of ACE inhibitor treatment [49]. Continued administration of ACE inhibitors despite episodes of angioedema may lead to progressively worsening attacks [50]. The pathophysiology is fairly well understood. Angioedema may occur after the first dose and is not immunologically mediated. It is also class – but not drug – specific. The specific mediator of the angioedema is the potent vasoactive nonapeptide bradykinin [51]. Apart from inhibiting conversion of angiotensin I to angiotensin II, ACE inhibitors also inhibit the enzymatic degradation of bradykinin by ACE, causing elevation of circulating and tissue bradykinin levels [52], which act on B2 receptors causing increased permeability of capillaries and post-capillary Table 4 Complement abnormalities in different forms of angioedema C'1 INH protein C'1 function C'4 C1q

95 kD C'1 INH

HAE type 1 HAE type 2 HAE type 3 Acquired type1 Acquired type 2

No No No No Present

Reduced Normal or increased Normal Reduced Reduced

Reduced Reduced Normal Reduced Reduced

Reduced Reduced Normal Reduced Reduced

Normal Normal Normal Reduced Reduced

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venules. Why only a tiny minority of patients receiving ACE inhibitors suffer this complication is unclear but may relate to genetic factors [53]. ACE inhibitor angioedema shows a predilection for the head and neck, the lips, tongue, larynx, pharynx and sub-glottal areas being commonly affected [54]. Dysarthria due to glossal or laryngeal oedema may prevent coherent drug historytaking. Sudden onset of abdominal pain, with or without visible mucocutaneous swelling in a middle aged or elderly patient should prompt enquiry about ACE inhibitor treatment. Like HAE, ACE inhibitor-evoked angioedema is rarely accompanied by urticaria. Both these conditions also share the common pathogenetic feature of mediation by bradykinin: over-production of bradykinin in the case of HAE, and suppressed degradation in the case of ACE inhibitor angioedema. Initially, the angiotensin receptor type 2 (AT 2) blockers were thought to be safe alternatives to ACE inhibitors for patients with ACE inhibitor angioedema, since they do not cause elevated bradykinin levels. However there have been several case reports of angioedema occurring in patients receiving AT 2 blockers [55, 56]. Although this would seem to challenge the notion that ACE inhibitor angioedema is caused by bradykinin, recent work has shown that AT 2 receptor antagonists cause a threefold increase in bradykinin B2 receptor expression leading to increased bradykinin binding [57]. Of interest is the recent report that omapatrilat, a dual inhibitor of ACE and endopeptidase showed 17% incidence of angioedema, frequency being especially high in Afro-Caribbean patients and smokers [58]. Overall, it would seem unwise to substitute an AT 2 receptor inhibitor in a patient with a history of angioedema due to an ACE inhibitor.

Idiopathic Recurrent Angioedema Without Urticaria These patients, who have no family history, get recurrent episodes of angioedema due to no obvious cause, and have a normal complement profile. Abdominal and laryngeal oedemas are rare in these patients. The pathogenesis is likely to be the same as for ‘idiopathic urticaria with angioedema’ in many cases, implying an autoimmune basis for some of these. However, it can be argued that others may have a different though as yet undefined aetiology [3].

Treatment of Angioedema The overall mortality of HAE and ACE inhibitor-induced angioedema is significant [29], but these fatalities are usually due to lack of access to appropriate emergency treatment, or mistaken use of inappropriate treatment. Fatalities are rare in angioedema accompanied by urticaria.

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Treatment of Acute Angioedema In cases where the mouth, pharynx or larynx is involved, the first priority should be to secure the airway. This can be done by intubation. Tracheostomy is rarely required. Once the airway is restored, oxygen should be given and an intravenous line established. All patients should receive subcutaneous or intramuscular adrenaline 0.3 mg (0.3 ml of a 1/1,000 solution) every 10–15 min until the angioedema is stabilized. Intravenous adrenaline should be avoided unless the patient is in shock. All patients with angioedema should be admitted for observation for at least 24 h as relapses are common. Except in patients with HAE, acquired C'1 INH deficiency and ACE inhibitor angioedema, chlorpheniramine 4 mg intramuscularly and hydrocortisone 100–200 mg intravenously are usefully given to lessen the possibility of a relapse. Angioedema due to NSAIDS should be treated in the emergency room as above.

Treatment of Acute Hereditary Angioedema Types 1 and 2 For mildly affected patients with no evidence of respiratory or abdominal complications subcutaneous or intramuscular adrenaline can be given, although of inconsistent effectiveness. Admission and close observation for at least 24 h should be advised, as relapses, which may be more severe than the original episode, are common. Intubation should be carried out for more severely affected patients with respiratory obstruction, followed by oxygen and an intravenous line. These patients and those with abdominal pain should be given C'1 inhibitor concentrate intravenously or, if this is unavailable, fresh frozen plasma. Fresh frozen plasma [59] 2 units (1 unit = 200 ml) should be given and repeated 1–2 hourly until the angioedema is stabilized [60]. It runs the theoretical risk of worsening the condition owing to its content of significant amounts of substrate for C'1 esterase. It is also not virally inactivated. Care must also be taken not to cause fluid overload. The preferred treatment is lyophilized vapor-heated C'1 inhibitor concentrate, which has been shown to be safe and effective in a double blind controlled trial [61] (Immuno, Aventis Behring, Vienna Austria or Berinert P injection; Centeon, Pharma GmbH, Vienna Austria) and which has largely obviated concerns about transmission of HIV, hepatitis B, hepatitis C and other viruses. One unit of C'1 inhibitor concentrate contains the amount of C'1 inhibitor in 1 ml of plasma. The saline-reconstituted concentrate contains 550 plasma units in a 10 ml vial. Twentyfive plasma units per kilogram body weight is administered to a total of 1,000 plasma units (500 units for children), repeated once if necessary. This treatment should reduce the swelling within 3–4 h. If there is no improvement the diagnosis of HAE should be reviewed. In the event that neither of these blood products is available, tranexamic acid (oral or intravenous) could be given in dosage 25 mg per kg up to 1 g, every 3–4 h (maximum 75 mg per kg per day) [62]. Systemic antihistamines and corticosteroids are

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ineffective in acute HAE. Recently, Icatibant, a bradykinin B2 receptor antagonist, has been licensed by the European Commission for the treatment of acute attacks of HAE [63]. It has a short half life (2–3 h) and is administered subcutaneously – a significant advantage over C'1 inhibitor concentrate, which is given intravenously. The product is not yet licensed for self-injection.

Treatment of Hereditary Angioedema Type 3 Women with this condition are unresponsive to C'1 inhibitor concentrate. Treatment is essentially confined to the emergency measures outlined above (Treatment of Acute Angioedema, vide supra).

Treatment of Angioedema Due to Acquired C'1 Inhibitor Deficiency The treatment is that of hereditary angioedema types 1 and 2. If the acquired C'1 inhibitor deficiency is due to autoantibodies directed against C'1 inhibitor, then rapid consumption of C'1 inhibitor occurs, and C1 inhibitor concentrate treatment may have to be supplemented by tranexamic acid [64]. Since the C'1 esterase inhibitor deficiency is secondary to an underlying cause, this should also be addressed.

Treatment of ACE Inhibitor-Induced Angioedema Emergency treatment is as outlined above (Treatment of Acute Angioedema, vide supra). Airway obstruction is common and fatal outcomes have been reported [65]. Therefore, prompt intubation may be necessary, or even occasionally admission to intensive care [66]. There is no specific treatment for this angioedema. Systemic antihistamines and corticosteroids are ineffective. Relapses following apparent recovery are common and patients should be routinely admitted for observation for at least 24 h. Patients with a history of idiopathic angioedema and with HAE types 1 and 2 (which may be formes frustes) are predisposed to ACE inhibitor-induced angioedema [67]. It is therefore prudent to check the C'4 level in all cases of ACE inhibitor-induced angioedema.

Treatment of Angioedema with Urticaria This has been recently reviewed [68]. When chronic urticaria is associated with angioedema serious and life-threatening complications such as laryngeal or severe

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glossal swelling and acute abdominal symptoms are very rare. However, acute allergic angioedema may affect the tongue palate and larynx, causing stridor and provoking panic in the patient, relatives and emergency room staff alike. In such cases the measures outlined above (Treatment of Acute Angioedema, vide supra) apply and adrenaline subcutaneously or intramuscularly is usually very effective. Signs and symptoms of anaphylaxis may also supervene requiring establishment of an intravenous line administration of adrenaline and maintenance of the airway. For chronic idiopathic angioedema with urticaria, oral H1 antihistamine such as chlorpheniramine 4 mg tds or diphenhydramine 50 mg tds with (in severer cases) prednisolone 30 mg daily with tapering is usually all that is required.

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44. Chevailler A, Arlaud G, Ponard D, et al. C1-inhibitor binding monoclonal immunoglobulins in three patients with acquired angioneurotic oedema. J Allergy Clin Immunol. 1996; 97: 998–1008. 45. Agah R, Bandi V, Gunturpalli KK. Angioedema: the role ofACE inhibitors and factors associated with poor outcome. Intensive Care Med. 1999; 23: 793–796. 46. Hedner T, Samuelsson O, Lunde H et al. Angioedema in relation to treatment with angiotensin converting enzyme inhibitors. Brit Med. J. 1992; 304: 941–946. 47. Lin RY, Shah SN, Karandish K. Increasing Hospitalisations due to angioedema in the united States. J Allergy Clin Immunol. 2008; Suppl. 1; 121: S99 (abs). 48. Pigman EC, Scott JL. Angioedema in the emergency department: the impact of angiotensin converting enzyme inhibitors. Am J Emerg Med. 1993; 11: 350–354. 49. Schiller PI, Langauer MS, Haefeli WE, et al. Angiotensin-converting enzyme inhibitor induced angioedema: late onset, irregular course and potential role of triggers. Allergy. 1997; 52: 432–435. 50. Brown NJ, Snowden M, Griffin MR. Recurrent angiotensin convertase enzyme inhibitorassociated angioedema. J Am Med Assoc. 1997; 278: 232–233. 51. Greaves MW, Shuster S. Responses of skin blood vessels to bradykinin, histamine and 5-hydroxytryptamine. J Physiol. 1967; 193: 255–267. 52. Nussberger J, Cugno M, Amstutz C, et al. Plasma bradykinin in angioedema. Lancet 1998; 351: 1693–1697. 53. Mathews KP, Pan M, Gardner MJ, Hugli T. Familial carboxypeptidase N deficiency. Ann Int Med. 1980; 93: 443–445. 54. Sabroe RA, Black AK. Angiotensin converting enzyme (ACE) inhibitors and angioedema. Br J Dermatol. 1997; 136: 153–158. 55. Irons BK, Kumar A. Valsartan-induced angioedema. Ann Pharmacother. 2003; 37: 1024–1027. 56. Abdi R, Dong VM, Lee CJ, Ntoso KA. Angiotensin 11 receptor blocker-associated angioedema: on the heels of ACE inhibitor angioedema. Pharmacotherapy. 2002; 22: 1173–1175. 57. Tan Y, Hutchinson FN, Jaffa AA. Mechanisms of angiotensin 11-induced expression of B2 kinin receptors. Am J Physiol Heart Circ Physiol. 2004; 286: H926–932. 58. Coats AJ. Omipatrilat – the story of Overture and Octave. Int J Cardiol. 2002; 86: 1–4. 59. Pickering RJ, Kelly JR, Good RA, Gerwuz H. Replacement therapy in hereditary angioedema: successful treatment of two patients with fresh frozen plasma. Lancet. 1969; 1: 326–330. 60. Gompels MM, Lock RJ. C1 inhibitor deficiency: management. Clin Exp Dermatol. 2005; 30: 737–740. 61. Waytes TA, Rosen FS, Frank MM. Treatment of hereditary angioedema with a vapor-heated C'1 inhibitor concentrate. N Engl J Med. 1996; 334: 1630–1634. 62. Bowen T, Cicardi M, Farkas H, et al. Canadian 2003 International Consensus Algorithm for the Diagnosis, Therapy and Management of Hereditary Angioedema. J Allergy Clin Immunol. 2004; 114: 629–646. 63. Bork K, Yasothan U, Kirkpatrick P. Icatibant. Nat Rev Drug Disc 2008; 7: 801–802. 64. Cugno M, Cicardi M, Agostoni A. Activation of the contact system and fibrinolysis in autoimmune acquired angioedema: a rationale for prophylactic use of tranexamic acid. J Allergy Clin Immunol. 1994; 93: 870–876. 65. OikeY, Ogata Y, Higashi D, et al. Fatal angioedema associated with enalapril. Int Med. 1993; 32: 308–310. 66. Thompson T, Frable M. Drug-induced life-threatening angioedema revisited. Laryngoscope. 1993; 103: 10–12. 67. Orfan, N, Patterson R, Dykewicz M. Severe angioedema related to ACE inhibitors in patients with a history of idiopathic angioedema. JAMA. 1990; 264: 1287–1289. 68. Kaplan AP. Clinical practice. Chronic urticaria and angioedema. N Engl J Med. 2002; 346: 175–179.

Diagnosis of Aspirin Sensitivity in Aspirin Exacerbated Respiratory Disease Marek L. Kowalski

Introduction The first patient with adverse reaction to aspirin – manifested as dyspnea and angioedema – was described in 1902 (already 3 years after aspirin was brought to the market) by Hirschberg from Poznan´ in Western Poland [1]. Two most common clinical manifestations of acetyl salicylic acid (ASA) hypersensitivity: aspirininduced asthma and aspirin-induced urticaria usually occur separately; however, in some patients both respiratory and cutaneous symptoms may appear following ingestion of aspirin [2]. This chapter will focus on respiratory type of aspirin hypersensitivity which affects a subpopulation of asthmatic patients presenting a typical triad: intolerance to aspirin (ASA) and other non-steroidal anti-inflammatory drug (NSAID), bronchial asthma and nasal polyposis [3]. These patients react with an acute dyspnea usually accompanied by nasal symptoms (rhinorrhea/nasal congestion) within 2 h after ingestion of aspirin or other NSAIDs [4]. Several various terms have been used to describe respiratory reactions to NSAIDs: aspirin sensitivity, pseudoallergy, idiosyncrasy or intolerance [5]. In agreement with current recommendation for nomenclature the term hypersensitivity seems to the most appropriate to describe reactions to aspirin and other NSAIDs [6]. The coexistence of aspirin hypersensitivity with upper airway (rhinosinusits/nasal polyps) and lower airway (asthma) inflammatory disease was referred to as aspirin triad, asthma triad, Samter’s syndrome, aspirin-induced asthma, aspirin-sensitive rhinosinusitis/asthma syndrome. The term aspirin-exacerbated respiratory disease (AERD) was proposed recently to describe this syndrome referring to the fact that the core issue in these patients is not ASA hypersensitivity but the underlying chronic inflammatory respiratory disease only occasionally exacerbated by aspirin or other NSAIDs [7]. M.L. Kowalski () Department of Immunology, Rheumatology and Allergy, Chair of Immunology, Faculty of Medicine, Medical University of Łodź, Poland; 251 Pomorska St., 92-213 Łódz´, Poland e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_20, © Springer 2009

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The prevalence of ASA hypersensitivity among adult asthmatics if assessed by history alone varies from 4.3% to 12% in various populations [8–10]. However, if oral provocation is used to determine aspirin hypersensitivity the incidence of AERD in general asthma population may increase up to 21.1% [11], and among patients with severe asthma the prevalence may be as high as 14–24% [12, 13]. Among asthmatics with chronic rhinosinusitis and/or nasal polyps the prevalence of aspirin hypersensitivity may reach 30–40% [14, 15]. Available data suggests that aspirin hypersensitivity among asthmatics remains still under-diagnosed [16].

Clinical Characteristics of Patients with AERD The typical patient with aspirin-exacerbated respiratory disease is a 30–40-year-old women with a history of chronic rhinosinusitis and/or asthma that usually precede the development of hypersensitivity to aspirin [5, 17] (Table 1). Some patients associate the beginning of the disease with flu-like infection which is followed by development of chronic intractable rhinosinusitis; already at this stage nasal polyps are frequently detected. Asthma is usually the second component of the triad precipitated in almost half of patients by upper respiratory tract infection – in the AIANE study aspirin or other NSAID were precipitating the first asthmatic attack only in 14% of patients [16]. Asthma once developed runs a protracted course which is independent of avoidance of aspirin and other NSAIDs. According to earlier reports ASA-hypersensitive asthmatics have been traditionally considered to have “intrinsic ” (i.e. non-allergic) type of asthma, but more recent studies documented that patients with AERD frequently present history of atopic allergy and have positive skin test results to common allergens [18–21]. Patients with AERD react not only to aspirin but also cross-react to other NSAIDs which have strong anti-inflammatory activity like naproxen, diclofenac or indomethacin. On the other hand weak inhibitors of prostaglandin synthesis like paracetamol and preferential COX-2 inhibitors are well tolerated. Table 1 Clinical features of patients with aspirin exacerbated airway disease Age of onset 20–40 years Gender F:M 2:1 Presence of atopy 30–70% Chronic underlying diseases: Eosinophylic infiltration within airways Chronic rhinosinusitis Pansinusitis on CT scans Watery rhinorrhea Nasal congestion anosmia, Nasal polyposis 60–90% High recurrence rate Bronchial asthma Moderate or difficult to treat/severe 30–50% on oral steroids Drug hypersensitivity Aspirin and other NSAIDs Cross-reactive type

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In majority of aspirin-hypersensitive asthmatics signs and symptoms of chronic rhinosinusitis are present [22, 23]. Rhinosinusitis has usually protracted course and in most cases is complicated by mucosal hypertrophy and polyp formation [24]. On computer tomography, mucosal hypertrophy is present in up to 100% of patients and the frequency of nasal polyps may be as high as 90% [25]. Nasal polyposis has a high tendency of recurrence after surgery; the recurrence rate in ASA-sensitive patients is almost three times higher than in intrinsic asthmatics and seven times higher than in atopic asthmatics [26]. A subgroup of ASA-sensitive patients manifests a reaction exclusively in the upper respiratory tract; they do not have asthma, but clinical picture of the nasal disease (hyperthrophic rhinosinusitis) in these patients is similar to that observed in patients with ASA-triad [23]. Although some of these patients may evolve with time to a full aspirin triad their risk of developing asthma in the future is not known. Patients hypersensitive to aspirin suffer from persistent asthma of greater-thanaverage severity and of higher-than-ordinary medication requirements, including dependence on steroids [16]. ASA-sensitive asthmatics are more likely to have severe asthma, to have been intubated and to have a steroid burst in the previous 3 months [27]. Although in hypersensitive patients ingestion of aspirin or other NSAID may aggravate respiratory symptoms both lower and upper airway disease persist and have protracted course even in the absence of exposure to ASA/ NSAIDs [5, 16]. Aspirin hypersensitivity is not only a significant risk factor for development of severe chronic asthma but is also strongly associated with nearfatal asthma and fatal outcome of asthma occurs more often than in asthmatics without the triad [21, 28, 29].

Pathomechanisms of AERD It has been postulated that the mystery of AERD has to be explained by two possibly distinctive, but to some extend overlapping mechanism: one is the mechanism of aspirin (NSAIDs)-induced acute hypersensitivity reaction and the second is the pathomechanism responsible for chronic eosinophylic inflammation underlying severe asthma and rhinosinusitis with nasal polyps [30]. Both mechanisms have been attributed to arachidonic acid metabolism although the putative biochemical defects have not been fully elucidated [17].

Patomechanism of Acute Aspirin-Induced Reaction In 1975, Szczeklik et al. [31] suggested that the mechanism of hypersensitivity to ASA and other NSAIDs, is not immunological, but is related to their pharmacological properties namely inhibition of prostaglandin synthesis. In a series of experiments they documented that only NSAIDs that are strong or at least moderate

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inhibitors of prostaglandin synthesis can evoke reaction in ASA-hypersensitive patients [31, 32]. It has been proposed, that ASA-induced reaction is the result of inhibition of cyclooxygenase, an enzyme that metabolizes arachidonic acid to prostaglandins, thromboxanes and prostacyclin. Further studies show that aspirin and NSAIDs trigger a chain of events leading to activation of inflammatory cells, release of mediators and eventually to clinical manifestation of acute hypersensitivity reaction: bronchial obstruction, rhinorrhea and nasal congestion. From practical standpoint it is important to stress that other NSAIDs like indomethacin, naproxen or ibuprofen, which are considered to be strong cyclooxygenase-1 inhibitors precipitate adverse symptoms in a significant (ranging from 30% to 80%) proportion of ASA-hypersensitive patients. On the other hand weak COX-1 inhibitors, for example, salicylic acid or acetaminophen and selective COX-2 inhibitors (e.g. celcoxib) are usually well tolerated by these patients (Table 2). Several inflammatory mediators were shown to be released in parallel to ASAinduced reaction including cysteinyl-leukotrienes, PGD2, histamine, mast cell tryptase and eosinophil cationic protein ECP, clearly indicating for activation of mast cells and eosinophils [30, 33]. Intrinsic deficiency of prostaglandin E2, increased susceptibility of cyclooxygenase-1 to inhibition by NSAIDs and/or decreased function of prostaglandin EP2 receptors may be responsible for distinctive response Table 2 Cross-reactivity of NSAIDs in patients with AERD NSAIDs with high crossreactivity (30–100%) Diclofenac Etololac Flurbiprofen Fenoprofen Ibuprofen Indomethacin Ketoprofen Ketorolac Mefanemic acid Meclofenamate Nabumetone Naproxen Piroxicam Sulindac NSAIDs with low crossreactivity (5–30%) Paracetamol (dose <1,000 mg) Diflunisal Nimesulide Meloxicam NSAIDs well tolerated (£ 1% crossreactors) Celecoxib Etoricoxib Lumiracoxib Parecoxib

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to NSAIDs by sensitive subjects [33, 34]. On the other hand, cysteinyl-leukotrienes (LTC4/D4/E4) are consistently increased after positive aspirin challenge in lower and upper airways as well as in urine and leukotriene LT1 receptor antagonists partially prevent ASA-induced reaction pointing at prominent role of leukotrienes in the pathophysiology of acute aspirin-induced reaction [35–37] (Fig. 1). Although the link between inhibition of cox-1 by aspirin and increased generation of cysteinyl-leukotrienes has not been elucidated further elaboration of a newly proposed ‘two compartment model’ of enhanced CysLT production and altered

Cell membrane phospholipids PLA

15-LOX

ASA 15-HETE

Arachidonic acid

Lipoxins COX-2

COX-1

5-LOX

PGG2

PGE2

PGH2

LTA4 EP-R

PGE2

LTC4s LTC4

EP-R

Eos

Mast cell

Asthma Rhinorrhea Congestion

LTD4

LTE4

Fig. 1 Pathomechanism of aspirin-induced asthma/rhinosinusitis according to the “Prostaglandin Hypothesis” In aspirin-hypersensitive asthmatics PGE2 acting via prostaglandin EP receptors is responsible for stabilization of inflammatory cells (mainly mast cells and eosinophils) and 5-lipoxygenase. Inhibition of Cyclooxygenase-1 by aspirin or other NSAIDs deprives the system of stabilizing activity of prostaglandin E2, leading to activation of 5-lipoxygenase, generation of cysteinylleukotrienes and activation of inflammatory cells. Mediators released from inflammatory cells, including cysteinyl-leukotriens, are responsible for development of symptoms. The role of 15-lipoxygenase pathway activation responsible for generation of 15-HETE and lipoxins is not clear and may be either pro-inflammatory and modualtory. PLA – Phospholipase ASA – Aspirin EP-R – Prostaglandin EP receptors Eos – Eosinophil

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end organ responsiveness may help to understand the pathophysiology of aspirin hypersensitivity [38]. In nasal polyp epithelial cells and peripheral blood leukocytes from ASAsensitive, but not ASA-tolerant patients aspirin triggers 15-HETE generation, suggesting the presence of a specific abnormality of 15-LO pathway in these patients [39, 40]. A correlation between expression of alternatively spliced variant of cyclooxygenase-1 and ASA-triggered 15-HETE release may suggest a potential link between cyclooxygenase and lypoxygenase pathways in inducing ASA-hypersensitivity reaction [41].

Pathomechanism of Chronic Eosinophylic Inflammation, Severe Asthma and Chronic Rhinosinusitis with Nasal Polyps in AERD Eosinophylic inflammation of the lower and upper airway mucosa, which is characteristic for AERD does not seem to be related to intake of aspirin or other NSAIDs. Complete avoidance of NSAIDs does not lead to clinical improvement and in most patients the presence of chronic eosinophylic inflammation in the upper or lower airways proceeds, even several years, development of hypersensitivity to aspirin. Viral infections have been proposed as primary triggers of aspirin hypersensitivity, but also as a cause of underlying chronic inflammation in the airways of ASAsensitive patient [42]. Autoimmunity may also play a role in aspirin hypersensitivity: autoimmune response markers have been found in ASA-sensitive patients [43, 44] and expression of specific HLA-DR antigens have been associated with aspirin sensitivity [45]. Recently IgE-antibodies to Staphylococcal enterotoxins (SAEs) were associated with eosinophylic inflammation in nasal polyps of ASAhypersensitive patients suggesting that Staphylococcus aureus enterotoxins acting as superantigen may trigger T-cell-mediated inflammatory reaction and/or exert direct effects on eosinophil proliferation and survival in the airway mucosa [46]. Role of Arachidonic Acid Metabolites Pathomechanism underlying airway inflammation in patients with AERD is thought to be different from that present in aspirin-tolerant asthmatics and have been associated with abnormalities of both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism [5, 47]. Lower generation of PGE2 by nasal polyps, nasal polyp epithelial cells and bronchial fibroblasts and corresponding decreased expression of COX-2mRNA were reported in patients with AERD [39, 48–50]. PGE2 deficiency may be accompanied by reduced expression of prostaglandin EP2 receptors in the airway mucosa of ASA-hypersensitive patients pointing at functional deficiency of this protective

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AA metabolite [51]. Intrinsic defect in local generation of PGE2 and/or dysfunction of relevant receptors could contribute to development of more severe eosinophylic inflammation in patients with AERD since PGE2 possesses anti-inflammatory activity and may inhibit eosinophil chemotaxis and activation. Basal levels of leukotrienes E4 in the urine of ASA-sensitive patients are elevated as compared to ASA-tolerant asthmatics, even in the absence of aspirin challenge suggesting that cysteinyl-leukotrienes may be also important mediators of persistent airway inflammation [35, 36]. However in the airways, the differences in basal levels of LTs between ASA-hypertensive and ASA-tolerant patients are less consistent [52–54]. An increased number of cell expressing LTC4synthase, an enzyme involved in transformation of arachidonic acid to cysteinyl-leukotrienes were also found in the bronchi of ASA-hypertensive asthmatics [55]. Although earlier studies demonstrated an increased production of cysteinyl-leukotrienes in nasal polyps of ASA-sensitive asthmatics as compared to aspirin-tolerant patients in vitro [56, 57], these observations could not be reproduced in vivo when nasal washes were analysed [37, 58]. Both basal and stimulated release of LTC4 was found to be similar in nasal polyp dispersed cells from ASA-sensitive and ASA-tolerant patients [59]. Although Perez-Novo et al. [60] found an increased generation of LTC4/D4/E4 and overexpression of enzymes involved in production of leukotrienes (5-LOX and LTC4 synthase) in nasal polyps tissue from ASA-sensitive patients production of cysteinyl-leukotriens correlated with tissue concentration of ECP both in ASA-sensitive and ASA-tolerant polyps. Furthermore, increased expression of leukotriene LT1 receptors that was found in the nasal mucosa of ASA-sensitive patients may result in local hyperresponsiveness to leukotrienes in this subpopulation of patients [61]. However, the role of cysteinyl-leukotrienes and their receptors in the pathogenesis of chronic mucosal inflammation in ASA-sensitive is not clear since chronic treatment with LT1 receptor antagonist do not demonstrate better effectiveness in relieving nasal and bronchial symptoms or reducing polyp size in ASA-hypersensitive as compared to ASA-tolerant patients [62, 63]. Abnormalities within arachidonic acid metabolites generated on 15-LOX pathway have been also associated with AERD. 15-Lipoxygenase seems to be upregulated in nasal mucosa of ASA-hypersensitive patients and decreased production of the anti-inflammatory lipoxin A4 found in peripheral blood leukocytes and in nasal polyp tissue from ASA-sensitive patients further point to an important role for 15-LOX metabolites in nasal polyps [60, 64].

Role of Cytokines and Growth Factors Increased number of eosinophils in the tissue and increased release of ECP have been linked to distinctive profile of cytokine expression with upregulation of several cytokines related to eosinophil activation and survival (e.g. IL-5, GMC-SF, RANTES, eotaxin) both in upper and lower airways and in nasal polyps [65–68]. It has been proposed that overproduction of IL-5 might be a major factor responsible for an increased survival of eosinophils in the nasal polyps resulting in

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increased intensity of the eosinophilic inflammation particularly in aspirin-sensitive patients [69, 70]. Also, mast cells are important component of the upper and lower airway inflammation in ASA-hypersensitive patients [47, 71, 72] and serum baseline levels of mast-cell-derived mediators tryptase and stable PGD2 metabolite are elevated [73]. In the nasal polyp tissue the density of mast cells was correlated with the number of polypectomies, implicating an important role for these cells in the pathogenesis of nasal polyposis [71]. In the same study stem cell factor (SCF), a cytokine critical for differentiation, survival, chemotaxis and activation, and of human mast cells was found to be increased in nasal polyps epithelial cells from aspirin-hypersensitive asthmatics and correlated closely with the density of mast cells and number of polypectomies. Increased release of cysteinyl-leukotrienes and differential regulation by PGE2 of leukotrienes release in mast cells cultured from peripheral blood of AERD patients has also been reported supporting the important role of these cells [74]. Potential role for mononuclear cells and activated T cells in perpetuating the inflammatory response in ASA-sensitive patients have also been proposed [75, 76].

Overall Diagnostic Approach to a Patient with AERD Presence of aspirin hypersensitivity is just a hallmark of widespread airway mucosal inflammation and should prompt a physician to perform extensive investigation of lower and upper airways. Majority of patients with AERD suffer from severe form of the disease and ASA-sensitivity is associated with increased risk of life-threatening asthma attacks. These patients had significantly lower postbronchodilator increase in FEV1 as compared with subjects with aspirin-tolerant asthma suggesting the presence of irreversible airway obstruction related to airway remodelling [27]. Virtually all patients with asthma and ASA hypersensitivity have hyperthrophic inflammation of sinus mucosa usually involving most sinuses and recurrent nasal polyposis reflects presence of chronic rhinosinusitis with typical symptoms. Presence or suspicion of AERD should prompt a physician to extend diagnosis of the upper airways. Flexible rhinoscopy and CT scans of sinuses should be performed in each case of suspected ASA hypersensitivity, allowing for assessment of the extent of upper airway inflammation. Patients with AERD on average have significantly higher sinus CT scan score although some overlap between ASAsensitive and tolerant is evident [25]. It has been demonstrated that the intensity of hypertrophy on CT may predict probability of AERDs, and sinus CT score <12 would support the likelihood of aspirin tolerance in a patient with an ambiguous history of adverse response to aspirin [77]. In earlier literature the presence of hypersensitivity to aspirin was associated with lack of allergic sensitization (atopy) and presence of atopy was considered to decrease the risk of aspirin hypersensitivity [4]. However, several more recent studies documented that majority of patients with AERD (50–70%) have multiple allergic sensitizations, thus atopy should not preclude the suspicion of ASA

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Table 3 Factors determining reliability of ASA-hypersensitivity history • Underlying chronic and often unstable disease • Concomitant treatment which may mask symptoms • NSAIDs are often hidden in composite preparations • Recent history of good tolerance of NSAIDs

hypersensitivity if other risk factors (e.g. severe asthma or nasal polyposis) exist [18, 20]. Presence of atopy was even suggested to be a risk factor for aspirin sensitivity among asthmatic patients challenged with oral aspirin [19]. Atopic sensitization to inhalant allergens may be an important mechanism contributing to the pathogenesis of the airway inflammation in a patient with AERD, thus it is fully justified to perform an allergic work-up in such a patient.

Anamnesis in Diagnosis of ASA Hypersensitivity Presence of hypersensitivity to ASA or other NSAIDs should always be suspected in a patient with moderate to severe asthma. Female gender, presence of chronic rhinosinusitis, nasal polyposis or history of near-fatal asthma exacerbation increases significantly the risk of AERD. On the other hand childhood onset of asthma, atopic background or absence of nasal polyposis in anamnesis does not exclude hypersensitivity to ASA. The diagnosis of ASA hypersensitivity in clinical setting is usually based on a history of adverse reaction precipitated with ASA or other non-steroidal anti-inflammatory drug. If a patient presents with convincing history of repeated drug-induced adverse reactions confirmation of hypersensitivity by provocation test may not be necessary. However, reliability of anamnesis related to adverse reaction to aspirin or other NSAIDs may be limited in patients with AERD (Table 3) and provocation testing is often necessary to confirm/exclude hypersensitivity. In asthmatic patients with negative history of reaction to NSAIDs and/or those who have never been exposed to NSAIDs, the presence of additional risk factors (rhinosinusitis, nasal polyposis, history of near-fatal reactions) increases the risk of AERD and provocation testing may be required. Thus, it is up to the clinician to consider in every individual case if circumstances justify necessity for extended diagnosis to confirm/exclude hypersensitivity to aspirin (Table 4). Recently published EAACI/ GA2LEN guideline may help to standardize diagnostic aspirin challenges performed in a clinical setting [78].

Aspirin Challenge Tests Although oral challenge is the gold standard for the diagnosis of aspirin hypersensitivity, nasal or bronchial provocation with lysine–ASA may be a valuable alternative to confirm/exclude drug hypersensitivity in asthmatic patients [79, 80].

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M.L. Kowalski Table 4 Indications and contraindications to aspirin challenge in patients with asthma Indications for aspirin challenge: 1. Ambiguous or not clear history of response to aspirin/NSAIDs 2. Exacerbation of asthma while on NSAIDs 3. Presence of risk factors but no recent history of safe aspirin intake 4. Beginning of desensitization Contraindications for aspirin challenge: 1. History of severe anaphylactic reactions induced by aspirin or other NSAIDs (as alternative nasal aspirin challenge can be considered) 2. Low baseline FEV1 (below 70% of the predicted value) 3. Not well controlled/not stable asthma 4. Severe disease of the heart, digestive tract, liver, kidney or cancer 5. Respiratory infection within 4 weeks prior to the challenge 6. Pregnancy

Oral Provocation Test Oral aspirin challenge test was introduced to clinical practice in the early 1970s and has been considered the gold standard for the diagnosis of hypersensitivity to aspirin and other NSAIDs [81]. Several protocols for oral aspirin provocation with different dose increments and various intervals between updosing have been developed, but so far none has been universally accepted as a standard [31, 32, 80, 82, 83]. More recently, an expert panel of EAACI/GA2LEN proposed a detailed protocol for oral aspirin challenge which merges experience of several groups and may become standard challenge protocol in the future [78]. Oral challenge should be performed in a hospital setting with immediate access to emergency equipment and service. Oral test is contraindicated if a patient has a history of severe anaphylactic reaction to NSAIDs or suffers from severe disease of heart, lung, kidney or digestive tract. Other contraindications include pregnancy, current treatment with beta-receptor blockers or recent respiratory infections. Before the challenge the patient should have well-controlled asthma with FEV1 exceeding 70% of the predicted values. Drug withdrawal periods recommended before an aspirin challenge are listed in Table 5 and regular treatment with oral corticosteroids should not exceed 10 mg of the prednisolone equivalent. On the first day placebo capsules are administered every 1.5–2 h and FEV1 is measured every 30 min following each challenge to establish baseline variability. On the second day the patient receives initially 10–30 mg of aspirin and the dose is doubled in 1.5–3 h intervals until positive reaction occurs. Currently recommended levels of updosing by EAACI/GALEN expert panel is shown in Table 6. The reaction is considered positive if at least 20% fall in FEV1 is measured and/or extrabronchial symptoms (nasal congestion, rhinorrhea, ocular injection, erythema of the skin, GI symptoms) appear. In most patients the reaction begins within 20–60 min and the likelihood of a positive reaction occurring after 90 min in a positive challenge was estimated to be as low as 5.2% [83].

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Table 5 Drug withdrawal periods recommended before aspirin challenge [78] Short-acting β2-agonists 6 h (if possible 8 h) Long-acting β2-agonists 24 h (if possible 48 h) Long-acting theophilline 24 h (if possible 48 h) Ipratropium bromide 24 h Short-acting antihistamines 3 days Cromolyn sodium 8h Nedocromil sodium 24 h Leukotriene modifiers 24 h Table 6 Protocol for oral aspirin challenge proposed by EAACI/GA2LEN guideline [78] Day 1 (placebo) – FEV1 is measured and the challenge is started if the baseline FEV1 is at least 70% of the predicted value for the patient – Three (or optionally four) capsules of placebo are administered every 1.5–2 h apart – FEV1 is measured every 30 min and the values are allowed to vary by <15% from baseline Day 2 (aspirin) Baseline FEV1 is measured and starting dose is chosen based on the past history of reaction to aspirin (a) History of moderate reaction Starting with 27 mg of aspirin exponentially increasing doses of aspirin (44, 117, 312 mg) are administered every 1.5–2 h until a cumulative dose of 500 mg is reached (b) History of severe reaction (severe dyspnea and/or anaphylactic shock) The test is started with 10 mg of aspirin and the next dose 17 mg is administered 1.5–2 h later, (i.e. the dose 27 mg is divided into two doses for safety reasons) and than 44, 117 and 312 mg are administered

When the positive reaction occurs the symptoms should be treated with nebulization of albuterol, intravenous antihistamine (e.g. clemastine) and oral or intravenous glucocorticosteroids to prevent development of delayed symptoms. Since late recurrence of respiratory symptoms may occur, if possible, the patient after positive oral reaction can be hospitalized for 24 h. Alternatively, the patient should stay in the clinic for at least 1–2 h after the completion of the challenge and can be discharged only after FEV1 returns close to baseline (at least 90% of prechallenge values). On discharge, the patient should receive peak flow metre and is instructed to monitor PEF every 2–3 h until late evening and then the next morning. Provocation aspirin dose inducing a 20% fall in FEV1 (PD20ASA) can be also calculated using the lowest FEV1 measured after the challenge. However, since in most cases the developing reaction is interrupted with bronchodilators the usefulness of PD20 calculations for oral aspirin challenge remains questionable. If the final dose 312 mg of aspirin (corresponding to 500 mg of cumulative dose) is ingested and well tolerated the patient is considered to be aspirin-tolerant. The negative challenge result excludes the presence of aspirin hypersensitivity but may also reflect development of ‘silent desensitization’ during the challenge procedure or sometimes blocks effects of medications used to control chronic inflammatory diseases [84, 85]. The use of asthma controller therapy (particularly

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antileukotrienes, but also long-acting β2-agonists and inhaled steroids) may alleviate the reaction and shift the response during aspirin challenge from bronchial (asthmatic symptoms and fall in FEV1) to nasocular reactions [86]. Similarly, the use of antihistamines may significantly decrease development not only of cutaneous but also respiratory symptoms thus increasing the rate of false negative aspirin challenge results. However, it is not advisable to discontinue controller medications before the aspirin challenge in patients with suspected. ASA hypersensitivity since it may increase the risk of severe reactions [87, 88]. Maintaining a routine anti-inflammatory treatment may prevent development of severe reactions even in patients with a history of hospitalization, admission to the intensive care unit or intubation after unintended use of ASA or NSAIDs [89]. Diagnostic oral challenge tests although the gold standard for confirmation of ASA-sensitivity, is a time-consuming procedure requiring experienced personnel and bearing the risk of severe systemic reaction. In certain situations oral provocation test may be substituted with alternative inhaled or intranasal aspirin challenges.

Bronchial Provocation Test Inhalation challenge with lysine–aspirin (a soluble form of acetylsalicylic acid) was introduced by Bianco et al. [90] in 1977 and can currently be considered the test of choice to confirm/exclude aspirin sensitivity in patients with AERD. According to the EAACI/GA2LEN protocol, following inhalation of diluent increasing doses of lysine–aspirin are inhaled by means of a dosimeter in 30 min intervals allowing to complete the procedure in an outpatient setting within less than 5 h [78]. The test is considered positive, and stopped if at least 20% drop in FEV1 is recorded at 10, 20 or 30 min after inhalation. If the positive reaction is observed PD20 ASA is calculated from the dose–response curve. Inhalation challenge in some patients induces also extrabronchial symptoms which are related to systemic recruitment of inflammatory cells progenitors from bone marrow [91]. Despite earlier observations inhaled challenge with lysine– aspirin may also induce late phase reaction, thus proper management of the early reaction and monitoring of patient at home with peak-flow metre for up to 24 h after the challenge is necessary. Generalized severe reaction following inhalation of lysine–aspirin was reported indicating that even this type of challenge is associated with a potential risk of dangerous reaction and should be performed in centres with experienced staff and with proper emergency assistance [92]. As compared to oral challenge, inhalation test is faster and safer to perform and the reaction is usually easily reversible with nebulized β2-agonists. In general, the management of patients after positive reaction occurred should be similar to that described after oral aspirin challenge. According to Dahlen and Zetterstrom [93] both tests have similar sensitivity and specificity (90%), and a study by Nizankowska et al. [79] demonstrated slightly lower sensitivity of the bronchial inhalation test (77% vs 89%) and identical

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specificity of both challenges (93%). These data confirm that aspirin inhalation test can be recommended as the standard diagnostic procedure in patients with AERD.

Nasal Provocation Test In some circumstances oral or inhaled aspirin provocation test can be substituted with intranasal aspirin provocation which is rapid and safe and can be performed in an outpatient setting. It is particularly recommended that asthmatic patients with low pulmonary function are not suitable for bronchial provocation. Before the test is performed careful rhinologic examination should exclude the presence of severe nasal pathology (e.g. prominent nasal polyposis, nasal septum deviation or septal perforation). Nasal provocation test with lysine aspirin is a reliable tool to diagnose hypersensitivity to aspirin providing the severe nasal obstruction is not present before the challenge and the clinical symptoms are combined with the objective and standardized technique of airflow measurement for assessment of the results [94]. Either acoustic rhinometry, active anterior rhinomanometry or peak nasal inspiratory flow measurement can be used for objective assessment of nasal obstruction during the challenge. The appearance of clinical symptoms (rhinorrhea, nasal congestion, sneezing, itching of the nose, palate or throat) should be recorded before and after each dose of aspirin and the intensity of clinical symptoms may be quantified with a visual analogue scale. Since intranasal aspirin challenge may also result in bronchoconstriction it is recommended that lower respiratory function is assessed by PEF or spirometry at the same time points as nasal function. Saline followed by increasing concentrations of lysine–aspirin solution are applied into each nostril via an Eppendorf pipette every 30 min with the patient’s head tilted back for 1 min, and all parameters (objective and subjective) are recorded every 10 min. The reaction is considered positive if clinical symptoms appear and/or a significant nasal obstruction is objectively documented. A 25% decrease in total nasal flow measured by acoustic rhinometry or 40% drop of inspiratory nasal flow as compared to baseline by rhinomanometry or by nasal peak flow metre demonstrates positive reaction. In experienced hands the sensitivity of intranasal aspirin provocation may exceed 80% and specificity is close to 95%, thus approaching performance of bronchial challenge [95–97]. Some limitations of this route of challenge are related to patients with significant nasal obstruction, turbulent nasal flow or unspecific nasal responsiveness.

Intravenous Challenge Test Intravenous challenge with indomethacin to diagnose aspirin hypersensitivity was proposed by Martelli [98]. Bronchial reaction started within 4–18 min after

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intravenous administration and the time-course of the reaction was similar to that after bronchial indomethacin challenge with maximum FEV1 decrease after 1 h. Dypirone (metamizol, a pyrazolone derivative) has been used by a Japanese group to substitute for oral/inhaled provocation to confirm diagnosis of aspirin hypersensitivity [99]. However, metamizol is not only strong cyclooxygenase-1 inhibitor mimicking the effect of aspirin but is also highly immunogenic and in some patients may evoke non-crossreactive, most likely IgE-mediated systemic reactions [100, 101]. A soluble form of acetylsalicylic acid, lysine–aspirin infused intravenously has been also employed to evoke bronchoconstriction in ASA-hypersensitive asthmatics [102]. More recently, intravenous route of lysine–aspirin challenge was used for the diagnosis and initiation of aspirin desensitization in patients with AERD [103]. Although the authors claim safety and convenience of intravenous challenge with NSAIDs the sensitivity, specificity and overall practicability of these challenge route have to be assessed.

