Journal of Allergy and Clinical Immunology

T HE J OURNAL OF
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Allergy Clinical AND
Immunology
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
Current reviews of allergy and clinical immunology

Y Innate immune responses to infection 241
w CME
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
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POSTMASTER: Send address changes to The Journal of Allergy and Clinical Immunology, Elsevier Periodicals Customer Service, 6277 Sea Harbor Drive,
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J ALLERGY CLIN IMMUNOL August 2005 5A

T HE J OURNAL OF
Contents
Immunology


w CME
About the cover


Orlando, FL 32887-4800.

T HE J OURNAL OF
Contents
Immunology


w CME
About the cover


Orlando, FL 32887-4800.

Contents
CONTENTS
CONTINUED
Continuing Medical Education examination: Innate immune responses 250

to infection
Y Molecular mechanisms in allergy and clinical immunology
wCME
EBV the prototypical human tumor virus—just how bad is it?
David A. Thorley-Lawson, PhD, Boston, Mass
Continuing Medical Education examination: EBV the prototypical

251
262
human tumor virus—just how bad is it?
Editorial
Infection versus immunity: What’s the balance? 263
Y 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
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.
d
EC Editors’ Choice (p 239)
d
OR Online Repository material
Y Theme issue
w CME CME examination article available online at www.mosby.com/jaci

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to infection
wCME

251
262
Editorial
Rostrum
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Y Theme issue

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to infection
wCME

251
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CONTINUED
Original articles
d
EC Is it traffic type, volume, or distance? Wheezing in infants living near 279
truck and bus traffic
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
d
EC Effect of low-dose ciclesonide on allergen-induced responses in subjects 285
with mild allergic asthma
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 292

allergen-induced asthmatic reactions
Emmerentia van Schalkwyk, MBChB, K. Strydom, MBChB, Zelda Williams, RN, Louis Venter, MSc,
Stefan Leichtl, PhD, Christine Schmid-Wirlitsch, PhD, Dirk Bredenbröker, 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 299

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
Mechanisms of asthma and allergic inflammation

d
OR 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 312

release in the human airway
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
d
OR 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 325

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

Contents
CONTENTS
CONTINUED
Differential effects of (S)- and (R)-enantiomers of albuterol in a mouse 332

asthma model
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 347

modified allergens are associated with reduced nasal allergen sensitivity
Jürgen Reisinger, MSc, Friedrich Horak, MD, Gabrielle Pauli, MD, Marianne van Hage, MD, Oliver Cromwell,
PhD, Franz König, Rudolf Valenta, MD, and Verena Niederberger, MD, Vienna, Austria, Stockholm, Sweden,
Strasbourg, France, and Reinbek, Germany
Levocetirizine: Pharmacokinetics and pharmacodynamics in children 355

age 6 to 11 years
F. Estelle R. Simons, MD, FRCPC, and Keith J. Simons, PhD, Winnipeg, Manitoba, Canada
Striking deposition of toxic eosinophil major basic protein in mucus: 362

Implications for chronic rhinosinusitis
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
d
OR Intranasal tolerance induction with polypeptides derived from 3 noncross- 370
reactive major aeroallergens prevents allergic polysensitization in mice
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

d
EC Prevalences of positive skin test responses to 10 common allergens in the 377
US population: Results from the Third National Health and Nutrition
d
OR
Examination Survey
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 384

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

Contents
CONTENTS
CONTINUED
Food allergy, dermatologic diseases, and anaphylaxis

d
EC COX-2 inhibition enhances the TH2 immune response to epicutaneous 390
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
d
EC Responsiveness to autologous sweat and serum in cholinergic urticaria 397
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
d
OR Lack of detectable allergenicity of transgenic maize and soya samples 403
Rita Batista, BSc, Baltazar Nunes, MSc, Manuela Carmo, Carlos Cardoso, PharmD, Helena São José,
António Bugalho de Almeida, MD, PhD, Alda Manique, MD, Leonor Bento, MD, PhD, Cândido
Pinto Ricardo, PhD, and Maria Margarida Oliveira, PhD, Lisboa, Oeiras, and Algés, 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

Saurabh Mehandru, MD, Klara Tenner-Racz, MD, Paul Racz, MD, PhD, and Martin Markowitz, MD,
419
New York, NY, and Hamburg, Germany
Editorial
Are you immunodeficient? 423
Y Francisco A. Bonilla, MD, PhD, and Raif S. Geha, MD, Boston, Mass
Rostrum
From idiopathic infectious diseases to novel primary immunodeficiencies 426
Y 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 Créteil,
France, and Frankfurt, Germany
Original articles
d
EC Infant home endotoxin is associated with reduced allergen-stimulated 431
lymphocyte proliferation and IL-13 production in childhood
d
OR
Joseph H. Abraham, ScD, Patricia W. Finn, MD, Donald K. Milton, MD, Louise M. Ryan, PhD,
Y David L. Perkins, MD, and Diane R. Gold, MD, Boston, Mass


Contents
CONTENTS
CONTINUED
Does early EBV infection protect against IgE sensitization? 438

Y Caroline Nilsson, MD, Annika Linde, MD, PhD, Scott M. Montgomery, BSc, PhD, Liselotte Gustafsson,
Per Näsman, 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 453

biomarkers in multiple sclerosis
Til Menge, MD, Patrice H. Lalive, MD, Hans-Christian von Büdingen, MD, Bruce Cree, MD, PhD,
Stephen L. Hauser, MD, and Claude P. Genain, MD, San Francisco, Calif, and Zürich, Switzerland
Letters to the Editor

Perilesional GM-CSF therapy of a chronic leg ulcer in a patient with common 460
variable immunodeficiency
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 Lemière, 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 463

corticosteroids for managing nighttime symptoms of allergic rhinitis
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

Toll-like receptors and atopy 467
Y Pierre Olivier Fiset, BSc, Meri Katarina Tulic, PhD, and Qutayba Hamid, MD, PhD, Editors
14A August 2005 J ALLERGY CLIN IMMUNOL

Contents
CONTENTS
CONTINUED
Chronic active Epstein-Barr virus infection of natural killer cells presenting 470
Y 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
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
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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
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T HE J OURNAL OF
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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
DANIEL L. HAMILOS, MD Boston, Mass 2009
ZUHAIR K. BALLAS, MD, PhD Iowa City, Iowa 2007
ANTHONY A. HORNER, MD La Jolla, Calif 2009
THOMAS BIEBER, MD, PhD Bonn, Germany 2007
HANS C. OETTGEN, MD, PhD Boston, Mass 2009
PEYTON A. EGGLESTON, MD Baltimore, Md 2007
DAVID P. HUSTON, MD Houston, Tex 2007 DEVENDRA K. AGRAWAL, PhD Omaha, Neb 2010
JOSHUA A. BOYCE, MD Boston, Mass 2010
PEDRO AVILA, MD Chicago, Ill 2008
JAVIER CHINEN, MD, PhD Bethesda, Md 2010
DENNIS LEDFORD, MD Tampa, Fla 2008
GURJIT K. KHURANA HERSHEY, MD, PhD Cincinnati, Ohio 2010
DAVID B. PEDEN, MD Chapel Hill, NC 2008
JOHN M. KELSO, MD San Diego, Calif 2010
HARALD RENZ, MD Marburg, Germany 2008
HANS-UWE SIMON, MD, PhD Bern, Switzerland 2010
HUGH A. SAMPSON, MD New York, NY 2008
ERIKA VON MUTIUS, MD, MSc Munich, Germany 2008
Board of Directors of the American Academy of Allergy, Asthma and Immunology

President F. ESTELLE R. SIMONS, MD, FAAAAI Secretary/Treasurer HUGH A. SAMPSON, MD, FAAAAI
Winnipeg, Manitoba, Canada New York, NY
President-Elect THOMAS A. E. PLATTS-MILLS, MD, PhD, FAAAAI Immediate Past President MICHAEL SCHATZ, MD, MS, FAAAAI
Charlottesville, Va San Diego, Calif
Vice President THOMAS B. CASALE, MD, FAAAAI Past Past President LANNY J. ROSENWASSER, MD, FAAAAI
Omaha, Neb Denver, Colo
DAVID H. BROIDE, MD, FAAAAI La Jolla, Calif RICHARD W. HONSINGER, MD, FAAAAI Los Alamos, NM
THOMAS A. FLEISHER, MD, FAAAAI Bethesda, Md DENNIS K. LEDFORD, MD, FAAAAI Tampa, Fla
SANDRA M. GAWCHIK, DO, FAAAAI Upland, Pa DONALD Y. M. LEUNG, MD, PhD, FAAAAI Denver, Colo
STANLEY GOLDSTEIN, MD, FAAAAI Rockville Centre, NY ARNOLD I. LEVINSON, MD, FAAAAI Philadelphia, Pa
PAUL A. GREENBERGER, MD, FAAAAI Chicago, Ill JAMES T. LI, MD, PhD, FAAAAI Rochester, Minn
REBECCA S. GRUCHALLA, MD, PhD, FAAAAI Dallas, Tex DENNIS R. OWNBY, MD, FAAAAI Augusta, Ga
Executive Vice President KAY WHALEN, CAE
Address of Executive Office

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J ALLERGY CLIN IMMUNOL AAAAI Developing Researcher Award 23A
VOLUME 116, NUMBER 2
T
he most frequently cited allergy/immunology journal in the
field, The Journal of Allergy and Clinical Immunology, con-
tinues 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.
for O AAAAI Award arch
The AAAAI Award for Outstanding Research Published in the JACI by
a Developing Researcher provides an unrestricted prize of $1,500, com- u se
memorative plaque, and recognition during the AAAAI’s Annual Busi- Publitsstanding ReJACI
h
by a D ed in the r
ness Meeting. Up to 4 awardees will be chosen during one calendar year. 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 pathogene-
sis 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, precep-
tor, 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 recom-
mendation 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 Fax
Country E-mail
Academic affiliation
for O AAAAI Award arch

Academic degree(s) Date degree(s) awarded
utstanding Rese
Publis I
Faculty appointments
he JAC
by a D hed in tAppointment date(s)
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 Academic affiliation:

(Name and address)
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 asso-
ciated 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 Sunday, March 5, 2006, designated as Anaphylaxis Day. On
n Anaphylaxis Day, the Presidential Symposium will be held
research, clinical practice concurrently with Sunday’s Plenary Session, and focus on
In the next year, the AAAAI will work to bridge the gap advances in research in basic and clinical science, relevant to
between basic science research in anaphylaxis and clinical the diagnosis and treatment of anaphylaxis.
practice through a comprehensive public education outreach The AAAAI will also host a press conference highlighting
initiative. The new Anaphylaxis Public Education Task Force anaphylaxis-related abstracts selected for presentation at the
is developing this educational effort to raise awareness of Annual Meeting.
anaphylaxis as a killer allergy with many potential triggers. 2006 AAAAI Primary Care Symposium
The initiative will also emphasize the importance of the role The 2006 Primary Care Symposium ‘‘Optimizing
of the allergist/immunologist in anaphylaxis management Treatment of Your Allergic Patients: An Update for the
and prevention. Primary Care Provider on Issues Affecting One-Third of Your
The new anaphylaxis public education campaign is one of Practice,’’ co-chaired by Joann Blessing-Moore, MD,
the initiatives of President F. Estelle R. Simons, MD, FAAAAI. FAAAAI and Richard F. Lockey, MD, FAAAAI, will also
The anaphylaxis initiative will include three major public include a segment on anaphylaxis. The annual symposium is
education campaigns: held immediately prior to the Annual Meeting and designed
d Back-to-school campaign, September 2005 to update local primary care physicians about the most
recent developments in allergic disease.
Elements of the back-to-school campaign will include Simons will also serve as one of ten AAAAI representatives
publicity to national magazines, and national daily and at the Second Symposium on the Definition and Management
weekly publications. The Task Force is also considering of Anaphylaxis, sponsored by the National Institutes of Health
a plan to reach school nurses, who may serve as a bridge to (NIH) and FAAN. The Symposium will build upon previous
educating a vast population of students. Regional, state and efforts and work toward developing a universally accepted
local allergy societies may be asked to work with their state definition for anaphylaxis. It will further expand its
and local school nurse associations. The Food Allergy & anaphylaxis research agenda, and identify educational needs
Anaphylaxis Network (FAAN) also offers a quarterly for health care professionals and patients. Hugh A. Sampson,
newsletter to school nurses, and the AAAAI Allergy and MD, FAAAAI, and Anne Munoz-Furlong will Co-Chair the
Asthma Tool Kit for School Nurses will also be utilized. Symposium.
d Holiday season campaign, late October through Anaphylaxis Retrospective
December 2005 The AAAAI is preparing a retrospective on anaphylaxis for
The holiday season campaign will focus on three main the 2006 Annual Meeting. If your research over the years has
holidays: Halloween, Thanksgiving and Christmas/ made a significant contribution to the understanding of
Hanukkah. Outreach will begin in October with publicity to anaphylaxis, the AAAAI History and Archives Committee,
national magazines, and daily and weekly media outlets. chaired by Michael A. Kaliner, MD, FAAAAI, wants to hear
from you. Please contact Audrey Mudek at the AAAAI
d Great outdoors campaign, March through May 2006 executive office, (414) 272-6071 or
The great outdoors campaign will conclude just before the e-mail amudek@aaaai.org.
Memorial Day weekend in May 2006. May is National Allergy
and Asthma Awareness Month, and also includes Food n American Academy of Allergy, Asthma
Allergy Awareness Week. Anaphylaxis publicity efforts will & Immunology Lifelong Learner Bill of
work hand-in-hand with these events.
Core messages for the public will include a brief description Rights
of anaphylaxis, and answers to basic questions including: Dear Colleagues and Friends:
d What is anaphylaxis? The American Academy of Asthma, Allergy &
d Who is at risk? Immunology provides outstanding continuing medical
d When can anaphylaxis occur? education through The Journal of Allergy and Clinical
d Where can anaphylaxis occur? Immunology (citation impact factor now 7.205), the AAAAI
d How is anaphylaxis treated? Annual Meeting, and other AAAAI programs, resources, and
d Why is follow-up needed? materials.
In order to emphasize our commitment to continuing
2006 Annual Meeting medical education, we have recently developed the AAAAI
The anaphylaxis public outreach effort will culminate Lifelong Learner Bill of Rights. We pledge to maintain the
during the 2006 Annual Meeting in Miami Beach, FL, with highest quality in all of our educational programs and to
J ALLERGY CLIN IMMUNOL August 2005 Page 25A

NEW SVIEW
newsview
from the AAAAI
fulfill our promise to provide you with a premier The CME program consists of a 15-minute didactic
educational experience. presentation with lecture notes highlighting the classification
Sincerely, of environmental triggers, followed by a series of pediatric
F. Estelle R. Simons, MD, FAAAAI and adult case studies with question and answer sections.
AAAAI President The didactic portion is presented with real-time audio
capabilities.
The American Academy of Allergy, Asthma & Immunology
The AAAAI designates this educational activity for a
(AAAAI) recognizes that you are a life-long learner who has
maximum of 1.0 category 1 credits towards the AMA
chosen to engage in continuing medical education to identify
Physician’s Recognition Award. The course is also
or fill a gap in knowledge, skill or performance. As part of the
approved by the California Board of Registered Nursing,
AAAAI’s duty to you as a learner, you have the right to expect
Provider #10704, for 1.2 contact hours and by the
that your continuing medical education experience with the
Commission for Case Manager Certification (CCMC) for
AAAAI includes:
1.0 contact hours.
Content that: For more information, visit the Professionals Center of
d promotes improvements or quality in healthcare; the AAAAI Web site, www.aaaai.org. Choose the
d is valid, reliable, and accurate; Professionals Center, click on the Education for You
d offers balanced presentations that are free of section and select the AAAAI Continuing Education
commercial bias for or against a product/service; Opportunities link.
d is vetted through a process that resolves any conflicts
of interests of planners, teachers, or authors; AWARDS AND GRANTS

d is driven and based on learning needs;
d addresses the stated objectives or purpose; and

n AAAAI/JACI Award for Outstanding
d is evaluated for its effectiveness in meeting the
identified educational needs. Research

David M. Fleischer MD, Denver, CO, has been awarded the
A learning environment that:
2005 AAAAI/JACI Award for Outstanding Research.
d supports learners’ ability to meet their individual needs;
d respects and attends to any special needs of the learners;
Fleischer was honored for his November 2004 paper, ‘‘Peanut
d respects the diversity of groups of learners; and
allergy: Recurrence and its management’’ (J Allergy Clin
d is free of promotional, commercial, and/or sales activities.
Immunol 2004 114(5):1195-201) designed and written during
his fellowship at Johns Hopkins, Baltimore MD.
Disclosure of:
d relevant financial relationships planners, teachers, n New FIT abstract awards
and authors have with commercial interests related to A new AAAAI Interest Section Fellow-in-Training Abstract
the content of the activity; and Award will honor seven fellows-in-training who submit the
d commercial support (funding or in-kind resources) of
best abstract in each Interest Section. The abstracts will
the activity. be selected by each Interest Section based on the highest
scored abstract. The AAAAI will award the presenter with
$500 and a plaque.
n Free online CME course No application is necessary for this award. All abstracts
The Environmental Management of Asthma, a free online submitted by an FIT will be considered. To submit an
Continuing Medical Education (CME) program, is available abstract, visit the Annual Meeting Web site,
on the AAAAI Web site, www.aaaai.org. The program provides www.annualmeeting.aaaai.org and choose the 2006 Annual
healthcare professionals and the insurance industry with Meeting link. Award recipients will be honored throughout
information and resources to incorporate environmental the 2006 Annual Meeting and during the Interest Section
management into clinical practices, and standards of care for Forums.
patients with asthma. AAAAI members and non-members For more information, please contact Mikelle Johnson
have an opportunity to earn 1.0 or 1.2 CME/CE credits. at the AAAAI executive office at (414) 272-6071 or e-mail
The program addresses the impact and management of mjohnson@aaaai.org.
environmental asthma triggers such as air pollutants, pollen,
environmental tobacco smoke, mold, dust mites, n 2006 AAAAI abstract awards available
cockroaches and warm-blooded pets. The course was Applications and details about all abstract awards
designed for physicians and allied health professionals, are available on the AAAAI Annual Meeting Web site,
including nurse case managers, in primary care specialties. www.annualmeeting.aaaai.org.
Page 26A August 2005 J ALLERGY CLIN IMMUNOL

NEW SVIEW
newsview
from the AAAAI
All applications are due September 7 with the exception Award: $1,000 with an accepted abstract or $500 without
of the Fellow-in-Training (FIT) Travel Scholarship. an abstract
AAAAI Allied Health Travel Scholarship Deadline: TBA
Purpose: Four scholarships are awarded to AAAAI allied Applications will be available on the Annual Meeting Web
health members who submit the best abstracts for site this fall. Please direct questions to Reaca Pearl at the
presentation at the 2006 Annual Meeting. AAAAI executive office at (414) 272-6071 or
Eligibility: AAAAI allied health members submitting e-mail rpearl@aaaai.org.
abstracts for presentation at the 2006 Annual Meeting 2006 ERT Allied Health Travel Grants
Award: Actual travel and hotel expenses incurred by the Purpose: The grants support education, research
abstract presenter, up to a limit of $750 per recipient. and travel for three AAAAI allied health members who submit
AAAAI Interest Section Fellow-in-Training Abstract Award the best abstracts for presentation at the 2006 Annual
Purpose: This new award will honor seven fellows-in- Meeting.
training who submit the best abstract in each Interest Section. Eligibility: AAAAI allied health members submitting
The abstracts will be selected by each Interest Section based abstracts for presentation at the 2006 Annual Meeting
on the highest scored abstract. No application is necessary for Award: $750 per recipient
this award. All abstracts submitted by an FIT will be International Fellow-in-Training (FIT) Travel
considered. The award recipients will be honored throughout Scholarship
the Annual Meeting and during the Interest Section Forums. Purpose: The scholarships are designed to financially
Eligibility: AAAAI fellows-in-training assist research and non-research fellows and residents-in-
Award: $500 and a plaque training in allergy and immunology in attending the AAAAI
2006 Annual Meeting.
AAAAI/Sepracor Research Excellence Awards
Eligibility: International FIT AAAAI members, or
Purpose: Three awards are given to abstract presenters
international FITs who have a completed membership
selected by the Abstract Review Committee. Abstracts will be
application on file at the time of scholarship application
judged by the novelty of their research development and the
submission, are eligible. Applicants must be post-doctoral
overall significance of their research toward the advancement
trainees in allergy/immunology who, at the time of
of allergy, asthma and immunology, and optimal patient care
application, are within seven years of their latest doctoral
Eligibility: The abstract first author must be an allergy and
degree, and are outside the United States and Canada.
immunology trainee (pre or post-doctoral), and the trainee
Award: To ensure greater equity, financial support reflects
must have conducted the vast majority of the abstract related
variable air travel costs based on location of award recipient.
work. The abstract co-author must be an AAAAI member.
Awards generally vary from $850 to $1,300.
The research should be investigator led and not sponsored by
any for-profit entity.
Award: $5,000 per recipient AAAAI MEMBER UPDATES
American Academy of Pediatrics Section on Allergy/
Immunology Outstanding Pediatric Allergy, Asthma and n 2006 Annual Meeting: call for abstracts
Immunology Abstract Awards for Fellows-in-Training The 2006 AAAAI abstract submission site is now open.
and Junior Faculty Take this important opportunity to make a significant
Purpose: Five awards are given, three to fellows-in- contribution to the overall scientific content of the 2006
training, and two to junior faculty members. All award Annual Meeting and share your findings with fellow Annual
recipients are eligible for a complimentary, one-year Meeting delegates.
membership in the AAP and SOAI. For more information, visit the AAAAI Annual Meeting Web
Eligibility: Fellows-in-training or junior faculty who site, www.annualmeeting.aaaai.org, or e-mail info@aaaai.org.
submit the best abstracts involving pediatric patients, birth The abstract submission deadline is September 7.
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 n Strategic Training in Allergy Research
each to two junior faculty members (ST*AR) Program
Domestic Fellow-in-Training (FIT) Travel Scholarship The new AAAAI Strategic Training in Allergy Research
Purpose: A grant to cover travel expenses to the Annual (ST*AR) Program will strengthen basic science research
Meeting in allergy/immunology and its translation to excellence
Eligibility: AAAAI FIT members in the United States and inclinical practice. Debuting at the 2006 Annual Meeting, the
Canada ST*AR Program will offer 40-50 PhD or post-doctoral

NEW SVIEW
newsview
from the AAAAI
students from the United States and Canada an in-depth d New Insights in Food Allergy
introduction to allergy/immunology research and an d New Paradigms in Upper and Lower Airways Diseases
opportunity to learn more about the specialty. The Plenary Session CD is $35. The CD may be purchased
Target audience: PhD trainees conducting research for up to two years following the 2005 Annual Meeting.
related to allergic diseases, asthma and immunology For more information or to order your CD, contact Alicia
Program overview: PhD trainees are invited to submit Josten at the AAAAI executive office at (414) 272-6071.
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 n AAAAI Annual Meeting dates and sites
ST*AR Program, which will include: 2006—Miami Beach, FL, March 3-7
d Presentation of original research at the ST*AR Program 2007—San Diego, CA, February 23-27
d Attendance at AAAAI Annual Meeting postgraduate 2008—Philadelphia, PA, March 14-18
program and symposia 2009—Washington, DC, March 13-17
d Participation in a special ST*AR Program that includes 2010—New Orleans, LA, February 26-March 2
basic research presentations in small groups by AAAAI
members who are NIH funded researchers in allergy/
immunology AAAAI WEB SITE RESOURCES
d Information about career opportunities in allergy,
Visit the AAAAI Web site, www.aaaai.org, for a variety of
asthma and immunology research professional and patient education resources.
d Funds for travel (including a stipend for ground
transportation), hotel accommodation and meeting

registration n Practice management tools available
The goal of the ST*AR Program is to provide PhD trainees Visit the AAAAI Web site, www.aaaai.org, for easy-to-use
with an opportunity to consider research opportunities in the resources that provide practice management assistance for
field of allergy and immunology. The AAAAI Annual Meeting established physicians, as well as those just starting out. A
provides an excellent forum for those interested in separate page will be developed for office managers and other
translating basic science advances into clinical practice to clinical staff that will include specific techniques and tools to
network with basic scientists, translational clinical support them in their day-to-day work.
researchers and clinical experts in allergy/ immunology. Current resource center topics include:
For more information, or to receive a ST*AR Program d 2004 Practice Management Workshop presentations
application, contact Reaca Pearl at the AAAAI executive d Financial performance
office at (414) 272-6071 or e-mail rpearl@aaaai.org. d HIPAA
d Medical office technology
n 2005 Annual Meeting Plenary Session on d Patient satisfaction
d Personal/time management
CD-ROM d Practice startup
Did you miss one of the six Plenary Sessions held during d Recruitment and staffing
the 2005 Annual Meeting? You still have an opportunity to d Resources and sample forms
learn about the newest research in the specialty and earn
AAAAI practice management resources are located in the
continuing education credits. AAAAI members and non-
Members Center of the AAAAI Web site. Visit the Members
members may order a CD of the six Annual Meeting Plenary
Center often, additional practice management information is
Sessions.
added continuously.
The Plenary Session CD will offer two types of
continuing education credit. Physicians may earn 10
Continuing Medical Education (CME) credits, and nurses n AAAAI mission statement
may earn 11.9 Continuing Education (CE) The mission of the American Academy of Allergy, Asthma
contact hours for designated sessions. & Immunology is the advancement of knowledge and practice
The six Plenary Sessions are: of allergy, asthma and immunology for optimal patient care.
d Atopy: Nature and Nurture For more information about the AAAAI, contact the executive
d New Paradigms in Cutaneous Inflammation office, 555 E. Wells Street, Suite 1100, Milwaukee, WI 53202-
d Optimizing Outcomes for Asthma 3823; phone (414) 272-6071; fax (414) 272-6070; e-mail
d New Insights in Primary Immune Deficiencies info@aaaai.org; or visit the Web site, www.aaaai.org.

