Journal Of Allergy And Clinical Immunology

Contents

T HE J OURNAL OF

Allergy Clinical Immunology AND

VOLUME 116 NUMBER 2 d

OFFICIAL JOURNAL OF THE AMERICAN ACADEMY OF ALLERGY, ASTHMA AND IMMUNOLOGY

The editors’ choice

239

Donald Y. M. Leung, MD, PhD, Harold S. Nelson, MD, and Stanley J. Szefler, MD

Reviews and feature articles

Y

w CME

Current reviews of allergy and clinical immunology Innate immune responses to infection

241

Michael F. Tosi, MD, New York, NY

Continued on page 7A

Y

This month’s theme: Infection and immunity

About the cover This month’s theme feature examines the fascinating interaction of infection and immunity. Our cover displays two exquisite images of the neutrophilic leukocyte’s response to infection in a mouse model. These leukocytes pass through the endothelium of blood vessels in response to a chemoattractant such as created by tissue infection. In the cover image, the left panel is a low power view of a small inflamed venule (pink) with numerous leukocytes (green) adhering to the endothelial lining. The right panel is a higher power view of a transmigrating leukocyte whose cell body lies beneath the pink endothelium, its amoeboid shape being easily appreciated. The leukocyte’s trailing tail or uropod (green) has not yet passed through the endothelium. Other articles in this issue that focus on the topic of infection and immunity are noted in the Table of Contents by the ‘‘theme’’ icon. Our sincere appreciation to Alan Burns, PhD, and C. Wayne Smith, MD, of the Departments of Pediatrics and Medicine, Baylor College of Medicine, who have captured these EM scanning images and made them available to us for presentation on our cover. Ó 2005 American Academy of Allergy, Asthma and Immunology The Journal of Allergy and Clinical Immunology (ISSN 00917-6749) is published monthly (12 issues per year) by Elsevier Inc., 360 Park Avenue South, New York, NY 10010-1710. Business and Editorial Offices: 1600 John F. Kennedy Boulevard, Suite 1800, Philadelphia, PA 19103-2899. Accounting and Circulation Offices: 6277 Sea Harbor Drive, Orlando, FL 32887-4800. Periodicals postage paid at Orlando, FL 32862 and additional mailing offices. POSTMASTER: Send address changes to The Journal of Allergy and Clinical Immunology, Elsevier Periodicals Customer Service, 6277 Sea Harbor Drive, Orlando, FL 32887-4800.

J ALLERGY CLIN IMMUNOL

August 2005 5A

Contents

T HE J OURNAL OF

Allergy Clinical Immunology AND

VOLUME 116 NUMBER 2 d

OFFICIAL JOURNAL OF THE AMERICAN ACADEMY OF ALLERGY, ASTHMA AND IMMUNOLOGY

The editors’ choice

239

Donald Y. M. Leung, MD, PhD, Harold S. Nelson, MD, and Stanley J. Szefler, MD

Reviews and feature articles

Y

w CME

Current reviews of allergy and clinical immunology Innate immune responses to infection

241

Michael F. Tosi, MD, New York, NY

Continued on page 7A

Y

This month’s theme: Infection and immunity

About the cover This month’s theme feature examines the fascinating interaction of infection and immunity. Our cover displays two exquisite images of the neutrophilic leukocyte’s response to infection in a mouse model. These leukocytes pass through the endothelium of blood vessels in response to a chemoattractant such as created by tissue infection. In the cover image, the left panel is a low power view of a small inflamed venule (pink) with numerous leukocytes (green) adhering to the endothelial lining. The right panel is a higher power view of a transmigrating leukocyte whose cell body lies beneath the pink endothelium, its amoeboid shape being easily appreciated. The leukocyte’s trailing tail or uropod (green) has not yet passed through the endothelium. Other articles in this issue that focus on the topic of infection and immunity are noted in the Table of Contents by the ‘‘theme’’ icon. Our sincere appreciation to Alan Burns, PhD, and C. Wayne Smith, MD, of the Departments of Pediatrics and Medicine, Baylor College of Medicine, who have captured these EM scanning images and made them available to us for presentation on our cover. Ó 2005 American Academy of Allergy, Asthma and Immunology The Journal of Allergy and Clinical Immunology (ISSN 00917-6749) is published monthly (12 issues per year) by Elsevier Inc., 360 Park Avenue South, New York, NY 10010-1710. Business and Editorial Offices: 1600 John F. Kennedy Boulevard, Suite 1800, Philadelphia, PA 19103-2899. Accounting and Circulation Offices: 6277 Sea Harbor Drive, Orlando, FL 32887-4800. Periodicals postage paid at Orlando, FL 32862 and additional mailing offices. POSTMASTER: Send address changes to The Journal of Allergy and Clinical Immunology, Elsevier Periodicals Customer Service, 6277 Sea Harbor Drive, Orlando, FL 32887-4800.

J ALLERGY CLIN IMMUNOL

August 2005 5A

Contents

T HE J OURNAL OF

Allergy Clinical Immunology AND

VOLUME 116 NUMBER 2 d

OFFICIAL JOURNAL OF THE AMERICAN ACADEMY OF ALLERGY, ASTHMA AND IMMUNOLOGY

The editors’ choice

239

Donald Y. M. Leung, MD, PhD, Harold S. Nelson, MD, and Stanley J. Szefler, MD

Reviews and feature articles

Y

w CME

Current reviews of allergy and clinical immunology Innate immune responses to infection

241

Michael F. Tosi, MD, New York, NY

Continued on page 7A

Y

This month’s theme: Infection and immunity

About the cover This month’s theme feature examines the fascinating interaction of infection and immunity. Our cover displays two exquisite images of the neutrophilic leukocyte’s response to infection in a mouse model. These leukocytes pass through the endothelium of blood vessels in response to a chemoattractant such as created by tissue infection. In the cover image, the left panel is a low power view of a small inflamed venule (pink) with numerous leukocytes (green) adhering to the endothelial lining. The right panel is a higher power view of a transmigrating leukocyte whose cell body lies beneath the pink endothelium, its amoeboid shape being easily appreciated. The leukocyte’s trailing tail or uropod (green) has not yet passed through the endothelium. Other articles in this issue that focus on the topic of infection and immunity are noted in the Table of Contents by the ‘‘theme’’ icon. Our sincere appreciation to Alan Burns, PhD, and C. Wayne Smith, MD, of the Departments of Pediatrics and Medicine, Baylor College of Medicine, who have captured these EM scanning images and made them available to us for presentation on our cover. Ó 2005 American Academy of Allergy, Asthma and Immunology The Journal of Allergy and Clinical Immunology (ISSN 00917-6749) is published monthly (12 issues per year) by Elsevier Inc., 360 Park Avenue South, New York, NY 10010-1710. Business and Editorial Offices: 1600 John F. Kennedy Boulevard, Suite 1800, Philadelphia, PA 19103-2899. Accounting and Circulation Offices: 6277 Sea Harbor Drive, Orlando, FL 32887-4800. Periodicals postage paid at Orlando, FL 32862 and additional mailing offices. POSTMASTER: Send address changes to The Journal of Allergy and Clinical Immunology, Elsevier Periodicals Customer Service, 6277 Sea Harbor Drive, Orlando, FL 32887-4800.

J ALLERGY CLIN IMMUNOL

August 2005 5A

Contents

CONTENTS CONTINUED

Continuing Medical Education examination: Innate immune responses to infection

Y

Molecular mechanisms in allergy and clinical immunology

w

EBV the prototypical human tumor virus—just how bad is it?

CME

250

251

David A. Thorley-Lawson, PhD, Boston, Mass

Continuing Medical Education examination: EBV the prototypical human tumor virus—just how bad is it?

262

Editorial

Y

Infection versus immunity: What’s the balance?

263

William T. Shearer, MD, PhD, Houston, Tex

Asthma diagnosis and treatment Rostrum The role of rhinovirus in asthma exacerbations

267

Samuel L. Friedlander, MD, and William W. Busse, MD, Madison, Wis

Perspectives in asthma Perspectives on the past decade of asthma genetics

274

Carole Ober, PhD, Chicago, Ill

Continued on page 9A The Journal of Allergy and Clinical Immunology posts in-press articles online in advance of their appearance in the print edition of the Journal. They are available at the JACI Web site at www.mosby.com/jaci at the ‘‘Articles in Press’’ link, as well as at Elsevier’s ScienceDirect Web site, www.sciencedirect.com. Each print article will acknowledge the e-publication date (the date when the article first appeared online). As soon as an article is published online, it is fully citable through use of its Digital Object Identifier (DOI). Please visit the JACI Web site and view our hot-off-the-wire articles through the ‘‘Articles in Press’’ link.

EC d

Editors’ Choice (p 239)

OR d

Online Repository material

Y

w CME

Theme issue CME examination article available online at www.mosby.com/jaci

J ALLERGY CLIN IMMUNOL

August 2005 7A

Contents

CONTENTS CONTINUED

Continuing Medical Education examination: Innate immune responses to infection

Y

Molecular mechanisms in allergy and clinical immunology

w

EBV the prototypical human tumor virus—just how bad is it?

CME

250

251

David A. Thorley-Lawson, PhD, Boston, Mass

Continuing Medical Education examination: EBV the prototypical human tumor virus—just how bad is it?

262

Editorial

Y

Infection versus immunity: What’s the balance?

263

William T. Shearer, MD, PhD, Houston, Tex

Asthma diagnosis and treatment Rostrum The role of rhinovirus in asthma exacerbations

267

Samuel L. Friedlander, MD, and William W. Busse, MD, Madison, Wis

Perspectives in asthma Perspectives on the past decade of asthma genetics

274

Carole Ober, PhD, Chicago, Ill

Continued on page 9A The Journal of Allergy and Clinical Immunology posts in-press articles online in advance of their appearance in the print edition of the Journal. They are available at the JACI Web site at www.mosby.com/jaci at the ‘‘Articles in Press’’ link, as well as at Elsevier’s ScienceDirect Web site, www.sciencedirect.com. Each print article will acknowledge the e-publication date (the date when the article first appeared online). As soon as an article is published online, it is fully citable through use of its Digital Object Identifier (DOI). Please visit the JACI Web site and view our hot-off-the-wire articles through the ‘‘Articles in Press’’ link.

EC d

Editors’ Choice (p 239)

OR d

Online Repository material

Y

w CME

Theme issue CME examination article available online at www.mosby.com/jaci

J ALLERGY CLIN IMMUNOL

August 2005 7A

Contents

CONTENTS CONTINUED

Continuing Medical Education examination: Innate immune responses to infection

Y

Molecular mechanisms in allergy and clinical immunology

w

EBV the prototypical human tumor virus—just how bad is it?

CME

250

251

David A. Thorley-Lawson, PhD, Boston, Mass

Continuing Medical Education examination: EBV the prototypical human tumor virus—just how bad is it?

262

Editorial

Y

Infection versus immunity: What’s the balance?

263

William T. Shearer, MD, PhD, Houston, Tex

Asthma diagnosis and treatment Rostrum The role of rhinovirus in asthma exacerbations

267

Samuel L. Friedlander, MD, and William W. Busse, MD, Madison, Wis

Perspectives in asthma Perspectives on the past decade of asthma genetics

274

Carole Ober, PhD, Chicago, Ill

Continued on page 9A The Journal of Allergy and Clinical Immunology posts in-press articles online in advance of their appearance in the print edition of the Journal. They are available at the JACI Web site at www.mosby.com/jaci at the ‘‘Articles in Press’’ link, as well as at Elsevier’s ScienceDirect Web site, www.sciencedirect.com. Each print article will acknowledge the e-publication date (the date when the article first appeared online). As soon as an article is published online, it is fully citable through use of its Digital Object Identifier (DOI). Please visit the JACI Web site and view our hot-off-the-wire articles through the ‘‘Articles in Press’’ link.

EC d

Editors’ Choice (p 239)

OR d

Online Repository material

Y

w CME

Theme issue CME examination article available online at www.mosby.com/jaci

J ALLERGY CLIN IMMUNOL

August 2005 7A

Contents

CONTENTS CONTINUED

Original articles EC d

Is it traffic type, volume, or distance? Wheezing in infants living near truck and bus traffic

279

Patrick H. Ryan, MS, Grace LeMasters, PhD, Jocelyn Biagini, MS, David Bernstein, MD, Sergey A. Grinshpun, PhD, Rakesh Shukla, PhD, Kimberly Wilson, MS, Manuel Villareal, MD, Jeff Burkle, BS, and James Lockey, MD, Cincinnati, Ohio EC d

Effect of low-dose ciclesonide on allergen-induced responses in subjects with mild allergic asthma

285

Gail M. Gauvreau, PhD, Louis Philippe Boulet, MD, Dirkje S. Postma, MD, PhD, Tomotaka Kawayama, MD, Richard M. Watson, BSc, MyLinh Duong, MD, Francine Deschesnes, BSc, Jan G. R. De Monchy, MD, PhD, and Paul M. O’Byrne, MD, Hamilton, Ontario, and Quebec City, Quebec, Canada, and Groningen, The Netherlands

Roflumilast, an oral, once-daily phosphodiesterase 4 inhibitor, attenuates allergen-induced asthmatic reactions

292

Emmerentia van Schalkwyk, MBChB, K. Strydom, MBChB, Zelda Williams, RN, Louis Venter, MSc, Stefan Leichtl, PhD, Christine Schmid-Wirlitsch, PhD, Dirk Bredenbro¨ker, MD, and Philip G. Bardin, FRACP, PhD, Cape Town and Rivonia, South Africa, Melbourne, Australia, and Konstanz, Germany

Duration of postviral airway hyperresponsiveness in children with asthma: Effect of atopy

299

Paraskevi Xepapadaki, MD, PhD, Nikolaos G. Papadopoulos, MD, PhD, Apostolos Bossios, MD, PhD, Emmanuel Manoussakis, MD, Theodoros Manousakas, MD, and Photini Saxoni-Papageorgiou, MD, PhD, Athens, Greece

Mechanisms of asthma and allergic inflammation OR d

Dissecting asthma using focused transgenic modeling and functional genomics

305

Douglas A. Kuperman, PhD, Christina C. Lewis, PhD, Prescott G. Woodruff, MD, Madeleine W. Rodriguez, BS, Yee Hwa Yang, PhD, Gregory M. Dolganov, PhD, John V. Fahy, MD, and David J. Erle, MD, Chicago, Ill, and San Francisco, Calif

Endobronchial adenosine monophosphate challenge causes tachykinin release in the human airway

312

Fionnuala Crummy, MD, MRCP, Mark Livingston, PhD, Joy E. S. Ardill, PhD, FCRPath, Catherine Adamson, MSc, Madeleine Ennis, PhD, and Liam G. Heaney, MD, MRCP, Belfast, Northern Ireland, United Kingdom OR d

Rat tracheal epithelial responses to water avoidance stress

318

Hiroshi Akiyama, PhD, Hiroo Amano, MD, PhD, and John Bienenstock, MD, Tokyo and Maebashi, Japan, and Hamilton, Ontario, Canada

Allergen-induced substance P synthesis in large-diameter sensory neurons innervating the lungs

325

Benjamas Chuaychoo, MD, Dawn D. Hunter, PhD, Allen C. Myers, PhD, Marian Kollarik, MD, PhD, and Bradley J. Undem, PhD, Baltimore, Md

Continued on page 11A J ALLERGY CLIN IMMUNOL

August 2005 9A

Contents

CONTENTS CONTINUED

Differential effects of (S)- and (R)-enantiomers of albuterol in a mouse asthma model

332

William R. Henderson, Jr, MD, Ena Ray Banerjee, PhD, and Emil Y. Chi, PhD, Seattle, Wash

Rhinitis, sinusitis, and ocular diseases Comparison of test devices for skin prick testing

341

Warner W. Carr, MD, Bryan Martin, DO, Robin S. Howard, MA, Linda Cox, MD, Larry Borish, MD, and the Immunotherapy Committee of the American Academy of Allergy, Asthma and Immunology, Silver Spring, Md, Fort Lauderdale, Fla, and Charlottesville, Va

Allergen-specific nasal IgG antibodies induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity

347

Ju¨rgen Reisinger, MSc, Friedrich Horak, MD, Gabrielle Pauli, MD, Marianne van Hage, MD, Oliver Cromwell, PhD, Franz Ko¨nig, Rudolf Valenta, MD, and Verena Niederberger, MD, Vienna, Austria, Stockholm, Sweden, Strasbourg, France, and Reinbek, Germany

Levocetirizine: Pharmacokinetics and pharmacodynamics in children age 6 to 11 years

355

F. Estelle R. Simons, MD, FRCPC, and Keith J. Simons, PhD, Winnipeg, Manitoba, Canada

Striking deposition of toxic eosinophil major basic protein in mucus: Implications for chronic rhinosinusitis

362

Jens U. Ponikau, MD, David A. Sherris, MD, Gail M. Kephart, BS, Eugene B. Kern, MD, David J. Congdon, MD, Cheryl R. Adolphson, MS, Margaret J. Springett, BS, Gerald J. Gleich, MD, and Hirohito Kita, MD, Rochester, Minn, Buffalo, NY, and Salt Lake City, Utah OR d

Intranasal tolerance induction with polypeptides derived from 3 noncrossreactive major aeroallergens prevents allergic polysensitization in mice

370

Karin Hufnagl, PhD, Birgit Winkler, MD, Margit Focke, PhD, Rudolf Valenta, MD, Otto Scheiner, PhD, Harald Renz, MD, and Ursula Wiedermann, MD, PhD, Vienna, Austria, and Marburg, Germany

Environmental and occupational respiratory disorders EC d OR d

Prevalences of positive skin test responses to 10 common allergens in the US population: Results from the Third National Health and Nutrition Examination Survey

377

Samuel J. Arbes, Jr, DDS, MPH, PhD, Peter J. Gergen, MD, MPH, Leslie Elliott, MPH, PhD, and Darryl C. Zeldin, MD, Research Triangle Park, NC, and Bethesda, Md

Airborne endotoxin in homes with domestic animals: Implications for cat-specific tolerance

384

James A. Platts-Mills, BA, Natalie J. Custis, BA, Judith A. Woodfolk, MD, PhD, and Thomas A. E. Platts-Mills, MD, PhD, Charlottesville, Va

Continued on page 13A J ALLERGY CLIN IMMUNOL

August 2005 11A

Contents

CONTENTS CONTINUED

Food allergy, dermatologic diseases, and anaphylaxis EC d

COX-2 inhibition enhances the TH2 immune response to epicutaneous sensitization

390

Dhafer Laouini, PhD, Abdala ElKhal, PhD, Ali Yalcindag, MD, Seiji Kawamoto, MD, PhD, Hans Oettgen, MD, PhD, and Raif S. Geha, MD, Boston, Mass EC d

Responsiveness to autologous sweat and serum in cholinergic urticaria classifies its clinical subtypes

397

Atsushi Fukunaga, MD, Toshinori Bito, MD, Kenta Tsuru, MD, Akiko Oohashi, MD, Xijun Yu, MD, Masamitsu Ichihashi, MD, Chikako Nishigori, MD, and Tatsuya Horikawa, MD, Kobe, Japan OR d

Lack of detectable allergenicity of transgenic maize and soya samples

403

Rita Batista, BSc, Baltazar Nunes, MSc, Manuela Carmo, Carlos Cardoso, PharmD, Helena Sa˜o Jose´, Anto´nio Bugalho de Almeida, MD, PhD, Alda Manique, MD, Leonor Bento, MD, PhD, Caˆndido Pinto Ricardo, PhD, and Maria Margarida Oliveira, PhD, Lisboa, Oeiras, and Alge´s, Portugal

Basic and clinical immunology Advances in Asthma, Allergy, and Immunology Series 2005 Basic and clinical immunology

411

Javier Chinen, MD, PhD, and William T. Shearer, MD, PhD, Bethesda, Md, and Houston, Tex

Current perspectives

Y

The gastrointestinal tract is critical to the pathogenesis of acute HIV-1 infection

419

Saurabh Mehandru, MD, Klara Tenner-Racz, MD, Paul Racz, MD, PhD, and Martin Markowitz, MD, New York, NY, and Hamburg, Germany

Editorial

Y

Are you immunodeficient?

423

Francisco A. Bonilla, MD, PhD, and Raif S. Geha, MD, Boston, Mass

Rostrum

Y

From idiopathic infectious diseases to novel primary immunodeficiencies

426

Jean-Laurent Casanova, MD, PhD, Claire Fieschi, MD, PhD, Jacinta Bustamante, MD, Janine Reichenbach, MD, Natasha Remus, MD, Horst von Bernuth, MD, and Capucine Picard, MD, PhD, Paris and Cre´teil, France, and Frankfurt, Germany

Original articles EC d OR d

Y

Infant home endotoxin is associated with reduced allergen-stimulated lymphocyte proliferation and IL-13 production in childhood

431

Joseph H. Abraham, ScD, Patricia W. Finn, MD, Donald K. Milton, MD, Louise M. Ryan, PhD, David L. Perkins, MD, and Diane R. Gold, MD, Boston, Mass

J ALLERGY CLIN IMMUNOL

Continued on page 14A August 2005 13A

Contents

CONTENTS CONTINUED

Y

Does early EBV infection protect against IgE sensitization?

438

Caroline Nilsson, MD, Annika Linde, MD, PhD, Scott M. Montgomery, BSc, PhD, Liselotte Gustafsson, Per Na¨sman, Ph Lic, Marita Troye Blomberg, PhD, and Gunnar Lilja, MD, PhD, Stockholm, Sweden

Biased use of VH5 IgE-positive B cells in the nasal mucosa in allergic rhinitis

445

Heather A. Coker, PhD, Helen E. Harries, MBiochem, Graham K. Banfield, FRCS, Victoria A. Carr, RGN, Stephen R. Durham, MD, Elfy Chevretton, FRCS, Paul Hobby, MSc, Brian J. Sutton, PhD, and Hannah J. Gould, PhD, London, United Kingdom

Antibody responses against galactocerebroside are potential stage-specific biomarkers in multiple sclerosis

453

Til Menge, MD, Patrice H. Lalive, MD, Hans-Christian von Bu¨dingen, MD, Bruce Cree, MD, PhD, Stephen L. Hauser, MD, and Claude P. Genain, MD, San Francisco, Calif, and Zu¨rich, Switzerland

Letters to the Editor Perilesional GM-CSF therapy of a chronic leg ulcer in a patient with common variable immunodeficiency

460

Ammar Z. Hatab, MD, Deanna McDanel, PharmD, BCPS, and Zuhair K. Ballas, MD, Iowa City, Iowa

Asthma caused by cyanoacrylate used in a leisure activity

462

Mona-Rita Yacoub, MD, Catherine Lemie`re, MD, MSc, and Jean-Luc Malo, MD, Montreal, Quebec, Canada

Correspondence Cystic fibrosis gene mutations and chronic rhinosinusitis

463

Clement L. Ren, MD, Rochester, NY

Leukotriene receptor antagonists are not as effective as intranasal corticosteroids for managing nighttime symptoms of allergic rhinitis

463

Robert A. Nathan, MD, Colorado Springs, Colo

Efficacy of ant venom immunotherapy and whole body extracts

464

Simon G. A. Brown, MBBS, PhD, FACEM, Robert J. Heddle, MBBS, PhD, FRACP, FRCPA, Michael D. Wiese, BPharm, MClinPharm, and Konrad E. Blackman, MBBS, FACEM, Fremantel, Bedford Park, and Hobart, Australia

Reply

465

David B. K. Golden, MD, Baltimore, Md

Images in allergy and immunology

Y

Toll-like receptors and atopy

467

Pierre Olivier Fiset, BSc, Meri Katarina Tulic, PhD, and Qutayba Hamid, MD, PhD, Editors

Continued on page 15A 14A August 2005

J ALLERGY CLIN IMMUNOL

Contents

CONTENTS CONTINUED

Y

Chronic active Epstein-Barr virus infection of natural killer cells presenting as severe skin reaction to mosquito bites

470

Susan E. Pacheco, MD, Stephen M. Gottschalk, MD, Mary V. Gresik, MD, Megan K. Dishop, MD, Takayuki Okmaura, MD, and Theron G. McCormick, MD, Guest Editors

Beyond our pages

473

Burton Zweiman, MD, and Marc E. Rothenberg, MD, PhD, Editors

Correction Physical activity and exercise in asthma: Relevance to etiology and treatment

298

(Lucas SR, Platts-Mills TAE. 2005;115:928-34)

Reader services Instructions for authors

www.mosby.com/jaci and July 2005, pages 15A-22A

Information for readers

19A

Newsview—American Academy of Allergy, Asthma and Immunology

25A

CME calendar—American Academy of Allergy, Asthma and Immunology

30A

CME activities information

32A

Professional opportunities

35A

Change of address

354

The Editors of The JACI are pleased to announce that Continuing Medical Education (CME) credit is now offered to readers who successfully complete examination questions accompanying monthly review articles in the Journal’s Current Reviews of Allergy and Clinical Immunology and Molecular Mechanisms in Allergy and Clinical Immunology series. This CME opportunity furthers the joint educational goals of the Journal and its sponsoring foundation, the American Academy of Allergy, Asthma and Immunology (AAAAI). Learning objectives, examination questions, and full details appear in each review article in the print and online Journal. The self-directed examinations can be taken at the JACI website (www.mosby.com/jaci). Credit is administered by the AAAAI.

Complimentary 1-year subscriptions to The Journal of Allergy and Clinical Immunology are available to AAAAI member FITs in the United States through an unrestricted educational grant from Alcon Laboratories, Inc.

Statements and opinions expressed in the articles and communications herein are those of the author(s) and not necessarily those of the Editor, publisher, or the American Academy of Allergy, Asthma and Immunology. The Editor, publisher, and the American Academy of Allergy, Asthma and Immunology disclaim any responsibility or liability for such material and do not guarantee, warrant, or endorse any product or service advertised in this publication, nor do they guarantee any claim made by the manufacturer of such product or service.

J ALLERGY CLIN IMMUNOL

August 2005 15A

T HE J OURNAL OF

Allergy Clinical Immunology AND

Editor in Chief DONALD Y. M. LEUNG, MD, PhD Denver, Colo Deputy Editors HAROLD S. NELSON, MD,

AND

STANLEY J. SZEFLER, MD Denver, Colo

Associate Editors ANDREA J. APTER, MD, MSc Philadelphia, Pa BRUCE BOCHNER, MD Baltimore, Md ROBERT K. BUSH, MD Madison, Wis FRED FINKELMAN, MD Cincinnati, Ohio QUTAYBA HAMID, MD, PhD Montreal, Quebec, Canada DAVID B. PEDEN, MD Chapel Hill, NC WILLIAM T. SHEARER, MD, PhD Houston, Tex SCOTT SICHERER, MD New York, NY AND DONATA VERCELLI, MD Tucson, Ariz Guest Editors BURTON ZWEIMAN, MD Philadelphia, Pa AND MARC E. ROTHENBERG, MD Cincinatti, Ohio Editorial Board LARRY BORISH, MD Charlottesville, Va

2006

CEZMI A. AKDIS, MD Davos, Switzerland

2009

REDWAN MOQBEL, PhD, FRCPath Edmonton, Alberta, Canada

2006

WILLIAM J. CALHOUN, MD Pittsburgh, Pa

2009

SANTA JEREMY ONO, PhD London, United Kingdom

2006

VERNON M. CHINCHILLI, PhD Hershey, Pa

2009

ZUHAIR K. BALLAS, MD, PhD Iowa City, Iowa

2007

THOMAS BIEBER, MD, PhD Bonn, Germany

2007

PEYTON A. EGGLESTON, MD Baltimore, Md

2007

DAVID P. HUSTON, MD Houston, Tex

2007

PEDRO AVILA, MD Chicago, Ill DENNIS LEDFORD, MD Tampa, Fla

2008 2008

DAVID B. PEDEN, MD Chapel Hill, NC

2008

HARALD RENZ, MD Marburg, Germany

2008

HUGH A. SAMPSON, MD New York, NY

2008

ERIKA VON MUTIUS, MD, MSc Munich, Germany

2008

DANIEL L. HAMILOS, MD Boston, Mass

2009

ANTHONY A. HORNER, MD La Jolla, Calif

2009

HANS C. OETTGEN, MD, PhD Boston, Mass

2009

DEVENDRA K. AGRAWAL, PhD Omaha, Neb

2010

JOSHUA A. BOYCE, MD Boston, Mass

2010

JAVIER CHINEN, MD, PhD Bethesda, Md

2010

GURJIT K. KHURANA HERSHEY, MD, PhD Cincinnati, Ohio

2010

JOHN M. KELSO, MD San Diego, Calif

2010

HANS-UWE SIMON, MD, PhD Bern, Switzerland

2010

Board of Directors of the American Academy of Allergy, Asthma and Immunology President F. ESTELLE R. SIMONS, MD, FAAAAI Winnipeg, Manitoba, Canada President-Elect THOMAS A. E. PLATTS-MILLS, MD, PhD, FAAAAI Charlottesville, Va Vice President THOMAS B. CASALE, MD, FAAAAI Omaha, Neb DAVID H. BROIDE, MD, FAAAAI La Jolla, Calif THOMAS A. FLEISHER, MD, FAAAAI Bethesda, Md SANDRA M. GAWCHIK, DO, FAAAAI Upland, Pa STANLEY GOLDSTEIN, MD, FAAAAI Rockville Centre, NY PAUL A. GREENBERGER, MD, FAAAAI Chicago, Ill REBECCA S. GRUCHALLA, MD, PhD, FAAAAI Dallas, Tex

Secretary/Treasurer HUGH A. SAMPSON, MD, FAAAAI New York, NY Immediate Past President MICHAEL SCHATZ, MD, MS, FAAAAI San Diego, Calif Past Past President LANNY J. ROSENWASSER, MD, FAAAAI Denver, Colo RICHARD W. HONSINGER, MD, FAAAAI Los Alamos, NM DENNIS K. LEDFORD, MD, FAAAAI Tampa, Fla DONALD Y. M. LEUNG, MD, PhD, FAAAAI Denver, Colo ARNOLD I. LEVINSON, MD, FAAAAI Philadelphia, Pa JAMES T. LI, MD, PhD, FAAAAI Rochester, Minn DENNIS R. OWNBY, MD, FAAAAI Augusta, Ga

Executive Vice President KAY WHALEN, CAE

Address of Executive Office AMERICAN ACADEMY OF ALLERGY, ASTHMA AND IMMUNOLOGY 555 East Wells Street, Suite 1100, Milwaukee, WI 53202-3823 (414) 272-6071 fax: (414) 272-6070 e-mail: [email protected]

16A August 2005

J ALLERGY CLIN IMMUNOL

T HE J OURNAL OF

Allergy Clinical Immunology AND

Information for Readers COMMUNICATION Communications regarding original articles and editorial management should be addressed to Donald Y. M. Leung, MD, PhD, Editor in Chief, The Journal of Allergy and Clinical Immunology, National Jewish Medical and Research Center, 1400 Jackson St (J324), Denver, CO 80206; phone 303-398-1963; fax 303-270-2269.

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J ALLERGY CLIN IMMUNOL

Ó 2005 American Academy of Allergy, Asthma and Immunology. All rights reserved. This journal and the individual contributions contained in it are protected under copyright by the American Academy of Allergy, Asthma and Immunology, and the following terms and conditions apply to their use: Photocopying Single photocopies of single articles may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for nonprofit education classroom use. Permissions may be sought directly from Elsevier’s Rights Department in Philadelphia, PA, USA: phone 215-239-3804, fax 215-239-3805, e-mail [email protected]. Requests may also be completed online via the Elsevier home page (http:// www.elsevier.com/locate/permissions). Users may clear permissions and make payments in the US through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone 978-750-8400; fax 978-750-4744; and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London W1P 0LP, UK; phone 44-20-7631-5555; fax 44-20-7631-5500. Other countries may have local reprographic rights agencies for payment. Derivative Works Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for internal circulation within their institutions. Permission of the Publisher is required for resale or distribution outside the institution. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this journal, including any article or part of an article. Except as outlined above, no part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Rights Department, at the fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher or the American Academy of Allergy, Asthma and Immunology for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. Although all advertising material is expected to conform to ethical (medical) standards, inclusion in this publication does not constitute a guarantee or endorsement of the quality or value of such product or of the claims made of it by its manufacturer. Indexed or Abstracted in Index Medicus, Science Citation Index, Current Contents/Clinical Medicine, Current Contents/Life Sciences. Available electronically from Ovid Technologies (800-950-2035). Microform edition available from ProQuest Information and Learning, 300 N Zeeb Rd, Ann Arbor, MI 48106-1346.

August 2005 19A

J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 2

AAAAI Developing Researcher Award 23A

T

he most frequently cited allergy/immunology journal in the field, The Journal of Allergy and Clinical Immunology, continues to grow in prominence because of the excellent research it showcases. The Academy is proud to announce this new JACI Award which will recognize innovative research and outstanding scientific writing by the new generation of allergy researchers and clinicians. The AAAAI Award for Outstanding Research Published in the JACI by a Developing Researcher provides an unrestricted prize of $1,500, commemorative plaque, and recognition during the AAAAI’s Annual Business Meeting. Up to 4 awardees will be chosen during one calendar year.

for O AAAAI Award arch u se Publitsstanding ReJACI h by a D ed in the r eveloping Researche

Objective • To encourage the submission to JACI of the highest quality articles on our science’s frontier • To recognize ground breaking research and excellent writing by the new generation of allergy researchers and clinicians Award • Formal presentation of an unrestricted prize of $1500 and a plaque for each award (funded by the AAAAI) will be made at the annual Academy Meeting • Announcement of the awards for the year will be featured in the published program for the annual Academy Meeting • Announcement and commendation of winners will be published in the Journal Title of the Award • The AAAAI Award for Outstanding Research Published in the JACI by a Developing Researcher Judgment Process • A chairman will be appointed by the JACI Editors to coordinate a panel whose task will be to review and evaluate nominated papers and reach a decision on recipients of the award • Judgement will be by a panel composed of the chairman of the award committee, one Editor, a member of the AAAAI Research Advisory Council, and at least two researchers or clinicians who are knowledgeable in the area of research being reported Conditions of Eligibility • Candidates must meet the following criteria for eligibility —can be from any training program world-wide —must have completed an MD and/or PhD or the equivalent within the 7 years prior to nomination for the award —must have conducted the research and written the paper during the post-doctoral fellowship training and not while holding a faculty position • The research must be considered outstanding and represent a conceptual advance in the treatment or pathogenesis of allergic disease • The article must have been published in JACI • The article must have appeared within 12 months of the nomination • The candidate must have conducted the majority of the research or have been the primary leader of the research team and appear as the first author • A written nomination of the candidate and paper must be submitted by a sponsor (the training director, preceptor, research mentor, or a current Editor of the Journal.) The sponsor must submit a completed Nomination Application Form, a statement briefly discussing the impact of the scientific observations on the field of allergy and immunology and justification of why the published work is deserving of the award, and 3 letters of recommendation for the nominated paper, 2 of which are from individuals at institutions with which the Fellow has never been associated. (Letters should address the significance and importance of the work in the paper and its relevance to the Fellow’s body of work, the quality of the written presentation, the Fellow’s personal characteristics and potential for a future in academic medicine.) • Up to 4 awards may be given within each calendar year; if no candidates are deemed of sufficient merit, no award will be given Criteria for Excellence in a Publication • The publication must present novel information that holds significant importance for the basic and clinical science of allergy, asthma or immunology • The article must meet the highest standards for scientific writing • The publication must represent a significant portion of the candidate’s fellowship work

*For application forms and further information, please contact the Editorial Office at 303 398-1963.

24A AAAAI Developing Researcher Award

J ALLERGY CLIN IMMUNOL AUGUST 2005

Nomination for AAAAI Developing Researcher Award SPONSOR

Name

Date

Relationship to FIT: ______TPD/Preceptor __________Research Mentor ________JACI Editor

DEVELOPING RESEARCHER

Name

Mailing Address

Phone

Country

E-mail

Fax

Academic affiliation

Academic degree(s)

Faculty appointments

Date degree(s) awarded for O AAAAI Award arch utstanding Rese Publis I date(s) he JAC by a D hed in tAppointment r eveloping Researche

Training program where work on paper was completed

Date of Training

____

to

____

Article submitted for consideration (MUST HAVE BEEN ACCEPTED FOR JACI PUBLICATION) (Provide title, authors & complete publication reference; if in press, provide planned publication date)

Attach 3 copies of the published article; if the article is “in press,” provide 2 copies of the manuscript, illustrations, and tables) 3 Letters of recommendation provided by (Name and address)

Academic affiliation:

1.

1.

2.

2.

3.

3.

(Attach the original copy of each letter in a sealed envelope. At least 2 letters should be provided by individuals who are not associated with the nominee’s training center) Attach a typewritten statement, no longer than 2 paragraphs, identifying the significance of the scientific observations in this publication and explaining why this published work is deserving of the AAAAI Developing Researcher publication award

NEW SVIEW newsview A Monthly Update of Developments from the AAAAI

Anaphylaxis: bridging the gap between research, clinical practice

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In the next year, the AAAAI will work to bridge the gap between basic science research in anaphylaxis and clinical practice through a comprehensive public education outreach initiative. The new Anaphylaxis Public Education Task Force is developing this educational effort to raise awareness of anaphylaxis as a killer allergy with many potential triggers. The initiative will also emphasize the importance of the role of the allergist/immunologist in anaphylaxis management and prevention. The new anaphylaxis public education campaign is one of the initiatives of President F. Estelle R. Simons, MD, FAAAAI. The anaphylaxis initiative will include three major public education campaigns: d

Back-to-school campaign, September 2005

Elements of the back-to-school campaign will include publicity to national magazines, and national daily and weekly publications. The Task Force is also considering a plan to reach school nurses, who may serve as a bridge to educating a vast population of students. Regional, state and local allergy societies may be asked to work with their state and local school nurse associations. The Food Allergy & Anaphylaxis Network (FAAN) also offers a quarterly newsletter to school nurses, and the AAAAI Allergy and Asthma Tool Kit for School Nurses will also be utilized. d

Holiday season campaign, late October through December 2005

The holiday season campaign will focus on three main holidays: Halloween, Thanksgiving and Christmas/ Hanukkah. Outreach will begin in October with publicity to national magazines, and daily and weekly media outlets. d

Great outdoors campaign, March through May 2006

The great outdoors campaign will conclude just before the Memorial Day weekend in May 2006. May is National Allergy and Asthma Awareness Month, and also includes Food Allergy Awareness Week. Anaphylaxis publicity efforts will work hand-in-hand with these events. Core messages for the public will include a brief description of anaphylaxis, and answers to basic questions including: d d d d d d

What is anaphylaxis? Who is at risk? When can anaphylaxis occur? Where can anaphylaxis occur? How is anaphylaxis treated? Why is follow-up needed?

2006 Annual Meeting The anaphylaxis public outreach effort will culminate during the 2006 Annual Meeting in Miami Beach, FL, with

J ALLERGY CLIN IMMUNOL

Sunday, March 5, 2006, designated as Anaphylaxis Day. On Anaphylaxis Day, the Presidential Symposium will be held concurrently with Sunday’s Plenary Session, and focus on advances in research in basic and clinical science, relevant to the diagnosis and treatment of anaphylaxis. The AAAAI will also host a press conference highlighting anaphylaxis-related abstracts selected for presentation at the Annual Meeting. 2006 AAAAI Primary Care Symposium The 2006 Primary Care Symposium ‘‘Optimizing Treatment of Your Allergic Patients: An Update for the Primary Care Provider on Issues Affecting One-Third of Your Practice,’’ co-chaired by Joann Blessing-Moore, MD, FAAAAI and Richard F. Lockey, MD, FAAAAI, will also include a segment on anaphylaxis. The annual symposium is held immediately prior to the Annual Meeting and designed to update local primary care physicians about the most recent developments in allergic disease. Simons will also serve as one of ten AAAAI representatives at the Second Symposium on the Definition and Management of Anaphylaxis, sponsored by the National Institutes of Health (NIH) and FAAN. The Symposium will build upon previous efforts and work toward developing a universally accepted definition for anaphylaxis. It will further expand its anaphylaxis research agenda, and identify educational needs for health care professionals and patients. Hugh A. Sampson, MD, FAAAAI, and Anne Munoz-Furlong will Co-Chair the Symposium. Anaphylaxis Retrospective The AAAAI is preparing a retrospective on anaphylaxis for the 2006 Annual Meeting. If your research over the years has made a significant contribution to the understanding of anaphylaxis, the AAAAI History and Archives Committee, chaired by Michael A. Kaliner, MD, FAAAAI, wants to hear from you. Please contact Audrey Mudek at the AAAAI executive office, (414) 272-6071 or e-mail [email protected].

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American Academy of Allergy, Asthma & Immunology Lifelong Learner Bill of Rights

Dear Colleagues and Friends: The American Academy of Asthma, Allergy & Immunology provides outstanding continuing medical education through The Journal of Allergy and Clinical Immunology (citation impact factor now 7.205), the AAAAI Annual Meeting, and other AAAAI programs, resources, and materials. In order to emphasize our commitment to continuing medical education, we have recently developed the AAAAI Lifelong Learner Bill of Rights. We pledge to maintain the highest quality in all of our educational programs and to

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NEW SVIEW newsview A Monthly Update of Developments from the AAAAI

fulfill our promise to provide you with a premier educational experience. Sincerely, F. Estelle R. Simons, MD, FAAAAI AAAAI President The American Academy of Allergy, Asthma & Immunology (AAAAI) recognizes that you are a life-long learner who has chosen to engage in continuing medical education to identify or fill a gap in knowledge, skill or performance. As part of the AAAAI’s duty to you as a learner, you have the right to expect that your continuing medical education experience with the AAAAI includes: Content that: d promotes improvements or quality in healthcare; d is valid, reliable, and accurate; d offers balanced presentations that are free of commercial bias for or against a product/service; d is vetted through a process that resolves any conflicts of interests of planners, teachers, or authors; d is driven and based on learning needs; d addresses the stated objectives or purpose; and d is evaluated for its effectiveness in meeting the identified educational needs. A learning environment that: supports learners’ ability to meet their individual needs; d respects and attends to any special needs of the learners; d respects the diversity of groups of learners; and d is free of promotional, commercial, and/or sales activities. d

Disclosure of: relevant financial relationships planners, teachers, and authors have with commercial interests related to the content of the activity; and d commercial support (funding or in-kind resources) of the activity. d

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Free online CME course

The Environmental Management of Asthma, a free online Continuing Medical Education (CME) program, is available on the AAAAI Web site, www.aaaai.org. The program provides healthcare professionals and the insurance industry with information and resources to incorporate environmental management into clinical practices, and standards of care for patients with asthma. AAAAI members and non-members have an opportunity to earn 1.0 or 1.2 CME/CE credits. The program addresses the impact and management of environmental asthma triggers such as air pollutants, pollen, environmental tobacco smoke, mold, dust mites, cockroaches and warm-blooded pets. The course was designed for physicians and allied health professionals, including nurse case managers, in primary care specialties.

Page 26A August 2005

The CME program consists of a 15-minute didactic presentation with lecture notes highlighting the classification of environmental triggers, followed by a series of pediatric and adult case studies with question and answer sections. The didactic portion is presented with real-time audio capabilities. The AAAAI designates this educational activity for a maximum of 1.0 category 1 credits towards the AMA Physician’s Recognition Award. The course is also approved by the California Board of Registered Nursing, Provider #10704, for 1.2 contact hours and by the Commission for Case Manager Certification (CCMC) for 1.0 contact hours. For more information, visit the Professionals Center of the AAAAI Web site, www.aaaai.org. Choose the Professionals Center, click on the Education for You section and select the AAAAI Continuing Education Opportunities link.

AWARDS AND GRANTS

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AAAAI/JACI Award for Outstanding Research

David M. Fleischer MD, Denver, CO, has been awarded the 2005 AAAAI/JACI Award for Outstanding Research. Fleischer was honored for his November 2004 paper, ‘‘Peanut allergy: Recurrence and its management’’ (J Allergy Clin Immunol 2004 114(5):1195-201) designed and written during his fellowship at Johns Hopkins, Baltimore MD.

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New FIT abstract awards

A new AAAAI Interest Section Fellow-in-Training Abstract Award will honor seven fellows-in-training who submit the best abstract in each Interest Section. The abstracts will be selected by each Interest Section based on the highest scored abstract. The AAAAI will award the presenter with $500 and a plaque. No application is necessary for this award. All abstracts submitted by an FIT will be considered. To submit an abstract, visit the Annual Meeting Web site, www.annualmeeting.aaaai.org and choose the 2006 Annual Meeting link. Award recipients will be honored throughout the 2006 Annual Meeting and during the Interest Section Forums. For more information, please contact Mikelle Johnson at the AAAAI executive office at (414) 272-6071 or e-mail [email protected].

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2006 AAAAI abstract awards available

Applications and details about all abstract awards are available on the AAAAI Annual Meeting Web site, www.annualmeeting.aaaai.org.

J ALLERGY CLIN IMMUNOL

NEW SVIEW newsview A Monthly Update of Developments from the AAAAI

All applications are due September 7 with the exception of the Fellow-in-Training (FIT) Travel Scholarship. AAAAI Allied Health Travel Scholarship Purpose: Four scholarships are awarded to AAAAI allied health members who submit the best abstracts for presentation at the 2006 Annual Meeting. Eligibility: AAAAI allied health members submitting abstracts for presentation at the 2006 Annual Meeting Award: Actual travel and hotel expenses incurred by the abstract presenter, up to a limit of $750 per recipient. AAAAI Interest Section Fellow-in-Training Abstract Award Purpose: This new award will honor seven fellows-intraining who submit the best abstract in each Interest Section. The abstracts will be selected by each Interest Section based on the highest scored abstract. No application is necessary for this award. All abstracts submitted by an FIT will be considered. The award recipients will be honored throughout the Annual Meeting and during the Interest Section Forums. Eligibility: AAAAI fellows-in-training Award: $500 and a plaque AAAAI/Sepracor Research Excellence Awards Purpose: Three awards are given to abstract presenters selected by the Abstract Review Committee. Abstracts will be judged by the novelty of their research development and the overall significance of their research toward the advancement of allergy, asthma and immunology, and optimal patient care Eligibility: The abstract first author must be an allergy and immunology trainee (pre or post-doctoral), and the trainee must have conducted the vast majority of the abstract related work. The abstract co-author must be an AAAAI member. The research should be investigator led and not sponsored by any for-profit entity. Award: $5,000 per recipient American Academy of Pediatrics Section on Allergy/ Immunology Outstanding Pediatric Allergy, Asthma and Immunology Abstract Awards for Fellows-in-Training and Junior Faculty Purpose: Five awards are given, three to fellows-intraining, and two to junior faculty members. All award recipients are eligible for a complimentary, one-year membership in the AAP and SOAI. Eligibility: Fellows-in-training or junior faculty who submit the best abstracts involving pediatric patients, birth to age 21 years, that are accepted for presentation at the 2006 Annual Meeting Award: $750 each to three fellows-in-training and $1,000 each to two junior faculty members Domestic Fellow-in-Training (FIT) Travel Scholarship Purpose: A grant to cover travel expenses to the Annual Meeting Eligibility: AAAAI FIT members in the United States and Canada

J ALLERGY CLIN IMMUNOL

Award: $1,000 with an accepted abstract or $500 without an abstract Deadline: TBA Applications will be available on the Annual Meeting Web site this fall. Please direct questions to Reaca Pearl at the AAAAI executive office at (414) 272-6071 or e-mail [email protected]. 2006 ERT Allied Health Travel Grants Purpose: The grants support education, research and travel for three AAAAI allied health members who submit the best abstracts for presentation at the 2006 Annual Meeting. Eligibility: AAAAI allied health members submitting abstracts for presentation at the 2006 Annual Meeting Award: $750 per recipient International Fellow-in-Training (FIT) Travel Scholarship Purpose: The scholarships are designed to financially assist research and non-research fellows and residents-intraining in allergy and immunology in attending the AAAAI 2006 Annual Meeting. Eligibility: International FIT AAAAI members, or international FITs who have a completed membership application on file at the time of scholarship application submission, are eligible. Applicants must be post-doctoral trainees in allergy/immunology who, at the time of application, are within seven years of their latest doctoral degree, and are outside the United States and Canada. Award: To ensure greater equity, financial support reflects variable air travel costs based on location of award recipient. Awards generally vary from $850 to $1,300.

AAAAI MEMBER UPDATES

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2006 Annual Meeting: call for abstracts

The 2006 AAAAI abstract submission site is now open. Take this important opportunity to make a significant contribution to the overall scientific content of the 2006 Annual Meeting and share your findings with fellow Annual Meeting delegates. For more information, visit the AAAAI Annual Meeting Web site, www.annualmeeting.aaaai.org, or e-mail [email protected]. The abstract submission deadline is September 7.

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Strategic Training in Allergy Research (ST*AR) Program

The new AAAAI Strategic Training in Allergy Research (ST*AR) Program will strengthen basic science research in allergy/immunology and its translation to excellence inclinical practice. Debuting at the 2006 Annual Meeting, the ST*AR Program will offer 40-50 PhD or post-doctoral

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NEW SVIEW newsview A Monthly Update of Developments from the AAAAI

students from the United States and Canada an in-depth introduction to allergy/immunology research and an opportunity to learn more about the specialty. Target audience: PhD trainees conducting research related to allergic diseases, asthma and immunology Program overview: PhD trainees are invited to submit abstracts for presentation at the 2006 AAAAI Annual Meeting, March 3-7, 2006, in Miami Beach, FL. Up to 40 selected participants will be invited to participate in the ST*AR Program, which will include: Presentation of original research at the ST*AR Program Attendance at AAAAI Annual Meeting postgraduate program and symposia d Participation in a special ST*AR Program that includes basic research presentations in small groups by AAAAI members who are NIH funded researchers in allergy/ immunology d Information about career opportunities in allergy, asthma and immunology research d Funds for travel (including a stipend for ground transportation), hotel accommodation and meeting registration The goal of the ST*AR Program is to provide PhD trainees with an opportunity to consider research opportunities in the field of allergy and immunology. The AAAAI Annual Meeting provides an excellent forum for those interested in translating basic science advances into clinical practice to network with basic scientists, translational clinical researchers and clinical experts in allergy/ immunology. For more information, or to receive a ST*AR Program application, contact Reaca Pearl at the AAAAI executive office at (414) 272-6071 or e-mail [email protected].

d d

The Plenary Session CD is $35. The CD may be purchased for up to two years following the 2005 Annual Meeting. For more information or to order your CD, contact Alicia Josten at the AAAAI executive office at (414) 272-6071.

AAAAI Annual Meeting dates and sites

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2006—Miami Beach, FL, March 3-7 2007—San Diego, CA, February 23-27 2008—Philadelphia, PA, March 14-18 2009—Washington, DC, March 13-17 2010—New Orleans, LA, February 26-March 2

d d

AAAAI WEB SITE RESOURCES Visit the AAAAI Web site, www.aaaai.org, for a variety of professional and patient education resources.

Practice management tools available

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Visit the AAAAI Web site, www.aaaai.org, for easy-to-use resources that provide practice management assistance for established physicians, as well as those just starting out. A separate page will be developed for office managers and other clinical staff that will include specific techniques and tools to support them in their day-to-day work. Current resource center topics include: d d d d

2005 Annual Meeting Plenary Session on CD-ROM

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Did you miss one of the six Plenary Sessions held during the 2005 Annual Meeting? You still have an opportunity to learn about the newest research in the specialty and earn continuing education credits. AAAAI members and nonmembers may order a CD of the six Annual Meeting Plenary Sessions. The Plenary Session CD will offer two types of continuing education credit. Physicians may earn 10 Continuing Medical Education (CME) credits, and nurses may earn 11.9 Continuing Education (CE) contact hours for designated sessions. The six Plenary Sessions are: d d d d

Atopy: Nature and Nurture New Paradigms in Cutaneous Inflammation Optimizing Outcomes for Asthma New Insights in Primary Immune Deficiencies

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New Insights in Food Allergy New Paradigms in Upper and Lower Airways Diseases

d d d d d

2004 Practice Management Workshop presentations Financial performance HIPAA Medical office technology Patient satisfaction Personal/time management Practice startup Recruitment and staffing Resources and sample forms

AAAAI practice management resources are located in the Members Center of the AAAAI Web site. Visit the Members Center often, additional practice management information is added continuously.

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AAAAI mission statement

The mission of the American Academy of Allergy, Asthma & Immunology is the advancement of knowledge and practice of allergy, asthma and immunology for optimal patient care. For more information about the AAAAI, contact the executive office, 555 E. Wells Street, Suite 1100, Milwaukee, WI 532023823; phone (414) 272-6071; fax (414) 272-6070; e-mail [email protected]; or visit the Web site, www.aaaai.org.

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cmeACTIVITIES activities CME calendar CALENDAR CME Mission Statement (Approved by the AAAAI’s CME Committee, March 18, 2001. Approved by Board of Directors, June 23, 2001.) The purpose of the AAAAI’s CME program is to provide educational activities that will stimulate, maintain, develop, and enhance the study and practice of allergy, asthma and immunology. The content areas of the AAAAI’s CME program include any topic within the field of allergy, asthma and immunology which impacts the study or practice of the specialty. The target audience for the AAAAI’s CME program includes allergists, immunologists, specialty and primary care physicians, and allied health professionals. The types of activities included as part of the AAAAI’s CME program include a national Annual Meeting; regional, local, and other live conferences; computer-assisted interactive activities; and an array of written, audio, video and multimedia enduring materials. The AAAAI supports the concept of jointly sponsoring activities with organizations whose goals are compatible with those listed in the Mission Statement. The expected results of the AAAAI’s CME program are to advance the science and practice within the specialty of allergy, asthma and immunology.

AAAAI SPONSORED ENDURING MATERIALS The activities listed below are directly sponsored by the AAAAI and are available in print form or on the AAAAI Web site, www.aaaai.org, as indicated by the descriptions below. Participants may review the materials at their leisure for credit; no attendance is necessary. CME credit will be awarded upon completion of each activity’s evaluation or post test. More specific instructions for claiming credits are included in the individual enduring material. Allergy-Immunology Medical Knowledge SelfAssessment Program Sponsored by the AAAAI, in partnership with the American College of Physicians (ACP). Funded through an unrestricted educational grant from sanofi aventis. For more information, e-mail [email protected]. Credits available: 50.0 CME. 2003 Postgraduate Plenary Session Webcast Allergy, Asthma & Immunology: 60 Years of Progress Sponsored by the AAAAI Location: www.aaaai.org

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More information: e-mail [email protected] Credits available: 2.5 CME Dinner Symposium 3906 The Suspension is Over: New Solutions for the Treatment of Asthma Sponsored by the AAAAI and funded through an unrestricted educational grant from sanofi aventis. Location: www.aaaai.org More information: e-mail [email protected] Credits available: 1.5 CME Dinner Symposium 4906 Webcast A Look to the Future: The Management of Allergic Diseases Sponsored by the AAAAI and funded through an unrestricted educational grant from sanofi aventis. Location: www.aaaai.org More information: e-mail [email protected] Credits available: 2.0 CME The JACI monthly review articles: Current Reviews of Allergy and Clinical Immunology Molecular Mechanisms in Allergy and Clinical Immunology Locations: JACI and online at www.mosby.com\jaci More information: e-mail [email protected] Credits available: 1.0 CME Respiratory Digest articles Jointly sponsored by AAAAI and Adelphi, Inc., and funded through an unrestricted educational grant from sanofi aventis. For more information, contact Joan Weiss at Adelphi, Inc. by phone at (646) 602-7060, Fax (646) 602-6071, or e-mail [email protected]. Credits available: 1.0 CME Respiratory Digest, Volume 3, Issue 3 Clinical Implications of the Allergic Rhinitis/Asthma Connection by Thomas B. Casale, MD, FAAAAI Respiratory Digest, Volume 3, Issue 4 Sleep-Disordered Breathing in Adults by Philip L. Smith, MD, and Alan R. Schwartz, MD Respiratory Digest, Volume 4, Issue 1 Evaluation of the Solitary Pulmonary Nodule by E. James Britt, MD

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Executive Office 555 East Wells Street Suite 1100 Milwaukee, WI 53202-3823 (414) 272-6071 www.aaaai.org Respiratory Digest, Volume 4, Issue 2 Management of Exercise-Induced Asthma by John M. Weiler, MD, and Julie A. Grant, MSPA-C Respiratory Digest, Volume 4, Issue 3 Treatment of Severe Asthma by Stephen P. Peters, MD, PhD Respiratory Digest, Volume 4, Issue 4 Management of Exacerbations of Chronic Bronchitis by Victor M. Pinto-Plata, MD, and Bartolome R. Celli, MD Respiratory Digest, Volume 5, Issue 1 Allergic Rhinitis and Its Comorbidities by Eli O. Meltzer, MD, FAAAAI Respiratory Digest, Volume 5, Issue 2 Managing Antihistamine Impairment in Allergic Rhinitis by Thomas B. Casale, MD, FAAAAI

J ALLERGY CLIN IMMUNOL

Respiratory Digest Special Report 1 (2003) A Look to the Future: The Management of Allergic Diseases by Thomas B. Casale, MD, FAAAAI, and Erwin W. Gelfand, MD, FAAAAI

Emerging Trends in the Management of Asthma and Other Allergic Diseases: A Focus on Anti-IgE Therapy Jointly sponsored by AAAAI and Synermed More information: E-mail [email protected] Credits available: 1.0 CME

The Clinical Benefits of Anti-Ige Therapy: A Targeted Treatment for Patients with Asthma and Other Allergic Airway Diseases Jointly sponsored by AAAAI and Synermed More information: E-mail [email protected] Credits available: 1.0 CME

August 2005 Page 31A

T HE J OURNAL OF

Allergy Clinical Immunology AND

INFORMATION FOR CATEGORY 1 CME CREDIT Credit can now be obtained, free for a limited time, by reading the review articles in this issue. Please note the following instructions. Method of Physician Participation in Learning Process: The core material for these activities can be read in this issue of the Journal or online at the JACI Web site: www.mosby.com/jaci. The accompanying tests may only be submitted online at www.mosby.com/jaci. Fax or other copies will not be accepted. Date of Original Release: August 2005. Credit may be obtained for these courses until July 31, 2006. Copyright Statement: Copyright Ó 2005-2006. All rights reserved. Overall Purpose/Goal: To provide excellent reviews on key aspects of allergic disease to those who research, treat, or manage allergic disease. Target Audience: Physicians and researchers within the field of allergic disease. Accreditation/Provider Statements and Credit Designation: The American Academy of Allergy, Asthma and Immunology (AAAAI) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. The AAAAI designates these educational activities for up to 1.0 hour in category 1 credit toward the AMA Physician’s Recognition Award. Each physician should claim only those hours of credit that he or she actually spent in the educational activity.

CME article

CME article

‘‘Innate immune responses to infection’’ (page 241)

‘‘EBV the prototypical human tumor virus—just how bad is it?’’ (page 251)

List of Design Committee Members: Author: Michael F. Tosi, MD Activity Objectives 1. To achieve a greater and more current understanding of the nature and scope of innate immune responses to infection. 2. To develop an enhanced appreciation for the range of interactions among various components of innate immunity. 3. To be able to appreciate the specific microbial targets of specific effector mechanisms of innate immunity. Recognition of Commercial Support: This CME activity has not received external commercial support. Disclosure of Significant Relationships with Relevant Commercial Companies/Organizations: Michael F. Tosi has no significant relationships to disclose.

List of Design Committee Members: Author: David A. Thorley-Lawson, PhD Activity Objectives 1. To understand that EBV uses mature B cell biology to establish latency, persist, and replicate. 2. To understand that even though EBV is so widespread and apparently benign, it is potentially life-threatening. 3. To understand that EBV evolved the capacity to make cells grow because it is an essential part of the mechanism for establishing latency in resting cells that are not pathogenic. 4. To understand that EBV-associated tumors arise from different stages in the life cycle of latently infected B cells and that disruption of the immune response is an important component in the development of all of the EBV-associated lymphomas. Recognition of Commercial Support: This CME activity has not received external commercial support. Disclosure of Significant Relationships with Relevant Commercial Companies/Organizations: David A. Thorley-Lawson has equity ownership in EBVax.

32A August 2005

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The Editors’ Choice Donald Y.M. Leung, MD, PhD Harold S. Nelson, MD Stanley J. Szefler, MD

Ciclesonide and allergen-induced asthmatic responses New-generation inhaled steroids having low systemic effects are being sought for the treatment of asthma. Ciclesonide (Alvesco) is a once-daily, nonhalogenated inhaled corticosteroid (ICS) that has recently been introduced into the United Kingdom and Germany for the treatment of persistent asthma at doses of 80 lg and 160 lg. This ICS remains inactive until cleaved by esterases present in the airway, where its active metabolite, desisobutyryl-ciclesonide, then binds glucocorticoid receptors. In this issue of the Journal, Gauvreau et al (p 285) have investigated the effects of ciclesonide in a multi-center, randomized, crossover study, comparing

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once-daily dosing of 40 lg, 80 lg, and placebo on allergeninduced airway responses of individuals with mild asthma. This study demonstrates that ciclesonide 80 lg attenuates the allergen-induced early and late changes in FEV1 as well as serum eosinophil cationic protein and sputum eosinophils measured at 24 hours after challenge (P < .025), whereas ciclesonide 40 lg attenuates the late asthmatic responses and sputum eosinophils measured at 24 hours after challenge (P < .025). This study provides new information regarding the minimally effective doses of inhaled ciclesonide for inhibition of allergen-induced airway responses and the apparent local anti-inflammatory effects on the airways. Further evaluation of ciclesonide will be required to address whether these low doses are clinically effective.

Wheezing in infants—associated with ‘‘stop-and-go’’ traffic? Recent research has suggested a possible link between diesel exhaust particulates (DEP) and respiratory and allergic diseases. In this issue of the Journal, Ryan et al (p 279) describe the association between wheezing in infants less than 1 year of age and exposure to stop-and-go truck and bus traffic. The investigators observed a dramatic increase in wheezing in infants who reside less than 100 m from stop-and-go truck and bus traffic compared with infants who reside farther from all sources of DEP. In addition, African American infants had the highest prevalence of wheezing in comparison with Caucasian infants, regardless of exposure category. These findings suggest that living very close to stop-and-go traffic early in infancy is a significant risk factor for wheezing. It also suggests that even within an urban environment, an infant’s risk for wheezing varies with exposure to different types and

Endotoxin and the pathway to allergy Endotoxin, a part of the cell wall of gram-negative bacteria, is present at higher levels in households with large animals (livestock or dogs). Infant endotoxin exposure has been proposed as a factor that might protect against allergy and early childhood immune responses (eg, production of the cytokine IL-13) that increase IgE production to allergens. Cross-sectional European studies have found that elevated endotoxin levels are associated with allergy protection in children of farmers, but the immunologic pathway to explain this association is uncertain and the relevance of the finding to children in the more urban US setting is unclear. In this issue of the Journal, Abraham

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Prevalence of wheeze (without cold) by distance from DEP source. Moving exposure, residing within 400 m of a highway with >1000 trucks daily; Stop-and-go exposure, residing within 100 m of either a bus route or an urban state route.

amounts of traffic. This study is the first report from the ongoing Cincinnati Childhood Allergy and Air Pollution Study, the goal of which is to elucidate the environmental and genetic contributions to the development of allergic diseases. et al. (p 431) assessed in a cohort of US children household dust endotoxin at age 2-3 months and PBMC-proliferative and cytokine responses to cockroach, dust mite, and cat allergens and to the nonspecific mitogen phytohemagglutinin at age 2-3 years. They found that increased endotoxin levels were associated with decreased IL-13 in response to cockroach, dust mite, and cat allergen but not in response to mitogen stimulation. An inverse, though nonsignificant, association was found between endotoxin and proliferative responses. Early-life endotoxinrelated reduction of IL-13 production might represent one pathway through which elevated endotoxin decreases the risk of allergic disease and allergy in later childhood.

August 2005 Page 239

Cholinergic urticaria patients: Sometimes allergic to their own sweat Cholinergic urticaria (CU) presents a characteristic picture of pinpoint-sized, highly pruritic wheals with surrounding erythema that occurs after sweating during physical exercise, bathing, or emotional stress. The pathogenesis of CU is not well defined. However, it has been reported that these patients have positive intradermal skin tests to their own sweat. In this issue of the Journal, Dr Fukunaga and colleagues (p 397) report on this sensitization in 18 subjects with CU and 10 controls. They performed intradermal skin testing and basophil histamine

release with autologous sweat and serum. Eleven of 17 patients with CU had positive skin test results, and 10 of 17 had basophil histamine release with autologous sweat, with a significant correlation between the 2 responses. Nine of 16 of the patients with CU had a positive intradermal skin test result with autologous serum. The authors proposed that there are 2 distinct subtypes of patients with CU: (a) those who have strong reactions to autologous sweat and negative reactions to autologous serum whose wheals are not associated with hair follicles and (b) those characterized by weak reactions to autologous sweat and positive reactions to autologous serum whose wheals are associated with hair follicles.

Sensitization to aeroallergens in the US population The National Health and Nutrition Examination Survey (NHANES) is a population-based survey undertaken periodically by the National Center for Health Statistics to determine the health and nutritional status of the US population. Prick/puncture skin testing (PPST) was performed in a subset of subjects to 8 aeroallergens in NHANES II (1976-80) and to 9 aeroallergens and peanuts in NHANES III (1988-94). The results of the skin testing in NHANES III are reported by Dr Arbes and colleagues in this issue of the Journal (p 377). Of 10,508 skin-tested subjects aged 6 to 59 years, more than half had at least 1 positive PPST result to the 9 aeroallergens. Those skin tests most commonly positive were to dust mite (positive in 27.5% of subjects), rye grass (in 26.9%), short ragweed (in 26.2%), and German cockroach (in 26.1%). The remainder— Bermuda grass, Russian thistle, white oak, cat, and Alternaria alternata—were positive in 10% to 20% of subjects. The least commonly positive skin test was to peanut (8.6%). The 3 most significant independent predictors of a positive PPST result to aeroallergens were age (maximal for the 20- to 29-years age group), male sex, and minority ethnicity, especially non-Hispanic black.

COX-2 inhibitors enhance allergic responses Mechanical injury to the skin by scratching is an important feature of atopic dermatitis (AD). In this issue of the Journal, Laouini et al (p 390) show that mechanical injury to mouse skin inflicted by tape stripping results in rapid induction of cyclooxygenase-2 (COX-2) mRNA and protein and in accumulation of the COX-2 product prostaglandin E2 (PGE2). The role of COX-2 was examined in a mouse model of AD elicited by repeated epicutaneous (EC) sensitization with ovalbumin (OVA) and characterized by eosinophil skin infiltration and a systemic TH2 response to

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Distribution of positive skin tests to 9 aeroallergens and peanuts in the US population, age 6 to 59 years.

Comparison of these results with the results of the skin testing performed in NHANES II was difficult because of numerous methodologic differences between the 2 studies. However, for the 6 aeroallergens tested in both surveys, a positive PPST result was 2.1 to 5.5 times more common in NHANES III than in NHANES II. Although the authors could not definitely conclude that these differences represent an increased sensitivity in the US population, they point out that such an increase would be consistent with reports from other countries. antigen. Administration of the COX-2 selective inhibitor NS-398 during EC sensitization resulted in enhanced skin infiltration by eosinophils and expression of IL-4 mRNA, enhanced OVA-specific IgE and IgG1 antibody responses, and increased IL-4 secretion by splenocytes following OVA stimulation. COX-2–deficient mice also exhibited an enhanced systemic TH2 response to EC sensitization. These results demonstrate that COX-2 products limit allergic skin inflammation, and they are consistent with the recent finding that engagement of the EP3 receptor by PGE2 inhibits allergic reactions (Nat Immunol 2005;6:524-31). More importantly, the work of Laouini et al suggests that COX-2 inhibitors might worsen allergic skin inflammation and should be avoided in patients with AD.

J ALLERGY CLIN IMMUNOL

Reviews and feature articles

Current reviews of allergy and clinical immunology Series editor: Harold S. Nelson, MD

Innate immune responses to infection Michael F. Tosi, MD New York, NY This activity is available for CME credit. See page 32A for important information.

The human host survives many infectious challenges in the absence of preexisting specific (adaptive) immunity because of the existence of a separate set of protective mechanisms that do not depend on specific antigenic recognition. These antigenindependent mechanisms constitute innate immunity. Antimicrobial peptides are released at epithelial surfaces and disrupt the membranes of many microbial pathogens. Toll-like receptors on epithelial cells and leukocytes recognize a range of microbial molecular patterns and generate intracellular signals for activation of a range of host responses. Cytokines released from leukocytes and other cells exhibit a vast array of regulatory functions in both adaptive and innate immunity. Chemokines released from infected tissues recruit diverse populations of leukocytes that express distinct chemokine receptors. Natural killer cells recognize and bind virus-infected host cells and tumor cells and induce their apoptosis. Complement, through the alternative and mannose-binding lectin pathways, mediates antibody-independent opsonization, phagocyte recruitment, and microbial lysis. Phagocytes migrate from the microcirculation into infected tissue and ingest and kill invading microbes. These innate immune mechanisms and their interactions in defense against infection provide the host with the time needed to mobilize the more slowly developing mechanisms of adaptive immunity, which might protect against subsequent challenges. (J Allergy Clin Immunol 2005;116:241-9.) Key words: Innate immunity, antimicrobial peptides, Toll-like receptors, chemokines, natural killer cells, complement, phagocytes

It is traditional to organize host responses to infection into separate arms or compartments, such as complement, phagocytes, cytokines, cell-mediated immunity, and humoral immunity. A more current approach has been to consider 2 larger categories: innate immunity, incorporat-

Abbreviations used CXCL: CXC ligand HBD: Human b-defensin ICAM-1: Intercellular adhesion molecule 1 LFA-1: Lymphocyte function-associated antigen 1 MAC: Membrane attack complex Mac-1: Macrophage antigen-1 MBL: Mannan-binding lectin NADPH: Reduced nicotinamide adenine dinucleotide phosphate NF: Nuclear factor NK: Natural killer PMN: Polymorphonuclear leukocyte TLR: Toll-like receptor

ing the more rapid and phylogenetically primitive nonspecific responses to infection, such as surface defenses, cytokine elaboration, complement activation, and phagocytic responses,1 and adaptive immunity, involving more slowly developing, long-lived, and highly evolved antigen-specific protective responses, such as antibody production and cell-mediated immunity, that exhibit extraordinarily diverse ranges of specificity.2,3 However, the components of innate and adaptive immunity engage in a range of interactions that is remarkably diverse and complex. This review attempts to provide an overview of the main innate responses to infection that are available to the human host, including relevant examples of such interactions.

INNATE IMMUNITY From the Department of Pediatrics, Mount Sinai School of Medicine, New York, and the Division of Pediatric Infectious Diseases, Maimonides Medical Center, Brooklyn. Disclosure of potential conflict of interst: M. F. Tosi—none disclosed. Received for publication May 17, 2005; accepted for publication May 18, 2005. Available online July 5, 2005. Reprint requests: Michael F. Tosi, MD, Division of Pediatric Infectious Diseases, Maimonides Medical Center, 977 48th St, Brooklyn, NY 11219. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.036

Epithelia, defensins, and other antimicrobial peptides The epithelium of skin and mucosal tissue functions as a mechanical barrier to the invasion of microbial pathogens. In the last 2 decades, it has become clear that epithelial cells also are a major source of antimicrobial peptides that play important roles in local host defense.4,5 Studies of their structure, sources, expression, and actions also have revealed an unexpected range of immunologic activities for these molecules, the functions of which once were considered mainly antimicrobial in nature.4 241

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Epithelial cells of mucous membranes of the airways and intestines, as well as keratinocytes, express the human b-defensins (HBD-1 through HBD-4). These small cationic peptides are similar to the a-defensins stored in the azurophilic granules of neutrophils, and they display antimicrobial activity against a broad range of bacteria, fungi, chlamydiae, and enveloped viruses.4,5 Their production by epithelial cells might be constitutive, as for HBD-1, or inducible, as for HBD-2, HBD-3, and HBD-4. For example, recent evidence indicates that epithelial cells of the airway or intestine can produce HBD-2 in response to activation by bacterial products through toll-like receptors (TLRs) 2 or 4 (see below) on the epithelial cells.6,7 Stimulation of epithelium by cytokines, including IL-1 or TNF-a, also can induce defensin production.4 Defensins have been reported to exert their antimicrobial action either through the creation of membrane pores or through membrane disruption resulting from electrostatic interaction with the polar head groups of membrane lipids, with more evidence now favoring the latter mechanism.4,8 Some microorganisms have evolved mechanisms for evading the action of defensins. For example, bacterial polysaccharide capsules might limit access of microbial peptides to the cell membrane,9 and an exoprotein of Staphylococcus aureus, staphylokinase, neutralizes the microbicidal action of neutrophil a-defensins.10 Several immunoregulatory properties of defensins and related peptides, distinct from their antimicrobial actions, have been documented.4 Several such peptides have been shown to facilitate posttranslational processing of IL-1b.11 Some of the b-defensins have been shown to function as chemoattractants for neutrophils, memory T cells, and immature dendritic cells by binding to the chemokine receptor CCR-6.5,12,13 Separately, HBD-2 has been shown to activate immature dendritic cells through a mechanism that requires TLR4.14 The activation of immature dendritic cells by these mechanisms also promotes their maturation. The b-defensins also act as a chemoattractant for mast cells through an undefined mechanism and can induce mast cell degranulation.15 HBD-2 and several other antimicrobial peptides can interfere with binding between bacterial LPS and LPS-binding protein.16 Additional antimicrobial peptides of epithelial cells include lysozyme and cathelicidin. Lysozyme, an antimicrobial peptide also found in neutrophil granules, attacks the peptidoglycan cell walls of bacteria and can be released from cells through mechanisms that involve TLR activation.17 Cathelicidin, or LL37, like lysozyme, is released from both neutrophils and epithelial cells. It exhibits broad antimicrobial activity and can inhibit lentiviral replication.5,18 Cathelicidin also exhibits chemotactic activity for neutrophils, monocytes, and T lymphocytes. This activity is mediated by a formyl peptide receptor-like molecule, FPRL1, rather than the chemokine receptor CCR6 bound by b-defensins.19 The release of defensins in response to activation of TLRs and the many actions of these peptides, including their direct antimicrobial activities, their chemoattractant actions for a wide range of immune cells, and their

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activation of dendritic cell maturation, already suggest a highly complex and regulatory role in the development of host defense and immunity. Recent genomic evidence for the possible existence of as many as 25 additional human defensins that have not yet been characterized suggests that current knowledge describes but a small sample of the overall contribution of these peptides to immune responses.20

TLRs Mononuclear phagocytes, including circulating monocytes and tissue macrophages, other phagocytic cells, and many epithelial cells, express a family of receptors that is highly homologous to the Drosophila receptor called Toll.6,7,21 These receptors mediate a phylogenetically primitive, nonclonal mechanism of pathogen recognition based on binding not to specific antigens but to structurally conserved pathogen-associated molecular patterns.21-23 There are at least 10 human TLRs with a range of microbial ligands, such as gram-negative bacterial LPS, bacterial lipoproteins, lipoteichoic acids of gram-positive bacteria, bacterial cell-wall peptidoglycans, cell-wall components of yeast and mycobacteria, unmethylated CpG dinucleotide motifs in bacterial DNA, and viral RNA.22-24 Gram-positive cell-wall components bind mainly to TLR2, and TLR2 also can bind components of herpes simplex virus.22-25 Gram-negative LPS activates TLR4 indirectly by first binding to LPS-binding protein, which binds in turn to CD14 at the cell surface. The bound CD14 has no transmembrane domain but associates directly with an extracellular domain of TLR4.23,24 TLR5 has been identified as the receptor for bacterial flagellin, TLR9 recognizes CpG motifs of bacterial DNA, and TLR3 has been shown to bind synthetic and viral double-stranded RNA.26-28 Signalling through TLRs occurs through a welldescribed pathway in which receptor binding generates a signal through an adaptor molecule, MyD88, that leads to intracellular association with IL-1 receptor-associated kinase. In turn, this leads to activation of TNF receptor– associated factor 6, which results in nuclear translocation of nuclear factor kB (NF-kB).23,24 NF-kB is an important transcription factor that activates the promoters of the genes for a broad range of cytokines and other proinflammatory products, such as TNF-a, IL-1, IL-6, and IL-8. This signalling pathway, on the basis of studies with TLR4, is similar but not identical to the signalling pathways activated by other TLRs.24 The activation of cytokine production by TLRs plays an important role in recruiting other components of innate host defense against bacterial pathogens. However, with large-scale cytokine release, the deleterious effects of sepsis or other forms of the systemic inflammatory response syndrome demonstrate that these pathways have both beneficial and potentially harmful effects for the host.24 Genetic polymorphisms in TLRs might play a role in determining the balance of these effects in certain individuals responding to the challenge of systemic infection.24,29,30

In addition to their first-responder roles in generating an inflammatory response to invading pathogens, TLRs can network with other components of innate and adaptive immunity. TLR4 function is suppressed by activation of cells through the chemokine receptor CXCR4.31 Activation of some TLRs also can induce expression of the costimulatory molecule B7 on antigen-presenting cells, which is required for activation of naive T cells.21

Cytokines A heterogeneous group of soluble small polypeptide or glycoprotein mediators, often collectively called cytokines, form part of a complex network that helps regulate the immune and inflammatory responses. Included in this group of mediators, the molecular weights of which range from about 8 to about 45 kd, are the ILs, IFNs, growth factors, and chemokines (see separately below). Most cells of the immune system, as well as many other host cell types, release cytokines, respond to cytokines through specific cytokine receptors, or both. The range of sources and effects of cytokines and their actions and interrelationships are of such complexity that they cannot be addressed here in detail. A number of them will be addressed individually in sections below, and several excellent reviews are available.32-34 However, 2 cytokines, IL-1 and TNF-a, are of such fundamental importance in acute host responses to infection that they warrant specific attention. IL-1 and TNF-a are small polypeptides, each with a molecular weight of approximately 17 kd, that exhibit a broad range of effects on immunologic responses, inflammation, metabolism, and hematopoiesis.34,35 IL-1 originally was described as ‘‘endogenous pyrogen,’’ referring to its ability to produce fever in experimental animals, and TNF-a, which produces some of the same effects produced by IL-1, was originally named ‘‘cachectin’’ after the wasting syndrome it produced when injected chronically in mice.34,35 Many of the physiologic changes associated with gram-negative sepsis can be reproduced by injecting experimental animals with these cytokines in the absence of microorganisms. Depending on the doses injected, these effects might include fever, hypotension, and either neutrophilia or leukopenia.34,35 In the production of endotoxic shock caused by gram-negative sepsis, IL-1 and TNF-a are produced by mononuclear phagocytes in response to activation of TLRs by bacterial LPS. They in turn activate the production of other cytokines and chemokines, lipid mediators (eg, platelet-activating factor and prostaglandins), and reactive oxygen species. They also induce expression of adhesion molecules of both endothelial cells and leukocytes, stimulating recruitment of leukocytes by inducing release of the chemokine IL-8 and activating neutrophils for phagocytosis, degranulation, and oxidative burst activity.24,35 These all are important and usually beneficial host responses to infection. However, at high levels of activation, there sometimes are pathophysiologic effects of this proinflammatory cascade, including vascular instability, decreased myocardial contractility, capillary leak, tissue hypoperfusion, coagulopathy, and

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multiple organ failure.24 For some systemic actions, notably the production of hemodynamic shock, IL-1 and TNF-a are synergistic. Both IL-1 and TNF-a also induce production of IL-6, a somewhat less potent cytokine that exhibits some of the actions of IL-1 and TNF-a.34 The human host produces several soluble antagonists of IL-1 and TNF-a that can modulate their effects, including IL-1 receptor antagonist, soluble TNF-a receptor, and anti-inflammatory cytokines, especially IL-10.24 The importance of effects mediated by IL-1 and TNF-a in the pathophysiology of septic shock has prompted much active research aimed at blocking their effects to reduce morbidity and mortality. Monoclonal antibodies against TNF-a and other inhibitors of TNF-a or IL-1 have showed early promise in vitro and in animal models of septic shock.24,34,36,37 However, they have been far more effective at preventing the effects of cytokines than reversing them. More recent attempts to address the issue of the timing of intervention have been directed at the intracellular signaling mechanisms activated through the TNF-a receptor or at mediators that appear later than TNF-a. Lipophilic inhibitors of protein tyrosine kinases, enzymes that propagate the cellular signals through TNF-a receptors, have been found to enhance survival in experimental animals, even when administered 2 hours after systemic injection with endotoxin.38 Additionally, mAbs against a cytokine-like nonhistone nucleoprotein product of macrophages, high-mobility group B1 (which appears much later than TNF-a or IL-1 after LPS stimulation), were found to rescue mice from endotoxin shock when given 2 hours after an otherwise lethal dose of LPS.39 More recently, clinical trials with activated protein C, a regulatory protein in the coagulation cascade, has demonstrated beneficial effects in selected patients with septic shock through mechanisms that might involve inhibition of NF-kB activation.40 To date, despite progress, clinical strategies to interfere with the cytokine-induced cascade that leads to endotoxin-induced shock have continued, overall, to meet with limited success.

Chemokines A specialized group of small cytokine-like polypeptides, chemokines, which all share the feature of being ligands for G protein–coupled, 7-transmembrane segment receptors, plays an increasingly complex role in the immune response as cellular activators that induce directed cell migration, mainly of immune and inflammatory cells.41-44 The chemokines and their receptors have been classified into 4 families on the basis of the motif displayed by the first 2 cysteine residues of the respective chemokine peptide sequence. Each of at least 16 CXC chemokines binds to 1 or more of the CXCRs (CXCR1 through CXCR6). Examples of CXC chemokines include IL-8 and Gro-a. Similarly, at least 28 CC chemokines, such as macrophage inflammatory protein 1a, RANTES, and eotaxin-1, 2, and 3, bind to one or more of the CCRs (CCR1 through CCR10). The sole CX3C chemokine, fractalkine, or neurotaxin, binds to CX3CR1, currently the only receptor in its family. The 2 XC chemokines,

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including lymphotaxin, bind to the sole receptor in this family, XCR1. A new nomenclature has been proposed to designate each of the chemokines as a numbered ligand for its respective receptor family. In this system Gro-a is CXC ligand (CXCL) 1 (or CXCL-1), and IL-8 now becomes CXCL-8. Similarly, RANTES becomes CCL-5, fractalkine is CX3CL-1, and lymphotactin is XCL-1.41,43 An update of this nomenclature system recently has been published, tabulating each of the families with their respective ligands and receptors, as well as the traditional names in both human and murine systems.43 Virtually every cell type of the immune system expresses receptors for one or more of the chemokines. The cells of most inflamed tissues can release a variety of chemokines, and tissues infected with different bacteria or viruses release chemokines that recruit characteristic sets of immune cells.44,45 For example, whereas rhinoviruses induce the release of chemokines that result mainly in recruitment of neutrophils (early in the course of infection), EBV induces a set of chemokines that result in recruitment of B cells, natural killer (NK) cells, and both CD41 and CD81 T cells.45 It is of interest that almost mutually exclusive sets of chemokines are induced by cytokines associated with TH1 (IL-12 and IFN-g) versus TH2 (IL-4 and IL-13) immune responses, indicating a tight interplay between cytokines and chemokines in determining the type of immune response to specific infectious challenges generated under different conditions.46 The specificity of such cellular responses is strongly influenced by the chemokines released from specific tissues, the vascular adhesion molecules expressed in those tissues, the chemokine receptors expressed by different populations of leukocytes, and the specific adhesion molecules expressed by leukocytes.44-46 Modulation of chemokine function can occur through several mechanisms. Chemokines themselves can be potentiated or inactivated by tissue proteases, including tissue peptidases and matrix metalloproteases.47 Heparin sulfate–related proteoglycans on endothelial cell surfaces tether chemokines locally, where they can most efficiently activate circulating leukocytes for adhesion (see below). However, similar proteoglycans free in the extracellular environment can bind and sequester chemokines, keeping them from interacting with their cellular receptors.48,49 Finally, in addition to the well-described use of chemokine receptors as coreceptors for viral entry by HIV-1, other viruses, especially members of the herpesvirus family, encode soluble decoy receptors that compete with native host receptors for chemokine binding, thereby disrupting normal host responses.49,50

NK cells NK cells are an important cellular feature of innate immunity. They are lymphoid cells that do not express clonally distributed receptors, such as T-cell receptors or surface immunoglobulin, for specific antigens.51 They respond in an antigen-independent manner to help contain viral infections before the development of adaptive immune responses, and they aid in the control of malignant

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tumors. NK cells are found in the peripheral circulation and in the spleen and bone marrow. Like many other leukocytes, they can be recruited to sites of inflammation by chemokines and other chemoattractants. They appear to be important for the control of tumors in vivo and serve a critical function in host defense against viral infections, especially those caused by members of the herpesvirus family.51,52 Activated NK cells also are an important source of IFN-g, which limits tumor angiogenesis and promotes the development of specific protective immune responses.51,52 Regulation of NK cell activity involves a balance between activating and inhibitory signals. Several cytokines can activate NK cell proliferation, cytotoxicity, or IFN-g production, including IL-12, IL-15, IL-18, IL-21, and IFN-ab.51 Activating signals through other receptors on NK cells, such as NKG2D, can lead to cytotoxicity, cytokine production, or both, depending on the receptor’s association with distinct intracellular adaptor proteins that signal through different kinases.51,53 Other molecules on NK cells can act as either costimulatory or adhesion receptors, including CD27, CD28, CD154 (CD40 ligand), and lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18).51-53 Additionally, FcgRIII (CD16) can contribute to NK cell cytotoxic activity through mechanisms that include antibody-dependent cell cytotoxicity.51 NK cells are able to distinguish normal cells of self origin through receptors that recognize specific MHC class I molecules. Activation of such receptors provides an inhibitory signal that protects healthy host cells from NK cell–mediated lysis. Virus-infected cells and malignant cells often express MHC class I molecules at reduced levels and thus are less able to generate inhibitory NK cell signals, rendering them more susceptible to attack by NK cells.51,52 NK cell inhibitory receptors, which are not well characterized, appear to contain intracytoplasmic tyrosine-based inhibition motifs and antagonize NK cell activation pathways through protein tyrosine phosphatases.51,54 Thus the regulation of the phosphorylation state of specific tyrosine residues by activating kinases and inhibitory phosphatases appears to be a pivotal determinant of NK cell activation. NK cells kill infected or malignant cells through the release of perforin and granzymes from granular storage compartments and through binding of the death receptors Fas and TRAIL-R on target cells through their respective NK cell ligands.51,55 The mechanisms by which perforin and granzymes mediate target cell death are not fully understood. The best available evidence suggests that perforin and one or more of 5 human granzymes released along with perforin from cytotoxic granules of NK cells associate with the cell membranes of target cells, either by binding through the mannose 6-phosphate receptor or through another mechanism that remains to be defined. One or more of the granzymes appears to activate intracellular pathways leading to target apoptosis through pathways that involve the mitochondria, caspases, or both.56 Separately, binding of the death receptors also activates caspases, causing target cell apoptosis.51 It is

notable that although some tumor cells do not express Fas, NK cells can induce Fas expression on these targets by releasing IFN-g and then proceed to kill them by binding to the newly expressed Fas.51 NK cells engage in several kinds of interactions with other cells of the immune system, including dendritic cells and other antigen-presenting cells. Dendritic cells can influence the proliferation and activation of NK cells both through release of cytokines, including IL-12, and through cell-surface interactions, including CD40/CD40 ligand, LFA-1/intercellular adhesion molecule 1 (ICAM-1), and CD27/CD70.57 In return, NK cells can provide signals that result in either dendritic cell maturation or apoptosis.51,52

The complement system The complement system is made up of at least 30 proteins in serum or at cell surfaces. Most of these proteins are made in the liver or, to a lesser extent, by mononuclear phagocytes. Activation of the complement cascade by one or more of 3 distinct pathways leads to the evolution of its main effector functions: microbial opsonization, phagocyte recruitment, and bacteriolysis. All 3 activation pathways act at a microbial surface to assemble a convertase that cleaves C3 to form C3b, which in turn binds to the target surface, either as an opsonin or to help activate C5 and the remainder of the cascade.58-61 Complement activation pathways. The classical pathway ordinarily is activated by IgG or IgM bound to microbial surface antigens. Antigen binding makes a site on the Fc portion of the antibody available to bind C1q, which in turn binds C1r and C1s. C1qrs catalyzes the cleavage and binding of C4 and C2, in the form of C4b2a, the classical pathway C3 convertase. This convertase then cleaves C3 to form C3a, which is released, and C3b, which remains bound on the target surface, and the cascade beyond C3 continues.59 The recently characterized mannan-binding lectin (MBL) pathway is similar to the classical pathway but does not involve antibodies.60 MBL is a serum protein of the collectin family with structural and functional similarities to C1q and binds to mannose-containing carbohydrates on microbial surfaces. Subsequently, MBL and the MBL-associated serine proteases, which have structural and functional similarities to C1r and C1s, form a complex that activates C4, with sequential binding of C4b and C2a and formation of C4b2a, the classical pathway C3 convertase.60 C3 is activated, and the cascade proceeds as described. The alternative pathway is vital to the host as a separate means by which C3 can be activated before development of specific antibodies.59,61 A low constitutive level of hydrolysis of the thioester of C3 in the fluid phase produces an activated form of C3, C3(H2O). This activated form of C3 can bind factor B, and the latter is cleaved by factor D to form the fluid-phase C3 convertase C3(H2O)Bb. The constitutive presence of small amounts of this convertase in the fluid phase ensures that some C3b always is available to initiate the alternative pathway at microbial surfaces.59,61 Factor B binds to surface

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C3b and undergoes proteolytic cleavage by factor D to form C3bBb, the alternative pathway C3 convertase. Properdin stabilizes the convertase, which produces more C3b, establishing the C3 amplification loop of the alternative pathway and activating the remainder of the cascade.59 Microbes that bear large amounts of surface sialic acid are usually poor activators of the alternative pathway because factor H outcompetes factor B for C3b binding at such surfaces.59,61 No convertase or amplification loop is created because factor H allows C3b cleavage by factor I to form iC3b, the only function of which is opsonic.59 Nonactivators of the alternative pathway are some of the most successful pathogens in nonimmune hosts. They include K1 Escherichia coli, groups A and B streptococci, Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b, and some salmonellae.62 Complement effector functions. Opsonization (from the Greek ‘‘to cater or prepare’’) facilitates the removal of microorganisms from the circulation by macrophages in the liver and spleen and from tissue sites by neutrophils and tissue macrophages.58,59 Recognition and attachment of surface-bound C3b and iC3b on microbes by the type 1 and type 3 phagocytic complement receptors, CR1 (CD35) and CR3 (CD11b/CD18), respectively, activates ingestion and intracellular killing of the organisms.59 Antibodies, themselves important opsonins, direct the localization of C3b binding on microorganisms through the classical pathway. This is important for encapsulated bacteria because the capsule’s presence as a barrier means that only C3b bound at the capsular surface by specific anticapsular antibody will be accessible to phagocyte receptors.63 The free cleavage fragments of C3 and C5 can promote host inflammatory responses. C3a stimulates marrow release of granulocytes, and C5a serves as a potent chemoattractant for monocytes, neutrophils, and eosinophils. C5a also stimulates expression of CR1 and CR3, aggregation, and microbicidal activity of phagocytes. C5a-induced neutrophil aggregation and stasis in the pulmonary circulation can contribute to the respiratory distress syndrome associated with sepsis.64 C4a, C3a, and especially C5a are anaphylotoxins that can induce release of histamine from mast cells and basophils, causing increased vascular dilatation and permeability.64 In large amounts, they can contribute to the pathophysiology of septic shock.64 When C5a is released by the classical or alternative pathway C5 convertase, C5b is bound at the target surface. The terminal complement proteins C5b, C6, C7, C8, and C9 assemble sequentially to form the membrane attack complex (MAC), which can kill and lyse target cells, especially gram-negative bacteria, by penetrating their outer membranes.59,61 The C5b-C8 complex serves as a polymerization site for several molecules of C9.61,65 Although C9 is not essential to membrane penetration, its presence as poly-C9 allows it to proceed more efficiently.59,65 The MAC also can lyse certain virus-infected host cells and some enveloped viruses directly.66

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Complement fragments can modulate other parts of the immune response, both directly by binding to CR1, CR2, and CR3 on the surfaces of T cells, B cells, and other cells involved in antigen recognition and indirectly by stimulating the synthesis and release of cytokines.67 For example, the C3b cleavage product, C3dg, when covalently bound to antigen, brings the antigen close to B cells by binding to B-cell CR2 (CD21); C3 influences antigen localization within germinal centers, anamnestic responses, and isotype switching; and C1, C2, C4, and C3 are important for normal antibody responses.68,69

Phagocytes The first recognized cellular mechanism of host defense was the accumulation of phagocytic host cells around a foreign body in starfish observed by Metchnikoff.70 Polymorphonuclear leukocytes (PMNs), the most abundant circulating phagocytes in the human host, will serve as a model for discussing phagocyte functions. These cells constitute a major line of defense against invading bacterial and fungi. The proliferation of myeloid marrow progenitors and their differentiation into mature progeny are regulated by specific growth factors and cytokines.71,72 The normal half-life of circulating PMNs is approximately 8 to 12 hours.73 In the absence of active infection, most PMNs leave the circulation through the gingival crevices and the lower gastrointestinal tract, where the resident flora stimulate ongoing local extravasation of PMNs, a process that helps maintain the integrity of these tissues.74 In response to invasive bacterial infection, circulating PMNs engage in 3 major functions: (1) migration to the site of infection, (2) recognition and ingestion of invading microorganisms, and (3) killing and digestion of these organisms. Phagocyte recruitment to infected sites. Activation of endothelial cells that line the microvessels of acutely infected tissue occurs through locally produced cytokines, eicosanoid compounds, and microbial products.75 As a result, the endothelial cells rapidly upregulate their surface expression of P-selectin and then E-selectin.75,76 These selectins engage in lectin-like interactions with the fucosylated tetrasaccharide moiety sialyl Lewis X, which is presented on constitutively expressed glycoproteins on PMNs, including L-selectin and P-selectin glycoprotein ligand 1.76 These early interactions slow the PMNs in this first adhesive phase of leukocyte recruitment, sometimes described as ‘‘slow rolling.’’75,77 Within several hours, newly synthesized ICAM-1 is expressed at the endothelial surface.75,77,78 The slowly rolling PMNs are activated by transient selectin-mediated interactions and locally produced mediators, especially endothelium-derived chemokines, such as IL-8.77 These chemokines are most effective in activating the PMNs when they are bound by complex proteoglycans at the endothelial cell surface.48 The activated PMNs then increase the surface expression, binding avidity, or both of the b2-integrins LFA-1 and macrophage antigen-1 (Mac-1) that interact with endothelial cell ICAM-1 in this second, firm adhesion phase mediated by integrin-ICAM interactions, which is also

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necessary for transendothelial migration of the PMNs.75,77,79,80 Other chemoattractants, such as C5a, N-formyl bacterial oligopeptides, and leukotrienes (eg, leukotriene B4) that diffuse from the site of infection further activate PMNs and provide a chemotactic gradient for PMN migration into tissue.41,80,81 The receptors for these chemoattractants, like the chemokine receptors, are G protein–coupled and share a 7-transmembrane domain structure.41,81 They constitute important sensory mechanisms of the PMNs for activating adhesion, directional orientation, and the contractile protein–dependent lateral movement of adhesion sites in the PMN membrane necessary for migration.41,81-83 Although the specific stimuli and adhesion molecules might vary, this general scheme applies to the local recruitment of virtually all circulating cells of the immune system.44-46 Phagocytosis. After PMNs reach the site of infection, they must recognize and ingest, or phagocytose, the invading bacteria. Opsonization, especially with IgG and fragments of C3, greatly enhances phagocytosis.58,63 Although nonopsonic phagocytosis can occur, only opsonin-mediated phagocytosis is considered here. CR1 and CR3 are the main phagocytic receptors for opsonic C3b and iC3b, respectively.79 When PMNs are activated by chemoattractants or other stimuli, CR1 and CR3 are rapidly translocated to the cell surface from intracellular storage compartments, increasing surface expression up to 10-fold.79 Note that CR3 is identical to the adhesionmediating integrin Mac-1.79 CR1 and CR3 act synergistically with receptors for the Fc portion of antibodies, especially IgG.58,59 Phagocytic cells can express up to 3 different types of IgG Fc receptors, or FcgRs, all of which can mediate phagocytosis.84 FcgRI (CD64) is a highaffinity receptor that is expressed mainly on mononuclear phagocytes.84 The 2 FcgRs ordinarily expressed on circulating PMNs are FcgRII (CD32) and FcgRIII (CD16).84 FcgRII is conventionally anchored in the cell membrane, exhibits polymorphisms that determine preferences for binding of certain IgG subclasses, and can directly activate PMN oxidative burst activity.84,85 FcgRIII is expressed on PMNs as a glycolipid-anchored protein, although it is anchored conventionally on NK cells and macrophages.84 Most phagocytes also express IgA receptors. The best characterized, CD89, binds monomeric IgA and promotes phagocytosis and killing of IgA-opsonized bacteria.86 The engagement of phagocyte receptors with opsonins bound on microbes locally activates cytoskeletal contractile elements, leading to invagination of the membrane at the site of initial engagement and extension of pseudopods around the microbe. The ligation of additional opsoninreceptor pairs leads to engulfment of the microbe within a sealed phagosome.87 This is followed by fusion of the phagosome with lysosomal compartments containing the phagocyte’s array of microbicidal products. Phagocyte microbicidal mechanisms. The microbicidal mechanisms of PMNs usually are categorized as either oxygen dependent or oxygen independent. Oxygendependent microbicidal mechanisms of phagocytes depend on a complex enzyme, reduced nicotinamide adenine

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FIG 1. Innate immunity: responses to first contact. Diagrammed are important host responses to infection that are independent of specific cell-mediated immunity or antibodies. Initial contact between the host and microbes or their products results in a range of activating signals that mobilize both cellular and humoral effectors for attack on their respective microbial targets. Components of the host response are highlighted in blue. MF, Macrophages; AP, alternative pathway; MBLP, mannose-binding lectin pathway.

dinucleotide phosphate (NADPH) oxidase, which converts molecular oxygen (O2) into superoxide anion (O22).88 This enzyme is assembled at the activated cell membrane from 6 or more components that include a cytochrome (a- and b-subunits, designated gp91phox and p22phox, respectively), a flavoprotein, and a quinone, all of which are associated with the cell membrane, and at least 2 cytoplasmic proteins, p47phox and p67phox (‘‘phox’’ refers to phagocyte oxidase), that assemble with the membraneassociated components to form the active enzyme complex.88,89 Each of the main oxidant products derived from NADPH activity and subsequent reactions exhibits microbicidal activity, including the earliest products, O22 and H2O2, which are less potent than the downstream products hypochlorite (OCl2) and chloramines (NH3Cl and RNH2Cl), with chloramines being the most stable.90,91 Oxygen-independent microbicidal activity of PMNs resides mainly in a group of proteins and peptides stored within primary (azurophilic) granules. Lysozyme is contained in both the primary and the secondary (specific) granules of PMNs.92 It cleaves important linkages in the peptidoglycan of bacterial cell walls and can act in concert with the complement MAC.58 The primary granules contain several cationic proteins with important microbicidal activity. A 59-kd protein, bactericidal/permeabilityincreasing protein, is active against only gram-negative bacteria.93 Smaller arginine- and cysteine-rich peptides, the a-defensins, similar to the b-defensins of epithelial cells, are active against a range of bacteria, fungi, chlamydiae, and enveloped viruses.6 Other related molecules include cathelicidin and a group of peptides called p15s.4,94

SUMMARY An overview of most of the main features of innate immunity discussed above, along with some of their important interactions, is diagrammed in Fig 1. Several levels of interaction are depicted, from initial host-pathogen contact, through a variety of activating signals, to the attack by host effector mechanisms on pathogenic targets. Initial contact between the host and microbes or their products might result in viral infection of cells, activation of TLRs on macrophages and epithelial cells, and activation of the alternative pathway or mannose-binding lectin pathway of complement. The resulting activation signals, including cytokines (eg, IL-12, TNF-a, and IL-1), chemokines, and products of the complement cascade mobilize both cellular (NK cells and phagocytes) and humoral (antimicrobial peptides and MAC) effectors that attack their respective microbial targets. REFERENCES 1. Janeway CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197-216. 2. Padlan EA. Anatomy of the antibody molecule. Mol Immunol 1994;31: 169-217. 3. Garcia KC, Teyton L, Wilson IA. Structural basis of T cell recognition. Annu Rev Immunol 1999;17:369-97. 4. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710-20. 5. Oppenheim JJ, Biragyn A, Kwak LW, Yang D. Roles of antimicrobial peptides such as defensins in innate and adaptive immunity. Ann Rheum Dis 2003;62(suppl 2):17-21. 6. Vora P, Youdim A, Thomas LS, Fukata M, Tesfay SY, Lukasek K, et al. Beta-defensin-2 expression is regulated by TLR signaling in intestinal epithelial cells. J Immunol 2004;173:5398-405.

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56. Trapani JA, Smyth MJ. Functional significance of the perforin/granzyme cell death pathway. Nat Rev Immunol 2002;2:735-47. 57. Degli-Esposti MA, Smyth MJ. Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nat Rev Immunol 2005;5: 112-24. 58. Joiner KA, Brown EJ, Frank MM. Complement and bacteria: chemistry and biology in host defense. Annu Rev Immunol 1984;2:461-91. 59. Berger M, Frank MM. The serum complement system. In: Stiehm ER, Ochs HD, Winkelstein JA, editors. Immunologic disorders in infants and children. 5th ed. Philadelphia: Elsevier Saunders; 2004. p. 157-87. 60. Peterson SV, Thiel S, Jensenius JC. The mannan-binding lectin pathway of complement activation: biology and disease association. Mol Immunol 2001;38:133-49. 61. Walport MJ. Complement. First of two parts. N Engl J Med 2001;344: 1058-66. 62. Joiner KA. Complement evasion by bacteria and parasites. Ann Rev Microbiol 1988;42:201-30. 63. Hostetter MK. Serotypic variations among virulent pneumococci in deposition and degradation of covalently bound C3b: implications for phagocytosis and antibody production. J Infect Dis 1986;153:682-93. 64. Hugli TE. Structure and function of the anaphylatoxins. Springer Semin Immunopathol 1984;7:193-220. 65. Bhakdi S, Tranum-Jensen J. Complement lysis: a hole is a hole. Immunol Today 1991;12:318-20. 66. Cooper NR, Nemerow GR. Complement-dependent mechanisms of virus neutralization. In: Ross GD, editor. Immunobiology of the complement system. Orlando (FL): Academic Press; 1986. p. 139-62. 67. Erdei A, Fust G, Gergely J. The role of C3 in the immune response. Immunol Today 1991;12:332-7. 68. Ochs HD, Nonoyama S, Zhu Q, Farrington M, Wedgwood RJ. Regulation of antibody responses: the role of complement and adhesion molecules. Clin Immunol Immunopathol 1993;67(suppl):S33-40. 69. Bottger EC, Bitter-Suermann D. Complement and the regulation of humoral immune responses. Immunol Today 1987;8:261-4. 70. Metchnikoff E. Immunity in the infectious diseases. 1st ed. New York: Macmillan Press; 1905. 71. Bainton DF. Developmental biology of neutrophils and eosinophils. In: Gallin JI, Goldstein IM, Snyderman R, editors. Inflammation: basic principles and clinical correlates. 2nd ed. New York: Raven Press; 1992. p. 303-24. 72. Tenen DG, Hromas R, Licht JD, Zhang DE. Transcription factors, normal myeloid development, and leukemia. Blood 1997;90:489-519. 73. Walker RI, Willemze R. Neutrophil kinetics and the regulation of granulopoiesis. Rev Infect Dis 1980;2:282-92. 74. Anderson DC, Schmalsteig F, Finegold MJ, Hughes BJ, Rothlein R, Miller LJ, et al. The severe and moderate phenotypes of heritable Mac-1, LFA-1, p150, 95 deficiency: Their quantitative definition and relation to leukocyte dysfunction and clinical features. J Infect Dis 1985;152:668-89. 75. Butcher EC. Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity. Cell 1991;67:1033-6. 76. Ley K. The role of selectins in inflammation and disease. Trends Mol Med 2003;9:263-8.

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77. Ley K. Integration of inflammatory signals by rolling neutrophils. Immunol Rev 2002;186:8-18. 78. Smith CW, Marlin SD, Rothlein R, Toman C, Anderson DC. Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J Clin Invest 1989;83:2008-17. 79. Berger M, O’Shea J, Cross AS, Folks TM, Chused TM, Brown EJ, et al. Human neutrophils increase expression of C3bi as well as C3b receptors upon activation. J Clin Invest 1984;74:1566-71. 80. Seo SM, McIntire LV, Smith CW. Effects of IL-8, Gro-alpha, and LTB4 on the adhesive kinetics of LFA-1 and Mac-1 on human neutrophils. Am J Physiol Cell Physiol 2001;281:C1568-78. 81. Gerard C, Gerard N. C5a anaphylatoxin and its seven transmembranesegment receptor. Annu Rev Immunol 1994;12:775-808. 82. Anderson DC, Hughes BJ, Smith CW. Abnormal mobility of neonatal polymorphonuclear leukocytes. Relationship to impaired redistribution of surface adhesion sites by chemotactic factor or colchicine. J Clin Invest 1981;68:863-74. 83. Stossel TP. The mechanical responses of white blood cells. In: Gallin JI, Goldstein IM, Snyderman R, editors. Basic principles and clinical correlates. New York: Raven Press; 1992. p. 459-75. 84. Unkeless JC, Shen Z, Lin CW, DeBeus E. Function of human Fc gamma RIIA and Fc gamma RIIIB. Semin Immunol 1995;7:37-44. 85. van der Pol WL, van de Winkel JGJ. IgG receptor polymorphisms: risk factors for disease. Immunogenetics 1998;48:222-32. 86. Hostoffer RW, Krukovets I, Berger M. Enhancement by tumor necrosis factor-a of Fca receptor expression and IgA-mediated superoxide generation and killing of Pseudomonas aeruginosa by polymorphonuclear leukocytes. J Infect Dis 1994;170:82-7. 87. Stossel TP. Phagocytosis. Prog Clin Biol Res 1977;13:87-102. 88. Clark RA, Leidal KG, Pearson DW, Nauseef WM. NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate activatable superoxide-generating system. J Biol Chem 1987;262:4065-74. 89. Borregaard N, Heiple JM, Simons ER, Clark RA. Subcellular localization of the b cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol 1983;97:52-61. 90. Root RK, Cohen MS. The microbicidal mechanisms of human neutrophils and eosinophils. Rev Infect Dis 1981;3:565-98. 91. Tosi MF. Immunologic and phagocytic responses to infection. In: Feigin RD, Cherry JD, Demmler GJ, Kaplan S, editors. Textbook of pediatric infectious diseases. 5th ed. New York: WB Saunders; 2004. p. 20-62. 92. Spitznagel JK, Dalldorf FG, Leffell MS, Folds JD, Welsh IR, Cooney MH, et al. Character of azurophil and specific granules purified from human polymorphonuclear leukocytes. Lab Invest 1974;30:774-85. 93. Weiss J, Victor M, Elsbach P. Role of charge and hydrophobic interactions in the action of the bactericidal/permeability-increasing protein of neutrophils on gram-negative bacteria. J Clin Invest 1983; 71:540-9. 94. Levy O, Weiss J, Zarember K, Ooi CE, Elsbach P. Antibacterial 15-kDa protein isoforms (p15s) are members of a novel family of leukocyte proteins. J Biol Chem 1993;268:6058-63.

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Continuing Medical Education examination

Innate immune responses to infection Instructions for category 1 Continuing Medical Education credit The American Academy of Allergy, Asthma and Immunology is accredited as a provider of Continuing Medical Education (CME) by the Accreditation Council for Continuing Medical Education. Test ID no.: mai0063 Contact hours: 1.0 Expiration date: July 31, 2006 Category 1 credit can be earned by reading the text material and taking this CME examination online. For complete instructions, visit the Journal’s Web site at www.mosby.com/jaci.

Learning objectives: ‘‘Innate immune responses to infection’’ 1. To achieve a greater and more current understanding of the nature and scope of innate immune responses to infection. 2. To develop an enhanced appreciation for the range of interactions among various components of innate immunity. 3. To be able to appreciate the specific microbial targets of specific effector mechanisms of innate immunity.

CME items Question 1. The main host defense functions of C5a and the membrane attack complex of the complement system are most closely reproduced by — A. TNF-a and phagocytes. B. chemokines and defensins. C. Toll-like receptors and NK cells. D. IL-12 and dendritic cells.

Question 3. The cellular nature of leukocytic infiltrates into infected tissue is most influenced by — A. the size of the microbial inoculum. B. preexisting specific antibodies. C. the chemokine receptors expressed by the infiltrating leukocytes. D. The defensins released at the infected site.

Question 2. NK cells are activated directly by virusinfected target cells — A. via specific receptors for viral antigens. B. through specific receptors for IL-12. C. because these targets express fewer molecules that bind to inhibitory receptors. D. in spite of increased MHC class I surface expression.

Question 4. The innate immune mechanisms that best epitomize rapid responses on first contact with microbes are — A. Toll-like receptors and complement. B. chemokines and NK cells. C. phagocytes and defensins. D. chemokines and phagocytes.

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Series editors: William T. Shearer, MD, PhD, Lanny J. Rosenwasser, MD, and Bruce S. Bochner, MD

EBV the prototypical human tumor virus—just how bad is it? David A. Thorley-Lawson, PhD Boston, Mass This activity is available for CME credit. See page 32A for important information.

EBV was the first candidate human tumor virus. It is found in several human cancers, particularly lymphomas and carcinomas, and has potent transforming activity in vitro. Yet the virus persists benignly for the lifetime of more than 90% of the human population. Thus it seems that EBV has the potential to be highly pathogenic yet rarely manifests this potential. Studies over the last several years show this is because the virus actually persists in resting memory B cells and not proliferating cells. EBV needs its growth-promoting ability to gain access to the memory compartment but has evolved to minimize its oncogenic potential. These studies also reveal that the different EBV-associated tumors apparently arise from different and discrete stages in the life cycle of B cells latently infected with EBV. This raises the question of how actively EBV participates in the development of human tumors. Does the virus cause the disease, or is it simply a passenger? In the case of immunoblastic lymphoma in the immunosuppressed patient, the virus almost certainly plays a causative role, but in other cases, such as Burkitt’s lymphoma, the contribution of EBV remains less clear. (J Allergy Clin Immunol 2005;116:251-61.) Key words: Epstein-Barr virus, carcinoma, lymphoma, persistent infection, latency, B cell, memory

EBV is well known because of its characteristic biology.1-3 If you define the success of a pathogen by the number and extent of hosts it infects, EBV is the most successful human pathogen because it latently infects virtually the whole human population and persists for life.4 In tissue culture EBV is one of the most potent transforming viruses,5,6 and it is found in several human cancers,1,3 yet for most of the population, it remains From the Department of Pathology, Tufts University School of Medicine. Supported by grants R01 CA65883, R01 A118757, and R01 A1062989. Disclosure of potential conflict of interest: D. Thorley-Lawson has equity ownership in EBVax. Received for publication April 11, 2005; accepted for publication May 16, 2005. Available online July 15, 2005. Reprint requests: David A. Thorley-Lawson, PhD, the Department of Pathology, Jaharis Building, Tufts University School of Medicine, 150 Harrison Ave, Boston, MA 02111. E-mail: David.Thorley-Lawson@ tufts.edu. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.038

Abbreviations used BL: Burkitt’s lymphoma CTL: Cytotoxic T lymphocyte EBNA: EBV nuclear antigen HD: Hodgkin’s disease IM: Infectious mononucleosis LMP: Latent membrane protein NPC: Nasopharyngeal carcinoma PTLD: Posttransplantation lymphoproliferative disease

benign. The collection of viral latent proteins expressed is different in each tumor type (Table I). Sometimes all of the known latent proteins are expressed, sometimes a limited subset, and sometimes only one. Despite the apparent robustness with which the human population deals with EBV (>95% of all adults carry the virus), the diseases caused by EBV indicate that the situation is finely balanced. The first indication comes from X-linked lymphoproliferative disease.7 In this disease persistent infection is not established because mutations in the SH2D1A gene8,9 cause acute EBV infection to become a fatal disease. Put melodramatically, a single nucleotide change in the SH2D1A gene is all that prevents the vast majority of the human race from dying of acute EBV infection. The second indication comes from the observation that immunologic disturbance, as a predisposing factor, is a unifying theme for all of the EBV B-cell lymphomas. This also suggests that the regulation of EBV infection in B cells is finely balanced. Disruptions can lead to deregulation and EBV-driven tumor development, even in otherwise healthy carriers of the virus. The clearest example of this is individuals who are immunosuppressed, such as patients undergoing organ transplantation, who are iatrogenically immunosuppressed, or patients with AIDS, who are immunosuppressed by HIV. These individuals are at risk for EBV lymphomas that are aggressive and often fatal.10 This means that it is only courtesy of an active immune response that we are protected from fatal EBVdriven lymphoma. Yet there are some curious properties of these tumors that suggest the risk is not as high as might be expected. For example, not every immunosuppressed 251

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TABLE I. The EBV transcription programs in normal B cells and tumors Transcription program

Growth

Genes expressed*

Default

EBNA1, 2, 3a, 3b, 3c, LP, LMP1, LMP2a, and LMP2b EBNA1, LMP1, and LMP2a

Latency EBNA1 only

None EBNA1

Lytic

All lytic genes

Infected normal B-cell typey

Function

Naive

Activate B cell

Germinal center

Differentiate activated B cell into memory Allow lifetime persistence Allow virus in latency program cell to divide Replicate the virus in plasma cell

Peripheral memory Dividing peripheral memory Plasma cell

Infected tumor type

Immunoblastic lymphoma HD

Burkitt’s lymphoma

*Does not include the noncoding EBER and BART RNAs that are assumed to be ubiquitous but have not been rigorously identified in all of the infected subtypes.  Except where indicated, the cell types are primarily restricted to the lymphoid tissue of the Waldeyer ring.

patient has the tumors, and the tumors are frequently oligoclonal. This is not the expected outcome. If it were simply a case of the immune system failing to control the EBV-infected cells, every immune-suppressed person should fill up with multiple tumors because everybody carries approximately 5 3 105 infected cells,11 and immunosuppressed individuals carry perhaps 50 times more.12 Taken together, these observations raise several questions. How does EBV persist benignly for the lifetime of a human despite its pathogenic potential? Why does EBV have such potent and pathogenic properties if it has evolved to persist for the lifetime of the human host it puts at risk by manifesting those properties? Where do the EBV-associated tumors come from, why do they have different patterns of latent gene expression, and why does disruption of the immune system predispose to EBV lymphoma development? Lastly, what goes wrong in the maintenance of persistence that leads to EBV-associated diseases? The key to answering these questions comes from a model of EBV persistence13,14 developed from the observation that despite EBV’s transforming ability, it persists in vivo in resting15 memory16 B cells that do not express any viral proteins.17 This article will first briefly review the complete life cycle of EBV infection and then discuss how the origins of EBV-associated tumors can be explained in the context of this model, with special emphasis on the role of an impaired immune response. Finally, the model will be used to attempt to answer the questions posed above.

EBV PERSISTENCE IN VIVO The essence of EBV’s behavior is that under normal conditions, it does not aberrantly deregulate the behavior of infected B cells in vivo. It initiates, establishes, and maintains persistent infection by subtly using virtually every aspect of normal B-cell biology. Ultimately, this allows the virus to persist within memory B cells for the lifetime of the host in a fashion that is nonpathogenic. The thesis of this review is that EBV is not a natural tumor

virus and that it has developed strategies to minimize its pathogenic potential to the host.

Establishment To understand EBV biology, it is first necessary to understand the biology of the B lymphocyte in the mucosal lymphoepithelium of the tonsil (Fig 1). A summary of normal mature B-cell biology and the proposed parallels with EBV is given in Figs 2 and 3, and a summary of information on the different viral latency programs is presented in Table I. The model has been described in detail elsewhere.13,14 The normal B-cell response. Environmental antigens entering the mouth are continuously sampled by the epithelium of the tonsil. Underneath the epithelium is a bed of lymphoid tissue including large numbers of naive lymphocytes.18,19 If antigen is recognized by the antibody on the surface of the naive B cell, it will bind and cause the B cell to become an activated blast and migrate into the follicle to form a germinal center (Fig 2).20 Here the cell undergoes rounds of rapid proliferation associated with isotype switching and mutation of the immunoglobulin genes, followed by competitive selection for those with the antibody that binds the antigen best. Those who lose in the competition to bind antigen die by apoptosis. Ultimately, the surviving cells leave the germinal center as memory cells primed to make a rapid response to rechallenge with the antigen. This process requires, in addition to the antigen, a signal to the B cell from an antigen-specific T helper cell. The parallel with EBV. EBV also transits the epithelium and infects naive B cells21 in the underlying tissue, where it expresses a set of latent genes that cause the cell to become activated and proliferate as though it were responding to antigen. This EBV transcription program (the growth program, Fig 2) involves 9 latent proteins, including nuclear antigens (EBV nuclear antigens [EBNAs]) and membrane proteins (latent membrane proteins [LMPs]).2 These proteins have all the necessary activities to push the B cell to become an activated blast without any necessity for external signaling. This cell migrates to the follicle, where the viral transcription program changes,22 such that

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FIG 1. The lymphoepithelium of the palatine tonsils from the Waldeyer ring. The tonsil consists of a highly involuted epithelium, creating a large surface area with deep invaginations. The epithelial surface is at the top of both micrographs. Antigen and EBV both enter through saliva and cross the epithelial barrier to activate or infect, respectively, the naive B cells below. The mantle zone (MZ), containing naive B cells (dark blue), is always facing the surface and is continuous with the epithelium. Naive cells enter the tonsil (black arrow) through the high endothelial venules (orange cuboidal cells). Numerous follicles containing germinal center B cells (GC) are arranged parallel to the surface. B cells leave the germinal center (red arrow) and enter the circulation through the efferent lymphatics. A higher magnification (expanded box) reveals the sponge-like structure of the epithelial cells in the lymphoepithelium that create spaces extending all the way to the mantle zone that are filled with infiltrating lymphocytes such that there is frequently only a single epithelial cell between the outer surface and the lymphocytes. (The micrographs were kindly provided by Dr Marta Perry).

only 3 of the latent proteins are expressed: EBNA1 (required to replicate the viral DNA) and 2 membrane proteins, LMP1 and LMP2 (the default program, Fig 2). The functions of LMP1 and LMP2 have evolved to steer the latently infected B cell through the germinal center environment. LMP2 alone will push B cells to form a germinal center in the mucosal follicle23; LMP1 and LMP2 can drive immunoglobulin gene mutation23 and isotype switching24 (the defining markers of the germinal center), respectively, and LMP1 downregulates expression of the germinal center regulatory transcription factor bcl-6,25 the signal for a memory cell to exit the germinal center.26 This implies coordinated expression of LMP1 and LMP2, where LMP2 is turned on before and LMP1 is turned on during the germinal center reaction. Thus constitutive expression of LMP1 in the absence of LMP2 blocks germinal center formation because the cells can never turn on bcl-6, an essential step in germinal center formation.27 This explains why EBV has the ability to make cells proliferate, despite the fact that this puts the host at risk for neoplastic disease. Essentially it has to because this is the mechanism, activation followed by differentiation, by which a normal B cell enters the B-cell memory pool.

Maintenance Once in the periphery, the latently infected cells shut down all viral protein expression (the latency program) and appear to be maintained as normal memory B cells.17 In the early stages of acute infectious mononucleosis (IM; primary EBV infection in the adult), the number of such cells in the blood can reach staggering proportions, with

50% or more of all memory cells being infected.28 However, the numbers decrease rapidly (half-life of 7 days; Hadinoto and Thorley-Lawson, unpublished data) for the first 2 months and then more steadily after that, until by 1 year there are typically only about 1 in 105 to 106 infected memory B cells. After this time, the level of infected cells appears to be relatively stable over many years.11 This presumably represents a balance between the replenishment of latently infected memory cells through cell division17 and their loss through viral replication (see below). This cell division must be regulated as part of normal memory B-cell homeostasis because there are no viral proteins expressed that could cause the cell to divide. When they divide, they express EBNA1 (the EBNA1-only program, Fig 2),17 which is needed to allow the viral DNA to replicate with the cells.29 Perhaps not surprisingly, because EBNA1 represents the only point of immune attack of the memory cells, EBNA1 has evolved to be poorly recognized by the immune system.30 By gaining entrance to normal memory B cells and shutting down viral protein expression, the virus is safe from immune surveillance. It is also benign because none of the latent proteins that drive growth are expressed. This explains why EBV is able to persist benignly in the vast majority of human subjects: EBV infection in vivo does not drive limitless proliferation. Rather it drives transient proliferation so that the cells can become resting memory cells. The virus persists in nonpathogenic resting cells not proliferating blasts. This also explains why EBVassociated tumors do not arise in every infected individual, even when they are immunosuppressed; something must go wrong with the normal biology that takes the latently

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Reviews and feature articles FIG 2. A model of how EBV uses normal B-cell biology to establish and maintain persistent infection in memory B cells. The response of a normal B cell to antigen, leading to the production of antigen-specific memory cells in the peripheral circulation, is diagrammed to the left, and the parallel series of steps by which EBV establishes latent infection in peripheral memory B cells is shown to the right. The specific viral transcription programs are labeled in blue to the right. For details, see the text and Table I.

FIG 3. A model of how EBV uses normal B-cell biology to replicate and be shed into saliva. The pathway by which antigen-specific B cells become activated and differentiate into antibody-producing plasma cells is shown to the left, and the parallels that lead to shedding of EBV are shown to the right. The EBV transcription program is indicated in blue to the right. For details, see text and Table I.

infected cells into a resting state before EBV could be involved in tumor development.

Release By accessing the memory compartment, EBV has a site for long-term persistence. However, it must replicate and be shed to spread to new hosts. The parallels between

normal B-cell biology and the mechanism of viral shedding are shown in Fig 3. Signals that cause the B cell to differentiate into an antibody-secreting plasma cell will in turn reactivate the virus.31 Because antibody-secreting plasma cells migrate into the mucosal epithelium,18,32 such a cell will be perfectly placed to release virus onto the mucosal surface, which, in the case of the tonsils,

is saliva. Thus infectious virus is spread through saliva contact.33

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Epithelial cells and viral shedding Although EBV is considered to be a B-lymphotropic virus, it can also infect epithelial cells because it is found in several important diseases of epithelial cells, including nasopharyngeal34 and gastric35 carcinomas and oral hairy leukoplakia.36 What is less clear is whether epithelial cells play a role in the normal biology of EBV. Early reports that claimed to find EBV in healthy nasopharyngeal epithelium have been discredited37; however, recent work has revisited this possibility. There is now evidence that normal epithelial cells in the nasopharynx express a distinct EBV receptor,38 that they can be infected in vitro, and that they are infected in vivo.39 However, it remains undetermined whether this infection occurs fortuitously because this epithelium is an area in which EBV happens to replicate or because it is an important component of the viral biology. The most likely role for epithelial cells is as a site for replication and amplification of the virus rather than as a site of persistent latent infection.40,41 Because the receptor is only expressed on the basolateral surface of epithelial cells, the virus can only infect from the lymphoid tissue and not from saliva. Thus if epithelial cells play an amplification role, it is during viral shedding and not primary infection. Perhaps the most compelling indirect evidence for epithelial cell infection comes from simple numbers. Estimates of the number of lymphocytes replicating EBV in the tonsils42 indicates that there are not nearly enough to account for the rates of viral shedding found in saliva (Hadinoto and Thorley-Lawson, unpublished data). This suggests that there must be a locationmechanism for amplifying the virus shed from plasma cells. The obvious candidates are epithelial cells because, from studies on oral leukoplakia, we know that epithelial cells replicate EBV to high copy numbers.

but in the case of EBV, it seems to ensure that newly infected cells are rapidly destroyed. This suggests that the new infection route might only be viable before the immune response arises (ie, in acute infection). Thereafter, the virus depends on homeostasis of the pool of latently infected memory cells for persistence and ensures that any new infected cells are rapidly killed because they might pose a lymphoproliferative threat to the host. Second, if EBV persists in normal antigen-selected B cells (unpublished results), why does it have LMP1 and LMP2, which can replace all the signaling necessary to produce a memory B cell? The answer to this is not yet clear. One possibility is that the role of LMP1 and LMP2 might be to give a selective advantage to the virus-infected cells in the highly competitive environment of the germinal center. This would give the latently infected cell a better chance of making it into the memory pool. Third, why does EBV not infect memory cells directly? A priori there seems no reason why EBV could not use the same mechanism to drive an infected memory cell back into memory; however, the evidence does not favor this alternative. First, there is no evidence that direct infection of memory cells occurs consistently in vivo.21,22 Second, when it does occur, it seems to lead to clonal proliferation46,47 and not differentiation, and third, the pool of latently infected memory cells is skewed (Sousa and Thorley-Lawson, unpublished data), which would not be expected if EBV infected memory cells at random. One possible explanation comes from the known biology of B cells. Activation of naive B cells through the germinal center leads predominantly to the production of memory cells over plasma cells,48 whereas activation of memory cells leads predominantly to the production of plasma cells.49 Therefore if the goal of EBV is to access the memory compartment, it will do so more efficiently by infecting and activating naive B cells rather than memory cells.

Unresolved questions about persistence in memory cells There are important unresolved questions relating to EBV persistence in memory B cells. First, what is the relative contribution of reinfection versus homeostatic cell division to the maintenance of stable levels of latently infected cells? We know that the host mounts a massive cytotoxic T-cell response against cells replicating EBV and newly infected cells43 and a neutralizing antibody response against the virus.4 It is therefore unclear whether newly infected cells are produced rapidly enough and survive long enough to contribute to the pool of latently infected memory cells once the immune response has begun. It is conceivable that new infection is only critical in establishing the pool of latently infected memory cells before the onset of the immune response and thereafter plays no role. A clue that this might be true comes from the observation that the epitopes recognized by cytotoxic T cells on newly infected B cells are conserved.44 Usually, a virus is continuously varying its sequence to avoid the immune response (eg, HIV45),

EBV AND DISEASE General considerations EBV has been associated with a number of human diseases. These generally fall into 2 categories: autoimmunity and cancer.1,3 The idea that EBV might be involved in autoimmunity stems from the knowledge that the virus can infect any B cell and cause it to proliferate indefinitely in culture. This raises the possibility that EBV could immortalize forbidden clones of B cells in vivo, perhaps allowing them to produce autoimmune antibodies in an uncontrolled fashion. This philosophic underpinning for a role of EBV in autoimmunity can now be seen to be incorrect. We know that EBV does not persist in vivo by immortalizing B cells but by establishing a true latency in normal resting memory B cells. There are also technical difficulties to proving a causal role for EBV in these diseases. First, EBV persists in circulating memory cells and therefore will be found in all tissues, irrespective of disease causality. Second, it is now apparent

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Reviews and feature articles FIG 4. The putative check points in the EBV life cycle that give rise to tumors. The events that occur normally in healthy carriers are denoted in black. For details, see Figs 2 and 3. EBV normally infects naive B cells in the Waldeyer’s ring, and these cells can differentiate into memory cells and out of the cell cycle (thick arrows), and therefore they are not pathogenic. PTLD: If a cell other than the naive B cell in the Waldeyer ring becomes infected, it will express the growth program and continue to proliferate because it cannot differentiate out of the cell cycle (thin dashed arrows). This is a very rare event, highlighting how carefully controlled EBV infection is. Normally, these bystander B-cell blasts would be destroyed by CTLs, but if the CTL response is suppressed, then they can grow into PTLD. Note: a bystander-type cell could also arise if a latently infected germinal center or memory cell fortuitously switched on the growth program. Hodgkin’s disease arises from an EBV-infected cell that is blocked at the germinal-center cell stage. This results in constitutive expression of the default program. Burkitt’s lymphoma evolves from a germinal-center cell that is entering the memory compartment but is stuck proliferating. Consequently, the cell expresses EBNA1 only. Nasopharyngeal carcinoma is hypothesized to arise from a latently infected epithelial cell blocked from terminal differentiation and viral replication. It is unclear why these cells would express the default program.

that EBV is extremely sensitive to the state of the immune system. This is because it relies on normal B-cell biology to establish and maintain persistence and T-cell responses to modulate the level of infection. Changes that affect the functionality of the immune system affect EBV by changing overall viral loads and states of infection. Because autoimmune diseases classically disrupt the immune system, it will be extremely difficult to dissect out causality of EBV from the background noise of changes occurring in the virus because of the disease. For all of these reasons, it has been difficult to establish a clear connection between EBV and any autoimmune diseases. Currently, such associations remain speculative, controversial, or both. The reason to believe EBV might cause cancer is apparent. EBV encodes genes that make B cells grow. Such genes will, of their nature, have potential as onco-

genic risk factors. However, as described above, EBV has evolved to minimize the risk that an infected cell will proliferate out of control. Therefore something must go wrong with the normal viral biology for EBV to play a causative role in tumor development. The plausibility of EBV as an oncogenic virus has led to claims of its association with many human tumors. Some, such as breast and hepatocellular carcinoma, have never been substantiated, but there are now several for which strong evidence exists, including immunoblastic lymphoma in immunosuppressed patients, Burkitt’s lymphoma, Hodgkin’s disease (HD), and nasopharyngeal carcinoma (NPC). The origins of all of these tumors can be understood as arising from specific stages in the EBV life cycle (Fig 4) and appear to be associated with disturbances of the immune system. This begs the following question: How convincing is the evidence that EBV

plays a causative role in these tumors and is not simply a passenger in a tumor cell that arose from an infected cell type?

Lymphoma in the immunosuppressed Individuals who are immunosuppressed are at risk for development of B-cell lymphoproliferative diseases, such as the immunoblastic lymphomas in patients with AIDS and the posttransplantation lymphoproliferative diseases (PTLDs) in patients undergoing organ transplantation.10 These are a heterogeneous collection of disorders that usually carry the virus and express the growth program (Table I).50 A wide range of factors (eg, organ type, immunosuppressive regime, location, and donor origin) influence the frequency with which these tumors arise. The explanation usually given for the origin of these tumors is that immunosuppression of the cytotoxic T-lymphocyte (CTL) response to EBV allows uninhibited growth of EBV-infected cells; however, it is not that simple. From the discussion above on the mechanism of EBV persistence, it is apparent that, under normal conditions, infected naive B cells in the tonsils do not give rise to lymphoma because they differentiate out of the cell cycle to become resting memory cells. For a cell to express the growth program, survive, and evolve into a neoplasm, 2 events must occur: the EBV-infected cell must be unable to respond to signals that drive it to differentiate into a resting memory cell, and the CTL response must be crippled so that these lymphoblasts can continue to proliferate. This could occur if any B cell that is not a naive B cell in the tonsil is exposed to the virus by chance—bystander infection (Fig 4). It could also occur if a latently infected germinal center or memory cell fortuitously received signals that caused it to inappropriately turn on the growth program. These cells can not exit the growth program, and therefore they continue to proliferate. Normally, they would be rapidly eliminated by CTLs because of the conserved CTL epitopes they express (see above); however, in the absence of effective T-cell immunity (immunosuppression), they will continue to proliferate. Direct evidence that this is indeed the case comes from studies of tonsils from acutely infected individuals. In these tonsils clonal expansions of directly infected germinal center51 and memory cells46 driven by the growth program can be found. Because these are bystander-infected cells, they are unable to differentiate into resting memory cells. Consequently, they proliferate until the immune response arises to eliminate them, explaining why such clones are never seen in healthy carriers of the virus but will appear if the immune response is subsequently suppressed. The origin of these tumors also explains their heterogeneity. They are derived from a mixture of B-cell types52 consistent with arising from a variety of bystander B cells that get infected by chance and not a specific subset of infected cells. This also explains why the tumors are relatively rare. The vast majority of infected cells differentiate into a resting memory state because they are naive;

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they will not be a cancer risk. Only the rare, atypical bystander infection is a risk for tumor development.

Hodgkin’s disease Acute EBV infection in the adolescent-adult can give rise to IM, long known to be a risk factor for HD. However, the strongest evidence directly linking EBV with HD came with the finding that approximately 40% of the tumors contain clonal EBV,53 which can approach 80% in developing countries and up to 100% in AIDSrelated HD.54 In addition, the tumor cells express the default transcription program (Table I),55-58 which includes 2 proteins (LMP1 and LMP2) that deliver survival and growth signals,59-61 at least one of which (LMP1) is known to act as an oncogene.62 A characteristic of IM, compared with the subclinical infection seen in children, is profound disruption of the immune system.63 This includes massive levels of virus-infected memory B cells (50%), a striking T-cell lymphocytosis caused, at least in part, by a very aggressive cytotoxic T-cell response, and tissue damage in the lymph nodes. The disease is almost certainly a product of an overreactive inflammatory response, and B-cell function is so badly disrupted that one of the characteristics of IM is the production of a broad range of nonspecific, low-affinity, so-called heteropohile antibodies. This suggests that HD is the consequence of deregulated EBV infection caused by the severe immunologic disturbance of IM. Nevertheless, the possibility that EBV is a passenger cannot be excluded. If the immunologic disruption of IM alone is the risk factor for HD, it is possible that the premalignant B cell will have EBV in it simply by chance. There is good evidence that EBV-positive HD arises from an infected germinal-center cell. As discussed above, one of the characteristics of germinal-center cells is that they actively mutate their immunoglobulin genes in a process termed hypermutation, which leaves a characteristic pattern of mutations. The immunoglobulin genes of HRS cells have this pattern of mutation.64 In addition, the default transcription program is used by EBV in latently infected germinal center B cells.22 Thus the immunoglobulin mutations and the viral gene expression data independently support the idea that EBV-positive HD arises from an EBV-infected germinal center B cell (Fig 4). Burkitt’s lymphoma EBV was discovered in cultured tumor cells from patients with the endemic form of Burkitt’s lymphoma (BL).65 It is sobering to realize that 40 years later, we still do not know how or even for sure whether EBV causes BL. This is despite the large volume of information we have acquired about EBV’s molecular and cellular biology, immunology, virology, epidemiology, clinical manifestations, and disease associations.1-3 The most compelling evidence of EBV’s involvement in BL is the high frequency (98%) of tumors carrying the virus66 in endemic areas and the presence of clonal EBV in all of the tumor cells.67 However, none of the growth-promoting latent genes are expressed. The only genes expressed

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encode for EBNA168 and the untranslated RNAs called EBERS and BARTS. It has been suggested that EBNA169 and the EBERS70 might have oncogenic potential, but the findings remain unsubstantiated and are controversial. Consequently, there is currently no broadly accepted understanding of the role of EBV in BL.71-74 What is apparent, however, is that malaria, which is chronically immunosuppressive,75 is classically known to be a risk factor for endemic (ie, EBV-positive) BL development.76 Once more, this supports the notion, discussed throughout this review, that EBV infection in the context of a compromised immune system is the risk factor for lymphoma development. Using the same arguments as for HD, we can surmise that BL is a tumor cell of a proliferating, latently infected memory B cell (Fig 4). BL has the same pattern of immunoglobulin gene hypermutations as memory B cells,77 and there is only one way known for producing an EBNA1-only phenotype in nontumor cells. This is when a latently infected memory cell expressing the latency program divides as part of normal B-cell homeostasis (Fig 2).17 One property of BL inconsistent with this idea is that the tumor cells have the surface phenotype of germinal-center cells.78 However, the cellular phenotype of tumor cells can be misleading. This is exemplified by HD, which is generally thought to be derived from a germinal-center cell, although it bears no phenotypic or morphologic resemblance to such cells. Thus it is difficult to know how directly the final cellular phenotype of BL relates to the original infected precursor. Possibly, BL is derived from a germinal-center cell on its way to becoming a resting memory cell expressing the latency program but through tumor-driven growth continues to proliferate and therefore expresses the EBNA1-only phenotype.

Nasopharyngeal carcinoma Given the B lymphotropism of EBV, it is surprising that one of the best candidates for a tumor caused by EBV is not a lymphoma but a carcinoma, NPC, responsible for 20% of all cancers in China and Taiwan79 and therefore an important world health problem. Virtually 100% of undifferentiated NPCs worldwide contain clonal EBV.34,80 The tumors express the viral default transcription program.81-83 Although only a subset, approximately 40%, express LMP1, it has been reported that the premalignant lesions of NPC all express LMP1.84 As with HD, the presence of LMP1 and LMP2 is additional evidence that the virus is playing a part in the cause of the tumor. Because LMP1 and LMP2 are potently and specifically evolved B cell–signaling molecules, their presence in the epithelial cells of NPC suggests the virus might be there fortuitously. An example of this is LMP2, which functions to cause B cells to migrate into mucosal follicles.23 This migratory ability, expressed in epithelial cells, might result in the invasive and metastatic activity of NPC. The potential role of EBV in NPC is clouded by our lack of certain knowledge about the role of epithelial cells in EBV biology. In Fig 4 the speculative assumption is made that EBV latently infects epithelial cells that then proceed

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to replicate the virus and shed it into saliva. NPC would derive from such a latently infected, undifferentiated epithelial cell, which was blocked from switching to viral replication and therefore continues to be latently infected. Why the default program, usually found in germinal center B cells, is expressed in NPC is completely unclear.