Testing for Tolerance of Alternative NSAIDs Other NSAIDs like naproxen, ibuprofen or ketoprofen, which are strong cyclooxygenase inhibitors with dominant preferential activity towards COX-1, may precipitate adverse symptoms in 30–80% of ASA-sensitive patients. Thus, history of adverse reaction to aspirin should prompt a strict avoidance of other NSAIDs with known moderate to strong cyclooxygenase inhibitory activity. Once ASA hypersensitivity is established, an alternative NSAID should be recommended to the patient. Even if no immediate indications for treatment with NSAIDs exist the doctor should anticipate the potential patient’s need for analgesic, antipyretic of anti-inflammatory treatment in the future [104]. NSAIDs with weaker inhibitory potency towards prostaglandin synthase (e.g. acetaminophen) and selective COX-2 inhibitors seem to be tolerated by majority of ASA-sensitive asthmatics. In clinical practice acetaminophen can be recommended for patients with asthma and NSAIDs hypersensitivity in moderate doses up to 1,000 mg. However, because in some patients even lower doses may evoke adverse reactions, oral tolerance test should be performed with 500 mg of acetaminophen before the drug is recommended. Preferential COX-2 inhibitors (nimesulide, meloxicam) are tolerated by a majority but not all hypersensitive patients and oral challenge test before administration is necessary. Most asthmatic patients with history of isolated bronchial reaction to aspirin or other NSAIDs can safely use selective COX-2 inhibitors (e.g. celecoxib). If the treatment with alternative NSAID is indicated and the choice of the drug with moderate or low preference for COX-1 has been made it seems reasonable to administer the first dose of the drug in the doctor’s office and watch the patient for 1–2 h. While planning the challenge with alternative drug one should bear in mind that a positive reaction to aspirin (both oral or inhaled) is followed by a state of

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refractoriness to further dosing of these drugs and full sensitivity reappears about 5–7 days after exposure [80].

In Vitro Tests for Diagnosis of ASA Hypersensitivity As in patients with AERD-specific antibodies to aspirin and NSAIDs were not found and immunological mechanism of hypersensitivity was excluded, since the 1970s, attempts have been made to develop an in vitro cell activation test to identify sensitive patients. Several cell activation tests which were proposed and tested (e.g. platelet activation tests or histamine release assay) turned out to be unreliable to distinguish aspirin/NSAIDs sensitive from insensitive individuals [105, 106]. More recently three in vitro tests measuring aspirin-specific peripheral blood leukocytes activation have been proposed for the diagnosis of aspirin hypersensitivity.

Sulfidoleukotrienes Release Assay Several studies demonstrated increased aspirin-triggered release of LTC4 from peripheral blood leukocytes from patients with AERD. The difference between leukotrienes release from ASA-sensitive and ASA-tolerant leukocytes were always quantitative and the sensitivity was usually low and varied between 18% and 82% while specificity approached 99% in some studies [107–110]. However, these studies involved small numbers of patients; only highly selected populations of sensitive asthmatics were tested and adequate control groups were lacking. The performance of sulphidoleukotriene test could be further improved if in addition to aspirin other NSAIDs were tested and the combined results analyzed [110, 111]. However, completely negative studies showing mostly non-specific release of leukotrienes from PBL upon stimulation with aspirin were also reported [39, 64, 112–116]. Taken together neither non-commercial nor commercial methods measuring aspirin-induced sulphidofleukotrienes release form PBL cannot be considered at present as a reliable tool to exclude or confirm hypersensitivity to ASA.

Basophil Activation Test Following earlier tests employing measurement of histamine release, more recently flow-assisted basophil activation test (BAT) relying upon anti-IgE to characterize basophils and anti-CD63 to assess activation of these cells were developed. Measurement of cell surface molecule CD63 expression upon in vitro challenge has been used for diagnosis of various hypersensitivities, including hypersensitivity

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to aspirin [117]. For metamizol (a pyrazolon derivate) which mostly induces skin reactions sensitivity and specificity of the test was 42% and 100%, respectively [118]. When the sensitivity for the different NSAIDs was assessed it varied from 15% to 55% with specificity generally exceeding 90%. In study of Sanz et al. [111] the commercially available test (Flow Cast; Buhlmann) resulted in sensitivity of 41% and specificity 100% with positive and negative predictive values approaching 100%. However, in these studies, heterogeneous population of patients with both respiratory and/or cutaneous type of hypersensitivity to different NSAIDs was analyzed, and the control population was not clinically characterized. Furthermore, a study not confirming that BAT may be reliable for the diagnosis of hypersensitivity to NSAID has also been published [119]. Studies in larger and more clearly defined patients’ population will be necessary to estimate the real performance of this test to diagnose AERD

HETE Generation Assay (ASPITest) It has been demonstrated that aspirin could trigger in vitro generation of arachidonic acid metabolite acid (15-HETE) from nasal polyp epithelial cells, and peripheral blood leukocytes from ASA-sensitive patients but not ASA-tolerant asthmatics or healthy subjects. Based on this observation measurement of 15-hydroxyicosatetrenoic (15-HETE) release from PBL has been proposed as Aspirin Sensitive Patient Identification Test (ASPITest®). In contrast to other in vitro tests ASPITest within the range of aspirin concentrations tested demonstrated mostly qualitative and not only quantitative differences between ASA-sensitive and ASA-tolerant asthmatics [39, 40]. Aspirin triggered 15-HETE release from PBL mimicked to some extent adverse reaction to aspirin observed in vivo in patients with AERD: in some patients release of 15-HETE could be triggered by another non-selective cyclooxygenase inhibitors naproxen or ibuprofen, but COX-2 selective NSAID celecoxib did not affect the release. The sensitivity of aspirin-triggered 15-HETE release tested in highly selected population of patients with AERD was 82% and the specificity was 83% with negative and positive predictive values 0.79 and 0.86, respectively [120]. These data indicate that measurement of 15-HETE release from PBL may be useful for detection of ASA hypersensitivity in asthmatic patients.

Prospect for New Diagnostic Measures Based on Biochemical and Molecular Abnormalities in AERD Several biochemical abnormalities may be detected in AERD patients although they are distinctive only for some but not all ASA-sensitive patients. Most of these abnormalities are related to arachidonic acid metabolism and can be detected in easily available biological materials like blood, urine or exhaled air.

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However, diagnostic value of these abnormalities to predict or confirm ASA-sensitivity have not been assessed, thus at present they cannot be recommended for clinical routine. The most characteristic abnormalities have been listed in (Table 7). Increased basal levels of leukotriene E4 in urine has been suggested to be most specific for AERD. LTE4 can be easily detected even in non-purified samples of urine by ELISA, although mass spectrometry method seems to be more sensitive [54, 79]. Urinary LTE4 excretion was consistently found to be increased in ASAsensitive patients as compared to ASA-tolerant asthmatic, even in the absence of aspirin challenge and the mean levels of LTE in urine of ASA-sensitive asthmatics were one- to twofold higher in sensitive patients [35, 36, 54, 121]. However, a significant overlap with non-sensitive asthmatics has been usually observed, thus not allowing for using a single urine LTE4 measurement to predict ASA-sensitivity [121, 122]. Furthermore, increased leukotrienuria has been associated with the presence and intensity of chronic hyperplastic rhinosinusits and polyposis in both ASA-sensitive and ASA-tolerant patients suggesting that basal levels of urinary cysteinyl-leukotrienes reflect severity of airway inflammation as expressed, for example, by increased number of tissue eosinophil and possibly mast cell numbers [123]. A significant decrease in urinary leukotrienes observed following nasal sinus surgery confirms that urinary leukotrienes reflect an overall increase in the number of leukotriene-producing cells in different tissues rather than AERDspecific biochemical abnormality. The differences in basal levels of cysteinyl LTs between ASA-hypersensitive and ASA-tolerant patients are less consistently observed in other organ compartments (see below).

Table 7 Baseline arachidonic acid abnormalities in patients with AERD Peripheral Abnormality blood cells Urine Exhaled airTissue

Reference

Decreased production of +/− * No NPEC+ [39, 50, 53, 54, 56, PGE2 108, 109] Decreased expression of * * * NP+ [51] PGE2 receptors Decreased expression No * * NP+ BM ? [48, 49] cyclooxygenase-2 Increased genera+/− + +/− NP+/− [35, 36, 53, 54, 59, tion of cysteinyl60, 122, 123] leukotrienes Increased expression of * * * NP+ [61] LT1 receptors LTC4 synthase * * * BM+ NP+ [55, 60] Decreased lipoxins + * * NPEC+ [60, 64] generation +well documented; −/+ not conclusive; * not relevant or information not available NP – nasal polyps; NPEC – nasal polyp epithelial cells; BM – bronchial mucosa

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Predictive value of other AA metabolism abnormalities (listed in Table 7) for aspirin sensitivity diagnosis is still speculative and far from being useful for clinical routine. New but still unexplored area of AERD diagnosis is related to possible genetic background of aspirin hypersensitivity. Although several single nucleotide polymorphisms (SNPs) were associated with hypersensitivity to aspirin the power of these associations is rather weak and does not allow their usage for detection of AERD [124]. Nevertheless, the predictive value of sets of polymorphisms remains to be determined.

Conclusions Aspirin Exacerbated Respiratory Disease is a distinct phenotype of bronchial asthma with increased risk of uncontrolled disease and severe upper airway inflammation. Confirmation of aspirin hypersensitivity and recommendation of alternative NSAID is important. The standard for the diagnosis of ASA hypersensitivity remains a combination of a positive history and placebo-controlled aspirin challenge by oral, bronchial or nasal route. Newly developed in vitro tests require further investigations and validation before becoming acceptable tools for confirming the presence of aspirin hypersensitivity.

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71. Kowalski -SCF Kowalski ML, Lewandowska-Polak A, Wozniak J, Ptasinska A, Jankowski A, Wagrowska-Danilewicz M, Danilewicz M, Pawliczak R (2005) Association of stem cell factor expression in nasal polyp epithelial cells with aspirin sensitivity and asthma. Allergy 60:631–637 72. Pawankar R, Lee KH, Nonaka M, Takizawa R (2007) Role of mast cells and basophils in chronic rhinosinusitis. Clin Allergy Immunol 20:93–101 73. Bochenek G, Nagraba K, Nizankowska E, Szczeklik A (2003) A controlled study of 9 alpha, 11beta-PGF2 (a prostaglandin D2 metabolite) in plasma and urine of patients with bronchial asthma and healthy controls after aspirin challenge. J Allergy Clin Immunol 111:743–749 74. Wang XS, Wu AY, Leung PS, Lau HY (2007) PGE suppresses excessive anti-IgE induced cysteinyl leucotrienes production in mast cells of patients with aspirin exacerbated respiratory disease. Allergy 62:620–627 75. Hamilos DL, Leung DYM, Wood R, Cunninghan L, Bean DK, Yasruel Z, Schotman E, Hamid Q (1995) Evidence for distinct cytokine expression in allergic versus nonallergic chronic sinusitis. J Allergy Clin Immunol 96:537–544 76. Bachert C, Wagenmann M., Hauser U, Rudack C (1997) IL-5 synthesis is upregulated in human nasal polyp tissue. J Allergy Clin Immunol 99:837–842 77. Mascia K, Borish L, Patrie J, Hunt J, Phillips CD, Steinke JW (2005) Chronic hyperplastic eosinophilic sinusitis as a predictor of aspirin-exacerbated respiratory disease. Ann Allergy Asthma Immunol 94:652–657 78. Nizankowska-Mogilnicka E, Bochenek G, Mastalerz L, Swierczynska M, Picado C, Scadding G, Kowalski ML, Setkowicz M, Ring J, Brockow K, Bachert C, Wohrl S, Dahlen B, Szczeklik A (2007) EAACI/GA2LEN guideline: aspirin provocation tests for diagnosis of aspirin hypersensitivity. Allergy 62:1111–1118 79. Nizankowska E, Bestyska-Krypel A, Cmiel, A. Szczeklik, A (2000) Oral and bronchial provocation tests with aspirin for diagnosis of aspirin-induced asthma. Eur Respir J 15:863–869 80. Melillo G, Balzano G, Bianco S, Dahlen B, Godard P, Kowalski ML, Picado C, Stevenson DD, Suetsugu S, Cheira C, Cocco G, Melillo E, Patriarca G, Vaghi A, Zanon P (2001) Oral and inhalation provocation tests for the diagnosis of aspirin-induced asthma. Allergy 56:899–911 81. McDonald JR, Mathison DA, Stevenson DD (1972) Aspirin intolerance in asthma. Detection by oral challenge. J Allergy Clin Immunol 50:198–207 82. Stevenson DD (2000) Approach to the patient with a history of adverse reactions to aspirin or NSAIDs: diagnosis and treatment. Allergy Asthma Proc 21:25–31. Review 83. Cormican LJ, Farooque S, Altmann DR, Lee TH (2005) Improvements in an oral aspirin challenge protocol for the diagnosis of aspirin hypersensitivity. Clin Exp Allergy 35:717–722 84. Szmidt M, Grzelewska-Rzymowska I, Kowalski ML, Rozniecki J (1987) Tolerance to acetylsalicylic acid (ASA) induced in ASA-sensitive asthmatics does not depend on initial adverse reaction. Allergy 42:182–185 85. Stevenson DD, Simon RA, Mathison DA, Christiansen SC (2000) Montelukast is only partially effective in inhibiting aspirin responses in aspirin-sensitive asthmatics. Ann Allergy Asthma Immunol 85:477–482 86. White AA, Stevenson DD, Simon RA (2005) Related articles, links the blocking effect of essential controller medications during aspirin challenges in patients with aspirin-exacerbated respiratory disease. Ann Allergy Asthma Immunol 95:330–335 87. Berges-Gimeno MP, Simon RA, Stevenson DD (2002) The effect of leukotriene-modifier drugs on aspirin-induced asthma and rhinitis reactions. Clin Exp Allergy 32:1491–1496 88. White A, Ludington E, Mehra P, Stevenson DD, Simon RA (2006) Effect of leukotriene modifier drugs on the safety of oral aspirin challenges. Ann Allergy Asthma Immunol 97:688–693 89. Williams AN, Simon RA, Woessner KM, Stevenson DD (2007) The relationship between historical aspirin-induced asthma and severity of asthma induced during oral aspirin challenges. J Allergy Clin Immunol 120:273–277 90. Bianco SR, Robuschi M, Petrini G (1977) Aspirin induced tolerance in aspirin asthma detected by a new challenge test. IRCS J Med Sci 5:129

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111. Sanz ML, Gamboa P, de Weck AL (2005) Related articles, links A new combined test with flowcytometric basophil activation and determination of sulfidoleukotrienes is useful for in vitro diagnosis of hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs. Int Arch Allergy Immunol 136:58–72 112. Pierzchalska M, Mastalerz L, Sanak M, Zazula M, Szczeklik A (2000) A moderate and unspecific release of cysteinyl leukotrienes by aspirin from peripheral blood leucocytes precludes its value for aspirin sensitivity testing in asthma. J Clin Exp Allergy 30:1785–1791 113. Lebel B, Messaad D, Kvedariene V, Rongier M, Bousquet J, Demoly P (2001) Cysteinylleukotriene release test (CAST) in the diagnosis of immediate drug reactions. Allergy 56:688–692 114. Abrahamsen O, Haas H, Schreiber J, Schlaak M (2001) Differential mediator release from basophils of allergic and non-allergic asthmatic patients after stimulation with anti-IgE and C5a. Clin Exp Allergy 31:368–378 115. Pierzchalska M, Mastalerz L, Sanak M, Zazula M, Szczeklik A (2000) A moderate and unspecific release of cysteinyl leukotrienes by aspirin from peripheral blood leucocytes precludes its value for aspirin sensitivity testing in asthma. J Clin Exp Allergy 30:1785–1791 116. Gray PA, Warner TD, Vojnovic I, Del Soldato P, Parikh A, Scadding GK, Mitchell JA (2002) Effects of non-steroidal anti-inflammatory drugs on cyclo-oxygenase and lipoxygenase activity in whole blood from aspirin-sensitive asthmatics vs healthy donors. Br J Pharmacol 137:1031–1038 117. Gamboa P, Sanz ML, Caballero MR, Urrutia I, Antepara I, Esparza R, de Weck AL (2004) The flow-cytometric determination of basophil activation induced by aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) is useful for in vitro diagnosis of the NSAID hypersensitivity syndrome. Clin Exp Allergy 34:1448–1457 118. Gamboa PM, Sanz ML, Caballero MR, Antepara I, Urrutia I, Jauregui I, Gonzalez G, Dieguez I, de Weck AL (2003) Use of CD63 expression as a marker of in vitro basophil activation and leukotriene determination in metamizol allergic patients. Allergy 58:312–317 119. Erdmann SM, Ventocilla S, Moll-Slodowy S, Sauer I, Merk HF (2005) Basophil activation tests in the diagnosis of drug reactions. Hautarzt 56:38–43 120. Kowalski ML, Ptasinska A, Jedrzejczak M, Bienkiewicz B, Cieslak M, Grzegorczyk J, Pawliczak R, Dubuske L (2005) Aspirin-triggered 15-HETE generation in peripheral blood leukocytes is a specific and sensitive Aspirin-Sensitive Patients Identification Test (ASPITest). Allergy 60:1139–1145 121. Higashi N, Taniguchi M, Mita H, Osame M, Akiyama K (2002) A comparative study of eicosanoid concentrations in sputum and urine in patients with aspirin-intolerant asthma. Clin Exp Allergy 32:1484–1490 122. Misso NL, Aggarwal S, Phelps S, Beard R, Thompson PJ (2004) Urinary leukotriene E4 and 9 alpha, 11 beta-prostaglandin F concentrations in mild, moderate and severe asthma, and in healthy subjects. Clin Exp Allergy 34:624–631 123. Higashi N, Taniguchi M, Mita H, Kawagishi Y, Ishii T, Higashi A, Osame M, Akiyama K (2004) Clinical features of asthmatic patients with increased urinary leukotriene E4 excretion (hyperleukotrienuria): involvement of chronic hyperplastic rhinosinusitis with nasal polyposis. J Allergy Clin Immunol 113:277–283 124. Kim SH, Park HS (2006) Genetic markers for differentiating aspirin-hypersensitivity. Yonsei Med J 47:15–21

Food Allergy: Diagnosis of Food Allergy Scott H. Sicherer

Introduction The most definitive test available to determine an adverse reaction to food is the physician-supervised oral food challenge (OFC). This test, in particular when performed in a double-blind, placebo-controlled (DBPC) manner, can confirm or refute adverse reactions or immunologic (allergic) reactions to foods. However, the test is labor-intensive and carries risks; therefore, ancillary tests are used to determine if a food allergy is likely. The primary tests include the clinical history, physical examination, serum and prick skin tests (PSTs) to detect IgE antibodies to particular suspected foods, and additional tests that are emerging with regard to their diagnostic value. The clinician must consider disease pathogenesis, epidemiology of food-related disorders, and the specific clinical history to appropriately select and interpret allergy tests. Table 1 shows essential factors to consider when proceeding toward the diagnosis of food allergy. Adverse reactions to foods may result from immunologic (food allergy) and non-immunologic responses [1, 2]. More than one in five persons alter their diet for a presumed food allergy, but nonimmune causes can often be identified [3]. Toxins or pharmacologically active components of the diet account for a number of nonimmune adverse reactions. The term hypersensitivity has been applied to exaggerated responses to any food component, even nonimmune reactions [1]. Therefore, lactose intolerance, resulting in abdominal discomfort from a reduced ability to digest lactose, may be defined as non-allergic hypersensitivity. A general overview of the approach to diagnosis is shown in Fig. 1.

S.H. Sicherer () The Elliot and Roslyn Jaffe Food Allergy Institute, Division of Allergy and Immunology, Department of Pediatrics, Mount Sinai School of Medicine, New York, NY, USA; Division of Allergy/Immunology, Mount Sinai Hospital, Box 1198, One Gustave L. Levy Place, New York, NY 10029-6574, USA e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_21, © Springer 2009

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Table 1 Clinical factors to consider when approaching the diagnosis of food allergy Factors Pathophysiology Intolerance (nonallergic hypersensitivity) Toxins Food allergy (immune responses) Neurologic and psychological/ psychiatric Pathophysiology of specific disorders

Epidemiology Common foods to cause severe reactions Common early childhood food allergies that usually resolve Associations of food allergy

Personal history

Examples Lactase deficiency, sensitivity to caffeine Food poisoning IgE-mediated, not IgE-associated (cellular), mixed IgE/non-IgE Anorexia nervosa, gustatory rhinitis (vasomotor rhinitis) IgE associated: Urticaria/angioedema, immediate gastrointestinal reaction (GI anaphylaxis), oral allergy syndrome (pollen-related), rhinitis, asthma, anaphylaxis, food-associated/exercise-induced anaphylaxis Non-IgE associated: Dietary protein enterocolitis, dietary protein proctitis, dietary protein enteropathy, celiac disease, dermatitis herpetiformis, contact dermatitis, pulmonary hemosiderosis Mixed IgE/non-IgE: Atopic dermatitis, eosinophilic gastroenteropathies Peanut, tree nuts, seafood, seeds Egg, milk, wheat, soybean Common: Anaphylaxis, acute urticaria Uncommon: Chronic asthma, chronic rhinitis, chronic urticaria Foods tolerated or suspected to cause a reaction, features of reactions, consistent/inconsistent tolerance, results of elimination

Clinical History The medical history is the most important diagnostic “test.” From a careful history, the clinician must determine whether the complaints are likely to be associated with food allergy, intolerance or toxic effects, or not related to foods. Furthermore, the history should reveal the likely pathophysiology of an adverse food response. For example, acute reactions are likely IgE-antibody mediated while chronic conditions and many gastrointestinal food allergic reactions may not be associated with IgE. The physical examination may confirm atopic dermatitis, growth problems, urticaria, and other symptoms of atopic disease. A careful history should focus upon: the symptoms attributed to food ingestion (type, acute vs. chronic), the food(s) involved, consistency of reactions, the quantity of food required to

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History/Physical examination -determine IgE/Not IgE associated -isolate suspect foods -consider non-immune causes

History consistent with intolerance or non-immune disorder

No IgE tests, pursue confirmatory tests, as indicated.

History consistent with nonIgE allergy

Confirmatory diagnostic tests if indicated (e.g., endoscopy)

Elimination diets and oral food challenges to confirm/identify triggers, if indicated

Atopic dermatitis, Eosinophilic gastroenteropathy

Likely IgE-mediated disorder

Tests for foodspecific IgE to suspect foods* Limited tests based upon history and epidemiology. Elimination diets and oral challenges to confirm, as indicated (see text).

Elimination of suspect food. Oral food challenge if history and test results are not otherwise adequately conclusive (see text)

Fig. 1 General diagnostic scheme. The history is the key starting point toward diagnosing a food allergy. This illustration provides a broad framework; see text for additional explanations. * Certain test results may be intrinsically likely to reflect clinical allergy (see text).

elicit symptoms, the timing between ingestion and onset of symptoms, the most recent reaction/patterns of reactivity, the manner in which the food was prepared (raw, cooked), potential contamination with allergens already identified as problematic for the patient, and any ancillary associated activity that may play a role (i.e., exercise, alcohol ingestion). Diet diaries may aid in disclosing specific details of importance. Consistent reactions raise prior probability that a suspect food is causal. If a food is an infrequent part of the diet, it is more likely a culprit than a food tolerated often. Attention must be paid to ancillary activities that can affect allergic outcomes such as exercise, or ingestion of medications such as nonsteroidal anti-inflammatory agents [4]. The importance of historical details is illustrated in the evaluation of fish allergy. A reaction to fish could be caused by scombroid

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poisoning (a toxin that results in symptoms that mimic anaphylaxis), or could be caused by an allergic reaction that may vary according to the method of heating [5], use of antacids that reduce digestion [6–8], the part of the fish consumed [9, 10], or could be attributed to fish parasites [11–14].

Tests for IgE Antibodies Both prick skin tests (PSTs) and serum IgE determinations are sensitive modalities to detect food-specific IgE antibodies. These tests detect sensitization, the presence of IgE to the tested protein, but this state may not necessarily reflect a current state of clinical allergy. Of course, for disorders where IgE is not central to the pathophysiology, a negative test does not exclude an allergy.

Prick Skin Tests For determination of food-specific IgE antibodies prick skin tests (PSTs), performed using a probe to introduce food protein into the superficial skin layer, are generally sensitive (~75–95%) and specific (~30–60%) with regard to clinical allergy in the context of a suspected disease [2]. Intradermal skin tests should not be used for evaluation of food allergy, because they are overly sensitive and may cause systemic allergic reactions. Though commercial extracts are available for performing prick skin tests for many foods, fresh extracts, particularly when testing fruits and vegetables whose proteins are prone to degradation, may be more sensitive [15]. PSTs are typically considered positive if there is a mean wheal diameter of 3 mm or greater, after subtraction of the saline control. However, additional diagnostic information may be learned by considering the actual size of the wheal response. Increasingly larger wheal diameters are associated with increasingly higher chances that the test reflects clinical reactions [16–18]. It has been observed by some investigators that particularly large PSTs may have 100% positive predictive value. This concept was demonstrated in a study [19] showing that for young infants, reactions to egg, milk, and peanut were certain to occur if the skin test wheal was ≥8 mm for cow’s milk and peanut and ≥7 mm for egg. These investigators [17] also evaluated children 4 months to 19 years of age referred for suspected nut allergy with a single lancet technique and commercial extracts (except whole food for sesame and pistachio) and compared skin test sizes to food challenge outcomes. Positive challenges were associated (>95% accuracy) with wheal sizes equal to or greater than 8 mm for cashew, hazel, walnut, and sesame. Correlation was poor for almond, pistachio, pecan, and Brazil nut, though fewer subjects were tested. Studies from various countries evaluating children of different ages and sometimes using different techniques (type of skin test probe, commercial versus raw

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extracts, methods of measuring wheal sizes) present varying predictive information from skin test wheal sizes [16, 18–20]. For example, highly predictive (~100%) wheal size for milk allergy was 8 mm for children with a median age of 3 years but 6 mm for children under age 2 [19]. For egg allergy, a wheal of 13 mm in a German study was associated with a 95% predictive value in children under 2 years of age, [20] while in an Australian study a wheal of 5–7 mm was 100% predictive [19]. These discrepancies could reflect subtle differences in patient selection, test materials, test interpretation, and factors used to decide the outcome of an oral food challenge. Studies also point out that reactions occur despite negative tests (~15%) [16, 18, 19]. Though PSTs introduce allergen, they are typically considered of low risk with regard to inducing systemic reactions. In a review of a database of 34,905 skin tests to foods in 1,138 patients, the systemic reaction rate was 0.008% and there were no severe reactions [21]. Devenney et al. [22] identified six infants with generalized reactions representing a rate of 521 per 100,000 tested children or 6,522 per 100,000 tested infants. All of the reactions identified were in infants under 6 months of age and they were tested with fresh foods rather than extracts. There is one fatality associated with prick skin testing; an adult with food allergy and with a recent asthma exacerbation was tested to 90 foods at one time and experienced a fatal respiratory arrest [23]. In general, these studies support the notion that PSTs are low risk but caution is needed for infants, use of undiluted extracts, and to avoid excessive numbers of tests. In summary, the tests have high utility because of their accuracy, safety, low cost, and prompt results.

Serum Food-Specific IgE Another means to detect food-specific IgE antibody is commonly referred to as the radioallergosorbent test (RAST), although the term is antiquated because modern assays typically do not use radiolabels. In a limited number of studies of a few foods in infants and/or children, diagnostic values associated with very high (≥95%) predictive values for reactions have been determined, though not universally confirmed. Studies determining the concentration of specific IgE antibody measured using a particular method (CAP-RAST FEIA® or UniCap® or ImmunoCAP® reported in kUA/l) showed a food-specific IgE concentration of ≥7 kUA/l to egg, ≥15 to milk, and ≥14 to peanut were 95% predictive for a reaction among 5-year-old children [24]. For walnut, a level of 18.5 kUA/l or greater was determined to have 95% predictive probability [25]. In another US study, about half of children reacted when levels to egg or milk were 2 kIU/l [26]. These results are not always applicable to different study populations [27]. For children under age 2 years, the values where most reacted were lower (e.g., ≥2 kUA/l for egg or milk) [28]. In a German study [29] 95% predictive value for egg was 15.9 kIU/l. In a large study of infants in Japan, the food-specific IgE concentration indicating a 95% risk of reaction were generally higher than previous studies but varied with age: for egg allergy,

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among children less than 1 year of age the value was 13.0 kUA/l and for 2 years or older it was 30.0 kUA/l [30]. It must be emphasized that diagnostic concentrations are undetermined for many foods, allergic disorders, and age groups. Additionally, reports of kUA/l from different test manufacturers may not be equivalent [31]. Foodspecific IgE typically declines, as a food allergy resolves [32, 33].

Additional Tests Additional diagnostic tests are being investigated. The Atopy Patch Test (APT), which is performed by placing the food allergen on the skin under occlusion for 48 h and assessing for a delayed rash at 24–72 h after removal, shows some promise for non-IgE-mediated disorders [4, 34]. In one study of children with atopic dermatitis [35], the APT had the highest positive predictive value for allergy to egg and milk compared to PST or serum food-specific IgE for both immediate and delayed reactions. However, the results vary widely among workers since much lower positive predictive accuracy (40–63%) and specificities (0.71–0.87) have been reported [36]. Spergel et al. [37, 38] showed utility of this test for detection of food allergies in children with eosinophilic esophagitis and food protein-induced enterocolitis syndrome, but confirmation by other investigators is needed [39]. In an analysis of 873 oral food challenges in children, Mehl et al. [29] noted that including the APT in addition to PST and serum resulted in excluding the need for oral food challenges in only 6–14% (considering 95% predictive values of the tests). There are also a host of tests that have been touted for the diagnosis of food allergy, but have never been found useful in blinded studies. These include measurement of IgG4 antibody, provocation–neutralization (drops placed under the tongue or injected to diagnose and treat various symptoms), and applied kinesiology (muscle strength testing) [4].

Elimination Diets In chronic disorders such as atopic dermatitis, eosinophilic gastroenteropathies or asthma, it is more difficult to pinpoint causal food(s) [40]. The history is helpful, but since these disorders have a waxing and waning course, and considering limitations in diagnostic laboratory tests, false-associations to food ingestion are common. The evaluation of these disorders may require a period of dietary elimination to observe for symptom resolution. This period of trial diet requires selection of foods to be eliminated (based usually on history, test results, epidemiology of the disorder, etc.) without confounding treatments such as steroid medications. Chronic symptoms should resolve during a period of elimination and, if they do, oral food challenges may be needed to determine which food(s) were causing the chronic symptoms. If symptoms do not resolve, then the eliminated foods are not

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likely causal. Elimination of foods to which IgE antibodies are demonstrable, but to which acute reactions are not observed, may carry a risk of loss of a desensitized state, where reintroduction later can trigger more evident acute reactions (e.g., urticaria, anaphylaxis) [41, 42]. The frequency of this occurrence is unknown and the length of time for elimination to warrant this concern is currently unclear.

Oral Food Challenges Oral food challenges are typically undertaken to identify a causal food when allergy is otherwise unclear (e.g., tests are equivocal or pathophysiologically irrelevant), or to monitor for resolution of an allergy. OFC is usually the only means to evaluate disorders or complaints where ancillary tests are irrelevant. For example, the evaluation of reactions to food dyes and preservatives usually requires oral food challenges. Food challenges can also be used to evaluate medical complaints associated by a patient with food ingestion in disorders that are not proven to be pathophysiologically linked to food allergy (e.g., arthritis, fatigue, behavioral problems, etc.). OFCs may help to confirm or refute such associations, which is crucial because patients may undertake unnecessary dietary alterations that can have nutritional and social consequences [43, 44]. With regard to undertaking an oral food challenge when there is supporting evidence of allergy, the decision requires consideration of risk, nutritional need, social need, and other factors. The issues to consider when deciding whether to undertake an oral challenge, and what challenge setting (e.g., open, single, or double-masked) are summarized in Table 2. Diagnostic test results (PST, serum food-specific IgE), results of elimination diets, and historical points are central to decision-making regarding risk, while social and nutritional factors are considered in reviewing benefits. There are three general modalities to perform OFCs and their selection depends upon various considerations [45–48]. Challenges can be done “openly” with the patient ingesting the food in its natural form, “single-blind” with the food masked

Table 2 Factors to consider in deciding to undertake, and performing an oral food challenge Factor Aspects to consider (examples) Risk of a reaction Severity of a reaction Motivation

Past history, test results Past severity, underlying asthma, past sensitivity Patient/parent’s wish to add the food to the diet, social benefits Nutrition Nutritional improvement in the diet Modality (open challenge, blinding, placebos) Likely bias, results of prior exposures/challenges with regard to subjective symptoms Location (office, hospital, monitored unit) Likely severity and risk in context of ability to treat and observe

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and the patient unaware if the test substance contains the target food or, double-blind and placebo-controlled (DBPCFC) where neither patient nor physician knows which challenges contain the food being tested. In the latter two formats, the food must be hidden in some way, such as in another food or opaque capsules. When challenges are undertaken for research purposes, the double-blind and placebo-controlled food challenge (DBPCFC) is the preferred format because there is the least chance for bias from either the patient or physician who must monitor for symptoms. The false positive and false negative rate for the DBPCFC, based primarily on studies in children with atopic dermatitis, is 0.7% and 3.2%, respectively [49, 50]. Because the food is masked, it is sometimes difficult to provide meal-sized portions in a foods’ natural state. To help exclude false negatives, it has long been suggested to include an open feeding under supervision of a meal-size portion of the tested food prepared in its usual manner as a follow-up to any negative DBPCFC [50]. Increasing the number of challenges (additional placebo and true foods) helps to diminish the possibility of a random association, but this can be a very labor-intensive approach [51, 52]. The specific procedures involved have not yet been standardized though there are aims to do so [4, 53–56]. Tolerance should only be considered confirmed when a meal-sized amount of food in its usual form is tolerated.

Integration of Tests/Avoiding Pitfalls Pitfalls in diagnosis are shown in Table 3. Because of their high sensitivity and the observation that persons with IgE may tolerate a food, a common problem can occur if IgE tests are used for screening. Conversely, a negative test should not be con-

Table 3 Pitfalls in food allergy diagnosis Pitfall Examples Masquerader of allergy

Screening test panels

Ignoring a negative test despite a convincing history

Missing historical details of importance

– Auriculotemporal syndrome (Gustatory flushing) – Scombroid fish poisoning – Gustatory rhinitis – Panic/anxiety – Performing large batteries of tests without a careful history or attention to epidemiology (identifying sensitization but not likely clinical reactivity) – Test may be inaccurate for IgE-associated reaction (e.g., missing a causal protein in an extract) – Reaction pathophysiology may not associated with IgE (e.g., enterocolitis) – Contamination of a food with a known allergen for the patient – Testing for a major allergen ingredient (e.g., peanut) that has been tolerated while ignoring less common allergens less frequently encountered (e.g., carmine)

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strued to exclude an allergy to a food when the pathophysiology of the disorder in not associated with IgE, for example, food protein-induced enterocolitis syndrome [57]. Studies correlating outcomes of skin and serum tests often indicate reactions of an acute nature despite negative tests [16, 24]. It is not clear why PSTs are occasionally negative despite apparent acute allergic reactions during a food challenge. As has been indicated for venom testing [58], it may be prudent to perform both serum IgE and PST to a food to improve sensitivity when suspicion of reactions are high based upon history. In addition, it has been suggested, when suspicion of allergy is high and before proceeding to OFC, to follow a negative PST performed with a commercial extract with a fresh extract of the same food [59]. Additional diagnostic utility may be determined when the skin and serum tests are used in a complementary fashion. For example, Knight et al. [18] challenged children to egg when their serum IgE concentrations to egg white was favorable, reflecting approximately a 50% chance to pass an oral food challenge, for example, around 2 kIU/l. The size of the wheal to a commercial egg extract PST correlated with the outcome of the food challenges: 20% of those with a negative skin test reacted to OFC while 90% with a wheal size of 9 mm reacted. The major point toward an accurate diagnosis is to apply prior probability reasoning based upon the personal medical history and epidemiology factors (and sometimes previous test results) when interpreting the results of IgE tests, and to evaluate the tests as a probability along a spectrum rather than simply as a dichotomous variable (positive/negative). These approaches are distilled when considering a test’s likelihood ratios on a probability nomogram, though more needs to be done to establish the likelihood ratios for many foods and clinical circumstances [4].

Future Tests With regard to IgE-mediated allergies, improvement in the diagnostic accuracy of tests for the future will require additional studies to better characterize the test utility over a broad spectrum of disease, patient age, and types of foods. Standardization of commercial extracts is needed, but development of extracts using bioengineered, well-characterized proteins may also prove beneficial. A single food represents a complex assortment of proteins. These proteins may vary in their stability and how the immune system recognizes them. For example, a relatively stable protein in cow’s milk or hen’s egg may elicit an IgE response that recognizes primarily stable epitopes that survive digestion (amino acids that comprise linear epitopes unchanges by unfolding a protein) or primarily conformational epitopes that are lost after digestion. Studies support the notion that patients reactive in the former manner are more likely to maintain an allergy than those in the latter group [60, 61]. It is apparent that microarray analysis of informative epitopes may provide insights to reactivity, natural course and possibly severity, using very small sample volumes [62, 63]. Evaluating binding profiles to specific proteins among many allergens within a single food may also provide important clinical information. This may

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be approached by analysis of binding to specific profiles of stable allergens [64] or by differentiating IgE binding to allergens known to be associated with mild reactions (e.g., Birch pollen-related proteins) compared to more stable allergens associated with more severe reactions (e.g., non-specific lipid transfer proteins); the latter approach being referred to as “component resolved diagnosis” [65–68]. Future IgE testing could combine aspects of which proteins are bound, where the binding occurs, the strength of binding, the diversity and frequency of binding to accomplish more specific diagnostic outcomes (Fig. 2). These studies might require additional information about biological sensitivity to become informative (e.g., patient characteristics such as asthma, cellular characteristics such as basophil

Determination of differential IgE binding to proteins associated with mild (labile) compared to severe (stable) outcomes

Determination of number of epitopes bound by IgE

Determination of specific epitopes recognized by IgE

Differential binding to digested or intact proteins

Avidity/affinity of binding

Tests to determine cellular reactivity

Determination of number of proteins bound

Automation/microarray/engineered proteins‡correlation with clinical outcomes

Improved diagnosis

Fig. 2 Future diagnostic tests for IgE-associated food allergies. Various modalities may provide improved diagnostic accuracy and clinical insights (severity, natural course) with regard to IgEassociated food allergies.

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responses and T cell profiles, non-IgE humoral immune profiles, etc.), and patient outcomes will likely still depend upon the dose ingested. Tests for food-specific IgE antibodies are not relevant for non-IgE-mediated food allergies, but alternative markers of inflammation [69] and studies mentioned previously may eventually provide insights.

Conclusions Currently, an accurate diagnosis of IgE-associated food allergy requires careful consideration of the medical history, epidemiology, and available tests for IgE antibodies. Elimination and physician-supervised oral food challenge are often needed to confirm a diagnosis, though the history and test results may be essentially definitive in many circumstances. For disorders that are not ever, or are not uniformly, associated with IgE, elimination and OFC are the primary modalities though studies are emerging about the use of additional tests, such as the atopy patch test. Future diagnostics may take advantage of a complex consideration of humoral and cellular aspects of immune responses to causal proteins to create a more definitive diagnosis with less reliance upon OFC.

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Evaluation of the Patient with Photosensitivity Ann K. Haylett and Lesley E. Rhodes

Introduction Photosensitivity is a term defining a range of conditions characterised by altered sensitivity to solar ultraviolet (UV) and/or visible radiation. Responses of normal skin to UV radiation (UVR) include sunburn, tanning and immunosuppression in the short term and photocarcinogenesis in the longer term, while in photosensitive individuals, exposure to low amounts of sunlight precipitates a range of abnormal skin reactions. Photosensitivity disorders can be subdivided into four broad categories: (i) idiopathic/immune-based dermatoses; (ii) drug and chemically induced reactions, including those exogenously mediated and the cutaneous porphyrias; (iii) disorders associated with defects in DNA excision repair; (iv) photoaggravated dermatoses, a wide range of disorders that may be worsened by light exposure. The skin chromophores, that is, the UV/visible radiation absorbing molecules initiating the responses, are known in only a limited number of conditions. Photosensitivity conditions differ in clinical presentation, severity and character of sensitivity, and a systematic evaluation, comprising detailed clinical assessment, phototesting and laboratory analysis, is required for accurate diagnosis. Clinical evaluation: Age of onset, family and medication history can be important indicators. Duration of sunlight exposure required, time of appearance of rash following exposure and time to resolve, help differentiate the conditions. Symptoms include itch and pain following light exposure, while appearance varies from erythema, to fragile skin, scaling, papules, blisters, nodules, plaques and scarring, depending on condition. Photosensitivity symptoms occur mainly from spring to autumn in temperate climes, although severe conditions may occur throughout the year, leading to the absence of a seasonal pattern. Induction of symptoms through window glass indicates precipitation by UVA (320–400 nm) or visible light (>400 nm), A.K. Haylett and L.E. Rhodes () Photobiology Unit, Dermatological Sciences, University of Manchester, Salford Royal Foundation Hospital, Stott Lane, Manchester, M6 8HD, UK e-mail: [email protected]

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since UVB (290–320 nm) is not transmitted by glass. Eruptions occur principally on light-exposed skin, often with sharp cut-off where clothing, watch-straps, etc. have protected the skin (Fig. 1). Phototesting: Responses are evaluated following application to the skin of defined wavelengths and doses of UVB, UVA and visible radiation, with attention to dosedependence, time course and morphology. Spectral emission and power output of light sources require regular calibration; this is achieved through collaboration with a medical physics department, who will normally also perform the major part of the phototesting procedure, while responses are read and interpreted by clinicians with subspecialty training in this branch of dermatology. A series of tests are performed over 4–5 days. Firstly, monochromated light testing involves exposing areas of the

Fig. 1 A photosensitive individual, illustrating sharp cut-off of the eruption at the edges of clothing

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skin of the back to narrow wavebands of radiation to determine the action spectrum (i.e. wavelengths of sensitivity) and severity of the condition. The lowest dose of radiation required to evoke an erythemal response at 24 h (the minimum erythemal dose, or MED) is noted and compared with the normal range (Fig. 2, Table 1). Secondly, sites on the body (usually forearm and back) are exposed to broader emission radiation sources in order to artificially provoke the photosensitivity rash (Fig. 3). This confirms the nature of the condition and may require repeated challenge on consecutive days. Thirdly, photopatch testing examines for the existence/coexistence of photocontact allergy. The latter is detected only when both candidate chemical and

Fig. 2 Phototesting to narrow wavebands of UVB, UVA and visible radiation (monochromator light testing). Assessment of erythemal thresholds permits determination of the action spectrum of a photosensitive disorder Table 1 Wavelengths and doses of UV and visible radiation used in the determination of erythemal thresholds 24h erythemal threshold, J/cm2 Wavelength Normal range nm (bandwidth) Senitive range 300 (5) 320 (10) 330 (10) 350 (20) 370 (20) 400 (20) 500 (20) 600 (20)

0.0018 0.13 0.31 0.44 0.63 0.9 1.3

0.0025 0.18 0.63 1.8

0.0035 0.25 0.9 2.5 1.8 3.5

0.005 0.35 1.3 3.5 3.5 7

0.007 0.5 1.8 5 7 14

0.01 0.7 2.5 7 14 28 50 50

0.014 1 3.5 10 20 40

0.02 1.4 5 14 28 57

0.028 2 7 20 57 113

0.04 2.5 10 25

0.08 4 14 40

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Fig. 3 Artificial provocation using broadband radiation sources confirms photosensitivity and allows clinical assessment of rash morphology

UVR are applied to the skin, and is of practical relevance to photosensitive patients since the principal causative agents are now the sunscreens. Laboratory tests: These include blood autoantibody screen, since both subacute lupus erythematosis and systemic lupus erythematosis can present with photosensitivity. Blood and urine are scanned for porphyrin fluorescence, and if positive, chemical analysis of blood, urine and on occasion faeces, is performed for confirmation and identification of the type of porphyria. Histocompatibility locus antigen (HLA) DR4 subtyping is performed when the diagnosis of actinic prurigo (AP) is suspected. Skin biopsies may be taken from naturally occurring or artificially provoked rash for diagnostic histology. When a DNA repair disorder is suspected, functional assays are performed at a specialist centre. Information gained from the detailed clinical appraisal, photoinvestigation and laboratory tests enable an accurate diagnosis to be made, which with knowledge of the action spectrum and severity, permit appropriate management of the patient.