cmeACTIVITIES
CME activities
CALENDAR
calendar
CME Mission Statement More information: e-mail cme@aaaai.org

Credits available: 2.5 CME
(Approved by the AAAAI’s CME Committee, March 18,
2001. Approved by Board of Directors, June 23, 2001.)
Dinner Symposium 3906
The purpose of the AAAAI’s CME program is to provide
The Suspension is Over: New Solutions for the
educational activities that will stimulate, maintain,
Treatment of Asthma
develop, and enhance the study and practice of allergy,
Sponsored by the AAAAI and funded through an
asthma and immunology. The content areas of the AAAAI’s
unrestricted educational grant from sanofi aventis.
CME program include any topic within the field of allergy,
Location: www.aaaai.org
asthma and immunology which impacts the study or
More information: e-mail cme@aaaai.org
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 Dinner Symposium 4906 Webcast
of activities included as part of the AAAAI’s CME program A Look to the Future: The Management of Allergic
include a national Annual Meeting; regional, local, and Diseases
other live conferences; computer-assisted interactive Sponsored by the AAAAI and funded through an
activities; and an array of written, audio, video and unrestricted educational grant from sanofi aventis.
multimedia enduring materials. Location: www.aaaai.org
The AAAAI supports the concept of jointly sponsoring More information: e-mail cme@aaaai.org
activities with organizations whose goals are compatible Credits available: 2.0 CME
with those listed in the Mission Statement. The expected
results of the AAAAI’s CME program are to advance the The JACI monthly review articles:
science and practice within the specialty of allergy, asthma
Current Reviews of Allergy and Clinical Immunology
and immunology.
Molecular Mechanisms in Allergy and Clinical
Immunology
AAAAI SPONSORED ENDURING Locations: JACI and online at www.mosby.com\jaci
MATERIALS More information: e-mail cme@aaaai.org
The activities listed below are directly sponsored by the
AAAAI and are available in print form or on the AAAAI Web Respiratory Digest articles
site, www.aaaai.org, as indicated by the descriptions below. Jointly sponsored by AAAAI and Adelphi, Inc., and
Participants may review the materials at their leisure for funded through an unrestricted educational grant from
credit; no attendance is necessary. CME credit will be sanofi aventis.
awarded upon completion of each activity’s evaluation or For more information, contact Joan Weiss at Adelphi,
post test. More specific instructions for claiming credits are Inc. by phone at (646) 602-7060, Fax (646) 602-6071, or
included in the individual enduring material. e-mail joan.weiss@adelphius.com.
Allergy-Immunology Medical Knowledge Self-
Assessment Program Respiratory Digest, Volume 3, Issue 3
Sponsored by the AAAAI, in partnership with the Clinical Implications of the Allergic Rhinitis/Asthma
American College of Physicians (ACP). Funded through Connection by Thomas B. Casale, MD, FAAAAI
an unrestricted educational grant from sanofi aventis.
For more information, e-mail cme@aaaai.org. Credits Respiratory Digest, Volume 3, Issue 4
available: 50.0 CME. Sleep-Disordered Breathing in Adults by Philip L. Smith,
MD, and Alan R. Schwartz, MD
2003 Postgraduate Plenary Session Webcast
Allergy, Asthma & Immunology: 60 Years of Progress Respiratory Digest, Volume 4, Issue 1
Sponsored by the AAAAI Evaluation of the Solitary Pulmonary Nodule by
Location: www.aaaai.org E. James Britt, MD

Executive Office
555 East Wells Street
Suite 1100
Milwaukee, WI 53202-3823
(414) 272-6071
www.aaaai.org
Respiratory Digest, Volume 4, Issue 2 Respiratory Digest Special Report 1 (2003)

Management of Exercise-Induced Asthma by John M. A Look to the Future: The Management of Allergic
Weiler, MD, and Julie A. Grant, MSPA-C Diseases by Thomas B. Casale, MD, FAAAAI, and
Erwin W. Gelfand, MD, FAAAAI
Respiratory Digest, Volume 4, Issue 3
Treatment of Severe Asthma by
Stephen P. Peters, MD, PhD Emerging Trends in the Management of Asthma and
Other Allergic Diseases: A Focus on Anti-IgE Therapy
Jointly sponsored by AAAAI and Synermed
Management of Exacerbations of Chronic
More information: E-mail cme@aaaai.org
Bronchitis by Victor M. Pinto-Plata, MD, and
Bartolome R. Celli, MD

Allergic Rhinitis and Its Comorbidities by The Clinical Benefits of Anti-Ige Therapy: A Targeted
Eli O. Meltzer, MD, FAAAAI Treatment for Patients with Asthma and Other
Allergic Airway Diseases
Respiratory Digest, Volume 5, Issue 2 Jointly sponsored by AAAAI and Synermed
Managing Antihistamine Impairment in Allergic Rhinitis More information: E-mail cme@aaaai.org
by Thomas B. Casale, MD, FAAAAI Credits available: 1.0 CME

T HE J OURNAL OF
Immunology
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’’ ‘‘EBV the prototypical human tumor virus—just
(page 241) how bad is it?’’ (page 251)
List of Design Committee Members: Author: List of Design Committee Members: Author:
Michael F. Tosi, MD David A. Thorley-Lawson, PhD
Activity Objectives Activity Objectives
1. To achieve a greater and more current understanding 1. To understand that EBV uses mature B cell biology to
of the nature and scope of innate immune responses to establish latency, persist, and replicate.
infection. 2. To understand that even though EBV is so widespread
2. To develop an enhanced appreciation for the range and apparently benign, it is potentially life-threatening.
of interactions among various components of innate 3. To understand that EBV evolved the capacity to make
immunity. cells grow because it is an essential part of the mechanism for
3. To be able to appreciate the specific microbial establishing latency in resting cells that are not pathogenic.
targets of specific effector mechanisms of innate 4. To understand that EBV-associated tumors arise from
immunity. different stages in the life cycle of latently infected B cells
Recognition of Commercial Support: This CME and that disruption of the immune response is an important
activity has not received external commercial support. component in the development of all of the EBV-associated
Disclosure of Significant Relationships with Relevant lymphomas.
Commercial Companies/Organizations: Recognition of Commercial Support: This CME
Michael F. Tosi has no significant relationships to activity has not received external commercial support.
disclose. Disclosure of Significant Relationships with Relevant
Commercial Companies/Organizations:
David A. Thorley-Lawson has equity ownership in
EBVax.

The Editors’ THE JOURNAL OF
Choice Allergy Clinical AND
Donald Y.M. Leung, MD, PhD

Harold S. Nelson, MD
Stanley J. Szefler, MD
Immunology
VOLUME 116 NUMBER 2
Ciclesonide and allergen-induced once-daily dosing of 40 lg, 80 lg, and placebo on allergen-
induced airway responses of individuals with mild
asthmatic responses asthma. This study demonstrates that ciclesonide 80 lg
attenuates the allergen-induced early and late changes in
New-generation inhaled steroids having low systemic FEV1 as well as serum eosinophil cationic protein and
effects are being sought for the treatment of asthma. sputum eosinophils measured at 24 hours after challenge
Ciclesonide (Alvesco) is a once-daily, nonhalogenated (P < .025), whereas ciclesonide 40 lg attenuates the late
inhaled corticosteroid (ICS) that has recently been asthmatic responses and sputum eosinophils measured at
introduced into the United Kingdom and Germany for 24 hours after challenge (P < .025). This study provides
the treatment of persistent asthma at doses of 80 lg new information regarding the minimally effective doses
and 160 lg. This ICS remains inactive until cleaved by of inhaled ciclesonide for inhibition of allergen-induced
esterases present in the airway, where its active metab- airway responses and the apparent local anti-inflamma-
olite, desisobutyryl-ciclesonide, then binds glucocorticoid tory effects on the airways. Further evaluation of
receptors. In this issue of the Journal, Gauvreau et al ciclesonide will be required to address whether these
(p 285) have investigated the effects of ciclesonide in a low doses are clinically effective.
multi-center, randomized, crossover study, comparing
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 Prevalence of wheeze (without cold) by distance from DEP source. Moving
infants who reside farther from all sources of DEP. In exposure, residing within 400 m of a highway with >1000 trucks daily;
addition, African American infants had the highest Stop-and-go exposure, residing within 100 m of either a bus route or an urban
state route.
prevalence of wheezing in comparison with Caucasian
infants, regardless of exposure category. These findings amounts of traffic. This study is the first report from the
suggest that living very close to stop-and-go traffic early ongoing Cincinnati Childhood Allergy and Air Pollution
in infancy is a significant risk factor for wheezing. It also Study, the goal of which is to elucidate the environmental
suggests that even within an urban environment, an infant’s and genetic contributions to the development of allergic
risk for wheezing varies with exposure to different types and diseases.
et al. (p 431) assessed in a cohort of US children

Endotoxin and the pathway to allergy household dust endotoxin at age 2-3 months and
PBMC-proliferative and cytokine responses to cockroach,
Endotoxin, a part of the cell wall of gram-negative
dust mite, and cat allergens and to the nonspecific mitogen
bacteria, is present at higher levels in households with large
phytohemagglutinin at age 2-3 years. They found that
animals (livestock or dogs). Infant endotoxin exposure has
increased endotoxin levels were associated with decreased
been proposed as a factor that might protect against allergy
IL-13 in response to cockroach, dust mite, and cat allergen
and early childhood immune responses (eg, production
but not in response to mitogen stimulation. An inverse,
of the cytokine IL-13) that increase IgE production to
though nonsignificant, association was found between
allergens. Cross-sectional European studies have found that
endotoxin and proliferative responses. Early-life endotoxin-
elevated endotoxin levels are associated with allergy
related reduction of IL-13 production might represent one
protection in children of farmers, but the immunologic
pathway through which elevated endotoxin decreases the
pathway to explain this association is uncertain and the
risk of allergic disease and allergy in later childhood.
relevance of the finding to children in the more urban
US setting is unclear. In this issue of the Journal, Abraham
J ALLERGY CLIN IMMUNOL August 2005 Page 239

Cholinergic urticaria patients: Sometimes release with autologous sweat and serum. Eleven of
17 patients with CU had positive skin test results, and
allergic to their own sweat 10 of 17 had basophil histamine release with autologous
sweat, with a significant correlation between the 2 re-
Cholinergic urticaria (CU) presents a characteristic sponses. Nine of 16 of the patients with CU had a positive
picture of pinpoint-sized, highly pruritic wheals with intradermal skin test result with autologous serum. The
surrounding erythema that occurs after sweating during authors proposed that there are 2 distinct subtypes of
physical exercise, bathing, or emotional stress. The path- patients with CU: (a) those who have strong reactions
ogenesis of CU is not well defined. However, it has been to autologous sweat and negative reactions to autologous
reported that these patients have positive intradermal serum whose wheals are not associated with hair follicles
skin tests to their own sweat. In this issue of the Journal, and (b) those characterized by weak reactions to autologous
Dr Fukunaga and colleagues (p 397) report on this sensi- sweat and positive reactions to autologous serum whose
tization in 18 subjects with CU and 10 controls. They wheals are associated with hair follicles.
performed intradermal skin testing and basophil histamine
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 Distribution of positive skin tests to 9 aeroallergens and peanuts in the
issue of the Journal (p 377). Of 10,508 skin-tested subjects US population, age 6 to 59 years.
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 Comparison of these results with the results of the skin
subjects), rye grass (in 26.9%), short ragweed (in 26.2%), testing performed in NHANES II was difficult because
and German cockroach (in 26.1%). The remainder— of numerous methodologic differences between the 2
Bermuda grass, Russian thistle, white oak, cat, and studies. However, for the 6 aeroallergens tested in both
Alternaria alternata—were positive in 10% to 20% of surveys, a positive PPST result was 2.1 to 5.5 times more
subjects. The least commonly positive skin test was to common in NHANES III than in NHANES II. Although the
peanut (8.6%). The 3 most significant independent authors could not definitely conclude that these differences
predictors of a positive PPST result to aeroallergens were represent an increased sensitivity in the US population,
age (maximal for the 20- to 29-years age group), male sex, they point out that such an increase would be consistent
and minority ethnicity, especially non-Hispanic black. with reports from other countries.
antigen. Administration of the COX-2 selective inhibitor

COX-2 inhibitors enhance allergic NS-398 during EC sensitization resulted in enhanced skin
responses infiltration by eosinophils and expression of IL-4 mRNA,
enhanced OVA-specific IgE and IgG1 antibody responses,
Mechanical injury to the skin by scratching is an and increased IL-4 secretion by splenocytes following
important feature of atopic dermatitis (AD). In this issue OVA stimulation. COX-2–deficient mice also exhibited an
of the Journal, Laouini et al (p 390) show that mechanical enhanced systemic TH2 response to EC sensitization. These
injury to mouse skin inflicted by tape stripping results in results demonstrate that COX-2 products limit allergic skin
rapid induction of cyclooxygenase-2 (COX-2) mRNA and inflammation, and they are consistent with the recent
protein and in accumulation of the COX-2 product prosta- finding that engagement of the EP3 receptor by PGE2
glandin E2 (PGE2). The role of COX-2 was examined in a inhibits allergic reactions (Nat Immunol 2005;6:524-31).
mouse model of AD elicited by repeated epicutaneous (EC) More importantly, the work of Laouini et al suggests that
sensitization with ovalbumin (OVA) and characterized by COX-2 inhibitors might worsen allergic skin inflammation
eosinophil skin infiltration and a systemic TH2 response to and should be avoided in patients with AD.
Page 240 August 2005 J ALLERGY CLIN IMMUNOL