CONCLUSION: ANSWERS TO THE QUESTIONS In the introduction to this article, several questions were raised about EBV. The answers to these questions can now be explained in light of the discussion above. First, why does EBV make cells proliferate when it puts the host at risk for neoplastic disease? Because it has to. The newly latently infected naive B cell has to become an activated blast before it can differentiate into a resting memory cell. Second, where do the EBV-positive tumors come from, why do the different tumors express different viral latent gene transcription programs, and why is disruption of the immune system a risk factor? The virus uses these different transcription programs to manipulate the biology of the infected B cell so that it can gain entry into and then persist in memory B cells. Any disruption of the immune system that interferes with the ability of the EBV-infected cells to become a resting memory cell will increase the risk of tumor development. Each tumor derives from a different step in this process and represents a cell that is blocked from progressing into a resting state and therefore continues to express the viral transcription program of its progenitor. Third, why are there so few EBV-infected tumors in the human population, even with immunosuppression, despite the large numbers of EBV-infected cells in each individual and the ability of EBV to make lymphocytes grow? This is because the viral biology is tightly regulated to ensure that an EBV-infected naive B cell that becomes activated and starts to proliferate will rapidly exit the cell cycle and become a resting memory cell expressing none of the dangerous growth-promoting genes. In addition, the virus has conserved the targets for CTLs to ensure that if a newly infected cell does not exit the cell cycle, it will be rapidly killed.

CONCLUSIONS AND FUTURE DIRECTIONS We now know the basic outlines of the EBV life cycle and have some understanding of where and why the tumors arise. For the basic scientist, the challenge remains to understand, at the molecular level, how EBV negotiates the changes between the different latency states in the different B-cell types. Because this is so dependent on B cells, it is likely that the mechanisms will only become clear when we learn how the processes are normally regulated in B cells. There is still also much to be learned about the role EBV plays at the molecular level in

tumorigenesis, particularly for BL. But perhaps the biggest gap in our knowledge is understanding what is different in the disruption of the immune response that leads to immunoblastic lymphoma in some cases, HD in others, and BL in yet others. Could timely immunologic intervention reduce the risk of subsequent development of these diseases? The identification of EBV within tumors provides a potentially unique opportunity to develop tumor-specific therapy targeted at the virus that will not hurt normal cells. A good example of this is recent work showing that in vitro expanded, EBV-specific CTLs can be effective therapy against PTLD,85 although they hold less promise for treatment of HD and NPC. An important and potentially fruitful area of clinical investigation will be the development of drugs specifically targeted against EBV. The best candidate latent protein might well be EBNA1, which allows replication of the viral DNA and therefore is essential for retention of the viral DNA in a proliferating (eg, tumor) cell. The crystal structure of EBNA1 bound to DNA is known, opening the path to the development of drugs that block this interaction. If the tumor requires EBV to grow, loss of the viral DNA should prevent tumor growth. Whether EBV truly plays a causative role in these tumors is therefore not an esoteric question. If the virus is not a key player, then therapies directed at the virus will be ineffective against the tumors. A hint of this comes from PTLD, in which restoration of the immune response leads to tumor regression. Eventually, however, the tumors become resistant. This raises the possibility that ultimately tumor growth might not be dependent on the virus. An interesting approach that does not require the virus to be essential for tumor growth would be the development of drugs that efficiently cause EBV in the tumors to begin replicating. Because replication of the virus kills the cell, this would be an indirect way to destroy EBV-positive tumors, irrespective of their dependence on the virus for growth. This would not need to lead to wholesale production of virus, however, because drugs that block this are already available (eg, valacyclovir).

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Continuing Medical Education examination

EBV the prototypical human tumor virus—just how bad is it? Instructions for category 1 Continuing Medical Education credit The American Academy of Allergy, Asthma and Immunology is accredited as a provider of Continuing Medical Education (CME) by the Accreditation Council for Continuing Medical Education. Test ID no.: mai0064 Contact hours: 1.0 Expiration date: July 31, 2006 Category 1 credit can be earned by reading the text material and taking this CME examination online. For complete instructions, visit the Journal’s Web site at www.mosby.com/jaci.

Learning objectives: ‘‘EBV the prototypical human tumor virus—just how bad is it?’’ 1. To understand that EBV uses mature B cell biology to establish latency, persist, and replicate. 2. To understand that even though EBV is so widespread and apparently benign, it is potentially life-threatening. 3. To understand that EBV evolved the capacity to make cells grow because it is an essential part of the mechanism for establishing latency in resting cells that are not pathogenic. 4. To understand that EBV-associated tumors arise from different stages in the life cycle of latently infected B cells and that disruption of the immune response is an important component in the development of all of the EBV-associated lymphomas.

CME items Question 1. EBV persists within what fraction of the healthy adult population? A. <1% B. ;10% C. ;50% D. >90% Question 2. To establish persistent infection, EBV primarily infects — A. naive B cells. B. memory cells. C. activated B cells. D. germinal center cells.

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Question 3. EBV-associated tumors are relatively rare because — A. EBV is not oncogenic. B. EBV replicates and kills the cells before they can grow into tumors. C. EBV has evolved to minimize its oncogenic potential. D. EBV cannot always be detected in tumors. Question 4. How many mutations in host genes are required to make EBV a fatal acute infection? A. 0 B. 1 C. 5-6 D. >10

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Infection versus immunity: What’s the balance? William T. Shearer, MD, PhD Houston, Tex

This issue of the Journal is devoted to the theme of infection and immunity and to attempts to describe the balance of host forces that protect against those infectious forces that would invade. For most of our lives we remain unaware of the moment-by-moment interplay of host resistance factors and infectious agents. Yet as described in the articles herein, a natural sequence of clonal expansion of microbes develops in human beings born with faulty immunity or in those whose immunity is temporarily overwhelmed by infection. There are 3 intersecting concepts drawn out for us in this month’s Journal— immunodeficiency, infection, and cancer—that meet in a common point in certain individuals, like the zero point of 3-dimensional axes. Most infections of humankind are attributed to being the result of chance, exposure, and dose of infectious agent. Compelling arguments are now being made, however, that at least in the case of the more severe forms of these infections, invading organisms have taken advantage of a hidden chink in the armament of presumed normal immunity. With special patients who need immunosuppressive treatments, there is often a manifestation of chronic infection and even the appearance of cancer. This issue of the Journal clarifies some of those mysterious mechanisms of immunity that enable the preservation of life. In the lead Current Reviews article, Tosi summarizes the rapidly expanding field of innate immunity that, though lacking immune memory and clonal expansion of lymphocytes, probably protects against more infections than does adaptive immunity.1 At epithelial skin surfaces, antimicrobial peptides disrupt the cell membranes of pathogens and prevent numerous skin infections.2 Atopic eczema is a clinical example of deficiency in From the Department of Pediatrics, Baylor College of Medicine, and the Department of Allergy and Immunology, Texas Children’s Hospital. Supported by the National Institutes of Health grants AI27551, AI36211, HD41983, RR0188, HD079533, HL72705, HD078522, contract 202PICL05; the Pediatric Research and Education Fund, Baylor College of Medicine; and the David Fund, Pediatrics AIDS Fund, and Immunology Research Fund, Texas Children’s Hospital. Received for publication May 31, 2005; accepted for publication June 1, 2005. Available online July 15, 2005. Reprint requests: William T. Shearer, MD, PhD, Department of Pediatrics, Section of Allergy and Immunology, Baylor College of Medicine, 6621 Fannin St (MC-FC330.01), Houston, TX 77030. E-mail: wtsheare@ TexasChildrensHospital.org. J Allergy Clin Immunol 2005;116:263-6. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.06.001

b-defensins that leads to repeated staphylococcal infections in eczematous patches of affected skin.3 Mononuclear phagocytes and other cells contain Tolllike receptors that react with bacterial products, such as endotoxin and DNA sequences, and become active secretors of cytokines that regulate subsequent inflammatory responses.4 Children with deficiencies of the Toll-like receptor signaling pathway involving the IL-1 receptor– associated kinase (IRAK) are subject to infections with pyogenic bacteria.5 Numerous cytokines are involved in immune reactions, both protecting the host from infections and contributing to the complications of infections. In the latter category, perhaps IL-1 and TNF-a are best known for their pathologic role in gram-negative bacterial toxininduced shock.6 Chemokines and their receptors are active in numerous immunologic reactions to infections, but none are more visible than the CCR5 and CXCR4 chemokine receptors that induce cognate receptor binding of the HIV-1 glycoprotein 120 and facilitate entry of the HIV-1 virion into target cells.7 The value of natural killer (NK) cells assumes more importance as we understand the primal role that they serve in host protection against viral infection and the development of cancer.8 Responding in an antigen-independent manner, NK cells bind and lyse virus-infected host and cancer cells by perforin formation or apoptosis induction. Complement9 and neutrophil-invaded immunity10 round out the repertoire of innate immunity, each contributing to that immediate response to infection that is so important prior to the engagement of the slower acquired immune responses. The illustration on the cover of this issue demonstrates how neutrophils police the vascular endothelium and, within seconds of detecting chemoattractants created by infections in the tissues, squeeze through intercellular spaces in pursuit of pathogens.11 On arrival at the site of infections, neutrophils engulf and kill microbes through the formation of superoxide. No prior memory of these pathogens is necessary for this bacteriocidal function of neutrophils. Thorley-Lawson12 writes in the Molecular Mechanisms article on how the Epstein-Barr virus (EBV) takes up longterm residence in almost all human beings and occasionally produces lymphomas. Virtually all individuals with EBV lymphomas are either immunosuppressed (eg, transplant patients) or lack components of immunity on a congenital basis (eg, X-linked immunoproliferative disease).13 EBV remains in a latent state in resting memory B cells that do not express EBV proteins on their cell 263

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surface.14 Thus, these EBV-containing B cells remain invisible to cytotoxic T cells. EBV is thought to play a role not only in lymphomas but also in Hodgkin’s disease, Burkitt’s lymphoma, and nasopharyngeal carcinoma. Thorley-Lawson acknowledges that these cells may just carry EBV rather than being the result of EBV-initated transformation events. He also questions why more EBVdriven tumors are not seen in human populations, even those immunosuppressed individuals. The answer seems to reside in the propensity of EBV-infected B cells to stop replicating under the influence of 2 viral genes—latent membrane protein (LMP) 1 and LMP2—and enter the latent resting memory cell condition.15 Circumstances that disrupt the immune system change this resting memory cell condition and favor tumor development. Thus, when human beings are given immunosuppressive drugs or have received therapeutic irradiation, immune forces are disrupted and the EBV B cell enters the replicating cell cycle. Unless killed by cytotoxic T cells, which respond to the newly expressed EBV cell surface antigens, these activated EBV cells could form oligoclonal tumor cells. These observations hold importance for immunologically normal human beings exposed to occupational or environmental conditions that cause immunosuppression, such as radiation in spaceflight.16 Mehandru and colleagues17 contribute a Perspectives/ Update article to this issue that details the rapidly unfolding discoveries of the crucial role of gastrointestinal lymphatic tissue in acute HIV-1 infection. In the face of relatively stable peripheral blood CD41 T (helper) cell concentrations in acute HIV-1 infection, there is a massive kill-off of tissue CD41 T cells, particularly those of the gastrointestinal tract.18-20 Moreover, these gastrointestinal CD41 T cells are of the memory phenotype and express the CCR5 chemokine receptor that attract the monocytotropic HIV-1 viral strains, as demonstrated in the simian model of HIV-1 infection.21,22 The emerging model of pathogenesis of acute HIV-1 infection suggests that the large pool of memory CD41 T cells in mucosal surfaces becomes preferentially infected and stimulates repetitive rounds of viral replication and CD41 T cell killing. Mehandru proposes that these discoveries will revamp the way clinicians decide when to intercede with antiretroviral agents (ie, immediate versus deferred therapy), rekindle the debate of using immunodulators to reduce the waves of inflammation and viral replication (eg, cyclosporine therapy during acute infection), accelerate the use of protective strategies for the gastrointestinal mucosal surfaces (eg, microbicides and CCR5 blockers), and redirect vaccine strategies to mucosal surfaces. In the Advances in Asthma, Allergy, and Immunology Series 2005: Basic and Clinical Immunology article in this issue, Chinen and Shearer mention several noteworthy, recent publications dealing with the interplay between immunity and infection.23 The importance of the HLA allele recognition system in viral infection is seen in human beings with certain HLA-B alleles with HIV-1 infection. HIV-1–infected individuals with HLAB57 and HLA-B*5801 select for variants with a specific

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mutation in the Gag epitope of HIV-1, but when this mutant viral strain is transmitted to another individual with different HLA alleles, this epitope reverts to the wild type.24 In HIV-1 discordant couples, the risk of HIV-1 transmission is 2-fold higher (independent of HIV-1 viral load) if the couples share one or both HLA-B alleles.25 In perinatal HIV-1 transmission, HLA-B*4901 and B*5301, alleles that inhibit motherto-infant HIV-1 transmission (despite high HIV-1 viral load), differ from otherwise identical HLA-B*5001 and B*3501 alleles by 5 amino acids encoding the ligand for the killer inhibitory receptor (KIR) 3DL1 for NK cells.26 The molecular basis for these 3 observations suggests strongly that recognition molecules on immune cells govern subsequent viral mutation and viral elimination through cytotoxic T cells and NK cells. Also summarized in the Advances article are the discoveries that mast cells participate in host defense via recognition of Toll-like receptors and viruses27 and secretion of cytokines that recruit effector cell.28 Articles in the area of primary immunodeficiency and infectious diseases are also cited, perhaps none more important than the identification of risks of malignancy when retroviral vectors are used to insert gene constructs in stem cells bone marrow derived in severe combined immunodeficiency.29 In this instance, the retroviral vector has the potential to insert into the human genome in the promotor region of oncogenes and to trigger the development of T-cell leukemia.30 Related to these observations in primary immunodeficiency is the Images in Allergy and Immunology article that pictures the case of a child who developed severe mosquito bite hypersensitivity, ulcerating skin lesions, enlarged and draining adjacent lymph nodes, and marked hepatosplenomegaly.31 Studies reveal that this child developed proliferation of EBV-containing NK cells similar, if not identical, to that seen in the few reported cases of chronic active EBV infection of NK cells that result in NK cell leukemia and lymphoma.32 It is possible that this child is an example of the atypical immunodeficiency that presents with a more common and less marked clinical phenotype that ultimately might be resolved by detection of causal genes, as proposed by Casanova33 and reviewed by Bonilla and Geha34 in an editorial in this issue. In addition to the interaction of viruses with immunodeficiency, there is strong evidence for a pathogenic role for viruses in allergic diseases. For example, rhinoviruses cause more than 50% of upper respiratory infections and are thought to be responsible for the induction of acute exacerbations of asthma in the lower airway. Friedlander and Busse35 review this evidence in this issue, and find that respiratory viruses are associated with approximately 80% of children and 50% of adults with wheezing episodes and that infection of the upper and lower respiratory mucosal surfaces induces increased airway hyperresponsiveness. This concept of rhinovirus induction of asthma includes (1) the attachment of the intercellular adhesion molecule 1 (ICAM-1) to the viral capsid molecules36 and (2) the stimulation of proinflammatory cytokines IL-6, IL-8, IL-16, and RANTES chemokine.37 The net result of

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FIG 1. Balance between immunity and infection. A, Normal immune system. B, Immunodeficiency.

upregulation of these mediators is an influx of eosinophils, monocytes, T cells, macrophages, and neutrophils into respiratory tissue.38 As a result of this increased inflammation of the airways, angiogenic growth factors might induce tissue remodeling of respiratory mucosa and could cause a permanent change in lower airway architecture and increased difficulties for treatment programs. All in all, the theme of this month’s Journal seems to have been substantially illustrated by the contributions of talented experts in immunity and infection. These interrelated concepts can be considered the 2 sides of a coin. Perhaps a better analogy is that of a teeter-totter: when the sitting board is horizontal, there is a balance between immunity and infection (Fig 1). When immunity is down, infections rise and immunity must be strengthened to gain balance, with the result that the inflammation of immunity often overshoots and infection drops. However, evidence is being gathered to strongly suggest that when this balance is upset, as is the case with immunodeficiency diseases, certain viruses are able to escape strong immune response and hide in a latent condition. When individuals who harbor latent viruses, such as patients receiving immunosuppressive drugs or therapeutic radiation, encounter an additional force, the latent virus is forced into its life cycle that yields outgrowths of clones of virus-containing transformed cells. More understanding of these balancing forces of immunity and infection is necessary so that we, as clinician-investigators, can intervene with the sometimes threatening consequences of imbalances in the forces of the immune system and infection. I thank Carolyn Jackson and Ruth Herrera for assistance with the preparation of this manuscript.

REFERENCES 1. Tosi M. Innate immune responses to infection. J Allergy Clin Immunol 2005;116:241-9. 2. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3:710-20. 3. Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, et al. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med 2002;347:1151-60. 4. Zarember KA, Godowski PJ. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 2002;168:554-61.

5. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, et al. Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 2003;299:2076-9. 6. Oppenheim JJ, Feldman M. Introduction to the role of cytokines in innate host defense and adaptive immunity. In: Oppenheim JJ, Feldman M, Durum SK, Hirano T, Vilcek J, Nicola N, editors. The cytokine reference. San Diego: Academic Press; 2001. p. 3-20. 7. Glass WG, Rosenberg HF, Murphy PM. Chemokine regulation of inflammation during acute viral infection. J Allergy Clin Immunol 2003;3:467-73. 8. Smyth MJ, Cretney E, Kelly JM, Westwood JA, Street SE, Yagita H, et al. Activation of NK cell cytotoxicity. Mol Immunol 2005;42:501-10. 9. Berger M, Frank MM. The serum complement system. In: Stiehm ER, Ochs HD, Winkelstein JA, editors. Immunologic disorders in infants and children. 5th ed. Philadelphia: Elsevier Saunders; 2004. p. 20-62. 10. Tosi MF. Immunologic and phagocytic responses to infection. In: Feigin RD, Cherry JD, Demmler GJ, Kaplan S, editors. Textbook of pediatric infectious diseases. 5th ed. New York: WB Saunders; 2004. p. 652-84. 11. Seo SM, McIntire LV, Smith CW. Effects of IL-8, Gro-alpha, and LTB(4) on the adhesive kinetics of LFA-1 and Mac-1 on human neutrophils. Am J Physiol Cell Physiol 2001;281:C1568-78. 12. Thorley-Lawson DA. EBV the protypical human tumor virus—just how bad is it? J Allergy Clin Immunol 2005;116:251-61. 13. Shearer WT, Ritz J, Finegold MJ, Guerra IC, Rosenblatt HM, Lewis DE, et al. Epstein-Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151-9. 14. Hochberg D, Middeldorp JM, Catalina M, Sullivan JL, Luzuriaga K, Thorley-Lawson DA. Demonstration of the Burkitt’s lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo. Proc Natl Acad Sci 2004;101:239-44. 15. Hochberg DR, Thorley-Lawson DA. Quantitative detection of viral gene expression in populations of Epstein-Barr virus-infected cells in vivo. Methods Mol Biol 2005;292:39-56. 16. Shearer WT, Zhang S, Reuben RM, Lee B, Butel JS. Effects of radiation and latent virus on immune responses in a space flight model. J Allergy Clin Immunol 2005;115:1297-303. 17. Mehandru S, Tenner-Racz K, Racz P, Markowitz M. The gastrointestinal tract is critical to the pathogenesis of acute HIV-1 infection. J Allergy Clin Immunol 2005;116:419-22. 18. Guadalupe M, Reay E, Sankaran S, Prindiville T, Flamm J, McNeil A, et al. Severe CD41 T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol 2003;77:11708-17. 19. Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, et al. Primary HIV-1 infection is associated with preferential depletion of CD41 T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 2004;200:761-70. 20. Brenchley JM, Schacker TW, Ruff LE, Price DA, Taylor JH, Beilman GJ, et al. CD41 T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 2004;200: 749-59.

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21. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. Massive infection and loss of memory CD41 T cells in multiple tissues during acute SIV infection. Nature 2005;434:1093-7. 22. Li Q, Duan L, Estes JD, Ma ZM, Rourke T, Wang Y, et al. Peak SIV replication in resting memory CD41 T cells depletes gut lamina propria CD41 T cells. Nature 2005;434:1148-52. 23. Chinen J, Shearer WT. Advances in asthma, allergy, and immunology series 2005: basic and clinical immunology. J Allergy Clin Immunol 2005;116:411-8. 24. Leslie AJ, Pfafferott KJ, Chetty P, Draenert R, Addo MM, Feeney M, et al. HIV evolution: CTL escape mutation and reversion after transmission. Nat Med 2004;10:282-9. 25. Dorak MT, Tang J, Penman-Aguilar A, Westfall AO, Zulu I, Lobashevsky ES, et al. Transmission of HIV-1 and HLA-B allele-sharing within serodiscordant heterosexual Zambian couples. Lancet 2004;363:2137-9. 26. Winchester R, Pitt J, Charurat M, Magder LS, Goring HH, Landay A, et al. Mother-to-child transmission of HIV-1: strong association with certain maternal HLA-B alleles independent of viral load implicates innate immune mechanisms. J Acquir Immune Defic Syndr 2004;36:659-70. 27. Kulka M, Alexopoulou L, Flavell RA, Metcalfe DD. Activation of mast cells by double-stranded RNA: evidence for activation through Toll-like receptor 3. J Allergy Clin Immunol 2004;114:174-82. 28. Marshall JS, Jawdat DM. Mast cells in innate immunity. J Allergy Clin Immunol 2004;114:21-7. 29. Chinen J, Puck JM. Successes and risks of gene therapy in primary immunodeficiencies. J Allergy Clin Immunol 2004;113:595-603.

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30. Cavazzana-Calvo M, Lagresle C, Hacein-Bey-Abina S, Fischer A. Gene therapy for severe combined immunodeficiency. Annu Rev Med 2005; 56:585-602. 31. Pacheco SE, Gottschalk SM, Gresik MV, Dishop MK, Okmaura T, McCormick TG. Chronic active Epstein-Barr virus (CAEBV) infection of NK cells presenting a severe skin reaction to mosquito bites. J Allergy Clin Immunol 2005;116:470-2. 32. Tokura Y, Ishihara S, Tagawa S, Seo N, Ohshima K, Takigawa M. Hypersensitivity to mosquito bites as the primary clinical manifestation of a juvenile type of Epstein-Barr virus-associated natural killer cell leukemia/lymphoma. J Am Acad Dermatol 2001;45:569-78. 33. Casanova JL, Fieschi C, Bustamante J, Reichenbach J, Remus N, von Bermuth H, et al. From ‘‘idiopathic’’ infectious diseases to ‘‘atypical’’ primary immunodeficiencies. J Allergy Clin Immunol 2005;116: 426-30. 34. Bonilla S, Geha RS. Are you immunodeficient? J Allergy Clin Immunol 2005;116:423-5. 35. Friedlander G, Busse WW. The role of rhinovirus in asthma exacerbations. J Allergy Clin Immunol 2005;116:267-73. 36. Yamaya M, Sasaki H. Rhinovirus and asthma. Viral Immunol 2003;16: 99-109. 37. Papadopoulos NG, Bates PJ, Bardin PG, Papi A, Leir SH, Fraenkel DJ, et al. Rhinoviruses infect the lower airways. J Infect Dis 2000;181: 1875-84. 38. Gern JE. Rhinovirus respiratory infections and asthma. Am J Med 2002; 112:19S-27S.

Asthma diagnosis and treatment

The role of rhinovirus in asthma exacerbations Samuel L. Friedlander, MD, and William W. Busse, MD Madison, Wis

Rhinoviruses are a major cause of asthma exacerbations in children and adults. With the use of sensitive RT-PCR methods, respiratory viruses are found in approximately 80% of wheezing episodes in children and in approximately one half of such episodes in adults. Rhinovirus is a member of the family Picornaviridae, and acute rhinovirus infections occur predominantly in the upper airway. This virus has also been identified in the lower airway, and it might cause acute wheezing through the production of proinflammatory mediators with a resulting neutrophilic inflammatory response. Precisely how this process leads to increases in airway hyperresponsiveness and airway obstruction is not fully established. However, risk factors for wheezing with colds include asthma and atopy, extremes in age, and perhaps having a deficient TH1 response to rhinovirus. With the use of in vitro models and experimental inoculation studies, significant advances have led to a better understanding of the mechanisms by which rhinovirus infections cause asthma exacerbations. Advances in our understanding of this interaction might provide knowledge that could ultimately lead to specific treatment modalities to prevent and/or treat this significant burden of asthma exacerbations. (J Allergy Clin Immunol 2005;116:267-73.) Key words: Rhinovirus, asthma exacerbations, virology, cytokine response profiles, mechanisms of asthma

Viral upper respiratory tract infections (URIs) are known to cause exacerbations of asthma. A significant and increasing body of evidence demonstrates that in large part the primary respiratory infection causing these exacerbations is rhinovirus (RV), the cause of more than 50% of URIs.1 The frequency of URI-provoked asthma makes it especially important to understand the role and mechanisms whereby RV infections lead to asthma exacerbations, the basic virologic features of RV, their ability to infect the lower airway, the host susceptibility factors, and mechanisms leading to airflow obstruction.

From the Division of Allergy and Immunology, Department of Medicine, University of Wisconsin, Madison. Received for publication April 7, 2005; revised June 3, 2005; accepted for publication June 7, 2005. Available online July 5, 2005. Reprint requests: William W. Busse, MD, Department of Medicine, K4/912 CSC-9988, 600 Highland Avenue, Madison, WI 53792. E-mail: wwb@ medicine.wisc.edu. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.06.003

Abbreviations used G-CSF: Granulocyte colony-stimulating factor ICAM: Intercellular adhesion molecule IL-1ra: IL-1 receptor antagonist RV: Rhinovirus URI: Upper respiratory tract infection

WHAT ROLE DOES RV PLAY IN ASTHMA EXACERBATIONS? Asthma exacerbations are most commonly precipitated by viral URIs, particularly with RV,2 and often occur despite concurrent use of appropriate controller medications. Detecting respiratory viruses—in particular, RV— by culture methodology alone has been insensitive and has previously underestimated the role of respiratory viruses in asthma exacerbations, especially in adults. Viral detection rates in asthma exacerbations have significantly increased with the use of sensitive methods and have thus underscored the overwhelming importance of respiratory viruses in asthma exacerbations. When RT-PCR is used to supplement conventional culture techniques, viruses have been found in approximately 80% of wheezing episodes in school-age children and in approximately one half of the acute wheezing episodes in adults. Of the respiratory viruses identified in these circumstances, RV is most commonly found and is detected 65% of the time.2,3 A pivotal study by Johnston and colleagues2 in children aged 9-11 years old with histories of asthma symptoms found that 80% to 85% of asthma exacerbations that were associated with reduced peak expiratory flow rates and wheezing were due to viral URIs. Without the use of RT-PCR, the authors reported, the viral detection rate in this study would have been only around 40%. Similarly, high rates of asthma attacks due to RV were found in adults. Nicholson et al3 reported on 138 young adults with asthma recruited from general practice, the hospital, and the community. In this longitudinal study, 80% of asthma episodes (223 of 280), described as symptoms of wheeze, chest tightness, or breathlessness, were associated with colds. Objectively, viruses were detected in 57% of people with symptomatic colds and asthma exacerbations. In more severe asthma exacerbations with reductions in peak flow measurements of 267

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increased polymorphonuclear neutrophils are seen in infected nasal epithelium,8 little or no mucosal damage occurs from the infection; this suggests that RV is likely to cause asthma exacerbations by mechanisms other than direct cellular killing.9 Even with large inoculating doses of virus, less than 10% of cells in primary airway epithelium cultures become infected. However, although RVinduced cytotoxicity is difficult to detect in vivo, an in vitro study has demonstrated cytopathic effects when high titers of virus are inoculated with sparsely seeded monolayer cultures of human bronchial epithelial cells.10 Moreover, the RV serotype might also be an important determinant of this in vitro–detected cytotoxicity.

ARE RV INFECTIONS LIMITED TO THE UPPER AIRWAY? FIG 1. Transverse section through the center of a pentamer depicting entry of its cellular receptor, ICAM-1, and the location of the drug-binding pocket just beneath the canyon floor. An ion, located at each pentamer center in RV-1A, 214, 216 is tentatively identified as calcium, which is necessary for attachment of some RVs. Modified with permission from Gwaltney JM. Rhinovirus. In: Mandell GL, Douglas RG, Bennett JE, Dolin R, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 6th ed. New York; Elsevier/Churchill Livingstone; 2005. p. 2185-94.

50 L/min, viruses were detected in 44% of episodes. In comparison with detection rates by cell culture, RT-PCR was 5 times more sensitive in identifying human RV in adults with respiratory infections.3 Further evidence to support the role of viral infection in asthma exacerbations also includes reports that peaks in hospital admissions for asthma significantly correlate with seasonal patterns of viral URIs.4 In the United States, RV infection occurs most commonly in the fall and spring.5 Thus, current evidence strongly supports the concept that RV respiratory infections are the major cause of acute asthma exacerbations.

WHAT ARE THE VIROLOGIC FEATURES OF RVS? The genera RV and Enterovirus are classified within the family Picornaviridae. There are more than 100 serotypes of RV; this explains, in part, the lack of an effective vaccine against the major etiologic agent causing the common cold. RV is a small, single-stranded RNA virus whose capsid contains 4 proteins (Fig 1). Three of these proteins, VP1, VP2, and VP3, are located on the surface of the capsid and are responsible for its antigenic diversity; the fourth, VP4, is located inside the virus and anchors the RNA core to the viral capsid.1 The majority of RV serotypes bind to intercellular adhesion molecule (ICAM) 1, whereas approximately 10% bind to the low-density lipoprotein receptor.6,7 Typically, RV infects small clusters of cells in the epithelial layer with little cellular cytotoxicity. Although

An infection with RV leads to symptoms of the common cold, which is primarily an upper airway illness. Because RV is primarily an infection of the upper airway, early research efforts were directed toward determining whether (a) RV infections could infect the lower airways directly and provoke asthma, (b) their actions on asthma occurred via indirect mechanisms due to the upper airway infection only, or (c) a combination of the 2 methods is responsible. Insight into these questions could suggest potential target areas to act therapeutically to prevent or treat an asthma exacerbation. Debate initially focused on whether RV could exist and replicate in the lung to directly cause lower airway inflammation. This was based on limited studies demonstrating that RV replication was optimal at 33°C, the temperature of the upper airways. To address this issue, direct thermal mapping of the lower airways was performed. While human subjects breathed room air (26°C), the temperature in the subjects averaged 32°C in the upper trachea and 35.5°C in the subsegmental bronchi. These findings refuted a possible limitation of RV growth due to higher temperatures in the lower airway.11 Moreover, with the use of multiple RV serotypes, it was possible to detect high viral titers in cell cultures at 37°C; little significant difference in replication was found when wild-type RV isolates were used at 33°C compared to 37°C. In fact, some serotypes grew more effectively at the higher temperature.10 In addition, when primary cultures of lower airway bronchial epithelial cells and upper airway adenoidal epithelial cells were used, RV appeared to infect both upper and lower segments of the respiratory tree with similar ability (Fig 2).9 Several additional lines of evidence support the ability of RV to infect the lower airways directly. When bronchoscopy was used to collect samples from subjects with symptomatic experimental infections, RV was detected from bronchial brush specimens.12 Furthermore, with the use of RT-PCR and Southern blotting, RV genetic material was found in higher amounts in cells of bronchial alveolar lavage fluid than in supernatant; this suggests that the virus was located intracellularly.13 However, the role

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FIG 2. Immunohistochemistry of airway tissue from experimentally infected normal human surgical specimens infected ex vivo. A, Bronchial tissue specimens were inoculated ex vivo and were incubated in tissue culture medium for 24 hours. After washing to eliminate extracellular virus, the tissue was embedded in paraffin, sectioned, and stained for the presence of RV serotype 16. B, Uninfected bronchial tissue specimen was processed as in panel A. C, Adenoidal tissue specimen infected ex vivo for 6 hours. Bar: 50 mm. Reprinted with permission from Mosser AG, Brockman-Schneider R, Amineva S, Burchell L, Sedgwick JB, Busse WW, et al. Similar frequency of rhinovirus-infectible cells in upper and lower airway epithelium. J Infect Dis 2002;185:734-43.

of contamination from the upper airway could not be definitively excluded in these studies. Another investigation found RV-16 RNA in 50% of bronchial biopsies in experimentally inoculated human volunteers.10 In this study, in situ hybridization was used to localize viral RNA by hybridizing the sequence of interest to the complementary replicative strand of the virus. This technique likely excludes the possibility of contamination of the lower airway with virus from the upper airway. This was further supported by the finding of viral replication in the lower airway as well as increases in viral RNA and the production of new viral proteins. Furthermore, the frequency of lower airway infection was similar to that observed in the upper airway; this indicates that infection of the lower airways might be relatively common as part of the natural history of RV infection. Finally, a recent study showed that an experimental RV infection was associated with virus detection in large lower airways biopsy samples by immunohistochemistry or qPCR in 17 of 19 subjects, but less so in the distal airways.14 Thus, RV is able to infect both the upper and lower airways. It is likely that the lower airways are infected as a result of self-inoculation from coughing, sneezing, or perhaps breathing. Whether the concentration of virus in the lower airways is large enough to produce clinically relevant effects is still not established. These studies support the concept that RV is a lower as well as an upper respiratory tract pathogen, and infection of the lower

airway directly likely contributes to viral-induced exacerbations of asthma.

WHAT ARE THE EFFECTS OF RV INFECTION ON THE MECHANISMS OF AIRWAY PHYSIOLOGY IN ASTHMA? Multiple studies demonstrate the adverse effects of RV on airway physiology in asthma. In school-age children, symptoms of either upper or lower respiratory tract infection were shown to last a week, and during these infectious episodes, the peak flow rates fell for a median duration of 2 weeks.2 In another study, asthmatic subjects were experimentally inoculated with RV-16 and found to demonstrate modest changes in increased airway hyperresponsiveness, airway obstruction, and inflammation.15 Experimental RV-16 infection also has been shown to reduce FEV1 in patients with mild asthma.16 In addition, increases in existing airway inflammation have occurred after segmental bronchoprovocation in atopic subjects, suggesting that enhanced airway inflammation is a feature of RV-associated asthma exacerbations.17 To support this possibility, subjects with allergic rhinitis, but not with active asthma, were inoculated with RV; they were found to have significantly increased airway hyperreactivity as well as a significantly increased incidence of late asthmatic reactions, defined as a 15% decrease in FEV1 approximately 6 hours after antigen

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Asthma diagnosis and treatment FIG 3. RV induces epithelial cells to produce proinflammatory cytokines leading to airway hyperresponsiveness, neurogenic inflammatory responses, mucous secretion, inflammatory cell recruitment and activation, and plasma leakage. Created with information from Gern JE. Rhinovirus respiratory infections and asthma. Am J Med 2002;112(Suppl 6A):19S-27S and from Yamaya M, Sasaki H. Rhinovirus and asthma. Viral Immunol 2003;16:99-109.

challenge.18 Before infection, only 1 patient in this study had a late asthmatic reaction. During the acute infection, this number increased to 8 of 10 subjects (P = .0085). The effect occurred independently of the enhancement in airway reactivity experienced during a cold alone. This demonstrates that in addition to causing airway hyperreactivity, RV also promotes the development of late-phase responses, even in nonasthmatic patients. RV infection also promotes eosinophil recruitment to airway segments after antigen challenges. Calhoun et al17 used segmental bronchoprovocation with antigen after inoculation with RV-16 in subjects with allergic rhinitis. After infection, bronchial alveolar lavage fluid revealed enhanced histamine release immediately and increased eosinophil recruitment 48 hours after antigen challenge. Interestingly, the increase in eosinophils persisted for up to 1 month after infection in some subjects. The effect of RV on airway inflammation appeared to be an augmentation of allergen-specific responses. Thus, enhancement of antigen-induced mediator release from pulmonary mast cells and basophils and eosinophilic recruitment, either directly or via cytokines, could provide one mechanism by which late allergic reactions and airway hyperresponsiveness are enhanced by viral uncoating and might act to intensify the airway inflammatory response to allergen. Conversely, when nasal allergen challenges in atopic patients were performed before experimental RV inoculation, the onset of cold symptoms was delayed and the responses were less severe in comparison with what was seen in patients without allergies.19 Delayed nasal inflammation, with attenuation of the increase in IL-6, IL-8, and neutrophils seen in infection, was also found in the group primed with nasal antigen challenge. This might have been due to cytokine profile changes with increased expression of IFN-g and IL-2, local production of nitric

oxide, or antiviral effects of eosinophil products. Thus, the timing and intensity of antigen exposure play an important role in the severity level and subsequent possible complications of a cold.

HOW DOES RV MODULATE INFLAMMATORY MEDIATORS OF EPITHELIAL CELLS CONTRIBUTING TO ASTHMA EXACERBATIONS? Epithelial cells are the principal targets of RV infections, allow viral replication, and likely initiate immune responses (Fig 3).20,21 Papadopoulos et al10 found local induction of proinflammatory mediators that could provide a mechanism to explain how lower airway infection can lead to inflammation and asthma. RV infection resulted in an increase in mRNA expression and subsequent translation of IL-6, IL-8, and IL-16. This also occurred with RANTES, a C-C chemokine with chemoattractant activity for eosinophils, monocytes, and T lymphocytes. IL-6 and IL-8 are proinflammatory cytokines, and IL-8 is a specifically potent chemoattractant for neutrophils. IL-16 is a powerful lymphocyte chemoattractant and activator of macrophages and eosinophils and appears to be an important mediator in the pathogenesis of asthma and lower airway inflammation due to RV.10 The inflammatory actions of RV appear to center on its ability to generate a variety of phlogistic mediators. Generation of these cytokines correlates with the worsening of respiratory physiology. For example, IL-1 enhances airway smooth muscle contraction in response to bronchospastic agents and attenuates smooth muscle dilation responses to bronchodilators.22,23 Differences in immune response, such as the modulation of costimulatory molecules and the induction of antigen presentation,

might explain how RV infections cause acute exacerbations in asthmatic patients. Virus-induced epithelial damage might cause increased permeability of the mucosal layer and thus increase allergen contact with immune cells to promote neurogenic inflammation. In addition, viruses can enhance vagally mediated reflex bronchoconstriction, possibly by limiting the function of the M2 muscarinic receptor.24 Viral replication activates epithelial cells to initiate innate and adaptive immune responses as well as the generation of oxidative stress.25 Also, double-stranded RNA synthesized in virus-infected cells induces the cytokines IL-8 and RANTES, which initiate proinflammatory and antiviral pathways within the cell.24 Upregulation, or activation, of ICAM-1, the principal receptor for RV, might increase tissue susceptibility to the major group RV and subsequent infection. The asthma phenotype, which is associated with increased ICAM-1 expression, might therefore be associated with increased susceptibility and complications from RV infection.21 Chronic antigen challenge can also increase ICAM-1 expression of the airway epithelium, and RV infection itself can increase ICAM-1 expression through production of IL-1b and a nuclear factor-kb–dependent mechanism. This might lead to the amplification of airway inflammation after RV infection.21,26 In addition, RV might enhance existing inflammation to a greater degree in asthmatic subjects than in those without the disease. For example, after inoculation with the virus, nasal lavage levels of IL-8 and the proinflammatory mediator IL-1b were increased in asthmatic patients.27 In this study, a small increase in the anti-inflammatory marker IL-1 receptor antagonist (IL-1ra), a competitive inhibitor of IL-1, also occurred in asthmatic subjects treated with budesonide, whereas lower levels were found in these patients at baseline. These findings suggest that RV might be able to alter the proinflammatory/antiinflammatory balance of IL-1b/IL-1ra toward inflammation more markedly in people with existing and active asthma. Cytokine response profiles generated by RV might translate into neutrophilic inflammation in both the upper and lower airways. Local RV infection is associated with increased levels of IL-8, a potent chemoattractant for neutrophils, and also granulocyte colony-stimulating factor (G-CSF) in nasal secretions and later in the circulation. Increased concentrations of circulatory G-CSF could act on the bone marrow to increase the circulating neutrophils. Thus, a local response in nasal epithelium to RV infection can result in a systemic inflammatory reaction.20 Elevated neutrophil counts are also found in the lower airways with RV infection. Through use of bronchoscopy and bronchial washes, significant increases in airway lumen neutrophils were found 96 hours after inoculation with RV-16 in patients with allergic asthma.28 Infected bronchial epithelium induces the secretion of proinflammatory cytokines, including IL-1, IL-8, TNF-a, IL-10, and IFN-a, as well. This stimulates the recruitment of inflammatory cells and neutrophilia. Products of neutro-

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TABLE I. Risk factors for severe lower respiratory tract complications from rhinovirus infection Asthma Atopy Elevated nasal eosinophils or eosinophil cationic protein Infants and elderly Low neutralizing antibody titers to rhinovirus Chronic lung diseases Smoking Low IFN-g producers High IL-5 producers Modified with permission from Gern JE. Rhinovirus respiratory infections and asthma. Am J Med 2002;112(Suppl 6A):19S-27S.

phil activation could cause airway obstruction through the production of elastase, which also upregulates goblet cell mucus secretion.29

WHAT ARE THE RISK FACTORS FOR WHEEZING WITH A COLD? Various risk factors increase the susceptibility of subjects for more severe lower respiratory complications from an RV infection, such as wheezing, bronchitis, and pneumonia (Table I). These include having low neutralizing antibody titers to RV, being an infant, being elderly, having chronic lung disease, being a smoker, and being an individual with existing asthma.22 In addition, subjects who are low producers of IFN-g in response to RV and are atopic appear to be more at risk for wheezing or having a severe respiratory infection. Brooks et al30 demonstrated that whereas RV induces IFN-g, which is consistent with a strong TH1-like immune response, those asthmatic patients with diminished, or deficient, TH1 responses to RV were characterized by increased airway hyperresponsiveness. Moreover, the ratio of RV-16–induced IFN-g:IL-5, a measure of TH1:TH2 balance, correlated with FEV1. These findings are similar, in general, to what is known about TH1 and TH2 responses in asthma. In another study, subjects with persistent and severe asthma displayed a defect in IFN-g production, whereas their increased IL-5 responses were felt to reflect the presence of atopy but were not specifically linked to asthma itself.31 Collectively, these findings demonstrate that a deficiency of the TH1 response, rather than an increased TH2 response, is responsible for RV’s adverse effect on the airways. The importance of IgE and eosinophilic airway inflammation was demonstrated by a study showing synergistic interactions between RV infection and allergic airway inflammation.32 In this study, which focused on children aged 2-16 years old, the odds ratios for wheezing with RV detected by RT-PCR in addition to positive radioallergosorbent test results, nasal eosinophilia, and elevated nasal eosinophil cationic protein were 17, 21, and 25, respectively. The odds ratios for wheezing with any of these 4 risk factors alone were much lower, between 3.2 and

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8; this shows the importance of IgE and eosinophil-driven inflammatory responses. Zambrano et al33 reported that the existence of airway inflammation prior to virus inoculation predisposed subjects to a more deleterious response to RV. Patients were inoculated with RV-16, and compared with those without asthma, those with mild asthma demonstrated increased airway hyperresponsiveness, decreased FEV1 at baseline, and increased upper and lower respiratory tract symptom scores in response to the infection. Asthmatic patients with elevated IgE profiles also demonstrated higher blood eosinophil counts, increased eosinophil cationic protein in nasal washes, and both an increased expired nitric oxide, a marker of inflammation, and decreased soluble ICAM-1 in nasal washes at baseline and during cold symptoms. These findings suggest that patients with asthma, who are highly atopic, might be more likely to have increased levels of airway inflammation and be at greater risk for asthma exacerbations in response to RV infection.

SUMMARY The importance of RV in asthma exacerbations is established in both adults and children. The complex mechanisms by which their interaction provokes asthma are becoming better understood. RV appears to have a direct and negative impact on the lower airways and causes an increase in obstructive airway symptoms and physiology. This effect on airway function is felt to occur as the virus upregulates proinflammatory cytokines and predisposes the asthmatic patient to more severe respiratory infections and hence to exacerbations. Defects in TH1-type immune responses appear to be an important factor in causing airway inflammation in people with asthma. Further work is needed to better explore the mechanisms behind the association between asthma exacerbations and RV infections. This might ultimately lead to treatment modalities to prevent and/or treat the significant burden of asthma exacerbations caused by RV infection.

REFERENCES 1. Greenberg SB. Respiratory consequences of rhinovirus infection. Arch Intern Med 2003;163:278-84. 2. Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, et al. Community study of role of viral infections in exacerbations of asthma in 9-11 year old children. BMJ 1995;310:1225-9. 3. Nicholson KG, Kent J, Ireland DC. Respiratory viruses and exacerbations of asthma in adults. BMJ 1993;307:982-6. 4. Johnston SL, Pattemore PK, Sanderson G, Smith S, Campbell MJ, Josephs LK, et al. The relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis. Am J Respir Crit Care Med 1996;154(3 Pt 1):654-60. 5. Arruda E, Pitkaranta A, Witek TJ Jr, Doyle CA, Hayden FG. Frequency and natural history of rhinovirus infections in adults during autumn. J Clin Microbiol 1997;35:2864-8. 6. Casasnovas JM. The dynamics of receptor recognition by human rhinoviruses. Trends Microbiol 2000;8:251-4.

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7. Gwaltney JM. Rhinovirus. In: Mandell GL, Douglas RG, Bennett JE, Dolin R, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 6th ed. New York: Elsevier/Churchill Livingstone; 2005. p. 2185-94. 8. Winther B, Farr B, Turner RB, Hendley JO, Gwaltney JM Jr, Mygind N. Histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds. Acta Otolaryngol Suppl 1984;413:19-24. 9. Mosser AG, Brockman-Schneider R, Amineva S, Burchell L, Sedgwick JB, Busse WW, et al. Similar frequency of rhinovirus-infectible cells in upper and lower airway epithelium. J Infect Dis 2002;185:734-43. 10. Papadopoulos NG, Bates PJ, Bardin PG, Papi A, Leir SH, Fraenkel DJ, et al. Rhinoviruses infect the lower airways. J Infect Dis 2000;181: 1875-84. 11. McFadden ER Jr, Pichurko BM, Bowman HF, Ingenito E, Burns S, Dowling N, et al. Thermal mapping of the airways in humans. J Appl Physiol 1985;58:564-70. 12. Halperin SA, Eggleston PA, Hendley JO, Suratt PM, Groschel DH, Gwaltney JM Jr. Pathogenesis of lower respiratory tract symptoms in experimental rhinovirus infection. Am Rev Respir Dis 1983;128:806-10. 13. Gern JE, Galagan DM, Jarjour NN, Dick EC, Busse WW. Detection of rhinovirus RNA in lower airway cells during experimentally induced infection. Am J Respir Crit Care Med 1997;155:1159-61. 14. Mosser AG, Vrtis R, Burchell L, Lee WM, Dick CR, Weisshaar E, et al. Quantitative and qualitative analysis of rhinovirus infection in bronchial tissues. Am J Respir Crit Care Med 2005;171:645-51. 15. Fraenkel DJ, Bardin PG, Sanderson G, Lampe F, Johnston SL, Holgate ST. Lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects. Am J Respir Crit Care Med 1995;151(3 Pt 1):879-86. 16. Grunberg K, Timmers MC, de Klerk EP, Dick EC, Sterk PJ. Experimental rhinovirus 16 infection causes variable airway obstruction in subjects with atopic asthma. Am J Respir Crit Care Med 1999;160:1375-80. 17. Calhoun WJ, Dick EC, Schwartz LB, Busse WW. A common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects. J Clin Invest 1994;94:2200-8. 18. Lemanske RF Jr, Dick EC, Swenson CA, Vrtis RF, Busse WW. Rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions. J Clin Invest 1989;83:1-10. 19. Avila PC, Abisheganaden JA, Wong H, Liu J, Yagi S, Schnurr D, et al. Effects of allergic inflammation of the nasal mucosa on the severity of rhinovirus 16 cold. J Allergy Clin Immunol 2000;105:923-32. 20. Gern JE. Rhinovirus respiratory infections and asthma. Am J Med 2002; 112(Suppl 6A):19S-27S. 21. Yamaya M, Sasaki H. Rhinovirus and asthma. Viral Immunol 2003;16: 99-109. 22. Gern JE, Busse WW. Association of rhinovirus infections with asthma. Clin Microbiol Rev 1999;12:9-18. 23. Hakonarson H, Carter C, Maskeri N, Hodinka R, Grunstein MM. Rhinovirus-mediated changes in airway smooth muscle responsiveness: induced autocrine role of interleukin-1beta. Am J Physiol 1999;277(1 Pt 1):13-21. 24. Gern JE. Mechanisms of virus-induced asthma. J Pediatr 2003;142: (2 Suppl):S9-13. 25. Kaul P, Biagioli MC, Singh I, Turner RB. Rhinovirus-induced oxidative stress and interleukin-8 elaboration involves p47-phox but is independent of attachment to intercellular adhesion molecule-1 and viral replication. J Infect Dis 2000;181:1885-90. 26. Terajima M, Yamaya M, Sekizawa K, Okinaga S, Suzuki T, Yamada N, et al. Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1beta. Am J Physiol 1997;273(4 Pt 1):749-59. 27. de Kluijver J, Grunberg K, Pons D, de Klerk EP, Dick CR, Sterk PJ, et al. Interleukin-1beta and interleukin-1ra levels in nasal lavages during experimental rhinovirus infection in asthmatic and non-asthmatic subjects. Clin Exp Allergy 2003;33:1415-8. 28. Jarjour NN, Gern JE, Kelly EA, Swenson CA, Dick CR, Busse WW. The effect of an experimental rhinovirus 16 infection on bronchial lavage neutrophils. J Allergy Clin Immunol 2000;105(6 Pt 1):1169-77. 29. Cardell LO, Agusti C, Takeyama K, Stjarne P, Nadel JA. LTB(4)induced nasal gland serous cell secretion mediated by neutrophil elastase. Am J Respir Crit Care Med 1999;160:411-4. 30. Brooks GD, Buchta KA, Swenson CA, Gern JE, Busse WW. Rhinovirus-induced interferon-gamma and airway responsiveness in asthma. Am J Respir Crit Care Med 2003;168:1091-4.

31. Smart JM, Horak E, Kemp AS, Robertson CF, Tang ML. Polyclonal and allergen-induced cytokine responses in adults with asthma: resolution of asthma is associated with normalization of IFN-gamma responses. J Allergy Clin Immunol 2002;110:450-6. 32. Rakes GP, Arruda E, Ingram JM, Hoover GE, Zambrano JC, Hayden FG, et al. Rhinovirus and respiratory syncytial virus in wheezing children

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requiring emergency care. IgE and eosinophil analyses. Am J Respir Crit Care Med 1999;159:785-90. 33. Zambrano JC, Carper HT, Rakes GP, Patrie J, Murphy DD, Platts-Mills TA, et al. Experimental rhinovirus challenges in adults with mild asthma: response to infection in relation to IgE. J Allergy Clin Immunol 2003; 111:1008-16.

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Perspectives in asthma Guest editor: William W. Busse, MD Asthma diagnosis and treatment

Perspectives on the past decade of asthma genetics Carole Ober, PhD Chicago, Ill

Although genetic linkage and association studies have identified more than 25 asthma or allergy susceptibility loci, replication of significant results remains a problem. Moreover, these approaches typically ignore the true complexity of these diseases, such as the role of gene-by-environment and gene-bygene interactions. As a result, many important associations might have been missed. Recent studies demonstrate not only that such interactions exist but also that the relationship between genotype and phenotype is more complex than previously thought. (J Allergy Clin Immunol 2005;116:274-8.) Key words: Asthma, allergy, genetics, gene-by-environment interactions

EXTENDING THE MENDELIAN PARADIGM TO COMPLEX DISEASES The search for genes that influence susceptibility to common diseases remains one of the greatest challenges in human genetics. With the recent completion of the human genome project,1 the tools are now available to fully meet this challenge and to redefine medicine in the 21st century. The ultimate goals of molecular medicine are both to identify genetically susceptible individuals and intervene before the onset of disease and to design drugs that are individualized and genotype specific. Although there have been countless successes with respect to defining the molecular basis of Mendelian (monogenic) diseases,2 genetic studies of common diseases with complex causes have turned out to be considerably more challenging than originally thought. In this perspective I will provide a brief update on the status of genetic studies of asthma and allergy and then discuss some of the insights that have

From the Departments of Human Genetics and Obstetrics and Gynecology, The University of Chicago. Supported in part by National Institutes of Health grants HL56399, HL66533, HL70831, and HL72414. Disclosure of potential conflict of interest: All authors—none disclosed. Received for publication April 19, 2005; accepted for publication April 25, 2005. Available online June 30, 2005. Reprint requests: Carole Ober, PhD, Department of Human Genetics, The University of Chicago, 920 East 58th St, CLSC 507C, Chicago, IL 60637-1463. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.039

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Abbreviations used AD: Atopic dermatitis ADRB2: b2-Adrenergic receptor CD14: Monocyte differentiation antigen 14 COAST: Childhood Onset of Asthma Study FCERB1: FceRb1 GSTM1: Glutathione S-transferase M1 GSTP1: Glutathione S-transferase P1 HLAG: Human leukocyte antigen G IL4RA: IL-4 receptor, a-chain LTA: Lymphotoxin a LTC4A: Leukotriene C4 synthase NOS3: Nitric oxide synthetase 3 TIM1: T-cell immunoglobulin- and mucin domain–containing molecule 1 TLR4: Toll-like receptor 4

been gained over the past 10 years on the genetic architecture of these traits.

LINKAGE AND ASSOCIATION STUDIES IDENTIFY SUSCEPTIBILITY GENES Fig 1 shows a common model of susceptibility to asthma and atopy, which implicates many genes and many environmental factors but implies that the effects of genes and environmental factors individually contribute to risk. However, the truth is much more complex, with genes interacting both with other genes and with environmental risk factors to confer susceptibility. In fact, few genes might have independent effects, as is typical for Mendelian diseases. Nonetheless, the approaches that have been used to find susceptibility genes, either through linkage or association studies, have for the most part considered one gene at a time (Fig 2). Despite this overly simplistic modeling of asthma and atopy genetics, many important discoveries have been made (Fig 3). In particular, 5 genes have been identified through family linkage studies, followed by positional cloning.3-7 These genes span a wide range of functions and in all cases were either unknown or would not have been considered as candidate asthma genes before their discovery. Among more than 100 genes that have been

FIG 1. Common model of the genetics of complex diseases. Several related disease and quantitative phenotypes result from the effects of many loci and many environmental factors. BHR, Bronchial hyperresponsiveness.

interrogated through association studies (reviewed in Hoffjan et al8), 8 genes have been replicated in more than 5 studies, and another 13 genes have been replicated but in fewer than 5 studies. These 26 genes are likely to be true susceptibility loci but represent just the tip of the iceberg because additional positionally cloned genes are soon to be reported, and many other candidate genes will be identified and replicated. Thus one could conclude that the field of asthma genetics has been quite successful and that many genes have been identified that contribute to risk.

THE PROBLEM OF REPLICATION Replicating results remains the gold standard for genetic association studies, but this has proved difficult for common diseases, such as asthma, irrespective of whether the initial association was identified through candidate gene association or positional cloning studies. Even among the most replicated genes, including those shown in Fig 3, there are many negative studies (for examples, see Table 1 in Hoffjan et al8). In fact, there are no genes that are associated with asthma, atopy, or a related phenotype in every study reported. Moreover, even when a gene is replicated, it is often with a different phenotype (eg, a polymorphism in intron 1 of the LTA gene is associated with asthma in some studies and IgE in others), with different polymorphisms in the same gene (eg, the 21112C/T promoter polymorphism in the IL13 gene is associated with atopic asthma in some studies, but the Arg130Gln polymorphism in exon 4 of the same gene is associated with asthma and atopic phenotypes in others), and even with different alleles of the same variant (eg, the 2159C allele in the promoter region of the CD14 gene is associated with atopic phenotypes in some populations, whereas the 2159T allele is associated in others). This level of complexity was unexpected and has suggested that models of susceptibility that consider one locus at a time, as is the paradigm for Mendelian diseases, are not adequate for discovering and characterizing asthma and allergy susceptibility loci. Rather, models that include interactions between genes and between genes and environmental risk factors might be required to fully elucidate the genetic architectures of asthma and atopy.

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FIG 2. Strategies for identifying disease genes. A, With the candidate gene approach, typically used in association studies, a gene is selected on the basis of its known function (ie, functional candidate gene). Variation in that gene is then examined for associations in patients and control subjects, cohorts of individuals, or families. Ultimately, the mechanism of the association is revealed through genotype-phenotype studies and functional studies of the associated variant. B, In positional cloning studies initially only information on the chromosomal location is known, usually from family linkage studies. All genes in the linked region become positional candidates, and association studies are performed as described above to identify the associated gene and variation that contributes to disease risk. Once the variation is identified by means of association studies, the mechanism of the association is studied as described above.

GENE-BY-ENVIRONMENT INTERACTIONS AND ASTHMA We and others have recently begun to examine interactions between individual genotypes and environmental exposures as a first step in developing more complex models of disease susceptibility. These models consider the possibility that specific genotypes might result in a phenotype only in certain environments or that a specific genotype might result in different phenotypes, depending on environmental exposures. Such interactions could mask associations if the study sample is heterogeneous with respect to the exposure or underlie discrepant results between samples drawn from populations that differ with respect to the exposure. A classic example of a gene-byenvironment interaction is that of the Mendelian disease a1-antitrypsin deficiency. The risk for respiratory diseases, such as emphysema and chronic obstructive pulmonary disease, among homozygotes for the PiZ null allele (ZZ genotype) is nearly 100% in the presence of cigarette smoke exposure. In this case the exposure is thought of as a trigger of disease in genetically susceptible individuals. Other examples of gene-by-environment interactions on asthma and atopy risk have been recently reported,7,9-16 and these suggest that such effects might be more the rule than the exception. These studies are summarized in Table I and in all cases provide examples in which genotypespecific effects are modified by environmental exposures. Although not all of these interaction effects have been replicated, they provide the basis for future studies and for characterizing the range of effects of important environmental exposures as modifiers of disease risk.

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Asthma diagnosis and treatment FIG 3. Approximate locations of asthma and atopy genes on human chromosomes. Five genes identified through linkage followed by positional cloning studies are shown in red. Twenty-one genes that were identified through association studies and replicated in subsequent studies are also shown (summarized from Hoffjan et al8). Eight genes that have been replicated in more than 5 studies are shown in blue, and 13 genes that have been replicated but in fewer than 5 studies are shown in black.

THE IMPORTANCE OF EARLY-LIFE EXPOSURES Epidemiologic studies have identified many environmental factors that influence risk for asthma and allergic disease, such as maternal asthma, birth order, and sibship size and early-life exposure to viral infections, endotoxin (LPS), day care, pets, and allergens. Yet few studies to date have examined how exposure to environmental risk factors during development modifies genotype-specific risks for asthma and allergic disease. The Childhood Onset of Asthma (COAST) Study is a prospective birth cohort study of high-risk children designed to evaluate the role of genes and environment on the development of immune responsiveness and allergic phenotypes.17 The first studies in this cohort to examine gene-by-environ-

ment interactions were recently reported, providing some intriguing examples of interactions. A study of the effects of dog ownership on the development of immune responsiveness and atopy in infancy revealed a protective effect of having a dog in the house at the time the child was born: only 30% of infants had atopic dermatitis (AD) if a pet was present in the home compared with 51% of infants in homes without a dog (P < .0001).14 However, this difference was even more striking among children with the 2159TT genotype at the locus encoding the receptor for LPS, CD14: only 5% of TT children exposed to a dog had AD compared with 43% of unexposed TT children (P =.04). In this example, even though both the polymorphism (CD14 2159C/ T) and the environmental exposure (dog) independently influenced risk for AD, the interaction between the 2 was

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TABLE I. Examples of gene-by-environment interaction effects on asthma and atopic disease Environmental Exposure

Phenotype

LTC4S

Aspirin exposure

Asthma

ADRB2

Cigarette smoke

Asthma

ADRB2

Physical activity

Asthma

TIM1

HAV

Atopy

TLR4

Endotoxin levels

Asthma

CD14

Dog ownership at birth

AD

GSTM1

Diesel exhaust particles

IgE and histamine response

GSTP1

Diesel exhaust particles

IgE and histamine response

NOS3

Day-care exposure in the first 6 mo of life

Change in TH2 cytokine (IL-5 and IL-13) response in first year of life

FCERB1

Day-care exposure in the first 6 mo of life

IL-5 response at 1 y of age

IL4RA

Day-care exposure in the first 6 mo of life

IFN- g response at 1 y of age

HLAG

Maternal BHR

Asthma-BHR in child

Comment

2444C allele is increased among individuals with aspirin-induced asthma compared with individuals with aspirin-tolerant asthma Increased risk of asthma among smokers with Arg16 genotype but not among nonsmokers Increased risk of asthma among sedentary women with Gly16 genotype but not among active women HAV protects against atopy in individuals with a 6-amino-acid insertion at residue 157 (157insMTTTVP) but not in individuals without the insertion At high levels of endotoxin exposure, carriers of the Gly299 and Ile399 alleles have reduced risk for asthma compared with other genotypes and other exposure groups 2159TT genotype is protective against AD in the first year among children with a dog in the home at birth Enhanced responses among GSTM1-null individuals but not among individuals with other genotypes Enhanced responses among individuals with the Ile105 allele but not among individuals without this allele Asp298 homozygosity associated with smallest changes in TH2 responses among children attending day care and largest changes among children not attending day care Gly237 allele associated with decreased IL-5 responsiveness among children attending day care and increased responsiveness among children not attending day care Val50 homozygosity associated with lowest response among children attending day care and highest response among children not attending day care 2964G allele is associated with asthma in children of mothers with BHR; 2964A allele is associated with atopy and asthma among children of mothers without BHR

Reference

Sanak et al9

Wang et al10

Barr et al11

McIntire et al12

Werner et al13

Gern et al14

Gilliland et al15

Gilliland15

Hoffjan et al16

Hoffjan et al16

Hoffjan et al16

Nicolae et al7

HAV, Hepatitis A; BHR, bronchial hyperresponsiveness.

significant (P = .0071), indicating that the risk associated with the TT genotype differs in different exposure groups. Interactions between CD14 genotype and levels of endotoxin exposure have been suggested as an explanation for the discrepant results of association studies with this polymorphism,18 as discussed earlier, and these data support that hypothesis. In a second study in this cohort, the effects of day-care attendance in the first 6 months of life on cytokine response profiles and allergic phenotypes were examined.16 Seventy-two polymorphisms in 35 genes were selected because of their putative role in immune re-

sponses or asthma and genotyped in 99 COAST children who attended day care and 109 COAST children who did not. Interestingly, neither day-care attendance nor genotype at these loci by themselves significantly influenced any of the first-year phenotypes examined. However, highly significant interaction effects (P < .001) were demonstrated with genotypes at 3 loci: NOS3, FCERB1, and IL4RA. In each case the effects of a particular genotype on the phenotype were opposite depending on whether the child attended day care (ie, the same genotype was associated with the highest cytokine responses or protection from disease among children attending day care

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but the lowest cytokine responses or risk for disease among children not attending day care). That is, the genotype effects at these loci were modified by the environment such that the same genotype was associated with protection from or risk for a phenotype depending on this early-life exposure! In the pooled sample (not stratified by day-care attendance) there were no detectable differences between genotypes (summarized in Table I). Interestingly, the interaction effects with the FCERB1 and IL4RA genes were likely accounted for by the increased number of viral infections among children attending day care; however, the interaction effects with NOS3 were independent of viral infections, suggesting that risk factors other than viruses but that are correlated with day-care exposure interact with the NOS3 genotype to determine risk. Complex interactions such as these could underlie some of the association studies in which one allele of a polymorphism is associated in some populations and the other allele of the same polymorphism is associated with the same phenotype in others.

CONCLUDING REMARKS The mechanisms underlying these interactions are not yet known. Nonetheless, these studies and others are beginning to reveal the true complexities of the genetics of asthma and allergy. The next phase of genetic investigation should continue to unravel the nature and overall importance of gene-by-environment and gene-by-gene interactions on the development of asthma and allergic phenotypes on disease progression and severity and on the response to therapeutic interventions. Thus the next 10 years of asthma genetic research will begin to meet the goals of the new molecular medicine. I thank Dr Nancy Cox and Dr Robert Lemanske for helpful discussions.

REFERENCES 1. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature 2004;431:931-45.

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2. Glazier AM, Nadeau JH, Aitman TJ. Finding genes that underlie complex traits. Science 2002;298:2345-9. 3. Van Eerdewegh P, Little RD, Dupuis J, Del Mastro RG, Falls K, Simon J, et al. Association of the ADAM33 gene with asthma and bronchial hyperresponsiveness. Nature 2002;418:426-30. 4. Zhang Y, Leaves NI, Anderson GG, Ponting CP, Broxholme J, Holt R, et al. Positional cloning of a quantitative trait locus on chromosome 13q14 that influences immunoglobulin E levels and asthma. Nat Genet 2003;34:181-6. 5. Allen M, Heinzmann A, Noguchi E, Abecasis G, Broxholme J, Ponting CP, et al. Positional cloning of a novel gene influencing asthma from chromosome 2q14. Nat Genet 2003;35:258-63. 6. Laitinen T, Polvi A, Rydman P, Vendelin J, Pulkkinen V, Salmikangas P, et al. Characterization of a common susceptibility locus for asthmarelated traits. Science 2004;304:300-4. 7. Nicolae D, Cox NJ, Lester LA, Schneider D, Tan Z, Billstrand C, et al. Fine mapping and positional candidate studies identify HLA-G as an asthma susceptibility gene on chromosome 6p21. Am J Hum Genet 2005;76:349-57. 8. Hoffjan S, Nicolae D, Ober C. Association studies for asthma and atopic diseases: a comprehensive review of the literature. Respir Res 2003;4:14-28. 9. Sanak M, Pierzchalska M, Bazan-Socha S, Szczeklik A. Enhanced expression of the leukotriene C(4) synthase due to overactive transcription of an allelic variant associated with aspirin-intolerant asthma. Am J Respir Cell Mol Biol 2000;23:290-6. 10. Wang Z, Chen C, Niu T, Wu D, Yang J, Wang B, et al. Association of asthma with beta(2)-adrenergic receptor gene polymorphism and cigarette smoking. Am J Respir Crit Care Med 2001;163:1404-9. 11. Barr RG, Cooper DM, Speizer FE, Drazen JM, Camargo CA Jr. Beta(2)adrenoceptor polymorphism and body mass index are associated with adult-onset asthma in sedentary but not active women. Chest 2001;120: 1474-9. 12. McIntire JJ, Umetsu SE, Macaubas C, Hoyte EG, Cinnioglu C, CavalliSforza LL, et al. Immunology: hepatitis A virus link to atopic disease. Nature 2003;425:576. 13. Werner M, Topp R, Wimmer K, Richter K, Bischof W, Wjst M, et al. TLR4 gene variants modify endotoxin effects on asthma. J Allergy Clin Immunol 2003;112:323-30. 14. Gern JE, Reardon CL, Hoffjan S, Nicolae D, Li Z, Roberg KA, et al. Effects of dog ownership and genotype on immune development and atopy in infancy. J Allergy Clin Immunol 2004;113:307-14. 15. Gilliland FD, Li YF, Saxon A, Diaz-Sanchez D. Effect of glutathioneS-transferase M1 and P1 genotypes on xenobiotic enhancement of allergic responses: randomised, placebo-controlled crossover study. Lancet 2004; 363:119-25. 16. Hoffjan S, Nicolae D, Ostrovnaya I, Roberg K, Evans M, Mirel DB, et al. Gene-environment interaction effects on the development of immune responses in the 1st year of life. Am J Hum Genet 2005;76:696-704. 17. Lemanske RF. The childhood origins of asthma (COAST) study. Pediatr Allergy Immunol 2002;15:1-6. 18. Vercelli D. Learning from discrepancies: CD14 polymorphisms, atopy and the endotoxin switch. Clin Exp Allergy 2003;33:153-5.

Is it traffic type, volume, or distance? Wheezing in infants living near truck and bus traffic Patrick H. Ryan, MS,a Grace LeMasters, PhD,a Jocelyn Biagini, MS,a David Bernstein, MD,b Sergey A. Grinshpun, PhD,a Rakesh Shukla, PhD,a Kimberly Wilson, MS,a Manuel Villareal, MD,b Jeff Burkle, BS,a and James Lockey, MDa Cincinnati, Ohio

Background: Previous studies of air pollution have not examined the association between exposure to varying types, distance, and amounts of traffic and wheezing in very young infants. Objective: We sought to determine the relationship between types of traffic, traffic volume, and distance and wheezing among infants less than 1 year of age. Methods: A geographic information system and a classification scheme were developed to categorize infants enrolled in the study as living near moving truck and bus traffic (highway >50 miles per hour, >1000 trucks daily, <400 m), stop-and-go truck and bus traffic (<50 miles per hour, <100 m), or unexposed and not residing near either. Symptom data were based on health questionnaires administered to parents when the infants were 6 months of age and monthly health diaries. Results: Infants living very near (<100 m) stop-and-go bus and truck traffic had a significantly increased prevalence of wheezing (adjusted odds ratio, 2.50; 95% CI, 1.15-5.42) when compared with unexposed infants. The prevalence of wheezing among nonwhite infants was at least twice that of white infants, regardless of exposure. Infants living less than 400 m from a high volume of moving traffic, however, did not have an increased prevalence of wheezing. Conclusion: These results suggest that the distance from and type of traffic exposures are more significant risk factors than traffic volume for wheezing in early infancy. (J Allergy Clin Immunol 2005;116:279-84.) Key words: Diesel, traffic, truck, bus, wheezing, Geographic Information System, infants

From athe Department of Environmental Health, and bthe Department of Internal Medicine, Division of Immunology, University of Cincinnati. Supported by grants ES11170 and ES10957 from the National Institute of Environmental Health Sciences. Disclosure of potential conflict of interest: S. A. Grinshpun has received grants–research support from the National Institute of Environmental Health Sciences. M. Villareal has consultant arrangements with Aventis, has stock or other equity ownership with Pfizer, and is on the speakers’ bureaus for AstraZeneca, UCB Pharma, Pfizer, Aventis, and GlaxoSmithKline. There are not other potential conflicts to disclose. Received for publication March 15, 2005; revised May 9, 2005; accepted for publication May 10, 2005. Available online June 17, 2005. Reprint requests: Patrick H. Ryan, MS, Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267-0056. E-mail: ryanph@ email.uc.edu. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.014

Abbreviations used CCAAPS: Cincinnati Childhood Allergy and Air Pollution Study DEP: Diesel exhaust particle GIS: Geographic information system mph: Miles per hour OR: Odds ratio PM: Particulate matter SPT: Skin prick test

Recent articles in this journal have reviewed the epidemiology and biology of air pollution and diesel exhaust and their effects on asthma risk and respiratory function.1,2 Diesel exhaust particles (DEPs) have been studied with respect to both the development and exacerbation of allergic rhinitis and asthma because of their unique capability to enhance those TH cells that direct allergic immune responses (ie, TH2 cells) and production of IgE.3 Although the mechanism by which DEPs might influence allergy and asthma development and exacerbation is still under investigation, the immunologic effects of DEPs have been recently reviewed.4,5 DEPs are respirable particles with a large surface area per unit mass that provide an excellent medium for absorbing and transporting proteins into the peripheral airways.6 Studies have demonstrated that DEPs are capable of binding with grass pollen allergen (Lol p 1), and this might be similar with other aeroallergens.7,8 In human studies exposure to DEPs has been shown to enhance allergic nasal cytokine and inflammatory responses after direct challenge with allergen extracts.9-11 Previous studies of traffic pollutants have focused on roadways with high truck and automobile traffic and minimal bus traffic as the source of air pollution, and these studies have been conducted primarily on school-age children. The purpose of this study was to determine whether distance, volume, and/or type of traffic might be associated with wheezing in infants younger than 1 year. The hypothesis was that infants who reside near major highways with heavy truck traffic, as well as infants who reside near local roads with stop-an-go truck and bus traffic will have a significantly increased risk of wheezing 279

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when compared with infants residing far from truck and bus traffic.

METHODS Asthma diagnosis and treatment

Subject recruitment The Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) is an ongoing birth cohort study. Infants enrolled in CCAAPS were identified from birth records, and the addresses obtained from these records were geocoded with EZLocate from TeleAtlas for the ArcView Geographic Information System (GIS) 3.2 (Environmental Systems Research Institute, Redlands, Calif). The distance to the nearest major highway or interstate (defined as >1000 trucks daily) was computed for all infants by using the Geoprocessing extension. Infants whose birth records indicated residency less than 400 m or greater than 1500 m from the nearest major highway or interstate were eligible. Parents were recruited when infants were 6 months of age or older and screened for allergy symptoms.12 Parents with likely atopy were subsequently tested with skin prick tests (SPTs) with a panel of 15 common indoor and outdoor regional aeroallergens. Infants with at least one atopic parent were enrolled.

Outcome variables At the time of the parent SPT, an interviewer administered the baseline health questionnaire at a physician’s office. This questionnaire gathered demographic information, occupants in the home and their smoking status, animal ownership, and information on other possible risk factors. The general health of the infant was queried from birth until the infants’ age at enrollment. These questions were based on the well-validated International Study of Allergy and Asthma in Children questionnaire for children ages 4 to 5 years, which was adapted for use with infants.13 In addition, monthly diaries were distributed to the parents of all enrolled infants at the time of the parental SPT. These diaries recorded parental observation of the infants’ illnesses and were returned by mail monthly until the child’s first visit at age 1 year. Wheeze with a cold and wheezing without a cold were assessed on both questionnaires. Wheezing without symptoms of a cold was the outcome variable for this study. To increase the reliability of parental report of wheezing, an infant whose parent reported wheezing (without a cold) on the parent questionnaire and returned at least one monthly diary indicating the identical symptom was designated to have wheezed.

Traffic exposure classification ArcView shapefiles containing the location of all state roads, interstates, and traffic counts (both truck and car) were obtained from the Ohio Department of Transportation and the Kentucky Transportation Cabinet. Shapefiles containing the location of public transportation (bus) routes and bus counts for the city of Cincinnati and Northern Kentucky were obtained from the Cincinnati Area Geographic Information Systems database, the Northern Kentucky Area Planning Commission, the Southwest Ohio Regional Transit Authority, and the Transit Authority of Northern Kentucky. The distance to the nearest federal interstate, state route, and bus route from the primary residence of the infant at the time of parent enrollment was derived by using the Geoprocessing extension for ArcView GIS 3.2. A traffic exposure classification scheme was subsequently applied to each infant by using the distance to the 3 types of traffic, the speed limit on the roadway closest to the infant, and the amount of DEPs producing traffic (trucks or buses) on the road type nearest each infant. Classification of exposure by distance was based on the methods of others, with distances of less than 100 m,14,15 150 m,16,17 200 m,18

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and 400 m.19 Fewer than 3% (n = 10) of residences of infants in this study, however, were within 100 m of an interstate; fewer than 10% (n = 88) were within 200 m of an interstate, whereas 28% (n = 248) of the population resided within 400 m of an interstate. Also, approximately 26% (n = 174) of the infants resided within 100 m of a state route or a bus route. Hence on the basis of our population’s geographic distribution and our pilot study, which revealed that the concentration of ultrafine particles decreased by one half between 50 m and 150 m downwind from a highway and an observable sulfur concentration gradient up to 400 m from a highway, infants were classified as exposed to interstate traffic if their residence was within 400 m.20 Exposure to a state or bus route was determined if their residence was within 100 m from one of these routes. If an infant resided greater than 400 m from the nearest interstate, greater than 100 m from the nearest state route, and greater than 100 m from the nearest bus route, the infant was placed in the unexposed category. Furthermore, exposure to moving traffic was determined if an infant’s residence was within 400 m of an interstate or within 100 m of a state route with a speed limit of greater than or equal to 50 miles per hour (mph). Fifty miles per hour was used as the cutoff because in Ohio this is the designation used for classification of an urban (greater traffic) or rural (less traffic) route. Exposure to stop-and-go traffic was determined if an infant’s residence was within 100 m of a bus route, within 100 m of a state route with a speed limit of less than 50 mph, or both.

Statistical analyses To determine the presence of an association between traffic exposure and wheezing, conditional logistic regression was performed with SAS software (version 8.2 for Windows; SAS Institute Inc, Cary, NC), adjusting for sex, race (white/nonwhite), breastfeeding (maternal report of breast-feeding <1 week, 1-4 weeks, or >5 weeks), pet ownership, income (<$40,000/$40,000), child care outside of the home (parent report of infant attending day care or babysitter), number of siblings, visible mold in the home, maternal and paternal self-report of asthma, and the number of monthly diaries returned.

RESULTS Recruitment for the CCAAPS study was completed on December 13, 2003. During year 1 of the study, 633 families had returned at least one monthly diary before January 1, 2004. Eleven (1.7%) of the 633 eligible families were excluded because of inaccuracy in the geocoding of their residence at the time of exposure classification. The average age of the infants in this study (at the time of their enrollment) was 7.5 months (6 2.4 months).

Exposure classification Table I displays the demographic characteristics of the infants and their families in the 3 exposure groups; 60.1% (n = 374) of the infants were unexposed, whereas the moving traffic category included 28.3% (n = 176) of the infants, and the stop-and-go category included 15.9% (n = 99) of the infants. As shown in Table I, the infants exposed to stop-and-go traffic were more likely to be African American, to have care outside their home, and to have had a father with asthma, whereas they were less likely to have been breast-fed. Because of these differences, these and other possible covariates were adjusted

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TABLE I. Prevalence of demographic characteristics by exposure categorization

White Income <$40,000 Male sex Current smoking mother Care outside home Owns dog Owns cat Weeks breast-fed 0 1-4 5 Diaries returned 1 2 3 No. of siblings 0 1 2 Has visible mold Paternal asthma Maternal asthma

Unexposed Moving Stop and go (n = 347), % (n = 176), % (n = 99), %

83.3 24.6 51.3 11.8 28.5 33.9 27.8

78.7 35.7 50.6 16.5 24.3 39.0 24.4

56.6 54.2 61.2 17.2 36.2 26.9 16.1

26.3 6.9 66.8

35.2 9.7 55.1

49.5 14.1 36.4

27.7 18.4 53.9

29.6 11.9 58.5

43.4 13.1 43.5

37.5 33.0 29.5 65.4 11.1 20.6

34.3 40.7 25.0 58.0 13.0 25.8

36.2 34.0 29.8 58.6 23.6 17.4

for in the logistic model. In the unexposed category the median distances to the nearest highway, state route, and bus route were 3287 m, 743 m, and 543 m, respectively. For those infants classified as exposed to moving traffic, the median distances to the nearest highway, state route, and bus route were 252 m, 696 m, and 341 m, respectively. Infants exposed to stop-and-go traffic resided a median distance of 2303 m, 439 m, and 43 m from the nearest highway, state route, and bus route, respectively. The median number of trucks on the highway nearest infants (n = 170) in the moving category was 11,820 daily. For those infants (n = 6) exposed to moving traffic on a state route, the median number of trucks per day was 1050. Infants exposed to buses (n = 71) or trucks (n = 9) only in the stop-and-go category had a median of 44 buses daily and 1250 trucks, respectively, on the route nearest their residence. Infants exposed to both buses and trucks (n = 19) had a median of 72 and 390, respectively.

Wheeze (without cold) Of the 622 infants, 50 (8.0%) reported wheezing without a cold. In the unexposed category 5.8% of infants reported wheezing without a cold compared with 7.4% in the moving category and 17.2% in the stop-and-go exposure category (P < .01). The prevalence of wheezing in the infants who were categorized into the 3 exposure categories was subsequently examined by distance from the nearest road and type of traffic (Fig 1). The prevalence of wheezing was 3 times higher (19%) in the infants who resided less than 50 m from stop-and-go traffic compared with those infants who were unexposed (6%). The prevalence of wheezing in infants who reside 200 to 300 m

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Characteristic

FIG 1. Prevalence of wheeze (without cold) among infants within exposure categories by distance from the nearest DEP source. SR, State route; BR, bus route; HW, highway.

from moving traffic (12%) was more than doubled when compared with that of infants who were classified as unexposed. Unadjusted and adjusted odds ratios (ORs) are shown in Table II. Living within 100 m of stop-and-go truck and bus traffic was the most important risk factor for early infant wheeze (adjusted OR, 2.50; 95% CI, 1.15-5.42). An infant with no siblings was at a decreased risk for wheezing (adjusted OR, 0.42; 95% CI, 0.19-0.93), and nonwhite infants were at an increased risk for wheezing (adjusted OR, 2.39; 95% CI, 1.20-4.76, respectively). Male sex and paternal self-report of asthma (although not maternal selfreport of asthma) were also significantly associated with wheezing (Table II). Infants classified as exposed to moving traffic did not have a significant association with wheezing without a cold when compared with infants classified as unexposed. A univariate analysis was conducted comparing wheezing without a cold and the season (winter [January-March], spring [April-June], summer [July-September], autumn [October-December]) in which it was first reported to address the possibility of a cold as a possible cause of wheezing. In this analysis there were no differences in the prevalence of wheezing among season (P = .50), and the same was true when season was added to the multivariate model.

DISCUSSION Infants exposed to stop-and-go bus and truck traffic had a significantly increased risk for wheezing without a cold compared with infants unexposed to truck or bus traffic or compared with infants exposed to moving truck traffic with a larger volume of trucks. Infants with immature lungs residing in close proximity to stop-and-go truck and bus traffic might be exposed to greater amounts of fine and ultrafine particulates.21-24 Sampling for fine particulate matter (PM 2.5 mm) and black carbon inside a bus and a car traveling ahead of the bus showed that the average DEP levels were approximately 20 mg/m3 and 5 mg/m3,

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TABLE II. Unadjusted and adjusted* ORs and 95% CIs for wheezing without a cold Unadjusted OR

Asthma diagnosis and treatment

Stop-and-go exposure  Nonwhite Paternal asthma Male sex Only childà

Adjusted OR

OR

95% CI

OR

95% CI

3.41 2.80 2.63 1.93 0.47

1.71-6.81 1.54-5.08 1.29-5.37 1.04-3.58 0.23-0.96

2.50 2.39 2.35 2.49 0.42

1.15-5.42 1.20-4.76 1.08-5.13 1.21-5.10 0.19-0.93

*Adjusted for maternal smoking, breast-feeding, pet ownership, visible mold, maternal asthma, care outside the home, and monthly diaries returned.  Reference category = unexposed. àReference category = 2 or more siblings. FIG 2. Prevalence of wheeze (without cold) among infants stratified by race.

but during stop-and-go traffic, the levels increased to more than 30 mg/m3 and 20 mg/m3, respectively.21 Other studies have also found acceleration, deceleration, and stop-andgo traffic to be associated with higher emissions of organic carbon, elemental carbon, carbon monoxide, nitric oxide, hydrocarbons, and soot when compared with cruising traffic.22-24 To our knowledge, the present study is the first to prospectively examine the effect of living in close proximity to roads with stop-and-go bus and truck traffic on infants’ respiratory health. Although our results are consistent with those of other investigations,14,15,18,25-28 this study improves on previous investigations. This prospective design during early infancy minimizes parental recall bias by allowing simultaneous measurements of exposure and outcome, whereas most previous studies have relied on parental recall of infant illness. The GIS database also accurately geocoded the infant’s residence, as well as the distance from the nearest traffic source. With the integration of county traffic data, the GIS minimized response bias by the parents who might inaccurately report the frequency or proximity of traffic. Although our a priori hypothesis also expected to find an effect with exposure to moving traffic, no association was found. Hence high intermittent exposures to pollutants might have a greater detrimental health effect on infants than exposure to lower continuous exposure. These findings, however, could be related to study limitations. Infants living near moving traffic were exposed to wide variations in the number of trucks. In addition, the median distance to stop-and-go traffic was 43 m, whereas the median distance to moving traffic was 252 m, and although state routes might have posted speed limits of greater than 50 mph and be classified as a rural route, the possibility exists that traffic might accelerate and decelerate at times of congestion. This scenario is also likely for short periods on highways where we have designated the traffic as moving. PM, a primary constituent of DEPs, has been significantly associated with emergency department visits for asthma, wheezing bronchitis, lower respiratory tract

symptoms, and physician visits for asthma.29-34 Others have found that fine and ultrafine particles (PM2.5 and PM1, respectively) have a greater association with respiratory symptoms than coarse particles (PM >2.5 mm) and are associated with pulmonary retention of particles.3,35,36 Induction of oxidative stress and mitochondrial damage by ultrafine particulates has been proposed.2,37 Thus whether the mechanism is total load or oxidative stress, infants who reside less than 100 m away are likely receiving a high dose of particulates. It is not possible, however, to separate the contributions of diesel and gasoline engines. Previous studies have found maternal asthma,38 paternal asthma,39 or both to be a significant risk factor for the development of asthma and wheezing in children.40,41 In our study only paternal asthma was significant. Although studies have found associations between parental smoking and wheezing in infants, the multivariate model showed no additional smoking effect. Also, multivariate analyses of maternal smoking found no associations with wheezing in the unexposed subpopulation. However, only 14% (n = 87) of the cohort were exposed to maternal smoking (Table I). Of particular interest are the findings regarding the high prevalence of wheezing among nonwhite infants in all exposure categories. As shown in Fig 2, the prevalence of wheezing in nonwhite infants was nearly 2 or more times higher in all groups, suggesting a health disparity beginning early in infancy. Thus increased susceptibility to wheezing is consistent with national statistics of asthma prevalence, as well as other studies.42-44 Because wheezing in the first year of life is generally a poor predictor of later development of childhood asthma, results must be interpreted cautiously.45 In conclusion, this is the first epidemiologic study to examine the risk of wheezing in infants younger than 1 year who are exposed to varying types and amounts of urban traffic. It demonstrated that even within an urban environment, the risk of wheezing varies with the type of and distance from traffic. A health disparity between races is also evident: nearly one fourth of African American infants exposed to stop-and-go traffic are wheezing before

age 1 year. This risk will be further investigated as the cohort ages and asthma and atopy can be diagnosed. We thank Stephanie Maier and Sherry Stanforth for their help with family interviews and testing.

REFERENCES 1. Peden DB. The epidemiology and genetics of asthma risk associated with air pollution. J Allergy Clin Immunol 2005;115:213-9. 2. Riedl M, Diaz-Sanchez D. Biology of diesel exhaust effects on respiratory function. J Allergy Clin Immunol 2005;115:221-8. 3. Peden DB. Development of atopy and asthma: candidate environmental influences and important periods of exposure. Environ Health Perspect 2000;108:475-82. 4. Pandya RJ, Solomon G, Kinner A, Balmes JR. Diesel exhaust and asthma: hypotheses and molecular mechanisms of action. Environ Health Perspect 2002;110:103-12. 5. Diaz-Sanchez D, Tsien A, Casillas A, Dotson AR, Saxon A. Enhanced nasal cytokine production in human beings after in vivo challenge with diesel exhaust particles. J Allergy Clin Immunol 1996;98:114-23. 6. US Environmental Protection Agency (EPA). Health assessment document for diesel engine exhaust. Prepared by the National Center for Environmental Assessment, Washington, DC, for the Office of Transportation and Air Quality; EPA/600/8–90/057F. 2002. Available at: http://www.epa.gov/ncea. 7. Knox RB, Suphioglu C, Taylor P, Desai R, Watson HC, Peng JL, et al. Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution. Clin Exp Allergy 1997;27:246-51. 8. Ormstad H. Suspended particulate matter in indoor air: adjuvants and allergen carriers. Toxicology 2000;152:53-68. 9. Diaz-Sanchez D, Tsien A, Fleming J, Saxon A. Combined diesel exhaust particulate and ragweed allergen challenge enhances human in vivo nasal ragweed-specific IgE and skews cytokine production to a T helper cell 2 type pattern. J Immunol 1997;158:2406-13. 10. Fujieda S, Diaz-Sanchez D, Saxon A. Combined nasal challenge with diesel exhaust particles and allergen induces in vivo IgE isotype switching. Am J Respir Cell Mol Biol 1998;19:507-12. 11. Fujimaki H, Nohara O, Ichinose T, Watanabe N, Saito S. IL-4 production in mediastinal lymph node cells in mice intratracheally instilled with diesel exhaust particulates and antigen. Toxicology 1994;92:261-8. 12. LeMasters GK, Wilson KA, Levin L, Bernstein DI, Lockey JE, Villareal M, et al. Validation of a population-based brief allergy symptoms questionnaire. Abstract presented at: American Thoracic Society International Conference; May 16-21, 2003; Seattle (WA). 13. Jenkins MA, Clarke JR, Carlin JB, Robertson CF, Hopper JL, Dalton MF, et al. Validation of questionnaire and bronchial hyperresponsiveness against respiratory physician assessment in the diagnosis of asthma. Int J Epidemiol 1996;25:609-16. 14. Brunekreef B, Janssen NAH, de Hartog J, Harssema H, Knape M van Vliet P. Air pollution from truck traffic and lung function in children living near motorways. Epidemiology 1997;8:298-303. 15. van Vliet P, Knape M, de Hartog J, Janssen N, Harssema H, Brunekreef B. Motor vehicle exhaust and chronic respiratory symptoms in children living near freeways. Environ Res 1997;74:122-32. 16. Venn AJ, Lewis SA, Cooper M, Hubbard R, Britton J. Living near a main road and the risk of wheezing illness in children. Am J Respir Crit Care Med 2001;164:2177-80. 17. Wilkinson P, Elliott P, Grundy C, Shaddick G, Thakrar B, Walls P, et al. Case-control study of hospital admission with asthma in children aged 5-14 years: relation with road traffic in north west London. Thorax 1999; 54:1070-4. 18. Lin S, Munsie JP, Hwang S, Fitzgerald E, Cayo MR. Childhood asthma: hospitalization and residential exposure to state route traffic. Environ Res 2002;88:73-81. 19. Janssen NAH, Brunekreef B, van Vliet P, Aarts F, Meliefste K, Harssema H, et al. The relationship between air pollution from heavy traffic and allergic sensitization, bronchial hyperresponsiveness, and respiratory symptoms in Dutch schoolchildren. Environ Health Perspect 2003;111: 1512-8.

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20. Reponen T, Grinshpun SA, Trakumas S, Martuzevicius D, Wang Z, LeMasters G, et al. Concentration gradient patterns of aerosol particles near interstate highways in the Greater Cincinnati airshed. J Environ Monit 2003;5:557-62. 21. Solomon G, Campbell T, Feuer G, Masters J, Samkian A, Paul K. No breathing in the aisles: Diesel exhaust inside school buses. Natural Resources Defense Council, Coalition for Clean Air. 2001. Available at: http://www.nrdc.org/air/transportation/schoolbus/sbusinx.asp. 22. Shah S, Cocker D, Miller JW, Norbeck JM. Emission rates of particulate matter and elemental and organic carbon from in-use diesel engines. Environ Sci Technol 2004;38:2544-50. 23. Tong HY, Hung WT, Cheung CS. On-road vehicle emissions and fuel consumption in urban driving conditions. J Air Waste Manag Assoc 2000;50:543-54. 24. Unal A, Frey HC, Rouphail NM. Quantification of highway vehicle emissions hot spots based upon on-board measurements. J Air Waste Manag Assoc 2004;54:130-40. 25. Hirsch T, Weiland SK, von Mutius E, Safeca AF, Grafe H, Csaplovics E, et al. Inner city air pollution and respiratory health and atopy in children. Eur Respir J 1999;14:669-77. 26. Ciccone G, Forastiere F, Agabiti N, Biggeri A, Bisanti L, Chellini E, et al. Road traffic and adverse respiratory effects in children. SIDRIA Collaborative Group. Occup Environ Med 1998;55:771-8. 27. Shima M, Nitta Y, Adachi M. Traffic related air pollution and respiratory symptoms in children living along trunk roads in Chiba Prefecture, Japan. J Epidemiol 2003;13:108-19. 28. Brauer M, Hoek G, Van Vliet P, Meliefste K, Fischer PH, Wijga A, et al. Air pollution from traffic and the development of respiratory infections and asthmatic and allergic symptoms in children. Am J Respir Crit Care Med 2002;166:1092-8. 29. Norris G, YoungPong S, Koenig J, Larson T, Sheppard L, Stout J. An association between fine particles and asthma emergency department visits for children in Seattle. Environ Health Perspect 1999;110:489-93. 30. Pino P, Walter T, Oyarzun M, Villegas R, Romieu I. Fine particulate matter and wheezing illnesses in the first year of life. Epidemiology 2004;15:702-8. 31. Shima M, Nitta Y, Ando M, Adachi M. Effects of air pollution on the prevalence and incidence of asthma in children. Arch Environ Health 2002;57:529-35. 32. van der Zee S, Hoek G, Boezen HM, Shouten J, van Wijnen JH, Brunekreef B. Acute effects of urban air pollution on respiratory health of children with and without chronic respiratory symptoms. Occup Environ Med 1999;56:802-13. 33. Jalaludin BB, O’Toole BI, Leeder SR. Acute effects of urban ambient air pollution on respiratory symptoms, asthma medication use, and doctor visits for asthma in a cohort of Australian children. Environ Res 2004;95: 32-42. 34. Penttinen P, Timonen KL, Tiittanen P, Mirme A, Ruuskanen J, Pekkanen J. Ultrafine particles in urban air and respiratory health among adult asthmatics. Eur Respir J 2001;17:428-35. 35. Schwartz J, Neas LM. Fine particles are more strongly associated than coarse particles with acute respiratory health effects in schoolchildren. Epidemiology 2000;11:6-10. 36. Brauer M, Avila-Casado C, Fortoul TI, Vedal S, Stevens B, Churg A. Air pollution and retained particles in the lung. Environ Health Perspect 2001;109:1039-43. 37. Li N, Sioutas C, Cho A, Schmitz D, Misra C, Sempf J, et al. Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ Health Perspect 2003;111:455-60. 38. Sheriff A, Peters TJ, Henderson J, Strachan D, ALSPAC Study Team. Risk factor associations with wheezing patterns in children followed longitudinally from birth to 3(1/2) years. Int J Epidemiol 2001;30: 1473-84. 39. El-Sharif N, Abdeen Z, Barghuthy F, Nemery B. Familial and environmental determinants for wheezing and asthma in a case-control study of school children in Palestine. Clin Exp Allergy 2003;33:176-86. 40. Arshad SH, Kurukulaaratchy RJ, Fenn M, Matthews S. Early life risk factors for current wheeze, asthma, and bronchial hyperresponsiveness at 10 years of age. Chest 2005;127:502-8. 41. London SJ, Gauderman J, Avol E, Rappaport EB, Peters JM. Family history and the risk of early-onset persistent, early onset transient, and late-onset asthma. Epidemiology 2001;12:577-83.

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42. Grant EN, Lyttle CS, Weiss KB. The relation of socioeconomic factors and racial/ethnic differences in the US asthma mortality. Am J Public Health 2000;90:1923-5. 43. Joseph CLM, Ownby DR, Peterson EL, Johnson CC. Racial differences in physiologic parameters related to asthma among middle-class children. Chest 2000;117:1336-44.

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44. Simon PA, Zeng Z, Wold CM, Haddock W, Fielding JE. Rate of childhood asthma and associated morbidity in Los Angeles County: impacts of race/ethnicity and income. J Asthma 2003;40:535-43. 45. Taussig L, Wright A, Holberg C, Halonen M, Morgan W, Martinez F. Tucson Children’s Respiratory Study: 1980 to present. J Allergy Clin Immunol 2003;111:661-75.

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Effect of low-dose ciclesonide on allergeninduced responses in subjects with mild allergic asthma Gail M. Gauvreau, PhD,a Louis Philippe Boulet, MD,b Dirkje S. Postma, MD, PhD,c Tomotaka Kawayama, MD,a Richard M. Watson, BSc,a MyLinh Duong, MD,a Francine Deschesnes, BSc,b Jan G. R. De Monchy, MD, PhD,c and Paul M. O’Byrne, MDa Hamilton, Ontario, and Quebec City, Quebec, Canada, and Groningen, The Netherlands

Background: Inhalation of allergens by sensitized patients with asthma induces reversible airway obstruction, airway hyperresponsiveness, and eosinophilic airway inflammation. Attenuation of allergen-induced bronchoconstriction and inflammation has been used to examine the efficacy of therapeutic agents such as inhaled corticosteroids in asthma. Ciclesonide, a nonhalogenated inhaled corticosteroid being developed for the treatment of persistent asthma, remains inactive until cleaved by esterases in the lung. Objective: This study examined the effect of low doses of inhaled ciclesonide, 40 mg and 80 mg, on allergen-induced bronchoconstriction, serum eosinophil cationic protein, and eosinophilic airway inflammation. Methods: Twenty-one nonsmokers with mild atopic asthma completed a multicenter, randomized, 3-way crossover study comparing the effects of 7-day treatment of ciclesonide or placebo. Allergen-induced responses, including the early and late fall in FEV1, peripheral blood eosinophils, serum eosinophil cationic protein levels, and eosinophils in induced sputum were measured. Results: Ciclesonide 80 mg attenuated the early and late asthmatic responses, including the change in FEV1, serum eosinophil cationic protein, and sputum eosinophils measured at 24 hours postchallenge (P < .025). Ciclesonide 40 mg attenuated the late asthmatic responses and sputum eosinophils measured at 24 hours postchallenge (P < .025), with no effect

From athe Department of Medicine, McMaster University, Hamilton; bInstitut de cardiologie et de pneumologie de l’Universite´ Laval, Hoˆpital Laval, Quebec City; and cthe Department of Pulmonology, University Hospital Groningen. Supported by Altana Pharma AG. Disclosure of potential conflict of interest: P. M. O’Byrne has consultant arrangements with AstraZeneca, GlaxoSmithKline, Topigen, and Altana, and has received grants/research support from AstraZeneca, GlaxoSmithKline, Pfizer, Altana, and Dynavax. L.-P. Boulet has been on Advisory Boards for AstraZeneca, Altana Novartis, GlaxoSmithKline, and Merck Frost, and received lecture fees from 3M, GlaxoSmithKline, AstraZeneca, and Merck Frosst. Sponsorship for basic research was received from 3M, Schering, Genentech, Dynavax, Roche, GlaxoSmithKline, Novartis, AstraZeneca, Altana, and Merck for participating in multicenter studies of the pharmacotherapy of asthma. D. Postma is on the Advisory Board of Altana. Received for publication March 29, 2005; revised May 13, 2005; accepted for publication May 17, 2005. Available online July 15, 2005. Reprint requests: Paul M. O’Byrne, MD, HSC 3W10, McMaster University, 1200 Main St West, Hamilton, Ontario, Canada L8N 3Z5. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.021

on the early allergen-induced bronchoconstriction, 24-hour FEV1, or serum eosinophil cationic protein levels (P < .025). Conclusion: With the exception of 24-hour postchallenge peripheral blood eosinophils, a low dose of ciclesonide, 80 mg, was effective in blocking all allergen-induced responses measured. (J Allergy Clin Immunol 2005;116:285-91.) Key words: Inhaled corticosteroid, allergen inhalation, airway inflammation

Asthma is characterized by reversible airway obstruction, airway hyperresponsiveness, and eosinophilic bronchial inflammation. Subjects with allergic asthma develop an immediate IgE-mediated early asthmatic response (EAR) after inhalation of an allergen to which they are sensitized. Approximately 50% of these subjects also develop a late asthmatic response (LAR), which begins 3 to 4 hours after allergen inhalation.1 The LAR is associated with elevated levels of airway inflammatory cells including eosinophils, basophils, and mast cells.2,3 This model of allergen-induced bronchoconstriction has been used successfully to assess drug efficacy in subjects with allergic asthma4-13 and is recommended for evaluation of inhaled corticosteroids (ICS).14 Inhaled corticosteroids, having potent anti-inflammatory properties, are indicated by the Global Initiative for Asthma as a primary controller for treatment of mildpersistent to severe asthma.15 Studies of ICS have consistently shown a significant attenuation of the allergeninduced LAR,5,16-18 with the proposed mechanism attenuation of the allergen-induced airway inflammation. However, undesirable side effects of ICS at higher doses have established a need to evaluate ICS properties at very low doses.14 Ciclesonide is a nonhalogenated ICS for the treatment of persistent asthma of all severities. This ICS remains inactive until cleaved by esterases present in the airway, where its active metabolite, desisobutyryl-ciclesonide, then binds glucocorticoid receptors. Before developing an optimal solution for metered dose inhaler (MDI) formulation for clinical use, early clinical studies were performed with ciclesonide by using a dry powder inhaler (DPI) device. One week of ciclesonide 800 mg DPI BID (twice daily; using Cyclohaler device [Pharmachemie, Haarlem, The Netherlands]) has been 285

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Abbreviations used AE: Adverse event AUC0-2h: Area under the curve of the early response AUC3-8h: Area under the curve of the late response BID: Twice daily DPI: Dry powder inhaler EAR: Early asthmatic response; maximum % fall in FEV1 from 0 to 2 hours after allergen challenge ECP: Eosinophil cationic protein ICS: Inhaled corticosteroids LAR: Late asthmatic response; maximum % fall in FEV1 from 3 to 8 hours after allergen challenge MDI: Metered dose inhaler

shown to attenuate significantly the allergen-induced EAR and LAR,19 confirming that this drug has biological activity in this model of allergic inflammation. The allergen-induced fall in FEV1 was shown to be a sensitive marker of dose-response effect in an earlier trial of mometasone furoate16; therefore, the current trial was performed to examine the dose-response of the LAR and to determine the efficacy of low-dose ciclesonide in the reduction of the allergen-induced EAR, LAR, and airway inflammation. In-house testing has led to the development of ciclesonide in MDI formulation with hydrofluoroalkane (HFA) propellent, delivering 40 mg, 80 mg, and 160 mg (ex-actuator) per puff. However, data are limited regarding what the lowest effective dose is with the MDI formulation of ciclesonide. This study, therefore, was designed to identify the lowest effective dose of ciclesonide in MDI formulation in an allergen inhalation model.

METHODS Subjects Thirty-five subjects were enrolled in the study. Of these, 13 patients did not meet randomization criteria. Twenty-two subjects (Groningen, n = 5; Laval, n = 7; McMaster, n = 10), 14 men and 8 women, age 19 to 58 years old (Table I), were randomized to one of the 6 treatment sequences. One subject discontinued the study prematurely for nonmedical reasons. Inclusion criteria required subjects to be nonsmokers with mild atopic asthma, free of other lung disease, and without lower respiratory tract infection for 6 weeks before entering the study. For randomization, subjects were required to have stable asthma with FEV1 > 70% of predicted; have baseline methacholine PC20 < 16 mg/mL; use no regular asthma medication during the study other than infrequent inhaled b2-agonist, which was withheld for 8 hours before each visit; and have no exposure to sensitizing allergens apart from house dust mite. Before entering the study, subjects could not have used systemic steroids or had an asthma exacerbation for 6 weeks and could not have used inhaled steroids for at least 4 weeks. Before morning visits to the lab, subjects were to refrain from tea or coffee.

Study Design This trial was a multicenter, double-blind, randomized, placebocontrolled, 3-period crossover study comparing 7 days of treatment with ciclesonide at 50 mg and 100 mg exvalve, corresponding to

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40 mg and 80 mg ex-actuator, with placebo. The study was approved by the ethics research board of the respective institutions, and signed informed consent was given to participate. Screening of subjects was performed over a period of 2 consecutive days and included a detailed history, physical examination, allergen skin test, FEV1, methacholine PC20, blood sampling for serum ECP levels, allergen inhalation challenge, and sputum induction. Subjects who developed an EAR (at least 20% fall in FEV1 within 2 hours after allergen inhalation) and LAR (at least 15% fall in FEV1 between 3 and 8 hours after allergen inhalation) during 1 screening allergen inhalation challenge were enrolled in the study. Subjects were screened once only, because we have shown the LAR to be a reproducible measurement.20,21 Subjects reported to the laboratory for 3 separate treatment periods separated by a minimum of 3 weeks (Fig 1). This washout time has been shown to be adequate in a previous study of ICS using this model.16 Each treatment period consisted of 4 morning visits. Day 1 consisted of pretreatment measurements of blood eosinophils, sputum inflammatory cells, and lung function; methacholine PC20 needed to be within 1 doubling dose of that measured during the screening period to continue with the treatment period. If this criterion was met, subjects then inhaled the first dose of study medication during the morning visit to the lab. The subsequent doses of study medication were inhaled for the next 6 consecutive mornings, immediately after waking, because ciclesonide has been shown to improve asthma control irrespective of the time of administration.22 Subjects returned to the laboratory on day 5 for measurement of preallergen challenge sputum inflammatory cells and on day 6 for allergen inhalation challenge. Measurements of FEV1 were taken at regular intervals until 8 hours after challenge. On day 7, subjects underwent measurements of sputum inflammatory cells, blood eosinophils, and serum ECP. Postallergen methacholine PC20 was not measured, because this study was not powered sufficiently for this comparison. All subjects were considered compliant with study medication according to the diary cards.

Laboratory procedures Methacholine inhalation test. Methacholine inhalation challenge was performed as described by Cockcroft.23 Subjects inhaled normal saline during 2 minutes of tidal breathing, nebulized at 0.13 mL/ minute from a Wright nebulizer, then doubling concentrations of methacholine chloride. FEV1 was measured at 30, 90, 180, and 300 seconds after each inhalation. The test was terminated when a fall in FEV1 of 20% of the baseline value occurred, and the methacholine PC20 was calculated. Allergen inhalation challenge. Allergen challenge was performed as described by O’Byrne et al.1 Subjects were skin tested for allergies to common aeroallergens. The allergen producing the largest skin wheal diameter was diluted in 0.9% saline and stored for subsequent allergen inhalation challenges. The concentration of allergen extract for inhalation was determined from a formula described by Cockcroft et al,24 and doubling concentrations of allergen were given until a <20% early fall in FEV1 at 10 minutes postallergen was reached. The FEV1 was then measured at regular intervals until 8 hours after allergen inhalation. The early area under the curve (AUC0-2h) and the late area under the curve (AUC3-8h) were calculated by using the trapezoidal rule, were normalized to 1 hour, and were expressed as liters 3 hour (L3h). Subjects inhaled the same dose of allergen for the 3 treatment periods. Sputum analysis. Sputum was induced and processed by using the method described by Pizzichini et al.25 Cell plugs were selected and mixed with 0.1% dithiothreitol (Sputolysin; Calbiochem Corp, San Diego, Calif) and Dulbecco PBS (Life Technologies Inc, Grand Island, NY) and filtered through a 48-mm nylon gauze (BNSH Thompson, Scarborough, Ontario, Canada), and cytospins were

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TABLE I. Subject characteristics at study screening visit

50001 50002 50004 50005 50008 50011 50012 50013 50014 50015 50025 50027 50028 50029 50031 50032 50033 50038 50039 50040 50041 50045 Mean 6 SEM

Age (y)

Sex

Predicted FEV1 (%)

FEV1/VC ratio

Methacholine PC20 (mg/mL)

25 21 42 20 26 54 33 54 19 39 26 28 25 23 30 26 38 19 58 40 49 33

F M M M F M M M F M F F F M M M M M M M F F

96 114 106 73 89 107 88 73 109 80 83 121 96 99 75 93 112 103 107 84 100 85

0.86 0.86 0.69 0.55 0.92 1.18 0.73 0.55 0.83 0.67 0.89 0.87 0.86 0.77 0.70 0.73 0.72 0.84 0.79 0.80 0.86 0.83

1.12 8.94 4.54 0.31 1.63 0.88 15.51 3.39 0.54 5.11 1.07 1.11 1.42 1.47 0.86 2.21 1.56 0.70 8.80 0.44 0.21 0.33

33.1 6 2.6

14 M 8F

95.1 6 3.0

0.80 6 0.03

1.46 (15.51-0.21)

Allergen

Ragweed HDM HDM HDM Ragweed Cat Ragweed HDM HDM HDM HDM Cat Cat Cat Cat HDM HDM HDM HDM HDM HDM HDM 14 HDM 5 Cat 3 RW

Final dose, median

1:64 1:16 1:32 1:1024 1:16 1:32 1:64 1:64 1:128 1:128 1:32 1:64 1:16 1:64 1:128 1:64 1:128 1:1024 1:2048 1:2048 1:2048 1:1024 1:64

HDM, House dust mite; VC, vital capacity.

prepared on glass slides. The total cell count was determined by using a Neubauer hemocytometer chamber (Hausser Scientific, Blue Bell, Pa) and expressed as the number of cells per milliliter sputum, and differential cell counts were obtained from slides stained with Diff Quik (American Scientific Products, McGaw Park, Ill). All slides were enumerated at 1 site. Slide preparation and enumeration were performed before unblinding. The study personnel collecting sputum samples and the technician enumerating the slides had no knowledge of the coding of the labels, nor of the airway physiology measured when these sputum samples were collected. All data were collected centrally and entered into a master database before the random code was distributed to participating sites. Serum eosinophil cationic protein and blood eosinophils. Blood was collected into 4-mL vacutainer hemogard SST tubes (Becton Dickinson, Franklin Lakes, NJ) for serum separation. Eosinophil cationic protein (ECP) was released by allowing blood to clot for 60 to 120 minutes at room temperature, and then samples were centrifuged at 1000g to 1300g for 10 minutes at room temperature. Serum was removed and stored at 220°C until analysis. All serum ECP was analyzed at 1 site by using the UniCAP system (Pharmacia, Uppsala, Sweden). Blood was also collected into 2-mL EDTA vacutainers, and eosinophil counts were performed by Coulter Counter (Beckman Coulter, Fullerton, Calif) at the respective institutions.

Statistical analysis The target sample size of 18 patients is sufficient to guarantee a power of 90% in correctly concluding superiority at the .0125 level, 1-sided, if the mean difference accounts for 90% of the SD (based on paired t test). The 21 subjects who completed the study were included in the statistical analyses. Summary statistics are expressed as mean

and SEM. Between-treatment differences in FEV1 during the LAR, FEV1 during the EAR, and sputum eosinophils 24 hours after allergen were analyzed by using ANOVA. The FEV1 measured 24 hours after allergen was analyzed with analysis of covariance by using the baseline value as a covariate to test both within-treatment and between-treatment differences. Within-treatment differences in sputum variables as well as between-treatment differences in blood eosinophils, ECP, and sputum variables were analyzed by means of the nonparametric test for 3 3 6 crossover design. One-sided P values were generated for all variables, and significance is shown at P < .025.

RESULTS All subjects inhaled the same dose of allergen for the 3 treatment periods. There were no serious adverse events (AEs), and 11 treatment-emergent AEs were reported. Of these, 7 AEs were mild and 4 were moderate. All AEs were assessed as unrelated to the study medication, except 1 case of thrombocytopenia, which was assessed unlikely related to the study medication. No adverse event led to premature discontinuation or a change in study medication. Before allergen challenge, the degree of airway hyperresponsiveness was within 1 doubling dose, and there was no significant difference in FEV1 between the 3 treatment periods. During placebo treatment, all subjects demonstrated early and late airway responses after allergen inhalation challenge; the maximum percent fall in FEV1 was 30.4% 6 2.2% during the early response

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Subject

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Asthma diagnosis and treatment FIG 1. Study schematic. BL, Pretreatment baseline.

TABLE II. Effect of ciclesonide on the maximum percent fall in FEV1 and area under the curve of the early and late airway responses Ciclesonide 40 mg

Placebo

EAR (%) AUC0-2h (L3h) LAR (%) AUC3-8h (L3h)

30.4 6 2.2 233.3 6 4.0

28.2 6 2.2 229.2 6 4.6

24.0 6 2.1 227.5 6 4.3

13.3 6 2.1** 211.8 6 4.0**

Ciclesonide 80 mg

23.6 6 2.2* 224.9 6 4.1* 10.7 6 2.1** 29.4 6 2.7**

*P < .025 compared with placebo. **P < .013 compared with placebo.

and 24.0% 6 2.1% during the late response, corresponding to AUC0-2h of 233.3 6 4.0 L3h and AUC3-8h of 227.5 6 4.3 L3h (Table II). During treatment with ciclesonide 80 mg, there was a significant reduction in the maximum percent fall in FEV1 during the EAR to 23.6% 6 2.2% (P = .016), the AUC0-2h (P = .013), the maximum percent fall in FEV1 during the LAR to 10.7% 6 2.1% (P < .001), and the AUC3-8h (P < .001). Treatment with ciclesonide 40 mg significantly reduced the maximum percent fall in FEV1 during the late response to 13.3% 6 2.1% (P = .0003) and AUC3-8h (P = .0003), with no significant effect on the early maximum percent fall in FEV1 or AUC0-2h (P > .025). There was no significant difference between ciclesonide 40 mg and 80 mg on the EAR, LAR, AUC0-2h, or AUC3-8h (P > .025; Table II). FEV1 was measured at pretreatment baseline (day 1), preallergen (day 6), and at 24 hours postallergen inhalation challenge (day 7). With placebo treatment, the FEV1 of 3.38 L 6 0.17 L at day 7 was significantly lower than the day 1 FEV1 of 3.60 L 6 0.15 L (P < .001) and the day 6 FEV1 of 3.56 L 6 0.16 L (P < .001). During treatment with ciclesonide 40 mg, the FEV1 of 3.50 L 6 0.16 L at day 7 was significantly lower than that of day 1 or day 6, 3.65 L 6 0.17 L and 3.62 L 6 0.15 L, respectively (P < .001). During treatment with ciclesonide 80 mg, the FEV1

of 3.59 L 6 0.18 L at day 7 was significantly lower than 3.67 L 6 0.18 L at day 1 (P = .013), but not significantly different from the FEV1 of 3.64 L 6 0.16 L at day 6 (P = 0.10). Compared with placebo, ciclesonide 80 mg significantly attenuated the day 7 allergen-induced decrease in FEV1 compared with day 1 (P = .002) and day 6 (P = .007; Fig 2). Compared with placebo, ciclesonide 40 mg did not attenuate day 7 allergen-induced decrease in FEV1 compared with day 1 or day 6 (P > .025). There was a trend toward a dose-response effect for ciclesonide, with greater attenuation of the 24-hour postallergen fall in FEV1 by ciclesonide 80 mg compared with ciclesonide 40 mg; however, this did not reach statistical significance (1-sided P = .028; Fig 2). At 24 hours after allergen inhalation challenge, serum ECP increased from a baseline of 20.7 mg/L 6 3.0 mg/L to 31.0 mg/L 6 5.8 mg/L with placebo, 33.0 mg/L 6 5.6 mg/L with ciclesonide 40 mg, and 26.0 mg/L 6 5.3 mg/L with ciclesonide 80 mg. There was significant attenuation of the allergen-induced increase in serum ECP with ciclesonide 80 mg versus placebo treatment (P = .024), but no effect of ciclesonide 40 mg. Peripheral blood eosinophils were not significantly reduced with ciclesonide 40 mg or 80 mg (P > .025). There was an allergeninduced increase in the number of peripheral blood eosinophils at 24 hours after challenge, increasing from a baseline of 0.294 3 106/mL 6 0.037 3 106/mL to 0.404 3 106/mL 6 0.040 3 106/mL with placebo, 0.385 3 106/mL 6 0.047 3 106/mL with 40 mg ciclesonide, and 0.347 3 106/mL 6 0.052 3 106/mL with 80 mg ciclesonide. There was an allergen-induced increase in the percent sputum eosinophils (Fig 3, A) and the number of eosinophils per milliliter sputum (Fig 3, B) on day 7 compared with day 5 with placebo, ciclesonide 40 mg, and ciclesonide 80 mg treatment (P < .002). However, both ciclesonide 40 mg and 80 mg significantly attenuated the allergen-induced increase in the percentage of sputum eosinophils (P < .001 and P = .006, respectively). Only ciclesonide 80 mg significantly attenuated the allergeninduced increase in the number of eosinophils per milliliter sputum, but the trend toward a dose-dependent effect

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FIG 2. A comparison of FEV1 measured at day 1 (before treatment), day 6 (after 6 days of treatment), and day 7 (24 hours after allergen inhalation challenge) with placebo (open bars), ciclesonide 40 mg (hatched bars), and ciclesonide 80 mg (solid bars) treatments.*P < .025 compared with day 1 pretreatment. àP < .025 compared with day 6 preallergen.  P < .025 attenuation of allergen-induced increase compared with placebo.

for ciclesonide did not reach statistical significance (1-sided P = .03).

DISCUSSION Ciclesonide is a new generation inhaled glucocorticosteroid that remains inactive until cleaved by esterases present in the lung. Ciclesonide has been shown to be effective to reduce allergen-induced early and late responses when administered at 800 mg twice daily by Cyclohaler (DPI),19 yet it was unknown whether lower doses of 80 mg or 40 mg administered by MDI would also be effective. In the current study, ciclesonide was administered by MDI once daily for 7 days at considerably lower doses than previously studied. This study has demonstrated that once-a-day treatment with 40 mg or 80 mg ciclesonide for 7 days is indeed efficacious in this model of allergen-induced bronchoconstriction and airway inflammation. Furthermore, we were able to determine the lowest effective dose of ciclesonide for attenuating all allergen-induced responses investigated in this study. In contrast with the 40 mg dose, ciclesonide at 80 mg per day attenuated the allergen-induced early airway response, the sustained fall in FEV1 measured 24 hours postchallenge, and the allergen-induced accumulation of eosinophils into the airways. Whether 80 mg represents a plateau beyond which there is no further attenuation of allergen-induced responses is unknown. Compared with the aforementioned study of 800 mg ciclesonide DPI BID resulting in approximately 51% attenuation in the late fall in FEV1,19 80 mg in the current

study resulted in 58% inhibition and 40 mg resulted in 45% inhibition of the fall in FEV1 during the late response. The early response was not attenuated as effectively with the lower doses of ciclesonide, with 40 mg and 80 mg providing approximately 8% and 23% inhibition of the early fall in FEV1, respectively, compared with the 45% inhibition with 800 mg ciclesonide BID previously studied.19 The deposition pattern of HFA-MDI formulations in the peripheral lung may not inhibit the EAR as effectively compared with the DPI formulation, which gets deposited in the large, central airways where the EAR is likely to be most active. This supports the consistent observation that multiple or single doses of inhaled steroids may significantly attenuate the LAR without significantly attenuating the EAR.5,18,26 Moreover, these data suggest that higher levels of HFA-MDI formulation steroids are necessary to inhibit IgE-mediated early responses to inhaled allergen, such as mast cell degranulation, compared with lower levels of steroids that appear to suppress the late response effectively, likely through inhibition of proinflammatory cytokine gene expression.27 Ciclesonide HFA-MDI 200 mg 4 times daily administered in a previous study28 did not affect urine cortisol levels after 4 weeks treatment, which suggests that the 80 mg dose in the current study would have a very low potential for systemic activity. We did not measure systemic safety markers, because no effect would have been expected. It is unknown, however, whether systemic activity in compartments such as the circulation and/or bone marrow is an unidentified yet important site of action of inhaled steroids. There is certainly evidence showing that steroids provide anti-inflammatory effects

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Asthma diagnosis and treatment FIG 3. A comparison of percent sputum eosinophils (top panel) and number of sputum eosinophils (bottom panel) measured on day 1 (before treatment), day 5 (after 5 days treatment), and day 7 (24 hours after allergen challenge) with placebo (open bars), ciclesonide 40 mg (hatched bars), and ciclesonide 80 mg (solid bars) treatments.*P < .025 compared with day 5 preallergen.  P < .025 attenuation of allergen-induced increase compared to placebo.

in these other compartments.17,29 The low potential for systemic activity with low-dose ciclesonide treatment supports the notion that steroid regulation of allergeninduced bone marrow responses is likely through suppression of proinflammatory mediators generated in airways that subsequently control the bone marrow, rather than the belief that steroids have a direct effect on cells in the bone marrow.30 Because new generation steroids are preferred as a result of their low systemic effects, this question of whether some systemic activity is also required will become an important issue that needs to be addressed. There was a numerical, though insignificant (P > .025), dose-response for low doses of ciclesonide during the late response, which is believed to be one of the most sensitive variables used to demonstrate a dose-response.16 Surprisingly, these data demonstrate dose-responses to measurements of airway obstruction and inflammation at 24 hours postchallenge. This is an unexpected finding, indicating these variables may be worthy of evaluation in subsequent trials evaluating dose-responses. Although results from this study suggest that 80 mg ciclesonide may be the minimally effective dose for protection against the EAR and serum ECP levels, it is noteworthy that there was a significant effect of both 80 mg and 40 mg ciclesonide on the LAR and percentage of sputum eosinophils. This implies that the minimally effective dose for protection against these parameters is likely to be less than 80 mg. However, during the course of the study, ciclesonide was always administered in the

morning immediately after waking, and approximately 1 hour preallergen challenge. Hence, the drug was present at the highest possible levels during challenges. Whether this degree of efficacy would have been observed had drug administration and allergen challenge been separated by a longer time interval is unknown, but is important to consider, because the protective effects of inhaled steroids against allergen-induced early responses, airway eosinophilia, and allergen-induced airway hyperresponsiveness are partially or completely lost as early as 12 hours after discontinuation of therapy.18 Direct comparisons between various ICS were not performed in this study, largely because of the difficulties associated with 4-way crossover studies. Although a direct comparison would need to be performed to indicate the relative potency of ciclesonide, the dose of 80 mg appears to be similar to proven effective doses of other inhaled steroids; 50 ug mometasone furoate BID has been shown to attenuate significantly the EAR, LAR, and 24-hour postallergen sputum eosinophils.16 This study has provided new information regarding the minimally effective doses of inhaled ciclesonide for inhibition of allergeninduced airway responses and the apparent local antiinflammatory effects on the airways. Further evaluation of ciclesonide will be required to address whether these low doses are clinically effective.

We thank Dr A. Widmann for logistic support of this study and Mrs C. Veltman for help in patient recruitment. We also thank Tara

Strinich, Irene Babirad, and Tracy Rerecich for help in sample preparation and enumeration. 16. REFERENCES 1. O’Byrne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am Rev Respir Dis 1987;136:740-51. 2. de Monchy JG, Kauffman HF, Venge P, Koeter GH, Jansen HM, Sluiter HJ, et al. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am Rev Respir Dis 1985;131:373-6. 3. Gauvreau GM, Parameswaran KN, Watson RM, O’Byrne PM. Inhaled leukotriene E(4), but not leukotriene D(4), increased airway inflammatory cells in subjects with atopic asthma. Am J Respir Crit Care Med 2001;164:1495-500. 4. Fahy JV, Fleming HE, Wong HH, Liu JT, Su JQ, Reimann J, et al. 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 1997;155:1828-34. 5. Gauvreau GM, Doctor J, Watson RM, Jordana M, O’Byrne PM. Effects of inhaled budesonide on allergen-induced airway responses and airway inflammation. Am J Respir Crit Care Med 1996;154:1267-71. 6. Gauvreau GM, Jordana M, Watson RM, Cockroft DW, O’Byrne PM. Effect of regular inhaled albuterol on allergen-induced late responses and sputum eosinophils in asthmatic subjects. Am J Respir Crit Care Med 1997;156:1738-45. 7. Gauvreau GM, Becker AB, Boulet LP, Chakir J, Fick RB, Greene WL, et al. The effects of an anti-CD11a mAb, efalizumab, on allergeninduced airway responses and airway inflammation in subjects with atopic asthma. J Allergy Clin Immunol 2003;112:331-8. 8. Hamilton A, Faiferman I, Stober P, Watson RM, O’Byrne PM. Pranlukast, a cysteinyl leukotriene receptor antagonist, attenuates allergen-induced early- and late-phase bronchoconstriction and airway hyperresponsiveness in asthmatic subjects. J Allergy Clin Immunol 1998;102:177-83. 9. Hamilton AL, Watson RM, Wyile G, O’Byrne PM. Attenuation of early and late phase allergen-induced bronchoconstriction in asthmatic subjects by a 5-lipoxygenase activating protein antagonist, BAYx 1005. Thorax 1997;52:348-54. 10. Leigh R, Vethanayagam D, Yoshida M, Watson RM, Rerecich T, Inman MD, et al. Effects of montelukast and budesonide on airway responses and airway inflammation in asthma. Am J Respir Crit Care Med 2002; 166:1212-7. 11. Parameswaran K, Watson R, Gauvreau GM, Sehmi R, O’Byrne PM. The effect of pranlukast on allergen-induced bone marrow eosinophilopoiesis in subjects with asthma. Am J Respir Crit Care Med 2004;169:915-20. 12. Kidney JC, Boulet LP, Hargreave FE, Deschesnes F, Swystun VA, O’Byrne PM, et al. Evaluation of single-dose inhaled corticosteroid activity with an allergen challenge model. J Allergy Clin Immunol 1997; 100:65-70. 13. Boulet LP, Chakir J, Milot J, Boutet M, Laviolette M. Effect of salmeterol on allergen-induced airway inflammation in mild allergic asthma. Clin Exp Allergy 2001;31:430-7. 14. Boulet LP, Cockcroft DW, Toogood J, Lacasse Y, Baskerville J, Hargreave FE. Comparative assessment of safety and efficacy of inhaled corticosteroids: report of a committee of the Canadian Thoracic Society. Eur Respir J 1998;11:1194-210. 15. Global strategy for asthma management and prevention. NHLBI/WHO workshop report. Global Initiative for Asthma. Bethesda: National

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Institutes of Health, National Heart Lung and Blood Institute; 2002. NIH publication number 02-3659. Inman MD, Watson RM, Rerecich T, Gauvreau GM, Lutsky BN, Stryszak P, et al. Dose-dependent effects of inhaled mometasone furoate on airway function and inflammation after allergen inhalation challenge. Am J Respir Crit Care Med 2001;164:569-74. Wood LJ, Sehmi R, Gauvreau GM, Watson RM, Foley R, Denburg JA, et al. An inhaled corticosteroid, budesonide, reduces baseline but not allergen-induced increases in bone marrow inflammatory cell progenitors in asthmatic subjects. Am J Respir Crit Care Med 1999;159:1457-63. Subbarao P, Dorman SC, Rerecich T, Watson RM, Gauvreau GM, O’Byrne PM. Protection by budesonide and fluticasone on allergeninduced airway responses following discontinuation of therapy. J Allergy Clin Immunol 2005;15:745-50. Larsen BB, Nielsen LP, Engelstatter R, Steinijans V, Dahl R. Effect of ciclesonide on allergen challenge in subjects with bronchial asthma. Allergy 2003;58:207-12. Gauvreau GM, Watson RM, Rerecich TJ, Baswick E, Inman MD, O’Byrne PM. Repeatability of allergen-induced airway inflammation. J Allergy Clin Immunol 1999;104:66-71. Inman MD, Watson R, Cockcroft DW, Wong BJ, Hargreave FE, O’Byrne PM. Reproducibility of allergen-induced early and late asthmatic responses. J Allergy Clin Immunol 1995;95:1191-5. Postma DS, Sevette C, Martinat Y, Schlosser N, Aumann J, Kafe H. Treatment of asthma by the inhaled corticosteroid ciclesonide given either in the morning or evening. Eur Respir J 2001;17:1083-8. Cockcroft DW. Measure of airway responsiveness to inhaled histamine or methacholine: method of continuous aerosol generation and tidal breathing inhalation. In: Hargreave FE, Woolcock AJ, editors. Airway responsiveness: measurement and interpretation. Mississauga: Astra Pharmaceuticals Canada Ltd; 1985. p. 22-8. Cockcroft DW, Murdock KY, Kirby J, Hargreave F. Prediction of airway responsiveness to allergen from skin sensitivity to allergen and airway responsiveness to histamine. Am Rev Respir Dis 1987;135:264-7. Pizzichini E, Pizzichini MM, Efthimiadis A, Evans S, Morris MM, Squillace D, et al. Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements. Am J Respir Crit Care Med 1996;154:308-17. Parameswaran K, Inman MD, Watson RM, Morris MM, Efthimiadis A, Ventresca PG, et al. Protective effects of fluticasone on allergen-induced airway responses and sputum inflammatory markers. Can Respir J 2000; 7:313-9. Masuyama K, Jacobson MR, Rak S, Meng Q, Sudderick RM, Kay AB, et al. Topical glucocorticosteroid (fluticasone propionate) inhibits cells expressing cytokine mRNA for interleukin-4 in the nasal mucosa in allergen-induced rhinitis. Immunology 1994;82:192-9. Lee DK, Fardon TC, Bates CE, Haggart K, McFarlane LC, Lipworth BJ. Airway and systemic effects of hydrofluoroalkane formulations of high-dose ciclesonide and fluticasone in moderate persistent asthma. Chest 2005;127:851-60. Gauvreau GM, Wood LJ, Sehmi R, Watson RM, Dorman SC, Schleimer RP, et al. The effects of inhaled budesonide on circulating eosinophil progenitors and their expression of cytokines after allergen challenge in subjects with atopic asthma. Am J Respir Crit Care Med 2000;162: 2139-44. Inman MD, Denburg JA, Ellis R, Dahlback M, O’Byrne PM. Allergeninduced increase in bone marrow progenitors in airway hyperresponsive dogs: regulation by a serum hemopoietic factor. Am J Respir Cell Mol Biol 1996;15:305-11.

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Asthma diagnosis and treatment

Roflumilast, an oral, once-daily phosphodiesterase 4 inhibitor, attenuates allergen-induced asthmatic reactions Emmerentia van Schalkwyk, MBChB,a K. Strydom, MBChB,a Zelda Williams, RN,b Louis Venter, MSc,c Stefan Leichtl, PhD,d Christine Schmid-Wirlitsch, PhD,d Dirk Bredenbro¨ker, MD,d and Philip G. Bardin, FRACP, PhDb Cape Town and Rivonia, South Africa, Melbourne, Australia, and Konstanz, Germany

Background: Asthma is a chronic inflammatory disease with increasing incidence worldwide. Roflumilast is an oral, oncedaily inhibitor of phosphodiesterase type 4 that prevents the breakdown of cyclic adenosine monophosphate levels, leading to inhibition of proinflammatory signaling. Objective: The objective of this study was to investigate the effects of repeated doses of 250 or 500 mg of roflumilast on asthmatic airway responses to allergen. Methods: Twenty-three patients with mild asthma with an FEV1 of 70% of predicted value or greater were enrolled in a randomized, double-blind, placebo-controlled, 3-period crossover study. Patients participated in 3 treatment periods (7-10 days) separated by washout periods (2-5 weeks). Patients received 250 mg of oral roflumilast, 500 mg of roflumilast, or placebo once daily. Allergen challenge was performed at the end of each treatment period, followed by FEV1 measurements over the ensuing 24 hours. Results: Late asthmatic reactions (LARs) were reduced by 27% (P = .0110) and 43% (P = .0009) in patients treated with 250 and 500 mg of roflumilast, respectively, versus placebo. Roflumilast, 250 and 500 mg, also attenuated early asthmatic reactions by 25% (P = .0038) and 28% (P = .0046), although not to the same extent as LAR attenuation. Roflumilast was well tolerated. No serious adverse events or discontinuations caused by adverse events were reported. Conclusion: Once-daily oral roflumilast modestly attenuated early asthmatic reactions and, to a greater extent, LARs to allergen in patients with mild allergic asthma. Pronounced suppression of late responses in an allergen challenge model suggests that roflumilast might have anti-inflammatory activity, which could provide clinical efficacy in chronic inflammatory

From athe Department of Internal Medicine, University of Stellenbosch, Cape Town; bMonash Centre for Inflammatory Diseases, Monash University and Medical Centre, Melbourne; cALTANA Madaus (Pty) Ltd, Rivonia; and d ALTANA Pharma AG, Konstanz. Disclosure of potential conflict of interest: Drs Bredenbro¨ker, SchmidWirlitsch, Leichtl, and Venter are employees of ALTANA Pharma, and Dr Bardin has served as a consultant to ALTANA Pharma and received research support from GlaxoSmithKline, AstraZeneca, Schering Plough, and Boehringer-Ingelheim. The other authors have no conflict of interest to disclose. Received for publication August 26, 2004; revised April 8, 2005; accepted for publication April 18, 2005. Available online June 1, 2005. Reprint requests: Philip G. Bardin, FRACP, PhD, Monash Medical Centre, 246 Clayton Rd, Clayton 3168, Melbourne, Australia. E-mail: p.bardin@ southernhealth.org.au. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.023

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pulmonary diseases, such as asthma. (J Allergy Clin Immunol 2005;116:292-8.) Key words: Asthma, roflumilast, phosphodiesterase type 4, allergen provocation, inflammation, late phase

Asthma is a worldwide public health concern that has been increasing in prevalence, particularly in developed countries.1,2 In the United States approximately 31 million persons have been given a diagnosis of asthma.3 Furthermore, the economic burden of asthma (eg, prescription drugs, hospitalization, and loss of productivity) has also increased over the past 20 years, with the economic costs associated with asthma estimated to exceed those of tuberculosis and HIV-AIDS combined.4 Asthma is characterized by chronic inflammation and airway hyperresponsiveness (AHR), leading to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing.5 A primary goal of asthma therapy is to achieve and maintain control of clinical symptoms by improving lung function and reducing AHR. In addition, reducing the frequency of asthmatic exacerbations and improving health-related quality of life are important therapeutic goals. There are several therapeutic options for long-term maintenance (ie, controllers) and symptom relief (ie, relievers) available to asthmatic patients. Commonly prescribed fixed-dose combination therapies based on inhaled corticosteroids (ICSs) provide effective relief to many patients with asthma and comprise the current standard of care. Unfortunately, this standard of care does not control asthma in all patients. Long-term use of ICSs can also potentially cause serious systemic side effects,6 and poor compliance is an additional concern.7 Furthermore, a small number of patients are unresponsive to ICS therapy and require alternative therapeutic options.8 Novel therapies that increase the natural anti-inflammatory response in inflammatory target cells have the potential to meet the current unmet medical need for asthmatic patients. Inhaled allergen challenge in patients with mild allergic asthma results in an early asthmatic reaction (EAR), followed by a late-phase response (the late asthmatic reaction [LAR]). The EAR is a consequence of the activation and degranulation of cells expressing allergenspecific IgE. Mediators are released that induce nerve stimulation, mucus hypersecretion, vasodilation, and microvascular leakage.

Abbreviations used AHR: Airway hyperresponsiveness AUC: Area under the curve cAMP: Cyclic adenosine monophosphate COPD: Chronic obstructive pulmonary disease EAR: Early asthmatic reaction ICS: Inhaled corticosteroid LAR: Late asthmatic reaction PC20FEV1: Provocative concentration resulting in a 20% decrease in FEV1 PDE4: Phosphodiesterase type 4

The LAR is believed to reflect mechanisms of asthmatic inflammation because in this response activated airway cells release cytokines and chemokines locally and into the circulation, thus stimulating the release of inflammatory leukocytes, particularly eosinophils and their precursors, from the bone marrow into the circulation. Inflammatory cells in the peripheral blood are then recruited into the inflamed airways, where they augment airway inflammation and increase AHR. Roflumilast (3-cyclo-propylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) is an oral, once-daily phosphodiesterase type 4 (PDE4) inhibitor in clinical development as long-term maintenance therapy for chronic obstructive pulmonary disease (COPD) and asthma. Phosphodiesterases hydrolyze the second messenger cyclic adenosine monophosphate (cAMP) to 5#-adenosine monophosphate, rendering it inactive. The PDE4 isozyme has localized activity in the lung, and PDE4 inhibitors, such as roflumilast, block cAMP hydrolysis in the airways. The inflammatory response is highly sensitive to levels of cAMP, and by preventing the breakdown of cAMP, PDE4 inhibitors are associated with a natural anti-inflammatory activity. As a second messenger, cAMP blocks proliferation and chemotaxis of inflammatory cells (eg, lymphocytes), inhibits proinflammatory cell activity (eg, phagocytosis and respiratory burst), and suppresses the release of inflammatory and cytotoxic mediators (eg, TNF-a) in the lungs.9 In previous in vitro and in vivo studies, roflumilast decreased inflammatory cell infiltration into the airways, total protein levels, and TNF-a release.10 Thus by maintaining levels of cAMP in key airway inflammatory cells, roflumilast acts as a steroid-free anti-inflammatory agent that might have utility in diseases such as asthma and COPD.11 This proof-of-concept study investigated the effect of repeat doses of roflumilast (250 and 500 mg) on allergeninduced asthmatic responses and AHR in patients with mild allergic asthma.

METHODS Patients Patients were included in the study if they had a history of wheezing consistent with mild asthma, had an FEV1 of 70% of

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predicted value or greater, were between 18 and 50 years of age, and were not receiving treatment with asthma medications other than short-acting bronchodilators for symptom relief. Inclusion criteria also required a positive allergen skin prick test response and hyperresponsiveness to methacholine, with a provocative concentration resulting in a 20% decrease in FEV1 (PC20FEV1) of 16 mg/mL or less. All patients provided informed written consent, and the Human Research Ethics Committee of the University of Stellenbosch (Cape Town, South Africa) approved the study.