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Idiopathic/Immune-Mediated Photosensitivity Disorders (i) Polymorphic light eruption (PLE) is the commonest photosensitivity disorder, with a reported prevalence of 21%, 15% and 5% in Scandinavia, the UK and Australia, respectively [1, 2]. It is more common in women, with a ratio of 3–4:1 [3], and while it typically has onset in teenage or the twenties, it can also present in childhood and later adult life. It occurs in a range of severities from mild, to moderate and severe. PLE is characterised by a pruritic skin eruption occurring in a delayed manner, usually several hours, following UVR exposure. Lesions comprise red papules and/or vesicles and/or plaques which resolve after a few days to weeks without scarring [4]. PLE occurs from spring to autumn, and during sunny holidays abroad. Most subjects experience ‘hardening’, that is, an increase in tolerance to sunlight towards the end of summer, and a similar process is responsible for the absence of facial involvement in around 60% patients. Patients attending hospital departments show a significant impact on their quality of life [3]. It was first suggested in 1942 that PLE might represent a type-IV delayed hypersensitivity response to a sunlight-induced cutaneous allergen [5]. Histologically PLE is characterised by a dense dermal perivascular lymphocytic infiltrate peaking by 72 h post-UVR; CD4+ cells increase first followed by a rise in CD8+ cells [6]. The expression pattern of adhesion molecules E-selectin and ICAM-1 post-UVR also support a type-IV hypersensitivity response [7]. Interestingly, epidermal CD1a+ Langerhans cells were found to be resistant to high-dose UVR in PLE patients, whereas those in healthy volunteers were reduced, as part of the ‘normal’ photoimmunosuppression response [8]. Evidence for a partial failure of UVRinduced immunosuppression in PLE was also seen in skin responses to the contact allergen 2,4-dinitrochlorobenzene compared to healthy controls [9]. This could theoretically allow a response to a putative photoallergen to emerge. Evidence of genetic clustering in PLE has also led to support for a genetic component in its aetiology [10]. It seems likely that both environmental and genetic factors contribute to the aetiology of PLE. Both UVA and UVB can provoke PLE eruptions, although generally UVA or solar-simulated light is more successful [11]. There is no standardised protocol for UV provocation and a wide range of variables may impact on the probability of successful elicitation of photosensitivity rash, including irradiation site, size of area, emission spectrum of light source and degree of UVR-adaptation. Monochromated light testing shows normal or borderline low erythemal thresholds to UVA and UVB, and several cases of coexistent sunscreen allergy and photoallergy have been reported. (ii) Chronic actinic dermatitis (CAD) is a severely photosensitive condition with eczematous morphology [12]. Incidence of CAD in the UK has been quoted as 1:6,000 with a male to female ratio of 9:1 [13]. Typically arising in older males, it has also been reported to occur in younger age groups [14, 15]. Due to the extreme sensitivity to UV/visible radiation found in CAD, the condition may be severe year-round, and the patient may be unaware of a relationship to sunlight. Coexistent contact allergies are frequently found, particularly to sesquiterpene

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lactone, found in plants [16, 17]. As with PLE the immunological findings are similar to a delayed-type hypersensitivity response, with a pattern of lymphocytic infiltration characterised by early peaks of CD4+ cells followed by CD8+ cells [18]. Phototesting in CAD reveals a marked reduction in erythemal thresholds, and eczematous lesions characteristic of the disease may also be induced [19]. Characteristically, patients show greatest sensitivity to UVB, followed by UVA, plus/minus sensitivity to visible light, an action spectrum resembling that of the sunburn response in healthy individuals. (iii) Actinic prurigo (AP) is an uncommon condition first described in the Navajo Native Americans in 1958 [20], although it has subsequently been observed in Europe and the Far East. AP occurs more frequently in females, and is usually characterised by childhood onset, although it can present in adult life. There is a seasonal pattern with deterioration in the spring and summer but lesions can also occur in the winter [21]. Eruptions appear on exposed skin although covered sites, particularly the buttocks, can also be involved. Lesions comprise nodules which resolve to leave scarring, and there is often a prominent eczematous component. Unlike PLE, sparing of chronically exposed sites such as the face, and summer ‘hardening’, is unusual. Immunohistochemistry of AP lesions reveals numerous Th1 lymphocytes [22], and CD20+ B lymphocytes clustered in the centre of lymphoid follicles [23]. Striking associations with HLA class I and II alleles are seen. Actinic prurigo is reportedly associated with HLA-A24 and HLA-Cw4 in Canada, HLA-Cw3 and HLA-B40 in Columbia, and HLA-DRB*14 in Inuit populations. In Europe, a strong association with HLA-DRB*0407 is reported; with 60% patients showing positivity compared with its scarcity in the healthy population, HLA DR4 sub-typing can be a useful supportive diagnostic test [24]. It has been speculated that PLE and AP may have a common genetic background with AP possibly representing an HLA restricted subset of PLE. The action spectrum of AP is characterised by reduced erythemal thresholds in approximately 60% of cases, most often to UVA [25]. The eruption can be provoked by repeated UVR exposure, although not as successfully as PLE. (iv) Solar urticaria (SU) is a rare form of urticaria characterised by itching, erythema and wheal formation occurring usually within a few minutes of sunlight exposure, and resolving within a few hours. Occasionally solar angioedema may also occur [26], and when large areas of the body are exposed additional symptoms can include dizziness, dyspnoea and syncope. SU is reportedly slightly more prevalent in women and peak onset is reported in the third decade of life [27]. Mechanisms underlying SU are incompletely understood. It is hypothesised that UV/visible radiation reacts with a precursor molecule to form an allergen, followed by generation of IgE against this allergen; the IgE is bound to the surface of mast cells which degranulate releasing histamine. Elevated histamine levels have been reported in SU [28–30]. Early work based on passive transfer techniques led to the distinction of type-I and type-II solar urticaria responses [31, 32], dependent on whether a wheal response in the healthy subject could be induced by injection of

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SU patients’, but not normal, irradiated serum (type-I response), or by both (type-II response). In the type-I response the precursor was hypothesised to occur only in the patient whilst in the type-II response the precursor occurred in both patient and healthy subject. The chromophore responsible for SU is unknown, and may vary with the action spectrum [33]. Action spectra for SU range from narrow to broad, encompassing UVB, UVA and visible radiation. Moreover, inhibition and augmentation spectra may occur. Hence the chances of successful provocation can be improved by employing a range of narrow and wideband radiation sources during diagnostic phototesting. (v) Hydroa vacciniforme (HV) is a rare condition, with no gender bias, that is nearly always associated with childhood onset. Sun exposure results in stinging erythema of exposed skin followed by formation of painful vesicles which become scabbed and necrotic, and heal to leave varioliform scars. Minimal sun exposure can precipitate the rash which occurs from spring to autumn and also on bright winter days. HV can considerably affect the quality of life in childhood. Although usually resolving in the teenage years, the scarring remains. Histologically it is characterised by focal reticulate degeneration of keratinocytes, spongiosis, vesiculation and a perivascular mononuclear infiltrate. It is speculated that HV reflects a phototoxic reaction, with, unlike other ‘idiopathic’ photodermatoses, no features indicating an immune basis. Clarification of HV pathogenesis has been complicated by descriptions of an ‘atypical variant HV’, associated with fever, wasting, hepatosplenomegaly and progression to malignant lymphoma/leukaemia [34]. These cases were all associated with Epstein-Barr virus (EBV) encoded nuclear RNA positive cells, and it was hypothesised that typical and atypical HV are variants within the same disease spectrum of EBV-associated T-cell lymphoproliferative disorders [35]. However, neither EBV nor malignancy is reported in association with HV in Europe, and the EBV association reported in South Asia and Japan could reflect its endemic distribution. The action spectrum of HV lies principally in the UVA region with repeated provocation testing eliciting typical lesions [36, 37].

Drug or Chemically Induced Photosensitivity Endogenous Disorders – The Porphyrias Five of the seven recognised porphyrias are associated with photosensitivity to visible light. Porphyrias are disorders of the haem metabolic pathway whereby glycine and succinyl coenzyme A are converted via a series of intermediates to haem. Deficiency of specific enzymes in the pathway results in the build up

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Table 2 Diagnostic porphyrin testing Disorder Biochemical diagnosis Variegate porphyria

Hereditary coproporphyria and harderoporphyria Congenital erythropoietic porphyria Erythropoietic protoporphyria

Increased urinary and faecal protoporphyrin IX and coproporphyrin Increased urinary porphobilinogen and aminolaevulinic acid during an acute porphyric attack Coproporphyrin III present in faeces Increased urinary porphobilinogen and aminolaevulinic acid during an acute porphyric attack Uroporphrin I and coproporphyrin I present in RBC, urine and faeces Protoporphyrin IX present in erythrocytes and faeces

of porphyrin intermediates (Table 2). These absorb light, with peak absorption around 405 nm, resulting in conversion to an excited state. As the excited state decays, reactive oxygen species and free radicals are produced, which damage cellular structures in their vicinity. The subcellular distribution of the porphyrins is related to their lipophilicity, and this in turn influences their manifestations. For example protoporphyrin IX is lipophilic, localising in endothelial cells and lysosomal membranes, and causes acute symptoms of photosensitivity, while the water-soluble uroporphyrin and coproporphyrin localise in the lower epidermis and basement membrane zone, and are associated with chronic features of photosensitivity, notably fragile skin and scarring [38]. Laboratory tests to assess porphyrin levels in blood, urine and faeces are the principal diagnostic tool in the porphyrias (Table 2) and phototesting is unnecessary. (i) Erythropoietic protoporphyria (EPP) is caused by a reduction in activity of the enzyme ferrochelatase which catalyses conversion of protoporphyrin IX to haem; a range of ferrochelatase mutations have been identified [39]. It is an autosomal dominant condition in most families, with variation in penetrance. EPP usually presents during childhood, although can escape diagnosis until later years. Features of acute photosensitivity occur, with skin pain and burning commencing within 5–30 min of sunlight exposure, and persisting from a few hours to several days. While erythema and oedema can occur, the skin often maintains a normal appearance, and a high index of suspicion is required for diagnosis. Later, linear scarring appears particularly around mouth and nose, and a weathered appearance may develop. (ii) Porphyria cutanea tarda (PCT) is the most common porphyria, with an estimated prevalence of 1 per 25,000 in the UK [40]. PCT is associated with chronic skin lesions, and since there are no acute symptoms on sunlight exposure the subject is unaware of its causation. Characteristically, sun exposed sites, particularly dorsal hands, display fragile skin and blisters, associated with scarring, milia formation, hypertrichosis and pigmentary changes. Decreased activity of the enzyme uroporphyrinogen decarboxylase occurs, principally catalysing conversion of uroporphyrinogen III to coproporphyrinogen III [41] and also uroporphyrinogen I

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to coproporphyrinogen I. PCT may be inherited (usually autosomal dominant) or acquired, and alcohol, oestrogens and hepatitis C contribute to the latter’s development. Abnormalities of iron metabolism occur, with elevation of total body iron stores in the majority of patients. A rare homozygous form of PCT, hepatoerythropoietic porphyria, is also reported, associated with childhood onset. (iii) Variegate porphyria is an autosomal dominant disorder generally manifesting after the second decade. Cutaneous features in VP and another autosomal dominant disorder, hereditary coproporphyria (HCP), below, are indistinguishable from PCT. It is important to biochemically differentiate these conditions since potentially lifethreatening acute porphyric (neurological) attacks can occur in VP and HCP, with prevention by avoidance of precipitating medications and dietary substances (a current list of these agents can be found at www.porphyria-europe.com). A decrease in protoporphyrinogen oxidase activity occurs, catalysing the conversion of protoporphyrinogen to protoporphyrin IX, and a range of mutations in the protoporphyrinogen oxidase gene have been identified. A rare homozygous variant is described [42], characterised by onset of lesions before 2 years, growth and mental retardation, flexion deformities of digits, convulsions and nystagmus. (iv) Hereditary coproporphyria and harderoporphyria Conversion of coproporphyrinogen III to protoporphyrinogen is catalysed by the enzyme coproporphyrinogen oxidase, a two-step decarboxylation process with harderoporphyrinogen formed as the intermediate species. Two conditions have been associated with defects of this process, i.e. HCP and harderoporphyria (HP). Both can be associated with photosensitivity, HC displaying features similar to those in PCT (see preceding subsection). (v) Congenital erythropoietic porphyria (CEP) is an extremely rare, severe autosomal recessive disorder. This presents in infancy, and causes erosions of the terminal phalanges, onycholysis, destruction of the ears, nose and eyelids, and erythrodontia. CEP is caused by deficiency of uroporphyrinogen III synthase, catalysing the conversion of hydroxymethylbilane to uroporphyrinogen III.

Exogenous Chemicals – Phototoxicity and Photoallergy Photosensitivity can arise through interactions between light and exogenous systemic or topical chemicals, and recognition is important since removal of the causative agent can be curative. The underlying mechanism can be allergic (photoallergy) or toxic (phototoxicity) in nature. A wide range of compounds have been associated with photosensitivity (Tables 3a and b). Reactions principally, although not exclusively, involve UVA [43]. Systemic reactions: Systemic agents (drugs or dietary) are activated by light exposure of the skin. In the more prevalent phototoxic reactions, reactive oxygen species cause cell damage and trigger inflammatory responses [44]. The typical clinical

Table 3a Drugs associated with photosensitivity side effects ACE inhibitors Captopril Enalapril maleate Fosinopril hydrochloride Linsinopril Moexipril hydrochloride Perindopril erbumine Quinapril Ramipril Tandolapril Anti-arrhythmic Flecainide acetate Antibiotics Pyrazinamide Clofazimine Trimethoprim Azithromycon Doxycycline Co-trimoxazole Quinolones Tetracyclines Antidepressants Venlafaxine Amitriptyline Citalopram Escitalopram Fluoxetine Fluvoxamine maleate Paroxetine Sertraline Anti-diabetic Chlorpropamide Glipizide

Antimuscarinic Darifenacin Duloxetine Flavoxate hydrochloride Oxybutin hydrochloride Propantheline bromide Propiverine hydrochloride Solifenacin succinate Tolterodine tartrate Trospium chloride Antiviral Tipranavir Efavirenz Ribavirin Aciclovir Asthma prophylaxis Omalizumab Atypical antipsychotic Zotepine Olanzapine Calcium channel antagonists Aprepitant Diltiazem hydrochloride Diuretics Triamterene Indapamide Benroflumethiazide Furosemide

Anti-epileptic Carbamazepine Lamotrigine

Hyperlipidaemia Gemfibrozil Bezafibrate Fenofibrate

Topical antihistamines Diphenhydramine

Urinary Incontinence Duloxetine

Antipsychotic Haloperidol

Hypnotics and anxiolytics Zalepion

Neurokinin receptor antagonists Aprepitant Non-steroidal anti-inflammatory Aceclofenac Acemetacin Asprin Celecoxib Dexibruprofen Dexketoprofen Diclofenac sodium Diflunisal Etodolac Etoricoxib Fenbufen Ibuprofen Indometacin Ketoprofen Lumiracoxib Mefenamic acid Meloxicam Nabumetone Naproxen Piroxicam Sulindac Tenoxicam Tiaprofenic acid Psoriasis Tar ointment, paste and bath preparations Calcipotriol Methotrexate Protein Kinase Inhibitor Dasatinib Imatinib Other Vardenafil Danazol Chloroquin Botulinum Acitretin Isotretinoin Oral contraceptive

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Table 3b Environmental and dietary agents associated with photosensitivity side effects Compound Found in Effect Furocoumarins

Quinine Hypericin Coal Tar Musk ambrette Chemical sunscreens

Rutaceae Moraceae Apiaceae Leguminoseae Soft drinks (e.g. Bitter Lemon) Hypericum perforatum Fragrances Sunscreen products

Phototoxicity

Phototoxicity Phototoxicity Phototoxicity Photoallergy Photoallergy

presentation is an exaggerated sunburn response, but a range of other manifestations occur including photoonycholysis, pseudoporphyria and pigmentary disturbances, patterns being specific to drug family. A wide range of drugs are associated with systemic phototoxicity, including non-steroidal anti-inflammatory drugs, antibiotics, anti-fungals, diuretics, cardiovascular and psychoactive agents. In photoallergy, interaction of light and drug results in formation of an antigen, followed by a typeIV delayed immune response. Clinical patterns include eczematous eruptions, with sulphanilamide and chlorpromazine as examples of precipitants. Drug reactions are suspected from time relationship and the agent’s proclivity to cause such responses, and confirmed by lowered erythemal thresholds on phototesting, with return to normal on re-testing several months after drug discontinuation. Topical reactions: Topical agents cause localised phototoxic and photoallergic reactions. Phototoxic reactions can arise due to exposure to polycyclic hydrocarbons, fragrances, dyes and topical medications, and characteristically present as erythema and blistering in patterns reflecting contact with the agent. Such reactions are diagnosed clinically, and resolve on reduced exposure to the agent. Photoallergic contact reactions are particularly troublesome since exposure to only small amounts of agent is required. The main culprits in recent years are sunscreen chemicals and non-steroidal anti-inflammatory drugs. Photoallergy is diagnosed by photopatch testing, where duplicate patches of chemical are applied to the skin, in the presence and absence of UVA [45]. Eczematous responses are seen at positive sites.

DNA Excision Repair Deficiency Disorders Solar UVR exposure causes repair deficiency disorders UVR generates cyclobutane 6–4 pyrimidine-pyrimidone

DNA damage to skin cells, but in the rare DNA there is limited ability to repair the damage. pyrimidine dimers (CPD) and to a lesser extent adducts (6–4 PP). Under normal circumstances

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nucleotide excision repair (NER) occurs via one of two pathways, the global genome NER (GGNER) and the transcription coupled NER (TCNER) pathways. Repair of damaged areas of DNA is achieved by coordinated actions of a specific sequence of protein complexes; in DNA excision repair deficiency disorders there are mutations in the genes encoding these proteins. There are four principal conditions with cutaneous features, all of which are rare autosomal recessive genodermatoses. (i) Xeroderma pigmentosum (XP) Photosensitivity symptoms often appear before 2 years of age, minimal sunlight exposure leading to severe and prolonged sunburn with blistering. Abnormal freckling, hypo- and hyper-pigmentation, skin atrophy and telangiectasia develop. Notably, there is an approximately 1,000fold increased skin cancer risk (comprising basal cell carcinoma, squamous cell carcinoma and malignant melanoma), with around 50% patients developing skin malignancy by 10 years of age [46]. Eye complications occur, including inflammatory lesions and malignancies of eyelids. Early diagnosis is essential, for early institution of protective measures. XP has been classified into seven complementation groups A to G, attributable to specific flaws in the excision repair process and exhibiting differences in clinical manifestations. Approximately 20% of XP patients, particularly XP-A and XP-D, show progressive neurological degeneration, while XP-C is associated with internal tumours, and XP-F has milder skin symptoms including delayed onset of skin cancers relative to other complementation groups. An XP variant with normal DNA repair rates also occurs [47]. Phototesting may show reduced erythemal thresholds, typically to UVB, and unusually prolonged erythema [48]. Diagnosis of XP is confirmed by measuring post-UV cell survival and DNA repair capacity in fibroblast cultures derived from the patient’s skin biopsy. (ii) Cockayne syndrome (CS) presents in infancy, with a scaly dermatitis of exposed sites and susceptibility to severe sunburn. Other clinical features include a bird-like facies, growth and mental retardation, neurological abnormalities and ocular lesions [49]. CS occurs in two complementation groups CS-A and CS-B. Diagnosis is confirmed by functional assay of DNA repair. (iii) Trichothiodystrophy (TTD) is associated with photosensitivity in approximately 50% of cases, with an XP-like pattern of severe and prolonged sunburn following minimal UVR exposure. Other characteristics include brittle sparse hair of scalp, eyelashes and eyebrows, with polarised light microscopy revealing a pattern of light and dark hair areas, the ‘tiger-tail’ appearance. An unusual facial appearance and variable degree of mental retardation may also occur. There are three complementation groups, associated with mutations in the XP-D (the majority), TTD-A and XP-B genes. As in CS, but in contrast to XP, there is not an increased risk of skin cancer. Clinical suspicion of TTD is supported by findings of low MCV and MCH of TTD-erythrocytes, and confirmed by functional assays.

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(iv) Blooms syndrome (BS) presents in infancy with telangiectasia and erythema exacerbated by sunlight exposure. BS is diagnosed by in vitro analysis including sister chromatid exchange frequency.

Photoaggravated Dermatoses The photoaggravated dermatoses are a broad range of conditions that can develop without UVR exposure but may be exacerbated by sunlight. Lupus erythematosus (LE) is the best known, and systemic, subacute and chronic discoid LE (SLE, SCLE and CDLE, respectively) are all typically provoked by sunlight, with worsening of cutaneous features during spring and summer months. SLE classically displays an erythematous ‘butterfly’ rash across nose and cheeks, but is also associated with more widespread erythema development following short spells of sunlight exposure. SCLE is characterised by distinctive superficial scaly red plaques occurring particularly on the mantle area. It can resemble acute photosensitivity disorders such as PLE, but the lesions are more prolonged and tend to be followed by post-inflammatory pigmentary changes taking several months to resolve. CDLE is characterised by discoid plaques on sun-exposed skin, which are prolonged and heal to leave prominent scarring. Diagnosis of SCLE is assisted by the presence of circulating antinuclear antibodies to Ro and LA in approximately 60% cases [50], while SLE may display antibodies to DNA. Immunohistochemical staining of lesional and non-lesional skin samples (lupus band test) characteristically reveals deposition of IgG, IgM, IgA, complement 3 and fibrinogen in the basement membrane zone. Phototesting with UVA or UVB may provoke lesions in patient with LE; SCLE has the greatest probability of a positive provocation test followed by SLE [51]. The UVR-induced erythemal thresholds are reportedly normal in all three LE subtypes [52]. Other conditions that may be photoaggravated include eczema, with a minority of atopic eczema patients developing UVR sensitivity. It is differentiated from CAD by its lesser severity and action spectrum principally involving UVA rather than UVB. Psoriasis is another common skin condition usually benefiting from UVR exposure, but with photoaggravation in a proportion. A photoaggravated rash also occurs in around 40% cases of dermatomyositis, as a violaceous erythema on photoexposed sites.

Conclusion Photosensitivity arises from a wide range of aetiologies, ranging from genetic disorders to immune and chemical reactions. The precise prevalence of photosensitivity disorders is unknown, but drug reactions and certain immune-based conditions are common, and together they cause a considerable disease burden. Evaluation of the photosensitive patient requires detailed clinical assessment,

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comprehensive photosensitivity testing and laboratory investigations. These confirm photosensitivity, indicate action spectrum and severity of the disorder, and enable the accurate diagnosis required for appropriate management.

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19. Menage H du P, Hawk JLM (1993) The red face: chronic actinic dermatitis. Clin Dermatol 11:297–305 20. Brandt R (1958) Dermatological observations on the Navaho reservation. AMA Arch Derm 77:581–585 21. National Radiological Protection Board (2002) Health effects from Ultraviolet Radiation. Report of an advisory group on non-ionizing radiation. National Radiation Protection Board 13:162–190 22. Umana A, Gomez A, Duran MM, Porras L (2002) Lymphocyte subtypes and adhesion molecules in actinic prurigo: observations with cyclosporin A. Int J Dermatol 41:139–145 23. Arrese JE, Dominguez-Soto L, Hojyo-Tomoka MT, Vega-Memije E, Cortes-Franco R, Guevara E, Pierard GE (2001) Effectors of inflammation in actinic prurigo. J Am Acad Dermatol 44:957–961 24. Grabczynska SA, McGregor JM, Kondeatis E, Vaughan RW, Hawk JL (1999) Actinic prurigo and polymorphic light eruption: common pathogenesis and the importance of HLA-DR4/ DRB1*0407. Br J Dermatol 140:232–236 25. Frain-Bell W. (1985) The idiopathic photodermatoses. In: Frain-Bell W (eds) Cutaneous Photobiology. Oxford University Press, Oxford, pp 24–59 26. Rose RF, Bhushan M, King CM, Rhodes LE (2005) Solar angioedema: an uncommonly recognized condition? Photodermatol Photoimmunol Photomed 2005 21:226–228 27. Beattie P (2006) Solar Urticaria. In Ferguson J. and Dover JS. (eds) Photodermatology. Manson Publishing, London, pp 45–50 28. Miyauchi H, Horio T (1995) Detection of action inhibition and augmentation spectra in solar urticaria. Dermatol 191:286–291 29. Hawk JLM, Eady RAJ, Challoner AVJ, Kobza-Black A, Keahey TM, Greaves MW (1980) Elevated blood histamine levels and mast cell degranulation in solar urticaria. Br J Clin Pharmacol 9:183–186 30. Soter NA, Wasserman SI, Pathak MA, Parish JA, Austen KF (1979) Solar urticaria release of mast cell mediators into the circulation after experimental challenge. J Invest Dermatol 72:282 31. Rajka E (1942) Passive transfer in light urticaria. Clin Immunol 13:327–345 32. Leenutaphong V, Holzle E, Plewig G (1989) Pathomechanisms and classification of solar urticaria: a new concept. J Am Acad Dermatol 21:237–240 33. Horio T, Hoelzle E (2007) Solar urticaria In: Lim HW, Hoenigsmann H, Hawk JLM (eds) Photodermatology. Informa Healthcare, New York 34. Magana M, Sangueza P, Gil-Beristain J, Sanchez-Sosa S, Salgado A, Ramon G, Sangueza OP (1998) Angiocentric cutaneous T-cell lymphoma of childhood (hydroa-like lymphoma): a distinctive type of cutaneous T-cell lymphoma. J Am Acad Dermatol 138:574–579 35. Iwatsuki K, Xu Z, Takata M, Iguchi M, Ohtsuka M, Akiba H, Mitsuhashi Y, Takenoshita H, Sugiuchi R, Tagami H, Kaneko F (1999) The association of latent Epstein-Barr virus infection with hydroa vacciniforme. Br J Dermatol 140:715–721 36. Halasz CL, Leach EE, Walther RR, Poh-Fitzpatrick MB (1983) Hydroa vacciniforme: induction of lesions with ultraviolet A. J Am Acad Dermatol 8:171–176 37. Rhodes LE, White SI, (1998) Dietary fish oil as a photoprotective agent in hydroa vacciniforme. Br J Dermatol 138:173–178 38. Elder GH (1999) The cutaneous porphyrias. In: Hawk JLM (ed) Photodermatology. Arnold, London, pp 171–197 39. Stenson PD, Ball EV, Mort M, Phillips AD, Shiel JA, Thomas NS, Abeysinghe S, Krawczak M, Cooper DN (2003) Human Gene Mutation Database (HGMD): 2003 update. Hum Mutat 21:577–581 40. Elder GH. (1990) The cutaneous porphyrias. Semin Dermatol 9:63–69 41. Nacht S, San Martin de Viale LC, Grinstein M (1970) Human porphyria cutanea tarda. Isolation and properties of the urinary porphyrins. Clin Chim Acta 27:445–452 42. Korda V, Deybach JC, Martasek P, Zeman J, da Silva V, Nordmann Y, Houstkova, H, Rubin A, Holub J (1984) Homozygous variegate porphyria. Lancet 1:851

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43. Gould JW, Mercurio MG, Elmets CA (1995) Cutaneous photosensitivity diseases induced by exogenous agents. J Am Acad Dermatol 33:551–573 44. Moore DE (2002) Drug induced cutaneous photosensitivity Incidence Mechanism Prevention and Management. Drug Safety 25:345–372 45. Ibbotson SH, Farr PM, Beck MH, Diffey BL, Ferguson J, George SA, Green C, Du P, Menage H, Murphy GM, Norris PG, Rhodes LE, White IR (1997) Workshop Report. Photopatch testing; methods and indications. Br J Dermatol 136:371–376 46. Kraemer KH, Lee MM. Scotto J (1987) Xeroderma Pigmentosum. Cutaneous ocular and neurological abnormalities in 830 published cases. Arch Dermatol 123:241–250 47. Johnson RE, Kondratick CM, Prakash S, Prakash L (1999) hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285:263–265 48. Norris PG, Lehmann AR (1999) The repair deficient photodermatoses. In: Hawk JML (ed) Photodermatology. Arnold, London 49. Nance MA, Berry SA (1992) Cockayne syndrome: review of 140 cases. Am J Med Genet 42:68–84 50. Deng JS, Sontheimer RD, Gilliam JN (1984) Relationships between antinuclear and anti-Ro/SS-A antibodies in subacute cutaneous lupus erythematosus. J Am Acad Dermatol 11:494–499 51. Werth VP, Honigsmann H (2007) Photoaggravated dermatoses. In: Lim HW Honigsmann H, Hawk JLM (eds) Photodermatology. Informa Healthcare, New York, pp 251–266 52. Sanders CJ, Van Weelden H, Kazzaz GA, Sigurdsson V, Toonstra J, Bruijnzeel-Koomen CA (2003) Photosensitivity in patients with lupus erythematosus: a clinical and photobiological study of 100 patients using a prolonged phototest protocol. Br J Dermatol 149:131–137

Clinical Manifestations of Allergic Diseases: Drug Hypersensitivity Benno Schnyder and Werner J. Pichler

Classification of Adverse Drug Reactions by Mechanism Adverse drug reactions are frequent and occur in 10–20% of hospitalized patients and in about 7% of the general population [1]. Often they stand for a serious health problem because of their severity and their impact of the use of medications. Adverse drug reactions have been classified by Rawlins and Thompson [2] in four types: • Type A reactions are common (ca. 80%), due to the pharmacologic or toxic propriety of the causative drug, and thus predictable; they may occur in any individual. • Type B reactions are uncommon (ca. 10–15%), not predictable, and occur only in susceptible individuals. • Type C reactions are associated with long-term therapy (e.g., benzodiazepine dependence). • Type D reactions have carcinogenic and teratogenic effects. Type B reactions comprise so-called idiosyncratic and immune reactions against the drug. The term idiosyncrasy is used differently: some authors use it for side effects, including immune-mediated reactions; others define idiosyncrasy as an unforeseen reaction due to some genetic defect (e.g., glucose-6-phosphatase deficiency and hemolysis after some drugs) and exclude immune-mediated reactions to drugs. To develop an immune reaction, antigens have to be presented by antigenpresenting cells (APC) on appropriate major histocompatibility complexes (MHC) and they have to be recognized by T cells. Most pharmaceutical agents are simple molecules of low molecular weight and not able to bind per se on the MHC. Actually, the hapten/prohapten concept is the prevailing explanation for primary sensitizations: drugs and their reactive metabolites, respectively, gain immunogenicity by forming stable hapten–carrier compounds [3]. These compounds are taken up as neo-antigens by dendritic cells, processed and transported to the local draining B. Schnyder () and W.J. Pichler Division of Allergology, Clinic for Rheumatology and Clinical Immunology/Allergology, Inselspital, University of Bern, 3010-Bern, Switzerland e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_23, © Springer 2009

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lymphoid tissue and presented there. If these compounds are recognized by specific naive T cell, they will induce a sensitization. It can be assumed that primary sensitizations for drug allergies may be induced not by the drug/drug-metabolite-carrier compounds only, but that also drug-independent antigens may induce T-cell reactions, which cross-react with drugs (e.g., viral infections, cf. Section on T-CellMediated, Delayed Drug Hypersensitivity Reactions). This primary sensitization is mostly asymptomatic. It may involve T cells alone or both T and B cells with consecutive forming of antibodies. On continued or renewed exposure affected T cells and/or antibodies determine the elicitation phase, which can be divided into four categories corresponding to type I–IV reactions according to Gell and Coombs [4]. The vast majority of real drug allergies are supposed to be either IgE-mediated (type I) or T-lymphocyte-mediated (type IV).

Type I (IgE-Mediated) Allergies If the primary sensitization leads to formation of drug-specific IgE, renewed contact with very small amounts of antigens and drugs, respectively, may already induce symptoms. This extraordinary sensitivity is achieved by the ubiquitous presence of mast cells armed with high-affinity Fc-IgE receptors, to which allergen-specific IgE is bound. A current paradigm postulates that also in the elicitation phase an antigen must be presented in multivalent form: For an allergic reaction, allergens must bind to Fab part of the IgE molecules. Binding of two or more cell-bound IgE molecules (cross-linking) leads to activation of the mast cell and the release of various factors such as histamine, leukotriens, prostaglandins, and cytokines. These released molecules elicit vasodilatation, increased vascular permeability, enhanced mucus production, bronchoconstriction, and contribute to eosinophilic recruitment. IgE-mediated reactions to drugs often manifest as urticaria or angioedema. But they may include respiratory symptoms, shock, and severe cardiac complications.

IgG-Mediated Cytotoxicity (Type II) Type II reactions comprise IgG-medicated cytotoxicity, which is predominantly directed to erythrocytes, leukocytes, platelets, and probably hematopoietic precursor cells in the bone marrow. The antibody-coated cells will be sequestrated to the reticulo-endothelial system in liver and spleen by Fc- or complement receptor binding or, more rarely intravascular destruction may occur by complementmediated lysis. Different pathways of antibody-recognition of target-cells have been supposed [5, 6]: 1. Structures of cell membranes were modified by the hapten and drug, respectively, which induce an immune response, which is directed to these target structures.

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Such a development of an IgG immune reaction to the hapten–carrier complex is rather rare and occurs mostly after longer duration of high-dose treatment. Some antibody reactivity may be directed to the carrier molecule itself (= autoantibodies). This autoimmune form is less abrupt, but longer lasting after cessation of the drug. 2. The drug induces conformational changes in structures of cell membranes, which induces nonspecific adherence with naturally occurring autoantibodies. Such reaction may occur only as long as drug is present in soluble form.

Immune Complex Deposition (Type III) Formation of immune complexes is a common event in the frame of a normal immune response and normally does not cause symptoms. In type III reaction, the immune complexes formed are activating endothelial cells with complement activation in small vessels and deposition due to Fc-IgG receptor interaction. Why and under which exact circumstances an immune complex disease develops is actually not clear. Clinical symptoms of a type III reaction comprise serum sickness, drug-induced lupus erythematosus (DILE), and/or vasculitis.

T-Cell-Mediated, Delayed Drug Hypersensitivity Reactions Hypersensitivity reactions to drugs are normally explained by the hapten/ prohapten concept, implying an immune reaction to a hapten–carrier complex. More recent findings suggest an additional pathway of immune stimulation by drugs, namely the direct stimulation of T cells by native drugs, which directly interact with T-cell receptors (p–i concept). This new concept may explain some hypersensitivity reactions in a more plausible way than the hapten/prohapten concept alone.

The Hapten/Prohapten Concept To be immunogenic, drugs act as haptens. Haptens are chemically reactive small molecules (mostly <1,000 D) that are able to undergo a stable binding to a larger protein or peptide. Certain drugs such as penicillins can, under physiologic conditions, directly bind covalently with macromolecules. These drug–protein complexes (hapten–carrier complexes) are processed and consecutively presented on MHC molecules as hapten-modified and thus immunogenic peptides. However, few drug molecules are chemically reactive in their native form. Thus, it is believed that inert drugs (prohaptens) undergo metabolism (bioactivation) and become reactive metabolites (haptens), which then covalently bind to proteins and thereby make

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them immunogenic [7]. The hapten/prohapten concept is a valid explanation for primary sensitizations to drugs. In the elicitation phase it is well documented for contact dermatitis.

The p–i Concept Investigations of drug-specific human T-cell clones (TCC) from drug allergic subjects revealed reactivity against the causative drug in its native form without stable binding to a carrier molecule or need of processing [8, 9]. Full reactivity of the TCC was observed only in the presence of both the inert drug and the APC with the appropriate MHC. The findings demonstrate that chemically poorly reactive, native drugs are capable of transmitting a stimulatory signal via the T-cell receptor for antigen, which activates T cells resulting in proliferation, cytokine production, and cytotoxicity. It is assumed that preferentially previously peptide primed, memory, or effector T cells are reactivated. Their stimulatory capacity may be due to some cross-reactivity, namely that the drugs are cross-stimulatory for the TCR of cells, which have been primed previously or simultaneously, e.g., in the frame of a generalized viral infection. The combined evaluation of the immunologic reactivity of drug-specific TCC and their way of drug “recognition” leads to the concept of a pharmacological interaction of drugs with immune receptors, briefly called p–i concept [10]. Drugs – in contrast to classical protein antigens – are not confined and concentrated in the lymph and lymphatic tissues, but are available throughout the body. If a sufficient drug concentration is reached in the tissue in vivo, drugs may not only fulfill their intended activity, e.g., block some receptor or enzyme activity, but also interact with the specific immune system, in particular T cells. Recent findings indicate that Teffector-memory cells are highly concentrated in the skin [11], where they may act as “sentinel” cells, rapidly stimulated by antigen penetrating the skin [12]. This feature makes this organ predestined to become affected in hypersensitivity reactions due to the p–i concept. Thus, the p–i concept may have the main role in (cutaneous) reactions to systemically applied drugs without previous sensitization and for drugs, which were not metabolized to reactive compounds.

Clinical Approach to the Diagnosis Adverse drug type B reactions address mainly two questions: • Is it an immune-mediated drug hypersensitivity? • What is the causative drug? There is no single standardized diagnostic test that will answer these questions. Thus, immune-mediated drug hypersensitivity reactions and their causative drugs

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have to be recognized by the constellation of exposure, timing, and clinical features including the pattern of organ manifestation. From a practical clinical approach adverse drug reactions (type B) are classified into immediate hypersensitivity and nonimmediate-type hypersensitivity.

Immediate-Type Hypersensitivity Reactions Clinical Aspects Immediate-type hypersensitivity reactions may comprise type I allergies or poorly understood mechanisms like “pseudoallergic reactions” (non-immune-mediated hypersensitivities). Type I allergies or “pseudoallergic reactions” manifest typically as urticaria (Fig. 1), angioedema, and/or anaphylaxis. Anaphylaxis refers to a severe allergic reaction involving multiple organs. Reactions typically follow a uniphasic course; however, 20% will be biphasic in nature [13]. Severe respiratory and cardiovascular manifestations such as arterial hypotension and cardiovascular collapse may be the primary manifestations, in particular, in perioperative reactions [14].

Fig. 1 Drug-induced urticaria

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B. Schnyder and W.J. Pichler Table 1 Types of immediate reaction and frequently involved drugs IgE-mediated reaction Intravenous administration of antibiotics such as penicillins and cephalosporins Latex Neuromuscular blocking agentsa Contrast mediaa Pseudoallergic reactions Asprin and other nonsteroidal antirheumatics Plasma volume expander (colloids) Neuromuscular blocking agentsa Contrast mediaa a Drugs which can induce IgE-mediated or pseudoallergic reactions

IgE-mediated allergies (type I) occur mostly within less than 1 h after drug administration [15]. Sometimes, especially after oral drug intake, mild reactions may even appear after a few hours. It has been a dogma that IgE reactions arise only on reexposure. However, recently, IgE reactions have been observed on the first exposure. This might be best explained by cross-reactivity between the involved drug and other xenobiotics, which the affected subject was exposed to beforehand. “Pseudoallergic reactions” tend to arise within 1–3 h after drug administration. They require higher dose than true IgE-mediated allergies and the typical initial symptoms for anaphylaxis, namely palmar and/or plantar itch, may be less common. They can be elicited by many drugs, but some drugs seem to elicit them more often (Table 1). Certain groups of people seem to show a higher susceptibility to react in this way – and they develop similar, mostly mild symptoms to a quite heterogeneous panel of drugs, with clearly distinct chemical and pharmacological features.

Laboratory Investigations and Skin Tests Acute Phase In the acute phase mast-cell activation can best be confirmed by assessment of the total tryptase in the serum 60–120 min after the onset of symptoms. A rise can be quantified as soon as 30 min after the onset of symptoms. Tryptase’s half-life is about 120 min. To enable comparison with baseline levels, a new sample should be collected >2 days after the reaction [16]. Serum tryptase does not differentiate between immunological and nonimmunological mast-cell activation. Identification of the Causative Drug In IgE-mediated reaction, skin test or the assessment of specific IgE may provide some help in the identification of the causative drug. Commonly, it is recommended to perform them within 1–6 months after the acute event, as effector cells may be exhausted or specific IgE antibodies are temporarily depleted. On the other hand, sensitivity of the tests might decrease over time and tests should be done in the

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first half-year after the reaction. In cases with severe anaphylactic reactions it is advisable to first perform skin test(s) with the presumably causative drug(s) only by prick. If responses are negative, intradermal tests may than be carried out. In cases with mild reactions testing might be started with intradermal tests, as these tests are more sensitive. For penicillin allergies standardized preparations with penicilloylpolylysine (PPL) and minor determinant mixture (MDM) are commercially available (Diater Laboratories, Madrid, Spain). While earlier American reports found a high sensitivity and specificity of skin tests with such standardized preparations, newer reports of Spanish researchers detected a sensitivity of only 70% [17]. Penicillin allergy may also be evaluated by in vitro test for specific IgE; however, these tests have a low sensitivity. The in vitro detection of IgE to other drugs has not been established, although some assays to suxamethonium, sufamethoxazol are offered. Reliable data on sensitivity and specificity of both skin tests and specific IgE for other drugs than betalactams are very limited. In pseudoallergic reactions skin tests and determination of specific antibodies are not helpful for identification of the causative drug. In selected cases diagnostic oral provocation tests may be performed. More frequent are tests with alternative drugs to establish a safe substitution. In vitro tests such as the basophil activation test (BAT) have been proposed as a tool to identify the relevant drug. This BAT is based on flow cytometric quantification of allergen/drug-induced CD63 expression, CD203 upregulation, or measurement of sulfo leukotriene release by enzymelinked immunosorbent assay (ELISA). The sensitivity appears to be superior to CAP-based IgE determinations, but lower than skin tests. But a role of BAT in the diagnosis of pseudoallergic reaction could not be established.

Nonimmediate-Type Hypersensitivity Reactions Clinical Aspects Nonimmediate reactions are those, which occur more than 1 h after drug administration. However, there is a certain overlap in the time of manifestations: Some IgE-mediated and pseudoallergic reactions occur after 1 h, some nonimmediate reactions may start prior to 1 h. Nonimmediate drug allergies are most frequently T-cell-mediated; whereas IgG-mediated responses are rare. Nonimmediate drug allergies may clinically manifest in a broad spectrum of diseases (sections from Maculopapular or “Morbilliform’ Exanthema” to Systemic Drug Reactions Drug Rash with Eosinophilia and Systemic Symptoms) (Table 2). Maculopapular or “Morbilliform” Exanthema The most frequent type B reactions are maculopapular or “morbilliform”, exanthema, which are observed in about 2% of hospitalized patients [18] (Fig. 2). They usually appear >2–9 days after the drug has been started. A large body of experimental

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Table 2 Types of nonimmediate reaction and frequently involved drugs Type II

Type III

Hemolytic anemia Leucopenia Thromopenia Serum sickness syndrome Drug-induced lupus Vasculitis

Type IV

Maculopapular exanthema (MPE) Fixed drug eruption Symmetrical drug-related intertriginous and flexural exanthema (SDRIFE) Acute generalized exanthematous pustulosis (AGEP) Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) Drug rash with eosinophilia and systemic symptoms (DRESS)

Methyldopa, levodopa, mefenamic acid, interferon-alpha, cyclosporin and fludarabin Aminopyrine Heparin Penicillins and cephalosporins Hydralazine, procainamide, quinidine, minocycline, statins, and anti-TNF-alpha agents Cefaclor, sulfa antibiotics, cephalexin amoxicillin, nonsteroidal anti-inflammatory drugs, and diuretics Betalactams, sulfa antibiotics, quinolon, diuretics Phenytoin, sulfa antibiotics, tetracycline, barbiturates Antibiotics like penicillin, streptomycin, corticosteroids, anticoagulants Aminopenicillins, sulfa antibiotics, diltiazem Nefirapine, allopurinol, oxicames, sulfa antibiotics, phenytoin, carbamazepine, barbiturates Anticonvulsants, allopurinol, sulfa antibiotics, dapsone, minocyclin, sulfasalazine

and clinical data indicates that these rashes correspond very often to T-cell-mediated (type IV) hypersensitivities. Fixed Drug Eruption The most characteristic finding of a fixed drug eruption is recurrence of similar lesions at the same sites and healing with residual hyperpigmentation. Diagnosis is not always easy; for example, as for nonpigmenting fixed drug eruptions, which have no residual hyperpigmentation. The reason for their recurrence at the same sites is still unknown. The validity of skin tests for identification of the causative drug is not known. To date, re-challenge is the most reliable method of identifying causative drugs, but skin tests, especially in the affected area, has gained the attention of investigators [19].

Symmetrical Drug-Related Intertriginous and Flexural Exanthema Rarely drug eruptions may present with a characteristic distribution pattern that is confined to a sharply defined symmetrical erythema of the gluteal area and the flexural or intertriginous folds without any systemic symptoms and signs. This con-

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Fig. 2 Maculopapular or “morbilliform” exanthema

dition has been called “baboon syndrome.” Originally, the baboon syndrome was described as a special form of systemic contact-type dermatitis (SCTD) that occurs after ingestion or systemic absorption of a contact allergen in individuals previously sensitized by topical exposure to the same allergen in the same areas. But frequently, a flare-up is not a reasonable explanation for the unusual localization [20]. Similar eruptions have been reported after systemic exposure to betalactam antibiotics and other drugs. Latency periods vary between a few hours and a few days after exposure. Thus, it is believed that it represents a type of recall phenomenon and that the characteristic localization and appearance of the eruption is determined by an earlier, unrelated dermatitis that had occurred in precisely the same areas.