feature articles
Reviews and
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 Abbreviations used
the existence of a separate set of protective mechanisms that do CXCL: CXC ligand
not depend on specific antigenic recognition. These antigen- HBD: Human b-defensin
independent mechanisms constitute innate immunity. ICAM-1: Intercellular adhesion molecule 1
Antimicrobial peptides are released at epithelial surfaces and LFA-1: Lymphocyte function-associated antigen 1
disrupt the membranes of many microbial pathogens. Toll-like MAC: Membrane attack complex
receptors on epithelial cells and leukocytes recognize a range of Mac-1: Macrophage antigen-1
microbial molecular patterns and generate intracellular signals MBL: Mannan-binding lectin
for activation of a range of host responses. Cytokines released NADPH: Reduced nicotinamide adenine dinucleotide
from leukocytes and other cells exhibit a vast array of phosphate
regulatory functions in both adaptive and innate immunity. NF: Nuclear factor
Chemokines released from infected tissues recruit diverse NK: Natural killer
populations of leukocytes that express distinct chemokine PMN: Polymorphonuclear leukocyte
receptors. Natural killer cells recognize and bind virus-infected TLR: Toll-like receptor
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 ing the more rapid and phylogenetically primitive non-
kill invading microbes. These innate immune mechanisms specific responses to infection, such as surface defenses,
and their interactions in defense against infection provide cytokine elaboration, complement activation, and phago-
the host with the time needed to mobilize the more slowly cytic responses,1 and adaptive immunity, involving more
developing mechanisms of adaptive immunity, which might slowly developing, long-lived, and highly evolved
protect against subsequent challenges. (J Allergy Clin
antigen-specific protective responses, such as antibody
Immunol 2005;116:241-9.)
production and cell-mediated immunity, that exhibit
extraordinarily diverse ranges of specificity.2,3 However,
Key words: Innate immunity, antimicrobial peptides, Toll-like
receptors, chemokines, natural killer cells, complement, phagocytes 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
It is traditional to organize host responses to infection the main innate responses to infection that are available
into separate arms or compartments, such as complement, to the human host, including relevant examples of such
phagocytes, cytokines, cell-mediated immunity, and interactions.
humoral immunity. A more current approach has been to
consider 2 larger categories: innate immunity, incorporat-
INNATE IMMUNITY
Epithelia, defensins, and other
From the Department of Pediatrics, Mount Sinai School of Medicine,
New York, and the Division of Pediatric Infectious Diseases, Maimonides antimicrobial peptides
Medical Center, Brooklyn. The epithelium of skin and mucosal tissue functions as
Disclosure of potential conflict of interst: M. F. Tosi—none disclosed.
a mechanical barrier to the invasion of microbial patho-
Received for publication May 17, 2005; accepted for publication May 18,
2005. gens. In the last 2 decades, it has become clear that
Available online July 5, 2005. epithelial cells also are a major source of antimicrobial
Reprint requests: Michael F. Tosi, MD, Division of Pediatric Infectious peptides that play important roles in local host defense.4,5
Diseases, Maimonides Medical Center, 977 48th St, Brooklyn, NY 11219. Studies of their structure, sources, expression, and actions
E-mail: mtosi@maimonidesmed.org.
0091-6749/$30.00
also have revealed an unexpected range of immunologic
Ó 2005 American Academy of Allergy, Asthma and Immunology activities for these molecules, the functions of which once
doi:10.1016/j.jaci.2005.05.036 were considered mainly antimicrobial in nature.4
241
242 Tosi J ALLERGY CLIN IMMUNOL
AUGUST 2005
feature articles
Reviews and
Epithelial cells of mucous membranes of the airways activation of dendritic cell maturation, already suggest a
and intestines, as well as keratinocytes, express the human highly complex and regulatory role in the development of
b-defensins (HBD-1 through HBD-4). These small cati- host defense and immunity. Recent genomic evidence for
onic peptides are similar to the a-defensins stored in the the possible existence of as many as 25 additional human
azurophilic granules of neutrophils, and they display anti- defensins that have not yet been characterized suggests
microbial activity against a broad range of bacteria, fungi, that current knowledge describes but a small sample of
chlamydiae, and enveloped viruses.4,5 Their production the overall contribution of these peptides to immune
by epithelial cells might be constitutive, as for HBD-1, or responses.20
inducible, as for HBD-2, HBD-3, and HBD-4. For exam-
ple, recent evidence indicates that epithelial cells of the
airway or intestine can produce HBD-2 in response to TLRs
activation by bacterial products through toll-like receptors Mononuclear phagocytes, including circulating mono-
(TLRs) 2 or 4 (see below) on the epithelial cells.6,7 cytes and tissue macrophages, other phagocytic cells, and
Stimulation of epithelium by cytokines, including IL-1 or many epithelial cells, express a family of receptors that is
TNF-a, also can induce defensin production.4 Defensins highly homologous to the Drosophila receptor called
have been reported to exert their antimicrobial action Toll.6,7,21 These receptors mediate a phylogenetically
either through the creation of membrane pores or through primitive, nonclonal mechanism of pathogen recognition
membrane disruption resulting from electrostatic interac- based on binding not to specific antigens but to structurally
tion with the polar head groups of membrane lipids, with conserved pathogen-associated molecular patterns.21-23
more evidence now favoring the latter mechanism.4,8 There are at least 10 human TLRs with a range of
Some microorganisms have evolved mechanisms for microbial ligands, such as gram-negative bacterial LPS,
evading the action of defensins. For example, bacterial bacterial lipoproteins, lipoteichoic acids of gram-positive
polysaccharide capsules might limit access of microbial bacteria, bacterial cell-wall peptidoglycans, cell-wall com-
peptides to the cell membrane,9 and an exoprotein of ponents of yeast and mycobacteria, unmethylated CpG
Staphylococcus aureus, staphylokinase, neutralizes the dinucleotide motifs in bacterial DNA, and viral RNA.22-24
microbicidal action of neutrophil a-defensins.10 Gram-positive cell-wall components bind mainly to
Several immunoregulatory properties of defensins and TLR2, and TLR2 also can bind components of herpes
related peptides, distinct from their antimicrobial actions, simplex virus.22-25 Gram-negative LPS activates TLR4
have been documented.4 Several such peptides have been indirectly by first binding to LPS-binding protein, which
shown to facilitate posttranslational processing of IL-1b.11 binds in turn to CD14 at the cell surface. The bound CD14
Some of the b-defensins have been shown to function as has no transmembrane domain but associates directly with
chemoattractants for neutrophils, memory T cells, and an extracellular domain of TLR4.23,24 TLR5 has been
immature dendritic cells by binding to the chemokine identified as the receptor for bacterial flagellin, TLR9
receptor CCR-6.5,12,13 Separately, HBD-2 has been shown recognizes CpG motifs of bacterial DNA, and TLR3 has
to activate immature dendritic cells through a mechanism been shown to bind synthetic and viral double-stranded
that requires TLR4.14 The activation of immature dendritic RNA.26-28
cells by these mechanisms also promotes their maturation. Signalling through TLRs occurs through a well-
The b-defensins also act as a chemoattractant for mast cells described pathway in which receptor binding generates
through an undefined mechanism and can induce mast cell a signal through an adaptor molecule, MyD88, that leads
degranulation.15 HBD-2 and several other antimicrobial to intracellular association with IL-1 receptor-associated
peptides can interfere with binding between bacterial LPS kinase. In turn, this leads to activation of TNF receptor–
and LPS-binding protein.16 associated factor 6, which results in nuclear translocation
Additional antimicrobial peptides of epithelial cells of nuclear factor kB (NF-kB).23,24 NF-kB is an important
include lysozyme and cathelicidin. Lysozyme, an antimi- transcription factor that activates the promoters of the
crobial peptide also found in neutrophil granules, attacks genes for a broad range of cytokines and other proin-
the peptidoglycan cell walls of bacteria and can be released flammatory products, such as TNF-a, IL-1, IL-6, and IL-8.
from cells through mechanisms that involve TLR activa- This signalling pathway, on the basis of studies with
tion.17 Cathelicidin, or LL37, like lysozyme, is released TLR4, is similar but not identical to the signalling
from both neutrophils and epithelial cells. It exhibits broad pathways activated by other TLRs.24 The activation of
antimicrobial activity and can inhibit lentiviral replica- cytokine production by TLRs plays an important role in
tion.5,18 Cathelicidin also exhibits chemotactic activity for recruiting other components of innate host defense against
neutrophils, monocytes, and T lymphocytes. This activity bacterial pathogens. However, with large-scale cytokine
is mediated by a formyl peptide receptor-like molecule, release, the deleterious effects of sepsis or other forms
FPRL1, rather than the chemokine receptor CCR6 bound of the systemic inflammatory response syndrome demon-
by b-defensins.19 strate that these pathways have both beneficial and
The release of defensins in response to activation of potentially harmful effects for the host.24 Genetic poly-
TLRs and the many actions of these peptides, including morphisms in TLRs might play a role in determining the
their direct antimicrobial activities, their chemoattractant balance of these effects in certain individuals responding
actions for a wide range of immune cells, and their to the challenge of systemic infection.24,29,30
J ALLERGY CLIN IMMUNOL Tosi 243
feature articles
Reviews and
In addition to their first-responder roles in generating multiple organ failure.24 For some systemic actions,
an inflammatory response to invading pathogens, TLRs notably the production of hemodynamic shock, IL-1 and
can network with other components of innate and adap- TNF-a are synergistic. Both IL-1 and TNF-a also induce
tive immunity. TLR4 function is suppressed by activa- production of IL-6, a somewhat less potent cytokine that
tion of cells through the chemokine receptor CXCR4.31 exhibits some of the actions of IL-1 and TNF-a.34 The
Activation of some TLRs also can induce expression of human host produces several soluble antagonists of
the costimulatory molecule B7 on antigen-presenting IL-1 and TNF-a that can modulate their effects, including
cells, which is required for activation of naive T cells.21 IL-1 receptor antagonist, soluble TNF-a receptor, and
anti-inflammatory cytokines, especially IL-10.24
Cytokines The importance of effects mediated by IL-1 and TNF-a
A heterogeneous group of soluble small polypeptide or in the pathophysiology of septic shock has prompted
glycoprotein mediators, often collectively called cyto- much active research aimed at blocking their effects to
kines, form part of a complex network that helps regulate reduce morbidity and mortality. Monoclonal antibodies
the immune and inflammatory responses. Included in this against TNF-a and other inhibitors of TNF-a or IL-1 have
group of mediators, the molecular weights of which range showed early promise in vitro and in animal models of
from about 8 to about 45 kd, are the ILs, IFNs, growth septic shock.24,34,36,37 However, they have been far more
factors, and chemokines (see separately below). Most cells effective at preventing the effects of cytokines than
of the immune system, as well as many other host cell reversing them. More recent attempts to address the issue
types, release cytokines, respond to cytokines through of the timing of intervention have been directed at the
specific cytokine receptors, or both. The range of sources intracellular signaling mechanisms activated through the
and effects of cytokines and their actions and interrela- TNF-a receptor or at mediators that appear later than
tionships are of such complexity that they cannot be TNF-a. Lipophilic inhibitors of protein tyrosine kinases,
addressed here in detail. A number of them will be enzymes that propagate the cellular signals through TNF-a
addressed individually in sections below, and several receptors, have been found to enhance survival in exper-
excellent reviews are available.32-34 However, 2 cyto- imental animals, even when administered 2 hours after
kines, IL-1 and TNF-a, are of such fundamental impor- systemic injection with endotoxin.38 Additionally, mAbs
tance in acute host responses to infection that they warrant against a cytokine-like nonhistone nucleoprotein product
specific attention. of macrophages, high-mobility group B1 (which appears
IL-1 and TNF-a are small polypeptides, each with a much later than TNF-a or IL-1 after LPS stimulation),
molecular weight of approximately 17 kd, that exhibit a were found to rescue mice from endotoxin shock when
broad range of effects on immunologic responses, inflam- given 2 hours after an otherwise lethal dose of LPS.39 More
mation, metabolism, and hematopoiesis.34,35 IL-1 origi- recently, clinical trials with activated protein C, a regula-
nally was described as ‘‘endogenous pyrogen,’’ referring tory protein in the coagulation cascade, has demonstrated
to its ability to produce fever in experimental animals, and beneficial effects in selected patients with septic shock
TNF-a, which produces some of the same effects produced through mechanisms that might involve inhibition of
by IL-1, was originally named ‘‘cachectin’’ after the NF-kB activation.40 To date, despite progress, clinical
wasting syndrome it produced when injected chronically strategies to interfere with the cytokine-induced cascade
in mice.34,35 Many of the physiologic changes associated that leads to endotoxin-induced shock have continued,
with gram-negative sepsis can be reproduced by injecting overall, to meet with limited success.
experimental animals with these cytokines in the absence
of microorganisms. Depending on the doses injected, these Chemokines
effects might include fever, hypotension, and either neu- A specialized group of small cytokine-like polypep-
trophilia or leukopenia.34,35 In the production of endotoxic tides, chemokines, which all share the feature of being
shock caused by gram-negative sepsis, IL-1 and TNF-a ligands for G protein–coupled, 7-transmembrane segment
are produced by mononuclear phagocytes in response to receptors, plays an increasingly complex role in the
activation of TLRs by bacterial LPS. They in turn activate immune response as cellular activators that induce directed
the production of other cytokines and chemokines, lipid cell migration, mainly of immune and inflammatory
mediators (eg, platelet-activating factor and prostaglan- cells.41-44 The chemokines and their receptors have been
dins), and reactive oxygen species. They also induce classified into 4 families on the basis of the motif displayed
expression of adhesion molecules of both endothelial cells by the first 2 cysteine residues of the respective chemokine
and leukocytes, stimulating recruitment of leukocytes by peptide sequence. Each of at least 16 CXC chemokines
inducing release of the chemokine IL-8 and activating binds to 1 or more of the CXCRs (CXCR1 through
neutrophils for phagocytosis, degranulation, and oxidative CXCR6). Examples of CXC chemokines include IL-8
burst activity.24,35 These all are important and usually and Gro-a. Similarly, at least 28 CC chemokines, such as
beneficial host responses to infection. However, at high macrophage inflammatory protein 1a, RANTES, and
levels of activation, there sometimes are pathophysio- eotaxin-1, 2, and 3, bind to one or more of the CCRs
logic effects of this proinflammatory cascade, including (CCR1 through CCR10). The sole CX3C chemokine,
vascular instability, decreased myocardial contractility, fractalkine, or neurotaxin, binds to CX3CR1, currently
capillary leak, tissue hypoperfusion, coagulopathy, and the only receptor in its family. The 2 XC chemokines,
AUGUST 2005
feature articles
Reviews and
including lymphotaxin, bind to the sole receptor in this tumors. NK cells are found in the peripheral circulation
family, XCR1. A new nomenclature has been proposed to and in the spleen and bone marrow. Like many other
designate each of the chemokines as a numbered ligand for leukocytes, they can be recruited to sites of inflammation
its respective receptor family. In this system Gro-a is CXC by chemokines and other chemoattractants. They appear
ligand (CXCL) 1 (or CXCL-1), and IL-8 now becomes to be important for the control of tumors in vivo and serve a
CXCL-8. Similarly, RANTES becomes CCL-5, fractal- critical function in host defense against viral infections,
kine is CX3CL-1, and lymphotactin is XCL-1.41,43 An especially those caused by members of the herpesvirus
update of this nomenclature system recently has been family.51,52 Activated NK cells also are an important
published, tabulating each of the families with their source of IFN-g, which limits tumor angiogenesis and
respective ligands and receptors, as well as the traditional promotes the development of specific protective immune
names in both human and murine systems.43 responses.51,52
Virtually every cell type of the immune system Regulation of NK cell activity involves a balance
expresses receptors for one or more of the chemokines. between activating and inhibitory signals. Several cyto-
The cells of most inflamed tissues can release a variety of kines can activate NK cell proliferation, cytotoxicity, or
chemokines, and tissues infected with different bacteria or IFN-g production, including IL-12, IL-15, IL-18, IL-21,
viruses release chemokines that recruit characteristic sets and IFN-ab.51 Activating signals through other receptors
of immune cells.44,45 For example, whereas rhinoviruses on NK cells, such as NKG2D, can lead to cytotoxicity,
induce the release of chemokines that result mainly in cytokine production, or both, depending on the receptor’s
recruitment of neutrophils (early in the course of infec- association with distinct intracellular adaptor proteins that
tion), EBV induces a set of chemokines that result in signal through different kinases.51,53 Other molecules on
recruitment of B cells, natural killer (NK) cells, and both NK cells can act as either costimulatory or adhesion
CD41 and CD81 T cells.45 It is of interest that almost receptors, including CD27, CD28, CD154 (CD40 ligand),
mutually exclusive sets of chemokines are induced by and lymphocyte function-associated antigen 1 (LFA-1)
cytokines associated with TH1 (IL-12 and IFN-g) versus (CD11a/CD18).51-53 Additionally, FcgRIII (CD16) can
TH2 (IL-4 and IL-13) immune responses, indicating a tight contribute to NK cell cytotoxic activity through mecha-
interplay between cytokines and chemokines in determin- nisms that include antibody-dependent cell cytotoxicity.51
ing the type of immune response to specific infectious NK cells are able to distinguish normal cells of self origin
challenges generated under different conditions.46 The through receptors that recognize specific MHC class I
specificity of such cellular responses is strongly influenced molecules. Activation of such receptors provides an
by the chemokines released from specific tissues, the inhibitory signal that protects healthy host cells from
vascular adhesion molecules expressed in those tissues, NK cell–mediated lysis. Virus-infected cells and malig-
the chemokine receptors expressed by different popula- nant cells often express MHC class I molecules at reduced
tions of leukocytes, and the specific adhesion molecules levels and thus are less able to generate inhibitory NK cell
expressed by leukocytes.44-46 signals, rendering them more susceptible to attack by NK
Modulation of chemokine function can occur through cells.51,52 NK cell inhibitory receptors, which are not well
several mechanisms. Chemokines themselves can be characterized, appear to contain intracytoplasmic tyro-
potentiated or inactivated by tissue proteases, including sine-based inhibition motifs and antagonize NK cell
tissue peptidases and matrix metalloproteases.47 Heparin activation pathways through protein tyrosine phospha-
sulfate–related proteoglycans on endothelial cell surfaces tases.51,54 Thus the regulation of the phosphorylation state
tether chemokines locally, where they can most efficiently of specific tyrosine residues by activating kinases and
activate circulating leukocytes for adhesion (see below). inhibitory phosphatases appears to be a pivotal determi-
However, similar proteoglycans free in the extracellular nant of NK cell activation.
environment can bind and sequester chemokines, keeping NK cells kill infected or malignant cells through the
them from interacting with their cellular receptors.48,49 release of perforin and granzymes from granular storage
Finally, in addition to the well-described use of chemokine compartments and through binding of the death receptors
receptors as coreceptors for viral entry by HIV-1, other Fas and TRAIL-R on target cells through their respective
viruses, especially members of the herpesvirus family, NK cell ligands.51,55 The mechanisms by which perforin
encode soluble decoy receptors that compete with native and granzymes mediate target cell death are not fully
host receptors for chemokine binding, thereby disrupting understood. The best available evidence suggests that
normal host responses.49,50 perforin and one or more of 5 human granzymes released
along with perforin from cytotoxic granules of NK cells
NK cells associate with the cell membranes of target cells, either
NK cells are an important cellular feature of innate by binding through the mannose 6-phosphate receptor or
immunity. They are lymphoid cells that do not express through another mechanism that remains to be defined.
clonally distributed receptors, such as T-cell receptors One or more of the granzymes appears to activate intra-
or surface immunoglobulin, for specific antigens.51 They cellular pathways leading to target apoptosis through
respond in an antigen-independent manner to help contain pathways that involve the mitochondria, caspases, or
viral infections before the development of adaptive im- both.56 Separately, binding of the death receptors also
mune responses, and they aid in the control of malignant activates caspases, causing target cell apoptosis.51 It is
feature articles
Reviews and
notable that although some tumor cells do not express Fas, C3b and undergoes proteolytic cleavage by factor D to
NK cells can induce Fas expression on these targets by form C3bBb, the alternative pathway C3 convertase.
releasing IFN-g and then proceed to kill them by binding Properdin stabilizes the convertase, which produces
to the newly expressed Fas.51 more C3b, establishing the C3 amplification loop of the
NK cells engage in several kinds of interactions with alternative pathway and activating the remainder of the
other cells of the immune system, including dendritic cells cascade.59 Microbes that bear large amounts of surface
and other antigen-presenting cells. Dendritic cells can sialic acid are usually poor activators of the alternative
influence the proliferation and activation of NK cells both pathway because factor H outcompetes factor B for C3b
through release of cytokines, including IL-12, and through binding at such surfaces.59,61 No convertase or amplifica-
cell-surface interactions, including CD40/CD40 ligand, tion loop is created because factor H allows C3b cleavage
LFA-1/intercellular adhesion molecule 1 (ICAM-1), and by factor I to form iC3b, the only function of which is
CD27/CD70.57 In return, NK cells can provide signals that opsonic.59 Nonactivators of the alternative pathway are
result in either dendritic cell maturation or apoptosis.51,52 some of the most successful pathogens in nonimmune
hosts. They include K1 Escherichia coli, groups A and
The complement system B streptococci, Streptococcus pneumoniae, Neisseria
The complement system is made up of at least 30 meningitidis, Haemophilus influenzae type b, and some
proteins in serum or at cell surfaces. Most of these proteins salmonellae.62
are made in the liver or, to a lesser extent, by mononuclear Complement effector functions. Opsonization (from
phagocytes. Activation of the complement cascade by one the Greek ‘‘to cater or prepare’’) facilitates the removal of
or more of 3 distinct pathways leads to the evolution of microorganisms from the circulation by macrophages in
its main effector functions: microbial opsonization, phago- the liver and spleen and from tissue sites by neutrophils
cyte recruitment, and bacteriolysis. All 3 activation path- and tissue macrophages.58,59 Recognition and attachment
ways act at a microbial surface to assemble a convertase of surface-bound C3b and iC3b on microbes by the type
that cleaves C3 to form C3b, which in turn binds to the 1 and type 3 phagocytic complement receptors, CR1
target surface, either as an opsonin or to help activate C5 (CD35) and CR3 (CD11b/CD18), respectively, activates
and the remainder of the cascade.58-61 ingestion and intracellular killing of the organisms.59
Complement activation pathways. The classical path- Antibodies, themselves important opsonins, direct the
way ordinarily is activated by IgG or IgM bound to localization of C3b binding on microorganisms through
microbial surface antigens. Antigen binding makes a site the classical pathway. This is important for encapsulated
on the Fc portion of the antibody available to bind C1q, bacteria because the capsule’s presence as a barrier means
which in turn binds C1r and C1s. C1qrs catalyzes the that only C3b bound at the capsular surface by specific
cleavage and binding of C4 and C2, in the form of C4b2a, anticapsular antibody will be accessible to phagocyte
the classical pathway C3 convertase. This convertase then receptors.63
cleaves C3 to form C3a, which is released, and C3b, which The free cleavage fragments of C3 and C5 can promote
remains bound on the target surface, and the cascade host inflammatory responses. C3a stimulates marrow
beyond C3 continues.59 release of granulocytes, and C5a serves as a potent
The recently characterized mannan-binding lectin chemoattractant for monocytes, neutrophils, and eosino-
(MBL) pathway is similar to the classical pathway but phils. C5a also stimulates expression of CR1 and CR3,
does not involve antibodies.60 MBL is a serum protein of aggregation, and microbicidal activity of phagocytes.
the collectin family with structural and functional simi- C5a-induced neutrophil aggregation and stasis in the
larities to C1q and binds to mannose-containing carbohy- pulmonary circulation can contribute to the respiratory
drates on microbial surfaces. Subsequently, MBL and the distress syndrome associated with sepsis.64 C4a, C3a, and
MBL-associated serine proteases, which have structural especially C5a are anaphylotoxins that can induce release
and functional similarities to C1r and C1s, form a complex of histamine from mast cells and basophils, causing
that activates C4, with sequential binding of C4b and increased vascular dilatation and permeability.64 In large
C2a and formation of C4b2a, the classical pathway C3 amounts, they can contribute to the pathophysiology of
convertase.60 C3 is activated, and the cascade proceeds septic shock.64
as described. When C5a is released by the classical or alternative
The alternative pathway is vital to the host as a separate pathway C5 convertase, C5b is bound at the target surface.
means by which C3 can be activated before development The terminal complement proteins C5b, C6, C7, C8, and
of specific antibodies.59,61 A low constitutive level of C9 assemble sequentially to form the membrane attack
hydrolysis of the thioester of C3 in the fluid phase complex (MAC), which can kill and lyse target cells,
produces an activated form of C3, C3(H2O). This acti- especially gram-negative bacteria, by penetrating their
vated form of C3 can bind factor B, and the latter is outer membranes.59,61 The C5b-C8 complex serves as a
cleaved by factor D to form the fluid-phase C3 convertase polymerization site for several molecules of C9.61,65
C3(H2O)Bb. The constitutive presence of small amounts Although C9 is not essential to membrane penetration,
of this convertase in the fluid phase ensures that some its presence as poly-C9 allows it to proceed more effi-
C3b always is available to initiate the alternative path- ciently.59,65 The MAC also can lyse certain virus-infected
way at microbial surfaces.59,61 Factor B binds to surface host cells and some enveloped viruses directly.66
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Reviews and
Complement fragments can modulate other parts of the necessary for transendothelial migration of the
immune response, both directly by binding to CR1, CR2, PMNs.75,77,79,80 Other chemoattractants, such as C5a,
and CR3 on the surfaces of T cells, B cells, and other cells N-formyl bacterial oligopeptides, and leukotrienes (eg,
involved in antigen recognition and indirectly by stimu- leukotriene B4) that diffuse from the site of infection fur-
lating the synthesis and release of cytokines.67 For exam- ther activate PMNs and provide a chemotactic gradient
ple, the C3b cleavage product, C3dg, when covalently for PMN migration into tissue.41,80,81 The receptors for
bound to antigen, brings the antigen close to B cells by these chemoattractants, like the chemokine receptors, are
binding to B-cell CR2 (CD21); C3 influences antigen G protein–coupled and share a 7-transmembrane domain
localization within germinal centers, anamnestic re- structure.41,81 They constitute important sensory mecha-
sponses, and isotype switching; and C1, C2, C4, and C3 nisms of the PMNs for activating adhesion, directional
are important for normal antibody responses.68,69 orientation, and the contractile protein–dependent lateral
movement of adhesion sites in the PMN membrane
Phagocytes necessary for migration.41,81-83 Although the specific
The first recognized cellular mechanism of host defense stimuli and adhesion molecules might vary, this general
was the accumulation of phagocytic host cells around a scheme applies to the local recruitment of virtually all
foreign body in starfish observed by Metchnikoff.70 circulating cells of the immune system.44-46
Polymorphonuclear leukocytes (PMNs), the most abun- Phagocytosis. After PMNs reach the site of infection,
dant circulating phagocytes in the human host, will serve they must recognize and ingest, or phagocytose, the
as a model for discussing phagocyte functions. These cells invading bacteria. Opsonization, especially with IgG and
constitute a major line of defense against invading bacte- fragments of C3, greatly enhances phagocytosis.58,63
rial and fungi. The proliferation of myeloid marrow Although nonopsonic phagocytosis can occur, only opso-
progenitors and their differentiation into mature progeny nin-mediated phagocytosis is considered here. CR1 and
are regulated by specific growth factors and cyto- CR3 are the main phagocytic receptors for opsonic C3b
kines.71,72 The normal half-life of circulating PMNs is and iC3b, respectively.79 When PMNs are activated by
approximately 8 to 12 hours.73 In the absence of active chemoattractants or other stimuli, CR1 and CR3 are
infection, most PMNs leave the circulation through the rapidly translocated to the cell surface from intracellular
gingival crevices and the lower gastrointestinal tract, storage compartments, increasing surface expression up to
where the resident flora stimulate ongoing local extrava- 10-fold.79 Note that CR3 is identical to the adhesion-
sation of PMNs, a process that helps maintain the integrity mediating integrin Mac-1.79 CR1 and CR3 act synergis-
of these tissues.74 In response to invasive bacterial infec- tically with receptors for the Fc portion of antibodies,
tion, circulating PMNs engage in 3 major functions: (1) especially IgG.58,59 Phagocytic cells can express up to 3
migration to the site of infection, (2) recognition and different types of IgG Fc receptors, or FcgRs, all of which
ingestion of invading microorganisms, and (3) killing and can mediate phagocytosis.84 FcgRI (CD64) is a high-
digestion of these organisms. affinity receptor that is expressed mainly on mononuclear
Phagocyte recruitment to infected sites. Activation phagocytes.84 The 2 FcgRs ordinarily expressed on circu-
of endothelial cells that line the microvessels of acutely lating PMNs are FcgRII (CD32) and FcgRIII (CD16).84
infected tissue occurs through locally produced cytokines, FcgRII is conventionally anchored in the cell membrane,
eicosanoid compounds, and microbial products.75 As a exhibits polymorphisms that determine preferences for
result, the endothelial cells rapidly upregulate their surface binding of certain IgG subclasses, and can directly activate
expression of P-selectin and then E-selectin.75,76 These PMN oxidative burst activity.84,85 FcgRIII is expressed on
selectins engage in lectin-like interactions with the PMNs as a glycolipid-anchored protein, although it is
fucosylated tetrasaccharide moiety sialyl Lewis X, which anchored conventionally on NK cells and macrophages.84
is presented on constitutively expressed glycoproteins on Most phagocytes also express IgA receptors. The best
PMNs, including L-selectin and P-selectin glycoprotein characterized, CD89, binds monomeric IgA and promotes
ligand 1.76 These early interactions slow the PMNs in this phagocytosis and killing of IgA-opsonized bacteria.86
first adhesive phase of leukocyte recruitment, sometimes The engagement of phagocyte receptors with opsonins
described as ‘‘slow rolling.’’75,77 Within several hours, bound on microbes locally activates cytoskeletal contrac-
newly synthesized ICAM-1 is expressed at the endothelial tile elements, leading to invagination of the membrane at
surface.75,77,78 The slowly rolling PMNs are activated by the site of initial engagement and extension of pseudopods
transient selectin-mediated interactions and locally pro- around the microbe. The ligation of additional opsonin-
duced mediators, especially endothelium-derived chemo- receptor pairs leads to engulfment of the microbe within a
kines, such as IL-8.77 These chemokines are most sealed phagosome.87 This is followed by fusion of the
effective in activating the PMNs when they are bound phagosome with lysosomal compartments containing the
by complex proteoglycans at the endothelial cell surface.48 phagocyte’s array of microbicidal products.
The activated PMNs then increase the surface expression, Phagocyte microbicidal mechanisms. The microbi-
binding avidity, or both of the b2-integrins LFA-1 and cidal mechanisms of PMNs usually are categorized as
macrophage antigen-1 (Mac-1) that interact with endo- either oxygen dependent or oxygen independent. Oxygen-
thelial cell ICAM-1 in this second, firm adhesion phase dependent microbicidal mechanisms of phagocytes de-
mediated by integrin-ICAM interactions, which is also pend on a complex enzyme, reduced nicotinamide adenine
feature articles
Reviews and
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 con- SUMMARY

verts molecular oxygen (O2) into superoxide anion
(O22).88 This enzyme is assembled at the activated cell An overview of most of the main features of innate
membrane from 6 or more components that include a immunity discussed above, along with some of their
cytochrome (a- and b-subunits, designated gp91phox and important interactions, is diagrammed in Fig 1. Several
p22phox, respectively), a flavoprotein, and a quinone, all of levels of interaction are depicted, from initial host-path-
which are associated with the cell membrane, and at least 2 ogen contact, through a variety of activating signals, to the
cytoplasmic proteins, p47phox and p67phox (‘‘phox’’ refers attack by host effector mechanisms on pathogenic targets.
to phagocyte oxidase), that assemble with the membrane- Initial contact between the host and microbes or their
associated components to form the active enzyme com- products might result in viral infection of cells, activation
plex.88,89 Each of the main oxidant products derived from of TLRs on macrophages and epithelial cells, and activa-
NADPH activity and subsequent reactions exhibits tion of the alternative pathway or mannose-binding lectin
microbicidal activity, including the earliest products, pathway of complement. The resulting activation signals,
O22 and H2O2, which are less potent than the downstream including cytokines (eg, IL-12, TNF-a, and IL-1), che-
products hypochlorite (OCl2) and chloramines (NH3Cl mokines, and products of the complement cascade mobi-
and RNH2Cl), with chloramines being the most stable.90,91 lize both cellular (NK cells and phagocytes) and humoral
Oxygen-independent microbicidal activity of PMNs (antimicrobial peptides and MAC) effectors that attack
resides mainly in a group of proteins and peptides stored their respective microbial targets.
within primary (azurophilic) granules. Lysozyme is con-
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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 Question 3. The cellular nature of leukocytic infiltrates
the membrane attack complex of the complement system into infected tissue is most influenced by —
are most closely reproduced by — A. the size of the microbial inoculum.
A. TNF-a and phagocytes. B. preexisting specific antibodies.
B. chemokines and defensins. C. the chemokine receptors expressed by the infil-
C. Toll-like receptors and NK cells. trating leukocytes.
D. IL-12 and dendritic cells. D. The defensins released at the infected site.
Question 2. NK cells are activated directly by virus-

infected target cells — Question 4. The innate immune mechanisms that best
A. via specific receptors for viral antigens. epitomize rapid responses on first contact with microbes
B. through specific receptors for IL-12. are —
C. because these targets express fewer molecules that A. Toll-like receptors and complement.
bind to inhibitory receptors. B. chemokines and NK cells.
D. in spite of increased MHC class I surface expres- C. phagocytes and defensins.
sion. D. chemokines and phagocytes.
250 August 2005 J ALLERGY CLIN IMMUNOL

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Molecular mechanisms in allergy and clinical immunology
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 Abbreviations used
carcinomas, and has potent transforming activity in vitro. Yet BL: Burkitt’s lymphoma
the virus persists benignly for the lifetime of more than 90% of CTL: Cytotoxic T lymphocyte
the human population. Thus it seems that EBV has the EBNA: EBV nuclear antigen
potential to be highly pathogenic yet rarely manifests this HD: Hodgkin’s disease
potential. Studies over the last several years show this is IM: Infectious mononucleosis
because the virus actually persists in resting memory B cells LMP: Latent membrane protein
and not proliferating cells. EBV needs its growth-promoting NPC: Nasopharyngeal carcinoma
ability to gain access to the memory compartment but has PTLD: Posttransplantation lymphoproliferative disease
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 benign. The collection of viral latent proteins expressed is
tumors. Does the virus cause the disease, or is it simply a different in each tumor type (Table I). Sometimes all of the
passenger? In the case of immunoblastic lymphoma in the known latent proteins are expressed, sometimes a limited
immunosuppressed patient, the virus almost certainly plays a subset, and sometimes only one.
causative role, but in other cases, such as Burkitt’s lymphoma, Despite the apparent robustness with which the human
the contribution of EBV remains less clear. (J Allergy Clin population deals with EBV (>95% of all adults carry the
Immunol 2005;116:251-61.) virus), the diseases caused by EBV indicate that the
situation is finely balanced. The first indication comes
Key words: Epstein-Barr virus, carcinoma, lymphoma, persistent
from X-linked lymphoproliferative disease.7 In this dis-
infection, latency, B cell, memory
ease persistent infection is not established because muta-
tions in the SH2D1A gene8,9 cause acute EBV infection to
EBV is well known because of its characteristic biol- become a fatal disease. Put melodramatically, a single
ogy.1-3 If you define the success of a pathogen by the nucleotide change in the SH2D1A gene is all that prevents
number and extent of hosts it infects, EBV is the most the vast majority of the human race from dying of acute
successful human pathogen because it latently infects EBV infection.
virtually the whole human population and persists for The second indication comes from the observation that
life.4 In tissue culture EBV is one of the most potent immunologic disturbance, as a predisposing factor, is a
transforming viruses,5,6 and it is found in several human unifying theme for all of the EBV B-cell lymphomas. This
cancers,1,3 yet for most of the population, it remains also suggests that the regulation of EBV infection in B
cells is finely balanced. Disruptions can lead to deregula-
From the Department of Pathology, Tufts University School of Medicine.
tion and EBV-driven tumor development, even in other-
Supported by grants R01 CA65883, R01 A118757, and R01 A1062989. wise healthy carriers of the virus. The clearest example of
Disclosure of potential conflict of interest: D. Thorley-Lawson has equity this is individuals who are immunosuppressed, such as
ownership in EBVax. patients undergoing organ transplantation, who are iatro-
Received for publication April 11, 2005; accepted for publication May 16,
genically immunosuppressed, or patients with AIDS, who
2005.
Available online July 15, 2005. are immunosuppressed by HIV. These individuals are at
Reprint requests: David A. Thorley-Lawson, PhD, the Department of risk for EBV lymphomas that are aggressive and often
Pathology, Jaharis Building, Tufts University School of Medicine, 150 fatal.10 This means that it is only courtesy of an active
Harrison Ave, Boston, MA 02111. E-mail: David.Thorley-Lawson@ immune response that we are protected from fatal EBV-
tufts.edu.
0091-6749/$30.00
driven lymphoma. Yet there are some curious properties
Ó 2005 American Academy of Allergy, Asthma and Immunology of these tumors that suggest the risk is not as high as might
doi:10.1016/j.jaci.2005.05.038 be expected. For example, not every immunosuppressed
251
252 Thorley-Lawson J ALLERGY CLIN IMMUNOL
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TABLE I. The EBV transcription programs in normal B cells and tumors
Transcription Infected normal Infected