Study design and measurements In a double-blind, placebo-controlled, 3-period crossover study, patients were randomized to 250 mg of roflumilast, 500 mg of roflumilast, or placebo once daily for 7 to 10 days, with washout periods of 2 to 5 weeks before each treatment period (Fig 1). Randomization was performed after the first washout period at the first treatment visit. Study medication was taken between 7 AM and 10 AM daily. At the first baseline visit, FEV1 was measured with a mobile spirometer (Vitalograph, Hamburg, Germany), and AHR to methacholine challenge was assessed for each patient. At the second baseline visit, allergen challenge was done to determine a provocative concentration causing an early response to allergen (FEV1 25% decrease) and late response (FEV1 15% decrease), as detailed below. During the baseline visits, patients were assessed for adherence to inclusion and exclusion criteria. In eligible patients methacholine challenge was performed on the first treatment visit, and allergen challenge was performed on the second treatment visit (ie, after taking study medication for 7-10 days). The FEV1 measurements were done over the ensuing 24 hours at 5, 10, 15, 30, 45, and 60 minutes; subsequently at 1-hour intervals for the next 11 hours; and at 4.5, 5.5, and 24 hours after allergen challenge. At the conclusion of the 24-hour period after allergen challenge, methacholine challenge was repeated. Allergen and methacholine challenges were performed according to the bronchial provocation technique described by Chai et al,12 as previously published by Bardin et al.13 The challenge tests were performed by using a Spira Elektra breath-actuated inhalation dosimeter (SPIRA OY; Ha¨meenlinna, Finland). The duration of delivery per actuation was 0.6 seconds, and the flow of compressed air was 8 L/min. Each inhalation was controlled to result in an inspiratory flow of 0.6 to 0.8 L/s. Provocation began with 5 breaths of saline inhalation, each lasting 5 seconds, followed by holding the breath for 2 seconds (from functional residual capacity to total lung capacity). Challenges were performed only if baseline FEV1 was 70% of predicted value or greater, if it was within 12% of the initial value measured at baseline, and if, after saline inhalation, the decrease in FEV1 was 10% or less. At initial assessment, a decrease in FEV1 of 25% or greater from the postsaline value within the first 2 hours after the challenge defined the EAR, whereas the LAR was characterized by a decrease of 15% or greater in FEV1 from the postsaline value at 2 or more time points after spontaneous reversal of the EAR (>2-12 hours after challenge) and with a typical gradual deterioration in FEV1. Each patient was challenged with a single allergen identified on the basis of reactivity during the skin prick test. The allergens used in this study were house dust mite, cat hair, South African grass pollen, and Bermuda grass pollen (dilutions: 1022, 1023, 1024, 1025, and 1026 SQU/mL; Bayer Miles, Inc, Cape Town, South Africa). Patients always started with the lowest allergen concentration, and FEV1 was recorded 5, 10, and 15 minutes after inhalation. If the decrease in FEV1 was less than 10% of the postsaline FEV1, a 10-fold higher allergen concentration was administered; if the decrease was 10% to 15%, a 5-fold higher concentration was administered; and if the

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Asthma diagnosis and treatment FIG 1. Study design. In this randomized, double-blind, 3-period crossover study, each treatment period lasted 7 to 10 days and was preceded by a 2- to 5-week washout period.

TABLE I. Baseline patient demographics and characteristics Parameter

Patients (n = 23)

Median age, y (range) Mean height 6 SD, cm Mean weight 6 SD, kg Sex distribution, n (%) Female Male Smoking habits, n (%) Never smoked Exsmokers Current smokers Mean FEV1 6 SD, L Mean FEV1 6 SD, % predicted Mean PC20FEV1 6 SD, mg/mL

28 (20-44) 168 6 10.7 76 6 19.3 12 (52) 11 (48) 19 (83) 3 (13) 1 (4) 3.17 6 0.82 89 6 10 4.09 6 3.79

decrease in FEV1 was greater than 15% to 25%, the same allergen concentration was administered a second time. The concentration of allergen causing a decrease in postsaline FEV1 of greater than 25% was used in each patient for allergen provocation on subsequent study days. If needed, patients received single doses of inhaled beclomethasone or budesonide (800 mg) for stabilization of their condition after allergen challenge. For methacholine challenge, if the decrease in postsaline FEV1 was 10% or less, 5 breaths of the lowest methacholine concentration (0.03 mg/mL) were inhaled. Doubling concentrations of methacholine were then administered until either FEV1 decreased to 20% or greater of the postsaline value or the highest available methacholine concentration (16 mg/mL) was reached. The PC20FEV1 was calculated by means of linear interpolation of the FEV1 dose-response curve.13 Clinical laboratory evaluations, electrocardiography, vital signs measurement, and physical examinations were performed at the beginning and end of the study. Adverse events were assessed throughout the study.

Statistical methods The primary efficacy variable of this study was inhibition of the LAR. Attenuation of the EAR and AHR were secondary end points. Pairwise tests were used to compare the effects of the 3 treatments on the area under the curve (AUC) from 2 to 12 hours of the FEV1,

corresponding to the primary variable, LAR, and on the AUC from 0 to 2 hours of the FEV1, corresponding to the EAR. The AUC of the FEV1 decrease over time was compared by using ANOVA for the 3-period crossover design for both the LAR and EAR. Geometric means and 95% CIs were calculated for the differences between population means. Point estimates and 95% CIs were calculated for the maximum decrease analysis. Percentage reductions in the EAR and LAR were calculated for the differences between population least-squares means for the per-protocol population. Other secondary end points were analyzed in a descriptive manner with geometric means, and 95% CIs were calculated where appropriate.

RESULTS Patients Patient demographics and characteristics at baseline are summarized in Table I. A total of 23 patients were randomized in this study; 2 patients terminated the study prematurely because of nonmedical reasons. The perprotocol population varied among the different efficacy analyses because of missing or invalid data. Safety data are reported for the intent-to-treat population that was actually exposed to each treatment. The numbers of patients exposed to 500 mg of roflumilast, 250 mg of roflumilast, and placebo were 23, 21, and 22, respectively. Three patients were exsmokers, and 1 patient was a current smoker. Median age was 28 years. At baseline, the mean FEV1 was 3.17 L (SD, 0.82), 89% of the predicted value. Lung function and response to allergen challenge Patients treated with 250 and 500 mg of roflumilast experienced a significant attenuation of the LAR after allergen challenge compared with those treated with placebo. Treatment with 250 mg of roflumilast led to a 27% reduction in the LAR by means of AUC analysis (P = .0110) compared with placebo, whereas 500 mg of roflumilast led to a 43% (P = .0009) reduction compared with placebo (Table II). Therefore roflumilast-induced attenuation of the LAR showed a dose-related trend. Significant attenuation of the LAR compared with placebo was maintained for 12 hours after allergen challenge

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TABLE II. Treatment differences in the LAR and EAR with AUC analysis

Roflumilast dose

Roflumilast, 250 mg,  vs placeboà Roflumilast, 500 mg,  vs placeboà Roflumilast, 500 mg,  vs roflumilast, 250 mgà

Patients,* n

21 19 19

EAR AUC0-2h

P value Reduction, %

20.148 (20.272 to 20.024) 20.243 (20.382 to 20.104) 20.084 (20.223 to 0.056)

.0110

27

.0009

43

.1113

21

Point estimate (95% CI)

20.146 (20.248 to 20.044) 20.179 (20.307 to 20.050) 20.015 (20.118 to 0.089)

P value Reduction, %

.0038

25

.0046

28

.3851

3

AUC2-12hr, Area under the curve for FEV1 between 2 and 12 hours; AUC0-2hr, area under the curve for FEV1 between 0 and 2 hours. *This summarizes the per-protocol population. Data for the comparisons between 500 mg of roflumilast versus placebo or 500 mg of roflumilast versus 250 mg of roflumilast were only available for 19 patients.  Test. àReference.

(Fig 2). Treatment with 250 and 500 mg of roflumilast resulted in a modest yet statistically significant inhibition of the maximum decrease in FEV1 during the LAR of 17% (P = .0321) and 33% (P = .0002), respectively, compared with placebo (Table III). Both measures of lung function (AUC and maximum decrease) demonstrated that roflumilast inhibits bronchoconstriction associated with antigen challenge. On the basis of the analysis of the AUC of FEV1, patients treated with 250 and 500 mg of roflumilast also experienced a statistically significant attenuation in the EAR compared with placebo. Treatment with 250 mg of roflumilast reduced the EAR by 25% (P = .0038) and 500 mg of roflumilast reduced the EAR by 28% (P = .0046) versus placebo, respectively (Fig 2). There were no statistically significant differences between the 250- and 500-mg roflumilast doses with respect to LAR and EAR attenuation (Table II). There was a modest reduction of 14% in the maximum decrease in FEV1 during the EAR for both 250 and 500 mg of roflumilast compared with placebo (Table III). AHR was only slightly modified by roflumilast. During roflumilast treatment, the PC20FEV1 ratio was 1.0 or greater, indicating that airway responsiveness did not increase despite the preceding allergen challenge (1.03 for 250 mg of roflumilast and 1.11 for 500 mg of roflumilast). The PC20FEV1 ratio was less than 1.0 (0.87) in patients treated with placebo, which reflects an increase in AHR (Table IV). There was an apparent trend in doubling doses between roflumilast and placebo treatment, but the difference between treatments did not reach statistical significance.

Safety Roflumilast was well tolerated at both dose levels tested. No serious adverse events or discontinuations because of adverse events occurred during the study. Most adverse events were mild to moderate in intensity and were related to the digestive tract (eg, diarrhea and gastrointestinal disorder) or the nervous system (eg, headache). Headache was the most common adverse event. During baseline, 6 (26%) of 23 patients reported headaches. During the

treatment period, headache was reported by 4 (18%) of 22 patients, 6 (29%) of 21 patients, and 8 (35%) of 23 patients treated with placebo, 250 mg of roflumilast, and 500 mg of roflumilast, respectively. The majority of headaches reported during the treatment period (73%) were considered by the investigator to be unlikely or not related to study medication. The 6 adverse events associated with the digestive tract were considered by the investigator to be likely related to study medication, but no adverse events were judged definitely related. There were no clinically relevant changes in vital signs, electrocardiographic results, or clinical laboratory parameters.

DISCUSSION Asthma is characterized by chronic inflammation of the airways that might be responsive to treatment with PDE4 inhibitors. We have assessed the benefits of the PDE4 inhibitor roflumilast in patients with mild asthma using an allergen challenge model. Roflumilast had only a modest effect on the EAR but demonstrated a more pronounced effect on the LAR. This suggests a potential anti-inflammatory role for roflumilast because it is believed that allergen-induced LARs are linked to an influx of inflammatory cells and mediators associated with an inflammatory response.14-16 In several in vivo and in vitro animal models, roflumilast has demonstrated multiple anti-inflammatory effects, including inhibition of inflammatory cell infiltration and reduction of TNF-a release in the lungs.11 In a previous study in patients with exercise-induced asthma, 500 mg of roflumilast reduced the decrease in FEV1 after exercise challenge by 41% and the median TNF-a levels by 21% versus placebo.17 Exercise-induced bronchoconstriction is regarded as an asthmatic airway reaction to nonspecific stimuli.18,19 As a common characteristic of asthma, exercise-induced bronchoconstriction is an appropriate model in which to study the efficacy of roflumilast; however, alternative models are needed to elucidate the mechanism of action of roflumilast in patients with asthma. The allergen challenge model (particularly the LAR) is a

Asthma diagnosis and treatment

LAR AUC2-12h Point estimate (95% CI)

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Asthma diagnosis and treatment FIG 2. Mean percentage decrease of FEV1 from postsaline value after allergen challenge. Roflumilast, 250 and 500 mg, significantly attenuated the EAR (0-2 hours) and LAR (2-12 hours) compared with placebo. Data are presented for the intent-to-treat population.

TABLE III. Treatment differences in the LAR and EAR with maximum decrease analysis* LAR Roflumilast dose

Roflumilast, 250 mg,  vs placeboà Roflumilast, 500 mg,  vs placeboà Roflumilast, 500 mg,  vs roflumilast 250 mgà

Point estimate (95% CI)

EAR

P value

Reduction, %

Point estimate (95% CI)

P value

Reduction, %

4.712 (0.427 to 8.997)

.0321

17

4.015 (0.082 to 7.948)

.0457

14

8.901 (4.507 to 13.294)

.0002

33

3.909 (20.140 to 7.958)

.0580

14

4.118 (20.209 to 8.586)

.0613

18

20.106 (24.161 to 3.949)

.9580

0

*This summarizes the per-protocol population (n = 20, 21, and 21 for patients administered 500 mg of roflumilast, 250 mg of roflumilast, and placebo, respectively).  Test. àReference.

validated technique used to reproduce amplified asthmatic airway inflammation and has previously been used to assess the influence of anti-inflammatory medications on the EAR and LAR.20 The significant inflammatory component of the LAR has been attributed to an influx of activated eosinophils and the release of cytokines and chemokines that lead to the recruitment of additional inflammatory cells, such as basophils, lymphocytes, and monocytes. In a similar challenge model montelukast and budesonide demonstrated a partial reduction of inflammation, as judged by changes in sputum inflammatory cells; however, budesonide did not significantly reduce the EAR.21 As a targeted PDE4 inhibitor with anti-inflamma-

tory actions, roflumilast might confer earlier benefits compared with corticosteroids and more comprehensive anti-inflammatory benefits compared with a cysteinyl leukotriene receptor antagonist. The objective of this study was to evaluate the effect of roflumilast by assessing its effect on the EAR and LAR in patients with allergen-induced asthma. Roflumilast at daily doses of 250 mg and 500 mg significantly diminished the LAR, confirming attenuation of allergen-induced responses to single-dose administration of roflumilast found in early studies.22 Roflumilast attenuation of the LAR showed a dose-related trend: compared with placebo, 250 mg of roflumilast resulted in a mean attenuation

TABLE IV. PC20FEV1 ratios* by dose groupy Placebo (n = 15)

0.87 Mean PC20 FEV1 ratio 95% CI 0.55 to 1.36 Mean doubling 20.20 factor 95% CI 20.85 to 0.45

Roflumilast, 250 mg (n = 18)

Roflumilast, 500 mg (n = 18)

1.03

1.11

0.59 to 1.81 0.04

0.67 to 1.85 0.15

20.77 to 0.86

20.59 to 0.89

*Ratio of second visit of treatment period versus first visit of treatment period.  This table summarizes PC20FEV1 data for the per-protocol population.

of 27%, whereas treatment with 500 mg of roflumilast compared with placebo resulted in a mean attenuation of 43%. There was a greater degree of protection against decreases in FEV1 during the LAR than the EAR, supported by both AUC and maximum decrease analyses. There was a more modest 14% reduction of the EAR with 250 and 500 mg of roflumilast, as determined by means of maximum decrease analysis; however, these data support the AUC analysis, which is a more comprehensive examination of the entire EAR and LAR. Roflumilast also has a modest effect on the EAR, which is mediated principally by mast cell degranulation. Corticosteroids do not have a significant effect on the EAR, which is likely because of their lack of effect on mast cell degranulation.21,23 The early effect of roflumilast on the EAR might suggest a different onset of anti-inflammatory effect than is achieved with corticosteroids. Although modest, the reduction of the EAR by roflumilast might result from mast cell stabilization, mediator release, or both. Roflumilast had more pronounced effects on the LAR, and the higher dose reduced responses by almost half. Because of the inflammatory processes underpinning the LAR, PDE4 inhibitors might be particularly suitable to prevent cellular recruitment and mediator activity in this phase. In line with these known characteristics, roflumilast had a more pronounced effect on the LAR than the EAR, possibly because of its ability to reduce inflammation. Roflumilast did not significantly attenuate AHR, although there was a trend toward reduction of AHR after allergen. Roflumilast-induced attenuation of the LAR in this study is similar in magnitude to that achieved with ICS treatment, the primary controller therapy used by patients with asthma. For example, 200 mg of budesonide attenuates the LAR by approximately 44%,24 and ciclesonide, a novel ICS under clinical development, attenuates the LAR by approximately 47% versus placebo.25 Thus in this study roflumilast appears to offer anti-inflammatory effects comparable with those achieved with ICSs in this model. Additionally, long-acting b-agonists do not express anti-inflammatory activity and have been shown to inhibit the LAR after a single dose.26,27 This effect is lost after multiple doses.28 In this study roflumilast maintained its inhibitory effect after multiple doses. This adds credence to the hypothesis that the effect on the LAR with roflumilast in this study is more likely caused by antiinflammatory mechanisms rather than a direct effect on

airway muscle tone. Indeed, previous studies have shown that roflumilast does not have direct bronchodilatory effects.29 Side effects have restricted the use of PDE inhibitors in asthma.20 Because the PDE4 inhibitors are more targeted, they also tend to produce fewer adverse effects, such as nausea and headache. Roflumilast was well tolerated in this study. Most side effects were mild to moderate in intensity. Headache, diarrhea, and mild nausea were the most common adverse events and appeared to be dose dependent. All were transient and did not result in treatment discontinuation. In the current study the frequency of headache is likely to be affected by the considerable proportion of patients who reported headache at baseline (26%) and the high incidence of headaches in the placebo group (18%). The incidence of adverse events in this study, notably headache, was higher than that reported by studies of the safety and efficacy of roflumilast conducted in larger populations.30,31 In conclusion, the current study demonstrates that roflumilast, a targeted PDE4 inhibitor, inhibits the LAR, which might result from the anti-inflammatory activity of roflumilast. Further clinical studies with surrogate markers of inflammation (eg, induced sputum) are needed to confirm the anti-inflammatory effects of roflumilast. Additionally, these data suggest that roflumilast shows promise as an oral, once-daily, steroid-free treatment for asthma. Additional studies to determine the clinical benefits of roflumilast in asthma and COPD are needed. We thank our patients who participated in the study. The nursing support of Dot Steyn and Wendy Lee is acknowledged, and we thank Marianne Koopman for her expert measurements of pulmonary function.

REFERENCES 1. Neri M, Spanevello A. Chronic bronchial asthma from challenge to treatment: epidemiology and social impact. Thorax 2000;55(suppl 2): S57-8. 2. Adams BK, Cydulka RK. Asthma evaluation and management. Emerg Med Clin North Am 2003;21:315-30. 3. American Lung Association, Epidemiology and Statistics Unit, Research and Scientific Affairs. Trends in asthma morbidity and mortality. Available at: http://www.lungusa.org/atf/cf/{7A8D42C2-FCCA-46048ADE-7F5D5E762256}/ASTHMA1.PDF. Accessed July 23, 2004. 4. World Health Organization. WHO fact sheet no. 206. Bronchial asthma. Available at: http://www.who.int/inf-fs/en/fact206.html. Accessed November 14, 2003. 5. National Institutes of Health, National Heart, Lung, and Blood Institute, Global Initiative for Asthma. Global strategy for asthma management and prevention. Available at: http://www.ginasthma.com/workshop.pdf. Accessed December 3, 2004. 6. Dykewicz MS. Newer and alternative non-steroidal treatments for asthmatic inflammation. Allergy Asthma Proc 2001;22:11-5. 7. Spector S. Noncompliance with asthma therapy—are there solutions? J Asthma 2000;37:381-8. 8. Adcock IM, Lane SJ. Corticosteroid-insensitive asthma: molecular mechanisms. J Endocrinol 2003;178:347-55. 9. Essayan DM. Cyclic nucleotide phosphodiesterase (PDE) inhibitors and immunomodulation. Biochem Pharmacol 1999;57:965-73. 10. Hatzelmann A, Schudt C. Anti-inflammatory and immunomodulatory potential of the novel PDE4 inhibitor roflumilast in vitro. J Pharmacol Exp Ther 2001;297:267-79.

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11. Bundschuh DS, Eltze M, Barsig J, Wollin L, Hatzelmann A, Beume R. In vivo efficacy in airway disease models of roflumilast, a novel orally active PDE4 inhibitor. J Pharmacol Exp Ther 2001;297:280-90. 12. Chai H, Farr RS, Froehlich LA, Mathison DA, McLean JA, Rosenthal RR, et al. Standardization of bronchial inhalation challenge procedures. J Allergy Clin Immunol 1975;56:323-7. 13. Bardin PG, Dorward MA, Lampe FC, Franke B, Holgate ST. Effect of selective phosphodiesterase 3 inhibition on the early and late asthmatic responses to inhaled allergen. Br J Clin Pharmacol 1998;45:387-91. 14. Rossi GA, Crimi E, Lantero S, Gianiorio P, Oddera S, Crimi P, et al. Late-phase asthmatic reaction to inhaled allergen is associated with early recruitment of eosinophils in the airways. Am Rev Respir Dis 1991;144: 379-83. 15. Niimi A, Amitani R, Yamada K, Tanaka K, Kuze F. Late respiratory response and associated eosinophilic inflammation induced by repeated exposure to toluene diisocyanate in guinea pigs. J Allergy Clin Immunol 1996;97:1308-19. 16. Larsen GL, Wilson MC, Clark RA, Behrens BL. The inflammatory reaction in the airways in an animal model of the late asthmatic response. Fed Proc 1987;46:105-12. 17. Timmer W, Leclerc V, Birraux G, Neuha¨user M, Hatzelmann A, Bethke T, et al. The new phosphodiesterase 4 inhibitor roflumilast is efficacious in exercise-induced asthma and leads to suppression of LPS-stimulated TNF-a ex vivo. J Clin Pharmacol 2002;42:297-303. 18. Hofstra WB, Sont JK, Sterk PJ, Neijens HJ, Kuethe MC, Duiverman EJ. Sample size estimation in studies monitoring exercise-induced bronchoconstriction in asthmatic children. Thorax 1997;52:739-41. 19. Cockcroft DW. Nonallergic airway responsiveness. J Allergy Clin Immunol 1988;81:111-9. 20. Torphy TJ. Phosphodiesterase isozymes: molecular targets for novel antiasthma agents. Am J Respir Crit Care Med 1998;157:351-70. 21. Leigh R, Vethanayagam D, Yoshida M, Watson RM, Rerecich T, Inman MD, et al. Effects of montelukast and budesonide on airway responses and airway inflammation in asthma. Am J Respir Crit Care Med 2002; 166:1212-7. 22. Nell H, Louw C, Leichtl S, Rathgeb F, Neuha¨user M, Bardin PG. Acute anti-inflammatory effect of the novel phosphodiesterase 4 inhibitor

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23.

24.

25.

26.

27.

28.

29.

30.

31.

roflumilast on allergen challenge in asthmatics after a single dose [abstract]. Am J Respir Crit Care Med 2000;161(suppl):A200. Palmqvist M, Bruce C, Sjostrand M, Arvidsson P, Lotvall J. Differential effects of fluticasone and montelukast on allergen-induced asthma. Allergy 2005;60:65-70. Kidney JC, Boulet LP, Hargreave FE, Deschesnes F, Swystun VA, O’Byrne PM, et al. Evaluation of single-dose inhaled corticosteroid activity with an allergen challenge model. J Allergy Clin Immunol 1997; 100:65-70. Larsen BB, Nielsen LP, Engelstatter R, Steinijans V, Dahl R. Effect of ciclesonide on allergen challenge in subjects with bronchial asthma. Allergy 2003;58:207-12. Pizzichini MMM, Kidney JC, Wong BJO, Morris MM, Efthimiadis A, Dolovich J, et al. Effect of salmeterol compared with beclomethasone on allergen-induced asthmatic and inflammatory responses. Eur Respir J 1996;9:449-55. Weersink EJ, Aalbers R, Koeter GH, Kauffman HF, De Monchy JG, Postma DS. Partial inhibition of the early and late asthmatic response by a single dose of salmeterol. Am J Respir Crit Care Med 1994;150:1262-7. Dente FL, Bacci E, Bartoli ML, Cianchetti S, Di Franco A, Giannini D, et al. One week treatment with salmeterol does not prevent early and late asthmatic responses and sputum eosinophilia induced by allergen challenge in asthmatics. Pulm Pharmacol Ther 2004;17:147-53. Engelsta¨tter R, Wingertzahn M, Schmid-Wirlitsch C, Leichtl S, Bredenbro¨ker D, Wurst W. Roflumilast, an oral, once-daily phosphodiesterase 4 (PDE4) inhibitor, does not exhibit bronchodilatory activity. Presented at: 2004 ACAAI Meeting; November 12-17, 2004; Boston, Mass. Leichtl S, Schmid-Wirlitsch C, Bredenbro¨ker D, Rathgeb F, Wurst W. Roflumilast, a new, orally active, selective phosphodiesterase 4 inhibitor, is effective in the treatment of asthma [abstract]. Eur Respir J 2002; 20(suppl 38):303s. Izquierdo JL, Bateman ED, Villasante C, Schmid-Wirlitsch C, Bredenbro¨ker D, Wurst W. Long-term efficacy and safety over one year of once-daily roflumilast, a new, orally active, selective, phosphodiesterase 4 inhibitor, in asthma [abstract]. Am J Respir Crit Care Med 2003;167:A765.

Correction With regard to the May 2005 article entitled ‘‘Physical activity and exercise in asthma: Relevance to etiology and treatment’’ (2005;115:928-34): In the abstract, the seventh sentence should have appeared as follows:

The allergy community has placed emphasis on medical therapy and allergen avoidance; in addition, exercise has not been formally incorporated into the National Asthma Education and Prevention Program guidelines.

298 van Schalkwyk

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11. Bundschuh DS, Eltze M, Barsig J, Wollin L, Hatzelmann A, Beume R. In vivo efficacy in airway disease models of roflumilast, a novel orally active PDE4 inhibitor. J Pharmacol Exp Ther 2001;297:280-90. 12. Chai H, Farr RS, Froehlich LA, Mathison DA, McLean JA, Rosenthal RR, et al. Standardization of bronchial inhalation challenge procedures. J Allergy Clin Immunol 1975;56:323-7. 13. Bardin PG, Dorward MA, Lampe FC, Franke B, Holgate ST. Effect of selective phosphodiesterase 3 inhibition on the early and late asthmatic responses to inhaled allergen. Br J Clin Pharmacol 1998;45:387-91. 14. Rossi GA, Crimi E, Lantero S, Gianiorio P, Oddera S, Crimi P, et al. Late-phase asthmatic reaction to inhaled allergen is associated with early recruitment of eosinophils in the airways. Am Rev Respir Dis 1991;144: 379-83. 15. Niimi A, Amitani R, Yamada K, Tanaka K, Kuze F. Late respiratory response and associated eosinophilic inflammation induced by repeated exposure to toluene diisocyanate in guinea pigs. J Allergy Clin Immunol 1996;97:1308-19. 16. Larsen GL, Wilson MC, Clark RA, Behrens BL. The inflammatory reaction in the airways in an animal model of the late asthmatic response. Fed Proc 1987;46:105-12. 17. Timmer W, Leclerc V, Birraux G, Neuha¨user M, Hatzelmann A, Bethke T, et al. The new phosphodiesterase 4 inhibitor roflumilast is efficacious in exercise-induced asthma and leads to suppression of LPS-stimulated TNF-a ex vivo. J Clin Pharmacol 2002;42:297-303. 18. Hofstra WB, Sont JK, Sterk PJ, Neijens HJ, Kuethe MC, Duiverman EJ. Sample size estimation in studies monitoring exercise-induced bronchoconstriction in asthmatic children. Thorax 1997;52:739-41. 19. Cockcroft DW. Nonallergic airway responsiveness. J Allergy Clin Immunol 1988;81:111-9. 20. Torphy TJ. Phosphodiesterase isozymes: molecular targets for novel antiasthma agents. Am J Respir Crit Care Med 1998;157:351-70. 21. Leigh R, Vethanayagam D, Yoshida M, Watson RM, Rerecich T, Inman MD, et al. Effects of montelukast and budesonide on airway responses and airway inflammation in asthma. Am J Respir Crit Care Med 2002; 166:1212-7. 22. Nell H, Louw C, Leichtl S, Rathgeb F, Neuha¨user M, Bardin PG. Acute anti-inflammatory effect of the novel phosphodiesterase 4 inhibitor

J ALLERGY CLIN IMMUNOL AUGUST 2005

23.

24.

25.

26.

27.

28.

29.

30.

31.

roflumilast on allergen challenge in asthmatics after a single dose [abstract]. Am J Respir Crit Care Med 2000;161(suppl):A200. Palmqvist M, Bruce C, Sjostrand M, Arvidsson P, Lotvall J. Differential effects of fluticasone and montelukast on allergen-induced asthma. Allergy 2005;60:65-70. Kidney JC, Boulet LP, Hargreave FE, Deschesnes F, Swystun VA, O’Byrne PM, et al. Evaluation of single-dose inhaled corticosteroid activity with an allergen challenge model. J Allergy Clin Immunol 1997; 100:65-70. Larsen BB, Nielsen LP, Engelstatter R, Steinijans V, Dahl R. Effect of ciclesonide on allergen challenge in subjects with bronchial asthma. Allergy 2003;58:207-12. Pizzichini MMM, Kidney JC, Wong BJO, Morris MM, Efthimiadis A, Dolovich J, et al. Effect of salmeterol compared with beclomethasone on allergen-induced asthmatic and inflammatory responses. Eur Respir J 1996;9:449-55. Weersink EJ, Aalbers R, Koeter GH, Kauffman HF, De Monchy JG, Postma DS. Partial inhibition of the early and late asthmatic response by a single dose of salmeterol. Am J Respir Crit Care Med 1994;150:1262-7. Dente FL, Bacci E, Bartoli ML, Cianchetti S, Di Franco A, Giannini D, et al. One week treatment with salmeterol does not prevent early and late asthmatic responses and sputum eosinophilia induced by allergen challenge in asthmatics. Pulm Pharmacol Ther 2004;17:147-53. Engelsta¨tter R, Wingertzahn M, Schmid-Wirlitsch C, Leichtl S, Bredenbro¨ker D, Wurst W. Roflumilast, an oral, once-daily phosphodiesterase 4 (PDE4) inhibitor, does not exhibit bronchodilatory activity. Presented at: 2004 ACAAI Meeting; November 12-17, 2004; Boston, Mass. Leichtl S, Schmid-Wirlitsch C, Bredenbro¨ker D, Rathgeb F, Wurst W. Roflumilast, a new, orally active, selective phosphodiesterase 4 inhibitor, is effective in the treatment of asthma [abstract]. Eur Respir J 2002; 20(suppl 38):303s. Izquierdo JL, Bateman ED, Villasante C, Schmid-Wirlitsch C, Bredenbro¨ker D, Wurst W. Long-term efficacy and safety over one year of once-daily roflumilast, a new, orally active, selective, phosphodiesterase 4 inhibitor, in asthma [abstract]. Am J Respir Crit Care Med 2003;167:A765.

Correction With regard to the May 2005 article entitled ‘‘Physical activity and exercise in asthma: Relevance to etiology and treatment’’ (2005;115:928-34): In the abstract, the seventh sentence should have appeared as follows:

The allergy community has placed emphasis on medical therapy and allergen avoidance; in addition, exercise has not been formally incorporated into the National Asthma Education and Prevention Program guidelines.

Asthma diagnosis and treatment

Duration of postviral airway hyperresponsiveness in children with asthma: Effect of atopy Paraskevi Xepapadaki, MD, PhD,* Nikolaos G. Papadopoulos, MD, PhD,* Apostolos Bossios, MD, PhD, Emmanuel Manoussakis, MD, Theodoros Manousakas, MD, and Photini Saxoni-Papageorgiou, MD, PhD Athens, Greece

Background: Respiratory viruses induce asthma exacerbations and airway hyperresponsiveness (AHR). Atopy is an important risk factor for asthma persistence. Objective: We sought to evaluate whether atopy is a risk factor for prolonged AHR after upper respiratory tract infections (URIs). Methods: Twenty-five children (13 atopic and 12 nonatopic children) with intermittent virus-induced asthma were studied. Clinical evaluation, skin prick tests, methacholine bronchoprovocation, questionnaires, and a nasal wash specimen were obtained at baseline. For 9 months, subjects completed diary cards with respiratory symptoms. During their first reported cold, a nasal wash specimen was obtained. Methacholine provocation was performed 10 days and 5, 7, 9, and 11 weeks later. In case a new cold developed, the provocation schedule was followed from the beginning. Results: Viruses were detected in 17 (68%) of 25 patients during their first cold, with rhinovirus being most commonly identified (82%). AHR increased significantly 10 days after the URI, equally in both groups (P = .67), and remained so up to the fifth week. Duration of AHR in subjects experiencing a single URI ranged from 5 to 11 weeks, without a significant difference between groups. In the duration of the study, atopic children experienced more colds and asthma exacerbations than nonatopic children. Thus for duration of AHR, significant prolongation was noted in the atopic group when assessed cumulatively. Conclusion: In asthmatic children the duration of AHR after a single natural cold is 5 to 11 weeks. However, an increased rate of symptomatic cold and asthma episodes in atopic children is associated with considerable cumulative prolongation of AHR, which might help explain the role of atopy as a risk factor for asthma persistence. (J Allergy Clin Immunol 2005;116: 299-304.) Key words: Asthma, airway hyperresponsiveness, atopy

From the Allergy Unit, 2nd Pediatric Clinic, University of Athens. *Drs Xepapadaki and Papadopoulos contributed equally to this work. Disclosure of potential conflict of interest: N. Papadopoulos has received grants–research support from GlaxoSmithKline and AstraZeneca. P. Saxoni-Papageorgiou has received grants–research support from Novartis and Schering-Plough. All other authors—none disclosed. Received for publication November 11, 2004; revised March 28, 2005; accepted for publication April 4, 2005. Available online May 24, 2005. Reprint requests: Paraskevi Xepapadaki, MD, PhD, UPC Research Laboratories, 13, Levadias 11527, Goudi, Greece. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.007

Abbreviations used AHR: Airway hyperresponsiveness NW: Nasal wash SPT: Skin prick test URI: Upper respiratory tract infection

Asthma is characterized by abnormalities in lung function, variable airway obstruction, and airway hyperresponsiveness (AHR)1; among others, a significant correlation exists between the degree of AHR and both clinical severity and medication needs for asthma.2 A number of stimuli, including respiratory viruses, are able to induce AHR.3 Increased AHR to histamine in healthy subjects after upper respiratory tract infections (URIs) lasting up to 6 weeks was observed more than 20 years ago.4 Human experimental infections with human rhinoviruses confirmed that increased AHR to nonspecific stimuli is observed for up to 4 weeks after a viral infection in allergic subjects.5-7 Furthermore, AHR after viral infections has been documented in animal models of paramyxovirus, respiratory syncytial virus, and influenza virus infections.8-10 Most asthma exacerbations in children are associated with URIs, attributed in their majority to rhinoviruses.11,12 Virus-induced AHR is increased in atopic individuals compared with in healthy control subjects.5,13 Moreover, atopy is one of the strongest risk factors for the development and persistence of asthma, especially in childhood.14 More than 60% of asthmatic children are atopic, whereas the presence of atopy at the age of 12 months increases 3 to 4 times the risk for asthma persistence during later childhood and adulthood.15,16 There are suggestions that genes responsible for atopy and AHR act together to develop the full asthma phenotype.17 Furthermore, it has been proposed that the defective epithelial repair cycle, which is characteristic of asthma and strongly correlates to AHR, is amplified by exposure to TH2 cytokines.18 Nevertheless, our current understanding of the mechanisms connecting atopy, AHR, and asthma is still incomplete. The classical model of sensitization and exposure to particular allergens19 can only partly explain the epidemiologic observations. More recently, we and others have shown that the response of atopic asthmatic individuals to rhinovirus infection is 299

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defective, with a suboptimal TH1 response and a shift toward a TH2 phenotype.20,21 Such a response might lead to incomplete viral clearance and inflammation persistence, potentially perpetuating AHR.22 On the basis of the above, we hypothesized that atopy could be a risk factor for prolongation of AHR after respiratory viral infections. The aim of this study was to prospectively evaluate the duration of AHR in children with asthma after naturally occurring respiratory infections and assess the role of atopy as a risk factor in this respect.

METHODS Patients Twenty-five children (16 boys), 7 to 12 years of age, with intermittent asthma and a history of disease exacerbations attributable to URI only, were recruited for the study. Asthma diagnosis was based on history, 12% or greater improvement in FEV1 after bronchodilation, and AHR (PC20  16 mg/mL). Classification of asthma severity was performed according to the Global Initiative for Asthma.23 Atopy was evaluated by using skin prick tests (SPTs) to a panel of 18 locally relevant allergens performed outside the pollen season. Exclusion criteria were a history of seasonal or allergendriven symptoms, current or previous use of specific immunotherapy, use of inhaled steroids within the previous 2 months, recent (<2 months) URI, and chronic conditions potentially affecting airway responsiveness.24 All parents provided written consent, and the study was approved by the hospital’s ethics committee.

Study design A prospective case-control design with 9 months’ follow-up (September-June) was used. Clinical evaluation, IgE determination, SPT, and methacholine bronchial provocation were performed at baseline. Questionnaires and a nasal wash (NW) specimen were also obtained. Patients were grouped according to the presence of atopy (with n = 13). Children (or their parents) were asked to prospectively complete diary cards with upper and lower respiratory tract symptoms.11 During the first reported URI, judged either subjectively or by means of increased daily symptom scores (4), an NW was performed. Methacholine provocation was performed 10 days, and 5, 7, 9, and 11 weeks later. In case a new cold developed within this time period, the above provocation schedule was followed from the beginning. Bronchodilators were used as relievers; in case of persistence or deterioration of asthma symptoms, subjects were instructed to receive systemic steroids, although these were not needed on any occasion.

Methacholine provocation Methacholine provocation was performed with the 2-minute breathing dosing protocol.25 The aerosols were generated with a nebulizer output of 0.13 mL/min (Air Dynaval Taema). The procedure was discontinued if FEV1 decreased 20% or greater from baseline values or when a 16 mg/mL concentration had been administered.26 Spirometry was performed according to the American Thoracic Society guidelines.27

Virus detection RT-PCR was used for detection of viral RNA in NW samples, as previously described.28 PCR reactions were performed for rhinoviruses; enteroviruses; respiratory syncytial virus; coronaviruses OC43 and 229E; influenza viruses A and B; parainfluenza viruses 1, 2, and 3; adenoviruses; Chlamydia pneumoniae; and Mycoplasma pneumoniae.29,30

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Statistical analysis Comparisons of baseline characteristics were performed with the Fisher exact test for categoric variables and t tests for continuous variables. A common cold and an asthma exacerbation were defined as an increase of the relevant symptom score totals over the personal median value for 2 days or more. The duration of each episode was defined as the time from symptom initiation to return to the median value. Severity of each was defined as the maximum symptom score. The Wilcoxon signed-rank test and the Mann-Whitney U test were used to compare independent and paired outcome variables, respectively. The time to PC20 restoration was displayed in Kaplan-Mayer curves compared with the log-rank test. P values of less than .05 were considered significant.

RESULTS Baseline characteristics Table I shows the demographic, socioeconomic, and disease characteristics of the 2 groups, as obtained at baseline. Atopic asthmatic children had statistically significantly higher levels of total IgE (P = .04). A trend toward more cases of positive family history of atopy was also noted in the atopic group (P = .08). In respect to all remaining characteristics, the groups were homogeneous, with no significant differences when statistically compared. Baseline spirometric values did not differ between the 2 groups (Table I). Detection of respiratory viruses with PCR All NW specimens obtained at baseline were negative for the presence of respiratory viruses. PCR revealed the presence of a virus in 17 (68%) of 25 patients during their subsequent first URI. The most commonly identified virus was rhinovirus (14/17 [82%]). Adenovirus was found in 4 (23%) of 17 positive samples, whereas one child had both rhinovirus and adenovirus. Virus identification rates did not differ significantly between groups (nonatopic, 58%; atopic, 77%; P = .34). Duration of AHR All subjects completed the study. Methacholine responsiveness at baseline was slightly, but not significantly, higher in atopic individuals (time = 0, atopic PC20 = 5.9 6 1.1 mg/mL, nonatopic PC20 = 8.4 6 1.5 mg/mL; P = .18). All subjects experienced at least one natural cold within the study period. AHR increased significantly 10 days after the first reported URI, equally in both groups (time = 10th day, atopic PC20 = 2.3 6 1.1 mg/mL, nonatopic PC20 = 2.6 6 0.9 mg/mL; P = .002 in comparison with their respective baseline values in both cases and P = .67 between groups). This increase remained statistically significant up to the fifth week after the onset of the cold, progressively decreasing, although still without statistically significant differences, between the groups (time = fifth week, atopic PC20 = 3.7 6 1.3 mg/mL, nonatopic PC20 = 4.7 6 1.3 mg/mL; P = .005 and P = .021, respectively, in comparison with baseline and P = .29 between groups). The above analysis was performed in subjects with no additional URI during that period (time = 0,

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TABLE I. Patient characteristics: Comparison of baseline characteristics between atopic and nonatopic asthmatic children included in the study

Age (y) Sex (boys) IgE (mg/dL) Residence (urban) Density (persons per room) Pet ownership Exposure to tobacco smoke School type (public) Paternal education level (secondary education) Maternal education level (secondary education) Father or mother with atopy Colds in the previous year (n) Asthma exacerbations in the previous year (n) Emergency visits for asthma in the previous year (n) Hospitalizations for asthma in the previous year (n) FVC (% mean) 6 SD FEV1 (% mean) 6 SD

Nonatopic (n = 12)

Atopic (n = 13)

10.4 6 1.8 10.3 6 1.2 50% 77% 78.9 6 14.5 339.4 6 117.1 66% 54% 1.5 1.2

P value

Asthma diagnosis and treatment

Baseline characteristics

NS NS .04 NS NS

12% 75%

13% 62%

NS NS

83% 73%

85% 69%

NS NS

58%

54%

NS

60%

85%

.08

3.5

3.6

NS

3.3

3.3

NS

2.3

2.7

NS

0.1

0

NS

92.9 6 11.3 81.4 6 6.7

88.5 6 9.3 78.2 6 7.4

NS NS

FVC, Forced vital capacity.

n = 25; time = 10 days, n = 24; time = 5 weeks, n = 23). After the fifth week, several children experienced additional colds, and therefore provocations were rescheduled according to the study design; comparisons were not done on fixed time points. Eleven weeks after the initial URI, 10 children (3 atopic and 7 nonatopic children) had no further cold, and their methacholine responsiveness had returned to their respective baseline value. The mean duration of AHR in these subjects was 7.0 6 2.0 weeks (median, 5 weeks; range, 5-11 weeks) for the atopic children and 7.3 6 1.0 weeks (median, 7 weeks; range, 5-11 weeks) for the nonatopic children. In addition, using a simple linear regression model based on AHR values from the 10th day on and including all subjects with a single URI, the predicted time for AHR to return to its respective baseline value was 5.6 to 8.9 weeks for the atopic children and 6.7 to 10.2 weeks for the nonatopic children (Fig 1). However, important differences, as shown in Fig 2, were observed when the cumulative duration of AHR was assessed prospectively in the complete cohort. All 12 nonatopic asthmatic children returned to their baseline PC20 values by day 120, after the first reported URI,

FIG 1. Linear regression model of AHR changes from the 10th day to the seventh week after a single URI in atopic (A) and nonatopic (B) children with intermittent virus-induced asthma. A significant and time-dependent AHR decrease is observed in both cases. The predicted time for AHR to return to baseline did not differ between the groups.

whereas only 9 (67%) of 13 of the atopic children returned to their baseline PC20 value by day 200 (P = .0068), showing that in assessing the natural history of the disease, increased airway responsiveness, possibly caused by repetitive colds, is considerably more prolonged in atopic asthmatic children and might in fact last more than 6 months.

Common cold and asthma exacerbation characteristics Atopic children had generally more disease episodes than nonatopic children (Fig 3). The average number of colds that each subject experienced during the whole study period was marginally higher in the atopic group (atopic group, 4.6 6 0.4; nonatopic group, 3.5 6 0.4; P = .06). The average duration of each cold did not differ between groups (atopic group, 10.4 6 1.8 days; nonatopic group, 9.4 6 1.3 days; P = .81), as was the case for cold severity (atopic group, 2.2 6 0.2; nonatopic group, 2.9 6 0.3; P = .12). Atopic asthmatic children experienced significantly more exacerbations during the study period (5.1 6 0.6 vs 3.2 6 0.3 of the nonatopic children, P = .01). There were no differences in the duration or severity of asthma exacerbations between the groups (duration: atopic group,

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TABLE II. Seasonal distribution of asthma exacerbation characteristics in atopic and nonatopic asthmatic children

Asthma diagnosis and treatment

Nonatopic Winter Spring P value Atopic Winter Spring P value

Number

Duration (d)

Severity (index)

1.50 (0.55) 2.00 (1.26) .52

10.17 (15.21) 4.67 (1.63) .79

1.43 (0.55) 1.10 (0.33) .29

2.67 (1.50) 1.42 (0.67) .02

9.25 (4.80) 7.08 (4.89) .15

1.52 (0.53) 1.31 (0.35) .24

Reported values are presented as means (SD). Winter, October through January; spring, March through June. FIG 2. Kaplan-Meyer diagram of PC20 return to its respective baseline value after one or more naturally occurring URIs during a 9-month period in atopic and nonatopic asthmatic children. The difference is significant (P = .0068, survival analysis).

FIG 3. Upper respiratory tract symptom exacerbations, colds, and asthma exacerbations experienced during a 9-month period in atopic (open bars) and nonatopic (filled bars) children with intermittent asthma. *P < .05, #P = .06.

8.5 6 0.9 days; nonatopic group, 11.4 6 2.3 days; P = .65; severity: atopic group, 4.4 6 0.8; nonatopic group, 4.6 6 0.9; P = .60). No statistically significant differences were found in either the duration or severity of symptom scores between patients with positive and negative virus identification (data not shown). All atopic children had positive SPT responses to common pollen allergens (with a wheal of 3 mm).31 One atopic child also presented with a positive SPT response to mites. To address the question of whether prolonged AHR in atopic children might be affected by exposure to allergens to which they were sensitized, we compared the characteristics of asthma exacerbations during the pollen season (March-June) and the cold season (October-January). There were no differences in any such characteristics within the nonatopic group (Table II). However, within the atopic group, the number of asthma exacerbations was significantly higher during the cold season, suggesting that exposure to allergens could not account for additional disease burden in these patients (Table II).

DISCUSSION This is the first study to prospectively evaluate the duration of AHR in children after naturally occurring colds for an extended period of time. Two major findings are reported: (1) the duration of postviral nonspecific AHR is considerably more prolonged than previously thought, and (2) the duration of AHR after a single cold is the same in atopic and nonatopic children; however, an increased number of symptomatic colds cumulatively lead the former to prolonged AHR. Most previous studies assessing the duration of AHR after viral infections have used human rhinovirus experimental infections, which usually result in mild-tomoderate colds and very mild, if any, asthma exacerbations.32 Furthermore, fixed time points for up to 8 weeks after infection have been used for outcome evaluation.7,33 In this respect these studies excluded, to a considerable extent, the possibility of interference from additional infections but on the other hand could not evaluate the duration of virus-induced AHR in real-life conditions. In our study, when the duration of AHR was assessed after a single infection, either in the subgroup of subjects who did not have additional infection for up to 11 weeks, or by means of a simple regression model, it was 7 weeks on average, ranging from 5 to 11 weeks. This time span is still longer than previously reported in human subjects (10 days to 6 weeks)4,34 and comparable with time spans reported studies prospectively evaluating postviral AHR in experimental animals (2-22 weeks).8,10,35,36 Nevertheless, when AHR was assessed in the long term after multiple colds, a considerable proportion of atopic subjects remained hyperresponsive for considerably longer, in some cases more than 6 months. This is consistent with the notion that additional, naturally occurring colds might further prolong AHR, as recently shown in an experimental animal setting,37 and might prove important in considering the natural history of the disease, as well as management of such patients. Therefore although our hypothesis that atopy might lead to postviral AHR prolongation was shown not to be directly true because there was no difference in AHR duration after a single infection, the total duration of AHR was in fact

significantly longer in atopic than nonatopic children and that was associated with an increased number of colds, as well as asthma episodes. Patient characteristics did not differ between the groups at baseline; it cannot be excluded, however, that subclinical inflammation might have been greater in atopic subjects, which were slightly, but not significantly, more hyperresponsive at baseline. Several studies have shown that viral infection induces greater changes in nonspecific AHR in patients with respiratory allergy than in healthy control subjects.5,7,38,39 Accordingly, it has been assumed that after a viral infection, the response to allergen exposure might be exaggerated, a notion elegantly confirmed by using segmental bronchial provocations with allergen.40,41 Furthermore, clinical studies have shown synergy between viral infection and allergen exposure in sensitized individuals.42,43 Nevertheless, other studies have shown that allergen exposure does not necessarily augment virusmediated responses.44,45 This might also be the case in the atopic population of our study, who, even though they were sensitized to pollen allergens, had more symptoms during the fall and winter months rather than during the pollen season. This was not completely unexpected because all our subjects were selected for a postinfectious asthma phenotype during the 2 previous years, apparently reporting symptoms only after colds and not after allergen exposure. Certainly, the possibility that unidentified allergens might have contributed to some exacerbations cannot be completely excluded. However, the fact that the identified allergens did not seem to influence the outcome, although the number of colds and subsequent asthma exacerbations during the study was higher in atopic children, suggests that an increased susceptibility to viral infection might be a more plausible mechanism for prolongation of AHR in that group. Previous evidence suggests that the immune response to rhinovirus is defective in atopic asthmatic individuals, with reduced IFN-g production,20,46 which might be associated with increased susceptibility to symptomatic virus infections. In fact, rhinovirus-induced IFN-g production is strongly associated with AHR in patients with asthma.21 There is a longstanding speculation that allergic subjects, asthmatic subjects, or both have more respiratory infections than the healthy population.47 Most studies have compared respiratory symptoms after URI in atopic and healthy individuals. In a recent longitudinal cohort study, Corne et al48 showed that subjects with atopic asthma are not at greater risk of a rhinovirus infection than healthy individuals but have more frequent, severe, and longer-lasting lower respiratory tract symptoms.48 On the other hand, even within an atopic population, the outcome of an experimental rhinovirus infection is closely associated with levels of TH1 and TH2 cytokines.46 It is possible that any effects of atopy on the susceptibility to respiratory viral infections might differ between adults and children, in which cytokine responses, including IFN-g responses, do not mature before late childhood.49,50 Furthermore, recent evidence suggests that primary epi-

thelial cells derived from atopic subjects are more susceptible to ex vivo rhinovirus infection than cells from healthy control subjects.51 A weakness of the present study is that PCR viral identification was performed only for the first reported cold and not for subsequent colds. Nevertheless, it is without doubt that respiratory viral infections are the cause of the common cold, whereas the association of colds with subsequent asthma exacerbations is also well established.11,12 The degree of airway responsiveness is indicative of asthma severity and counts as an indirect marker of airway inflammation.2,52 In this respect prolongation of virusinduced AHR might well reflect persistent airway inflammation after multiple insults. Perpetuation of subclinical airway inflammation could have a substantial effect on the risk of asthma persistence, relapse later in life, or both, providing a possible mechanism for the well-established role of atopy as a major risk factor for the persistence of asthma and AHR from childhood to adulthood.53,54 In conclusion, the duration of AHR in children with intermittent virus-induced asthma ranges from 5 to 11 weeks after a single infection but might be considerably prolonged with repeated infections. Atopic subjects present with more symptomatic colds and thus cumulatively have significantly more prolonged AHR. Prolongation of virus-induced AHR in atopic subjects might help explain the well-established role of atopy as a risk factor for asthma persistence.

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11. Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, et al. Community study of role of viral infections in exacerbations of asthma in 9-11 year old children. BMJ 1995;310:1225-9. 12. Papadopoulos NG, Psarras S, Manoussakis E, Saxoni-Papageorgiou P. The role of respiratory viruses in the origin and exacerbations of asthma. Curr Opin Allergy Clin Immunol 2003;3:39-44. 13. Bardin PG, Sanderson G, Robinson BS, Holgate ST, Tyrrell DA. Experimental rhinovirus infection in volunteers. Eur Respir J 1996;9: 2250-5. 14. Christodoulopoulos P, Cameron L, Nakamura Y, Lemiere C, Muro S, Dugas M, et al. TH2 cytokine-associated transcription factors in atopic and nonatopic asthma: evidence for differential signal transducer and activator of transcription 6 expression. J Allergy Clin Immunol 2001; 107:586-91. 15. Martinez FD. Links between pediatric and adult asthma. J Allergy Clin Immunol 2001;107(suppl):S449-55. 16. Warner JO. Bronchial hyperresponsiveness, atopy, airway inflammation and asthma. Pediatr Allergy Immunol 1998;9:55-6. 17. Postma DS, Koppelman GH, Meyers DA. The genetics of atopy and airway hyperresponsiveness. Am J Respir Crit Care Med 2000;162 (suppl):S118-23. 18. Davies DE, Wicks J, Powell RM, Puddicombe SM, Holgate ST. Airway remodeling in asthma: new insights. J Allergy Clin Immunol 2003;111: 215-26. 19. Nelson HS, Szefler SJ, Jacobs J, Huss K, Shapiro G, Sternberg AL. The relationships among environmental allergen sensitization, allergen exposure, pulmonary function, and bronchial hyperresponsiveness in the Childhood Asthma Management Program. J Allergy Clin Immunol 1999;104:775-85. 20. Papadopoulos NG, Stanciu LA, Papi A, Holgate ST, Johnston SL. A defective type 1 response to rhinovirus in atopic asthma. Thorax 2002; 57:328-32. 21. Brooks GD, Buchta KA, Swenson CA, Gern JE, Busse WW. Rhinovirusinduced interferon-g and airway responsiveness in asthma. Am J Respir Crit Care Med 2003;168:1091-4. 22. Papadopoulos NG, Stanciu LA, Papi A, Holgate ST, Johnston SL. Rhinovirus-induced alterations on peripheral blood mononuclear cell phenotype and costimulatory molecule expression in normal and atopic asthmatic subjects. Clin Exp Allergy 2002;32:537-42. 23. Global Strategy for Asthma Management and Prevention. Bethesda (MD): National Institutes of Health, National Heart, Lung and Blood Institute; 2002. Publication no. 02-3659. 24. Cain H. Bronchoprovocation testing. Clin Chest Med 2001;22:651-9. 25. American Thoracic Society. Guidelines for methacholine and exercise challenge testing-1999. Am J Respir Crit Care Med 2000;161:309-29. 26. Cockcroft DW. How best to measure airway responsiveness. Am J Respir Crit Care Med 2001;163:1514-5. 27. American Thoracic Society. Standarization of spirometry: 1994 update. Am J Respir Crit Care Med 1995;152:1107-36. 28. Papadopoulos NG, Sanderson G, Hunter J, Johnston SL. Rhinovirus replicate effectively at lower airway temperatures. J Med Virol 1999;58: 100-4. 29. Papadopoulos NG, Hunter J, Sanderson G, Meyer J, Johnston SL. Rhinovirus identification by BglI digestion of picornavirus RT-PCR amplicons. J Virol Methods 1999;80:179-85. 30. Papadopoulos NG, Moustaki M, Tsolia M, Bossios A, Astra E, Prezerakou A, et al. Association of rhinovirus infection with increased disease severity in acute bronchiolitis. Am Respir Crit Care Med 2002; 165:1285-9. 31. Sub-Committee on Skin Tests of the European Academy of Allergology and Clinical Immunology. Skin tests used in type I allergy testing [position paper]. Allergy 1989;44 Suppl 10:1-59. 32. Skoner DP, Doyle WJ, Seroky J, Fireman P. Lower airway responses to influenza A virus in healthy allergic and nonallergic subjects. Am J Respir Crit Care Med 1996;154:661-4. 33. Fleming HE, Little FF, Schnurr D, Avila PC, Wong H, Liu J, et al. Rhinovirus-16 colds in healthy and in asthmatic subjects: similar

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changes in upper and lower airways. Am J Respir Crit Care Med 1999; 160:100-8. Grunberg K, Timmers MC, Smits HH, de Klerk EP, Dick EC, Spaan WJ, et al. Effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo. Clin Exp Allergy 1997;27:36-45. Schwarze J, Gelfand EW. Respiratory viral infections as promoters of allergic sensitization and asthma in animal models. Eur Respir J 2002;19: 341-9. Folkerts G, Nijkamp FP. Virus-induced airway hyperresponsiveness. Role of inflammatory cells and mediators. Am J Respir Crit Care Med 1995;151:1666-74. Matsuse H, Behera AK, Kumar M, Rabb H, Lockey RF, Mohapatra SS. Recurrent respiratory syncytial virus infections in allergen-sensitized mice lead to persistent airway inflammation and hyperresponsiveness. J Immunol 2000;164:6583-92. Trigg CJ, Nicholson KG, Wang JH, Ireland DC, Jordan S, Duddle JM, et al. Bronchial inflammation and the common cold: a comparison of atopic and non-atopic individuals. Clin Exp Allergy 1996;26:665-76. Sears MR, Burrows B, Herbison GP, Holdaway MD, Flannery EM. Atopy in childhood. II. Relationship to airway responsiveness, hay fever and asthma. Clin Exp Allergy 1993;23:949-56. Lemanske RF, Dick EC, Swenson CA, Vrtis RF, Buse WW. Rhinovirus upper respiratory tract infection increases airway hyperreactivity and late asthmatic reactions. J Clin Invest 1989;83:1-10. Calhoun WJ, Dick EC, Schwartz LB, Busse WW. A common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects. J Clin Invest 1994;94: 2200-8. Green RM, Custovic A, Sanderson G, Hunter J, Johnston SL, Woodcock A. Synergism between allergens and viruses and risk of hospital admission with asthma: case-control study. BMJ 2002;324:763-8. Papadopoulos NG, Psarros F, Manoussakis E, Hatzipsalti M, Bossios A, Syrigou E, et al. Association of respiratory viral infections with disease exacerbations in patients with allergen-driven seasonal asthma and hay fever [abstract]. Allergy 2002;57:295. Avila PC. Interactions between allergic inflammation and respiratory viral infections. J Allergy Clin Immunol 2000;106:829-31. de Kluijver J, Evertse CE, Sont JK, Schrumpf JA, van Zeijl-van der Ham CJ, Dick CR, et al. Are rhinovirus-induced airway responses in asthma aggravated by chronic allergen exposure? Am J Respir Crit Care Med 2003;168:1174-80. Parry DE, Busse WW, Sukow KA, Dick CR, Swenson C, Gern JE. Rhinovirus-induced PBMC responses and outcome of experimental infection in allergic subjects. J Allergy Clin Immunol 2000;105:692-8. Busse WW, Gern JE. Do allergies protect against the effects of a rhinovirus cold? J Allergy Clin Immunol 2000;105:889-91. Corne JM, Marshall C, Smith S, Schreiber J, Sanderson G, Holgate ST, et al. Frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study. Lancet 2002;359:831-4. Prescott SL. Allergy: when does it begin and where will it end? Allergy 2003;58:864-7. Smart JM, Kemp AS. Ontogeny of T-helper 1 and T-helper 2 cytokine production in childhood. Pediatr Allergy Immunol 2001;12:181-7. Wark PA, Johnston SL, Bucchieri F, Powell R, Puddicombe S, Laza-Stanca V, et al. Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus. J Exp Med 2005;201:937-47. Boulet LP. Asymptomatic airway hyperresponsiveness: a curiosity or an opportunity to prevent asthma? Am J Respir Crit Care Med 2003;167: 371-8. Arruda LK, Sole D, Baena-Cagnani CE, Naspitz CK. Risk factors for asthma and atopy. Curr Opin Allergy Clin Immunol 2005;5:153-9. Van den Toorn LM, Overbeek SE, de Jongste JC, Leman K, Hoogsteden HC, Prins JB. Airway inflammation is present during clinical remission of atopic asthma. Am J Respir Crit Care Med 2001;164:2107-13.

Mechanisms of asthma and allergic inflammation

Douglas A. Kuperman, PhD,a,b,c Christina C. Lewis, PhD,b,c Prescott G. Woodruff, MD,b,d,e Madeleine W. Rodriguez, BS,b,c Yee Hwa Yang, PhD,b,c Gregory M. Dolganov, PhD,b,d,e John V. Fahy, MD,b,d,e and David J. Erle, MDb,c,d,e,f Chicago, Ill, and San Francisco, Calif

Background: Asthma functional genomics studies are challenging because it is difficult to relate gene expression changes to specific disease mechanisms or pathophysiologic features. Use of simplified model systems might help to address this problem. One such model is the IL-13/Epi (IL-13–overexpressing transgenic mice with STAT6 expression limited to epithelial cells) focused transgenic mouse, which isolates the effects of a single mediator, IL-13, on a single cell type, the airway epithelial cell. These mice develop airway hyperreactivity and mucus overproduction but not airway inflammation. Objective: To identify how effects of IL-13 on airway epithelial cells contribute to gene expression changes in murine asthma models and determine whether similar changes are seen in people with asthma. Methods: We analyzed gene expression in ovalbumin allergic mice, IL-13–overexpressing mice, and IL-13/Epi mice with microarrays. We analyzed the expression of human orthologues of genes identified in the mouse studies in airway epithelial cells from subjects with asthma and control subjects. Results: In comparison with the other 2 models, IL-13/Epi mice had a remarkably small subset of gene expression changes. Human orthologues of some genes identified as increased in the mouse models were more highly expressed in airway epithelial cells from subjects with asthma than in controls. These included calcium-activated chloride channel 1, 15-lipoxygenase, trefoil factor 2, and intelectin. Conclusion: The combination of focused transgenic models, DNA microarray analyses, and translational studies provides a powerful approach for analyzing the contributions of specific mediators and cell types and for focusing attention on a limited number of genes associated with specific pathophysiologic From athe Department of Medicine, Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago; and bthe Department of Medicine, cthe Lung Biology Center, dthe Division of Pulmonary and Critical Care Medicine, ethe Cardiovascular Research Institute, and fthe Program in Immunology, University of California San Francisco School of Medicine. Supported by National Institute of Health grants HL56835 and HL72301 and by the UCSF Sandler Center for Basic Research in Asthma. Disclosure of potential conflict of interest: D. A. Kuperman, none disclosed. C. A. Lewis, none disclosed. P. G. Woodruff, none disclosed. M. W. Rodriguez, none disclosed. Y. H. Yang, none disclosed. G. M. Dolganov, none disclosed. J. V. Fahy, none disclosed. D. J. Erle, none disclosed. Received for publication January 6, 2005; revised February 28, 2005; accepted for publication March 9, 2005. Available online May 2, 2005. Reprint requests: David J. Erle, MD, UCSF Box 2922, San Francisco, CA 94143-2922. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.03.024

aspects of asthma. (J Allergy Clin Immunol 2005;116: 305-11.) Key words: Asthma, IL-13, calcium-activated chloride channel, trefoil factor, 15-lipoxygenase, intelectin

Asthma results from a complex interplay between genetic and environmental factors.1 Microarray studies of lung tissue from subjects with asthma and from animals with experimental allergic asthma typically reveal hundreds of genes that are differentially expressed in comparison with normal lung.2-4 However, it has been difficult to relate gene expression changes to specific mediators, cell types, or pathophysiologic features. Models that focus on one particular mediator or cell type or on specific pathophysiologic features might help overcome this obstacle. We developed a transgenic mouse model to analyze effects of a single cytokine, IL-13, on a single cell type, the nonciliated airway epithelial cell.5 IL-13 expression is increased in the airways of subjects with asthma,6,7 and IL-13 is necessary and sufficient for the development of experimental asthma.8-10 IL-13 activates the signaling molecule signal transducer and activator of transcription factor 6 (STAT6) in many cell types.11 Overexpression of IL-13 in the airways of mice with normal STAT6 expression (tg–IL-13 mice) causes mucus overproduction, airway hyperreactivity, inflammation, fibrosis, and emphysema.10 To isolate the effects of IL-13 on airway epithelial cells, we produced mice that overexpress IL-13 in the airway and express STAT6 only in nonciliated airway epithelial cells. These IL-13/Epi mice developed mucus overproduction and airway hyperreactivity but not inflammation, fibrosis, or emphysema.5 Here we apply a genomics-based approach to identify gene expression changes in the IL-13/Epi focused model, the tg–IL-13 model, and a conventional allergic asthma model. Inclusion of the focused model allowed us to pinpoint a remarkably small number of gene expression changes that were consistently associated with airway hyperreactivity and mucus production. Furthermore, we used these results to guide translational studies that show the relevance of some of these gene expression changes in people with asthma. These experiments demonstrate that focused transgenic models combined with microarrays can lead to an improved understanding of the pathogenesis of complex diseases such as asthma. 305

Mechanisms of asthma and allergic inflammation

Dissecting asthma using focused transgenic modeling and functional genomics

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Microarray analysis Abbreviations used GAPD: Glyceraldehyde-3-phosphate dehydrogenase IL-13/Epi: IL-13–overexpressing transgenic mice with STAT6 expression limited to epithelial cells tg–IL-13: IL-13–overexpressing transgenic mice STAT6: Signal transducer and activator of transcription factor 6 UCSF: University of California San Francisco

Mechanisms of asthma and allergic inflammation

METHODS Mice The University of California San Francisco (UCSF) Committee on Animal Research approved the use of mice for these experiments. Care and use of animals complied with the United States Public Health Service’s Policy on Humane Care and Use of Laboratory Animals by Awardee Institutions (#3400-01). Three groups of transgenic mice were used in these experiments: (1) CC10-IL-131, Stat61/2 mice (tg– IL-13 mice); (2) CC10–IL-131 Stat62/2 mice; and (3) CC10–IL-131, Stat62/2, CC10-hStat61 mice (IL-13/Epi mice). CC10 refers to the Clara cell specific promoter used to express IL-13 and human STAT6 (hSTAT6).12 The development and characterization of these transgenic mice have been previously described.5 Mice used here were backcrossed 5 times onto the Balb/c genetic background. tg–IL-13 mice have an intact Stat6 gene and IL-13–driven activation of STAT6 in a wide range of cells in the lung, resulting in airway inflammation, mucus overproduction, airway hyperreactivity, subepithelial fibrosis, and emphysema. IL-131 Stat62/2 mice (used as negative controls) lack STAT6 and did not develop any detectable IL-13–induced lung pathology. IL-13/Epi mice also lack mouse STAT6 but express human STAT6 selectively in airway epithelial cells. hSTAT6 is functional in mice, and epithelial-restricted activation of Stat6 by IL-13 induced airway hyperreactivity and mucus production without airway inflammation, subepithelial fibrosis, or emphysema. Wild-type Balb/c mice 6 to 8 weeks old were used for the ovalbumin challenge model. There were 5 mice in each experimental and control group.

Antigen sensitization and challenge Mice were sensitized by intraperitoneal administration of 50 mg grade V ovalbumin mixed with adjuvant (10 mg aluminum potassium sulfate) 3 times at weekly intervals. Control mice received adjuvant alone. Beginning 1 week after the last injection, mice were challenged 3 times by intranasal administration of ovalbumin (1 mg in 50 mL PBS) at daily intervals. Control mice were challenged with PBS alone. Tissues were harvested for isolation of RNA 24 hours after the last challenge.

Isolation and labeling of RNA from mice Whole-lung RNAs were purified by using Trizol (Invitrogen, Carlsbad, Calif). Integrity of all RNA samples used in this study was confirmed with a model 2100 bioanalyzer (Agilent Technologies, Inc, Palo Alto, Calif). Cy3-labeled and Cy5-labeled lung cDNAs were prepared as described.13 To obtain samples enriched for airway epithelial cell RNA, the superior portion of the trachea was cannulated, and the trachea and proximal major bronchi were excised from the thorax and slowly perfused with 0.35 mL lysis buffer (RNeasy kit; Qiagen Inc, Valencia, Calif). RNA was isolated from perfusate according to the manufacturer’s instructions. Because the amount of RNA obtained from tracheal perfusates was only ~1 mg, a T7 RNA polymerase-based method13 was used to prepare Cy3labeled and Cy5-labeled amplified cRNAs for array hybridizations.

Lung gene expression was analyzed by hybridizing Cy5-labeled cDNA from mouse lungs (5 mice per group, each hybridized separately) along with Cy3-labeled reference lung cDNA pooled from wild-type mice. Tracheal perfusate samples were analyzed similarly, except that amplified cRNA from each mouse was compared with an amplified cRNA reference pool made by using tracheal perfusate samples from wild-type mice. DNA microarrays used in these experiments were produced by using the Operon Biotechnologies (Huntsville, Ala) Mouse Genome Oligo 2.0 set of 70-mer oligonucleotides, supplemented by some additional 70-mers. A MIAME-compliant description of the array experiments and the raw array data are available from Gene Expression Omnibus (http:// www.ncbi.nlm.nih.gov/geo, accession number GSE1438). We used an approach that allowed us to estimate differential gene expression between the various groups we studied on the basis of linear models, as previously described.14,15 To determine whether there were significant differences in gene expression between groups, we calculated the odds ratio (probability of being differentially expressed/probability of not being differentially expressed). When the log2 of the odds ratios (known as the B-statistic) was greater than 0, we classified the gene as differentially expressed.16,17 Hierarchical clustering, a method for grouping together genes with similar expression patterns, was performed by using Acuity 4.0 software (Axon Instruments, Union City, Calif). In addition, genes were classified on the basis of expression patterns to determine whether they were increased relative to controls in 1, 2, or all 3 of the experimental groups (ovalbumin, tg–IL-13, and IL-13/Epi) as follows. Seven pseudogene vectors were created to represent genes that were increased only in 1 of the 3 models ([0,0,1]; [0,1,0]; and [1,0,0]), genes increased equally in 2 models ([0,1,1]; [1,0,1]; [1,1,0]), and genes increased equally in all 3 models ([1,1,1]). Each differentially expressed gene was assigned a vector in 3-dimensional space according to the median log fold-change gene expression values determined for that gene in the 3 experimental groups. All vectors were scaled to unit length, and each gene was matched to the closest pseudogene, as determined by Euclidean distance.

Human subjects These studies were approved by the UCSF Committee on Human Research and conducted in compliance with the Declaration of Helsinki principles. Written informed consent was obtained from all subjects. All subjects were adult nonsmokers (,10 pack-year total smoking history with last cigarette .1 year before the study). Medical histories were obtained, physical examinations were performed, symptom questionnaires were collected, and spirometry was performed as described.18 Airway reactivity was measured by determining the PC20.19 The 28 healthy control subjects had no history of lung disease and were not hyperreactive (PC20 . 16 mg/mL). All 30 subjects with asthma had a previous physician diagnosis of asthma, were hyperreactive (PC20 , 8 mg/mL), and used only short-acting inhaled b-adrenergic–agonist medications for therapy. Individuals with an asthma exacerbation or respiratory infection within the previous 6 weeks or significant medical problems other than asthma and those using inhaled or systemic corticosteroids or leukotriene antagonists were excluded.

Bronchial epithelial brushings Bronchoscopy was performed, and bronchial brushings were obtained randomly from right or left lower lobe bronchial segments by using 4 disposable cytology brushes. The brushes were gently vortexed in sterile saline. Cells from all brushes were pooled, yielding a single sample for each subject. An aliquot was removed for cytocentrifugation, stained with Diff-Quik (Baxter, McGraw Park,

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FIG 1. Lung gene expression changes in 3 mouse asthma models. Differentially expressed genes were arranged by hierarchical clustering. Each column represents data from 1 of 5 individual mice in each group. Colors represent fold-change compared with the appropriate controls. The arrow indicates a small group of genes that were increased in all 3 models. Ova, Ovalbumin.

Ill), and examined by light microscopy. On average, the bronchial brushings contained 97% epithelial cells. Total RNA was extracted by using the RNeasy kit. Thirty-four of the 58 human epithelial cell RNA samples analyzed for this study were analyzed separately for another study (Woodruff et al, unpublished data).

Cultured human bronchial epithelial cells Air-liquid interface cultures were established by using published protocols.20 Primary normal human bronchial epithelial cells (lot 3F1191; Cambrex Bio Science, Baltimore, Md) were seeded onto 12-mm–diameter Corning Transwells (Corning, NY) containing 0.4-mm pores (4-5 replicates per condition). Cells were submerged in bronchial epithelial growth media (Clonetics) until confluent (3 days), and then the apical media was removed and the basolateral media was changed to differentiation media (1:1 of Dulbecco modified Eagle media to bronchial epithelial growth media) for 8 days. After the differentiation period, IL-13 (0-10 ng/mL) was maintained in the basolateral media over the period of the next 4 days. At the end of the IL-13 exposure, total RNA was isolated by using the RNeasy kit.

Analysis of gene expression by real-time PCR Primers and a probe for mouse genes (see Table E1 in the Journal’s Online Repository at www.mosby.com/jaci) and human genes (see Table E2 in the Online Repository at www.mosby.com/jaci) were designed by using Primer Express software (Perkin Elmer, Boston, Mass). First-strand cDNA synthesis and PCR were performed by using ABI Prizm 7700 or 7900 Sequence Detection Systems (Applied Biosystems, Foster City, Calif). For mouse lung and cultured human airway epithelial cell studies, cycle thresholds for each gene were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPD). For human bronchial brushing samples, a 2-step PCR approach21 was used, and transcript copy numbers were normalized on the basis of the geometric mean expression values of 3 housekeeping genes (GAPD, elongation factor 1a1, and cyclophilin A) as described.22 To identify genes that were differentially expressed between subjects with asthma and normal subjects, we performed a Wilcoxon rank-sum test and calculated the corresponding adjusted P value by using the Westfall and Young23 maxT algorithm implemented in Bioconductor’s multtest package (Bioconductor; open source software for bioinformatics, www.bioconductor.org).24

FIG 2. Gene expression patterns. Grouping revealed genes with increased expression in (A) the ovalbumin (Ova) model only, (B) the Ova and tg–IL-13 models, (C) the tg–IL-13 model only, and (D) all 3 models. The number of genes (left) and representative genes (right) is shown for each group. Phenotypic attributes of each model are shown at the bottom. AHR, Airway hyperreactivity.

RESULTS Microarray analysis of gene expression in mouse asthma models We used DNA microarrays to analyze gene expression in 3 murine models of asthma. The first model was composed of mice that were sensitized and challenged with ovalbumin (ovalbumin model). The second model was composed of mice with transgenic overexpression of IL-13 in the lung (tg–IL-13 model); these mice had an intact gene for STAT6, a key signaling molecule required for IL-13 activity. The third model was composed of IL-13 transgenic mice with STAT6 expression limited to nonciliated airway epithelial cells (IL-13/Epi model). In whole-lung samples, 805 gene transcripts were differentially expressed (increased or decreased) in at least 1 model (Fig 1). Of these, 509 genes were increased or decreased by 2-fold in at least 1 model. There were 583 gene expression changes in the ovalbumin model and 351 changes in the tg–IL-13 model. In contrast, only 18 changes were seen in the IL-13/Epi focused transgenic model. Comparison of the focused transgenic model to the other models allowed for the identification of a small subset of gene expression changes that are attributable to a specific mechanism and also are relevant to pathogenesis in complex systems.

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TABLE I. Genes induced by allergen and by direct effects of IL-13 on airway epithelial cells* Fold-change by arrays Symbol

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Clca3 Slc26a4 Retnla Itln Nadsyn Atp2a1 AMCase Muc5ac Agr2 Tff1 Reg3g Pigr Muc5b D630002J15Rik Scin Alox15 H2-Q7 1110001D15Rik Tff2

Fold-change by PCR

Description

Ova

tg–IL-13

IL-13/Epi

Ova

tg–IL-13

IL-13/Epi

Chloride channel calcium activated 3 Solute carrier family 26, member 4 Resistin-like a Intelectin NAD synthetase 1 Ca11 transporting ATPase Acidic mammalian chitinase Mucin 5, subtypes A and C Anterior gradient 2 Trefoil factor 1 Regenerating islet-derived 3g Polymeric immunoglobulin receptor Mucin 5, subtype B RIKEN cDNA D630002J15 gene Scinderin 15-lipoxygenase Histocompatibility 2, Q region locus 7 RIKEN cDNA 1110001D15 gene Trefoil factor 2

78.3 9.9 4.3 7.6 17.2 15.3 2.5 12.1 3.8 3.3 6.4 2.5 4.0 2.8 2.8 3.9 2.4 2.1 4.5

59.8 29.8 23.8 10.8 12.5 8.0 12.3 6.8 7.7 6.4 4.1 8.4 5.1 4.3 2.9 1.9 2.1 2.1 4.5

28.2 20.0 23.7 19.2 6.4 3.9 8.6 3.8 4.3 5.7 3.2 2.3 2.8 2.0 3.0 2.0 2.1 2.4 1.8

820.3 18.7 ND  9.5 0.7 0.4 3.7 33.5 11.1 52.6 ND 1.5 3.6 ND 21.9 5.2 ND ND 3.5

1530.7 40.1 ND 73.8 0.5 0.1 7.4 23.4 21.6 592.2 ND 12.3 7.4 ND 21.8 1.6 ND ND 8.6

1448.2 10.3 ND 146.2 0.8 0.9 4.4 7.8 10.2 224.7 ND 2.2 13.2 ND 34.5 2.8 ND ND 4.9

*Genes with a 2.0-fold or greater increase in expression in both the ovalbumin (Ova) allergic model and the IL-13/Epi model are included. Array fold-change values represent the larger median fold change from lung or tracheal perfusate compared with the appropriate control group.  ND indicates that we did not use PCR to determine fold change for these mouse genes, which do not have obvious human orthologues.

TABLE II. Characteristics of human subjects*

Number Age Sex PC20 (mg/mL) FEV1/forced vital capacity (%) FEV1 (% predicted)

Control subjects

Subjects with asthma

P

28 36 6 8 16 F/12 M 60.8 6 1.4 80.6 6 1.4

30 38 6 13 18 F/12 M 0.9 6 1.3 71.1 6 1.4

— NS NS ,.0001 ,.0001

106.5 6 13.2

85.2 6 13.9

,.0001

*Values represent means 6 SDs.

To aid in interpretation, we grouped transcripts according to the magnitude of change in expression across the 3 models. Transcripts were grouped by determining whether the expression changes were seen in 1, 2, or all 3 of the models (see Table E3 in the Online Repository at www.mosby.com/jaci). There were 17 transcripts with at least 2-fold increases in the IL-13/Epi model but only 3 transcripts with at least 2-fold decreases; therefore, we focused our subsequent analyses on increased genes. A total of 239 transcripts were increased in the ovalbumin model but not induced (or induced to a much lesser extent) in the tg–IL-13 or IL-13/Epi models (Fig 2, A). These changes are likely largely attributable to allergen-induced lymphocyte activation and to production of mediators other than IL-13. There were 247 genes increased similarly in the ovalbumin and tg–IL-13 models but not in the IL-13/Epi model (Fig 2, B). These allergen-induced genes are apparently increased because of IL-13 effects on cells other than nonciliated airway epithelial cells, and the large

number of gene transcripts is consistent with the idea that effects of IL-13 on these cells are important in allergic inflammatory responses.8,9 A total of 73 transcripts were increased in the tg–IL-13 model but not increased (or increased to a much lesser extent) in the other 2 models (Fig 2, C). These represent genes that are induced by prolonged high-level overexpression of IL-13 but not by acute allergen challenge and may be involved in the pathogenesis of subepithelial fibrosis and emphysema, pathologic features present in the tg–IL-13 model but not the other 2 models. We were especially interested in identifying genes that were increased in all 3 models. Analysis of whole-lung samples revealed 35 genes that had similar fold increases in all models (Fig 2, D). Because some epithelial gene expression changes might have been undetectable in whole lung samples, we also analyzed gene expression in tracheal perfusate samples enriched for airway epithelial cell RNA. In these samples, we detected 276 genes with altered expression (increased or decreased) in at least 1 model (see Table E4 in the Online Repository at www.mosby.com/jaci). Of these, 107 genes were changed by 2-fold. There were 43 genes changed in the ovalbumin model and 239 in the tg–IL-13 model. There were 76 genes changed in the IL-13/Epi model, but only 22 of these were changed by 2-fold or more. Some genes had similar expression changes in both analyses, but many other changes were identified only in lung samples or only in tracheal samples. In part, this reflects the fact that the tracheal sample was enriched for large airway epithelial cells, whereas the whole-lung sample contained smaller airway epithelial cells as well as many other cell types. In

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TABLE III. Gene expression in airway epithelial cells from control subjects and subjects with asthma

Clca1  Itln Alox15 Tff2 Muc5ac Tff1 Agr2 Slc26a4 Scin Muc5b AMCase

Description

Fold Difference*

Chloride channel calcium activated 1 Intelectin 15-lipoxygenase Trefoil factor 2 Mucin 5, subtypes A and C Trefoil factor 1 Anterior gradient 2 Solute carrier family 26, member 4 Scinderin Mucin 5, subtype B Acidic mammalian chitinase

216.5 3.4 1.3 2.7 1.5 1.4 1.1 0.8 1.0 25.0 —

Median gene copy number Control

Asthmatic 4

0.06 3 10 1.73 3 105 2.28 3 106 0.55 3 104 1.67 3 107 1.47 3 105 0.90 3 107 3.72 3 104 4.70 3 105 1.84 3 106 NDà

4

8.21 3 10 6.12 3 105 3.27 3 106 1.00 3 104 2.07 3 107 2.17 3 105 1.04 3 107 3.35 3 104 4.99 3 105 0.42 3 106 ND

Adjusted P

.0001 .0002 .06 .06 NS NS NS NS NS .0001 —

*Fold difference between subjects with asthma and control subjects, adjusted for age and sex.  Human Clca1 is orthologous to mouse Clca3. àND indicates that this transcript was not detected in most samples from both groups.

addition, we expect that there were differences in the sensitivity of the lung and tracheal sample analyses because we did not need to use RNA amplification for lung samples but did need to amplify tracheal samples. By combining microarray results from whole-lung and tracheal perfusate samples, we identified 18 genes that were increased at least 2-fold by allergen and by direct effects of IL-13 on airway epithelial cells (Table I). We also included trefoil factor 2 because previous PCR analysis5 suggested that the microarrays underestimated changes in expression of this gene.

PCR validation of gene expression changes in mouse models In preparation for translational studies, we generated a validated list of genes with human orthologues that were increased in both ovalbumin and IL-13/Epi mice. Five transcripts listed in Table I did not have obvious human orthologues. Eleven of the remaining 14 transcripts were increased by at least 2-fold in both ovalbumin allergic and IL-13/Epi mice by PCR (Table I). Our mouse modeling approach therefore resulted in the selection of 11 genes that could be studied in people with asthma. Airway epithelial gene expression in human subjects with and without asthma We used real-time PCR to analyze gene expression in airway epithelial cells from 30 subjects with mild to moderate asthma and 28 controls (Table II). There were substantial and highly significant increases in the expression of the calcium activated chloride channel Clca1 (orthologue of murine Clca3) and intelectin in subjects with asthma (Table III and Fig 3, A). There were smaller increases in expression of 15-lipoxygenase and trefoil factor 2 in subjects with asthma. One transcript, mucin 5b, was less abundant in subjects with asthma, and other transcripts examined were not significantly different. We were unable to consistently detect acidic mammalian chitinase transcripts in either group by using 2 different sets of PCR primers and probes.

FIG 3. Intelectin (Itln) expression in human airway epithelial cells. A, Intelectin gene expression by airway epithelial cells from control subjects and subjects with asthma. Medians and interquartile ranges are shown. B, IL-13 treatment of cultured human airway epithelial cells induced increased expression of intelectin transcripts.

IL-13 induces intelectin expression in cultured human airway epithelial cells One of the transcripts substantially increased in airway epithelial cells from subjects with asthma was intelectin, a pattern recognition molecule that has not been previously implicated in asthma. IL-13 treatment of cultured primary human bronchial epithelial cells led to a large increase in intelectin transcripts (Fig 3, B), demonstrating that IL-13 directly increases intelectin expression and that other cell types are not required. DISCUSSION We combined focused transgenic modeling, functional genomics, and translational studies in human subjects to help understand important aspects of the complex pathogenesis of asthma. As expected, microarray analysis of an allergic model of asthma revealed hundreds of gene expression changes. Analysis of the tg–IL-13 model, where IL-13 acts on many different cell types to produce extensive lung pathology, also showed hundreds of changes, many of which were similar to those seen in the allergic model. By using the data from these 2 models,

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Symbol

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it would have been extremely challenging to determine how particular gene expression changes relate to specific disease mechanisms or to select a reasonable number of candidate genes for further study. By analyzing a third model (IL-13/Epi) that focused on the effects of a single mediator, IL-13, on a single cell type, the airway epithelial cell, we were able to generate a dramatically shorter list of differentially expressed genes. These expression changes were associated with airway hyperreactivity and mucus overproduction, which are features of all 3 models, but not with inflammation, fibrosis, or emphysema, which are absent in the IL-13/Epi model. Our approach was designed to reduce the complexity inherent in microarray studies of disease pathogenesis. Other microarray studies have reduced complexity by analyzing homogeneous populations of cultured cells or cells isolated from tissues by laser capture microscopy or flow-cytometric sorting. Although those approaches can be useful, an important advantage of the focused transgenic model approach that we describe here is that it isolates the effects that a single mediator exerts on a single cell type in vivo. We used translational studies to determine whether the gene expression changes first identified by using mouse models were also seen in human disease. Four of the orthologues identified in the models (the chloride channel Clca1, intelectin, 15-lipoxygenase, and trefoil factor 2) were increased in airway epithelial cells from people with asthma. In another study involving a subset of the human subjects used for this study, we found that expression of Clca1 transcripts and protein was increased in airway epithelial cells from subjects with asthma (Woodruff et al, unpublished data). Previous reports suggest that induction of the mClca3/hClca1 chloride channel in airway epithelial cells is important for mucus production.25-27 15Lipoxygenase produces 15S-hydroxyeicosatetraenoic acid, which has been reported to trigger mucus secretion in dogs,28 promote contraction of human airway smooth muscle,29 and potentiate increases in allergen-induced early asthmatic responses in human beings.30 Trefoil factor 2 was shown to be increased in an allergic mouse model of asthma,31 and we now show that trefoil factor 2 is increased in airway epithelial cells from people with asthma. Trefoil factor 2 promotes migration of cultured bronchial epithelial cells32 and contributes to repair of injured intestinal epithelium.33 Intelectin is a recently described pattern recognition molecule not previously implicated in asthma. There are 2 related intelectins, Itln1 and Itln2. The probes and primers that we used were designed to recognize Itln1, but they might not distinguish between these 2 very closely related sequences. The molecular patterns recognized by intelectin include furanosides such as galactofuranose.34 Galactofuranosyl residues are present in bacterial and fungal cell walls and in protozoan parasites but not in mammalian cells. The possible contributions of intelectin to asthma pathogenesis require further exploration, but intelectin in the airway might alter the response of subjects with asthma to infection or colonization with bacterial or fungal pathogens.

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We did not find significant increases in expression of the other human homologues that we examined. To some extent, this likely reflects difficulties inherent in detecting gene expression changes in limited samples (epithelial brushings) taken from human subjects with stable mild to moderate disease, as opposed to whole-lung analysis of mice with homogeneous genetic backgrounds housed in a controlled environment and subjected to a potent disease-inducing stimulus. One transcript, Muc5b, was significantly increased in each of the mouse models, but expression of both the transcript and the protein is decreased in people with asthma (Woodruff et al, unpublished data). Differences in Muc5b expression might relate to the fact that human Muc5b is expressed predominantly in submucosal glands.35 These glands are abundant in human airways but are absent in the mouse, with the exception of a single gland in the trachea. This is an example of the limits of mouse modeling. Our PCR assay was unable consistently to detect the transcript for another orthologue, AMCase, but a recent report demonstrated that airway epithelial production of AMCase is also increased in subjects with asthma.36 Including translational studies helped to clarify the relevance of the animal models to human disease and allowed us to draw novel inferences about the activity of a specific mechanism in human disease. We conclude that the combination of focused transgenic models, DNA microarray analyses, and translational studies provides a powerful approach for analyzing the contributions of specific mediators and cell types and for focusing attention on a limited number of genes associated with specific pathophysiologic aspects of complex diseases like asthma. We thank Dean Sheppard and Andrea Barczak for their advice and Xiaozhu Huang, Louis Nguyenvu, Michael Salazar, and the staffs of the Sandler Center Animal Physiology and Microscopy Core and the UCSF National Heart, Lung, and Blood Institute Shared Microarray Facility for technical assistance.

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7. Humbert M, Durham SR, Kimmitt P, Powell N, Assoufi B, Pfister R, et al. Elevated expression of messenger ribonucleic acid encoding IL-13 in the bronchial mucosa of atopic and nonatopic subjects with asthma. J Allergy Clin Immunol 1997;99:657-65. 8. Grunig G, Warnock M, Wakil AE, Venkayya R, Brombacher F, Rennick DM, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998;282:2261-3. 9. Wills-Karp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL, et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282:2258-61. 10. Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, et al. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999;103:779-88. 11. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. The IL-4 receptor: signaling mechanisms and biologic functions. Annu Rev Immunol 1999;17:701-38. 12. Stripp BR, Sawaya PL, Luse DS, Wikenheiser KA, Wert SE, Huffman JA, et al. cis-acting elements that confer lung epithelial cell expression of the CC10 gene. J Biol Chem 1992;267:14703-12. 13. Barczak A, Rodriguez MW, Hanspers K, Koth LL, Tai YC, Bolstad BM, et al. Spotted long oligonucleotide arrays for human gene expression analysis. Genome Res 2003;13:1775-85. 14. Diaz E, Yang YH, Ferreira T, Loh KC, Okazaki Y, Hayashizaki Y, et al. Analysis of gene expression in the developing mouse retina. Proc Natl Acad Sci U S A 2003;100:5491-6. 15. Jin W, Riley RM, Wolfinger RD, White KP, Passador-Gurgel G, Gibson G. The contributions of sex, genotype and age to transcriptional variance in Drosophila melanogaster. Nat Genet 2001;29:389-95. 16. Lonnstedt I, Speed TP. Replicated microarray data. Statistica Sinica 2002;12:31-46. 17. Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 2004;3:3. Available at: http://www.bepress.com/sagmb/vol3/ iss1/art3/. 18. Claman DM, Boushey HA, Liu J, Wong H, Fahy JV. Analysis of induced sputum to examine the effects of prednisone on airway inflammation in asthmatic subjects. J Allergy Clin Immunol 1994;94: 861-9. 19. Chai H, Farr RS, Froehlich LA, Mathison DA, McLean JA, Rosenthal RR, et al. Standardization of bronchial inhalation challenge procedures. J Allergy Clin Immunol 1975;56:323-7. 20. Atherton HC, Jones G, Danahay H. IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation. Am J Physiol Lung Cell Mol Physiol 2003;285:L730-9. 21. Dolganov GM, Woodruff PG, Novikov AA, Zhang Y, Ferrando RE, Szubin R, et al. A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na1-K1-Clcotransporter (NKCC1) in asthmatic subjects. Genome Res 2001;11: 1473-83.

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22. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3:research0034.1-0034.11. 23. Westfall PH, Young SS. Resampling-based multiple testing: examples and methods for p-value adjustment. New York: John Wiley & Sons; 1993. 24. Ge Y, Dudoit S, Speed TP. Resampling-based multiple testing for microarray data hypothesis. Test 2003;12:1-44. 25. Hoshino M, Morita S, Iwashita H, Sagiya Y, Nagi T, Nakanishi A, et al. Increased expression of the human Ca21-activated Cl- channel 1 (CaCC1) gene in the asthmatic airway. Am J Respir Crit Care Med 2002;165:1132-6. 26. Nakanishi A, Morita S, Iwashita H, Sagiya Y, Ashida Y, Shirafuji H, et al. Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proc Natl Acad Sci U S A 2001;98:5175-80. 27. Zhou Y, Shapiro M, Dong Q, Louahed J, Weiss C, Wan S, et al. A calcium-activated chloride channel blocker inhibits goblet cell metaplasia and mucus overproduction. Novartis Found Symp 2002;248:150-65; discussion 65-70, 277-82. 28. Johnson HG, McNee ML, Sun FF. 15-Hydroxyeicosatetraenoic acid is a potent inflammatory mediator and agonist of canine tracheal mucus secretion. Am Rev Respir Dis 1985;131:917-22. 29. Copas JL, Borgeat P, Gardiner PJ. The actions of 5-, 12-, and 15-HETE on tracheobronchial smooth muscle. Prostaglandins Leukot Med 1982;8: 105-14. 30. Lai CK, Polosa R, Holgate ST. Effect of 15-(s)-hydroxyeicosatetraenoic acid on allergen-induced asthmatic responses. Am Rev Respir Dis 1990; 141:1423-7. 31. Nikolaidis NM, Zimmermann N, King NE, Mishra A, Pope SM, Finkelman FD, et al. Trefoil factor-2 is an allergen-induced gene regulated by Th2 cytokines and STAT6 in the lung. Am J Respir Cell Mol Biol 2003;29:458-64. 32. Oertel M, Graness A, Thim L, Buhling F, Kalbacher H, Hoffmann W. Trefoil factor family-peptides promote migration of human bronchial epithelial cells: synergistic effect with epidermal growth factor. Am J Respir Cell Mol Biol 2001;25:418-24. 33. Farrell JJ, Taupin D, Koh TJ, Chen D, Zhao CM, Podolsky DK, et al. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. J Clin Invest 2002;109:193-204. 34. Tsuji S, Uehori J, Matsumoto M, Suzuki Y, Matsuhisa A, Toyoshima K, et al. Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall. J Biol Chem 2001;276:23456-63. 35. Sharma P, Dudus L, Nielsen PA, Clausen H, Yankaskas JR, Hollingsworth MA, et al. MUC5B and MUC7 are differentially expressed in mucous and serous cells of submucosal glands in human bronchial airways. Am J Respir Cell Mol Biol 1998;19:30-7. 36. Zhu Z, Zheng T, Homer RJ, Kim YK, Chen NY, Cohn L, et al. Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation. Science 2004;304:1678-82.

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Endobronchial adenosine monophosphate challenge causes tachykinin release in the human airway Fionnuala Crummy, MD, MRCP,a,c Mark Livingston, PhD,a,b Joy E. S. Ardill, PhD, FCRPath,c Catherine Adamson, MSc,a,b Madeleine Ennis, PhD,a,b and Liam G. Heaney, MD, MRCPa,c Belfast, Northern Ireland, United Kingdom Mechanisms of asthma and allergic inflammation

Background: Adenosine 5 monophosphate (AMP) has been shown to cause bronchoconstriction and a sensation of chest tightness when inhaled by asthmatic subjects. This response is attenuated after repeated inhalation of bradykinin, suggesting that AMP may act in part by the release of neuropeptides. Objective: This study examined neuropeptide release in the human airway after endobronchial AMP challenge. Methods: Endobronchial AMP challenge was performed in 20 subjects and tachykinin levels were measured after endobronchial AMP challenge and after placebo endobronchial challenge with saline. Results: All subjects coughed immediately after adenosine challenge. There was a significant increase in neurokinin A and substance P levels (P < .01, P< .01 respectively) when post-saline and post-AMP levels were compared. There was, however, no significant change in calcitonin gene related peptide levels (P=.37). Conclusion: This study demonstrates that endobronchial AMP challenge causes tachykinin release in the human airway in vivo. (J Allergy Clin Immunol 2005;116:312-7.) Key words: Tachykinin, neuropeptide, adenosine, endobronchial challenge

Adenosine is a naturally occurring purine nucleoside that functions as a constituent of nucleic acid, as an intracellular and autocoid mediator.1 Elevated levels have been found in bronchoalveolar lavage fluid of asthmatic subjects as compared to normal subjects, suggesting that adenosine may be a mediator in asthma.2 Inhalation of adenosine monophosphate (AMP), which is rapidly dephosphorylated to adenosine in vivo,3 causes bronchoconstriction in atopic asthmatic and non-asthmatic subjects but not in non-atopic, non-asthmatic subjects.4 The related nucleoside guanosine has no effect, suggesting that this is a specific receptor mediated effect.5

From aRespiratory Research Group, School of Medicine, Queen’s University of Belfast and Departments of bClinical Biochemistry and Metabolic Medicine and cMedicine, Queens University, Belfast. Funding: Northern Ireland Chest Heart and Stroke Association. Received for publication November 18, 2004; revised March 10, 2005; accepted for publication March 28, 2005. Available online June 17, 2005. Reprint requests: Liam G. Heaney, MD, MRCP, Regional Respiratory Centre, Level 8, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, UK. BT9 7AB. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.03.034

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Abbreviations used AMP: Adenosine 5# monophosphate CGRP: Calcitonin gene related peptide NEP: Neutral endopeptidase NKA: Neurokinin A NKB: Neurokinin B NPK: Neuropeptide K PC20 AMP: Provocative concentration of AMP causing a 20% fall in FEV1 SP: Substance P

Atopic asthmatic and non-asthmatic subjects cough and bronchoconstrict in response to stimuli such as inhaled AMP and sulphur dioxide; however, asthmatics tend to respond to lower concentrations.6 Cough and chest tightness are both common symptoms in asthma and are related to stimulation of sensory nerves.6 Evidence exists that while AMP acts mainly via primed mast cells, the agent also stimulates vagal nerves. Pretreatment with ipratropium (an anti-cholinergic agent) has a bronchoprotective effect on the response to AMP, suggesting activation of cholinergic nerves in the response to AMP.7,8 Inhalation of AMP and bradykinin cause a greater sensation of chest tightness than does inhalation of methacholine, for the same degree of bronchoconstriction, suggesting that the former acts on sensory pathways.9 Repeated inhalation of bradykinin attenuates the response to inhaled AMP suggesting that both of these agents act in part via liberation of neuropeptides from sensory nerves.10 Hong et al11 have shown that pulmonary C fibers (the nerve fibers containing neuropeptides) in the rat are activated after right atrial injection of adenosine, implicating these nerves in the response to AMP in this model. The purpose of this study was to examine neuropeptide release in vivo in the human airway after endobronchial AMP challenge.

METHODS Subjects Ethical approval was granted by the Research Ethics Committee of the Queen’s University of Belfast. All subjects gave written informed consent. All subjects were non-smokers and had not received any anti-histamines or inhaled or oral steroids in the preceding six months. Asthmatics were recruited if they (1) had a prior clinical

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Skin prick testing Skin prick testing was performed using a standardized puncture technique,12 using allergen preparations of house dust mite, cat, and dog protein and grass pollen (Dome-Hollister-Stier, Epernon Cedex, France). A positive reaction was taken as a wheal size of 3 mm or more.

Inhalational challenging Spirometry was performed according to American Thoracic Society Guidelines13 using a Vitalograph spirometer (Buckingham, UK). AMP (Sigma-Aldrich Ltd., Poole, UK) was freshly prepared in 0.9% saline in doubling concentrations ranging from 0.391 mg/mL to 400 mg/mL. The AMP provocation test was performed using the twominute tidal breathing method of Cockcroft et al14 using a Medix Turbonebuliser (Leicestershire, UK) with an output of 0.13 mL/min. PC20 AMP was calculated by linear interpolation.

Bronchoscopy AMP was freshly made up on the morning of the bronchoscopy in 0.9% saline from a stock solution of 400 mg/mL. At bronchoscopy, subjects were given intravenous Midazolam (up to 14 mg) to achieve mild sedation and the hypopharynx was anaesthetised using 4% lignocaine spray. Vocal cord and tracheal anaesthesia was achieved using 4 mL of 4% lignocaine introduced trans-cricoidally. Oxygen was routinely applied at 2 L/min via nasal cannulae. Heart rate, ECG, and oxygen saturations were monitored throughout the procedure. The bronchoscope (240 IT Olympus Optical Co. Ltd. Tokyo, Japan) was introduced orally and 2-mL aliquots of 2% lignocaine were used as necessary to anesthetize the airways below the carina to suppress coughing. The site of the subsequent endobronchial challenge was randomized prior to bronchoscopy. Subjects were randomly assigned a number, which determined the site of the active challenge to either the right middle or upper lobes, and randomization was constrained to achieve balance. The placebo challenge was automatically assigned to the opposite site from the active challenge. The bronchoscope was initially wedged in a segmental orifice of the site randomized for the placebo challenge and a baseline bronchial wash with 20 mL of saline was performed and aspirated back after minimum dwell time. A placebo challenge of 5 mL of saline was administered to the same segment and the segment closely observed for any visible reaction. After 3 minutes, a second bronchial wash using 20 mL of saline was performed and aspirated back after minimal dwell time. The active (AMP) challenge was then performed in the other site. Again a baseline bronchial wash was performed using 20 mL saline and immediately aspirated back under gentle suction. Then the active challenge with 5 mL AMP was performed. The initial AMP concentration administered was one tenth that which caused a 20% fall in FEV1 on the prior inhalational challenge or if the subject had been unresponsive to adenosine one tenth of the maximum concentration during the inhalation challenge (400 mg/mL). Up to two subsequent AMP doses were given at quadrupling concentrations, the maximum

TABLE I. Visual analogue reaction for grading of response after endobronchial challenge Analogue score

0 1 2

3

Reaction observed

No reaction Subject coughs after instillation of AMP (no coughing after saline challenge) Subject coughs/immediate bronchial pallor then hyperemia/increased mucus secretion after instillation of AMP Bronchoconstriction observed after instillation of AMP

Adapted from Polosa et al.1

administered endobronchial dose being 400 mg/mL. There was a time lapse of 3 minutes after each concentration given to observe for any visual reaction using the analogue outlined in Table I. The endobronchial challenge was terminated either when there was a visible reaction to AMP, when the maximum concentration of AMP had been administered or when it was necessary to terminate the challenge for reasons of patient comfort. Three minutes after the final concentration of adenosine had been administered, a further bronchial wash of 20 mL of saline was performed and aspirated after a minimum dwell time. Subjects remained under observation for a period of at least two hours after the procedure.

Processing of samples A total cell count was measured using a modified Neubauer hemocytometer and was expressed as the number of cells 3105/mL of BAL. Cell viability was assessed by Trypan blue exclusion staining. Viable cells are expressed as a percentage of total cell numbers. Samples were centrifuged at 200 3 g for 10 minutes at 4°C to separate any debris and added to a protease inhibitor cocktail (see Appendix) and stored at 270°C for subsequent analysis.

Neuropeptide measurement NKA was measured using radioimmunoassay, utilizing a N-terminal specific anti-serum that was raised in guinea pigs to synthetic human NKA (Amersham Bioscience UK Ltd product number IM168, Buckinghamshire, UK). It cross-reacts fully with NKB and NPK but less than 0.1% with SP. The detection limit for the assay is 2 ng/L. CGRP immunoreactivity was measured using a commercial CGRP human radioimmunoassay (RIA) kit (catalogue number RIK009, Peninsula Laboratories, San Carlos, Calif). This antibody is a rabbit anti-human CGRP peptide (II) antibody. The label was 125 I-Tyr0-CGRP (catalog number Y6011). The limit of detection for this assay is 2 ng/L and the antibody cross-reacts 100% with human CGRP (II), human CGRP, and rat CGRP. It cross-reacts <0.001% with rat calcitonin C-terminal adjacent peptide and less than 0.02% with insulin, glucagon, somatostatin, SP, vasoactive intestinal peptide, and gastrin releasing peptide. Substance P (SP) was measured using a commercially available ELISA (catolog number DE1400, R&D Systems, Minneapolis, Minn). It shows no significant cross-reactivity with NKA, neurokinin B (NKB), and neuropeptide K (NPK). The limit of detection of this assay is 8 pg/mL. For radioimmunoassays, lavage fluid was extracted using a previously validated technique.16 In brief, cleared plasma and traysolol were added to equal volume of lavage fluid followed by precipitation of large molecular weight proteins in 60% alcohol and the sample centrifuged (30 min, 3 1500 g). Thiomersal was added to the supernatant. This was then decanted, the extract was evaporated to

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diagnosis of asthma and a history of intermittent shortness of breath or wheeze, (2) were atopic (reacted to at least one allergen on skin prick testing), and (3) had FEV1 greater than 60% predicted. All other subjects had no symptoms suggestive of asthma; atopic non-asthmatic subjects had at least one positive skin prick test as aforementioned. All subjects attended on two occasions. At the screening visit, informed consent was obtained and clinical assessment, skin prick testing, and AMP inhalation challenge were performed. At the subsequent visit (at least 72 hours after screening visit), bronchoscopy and endobronchial AMP challenge were performed.

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TABLE II. Demographic details of subjects

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Normals (N)

Atopics (AT)

Asthmatics (AS)

Number Age (y), Median (IQR) Sex (n = male) FEV1% predicted, Mean (SD) FVC% predicted, Mean (SD) PC20 AMP (mg/mL), Median (IQR) Maximum dose of endobronchial AMP (mg/mL), Median (range) Number of aliquots of endobronchial AMP, Median (range)

7 21 (21-24) 4 114.0 (8.4) 97.7 (11.0) 640.0 160.0 (40.0-400.0) 2 (1.0-3.0)

6 21 (21-24) 3 99.8 (14.2) 99.2 (15.4) 413.9 (144.8-640.0) 40.0 (10.0-160.0) 11 (1.0-2.0)

7 26 (21-30) 2 90.0 (14.8) 95.6 (16.4) 3.6 (2.3-7.9) 1.3 (0.15-10.0) 11 (1.0-3.0)

dryness and the sample assayed. For SP ELISA, samples were Seppakked (C18 Sep-pak; Waters, Milford, Mass) and eluted using a 60:40 solution of acetontirile and 1% trifluoro-acetic acid, dried down and reconstituted in buffer prior to assay. Using these extraction and assay methods, peptide recovery was >90%. We have previously characterised NKA, SP, and CGRP immunoreactivity in bronchoalveolar lavage fluid using HPLC, confirming it to be target peptide.17

12.5 mg/mL, 326 mOsm; 40 mg/mL, 403 mOsm; 100 mg/mL, 567 mOsm; 400 mg/mL, 1292 mOsm. Endobronchial responses to AMP challenge are shown in Figure 1. Instillation of endobronchial AMP led to immediate coughing in all subjects after administration of adenosine, which was not seen after instillation of the saline placebo challenge. However, there was no evidence of generalised lung involvement (eg, wheeze, hypoxia) in response to the endobronchial AMP challenge in any subject. As immediate coughing was observed in all groups, for the purposes of analysis all subjects were considered together. There was a significant increase in median (IQR) NKA and substance P levels [10.0 (5.0–15.0) vs. 20.0 (10.6–25.0) pg/mL, P < .01 and 227. 8 (176.9–278.6) vs. 318.1 (190.6–422.9) pg/mL, P < .01] respectively when post-saline and post-AMP levels were compared (Fig 2). There was no significant change in CGRP levels (P=.37, Fig 2). There was no significant difference between groups in NKA, CGRP, or substance P levels at baseline or post-challenge. There was no correlation between the change (postAMP–post-saline) in NKA and substance P after AMP challenging (r = 0.27, P=.25) or between the change in CGRP and substance P (r = 0.25, P=.34). There was no significant correlation between the change in NKA or substance P and the PC20 AMP for the group as a whole (r = 0.09, P=.71, r = 0.26, P=.31 respectively). There was no difference between the changes in NKA observed between those who bronchoconstricted after endobronchial challenge (visual reaction 3) and those who did not (P=.30).

Measurement of osmolality of AMP solutions AMP was freshly made up in 0.9% normal saline in concentrations ranging from 0.39 mg/mL to 400 mg/mL and osmolality measured (Advanced Micro-osmometer 3300; Advanced Instruments Inc, Pomona, Calif).

Statistical analysis All statistical analysis was performed using SPSS version 11.0 (Statistical Package for Social Sciences, Chicago, Ill). All data were tested for normality of distribution using Shapiro-Wilk W tests. Where the data were skewed values are quoted as median and interquartile range (IQR), unless otherwise stated. For non-parametric data, comparisons were made between paired samples using the Wilcoxon analysis and for unpaired samples using the MannWhitney U test. Comparison between more than two groups were performed using Kruskall-Wallis analysis. Post hoc multiple comparisons were then performed to demonstrate the underlying statistical differences (Stats Direct, Cambridge, UK). Non–parametric correlations were calculated using Spearman’s rank correlations. Subjects who did not achieve at least a 20% drop in FEV1 after inhaling 400 mg/mL of AMP were given an assigned PC20 value of 640 mg/mL for statistical analysis. P values <.05 were regarded as statistically significant.

RESULTS A total of 24 subjects were recruited. Three subjects (1 normal, 1 atopic, and 1 asthmatic) did not complete the endobronchial challenge. Twenty-one subjects completed the endobronchial challenge protocol (1 sample was inadequate for processing). The demographic details for the remaining 20 subjects (7 normals, 6 atopic non-asthmatics, and 7 atopic asthmatics) are shown in Table II. There was no difference between the groups in terms of age or baseline spirometry. Measurements of osmolality of saline and AMP solutions showed the following: 0.9% saline, 281 mOsm; 0.39 mg/mL, 282 mOsm; 3.125 mg/mL, 297 mOsm;

DISCUSSION This study provides the first evidence of in vivo tachykinin release after a chemical airway challenge in humans. During the study it was observed that many of the subjects who were sedated and who did not cough during the placebo saline challenge or baseline lavage at the active challenge site, started to cough immediately after the instillation of endobronchial AMP. In three cases this coughing induced by AMP was believed to be distressing enough to necessitate termination of the procedure.

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FIG 1. The frequency of endobronchial response to AMP challenge in normals, atopics, and asthmatics.

The immediacy of the cough after AMP challenge suggests that the cough is mediated by afferent sensory nerves. It has previously been shown that AMP acts on vagal nerves.7,8,9 The co-existent increase in NKA and substance P suggest the mechanism may involve stimulation of sensory C fibres with antidromic release of tachykinins. Stimulation of sensory C fibers and release of neuropeptides have previously been implicated in cough. It is known that inhalation of capsaicin acting through the VR-1 receptor causes cough.18,19 Capsaicin also liberates neuropeptides from sensory nerves and after repeated inhalation the cough diminishes, possibly due to depletion of neuropeptides and suggesting propagation of cough by neuropeptides in this model. There is evidence, in animal models, that AMP can act directly on pulmonary C fibers11; however, the exact receptor involved seems to vary between species. In rats it was found that adenosine potentiated the response of pulmonary C fibers to chemical stimuli and that this response was attenuated by pretreatment with an A1 receptor antagonist20; while in guinea pig lung A2 agonists decreased the release of SP from pulmonary C fibers.21 Thus, it is possible in this study that AMP was acting directly on nerve endings to cause cough via a central mechanism rather than directly by neuropeptide release, although this potential mechanism has not been well studied in the human airway. The coughing after endobronchial AMP challenge and subsequent release of tachykinins was observed across all groups of subjects. While it has been suggested that the nerves of asthmatics may be more sensitive to stimuli than

those of non-asthmatics, the asthmatics in this study were mild both in terms of airway hyperresponsiveness and treatment requirements and may not reflect differences that may occur with more significant asthma. There was no correlation between the changes in NKA, CGRP, or substance P. This is perhaps not surprising in the case of NKA, as this neuropeptide may be located independently of the other two peptides.22 However, it is surprising that there was no correlation between the changes seen in substance P and CGRP because they are often co-localized in the same neurones. There are a number of possible explanations for this discrepancy. While neuropeptides are co-localized the exact amounts present may vary depending on the location of the tissue.23 There may also be differential paths of degradation. Substance P and NKA are primarily degraded in the airways by the enzyme neutral endopeptidase (NEP). However, the exact degradation pathway of CGRP has not yet been elucidated. It has been shown that CGRP is subject to degradation by tryptase while the tachykinins NKA and substance P are not.24 Most of the subjects in this study had tryptase release after endobronchial AMP challenging,15 which may explain why there was no difference in CGRP levels after AMP challenge and also why there is no relationship between CGRP and SP levels. In addition, it may be that differential release accounts for the patterns of neuropeptide release observed. The relative amount of neuropeptide released may be dependent on the frequency with which the nerve is stimulated as well as the quantity of each peptide in the nerve ending.25 However, the precise mechanisms remain to be established.

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that these non-neuronal sources are responsible for the neuropeptide release induced by AMP in this study. Thus, the neuropeptide release and the cough may be two concomitant but separate events in the airway after AMP challenge. Nasal challenge with hypertonic saline induces neuropeptide release31 and mannitol challenge in asthmatics causes cough that is independent of bronchoconstriction.32 Osmolality of the adenosine concentrations demonstrated that the challenges used in normal subjects were relatively hyperosmolar compared to normal saline/ plasma; however, this was not the case for the concentrations used in asthmatic subjects (Table II). Thus, while we cannot completely exclude an osmotic effect in normal subjects, given this mechanism is not applicable in asthmatic subjects, we believe it is unlikely that two entirely separate mechanisms are producing cough and causing tachykinin release. There was no relationship between PC20 AMP and the change in NKA, SP, or CGRP levels. Bronchoconstriction after inhalation of AMP is mediated by multiple mechanisms including the release of mast cell mediators, which were present in many of these subjects.15 Thus a simple correlation between changes in airway physiology and individual endobronchial tachykinin or other mediator release would seem unlikely. This study has provided evidence that endobronchial AMP challenge causes immediate cough and significant NKA and substance P release in non-atopic non-asthmatic, atopic non-asthmatic, and atopic asthmatic subjects, which occurs in the absence of significant CGRP release. This is the first demonstration of in vivo tachykinin release, after chemical stimulation, in the human airway. It therefore seems likely that AMP can act through a number of mechanisms, in addition to mast cell mediator release, to generate responses in the human airway. REFERENCES

FIG 2. The change in post-saline and post-AMP levels of (A) NKA, (B) Substance P, and (C) CGRP.

Another possibility is that while AMP mediates the cough through afferent sensory nerves, the tachykinin release is from a non-neuronal source. Sensory nerves containing neuropeptides account for around 1% of all the nerve fibers found in human airways.22 However, much higher levels of neuropeptides have been found in induced sputum 26 and in BAL17 than would be expected from this source alone. It has been shown that tachykinins may be produced by eosinophils, macrophages, lymphocytes, neutrophils, and epithelial cells,27,28,29,30 and it may be

1. Polosa R, Ng WH, Crimi N, Vancheri C, Holgate ST, Church MK, et al. Release of mast-cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med 1995;151:624-9. 2. Driver AG, Kukoly CA, Ali S, Mustafa SJ. Adenosine in bronchoalveolar lavage fluid in asthma. Am Rev Respir Dis 1993;148:91-7. 3. Mann JS, Holgate ST, Renwick AG, Cushley MJ. Airway effects of purine nucleosides and nucleotides and release with bronchial provocation in asthma. J Appl Physiol 1986;61:1667-76. 4. Cushley MJ, Tattersfield AE, Holgate ST. Inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. Br J Clin Pharmacol 1983;15:161-5. 5. Holgate ST, Cushley MJ, Mann JS, Hughes P, Church MK. The action of purines on human airways. Arch Int Pharmacodyn Ther 1986;280(2 Suppl):240-52. 6. Spina D. Airway sensory nerves: a burning issue in asthma? Thorax 1996;51:335-7. 7. Crimi N, Palermo F, Oliveri R, Polosa R, Settinieri I, Mistretta A. Protective effects of inhaled ipratropium bromide on bronchoconstriction induced by adenosine and methacholine in asthma. Eur Respir J 1992;5: 560-5. 8. Polosa R, Phillips GD, Rajakulasingam K, Holgate ST. The effect of inhaled ipratropium bromide alone and in combination with oral terfenadine on bronchoconstriction provoked by adenosine 5#- monophosphate and histamine in asthma. J Allergy Clin Immunol 1991;87:939-47.

9. Marks GB, Yates DH, Sist M, Ceyhan B, De Campos M, Scott DM, et al. Respiratory sensation during bronchial challenge testing with methacholine, sodium metabisulphite, and adenosine monophosphate. Thorax 1996;51:793-8. 10. Polosa R, Rajakulasingam K, Church MK, Holgate ST. Repeated inhalation of bradykinin attenuates adenosine 5#-monophosphate (AMP) induced bronchoconstriction in asthmatic airways. Eur Respir J 1992;5: 700-6. 11. Hong JL, Ho CY, Kwong K, Lee LY. Activation of pulmonary C fibres by adenosine in anaesthetized rats: role of adenosine A1 receptors. J Physiol 1998;508:109-18. 12. Skin tests used in type I allergy testing Position paper. Sub-Committee on Skin Tests of the European Academy of Allergology and Clinical Immunology. Allergy 1989;44(Suppl 10):1-59. 13. Standardization of spirometry–1987 update. Statement of the American Thoracic Society. Am Rev Respir Dis 1987;136:1285-98. 14. Cockcroft DW, Killian DN, Mellon JJ, Hargreave FE. Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy 1977;7:235-43. 15. Crummy F, Livingston M, Ennis M, Heaney LG. Mast cell mediator release in non-asthmatic subjects after endobronchial adenosine challenge. J Allergy Clin Immunol 2004;114:34-9. 16. Heding LG. Radioimmunological determination of pancreatic and gut glucagon in plasma. Diabetologia 1971;7:10-9. 17. Heaney LG, Cross LJ, McGarvey LP, Buchanan KD, Ennis M, Shaw C. Neurokinin A is the predominant tachykinin in human bronchoalveolar lavage fluid in normal and asthmatic subjects. Thorax 1998;53:357-62. 18. Bevan S, Szolcsanyi J. Sensory neuron -specific actions of capsiacin: mechanisms and applications. Trends Pharmacol Sci 1990;11:330-3. 19. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat activated ion channel in the pain pathway. Nature 1997;389:816-24. 20. Gu Q, Ruan T, Hong JL, Burki N, Lee LY. Hypersensitivity of pulmonary C fibres induced by adenosine in anaesthetized rats. J Appl Physiol 2003;95:1315-24. 21. Morimoto H, Yamashita M, Imazumi K, Matsuda A, Ochi T, Seki N, et al. Effects of adensoine A2 receptor agonists on the excitation of capsaicinsensitive afferent sensory nerves in airway tissues. Eur J Pharmacol 1993; 240:121-6. 22. Joos GF, Germonpre PR, Pauwels RA. Role of tachykinins in asthma. Allergy 2000;55:321-37. 23. Bakhle YS, Bell C. Neurokinin A and substance P vary independently in different regions of rat sensory neurons. Neuropeptides 1995;28:237-41.

24. Sommerhoff CP. Mast cell tryptases and airway remodeling. Am J Respir Crit Care Med 2001;164:S52-8. 25. Widdicombe JG. Autonomic regulation. i-NANC/e-NANC. Am J Respir Crit Care Med 1998;158:S171-5. 26. Tomaki M, Ichinose M, Miura M, Hirayama Y, Yamauchi H, Nakajima N, et al. Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. Am J Respir Crit Care Med 1995;151:613-7. 27. Maggi CA. The effects of tachykinins on inflammatory and immune cells. Regul Pept 1997;70:75-90. 28. Joos GF, Germonpre PR, Pauwels RA. Neural mechanisms in asthma. Clin Exp Allergy 2000;30(Suppl 1):60-5. 29. Joos GF, De Swert KO, Pauwels RA. Airway inflammation and tachykinins: prospects for the development of tachykinin receptor antagonists. Eur J Pharmacol 2001;429:239-50. 30. Reynolds PN, Scicchitano R, Holmes MD. Pre-protachykinin-A mRNA is increased in the airway epithelium of smokers with chronic bronchitis. Respirology 2001;6:187-97. 31. Baraniuk JN, Mushtaq A, Atsushi Y, Sheen-Yie F, Naranch K. Hypertonic saline nasal provocation stimulates nocioceptive nerves, substance P release and glandular mucus exocytosis in normal humans. Am J Respir Crit Care Med 1999;160:655-62. 32. Koskela HO, Hyvarinen L, Brannan JD, Chan HK, Anderson SD. Coughing during mannitol challenge is associated with asthma. Chest 2004;125:1985-92.

APPENDIX Protease inhibitor cocktail contained the following reagents at recommended concentrations: Soybean Trypsin Inhibitor - BDH Laboratory Supplies, Poole, UK Aprotinin - Sigma-Aldrich Ltd., Poole, UK Alpha-1-antitrypsin - Sigma-Aldrich Ltd., Poole, UK Pepstatin A - BDH Laboratory Supplies, Poole, UK Phenanthroline - Acros Organics, Geel, Belgium EDTA acid - Acros Organics, Geel, Belgium Benzamidine - Sigma-Aldrich Ltd., Poole, UK

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Rat tracheal epithelial responses to water avoidance stress Hiroshi Akiyama, PhD,a Hiroo Amano, MD, PhD,b and John Bienenstock, MDc Tokyo and Maebashi, Japan, and Hamilton, Ontario, Canada

Mechanisms of asthma and allergic inflammation

Background: Psychologic stress has major effects on many organs and cellular systems. The hypothalamic-pituitaryadrenal axis, corticotropin-releasing factor (CRF), mast cells, and nerves have all been shown to be involved in intestinal epithelial responses to stress. There has been little information in the literature on stress and the lung. Objective: To investigate Wistar rat tracheal epithelial responses to acute water avoidance stress (1 hour). Methods: Tracheal tissue was examined in Ussing chambers. Results: Increases in short-circuit current, but not in conductance, occurred after stress and were inhibited by previous injection of the CRF 1 and 2 receptor antagonist, a-helical CRF–(9-41). Electron microscopic morphologic evidence for tracheal mast cell activation and degranulation was found after stress. Stress and CRF injection both enhanced responses to substance P, but these effects were not inhibited by a-helical CRF. Conclusion: The data suggest that acute stress affects tracheal epithelium and sensitizes it to enhanced responses to substance P, partly through mast cell activation. Many but not all of these effects are mediated by CRF. These results offer the possibility that stress may be involved in inflammatory diseases of the lung such as asthma. (J Allergy Clin Immunol 2005;116:318-24.) Key words: Mast cell, hypothalamic-pituitary-adrenal axis, shortcircuit current, corticotropin-releasing factor, substance P

The role of stress in asthma is a controversial subject, which is reflected by the literature on this subject.1 A recent investigation of college students clearly showed that stress associated with examinations enhanced several outcome measures such as sputum eosinophils, eosinophil-derived neurotoxin levels, and IL-5. The authors concluded that stress could act as a cofactor with inhaled antigen to enhance inflammation and asthma severity.2 Asthma exacerbations in children increase significantly within 1 to 2 days of a major stressful event. This effect From aDivision of Foods, National Institute of Health Sciences, Tokyo; bthe Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi; and cthe Departments of Medicine and Pathology and Molecular Medicine, McMaster University and the Brain-Body Institute, St Joseph’s Healthcare, Hamilton. Dr Akiyama and Dr Amano contributed equally to this work. Supported by the Medical Research Council of Canada and St Joseph’s Hospital Foundation, Hamilton, Ontario, Canada. Received for publication November 22, 2004; revised March 21, 2005; accepted for publication March 28, 2005. Available online May 24, 2005. Reprint requests: Hiroshi Akiyama, PhD, Division of Foods, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501 Japan. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.03.040

318

Abbreviations used a-Helical CRF: a-Helical CRF–(9-41) CRF: Corticotropin-releasing factor Isc: Short-circuit current PD: Potential difference SP: Substance P WAS: Water avoidance stress

was statistically evident for as long as 5 to 7 weeks.3 Indeed, it has become increasingly evident in recent years that the effects of acute and chronic stress on physiologic function can be important in the initiation, development, and/or perpetuation of many human diseases.4 The intestine has long been thought of as a target for stress. The role of stress as a determinant in intestinal disease is well established both experimentally and clinically5-7 and may well be relevant to the study of asthma.8 Irritable bowel syndrome, as in asthma, is characterized by smooth muscle hyperreactivity.9 The major prospective determinant for irritable bowel syndrome after acute infectious diarrhea is accompanying poor psychosocial circumstances.10 Activation of mast cells colocalized with enteric nerves11,12 is a significant hallmark of disease. Finally, experimental acute and chronic stress causes intestinal hypersecretion, permeability, mucin release, and smooth muscle hyperreactivity.5-7,13-16 These effects seem to be mediated peripherally by neuropeptides such as substance P and neurotensin, as well as by the autonomic nervous system, and centrally by corticotrophin-releasing factor (CRF). Despite this evidence for stress effects on the intestine, little attention has been paid experimentally to the possible effects of stress on lung function,17 although the effect of stress on the development and expression of atopy has begun to receive attention.18 This report represents one of the first such attempts. In it we show that acute stress, mediated in part through the action of CRF, has a stimulatory effect on ion secretion by rat tracheal epithelium, and is accompanied by evidence for mast cell activation and degranulation. At the same time, both stress and CRF prepare (sensitize) tissues for enhanced subsequent responses to substance P (SP). These studies may have important implications for the role of stress in lung diseases such as asthma.

METHODS Details of the methods can be found in the Journal’s Online Repository (www.mosby.com/jaci).

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Animals Specific pathogen-free male Wistar rats (300-420 g; Charles River, St Constant, Quebec, Canada) were maintained on a 12-hour: 12-hour light-dark cycle. Rats were provided with food and water ad libitum and handled daily for 1 week before the study to minimize effects of stress occasioned by human handling. All experiments were approved by the Animal Care Committee at McMaster University.

Water avoidance stress (WAS) was chosen as the model of stress. This model appears to have similar effects and modes of action insofar as intestinal responses and the hypothalamic-pituitary-adrenal axis are concerned.5,19 Stress sessions were performed between 1000 and 1300 hours to minimize diurnal variations in response.

FIG 1. Effect of WAS on tracheal baseline Isc. Rats were subjected to WAS or sham stress for 1 hour or injected intraperitoneally with CRF (50 mg/kg) or a-helical CRF (250 mg/kg) 30 minutes before stress or CRF protocol. Bars represent means 6 SEMs; n = 4 to 12 rats/group. **P < .01 vs control;  P < .05 vs stress; CP < .05 vs CRF.

Drugs Corticotrophin-releasing factor and a-helical CRF–(9-41) (a-helical CRF), a CRF 1 and 2 antagonist (Peninsula Lab, Belmont, Calif), were dissolved in PBS according to the manufacturer’s instructions, aliquoted, and kept frozen at 270°C until used. The neutral endopeptidase inhibitor, phosphoramidon, was obtained from Sigma Chemical Co (St Louis, Mo) and dissolved in PBS. SP (Peninsula Lab) was dissolved in PBS, aliquoted, and stored at 0°C before use. All drugs were used fresh. Immediately before the experiments, the drugs were diluted in PBS to the appropriate concentrations.

and then washed 3 times in the same buffer. Samples were postfixed in 2% osmium tetroxide for 1 hour, dehydrated in graded ethanol, and embedded in Spurr resin. The sections were photographed at a magnification of 30003 in a transmission electron microscope (JEOL 1200 EX, Tokyo, Japan). At least 20 mast cells from each rat were analyzed by 2 investigators who were blind to the experiments. Interobserver error did not vary by more than 3%. Activation of mast cells was defined by the presence of granules with altered or absent electron-dense content.

Ussing chamber experiments

Fecal pellet output

Trachea. The trachea was removed, immersed in 37°C oxygenated Krebs buffer, opened along the anterior margin, and immediately mounted in Ussing chambers. Ussing chambers measure current across living tissue. In the case of tracheal tissue, the current measured reflects chloride ion secretion by the epithelium. Tissue was mounted in an Ussing chamber and the spontaneous potential difference (PD) clamped at 0 volts (WP Instruments automated voltage clamp; WP Instruments, Nacro Scientific, Mississauga, Ontario, Canada). The injected current, the short-circuit current (Isc in mA/ cm2), required to maintain 0 PD, was continuously measured and indicates net active ion transport. At intervals, the PD across the tissue was measured to allow calculation of tissue ion conduction (indicates net passive ion flux across the preparation) using the Ohm law and the PD and Isc values. Baseline values for Isc, an indicator of ion secretion, and G, an indicator of ion permeability, were measured 15 minutes after mounting the tissues, and then every 5 minutes for 40 minutes. The tissue was considered equilibrated after 15 minutes. Abnormal baseline values of G > 50 millisiemens/cm2 were considered damaged and were excluded. Colon. Tissue was treated as described elsewhere.5

The number of fecal pellets expelled by each rat per hour was used as an indirect measure of colonic motility, as previously described.13,20

A pilot study showed that colonic epithelial abnormalities were maximal at 1 hour after initiation of WAS. The rats were injected intraperitoneally with CRF (50 mg/kg) 1 hour before mounting the tissue. Others had previously determined that this dose was maximal in intestinal Isc responses.5 The dose (250 mg/kg) of a-helical CRF has been shown to block intestinal responses to WAS effectively5 and was injected intraperitoneally 30 minutes before the WAS protocol.

Tracheal responses to SP in vitro

The data were expressed as means 6 SEMs. A P value of less than .05 was considered significant. Differences between the values were tested by using the Student t test or the Scheffe method after ANOVA.22

Substance P in amounts of 10 mL to a final concentration of 1026 mol/L was added to the luminal or the serosal buffer of the Ussing chambers 50 minutes after mounting. After adding SP to the luminal buffer, immediate, sharp increases in Isc were observed. The Isc was calculated as the difference between the baseline Isc value and the maximum value within 10 seconds of addition of SP. In all experiments, phosphoramidon (final concentration: 1025 mol/L) was added to the chamber buffer 10 minutes before adding SP.

Electron microscopy The tissue was fixed in 2% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4) for 2 hours at room temperature, washed, left overnight at 4°C in 0.2 mol/L sodium cacodylate buffer, pH 7.4,

Experimental design and pharmacological treatments

Measurement of serum corticosterone Levels were determined by the HPLC method of Wong et al21 with some minor modifications.

Statistics

RESULTS Corticosterone levels For corticosterone levels, see Fig E1 in the Online Repository (www.mosby.com/jaci). The results from the HPLC standards were linear over a dose range of 10 to 1000 ng/mL carbon-treated serum. Control values in normal rats were 102.6 6 19.5. It was evident that the sham animals were undergoing mild stress, because their levels were 270 6 56.1, which differed significantly from

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Stress model

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FIG 2. Effect of SP (1026 mol/L) added to the luminal or serosal sides of trachea on Isc generation in Ussing chamber. SP was added 50 minutes after mounting. These tracings are representative of 3 similar experiments.

the controls (P < .05). The levels in stressed animals (785.1 6 121.8) differed significantly both from sham (P < .01) and control (P < .01) groups. The responses to an intraperitoneal injection of CRF (50 mg/kg) in PBS 1 hour before mounting the tissues in Ussing chambers were 912.6 6 201.8, which again differed significantly from control (P < .01). The injection of a-helical CRF before the stress or CRF protocol reduced the levels to approximately the same as those of the sham (234.2 6 29.4). This level, however, differed significantly from the control values (P < .05).

Defecation during stress and response to a-helical CRF See Fig E2 in the Online Repository (www.mosby.com/ jaci). Measurement of the number of fecal pellets per hour corresponded to values expected in controls (0.5 6 0.3), sham (1.1 6 1.0), or stress (6.4 6 0.7) and those animals pretreated with a-helical CRF (3.8 6 1.0). The number seen in the stress group differed significantly from both control and sham groups (P < .01), whereas the animals pretreated with a-helical CRF had a reduced but not significantly reduced number of pellets. Effects of stress, CRF, and a-helical CRF on colonic epithelial physiology See Fig E3 in the Online Repository (www.mosby.com/ jaci). Baseline Isc responses 15 minutes after mounting were approximately the same in control (30.7 6 6.3) and sham groups (37.7 6 6.1). WAS significantly increased Isc relative to sham (P < .05) and control groups (P < .01). Intraperitoneal injection of CRF (25 mg/kg in saline) confirmed that this produced significant increases in Isc responses. a-Helical CRF had no effect on colonic Isc5 and reduced the effects of stress to baseline.5 Effects of stress, CRF, and a-helical CRF on tracheal epithelial physiology Stress caused increases in Isc compared with both sham (P < .05) and control groups (P < .01; Fig 1). The effect of intraperitoneal injection of CRF (50 mg/kg) 1 hour before the tissue was mounted in the Ussing chamber was significant compared with sham (P < .01) and control (P < .01) groups. This concentration has been shown by others5 to have maximal effects on colon epithelial

physiology. The time point of 1 hour was chosen to examine the effect of CRF on the trachea because the stress exposure lasted for 1 hour. We saw no difference at 2 or 4 hours from that seen at 1 hour. There was no significant difference between any of the groups in conductance values (see Table E1 in the Online Repository at www.mosby.com/jaci). Pretreatment with a-helical CRF reduced the Isc seen in stress to the levels of control and sham (P < 0.05). The effects of a-helical CRF (42.8 6 6.4) alone did not differ significantly between control (43.5 6 1.9) and sham (41.8 6 3.4) groups. No changes in conductance were seen as a result of treatment with a-helical CRF in any of the groups examined.

Effects of stress and CRF on SP-induced tracheal epithelial Isc We wished to establish whether stress sensitized tissues for responses to other agonists such as SP. SP effects on the generation of tracheal epithelial Isc have been shown by others to be effective only if delivered on the luminal side.23-25 We confirmed these results (Fig 2) and established that the effect of SP at a concentration of 1026 mol/L was optimal on tissue equilibrated for 15 minutes. No differences were seen with or without the addition of phosphoramidon. As seen in Table I, both stress (P < .01) and sham stress (P < .05) significantly increased the response to standardized amounts of SP compared with controls. Similarly, CRF injection (50 mg/ kg) intraperitoneally 1 hour before the experiment significantly increased responsiveness of the tissue to SP (P < .01). No changes in conductance were seen at any time in these experiments. Effects of a-helical CRF on stress and CRF effects Results of experiments with a-helical CRF are shown in Fig 3. a-Helical CRF, which in earlier experiments at this concentration (250 mg/kg) was effective in reducing stress effects, had no effect on the sensitization induced by stress for subsequent SP responses. Surprisingly, the control experiment of injection of a-helical CRF by itself sensitized the trachea equally as well as stress or CRF so that there were no significant differences compared with control between the stress, CRF, a-helical CRF plus

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TABLE I. Effect of stress and intraperitoneal CRF injection on tracheal responses to SPy DIsc (mA/cm2)

47.9 105.1 107.1 129.5

6 6 6 6

4.4 21.7* 6.4** 4.1**

 Values are means 6 SEMs. The DIsc was calculated as the difference between the baseline Isc value and the maximum value within 10 seconds of addition of SP. **P < .01; *P < .05 vs control.

stress, and a-helical CRF groups. Pretreatment with a-helical CRF before injection with CRF did not reduce the enhanced response to SP. In fact, the response of this group showed a significant enhancement over CRF itself (P < .05), but not over the a-helical CRF alone.

Effects of stress on mast cell morphology Acute stress caused mast cell activation (Table II) and in some cases degranulation (Fig 4). There were significant differences between control and stressed animals (P < .05) in the trachea as well as in the colon (P < .01). Nevertheless, some loss of density of granule contents was also found in both colon and trachea of control animals. If this criterion is used to establish activation, an average of 22% of total mast cell granules in various normal tissues, such as skin, ileum, heart, and cranial dura mater,26-30 show signs of activation under normal physiologic conditions. DISCUSSION To the best of our knowledge, this is the first report of the effects of psychological stress on lung epithelial physiology. We have shown that in Wistar rats, acute water avoidance stress caused increased tracheal epithelial Isc activity, and that this was accompanied by morphologic evidence of mast cell activation and degranulation, was mimicked by systemic CRF injection, and could be largely reversed by a CRF receptor competitive inhibitor, a-helical CRF. There are many reports now that have focused on the effects of different forms of acute or chronic psychological stress on various intestinal physiologic parameters.6,7,14,15,20,31,32 These include experimental results, obtained mostly16 (but not exclusively) with animal tissues, and include effects on fluid or ion secretion and absorption, permeability,5,15,23,33,34 motility,6,13,20 and pain perception.32 Tissue responses have been shown to vary according to the experimental animal, strain, and tissue source, ie, stomach,35 jejunum,15,31,33 ileum,36 and colon.5,6,13,32 Thus, a significant body of literature now exists that suggests acute stress effects in the intestine are largely mediated by CRF,5,6,37,38 because many of the physiological consequences can be reproduced equally either by intracerebral injection of CRF6,7,32,38 or through its

FIG 3. Effect of WAS, CRF, or a-helical CRF injection on SP responses of the trachea in Wistar rats. Rats were subjected to WAS or sham stress for 1 hour or injected intraperitoneally with CRF (50 mg/kg). One hour after the initiation of stress, 90 minutes after a-helical CRF, or 1 hour after CRF injection alone, tracheas were mounted in Ussing chambers. SP (1026 mol/L) was added to the luminal side of the chamber at 50 minutes after mounting. Bars represent means 6 SEMs; **P < .01, *P < .05 vs control;  P < .05 vs CRF.

peripheral systemic administration.5-7,37,38 These effects are mostly mediated through mast cell activation, because pharmacological inhibition of mast cell degranulation prevents many of the consequences of acute stress,5,6,32 and mast cell deficient rats fail to exhibit these findings.14,15 Mast cell–nerve interactions and communication11,39,40 have been shown to be involved in the generation of a variety of intestinal and lung physiologic responses to antigen.16,25,39-41 These interactions35 appear to be crucial for the generation of many of the functional intestinal effects of stress, because SP antagonists6 block these effects. They are also inhibited by cholinergic, adrenergic, and autonomic ganglion blockade through the use of a variety of well defined pharmacologic agents.5 At no time did we see any increase in conductance, a reflection of the integrity of the tracheal epithelium, whereas this is invariably seen as a stress response in the intestine.5,34 This probably reflects a general difference between trachea and intestine, because antigen exposure of tracheal tissue from sensitized rats, despite causing large increases in Isc25 and mast cell degranulation,21 never showed increased conductance.25,30 Stress effects are complex end results of a range of interacting signals between a variety of systems and cells of different types. Indeed, the results obtained with a-helical CRF inhibition of stress on corticosterone levels are instructive, because the levels of corticosterone were reduced only to those found in sham animals, and still differed significantly from those of controls. This suggests that factors in addition to CRF and its pathway are operative in the production of corticosterone and not inhibited by this antagonist of CRF receptors 1 and 2.42,43 Indeed, behavioral changes apparently mediated by CRF 1 receptors can still be seen in transgenic CRF knockout mice.44 Furthermore, stress-enhanced inflammation in a hapten sensitization model of colitis was shown not to be caused by CRF.45 Corticotropin-releasing factor is known to have both proinflammatory29,46 and anti-inflammatory47-50 effects.

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Control Sham Stress CRF

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TABLE II. Mast cell activation during stressy

Tissue

Mast cell activation Total granular change Condition granules Total low densityz

Trachea Control Stress Colon Control Stress

2631 3234 2728 2352

600 1163 619 940

% Total activation§

22.5 35.3 22.0 41.0

6 6 6 6

2.0 2.9* 2.0 1.7**

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 At least 20 mast cells from each rat were evaluated (n = 4 in each group). àLoss of granule electron density content. §Indicates the percentage of changed granules (means 6 SEMs). **P < .01; *P < .05 vs control.