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Symmetrical drug-related intertriginous and flexural exanthema (SDRIFE) specifically refers to the distinctive clinical pattern of this drug eruption, and the following diagnostic criteria are proposed: (1) exposure to a systemically administered drug either at the first or repeated dose (excluding contact allergens); (2) sharply demarcated erythema of the gluteal/perianal area and/or V-shaped erythema of the inguinal/perigenital area; (3) involvement of at least one other intertriginous/ flexural localization; (4) symmetry of affected areas; and (5) absence of systemic symptoms and signs. It is a prognostically benign and often underdiagnosed drug eruption. Adverse drug reactions should be considered in the differential diagnosis of intertrigo, especially in atypical and therapy-resistant cases [21].

Acute Generalized Exanthematous Pustulosis Acute generalized exanthematous pustulosis (AGEP) is a rare disease [22, 23]. Its clinical hallmark is the presence of myriads of disseminated, sterile pustules in the skin, which appear rather rapidly, namely after 3–5 days of treatment. The patients have fever and massive leukocytosis in the blood, sometimes with eosinophilia. Epicutaneous patch test reactions can cause a similar pustular reaction locally (Fig. 3). Some pustules coalesce together and may form bullae. The lethality is about 4%, particularly in older people. This disease and the underlying T-cell reaction seems to be a model for sterile neutrophilic inflammations like pustular psoriasis and Behcet’s disease [24].

Fig. 3 Acute generalized exanthematous pustulosis (AGEP): positive reaction in patch testing

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Bullous Exanthema: Stevens–Johnson Syndrome and Toxic Epidermal Necrolysis The most severe forms of drug-induced skin reactions go along with formation of bullae. The most severe bullous skin diseases are Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). These diseases differ solely in the extent of affected skin (<10% in SJS and >30% in TEN, and SJS/TEN overlap syndromes with 10–30% skin detachment). They are clearly different from erythema exsudativum multiforme, which is mainly caused by viral infections [25], is often recurrent, and affects younger persons (mean age 24 years). However, also in SJS/ TEN an infectious origin (Mycoplasma sp., Klebsiella sp.) is suspected in ca. 5% of the cases, as no drug treatment was given the week before the disease started. Most reactions start within the first 3 weeks of treatment, but SJS/TEN induced by sulfonamide antibiotics may appear later. The bullous disease may progress very rapidly: SJS and TEN may start with a macular, purple-red exanthem, which can be painful – an ominous sign. Within 12 h bullae might develop – even after stopping the drug. Mucous membranes (mouth, genitalia) are involved with blister formation, and a purulent keratoconjunctivitis with formation of synechiae may result in permanent damage. Lethality is dependent on age and extent of skin detachment: In SJS it is 1–5%, and in TEN >25–35% [26]. Systemic Drug Reactions Drug Rash with Eosinophilia and Systemic Symptoms Some drugs (cf. Table 2), in particular anticonvulsants, are known to cause a severe systemic disease with fever, lymph-node swelling, massive hepatitis, and various forms of exanthemas: More rarely, patients develop colitis, pancreatitis, and interstitial lung disease [27]. Over 70% of these cases have a marked eosinophilia (often >1 G/l), and activated T cells are often found in the circulation, similar to acute HIV or generalized herpes virus infections. This syndrome has many names, the most frequently used are drug (induced) hypersensitivity syndrome (DHS or DiHS) or drug rash with eosinophilia and systemic symptoms (DRESS). Importantly, the symptoms can start 12 weeks after the start of treatment, often after increasing the dose, and it may also persist and recur for many weeks even after cessation of drug treatment. Many patients have a facial swelling; some have signs of a capillary leak syndrome. The lethality is ca. 10%, and mainly due to liver failure. Typical is a recurrence of symptoms often in the third week. It is related to reactivation of herpes viruses, in particular, human herpes virus 6, EBV, or CMV. Japanese researchers have recently proposed diagnostic criteria for the diagnosis of this disease, which is still often not recognized [28] (Table 3). Cytopenia Drug-induced IgG-mediated immune cytopenia may manifest as hemolytic anemia, unexpected precipitous fall in peripheral leucocyte count, or thrombocytopenia. The diagnosis consists of identifying clinical symptoms, general laboratory inves-

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Table 3 Diagnostic criteria for DIHS established by a Japanese consensus croup [28] • Maculopapular rash developing >3 weeks after starting with a limited number of drugs • Prolonged clinical symptoms after discontinuation of the causative drug • Fever (>38°C) • Liver abnormalities (ALAT > 100 U/l) or other organ involvement • Leukocyte abnormalities (at least one present): 0 Leukocytosis (>11 × 109/l) 0 Atypical lymphocytosis (>5%) 0 Eosinophilia (>1.5 × 109/l) • Lymphadenopathy • HHV-6-reactivation (detected in the second to third week after start of symptoms) The diagnosis is confirmed by the presence of all seven criteria given above (typical DIHS) or five (1–5) of them (atypical DIHS).

tigations, such as total blood count and peripheral blood smear (e.g., to rule out pseudothrombocytopenia), and the clinical suspicion of a causal relationship with the culprit drug. Serology tests are only of limited diagnostic value (cf. section on Acute phase). Frequently involved drugs are mentioned in Table 2. Serum Sickness Syndrome In the past, the widespread use of heterologous serum frequently produced the full serum sickness syndrome 1–3 weeks after antiserum administration. Currently, non-protein drugs such as penicillins and cephalosporins are the most common causes of serum sickness. The typical clinical manifestations include skin eruptions (urticarial, maculopapular), fever, and multiple, large-joint arthralgias. Druginduced serum sickness is usually self-limited, with symptoms lasting 1–2 weeks before resolving. Drug-Induced Lupus Drug-induced lupus erythematosus (DILE) resembles naturally occurring systemic lupus erythematosus. The average age of patients with DILE is nearly twice that of patients with idiopathic SLE. Approximately half the patients with drug-induced SLE are women, compared with 90% of patients with idiopathic SLE. Similar to idiopathic lupus, DILE can be divided into systemic, subacute cutaneous, and chronic cutaneous lupus. The syndrome is characterized by arthralgia, myalgia, pleurisy, rash, and fever in association with antinuclear antibodies (antihistone) in the serum. The clinical and laboratory manifestations of drug-induced SLE are similar to those of idiopathic SLE, but central nervous system and renal involvement are rare in DILE. Recognition of DILE is important because it usually reverts spontaneously within a few weeks after stopping the drug [29].

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Vasculitis Drug-induced hypersensitivity vasculitis reportedly has an incidence of 10–30 cases per million people per year. Clinical manifestations range from small vessel hypersensitivity vasculitis and leukocytoclastic vasculitis to clinical syndromes indistinguishable from classical systemic forms of vasculitis such as Wegener’s granulomatosis, polyarteritis nodosa, and Churg–Strauss syndrome (CSS). Withdrawal of the offending agent alone is often sufficient to induce prompt resolution of clinical manifestations, obviating the need for systemic therapy [30].

Laboratory Assessment and Skin Tests in Nonimmediate-Type Hypersensitivity Reactions Acute phase During the acute phase of a suspected drug hypersensitivity reaction some laboratory tests are useful to determine the severity and involvement of internal organs. Patients with generalized exanthemas should be examined in regard to the peripheral blood cell count and liver enzyme. The finding of an eosinophilia may support the diagnosis of an immune-mediated hypersensitivity reaction. For atypical skin lesions a biopsy with histological examination may help to recognize or exclude an important differential diagnosis. In the rare cases with a presumed type II or III reaction the IgG-mediated mechanism may be supported by additional laboratory investigations: Positive direct anti-globulin test (Coombs’ test) represents a central criterion in diagnosing immune hemolytic anemia, leading to further detailed analyses, often requiring close collaboration of a number of disciplines [31]. Platelet serology in druginduced immune-thrombocytopenia is only of limited diagnostic value: The sensitivity of these tests is dependent on factors such as the concentration of the drug in the test and the potential sensitization of the patient by metabolites instead of the parent drug [32]. On the other hand, such antibodies are also detectable in a high percentage of patients not developing thrombocytopenia [33]. Assessment of complement levels (C3, C4, CH50) and immune complexes (C1q binding or Raji cell assays) can support the diagnosis in cases with serum sickness syndrome, but negative values do not rule it out.

Identification of the Causative Drug in T-Cell-Mediated Reactions Lymphocyte transformation tests (LTT) and/or skin tests (patch testing or also intradermal testing with reading at 24, 48 h, and later) with the suspected drug(s) may help to identify the causative agent. As provocation tests in nonimmediate-type hypersensitivity reactions are time-consuming and arduous for the patients, valuable data about the sensitivity and specificity of these tests are very limited. The LTT is complex and requires skilled personnel. Thus, its role

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in routine diagnostic procedure has not been established. Drug skin tests are best preformed 6 weeks to 6 months after complete healing of the skin eruption. Patch tests are performed with the commercialized form of the drug used by the patient diluted in petrolatum or water, intradermal tests with sterile sequential dilutions. The methods are described in more details in guidelines for performing skins tests with drugs, which has been proposed by the working party of the European Society of Contact Dermatitis [34].

Management of Drug Allergy

Acute Treatment Immediate-Type Hypersensitivity Reactions The management of acute allergic reaction involves withdrawal of the offending agent. In severe reactions with prominent respiratory and/or circulatory signs, corresponding to an anaphylaxis, adrenaline (10 mcg/kg) via the intramuscular route is the first-line drug of choice. In clinical practice, however, antihistamines and glucocorticoids are widely used, as the potential side effects of adrenalin (tachyarythmia, hypertension) are too much feared. Antihistamines cannot antagonize histamine that has already been bound on Histamin H1-receptors, therefore making it a secondary drug with little benefit in the initial treatment of anaphylaxis, particularly anaphylactic shock [35]. Glucocorticoids, when given in pharmacological quantities, have an anti-inflammatory effect, and may reduce the risk of late relapse [36]. However, the commencement of glucocorticoid’s action is late (>45 min) as it depends on new gene products.

Nonimmediate-Type Hypersensitivity Reactions In patients with systemic drug allergies the T-cell immune system is massively activated, similar to acute viral infections: These patients seem to have a higher tendency to react to a new drug. Sometimes they may also react with a flare up of their rash, e.g., to the alternative antibiotic given – but the second is later well tolerated, if the costimulatory conditions are not present any more. Thus, withdrawal of the causative, potentially causative, and not urgently needed drug is almost always prudent. However, there are some exceptions to this rule. In lateoccurring mild maculopapular exanthema with only little pruritus treatment may be continued, provided there is compelling clinical need to do so. Such “treating through” requires careful monitoring for systemic involvement (fever, eosinophilia, lymphadenopathia, hepatitis) or involvement of mucosal surfaces. Mild nonimmediate drug reactions are self-limited diseases and treatment is symptomatic. Corticosteroids (topical, sometimes systemic) and/or antihistamines

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are used to block or reduce prolonged or late phase reactions. However, there are scarce data, which support the benefit of such a treatment. The management of SJS/TEN patients should best be undertaken in intensive care units, best with experience in therapy for burns: warming of the environment, correction of electrolyte disturbances, administration of a high caloric enteral intake, and prevention of sepsis. Efficacy of drugs used in some case reports is difficult to evaluate: cyclosporin, cyclophosphamide, pentoxyfilline, and thalidomide have all been tried [37]. Recent data support corticosteroid use [38] but its value has not been established yet. Intravenous immunoglobulins are widely used but their effectiveness is still debated [39]. Specific nursing care and adequate topical management reduce associated morbidity and allow a more rapid re-epithelialization of skin lesions. After healing, follow-up is needed for ophthalmologic and mucous membrane sequelae. Sun blocks are recommended. Avoidance of the responsible drug and chemically related compounds is essential not only for the patients, but also for first-degree relatives [37]. For DRESS, corticoids are used in cases of life-threatening systemic impairment, which is often heaptitis. Occasionally, liver transplantation is necessary.

Cross-Reactivity and Recommendations of Alternative Drugs Cross-reactivity among drugs may be either mediated by immunologic mechanisms or other pathways. Immunologic cross-reactivity is due to the presence of common antigenic determinants in involved drugs. In the case of compounds provoking non-allergic hypersensitivity reactions, cross-reactivity is explained by a common pharmacological characteristic, such as the inhibitory effect of nonsteroidal anti-inflammatory drugs on cyclooxygenase-1 and the assumed ability of muscle relaxants or contrast media to release histamine through a non-immunologic mechanism. In choosing alternative compounds, skin testing has been used in evaluating IgE-mediated cross-reactivity between penicillins and cephalosporins, as well as among muscle relaxants. In assessing T-cell-mediated cross-reactivity among contrast media, corticosteroids, anticonvulsants, and heparins, delayed-reading intradermal tests and patch tests, together with lymphocyte transformation tests, can be performed. However, the sensitivity of in vivo and in vitro testing remains limited. In patients who especially require one specific drug also a graded challenge with the alternative drug may be performed. Betalactams IgE-mediated allergy: When a patient with immediate penicillin allergy requires an alternative betalactam drug, consideration can be given to prescribing a cephalosporin. A review of several studies of cephalosporin administration to patients with a history of penicillin allergy found cephalosporin reactions in

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4.4% of patients with positive skin tests for penicillin [40]. Attention should be given to avoid an alternative with an identical side chain (e.g., amoxicillin and cephadroxil). The incidence of cross-hypersensitivity reactions between penicillins and carbapenems may be lower than previously reported and lies at about 10%. [41]. Thus, carbapenem may be used in penicillin-allergic patients. T-cell-mediated allergy: In contrast to IgE-mediated reactions only very few data exist about nonimmediate reactions. In vitro data indicate that cross-reactivity of T cells between penicillins and cephalosporins seems to be very rare [42]. This is in agreement with our clinical experience. Sulfonamides The putative cross-reactivity among sulfonamides is a common clinical problem. Indeed, there has been demonstrated in vitro certain cross-reactivity on TCR level [43, 44]. Two regions of the sulfonamide antibiotic chemical structure are implicated in the hypersensitivity reactions associated with the class: • The first is the N1 heterocyclic ring, which causes a type I hypersensitivity reaction. • The second is the N4 amino nitrogen that, in a stereospecific process, forms reactive metabolites that cause either direct cytotoxicity or immunologic response. Most of the nonantibiotic sulfonamides lack both of these structures. Therefore, crossreactivity between sulfonamide antibiotics and nonantibiotics (e.g., diuretics, sulfonylureas, celecoxib, and sumatriptan) are highly unlikely [45, 46]. However, such cross-reactions occur with the anti-inflammatory compound sulfasalazine, which is metabolized to the sulfonamide antibiotic sulfapyridine. There is no relationship between sulfonamide allergy and intolerance to sulfite preservatives in food [46]. Contrast Media and Iodinated Drug IgE-mediated cross-reactivity between different contrast media is rather rare. However, cross-reactivity in delayed-type reaction is frequent and may be a result of the presence of contrast media-specific T cells, some of which show a broad cross-reactivity pattern. Iodide ions, known to be present at low concentration in contrast media solutions, do not seem to be the causative moiety. Detailed intradermal skin testing or in vitro analysis might help identify non-cross-reactive contrast media [47]. Local Anesthetics Adverse reactions to local anesthetics are common and are mostly of pharmacological or toxic origin. True allergic reactions with local anesthetics are rare and are mostly involving specific T cells. Sensitization with contact dermatitis occurs mostly via lidocain-containing ointments. Immediate allergy to local anesthetics

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remains extremely rare with only few authentic documented cases being published to date. Skin tests offer a quite reliable method for exploring delayed allergy, for immediate allergy their specificity is rather low [48].

Corticosteroids Topical corticosteroid sensitivity produces classic allergic contact dermatitis. Very rarely, allergy to a topical corticosteroid ointment or crème is associated with allergy to oral or injected corticosteroids, while an associated allergy to a nasal or bronchial corticosteroid can occasionally be observed. Allergy to a specific topical steroid is frequently associated with allergy to other corticosteroids [49] (Table 4).

Multiple Drug Hypersensitivity Syndromes The term multiple drug hypersensitivity is widely used for different forms of side effects to various drugs: Some use it to characterize patients with multiple drug intolerance (pseudoallergy), others for well-documented repeated immune-mediated reactions to structurally not related drugs [50]. Cross-reactivity due to a structural similarity is not included. About 10% of our patients with well-documented drug hypersensitivity (skin and/or lymphocyte transformation test positive) have multiple drug allergies [51]: They may have reacted to injected lidocain with massive angioedema, years later the same patient develops a contact allergy to corticosteroids, or a patient reacts to amoxicillin, phenytoin, and sulfamethoxazole within a few months, but with different symptoms (MPE, DRESS, erythrodermia). Most patients have had rather severe reactions to at least

Table 4 Cross-reactivity among different steroids [49] Group A: hydrocortisone and tixocortol type

Group B: triamcinalone acetonide type

Group C: betamethasone type

Group D: hydrocortisone and clobetasone 17 butyrate type

Hydrocortisone acetate, prednisone, prednisolone acetate, methylprednisolone, meprednisone, cortisone, cortisone acetate, tixocortol pivalate Triamcinalone, budesonide, amcinonide, desonide, halcinonide, fluocinonide, fluocinolone acetonide Betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone Hydrocortisone butyrate, hydrocortisone17-valerate, clobetasol propionate, clobetasone 17 butyrate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, fluocortolone pivalate, mometasone furorate, fluticasone propionate, fluprenidene acetate

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one drug. An IgE-mediated reaction might be followed by a T-cell-mediated reaction. The pathomechansim of this syndrome is unknown, but a clear sensitization to the various drugs has been documented repeatedly in single cases. One explanation might be a deficient tolerance mechanism to small compounds. Preliminary data suggest indeed that a previous drug allergy might be a risk factor for the development of a delayed hypersensitivity reaction to contrast media [52].

Drug Desensitization Desensitization for drug allergy may have a role in those patients for whom no alternative drugs exists. Gradual reintroduction of small doses of the drug at fixed time intervals allows for the delivery of full therapeutic doses, protecting patients from anaphylaxis [53]. The desensitized state is not permanent – in contrast to some allergen immunotherapy – and is sustained only with a daily maintenance dose of the drug. While most of the studies have been performed in penicillin-allergic patients, this procedure has been used safely with modified desensitization protocols in patients with sensitivity to other agents. Classically, the procedure is applied in IgE-mediated reactions, but its use has been extended to other drug reactions [54]. Drugs for which desensitization may be successful include allopurinol, cotrimoxazole, betalactam antibiotics, and cisplatin. Patients with aspirin intolerance can also be desensitized with occasional beneficial effect in asthma and polyposis symptoms [55]. The mechanism of drug desensitization is not well understood.

References 1. Gomes ER, Demoly P. Epidemiology of hypersensitivity drug reactions. Curr Opin Allergy Clin Immunol 2005; 5:309–316 2. Rawlins M, Thompson W. Mechanisms of adverse drug reactions. In: Davies D, ed. Textbook of adverse drug reactions. New York: Oxford University Press; 1991, pp. 18–45 3. Naisbitt DJ, Grodon SF, Pirmohamed M, Park BK. Immunological principles of adverse drug reactions: the initiation and propagation of immune responses elicited by drug treatment. Drug Saf 2000; 23:483–507 4. Gerber BO, Pichler WJ. Cellular mechanisms of T cell mediated drug hypersensitivity. Curr Opin Immunol 2004; 16:732–737 5. Aster RH. Drug-induced immune cytopenias. Toxicology 2005 Apr 15; 209(2):149–153 6. Warkentin TE, Levine MN, Hirsh J, Horsewood PD, Roberts S, Gen M, Kleton JG. NJEM, heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. N Engl J Med 2007; 356:891–893 7. Riley R, Leeder J. In vitro analysis of metabolic predisposition to drug hypersensitivity reactions. Clin Exp Immunol 1995; 99:1–6 8. Schnyder B, Mauri-Hellweg D, Zanni M, Bettens F, Pichler WJ. Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alpha-beta T cell clones. J Clin Invest 1997; 100:136–141 9. Zanni MP, von Greyerz S, Schnyder B, Brander KA, Frutig K, Hari Y, Valitutti S, Pichler WJ. HLA-restricted, processing- and metabolism-independent pathway of drug recognition by human alpha beta T lymphocytes. J Clin Invest 1998; 102:1591–1598

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10. Pichler WJ. Delayed drug hypersensitivity reactions. Ann Intern Med 2003 Oct 21; 139(8):683–693 11. Clark RA, Chong B, Mirchandani N, Brinster NK, Yamakana K, Dowgiert RK, Kupper TS. The vast majority of CLA+ T cells are resident in normal skin. J Immunol 2006; 176:4431–4439 12. Schaerli P, Moser B. Chemokines: control of primary and memory T-cell traffic. Immunol Res 2005; 31:57–74 13. Ellis AK, Day JH. Diagnosis and management of anaphylaxis. Can Med Assoc J 2003; 169:307–311 14. Mertes PM, Laxenaire MC. Allergic reactions occurring during anaesthesia. Eur J Anaesthesiol 2002; 19:240–262 15. Weiss ME, Adkinson NF. Immediate hypersensitivity reactions to beta-lactam antibiotics. Ann Int Med 1987; 107:204–215 16. Ebo DG, Fisher MM, Hagendorens MM, Bridts CH, Stevens WJ. Anaphylaxis during anaesthesia: diagnostic approach. Allergy 2007; 62:471–487 17. Torres MJ, Romano A, Mayorga C, Moya MC, Guzman AE, Reche M, Juarez C, Blanca M. Diagnostic evaluation of a large group of patients with immediate allergy to penicillins: the role of skin testing. Allergy 2001 Sept; 56(9):850–856 18. Fiszenson-Albala F, Auzerie V, Mahe E, Farinotti R, Durand-Stocco C, Crickx B, DescampsV. A 6-month prospective survey of cutaneous drug reactions in a hospital setting. Br J Dermatol 2003 Nov; 149(5):1018–1022 19. Lee AY. Fixed drug eruptions. Incidence, recognition, and avoidance. Am J Clin Dermatol 2000 Sept–Oct; 1(5):277–285 20. Wolf R, Orion E, Matz H. The baboon syndrome or intertriginous drug eruption: a report of 11 cases and a second look at its pathomechanism. Dermatol Online J 2003; 3(9):2 21. Hausermann P, Harr T, Bircher AJ. Baboon syndrome resulting from systemic drugs: is there strife between SDRIFE and allergic contact dermatitis syndrome? Contact Dermatitis 2004 Nov–Dec; 51:297–310 22. Britschgi M, Steiner UC, Schmid S, Depta JP, Senti G, Bircher A, Burkhart C, Yawalkar N, Pichler WJ, et al. T-cell involvement in drug-induced acute generalized exanthematous pustulosis. J Clin Invest 2001; 107:1433–1441 23. Roujeau J, Bioulac-Sage P, Bourseau C. Acute generalized exanthematous pustulosis: analysis of 63 cases. Arch Dermatol 1991; 127:1333–1338 24. Keller M, Spanou Z, Schaerli P, Britschgi M, Yawalkar N, Seitz M, Villiger PM, Pichler WJ. T cell-regulated neutrophilic inflammation in autoinflammatory diseases. J Immunol 2005; 175:7678–7686 25. Roujeau JC, Stern RS. Severe adverse cutaneous reactions to drugs. N Engl J Med 1994; 331:1272–1285 26. French LE. Toxic epidermal necrolysis and Stevens Johnson syndrome: our current understanding. Allergol Int 2006; 55:9–16 27. Knowles SR, Shapiro LE, Shear NH. Anticonvulsant hypersensitivity syndrome: incidence, prevention and management. Drug Saf 1999; 21:489–501 28. Shiohara T, Inaoka M, Kano Y. Drug induced hypersensitivity syndrome (DIHS): a reaction induced by a complex interplay among herpesvirus and antiviral and antidrug immune responses. Allergol Int 2006; 55:1–8 29. Sarzi-Puttini P, Atzeni F, Capsoni F, Lubrano E, Doria A. Drug-induced lupus erythematosus. Autoimmunity 2005 Nov; 38(7):507–518 30. Doyle MK, Cuellar ML. Drug-induced vasculitis. Expert Opin Drug Saf 2003; 2:401–409 31. Pruss A, Salama A, Ahrens N, Hansen A, Kiesewetter H, Koscielny J, Dorner T. Immune hemolysis-serological and clinical aspects. Clin Exp Med 2003 Sept; 3(2):55–64. 32. Van den Bernt PM, Meyboom RH, Egberts AC. Drug-induced immune thrombocytopenia. Drug Saf 2004; 27(15):1243–1252 33. Greinacher A, Eichler P, Lubenow N, Kiefel V. Drug-induced and drug-dependent immune thrombocytopenias. Rev Clin Exp Hematol 2001 Sept; 5(3):166–200

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34. Barbaud A, Concalo M, Bruynzeel D, Bircher A. European Society of Contact Dermatitis. Guidelines for performing skin tests with drugs in the investigation of cutaneous adverse drug reactions. Contact Dermatitis 2001 Dec; 45(6):321–328 35. Evans C, Tippins E. Emergency treatment of anaphylaxis. Accid Emerg Nurs 2005 Oct; 13(4):232–237. Epub 2005 Sept 21 36. Ewan PW, Anaphylaxis. Br Med J 1998; 316:1442–1445 37. Ghislain PD, Roujeau JC. Treatment of severe drug reactions: Stevens-Johnson syndrome, toxic epidermal necrolysis and hypersensitivity syndrome. Dermatol Online J 2002 June; 8:5 38. Karaun SH, Jonkman MF. Dexamethasone pulse therapy for Stevens-Johnson syndrome/toxic epidermal necrolysis. Acta Derm Venereol 2007; 87(2):144–148 39. Mittmann N, Chan BC, Knowles S, Shear NH. IVIG for the treatment of toxic epidermal necrolysis. Skin Ther Lett 2007 Feb; 12(1):7–9 40. Kelkar PS, Li JT. Cephalosporin allergy. N Engl J Med 2001; 345:804–809 41. Sodhi M, Axtell SS, Callahan J, Shekar R. Is it safe to use carbapenems in patients of allergy to penicillin? J Antimicrob Chemother 2004; 54:1155–1157 42. Mauri-Hellweg D, Zann M, Frei E, Bettens F, Brander C, Mauri D, Padovan E, Weltzin HU, Pichler WJ. Cross-reactivity of T cell lines and clones to beta-lactam antibiotics. J Immunol 1996; 157:1071–1079 43. von Greyerz S, Zanni MP, Frutig K, Schnyder B, Burkhart C, Pichler WJ. Interaction of sulfonamide derivatives with the TCR of sulfamethoxazole-specific human αß+ T cell clones. J Immunol 1999; 162:595–602 44. Schmid DA, Depta JP, Lüthi M, Pichler WJ. Transfection of drug-specific T-cell receptors into hybridoma cells: tools to monitor drug interaction with T-cell receptors and evaluate crossreactivity to related compounds. Mol Pharmacol 2006 July; 70(1):356–365 45. Brackett CC. Likelihood and mechanisms of cross-allergenicity between sulfonamide antibiotics and other drugs containing a sulfonamide functional group. Pharmacotherapy 2004; 24(7):856–870 46. Strom BL, Schinnar R, Apter AJ, et al. Absence of cross-reactivity between sulfonamide antibiotics and sulfonamide nonantibiotics. N Engl J Med 2003; 349:1628–1635 47. Lerch M, Keller M, Britschgi M, Kanny G, Tache V, Schmid DA, Beeler A, Gerber BO, Luethi M, Bircher AJ, Christiansen C, Pichler WJ. Cross-reactivity patterns of T cells specific for iodinated contrast media. J Allergy Clin Immunol 2007 June; 119(6):1529–1536. Epub 2007 48. Thyssen JP, Menné T, Elberling J, Plaschke P, Johansen JD. Hypersensitivity to local anaesthetics–update and proposal of evaluation algorithm.Contact Dermatitis 2008 Aug; 59(2):69–78 49. Lutz EM, El-Azhary AR. Allergic contact dermatitis due to topical application of corticosteroids: review and clinical implications. Mayo Clin Proc 1997; 72:1141–1144 50. Sullivan T. Studies of the multiple drug allergy syndrome. J Allergy Clin Immunol 1989; 83:270 51. Gex-Collet C, Helbling A, Pichler WJ. Multiple drug hypersensitivity–proof of multiple drug hypersensitivity by patch and lymphocyte transformation tests. J Investig Allergol Clin Immunol 2005; 15:293–296 52. Kanny G, Pichler W, Morisset M, Franck P, Marie B, Kohler C, Renaudin JM, Beaudouin E, Laudy JS, Moneret-Vautrin DA. T cell-mediated reactions to iodinated contrast media: evaluation by skin and lymphocyte activation tests. J Allergy Clin Immunol 2005; 115:179–185 53. Castells M. Desensitization for drug allergy. Curr Opin Allergy Clin Immunol 2006; 6:476–481 54. Solensky R. Drug desensitization. Immunol. Allergy Clin N Am 2004; 24:425–443 55. Stevenson DD. Aspirin and NSAID sensitivity. Immunol Allergy Clin N Am 2004 Aug; 24(3):491–505

State of the Art: Multiple Chemical Sensitivity Michael Lacour, Klaus Schmidtke, Peter Vaith, and Carl Scheidt

The ‘multiple chemical sensitivity syndrome’ (MCS) is defined descriptively as a (1) chronic condition (2) with symptoms that recur reproducibly (3) in multiple organ systems (4) in response to low levels of exposures (5) to multiple unrelated chemicals and which (6) improve or are resolved when incitants are removed. Patients’ symptom profiles indicate that two different symptom clusters exist with predominant ‘central’, to the central nervous system (CNS)-related, and ‘peripheral’, mucosa-associated chemical sensitivities. The latter is identical with occupational rhinitis and asthma. Thus, an MCS diagnosis should only be made in cases with predominant exposure-related, nonspecific complaints of the CNS. Our understanding of the aetiology and pathogenesis of MCS is still incomplete. There is sufficient evidence that psychological, but not neurophysiologic or biological factors contribute to the induction and perpetuation of MCS. There are no specific laboratory tests to establish an MCS diagnosis. Differential diagnostic procedures encompass a clarification analogous to chronic fatigue syndrome (CFS) standards, evaluation of additional symptoms and a psychiatric evaluation. At present, no evidence-based treatment options exist.

Introduction The first attempts to describe the phenomenon of multiple chemical sensitivities were made by Randolph in 1954 and Rea in 1978 [1, 2]. The syndrome they intended to specify is characterized by a wide range of symptoms resulting from M. Lacour () and C. Scheidt Department of Psychotherapy and Psychosomatic Medicine, Freiburg University Hospital, Hauptstraße 8, D-79104 Freiburg, Germany e-mail: [email protected] K. Schmidtke Center for Geriatric Medicine and Gerontology, Freiburg University Hospital, Lehener Straße 88, D-79106 Freiburg, Germany P. Vaith Department of Rheumatology and Clinical Immunology, Freiburg University Hospital, Hugstetter Straße 55, D-79106 Freiburg, Germany

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functional disturbances in different organ systems. Randolph and Rea thought that multiple chemical sensitivities are triggered by hazardous substances and other environmental factors that induce ‘environmental overload’. As a result, they developed a new medical discipline called ‘clinical ecology’. In 1987, Cullen made an attempt to develop a case definition of the ‘multiple chemical sensitivity syndrome’ (MCS) [3, 4]. He described a chronic condition of symptoms that recur reproducibly in multiple organ systems in response to low levels of exposures to multiple unrelated chemicals. Others question the idea of MCS being a distinct syndrome with a specific underlying aetiology and pathogenesis [5–8]. In the following review, essentials of the scientific literature are reviewed.

Age and Sex Distribution, Prevalence Population-based investigations suggest that persons reporting chemical sensitivities come from all age, income and occupational groups [9–11]. Depending on the population surveyed, the prevalence of self-reported multiple chemical sensitivities lies between 0.3% and 13.1% [11–13]. Physician-diagnosed MCS was found in 0.5–6.3% of the subjects in the general population and in 0.2–3.4% of deployed Gulf War veterans [9–11, 14–16].

Common ‘Triggers and Stimuli’ The medical history of patients with self-reported multiple chemical sensitivities frequently shows that they have previously undergone exposure to various or distinct chemical compounds [17]. These may be cleaning products, tobacco smoke, perfume, pesticides, especially organophosphates, carbamates, pyrethroides, a variety of organic solvents, car exhaust or complex mixtures of substances related to ‘sick building syndrome’ [18–23]. The degree of chemical exposures eliciting symptoms lies below known toxic thresholds and in some circumstances may be less than 1% of established threshold values [3]. The majority of chemical sensitive individuals respond to these exposures immediately [24]. In Gulf War veterans different threats from chemical and biological weapons, medical countermeasures and vaccinations are regarded as possible triggers of MCS [25, 26]. Other environmental stimuli such as electromagnetic fields, radiation or noise may also elicit symptoms [27–29]. There is no proof for the pathogenic significance of all these triggers. As a result it was proposed to speak descriptively of idiopathic environmental intolerance (IEI) [30].

‘Central’ Symptoms of Chemical Sensitivity The symptom complex of MCS includes exposure-related disorders that affect multiple organ systems [3, 4]. Exposure-related symptoms frequently occur with self-reported odour hypersensitivity, but in rare cases hyposmia occurs [31, 32].

Clinical cross-sectional study

Clinical follow-up study

Clinical cross-sectional study

Clinical cross-sectional and follow-up study Retrospective clinical evaluation

Black et al. (1990) [34]

Black et al. (2000) [35]

Buchwald and Garrity (1994) [36]

Lax and Hennenberger (1995) [37]

Lohmann et al. (1996) [38]

MCS diagnosis according to modified Cullencriteria (n = 35) MCS diagnosis by clinical ecologist (n = 136)

MCS diagnosis by allergist/clinical ecologist (n = 30)

MCSb-diagnosis by clinical ecologist (n = 18)

Diagnosis of EIa by clinical ecologist (n = 26)

Headaches (83.8%) Vertigo (83.1%) Sensation of coldness of extremities (75%)

Respiratory (58%) Neurologic (inclusive headaches) (38%) Fatigue/weakness (35%) Headaches (61%) Gastrointestinal (44%) Dermatologic (44%) pain (44%) Fatigue (90%) Muscle weakness (67%) Headaches (63%) Myalgias (63%) Symptoms of nervous system (majority) Diagnosis of neurotoxic disorders by neurologist

Non-MCS (n = 557)

CFSc-diagnosis (n = 30)

None

Normal subjects (n = 33)

(continued)

Headaches (76.6%) Vertigo (57.1%) Coldness of extremities (64.1%)

n.d.

Fatigue (100%) Muscle weakness (67%) Headaches (83%) Myalgias (77%)

n.d.

Table 1 Overview of the original literature of symptom profiles in patients with self-reported multiple chemical sensitivities/MCS (From [33]. With permission) Literature Study type Cases Main symptoms Main controls Main symptoms

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Cross-sectional study by questionnaire

Cross-sectional and follow-up study by questionnaire Cross-sectional study by questionnaire

Cross-sectional study by questionnaire

Cross-sectional study by questionnaire

Maschewsky and Oppl (2000) [39]

McKeown-Eyssen et al. (2000) [40]

Miller and Prihoda (2000) [42]

Ziem and Tamney (1997) [43]

b

EI: Environmental illness MCS: Multiple chemical sensitivity syndrome c CFS: Chronic fatigue syndrome d sr-mcs: Self-reported multiple chemical sensitivities e CNS: Central nervous system

a

Miller and Mitzel (1995) [41]

Study type

Table 1 (continued) Literature

sr-mcs (n = 91)

sr-mcs with reported exposures (n = 96)

sr-mcs attributed to pesticide exposures (n = 37)

sr-mcsd (n = 134)

sr-mcs (n = 613)

Cases

Fatigue, confusion, memory problems (60–70% daily or several days/week)

Cognitive symptoms (highest severity)

Fatigue (82%) Cognitive deficits (77%) Headaches (76%) Exposure-dependent CNSe symptoms (majority) Cognitive symptoms (highest severity)

Main symptoms

Follow-up of sr-mcs (n = 134) sr-mcs attributed to remodeling of buildings (n = 75) Female conference members and others None

None

Main controls

Cognitive symptoms (less severity, p = 0.0001)

Cognitive symptoms (highest severity)

Agreement (76.1%)

Main symptoms

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Non-specific complaints of the central nervous system (CNS) are the symptoms that are most frequently described by MCS patients as a response to chemical exposures [33]. These complaints include concentration problems, forgetfulness, headaches and other unspecific neurological or psychological symptoms [23]. This has been reported in several studies (see Table 1) [34–43], reviews and expert panels [44]. Non-specific CNS symptoms are showing exposure-related circumstances, highest severity rates and highest symptoms total agreement in test–retest investigations [40–42]. Computed severity scores ranked non-specific CNS symptoms after chemical exposures highest in a sample of 351 individuals reporting multiple chemical sensitivities [32]. Exposure-related circumstances and symptoms are critically based on the patients self-reports and may not be confirmed by provocation challenges or neuropsychological tests [45, 46]. Non-specific complaints of the CNS were also reported by Gulf War veterans who were suffering from ill health or who reported chemical sensitivities [14, 26, 47, 48]. In a survey of 42,818 UK military personnel, strongest association with Gulf War deployment were for mood swings and memory loss or lack of concentration [48]. In a factor analysis of symptoms, three symptom complexes (called ‘impaired cognition’, ‘confusion ataxia’ and ‘central pain’) were identified in ill Gulf War veterans [49]. Being confirmed by another factor analysis, the symptom clusters described show marked similarities with the non-specific complaints of the CNS frequently seen in MCS patients and highlight the importance of an overlap with multiple pain conditions [50]. On the contrary, two factor analyses did not support the existence of a unique syndrome affecting a subgroup of Gulf War veterans [51, 52]. In addition to the exposure-related aspects of MCS, patients also claim to be suffering from chronic, non-exposure-related fatigue, tiredness not relieved by rest or sleep and difficulties to concentrate [32]. Thus, there is a significant overlap between the complaints attributed to multiple chemical sensitivities and chronic fatigue syndrome (CFS) [53, 54]. These findings were confirmed by a systematic review which described high prevalence rates of fatigue and nearly identical frequencies of CFS and multiple chemical sensitivities in Gulf War veterans [13]. There is also significant overlap between symptoms of MCS and fibromyalgia (FM) [54]. Consequently, it was proposed to subsume MCS together with CFS and FM within the spectrum of chronic multi-symptom illnesses [55]. An overview on the prevalence of symptoms in MCS is shown in Fig. 1 [34, 38, 39].

‘Peripheral’ Symptoms of Chemical Sensitivity Some authors emphasize that symptoms related to mucosal irritations are more common in MCS patients after exposures to environmental irritants than symptoms of the CNS [12]. Frequencies of chemical sensitive patients’ non-specific complaints of the CNS and symptoms of mucosal irritations were equal in two studies [23, 56]. In a population-based study significant overlap between asthma and chemical sensitivities was found [57]. There might exist two distinct symptom clusters of a ‘central’, CNSrelated and a ‘peripheral’, mucosa-related symptom complex of chemical sensitivity. Other common features in patients with self-reported multiple chemical sensitivities

CNS

626

musculoskeletal

565

Symptoms

gastrointestinal

471

dermal

435

auditory

405

mucosal and respiratory

390

polyneuropathy-like

102

cardiovascular

44 0

100

200

300

400

500

600

700

Number of patients

Fig. 1 Self-reported complaints of 777 MCS patients (From [33]. With permission)

are allergic rhinitis, asthma and dermatitis, food allergy and intolerance, alcohol intolerance, or poor tolerance to prescription and recreational drugs [31, 58].

Course of Disease, Quality of Life Many scientists believe that the condition associated with MCS is chronic and symptoms show only a slight tendency towards spontaneous resolution [15, 35, 40, 44]. The majority of hypersensitive respondents characterize their symptoms as severe or moderately severe [9, 24]. Patients with self-reported multiple chemical sensitivities experience impairments of lifestyle and function, which is in accordance with higher rates of health care utilization [35, 59]. More than 13% of sensitive respondents reported losing their jobs [9, 10]. In Gulf War veterans, MCS and other multi-symptom conditions are highly correlated with multiple medical problems [15]. Severity of the initial symptoms, psychological distress and attributions proved to be important risk factors for the persistence of the complaints in Gulf War veterans [60].

Aetiology and Pathogenesis There is still incomplete understanding of the aetiology and pathogenesis of MCS [5, 6, 30, 61]. Concerning the possible central aspects of MCS with regard to nonspecific complaints of the CNS, the discussion centres on sensitization models of

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clinical ecology, biological factors of susceptibility, neurophysiologic models of sensitization, models of neurotoxicity as a result of low-level chemical exposures, psychological models of sensitization and other psychological or psychiatric factors that might contribute to the aetiology and pathogenesis of MCS [44, 62, 63]. All these mechanisms discussed are more or less hypothetical. Only high-level exposures show distinct and characteristic clinical findings of neurotoxicity, and the causal role of biological factors of susceptibility, sensitization and psychological factors is difficult to assess [64, 65]. Possible peripheral, mucosa-associated aspects of chemical sensitivity were linked to reactive airway dysfunction syndrome (RADS) and reactive upper airway dysfunction syndrome (RUDS) [66, 67]. Whether RADS and RUDS are distinct from occupational rhinitis and asthma is questionable [68, 69].

CNS-Related Health Effects by Chemical Exposures In recent research of occupational medicine, increasing data indicate that moderate exposures to organophosphates and other pesticides lead to non-specific symptoms of the CNS, changes in mood and affect in exposed individuals [65, 70]. One first indication that not only stress of war, but also different threats from chemical and biological weapons, vaccinations and medical countermeasures might have an influence in symptomatic ill health indicative for multiple chemical sensitivities came from the comparison of the health status of UK service personnel after the Gulf and Iraq wars [25, 26, 71]. Prevalence of symptoms that range from non-specific complaints of the CNS to multiple functional disturbances in a variety of other organ systems was only significantly increased in deployed military personnel in the Gulf War in 1991, but not in the Iraq war in 2003. An interpretation of these findings should take into account that improved health surveillance during and after the Iraq war may have reduced some health concerns and improved health status. In addition, it is controversial as to whether exposure levels to toxins encountered during the Persian Gulf war were effectual to induce Gulf War syndrome [25, 72]. In a causation analysis, the theory of low-level chemical neurotoxicity to induce MCS failed to meet the extended Bradford Hills’ criteria, which are recommended for use in the assessment of environmental exposures by the Centers for Disease Control and Prevention (CDC) [73, 74]. In conclusion, at present there is no proof for the existence of a low-level chemical neurotoxicity that induces CNS-related health effects.

Hypotheses on Susceptibility to Chemicals Clinical ecology explains susceptibility to chemicals by the chemical overload theory [1, 2]. In the meantime, pathogenic mechanisms were described more precisely and cover altered phase I cytochrome P450-mediated oxidative metabolism and

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phase II alterations in detoxifying enzyme systems [75, 76]. In a recent case-control study MCS was linked to higher cytochrome P4502D6 (CYP2D6) activity in connection with rapid acetylators, mediated by N-acetyltransferase (NAT2*6/*6) probably leading to toxicity by intermediates [77]. Others favour a nitric oxide (NO)-mediated reduction of cytochrome P450 activity to explain susceptibility to chemicals in MCS patients [78]. A cross-sectional study of 521 subjects (of whom 273 individuals were classified as having chemical-related sensitivity) showed that individuals with a deletion of glutathione-S-transferase (genotype GSTM1 or GSTT1) and slow acetylators (genotype NAT2*4/*4) are at high risk for developing self-reported chemical sensitivity [79]. All these investigations suffer methodically from the lack of a unique case definition that is sufficient for an MCS diagnosis and the problem to distinguish between a possible pathogenic role and epiphenomenon not related to complaints. In conclusion, at present there is no proof that altered biotransformation of ubiquitous chemical substances contribute to the development of chemical sensitivity.