program Genes expressed* B-cell typey Function tumor type
Growth EBNA1, 2, 3a, 3b, 3c, LP, LMP1, Naive Activate B cell Immunoblastic
LMP2a, and LMP2b lymphoma
Default EBNA1, LMP1, and LMP2a Germinal center Differentiate activated B HD
cell into memory
Latency None Peripheral memory Allow lifetime persistence
EBNA1 only EBNA1 Dividing peripheral Allow virus in latency program Burkitt’s
memory cell to divide lymphoma
Lytic All lytic genes Plasma cell Replicate the virus in plasma cell
*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 virus and that it has developed strategies to minimize its
oligoclonal. This is not the expected outcome. If it were pathogenic potential to the host.
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 Establishment
carries approximately 5 3 105 infected cells,11 and To understand EBV biology, it is first necessary to
immunosuppressed individuals carry perhaps 50 times understand the biology of the B lymphocyte in the
more.12 mucosal lymphoepithelium of the tonsil (Fig 1). A sum-
Taken together, these observations raise several ques- mary of normal mature B-cell biology and the proposed
tions. How does EBV persist benignly for the lifetime of a parallels with EBV is given in Figs 2 and 3, and a summary
human despite its pathogenic potential? Why does EBV of information on the different viral latency programs is
have such potent and pathogenic properties if it has presented in Table I. The model has been described in
evolved to persist for the lifetime of the human host it detail elsewhere.13,14
puts at risk by manifesting those properties? Where do the The normal B-cell response. Environmental antigens
EBV-associated tumors come from, why do they have entering the mouth are continuously sampled by the
different patterns of latent gene expression, and why does epithelium of the tonsil. Underneath the epithelium is a
disruption of the immune system predispose to EBV bed of lymphoid tissue including large numbers of naive
lymphoma development? Lastly, what goes wrong in the lymphocytes.18,19 If antigen is recognized by the antibody
maintenance of persistence that leads to EBV-associated on the surface of the naive B cell, it will bind and cause the
diseases? B cell to become an activated blast and migrate into the
The key to answering these questions comes from a follicle to form a germinal center (Fig 2).20 Here the cell
model of EBV persistence13,14 developed from the obser- undergoes rounds of rapid proliferation associated with
vation that despite EBV’s transforming ability, it persists isotype switching and mutation of the immunoglobulin
in vivo in resting15 memory16 B cells that do not express genes, followed by competitive selection for those with
any viral proteins.17 This article will first briefly review the the antibody that binds the antigen best. Those who lose in
complete life cycle of EBV infection and then discuss how the competition to bind antigen die by apoptosis.
the origins of EBV-associated tumors can be explained in Ultimately, the surviving cells leave the germinal center
the context of this model, with special emphasis on the role as memory cells primed to make a rapid response to
of an impaired immune response. Finally, the model will rechallenge with the antigen. This process requires, in
be used to attempt to answer the questions posed above. 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 epithe-
lium and infects naive B cells21 in the underlying tissue,
EBV PERSISTENCE IN VIVO where it expresses a set of latent genes that cause the cell
to become activated and proliferate as though it were re-
The essence of EBV’s behavior is that under normal sponding to antigen. This EBV transcription program (the
conditions, it does not aberrantly deregulate the behavior growth program, Fig 2) involves 9 latent proteins, includ-
of infected B cells in vivo. It initiates, establishes, and ing nuclear antigens (EBV nuclear antigens [EBNAs]) and
maintains persistent infection by subtly using virtually membrane proteins (latent membrane proteins [LMPs]).2
every aspect of normal B-cell biology. Ultimately, this These proteins have all the necessary activities to push the
allows the virus to persist within memory B cells for the B cell to become an activated blast without any necessity
lifetime of the host in a fashion that is nonpathogenic. The for external signaling. This cell migrates to the follicle,
thesis of this review is that EBV is not a natural tumor where the viral transcription program changes,22 such that
J ALLERGY CLIN IMMUNOL Thorley-Lawson 253
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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 50% or more of all memory cells being infected.28
(required to replicate the viral DNA) and 2 membrane However, the numbers decrease rapidly (half-life of 7
proteins, LMP1 and LMP2 (the default program, Fig 2). days; Hadinoto and Thorley-Lawson, unpublished data)
The functions of LMP1 and LMP2 have evolved to for the first 2 months and then more steadily after that,
steer the latently infected B cell through the germinal until by 1 year there are typically only about 1 in 105 to 106
center environment. LMP2 alone will push B cells to form infected memory B cells. After this time, the level of
a germinal center in the mucosal follicle23; LMP1 and infected cells appears to be relatively stable over many
LMP2 can drive immunoglobulin gene mutation23 and years.11 This presumably represents a balance between the
isotype switching24 (the defining markers of the germinal replenishment of latently infected memory cells through
center), respectively, and LMP1 downregulates expres- cell division17 and their loss through viral replication (see
sion of the germinal center regulatory transcription factor below). This cell division must be regulated as part of
bcl-6,25 the signal for a memory cell to exit the germinal normal memory B-cell homeostasis because there are no
center.26 This implies coordinated expression of LMP1 viral proteins expressed that could cause the cell to divide.
and LMP2, where LMP2 is turned on before and LMP1 When they divide, they express EBNA1 (the EBNA1-only
is turned on during the germinal center reaction. Thus program, Fig 2),17 which is needed to allow the viral DNA
constitutive expression of LMP1 in the absence of to replicate with the cells.29 Perhaps not surprisingly,
LMP2 blocks germinal center formation because the cells because EBNA1 represents the only point of immune
can never turn on bcl-6, an essential step in germinal attack of the memory cells, EBNA1 has evolved to be
center formation.27 poorly recognized by the immune system.30
This explains why EBV has the ability to make cells By gaining entrance to normal memory B cells and
proliferate, despite the fact that this puts the host at risk for shutting down viral protein expression, the virus is safe
neoplastic disease. Essentially it has to because this is from immune surveillance. It is also benign because none
the mechanism, activation followed by differentiation, by of the latent proteins that drive growth are expressed. This
which a normal B cell enters the B-cell memory pool. explains why EBV is able to persist benignly in the vast
majority of human subjects: EBV infection in vivo does
Maintenance not drive limitless proliferation. Rather it drives transient
Once in the periphery, the latently infected cells shut proliferation so that the cells can become resting memory
down all viral protein expression (the latency program) cells. The virus persists in nonpathogenic resting cells
and appear to be maintained as normal memory B cells.17 not proliferating blasts. This also explains why EBV-
In the early stages of acute infectious mononucleosis (IM; associated tumors do not arise in every infected individual,
primary EBV infection in the adult), the number of such even when they are immunosuppressed; something must
cells in the blood can reach staggering proportions, with go wrong with the normal biology that takes the latently
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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 normal B-cell biology and the mechanism of viral shed-
involved in tumor development. ding are shown in Fig 3. Signals that cause the B cell to
differentiate into an antibody-secreting plasma cell will
Release in turn reactivate the virus.31 Because antibody-secreting
By accessing the memory compartment, EBV has a plasma cells migrate into the mucosal epithelium,18,32
site for long-term persistence. However, it must replicate such a cell will be perfectly placed to release virus onto
and be shed to spread to new hosts. The parallels between the mucosal surface, which, in the case of the tonsils,
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is saliva. Thus infectious virus is spread through saliva but in the case of EBV, it seems to ensure that newly
contact.33 infected cells are rapidly destroyed. This suggests that the
new infection route might only be viable before the
Epithelial cells and viral shedding immune response arises (ie, in acute infection). There-
Although EBV is considered to be a B-lymphotropic after, the virus depends on homeostasis of the pool of
virus, it can also infect epithelial cells because it is found latently infected memory cells for persistence and ensures
in several important diseases of epithelial cells, including that any new infected cells are rapidly killed because they
nasopharyngeal34 and gastric35 carcinomas and oral hairy might pose a lymphoproliferative threat to the host.
leukoplakia.36 What is less clear is whether epithelial cells Second, if EBV persists in normal antigen-selected
play a role in the normal biology of EBV. Early reports B cells (unpublished results), why does it have LMP1 and
that claimed to find EBV in healthy nasopharyngeal LMP2, which can replace all the signaling necessary to
epithelium have been discredited37; however, recent produce a memory B cell? The answer to this is not yet
work has revisited this possibility. There is now evidence clear. One possibility is that the role of LMP1 and LMP2
that normal epithelial cells in the nasopharynx express a might be to give a selective advantage to the virus-infected
distinct EBV receptor,38 that they can be infected in vitro, cells in the highly competitive environment of the germi-
and that they are infected in vivo.39 However, it remains nal center. This would give the latently infected cell a
undetermined whether this infection occurs fortuitously better chance of making it into the memory pool.
because this epithelium is an area in which EBV happens Third, why does EBV not infect memory cells directly?
to replicate or because it is an important component of the A priori there seems no reason why EBV could not use the
viral biology. The most likely role for epithelial cells is as same mechanism to drive an infected memory cell back
a site for replication and amplification of the virus rather into memory; however, the evidence does not favor this
than as a site of persistent latent infection.40,41 Because the alternative. First, there is no evidence that direct infection
receptor is only expressed on the basolateral surface of of memory cells occurs consistently in vivo.21,22 Second,
epithelial cells, the virus can only infect from the lym- when it does occur, it seems to lead to clonal prolifera-
phoid tissue and not from saliva. Thus if epithelial cells tion46,47 and not differentiation, and third, the pool of
play an amplification role, it is during viral shedding and latently infected memory cells is skewed (Sousa and
not primary infection. Perhaps the most compelling indi- Thorley-Lawson, unpublished data), which would not
rect evidence for epithelial cell infection comes from be expected if EBV infected memory cells at random.
simple numbers. Estimates of the number of lymphocytes One possible explanation comes from the known biology
replicating EBV in the tonsils42 indicates that there are not of B cells. Activation of naive B cells through the ger-
nearly enough to account for the rates of viral shedding minal center leads predominantly to the production of
found in saliva (Hadinoto and Thorley-Lawson, unpub- memory cells over plasma cells,48 whereas activation
lished data). This suggests that there must be a location- of memory cells leads predominantly to the production of
mechanism for amplifying the virus shed from plasma plasma cells.49 Therefore if the goal of EBV is to access
cells. The obvious candidates are epithelial cells because, the memory compartment, it will do so more efficiently by
from studies on oral leukoplakia, we know that epithelial infecting and activating naive B cells rather than memory
cells replicate EBV to high copy numbers. cells.
Unresolved questions about persistence
in memory cells
EBV AND DISEASE
There are important unresolved questions relating to
EBV persistence in memory B cells. General considerations
First, what is the relative contribution of reinfection EBV has been associated with a number of human
versus homeostatic cell division to the maintenance of diseases. These generally fall into 2 categories: autoim-
stable levels of latently infected cells? We know that the munity and cancer.1,3 The idea that EBV might be
host mounts a massive cytotoxic T-cell response against involved in autoimmunity stems from the knowledge
cells replicating EBV and newly infected cells43 and a that the virus can infect any B cell and cause it to
neutralizing antibody response against the virus.4 It is proliferate indefinitely in culture. This raises the possibil-
therefore unclear whether newly infected cells are pro- ity that EBV could immortalize forbidden clones of B cells
duced rapidly enough and survive long enough to con- in vivo, perhaps allowing them to produce autoimmune
tribute to the pool of latently infected memory cells once antibodies in an uncontrolled fashion. This philosophic
the immune response has begun. It is conceivable that new underpinning for a role of EBV in autoimmunity can now
infection is only critical in establishing the pool of latently be seen to be incorrect. We know that EBV does not
infected memory cells before the onset of the immune persist in vivo by immortalizing B cells but by establishing
response and thereafter plays no role. A clue that this a true latency in normal resting memory B cells. There are
might be true comes from the observation that the epitopes also technical difficulties to proving a causal role for EBV
recognized by cytotoxic T cells on newly infected B cells in these diseases. First, EBV persists in circulating mem-
are conserved.44 Usually, a virus is continuously varying ory cells and therefore will be found in all tissues, irres-
its sequence to avoid the immune response (eg, HIV45), pective of disease causality. Second, it is now apparent
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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 genic risk factors. However, as described above, EBV has
system. This is because it relies on normal B-cell biology evolved to minimize the risk that an infected cell will
to establish and maintain persistence and T-cell responses proliferate out of control. Therefore something must go
to modulate the level of infection. Changes that affect wrong with the normal viral biology for EBV to play a
the functionality of the immune system affect EBV by causative role in tumor development. The plausibility of
changing overall viral loads and states of infection. EBV as an oncogenic virus has led to claims of its
Because autoimmune diseases classically disrupt the association with many human tumors. Some, such as
immune system, it will be extremely difficult to dissect breast and hepatocellular carcinoma, have never been
out causality of EBV from the background noise of substantiated, but there are now several for which
changes occurring in the virus because of the disease. strong evidence exists, including immunoblastic lym-
For all of these reasons, it has been difficult to establish a phoma in immunosuppressed patients, Burkitt’s lym-
clear connection between EBV and any autoimmune phoma, Hodgkin’s disease (HD), and nasopharyngeal
diseases. Currently, such associations remain speculative, carcinoma (NPC). The origins of all of these tumors can
controversial, or both. be understood as arising from specific stages in the EBV
The reason to believe EBV might cause cancer is life cycle (Fig 4) and appear to be associated with
apparent. EBV encodes genes that make B cells grow. disturbances of the immune system. This begs the follow-
Such genes will, of their nature, have potential as onco- ing question: How convincing is the evidence that EBV
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plays a causative role in these tumors and is not simply a they will not be a cancer risk. Only the rare, atypical
passenger in a tumor cell that arose from an infected cell bystander infection is a risk for tumor development.
type?
Hodgkin’s disease
Lymphoma in the immunosuppressed Acute EBV infection in the adolescent-adult can give
Individuals who are immunosuppressed are at risk for rise to IM, long known to be a risk factor for HD.
development of B-cell lymphoproliferative diseases, such However, the strongest evidence directly linking EBV
as the immunoblastic lymphomas in patients with AIDS with HD came with the finding that approximately 40%
and the posttransplantation lymphoproliferative diseases of the tumors contain clonal EBV,53 which can approach
(PTLDs) in patients undergoing organ transplantation.10 80% in developing countries and up to 100% in AIDS-
These are a heterogeneous collection of disorders that related HD.54 In addition, the tumor cells express the
usually carry the virus and express the growth program default transcription program (Table I),55-58 which in-
(Table I).50 A wide range of factors (eg, organ type, cludes 2 proteins (LMP1 and LMP2) that deliver survival
immunosuppressive regime, location, and donor origin) and growth signals,59-61 at least one of which (LMP1) is
influence the frequency with which these tumors arise. known to act as an oncogene.62 A characteristic of IM,
The explanation usually given for the origin of these compared with the subclinical infection seen in children,
tumors is that immunosuppression of the cytotoxic is profound disruption of the immune system.63 This in-
T-lymphocyte (CTL) response to EBV allows uninhibited cludes massive levels of virus-infected memory B cells
growth of EBV-infected cells; however, it is not that (50%), a striking T-cell lymphocytosis caused, at least in
simple. part, by a very aggressive cytotoxic T-cell response, and
From the discussion above on the mechanism of EBV tissue damage in the lymph nodes. The disease is almost
persistence, it is apparent that, under normal conditions, certainly a product of an overreactive inflammatory
infected naive B cells in the tonsils do not give rise to response, and B-cell function is so badly disrupted that
lymphoma because they differentiate out of the cell cycle one of the characteristics of IM is the production of a broad
to become resting memory cells. For a cell to express the range of nonspecific, low-affinity, so-called heteropohile
growth program, survive, and evolve into a neoplasm, 2 antibodies. This suggests that HD is the consequence of
events must occur: the EBV-infected cell must be unable deregulated EBV infection caused by the severe immu-
to respond to signals that drive it to differentiate into a nologic disturbance of IM. Nevertheless, the possibility
resting memory cell, and the CTL response must be that EBV is a passenger cannot be excluded. If the
crippled so that these lymphoblasts can continue to immunologic disruption of IM alone is the risk factor for
proliferate. This could occur if any B cell that is not a HD, it is possible that the premalignant B cell will have
naive B cell in the tonsil is exposed to the virus by EBV in it simply by chance.
chance—bystander infection (Fig 4). It could also occur if There is good evidence that EBV-positive HD arises
a latently infected germinal center or memory cell fortu- from an infected germinal-center cell. As discussed above,
itously received signals that caused it to inappropriately one of the characteristics of germinal-center cells is that
turn on the growth program. These cells can not exit the they actively mutate their immunoglobulin genes in a
growth program, and therefore they continue to prolifer- process termed hypermutation, which leaves a character-
ate. Normally, they would be rapidly eliminated by CTLs istic pattern of mutations. The immunoglobulin genes of
because of the conserved CTL epitopes they express (see HRS cells have this pattern of mutation.64 In addition, the
above); however, in the absence of effective T-cell default transcription program is used by EBV in latently
immunity (immunosuppression), they will continue to infected germinal center B cells.22 Thus the immunoglob-
proliferate. Direct evidence that this is indeed the case ulin mutations and the viral gene expression data inde-
comes from studies of tonsils from acutely infected pendently support the idea that EBV-positive HD arises
individuals. In these tonsils clonal expansions of directly from an EBV-infected germinal center B cell (Fig 4).
infected germinal center51 and memory cells46 driven by
the growth program can be found. Because these are Burkitt’s lymphoma
bystander-infected cells, they are unable to differentiate EBV was discovered in cultured tumor cells from
into resting memory cells. Consequently, they proliferate patients with the endemic form of Burkitt’s lymphoma
until the immune response arises to eliminate them, (BL).65 It is sobering to realize that 40 years later, we still
explaining why such clones are never seen in healthy do not know how or even for sure whether EBV causes
carriers of the virus but will appear if the immune response BL. This is despite the large volume of information
is subsequently suppressed. we have acquired about EBV’s molecular and cellular
The origin of these tumors also explains their hetero- biology, immunology, virology, epidemiology, clinical
geneity. They are derived from a mixture of B-cell types52 manifestations, and disease associations.1-3 The most
consistent with arising from a variety of bystander B cells compelling evidence of EBV’s involvement in BL is the
that get infected by chance and not a specific subset of high frequency (98%) of tumors carrying the virus66 in
infected cells. This also explains why the tumors are endemic areas and the presence of clonal EBV in all of the
relatively rare. The vast majority of infected cells differ- tumor cells.67 However, none of the growth-promoting
entiate into a resting memory state because they are naive; latent genes are expressed. The only genes expressed
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encode for EBNA168 and the untranslated RNAs called to replicate the virus and shed it into saliva. NPC would
EBERS and BARTS. It has been suggested that EBNA169 derive from such a latently infected, undifferentiated
and the EBERS70 might have oncogenic potential, but epithelial cell, which was blocked from switching to viral
the findings remain unsubstantiated and are controversial. replication and therefore continues to be latently infected.
Consequently, there is currently no broadly accepted Why the default program, usually found in germinal center
understanding of the role of EBV in BL.71-74 What is B cells, is expressed in NPC is completely unclear.
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 CONCLUSION: ANSWERS TO THE
Once more, this supports the notion, discussed throughout QUESTIONS
this review, that EBV infection in the context of a
compromised immune system is the risk factor for lym- In the introduction to this article, several questions were
phoma development. raised about EBV. The answers to these questions can now
Using the same arguments as for HD, we can surmise be explained in light of the discussion above.
that BL is a tumor cell of a proliferating, latently infected First, why does EBV make cells proliferate when it puts
memory B cell (Fig 4). BL has the same pattern of the host at risk for neoplastic disease?
immunoglobulin gene hypermutations as memory B Because it has to. The newly latently infected naive
cells,77 and there is only one way known for producing B cell has to become an activated blast before it can
an EBNA1-only phenotype in nontumor cells. This is differentiate into a resting memory cell.
when a latently infected memory cell expressing the Second, where do the EBV-positive tumors come from,
latency program divides as part of normal B-cell homeo- why do the different tumors express different viral latent
stasis (Fig 2).17 One property of BL inconsistent with this gene transcription programs, and why is disruption of
idea is that the tumor cells have the surface phenotype of the immune system a risk factor? The virus uses these
germinal-center cells.78 However, the cellular phenotype different transcription programs to manipulate the biology
of tumor cells can be misleading. This is exemplified by of the infected B cell so that it can gain entry into and then
HD, which is generally thought to be derived from a persist in memory B cells. Any disruption of the immune
germinal-center cell, although it bears no phenotypic or system that interferes with the ability of the EBV-infected
morphologic resemblance to such cells. Thus it is difficult cells to become a resting memory cell will increase the risk
to know how directly the final cellular phenotype of BL of tumor development. Each tumor derives from a differ-
relates to the original infected precursor. Possibly, BL is ent step in this process and represents a cell that is blocked
derived from a germinal-center cell on its way to becom- from progressing into a resting state and therefore con-
ing a resting memory cell expressing the latency program tinues to express the viral transcription program of its
but through tumor-driven growth continues to proliferate progenitor.
and therefore expresses the EBNA1-only phenotype. Third, why are there so few EBV-infected tumors in the
human population, even with immunosuppression, despite
Nasopharyngeal carcinoma the large numbers of EBV-infected cells in each individual
Given the B lymphotropism of EBV, it is surprising that and the ability of EBV to make lymphocytes grow? This is
one of the best candidates for a tumor caused by EBV is because the viral biology is tightly regulated to ensure that
not a lymphoma but a carcinoma, NPC, responsible for an EBV-infected naive B cell that becomes activated and
20% of all cancers in China and Taiwan79 and therefore starts to proliferate will rapidly exit the cell cycle and
an important world health problem. Virtually 100% of become a resting memory cell expressing none of the
undifferentiated NPCs worldwide contain clonal dangerous growth-promoting genes. In addition, the virus
EBV.34,80 The tumors express the viral default transcrip- has conserved the targets for CTLs to ensure that if a newly
tion program.81-83 Although only a subset, approximately infected cell does not exit the cell cycle, it will be rapidly
40%, express LMP1, it has been reported that the prema- killed.
lignant 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. CONCLUSIONS AND FUTURE DIRECTIONS
Because LMP1 and LMP2 are potently and specifically
evolved B cell–signaling molecules, their presence in the We now know the basic outlines of the EBV life cycle
epithelial cells of NPC suggests the virus might be there and have some understanding of where and why the
fortuitously. An example of this is LMP2, which functions tumors arise. For the basic scientist, the challenge remains
to cause B cells to migrate into mucosal follicles.23 This to understand, at the molecular level, how EBV negotiates
migratory ability, expressed in epithelial cells, might the changes between the different latency states in the
result in the invasive and metastatic activity of NPC. different B-cell types. Because this is so dependent on
The potential role of EBV in NPC is clouded by our lack B cells, it is likely that the mechanisms will only become
of certain knowledge about the role of epithelial cells in clear when we learn how the processes are normally
EBV biology. In Fig 4 the speculative assumption is made regulated in B cells. There is still also much to be learned
that EBV latently infects epithelial cells that then proceed about the role EBV plays at the molecular level in
feature articles
Reviews and
tumorigenesis, particularly for BL. But perhaps the big- 7. Seemayer TA, Gross TG, Egeler RM, Pirruccello SJ, Davis JR, Kelly
gest gap in our knowledge is understanding what is CM, et al. X-linked lymphoproliferative disease: twenty-five years after
the discovery. Pediatr Res 1995;38:471-8.
different in the disruption of the immune response that 8. Coffey AJ, Brooksbank RA, Brandau O, Oohashi T, Howell GR, Bye
leads to immunoblastic lymphoma in some cases, HD in JM, et al. Host response to EBV infection in X-linked lymphoprolifer-
others, and BL in yet others. Could timely immunologic ative disease results from mutations in an SH2-domain encoding gene.
intervention reduce the risk of subsequent development of Nat Genet 1998;20:129-35.
9. Sayos J, Wu C, Morra M, Wang N, Zhang X, Allen D, et al. The
these diseases?
X-linked lymphoproliferative-disease gene product SAP regulates signals
The identification of EBV within tumors provides a induced through the co-receptor SLAM. Nature 1998;395:462-9.
potentially unique opportunity to develop tumor-specific 10. Hopwood P, Crawford DH. The role of EBV in post-transplant malig-
therapy targeted at the virus that will not hurt normal cells. nancies: a review. J Clin Pathol 2000;53:248-54.
A good example of this is recent work showing that 11. Khan G, Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson DA.
Is EBV persistence in vivo a model for B cell homeostasis? Immunity
in vitro expanded, EBV-specific CTLs can be effective
1996;5:173-9.
therapy against PTLD,85 although they hold less promise 12. Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA. Epstein-
for treatment of HD and NPC. An important and poten- Barr virus-infected resting memory B cells, not proliferating lympho-
tially fruitful area of clinical investigation will be the blasts, accumulate in the peripheral blood of immunosuppressed patients.
development of drugs specifically targeted against EBV. J Exp Med 1999;190:567-76.
13. Thorley-Lawson DA, Gross A. Persistence of the Epstein-Barr virus and
The best candidate latent protein might well be EBNA1,
the origins of associated lymphomas. N Engl J Med 2004;350:1328-37.
which allows replication of the viral DNA and therefore is 14. Thorley-Lawson DA. Epstein-Barr virus: exploiting the immune system.
essential for retention of the viral DNA in a proliferating Nat Rev Immunol 2001;1:75-82.
(eg, tumor) cell. The crystal structure of EBNA1 bound to 15. Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson DA. Identification
DNA is known, opening the path to the development of of the site of Epstein-Barr virus persistence in vivo as a resting B cell.
J Virol 1997;71:4882-91.
drugs that block this interaction. If the tumor requires EBV
16. Babcock GJ, Decker LL, Volk M, Thorley-Lawson DA. EBV persistence
to grow, loss of the viral DNA should prevent tumor in memory B cells in vivo. Immunity 1998;9:395-404.
growth. Whether EBV truly plays a causative role in these 17. Hochberg D, Middeldorp JM, Catalina M, Sullivan JL, Luzuriaga K,
tumors is therefore not an esoteric question. If the virus is Thorley-Lawson DA. Demonstration of the Burkitt’s lymphoma Epstein-
not a key player, then therapies directed at the virus will be Barr virus phenotype in dividing latently infected memory cells in vivo.
Proc Natl Acad Sci U S A 2003;101:239-44.
ineffective against the tumors. A hint of this comes from 18. Brandtzaeg P, Farstad IN, Haraldsen G. Regional specialization in the
PTLD, in which restoration of the immune response leads mucosal immune system: primed cells do not always home along the
to tumor regression. Eventually, however, the tumors same track. Immunol Today 1999;20:267-77.
become resistant. This raises the possibility that ultimately 19. Perry M, Whyte A. Immunology of the tonsils. Immunol Today 1998;19:
tumor growth might not be dependent on the virus. 414-21.
20. Liu YJ, Arpin C. Germinal center development. Immunol Rev 1997;156:
An interesting approach that does not require the virus 111-26.
to be essential for tumor growth would be the development 21. Joseph AM, Babcock GJ, Thorley-Lawson DA. Cells expressing the
of drugs that efficiently cause EBV in the tumors to begin Epstein-Barr virus growth program are present in and restricted to the
replicating. Because replication of the virus kills the cell, naive B-cell subset of healthy tonsils. J Virol 2000;74:9964-71.
this would be an indirect way to destroy EBV-positive 22. Babcock GJ, Hochberg D, Thorley-Lawson AD. The expression pattern
of Epstein-Barr virus latent genes in vivo is dependent upon the
tumors, irrespective of their dependence on the virus for differentiation stage of the infected B cell. Immunity 2000;13:497-506.
growth. This would not need to lead to wholesale 23. Casola S, Otipoby KL, Alimzhanov M, Humme S, Uyttersprot N, Kutok
production of virus, however, because drugs that block JL, et al. B cell receptor signal strength determines B cell fate.
this are already available (eg, valacyclovir). Nat Immunol 2004;5:317-27.
24. He B, Raab-Traub N, Casali P, Cerutti A. EBV-encoded latent membrane
protein 1 cooperates with BAFF/BLyS and APRIL to induce T cell-
independent Ig heavy chain class switching. J Immunol 2003;171:
5215-24.
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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 Question 3. EBV-associated tumors are relatively rare
healthy adult population? because —
A. <1% A. EBV is not oncogenic.
B. ;10% B. EBV replicates and kills the cells before they
C. ;50% can grow into tumors.
D. >90% C. EBV has evolved to minimize its oncogenic potential.
D. EBV cannot always be detected in tumors.
Question 2. To establish persistent infection, EBV Question 4. How many mutations in host genes are
primarily infects — required to make EBV a fatal acute infection?
A. naive B cells. A. 0
B. memory cells. B. 1
C. activated B cells. C. 5-6
D. germinal center cells. D. >10
262 August 2005 J ALLERGY CLIN IMMUNOL