It has been shown to cause mast cell degranulation when injected into the skin.29 It also appears to be involved in stress-induced cardiac mast cell degranulation,51 degranulation of cranial dura mater mast cells,52 and those of the urinary bladder.53 However, CRF has not previously been shown to be involved in tracheal or lung mast cell activation. Because we have shown that the tracheal Isc responses to stress are inhibited by the a-helical CRF, and because stress increased tracheal mast cell degranulation (Table II; Fig 4), it appears likely that directly or indirectly, CRF causes tracheal mast cell degranulation. Mast cell degranulation is one of the first effects of allergen inhalation. Stress mediated lung mast cell degranulation is another mechanism for enhancement of the consequence of allergen inhalation in the sensitized host and would further promote airways inflammation. Although rat tracheal and lung mast cells are a mixture of mucosal and connective tissue types,54 both seem to respond to stress and CRF, as evidenced by the fact that injection of CRF into rat skin caused degranulation of primarily connective tissue–type mast cells,29 and stress caused elevations of rat mast cell protease II,6 a specific marker of mucosal mast cells. Our results also showed that both acute stress and the peripheral systemic injection of CRF enhanced the action of SP on epithelial cell secretion. Joachim et al55 recently showed that stressed mice exhibited increased bronchial hyperreactivity and that this and the increase in airways inflammation was caused by SP. Our experimental results were anticipated in view of the data of McAlexander and Undem,56 who showed that CRF caused enhancement of tachykinin-induced contractions of guinea pig tracheobronchial smooth muscle. However, our results could not be predicted, because there are now several reports showing that CRF can have a protective effect.47-50 These results range from experiments involving antigeninduced plasma extravasation and that induced by SP or vagal stimulation, all the way to stress-induced worsening of a hapten model of colitis48 both in hypo Lewis (LEW/N) and hyper (Fischer, 344/N) CRF responders to stress.57,58 Interpretation of the role of CRF in the modulation of stress effects is complicated by our own results, which clearly indicated not only that effects on stress-induced Isc increases were inhibited totally by the

FIG 4. Representative electron micrographs of mast cells in rat tracheal epithelium from control (A) and stress (B) rats. Tracheas were fixed 15 minutes after mounting in Ussing chambers. Mast cells from control groups showed some signs of activation. Mast cells from stress groups were more activated, as indicated by loss of granule density (arrows point to such granules in B). Magnification: 30003.

a-helical CRF inhibitor but also that the stress-induced enhancement of the effect of SP was not inhibited at all by the a-helical CRF inhibitor. Further complexity to interpretation was added by the observation that the a-helical CRF inhibitor alone enhanced the SP effect but notably had no effect itself on Isc generation either in the colon or trachea. Last, a-helical CRF did not reduce the activity of CRF in this model but actually enhanced it, confirming its activity independent of CRF. We can conclude that although stress effects on Isc may be mediated largely by CRF, its effects on enhancement of SP may be through additional and different mechanisms. Several possible explanations may be entertained to explain these unexpected findings. First, a-helical CRF has weak intrinsic activity37,42 in several different systems.43,59 It might have influenced SP receptor number or affinity or have agonist activity on new, as yet undiscovered CRF receptors expressed in target tissue. That the stress effect itself is, however, not wholly dependent on CRF is further evidenced by our observations that corticosterone levels after stress in a-helical CRF pretreated animals were not reduced to levels seen in controls, but rather only to those seen in sham animals. The biological significance and relevance of these experiments to asthma lie in the primary observation that acute stress can cause moderate effects on tracheal function. It seems reasonable to argue teleologically that it would be detrimental to the host if hypersecretion by the

tracheal epithelium occurred in response to acute stress, whereas intestinal hypersecretion would not be deleterious to the host’s survival. Therefore, it may be expected that these tracheal responses would indeed be moderate and likely to be subject to a significant array of physiologic mechanisms of inhibition and regulation. However, stress activation of epithelium15 may cause upregulation of cytokines such as IL-8 and possibly eotaxin to account for the findings of Liu et al2 that stress enhances sputum eosinophil numbers and products. Our observation of profound enhancement of the effect of SP as a result of stress may have great significance in terms of understanding the mechanisms of lung diseases such as asthma. SP and other neuropeptides may have major effect in the generation of bronchial smooth muscle contraction experimentally and in asthma.60 Extensive reduction of thresholds for this activity would likely have profound deleterious effects in asthma. It is becoming increasingly recognized that several signals may have to occur simultaneously, or in prescribed succession, and only when these occur in genetically vulnerable individuals may this constellation of stimuli lead to disease.61,62 In this scenario, acute stress occurring in a particular context of infection and genetic susceptibility could have significant detrimental consequences to the host.63 We thank Linda Builder, Laurie Nielsen, and Todd Prior for technical assistance and advice and Professor Yuzo Endo, Dr PingChang Yang, and the staff of the Electron Microscopy Unit, McMaster University, for expert assistance. The help of Professor Jack Rosenfeld with development of the corticosterone assay is gratefully acknowledged. REFERENCES 1. Busse WW, Kiecolt-Glaser JK, Coe C, Martin RJ, Weiss ST, Parker SR. NHLBI Workshop summary: stress and asthma. Am J Respir Crit Care Med 1995;151:249-52. 2. Liu LY, Coe CL, Swenson CA, Kelly EA, Kita H, Busse WW. School examinations enhance airway inflammation to antigen challenge. Am J Respir Crit Care Med 2002;165:1062-7. 3. Sandberg S, Jarvenpaa S, Penttinen A, Paton JY, McCann DC. Asthma exacerbations in children immediately following stressful life events: a Cox’s hierarchical regression. Thorax 2004;59:1046-51. 4. Csermely P. Stress of life: from molecules to man: proceedings of a conference: Budapest, Hungary, July 1-5, 1997. Ann N Y Acad Sci 1998;851:1-47. 5. Santos J, Saunders PR, Hanssen NP, Yang PC, Yates D, Groot GA, et al. Corticotropin-releasing hormone mimics stress-induced colonic epithelial pathophysiology in the rat. Am J Physiol 1999;277:G391-9. 6. Castagliuolo I, LaMont JT, Qiu B, Fleming SM, Bhaskar KR, Nikulasson ST, et al. Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells. Am J Physiol 1996; 271:G884-92. 7. Pothoulakis C, Castagliuolo I, Leeman SE. Neuroimmune mechanisms of intestinal responses to stress: role of corticotropin-releasing factor and neurotensin. Ann N Y Acad Sci 1998;840:635-48. 8. Bienenstock J. Stress and asthma: the plot thickens. Am J Respir Crit Care Med 2002;165:1034-5. 9. Verdu EF, Collins SM. Microbial-gut interactions in health and disease: irritable bowel syndrome. Best Pract Res Clin Gastroenterol 2004;18: 315-21. 10. Bennett EJ, Piesse C, Palmer K, Badcock CA, Tennant CC, Kellow JE. Functional gastrointestinal disorders: psychological, social, and somatic features. Gut 1998;43:256-61.

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11. Stead RH, Tomioka M, Quinonez G, Simon GT, Felten SY, Bienenstock J. Intestinal mucosal mast cells in normal and nematode-infected rat intestines are in intimate contact with peptidergic nerves. Proc Natl Acad Sci U S A 1987;84:2975-9. 12. Barbara G, Stanghellini V, De Giorgio R, Cremon C, Cottrell GS, Santini D, et al. Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome. Gastroenterology 2004;126: 693-702. 13. Monnikes H, Schmidt BG, Tache Y. Psychological stress-induced accelerated colonic transit in rats involves hypothalamic corticotropinreleasing factor. Gastroenterology 1993;104:716-23. 14. Castagliuolo I, Wershil BK, Karalis K, Pasha A, Nikulasson ST, Pothoulakis C. Colonic mucin release in response to immobilization stress is mast cell dependent. Am J Physiol 1998;274:G1094-100. 15. Santos J, Benjamin M, Yang PC, Prior T, Perdue MH. Chronic stress impairs rat growth and jejunal epithelial barrier function: role of mast cells. Am J Physiol Gastrointest Liver Physiol 2000;278:G847-54. 16. Santos J, Saperas E, Nogueiras C, Mourelle M, Antolin M, Cadahia A, et al. Release of mast cell mediators into the jejunum by cold pain stress in humans. Gastroenterology 1998;114:640-8. 17. Joachim RA, Sagach V, Quarcoo D, Dinh QT, Arck PC, Klapp BF. Neurokinin-1 receptor mediates stress-exacerbated allergic airway inflammation and airway hyperresponsiveness in mice. Psychosom Med 2004;66:564-71. 18. Wright RJ, Cohen RT, Cohen S. The impact of stress on the development and expression of atopy. Curr Opin Allergy Clin Immunol 2005;5: 23-9. 19. Bonaz B, Tache Y. Water-avoidance stress-induced c-fos expression in the rat brain and stimulation of fecal output: role of corticotropinreleasing factor. Brain Res 1994;641:21-8. 20. Barone FC, Deegan JF, Price WJ, Fowler PJ, Fondacaro JD, Ormsbee HSI. Cold-restraint stress increases rat fecal pellet output and colonic transit. Am J Physiol 1990;258:G329-37. 21. Wong YN, Chien BM, D’mello AP. Analysis of corticosterone in rat plasma by high-performance liquid chromatography. J Chromatogr B Biomed Appl 1994;661:211-8. 22. Hopkins KD, Glass GV, Hopkins BR. Basic statistics for the behavioral sciences. 2nd ed. Englewood Cliffs (NJ): Prentice-Hall; 1987. 23. Al-Bazzaz FJ, Kelsey G, Kaage WD. Substance P stimulation of chloride secretion by canine tracheal mucosa. Am Rev Respir Dis 1985;131:86-9. 24. Rangachari PK, McWade D. Effects of tachykinins on the electrical activity of isolated canine tracheal epithelium: an exploratory study. Regul Pept 1985;12:9-19. 25. Sestini P, Bienenstock J, Crowe SE, Marshall JS, Stead RH, Kakuta Y, et al. Ion transport in rat tracheal epithelium in vitro: role of capsaicinsensitive nerves in allergic reactions. Am Rev Respir Dis 1990;141:393-7. 26. Rozniecki JJ, Dimitriadou V, Lambracht-Hall M, Pang X, Theoharides TC. Morphological and functional demonstration of rat dura mater mast cell-neuron interactions in vitro and in vivo. Brain Res 1999;849:1-15. 27. Singh LK, Pang X, Alexacos N, Letourneau R, Theoharides TC. Acute immobilization stress triggers skin mast cell degranulation via corticotropin releasing hormone, neurotensin, and substance P: a link to neurogenic skin disorders. Brain Behav Immun 1999;13:225-39. 28. Spanos C, Pang X, Ligris K, Letourneau R, Alferes L, Alexacos N, et al. Stress-induced bladder mast cell activation: implications for interstitial cystitis. J Urol 1997;157:669-72. 29. Theoharides TC, Singh LK, Boucher W, Pang X, Letourneau R, Webster E, et al. Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects. Endocrinology 1998;139:403-13. 30. Yang PC, Berin MC, Perdue MH. Enhanced antigen transport across rat tracheal epithelium induced by sensitization and mast cell activation. J Immunol 1999;163:2769-76. 31. Yates DA, Santos J, Soderholm JD, Perdue MH. Adaptation of stressinduced mucosal pathophysiology in rat colon involves opioid pathways. Am J Physiol Gastrointest Liver Physiol 2001;281:124-8. 32. Gue M, Del Rio-Lacheze C, Eutamene H, Theodorou V, Fioramonti J, Bueno L. Stress-induced visceral hypersensitivity to rectal distension in rats: role of CRF and mast cells. Neurogastroenterol Motil 1997;9:271-9. 33. Kiliaan AJ, Saunders PR, Bijlsma PB, Berin MC, Taminiau JA, Groot JA, et al. Stress stimulates transepithelial macromolecular uptake in rat jejunum. Am J Physiol 1998;275:G1037-44.

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34. Saunders PR, Kosecka U, McKay DM, Perdue MH. Acute stressors stimulate ion secretion and increase epithelial permeability in rat intestine. Am J Physiol 1994;267:G794-9. 35. Pare WP. Stress ulcer susceptibility and depression in Wistar Kyoto (WKY) rats. Physiol Behav 1989;46:993-8. 36. Theoharides TC, Letourneau R, Patra P, Hesse L, Pang X, Boucher W, et al. Stress-induced rat intestinal mast cell intragranular activation and inhibitory effect of sulfated proteoglycans. Dig Dis Sci 1999;44:87S-93S. 37. Tache Y, Martinez V, Million M, Rivier J. Corticotropin-releasing factor and the brain-gut motor response to stress. Can J Gastroenterol 1999; 13(suppl A):18A-25A. 38. Williams CL, Peterson JM, Villar RG, Burks TF. Corticotropin-releasing factor directly mediates colonic responses to stress. Am J Physiol 1987; 253:G582-6. 39. McKay DM, Bienenstock J. The interaction between mast cells and nerves in the gastrointestinal tract. Immunol Today 1994;15:533-8. 40. Marshall JS, Bienenstock J. The role of mast cells in inflammatory reactions of the airways, skin and intestine. Curr Opin Immunol 1994;6: 853-9. 41. Sestini P, Dolovich M, Vancheri C, Stead RH, Marshall JS, Perdue M, et al. Antigen-induced lung solute clearance in rats is dependent on capsaicin-sensitive nerves. Am Rev Respir Dis 1989;139:401-6. 42. Fisher L, Rivier C, Rivier J, Brown M. Differential antagonist activity of alpha-helical corticotropin-releasing factor 9-41 in three bioassay systems. Endocrinology 1991;129:1312-6. 43. Gulyas J, Rivier C, Perrin M, Koerber SC, Sutton S, Corrigan A, et al. Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor. Proc Natl Acad Sci U S A 1995;92: 10575-9. 44. Weninger SC, Dunn AJ, Muglia LJ, Dikkes P, Miczek KA, Swiergiel AH, et al. Stress-induced behaviors require the corticotropin-releasing hormone (CRH) receptor, but not CRH. Proc Natl Acad Sci U S A 1999; 96:8283-8. 45. Gue M, Bonbonne C, Fioramonti J, More J, Del Rio-Lacheze C, Comera C, et al. Stress-induced enhancement of colitis in rats: CRF and arginine vasopressin are not involved. Am J Physiol 1997;272:G84-91. 46. Karalis K, Sano H, Redwine J, Listwak S, Wilder RL, Chrousos GP. Autocrine or paracrine inflammatory actions of corticotropin-releasing hormone in vivo. Science 1991;254:421-3. 47. Gao GC, Dashwood MR, Wei ET. Corticotropin-releasing factor inhibition of substance P-induced vascular leakage in rats: possible sites of action. Peptides 1991;12:639-44. 48. Million M, Tache Y, Anton P. Susceptibility of Lewis and Fischer rats to stress-induced worsening of TNB-colitis: protective role of brain CRF. Am J Physiol 1999;276:G1027-36.

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49. Wei ET, Kiang JG. Inhibition of protein exudation from the trachea by corticotropin-releasing factor. Eur J Pharmacol 1987;140:63-7. 50. Yoshihara S, Ricciardolo FL, Geppetti P, Linden A, Hara M, Chan B, et al. Corticotropin-releasing factor inhibits antigen-induced plasma extravasation in airways. Eur J Pharmacol 1995;280:113-8. 51. Pang X, Alexacos N, Letourneau R, Seretakis D, Gao W, Boucher W, et al. A neurotensin receptor antagonist inhibits acute immobilization stress-induced cardiac mast cell degranulation, a corticotropin-releasing hormone-dependent process. J Pharmacol Exp Ther 1998;287:307-14. 52. Theoharides TC, Spanos C, Pang X, Alferes L, Ligris K, Letourneau R, et al. Stress-induced intracranial mast cell degranulation: a corticotropinreleasing hormone-mediated effect. Endocrinology 1995;136:5745-50. 53. Alexacos N, Pang X, Boucher W, Cochrane DE, Sant GR, Theoharides TC. Neurotensin mediates rat bladder mast cell degranulation triggered by acute psychological stress. Urology 1999;53:1035-40. 54. Bienenstock J, Befus D, Denburg J, Goto T, Lee T, Otsuka H, et al. Comparative aspects of mast cell heterogeneity in different species and sites. Int Arch Allergy Appl Immunol 1985;77:126-9. 55. Joachim RA, Quarcoo D, Arck PC, Herz U, Renz H, Klapp BF. Stress enhances airway reactivity and airway inflammation in an animal model of allergic bronchial asthma. Psychosom Med 2003;65:811-5. 56. McAlexander MA, Undem BJ. Enhancement of tachykinin-induced contractions of guinea pig isolated bronchus by corticotropin-releasing factor. Neuropeptides 1997;31:293-9. 57. Sternberg EM, Glowa JR, Smith MA, Calogero AE, Listwak SJ, Aksentijevich S, et al. Corticotropin releasing hormone related behavioral and neuroendocrine responses to stress in Lewis and Fischer rats. Brain Res 1992;570:54-60. 58. Sternberg EM, Zareie M, Young WS III, Alogero AE, Chrousos GP, Gold PW, et al. A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wall-induced arthritis in Lewis rats. Proc Natl Acad Sci U S A 1989;86:4771-5. 59. Menzaghi F, Howard RL, Heinrichs SC, Vale W, Rivier J, Koob GF. Characterization of a novel and potent corticotropin-releasing factor antagonist in rats. J Pharmacol Exp Ther 1994;269:564-72. 60. van der Kleij H, Kraneveld AD, Redegeld FA, Gerard NP, Morteau O, Nijkamp FP. The tachykinin NK1 receptor is crucial for the development of non-atopic airway inflammation and hyperresponsiveness. Eur J Pharmacol 2003;476:249-55. 61. Holgate ST. The epidemic of allergy and asthma. Nature 1999;402:B2-4. 62. Holgate ST. Science, medicine, and the future: allergic disorders. BMJ 2000;320:231-4. 63. Christiansen SC. Day care, siblings, and asthma: please, sneeze on my child. N Engl J Med 2000;343:574-5.

Allergen-induced substance P synthesis in large-diameter sensory neurons innervating the lungs

Background: Tachykinins such as substance P are localized in unmyelinated slow-conducting C fibers that can be activated by noxious stimuli and tissue inflammation. Substance P is seldom expressed in fast-conducting large-diameter (A-fiber) vagal sensory neurons. We have previously found that allergic inflammation causes a phenotypic change in tachykinergic innervation of the trachea such that the production of substance P is induced in large-diameter sensory neurons projecting mechanosensitive A fibers to the trachea. Objective: To evaluate whether allergic inflammation also induces substance P synthesis in large-diameter sensory stretch-receptor neurons innervating guinea pig lungs, and to investigate potential mechanisms by which this may occur. Methods: Sensitized guinea pigs were exposed to allergen (ovalbumin) aerosol. One day later, immunohistochemical analysis was performed on vagal sensory neurons that had been retrogradely labeled from the lungs. Results: Ovalbumin inhalation caused a significant increase in substance P expression in large-diameter neurofilamentpositive nodose ganglion neurons that innervate the lungs (P < .05). This effect was decreased by ipsilateral vagotomy. Exposing isolated nodose ganglia to the sensitizing antigen, ovalbumin, also significantly increased substance P expression compared with control. Conclusion: Allergic inflammation induces substance P synthesis in large-diameter (A-fiber) nodose ganglion neurons innervating guinea pig lungs. This could contribute to the hyperreflexia seen in allergic airway disease. The full expression of this phenotypic switch in vagus nodose ganglion neurons requires intact vagus nerve, but if allergen reached the systemic circulation in sufficient quantities, it could also affect substance P synthesis by local activation of vagal ganglionic mast cells. (J Allergy Clin Immunol 2005;116:325-31.)

From the Johns Hopkins University School of Medicine and Bloomberg School of Public Health. Supported by the Heart, Lung and Blood Institute of the National Institutes of Health (Bethesda, Md) and scholarship funding to Dr Chuaychoo from the Faculty of Medicine, Siriraj Hospital, Mahidol University (Bangkok, Thailand). Received for publication February 13, 2005; revised March 30, 2005; accepted for publication April 4, 2005. Available online June 1, 2005. Reprint requests: Bradley J. Undem, PhD, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.005

Key words: Allergic inflammation, phenotypic switch, neurogenic inflammation, substance P, nodose ganglion, sensory nerve, vagus nerve

In both the somatosensory and visceral-sensory system, sensory neuropeptides, exemplified by the tachykinin substance P (SP), are commonly found stored in the peripheral and central terminals of unmyelinated sensory C fibers.1-3 Inflammatory reactions in the rat paw have been found to evoke a phenotypic switch in the nature of tachykinergic innervation.4,5 After a local inflammatory reaction in the paw, the preprotachykinin gene is induced de novo in large-diameter, low-threshold, touch-sensitive Ab fibers.4 Simply touching the paw can then lead to neuropeptide release in the spinal cord that, in theory, could lead to an increase in neurotransmission to the extent that this innocuous touch stimulus is interpreted as a noxious pain-producing stimulus (ie, an allodynia). A similar inflammation-induced phenotypic switch can occur in vagal sensory innervation of the airways. In the respiratory tract of most mammals, including guinea pigs and human beings, SP-containing fibers are located mainly in the upper airways, trachea, and main bronchi.1,6 There are relatively few SP containing fibers in the lungs. In healthy guinea pig trachea and large bronchi, tachykinins are localized nearly exclusively in nociceptive C fibers derived from small-diameter cell bodies in jugular sensory ganglia.2,3 One day after allergic inflammation in the lungs, SP production is increased in the lungs and in vagal sensory neurons.7 On further analysis, it was determined that part of the increase in SP production occurred de novo in largediameter neurons that project mechanosensitive Ad fibers to the trachea.8 This same type of phenotypic switch occurs 2 to 3 days after respiratory tract viral infection.9 The touch-sensitive Ad fibers in the guinea pig trachea represent a unique subtype of vagal afferent mechanosensors apparently designed to evoke cough on activation.10 They differ from classically described intrapulmonary stretch-sensitive fibers in neurophysiology, activation profile, and distribution in the lungs.10 The intrapulmonary stretch-sensitive Ab fibers are further subclassified as either rapidly adapting receptors (RARs) or slowly adapting receptors (SARs). Whether the stretch-sensitive fibers in the lungs can be induced by inflammation to produce neuropeptides in a fashion similar the tracheal touchsensitive fibers is unknown. There is also little known about the mechanism by which airway inflammation leads to phenotypic changes 325

Mechanisms of asthma and allergic inflammation

Benjamas Chuaychoo, MD, Dawn D. Hunter, PhD, Allen C. Myers, PhD, Marian Kollarik, MD, PhD, and Bradley J. Undem, PhD Baltimore, Md

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Abbreviations used NGF: RAR: SAR: SP: Triton-BSA-PBS:

Nerve growth factor Rapidly adapting receptor Slowly adapting receptor Substance P PBS containing 0.3 % TritonX-100 and 1% BSA Trk: Tyrosine kinase receptor

Mechanisms of asthma and allergic inflammation

of the neurons located in the remote vagal sensory ganglia (nodose and jugular ganglia situated near the base of the brain). A logical scenario is that the allergic reaction leads to the release of certain neurotrophic factors such as nerve growth factor (NGF) that interact with tyrosine kinase receptor (Trk) A on the terminals. Activation of neurotrophin Trk receptors at nerve terminals can signal events to the cell body via internalization and transport of the activated receptors along the nerve axon.11 However, there are alternative mechanisms to consider. The vagal sensory ganglia that contain the cell bodies of vagal sensory neurons are enriched with mast cells. In sensitized animals, antigen can activate these ganglionic mast cells, leading to local mediator release and change in neuronal excitability.12 It is possible, therefore, that the inhaled allergen is not triggering tachykinin synthesis from within the lungs, but rather, allergen is systemically absorbed and interacts with mast cells locally within the vagal sensory ganglia. In the current study, experiments were performed to address 2 hypotheses. First, allergic inflammation can lead to induction of tachykinin synthesis not only in tracheal Ad neurons but also in neurons that project Ab stretch-sensitive fibers to the lungs. Second, the mechanism for allergen-induced SP production in bronchopulmonary A-fiber nodose neurons requires intact vagal nerve fibers.

METHODS All experiments were approved by the Johns Hopkins Animal Care and Use Committee. For active sensitization, male Hartley guinea pigs (Hilltop, Scottsdale, Pa) weighing 100 to 300 g were immunized by intraperitoneal injection (10 mg/kg) of ovalbumin (10 mg/mL) dissolved in saline on day 1, day 3, and day 5. On day 21 after the final sensitization, the actively sensitized animals were killed by CO2 asphyxiation and exsanguinated. The blood was collected, and serum containing ovalbumin-specific IgG1 was isolated. For passive sensitization to ovalbumin, male Hartley guinea pigs weighing 100 to 300 g were sensitized by intraperitoneal injection of serum (2 mL/kg) containing IgG1 that was collected from guinea pigs actively sensitized to ovalbumin13 and challenged 1 day after sensitization. Control guinea pigs were not injected with serum but challenged in a similar fashion.13 The studies evaluating lung-labeled afferent neurons, and the ex vivo studies were performed by using actively sensitized animals. The passive sensitization protocol was used in the vagotomy studies as a means to decrease the intra-animal variation in the allergen response. In the in vivo studies, sensitized (active or passive) and unsensitized (control) animals were exposed to aerosolized antigen in a Plexiglas chamber (volume, 8 L) with

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consecutive increasing concentrations of ovalbumin (0.01%, 0.03%, 0.1% ovalbumin diluted in saline), with 10 minutes of exposure for each concentration to the maximum dose unless the animal developed signs of allergic response (gasping; rapid, shallow breathing; or coughing), whereupon it was removed to breathe ambient air. After 24 hours, the animals were killed with CO2 inhalation and exsanguinated. We found no difference in the overresponse of the animal to ovalbumin between actively and passively sensitized animals. Nonsensitized animals did not respond to the ovalbumin treatment. The ovalbumin treatment resulted in an eosinophilic bronchitis within 24 hours of exposure (data not shown).

Identification of nonretrogradely labeled large-diameter nodose neurons We previously reported that allergen challenge induced a phenotypic switch to SP production in large-diameter nodose ganglion neurons (>25 mm diameter) that project nerve fibers specifically to the trachea.8 To evaluate whether allergic inflammation induces a similar phenotypic switch in large-diameter neurons innervating beyond the trachea, we first evaluated all nodose ganglion neurons with diameters larger than 25 mm for SP immunoreactivity in control animals and after antigen challenge. Nodose ganglia were removed and fixed in 4% formaldehyde (fixative) for 2 hours at 4°C, rinsed 3 times in 0.1 M PBS (pH 7.4) and then cryoprotected with 18% sucrose in PBS for 18 to 24 hours. Serial cryostat sections of the nodose ganglia (12 mm thickness) were mounted on 4 consecutive slides, such that the first slide had sections 1, 5, 9., the second 2, 6, 10, and so forth, and the alternate slides were used for the analysis. The sections were dried at room temperature for 30 minutes and rinsed with water and PBS and incubated with blocking solution containing 1% BSA, 10% goat serum, and 0.5% Tween 20 in PBS at room temperature for 1 hour. The slides were then processed for double immunofluorescence staining with a mixture of rabbit polyclonal antibody to SP (1 mg/mL; Peninsula Laboratories Inc, San Carlos, Calif) and mouse mAb to 160 kd neurofilament protein (13 mg/mL; Chemicon, Temecula, Calif) diluted in PBS containing 0.3 % TritonX-100 and 1% BSA (Triton-BSA-PBS; Sigma Chemical Co, St Louis, Mo) for 24 hours at 4°C. The sections were washed 3 times with Triton-BSA-PBS and covered with a mixture of goat antirabbit fluorescein (20 mg/mL diluted in Triton-BSA-PBS; Vector Laboratories, Burlingame, Calif) and goat antimouse Texas red (30 mg/mL diluted in Triton-BSA-PBS; Vector Lab) for 2 hours at room temperature. The sections were then rinsed twice with PBS and once with higher pH PBS (pH 8.6), then coverslipped with antifade glycerol (Fluoromount, Molecular Probes, Eugene, Ore). Slides were examined under epifluorescence (Olympus DX60 microscope; Olympus Corp, Melville, NY) by using appropriate filter combinations for fluorescein (excitation filter 450-480 nm; barrier filter 500515 nm) and Texas red (excitation filter 510-550 nm; barrier filter 570-590 nm). The 160-kd neurofilament protein has been reported to be a selective marker for myelinated neurons,2,3 most of which are large-diameter neurons (>25 mm and mean diameter ;40 mm3,8); only large-diameter, neurofilament-positive, nodose ganglion neurons were evaluated for the expression of SP. Negative controls included slides treated with only secondary antibodies and slides in which available commercially obtained isotype controls were used as the primary antibodies for rabbit and mouse (Vector Lab). These resulted in categorically negative staining.

Identification of lung-projecting large-diameter neurons Nodose ganglion neurons were retrogradely labeled from the lung with True Blue (Chemicon) a week before sensitization and allergen challenge. True Blue is fluorescent dye that is taken up by the nerve

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Isolated ganglia studies The animals were actively sensitized with intraperitoneal injections of ovalbumin as described. Three weeks after the sensitization, the animals were killed by CO2 inhalation and exsanguinated. Nodose ganglia from both sides were isolated, cut nearly in half to expose the neurons to the antigen ex vivo, and placed immediately into Krebs bicarbonate solution (composed of NaCl, 118 mM; KCl, 5.4 mM; NaH2PO4, 1.0 mM; MgSO4, 1.2 mM; CaCl2, 1.9 mM; NaHCO3, 25.0 mM; dextrose, 11.1 mM; and gassed with 95% O2, 5% CO2, pH 7.4). Each individual nodose ganglion was incubated (37 °C) in 2 mL Krebs bicarbonate solution containing antibiotics (penicillin/streptomycin, 100 U/mL) and continuously oxygenated (95% O2, 5% CO2). The solution was changed every 15 minutes 4 times to wash and equilibrate the tissues. The nodose ganglia from the same animal were divided into a control and an antigen challenge group. In the antigen challenge group, ovalbumin (10 mg/mL) was added to the solution. The ganglia were incubated for various periods (0, 4, 8, 12, 18 hours) at 37 °C and then removed from the test tubes and fixed and processed for double immunofluorescence staining for SP and neurofilament as described in the nonlabeled large-diameter nodose neuron study. We evaluated all large-diameter, neurofilament-positive nodose ganglion

neurons to determine whether antigen induced the expression of SP ex vivo.

Vagotomy studies The animals were anesthetized as described, and 25 mL 5% Fast Blue dye (Sigma Chemical Co, St Louis, Mo) was instilled into the trachea as describe previously.14 A week later, the animals were divided into unsensitized (control) and sensitized groups. In the sensitized group, the animals were passively sensitized with intraperitoneal injection of ovalbumin-sensitized serum as described. A day after the sensitization, under general anesthesia as described, the vagus nerve was unilaterally ligated caudal to the nodose ganglion with suture in 2 places approximately 1 cm apart, and the section of vagus nerve between sutures was removed. On the following day, the animals were challenged with ovalbumin inhalation as described. Unsensitized (control) animals had a similar unilateral vagotomy and were challenged with ovalbumin. Twenty-four hours after the antigen challenge, the animals were killed with CO2 inhalation and exsanguinated. The nodose ganglia were removed bilaterally and fixed for double immunofluorescence staining with antibodies to neurofilament (160 kd) and SP as described in the nonlabeled largediameter nodose neuron study. Only Fast Blue–labeled neurons that were neurofilament-positive were evaluated. For vagotomy, Fast Blue was used; no differences in labeling were noted between Fast Blue and True Blue, and the latter was used for the lung labeling procedure described.

Statistical analysis The data were analyzed by using the Student paired or nonpaired t tests. Data were expressed as means 6 SEMs. P < .05 was considered significant.

RESULTS Nonretrogradely labeled large-diameter nodose neuron study We have previously made exhaustive studies of cell diameters and neurofilament staining in vagal sensory neurons projecting to the respiratory tract.3,9 The cell diameters of neurofilament-negative neurons form a unimodal population with a mean diameter of about 20 mm. The cell diameters of neurofilament-positive neurons also form a unimodal distribution with an average diameter of 43 6 8 (n = 713) in one study and 40 6 3 (n = 307) in another study. In the current study, we evaluated 781 nodose ganglion neurons that were <25 mm in diameter and found that >99% were neurofilament-negative, whereas among the 792 neurons with diameters >30 mM, all were neurofilament-positive. On the basis of these studies, and in accordance with conclusions drawn from more direct studies in the somatosensory system,15 we conclude that the large diameter neurofilament-positive population of neurons project myelinated A fibers to peripheral tissues. In unsensitized (control) animals, we found that only 78 of 2034 large neurons were SP-immunoreactive (average of 3.8% 6 0.6%; n = 4 animals). In ovalbumin-sensitized animals, the number of large-diameter neurons positive for SP was substantially higher, 712 of 1939 (average of 36.7% 6 3.1%; n = 4 animals; P < .01; compared with unsensitized animals, Fig 1, C; part of these data was

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terminals and travels in membranous vesicles back to the cell soma by retrograde axoplasmic flow. Accordingly, it is extensively used in neurobiology to retrogradely backfill neurons. Guinea pigs were anesthetized with intramuscular injection of ketamine (50 mg/kg) and xylazine (2.5 mg/kg) and received two 1-mL (3%) transdermal injections of True Blue dye (in PBS containing 1% dimethyl sulfoxide; Molecular Probes) into the right lung using a 5-mL Hamilton syringe. One week after the retrograde labeling, the animals were exposed to aerosolized antigen as described. Twenty-four hours after antigen challenge, the animals were killed by intraperitoneal injection of an overdose of sodium pentobarbital (150 mg/kg) and perfused via the ascending aorta with a rinsing solution containing procaine (100 mg/mL) and heparin (10,000 IU/L) in PBS followed by fixative. Intrathoracic organs (lung, trachea, esophagus, heart, and surrounding soft tissues) and nodose ganglia were removed and put into fixative for 2 hours at 4°C, then rinsed 3 times in PBS. Lungs were cut in sections of 1 to 2 mm to identify the dye distribution in parenchyma, blood vessels, and airways. Trachea and esophagus were examined inside and outside the lumens. Ganglia from animals were used only if the dye was delimited primarily to the lung parenchyma and distal airways, with no dye diffusion into the large airways or other extrapulmonary sites. These nodose ganglia from the allergen challenged animals were prepared and sectioned as described. The alternate slides were examined, and all True Blue–labeled neurons were photographed to identify labeled cells in case some labeled neurons faded after immunostaining. The sections were double stained as described with the exception of using a rat mAb to SP (5 mg/mL diluted in TritonBSA-PBS; Chemicon) with the mouse mAb to 160-kd neurofilament (13 mg/mL diluted in Triton-BSA-PBS; Chemicon) for 24 hours at 4°C as primary antibodies, followed by a mixture of goat antirat antibody labeled with Alexa Fluor 594 (20 mg/ mL diluted in TritonBSA-PBS; Molecular Probes) and goat antimouse antibody labeled with Alexa Fluor 488 (10 mg/mL diluted in Triton-BSA-PBS; Molecular Probes) for 2 hours at room temperature as secondary antibodies. The slides were examined as described, except the True Blue was visualized with an UV filter (excitation filter 330-385 nm; barrier filter 400-420 nm). Only True Blue–labeled neurons with neurofilament-positive immunofluorescence were evaluated to determine whether allergic inflammation induces SP production. We tested both SP antibodies in the same ganglion sections and found there was no difference in staining (not shown).

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FIG 1. SP immunoreactivity (SP-IR) in nodose ganglion neurons 1 day after the antigen challenge with ovalbumin (OVA) inhalation in (A) unsensitized (control) and (B) sensitized guinea pigs. Representative photomicrographs of lung neurons labeled with True Blue (TB) dye with double immunofluorescence staining of SP and neurofilament (NF; 160-kd) antibodies (scale bar = 50 mm). C, Histogram showing the percentage of SP immunoreactivity in large-diameter NF-positive neurons in nodose ganglia after OVA challenge in unlabeled (n = 4), tracheal-labeled (n = 3), and lunglabeled (n = 8, unsensitized; n = 9, sensitized). Note, tracheal labeled neuron data taken from our previous study.8 Data are presented as the means 6 SEMs. *P < .01 between sensitized and unsensitized animals).

obtained from our previous study8 and is included here for comparison purposes).

Lung-labeling experiment To address more specifically the hypothesis that SP synthesis could be induced in intrapulmonary A-fiber neurons, we used a retrograde labeling strategy. The retrograde tracer True Blue was injected into right lung. One week after the injection, both actively sensitized and unsensitized (control) groups were challenged with inhaled ovalbumin. The animals were killed 1 day after antigen challenge, and the distribution of the dye was examined in the thoracic tissues. Thirty-two animals were used for this experiment. In 17 animals, we confirmed that the dye was delimited primarily to the lung parenchyma and distal airways, with no dye diffusion into the large airways or extrapulmonary sites. Fifteen animals were excluded from further analysis as a result of diffusion of the dye to the large airways and/or cardiac tissue. Among the successfully labeled animals, 9 animals were unsensitized (controls) and 8 were sensitized. All animals were challenged with ovalbumin, and 1 day later, the neurons were evaluated for SP immunoreactivity. In control animals, an average of 18% 6 4% of the large-diameter neurofilament-positive neurons were SPimmunoreactive. In sensitized animals challenged with ovalbumin, 30% 6 6% of large-diameter neurofilamentpositive neurons were SP-positive (P < .05 compared with control animals; Fig 1, C). Isolated ganglia studies We have previously noted that mast cells located within the nodose ganglia isolated from ovalbumin-sensitized

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FIG 2. Nodose ganglia (ex vivo) were treated ovalbumin (OVA; 10 mg/mL) or saline for the times indicated (n 5 1-2 for 0, 4, 8, 12 h and n 5 8 for 18 h). At 18 hours after OVA treatment, the percentage of SP immunoreactivity (SP-IR) is significantly increased in large-diameter (>25 mm) neurofilament (NF)–positive neurons (16% 6 2%; n = 8) compared with the control (2% 6 0.8%; n = 6). Data are presented as means 6 SEMs. *P < .01.

animals degranulate and release inflammatory mediators on antigen challenge.12 We therefore next addressed the question whether a local allergic reaction within the nodose ganglion is sufficient to induce SP expression in large-diameter neurofilament-positive neurons. The nodose ganglia were isolated from actively sensitized animals and exposed to 10 mg/mL ovalbumin or vehicle (Krebs bicarbonate solution) ex vivo. Ovalbumin effectively induced SP synthesis in neurofilament-positive neurons beginning at between 8 and 12 hours of exposure. After 18 hours of ovalbumin treatment, approximately 16 % of neurofilament-positive neurons were SP-positive (n = 8), compared with ;2% in the vehicle treated ganglia (n = 6; P < .01; Fig 2).

Vagotomy studies The data from the isolated ganglia studies suggest that if inhaled ovalbumin can reach the vagal sensory ganglia in sensitized animals, it may be capable of acting locally to induce SP synthesis in neurofilament-positive neurons. To address this question further, in vivo experiments were conducted in passively sensitized (or control) animals with unilateral vagotomy performed before ovalbumin inhalation. The induction of SP immunoreactivity in the nodose ganglion neurons was compared between the vagotomy side and contralateral side with intact vagus. If the allergen-inducing signal for SP synthesis travels to the cell body via the nerve fiber, then the induction will be noted only in the ganglia associated with an intact vagus nerve. In these studies, either the right or left vagus nerve was severed in a given animal. The data obtained were the same whether the right or left ganglia was studied, and therefore, the results were pooled. Fast Blue dye (25 mL) was instilled into cervical trachea 1 week before the antigen challenge. This procedure labeled both extrapulmonary and intrapulmonary compartments. All animals were then challenged with aerosolized ovalbumin. Twenty-four hours after the antigen challenge, the animals were killed, and labeled neurofilament-positive neurons were evaluated for the SP immunoreactivity.

Vagus nerve intact side. In unsensitized animals, 3% 6 1% (n = 4) of neurofilament-positive neurons expressed SP in nodose ganglia of an intact vagus nerve. As expected, in sensitized animals, the percentage of neurofilament-positive neurons expressing SP in nodose ganglia with intact vagus averaged 29% 6 3 % (n = 4; Fig 3). Vagus nerve severed side. In unsensitized animals, 9% 6 1% of the neurofilament-positive neurons were SPpositive in nodose ganglia on the side in which the vagus nerve was cut. Comparing these results with those observed in ganglia obtained from the side with an intact vagus (3% 6 1%) suggests that severing the vagus per se signals the induction of tachykinin synthesis in a subset of neurofilament-positive nodose neurons. In sensitized and challenged animals, there was a significant (P < .05) but slight increase in the percentage of the neurofilamentpositive neurons that were SP-positive in nodose ganglia on the side of the vagotomy (Fig 3). However, this apparent ovalbumin effect in ganglia ipsilateral to the vagotomy (1.8-fold increase) was significantly less than that observed on the vagus intact side (9.7-fold increase). DISCUSSION The results support the hypothesis that allergic inflammation in guinea pig lungs can lead to a switch in sensory SP innervation such that, in addition to sensory C fibers, SP is found in cough-causing A fibers in the trachea8 and in stretch-sensitive A fibers in the lungs (current study). In addition, the results are consistent with the hypothesis that this phenotypic switch in tachykinergic innervation requires signals that, at least in part, reach the cell bodies in the distant sensory ganglia via the vagal nerve fibers. In healthy mammals, most neuronal tachykinins (SP and neurokinin A) in the mammalian respiratory tract are found in sensory C fibers.1,2 The vagal C-fiber neurons in the guinea pig respiratory tract are derived from cell bodies situated in the jugular and nodose ganglia.2,3,16 The jugular C fibers, found in extrapulmonary airways and intrapulmonary bronchi, are more likely to contain SP than the nodose C fibers located within the lungs.16 Regardless of the type of C fiber, these nerves are typically not thought to be activated in healthy animals under normal circumstances. Rather, they are recruited to action by noxious stimuli or pathological conditions such as tissue inflammation.17 On activation of a tachykinin-containing nerve, the tachykinins are released into synapses in the central nervous system, where they serve to evoke pain in the somatosensory system4,18 and augment cough19,20 and parasympathetic reflexes in the respiratory system.21-24 When released from the peripheral nerve terminals via axon reflexes, the tachykinins may cause or contribute to local inflammatory reactions.25-27 Substance P is seldom expressed in large-diameter (A-fiber) sensory neurons innervating the trachea or lungs.2,3,28 The large-diameter neurons innervating the guinea pig respiratory system are located mainly in the nodose ganglia and project touch-sensitive and stretch-

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FIG 3. Histogram demonstrating the percentage of SP immunoreactivity (SP-IR) in Fast Blue–labeled neurofilament (NF)–positive nodose neurons in intact versus severed vagus nerves 1 day after the ovalbumin inhalation. Data are presented as means 6 SEMs; n = 4 per group. *P  .05.

sensitive A fibers to the trachea and lungs, respectively.3,10,16 Fischer et al7 were first to publish that allergic inflammation in guinea pig airways is associated with an increase in preprotachykinin gene transcription and SP synthesis in nodose ganglion neurons. Subsequently, we showed that this could occur in large-diameter neurofilament-positive neurons that projected low-threshold touchsensitive Ad fibers to the trachea.8 Further investigation indicated that tracheal nodose Ad fibers in the guinea pig represent a unique type of sensory nerve that evokes cough on activation.10 In the current study, we designed experiments to determine the effects of allergic inflammation in the lung on SP content of neurons that project A fibers to intrapulmonary structures (ie, RAR/SAR population). We injected small volumes of dye directly into the lung parenchyma and subsequently evaluated retrogradely labeled neurofilament-positive neurons. In the allergenchallenged animals, about 30% of the lung-specific neurofilament-positive neurons expressed SP immunoreactivity. This value is similar to that observed in tracheallabeled neurons. We have previously reported that under control conditions, very few (1% to 3%) of neurofilamentpositive neurons in the nodose ganglia express SP immunoreactivity.8 Likewise, Kummer et al2 have reported that nodose neurons labeled from healthy guinea pig lungs are essentially all SP-negative. We were therefore surprised by our finding that 18% of the neurofilament-positive neurons labeled from the lung were SP-positive in the control animals. We suspect that this may be a result of some inflammation caused by our intraparenchymal injections of the dye itself. In any event, the results support the hypothesis that allergic inflammation can lead to induction of tachykinin synthesis in stretch-sensitive A fibers within the lungs. In our previous electrophysiological analysis of nodose A fibers projecting to the guinea pig lungs, we found that they represented a rather homogenous population of fastconducting stretch-sensitive nerves.16 All of the fibers had conduction velocities in the range of 9 to 25 m/s and could be segregated on the basis of their adaptation to prolonged lung inflation into either RAR or SAR phenotypes. The finding that 30% of nodose A-fiber neurons express SP after allergen challenge indicates that SP production can

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be induced in some vagal stretch-sensitive nerves. This raises the possibility that SP could be released from their central terminals in the brain stem during breathing, independently of noxious stimuli. This would be analogous to the phenotypic switch described in touch-sensitive Ab fibers of the somatosensory system, in which such an effect has been suggested to underlie sensations of allodynia.4 How allergic inflammation causes increased SP immunoreactivity in nodose neurons is not known. By using a similar model system, Fischer et al7 provided direct evidence that allergen-induced increase in tachykinin synthesis occurs at the level of preprotachykinin gene transcription in the cell nucleus. We considered 2 pathways by which inhaled allergen can influence gene transcription in the cell bodies located at a distant vagal sensory ganglion. First, the allergen could reach the systemic circulation and activate mast cells within the ganglion, causing a local effect on the neighboring neurons. Second, the allergic inflammation within the airway wall could influence the nerve terminals in a manner that sends long-distance signals to the cell nucleus via the afferent nerve fiber. When the nodose ganglia isolated from sensitized guinea pig are subsequently exposed to the sensitizing antigen, ganglionic mast cells degranulate, mast cell– associated mediators are released, and the electrical excitability of resident neurons is altered.12 In the current study, we found that antigen administered to the nodose ganglia ex vivo caused a ;10-fold increase in the percentage of SP-expressing neurofilament-positive neurons. This effect is presumably secondary to the activation of resident mast cells, although this hypothesis was not further addressed. On the basis of these findings, it can be surmised that if inhaled ovalbumin reached the systemic circulation in sufficient quantities, it could affect SP synthesis in nodose neurons via a local activation of ganglionic mast cells. The experiment in which the vagus nerve was unilaterally severed 1 day before the allergen inhalation more directly addresses this issue. Regrettably, severing the nerve itself appeared to increase the SP production in neurofilament-positive neurons to nearly 10% (compared with 3% with the vagus nerve intact). This is consistent with the findings of Noguchi et al,29,30 who reported that severing the peripheral fibers in vivo in the somatosensory system leads to the induction of neuropeptides production in dorsal root ganglion neurons. This vagotomy-induced increase in SP immunoreactivity in neurons clouds the interpretation of the data. Nevertheless, allergen inhalation caused the expected robust increase (9.7-fold) in the percentage of neurofilament-positive, SP-positive nodose neurons only in the side with the vagus nerve intact. On the side in which the vagus was severed, ovalbumin inhalation was associated with only a marginal increase (1.8-fold) in the percentage of neurofilament-positive neurons expressing SP. This is the expected result if the site of action of the allergen is within the lung, but it would not be expected if the allergen were acting at the level of the sensory ganglia.

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Also supporting the hypothesis that the site of initiation of the phenotypic switch is within the lung is the finding that the effect of allergic inflammation on SP induction in large-diameter neurons can be mimicked with respiratory tract viral infection.9 The virus infection in this model is thought not to extend beyond the airway epithelium. The small ovalbumin-induced effect observed in nodose neurons ipsilateral to the vagotomy may have been a result of the fact that the vagus was cut caudal to the point where the superior laryngeal nerve meets the vagus. Thus, the pathway remained intact for that population of A fibers that project to the larynx and pharynx via the superior laryngeal nerve. The question remains as to the nature of the chemical mediators capable of being released after allergic inflammation that can lead to long-distance nuclear signaling via the vagal axons. On the basis of the literature, it seems likely that a neurotrophin molecule such as NGF may be causally involved.31 Neurotrophins and their Trk receptors are, in fact, designed to signal gene transcriptional events from interactions occurring at distant terminals.11 In addition, NGF is associated with allergic reactions,32 and we have previously reported that microinjection of NGF into the tracheal wall leads to SP production in largediameter neurofilament-positive nodose neurons innervating the trachea.33 Similar results have also been observed in the mouse.34 We thank Ms Holly K. Rohde for her technical assistance.

REFERENCES 1. Lundberg JM, Hokfelt T, Martling CR, Saria A, Cuello C. Substance Pimmunoreactive sensory nerves in the lower respiratory tract of various mammals including man. Cell Tissue Res 1984;235:251-61. 2. Kummer W, Fischer A, Kurkowski R, Heym C. The sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry. Neuroscience 1992;49:715-37. 3. Riccio MM, Kummer W, Biglari B, Myers AC, Undem BJ. Interganglionic segregation of distinct vagal afferent fibre phenotypes in guineapig airways. J Physiol 1996;496:521-30. 4. Neumann S, Doubell TP, Leslie T, Woolf CJ. Inflammatory pain hypersensitivity mediated by phenotypic switch in myelinated primary sensory neurons. Nature 1996;384:360-4. 5. Ma QP, Woolf CJ. Progressive tactile hypersensitivity: an inflammationinduced incremental increase in the excitability of the spinal cord. Pain 1996;67:97-106. 6. Lilly CM, Bai TR, Shore SA, Hall AE, Drazen JM. Neuropeptide content of lungs from asthmatic and nonasthmatic patients. Am J Respir Crit Care Med 1995;151:548-53. 7. Fischer A, McGregor GP, Saria A, Philippin B, Kummer W. Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation. J Clin Invest 1996;98: 2284-91. 8. Myers AC, Kajekar R, Undem BJ. Allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways. Am J Physiol Lung Cell Mol Physiol 2002;282:L775-81. 9. Carr MJ, Hunter DD, Jacoby DB, Undem BJ. Expression of tachykinins in nonnociceptive vagal afferent neurons during respiratory viral infection in guinea pigs. Am J Respir Crit Care Med 2002;165:1071-5. 10. Canning BJ, Mazzone SB, Meeker SN, Mori N, Reynolds SM, Undem BJ. Identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea-pigs. J Physiol 2004;557:543-58.

11. Neet KE, Campenot RB. Receptor binding, internalization, and retrograde transport of neurotrophic factors. Cell Mol Life Sci 2001;58:1021-35. 12. Undem BJ, Hubbard W, Weinreich D. Immunologically induced neuromodulation of guinea pig nodose ganglion neurons. J Auton Nerv Syst 1993;44:35-44. 13. Undem BJ, Buckner CK, Harley P, Graziano FM. Smooth muscle contraction and release of histamine and slow-reacting substance of anaphylaxis in pulmonary tissues isolated from guinea pigs passively sensitized with IgG1 or IgE antibodies. Am Rev Respir Dis 1985;131: 260-6. 14. Hunter DD, Undem BJ. Identification and substance P content of vagal afferent neurons innervating the epithelium of the guinea pig trachea. Am J Respir Crit Care Med 1999;159:1943-8. 15. Lawson SN, Perry MJ, Prabhakar E, McCarthy PW. Primary sensory neurones: neurofilament, neuropeptides, and conduction velocity. Brain Res Bull 1993;30:239-43. 16. Undem BJ, Chuaychoo B, Lee MG, Weinreich D, Myers AC, Kollarik M. Subtypes of vagal afferent C-fibres in guinea-pig lungs. J Physiol 2004;556:905-17. 17. Coleridge JC, Coleridge HM. Afferent vagal C fibre innervation of the lungs and airways and its functional significance. Rev Physiol Biochem Pharmacol 1984;99:1-110. 18. Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science 2000;288:1765-9. 19. Mazzone SB. Sensory regulation of the cough reflex. Pulm Pharmacol Ther 2004;17:361-8. 20. Bonham AC, Sekizawa SI, Joad JP. Plasticity of central mechanisms for cough. Pulm Pharmacol Ther 2004;17:453-7. 21. Carr MJ, Undem BJ. Inflammation-induced plasticity of the afferent innervation of the airways. Environ Health Perspect 2001;109(suppl 4):567-71. 22. Myers AC, Undem BJ. Functional interactions between capsaicinsensitive and cholinergic nerves in the guinea pig bronchus. J Pharmacol Exp Ther 1991;259:104-9. 23. Rumsey WL, Aharony D, Bialecki RA, Abbott BM, Barthlow HG, Caccese R, et al. Pharmacological characterization of ZD6021: a novel,

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orally active antagonist of the tachykinin receptors. J Pharmacol Exp Ther 2001;298:307-15. Canning BJ, Fischer A. Neural regulation of airway smooth muscle tone. Respir Physiol 2001;125:113-27. Groneberg DA, Quarcoo D, Frossard N, Fischer A. Neurogenic mechanisms in bronchial inflammatory diseases. Allergy 2004;59: 1139-52. Barnes PJ. Neurogenic inflammation in the airways. Respir Physiol 2001; 125:145-54. Lundberg JM. Tachykinins, sensory nerves, and asthma: an overview. Can J Physiol Pharmacol 1995;73:908-14. Springall DR, Cadieux A, Oliveira H, Su H, Royston D, Polak JM. Retrograde tracing shows that CGRP-immunoreactive nerves of rat trachea and lung originate from vagal and dorsal root ganglia. J Auton Nerv Syst 1987;20:155-66. Noguchi K, Dubner R, De Leon M, Senba E, Ruda MA. Axotomy induces preprotachykinin gene expression in a subpopulation of dorsal root ganglion neurons. J Neurosci Res 1994;37:596-603. Noguchi K, Kawai Y, Fukuoka T, Senba E, Miki K. Substance P induced by peripheral nerve injury in primary afferent sensory neurons and its effect on dorsal column nucleus neurons. J Neurosci 1995;15: 7633-43. Wilfong ER, Dey RD. Nerve growth factor and substance P regulation in nasal sensory neurons after toluene diisocyanate exposure. Am J Respir Cell Mol Biol 2004;30:793-800. Bonini S, Lambiase A, Levi-Schaffer F, Aloe L. Nerve growth factor: an important molecule in allergic inflammation and tissue remodelling. Int Arch Allergy Immunol 1999;118:159-62. Hunter DD, Myers AC, Undem BJ. Nerve growth factor-induced phenotypic switch in guinea pig airway sensory neurons. Am J Respir Crit Care Med 2000;161:1985-90. Dinh QT, Groneberg DA, Peiser C, Springer J, Joachim RA, Arck PC, et al. Nerve growth factor-induced substance P in capsaicin-insensitive vagal neurons innervating the lower mouse airway. Clin Exp Allergy 2004;34:1474-9.

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Differential effects of (S)- and (R)-enantiomers of albuterol in a mouse asthma model William R. Henderson, Jr, MD, Ena Ray Banerjee, PhD, and Emil Y. Chi, PhD Seattle, Wash

Mechanisms of asthma and allergic inflammation

Background: (R)- and (S)-Enantiomers of albuterol likely exert differential effects in patients with asthma. The (R)-enantiomer binds to the b2-adrenergic receptor with greater affinity than the (S)-enantiomer and is responsible for albuterol’s bronchodilating activity. (S)-Albuterol augments bronchospasm and has proinflammatory actions. Objective: The study aim was to determine whether the (S)-enantiomer, in contrast to the (R)-enantiomer, has adverse effects on allergic airway inflammation and hyperresponsiveness in a mouse asthma model. Methods: Mice sensitized to ovalbumin (OVA) intraperitoneally on days 0 and 14 were challenged with OVA intranasally on days 14, 25, and 35. On day 36, 24 hours after the final allergen challenge, the effect of the (R)- and (S)-enantiomers of albuterol (1 mg
kg21
d21 administered by means of a miniosmotic pump from days 13-36) on airway inflammation and hyperreactivity was determined. Results: In OVA-sensitized/OVA-challenged mice, (R)-albuterol significantly reduced the influx of eosinophils into the bronchoalveolar lavage fluid and airway tissue. (R)-Albuterol also significantly decreased airway goblet cell hyperplasia and mucus occlusion and levels of IL-4 in bronchoalveolar lavage fluid and OVA-specific IgE in plasma. Although (S)-albuterol significantly reduced airway eosinophil infiltration, goblet cell hyperplasia, and mucus occlusion, it increased airway edema and responsiveness to methacholine in OVA-sensitized/ OVA-challenged mice. Allergen-induced airway edema and pulmonary mechanics were unaffected by (R)-albuterol. Conclusion: Both (R)- and (S)-enantiomers of albuterol reduce airway eosinophil trafficking and mucus hypersecretion in a mouse model of asthma. However, (S)-albuterol increases allergen-induced airway edema and hyperresponsiveness. These adverse effects of the (S)-enantiomer on lung function might limit the clinical efficacy of racemic albuterol. (J Allergy Clin Immunol 2005;116:332-40.)

From the Departments of Medicine and Pathology, University of Washington. Supported by National Institutes of Health grants AI04989 and HL073722 and by a grant from Sepracor Inc. Disclosure of potential conflict of interest: E. Chi and E. R. Banerjee—none disclosed. W. R. Henderson, Jr, receives grants–research support from Sepracor, Inc. Received for publication August 4, 2004; revised April 1, 2005; accepted for publication April 12, 2005. Available online June 1, 2005. Reprint requests: William R. Henderson, Jr, MD, Department of Medicine, Center for Allergy and Inflammation, Box 358050, University of Washington, 815 Mercer Street, Seattle, WA 98109. E-mail: joangb@ u.washington.edu. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.013

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Key words: b2-adrenergic agonist enantiomers, airways, mucus, edema, inflammation, hyperresponsiveness

Adrenergic receptors are composed of a- and breceptors that bind endogenous catecholamines, such as epinephrine. Although 3 subtypes of b-adrenergic receptors exist, smooth muscle relaxation producing vasodilation and bronchodilation is mediated by the b2-receptor. Short-acting b2-adrenergic receptor agonists rapidly induce bronchodilation in patients with asthma and are used for relief of acute symptoms, prevention of exerciseinduced asthma, and management of acute severe asthma. Racemic albuterol contains equal concentrations (50:50) of the (R)- and (S)-enantiomers (ie, enantiomers that are nonsuperimposable mirror images).1 The (R)enantiomer of albuterol binds to b2-adrenergic receptors with nearly 100-fold greater affinity than the (S)-enantiomer, suggesting that the (S)-enantiomer does not act through b-adrenergic receptor activation.1 Whereas the (R)-enantiomer of albuterol (levalbuterol) exerts the bronchodilating properties of albuterol, the (S)-enantiomer has adverse effects, including augmentation of bronchospasm and proinflammatory activities.2-5 In murine mast cells (S)-albuterol increases IgE-induced histamine and IL-4 production, whereas the (R)-enantiomer lacks these effects.2 Anti-inflammatory effects of (R)-albuterol, such as inhibition of T-cell proliferation, might be negated by the presence of the (S)-enantiomer.3 Differential effects of the enantiomers might result from differences in pharmacokinetics.1 The initial step in the metabolism of the (S)- and (R)-enantiomers is sulfate conjugation, a stereospecific process in human airway epithelial cells and other cells and tissues.6 The greater rate of sulfate conjugation of (R)-albuterol might lead to lower plasma levels of (R)- than (S)-albuterol in human subjects.7 Potential adverse effects of (S)-albuterol on asthma control might also be augmented by increased binding to lung tissue. In this study we characterized the effects of the (R)- and (S)-enantiomers of albuterol on allergic airway inflammation and hyperresponsiveness in a mouse asthma model that mimics key features of human asthma.8 Although prior studies in guinea pigs and human subjects have demonstrated that the (S)-enantiomer of albuterol can induce airway hyperreactivity, there are no prior studies examining the effect of (S)-albuterol versus (R)-albuterol on both airway hyperresponsiveness and the TH2 phenotype (ie, allergen-induced airway eosinophil trafficking, mucus metaplasia, edema, and TH2 cytokine release) in an in vivo asthma model. We report that both

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(R)- and (S)-enantiomers reduce allergen-induced airway eosinophil and mucus gland hyperplasia. However, only (S)-albuterol increases airway edema and responsiveness to methacholine, effects that would decrease the therapeutic efficacy of racemic albuterol.

METHODS Study protocol All animal use procedures were approved by the University of Washington Animal Care Committee. Female BALB/c mice (6-8 weeks of age; The Jackson Laboratory, Bar Harbor, Me) received an intraperitoneal injection of 100 mg of ovalbumin (OVA; 0.2 mL of 500 mg/mL) complexed with alum on days 0 and 14 (Fig 1). Mice were anesthetized with 0.2 mL of ketamine (6.5 mg/mL)/xylazine (0.44 mg/mL) in normal saline administered intraperitoneally before receiving an intranasal dose of 50 mg of OVA (50 mL of 1 mg/mL) on days 14, 25, and 35 (Fig 1). The control group received 0.2 mL of normal saline with alum administered intraperitoneally on days 0 and 14 and 0.4 mL of saline without alum administered intranasally on days 14, 25, and 35. In both the saline- and OVA-treated groups, miniosmotic pumps (200 mL, Alzet Model 2004; Durect Corp, Cupertino, Calif) containing either (R)- or (S)-albuterol (1 mg
kg21
d21, 6 mL/d delivery administration) were inserted subcutaneously on the back slightly posterior to the scapulae on day 13 and remained in place until study conclusion on day 36 (Fig 1). Absorption of the compounds by local capillaries results in systemic administration. Each study group consisted of 4 to 6 animals. The 1 mg
kg21
d21 dose of albuterol enantiomer infusion was selected on the basis of prior work by Sartori et al,9 demonstrating that continuous release of racemic albuterol (2 mg
kg21
d21) subcutaneously by means of miniosmotic pump produced steady-state, high-plasma levels of albuterol (1025 M) in mice.

Pulmonary function testing In vivo airway responsiveness to methacholine was determined on day 36 in conscious, freely moving, spontaneously breathing mice by using whole-body plethysmography (Model PLY 3211; Buxco Electronics Inc, Sharon, Conn), as described by Hamelmann et al.10 Mice were challenged with aerosolized saline or increasing doses (2 and 10 mg/mL) of methacholine generated by an ultrasonic nebulizer (DeVilbiss Health Care, Inc, Somerset, Pa) for 2 minutes. The degree of bronchoconstriction was expressed as enhanced pause (Penh), a calculated dimensionless value that correlates with measurement of airway resistance, impedance, and intrapleural pressure.9-11 Penh readings were taken and averaged for 4 minutes after each nebulization challenge. Penh is calculated as follows: Penh ¼ ½ðTe =Tr 21Þ3ðPEF=PIFÞ, where Te is expiration time, Tr is relaxation time, PEF is peak expiratory flow, and PIF is peak inspiratory flow 3 0.67 coefficient. The time for the box pressure to change from a maximum to a user-defined percentage of the maximum represents the relaxation time. The Tr measurement begins at the maximum box pressure and ends at 40%. Because Penh is the ratio of measurements obtained during the same breath, it is mainly

independent of functional residual capacity, tidal volume, and respiratory rate.

Light microscopy-morphometry After pulmonary function testing, bronchoalveolar lavage (BAL) was performed on the right lung, with total BAL fluid cells counted and eosinophils identified by means of eosin staining.12 Left lung tissue was obtained for histopathology, and plasma was obtained for OVA-specific IgE levels. Ten lung sections per animal were randomly selected and examined in a blinded manner. Sections were stained with hematoxylin and eosin, the total inflammatory cell infiltrate was assessed on a semiquantitative scale (0-41), the number of eosinophils per unit of airway area (2200 mm2) was determined by using a point-counting system (Image-Pro Plus point-counting system software, Version 1.2 for Windows; Media Cybernetics, Silver Spring, Md),12 and interstitial and perivascular airway edema were assessed.13,14 Airway goblet cells (as a percentage of total airway cells) were identified by means of Alcian blue staining,12 and the degree of mucus plugging of the airways (0.5 mm to 0.8 mm in diameter) with the percentage occlusion of the airway diameter was classified on a 0 to 41 scale on the basis of the following criteria: 0, no mucus; 1, approximately 10% occlusion; 2, approximately 30% occlusion; 3, approximately 50% occlusion; 4, greater than approximately 80% occlusion.12

Cytokine assays IL-4, IL-5, IL-10, GM-CSF, TNF-a, IL-2, and IFN-g were assayed in BAL fluid with Bio-Plex Mouse Cytokine assays (BioRad Laboratories, Hercules, Calif) that are bead-based multiplex sandwich immunoassays with a limit of detection of less than 10 pg/ mL. IL-13 was assayed in BAL fluid with a mouse IL-13 immunoassay (Quantikine M; R&D Systems, Minneapolis, Minn), with a limit of detection of less than 1.5 pg/mL.

OVA-specific IgE assay OVA-specific IgE was assayed by modification of the method of Iio et al.15 Nunc 96-well flat-bottom plates (Nalge Nunc International, Rochester, NY) were coated with 50 mg/mL OVA in 13 PBS overnight at room temperature, washed 3 times with 13 PBS plus 0.05% Tween-20 (wash buffer), blocked with 3% BSA in 13 PBS for 1 hour at room temperature, and washed 4 times with wash buffer. Fifty-microliter plasma samples (1:1 in 13 PBS) were added per well and incubated for 90 minutes at 37°C, then washed 4 times with wash buffer, and blotted dry by inverting over paper towels. One hundred microliters (1:100 in 13 PBS) of biotinconjugated rat anti-mouse IgE mAb (clone R35-72; BD Biosciences, San Diego, Calif) was added to each well and incubated overnight at 4°C and then washed 4 times with wash buffer and blotted dry. Then 100 mL per well (1:1000 in 13 PBS) streptavidin-horseradish peroxidase–conjugated secondary antibody (BD Biosciences) was added, and samples were incubated at 37°C for 90 minutes, then washed 4 times with wash buffer, and blotted dry. One hundred microliters of substrate solution (ie, 1 tablet of 2,2#-azinobis [3ethylbenzthiazoline-sulfonic acid, ABTS; Sigma Chemical Co, St Louis, Mo] dissolved in 100 mL of 0.05 M phosphate-citrate buffer, pH 5.0, and 25 mL of 30% H2O2) was added per well, and color was developed for 30 minutes at room temperature. OD 405 nm was measured by using an AD 340C absorbance detector (Beckman Coulter Inc, Fullerton, Calif). For the IgE standard curve, a sandwich ELISA was used in which in separate assay plates biotin-conjugated rat anti-mouse IgE mAb (clone R35-72, BD Biosciences) was used to coat the wells, and instead of plasma samples, known concentrations of purified anti-mouse IgE (clone C38-2, BD Biosciences)

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Abbreviations used AP-1: Activator protein 1 BAL: Bronchoalveolar lavage OVA: Ovalbumin Penh: Enhanced pause

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FIG 1. Study protocol. i.p., Intraperitoneal; i.n., intranasal

were incubated; assays were run as described above. The standard curve was constructed by using a linear regression analysis of the absorbances against serial dilutions of known concentrations of mouse IgE. Pooled mouse plasma from OVA-sensitized/OVAchallenged mice was used as a positive control.

Statistical analysis The data are reported as the means 6 SE of the combined experiments. Differences were analyzed for significance (P < .05) by means of ANOVA with the protected least-significant-difference method (Statview II; Abacus Concepts, Berkeley, Calif).

RESULTS Effect of (R)- and (S)-enantiomers of albuterol on allergen-induced airway inflammation Airway infiltration by eosinophils. A marked infiltration of inflammatory cells that were predominantly eosinophils around the airways and pulmonary blood vessels was observed in the lung interstitium of OVAtreated mice (Fig 2, B) compared with that seen in salinetreated control mice (Fig 2, A) on day 36, 24 hours after the last intranasal OVA or saline challenge. By means of morphometric analysis (Fig 3, A and B) of the histologic sections (Fig 2, C and D vs B), administration of (R)and (S)-albuterol by means of miniosmotic pumps (1 mg
kg21
d21 dose from days 13-36) significantly decreased the influx of total inflammatory cells (P = .0035, R-albuterol/OVA vs OVA; P = .0226, S-albuterol/ OVA vs OVA; Fig 3, A) and eosinophils (P = .021, Ralbuterol/OVA vs OVA; P = .008; S-albuterol/OVA vs OVA; Fig 3, B) into the lung interstitium. Compared with the saline group (Fig 3, C), OVA-sensitized/OVA-challenged mice exhibited a marked increase in BAL fluid eosinophils to 2.5 6 0.5 3 105 eosinophils/mL (P < .0001, OVA vs saline; Fig 3, C), which represented 41.8% of total BAL fluid cells. In OVA-sensitized/OVA-challenged mice, treatment with (R)-albuterol significantly inhibited the influx of eosinophils into BAL fluid by 40.6% (P = .0043, R-albuterol/OVA vs OVA; Fig 3, C). In contrast, (S)-albuterol had no significant effect on eosinophil influx into the BAL fluid of OVA-treated mice (Fig 3, C). Airway mucus hypersecretion. Hyperplasia of airway goblet cells and hypersecretion of mucus were observed in OVA-treated mice (Fig 2, B) compared with in control

mice (Fig 2, A). Airway goblet cells increased to 38.0% of total airway cells in OVA-treated mice compared with 0.4% in saline control mice (P = .0001, OVA vs saline; Fig 4, A). The mucus occlusion of the airway diameter morphometric score increased 15-fold in the OVA-treated mice compared with control mice (P < .0001, OVA vs saline; Fig 4, B). Allergen-induced goblet cell hyperplasia and mucus occlusion of airway diameter were inhibited by both the (R)-enantiomer (Fig 2, C) and (S)-enantiomer (Fig 2, D) of albuterol. By means of morphometric analysis, (R)-albuterol decreased goblet cell hyperplasia by 48.9% (P = .0066, R-albuterol/OVA vs OVA; Fig 4, A) and airway mucus occlusion by 41.4% (P = .0042, R-albuterol/OVA vs OVA; Fig 4, B). (S)-Albuterol reduced goblet cell hyperplasia by 44.8% (P = .0095, S-albuterol/OVA vs OVA; Fig 4, A) and mucus occlusion of the airways by 35.7% (P = .0088, S-albuterol/OVA vs OVA; Fig 4, B). Airway edema. Airway edema was observed in the lungs of OVA-treated mice (Fig 2, B) compared with that seen in saline-treated control mice (Fig 2, A). (S)-Albuterol markedly increased airway edema in OVAtreated mice (Fig 2, D vs B). In contrast, (R)-albuterol had no effect on allergen-induced edema in the airways of OVA-treated mice (Fig 2, C). Cytokine release. Significant levels (P < .05) of IL-4, IL-5, IL-13, and GM-CSF were found in the BAL fluid of OVA-treated mice compared with levels in the saline control group (Fig 5). The increased levels of IL-4 in OVA-treated mice were reduced 70.5% and 52.2% by (R)-albuterol and (S)-albuterol, respectively; the reduction was statistically significant only for the (R)-enantiomer (P = .043, R-albuterol/OVA vs OVA; Fig 5). There was no significant effect of either the (R)- or (S)-enantiomer of albuterol on the increased BAL fluid levels of IL-5, IL-13, and GM-CSF in the OVA-treated animals. The levels of IL-10, TNF-a, IL-2, and IFN-g were not significantly increased in the BAL fluid of OVA-treated mice compared with levels seen in saline-treated control mice; neither enantiomer affected the levels of these cytokines in OVAtreated animals (Fig 5). OVA-specific IgE. Plasma OVA-specific IgE was absent in saline-treated control mice and present in OVA-treated control mice (Fig 6). (R)-Albuterol and (S)-albuterol reduced OVA-specific IgE levels in OVAtreated mice; the reduction was statistically significant

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FIG 2. Effect of (R)- and (S)-albuterol enantiomers on airway histopathology in a mouse asthma model. Lung tissue was obtained on day 36 from saline-treated control animals (A), OVA-treated control animals (B), OVAtreated mice administered (R)-albuterol (C), and OVA-treated mice administered (S)-albuterol (D), and sections were stained with hematoxylin and eosin. Arrows indicate eosinophils and other inflammatory cells, arrowheads indicate mucus, and asterisks indicate edema. AW, Airway; BV, blood vessel. Bars = 100 mm.

only for the (R)-enantiomer (P = .0120, R-albuterol/OVA vs OVA; Fig 6).

Effect of (S)- and (R)-enantiomers of albuterol on allergen-induced airway hyperresponsiveness Pulmonary mechanics were assessed in response to aerosolized methacholine by means of noninvasive in vivo plethysmography on day 36, 24 hours after the last intranasal OVA challenge. The OVA-sensitized mice

had been challenged with 3 intranasal doses of OVA, a protocol that we have previously shown to induce airway inflammation and mucus hypersecretion but that is suboptimal for inducing airway hyperresponsiveness.16,17 This protocol was used because an augmenting effect of (S)-albuterol on airway hyperresponsiveness could have been masked in our mouse asthma model protocol, in which bronchial hyperresponsiveness is achieved through administration of 4 intranasal doses of OVA in mice sensitized by 2 intraperitoneal OVA doses.16,17

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Mechanisms of asthma and allergic inflammation FIG 3. Effect of (R)- and (S)-albuterol enantiomers on allergen-induced airway inflammatory cell infiltration. The total inflammatory cell infiltration of the airways (A), the number of eosinophils per unit area (2200 mm2) of lung tissue (B), and eosinophils per milliliter of BAL fluid (C) were determined. *P < .05 versus OVA.

In OVA-treated mice (S)-albuterol significantly increased bronchial responsiveness to methacholine (Fig 7). In contrast, (R)-albuterol did not alter airway responsiveness to methacholine in OVA-sensitized/OVA-challenged mice (Fig 7).

Effect of (R)- and (S)-enantiomers of albuterol in non–OVA-sensitized/OVA-challenged mice Miniosmotic pumps containing either (R)- or (S)albuterol were placed subcutaneously in saline control animals for a 24-day treatment period before pulmonary function testing and assessment of lung histopathology (Fig 8) to examine the effect of the albuterol enantiomers independently of a modification of the allergic inflammatory response. Lung morphology. The (S)-enantiomer of albuterol did not induce airway edema independently of an allergic response. No airway edema or inflammation was seen in saline-treated mice administered either (S)-albuterol (Fig 8, B) or (R)-albuterol (Fig 8, C). Pulmonary mechanics. The (S)-enantiomer of albuterol did not affect airway reactivity in non–OVA-sensitized/ OVA-challenged mice. At 0, 2, and 10 mg/mL methacholine challenge doses, Penh (percentage of air) values were, respectively, 105.1%, 92.3%, and 100.0% of the values of saline control animals. Similarly, (R)-albuterol

had no effect on Penh (percentage of air) values of the saline control group. At 0, 2, and 10 mg/mL methacholine doses, non–OVA-sensitized/OVA-challenged mice administered (R)-albuterol had 106.4%, 95.7%, and 101.1% Penh (percentage of air) values of the saline control animals.

DISCUSSION In this mouse model of asthma, we found both overlapping and distinct actions of the (S)- and (R)-enantiomers of albuterol on key features of allergen-induced airway inflammation and responsiveness to methacholine. (R)-Albuterol significantly reduced the following features of allergen-induced airway inflammation: BAL fluid levels of IL-4 and eosinophils, airway eosinophil infiltration, goblet cell hyperplasia, and mucus occlusion and circulating levels of OVA-specific IgE. Although (S)albuterol also decreased airway tissue eosinophilia, goblet cell hyperplasia, and mucus plugging of the airways, no significant effect on the influx of eosinophils into the BAL fluid was observed. (S)-Albuterol, but not (R)-albuterol, had adverse effects on airway function by increasing airway edema and hyperresponsiveness in OVA-treated mice.

Henderson, Banerjee, and Chi 337

FIG 4. Effect of (R)- and (S)-albuterol enantiomers on allergen-induced airway mucus hypersecretion. The number of goblet cells (A) and mucus occlusion of airway diameter (B) were determined. *P < .05 versus OVA.

FIG 5. Effect of (R)- and (S)-albuterol enantiomers on BAL fluid cytokine levels in OVA-treated mice. BAL fluid was assayed for TH1 and TH2 cytokines. *P < .05 versus OVA.

We found in this mouse asthma model that both (S)- and (R)-albuterol inhibited infiltration of eosinophils into airway tissue, but only (R)-albuterol significantly reduced eosinophil influx into BAL fluid. Although a correlation between BAL fluid levels of IL-5 and eosinophilia is typically seen, there was no reduction in the increased IL-5 levels of OVA-treated mice administered either albuterol enantiomer. Short- and long-acting b2-adrenergic agonists might facilitate eosinophil apoptosis, thereby reducing airway eosinophilia.18 Albuterol (0.1-10 mM) in vitro dose dependently decreases colony numbers and increases apoptosis of eosinophil progenitor cells from the blood of patients with asthma.19 In contrast, racemic albuterol, (R)-albuterol, and (S)-albuterol do not affect apoptosis of antigen-specific human T-cell lines.3 An unexpected finding of this study was the reduction in airway goblet cell hyperplasia and mucus hypersecretion by both the (R)- and (S)-enantiomers of albuterol. Limited data exist regarding the effect of (R)- and (S)-enantiomers of albuterol on airway mucus gland

function. Sympathetic (ie, adrenergic), parasympathetic (ie, cholinergic), and sensory-efferent (ie, tachykininmediated) pathways regulate mucus secretion from airway epithelial goblet cells and submucosal glands.20 In human airways the cholinergic response is predominant and is mediated by muscarinic M3-receptors on the mucus secretory cells.20 In ovine tracheal epithelial cells (R)-albuterol increased ciliary beat frequency, whereas (S)-albuterol had no significant effect.21 Increased mucociliary clearance rates by b2-adrenergic agonists have been reported in some patients with asthma.22 The TH2 cytokines IL-4 and IL-13 have potent effects on mucus secretion.23,24 Administration of each cytokine independently stimulates airway mucus accumulation in mice.24 MUC5AC gene expression and BAL fluid mucus protein release are increased in IL-4 transgenic mice.23 In addition, inhibition of IL-4 by administration of soluble IL-4 receptor reduces airway mucus hypersecretion and inflammatory cell trafficking to the lungs in OVA-treated mice.25 We found that the increased BAL fluid levels of IL-4, but not IL-13,

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FIG 6. (R)-Albuterol decreases OVA-specific IgE levels in OVA-treated mice. Plasma OVA-specific IgE levels were determined. *P < .05 versus OVA.

FIG 7. (S)-Albuterol increases allergen-induced airway hyperresponsiveness. The degree of bronchoconstriction to aerosolized methacholine (0, 2, and 10 mg/mL) was expressed as Penh (percentage of air as control). *P < .05 versus OVA.

in OVA-treated mice were significantly reduced by (R)-albuterol, with a trend toward IL-4 reduction by (S)-albuterol. Thus the albuterol enantiomers might modulate allergen-induced airway inflammation and mucus hypersecretion through IL-4, rather than IL-5 or IL-13, signaling. We have recently demonstrated a similar discordance between IL-4 and IL-5/IL-13 in mediation of allergen-induced airway eosinophilia and mucus hypersecretion.14 In a mouse asthma model the selective redox effector factor 1 inhibitor PNRI-299, which inhibits the transcription factor activator protein-1 (AP-1), significantly decreased airway eosinophil infiltration and mucus occlusion and lung gene expression of IL-4 but not IL-5 or IL-13.14 During T-cell activation, a complex interaction between nuclear factor of activated T cells and AP-1 is necessary for inducible expression of IL-4.26 In contrast, transcriptional regulation of IL-5 and IL-13 might be independent of AP-1 binding.27 The induction of goblet cell hyperplasia and eosinophilia by IL-4 in triple IL-5/IL-9/IL-13–knockout mice further demonstrates the key role IL-4 exerts in the development of the TH2 phenotype.28 In our studies the reduction in IL-4 in OVA-treated mice administered (R)-albuterol correlated with a decrease in circulating OVA-specific IgE. We found that (S)-albuterol, but not (R)-albuterol, augmented the interstitial edema observed in OVA-treated

mice, suggesting a proinflammatory effect unique to (S)-albuterol. This effect of (S)-albuterol was not observed in nonsensitized/nonchallenged mice. In 2 sheep models of altered lung fluid balance, the effect of aerosolized racemic albuterol and its (R)- and (S)-enantiomers on lung epithelial permeability has been examined.29 Pretreatment with (S)-albuterol increased the level of albumin in the epithelial lining fluid in sheep receiving histamine to increase lung permeability. This effect was not observed after pretreatment with either (R)-albuterol or its racemate. In sheep receiving an increase in left atrial pressure to increase hydrostatic forces, only (S)-albuterol increased lung water volume. Thus in the presence of altered lung fluid balance, (S)-albuterol, but not (R)-albuterol, might increase lung epithelial permeability. Our finding that (S)-albuterol increases allergen-induced interstitial edema indicates a potential adverse effect of this enantiomer in patients with asthma. (S)-Albuterol significantly increased bronchial responsiveness to methacholine challenge in OVA-sensitized/OVA-challenged mice, an adverse effect on airway function not shared by (R)-albuterol. In nonsensitized/ nonchallenged mice (S)-albuterol did not independently affect the airway response to methacholine. We used whole-body plethysmography to assess airway hyperreactivity to methacholine in the study groups. Although

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FIG 8. Effect of (R)- and (S)-albuterol enantiomers on airway histology in non–OVA-sensitized/non–OVAchallenged mice. Lung tissue was obtained on day 36 from saline control animals (A), saline-treated mice administered (S)-albuterol (B), and saline-treated mice administered (R)-albuterol (C), and sections were stained with hematoxylin and eosin. AW, Airway. Bars = 100 mm.

there has been recent controversy regarding the use of Penh as an indirect measure of pulmonary mechanics,30-32 Penh values correlate well with airway resistance measured directly in anesthetized, tracheotomized, and mechanically ventilated mice.10,11 A strong correlation also exists between Penh values and the intensity of the allergen-induced airway eosinophil infiltration in the mouse asthma model.33 (S)-Albuterol might mediate its augmenting effect on bronchial hyperresponsiveness through several modes of action, including parasympathetic and sympathetic pathways. In OVA-sensitized guinea pigs continuous exposure to (R,S)- and (S)-albuterol, but not (R)-albuterol,

for a 10-day period increased bronchial hyperresponsiveness to both histamine and OVA.5 Chronic capsaicin treatment prevented the (R,S)- and (S)-albuterol–induced bronchial hyperresponsiveness in this model to indicate the importance of capsaicin-sensitive sensory nerves in (S)-albuterol-mediated development of airway hyperresponsiveness.5 Recent studies by Agrawal et al4 indicate that (S)-albuterol activates proconstrictor and proinflammatory pathways in human bronchial smooth muscle cells. (S)-Albuterol significantly increased the expression and activity of Gia-1 protein and reduced Gs protein in these cells.4 (S)-Albuterol also increased the intracellular free calcium concentration in the bronchial smooth

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muscle cells after methacholine stimulation.4 These proconstrictor effects of (S)-albuterol were accompanied by stimulation of phosphatidylinositol 3#-OH-kinase and nuclear factor kB proinflammatory pathways in the smooth muscle cells. (R)-Albuterol induced the opposite effects on Gia-1, Gs, and intracellular free calcium concentration in the bronchial smooth muscle cells, which indicates separate mechanisms of action of the enantiomers.4 In summary, the actions of the (R)- and (S)-enantiomers of albuterol in the lungs of allergen-sensitized/allergenchallenged mice are complex. Although both enantiomers reduce mucus hypersecretion and trafficking of eosinophils to the lungs after allergen challenge, only the (S)-enantiomer induces airway edema and hyperresponsiveness to methacholine. Additional studies are needed to delineate the specific effects of the (R)- and (S)enantiomers of albuterol on airway inflammation and hyperresponsiveness in patients with asthma.

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12.

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We thank Gertrude Chiang, Falaah Jones, and Ying-tzang Tien for excellent technical assistance and Rachel Norris for typing this manuscript. 20. 21. REFERENCES 1. Slattery D, Wong SW, Colin AA. Levalbuterol hydrochloride. Pediatr Pulmonol 2002;33:151-7. 2. Cho SH, Hartleroad JY, Oh CK. (S)-Albuterol increases the production of histamine and IL-4 in mast cells. Int Arch Allergy Immunol 2001;124: 478-84. 3. Baramki D, Koester J, Anderson AJ, Borish L. Modulation of T-cell function by (R)- and (S)-isomers of albuterol: anti-inflammatory influences of (R)-isomers are negated in the presence of the (S)-isomer. J Allergy Clin Immunol 2002;109:449-54. 4. Agrawal DK, Ariyarathna K, Kelbe PW. (S)-Albuterol activates proconstrictory and pro-inflammatory pathways in human bronchial smooth muscle cells. J Allergy Clin Immunol 2004;113:503-10. 5. Keir S, Page C, Spina D. Bronchial hyperresponsiveness induced by chronic treatment with albuterol: role of sensory nerves. J Allergy Clin Immunol 2002;110:388-94. 6. Eaton EA, Walle UK, Wilson HM, Aberg G, Walle T. Stereoselective sulphate conjugation of salbutamol by human lung and bronchial epithelial cells. Br J Clin Pharmacol 1996;41:201-6. 7. Boulton DW, Fawcett JP. Enantioselective disposition of salbutamol in man following oral and intravenous administration. Br J Clin Pharmacol 1996;41:35-40. 8. Henderson WR Jr, Lodewick MJ. Animal models of asthma. In: Adkinson NF Jr, Yuninger JW, Busse WW, Bochner BS, Holgate ST, Simons FER, editors. Middleton’s allergy: principles and practice. 6th ed. St Louis: Mosby; 2003. p. 465-81. 9. Sartori C, Fang X, McGraw DW, Koch P, Snider ME, Folkesson HG, et al. Selected contribution: long-term effects of b2-adrenergic receptor stimulation on alveolar fluid clearance in mice. J Appl Physiol 2002;93: 1875-80. 10. Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG, et al. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 1997;156:766-75. 11. Justice JP, Shibata Y, Sur S, Mustafa J, Fan M, Van Scott MR. IL-10 gene knockout attenuates allergen-induced airway hyperresponsiveness

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in C57BL/6 mice. Am J Physiol Lung Cell Mol Physiol 2001;280: L363-8. Henderson WR Jr, Tang L-O, Chu S-J, Tsao S-M, Chiang GKS, Jones F, et al. A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am J Respir Crit Care Med 2002;165:108-16. Oh SW, Chong IP, Dong Keun L, Jones F, Chiang GKS, Kim HO, et al. Tryptase inhibition blocks airway inflammation in a mouse asthma model. J Immunol 2002;168:1992-2000. Nguyen C, Teo J-L, Matsuda A, Eguchi M, Chi E, Henderson WR Jr, et al. Chemogenomic identification of Ref-1/AP-1 as a novel therapeutic target for asthma. Proc Natl Acad Sci U S A 2003;100:1169-73. Iio J, Katamura K, Takeda H, Ohmura K, Yasumi T, Meguro TA, et al. Lipid A analogue, ONO-4007, inhibits IgE response and antigen-induced eosinophilic recruitment into airways in BALB/c mice. Int Arch Allergy Immunol 2002;127:217-25. Henderson WR Jr, Lewis DB, Albert RK, Zhang Y, Lamm WJE, Chiang GKS, et al. The importance of leukotrienes in airway inflammation in a mouse model of asthma. J Exp Med 1996;184:1483-94. Zhang Y, Lamm WJE, Albert RK, Chi EY, Henderson WR Jr, Lewis DB. Influence of the route of allergen administration and genetic background on the murine allergic pulmonary response. Am J Respir Crit Care Med 1997;155:661-9. Johnson M. Effects of b2-agonists on resident and infiltrating inflammatory cells. J Allergy Clin Immunol 2002;110(suppl):S282-90. Wang CH, Lin HC, Lin CH, Yu CT, Liu SL, Huang KH, et al. Effect of theophylline and specific phosphodiesterase IV inhibition on proliferation and apoptosis of progenitor cells in bronchial asthma. Br J Pharmacol 2003;138:1147-55. Rogers DF. Pharmacological regulation of the neuronal control of airway mucus secretion. Curr Opin Pharmacol 2002;2:249-55. Frohock JI, Wijkstrom-Frei C, Salathe M. Effects of albuterol enantiomers on ciliary beat frequency in ovine tracheal epithelial cells. J Appl Physiol 2002;92:2396-402. Bennett WD. Effect of b-adrenergic agonists on mucociliary clearance. J Allergy Clin Immunol 2002;110(suppl):S291-7. Temann U-A, Prasad B, Gallup MW, Basbaum C, Ho SB, Flavell RA, et al. A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion. Am J Respir Cell Mol Biol 1997; 16:471-8. Grunig G, Warnock M, Wakil AE, Venkayya R, Brombacher F, Rennick DM, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998;282:2261-3. Henderson WR Jr, Chi EY, Maliszewski CR. Soluble IL-4 receptor inhibits airway inflammation following allergen challenge in a mouse model of asthma. J Immunol 2000;164:1086-95. Burke TF, Casolaro V, Georas SN. Characterization of P5, a novel NFAT/AP-1 site in the human IL-4 promoter. Biochem Biophys Res Commun 2000;270:1016-23. Macian F, Garcia-Rodriguez C, Rao A. Gene expression elicited by NFAT in the presence or absence of cooperative recruitment of Fos and Jun. EMBO J 2000;19:4783-5. Fallon PG, Jolin HE, Smith P, Emson CL, Townsend MJ, Fallon R, et al. IL-4 induces characteristic Th2 responses even in the combined absence of IL-5, IL-9, and IL-13. Immunity 2002;17:7-17. Peterson BT, Miller EJ. Effects of enantiomers of albuterol on lung epithelial permeability [abstract]. Am J Respir Crit Care Med 2000;161: A416. Petak F, Habre W, Donati YR, Hantos Z, Barazzone-Argiroffo C. Hyperoxia-induced changes in mouse lung mechanics: forced oscillations vs. barometric plethysmography. J Appl Physiol 2001;90:2221-30. Lundblad LK, Irvin CG, Adler A, Bates JH. A reevaluation of the validity of unrestrained plethysmography in mice. J Appl Physiol 2002; 93:1198-207. Mitzner W, Tankersley C. Interpreting Penh in mice. J Appl Physiol 2003;94:828-31. Dohi M, Tsukamoto S, Nagahori T, Shinagawa K, Saitoh K, Tanaka Y, et al. Noninvasive system for evaluating the allergen-specific airway response in a murine model of asthma. Lab Invest 1999;79:1559-71.

Rhinitis, sinusitis, and ocular diseases Comparison of test devices for skin prick testing Warner W. Carr, MD,a Bryan Martin, DO,a Robin S. Howard, MA,b Linda Cox, MD,c Larry Borish, MD,d and the Immunotherapy Committee of the American Academy of Allergy, Asthma and Immunology Silver Spring, Md, Fort Lauderdale, Fla, and Charlottesville, Va

From athe Department of Allergy and Immunology and bthe Department of Clinical Investigations, Walter Reed Army Medical Center, Silver Spring; c private practice, Fort Lauderdale; and dthe Asthma and Allergic Disease Center, University of Virginia Health System. Supported by the Walter Reed Army Medical Center, Department of Clinical Investigations. Disclosure of potential conflict of interest: L. Borish has consultant arrangements with PDL, Syngenta, and Sepracor. No other relevant conflicts of interest to disclose. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Received for publication September 3, 2004; revised March 28, 2005; accepted for publication March 28, 2005. Available online May 16, 2005. Reprint requests: Warner W. Carr, MD, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.03.035

that technicians are sufficiently trained on the correct use of that device. (J Allergy Clin Immunol 2005;116:341-6.) Key words: Skin testing, device, performance, variability, pain

The US Joint Council of Allergy, Asthma and Immunology1 and the European Academy of Allergology and Clinical Immunology2 recommend percutaneous testing as the primary test for diagnosis of IgE mediated allergic disease. Skin testing is also the preferred method for selecting allergens to be included in immunotherapy.3 Given this, the findings on the initial skin test panel are very important clinical data. If a particular device is too sensitive (resulting in false-positive findings), the patient may receive an antigen that is not required to achieve clinical benefit. On the other hand, a high false-negative rate for a particular device will result in a patient not receiving a needed antigen while undergoing immunotherapy. The goal for the allergist is to perform allergen skin testing in an appropriate patient population by using a device that minimizes both false-negative and falsepositive findings. In addition, it is desirable to use a device that results in minimal patient discomfort. Previous studies comparing devices for skin prick (ie, prick and puncture) testing have revealed significant differences in the size of wheal and flare reactions. These differences have been seen at both positive (allergen extract or histamine) and negative (saline) sites.4-7 In these studies, the difference appeared to result from the degree of trauma imparted to the skin by the device, an interpretation that was reinforced by the fact that those producing larger wheals also caused more patient discomfort.6 New skin devices continue to be developed, with a current trend toward devices that allow for application of several antigens simultaneously, referred to as multiheaded. This may limit technician time and increase efficiency. In addition, multiheaded devices have increasing popularity in children, in whom the acceptance of a few multiple test devices tends to be better than many individually applied devices. In a recent letter to the editor, Nelson et al7 compared 3 new multidevices with previously reviewed devices. In this communication, significant differences were noted with the smallpox needle on the back and the Greer Track (Greer Labs, Lenoir, NC) on the arm. Given this, we reviewed 4 devices that allow 341

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Background: Allergy skin testing guides developing avoidance plans and writing an immunotherapy prescription. The goal for the allergist is to apply allergen skin testing to the appropriate patient population by using a device that minimizes both false-negative and false-positive findings while minimizing patient discomfort. New skin testing devices continue to be developed with a trend toward production of multiheaded devices. Data on the performance of these devices in a head-to-head prospective fashion are limited. Objective: Our goal was to study 8 commonly used devices to compare their performance in a head-to-head fashion. Methods: In a prospective, double-blind fashion, the performance of 8 skin test devices was evaluated. Devices were tested with histamine and saline on both the arms and back of each subject. Devices were rotated over 4 testing sessions, at least a week apart, so each device was tested in each anatomic testing location. Performance elements examined included wheal, flare, pain, sensitivity, specificity, and intradevice variability. Results: We found significant differences in all areas of device performance among all devices examined. Multiheaded devices also demonstrated significant intradevice variability and were more painful than single devices. Furthermore, multiheaded devices had larger reactions on the back, whereas single devices had larger reactions on the arms. Conclusion: Statistically significant differences exist among all devices tested. Providers should consider this data when choosing a device that suits their practice setting and ensure

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Abbreviation used CV: Coefficient of variation

application of multiple antigens at once (multiheaded) and 4 devices that allow application of only 1 antigen at a time (single devices). Our goal was to study a cohort of devices to compare their performance in a head-to-head fashion. We specifically intended to determine sensitivity, specificity, variability, and pain. With these results, providers will be able to determine which device is best suited for their practices.

FIG 1. The left image illustrates the 4 test zones of the back, and the image on the right represents the 2 left arm test zones. Right arm test zones are not shown but mirror those of the left arm. LLA, Left lower arm; LLB, left lower back; LUA, left upper arm; LUB, left upper back; RLB, right lower back; RUB, right upper back.

Rhinitis, sinusitis, and ocular diseases

METHODS Study design The study was a prospective, double-blind clinical trial and was reviewed and approved by the Walter Reed Army Medical Center Clinical Investigation Committee and the Human Use Committee. All subjects enrolled into the study voluntarily agreed to participate and gave written informed consent. Each subject underwent testing in 4 sessions, with each at least 1 week apart. Each device was tested both on the arm and the back, with histamine (10 mg/mL; Hollister-Stier, Spokane, Wash) and glycerol-saline (Hollister-Stier) during each session. During the course of 4 sessions, the locations on the arm and back were rotated to ensure each device was tested on the upper and lower arm and upper and lower back. Therefore, each session yielded 4 test sites per device: 2 histamine tests (1 back and 1 arm) and 2 glycerol-saline tests (1 back and 1 arm). Fig 1 illustrates the back and left arm test regions for one test session. Over the course of the study, sites were rotated to give an equal number of tests in all areas to offset any differences in reactivity.6 At the end of the study, each device had been tested on the upper and lower back and the upper and lower arms, with an even distribution between left and right. All heads of a multiheaded device were tested with histamine at the histamine site, and all heads were tested with saline at the saline site. At the conclusion of the fourth session, a mean result was determined for each head of the multiheaded devices, and from this, intradevice variability was determined. Single device test sites were spaced at least 30 mm apart, and multiheaded spacing was fixed at 20 mm to 30 mm on the basis of the design of the device. With the devices examined, this resulted in 132 individual pricks per subject per session. The total number of individual pricks over the course of the study for each subject was 528. With 13 subjects completing the study, this yielded 6864 individual prick sites for examination. Before each session, antihistamines were withheld for at least 1 week, and H2 antagonists and leukotriene antagonists were withheld for 72 hours. To maintain objectivity, the technician who performed all of the tests was blind to the contents of the test solution, either histamine or saline. A second technician who was not in the room during application of each device recorded the results. This technician was blind to the device used as well as to the solution used. Before the study was initiated, a representative of the manufacturer trained the technician who performed the skin tests on each device. This step was taken to achieve the best possible results by using the manufacturer’s recommended skin testing technique. Pain assessment was performed by using the Wong-Baker FACES pain rating scale8 immediately after application of each skin test

device (measured on a scale from 0-10). On the basis of this scale, a level of 1 to 2 is considered minimal pain. The greatest reported pain was recorded for that particular test site and session. Pain was recorded within seconds of application of the skin test device to minimize the influence of histamine on pain perception. Results were recorded for pain sensation on the arm and on the back.

Subjects Male or female subjects age 18 to 70 years, with or without allergies, were eligible for the study. Subjects were excluded if they had dermatographism, severe atopic dermatitis, or asthma, or were taking antidepressants. Antihistamines were withheld for 1 week before testing. Leukotriene antagonists and H2 antagonists were withheld for 72 hours before testing.

Devices Four single-headed devices and 4 multiheaded devices were tested. Single headed devices included the Greer Pick (Greer Labs), Accuset (ALK-Abello´, Inc, Round Rock, Tex), Sharptest (Panatrex, Inc, Placentia, Calif), and Quintip (Hollister-Stier). Multiheaded devices tested were the Quintest (Hollister-Stier), Quantitest (Panatrex, Inc), Greer Track, and Multi-Test II (Lincoln Diagnostics, Inc, Decatur, Ill; Fig 2).

Skin testing All testing was performed first on the arms, and once results were obtained and recorded, testing proceeded on the back. The wheal and flare results were recorded at 15 minutes by obtaining the longest orthogonal diameters. Mean diameters were used for statistical analyses. Pain was recorded immediately after application of each skin test device. Positive test solution consisted of 10 mg/mL histamine (Hollister-Stier), with standard glycerol saline (HollisterStier) used as a negative solution.

Statistical analysis Results were analyzed by using repeated-measures ANOVA, with the within-subject factors body site (upper arm, lower arm, upper back, lower back) and device. Thirteen subjects were needed to power the study adequately to detect a minimum difference of 2 mm between each device. When calculating sensitivity and specificity, a true-positive result was considered a histamine wheal of 3 mm or greater, and a true-negative result was a glycerol-saline wheal less than 3 mm. A result was considered false-negative if a histamine

Carr et al 343

FIG 2. Skin test devices investigated. Multiheaded devices from top left to right followed by midleft to right: Quintest, Greer Track, Multi-Test II, and Quantitest. Single devices from bottom left to right: Accuset, Quintip, Sharptest, Greer Pick.

wheal was less than 3 mm, and a result was considered false-positive if the glycerol-saline site was 3 mm or greater. Results are presented as the means 6 SDs, and for multiheaded devices, the average of all heads was used in the calculation of sensitivity and specificity. Sensitivity and specificity of each device are presented as proportions with 95% CIs, and devices were compared by using the Fisher exact test (2-tailed). Sensitivity was calculated by dividing true-positive results by the sum of true-positive and false-negative results. Specificity was calculated by dividing true-negative results by the sum of true-negative and false-positive results. When multiheaded devices were analyzed, intradevice variability was described by using the coefficients of variation (CVs; presented as medians with the interquartile range) for each device. For each multiheaded device, the wheal produced by each head was compared by using repeated-measures ANOVA. Pain scores were compared among devices by using the Wilcoxon signed-rank test: median pain scores were presented as well as the proportion of pain scores above a value of 2 (representing mild pain on the Wong-Baker FACES pain rating scale). For interdevice comparisons of pain, wheal, and flare size within the single-headed or multiheaded groups, there are 6 possible pairwise analyses. Using a Bonferroni correction of the overall experimental P value of .05, a P value of .008 (.05/6) or less is considered significant.

RESULTS Twenty subjects were recruited for the study, and 7 subjects did not complete because of pregnancy (1) and military operational requirements (6). Eight men and 5 women completed the study. The mean age was

32.2 years (range, 22-57), and 7 subjects had a history of atopy.

Interdevice comparisons Histamine and saline reactions are presented in Table I. Controlling for site (arm vs back), there was a significant difference in histamine wheal size among all devices in each device group (P < .008 for all comparisons), except for no significant difference between the Accuset and the Quintip (P = .28) and the Multi-Test II and Quantitest (P = .27). The largest reactions to histamine base were found with 2 single devices, Sharptest and Greer Pick. There were no significant differences in saline wheal reactions. In addition, all mean histamine flares were greater than 10 mm, and mean saline flares were below 5 mm. Table II gives the number of results that exceeded the limits for positive and negative reactions set for histamine and saline. For histamine wheal reactions, the Greer Pick gave the lowest number of false-negative results (2/208 or 0.96%); the range for single devices was 0.96% to 3.8% (Accuset). The range for multiheaded devices was 57/1664 (3.4%) with the Multi-Test II and 366/1664 (22%) with Greer Track. Single devices demonstrated a high degree of reproducibility, with CVs ranging from 0.22 to 0.37 (Table I). The CV reported in Table I for the multiheaded devices represents a CV of the mean of all heads. For intradevice variability, or differences between each head of a multiheaded device, see Table III.

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TABLE I. Outcome measures for 8 devices* Histamine wheal, mean 6 SD

Single devices Sharptest Greer Pick Accuset Quintip Multidevices Multi-Test II Quantitest Quintest Greer Track

Histamine flare, mean 6 SD

CV

Saline wheal, mean 6 SD

Saline flare, mean 6 SD

Sensitivity % (95% CI)

Specificity % (95% CI)

7.1 6.6 5.1 4.8

6 6 6 6

1.7 1.8 1.9 1.7

31.6 33.3 24.3 22.6

6 6 6 6

8.4 9.5 10.7 9.3

0.22 0.37 0.34 0.36

0.03 0.0 0.1 0.0

6 6 6 6

0.3 0.0 0.5 0.0

3.2 2.7 1.5 1.1

6 6 6 6

2.8 2.4 2.4 2.6

97 98 92 95

(91-991) (93-991) (85-97) (89-99)

99 100 98 100

(94-991) (97-100) (93-991) (97-100)

5.9 5.7 4.3 3.2

6 6 6 6

1.3 1.6 1.4 1.3

26.0 25.6 19.9 16.5

6 6 6 6

5.7 7.3 7.8 6.4

0.23 0.34 0.25 0.42

0.02 0.01 0.0 0.012

6 6 6 6

0.2 1.6 0.0 0.1

3.3 3.2 0.8 3.4

6 6 6 6

1.5 1.8 1.5 1.4

93 89 86 56

(91-95) (86-91) (82-89) (52-60)

99 99 100 99

(98-991) (98-991) (99-100) (98-991)

*Values for wheal and flare expressed in millimeters.

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TABLE II. Number of tests that exceed 3 mm for saline wheal and 10 mm for saline flare, and number of tests that are below 3 mm for histamine wheal and 10 mm for histamine flare Histamine wheal, mm Total test

Single devices Sharptest Greer Pick Accuset Quintip Multidevices Multi-Test II Quantitest Quintest Greer Track

<3

Histamine flare, mm

Multi-Test II Quantitest Quintest GreerTrack

Saline flare, mm

<10

Range

>3

Range

>10

Range

208 208 208 208

3 2 8 5

0-10 0-12 0-8 0-10

1 2 11 9

9-60 7-75 0-50 0-40

0 0 1 0

0-3 0 0-4 0

1 0 1 2

0-15 0-10 0-12 0-15

1664 1664 1040 1664

57 94 73 366

0-12 0-11 0-11 0-11

42 85 93 361

0-50 0-62 0-50 0-47

4 1 0 2

0-4 0-5 0 0-5

4 3 1 1

0-20 0-32 0-15 0-22

TABLE III. Intradevice variability for multiheaded devices expressed as CV Multidevices

Saline wheal, mm

Range

Histamine wheal,* mean 6 SD

5.9 5.7 4.3 3.2

6 6 6 6

1.3 1.6 1.4 1.3

CVy (interquartile range)

0.20 0.23 0.25 0.93

(0.14-0.44) (0.14-0.50) (0.18-0.59) (0.70-1.39)

*Values expressed in millimeters.  CV median (interquartile range), intradevice variability.

Sensitivity and specificity The results of device sensitivity and specificity are listed in Table I. All single devices and the Multi-Test II had sensitivities >90%, and there was no significant difference in sensitivity among the single devices and the Multi-Test II. The Multi-Test II was more sensitive compared with all other multiheaded devices (P < .002). The Quintest was less sensitive than the Greer Pick, Sharptest, and Multi-Test II. The Greer Track was less sensitive than all other devices (P < .0005).

Arm versus back comparisons There was a significant difference in histamine wheal sizes between the arms and backs for all devices (P < .0005; Fig 3). Histamine wheals for all single devices were significantly larger on the arms (P < .05 for all comparisons), and wheals for all multiheaded devices (except the Quintest) were larger on the back (P < .0013). The Quintest device was larger on the back, but this difference did not reach statistical significance (P = .17). There was no significant difference between upper and lower arm. There also was no significant difference between upper and lower back. Multidevices: intradevice variability Intradevice variability reactions are presented in Table III. Analyzing the multiheaded devices for intradevice variability, there were significant differences in the wheal sizes between the various heads for each device (P < .009 for all devices). Fig 4 illustrates the intradevice variability for the Greer Track. With the 8-headed devices (Greer Track, Multi-Test II, and Quantitest), the greatest degree of variability was found comparing the interior heads (S2, S3, S6, S7) with the corner heads (S1, S4, S5, S8) for all of them.

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FIG 3. Mean histamine wheal size in millimeters for all devices.

TABLE IV. Pain outcome measures for 8 devices

The greatest degree of intradevice variability was found within the Greer Track.

Pain Median pain scores for all of the devices was 1.0, except for the Greer Track, with a median of 2.0 (Table IV). Reports of pain were considered minor, with only 1 pain rating reported above 6 on a scale from 0 to 10 on the Wong-Baker FACES pain rating scale.8 The highest pain rating was for the Greer Track (34% of pain scores above 2), and the minimum pain reported was for the Greer Pick (5% of pain scores above 2). All single devices were significantly less painful than the multiheaded devices (P < .0005). Comparing the single devices, Sharptest pain scores were significantly higher than Greer Pick (P < .0005) and Accuset (P = .001). For the multidevices, Greer Track scores were significantly higher than all other devices (P < .0005 for all comparisons), and the Quantitest was more painful than the Quintest (P = .001). In addition, pain was negatively associated with sensitivity (r = 20.77; P = .027), because the devices with greater sensitivity had lower pain scores. For the Greer Track multidevice with 56% sensitivity, 34% of pain scores were above 2. For the Greer Pick single device with 98% sensitivity, only 5% of pain scores were above 2. DISCUSSION We have concluded a head-to-head prospective comparative study of 8 skin test devices and found that there

Median pain

1.17 0.88 0.94 1.0

1 1 1 1

13% 5% 9% 7%

(7-22) (1-11) (4-16) (2-14)

1.62 1.74 1.45 2.04

1 1 1 2

26% 26% 17% 34%

(17-36) (17-36) (10-26) (23-43)

*Percentage of values above 2 on the Wong-Baker FACES pain rating scale (percentage of values interpreted as greater than mild pain).

are statistically significant differences among virtually all devices tested. One device that stands out with the lowest performance in all areas is the Greer Track. This device was the most painful, had the smallest mean histamine wheals and flares, was the least sensitive, and had the greatest degree of intradevice variability. These statistical findings of performance may very well equate to clinically significant differences in performance. Excluding the Greer Track, it is unknown whether the statistical differences among the remaining 7 devices will equate to clinically significant differences in performance. Of the remaining 7 devices, all had mean histamine wheals greater than 3 mm and mean histamine flares greater than 10 mm, with sensitivities from 86% to 97%. In addition, all of the remaining 7 devices had specificities of 98% or greater. Therefore, each individual provider should determine which device is best for that provider’s practice. Keep in mind that these studies were performed under the best of circumstances, with all tests conducted by 1 technician who was certified by a representative of the manufacturer on the proper use of each device. We would recommend that technicians within a given practice undergo this same type of training before using a given device. In addition, these findings may not be directly applicable to allergen skin testing, because we looked only at histamine and glycerol-saline responses. A separate study may be required to compare devices for this purpose.

Rhinitis, sinusitis, and ocular diseases

FIG 4. Mean intradevice variability of the Greer Track. Sites are labeled S1 through S8, with sites S1, S4, S5, and S8 representing the corners and S2, S3, S6, and S7 representing the interior heads.

Single devices Sharptest Greer Pick Accuset Quintip Multidevices Multi-Test II Quantitest Quintest Greer Track

Pain %* (95% CI)

Mean pain

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When choosing a skin test device, a few points are worth consideration. First, in our study, single devices had larger reactions on the arm, and multidevices had larger reactions on the back. Historically, it has been thought that the back has been more reactive than the arms, and although our multiheaded device data concur with this, our single-headed device data do not. There are several possibilities for this observation, including intraoperator variability, inadvertent operator bias, or a true difference. With regard to the difference between single and multiheaded devices, we think that this difference is related to the back being a flatter surface; therefore, better contact is made with all of the test sites on a multiheaded device. The arms are technically more challenging when placing a multiheaded device, given natural curvatures. To compensate for the curvatures and to ensure contact with all of the device heads, manufacturers have recommended a rocking motion. With this motion, contact between all heads on a multiheaded device and the skin is achieved. However, we think that this rocking motion is responsible for the differences noted between individual test sites within a given multiheaded device, or intradevice variability (Fig 4). With this rocking motion, more pressure is exerted on the skin from the corner test sites. It is important to note that single devices also have differences in the recommended technique of application. The Quintip and Sharptest use a simple downward perpendicular pressure, and both of these devices have a depth control feature. Manufacturer-recommended techniques for the Greer Pick and Accuset are slightly more complicated. The skin surface is penetrated at an angle, and then a flick, pricknot-puncture technique is used. Neither of these last 2 devices has a depth control feature. Given the technique and lack of depth control, the Greer Pick and Accuset may result in greater intertechnician variability if care is not taken to control for these features. However, with correct technique, and by using 1 technician, all single devices had sensitivities greater than 90% while maintaining specificities of 98% or greater. Another observation from our study is that skin testing is not a painful procedure on average. The mean pain scores for all devices ranged from 0.88 to 2.04. Using the Wong-Baker FACES pain rating scale,8 this is considered mild pain. The degree of pain was significantly associated with the type of device used, with the multiheaded devices more painful than the single devices. However, when making this comparison, it is noteworthy that with a minimal increase in pain, as many as 8 times more tests are applied. Therefore, with a pain score of 0.88, the Greer Pick applied 1 skin test, and with a pain score of 1.62, the Multi-Test II applied 8 skin tests. It is unclear whether these observed statistical differences in pain would equate to significant clinical differences, because all pain was considered mild.

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In contrast with previous studies, we did not find a clear relationship between pain and wheal size.6 In fact, the device that resulted in the greatest degree of pain had the smallest mean histamine wheal size. Again, we think it is up to the individual practitioner to consider these differences when using a given device in practice. Finally, when comparing sensitivity and specificity, there are few differences among devices, with 2 exceptions. The Greer Track was less sensitive than all other devices, and the Quintest was less sensitive than the Greer Pick, Sharptest, and Multi-Test II. With the 6 remaining devices, there was no significant difference among sensitivities. In addition, we found no significant differences in specificity among devices. Overall, we found a very low false-positive rate in all devices when using the manufacturer’s recommended skin testing technique.

CONCLUSION We have completed a prospective, head-to-head comparison of the performance of 8 skin test devices. This study was performed under the best of clinical circumstances, with 1 technician, trained by a representative of the manufacturer, who performed all skin testing, and another technician who read all of the results. We have found significant differences among all devices tested. Whether this equates to clinical differences is yet to be determined. Overall, skin testing is associated with minimal pain, and individual providers should choose a skin test device on the basis of their own practice setting. As new devices are being produced, this study suggests the need for continued evaluation of these devices in a prospective, nonbiased fashion. REFERENCES 1. Li JT, Lockey RF, Bernstein IL, Portnoy JM, Nicklas RA. Allergen immunotherapy: a practice parameter. Ann Allergy Asthma Immunol 2003;90(suppl):1-40. 2. Position paper: immunotherapy. (EAACI) The European Academy of Allergology and Clinical Immunology. Allergy 1993;48(suppl 14):7-35. 3. Spector SL, Nicklas RA. Practice parameters for the diagnosis and treatment of asthma. J Allergy Clin Immunol 1995;96:707-870. 4. Adinoff AD, Rosloniec DM, McCall LI, Nelson HS. A comparison of six epicutaneous devices in the performance of immediate hypersensitivity skin testing. J Allergy Clin Immunol 1989;84:168-74. 5. Nelson HS, Rosloniec DM, McCall LI, Ikle D. Comparative performance of five commercial prick skin test devices. J Allergy Clin Immunol 1993; 92:750-6. 6. Nelson HS, Lahr J, Buchmeier A, McCormick D. Evaluation of devices for skin prick testing. J Allergy Clin Immunol 1998;101:153-6. 7. Nelson HS, Kolehmainen C, Lahr J, Murphy J, Buchmeier A. A comparison of multiheaded devices for allergy skin testing. J Allergy Clin Immunol 2004;113:1218-9. 8. Wong-Baker FACES Pain Rating Scale. In: Wong DL, HockenberryEaton M, Wilson D, Winkelstein ML, Schwartz P. Wong’s essentials of pediatric nursing. 6th ed. St Louis: Elsevier; 2001. p. 1301-2.

Allergen-specific nasal IgG antibodies induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity

Background: We have performed a double-blind, placebocontrolled injection immunotherapy study with genetically modified derivatives of the major birch pollen allergen, Bet v 1 (Bet v 1–trimer, Bet v 1–fragments). Objective: To investigate whether vaccination with genetically modified allergens induces allergen-specific antibodies in nasal secretions and to study whether these antibodies affect nasal allergen sensitivity. Methods: A randomly picked subgroup of patients (n = 23; placebo, n = 10; trimer, n = 10; fragments, n = 3) was subjected to an extensive analysis of serum samples and nasal lavage fluids and to nasal provocation testing. Bet v 1–specific IgG1-4 and IgA antibodies were determined in serum samples obtained before and after vaccination, after the birch pollen season, and 1 year after start of vaccination as well as in nasal lavage fluids obtained after the birch pollen season and 1 year after start of vaccination by ELISA. Nasal sensitivity to natural, birch pollen–derived Bet v 1 was determined by active anterior rhinomanometry after the birch pollen season and 1 year after start of vaccination. Results: Vaccination with genetically modified Bet v 1 derivatives, but not with placebo, induced Bet v 1–specific IgG1, IgG2, and IgG4, and low IgA antibodies in serum, which also appeared in nasal secretions, but no IgG3 antibodies. The levels of therapy-induced Bet v 1–specific IgG4 antibodies in nasal secretions were significantly (P < .05) associated with reduced nasal sensitivity to natural, birch pollen–derived Bet v 1 as objectively determined by controlled nasal provocation experiments.

From athe Department of Otorhinolaryngology, and bthe Department of Pathophysiology, Center for Physiology and Pathophysiology, Vienna General Hospital, and cthe Department of Medical Statistics, Medical University of Vienna; dthe Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet and Hospital, Stockholm; eService de Pneumologie, Hoˆpitaux Universitaires de Strasbourg; and fAllergopharma Joachim-Ganzer KG, Reinbek. Supported by grants F01811 and F01815 of the Austrian Science Fund and by a research grant from Biomay, Vienna. Received for publication February 3, 2005; revised March 23, 2005; accepted for publication April 1, 2005. Available online June 1, 2005. Reprint requests: Verena Niederberger, MD, Department of Otorhinolaryngology, Vienna General Hospital, AKH, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: Verena. [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.003

Conclusion: Our data demonstrate that vaccination with genetically modified allergens induces IgG antibody responses against the corresponding natural allergen not only in serum but also in mucosal fluids, where they may protect against allergen-induced inflammation. (J Allergy Clin Immunol 2005;116:347-54.) Key words: Allergen-specific immunotherapy, IgG antibodies, genetically modified allergens, nasal provocation

In the last 15 years, considerable progress has been made in the field of molecular allergen characterization. The repertoire of the disease-eliciting allergens has been recreated in the form of recombinant allergens for the most common allergen sources and can be used for diagnostic tests that allow dissection of the reactivity profiles of patients down to a molecular level.1,2 In attempts to reduce IgE-mediated side effects during allergen-specific immunotherapy, several research groups have used genetic engineering and peptide chemistry to develop allergen derivatives with reduced allergenic activity.3-7 By using genetically engineered derivatives of the major birch pollen allergen, Bet v 1, we have conducted a first doubleblind, placebo-controlled immunotherapy study in patients allergic to birch pollen.8 One group of patients was treated with a mixture of 2 recombinant Bet v 1 fragments that exhibited strongly reduced IgE reactivity and allergenic activity; a second group received a recombinant Bet v 1 trimer, also characterized by reduced allergenic activity; and the third group was treated with aluminium hydroxide alone, which was used as adjuvant for the subcutaneous injections.8-12 The analysis of the immunological mechanisms showed that vaccination with the genetically modified Bet v 1 derivatives induced de novo serum IgG antibodies that recognized the Bet v 1–wildtype allergen and inhibited Bet v 1–induced histamine release from basophils.8 Furthermore, we obtained evidence that boosts of the IgE memory responses caused by seasonal allergen exposure were reduced in actively vaccinated patients.8 In this sub-study, we asked whether vaccination with the genetically engineered allergens also leads to an induction of allergen-specific antibodies in the nasal mucosa, the main site of allergic inflammation, and if so, whether such antibodies are associated with reduced 347

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Ju¨rgen Reisinger, MSc,a Friedrich Horak, MD,a Gabrielle Pauli, MD,e Marianne van Hage, MD,d Oliver Cromwell, PhD,f Franz Ko¨nig,c Rudolf Valenta, MD,b and Verena Niederberger, MDa Vienna, Austria, Stockholm, Sweden, Strasbourg, France, and Reinbek, Germany

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TABLE I. Demographic and clinical data of the 23 patients (trimer, n = 10; fragments, n = 3; placebo, n = 10) included in this study. The numbering of the immunotherapy trial was preserved and indicates that the patients were randomly picked when the study was still blind. Pat.-Nr.

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Age

Sex

Sensitized to

Symptoms

Treatment

Nr. of injections

Cumulative injected dose (mg)

33 39 42 36 27 51 28 45 37 33 45 51 36 28 38 23 26 30 55 36 40 43 35

m m m f f f m m m m f m m f m m f m f m f m m

b, g b b b b, g, w b, a b, g, w, a b b b b, w, a b, g, w, a b, g b, g, w b, w b, g, a b b, g, w, a, m b, w, a b b b, g, w, a b, g

r, c r, c, a r, c, a r, c, a r, a r, c, a r, c, a r, c, a r, c r, c r, c, a, d r, c, a r, c r, c r, c, a r, c r, c r, c r, c r, c r, c, a r, c, a r, c, a

trimer trimer trimer trimer trimer trimer trimer trimer trimer trimer fragments fragments fragments placebo placebo placebo placebo placebo placebo placebo placebo placebo placebo

10 9 8 10 8 8 8 9 8 8 9 7 9 8 8 8 9 9 9 8 8 9 9

265 185 165 169 165 163 165 245 69 165 103 85 245 0 0 0 0 0 0 0 0 0 0

2 8 15 18 26 33 34 41 44 49 36 42 64 3 5 6 28 37 51 56 57 58 61

Abbreviations used: m: male, f: female; b: birch pollen, g: grass pollen, w: weed pollen, a: animal dander, m: mites; symptoms: r: rhinitis, c: conjunctivitis, a: asthma, d: dermatitis

Abbreviation used r: Recombinant

allergen sensitivity in the nose as evaluated objectively by nasal provocation. For this purpose, we performed an indepth analysis of serum and nasal lavage fluids regarding allergen-specific antibodies and extensive nasal provocation in a subgroup of 23 subjects recruited at random from patients participating in the immunotherapy trial.

METHODS Recombinant allergens and vaccine formulation Recombinant major birch pollen allergen, Bet v 1,13 mimicking natural birch pollen–derived Bet v 1 was obtained from Biomay (Vienna, Austria). Recombinant Bet v 1 fragments and trimer were expressed in Escherichia coli and purified as described.9,10 Aluminium hydroxide adsorbates containing 100 mg protein/mL adsorbate trimer or aluminium hydroxide alone (placebo) were formulated as described14 following Good Manufacturing Practice guidelines.

Vaccination of patients allergic to birch pollen with Bet v 1 derivatives or placebo The immunotherapy study was conducted as a placebo-controlled, double-blind, randomized vaccination trial over a period of 12 months with 1 preseasonal treatment course in 3 study centers

FIG 1. Experimental protocol. Patients received a single course of subcutaneous injections (therapy) before the birch pollen season and were monitored for almost 1 year. Times of blood sampling, nasal washings, and provocations are indicated.

(Stockholm, Strasbourg, Vienna). Patients were included if they had a positive result with skin prick test and ImmunoCAP (3.5 kU/L; Pharmacia, Uppsala, Sweden) for Bet v 1 and natural birch pollen extract and had a history of moderate to severe seasonal allergic rhinoconjunctivitis attributable to birch pollen. Subcutaneous injections of aluminium hydroxide–adsorbed allergen derivatives containing increasing doses (1-80 mg) of recombinant Bet v 1 derivatives (Bet v 1–trimer, Bet v 1–fragments) or placebo were administered at intervals of 1 to 2 weeks as a preseasonal treatment. After reaching the maximal dose, the treatment was continued at 4-week intervals until before the flowering season. Clinical outcome was monitored by questionnaires, skin prick tests, and nasal provocation tests. The study was approved by the local ethical committees, and written informed consent was obtained from each patient. In a substudy, nasal lavage fluids were collected in 23 randomly picked patients from a total of 71 at the Vienna center. Serum samples

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FIG 2. Bet v 1–specific antibody levels in nasal lavage fluids (diluted 1:2) and serum (diluted 1:50). IgG1 (A), IgG2 (B), IgG4 (C), and IgA (D) levels (y-axis, OD values) were measured in nasal secretions (circles, left panel) and sera (rhombuses, right panel) from patients treated with trimer (black circles or rhombuses), fragments (grey circles or rhombuses), or placebo (white circles or rhombuses) at different times (x-axis). Bars indicate mean values. Statistically significant differences for nasal lavage fluid data are indicated with P values.

and nasal lavage fluids as well the results from the nasal provocation tests were analyzed after deblinding of the immunotherapy study (Table I).

Experimental protocol The sequence of serum sampling, nasal washings, and nasal provocation tests is illustrated in Fig 1.

Collection of nasal lavage fluids and serum samples Nasal lavage fluids were obtained from 23 patients (treated with Bet v 1 fragments, n = 3; Bet v 1 trimer, n = 10; placebo, n = 10) after the birch pollen season (May 2001) and 1 year after beginning of the study (autumn/winter 2001). To obtain nasal secretions, patients sat with their head bent forward, and 1 nostril was sealed with a foam plastic plug encircling

the end piece of a narrow plastic tube. The respective side of the nose was slowly filled and emptied 5 times with 5 mL prewarmed 0.9% saline from a syringe attached to the other end of the plastic tube. Subsequently, lavages were obtained from the other nostril. Serum samples were obtained before treatment (autumn/winter 2000), after treatment (February/March 2001), after the birch pollen season (May 2001), and in autumn/winter 2001. Nasal lavage fluids and serum samples were stored at 220°C until analysis.

Nasal provocation tests Nasal provocation tests were performed with natural birch pollen extract containing defined Bet v 1 concentrations before treatment (November/December 2000), after the birch pollen season (May 2001), and after 1 year (October 2001). Allergen solutions were freshly prepared from the standardized lyophilized birch pollen extract, were kept at 4°C between tests, and were discarded after 48 hours.

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Diego, Calif) that recognizes IgA1, IgA2, and secretory IgA. The alkaline phosphatase–labeled detection antibody was developed as described.16 Nasal lavage fluids were diluted 1:2 in PBS, 0.05% vol/vol Tween 20, 0.5 % wt/vol BSA (for IgG1, IgG2, IgG3, and IgG4 determinations); or in TRIS-buffered saline, 0.05% vol/vol Tween 20, 0.5 % wt/vol BSA (for IgA determinations). ELISA measurements were performed as described for serum samples. All determinations were performed in duplicate with less than 5% deviation, and results are displayed as means.

Statistical analyses

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FIG 3. Association of vaccination-induced nasal Bet v 1–specific IgG1 antibody concentrations with the cumulative injected dose (mg) of either Bet v 1 trimer or fragments. The cumulative injected dose (mg) received during treatment is shown on the x-axis. IgG1 levels specific for Bet v 1 are indicated as OD values on the y-axis. Black spots, treatment with Bet v 1–trimer; grey spots, treatment with Bet v 1–fragments; white spots, placebo treatment.

The baseline levels of nasal parameters (nasal flow and resistance) were established by active anterior rhinomanometry (Allergopharma Rhinomanometer; Allergopharma, Reinbek, Germany) without any interaction and 10 minutes after application of 0.9% sodium chloride. Thereafter, the patient received increasing doses of birch pollen extract solution containing 0.0064 mg/mL, 0.064 mg/mL, 0.64 mg/ mL, 6.4 mg/mL, and 64 mg/mL Bet v 1, or placebo. Approximately 0.05 mL (1 pump action) of allergen solutions was administered into both nostrils by using a metered dose pump. During application of test solutions, patients had to hold their breath in full inspiration to avoid bronchial provocation. Changes in nasal parameters were determined 15 and 20 minutes after allergen application. The nasal flow and resistance values with saline were used as a reference for calculations of changes of flow and resistance with birch pollen allergen. Local and systemic symptoms were recorded 15 minutes after provocation (local symptoms—secretion: mild = 1 score, intensive = 2 scores; sneezing: 3-53 = 1 score, >53 = 2 scores; systemic symptoms— tear flow and/or itching of throat and/or ears = 1 score, conjunctivitis and/or chemosis and/or urticaria and/or cough and/or dyspnea = 2 scores). The test was regarded as positive if a 40% or more reduction of nasal air flow was obtained or if a symptom score of 3 was exceeded. If the test was negative after 15 and 20 minutes, the testing procedure was repeated with a 10-fold dose increase. When the test became positive, the maximum allergen concentration tolerated by the patient was recorded, and the provocation test was concluded. Reduction of nasal sensitivity to birch pollen extract in nasal provocation experiments was expressed as change in tolerated concentration steps.

Measurement of antibodies specific for wild-type Bet v 1, Bet v 1–fragments, and Bet v 1–trimer by ELISA Serum IgG1, IgG2, IgG3, and IgG4 subclass antibodies (serum dilution, 1:50) specific for recombinant (r) Bet v 1, Bet v 1–fragments, and Bet v 1–trimer were measured as described.15 Bet v 1–specific serum IgA antibodies (serum dilution, 1:50) were detected by using an alkaline phosphatase–conjugated mouse monoclonal antihuman IgA antibody (G20-359; dilution, 1:1000; BD Pharmingen, San

Spearman rank correlation coefficient r was used to assess correlations between parameters. Wilcoxon 2-sample tests were used to check for differences in IgG1, IgG2, IgG4, and IgA levels between actively treated and placebo-treated patients. P values <.05 were considered statistically significant.

RESULTS Demographic, clinical, and immunological characterization of patients allergic to birch pollen Thirteen men and 10 women with a mean age of 38 years were analyzed. Nine of these patients were allergic exclusively to birch pollen, whereas the other 14 patients also had allergic symptoms to other allergen sources. The patients were sensitized exclusively to the major birch pollen allergen, Bet v 1, within birch pollen. All patients had allergic rhinitis, all but 1 also had allergic conjunctivitis, and 13 had mild seasonal allergic asthma. On an average, patients had received 8.52 injections (placebo = 8.50, range = 8-9; treatment = 8.54, range = 7-10). Actively treated patients had received a cumulative injected dose of 164.4 mg recombinant protein on average (range = 69-265 mg). Vaccination with recombinant Bet v 1 derivatives induces Bet v 1–specific IgG1, IgG2, and IgG4 but not IgG3 and IgA antibodies in nasal secretions Levels of IgG1-4 subclass and IgA antibodies specific for Bet v 1 were measured in nasal secretions obtained in May (after the birch pollen season) and in October 2001 (1 year after the beginning of immunotherapy). In May 2001, IgG1 levels to Bet v 1 were significantly higher in nasal lavage fluids from actively treated patients than in nasal lavage fluids from patients who had received placebo (P < .05; Fig 2, A, left panel). We also found higher Bet v 1–specific IgG2 and IgG4 levels in nasal secretions from actively treated patients compared with the placebo group, but these differences did not reach significance (IgG2, Fig 2, B, left panel; IgG4, Fig 2, C, left panel). In October 2001, 1 year after treatment, antibody concentrations declined in the nasal lavage samples, indicating that vaccination-induced elevations of Bet v 1– specific IgG levels in nasal secretions show a kinetic similar to that of serum antibody levels (Fig 2, A-C, left

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FIG 4. Vaccination with Bet v 1 derivatives (trimer, fragments) induces IgG1 (A) and IgG4 (B) antibodies to wildtype Bet v 1 and to the derivatives. Levels of IgG1 and IgG4 to wild-type Bet v 1, Bet v 1–fragment 1, Bet v 1– fragment 2, and Bet v 1–trimer (x-axis) were determined in the serum of the 13 patients who had received active treatment at 4 different time points. IgG1 (A) and IgG4 (B) levels are indicated as OD values on the y-axis.

FIG 5. Immunotherapy-induced nasal Bet v 1–specific IgG1 (A), IgG2 (B), and IgG4 (C) antibody levels in May 2001 and Bet v 1–specific nasal IgG4 levels in October 2001 (D) are associated with the respective serum antibody concentrations. IgG1,2,4 antibody levels in nasal lavage fluids are shown as OD values on the x-axis. The serum IgG1,2,4 concentrations are indicated as OD values on the y-axis. Black spots, treatment with Bet v 1–trimer; grey spots, treatment with Bet v 1–fragments; white spots, placebo treatment.

panels).8 Active treatment did not lead to increased nasal IgG3 (data not shown) and IgA (Fig 2, D, left panel) levels. Furthermore, we noted no relevant difference between May 2001 and October 2001 measurements of nasal IgG3 and IgA levels. The levels of Bet v 1–specific IgG1 in nasal secretions were dependent on the dose of genetically modified allergens injected during immunotherapy. A statistically significant correlation (r = 0.446; P < .05) was found

between the cumulative injected dose of the derivatives and IgG1 (May 2001) in nasal lavage fluids (Fig 3).

Bet v 1–specific nasal antibodies mirror serum antibody responses Sera had been collected at several time points (Fig 1; before therapy, autumn 2000; after therapy, spring 2001; after the birch pollen season, May 2001; and 1 year after the beginning of the study, autumn 2001).

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FIG 6. Association of vaccination-induced IgG1 and IgG4 antibody levels with changes in nasal sensitivity to birch pollen–derived Bet v 1. Differences between the concentration of birch pollen extract tolerated in nasal provocation experiments before (November 2000) and after (May 2001) immunotherapy are shown on the x-axis as concentration steps. A value of 11 indicates that a 10-fold higher concentration of Bet v 1 was tolerated in May 2001 compared with November 2000. IgG1 (A) and IgG4 (B) levels specific for Bet v 1 are displayed as OD values on the y-axis. Two trimer-treated patients and 1 placebo-treated patient did not undergo the second (May 2001) provocation. Black spots, treatment with Bet v 1–trimer; grey spots, treatment with Bet v 1–fragments; white spots, placebo treatment.

Actively treated patients exhibited significantly higher Bet v 1–specific serum IgG1 (Fig 2, A, right panel), IgG2 (Fig 2, B, right panel), and IgG4 (Fig 2, C, right panel) antibody levels than placebo-treated patients in February 2001 and May 2001 (all P values <.05). No relevant alterations in IgG3 (data not shown) and IgA (Fig 2, D, right panel) levels were observed after active treatment. Active treatment induced IgG1 and IgG4 antibodies specific for intact Bet v 1 as well as for Bet v 1 derivatives (Fig 4). Significant correlations were found between serum and nasal lavage fluid IgG1 (r = 0.572; P < .01; Fig 5, A), IgG2 (r = 0.498; P < .05; Fig 5, B), and IgG4 (r = 0.623; P < .01; Fig 5, C) antibody levels in May and between IgG4 levels in October 2001 (r = 0.461; P < .05; Fig 5, D). No such correlations were found concerning IgA levels (data not shown).

Vaccination-induced Bet v 1–specific IgG antibody levels are associated with reduced nasal sensitivity to Bet v 1 All 23 patients completed the first (November/ December 2000) and third (October 2001) provocation test, but 2 trimer-treated patients and 1 placebo-treated patient did not undergo the second (May 2001) provocation. Therapy-induced Bet v 1–specific IgG4 antibody levels (May 2001) in nasal secretions were significantly correlated with a reduced nasal sensitivity to birch pollen– derived Bet v 1 allergen (r = 0.590; P < .01; Fig 6, B). Furthermore, there is a weak correlation between nasal IgG1 levels and reduced nasal sensitivity (r = 0.425), but this did not achieve statistical significance (P = .062; Fig 6, A). By contrast, there were no associations between

either the nasal IgG2, IgG3, and IgA levels and the results from the May nasal provocation tests, or the October nasal antibody levels and the outcomes of the October nasal provocation tests. None of the patients reacted to saline in nasal provocation tests.

DISCUSSION We have recently described that injection immunotherapy with genetically modified derivatives (ie, rBet v 1 fragments, rBet v 1 trimer) of the major birch pollen allergen, Bet v 1, induces serum IgG antibody responses recognizing the wild-type allergen, which inhibit allergen induced histamine release from basophils in vitro.8 In this substudy, we demonstrate that IgG antibodies appear also in nasal secretions and that their presence is associated with reduced allergen-specific nasal sensitivity. Treatment of patients with genetically modified versions of Bet v 1 induced the generation of antibodies, not only against the Bet v 1–fragments and Bet v 1–trimer, but also against wild-type Bet v 1 allergen in serum and nasal lavage fluids. This finding can be explained by the fact that both types of genetically engineered Bet v 1 derivatives were found to induce IgG antibodies in animals. These IgG antibodies recognized natural, pollen-derived Bet v 1 and inhibited allergic patients’ IgE binding to Bet v 1.9,17 Because the therapy-induced responses to wild-type Bet v 1 were as strong as (ie, trimer) and sometimes even stronger than to the derivatives (ie, fragments), it appears that most of the IgG response induced by vaccination with the derivatives is directed against the wildtype allergen. The composition of the Bet v 1–specific

antibodies in nasal lavage fluids mirrored that of the serum antibodies regarding subclasses and kinetics. In serum as well as in nasal lavage fluids, Bet v 1–specific IgG1, IgG4, and IgG2, low IgA, and no IgG3 responses were induced by vaccination, and the kinetics of antibody levels were similar in serum and nasal lavage fluids. Bet v 1–specific IgG levels were high after vaccination (May) and declined almost to baseline by October of the same year. The latter observations and previous studies18,19 on allergen-specific antibody reactivities in mucosal fluids support the assumption that the nasal allergen-specific IgG may result from transudation. The importance of the vaccine-induced Bet v 1–specific IgG antibodies is indicated by the fact that their concentrations were associated with a reduction of nasal allergen sensitivity and by the observation that this protective effect disappeared after their decline in October. We and others have found that the vaccine-induced IgG antibodies inhibited allergen-induced degranulation of basophils20-23 in vitro and hence assume also that the nasal Bet v 1– specific IgG may act as blocking antibodies that mask IgE epitopes of Bet v 1 and thus prevent mast cell degranulation in the submucosal tissues. In addition, it is possible that the Bet v 1–specific IgG acts as protective shield preventing the intrusion of allergen into the submucosa. In fact, several recent studies point to the importance of allergen-specific IgG antibodies for the success of immunotherapy.20,24 For example, it has been demonstrated that allergen-specific IgG can inhibit IgE-mediated allergen presentation to T cells.25 The nasal mucosa is one of the most important sites for allergic inflammation, and there is also increasing evidence that local IgE production in the nasal mucosa plays an important role in systemic IgE responses.26 In this context, it should be noted that patients vaccinated with the genetically modified Bet v 1 derivatives had exhibited lower boosts of systemic IgE production after seasonal birch pollen exposure than the placebo-treated group.8 It is hence tempting to speculate that vaccine-induced nasal IgG antibodies may inhibit the boosting of IgE memory cells in the nasal mucosa by allergen contact in vaccinated patients. Should further studies provide evidence for the importance of allergen-specific nasal IgG antibody responses for the success of immunotherapy, it will be possible to develop more effective protocols for allergen-specific immunotherapy.