Hypotheses on Allergic Mechanisms of Chemical Sensitivity Clinical ecology used to postulate an allergic mechanism is not yet precisely characterized, which causes sensitivity to environmental chemicals and foodstuff [1, 80]. However, no pathogenic linkage between MCS and specific mechanisms of type I–IV allergic reactions formerly described by Coombs and Gell was found [5, 6]. Despite these analyses, co-morbid IgE-mediated allergies as in allergic rhinitis and asthma (as well as other forms of allergies and intolerances) are common in MCS patients [57, 58]. Various pollutants (e.g. car exhaust) seem to play a role in the rising prevalence of allergic rhinitis and asthma over the last decades [81]. In addition, persons with allergic rhinitis are more sensitive to occupational exposures to irritant agents, even at sub-toxic levels [82]. The exact mechanism of occupational-induced airway inflammation remains unclear. An increasing data of evidence indicates that alterations in neuropeptide profiles, such as an increase in vasoactive intestinal peptide (VIP) and nerve growth factor (NGF), are of pathogenic importance in this type of airway inflammation [83]. Other possible mechanisms of occupational-induced airway inflammation are IgEmediated respiratory sensitization in response to low-molecular weight chemicals (e.g. formaldehyde, toluene diisocyanates, potassium dichromates) and IgG1 or IgG4-mediated allergen-specific airway reactions in response to high-molecular weight compounds (e.g. latex) [84]. A neurogenic mechanism of airway inflammation (‘neurogenic inflammation’) as a result of chemical sensitivity mediated by substance P was previously described in association with the so-called reactive airway dysfunction syndrome (RADS) and the reactive upper airway dysfunction syndrome (RUDS) that is probably identical with occupational rhinitis and asthma [66, 67]. RADS and RUDS may be mediated by chemical stimulation of

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vanilloid receptor (TRPV1) of C-fibres of trigeminal nerve ends, the major target of capsaicin, which consecutively induce nitric oxide (NO) release and increased N-methyl-d-aspartate (NMDA) receptor activity leading to further excessive NO and peroxynitrite formation that causes neurogenic inflammation, sensitization and tissue damage [66, 67, 85]. This hypothesis is supported by the evidence of NO and peroxynitrite elevation, increase of neopterin levels (which is an indicator of the activity of the inducible NO synthase), elevated oxidative stress, and elevated plasma levels of substance P, NGF and VIP in patients with self-reported multiple chemical sensitivities [86–88]. Further support comes from studies that found an increase of internal nose airflow resistance and cough frequency in association with capsaicin provocation or odorant challenges in patients with self-reported odour intolerance or multiple chemical sensitivities [89–91]. In summary, there is no evidence that specific allergic mechanisms contribute to the aetiology and pathogenesis of MCS. RADS and RUDS share pathomechanisms described in occupational rhinitis and asthma and should not be regarded as distinct MCS-related disorders.

Hypotheses on Immunological Mechanisms of Chemical Sensitivity Several investigations tried to determine the significance of immunological alterations in individuals reporting chemical sensitivity without conclusive results [5, 6, 17, 92]. In a recent study lower mean lymphocyte counts, and a higher percentage of cases with increased eosinophils counts were detected in patients suffering from multiple chemical sensitivities as compared to controls [93]. Another study found several cellular and humoral immune abnormalities in Gulf War veterans [94]. These encompass a higher percentage of individuals that fell outside of the expected range of T cells (CD3), a greater mean percentage of B cells (CD19), an elevation of CD4/CD8 ratio, a reduced lytic activity of natural killer (NK) cells, an altered distribution of mitogen stimulation of peripheral lymphocytes, greater autoantibody levels for several autoantigens, an elevation of immune complexes, and higher mean levels of antibodies to viruses of the herpes group. Based on these findings, a model was proposed that describes initial immune dysregulation by individuals with genetic susceptibility to chemicals that leads to a viral reactivation and consecutive alterations in the immune system referring to the symptoms of Gulf War syndrome. Other authors proposed that autoimmunity to vasoactive neuropeptides as a part of neurogenic inflammatory response or their receptors is involved in xenobiotic-associated neurotoxicity, implicating Gulf War syndrome and other fatigue-related disorders [95]. Concerning the described immunological findings, there are several methodical problems, e.g. selection bias, the problem of multiple testing and to distinguish between a possible pathogenic role and epiphenomenon not related to complaints, and the lack of reliability of immunological tests to

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evaluate MCS [96]. Thus, the significance of the described immune dysregulation and autoimmunity remains unclear.

Hypotheses on Psychoneuroimmunological Interactions and Neuroendocrinologic Alterations in Response to Low-Level Chemical Exposures The relevance of psychoimmunological interactions and neuroendocrinologic alterations in the pathogenesis of CFS, sick building syndrome and MCS are widely discussed [97]. Concerning psychological stress factors, two pathways were described in which these stressors might alter immune function. The first pathway is the hypothalamic–pituitary–adrenal (HPA) axis and the second is the sympathetic branch of the autonomic nervous system. Immunological alterations of the first pathway have been linked to pro-inflammatory cytokine release of interleukin-1 and a shift in immune system functioning towards a T-helper (Th)1 profile [98]. The sympathetic branch was linked to an activation of β2 adrenergic receptors on the surface of Th1 and B cells that increases intracellular cyclic adenosine-monophosphate (cAMP) and inhibits cell function or NK cell activity [97]. In a recent study, environmentally annoyed groups showed no signs of increased HPA-axis activation [99]. Whether these two pathways of stress-mediation are of significance for the pathogenesis of MCS remains unclear.

Hypotheses on Learning Models and NeurophysiologicBased Mechanisms of Sensitization in Response to Chemical Exposures Possible models of sensitization contributing to the MCS pathogenesis encompass allostatic load (dynamic adjustment to cope with the impact of stressors), cognitive arousal theory of stress (sustained arousal when there is no possibility to keep homeostasis), learning models (classical and operant conditioning or associative learning mechanisms) and neurophysiologic-based mechanisms of sensitization (limbic kindling and time-dependent sensitization) [100–108]. The latter are neural sensitization models involving the olfactory bulb, hypothalamus, temporal lobe and limbic system. The limbic system produces long-term synaptic sensitivity induced by previous electrical and chemical stimuli thought to be the central sensitization mechanism of long-term potentiation (LTP). LTP studies focus on hippocampus, where LTP involves stimulation of the NMDA receptor [78]. Hippocampus is also rich in density of vanilloid receptor [85, 109, 110]. Both receptors possibly link LTP neural sensitization mechanisms to the vanilloid receptor/NMDA/NO/ peroxynitrite hypothesis of low-level chemical-mediated neurotoxicity [85]. All these mechanisms of sensitization are still hypothetical.

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Hypotheses on Neurobiological Mechanisms of Sensitization in Response to Low-Level Chemical Exposures Recent studies point to the vanilloid receptor (TRPV1/VR1) as the major target of chemicals, with consecutive increase of NO levels and NMDA receptor activity [85]. There are many compounds (e.g. toluene, benzene, formaldehyde, diisocyanate, chlorine, capsaicin) that induce vanilloid receptor activity and chemical-induced inflammation. Vanilloid receptors are widely distributed in the peripheral (C-fibres of the trigeminal nerve ends) and central nervous system (e.g. hippocampus, hypothalamus, amygdale, cerebral cortex) [109–112]. Activation of the receptor leads to intracellular calcium influx, consecutive nerve action potentials, NO and peroxynitrite formation, oxidative stress, release of substance P, vasoactive neuropeptides (calcitonine gene-related peptide – CGRP, VIP), adenosine and pro-inflammatory cytokines (TNF-α, IL-1, IL-6, IFN-γ). This probably induces neurogenic inflammation, peripheral or central chemical sensitivity and neurotoxicity. Vanilloid receptor activation further leads to glutamate release with consecutive NMDA receptor stimulation, followed by excessive NO release and formation of peroxynitride. Apart from glutamate, NMDA receptors are activated by organic solvents including formaldehyde [113]. Organophosphates and carbamates are also discussed to trigger MCS symptoms by an increase of acetylcholine and activation of muscarinic receptors, which leads to additional NO release and peroxynitrite formation. Peroxynitrite depletes adenosine-tri-phosphate (ATP) pools, which further increases NMDA sensitivity [78]. Subsequently, peroxynitrite-mediated increased blood–brain barrier permeability may lead to increased access of chemicals to the CNS. When interpretating the data described above one should keep in mind that the majority of the investigations were based only on in vitro studies and animal models. Thus, there is no clinical proof for the vanilloid receptor/NMDA/NO/ peroxynitrite hypothesis of low-level chemical-mediated sensitivity.

Hypotheses on Low-Level Chemical-Induced Neurotoxicity Some evidence for neuronal dysfunction or injury as a result of chemical exposures comes from investigations in Gulf War veterans. Deployed military personnel have a lower N-acetyl aspartate to creatine ratio (NAA/Cr) in basal ganglia and brain stem as compared to controls, as assessed by proton magnetic resonance (MR) spectroscopy [114]. A decrease in the NAA/Cr ratio may reflect a reduction of functioning neuronal mass that cannot be detected in conventional MR imaging studies [115]. Severity of NAA/Cr-indicated brain cell functional impairment was associated to a decrease of central dopamine activity (left basal ganglia), severity of CNS symptoms and blood level of type Q paraoxonase (PON)1 (right basal ganglia) that is an important enzyme system to react with organophosphates [72, 116]. Recently, a lower NAA/Cr ratio for Gulf War veterans has been linked

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to the hippocampus indicating hippocampal dysfunction [117]. As LTP-based neural sensitization models of MCS focus on the hippocampal region, this finding is another support for the neurobiological basis of LTP sensitization models. As the exact physiological role of NAA is unknown, there is no proof of MR spectroscopy to determine low-level chemical-induced neurotoxicity.

Other Medical Conditions that Might Produce MCS-Like Symptoms In rare cases intracranial mass lesions can affect olfaction and produce MCS-like symptoms [118]. Other possible conditions that may contribute to the MCSassociated symptoms are sleep apnea or sleep disturbances, hypoxia and hypercapnia, alterations of the vomeronasal organ, autonomic nervous system dysfunction and disturbances of heme synthesis [119–124]. The latter may have an association with the NMDA/NO/peroxynitrite hypothesis of MCS by the interactions of NO and peroxynitrite with the regulation and synthesis of porphyrin intermediates in the liver and blood-forming tissue [78]. The conditions described above are still hypothetical and their significance for the pathogenesis of MCS remains unclear.

Psychological Factors, Cognitive Processing, Psychiatric Co-morbidity Chemically sensitive persons frequently show pathological scores in psychometric tests [64, 125, 126]. Scales for depression, anxiety, somatization and general psychopathology are elevated. Subscales for physical and mental health also show significant deficits in military personnel reporting multiple chemical sensitivities [14]. MCS patients reach significant higher scores on negative mood states, somatization and anxiety following odorant exposures than asymptomatic controls, but they did not have somatic or psychological symptoms under chemical-free conditions [23, 127]. Recent studies point to alterations in the cognitive processing of olfactory information by non-sensorial factors, such as attentiveness in patients reporting multiple chemical sensitivities [31, 128]. MCS patients rate trigger words that remind them of exposures to chemicals as more unpleasant and more arousing than patients with somatoform disorders or healthy controls [129]. A large number of investigations have revealed axis I psychiatric disorders in patients with self-reported multiple chemical sensitivities or MCS [64, 126, 130–134]. These encompass somatoform, depressive and anxiety disorders, especially agoraphobia, panic disorder and posttraumatic distress disorder. There may be a shift from mood and anxiety disorders towards somatoform disorders during the course of disease as reported by a controlled study and a 9-year follow-up investigation of MCS subjects [34, 35]. A recent investigation confirmed a significant phenomenological overlap between multiple chemical sensitivity and somatoform disorders [135]. The importance of psychiatric disorders was also supported by several studies in Gulf

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War veterans suffering from MCS-like symptoms [71, 136]. In addition, previous professional psychiatric treatment and previous psychotropic medication use show a robust association with symptoms suggestive of MCS in a military population [14]. This corresponds to an elevated prevalence of self-reported sensitivities to various chemicals in patients with previously diagnosed seasonal affective disorders and obsessive-compulsive disorders [137]. However, psychiatric disorders could involve etiologically and pathogenetically independent phenomena reflecting co-morbid disturbances [138]. A reactive genesis of psychiatric symptoms to the severe stress associated with the underlying illness is also conceivable [23]. To clarify some aspects of the situation, MCS patients have been challenged by sodium lactate infusion or single-breath inhalation of carbon dioxide [45, 139, 140]. Both methods frequently provoke panic response in patients with panic disorder. The majority of MCS patients fulfilled panic attack criteria according to DSM IIIR/IV as compared to placebo infusion or in comparison to controls. Other authors found an increased prevalence of panic disorder-associated cholecystokinin B receptor allele 7 or elevated nitric oxide/peroxynitrite in MCS patients, which is also found in posttraumatic stress disorder [88, 141]. There are also arguments for the importance of psychic co-factors contributing to the aetiology and pathogenesis of MCS by means of a heightened vulnerability. One study of first-degree relatives of patients with self-reported multiple chemical sensitivities revealed a heightened incidence of depression, panic disorders, obsessive-compulsive disorders, personality traits and alcoholism [142]. Histories of endorsed family problems, especially paternal alcohol and drug problems are frequently reported by chemical intolerant individuals and score higher than controls on questionnaires [100, 143, 144]. Several studies found early increased life stress ratings for persons with chemical intolerance [143, 145, 146]. Two studies described traumatic events and conditions like sexual and other types of abuse in the history of the patients’ childhood as predictors for MCS [86, 147]. A causation analysis which applied extended Bradford Hills’ criteria to test the psychogenic theory of MCS supports the relevance of psychic factors [134]. Thus, there is sufficient evidence that psychological conditions are important (co-)factors in MCS.

MCS Case Definitions At present, there exist at least ten case definitions for MCS, a structured interview for IEI diagnosis, as well as self-administered questionnaires to assess environmental sensitivity [40, 148, 149]. One new case definition, which was developed in the USA in 1999 by 89 MCS experts within the scope of a consensus conference shows a high discriminant validity as determined by the Toronto’s Health Survey self-administered questionnaire [150, 151]. The case definition takes into account the special self-reported, exposure-related circumstances that patients experience as multiple chemical sensitivities. A differentiation of symptoms, symptom complexes and recommendations for clinical evaluation is not offered. Thus, an operationalization of the MCS case definition that enables physicians to apply a comprehensive

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Table 2 Proposals for a further extension of the US MCS case definition from 1999 (From [33]. With permission) US MCS case definition MCS is: Proposals 1. A chronic condition 2. With symptoms that recur reproducibly 3. In multiple organ systems

Of at least 6 months, that causes significant lifestyle or functional impairments In the CNS in association with self-reported odour hypersensitivity Obligatory Fig. 1 in the CNS and at least one symptom of an other organ system (see Fig. 1)

4. In response to low levels of exposure 5. To multiple unrelated chemicals and which 6. Improve or are resolved when incitants are removed

diagnostic procedure is urgently needed. In 2005, we suggested an extension of the US MCS case definition [33]. From a systematic literature search and data analysis we concluded that MCS should only be diagnosed in patients who are mainly suffering from exposure-related non-specific complaints of the CNS (see Table 2). In the presence of predominant mucosa-associated symptoms not only MCS, but also irritant toxic (occupational) or other forms of ‘nonallergic noninfectious’ rhinitis or asthma should be diagnosed [68, 69].

Verification of Low-Level Chemical Exposures by Biomarkers Biomarkers to evaluate past as well as current low-level chemical exposures are best established for pesticides, namely organochlorines and organophosphates [65]. Organochlorines have a long half-life, so serum levels and whole-blood analyses can be used as a marker of exposure. As organophosphates inhibit acetylcholinesterase (AChE), erythrocyte AChE can be used as an exposure marker in a 4-month time frame. Urine and whole-blood analyses are screening tests for heavy metals. The demonstration of low-level chemical exposures by biomarkers is no proof for physical harm. Even under moderate occupational exposure conditions, a clear association between organophosphate exposures, as assessed by biomarkers, and increased symptom prevalence was not possible [65].

Specific Laboratory Tests The problem with laboratory tests to establish an MCS diagnosis is the lack of reliability and specificity [5, 92]. So far, only a few reproducibility studies of immunological tests used to assess MCS are available [96]. Other investigations

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(e.g. intraerythrocytic mineral levels) found no significant abnormalities in MCS patients [152]. Thus, at present specific laboratory tests to establish an MCS diagnosis do not exist.

In Vitro and In Vivo Tests to Determine Chemical Sensitivity Attempts to test for chemical sensitivity were made by paralleling the diagnostic procedures of food allergy [153]. Specific IgE in vitro tests can be useful to detect common allergens like formaldehyde and latex, as well as food allergy [154–156]. Prick tests and atopy patch tests can be used in IgE-mediated allergies (e.g. latex, food allergy), when specific IgE in vitro tests did not show significant results, and in contact dermatitis (e.g. nickel) [154, 156–158]. Only a few investigations concerning intradermal provocation-neutralization testing to verify chemical sensitivities or effects of other environmental factors are available and do not show conclusive results [159, 160]. Thus, there is no proof of reliability and validity for such intradermal tests to verify multiple chemical sensitivities [154].

Verification of Odour Hypersensitivity Similar to healthy controls, patients with self-reported chemical sensitivities show a normal odour threshold in experimental tests, and in some cases it may even be higher [31, 127, 161]. Thus, self-reported odour hypersensitivity is exclusively based on the patients’ self-reports.

Verification of Symptom Profiles Several studies have been conducted for the purpose of verifying sensitivity to chemicals and resulting symptom profiles by intranasal challenges or provocation chamber challenges, and these have shown no conclusive results or did not survey a representative symptom profile [45, 162–165]. Thus, the MCS symptom profile is principally determined by patients’ self-reports that cannot be objectified. Former investigations may have been biased by the lack of an adaptation period in testing individuals with reported chemical sensitivities [56]. Another problem is the need to control psychological reactions overlapping symptom reports [127].

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Electrophysiological and Neuroimaging Investigations Quantitative electro encephalographic (EEG) brain electrical activity mapping (BEAM), evoked potentials and chemosensory event-related potentials (ERPs), single photon emission computed tomography (SPECT) and positron emission tomography (PET) are insufficiently validated and studies are not conclusive to cover the problems addressed with the MCS phenomenon [166–169]. Some advance was made by a PET study in MCS subjects that investigated odour processing [170]. At baseline, the pattern of activation was similar in MCS and controls. After application of several different odorants MCS subjects activated odour-processing brain regions less than controls, but showed an odorant-related increase in activation of the anterior cingulate cortex and cuneus/ precuneus. The findings were interpreted as a top-down regulation of odour response via the cingulate cortex. Further advance in neuroimaging techniques are expected by proton MR spectroscopy. In previous studies a lower NAA/Cr ratio in basal ganglia, brain stem or the hippocampal region of Gulf War veterans has been reported [114, 115, 117].

Differential Diagnostic Procedures Differential diagnostic clarification in patients with self-reported multiple chemical sensitivities was not the focus of former investigations and recommendations. The clarification procedures described below in detail encompass clarification procedures analogous to CFS standards as recommended by Fukuda et al. (1994), optional evaluation of additional symptoms, and psychiatric evaluation according to DSM-IV [33, 171]. First Step: Syndromes showing overlap with MCS require separate definition and should exclude an MCS diagnosis. These include CFS and fibromyalgia if they precede the MCS symptomatics. Second Step: The employment of standardized procedures must exclude diseases that can explain functional symptoms of the CNS. These procedures include an evaluation in accordance with the standards proposed by Fukuda et al. (1994) for CFS, which shows significant overlap with the non-specific symptoms of the CNS reported by patients suffering from self-reported multiple chemical sensitivities [171]. Some of the differential diagnoses, which have to be ruled out, are shown in Table 3. In general, the evaluation of the case history and anamnesis, a psychiatric and physical examination, as well as routine laboratory tests, are sufficient to rule out these conditions [33]. Diagnostic procedures should be extended according to the clinical needs (laboratory test to rule out infectious diseases and immunological disorders, allergologic, ear–nose–throat and neurological examinations, electro encephalogram and imaging procedure of the CNS). Regarding the psychiatric

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Table 3 Examples of diseases or disorders that may overlap with MCS-defining symptoms (From [33]. With permission) 1. Lifetime or at present point in time Psychosomatic medicine/psychiatry – Any type of schizophrenia, schizophreniform disorder, schizoaffective disorder, paranoia or other mental illness in the psychotic realm of illness – Major depression with psychotic, catatonic or melancholic components or any bipolar disorder – Anorexia nervosa or bulimia – Delirium, dementia or amnestic disorder 2. Two years before onset of illness or at any point thereafter – Drug-abuse or drug-dependence 3. Manifest, inadequately treated or incompletely cured neurology – Cerebrovascular diseases – Degenerative diseases of the CNS or dementia – Inflammatory diseases of the CNS Pneumology – Sleep-apnea syndrome – Narcolepsy – Chronic disease of the bronchial system analogous to NYHA class II or above a frequency of attacks > 1×/day – Chronic lung disease analogous to NYHA class II Cardiology – Chronic disease of the coronary vessels – Coronary insufficiency from NYHA stage II – Arterial hypertonia for which medication does not provide a satisfactory treatment – Insulin-dependent diabetes mellitus – Obesity (body mass index > 45) Gastroenterology – Chronic hepatopathy – Chronic inflammatory bowel disease Nephrology – Chronic kidney disease: creatinine > 1.5 mg/dl Endocrinology and metabolic disturbances – Hypothyroidism and hyperthyroidism – Adrenal insufficiency – Pituitary insufficiency – Cushing’s syndrome – Porphyrias Hematology/oncology – Anemia – Oncological disease Rheumatology/immunology – Collagen vascular diseases – Primary systemic vasculitides – Other immunopathies Infectious diseases (continued)

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Table 3 (continued) – Chronic hepatitis C (or B) virus infection – HIV infection – Lues – Chronic Borrelia infection – Toxoplasmosis – Tuberculosis – Other chronic infections 4. Present point in time Other conditions – Intake of sedating medication and psychopharmacologic drugs (benzodiazepine, barbiturates, antidepressants with sedating components, neuroleptic drugs) NYHA: New Yorker Heart Association

evaluation, psychiatric diagnoses of somatoform disorders, somatization disorders, dissociative disorders or anxiety disorders should be regarded as overlapping MCS or treated as co-morbidity (like in depression). Confirmation should be carefully investigated in each individual case [172]. Third Step: Procedures that are recommended for diagnostic clarification of additional functional symptoms of organ systems other than the CNS are shown in Table 4. Some diseases or functional disturbances do not rule out a diagnosis of MCS (Table 5).

Therapy The self-help strategies of multiple chemical sensitivity sufferers include prevention/avoidance, detoxification and emotional self-care [10, 173, 174]. Treatment outcome critically depends on the patients’ health beliefs, chemical sensitivities treatment preference and the context of contested causation [175, 176]. Depending on the clinical presentation of chemical sensitivity-associated complaints and the attending physician’s belief in the aetiology and pathogenesis of the condition, clinical ecology and complementary methods, conventional medication and behavioral treatment are promulgated [177–180]. Treatment options of clinical ecology cover detoxification, nutritional supplementation, application of antioxidants, intradermal desensitization and rotatory-elimination diets. Antidepressants are preferentially from the group of selective serotonin reuptake inhibitors (SSRI). Cognitive-behavioral treatment has been practised in single and group interventions. An interdisciplinary treatment attempt covered not only psychological support, but also sports activity and acupuncture [178]. None of the treatment options are evidence-based so far, nor can they be considered as specific [181, 182].

Case history: ascertain

Suprapubic and hypogastric tenderness, rebound tenderness, guarding, renal beds sensitivity, rectal examination

3 × test for occult blood, α-amylase, lipase, IgE

Palpation and auscultation of the abdomen, rectal examination Inspection of the oral cavity, the skine, the nails and check for dermographism Inspection of the ear, the external auditory, the tympanic membrane, Weber’sand Rinne’s-hearing test, examination of the vestibular system Inspection of the oro-pharyngeal mucosa, auscultation of the lung Sensitivity testing, percussion of the retinaculum of the wrist, test for muscle strength, reflex status, pathological reflexes Pulse check, auscultation of the heart

b

If necessary: ECGg, 24-h ECG, exercise ECG, echocardiography If necessary: microbiologic examination of the urine, sonography of the kidneys and lower abdomen, plain abdominal radiography, urological and gynecological examination by a specialist

If necessary: neurological and electrophysiological examination by specialist

IgE

IgE if necessary: C1-esterase inhibitor concentration and function, dermatologic or allergologic examination by a specialist If necessary: ENTf-examination

If necessary: rheumatologic, neurologic and angiologic examinations by a specialist

Laboratory tests: complete by

Rheumatologic, neurologic and angiologic examination status

Physical examination: carry out

For example, nickel, pyrethroids House dust mites, pollen c Molds, isozyanates, formaldehyde, -3-carenes, methyl benzoate, dimethyl maloate, foodstuffs d Solvents, aldehydes, ketones, pyrethroids e Face, hairline, scalp, wrists, popliteal cavities, extensor side of the extremities, rima ani f Ear, nose, throat g Electrocardiogram

a

Anxiety-related situations

Cardiac arrhythmia, palpations or other cardiac complaints Pain and other disturbances of the urogenital tract Infections, cycle-associated complaints, kidney stones, appendicitis

Common allergens, environmental allergens, irritants Anxiety-related situations and hyperventilation

Mucosal irritation or other respiratory complaints Dysesthesia, muscle weakness or other complaints of the extremities

Multiple arthralgias, soft-tissue Assessment according to American College rheumatic disorders or other of Rheumatology (ACR) criteria, rheumatic complaints checklist for specific symptoms of systemic rheumatic diseases Stool irregularities or other Food intolerance abdominal complaints Erythema, urticarial changes, Contact allergiesa, common allergensb, (facial) swelling or other environmental allergensc, irritantsd skin eruptions Auditory complaints Hypacusia, otitis media, tinnitus, Mennière’s syndrome

Complaints

Table 4 Standardized diagnostics on the facultative, chemical-exposure-related symptoms of MCS to exclude relevant organic disease (From [33]. With permission) Standardized diagnostic procedure encompasses the general case history, physical examination and laboratory tests as proposed by Fukuda et al. (1994) [171]. Certain complaints need special investigations

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Table 5 Examples of diseases that do not rule out a diagnosis of MCS (From [33]. With permission) If one of these diseases or functional disturbances is detected, the symptoms should not be attributed to self-reported multiple chemical sensitivities Disorders of the cervical spine CNS symptoms Sinusitis as a result of: Orthostatic collapse Locomotor system Gastrointestinal tract

Skin Auditory system

Mucosa/respiratory tract

Symptoms of the peripheral nervous system

Cardiovascular system

Urogenital tract and systemic complaints

Arthrotic conditions Chronic gastritis Intolerance to foodstuffs without additives Lactose intolerance Gluten-sensitive enteropathy Mild pancreas insufficiency without evidence of florid Pancreatitis or alcohol abuse Atatus following surgery of the gastrointestinal tract for non-malignant disease, which may lead to mild stool irregularities but without evidence of dumping syndrome Mild dermatologic diseases without requiring therapy, which has a systemic effect and where there is no underlying systemic disease Tinnitus Hypacusis Otitis media Allergic conjunctivitis Allergic rhinitis Mild bronchial asthma (asthmatic symptoms maximally 1×/day) Sicca symptoms without evidence of rheumatologic systemic disease or untreated hyperthyroidism Hyperventilation syndrome Carpal tunnel syndrome without evidence of other severe underlying causes, such as insulin-dependent diabetes mellitus or rheumatological systemic disease Mild idiopathic polyneuropathy Mitral valve prolapse Paroxysmal supraventricular tachycardia Ventricular arrhythmia up to Lown IV b without evidence of other underlying cardiac diseases Perimenstrual syndrome Menopausal complaints Known kidney stones

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Monitoring the Allergic Inflammation Per Venge

Introduction Allergy is essentially an inflammatory disease [1] and our knowledge of the cells and mediators that are involved in the allergic inflammation has increased immensely during the last decade and are thoroughly described in this volume. This knowledge provides the basis of more rational ways to develop therapeutic principles that may relieve or prevent the development of allergic symptoms. This knowledge also provides the basis for the development of diagnostic assays that reflect the mechanisms underlying the symptoms of the allergic patient. This chapter will describe some of the diagnostic means that are used today in the clinical setting and also touch upon some other possibilities that may be used in the near future.

Why Therapy Stratification and Objective Disease – Treatment Monitoring? Treatment of inflammatory disease is largely based on trial-and-error experience guided by the symptoms of the patients. Treatment of allergic disease is no exception and is today often quite stereotyped based on national or international recommendations. In most cases the treatment works well; however, in many cases it is quite obvious that treatment is less successful. It is well documented that there is a great interindividual variation as to the inflammatory cells and mediators involved in the disease process [2]. Modern treatment of inflammatory disease should therefore not only consider the disease entity and symptoms, but also the underlying pathophysiology in order to be optimal to the patient. The scientific bases for the trial-and-error treatment strategy are in most cases controlled and well-performed clinical trials conducted by drug companies in P. Venge () Department of Medical Sciences, Clinical Chemistry and Pharmacology, University Hospital, SE-751 85 Uppsala, Sweden e-mail: [email protected]

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collaboration with expert clinicians. The results of successful trials show on average statistically significant differences between the compared populations, and the success rate is often higher if the patient populations have been highly selected as to gender, age, life style, specific symptoms, etc. It is, however, obvious from most such trials that the individual responses to the treatment vary immensely, from nonresponders to high-responders. Consequently, one should expect the same variation in treatment response in the daily management and care of similar patients. Indeed, treatment failures are common in major chronic inflammatory disease such as asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease, and rheumatic disease. In bronchial asthma and COPD it has been shown that responsiveness to glucocorticosteroids is related to the involvement of eosinophils in the disease process [3] suggesting that phenotyping of the individual patient as to the involving inflammatory cells and mediators can be helpful in stratifying the patient to the correct and successful treatment. Thus, studies in asthma have shown that treatment guided by specific markers of inflammation more efficiently controls symptoms and reduces the need of anti-inflammatory treatment. This approach has become and will become even more important with the advent of the more selective anti-inflammatory drugs such as anti-leukotrienes, TNF-α blockers [4, 5]. Thus, therapy stratification based on clinical symptoms as well as the knowledge of the specific pathophysiology of the inflammatory process underlying the signs and symptoms of the patient should reduce the number of trial-and-errors and form the rationale for a more successful treatment. The development of objective means, therefore, to monitor disease activity and treatment efficacy should reduce treatment costs and patient suffering.

Monitoring of Inflammation Many different cells are involved in the process of allergic inflammation. As noted above, the interindividual variation in the presence and activity of the different cells is considerable and the mix of cells and mediators varies between the affected organs, i.e., lung, nose, skin, GI-tract, middle ear, etc. The cells of particular interest in the allergic inflammation are the eosinophils, mast cells, Th2-type lymphocytes [1, 6] and possibly basophils with the eosinophils and mast cells (and possibly basophils) being the major effector cells, whereas the Th2-cell seems to be the major director of the process. However, many other cells should also be considered such as neutrophil granulocytes, monocytes/macrophages, epithelial cells, dendritic cells, and endothelial cells and platelets. In monitoring allergic disease classic expressions of inflammation such as the erythrocyte sedimentation rate, plasma protein electrophoresis, CRP (C-reactive protein), or other so-called acute phase reactants offer poor guidance, since these expressions largely are induced by IL-6, a cytokine that is rarely operative in allergic inflammation. Blood cell counts and differentials are other classic assays and the numbers of blood eosinophils are typically raised in allergy and even to some extent reflect the severity of the disease such as in asthma. Eosinophilia is so commonly seen in allergic disease, so the first question to ask the patient with blood eosinophilia is whether she

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suffers from allergy. Even if the circulating numbers of cells, such as the eosinophils, give us a hint of the involvement in the allergic process of this particular cell, they provide us with limited understanding of the extent of involvement, i.e., to what extent they secrete their often harmful molecules in the local tissue. Thus, in order to be able to correctly evaluate the participation of the individual inflammatory cells we need to know which are the participating cells and to what extent do they participate. In the last decades we and others have identified secretory molecules of various inflammatory cells and developed sensitive immunoassays for these molecules to be used in the monitoring of the presence and activity of specific inflammatory cells in the allergic processes [7–9]. In Table 1 some of these molecules are shown. Not all of these markers are entirely cell specific [10]. ECP and EPO (eosinophil peroxidase) are the most eosinophil-specific secretory proteins, whereas EPX/EDN (eosinophil protein x/eosinophil derived neurotoxin) and MBP (major basic protein) Table 1 Inflammatory cells and some of their secretory molecules that have been measured in various biological materials in allergic subjects Cells Molecules Biological materials Eosinophils

ECP (eosinophil cationic protein) EPO (eosinophil peroxidase)

Mast cells

EPX/EDN (eosinophil protein x/eosinophil derived neurotoxin) MBP (major basic protein) NO (nitric oxide) LTE4 (leukotriene E4) Tryptase PGD2 (prostaglandin D2) Histamine

Neutrophils

Elastase HNL (human neutrophil lipocalin) Lactoferrin MPO (myeloperoxidase)

Monocytes/ macrophages

Lysozyme

Serum, plasma, sputum, saliva, BAL, NAL, ear fluid, tear fluid, faeces, GI-lavage Serum, plasma, sputum, BAL, GI-lavage Serum, plasma, urine, faeces, GI-lavage Serum, plasma, sputum, BAL Exhaled air, GI-tract Urine, sputum, breath condensate Serum, plasma, sputum, BAL, NAL, ear fluid, tear fluid, GI-lavage Urine, sputum, BAL, NAL, breath condensate Serum, plasma, BAL, NAL, urine, tear fluid, GI-lavage Serum, plasma, sputum, BAL, NAL Serum, plasma, sputum, BAL, NAL, urine, GI-lavage, faeces Serum, plasma Serum, plasma, sputum, saliva, BAL, NAL, ear fluid, faeces, GI-lavage Serum, plasma

IL-6 Serum, plasma, BAL sCD86 Serum T-lymphocytes sIL-2r (soluble interleukin-2 Serum, plasma, sputum, BAL, NAL receptor) Endothelial cells E-selectin Serum, plasma, sputum, BAL BAL: bronchoalveolar lavage, NAL: nasal lavage, GI: gastrointestinal

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are produced also in small amounts by neutrophils [11] and basophils, respectively. Myeloperoxidase (MPO) and elastase are major components of neutrophil primary granules, but also found in monocyte secretory granules although at somewhat lower amounts. At differentiation into macrophages, though, myeloperoxidase disappears [12]. Among blood cells lactoferrin and HNL (human neutrophil lipocalin) are only found in the secondary granules of neutrophils. Lactoferrin is constitutively produced by exocrine glands [13] and the production of HNL may be induced by cytokines such as IL1β in certain epithelial cells [14]. HNL is also known as NGAL (neutrophil gelatinase-associated Lipocalin) [15]. No marker seems to be entirely specific to monocytes/macrophages. The assay of lysozyme has been used to reflect these cells although neutrophils are also major producers. A rough estimate has been that 90% of lysozyme in blood is derived from monocytes/macrophages [16]. In other body fluids lysozyme is unsuitable as a monocyte/macrophage marker because of the secretion from exocrine glands. Alternatively, IL-6 could be used since monocytes are major producers of this cytokine. Histamine is secreted by both basophils and mast cells, whereas the secretion of tryptase is rather unique to mast cells. Mast cells also produce large amounts of prostaglandin D2 (PGD2), which can be measured in urine [17]. The markers listed in Table 1 have been analyzed in allergic subjects as indices of inflammatory activity in several different body fluids. It is true to say that measurement in local fluids better reflect local processes than measurements in blood. Measurements in blood, especially in serum, put special requirements on the handling of blood in order to avoid false levels due to leakage from the leukocytes [18]. Measurements in blood, however, may under standardized pre-analytical conditions provide us with important and useful information about the inflammatory process ongoing in our allergic patient. In addition to the cell-specific markers it is possible to measure a number of other inflammatory markers among which are cytokines and chemokines. Although not cell specific, the levels of cytokines and chemokines may provide information of the mechanisms that are involved in the regulation of a particular inflammatory process. In allergic conditions the cytokines that reflect the Th2-driven process are of particular interest such as IL-4, 5 and 13, and GM-CSF [1]. Because of the very low levels in body fluids of these cytokines the assays have to be extremely sensitive to catch a clinically useful signal, which has limited their use in the clinical setting. Another inflammation marker which is produced by many cells and is increasingly used as a tool to reflect inflammation in the airways is NO in exhaled air [19, 20]. Its position in the clinical routine is still uncertain mostly because of methodological problems and difficulties in the interpretation of the results [20, 21].

Monitoring Eosinophil Activity and Turnover The marker that so far has become most widely used in the everyday clinical routine of the allergist is ECP [22]. ECP is an indicator of the activity and turnover of the eosinophil granulocyte. Currently it is measured in serum/plasma, but

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measurements in sputum and possibly saliva are interesting alternatives, since ECP in sputum and saliva more accurately reflects the local process. Below, the advantages and disadvantages of measuring ECP in various biological fluids will be discussed and the current evidence that ECP may be a useful complement to the diagnostic armamentarium for monitoring and characterization of disease activity in the allergic patient. The emerging evidence of the clinical usefulness of urine measurement of EPX/EDN or NO in exhaled air as alternatives to ECP measurements as reflectors of eosinophil turnover and activity will also be discussed [23–27].

ECP in Sputum and Other Secretions Numerous reports [22] show that the assays of ECP in sputum, saliva, and nasal lavage have the potential of becoming clinical instruments for the characterization and monitoring of inflammatory processes in the airways [28, 29]. This in particular has been shown for patients with asthma, COPD, and cystic fibrosis [28, 30–42]. In many cases sputum has to be induced by hypertonic saline and in order to analyze mediators released from inflammatory cells one needs to separate cells in the sputum from the supernatant. Procedures which are relatively time-consuming and complicated [29] probably are the main obstacles for a more widespread use of sputum measurement as a clinical tool. An alternative, however, which makes the procedure much simpler, is the extraction of the whole sputum sample and the subsequent measurement in the extract of specific markers of various cells. The numbers of eosinophil granulocytes in sputum have been estimated in this way by the means of ECP and there are a large number of publications showing that the numbers of eosinophils measured in this way are well correlated to disease activity in asthma and how the numbers are reduced as a consequence of corticosteroid treatment. In patients with COPD it was shown that treatment success with corticosteroids is related to the eosinophilic inflammation in the bronchi as measured by sputum ECP [43, 44]. An interesting alternative to sputum is saliva, since we showed recently that asthmatics had significantly raised levels of ECP in saliva and that the levels were reduced by corticosteroid treatment [45]. Still, however, we do not know what the ECP levels in saliva actually reflect – systemic or local eosinophil activity? Also the measurements of specific cell markers in nasal lavage fluids [46] or ear secretions [47] have been done quite extensively and clearly indicated the usefulness of such measurements in the understanding of cellular involvement. However, the clinical application is still not established.

ECP in Serum/Plasma ECP may be measured in both serum and plasma [18, 22]. If plasma is chosen the blood should be anticoagulated with EDTA or citrate in order to prevent spontaneous

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extracellular release of ECP and interaction with heparin [18]. The levels of ECP in serum are consistently higher than in plasma, which is due to the fact that eosinophils in the test tube continue their extracellular release of ECP. This is an active process that is both time- and temperature- dependent which means that higher extracellular levels are achieved by time and at higher ambient temperature or vice versa. Thus, if ECP is measured in serum, strict standardization of the blood sampling procedure and the handling of the blood sample are necessary in order to avoid artificial and unacceptable variations in the ECP levels [18]. Our recommendation is that blood is taken in tubes with gel separator and that coagulation is allowed for 1 h at room temperature (+22°C) before centrifugation and separation of serum. Both plastic and glass tubes may be used. However, differences in the material and the inclusion of coagulation activators in the tubes may affect the levels. This means that normal ranges of ECP have to be prepared at each laboratory that does not follow the recommendation of the manufacturer. The levels of ECP in EDTA-plasma probably correctly reflect the circulating levels of ECP at the time of blood sampling. These levels are the consequences of production and elimination of ECP, that is, local or systemic release of ECP to the circulation as well as variations in the turnover rate of ECP. Normally turnover is quite rapid with a t½ of about 45 min [48]. For several reasons we can assume that the turnover is more rapid in subjects with ongoing inflammation. This means that an increased release of ECP to the circulation in certain diseases does not always lead to the anticipated increase in plasma levels, since the increased release may be partly or fully counteracted by an increased elimination rate. This dynamics of counteracting principles may be the main explanation of the fact that the clinical information that is obtained by EDTAplasma measurements of ECP in most cases is much less clear and useful than the information obtained by serum measurements. The serum levels of ECP reflect in addition to the circulating levels also the secretory activity of the eosinophil population in the blood, and since the levels in serum are often 5–10 times those in plasma we may draw the conclusion that it is above all the secretory activity of the eosinophils that determines the levels. The propensity of blood eosinophils to release ECP is increased in subjects exposed to allergen [49]. My own interpretation of serum ECP levels is that they reflect the propensity of the eosinophil population to release ECP in the local process, e.g., in the lung of asthmatics. The higher this propensity the more damage is inflicted on the patient [50]. In order to eliminate the influence of eosinophil counts on the serum levels of ECP and thereby obtain a purer reflection of eosinophil activity, the serum levels may be divided by the eosinophil count, thus forming an ECP/eosinophil ratio. In some studies such a ratio was found to be more closely related to disease severity in asthma [51]. Almost thousand papers have been published dealing with the relation between ECP levels and allergic or other inflammatory diseases [22]. The majority of these publications indicate that ECP provides novel information about the process and that the information may be used in the treatment stratification and monitoring of the disease [52], since the ECP levels are closely related to exacerbation propensity and severity in diseases such as asthma, atopic dermatitis. Several publications, though, have questioned and sometimes rejected ECP as a clinically useful marker.

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One reason for this may be the simplified view that asthma is one disease and that the disease is caused by one cell, that is, the eosinophil and that one marker such as ECP will solve all clinical problems. This is obviously not true, since we know today that the involvement of eosinophils in the asthmatic process is very variable between individuals [2]. Another reason to the variations in the results is probably the unawareness of the importance of correct sample handling [18]. Still another possibility may be related to the new knowledge of several genetic variants of ECP and the fact that these variants are related to the expression of allergic symptoms [53] and the serum levels of ECP [54, 55]. Thus, the levels of ECP in blood or any other biological fluid are not disease specific, but provide us with information about the extent to which eosinophils are involved in the particular disease process.

EPX/EDN in Urine In a search for noninvasive means to monitor eosinophil involving inflammation urine measurement of EPX/EDN has emerged as a promising candidate particularly in children [23, 25, 37]. In order to minimize the influence of differences in water dilution of the urine the EPX/EDN levels have to be adjusted by the creatinine concentrations unless 24-h samplings are carried out. It is also useful to bear in mind that all eosinophil markers including the excretion of EPX/EDN show circadian rhythms with the highest levels occurring at night [56]. Thus, for the sake of standardization of blood or urine measurements of eosinophil markers sampling should be carried out at the same time of the day. Several studies have shown that EPX/EDN in urine is elevated in atopic dermatitis [57] and asthma and also shown relations to disease activity and reductions in relation to anti-inflammatory treatment, i.e., corticosteroid treatment [23, 58]. Elevated levels of EPX/EDN in urine in wheezy children also seem to predict the development of asthma [26, 59]. EPX/EDN was also measured in serum in asthmatics and showed similar results as ECP [60].

Exhaled NO as a Marker of Eosinophil Activity High levels of NO are found in the exhaled air of patients with allergic asthma and several studies show strong correlations to different expressions of eosinophil activity [27, 61, 62]. The conclusion, therefore, is that exhaled NO largely reflects the involvement of eosinophils in the allergic inflammation and thereby may act as a surrogate marker of these cells in asthma. The clinical usefulness of exhaled NO has also been suggested by studies in which NO levels were used to guide corticosteroid therapy and reduce corticosteroid dosages without any negative clinical consequences [63]. Because of the simplicity in measuring exhaled NO the procedure has gained a certain degree of popularity among clinicians taking care of asthma patients. The weakness of exhaled NO as compared to specific

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markers of eosinophils mentioned above is obviously the lack of cellular specificity, since NO may be produced by many other cells as well. The levels of exhaled NO are also reduced by smoking and infectious processes in the lung involving neutrophils. Thus, measurement of NO in exhaled air may be a practical tool for the clinician in the evaluation of the therapy responsiveness and compliance of the asthmatic patient, but should probably be used in conjunction with more cellspecific diagnostic procedures such as blood or sputum measurements in the initial investigation of the patient [64, 65].

Monitoring Neutrophil Activity The involvement of neutrophils in the allergic inflammation is somewhat controversial and their presence may reflect the addition to the process of other inciting agents such as smoke in allergic asthmatics and bacteria in the nose of allergic rhinitics. Their presence, however, may also reflect the fact that allergic inflammation can acquire different characteristics under certain circumstances for reasons poorly understood [4, 50]. Thus, endotoxin was shown to aggravate allergy through a process that probably includes the activity of the neutrophils [66]. The most widely used markers of neutrophil activity have been elastase and myeloperoxidase, although their specificities as neutrophil markers are not absolute, since they are also produced and secreted by monocytes. Other markers of interest are HNL and lactoferrin. HNL or NGAL (neutrophil gelatinase-associated lipocalin) is stored in the secondary granules of neutrophils and is as such a unique marker of the activation of neutrophils [15, 67]. However, under certain conditions the gene for HNL/NGAL production may be turned on also in other cells, of which epithelial cells are the best described [14]. Thus, when measured in blood HNL probably uniquely reflects the activity of neutrophils, whereas when measured in other body fluids, some of the protein may originate from epithelial cells [68]. Blood measurements of HNL were shown to be highly elevated in subjects with acute infections and to discriminate with high sensitivity and specificity between a bacterial or viral cause of the infection. HNL was also found elevated in blood and sputum in COPD [69], but not in allergy lending support to the absence of neutrophil participation in most allergic processes.