feature articles
Reviews and
Editorial
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 b-defensins that leads to repeated staphylococcal in-
infection and immunity and to attempts to describe the fections in eczematous patches of affected skin.3
balance of host forces that protect against those infectious Mononuclear phagocytes and other cells contain Toll-
forces that would invade. For most of our lives we remain like receptors that react with bacterial products, such as
unaware of the moment-by-moment interplay of host endotoxin and DNA sequences, and become active secre-
resistance factors and infectious agents. Yet as described tors of cytokines that regulate subsequent inflammatory
in the articles herein, a natural sequence of clonal expan- responses.4 Children with deficiencies of the Toll-like
sion of microbes develops in human beings born with receptor signaling pathway involving the IL-1 receptor–
faulty immunity or in those whose immunity is temporar- associated kinase (IRAK) are subject to infections with
ily overwhelmed by infection. There are 3 intersecting pyogenic bacteria.5 Numerous cytokines are involved in
concepts drawn out for us in this month’s Journal— immune reactions, both protecting the host from infections
immunodeficiency, infection, and cancer—that meet in a and contributing to the complications of infections. In the
common point in certain individuals, like the zero point of latter category, perhaps IL-1 and TNF-a are best known
3-dimensional axes. Most infections of humankind are for their pathologic role in gram-negative bacterial toxin-
attributed to being the result of chance, exposure, and dose induced shock.6 Chemokines and their receptors are ac-
of infectious agent. Compelling arguments are now being tive in numerous immunologic reactions to infections,
made, however, that at least in the case of the more severe but none are more visible than the CCR5 and CXCR4
forms of these infections, invading organisms have taken chemokine receptors that induce cognate receptor binding
advantage of a hidden chink in the armament of presumed of the HIV-1 glycoprotein 120 and facilitate entry of
normal immunity. With special patients who need immu- the HIV-1 virion into target cells.7 The value of natural
nosuppressive treatments, there is often a manifestation of killer (NK) cells assumes more importance as we under-
chronic infection and even the appearance of cancer. This stand the primal role that they serve in host protection
issue of the Journal clarifies some of those mysterious against viral infection and the development of cancer.8
mechanisms of immunity that enable the preservation of Responding in an antigen-independent manner, NK cells
life. bind and lyse virus-infected host and cancer cells by
In the lead Current Reviews article, Tosi summarizes perforin formation or apoptosis induction. Complement9
the rapidly expanding field of innate immunity that, and neutrophil-invaded immunity10 round out the reper-
though lacking immune memory and clonal expansion toire of innate immunity, each contributing to that imme-
of lymphocytes, probably protects against more infections diate response to infection that is so important prior to the
than does adaptive immunity.1 At epithelial skin surfaces, engagement of the slower acquired immune responses.
antimicrobial peptides disrupt the cell membranes of The illustration on the cover of this issue demonstrates
pathogens and prevent numerous skin infections.2 how neutrophils police the vascular endothelium and,
Atopic eczema is a clinical example of deficiency in within seconds of detecting chemoattractants created by
infections in the tissues, squeeze through intercellular
From the Department of Pediatrics, Baylor College of Medicine, and the spaces in pursuit of pathogens.11 On arrival at the site of
Department of Allergy and Immunology, Texas Children’s Hospital. infections, neutrophils engulf and kill microbes through
Supported by the National Institutes of Health grants AI27551, AI36211,
HD41983, RR0188, HD079533, HL72705, HD078522, contract
the formation of superoxide. No prior memory of these
202PICL05; the Pediatric Research and Education Fund, Baylor College pathogens is necessary for this bacteriocidal function of
of Medicine; and the David Fund, Pediatrics AIDS Fund, and Immunology neutrophils.
Research Fund, Texas Children’s Hospital. Thorley-Lawson12 writes in the Molecular Mechanisms
Received for publication May 31, 2005; accepted for publication June 1, 2005.
article on how the Epstein-Barr virus (EBV) takes up long-
Available online July 15, 2005.
Reprint requests: William T. Shearer, MD, PhD, Department of Pediatrics, term residence in almost all human beings and occasion-
Section of Allergy and Immunology, Baylor College of Medicine, 6621 ally produces lymphomas. Virtually all individuals with
Fannin St (MC-FC330.01), Houston, TX 77030. E-mail: wtsheare@ EBV lymphomas are either immunosuppressed (eg, trans-
TexasChildrensHospital.org. plant patients) or lack components of immunity on a
J Allergy Clin Immunol 2005;116:263-6.
0091-6749/$30.00
congenital basis (eg, X-linked immunoproliferative dis-
Ó 2005 American Academy of Allergy, Asthma and Immunology ease).13 EBV remains in a latent state in resting memory
doi:10.1016/j.jaci.2005.06.001 B cells that do not express EBV proteins on their cell
263
264 Shearer J ALLERGY CLIN IMMUNOL
AUGUST 2005
feature articles
Reviews and
surface.14 Thus, these EBV-containing B cells remain mutation in the Gag epitope of HIV-1, but when this
invisible to cytotoxic T cells. EBV is thought to play a role mutant viral strain is transmitted to another individual
not only in lymphomas but also in Hodgkin’s disease, with different HLA alleles, this epitope reverts to the
Burkitt’s lymphoma, and nasopharyngeal carcinoma. wild type.24 In HIV-1 discordant couples, the risk of
Thorley-Lawson acknowledges that these cells may just HIV-1 transmission is 2-fold higher (independent of
carry EBV rather than being the result of EBV-initated HIV-1 viral load) if the couples share one or both
transformation events. He also questions why more EBV- HLA-B alleles.25 In perinatal HIV-1 transmission,
driven tumors are not seen in human populations, even HLA-B*4901 and B*5301, alleles that inhibit mother-
those immunosuppressed individuals. The answer seems to-infant HIV-1 transmission (despite high HIV-1 viral
to reside in the propensity of EBV-infected B cells to stop load), differ from otherwise identical HLA-B*5001 and
replicating under the influence of 2 viral genes—latent B*3501 alleles by 5 amino acids encoding the ligand for
membrane protein (LMP) 1 and LMP2—and enter the the killer inhibitory receptor (KIR) 3DL1 for NK cells.26
latent resting memory cell condition.15 Circumstances that The molecular basis for these 3 observations suggests
disrupt the immune system change this resting memory strongly that recognition molecules on immune cells
cell condition and favor tumor development. Thus, when govern subsequent viral mutation and viral elimination
human beings are given immunosuppressive drugs or have through cytotoxic T cells and NK cells. Also summarized
received therapeutic irradiation, immune forces are dis- in the Advances article are the discoveries that mast cells
rupted and the EBV B cell enters the replicating cell cycle. participate in host defense via recognition of Toll-like
Unless killed by cytotoxic T cells, which respond to the receptors and viruses27 and secretion of cytokines that
newly expressed EBV cell surface antigens, these acti- recruit effector cell.28 Articles in the area of primary
vated EBV cells could form oligoclonal tumor cells. These immunodeficiency and infectious diseases are also cited,
observations hold importance for immunologically nor- perhaps none more important than the identification of
mal human beings exposed to occupational or environ- risks of malignancy when retroviral vectors are used to
mental conditions that cause immunosuppression, such as insert gene constructs in stem cells bone marrow derived
radiation in spaceflight.16 in severe combined immunodeficiency.29 In this instance,
Mehandru and colleagues17 contribute a Perspectives/ the retroviral vector has the potential to insert into the
Update article to this issue that details the rapidly human genome in the promotor region of oncogenes and
unfolding discoveries of the crucial role of gastrointestinal to trigger the development of T-cell leukemia.30 Related to
lymphatic tissue in acute HIV-1 infection. In the face of these observations in primary immunodeficiency is the
relatively stable peripheral blood CD41 T (helper) cell Images in Allergy and Immunology article that pictures the
concentrations in acute HIV-1 infection, there is a massive case of a child who developed severe mosquito bite
kill-off of tissue CD41 T cells, particularly those of the hypersensitivity, ulcerating skin lesions, enlarged and
gastrointestinal tract.18-20 Moreover, these gastrointestinal draining adjacent lymph nodes, and marked hepatosplen-
CD41 T cells are of the memory phenotype and express omegaly.31 Studies reveal that this child developed pro-
the CCR5 chemokine receptor that attract the monocyto- liferation of EBV-containing NK cells similar, if not
tropic HIV-1 viral strains, as demonstrated in the simian identical, to that seen in the few reported cases of chronic
model of HIV-1 infection.21,22 The emerging model of active EBV infection of NK cells that result in NK cell
pathogenesis of acute HIV-1 infection suggests that the leukemia and lymphoma.32 It is possible that this child is
large pool of memory CD41 T cells in mucosal surfaces an example of the atypical immunodeficiency that presents
becomes preferentially infected and stimulates repetitive with a more common and less marked clinical phenotype
rounds of viral replication and CD41 T cell killing. that ultimately might be resolved by detection of causal
Mehandru proposes that these discoveries will revamp genes, as proposed by Casanova33 and reviewed by
the way clinicians decide when to intercede with anti- Bonilla and Geha34 in an editorial in this issue.
retroviral agents (ie, immediate versus deferred therapy), In addition to the interaction of viruses with immuno-
rekindle the debate of using immunodulators to reduce the deficiency, there is strong evidence for a pathogenic role
waves of inflammation and viral replication (eg, cyclo- for viruses in allergic diseases. For example, rhinoviruses
sporine therapy during acute infection), accelerate the use cause more than 50% of upper respiratory infections and
of protective strategies for the gastrointestinal mucosal are thought to be responsible for the induction of acute
surfaces (eg, microbicides and CCR5 blockers), and exacerbations of asthma in the lower airway. Friedlander
redirect vaccine strategies to mucosal surfaces. and Busse35 review this evidence in this issue, and find
In the Advances in Asthma, Allergy, and Immunology that respiratory viruses are associated with approximately
Series 2005: Basic and Clinical Immunology article in 80% of children and 50% of adults with wheezing epi-
this issue, Chinen and Shearer mention several note- sodes and that infection of the upper and lower respiratory
worthy, recent publications dealing with the interplay be- mucosal surfaces induces increased airway hyperrespon-
tween immunity and infection.23 The importance of the siveness. This concept of rhinovirus induction of asthma
HLA allele recognition system in viral infection is seen includes (1) the attachment of the intercellular adhesion
in human beings with certain HLA-B alleles with molecule 1 (ICAM-1) to the viral capsid molecules36 and
HIV-1 infection. HIV-1–infected individuals with HLA- (2) the stimulation of proinflammatory cytokines IL-6,
B57 and HLA-B*5801 select for variants with a specific IL-8, IL-16, and RANTES chemokine.37 The net result of
J ALLERGY CLIN IMMUNOL Shearer 265
feature articles
Reviews and
FIG 1. Balance between immunity and infection. A, Normal immune system. B, Immunodeficiency.
upregulation of these mediators is an influx of eosinophils, 5. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, et al.
Pyogenic bacterial infections in humans with IRAK-4 deficiency.
monocytes, T cells, macrophages, and neutrophils into
Science 2003;299:2076-9.
respiratory tissue.38 As a result of this increased inflam- 6. Oppenheim JJ, Feldman M. Introduction to the role of cytokines
mation of the airways, angiogenic growth factors might in innate host defense and adaptive immunity. In: Oppenheim JJ,
induce tissue remodeling of respiratory mucosa and could Feldman M, Durum SK, Hirano T, Vilcek J, Nicola N, editors. The
cause a permanent change in lower airway architecture cytokine reference. San Diego: Academic Press; 2001. p. 3-20.
7. Glass WG, Rosenberg HF, Murphy PM. Chemokine regulation of
and increased difficulties for treatment programs. inflammation during acute viral infection. J Allergy Clin Immunol
All in all, the theme of this month’s Journal seems to 2003;3:467-73.
have been substantially illustrated by the contributions of 8. Smyth MJ, Cretney E, Kelly JM, Westwood JA, Street SE, Yagita H,
talented experts in immunity and infection. These inter- 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,
related concepts can be considered the 2 sides of a coin.
Ochs HD, Winkelstein JA, editors. Immunologic disorders in infants and
Perhaps a better analogy is that of a teeter-totter: when the children. 5th ed. Philadelphia: Elsevier Saunders; 2004. p. 20-62.
sitting board is horizontal, there is a balance between 10. Tosi MF. Immunologic and phagocytic responses to infection. In: Feigin
immunity and infection (Fig 1). When immunity is down, RD, Cherry JD, Demmler GJ, Kaplan S, editors. Textbook of
infections rise and immunity must be strengthened to gain pediatric infectious diseases. 5th ed. New York: WB Saunders; 2004.
p. 652-84.
balance, with the result that the inflammation of immunity 11. Seo SM, McIntire LV, Smith CW. Effects of IL-8, Gro-alpha, and
often overshoots and infection drops. However, evidence LTB(4) on the adhesive kinetics of LFA-1 and Mac-1 on human
is being gathered to strongly suggest that when this neutrophils. Am J Physiol Cell Physiol 2001;281:C1568-78.
balance is upset, as is the case with immunodeficiency 12. Thorley-Lawson DA. EBV the protypical human tumor virus—just how
bad is it? J Allergy Clin Immunol 2005;116:251-61.
diseases, certain viruses are able to escape strong immune
13. Shearer WT, Ritz J, Finegold MJ, Guerra IC, Rosenblatt HM, Lewis DE,
response and hide in a latent condition. When individuals et al. Epstein-Barr virus-associated B-cell proliferations of diverse clonal
who harbor latent viruses, such as patients receiving im- origins after bone marrow transplantation in a 12-year-old patient with
munosuppressive drugs or therapeutic radiation, encoun- severe combined immunodeficiency. N Engl J Med 1985;312:1151-9.
ter an additional force, the latent virus is forced into its life 14. Hochberg D, Middeldorp JM, Catalina M, Sullivan JL, Luzuriaga K,
Thorley-Lawson DA. Demonstration of the Burkitt’s lymphoma
cycle that yields outgrowths of clones of virus-containing Epstein-Barr virus phenotype in dividing latently infected memory cells
transformed cells. More understanding of these balancing in vivo. Proc Natl Acad Sci 2004;101:239-44.
forces of immunity and infection is necessary so that we, 15. Hochberg DR, Thorley-Lawson DA. Quantitative detection of viral gene
as clinician-investigators, can intervene with the some- expression in populations of Epstein-Barr virus-infected cells in vivo.
Methods Mol Biol 2005;292:39-56.
times threatening consequences of imbalances in the
16. Shearer WT, Zhang S, Reuben RM, Lee B, Butel JS. Effects of radiation
forces of the immune system and infection. and latent virus on immune responses in a space flight model. J Allergy
I thank Carolyn Jackson and Ruth Herrera for assistance with the Clin Immunol 2005;115:1297-303.
preparation of this manuscript. 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,
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2. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev 2003;77:11708-17.
Immunol 2003;3:710-20. 19. Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan
3. Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, C, et al. Primary HIV-1 infection is associated with preferential depletion
et al. Endogenous antimicrobial peptides and skin infections in atopic of CD41 T lymphocytes from effector sites in the gastrointestinal tract.
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receptors and differential regulation of Toll-like receptor mRNAs in GJ, et al. CD41 T cell depletion during all stages of HIV disease
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Reviews and
21. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. 30. Cavazzana-Calvo M, Lagresle C, Hacein-Bey-Abina S, Fischer A. Gene
Massive infection and loss of memory CD41 T cells in multiple tissues therapy for severe combined immunodeficiency. Annu Rev Med 2005;
during acute SIV infection. Nature 2005;434:1093-7. 56:585-602.
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replication in resting memory CD41 T cells depletes gut lamina propria McCormick TG. Chronic active Epstein-Barr virus (CAEBV) infection
CD41 T cells. Nature 2005;434:1148-52. of NK cells presenting a severe skin reaction to mosquito bites. J Allergy
23. Chinen J, Shearer WT. Advances in asthma, allergy, and immunology Clin Immunol 2005;116:470-2.
series 2005: basic and clinical immunology. J Allergy Clin Immunol 32. Tokura Y, Ishihara S, Tagawa S, Seo N, Ohshima K, Takigawa M.
2005;116:411-8. Hypersensitivity to mosquito bites as the primary clinical manifestation
24. Leslie AJ, Pfafferott KJ, Chetty P, Draenert R, Addo MM, Feeney M, of a juvenile type of Epstein-Barr virus-associated natural killer cell
et al. HIV evolution: CTL escape mutation and reversion after transmis- leukemia/lymphoma. J Am Acad Dermatol 2001;45:569-78.
sion. Nat Med 2004;10:282-9. 33. Casanova JL, Fieschi C, Bustamante J, Reichenbach J, Remus N,
25. Dorak MT, Tang J, Penman-Aguilar A, Westfall AO, Zulu I, Lobashevsky von Bermuth H, et al. From ‘‘idiopathic’’ infectious diseases to ‘‘atyp-
ES, et al. Transmission of HIV-1 and HLA-B allele-sharing within ical’’ primary immunodeficiencies. J Allergy Clin Immunol 2005;116:
serodiscordant heterosexual Zambian couples. Lancet 2004;363:2137-9. 426-30.
26. Winchester R, Pitt J, Charurat M, Magder LS, Goring HH, Landay A, 34. Bonilla S, Geha RS. Are you immunodeficient? J Allergy Clin Immunol
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certain maternal HLA-B alleles independent of viral load implicates innate 35. Friedlander G, Busse WW. The role of rhinovirus in asthma exacerba-
immune mechanisms. J Acquir Immune Defic Syndr 2004;36:659-70. tions. J Allergy Clin Immunol 2005;116:267-73.
27. Kulka M, Alexopoulou L, Flavell RA, Metcalfe DD. Activation of mast 36. Yamaya M, Sasaki H. Rhinovirus and asthma. Viral Immunol 2003;16:
cells by double-stranded RNA: evidence for activation through Toll-like 99-109.
receptor 3. J Allergy Clin Immunol 2004;114:174-82. 37. Papadopoulos NG, Bates PJ, Bardin PG, Papi A, Leir SH, Fraenkel DJ,
28. Marshall JS, Jawdat DM. Mast cells in innate immunity. J Allergy Clin et al. Rhinoviruses infect the lower airways. J Infect Dis 2000;181:
Immunol 2004;114:21-7. 1875-84.
29. Chinen J, Puck JM. Successes and risks of gene therapy in primary 38. Gern JE. Rhinovirus respiratory infections and asthma. Am J Med 2002;
immunodeficiencies. J Allergy Clin Immunol 2004;113:595-603. 112:19S-27S.
Rostrum
Asthma diagnosis and

The role of rhinovirus in asthma exacerbations
treatment
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, Abbreviations used
respiratory viruses are found in approximately 80% of G-CSF: Granulocyte colony-stimulating factor
wheezing episodes in children and in approximately one half of ICAM: Intercellular adhesion molecule
such episodes in adults. Rhinovirus is a member of the family IL-1ra: IL-1 receptor antagonist
Picornaviridae, and acute rhinovirus infections occur RV: Rhinovirus
predominantly in the upper airway. This virus has also been URI: Upper respiratory tract infection
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 WHAT ROLE DOES RV PLAY IN ASTHMA
include asthma and atopy, extremes in age, and perhaps having EXACERBATIONS?
a deficient TH1 response to rhinovirus. With the use of in vitro
models and experimental inoculation studies, significant
Asthma exacerbations are most commonly precipitated
advances have led to a better understanding of the mechanisms
by which rhinovirus infections cause asthma exacerbations.
by viral URIs, particularly with RV,2 and often occur
Advances in our understanding of this interaction might despite concurrent use of appropriate controller medica-
provide knowledge that could ultimately lead to specific tions. Detecting respiratory viruses—in particular, RV—
treatment modalities to prevent and/or treat this significant by culture methodology alone has been insensitive and has
burden of asthma exacerbations. (J Allergy Clin Immunol previously underestimated the role of respiratory viruses in
2005;116:267-73.) asthma exacerbations, especially in adults. Viral detection
rates in asthma exacerbations have significantly increased
Key words: Rhinovirus, asthma exacerbations, virology, cytokine with the use of sensitive methods and have thus under-
response profiles, mechanisms of asthma scored the overwhelming importance of respiratory vi-
ruses in asthma exacerbations. When RT-PCR is used to
Viral upper respiratory tract infections (URIs) are supplement conventional culture techniques, viruses have
known to cause exacerbations of asthma. A significant been found in approximately 80% of wheezing episodes
and increasing body of evidence demonstrates that in large in school-age children and in approximately one half of the
part the primary respiratory infection causing these exac- acute wheezing episodes in adults. Of the respiratory
erbations is rhinovirus (RV), the cause of more than 50% viruses identified in these circumstances, RV is most
of URIs.1 The frequency of URI-provoked asthma makes commonly found and is detected 65% of the time.2,3
it especially important to understand the role and mech- A pivotal study by Johnston and colleagues2 in children
anisms whereby RV infections lead to asthma exacerba- aged 9-11 years old with histories of asthma symptoms
tions, the basic virologic features of RV, their ability to found that 80% to 85% of asthma exacerbations that were
infect the lower airway, the host susceptibility factors, and associated with reduced peak expiratory flow rates and
mechanisms leading to airflow obstruction. 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
From the Division of Allergy and Immunology, Department of Medicine, found in adults. Nicholson et al3 reported on 138 young
University of Wisconsin, Madison.
adults with asthma recruited from general practice, the
Received for publication April 7, 2005; revised June 3, 2005; accepted for
publication June 7, 2005. hospital, and the community. In this longitudinal study,
Available online July 5, 2005. 80% of asthma episodes (223 of 280), described as
Reprint requests: William W. Busse, MD, Department of Medicine, K4/912 symptoms of wheeze, chest tightness, or breathlessness,
CSC-9988, 600 Highland Avenue, Madison, WI 53792. E-mail: wwb@ were associated with colds. Objectively, viruses were
medicine.wisc.edu.
0091-6749/$30.00
detected in 57% of people with symptomatic colds and
Ó 2005 American Academy of Allergy, Asthma and Immunology asthma exacerbations. In more severe asthma exacerba-
doi:10.1016/j.jaci.2005.06.003 tions with reductions in peak flow measurements of
267
268 Friedlander and Busse J ALLERGY CLIN IMMUNOL
AUGUST 2005
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 epithe-