REFERENCES 1. Valenta R, Kraft D. From allergen structure to new forms of allergenspecific immunotherapy. Curr Opin Immunol 2002;14:718-27. 2. Hiller R, Laffer S, Harwanegg C, Huber M, Schmidt WM, Twardosz A, et al. Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment. FASEB J 2002;16:414-6. 3. Valenta R. The future of antigen-specific immunotherapy of allergy. Nat Rev Immunol 2002;2:446-53. 4. Singh MB, de Weerd N, Bhalla PL. Genetically engineered plant allergens with reduced anaphylactic activity. Int Arch Allergy Immunol 1999;119:75-85.

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5. Ferreira F, Wallner M, Breiteneder H, Hartl A, Thalhammer J, Ebner C. Genetic engineering of allergens: future therapeutic products. Int Arch Allergy Immunol 2002;128:171-8. 6. Vrtala S, Focke-Tejkl M, Swoboda I, Kraft D, Valenta R. Strategies for converting allergens into hypoallergenic vaccine candidates. Methods 2004;32:313-20. 7. Drew AC, Eusebius NP, Kenins L, de Silva HD, Suphioglu C, Rollan JM, et al. Hypoallergenic variants of the major latex allergen Hev b 6 retaining human T lymphocyte reactivity. J Immunol 2004;173:5872-9. 8. Niederberger V, Horak F, Vrtala S, Spitzauer S, Krauth MT, Valent P, et al. Development of a novel allergy vaccine: genetically engineered allergens prevent progression of allergic disease. Proc Nat Acad Sci U S A 2004;101(suppl 2):14677-82. 9. Vrtala S, Hirtenlehner K, Vangelista L, Pastore A, Eichler HG, Sperr WR, et al. Conversion of the major birch pollen allergen, Bet v 1 into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy. J Clin Invest 1997;99:1673-81. 10. Vrtala S, Hirtenlehner K, Susani M, Akdis M, Kussebi F, Akdis CA, et al. Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen Bet v 1. FASEB J 2001;15:2045-7. (Express article Available at: www.fasebj.org/cgi/reprint/00-0767fjev1). 11. Pauli G, Purohit A, Oster JP, De Blay F, Vrtala S, Niederberger V, et al. Comparison of genetically engineered hypoallergenic rBet v 1 derivatives with rBet v 1 wild-type by skin prick and intradermal testing: results obtained in a French population. Clin Exp Allergy 2000;30:1076-84. 12. van Hage-Hamsten M, Kronqvist M, Zetterstrom O, Johansson E, Niederberger V, Vrtala S, et al. Skin test evaluation of genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1: results obtained with a mix of two recombinant Bet v 1 fragments and recombinant Bet v 1 trimer in a Swedish population before the birch pollen season. J Allergy Clin Immunol 1999;104:969-77. 13. Ferreira FD, Hoffmann-Sommergruber K, Breiteneder H, Pettenburger K, Ebner C, Sommergruber W. Purification and characterization of recombinant Bet v 1, the major birch pollen allergen: immunological equivalence to natural Bet v 1. J Biol Chem 1993;268:19574-80. 14. Mahler V, Vrtala S, Kuss O, Diepgen TL, Suck R, Cromwell O, et al. Vaccines for birch pollen allergy based on genetically-engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1. Clin Exp Allergy 2004;34:510-2. 15. Vrtala S, Susani M, Sperr WR, Valent P, Laffer S, Dolecek C, et al. Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p 2, Phl p 5). J Allergy Clin Immunol 1996;97:781-7. 16. Denepoux S, Eibensteiner PB, Steinberger P, Vrtala S, Visco V, Weyer A, et al. Molecular characterization of human IgG monoclonal antibodies specific for the major birch pollen allergen Bet v 1: antiallergen IgG can enhance the anaphylactic reaction. FEBS Lett 2000; 465:39-46. 17. Vrtala S, Akdis CA, Budak F, Akdis M, Blaser K, Kraft D, et al. T cell epitope-containing hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1, induce blocking antibodies. J Immunol 2000;165:6653-9. 18. Hoffmann-Sommergruber K, Ferreira ED, Ebner C, Barisani T, Korninger L, Kraft D, et al. Detection of allergen-specific IgE in tears of grass pollen-allergic patients with allergic rhinoconjunctivitis. Clin Exp Allergy 1996;26:79-87. 19. Aghayan-Ugurluoglu R, Ball T, Vrtala S, Schweiger C, Kraft D, Valenta R. Dissociation of allergen-specific IgE and IgA responses in sera and tears of pollen-allergic patients: a study performed with purified recombinant pollen allergens. J Allergy Clin Immunol 2000; 105:803-13. 20. Flicker S, Valenta R. Renaissance of the blocking antibody concept in type I allergy. Int Arch Allergy Immunol 2003;132:13-24. 21. Mothes N, Heinzkill M, Drachenberg KJ, Sperr WR, Krauth MT, Majlesi Y, et al. Allergen-specific immunotherapy with a monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted IgE production and inhibition of basophil histamine release by therapy-induced blocking antibodies. Clin Exp Allergy 2003;33:1-11. 22. Ball T, Sperr WR, Valent P, Lidholm J, Spitzauer S, Ebner C, et al. Induction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy. Eur J Immunol 1999; 29:2026-36.

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23. Clinton PM, Kemeny DM, Youlten LJ, Lessof MH. Histamine release from peripheral blood leukocytes with purified bee venom allergens: effect of hyperimmune beekeeper plasma. Int Arch Allergy Immunol 1989;89:43-8. 24. Wachholz PA, Durham SR. Mechanisms of immunotherapy: IgG revisited. Curr Opin Allergy Clin Immunol 2004;4:313-8.

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25. Wachholz PA, Kristensen Soni N, Till SJ, Durham SR. Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Immunol 2003;112:915-22. 26. Durham SR, Gould HJ, Hamid QA. Local IgE production in nasal allergy. Int Arch Allergy Immunol 1997;113:128-30.

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23. Clinton PM, Kemeny DM, Youlten LJ, Lessof MH. Histamine release from peripheral blood leukocytes with purified bee venom allergens: effect of hyperimmune beekeeper plasma. Int Arch Allergy Immunol 1989;89:43-8. 24. Wachholz PA, Durham SR. Mechanisms of immunotherapy: IgG revisited. Curr Opin Allergy Clin Immunol 2004;4:313-8.

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25. Wachholz PA, Kristensen Soni N, Till SJ, Durham SR. Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Immunol 2003;112:915-22. 26. Durham SR, Gould HJ, Hamid QA. Local IgE production in nasal allergy. Int Arch Allergy Immunol 1997;113:128-30.

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Levocetirizine: Pharmacokinetics and pharmacodynamics in children age 6 to 11 years F. Estelle R. Simons, MD, FRCPC,a and Keith J. Simons, PhDa,b Winnipeg, Manitoba, Canada

From athe Department of Pediatrics and Child Health, Department of Immunology, Canadian Institutes of Health Research National Training Program in Allergy and Asthma, Faculty of Medicine, and bthe Faculty of Pharmacy, Department of Pediatrics and Child Health, Faculty of Medicine, The University of Manitoba. Supported by an Institutional Grant from UCB Pharma, Inc (Belgium) to the University of Manitoba and the Health Sciences Centre. Disclosure of potential conflict of interest: Grants and research support from UCB Pharma. Received for publication December 22, 2004; revised April 4, 2005; accepted for publication April 11, 2005. Available online June 1, 2005. Reprint requests: F. Estelle R. Simons, MD, FRCPC, 820 Sherbrook St, Winnipeg, Manitoba, Canada R3A 1R9. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.010

Key words: H1-antihistamine, levocetirizine, pharmacokinetics, pharmacodynamics, wheal, flare, allergic rhinitis, urticaria, children

The pharmacokinetics (absorption, distribution, metabolism, and excretion) and pharmacodynamics of many medications, including those used in the treatment of allergic diseases, have not been optimally investigated in the pediatric population.1 In the absence of such clinical pharmacology data, drug doses and dose intervals have to be extrapolated from those recommended for adults, and the dose and dose interval selected may not be optimally efficacious or safe in children. Indeed, many drug regulatory agencies now mandate clinical pharmacology studies in the pediatric population.2 More than 40 H1-antihistamines are used in the treatment of allergic rhinitis, urticaria, and other diseases.3 Most of the orally administered H1-antihistamines are available in dosage formulations suitable for administration to children and even to infants; however, only 11 of the 40 H1-antihistamines have been studied prospectively in children with regard to their pharmacokinetics and pharmacodynamics.4-23 These studies have generally been conducted after administration of a single dose,5-10,12-20 but 3 studies have been performed at steady state,11,12,20 and in a few studies, a population pharmacokinetic design21-23 has been used. The clinical pharmacology of a few of the first-generation H1-antihistamines, such as chlorpheniramine, brompheniramine, diphenhydramine, and hydroxyzine, was investigated after they had been used in children for several decades. In contrast, the pharmacokinetics and pharmacodynamics of the secondgeneration H1-antihistamines cetirizine, fexofenadine, ebastine, loratadine, levocetirizine, and mizolastine have been investigated in the pediatric population relatively early in drug development. In the present study our objective was to characterize the pharmacokinetics and pharmacodynamics of the new H1-antihistamine levocetirizine in children aged 6 to 11 years. Levocetirizine24-29 (Fig 1) is the active Renantiomer of the racemate cetirizine. It is highly selective for the human histamine H1-receptor, at which it has twice the binding affinity of cetirizine. Levocetirizine has conformational stability and is not converted to dextrocetirizine, the S-enantiomer, which has 30-fold less binding affinity than cetirizine at the H1-receptor. Levocetirizine is minimally metabolized; during the week after administration of a single oral 14C-labeled dose 355

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Background: The pharmacokinetics and pharmacodynamics of medications may differ between children and adults, necessitating different dose regimens for different age groups. Levocetirizine, the active enantiomer of cetirizine, is used in the treatment of allergic rhinitis and chronic urticaria in Europe. Its pharmacokinetics and pharmacodynamics have not yet been studied prospectively in school-age children. Objectives: This study was performed to investigate levocetirizine pharmacokinetic disposition and pharmacodynamics in relation to skin reactivity to histamine in children aged 6 to 11 years. Methods: Blood samples were obtained at predose baseline and at defined intervals up to and including 28 hours after a 5-mg levocetirizine dose. Concurrently, epicutaneous tests with histamine phosphate, 1 mg/mL, were performed. Wheals and flares were traced at 10 minutes, and the areas were measured with a computerized digitizing system. Results: In children aged 8.6 6 0.4 years (6 SEM), the peak levocetirizine concentration was 450 6 37 ng/mL, and the time at which peak concentrations occurred was 1.2 6 0.2 hours. The terminal elimination half-life was 5.7 6 0.2 hours, the oral clearance was 0.82 6 0.05 mL/min/kg, and the volume of distribution was 0.4 6 0.02 L/kg. Compared with predose areas, the wheals and flares produced by histamine phosphate were significantly decreased from 1 to 28 hours, inclusive (P < .05). Mean maximum inhibition of wheals and flares occurred from 2 to 10 hours (97% 6 1%) and from 2 to 24 hours (93% 6 1%), respectively. Conclusions: Levocetirizine had an onset of action within 1 hour and provided significant peripheral antihistaminic activity for 28 hours after a single dose. Once-daily dosing may be optimal in children aged 6 to 11 years, as it is in adults. (J Allergy Clin Immunol 2005;116:355-61.)

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Abbreviations used EC50: Plasma concentration producing 50% of Emax Emax: Maximum effect attributable to medication

to adults, 85.4% and 12.9% of the drug can be recovered unchanged in urine and feces, respectively.26 Like enantiomers of other medications, levocetirizine is considered to be a new chemical entity, and as such, its pharmacokinetics, pharmacodynamics, efficacy, and safety need to be defined in individuals in various age groups. We hypothesized that in children aged 6 to 11 years, as in adults, it would have prompt onset of action and would also have peripheral H1-antihistaminic activity lasting at least 24 hours after a single dose. Rhinitis, sinusitis, and ocular diseases

METHODS To test the hypothesis stated above, we performed a prospective, open-label, single-dose study of levocetirizine involving objective pharmacokinetic and pharmacodynamic measurements. Approval for levocetirizine administration was obtained through a New Drug Submission to Health Canada. The study protocol was approved by the University of Manitoba Research Ethics Board on the Use of Human Subjects in Research. Before study entry, written assent was obtained from each child, and written informed consent was obtained from the parent or parents of each child.

Selection of participants Children were eligible to participate if they were 6 to 11 years of age, weighed 20 to 40 kg, and had mild allergic rhinitis. They were excluded if they had any recent acute illness or any other health problem except for mild intermittent or persistent asthma or if they required any oral medication, including any oral H1-antihistamines, in the week before study entry or during the study. The only medications permitted before and during the study were as follows: lowdose (100 mg) intranasal glucocorticoids for rhinitis and low-dose (
250 mg) inhaled glucocorticoids and as-needed inhaled albuterol for asthma.

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26, and 28 hours afterward. The first 1 mL of blood was discarded. After each sample was obtained, the catheter was rinsed with 1.5 mL of 0.9% saline. Blood samples were centrifuged at room temperature at 3700 rpm for 10 minutes. The plasma was transferred to polypropylene tubes, which were sealed and frozen at 220°C until measurement of levocetirizine concentrations was performed.22 After each blood sample was collected at the times stated above, peripheral H1-antihistaminic activity was evaluated by one investigator who performed epicutaneous tests with histamine phosphate, 1 mg/mL, on the volar surfaces of the forearms by using sterilized disposable straight needles (Coates & Clark, Greer, SC) and the prickthrough drop technique. All skin tests were performed in duplicate. A different site on the volar surfaces of the forearms was used for each skin test. The sequence of test sites was identical in all children.

Analytic methods Levocetirizine concentrations were determined in plasma samples by using chiral HPLC with tandem mass spectrometric detection after online processing through the column-switching method.22 Quality control samples at 7.5, 150, and 750 ng/mL were assayed in duplicate with each batch of clinical samples. Between-run accuracy and precision were better than 10% throughout the range. The lower limit of quantification for the assay was 12 ng/mL. Wheal-and-flare circumferences were traced with a pen at 10 minutes and transferred to paper by using transparent tape. The tracings were scanned, and the areas were calculated with SigmaScan (Jandel Scientific, San Rafael, Calif). By using this system, with wheal-and-flare sizes ranging from 0.05 to 5.0 cm2 and a sample size of 14 children, differences of 20% could be detected with a 95% level of confidence.

Data analysis Pharmacokinetics. The pharmacokinetic parameters were calculated by using the noncompartmental analysis approach. The elimination rate constant (Ke) was calculated from the plasma levocetirizine concentration (C) versus time (t) data measured after Cmax had occurred, within 0.5 to 2 hours after dosing, by using equation 1: C ¼ C°e-Ket where C° is the plasma concentration extrapolated to zero time after application of equation 1 by using WIN-NONLIN (Scientific Consulting, Apex, NC). The elimination half-life (t1/2) was calculated by using equation 2: t1=2 ¼ ln 2=Ke

Study outline During a preliminary visit to the Manitoba Institute of Child Health Pediatric Allergy Laboratory, the children were assessed for their ability to meet the inclusion criteria of the study. Medical history was obtained, and physical examination, complete blood count, urinalysis, and assessment of hepatic and renal function were performed. The children were given the opportunity to become familiar with the test procedures. In addition to the medication restrictions noted previously, before the levocetirizine dose and for 28 hours afterward, study participants refrained from ingesting methylxanthine-containing substances (eg, cola, chocolate, or cocoa). After an 8- to 10-hour overnight fast, at 8 AM, a single dose of levocetirizine was administered as a 5-mg tablet, followed by 150 mL of water. For the first 1.5 to 2 hours after dosing, only clear juice or water was permitted. EMLA local anesthetic cream (Astra, Mississauga, ON, Canada) was applied to potential venipuncture sites. An indwelling intravenous catheter (Critikon, Tampa, FL) was inserted, and 2.5-mL blood samples were obtained before dosing and at 0.5, 1, 2, 3, 4, 6, 8, 10, 24,

Although levocetirizine appears to be well absorbed, there is no intravenous formulation, and therefore the absolute bioavailability (F) is unknown. Total body clearance (Cl) and apparent volume of distribution (Vd) were calculated as Cl/F and Vd/F, as shown in equation 3: Cl=F ¼ AUC=Dose and equation 4: Vd=F ¼

Cl=F Ke

where AUC is the area under the plasma levocetirizine concentration versus time curve from time zero to 28 hours. Pharmacodynamics. The pharmacodynamic parameters maximum effect attributable to medication (Emax) and plasma concentration producing 50% of Emax (EC50) were calculated by using WIN-NONLIN (Scientific Consulting) and equation 5:

FIG 1. Structure of levocetirizine. The asterisk indicates the position of the chiral center. The molecular weight is 461.8 g/mole. The formula is C21H25CIN2O3.2HCl.

E¼

Emax C EC50 1C

where E is the clinical effect, percentage suppression of histamineinduced wheal or flare, and C is the levocetirizine plasma concentration at which E occurs.30 Statistical analysis. Absolute wheal-and-flare areas over time were analyzed by using 1-way ANOVA, with subject and time as variates, analysis of covariance with predose wheal-or-flare areas as the covariates, and the Tukey and Bonferroni multiple-range tests. Differences were considered to be significant at P values of less than or equal to .05.

TABLE I. Demographics N = 14* (9 boys) Age: 8.6 6 0.4 y Weight: 30.4 6 2.2 kg Height: 132.5 6 3.3 cm Body mass index: 16.9 6 0.6 Levocetirizine dose: 0.18 6 0.01 mg/kg All values are presented as means 6 SEM. *At the time of the study, 4 children were using an intranasal glucocorticoid for mild persistent allergic rhinitis, and 2 were using an orally inhaled glucocorticoid for mild persistent asthma.

RESULTS The 14 Caucasian children (9 boys) with mild allergic rhinitis enrolled in the study had a mean (6 SEM) age of 8.6 6 0.4 years, a mean weight of 30.4 6 2.2 kg, a mean height of 132.5 6 3.3 cm, and a mean body mass index of 16.9 6 0.6. They received a single 5-mg levocetirizine dose, which was equivalent to a mean dose of 0.18 6 0.01 mg/kg (Table I). Complete pharmacokinetic data were available on only 13 of the 14 children because of missing blood samples in 1 child. The mean plasma levocetirizine concentration versus time plot is shown in Fig 2. The pharmacokinetic parameters, including elimination rate constants, area under the plasma concentration versus time curve, oral clearance, and apparent volume of distribution, were calculated by using noncompartmental analysis. The mean maximum plasma levocetirizine concentration of 450 6 37 ng/mL occurred at a mean time of 1.2 6 0.2 hours (Table II). The mean terminal elimination half-life was 5.7 6 0.2 hours, the mean area under the plasma levocetirizine concentration versus time plot was 3549 6 342 ng/mL/h, the mean oral clearance rate was

0.82 6 0.05 mL/min/kg, and the mean apparent volume of distribution was 0.4 6 0.02 L/kg. The mean residence time was 6.8 6 0.3 hours. Pharmacodynamic data were available on all 14 children. Wheal-and-flare areas after testing with histamine phosphate, 1 mg/mL, are shown in Fig 3. Compared with predose values, the wheals were significantly suppressed (P < .05) from 1 to 28 hours, inclusive, with the mean maximum suppression of 97% 6 1% occurring from 2 to 10 hours. Compared with predose values, the flares were significantly suppressed (P < .05) from 1 to 28 hours, inclusive, with the mean maximum suppression of 93% 6 1% occurring from 2 to 24 hours. The relationships between the plasma levocetirizine concentrations and percentage suppression of wheal-and-flare responses compared with predose values versus time are shown in Fig 4. Pharmacodynamic analysis resulted in calculation of an EC50 estimate of 16.1 6 2.2 ng/mL and an Emax estimate of 104.3% 6 2.6% for wheal suppression, and an EC50 of 1.4 6 0.1 ng/mL and an Emax of 94.5% 6 0.4% for flare suppression.

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FIG 2. Plasma levocetirizine concentration (mean 1 SEM) versus time plot after ingestion of levocetirizine, 5 mg.

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TABLE II. Levocetirizine pharmacokinetics Cmax (ng/mL) tmax (h) t1/2 (h) AUC (ng/mL/h) Cl/F (mL/min/kg) Vd/F (L/kg)

450 1.2 5.7 3549 0.82 0.4

6 6 6 6 6 6

37 0.2 0.2 342 0.05 0.02

Values are presented as means 6 SEM. Cmax, Maximum plasma concentration; tmax, time of maximum plasma concentration; t1/2, terminal elimination half-life; AUC, area under the plasma concentration versus time plot; Cl, oral clearance; F, oral bioavailability; Vd, apparent volume of distribution.

There were no serious adverse effects. Two children experienced some sneezing, nasal congestion, and discharge, and 1 child had intermittent coughing. The nasal symptoms were attributed to allergic rhinitis, and the cough was attributed to asthma; that is, the respiratory symptoms were considered to be due to underlying allergic diseases and to be unrelated to administration of the study drug. Two hours after dosing, one child had nausea that was relieved by eating, and 23 hours after dosing, another child had a sore stomach that was relieved by eating. These gastrointestinal symptoms were attributed either to overnight fasting or to anxiety about test procedures and were considered to be probably unrelated to the study drug. One child was more tired than usual 6 hours after the dose of the study drug, and 2 children were more tired than usual 12 hours after dosing. Although this fatigue might have been due to the intensity of the procedures during the first 12 to 13 hours of the study, it was considered to be possibly related to the study drug.

DISCUSSION In this study levocetirizine appeared to be well absorbed, with peak plasma concentrations occurring at

about 1 hour. In the absence of an intravenous levocetirizine formulation, true bioavailability cannot be determined. On the basis of the mean levocetirizine terminal elimination half-life of 5.7 hours that was found, dosing every 24 hours on a regular basis would be expected to lead to minimal or no levocetirizine accumulation in plasma. On the basis of significant wheal-and-flare suppression from 1 to 28 hours after dosing, levocetirizine would be expected to have significant H1-antihistaminic activity throughout the dosing interval. In pharmacokinetic and pharmacodynamic studies of H1-antihistamines, although outcome measures such as blood tests and skin tests are highly objective, they are inherently invasive, and the studies therefore present unique challenges in children.2,4 Study designs do not usually involve a placebo control,7-20 not only because of ethical constraints and parental concerns about the use of placebo, but also because a potent H1-antihistamine suppresses wheals and flares by up to 100%,5,6,12,13 thus making it difficult to maintain double-masked observations and measurements. The objective, standardized, histamine-induced whealand-flare bioassay is useful for studying the onset, amount, and duration of activity of H1-antihistamines. Skin tests with histamine relate to the suppression of wheals, a primary symptom and sign in urticaria, and flares, which are caused by an axon reflex and are thus related to histamine indirectly rather than directly. Whether skin test suppression correlates with events in the airways remains controversial31; however, it is noteworthy that in allergy practice worldwide, skin tests with allergen are performed in lieu of nasal and bronchial allergen challenges to ascertain the relevance of allergens to allergic rhinitis and asthma symptoms, and in the clinical setting cutaneous responses are assumed to reflect airways responses. The pharmacokinetics and pharmacodynamics of levocetirizine, reported here in children aged 6 to 11 years, differ slightly from those reported previously in adults with a mean age of 35 6 2 years and a mean weight of

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FIG 3. The effect of levocetirizine, 5 mg, on the wheals and flares produced by epicutaneous tests with histamine phosphate, 1 mg/mL. A, The wheals (1 SEM) were suppressed from 1 to 28 hours, inclusive (P
.05). B, The flares (1 SEM) were suppressed from 1 to 28 hours, inclusive (P
.05).

FIG 4. Mean plasma levocetirizine concentrations and mean wheal-and-flare percentage suppression compared with predose values, plotted versus time.

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67.3 6 2.3 kg, in whom the time of maximum plasma concentration is 0.73 6 0.7 hours, the terminal elimination half-life value is 7.8 6 0.3 hours, the clearance rate is 0.62 6 0.02 mL/min/kg, and the apparent volume of distribution is 0.41 6 0.02 L/kg.24,25 As noted previously, after administration of radioactively labeled levocetirizine to adults, 85.4% of the drug is eliminated unchanged in the urine, and 12.9% is eliminated unchanged in the feces within 1 week.26 The duration of action of a single levocetirizine dose is greater than 24 hours in adults.27 The pharmacokinetics and pharmacodynamics of levocetirizine reported here in children aged 6 to 11 years also differ from those reported previously in very young children. In a prospective study in children with a mean age of 20.7 6 3.7 months and a mean weight of 11.6 6 1.8 kg, the time of maximum plasma concentration was 1 hour, the terminal elimination half-life was 4.1 6 0.67 hours, the clearance was 1.05 6 0.10 mL/min/kg, and the apparent volume of distribution was 0.37 6 0.06 L/kg.20 Rapid elimination of levocetirizine was also found in a population pharmacokinetic study in which cetirizine was given to 343 children aged 14 to 46 months, and timed sparse blood samples were obtained at steady state for measurement of plasma levocetirizine (the active enantiomer or eutomer) and dextrocetirizine (the inactive enantiomer or distomer) values.22,23 The population pharmacokinetic model used predicted that with increasing body weight, levocetirizine oral clearance would increase by 0.044 L/h/kg, and levocetirizine volume of distribution would increase by 0.639 L/kg. Taken together, the results of these 2 studies indicate that in very young children, compared with older children and adults, higher levocetirizine doses may be needed on a milligram per kilogram basis, and twice-daily dosing may be required. Development of organ function and many of the maturational changes affecting pharmacokinetic disposition of drugs is ongoing throughout infancy and childhood, but elimination through the renal route is largely completed by age 4 to 5 years.1 This study provides a rationale for administration of levocetirizine, 5 mg, once daily in children aged 6 to 11 years, as in adults, with the expectation of prompt onset of action and significant longlasting antihistaminic activity at the H1-receptor. We thank Dr Marc de Longueville and Dr Eugene Baltes for their collaboration. We also thank Dr Nestor Cisneros; Gail Bonin, RN; and especially Sandra S. Goritz, RN, for their contributions to this study.

REFERENCES 1. Kearns GL, Abdel-Rahman SM, Alander SW, Blowey DL, Leeder JS, Kauffman RE. Developmental pharmacology—drug disposition, action and therapy in infants and children. N Engl J Med 2003;349: 1157-67. 2. Steinbrook R. Testing medications in children. N Engl J Med 2002;347: 1462-70. 3. Simons FER. Advances in H1-antihistamines. N Engl J Med 2004;351: 2203-17. 4. Simons FER. H1-antihistamines in children. In: Simons FER, editor. Histamine and H1-antihistamines in allergic disease. 2nd Ed. New York: Marcel Dekker, Inc; 2002. p. 437-64.

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5. Simons FER, Johnston L, Simons KJ. Clinical pharmacology of the H1-receptor antagonists cetirizine and loratadine in children. Pediatr Allergy Immunol 2000;11:116-9. 6. Simons FER, Semus MJ, Goritz SS, Simons KJ. H1-antihistaminic activity of cetirizine and fexofenadine in allergic children. Pediatr Allergy Immunol 2003;111:1244-8. 7. Simons FER, Luciuk GH, Simons KJ. Pharmacokinetics and efficacy of chlorpheniramine in children. J Allergy Clin Immunol 1982;69:376-81. 8. Simons FER, Simons KJ, Becker AB, Haydey RP. Pharmacokinetics and antipruritic effects of hydroxyzine in children with atopic dermatitis. J Pediatr 1984;104:123-7. 9. Simons KJ, Watson WTA, Martin TJ, Chen XY, Simons FER. Diphenhydramine: pharmacokinetics and pharmacodynamics in elderly adults, young adults, and children. J Clin Pharmacol 1990;30:665-71. 10. Simons FER, Roberts JR, Gu X, Kapur S, Simons KJ. The clinical pharmacology of brompheniramine in children. J Allergy Clin Immunol 1999;103:223-6. 11. Schmidt-Redemann B, Brenneisen P, Schmidt-Redemann W, Gonda S. The determination of pharmacokinetic parameters of ketotifen in steady state in young children. Int J Clin Pharmacol Ther Toxicol 1986;24:496-8. 12. Watson WTA, Simons KJ, Chen XY, Simons FER. Cetirizine: a pharmacokinetic and pharmacodynamic evaluation in children with seasonal allergic rhinitis. J Allergy Clin Immunol 1989;84:457-64. 13. Simons FER, Watson WTA, Simons KJ. Pharmacokinetics and pharmacodynamics of ebastine in children. J Pediatr 1993;122:641-6. 14. Simons FER, Bergman JN, Watson WTA, Simons KJ. The clinical pharmacology of fexofenadine in children. J Allergy Clin Immunol 1996; 98:1062-4. 15. Lin CC, Radwanski E, Affrime MB, Cayen MN. Pharmacokinetics of loratadine in pediatric subjects. Am J Ther 1995;2:504-8. 16. Salmun LM, Herron JM, Banfield C, Padhi D, Lorber R, Affrime MB. The pharmacokinetics, electrocardiographic effects, and tolerability of loratadine syrup in children aged 2 to 5 years. Clin Ther 2000;22:613-21. 17. Desager JP, Dab I, Horsmans Y, Harvengt C. A pharmacokinetic evaluation of the second-generation H1-receptor antagonist cetirizine in very young children. Clin Pharmacol Ther 1993;53:431-5. 18. Pariente-Khayat A, Rey E, Dubois MC, Vauzelle-Kervroedan F, Pons G, D’Athis P, et al. Pharmacokinetics of cetirizine in 2- to 6-year-old children. Int J Clin Pharmacol Ther 1995;33:340-4. 19. Spicak V, Dab I, Hulhoven R, Desager J-P, Klanova M, de Longueville M, et al. Pharmacokinetics and pharmacodynamics of cetirizine in infants and toddlers. Clin Pharmacol Ther 1997;61:325-30. 20. Cranswick NE, Fuchs M, Turzikova J. Efficacy, safety and pharmacokinetics of levocetirizine in allergic children age 1-2 years. Clin Pharmacol 2004;75:P46. 21. Mentre´ F, Dubruc C, The´not J-P. Population pharmacokinetic analysis and optimization of the experimental design for mizolastine solution in children. J Pharmacokinet Pharmacodyn 2001;28:299-319. 22. Hussein Z, Pitsiu M, Majid O, Aarons L, de Longueville M, Stockis A, et al. Retrospective population pharmacokinetics of levocetirizine in atopic children receiving cetirizine: the ETAC study. Br J Clin Pharmacol 2005;59:28-37. 23. Simons FER, on behalf of the ETAC Study Group. Population pharmacokinetics of levocetirizine in very young children: the pediatricians’ perspective. Pediatr Allergy Immunol 2005;16:97-103. 24. Baltes E, Coupez R, Giezek H, Voss G, Meyerhoff C, Strolin Benedetti M. Absorption and disposition of levocetirizine, the eutomer of cetirizine, administered alone or as cetirizine to healthy volunteers. Fundam Clin Pharmacol 2001;15:269-77. 25. Tillement JP, Testa B, Bree F. Compared pharmacological characteristics in humans of racemic cetirizine and levocetirizine, two histamine H1-receptor antagonists. Biochem Pharmacol 2003;66:1123-6. 26. Benedetti MS, Plisnier M, Kaise J, Maier L, Baltes E, Arendt C, et al. Absorption, distribution, metabolism and excretion of [14C]levocetirizine, the R enantiomer of cetirizine, in healthy volunteers. Eur J Clin Pharmacol 2001;57:571-82. 27. Devalia JL, De Vos C, Hanotte F, Baltes E. A randomized, double-blind, crossover comparison among cetirizine, levocetirizine, and ucb 28557 on histamine-induced cutaneous responses in healthy adult volunteers. Allergy 2001;56:50-7. 28. Bachert C, Bousquet J, Canonica GW, Durham SR, Klimek L, Mullol J, et al. Levocetirizine improves quality of life and reduces costs in

long-term management of persistent allergic rhinitis. J Allergy Clin Immunol 2004;114:838-44. 29. Potter PC, for the Pediatric Levocetirizine Study Group. Efficacy and safety of levocetirizine on symptoms and health-related quality of life of children with perennial allergic rhinitis. Ann Allergy Asthma Immunol. In press 2005.

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30. Holford NHG, Sheiner LB. Understanding the dose-effect relationship: clinical application of pharmacokinetic-pharmacodynamic models. Clin Pharmacokinet 1981;6:429-53. 31. Simons FER. Antihistamines. In: Adkinson NF Jr, Yunginger JW, Busse WW, Bochner BS, Holgate ST, Simons FER, editors. Middleton’s allergy: principles and practice. 6th ed. St Louis: Mosby, Inc; 2003. p. 834-69.

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Striking deposition of toxic eosinophil major basic protein in mucus: Implications for chronic rhinosinusitis Jens U. Ponikau, MD,a David A. Sherris, MD,b Gail M. Kephart, BS,c Eugene B. Kern, MD,b David J. Congdon, MD,a Cheryl R. Adolphson, MS,c Margaret J. Springett, BS,d Gerald J. Gleich, MD,e and Hirohito Kita, MDc Rochester, Minn, Buffalo, NY, and Salt Lake City, Utah

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Background: The mechanisms by which eosinophilic inflammation damages the epithelium and contributes to recurrent acute exacerbations in chronic rhinosinusitis (CRS) have not been fully elucidated. Objective: We tested the hypotheses that eosinophils deposit toxic major basic protein (MBP) in the mucus and that MBP reaches concentrations able to damage the sinonasal epithelium. Methods: Tissue specimens with mucus attached to the tissue were carefully collected from 22 patients with CRS and examined by using immunofluorescence staining for MBP. This immunofluorescence was digitally analyzed to determine the area covered by MBP and the intensity of the staining (estimating MBP concentration). Levels of MBP in extracts from nasal mucus were quantitated by means of RIA. Results: Heterogeneous eosinophilia was evident within tissue and mucus specimens. All tissue specimens showed intact eosinophils, but diffuse extracellular MBP deposition, as a marker of eosinophil degranulation, was rare. In contrast, all mucus specimens showed diffuse MBP throughout and abundant diffuse extracellular MBP deposition within clusters of eosinophils. Digitized analyses of MBP immunofluorescence revealed increased area coverage (P < .0001) in mucus compared with that seen in tissue. Estimated concentrations of MBP within the clusters suggested toxic levels. MBP concentrations in mucus extract reached 11.7 mg/mL; MBP was not detectable in healthy control subjects. Conclusion: In patients with CRS, eosinophils form clusters in the mucus where they release MBP, which is diffusely deposited on the epithelium, a process not observed in the tissue. Estimated MBP levels far exceed those needed to damage epithelium from the luminal side and could predispose

From athe Department of Otorhinolaryngology–Head and Neck Surgery, cthe Department of Internal Medicine, Division of Allergic Diseases, and dthe Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester; bthe Department of Otorhinolaryngology, University at Buffalo, The State University of New York; and ethe Departments of Dermatology and Medicine, University of Utah, Salt Lake City. Supported by grants from the National Institutes of Health (AI 49235, AI 09728) and from the Mayo Foundation. Received for publication May 7, 2004; revised March 4, 2005; accepted for publication March 31, 2005. Available online June 17, 2005. Reprint requests: Jens Uwe Ponikau, MD, Department of Otorhinolaryngology, Mayo Clinic Rochester, 200 First St SW, Rochester, MN 55905. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.03.049

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patients with CRS to secondary bacterial infections. (J Allergy Clin Immunol 2005;116:362-9.) Key words: Eosinophils, chronic rhinosinusitis, mucus, degranulation, major basic protein

A recent survey by the National Center for Health Statistics reported that 14.2% (29.2 million patients) of the US adult population recalled a health professional’s diagnosis of sinusitis.1 Rhinosinusitis is now preferred to the previous term sinusitis ‘‘. because sinusitis is almost always accompanied by concurrent nasal airway inflammation. .’’2 The economic effect of chronic rhinosinusitis (CRS) is huge; in the US the direct cost was estimated in 1996 at $5.6 billion per year, and the indirect cost was estimated as more than 70 million lost activity days per year.3 Patients with CRS have long-term nasal congestion, thick mucus production, loss of sense of smell, and intermittent acute exacerbations secondary to bacterial infections; they also experience severe quality-of-life impairment.2,4 As an additional burden, CRS lacks a plausible cause. To date, the US Food and Drug Administration has not approved any drug or treatment for CRS; no medical intervention has ever been efficacious in a controlled clinical trial. CRS is an inflammatory disease of the nasal and paranasal mucosa with persistent symptoms for longer than 3 months; its ultimate end stage is inflammatory mucosal thickening and, in a subset of patients, polypoid changes.2,5 The histologic hallmark of CRS is persistent underlying eosinophilic inflammation.5-7 Eosinophil granules contain several cytotoxic proteins,8 and eosinophil granule major basic protein (MBP) is directly toxic to extracellular microorganisms as well as host tissue, including respiratory mucosa.9 CRS specimens show epithelial damage that is colocalized with MBP deposition.6,7,10 In vitro, MBP directly damages respiratory and sinus epithelium in a time- and dose-dependent manner.11,12 Recent histologic analyses of CRS specimens suggested that intact eosinophils migrate from the tissue into the mucus to form distinct and characteristic clusters.13,14 Therefore we tested whether eosinophils release MBP in the mucus, but not in the tissue, and whether MBP reaches concentrations capable of damaging the sinonasal epithelium in patients with CRS.

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METHODS Patient selection

The tissue and the attached mucus were each evaluated with the following scoring system, which was previously used to describe eosinophil7 and neutrophil10 infiltration and degranulation. Because of the heterogeneity of the eosinophilic and neutrophilic inflammation, only those areas of tissue and attached mucus with the most prominent cellular infiltrate for these leukocytes were scored in this semiquantitative scheme. For each specimen, we calculated the means of the examiners’ scores (0, not present; 1, few present/ scattered; 2, many present/abundant) for tissue and for attached mucus using the criteria listed below: d

The diagnostic guidelines and criteria for CRS were consistent with those adopted at the recent Rhinosinusitis Consensus Conference.2 All patients had symptoms consistent with CRS for longer than 3 months, inflamed mucosa on endoscopy, and a coronal computed tomographic scan demonstrating mucosal thickening of greater than 5 mm in more than 2 sinuses. Retention cysts and cystic fibrosis are differential diagnoses to CRS, and if these diseases were diagnosed, those patients were excluded from the study. Because complete immunologic evaluations were not performed in all patients, we have not excluded patients (if any) with immunodeficiencies. With regard to noneosinophilic inflammatory sinusitis, we found eosinophilia in tissues from all patients of our otherwise unselected patient population. Patients were not preselected for having eosinophilic CRS.

Histologic analyses of specimens For the histologic analyses, specimens were collected from 22 consecutive patients with CRS undergoing endoscopic sinus surgery. During surgical intervention, we used Blakesley surgical forceps to carefully and gently collect the maximum amounts of tissue and mucus, and we ensured that the mucus remained attached to the tissue, which was immediately fixed in formalin. Four specimens from the ethmoid sinuses of healthy individuals (nonallergic and no asthma) undergoing septoplasty procedures served as negative controls. The Institutional Review Board of the Mayo Clinic approved the study. Paraffin-embedded tissue blocks with attached mucus were cut in 5-mm-thick serial sections, mounted on positively charged slides, and stained with the following: (1) hematoxylin and eosin; (2) antibody to eosinophil MBP using rabbit antihuman MBP15-17; (3) antibody to neutrophil elastase10 using rabbit antihuman elastase (IgG fraction; Cortex Biochem, San Leandro, Calif); and (4) negative control for MBP and elastase (normal rabbit IgG). All specimens were incubated in 10% normal goat serum to block nonspecific binding by the second-stage antibody and in 1% chromotrope 2R to block nonspecific binding of fluorescein dye to the eosinophils.6 Fluorescein isothiocyanate–conjugated goat anti-rabbit IgG was used as the secondary antibody.15-17 MBP was chosen to assess eosinophil infiltration and degranulation because it is the predominant eosinophil granule protein. It accounts for roughly 50% of the total protein mass in the eosinophil granule,18 and the release of MBP is highly correlated with the release of the other granule proteins.19 Elastase, the predominant neutrophil granule protein, was studied to assess neutrophil infiltration and degranulation.

Semiquantitative analysis of MBP and elastase immunofluorescence in tissue and mucus Three examiners independently examined the entire specimen. To exclude artifacts from trauma caused through the removal of the specimen during surgery, only areas of untouched mucosa covered with mucus were evaluated. In contrast, areas with obvious tears or influx of red blood cells, indicating trauma with bleeding in the areas touched by the forceps, were excluded.

d

d

intact eosinophils or neutrophils; punctate staining (MBP or elastase within intact extracellular granules); and diffuse staining (extracellular MBP or elastase not in granules).

In addition, eosinophils forming clusters within the mucus (eosinophilic mucin) were noted as present (1) or absent (2), and diffuse MBP staining within the clusters was noted as present (1) or absent (2). Neutrophil clusters and elastase deposition were evaluated similarly.

Digital analyses of MBP and elastase immunofluorescence in tissue and mucus Computer analysis of sections stained for MBP or elastase by means of immunofluorescence was performed to evaluate objectively the overall areas covered by MBP or elastase, as well as the intensities of the staining (as indirect markers for MBP or elastase concentrations).6 Briefly, we used a confocal microscope (LSM510 Confocal Microscope; Carl Zeiss, Inc, Oberkochen, Germany) to survey and select both the least and most intense areas of MBP or elastase staining in the tissue and the mucus. First, digital images (512 3 512 pixels, 4003 magnification, 488-nm excitation wavelength) of areas that showed maximal fluorescence staining (most intense accumulation of either eosinophils or neutrophils or diffuse extracellular MBP or elastase deposition) were obtained. Second, digital images of the corresponding areas on the serial section, which was stained with normal rabbit IgG, as the negative control, were recorded. Third, by using image-analysis software (KS400 Image Analysis System, Carl Zeiss, Inc), the threshold for each negative control image was calibrated to a baseline value that showed no positive pixels. Fourth, this background threshold was used to analyze the corresponding area on the MBP or elastase immunofluorescencestained specimen. Any pixels recorded were quantitated as a percentage of an area (512 3 512 pixels) positive for MBP or elastase. The image-analysis software then compared the different areas within the specimen and determined the area with the highest percentage of positive pixels; this percentage indirectly indicated the area of maximal inflammatory eosinophilic or neutrophilic infiltrate (tissue) or maximal MBP or elastase deposition (mucus). A similar survey was made to find the area in the MBP or elastase immunofluorescence specimen with the least fluorescence in the tissue and in the mucus; once located, these areas were compared with the respective corresponding areas in the negative control serial section and analyzed as above.

Electron microscopy and immunogold labeling for MBP We also used electron microscopy to investigate the morphology of eosinophils and eosinophil granules and to localize MBP in tissues from 3 patients with CRS, as described earlier.20 After the primary antibody, affinity-purified antibody to MBP, we used a secondary antibody conjugated to 15 nm colloidal gold particles.17

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Abbreviations used CRS: Chronic rhinosinusitis MBP: Major basic protein

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Quantitative analysis of MBP in mucus Nasal mucus was collected by means of direct trap suctioning from the nasal cavity (middle meatus region) and from one maxillary sinus under endoscopic guidance with a sinus secretion collector (Xomed Surgical Products, Jacksonville, Fla) from 12 additional patients with CRS who met the same diagnostic criteria as described above and from 9 healthy control subjects. The mass of each mucus specimen was obtained, and a 3-fold excess of normal saline (0.15 M NaCl) was added. After vigorous vortexing for 10 seconds (33), the mucus suspension was centrifuged, and the resulting supernatant fluid was frozen at 270°C. (This vortexing step probably did not release the intracellular intragranular MBP, which requires more severe conditions. Specifically, to extract MBP, eosinophils need to be incubated for 30 minutes at room temperature in 0.5% NP-40 [Sigma-Aldrich, St Louis, Mo] and 0.01 M HCl8). Finally, MBP levels in the mucus supernatant fluid were determined by means of RIA, essentially as described earlier.21

Statistical analysis Rhinitis, sinusitis, and ocular diseases

The groups were compared with a 2-sided Student t test or the Wilcoxon matched-pairs signed-rank test, and a P value of less than .05 was considered significant. Data are presented as means (6 SD) or medians (ranges).

RESULTS Patient demographics Patient demographics have been previously described.6 Briefly, the mean age of the 22 patients with CRS was 47 years (range, 16-86 years); 11 were women; the mean number of sinus operations was 1.8 (range, 0-7); the mean duration of disease was 8.6 years (range, 2-27 years); the incidence of aspirin idiosyncrasy was 41%; 11 had increased serum levels of total IgE (>128 U/mL, 2 SDs above the mean value of healthy adult control subjects); and 10 were considered allergic, as defined by a positive skin prick test response to at least one allergen from a panel of 16 common aeroallergens. The incidence of physician-diagnosed asthma was 68% (15/22); the other 7 patients underwent a methacholine challenge, and 5 had positive responses. Eosinophil and neutrophil infiltration and degranulation in tissue and mucus Although intact eosinophils were abundant in numerous areas in the tissue (Fig 1, A and B), the most striking observation was the abundance of diffuse MBP staining (not in cells and not in granules) in the mucus compared with its absence in the tissue (Fig 1, A-D). Eosinophils formed cell clusters in the mucus, and diffuse MBP staining was observed within and around these clusters (Fig 1, A-D and F). Punctate staining, indicating intact extracellular eosinophil granules, was frequently observed in both tissue and mucus (Fig 1, E and F). Compared with MBP staining, only isolated areas in tissue and mucus stained for neutrophil elastase (results not shown). As summarized in Fig 2 (top), tissue in all specimens showed abundant intact eosinophils (22/22) compared with mucus (11/22). The numbers of patient specimens

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showing various amounts of punctate staining were similar between tissue and mucus. Diffuse MBP deposition in the mucus was abundant in all (22/22) CRS specimens but was not observed in the tissue, except in one small area from 1 specimen (1/22). Intact neutrophils were evident in both tissue (10/22) and mucus (14/22) specimens. In contrast to the numerous areas showing diffuse MBP deposition in the mucus of all 22 specimens (Fig 2, A), isolated areas of diffuse elastase deposition were noted in the mucus of only 9 of 22 patients. The majority of specimens showed a virtual absence of diffuse elastase in the mucus (Fig 2, B). Eosinophils forming clusters within the mucus (eosinophilic mucin) were present in 22 of 22 specimens, and diffuse deposition of MBP in and around these clusters was seen in 22 of 22 specimens. In contrast, neutrophil cluster formation was observed in only 5 of 22 specimens, and elastase deposition in these clusters was observed in only 4 of 22 specimens. None of the specimens from healthy control subjects (0/4) were positive for intact eosinophils or neutrophils or punctate or diffuse staining for MBP or elastase (results not shown).

Digital analyses of MBP and elastase immunofluorescence In the tissue the maximum area positive for MBP immunofluorescence staining had a median of 18.35% (range, 0.64% to 56.63%); by contrast, the maximum area positive for elastase immunofluorescence was significantly less (median, 3.11%; range, 0.05% to 46.2%; P < .002; Fig 3). In the mucus the maximum area positive for MBP immunofluorescence staining had a median of 93.28% (range, 3.62% to 100%); by contrast, the maximum area positive for elastase immunofluorescence was also significantly less (median, 29.30%; range, 0.10% to 85.42%; P < .001; Fig 3). Furthermore, the maximum area positive for MBP immunofluorescence in the mucus (median, 93.28%) was significantly increased compared with the maximum mean area in tissue (median, 18.35%; P < .0001). Electron microscopy and immunogold labeling for MBP In tissue from a patient with CRS, both an intact eosinophil containing the characteristic electron-dense granule cores with MBP and extracellular granules are visible (Fig 4, A). However, the immunogold MBP stain shows MBP confined within the intact granules (Fig 4, B), corresponding with the punctate staining in MBP immunofluorescence (see Fig 1, E). Diffuse MBP immunogold staining is not seen in the tissue (Fig 4, B), which is consistent with the lack of diffuse MBP immunofluorescence staining seen in the tissue (see Fig 1, E). Measurement of MBP in the mucus As shown in Fig 5, the mean concentration of detectable MBP in mucus extracts from the maxillary sinuses in patients with CRS was 4.2 mg/mL (6 3.1 mg/mL; range,

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FIG 1. Photomicrographs of CRS specimens stained for MBP by means of immunofluorescence or stained with hematoxylin and eosin. Panel a demonstrates eosinophilic inflammation in tissue, eosinophil clusters (black arrows) in mucus, subepithelial basement membrane thickening, and damaged epithelium (yellow arrows) (hematoxylin and eosin counterstain of Panel b; original magnification, 1603). Panel b shows MBP in tissue is contained within the cells or in intact granules (punctate staining) outside the cells. In mucus, diffuse MBP staining is in eosinophil clusters (white arrows) and outside of clusters (anti-MBP; original magnification, 1603). Panel c shows minimal tissue eosinophilia, massive eosinophilia in mucus, subepithelial basement membrane thickening, and the damaged epithelium (yellow arrows) (hematoxylin and eosin; original magnification, 4003). Panel d (serial section of Panel c) shows few intact eosinophils in tissue, intense diffuse MBP deposition within the mucus, and MBP adjacent to the epithelial surface (anti-MBP; original magnification, 4003). Panels e (tissue) and f (mucus) show intact eosinophils (white arrows) and free granules (punctate staining, blue arrows); diffuse extracellular MBP staining (orange arrows) appears unique to mucus (anti-MBP; original magnification, 14003). Serial sections stained with normal rabbit IgG were negative (results not shown).

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FIG 2. Comparisons of eosinophilic and neutrophilic inflammation in tissue versus mucus. The graphs show the mean MBP and elastase immunofluorescence scores for intact eosinophils and neutrophils, punctate staining (MBP and elastase within intact extracellular granules), and diffuse staining (extracellular MBP and elastase not in granules) in tissue and mucus from 22 patients with CRS. Each dot represents the mean score of 3 independent examiners for each patient (scoring on vertical axis follows the grading system presented in the Methods section). Panel a shows a CRS specimen stained with anti-MBP; note the abundant diffuse MBP in mucus that is absent in tissue. Panel b shows a CRS specimen stained with anti-elastase; note the virtual absence of diffuse elastase in both mucus and tissue (original magnification, 4003).

0.3-11.7 mg/mL; n = 12). In 9 of 12 patients with CRS, we were also able to harvest sufficient mucus from the nasal cavity to detect a mean MBP concentration of 4.1 mg/mL (6 2.6 mg/mL; range, 0-8.0 mg/mL), which did not differ significantly from the mean concentration in the maxillary sinuses. In contrast, in mucus specimens from the nasal cavities of the healthy control subjects, no MBP could be detected above the sensitivity of the assay (0.010 mg/mL). Thus even the lowest MBP concentration detected in mucus from the maxillary sinus of a patient with CRS (0.31 mg/mL) was at least 30-fold greater than that of healthy control subjects.

DISCUSSION The underlying eosinophilic inflammation is increasingly recognized to play an important role in the pathogenesis of CRS, and its association with epithelial damage has been suspected.6,7 How the eosinophil actually mediates the pathophysiology, such as damaging the epithelium, remains unclear. Earlier studies in patients with CRS used tissue biopsy specimens without mucus7,10 and showed MBP deposition within damaged sinus epi-

thelium; in contrast, our specimens were carefully collected to ensure that the mucus remained attached to the harvested tissue. Thus we could document the extent, localization, and degranulation pattern of the heterogeneous eosinophilia and neutrophilia in both the tissue and the mucus. Eosinophil, rather than neutrophil, inflammation was predominant in both tissue and mucus. Striking extracellular deposition of diffuse MBP (especially in clusters) was unique to the mucus in all 22 patients with CRS and was not found in the tissue (with the exception of one small area in 1 patient). In contrast, extracellular deposition of diffuse elastase was found in less than half of the patients (9/22) and only in isolated areas. The pattern of eosinophil degranulation described in this study could explain the sinonasal epithelial erosion observed in patients with CRS.6 As seen in Fig 1, A and C, the outer layers of the epithelium are eroded away, but a layer of basal epithelial cells still remains. This observation, as well as the marked deposition of toxic eosinophil granule MBP in the mucus, suggests that the damage to the epithelium occurs from the outside (luminal side). The damaged epithelium might provide an entry port for colonizing bacteria that are present in the sinuses of patients with CRS, as well as healthy control subjects.22,23 Thus

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FIG 3. Comparison of digitized areas of minimal and maximal MBP or elastase staining in the tissue and mucus of patients with CRS. To demonstrate the heterogenicity within the 22 specimens, these data points represent the minimal and maximal percentages of area positive for MBP or elastase immunofluorescence. The horizontal lines show the median values in each group.

the release of toxic eosinophil granule proteins, such as MBP, in the mucus could be a crucial predisposing factor for the secondary bacterial infections that likely mediate the acute exacerbations of CRS. In healthy control subjects, the absence of MBP in the mucus might also explain the lack of epithelial damage and, consequently, the lack of bacterial infections, despite the presence of bacteria.22,23 Taken together, these findings might explain the complex clinical course of CRS with its occasional bacterial exacerbations; indeed, the numbers of infiltrating tissue neutrophils have been directly correlated to the numbers of bacteria present.24 Overall, these exacerbations are superimposed on the persistent and underlying eosinophilic inflammation uniformly seen in all patients. Although we used an RIA for MBP in extracts from mucus specimens, this procedure likely underestimated the actual local concentrations for the following reasons. First, we attempted to extract MBP from the thick mucus through vortexing in saline, and thus only the MBP that actually dissolved in the saline could be measured. The remaining mucus and probably a large amount of undissolved MBP had to be discarded. Second, the concentration of MBP was based on the entire volume of the mucus specimen; because portions of the mucus do not contain MBP (Fig 3), our results underestimate the local maximal MBP concentrations. To address this potential underestimation, we calculated the approximate MBP concentration in an eosinophil to be 33 mg/mL or 2.1 3 1023 M (8.98 3 1029 mg [mass MBP/eosinophil]/2.68 3 10210 mL [volume/eosinophil]).8 Although the immunogenic epitopes might not be equally available for MBP in mucus compared with MBP in the granules of intact cells, we used digital

analyses to compare the brightness of anti-MBP staining in confocal microscopic images. Overall, 17 of 22 specimens showed brighter immunofluorescence staining for MBP in mucus compared with that seen in tissue; no brightness differences were noted for intact cells in tissue, blood vessels, or mucus. Thus we estimate that areas of diffuse MBP in the mucus might exceed 33 mg of MBP/mL. Because MBP is toxic to and causes erosion of the epithelium at concentrations less than 10 mg/mL,12 our MBP immunofluorescence results suggest that MBP reaches local concentrations in the mucus of patients with CRS far exceeding those necessary to mediate epithelial damage. In addition, inspissation (dessication) of mucus at mucosal surfaces in vivo might lead to a further increase in the local MBP concentration. We observed striking MBP deposition directly adjacent to the damaged epithelium (Fig 1, A-D). In the tissue, extracellular MBP is confined to intact eosinophil granules (ie, punctate staining), as shown by means of MBP immunofluorescence staining (Fig 1, B, D, and E) and by means of electron microscopy and immunogold labeling for MBP (Fig 4). The apparent lack of subepithelial tissue damage, as assessed with hematoxylin-and-eosin staining of specimens from patients with CRS, suggests that intact granules containing MBP might not have physiologic or damaging actions in the tissue. The accumulation of diffuse MBP immunofluorescence in the mucus (and not in the tissue) suggests that the cells found in the tissue are in transit toward their final target in the mucus, with ongoing deposition of MBP (ie, the cluster of eosinophils surrounded by the diffuse cloud of MBP). This pattern of cluster-forming eosinophils appears strikingly similar to the eosinophils’ role in their immune

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FIG 4. Transmission electron micrographs of sinus tissue from a patient with CRS. Panel a shows the characteristic electron-dense secondary granules within an intact cell (white arrows) and intact extracellular granules in the tissue (black arrows; original magnification, 10,0003). Panel b shows immunogold labeling (black dots) for MBP and demonstrates that MBP is localized within the intact granules; note the lack of MBP labeling in the surrounding tissue (original magnification, 33,0003).

FIG 5. MBP concentrations in mucus specimens from patients with CRS and healthy control subjects. Mucus specimens were extracted with 0.15 M NaCl, and MBP was measured in the supernatants by means of RIA. MBP was detected in the maxillary sinus mucus and in the nasal cavity mucus of patients with CRS but not in mucus from the healthy control subjects. Horizontal bars indicate mean values for each group.

defense against parasites when they cluster around the organisms, subsequently releasing granular proteins (including MBP) that destroy the parasite.25 It is generally assumed that eosinophil cytolysis and piecemeal degranulation are distinct mechanisms by which granules and, subsequently, granule proteins are released in diseased airway tissue.26 Instead, we found that

eosinophils release cytotoxic MBP in the mucus, but not in the tissue, at concentrations likely exceeding those needed to damage the epithelium in patients with CRS. Therefore one might need to take not only the tissue but also the mucus into account when trying to understand the pathophysiology of CRS and probably other airway diseases. In addition, this new understanding suggests a beneficial

effect in therapies that target primarily the underlying and presumably damage-inflicting eosinophilic inflammation instead of the secondary bacterial infection. REFERENCES 1. Lethbridge-Cejku M, Schiller JS, Bernadel L. Summary health statistics for U.S. Adults; National Health Interview Survey, 2002. National Center for Health Statistics. Vital Health Stat 2004;10:23. 2. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas RA, et al. Rhinosinusitis: establishing definitions for clinical research and patient care. J Allergy Clin Immunol 2004;114(suppl):S155-212. 3. Ray NF, Baraniuk JN, Thamer M, Rhinehart CS, Gergen PJ, Kaliner M, et al. Healthcare expenditures for sinusitis in 1996: contributions of asthma, rhinitis, and other airway disorders. J Allergy Clin Immunol 1999;103:408-14. 4. Gliklich RE, Metson R. The health impact of chronic sinusitis in patients seeking otolaryngologic care. Otolaryngol Head Neck Surg 1995;113: 104-9. 5. Kaliner MA, Osguthorpe JD, Fireman P, Anon J, Georgitis J, Davis JL. Sinusitis: bench to bedside. Current findings, future directions. Otolaryngol Head Neck Surg 1997;116(suppl):S1-20. 6. Ponikau JU, Sherris DA, Kephart GM, Kern EB, Gaffey TA, Tarara JE, et al. Features of airway remodeling and eosinophilic inflammation in chronic rhinosinusitis: is the histopathology similar to asthma? J Allergy Clin Immunol 2003;112:877-82. 7. Harlin SL, Ansel DG, Lane SR, Myers J, Kephart GM, Gleich GJ. A clinical and pathologic study of chronic sinusitis: the role of the eosinophil. J Allergy Clin Immunol 1988;31:867-75. 8. Abu-Ghazaleh RI, Dunnette SL, Loegering DA, Checkel JL, Kita H, Thomas LL, et al. Eosinophil granule proteins in peripheral blood granulocytes. J Leukoc Biol 1992;52:611-8. 9. Gleich GJ, Adolphson CR, Leiferman KM. The biology of the eosinophilic leukocyte. Annu Rev Med 1993;44:85-101. 10. Fujisawa T, Kephart GM, Gray BH, Gleich GJ. The neutrophil and chronic allergic inflammation: immunochemical localization of neutrophil elastase. Am Rev Respir Dis 1990;141:689-97. 11. Frigas E, Loegering DA, Gleich GJ. Cytotoxic effects of the guinea pig eosinophil major basic protein on tracheal epithelium. Lab Invest 1980; 42:35-43. 12. Hisamatsu K, Ganbo T, Nakazawa T, Murakami Y, Gleich GJ, Makiyama K, et al. Cytotoxicity of human eosinophil granule major basic protein to human nasal sinus mucosa in vitro. J Allergy Clin Immunol 1990;86:52-63.

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13. Ponikau JU, Sherris DA, Kern EB, Homburger HA, Frigas E, Gaffey TA, et al. The diagnosis and incidence of allergic fungal sinusitis. Mayo Clin Proc 1999;74:877-84. 14. Braun H, Busina W, Freudenschuss K, Beham A, Stammberger H. ‘‘Eosinophilic fungal rhinosinusitis’’: a common disorder in Europe? Laryngoscope 2003;113:264-9. 15. Filley WV, Ackerman SJ, Gleich GJ. An immunofluorescent method for specific staining of eosinophil granule major basic protein. J Immunol Methods 1981;47:227-38. 16. Filley WV, Holley KE, Kephart GM, Gleich GJ. Identification by immunofluorescence of eosinophil granule major basic protein in lung tissues of patients with bronchial asthma. Lancet 1982;2:11-6. 17. Peters MS, Schroeter AL, Kephart GM, Gleich GJ. Localization of eosinophil granule major basic protein in chronic urticaria. J Invest Dermatol 1983;81:39-43. 18. Kita H, Adolphson CR, Gleich GJ. Biology of eosinophils. In: Adkinson NF Jr, Bochner BS, Yunginger JW, Holgate ST, Busse WW, Simons FE, editors. Middleton’s allergy: principles and practice. 6th ed. Philadelphia: Mosby; 2003. p. 305-32. 19. Ott NL, Gleich GJ, Peterson EA, Fujisawa T, Sur S, Leiferman KM. Assessment of eosinophil and neutrophil participation in atopic dermatitis: comparison with the IgE-mediated late-phase reaction. J Allergy Clin Immunol 1994;94:120-8. 20. Popken-Harris P, Checkel J, Loegering D, Madden B, Springett M, Kephart G, et al. Regulation and processing of a precursor form of eosinophil granule major basic protein (proMBP) in differentiating eosinophils. Blood 1998;92:623-31. 21. Wagner JM, Bartemes K, Vernof KK, Dunnette S, Offord KP, Checkel JL. Analysis of pregnancy-associated major basic protein levels throughout gestation. Placenta 1993;14:671-81. 22. Kalcioglu MT, Durmaz B, Aktas E, Ozturano O, Durmaz R. Bacteriology of chronic maxillary sinusitis and normal maxillary sinuses: using culture and multiplex polymerase chain reaction. Am J Rhinol 2003;17:143-7. 23. Nadel DM, Lanza DC, Kennedy DW. Endoscopically guided cultures in chronic sinusitis. Am J Rhinol 1998;12:233-41. 24. Dunnette SL, Hall MM, Washington JA 2nd, Kern EB, McDonald TJ, Facer GW, et al. Microbiologic analyses of nasal polyp tissue. J Allergy Clin Immunol 1986;78:102-8. 25. Kephart GM, Gleich GJ, Connor DH, Gibson DW, Ackerman SJ. Deposition of eosinophil granule major basic protein onto microfilariae of Onchocerca volvulus in the skin of patients treated with diethylcarbamazine. Lab Invest 1984;50:51-61. 26. Erjefalt JS, Persson CG. New aspects of degranulation and fates of airway mucosal eosinophils. Am J Respir Crit Care Med 2000;161: 2074-8.

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Intranasal tolerance induction with polypeptides derived from 3 noncrossreactive major aeroallergens prevents allergic polysensitization in mice Karin Hufnagl, PhD,a Birgit Winkler, MD,a Margit Focke, PhD,a Rudolf Valenta, MD,a Otto Scheiner, PhD,a Harald Renz, MD,b and Ursula Wiedermann, MD, PhDa Vienna, Austria, and Marburg, Germany

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Background: Specific immunotherapy is less effective in patients with multiple allergic sensitizations compared with monosensitized patients. Objective: We therefore established a mouse model of polysensitization to the major birch and timothy grass pollen allergens to test whether allergic polysensitization can be prevented by multiple allergen application via the mucosal route. Methods: Female BALB/c mice were immunized intraperitoneally with recombinant (r) Bet v 1, rPhl p 1, and rPhl p 5. For intranasal tolerance induction, a mixture of the complete allergens was compared with allergen-derived immunodominant peptides applied either as a mixture or as a synthetic hybrid peptide composed of the T-cell epitopes of the 3 allergens. Results: Intranasal application of the mixture of the complete allergen molecules did not prevent polysensitization to the same allergens. In contrast, pretreatment with a mixture of the immunodominant peptides or the hybrid peptide led to significantly reduced allergen-specific IgE responses in sera, IL-4 production in vitro, and suppressed airway inflammation. TGF-b mRNA levels did not change, and IL-10 production was significantly suppressed after the pretreatment. The fact that the reduction of IL-10 was not abrogated after IL-10 receptor neutralization and that tolerance was not transferable with splenocytes indicates that the suppression of TH2 responses in polysensitized mice might not be mediated by immunosuppressive cytokines. Conclusion: Our study demonstrates that it is possible to suppress allergic immune responses simultaneously to several clinical important allergens. Thus, mucosal coapplication of selected peptides/hybrid peptides could be the basis of a

From athe Department of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology, Medical University of Vienna; and b the Department of Clinical Chemistry and Molecular Diagnostics, Hospital of the Philipps University Marburg. Supported by grants from the Austrian Science Fund (F01814, P14634-PAT, Y0784GEN, FWF T163). Received for publication July 9, 2004; revised March 10, 2005; accepted for publication April 1, 2005. Available online May 24, 2005. Reprint requests: Ursula Wiedermann, MD, PhD, Department of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.002

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mucosal polyvalent vaccine to prevent multiple sensitivities in atopic patients. (J Allergy Clin Immunol 2005;116:370-6.) Key words: Animal model, type I allergy, polysensitization, mucosal tolerance, polypeptides, hybrid, IL-10, TGF-b

About 25% of the population in industrialized countries has type I allergy, a genetically determined immunological disorder. In Europe, the most common seasonal airborne allergens are derived from white birch (Betula verrucosa) and timothy grass (Phleum pratense).1 More than 90% of the patients allergic to birch pollen (BP) react to the major allergen Bet v 1, and in patients allergic to grass pollen, Phl p 1 and Phl p 5 represent major allergens.2-4 There is substantial clinical evidence that many patients with allergy are cosensitized to several unrelated airborne allergens. In this context, recent studies demonstrated that BP allergy is frequently associated with sensitization to other inhalant allergens, including grass pollen, and that multiallergies often develop with increasing age.5,6 One of the most effective treatments for type I allergy, especially in young and monosensitized patients, is specific immunotherapy, performed by repeated subcutaneous injections of increasing amounts of allergen extracts.7 In patients with multiple sensitivities, specific immunotherapy is known to be of low efficacy and is associated with an increased risk of anaphylactic side reactions.7,8 Thus, there is a clear necessity to improve or develop new treatment strategies particularly for polysensitized individuals. Such improvements could be achieved by the use of recombinant allergens or allergen-derived peptides according to the patient’s sensitization profile,9-11 instead of natural allergen extracts containing a variety of allergenic molecules. Recently the possibility of using prophylactic allergy vaccines has been discussed.12 Following the concept of vaccination against many infectious diseases, prophylactic treatment to most common allergens could be a possible strategy for early allergy prevention. In addition, the change to a less invasive route of administration, such as application of allergens via the mucosal surfaces, might even enhance

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the patient’s compliance to treatment. In several experimental animal studies, it has been shown that mucosal tolerance is highly effective in preventing allergic disease.13-17 A recent study in mice demonstrated that the intranasal route of desensitization can be even more effective than the intradermal route.18 We previously demonstrated in a murine model of allergic sensitization to BP that mucosal administration of recombinant (r) Bet v 1 suppressed allergic sensitization and airway inflammation in naive and in sensitized mice.16,17,19 In the current study, we established a murine model of polysensitization to the noncross-reactive major birch and grass pollen allergens Bet v 1, Phl p 1, and Phl p 5. By using this model, we tested whether it is possible to induce tolerance simultaneously against these different pollen allergens by intranasal application of either the allergen proteins or the allergen-derived peptides composed of the immunodominant epitopes thereof. Our results show that intranasal application of polypeptides successfully prevented allergic polysensitization.

METHODS Animals Female inbred 7-week-old BALB/c mice (n = 5 per group) were obtained from Charles River (Sulzfeld, Germany). All experiments were approved by the Animal Experimentation Committee of the University of Vienna and the Federal Ministry of Education, Science and Culture.

Synthesis, purification, and characterization of peptides for intranasal pretreatment Subsequent to the identification of the immunodominant regions of Bet v 1, Phl p 1, and Phl p 5 by epitope mapping, the respective peptides (see Results and Tables E1-E3 in the Journal’s Online Repository at www.mosby.com/jaci) for intranasal pretreatment were synthesized by using a 9-fluorenylmethoxycarbonyl strategy with 2-(1H-benzotriazol-1-yl)1,1,3,3 tetramethyluronium hexafluorophosphat activation (0.1-mmol small-scale cycles) on the Applied Biosystems (Foster City, Calif) peptide synthesizer Model 433A.20 The identity to the peptides was checked by mass spectrometry, and the peptides were purified to >90% purity by preparative HPLC (Pichem, Graz, Austria).

Dose-finding and kinetic studies for intranasal pretreatment Dose-finding experiments and kinetic studies with Bet v 1, Phl p 1, and Phl p 5 proteins were performed by using 0.1 to 100 mg/antigen applied 3 times at 7-day intervals or every third day for 3 weeks. In line with previous studies in monosensitized mice, a treatment regimen with 10 mg applied 3 times every 7 days was chosen.16,17 Dose-finding experiments with the immunodominant peptides (1-100 mg/peptide) revealed that low-dose application (ie, 5 mg/peptide) was optimal for intranasal tolerance induction.

Polysensitization and intranasal pretreatment Polysensitization was performed by 3 intraperitoneal injections (days 22, 36, and 50) of a mixture of 5 mg rBet v 1, 5 mg rPhl p 1, and 5 mg rPhl p 5 adsorbed to aluminium hydroxide (Al[OH]3; Serva, Heidelberg, Germany) at 14-day intervals (group 1). For intranasal pretreatment, a mixture of the 3 allergens, Bet v 1, Phl p 1, and Phl p 5 (10 mg each), was applied intranasally in 30 mL 0.9% NaCl 3 times at 7-day intervals (days 0, 7, and 14) before polysensitization (group 2). Peptide pretreatment was performed by applying a mixture of 5 mg Bet v 1 peptide, 5 mg Phl p 1 peptide, and 5 mg Phl p 5 peptide 2 in a volume of 30 mL (group 3). Intranasal pretreatment with the hybrid peptide was performed by using a concentration of 20 mg construct in 30 mL per application (group 4). Control mice were intranasally shamtreated with 30 mL 0.9% NaCl before polysensitization (group 1). One week after the last intraperitoneal immunization, an aerosol challenge with 1% wt/vol BP and Phleum extract was performed on 2 consecutive days, as previously described.17

Sampling Recombinant allergens and natural allergen extracts rBet v 1, rPhl p 1, and rPhl p5 were obtained from Biomay AG (Vienna, Austria). Birch pollen (B verrucosa) and timothy grass pollen (P pratense) were purchased from Allergon (Va¨linge, Sweden), and extracts were prepared as previously described.16

Epitope mapping studies For T-cell epitope mapping, a panel of 50 peptides of the Bet v 1 molecule (Cambridge Research Biochemicals Limited, Cambridge, United Kingdom), 77 peptides of Phl p 1 (Cambridge Research Biochemicals Limited), and 92 peptides of Phl p 5 (provided by Dr Helmut Fiebig, Reinbek, Germany) were used. Spleen cell suspensions from Bet v 1, Phl p 1, and Phl p 5 immunized mice were incubated with each of the dodecapeptides (2 mg/well), spanning the whole amino acid (aa) sequence of the respective antigens. The dodecapeptides overlapped by 9 residues. Proliferative responses were measured according to a previous description.16

Blood samples were taken before treatment and 2 days after the aerosol challenge (day 60) by tail bleeding. After the bleeding, the mice were sacrificed, spleen cell suspensions were prepared, and bronchoalveolar lavages were collected, as previously described.16,17

Airway eosinophilia Bronchoalveolar lavage samples were spun onto microscope slides and stained with hematoxylin and eosin (Hemacolor; Merck, Darmstadt, Germany). The number of eosinophils was expressed as percentage of the total counted cell number, as previously described.19

Detection of allergen-specific antibody levels in serum Microtiter plates (Nunc, Roskilde, Denmark) were coated with each of the recombinant allergens (5 mg/mL) before incubation with sera. Rat antimouse IgG1, IgG2a, and IgE antibodies (1/500; Pharmingen, San Diego, Calif) were used, followed by peroxidaseconjugated mouse antirat IgG antibodies (1/2000; Jackson Immuno

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Abbreviations used aa: Amino acid BP: Birch pollen r: Recombinant RBL: Rat basophil leukemia

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TABLE I. Allergen-specific antibody levels, T-cell proliferation, and cytokine production in polysensitized mice rBet v 1

IgG1* IgE* IgG2a* Stimulation index 

1.98 0.85 0.51 2.97

6 6 6 6

0.28 0.21 0.35 0.51

rPhl p 1

1.92 0.48 0.31 2.63

6 6 6 6

0.05 0.05 0.12 1.02

rPhl p 5

1.61 0.53 0.75 4.41

6 6 6 6

0.27 0.02 0.37 2.38

IFN-gà IL-4à IL-5à

BP

Phleum

1501.9 6 621.1 28.6 6 14.6 51.7 6 42.52

1307.9 6 372.7 65.8 6 17.3 246.7 6 105.1

*Serum antibody levels (OD) were measured by ELISA.  Spleen cells from polysensitized mice were cultured with the respective antigens for 4 days (stimulation index, background medium values 5820.2 6 1519.3 cpm). àCytokine levels (pg/mL) were measured by ELISA. All results are mean values (6SDs) from 3 independent experiments with 5 animals per experiment.

Lab, West Grove, Pa).16 Results show the OD values after subtraction of baseline levels (0.058 6 0.025) from preimmune sera.

Rat basophil leukemia cell mediator release assay Rhinitis, sinusitis, and ocular diseases

Rat basophil leukemia (RBL)-2H3 cells were incubated with sera obtained from pretreated and polysensitized mice at dilutions of 1/30 to 1/300. Degranulation of RBL cells was induced by adding 0.03 mg of each allergen diluted in 100 mL Tyrode’s buffer. Supernatants were analyzed for b-hexosaminidase activity as previously described.21 Results are reported as percentages of total b-hexosaminidase released after addition of 1% Triton X-100 and are shown after subtraction of baseline release levels (1.13 6 0.81) obtained with preimmune sera.

Lymphocyte proliferation and cytokine production Proliferation of splenocytes (2 3 105 cells/well) after stimulation with recombinant allergens (2 mg/well) was measured as previously described.16 IFN-g, IL-4, IL-5, and IL-10 production was measured in spleen cell suspensions incubated for 40 hours with BP (25 mg/well) or Phleum extract (25 mg/well) as described.16 IL-5 was measured in bronchoalveolar lavage fluids as previously described.17 For neutralization of the IL-10 receptor in vitro pooled splenocytes were cultured in the presence of rat antimouse IL-10 receptor mAb (60 mg/mL; BD Biosciences, Heidelberg, Germany) or control rat IgG1, k isotype antibody (BD Biosciences).22 Cytokine levels are shown in pg/mL after subtraction of baseline levels (IFN-g, 147.23 6 61.6 pg/mL; IL-4/5, 4.8 6 4.1 pg/mL; IL-10, 27.75 6 23.3 pg/mL) of unstimulated cultures.

Quantification of TGF-b mRNA expression by real-time RT-PCR Total RNA was isolated from pooled spleen cell suspensions on the day of sacrifice by using RNeasy Minikit (Quiagen, Valencia, Calif), treated with DNase (Quiagen), and then reverse-transcribed into cDNA by using random hexamers (GeneAmp RT-PCR kit; Perkin Elmer, Boston, Mass). Gene expression was determined by quantitative real-time PCR by using predesigned TaqMan Gene Expression Assays according to the manufacturer’s protocol on an ABI 7700 sequence detection system (Applied Biosystems, Foster City, Calif). Amplification of the endogenous control 18S rRNA (Applied Biosystems) was performed to standardize the amount of sample cDNA added, and relative quantitation was performed by using the standard curve method (Applied Biosystems). The thermal cycle conditions were 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of amplification at 95°C for 15 seconds and 60°C for 1 minute.

Adoptive cell transfer Pretreatment with the peptide mixture or the hybrid peptide was performed as described. Eight days after the last intranasal application, donor spleen cells (1 3 107) were intravenously injected into naive recipients.17 Four hours after the cell transfer, the recipients were polysensitized as described, and immune responses were evaluated 7 days after the last immunization.

Statistics Data are expressed as means 6 SEMs from 3 independent experiments. Differences between groups were tested by KruskalWallis tests. Post hoc comparisons for all pairs of groups were performed applying Tukey-Kramer tests. P values below .05 were considered significant. Pairwise comparison of sensitized versus pretreated groups was performed by using the Mann-Whitney U test.

RESULTS Polysensitized mice displayed comparable immune responses to each of the 3 allergens Immunization with rBet v 1, rPhl p 1, and rPhl p 5 induced comparably high IgG1, IgE, and IgG2a levels and strong lymphocyte proliferative responses to each of the allergens (Table I). In addition, similar levels of IL-4, IL-5, and IFN-g were detected after stimulation of spleen cell suspensions from polysensitized mice with BP and Phleum extract (Table I). Naive splenocytes stimulated with rBet v 1, rPhl p 1, rPhl p 5, BP, or Phleum extract did not differ in their proliferative responses or in cytokine levels from medium control levels (data not shown). Epitope mapping studies for characterization of the immunodominant peptides of Bet v 1, Phl p 1, and Phl p 5 In Bet v 1 immunized mice, 1 immunodominant T-cell epitope, MGETLLRAVESY, was located at the C-terminus corresponding to the aa sequence position 139-150.23 One immunodominant region of Phl p 1, AGELELQFRRVKCKY, corresponding to the aa sequence position 127-141, was identified, also described as a T-cell– reactive region in human beings.24 Concerning Phl p 5, 2 immunodominant regions, KVDAAFKVAATAANA corresponding to the aa sequence 166-180 (peptide 1) and TVATAPEVKYTVFETALK corresponding to the aa

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Synthesis of peptides for intranasal tolerance induction According to the epitope mapping studies, we synthesized single peptides from Bet v 1 (aa: SKEMGETLLRAVESYLLAHSDE), Phl p 1 (aa: LRSAGELELQFRRVKCKYPEG), and Phl p 5 (aa sequence: YAATVATAPEVKYTVFETALKKAI) for intranasal application as peptide mixture, or a hybrid peptide composed of the immunodominant regions of these 3 allergens (aa: MGETLLRAVESYAGELELQFRRVKCKYTVATAPEVKYTVFETALK; see Tables E1-E3 in the Online Repository at www.mosby.com/jaci). Intranasal tolerance induction with the peptide mixture and the hybrid peptide, but not with the protein mixture, suppressed TH2 immune responses and airway inflammation in polysensitized mice Sera from peptide mixture as well as from hybrid peptide–pretreated mice, but not from mice pretreated with the complete proteins, induced significantly lower IgE-induced basophil degranulation in vitro than sera from polysensitized mice (Fig 1, A). In accordance with this reduction, IL-4 levels were significantly suppressed in spleen cell cultures from mice pretreated with the peptide mixture or the hybrid peptide. In contrast, IL-4 levels were enhanced or remained unchanged in the protein-pretreated group (Fig 1, B). Unlike the allergen-specific TH2 responses, IgG2a antibody production was not significantly changed by either of the pretreatments (Table II). Similarly, IFN-g levels in vitro remained unchanged except for a significant enhancement in BP-stimulated splenocytes from the hybrid peptide–pretreated mice (Table II). Airway inflammation, characterized by eosinophils and IL-5 production in bronchoalveolar lavages, was significantly reduced after pretreatment with the peptide mixture or the hybrid peptide (Fig 2). Polytolerance induction is not regulated by TGF-b or IL-10 No significant changes in TGF-b mRNA expression were detected after pretreatment with the peptide mixture or the hybrid peptide compared with polysensitized controls (Fig 3, A). IL-10 levels were significantly suppressed in peptide mixture as well as hybrid peptide– pretreated mice (Fig 3, B). Neutralization of IL-10 receptor in vitro did not abrogate the suppression of IL-10 levels in the pretreated groups (Table III). Moreover, adoptive cell transfer experiments showed that tolerance was not transferable by spleen cells, because IgE-induced basophil

FIG 1. IgE-dependent allergen-specific basophil degranulation (RBL assay) (A), and IL-4 production (B) in vitro from protein mixture (hatched bars), peptide mixture (gray bars), and hybrid peptide– pretreated mice (black bars) compared with polysensitized controls (white bars). *P < .05, **P < .01, pretreated vs polysensitized.

degranulation remained unchanged in polysensitized recipients (Fig 3, C).

DISCUSSION In the current study, we demonstrate that it is possible to prevent the development of multiple sensitivities to birch and grass pollen allergens by intranasal antigen application. Successful tolerance induction was achieved by intranasal administration of the immunodominant peptides of Bet v 1, Phl p 1, and Phl p 5, but not by the mixture of the allergen proteins. It was recently shown that the dose of coadministered antigens is most important for the establishment of a balanced TH2 immune response.25 Accordingly, in the current study, we demonstrated that immunization with the optimal doses of Bet v 1, Phl p 1, and Phl p 5 (ie, 5 mg each) led to comparable humoral and cellular immune responses to all 3 allergens/antigens (Table I). Moreover, the immunodominant epitopes recognized by T cells from polysensitized mice were identical to some of the T-cell epitopes in patients allergic to birch and grass pollen.24,26,27 Thus, our murine model of multiple allergen sensitivity showed similar immunological characteristics to those of human pollinosis. From previous studies we know that rBet v 1 acts as a potent mucosal tolerogen in monosensitized mice.16,17,19 However, when rBet v 1 was intranasally applied before polysensitization with the 3 allergens, the tolerizing effects of Bet v 1 toward itself were considerably impaired.28 In addition, in polysensitized mice, mucosal pretreatment with Bet v 1 alone did not alter the immune response toward the coapplied grass pollen allergens, indicating that no bystander or linked suppression can be

Rhinitis, sinusitis, and ocular diseases

sequence 226-243 (peptide 2), were identified. Comparison of the tolerogenicity of these peptides revealed that peptide 2 led to the highest immunosuppression in Phl p 5–sensitized mice (data not shown). Peptide 2 was therefore selected for tolerance induction in polysensitized mice.

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TABLE II. Allergen-specific IgG2a antibody levels and IFN-g production in vitro in polysensitized and pretreated mice IgG2a (OD)* rBet v 1

Polysensitized Protein-tolerized Peptide-tolerized Hybrid-tolerized

0.51 0.29 0.93 1.25

6 6 6 6

0.35 0.19 0.71 1.03

rPhl p 1

0.31 0.29 1.02 1.07

6 6 6 6

0.12 0.26 0.77 0.82

IFN-g (pg/mL)y rPhl p 5

0.75 0.71 1.56 1.38

6 6 6 6

0.37 0.51 0.46 0.61

BP

1501.9 2160.7 2305.7 3486.9

6 6 6 6

621.1 752.1 798.5 184.1**

Phleum

1307.9 1885.6 1753.5 1869.7

6 6 6 6

372.7 553.3 657.1 204.1

*IgG2a antibody levels were measured by ELISA.  IFN-g levels were measured in spleen cell cultures after stimulation with BP or Phleum extract. **P < .01 hybrid-tolerized vs polysensitized as determined by Mann-Whitney U test.

Rhinitis, sinusitis, and ocular diseases FIG 2. Number of eosinophils (A) and IL-5 levels (B) in bronchoalveolar lavage fluids from polysensitized mice (white bars), from peptide mixture–pretreated mice (gray bars), and from mice pretreated with the hybrid peptide (black bars). *P < .05, **P < .01, pretreated vs polysensitized. BALF, Bronchoalveolar lavage fluid.

achieved in mice with multiple sensitivities (data not shown).22,29 Therefore, a mixture of all 3 allergen proteins was used for intranasal pretreatment. However, it was not possible to reduce IgE-dependent basophil degranulation, allergen-specific antibody responses, or cytokine production with the mixture of the proteins in polysensitized mice. These results are in line with other studies showing that the tolerogenic efficacy was impaired when more than 1 protein antigen was ingested via the nasal or oral route.30,31 Several studies have demonstrated that allergenderived immunodominant peptides applied via mucosal surfaces have a high tolerogenicity in naive and in primed animals.15,32,33 It was further shown that treatment of mice with a peptide containing a single immunodominant epitope led to inhibition of immune responses directed to the other epitopes on the natural allergen, a phenomenon called intramolecular suppression.33 Therefore, to enhance the efficacy of tolerance induction in the polysensitized mice, we applied a mixture of the immunodominant peptides or a synthetic hybrid composed of the major T-cell epitopes of Bet v 1, Phl p 1, and Phl p 5.

FIG 3. A, TGF-b mRNA expression from peptide mixture (gray bar) and hybrid peptide–pretreated mice (black bar) shown as relative values in comparison with polysensitized controls (white bar). B, IL-10 production in vitro from polysensitized (white bars), peptide mixture (gray bars), and hybrid peptide–pretreated (black bars) mice. *P < .05, **P < .01, pretreated vs polysensitized. C, RBL assay after adoptive transfer of spleen cells from peptide mixture (transfer, gray bars) or hybrid peptide–pretreated mice (transfer, black bars) compared with polysensitized controls (white bars).

Indeed, intranasal administration of either of the peptide mixture or the hybrid peptide led to a highly significant reduction of allergen-specific TH2 responses (Fig 1) and airway inflammation (Fig 2). Other studies showing that application of a panel of peptides suppressed TH2 immune responses were directed only against single allergens,32,34 whereas this is the first report showing that peptideinduced tolerance can be directed against 3—and probably more—different antigens/allergens. Our observation that the mixture of protein allergens was unable to induce tolerance, whereas the polypeptides

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Control Ab

Poly-sens Peptide-tol Hybrid-tol

aIL-10 receptor Ab

BP

Phleum

BP

Phleum

241.82 138.63 165.84

940.66 472.41 630.72

283.94 158.91 283.74

1095.78 661.14 790.85

*Pooled splenocytes from polysensitized (poly-sens), peptide mixture (peptide-tol), or hybrid peptide (hybrid-tol) pretreated mice were stimulated with BP or Phleum extract in the presence of a neutralizing anti IL-10 receptor antibody or an isotype control. IL-10 levels (pg/mL) in supernatants were measured by ELISA.

were able to do so, leads to the speculation that because of the different conformation of the allergens, distinct antigenpresenting cells were targeted.35 It might further be speculated that the reduced/nontolerogenic capacity of the protein mixture is a result of an intermolecular competition among a higher number of epitopes for the accessibility of MHC molecules than might be the case with only 3 (or few) immunodominant peptides.36 In studies on secondary prevention, the efficacy of the peptide treatment depending on their length was evaluated: whereas short peptides (20 aa or less) have the advantage that they cannot cross-link IgE on mast cells, they may require characterization of major HLA-restricted T-cell epitopes on a patient basis.37 Long peptides (50 aa or more) might overcome this problem, but at the cost of safety.32 The obvious advantage of primary prevention, as performed in this study, lies in the risk-free application of selected peptides, but limitations might still be based on the polymorphisms of the HLA molecules. However, it is also known that peptides capable of binding multiple HLA types and of being immunogenic in context with different HLA molecules do exist.38 This has been also shown for several allergens.27,39 The fact that the immunodominant peptides of the 3 allergens—representing major T-cell epitopes in a high number of patients allergic to birch and grass pollen24,26,27—were also tolerogenic in another mouse strain, ie, C57Bl/6J (data not shown), may indicate that tolerance induction was not restricted to a particular MHC haplotype. Concerning the underlying mechanisms, we previously demonstrated in Bet v 1–monosensitized mice that tolerance induction depends on the conformation of the antigen: whereas tolerance induction with the whole Bet v 1 molecule led to enhanced TGF-b and IL-10 mRNA levels and tolerance was transferable with spleen cells, tolerance induction with a fragment of Bet v 1—including the immunodominant T-cell epitope—was not mediated by regulatory mechanisms.16,17 Similarly, in this study of polypeptide-induced tolerance, we found significantly reduced IL-10 levels along with unchanged TGF-b mRNA levels. Neutralization of the IL-10 receptor in vitro did not abrogate the reduction of IL-10 (Table III). Moreover, because tolerance was not transferable with splenocytes, it seems unlikely that regulatory cytokines mediated tolerance in polysensitized mice. Thus, mecha-

nisms such as clonal anergy, described in a model of peptide-induced tolerance,32 or cytokine-independent mechanisms, such as cell-cell contact interaction via the Notch signalling pathway40 or via cytotoxic T lymphocyte-associated antigen 4 expression on antigen-specific regulatory T cells,41 might rather play a role in our model of polytolerance induction. In conclusion, this is the first report showing that mucosal application of polypeptide constructs inhibited polysensitization to several antigens/allergens. Thus, these data could be the basis for the development of a mucosal polyvalent allergy vaccine for primary prevention of multiple sensitivities in atopic individuals. It remains to be investigated whether polytolerance induction can be used for treatment of already established multisensitivities. We thank Karin Baier for technical assistance, Dr Michael Kundi for statistical analysis, and Dr Helmut Fiebig for providing the Phl p 5 peptides.

REFERENCES 1. D’Amato G, Spieksma FT, Liccardi G, Jager S, Russo M, Kontou-Fili K, et al. Pollen-related allergy in Europe. Allergy 1998;53:567-78. 2. Ferreira FD, Hoffmann-Sommergruber K, Breiteneder H, Pettenburger K, Ebner C, Sommergruber W, et al. Purification and characterization of recombinant Bet v 1, the major birch pollen allergen: immunological equivalence to natural Bet v 1. J Biol Chem 1993;268:19574-80. 3. Laffer S, Valenta R, Vrtala S, Susani M, van Ree R, Kraft D, et al. Complementary DNA cloning of the major allergen Phl p I from timothy grass (Phleum pratense); recombinant Phl p I inhibits IgE binding to group I allergenes from eight different grass species. J Allergy Clin Immunol 1994;94:689-98. 4. Vrtala S, Sperr WR, Reimitzer I, van Ree R, Laffer S, Muller WD, et al. cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl p V Allergen. J Immunol 1993;151:4773-81. 5. Eriksson NE, Holmen A. Skin prick tests with standardized extracts of inhalant allergens in 7099 adult patients with asthma or rhinitis: crosssensitizations and relationships to age, sex, month of birth and year of testing. J Investig Allergol Clin Immunol 1996;6:36-46. 6. Silvestri M, Rossi GA, Cozzani S, Pulvirenti G, Fasce L. Age-dependent tendency to become sensitized to other classes of aeroallergens in atopic asthmatic children. Ann Allergy Asthma Immunol 1999;83:335-40. 7. Bousquet J, Lockey R, Malling HJ. Allergen immunotherapy: therapeutic vaccines for allergic diseases: a WHO position paper. J Allergy Clin Immunol 1998;102:558-62. 8. Bousquet J, Van Cauwenberge P, Khaltaev N. Aria Workshop Group, World Health Organization. Allergic rhinitis and its impact on asthma. J Allergy Clin Immunol 2001;108:S147-334. 9. Scheiner O, Kraft D. Basic and practical aspects of recombinant allergens. Allergy 1995;50:384-91. 10. Valenta R, Lidholm J, Niederberger V, Hayek B, Kraft D, Gronlund H. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT). Clin Exp Allergy 1999;29: 896-904. 11. Alexander C, Kay AB, Larche M. Peptide-based vaccines in the treatment of specific allergy. Curr Drug Targets Inflamm Allergy 2002; 1:353-61. 12. Valenta R. The future of antigen-specific immunotherapy of allergy. Nat Rev Immunol 2002;2:446-53. 13. Holt PG, Patty JE, Turner KG. Inhibition of specific IgE responses in mice by pre-exposure to inhaled antigen. Immunology 1981;42:409-17. 14. Tsitoura DC, DeKruyff RH, Lamb JR, Umetsu DT. Intranasal exposure to protein antigen induces immunological tolerance mediated by functionally disabled CD41 T cells. J Immunol 1999;163:2592-600.

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TABLE III. IL-10 levels after neutralization of IL-10 receptor in vitro*

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15. Jarnicki AG, Tsuji T, Thomas WR. Inhibition of mucosal and systemic T(h)2-type immune responses by intranasal peptides containing a dominant T cell epitope of the allergen Der p 1. Int Immunol 2001;13: 1223-31. 16. Wiedermann U, Jahn-Schmid B, Bohle B, Repa A, Renz H, Kraft D, et al. Suppression of antigen-specific T-and B-cell responses by intranasal or oral administration of recombinant Bet v 1, the major birch pollen allergen, in a murine model of type I allergy. J Allergy Clin Immunol 1999;103:1202-10. 17. Wiedermann U, Herz U, Baier K, Vrtala S, Neuhaus-Steinmetz U, Bohle B, et al. Intranasal treatment with a recombinant hypoallergenic derivative of the major birch pollen allergen Bet v 1 prevents allergic sensitization and airway inflammation in mice. Int Arch Allergy Immunol 2001;126:68-77. 18. Takabayashi K, Libet L, Chisholm D, Zubeldia J, Horner AA. Intranasal immunotherapy is more effective than intradermal immunotherapy for the induction of airway allergen tolerance in Th2-sensitized mice. J Immunol 2003;170:3898-905. 19. Winkler B, Baier K, Wagner S, Repa A, Scheiner O, Kraft D, et al. Mucosal tolerance as therapy of type I allergy: intranasal application of recombinant Bet v 1, the major birch pollen allergen, leads to the suppression of allergic immune responses and airway inflammation in sensitized mice. Clin Exp Allergy 2002;32:30-6. 20. Focke M, Mahler V, Ball T, Sperr WR, Majlesi Y, Valent P, et al. Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination. FASEB J 2001;15:2042-4. 21. Hufnagl K, Wagner B, Winkler B, Baier K, Hochreiter R, Thalhamer J, et al. Induction of mucosal tolerance with recombinant Hev b 1 and recombinant Hev b 3 for prevention of latex allergy in BALB/c mice. Clin Exp Immunol 2003;133:170-6. 22. Winkler B, Bolwig C, Seppala U, Spangfort MD, Ebner C, Wiedermann U. Allergen-specific immunosuppression by mucosal treatment with recombinant Ves v 5, a major allergen of Vespula vulgaris venom, in a murine model of wasp venom allergy. Immunology 2003;110:376-85. 23. Bauer L, Bohle B, Jahn-Schmid B, Wiedermann U, Daser A, Renz H, et al. Modulation of the allergic immune response in BALB/c mice by subcutaneous injection of high doses of the dominant T cell epitope from the major birch pollen allergen Bet v 1. Clin Exp Immunol 1997;107: 536-41. 24. Schenk S, Breiteneder H, Susani M, Najafian N, Laffer S, Duchene M, et al. T-cell epitopes of Phl p 1, major pollen allergen of timothy grass (Phleum pratense): evidence for crossreacting and non-crossreacting T-cell epitopes within grass group I allergens. J Allergy Clin Immunol 1995;96:986-96. 25. Von Garnier C, Astori M, Kettner A, Dufour N, Corradin G, Spertini F. In vivo kinetics of the immunoglobulin E response to allergen: bystander effect of coimmunization and relationship with anaphylaxis. Clin Exp Allergy 2002;32:401-10. 26. Mu¨ller WD, Karamfilov T, Kahlert H, Stuwe HT, Fahlbusch B, Cromwell O, et al. Mapping of T-cell epitopes of Phl p 5: evidence for

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crossreacting and non-crossreacting T-cell epitopes within Phl p 5 isoallergens. Clin Exp Allergy 1998;28:1538-48. Ebner C, Schenk S, Najafian N, Siemann U, Steiner R, Fischer GW, et al. Nonallergic individuals recognize the same T cell epitopes of Bet v 1, the major birch pollen allergen, as atopic patients. J Immunol 1995;154: 1932-40. Hufnagl K, Winkler B, Baier K, Valenta R, Kraft D, Wiedermann U. Induction of mucosal tolerance by coapplication of recombinant Bet v 1, the major birch pollen allergen, recombinant Phl p 1 and Phl p 5, major grass pollen allergens, in polysensitized mice. Allergy 2002;57(suppl 73):49. Miller A, Lider O, Weiner HL. Antigen-driven bystander suppression after oral administration of antigens. J Exp Med 1991;174:791-8. Mowat AM. The regulation of immune responses to dietary protein antigens. Immunol Today 1987;8:93-8. Shi FD, Bai XF, Xiao BG, van der Meide PH, Link H. Nasal administration of multiple antigens suppresses experimental autoimmune myasthenia gravis, encephalomyelitis and neuritis. J Neurol Sci 1998; 155:1-12. Astori M, von Garnier C, Kettner A, Dufour N, Corradin G, Spertini F. Inducing tolerance by intranasal administration of long peptides in naive and primed CBA/J mice. J Immunol 2000;165:3497-505. Hoyne GF, Jarnicki AG, Thomas WR, Lamb JR. Characterization of the specificity and duration of T cell tolerance to intranasally administered peptides in mice: a role for intramolecular epitope suppression. Int Immunol 1997;9:1165-73. Hirahara K, Tatsuta T, Takatori T, Ohtsuki M, Kirinaka H, Kawaguchi J, et al. Preclinical evaluation of an immunotherapeutic peptide comprising 7 T-cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen allergens. J Allergy Clin Immunol 2001;108:94-100. Akdis CA, Blesken T, Wymann D, Akdis M, Blaser K. Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants. Eur J Immunol 1998;28:914-25. Lo-Man R, Leclerc C. Parameters affecting the immunogenicity of recombinant T cell epitopes inserted into hybrid proteins. Hum Immunol 1997;54:180-8. Smith TR, Larche M. Investigating T cell activation and tolerance in vivo: peptide challenge in allergic asthmatics. Cytokine 2004;28:49-54. Southwood S, Sidney J, Kondo A, del Guercio MF, Appella E, Hoffman S, et al. Several common HLA-DR types share largely overlapping peptide binding repertoires. J Immunol 1998;160:3363-73. Bohle B, Radakovics A, Jahn-Schmid B, Hoffmann-Sommergruber K, Fischer GF, Ebner C. Bet v 1, the major birch pollen allergen, initiates sensitization to Api g 1, the major allergen in celery: evidence at the T cell level. Eur J Immunol 2003;33:3303-10. Hoyne GF, Le Roux I, Corsin-Jimenez M, Tan K, Dunne J, Forsyth LM, et al. Serrate1-induced notch signalling regulates the decision between immunity and tolerance made by peripheral CD4(1) T cells. Int Immunol 2000;12:177-85. Fowler S, Powrie F. CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required for oral tolerance. Eur J Immunol 2002;32: 2997-3006.

Environmental and occupational respiratory disorders Prevalences of positive skin test responses to 10 common allergens in the US population: Results from the Third National Health and Nutrition Examination Survey Samuel J. Arbes, Jr, DDS, MPH, PhD,a Peter J. Gergen, MD, MPH,b Leslie Elliott, MPH, PhD,a and Darryl C. Zeldin, MDa Research Triangle Park, NC, and Bethesda, Md Key words: Allergens, allergic sensitization, allergy skin test, epidemiology, NHANES II, NHANES III, survey

Over the last 2 or more decades, rates of asthma have increased in the United States and worldwide, although there is some evidence that asthma rates might have peaked.1-3 One of the most important risk factors for asthma is sensitization to one or more allergens. The National Center for Health Statistics included allergy skin testing in the second and third National Health and Nutrition Examination Surveys (NHANES II and III), which were conducted from 1976 through 1980 and 1988 through 1994, respectively, to estimate and monitor the prevalence of allergic sensitization in the United States. Although skin test results from NHANES II have been published,4 a comprehensive summary of skin test results from NHANES III has not been published, nor has a comparison between NHANES II and III data been published. The primary objectives were to estimate rates of positive skin test responses in NHANES III and to identify predictors of a positive test response to 1 or more allergens. A secondary objective was to compare positive skin test response rates between NHANES II and III; however, methodological differences between the 2 surveys, which this article describes in detail, provide challenges for comparing and interpreting rate differences between the 2 surveys.

METHODS a

From the Laboratory of Respiratory Biology, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, and bthe Division of Allergy, Immunology, and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda. This analysis of National Health and Nutrition Examination Survey (NHANES III) data was funded by the National Institute of Environmental Health Sciences and the National Institute of Allergy and Infectious Diseases. Received for publication March 1, 2005; revised April 25, 2005; accepted for publication May 12, 2005. Available online July 15, 2005. Reprint requests: Darryl C. Zeldin, MD, NIEHS/NIH, PO Box 12233, MD D2-01, Research Triangle Park, NC 27709. E-mail: [email protected]. 0091-6749 doi:10.1016/j.jaci.2005.05.017

NHANES II and III NHANES II and III were two in a series of population-based surveys conducted by the National Center for Health Statistics to determine the health and nutritional status of the US population. Both surveys used a complex design to sample the civilian, noninstitutionalized population. In NHANES II, questionnaires and medical examinations were administered to 20,322 individuals aged 6 months to 74 years, whereas in NHANES III, 31,311 individuals aged 2 months to 90 years were interviewed and examined.

Allergy skin testing in NHANES II and III Prick-puncture allergy skin testing was performed in NHANES II and III; however, there were important differences in age eligibility, 377

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Background: Allergy skin tests were administered in the second and third National Health and Nutrition Examination Surveys (NHANES II and III) conducted in the United States from 1976 through 1980 and 1988 through 1994, respectively. Objectives: This study estimated positive skin test response rates in NHANES III and identified predictors of one or more positive test responses. Comparisons with NHANES II were also made. Methods: In NHANES III, 10 allergens and 2 controls were tested in all subjects aged 6 to 19 years and a random halfsample of subjects aged 20 to 59 years. A wheal-based definition of a positive test response was used. Results: In NHANES III, 54.3% of the population had positive test responses to 1 or more allergens. Prevalences were 27.5% for dust mite, 26.9% for perennial rye, 26.2% for short ragweed, 26.1% for German cockroach, 18.1% for Bermuda grass, 17.0% for cat, 15.2% for Russian thistle, 13.2% for white oak, 12.9% for Alternaria alternata, and 8.6% for peanut. Among those with positive test responses, the median number of positive responses was 3.0. Adjusted odds of a positive test response were higher for the following variables: age of 20 to 29 years, male sex, minority race, western region, old homes, and lower serum cotinine levels. For the 6 allergens common to NHANES II and III, prevalences were 2.1 to 5.5 times higher in NHANES III. Conclusions: The majority of the US population represented in NHANES III was sensitized to 1 or more allergens. Whether the higher prevalences observed in NHANES III reflect true changes in prevalence or methodological differences between the surveys cannot be determined with certainty. (J Allergy Clin Immunol 2005;116:377-83.)

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Abbreviations used NHANES II: The second National Health and Nutrition Examination Survey NHANES III: The third National Health and Nutrition Examination Survey

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medical exclusion criteria, number and types of allergens tested, standardization of allergen extracts, and reading times for the reactions. Overviews of the allergy skin test protocols for both surveys are presented here; however, details of the protocols can be found elsewhere.5,6 In NHANES II, prick-puncture allergy skin tests to 8 allergens (house dust, cat, dog, Alternaria alternata, mixed giant-short ragweed, oak, perennial ryegrass, and Bermuda grass) and 2 controls (positive and negative) were administered to all subjects aged 6 to 74 years. Each of the allergens was commercially available and US Food and Drug Administration licensed, but none was standardized. A standardized extract is one for which a reference standard for potency exists. The positive control was histamine phosphate, and the negative control was 50% glycerol saline. Subjects in 48 of the 64 primary sampling units were tested with a histamine base concentration of 0.1 mg/mL, a less than optimal concentration, whereas the rest were tested with the optimal concentration of 1.0 mg/mL.7 Lengths and widths of wheals (raised area in the middle of the reaction) and flares (reddish area around the wheal) were measured at 10 and 20 minutes. Subjects with a history of allergy to cats, dogs, or ragweed were not initially tested for those allergens. At the 10-minute reading, if the subject reacted to fewer than 3 of the remaining allergens, then dog, cat, and ragweed were tested on the other arm. If 3 or more responses were positive, then only ragweed was tested on the other arm. In NHANES III, prick-puncture allergy skin tests to 10 allergens (Table I) and 2 controls (positive and negative) were administered to all subjects aged 6 to 19 years and a random half-sample of subjects aged 20 to 59 years. The positive control was histamine phosphate (concentration is not published), and the negative control was 50% glycerol saline.8 Only house dust mite, cat, and short ragweed allergens were standardized (personal communication with Paul Turkeltaub, MD, December 2, 2004). Lengths and widths of wheals and flares were measured after 15 minutes (6 5 minutes). Subjects were medically excluded from skin testing if they usually did not have trouble breathing in their chest or lungs but were having trouble breathing at the time of the examination, although not from a cold; if they usually had trouble breathing in their chest or lungs and had more trouble breathing at the time of the examination; if they had a severe response to allergen skin testing previously; or if they had severe eczema or infection on both arms. For comparisons between NHANES II and III, prevalences of positive skin test reactions in NHANES II were estimated for the 6 allergens and ages (6-59 years) common to both surveys. The 6 allergens were cat, ragweed (mixed giant and short in NHANES II and short in NHANES III), perennial rye, oak (oak in NHANES II and white oak in NHANES III), Bermuda grass, and A alternata.

Definition of a positive skin test response For our analyses of NHANES II and III skin test data, we considered an allergen-specific skin test response positive if the skin test panel was valid and the difference between the mean of the wheal’s length and width for the allergen-specific test and the negative control was at least 3 mm. A skin test panel was considered valid if the difference between the mean wheal diameters of the

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TABLE I. Prevalences of positive skin test responses among the US population aged 6 to 59 years represented in NHANES III Allergen tested

Indoor allergens Dust mite German cockroach Cat At least one indoor allergen Outdoor allergens Perennial rye Short ragweed Bermuda grass Russian thistle White oak Alternaria alternata At least one outdoor allergen Food allergen: peanut At least one indoor or outdoor allergen At least one of any type

Percentage (SE)

27.5 26.1 17.0 43.0

(1.02) (0.82) (1.00) (1.12)

26.9 26.2 18.1 15.2 13.2 12.9 40.0 8.6 53.9 54.3

(0.88) (1.03) (0.81) (0.92) (0.78) (0.69) (1.22) (0.51) (1.02) (1.00)

positive and negative controls was at least 1 mm. For NHANES II results, measurements from the 20-minute reading were used. In NHANES II, 11,769 of the 16,204 subjects who were age eligible for skin testing were aged 6 to 59 years, and of those, 11,062 had a wheal-based result for the 6 allergens common to both surveys. Of the 11,062 subjects, 7230 had a valid skin test panel, 3024 had an invalid panel, and 808 were missing a positive control result. The NHANES II analysis was limited to the 7230 subjects; however, a secondary analysis was conducted without regard to the valid panel criterion (n = 11,062). In NHANES III, there were 12,106 age-eligible subjects, and of those, 10,863 participated in skin testing, 174 were excluded for medical reasons, and 1069 refused or were unavailable for testing. Of the 10,863 subjects, 10,841 had a result for all 10 allergens, and of those, 10,508 had a valid skin test panel, 332 had an invalid panel, and 1 subject was missing a positive control result. The NHANES III analysis was limited to the 10,508 subjects.

Statistical analyses Percentages (with SEs) of the population with positive skin test responses were estimated among the populations aged 6 to 59 years represented by the surveys. Sociodemographic or medical examination variables were assessed as potential predictors of one or more positive skin test responses in NHANES III. The complete list can be viewed in Table E1 in the Online Repository in the online version of this article at www.mosby.com/jaci. Potential predictors were evaluated first with x2 statistics and then with multivariable logistic regression by using a backward selection process. The process began with all potential predictors in the model and ended with variables at a P value of .050 or less. Education, rather than poverty/income ratio, was modeled as an indicator of socioeconomic status because the latter had a large number of missing values, and serum cotinine level was modeled in place of smoker in the home because serum cotinine level is a biomarker for tobacco smoke exposure. Two-way interactions between sex, age, and race-ethnicity were evaluated and adjusted for the other predictors in the model. Only interactions significant at the .050 level were reported. Statistical analyses were conducted with SAS Version 9.1 (SAS Institute, Cary, NC) or SUDAAN Release 9.0 (RTI International, Research Triangle Park, NC) software. All percentages and odds

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TABLE II. Distribution of positive skin test responses by allergen classification among the US population aged 6 to 59 years represented in NHANES III Percentage (SE)

ratios reported in this article were weighted to represent population estimates, and all SEs were adjusted for the complex survey design. Numbers of subjects reported in this article are unweighted.

RESULTS NHANES III: Prevalences of positive skin test responses Table I shows the prevalences of positive skin test responses among the US population aged 6 to 59 years. More than half of the population had positive test responses to one or more allergens. The highest prevalences were for dust mite, rye, ragweed, and cockroach, and the lowest prevalence was for peanut. A positive test response to at least 1 indoor allergen was slightly more common than a positive test response to at least 1 outdoor allergen (43.0% vs 40.0%), even though twice as many outdoor allergens were tested. The percentage of the population with a positive test response decreased as the number of positive test responses increased from 1 to 10 (Fig 1). A solitary positive skin test response was seen in 15.5% (SE = 0.48) of the total population and 28.7% (SE = 0.95) of the population with positive test responses. The 2 most common solitary reactions were to cockroach (4.3% [SE = 0.42] of the total population) and dust mite (4.2%, SE = 0.24). The prevalences of a solitary reaction to the other allergens ranged from 0.10% to 1.70% of the total population. The mean and median numbers of positive test responses among those with positive test responses were 3.5 (SE = 0.06) and 3.0, respectively. Table II shows how positive skin test responses— classified as indoor, outdoor, and peanut—were distributed among the total US population and among those with positive test responses. Among those with positive test responses, 41% reacted to a combination of indoor and outdoor allergens (but had negative test responses to peanut). A positive test response to peanut alone was quite rare (0.6%), as were positive test responses to indoor allergens and peanut (0.3%) and outdoor allergens and peanut (2.3%).

Indoor only* Outdoor only  Peanut only Indoor and outdoor only Indoor and peanut only Outdoor and peanut only Indoor, outdoor, and peanut None Total

13.7 9.7 0.3 22.2 0.2 1.2 6.9 45.7 100.0

(0.55) (0.68) (0.11) (1.10) (0.06) (0.18) (0.50) (1.00) (0.00)

Among the population with positive test responses

25.3 (1.13) 17.9 (1.26) 0.6 (0.20) 41.0 (1.57) 0.3 (0.11) 2.3 (0.32) 12.6 (0.88) – 100.0 (0.00)

*House dust mite, cat, or German cockroach.  Short ragweed, perennial rye, Alternaria alternata, Bermuda grass, Russian thistle, or white oak.

NHANES III: Predictors of 1 or more positive test responses The independent predictors of 1 or more positive test responses were sex, age, race-ethnicity, census region, home construction year, and serum cotinine level. The distributions of these predictors in the US population and their adjusted odds ratios are shown in Table III. The distributions of all tested predictors and their bivariate associations with each of the 10 allergen skin tests can be found in Table E1 in the Online Repository in the online version of this article at www.mosby.com/jaci. Age was bivariately associated with each allergen test (Table E1). The prevalence of 1 or more positive test responses, as well as the adjusted odds ratio, increased from the first decade of age to the second, peaked in the third decade, and then decreased through the sixth decade (Table III). For each allergen tested, male subjects were more likely than female subjects to have positive test responses (Table E1). The adjusted odds of having 1 or more positive test responses were 1.6 times greater in male subjects (Table III). The odds ratio for sex did not differ by age (P value for sex-age interaction term = .518); however, it did differ by race-ethnicity (P value for sex-race interaction term = .027). With the sex-race interaction term in the model (model not shown), the adjusted odds ratios comparing male subjects with female subjects were 1.6 (95% CI, 1.3-2.0) for non-Hispanic whites, 1.4 (95% CI, 1.11.8) for non-Hispanic blacks, 1.1 (95% CI, 0.9-1.4) for Mexican Americans, and 2.0 (95% CI, 1.1-3.8) for others. Race-ethnicity was bivariately associated with a positive test response to 7 of the 10 allergens (Table E1). Compared with non-Hispanic whites, the adjusted odds of having 1 or more positive test responses were greater for each of the other 3 race-ethnicity categories (Table III). However, as mentioned in the previous paragraph,

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FIG 1. Percentage of the US population aged 6 to 59 years (NHANES III) by numbers of positive skin test responses.

Allergen type

Among the total population

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TABLE III. Prevalences and odds ratios for the independent predictors of 1 or more positive skin test responses among the US population aged 6 to 59 years represented in NHANES III

Predictor

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Age (y) 6-9 10-19 20-29 30-39 40-49 50-59 Sex Female Male Race-ethnicity Non-Hispanic white Non-Hispanic black Mexican American Other Census region South West Northeast Midwest Year home constructed 1974 to present 1946-1973 Before 1946 Cotinine (ng/mL) 0.035-0.100 0.100-10.00 10.00-1080.00

Percentage (SE)

45.6 55.5 60.0 56.5 50.5 49.1

Adjusted* odds ratio (95% CI)

Wald F test, P value

(2.19) (1.33) (1.79) (1.96) (2.71) (2.70)

1.0 (reference) 1.7 (1.3-2.1) 2.1 (1.6-2.8) 1.8 (1.4-2.4) 1.5 (1.1-2.0) 1.4 (1.0-1.8)

<.001

49.2 (1.23) 59.4 (1.21)

1.0 (reference) 1.6 (1.4-1.8)

<.001

51.3 62.0 57.1 64.0

(1.17) (1.26) (1.28) (2.85)

1.0 (reference) 1.6 (1.4-1.9) 1.2 (1.0-1.4) 1.5 (1.2-2.0)

<.001

50.8 58.0 57.9 52.8

(1.40) (1.51) (2.78) (2.57)

1.0 (reference) 1.3 (1.1-1.6) 1.2 (0.9-1.8) 1.1 (0.9-1.5)

.042

53.1 (1.40) 52.1 (1.60) 59.4 (1.69)

1.0 (reference) 0.9 (0.8-1.1) 1.3 (1.1-1.6)

.002

56.9 (2.17) 55.4 (1.63) 51.0 (1.48)

1.0 (reference) 0.9 (0.7-1.1) 0.7 (0.5-0.9)

.012

*Adjusted for each variable in the table.

there was a significant interaction between sex and race-ethnicity. Among female subjects, the adjusted odds ratios for race-ethnicity (with non-Hispanic whites as the referent) were 1.8 (95% CI, 1.4-2.2) for nonHispanic blacks, 1.4 (95% CI, 1.2-2.7) for Mexican Americans, and 1.4 (95% CI, 0.9-2.1) for others. Among male subjects, those adjusted odds ratios were 1.5 (95% CI, 1.2-1.8), 1.0 (95% CI, 0.8-1.2), and 1.7 (95% CI, 1.22.5), respectively. Census region was bivariately associated with tests to the outdoor allergens ragweed, rye, grass, and thistle (Table E1). For 1 or more positive test responses, the adjusted odds ratio was lowest for the south and highest for the west (Table III). Home construction year was bivariately associated with a positive test response to dust mite, cockroach, ragweed, and peanut (Table E1). The prevalence of one or more positive test responses, as well as the adjusted odds ratio, was greatest in the oldest homes (Table III). Serum cotinine levels were bivariately associated with 4 of the 6 outdoor allergens (Table E1); however, for those allergens, the lowest cotinine level was associated with the highest prevalence of a positive test response. That same

pattern remained in the adjusted model for 1 or more positive test responses (Table III).

Comparisons between NHANES II and III The prevalences in NHANES II for positive test responses to the 6 allergens and ages (6-59 years) common to both surveys were 12.5% (SE = 0.74) for ragweed, 11.9% (SE = 0.62) for rye, 5.8% (SE = 0.54) for oak, 5.2% (SE = 0.49) for Bermuda grass, 4.5% (SE = 0.29) for A alternata, and 3.1% (SE = 0.32) for cat. The NHANES III prevalences for those 6 allergens were 2.1 to 5.5 times higher (Table I), and the NHANES III population was much more likely to react to at least 1 of the 6 allergens (41.9% [SE = 1.23] vs 21.8% [SE = 0.94]). As shown in Fig 2, rates of positive test responses were consistently higher in NHANES III than in NHANES II at each age group. For both surveys, the prevalences without the validpanel criterion were systematically less, although only slightly less. For example, the rate for a positive test response to 1 or more of the 6 allergens decreased from 41.9% to 41.4% in NHANES III and 21.8% to 19.6% in NHANES II. DISCUSSION The main finding of this study was that 54.3% of the population represented by NHANES III had 1 or more positive skin test responses to 10 common allergens. With the limited number of allergens tested, this might be an underestimation of the prevalence of allergic sensitization in the US population. On average, an individual with a positive test response reacted to 3 to 4 allergens, and most with positive test responses reacted to a combination of indoor and outdoor allergens as opposed to indoor, outdoor, or peanut allergens alone. For each of the 6 allergens tested in both NHANES II and III, the prevalence of a positive test response was higher in NHANES III at each decade of age. Even though we analyzed positive skin test response rates for the allergens and ages common to both surveys, there were differences between the surveys that could not be controlled, such as differences in medical exclusion criteria, in reading times of the reactions, in the histamine concentrations for the positive controls, and in the quality of the allergen extracts. It would seem doubtful that differences in medical exclusion criteria, reading times, and histamine concentrations would have contributed significantly to the differences in rates because only a small percentage of subjects were excluded for medical reasons in each survey, reading times overlapped somewhat, and results remained essentially the same irrespective of whether the histamine control was used in the definition of a positive test response. One methodological difference that could potentially explain the differences in positive skin test response rates is the potency of the allergens used. Only the cat and ragweed allergens tested in NHANES III were standardized, and without standard-

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ization, it cannot be assumed that the potencies of the allergens were the same between surveys. In fact, unstandardized allergen extracts can vary greatly in their potencies.9 The relative potencies of the allergens used in these 2 surveys are unknown, and because of this, it cannot be stated with any certainty that the increases in positive skin test response rates observed between NHANES II and III were due to true increases in reactivity in the US population. However, we would like to present 2 arguments that support true increases. First, potency between unstandardized allergens could be greater, less, or the same, and it would seem unlikely that the potency would have been systematically greater for all 6 of the NHANES III allergens. An example of the variability one might expect between allergen preparations can be found within NHANES II itself. Within NHANES II, complete panels of allergens were purchased from 2 different manufacturers, and subjects were tested with one panel or the other.7 In a comparison of positive skin test response rates between these 2 panels of allergens, Gergen and Turkeltaub7 found that one panel gave higher rates for 3 allergens, lower rates for 1 allergen, and similar rates for 4 allergens; however, none of the absolute differences was greater than 4.7%. Second, the increases seen between NHANES II and III are consistent with reports from other countries, such as Japan,10 the United Kingdom,11 and Denmark.12 In NHANES III, sex, race-ethnicity, age, census region, home construction date, and serum cotinine level were independent predictors of 1 or more positive skin test responses. Age was the strongest independent predictor of

1 or more positive skin test responses, with rates peaking at age 20 to 29 years. In cross-sectional studies it is often difficult to determine whether age effects are real or are due to a cohort effect (ie, the effect of capturing a high-risk cohort at a point in time). However, the age-specific comparisons between NHANES II and III (Fig 2) provide strong evidence that the prevalence of allergic sensitization truly peaks in the third decade of life. If the NHANES III finding had been due to a cohort effect, then NHANES II rates would have peaked at a younger age. The prevalence of 1 or more positive test responses was higher among male than female subjects, and the prevalence was higher for male subjects at each decade of life. In the general population, male subjects have higher levels of serum IgE than female subjects at any given age,13 but whether sex influences sensitization primarily through a genetic or an environmental pathway is not known. The higher prevalence of allergic sensitization among male subjects at any age is in contrast to the pattern seen with asthma. For asthma, the prevalence is greater in male subjects during childhood but greater in female subjects during the teenage and adult years.14 This contrast suggests that factors other than allergic sensitization are responsible for the sex-related shift in asthma prevalence observed at or near puberty. Compared with non-Hispanic whites, the odds of having 1 or more positive skin test responses were increased for the other 3 race-ethnicity categories. For NHANES II, Gergen et al4 reported that the age-adjusted prevalence of 1 or more positive test responses was higher in blacks than whites; however, the difference was not statistically significant. In a study of allergic sensitization among children

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FIG 2. Age-specific comparisons of positive skin test response rates for the 6 allergens tested in NHANES II (dashed lines) and NHANES III (solid lines).

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in NHANES III, Stevenson et al15 argued that race or ethnicity differences in sensitization likely reflect differences in environmental exposures rather than genetics. In finding race-ethnicity a strong predictor of positive test responses to dust mite, cockroach, and A alternata, those authors reasoned that the association was most likely to be due to differences in housing and community environments, which would lead to differences in allergen exposures. For census region and home construction date, the allergen-specific results suggest that these predictors affect sensitization primarily through an environmental pathway. Census region was bivariately associated with positive test responses to outdoor allergens only, which likely reflects geographic differences in exposures to those allergens. Consistent with the NHANES III results, Gergen et al4 reported that positive test response rates in NHANES II were lowest in the south. Older homes were bivariately associated with positive test responses to the indoor allergens dust mite and cockroach. In the National Survey of Lead and Allergens in Housing, a representative survey of US housing, it was shown that older homes had higher levels of dust mite, cockroach, and mouse allergens than newer homes.16,17 Higher serum cotinine levels predicted lower prevalences of 1 or more positive test responses. Active smoking has been associated with increased serum levels of total IgE; however, the published literature on the relationship between either active or passive smoking and skin test response positivity is inconclusive.18,19 Chronic tobacco smoke exposure can suppress the immune system and impair host defenses,20 which could potentially lead to lower sensitization rates; however, smoke avoidance among persons with allergies and asthma would also lead to lower rates. Two potential predictors worth discussing that did not remain in the final prediction model were the presence of an indoor cat and the presence of an indoor dog. The role of pet exposure in the cause of allergic sensitization and disease is controversial. One limitation to this crosssectional analysis was the inability to assess the timing of exposures and the development of allergic sensitization, which could be an explanation for the lack of association with indoor cat and dog. Interestingly, the presence of an indoor cat was not associated with a positive skin test response to cat allergen. One potential explanation for this null result could be the pervasiveness of cat allergen in US homes. In the National Survey of Lead and Allergens in Housing, 99% of homes with an indoor cat and 56% of homes without an indoor cat had cat allergen levels that exceeded the proposed threshold for allergic sensitization.21 In epidemiologic studies the more widespread an exposure is within a population, the more difficult it becomes to demonstrate its effects.22 Another potential explanation could be cat avoidance among persons who are sensitized to cats. In conclusion, the majority of the US population represented in NHANES III was sensitized to 1 or more allergens. Although it cannot be definitively concluded

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that the increases in positive skin test response rates observed between NHANES II and III represent an increase in the reactivity of the US population, such an increase would be consistent with studies from other countries. In NHANES 2005-2006, total and allergenspecific IgE levels are being measured in all subjects, along with levels of indoor allergens in their homes. We thank Drs Stephanie London and Donna Baird (Epidemiology Branch, National Institute of Environmental Health Sciences) for providing helpful comments during the preparation of this manuscript.

REFERENCES 1. Mannino DM, Homa DM, Akinbami LJ, Moorman JE, Gwynn C, Redd SC. Surveillance for asthma—United States, 1980-1999. MMWR Surveill Summ 2002;51:1-13. 2. Robertson CF, Roberts MF, Kappers JH. Asthma prevalence in Melbourne schoolchildren: have we reached the peak? Med J Aust 2004;180:273-6. 3. Verlato G, Corsico A, Villani S, Cerveri I, Migliore E, Accordini S, et al. Is the prevalence of adult asthma and allergic rhinitis still increasing? Results of an Italian study. J Allergy Clin Immunol 2003;111:1232-8. 4. Gergen PJ, Turkeltaub PC, Kovar MG. The prevalence of allergic skin test reactivity to eight common aeroallergens in the U.S. population: results from the second National Health and Nutrition Examination Survey. J Allergy Clin Immunol 1987;80:669-79. 5. National Health and Nutrition Examination Survey III. Training manual for allergy component. Available at: http://www.cdc.gov/nchs/data/ nhanes/nhanes3/cdrom/nchs/manuals/train.pdf. Accessed November 9, 2004. 6. National Center for Health Statistics (U.S.). Public use data tape documentation: allergy skin testing: tape number 5309: National Health and Nutrition Examination Survey, 1976-80. Hyattsville (MD): US Department of Health and Human Services, Public Health Service, National Center for Health Statistics; 1986. 7. Gergen PJ, Turkeltaub PC. National Center for Health Statistics (US). Percutaneous immediate hypersensitivity to eight allergens, United States, 1976-80. Washington (DC): US Department of Health and Human Services Public Health Service, National Center for Health Statistics; 1986. 8. Plan and operation of the Third National Health and Nutrition Examination Survey, 1988-94. Series 1: programs and collection procedures. Vital Health Stat 1 1994:(32)1-407. 9. Gleich GJ, Leiferman KM, Jones RT, Hooton ML, Baer H. Analysis of the potency of extracts of June grass pollen by their inhibitory capacities in the radioallergosorbent test. J Allergy Clin Immunol 1976;58:31-8. 10. Nakagomi T, Itaya H, Tominaga T, Yamaki M, Hisamatsu S, Nakagomi O. Is atopy increasing? Lancet 1994;343:121-2. 11. Sibbald B, Rink E, D’Souza M. Is the prevalence of atopy increasing? Br J Gen Pract 1990;40:338-40. 12. Linneberg A, Nielsen NH, Madsen F, Frolund L, Dirksen A, Jorgensen T. Increasing prevalence of specific IgE to aeroallergens in an adult population: two cross-sectional surveys 8 years apart: the Copenhagen Allergy Study. J Allergy Clin Immunol 2000;106:247-52. 13. Barbee RA, Halonen M, Lebowitz M, Burrows B. Distribution of IgE in a community population sample: correlations with age, sex, and allergen skin test reactivity. J Allergy Clin Immunol 1981;68:106-11. 14. Asthma Prevalence, Health Care Use and Mortality, 2000-2001. Available at: http://www.cdc.gov/nchs/products/pubs/pubd/hestats/asthma/ asthma.htm. Accessed April 29, 2003. 15. Stevenson LA, Gergen PJ, Hoover DR, Rosenstreich D, Mannino DM, Matte TD. Sociodemographic correlates of indoor allergen sensitivity among United States children. J Allergy Clin Immunol 2001;108:747-52. 16. Arbes SJ Jr, Cohn RD, Yin M, Muilenberg ML, Burge HA, Friedman W, et al. House dust mite allergen in US beds: results from the First National Survey of Lead and Allergens in Housing. J Allergy Clin Immunol 2003; 111:408-14.

17. Cohn RD, Arbes SJ Jr, Yin M, Jaramillo R, Zeldin DC. National prevalence and exposure risk for mouse allergen in US households. J Allergy Clin Immunol 2004;113:1167-71. 18. Burrows B, Halonen M, Lebowitz MD, Knudson RJ, Barbee RA. The relationship of serum immunoglobulin E, allergy skin tests, and smoking to respiratory disorders. J Allergy Clin Immunol 1982;70:199-204. 19. Strachan DP, Cook DG. Health effects of passive smoking. 5. Parental smoking and allergic sensitisation in children. Thorax 1998;53:117-23.

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20. Sopori ML, Kozak W. Immunomodulatory effects of cigarette smoke. J Neuroimmunol 1998;83:148-56. 21. Arbes SJ Jr, Cohn RD, Yin M, Muilenberg ML, Friedman W, Zeldin DC. Dog allergen (Can f 1) and cat allergen (Fel d 1) in US homes: results from the National Survey of Lead and Allergens in Housing. J Allergy Clin Immunol 2004;114:111-7. 22. Rose G. Sick individuals and sick populations 1985. Bull World Health Organ 2001;79:990-6.

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Airborne endotoxin in homes with domestic animals: Implications for cat-specific tolerance James A. Platts-Mills, BA, Natalie J. Custis, BA, Judith A. Woodfolk, MD, PhD, and Thomas A. E. Platts-Mills, MD, PhD Charlottesville, Va

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Background: Although endotoxin is known to increase symptoms in allergic individuals, early exposure might decrease sensitization. Similarly, the presence of an animal in the home has been associated with decreased sensitization to animal allergens. It has been suggested that the effect of animals could be explained by increased endotoxin exposure. Objective: We sought to investigate the effects of domestic animals on airborne endotoxin. Methods: By using a silent particle collector, air was sampled over 24 hours in homes with or without animals. The total volume sampled was approximately 1000 m3, which provides quantities of allergen and endotoxin that can easily be measured with standard assays. Results: The quantity of endotoxin ranged from less than 0.5 to more than 500 pg/m3, whereas cat and dog allergen ranged from less than 0.002 to more than 5 ng/m3. Overall, the quantity of airborne endotoxin was not higher in homes with at least one animal. However, airborne endotoxin levels were significantly lower in homes with a cat compared with homes with a dog (P < .001). In keeping with this, there was a significant correlation between airborne Can f 1 and airborne endotoxin (r = 0.50, P < .01) but not between endotoxin and Fel d 1 (r = 0.17, P = .27). Conclusions: The results demonstrate that endotoxin is present in the air of almost all homes. Although higher levels were seen in homes with a dog, similar levels might be present in homes with no animals. The results argue that the effects of cat ownership cannot be explained by increased exposure to endotoxin. (J Allergy Clin Immunol 2005;116:384-9.) Key words: Airborne endotoxin, cats, dogs

Respiratory symptoms related to domestic animals are a significant health issue. However, in many studies the prevalence of IgE specific for cats or dogs is lower than for other major allergens, such as pollens or dust mites.1-3 This is not due to inadequate exposure because the major allergens Fel d 1 and Can f 1 are found in schools, public

From the Asthma and Allergic Diseases Center, University of Virginia. Supported by National Institutes of Health grants AI-20565 and AID/EHS grant P01-AI-50989. In addition, J.P.M received an unrestricted educational grant from The Sharper Image. Disclosure of potential conflict of interest: None disclosed. Received for publication January 10, 2005; revised April 29, 2005; accepted for publication May 9, 2005. Available online June 29, 2005. Reprint requests: Thomas A. E. Platts-Mills, MD, PhD, University of Virginia Health Systems, Asthma and Allergic Diseases Center, PO Box 801355, Charlottesville, VA 22908-1355. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.012

384

Abbreviations used EU: Endotoxin units ICD: Ion-charging device

places, and houses without a cat, as well as in those with an animal.4-7 Despite (or because of) this high allergen exposure, children raised in a house with an animal are less likely to become sensitized to animal allergens.8-12 One possible explanation for this paradoxical finding is that allergic families choose to avoid owning animals because of the perceived risk of sensitization.10,13 We have previously reported that high exposure to the cat allergen Fel d 1 induces a form of immune tolerance that is allergen specific.9,14,15 Alternatively, it has been argued that cats and other animals might increase agents such as bacterial LPS (endotoxin) in the home.12 Endotoxin is known to favor a shift away from TH2 responses in mice and might have the same effect on children.16,17 In keeping with this, children raised in close contact with farm animals (ie, with high endotoxin exposure) have less allergic disease.18,19 However, published reports are not consistent about the effects of pets on either floor or airborne endotoxin levels.15,20,21 Measuring airborne allergen or endotoxin requires both sensitive assays and a technique for collecting airborne particles.22,23 The quantity measured is a function of the airborne concentration and the volume of air sampled. If the concentration in the air is low, low-volume samplers will require very sensitive assays and might still provide inadequate samples.22,24 On the other hand, a highvolume collector may sample the air repeatedly and thus underestimate the airborne concentration, whereas collectors with a fan are at risk of artificially increasing the flux of particles into the air.23,25 The ion-charging device (ICD) used here is silent but has a moderately high flow rate. The device has 3 stainless-steel collection plates from which the particles can be removed and analyzed. We have used this technique to measure airborne allergen levels in homes and airborne endotoxin levels in animal facilities.26,27 Those studies included validating the assay techniques and the collection efficiency of the device.

METHODS Airborne sampling The machines used for airborne sampling (Ionic Breeze Quadras from The Sharper Image, San Francisco, Calif) cycle between 2

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distinct flow rates. For each device, we timed the periods P1 and P2 of each rate. By using a vanometer, the wind speed was measured at the center of 114 squares, each with an area of 9 cm2, on a 2-dimensional grid placed orthogonal to and directly in front of the machine. These speeds were averaged and multiplied by the measurement area to determine the flow rate at each speed, V1 and V2. The flow rate of each device was calculated as follows: ðP1 V1 1P2 V2 Þ=ðP1 1P2 Þ in cubic meters per minute. The average flow rate of the 14 devices used in this study was 1.48 6 0.09 m3/min.26,27 A 20 L/min air-sampling pump and an ICD were run in parallel in a room with artificially disturbed dust (by using a vacuum cleaner without a filter) for 10-minute periods (n = 8) to determine the collection efficiency of the devices. The 20 L/min pump collected airborne particles by using the same prefilter as was used to clean the collection plates of the ICDs. All samples were extracted overnight in 2 mL of PBS and assayed for endotoxin and Fel d 1. The flow rates of the 2 devices and the amount of endotoxin and cat allergen measured were used to determine the collection efficiency for ICDs. The mean collection efficiency was 40.6% 6 9.0% for endotoxin and 51.7% 6 12.0% for Fel d 1, which were not significantly different. For the purposes of comparison with other studies, an estimated sampling rate of 0.67 m3/min was used, the product of the flow rate and a mean particle collection efficiency of 45%. Using this estimated sampling rate, we can convert values for the total quantities of airborne endotoxin or allergen collected to airborne concentrations (Figs 1-3). All sampling used 2 ICDs in parallel running for 24 hours and placed at least 6 feet apart and at least 4 feet from the wall. In each case, the stainless-steel plates of the 2 ICDs were removed and cleaned with a series of 3 filters (Millipore prefilters, AP20, 35 mm; Millipore Corporation, Bedford, Mass) dampened with sterile water. Each filter was placed in a 3-mL syringe and extracted overnight at 4°C. The 3 filters from the first ICD were extracted in 2 mL of 1% BSA in PBS-Tween for measuring Can f 1 and Fel d 1, whereas those from the latter were extracted in 2 mL of endotoxin-free PBS for measuring endotoxin. In preliminary experiments 2 ICDs were run in parallel and both were assayed for either endotoxin or Fel d 1, and

there was a close correlation between samples for both endotoxin (n = 56, r = 0.91) and Fel d 1 (n = 44, r = 0.93).

Domestic sampling A total of 71 homes in Central Virginia were studied between November 2003 and May 2004 to collect dust and carry out air sampling for 24 hours. Because seasonal variation has been reported for endotoxin, 43 homes were studied both in November-December and April-May. A floor dust sample was also collected with a Hoover handheld vacuum.

Animal room sampling We sampled 20 mouse rooms from 4 different animal facilities (vivariums) at the University of Virginia. Each room was sampled at least twice. A variety of cage types are used, but for the purposes of this study, we have distinguished only between open cages, which have only a metal grill to prevent the animals from exiting the cage, and filter-topped cages of any configuration. The detailed methods have been published elsewhere.27 All animals were used for research studies that had been approved by the University of Virginia Institutional Animal Care and Use Committee.

Assays Samples were assayed for Fel d 1 and Can f 1 by using 2-site mAbbased ELISAs (Indoor Biotechnologies, Inc, Charlottesville, Va), which are sensitive to 1 ng/mL, and for endotoxin with the Limulus Amoebocyte Lysate test QCL 1000, which is sensitive to 0.3 endotoxin units (EU)/mL (equivalent to 30 pg of endotoxin/mL; Bio-Whittaker/Cambrex). Because extract freeze-thaw cycles are associated with a significant decrease in endotoxin concentration, samples were assayed for endotoxin immediately on extraction. The buffer used for extraction was used in each assay as a negative control and was less than the level of detection in all cases.

Statistical analysis Because the exposure data had a log-normal distribution, all values were reported as geometric means with 95% CIs, and statistics

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FIG 1. Airborne endotoxin concentrations in picograms per cubic meter calculated from quantities collected on the basis of 1 EU = 100 pg and a sampling rate of 0.67 m3/min (see the ‘‘Methods’’ section). Geometric means are indicated. Any subset of homes with dogs had significantly more airborne endotoxin than any subset without dogs.

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FIG 2. Airborne endotoxin was measured in 43 homes on 2 separate occasions 4 months apart. Although there was considerable variation between the 2 samples, the correlation was: r = 0.57, P < .001. For homes with cats, shown with open circles, each sample was less than 30 pg/m3.

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were performed on log-transformed data. Means were compared by using independent-sample t tests, whereas a least squares linear regression was used to assess the seasonal variation in airborne endotoxin, the correlation between airborne allergens and endotoxin, and the correlation between duplicate measurements for endotoxin and cat allergen. A P value of less than .05 was considered significant. All statistical analyses were performed with SPSS 11 (SPSS Inc, Chicago, Ill).

Presenting the results as endotoxin collected over 24 hours allows comparison between different homes and with animal facilities sampled in the same way. The results show that endotoxin exposure in homes is lower than in animal rooms where rats or mice are kept without a filter top on the cage. However, in animal rooms where cage tops are present, which is the case for the majority of animal facilities, the mean endotoxin airborne concentration was lower than that for homes (Table III).

RESULTS Easily measurable concentrations of endotoxin and animal allergens were present in the floor dust samples. There was a wide range of values, from 0.82 EU/mg to 660 EU/mg, and overall, there was no significant effect of animal ownership (Table I). There were large differences in the concentration of cat and dog allergens, which was in keeping with the presence of animals. The results for airborne endotoxin also showed no overall difference between homes with animals and homes without animals (Table II). The same data are presented as airborne concentrations (Fig 1). When the results were analyzed by the species of animal present, there were highly significant differences. Homes with a cat or cats had significantly lower airborne endotoxin levels than homes with a dog or with both species (Fig 1). In 43 houses airborne sampling for endotoxin was carried out twice, first in November-December and again in AprilMay (Fig 2). Overall, there was a good correlation between the 2 measurements (r = 0.57, P < .001). In particular, the values in homes with a cat were consistently lower (ie, <30 pg/m3). Comparing airborne dog allergen levels with airborne endotoxin levels showed a significant positive correlation (r = 0.56, P < .001; Fig 3, A). The association was not significant between endotoxin and cat allergen (r = 0.13, P = .33; Fig 3, B).