Monitoring Basophil Activity The involvement of basophils in the allergic inflammation has received increasing support [1]. Basophils release histamine upon activation. This property has been taken advantage of in the so-called histamine-release assay in which patient’s whole blood is exposed to different allergens after which histamine is measured as a sign of specific IgE-sensitization of the basophils [70]. Thus, an ex vivo functional test of basophils and the presence of specific IgE bound to the cell membrane.

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Recently, cell surface expressions of CD63 and CD203c on basophils have been used as signs of basophil activation in allergic subjects [71, 72]. The sensitivity and specificity of these reactions, however, have been questioned, although it seems that these markers could be of clinical value in certain situations. No secretory molecule specific to basophil activation in vivo has yet been identified. Histamine in the body is derived from both basophils and mast cells. When measured in blood care has to be taken not to activate the basophils during blood sampling, since such activation inevitably will produce falsely elevated levels. Interpretation of elevated levels as to cellular origin is often difficult, but elevated levels are seen in patients with acute allergic reactions [73].

Monitoring Mast Cell Activity A role of mast cells in allergic inflammation is well established. Upon activation the mast cells release various secretory proteins such as tryptase and chymase, arachidonicacid-derived products as PGD2, leukotrienes, and histamine [74]. Tryptase is contained to a certain extent in basophils too, but when measured in serum or plasma the levels are in most cases reflecting mast cell activation [75]. Elevations in blood require extensive activation and involvement of mast cells such as seen in anaphylaxis, acute allergic reactions to bee venom, or mastocytosis [73, 75], whereas increased levels are seldom seen in patients with allergic rhinitis or asthma. The low and often undetectable levels in serum or plasma in most subjects with allergic disease has limited the clinical use of tryptase as a monitoring tool in these diseases. It was, however, shown that different assay configurations as to antibody choice may detect subunits of tryptase and that such assays may have a greater capacity to detect elevations in plasma or serum even in patients with mild allergic disease [76]. In several studies tryptase measurement in local fluids such as nasal fluid and sputum has been carried out with greater success [77–79]. Tryptase levels in most of these studies were increased in allergen exposed subjects and reduced upon treatment with specific allergen immunotherapy or corticosteroids. Tryptase measurements in nasal fluids or sputum could therefore be useful objective markers of treatment monitoring. An alternative to tryptase measurements in various body fluids is histamine measurement. However, as noted above the cellular origin, being the mast cell or the basophil is in most cases impossible to judge unless parallel rises in tryptase are seen as well. As an objective means to monitor the response to local allergen challenge in the nose histamine could be of some value [79, 80].

Monitoring T-Cell Activity Allergy is mainly associated to a Th2-response, which means that specific cytokines produced by these particular lymphocytes are candidate molecules for diagnosis and monitoring. Some of these cytokines are IL-4, IL-5, and IL-13

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and indeed commercial assays are available for all three. IL-4 is best assayed in supernatants of T-lymphocytes harvested from patients with allergic disease and the production has generally been shown to be increased [81]. However, in fluid phase such as blood or sputum IL-4 is mostly immeasurable. The measurement of IL-5 in serum and sputum has been reported and been related to the eosinophil involvement in the disease [82–84]. However, its usefulness as clinical tool in monitoring the allergic inflammation has not been established. Increased levels of IL-13 were found in allergic disease both in serum/plasma [85] and in sputum [82, 86, 87] although in most cases nonmeasurable levels were obtained. In sputum the levels were decreased by corticosteroid treatment and also correlated to lung function [82, 87]. Reduction in IL-13 levels after corticosteroids was also seen in nasal secretions [88]. The plasma levels were dependent on certain genetic polymorphisms in the IL-13 gene [89]. The IL-2 receptor is exposed on many cells such as T-lymphocytes and eosinophils. The IL-2 receptor is shed from the cells and may be measured in a soluble form in blood and other biological fluids. The levels of sIL-2r (sCD25) are generally believed to reflect T-cell involvement in the process and raised levels were found in patients with allergic disease and shown to be related to the severity of asthma and atopic dermatitis [84, 90]. Also the levels were reduced by corticosteroid treatment as a likely reflection of the down regulation of the activity of the T-cells [84]. Thus, the measurements of sIL-2r can be used to suggest the involvement of T-cells in the disease. It is noteworthy that CD25 expression in lung cells uniquely distinguishes allergic from nonallergic asthma, since CD25 expressing cells were only found in the bronchial biopsies of allergic asthmatics [2].

Monitoring of Endothelial Cell Activity The adhesion molecules ICAM-1, VCAM-1, and E-selectin are all measurable as soluble proteins in blood. E-selectin is probably a unique marker of endothelial cell activity, whereas the origin of the soluble forms of ICAM-1 and VCAM-1 in blood is uncertain although a major source probably is the endothelial cells. Elevated levels of all three adhesion molecules were shown in patients with atopic dermatitis, with E-selectin most closely related to disease severity [91]. In patients with allergic asthma only E-selectin was found elevated and closely related to airways conductance. Also in nonallergic asthmatics such a relationship was found. However, in nonallergic asthmatics, but not in allergic asthma, E-selectin was found closely related to measures of airflow variability and airways hyperresponsiveness, suggesting the involvement of endothelial cells in these processes in nonallergic asthma [92]. Thus, the measurements of E-selectin, as a reflection of endothelial cell involvement, should be of interest in the characterization of the inflammatory process of the asthmatic patient and in atopic dermatitis.

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Monitoring of Neurotrophins Neurotrophins comprise a family of proteins, which includes among others NGF (nerve growth factor) and BDNF (brain-derived neurotrophic factor). Neurotrophins are produced by most immune cells including mast cells, T-cells, and eosinophils [93]. The neurotrophins are anti-apoptotic to many cells including nerve cells and were also shown to activate eosinophils with regard to granule protein release and chemotaxis. High circulating levels of NGF were found in subjects with a variety of allergic disease such as asthma, rhinoconjunctivitis, and atopic dermatitis [94, 95]. The levels were related to the severity of the disease. In vernal keratoconjunctivitis the levels of NGF were closely related to the numbers of mast cells infiltrating the conjunctiva [96]. Circulating levels of BDNF have been shown to be raised in atopic dermatitis [97] with a close relationship to numbers of blood eosinophils and Th2 lymphocytes [98]. The levels of BNDF decreased at remission of the disease in parallel to the decrease in blood eosinophil and Th2 cell counts, which suggested these cells to be the major sources of BDNF. BNDF affected several functions of eosinophils obtained from patients with atopic dermatitis, but not those obtained from non-atopic subjects [97]. No information about circulating levels of BNDF in other allergic disease is available although a role of BNDF in asthma is suggested from animal data [93]. Neurotrophins seem to be uniquely involved in several aspects of allergic inflammation and should therefore be of clinical interest in understanding the underlying mechanisms of the allergic patient.

Cytokines and Chemokines in the Monitoring of Allergic Inflammation In addition to the specific Th2-derived cytokines discussed above, a number of other cytokines and chemokines have been measured in allergic disease. Some of these, i.e., eotaxins, are related to the eosinophil component of the process [99–103] and others to the Th2 component such as IL-16 and MDC (macrophagederived chemokine) [104, 105], and still others have a less clear relation to the allergic inflammation such as IL-12 and IL-18 [106–108], although these latter cytokines were shown to relate to the severity of the allergic disease. One report showed highly raised levels of TNF-α and IL-8 in severe asthma together with raised blood numbers of eosinophils and neutrophils [4], suggesting a systemic inflammation in those patients with the additional involvement of neutrophils in the process. In this study, however, the levels of IL-13 and IL-16 were unaltered in contrast to findings in atopic dermatitis [104]. In the study on severe asthma the measurement of exhaled NO was not helpful, since the levels tended to be lower among these patients, thus emphasizing the notion of different pathophysiologic mechanisms operative at different severity stages of allergic disease. The plasma

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levels of monocyte chemotactic protein-4 (MCP-4) were increased in asthma and found associated to asthma exacerbations. By logistic regression analysis MCP-4 was found to be an independent predictor of asthma diagnosis and the interpretation was that MCP-4 is a systemically expressed biomarker that independently predicts susceptibility to asthma and directly associated with exacerbations [109].

Other Biological Markers of Allergic Inflammation The arachidonic acid-derived products are important mediators of allergic inflammation and are discussed in detail in other chapters of this volume. In many studies such products including leukotrienes, prostaglandins, and PAF (platelet-activating factors) have been measured in various biological fluids such as sputum and urine, in patients with allergic inflammation [110]. However, with the possible exception of PGD2 measurement in urine as a marker of mast cell activity as described above and LTE4 in urine and sputum as a likely reflection of eosinophil activity, these assays have mostly been used in experimental settings to investigate the importance of such products in the allergic inflammation [111–116]. Both PGD2 and LTE4 are elevated in urine in patients with allergic disease and reflect disease severity. With the advent of leukotriene antagonists as therapeutic principles in asthma, leukotriene measurement might become useful in the therapy stratification of the asthmatic patient [117]. As discussed above certain cell surface markers may identify basophils and the extent of their activation. Alterations of cell surface markers are also seen on B-cells, T-cells, and eosinophils in patients with allergic disease. B-cell surface expression of CD23, CD40, and HLA-DR were all increased in allergic subjects, but downregulated at successful immunotherapy [118]. Eosinophils undergoing apoptosis expressed CD25, CD122, CD28 (B7-ligand), and CD86 (B7-2), possibly as a sign of transformation into an antigen presenting cells [119] and eosinophils obtained from asthmatic subjects had increased expression of CD45 [120]. Furthermore, atopic eosinophils have increased expression of chemokine receptors [121]. These few examples serve to illustrate the potential of measuring cell surface expression of immune cells in the monitoring of allergic inflammation. Soluble CD86 was found increased in serum in asthma during an exacerbation [122]. The levels were correlated to eosinophil, lymphocyte, and monocyte numbers in blood, but apparently only produced at increased amounts by monocytes. The conclusion was that soluble CD86 in the circulation might be a useful marker of monocyte involvement in bronchial asthma.

Conclusions Individual symptoms of allergy such as asthma, dermatitis, and rhinitis have many different underlying mechanisms. The detailed characterization of the inflammatory mechanisms underlying symptom development in the individual patient (phenotyping

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of inflammation) is important in order to stratify the patient to the optimal treatment, but also for monitoring treatment efficacy and compliance [5]. With today’s knowledge and development numerous possibilities are at hand. The combined assays of markers reflecting individual cells and their extent of activation will provide us with a deeper understanding of the inflammatory process ongoing in the patient. In this chapter I have briefly discussed some of these possibilities and most certainly forgotten some, since any aspect of the allergic inflammation is a potential candidate, be it soluble markers found in blood, urine, sputum or any other biological fluid, or molecules expressed on cell surfaces or present in exhaled air in the breath. A modern and different approach is the search for markers by different array approaches [123].

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37. Koller DY, Nilsson M, Enander I, Venge P, Eichler I (1998) Serum eosinophil cationic protein, eosinophil protein X and eosinophil peroxidase in relation to pulmonary function in cystic fibrosis. Clin Exp Allergy 28:241–248 38. Dahlen I, Janson C, Bjornsson E, Stalenheim G, Peterson CG, Venge P (1999) Inflammatory markers in acute exacerbations of obstructive pulmonary disease: predictive value in relation to smoking history. Respir Med 93:744–751 39. Maestrelli P, Richeldi L, Moretti M, Fabbri LM (2001) Analysis of sputum in COPD. Thorax 56:420–422 40. Woodruff PG, Khashayar R, Lazarus SC, Janson S, Avila P, Boushey HA, Segal M, Fahy JV (2001) Relationship between airway inflammation, hyperresponsiveness, and obstruction in asthma. J Allergy Clin Immunol 108:753–758 41. Gibson PG, Simpson JL, Saltos N (2001) Heterogeneity of airway inflammation in persistent asthma: evidence of neutrophilic inflammation and increased sputum interleukin-8. Chest 119:1329–1336 42. Metso T, Venge P, Haahtela T, Peterson CG, Seveus L (2002) Cell specific markers for eosinophils and neutrophils in sputum and bronchoalveolar lavage fluid of patients with respiratory conditions and healthy subjects. Thorax 57:449–451 43. Keatings VM, Jatakanon A, Worsdell YM, Barnes PJ (1997) Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 155:542–548 44. Dahlen I, Janson C, Bjornsson E, Stalenheim G, Peterson CG, Venge P (2001) Changes in inflammatory markers following treatment of acute exacerbations of obstructive pulmonary disease. Respir Med 95:891–897 45. Schmekel B, Ahlner J, Malmstrom M, Venge P (2001) Eosinophil cationic protein (ECP) in saliva: a new marker of disease activity in bronchial asthma. Respir Med 95:670–675 46. Rasp G, Thomas PA, Bujia J (1994) Eosinophil inflammation of the nasal mucosa in allergic and non-allergic rhinitis measured by eosinophil cationic protein levels in native nasal fluid and serum. Clin Exp Allergy 24:1151–1156 47. Hurst DS, Venge P (2000) Evidence of eosinophil, neutrophil, and mast-cell mediators in the effusion of OME patients with and without atopy. Allergy 55:435–441 48. Venge P, Nilsson L, Nyström S-O, Åberg T (1987) Serum and plasma measurements of neutrophil granule proteins during cardiopulmonary bypass. A model to estimate human turnover of lactoferrin and myeloperoxidase. Eur J Haematol 39:339–345 49. Carlson M, Håkansson L, Kämpe M, Stålenheim G, Peterson C, Venge P (1994) Degranulation of eosinophils from pollen-atopic patients with asthma is increased during pollen season. J Allergy Clin Immunol 89 (No.1, Part 1):131–139 50. Venge P (2004) Monitoring the allergic inflammation. Allergy 59:26–32 51. Stelmach I, Majak P, Grzelewski T, Jerzynska J, Juralowicz D, Stelmach W, BorzeckaPodsiadlowicz M, Korzeniewska A, Kuna P (2004) The ECP/Eo count ratio in children with asthma. J Asthma 41:539–546 52. Lowhagen O, Wever AM, Lusuardi M, Moscato G, De Backer WA, Gandola L, Donner CF, Ahlstedt S, Larsson L, Holgate ST (2002) The inflammatory marker serum eosinophil cationic protein (ECP) compared with PEF as a tool to decide inhaled corticosteroid dose in asthmatic patients. Respir Med 96:95–101 53. Jonsson UB, Bystrom J, Stalenheim G, Venge P (2002) Polymorphism of the eosinophil cationic protein-gene is related to the expression of allergic symptoms. Clin Exp Allergy 32:1092–1095 54. Munthe-Kaas MC, Gerritsen J, Carlsen KH, Undlien D, Egeland T, Skinningsrud B, Torres T, Carlsen KL (2007) Eosinophil cationic protein (ECP) polymorphisms and association with asthma, s-ECP levels and related phenotypes. Allergy 62:429–436 55. Noguchi E, Iwama A, Takeda K, Takeda T, Kamioka M, Ichikawa K, Akiba T, Arinami T, Shibasaki M (2003) The promoter polymorphism in the eosinophil cationic protein gene and its influence on the serum eosinophil cationic protein level. Am J Respir Crit Care Med 167:180–184

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77. Marcucci F, Sensi L, Di CG, Salvatori S, Bernini M, Pecora S, Burastero SE (2005) Threeyear follow-up of clinical and inflammation parameters in children monosensitized to mites undergoing sub-lingual immunotherapy. Pediatr Allergy Immunol 16:519–526 78. Louis R, Lau LC, Bron AO, Roldaan AC, Radermecker M, Djukanovic R (2000) The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med 161:9–16 79. Wilson SJ, Lau L, Howarth PH (1998) Inflammatory mediators in naturally occurring rhinitis. Clin Exp Allergy 28:220–227 80. Di Lorenzo G, Drago A, Esposito PM, Candore G, Colombo A, Gervasi F, Pacor ML, Purello DF, Caruso C (2001) Measurement of inflammatory mediators of mast cells and eosinophils in native nasal lavage fluid in nasal polyposis. Int Arch Allergy Immunol 125:164–175 81. Hoekstra MO, Hoekstra Y, De Reus D, Rutgers B, Gerritsen J, Kauffman HF (1997) Interleukin-4, interferon-gamma and interleukin-5 in peripheral blood of children with moderate atopic asthma. Clin Exp Allergy 27:1254–1260 82. Park SW, Jangm HK, An MH, Min JW, Jang AS, Lee JH, Park CS (2005) Interleukin-13 and interleukin-5 in induced sputum of eosinophilic bronchitis: comparison with asthma. Chest 128:1921–1927 83. Lampinen M, Carlson M, Hakansson LD, Venge P (2004) Cytokine-regulated accumulation of eosinophils in inflammatory disease. Allergy 59:793–805 84. Sahid El-Radhi A, Hogg CL, Bungre JK, Bush A, Corrigan CJ (2000) Effect of oral glucocorticoid treatment on serum inflammatory markers in acute asthma. Arch Dis Child 83:158–162 85. Lee YC, Lee KH, Lee HB, Rhee YK (2001) Serum levels of interleukins (IL)-4, IL-5, IL-13, and interferon-gamma in acute asthma. J Asthma 38:665–671 86. Berry MA, Parker D, Neale N, Woodman L, Morgan A, Monk P, Bradding P, Wardlaw AJ, Pavord ID, Brightling CE (2004) Sputum and bronchial submucosal IL-13 expression in asthma and eosinophilic bronchitis. J Allergy Clin Immunol 114:1106–1109 87. Komai-Koma M, McKay A, Thomson L, McSharry C, Chalmers GW, Liew FY, Thomson NC (2001) Immuno-regulatory cytokines in asthma: IL-15 and IL-13 in induced sputum. Clin Exp Allergy 31:1441–1448 88. Erin EM, Leaker BR, Zacharasiewicz AS, Higgins LA, Williams TJ, Boyce MJ, de BP, Durham SR, Barnes PJ, Hansel TT (2005) Single dose topical corticosteroid inhibits IL-5 and IL-13 in nasal lavage following grass pollen challenge. Allergy 60:1524–1529 89. Arima K, Umeshita-Suyama R, Sakata Y, Akaiwa M, Mao XQ, Enomoto T, Dake Y, Shimazu S, Yamashita T, Sugawara N, Brodeur S, Geha R, Puri RK, Sayegh MH, Adra CN, Hamasaki N, Hopkin JM, Shirakawa T, Izuhara K (2002) Upregulation of IL-13 concentration in vivo by the IL13 variant associated with bronchial asthma. J Allergy Clin Immunol 109:980–987 90. Pumputiene I, Emuzyte R, Dubakiene R, Firantiene R, Tamosiunas V (2006) T cell and eosinophil activation in mild and moderate atopic and nonatopic children’s asthma in remission. Allergy 61:43–48 91. Wolkerstorfer A, Savelkoul HF, de Waard van der Spek FB, Neijens HJ, van MT, Oranje AP (2003) Soluble E-selectin and soluble ICAM-1 levels as markers of the activity of atopic dermatitis in children. Pediatr Allergy Immunol 14:302–306 92. Janson C, Ludviksdottir D, Gunnbjornsdottir M, Bjornsson EH, Hakansson L, Venge P (2005) Circulating adhesion molecules in allergic and non-allergic asthma. Respir Med 99:45–51 93. Rochlitzer S, Nassenstein C, Braun A (2006) The contribution of neurotrophins to the pathogenesis of allergic asthma. Biochem Soc Trans 34:594–599 94. Bonini S, Lambiase A, Bonini S, Angelucci F, Magrini L, Manni L, Aloe L (1996) Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma. Proc Natl Acad Sci USA 93:10955–10960 95. Toyoda M, Nakamura M, Makino T, Hino T, Kagoura M, Morohashi M (2002) Nerve growth factor and substance P are useful plasma markers of disease activity in atopic dermatitis. Br J Dermatol 147:71–79

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96. Lambiase A, Bonini S, Micera A, Magrini L, Bracci Laudiero L, Aloe L (1995) Increased plasma levels of nerve growth factor in vernal keratoconjunctivitis and relationship to conjunctival mast cells. Invest Ophthalmol Vis Sci 36:2127–2132 97. Raap U, Werfel T, Goltz C, Deneka N, Langer K, Bruder M, Kapp A, Schmid-Ott G, Wedi B (2006) Circulating levels of brain-derived neurotrophic factor correlate with disease severity in the intrinsic type of atopic dermatitis. Allergy 61:1416–1418 98. Namura K, Hasegawa G, Egawa M, Matsumoto T, Kobayashi R, Yano T, Katoh N, Kishimoto S, Ohta M, Obayashi H, Ose H, Fukui M, Nakamura N, Yoshikawa T (2007) Relationship of serum brain-derived neurotrophic factor level with other markers of disease severity in patients with atopic dermatitis. Clin Immunol 122:181–186 99. Feltis BN, Reid DW, Ward C, Walters EH (2004) BAL eotaxin and IL-5 in asthma, and the effects of inhaled corticosteroid and beta2 agonist. Respirology 9:507–513 100. Tateno H, Nakamura H, Minematsu N, Nakajima T, Takahashi S, Nakamura M, Fukunaga K, Asano K, Lilly CM, Yamaguchi K (2004) Plasma eotaxin level and severity of asthma treated with corticosteroid. Respir Med 98:782–790 101. Yamamoto K, Takanashi S, Hasegawa Y, Kanehira Y, Kaizuka M, Okumura K (2003) Eotaxin level in induced sputum is increased in patients with bronchial asthma and in smokers. Respiration 70:600–605 102. Taha RA, Laberge S, Hamid Q, Olivenstein R (2001) Increased expression of the chemoattractant cytokines eotaxin, monocyte chemotactic protein-4, and interleukin-16 in induced sputum in asthmatic patients. Chest 120:595–601 103. Lilly CM, Woodruff PG, Camargo CA, Jr., Nakamura H, Drazen JM, Nadel ES, Hanrahan JP (1999) Elevated plasma eotaxin levels in patients with acute asthma. J Allergy Clin Immunol 104:786–790 104. Ngelova-Fischer I, Hipler UC, Bauer A, Fluhr JW, Tsankov N, Fischer TW, Elsner P (2006) Significance of interleukin-16, macrophage-derived chemokine, eosinophil cationic protein and soluble E-selectin in reflecting disease activity of atopic dermatitis – from laboratory parameters to clinical scores. Br J Dermatol 154:1112–1117 105. Leung TF, Ma KC, Hon KL, Lam CW, Wan H, Li CY, Chan IH (2003) Serum concentration of macrophage-derived chemokine may be a useful inflammatory marker for assessing severity of atopic dermatitis in infants and young children. Pediatr Allergy Immunol 14:296–301 106. Ando M, Shima M (2007) Serum interleukins 12 and 18 and immunoglobulin E concentrations and allergic symptoms in Japanese schoolchildren. J Investig Allergol Clin Immunol 17:14–19 107. Hon KL, Leung TF, Ma KC, Wong CK, Wan H, Lam CW (2004) Serum concentration of IL-18 correlates with disease extent in young children with atopic dermatitis. Pediatr Dermatol 21:619–622 108. Yoshizawa Y, Nomaguchi H, Izaki S, Kitamura K (2002) Serum cytokine levels in atopic dermatitis. Clin Exp Dermatol 27:225–229 109. Kalayci O, Sonna LA, Woodruff PG, Camargo CA, Jr., Luster AD, Lilly CM (2004) Monocyte chemotactic protein-4 (MCP-4; CCL-13): a biomarker of asthma. J Asthma 41: 27–33 110. Kumlin M (2000) Measurement of leukotrienes in humans. Am J Respir Crit Care Med 161:S102–S106 111. Macfarlane AJ, Dworski R, Sheller JR, Pavord ID, Kay AB, Barnes NC (2000) Sputum cysteinyl leukotrienes increase 24 hours after allergen inhalation in atopic asthmatics. Am J Respir Crit Care Med 161:1553–1558 112. Pavord ID, Ward R, Woltmann G, Wardlaw AJ, Sheller JR, Dworski R (1999) Induced sputum eicosanoid concentrations in asthma. Am J Respir Crit Care Med 160:1905–1909 113. Vachier I, Kumlin M, Dahlen SE, Bousquet J, Godard P, Chanez P (2003) High levels of urinary leukotriene E4 excretion in steroid treated patients with severe asthma. Respir Med 97:1225–1229 114. Severien C, Artlich A, Jonas S, Becher G (2000) Urinary excretion of leukotriene E4 and eosinophil protein X in children with atopic asthma. Eur Respir J 16:588–592

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115. Oymar K, Aksnes L (2005) Increased levels of urinary leukotriene E4 in children with severe atopic eczema/dermatitis syndrome. Allergy 60:86–89 116. Misso NL, Aggarwal S, Phelps S, Beard R, Thompson PJ (2004) Urinary leukotriene E4 and 9 alpha, 11 beta-prostaglandin F concentrations in mild, moderate and severe asthma, and in healthy subjects. Clin Exp Allergy 34:624–631 117. Cai C, Yang J, Hu S, Zhou M, Guo W (2007) Relationship between urinary cysteinyl leukotriene E4 levels and clinical response to antileukotriene treatment in patients with asthma. Lung 185:105–112 118. Hakansson L, Heinrich C, Rak S, Venge P (1998) Activation of B-lymphocytes during pollen season. Effect of immunotherapy [see comments]. Clin Exp Allergy 28:791–798 119. Seton K, Hakansson L, Carlson M, Stalenheim G, Venge P (2003) Apoptotic eosinophils express IL-2R chains alpha and beta and co-stimulatory molecules CD28 and CD86. Respir Med 97:893–902 120. Blaylock MG, Lipworth BJ, Dempsey OJ, Duncan CJ, Lee DK, Lawrie A, Douglas JG, Walsh GM (2003) Eosinophils from patients with asthma express higher levels of the panleucocyte receptor CD45 and the isoform CD45RO. Clin Exp Allergy 33:936–941 121. Phillips RM, Stubbs VE, Henson MR, Williams TJ, Pease JE, Sabroe I (2003) Variations in eosinophil chemokine responses: an investigation of CCR1 and CCR3 function, expression in atopy, and identification of a functional CCR1 promoter. J Immunol 170:6190–6201 122. Shi HZ, Xie ZF, Deng JM, Chen YQ, Xiao CQ (2004) Soluble CD86 protein in serum samples of patients with asthma. Thorax 59:870–875 123. Izuhara K, Saito H (2006) Microarray-based identification of novel biomarkers in asthma. Allergol Int 55:361–367

Asthma Patient Education Vanessa M. McDonald and Peter G. Gibson

Introduction Patient education is an integral component of effective asthma management that is necessary for all age groups [1]. It can be viewed as a two-staged process, which involves not only the acquisition of knowledge, but also the integration of skills and attitudes that leads to a change in behaviour. Effectively this is asthma selfmanagement education (SME). People with asthma often have poor asthma knowledge and management skills [2]. Effective self-management education can improve asthma knowledge and skills and reduce asthma exacerbations [3]. Base level or traditional patient education imparts disease-specific information and technical skills [4]. This can be further developed into SME that in addition to base-level education teaches problem-solving skills. Whilst this base-level education approach seeks to define the problem, SME allows patients to identify the problem and provides them with techniques and skills to help make decisions and implement or change treatment as they recognize changes in their disease control [5]. This is clearly a more powerful approach to patient education and one that is associated with definite improvement in patient health care outcomes. Asthma education may take many forms, and be conducted in many different settings. At its simplest level, it is limited to the transfer of information about asthma, its causes and its management. More complex interventions have been described, which are designed to develop self-management skills, alter attitudes and/or behaviours concerning asthma and improve medical management. Systematic reviews of randomized controlled trials (RCTs) of patient education have been performed to V.M. McDonald and P.G. Gibson Department of Respiratory and Sleep Medicine, John Hunter Hospital, Lookout Rd, New Lambton, NSW 2305, Australia; Hunter Medical Research Institute, Lookout Rd, New Lambton, NSW 2305, Australia; University of Newcastle, University Drive, Callaghan, NSW 2308, Australia P.G. Gibson () Department of Respiratory and Sleep Medicine, John Hunter Hospital, Locked Bag 1, Hunter Region Mail Centre, NSW 2310, Australia e-mail: [email protected]

R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, DOI 10.1007/978-4-431-98349-1_26, © Springer 2009

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evaluate the effects of different educational approaches and these demonstrate that the most effective form of patient education in asthma is self-management education [3]. Successful self-management education programmes incorporate a number of essential components, these are: the provision of information, self-monitoring, regular review with optimization of pharmacotherapy and a written plan for the management of unstable asthma [3].

Review of the Literature It is well established that education in asthma management is associated with improved patient knowledge [6] and patient satisfaction [7]; however, the effect that this has on health outcomes depends on the type of education provided [3]. Many RCTs have been conducted to evaluate the effectiveness of asthma education programmes. These trials have investigated several different types of education. More specifically, the provision of information alone and, secondly, education programmes that deliver self-management interventions. A Cochrane systematic review of 12 RCTs examining the provision of information about asthma alone concluded that this form of education led to improved knowledge, but did not reduce hospital admissions or unscheduled doctors’ visits for asthma; nor did it improve lung function or change medication use. One study showed a reduction in emergency department visits and two studies demonstrated an improvement in perceived asthma symptoms. The conclusions drawn from this analysis are that the use of limited asthma education does not appear to improve health outcomes in adults with asthma [6]. Another Cochrane systematic review evaluated 36 RCTs that compared selfmanagement education and usual medical care. The results of this analysis demonstrated that self-management education reduced hospitalizations, emergency department visits, unscheduled visits to the doctor, days off work or school, nocturnal asthma and improved quality of life. The conclusions drawn from this review indicate that education in asthma self-management, which involves the provision of information, self-monitoring by either peak expiratory flow (PEF) or symptoms and regular medical review coupled with a written asthma action plan (WAAP) improves health outcomes for adults with asthma [3], and are more successful than other education programmes. Further studies have examined different ways to deliver self-management education. In another Cochrane review six studies compared optimal self-management allowing self-adjustment of medications using individualized written action plans to adjustment of medications by a doctor. These two styles of asthma management gave equivalent effects using hospitalization as the outcome. Self-management using a written action plan based on peak expiratory flow (PEF) was found to be equivalent to self-management using a symptom-based written action plan in the six studies, which compared these interventions. Three studies compared different self-management options. One provided optimal therapy but tested the omission of regular review. The omission of regular review was associated with more health care

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utilization and days of sickness. In another study, comparing high- and low-intensity education, the latter was associated with more unscheduled doctor visits. In a third study, no difference in health care utilization or lung function was reported between verbal instruction and written action plans. Whilst most of the options tested were comparable, it is important to recognize that reducing the intensity of selfmanagement education or level of clinical review may reduce its effectiveness [8].

Delivery of Asthma Education Asthma education can be delivered using either an interactive or non-interactive approach [6, 9]. Methods of interactive delivery include individual consultations with a health professional, group education sessions, computerized interactive education, lectures and audiovisual display of material to encourage discussion, participatory learning, demonstration of practical skills and role play [9]. Non-interactive interventions include written information, audiovisual and audiotapes and non-interactive computer education delivered without interaction with an educator. A number of studies examined the effect of this type of education and found that overall they improved knowledge of asthma but had no effect on asthma morbidity [6]. Asthma self-management programmes have been evaluated in many different settings such as hospital ambulatory care centres, hospital emergency departments and primary care settings [3]. In addition, programmes have targeted diverse audiences with specific needs including pregnant women [10], children and adolescents [11, 12] and culturally and linguistically diverse groups [13]. These different studies describe how asthma SME can be adapted to different settings and patient groups.

Asthma Education in Children There have been many trials investigating the effect of self-management education in children. Wolf et al. evaluated 32 randomized trials in a Cochrane systematic review and found that asthma SME in children was associated with improvements in airflow obstruction and self-efficacy, as well as a reduction in asthma symptoms, school absenteeism and emergency department attendance [14].

Asthma Education in Pregnancy Achieving good control in this group is essential, pregnant women may have concerns about the use and safety of asthma medications during pregnancy and this may lead to non-adherence to treatment, poor control and frequent exacerbations [15]. Uncontrolled asthma during pregnancy can have an adverse effect on

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the foetus [16, 17]. Providing asthma education to this group may overcome this issue. A study by Murphy et al. examined the effect of self-management education in pregnant women. The participants in this study had poor baseline asthma knowledge, skills and adherence rates. Only 40% of participants reported adherence to their asthma treatment. During the intervention participants were provided with information about asthma and its management, instruction in self-monitoring and prescription and education relating to an asthma action plan. This resulted in a significant improvement in asthma knowledge, skills and adherence, suggesting that SME should be integrated into standard antenatal care [10].

Asthma Education in Cultural and Linguistically Diverse Groups Griffiths et al. conducted a RCT to determine whether asthma specialist nurses, using a liaison model of care, could reduce unscheduled health care in a deprived multiethnic area. Participants randomized to the intervention group were reviewed by specialist nurses who conducted an assessment of asthma control, provided a written asthma action plan and, when assessed as appropriate gave instruction in self-monitoring. The control group received a single visit from the nurses to discuss standard guidelines for asthma and had their inhaler techniques assessed. This study demonstrated a reduction in health care utilization in the intervention group [13].

Components of Asthma Education Programmes Developing Partnerships The delivery of effective education involves engaging the patient in the intervention, communicating information and building effective clinician/patient relationships or partnerships. Improved communication between the patient and clinician is associated with better patient satisfaction, increased adherence to treatment regimes and improved health outcomes [7, 18–20]. At the core of good communication and effective partnerships is person-centred care. Person-centred care has been defined as ‘treatment and care provided by health services that places the person at the centre of their own care.’ Developing good communication and practicing personcentred care involves treating patients as partners by involving them in decision making, improving their autonomy and respecting their beliefs and concerns [21]. The following are some guiding principles to facilitate good communication and partnerships: • Listen actively. • Listen to the patient’s perspective, values beliefs and past experiences.

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• Identify immediate and long-term concerns and beliefs that may lead to nonadherence. • Share in decision making, acknowledging the patient as ‘experts their own lives’. • Set short-term goals in partnership with your patient. • Give honest but reassuring messages. • Use open-ended, nonjudgmental questions. • Use appropriate verbal and nonverbal encouragement. [4, 22, 23] A recent RCT of ‘physician asthma care education’ (PACE) found that providing interactive sessions to paediatric primary care physicians led to positive effects on physician communication and patient outcomes. The intervention group was provided with two 2.5-h sessions that included short lectures, case discussions and video modelling communication techniques. The content of the sessions reviewed national asthma guidelines, communication skills and key educational messages. Parents of the children reviewed in this study participated in a phone interview 1 year after the intervention. Compared with the control group, the parents of the children in the intervention group reported that the physicians who reviewed their children were more likely to inquire about patients’ concerns about asthma, encourage patients to be physically active and set goals for successful treatment. The children reviewed by intervention physicians also had a greater decrease in days limited by asthma symptoms and fewer emergency department asthma visits [24]. This study demonstrated the positive effects of improving communication on not only patient satisfaction, but also how communication can effectively reduce health care utilization and improve patient outcomes.

Provision of Information About Asthma and Its Management There are a number of core information items and skill domains that all people with asthma require; however, the education process must be personalized to reflect the patients’ beliefs and values, learning needs and special considerations. Asthma education should include the provision of information relating to diagnosis, mechanisms and management of asthma, the role of asthma medications and the importance of regular long-term therapy. It should also include practical information about how to use inhaled therapy, the care of devices, information specific to the patients’ medication regime, signs and symptoms of worsening asthma, the identification, avoidance and management of trigger factors, an assessment of adherence and discussions focusing on adherence aiding strategies. The importance and benefits of self-monitoring, written asthma action plans and the need for regular follow-up should also be addressed [1, 25]. A checklist for an asthma education consult is illustrated in Fig. 1 [25].

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Information items and skills domains

Yes

N/A

Date and signature

Concept of asthma as an inflammatory Disease Concept of airway narrowing – mechanisms Concept of asthma medication classes Medications - action, when to use - side effects (how to reduce risk) Assess/demonstrate use of inhaler devices. Minimise the number of different devices Discuss care of devices Commence/review self monitoring (symptoms or peak flow) Prescription of Written Asthma Action Plan (WAAP) Assessment of understanding and acceptance of WAAP Assess adherence to treatment Exposure to tobacco smoke Quit advice given – specify to whom: Identification and management of triggers Myths about asthma and medications Assess asthma knowledge and skills Answer any questions Assessment of asthma knowledge and skills Encourage patients to answer in their own words. Give reinforcement, or further education if necessary. Good knowledge or skill?

Yes/No N/A

Date and signature

What is the Reliever that you will be using? When will you use it? What is the Preventer that you will be using? When will you use it? How will you tell when your asthma is getting worse? Explain to me how and when you will use the Action Plan Show me how you will record your asthma symptoms/peak flows Show me how you use your puffer. What is the major goal of asthma management?

Fig. 1 Asthma education consultation checklist. This figure illustrates a checklist that can be used to guide an asthma education consultation (From [25]. With permission)

Instructions should also be provided to assist the patient in the acquisition of skills. These include: • Assessment and correction of inhaled therapy • Explanation, assessment of technique and initiation of self-monitoring

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Fig. 2 Example of written asthma action plan. This figure illustrates an example of a ‘traffic light configuration’ written asthma action plan

• Prescription and agreement on course of action to take following an increase in signs and symptoms, namely the prescription of a WAAP (Self-monitoring and written asthma action plans will be discussed later in this chapter). When prescribing inhaled therapy it is important to demonstrate a number of devices and have the patient participate in the chose of device based on both the prescriber and the patient’s preference, considering their ability to adequately master the technique.

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Inhaler and peak flow technique should also be subsequently and repeatedly rechecked during follow-up consultations. Figure 2 provides an assessment tool to rank the skill proficiency for the most commonly used inhaler devices. Consideration should also be given to the number of different inhaled devices prescribed. A study by the present authors demonstrated the existence of inhaler device poly-pharmacy (defined by the use of more than two different devices), and its effect on the patient’s ability to adequately use their inhaled medication. The study reported that device technique adequacy decreased as the number of prescribed inhaler devices increased. There was no justification for the use of more than two inhalation delivery devices in asthma management [26]. Reinforcement of the messages given during the consultation is essential. The literature reports poor recall of the decisions and content of medical consultations [27]. Studies have demonstrated that patients only recall around 50% of what they have been told during the consultation. Importantly though, retention increases as the messages are subsequently repeated [28, 29]. Supplementing the education with written or pictorial information can improve this and is a recommended practice [30, 31].

Self-monitoring Self-monitoring enables the patients to assess their level of asthma control and recognize worsening symptoms. It is essential to the effective provision and use of written action plans as it determines the action points during exacerbations. The instruments available for self-monitoring include self-recording of symptoms and medication use using symptom diaries and self-recording of airflow obstruction by means of peak flow monitoring. These methods may be electronic or paper-based. Several studies have compared the effect of symptom versus peak flow monitoring in people with asthma and have found that either method offers equivalent results when coupled with a WAAP [8, 32]. Therefore, patients can choose to monitor symptoms, peak flow or both. There are, however, several exceptions. Peak flow monitoring may be advantageous in people who poorly perceive their symptoms. The peak flow may be more sensitive in determining asthma exacerbations and should be encouraged in this group. Conversely, symptoms may often deteriorate before peak flow during a mild exacerbation and this suggests that symptom-monitoring maybe of greater benefit in mild asthma [33]. When commencing patients on self-monitoring, consideration should also be given to literacy, cognition and ability to record symptoms or peak flow. Patients should be involved in the decision to commence self-monitoring and choose the form of monitoring based on their understanding, agreement, ability, lifestyle and financial implications of each method. Included in the consultation should be instruction regarding the rationale and benefits of self-monitoring, interpretation and the clinical relevance of the results on asthma control.

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When recommending peak flow monitoring there are a number of limitations to be considered. Only a very small proportion of people with asthma have or use a peak flow meter [34] and it is very effort- and technique-dependent. Clinicians should provide clear written instructions on how to perform and record peak flow and continue to regularly check technique as effort and skill may reduce over time. Additionally, there is little standardization between different meters [35], therefore it is important to inform patients of the necessity to use the same meter when assessing asthma control. Peak flow meter reproducibility may also reduce over time and frequently used meter should be replaced every 2 years [36].

Regular Review The aim of the medical review is to determine asthma control, prescribe pharmacological treatments and assess response to treatment and to provide instructions for the self-adjustment of medications during an exacerbation. Asthma education can improve treatment adherence [37] and hence provides an effective link to optimal pharmacotherapy. An asthma review can be structured systematically to measure current control through assessment of nocturnal wakening, diurnal symptoms, activity limitation, beta2 agonist use and lung function using spirometry. The review also provides an opportunity to assess inhaler technique, self-monitoring skills, knowledge of treatments and treatment plans, adherence and importantly for prescription and review of a written asthma action plan and reinforcement of previous education. Time for patient education is often a limiting factor; achievement of effective education can be accomplished through a multidisciplinary approach of several health professionals. Examples include a team approach combining a doctor/nurse, a doctor/pharmacist team or a doctor/asthma educator. Each of the participating health professionals should have specific training to achieve competency in asthma self-management education.

Written Asthma Action Plans A written action plan is a set of written instructions that helps people with asthma detect early signs and symptoms of worsening asthma and provides instructions about how to manage exacerbations. The plan should include instructions for maintenance therapy, early exacerbation management and crisis management. An example pro forma of a WAAP is illustrated in Fig. 3. Exacerbations of asthma rarely occur without warning. Whilst these exacerbations are often severe they are not necessarily acute, and will usually occur over a period of days or weeks or on a background of poor asthma control [38, 39]. This suggests that there is time for patients to make changes to their asthma management at the initial onset of symptoms or deterioration in control.

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WAAPs are essential to effective self-management [3], and their prescription is a key recommendation of international clinical practice guidelines for asthma [1, 40]. There is a large body of evidence that supports the use of WAAPs when packaged with self-management education [3]. Despite this evidence and guideline recommendations only a small number of people report ownership or use of a written action plan [41–44]. A population survey in South Australia demonstrated that the proportion of people who stated that they had a written action plan fell from 42% in 1995 to 20.8% in 2003 [42]. The low-level uptake of WAAPs may reflect poor training of doctors and health professionals in creating and delivering negotiated action plans in partnership with people with asthma [45]. Studies have shown that WAAPs are viewed as useful and desirable by patients, however in practice, they modify the plans to their perception and experience [46], whereas other studies have reported that patients do not wish to take control of their disease [47]. It is therefore necessary not only to improve knowledge of the efficiacy and benefits of WAAPs in both clinicians and patients, but also to improve clinician’s skills in WAAP prescription and skill transfer. There are five essential components of a WAAP, four of these include written information about [48]: 1. When to increase treatment (action point)? (a) Symptoms versus peak flow (b) PEF: percentage predicted versus personal best (c) Number of action points: 2, 3 or 4 (d) Presentation; “traffic light” 2. How to increase treatment? (a) Increase inhaled corticosteroid (ICS) and long-acing beta agonist (b) Oral corticosteroids (OCS) (c) Combination 3. How long to stay on increased treatment? (a) Duration of treatment 4. When to seek further medical attention? 5. The fifth of these components is not written but refers is the delivery of the action plan instruction to the patient and the assessment of their understanding and agreement. The first component (when to increase treatment) represents the action point or the point when the action plan should be initiated. The action point is established on the result of the patient’s self-monitoring and may be based on either symptoms or peak expiratory flow measurements. A Cochrane systematic review pooled the results of six RCTs that compared the effect of symptom self-monitoring versus peak flow self-monitoring. This review demonstrated no significant difference in health outcome using either form of self-monitoring, suggesting that the use of either method is effective. This important observation allows the clinician to confidently tailor self-monitoring to patient preference, skill and available resources [8].