lium cultures become infected. However, although RV-
treatment
induced 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 An infection with RV leads to symptoms of the
of the drug-binding pocket just beneath the canyon floor. An ion,
located at each pentamer center in RV-1A, 214, 216 is tentatively
common cold, which is primarily an upper airway illness.
identified as calcium, which is necessary for attachment of some Because RV is primarily an infection of the upper airway,
RVs. Modified with permission from Gwaltney JM. Rhinovirus. In: early research efforts were directed toward determining
Mandell GL, Douglas RG, Bennett JE, Dolin R, editors. Mandell, whether (a) RV infections could infect the lower airways
Douglas, and Bennett’s principles and practice of infectious dis-
directly and provoke asthma, (b) their actions on asthma
eases. 6th ed. New York; Elsevier/Churchill Livingstone; 2005. p.
2185-94. 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
50 L/min, viruses were detected in 44% of episodes. In potential target areas to act therapeutically to prevent or
comparison with detection rates by cell culture, RT-PCR treat an asthma exacerbation.
was 5 times more sensitive in identifying human RV in Debate initially focused on whether RV could exist
adults with respiratory infections.3 and replicate in the lung to directly cause lower airway
Further evidence to support the role of viral infection in inflammation. This was based on limited studies dem-
asthma exacerbations also includes reports that peaks in onstrating that RV replication was optimal at 33°C, the
hospital admissions for asthma significantly correlate with temperature of the upper airways. To address this issue,
seasonal patterns of viral URIs.4 In the United States, RV direct thermal mapping of the lower airways was per-
infection occurs most commonly in the fall and spring.5 formed. While human subjects breathed room air (26°C),
Thus, current evidence strongly supports the concept that the temperature in the subjects averaged 32°C in the upper
RV respiratory infections are the major cause of acute trachea and 35.5°C in the subsegmental bronchi. These
asthma exacerbations. 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
WHAT ARE THE VIROLOGIC FEATURES high viral titers in cell cultures at 37°C; little significant
OF RVS? difference in replication was found when wild-type RV
isolates were used at 33°C compared to 37°C. In fact,
The genera RV and Enterovirus are classified within the some serotypes grew more effectively at the higher
family Picornaviridae. There are more than 100 serotypes temperature.10 In addition, when primary cultures of
of RV; this explains, in part, the lack of an effective lower airway bronchial epithelial cells and upper airway
vaccine against the major etiologic agent causing the adenoidal epithelial cells were used, RV appeared to infect
common cold. RV is a small, single-stranded RNA virus both upper and lower segments of the respiratory tree with
whose capsid contains 4 proteins (Fig 1). Three of these similar ability (Fig 2).9
proteins, VP1, VP2, and VP3, are located on the surface Several additional lines of evidence support the ability
of the capsid and are responsible for its antigenic diversity; of RV to infect the lower airways directly. When bron-
the fourth, VP4, is located inside the virus and anchors choscopy was used to collect samples from subjects with
the RNA core to the viral capsid.1 The majority of RV symptomatic experimental infections, RV was detected
serotypes bind to intercellular adhesion molecule (ICAM) from bronchial brush specimens.12 Furthermore, with the
1, whereas approximately 10% bind to the low-density use of RT-PCR and Southern blotting, RV genetic mate-
lipoprotein receptor.6,7 rial was found in higher amounts in cells of bronchial
Typically, RV infects small clusters of cells in the alveolar lavage fluid than in supernatant; this suggests that
epithelial layer with little cellular cytotoxicity. Although the virus was located intracellularly.13 However, the role
J ALLERGY CLIN IMMUNOL Friedlander and Busse 269

treatment
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 airway directly likely contributes to viral-induced exacer-
definitively excluded in these studies. bations of asthma.
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 WHAT ARE THE EFFECTS OF RV INFECTION
to localize viral RNA by hybridizing the sequence of ON THE MECHANISMS OF AIRWAY
interest to the complementary replicative strand of the PHYSIOLOGY IN ASTHMA?
virus. This technique likely excludes the possibility of
contamination of the lower airway with virus from the Multiple studies demonstrate the adverse effects of RV
upper airway. This was further supported by the finding of on airway physiology in asthma. In school-age children,
viral replication in the lower airway as well as increases in symptoms of either upper or lower respiratory tract in-
viral RNA and the production of new viral proteins. fection were shown to last a week, and during these infec-
Furthermore, the frequency of lower airway infection was tious episodes, the peak flow rates fell for a median
similar to that observed in the upper airway; this indicates duration of 2 weeks.2 In another study, asthmatic subjects
that infection of the lower airways might be relatively were experimentally inoculated with RV-16 and found to
common as part of the natural history of RV infection. demonstrate modest changes in increased airway hyper-
Finally, a recent study showed that an experimental responsiveness, airway obstruction, and inflammation.15
RV infection was associated with virus detection in large Experimental RV-16 infection also has been shown to
lower airways biopsy samples by immunohistochemistry reduce FEV1 in patients with mild asthma.16 In addition,
or qPCR in 17 of 19 subjects, but less so in the distal increases in existing airway inflammation have occurred
airways.14 Thus, RV is able to infect both the upper and after segmental bronchoprovocation in atopic subjects,
lower airways. suggesting that enhanced airway inflammation is a feature
It is likely that the lower airways are infected as a result of RV-associated asthma exacerbations.17
of self-inoculation from coughing, sneezing, or perhaps To support this possibility, subjects with allergic rhini-
breathing. Whether the concentration of virus in the lower tis, but not with active asthma, were inoculated with RV;
airways is large enough to produce clinically relevant they were found to have significantly increased airway
effects is still not established. These studies support hyperreactivity as well as a significantly increased inci-
the concept that RV is a lower as well as an upper dence of late asthmatic reactions, defined as a 15%
respiratory tract pathogen, and infection of the lower decrease in FEV1 approximately 6 hours after antigen
AUGUST 2005
treatment
FIG 3. RV induces epithelial cells to produce proinflammatory cytokines leading to airway hyperresponsive-
ness, 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 oxide, or antiviral effects of eosinophil products. Thus, the
had a late asthmatic reaction. During the acute infection, timing and intensity of antigen exposure play an important
this number increased to 8 of 10 subjects (P = .0085). The role in the severity level and subsequent possible compli-
effect occurred independently of the enhancement in cations of a cold.
airway reactivity experienced during a cold alone. This
demonstrates that in addition to causing airway hyperre-
activity, RV also promotes the development of late-phase HOW DOES RV MODULATE INFLAMMATORY
responses, even in nonasthmatic patients. MEDIATORS OF EPITHELIAL CELLS
RV infection also promotes eosinophil recruitment to CONTRIBUTING TO ASTHMA
airway segments after antigen challenges. Calhoun et al17 EXACERBATIONS?
used segmental bronchoprovocation with antigen after
inoculation with RV-16 in subjects with allergic rhinitis. Epithelial cells are the principal targets of RV infec-
After infection, bronchial alveolar lavage fluid revealed tions, allow viral replication, and likely initiate immune
enhanced histamine release immediately and increased responses (Fig 3).20,21 Papadopoulos et al10 found local
eosinophil recruitment 48 hours after antigen challenge. induction of proinflammatory mediators that could pro-
Interestingly, the increase in eosinophils persisted for up vide a mechanism to explain how lower airway infection
to 1 month after infection in some subjects. The effect of can lead to inflammation and asthma. RV infection re-
RV on airway inflammation appeared to be an augmen- sulted in an increase in mRNA expression and subsequent
tation of allergen-specific responses. Thus, enhancement translation of IL-6, IL-8, and IL-16. This also occurred
of antigen-induced mediator release from pulmonary with RANTES, a C-C chemokine with chemoattractant
mast cells and basophils and eosinophilic recruitment, activity for eosinophils, monocytes, and T lymphocytes.
either directly or via cytokines, could provide one IL-6 and IL-8 are proinflammatory cytokines, and IL-8 is a
mechanism by which late allergic reactions and airway specifically potent chemoattractant for neutrophils. IL-16
hyperresponsiveness are enhanced by viral uncoating and is a powerful lymphocyte chemoattractant and activator
might act to intensify the airway inflammatory response of macrophages and eosinophils and appears to be an
to allergen. important mediator in the pathogenesis of asthma and
Conversely, when nasal allergen challenges in atopic lower airway inflammation due to RV.10 The inflamma-
patients were performed before experimental RV inocu- tory actions of RV appear to center on its ability to
lation, the onset of cold symptoms was delayed and the generate a variety of phlogistic mediators.
responses were less severe in comparison with what Generation of these cytokines correlates with the
was seen in patients without allergies.19 Delayed nasal worsening of respiratory physiology. For example, IL-1
inflammation, with attenuation of the increase in IL-6, enhances airway smooth muscle contraction in response to
IL-8, and neutrophils seen in infection, was also found in bronchospastic agents and attenuates smooth muscle
the group primed with nasal antigen challenge. This might dilation responses to bronchodilators.22,23 Differences in
have been due to cytokine profile changes with increased immune response, such as the modulation of costimula-
expression of IFN-g and IL-2, local production of nitric tory molecules and the induction of antigen presentation,
might explain how RV infections cause acute exacerba- TABLE I. Risk factors for severe lower respiratory tract
tions in asthmatic patients. complications from rhinovirus infection
Virus-induced epithelial damage might cause increased
Asthma
permeability of the mucosal layer and thus increase Atopy
allergen contact with immune cells to promote neurogenic Elevated nasal eosinophils or eosinophil cationic protein

inflammation. In addition, viruses can enhance vagally Infants and elderly
mediated reflex bronchoconstriction, possibly by limiting Low neutralizing antibody titers to rhinovirus
treatment
the function of the M2 muscarinic receptor.24 Viral Chronic lung diseases
replication activates epithelial cells to initiate innate and Smoking
adaptive immune responses as well as the generation of Low IFN-g producers
oxidative stress.25 Also, double-stranded RNA synthe- High IL-5 producers
sized in virus-infected cells induces the cytokines IL-8 and
Modified with permission from Gern JE. Rhinovirus respiratory infections
RANTES, which initiate proinflammatory and antiviral and asthma. Am J Med 2002;112(Suppl 6A):19S-27S.
pathways within the cell.24
Upregulation, or activation, of ICAM-1, the principal
receptor for RV, might increase tissue susceptibility to the phil activation could cause airway obstruction through the
major group RV and subsequent infection. The asthma production of elastase, which also upregulates goblet cell
phenotype, which is associated with increased ICAM-1 mucus secretion.29
expression, might therefore be associated with increased
susceptibility and complications from RV infection.21
Chronic antigen challenge can also increase ICAM-1 ex- WHAT ARE THE RISK FACTORS FOR
pression of the airway epithelium, and RV infection itself WHEEZING WITH A COLD?
can increase ICAM-1 expression through production of
IL-1b and a nuclear factor-kb–dependent mechanism. Various risk factors increase the susceptibility of sub-
This might lead to the amplification of airway inflamma- jects for more severe lower respiratory complications
tion after RV infection.21,26 from an RV infection, such as wheezing, bronchitis, and
In addition, RV might enhance existing inflammation to pneumonia (Table I). These include having low neutral-
a greater degree in asthmatic subjects than in those without izing antibody titers to RV, being an infant, being elderly,
the disease. For example, after inoculation with the virus, having chronic lung disease, being a smoker, and being an
nasal lavage levels of IL-8 and the proinflammatory individual with existing asthma.22
mediator IL-1b were increased in asthmatic patients.27 In addition, subjects who are low producers of IFN-g in
In this study, a small increase in the anti-inflammatory response to RV and are atopic appear to be more at risk for
marker IL-1 receptor antagonist (IL-1ra), a competitive wheezing or having a severe respiratory infection. Brooks
inhibitor of IL-1, also occurred in asthmatic subjects et al30 demonstrated that whereas RV induces IFN-g,
treated with budesonide, whereas lower levels were found which is consistent with a strong TH1-like immune re-
in these patients at baseline. These findings suggest that sponse, those asthmatic patients with diminished, or
RV might be able to alter the proinflammatory/anti- deficient, TH1 responses to RV were characterized by
inflammatory balance of IL-1b/IL-1ra toward inflamma- increased airway hyperresponsiveness. Moreover, the
tion more markedly in people with existing and active ratio of RV-16–induced IFN-g:IL-5, a measure of
asthma. TH1:TH2 balance, correlated with FEV1. These findings
Cytokine response profiles generated by RV might are similar, in general, to what is known about TH1 and
translate into neutrophilic inflammation in both the upper TH2 responses in asthma. In another study, subjects with
and lower airways. Local RV infection is associated with persistent and severe asthma displayed a defect in IFN-g
increased levels of IL-8, a potent chemoattractant for production, whereas their increased IL-5 responses were
neutrophils, and also granulocyte colony-stimulating fac- felt to reflect the presence of atopy but were not specif-
tor (G-CSF) in nasal secretions and later in the circulation. ically linked to asthma itself.31 Collectively, these findings
Increased concentrations of circulatory G-CSF could act demonstrate that a deficiency of the TH1 response, rather
on the bone marrow to increase the circulating neutrophils. than an increased TH2 response, is responsible for RV’s
Thus, a local response in nasal epithelium to RV infection adverse effect on the airways.
can result in a systemic inflammatory reaction.20 The importance of IgE and eosinophilic airway inflam-
Elevated neutrophil counts are also found in the lower mation was demonstrated by a study showing synergistic
airways with RV infection. Through use of bronchoscopy interactions between RV infection and allergic airway
and bronchial washes, significant increases in airway inflammation.32 In this study, which focused on children
lumen neutrophils were found 96 hours after inoculation aged 2-16 years old, the odds ratios for wheezing with RV
with RV-16 in patients with allergic asthma.28 Infected detected by RT-PCR in addition to positive radioallergo-
bronchial epithelium induces the secretion of proinflam- sorbent test results, nasal eosinophilia, and elevated nasal
matory cytokines, including IL-1, IL-8, TNF-a, IL-10, eosinophil cationic protein were 17, 21, and 25, respec-
and IFN-a, as well. This stimulates the recruitment of tively. The odds ratios for wheezing with any of these
inflammatory cells and neutrophilia. Products of neutro- 4 risk factors alone were much lower, between 3.2 and
AUGUST 2005
8; this shows the importance of IgE and eosinophil-driven 7. Gwaltney JM. Rhinovirus. In: Mandell GL, Douglas RG, Bennett JE,
Dolin R, editors. Mandell, Douglas, and Bennett’s principles and practice
inflammatory responses.
of infectious diseases. 6th ed. New York: Elsevier/Churchill Livingstone;
Zambrano et al33 reported that the existence of airway 2005. p. 2185-94.
inflammation prior to virus inoculation predisposed sub- 8. Winther B, Farr B, Turner RB, Hendley JO, Gwaltney JM Jr, Mygind N.
jects to a more deleterious response to RV. Patients were Histopathologic examination and enumeration of polymorphonuclear
inoculated with RV-16, and compared with those without leukocytes in the nasal mucosa during experimental rhinovirus colds.
Acta Otolaryngol Suppl 1984;413:19-24.
asthma, those with mild asthma demonstrated increased 9. Mosser AG, Brockman-Schneider R, Amineva S, Burchell L, Sedgwick
treatment
airway hyperresponsiveness, decreased FEV1 at baseline, JB, Busse WW, et al. Similar frequency of rhinovirus-infectible cells in
and increased upper and lower respiratory tract symptom upper and lower airway epithelium. J Infect Dis 2002;185:734-43.
scores in response to the infection. Asthmatic patients with 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:
elevated IgE profiles also demonstrated higher blood
1875-84.
eosinophil counts, increased eosinophil cationic protein 11. McFadden ER Jr, Pichurko BM, Bowman HF, Ingenito E, Burns S,
in nasal washes, and both an increased expired nitric Dowling N, et al. Thermal mapping of the airways in humans. J Appl
oxide, a marker of inflammation, and decreased soluble Physiol 1985;58:564-70.
ICAM-1 in nasal washes at baseline and during cold 12. Halperin SA, Eggleston PA, Hendley JO, Suratt PM, Groschel DH,
Gwaltney JM Jr. Pathogenesis of lower respiratory tract symptoms in
symptoms. These findings suggest that patients with experimental rhinovirus infection. Am Rev Respir Dis 1983;128:806-10.
asthma, who are highly atopic, might be more likely to 13. Gern JE, Galagan DM, Jarjour NN, Dick EC, Busse WW. Detection of
have increased levels of airway inflammation and be at rhinovirus RNA in lower airway cells during experimentally induced
greater risk for asthma exacerbations in response to RV 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.
infection.
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
SUMMARY 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
The importance of RV in asthma exacerbations is rhinovirus 16 infection causes variable airway obstruction in subjects with
established in both adults and children. The complex 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,
mechanisms by which their interaction provokes asthma rhinovirus 16, potentiates airway inflammation after segmental antigen
are becoming better understood. RV appears to have a bronchoprovocation in allergic subjects. J Clin Invest 1994;94:2200-8.
direct and negative impact on the lower airways and 18. Lemanske RF Jr, Dick EC, Swenson CA, Vrtis RF, Busse WW.
causes an increase in obstructive airway symptoms and Rhinovirus upper respiratory infection increases airway hyperreactivity
and late asthmatic reactions. J Clin Invest 1989;83:1-10.
physiology. This effect on airway function is felt to occur
19. Avila PC, Abisheganaden JA, Wong H, Liu J, Yagi S, Schnurr D, et al.
as the virus upregulates proinflammatory cytokines and Effects of allergic inflammation of the nasal mucosa on the severity of
predisposes the asthmatic patient to more severe respira- rhinovirus 16 cold. J Allergy Clin Immunol 2000;105:923-32.
tory infections and hence to exacerbations. Defects in 20. Gern JE. Rhinovirus respiratory infections and asthma. Am J Med 2002;
TH1-type immune responses appear to be an important 112(Suppl 6A):19S-27S.
21. Yamaya M, Sasaki H. Rhinovirus and asthma. Viral Immunol 2003;16:
factor in causing airway inflammation in people with 99-109.
asthma. Further work is needed to better explore the 22. Gern JE, Busse WW. Association of rhinovirus infections with asthma.
mechanisms behind the association between asthma Clin Microbiol Rev 1999;12:9-18.
exacerbations and RV infections. This might ultimately 23. Hakonarson H, Carter C, Maskeri N, Hodinka R, Grunstein MM. Rhino-
virus-mediated changes in airway smooth muscle responsiveness: induced
lead to treatment modalities to prevent and/or treat the
autocrine role of interleukin-1beta. Am J Physiol 1999;277(1 Pt 1):13-21.
significant burden of asthma exacerbations caused by 24. Gern JE. Mechanisms of virus-induced asthma. J Pediatr 2003;142:
RV infection. (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.
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1. Greenberg SB. Respiratory consequences of rhinovirus infection. Arch 26. Terajima M, Yamaya M, Sekizawa K, Okinaga S, Suzuki T, Yamada N,
Intern Med 2003;163:278-84. et al. Rhinovirus infection of primary cultures of human tracheal epithe-
2. Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, lium: role of ICAM-1 and IL-1beta. Am J Physiol 1997;273(4 Pt 1):749-59.
et al. Community study of role of viral infections in exacerbations of 27. de Kluijver J, Grunberg K, Pons D, de Klerk EP, Dick CR, Sterk PJ, et al.
asthma in 9-11 year old children. BMJ 1995;310:1225-9. Interleukin-1beta and interleukin-1ra levels in nasal lavages during
3. Nicholson KG, Kent J, Ireland DC. Respiratory viruses and exacerba- experimental rhinovirus infection in asthmatic and non-asthmatic sub-
tions of asthma in adults. BMJ 1993;307:982-6. jects. Clin Exp Allergy 2003;33:1415-8.
4. Johnston SL, Pattemore PK, Sanderson G, Smith S, Campbell MJ, 28. Jarjour NN, Gern JE, Kelly EA, Swenson CA, Dick CR, Busse WW. The
Josephs LK, et al. The relationship between upper respiratory infections effect of an experimental rhinovirus 16 infection on bronchial lavage
and hospital admissions for asthma: a time-trend analysis. Am J Respir neutrophils. J Allergy Clin Immunol 2000;105(6 Pt 1):1169-77.
Crit Care Med 1996;154(3 Pt 1):654-60. 29. Cardell LO, Agusti C, Takeyama K, Stjarne P, Nadel JA. LTB(4)-
5. Arruda E, Pitkaranta A, Witek TJ Jr, Doyle CA, Hayden FG. Frequency induced nasal gland serous cell secretion mediated by neutrophil elastase.
and natural history of rhinovirus infections in adults during autumn. Am J Respir Crit Care Med 1999;160:411-4.
J Clin Microbiol 1997;35:2864-8. 30. Brooks GD, Buchta KA, Swenson CA, Gern JE, Busse WW. Rhino-
6. Casasnovas JM. The dynamics of receptor recognition by human virus-induced interferon-gamma and airway responsiveness in asthma.
rhinoviruses. Trends Microbiol 2000;8:251-4. Am J Respir Crit Care Med 2003;168:1091-4.
31. Smart JM, Horak E, Kemp AS, Robertson CF, Tang ML. Polyclonal and requiring emergency care. IgE and eosinophil analyses. Am J Respir Crit
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J Allergy Clin Immunol 2002;110:450-6. TA, et al. Experimental rhinovirus challenges in adults with mild asthma:
32. Rakes GP, Arruda E, Ingram JM, Hoover GE, Zambrano JC, Hayden FG, response to infection in relation to IgE. J Allergy Clin Immunol 2003;
et al. Rhinovirus and respiratory syncytial virus in wheezing children 111:1008-16.

treatment
Guest editor: William W. Busse, MD
Perspectives on the past decade of asthma

genetics
treatment
Carole Ober, PhD Chicago, Ill
Although genetic linkage and association studies have identified

more than 25 asthma or allergy susceptibility loci, replication Abbreviations used
of significant results remains a problem. Moreover, these AD: Atopic dermatitis
approaches typically ignore the true complexity of these ADRB2: b2-Adrenergic receptor
diseases, such as the role of gene-by-environment and gene-by- CD14: Monocyte differentiation antigen 14
gene interactions. As a result, many important associations COAST: Childhood Onset of Asthma Study
might have been missed. Recent studies demonstrate not only FCERB1: FceRb1
that such interactions exist but also that the relationship GSTM1: Glutathione S-transferase M1
between genotype and phenotype is more complex than GSTP1: Glutathione S-transferase P1
previously thought. (J Allergy Clin Immunol 2005;116:274-8.) HLAG: Human leukocyte antigen G
IL4RA: IL-4 receptor, a-chain
Key words: Asthma, allergy, genetics, gene-by-environment inter- LTA: Lymphotoxin a
actions LTC4A: Leukotriene C4 synthase
NOS3: Nitric oxide synthetase 3
TIM1: T-cell immunoglobulin- and mucin
EXTENDING THE MENDELIAN PARADIGM domain–containing molecule 1
TO COMPLEX DISEASES TLR4: Toll-like receptor 4
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 been gained over the past 10 years on the genetic archi-
genome project,1 the tools are now available to fully meet tecture of these traits.
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 LINKAGE AND ASSOCIATION STUDIES
before the onset of disease and to design drugs that are IDENTIFY SUSCEPTIBILITY GENES
individualized and genotype specific. Although there have
been countless successes with respect to defining the
Fig 1 shows a common model of susceptibility to
molecular basis of Mendelian (monogenic) diseases,2
asthma and atopy, which implicates many genes and many
genetic studies of common diseases with complex causes
environmental factors but implies that the effects of genes
have turned out to be considerably more challenging than
and environmental factors individually contribute to risk.
originally thought. In this perspective I will provide a brief
However, the truth is much more complex, with genes
update on the status of genetic studies of asthma and
interacting both with other genes and with environmental
allergy and then discuss some of the insights that have
risk factors to confer susceptibility. In fact, few genes
might have independent effects, as is typical for Mende-
From the Departments of Human Genetics and Obstetrics and Gynecology,
lian diseases. Nonetheless, the approaches that have been
The University of Chicago. used to find susceptibility genes, either through linkage or
Supported in part by National Institutes of Health grants HL56399, HL66533, association studies, have for the most part considered one
HL70831, and HL72414. gene at a time (Fig 2).
Disclosure of potential conflict of interest: All authors—none disclosed.
Despite this overly simplistic modeling of asthma and
Received for publication April 19, 2005; accepted for publication April 25,
2005. atopy genetics, many important discoveries have been
Available online June 30, 2005. made (Fig 3). In particular, 5 genes have been identified
Reprint requests: Carole Ober, PhD, Department of Human Genetics, The through family linkage studies, followed by positional
University of Chicago, 920 East 58th St, CLSC 507C, Chicago, IL cloning.3-7 These genes span a wide range of functions
60637-1463. E-mail: c-ober@genetics.uchicago.edu.
0091-6749/$30.00
and in all cases were either unknown or would not have
Ó 2005 American Academy of Allergy, Asthma and Immunology been considered as candidate asthma genes before their
doi:10.1016/j.jaci.2005.04.039 discovery. Among more than 100 genes that have been
274
J ALLERGY CLIN IMMUNOL Ober 275

treatment
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, FIG 2. Strategies for identifying disease genes. A, With the candi-
Bronchial hyperresponsiveness. date 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
interrogated through association studies (reviewed in patients and control subjects, cohorts of individuals, or families.
Hoffjan et al8), 8 genes have been replicated in more Ultimately, the mechanism of the association is revealed through
genotype-phenotype studies and functional studies of the associ-
than 5 studies, and another 13 genes have been replicated
ated variant. B, In positional cloning studies initially only informa-
but in fewer than 5 studies. These 26 genes are likely to be tion on the chromosomal location is known, usually from family
true susceptibility loci but represent just the tip of the linkage studies. All genes in the linked region become positional
iceberg because additional positionally cloned genes are candidates, and association studies are performed as described
soon to be reported, and many other candidate genes will above to identify the associated gene and variation that contributes
to disease risk. Once the variation is identified by means of
be identified and replicated. Thus one could conclude that association studies, the mechanism of the association is studied
the field of asthma genetics has been quite successful and as described above.
that many genes have been identified that contribute to
risk.
THE PROBLEM OF REPLICATION GENE-BY-ENVIRONMENT INTERACTIONS