DISCUSSION In many studies the prevalence of IgE antibodies specific for cat or dog allergen is lower than for other major allergens, such as pollens or dust mites.1-3 This is not due to inadequate exposure because (1) the phenomenon is more marked among children living in a house with a cat,8-12 (2) the allergens are present and airborne continuously, and (3) the major allergens Fel d 1 and Can f 1 are found in schools, public places, and houses without animals, as well as those with an animal. Our data show that the presence of a cat in the home does not increase airborne endotoxin levels. Thus the allergenspecific tolerance to cat allergens cannot be attributed to increased endotoxin exposure in homes with a cat. Inevitably, any sampling technique that collects a quantity of allergen or endotoxin that can be confidently measured will alter the particles present in the air. In addition, it is well established that the airflow created by a high-volume air filter can increase airborne allergen levels.23 Thus all methods of measuring airborne concentrations of allergen or endotoxin involve a compromise. The particle collector used here has several disadvantages and some major advantages. The disadvantages are (1) those associated with high-volume sampling and (2) that

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FIG 3. A, Airborne dog allergen concentration in nanograms per cubic meter compared with airborne endotoxin concentration in picograms per cubic meter (r = 0.56, P < .001). B, Airborne cat allergen concentration in nanograms per cubic meter compared with airborne endotoxin concentration in nanograms per cubic meter (r = 0.17, P = .27).

the interpretation of the quantity collected requires an estimate of the efficiency with which particles are collected. The advantages of the device are that (1) it is silent and therefore well accepted for use in any room, including bedrooms; (2) sampling of particles off the stainless-steel plates is simple and very consistent (this is not possible with most devices designed to clean the air, including all high-efficiency particulate air filters); and (3) it collects particles from large volumes of air, but because of the wide aperture, the velocity of air coming out is relatively low. In preliminary experiments sampling air at 18 L/min for 2 to 6 hours, we were not able to detect significant endotoxin levels in most houses. Other groups have

successfully measured airborne endotoxin levels by using very low-volume sampling (ie, 2 L/min).22,28 However, that required extrasensitive assays and extensive precautions to avoid endotoxin contamination. The collector used here has no electrical safety concerns. Most pumps used for collecting airborne allergen are not approved as domestic appliances and therefore should not be left in a home without the presence of an investigator. The range of results observed (ie, from <5 to >5000 EU/24 hours or <0.5 to >500 pg/m3) is such that small differences in the estimated collection efficiency (ie, between 41% and 52%) would not affect the interpretation of our results. In an additional experiment (data not shown), airborne endotoxin and Fel d 1 levels were measured before and

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TABLE I. Floor dust concentration of endotoxin and allergens* N

No animals Animals Cat(s) only Dog(s) only Both species

Endotoxin (EU/mg)y

23 39 13 15 11

49.8 (29.9-82.9) 63.9 (47.1-86.8) 82.8 (45.3-151) 49.9 (31.3-79.5) 66.1 (39.5-110.7)

Fel d 1 (mg/g)

Can f 1 (mg/g)

2.5 (1.3-4.7) 25.1 (7.9-79.9) 583 (201-1690) 0.52 (0.24-1.1) 121 (36.1-405)

2.3 (1.4-4.0) 49.4 (17.2-142) 0.65 (0.24-1.7) 405 (226-728) 315 (169-586)

*All values are presented as geometric means (95% CIs).  Endotoxin levels were not significantly different between cats and dogs (P = .189) or cats and no animals (P = .221).

TABLE II. Airborne quantity of endotoxin and allergens collected in 24 hours* n

No animals Animals Cat(s) only Dog(s) only Both species

Endotoxin (EU/24 h)y

28 43 16 15 12

115 (61-217) 240 (155-372) 68 (44-105) 447 (250-800) 588 (282-1230)

Fel d 1 (ng/24 h)

6.6 (4.9-8.8) 102 (64-162) 488 (361-660) 4.7 (3.6-6.1) 1076 (545-2126)

Can f 1 (ng/24 h)

3.3 (2.7-4) 138 (87-219) 3.4 (2.3-4.9) 777 (566-1067) 1030 (790-1342)

*All values are presented as geometric means (95% CIs).  Homes with dogs had higher airborne endotoxin than homes with cats (P < .001) or homes with no animals (P = .003), as did homes with both animals (vs homes with cats, P < .001; vs homes with no animals, P = .005).

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TABLE III. Airborne endotoxin in homes and animal rooms n

Homes without animals Homes with animals Mouse rooms (open cages) Mouse rooms (filter tops on cages)

28 43 8 20

EU/24 h (GM)

115 240 1930 47.8

(61-217) (155-372) (752–4940)* (31.9-71.6) 

GM, Geometric mean. *Significantly higher than homes with or without animals (P < .001).  Significantly lower than homes with animals (P < .001) or without animals (P = .024).

after disturbance of dust in an experimental room by using a domestic vacuum cleaner without a filter. The results suggested that 90% of both airborne endotoxin and cat allergen levels fell within 10 minutes. The rapid falling rates of both endotoxin and cat allergen suggest that the level of disturbance is as important as the concentration in reservoirs of dust. The observation that endotoxin levels in the floor dust of houses with cats or dogs are similar, yet the airborne levels are significantly different, might suggest that dogs were a greater cause of disturbance. Differences in the quantity of endotoxin either airborne or in the floor dust of houses with a cat cannot explain why the presence of a cat in the house is associated with a form of immune tolerance. However, this is not to say that airborne endotoxin is irrelevant to the response. In mice the immune response to inhaled ovalbumin can be suppressed or changed by high exposure to endotoxin, but the IgE antibody response is enhanced by small quantities of endotoxin.16 We have found airborne endotoxin in almost all homes, and this might be sufficient to act as an adjuvant for responses to inhaled allergens.29 At

present, it remains unclear whether inhaled endotoxin in homes contributes either to sensitization or symptoms. The concentrations reported here in homes are higher than the concentrations reported to give rise to symptoms among animal handlers.28 However, those studies used low-volume sampling and reported a much smaller range of results than we have found here or in animal facilities.27 Furthermore, recent short-term challenge studies found that doses of less than 10,000 EU produced very little immediate change in the lungs.30 Some authors have implied that both the effects of cow ownership in Europe and the paradoxical effects of cat ownership are in keeping with the hygiene hypothesis. Our data argue in favor of a completely different interpretation of the effects of cat ownership. In our studies on 4 different cohorts, the effect of cat ownership has been cat specific. In particular, cat ownership has no effect on the IgE antibody response to dust mite allergens.9,15 In addition, the immune response to Fel d 1 includes the IL-4–dependent isotype IgG4.9,10 Thus the nonallergic response to cat has the features of a modified TH2 response and not the TH1 response that would be predicted if the tolerant response was related to increased endotoxin exposure.16-18 It is important to recognize that there are major differences in the ways that cats and dogs are kept, and our results might be relevant to specific housing conditions. In New Zealand the floor dust levels of endotoxin in homes with or without cats (13.7 vs 17.4 EU/mg, not significant) were lower than the levels seen here.15 Thus we could be looking at 2 different phenomena overlapping in different studies. The first effect is tolerance induced specifically by cat allergen exposure, whereas the second, a nonspecific effect of animal ownership, including dog ownership,

could reflect increased exposure to endotoxin or other bacterial products.

15.

REFERENCES 1. Roost HP, Kunzli N, Schindler C, Jarvis D, Chinn S, Perruchoud AP. Role of current and childhood exposure to cat and atopic sensitization. European Community Respiratory Health Survey. J Allergy Clin Immunol 1999;104:941-7. 2. Sears MR, Herbison GP, Holdaway MD, Hewitt CJ, Flannery EM, Silva PA. The relative risks of sensitivity to grass pollen, house dust mite and cat dander in the development of childhood asthma. Clin Exp Allergy 1989;19:419-24. 3. Lewis SA, Weiss ST, Platts-Mills TA, Syring M, Gold DR. Association of specific allergen sensitization with socioeconomic factors and allergic disease in a population of Boston women. J Allergy Clin Immunol 2001; 107:615-22. 4. Sporik R, Ingram JM, Price W, Sussman JH, Honsinger RW, Platts-Mills TA. Association of asthma with serum IgE and skin test reactivity to allergens among children living at high altitude. Tickling the dragons breath. Am J Respir Crit Care Med 1995;151:1388-92. 5. Almquist C, Larsson PH, Egmar AC, Hedren M, Malmberg P, Wickman M. School as a risk environment for children allergic to cats and a site for transfer of cat allergen to homes. J Allergy Clin Immunol 1999;103: 1012-7. 6. Custovic A, Green R, Taggart SC, Smith A, Pickering CA, Chapman MD, et al. Domestic allergens in public places. II: Dog (Can f1) and cockroach (Bla g 2) allergens in dust and mite, cat, dog and cockroach allergens in the air in public buildings. Clin Exp Allergy 1996;26:1246-52. 7. Perzanowski MS, Ronmark E, Nold B, Lundback B, Platts-Mills TA. Relevance of allergens from cats and dogs to asthma in the northernmost province of Sweden: schools as a major site of exposure. J Allergy Clin Immunol 1999;103:1018-24. 8. Hesselmar B, Aberg N, Aberg B, Eriksson B, Bjorksten B. Does early exposure to cat or dog protect against later allergy development? Clin Exp Allergy 1999;29:611-7. 9. Platts-Mills T, Vaughan J, Squillace S, Woodfolk J, Sporik R. Sensitization, asthma, and a modified Th2 response in children exposed to cat allergen: a population-based cross-sectional study. Lancet 2001;357: 752-6. 10. Perzanowski MS, Ronmark E, Platts-Mills TA, Lundback B. Effect of cat and dog ownership on sensitization and development of asthma among preteenage children. Am J Respir Crit Care Med 2002;166: 696-702. 11. Custovic A, Hallam CL, Simpson BM, Craven M, Simpson A, Woodcock A. Decreased prevalence of sensitization to cats with high exposure to cat allergen. J Allergy Clin Immunol 2001;108:537-9. 12. Ownby DR, Johnson CC, Peterson EL. Exposure to dogs and cats in the first year of life and risk of allergic sensitization at 6 to 7 years of age. JAMA 2002;288:963-72. 13. Anyo G, Brunekreef B, de Meer G, Aarts F, Janssen NA, van Vliet P. Early, current and past pet ownership: associations with sensitization, bronchial responsiveness and allergic symptoms in school children. Clin Exp Allergy 2002;32:361-6. 14. Reefer AJ, Carneiro RM, Custis NJ, Platts-Mills TA, Sung SS, Hammer J, et al. A role for IL-10-mediated HLA-DR7-restricted T cell-dependent

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events in development of the modified Th2 response to cat allergen. J Immunol 2004;172:2763-72. Erwin EA, Wickens K, Custis NJ, Siebers R, Woodfolk JA, Barry D, et al. Cat and dust mite sensitivity and tolerance in relation to wheezing among children with high exposure to allergens. J Allergy Clin Immunol 2005;115:74-9. Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K. Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 2002; 196:1645-51. Gereda JE, Leung DY, Thatayatikom A, Strieb JE, Price MR, Klinnert MD, et al. Relation between house-dust endotoxin exposure, type 1 T-cell development, and allergen sensitization in infants at high risk of asthma. Lancet 2000;355:1680-3. Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 2002;347:869-77. Gehring U, Bischof W, Fahlbusch B, Wichmann H-E, Heinrich J. House dust endotoxin and allergic sensitization in children. Am J Respir Crit Care Med 2002;166:939-44. Heinrich J, Gehring U, Douwes J, Koch A, Fahlbusch B, Bischof W, et al. Pets and vermin are associated with high endotoxin levels in house dust. Clin Exp Allergy 2001;31:1839-45. El Sharif N, Douwes J, Hoet PHM, Doekes G, Nemery B. Concentrations of domestic mite and pet allergens and endotoxin in Palestine. Allergy 2004;59:623-31. Park JH, Spiegelman DL, Gold DR, Burge HA, Milton DK. Predictors of airborne endotoxin in the home. Environ Health Perspect 2001;109: 859-64. Luczynska CM, Li Y, Chapman MD, Platts-Mills TA. Airborne concentrations and particle size distribution of allergen derived from domestic cats (Felis domesticus). Measurements using cascade impactor, liquid impinger, and a two-site monoclonal antibody assay for Fel d 1. Am Rev Respir Dis 1990;141:361-7. Schweitzer IB, Smith E, Harrison DJ, Myers DD, Eggleston PA, Stockwell JD, et al. Reducing exposure to laboratory animal allergens. Comp Med 2003;53:486-92. Swanson MC, Agarwal MK, Reed CE. An immunological approach to indoor aeroallergen quantitation with a new volumetric air sampler: studies with mite, roach, cat, mouse, and guinea pig antigens. J Allergy Clin Immunol 1985;76:724-9. Custis NJ, Woodfolk JA, Vaughan JW, Platts-Mills TAE. Quantitive measurement of airborne allergens from dust mites, dogs, and cats using an ion charging device. Clin Exp Allergy 2003;33:986-91. Platts-Mills J, Custis N, Kenney A, Tsay A, Chapman M, Feldman S, et al. The effects of cage design on airborne allergens and endotoxin in animal rooms: high-volume measurements with an ion-charging device. Contemp Top Lab Anim Sci 2005;44:12-6. Pacheco KA, McCammon C, Liu AH, Thorne PS, O’Neill M, Martyny J, et al. Airborne endotoxin predicts symptoms in non-mouse-sensitized technicians and research scientists exposed to laboratory mice. Am J Respir Crit Care Med 2003;167:983-90. Liu AH. Endotoxin exposure in allergy and asthma: reconciling a paradox. J Allergy Clin Immunol 2002;109:379-92. Alexis NE, Lay JC, Almond M, Peden DB. Inhalation of low-dose endotoxin favors local T(H)2 response and primes airway phagocytes in vivo. J Allergy Clin Immunol 2004;114:1325-31.

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Food allergy, dermatologic diseases, and anaphylaxis COX-2 inhibition enhances the TH2 immune response to epicutaneous sensitization Dhafer Laouini, PhD, Abdala ElKhal, PhD, Ali Yalcindag, MD, Seiji Kawamoto, MD, PhD, Hans Oettgen, MD, PhD, and Raif S. Geha, MD Boston, Mass

Food allergy, dermatologic diseases, and anaphylaxis

Background: Mechanical injury to the skin by scratching is an important feature of atopic dermatitis (AD). Objective: To investigate the role of COX-2 in allergic skin inflammation elicited by epicutaneous (EC) sensitization via introduction of ovalbumin through shaved tape-stripped skin. Methods: COX-2 mRNA was measured by quantitative PCR, and COX-2 protein was measured by Western blotting. We investigated the effect of administration of the COX-2 selective inhibitor NS-398 during EC sensitization with ovalbumin in a mouse model of AD characterized by eosinophil skin infiltration, elevated total and antigen specific IgE, and a systemic TH2 response to antigen. We further examined the response of COX-2–deficient mice to EC immunization with ovalbumin. Results: Tape stripping caused a transient increase in skin COX-2 mRNA. In contrast, COX-2 mRNA was not increased after ovalbumin sensitization. Infiltration by eosinophils and expression of IL-4 mRNA in ovalbumin-sensitized skin sites, ovalbumin specific IgE and IgG1 antibody responses, and IL-4 secretion by splenocytes after ovalbumin stimulation were all significantly increased in EC mice that received NS-398. In contrast, ovalbumin specific IgG2a antibody response and IFN-g secretion by splenocytes after ovalbumin stimulation were significantly decreased in these mice. COX-2–deficient mice also exhibited an enhanced systemic TH2 response to EC sensitization. Conclusion: These results demonstrate that COX-2 limits the TH2 response to EC sensitization and suggest that COX inhibitors may worsen allergic skin inflammation in patients with AD. (J Allergy Clin Immunol 2005;116:390-6.) Key words: Atopic dermatitis, allergic skin Inflammation, NS-398, COX-2, TH1, TH2

Prostaglandins are formed by the oxidative cyclization of the central carbons within 20 carbon polyunsaturated fatty acids. 5-COX is the key enzyme involved in the

From the Division of Immunology, Children’s Hospital; and the Department of Pediatrics, Harvard Medical School. Dr Laouini and Dr ElKhal contributed equally to the article. Supported by National Institutes of Health grant AI-31541. Received for publication July 27, 2004; revised March 31, 2005; accepted for publication March 31, 2005. Available online June 1, 2005. Reprint requests: Raif S. Geha, MD, Enders 8, Division of Immunology, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.03.042

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Abbreviations used AD: Atopic dermatitis BAL: Bronchoalveolar lavage CysLT: Cysteinyl leukotriene DC: Dendritic cell EC: Epicutaneous HPF: High-power field PG: Prostaglandin PPAR: Peroxisome proliferator-activated receptor WT: Wild type

conversion of arachidonic acid to prostaglandin (PG) G2 and PGH2. PGH2 is subsequently converted to a variety of eicosanoids that include PGE2, PGD2, PGF2a, PGI2, and thromboxane A2.1 The spectrum of prostaglandins produced depends on the downstream enzymatic machinery expressed in a particular cell type. Prostaglandins have both autocrine and paracrine effects. These are mediated by an array of receptors, which are differentially expressed by various cell types. Two classes of prostaglandin receptors exist: the membrane G-coupled receptor class, ie, E-prostanoid 1-4 receptors for PGE2; and the nuclear peroxisome proliferator-activated receptor (PPAR) class, ie, PPARa, PPARg, and PPARd, which acts as a transcription factor on ligand binding.2 Nonsteroidal antiinflammatory drugs inhibit COX, leading to a marked decrease in prostaglandin synthesis and inflammation.3 Two COX isoforms, COX-1 and COX-2, have been identified and are encoded by distinct genes.4 COX-1 is expressed in nearly all tissues under basal conditions, suggesting that its major function is to generate prostaglandin precursors for homeostatic regulation.5 COX-2 is mainly an inducible enzyme. Inflammatory cytokines, which include IL-1 and TNF-a, and growth factors, which include TGF-a, platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor, all have been shown to induce COX-2 expression.6-9 Prostaglandins have profound effects on the immune response. A large body of data suggests that addition of PGE2 in vitro inhibits IL-12 production and promotes IL-10 production by antigen-presenting cells, inhibits the production of TH1 cytokines, and promotes TH2 cell differentiation.10,11 Furthermore, PGE2 was shown to enhance IL-4–driven isotype switching to IgE.12 Topical application of PGE2 suppresses the cutaneous immune

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METHODS Mice BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, Me). COX-2+/2 mice on C57BL6x129/SvlmJ background and genetically matched controls were obtained from Taconic (Germantown, NY). Homozygous COX-2–deficient mice were obtained by genotyping the offspring of COX-2+/2 parents, and, as previously described, were not fertile.26 All mice were kept in a pathogen-free environment. All procedures performed on the mice were in accordance with the Animal Care and Use Committee of the Children’s Hospital.

EC sensitization EC sensitization of female mice 4 to 6 weeks old was performed as described previously.25 Briefly, the skin of anesthetized mice was shaved and tape-stripped 6 times. Ovalbumin (grade V; Sigma Chemical Co, St Louis, Mo) 100 mg in 100 mL normal saline, or placebo (100 mL normal saline), was placed on a patch of sterile gauze (1 cm 3 1 cm), which was secured to the skin with a transparent bio-occlusive dressing (Tegaderm, Owens & Minor Inc, Franklin, Mass). Each mouse had a total of three 1-week exposures to the patch separated by 2-week intervals. On day 49, the mice were killed and their tissues examined.

Treatment with COX-2 inhibitor Mice were given 1 mg/kg of the selective COX-2 inhibitor NS-398 (Biomol Research Laboratories, Inc, Plymouth Meeting, Pa) intra-

peritoneally daily for the duration of the sensitization period. NS-398 (25 mg/mL in dimethyl sulfoxide) was diluted in a 5% NaHCO3 solution before injection.

Histological analysis Specimens were fixed in 10% buffered formalin and embedded in paraffin. Multiple 4-mm sections were stained with hematoxylin and eosin. Individual cell types were counted blinded in 15 to 20 highpower fields (HPFs) at 10003.

Quantitative RT-PCR for COX enzyme mRNA expression Five hundred milligrams of skin was homogenized by using a Polytron RT-3000 (Kinematica AG, Brinkmann Instruments Inc) in lysis buffer solution provided in the RNAqueous extraction kit (Ambion Inc, Austin, Tex). RT was performed by using transcriptor first-strand cDNA synthesis kit (Roche Diagnostic, Foster City, Calif). PCR reactions were run on an ABI Prism 7700 (Applied Biosystems, Foster City, Calif) sequence detection system platform. Taqman primers with 6-carboxyfluorescein-labeled probe were obtained from Applied Biosystems. The housekeeping gene b2microglobulin was used as a control. The relative gene expression among the different samples was determined by using the method described by Pfaffl.27

Determination of COX-2 protein expression and PGE2 levels in skin Five hundred milligrams of skin was homogenized in 1 mL 0.1 mol/L PBS solution (pH = 7.4) containing 1 mmol/L EDTA, 0.1 mmol/L indomethacin, and a cocktail of protease inhibitors. Fifteen microliters of this solution was used for Western blotting for COX-2, with a rabbit anti–COX-2 antiserum (Abcam Inc, Cambridge, Mass) followed by horseradish peroxidase–conjugated donkey antirabbit antibody (Amersham Bioscience, Temecula, Calif). The blots were reprobed with mAb to actin (Chemicon International, Piscataway, NJ) followed by horseradish peroxidase–conjugated sheep antimouse antibody (Amersham Bioscience) for loading control. The rest of the material was extracted as described,28 and the extract used to determine PGE2 concentration by ELISA (Cayman Chemicals, Ann Arbor, Mich).

Competitive RT-PCR evaluation of cytokine mRNA in skin Competitive RT-PCR evaluation of cytokine mRNA in skin was performed as described previously.29 Skin biopsies were immediately frozen in dry ice. The samples were homogenized in Trizol (GIBCO BRL, Carlsbad, Calif) by using a Polytron RT-3000. RNA extraction was performed following the manufacturer’s instructions. cDNA was synthesized from 10 mg total RNA in a 40-mL reaction mix by using Superscript II (GIBCO BRL). The primers used to amplify cDNA for b2-microglobulin, IL-4, and IFN-g and DNA amplification were as described previously.29 To quantify cytokine mRNA, a fixed amount of reverse-transcribed cellular mRNA was coamplified in the presence of serial dilutions of a multispecific internal plasmid control (pMUS3), which contains nucleotide sequences of multiple cytokines.30 Results were expressed as a ratio of cytokine cDNA to b2-microglobulin cDNA. We have recently found that the results of competitive RT-PCR for determination of cytokine mRNA in skin compare favorably with those of quantitative RT-PCR. In 2 experiments in BALB/c mice, each using 6 mice with EC with ovalbumin and 6 mice with EC with saline, we found that the mean increase in skin IL-4 mRNA expression after ovalbumin sensitization was 4.9fold using competitive PCR compared with 4.3-fold using quantitative RT-PCR.

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response.13 The PPARg agonist 15-deoxy-d(12,14)-prostaglandin J(2) also inhibits IL-12 production by macrophages14 and ameliorates experimental autoimmune encephalomyelitis.15 In a model of allergic airway inflammation, COX inhibition by nonsteroidal anti-inflammatory drug increased IL-5 and IL-13 production in bronchoalveolar lavage (BAL) fluid and airway hyperresponsivenessAHR.16 Furthermore, lung inflammatory indices, which include BAL cells, proteins, and IgE as well as lung inflammation as determined by histopathology, were significantly increased in the absence of either COX-1 or COX-2.17 COX-1 is basally expressed at low levels in skin.18 Skin injury by UV light has been shown to induce COX-2 expression,19 whereas mechanical injury increases PGE2 in the skin.20 This may be mediated by IL-1 and TNF-a released from keratinocytes, fibroblasts, and mast cells.21-23 Atopic dermatitis (AD) is an inflammatory skin disease that frequently occurs in subjects with personal or family history of atopic disease.24 Mechanical injury to the skin by scratching is an important feature of AD. We have developed a mouse model of allergic skin inflammation elicited by epicutaneous (EC) sensitization with ovalbumin. This model displays many of the features of human AD, including a dermatitis characterized by dermal infiltration of T cells and eosinophils and increased local expression of TH2 cytokines and by a systemic allergen specific TH2 response characterized by IgG1 and IgE antibodies and IL-4 secretion by splenocytes after in vitro stimulation with ovalbumin.25 We used this model to assess the role of COX-2 in allergic skin inflammation.

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FIG 1. COX-2 and COX-1 mRNA expression in the skin of BALB/C mice after tape stripping (n = 2 per group; A) and after EC sensitization with ovalbumin (OVA) and saline (SAL) sensitization (n = 5 per group; B), expressed as fold induction over levels in unmanipulated skin. C, COX-2 protein expression in skin after tape stripping (left panel) and EC sensitization (right panel). Results are representative of 3 experiments. D, PGE2 levels in skin (n = 4 per group). Columns and bars represent means and SEMs. *P < .05.

Serum antibody determinations IgG1, IgG2a, and IgE antiovalbumin antibodies were determined by ELISA following the procedures we previously described.25

IL-4 and IFN-g synthesis by spleen cells Single cell suspensions of spleen cells were prepared and cultured at 2 3 106/mL in 24-well plates in the presence of ovalbumin (50 mg/mL) as previously described.31 Supernatants were collected after 96 hours. IL-4 and IFN-g were determined by ELISA (Pharmingen, San Diego, Calif).

Statistical analysis Food allergy, dermatologic diseases, and anaphylaxis

The nonparametric Mann-Whitney test was used to compare the different mice groups.

RESULTS Tape stripping induces expression of COX-2 mRNA in normal mouse skin We used quantitative RT-PCR to examine the effect of tape stripping on COX-2 mRNA expression in mouse skin. Low levels of COX-2 mRNA were detectable in uninjured skin. After tape stripping 6 times, COX-2 mRNA expression increased, with peak levels 8 hours poststripping, and returning to normal 48 hours later (Fig 1, A). In contrast, there was no detectable increase in the levels of COX-1 mRNA levels in the skin after tape stripping. COX-2 and COX-1 mRNA levels in ovalbumin-sensitized skin sites did not significantly differ from those in saline-sensitized or unmanipulated skin sites (Fig 1, B). Western blotting analysis demonstrated that COX-2 protein expression in the skin increased 8 hours after tape stripping (Fig 1, C). In contrast, there was no detectable increase in COX-2 protein expression in ovalbumin-

sensitized skin sites compared with saline-sensitized skin sites or with unmanipulated skin (Fig 1, C). The increased COX-2 mRNA expression observed 8 hours after stripping was associated with significantly increased level of the COX metabolite PGE2 (Fig 1, D). There was no increase in PGE2 levels in either ovalbumin-sensitized or saline-sensitized skin sites.

Eosinophil infiltration is increased in ovalbumin-sensitized skin sites of mice treated with COX-2 inhibitor There was no difference in the numbers of eosinophils in saline sensitized sites of untreated mice and mice treated with NS-398 (Fig 2, A). Ovalbumin sensitization caused a significant increase in the number of eosinophils in the skin of control untreated BALB/c mice, consistent with previous observations.25 There were significantly more eosinophils in ovalbumin-sensitized skin of mice treated with NS-398 than in ovalbumin-sensitized skin of untreated controls (Fig 2, A). Ovalbumin sensitization caused an increase in skin mononuclear cells that was modestly but significantly higher in mice treated with NS-398 compared with untreated controls (Fig 2, B). IL-4 expression is increased in EC skin sites of mice treated with COX-2 inhibitor We used competitive PCR to measure cytokine mRNA in skin. Low and comparable levels of IL-4 and IFN-g mRNA were detected in saline sensitized skin from untreated mice and mice treated with NS-398 (Fig 3). Consistent with previous results,25 expression of IL-4 mRNA, but not IFN-g mRNA, markedly increased in ovalbumin-sensitized skin sites of untreated control BALB/c mice. IL-4 mRNA was significantly increased

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FIG 2. Effect of the COX-2 inhibitor NS-398 on the number of infiltrating eosinophils (A) and mononuclear cells (B) in ovalbumin (OVA)–sensitized and saline-sensitized skin sites of untreated mice and in mice treated with NS-398. The columns and error bars represent means 6 SEMs/HPF of cells calculated by examining 15 to 20 HPFs per mouse (n = 6). *P < .05. **P < .01.

FIG 4. Effect of the COX-2 inhibitor NS-398 on (A) serum levels of ovalbumin (OVA) specific IgG, IgE, and IgG2a and (B) cytokine production by spleen cells from EC mice (n = 6 for each group). Columns and error bars represent means 6 SEMs. *P < .05. **P < .01. SAL., Saline; Sens., sensitization. FIG 3. Effect of the COX-2 inhibitor NS-398 on IL-4 (A) and IFN-g (B) mRNA expression in saline and ovalbumin (OVA)–sensitized skin. Levels were normalized to b2-microglobulin. Pooled results of experiments using 6 mice per group. Bars represent means 6 SEMs. *P < .05. **P < .01.

Treatment with COX-2 inhibitor enhances antigen specific IgE and IgG1 antibody responses to EC sensitization with ovalbumin The TH2 cytokine IL-4 plays an important role in isotype switching to IgE and IgG1, whereas the TH1 cytokine IFN-g plays an important role in isotype switching to IgG2a.32 To investigate whether COX-2 inhibition enhanced the systemic TH2 response to EC sensitization with ovalbumin, we measured total and ovalbumin specific IgE and IgG1 in serum. Fig 4, A, shows that ovalbumin specific IgG1 and IgE levels were significantly higher in mice treated with NS-398. Treatment with NS-398 had no effect on the IgG2a antibody response. These results suggest that COX products normally downregulate the systemic IgE and IgG1 antibody response to EC-introduced antigen. COX-2 inhibition causes increased systemic TH2 response and decreased systemic TH1 response to EC sensitization We have previously shown that splenocytes from BALB/c mice with EC with ovalbumin secrete IL-4, and

FIG 5. Serum levels of ovalbumin (OVA) specific IgG1 (A), IgE (B), and IgG2a (C) in EC mice, COX-22/2 mice, and WT controls (n = 6 for each group). Columns and error bars represent means 6 SEMs. *P < .05.

IFN-g after ovalbumin stimulation in vitro.31 Fig 4, B, shows that splenocytes from EC mice treated with NS-398 secreted significantly higher amounts of IL-4, and significantly less IFN-g, than splenocytes of unsensitized, untreated controls. These results suggest that COX products normally limit the systemic TH2 response to EC-introduced antigen and promote the systemic TH1 response.

Increased systemic TH2 response and decreased systemic TH1 response in COX-2–deficient mice NS-398 may have effects other than COX-2 enzyme inhibition. To ascertain that the effect of NS-398 on the TH response to EC sensitization was a result of COX-2 inhibition, we examined the response of COX-22/2 mice to EC immunization. Fig 5 shows that ovalbumin specific IgE levels were significantly higher and ovalbumin specific IgG2a levels were significantly lower in

Food allergy, dermatologic diseases, and anaphylaxis

in ovalbumin-sensitized skin of mice treated with NS-398 compared with untreated ovalbumin-sensitized controls. These results suggest that COX-2 products normally downregulate the TH2 cytokine profile of infiltrating T cells.

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FIG 6. Cytokine production by spleen cells from EC mice, COX-22/2 mice, and WT controls (n = 6 for each group). Columns and error bars represent means 6 SEMs. *P < .05. SAL., Saline.

COX-2–deficient mice than in wild-type (WT) controls. COX-2 deficiency had no detectable effect on the IgG1 antibody response. Fig 6 shows that splenocytes from ECimmunized COX-22/2 mice secreted significantly more IL-4 and significantly less IFN-g than splenocytes of genetically matched WT controls.

DISCUSSION

Food allergy, dermatologic diseases, and anaphylaxis

The results of this study suggest that COX-2 products limit the systemic TH2 response and enhance the systemic TH1 response to epicutaneously introduced antigen and promote skin infiltration with eosinophils in a mouse model of allergic dermatitis. Both COX-1 and COX-2 were expressed in unmanipulated mouse skin (Fig 1, A), consistent with previous reports on mouse and human skin.18,33,34 Mechanical injury by tape stripping transiently upregulated COX-2 mRNA but not COX-1 mRNA expression in mouse skin. This finding is consistent with the observation that PGE2 levels increase in human skin after tape stripping,20 which we confirmed in this mouse study (Fig 1, C). Keratinocytes, fibroblasts, mast cells, endothelial cells, and tissue macrophages have all been reported to express COX-2 after activation.21-23 IL-1b and TNF-a are known inducers of COX-2 mRNA expression.6,7 A correlation has been observed between levels of PGE2 and IL-1a in tape-stripped skin.20 The COX-2 selective inhibitor NS-398 enhanced dermal infiltration with eosinophils in our model (Fig 2, A). Eosinophil infiltration of the skin in our model is dependent on their expression of the eotaxin receptor CCR3,31 and that eotaxin expression is dependent on IL-4.29 Expression of mRNA for the TH2 cytokine IL-4, but not for the TH1 cytokine IFN-g, in ovalbumin-sensitized skin sites was significantly enhanced in NS-398–treated mice (Fig 3). This may have contributed to increased eosinophil skin infiltration. The TH2 cytokine IL-5 is also important for eosinophil infiltration of the skin in our model. Although we did not measure IL-5 expression, it is likely that it was also enhanced in NS-398–treated mice and contributed to their exaggerated skin eosinophilia, because IL-5 and IL-4 expression by TH2 cells is usually concordant.

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The increased infiltration by mononuclear cells observed in ovalbumin-sensitized skin of NS-398–treated mice (Fig 2, B) may reflect the stronger TH2 response exhibited by these mice. Tape stripping induces the expression of the TH2 selective chemokines thymus and activation-regulated chemokine and cutaneous T cell– attracting chemokine, which attract TH2 cells in the skin (unpublished data). Secretion of the TH2 cytokines IL-4 and IL-13 by infiltrating TH2 cells further upregulates the expression of TH2 selective chemokines by skin cells35 and enhances TH2 cell infiltration. Increased secretion of TH2 cytokines in the skin of NS-398–treated mice may contribute to the enhanced infiltration of T cells in the skin of these mice. Treatment of mice with the COX-2 inhibitor NS-398 promoted the TH2 systemic response to EC sensitization. This was evidenced by enhanced serum IgG1 and IgE antibody levels to ovalbumin mice (Fig 4, A) and increased IL-4 secretion by splenocytes in response to stimulation with ovalbumin (Fig 4, B). In contrast, IFN-g secretion was significantly decreased (Fig 4, B). Studies with COX22/2 mice strongly supported the conclusion that the enhancing effect of NS-398 on the TH2 response to EC immunization was a result of inhibition of COX-2 activity. After EC immunization, these mice mounted a significantly higher ovalbumin specific IgE antibody response and their splenocytes secreted significantly more IL-4 and significantly less IFN-g than splenocytes from WT controls (Figs 5 and 6). Taken together, our results suggest that COX-2 products normally limit the development of TH2 cytokines and promote the development of TH1 cytokines in response to EC sensitization with antigen. The fact that there was no detectable increase in COX-2 or COX-1 mRNA expression, COX-2 protein expression, or PGE2 levels in ovalbumin-sensitized skin compared with either saline sensitized or unmanipulated skin (Fig 1, B-D) suggests that the effects of COX-2 inhibition may be exerted early in the sensitization phase of our model, which is dependent on tape stripping, because we are unable to sensitize the mice without it. However, we cannot rule out an effect on later events via the inhibition of baseline COX-2 activity in the skin. Our findings of increased IL-4 response to EC sensitization with inhibition or lack of COX-2 is in agreement with previous findings that administration of the COX inhibitor indomethacin 2 days before and during intraperitoneal immunization enhances mRNA expression and secretion of the TH2 cytokines IL-5 and IL-13 in the lung after allergen challenge.16 Similarly, allergic lung inflammation as evidenced by eosinophils in BAL fluid and lung histopathology and TH2 cytokine secretion are enhanced in intraperitoneally immunized mice deficient in COX-1 or COX-2.17,36 In these studies, COX inhibition did not result in a significant increase in the IgE antibody response to intraperitoneal immunization, and TH cytokine secretion by splenic T cells was not examined. There is a plethora of evidence that the COX-2 product PGE2, which is present in skin of patients with AD,37

inhibits the development of TH1 cells and promotes the development of TH2 cells in vitro,10,38 although discrepant results have also been reported.39,40 This inhibitory effect is exerted in a large part at the level of the dendritic cells, because PGE2 is a potent inhibitor of IL-12 production by these cells10 and an inhibitor of IL-12b1 receptor and IL-12b2 receptor expression.41 On the basis of these in vitro results, one would expect that decreased PGE2 generation in the skin subsequent to inhibition or lack of COX-2 may promote the TH1 response and inhibit the TH2 response to EC sensitization. In fact, the reverse was observed. However, PGE2 may not be the most abundant or most relevant prostaglandin generated in injured skin. Langerhans cells generate high amounts of PGD2 but very little amounts of other prostaglandins.42 The prostanoid PGI2 limits lung allergic inflammation,43 and mice deficient in the PGI2 receptor mount an exaggerated TH2 response.43,44 Further studies are needed to examine the nature of prostaglandins that accumulate in the skin after mechanical injury and the roles of individual prostaglandins in modulating the TH response to EC sensitization. Inhibition or lack of COX-2 activity may result in enhanced leukotriene synthesis because of both increased availability of arachidonic acid substrate and release from the inhibitory effect of prostaglandins on 5-lipooxygenase–activating protein expression.45 Increased amounts of leukotriene C4 in the skin may promote CC chemokine ligand 19-dependent mobilization of antigen bearing dendritic cells (DC) to lymph nodes,46 resulting in the exaggeration of what is already a TH2-skewed response to EC sensitization. Cysteinyl leukotrienes (cysLTs) may promote the induction of TH2 responses by DCs, as suggested by the observation that intranasal administration of DCs pulsed with antigen and cysLTs enhance allergic inflammation compared with DCs pulsed with antigen alone.47 CysLTs also promote eosinophil locomotion and hence infiltration at skin sites of allergic sensitization.48 EC sensitization of mice is relevant to human sensitization because it mimics allergen sensitization via abraded skin in patients with AD. Although there are no data on COX-2 expression in human AD, COX products are increased in the skin of patients with AD,37 and COX-2 gene expression is induced by IL-13, which is expressed in AD lesions.49 Our results clearly show that COX-2 inhibition may exacerbate AD by promoting the systemic and cutaneous TH2 response and are best avoided in this disease. REFERENCES 1. Smith W, Marnett L, DeWitt D. Prostaglandin and thromboxane biosynthesis. Pharmacol Ther 1991;49:153-79. 2. Forman BM, Chen J, Evans RM. The peroxisome proliferator-activated receptors: ligands and activators. Ann N Y Acad Sci 1996;804:266-75. 3. Simon LS. Actions and toxicity of nonsteroidal anti-inflammatory drugs. Curr Opin Rheumatol 1996;8:169-75. 4. Smith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 1996;271:33157-60. 5. Crofford LJ. COX-1 and COX-2 tissue expression: implications and predictions. J Rheumatol 1997;24(suppl 49):15-9.

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6. Raz A, Wyche A, Siegel N, Needleman P. Regulation of fibroblast cyclooxygenase synthesis by interleukin-1. J Biol Chem 1988;263: 3022-8. 7. Diaz A, Chepenik KP, Korn JH, Reginato AM, Jimenez SA. Differential regulation of cyclooxygenases 1 and 2 by interleukin-1 beta, tumor necrosis factor-alpha, and transforming growth factor-beta 1 in human lung fibroblasts. Exp Cell Res 1998;241:222-9. 8. Saha D, Datta PK, Sheng H, Morrow JD, Wada M, Moses HL, et al. Synergistic induction of cyclooxygenase-2 by transforming growth factor-beta1 and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia 1999;1:508-17. 9. Goppelt-Struebe M, Rehm M, Schaefers HJ. Induction of cyclooxygenase-2 by platelet-derived growth factor (PDGF) and its inhibition by dexamethasone are independent of NF-kappaB/IkappaB transcription factors. Naunyn Schmiedebergs Arch Pharmacol 2000;361:636-45. 10. van der Pouw Kraan TC, Boeije LC, Smeenk RJ, Wijdenes J, Aarden LA. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J Exp Med 1995;181:775-9. 11. Kalinski P, Hilkens CM, Snijders A, Snijdewint FG, Kapsenberg ML. IL-12-deficient dendritic cells, generated in the presence of prostaglandin E2, promote type 2 cytokine production in maturing human naive T helper cells. J Immunol 1997;159:28-35. 12. Roper RL, Brown DM, Phipps RP. Prostaglandin E2 promotes B lymphocyte Ig isotype switching to IgE. J Immunol 1995;154:162-70. 13. Rheins LA, Barnes L, Amornsiripanitch S, Collins CE, Nordlund JJ. Suppression of the cutaneous immune response following topical application of the prostaglandin PGE2. Cell Immunol 1987;106:33-42. 14. Azuma Y, Shinohara M, Wang PL, Ohura K. 15-Deoxy-delta(12,14)prostaglandin J(2) inhibits IL-10 and IL-12 production by macrophages. Biochem Biophys Res Commun 2001;283:344-6. 15. Diab A, Deng C, Smith JD, Hussain RZ, Phanavanh B, Lovett-Racke AE, et al. Peroxisome proliferator-activated receptor-gamma agonist 15-deoxy-delta(12,14)-prostaglandin J(2) ameliorates experimental autoimmune encephalomyelitis. J Immunol 2002;168:2508-15. 16. Peebles RS Jr, Dworski R, Collins RD, Jarzecka K, Mitchell DB, Graham BS, et al. Cyclooxygenase inhibition increases interleukin 5 and interleukin 13 production and airway hyperresponsiveness in allergic mice. Am J Respir Crit Care Med 2000;162:676-81. 17. Gavett SH, Madison SL, Chulada PC, Scarborough PE, Qu W, Boyle JE, et al. Allergic lung responses are increased in prostaglandin H synthasedeficient mice. J Clin Invest 1999;104:721-32. 18. Abd-El-Aleem SA, Ferguson MW, Appleton I, Bhowmick A, McCollum CN, Ireland GW. Expression of cyclooxygenase isoforms in normal human skin and chronic venous ulcers. J Pathol 2001;195:616-23. 19. Athar M, An KP, Morel KD, Kim AL, Aszterbaum M, Longley J, et al. Ultraviolet B(UVB)-induced cox-2 expression in murine skin: an immunohistochemical study. Biochem Biophys Res Commun 2001; 280:1042-7. 20. Reilly DM, Green MR. Eicosanoid and cytokine levels in acute skin irritation in response to tape stripping and capsaicin. Acta Derm Venereol 1999;79:187-90. 21. Scholz K, Furstenberger G, Muller-Decker K, Marks F. Differential expression of prostaglandin-H synthase isoenzymes in normal and activated keratinocytes in vivo and in vitro. Biochem J 1995;309:263-9. 22. Warnock LJ, Hunninghake GW. Multiple second messenger pathways regulate IL-1 beta-induced expression of PGHS-2 mRNA in normal human skin fibroblasts. J Cell Physiol 1995;163:172-8. 23. Leong J, Hughes-Fulford M, Rakhlin N, Habib A, Maclouf J, Goldyne ME. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation. Exp Cell Res 1996;224:79-87. 24. Leung DY. Pathogenesis of atopic dermatitis. J Allergy Clin Immunol 1999;104:S99-108. 25. Spergel J, Mizoguchi E, Brewer J, Martin T, Bhan A, Geha R. Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolized antigen in mice. J Clin Invest 1998;101:1614-22. 26. Morham SG, Langenbach R, Loftin CD, Tiano HF, Vouloumanos N, Jennette JC, et al. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 1995;83:473-82. 27. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.

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28. Takemura N, Takahashi K, Tanaka H, Ihara Y, Ikemoto A, Fujii Y, et al. Dietary, but not topical, alpha-linolenic acid suppresses UVB-induced skin injury in hairless mice when compared with linoleic acids. Photochem Photobiol 2002;76:657-63. 29. Spergel JM, Mizoguchi E, Oettgen H, Bhan AK, Geha RS. Roles of TH1 and TH2 cytokines in a murine model of allergic dermatitis. J Clin Invest 1999;103:1103-11. 30. Shire D, Legoux P. Gene expression analysis using competitive reverse transcription polymerase chain reaction and multispecific internal control. Totowa (NJ): Humana Press, Inc; 1995. 31. Ma W, Bryce PJ, Humbles AA, Laouini D, Yalcindag A, Alenius H, et al. CCR3 is essential for skin eosinophilia and airway hyperresponsiveness in a murine model of allergic skin inflammation. J Clin Invest 2002;109:621-8. 32. Snapper CM, Paul WE. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 1987;236: 944-7. 33. Muller-Decker K, Reinerth G, Krieg P, Zimmermann R, Heise H, Bayerl C, et al. Prostaglandin-H-synthase isozyme expression in normal and neoplastic human skin. Int J Cancer 1999;82:648-56. 34. Muller-Decker K, Scholz K, Neufang G, Marks F, Furstenberger G. Localization of prostaglandin-H synthase-1 and -2 in mouse skin: implications for cutaneous function. Exp Cell Res 1998;242:84-91. 35. Romagnani S. Cytokines and chemoattractants in allergic inflammation. Mol Immunol 2002;38:881-5. 36. Carey MA, Germolec DR, Bradbury JA, Gooch RA, Moorman MP, Flake GP, et al. Accentuated T helper type 2 airway response after allergen challenge in cyclooxygenase-1-/- but not cyclooxygenase-2-/mice. Am J Respir Crit Care Med 2003;167:1509-15. 37. Fogh K, Herlin T, Kragballe K. Eicosanoids in skin of patients with atopic dermatitis: prostaglandin E2 and leukotriene B4 are present in biologically active concentrations. J Allergy Clin Immunol 1989;83: 450-5. 38. Katamura K, Shintaku N, Yamauchi Y, Fukui T, Ohshima Y, Mayumi M, et al. Prostaglandin E2 at priming of naive CD4+ T cells inhibits acquisition of ability to produce IFN-gamma and IL-2, but not IL-4 and IL-5. J Immunol 1995;155:4604-12.

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39. Rieser C, Bock G, Klocker H, Bartsch G, Thurnher M. Prostaglandin E2 and tumor necrosis factor alpha cooperate to activate human dendritic cells: synergistic activation of interleukin 12 production. J Exp Med 1997;186:1603-8. 40. Parker CW, Huber MG, Godt SM. Modulation of IL-4 production in murine spleen cells by prostaglandins. Cell Immunol 1995;160:278-85. 41. Wu CY, Wang K, McDyer JF, Seder RA. Prostaglandin E2 and dexamethasone inhibit IL-12 receptor expression and IL-12 responsiveness. J Immunol 1998;161:2723-30. 42. Ruzicka T, Aubock J. Arachidonic acid metabolism in guinea pig Langerhans cells: studies on cyclooxygenase and lipoxygenase pathways. J Immunol 1987;138:539-43. 43. Jaffar Z, Wan KS, Roberts K. A key role for prostaglandin I2 in limiting lung mucosal Th2, but not Th1, responses to inhaled allergen. J Immunol 2002;169:5997-6004. 44. Takahashi Y, Tokuoka S, Masuda T, Hirano Y, Nagao M, Tanaka H, et al. Augmentation of allergic inflammation in prostanoid IP receptor deficient mice. Br J Pharmacol 2002;137:315-22. 45. Harizi H, Juzan M, Moreau JF, Gualde N. Prostaglandins inhibit 5lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism. J Immunol 2003;170:139-46. 46. Robbiani DF, Finch RA, Jager D, Muller WA, Sartorelli AC, Randolph GJ. The leukotriene C(4) transporter MRP1 regulates CCL19 (MIP3beta, ELC)-dependent mobilization of dendritic cells to lymph nodes. Cell 2000;103:757-68. 47. Machida I, Matsuse H, Kondo Y, Kawano T, Saeki S, Tomari S, et al. Cysteinyl leukotrienes regulate dendritic cell functions in a murine model of asthma. J Immunol 2004;172:1833-8. 48. Fregonese L, Silvestri M, Sabatini F, Rossi GA. Cysteinyl leukotrienes induce human eosinophil locomotion and adhesion molecule expression via a CysLT1 receptor-mediated mechanism. Clin Exp Allergy 2002;32: 745-50. 49. Yu CL, Huang MH, Kung YY, Tsai CY, Tsai YY, Tsai ST, et al. Interleukin-13 increases prostaglandin E2 (PGE2) production by normal human polymorphonuclear neutrophils by enhancing cyclooxygenase 2 (COX-2) gene expression. Inflamm Res 1998;47:167-73.

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Responsiveness to autologous sweat and serum in cholinergic urticaria classifies its clinical subtypes Atsushi Fukunaga, MD, Toshinori Bito, MD, Kenta Tsuru, MD, Akiko Oohashi, MD, Xijun Yu, MD, Masamitsu Ichihashi, MD, Chikako Nishigori, MD, and Tatsuya Horikawa, MD Kobe, Japan

Key words: Cholinergic urticaria, sweat, autologous serum, skin test, histamine release test, acetylcholine test

From the Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine. Disclosure of potential conflict of interest: A. Fukunaga, none disclosed; T. Bito, none disclosed; K. Tsuru, none disclosed; A. Oohashi, none disclosed; X. Yu, none disclosed; M. Ichihashi, none disclosed; C. Nishigori, none disclosed; T. Horikawa, none disclosed. Received for publication November 11, 2004; revised May 13, 2005; accepted for publication May 17, 2005. Available online July 15, 2005. Reprint requests: Tatsuya Horikawa, MD, Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.024

Abbreviations used CU: Cholinergic urticaria ASST: Autologous serum skin test

Cholinergic urticaria (CU), which was first described by Duke1 in 1924, is characterized by unique clinical features: pinpoint sized, highly pruritic wheals with surrounding erythema that occur after sweating during physical exercise, taking a bath, raising the body temperature, and emotional stress. In typical cases, this disorder usually occurs in young adults. Occasionally, this disorder is accompanied with angioedema and anaphylactic reactions.2,3 The pathogenesis of CU has not yet been well clarified despite the fact that numerous investigators have described its clinical characteristics and possible pathogenesis. In patients with CU, injection of acetylcholine (mecholyl) into normal-appearing skin produces a wheal and flare reaction, often surrounded by smaller satellite lesions that are similar to the skin symptoms of CU.4 Acetylcholine is thus believed to play a significant role in the development of the symptoms of CU. Another aspect of the pathogenesis of CU has focused on sweat itself on the basis of evidence that this unique eruption occurs after sweating. Adachi et al5 found that 20 patients with CU showed immediate-type reactions after an intradermal skin test with autologous sweat. Kobayashi et al6 presumed that the leakage of sweat into the dermis because of ductal occlusion at the superficial acrosyringium causes CU. Kaplan et al7 and Sigler et al8 demonstrated plasma histamine elevations after exercise challenge of patients with CU. We performed this study to clarify further the possible involvement of sweat-mediated and autoimmunemediated mechanisms in CU and in its clinical features. Skin responsiveness was evaluated after the intracutaneous injection of autologous sweat and serum. We assessed the correlation between the degrees of skin reactions and amounts of in vitro histamine released from basophils after stimulation with autologous sweat. We further analyzed the relationship between the clinical symptoms of CU and the characteristics of these tests. 397

Food allergy, dermatologic diseases, and anaphylaxis

Background: It has been reported that patients with cholinergic urticaria have a type 1 allergy to autologous sweat; however, the pathogenesis of that disorder has not been fully elucidated. Objective: We investigated the responsiveness to autologous sweat and serum in patients with cholinergic urticaria in relation to their clinical characteristics. We further classified the clinical subtypes that are clearly characterized by responsiveness to in vivo and in vitro tests as well as their clinical features. Methods: Intradermal tests with autologous sweat and serum were performed in 18 patients with cholinergic urticaria. Histamine release from peripheral blood basophils induced by autologous sweat was measured. Results: Eleven of 17 patients with cholinergic urticaria showed positive reactions in skin tests with their own diluted sweat. Substantial amounts of sweat-induced histamine release from autologous basophils were observed in 10 of 17 patients. Eight of 15 patients with cholinergic urticaria showed positive reactions in the autologous serum skin tests. All 6 patients who developed satellite wheals after the acetylcholine test showed hypersensitivity to sweat. Further, patients whose eruptions were coincident with hair follicles showed positive responses to the skin test with autologous serum, whereas patients whose eruptions were not coincident with hair follicles did not. Conclusion: On the basis of these findings, we propose that cholinergic urticaria should be classified into 2 distinct subtypes. The first (nonfollicular) subtype shows strong positive reactions to autologous sweat and negative reactions to autologous serum. The second (follicular) subtype shows weak reactions to autologous sweat and positive reactions to autologous serum. (J Allergy Clin Immunol 2005;116: 397-402.)

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METHODS Subjects Eighteen patients with CU were enrolled at the Dermatological Institute of Kobe University Hospital. CU was confirmed by the development of numerous small wheals after exercise until sweating.9 All of the patients had no aquagenic urticaria. The characteristics of patients with CU are described in Table I. Their age ranged between 15 and 31 years (mean, 21.8 years). Nine patients had a past history of atopic diseases (6 atopic dermatitis and 3 allergic rhinitis). Five patients were accompanied with cold urticaria. Healthy control subjects were enrolled from the staff at Kobe University Hospital. All subjects provided oral consent for this study after oral and written explanations. Relevant drugs such as histamine H1-receptor antagonists were withdrawn for at least 24 hours before the examination. All of the patients had never had systemic corticosteroids for at least 3 months before the examination.

Materials Venous blood was taken into sterile glass tubes and allowed to clot at room temperature for 30 minutes. Serum was separated by centrifugation at 500g for 20 minutes and passed through a 0.45-mm MILLEX HV membrane (Millipore, Molsheim, France). Sweat was collected from each patient’s forearm after exercise. The sweat was sterilized after collection by using a 0.45-mm MILLEX HV membrane, and it was preserved at 280°C before use. Sweat samples were diluted with saline (1/100 dilution) before the skin test. Histamine contents of sweat samples (1/100 dilution) from healthy control subjects and patients with CU were less than 10 nmol/L.

Skin test technique Samples of autologous diluted sweat (0.02 mL), autologous serum (0.05 mL), and 0.9% sterile saline (0.02 or 0.05 mL) were separately injected intradermally into the volar aspect of the forearm of each subjects when they were quiet and with no wheal. The diameters of wheals and erythema were measured after 15 minutes. Reactions were assessed as positive if the diameter of the wheal induced by sweat and serum was equal to or larger than 6 mm. The sterile salineinduced wheals of all subjects were below 4 mm and 2 mm when the amounts of 0.05 and 0.02 mL were injected, respectively. Food allergy, dermatologic diseases, and anaphylaxis

Local provocation test Responses to acetylcholine chloride (Ovisot; Daiichi, Tokyo, Japan) were evaluated. Acetylcholine (0.1 mL) was intradermally injected at a concentration of 100 mg/mL diluted with saline. The development of satellite wheals around the injection site was considered as positive. Simultaneously, we checked sweating around the injection site by the iodine-starch technique, and all patients tested showed sweating by this test.10 All of the normal controls tested showed a significant number of tiny sweating points by this method.

Basophil histamine release test A histamine release test was performed in vitro by using HRT (Shionogi, Osaka, Japan) as previously described.11 Venous peripheral blood samples from patients with CU and normal healthy controls, 20 mL, and antibasophil antibodies conjugated to magnetic beads were added to each well of a 96-well plate and incubated for 10 minutes at room temperature on a plate mixer. Antibody-binding basophils in each well were then trapped with a chandelier-shaped magnet and transferred to another microplate, where the basophils were stimulated at 37°C for 1 hour with autologous sweat, anti-IgE antibody, and digitonin, respectively. Histamine released into the

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medium was measured by an ELISA with a characteristic detection profile.12

Statistical analysis The statistical significance of differences was determined by using the Student t test. Some data were analyzed by regression analysis by using the statistical package StatView J (Abacus Concepts Inc, Palo Alto, CA). A difference was considered statistically significant at P < .05.

RESULTS Skin tests for autologous sweat Of 17 patients with CU, except for 1 patient who showed mechanical urticaria in skin test for autologous sweat, 11 (64.7%) showed positive reactions to their own 1/100 diluted sweat by measuring the diameter of wheals (Table II). In contrast, all 10 healthy controls showed negative reactions to their own 1/100 diluted sweat, whereas a few healthy controls had positive reactions to their own 1/10 diluted sweat (data not shown). Sweatinduced wheals in the skin tests were significantly greater for patients with CU than for healthy control subjects (Fig 1, A). Sweat-induced histamine release from basophils We investigated the histamine release from basophils of 17 patients with CU and of 10 healthy controls after incubation with autologous sweat. Of 17 CU patients’ basophils, 10 (58.8%) showed positive responses (more than 5% histamine release) after incubation with 1/100 diluted autologous CU sweat, whereas none of the 10 healthy controls’ basophils did with 1/100 diluted autologous normal sweat. Four of the CU patients’ basophils showed positive responses to 1/1000 diluted CU sweat. The overall values of percent histamine release from basophils of patients with CU were significantly larger than those from healthy control subjects (Fig 1, B). Correlation of skin tests for autologous sweat with sweat-induced histamine release from basophils We examined whether sweat-induced histamine release from CU basophils correlates with skin tests for autologous CU sweat in 16 patients. As shown in Fig 1, C, % histamine release correlated positively with the area in wheal using skin tests on 1/100 diluted sweat. These results indicate that the degree of percent histamine release for autologous sweat represents the responsiveness of skin tests for autologous sweat. Autologous serum skin tests Of 15 patients with CU, 8 (53.3%) showed a positive response in the autologous serum skin test (ASST; Table II). In contrast, all 6 healthy controls showed a negative response for ASST. Most patients with CU who had a negative response for ASST tended to show a positive

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TABLE I. Clinical characteristics of patients with cholinergic urticaria Patient

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Age (y)

Sex

History

Accompanying symptoms

Total IgE (IU/mL)

IgE RAST

26 25 22 20 21 20 22 19 22 26 31 19 24 15 26 18 19 17

Female Female Male Male Male Male Female Male Male Male Male Female Female Male Female Female Male Male

Atopic dermatitis Atopic dermatitis None None Atopic dermatitis None Allergic rhinitis None None None Allergic rhinitis Atopic dermatitis Atopic dermatitis None None Atopic dermatitis Urticaria Allergic rhinitis

None Asthma Cold urticaria None None None Cold urticaria None None None None Cold urticaria, angioedema None Cold urticaria None None Cold urticaria None

919 5518 432 85 4080 250 1842 Not done 116 Not done 194 342 907 1302 144 118 Not done 423

Mite, Candida Cedar, orchard grass Wheat Not done Mite, Candida, wheat Not done Mite, Candida Not done Not done Not done Not done Mite Mite Mite, Candida, orchard grass Negative Mite Not done Not done

% Histamine release by sweat

Autologous sweat skin test Patient

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

(Erythema*)

31 3 16 25 3 20 11 3 10 23 3 20 11 3 10 10 3 10 24 3 23 25 3 22 20 3 15 15 3 13 030 10 3 8 939 Mechanical urticaria 030 736 030 737

(Whealy)

1/100z

1/1000z

Autologous serum skin test§

Acetylcholine testk

Characteristics of eruption

20 10 9 8 11 7 10 9 7 6 0 10 0

3 3 3 3 3 3 3 3 3 3 3 3 3

14 10 8 8 10 7 8 9 6 5 0 8 0

0 0 0 4

3 3 3 3

0 0 0 4

100 89.6 0.5 7.3 21.4 Not done 40 48.8 54.8 63.2 10.6 2.1 1 0.2 0.4 0 0.4 1.1

36.6 23.6 0 0.4 6.5 Not done 3.9 0.2 6.7 4.6 1.3 0.2 0 0 0 0 0.4 0

Negative Negative Negative Negative Negative Negative Not done Not done Positive Positive Positive Positive Positive Not done Positive Positive Positive Negative

Not done Positive Positive Positive Positive Positive Positive Not done Not done Not done Not done Negative Negative Not done Negative Negative Negative Negative

Nonfollicular Nonfollicular Nonfollicular Nonfollicular Undetermined Nonfollicular Nonfollicular Nonfollicular Undetermined Undetermined Follicular Follicular Follicular Follicular Follicular Nonfollicular Follicular Nonfollicular

* Long axis and short axis of oval area are presented. 1/100 diluted sweat is used in autologous sweat skin test. àDilution of sweat. §Autologous serum was injected intradermally into the volar aspect of the forearm. kAcetylcholine was intradermally injected 0.1 mL in concentration of 100 mg/mL diluted with saline.

response for skin tests and for the histamine release test with autologous CU sweat. In contrast, a few patients with a positive response for ASST tended to show hypersensitivity for sweat (Table II).

Intradermal acetylcholine test After intradermal injections of relatively high concentrations of cholinergic agents, the typical satellite pinpoint

wheals around the central large wheal were seen only in patients with CU.4 However, these satellite wheals seemed to develop only in a few patients with CU.13 We therefore examined whether the patients with CU showed satellite wheals by acetylcholine injection. In this study, 6 (50%) of the 12 patients with CU tested showed a positive response for acetylcholine (Table II). Almost all of the patients who were checked for sweating by iodine-starch method

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TABLE II. Details of results for skin tests, histamine release test, acetylcholine test, and clinical chracterization

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those from patients who showed negative responses for the acetylcholine test (Fig 2, B). In addition, certain satellite wheals after the acetylcholine test were recognizable as coincident with perspiration points when sweating points were detected by the starch-iodine method (Fig 2, C).

Characterization of the clinical phenotype When patients with CU developed wheals after exercise, we observed that the wheals sometimes coincided with hair follicles. Of 16 patients with CU, 6 (37.5%) had wheals coincident with follicles (the follicular type), and 8 (50%) had wheals that were not coincident with follicles (the nonfollicular type). Compared with the follicular type, the areas of CU sweat-induced wheals in the skin test were significantly greater in the nonfollicular type (Fig 3, A). The values of CU sweat-induced histamine released from basophils of the nonfollicular type tended to be greater than those released from basophils of the follicular type (Fig 3, B). A representative clinical picture of wheals consisting of follicles is shown (Fig 3, C). DISCUSSION

Food allergy, dermatologic diseases, and anaphylaxis

FIG 1. Differences in responses to sweat in patients with CU and normal controls. A, The areas of wheals induced by intradermal injection with autologous sweat. B, Values of the histamine release from basophils stimulated with CU sweat or normal sweat. C, The relationship of responsiveness of skin tests with CU sweat and CU sweat-induced histamine release from CU basophils.

showed sweating after acetylcholine injection in our series, indicating that the absence of the satellite wheals by the agent is not attributed to the dyshidrosis. Compared with patients who showed a negative response for the acetylcholine test, the areas of CU sweat-induced wheals in the skin tests were significantly greater in patients who showed satellite wheals for the acetylcholine test (Fig 2, A). The values of CU sweat-induced histamine released from basophils of patients who showed positive responses for the acetylcholine test were significantly greater than

We demonstrated that the majority of patients with CU are highly sensitive to autologous sweat. The heterogeneous responses in skin tests with autologous sweat suggest that patients with CU have various degrees of hypersensitivity to sweat. We further observed that various amounts of histamine were detected in the medium when basophils were incubated in the presence of autologous sweat, which suggests that autologous sweat itself contains factors that can induce histamine release. The amounts of histamine released from CU basophils correlated relatively well with the degree of response in the skin tests to autologous CU sweat. In contrast, normal healthy controls did not respond to intracutaneous challenge with autologous sweat and did not show histamine release from basophils by stimulation with autologous sweat. These results indicate that patients with CU have various degrees of hypersensitivity to sweat and that in vitro histamine release tests using autologous sweat correlate with the intracutaneous test. Previously, Adachi et al5 reported that all patients with CU examined showed immediate-type skin reactions to intradermal tests with sweat at various dilutions (20-29). We observed that a few healthy controls had positive reactions after intradermal injection of 1/10 autologous sweat, whereas no healthy controls showed a positive reaction to their own 1/100 diluted sweat. Certain patients who showed a negative response at 1/100 dilution might have shown a positive response at higher concentrations (1/10 and higher dilutions). In other words, those patients with CU who showed positive skin responses in this study might represent the presence of strong hypersensitivity to sweat. Interestingly, Hide et al14 recently reported that patients with atopic dermatitis show hypersensitivity to autologous sweat antigens. They speculate that this phenomenon is

FIG 2. The relationship between responses to sweat and acetylcholine tests in patients with CU. A, The areas of wheals induced by intradermal injection with CU sweat in patients with CUs with or without satellite wheals. B, The CU sweat-induced histamine release from basophils in patients with CU with or without satellite wheals. C, A representative clinical picture that satellite wheals are coincident with perspiration points.

an IgE-mediated response, because histamine release was impaired by removal of IgE from patients’ basophils and myeloma IgE blocked the sensitization of basophils with the patient’s serum. It is of interest to note that patients with CU frequently have atopic dermatitis.5,15 Adachi et al5 have shown that leukocytes from a normal healthy donor did release histamine on sweat challenging after being sensitized with a patient’s serum. Therefore, it might be possible that, similar to atopic dermatitis, hypersensitivity to sweat in patients with CU could be an IgE-mediated response. An attractive hypothesis for the pathomechanisms of CU is that sweat leaks from the sweat duct into the dermis.6,16 Several cases of CU have been described that are associated with hypohidrosis/anhidrosis.6,16 Occlusion of the superficial acrosyringium might result in sweat leakage into the dermis in patients with CU and anhidrosis.6 If those patients with CU are hypersensitive to sweat, the leaking sweat possibly induces urticarial symptoms around the sweat ducts, resulting in small pinpoint wheals. Commens and Greaves4 examined 12 patients with CU by intradermal testing with methacholine and found that satellite wheals were induced in only 6 of them. It is not yet clear why only some patients with CU develop satellite wheals after injection of cholinergic agents. We showed

Fukunaga et al 401

FIG 3. The difference of response to sweat in the relationship between follicles and eruption in patients with CU. A, The areas of wheals induced by intradermal injection with CU sweat in patients with CU with nonfollicular or follicular wheals. B, The CU sweatinduced histamine release from basophils in CU patients with nonfollicular or follicular wheals. C, A representative picture of wheals consisting of follicles.

here that patients developing satellite wheals in the acetylcholine test had significantly enhanced responses to sweat in the skin tests and in histamine release tests (Fig 2). This means that those with hypersensitivity to sweat tend to develop satellite wheals after stimulation with acetylcholine, a sweat inducer. Moreover, we observed that satellite wheals after the acetylcholine test were coincident with perspiration points by the iodine-starch method (Fig 2, C). These results are compatible with the idea that sweat leakage from sweat ducts induces small wheals in certain patients with CU. Circulating functional histamine-releasing autoantibodies reactive against either the a-subunit of the highaffinity IgE receptor (FceRIa) or IgE have been identified in more than one third of patients with chronic idiopathic urticaria.17-22 The ASST is now recognized as a suitable screening test for such autoantibodies in such patients.4 However, it is still unclear whether the wheal-inducing factors in the patient’s sera in this study are these autoantibodies, and this issue should be further clarified in the future. Sabroe et al23 have reported that only 1 of 9 patients with CU had a positive ASST. In contrast, we showed here that 8 of 15 patients had a positive ASST. The discrepancy of the ratio of responsiveness in ASST between their findings and ours might be attributed to the patient population; that is, the majority of the patients in their study might have had the nonfollicular type. So far, it is unclear whether we might enroll more follicular-type

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patients than the usual population in CU. Therefore, this issue should be further studied in the future. We observed that certain patients with CU develop wheals in association with hair follicles, whereas the other patients do not. This phenomenon is similar to that seen in aquagenic urticaria, in which follicular wheals develop after contact with water. We found that patients with nonfollicular-type CU tend not only to show satellite wheals after the acetylcholine test but also to have hypersensitivity to sweat as determined by skin tests and by histamine release tests (Table II). On the other hand, most of the patients with follicular-type CU showed a positive reaction to ASST and no satellite wheals by acetylcholine or hypersensitivity to sweat (Table II). On the basis of these findings, we strongly believe that CU should be classified into 2 subtypes from the clinical and pathological aspects. The relationship between CU and hair follicles should be examined further. In summary, 2 subtypes were identified in patients with CU. The first subtype shows nonfollicular wheals, a hypersensitivity to autologous sweat, satellite wheals in the acetylcholine test, and negative reactions to autologous serum. The second subtype shows follicular wheals, a very weak hypersensitivity to sweat, no satellite wheals in the acetylcholine test, and positive reactions to autologous serum. Thus, we suggest that the pathogenesis of CU involves hypersensitivity or autoimmunity to sweat. In considering the pathogenesis of CU, the classification presented here may be useful to this unique disorder. REFERENCES

Food allergy, dermatologic diseases, and anaphylaxis

1. Duke WW. Urticaria caused specifically by the action of physical agents. JAMA 1924;83:3-9. 2. Kaplan AP, Natbony SF, Tawil AP, Fruchter L, Foster M. Exerciseinduced anaphylaxis as a manifestation of cholinergic urticaria. J Allergy Clin Immunol 1981;68:319-24. 3. Lawrence CM, Jorizzo JL, Kobza-Black A, Coutts A, Greaves MW. Cholinergic urticaria with associated angio-oedema. Br J Dermatol 1981; 105:543-50. 4. Commens CA, Greaves MW. Tests to establish the diagnosis in cholinergic urticaria. Br J Dermatol 1978;98:47-51. 5. Adachi J, Aoki T, Yamatodani A. Demonstration of sweat allergy in cholinergic urticaria. J Dermatol Sci 1994;7:142-9. 6. Kobayashi H, Aiba S, Yamagishi T, Tanita M, Hara M, Saito H, et al. Cholinergic urticaria, a new pathogenic concept: hypohidrosis due to interference with the delivery of sweat to the skin surface. Dermatology 2001;204:173-8.

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7. Kaplan AP, Gray L, Shaff RE, Horakova Z, Beaven MA. In vivo studies of mediator release in cold urticaria and cholinergic urticaria. J Allergy Clin Immunol 1975;55:394-402. 8. Sigler RW, Levinson AL, Evans R III, Horakova Z, Kaplan AP. Evaluation of patients with cold and cholinergic urticaria. J Allergy Clin Immunol 1979;63:35-8. 9. Kobza Black A, Lawlor F, Greaves MW. Consensus meeting on the definition of physical urticarias and urticarial vasculitis. Clin Exp Dermatol 1996;21:424-6. 10. Wada M, Takagaki T. A simple and accurate method for detecting the secretion of sweat. Tohoku J Exp Med 1948;49:284. 11. Adachi A, Fukunaga A, Hayashi K, Kunisada M, Horikawa T. Anaphylaxis to polyvinylpyrrolidone after vaginal application of povidoneiodine. Contact Dermatitis 2003;48:133-6. 12. Nishi H, Nishimura S, Higashiura M, Ikeya N, Ohta H, Tsuji T, et al. A new method for histamine release from purified peripheral blood basophils using monoclonal antibody-coated magnetic beads. J Immunol Methods 2000;240:39-46. 13. Commens CA, Greaves MW. Tests to establish the diagnosis in cholinergic urticaria. Br J Dermatol 1978;98:47-51. 14. Hide M, Tanaka T, Yamamura Y, Koro O, Yamamoto S. IgE-mediated hypersensitivity against human sweat antigen in patients with atopic dermatitis. Acta Derm Venereol 2002;82:335-40. 15. Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI. Fitzpatrick’s dermatology in general medicine. 6th ed. New York: McGraw-Hill; 2003. p. 1132-3. 16. Itakura E, Urabe K, Yasumoto S, Nakayama J, Furue M. Cholinergic urticaria associated with acquired generalized hypohidrosis: report of a case and review of literature. Br J Dermatol 2000;143:1064-6. 17. Hide M, Francis DM, Grattran CE, Hikimi J, Kochan JP, Greaves MW. Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria. N Engl J Med 1993;328: 1599-604. 18. Niimi N, Francis DM, Kermani F, O’Donnell BF, Hide M, Kobza Black A, et al. Dermal mast cell activation by autoantibodies against the high affinity IgE receptor in chronic urticaria. J Invest Dermatol 1996;106: 1001-6. 19. Fiebiger E, Maurer D, Holub H, Reininger B, Hartmann G, Woisetschlager M, et al. Serum IgG autoantibodies directed against the alpha chain of Fc epsilon RI: a selective marker and pathogenetic factor for a distinct subset of chronic urticaria patients? J Clin Invest 1995;96: 2606-12. 20. Tong LJ, Balakrishnan G, Kochan JP, Kinet JP, Kaplan AP. Assessment of autoimmunity in patients with chronic urticaria. J Allergy Clin Immunol 1997;99:461-5. 21. Zweiman B, Valenzazo M, Atkins PC, Tanus T, Getsy JA. Characteristics of histamine-releasing activity in the sera of patients with chronic idiopathic urticaria. J Allergy Clin Immunol 1996;98:89-98. 22. Ferrer M, Kinet JP, Kaplan AP. Comparative studies of functional and binding assays for IgG anti-Fc(epsilon)RIalpha (alpha-subunit) in chronic urticaria. J Allergy Clin Immunol 1998;101:672-6. 23. Sabroe RA, Grattan CEH, Francis DM, Barr RM, Black AK, Graves MW. The autologous serum skin test: a screening test for autoantibodies in chronic idiopathic urticaria. Br J Dermatol 1999;140:446-52.

Lack of detectable allergenicity of transgenic maize and soya samples Rita Batista, BSc,a,b Baltazar Nunes, MSc,a Manuela Carmo,a Carlos Cardoso, PharmD,c Helena Sa˜o Jose´,c Anto´nio Bugalho de Almeida, MD, PhD,d Alda Manique, MD,d Leonor Bento, MD, PhD,e Caˆndido Pinto Ricardo, PhD,b,f and Maria Margarida Oliveira, PhDb,g Lisboa, Oeiras, and Alge´s, Portugal

Key words: Transgenic food, allergenicity, immune response, public health, food safety, recombinant DNA technology

From aInstituto Nacional de Sau´de Dr Ricardo Jorge, Lisboa; bInstituto de Tecnologia Quı´mica e Biolo´gica/Instituto de Biologia Experimental e Tecnolo´gica, Oeiras; cClı´nica Me´dica e de Diagno´stico Dr Joaquim Chaves, Alge´s; dClı´nica Universita´ria de Pneumologia do Hospital de Santa Maria, Lisboa; eDepartamento de Clı´nica Pedia´trica do Hospital de Santa Maria, Lisboa; fInstituto Superior de Agronomia, Tapada da Ajuda, Lisboa; and gDepartamento Biologia Vegetal, Faculdade de Cieˆncias de Lisboa, Lisboa. Supported by Fundacxa˜o Calouste Gulbenkian, research project SDH.SP.I.01.11 and by Comissa˜o de Fomento da Investigacxa˜o em Cuidados de Sau´de, research project no. 186/01. Received for publication January 11, 2005; revised March 22, 2005; accepted for publication April 12, 2005. Available online June 1, 2005. Reprint requests: Rita Batista, BSc, Instituto Nacional de Sau´de Dr Ricardo Jorge, Av Padre Cruz, 1649-016 Lisboa, Portugal; E-mail: rbatista@ itqb.unl.pt. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.014

Abbreviations used Bt: Bacillus thuringiensis EPSPS: 5-Enolpyruvylshikimate-3-phosphate synthase GM: Genetically modified GMO: Genetically modified organism PAT: Phosphinotricine acetyl transferase RUR: Roundup Ready SPT: Skin prick test

Recombinant DNA technology or genetic engineering allows the transfer of single genes from one organism to another, even if distantly related, a feat impossible through conventional plant breeding. As a result, a genetically modified organism (GMO) will contain a modified or additional trait encoded by the introduced gene or genes, which generally results in additional proteins. Potential benefits for world agriculture derived from GMOs could be enormous, including the possibility of producing higher yields of more nutritious food in more sustainable regimens.1-5 With the development of the new modification techniques, there is the increasing concern of emergence of new food allergies. An example of such a situation is the Brazil nut allergen (2S protein), which when overexpressed in soybean was found to retain its allergenicity and was therefore never commercialized.6 Food allergy is a term that should be used to describe adverse reactions to certain foods because of immunologic mechanisms.7 The majority of individuals with documented immunologic reactions to foods exhibit IgE-mediated hypersensitivity reactions that can be sudden, severe, and life-threatening.8 The best estimates are that IgE-mediated food allergies affect approximately 1% to 2% of the adult population9,10; in children this value is estimated to be 2% to 8%.11,12 Before market introduction, genetically modified (GM) food products are subjected to extensive assessment of potential effects to human health, including toxicity and potential allergenicity. When the gene source is an allergenic food, in vitro and clinical tests are available to assess the allergenicity of the transferred protein or proteins. However, most genes transferred through genetic engineering are obtained from organisms with no allergenic history. In such cases the assessment of allergenicity becomes more difficult to obtain because of the absence of valid methods and models.13-16 403

Food allergy, dermatologic diseases, and anaphylaxis

Background: The safety issues regarding foods derived from genetically modified (GM) plants are central to their acceptance into the food supply. The potential allergenicity of proteins newly introduced in GM foods is a major safety concern. Objective: We sought to monitor, in potentially sensitive human populations, the allergenicity effects of 5 GM materials obtained from sources with no allergenic potential and already under commercialization in the European Union. Methods: We have performed skin prick tests with protein extracts prepared from transgenic maize (MON810, Bt11, T25, Bt176) and soya (Roundup Ready) samples and from nontransgenic control samples in 2 sensitive groups: children with food and inhalant allergy and individuals with asthmarhinitis. We have also tested IgE immunoblot reactivity of sera from patients with food allergy to soya (Roundup Ready) and maize (MON810, Bt11, Bt176) samples, as well as to the pure transgenic proteins (CryIA[b] and CP4 5-enolpyruvylshikimate3-phosphate synthase). Results: None of the individuals undergoing tests reacted differentially to the transgenic and nontransgenic samples under study. None of the volunteers tested presented detectable IgE antibodies against pure transgenic proteins. Conclusion: The transgenic products under testing seem to be safe in terms of allergenic potential. We propose postmarket testing as an important screening strategy for putative allergic sensitization to proteins introduced in transgenic plants. (J Allergy Clin Immunol 2005;116:403-10.)

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TABLE I. Transgenic flour products tested in SPTs and in IgE Immunoblot reactivity assays

Material

2% GM Bt11 maize

Characteristics

1998

Syngenta

100% GM Insect resistance (CryIA[b] Bt176 maize gene) and ammonium glufosinate tolerance (PAT gene); 35S pro; 35S t

1997

Syngenta

100% GM T25 maize

1998

Bayer Crop Sciences

1998

Monsanto

1996

Monsanto

100% GM MON810 maize

5% GM RUR soya

Insect resistance (CryIA[b] gene) and ammonium glufosinate tolerance (PAT gene); 35S pro; NOS 3# t

Date of Responsible commercialization company

Ammonium glufosinate tolerance (PAT gene); 35S pro; 35S t Insect resistance (CryIA[b] gene); 35S pro; NOS3# t

Gliphosate resistance (CP4EPSPS gene); 35S pro; NOS 3# t

Origin and certification of the material

Method of testing and human population studied

Institute of Reference Materials and Measurements certified

SPTs in a population of allergic children (27 individuals); IgE immunoblot reactivity assay with sera from patients with food allergy (57 individuals) SPTs in a population of allergic children (27 individuals); IgE immunoblot reactivity assay with sera from patients with food allergy (57 individuals) National Service of SPTs in a population of Plant Protection patients with asthma(DGPC); not certified rhinitis (50 individuals) SPTs in a population with asthma-rhinitis (50 individuals); IgE immunoblot reactivity assay with sera from patients with food allergy (24 of the 57 individuals) Institute of Reference SPTs in a population of Materials and allergic children (27 Measurements individuals); IgE certified immunoblot reactivity assay with sera from patients with food allergy (57 individuals)

35S pro, 35S Cauliflower Mosaic Virus promoter; 35S t, 35S Cauliflower Mosaic Virus terminator; NOS 3# t, Agrobacterium tumefaciens nopaline synthase terminator; DGPC, Direccxao Geral de Proteccxa˜o de Culturas.

Food allergy, dermatologic diseases, and anaphylaxis

In this study we have monitored the IgE response of allergy-sensitive populations to GM maize and soya products (Table I). The transgenes in maize and soya were obtained from sources with no allergenic history and approved for human consumption in the European Union. The IgE response of the same individuals to nonmodified products was also analyzed for comparison.

METHODS This study was evaluated and approved by the Research Ethic Committees of the Hospital of Santa Maria and the National Institute of Health, Lisbon, Portugal. All individuals participating in this study or their parents also provided informed consent.

Food inquiry Because of the fact that IgE-mediated allergic reactions require prior exposure, resulting in sensitization, we have performed a food inquiry to evaluate the consumption of soya and maize food-derived products. Bearing in mind that since 1998 all the GM products under testing were approved for commercialization in the European Union (Table I), we assumed that consumption of maize and soya

food-derived products implied a consumption of GM soya and maize. The food inquiry was performed on 106 healthy volunteers to find out which maize- and soya-derived products (from a list of 205 different products) they had already consumed. The population studied included individuals with ages from 1 to 41 years, with an average of 12.4 years (48 male and 58 subjects).

Transgenic quality of the noncertified flour samples In addition to the 3 noncertified transgenic products listed in Table I, nontransgenic analogues were also tested as controls. For the noncertified material (Table I), we have first confirmed the transformation event and the absence of cross-contamination among them. For these analyses, DNA was isolated by using the cetyltrimethylammonium bromide method,17 with 3 replicas per sample. DNA quality and concentration were analyzed by means of agarose gel electrophoresis, and maize-specific amplifiable DNA was detected by using PCR amplification of a 226-bp sequence from the maize invertase gene.18 The presence or absence of the 35S Cauliflower Mosaic Virus promoter in the transgenic (Table I) and control samples was checked

by using standard protocols for the amplification of a 195-bp DNA sequence.18 Transformation event–specific PCR reactions were performed to verify the presence of MON810, T25, and Bt176 transgenic events.18 Different internal controls were always used to detect putative contaminations. In each case whole or digested (HaeIII or Hinf I) PCR product size was compared with expected values.18

Preparation of protein extracts for human skin prick testing and IgE immunoblot reactivity Maize and soya protein extracts were made by Laboratorios Leti, SL (Madrid, Spain) according to approved pharmaceutical preparative and safety procedures for the production of diagnostic skin prick test (SPT) materials. About 10 g of each of the maize and soya flour samples was extracted for 16 hours in 1:20 (wt/vol) PBS (pH 7.4). After centrifugation, the pellet was discarded, and the supernatant was extensively dialyzed against bidistilled water. The extracts were centrifuged, filter sterilized, and freeze-dried. For human SPTs, the extracts were resuspended to 10 mg/mL maize or soya freeze-dried material (approximately 2 mg of total protein/mL for MON810, Bt11, Bt176, and control samples; approximately 3 mg of total protein/mL for T25 and control samples; and approximately 3.5 mg of total protein/mL for Roundup Ready [RUR] and control samples). For the IgE immunoblot reactivity assay conducted with sera from patients with food allergy, we used an extract prepared with food to which the person undergoing the test was allergic as a positive control extract. Four grams of food material was homogenized in liquid nitrogen and precipitated with 20 mL of 10% trichloroacetic acid (wt/vol) in cold acetone containing 20 mM dithiothreitol for 1 hour at 220°C. The precipitate was collected by means of centrifugation (15 minutes at 14,000g at 4°C), washed twice with 20 mM dithiothreitol in cold acetone, and allowed to dry completely.

Quality of transgenic proteins in maize and soya extracts ELISA GMO Check Bt maize test kit (SDI Europe, London, United Kingdom) was used to evaluate the presence-absence of Bt CryIA(b) protein in the lyophilized extracts prepared by Laboratorios Leti. Ten milligrams of dry extract was resuspended in 200 mL of the kit extraction buffer provided, and all nonsoluble material was removed by means of centrifugation (10 minutes at 11,000g). The manufacturer’s instructions were followed thereafter, using approximately 200 mg of total protein. To evaluate the presence or absence of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4EPSPS) protein in RUR soya and nontransgenic analogues, the lyophilized materials were tested with an ELISA GMO Check RUR Soya Grain test kit (Strategic Diagnostics Inc). Five milligrams of dry extract was diluted in 200 mL of kit extraction buffer. The nonsoluble material was removed by means of centrifugation (10 minutes at 11,000g). The manufacturer’s instructions were followed thereafter, using approximately 2.5 mg of total protein. Thirty micrograms of each sample was also run by means of SDS-PAGE and immunobloted with rabbit anti-Bt CryIA(b) polyclonal antibodies (RDI, Flanders, NJ) or goat anti-CP4EPSPS serum (Monsanto Co, St Louis, Mo; see description below). It was impossible to obtain commercial anti-phosphinotricine acetyl transferase (anti-PAT) antibodies, and there is no commercially available ELISA kit for PAT. We therefore decided to use the Trait LL corn grain test kit (Strategic Diagnostics Inc) to evaluate the presence or absence of PAT in Bt176, Bt11, T25, and nontransgenic analogues. This kit uses PAT-specific antibodies coupled to a color reagent and incorporated into strips, allowing the detection of PAT in an extract

Batista et al 405

through color development. Ten milligrams of lyophilized samples was diluted in the kit buffer provided, and 100 mL of each sample (approximately 200 mg of total protein) was eluted along the strip. For protein quantification, the Bio-Rad protein assay (Bio-Rad laboratories) was used, with turkey albumin (Merck) as a standard.

Skin testing of the 2 populations Skin tests were performed in 2 human populations with positive histories of food allergy, inhalant allergy, or both, as well as a positive SPT response for related allergens; one group was composed of 27 children with food and inhalant allergy from the Paediatrics Allergy Department of the Hospital of Santa Maria, and the other was composed of 50 patients with asthma-rhinitis from the University Clinic of Pneumology from the Hospital of Santa Maria (see Tables E1 and E2 in the Online Repository in the online version of this article at www.mosby.com/jaci). The children were tested with the extracts of Bt176, Bt11, RUR, and nontransgenic analogues; for the asthma-rhinitis population, we used the extracts of MON810, T25, and nontransgenic analogues for testing (Table I). Skin tests were performed by using the prick procedure,19 and results were read after 20 minutes. The results were classified as positive when the larger diameter of the wheal exceeded 3 mm. Histamine hydrochloride, 10 mg/mL (Leti), was used as a positive control, and Phenolate saline serum with glycerine (Leti) was used as a negative control. All the protein extracts were first tested on a control population of 20 nonallergic healthy individuals.

Sera for the IgE immunoblot reactivity assay Patient sera were provided by the Joaquim Chaves Clinic and were obtained from 57 individuals who had a positive history of documented food allergy, as well as a positive value equal to or higher than class 3 on specific UniCAP test (Pharmacia Diagnostics, Seixal, Portugal; see Table E3 in the Online Repository in the online version of this article at www.mosby.com/jaci). All 57 sera were first assayed for reactivity against nontransgenic maize and soya by means of specific IgE UniCAP testing. The sera were then tested for IgE immunoblot reactivity against Bt11, Bt176 maize, and RUR soya, as well as against nontransgenic analogues (Table I). MON810 maize and its nontransgenic analogue, as well as pure CryIA(b) (Research Diagnostics, Inc) and CP4EPSPS (Monsanto Co), were used to test the IgE immunoblot reactivity of sera of the 24 more sensitive patients (Table I).

SDS-PAGE and protein transfer to nitrocellulose membranes Samples were diluted 1:2 in sample buffer (0.125 M Tris-HCl [pH 6.8], 4% SDS, 20% vol/vol glycerol, 0.2 M dithiothreitol, and 0.02% bromophenol blue) and boiled for 5 minutes before electrophoresis in a 0.75-mm-thick 10% acrylamide gel with 4% stacking gel.20 After electrophoresis, the proteins were blotted onto hybond ECL nitrocellulose membranes (Amersham Biosciences, Carnaxide, Portugal) by means of wet transfer in 25 mM Tris, 192 mM glycine, 0.1% SDS, and 20% methanol for 1 hour at 75V at room temperature.

IgE immunoblot reactivity assay of sera from patients with food allergy The detection of patient sera IgE reactivity was carried out after electrophoresis of 30 mg (60 mg/cm gel width) of MON810, Bt11, Bt176, and RUR transgenic samples and nontransgenic analogues and 25 ng (50 ng/cm gel width) of pure CryIA(b) and CP4EPSPS and transfer to nitrocellulose membrane. Blots were blocked overnight at 4°C with PBS-T (58 mM Na2HPO4, 17 mM NaH2PO4.H2O, 68 mM NaCl, and 0.2% Tween

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TABLE II. Results of the food inquiry regarding the probability of an individual having consumed a transgenic maize or soya sample Mean number of consumed products with maize or soya (l estimates) n

Total Sex Male Female Age group (y) <5 5-10 10-25 25

Probability of consumption of products with transgenic protein

95% CI

P = .235

95% CI

106

39.3

35.2-43.4

0.999902

0.99974-0.99999

48 58

34.8 43.0

29.2-40.4 37.1-48.2

0.999959 0.999719

0.99895-0.99993 0.99983-0.99999

20 56 11 19

29.5 41.1 48.8 38.9

22.6-36.3 35.2-46.9 35.6-62.0 27.5-50.3

0.999024 0.999936 0.999990 0.999893

0.99506-0.99980 0.99974-0.99998 0.99976-1.00000 0.99844-0.99999

n, Number of valid responses; P, probability of one product with maize or soya having transgenic proteins (Instituto de Biologia Experimental e Tecnolo´gica Good Laboratory Practices Microbiology laboratory data).

20) and 5% skimmed milk powder (or 3% BSA for patients with milk allergy) and washed with PBS-T before incubation in serum diluted 1:10 in blocking solution for 1 hour and 30 minutes at room temperature. After washing with PBS-T, the membranes were incubated for 1 hour at room temperature in alkaline phosphatase– conjugated monoclonal anti-human IgE (Southern Biotechnology Associates, Birmingham, Ala) diluted 1:2000 in blocking solution, washed with PBS-T and assay buffer, and incubated for 5 minutes with CDP-Star solution with Nitro-Block II enhancer (Tropix Western-Star Immunodetection System). Blots were observed after exposure (5 seconds-30 minutes) to a high-performance chemiluminescence Hyperfilm ECL (Amersham Biosciences).

Immunoblot detection of Bt CryIA(b) and CP4EPSPS

Food allergy, dermatologic diseases, and anaphylaxis

The procedure was identical to the one described for IgE immunobloting of patient’s sera, with the following differences. For Bt CryIA(b), the first antibody incubation was performed in rabbit anti-Bt CryIA(b) polyclonal (Research Diagnostics, Inc) diluted 1:1400 in blocking solution, and the second antibody incubation was performed in goat anti-rabbit IgG-AP conjugate (Tropix-Applied Biosystems, Porto, Portugal) diluted 1:2800 in blocking solution. For CP4EPSPS, the first antibody incubation was performed in goat antiCP4EPSPS serum (Monsanto Co) diluted 1:5000 in blocking solution, and the second antibody incubation was performed in anti-goat IgG-alkaline phosphatase conjugate (Sigma, Sintra, Portugal) diluted 1:2500 in blocking solution.

Statistical analysis To estimate the probability of one individual from the Portuguese population having once been in contact with transgenic proteins present in maize or soya foods, we used (1) the results from the food inquiry and (2) the percentage data of maize and soya products with detectable transgenic proteins provided by Instituto de Biologia Experimental e Tecnolo´gica Good Laboratory Practices Microbiology laboratory. This laboratory is one of the 2 national laboratories responsible for food GMO detection. Assuming that the number of products with maize or soya consumed by the population is a Poisson random variable with the expected value l and that the probability of an individual having consumed a product with transgenic proteins (provided he or she had consumed n products with maize or soya) is modeled by using binomial distributions21 (n = number of experiences, p = probability

of one product with maize or soya having transgenic proteins), we can then calculate the probability of one individual having been in contact with transgenic proteins, which is 12e2lp . To estimate this probability, we used as l the mean number of consumed products with maize or soya obtained in the survey, and as p the proportion of maize and soya products detected with transgenic proteins calculated by using the Instituto de Biologia Experimental e Tecnolo´gica Good Laboratory Practices Microbiology laboratory data during the last 2 years.

RESULTS Food inquiry All 106 individuals participating in this inquiry consumed some of the 205 products presented. The extreme cases, with lower and higher numbers of consumed products, were relative to a 1-year-old and 9-year-old girl with 4 and 129 consumed products, respectively. The mean of consumed products with maize and soya was 39.3 (95% CI, 35.2-43.4), and the probability of an individual having eaten GM food was near 100% (Table II). Transgenic quality of the noncertified flour samples All 6 tested samples (Bt176, T25, MON810, and the nontransgenic analogues) showed the expected bands when checking for amplifiable maize DNA (data not shown), and only the 3 transgenic samples showed the expected amplicon of the 35S promoter (data not shown). The final confirmation that all the samples tested were correctly labeled and that there was no cross-contamination among them was obtained from construct-specific PCR (Fig 1). As expected, the digestion of the obtained amplicons confirmed the accuracy of the specific PCR (data not shown). Quality of transgenic proteins in maize and soya extracts As described in the Methods section, Laboratorios Leti protein extracts were tested for the presence of the

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FIG 2. Western blot for the detection of CryIA(b) protein in Laboratorios Leti protein extracts. I, 10% Acrylamide SDS-PAGE; II, immunoblot with rabbit anti-Bt CryIA(b) polyclonal. M, Molecular weight marker; Bt112, MON8102, Bt1762, non-GM controls; Bt111, MON8101, Bt1761, GM material 2% Bt11, 100% MON810, and 100% Bt176, respectively.

transgenic proteins under testing. CryIA(b) was detected by using ELISA (data not shown) and Western blotting (Fig 2) in MON810, Bt11, and Bt176 extracts and was absent from the nontransgenic control analogues. CP4EPSPS was also detected by means of ELISA (data not shown) and Western blotting (Fig 3) in RUR extract and was absent from the nontransgenic analogue. Both pure CryIA(b) and CP4EPSPS proteins were detected with the respective specific antibodies (data not shown). In T25, Bt11, and Bt176 samples PAT protein was detected in 200 mg of total protein solutions by using the

Trait LL corn grain test kit. With this system, we have also confirmed the absence of PAT in nontransgenic analogues (data not shown).

Allergenicity tests Skin testing of the 2 populations. Only individuals with maize sensitivity, soybean sensitivity, or both had positive results against the protein extracts under testing; however, none of the volunteers reacted differentially to GM versus non-GM samples (Table III and Tables E1 and E2 in the Online Repository in the online version of this article at www.mosby.com/jaci).

Food allergy, dermatologic diseases, and anaphylaxis

FIG 1. Construct-specific PCR for the detection of modified DNA sequences from T25, Bt176, and MON810 maize. M, 100-bp DNA ladder; MM, Mastermix; Bl, DNA extraction blank; T252, Bt1762, MON8102, non-GM controls; T251, Bt1761, MON8101, 100% GM T25, Bt176, and MON810 maize, respectively.

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FIG 3. Western blot for the detection of CP4EPSPS protein in Laboratorios Leti protein extracts. I, 10% Acrylamide SDS-PAGE; II, immunoblot with anti-CP4EPSPS goat serum (IgG). M, Molecular weight marker; Bt112, Bt1762, RUR2, non-GM controls; Bt111, Bt1761, RUR1, GM material 2% Bt11, 100% Bt176, and 5% RUR, respectively.

TABLE III. Results obtained with the allergenicity tests performed by using SPTs and IgE immunoblot reactivity assays IgE immunoblot reactivity assay

SPTs No. of individuals tested

Positive responses (%)

No. of individuals tested

Positive responses (%)

77 77 27

0 0 0

NT 57 57

2 0 0

GM protein PAT CRY1A(b) CP4EPSPS NT, Not tested.

All the patients had wheals larger than 3 mm for histamine, and none of them reacted against the negative control.

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IgE Immunoblot reactivity assay of sera from patients with food allergy. Two types of Western blotting results (Figs 4 and 5) were observed. In Fig 4 serum from an individual with octopus allergy of class 4 on specific UniCAP testing reacted only against positive controls. In Fig 5, concerning an individual with peanut allergy of class 6 on a specific UniCAP test, positive signals were observed against the positive control but also against other maize and soya protein extracts. None of the volunteers tested presented differential signals against nontransgenic versus transgenic protein extracts (Table III). All 24 individuals tested against pure transgenic proteins (CP4EPSPS and CryIAb) presented no detectable reactions against these controls. DISCUSSION Although absolute certainties regarding GM food risks to health and the environment will hardly be obtained, reports regarding potential problems have raised public concern. Some of the concerning issues include the putative toxicity-allergenicity of crops expressing foreign proteins,22-25 although these fears have not been confirmed in some later studies,26,27 and the adequacy of the

methods of testing have been questioned.28 Considering that the past few decades have witnessed a significant increase in IgE-mediated allergic diseases, the allergenic potential of these novel foods is a major concern in public health. The food inquiry performed in this study indicated that the probability of an individual having eaten GM food was near 100% (Table II). This value is probably underestimated because each individual probably consumed each product several times, which was not considered in statistical calculations. Also, it is possible that the first sensitization occurred during breast-feeding in the individuals submitted to SPTs and Western blot analyses who were younger than 6 years (the time between the first commercialization in 1998 and 2004).29 It therefore seems reasonable to assume that all the individuals participating in this study had already been in contact with the products tested. The DNA and protein quality analysis performed in this study confirmed the quality of flour samples and maize and soya protein extracts (Figs 1-3). In the Western assay for the detection of CryIA(b) in Bt11, Bt176, MON810, and nontransgenic control analogues (Fig 2), the multiple bands approximately equal to the CryIA(b) trypsin resistant core observed are likely the products of endogenous grain proteases.30 Some of the protein is degraded further to produce lower-molecular-weight bands, including a 30-kd product previously reported.30 As already mentioned, we have performed this study on sensitive populations. The population submitted to SPTs and immunoblot analyses was composed of individuals with food allergy and inhalant allergy, many of them children. Children are more susceptible to food allergies than adults. This higher susceptibility is probably the result of immunologic immaturity and, to some extent, immaturity of the gut.31,32 In addition, children who have preexisting food allergies are more likely to experience allergic reactions to other foods introduced in their diets. The absence of detectable differences in IgE reactivity between GM maize and soya samples and the corresponding wild-type samples obtained in this study is in accordance with some previously published results.33,34 The appearance of nondifferential bands on some chemiluminescence films for maize and soya protein

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FIG 5. IgE antibody reactivity assay from a peanut-sensitive patient. I, 10% Acrylamide SDS-PAGE; II, immunoblot. M, Molecular weight marker; Bt112, Bt1762, RUR2, non-GM controls; Bt111, Bt1761, RUR1, GM material 2% Bt11, 100% Bt176, and 5% RUR, respectively; Pnt, Peanut protein extract.

extract lanes (Fig 5) might be due to the phenomenon of cross-reactivity among various plant and animal proteins.35,36 In the example presented in Fig 5, although the patient tested had only documented peanut allergy (class 6 on UniCAP test), it was shown that he also had IgE binding to other foods, such as almond (class 2), hazelnut (class 3), walnut (class 2), cashew (class 4), soybean (class 3), and maize (class 3). This fact justifies the appearance of the nondifferential bands on maize and soya lanes. Although IgE detection (either SPT or specific IgE) serves as a good indicator of sensitization but not necessarily of disease, in the clinical setting the absence of detectable IgE was found to have excellent negative predictive accuracy indices and therefore might be very useful in excluding the presence of immediate food hypersensitivity.37

In this study we did not obtain any differential positive results, which allows us to conclude that the transgenic products under testing seem to be safe regarding their allergenic potential. Although we succeeded in integrating a private clinic and a hospital in this study, it would be desirable to increase the size of the analyzed population and eventually extend this work to other countries. We also propose the development and use of clinical testing with specific IgE in the postmarketing surveillance of foods produced through biotechnology. Positive test results should be followed by double-blind, placebocontrolled food challenges under appropriate clinical observation to identify true clinical reactions.38 We gratefully acknowledge the National Service of Plant Protection (DGPC) for providing BT176, T25, MON810, and nontransgenic analogue maize samples; Laboratorios Leti for the

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FIG 4. IgE antibody reactivity assay from an octopus-sensitive patient. I, 10% Acrylamide SDS-PAGE; II, immunoblot. M, Molecular weight marker; Bt112, Bt1762, RUR2, MON8102, non-GM controls; Bt111, Bt1761, RUR1, MON8101, GM material 2% Bt11, 100% Bt176, 5% RUR, and 100% MON810, respectively; Otp, Octopus protein extract; Cry, CryIA(b); CP4, CP4EPSPS.

410 Batista et al

preparation of maize and soya protein extracts; and Monsanto, especially Dr Richard Goodman, for providing the CP4EPSPS protein and the corresponding antiserum. Fernanda Spı´nola and Ca´tia Peres are gratefully acknowledged for their advice regarding GMO detection. We also thank Margarida Santos, Helena Raquel, Madalena Martins, and Sara Silva for help in the preparation of the food inquiry. Finally, we thank Phil Jackson for the final revision of the manuscript and Fundac xa˜o Calouste Gulbenkian and Comissa˜o de Fomento da Investigac xa˜o em Cuidados de Sau´de for funding.

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19.

20. 21. 22. 23.

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1. Alam MF, Datta K, Abrigo E, Vasquez A, Senadhira D, Datta SK. Production of transgenic deepwater indica rice plants expressing a synthetic Bacillus thuringiensis cryIA(b) gene with enhanced resistance to yellow stem borer. Plant Sci 1998;135:25-30. 2. Shintani D, Della Penna D. Elevating the vitamin E content of plants through metabolic engineering. Science 1998;282:2098-100. 3. De la Fuente JM, Ramirez Rodriguez V, Cabrera Ponce JL, Herrera Estrella L. Aluminium tolerance in transgenic plants by alteration of citrate synthesis. Science 1997;276:1566-8. 4. Ye X, Al Babili S, Kloeti A, Zhang J, Lucca P, Beyer P, et al. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 2000;287:303-5. 5. Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F. Iron fortification of rice seed by soybean ferritin gene. Nat Biotechnol 1999;17:282-6. 6. Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK. Identification of a Brazil-nut allergen in transgenic soybeans. N Engl J Med 1996;334:688-92. 7. Matsuda T, Nakamura R. Molecular structure and immunological properties of food allergens. Trends Food Sci Technol 1993;4:289-93. 8. Taylor SL, Leher SB. Principles and characteristics of food allergens. Crit Rev Food Sci Nutr 1996;36(suppl):S91-118. 9. Sampson HA. Food Allergy. JAMA 1997;278:1888-94. 10. Anderson JA. Allergic reactions to foods. Crit Rev Food Sci Nutr 1996; 36(suppl):S19-38. 11. Bock SA. Prospective appraisal of complaints of adverse reactions to foods in children during the first three years of life. Paediatrics 1987;79: 683-8. 12. Helm RM, Burks AW. Mechanisms of food allergy. Curr Opin Immunol 2000;12:647-53. 13. Metcalfe DD, Astwood JD, Townsend R, Sampson HA, Taylor SL, Fuchs RL. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit Rev Food Sci Nutr 1996; 36(suppl):S165-86. 14. Mendieta NLR, Nagy AM, Lints FA. The potential allergenicity of novel foods. J Sci Food Agric 1997;75:405-11. 15. Taylor SL. Assessment of the allergenicity of genetically modified foods. Nutr Abstracts Rev (Series A) 1997;67:1163-8. 16. Lack G, Chapman M, Kalsheker N, King V, Robinson C, Venables K. Report on the potential allergenicity of genetically modified organisms and their products. Clin Exp Allergy 2002;32:1131-43. 17. Draft International Standard ISO/DIS 21571. Foodstuffs—methods of analysis for the detection of genetically modified organisms and derived products—nucleic acid extraction. Nov 2002. p. 25-8. 18. Draft International Standard ISO/DIS 21569. Foodstuffs—methods of analysis for the detection of genetically modified organisms and

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derived products—qualitative nucleic acid based methods. Nov 2002. p. 23-66. Sub-Committee on Skin Tests of the European Academy of Allergology and Clinical Immunology. Skin tests used in type I allergy testing. Allergy 1989;44(suppl 10):1-59. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5. Bliss CI. Statistics in biology. Vol 1. New York: MacGraw-Hill; 1967. p. 558. Losey JE, Rayon LS, Carter ME. Transgenic pollen harms monarch larvae. Nature 1999;395:214. Ewen SWB, Pusztai A. Effect of diets containing genetically modified potatoes expressing Galanthus nivalis lectin on rat small intestine. Lancet 1999;354:1353-4. Taco Bell taco shells sold in grocery stores contain banned corn [transcript]. CNN. September 18, 2000. Available at: http://europe.cnn.com/2000/ FOOD/news/09/18/food.corn.reut/. Accessed January 10, 2005. EPA Assessment of Scientific Information concerning Starlink Corn Cry9C Bt Corn Plant-pesticide. Federal Register 65 (October 31, 2000). Environmental Protection Agency publication no. 65246-65251. Available at: http://www.access.gpo.gov/su_docs/fedreg/frcont00.html. Accessed January 10, 2005. Sears MK, Hellmich RL, Stanley-Horn DE, Oberhauser KS, Pleasants JM, Matilla HR, et al. Impact of Bt corn pollen on monarch butterfly populations: a risk assessment. Proc Natl Acad Sci U S A 2001;21: 11937-42. Sutton SA, Assa’ad AH, Steinmetz C, Rothenberg ME. A negative, double-blind, placebo-controlled challenge to genetically modified corn. J Allergy Clin Immunol 2003;112:1011-2. Kuiper HA, Noteborn HPJM, Peijnenburg ACM. Adequacy of methods for testing the safety of genetically modified foods. Lancet 1999;354: 1315-6. Vadas P, Wai Y, Burks W, Perelman B. Detection of peanut allergens in breast milk of lactating women. JAMA 2001;285:1746-8. Miranda R, Zamudio FZ, Bravo A. Processing of Cry1Ab d-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation. Insect Biochem Mol Biol 2001;31:1155-63. Sampson HA, Metcalfe DD. Food allergies. JAMA 1992;268:2840-4. Sampson HA. Food allergy. Part 2: diagnosis and management. J Allergy Clin Immunol 1999;103:981-9. Sten E, Skov PS, Andersen SB, Torp AM, Olesen A, Bindslev-Jensen U, et al. A comparative study of the allergenic potency of wild-type and glyphosate-tolerant gene-modified soybean cultivars. APMIS 2004;112: 21-8. Burks AW, Fuchs RL. Assessment of the endogenous allergens in glyphosate-tolerant and commercial soybean varieties. J Allergy Clin Immunol 1995;96:1008-10. Sicherer SH. Clinical implications of cross-reactive food allergens. J Allergy Clin Immunol 2001;108:881-90. Vieths S, Scheurer S, Ballmer-Weber B. Current understanding of crossreactivity of food allergens and pollen. Ann N Y Acad Sci 2002;964: 47-68. Sampson HA, Albergo R. Comparison of results of skin tests, RAST and double-blind, placebo-controlled food challenges in children with atopic dermatitis. J Allergy Clin Immunol 1984;74:26-33. Bindslev-Jensen C, Poulsen LK. Accuracy of in vivo and in vitro tests. Allergy 1998;53:72-4.

Basic and clinical immunology Advances in Asthma, Allergy, and Immunology Series 2005 Basic and clinical immunology Javier Chinen, MD, PhD,a and William T. Shearer, MD, PhDb Bethesda, Md, and Houston, Tex

Key words: Immunoregulation, HIV, immunodeficiency, innate immunity, complement

Abbreviations used APC: Antigen-presenting cell CTL: Cytotoxic T cell CVID: Common variable immunodeficiency DGS: DiGeorge syndrome DSS: Dextran sulfate sodium HIGM: Hyper-IgM syndrome ICOS: Inducible costimulatory molecule IRD: Immune restoration disease NEMO: NF-kB essential modifier NK: Natural killer PID: Primary immunodeficiency TCR: T-cell receptor TLR: Toll-like receptor

The areas of basic and clinical immunology continue to develop at a fast pace, with numerous reports exploring relatively new and old areas of these fields, such as the biology of Toll-like receptors (TLRs) and the description of primary immunodeficiencies (PIDs), respectively. The goal of this article is to review some of the significant progress in basic and clinical immunology published in 2004, with focus on articles that the authors considered of interest to the readers of The Journal of Allergy and Clinical Immunology.

BASIC IMMUNOLOGY Some key advances in basic immunology are listed in Table I.

From aGenetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, and bthe Department of Allergy and Immunology, Texas Children’s Hospital, and the Departments of Pediatrics and Immunology, Baylor College of Medicine, Houston. The opinions expressed in this article do not necessarily represent the views of the National Human Genome Research Institute or the National Institutes of Health. Disclosure of potential conflict of interest: None disclosed. Received for publication May 4, 2005; accepted for publication May 6, 2005. Available online July 5, 2005. Reprint requests: Javier Chinen, MD, PhD, National Human Genome Research Institute, 10 Center Drive, MSC 1611, Building 10/CRC Room 6-3340, Bethesda, MD 20892. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.010

Regulation of the T-cell response: TH1 versus TH2 Wittmann et al1 investigated the cytokine secretion profile resulting from the interaction of monocytes derived from peripheral blood and autologous CD41 T cells isolated from inflammatory skin lesions induced by an allergen patch test. These activated T cells induced IL-12 secretion by monocytes that were stimulated with IFN-g. However, when the T cells were incubated with resting monocytes, IL-12 secretion was not induced. In contrast, resting T cells did not inhibit IL-12 secretion in resting monocytes. The authors further determined that this effect on IL-12 secretion was cell-contact specific and dependent 411

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The authors selected articles published in the literature from January 2004 through December 2004 that were relevant to the areas of basic and clinical immunology. Several articles explored the development of TH1 or TH2 response and the role of the monocyte–T cell interaction. Others were articles describing the action of drugs commonly used in asthma to inhibit cytokine responses and the anti-inflammatory role of nonimmune pulmonary cells present in the lung. Several reports show how dendritic cells are being developed as vehicles for DNA vaccines aimed at stimulating cellular responses, an advance of great importance for HIV researchers working on vaccines, who are concerned about the different ways HIV evades the immune response. Other publications described Toll-like receptors in diverse cells, including mast cells and CD41 T cells, for the recognition of viruses and bacteria. In the area of clinical immunology, an updated classification for primary immunodeficiencies with more than 100 identified genes responsible for these diseases and the report on the second clinical trial of gene therapy for X-linked severe combined immunodeficiency syndrome were published. Significant advances included the clinical prognosis in common variable immunodeficiency for patients presenting with lung pathology, the safety of live vaccines in partial DiGeorge syndrome, the report of patients with complete DiGeorge syndrome with the presence of peripheral blood T cells, the clinical spectrum of patients with NF-kB essential modifier (NEMO) gene deficiency, the publication of a consensus algorithm for the management of hereditary angioedema, and the report of immune restoration syndrome in pediatric HIV infection. (J Allergy Clin Immunol 2005;116:411-8.)

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TABLE I. Key advances in basic immunology 1. Monocyte activation is necessary for induction of a TH1 response. 2. Salmeterol and fluticasone act synergistically to inhibit secretion of inflammatory mediators in asthma. 3. A 10-kd protein from pulmonary Clara cells and IL-17F from bronchial epithelial cells are potent anti-inflammatory cytokines. 4. HLA alleles influence the generation of HIV CTL escape mutants and the risk of HIV transmission. 5. Mast cells recognize viruses and are activated through Toll receptors. 6. CD4 T cells can be directly activated through Toll receptors. 7. Alternative splicing might be responsible for breaking immune tolerance and the development of autoimmune disorders.

Basic and clinical immunology

on the induction of T-bet expression in monocytes. T-bet is a signal transduction factor that is essential for the development of the TH1 response. On the basis of these findings, the authors suggest that initial events in skin atopic disease might involve the infiltration of activated T cells into the skin and interaction with resting antigenpresenting cells (APCs), resulting in absence of IL-12 and development of a TH2 environment. The role of T-bet as the key protein for the induction of the TH1 response was supported by the work of Lametschwandtner et al,2 who induced T-bet expression in TH2 cells obtained from skin biopsy specimens of atopic individuals. TH2 cells expressing T-bet were able to secrete and express high levels of IFN-g, TNF-a, IL-2, and IL-12, with a decrease of the expression levels of IL-4 and IL-5. In addition, they showed a reversion of chemokine expression profile from TH2 to TH1. The immunologic events early in infancy that can lead to atopic disease were investigated by Upham et al,3 who described that the HLA-DR expression in monocytes obtained from cord blood stimulated with IFN-g correlated with IL-12 secretion induced by endotoxin and had an inverted association with IL-13 secretion induced by ovalbumin or dust mite. HLA-DR expression in unstimulated monocytes was inversely associated with allergic disease at the 2-year follow up. These findings suggest that early activation of APCs might decrease the TH2 response and the risk of development of atopic disease. Two drugs commonly used in asthma, fluticasone and salmeterol, were shown to synergistically inhibit cytokine secretion and to influence the TH2 to TH1 balance.4 The drug combination inhibited the secretion of the TH1 cytokines TNF-a and IFN-g by mitogen-stimulated PBMCs from normal and asthmatic patients. In contrast, the secretion of the TH2 cytokines IL-5 and IL-13 was inhibited only in PBMC cultures from control subjects but not from asthmatic patients. The addition of a phosphodiesterase inhibitor to the combination suppressed IL-13 secretion in PBMCs from asthmatic patients, an effect that can be explained by the maintenance of high cyclic adenosine monophosphate levels. Pace et al5 were also interested in the mechanism of action of the combination of fluticasone and salmeterol in T cells. These investigators found that the induction of apoptosis in peripheral

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blood T cells by fluticasone increases synergistically with the addition of salmeterol. Increased apoptosis was associated with a more efficient caspase processing, increased translocation of the glucocorticoid receptor, and reduction of the expression of phosphorylated IkBa. Salmeterol alone did not produce apoptosis in the T cells. A factor that affects the immune regulation of the allergic response is the 10-kd protein secreted by the pulmonary Clara cells.6 This protein decreased the expression of the TH2 cytokines IL-4, IL-5, and IL-13 of ovalbumin-sensitized mouse splenocytes and of CD41 T cells that had been polarized into TH2 cells. This potent effect was dose dependent and associated with a reduction of intracellular GATA-3, the transcription factor that mediates TH2 response. This result underscores the dynamic interaction between immune cells and highly specialized lung cells in lung inflammation and asthma pathogenesis.

DNA vaccines DNA vaccination is being actively explored as an alternative for the treatment of allergic diseases. DNAbased immunomodulation has been shown to switch the immune response from a TH2- to a TH1-dominant response in several mouse models.7 Klostermann et al8 used human dendritic cells transduced with an adenovirus vector carrying the expression cassette for the grass pollen Phl p 1 protein. When these cells were cocultured with T cells, they induced a TH1-like response, with proliferation of the CD81 T-cell subtype, increased IFN-g secretion, and less IL-4 and IL-5 secretion than when nontransduced dendritic cells were pulsed with the Phl p 1 protein. A different delivery strategy for DNA vaccination in allergy was shown by Ludwig-Portugall et al9 using a gene gun–mediated delivery in vivo. Although this method requires 100- to 1000-fold more DNA, it does not involve a viral vector. The authors used the gene gun to immunize mice percutaneously with a b-galactosidase– encoding plasmid and then sensitized the animal to b-galactosidase protein. IgG2a was 10-fold increased, and specific IgE was not detectable. In contrast, mice that were immunized intraperitoneally with b-galactosidase had high specific IgE levels. To increase the immunogenicity of DNA vaccines, Jilek et al10 examined the use of biodegradable microspheres as DNA carriers for prophylaxis against anaphylaxis. They used microspheres made of polylactidecoglycolide, a biodegradable material that is readily phagocytosed by dendritic cells.11 When mice received subcutaneous polylactidecoglycolide microspheres with DNA encoding phospholipase A, the bee venom major allergenic protein, and then were sensitized and challenged with a lethal dose of the phospholipase A protein, anaphylaxis was prevented in 50 of 54 experimental mice. Interestingly, the preventive effect was also achieved in animals that received microspheres with nonspecific DNA. The authors also demonstrated similar production of IgG2a, IL-4, IFN-g, and IL-10, suggesting that polylactidecoglycolide microspheres drive APCs toward the TH1 phenotype and could be considered for

therapeutic use in atopic patients. Another molecule studied was the CpG oligonucleotide, which was shown to drive TH1 cytokine expression in plasmacytoid dendritic dells obtained from patients with allergic rhinitis.12 When a CpG oligonucleotide was introduced to cocultures of dendritic cells and CD41 T cells, the cytokine secretion profile changed from being predominantly composed of IL-4 and IL-5 to being mostly IFN-a and TNF-a. This study suggests that mucosal dendritic cells can be considered as targets for DNA-based immunomodulation of the T-cell response.

Advances in cytokine research The production of IL-13 by human B lymphocytes and its role in IgE synthesis was investigated by Hajoui et al13 using B cells isolated from the tonsils of healthy volunteers. They reported a 10-fold increase of IL-13 secretion when these cells were stimulated with anti-CD40 antibody and IL-4. When they added neutralizing anti-IL-13 antibodies, IgE levels decreased by 80%, and IgE transcripts decreased by 50%, suggesting that B-cell secretion of IgE is regulated in part by IL-13 produced by the same B-cell population. Two articles published in the Journal focused on the IL-17 family of inflammation proteins and the role of nonimmune cells in lung inflammation. Kawaguchi et al14 reported that a function of a newly identified IL-17F protein in primary bronchial epithelial cells was to induce GM-CSF secretion through activation of Raf-1/MEKERK1/2, and therefore IL-17F participates in the pathophysiology of allergic inflammation. A second article examined the regulation of IL-17A in human airway smooth cells obtained from patients undergoing lung surgery. IL-17A induced secretion of IL-6 after stimulation with TNF-a but not after stimulation with IL-1b. Of note, there was no induction of other inflammation markers, such as intercellular adhesion molecule expression or GM-CSF secretion.15 The expression of IL-10 and FoxP3, which phenotypically define regulatory T cells, was compared in CD41 T cells obtained from patients with moderate and severe asthma and from healthy control subjects.16 This study found that FoxP3 mRNA expression correlated with IL-10 mRNA expression, and it was 2-fold higher in asthmatic patients receiving steroids than in healthy control subjects or patients with mild asthma. In addition, it was shown that CD41CD251 T cells expressed 11-fold more IL-10 and FoxP3 than total CD41 T cells after being exposed to corticosteroids in vitro. These results suggest that the anti-inflammatory effect of corticosteroids include the development of regulatory T cells secreting IL-10. HIV immunopathogenesis The mechanisms by which HIV evades the immune system are far from being completely elucidated. Leslie et al17 studied the association of a specific cytotoxic T-cell (CTL) epitope in the HIV1 Gag protein and HLA alleles in an HIV-infected population. They found that HIVinfected individuals expressing the HLA alleles B57 and B*5801 had selected for variants with a specific mutation

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in this Gag epitope. However, when this HIV strain was transmitted to an individual with different HLA alleles, this epitope reverted to the wild type. Supporting their finding, they demonstrated that a second mutation in the same epitope did not revert. The reasons why some epitope mutations persist and others revert to the wild type are not clearly related to virus fitness, but certainly this is of concern for the design of vaccines targeting the anti-HIV CTL response. A related article by Dorak et al18 examined 125 couples who were initially HIV discordant after which the spouse converted and 104 persistent HIVdiscordant couples. They found that the risk of HIV transmission to the spouse was 2-fold higher, independent of viral load, if the couples shared one or both HLA-B alleles than if they had different HLA-B alleles. This suggests that HIV CTL escape mutants are transmitted more efficiently in a homogenous population and that they will vary from population to population (Fig 1). Adding one more strategy for HIV immune evasion, Draenert et al19 reported mutations in the Gag protein outside a particular epitope that altered a target site for protein processing in APCs and therefore cannot be presented, making CTLs unable to lyse infected cells. Winchester et al20 investigated innate immunity in mother-to-child transmission of HIV, mediated by maternal natural killer (NK) cells. The expression of HLAB*4901 and B*5301 alleles inhibited mother-to-infant HIV transmission despite high maternal viral loads. They also bound the KIR30L1 NK receptor. The HLA-B*5001 and B*3501 alleles, which differ from B*4901 and B*5301 by only 5 amino acids, did not bind the KIR30L1 receptor and were associated with enhanced vertical transmission. The authors proposed that the molecular basis of this observation involved maternal NK cell recognition by engagement of NK cell receptors with polymorphic ligands encoded by maternal HLA-B alleles. Moreover, they believe that the placenta is the site where protection against vertical HIV transmission occurs, mediated by interrelating adoptive and innate immune recognition mechanisms. In the B-cell compartment, Moir et al21 found 42 genes upregulated in B cells from HIV-viremic patients compared with B cells from healthy control subjects. Most of these genes were associated with the activation of the IFN-g pathway or with terminal differentiation of B cells. In addition, they showed that CD95 expression in B cells correlated with HIV viremia. This report is valuable for the identification of genes involved in the mechanisms of B-cell dysfunction in HIV infection.

Innate immunity The role of TLRs in innate immunity continues to expand and involve many different immune cells and processes. Kulka et al22 added mast cells to the list of effector cells participating in the recognition of viruses through TLRs. Mast cells had already been shown to respond to LPS and peptidoglycan through TLR-1, TLR-2, TLR-4, and TLR-6. The importance of mast cells on shaping the innate immunity response to infection was

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FIG 1. HIV escape mutants (blue particles) appear over time in HIV-infected individuals with effective specific CTL responses because of survival selective pressure and depending on viral fitness of the new mutants. They persist after transmission between individuals sharing the same HLA alleles but might revert to the wild type (red particles) in the newly infected individual with different HLA alleles. There is a higher transmission rate when HIV-infected couples share the same HLA alleles.

Basic and clinical immunology

reviewed by Marshall and Jawdat.23 Mast cells are not only activated by TLRs but also by complement components, leading to the secretion of cytokines important for selective recruitment of effector cells. The expression of TLR-3 in mast cells derived from peripheral blood and 2 mast cell lines was newly reported, and their production of IFN-a and IFN-b in response to exposure to dsDNA and to PolyI:C was also reported. A similar response was obtained when mast cells were exposed to UV light–inactivated influenza virus and to type 1 reovirus. Flo et al24 demonstrated that TLR-4 stimulation with LPS in macrophages helped to control bacterial replication by increasing levels of lipocalin 2 expression. This protein inhibits iron uptake by Escherichia coli. Lipocalin 2 knockout mice became highly bacteremic after infection with E coli but not after infection with other bacteria less dependent on iron. In the gut the existence of commensal bacteria has prompted the question of how the gut controls inflammation, and it has been thought that inflammatory bowel disease could be caused by inappropriate recognition of antigens. Rakoff-Nahoum et al25 showed that mice deficient in TLRs have increased mortality than wild-type mice after receiving dextran sulfate sodium (DSS) as a model for inflammation. These mice were deficient on MYD88, a signal transduction factor essential for several TLR-mediated responses. The mice presented with epithelial injury and severe colonic bleeding but not leukocyte infiltration or bacterial overload. Previous administration of antibiotics did not modify the pathologic changes. The TLR-deficient mice had increased prolifer-

ation of colonic cells and were more susceptible to injury caused by DSS or radiation. Similar mortality occurred when wild-type mice were deprived of commensal bacteria and then were treated with DSS. When these animals were given LPS, the animals were protected, suggesting that TLR stimulation might mediate epithelial barrier repair. To provide a direct link between innate and adaptive immunity, Gelman et al26 reported that mouse CD41 T cells expressed TLR-3 and TLR-9 and responded to CpG and polyI:C stimulation with increased survival and nuclear factor kB (NF-kB) activation. This observation might represent the response of the immune system for infectious organisms that impair APCs by directly activating the adaptive immune cells.

Immune mechanisms of drug allergy Depta et al27 challenged the classical notion that haptens stimulate specific T cells only when they are covalently bound to proteins. The investigators transfected a mouse T-cell hybridoma to express a plasmid encoding a T-cell receptor (TCR) specific to sulfametoxazole. When these cells were exposed to the drug in the presence of fixed EBV-transformed B cells, they proliferated with a reactivity that was dependent on the level of the specific TCR expression. Increased TCR expression also correlated with cross-reactivity with other drugs that share the sulfanilamide core structure but not with other sulfonamides, like furosemide or celecoxib. Because fixed B cells were used as APCs, these results showed that drugs can directly interact with TCRs and that there is no need for antigen processing to obtain T-cell reactivity to

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Applications of the human genome sequence in immunology Ng et al29 took advantage of the completion of the mapping and sequencing of the human genome to support their hypothesis that alternative splicing of self-antigens might play a role for the generation of autoantigens in autoimmune disorders. Alternative splicing might disturb peripheral tolerance that has already been attained for the normal spliced protein. The authors randomly chose 45 self-proteins that have been implicated in autoimmunity and compared their alternative splicing frequency with 9554 random proteins of the human genome. Forty-two percent of the random proteins present alternative splicing in contrast to 100% of the 45 proteins implicated in autoimmunity. Eighty percent of these proteins might undergo noncanonical alternative splicing, which was much higher than the 1% of randomly selected proteins. More experimental data are needed to confirm this original hypothesis, which would provide insight into the pathogenesis of autoimmunity disorders and novel therapy development. In an example of high-yield gene search studies, Nakajima et al30 used a gene chip containing about 22,000 gene probes to compare transcripts expressed in CD41 cells, CD81 cells, basophils, eosinophils, neutrophils, CD141 cells, and CD191 cells, focusing on the expression of granulocyte-selective genes for ion channels. The authors found 17 novel transcripts from 51 with 5-fold greater expression than other leukocyte lineages. Six of these 17 were eosinophil and basophil specific. The authors reported the list of genes with specific expression and stressed their importance for drug targets in allergic and inflammatory processes and their significance for drug development in allergic disease and inflammation. Genetic variations influencing allergy were described by Hoffjan et al31 by screening 200 children for 61 polymorphisms in 35 immunoregulatory genes. The polymorphisms were analyzed in regard to cytokine production and allergic sensitization, as well as interaction between the polymorphisms. The authors found 5 associations that involved a reduced IL-13 secretion, including polymorphisms in the genes for IL-13, TGF-b, IgE receptor, and nitric oxide. None of the genes were associated with atopic dermatitis. The authors concluded that variations in immunoregulatory genes might be risk factors for the development of allergic disease and childhood asthma.

CLINICAL IMMUNOLOGY Some key advances in clinical immunology are listed in Table II.

Asthma and the immune response Hanania et al32 asked whether the corticosteroid therapy in patients with asthma affects the immune response to influenza vaccine. Asthmatic subjects (n = 294) who were randomized to receive either placebo or inactivated influenza vaccine were divided in 2 groups, one that received medium- or high-dose inhaled corticosteroids and another that received none or only low corticosteroid doses. The serologic response to influenza serotype A was not impaired with the use of corticosteroids, but the response to serotype B was slightly decreased, with a 2.1-fold increase of the titers compared with an increase of 2.5-fold in the group receiving no steroids or only low-dose steroids. Although actual protection against influenza infection was not measured, the study places a word of caution on the possible decreased immune response in asthmatic patients taking steroids. PIDs A must-read article is the update in PIDs written by Notarangelo et al33 representing the International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee. The authors reviewed and classified PIDs reported up to their last meeting in 2003. More than 100 PIDs have been defined and characterized. Although rare, PIDs are diagnosed with more frequency and in more diverse ethnic groups. However, more efforts are needed for PID awareness in minority groups, as noted by Cunningham-Rundles et al,34 who looked in a database of over 120,000 inpatients of a general hospital for conditions suggestive of immunodeficiency. Fifty-nine patients were identified, and 17 of them had an undiagnosed PID. Eighty-six percent of these previously undiagnosed patients with PIDs were African American or Hispanic. It is common to think that the de novo genetic defects of PIDs should occur during egg fertilization and embryo formation. This idea was challenged by Holzelova et al.35 They investigated patients with the autoimmune lymphoproliferative syndrome but without identified mutations in the causative genes Fas, Fas L, Casp8, and Casp10. The authors cleverly explored the double-negative T cells that accumulate in these patients. They identified Fas mutations in these cells and subsequently in monocytes and CD341 cells, but the mutations were not present in mucosal cells or B cells or when total T cells were tested. The authors were able to demonstrate these somatic mutations in 2 of 6 patients studied and showed that the survival advantage conferred to a subset of lymphocytes was enough to produce autoimmune lymphoproliferative syndrome. Chinen and Puck36 reviewed the progress and current hurdles of gene therapy for PIDs. The French clinical trial for X-linked severe combined immunodeficiency has now

Basic and clinical immunology

sulfonamides. A study by Nassif et al28 examined the phenotypes of lymphocytes obtained from 6 patients with toxic epidermal necrolysis caused by hypersensitivity to a single drug, either cotrimoxazole and carbamazepine, tetrazepam, or piroxicam. These lymphocytes were 70% to 90% CD81 CTLs and were specific for the offending drug in the cases of cotrimoxazole and carbamazepine but not for tetrazepam or piroxicam. The authors believe that the lack of specificity for the last 2 drugs can be explained by the uncertainty of the offending drug, but overall, the data support the hypothesis that specific CTLs are responsible for toxic epidermal necrolysis pathology.

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TABLE II. Key advances in clinical immunology 1. The immune response to influenza vaccine in patients with moderate-to-severe asthma taking inhaled or oral corticosteroids is slightly decreased. 2. Somatic mutations in T-cell progenitors might cause ALPS. 3. Four infants with XSCID were successfully treated with gene therapy in England. 4. Patients with CVID with granulomatous lung disease, lymphoid hyperplasia, and lymphoid interstitial pneumonia have poor survival prognosis. 5. Some patients with complete DGS might have detectable numbers of T cells in peripheral blood, although oligoclonal in nature and with poor function. 6. NEMO-deficient patients have increased susceptibility to pyogenic and mycobacterial infections. Some of these patients might not have the ectodermal component of this syndrome. 7. IRD occurs in pediatric HIV infection. ALPS, Autoimmune lymphoproliferative syndrome; XSCID, X-linked severe combined immunodeficiency.

Basic and clinical immunology

treated 11 infants, with successful T-cell restoration in 9 of them; the other 2 had only partial reconstitution and subsequently received a conventional bone marrow transplantation.37 In addition, Gaspar et al38 published their experience in 4 infants with X-linked severe combined immunodeficiency in England who received gene therapy and achieved normal T-cell numbers. However, these successes were tempered with the occurrence of T-cell leukemia in 3 of the children from the French trial.37 These malignancies were proved to be caused, at least in part, by insertion of the retroviral vector in oncogenes. New gene vector designs are being investigated to reduce the risk of cancer caused by insertional mutagenesis. Gardulf et al39 reported their experience with the subcutaneous self-administration of immunoglobulins for patients with PID done at home, suggesting that the quality of life of these patients might increase with this modality. Chinen and Shearer40 reviewed the pros and cons of subcutaneous administration of immunoglobulins for immunodeficiency, which is being established as a viable alternative to traditional intravenous infusions. Regarding specific PIDs, several advances have been made in common variable immunodeficiency (CVID), hyper-IgM syndrome (HIGM), and DiGeorge syndrome (DGS). Bates et al41 investigated the clinical features of noninfectious pulmonary disease in patients with CVID. They found that 29 of 69 patients with CVID presented with none of these abnormalities, 23 had respiratory symptoms but were radiologically normal, and 18 had respiratory symptoms and radiologic diffuse abnormalities. Within this last group, those who had granulomatous lung disease, follicular bronchiolitis, lymphoid hyperplasia, and lymphoid interstitial pneumonia (13/18 patients) had worse prognosis and survival than the other groups, with a survival of 13.7 years since the time of diagnosis compared with 28.8 years in the other groups. In addition, these patients also were at higher risk of lymphoproliferative disease. Salzer et al42 investigated the role of

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inducible costimulatory molecule (ICOS) in their cohort of patients with CVID and found mutations in the ICOS gene in 2 of 9 families with autosomal-recessive CVID and no mutations in the ICOS ligand gene. No polymorphic variants found in the ICOS gene sequence were more common in patients with CVID than in the general population. An additional 181 patients with sporadic CVID were examined, and no mutations were found. This report confirms that an ICOS gene defect might cause CVID, although it is responsible for only a minority of cases. A comprehensive review of DGS or chromosome 22q11.2 deletion syndrome by Sullivan43 emphasizes the spectrum of severity of each of the clinical findings of this condition, including T-cell deficiency. A few cases might remain undiagnosed until the patient reached childhood and receives live vaccines before immunologic status is assessed. Moylett et al44 studied a cohort of 53 such patients and found that 25 of them had received a live vaccine. However, no serious adverse effects related to live vaccines occurred. Although reassuring, it is important to note that this cohort of patients had only a mildto-moderate decrease of T cells. The recommendation on the use of live vaccines in this group of patients is still controversial, and a cautious approach is advised against live vaccines administration until more data are available. Markert et al45 reported an unusual presentation of 5 patients with DGS who had heart, parathyroid, and immune defects. In addition, they presented with rash and lymphadenopathy. Although they had T cells and some had mitogen proliferative responses, these T cells were oligoclonal and there was no evidence of thymus activity, as measured by the absence and low output of naive cells. The authors concluded that the presence of T cells did not necessarily mean the presence of thymus activity and recommended that patients with DGS should undergo an evaluation of thymus activity in addition to the assessment of T-cell numbers. Two review articles by Etzioni and Ochs46 and Luo et al47 described and summarized the recent developments on HIGM and the biology of the activation-induced cytosine deaminase, one of the proteins that, when missing, results in HIGM. Orange et al48 described 7 patients with anhydrotic ectodermal dysplasia with immunodeficiency caused by mutations in the NF-kB essential modifier (NEMO) gene. They demonstrated a particular susceptibility to pyogenic and mycobacterial infections. NEMO deficiency was initially described as a form of HIGM but is currently classified as an innate immunity defect.33 In a follow-up article, the authors described a 16-year-old patient who had a mutation in the Ikb kinase portion of the NEMO gene, and although presenting with immunologic defects, the patient did not have the ectodermal defect components of this condition.49 Niehues et al50 reported a similar patient, although with a mutation in exon 2 inducing a premature stop codon. These 2 cases underscore the variety of presentations of these rare disorders and the need for continuing awareness for diagnosis.

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Immunorestoration syndrome in HIV infection Seven cases of immune restoration disease (IRD) were described in a cohort of 69 perinatally acquired HIVinfected children.54 IRD presents as a severe inflammatory reaction to opportunistic infections in HIV-infected patients who received highly active antiretroviral therapy and have a good response with recovery of normal T-cell counts. This is the first report of IRD in children. Interestingly, all 7 cases were caused by herpes zoster and occurred in those with the most severe immunodeficiency at baseline.

CONCLUSIONS In 2004, several exciting developments were reported in the areas of basic and clinical immunology. The role of monocyte activation in the development of TH1 responses was characterized, as well as the action of drugs commonly used in asthma to inhibit cytokine responses and induce regulatory T-cell differentiation. The regulatory effect of nonimmune cells present in the lung in the development of a TH1 or TH2 response has been established, with reports of pulmonary Clara cells inhibiting IL-4 and IL-13 expression and bronchial epithelial cells secreting the newly described IL-17F, a cytokine that participates in controlling allergic inflammation. The use of dendritic cells genetically engineered to favor the expression of TH1 cytokines on specific allergen stimulation has proved to be effective in in vitro models. In the area of HIV immunology, several investigators have described several methods used by HIV to escape CTL surveillance and control. This work is of importance to researchers developing vaccines on the basis of cellular response because it suggests that multiple epitopes specific for target populations need to be included. Research on Toll receptors continues to reveal more mechanisms of

innate immunity. They are present in mast cells and CD41 T cells for the recognition of viruses and bacteria, and they are involved in the control of iron metabolism that is essential for some bacteria species. Clinical research in immunologic diseases continues to show remarkable progress. An updated classification for PIDs is now available, with more than 100 genes identified as responsible for these diseases. ICOS is known now to be responsible for a minority of patients with CVID. With the advances in the field of genetics, a second successful trial of gene therapy has been published, although 3 cases of leukemia have occurred in patients from the first trial. Other advances were the definition of poor prognosis in CVID for patients presenting with specific inflammatory lung pathology, the estimation of safety for administration of live vaccines in patients with partial DGS, the unusual presentation of complete DGS with the presence of host T cells in peripheral blood, the presentation of the clinical spectrum of patients with NEMO deficiency, the elaboration of a consensus algorithm for the management of hereditary angioedema, and the description of immune restoration syndrome in HIV-infected children with predominance of herpes zoster infections.