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If using peak flow-based action points the instructions for treatment initiation are often based on a percentage of predicted PEF or percentage of personal best PEF. A number of studies have compared the use of both types of action plans to usual care. The present author (Peter G. Gibson) performed an evidenced-based review of the key components of written action plans, which included a metaanalysis of the studies using percentage predicted PEF (n = 4) or personal best PEF (n = 5) against usual care [48]. The pooled result indicates that both types of action plans reduced hospital admission, however, only the personal best PEF plans led to reduced emergency department attendance and improved airway calibre, suggesting that percentage of personal best PEF action points are superior [48]. A further variation of WAAPs is the number of action points included. Studies have been performed using 2, 3 or 4 action points. The same review conducted a comparison of these trials and found that using 2, 3 or 4 action points produced similar results [48]. This is another clinically significant finding to allow clinicians to tailor the plan to patient preference, knowledge, ability and skill. WAAP may be presented in different forms. Commonly used forms include standard written instructions and ‘traffic light’ configurations (Fig. 3), where green indicates maintenance therapy, orange indicates early exacerbation management and red indicates crisis management. A review of trials comparing this type of plan with standard written instruction demonstrated very similar outcomes [48]. The second key component of a WAAP is the instruction for how to increase treatment. Reddel and Barnes [49] performed a review of the pharmacological strategies for self-management of asthma exacerbations to provide clinicians with recommendations for the ‘how to increase treatment’ component of the plan. The overall recommendations resulting from this review are that oral corticosteroids (OCS) are effective in managing acute exacerbations of asthma. Doubling the dose of inhaled corticosteroid (ICS) was not supported by the literature, whereas increasing to high-dose ICS equivalent to 2,400–4,000 mcg beclomethasone was an effective alternative to OCS in mild to moderate exacerbations. The use of budesonide/formoterol as single maintenance and reliever therapy (SMART) is also supported by evidence [49]. The variation in treatment recommendations for acute exacerbations described here is important when both prescribing and conveying the WAAP, and highlights that there is no one-size-fits-all approach to WAAPs. Patient education is important to ensure that the patient has a thorough understanding of what is being asked of them during exacerbations. The third key component relates to how long the patient should remain on increased therapy. There is a risk of patents stopping their increased treatment once they notice some resolution of symptoms. The WAAP should clearly state the duration of treatment irrespective of symptom resolution. Oral corticosteroids are effective in treating acute exacerbations. A number of studies have compared the duration of OCS treatment and the effect of abrupt cessation or tapering. The evidence supports the use of OCS in the treatment of exacerbations for at least 7 days. There does not appear to be any benefit in continuing OCS treatment beyond 10 days [49]. Tapering OCSs does not reduce the incidence of relapse and is therefore not required; however, studies comparing tapering to abrupt cessation using side effects as an outcome have not been performed [49].

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The final step describes the time to seek further medical attention or the crisis management. This should adhere to national asthma guidelines that have published first aid for asthma plans, and be adapted to individual circumstances [40]. These four steps should be written in a language and text the patient will understand, carefully avoiding the use of medical terminology. There have been studies using pictography to assist in patient education and recall of medical instructions [30] and this may be a useful alternative to WAAPs in an illiterate population. Examples of these have been suggested for use in asthma management [50], although they have not been evaluated in practice. The fifth essential ingredient for an action plan relates to the translation of the written text into the understanding and acceptance of the treatment plan by the patient and the subsequent change in behaviour. When designing the action plan the patient should be involved in the decision-making process, taking their needs, beliefs and concerns into consideration. Once treatment strategies have been agreed upon the clinician should assess the patient’s level of understanding and acceptance by asking open-ended questions and using problem-solving scenarios. The partnership that is build between the patient and clinician will be paramount to the successful acceptance and utilization of the plan. Asthma education is necessary to the successful conveyance of a WAAP.

Conclusions Asthma education is essential to asthma management. The most effective form is self-management education, which combines the provision of information about asthma and its management, instruction in self-monitoring, regular review and prescription of a WAAP. When this is done with effective clinician/patient partnerships, involving patients in decision-making and ongoing management, it results in improved disease knowledge, improved asthma management skills and reduced symptoms from asthma and reduced health care utilization. The delivery of asthma education can be tailored to a variety of settings involving a team of health professionals working together with the patient to achieve effective asthma control.

References 1. GINA: Global strategy for asthma management and prevention: Global Strategy for Asthma Management and Prevention, NHLBI/WHO Workshop Report, 2006. 2. Gibson PG, Talbot PI, Hancock J, Hensley MJ: A prospective audit of asthma management following emergency treatment at a teaching hospital. Med J Aust 1993; 158:775–778. 3. Gibson PG, Powell H, Coughlin J, Wilson AJ, Abramson M, Haywood P, Bauman A, Hensley MJ, Walters EH: Self-management education and regular practitioner review for adults with asthma. The Cochrane Database of Systematic Reviews 2003. 4. Bodenheimer T, Lorig K, Holman H, Grumbach K: Patient self management of chronic disease in primary care. JAMA 2002;288:2469–2475. 5. D’Zurilla T: Problem solving therapy. New York, Springer, 1986.

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6. Gibson PG, Powell H, Coughlan J, Wilson AJ, Hensley MJ, Abramson M, Bauman A, Walters EH: Limited (information only) patient education programs for adults with asthma. The Cochrane Database Systematic Review 2002 7. Clark NM, Gong M, Schork MA, Kaciroti N, Evans D, Roloff D, Hurwitz M, Maiman LA, Mellins RB: Long-term effects of asthma education for physicians on patient satisfaction and use of health services. Eur Respir J 2000; 16:15–21. 8. Powell H, Gibson PG: Options for self-management education for adults with asthma. The Cochrane Database of Systematic Reviews 2003 9. Wagner CW, Karpel SK: The educational aspect of asthma management. Int Rev Asthma 2001; 3:73–85. 10. Murphy VE, Gibson PG, Talbot PI, Kessell CG, Clifton VL: Asthma self-management skills and the use of asthma education during pregnancy. Eur Respir J 2005; 26:435–441. 11. Guevara JP, Wolf FM, Grum CM, Clark NM,. Effects of educational interventions for self management of asthma in children and adolescents: Systematic review and meta-analysis. BMJ 2003; 326:1308–1309. 12. Shah S, Peat JK, Mazurski EJ, Wang H, Sindhusake D, Bruce C, Henry RL, Gibson PG: Effect of peer led programme for asthma education in adolescents: Cluster randomised controlled trial. BMJ 2001; 322:583–585. 13. Griffiths C, Foster G, Barnes N, Eldridge S, Tate H, Begum S, et al.: Specialist nurse intervention to reduce unscheduled asthma care in a deprived multiethnic area: The east London randomised controlled trial for high risk asthma (electra). BMJ 2004; 328:144. 14. Wolf FM, Guevara JP, Grum CM, Clark NM, Cates CJ: Educational interventions for asthma in children. The Cochrane Database of Systematic Reviews 2002 15. Jana N, Vasishta K, Saha SC, Khunnu B: Effect of bronchial asthma on the course of pregnancy, labour and perinatal outcome. J Obstet Gynaecol 1995; 21:227–232. 16. Schatz MJ: Interrelationships between asthma and pregnancy: A literature review. Allergy Clin Immunol 1999; 103(2 Pt 2):S330–336. 17. Demissie K, Breckenridge MB, Rhoads GG: Infant and maternal outcomes in the pregnancies of asthmatic women. Am J Respir Crit Care Med 1998; 158:1091–1095. 18. Neuwirth ZE: An essential understanding of physician-patient communication. Part II. J Med Pract Manage 1999; 15:68–72. 19. Beckman H, Frankel R: The effect of physician behavior on the collection of data. Ann Intern Med 1984; 101:692–696. 20. Roter DL, Stewart M, Putnam SM, Lipkin M Jr, Stiles W, Inui TS: Communication patterns of primary care physicians. JAMA 1997; 277:350–356. 21. National Ageing Research Institute: What is person-centred health care? A literature review. Melbourne, Australia, 2006. 22. National Asthma Campaign: Asthma adherence: A guide for health professionals. Australia, National Asthma Campaign, 1999. 23. Partridge MR, Hill SR: Enhancing care for people with asthma: The role of communication, education, training and self-management. Eur Respir J 2000; 16:334–348. 24. Cabana MD, Slish KK, Evans B, Mellins RB, Brown RW, Lin X, Kaciroti N, Clark NM: Impact of physician asthma care education on patient outcomes. Pediatrics 2006; 117:2149–2157. 25. McDonald VM, Gibson PG: Asthma self-management education. Chron Respir Dis 2006; 3:29–37. 26. McDonald VM, Gibson PG: Inhalation device polypharmacy in asthma. Med J Aust 2005; 182:250–251. 27. Page P, Verstraete DG, Robb JR, Etzwiler DD: Patient recall of self-care recommendations in diabetes. Diabetes Care 1981; 4:96–98. 28. Falvo D, Tippy P: Communicating information to patients: Patient satisfaction and adherence as associated with resident skill. J Fam Pract 1988; 26:643–647. 29. Pederson S: Ensuring compliance in children. Eur Respir J 1992; 5:143–145. 30. Houts PS, Bachrach R, Witmer JT, Tringali CA, Bucher JA, Localio RA: Using pictographs to enhance recall of spoken medical instructions. Patient Educ Couns 1998; 35:83–88.

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31. Sandler DA, Heaton C, Garner ST, Mitchell JR: Patients’ and general practitioners’ satisfaction with information given on discharge from hospital: Audit of a new information card. BMJ 1989; 16:1511–1513. 32. Buist SA, Vollmer WM, Wilson SR, Frazier EA, Hayward AD: A randomized clinical trial of peak flow versus symptom monitoring in older adults with asthma. AJRCCM 2006; 174:1077–1088. 33. Gibson PG: Monitoring the patient with asthma: An evidenced-based approach. J Allergy Clin Immunol 2000;106:17–26. 34. Chowienczyk PJ, Parkin DH, Lawson CP, Cochrane GM: Do asthmatic patients correctly record home spirometry measurements? BMJ 1994; 309:1618. 35. Hancox B, Whyte KA: Pocket guide to lung function tests. Australia, McGraw-Hill, 2001. 36. The Thoracic Society of Australia and New Zealand: Peak flow meter use in asthma management. Med J Aust 1996; 164:727–730. 37. Bailey WC, Richards JM, Brooks CM, Soong SJ, Windsor RA, Manzella MPH: A randomised trial to improve self management practices of adults with asthma. Arch Int Med 1990; 150:1664–1668. 38. Chan-Yeung M, Chang JH, Manfreda J, Ferguson A, Becker A: Changes in peak flow, symptom score and the use of medications during acute exacerbations of asthma. Am J Respir Crit Care Med 1996; 154:889–893. 39. Turner MO, Noertjojo K, Vedal S, Bai T, Crump S, Fitzgerald M: Risk factors for near fatal asthma. Am J Respir Crit Care Med 1998;157:1804–1809. 40. National Asthma Council: Asthma management handbook. Melbourne Australia, 2006. 41. Price D, Wolfe S: Delivery of asthma care: Patients’ use of and views on healthcare services as determined from a nationwide interview survey. Asthma J 2000; 5:141–144. 42. Wilson DH, Adams RJ, Tucker G, Appleton S, Taylor AW, Ruffin RE: Trends in asthma prevalence and population changes in south Australia, 1990–2003. Med J Aust 2006; 184(5):226–229. 43. Wilson DH, Adams RJ, Appleton SL, Hugo G, Wilkinson D, Hiller J, Ryan P, J C, RE R: Prevalence of asthma and asthma action plans in south Australia: Population surveys from 1990 to 2001. MJA 2003; 178:483–485. 44. Matheson M, Wicking J, Raven J, Woods R, Thien F, Abramson M, Walters EH: Asthma management: How effective is it in the community? Intern Med J 2002; 32:451–456. 45. Walters EH, Walters JAE, Wood-Baker R: Why have asthma action plans failed the consumer test? Med J Aust 2003; 178:477–478. 46. Douglass J, Aroni R, Goeman D, Stewart K, Thien F, Abramson M: A qualitative study of action plans for asthma. BMJ 2002; 324:1003–1005. 47. Jones A, Pill R, Adams S: Qualitative study of use of health professionals and patients on guided self management plans for asthma. BMJ 2000; 321:1507–1510. 48. Gibson PG, Powell H: Written action plans for asthma: An evidence-based review of the key components. Thorax 2004; 59:94–99. 49. Reddel HK, Barnes DJ: Pharmacological strategies for self-management of asthma exacerbations. Eur Respir J 2006; 28:182–199. 50. Roberts NJ, Partridge MR: Optimal consultations for those with respiratory illness. Breathe 2007; 3:331–337.

The Costs of Allergy and Asthma and the Potential Benefit of Prevention Strategies Jay M. Portnoy and Mercedes C. Amado

Introduction Asthma and allergic diseases account for a significant proportion of the chronic illnesses that afflict human beings. Worldwide, asthma has been described as an epidemic that has increased both in prevalence and incidence over the last 20 years despite improved pharmacotherapy and environmental control. In the same way, allergic diseases such as rhinitis, food allergy, atopic dermatitis, and asthma triggered by allergies have also increased. The total burden of these chronic diseases is staggering. Recent research has identified the details of both the development and the persistence of allergic pathways (otherwise known as the “Atopic March”) and how allergies develop from the earliest periods of life through early childhood and into adulthood. Despite this information, methods for prevention or control have not been widely identified and implemented in clinical practice. The purpose of this review is to describe the burden of asthma in the human population and to review various interventions that have been proposed and in some cases tested to accomplish either primary or secondary prevention.

The Costs of Asthma and Allergic Diseases Asthma is one of the most common medical conditions afflicting both children and adults. In 2005, an estimated 7.7% of the US population or 22.2 million persons had asthma, of which 6.5 million (8.9%) were children and 15.7 million (7.2%) were adults [1]. In childhood, asthma is more common in boys than girls though it is more common in adult women than adult men [2]. It disproportionately affects African Americans although Hispanics have lower rates of asthma than non-Hispanic blacks and whites, Puerto Ricans have higher rates of asthma than other Hispanic subgroups J.M. Portnoy () and M.C. Amado Children’s Mercy Hospitals and Clinics, 2401 Gillham Road, Kansas City, MO 64108, USA e-mail: [email protected]

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and non-Hispanic whites, as well as higher death rates than other Hispanic subgroups, non-Hispanic whites, and blacks [3, 4]. Asthma produces a significant burden upon the individual, family, and society in terms of physical illness, psychological stress, decreased productivity, and cost of care [5]. It is the major cause of school absenteeism in children, contributing to an estimated ten million missed school days a year [6]. In 2003, 10.1 million work days were missed secondary to asthma by employed adults of 18 years and older. This number does not include parents who missed work to care for a sick child with asthma. In one study of children and adolescents, more than half were inadequately controlled as measured using the Asthma Control Test. Such inadequately controlled asthma significantly affected asthma-specific QOL, school productivity and attendance, and work productivity of children and their caregivers. More specifically, caregivers reported missing 1.4 days of work due to their child’s asthma with the child missing 4.1 school days [7]. In 2004, there were 14.7 million outpatient ambulatory visits, 1.8 million ED visits, and 497,000 hospitalizations for asthma. The highest asthma hospitalization rates among children were for those aged 0–4 years. In 2003, 4,055 persons died of asthma, of which the majority were adults of 18 years and over [1]. In terms of monetary costs, in 1998, asthma in the USA accounted for an estimated cost of $12.7 billion annually and 58% of these costs were direct medical expenditures and 42% were indirect costs such as child care expenses, transportation, and loss of workforce participation [8]. More recent estimates of the annual cost of asthma are nearly $18 billion per year with direct costs and nearly $10 billion with hospitalizations, the single largest portion of direct costs [9]. In addition to asthma, it has been estimated that one in five Americans or 50 million persons experience allergies including nasal allergies, food allergy, drug allergy atopic dermatitis, and insect allergy, and the incidence of allergic diseases has been increasing in all ages for the past 20 years. Nasal allergies affect 75%, skin allergies 7%, food and drug allergy 6%, and insect allergy 4% of allergies sufferers. Some surveys even suggest that atopic dermatitis imposes an economic burden with overall costs similar to those for treatment of asthma [10]. The annual cost of allergies is estimated to be nearly $7 billion.

Prevention of Asthma and Allergic Diseases [11] It has been said that an ounce of prevention is worth a pound of cure. Given the high costs of asthma and allergic diseases described above, this saying certainly is germane. Clearly, the most desirable approach to treatment of allergic diseases would be to prevent them from occurring in the first place. This type of strategy can be divided into primary and secondary prevention [12, 13]. The US Preventative Services Task Forces’ Guide to Clinical Preventive Services [14] defines primary prevention measures as “those provided to individuals to prevent the onset of a targeted condition.” Primary prevention measures include

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activities that help avoid a given health care problem. Examples include passive and active immunization against disease as well as health-protecting education and counseling. Since successful primary prevention helps avoid the suffering, cost, and burden associated with disease, it is considered to be the most cost-effective form of health care. Secondary prevention measures are those that “identify and treat asymptomatic persons who have already developed risk factors or preclinical disease but in whom the condition is not clinically apparent” [14]. These activities focus on early identification of asymptomatic persons who have significant risk for negative outcome without treatment. Screening tests are examples of secondary prevention activities, particularly for diseases that have a significant latency period. Early identification may permit alteration of the natural history of the disease to maximize well-being and minimize suffering. Tertiary prevention involves the care of persons with established disease. As such, it often is considered to be treatment rather than prevention and involves interventions that attempt to restore to highest function, minimize the negative effects of disease, and prevent disease-related complications. Once the disease is established, the opportunity for primary prevention has been missed. Early detection through secondary prevention may minimize the impact of the disease. Since the development of atopic diseases depends on an interaction between genetic factors and environmental factors (both specific and nonspecific), prevention may include both exposure to allergens and/or adjuvants and pharmacological treatments. Depending on the type of prevention, it therefore is necessary to address the general population or at least children at risk for developing atopic diseases if they can be identified for primary prevention, children with early symptoms of allergic disease for secondary prevention or children with established chronic disease for tertiary prevention.

Primary Prevention For the purpose of this review, primary prevention refers to any strategy that avoids the development of the disease. Primary strategies for atopy would prevent the development of IgE responses to environmental exposures and lead to a predominantly Th1 phenotype. A primary strategy for asthma would prevent the development of airways inflammation leading to a clinical illness that would be recognized as asthma. Both of these strategies require either universal implementation to prevent anyone from developing the diseases, or an effective means for identifying individuals at increased risk for their development who would receive a targeted intervention. The effectiveness of prevention measures is in part dependent on the ability to identify individuals who would benefit from the measures as well as the consistency with which they are applied to those individuals. Dietary restriction, for example, is unlikely to be accepted by the majority of the world’s population due to its inconvenience and potentially increased cost. Yet, if individuals who would

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benefit from such restriction are not identified and treated early enough (i.e., prior to development of allergic sensitivities) primary prevention will not be as effective as it could be. For example, preadolescent boys are more likely to be diagnosed with asthma than girls (32% vs 18%, respectively). In one study, 4 more weeks of breast-feeding reduced the number of wheezing episodes and shortness of breath in boys but not in girls suggesting that a high risk for developing asthma might be gender-specific as is the effectiveness of the intervention [15]. Another consideration when looking for primary intervention strategies is whether complete avoidance of potential sensitizers is really such a good idea. For example, though bronchiolitis due to respiratory syncytial virus is suspected to be a triggering event for the onset of asthma manifestations but not atopy in infancy, other infections in addition to the use of probiotics may actually prevent the development of allergic respiratory tract diseases, including asthma [16, 17]. This gets to the concept of the hygiene hypothesis, which states that exposure to environmental substances such as endotoxins, bacteria, viruses, and other exposures is necessary for the development of the immune system. Patients who avoid exposure to normal environmental substances have a higher likelihood of developing asthma and allergic sensitivities. Therefore, complete control of environmental exposures may not be an effective intervention.

Dietary Interventions Dietary interventions have long been used for primary prevention of allergic sensitization to foods. This is relevant for food allergies as well as for atopic dermatitis, which commonly is triggered by ingestion of certain foods. The American Academy of Pediatrics has recommended that infants be exclusively breast-fed for the first 6–12 months and that they avoid common allergic sensitizers such as peanuts for 2–4 years. While this approach undoubtedly delays the onset of allergic sensitization, it is not clear whether the development of specific IgE to these foods is prevented in the long term. In other words, to patients who avoid peanuts when they are young simply develop peanut allergy when they get older? It is unclear whether dietary interventions can actually prevent the development of allergies and asthma. In a systematic review of the literature, the most effective method for primary prevention of food allergy was found to include exclusive breast-feeding for at least 6 months, delayed introduction of solid foods for at least 6 months, and use of “hypoallergenic” formulae in high-risk patients [18]. Infants with a family history of atopy and a cord blood IgE greater than or equal to 0.3 kU/l have been shown to have an increased risk of developing atopic diseases. This was particularly true if both parents had atopy. The effect of feeding high-risk infants breast milk and/or hydrolyzed cow’s milk-based formula for the first 4–6 months has been studied prospectively in this group of high-risk infants. Infants who were exclusively fed the restricted diet for at least 4 months and who were followed for

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5 years were found to have a 17% reduction in development of atopy than a control group fed a normal diet. The preventative effect of breast-feeding and use of hydrolyzed formula were similar. Avoidance of other factors such as tobacco smoke, exposure to pets, delayed introduction of solid foods, and higher socioeconomic status was significantly less effective at preventing development of milk allergy than the restricted diets. Interestingly, a restricted diet lasting for 4 months was just as effective for preventing food allergy as a diet lasting 6 months or longer [19]. No significant differences were found in infant allergy or childhood cow’s milk allergy in two trials comparing early, short-term hydrolyzed formula with exclusive breast-feeding. It is not clear whether prolonged feeding of hydrolyzed formula compared human milk feeding would show a difference as such a trial has not been performed to date. A number of studies have compared prolonged feeding with hydrolyzed formula with cow’s milk formula in high-risk infants. A meta-analysis found a significant reduction in the development of infant cow’s milk allergy, but not in the incidence of childhood allergy and no significant difference in the development of infant or childhood eczema, asthma, rhinitis, and other food allergies. Infants fed extensively hydrolyzed formula compared with partially hydrolyzed formula had a significant reduction in food allergy, but not in other types of allergies. The bottom line is that exclusive breast-feeding or feeding with extensively hydrolyzed formula is helpful for primary prevention of food allergy in high-risk infants though there is limited evidence that prolonged feeding reduces infant and childhood allergy [20]. Another meta-analysis comparing prolonged soy formula to cow’s milk formula feeding found no significant differences in the development of infant cow’s milk protein intolerance or in the incidence or prevalence of infant or childhood allergy, asthma, eczema, rhinitis, or urticaria. Clearly, exclusive feeding with a soy formula is not useful for prevention of allergy or food intolerance in high-risk infants [21]. Another approach to primary prevention involves maternal dietary antigen avoidance either during pregnancy or lactation. A systematic review found that such a restricted diet during pregnancy was associated with a slightly lower mean gestational weight gain, a higher risk of preterm birth, and a reduction in mean birthweight without any protective effect on the infant. Maternal antigen avoidance during lactation had no effect on the incidence of eczema during the first 18 months though there was a slight reduction in severity of established eczema [22]. Though it may be desirable to prevent development of food allergy in young children, the feasibility of such an approach has been called into question. In an attempt to study complete avoidance of allergenic foods in children starting birth in one study, dietary avoidance was found to be incomplete and not feasible in most study subjects despite substantial attempts to restrict those diets [23]. The difficulty of avoiding allergenic foods can limit the availability of this type of intervention. For example, in Hungary, socioeconomic status was found to be directly related to implementation of primary allergy prevention in infant feeding, presumably due to increased cost of such diets [24]. Reduced consumption of omega3-polyunsaturated fatty acids observed especially in the Western societies has been suspected to contribute to the increasing

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prevalence of asthma and allergies. Though clinical trials of omega3-fatty acid supplementation in adults with allergies and asthma have been disappointing, supplementation in the perinatal and early childhood years has shown some beneficial effects on clinical phenotypes of the atopic syndrome [25]. The risk of developing symptoms, but not of sensitization has even been shown to be affected by differences in the intake of n-3 long chain polyunsaturated fatty acids through breast milk among infants who do breast-feed [26]. Clearly, improved understanding of the association between early life lipid intake and the development of allergic diseases is needed.

Weight Management Obesity has been shown to correlate with the prevalence, incidence, and severity of asthma [27]. Evidence for causality includes the observation that weight loss in the individuals improves asthma outcomes. A number of hypotheses about the biological basis for this relationship have been proposed including common comorbidities, the effect of obesity on lung function, and inflammatory mediators produced by adipose tissue. Given the epidemic of obesity in the USA and more recently on a global basis, primary prevention of atopy may also require prevention of obesity at the same time [28]. One factor that may affect development of obesity is lack of breast-feeding. In a longitudinal Dutch study of body mass index (BMI), children breast-fed for more than 16 weeks had a lower BMI at 1 year than their bottle-fed cohorts. When BMI was followed for 7 years, no association was found between breastfeeding and BMI. On the other hand, an elevated BMI at 1 year of age was strongly associated with a high BMI between 1 and 7 years of age suggesting that breast-fed children with lower BMI at 1 year have a lower risk of becoming overweight later in life [29]. A systematic review comparing BMI and asthma identified a dose–response effect of elevated BMI on asthma incidence that was similar both for men and women. This further suggests that the incidence of asthma could be reduced by interventions targeting overweight and obesity [30]. In a cross-sectional study of Canadian adults, one unit of increased BMI was associated with approximately 6% increase in asthma risk in women, and 3% in men. Interestingly, obesity seemed to have a larger effect on nonallergic asthma, possibly explaining the stronger association seen in women since they have a lower prevalence of allergy [31]. To determine whether asthma precedes increased body mass index or vice versa, investigators with the First National Health and Nutrition Examination Survey collected height and weight data, and information about doctor-diagnosed asthma from 14,407 subjects aged 25–74 with a mean follow-up of 10 years. Increasing BMI was associated with increased prevalence of asthma though no associations were observed for the incidence of asthma. Though African Americans had an increased risk of asthma, this risk was not associated with BMI [32].

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Several genes have been hypothesized to contribute to the development of both obesity and asthma including the beta2-adrenergic receptor, tumor necrosis factoralpha (TNF-alpha) and the insulin-like growth factor-1 (IGF-1) [33].

Environmental Interventions Development of respiratory allergies such as rhinitis and asthma requires sensitization to environmental allergens, subsequent exposure to the allergens, and increased sensitivity of the target organ to the resulting allergic inflammation. If any of these three compliments are missing, the patient is unlikely to have clinical symptoms. There is some evidence that the development of allergic inflammation and increased sensitivity of the target organ are related to the initial sensitization. For that reason, primary intervention using environmental control must rely on prevention of the original sensitization. Prevention of sensitization to aeroallergens depends in part on the specific allergen being considered. In general, exposure to 10 mcg per gram of dust has been shown to be sufficient to sensitize a predisposed individual to dust mite. Once this sensitization has taken place, subsequent exposures of as little as 2 mcg per gram of dust seem to be adequate for triggering symptoms. This is different for cat allergen in which exposure to significantly higher concentrations prior to sensitization seems to protect a predisposed individual from becoming cat allergic. It is not known why one allergen would cause sensitization, while the other would prevent it, but clearly once the patient has become sensitized it is too late for primary prevention. The hygiene hypothesis predicts that exposure to microbial agents might inhibit the development of atopy and asthma. When Endotoxin, fungal (1–> 3)-beta-dglucans, extracellular polysaccharides from several fungi, and dust on living room floors were measured, persistent wheeze was less common in the high-exposure group, particularly if exposed to fungi. Though this suggests that microbial exposure in early life might protect against asthma, the total value of such interventions has not yet been proven [34]. Several intervention studies have provided evidence that avoidance of indoor allergens such as house dust mite high-risk infants may reduce the incidence of asthma and sensitization during the first 4 years of life. In particular, semipermeable polyurethane mattress and pillow encasings (allergy control) when compared with placebo encasings resulted in a significant reduction of dust mite exposure leading to a reduction in the need for inhaled steroids after 1 year [19]. Sensitization to house dust mite is recognized as an important risk factor for the development of asthma and allergic disease in childhood with higher levels of exposure corresponding to increased risk of sensitization. The most common avoidance procedure is with the use of mite-impermeable mattress encasings. Unfortunately, studies looking at this procedure alone have not reliably demonstrated significant improvement in symptoms or prevention of sensitization [35]. In one study there was a 7.2% reduction in sensitization to house dust mite in children at high risk for atopy with avoidance (NNT 14) [36].

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It is likely that since allergic diseases and asthma are multifactorial diseases, prevention by addressing only one risk factor is unlikely to be effective. Instead, a multifaceted approach is likely to be required. This is in fact what has been observed as studies with a multifaceted approach do have a greater chance of being successful than studies using a monofaceted approach [37]. Overall, most studies of dust mite avoidance suggest that use of acaricides and extensive bedroom-based environmental control may be of some benefit in reducing rhinitis symptoms while isolated use of dust mite-impermeable covers has not proven to be effective [38]. Another study tested the effectiveness of dust mite avoidance and dietary fatty acid modification when implemented during the first 5 years of life. Even though dust mite avoidance led to a 61% reduction in exposure in the child’s bed, no difference was found in the prevalence of asthma or atopy. Increases in plasma omega-3 fatty acids also did not reduce the prevalence of asthma, eczema, or atopy [39].

Vaccines Vaccines have been suspected of contributing to the increased prevalence of allergic diseases. Several mechanisms have been proposed including the use of alum as an adjuvant since it is suspected of contributing to a TH2 phenotype response. For this reason a great deal of research has emphasized adjuvants that could lead to a TH1 type of response. One potential candidate for this consists of immunostimulatory sequences of DNA containing a CPG motif. This sequence has been identified in mycobacteria that the human body recognizes as strongly immunogenic. Initial evidence from clinical trials of ragweed antigen suggests that this sequence can augment an IgG immune response even in a patient with an allergic sensitization [40]. Currently, similar trials are being done to see whether common childhood vaccines when combined with this type of sequence can be used to reduce the likelihood of allergy sensitization. The hygiene hypothesis suggests that early life environmental exposure to microbes, other pathogens, and their products promotes immune responses that suppress the development of atopy. Since asthma and atopy are characterized by Th2 patterns of inflammation, the hygiene hypothesis predicts that the currently observed increases in atopy and asthma are related to a lack of microbial stimuli. In addition, by providing the missing stumuli it should be possible to convert this undesirable pattern to a Th1 phenotype. The regulation of this switch is complex and includes interactions between antigen-presenting cells as well cytokines such as IL-10 and TGF-beta effects. Toll-like receptors appear to have evolved to recognize specific ligands on immune cells that control the development of these responses. Ongoing clinical trials for some of the compounds that activate these receptors such as cytosine-guanine dinucleotide DNA and Mycobacterium vaccae are being conducted at the present time [41]. The association between exposure to mycobacterial components and prevention of atopic diseases suggests that mycobacterial adjuvants may also be useful for prevention of allergic diseases [42].

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Another approach to immunization involves alterations in the intestinal microflora since it is an important influence on maturation of the immune system after birth. Interestingly, within the first week of life the composition of the microflora differs between healthy and allergic infants and in countries with a high and low prevalence of allergies. For this reason, alterations of the microflora using live microorganisms such as probiotics that might be beneficial have yielded encouraging results [43].

Immunotherapy Though numerous pharmacotherapeutic options are available for treatment of allergic diseases, there is little evidence that any of them can effectively prevent or alter their natural history. On the other hand, there is increasing evidence that allergen immunotherapy may be useful for the prevention of atopy and allergic asthma [44, 45]. In one study, a 3-year course of specific immunotherapy in children with allergic rhinitis and sensitivity to grass and/or birch pollen significantly reduced their risk of developing asthma. This improvement persisted for at least 5 years after immunotherapy was discontinued. More important, immunotherapy-treated children were significantly less likely to develop asthma and new sensitivities after 5 years than matched controls [46].

Combinations Since avoidance of individual risk factors for developing childhood asthma has not proven to be effective, multifaceted interventions have been studied in high-risk infants. Such interventions generally include avoidance of house dust, pets, and environmental tobacco smoke along with encouragement of breast-feeding and delayed introduction of solid foods. In one such study, the prevalence of asthma at 7 years was significantly lower in the intervention group than in a control group though the investigators were unable to demonstrate a reduced prevalence of allergic rhinitis, atopic dermatitis, positive skin tests, or bronchial hyperresponsiveness [47]. In another study, allergen avoidance measures such as use of dust mite covers and acaracides were instituted from birth and infants were either breast-fed with mother on a low allergen diet or given an extensively hydrolyzed formula. This combined prevention strategy led to a significantly decreased risk for development of asthma and dust mite sensitivity [48]. Dietary supplementation with omega-3 fatty acids and house dust mite allergen avoidance in children at high risk of asthma showed a 10% reduction in cough in the active diet group (NNT 10) but a negligible reduction among nonatopic children [36]. Perhaps the most comprehensive review of primary prevention was found in a position statement issued by The Australasian Society of Clinical Immunology and Allergy [49]. The conclusions of that review are shown in Table 1. Similar conclusions were made in another systematic review [50].

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Table 1 The Australasian Society of Clinical Immunology and Allergy position statement: summary of allergy prevention in children [49] • Dietary restrictions in pregnancy are not recommended • Avoidance of inhalant allergens during pregnancy has not been shown to reduce allergic disease, and is not recommended • Breast-feeding should be recommended because of other beneficial effects, but if it is not possible, a hydrolyzed formula is recommended (rather than conventional cow’s milk formulae) in high-risk infants only • Maternal dietary restrictions during breast-feeding are not recommended • Soy formulae and other formulae (e.g., goat’s milk) are not recommended for reducing food allergy risk • Complementary foods (including normal cow’s milk formulae) should be delayed until a child is aged at least 4–6 months, but a preventive effect from this measure has only been demonstrated in high-risk infants • There is no evidence that an elimination diet after age 4–6 months has a protective effect, although this needs additional investigation • Further research is needed to determine the relationship between house dust mite exposure at an early age and the development of sensitization and disease; no recommendation can yet be made about avoidance measures for preventing allergic disease • No recommendations can be made about exposure to pets in early life and the development of allergic disease. If a family already has pets it is not necessary to remove them, unless the child develops evidence of pet allergy (as assessed by an allergy specialist) • Women should be advised not to smoke while pregnant, and parents should be advised not to smoke • No recommendations can be made on the use of probiotic supplements (or other microbial agents) for preventing allergic disease at this time • Immunotherapy may be considered as a treatment option for children with allergic rhinitis, and may prevent the subsequent development of asthma

Secondary Prevention Pharmacotherapy Pharmacotherapy is the most widely used means of controlling asthma and allergies; yet it is unclear whether there is any long-term beneficial effect. For asthma this usually involves questions related to airways remodeling and potential longterm declines in lung function. Recently, a pediatric study was performed that evaluated the potential effectiveness of inhaled corticosteroids for long-term prevention of asthma. Patients received either inhaled steroids continuously for 2 years or a placebo and then they were observed for an additional 2 years. The patients who received steroids had a rapid decrease in their lung function once the steroids were stopped such that they were similar to the placebo group. This suggested that treatment with inhaled corticosteroids had no preventative effect on asthma. To date, there has been no clear-cut pharmacotherapy that has been suggested for either reduction or prevention of the tendency to develop IgE. Therefore, drug therapy is considered to be purely symptomatic once a patient has developed allergies.

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Immunotherapy Immunotherapy has been used to prevent the development of allergies and asthma, as well as a means to shorten the natural history of the disease in patients who already have developed sensitivities and who therefore have missed the opportunity for primary intervention. Immunotherapy has the potential to prevent the onset of new sensitizations in children with rhinitis and/or asthma who are monosensitized to house dust mite. In a study of children with rhinitis and/or asthma monosensitized to house dust mite given immunotherapy to dust mite over 5 years, 75.3% in the SIT group showed no new sensitization compared to 46.7% in the control group. The patients who developed new sensitizations were more atopic and required more medications for treatment of rhinitis and asthma [51]. In addition to preventing new sensitizations, immunotherapy also has been shown to reduce the likelihood of monosensitized children from developing asthma. The largest clinical trial to date evaluating this effect was the PAT study performed in Europe. In this study, a large number of young children were randomized to receive either specific immunotherapy or placebo and were monitored for the development of asthma as well as new sensitizations. The patients in the active treatment group had a significantly decreased risk of developing asthma than those who received placebo [46].

Environmental Control There has been a suspicion that if patients can fully control their environmental exposure, their allergy sensitivity would wane over time. Though a number of anecdotal reports have indeed suggested that this could happen, the majority of controlled studies have had difficulty even documenting an improvement in symptoms with environmental control. It is clear that a comprehensive program of dust mite avoidance can lead to reduced asthma symptoms, but this has not been shown to put the asthma into remission. A randomized trial involving home environmental allergen and irritant remediation among children aged 6–11 years with moderate-to-severe asthma successfully reduced asthma symptoms. The intervention cost $1,469 per family and led to statistically significant reductions in symptom days, unscheduled clinic visits, and use of beta-agonist inhalers. The intervention cost was $27.57 per additional symptom-free day [52].

Vaccines Since respiratory viruses are common triggers of asthma episodes, development of effective vaccines could be highly effective for preventing asthma exacerbations and other complications. Influenza is the only agent for which a vaccine is currently

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available though its cost-effectiveness for patients with asthma is questionable. A vaccine also is under development for respiratory syncytial virus. Unfortunately, the most common trigger of asthma, Rhinovirus, has eluded efforts to develop an effective vaccine [53].

Dietary Interventions Dietary salt consumption has been hypothesized as an explanation for the wide geographical variation in asthma prevalence. In a systematic review of six randomized controlled trials, low-sodium diet was associated with a significantly lower urine sodium excretion than normal or high-salt diets. There were no significant differences in any asthma outcome between low-salt and normal or high-salt diets. Reliever bronchodilator use was slightly lower with low salt intake as were improvements in pulmonary function though neither of these were very large [54].

Conclusion The most promising asthma prevention strategies to date have been those that use a multi-interventional approach employing both dietary and environmental manipulations. More research is needed to assess the long-term follow-up of multiinterventional trials and to evaluate novel intervention strategies in the primary or secondary prevention of asthma in childhood [55]. Given the significant burden of both asthma and allergies, methods to prevent their development would be a huge step forward. Though the evidence is encouraging for several prevention strategies, none of them are convincing enough to be recommended for widespread clinical use. Even so, some are currently being used in routine practice because they can reduce symptoms and prevent morbidity whether they prevent the underlying disease or not. This seems to be sufficient justification for their use at this time. If at some future date they are shown to be effective for primary or secondary prevention, then their use is much more effective.

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Outcome Measures in Asthma Management Michael Schatz

Introduction Outcome measures can be defined as health status and physiologic manifestations that result from a clinical illness. These measures can be used in clinical trials to assess the efficacy of an intervention; in clinical practice to assess the severity and progress of individual patients; for population assessment and management; and as measures of the quality of care. There are many types of asthma outcome measures, including patient-centered outcomes, physiologic outcomes, health care utilization outcomes, and economic outcomes. No single outcome describes the entire spectrum of asthma impact on individuals, health plans, or society. Some outcomes are more relevant to patients (e.g., quality of life), while others are more relevant to providers (physiologic measures), insurers (health care utilization), payers (work productivity), or society in general (direct and indirect costs). The many types of asthma outcomes have variable relationships to each other. One factor analysis of three clinical trials reported that the asthma outcomes assessed represented four separate constructs: quality of life, pulmonary function, daytime symptoms, and nighttime symptoms [1]. Another factor analysis of survey data from 2,854 patients revealed four separate themes underlying the subjective patient experience: symptom frequency, symptom bother, physical activity, and exacerbations [2]. A third factor analysis from a large clinical trial of children with mild to moderate asthma (n = 1,043) identified separate factors for inflammatory markers, symptoms/medication use, asthma exacerbations, and measures of lung function [3]. Finally, a recent factor analysis has shown that asthma control as determined by a validated questionnaire is a separate factor from spirometric measures [4]. Other studies have confirmed poor correlations between pulmonary function and symptoms [5, 6] or functional impairment [7, 8]. These data together suggest at least five unique asthma outcome domains: symptoms, quality of life (including bother and functional impairment), pulmonary function, inflammatory markers, and exacerbations.

M. Schatz () Department of Allergy, Kaiser Permanente Medical Center, San Diego, CA, USA e-mail: [email protected]

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This chapter reviews the major types of asthma outcome measures. For each measure, definitions will be provided, and, if available, prevalence data, predictors of that outcome, and relationships of that outcome to other outcomes.

Patient-Reported Outcomes Symptoms Asthma symptoms have probably been the most commonly used outcomes in clinical trials and clinical practice. Sometimes specific symptoms may be monitored, such as wheezing or shortness of breath, the latter of which appears to be particularly related to asthma control [9] and quality of life [10]. Asthma symptoms are often divided into daytime symptoms and nighttime symptoms, which have been reported to track separately [1]. Nocturnal symptoms are considered separately in the NAEPP guideline classification of asthma severity and control [11]. Nocturnal asthma symptoms are independently related to asthma control in validated tools [9, 12]. Asthma symptoms may not be a good reflection of functional impairment [1, 6], which may be underestimated by assessing symptoms alone [13, 14]. These symptoms may be assessed by daily diary or interval patient report. Symptom diary scales have been developed for use in clinical trials [15], and symptom-free days is also a commonly used outcome measure in clinical trials. Evaluation of symptoms in pediatric studies presents an additional challenge not encountered in adults. Children may not effectively communicate their symptoms to a caregiver or a physician, resulting in misperceptions of asthma severity. Although both children with asthma and their caregivers have been reported to overestimate and underestimate asthma symptom severity, caregivers seem more likely than children to underestimate symptoms in children with impaired pulmonary function [16]. Parents also frequently report good asthma control despite the presence of daily symptoms in a child [17]. In children older than age 11, parentreported symptoms may be particularly misleading [18].

Rescue Bronchodilator Therapy Use Use of rescue bronchodilator therapy is often separately assessed from asthma symptoms, although it often tracks with asthma symptoms [1, 5, 6]. Specifically, use of short-acting beta agonists is usually monitored. Use of rescue therapy may be a less reliable surrogate for asthma symptoms in patients who routinely use shortacting beta agonists, such as before exercise, unless prophylactic use is specifically distinguished from rescue use. Rescue therapy use may also reflect asthma symptom frequency and severity less well in patients who perceive dyspnea poorly

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(underestimate) or in those with high panic-fear personalities (overestimate). Males have been reported to use more rescue therapy than females [19, 20], even though adult females have more exacerbations and poorer quality of life (see below).

Functional Measures An important measure of asthma impact is its effect on patient functional capacity. This includes missed work, missed school, reduced productivity at work or school, interference with other normal activities, and interference with exercise. In 2003 in the USA, children missed 12.8 million days of school and adults missed 10.1 million days of work due to asthma [21]. As noted above, asthma severity is classified as substantially greater when functional limitations as well as symptoms are considered [13, 14]. It has been reported that children or adults who report uncontrolled asthma are more likely to miss school or work than children or adults who report controlled asthma [22, 23]. One study has used an asthma-specific adaptation of the Work Productivity and Activity Impairment Questionnaire (WPAI) to show that [1] more asthma control problems correlated with higher levels of WPAI impairment [2], improved quality of life over the 12-month follow-up period correlated with decreased levels of WPAI impairment, and [3] more WPAI impairment at baseline predicted subsequent asthma emergency department visits and hospitalizations [24]. A recent report has shown that asthma severity, as determined by GINA classification, and a history of serious exacerbations are independent risk factors for missed school in children [25].

Validated Multidimensional Questionnaires Asthma-Specific Quality of Life The first validated multidimensional questionnaires applied to asthma measured asthma-specific quality of life [26]. Several instruments have been developed and validated (Table 1), but there are few comparisons between them. Most of them include least 15 questions and measure several domains. For example, the most commonly cited instruments are the Juniper tools [27], which include the domains of symptoms, emotions, activities, and environment. Recently, a brief six-question tool has been presented, which correlates well (r = 0.84) with the mini-AQLQ and appears to reflect the same factors [28]. Women [28–31], racial/ethnic minorities [32], smokers [28, 33, 34], patients with lower income [28, 32, 35, 36], patients with lower educational levels [28, 30, 32], unemployed patients [29], obese patients [28, 37], patients with anxiety or depressive disorders [38, 39], patients with more negative life events [36], and patients with

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Table 1 Representative adult and pediatric asthma-specific quality of life tools Author Number of Tool (reference) questions Domains Adult tools Asthma Quality of Life Questionnaire (AQLQ) – original version AQLQ – standardized activity version Mini-AQLQ

Juniper [26]

32

Symptoms; emotions; activity limitation; environment

Juniper [43]

32

Juniper [209]

15

Marks Asthma Quality of Life Questionnaire

Katz [41]

20

ITG Asthma Short Form

Bayliss [40]

15

Asthma Impact Survey Pediatric tools Pediatric Asthma Quality of Life Questionnaire Pediatric Asthma Caregivers Quality of Life Questionnaire

Schatz [28]

Symptoms; emotions; activity limitation; environment Symptoms; emotions; activity limitation; environment Physical impact; emotional impact; social impact; health concerns Symptom-free index; functioning with asthma; psychosocial impact of asthma; asthma energy; asthma confidence in health None

Juniper [210]

23

Juniper [211]

13

Symptoms; activity limitation; emotional function Emotional function; activity limitation

more severe asthma [28, 29, 32, 36, 40–42] report lower asthma-specific quality of life. Asthma-specific quality of life correlates strongly (r = 0.57–0.66) with generic physical quality of life scores and significantly but less strongly (r = 0.40–0.48) with generic mental quality of life scores [41, 43]. It has been shown to correlate well with or predict other asthma outcomes, such as asthma control [43–48], missed work [40], missed school [49], disability in valued life activities [50], asthma exacerbations [51–54], and direct health care costs [52]. Correlations between asthmaspecific quality of life and pulmonary function are much lower (e.g., r = 0.14–0.18) [10, 30, 41, 43]. Patients with less bronchial hyperreactivity based on methacholine PD20 have been reported to have significantly higher quality of life scores than patients with more bronchial hyperreactivity [55].