AND ASTHMA
Replicating results remains the gold standard for
genetic association studies, but this has proved difficult We and others have recently begun to examine interac-
for common diseases, such as asthma, irrespective of tions between individual genotypes and environmental
whether the initial association was identified through exposures as a first step in developing more complex
candidate gene association or positional cloning studies. models of disease susceptibility. These models consider
Even among the most replicated genes, including those the possibility that specific genotypes might result in a
shown in Fig 3, there are many negative studies (for phenotype only in certain environments or that a specific
examples, see Table 1 in Hoffjan et al8). In fact, there are genotype might result in different phenotypes, depending
no genes that are associated with asthma, atopy, or a on environmental exposures. Such interactions could mask
related phenotype in every study reported. Moreover, even associations if the study sample is heterogeneous with
when a gene is replicated, it is often with a different respect to the exposure or underlie discrepant results
phenotype (eg, a polymorphism in intron 1 of the LTA between samples drawn from populations that differ with
gene is associated with asthma in some studies and IgE in respect to the exposure. A classic example of a gene-by-
others), with different polymorphisms in the same gene environment interaction is that of the Mendelian disease
(eg, the 21112C/T promoter polymorphism in the IL13 a1-antitrypsin deficiency. The risk for respiratory diseases,
gene is associated with atopic asthma in some studies, but such as emphysema and chronic obstructive pulmonary
the Arg130Gln polymorphism in exon 4 of the same gene disease, among homozygotes for the PiZ null allele (ZZ
is associated with asthma and atopic phenotypes in others), genotype) is nearly 100% in the presence of cigarette
and even with different alleles of the same variant (eg, the smoke exposure. In this case the exposure is thought of as a
2159C allele in the promoter region of the CD14 gene is trigger of disease in genetically susceptible individuals.
associated with atopic phenotypes in some populations, Other examples of gene-by-environment interactions on
whereas the 2159T allele is associated in others). This asthma and atopy risk have been recently reported,7,9-16
level of complexity was unexpected and has suggested and these suggest that such effects might be more the rule
that models of susceptibility that consider one locus at a than the exception. These studies are summarized in Table
time, as is the paradigm for Mendelian diseases, are not I and in all cases provide examples in which genotype-
adequate for discovering and characterizing asthma and specific effects are modified by environmental exposures.
allergy susceptibility loci. Rather, models that include Although not all of these interaction effects have been
interactions between genes and between genes and envi- replicated, they provide the basis for future studies and for
ronmental risk factors might be required to fully elucidate characterizing the range of effects of important environ-
the genetic architectures of asthma and atopy. mental exposures as modifiers of disease risk.
276 Ober J ALLERGY CLIN IMMUNOL
AUGUST 2005
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 ment interactions were recently reported, providing some
EXPOSURES intriguing examples of interactions. A study of the
effects of dog ownership on the development of immune
Epidemiologic studies have identified many environ- responsiveness and atopy in infancy revealed a protec-
mental factors that influence risk for asthma and allergic tive effect of having a dog in the house at the time the
disease, such as maternal asthma, birth order, and sibship child was born: only 30% of infants had atopic derma-
size and early-life exposure to viral infections, endotoxin titis (AD) if a pet was present in the home compared
(LPS), day care, pets, and allergens. Yet few studies to with 51% of infants in homes without a dog (P <
date have examined how exposure to environmental risk .0001).14 However, this difference was even more strik-
factors during development modifies genotype-specific ing among children with the 2159TT genotype at the
risks for asthma and allergic disease. The Childhood locus encoding the receptor for LPS, CD14: only 5% of
Onset of Asthma (COAST) Study is a prospective birth TT children exposed to a dog had AD compared with
cohort study of high-risk children designed to evaluate 43% of unexposed TT children (P =.04). In this exam-
the role of genes and environment on the development of ple, even though both the polymorphism (CD14 2159C/
immune responsiveness and allergic phenotypes.17 The T) and the environmental exposure (dog) independently
first studies in this cohort to examine gene-by-environ- influenced risk for AD, the interaction between the 2 was
J ALLERGY CLIN IMMUNOL Ober 277
TABLE I. Examples of gene-by-environment interaction effects on asthma and atopic disease
Environmental
Gene Exposure Phenotype Comment Reference
LTC4S Aspirin exposure Asthma 2444C allele is increased among individuals Sanak et al9

with aspirin-induced asthma compared
with individuals with aspirin-tolerant
asthma
treatment
ADRB2 Cigarette smoke Asthma Increased risk of asthma among smokers Wang et al10
with Arg16 genotype but not among
nonsmokers
ADRB2 Physical activity Asthma Increased risk of asthma among sedentary Barr et al11
women with Gly16 genotype but not
among active women
TIM1 HAV Atopy HAV protects against atopy in individuals McIntire et al12
with a 6-amino-acid insertion at residue
157 (157insMTTTVP) but not in
individuals without the insertion
TLR4 Endotoxin levels Asthma At high levels of endotoxin exposure, carriers Werner et al13
of the Gly299 and Ile399 alleles have
reduced risk for asthma compared with
other genotypes and other exposure groups
CD14 Dog ownership at birth AD 2159TT genotype is protective against AD Gern et al14
in the first year among children with a dog
in the home at birth
GSTM1 Diesel exhaust particles IgE and histamine response Enhanced responses among GSTM1-null Gilliland et al15
individuals but not among individuals with
other genotypes
GSTP1 Diesel exhaust particles IgE and histamine response Enhanced responses among individuals with Gilliland15
the Ile105 allele but not among individuals
without this allele
NOS3 Day-care exposure in Change in TH2 cytokine Asp298 homozygosity associated with Hoffjan et al16
the first 6 mo of life (IL-5 and IL-13) response smallest changes in TH2 responses among
in first year of life children attending day care and largest
changes among children not attending day
care
FCERB1 Day-care exposure in IL-5 response at 1 y of age Gly237 allele associated with decreased IL-5 Hoffjan et al16
the first 6 mo of life responsiveness among children attending
day care and increased responsiveness
among children not attending day care
IL4RA Day-care exposure in the IFN- g response at 1 y of age Val50 homozygosity associated with lowest Hoffjan et al16
first 6 mo of life response among children attending day
care and highest response among children
not attending day care
HLAG Maternal BHR Asthma-BHR in child 2964G allele is associated with asthma in Nicolae et al7
children of mothers with BHR; 2964A
allele is associated with atopy and asthma
among children of mothers without BHR
HAV, Hepatitis A; BHR, bronchial hyperresponsiveness.
significant (P = .0071), indicating that the risk associated sponses or asthma and genotyped in 99 COAST children
with the TT genotype differs in different exposure who attended day care and 109 COAST children who did
groups. Interactions between CD14 genotype and levels not. Interestingly, neither day-care attendance nor geno-
of endotoxin exposure have been suggested as an type at these loci by themselves significantly influenced
explanation for the discrepant results of association any of the first-year phenotypes examined. However,
studies with this polymorphism,18 as discussed earlier, highly significant interaction effects (P < .001) were
and these data support that hypothesis. demonstrated with genotypes at 3 loci: NOS3, FCERB1,
In a second study in this cohort, the effects of day-care and IL4RA. In each case the effects of a particular
attendance in the first 6 months of life on cytokine genotype on the phenotype were opposite depending on
response profiles and allergic phenotypes were exam- whether the child attended day care (ie, the same genotype
ined.16 Seventy-two polymorphisms in 35 genes were was associated with the highest cytokine responses or
selected because of their putative role in immune re- protection from disease among children attending day care
278 Ober J ALLERGY CLIN IMMUNOL
AUGUST 2005
but the lowest cytokine responses or risk for disease 2. Glazier AM, Nadeau JH, Aitman TJ. Finding genes that underlie
complex traits. Science 2002;298:2345-9.
among children not attending day care). That is, the
3. Van Eerdewegh P, Little RD, Dupuis J, Del Mastro RG, Falls K, Simon J,
genotype effects at these loci were modified by the et al. Association of the ADAM33 gene with asthma and bronchial
environment such that the same genotype was associated hyperresponsiveness. Nature 2002;418:426-30.
with protection from or risk for a phenotype depending on 4. Zhang Y, Leaves NI, Anderson GG, Ponting CP, Broxholme J, Holt R,
this early-life exposure! In the pooled sample (not strat- et al. Positional cloning of a quantitative trait locus on chromosome
13q14 that influences immunoglobulin E levels and asthma. Nat Genet
ified by day-care attendance) there were no detectable 2003;34:181-6.
treatment
differences between genotypes (summarized in Table I). 5. Allen M, Heinzmann A, Noguchi E, Abecasis G, Broxholme J, Ponting
Interestingly, the interaction effects with the FCERB1 and CP, et al. Positional cloning of a novel gene influencing asthma from
IL4RA genes were likely accounted for by the increased chromosome 2q14. Nat Genet 2003;35:258-63.
6. Laitinen T, Polvi A, Rydman P, Vendelin J, Pulkkinen V, Salmikangas
number of viral infections among children attending day
P, et al. Characterization of a common susceptibility locus for asthma-
care; however, the interaction effects with NOS3 were related traits. Science 2004;304:300-4.
independent of viral infections, suggesting that risk factors 7. Nicolae D, Cox NJ, Lester LA, Schneider D, Tan Z, Billstrand C, et al.
other than viruses but that are correlated with day-care Fine mapping and positional candidate studies identify HLA-G as an
exposure interact with the NOS3 genotype to determine asthma susceptibility gene on chromosome 6p21. Am J Hum Genet
2005;76:349-57.
risk. Complex interactions such as these could underlie 8. Hoffjan S, Nicolae D, Ober C. Association studies for asthma and atopic
some of the association studies in which one allele of a diseases: a comprehensive review of the literature. Respir Res 2003;4:14-28.
polymorphism is associated in some populations and the 9. Sanak M, Pierzchalska M, Bazan-Socha S, Szczeklik A. Enhanced
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1474-9.
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tion should continue to unravel the nature and overall Nature 2003;425:576.
importance of gene-by-environment and gene-by-gene 13. Werner M, Topp R, Wimmer K, Richter K, Bischof W, Wjst M, et al.
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S-transferase M1 and P1 genotypes on xenobiotic enhancement of allergic
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Original articles
Is it traffic type, volume, or distance? Wheezing

in infants living near truck and bus traffic
treatment
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, Abbreviations used
distance, and amounts of traffic and wheezing in very young CCAAPS: Cincinnati Childhood Allergy and Air
infants. Pollution Study
Objective: We sought to determine the relationship between DEP: Diesel exhaust particle
types of traffic, traffic volume, and distance and wheezing GIS: Geographic information system
among infants less than 1 year of age. mph: Miles per hour
Methods: A geographic information system and a classification OR: Odds ratio
scheme were developed to categorize infants enrolled in the PM: Particulate matter
study as living near moving truck and bus traffic (highway >50 SPT: Skin prick test
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. Recent articles in this journal have reviewed the
Results: Infants living very near (<100 m) stop-and-go bus
epidemiology and biology of air pollution and diesel
and truck traffic had a significantly increased prevalence of
exhaust and their effects on asthma risk and respiratory
wheezing (adjusted odds ratio, 2.50; 95% CI, 1.15-5.42) when
compared with unexposed infants. The prevalence of wheezing function.1,2 Diesel exhaust particles (DEPs) have been
among nonwhite infants was at least twice that of white infants, studied with respect to both the development and exacer-
regardless of exposure. Infants living less than 400 m from a bation of allergic rhinitis and asthma because of their
high volume of moving traffic, however, did not have an unique capability to enhance those TH cells that direct
increased prevalence of wheezing. allergic immune responses (ie, TH2 cells) and production
Conclusion: These results suggest that the distance from and of IgE.3 Although the mechanism by which DEPs might
type of traffic exposures are more significant risk factors than influence allergy and asthma development and exacerba-
traffic volume for wheezing in early infancy. (J Allergy Clin tion is still under investigation, the immunologic effects of
Immunol 2005;116:279-84.)
DEPs have been recently reviewed.4,5 DEPs are respirable
particles with a large surface area per unit mass that
Key words: Diesel, traffic, truck, bus, wheezing, Geographic Infor-
provide an excellent medium for absorbing and trans-
mation System, infants
porting proteins into the peripheral airways.6 Studies have
demonstrated that DEPs are capable of binding with grass
From athe Department of Environmental Health, and bthe Department of pollen allergen (Lol p 1), and this might be similar with
Internal Medicine, Division of Immunology, University of Cincinnati. other aeroallergens.7,8 In human studies exposure to DEPs
Supported by grants ES11170 and ES10957 from the National Institute of has been shown to enhance allergic nasal cytokine and
Environmental Health Sciences. inflammatory responses after direct challenge with aller-
Disclosure of potential conflict of interest: S. A. Grinshpun has received
grants–research support from the National Institute of Environmental Health
gen extracts.9-11
Sciences. M. Villareal has consultant arrangements with Aventis, has stock Previous studies of traffic pollutants have focused on
or other equity ownership with Pfizer, and is on the speakers’ bureaus for roadways with high truck and automobile traffic and
AstraZeneca, UCB Pharma, Pfizer, Aventis, and GlaxoSmithKline. There minimal bus traffic as the source of air pollution, and these
are not other potential conflicts to disclose.
studies have been conducted primarily on school-age
Received for publication March 15, 2005; revised May 9, 2005; accepted for
publication May 10, 2005. children. The purpose of this study was to determine
Available online June 17, 2005. whether distance, volume, and/or type of traffic might be
Reprint requests: Patrick H. Ryan, MS, Department of Environmental Health, associated with wheezing in infants younger than 1 year.
University of Cincinnati, Cincinnati, OH 45267-0056. E-mail: ryanph@ The hypothesis was that infants who reside near major
email.uc.edu.
0091-6749/$30.00
highways with heavy truck traffic, as well as infants who
Ó 2005 American Academy of Allergy, Asthma and Immunology reside near local roads with stop-an-go truck and bus
doi:10.1016/j.jaci.2005.05.014 traffic will have a significantly increased risk of wheezing
279
280 Ryan et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
when compared with infants residing far from truck and and 400 m.19 Fewer than 3% (n = 10) of residences of infants in this
bus traffic. 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,
METHODS 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

Subject recruitment geographic distribution and our pilot study, which revealed that the
The Cincinnati Childhood Allergy and Air Pollution Study concentration of ultrafine particles decreased by one half between
treatment
(CCAAPS) is an ongoing birth cohort study. Infants enrolled in 50 m and 150 m downwind from a highway and an observable sulfur
CCAAPS were identified from birth records, and the addresses concentration gradient up to 400 m from a highway, infants were
obtained from these records were geocoded with EZLocate from classified as exposed to interstate traffic if their residence was within
TeleAtlas for the ArcView Geographic Information System (GIS) 3.2 400 m.20 Exposure to a state or bus route was determined if their
(Environmental Systems Research Institute, Redlands, Calif). The residence was within 100 m from one of these routes. If an infant
distance to the nearest major highway or interstate (defined as >1000 resided greater than 400 m from the nearest interstate, greater than
trucks daily) was computed for all infants by using the Geoprocessing 100 m from the nearest state route, and greater than 100 m from the
extension. Infants whose birth records indicated residency less than nearest bus route, the infant was placed in the unexposed category.
400 m or greater than 1500 m from the nearest major highway or Furthermore, exposure to moving traffic was determined if an infant’s
interstate were eligible. residence was within 400 m of an interstate or within 100 m of a state
Parents were recruited when infants were 6 months of age or older route with a speed limit of greater than or equal to 50 miles per hour
and screened for allergy symptoms.12 Parents with likely atopy were (mph). Fifty miles per hour was used as the cutoff because in Ohio
subsequently tested with skin prick tests (SPTs) with a panel of 15 this is the designation used for classification of an urban (greater
common indoor and outdoor regional aeroallergens. Infants with at traffic) or rural (less traffic) route. Exposure to stop-and-go traffic was
least one atopic parent were enrolled. 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,
Outcome variables or both.
At the time of the parent SPT, an interviewer administered the
baseline health questionnaire at a physician’s office. This question- Statistical analyses
naire gathered demographic information, occupants in the home and
To determine the presence of an association between traffic
their smoking status, animal ownership, and information on other
exposure and wheezing, conditional logistic regression was per-
possible risk factors. The general health of the infant was queried
formed with SAS software (version 8.2 for Windows; SAS Institute
from birth until the infants’ age at enrollment. These questions were
Inc, Cary, NC), adjusting for sex, race (white/nonwhite), breast-
based on the well-validated International Study of Allergy and
feeding (maternal report of breast-feeding <1 week, 1-4 weeks, or >5
Asthma in Children questionnaire for children ages 4 to 5 years,
weeks), pet ownership, income (<$40,000/$40,000), child care
which was adapted for use with infants.13 In addition, monthly diaries
outside of the home (parent report of infant attending day care or
were distributed to the parents of all enrolled infants at the time of the
babysitter), number of siblings, visible mold in the home, maternal
parental SPT. These diaries recorded parental observation of the
and paternal self-report of asthma, and the number of monthly diaries
infants’ illnesses and were returned by mail monthly until the child’s
returned.
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 RESULTS
parent reported wheezing (without a cold) on the parent questionnaire
and returned at least one monthly diary indicating the identical Recruitment for the CCAAPS study was completed on
symptom was designated to have wheezed. December 13, 2003. During year 1 of the study, 633
families had returned at least one monthly diary before
Traffic exposure classification January 1, 2004. Eleven (1.7%) of the 633 eligible families
ArcView shapefiles containing the location of all state roads, were excluded because of inaccuracy in the geocoding of
interstates, and traffic counts (both truck and car) were obtained their residence at the time of exposure classification. The
from the Ohio Department of Transportation and the Kentucky
average age of the infants in this study (at the time of their
Transportation Cabinet. Shapefiles containing the location of public
transportation (bus) routes and bus counts for the city of Cincinnati enrollment) was 7.5 months (6 2.4 months).
and Northern Kentucky were obtained from the Cincinnati Area
Geographic Information Systems database, the Northern Kentucky
Exposure classification
Area Planning Commission, the Southwest Ohio Regional Transit Table I displays the demographic characteristics of the
Authority, and the Transit Authority of Northern Kentucky. The infants and their families in the 3 exposure groups; 60.1%
distance to the nearest federal interstate, state route, and bus route (n = 374) of the infants were unexposed, whereas the
from the primary residence of the infant at the time of parent moving traffic category included 28.3% (n = 176) of the
enrollment was derived by using the Geoprocessing extension for infants, and the stop-and-go category included 15.9%
ArcView GIS 3.2. A traffic exposure classification scheme was
(n = 99) of the infants. As shown in Table I, the infants
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, exposed to stop-and-go traffic were more likely to be
and the amount of DEPs producing traffic (trucks or buses) on the African American, to have care outside their home, and to
road type nearest each infant. have had a father with asthma, whereas they were less
Classification of exposure by distance was based on the methods likely to have been breast-fed. Because of these differ-
of others, with distances of less than 100 m,14,15 150 m,16,17 200 m,18 ences, these and other possible covariates were adjusted
J ALLERGY CLIN IMMUNOL Ryan et al 281
TABLE I. Prevalence of demographic characteristics by

exposure categorization
Unexposed Moving Stop and go

Characteristic (n = 347), % (n = 176), % (n = 99), %

White 83.3 78.7 56.6
Income <$40,000 24.6 35.7 54.2
Male sex 51.3 50.6 61.2
treatment
Current smoking mother 11.8 16.5 17.2
Care outside home 28.5 24.3 36.2
Owns dog 33.9 39.0 26.9
Owns cat 27.8 24.4 16.1
Weeks breast-fed
0 26.3 35.2 49.5
1-4 6.9 9.7 14.1
5 66.8 55.1 36.4
Diaries returned
FIG 1. Prevalence of wheeze (without cold) among infants within
1 27.7 29.6 43.4
exposure categories by distance from the nearest DEP source.
2 18.4 11.9 13.1 SR, State route; BR, bus route; HW, highway.
3 53.9 58.5 43.5
No. of siblings
0 37.5 34.3 36.2 from moving traffic (12%) was more than doubled when
1 33.0 40.7 34.0 compared with that of infants who were classified as
2 29.5 25.0 29.8 unexposed.
Has visible mold 65.4 58.0 58.6 Unadjusted and adjusted odds ratios (ORs) are shown in
Paternal asthma 11.1 13.0 23.6
Table II. Living within 100 m of stop-and-go truck and bus
Maternal asthma 20.6 25.8 17.4
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
for in the logistic model. In the unexposed category the (adjusted OR, 0.42; 95% CI, 0.19-0.93), and nonwhite
median distances to the nearest highway, state route, and infants were at an increased risk for wheezing (adjusted
bus route were 3287 m, 743 m, and 543 m, respectively. OR, 2.39; 95% CI, 1.20-4.76, respectively). Male sex and
For those infants classified as exposed to moving traffic, paternal self-report of asthma (although not maternal self-
the median distances to the nearest highway, state route, report of asthma) were also significantly associated with
and bus route were 252 m, 696 m, and 341 m, respectively. wheezing (Table II). Infants classified as exposed to
Infants exposed to stop-and-go traffic resided a median moving traffic did not have a significant association with
distance of 2303 m, 439 m, and 43 m from the nearest wheezing without a cold when compared with infants
highway, state route, and bus route, respectively. The classified as unexposed. A univariate analysis was con-
median number of trucks on the highway nearest infants ducted comparing wheezing without a cold and the season
(n = 170) in the moving category was 11,820 daily. For (winter [January-March], spring [April-June], summer
those infants (n = 6) exposed to moving traffic on a state [July-September], autumn [October-December]) in which
route, the median number of trucks per day was 1050. it was first reported to address the possibility of a cold as a
Infants exposed to buses (n = 71) or trucks (n = 9) only in possible cause of wheezing. In this analysis there were no
the stop-and-go category had a median of 44 buses daily differences in the prevalence of wheezing among season
and 1250 trucks, respectively, on the route nearest their (P = .50), and the same was true when season was added to
residence. Infants exposed to both buses and trucks the multivariate model.
(n = 19) had a median of 72 and 390, respectively.
Wheeze (without cold) DISCUSSION
Of the 622 infants, 50 (8.0%) reported wheezing
without a cold. In the unexposed category 5.8% of infants Infants exposed to stop-and-go bus and truck traffic had
reported wheezing without a cold compared with 7.4% a significantly increased risk for wheezing without a cold
in the moving category and 17.2% in the stop-and-go compared with infants unexposed to truck or bus traffic or
exposure category (P < .01). The prevalence of wheezing compared with infants exposed to moving truck traffic
in the infants who were categorized into the 3 exposure with a larger volume of trucks. Infants with immature
categories was subsequently examined by distance from lungs residing in close proximity to stop-and-go truck and
the nearest road and type of traffic (Fig 1). The prevalence bus traffic might be exposed to greater amounts of fine and
of wheezing was 3 times higher (19%) in the infants who ultrafine particulates.21-24 Sampling for fine particulate
resided less than 50 m from stop-and-go traffic compared matter (PM 2.5 mm) and black carbon inside a bus and a
with those infants who were unexposed (6%). The prev- car traveling ahead of the bus showed that the average
alence of wheezing in infants who reside 200 to 300 m DEP levels were approximately 20 mg/m3 and 5 mg/m3,
AUGUST 2005
TABLE II. Unadjusted and adjusted* ORs and 95% CIs

for wheezing without a cold
Unadjusted OR Adjusted OR
OR 95% CI OR 95% CI
Stop-and-go exposure 3.41 1.71-6.81 2.50 1.15-5.42

Nonwhite 2.80 1.54-5.08 2.39 1.20-4.76
treatment
Paternal asthma 2.63 1.29-5.37 2.35 1.08-5.13