REFERENCES 1. Wittmann M, Alter M, Stunkel T, Kapp A, Werfel T. Cell-to-cell contact between activated CD41 T lymphocytes and unprimed monocytes interferes with a TH1 response. J Allergy Clin Immunol 2004;114: 965-73. 2. Lametschwandtner G, Biedermann T, Schwarzler C, Gunther C, Kund J, Fassl S, et al. Sustained T-bet expression confers polarized human TH2 cells with TH1-like cytokine production and migratory capacities. J Allergy Clin Immunol 2004;113:987-9. 3. Upham JW, Holt PG, Taylor A, Thornton CA, Prescott SL. HLA-DR expression on neonatal monocytes is associated with allergen-specific immune responses. J Allergy Clin Immunol 2004;114:1202-8. 4. Goleva E, Dunlap A, Leung DY. Differential control of TH1 versus TH2 cell responses by the combination of low-dose steroids with beta2-adrenergic agonists. J Allergy Clin Immunol 2004;114:183-91. 5. Pace E, Gagliardo R, Melis M, La Grutta S, Ferraro M, Siena L, et al. Synergistic effects of fluticasone propionate and salmeterol on in vitro T-cell activation and apoptosis in asthma. J Allergy Clin Immunol 2004;114:1216-23. 6. Hung CH, Chen LC, Zhang Z, Chowdhury B, Lee WL, Plunkett B, et al. Regulation of TH2 responses by the pulmonary Clara cell secretory 10-kd protein. J Allergy Clin Immunol 2004;114:664-70. 7. Spiegelberg HL, Raz E. DNA-based approaches to the treatment of allergies. Curr Opin Mol Ther 2002;4:64-71. 8. Klostermann B, Bellinghausen I, Bottcher I, Petersen A, Becker WM, Knop J, et al. Modification of the human allergic immune response by allergen-DNA-transfected dendritic cells in vitro. J Allergy Clin Immunol 2004;113:327-33. 9. Ludwig-Portugall I, Montermann E, Kremer A, Reske-Kunz AB, Sudowe S. Prevention of long-term IgE antibody production by gene gun-mediated DNA vaccination. J Allergy Clin Immunol 2004;114: 951-7. 10. Jilek S, Walter E, Merkle HP, Corthesy B. Modulation of allergic responses in mice by using biodegradable poly(lactide-co-glycolide) microspheres. J Allergy Clin Immunol 2004;114:943-50. 11. O’Hagan DT, Singh M, Ulmer JB. Microparticles for the delivery of DNA vaccines. Immunol Rev 2004;199:191-200. 12. Farkas L, Kvale EO, Johansen FE, Jahnsen FL, Lund-Johansen F. Plasmacytoid dendritic cells activate allergen-specific TH2 memory cells: modulation by CpG oligodeoxynucleotides. J Allergy Clin Immunol 2004;114:436-43.

Basic and clinical immunology

Complement deficiency Three highly recommended reference articles for the management of complement deficiencies were published in 2004. One is a comprehensive review for the clinical evaluation of complement deficiencies addressed for the clinician and containing useful algorithms for the evaluation of patients with suspected complement deficiency.51 This article explains the genetics of these deficiencies, clinical manifestations, and current therapeutic advances. The second article is a review of the history, genetics, clinical presentation, and current management of hereditary angioedema caused by deficiency of C1 inhibitor, written by Frank.52 In an effort to gain consensus in the management of hereditary angioedema, an international conference was held in Ontario, Canada, in 2003 with the participation of European and American researchers. A summarizing consensus algorithm was drafted and published.53 Diagnostics and managements available were reviewed, including appropriate use of tranexamic acid, androgens, and C1 inhibitor concentrate.

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13. Hajoui O, Janani R, Tulic M, Joubert P, Ronis T, Hamid Q, et al. Synthesis of IL-13 by human B lymphocytes: regulation and role in IgE production. J Allergy Clin Immunol 2004;114:657-63. 14. Kawaguchi M, Kokubu F, Odaka M, Watanabe S, Suzuki S, Ieki K, et al. Induction of granulocyte-macrophage colony-stimulating factor by a new cytokine, ML-1 (IL-17F), via Raf I-MEK-ERK pathway. J Allergy Clin Immunol 2004;114:444-50. 15. Henness S, Johnson CK, Ge Q, Armour CL, Hughes JM, Ammit AJ. IL-17A augments TNF-alpha-induced IL-6 expression in airway smooth muscle by enhancing mRNA stability. J Allergy Clin Immunol 2004; 114:958-64. 16. Karagiannidis C, Akdis M, Holopainen P, Woolley NJ, Hense G, Ruckert B, et al. Glucocorticoids upregulate FOXP3 expression and regulatory T cells in asthma. J Allergy Clin Immunol 2004;114:1425-33. 17. Leslie AJ, Pfafferott KJ, Chetty P, Draenert R, Addo MM, Feeney M, et al. HIV evolution: CTL escape mutation and reversion after transmission. Nat Med 2004;10:282-9. 18. Dorak MT, Tang J, Penman-Aguilar A, Westfall AO, Zulu I, Lobashevsky ES, et al. Transmission of HIV-1 and HLA-B allelesharing within serodiscordant heterosexual Zambian couples. Lancet 2004;363:2137-9. 19. Draenert R, Le Gall S, Pfafferott KJ, Leslie AJ, Chetty P, Brander C, et al. Immune selection for altered antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection. J Exp Med 2004;199: 905-15. 20. Winchester R, Pitt J, Charurat M, Magder LS, Goring HH, Landay A, Read JS, et al. Mother-to-child transmission of HIV-1: strong association with certain maternal HLA-B alleles independent of viral load implicates innate immune mechanisms. J Acquir Immune Defic Syndr 2004;36: 659-70. 21. Moir S, Malaspina A, Pickeral OK, Donoghue ET, Vasquez J, Miller NJ, et al. Decreased survival of B cells of HIV-viremic patients mediated by altered expression of receptors of the TNF superfamily. J Exp Med 2004; 200:587-92. 22. Kulka M, Alexopoulou L, Flavell RA, Metcalfe DD. Activation of mast cells by double-stranded RNA: evidence for activation through Toll-like receptor 3. J Allergy Clin Immunol 2004;114:174-8. 23. Marshall JS, Jawdat DM. Mast cells in innate immunity. J Allergy Clin Immunol 2004;114:21-7. 24. Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 2004;432:917-21. 25. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 2004;118:229-41. 26. Gelman AE, Zhang J, Choi Y, Turka LA. Toll-like receptor ligands directly promote activated CD41 T cell survival. J Immunol 2004;172: 6065-73. 27. Depta JP, Altznauer F, Gamerdinger K, Burkhart C, Weltzien HU, Pichler WJ. Drug interaction with T-cell receptors: T-cell receptor density determines degree of cross-reactivity. J Allergy Clin Immunol 2004;113:519-27. 28. Nassif A, Bensussan A, Boumsell L, Deniaud A, Moslehi H, Wolkenstein P, et al. Toxic epidermal necrolysis: effector cells are drug-specific cytotoxic T cells. J Allergy Clin Immunol 2004;114: 1209-12. 29. Ng B, Yang F, Huston DP, Yan Y, Yang Y, Xiong Z, Peterson LE, et al. Increased noncanonical splicing of autoantigen transcripts provides the structural basis for expression of untolerized epitopes. J Allergy Clin Immunol 2004;114:1463-70. 30. Nakajima T, Iikura M, Okayama Y, Matsumoto K, Uchiyama C, Shirakawa T, et al. Identification of granulocyte subtype-selective receptors and ion channels by using a high-density oligonucleotide probe array. J Allergy Clin Immunol 2004;113:528-35. 31. Hoffjan S, Ostrovnaja I, Nicolae D, Newman DL, Nicolae R, Gangnon R, et al. Genetic variation in immunoregulatory pathways and atopic phenotypes in infancy. J Allergy Clin Immunol 2004;113:511-8. 32. Hanania NA, Sockrider M, Castro M, Holbrook JT, Tonascia J, Wise R, et al. Immune response to influenza vaccination in children and adults with asthma: effect of corticosteroid therapy. J Allergy Clin Immunol 2004;113:717-24.

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33. Notarangelo L, Casanova JL, Fischer A, Puck J, Rosen F, Seger R, et al. Primary immunodeficiency diseases: an update. J Allergy Clin Immunol 2004;114:677-87. 34. Cunningham-Rundles C, Sidi P, Estrella L, Doucette J. Identifying undiagnosed primary immunodeficiency diseases in minority subjects by using computer sorting of diagnosis codes. J Allergy Clin Immunol 2004; 113:747-55. 35. Holzelova E, Vonarbourg C, Stolzenberg MC, Arkwright PD, Selz F, Prieur AM, et al. Autoimmune lymphoproliferative syndrome with somatic Fas mutations. N Engl J Med 2004;351:1409-18. 36. Chinen J, Puck JM. Successes and risks of gene therapy in primary immunodeficiencies. J Allergy Clin Immunol 2004;113:595-603. 37. Cavazzana-Calvo M, Lagresle C, Hacein-Bey-Abina S, Fischer A. Gene therapy for severe combined immunodeficiency. Annu Rev Med 2005; 56:585-602. 38. Gaspar HB, Parsley KL, Howe S, King D, Gilmour KC, Sinclair J, et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 2004;364:2181-7. 39. Gardulf A, Nicolay U, Math D, Asensio O, Bernatowska E, Bock A, et al. Children and adults with primary antibody deficiencies gain quality of life by subcutaneous IgG self-infusions at home. J Allergy Clin Immunol 2004;114:936-42. 40. Chinen J, Shearer WT. Subcutaneous immunoglobulins: alternative for the hypogammaglobulinemic patient? J Allergy Clin Immunol 2004;114: 934-5. 41. Bates CA, Ellison MC, Lynch DA, Cool CD, Brown KK, Routes JM. Granulomatous-lymphocytic lung disease shortens survival in common variable immunodeficiency. J Allergy Clin Immunol 2004; 114:415-6. 42. Salzer U, Maul-Pavicic A, Cunningham-Rundles C, Urschel S, Belohradsky BH, Litzman J, et al. ICOS deficiency in patients with common variable immunodeficiency. Clin Immunol 2004;113:234-40. 43. Sullivan KE. The clinical, immunological, and molecular spectrum of chromosome 22q11.2 deletion syndrome and DiGeorge syndrome. Curr Opin Allergy Clin Immunol 2004;4:505-12. 44. Moylett EH, Wasan AN, Noroski LM, Shearer WT. Live viral vaccines in patients with partial DiGeorge syndrome: clinical experience and cellular immunity. Clin Immunol 2004;112:106-24. 45. Markert ML, Alexieff MJ, Li J, Sarzotti M, Ozaki DA, Devlin BH, et al. Complete DiGeorge syndrome: development of rash, lymphadenopathy, and oligoclonal T cells in 5 cases. J Allergy Clin Immunol 2004;113: 734-41. 46. Etzioni A, Ochs HD. The Hyper IgM syndrome—an evolving story. Pediatr Res 2004;56:519-25. 47. Luo Z, Ronai D, Scharff MD. The role of activation-induced cytidine deaminase in antibody diversification, immunodeficiency, and B-cell malignancies. J Allergy Clin Immunol 2004;114:726-35. 48. Orange JS, Jain A, Ballas ZK, Schneider LC, Geha RS, Bonilla FA. The presentation and natural history of immunodeficiency caused by nuclear factor kappaB essential modulator mutation. J Allergy Clin Immunol 2004;113:725-33. 49. Orange JS, Levy O, Brodeur SR, Krzewski K, Roy RM, Niemela JE, et al. Human nuclear factor kappa B essential modulator mutation can result in immunodeficiency without ectodermal dysplasia. J Allergy Clin Immunol 2004;114:650-6. 50. Niehues T, Reichenbach J, Neubert J, Gudowius S, Puel A, Horneff G, et al. Nuclear factor kappaB essential modulator-deficient child with immunodeficiency yet without anhidrotic ectodermal dysplasia. J Allergy Clin Immunol 2004;114:1456-62. 51. Wen L, Atkinson JP, Giclas PC. Clinical and laboratory evaluation of complement deficiency. J Allergy Clin Immunol 2004;113:585-93. 52. Frank MM. Hereditary angioedema: a half century of progress. J Allergy Clin Immunol 2004;114:626-8. 53. Bowen T, Cicardi M, Farkas H, Bork K, Kreuz W, Zingale L, et al. Canadian 2003 international consensus algorithm for the diagnosis, therapy, and management of hereditary angioedema. J Allergy Clin Immunol 2004;114:629-37. 54. Tangsinmankong N, Kamchaisatian W, Lujan-Zilbermann J, Brown CL, Sleasman JW, Emmanuel PJ. Varicella zoster as a manifestation of immune restoration disease in HIV-infected children. J Allergy Clin Immunol 2004;113:742-63.

Current perspectives The gastrointestinal tract is critical to the pathogenesis of acute HIV-1 infection Saurabh Mehandru, MD,a Klara Tenner-Racz, MD,b Paul Racz, MD, PhD,b and Martin Markowitz, MDa New York, NY, and Hamburg, Germany

Key words: Acute HIV-1, gastrointestinal tract, CD41 T cells

Acute infection is a critical time in the course of HIV-1 infection. During this phase, the virus gains access to its target cells, infects, replicates, disseminates, and simultaneously establishes a pool of latently infected cells. As evidenced by the explosive growth of the epidemic in the last 2 decades, it is clear that HIV-1 is extremely adept at accomplishing these tasks. Once established, untreated HIV-1 infection culminates in profound immunodeficiency and death in the majority of individuals. Recent evidence derived from human subjects with acute HIV-1 infection and macaques with acute simian immunodeficiency virus (SIV) infection suggests that the course of these infections might be determined during the acute phase of infection. Because the virus entering a new host must negotiate a series of obstacles between entry, amplification, and dissemination, it is plausible that interventions made during acute HIV-1 infection have the potential to change the natural history of this disease. To date, much work has focused on understanding these events and have described changes exclusively within the peripheral blood. Until recently, mucosal sites, such as the gastrointestinal (GI) tract, have been relatively underFrom aAaron Diamond AIDS Research Center and Rockefeller University, New York, and bBernhard-Nocht Institut fur Tropenmedizin, Hamburg. Disclosure of potential conflict of interest: All authors—none disclosed. Received for publication May 20, 2005; accepted for publication May 24, 2005. Available online July 5, 2005. Reprint requests: Saurabh Mehandru, MD, Aaron Diamond AIDS Research Center, The Rockefeller University, 455 First Ave, 7th Floor, New York, NY 10016. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.040

Abbreviations used GI: Gastrointestinal SIV: Simian immunodeficiency virus

emphasized in the study of acute HIV-1 infection. Here we will summarize the recent findings suggesting the critical role of the lymphoid system of the GI tract during acute HIV and SIV infection. It has been well established that the GI tract harbors the majority of the body’s complement of immune cells.1 GI tract lymphocytes, placed in close proximity to the external environment, are phenotypically distinct from peripheral blood lymphocytes; the majority of the intestinal lymphocytes (>90%) exhibit a memory phenotype.2 In addition, because of constant exposure to a myriad of food and microbial antigens, GI tract lymphocytes are significantly more activated than peripheral blood lymphocytes.2 Furthermore, up to 70% of GI tract lymphocytes express CCR5, a chemokine receptor that serves as an essential coreceptor for the entry of CCR5-tropic HIV-1 into CD41 T cells.3 (In contrast, approximately 20% of peripheral blood lymphocytes express CCR5.) Thus the vast population of activated memory CD41 T cells with abundant expression of chemokine receptors provides HIV-1 with an ideal environment to establish infection. Unfortunately, the study of the human GI tract during acute HIV-1 infection is challenging. It is fraught with difficulties in identifying individuals during acute infection and the added complexity of obtaining GI tract biopsy specimens in the face of psychological and physical complications associated with acute HIV-1 infection. Consequently, initial work in this area emerged from the SIV macaque model. A striking depletion of intestinal CD41 T cells was noted in macaques within days of SIV infection, at a time when little or no CD41 T-cell depletion was evident in the peripheral blood.4,5 Furthermore, intestinal CD41 T-cell depletion occurred regardless of whether viral inoculum was delivered intrarectally or intravenously.5 These studies were subsequently extended to demonstrate that GI tract lymphocyte depletion occurs in all stages of SIV infection, including acute infection.4 Studies conducted during the 1990s indicated that intestinal CD41 T-cell depletion might be an early feature of HIV-1 infection as well6 and that intestinal CD41 T-cell 419

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It has become evident that the gastrointestinal tract is preferentially and profoundly depleted of CD41 T cells during acute HIV-1 infection. The enhanced susceptibility of gastrointestinal lymphoid tissue to HIV-1 is in part due to the large complement of CCR51 memory CD41 T cells resident at this site. Here we summarize the recent findings demonstrating that the gastrointestinal tract plays a critical role in the pathogenesis of acute HIV-1 and simian immunodeficiency virus infections. Ongoing work in this field is likely to have a significant effect on HIV research in the near future. (J Allergy Clin Immunol 2005;116:419-22.)

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FIG 1. Effector sites (lamina propria) of the GI tract show pronounced CD41 T-cell depletion in acute and early HIV-1 infection. At magnifications of 253 (A) and 803 (B), CD41 T cells (stained red) are seen in a representative biopsy specimen from an HIV-uninfected individual. In comparison, profound CD41 T-cell depletion is noted in low-power (C, 253) and high-power (D, 803) views in a specimen from a subject with acute HIV-1 infection.

Basic and clinical immunology

depletion is more significant than depletion of CD41 T cells in the peripheral blood.7 However, such studies were not conducted in patients identified during acute infection. Along with 2 other groups, we have recently demonstrated that during acute HIV-1 infection, a preferential and profound depletion of CD41 T cells occurs within the GI tract.8-10 In the first of these studies, Guadalupe et al8 described 2 individuals with acute HIV-1 infection in which significant CD41 T-cell depletion occurred within approximately 4 to 6 weeks of infection. Brenchley et al10 studied one individual with acute HIV-1 infection (infected for <1 month) and 4 individuals with early HIV-1 infection (duration of infection was 4-9 months). In all 5 subjects, a preferential CD41 T-cell depletion was noted in the intestines. In fact, significant GI tract CD41 T-cell depletion was characteristic of all stages of HIV-1 infection.9,10 Our initial focus was to describe changes within the GI tract of individuals with acute and early HIV-1 infection. To this end, we studied 13 individuals identified during acute (n = 7) and early (n = 6) infection. Mean CD41 Tcell percentage in the GI tract was 15.7% 6 3.6% compared with a mean of 42.3% 6 14.7% in the blood (P < .001). Thus in all of our subjects, profound CD41

T-cell depletion was observed in the GI tract and was significantly greater than depletion in the peripheral blood (Fig 1). Since then, we have extended our studies to 26 subjects with acute and early HIV-1 infection and have observed far greater CD41 T-cell depletion in the intestines compared with in the peripheral blood (unpublished data). Having demonstrated a striking CD41 T-cell depletion within the GI tract, we have examined the subsets of CD41 T cells in which this depletion was most prominent. Because a majority of intestinal CD41 T cells express CCR53 and given that viruses during the early stages of HIV-1 infection are predominantly CCR5tropic,11 it was not surprising that Brenchley et al10 and we9 both observed that GI tract lymphocyte depletion was most significant among the CCR51 subsets of CD41 T cells. In addition, we have observed marked CD41 T-cell depletion in the effector sites (lamina propria) of the GI tract, with relative sparing of the inductive sites (organized lymphoid tissue). In contrast, however, HIV-1 RNA was localized to the inductive sites. We believe that this discrepancy is best explained by loss of target cells in the effector compartment. We hypothesize that if we could examine a subject within the first 7 to 10 days of infection, viral RNA would be evident in the effector compartment

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subjects and SIV-infected macaques suggest an emerging model for the pathogenesis of HIV-1 infection. The large complement of resident memory cells in mucosal surfaces get preferentially infected and fuel subsequent rounds of replication and infection. Therefore in acute stages mucosal sites appear to propel the infection forward. What are the implications of these findings? A number of significant issues emerge from these studies: 1. By demonstrating that significant viral replication and immune depletion occurs at mucosal sites during acute infection, these findings provide compelling evidence to the argument that mucosal sites should be the focus of further examination and should be considered in the monitoring of patients on therapy. 2. These findings shatter the dogma that during acute HIV-1 infection, there is little CD41 T-cell depletion in the body. 3. These findings reinvigorate the debate regarding the timing of therapy in HIV-1 infection. Guidelines recommending that treatment need not be initiated until CD41 T-cell count decreases to less than 350 cells/mm3 or until plasma viral load is more than 100,000 copies/mL14 should take into account these recent data demonstrating severe mucosal CD41 T-cell depletion in acute and early HIV-1 infection. This said, however, it must be mentioned that the clinical significance of mucosal immune depletion remains uncertain. Redundancy in our immune system might prevent long-term consequences, yet the possibility of early immune senescence exists, and the long-term consequences are not clear. 4. These findings suggest a potential for the use of immunomodulators, such as cyclosporine, during acute HIV-1 infection with the goal of curtailing successive rounds of viral infection and CD41 T-cell depletion within the GI tract. 5. With regard to preventive efforts directed against HIV-1, recent data underscore the need to develop strategies to protect mucosal surfaces from infection. The use of microbicides and CCR5 blockers would represent such interventions. 6. Finally and perhaps most importantly, these findings reemphasize the fact that mucosal immune responses must be targeted in the development of effective HIV-1 vaccines. The weight of current evidence places mucosal lymphoid tissue as pivotal in HIV-1 pathogenesis. These findings are likely to have a significant effect on HIV research in the near future.

REFERENCES 1. Mowat AM. Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol 2003;3:331-41. 2. Schieferdecker HL, Ullrich R, Hirseland H, Zeitz M. T cell differentiation antigens on lymphocytes in the human intestinal lamina propria. J Immunol 1992;149:2816-22. 3. Anton PA, Elliott J, Poles MA, McGowan IM, Matud J, Hultin LE, et al. Enhanced levels of functional HIV-1 co-receptors on human mucosal

Basic and clinical immunology

as well before the annihilation of the resident CD41 cells. In a small cross-sectional cohort, we have observed that in contrast to the peripheral blood, restoration of CD41 T cells in the GI compartment with highly active antiretroviral therapy is incomplete. We are currently examining patients longitudinally to determine whether the timing of onset of antiretroviral therapy is associated with better GI tract CD41 T-cell reconstitution, as has been suggested by Guadalupe et al8 in a limited cohort comprised of 2 subjects. Recent data obtained from the SIV macaque model provide startling insights into the degree of GI tract CD41 T-cell infection and depletion as well. Studies by Mattapallil et al12 and Li et al13 demonstrate rapid infection and destruction of GI tract memory CD41 T cells within days of infection with SIV. The study by Mattapallil et al12 suggests that at peak viremia (day 10), as many as 60% of GI tract memory CD41 T cells might be infected with SIV and that these cells are lost within 14 days of infection. This results in profound immunodeficiency that begins within days of infection, not months to years as was previously thought. The authors put forth the notion that memory CD41 T cells are killed by means of direct, virus-mediated destruction rather than bystander effects or suppression of CD41 T-cell production. Li et al13 showed, somewhat surprisingly, that GI tract cells infected initially with SIV have a nonactivated (CD692CD252Ki672) phenotype. Peak infection of memory CD41 T cells within the GI tract corresponded to peak viremia, and depletion of GI tract CD41 T cells coincided with a decrease in the peripheral viral load. Li et al therefore suggested the concept of ‘‘substrate depletion,’’ resulting in viral load reduction in the host. In contrast to Mattapallil et al,12 Li et al13 propose that CD41 T-cell depletion is caused by virus-triggered, Fas–Fas ligand–mediated apoptosis. It is likely that CD41 T-cell destruction is multifactorial, caused by virus-induced cytolysis, apoptosis, and the host’s own cytotoxic Tlymphocyte, natural killer cell responses. Further work needs to be done to resolve this issue. Concurrent studies in our laboratory have focused on determining virologic and immunologic correlates of GI tract CD41 T-cell depletion. Our data (unpublished) suggest that acute HIV-1 infection results in immunologic activation and proliferation of GI tract CD41 T cells, creating a local niche of viral replication. We have observed a highly significant difference between the level of CD41 T-cell infection in the GI tract and peripheral blood. In addition, we have observed a striking difference in HIV-1 viral RNA production within CD41 T cells derived from the GI tract compared with from the peripheral blood. Thus we hypothesize that during acute infection, HIV-1 encounters a vast population of susceptible cells within the GI tract and preferentially infects them. Viral infection results in immune activation and CD41 T-cell proliferation, both of which augment viral production, setting up the next round of infection. The end result is profound CD41 T-cell depletion within the GI tract. Combined, the results from recent studies in human

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4.

5.

6.

7.

8.

T cells demonstrated using intestinal biopsy tissue. AIDS 2000;14: 1761-5. Smit-McBride Z, Mattapallil JJ, McChesney M, Ferrick D, Dandekar S. Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(1) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes. J Virol 1998;72:6646-56. Veazey RS, DeMaria M, Chalifoux LV, Shvetz DE, Pauley DR, Knight HL, et al. Gastrointestinal tract as a major site of CD41 T cell depletion and viral replication in SIV infection. Science 1998;280:427-31. Lim SG, Condez A, Lee CA, Johnson MA, Elia C, Poulter LW. Loss of mucosal CD4 lymphocytes is an early feature of HIV infection. Clin Exp Immunol 1993;92:448-54. Schneider T, Jahn HU, Schmidt W, Riecken EO, Zeitz M, Ullrich R. Loss of CD4 T lymphocytes in patients infected with human immunodeficiency virus type 1 is more pronounced in the duodenal mucosa than in the peripheral blood. Berlin Diarrhea/Wasting Syndrome Study Group. Gut 1995;37:524-9. Guadalupe M, Reay E, Sankaran S, Prindiville T, Flamm J, McNeil A, et al. Severe CD41 T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial

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9.

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11.

12.

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14.

delay in restoration following highly active antiretroviral therapy. J Virol 2003;77:11708-17. Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, et al. Primary HIV-1 infection is associated with preferential depletion of CD41 T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 2004;200:761-70. Brenchley JM, Schacker TW, Ruff LE, Price DA, Taylor JH, Beilman GJ, et al. CD41 T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 2004;200:749-59. Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR. Change in coreceptor use coreceptor use correlates with disease progression in HIV-1–infected individuals. J Exp Med 1997;185:621-8. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. Massive infection and loss of memory CD41 T cells in multiple tissues during acute SIV infection. Nature 2005;434:1093-7. Li Q, Duan L, Estes JD, Ma ZM, Rourke T, Wang Y, et al. Peak SIV replication in resting memory CD41 T cells depletes gut lamina propria CD41 T cells. Nature 2005;434:1148-52. Guidelines for the use of antiretroviral agents in HIV-infected adults and adolescents. Washington (DC): Department of health and Human Services; 2005.

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Editorial Are you immunodeficient? Francisco A. Bonilla, MD, PhD, and Raif S. Geha, MD Boston, Mass

In their Rostrum monograph, Casanova et al1 consider the problems of definition and classification of primary immunodeficiencies (PIs). They begin with a standard and straightforward premise: ‘‘immunodeficiency is a failure to achieve immune function to provide efficient, selflimited host defense against the biotic and abiotic environment while preserving tolerance to self.’’ The challenge to practitioners is to translate this axiom into principles that answer specific clinical and academic needs. One essential message of Casanova et al is to consider the susceptibility to infection limited to one or a few pathogens and having Mendelian inheritance to be within the spectrum of PI, regardless of the immunologic phenotype. They further argue that in light of this, academic and clinical needs will best be met by a classification system on the basis of clinical phenotype, in contrast to the classic, or perhaps traditional, system on the basis of immunologic phenotype.2,3 According to their scheme, Casanova et al1 distinguish conventional and unconventional immunodeficiencies as those that do or do not have clearly defined immunologic phenotypes, respectively. The authors consider these to be end points of a spectrum rather than dichotomous. They further state there has ‘‘never been a fully satisfactory classification of PID.’’ However, it is worth discussing whether any single system on the basis of clinical phenotype or immunologic phenotype could ever be so. Regardless of how we might classify their diseases, immunodeficient patients come to clinical attention predominantly as a result of a predisposition to infection. This predisposition is manifested in one or more of the clinical dimensions of infection: the inherent virulence of the From the Division of Immunology, Children’s Hospital, and the Department of Pediatrics, Harvard Medical School, Boston, Mass. Disclosure of potential conflict of interest: F. A. Bonilla has consultant arrangements with Talecris Biotherapeutics and is on the speakers’ bureau for Accredo Therapeutics. R. Geha has no conflicts of interest to disclose. Received for publication May 18, 2005; accepted for publication May 19, 2005. Available online July 5, 2005. Reprint requests: Francisco A. Bonilla, MD, PhD, Children’s Hospital, Immunology, Enders 809, 300 Longwood Ave, Boston, MA 02115. E-mail: [email protected]. J Allergy Clin Immunol 2005;116:423-5. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.026

Abbreviation used PI: Primary immunodeficiency

organism, the site of infection (localized vs disseminated), the infection’s severity (degree of tissue or organ damage), the infection’s persistence or resistance to therapy, and the frequency of relapse or reinfection. In spite of the commonality of these considerations early in the approach to the potentially immunodeficient patient, there are very few data or agreement on where to best draw the dividing line between normal and abnormal along any of these dimensions. It might also be important to consider that this line can be drawn differently under circumstances that differ with respect to, for example, the level of public hygiene, the prevalence of particular pathogens, or the availability of vaccinations. (Casanova et al,1 in fact, consider these factors as masking the true prevalence of PI.) In addition, Casanova et al consider the importance of Mendelian (single gene) inheritance in a clinical definition. Perhaps this element could be generalized to any definable genetic component to include interactions among mutations, polymorphisms, or both that might determine a phenotype, as has been observed in some PI diseases.4 Whether one is prepared to alter one’s conceptualization of immune deficiency to include unconventional forms, the matter of definition requires further study from all sides (epidemiology, immunology, and genetics) to provide a more solid framework for further discussion. Ultimately, the issue of where to draw the line between normal and abnormal is critical if the search for ‘‘currently unknown Mendelian primary immunodeficiencies’’1 is to set out with hope for meaningful discovery. The criteria of normalcy in a system based on immunologic phenotype relate to population distributions of screening laboratory studies of immune function (eg, serum immunoglobulin levels and specific antibody titers, peripheral blood lymphocyte subpopulations, in vivo or in vitro measures of T-cell function, assays of phagocyte oxidative burst or adhesion, and complement function or serum component levels).2 This system affords convenient statistical labels for normal and abnormal. Unfortunately, the biologic and clinical correlates are also largely lacking here, as in the discussion of predisposition to infection above. This sometimes leads to an important point raised by Casanova et al1 that certainly bears repeating: ‘‘patients with specific clinical infectious 423

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Key words: Clinical immunology, primary immunodeficiency, infectious diseases, genetics

424 Bonilla and Geha

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diseases but no overt immunological phenotype [are] being largely neglected.’’ However, this is not precisely a failure of a system or classification based on immunologic phenotype, as much as it reflects 3 distinct elements. First, one might choose simply not to categorize a limited infection predisposition as a PI, one of the main points of Casanova et al1 (ie, if it is definable genetically, then we should). Second, we must consider further what is meant by immunologic phenotype. Casanova et al use the adjective ‘‘overt’’ to denote an abnormality detectable by the battery of screening tests listed above. However, at some level, all genetically definable PIs must have some immunologic phenotype; it is the clinical phenotype that dictates the specific evaluation that will ultimately identify it (therefore we can agree on the value of the clinical classification here). And finally, it is clear that we have incomplete knowledge regarding how the organism as a whole (including the immune system) protects itself from infection. Only in recent years have the importance of defects of natural killer cell function5 and toll-like receptor signaling6,7 become the foci of attention in PI. It is inevitable that the clinical screening immunologic evaluation of patients will continue to develop along with expanding knowledge of immune mechanisms. In the final analysis, the matter of classification might truly be secondary. Consider Table I, which shows a very simple scheme outlining the biologic and clinical elements of PI and the classification systems that might be proposed in response to or motivated by these different aspects of the interaction of a pathogen with a susceptible host. No system is superior in all cases, and some might even be frankly cumbersome in some situations, but there is no single system that optimally serves all needs. We all work together toward the determination of complete sets or elements of a PI knowledge base, such as the complete set of gene alterations that lead to susceptibility to infection, the complete set of clinical phenotypes of PIs, and the complete set of immunologic phenotypes. These will not be conveniently ordered along 1 or 2 dimensions that will be useful in every situation, as outlined above. What might serve best is a multisegmented database in which each segment orders the information according to a distinct classification system and every entry is linked to its entries in the other segments. This type of organization is legion on the Internet (see, for example, the National Center for Biotechnology Information of the National Institutes of Health, Bethesda, Maryland, at http://www.ncbi.nih.gov/, and the Institute for Medical Technology Bioinformatics Group of the University of Tampere, Tampere, Finland, at http:// bioinf.uta.fi/). The adaptive immune system might be dispensable for human development and survival in a germ-free environment.8 On the other hand, elements of innate immunity interact with commensal flora and are required for normal function of some systems.9 In light of the interrelations of immunity with other organ systems, the boundaries of the immune system, as a whole, become less distinct. Whether

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TABLE I. Clinical-academic considerations or questions in PI diseases and potential classification schemes to address them Biomedical aspects of immunodeficiency

Infecting microbe(s) Host with genetic lesion(s)-polymorphism(s) Altered immunopathogenesis of infection Immune dysregulation (atopy, autoimmunity, lymphoproliferation, malignancy) Clinical syndrome of immunodeficiency Diagnostic evaluation Therapy

Outcome

Basis for classification of PI

Microbial taxonomy Genetic catalog, mode of inheritance Biochemical and cell biologic mechanisms Biochemical and cell biologic mechanisms All clinical features of the disease Immunologic phenotype Anti-infective, immune reconstitution, response to therapy Natural history, prognosis

it is truly ‘‘the least efficient physiological system at the individual level’’1 and whether we might discover that a large fraction of human subjects are immunodeficient depends on one’s perspective, as we have discussed, notwithstanding the benefits of public hygiene, immunization, and antibiotics. This is apparent if one shifts focus from the anthropocentric view and remembers that human pathogens and human beings evolve together. At the bedside, the clinical immunologist’s interest is piqued by the constellation of history, symptoms, and findings. Some version of the clinical classification of which Casanova et al1 speak is foremost in our minds while we determine the most efficient path toward defining the immunologic phenotype, making a definitive diagnosis, or both. Experience informs us that our ability to define the immunologic phenotype with precision depends utterly on the sophistication of the laboratory methods available for study of the individual patient. We note in passing that recent advances in molecular methods can, in some instances, divorce the processes of defining the immunologic phenotype from making a diagnosis in the case of a PI that has already been defined at the molecular level. For example, a 15-month-old boy presents with severe recurrent respiratory tract infections with encapsulated bacteria. We sequence his BTK gene and find a mutation or deletion consistent with X-linked agammaglobulinemia. We have established a diagnosis without knowing whether he is agammaglobulinemic or B lymphopenic. We do not advocate such an approach, however, because it perpetuates or even creates critical gaps in our knowledge base. As we stated, the example applies only where the molecular defect is known. The situation is different for the patient with a less well-understood form of PI. The level of laboratory sophistication required for the definition of new forms of PI is an order of magnitude

beyond what suffices for diagnosis of known entities. Technologic advances might soon provide us with the ability to automate functional studies of pathogen-specific immune responses and to link these to genomics-derived strategies to identify loci for study.10 Wherever they exist, these resources must be made available in some way to the larger community of clinical immunologists. The importance of this cannot be overstated. We agree with Casanova et al1 that the full spectrum of human susceptibility to infection is largely waiting to be discovered. For those with PI and those who study it, hope derives from a chance encounter with ‘‘a prepared mind,’’ timely recognition, and the technology to repair it.11,12 REFERENCES 1. Casanova J-L, Fieschi C, Bustamante J, Reichenbach J, Remus N, von Bernuth H, et al. From idiopathic infectious diseases to novel primary immunodeficiencies. J Allergy Clin Immunol 2005;116:426-30. 2. Bonilla FA, Bernstein IL, Khan DA, Ballas ZK, Chinen J, Frank MM, et al. Practice Parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol 2005;94(suppl):S1-63. 3. Notarangelo L, Casanova JL, Fischer A, Puck J, Rosen F, Seger R, et al. Primary immunodeficiency diseases: an update. J Allergy Clin Immunol 2004;114:677-87.

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4. Foster CB, Lehrnbecher T, Mol F, Steinberg SM, Venzon DJ, Walsh TJ, et al. Host defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease. J Clin Invest 1998;102:2146-55. 5. Orange JS. Human natural killer cell deficiencies and susceptibility to infection. Microbes Infect 2002;4:1545-58. 6. Orange JS, Levy O, Brodeur SR, Krzewski K, Roy RM, Niemela JE, et al. Human nuclear factor kappa B essential modulator mutation can result in immunodeficiency without ectodermal dysplasia. J Allergy Clin Immunol 2004;114:650-6. 7. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, et al. Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 2003;299:2076-9. 8. Guerra IC, Shearer WT. Environmental control in management of immunodeficient patients: experience with ‘‘David’’. Clin Immunol Immunopathol 1986;40:128-35. 9. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 2004;118:229-41. 10. Tian Q, Stepaniants SB, Mao M, Weng L, Feetham MC, Doyle MJ, et al. Integrated genomic and proteomic analyses of gene expression in Mammalian cells. Mol Cell Proteomics 2004;3:960-9. 11. Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002;296:2410-3. 12. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000;288:669-72.

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J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 2

Rostrum From idiopathic infectious diseases to novel primary immunodeficiencies Jean-Laurent Casanova, MD, PhD,a,b Claire Fieschi, MD, PhD,a,c Jacinta Bustamante, MD,a Janine Reichenbach, MD,a,d Natasha Remus, MD,a,e Horst von Bernuth, MD,a and Capucine Picard, MD, PhDa,b Paris and Cre´teil, France, and Frankfurt, Germany

Primary immunodeficiencies are typically seen as rare monogenic conditions associated with detectable immunologic abnormalities, resulting in a broad susceptibility to multiple and recurrent infections caused by weakly pathogenic and more virulent microorganisms. By opposition to these conventional primary immunodeficiencies, we describe nonconventional primary immunodeficiencies as Mendelian conditions manifesting in otherwise healthy patients as a narrow susceptibility to infections, recurrent or otherwise, caused by weakly pathogenic or more virulent microbes. Conventional primary immunodeficiencies are suspected on the basis of a rare, striking, clinical phenotype and are defined on the basis of an overt immunologic phenotype, often leading to identification of the disease-causing gene. Nonconventional primary immunodeficiencies are defined on the basis of a more common and less marked clinical phenotype, which remains isolated until molecular cloning of the causal gene reveals a hitherto undetected immunologic phenotype. Similar concepts can be applied to primary immunodeficiencies presenting other clinical features, such as allergy and autoimmunity. Nonconventional primary immunodeficiencies thus expand the clinical boundaries of this group of inherited disorders considerably, suggesting that Mendelian primary immunodeficiencies are more common in the general population than previously thought and might affect children with a single infectious, allergic, or autoimmune disease. (J Allergy Clin Immunol 2005;116:426-30.) Key words: Primary immunodeficiency, infectious diseases, idiopathic infections, inborn errors, Mendelian disorders, predisposition to infection

Basic and clinical immunology

From aLaboratoire de Ge´ne´tique Humaine des Maladies Infectieuses, Universite´ de Paris Rene´ Descartes-INSERM U550, Faculte´ de Me´decine Necker, Paris; bUnite´ d’Immunologie et d’He´matologie Pe´diatriques, Hoˆpital Necker Enfants Malades, Paris; cService d’Immunologie Clinique, Hoˆpital Saint Louis, Paris; dKlinik fu¨r Kinderheilkunde, Klinikum der J.W. Goethe Universita¨t, Frankfurt; and eService de Pe´diatrie, Centre Hospitalier Intercommunal de Cre´teil, Cre´teil. Our laboratory is supported in part by grants from the BNP-Paribas and Schlumberger foundations, the Institut Universitaire de France, and the EU grant QLK2-CT-2002-00846. Received for publication February 21, 2005; revised March 29, 2005; accepted for publication March 30, 2005. Available online July 5, 2005. Reprint requests: Jean-Laurent Casanova, MD, PhD, Laboratoire de Ge´ne´tique Humaine des Maladies Infectieuses, Universite´ de Paris Rene´ Descartes-INSERM U550, Faculte´ de Me´decine Necker, Paris 75015, France, EU. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.03.053

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An immunologic definition and classification of primary immunodeficiencies currently prevails and is expected to do so for the foreseeable future.1 Unfortunately, this has resulted in studies of otherwise healthy patients with specific infectious clinical diseases but no overt immunologic phenotype being largely neglected. The attention of most investigators and clinicians has remained focused on the tip of the iceberg: those rare patients with a noisy clinical phenotype (multiple, recurrent, and severe infections) and a visible immunologic phenotype (defining the primary immunodeficiency). The most striking example of such conventional primary immunodeficiencies is reticular dysgenesia, an exceedingly rare disorder associated with agranulocytosis and alymphocytosis, resulting in early-onset vulnerability to virtually all microorganisms and a rapidly fatal outcome in the absence of hematopoietic stem cell transplantation.2 Immunodeficiency is commonly ruled out in patients with a single severe infectious disease (even if recurrent or life-threatening) and normal routine immunologic workup (searching for signs of inherited or acquired immunodeficiency). Self-contradictory titles in the medical literature, such as ‘‘Fatal infection in an immunocompetent individual,’’ remain common. Unusual infectious diseases are often described as idiopathic, demonstrating caution and a desire to avoid the direct incrimination of the patient’s genetic background. However, some infections typically caused by weakly virulent (opportunist) microbes have been found to be associated with a high frequency of familial forms, parental consanguinity, or both, suggesting Mendelian predisposition. This group of nonconventional primary immunodeficiencies is characterized by a very narrow spectrum of opportunistic infections limited to one microbial genus or species possibly, but not necessarily, recurrent in otherwise healthy patients with no detectable immunologic abnormality on initial investigation.3 These diseases do not fit easily into the classical classification of primary immunodeficiencies. Nonconventional primary immunodeficiencies include the syndromes of Mendelian susceptibility to mycobacterial diseases (OMIM 209950,4 first described in 1951) in patients with mutations in the IL-12/23–IFN-g circuit (first identified in 1996)5-8; recurrent invasive disease caused by Neisseiria species in patients with mutations affecting the terminal components of complement (C5 to C9) forming the membrane attack complex (first described

in 1974)9,10; isolated chronic mucocutaneous candidiasis (OMIM 114580, first described in 1969), which remains unexplained genetically11,12; epidermodysplasia verruciformis with disseminated warts caused by human papillomaviruses belonging to group B1 (OMIM 226400, first described clinically in 1922 and subsequently shown to be a Mendelian trait [1939] conferring susceptibility to papillomaviruses [1946-1966]) in patients with mutations in EVER1 and EVER2 (first described in 2002)13,14; and X-linked lymphoproliferative syndrome caused by Epstein-Barr virus (OMIM 308240, first described in 1975) in patients with mutations in SAP (first described in 1998).15,16 Needless to say, the dichotomy between conventional and nonconventional conditions is somewhat artificial because there is really a continuum between these 2 extremes.17 Patients with a recently described conventional primary immunodeficiency, IL-1 receptorassociated kinase 4 deficiency, are particularly susceptible to Streptococcus pneumoniae,18,19 and conversely, patients with mutations in the IL-12–IFN-g axis are also susceptible to Salmonella species.20 In any event, neither the identification of a cellular phenotype nor that of the causal gene suggested the existence of an underlying primary immunodeficiency in patients with nonconventional primary immunodeficiencies. Instead, primary immunodeficiency diagnosis was based on the relatively low virulence of the microbe and the seemingly Mendelian inheritance of predisposition to severe disease. It would not be wise to limit the group of nonconventional primary immunodeficiencies to these 5 Mendelian syndromes, to patients presenting unexplained infections caused by weakly virulent opportunistic microorganisms, or even to patients with recurrent infections caused by more virulent pathogens. There is good reason to believe that other human conditions reflect currently unknown Mendelian primary immunodeficiencies. First, genetic epidemiologic studies searching for familial forms and parental consanguinity have not been carried out for most infectious, autoimmune, and allergic clinical syndromes. Second, neither the absence of familial cases nor the lack of consanguinity are sufficient to exclude Mendelian defects, and sporadic cases might reflect a genetic lesion, as illustrated by the first genetic lesion discovered in human subjects, trisomy 21, in patients with Down syndrome.21 Third, the virulence of microorganisms is also a continuum, and many pathogenic microbes, such as Mycobacterium tuberculosis, are actually innocuous in most human beings. A number of common infectious diseases are likely to reflect nonconventional primary immunodeficiencies in at least a fraction of patients. Consistent with this view, mycobacterial diseases caused by weakly virulent BCG species were described as idiopathic infections before the identification of defects in the IL-12–IFN-g circuit.22,23 The identification of these defects has led to the recent description of 3 unrelated families with a purely Mendelian form of predisposition to bona fide tuberculosis,24-26 following on from the observation that IL-12Rb1 deficiency had low penetrance for the case-definition phenotype of clinical disease caused by

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weakly virulent mycobacteria.20,27 Currently unexplained candidate infectious diseases include invasive pneumococcal disease18,19 and herpes simplex encephalitis,28,29 which have been diagnosed in at least a few patients with conventional immunodeficiencies. Many life-threatening infectious diseases might well turn out to result from the Mendelian inheritance of a specific predisposition, reflecting a nonconventional primary immunodeficiency. Nonconventional primary immunodeficiencies are defined on clinical grounds, raising the issue of the classification of primary immunodeficiencies. In fact, there has never been a fully satisfactory classification of primary immunodeficiencies.1,30,31 This problem has become increasingly acute because of the explosion of knowledge in the field in the last 20 years, with at least 200 conditions described clinically and more than 100 disease-causing genes identified. Moreover, many more conventional and nonconventional primary immunodeficiencies are likely to be identified in the near future. A genetic classification was impossible in the early days before identification of the disease-causing genes. Even today, genetic classification would be hindered by the lack of a well-defined temporal and spatial expression pattern for the disease-causing genes, limiting our understanding of pathogenesis. Furthermore, different clinical syndromes might be caused by different mutations in the same gene, and the same syndrome might be caused by different genetic causes. Even if it were possible, a genetic classification would not actually be sufficient because phenotypes are obviously more important than genotypes; the chief value of a genotype lies in its ability to account for a given phenotype. Accordingly, the McKusick catalog of human genetic disorders is merely a catalog and not a classification.4 Any classification system for academic and clinical purposes must therefore be primarily phenotypic, although improvements in our understanding of the genetic basis of primary immunodeficiencies might lead to changes in phenotypic classification. The phenotypic definition of primary immunodeficiencies is clearly the necessary starting point for phenotypic classification. Historically, the identification of agammaglobulinemia in 1952 by Ogden Bruton32 and the subsequent discovery that its inheritance was X-linked and recessive33 was the origin of current classifications of primary immunodeficiencies on the basis of a combination of immunologic phenotypes and modes of inheritance.1,30,31,34-39 Primary immunodeficiencies are commonly classified into disorders of T cells, B cells, phagocytes, and complement. They are then further classified according to the mode of inheritance and, when known, genetic cause. This method of classification poses a serious problem of definition because it tightly links the concept of primary immunodeficiency with the observation of an immunologic phenotype. According to this view, even asymptomatic IgA-deficient individuals are immunodeficient, unlike, paradoxically, patients dying of infectious disease without immunologic abnormality. Moreover, because many disease-causing genes are expressed in different cell types in which mutant alleles might have different effects,

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clinical phenotypes, whether infectious, allergic, or autoimmune, are far from being consistent within each of the 4 cell type–based groups. What is the clinical similarity between defects of C1 inhibitor and C9? Conversely, X-linked agammaglobulinemia (primarily, but not exclusively, a B-cell defect) clinically resembles HLA class I deficiency (often improperly classified as a T-cell defect). This situation also results in paradoxical classifications, with an immunologic phenotype attributed to one cell type but a genetic defect actually affecting another cell type. For example, CD40L deficiency is generally described as a B-cell defect because of the hyper-IgM syndrome,40 but CD40L is expressed on T cells and is involved in the interaction of T cells with both B cells and macrophages– dendritic cells. The focus on certain immunologic phenotypes, such as hyper-IgM syndrome, is misleading in itself: there are more differences than common points between patients with mutations in CD40L (expressed on T cells), AID (expressed in B cells), and NEMO (expressed ubiquitously), all of which can result in hyperIgM syndrome. The current immunologic classification of primary immunodeficiencies is thus imperfect both immunologically and clinically. An ideal definition and classification of primary immunodeficiencies and inborn deficiencies should evidently rely on clinical phenotype because this best reflects the physiologic effect of any deleterious genotype. Indeed, immunodeficiencies in general, whether inherited or acquired, should be defined clinically, as opposed to immunologically. Would anyone seriously suggest that it would be better to define respiratory failure in terms of epithelial abnormalities rather than the physiologic consequences of insufficient oxygen inhalation? It is certainly useful to assess various parameters in the course of any organ failure, but the definition and monitoring of organ failure must be physiologic. Immunodeficiency is a failure to achieve immune function to provide efficient, self-limited host defense against the biotic and abiotic environment while preserving tolerance to self. Immunodeficiencies are thus best defined in terms of the diverse forms of lifethreatening infections, allergies, or autoimmune reactions. The detection of an identifiable immunologic abnormality is less important and depends on the tools available to the investigator. Immunodeficiencies might also be considered in terms of whether they are inherited or acquired. Most immunodeficiencies are actually idiosyncratic, reflecting both nature (genetic background) and nurture (the effect of the environment on the host). An ideal classification of primary immunodeficiencies should take this into account, considering infectious syndromes one by one (and possibly autoimmune and allergic syndromes as well). For example, primary immunodeficiencies associated with mycobacterial41 or pneumococcal42 diseases are specifically associated with defects of interaction between T-cells and phagocytes (involving in particular the IL-12/ 23–IFN-g circuit and the respiratory burst) and bacterial sensing and opsonization (involving mucosal inflammation, complement, carbohydrate-specific antibodies, and splenic macrophages), respectively. This classification is

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not operational yet because it awaits investigators in the field to review all pathogens one by one, perhaps in a collaborative effort (eg, primary immunodeficiencies associated with, for example, Pneumocystis species infection or Toxoplasma species infection). This classification will address the most relevant clinical question at the bedside (Which immunodeficiency should the physician consider in a given infected patient?) and the most relevant immunologic question at the bench (What role does a particular molecule play in immunity to infection in vivo?). Of course, the genotype and immunologic phenotype are invaluable both clinically to tailor treatment options to individual patients and immunologically to decipher the molecular basis of immune responses. A corollary of this purely clinical definition and classification of primary immunodeficiencies is that inborn Mendelian deficiencies of immunity are more common than initially thought. Accordingly, newly described primary immunodeficiencies, such as partial IFN-gR1 and signal transducer and activator of transcription 1 deficiencies, have been shown to be transmitted as autosomal dominant traits in multiplex families, at odds with the classical view that primary immunodeficiencies are necessarily recessive traits because of their severity.43 Not all severe infectious diseases will be found to reflect a Mendelian primary immunodeficiency or to be due to the inheritance of a major susceptibility gene, as seen in leprosy with mutations in Parkin,44-46 because predisposition to infection might display truly polygenic determinism. Nevertheless, it will be important in the future to decipher the Mendelian genetic basis of infectious diseases. Studies of autoimmune and allergic syndromes are also likely to reveal novel Mendelian disorders. Once a clinical definition of immunodeficiency is accepted, patients with infectious diseases, allergy, or autoimmunity (in the broad sense of these terms, including angioedema, hemophagocytosis, and autoinflammation) should be considered as potential bearers of Mendelian primary immunodeficiencies. Accordingly, several primary immunodeficiencies were recently shown to present purely as autoimmune,47 autoinflammatory,48 and hemophagocytosis49 syndromes. Intriguingly, only one Mendelian disorder, C1 inhibitor deficiency, has thus far been found to be purely associated with autosomal-dominant angioedema, a syndrome related to (but possibly different from) allergy.50 Noninfectious immunologic diseases have only recently emerged as a public health problem and do not threaten mankind as acutely as infections. Records show that life expectancy in Western Europe in the 18th century was about 25 years, whereas life expectancy is currently about 40 years in Sub-Saharan Africa, largely because of the burden of infection.17,51 The current longer life expectancy in developed countries primarily reflects recent developments in hygiene (preventing infection), vaccines (preventing disease), and antibiotics (preventing a fatal outcome), rather than the intrinsic efficiency of our immune system.51 Although the immune system serves well at the population level, ensuring the reproduction of species, it is the least efficient physiologic system at the

individual level. As indicated by medical and demographic data, most human subjects are immunodeficient and exposed to life-threatening infectious diseases.51 Many might carry a Mendelian primary immunodeficiency, being thus perhaps the rule rather than the exception and paradoxically raising hope for scientists, physicians, and patients. We thank Laurent Abel for critical reading of the manuscript and other members of the laboratory of Human Genetics of Infectious Diseases for helpful discussions. We also thank Ge´rard Orth for helpful discussions, and we thank 2 anonymous reviewers for their constructive criticisms.

REFERENCES 1. Notarangelo L, Casanova JL, Fischer A, Puck J, Rosen F, Seger R, et al. Primary immunodeficiency diseases: an update. J Allergy Clin Immunol 2004;114:677-87. 2. Bertrand Y, Muller SM, Casanova JL, Morgan G, Fischer A, Friedrich W. Reticular dysgenesis: HLA non-identical bone marrow transplants in a series of 10 patients. Bone Marrow Transplant 2002;29:759-62. 3. Casanova JL, Schurr E, Abel L, Skamene E. Forward genetics of infectious diseases: immunological impact. Trends Immunol 2002;23: 469-72. 4. McKusick VA. Mendelian inheritance in man. Catalogs of human genes and genetic disorders. Baltimore (MD): Johns Hopkins University Press; 1998. 5. Newport MJ, Huxley CM, Huston S, Hawrylowicz CM, Oostra BA, Williamson R, et al. A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 1996;335: 1941-9. 6. Jouanguy E, Altare F, Lamhamedi S, Revy P, Emile JF, Newport M, et al. Interferon-gamma-receptor deficiency in an infant with fatal bacille Calmette-Guerin infection. N Engl J Med 1996;335:1956-61. 7. Dorman SE, Holland SM. Interferon-gamma and interleukin-12 pathway defects and human disease. Cytokine Growth Factor Rev 2000;11: 321-33. 8. Casanova JL, Abel L. Genetic dissection of immunity to mycobacteria: the human model. Annu Rev Immunol 2002;20:581-620. 9. Wu¨rzner R, Orren A, Lachmann PJ. Inherited deficiencies of the terminal components of human complement. Immunodef Rev 1992;3: 123-47. 10. Wu¨rzner R, Witzel-Schlomp K, Tokunaga K, Fernie BA, Hobart MJ, Orren A. Reference typing report for complement components C6, C7 and C9 including mutations leading to deficiencies. Exp Clin Immunogenet 1998;15:268-85. 11. Kirkpatrick CH. Chronic mucocutaneous candidiasis. Pediatr Infect Dis J 2001;20:197-206. 12. Lilic D. New perspectives on the immunology of chronic mucocutaneous candidiasis. Curr Opin Infect Dis 2002;15:143-7. 13. Ramoz N, Rueda LA, Bouadjar B, Montoya LS, Orth G, Favre M. Mutations in two adjacent novel genes are associated with epidermodysplasia verruciformis. Nat Genet 2002;32:579-81. 14. Orth G. Human papillomaviruses and the skin: more to be learned. J Invest Dermatol 2004;123:XI-XIII. 15. Morra M, Howie D, Grande MS, Sayos J, Wang N, Wu C, et al. X-linked lymphoproliferative disease: a progressive immunodeficiency. Annu Rev Immunol 2001;19:657-82. 16. Nichols KE, Ma CS, Cannons JL, Schwartzberg PL, Tangye SG. Molecular and cellular pathogenesis of X-linked lymphoproliferative disease. Immunol Rev 2005;203:180-99. 17. Casanova JL, Abel L. The human model: a genetic dissection of immunity to infection in natural conditions. Nat Rev Immunol 2004;4: 55-66. 18. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, et al. Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 2003;299:2076-9.

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19. Ku CL, Yang K, Bustamante J, Puel A, von Bernuth H, Dos Santos O, et al. Inherited disorders of human Toll-like receptor signalling: immunological implications. Immunol Rev 2005;203:10-20. 20. Fieschi C, Casanova JL. The role of interleukin-12 in human infectious diseases: only a faint signature. Eur J Immunol 2003;33:1461-4. 21. Lejeune J, Gautier M, Turpin R. [Study of somatic chromosomes from 9 mongoloid children]. C R Hebd Seances Acad Sci 1959;248:1721-2. 22. Casanova JL, Jouanguy E, Lamhamedi S, Blanche S, Fischer A. Immunological conditions of children with BCG disseminated infection. Lancet 1995;346:581. 23. Casanova JL, Blanche S, Emile JF, Jouanguy E, Lamhamedi S, Altare F, et al. Idiopathic disseminated bacillus Calmette-Guerin infection: a French national retrospective study. Pediatrics 1996;98: 774-8. 24. Altare F, Ensser A, Breiman A, Reichenbach J, Baghdadi JE, Fischer A, et al. Interleukin-12 receptor beta1 deficiency in a patient with abdominal tuberculosis. J Infect Dis 2001;184:231-6. 25. Caragol I, Raspall M, Fieschi C, Feinberg J, Larrosa MN, Hernandez M, et al. Clinical tuberculosis in 2 of 3 siblings with interleukin-12 receptor beta1 deficiency. Clin Infect Dis 2003;37:302-6. ¨ zbek N, Fieschi C, Yilmaz BT, De Beaucoudrey L, Bikmaz YE, 26. O Feinberg J, et al. Interleukin-12 receptor beta 1 chain deficiency in a child with disseminated tuberculosis. Clin Infect Dis 2005;40:e55-8. 27. Fieschi C, Dupuis S, Catherinot E, Feinberg J, Bustamante J, Breiman A, et al. Low penetrance, broad resistance, and favorable outcome of interleukin 12 receptor beta1 deficiency: medical and immunological implications. J Exp Med 2003;197:527-35. 28. Dupuis S, Jouanguy E, Al-Hajjar S, Fieschi C, Al-Mohsen IZ, Al-Jumaah S, et al. Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency. Nat Genet 2003; 33:388-91. 29. Niehues T, Reichenbach J, Neubert J, Gudowius S, Puel A, Horneff G, et al. A NEMO-deficient child with immunodeficiency yet without anhidrotic ectodermal dysplasia. J Allergy Clin Immunol 2004;114: 1456-62. 30. Stiehm ER, Ochs HD, Winkelstein JA. Immunologic disorders in infants and children. Philadelphia: Elsevier Saunders; 2004. 31. Ochs H, Smith CIE, Puck J. Primary Immunodeficiencies: a molecular and genetic approach. New York: Oxford University Press; 2005. 32. Bruton OC. Agammaglobulinemia. Pediatrics 1952;9:722-8. 33. Bruton OC. A decade with agammaglobulinemia. J Pediatr 1962;60: 672-6. 34. Good RA. Historical aspects of immunologic deficiency diseases. In: Kagen BM, Stiehm ER, editors. Immunologic incompetence. Chicago: Year Book Medical Publishing; 1971. p. 149-77. 35. Hitzig WH. The discovery of agammaglobulinaemia in 1952. Eur J Pediatr 2003;162:289-304. 36. Stiehm ER, Johnston RB Jr. A history of pediatric immunology. Pediatr Res. In press 2005. 37. Stiehm ER. New and old immunodeficiencies. Pediatr Res 1993; 33(suppl):S2-8. 38. Fischer A. Primary immunodeficiency diseases: an experimental model for molecular medicine. Lancet 2001;357:1863-9. 39. Conley ME. Molecular basis of immunodeficiency. Immunol Rev 2005; 203:5-9. 40. Etzioni A, Ochs HD. The hyper IgM syndrome—an evolving story. Pediatr Res 2004;56:519-25. 41. Reichenbach J, Rosenzweig S, Doffinger R, Dupuis S, Holland SM, Casanova JL. Mycobacterial diseases in primary immunodeficiencies. Curr Opin Allergy Clin Immunol 2001;1:503-11. 42. Picard C, Puel A, Ku CL, Casanova JL. Primary immunodeficiencies associated with pneumococcal disease. Curr Opin Allergy Clin Immunol 2003;3:451-9. 43. Lawrence T, Puel A, Reichenbach J, Ku CL, Chapgier A, Renner E, et al. Autosomal-dominant primary immunodeficiencies. Curr Opin Hematol 2005;12:22-30. 44. Mira MT, Alcais A, Nguyen VT, Moraes MO, Di Flumeri C, Vu HT, et al. Susceptibility to leprosy is associated with PARK2 and PACRG. Nature 2004;427:636-40. 45. Mira MT, Alcais A, Van Thuc N, Thai VH, Huong NT, Ba NN, et al. Chromosome 6q25 is linked to susceptibility to leprosy in a Vietnamese population. Nat Genet 2003;33:412-5.

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46. Alcais A, Mira M, Casanova JL, Schurr E, Abel L. Genetic dissection of immunity in leprosy. Curr Opin Immunol 2005;17:44-8. 47. Arkwright PD, Abinun M, Cant AJ. Autoimmunity in human primary immunodeficiency diseases. Blood 2002;99:2694-702. 48. Hull KM, Shoham N, Chae JJ, Aksentijevich I, Kastner DL. The expanding spectrum of systemic autoinflammatory disorders and their rheumatic manifestations. Curr Opin Rheumatol 2003;15:61-9.

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49. de Saint Basile G, Fischer A. Defective cytotoxic granule-mediated cell death pathway impairs T lymphocyte homeostasis. Curr Opin Rheumatol 2003;15:436-45. 50. Davis AE 3rd. The pathophysiology of hereditary angioedema. Clin Immunol 2005;114:3-9. 51. Cairns J. Matters of life and death. Princeton: Princeton University Press; 1997.

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Original articles Infant home endotoxin is associated with reduced allergen-stimulated lymphocyte proliferation and IL-13 production in childhood Joseph H. Abraham, ScD,a,b,c Patricia W. Finn, MD,d Donald K. Milton, MD,b,c Louise M. Ryan, PhD,e David L. Perkins, MD,d and Diane R. Gold, MDb,c Boston, Mass

Key words: Endotoxin, lymphocyte proliferation, cytokine, childhood, allergy From the Departments of aEpidemiology, bEnvironmental Health, and e Biostatistics, Harvard School of Public Health, and cThe Channing Laboratory, Department of Medicine, and dthe Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School. Disclosure of potential conflict of interest: None disclosed. Supported by NIEHS R01 ES-07036; NIEHS 2P30ES00002; NIH/NHLBI HL07427-23; and AI/EHS35786. Received for publication December 10, 2004; revised March 28, 2005; accepted for publication May 9, 2005. Available online July 5, 2005. Reprint requests: Diane R. Gold, MD, Channing Laboratory, 181 Longwood Ave, Boston, MA 02115. E-mail: [email protected] 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.05.015

Abbreviations used EU: Endotoxin units OR: Odds ratio SI: Stimulation index TLR4: Toll-like receptor 4

Childhood allergic diseases, such as asthma and hay fever, are increasing in prevalence, cause chronic ill health, and are a substantial public health concern in developed countries.1,2 Measurable childhood sensitization to inhaled allergens occurs primarily after the age of 3 years,3 but the immunologic underpinnings of allergic disease and airway inflammation likely develop far earlier in life.4,5 Manifestation of an allergic phenotype likely results from the complex interplay of genetic, developmental, and environmental influences. Adaptive immune processes, including activation of helper T lymphocytes and subsequent B-lymphocyte activation with IgE isotype switching, underlie the process of allergic sensitization in individuals genetically predisposed to development of allergic responses.6 Although still controversial,7,8 there is mounting evidence from animal models9-13 and the epidemiologic literature14,15 suggesting that exposure to endotoxin, a potent activator of innate immunity, might influence subsequent adaptive immune responses to allergen. Furthermore, these studies suggest that the timing and dose of endotoxin exposure influence the nature of the immune response, leading to the hypothesis that early life might be a crucial time window during which endotoxin might reduce the risk of allergy through its influence on innate immunity and downstream T-cell and B-cell regulation of cytokine and IgE expression. The immunologic pathway linking endotoxin exposure to adaptive immunity and evidence for its effects on the development of allergic diseases have recently been reviewed.7,16,17 Briefly, endotoxin is biologically active LPS, a primary component of the outer cell membrane of gram-negative bacteria.18,19 Even minute amounts of endotoxin provoke innate immune responses in vitro and in vivo.20 The nature of that response, which is only partially understood, might depend on the developmental 431

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Background: Infant endotoxin exposure has been proposed as a factor that might protect against allergy and the early childhood immune responses that increase the risk of IgE production to allergens. Objective: Using a prospective study design, we tested the hypothesis that early-life endotoxin exposure is associated with allergen- and mitogen-induced cytokine production and proliferative responses of PBMCs isolated from infants with a parental history of physician-diagnosed asthma or allergy. Methods: We assessed household dust endotoxin at age 2 to 3 months and PBMC proliferative and cytokine responses to cockroach allergen (Bla g 2), dust mite allergen (Der f 1), cat allergen (Fel d 1), and the nonspecific mitogen PHA at age 2 to 3 years. Results: We found that increased endotoxin levels were associated with decreased IL-13 levels in response to cockroach, dust mite, and cat allergens, but not mitogen stimulation. Endotoxin levels were not correlated with allergen- or mitogeninduced IFN-g, TNF-a, or IL-10. Increased endotoxin levels were associated with decreased lymphocyte proliferation after cockroach allergen stimulation. An inverse, although nonsignificant, association was also found between endotoxin and proliferation to the other tested stimuli. Conclusion: Increased early-life exposure to household endotoxin was associated with reduced allergen-induced production of the TH2 cytokine IL-13 and reduced lymphoproliferative responses at age 2 to 3 years in children at risk for allergy and asthma. Early-life endotoxin-related reduction of IL-13 production might represent one pathway through which increased endotoxin decreases the risk of allergic disease and allergy in later childhood. (J Allergy Clin Immunol 2005;116:431-7.)

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stage of the organism (eg, infant or adult) and on the sequence and mode of exposure to endotoxin and allergen. Inhaled endotoxin activates the innate immune system by binding transmembrane toll-like receptors (TLRs) expressed on macrophages and dendritic cells21 through IL-12 signaling.22 Given the right timing of exposure and genotype, these endotoxin-activated antigen-presenting cells might stimulate production of IFN-g and other TH1 cytokines.23 Through production of IFN-g by TH1-biased lymphocytes or through alternative pathways, endotoxin might downregulate TH2 cytokine (including IL-13) secretion, IgE production, and consequent allergic disease. Although studied extensively in animal models, few prospective data are available on the effects of home LPS exposure on cytokine production in young children. Using a prospective birth cohort with a parental history of allergy or asthma, we explored the hypothesis that early-life endotoxin exposure alters immune system responsiveness by examining the relationship between house dust endotoxin and PBMC responses to allergen stimulation. Specifically, we examined associations between house dust endotoxin levels measured 2 to 3 months after the child’s birth and allergen- and mitogen-induced cytokine and proliferative responses of mononuclear cells isolated from peripheral blood sampled at age 2 to 3 years. Previously, we had found that increased endotoxin levels in infancy were associated with decreased risk of eczema in the first year of life, suggesting that endotoxin might have a protective effect against allergic disease and the biologic pathways influencing the risk of allergy.24 We hypothesized that endotoxin levels would be positively correlated with levels of IFN-g, a TH1 cytokine, and inversely associated with levels of the TH2 cytokine IL-13, which can mediate isotype switching to IgE.25 We further hypothesized that endotoxin levels would be associated with decreased proliferative responses after allergen stimulation.

METHODS Description of cohort

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The Epidemiology of Home Allergens and Asthma study is a longitudinal birth cohort study of environmental predictors of allergy and asthma development. A description of the recruitment of study participants and study protocol has been previously published.26 In brief, 505 children from 499 families with a parental history of asthma or allergy were enrolled in a birth cohort study designed to examine the effects of allergen exposure in early life on the development of asthma. The Brigham and Women’s Hospital Human Research Committee approved the study. Informed consent was obtained from the parents for blood collection and longitudinal follow-up. Mothers in the greater Boston metropolitan area delivered at a large Boston hospital were screened with the following questions: (1) Have you ever had asthma, hay fever, or allergies? (2) Has the biologic father of your child ever had asthma, hay fever, or allergies? Mothers responding yes to either question were asked to complete a screening questionnaire. Families were not approached if the index child was premature, had a major congenital anomaly, or was in the neonatal intensive care unit or if the mother was less than 18 years old or could not speak English or Spanish. Informed consent was obtained from

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the parents for blood collection and longitudinal follow-up. The Brigham and Women’s Hospital Institutional Review Board approved the study protocol.

Environmental sampling and endotoxin measurements The home sampling protocol has been described previously.27 A trained research assistant visited participants’ homes within 2 to 3 months of the child’s birth. During these visits, conducted between 1994 and 1996, detailed demographic, socioeconomic, parental disease history, and home characteristics questionnaires were completed, and standardized dust sampling was conducted in various sites within the home, including the family room. Dust samples to be used for endotoxin assays were stored desiccated at 220°C until extraction. Endotoxin activity of dust samples was determined by using the kinetic Limulus amebocyte lysate assay with resistant-parallel-line estimation, as previously described.28-30 The Limulus amebocyte lysate was supplied by BioWhittaker (Walkersville, Md). Reference standard endotoxin was obtained from the United States Pharmacopoeia, Inc (Rockville, Md), and control standard endotoxin was supplied by Associates of Cape Cod (Woods Hole, Mass). Results were reported in endotoxin units (EU) per milligram of dust adjusted to account for lot-to-lot variation in Limulus amebocyte lysate sensitivity to house dust endotoxin and referenced to the reference standard endotoxin EC5 and EC6 (US Pharmacopoeia, Inc; 1 ng of EC5 and EC6 = 10 EU).30 Because of the prioritization schema for assaying dust samples, the availability of endotoxin measurements was conditional on there being sufficient dust to first assay for home allergens and fungi. As such, for 19 of the 115 subjects with biomarker outcome data, family room dust endotoxin levels were unavailable.

PBMC responses At 2 to 3 years of age, blood sampling and analysis was conducted in a subgroup of the study participants (n = 115). As previously described, selection of this subgroup was based on the home allergen levels measured during the initial home visit.31,32 The goal in choosing these subjects was to maximize variability in early-life exposure to the allergens with which their cells were to be stimulated. PBMCs were isolated from this blood sample by using FicollHypaque centrifugation.33 Fresh cells were incubated in media; media containing either 30 mg/mL cockroach allergen (Bla g 2), 30 mg/mL house dust mite allergen (Der f 1), or 1000 U/mL cat allergen (Fel d 1); or media plus 10 mg/mL PHA. Optimal stimulant concentrations used for the assay were determined in a prior doseresponse analysis.31 At 24 and 60 hours after the initiation of stimulation, supernatants were harvested, and cytokine concentrations were quantified by means of ELISA (Endogen, Woburn, Mass). On the basis of prior optimization for detection of cytokine levels, IL-10 and TNF-a were measured in the 24-hour samples, and IFN-g and IL-13 were measured in the 60-hour samples. The lower limits of detection for cytokine assays were as follows: IFN-g, less than 2 pg/mL; IL-13, less than 7 pg/mL; IL-10, less than 3 pg/mL; and TNF-a, less than 5 pg/mL. Because supernatant quantities were limited, cytokine assays were prioritized, with IFN-g levels being measured first. As a result, fewer subjects have observations of IL-13, IL-10, and TNF-a levels.32 After incubation of PBMCs with allergen or mitogen for 72 hours, 1 mCi of tritiated thymidine was added to each well. After incubation for an additional 8 hours, the cells were harvested, and tritiated thymidine uptake was determined by means of b-counting.

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TABLE I. Characteristics of children in the cohort

Variable

Sex, n Male Female Race-ethnicity, n White or Asian Other Income, n <$50,000
$50,000 Family room Bla g (1 or 2), n
0.05 U/g <0.05 U/g Family room Der f 1, n
2 mg/g <2 mg/g Family room Fel d 1, n
1 mg/g <1 mg/g Cold before blood draw, n Yes No

Subjects with measurement of endotoxin and PBMC outcomes* (n = 96)

Subjects with no measurement of both endotoxin and PBMC outcomes (n = 402)

Total (n = 498)

60 (63%) 36 (38%)

208 (52%) 194 (48%)

268 (54%) 230 (46%)

79 (82%) 17 (18%)

324 (81%) 78 (19%)

403 (81%) 95 (19%)

27 (28%) 68 (71%)

106 (26%) 283 (70%)

133 (27%) 351 (70%)

25 (26%) 69 (72%)

84 (21%) 291 (72%)

109 (22%) 360 (72%)

43 (45%) 53 (55%)

190 (47%) 206 (51%)

233 (47%) 259 (52%)

61 (64%) 34 (35%)

234 (58%) 147 (37%)

295 (59%) 181 (36%)

24 (25%) 71 (75%)

– –

– –

*The total number varied according to specific outcomes, but 96 children had endotoxin and lymphocyte proliferation data.

SI ¼ðMean valueof tritiatedthymidineuptakeforstimulatedsamplesÞ= ðMean valueof tritiatedthymidineuptakeforunstimulatedsamplesÞ:31

IgE measurements Serum samples were assayed for total IgE and specific IgE antibodies to dust mite, cat, cockroach, and ovalbumin by using the UNICAP System (Pharmacia, Uppsala, Sweden). IgE increase was defined either as an IgE specific response (
0.35 IU/mL) to at least one allergen or a total IgE level of greater than 60 IU/mL for age 2 to 3 years.

Statistical methods All statistical analyses were conducted with SAS version 8.2 (SAS Institute Inc, Cary, NC). In primary analyses investigating lymphoproliferative response as an outcome, both endotoxin and SI were treated as continuous variables. The distribution of SIs and endotoxin levels were log10 transformed for linear regression analyses to improve the normality of residuals and facilitate interpretation of model results. The relationships between log10-transformed SIs, log10-transformed endotoxin, and potential covariates were assessed by using ordinary least-squares univariate and multivariate regression models. The estimates for the relationship of the log of endotoxin to SI were used to calculate the percentage difference in SI for a doubling of endotoxin. This enabled us to translate the estimates into an easier-to-understand outcome that was scaled by a realistic (within the range of our data) increase in endotoxin level. We also considered SI as a dichotomous categorical by using cutoff points that have been considered as positive PBMC responses to allergen or mitogen stimuli in other studies (allergen responses, SI > 3; PHA responses, SI > 153).31

Our focus on the relation of endotoxin to IL-13 production presented statistical challenges because of the skewed distribution of the cytokine levels and because of the significant proportion of values below the limit of detection. First, we used nonparametric correlation analyses to relate continuous dust endotoxin with continuous cytokine levels. For nonparametric testing, cytokine observations below the lower limits of detection were assigned very low but nonmissing values (0.001 pg/mL) to be included in the analysis and tested the sensitivity of our findings to the choice of number assigned to the observations below the limit of detection. We then assessed the relation between endotoxin and the odds of having measurable IL-13. Finally, in secondary analyses we tested for trends in the proportion of cytokine observations above the lower limit of detection when grouped by tertile of endotoxin level. Cockroach (Bla g 1 or 2), dust mite (Der f 1), and cat (Fel d 1) allergen levels were classified as follows: Bla g 1 or 2 of 0.05 U/g or greater, Fel d 1 of 1 mg/g or greater, and Der f 1 of 2 mg/g or greater).31 Reported household income was classified as being less than or greater than $50,000.

RESULTS Of the 505 children initially enrolled in the study, 7 were followed for less than 5 months in the first year of life. Of the 498 with follow-ups, 115 had lymphocyte proliferation measurements at age 2 to 3 years, of whom 96 also had family room dust endotoxin measurements. Compared with the 402 subjects without both lymphocyte proliferation and endotoxin data, the subsample with both (n = 96) had a greater proportion of boys (Table I). No other selection bias was detected. Endotoxin in the 96 family room dust samples had a geometric mean of 95 EU/mg (geometric SD, 1.8 EU/mg) and a median level of

Basic and clinical immunology

Proliferation was quantified by calculating the stimulation index (SI) for each stimulant with the following formula:

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TABLE II. Distribution of endotoxin, IL-13, and PBMC lymphocyte proliferative response levels* Variable

Endotoxin (EU/mg), n = 96 IL-13 (pg/mg), n = 67 Bla g 2 Der f 1 Fel d 1 PHA SI Bla g 2 Der f 1 Fel d 1 PHA

Minimum

Median

75th Percentile

Maximum

Detectable, n

19.3

101.2

138.4

518.8

96 (100%)



9.6 30.5 4.7 2255

207.2 397.3 137.6 3812

32 47 26 64

(48%) (70%) (39%) (96%)

0.9 0.4 0.3 14.4

4.1 3.3 2.2 138.6

5.7 5.9 4.4 285.5

53.6 32.5 27.5 1205.1

94 95 96 96

(100%) (100%) (100%) (100%)

< LD, Below the limits of detection. *For the purpose of ranking/nonparametric testing, values below the limits of detection were assigned the value 0.001 pg/mL.

101 EU/mg (Table II). The distributions of lymphocyte proliferation and cytokine levels for this subsample were similar to those previously demonstrated for the larger sample (Table II).31,32 We found no confounders of the association of endotoxin with either the lymphoproliferative response or the cytokine production by stimulated PBMCs after considering living room allergen level, report of dog or cat, raceethnicity, household income, or cold before blood draw (data not shown). As previously reported for the larger cohort, cockroach allergen (Bla g 1 or 2), cat allergen (Fel d 1), and dust mite allergen (Der f 1) levels measured in family room dust samples were not significantly associated with home endotoxin levels.34

Basic and clinical immunology

Association of house dust endotoxin and SIs We observed a consistent association between increased dust endotoxin measured in the first months of a baby’s life and decreased SIs at age 2 to 3 years, although this association was only significant for the response to cockroach allergen (Table III). Despite the absence of measurable confounders, to adjust for potential independent influences on the lymphoproliferative responses, we adjusted for indicators of age at the time of blood draw, report of a cold in the week before blood draw, household income level, and house dust allergen levels in the allergenspecific proliferation models in addition to reporting the univariate models.31 The multivariate-adjusted relationships between endotoxin and the SIs ranged from 24% to 217%, being strongest and statistically significant for the SI after cockroach allergen stimulation. When dichotomized at previously defined cutoff points,31 positive cockroach-induced lymphocyte proliferative responses (SI > 3) were also inversely associated with endotoxin levels (odds ratio [OR] of Bla g 2 SI
3 for a doubling in family room endotoxin level: OR, 0.53; 95% CI, 0.271.049; P = .07). Although the estimates for Der f 1 SI were negative, the analyses using SI as a dichotomous outcome were not supportive of a significant association of endotoxin with a lower odds of Der f 1 SI of greater than 3 (P = .9) or Fel d 1 SI of greater than 3 (P = .7).

Association of house dust endotoxin and stimulation-induced cytokine production By using rank-based correlations, increasing house dust endotoxin levels measured early in life were inversely correlated with allergen-induced IL-13 but not IL-13 induced by PHA (Table IV). For a doubling of endotoxin levels, children had significantly reduced odds of having measurable-detectable allergen-induced IL-13 levels in response to all 3 allergens, with no association between endotoxin- and PHA-induced IL-13 (Table V). With increasing tertiles of endotoxin, a smaller percentage of children had measurable allergen-induced IL-13 levels (above the limits of detection), although this trend was only statistically significant for the response to Der f 1 (see Fig E1 in the Online Repository in the online version of this article at www.mosby.com/jaci). We did not observe correlations between allergen- or mitogen-induced levels of IFN-g, TNF-a, and IL-10 in the supernatants of the isolated PBMCs and endotoxin (data not shown). We found no evidence of an association between PHAinduced IL-13 and endotoxin. DISCUSSION In this prospective cohort study of young children with a family history of allergic disease, we observed that increased levels of family room dust endotoxin in infancy were associated with decreased allergen-induced IL-13 production by mononuclear cells isolated from peripheral blood at age 2 to 3 years. We also found that endotoxin levels were associated with a downregulation of allergen- and mitogen-stimulated lymphocyte proliferation. Through the interactions of its Lipid A moiety with receptors such as TLR4, endotoxin is hypothesized to modulate the innate immunity pathway and, through those pathways, to influence adaptive immunity16,17 with reduced production of cytokines, such as IL-13, as seen in our study. This in turn might result in subsequent reduction of IgE levels, allergy, and allergic disease. In our cohort at this age, we have previously demonstrated an

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TABLE III. Percentage change in lymphocyte proliferative response (SI) for a doubling of family room dust endotoxin levels Univariate* Stimulus

Cockroach allergen (Bla g 2) Dust mite allergen (Der f 1) Cat allergen (Fel d 1) PHA

% Difference

215 213 25 211

Multivariatey 95% CI

227 230 221 228

to to to to

21 8 15 10

% Difference

217 215 24 29

95% CI

228 231 221 227

to to to to

24 5 16 12

association between lower IL-13 levels and reduced risk of allergy/increased IgE levels.32 Increased endotoxin levels were also associated with reduced risk of allergy, but this association was not statistically significant. A doubling of endotoxin in family room dust was associated with an OR of 0.71 (95% CI, 0.39-1.31) for increased IgE levels, as determined by positive RAST results or high total IgE levels among 97 children with data on endotoxin in early life and IgE levels at age 2 to 3 years. In a previous report from this study, we demonstrated that higher infant endotoxin levels were associated with a significantly lower risk of eczema in the first year of life.24 The data we present in this article support the hypothesis that the association of increased home endotoxin exposure with protection against childhood allergic disease might be mediated in part through reduced TH2 cytokine expression in early life. Researchers in Europe have observed an association between a farming lifestyle and lower prevalence of allergic disease in children and hypothesized that increased exposure to endotoxin in infancy might be responsible for this association through an early-life influence on innate immune development and its relation to adaptive immunity, cytokine production, and IgE production.14,35,36 In a cross-sectional study of German, Austrian, and Swiss children ages 6 to 13 years, endotoxin measured in the dust of children’s mattresses was inversely associated with asthma, atopic sensitization, and hay fever.37 Increased bed dust endotoxin levels were associated with decreased LPS-induced production of IFN-g, IL-10, IL-12, and TNF-a. The researchers interpreted these findings as a global downregulation of the immune response. In contrast, we found a specific endotoxin-related downregulation of a TH2 cytokine, IL-13, which can mediate isotype switching to IgE25,38 without an influence on IFN-g, IL-10, or TNF-a production. Our study contrasts with the multicenter European study in many ways. We stimulated our PBMCs with allergen, our study was prospective, our cohort was predisposed to allergy by virtue of family history, and our endotoxin levels, measured in infancy, were relatively low. In a small US cross-sectional study of infants with repeated wheeze, allergen-sensitized children came from homes with higher endotoxin levels, and higher home endotoxin levels were correlated with increased

proportions of IFN-g–producing CD4 T cells.15 In this study the investigators did not evaluate cytokine secretion of lymphocyte proliferative response of antigen- or mitogen-stimulated PBMCs. Although this is, to the best of our knowledge, the first report of a prospective association of endotoxin with diminished IL-13 production in children, some animal models have demonstrated similar associations. However, in animal models endotoxin exposure with TLR4 agonists has been shown to both diminish and enhance IFN-g production, IL-13 production, and allergic responses.12 The directionality and nature of the immune and allergic responses appears to be dependent on many factors, including the type of model, dose of allergen or TLR4 agonist, timing of allergen or TLR4 agonist, use or not of adjuvant, and type of species or murine strain.9,11,39-42 In one murine model prenatal exposure to LPS resulted in increased neonatal IFN-g secretion and decreased neonatal IL-13 production after OVA exposure, with no protection against airway responsiveness.43 In another murine model, Velasco et al12 showed that pulmonary administration of Lipid A before allergen sensitization decreased eosinophilia, bronchoalveolar lavage fluid IL-13 levels, serum IgE levels, airway hyperresponsiveness, and the number of CD41 cells in the lung. Lipid A administration during allergen challenge diminished eosinophilia. In contrast, Delayre-Orthez et al42 showed that LPS during challenge enhanced allergen-induced eosinophilia but did not enhance allergen-induced IgE or airway hyperresponsiveness. A recent study compared the effects of Lipid A in terms of maturity of the immune system, species, dose, and timing by examination of human cord and adult PBMCs and murine cells in vitro and in vivo.13 Lipid A induced primarily a time- and dose-independent production of IFN-g. In both human and animal models, the effects of environmental endotoxin exposure, are likely to be dependent on the dose and timing of exposure and on the developmental stage and genotype.44 These factors might explain seemingly contradictory epidemiologic findings of endotoxin effects on allergic disease.7,16 As an irritant, in those with or without asthma, endotoxin might increase the severity of symptoms.31,45 Previously, we found that endotoxin was associated with increased risk of wheeze

Basic and clinical immunology

*The percentage difference in SI for a doubling of endotoxin was calculated as follows: ð10ˆ ½log10 ð2Þ  ðbÞ21Þ  100, where b is the endotoxin effect estimate for the univariate model: log10 ðSIÞ ¼ b0 1b1 ½log10 ðEndotoxinÞ. Also see the Methods section.  Same as the univariate model: adjusted for age at blood draw, report of a cold before blood draw, household income, and family room dust allergen level for allergen-induced proliferation models.

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TABLE IV. Correlations between allergen- and mitogeninduced cytokine levels and family room dust endotoxin levels* Stimulus

Bla g 2

Der f 1

Fel d 1

PHA

Cytokine (pg/mL)

IFN-g IL-13 IL-10 TNF-a IFN-g IL-13 IL-10 TNF-a IFN-g IL-13 IL-10 TNF-a IFN-g IL-13 IL-10 TNF-a

Obs.

Correlation coefficient

P value

80 67 64 50 85 67 69 50 80 67 64 50 85 67 69 50

20.05 20.31 0.11 20.01 0.01 20.27 20.08 20.09 0.06 20.21 20.02 20.03 0.08 0.12 20.05 20.14

.66 .01 .38 .94 .91 .02 .51 .54 .57 .08 .89 .83 .46 .34 .67 .35

TABLE V. Relative odds of detectable versus nondetectable allergen- and mitogen-induced IL-13 for a doubling of family room endotoxin levels Cytokine

Obs., Number of observations. *Rank-based (Spearman) correlation coefficients.

Basic and clinical immunology

and repeated wheeze in the first year of life in this cohort; this might be secondary to its irritant effects.29 In the siblings of the subjects in this cohort, the association of endotoxin with wheeze decreased over time, and as the child grew older, earlier-life endotoxin appears to have a null or even protective effect on wheeze risk, perhaps because the wheeze was more related to allergic inflammation.46 This is consistent with the hypothesis that endotoxin might increase the risk of irritant wheeze at the same time as decreasing the risk of allergy and its pulmonary and extrapulmonary manifestations. In their studies of children growing up on farms, Braun-Fahrlander et al37 have found consistent protective effects of endotoxin on allergy but an increased risk of wheeze with increasing endotoxin levels among those without allergy. We had limited power to fully elucidate pathways leading from endotoxin exposure to cytokine production, early allergy, and subsequent allergic disease expression. In this group of young children, when allergy, particularly allergy to inhaled allergens, is just developing, we found that increased levels of endotoxin were associated with a lower risk of high IgE levels but that association was not statistically significant. This might be due to small numbers (low power) because we were only able to measure both endotoxin and cytokines on a subset of the cohort or due to the age of the children because the allergic phenotype is not fully evolved by age 2 to 3 years. It is quite possible that a skewing toward TH2 cytokine production is an earlier step in the pathway toward full expression of the allergic phenotype. The associations between observed family room dust endotoxin and allergenand mitogen-induced proliferation of lymphocytes were small and achieved statistical significance only after stimulation with cockroach allergen. Therefore we cannot

Stimulus

Obs.

OR

95% CI

67 67 67 67

0.36 0.27 0.44 0.89

0.16-0.81 0.11-0.70 0.20-0.94 0.19-4.17

IL-13 Bla g 2 Der f 1 Fel d 1 PHA Obs., Number of observations.

exclude the role of chance in determining the observed association between endotoxin and proliferation after stimulation with the other allergens and the mitogen. However, the consistently negative associations suggest that this is not a chance observation. Although confounding bias by unmeasured factors is always a possibility, we found no confounding by other measured exposures (including dog) in our analyses evaluating the association of endotoxin with detectable IL-13, and we adjusted for confounders in our analyses, with SI as our outcome. Ideally, an estimate of each subject’s endotoxin exposure would capture the true temporally and spatially integrated exposure that occurs within the first year of life. Our single measure of endotoxin in the home is likely to represent this true exposure with error. Because of the prospective study design, differences between the observed measurements of endotoxin and the subjects’ true endotoxin exposure are most likely nondifferential with respect to PBMC responses. This measurement error would most likely have attenuated the exposure effect estimates. A final limitation is that we did not evaluate the allergens that we used to stimulate the PBMCs for the possible presence of endotoxin. Production of IL-13 is likely to be less influenced by trace LPS than production of cytokines involved in innate immunity, such as TNF-a. The possibility remains, however, that increased home allergen levels in infancy are associated with reduced IL-13 production not only in response to allergens but also in response to a combination of allergen and LPS. In conclusion, in a cohort of children at risk of allergy and asthma, we conducted a prospective study on the relation of home endotoxin levels in infancy to subsequent allergen-stimulated lymphocyte proliferative responses and cytokine production. Our data suggest that increased early-life endotoxin exposure might be associated with a reduction in subsequent allergen-stimulated lymphocyte proliferation and IL-13 secretion. Endotoxin-induced reduction in IL-13 secretion might be one early-life step in the pathway to endotoxin-associated reduction in allergy and allergic disease.

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2. Holgate ST. The epidemic of allergy and asthma. Nature 1999;402:B2-4. 3. Kulig M, Bergmann R, Klettke U, Wahn V, Tacke U, Wahn U. Natural course of sensitization to food and inhalant allergens during the first 6 years of life. J Allergy Clin Immunol 1999;103:1173-9. 4. Croner S, Kjellman NI. Natural history of bronchial asthma in childhood. A prospective study from birth up to 12-14 years of age. Allergy 1992; 47:150-7. 5. Wahn U, von Mutius E. Childhood risk factors for atopy and the importance of early intervention. J Allergy Clin Immunol 2001;107: 567-74. 6. Donovan CE, Finn PW. Immune mechanisms of childhood asthma. Thorax 1999;54:938-46. 7. Liu AH. Endotoxin exposure in allergy and asthma: reconciling a paradox. J Allergy Clin Immunol 2002;109:379-92. 8. Douwes J, Pearce N, Heederik D. Does environmental endotoxin exposure prevent asthma? Thorax 2002;57:86-90. 9. Tulic MK, Wale JL, Holt PG, Sly PD. Modification of the inflammatory response to allergen challenge after exposure to bacterial lipopolysaccharide. Am J Respir Cell Mol Biol 2000;22:604-12. 10. Wan GH, Li CS, Lin RH. Airborne endotoxin exposure and the development of airway antigen-specific allergic responses. Clin Exp Allergy 2000;30:426-32. 11. Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K. Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 2002; 196:1645-51. 12. Velasco G, Campo M, Manrique OJ, Bellou A, He H, Arestides RS, et al. Toll-like receptor 4 or 2 agonists decrease allergic inflammation. Am J Respir Cell Mol Biol 2005;32:218-24. 13. Schaub B, Bellou A, Gibbons FK, Velasco G, Campo M, He H, et al. TLR2 and TLR4 stimulation differentially induce cytokine secretion in human neonatal, adult, and murine mononuclear cells. J Interferon Cytokine Res 2004;24:543-52. 14. Von Ehrenstein OS, Von Mutius E, Illi S, Baumann L, Bohm O, von Kries R. Reduced risk of hay fever and asthma among children of farmers. Clin Exp Allergy 2000;30:187-93. 15. Gereda JE, Leung DY, Thatayatikom A, Streib JE, Price MR, Klinnert MD, et al. Relation between house-dust endotoxin exposure, type 1 T-cell development, and allergen sensitisation in infants at high risk of asthma. Lancet 2000;355:1680-3. 16. Reed CE, Milton DK. Endotoxin-stimulated innate immunity: a contributing factor for asthma. J Allergy Clin Immunol 2001;108:157-66. 17. Bellou A, Schaub B, Ting L, Finn PW. Toll receptors modulate allergic responses: interaction with dendritic cells, T cells and mast cells. Curr Opin Allergy Clin Immunol 2003;3:487-94. 18. Rietschel ET, Brade H. Bacterial endotoxins. Sci Am 1992;267:54-61. 19. Reeves PR, Hobbs M, Valvano MA, Skurnik M, Whitfield C, Coplin D, et al. Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol 1996;4:495-503. 20. Imrich A, Ning YY, Koziel H, Coull B, Kobzik L. Lipopolysaccharide priming amplifies lung macrophage tumor necrosis factor production in response to air particles. Toxicol Appl Pharmacol 1999;159:117-24. 21. Beutler B, Poltorak A. Positional cloning of Lps, and the general role of toll-like receptors in the innate immune response. Eur Cytokine Netw 2000;11:143-52. 22. D’Andrea A, Rengaraju M, Valiante NM, Chehimi J, Kubin M, Aste M, et al. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J Exp Med 1992;176: 1387-98. 23. Le J, Lin JX, Henriksen-DeStefano D, Vilcek J. Bacterial lipopolysaccharide-induced interferon-gamma production: roles of interleukin 1 and interleukin 2. J Immunol 1986;136:4525-30. 24. Phipatanakul W, Celedon JC, Raby BA, Litonjua AA, Milton DK, Sredl D, et al. Endotoxin exposure and eczema in the first year of life. Pediatrics 2004;114:13-8. 25. de Vries JE. The role of IL-13 and its receptor in allergy and inflammatory responses. J Allergy Clin Immunol 1998;102:165-9.

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26. Litonjua AA, Carey VJ, Burge HA, Weiss ST, Gold DR. Parental history and the risk for childhood asthma. Does mother confer more risk than father? Am J Respir Crit Care Med 1998;158:176-81. 27. Chew GL, Burge HA, Dockery DW, Muilenberg ML, Weiss ST, Gold DR. Limitations of a home characteristics questionnaire as a predictor of indoor allergen levels. Am J Respir Crit Care Med 1998;157:1536-41. 28. Milton DK, Johnson DK, Park JH. Environmental endotoxin measurement: interference and sources of variation in the Limulus assay of house dust. Am Ind Hyg Assoc J 1997;58:861-7. 29. Park JH, Spiegelman DL, Gold DR, Burge HA, Milton DK. Predictors of airborne endotoxin in the home. Environ Health Perspect 2001;109:859-64. 30. Milton DK, Feldman HA, Neuberg DS, Bruckner RJ, Greaves IA. Environmental endotoxin measurement: the Kinetic Limulus Assay with Resistant-parallel-line Estimation. Environ Res 1992;57:212-30. 31. Finn PW, Boudreau JO, He H, Wang Y, Chapman MD, Vincent C, et al. Children at risk for asthma: home allergen levels, lymphocyte proliferation, and wheeze. J Allergy Clin Immunol 2000;105:933-42. 32. Contreras JP, Ly NP, Gold DR, He H, Wand M, Weiss ST, et al. Allergen-induced cytokine production, atopic disease, IgE, and wheeze in children. J Allergy Clin Immunol 2003;112:1072-7. 33. Lara-Marquez ML, Deykin A, Krinzman S, Listman J, Israel E, He H, et al. Analysis of T-cell activation after bronchial allergen challenge in patients with atopic asthma. J Allergy Clin Immunol 1998;101:699-708. 34. Park JH, Spiegelman DL, Burge HA, Gold DR, Chew GL, Milton DK. Longitudinal study of dust and airborne endotoxin in the home. Environ Health Perspect 2000;108:1023-8. 35. Braun-Fahrlander C, Gassner M, Grize L, Neu U, Sennhauser FH, Varonier HS, et al. Prevalence of hay fever and allergic sensitization in farmer’s children and their peers living in the same rural community. SCARPOL team. Swiss Study on Childhood Allergy and Respiratory Symptoms with Respect to Air Pollution. Clin Exp Allergy 1999;29:28-34. 36. Riedler J, Braun-Fahrlander C, Eder W, Schreuer M, Waser M, Maisch S, et al. Exposure to farming in early life and development of asthma and allergy: a cross-sectional survey. Lancet 2001;358:1129-33. 37. Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 2002;347:869-77. 38. Zimmermann N, Hershey GK, Foster PS, Rothenberg ME. Chemokines in asthma: cooperative interaction between chemokines and IL-13. J Allergy Clin Immunol 2003;111:227-43. 39. Gerhold K, Blumchen K, Bock A, Seib C, Stock P, Kallinich T, et al. Endotoxins prevent murine IgE production, T(H)2 immune responses, and development of airway eosinophilia but not airway hyperreactivity. J Allergy Clin Immunol 2002;110:110-6. 40. Dabbagh K, Lewis DB. Toll-like receptors and T-helper-1/T-helper-2 responses. Curr Opin Infect Dis 2003;16:199-204. 41. Rodriguez D, Keller AC, Faquim-Mauro EL, de Macedo MS, Cunha FQ, Lefort J, et al. Bacterial lipopolysaccharide signaling through Toll-like receptor 4 suppresses asthma-like responses via nitric oxide synthase 2 activity. J Immunol 2003;171:1001-8. 42. Delayre-Orthez C, de Blay F, Frossard N, Pons F. Dose-dependent effects of endotoxins on allergen sensitization and challenge in the mouse. Clin Exp Allergy 2004;34:1789-95. 43. Blumer N, Herz U, Wegmann M, Renz H. Prenatal lipopolysaccharideexposure prevents allergic sensitization and airway inflammation, but not airway responsiveness in a murine model of experimental asthma. Clin Exp Allergy 2005;35:397-402. 44. Cook DN, Wang S, Wang Y, Howles GP, Whitehead GS, Berman KG, et al. Genetic regulation of endotoxin-induced airway disease. Genomics 2004;83:961-9. 45. Michel O, Ginanni R, Duchateau J, Vertongen F, Le Bon B, Sergysels R. Domestic endotoxin exposure and clinical severity of asthma. Clin Exp Allergy 1991;21:441-8. 46. Litonjua AA, Milton D, Celedon JC, Ryan L, Weiss S, Gold D. A longitudinal analysis of wheezing in young children: the independent effects of early life exposure to house dust endotoxin, allergens, and pets. J Allergy Clin Immunol 2002;110:736-42.

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J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 2

Does early EBV infection protect against IgE sensitization? Caroline Nilsson, MD,a Annika Linde, MD, PhD,b Scott M. Montgomery, BSc, PhD,c Liselotte Gustafsson,b Per Na¨sman, Ph Lic,d Marita Troye Blomberg, PhD,e and Gunnar Lilja, MD, PhDa Stockholm, Sweden

Background: There is indirect evidence that an increased infectious burden is associated with a decreased prevalence of IgE-mediated allergy during childhood. Objective: To determine whether there is a relation between the serostatus of 13 different viruses and parentally reported infections and IgE sensitization in 2-year-old children. To investigate whether there is an interaction between cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in relation to IgE sensitization. Methods: A total of 246 infants were followed prospectively to 2 years of age with clinical examinations, skin prick test, and specific IgE analyses and through analysis of seropositivity against adenovirus, influenza, parainfluenza, respiratory syncytial virus, CMV, EBV, herpes simplex virus, human herpesvirus 6, and varicella-zoster virus. Results: There was some evidence that IgE sensitization (24%) tended to be more common among children who were seropositive against few compared with children who were seropositive against many viruses, but this was not statistically significant, and there was no consistent trend across the groups. IgE sensitization was statistically significantly less prevalent at 2 years of age among infants who were seropositive against EBV but not other viruses (adjusted odds ratio, 0.34; 95% CI, 0.14-0.86). The interaction of seropositivity against both CMV and EBV antibodies indicated a further reduction in the risk for IgE sensitization (adjusted odds ratio for interaction, 0.10; 95% CI, 0.01-0.92), indicating effect modification associated with seropositivity against CMV. Conclusion: Our results indicate that acquisition of EBV infection during the first 2 years of life is associated with a reduced risk of IgE sensitization, and this effect is enhanced

Basic and clinical immunology

From athe Department of Pediatrics, Sachs’ Children’s Hospital, and bthe Swedish Institute for Infectious Disease Control, Microbiology and Tumor Biology Center, Karolinska Institute; cthe Clinical Epidemiology Unit, Department of Medicine, Karolinska Hospital, Karolinska Institute and ¨ rebro University Hospital; dthe Royal Institute Clinical Research Centre, O of Technology; and ethe Department of Immunology, Stockholm University. Disclosure of potential conflict of interest: None to disclose. Supported by the Swedish Asthma and Allergy Association, Consul Th C Berg’s Foundation, the Samariten Foundation, Mjo¨lkdroppen, the Va˚rdal Foundation, the Heart and Lung Foundation, GlaxoSmithKline, Brio AB, and the Karolinska Institute. Pharmacia Diagnostics AB supplied reagents for plasma IgE analyses. Received for publication October 25, 2004; revised April 20, 2005; accepted for publication April 21, 2005. Available online July 5, 2005. Reprint requests: Caroline Nilsson, MD, Department of Pediatrics, Sachs’ Children’s Hospital, Stockholm South Hospital, S-118 83 Stockholm, Sweden. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.027

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by CMV coinfection. (J Allergy Clin Immunol 2005;116: 438-44.) Key words: Childhood, CMV, EBV, IgE, infections, sensitization, serology, viral infections

The increasing prevalence of allergic disease has become a major health problem in the industrialized parts of the world.1 Epidemiological studies have shown that various markers of increased burden of infections are associated with a decreased prevalence of allergy and asthma during childhood.2,3 Viral infections have been implicated in influencing IgE-mediated sensitization, but their exact role remains controversial.3,4 It has been sugested that many viruses influence the differentiation of T cells, thus causing an imbalance between TH1 and TH2 immune responses.5 Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are persistent viral infections, as demonstrated by frequent presence of the virus in saliva and urine of healthy individuals,6 and may influence the immune system with respect to the development of allergy, as recently proposed by Sidorchuk et al.7 The aim of this study was therefore to elucidate the interplay between CMV and EBV in relation to IgE sensitization among children at 24 months of age. The study was also designed to investigate the association of IgE sensitization with infectious burden measured as seroprevalence against 13 different viruses, including CMV, EBV, and respiratory syncytial virus (RSV), as well as parentally reported infections.

METHODS Subjects Families who where expecting a child were asked by the midwife at the maternity ward whether they were interested in participating in the study. Only parents who reported they had a history of allergy in the mother, in both parents, or in neither parent were eligible. The parents provided a blood sample and underwent skin prick tests (SPTs). Only parents whose SPT results confirmed their positive or negative history of respiratory allergy to pollen and/or furred pets were invited to continue. When evaluated at 24 months of age, 246 children (126 boys and 120 girls) born to the selected parents participated in the study. One hundred two children had 2 allergic parents, 75 children had an allergic mother, and 69 children had no parental history of allergy. All infants were born full-term (>35 weeks of gestation) at hospitals in Stockholm and had birth weights within the normal range (data not shown). The socioeconomic status of the

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families was estimated through the father’s occupation grouped according to the classification used by Statistics Sweden. Demographic data are presented in Table I. The study was approved by the Human Ethics Committee at Huddinge University Hospital, Stockholm (Dnr 75/97, 113/97), and the parents provided informed consent.

Clinical evaluation The children were followed from birth to 2 years of age and were clinically evaluated at ages 6, 12, 18, and 24 months by 1 pediatrician (C. N.).

Skin prick testing Skin prick tests against food and inhalant allergens were performed among the children at 24 months of age according to the manufacturer’s recommendation (ALK, Copenhagen, Denmark). The SPT included food allergens: egg white (Soluprick weight to volume ratio, 1/100), cod (Soluprick 1/20), peanut, (Soluprick 1/20), cow’s milk (3% fat, standard milk), and soybean protein (Soja Semp; Semper AB, Stockholm, Sweden). SPTs were also performed for inhalant allergens: cat, dog, Dermatophagoides farinae, birch, and timothy (Soluprick 10 Histamine Equivalent Prick test). All parents were skin prick tested against the same inhalant allergens as the children but also against horse, rabbit, and mugwort. Histamine chloride (10 mg/mL) was the positive control and the allergen diluent the negative control. The SPT was considered positive if the wheal diameter was 3 mm after 15 minutes.

Parents’ report of infections During the observation period, the parents were asked to record every infection that their child had in a structured diary. This included symptoms—runny nose, cough, vomiting, diarrhea, fever—and a doctor’s diagnosis where relevant. The diary consisted of sets of structured questions, 1 set for each illness. The parents filled in the form every time the child was ill, marking the correct squares with an X and recording the date of the illness. Parents confirmed the events recorded in the diary when they visited the outpatient clinic with the child at ages 6, 12, 18, and 24 months. In an attempt to record missed illnesses, at each visit the parents were asked, ‘‘Has your child had any illness since your last visit?’’ If additional illnesses were mentioned, they were added to the diary.

Blood sampling Venous blood samples were collected when the children were 24 months old. Plasma was separated by centrifugation and stored at 270°C pending analysis.

Specific IgE Circulating IgE antibodies against cow’s milk, egg white, peanut, cod fish, soy bean, wheat, cat dander, dog dander, birch pollen, timothy pollen, and Dermatophagoides farinae were determined in

plasma with Pharmacia CAP-FEIA (Pharmacia-Upjohn, Uppsala, Sweden). A positive test was defined as an IgE antibody level 0.35 kilo Units allergen-specific antibodies (kUA) per liter.

Classification of the children In accordance with Johansson et al,8 the child was classified as IgE-sensitized if at least 1 SPT was positive (3 mm) and/or if specific IgE against at least 1 of the selected allergens was 0.35kUA/L. To optimize the classification in IgE-sensitized and non–IgEsensitized children, the results of the in vivo and in vitro analysis were combined.

Viral infections/serological methods The serostatus against 13 viruses was investigated: respiratory tract infections, including adenovirus, influenza (A/H1, A/H3, and influenza B), parainfluenza (types 1, 2, 3), and RSV; and herpesvirus, including CMV, EBV, herpes simplex virus (HSV), human herpesvirus 6 (HHV6), and varicella-zoster virus (VZV). IgG against the EBV capsid antigen and HHV6 was determined according to previously published immunofluorescence assays.9,10 A specific fluorescence in dilution of 1/20 was regarded as a sign of seropositivity. For HSV, CMV, and VZV IgG ELISA with purified nuclear antigens from the respective viruses cultivated in human fetal lung fibroblasts were used.11,12 The cutoff for seropositivity was an absorbance of >0.2 at a dilution of 1/100. IgG antibodies against influenza A/H1, A/H3, and influenza B were determined with ELISAs by using recombinant influenza antigens.13 IgG antibodies against parainfluenza (serotypes 1, 2, 3), RSV, and adenoviruses were measured by ELISA. The viruses were cultured to full cytopathogenic effect either in human fetal lung fibroblasts (RSV, adenovirus) or MA 104 cells (monkey kidney cells, parainfluenza) and prepared mainly by ultracentrifugation and sonification of clarified supernatants. For adenovirus, sonicated, infected cells were used. Preparations from the respective cell lines were used as control antigen in the assays using cell culture antigen. Optical densities above 0.3 after subtraction of control antigen activity were regarded as a sign of past infection for the respiratory viruses.

Statistics Descriptive statistics were used to characterize the data. x2 Analysis and the Student t test (2-tailed) were used for comparison of IgE-sensitized and non–IgE-sensitized children where appropriate. The number of serologically verified infections was normally distributed (Fig 1), and the parentally reported infections were close to normally distributed, and they were divided into quarters defined by quartiles by using the statistical program SPSS 11.0 for Windows (SPSS Inc, Chicago, Ill). There was variation in the size of the groups as a result of characteristics of the distribution. Odds ratios and 95% CIs were calculated for the development of IgE sensitization. Data were adjusted for background variables by using multiple logistic regression analysis. Adjustments were made for sex, parental allergy (none, single-heredity, or double-heredity), maternal age, parental smoking, furred pets at home, months of birth, older siblings, duration of breast-feeding, socioeconomic status, parentally reported infections, and seropositivity against viruses. All of the measures were modeled as series of binary dummy variables. The interaction of seropositivity for CMV and EBV was investigated by using logistic regression, with adjustment for the main effects. P values <.05 were considered statistically significant. The data were analyzed by using Stata 7.0 (Stata Corp, College Station, Tex), SPSS 11.0 for Windows, and the SAS System for Windows release 8.02 (SAS Institute, Cary, NC).

Basic and clinical immunology

Abbreviations used EBV: Epstein-Barr virus CMV: Cytomegalovirus HHV6: Human herpesvirus 6 HSV: Herpes simplex virus kUA: Kilo Units allergen-specific antibodies RSV: Respiratory syncytial virus SPT: Skin prick test VZV: Varicella-zoster virus

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TABLE I. Data among infants with positive SPT and/or positive specific IgE and infants with negative SPT/specific IgE at 24 months of age

n (%) Sex Boy, n (%) Girl, n (%) Heredity Nonheredity; n (%) Double-heredity; n (%) Maternal heredity; n (%) Maternal age at delivery Maternal age, mean (range) 21-30 y, n (%) 31-44 y, n (%) Month of birth Born April-September, n (%) Born October-March, n (%) Exposure Exclusive breast-feeding 0 mo, n (%) 0.5-3.9 mo, n (%) 4-5 mo, n (%)* 5.1-10 mo, n (%) Mothers smoking, n (%) Fathers smoking, n (%) Furred pets at home, n (%) Number of older siblings 0, n (%) 1, n (%) >2, n (%) Socioeconomic status  High, n (%) Medium, n (%) Low, n (%) Studying, n (%) Not specified, n (%)

Whole cohort

IgE-sensitized

Nonsensitized

246

59 (24.0)

187 (76.0)

OR; 95% CI

ORadj; 95% CI

126 (51.2) 120 (48.8)

34 (27.0) 25 (20.8)

92 (73.0) 95 (79.2)

1 0.71; 0.39-1.28

1 0.65; 0.32-1.31

69 (28.0) 102 (41.5) 75 (30.5)

11 (15.9) 32 (31.4) 16 (21.3)

58 (84.1) 70 (68.6) 59 (78.7)

1 2.41; 1.12-5.20 1.43; 0.61-3.34

1 3.79; 1.50-9.60 2.29; 0.83-6.31

31.4 (21-44)

31.5 (22-44)

31.3 (21-43)

103 (41.9) 143 (58.1)

22 (21.6) 37 (25.9)

81 (78.4) 106 (74.1)

1 1.29; 0.70-2.35

1 1.42; 0.67-3.01

148 (60.2) 98 (39.8)

27 (18.2) 32 (32.7)

121 (81.8) 66 (67.3)

1 2.17; 1.20-3.93

1 2.23; 1.10-4.52

13 34 166 33 10 19 45

(5.3) (13.8) (67.5) (13.4) (4.1) (7.7) (18.3)

2 (15.4) 10 (29.4) 38 (22.9) 9 (27.3) 1 (1.7) 7 (11.9) 9 (15.3)

11 24 128 24 9 12 36

0.61; 1.40; 1 1.26; 0.34; 1.96; 0.76;

0.91; 3.11; 1 1.21; 0.32; 2.24; 0.74;

135 (54.9) 81 (32.9) 30 (12.2)

34 (25.2) 24 (29.6) 1 (3.3)

101 (74.8) 57 (70.4) 29 (96.7)

1 1.25; 0.68-2.31 0.10; 0.01-0.78

1 1.35; 0.65-2.80 0.07; 0.01-0.65

141 39 41 11 14

34 9 10 4 2

107 30 31 7 12

1 0.94; 1.02; 1.80; 0.52;

1 0.97; 1.35; 3.39; 0.52;

(57.3) (15.9) (16.7) (4.5) (5.7)

(24.1) (23.1) (24.4) (36.4) (14.3)

(84.6) (70.6) (77.1) (72.7) (4.8) (6.4) (19.3)

(75.9) (76.9) (75.6) (63.6) (85.7)

0.13-2.89 0.62-3.19 0.54-2.95 0.04-2.75 0.73-5.24 0.34-1.68

0.41-2.19 0.45-2.28 0.50-6.52 0.11-2.46

0.13-6.60 1.10-8.85 0.45-3.21 0.03-3.19 0.68-7.34 0.28-1.94

0.36-2.64 0.49-3.75 0.67-17.06 0.09-3.14

OR, Odds ratio; ORadj, adjusted odds ratio. *The recommended time for exclusively breast-feeding is at least 4 months in Sweden.  Grouped by the father’s occupation according to Statistics Sweden (Swedish government for official statistics).

RESULTS Basic and clinical immunology

IgE sensitization The children had an average age of 24.1 months (range, 22-29) at the 24-month evaluation. Fifty-nine (24%) children were classified as IgE-sensitized. The in vitro test (allergen-specific IgE in plasma) was positive in 52 (22%) infants, whereas 35 (14%) infants had at least 1 positive SPT against the selected food and inhalant allergens. The majority (n = 49; 83%) were sensitized against food allergens, and sensitization toward individual allergens was for milk, 13.4%; egg, 7.7%; peanut, 6.1%; dog, 4.9%; wheat, 4.1%; cat, 3.7%; birch, 3.7%; soy, 2.0%; fish, 1.2%; timothy, 0.8%; and mite, 0.8%. There were no statistically significant differences in sex, having a furred pet at home, or having smoking parents between IgE-sensitized and nonsensitized children (Table I).

However, the nonsensitized children were statistically significantly more likely to have been born during the summer than sensitized children and more frequently had more than 1 sibling. The sensitized children were statistically significantly more likely to have 2 atopic parents. The statistically significant association with short duration of breast-feeding was observed only in the adjusted analyses, suggesting a complex set of associations or a chance finding.

IgE sensitization and the parental report of infections The median number of parental reported infections was 13 (range, 4-24) during the first 24 months of life. For evaluation, the infants were divided into 4 groups defined by quartiles: 4 to 9, 10 to 13, 14 to 16, and 17 to 24 reported infections. The association with IgE sensitization

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for each group was evaluated. The sensitized children were fewer in the group with 10 to 13 parentally reported infections, but there were no statistically significant difference between the groups, and the trend across the groups was not consistent (Table II).

IgE sensitization and seropositivity to individual viruses Respiratory viruses. The association between presence of antibodies against the individual viruses and IgE sensitization is presented in Table II. There were no statistically significant associations between IgE sensitization and seropositivity against the 8 airborne viruses—adenovirus, influenza (A/H1, A/H3, and B), parainfluenza (types 1, 2, 3), and RSV. Sixty-three percent (154 children) were seropositive against RSV at 24 months of age, and among these, 21% (n = 33) were IgE-sensitized. Herpesviruses. Seropositivity against the viruses belonging to the herpesvirus family—CMV, EBV, HSV, HHV6 and VZV—and IgE sensitization are also presented in Table II. Seronegativity against EBV was statistically significantly associated with IgE sensitization. Sixty-four children (26%) were seropositive against EBV. Among these, 8 (12 %) were sensitized. This was significantly less than in the EBV seronegative group (odds ratio, 0.37; 95% CI, 0.16-0.82). The association remained statistically significant after adjustment for all of the potential confounding factors (Table II). Cytomegalovirus IgG antibodies were detectable in 96 (39%) children. Among these, 27 (28%) were IgE-sensitized. There were no statistically significant differences in the numbers of seropositivity or seronegativity against CMV when comparing the IgE sensitized and nonsensitized infants. However, seropositivity for EBV was more negatively associated with sensitization among subjects who were also seropositive for CMV. The odds ratio for

FIG 1. Frequency of seropositivity against the 13 selected viruses at 24 months of age in IgE sensitized* (n = 59) and nonsensitized (n = 187) infants. *At least 1 positive SPT 3 mm and/or at least 1 specific IgE against the selected allergens 0.35 kUA/L.

IgE sensitization associated with seropositivity against EBV among children seronegative against CMV was 0.75 (95% CI, 0.30-1.91) but was reduced to 0.07 (95% CI, 0.01-0.57) among children who were seropositive against both viruses. Interaction testing revealed a statistically significant synergistic effect (effect modification), producing an odds ratio for the interaction of both viruses with IgE sensitization of 0.10 (95% CI, 0.01-0.92) after adjustment for the main effects. We also observed an association with sensitization among subjects who were seropositive against CMV and seronegative against EBV. Among those seronegative against EBV (n = 182), 26 of 71 were sensitized among those infected with CMV, compared with 25 of 111 who were not infected with CMV. This produces an odds ratio of 1.99 (95% CI, 1.03-3.83), suggesting a modest increased risk associated with CMV infection in subjects who are seronegative for EBV. The serostatus against the other herpesviruses was not significantly associated with IgE sensitivity.

DISCUSSION The hypothesis that infectious diseases during early childhood may have a protective role against the development of allergy, the hygiene hypothesis, was raised in the late 1980s.5 In the current study, we did not find strong evidence of an association between IgE sensitization and the number of previous infections indicated either by parental report or by seropositivity. However, IgE sensitization was less prevalent at 2 years of age among infants who were seropositive against EBV. The combination of having both CMV and EBV antibodies was more strongly negatively associated with sensitization than would be predicted by the individual associations of EBV or CMV antibodies alone, indicative of an interactive effect. The prevalence of EBV antibodies in our study is comparable with that of other industrialized countries.14 Previous studies of EBV are contradictory in relation to development of atopy. Increased levels of antibodies against EBV were found in children 5 to 18 years old

Basic and clinical immunology

IgE sensitization and the frequency of seropositivity The frequency of seropositivity at 24 months of age against the selected viruses is presented in Table II. All children apart from 1 had detectable IgG antibodies against at least 1 of the viruses studied, and 1 child had detectable IgG against all 13. There were no significant associations between the number of parentally reported infections and the number of viruses identified through serology (data not shown). The mean number of viruses identified through serology was 5 (Fig 1). The children were divided into 4 groups defined by quartiles. Fifty-five children had antibodies against 0 to 3 viruses, 95 children against 4 to 5 viruses, 42 against 6 viruses, and 54 against 7 to 13 viruses, respectively. There was some suggestion that children with fewer antibodies were more often sensitized than children with antibodies against many viruses, but this result was not statistical significant (Table II), and the pattern did not show a consistent trend across the groups.

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TABLE II. Seropositivity against the investigated viruses and parentally reported infections in IgE-sensitized and nonsensitized children Whole cohort

n Parainfluenza 1, n (%) Parainfluenza 2, n (%) Parainfluenza 3, n (%) Influenza Panama, n (%) Influenza Texas, n (%) Influenza Beijing, n (%) Adenovirus, n (%) RSV, n (%) HHV6, n (%) HSV, n (%) CMV, n (%) VZV, n (%) EBV, n (%) Frequency of seropositivity 0-3 viruses, n (%) 4-5 viruses, n (%) 6 viruses, n (%) 7-13 viruses, n (%) Parentally reported infections 4-9 infections, n (%) 10-13 infections, n (%) 14-16 infections, n (%) 17-24 infections, n (%)

246 70 (28.5) 30 (12.2) 166 (67.5) 47 (19.1) 30 (12.2) 92 (37.4) 200 (81.2) 154 (62.6) 207 (84.2) 24 (9.8) 96 (39.0) 45 (18.3) 64 (26.0)

IgE-sensitized

16 7 42 13 6 16 46 33 47 6 27 8 8

59 (27.1) (11.9) (71.2) (22.0) (10.2) (27.1) (78.0) (55.9) (79.7) (10.2) (45.8) (13.6) (13.6)

Nonsensitized

187 54 (28.9) 23 (12.3) 124 (66.3) 34 (18.2) 24 (12.8) 76 (43.8) 154 (82.4) 121 (64.7) 160 (85.6) 18 (9.6) 69 (36.9) 37 (19.8) 56 (29.9)

OR; 95% CI

0.92; 0.96; 1.26; 1.27; 0.76; 0.54; 0.76; 0.69; 0.66; 1.06; 1.44; 0.64; 0.37;

0.48-1.76 0.39-2.36 0.66-2.38 0.62-2.61 0.30-1.97 0.28-1.03 0.37-1.56 0.38-1.26 0.31-1.40 0.40-2.81 0.80-2.60 0.28-1.45 0.16-0.82

ORadj; 95% CI

0.93; 1.14; 1.13; 1.32; 0.63; 0.52; 0.77; 0.71; 0.66; 1.10; 1.20; 0.60; 0.34;

0.46-1.86 0.44-2.99 0.58-2.21 0.62-2.82 0.23-1.76 0.26-1.01 0.36-1.63 0.37-1.36 0.30-1.46 0.40-3.02 0.64-2.27 0.25-1.42 0.14-0.86

55 95 42 54

(22.4) (38.6) (17.1) (22.0)

16 25 7 11

(27.1) (42.4) (11.9) (18.6)

39 70 35 43

(20.9) (37.4) (18.7) (23.0)

1.57; 0.71-3.50 1.15; 0.47-2.84 1.02; 0.42-2.51 1

1.53; 0.59-3.97 0.78; 0.28-2.21 0.99; 0.35-2.80 1

54 75 62 55

(22) (30.5) (25.2) (22.3)

17 13 16 13

(28.8) (22.0) (27.1) (22.0)

37 62 46 42

(19.8) (33.2) (24.6) (22.5)

1.48; 0.64-3.46 0.68; 0.29-1.61 1.12; 0.48-2.61 1

1.40; 0.50-3.95 0.57; 0.21-1.54 1.11; 0.40-3.08 1

OR, Odds ratio; ORadj, adjusted odds ratio.

Basic and clinical immunology

with clinical signs of atopy compared with their nonatopic counterparts.15 However, Calvani et al14 reported a higher prevalence of atopy among EBV seronegative children in the age group 0 to 6 years, corroborating our results. A recent Swedish study (BAMSE) failed to demonstrate that the EBV serostatus in 4-year-old children correlated with IgE sensitization.16 In developing countries, where EBV asymptomatically infects the majority of children before 3 years of age,17 the prevalence of atopy has been reported to be lower than in industrialized countries.18 In combination with our findings, these observations might indicate an age-dependent role of EBV, rather than EBV infection per se, in relation to IgE sensitization. Cytomegalovirus becomes persistent after primary infection and seems to induce a TH1 cytokines response.19 CMV is frequently transmitted from mothers to infants during pregnancy, at delivery, or via breast milk.20 The few studies published on the relation between CMV and allergies are inconclusive.21 In the Swedish BAMSE study, no association was found between CMV seropositivity and IgE sensitization in 4-year-old children. However, among children with seropositivity against CMV and seronegativity against EBV, there was a positive association with sensitization to food allergens.7 This led us to test the interaction of seropositivity against CMV and EBV in relation to IgE sensitization. This indicated effect modification such that the negative association of EBV seropositivity with sensitization was further enhanced in children who were also seropositive for CMV. The mechanism for this putative interaction is unknown.

Several plausible explanations are possible, including the idea that IL-10 homologues present in the viruses might downregulate the antigen processing/presentation capacity of dendritic cells/macrophages and thereby switch off the host T-cell system, similar to the downregulation observed for T regulatory cells.22,23 Alternately, both EBV and CMV can polyclonally activate B cells to produce antibodies with many different specificities and thereby hinder the capacity of allergens to cross-link the B-cell receptor as seen for helmintic infections.24 Thus, these data and our results provide further support for the hypothesis that specific characteristics of EBV and/or CMV infection, rather than infection per se, might influence the risk of IgE sensitization. Interestingly, RSV infection, according to serology, was not associated with IgE sensitization at 24 months of age. Previous studies have suggested that hospitalization because of RSV infection is linked with atopy and induction of IgE synthesis.25 Discrepancies between different studies indicate that the vulnerability to RSV might be associated with a propensity for asthma and allergy, and not that RSV per se causes asthma. Again, the characteristics of infection may be important, and identification through hospitalization suggests more severe acute infection than the majority of children in our study would have had. Blanco-Quiros et al26 reported that infants who developed severe RSV bronchiolitis had low levels of IL-12, a strong TH1 inducer, in cord blood. We have recently published similar data showing low levels of IL-12 in cord blood of IgE sensitized infants at 24 months of age.27

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confer greater protection, such as EBV infection in the age-defined window of susceptibility with possible modification through interaction with CMV. Development of more precise measures of patterns of acute infection and their immunological sequel will assist in our understanding of how early in life viral infections are implicated in the etiology of IgE sensitization. We thank the families who participated in the study. We also thank Anna Stina Ander, Johan Berggren, Jeanette Harrysson, Lena Ja¨gdahl, and Monica Nordlund for assistance.

REFERENCES 1. Aberg N, Hesselmar B, Aberg B, Eriksson B. Increase of asthma, allergic rhinitis and eczema in Swedish schoolchildren between 1979 and 1991. Clin Exp Allergy 1995;25:815-9. 2. Matricardi PM, Franzinelli F, Franco A, Caprio G, Murru F, Cioffi D, et al. Sibship size, birth order, and atopy in 11,371 Italian young men. J Allergy Clin Immunol 1998;101:439-44. 3. Matricardi PM, Rosmini F, Ferrigno L, Nisini R, Rapicetta M, Chionne P, et al. Cross sectional retrospective study of prevalence of atopy among Italian military students with antibodies against hepatitis A virus. BMJ 1997;314:999-1003. 4. Benn CS, Melbye M, Wohlfahrt J, Bjorksten B, Aaby P. Cohort study of sibling effect, infectious diseases, and risk of atopic dermatitis during first 18 months of life. BMJ 2004;328:1223-8. 5. Strachan DP. Hay fever, hygiene, and household size. BMJ 1989;299: 1259-60. 6. Grillner L, Strangert K. A prospective molecular epidemiological study of cytomegalovirus infections in two day care centers in Sweden: no evidence for horizontal transmission within the centers. J Infect Dis 1988;157:1080-3. 7. Sidorchuk A, Wickman M, Pershagen G, Lagarde F, Linde A. Cytomegalovirus infection and development of allergic diseases in early childhood: interaction with EBV infection? J Allergy Clin Immunol 2004;114:1434-40. 8. Johansson SG, Hourihane JO, Bousquet J, Bruijnzeel-Koomen C, Dreborg S, Haahtela T, et al. A revised nomenclature for allergy: an EAACI position statement from the EAACI nomenclature task force. Allergy 2001;56:813-24. 9. Linde A, Fridell E, Dahl H, Andersson J, Biberfeld P, Wahren B. Effect of primary Epstein-Barr virus infection on human herpesvirus 6, cytomegalovirus, and measles virus immunoglobulin G titers. J Clin Microbiol 1990;28:211-5. 10. Linde A, Andersson J, Lundgren G, Wahren B. Subclass reactivity to Epstein- Barr virus capsid antigen in primary and reactivated EBV infections. J Med Virol 1987;21:109-21. 11. Sundqvist VA, Linde A, Wahren B. Virus-specific immunoglobulin G subclasses in herpes simplex and varicella-zoster virus infections. J Clin Microbiol 1984;20:94-8. 12. Sundqvist VA, Wahren B. An interchangeable ELISA for cytomegalovirus antigen and antibody. J Virol Methods 1981;2:301-12. 13. Stepanova L, Naykhin A, Kolmskog C, Jonson G, Barantceva I, Bichurina M, et al. The humoral response to live and inactivated influenza vaccines administered alone and in combination to young adults and elderly. J Clin Virol 2002;24:193-201. 14. Calvani M, Alessandri C, Paolone G, Rosengard L, Di Caro A, De Franco D. Correlation between Epstein Barr virus antibodies, serum IgE and atopic disease. Pediatr Allergy Immunol 1997;8:91-6. 15. Strannegard IL, Strannegard O. Epstein-Barr virus antibodies in children with atopic disease. Int Arch Allergy Appl Immunol 1981;64:314-9. 16. Sidorchuk A, Lagarde F, Pershagen G, Wickman M, Linde A. EpsteinBarr virus infection is not associated with development of allergy in children. Pediatr Infect Dis J 2003;22:642-7. 17. Cohen JI. Epstein-Barr virus infection. N Engl J Med 2000;343:481-92. 18. Addo Yobo EO, Custovic A, Taggart SC, Asafo-Agyei AP, Woodcock A. Exercise induced bronchospasm in Ghana: differences

Basic and clinical immunology

These observations might indicate an interaction among cytokines, RSV, and sensitization during early infancy. There was some indication that the numbers of serologically verified viral infections were inversely correlated to IgE sensitization, but there was no consistent trend across the groups and no statistically significant association. The number of parentally reported infections and seropositivity against the selected viruses did not correlate. This is not entirely surprising, because many viruses, eg, rhinovirus and corona virus, which are proposed to be the major causes of upper respiratory infections in infants, were not included in our analysis.28 There is not always a relation between viral infections and diseases, because asymptomatic infections are common. The suggestion of an inverse association between the frequencies of parentally reported and serologically verified viral infections with sensitization is in agreement with previous publications,29,30 although these authors have mainly studied clinical signs of allergy. It is possible that the suggested protective effect of infections continues to influence the nascent immune system beyond age 2 years, and our follow-up was conducted at too young an age to observe the protective effect against allergic disease. Previous studies have shown mainly indirect evidence for the importance of infections for the development of allergy,3,5,29 and these studies have often been retrospective. The strength of our study is that the atopic status of the parents was characterized, and information on infections was collected prospectively by using diaries. Importantly, objective serological measurements against antiviral antibodies were made, and the results were adjusted for factors that might bias the evaluation. However, we recognize some limitations. The study population was selected and not population-based. Because we selected children with different family histories of allergy, we believe that the group of children in our study is fairly comparable with children in the general population. Furthermore, the exact sensitivity for seropositivity in some of the assays used to evaluate the respiratory infections is not fully known because they have been used mainly to detect ongoing infections, so the rate of seropositivity is somewhat underestimated, but this should not introduce bias. The optimal approach would have been to perform neutralization assays, but serum samples from infants are inevitably insufficient for tests using low serum dilutions. In contrast, the sensitivity and specificity for seropositivity of the assays used for the herpesvirus infections have been proven reliable, as best demonstrated by the correlation between initial serostatus and clinical outcome found in transplant recipients.31 It could also be argued that analyzing 13 different viruses could produce some associations with IgE sensitization by chance. However, because an association specifically with EBV was evaluated as a result of an a priori hypothesis based on the results of previous studies,14 the associations reported for EBV are unlikely to be caused by chance. In summary, our results indicate that an EBV infection during the first 2 years of life is associated with a reduced risk of IgE sensitization. Some patterns of infection may

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19.

20.

21.

22.

23.

24.

in prevalence between urban and rural schoolchildren. Thorax 1997; 52:161-5. Rentenaar RJ, Gamadia LE, van DerHoek N, van Diepen FN, Boom R, Weel JF, et al. Development of virus-specific CD4(1) T cells during primary cytomegalovirus infection. J Clin Invest 2000;105:541-8. Ahlfors K, Ivarsson SA, Harris S. Report on a long-term study of maternal and congenital cytomegalovirus infection in Sweden: review of prospective studies available in the literature. Scand J Infect Dis 1999;31: 443-57. Wu CA, Puddington L, Whiteley HE, Yiamouyiannis CA, Schramm CM, Mohammadu F, et al. Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease. J Immunol 2001;167:2798-807. Salek-Ardakani S, Arrand JR, Mackett M. Epstein-Barr virus encoded interleukin-10 inhibits HLA-class I, ICAM-1, and B7 expression on human monocytes: implications for immune evasion by EBV. Virology 2002;304:342-51. Raftery MJ, Wieland D, Gronewald S, Kraus AA, Giese T, Schonrich G. Shaping phenotype, function, and survival of dendritic cells by cytomegalovirus-encoded IL-10. J Immunol 2004;173:3383-91. Smits HH, Hartgers FC, Yazdanbakhsh M. Helminth infections: protection from atopic disorders. Curr Allergy Asthma Rep 2005;5:42-50.

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25. Schauer U, Hoffjan S, Bittscheidt J, Kochling A, Hemmis S, Bongartz S, et al. RSV bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life. Eur Respir J 2002;20:1277-83. 26. Blanco-Quiros A, Gonzalez H, Arranz E, Lapena S. Decreased interleukin-12 levels in umbilical cord blood in children who developed acute bronchiolitis. Pediatr Pulmonol 1999;28:175-80. 27. Nilsson C, Larsson A-K, So¨derlund A, Gabrielsson S, Troye Blomberg M, Lilja G. Low numbers of IL-12-producing cord blood mononuclear cells and immunoglobulin E sensitization in early childhood. Clin Exp Allergy 2004;34:373-80. 28. Johnston SL. Natural and experimental rhinovirus infections of the lower respiratory tract [review]. Am J Respir Crit Care Med 1995;152: S46-52. 29. Celedon JC, Wright RJ, Litonjua AA, Sredl D, Ryan L, Weiss ST, et al. Day care attendance in early life, maternal history of asthma, and asthma at the age of 6 years. Am J Respir Crit Care Med 2003;167:1239-43. 30. Lau S, Nickel R, Niggemann B, Gruber C, Sommerfeld C, Illi S, et al. The development of childhood asthma: lessons from the German Multicentre Allergy Study (MAS). Paediatr Respir Rev 2002;3:265-72. 31. Ljungman P, Aschan J, Lewensohn-Fuchs I, Carlens S, Larsson K, Lonnqvist B, et al. Results of different strategies for reducing cytomegalovirus-associated mortality in allogeneic stem cell transplant recipients. Transplantation 1998;66:1330-4.

Basic and clinical immunology

Biased use of VH5 IgE-positive B cells in the nasal mucosa in allergic rhinitis Heather A. Coker, PhD,a* Helen E. Harries, MBiochem,a Graham K. Banfield, FRCS,b Victoria A. Carr, RGN,b Stephen R. Durham, MD,b Elfy Chevretton, FRCS,c Paul Hobby, MSc,a Brian J. Sutton, PhD,a and Hannah J. Gould, PhDa London, United Kingdom

Key words: Human, allergic rhinitis, VH5, superantigen, B lymphocyte, mucosa

From aThe Randall Division of Cell and Molecular Biophysics, King’s College London; bUpper Respiratory Medicine, National Heart and Lung Institute, Imperial College, London; and cthe Department of Respiratory Medicine and Allergy, Guy’s Hospital, London. *Dr Coker is currently affiliated with Clare Hall Laboratories, Cancer Research UK, South Mimms, Hertfordshire, United Kingdom. Supported by the Clinical Research Committee Royal Brompton and Harefield Hospitals NHS Trust. HAC and HEH were supported by BBSRC PhD studentships, and HG and SRD were supported by project grants from Asthma UK (grant no. 03/055) and the MRC (grant no. G0200485). Received for publication August 25, 2004; revised March 23, 2005; accepted for publication April 22, 2005. Available online June 17, 2005. Reprints of this article are not available from the authors. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.032

Abbreviations used CDR: Complementarity-determining region FWR: Framework region R/S: Replacement/silent mutation ratio

IgE and its receptors are central to allergic disease, manifested in different target organs, including the nose (allergic rhinitis), the lung (allergic asthma), the skin (atopic dermatitis), and the gut (allergic gastroenteritis). IgE binds to effector and antigen-presenting cells bearing IgE receptors (FceRI, CD23, or both) in mucosal tissues associated with the target organs mediating the allergic response. In addition, IgE-mediated allergen presentation to T helper cells might lead to renewed IgE antibody synthesis, epitope spreading, and exacerbation of allergic sensitivity. A large number of genetic and environmental risk factors for allergy have been identified, providing broad insight into the pathogenesis of allergic disease. The factors that determine the susceptible target organ in different individuals exposed to the same allergens are, however, unknown. We suggest that these factors might be localized in the target organ and might include superantigens. Snow and coworkers1-5 have observed a bias in the repertoire of IgE heavy-chain variable (IgE VH) regions in asthma, which exhibited the hallmarks of a superantigen. There are 51 VH genes grouped into 7 gene families (VH1VH7), varying in size from 22 members in VH3 to 1 or 2 members in VH5, VH6, and VH7.6 Each VH region has 3 framework regions (FWR1-FWR3) alternating with 3 complementarity-determining regions (CDR1-CDR3). The FWRs determine the structural framework for the antigen-binding sites of the CDRs. The CDR sequences are inherently more prone to somatic hypermutation during affinity maturation of antibodies in the immune response, whereas the FWR sequences are relatively conserved.7 In peripheral blood B cells of healthy individuals, the proportion of expressed VH regions from different VH families generally reflects the size of the family.8 In asthma, however, Snow and coworkers found an overabundant use of the minor VH5 family in IgE in the blood, lung mucosa, and spleen of asthmatic patients1-3 445

Basic and clinical immunology

Background: IgE antibody-producing B cells are enriched in the nasal mucosa in patients with allergic rhinitis because of local class switching to IgE. The expressed IgE VH genes also undergo somatic hypermutation in situ to generate clonal families. The antigenic driving force behind these events is unknown. Objective: To examine the possible involvement of a superantigen in allergic rhinitis, we compared the variable (VH) gene use and patterns of somatic mutation in the expressed IgE heavy-chain genes in nasal biopsy specimens and blood from allergic patients and the IgA VH use in the same biopsy specimens and also those from nonallergic controls. Methods: We extracted mRNA from the nasal biopsy specimens of 13 patients and 4 nonallergic control subjects and PBMCs from 7 allergic patients. IgE and IgA VH regions were RT-PCR amplified, and the DNA sequences were compared with those of control subjects. We constructed a molecular model of VH5 to locate amino acids of interest. Results: We observed a significantly increased frequency of IgE and IgA VH5 transcripts in the nasal mucosa of the allergic patients compared with the normal PBMC repertoire. Within IgE and IgA VH5 sequences in the nasal mucosa, the distribution of replacement amino acids was skewed toward the immunoglobulin framework regions. Three of 4 nonintrinsic hotspots of mutation identified in the VH5 sequences were in framework region 1. The hotspots and a conserved VH5-specific framework residue form a tight cluster on the surface of VH5. Conclusion: Our results provide evidence for the activity of a superantigen in the nasal mucosa in patients with allergic rhinitis. (J Allergy Clin Immunol 2005;116:445-52.)