Asthma Control Asthma control, the degree to which the adverse impact of asthma on the patient is mitigated, is being increasingly recognized as an important asthma outcome [11, 56, 57]. Well-controlled asthma may be defined based on the combination of few or minimal daytime and nighttime symptoms and rescue therapy use, no activity limitation, optimal pulmonary function, and no or infrequent exacerbations [11, 57, 58].

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Recent surveys from several countries suggest that well-controlled asthma is not being achieved by the majority of patients [22, 59–72]. Several questionnaires have been validated to reflect the multidimensional construct of asthma control (Table 2). Moderate to strong correlations between the ACQ and the ACT [73, 74], between the ATAQ and the ACT [75], and between the ACSS and the ACQ [76] have been demonstrated. Specific cut-offs for uncontrolled asthma [9, 12, 77] and minimal important differences [12, 78] have been defined for some of the tools. Most of the tools include an assessment of nocturnal symptoms, rescue therapy, and activity limitation (Table 2). All of Table 2 Validated asthma control tools

Tool (reference)

Items included in tool Rescue Number Nocturnal therapy Activity Physiologic of items symptoms use limitation measures

Exacerbations

Adult tools Asthma Therapy 4 Yes Yes Yes No No Assessment Questionnaire (ATAQ) [12] Yes Yesa Yes FEV1a No Asthma Control 7a Questionnaire (ACQ) [46] Asthma Control 5 Yes Yes Yes No No Test (ACT) [9] Asthma Control 6 Yes Yes Yes FEV1, Sputum No eosinophilia Scoring System (ACSS) [76] Guideline-based 7 Yes Yes No PEFR Yes Composite Outcome Assessment Tool [80] Pediatric tools Asthma Quiz [79] 6 Yes Yes Yes No Yes Asthma Therapy 7 Yes Yes Yes No No Assessment Questionnaire for Children and Adolescents [212] Pediatric Asthma 10 Yes Yes Yes No Yes Control Tool [45] Childhood Asthma 7 Yes No Yes No No Control Test [213] a ACQ versions without FEV1 and rescue therapy retain the measurement properties of the original tool [78].

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the tools reflect the impairment domain of asthma control [11], but only three [45, 79, 80] also address the risk domain of exacerbations. Three of the tools include physiologic measures. However, the ACQ performs as well without the FEV1 assessment [78], and the inclusion of sputum eosinophilia in the ACSS tool [76] limits its applicability to settings in which this test is available. The ACT has been validated for use on the telephone using speech recognition technology [81]. Women [67], racial/ethnic minorities [69, 82], smokers [67, 68, 82–86], patients with lower socioeconomic status [69, 83–85], obese patients [37, 67, 84], patients with more identified asthma triggers [83], patients with more severe asthma [67, 83, 84, 87], patients with poor adherence [85], and patients with co-morbidities (chronic sinusitis [69], reflux [69, 84], hypertension [69], and depressive disorders [38]) report poorer asthma control as assessed by validated tools. In contrast, specialty care [84] has been associated with improved asthma control. Asthma control has been shown to moderately correlate with asthma-specific quality of life [22, 43, 44, 46–48, 75, 76, 80, 88]; to be related to missed school and missed work [22, 23, 59]; to independently predict subsequent asthma exacerbations [12, 51, 81, 89–91]; and to be directly related to asthma costs [59, 92–94].

Patient Satisfaction Patient satisfaction with asthma care or treatment has been a less frequently reported asthma outcome. However, it is now an important part of some pay for performance systems that assess the quality of asthma care [95]. Satisfaction with asthma care may include satisfaction with access to care, satisfaction with provider skill and communication, satisfaction with treatment, and overall satisfaction with care. One study has shown that satisfaction with asthma care is influenced by demographic characteristics [96], and another study demonstrated a relationship between asthma control and patient satisfaction [23]. A tool to measure patient satisfaction with inhaled asthma therapy has been developed [97]. It has four domains (effectiveness of treatment, ease of use, medication burden, and side effects and worries) and appears to have good measurement properties [97]. Another tool has been developed as a general measure of treatment satisfaction, the Treatment Satisfaction Questionnaire for Medication (TSQM) [98]. It includes four scales derived from factor analysis: global satisfaction, side effects, effectiveness, and convenience. It appears to be a psychometrically sound and valid measure of the major dimensions of patients’ satisfaction with medication.

Change in Medication Requirement Change in medication requirement may be assessed in clinical trials or clinical practice as an outcome measure of treatment efficacy or effectiveness. Reduction in use

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of rescue bronchodilator therapy would be one example, which has been discussed above. Reduction in use of oral or inhaled corticosteroid therapy has also been used as an outcome measure to test the value of non-corticosteroid interventions. However, this approach is limited by the necessity to define the minimally effective oral or inhaled corticosteroid dose before adding in the intervention in order to be able to ascertain that the dose reduction was due to the intervention [99]. Using reduction in long-term control medication as a valid outcome measure also requires a long-enough follow-up period to assure that the medication reduction is tolerated without a loss of asthma control [99].

Pulmonary Function Outcomes The most common pulmonary function asthma outcomes are the forced expiratory volume in one second (FEV1) derived from spirometry and the peak expiratory flow rate (PEFR). Other potential pulmonary function asthma outcome measures include maximum mid-expiratory flow rates (MMEF), which can also be determined from spirometry, and lung volume measurements, which are usually obtained from body plethysmography.

Spirometry The FEV1 is a well-standardized measure of airway obstruction derived from spirometry. It is obtained from a forced expiratory maneuver, which follows a maximal inspiration and continues to maximum expiration. The total volume expelled during this maneuver is the forced vital capacity (FVC), and the amount of air expelled in the first second is the FEV1. It is usually expressed as percentage predicted, and predicted values based on age, height, sex, and race have been well established [100]. Although the FEV1 is reduced with airway obstruction, it may also be reduced due to restrictive lung disease, in which case the FVC is reduced as well. The ratio of FEV1 to total FVC is considered the best measure of airway obstruction, especially in children [11]. An abnormal FEV1 to FVC ratio is optimally defined as a value below the statistically determined fifth percentile of normal [101]. In severe cases of asthma, though, the FVC may be reduced due to trapping of air in the lungs [11]. Significant reversibility of airway obstruction as defined by spirometry is the most specific test for asthma diagnosis (95% in one study), but it has low sensitivity (32% in that study) [102]. Significant reversibility is indicated by ATS standards as an increase in FEV1 of >200 ml and >12% from the baseline measure after inhalation of a short-acting bronchodilator [11]. In one study, the correlation between airflow reversibility and airway inflammation (sputum eosinophilia) was stronger (r = 0.40–0.47) than the correlation between baseline FEV1 and airway

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inflammation (r = 0.20–0.23) [103]. The post-bronchodilator FEV1 measure can be used to follow lung growth patterns over time [104]. Spirometry is generally valuable in adults and in children over age 4, although some children cannot conduct the maneuver adequately until after age 7 [11]. Healthy young children complete exhalation of their entire vital capacity in a few seconds, but it can take older patients much longer, especially patients who have airflow obstruction, because expiratory flow is so low at low lung volumes [11]. In these patients, sustaining a maximal expiratory effort for the time necessary for complete exhalation, which may be more than 12 or 15 s is long enough for some patients to find the maneuver uncomfortable or associated with light-headedness [11]. This accounts for the interest in measuring FEV6 as a substitute for the FVC in adults. In adults, the FEV6 has been shown to be equivalent to FVC for identifying obstructive and restrictive patterns and to be more reproducible and less physically demanding than the FVC [105]. As noted above, correlations are low between FEV1 and other patient-reported measures, such as symptoms [106–109], rescue therapy [108], and quality of life [1]. For example, one study reported that nearly one third of the children who had moderate to severe asthma were only appropriately classified when pulmonary function reports of FEV1 were considered in addition to symptom frequency [110]. FEV1 has been independently associated with subsequent asthma exacerbations [111–114]. For example, in one study, Fuhlbrigge et al. [112] reported that, compared with children with an FEV1% predicted ≥100%, children with FEV1% of 80–90%, 60–79%, and <60% were 1.3, 1.8, and 4.8, respectively, more likely to have a serious asthma exacerbation during the ensuing 4 months. In another recent study, patients with FEV1 60–80% and <60% predicted were 2.5 and 4.6 times, respectively more likely than patients with FEV1 >80% predicted to experience subsequent asthma emergency department visits or hospitalizations, even after accounting for other significant demographic, environmental, allergic, and asthma severity risk factors [114]. Pulmonary function measures have also been shown to predict mortality in asthmatic patients [115, 116], with the FVC being most robust in this regard [117]. The risk of respiratory death was eight times greater in the lowest compared to highest quartile of best FVC [117]. Another measure derived from spirometry is the flow rate at lower lung volumes, such as the maximum mid-expiratory flow rate from 25% to 75% of the FVC (MMEF25–75). This measure has been utilized because it has been thought to better reflect small airway function than the FEV1, although this interpretation has been recently questioned [118]. In addition, a decreased MMEF has been shown to have no predictive value for development of chronic airways disease [119].

Peak Expiratory Flow Rate The PEFR is the highest air flow rate achieved from a forced expiration. It is an easier maneuver to perform and requires less expensive tools to measure than FEV1.

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However, it is less well-standardized than the FEV1, and thus, for the purposes of diagnosing reversible airway obstruction, spirometry is generally recommended over PEFR [11]. There is wide variability in the published predicted reference values for PEFR. Moreover, reference values need to be specific to each brand of peak flow meter, and such normative brand-specific values currently are not available for most brands [11]. Thus, PEFR is best used as a monitoring measure, not as a diagnostic tool. PEFR can be monitored by patients at home, and PEFR values measured at home compared to a patient’s personal best PEFR form the basis for peak flow-based action plans. When measured at home for a clinical trial, PEFR is usually measured in the morning and evening, with outcomes including mean morning PEFR, mean evening PEFR, and percentage diurnal variability. The latter had previously been considered a reliable measure of airway hyperreactivity, but recently this concept has been questioned [120, 121], and PEFR diurnal variability is no longer included as a measure of severity in the NAEPP asthma guidelines [11]. PEFR measures at home or in the office correlate at least modestly with FEV1 [108]. However, like FEV1, correlations between office or home PEFR measurements and symptoms or rescue therapy use are low (r = 0.13–0.23) [108]. Although symptoms and PEFR measures may correlate poorly, symptom-based, and PEFR-based action plans appear to be equally effective in increasing patients’ awareness of their disease status and control, enhancing clinician–patient communication, and preventing exacerbations [11].

Fixed Airways Obstruction Although the hallmark of asthma is reversible airways obstruction, the decline in FEV1 over time is accelerated in patients with asthma compared with subjects without asthma [122], and some patients with asthma develop airway obstruction that is only partially reversible. The pathology of this fixed airway obstruction in patients with asthma is different from the pathology of the irreversible airway obstruction seen in patients with emphysema. Patients with asthma manifest more eosinophils in peripheral blood, bronchoalveolar lavage fluid, and airway mucosa; fewer neutrophils in sputum and bonchoalveolar lavage fluid; a higher CD4+/CD8+ ratio of T cells infiltrating the bronchial mucosa; and a thicker reticular layer of the epithelial basement membrane [123]. The features of airway remodeling in asthma include epithelial denudation, subepithelial fibrosis, mucus gland hypertrophy, increased airway smooth muscle mass, and bronchial wall angiogenesis [124, 125]. Of these changes, the subpeithelial fibrosis and angiogenesis appear to correlate best with asthma severity and FEV1 [124]. It is not clear what proportion of patients with asthma develops fixed airways obstruction, but one birth cohort study has suggested that impaired lung function due to airway remodeling in patients with asthma begins in childhood and continues into adult life [126]. Patient and disease characteristics that have been associated with fixed airway obstruction include older age [127–130], male gender [128],

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black ethnicity [128], lower BMI [129], later age of asthma onset [115, 130], longer disease duration [127–130], more smoking [115, 128], aspirin sensitivity [128], more severe chronic asthma [129], more severe asthma exacerbations [129, 131], more bronchial hyperreactivity [130], higher eNO [127], higher peripheral blood eosinophilia [127], and higher sputum eosinophilia [130]. In addition, persistent airflow limitation has been recently associated with antibodies to Chlamydia, but only in patients with adult-onset, non-atopic asthma [132]. It has also been shown that the presence of fixed airway obstruction contributes to the poor correlation between asthma symptoms and FEV1 [106]. Several observational studies have suggested that inhaled corticosteroid therapy may reduce lung function decline in patients with asthma [133–135], but the data are not consistent on this subject. For example, one study did not find any difference in the amount of inhaled steroid treatment reported by patients with severe fixed airways obstruction compared with patients with no evidence of fixed airway obstruction [127]. In addition, one study showed that the beneficial effect of inhaled corticosteroids on decline in lung function was only significant in patients with high (>100 kU/l) IgE levels [134], and another study showed that this beneficial effect was not seen in men with ≥5 years of smoking or in women [136]. In contrast, Lange et al. [135] reported that a less steep decline in FEV1 was associated with inhaled corticosteroid treatment, even with adjustment for sex and smoking. One large 3-year clinical trial showed that treatment with an inhaled corticosteroid was associated with a reduced decline from baseline of the post-bronchodilator FEV1 [137]. Further long-term clinical trials will be necessary to confirm that inhaled corticosteroid therapy or other pharmacologic modalities reduce clinically significant fixed airways obstruction in patients with asthma. Another way to identify stigmata of airway remodeling is by means of highresolution computed tomography (HRCT). Bumbacea et al. [127] found higher HRCT scores for bronchial wall thickening and dilation in asthmatic patients with severe fixed airways obstruction defined based on pulmonary function tests compared to patients without fixed airways obstruction. One study [133] has reported that early onset of disease was an independent risk factor for the presence of irreversible HRCT scan abnormalities in elderly patients with asthma. Finally, decreased bronchial wall thickness and reticular basement membrane thickness of the subsegmental airways as assessed by HRCT has been reported after treatment with inhaled formoterol and budesonide for 3 months [138]. Longer-term studies will be necessary to define the overall clinical significance of this observation.

Lung Volumes Total lung capacity, usually measured by body plethysmography, has been used to reflect hyperinflation in asthmatic patients. In addition, total lung capacity has been reported to be strongly correlated with the degree of eosinophilic distal lung inflammation [118]. The closing volume and closing capacity are considered measures

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of small airway disease [139]. These measures, obtained from a single breath nitrogen test, have been shown to be the only pulmonary function measures to differentiate patients with severe asthma with histories of recent exacerbations from patients with severe asthma without such a history [139]. The residual volume, also obtained from body plethysmography, has been considered to reflect air trapping, which may also reflect distal lung physiology [140]. In one study, improvement in asthma symptoms with therapy correlated significantly with changes in residual volume, but not with changes in FEV1 or FEV1 to FVC ratio [141]. Assessment of air trapping by means of high-resolution computerized tomography scans has been recently introduced [142].

Markers of Airway Inflammation Although the actual cause of asthma remains unknown, the pathophysiological mechanism has been established to be airway inflammation. Thus, measurements of airway inflammation could be the penultimate asthma outcome. However, direct measurement of airway inflammation requires histological samples of airway tissue, which are unavailable under most circumstances. There has thus been an interest in the identification of indirect markers of airway inflammation. The best studied of these are airway hyperresponsiveness, sputum eosinophilia (and eosinophilic cationic protein [ECP]), and the fraction of exhaled nitric oxide (eNO) [143], which will be discussed below. Other less sensitive or less studied markers include peripheral blood eosinophilia, exhaled breath condensates (including eicosanoids, isoproatanes, and hydrogen peroxide), and urinary leukotrienes [143].

Airway Hyperresponsiveness Airway hyperresponsiveness to agents such as histamine, methacholine, and adenosine monophosphate (AMP) is characteristic of asthma and has been used for years to help establish the diagnosis of asthma. More recently, it has been demonstrated that the degree of hyperreactivity to these agents is directly linked to underlying bronchial inflammation and the potential for airway remodeling [144].

Methacholine Challenge Responsiveness to inhaled methacholine, usually expressed as the cumulative dose of methacholine that causes a 20% reduction in FEV1 (PD20) or the concentration of methacholine that causes a 20% reduction in FEV1 (PC20), is probably the most commonly used test of bronchial hyperreactivity [145]. Methachloline PD20 has been reported to correlate with airway inflammation as reflected by sputum eosinophilia,

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endotracheal biopsy eosinophilia, reticular basement membrane thickening, BAL mast cells, and BAL epithelial cells [144, 146, 147]. Methacholine PD20 has also been recently shown to be associated with a history of serious asthma exacerbations [25]. One study reported a significant correlation (r = −0.48, p = 0.003) between improvement in methacholine PD20 and change in bronchial biopsy eosinophilia [148]. In the latter study, it was also shown that using methacholine sensitivity as an adjunct to guide the prescribed dose of inhaled corticosteroids in patients with mild to moderate asthma led to a significant 80% reduction in asthma exacerbations and improved FEV1 when compared to the conventional clinical approach [148]. However, methacholine challenges are generally impractical outcomes to use for usual clinical practice because of the time and resources required to carry out the challenge test.

AMP and Mannitol Challenges Challenge testing with indirect stimuli, such as AMP and mannitol, may confer theoretical and practical advantages over direct stimuli, such as methacholine [143]. These tests intrinsically depend on airway inflammation to mediate their constrictor effects and thus represent a more physiologic model of asthmatic airway narrowing than their directly acting counterparts of methacholine and histamine challenges [143]. Indeed, it has been demonstrated that AMP PC20 reflected sputum eosinophilia more closely than methacholine PC20 [149] and that airway hyperreactivity in response to mannitol was more closely associated with sputum eosinophilia and eNO than methacholine sensitivity [150]. Mannitol challenges correlate well with responses to histamine as well as AMP and other indirect challenge tests [143]. Mannotol challenge-determined bronchial hyperreactivity has also been shown to be a good predictor for the failure of inhaled corticosteroid dose reduction among asthmatic subjects [151]. In addition, mannitol has practical advantages because it requires little specialized equipment apart from a spirometer and a hand-held dry powder inhaler [143]. However, no longitudinal studies have been conducted to evaluate the use of this tool to monitor or guide asthma therapy.

Sputum Eosinophilia Eosinophils are strongly implicated in the pathogenesis of asthma. Sputum eosinophilia has been shown to correlate with symptoms, FEV1, and bronchial hyperreactivity in asthmatic patients [147, 149, 152]. The eosinophil count in induced sputum is the most robust predictor of response to corticosteroid therapy in patients with asthma [143] and asthma deterioration when inhaled corticosteroids are discontinued [153–155]. Green et al. [156] showed that treatment based on achieving a sputum eosinophilia ≤3% was associated with a nearly fivefold reduction in exacerbation rate compared with conventional management. The sputum management group also had greater improvement in other surrogate inflammatory markers (eNO and methacholine PC20) with no difference in daily dose of inhaled or oral corticosteroids between

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groups. Two additional studies have confirmed these observations of reduced exacerbations with no increase in inhaled corticosteroid dose using a management strategy based on sputum eosinophilia [157, 158] Despite these impressive results, induced sputum analysis is not a practical outcome for clinical management. Degranulation of eosinophils leads to the release of numerous pro-inflammatory mediators, including eosinophilic cationic protein (ECP). ECP has been measured in serum, sputum, and broncho-alveolar specimens, where levels have been shown to correlate with each other as well as with lung function, overall disease severity, and bronchial hyperreactivity [143, 152]. ECP levels may also predict exacerbations and response to anti-inflammatory treatment [143]. ECP measurements may be less technically demanding than induced sputum eosinophil counts, but ECP measurements have not yet been shown to be useful as a marker to guide therapy [143].

Exhaled Nitric Oxide eNO measured in the expired breath of asthmatic patients is elevated due to effects of inflammation on inducible nitric oxide synthase [159]. eNO has been shown to correlate with other markers of asthmatic inflammation including sputum and peripheral eosinophil count, bronchial hyperresponsiveness, ECP, and bronchial wall thickening on HRCT scans [143, 160–162]. eNO has also been shown to correlate with asthma severity [163] and asthma control in some [163–165] but not all [74] studies. eNO reliably varies with corticosteroid therapy such that it can be used as a marker of compliance with corticosteroids [163]. Elevated levels of eNO in treated patients have been shown to herald the subsequent loss of asthma control [143, 160, 166, 167] or asthma exacerbations [133, 168]. Measurement of eNO is quick and painless and has a high degree of reproducibility and tolerability, making it an ideal clinical outcome measure for routine practice [143]. However, clinical trials to date have not demonstrated an improvement in exacerbation rate, symptoms, or pulmonary function in patients whose management was guided by eNO compared to patients managed by standard clinical criteria [169–172]. Further studies will be necessary to define the clinical role of eNO measurements as an asthma outcome, especially as a guide to therapy.

Asthma Exacerbations Asthma exacerbations can be defined as acute or subacute episodes of progressively worsening shortness of breath, cough, wheezing, and chest tightness, or some combination of these symptoms [11]. A more practical working definition is a worsening of asthma of sufficient severity to require intervention from a medical professional or self-administration of oral corticosteroids [173]. The recent NAEPP guidelines have classified asthma exacerbations as mild, moderate, or severe, or life threatening [11] (Table 3). These guidelines have

Too dyspneic to speak; perspiring

Subset: life threatening PEF <25% predicted or personal best

PEF <40% predicted or personal best

Relief from frequent inhaled SABA. Symptoms for 1–2 days after oral corticosteroids begun Partial relief from frequent inhaled SABA. Symptoms for ≥3 days after oral corticosteroids begun Minimal or no relief from frequent inhaled SABA

Prompt relief with inhaled SABA

PEF ≥ 70% predicted or personal best PEF 40–69% predicted or personal best

Response to treatment

Initial PEF (or FEV1)

→ ED/hospitalization, possible ICU admission. Course of intravenous corticosteroids

→ ED visit and likely hospitalization. Course of oral corticosteroids

Contact clinician → likely office or ED visit. Course of oral corticosteroids

Contact clinician → consider short course of oral corticosteroids

Recommended medical intervention

ED, emergency department; FEV1, forced expiratory volume in one second; ICU, intensive care unit; PEF, peak expiratory flow; SABA, short-acting beta2agonist

Dyspnea at rest; interferes with conversation

Dyspnea only with activity (assess tachypnea in young children) Dyspnea interferes with or limits usual activity

Severe

Moderate

Mild

Symptoms and signs

Table 3 NAEPP Expert Panel 3 report classifications of asthma exacerbations [11]

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also included the number of exacerbations requiring oral corticosteroids in the prior year as part of the risk domain of asthma severity and control [11].

Asthma Mortality The ultimate severe asthma exacerbation is a fatal one. Fortunately, after a rise in mortality between 1978 and 1996, with a peak of 5,667 asthma deaths in 1996, asthma mortality has fallen [174, 175] such that there were 4,055 asthma deaths in 2003 [21]. In-hospital asthma mortality was 0.5% of all admissions for asthma exacerbations among patients greater than 5 years of age in 2000 and accounted for about one third of all asthma deaths reported in the USA [176]. Identified risk factors for asthma deaths or near-fatal episodes are shown in Table 4. Treatment with inhaled corticosteroids [116, 177], written action plans [178, 179], and recent use of oral corticosteroids for an attack [178, 180] have been reported to be associated with reduced asthma mortality or severe lifethreatening asthma episodes.

Table 4 Risk factors for asthma mortality or near-fatal asthma episodes Patient factors Older age [115, 178, 179] Lower education level [214, 215] Lower income [116, 215] African American race [215, 216] Family history of asthma death [217] Asthma characteristics Severe persistent asthma [178, 214] Increased asthma medication treatment (beta agonist, theophylline, oral corticosteroids) [115, 180, 218] Poor compliance with treatment [219] More prior hospital admissions [218], especially in prior year [116] or prior month [178] History of prior very severe exacerbations, e.g., requiring intensive care unit admission and/or intubation [218] Sensitivity to Alternaria [220] Decreased pulmonary function (FVC, FEV1) [115–117] Reduced perception of dyspnea [214, 221–223] Co-morbidities Smoking [178, 219] Drinking excessive alcohol [178, 219] Illicit drug use [224] Family problems [178, 225] Psychiatric co-morbidity [214, 219, 225]

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Other Asthma Exacerbations Asthma exacerbations requiring hospitalizations, emergency department visits, or other unscheduled medical visits exact a high humanistic and economic cost. Although the asthma hospitalization rate has decreased since 1995 [174, 181], there were still 497,000 hospitalizations and 1.8 million ED visits for asthma in the USA in 2004 [21]. Risk factors that have been identified for asthma exacerbations requiring emergency hospital care include demographic, socioeconomic, clinical, laboratory, utilization, medication, environmental, and psychosocial characteristics (Table 5). Interventions associated with a decreased risk of emergency hospital care include action plans, specialist care, self-management education, controller therapy (especially inhaled corticosteroids), and treatment of allergic rhinitis (Table 5). Table 5 Patient risk factors identified for asthma emergency department visits or hospitalizations Age/gender Younger age (children) [226–235] Male gender (age 15) [31, 236, 238, 240–248] Age 18–44 (versus older) [245, 248] Race/ethnicity African American [52, 228, 229, 232, 238, 243, 246, 249–254] Hispanic [226, 232, 243, 249, 251] Socioeconomic Lower income [226, 230, 232, 234, 237, 251, 252, 255, 256] Subsidized insurance/Medicaid/public aid [185, 228, 257, 258] Unemployment [245, 252, 259] Lower educational level [52, 226, 232, 235, 237, 245, 246, 252, 255] Less health literacy [260] Clinical Increased risk Maternal asthma [261, 262] Younger age at onset of symptoms [239, 261] Increased symptom severity or frequency [25, 91, 231, 235, 243, 245–247, 255, 263–267] Nocturnal symptoms [231, 263, 268] Asthma affected activities in last month [247, 269, 270] Missed school or work [24, 264, 270] Low perception of dyspnea [271] Decreased generic quality of life [51, 54] Decreased asthma-specific quality of life [51, 52, 54] Decreased asthma control [12, 81, 89, 90, 272–276] Concomitant allergic rhinitis [277–279] Gastroesophageal reflux disease [253] Asthma triggered by perfumes or odors [262] History of pneumonia [270] (continued)

Asthma Outcomes Table 5 (continued) History of intubation for asthma [248] Elevated BMI [248] Smoking [33, 34, 243, 248, 251, 269, 275] Decreased risk Having personal MD [183] Action plan [240, 255, 280, 281] Correct use of metered dose inhaler [269, 270] Allergy specialist care [282–288] Self-management education [289] Laboratory Decreased FEV1 [114, 239, 268, 290] Increased bronchial hyperreactivity [239] Allergen sensitization [114, 228, 239, 261, 291, 292] Higher IgE level [239] High eosinophil level [239] IL4Rα mutations [293] Utilization Prior emergency department visits [91, 183–185, 231, 234, 247, 255–257, 268–270, 294] Prior hospitalizations [91, 183, 185, 234, 247, 256–258, 261, 263, 269, 270, 281, 290, 294], especially with ICU admission [295] More asthma MD visit past 6–12 months [231, 257, 264] Decreased continuity of care [296] Defaulted follow-up visits [270] Medication Increased risk High beta agonist use [183, 185, 187, 234, 253, 255, 268, 277, 294, 297–299] Poor adherence to inhaled corticosteroids [300, 301] Low inhaled corticosteroid to beta agonist ratio [184, 192–195, 298, 302, 303] Use of oral corticosteroids [182–185, 234, 245, 248, 257, 263, 277, 281, 290] Use of theophylline [253, 294] Dispensing of proton pump inhibitor medication [304] Decreased risk Use of cromolyn [183, 233, 297] Use of inhaled corticosteroids [262, 265, 269, 288, 292, 297, 299, 305–311] Use of leukotriene receptor antagonists [312, 313] Use of omalizumab [314–316] Use of intranasal corticosteroids [317, 318] or other treatment for allergic rhinitis [294] Environmental Environmental tobacco smoke exposure [235, 261, 319] No mattress cover [264, 313] or did not wash bedsheets more than twice per month [255] Proximity to industrial complex [262] Work-related asthma [320] Psychosocial Low social support [264] Maternal depressive symptoms [265, 321] or anxiety disorder [321] Anxiety/depression [322–324] History of leaving the hospital against medical advice [325]

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Administrative Data Computerized utilization or claims data that include hospitalizations, outpatient visits, and pharmacy dispensings are available to health plans. Although not primarily intended for clinical use, such information can be used to derive asthma outcomes for population management to identify higher-risk patients who need intervention. Such data have also been used as a surrogate for quality of care. Several authors have used administrative data to develop risk stratification schemes to identify patients at higher risk of subsequent emergency hospital care (emergency department visits or hospitalizations for asthma) [182–185]. One relatively simple scheme that has been validated in several populations identifies high-, medium-, and low-risk patients based on their need for emergency hospital care, oral corticosteroid dispensings, and short-acting beta agonist canister dispensings in the prior year [185, 186] (Table 6). Administrative data have also been reported to reflect long-term asthma control. A four-level scale based on the number of short-acting beta agonist canisters dispensed in a 12-month period was shown to correlate with measures of short-term asthma control and quality of life, as well as predict subsequent asthma exacerbations [187]. Independent predictors of poorer long-term control defined in this way included male sex, prior exacerbations, smoking, African American race, and lower educational level [20]. Independent predictors of better long-term control included regular inhaled corticosteroid use, regular long-acting beta agonist use, and specialty care [20]. Finally, administrative data have been used for a number of years to reflect quality of care by health plans. The Health Plan Employer Data and Information Set (HEDIS) was developed by the National Committee for Quality Assurance (NCQA) and applied to many diseases, including asthma [188]. The HEDIS asthma measure identifies patients with “persistent asthma” based on inpatient, outpatient, and pharmacy utilization data, and assesses the proportion of such patients dispensed at least one controller medication. This is actually a process measure, rather than an outcome measure. Problems with this measure include the fact that this administrative definition of persistent asthma may not adequately Table 6 Risk stratification scheme for subsequent asthma emergency department visits or hospitalizations based on administrative utilization data from the prior year [185] HIGH RISK Asthma hospitalization or emergency department visit OR Oral corticosteroid dispensing AND dispensing of ≥15 short-acting beta agonist canisters MEDIUM RISK Oral corticosteroid dispensing OR Dispensing of ≥15 short-acting beta agonist canisters LOW RISK None of the above

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reflect persistent asthma as defined clinically [189, 190] and that patients who meet the criteria for appropriate treatment may actually be at increased risk of subsequent emergency hospital care [191, 192]. The ratio of controller medication to total (controller plus rescue medications) has been proposed as an alternative administrative quality of care process measure that better reflects asthma outcomes. This measure has been shown to be significantly related to survey outcome measures, such as asthma quality of life and control [193]. In addition, patients with a higher ratio experience significantly fewer subsequent asthma exacerbations defined by administrative data [192–195].

Economic Outcomes Economic outcomes include total costs, which combine direct medical costs (inpatient, outpatient, pharmaceutical) and indirect costs (costs of lost productivity). Cost estimates from developed countries indicate that asthma accounts for up to 2% of the economic costs of all diseases [196]. Direct medical costs represent 58–65% of total costs in reports from the USA [197]. The largest component of direct asthma costs is currently for pharmaceuticals [93, 197, 198], and the largest component of indirect costs is for lost work productivity [197]. Asthma is the fifth most common cause of workplace limitation [196]. A study of direct and indirect costs of asthma to an employer confirmed that the use of health care services, as well as the rate of disability, was substantially higher among asthmatic patients than among control subjects [199]. Similarly, direct costs for asthmatic children enrolled in a health maintenance organization were substantially higher compared to the general population of children using services [200]. Asthma costs have been shown to be directly related to asthma severity [197, 201, 202] and inversely related to asthma control [59, 92–94]. Several types of economic analyses can be performed in order to determine the economic value of a given intervention. Cost effectiveness studies relate cost in dollars to a clinical outcome measure of effectiveness. A standard effectiveness outcome in cost-effectiveness studies that can be compared across disease areas is the quality-adjusted life year (QALY). This utility weight is anchored at 1, representing perfect health, and 0, representing death. Methods for gathering utilities include direct measures, such as the visual analogue scale (VAS), time trade-off (TTO), or standard gamble (SG), or questionnaires with empirically based weighting schemes to reflect patient preferences [203, 204]. The latter questionnaires include generic instruments, such as the Health Utilities Index (HUI), EuroQoL, or Short-form 6D, and the asthma-specific instrument, the Multi-attribute Asthma Symptom Utility Instrument (ASUI) [203]. The VAS and TTO have been shown to be significantly associated with pulmonary function and the frequency of several asthma symptoms [204]. The SG was not related to pulmonary function and was less strongly related to asthma symptom frequency than the other direct preference measures [204]. The generic HUI has

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been shown to be significantly related to the frequency of all asthma symptoms evaluated [175], but not to pulmonary function [203, 204]. Two other generic utility instruments (EuroQoL and Short-form 6D) have been shown to correlate well with asthma control [205]. The ASUI has been shown to correlate significantly with asthma-specific quality of life [203, 206], the asthma control questionnaire [206], the generic HUI [203], and pulmonary function [203, 204]. In addition to the USA, country-specific preference weighting algorithms for the ASIU have been derived for France, Italy, and the UK [206]. Asthma-specific utility weights can also be translated from asthmaspecific quality of life scores using a mapping algorithm [207]. Cost–benefit analyses express both the cost and the clinical benefit of an intervention in monetary terms [198]. The ratio of monetary benefits to overall costs determines whether the value produced by the intervention is worth the costs. The drawback to this method is that it is often difficult to express health outcomes in monetary terms [198]. One approach to expressing changes in health in monetary terms uses the contingent valuation method. This method, also known as the willingness to pay method, consists of surveys that elicit the amount of money an individual is willing to pay for a specific health change [208]. The premise underlying this approach is that an individual’s willingness to trade dollars for a defined health status change is that person’s net assessment of the monetary value of that health care item [208]. In one study, the willingness to pay for a cure for asthma was directly related to both objective and subjective measures of disease severity [208].

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Index

A Acceptance, 487 ACE inhibitor, 335 Adenosine monophosphate challenge, 518 Adherence/compliance, 81 Administrative data, 524 Age, 321 Airway hyperreactivity, 81 Airway hyperresponsiveness, 63, 517 Airway inflammation, 81 Allergen, 3 bronchial challenge, 49 definition of, 319 nasal challenge, 49 PC20, 49 Allergen-induced hyperresponsiveness, 49 Allergen-induced inflammation, 49 Allergic asthma, 21 Allergic inflammation, 127 Allergic rhinitis, 21 Allergy, 3, 201, 491 diagnosis, 223 prevention, 491 Alpha tryptase, 331 AMP, 63 Angioedema, 323, 335 Aretaeus of Cappadocia, 247 Aspirin, 349 challenge, 357 exacerbated respiratory disease (AERD), 349 ASPITest, 364 Assessment, 480 Asthma, 49, 63, 81, 223, 295, 475, 491 clinical description, 249 clinical types, 253 control, 483, 510, 511, 524 control predictors, 512

diagnosis, 141 emergency department risk factors, 523 emergency department visits, 522 exacerbations, 519, 522 hospitalization risk factors, 523 hospitalizations, 522 monitoring, 141 mortality, 521 mortality risk factors, 521 outcome definitions, 507 outcomes, 476, 507 phenotypes, 255 quality of care, 524 risk stratification, 524 subtypes, 251 treatment, 141 varieties, 250 Asthma-specific quality of life, 509, 510 Asthma-specific quality of life tools, 510 Atopic adolescent and adult phase, 298 cheilitis, 304 childhood phase, 296 Dennie-Morgan fold, 305 dermatographism, 306 diagnostic criteria, 307 - 309 differential diagnosis, 309, 311 discoid variant, 301, 302 eczema, 295–317 eyelash, 306 eyelid, 303, 304 facial pallor, 306 fingertip, 301 follicular variant, 299, 300 genetics, 313 hand, 301, 303 herthoghe’s sign, 306

543

544 Atopic (cont.) hyperlinearity, 305 IgE, 310–312 impetiginization, 296 infantile phase, 296 infraorbital fold, 305 keratosis pilaris, 306 lichenification, 296 nails, 298 nipple, 303 nummular variant, 301, 302 papular lichenoid variant, 299, 300 patch test, 312 periorbital darkening, 306 pityriasis alba, 305 prognosis, 299 prurigo type, 301 SCORAD index, 309 skin prick test, 310 winter feet, 302 xerosis, 305 Atopic dermatitis, see Atopic eczema Atopy, 295, 321 Atopy patch test, 378 Autonomic epilepsy, 327

B Basophil, 201, 453 activation, 161 analysis of results, 194 CD63 and flow cytometry, 185, 186 cut-off values, 187–189 methods, 184 test, 363 Beta tryptase, 331 Betalactams, 161 Bezold-Jarisch reflex, 324 Biphasic, 324 Body plethysmography, 101 Bradycardia, 325 Bronchial asthma, 127 Bronchial biopsy, 127 Bronchial provocation test, 360 Bronchoalveolar lavage, 275 Bronchoscopy, 127

C C1 INH concentrate, 335 C1 inhibitor, 335 CAMP, 81 Capillary leak syndrome, 326 Carcinoid, 326

Index Carcinoid syndrome, 327 Cardiovascular, 324 CD63, 183, 190, 201, 330 CD203c, 183, 190, 201 allergen issues, 193 methodological issues, 191, 192 Cellular tests, 21 Central and peripheral symptoms, 423 Change in medication requirement, 512, 513 Chemical exposure and sensitivity, 423 Chemoattractants, 322 Children and pregnancy, 477 Chronobiology, 321 CLIC, 81 Communication, 479 Comparing potency, 81 Component resolved diagnosis, 382 Components, 485 Corticosteroid, 486 inhaled (ICS), 81 Cost-benefit analysis, 491, 526 Cost-effectiveness, 525, 526 Cox-2 ibhibitors, 350 Cross-reactivity, 403 Cross-reactivity of NSAIDs, 352 CT scans, 356 Culturally and linguistically diverse, 478 Cutaneous, 323 Cytokine, 453

D Decision making, 487 Determination variability, 81 Determining factors, 81 Diagnosis, 81, 201, 257 Diet diaries, 375 Differential diagnosis, 325 Direct cost, 525 Disseminated intravascular coagulation, 323 Distal (small airways), 81 Drug, 320 Drug hypersensitivity, 161, 403 Drug rash with eosinophilia and systemic symptoms (DRESS), 403 Drug reactions, 387 Dyspnea, 323

E Early response, 49 Economic outcomes, 525 Eczema, 295 Education settings, 477

Index Elimination diet, 378 Eosinophil, 453 Eosinophil-cationic protein, 519 EP receptors, 353 Eucapnic voluntary hperpnoea (EVH), 63 Evidence, 476 Exacerbations (loss of control), 81, 486 Exercise, 63 Exhaled breath condensate, 101 Exhaled nitric oxide, 141, 519 Extrinsic allergic alveolitis, 275

F Factor analysis, 507 FENO see Fractional exhaled nitric oxide Fixed airway obstruction, 515, 516 Flow cytometry, 161, 201 Floyer, John, 247 Flush, 323 Flushing, 325 Flush syndromes, 326 Food allergy, 161 Food allergy diagnosis, 373 Foods, 320 Food-specific IgE antibodies, 376, 377 Forced expiratory volume in 1 second (FEV1), 514 Forced vital capacity, 514 Fractional exhaled nitric oxide (FENO), 81, 101 Functional measures, 509

G Galen, 247 Gastroesophageal reflux disease (GERD), 81 Gastrointestinal vasopeptides, 329 Gender, 321 Genodermatoses, 387

H Health outcome, 477 Hereditary angioedema, 335 15-HETE generation assay, 364 15-HETE lipoxins, 353 Heterogeneity, 81 High resolution computed tomography, 516 Hippocrates, 247 Histamine, 329 mediators of, 322 metabolites, 329 poisoning, 327 History, 3

545 HMW agents, 233 Homer, 247 Hymenoptera, 161 Hypersensitivity pneumonitis, 275 Hypertonic saline, 63

I IFN γ, 81 IFWIN, 81 IgE, 320, 403 IgG, 403 IL1β, 81 Immunotherapy, 223 In vitro tests, 363 Incidence, 320, 321 Indirect cost, 525 Induced sputum eosinophil analysis, 81 Information, 479 Inhalant, 161 Inhaled corticosteroids and fixed airway obstruction, 516 Inhaler device, 484 Inhaler device polypharmacy, 482 Inhaler device therapy, 480 Intravenous challenge, 361 Isoforms, 81

K Knowledge and skill acquisition, 479

L Lactose intolerance, 373 Laennec, 248 Language, 487 Late response, 49 Latex, 161 Leukotrienes, 355 Leukotrienes release assay, 363 Life-threatening asthma exacerbations, 520 LMW agents, 233 Lung volumes, 516, 517

M Maculo-papular exanthema (MPE), 403 Manifestations, 323 Mannitol, 63 Mannitol challenge, 518 Markers of airway inflammation, 517 Markers of inflammation, 81 Mast cells, 453

546 Mastocytosis, 331 Maximum mid-expiratory flow rate, 514 Medical review, 483 Medullary carcinoma of thyroid, 327 Methacholine, 63 Methacholine challenge, 517 Microarray, 382 Mild asthma exacerbations, 520 Mites, 223 Moderate asthma exacerbations, 520 Monosodium glutamate, 327 Myorelaxants, 161

N Nasal challenge test, 233 Nasal nonselective provocation, 63 Nasal polyps, 350 Nasal provocation test, 361 Neurologic manifestations, 328 Neutral proteases, 322 Nitric oxide (NO), 81, 322, 453 Nitric oxide synthase, 81 Non-immunologic, 320 Non-steroidal anti-inflammatory drug (NSAID), 161, 349

O Objective assessment, 484 Occupational asthma, 257 Occupational rhinitis, 233 Oral food challenge, 373, 379 Oral provocation, 358

P p 38 MAPK, 201 Pancreatic tumors, 327 Patient – clinician partnerships, 478 Patient education, 475 Patient reported outcomes, 508 Patient satisfaction, 512 Patient understanding, 487 Peak expiratory flow rate, 515 Peak flow, 101, 482 PEAK study, 81 Perioperative, 324 Pharmacological strategies, 486 Phenotyping, 453 Photodermatoses, 387 Photosensitivity, 387 Phototesting, 387

Index Physical factors, 326 p-i concept (pharmacological interaction of drugs with immune receptors), 403 Plasma histamine, 330 Plasma-free metanephrine, 329 Platelet activating factor, 322 Polymorphisms, 81 Practical applications, 81 Prausnitz, 3 Predicting response, 81 Prevalence, 257 Prevention and control, 491 Prevention strategies, 491 Preventive measurements, 257 Prick skin test, 373 Prostaglandin, 322, 351 Prostaglandin hypothesis, 353 Proteoglycans, 322 Provocation tests, 21 Pruritus, 296, 323 Pseudoallergy, 403 Psychologic factors, 423 Pulmonary fibrosis, 275 Pulmonary function outcomes, 513

R Rackemann, 251 Randomized control trials, 476 Recombinant allergens, 223 Remission, 81 Remodeling, 81 Rescue bronchodilator use, 508 Rhinitis, 49, 63 Rhinoconjunctivitis, 295 Rhinosinusitis, 350, 356

S Salter, Henry Hyde, 248 Scombroid, 375 Scombroidosis, 328 Seizure, 323 Self management education, 475 Self monitoring, 482 Serum serotonin, 329 Severe asthma exacerbations, 520 Skin test, 21, 403 Socioeconomic status, 321 Specific IgE, 21 Spirometry, 101, 513, 514 Sputum eosinophilia, 518 Subcutaneous, 323

Index Sun allergy, 387 Symptoms, 482, 508 Systematic review, 476 Systemic mastocytosis, 326

T Tailored, 487 Target groups, 477 T cells, 403 Team, 487 Team approach, 483 Technique adequacy, 484 Template, 481 Th2-lymphocyte, 453 Titration, 81 T lymphocytes, 275 Tools, 480 Total cost, 525 Treatment escalation, 485 Tryptase, 329, 330 Tumor necrosis factor alpha (TNFα), 81, 322

547 U Ultraviolet radiation, 387 Unmet need, 81 Unusual manifestations, 325 Urinary LTE4, 365 Urinary vanilmandelic acid, 329 Urticaria, 323 Utility, 526

V Validated asthma control tools, 511 Validated multidimensional questionnaires, 509 Visible light, 387 Vocal cord dysfunction syndrome, 326 von Pirquet, 3

W Wheeze, 323 Written Asthma Action Plan, 481, 485