446 Coker et al

and in the blood of the majority of 6 asthmatic patients.5 Overabundant use of VH5 was also reported in the blood of 3 patients with atopic dermatitis9 but not in another study of 2 patients with atopic dermatitis.10 By contrast, in the blood of 2 subjects with peanut allergy, Snow and coworkers observed an overabundant use of VH1, suggesting the activity of a peanut-associated superantigen.11 B-cell superantigens, similar to T-cell superantigens, act by binding to immunoglobulin FWRs, leading to clonal amplification of all members of the family. Because the number of CDRs in the B-cell repertoire (millions) is vastly greater than the number of VH families (7), this results in biased use of the selected family.12 Two previously identified B-cell superantigens, Staphylococcus aureus protein A and HIV gp120, selectively expand B cells expressing VH3.12-14 The interaction of protein A with the FWR of VH3 can be seen in the crystal structure of a complex with the VH3 Fab fragment of an IgM antibody.15 Superantigen-selected B cells might also exhibit a skewed distribution of amino acid substitutions away from CDRs toward the FWR.7,16 The frequent occurrence of point mutation hotspots in DNA sequences that are intrinsically more susceptible to hypermutation is a further criterion for CDR- versus FWR-oriented selection; the sequences WRC and WA, which occur more frequently in the CDRs, are recognized as intrinsic hotspots of mutation.17 These 2 features of superantigen selection of B cells were also linked to the VH5 overabundance in asthma and atopic dermatitis,2,9 which is consistent with the influence of a superantigen and in contrast to the lack of such influence observed in VH5 B cells from normal spleen.18 In a previous study of the expressed VH regions in allergic rhinitis, we presented evidence of local clonal expansion, somatic hypermutation, and class switching in the nasal mucosa in patients with allergic rhinitis.19 In this study we present a detailed analysis of IgE and IgA VH family use in the nasal mucosa in patients with allergic rhinitis.

METHODS Basic and clinical immunology

Samples from patients with allergic rhinitis and nonallergic control subjects Male and female donors with allergic rhinitis aged between 18 and 55 years were recruited for this study. The allergic status of the donors was assessed on the basis of medical history, skin prick tests, and, where possible, serum allergen-specific IgE (RAST). Of the 11 tissue samples from the nasal mucosa, 10 originated from patients with multiple allergies (CD6, JB7, CM10, HD14, SO16, AP19, SJ24, TL25, CA30, and SLT1) who were allergic to grass and also allergens such as animal dander or house dust mite, to which they could be perennially exposed. One tissue sample (HD17) originated from a patient with only grass pollen allergy. The samples were taken throughout the year, with the patient with only grass pollen allergy undergoing biopsy within the grass pollen season. Of the 11 nasal mucosa samples analyzed, 10 were biopsy specimens taken from the inferior turbinate, and one (SLT1) consisted of a piece of an inferior turbinate removed by surgery to alleviate nasal obstruction. In

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agreement with previous authors,9,19 we were unable to use healthy subjects as a control group for IgE because PCR amplification of IgE was only successful in the allergic subjects. Instead, we recruited 4 nonallergic subjects (AA2, MTS3, KB5, and SK6) and used biopsy specimens from 2 of the allergic patients (TL25 and CA30) plus 2 additional patients with multiple allergies (JC1 and IB4) to examine VH use in IgA. Volunteers were recruited from the Royal Brompton Hospital Allergy Clinic or by advertisement in the local press to donate a nasal biopsy specimen. None had received immunotherapy, and any medication was discontinued at least 2 weeks before nasal biopsy. Biopsies were performed at the Royal Brompton Hospital, London, United Kingdom, and processed as described previously.20 All such work had the approval of the local ethics committee and the patients’ written informed consent. Blood samples were taken from 7 of the 16 patients who also donated nasal biopsy specimens (CD6, JB7, CM10, HD14, SO16, HD17, and AP19). PBMCs, including B cells, were isolated from these samples, as described previously.19 The tissue sample from the inferior turbinate resulted from operations performed at Guy’s Hospital, London, United Kingdom, with the approval of the Guy’s Research Ethics Committee and also with the patient’s written informed consent.

Amplification and analysis of VH region sequences Total RNA was extracted from both the nasal mucosa and PBMC samples, cDNA was produced, and VH-Ce sequences were PCR amplified by using the proofreading Pfu DNA polymerase (Promega, Madison, Wis) from IgE-positive B cells. The VH-Ce PCR products were then cloned and sequenced. This entire procedure has been described in detail previously.19 VH-Ca sequences were amplified from cDNA in the same way as VH-Ce sequences, replacing the Ce-specific primers with nested primers specific for Ca: Ca1, 5#TTTCGCTCCAGGTCACAC-3#; Ca2, 5#-GGGAAGAAGCCCTGGACCAGGC-3#. The annealing temperature for the second round of PCR was adjusted from 65°C to 69°C. Assignment of the VH genes and their somatic mutations was carried out according to their homology with the germline sequences detailed on the VBase database (www.mrc-cpe.cam.ac.uk). Only unique sequences were included in the analysis, meaning that repeat copies of identical sequences and also sequences that originated from clonally related B cells (determined by an identical CDR3/FWR4 signature region) were included only once in the analysis. However, when sequences did originate from related B cells, each unique mutation isolated from the family members was included in the mutational analysis. Statistical significance was determined by the use of x2 analysis with the Yates correction for continuity (generating a more conservative calculation of significance when smaller data values are used).

Molecular modeling of VH5 antibody structure Because no VH5 antibody crystal structure is available, a model was generated for the VH5-51 germline sequence on the basis of the known structure of the most closely matched antibody (PDB code: 1CGS). The heavy-chain CDR1 and CDR2 (H1 and H2) loop lengths were identical in both antibody sequences, and the heavy-chain CDR3 (H3) loop and the light chain (VL) domain structures were taken directly from 1CGS to produce a complete Fv model. No steric clashes were detected after substitution of the VH5-51 sequence. The model was generated by using HOMOLOGY and displayed with INSIGHT II (Accelrys, Cambridge, United Kingdom).

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Coker et al 447

FIG 1. VH gene use in allergic nasal mucosa IgE-positive B cells. Sixty-two sequences from 11 allergic nasal mucosa samples (filled bars) and 50 sequences from 7 allergic PBMC samples (gray bars) were pooled to compare VH gene use. VH gene use in normal PBMCs (open bars) was also included for comparison.21

*Nucleotide sequences submitted to Genbank, accession numbers AY640472– AY640533.  Nucleotide sequences submitted to Genbank, accession numbers AY640534– AY640583.

These data were taken from sequences pooled from multiple patients. The occurrence of VH5 sequences across the PBMC samples appeared to be evenly distributed (with the 4 VH5 sequences occurring in 4 of the 7 different PBMC samples). This was in contrast to the distribution of VH5 sequences in the samples from the nasal mucosa, in which the 18 VH5 sequences were isolated from only 5 of the 11 patients. There was a particularly high level of VH5 use in the nasal mucosa of patient CA30, in whom 11 of 18 sequences were from unrelated B cells expressing VH5 IgE-positive sequences. However, even when this patient was excluded from the analysis, the increased VH5 IgE use (14%) in the allergic nasal mucosa compared with that expected on the basis of the normal PBMC repertoire was still highly significant (P < .005, x2 analysis), although the significance between the allergic nasal mucosa and allergic PBMCs was lost (P > .1, x2 analysis).

Hotspots of mutation in IgE VH5 sequences from the allergic nasal mucosa All of the sequences isolated from nasal biopsy specimens exhibited evidence of somatic hypermutation. Somatic mutations evident within 17 distinct VH5 sequences were compared with those from 19 randomly chosen non-VH5 sequences to determine whether there was evidence to suggest that the overuse of VH5 in the allergic nasal mucosa might have been a consequence of B-cell selection (eg, by the presence of nonintrinsic hotspots of mutation). The percentage variability of each codon was used to identify apparent hotspots of somatic hypermutation (Fig 2). The percentage mutation (including both silent and replacement mutations) and the percentage mutation at each nucleotide were also analyzed (data not shown),

Basic and clinical immunology

RESULTS IgE VH gene use We examined a total of 62 in-frame VH region sequences derived from the nasal mucosa of the 11 patients with allergic rhinitis* and 50 VH region sequences from the PBMCs of 7 of these patients.  There was a clear difference in the VH gene use observed in IgE B cells from the nasal mucosa of the allergic patients compared with that expected on the basis of the normal genomic PBMC repertoire21 and also compared with that observed in IgEpositive PBMCs from the allergic patients (Fig 1).21 There was a highly significant decrease in the use of VH3 by IgE-positive B cells in the allergic nasal mucosa (34%) compared with that expected on the basis of the normal repertoire in PBMCs (55%) observed by previous workers (P < .005, x2 analysis).21 This was not, however, significant when compared with that observed in the allergic PBMCs (50%; P > .1, x2 analysis). There was also a highly significant increase in the use of VH5 in the nasal mucosa (29%) compared with that observed by previous researchers in normal PBMCs (2.9%; P < .005, x2 analysis). The increased use of VH5 in the allergic nasal mucosa was significant when compared with the 8% VH5 use observed in the allergic IgEpositive PBMCs (P < .025, x2 analysis). There was no significant difference in the use of VH5 in the allergic PBMCs compared with that expected on the basis of the normal PBMC repertoire (8% vs 2.9%; P > .1, x2 analysis).

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Basic and clinical immunology

FIG 2. Distribution of somatic mutations across IgE VH. A, Somatic mutations identified in 17 allergic nasal mucosa VH5 sequences were pooled. Hotspots of mutation were identified on the basis of the percentage variability at each codon. B, Nineteen non-VH5 sequences randomly chosen from the same cohort of allergic patients were subjected to the same analysis.

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Analysis of replacement/silent mutation ratio values Because 3 of the 4 apparently nonintrinsic hotspots of mutation in the VH5 sequences were unconventionally present in the framework region, further analysis of the ratio of replacement to silent mutations (R/S values) in the different sequences was carried out. The non-VH5 group of sequences exhibited R/S values that were consistent with conventional antigen selection, with a significant difference between the CDR and the FWR (CDR, 3.46; FWR, 1.67; P < .025, x2 analysis). In stark contrast, there was no significant difference between the R/S values in the CDR and the FWR of the VH5 group of sequences (CDR, 2.38; FWR, 2.06; P > .1, x2 analysis). Because the VH5 FWR R/S value was higher than expected and also because 3 of the 4 nonintrinsic muta-

TABLE I. The direction of somatic mutation at hotspots in VH IgE sequences from the allergic nasal mucosa suggests the presence of 4 nonintrinsic hotspots in VH5 sequences Hotspot

From

To

VH 5 Lys 23(1)

AAG

Gly 24(2)

GGT

Ser 28(3)

AGC

Thr 30(2)

ACC

Ser 31(2)

AGC

Ser 31(3)

AGC

Tyr 32(3)

TAC

Tyr 52(2)

TAT

Asp 53(3)

GAT

Ser 57 (3)

AGC

Ala 71(2)

GCC

Ser 76(2)

AGC

Met 89(3)

ATG

CAG GAG TAG GAT GCT GTT AGA AGG AGT AAC AGC ATC AAC ACC ATC AGA AGG AGT TAA TAG TAT TCT TGT TTT GAA GAC GAG AGA AGG AGT GAC GGC GTC AAC ACC ATC ATA ATC ATT

Non-VH5 Ser 31(2)

AGC

AAC ACC ATC

O*

Ey

Gln Glu Stop Asp Ala Val Arg Arg Ser Asn Ser Ile Asn Thr Ile Arg Arg Ser Stop Stop Tyr Ser Cys Phe Glu Asp Glu Arg Arg Ser Asp Gly Val Asn Thr Ile Ile Ile Ile

6 2 0 0 5 1 3 2 4 1 4 2 7 5 0 1 0 6 0 0 4 1 1 5 4 2 1 0 0 7 0 0 6 6 1 0 4 0 1

2 4 2 2 0 4 1 2 6 1 1 5 7 4 1 1 1 5 0 1 3 1 4 2 2 4 1 1 1 5 1 1 4 4 2 1 3 2 0

Asn Thr Ile

5 0 0

3 2 0

Nonintrinsic

Nonintrinsic

Nonintrinsic

Nonintrinsic

*O denotes the observed number of somatic mutations.  E denotes the expected direction of mutation on the basis of previous research.22

tions were present in FWR1, analysis of the R/S values of the individual FWRs in the VH5 group of sequences was also carried out. FWR1 exhibited an R/S value of 2.16, FWR2 exhibited an R/S value of 1.06, and FWR3 exhibited an R/S value of 2.61, suggesting that although FWR2 was conventionally conserved in the VH5 sequences, FWR1 and FWR3 contributed to the unusual overall FWR R/S value observed in the VH5 sequences.

Basic and clinical immunology

although neither identified any further hotspots of mutation to that evident from Fig 2. Of the hotspots of mutation identified in Fig 2, those at Met 40 and Tyr 56 in the VH5 sequences and at codons 52a, 56, and 84 in the non-VH5 sequences, when examined in more detail, were disregarded because the mutations were spread evenly across the 3 nucleotide positions of the codon and had very different effects on the amino acid. The apparent hotspots at codon 52b and also codon 82a in the non-VH5 sequences were also disregarded because codon 52b was mutated in both of only 2 sequences that used this polymorphic codon, and the mutations at codon 82a were divided between 3 Asn and 3 Ser residues, such that there were insufficient mutations of each for analysis. The exact nature of the remaining apparent hotspots of mutation was examined in detail to determine whether the direction of mutation at each codon reflected the accepted trends of somatic hypermutation, as defined by previous authors.22 Those mutations that conformed to such a profile were defined as intrinsic, whereas those that clearly differed were defined as nonintrinsic and therefore likely to have been selected in response to antigen (Table I).22 The only hotspot apparent in the non-VH5 sequences was a commonly identified hotspot, Ser 31,22 which was classified as intrinsic. In contrast, although 9 of the 13 remaining hotspots identified in the VH5 sequences also appeared to be intrinsic in nature, importantly, 4 apparent nonintrinsic hotspots were also identified. This is consistent with the non-VH5 IgE molecules having been targeted toward a wide range of antigens, whereas the identification of nonintrinsic hotspots of mutation in the VH5 sequences suggests that the VH5 IgE molecules were targeted toward a limited number of antigens. The 4 nonintrinsic mutations evident in the VH5 sequences were present at Lys 23(1), Gly 24(2), and Thr 30(2), each in FWR1, and also at Tyr 52(2), which was found in CDR2. According to Kabat’s numbering, CDR1 incorporates codons 31 through 35, and CDR2 incorporates codons 50 through 65. FWR1 incorporates codons 1 through 30, FWR2 incorporates codons 36 through 49, and FWR3 incorporates codons 66 through 95.

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TABLE II. Regional diversification of IgE and IgA VH sequences Number of sequences Biopsy Isotype specimen VH1 VH2 VH3 VH4 VH5 VH6 VH7 Total

IgE IgA

CA30A CA30B CA30A CA30B

0 0 1 2

0 0 0 0

0 1 8 6

0 3 1 1

11 0 2 0

0 0 0 1

0 0 0 0

11 4 12 10

Basic and clinical immunology

IgA VH gene use If a superantigen was involved in the biased use of VH5, this might be apparent in isotypes other than IgE. We first examined this possibility by using 2 adjacent biopsy specimens (CA30A and CA30B) from patient CA30. As shown in Table II, all 11 IgE VH sequences from biopsy specimen A but none of the 4 sequences from biopsy specimen B were VH5. IgA exhibited a similar pattern, with 2 of 12 IgA VH sequences in biopsy specimen A but none of 10 in biopsy specimen B being VH5. Combining IgE and IgA results, 57% (13/37) of sequences in biopsy specimen A were VH5, whereas 0% (0/14) of sequences in biopsy specimen B was VH5. None of 13 VH5 sequences in CA30 were related to any of the others, suggesting polyclonal activation of B cells expressing VH5 sequences locally in the region of biopsy specimen A. The results for CA30 demonstrate the linkage between VH5 bias in IgE and IgA and suggest that a B-cell superantigen might influence the selection of VH in a local manner. The FWR and CDR R/S values for IgA VH5 and nonVH5 were determined and compared. R/S values for IgA non-VH5 sequences in CA30 were comparable with those of IgE non-VH5 sequences (CDR, 4; FWR, 1.4) and thus indicative of normal antigen selection. However, as observed for IgE VH5, the FWR and CDR R/S values for IgA VH5 sequences were similar (CDR, 4; FWR, 4.3). On analysis of 73 IgA VH sequencesà in 2 of the biopsy specimens from the original cohort (TL25 and CA30) plus 2 additional biopsy specimens (JC1 and IB4) from allergic patients, we observed VH5 to be significantly higher than in normal PBMCs (14% vs 2.9%; P < .005, x2 analysis).21 The greater abundance of IgA-expressing compared with IgE-expressing B cells in the nasal mucosa allowed us to determine the IgA VH use in the nasal mucosa of healthy nonallergic subjects. Of 55 sequences§ from 4 nonallergic donors, we found a 9% frequency of VH5, which is also significantly different from that seen in normal PBMCs (P <.05, x2 analysis).21

FIG 3. Location of nonintrinsic hotspots in the 3-dimensional structure. Hotspots Lys23, Gly24 and Thr30 (FWR1), and Tyr52 (CDR2) in VH5 are shown in yellow on a homology model of VH5-51. Exposed hydrophobic residue Ile75 is shown in green. CDRs are indicated, encompassing both Kabat and Chothia24 definitions. The remaining VH and VL FWRs are shown in dark and light blue.

Location of nonintrinsic hotspots in the structure of IgE VH5 The 4 nonintrinsic hotspots occurred at Lys 23(1), Gly 24(2), Thr 30(2), and Tyr 52(2). Of these, 3 were unusually situated within FWR1, with only Tyr 52(2) in CDR2. Therefore it would seem likely that the putative superantigen has contact with FWR1 and CDR2, although the R/S values imply that FWR3 might also be involved. The B-cell superantigen protein A has been shown to bind similarly to VH3, interacting with FWR1, FWR3, and CDR2.12,13,15,23 Because no crystal structure of a VH5 is available, we constructed a model on the basis of the known structure of the most closely matched antibody. There are 2 VH5 genes, VH5-51 and VH5-a, but only the former is expressed in the majority of the population. Fig 324 shows Lys 23, Gly 24, and Thr 30 in FWR1 and Tyr 52 in CDR2 in our model of VH5-51. When the locations of the 4 nonintrinsic hotspots are identified within this model, it is immediately apparent that they are clustered together at the edge of the conventional antigen-binding site, as defined by the 6 CDRs. Lys 23 and Gly 24 are adjacent to CDR1, and Thr 30 lies at the very boundary between FWR1 and CDR1; Tyr 52 lies at the boundary between FWR2 and CDR2. Also shown in Fig 3 is residue Ile 75, a hydrophobic surface residue in an unusually exposed location but conserved in the vast majority of VH5 sequences. In fact, all VH2, VH3, VH4, and VH6 germline sequences have lysine at this position, whereas it is isoleucine only in VH5. This cluster of residues might thus define at least a part of the site at which a putative superantigen might interact with IgE VH5. DISCUSSION

àNucleotide sequences submitted to Genbank, accession numbers AY971069AY971142 §Nucleotide sequences submitted to Genbank, accession numbers AY971012AY971068

In our previous study of somatic hypermutation in VH sequences in IgE-expressing B cells in the nasal mucosa of patients with allergic rhinitis, we observed that 2 of a total

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antibodies that recognize the carbohydrate I/i red blood cell antigen are restricted to the VH4-34 gene, and residues 23-25 in FWR1 are implicated in binding.25 In addition, the crystal structure of the complex between a rheumatoid factor antibody and its autoantigen, IgG Fc, involved residues at the FWR1/CDR1 and FWR2/CDR2 boundaries, enabling the CDR to remain available for conventional antigen binding.26,27 Remarkably, exactly the same features that we have observed in IgE VH5 in allergic rhinitis were previously reported in asthma.1-5 For example, VH5 represented 29% and 33% of the IgE sequences in this allergic rhinitis study and a previous asthma study,2 respectively. Exactly the same nonintrinsic hotspots, Lys 23(1), Gly 24(2), Thr 30(2), and Tyr 52(2), were observed in asthma1 as in this study of allergic rhinitis but differing from those in atopic dermatitis (Gly 35 and Thr 95).22 The similarities between allergic rhinitis and asthma might not be surprising, given that the upper and lower airways are physically contiguous and exposed to some of the same aeroallergens, but are important, given the differing susceptibility of some individuals to asthma and others to rhinitis and that many individuals have both conditions. It might be fortuitous that the same minor VH family appears to be overexpressed in both allergic rhinitis–asthma and (at least in one of the 2 studies) atopic dermatitis. The selection of different FWR mutations in the IgE VH5 in atopic dermatitis, however, might point to the action of different superantigens. What might be the putative B-cell superantigen associated with allergic rhinitis? About 37% of the population carries Staphylococcus aureus in the nasal mucosa.28 Certain S aureus enterotoxins are characterized as T-cell superantigens that stimulate B-cell proliferation, IgE synthesis, or both.29-31 Some of these or others might act as B-cell superantigens. If a B-cell superantigen binds as we suspect to FWR1 of VH5, an allergen might still bind to the CDR on the immunoglobulin. This could have important consequences for the activation of B cells expressing VH5 and also the mast cells and dendritic cells that capture IgE VH5 in the tissue. We thank Drs Rebecca Beavil, Pooja Takhar, Lyn Smurthwaite, and Graham Dunn (King’s College London) for helpful discussions and practical advice.

REFERENCES 1. Snow RE, Chapman CJ, Frew AJ, Holgate ST, Stevenson FK. Pattern of usage and somatic hypermutation in the VH5 gene segments of a patient with asthma: implications for IgE. Eur J Immunol 1997;27:162-70. 2. Snow RE, Djukanovic R, Stevenson FK. Analysis of immunoglobulin E VH transcripts in a bronchial biopsy of an asthmatic patient confirms bias towards VH5, and indicates local clonal expansion, somatic mutation and isotype switch events. Immunology 1999;98:646-51. 3. Snow RE, Chapman, Frew AJ, Holgate ST, Stevenson FK. Analysis of Ig VH region genes encoding IgE antibodies in splenic B lymphocytes of a patient with asthma. J Immunol 1995;54:5576-81. 4. Snow RE, Chapman CJ, Holgate ST, Stevenson FK. Clonally related IgE and IgG4 transcripts in blood lymphocytes of patients with asthma reveal differing patterns of somatic mutation. Eur J Immunol 1998;26:3354-61.

Basic and clinical immunology

of 3 clonal families were derived from the VH5 family, and we detected an IgA VH5 clone related to one of the families.19 This hinted at a biased use of VH5 in the tissue similar to that found in asthma and atopic dermatitis.1-5,9 We have therefore analyzed VH sequences from a large cohort of patients with allergic rhinitis to test this hypothesis. Our results have revealed a significant bias toward VH5, even when one biopsy (CA30), which exhibited the most extreme bias, was excluded. A similar trend in the PBMCs from a subgroup of these patients did not reach statistical significance. The greater bias in tissue suggests that a local event caused the VH5 bias. The similar trend in PBMCs might result from the displacement of VH5-expressing B cells from the tissue and dilution in the pool of normal B cells in the circulation. IgA VH sequences also exhibited a significant but less extreme bias toward VH5 compared with that seen in IgE sequences. The demonstration of VH5 bias in both IgE and IgA and colocalization in the nasal mucosa support the superantigen hypothesis. The putative superantigen might bind to the subset of B cells expressing VH5, leading to selective proliferation or rescue from apoptosis. The smaller bias in IgA VH5 might be due to the TH2 environment of the nasal mucosa, which favors class switching to IgE in rapidly dividing B cells. We observed an even smaller IgA VH5 bias in the normal nasal mucosa, which might hint that the bias precedes the development of allergy. Allergen-specific B cells might be further selected from this population. Although there was a significant IgE VH5 bias in the 62 sequences analyzed, these sequences came from a minority (5/11) of the patient population. This must not be taken to indicate that a superantigen is likely to have been acting in only the 5 patients, however. We have demonstrated previously19 and again here, exemplified by the comparison of biopsy specimens A and B from patient CA30, that there are not necessarily any identical sequences or even clonal relationships between cells in adjacent 1- to 2-mm3 biopsy specimens. These biopsy specimens (10-20 mg) are less than 1% of the inferior turbinates (average weight, approximately 2 g). Thus sequences derived from a single biopsy specimen are not necessarily representative of the tissues in individual patients. Our VH5 bias is based on sampling a population of patients. The absence of VH5 sequences in 6 of 11 of the biopsy specimens does not imply that there are none elsewhere in the tissue. Superantigens acting in other regions of the tissue might still contribute to the ‘‘VH5-negative patients’’’ symptoms of allergy. Our comparison of VH5 and non-VH5 R/S values and the positions of the nonintrinsic mutations within FWR, notably FWR1, provide additional evidence to support the superantigen hypothesis. Moreover, the 3 nonintrinsic hotspots in FWR1, along with one at the edge of CDR2, are clustered in our model of VH5. These mutations might increase the affinity of the putative superantigen for VH5. Similar interactions involving the edge of the conventional antigen-binding site and adjacent FWR residues have also been observed in other superantigen-antibody complexes:

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5. Snow RE, Chapman CJ, Stevenson FK. Allergen recognition sites in immunoglobulin E from patients with asthma. In: Holgate ST, Busse WW, editors. Lung biology in health and disease. New York: Marcel Dekker Inc; 1989. p. 323-41. 6. Cook GP, Tomlinson IM. The human immunoglobulin VH repertoire. Immunol Today 1995;16:237-42. 7. Chang B, Casali P. The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement. Immunol Today 1994;15:367-73. 8. Brezinschek H-P, Brezinschek RI, Lipsky PE. Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J Immunol 1995;155P:190-202. 9. Van der Stoep N, Van der Linden J, Logtenberg T. Molecular evolution of the human immunoglobulin E response: high incidence of shared mutations and clonal relatedness among e VH5 transcripts from three unrelated patients with atopic dermatitis. J Exp Med 1993;177:99-107. 10. Edwards MR, Brouwer W, Choi CHY, Ruhno J, Ward RL, Collins AM. Analysis of IgE antibodies from a patient with atopic dermatitis; biased V gene usage and evidence for polyreactive IgE heavy chain complementarity-determining region 3. J Immunol 2002;168:6305-13. 11. Janezic A, Chapman CJ, Snow RE, Hourihane JO, Warner JO, Stevenson FK. Immunogenetic analysis of heavy chain variable regions of IgE from patients allergic to peanuts. J Allergy Clin Immunol 1998;101:391-6. 12. Silverman GJ, Goodyear CS. A model B-cell superantigen and the immunobiology of B lymphocytes. Clin Immunol 2002;102:117-34. 13. Potter KN, Li Y-C, Capra JD. Staphylococcal protein A simultaneously interacts with framework region 1, complementarity determining region 2, and framework region 3 on human VH3 encoded immunoglobulins. J Immunol 1996;157:2982-8. 14. Berberian L, Goodglick L, Kipps TJ, Braun J. Immunoglobulin VH3 gene products: natural ligands for HIV gp120. Science 1993;261:1588-91. 15. Graille ME, Stura A, Corper AL, Sutton BJ, Taussig MJ, Charbonnier J-B, et al. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity. Proc Natl Acad Sci U S A 2000;97:5399-404. 16. Shlomchik MJ, Marshak-Rothstein A, Wolfowicz CB, Rothstein TL, Weigert MG. The role of clonal selection and somatic mutation in autoimmunity. Nature 1987;328:805-11. 17. Rogozin IB, Pavlov YI. Theoretical analysis of mutation hotspots and their DNA sequence context specificity. Mut Res 2003;544:65-85. 18. Ellyard JI, Avery DT, Phan TG, Hare NJ, Hodgkin PD, Tangye SG. Antigen-selected, immunoglobulin-secreting cells persist in human spleen and bone marrow. Blood 2004;103:3805-12.

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19. Coker HA, Durham SR, Gould HJ. Local somatic hypermutation and class switch recombination in the nasal mucosa of allergic rhinitis patients. J Immunol 2003;171:5602-10. 20. Durham SR, Ying S, Varney VA, Jacobson MR, Sudderick RM, Mackay IS, et al. Cytokine messenger RNA expression for IL-3, IL-4, IL-5 and granulocyte/macrophage-colony-stimulating factor in the nasal mucosa after local allergen provocation: relationship to tissue eosinophilia. J Immunol 1992;48:2390-4. 21. Brezinschek H-P, Dorner T, Monson NL, Brezinshek RI, Lipsky PE. The influence of CD40-CD154 interactions on the expressed human VH repertoire: analysis of VH genes expressed by individual B cells of a patient with X-linked hyper-IgM syndrome. Int Immunol 2000;12: 767-75. 22. Betz AG, Neuberger MS, Milstein C. Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes. Immunol Today 1993;14:405-11. 23. Randen I, Potter KN, Li Y, Thompson KM, Pascual V, Forre O, et al. Complementarity-determining region 2 is implicated in the binding of staphylococcal protein A to human immunoglobulin VHIII variable regions. Eur J Immunol 1993;23:2682-6. 24. Chothia C, Lesk AM. Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol 1987;196:901-17. 25. Potter KN, Hobby P, Klijn S, Stevenson FK, Sutton BJ. Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded immunoglobulins in recognition of the red cell I antigen. J Immunol 2002;16:3777-82. 26. Corper AL, Sohi MK, Bonagura VR, Steinitz M, Jefferis R, Feinstein A, et al. Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction. Nat Struct Biol 1997;4:374-81. 27. Sutton BJ, Corper AL, Bonagura V, Taussig MJ. The structure and origin of rheumatoid factors. Immunol Today 2000;21:177-83. 28. Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev 1997;10:505-20. 29. Hofer MF, Harbeck RJ, Schlievert PM, Leung DYM. Staphylococcal toxins augment specific IgE responses by atopic patients exposed to allergen. J Invest Dermatol 1999;112:171-6. 30. Jabara HH, Geha RS. The superantigen syndrome toxin-1 induces CD40 ligand expression and modulates IgE isotype switching. Int Immunol 1996;8:1503-10. 31. Bachert C, Van Zele T, Gavaert P, De Schrijver L, Van Cauwenberge P. Superantigens in nasal polyps. Curr Allergy Asthma Rep 2003;3: 523-31.

Basic and clinical immunology

Antibody responses against galactocerebroside are potential stage-specific biomarkers in multiple sclerosis Til Menge, MD,a Patrice H. Lalive, MD,a Hans-Christian von Bu¨dingen, MD,a,b Bruce Cree, MD, PhD,a Stephen L. Hauser, MD,a and Claude P. Genain, MDa San Francisco, Calif, and Zu¨rich, Switzerland

Background: Galactocerebroside, the major glycolipid of central nervous system myelin, is a known target for pathogenic demyelinating antibody responses in experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Objective: To address the importance of anti-galactocerebroside (a-GalC) antibodies in MS and to evaluate them as biomarkers of disease. Methods: a-GalC IgGs were quantified from sera of patients with MS and in marmoset EAE by a new immunosorbent assay. Results: We report a significant difference in serum a-GalC IgG titers between patients with relapsing-remitting (RR)–MS and healthy controls (HCs; P < .001). The frequencies of a-GalC antibody-positive subjects (a-GalC titers $ mean HC titers 1 3 SD) are also significantly elevated in RR-MS compared with HC (40% vs 0%; P = .0033). Immunoaffinity purified a-GalC IgGs from human serum bind to cultured human oligodendrocytes, indicating that the ELISA detects a biologically relevant epitope. Corroborating these findings, a-GalC antibody responses in marmoset EAE were similarly found to be specifically associated with the RR forms and not the peracute or progressive forms, in contrast with other anti-myelin antibodies (P = .0256). Conclusion: (1) a-GalC antibodies appear MS-specific and are not found in healthy subjects, unlike antibodies against myelin proteins; (2) when present, a-GalC antibodies identify mostly RR-MS and may be an indicator of ongoing disease activity. This novel assay is a suitable and valuable method to increase accuracy of diagnosis and disease staging in MS. (J Allergy Clin Immunol 2005;116:453-9.)

Key words: Galactocerebroside, myelin antigens, autoantibody, multiple sclerosis, experimental allergic encephalomyelitis

From athe Department of Neurology, University of California San Francisco; and bthe Neurologische Klinik der Universita¨t Zu¨rich. Supported by grants from the National Institutes of Health (NS4678-01 to Dr Genain and AI43073-11 to Dr Hauser), the National Multiple Sclerosis Society (RG3370-A-3 and 3438-A-7 to Dr Genain), the Cure MS Now fund, the Lunardi Supermarkets, Inc, the Nancy Davis Center Without Walls, and Aventis Pharmaceuticals. Dr Menge and Dr Lalive are postdoctoral research fellows of the National Multiple Sclerosis Society. Disclosure of potential conflict of interest: T. Menge: named as inventor on patent application ‘‘Methods to diagnose and prognose multiple sclerosis,’’ filed by University of California San Francisco, which includes data from this work; received postdoctoral fellowship of the National Multiple Sclerosis Society (FG 1476-A-1); employed by University of California San Francisco. P. H. Lalive: named as inventor on patent application ‘‘Methods to diagnose and prognose multiple sclerosis,’’ filed by University of California San Francisco, which includes data from this work; received postdoctoral fellowship of the National Multiple Sclerosis Society (FG 1476-A-1); received grant/support from Swiss National Foundation (PBGEB-102918); employed by University of California San Francisco. H.-C. von Bu¨dingen: none disclosed. B. Cree: none disclosed. S. L. Hauser:

none disclosed. C. Genain: has done consulting work for Aventis Pharmaceuticals; named as the main inventor on a patent application ‘‘Methods to diagnose and prognose multiple sclerosis,’’ filed by University of California San Francisco, which includes data from this work; received grants/support from National Institutes of Health (NS4678-01), National Multiple Sclerosis Society (RG3370-A-3 and 3438-A-7); research contract with Aventis Pharmaceuticals; donations from the Cure MS Now Foundation and the Lunardi Supermarkets, Inc; employed by University of California San Francisco; on the speakers’ bureau for Biogenidec, Teva Pharmaceuticals, Serono, Inc. Received for publication January 7, 2005; revised March 9, 2005; accepted for publication March 11, 2005. Available online May 16, 2005. Reprint requests: Claude Genain, MD, Department of Neurology, Neuroimmunological Laboratories, C-440, University of California San Francisco, 513 Parnassus Ave, San Francisco CA 94143-0114. E-mail: [email protected]. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.03.023

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Multiple sclerosis (MS) is a chronic immune-mediated inflammatory demyelinating disease of the central nervous system (CNS) characterized by heterogeneity in clinical presentation and underlying pathological mechanisms.1 There is currently no easy paraclinical marker to diagnose MS subtypes and predict disease course accurately without lengthy periods of clinical follow-up. Several myelin autoantigens may serve as targets for the autoaggressive attack in MS—for example, myelin protein myelin/oligodendrocyte glycoprotein (MOG), expressed on the outermost lamellae of the myelin sheath and thus readily accessible to the immune machinery; and a major CNS myelin glycolipid, galactocerebroside (GalC), which accounts for 32% of the myelin lipid content. Both MOG and galactocerebroside are highly encephalogenic in various models of experimental autoimmune encephalomyelitis (EAE), the prototypic animal model for MS.2-4 Furthermore, passive antibody transfers in myelin basic protein (MBP)–primed animals5-9 and in vitro models have demonstrated the demyelinating properties of anti-galactocerebroside (a-GalC) and a-MOG antibodies.10-13 Antibody responses against these myelin targets are thus factors that potentially regulate

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Animals Abbreviations used AM: Acute monophasic CIS: Clinically isolated syndrome CNS: Central nervous system EAE: Experimental allergic encephalomyelitis GalC: Galactocerebroside a-GalC: Anti-galactocerebroside HC: Healthy control HR: Hazard ratio MBP: Myelin basic protein MOG: Myelin/oligodendrocyte glycoprotein MRI: Magnetic resonance imaging MS: Multiple sclerosis PP: Primary-progressive rMOG: Recombinant rat myelin/oligodendrocyte glycoprotein (extracellular domain) RR: Relapsing-remitting RT: Room temperature SP: Secondary-progressive

disease phenotype expression in the context of established CNS inflammation. The pathogenic involvement of anti-myelin antibodies in human MS is less well established, because antibody titers against the myelin proteins do not unequivocally differ between control populations and patients with MS.14-19 However, regardless of pathogenicity, antimyelin antibodies have recently been proposed as predictive disease markers.20 Here, we examined whether a-GalC antibodies could serve as disease markers in MS. We demonstrate for the first time that significantly elevated titers of a-GalC antibodies are specifically found in relapsing-remitting (RR)–MS, and not in early or progressive forms of the disease. In strong support of our clinical observations, longitudinal assessment of galactocerebroside reactivity during the course of relapsing EAE in marmosets indicates that appearance of antibodies against galactocerebroside is delayed with respect to disease onset.

Basic and clinical immunology

METHODS Patients and controls Sixty-five consecutive patients seen in our MS center, 51 meeting the diagnostic criteria for clinically definite MS,21 were recruited for this study: 20 with RR-MS, 15 secondary-progressive (SP)–MS, and 16 primary-progressive (PP)–MS (Table I). In addition, 14 patients had a clinically isolated syndrome (CIS), ie, a single clinical attack suggestive of CNS demyelination. Twenty volunteers served as healthy controls (HCs). Both untreated patients and patients treated with IFN-b and glatiramer acetate were included in this study, but those treated with glucocorticoids within 3 months or on immunosuppressive therapy within 6 months of phlebotomy were excluded. Blood was drawn by venipuncture and clotted serum stored at 240°C. Informed consent was obtained from the patients and HCs, and the study was conducted in accordance with Institutional Review Board approval.

Callithrix jacchus marmosets were cared for in accordance with the guidelines of the Institutional Animal Care and Usage Committee. EAE was induced by immunization with 100 mg human white matter homogenate as described.22 Plasma samples were obtained from EDTA-anticoagulated blood at baseline and at intervals of 2 to 4 weeks and stored at 240°C. The animals were scored every other day for the development of clinical signs and disability using a previously published scale.22

a-GalC ELISA Bovine brain–derived galactocerebroside (Matreya, Pleasant Gap, Pa) was dissolved in chloroform-methanol (2:1). For coating, galactocerebroside was air-dried, stepwise resuspended in 65°C hot ethanol (50% vol/vol) at a final concentration of 50 ug/mL, with 100 uL added to wells of Polysorb 96-well microtiter plates (Nunc, Rochester, NY), and incubated uncovered overnight at room temperature (RT) for solvent evaporation. Plates were washed with double-distilled H2O and blocked with 1% BSA (A7030; Sigma, St Louis, Mo) in PBS (ELISA buffer) for 2 hours at RT. After washing with PBS and ddH2O, 100 uL of either human serum samples, diluted 1:40 in ELISA buffer, or C jacchus samples, diluted 1:100, were incubated in triplicate overnight at 4°C. Background binding of each sample was controlled for on blocked wells without coated antigen. After washing, specific antibody binding was detected by an alkaline phosphate–labeled goat-anti-human IgG (A9544; Sigma) or by a horseradish peroxidase–conjugated rabbit-anti-monkey IgG (A2054; Sigma), diluted in ELISA buffer and incubated for 1 hour at RT. For human sera, binding was detected by reading the OD at 405 nm in a microplate reader (SpectraMax; Molecular Devices, Sunnyvale, Calif) after incubation with paranitrophenyl phosphate (Moss, Pasadena, Md) for 30 minutes in the dark at RT. The marmoset assay was developed with 3,3#,5,5#-tetramethylbenzidine (Pierce, Rockford, Ill) for 15 minutes at RT and the OD read at 450 nm wavelength. For specificity and sensitivity controls, a polyclonal rabbitanti-bovine galactocerebroside antiserum (G9152; Sigma) was used and antibody binding detected by a horseradish peroxidase–labeled goat-anti-rabbit IgG (A0545; Sigma). Quenching experiments were performed by overnight pre-incubation with solubilized galactocerebroside; galactocerebroside was air-dried and resuspended in 65°C hot ethanol at 200 ug/mL and further diluted in ELISA buffer to a final concentration of 2 ug/mL.

Anti-myelin protein antibody ELISA C jacchus antibodies against human MBP and recombinant rat (r)MOG, amino acids 1-12523 were coated to microtiter plates (Maxisorb; Nunc) overnight with 1 ug antigen per well. After washing and blocking with 3% BSA in PBS plus .05% Tween for 1 hour at 37°C, marmoset samples were incubated for 1 hour at 37°C and diluted 1:100 in 3% BSA in PBS plus .05% Tween. Antibody binding was detected by a peroxidase-labeled rabbit-anti-monkey IgG for 1 hour at 37°C.

Statistical analysis To express the results of the galactocerebroside assay, a signalto-background binding ratio was calculated as the ratio of OD (signal) over OD (background). Positive controls, ie, a human sample with strong binding signal, and negative controls, ie, ELISA buffer only, omitting serum, were included on each plate. For human samples, samples above the mean binding ratio 1 3 SD for the HC group were considered positive. In the marmoset assay, samples were considered positive for a binding ratio above 3 with ODGalC >0.1 and greater than 3-fold the baseline (unimmunized) sample. Statistical

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TABLE I. Characteristics of patients with MS and HCs Variable

HC

CIS

RR-MS

SP-MS

PP-MS

N Sex Female Male Age (y) Median Range Disease duration (mo) Median Range Expanded Disability Status Scale Median SD

20

14

20

15

16

10 10

9 5

15 5

9 6

10 6

51.0 28-71

36.0* 23-50

43.0 25-61

45.0 37-60

51.0 40-65

NA NA

NA NA

120 9-266

120 24-384

48 9-216

NA NA

1.5  0.9

2.0à 1.5

5.5 1.5

4.5 1.2

NA, Not applicable. *P < .05 if compared with HC and P < .01 if compared with PP-MS (ANOVA with Bonferroni correction for multiple comparisons).  P < .001 and àP < .01 if compared with SP-MS or PP-MS, respectively (Kruskal-Wallis test with Dunn post hoc test for multiple comparisons).

Antibody affinity purification Human serum was diluted in 10 mmol/L sodium phosphate buffer, pH 7.0 (SP buffer), and IgG was purified over a protein G column (HiTrap HP; Amersham, Piscataway, NJ). Bound IgG was eluted with 100 mmol/L glycine-HCl, pH 2.7, and dialyzed against the sodium phosphate buffer. For immunoaffinity purification of a-GalC antibodies, galactocerebroside was dissolved at 5.0 mg/mL in 65°C hot methanol and hydrophobically bound to a FF-octyl column (HiTrap; Amersham) as previously described.24 The IgG fraction was applied to this column and bound IgG eluted and dialyzed into PBS as described.

Immunohistochemistry The human oligodendrocytoma cell line HOG (kind gift of Dr Glyn Dawson), known to express galactocerebroside,25 was grown in monolayers. Cells were trypsinized and plated at a density of 20,000 cells/well onto chamber glass slides (Nunc); fixed in icecold methanol; blocked with 2% BSA and 2% FBS in PBS; and stained with human serum (1:50), rabbit antiserum (1:50), or 1006GalC (30 ug/mL), respectively, diluted in 1% BSA-PBS for 1 hour at RT and developed with fluorescein isothiocyanate–labeled anti-IgG secondary antibodies (F3512 for human, F9887 for rabbit; Sigma). Control slides omitting the first antibodies were included.

RESULTS Validation of the a-GalC assay The assay was validated by a rabbit antiserum reactive to bovine galactocerebroside, with reactivity detectable to

a titer of 1:12,800. Preincubation of the rabbit antiserum with galactocerebroside solubilized in ELISA buffer (maximal solubility concentration, 2 ug/mL in aqueous buffer) led to an 85% reduction in signal, proving specificity of the assay. A mAb reactive against MOG (8.18C5) did not react with the coated galactocerebroside, confirming the purity of the antigen. In serial dilutions of total IgG purified on protein G from either the human positive control or pooled immune C jacchus sera, the threshold of detection was 6.25 ug IgG per well. The interplate and intraplate coefficients of variation were 15% and 4%, respectively.

Detection of a-GalC IgG in patients with MS Quantitatively, significant differences in a-GalC antibody titers were found between HC and RR-MS (P < .001) as well as between patients with CIS and RR-MS (P < .05; ANOVA with Bonferroni correction for multiple comparisons; Fig 1, A). There was a trend suggesting a difference for the antibody titers between SP-MS and HC (P = .092). In contrast, there were no significant differences for a-GalC reactivity among the HC, CIS, and PP-MS subgroups. Even if the 2 patients with the highest binding ratios in the RR-MS group were excluded from the calculations, the difference in the reciprocal binding ratio compared with the HC group remained highly significant (P < .01). The threshold for positivity was 3.23 and is indicated in Fig 1, A (dashed line; see Methods). The frequencies of patients with RR-MS identified as a-GalC antibody– positive by this analysis were significantly higher compared with HC (40% vs 0%; P = .0033; Fisher exact test

Basic and clinical immunology

analysis was conducted by using STATA 7.0 (StataCorp LP, College Station, Tex) and GraphPad Prism 3.0 (GraphPad Software, San Diego, Calif). Categorical variables were compared by using the x2 test, continuous variables by using ANOVA, and ordinal variables by using the Kruskal-Wallis test. The Bonferroni method and the Dunn test were used to determine differences in between groups. Survival analysis was used to assess time-dependent variables. Because the binding ratios are not normally distributed, the binding ratio was transformed by using an inverse ratio to generate a normal distribution for parametric analysis.

FIG 1. Binding ratios and frequencies of a-GalC IgG responses in human MS and HCs. A, a-GalC IgG binding ratios for each disease subgroup. Solid lines (—) denote mean binding ratios; dashed line (--) denotes threshold of detection (mean binding ratio of HC 1 3 SD (see B). B, Frequencies of a-GalC IgG seropositivity in human sera.

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FIG 2. Immunostaining of HOG cells with affinity-purified human a-GalC IgG. A, Affinity purified a-GalC IgG (1006-GalC) at 30 ug/mL. B, Positive control (rabbit a-GalC antiserum) at 1:50 dilution. C, Staining with serum of 1006-GalC at dilution 1:50. D and E, Negative controls: fluorescein isothiocyanate–labeled anti-human and anti-rabbit IgG.

with Bonferroni correction for multiple independent comparisons). Again, there was a trend observed for a-GalC antibody positivity in SP-MS compared with HC (26.7% vs 0%; P = .026; not significant after correction for multiple independent comparisons). Other pairwise comparisons were not significant (Fig 1, B).

Immunoaffinity purification of a-GalC IgG and immunohistochemistry To assess the specificity and biological relevance of the ELISA assay, serum of 1 patient demonstrating a high a-GalC response (#1006) was subjected to immunoaffinity purification of a-GalC IgG. From 50 mL serum, 190 ug a-GalC IgG (1006-GalC) was extracted by a custommade galactocerebroside column. 1006-GalC reacted in the ELISA with a detection limit 62.5 ng specific IgG per well (0.625 ug/mL), and the signal could be quenched by soluble galactocerebroside (45% signal reduction). These galactocerebroside-purified IgGs showed staining of the human oligodendrocytoma cell line HOG identical to the control rabbit a-GalC specific antiserum (Fig 2, A and B). These results unequivocally show that galactocerebroside specific antibodies purified from human serum are cellsurface binding on oligodendrocytes, and indicate that the newly implemented ELISA assay system likely detects biologically relevant antibodies.

Basic and clinical immunology

Detection of a-GalC IgG responses in marmoset EAE Sequential sera of 20 animals immunized with human white matter homogenate were studied. Because of the outbred nature of the animals, the clinical course of EAE is not uniform: 9 animals displayed a RR-EAE disease course, and 2 animals did not remit during attacks but progressively worsened over time (similar to a PP course). Six animals were euthanized at onset of the first attack, termed acute monophasic (AM), and 2 of these had a peracute disease course rapidly progressing to a score of 4. An additional 3 animals were euthanized before the onset of clinical disease, at the time when pleocytosis was present in the cerebrospinal fluid, demonstrating presence of CNS inflammation. Clinical information is summarized in Table II. Antibodies against rMOG and MBP were detected in all but 1 of the animals regardless of their disease course,

including the preclinical animals (Table II). In contrast, a-GalC antibodies were detected only in animals with RR-EAE, and not during the first attack of AM-EAE, even in the severely affected animals or in animals displaying a progressive course (Table II). However, this could have resulted from the overall shorter observation period for these animals (median, 28 and 60 days postimmunization vs 70 days postimmunization for RR-EAE; Table II). The a-GalC antibody response appeared significantly later compared with antibody responses against the myelin protein rMOG and MBP in RR-EAE: median time lapse between immunization and appearance, 70 days for a-GalC vs 45 days for a-rMOG and 27 days for a-MBP (P = .0256; log rank test for equivalence of survival functions). A Cox proportional hazard model showed that the hazard ratios (HRs) for a-rMOG and a-MBP antibody responses were significantly different from the HR for a-GalC (HR a-rMOG = 5.56, P = .013; HR a-MBP = 12.76, P = .001; Fig 3), indicating that a-GalC antibodies occurred most distant from immunizations and thus onset of EAE in these animals.

DISCUSSION We present here a reproducible solid-phase assay for detection of galactocerebroside-specific IgG in human sera. These specific IgG were purified by means of a galactocerebroside immunoaffinity chromatography column and were shown to retain the ability to bind to a galactocerebroside epitope expressed on human oligodendrocytes and in vitro by ELISA. The assays previously described to measure such antibodies in MS18,19,26 identified differences between controls and MS for cerebrospinal fluid, but not serum, even with undiluted serum in a solid-phase radioimmunoassay.18 The most likely explanation for the differences we find between HC and MS is the stratification for MS subgroups, which was not examined in previous studies.18,19 Indeed, comparing all our 65 patients with MS as 1 group with HC showed no significant difference in the frequency of antibody-positive patients. The current a-GalC IgG assay is performed in serum at dilutions of 1:40 and above, which is considerably easier to access than cerebrospinal fluid and can be repeated

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TABLE II. Characteristics of C jacchus marmoset EAE and antibody status Animal

Disease Clinical onset Sacrifice Maximal clinical a-GalC First day a-rMOG First day a-MBP First day course (day PI) (day PI)y score (day PI)y IgG detected (PI)z IgG detected (PI)z IgG detected (PI)z

326-91 106-90 191-92 378-85 U062-02 185-99 U050-01 U057-02 U050-00 Median 6 SD

RR RR RR RR RR RR RR RR RR RR

14 16 16 16 43 7 21 32 21 21 6 11

120 112 112 112 97 86 86 82 78 98 6 16

127-93 U052-01 U025-00 U023-00 273-93 274-93 U021-99 346-92 Median 6 SD

CP CP AM AM AM AM AM AM AM

16 21 21 16 12 16 18 17 16 6 3

68 52 61 31 28 28 23 22 28 6 15

Preclinical Preclinical Preclinical

NA NA NA

38 31 23

U053-01 U061-02 U030-00

4 (120) 3 (96) 2 (20) 2 (40) 3 (84) 2 (56) 2 (57) 1.5 (73) 3 (38) 2.0 6 0.8 (97 6 16) 4 (56) 3 (40) 2 (57) 2 (28) 3.5 (24) 2 (20) 1.5 (23) 3 (22) 2.0 6 0.8 (23 6 14) 0 0 0

1 1 1 1 1 1 1 1 1

23 80 81* 26* 96 70 70 62 78 70 6 25

1 1 1 1 1 1 1 1 1

63 53 53* 26* 36 15 45 62 29 42 6 17

1 1 1 1 1 1 1 1 1

23 25 27* 56* 36 28 15 18 29 29 6 12

2 2 2 2 2 2 2 2

2 2 2 2 2 2 2 2

1 1 1 1 1 1 1 1

54* 36 42 28 28* 30* 20 23 28 6 8

1 1 1 1 1 1 1 1

26* 18 28 28 28* 30* 20 23 28 6 4

2 2 2

2 2 2

1 2 1

35 2 13

1 1 1

35 31 20

multiple times. Most significant, serum a-GalC are specific for MS, because they are not encountered in any of the controls, and practically never if at all in CIS. Although other neurological diseases were not examined, this finding at least indicates that, unlike for myelin proteins like MOG, serum positivity helps to distinguish patients with MS from healthy individuals. The intergroup differences are very significant, despite the relatively small number of subjects studied. The 65 patients were chosen randomly in consecutive order of presentation, and a-GalC measurements were performed in a blind fashion. In addition, we could rule out any confounding variable for age, sex, or disease duration. These observations imply that a-GalC antibodies can help stratify different MS subgroups, namely RR-MS, a novel finding with high clinical relevance. Patients with CIS by definition have had 1 apparent clinical attack, whereas patients with RR-MS are characterized by disease dissemination in time and space. A high proportion of CIS who present with brain magnetic resonance imaging (MRI) abnormalities will proceed to develop RRMS,27,28 and indeed, for many of those patients, subclinical MS or minor attacks may have been present for a considerable period. Thus it can be envisioned that detection of a-GalC antibodies may permit staging of MS forms according to time from the first demyelinating event. Because these antibodies appear to be characteristic

FIG 3. Time course of a-GalC and a-myelin protein IgG responses in immunized C jacchus. Serum dilutions, 1:100. (.) denotes onset of clinical signs; (-;-) denotes a-MBP positivity; (-*-) denotes a-rMOG positivity; (-n-) denotes a-GalC positivity. Significant levels for median onset postimmunization (pi) of antibody positivity were determined by a Cox proportional hazard model.

of established MS, their detection in patients with early MS and CIS could potentially help correct and achieve an earlier diagnosis of definite MS than with conventional criteria. The anti-myelin protein antibodies, on the other hand, have recently been described as potential predictors of early conversion in patients with CIS.20 Critical for interpretation of our clinical findings in the absence of longitudinal measurements in human MS was

Basic and clinical immunology

NA, Not applicable; PI, postimmunization. *Monthly blood draw only.  P < .001 for timing of euthanasia and maximal clinical scores between RR-EAE vs AM, respectively (2-tailed t test); maximal clinical score or time of clinical onset were not significantly different (P = .69 and .39, respectively; 2-tailed t test) àStatistical analysis provided in Fig 3.

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the study of chronic relapsing marmoset EAE, which best approximates MS complex pathophysiology. Specifically, we found that the a-GalC responses occurred distinctly after disease onset only in animals with RR forms of EAE (Table II; Fig 3). This was in contrast to the a-rMOG and a-MBP responses that occurred in all animals tested, in some cases even before clinical onset. These findings are in line with results from 2 other EAE models,29,30 in which a-GalC antibodies were also present in the early chronic stage of guinea pig EAE29 and occurred after the clinical onset and after the development of a-MBP antibodies.30 Reactivity against rMOG was not tested in either of these studies. Although the pathophysiological explanation for the delayed antibody response to galactocerebroside in MS and EAE is not known, several mechanisms may be postulated. First, glycolipids are not classic, MHCrestricted T-cell antigens but may elicit a TH1 response via CD1 presentation.31,32 CD1 expression has been demonstrated on astrocytes within MS lesions.33 Glycolipid antigens may be presented to T cells only once detached from the membrane bilayer, yet the degradation of myelin glycolipids by macrophages takes considerably longer than the breakdown of myelin proteins.34 Second, lipids as such may be haptens and have to be attached to carrier proteins to elicit an immune response.4 It has been proposed that MOG may serve as a carrier protein interacting with galactocerebroside within the cell membrane.35 These possibilities all may also explain the low titers of a-GalC antibodies, which are considerably lower in human beings compared with titers of antibodies against myelin proteins (Dr Menge, personal observation, ref 15), as in EAE models.29 Antibodies reactive against galactocerebroside may have demyelinating properties, at least experimentally in vitro10,12,13 and in vivo.6,9,36 Although our study did not aim at proving any functional disability associated with the presence of a-GalC antibodies in human beings, it is interesting to note that 40% of RR-MS cases studied have detectable a-GalC reactivity, which could potentially be indicative of a particular RR-MS group in terms of disease course and severity. In addition, a lesser proportion of patients with SP-MS than with RR-MS appear to be a-GalC antibody–positive. This could mean that a-GalC autoantibodies predominate during a yet to be defined window of time that overlaps between RR-MS and SP-MS, with a tendency to decrease during the neurodegenerative stage of SP-MS. Future studies with larger numbers of subjects and longitudinal measurements are needed to address whether these antibody responses are associated with clinical (Expanded Disability Status Scale, progression rate, treatment response) or paraclinical (magnetic resonance imaging burden of lesion) parameters, and to establish their prognostic significance. In conclusion, we have demonstrated that a-GalC antibodies are a predominant phenomenon of RR-MS, and that in a primate disease model, the a-GalC response occurs significantly later than a-myelin protein responses. This galactocerebroside assay is available as a paraclinical

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investigation, in combination with MRI. In line with other recent reports on humoral immunity in MS and EAE,20,37 these novel findings continue to underscore the value of a-myelin antibody assessment—both protein and glycolipid—as biomarkers that will be used in the near future for MS diagnostics, staging, and prognosis. We thank Salomon Martinez for expert animal work; Jerry Hernandez, Drew Dover, and Kevin Morgan for help analyzing the samples, and the clinical coordinators and neurologists at the University of California San Francisco MS Center for sample collection.

REFERENCES 1. Steinman L. Multiple sclerosis: a two-stage disease. Nat Immunol 2001; 2:762-4. 2. von Bu¨dingen HC, Villoslada P, Ouallet JC, Hauser SL, Genain CP. Immune responses against the myelin/oligodendrocyte glycoprotein in experimental autoimmune demyelination. J Clin Immunol 2001;21: 155-70. 3. Moore GR, Traugott U, Farooq M, Norton WT, Raine CS. Experimental autoimmune encephalomyelitis: augmentation of demyelination by different myelin lipids. Lab Invest 1984;51:416-24. 4. Raine CS, Traugott U, Farooq M, Bornstein MB, Norton WT. Augmentation of immune-mediated demyelination by lipid haptens. Lab Invest 1981;45:174-82. 5. Genain CP, Nguyen MH, Letvin NL, Pearl R, Davis RL, Adelman M, et al. Antibody facilitation of multiple sclerosis-like lesions in a nonhuman primate. J Clin Invest 1995;96:2966-74. 6. Fierz W, Heininger K, Schaefer B, Toyka KV, Linington C, Lassmann H. Synergism in the pathogenesis of EAE induced by an MBP-specific T-cell line and monoclonal antibodies to galactocerebroside or a myelin oligodendroglial glycoprotein. Ann N Y Acad Sci 1988;540:360-3. 7. Schluesener HJ, Sobel RA, Linington C, Weiner HL. A monoclonal antibody against a myelin oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease. J Immunol 1987;139:4016-21. 8. Linington C, Bradl M, Lassmann H, Brunner C, Vass K. Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein. Am J Pathol 1988;130:443-54. 9. Morris-Downes MM, Smith PA, Rundle JL, Piddlesden SJ, Baker D, Pham-Dinh D, et al. Pathological and regulatory effects of anti-myelin antibodies in experimental allergic encephalomyelitis in mice. J Neuroimmunol 2002;125:114-24. 10. Fry JM, Weissbarth S, Lehrer GM, Bornstein MB. Cerebroside antibody inhibits sulfatide synthesis and myelination and demyelinates in cord tissue cultures. Science 1974;183:540-2. 11. Raine CS, Johnson AB, Marcus DM, Suzuki A, Bornstein MB. Demyelination in vitro: absorption studies demonstrate that galactocerebroside is a major target. J Neurol Sci 1981;52:117-31. 12. Saida T, Saida K, Silberberg DH. Demyelination produced by experimental allergic neuritis serum and anti-galactocerebroside antiserum in CNS cultures: an ultrastructural study. Acta Neuropathol (Berl) 1979;48:19-25. 13. Menon KK, Piddlesden SJ, Bernard CC. Demyelinating antibodies to myelin oligodendrocyte glycoprotein and galactocerebroside induce degradation of myelin basic protein in isolated human myelin. J Neurochem 1997;69:214-22. 14. Lindert RB, Haase CG, Brehm U, Linington C, Wekerle H, Hohlfeld R. Multiple sclerosis: B- and T-cell responses to the extracellular domain of the myelin oligodendrocyte glycoprotein. Brain 1999;122:2089-100. 15. Reindl M, Linington C, Brehm U, Egg R, Dilitz E, Deisenhammer F, et al. Antibodies against the myelin oligodendrocyte glycoprotein and the myelin basic protein in multiple sclerosis and other neurological diseases: a comparative study. Brain 1999;122:2047-56. 16. Lampasona V, Franciotta D, Furlan R, Zanaboni S, Fazio R, Bonifacio E, et al. Similar low frequency of anti-MOG IgG and IgM in MS patients and healthy subjects. Neurology 2004;62:2092-4.

17. Mantegazza R, Cristaldini P, Bernasconi P, Baggi F, Pedotti R, Piccini I, et al. Anti-MOG autoantibodies in Italian multiple sclerosis patients: specificity, sensitivity and clinical association. Int Immunol 2004;16: 559-65. 18. Rostami AM, Burns JB, Eccleston PA, Manning MC, Lisak RP, Silberberg DH. Search for antibodies to galactocerebroside in the serum and cerebrospinal fluid in human demyelinating disorders. Ann Neurol 1987;22:381-3. 19. Kasai N, Pachner AR, Yu RK. Anti-glycolipid antibodies and their immune complexes in multiple sclerosis. J Neurol Sci 1986;75:33-42. 20. Berger T, Rubner P, Schautzer F, Egg R, Ulmer H, Mayringer I, et al. Antimyelin antibodies as a predictor of clinically definite multiple sclerosis after a first demyelinating event. N Engl J Med 2003;349:139-45. 21. Poser CM, Paty DW, Scheinberg L, McDonald WI, Davis FA, Ebers GC, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983;13:227-31. 22. Massacesi L, Genain CP, Lee-Parritz D, Letvin NL, Canfield D, Hauser SL. Active and passively induced experimental autoimmune encephalomyelitis in common marmosets: a new model for multiple sclerosis. Ann Neurol 1995;37:519-30. 23. Amor S, Groome N, Linington C, Morris MM, Dornmair K, Gardinier MV, et al. Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice. J Immunol 1994;153:4349-56. 24. Nakajima H, Katagiri YU, Kiyokawa N, Taguchi T, Suzuki T, Sekino T, et al. Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramideconjugated octyl sepharose CL-4B. Protein Expr Purif 2001;22:267-75. 25. Lily O, Palace J, Vincent A. Serum autoantibodies to cell surface determinants in multiple sclerosis: a flow cytometric study. Brain 2004; 127:269-79. 26. Ichioka T, Uobe K, Stoskopf M, Kishimoto Y, Tennekoon G, Tourtellotte WW. Anti-galactocerebroside antibodies in human cerebrospinal fluids determined by enzyme-linked immunosorbent assay (ELISA). Neurochem Res 1988;13:203-7.

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27. Frohman EM, Goodin DS, Calabresi PA, Corboy JR, Coyle PK, Filippi M, et al. The utility of MRI in suspected MS: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. Neurology 2003;61:602-11. 28. Brex PA, Ciccarelli O, O’Riordan JI, Sailer M, Thompson AJ, Miller DH. A longitudinal study of abnormalities on MRI and disability from multiple sclerosis. N Engl J Med 2002;346:158-64. 29. Tabira T, Endoh M. Humoral immune responses to myelin basic protein, cerebroside and ganglioside in chronic relapsing experimental allergic encephalomyelitis of the guinea pig. J Neurol Sci 1985;67: 201-12. 30. Lolli F, Liuzzi GM, Vergelli M, Massacesi L, Ballerini C, Amaducci L, et al. Antibodies specific for the lipid-bound form of myelin basic protein during experimental autoimmune encephalomyelitis. J Neuroimmunol 1993;44:69-75. 31. Beckman EM, Porcelli SA, Morita CT, Behar SM, Furlong ST, Brenner MB. Recognition of a lipid antigen by CD1-restricted alpha beta1 T cells. Nature 1994;372:691-4. 32. Shamshiev A, Donda A, Carena I, Mori L, Kappos L, De Libero G. Self glycolipids as T-cell autoantigens. Eur J Immunol 1999;29: 1667-75. 33. Battistini L, Fischer FR, Raine CS, Brosnan CF. CD1b is expressed in multiple sclerosis lesions. J Neuroimmunol 1996;67:145-51. 34. Lumsden CE. The neuropathology of multiple sclerosis. In: Vinken PI, Bruyn GW, editors. Handbook of clinical neurology. New York: Elsevier; 1970. p. 217-309. 35. Bernard CC, Johns TG, Slavin A, Ichikawa M, Ewing C, Liu J, et al. Myelin oligodendrocyte glycoprotein: a novel candidate autoantigen in multiple sclerosis. J Mol Med 1997;75:77-88. 36. Rosenbluth J, Schiff R, Liang WL, Dou WK, Moon D. Antibodymediated CNS demyelination: focal spinal cord lesions induced by implantation of an IgM anti-galactocerebroside-secreting hybridoma. J Neurocytol 1999;28:397-416. 37. Robinson WH, Steinman L, Utz PJ. Protein arrays for autoantibody profiling and fine-specificity mapping. Proteomics 2003;3:2077-84.

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Letters to the Editor Perilesional GM-CSF therapy of a chronic leg ulcer in a patient with common variable immunodeficiency

Letters to the Editor

To the Editor: Impaired wound healing characterizes multiple immunodeficiency states, including common variable immunodeficiency (CVID). Chronic, nonhealing wounds often ensue, with a considerable associated toll of pain, disfigurement, disability, and increased medical costs. These chronic wounds are notoriously difficult to treat, often prompting debridement and skin grafting. Here we report a patient with CVID with chronic leg ulcers who responded to perilesional GM-CSF therapy. The patient was a 68-year-old white man with CVID who had been on intravenous immunoglobulin replacement for 15 years. He also had diabetes mellitus (type 2), was on chronic steroid therapy for several years (for steroid-dependent asthma), and more recently was on methotrexate for chronic inflammatory myositis. In May 2002, he developed a right leg ulcer that did not heal and required skin grafting. In January 2004, he developed another ulcer over his right leg, after minor trauma, in an area distinct from the previous ulcer. The wound continued to progress despite debridement and local care, reaching a maximum size of 7 cm 3 5 cm (Fig 1, A). Bacterial culture from the ulcer (March 2004) grew Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Standard therapy, including local care and topical and systemic antibiotics, was ineffective. GM-CSF treatment (sargramostim, Leukine; Immunex Corp, Seattle, Wash) was begun in March 2004. He received 125 mg in 0.25 mL subcutaneously at each of 4 sites distributed evenly around the healthy edge of the ulcer (3, 6, 9 and 12 o’clock). This was repeated weekly for 4 weeks. The ulcer started improving a few days after the first injection. After 4 weeks, the patient was instructed to wash the ulcer with GM-CSF (3 mL saline containing 15 mg GM-CSF) twice daily for another 4 weeks (Fig 1, B). The ulcer healed completely in September 2004 (Fig 1, C). The patient reported no side effects from GM-CSF treatment. It is worth noting that his methotrexate dose was actually increased during this period, thus ruling out the possibility that the accelerated wound healing was a result of removal of immunosuppression. Normal wound healing can be divided into 3 different phases: inflammation, proliferation, and maturation.1 Inflammation is characterized by an influx of monocytes and neutrophils that remove the debris. This is followed by the proliferation of epithelial cells, fibroblasts, and endothelial cells. The resultant effect is to lay down connective tissue and restore tissue architecture. During maturation, the wound contracts and gains tensile strength. GM-CSF has been shown to influence all phases of wound healing. In addition to its well characterized effects on neutrophil and monocyte proliferation, migration, and 460

phagocytosis, GM-CSF stimulates the proliferation of keratinocytes and the differentiation of myofibroblasts.1-3 GM-CSF also increases wound tensile strength.4 The role of bacterial infections in chronic wounds is not well established. However, GM-CSF might prove useful in chronically infected wounds. Previous studies have demonstrated that GM-CSF enhances phagocyte killing of several microorganisms.5,6 In addition, GM-CSF was shown in animal studies to promote the healing of infected wounds.7 Interestingly, GM-CSF appears to be effective in chronic venous stasis ulcers, which one would not expect to be associated with decreased immune defense. Da Costa et al8 administered perilesional placebo or GMCSF in 200-mg or 400-mg doses. The healing rates were 57%, 61%, and 19% for the low-dose GM-CSF, high-dose GM-CSF, and placebo, respectively. GM-CSF was also used in a patient with CVID with bilateral leg ulcers caused by dermatofibromas, with a dramatic response.9 Our patient illustrates that in addition to venous stasis ulcers and dermatofibroma-associated ulcers, perilesional GM-CSF can be very effective in the treatment of infected leg ulcers in patients with CVID. Ammar Z. Hatab, MDa Deanna McDanel, PharmD, BCPSb Zuhair K. Ballas, MDa,c a Division of Allergy/Immunology Department of Internal Medicine C42/E-13, GH Carver College of Medicine University of Iowa b Department of Pharmaceutical Care University of Iowa Hospitals and Clinics and the College of Pharmacy University of Iowa c Iowa City VA Medical Center Iowa City, Iowa REFERENCES 1. Groves RW, Schmidt-Lucke JA. Recombinant human GM-CSF in the treatment of poorly healing wounds. Adv Skin Wound Care 2000;13:107-12. 2. Hancock GE, Kaplan G, Cohn ZA. Keratinocyte growth regulation by the products of immune cells. J Exp Med 1988;168:1395-402. 3. Gabbiani G. Modulation of fibroblastic cytoskeletal features during wound healing and fibrosis. Pathol Res Pract 1994;190:851-3. 4. Jyung RW, Wu L, Pierce GF, Mustoe TA. Granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor: differential action on incisional wound healing. Surgery 1994;115:325-34. 5. Reed SG, Nathan CF, Pihl DL, Rodricks P, Shanebeck K, Conlon PJ, et al. Recombinant granulocyte/macrophage colony-stimulating factor activates macrophages to inhibit Trypanosoma cruzi and release hydrogen peroxide: comparison with interferon gamma. J Exp Med 1987;166:1734-46. 6. Weiser WY, Van Niel A, Clark SC, David JR, Remold HG. Recombinant human granulocyte/macrophage colony-stimulating factor activates intracellular killing of Leishmania donovani by human monocyte-derived macrophages. J Exp Med 1987;166:1436-46. 7. Robson M, Kucukcelebi A, Carp SS, Hayward PG, Hui PS, Cowan WT, et al. Effects of granulocyte-macrophage colony-stimulating factor on wound contraction. Eur J Clin Microbiol Infect Dis 1994;13(suppl 2):S41-6. 8. Da Costa RM, Ribeiro Jesus FM, Aniceto C, Mendes M. Randomized, double-blind, placebo-controlled, dose-ranging study of granulocytemacrophage colony stimulating factor in patients with chronic venous leg ulcers. Wound Repair Regen 1999;7:17-25.

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FIG 1. A, Leg ulcer at initiation of GM-CSF therapy. B, Marked improvement after 4 weeks of GM-CSF. C, Complete healing after completion of therapy.

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9. Siddiqui FH, Biundo JJ Jr, Moore C, Ermitano ML, Ortigas AP, DeFrancesch F. Recombinant granulocyte macrophage colony stimulating factor (rhu-GM-CSF) in the treatment of extensive leg ulcers: a case report. Surgery 2000;127:589-92. Available online May 24, 2005. doi:10.1016/j.jaci.2005.04.008

Asthma caused by cyanoacrylate used in a leisure activity

Letters to the Editor

To the Editor: Acrylic compounds (acrylates, methacrylates, and cyanoacrylates) are volatile and chemically reactive agents used extensively in the manufacture of such products as adhesives, resins, solvents, and glues and in the health profession (dental prostheses and bone cement in orthopedics).1 These agents are well known to cause occupational asthma,2,3 as well as skin sensitization and irritation.4 Although acrylate glues are widely used in several activities of daily life, to our knowledge, there has been only one case reported of their causing respiratory symptoms out of the workplace.5 We report bronchial asthma caused by cyanoacrylate in a 55-year-old man whose hobby was making miniature planes, an activity that required the use of a cyanoacrylate adhesive paste. This exsmoker had never experienced asthmatic or rhinitis symptoms. A year before being seen at the clinic, he reported acute dyspnea during a weekend, which is when he normally worked on his model planes; this episode required emergency care, followed by a short course of oral and inhaled corticosteroid therapy. After this occasion, he stopped practicing his hobby and did not require medication, except for short-acting bronchodilator occasionally when his respiratory symptoms were exacerbated by physical exercise, cold temperature, and heavy smells. The results of skin prick tests to common aeroallergens were negative; spirometry showed an FEV1 of 2.9 L (100% of predicted value), a forced vital capacity of 3.5 L (100% of predicted value), and an FEV1/forced vital capacity ratio of 83% (normal). Methacholine bronchial responsiveness was normal (PC20 5 128 mg/mL; normal value >16 mg/mL in our laboratory). The subject underwent a specific inhalation challenge (SIC) according to a standardized procedure.6 Results are shown in Fig 1. On a control day, the patient was exposed to diluent paint by means of nebulization for 30 minutes. Spirometry, methacholine testing, and induced sputum performed after diluent exposure produced normal results. On 2 subsequent days, exposure to cyanoacrylate was carried out by asking the patient to mimic his leisure activity in a challenge room, spreading cyanoacrylate glue on a piece of cardboard for progressively longer periods of time (totals of 4 and 30 minutes of exposure on the 2 days). The test revealed a typical early late response. Induced sputum performed before and after SIC demonstrated pronounced eosinophlia after the cyanoacrylate challenge: eosinophil counts switched from 0.5% before SIC to 63% at the end of the last day of exposure.

FIG 1. Results of SIC.

Occupational asthma caused by acrylates has been described often, but in this case exposure took place only on occasion during a leisure activity; it is relevant to highlight that in this nonatopic subject specific reactivity to the sensitizer persisted even 1 year after cessation of exposure. This case underlines the sensitization strength of acrylates, proved as well by the fact that very intermittent exposure was enough to trigger bronchial asthma. This can also reasonably explain why the subject was cured after stopping exposure. Dr Mona-Rita Yacoub is a postdoctoral fellow supported by Asthma in the Workplace (Canadian Institutes of Health Research, Canadian Lung Association, Institut de recherche Robert-Sauve´ en sante´ et se´curite´ du travail du Que´bec). We thank L. Schubert for reviewing the manuscript. Mona-Rita Yacoub, MD Catherine Lemie`re, MD, MSc Jean-Luc Malo, MD Department of Chest Medicine Sacre´-Coeur Hospital 5400 West Gouin Blvd Montreal, Quebec, Canada H4J 1C5

REFERENCES 1. Piirila¨ P, Kanerva L, Keskinen H, Estlander T, Hyto¨nen M, Tuppurainen M. Occupational respiratory hypersensitivity caused by preparations containing acrylates in dental personnel. Clin Exp Allergy 1998;28:1404-11. 2. Quirce S, Baeza ML, Tornero P, Blasco A, Barranco R, Sastre J. Occupational asthma caused by exposure to cyanoacrylate. Allergy 2001;56:446-9. 3. Weytjens K, Cartier A, Lemiere C, Malo JL. Occupational asthma to diacrylate. Allergy 1999;54:289-90. 4. Kopferschmit-Kubler MC, Stenger R, Blaumeiser M, Eveilleau C, Bessot JC, Pauli G. Asthma, rhinitis and urticaria following occupational exposure to cyanoacrylate glues. Rev Mal Respir 1996;13:305-7. 5. Kopp SK, McKay RT, Moller DR, Cassedy K, Brooks SM. Asthma and rhinitis due to ethylcyanoacrylate instant glue. Ann Intern Med 1985;102: 613-5. 6. Cartier A, Bernstein IL, Burge PS, Cohn JR, Fabbri LM, Hargreave FE, et al. Guidelines for bronchoprovocation on the investigation of occupational asthma. J Allergy Clin Immunol 1989;84(suppl):823-9. Available online June 1, 2005. doi:10.1016/j.jaci.2005.04.015

462 Letters to the Editor

J ALLERGY CLIN IMMUNOL AUGUST 2005

9. Siddiqui FH, Biundo JJ Jr, Moore C, Ermitano ML, Ortigas AP, DeFrancesch F. Recombinant granulocyte macrophage colony stimulating factor (rhu-GM-CSF) in the treatment of extensive leg ulcers: a case report. Surgery 2000;127:589-92. Available online May 24, 2005. doi:10.1016/j.jaci.2005.04.008

Asthma caused by cyanoacrylate used in a leisure activity

Letters to the Editor

To the Editor: Acrylic compounds (acrylates, methacrylates, and cyanoacrylates) are volatile and chemically reactive agents used extensively in the manufacture of such products as adhesives, resins, solvents, and glues and in the health profession (dental prostheses and bone cement in orthopedics).1 These agents are well known to cause occupational asthma,2,3 as well as skin sensitization and irritation.4 Although acrylate glues are widely used in several activities of daily life, to our knowledge, there has been only one case reported of their causing respiratory symptoms out of the workplace.5 We report bronchial asthma caused by cyanoacrylate in a 55-year-old man whose hobby was making miniature planes, an activity that required the use of a cyanoacrylate adhesive paste. This exsmoker had never experienced asthmatic or rhinitis symptoms. A year before being seen at the clinic, he reported acute dyspnea during a weekend, which is when he normally worked on his model planes; this episode required emergency care, followed by a short course of oral and inhaled corticosteroid therapy. After this occasion, he stopped practicing his hobby and did not require medication, except for short-acting bronchodilator occasionally when his respiratory symptoms were exacerbated by physical exercise, cold temperature, and heavy smells. The results of skin prick tests to common aeroallergens were negative; spirometry showed an FEV1 of 2.9 L (100% of predicted value), a forced vital capacity of 3.5 L (100% of predicted value), and an FEV1/forced vital capacity ratio of 83% (normal). Methacholine bronchial responsiveness was normal (PC20 5 128 mg/mL; normal value >16 mg/mL in our laboratory). The subject underwent a specific inhalation challenge (SIC) according to a standardized procedure.6 Results are shown in Fig 1. On a control day, the patient was exposed to diluent paint by means of nebulization for 30 minutes. Spirometry, methacholine testing, and induced sputum performed after diluent exposure produced normal results. On 2 subsequent days, exposure to cyanoacrylate was carried out by asking the patient to mimic his leisure activity in a challenge room, spreading cyanoacrylate glue on a piece of cardboard for progressively longer periods of time (totals of 4 and 30 minutes of exposure on the 2 days). The test revealed a typical early late response. Induced sputum performed before and after SIC demonstrated pronounced eosinophlia after the cyanoacrylate challenge: eosinophil counts switched from 0.5% before SIC to 63% at the end of the last day of exposure.

FIG 1. Results of SIC.

Occupational asthma caused by acrylates has been described often, but in this case exposure took place only on occasion during a leisure activity; it is relevant to highlight that in this nonatopic subject specific reactivity to the sensitizer persisted even 1 year after cessation of exposure. This case underlines the sensitization strength of acrylates, proved as well by the fact that very intermittent exposure was enough to trigger bronchial asthma. This can also reasonably explain why the subject was cured after stopping exposure. Dr Mona-Rita Yacoub is a postdoctoral fellow supported by Asthma in the Workplace (Canadian Institutes of Health Research, Canadian Lung Association, Institut de recherche Robert-Sauve´ en sante´ et se´curite´ du travail du Que´bec). We thank L. Schubert for reviewing the manuscript. Mona-Rita Yacoub, MD Catherine Lemie`re, MD, MSc Jean-Luc Malo, MD Department of Chest Medicine Sacre´-Coeur Hospital 5400 West Gouin Blvd Montreal, Quebec, Canada H4J 1C5

REFERENCES 1. Piirila¨ P, Kanerva L, Keskinen H, Estlander T, Hyto¨nen M, Tuppurainen M. Occupational respiratory hypersensitivity caused by preparations containing acrylates in dental personnel. Clin Exp Allergy 1998;28:1404-11. 2. Quirce S, Baeza ML, Tornero P, Blasco A, Barranco R, Sastre J. Occupational asthma caused by exposure to cyanoacrylate. Allergy 2001;56:446-9. 3. Weytjens K, Cartier A, Lemiere C, Malo JL. Occupational asthma to diacrylate. Allergy 1999;54:289-90. 4. Kopferschmit-Kubler MC, Stenger R, Blaumeiser M, Eveilleau C, Bessot JC, Pauli G. Asthma, rhinitis and urticaria following occupational exposure to cyanoacrylate glues. Rev Mal Respir 1996;13:305-7. 5. Kopp SK, McKay RT, Moller DR, Cassedy K, Brooks SM. Asthma and rhinitis due to ethylcyanoacrylate instant glue. Ann Intern Med 1985;102: 613-5. 6. Cartier A, Bernstein IL, Burge PS, Cohn JR, Fabbri LM, Hargreave FE, et al. Guidelines for bronchoprovocation on the investigation of occupational asthma. J Allergy Clin Immunol 1989;84(suppl):823-9. Available online June 1, 2005. doi:10.1016/j.jaci.2005.04.015

Correspondence Cystic fibrosis gene mutations and chronic rhinosinusitis To the Editor: In their review of the definition, pathophysiology, treatment of rhinosinusitis by Meltzer et al,1 there is no discussion of the association between mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and chronic rhinosinusitis.2 Most of these patients do not meet diagnostic criteria for cystic fibrosis because their sweat chloride is not elevated above 60 mmol/L and they do not have evidence of 2 diseasecausing CFTR gene mutations. However, some of the patients with CFTR gene mutations and chronic rhinosinusitis reported by Wang et al2 did have other features of cystic fibrosis, such as infertility and infection with Pseudomonas aeruginosa. Identification of CFTR gene mutations in patient with chronic rhinosinusitis may potentially be of clinical importance in the future, because pharmacologic therapies to overcome defective CFTR function are under development.3 Clement L. Ren, MD Division of Pediatric Pulmonology and Allergy Room 4-3236 601 Elmwood Avenue University of Rochester Rochester, NY 14642 Editor’s note: This Correspondence has no accompanying reply. The authors of the Meltzer article chose not to reply, saying that the correspondence provides interesting information and is worthy of publication. REFERENCES 1. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas RA. Rhinosinusitis: establishing definitions for clinical research and patient care. J Allergy Clin Immunol 2004;114:S156-212. 2. Wang X, Moylan B, Leopold DA, Kim J, Rubenstein RC, Togias A, et al. Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population. JAMA 2000; 284:1814-9. 3. Zeitlin PL. Novel pharmacologic therapies for cystic fibrosis. J Clin Invest 1999;103:447-52. Available online May 16, 2005. doi:10.1016/j.jaci.2005.03.032

Leukotriene receptor antagonists are not as effective as intranasal corticosteroids for managing nighttime symptoms of allergic rhinitis To the Editor: I wish to comment on the supplement published in the November 2004 issue titled ‘‘Allergic Rhinitis After Hours: The Relevance and Consequences of Nighttime Symptoms.’’1 Nasal congestion associated with allergic rhinitis was identified as an important risk factor for sleep-disordered breathing, sleep fragmentation, daytime

somnolence, and fatigue, and it was noted that nasal congestion and other rhinitis symptoms follow a circadian rhythm, being more severe at night and early in the morning. Chronotherapy, the timed dosing of rhinitis medications to manage optimally the diurnal variation in nasal congestion and other rhinitis symptoms, was discussed, as well as the advantages and disadvantages of each medication class in relation to nighttime rhinitis symptoms; however, readers must draw their own conclusions regarding the most effective medication class. Three large, double-blind, randomized, placebo-controlled trials of montelukast 10 mg (MON), the only leukotriene receptor antagonist approved in the United States for seasonal allergic rhinitis, were discussed in the supplement.2-4 Compared with placebo, MON administered once daily at bedtime significantly reduced the nighttime symptom scores and peripheral blood eosinophil counts in all 3 trials. However, in contrast with the 3 large trials cited for montelukast, the intranasal corticosteroid trials discussed varied considerably in size, scope, and design. The supplement did not include a discussion of a recent large, randomized, double-blind, double-dummy, parallelgroup trial in 705 subjects with seasonal allergic rhinitis that compared the effectiveness of a 15-day course of intranasal fluticasone propionate aqueous nasal spray 200 mg (FPANS) with MON, both administered once daily in the evening.5 The results of this study showed that FPANS was consistently superior to MON for all daytime and nighttime symptoms, including nasal congestion. The nighttime symptoms (difficulty going to sleep, nighttime awakenings, nasal congestion on awakening) and scoring in this trial were the same as in the 3 montelukast trials cited. In addition, a randomized, double-blind, double-dummy, placebo-controlled, parallel-group trial in 62 subjects with seasonal allergic rhinitis compared subjects treated with FPANS, MON, MON 1 loratadine 10 mg (LOR), or placebo throughout the grass pollen season.6 Subjects assessed their rhinitis symptoms during the study and also underwent nasal biopsy before and during the season for evaluation of local eosinophilic inflammation. Both MON and MON 1 LOR were less effective than FPANS for control of daytime and nighttime symptoms, including nasal blockage, and for reduction of pollen-induced nasal eosinophilic inflammation on biopsy. Furthermore, when evaluating nighttime rhinitis treatment options, another important consideration is that many patients with rhinitis have mixed rhinitis, which may include a combination of seasonal allergic, perennial allergic, or perennial nonallergic rhinitis. Whereas MON is currently indicated only for relief of seasonal allergic rhinitis, FPANS is indicated for the management of nasal symptoms of all 3 of these types of rhinitis. I agree with the premise that therapy aimed at reducing nighttime nasal congestion is paramount for improving 463

Correspondence Cystic fibrosis gene mutations and chronic rhinosinusitis To the Editor: In their review of the definition, pathophysiology, treatment of rhinosinusitis by Meltzer et al,1 there is no discussion of the association between mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and chronic rhinosinusitis.2 Most of these patients do not meet diagnostic criteria for cystic fibrosis because their sweat chloride is not elevated above 60 mmol/L and they do not have evidence of 2 diseasecausing CFTR gene mutations. However, some of the patients with CFTR gene mutations and chronic rhinosinusitis reported by Wang et al2 did have other features of cystic fibrosis, such as infertility and infection with Pseudomonas aeruginosa. Identification of CFTR gene mutations in patient with chronic rhinosinusitis may potentially be of clinical importance in the future, because pharmacologic therapies to overcome defective CFTR function are under development.3 Clement L. Ren, MD Division of Pediatric Pulmonology and Allergy Room 4-3236 601 Elmwood Avenue University of Rochester Rochester, NY 14642 Editor’s note: This Correspondence has no accompanying reply. The authors of the Meltzer article chose not to reply, saying that the correspondence provides interesting information and is worthy of publication. REFERENCES 1. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas RA. Rhinosinusitis: establishing definitions for clinical research and patient care. J Allergy Clin Immunol 2004;114:S156-212. 2. Wang X, Moylan B, Leopold DA, Kim J, Rubenstein RC, Togias A, et al. Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population. JAMA 2000; 284:1814-9. 3. Zeitlin PL. Novel pharmacologic therapies for cystic fibrosis. J Clin Invest 1999;103:447-52. Available online May 16, 2005. doi:10.1016/j.jaci.2005.03.032

Leukotriene receptor antagonists are not as effective as intranasal corticosteroids for managing nighttime symptoms of allergic rhinitis To the Editor: I wish to comment on the supplement published in the November 2004 issue titled ‘‘Allergic Rhinitis After Hours: The Relevance and Consequences of Nighttime Symptoms.’’1 Nasal congestion associated with allergic rhinitis was identified as an important risk factor for sleep-disordered breathing, sleep fragmentation, daytime

somnolence, and fatigue, and it was noted that nasal congestion and other rhinitis symptoms follow a circadian rhythm, being more severe at night and early in the morning. Chronotherapy, the timed dosing of rhinitis medications to manage optimally the diurnal variation in nasal congestion and other rhinitis symptoms, was discussed, as well as the advantages and disadvantages of each medication class in relation to nighttime rhinitis symptoms; however, readers must draw their own conclusions regarding the most effective medication class. Three large, double-blind, randomized, placebo-controlled trials of montelukast 10 mg (MON), the only leukotriene receptor antagonist approved in the United States for seasonal allergic rhinitis, were discussed in the supplement.2-4 Compared with placebo, MON administered once daily at bedtime significantly reduced the nighttime symptom scores and peripheral blood eosinophil counts in all 3 trials. However, in contrast with the 3 large trials cited for montelukast, the intranasal corticosteroid trials discussed varied considerably in size, scope, and design. The supplement did not include a discussion of a recent large, randomized, double-blind, double-dummy, parallelgroup trial in 705 subjects with seasonal allergic rhinitis that compared the effectiveness of a 15-day course of intranasal fluticasone propionate aqueous nasal spray 200 mg (FPANS) with MON, both administered once daily in the evening.5 The results of this study showed that FPANS was consistently superior to MON for all daytime and nighttime symptoms, including nasal congestion. The nighttime symptoms (difficulty going to sleep, nighttime awakenings, nasal congestion on awakening) and scoring in this trial were the same as in the 3 montelukast trials cited. In addition, a randomized, double-blind, double-dummy, placebo-controlled, parallel-group trial in 62 subjects with seasonal allergic rhinitis compared subjects treated with FPANS, MON, MON 1 loratadine 10 mg (LOR), or placebo throughout the grass pollen season.6 Subjects assessed their rhinitis symptoms during the study and also underwent nasal biopsy before and during the season for evaluation of local eosinophilic inflammation. Both MON and MON 1 LOR were less effective than FPANS for control of daytime and nighttime symptoms, including nasal blockage, and for reduction of pollen-induced nasal eosinophilic inflammation on biopsy. Furthermore, when evaluating nighttime rhinitis treatment options, another important consideration is that many patients with rhinitis have mixed rhinitis, which may include a combination of seasonal allergic, perennial allergic, or perennial nonallergic rhinitis. Whereas MON is currently indicated only for relief of seasonal allergic rhinitis, FPANS is indicated for the management of nasal symptoms of all 3 of these types of rhinitis. I agree with the premise that therapy aimed at reducing nighttime nasal congestion is paramount for improving 463

464 Correspondence

J ALLERGY CLIN IMMUNOL AUGUST 2005

sleep and quality of life, but also that the most effective class of medication (ie, intranasal steroids) should be considered as initial therapy for relief of nighttime rhinitis symptoms. Robert A. Nathan, MD University of Colorado Health Sciences Center 2709 North Tejon Colorado Springs, CO 80907 Disclosure of potential conflict of interest: Dr Nathan receives grants/research support from Abbott, Altana, Aventis, AstraZeneca, Bayer, Berlex, Boehringer Ingelheim, Bristol-Myers Squibb, CIBA Geigy, Dura, Forest, GlaxoSmithKline, Immunex, Janssen, ParkeDavis, Pfizer, 3-M Pharmaceuticals, Proctor & Gamble, Roberts, Sandoz, Sanofi, Schering/Key, Sepracor, Sterling, Tap Pharm, Wallace, and Wyeth; is a consultant/scientific advisor for AMGEN, Altana, AstraZeneca, Aventis, Genentech, GlaxoSmithKline, Merck, Novartis, Pfizer, Schering/Key, Sepracor, and Viropharm; and is on the speakers’ bureau for AstraZeneca, Aventis, Genentech/Novartis, GlaxoSmithKline, Pfizer, and Schering/Key. Editor’s note: This Correspondence has no accompanying reply.

REFERENCES 1. Meltzer EO, editor. Allergic rhinitis after hours: the relevance and consequence of nighttime symptoms. J Allergy Clin Immunol 2004;114:S133-53. 2. Philip G, Malmstrom K, Hampel FC, Weinstein SF, LaForce CF, Ratner PH, et al. Montelukast for treating seasonal allergic rhinitis: a randomized, double-blind, placebo-controlled trial performed in the spring. Clin Exp Allergy 2002;32:1020-8. 3. Nayak AS, Philip G, Lu S, Malice M-P, Reiss TF. Efficacy and tolerability of montelukast alone or in combination with loratadine in seasonal allergic rhinitis: a multicenter, randomized, double-blind, placebocontrolled trial performed in the fall. Ann Allergy Asthma Immunol 2002;88:592-600. 4. van Adelsberg J, Phillip G, LaForce CF, Weinstein SF, Menten J, Malice M-P, et al. Randomized controlled trial evaluating the clinical benefit of montelukast for treating spring seasonal allergic rhinitis. Ann Allergy Asthma Immunol 2003;90:214-22. 5. Ratner PH, Howland WC, Arastu R, Philpot EE, Klein KC, Baidoo CA, et al. Fluticasone propionate aqueous nasal spray provided greater improvement in daytime and nighttime nasal symptoms of seasonal allergic rhinitis compared with montelukast. Ann Allergy Asthma Immunol 2003; 90:536-42. 6. Pullerits T, Praks L, Ristioja V, Lotvall J. Comparison of a nasal glucocorticoid, antileukotriene, and a combination of antileukotriene and antihistamine in the treatment of seasonal allergic rhinitis. J Allergy Clin Immunol 2002;109:949-55. Available online May 24, 2005. doi:10.1016/j.jaci.2005.03.045

Efficacy of ant venom immunotherapy and whole body extracts To the Editor: Golden1 presents a useful review of insect venom immunotherapy, but we disagree with his conclusion that imported fire ant (IFA) whole body extract (WBE) has been proven efficacious. Golden1 stresses the need to understand the natural history of sting allergy and the importance of controlled studies. We add to this the need for prospective design, adequate randomization, and double-blinding.

No prospective controlled study of IFA WBE treatment efficacy or prospective study of the natural history of IFA allergy has been published. Retrospective studies have selection bias, and the natural history of allergy can vary enormously between species. Large prospective studies have found reaction rates on re-exposure to range from 70% for the jack jumper ant through 50% for the honeybee and to 25% for the yellow jacket.2,3 Individuals allergic to IFA who react to multiple simultaneous stings (as often occurs) may experience few reactions when exposed to smaller doses of venom. Our randomized, double-blind, placebo-controlled trial of venom immunotherapy (VIT) provides a model that could be used to assess IFA WBE.3 How did we justify our study, and why did 2 respected university ethics committees approve? First, large studies that have demonstrated the safety of sting challenges after applying health and age exclusion criteria included a total of 238 patients with severe (Mueller grade IV) allergy. Second, the efficacy data from 2 controlled trials of VIT to prevent honey bee and vespid sting anaphylaxis are suboptimal by contemporary standards. One allocated treatment according to patient choice, with outcomes determined by reactions occurring outside hospital that were unobserved by the investigators. The other was single-blind and stratified by using factors that do not influence reaction risk. Finally, the efficacy of immunotherapy varies between species, and we could not be sure of the efficacy of jack jumper ant VIT. Without double-blinding, investigators can be misled by personal bias and subjective features such as itch, mild flushing, breathlessness, anxiety, and hypotension associated with bradycardia and anxiety. It is notable that we gave epinephrine to a patient who appeared to have a moderate reaction to an injection that was later revealed to be placebo.3 The inclusion of a blind placebo group is the only way to be certain that a sting challenge adequately tests treatment efficacy. This is an important ethical issue; we have observed the death of a man who believed he was protected by ant WBE, reinforcing the risks of a flawed evidence base. As explained, the exposure to a small number of IFA in a sting challenge may be insufficient to provoke anaphylaxis in many people with allergy. Furthermore, insect handling procedures may lead to a depletion of venom as assessed at the time of venom sac dissection.3 Imported fire ant WBE may be an effective treatment, because extracts contain venom proteins and quality control methods have been developed. However, uncertainties remain with regard to the natural history of IFA allergy and whether the dose of venom delivered by IFA WBE extracts is sufficient to confer protection. Simon G. A. Brown, MBBS, PhD, FACEMa Robert J. Heddle, MBBS, PhD, FRACP, FRCPAb Michael D. Wiese, BPharm, MClinPharmc Konrad E. Blackman, MBBS, FACEMc a University of Western Australia Fremantle Hospital

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J ALLERGY CLIN IMMUNOL AUGUST 2005

sleep and quality of life, but also that the most effective class of medication (ie, intranasal steroids) should be considered as initial therapy for relief of nighttime rhinitis symptoms. Robert A. Nathan, MD University of Colorado Health Sciences Center 2709 North Tejon Colorado Springs, CO 80907 Disclosure of potential conflict of interest: Dr Nathan receives grants/research support from Abbott, Altana, Aventis, AstraZeneca, Bayer, Berlex, Boehringer Ingelheim, Bristol-Myers Squibb, CIBA Geigy, Dura, Forest, GlaxoSmithKline, Immunex, Janssen, ParkeDavis, Pfizer, 3-M Pharmaceuticals, Proctor & Gamble, Roberts, Sandoz, Sanofi, Schering/Key, Sepracor, Sterling, Tap Pharm, Wallace, and Wyeth; is a consultant/scientific advisor for AMGEN, Altana, AstraZeneca, Aventis, Genentech, GlaxoSmithKline, Merck, Novartis, Pfizer, Schering/Key, Sepracor, and Viropharm; and is on the speakers’ bureau for AstraZeneca, Aventis, Genentech/Novartis, GlaxoSmithKline, Pfizer, and Schering/Key. Editor’s note: This Correspondence has no accompanying reply.

REFERENCES 1. Meltzer EO, editor. Allergic rhinitis after hours: the relevance and consequence of nighttime symptoms. J Allergy Clin Immunol 2004;114:S133-53. 2. Philip G, Malmstrom K, Hampel FC, Weinstein SF, LaForce CF, Ratner PH, et al. Montelukast for treating seasonal allergic rhinitis: a randomized, double-blind, placebo-controlled trial performed in the spring. Clin Exp Allergy 2002;32:1020-8. 3. Nayak AS, Philip G, Lu S, Malice M-P, Reiss TF. Efficacy and tolerability of montelukast alone or in combination with loratadine in seasonal allergic rhinitis: a multicenter, randomized, double-blind, placebocontrolled trial performed in the fall. Ann Allergy Asthma Immunol 2002;88:592-600. 4. van Adelsberg J, Phillip G, LaForce CF, Weinstein SF, Menten J, Malice M-P, et al. Randomized controlled trial evaluating the clinical benefit of montelukast for treating spring seasonal allergic rhinitis. Ann Allergy Asthma Immunol 2003;90:214-22. 5. Ratner PH, Howland WC, Arastu R, Philpot EE, Klein KC, Baidoo CA, et al. Fluticasone propionate aqueous nasal spray provided greater improvement in daytime and nighttime nasal symptoms of seasonal allergic rhinitis compared with montelukast. Ann Allergy Asthma Immunol 2003; 90:536-42. 6. Pullerits T, Praks L, Ristioja V, Lotvall J. Comparison of a nasal glucocorticoid, antileukotriene, and a combination of antileukotriene and antihistamine in the treatment of seasonal allergic rhinitis. J Allergy Clin Immunol 2002;109:949-55. Available online May 24, 2005. doi:10.1016/j.jaci.2005.03.045

Efficacy of ant venom immunotherapy and whole body extracts To the Editor: Golden1 presents a useful review of insect venom immunotherapy, but we disagree with his conclusion that imported fire ant (IFA) whole body extract (WBE) has been proven efficacious. Golden1 stresses the need to understand the natural history of sting allergy and the importance of controlled studies. We add to this the need for prospective design, adequate randomization, and double-blinding.

No prospective controlled study of IFA WBE treatment efficacy or prospective study of the natural history of IFA allergy has been published. Retrospective studies have selection bias, and the natural history of allergy can vary enormously between species. Large prospective studies have found reaction rates on re-exposure to range from 70% for the jack jumper ant through 50% for the honeybee and to 25% for the yellow jacket.2,3 Individuals allergic to IFA who react to multiple simultaneous stings (as often occurs) may experience few reactions when exposed to smaller doses of venom. Our randomized, double-blind, placebo-controlled trial of venom immunotherapy (VIT) provides a model that could be used to assess IFA WBE.3 How did we justify our study, and why did 2 respected university ethics committees approve? First, large studies that have demonstrated the safety of sting challenges after applying health and age exclusion criteria included a total of 238 patients with severe (Mueller grade IV) allergy. Second, the efficacy data from 2 controlled trials of VIT to prevent honey bee and vespid sting anaphylaxis are suboptimal by contemporary standards. One allocated treatment according to patient choice, with outcomes determined by reactions occurring outside hospital that were unobserved by the investigators. The other was single-blind and stratified by using factors that do not influence reaction risk. Finally, the efficacy of immunotherapy varies between species, and we could not be sure of the efficacy of jack jumper ant VIT. Without double-blinding, investigators can be misled by personal bias and subjective features such as itch, mild flushing, breathlessness, anxiety, and hypotension associated with bradycardia and anxiety. It is notable that we gave epinephrine to a patient who appeared to have a moderate reaction to an injection that was later revealed to be placebo.3 The inclusion of a blind placebo group is the only way to be certain that a sting challenge adequately tests treatment efficacy. This is an important ethical issue; we have observed the death of a man who believed he was protected by ant WBE, reinforcing the risks of a flawed evidence base. As explained, the exposure to a small number of IFA in a sting challenge may be insufficient to provoke anaphylaxis in many people with allergy. Furthermore, insect handling procedures may lead to a depletion of venom as assessed at the time of venom sac dissection.3 Imported fire ant WBE may be an effective treatment, because extracts contain venom proteins and quality control methods have been developed. However, uncertainties remain with regard to the natural history of IFA allergy and whether the dose of venom delivered by IFA WBE extracts is sufficient to confer protection. Simon G. A. Brown, MBBS, PhD, FACEMa Robert J. Heddle, MBBS, PhD, FRACP, FRCPAb Michael D. Wiese, BPharm, MClinPharmc Konrad E. Blackman, MBBS, FACEMc a University of Western Australia Fremantle Hospital

Correspondence 465

J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 2

Alma Street Fremantle, WA 6160, Australia b Department of Respiratory Medicine Flinders Medical Centre Bedford Park, Australia c Royal Hobart Hospital Hobart, Australia

REFERENCES 1. Golden DB. Insect sting allergy and venom immunotherapy: a model and a mystery. J Allergy Clin Immunol 2005;115:439-47. 2. Brown SGA, Franks RW, Baldo BA, Heddle RJ. Prevalence, severity, and natural history of jack jumper ant venom allergy in Tasmania. J Allergy Clin Immunol 2003;111:187-92. 3. Brown SGA, Wiese MD, Blackman KE, Heddle RJ. Ant venom immunotherapy: a double-blind, placebo-controlled, crossover trial. Lancet 2003;361:1001-6. Available online June 17, 2005. doi:10.1016/j.jaci.2005.04.025

Reply To the Editor: I appreciate the comments of Brown et al.1 It was clearly only by unintended oversight that the exemplary work of the authors was not cited in my review that focused on treatment strategies in the United States.2 Their work on the natural history of ant venom allergy and their controlled trial of ant venom immunotherapy are a model to which we should aspire.3,4 In contrast, there has been no controlled trial of imported fire ant (IFA) whole body extract (WBE) immunotherapy, and the natural history of IFA allergy is unknown. I am dismayed that my statements were construed to imply that IFA WBE has been proven efficacious. Comparison of IFA WBE and venom in vitro and by skin test suggests that although inferior to the venom, WBE contains sufficient allergen to provide reasonable diagnostic accuracy.5-8 For this reason, I stated, ‘‘For fire ant allergy, venom is the most accurate diagnostic material, but WBE have shown adequate diagnostic sensitivity.’’ My statement that ‘‘Fire ant immunotherapy is performed with WBE that contains sufficient venom allergens to provide reasonable clinical protection’’ was based on the reported content of venom allergens in IFA WBE. I did not state that WBE was as potent as venom.8 The report of clinical efficacy of IFA WBE by Freeman et al9 had the strengths of prospective sting challenge (instead of retrospective field sting reports) and a limited (but highly significant) control group. Still, Freeman et al9 concluded that ‘‘A controlled prospective trial of WBE versus placebo is needed.to help define the natural history of IFA hypersensitivity.’’ Of recent interest are reports of rush immunotherapy with IFA WBE to prevent systemic and large local reactions, but these too were uncontrolled.10,11 Together, the in vivo and in vitro evidence led to the belief that unlike the other Hymenoptera, IFA WBE does

contain sufficient venom allergens to have acceptable, albeit suboptimal, efficacy. Also, unlike the winged Hymenoptera WBE, there are relatively few reports of treatment failure with IFA WBE, and no fatalities. Brown et al1 mention a fatal reaction but not the species of WBE used for treatment or the dose, schedule, and duration of treatment. However, our experience with the winged Hymenoptera WBEs ‘‘demonstrates the value of a complete understanding of the natural history of the disease in determining the efficacy and indications for treatment and the importance of clinical trials.’’2 As Brown et al1 point out, prospective design and blind treatment are also critical to the strength of the evidence. The lack of IFA venom products for diagnosis and immunotherapy in the United States is a continuing gap in our repertoire. It is not the ethics but the economics of clinical trials that has deterred the performance of doubleblind, placebo-controlled clinical trials of IFA WBE and venom products and encouraged the acceptance of the WBEs as the only option in practice. However, the dilemma posed by the authors has attracted the attention of the Insect Committee of the American Academy of Allergy, Asthma and Immunology, who have now resolved to explore the development of a controlled trial of IFA WBE immunotherapy. (Nelson, personal communication, March 2005). When the efficacy of current treatment has not been proven up to current standards and the risk of treatment failure is a life-threatening reaction, a controlled trial is clearly justified. Our thanks to Brown et al1 for exposing this important issue. David B. K. Golden, MD Johns Hopkins Asthma and Allergy Center 5501 Hopkins Bayview Blvd Baltimore, MD 21224

REFERENCES 1. Brown SGA, Heddle RJ, Wiese MD, Blackman KE. Efficacy of ant venom immunotherapy and whole body extracts. J Allergy Clin Immunol 2005;116:464-5. 2. Golden DBK. Insect sting allergy and venom immunotherapy: a model and a mystery. J Allergy Clin Immunol 2005;115:439-47. 3. Brown SG, Franks RW, Baldo BA, Heddle RJ. Prevalence, severity and natural history of jack jumper ant venom allergy in Tasmania. J Allergy Clin Immunol 2003;111:187-92. 4. Brown SG, Wiese MD, Blackman KE, Heddle RJ. Ant venom immunotherapy: a double-blind placebo-controlled crossover trial. Lancet 2003;361:1001-6. 5. Strom GB, Boswell MD, Jacobs RL. In vivo and in vitro comparison of fire ant venom and fire ant whole body extract. J Allergy Clin Immunol 1983;72:46-53. 6. Paull BR, Coghlan TH, Vinson SB. Fire ant venom hypersensitivity, I: comparison of fire ant venom and whole body extract in the diagnosis of fire ant allergy. J Allergy Clin Immunol 1983;71:448-53. 7. Butcher BT, deShazo RD, Ortiz AA, Reed MA. Superiority of Solenopsis invicta venom to whole body extract in RAST for diagnosis of imported fire ant allergy. Int Arch Allergy Appl Immunol 1988;85: 458-61. 8. Hoffman DR, Jacobson RS, Schmidt M, Smith AM. Allergens in Hymenoptera venoms, XXIII: venom content of imported fire ant whole body extracts. Ann Allergy 1991;66:29-31.

Correspondence 465

J ALLERGY CLIN IMMUNOL VOLUME 116, NUMBER 2

Alma Street Fremantle, WA 6160, Australia b Department of Respiratory Medicine Flinders Medical Centre Bedford Park, Australia c Royal Hobart Hospital Hobart, Australia

REFERENCES 1. Golden DB. Insect sting allergy and venom immunotherapy: a model and a mystery. J Allergy Clin Immunol 2005;115:439-47. 2. Brown SGA, Franks RW, Baldo BA, Heddle RJ. Prevalence, severity, and natural history of jack jumper ant venom allergy in Tasmania. J Allergy Clin Immunol 2003;111:187-92. 3. Brown SGA, Wiese MD, Blackman KE, Heddle RJ. Ant venom immunotherapy: a double-blind, placebo-controlled, crossover trial. Lancet 2003;361:1001-6. Available online June 17, 2005. doi:10.1016/j.jaci.2005.04.025

Reply To the Editor: I appreciate the comments of Brown et al.1 It was clearly only by unintended oversight that the exemplary work of the authors was not cited in my review that focused on treatment strategies in the United States.2 Their work on the natural history of ant venom allergy and their controlled trial of ant venom immunotherapy are a model to which we should aspire.3,4 In contrast, there has been no controlled trial of imported fire ant (IFA) whole body extract (WBE) immunotherapy, and the natural history of IFA allergy is unknown. I am dismayed that my statements were construed to imply that IFA WBE has been proven efficacious. Comparison of IFA WBE and venom in vitro and by skin test suggests that although inferior to the venom, WBE contains sufficient allergen to provide reasonable diagnostic accuracy.5-8 For this reason, I stated, ‘‘For fire ant allergy, venom is the most accurate diagnostic material, but WBE have shown adequate diagnostic sensitivity.’’ My statement that ‘‘Fire ant immunotherapy is performed with WBE that contains sufficient venom allergens to provide reasonable clinical protection’’ was based on the reported content of venom allergens in IFA WBE. I did not state that WBE was as potent as venom.8 The report of clinical efficacy of IFA WBE by Freeman et al9 had the strengths of prospective sting challenge (instead of retrospective field sting reports) and a limited (but highly significant) control group. Still, Freeman et al9 concluded that ‘‘A controlled prospective trial of WBE versus placebo is needed.to help define the natural history of IFA hypersensitivity.’’ Of recent interest are reports of rush immunotherapy with IFA WBE to prevent systemic and large local reactions, but these too were uncontrolled.10,11 Together, the in vivo and in vitro evidence led to the belief that unlike the other Hymenoptera, IFA WBE does

contain sufficient venom allergens to have acceptable, albeit suboptimal, efficacy. Also, unlike the winged Hymenoptera WBE, there are relatively few reports of treatment failure with IFA WBE, and no fatalities. Brown et al1 mention a fatal reaction but not the species of WBE used for treatment or the dose, schedule, and duration of treatment. However, our experience with the winged Hymenoptera WBEs ‘‘demonstrates the value of a complete understanding of the natural history of the disease in determining the efficacy and indications for treatment and the importance of clinical trials.’’2 As Brown et al1 point out, prospective design and blind treatment are also critical to the strength of the evidence. The lack of IFA venom products for diagnosis and immunotherapy in the United States is a continuing gap in our repertoire. It is not the ethics but the economics of clinical trials that has deterred the performance of doubleblind, placebo-controlled clinical trials of IFA WBE and venom products and encouraged the acceptance of the WBEs as the only option in practice. However, the dilemma posed by the authors has attracted the attention of the Insect Committee of the American Academy of Allergy, Asthma and Immunology, who have now resolved to explore the development of a controlled trial of IFA WBE immunotherapy. (Nelson, personal communication, March 2005). When the efficacy of current treatment has not been proven up to current standards and the risk of treatment failure is a life-threatening reaction, a controlled trial is clearly justified. Our thanks to Brown et al1 for exposing this important issue. David B. K. Golden, MD Johns Hopkins Asthma and Allergy Center 5501 Hopkins Bayview Blvd Baltimore, MD 21224

REFERENCES 1. Brown SGA, Heddle RJ, Wiese MD, Blackman KE. Efficacy of ant venom immunotherapy and whole body extracts. J Allergy Clin Immunol 2005;116:464-5. 2. Golden DBK. Insect sting allergy and venom immunotherapy: a model and a mystery. J Allergy Clin Immunol 2005;115:439-47. 3. Brown SG, Franks RW, Baldo BA, Heddle RJ. Prevalence, severity and natural history of jack jumper ant venom allergy in Tasmania. J Allergy Clin Immunol 2003;111:187-92. 4. Brown SG, Wiese MD, Blackman KE, Heddle RJ. Ant venom immunotherapy: a double-blind placebo-controlled crossover trial. Lancet 2003;361:1001-6. 5. Strom GB, Boswell MD, Jacobs RL. In vivo and in vitro comparison of fire ant venom and fire ant whole body extract. J Allergy Clin Immunol 1983;72:46-53. 6. Paull BR, Coghlan TH, Vinson SB. Fire ant venom hypersensitivity, I: comparison of fire ant venom and whole body extract in the diagnosis of fire ant allergy. J Allergy Clin Immunol 1983;71:448-53. 7. Butcher BT, deShazo RD, Ortiz AA, Reed MA. Superiority of Solenopsis invicta venom to whole body extract in RAST for diagnosis of imported fire ant allergy. Int Arch Allergy Appl Immunol 1988;85: 458-61. 8. Hoffman DR, Jacobson RS, Schmidt M, Smith AM. Allergens in Hymenoptera venoms, XXIII: venom content of imported fire ant whole body extracts. Ann Allergy 1991;66:29-31.

466 Correspondence

9. Freeman TM, Hyghlander R, Ortiz A, Martin ME. Imported fire ant immunotherapy: effectiveness of whole body extracts. J Allergy Clin Immunol 1992;90:210-5. 10. Tankersley MS, Walker RL, Butler WK, Hagan LL, Napoli DC, Freeman TM. Safety and efficacy of an imported fire ant rush immunotherapy protocol with and without prophylactic treatment. J Allergy Clin Immunol 2002;109:556-62.

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11. Walker R, Jacobs J, Tankersly M, Hagan L, Freeman T. Rush immunotherapy for the prevention of large local reactions secondary to imported fire ant stings [abstract]. J Allergy Clin Immunol 1999;103: S180. Available online June 17, 2005. doi:10.1016/j.jaci.2005.04.026

Images in

Allergy and

Immunology Toll-like receptors and atopy Pierre Olivier Fiset, BSc, Meri Katarina Tulic, PhD, and Qutayba Hamid, MD, PhD, Editors Editor’s note: This feature, Images in allergy and immunology, is designed to highlight current concepts of the immunopathology of allergic diseases and other common immunologically mediated diseases. The presentation will appear as sets of images that involve cross-pathology, histopathology, and molecular pathology and will cover a range of topics of interest to allergists and immunologists.

The Toll-like receptors (TLRs) are a recently discovered family of receptors involved in the innate recognition of pathogens. TLRs have much homology to the IL-1 receptor family and the Drosophila Toll protein, and at least 10 distinct TLRs have now been identified in human subjects (Fig 1). TLR ligands are highly conserved structures and molecules present on many pathogens, the so-called pathogen-associated

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FIG 2. A, Prevalence of asthma, atopy, and current hay fever symptoms in TLR2/216934 in farmers’ children. B, Prevalence of asthma, atopy, and current hay fever symptoms in TLR4/ 14434 in children exposed to high endotoxin concentrations.1

FIG 1. Ten distinct TLRs exist in human subjects, recognizing many PAMPs. TLRs can associate as heterodimers changing their ligand specificity.

From Meakins-Christie Laboratories, Department of Pathology and Medicine, McGill University, Montreal, Canada. Received for publication April 21, 2005; accepted for publication April 22, 2005. Available online June 17, 2005. Reprint requests: Qutayba Hamid, MD, PhD, McGill University, Meakins-Christie Laboratory, 3626 St Urbain St, Montreal, Canada H2X 2P2. E-mail: [email protected]. J Allergy Clin Immunol 2005;116:467-70. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.034

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molecular patterns (PAMPs). Some PAMPs are bacterial molecules, such as lipopeptides, mannans, LPSs, flagellin, and CpG DNA. Other PAMPs recognized by TLRs include virus- and fungus-associated molecules. Triggering of TLRs leads to expression of many genes involved in inflammatory responses to pathogens, leading to cell activation, differentiation, proliferation, and cell recruitment. Because of their strong immunostimulatory capacities, many PAMPs are currently studied as potential treatment agents for allergic diseases. Because the TLRs are part of the innate immune system, they are not modified during an immune response and are passed on to the progeny with little genetic change. This has prompted genetic studies to determine whether specific single nucleotide polymorphisms in the TLR genes are associated with atopy. A recent study has suggested a polymorphism in TLR2 and TLR4 in Europeans to be associated with decreased atopy, dependent on PAMP exposure

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FIG 3. The hygiene hypothesis states that exposure to microorganisms (decreased hygiene) during early age is important for the development of a balanced immune system. Increased hygiene leads to an uncontrolled TH2 immune response to allergens, resulting in atopic diseases.

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FIG 4. Detection of bromodeoxyuridine-positive proliferating cells colocalized with CD3-positive cells in explants of nasal mucosa of children. The explants were stimulated with LPS (0.1 mg/ mL) for 2 hours (A) and 24 hours (B). Ten percent colocalization was seen at 2 hours, and 70% colocalization was seen at 24 hours.3

August 2005

(Fig 2).1 On the other hand, another study showed no association of atopy and polymorphisms in TLR2, TLR3, TLR4, and TLR9 in Japanese populations.2 Evidence from epidemiologic studies has shown an association between high exposure to PAMPs during early life with decreased levels of atopic diseases and asthma. This has led to the proposal of the hygiene hypothesis, which states that lack of a ‘‘pathogenic pressure’’ (increased hygiene) in early childhood results in an imbalanced immune system hypersensitive to allergens (Fig 3). Thus atopy is associated with increases in TH2 cytokines compared with TH1 or immunoregulatory cytokines. Tulic et al3 have shown that LPS can cause a proliferation of CD3-positive cells in the nasal mucosa of children (Fig 4). This was associated with increases in IL-2–positive, IL-12– positive, and IFN-g–positive cells without increases in TH2 cytokines and MBP–positive cells (Fig 5). It has been also shown that LPS can inhibit allergeninduced increases in IL-4–positive, IL-5–positive, and IL-13–positive cells, as well as in MBP-positive and tryptase-positive cells.4 These effects were determined to be due to the increase of IL-10, IL-12, and IFN-g induced by LPS. Additionally, in this same study TLR4-positive cells were higher and more responsive to LPS in children compared with adults. PAMPs are also studied to treat patients with atopic diseases. In this context PAMPs are used as adjuvants for current immunotherapy regimens to reduce the antigen dose needed for therapy and to promote the development of immunoregulatory mechanisms. For example, combining CpG DNA with ragweed immunotherapy has been shown to provide potential clinical benefits. In a mouse model of allergy, physical linking of CpG DNA to ragweed protein inhibited IgE expression, inhibited IL-5 expression, and promoted IFN-g expression.5 Physical linking of the CpG DNA to the ragweed protein enhanced the effects, suggesting that cells reacting to the allergen also have TLR9

J ALLERGY CLIN IMMUNOL

FIG 6. Linking of the allergen to an immunostimulatory CpG DNA sequence increases the potency of the ragweed CpG DNA vaccine for immunotherapy. The same antigen-presenting cell is activated by the complex, through TLR9, to synthesize cytokines and increase antigen presentation of the allergen. Allergen presentation and cytokines activate allergen-specific T cells to change their cytokine profile.

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FIG 5. IL-12 in situ hybridization of sections taken from nasal explants without stimulation (A) and stimulated with 0.1 mg/mL LPS (B). IL-2, IL-12, and IFN-g profile after stimulation with 0.1 mg/mL LPS (C). Open bars indicate no stimulation, and filled bars indicate LPS stimulation.3

activated by the CpG DNA (Fig 6). Ragweed CpG DNA injections in human clinical trials has been shown to inhibit allergen-induced IL-4 mRNA expression, IL-5 mRNA expression, and MBP-positive cell numbers in the nasal mucosa of allergic patients (Fig 7) and to reduce chest and nasal symptom scores.6 The compound can also increase the number of TLR9positive cells in the nasal mucosa (Fig 8). As the role of PAMPs and TLRs is clarified in atopy and TLRs are better characterized, development of new therapies to both prevent atopic diseases and treat existing disease will be possible.

J ALLERGY CLIN IMMUNOL

FIG 7. Horseradish peroxidase immunocytochemistry for MBPpositive cells in sections of patients receiving the ragweed CpG DNA vaccine for immunotherapy (A) or placebo (B).

REFERENCES 1. Eder W, Klimecki W, Yu L, von Mutius E, Riedler J, Braun-Fahrlander C, et al. Toll-like receptor 2 as a major gene for asthma in children of European farmers. J Allergy Clin Immunol 2004;113:482-8. 2. Noguchi E, Nishimura F, Fukai H, Kim J, Ichikawa K, Shibasaki M, et al. An association study of asthma and total serum immunoglobin E levels for Toll-like receptor polymorphisms in a Japanese population. Clin Exp Allergy 2004;34:177-83. 3. Tulic MK, Manoukian JJ, Eidelman DH, Hamid Q. T-cell proliferation induced by local application of LPS in the nasal mucosa of nonatopic children. J Allergy Clin Immunol 2002;110:771-6. 4. Tulic MK, Fiset PO, Manoukian JJ, Frenkiel S, Lavigne F, Eidelman DH, et al. Role of toll-like receptor 4 in protection by bacterial lipopolysaccharide in the nasal mucosa of atopic children but not adults. Lancet 2004;363:1689-97.

August 2005

producing latent infections that might lead to B-cell and other lymphoproliferative diseases. A relatively unusual target of EBV infection involves natural killer (NK) cells. Despite varying classifications, a form of chronic active EBV infection (CAEBV) involving NK cells presents with severe inflammatory and necrotic skin reactions considered pathognomonic of EBV1 NK cell lymphoproliferative disease.1-3 Most patients presenting with this condition are of Asian descent, and there is no sex predominance.

FIG 8. In situ hybridization for TLR9 mRNA–positive cells in sections of patients receiving placebo (A) or the ragweed CpG DNA vaccine for immunotherapy (B).

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5. Tighe H, Takabayashi K, Schwartz D, Van Nest G, Tuck S, Eiden JJ, et al. Conjugation of immunostimulatory DNA to the short ragweed allergen Amb a 1 enhances its immunogenicity and reduces its allergenicity. J Allergy Clin Immunol 2000;106:124-34. 6. Tulic MK, Fiset PO, Christodoulopoulos P, Vaillancourt P, Desrosiers M, Lavigne F, et al. Amb a 1-immunostimulatory oligodeoxynucleotide conjugate immunotherapy decreases the nasal inflammatory response. J Allergy Clin Immunol 2004;113:235-41.

FIG 1.

Chronic active Epstein-Barr virus infection of natural killer cells presenting as severe skin reaction to mosquito bites Susan E. Pacheco, MD, Stephen M. Gottschalk, MD, Mary V. Gresik, MD, Megan K. Dishop, MD, Takayuki Okmaura, MD, and Theron G. McCormick, MD, Guest Editors

Fig 1 shows a 7-year-old Latin American boy with NK cell CAEBV. Typical of this condition is the presence of bullous and ulcerative skin lesions after exposure to mosquito bites. In addition, patients develop high fever, lymphadenopathy in draining nodes, and marked hepatosplenomegaly. Bullous

Discovered more than 40 years ago, EBV is known to exhibit tropism for lymphocytes, especially B-cells. This g herpes virus is capable of immune evasion, From Baylor College of Medicine, Pediatric Allergy and Immunology Service, Texas Children’s Hospital, Houston, Tex. Received for publication February 25, 2005; revised April 11, 2005; accepted for publication April 20, 2005. Available online July 15, 2005. Reprint requests: Theron G. McCormick, MD, Baylor College of Medicine, Pediatric Allergy and Immunology Service, Texas Children’s Hospital, 6621 Fannin St. FC330.01, Houston, TX 77030-2399. E-mail: [email protected]. J Allergy Clin Immunol 2005;116:470-2. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.044

August 2005

FIG 2.

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producing latent infections that might lead to B-cell and other lymphoproliferative diseases. A relatively unusual target of EBV infection involves natural killer (NK) cells. Despite varying classifications, a form of chronic active EBV infection (CAEBV) involving NK cells presents with severe inflammatory and necrotic skin reactions considered pathognomonic of EBV1 NK cell lymphoproliferative disease.1-3 Most patients presenting with this condition are of Asian descent, and there is no sex predominance.

FIG 8. In situ hybridization for TLR9 mRNA–positive cells in sections of patients receiving placebo (A) or the ragweed CpG DNA vaccine for immunotherapy (B).

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5. Tighe H, Takabayashi K, Schwartz D, Van Nest G, Tuck S, Eiden JJ, et al. Conjugation of immunostimulatory DNA to the short ragweed allergen Amb a 1 enhances its immunogenicity and reduces its allergenicity. J Allergy Clin Immunol 2000;106:124-34. 6. Tulic MK, Fiset PO, Christodoulopoulos P, Vaillancourt P, Desrosiers M, Lavigne F, et al. Amb a 1-immunostimulatory oligodeoxynucleotide conjugate immunotherapy decreases the nasal inflammatory response. J Allergy Clin Immunol 2004;113:235-41.

FIG 1.

Chronic active Epstein-Barr virus infection of natural killer cells presenting as severe skin reaction to mosquito bites Susan E. Pacheco, MD, Stephen M. Gottschalk, MD, Mary V. Gresik, MD, Megan K. Dishop, MD, Takayuki Okmaura, MD, and Theron G. McCormick, MD, Guest Editors

Fig 1 shows a 7-year-old Latin American boy with NK cell CAEBV. Typical of this condition is the presence of bullous and ulcerative skin lesions after exposure to mosquito bites. In addition, patients develop high fever, lymphadenopathy in draining nodes, and marked hepatosplenomegaly. Bullous

Discovered more than 40 years ago, EBV is known to exhibit tropism for lymphocytes, especially B-cells. This g herpes virus is capable of immune evasion, From Baylor College of Medicine, Pediatric Allergy and Immunology Service, Texas Children’s Hospital, Houston, Tex. Received for publication February 25, 2005; revised April 11, 2005; accepted for publication April 20, 2005. Available online July 15, 2005. Reprint requests: Theron G. McCormick, MD, Baylor College of Medicine, Pediatric Allergy and Immunology Service, Texas Children’s Hospital, 6621 Fannin St. FC330.01, Houston, TX 77030-2399. E-mail: [email protected]. J Allergy Clin Immunol 2005;116:470-2. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.04.044

August 2005

FIG 2.

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FIG 3. FIG 6.

Skin biopsy from a bullous lesion revealed subepidermal bullae with a dense dermal infiltrate of eosinophils, lymphocytes, and histiocytes and a negative gram stain analysis (hematoxylin-eosin stain; Figs 5 and 6). The unusual reaction to mosquito bites, very high IgE level (often >10,000 IU/mL), and significant eosinophilia has prompted the nomenclature of ‘‘hypersensitivity reaction.’’ However, this condition does not meet criteria for an immunologic allergic or hypersensitivity reaction to mosquitoes on laboratory or clinical grounds.

471

FIG 4.

lesions develop within 24 hours after mosquito exposure and are filled with a sterile fluid; this is followed by necrotic ulcerations (Figs 2 through 4).

FIG 7.

TABLE I. Characteristics of NK cell CAEBV

FIG 5.

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Cell markers

CD32, CD561, and/or CD161

Receptor for infection Main transforming protein Mosquito bite reactions Target population Associated malignancies

Unknown Unknown Often present First 2 decades of life Hemophagocytic lymphohistiocytosis, NK cell leukemia, NK cell lymphoma

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By current parameters, patients with NK cell CAEBV disease seem to have normal immunity before development of the disease. An immunologic evaluation in this patient revealed normal lymphocyte proliferation to antigen and mitogens and functional antibodies to polysaccharide and protein antigens. Skin biopsy specimens from patients with NK cell CAEBV related to mosquito bites are significant for an inflammatory reaction composed primarily of NK cells (CD561CD3–) expressing EBV DNA by in situ hybridization (Fig 7). PBMCs from affected patients often demonstrate 30% to 70% NK cells, most infected with monoclonal or oligoclonal EBV. In addition, EBV DNA PCR levels from PBMCs are significantly elevated, with mean levels of 104
2 copies/mg.4 A table distinguishing NK cell CAEBV is provided (Table I). Aside from

symptomatic care, the optimal treatment option is bone marrow transplantation.5 REFERENCES 1. Miyazato H, Nakasuka S, Dong Z, Takakuwa T, Oka K, Hanamoto H, et al. NK-cell related neoplasms in Osaka, Japan. Am J Hematol 2004;76:230-5. 2. Tokura Y, Ishihara S, Tagawa S, Naoshiro S, Ohshima K, Takigawa M. Hypersensitivity to mosquito bites as the primary clinical manifestation of a juvenile type of Epstein-Barr virus-associated natural killer cell leukemia/lymphoma. J Am Acad Dermatol 2001;45:569-78. 3. Ohga S, Nomura A, Takada H, Hara T. Immunological aspects of Epstein-Barr virus infection. Crit Rev Oncol Hematol 2002;44:203-15. 4. Kimura H, Hoshino Y, Kanegane H, Tsuge I, Okamura T, Kawa K, et al. Clinical and virologic characteristics of chronic active Epstein-Barr virus infection. Blood 2001;98:280-6. 5. Fujii N, Takenaka K, Hiraki A, Maeda Y, Ikeda K, Shingawa K, et al. Allogeneic peripheral blood stem cell transplantation for the treatment of chronic active Epstein-Barr virus infection. Bone Marrow Transplant 2000;26:805-8.

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Articles of note. . . Adverse events after influenza immunization in young children Because influenza (Flu) infections cause considerable morbidity in young children, the Advisory Committee on Immunization Practices has encouraged health care providers to give healthy 6- to 23-month-old children the trivalent Flu influenza vaccine (TFV). However, concerns have been raised by some parents about adverse effects of such immunization. This study reviewed records of the Vaccine Adverse Event Reporting System (VAERS), a passive surveillance system begun by the US Food and Drug Administration and the Centers for Disease Control and Prevention in 1990. Since 1990, there were 166 reports of adverse events (AEs) following receipt of the TFV alone or along with other vaccines in children less than 2 years old. These AEs have generally been quite mild, (fever, transient rash). Seizures were the most common serious AE, reported in 28 cases. Most of the seizures occurred along with fever with onset within 2 days after immunization. No sequelae were reported. Although there is probably some underreporting of AE in the voluntary reporting in the VAERS, these findings suggest that TFV immunization is generally very safe and well tolerated by young children. (McMahon et al. Pediatrics 2005;115:453-9.)

Respiratory syncytial virus infection in elderly and high-risk adults Respiratory syncytial virus (RSV) infection, extensively investigated in children, is increasingly recognized as a cause of illness in adults. This study prospectively investigated all respiratory illnesses in cohorts of (1) healthy elderly patients (65 years of age or older), (2) high-risk adults (those with chronic heart, lung, or airways disease), and (3) patients hospitalized with acute cardiopulmonary conditions. RSV infections occurred annually in 3% to 7% of healthy elderly patients and in 4% to 10% of high-risk adults. The frequency of RSV infection was at least as great as that of influenza A in these populations. In the hospitalized patients, RSV infection and influenza A resulted in similar lengths of stay, rates of use of intensive care (15% and 12%, respectively), and mortality (8% and 7%, respectively). RSV infection accounted for 10.6% of hospitalizations for pneumonia, 11.4% of hospitalizations for chronic obstructive pulmonary disease, and 7.2% of hospitalizations for asthma. The authors concluded that RSV infection is an important illness in elderly and high-risk adults, with a disease burden similar to that of nonpandemic influenza A in a population in which the prevalence of immunization for influenza is great. One would hope that these

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Beyond Our Pages Burton Zweiman, MD, & Marc E. Rothenberg, MD, PhD, Editors

findings will help stimulate a continued search for an effective, safe RSV vaccine. (Falsey et al. N Engl J Med 2005;352:1749-59.)

Anaphylactic reaction to lupin flour Flour made from ground-up lupin beans is being used increasingly as a wheat flour substitute in some European countries. This case report described a severe anaphylactic reaction in a 25-year-old woman shortly after ingestion of a meal containing chicken, fried potatoes, and onion rings with recovery after intensive therapy. There was a past history of transient asthma at age 15 years and a prior anaphylactic reaction to peanuts. It was found that the breading on the onion rings contained lupin flour. Subsequent skin tests to peanuts and a crude extract of lupin flour were strongly positive. There is probably a 20% to 40% cross-reacting homology between lupin and one of the allergens in peanuts. Lupin flour allergy has been reported mainly in European patients known to be allergic to other legumes, particularly peanut, soy, or pea. Indeed, reactions to lupin are one of the most common types of food-induced anaphylaxis in France. This report should be kept in mind, because lupin beans and lupin flour are now becoming available for consumption in the United States. (Radcliffe et al. Lancet 2005;365:1360.)

A new mechanism of nonatopic asthma elicited by immunoglobulin free light chains A significant proportion of asthmatic individuals are nonatopic, yet the mechanism by which an asthma exacerbation is triggered in these individuals is not known. In this report, the investigators extended their prior observation that antigen-specific immunoglobulin free light chains (LCs) mediate mast cell–dependent hypersensitivity by examining the role of LCs in a murine model of nonatopic asthma. In particular, they use a LC antagonist, the 9-mer F991, and abrogate the development of airway hyperresponsiveness and pulmonary infiltration. Using mast cell–deficient mice, they show that the role of LCs is dependent on mast cells. Finally, they demonstrate that asthmatic individuals (both atopic and nonatopic) have elevated sera

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levels of jLC (but not kLC) compared with control individuals. These results substantiate a new mechanism that might be involved in triggering allergic, but not IgE-mediated, asthma and suggest that inhibition of LC-mediated mast cell activation might be therapeutically useful. (Kraneveld et al. Proc Natl Acad Sci U S A 2005;102: 1578-83.)

Suppressive effects of prostaglandin E receptor subtype EP3—the mechanism of aspirin sensitivity? Prostaglandins (PGs), including PGD2 and PGE2, are produced during allergic responses, yet PG synthesis inhibitors (eg, aspirin) are generally ineffective for asthma. Inasmuch as PGD2 is a potent proinflammatory mediator and smooth muscle constrictor, this suggests that PGE2 might have an important regulatory (or protective) role in allergy. To address this possibility, the investigators subjected mice with specific deficiencies in each of the 4 PGE2 receptors (EP1 through EP4) to an OVA-induced model of asthma. Notably, mice deficient in EP3 had a marked increase in multiple aspects of asthma (including inflammation and TH2 cytokine production), whereas mice deficient in the other receptors were comparable to wild-type mice. On the basis of these results suggesting a suppressive role for EP3 signaling, the investigators examined the impact of an EP3-selective agonist on the development of asthma. Indeed, the EP3 agonist inhibited airway inflammation, TH2 cytokine production, and bronchoconstriction, even when it was administered 3 hours after antigen challenge. In addition, a significant number of allergen-induced genes were inhibited by the EP3 agonist. Taken together, these results call attention to the anti-allergic role of PGE2 and its EP3 receptor, providing a new paradigm for therapeutic intervention. Furthermore, the results provide a possible explanation for aspirinsensitive asthma by suggesting that such individuals might preferentially require the protective effects of the EP3 pathway (and are thus sensitive to aspirin). (Kunikata et al. Nat Immunol 2005;6:524-31.)

Dendritic cells are critical for experimental asthma Dendritic cells (DCs) have been shown to have an important role in sensitization to inhaled allergens, but their function in ongoing TH2 cell–mediated lung inflammation is currently unknown. Using an OVAinduced murine asthma model, the investigators show that airway DCs acquire a mature phenotype and interact with CD4 T cells within the lung tissue. To study whether the DCs contributed to inflammation, they subsequently depleted the DCs from the airways using CD11c-diphtheria toxin (DT) receptor transgenic mice during the OVA aerosol challenge. In these

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mice, DT is only active on CD11c+ cells (primarily DCs and macrophages). Indeed, administration of DT to the lungs of these transgenic mice depleted CD11c+ DCs and alveolar macrophages. Notably, DT abolished the characteristic features of asthma, including eosinophilic inflammation, goblet cell hyperplasia, and bronchial hyperreactivity. Furthermore, in the absence of CD11c+ cells, TH2 cells did not produce IL-4, IL-5, and IL-13 in response to OVA aerosol. Importantly, in CD11c-depleted mice, eosinophilic inflammation and TH2 cytokine secretion were restored by adoptive transfer of CD11c+ DCs, but not by transfer of alveolar macrophages. These findings identify lung DCs as key pro-inflammatory cells that are necessary and sufficient for TH2 cell stimulation during ongoing lung inflammation. (van Rijt et al. J Exp Med 2005;201:981-91.)

Daily versus as-needed inhaled corticosteroid treatment of mild, persistent asthma Most current national guidelines recommend daily use of controller medications, such as an inhaled corticosteroid (ICS), in the treatment of persistent asthma (PA), even of mild degree. This randomized, doubleblind study investigated whether treatment with daily ICS (budesonide 200 micrograms bid), or daily leukotriene antagonist (zafirlukast 20 mg bid) was more effective than daily placebo with as-needed ICS use in the treatment of mild PA in 199 adults. After 1 year of treatment, there were no significant differences in the major asthma outcome (increases in average morning peak expiratory flow) among the 3 treatment groups. The frequency of acute asthma exacerbations requiring corticosteroid therapy was also not significantly different in the 3 groups. There were greater improvements in patients using ICS daily than in placebo-treated individuals in prebronchodilator FEV1 (P = .005), PC20 bronchial reactivity (P < .001), asthma control score (P < .001), sputum eosinophils (P = .007), and number of symptom-free days (P = .03). However, the postbronchodilator FEV1 and quality of life scores were not significantly different between those treated daily with ICS and those receiving placebo. There were no differences in any asthma outcome scores between those treated with daily zafirlukast and those receiving placebo. These findings indicate that daily ICS treatment improves some manifestations of mild PA in adults. However, the authors concluded that daily ICS might not be needed in such asthmatic individuals; they can instead be treated with short intermittent courses of inhaled or oral corticosteroids taken when asthma symptoms worsen significantly. It is still uncertain whether these findings can be extended to mild PA in children. (Boushey et al. N Engl J Med 2005;352:1519-28.)

J ALLERGY CLIN IMMUNOL