Male sex 1.93 1.04-3.58 2.49 1.21-5.10
Only childà 0.47 0.23-0.96 0.42 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 symptoms, and physician visits for asthma.29-34 Others
than 30 mg/m3 and 20 mg/m3, respectively.21 Other studies have found that fine and ultrafine particles (PM2.5 and
have also found acceleration, deceleration, and stop-and- PM1, respectively) have a greater association with respi-
go traffic to be associated with higher emissions of organic ratory symptoms than coarse particles (PM >2.5 mm) and
carbon, elemental carbon, carbon monoxide, nitric oxide, are associated with pulmonary retention of particles.3,35,36
hydrocarbons, and soot when compared with cruising Induction of oxidative stress and mitochondrial damage
traffic.22-24 by ultrafine particulates has been proposed.2,37 Thus
To our knowledge, the present study is the first to whether the mechanism is total load or oxidative stress,
prospectively examine the effect of living in close prox- infants who reside less than 100 m away are likely
imity to roads with stop-and-go bus and truck traffic on receiving a high dose of particulates. It is not possible,
infants’ respiratory health. Although our results are con- however, to separate the contributions of diesel and
sistent with those of other investigations,14,15,18,25-28 this gasoline engines.
study improves on previous investigations. This prospec- Previous studies have found maternal asthma,38 pater-
tive design during early infancy minimizes parental recall nal asthma,39 or both to be a significant risk factor for the
bias by allowing simultaneous measurements of exposure development of asthma and wheezing in children.40,41 In
and outcome, whereas most previous studies have relied our study only paternal asthma was significant. Although
on parental recall of infant illness. The GIS database also studies have found associations between parental smoking
accurately geocoded the infant’s residence, as well as the and wheezing in infants, the multivariate model showed
distance from the nearest traffic source. With the integra- no additional smoking effect. Also, multivariate analyses
tion of county traffic data, the GIS minimized response of maternal smoking found no associations with wheezing
bias by the parents who might inaccurately report the in the unexposed subpopulation. However, only 14%
frequency or proximity of traffic. (n = 87) of the cohort were exposed to maternal smoking
Although our a priori hypothesis also expected to find (Table I). Of particular interest are the findings regard-
an effect with exposure to moving traffic, no association ing the high prevalence of wheezing among nonwhite
was found. Hence high intermittent exposures to pollu- infants in all exposure categories. As shown in Fig 2, the
tants might have a greater detrimental health effect on prevalence of wheezing in nonwhite infants was nearly 2
infants than exposure to lower continuous exposure. or more times higher in all groups, suggesting a health
These findings, however, could be related to study limi- disparity beginning early in infancy. Thus increased
tations. Infants living near moving traffic were exposed to susceptibility to wheezing is consistent with national
wide variations in the number of trucks. In addition, the statistics of asthma prevalence, as well as other stud-
median distance to stop-and-go traffic was 43 m, whereas ies.42-44 Because wheezing in the first year of life is
the median distance to moving traffic was 252 m, and generally a poor predictor of later development of child-
although state routes might have posted speed limits of hood asthma, results must be interpreted cautiously.45
greater than 50 mph and be classified as a rural route, the In conclusion, this is the first epidemiologic study to
possibility exists that traffic might accelerate and deceler- examine the risk of wheezing in infants younger than
ate at times of congestion. This scenario is also likely for 1 year who are exposed to varying types and amounts of
short periods on highways where we have designated the urban traffic. It demonstrated that even within an urban
traffic as moving. environment, the risk of wheezing varies with the type of
PM, a primary constituent of DEPs, has been signifi- and distance from traffic. A health disparity between races
cantly associated with emergency department visits for is also evident: nearly one fourth of African American
asthma, wheezing bronchitis, lower respiratory tract infants exposed to stop-and-go traffic are wheezing before
J ALLERGY CLIN IMMUNOL Ryan et al 283
age 1 year. This risk will be further investigated as the 20. Reponen T, Grinshpun SA, Trakumas S, Martuzevicius D, Wang Z,
LeMasters G, et al. Concentration gradient patterns of aerosol particles
cohort ages and asthma and atopy can be diagnosed.
near interstate highways in the Greater Cincinnati airshed. J Environ
Monit 2003;5:557-62.
We thank Stephanie Maier and Sherry Stanforth for their help with 21. Solomon G, Campbell T, Feuer G, Masters J, Samkian A, Paul K.
family interviews and testing. 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
treatment
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main road and the risk of wheezing illness in children. Am J Respir Crit Environ Health Perspect 2003;111:455-60.
Care Med 2001;164:2177-80. 38. Sheriff A, Peters TJ, Henderson J, Strachan D, ALSPAC Study Team.
17. Wilkinson P, Elliott P, Grundy C, Shaddick G, Thakrar B, Walls P, et al. Risk factor associations with wheezing patterns in children followed
Case-control study of hospital admission with asthma in children aged longitudinally from birth to 3(1/2) years. Int J Epidemiol 2001;30:
5-14 years: relation with road traffic in north west London. Thorax 1999; 1473-84.
54:1070-4. 39. El-Sharif N, Abdeen Z, Barghuthy F, Nemery B. Familial and environ-
18. Lin S, Munsie JP, Hwang S, Fitzgerald E, Cayo MR. Childhood asthma: mental determinants for wheezing and asthma in a case-control study of
hospitalization and residential exposure to state route traffic. Environ Res school children in Palestine. Clin Exp Allergy 2003;33:176-86.
2002;88:73-81. 40. Arshad SH, Kurukulaaratchy RJ, Fenn M, Matthews S. Early life risk
19. Janssen NAH, Brunekreef B, van Vliet P, Aarts F, Meliefste K, Harssema factors for current wheeze, asthma, and bronchial hyperresponsiveness at
H, et al. The relationship between air pollution from heavy traffic and 10 years of age. Chest 2005;127:502-8.
allergic sensitization, bronchial hyperresponsiveness, and respiratory 41. London SJ, Gauderman J, Avol E, Rappaport EB, Peters JM. Family
symptoms in Dutch schoolchildren. Environ Health Perspect 2003;111: history and the risk of early-onset persistent, early onset transient, and
1512-8. late-onset asthma. Epidemiology 2001;12:577-83.
AUGUST 2005
42. Grant EN, Lyttle CS, Weiss KB. The relation of socioeconomic factors 44. Simon PA, Zeng Z, Wold CM, Haddock W, Fielding JE. Rate of
and racial/ethnic differences in the US asthma mortality. Am J Public childhood asthma and associated morbidity in Los Angeles County:
Health 2000;90:1923-5. impacts of race/ethnicity and income. J Asthma 2003;40:535-43.
43. Joseph CLM, Ownby DR, Peterson EL, Johnson CC. Racial differences 45. Taussig L, Wright A, Holberg C, Halonen M, Morgan W, Martinez F.
in physiologic parameters related to asthma among middle-class children. Tucson Children’s Respiratory Study: 1980 to present. J Allergy Clin
Chest 2000;117:1336-44. Immunol 2003;111:661-75.
treatment
Effect of low-dose ciclesonide on allergen-
induced responses in subjects with mild
allergic asthma

Gail M. Gauvreau, PhD,a Louis Philippe Boulet, MD,b Dirkje S. Postma, MD, PhD,c
treatment
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 on the early allergen-induced bronchoconstriction, 24-hour
with asthma induces reversible airway obstruction, airway FEV1, or serum eosinophil cationic protein levels (P < .025).
hyperresponsiveness, and eosinophilic airway inflammation. Conclusion: With the exception of 24-hour postchallenge
Attenuation of allergen-induced bronchoconstriction and peripheral blood eosinophils, a low dose of ciclesonide, 80 mg,
inflammation has been used to examine the efficacy of was effective in blocking all allergen-induced responses
therapeutic agents such as inhaled corticosteroids in asthma. measured. (J Allergy Clin Immunol 2005;116:285-91.)
Ciclesonide, a nonhalogenated inhaled corticosteroid being
developed for the treatment of persistent asthma, remains Key words: Inhaled corticosteroid, allergen inhalation, airway
inactive until cleaved by esterases in the lung. inflammation
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. Asthma is characterized by reversible airway obstruc-
Methods: Twenty-one nonsmokers with mild atopic asthma
tion, airway hyperresponsiveness, and eosinophilic bron-
completed a multicenter, randomized, 3-way crossover study
comparing the effects of 7-day treatment of ciclesonide or
chial inflammation. Subjects with allergic asthma develop
placebo. Allergen-induced responses, including the early and an immediate IgE-mediated early asthmatic response
late fall in FEV1, peripheral blood eosinophils, serum (EAR) after inhalation of an allergen to which they are
eosinophil cationic protein levels, and eosinophils in induced sensitized. Approximately 50% of these subjects also
sputum were measured. develop a late asthmatic response (LAR), which begins
Results: Ciclesonide 80 mg attenuated the early and late 3 to 4 hours after allergen inhalation.1 The LAR is
asthmatic responses, including the change in FEV1, serum associated with elevated levels of airway inflammatory
eosinophil cationic protein, and sputum eosinophils measured cells including eosinophils, basophils, and mast cells.2,3
at 24 hours postchallenge (P < .025). Ciclesonide 40 mg This model of allergen-induced bronchoconstriction has
attenuated the late asthmatic responses and sputum eosinophils
been used successfully to assess drug efficacy in subjects
measured at 24 hours postchallenge (P < .025), with no effect
with allergic asthma4-13 and is recommended for evalua-
tion of inhaled corticosteroids (ICS).14
Inhaled corticosteroids, having potent anti-inflamma-
From athe Department of Medicine, McMaster University, Hamilton; bInstitut tory properties, are indicated by the Global Initiative
de cardiologie et de pneumologie de l’Université Laval, Hôpital Laval, for Asthma as a primary controller for treatment of mild-
Quebec City; and cthe Department of Pulmonology, University Hospital persistent to severe asthma.15 Studies of ICS have
Groningen.
consistently shown a significant attenuation of the allergen-
Supported by Altana Pharma AG.
Disclosure of potential conflict of interest: P. M. O’Byrne has consultant induced LAR,5,16-18 with the proposed mechanism atten-
arrangements with AstraZeneca, GlaxoSmithKline, Topigen, and Altana, uation of the allergen-induced airway inflammation.
and has received grants/research support from AstraZeneca, GlaxoSmith- However, undesirable side effects of ICS at higher doses
Kline, Pfizer, Altana, and Dynavax. L.-P. Boulet has been on Advisory have established a need to evaluate ICS properties at very
Boards for AstraZeneca, Altana Novartis, GlaxoSmithKline, and Merck
Frost, and received lecture fees from 3M, GlaxoSmithKline, AstraZeneca,
low doses.14
and Merck Frosst. Sponsorship for basic research was received from 3M, Ciclesonide is a nonhalogenated ICS for the treatment
Schering, Genentech, Dynavax, Roche, GlaxoSmithKline, Novartis, As- of persistent asthma of all severities. This ICS remains
traZeneca, Altana, and Merck for participating in multicenter studies of the inactive until cleaved by esterases present in the airway,
pharmacotherapy of asthma. D. Postma is on the Advisory Board of Altana.
where its active metabolite, desisobutyryl-ciclesonide, then
Received for publication March 29, 2005; revised May 13, 2005; accepted for
publication May 17, 2005. binds glucocorticoid receptors.
Available online July 15, 2005. Before developing an optimal solution for metered dose
Reprint requests: Paul M. O’Byrne, MD, HSC 3W10, McMaster University, inhaler (MDI) formulation for clinical use, early clinical
1200 Main St West, Hamilton, Ontario, Canada L8N 3Z5. E-mail: studies were performed with ciclesonide by using a dry
obyrnep@mcmaster.ca.
0091-6749/$30.00
powder inhaler (DPI) device. One week of ciclesonide
Ó 2005 American Academy of Allergy, Asthma and Immunology 800 mg DPI BID (twice daily; using Cyclohaler device
doi:10.1016/j.jaci.2005.05.021 [Pharmachemie, Haarlem, The Netherlands]) has been
285
286 Gauvreau et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
40 mg and 80 mg ex-actuator, with placebo. The study was approved

Abbreviations used by the ethics research board of the respective institutions, and signed
AE: Adverse event informed consent was given to participate. Screening of subjects was
AUC0-2h: Area under the curve of the early response performed over a period of 2 consecutive days and included a detailed
AUC3-8h: Area under the curve of the late response history, physical examination, allergen skin test, FEV1, methacholine
BID: Twice daily PC20, blood sampling for serum ECP levels, allergen inhalation
DPI: Dry powder inhaler challenge, and sputum induction. Subjects who developed an EAR (at
EAR: Early asthmatic response; maximum % fall in least 20% fall in FEV1 within 2 hours after allergen inhalation) and
treatment
FEV1 from 0 to 2 hours after allergen challenge LAR (at least 15% fall in FEV1 between 3 and 8 hours after allergen
ECP: Eosinophil cationic protein inhalation) during 1 screening allergen inhalation challenge were
ICS: Inhaled corticosteroids enrolled in the study. Subjects were screened once only, because we
LAR: Late asthmatic response; maximum % fall in have shown the LAR to be a reproducible measurement.20,21 Subjects
FEV1 from 3 to 8 hours after allergen challenge reported to the laboratory for 3 separate treatment periods separated
MDI: Metered dose inhaler 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 inflamma-
tory cells, and lung function; methacholine PC20 needed to be within
shown to attenuate significantly the allergen-induced 1 doubling dose of that measured during the screening period to
EAR and LAR,19 confirming that this drug has biological continue with the treatment period. If this criterion was met, subjects
activity in this model of allergic inflammation. The then inhaled the first dose of study medication during the morning
allergen-induced fall in FEV1 was shown to be a sensitive visit to the lab. The subsequent doses of study medication were
inhaled for the next 6 consecutive mornings, immediately after
marker of dose-response effect in an earlier trial of
waking, because ciclesonide has been shown to improve asthma
mometasone furoate16; therefore, the current trial was
control irrespective of the time of administration.22 Subjects returned
performed to examine the dose-response of the LAR and to the laboratory on day 5 for measurement of preallergen challenge
to determine the efficacy of low-dose ciclesonide in the sputum inflammatory cells and on day 6 for allergen inhalation
reduction of the allergen-induced EAR, LAR, and airway challenge. Measurements of FEV1 were taken at regular intervals
inflammation. In-house testing has led to the development until 8 hours after challenge. On day 7, subjects underwent measure-
of ciclesonide in MDI formulation with hydrofluoroalkane ments of sputum inflammatory cells, blood eosinophils, and serum
(HFA) propellent, delivering 40 mg, 80 mg, and 160 mg ECP. Postallergen methacholine PC20 was not measured, because this
(ex-actuator) per puff. However, data are limited regarding study was not powered sufficiently for this comparison. All subjects
what the lowest effective dose is with the MDI formulation were considered compliant with study medication according to the
diary cards.
of ciclesonide. This study, therefore, was designed to
identify the lowest effective dose of ciclesonide in MDI
formulation in an allergen inhalation model. Laboratory procedures
Methacholine inhalation test. Methacholine inhalation challenge
was performed as described by Cockcroft.23 Subjects inhaled normal
METHODS saline during 2 minutes of tidal breathing, nebulized at 0.13 mL/
minute from a Wright nebulizer, then doubling concentrations of
Subjects
methacholine chloride. FEV1 was measured at 30, 90, 180, and 300
Thirty-five subjects were enrolled in the study. Of these, seconds after each inhalation. The test was terminated when a fall in
13 patients did not meet randomization criteria. Twenty-two subjects FEV1 of 20% of the baseline value occurred, and the methacholine
(Groningen, n = 5; Laval, n = 7; McMaster, n = 10), 14 men and PC20 was calculated.
8 women, age 19 to 58 years old (Table I), were randomized to one Allergen inhalation challenge. Allergen challenge was performed
of the 6 treatment sequences. One subject discontinued the study as described by O’Byrne et al.1 Subjects were skin tested for allergies
prematurely for nonmedical reasons. Inclusion criteria required to common aeroallergens. The allergen producing the largest skin
subjects to be nonsmokers with mild atopic asthma, free of other wheal diameter was diluted in 0.9% saline and stored for subsequent
lung disease, and without lower respiratory tract infection for 6 weeks allergen inhalation challenges. The concentration of allergen extract
before entering the study. For randomization, subjects were required for inhalation was determined from a formula described by Cockcroft
to have stable asthma with FEV1 > 70% of predicted; have baseline et al,24 and doubling concentrations of allergen were given until a
methacholine PC20 < 16 mg/mL; use no regular asthma medication <20% early fall in FEV1 at 10 minutes postallergen was reached. The
during the study other than infrequent inhaled b2-agonist, which was FEV1 was then measured at regular intervals until 8 hours after
withheld for 8 hours before each visit; and have no exposure to allergen inhalation. The early area under the curve (AUC0-2h) and the
sensitizing allergens apart from house dust mite. Before entering the late area under the curve (AUC3-8h) were calculated by using the
study, subjects could not have used systemic steroids or had an trapezoidal rule, were normalized to 1 hour, and were expressed as
asthma exacerbation for 6 weeks and could not have used inhaled liters 3 hour (L3h). Subjects inhaled the same dose of allergen for
steroids for at least 4 weeks. Before morning visits to the lab, subjects the 3 treatment periods.
were to refrain from tea or coffee. Sputum analysis. Sputum was induced and processed by using the
method described by Pizzichini et al.25 Cell plugs were selected and
Study Design mixed with 0.1% dithiothreitol (Sputolysin; Calbiochem Corp, San
This trial was a multicenter, double-blind, randomized, placebo- Diego, Calif) and Dulbecco PBS (Life Technologies Inc, Grand
controlled, 3-period crossover study comparing 7 days of treatment Island, NY) and filtered through a 48-mm nylon gauze (BNSH
with ciclesonide at 50 mg and 100 mg exvalve, corresponding to Thompson, Scarborough, Ontario, Canada), and cytospins were
J ALLERGY CLIN IMMUNOL Gauvreau et al 287
TABLE I. Subject characteristics at study screening visit
Age Methacholine Final dose,

Subject (y) Sex Predicted FEV1 (%) FEV1/VC ratio PC20 (mg/mL) Allergen median
50001 25 F 96 0.86 1.12 Ragweed 1:64

50002 21 M 114 0.86 8.94 HDM 1:16
50004 42 M 106 0.69 4.54 HDM 1:32
50005 20 M 73 0.55 0.31 HDM 1:1024
treatment
50008 26 F 89 0.92 1.63 Ragweed 1:16
50011 54 M 107 1.18 0.88 Cat 1:32
50012 33 M 88 0.73 15.51 Ragweed 1:64
50013 54 M 73 0.55 3.39 HDM 1:64
50014 19 F 109 0.83 0.54 HDM 1:128
50015 39 M 80 0.67 5.11 HDM 1:128
50025 26 F 83 0.89 1.07 HDM 1:32
50027 28 F 121 0.87 1.11 Cat 1:64
50028 25 F 96 0.86 1.42 Cat 1:16
50029 23 M 99 0.77 1.47 Cat 1:64
50031 30 M 75 0.70 0.86 Cat 1:128
50032 26 M 93 0.73 2.21 HDM 1:64
50033 38 M 112 0.72 1.56 HDM 1:128
50038 19 M 103 0.84 0.70 HDM 1:1024
50039 58 M 107 0.79 8.80 HDM 1:2048
50040 40 M 84 0.80 0.44 HDM 1:2048
50041 49 F 100 0.86 0.21 HDM 1:2048
50045 33 F 85 0.83 0.33 HDM 1:1024
14 HDM
Mean 6 SEM 33.1 6 2.6 14 M 95.1 6 3.0 0.80 6 0.03 1.46 5 Cat 1:64
8F (15.51-0.21) 3 RW
HDM, House dust mite; VC, vital capacity.
prepared on glass slides. The total cell count was determined by using and SEM. Between-treatment differences in FEV1 during the LAR,
a Neubauer hemocytometer chamber (Hausser Scientific, Blue Bell, FEV1 during the EAR, and sputum eosinophils 24 hours after allergen
Pa) and expressed as the number of cells per milliliter sputum, and were analyzed by using ANOVA. The FEV1 measured 24 hours after
differential cell counts were obtained from slides stained with Diff allergen was analyzed with analysis of covariance by using the
Quik (American Scientific Products, McGaw Park, Ill). All slides baseline value as a covariate to test both within-treatment and
were enumerated at 1 site. Slide preparation and enumeration were between-treatment differences. Within-treatment differences in spu-
performed before unblinding. The study personnel collecting sputum tum variables as well as between-treatment differences in blood
samples and the technician enumerating the slides had no knowledge eosinophils, ECP, and sputum variables were analyzed by means of
of the coding of the labels, nor of the airway physiology measured the nonparametric test for 3 3 6 crossover design. One-sided P values
when these sputum samples were collected. All data were collected were generated for all variables, and significance is shown at
centrally and entered into a master database before the random code P < .025.
was distributed to participating sites.
Serum eosinophil cationic protein and blood eosinophils. Blood
was collected into 4-mL vacutainer hemogard SST tubes (Becton RESULTS
Dickinson, Franklin Lakes, NJ) for serum separation. Eosinophil
cationic protein (ECP) was released by allowing blood to clot for All subjects inhaled the same dose of allergen for the 3
60 to 120 minutes at room temperature, and then samples were
treatment periods. There were no serious adverse events
centrifuged at 1000g to 1300g for 10 minutes at room temperature.
(AEs), and 11 treatment-emergent AEs were reported. Of
Serum was removed and stored at 220°C until analysis. All serum
ECP was analyzed at 1 site by using the UniCAP system (Pharmacia,
these, 7 AEs were mild and 4 were moderate. All
Uppsala, Sweden). Blood was also collected into 2-mL EDTA AEs were assessed as unrelated to the study medication,
vacutainers, and eosinophil counts were performed by Coulter except 1 case of thrombocytopenia, which was assessed
Counter (Beckman Coulter, Fullerton, Calif) at the respective unlikely related to the study medication. No adverse event
institutions. led to premature discontinuation or a change in study
medication. Before allergen challenge, the degree of
Statistical analysis airway hyperresponsiveness was within 1 doubling dose,
The target sample size of 18 patients is sufficient to guarantee a
and there was no significant difference in FEV1 between
power of 90% in correctly concluding superiority at the .0125 level, the 3 treatment periods. During placebo treatment, all
1-sided, if the mean difference accounts for 90% of the SD (based on subjects demonstrated early and late airway responses
paired t test). The 21 subjects who completed the study were included after allergen inhalation challenge; the maximum percent
in the statistical analyses. Summary statistics are expressed as mean fall in FEV1 was 30.4% 6 2.2% during the early response
AUGUST 2005
treatment
FIG 1. Study schematic. BL, Pretreatment baseline.
TABLE II. Effect of ciclesonide on the maximum percent of 3.59 L 6 0.18 L at day 7 was significantly lower than
fall in FEV1 and area under the curve of the early and 3.67 L 6 0.18 L at day 1 (P = .013), but not significantly
late airway responses different from the FEV1 of 3.64 L 6 0.16 L at day 6
Ciclesonide Ciclesonide
(P = 0.10). Compared with placebo, ciclesonide 80 mg
Placebo 40 mg 80 mg significantly attenuated the day 7 allergen-induced de-
crease in FEV1 compared with day 1 (P = .002) and day 6
EAR (%) 30.4 6 2.2 28.2 6 2.2 23.6 6 2.2*
(P = .007; Fig 2). Compared with placebo, ciclesonide
AUC0-2h 233.3 6 4.0 229.2 6 4.6 224.9 6 4.1*
(L3h)
40 mg did not attenuate day 7 allergen-induced decrease in
LAR (%) 24.0 6 2.1 13.3 6 2.1** 10.7 6 2.1** FEV1 compared with day 1 or day 6 (P > .025). There was
AUC3-8h 227.5 6 4.3 211.8 6 4.0** 29.4 6 2.7** a trend toward a dose-response effect for ciclesonide, with
(L3h) greater attenuation of the 24-hour postallergen fall in
FEV1 by ciclesonide 80 mg compared with ciclesonide
*P < .025 compared with placebo. 40 mg; however, this did not reach statistical significance
**P < .013 compared with placebo.
(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
and 24.0% 6 2.1% during the late response, correspond- to 31.0 mg/L 6 5.8 mg/L with placebo, 33.0 mg/L 6 5.6
ing to AUC0-2h of 233.3 6 4.0 L3h and AUC3-8h of mg/L with ciclesonide 40 mg, and 26.0 mg/L 6 5.3 mg/L
227.5 6 4.3 L3h (Table II). with ciclesonide 80 mg. There was significant attenuation
During treatment with ciclesonide 80 mg, there was a of the allergen-induced increase in serum ECP with
significant reduction in the maximum percent fall in ciclesonide 80 mg versus placebo treatment (P = .024),
FEV1 during the EAR to 23.6% 6 2.2% (P = .016), the but no effect of ciclesonide 40 mg. Peripheral blood
AUC0-2h (P = .013), the maximum percent fall in FEV1 eosinophils were not significantly reduced with cicleso-
during the LAR to 10.7% 6 2.1% (P < .001), and the nide 40 mg or 80 mg (P > .025). There was an allergen-
AUC3-8h (P < .001). Treatment with ciclesonide 40 mg induced increase in the number of peripheral blood
significantly reduced the maximum percent fall in FEV1 eosinophils at 24 hours after challenge, increasing from
during the late response to 13.3% 6 2.1% (P = .0003) a baseline of 0.294 3 106/mL 6 0.037 3 106/mL to
and AUC3-8h (P = .0003), with no significant effect on 0.404 3 106/mL 6 0.040 3 106/mL with placebo, 0.385 3
the early maximum percent fall in FEV1 or AUC0-2h 106/mL 6 0.047 3 106/mL with 40 mg ciclesonide,
(P > .025). There was no significant difference between and 0.347 3 106/mL 6 0.052 3 106/mL with 80 mg
ciclesonide 40 mg and 80 mg on the EAR, LAR, AUC0-2h, ciclesonide.
or AUC3-8h (P > .025; Table II). There was an allergen-induced increase in the percent
FEV1 was measured at pretreatment baseline (day 1), sputum eosinophils (Fig 3, A) and the number of eosin-
preallergen (day 6), and at 24 hours postallergen inhala- ophils per milliliter sputum (Fig 3, B) on day 7 compared
tion challenge (day 7). With placebo treatment, the FEV1 with day 5 with placebo, ciclesonide 40 mg, and
of 3.38 L 6 0.17 L at day 7 was significantly lower than ciclesonide 80 mg treatment (P < .002). However, both
the day 1 FEV1 of 3.60 L 6 0.15 L (P < .001) and the day ciclesonide 40 mg and 80 mg significantly attenuated the
6 FEV1 of 3.56 L 6 0.16 L (P < .001). During treatment allergen-induced increase in the percentage of sputum
with ciclesonide 40 mg, the FEV1 of 3.50 L 6 0.16 L eosinophils (P < .001 and P = .006, respectively). Only
at day 7 was significantly lower than that of day 1 or day 6, ciclesonide 80 mg significantly attenuated the allergen-
3.65 L 6 0.17 L and 3.62 L 6 0.15 L, respectively (P < induced increase in the number of eosinophils per milli-
.001). During treatment with ciclesonide 80 mg, the FEV1 liter sputum, but the trend toward a dose-dependent effect

treatment
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 study resulted in 58% inhibition and 40 mg resulted in
(1-sided P = .03). 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
DISCUSSION providing approximately 8% and 23% inhibition of the
early fall in FEV1, respectively, compared with the 45%
Ciclesonide is a new generation inhaled glucocortico- inhibition with 800 mg ciclesonide BID previously stud-
steroid that remains inactive until cleaved by esterases pre- ied.19 The deposition pattern of HFA-MDI formulations in
sent in the lung. Ciclesonide has been shown to be effective the peripheral lung may not inhibit the EAR as effectively
to reduce allergen-induced early and late responses when compared with the DPI formulation, which gets deposited
administered at 800 mg twice daily by Cyclohaler (DPI),19 in the large, central airways where the EAR is likely to be
yet it was unknown whether lower doses of 80 mg or most active. This supports the consistent observation that
40 mg administered by MDI would also be effective. In multiple or single doses of inhaled steroids may signifi-
the current study, ciclesonide was administered by MDI cantly attenuate the LAR without significantly attenuating
once daily for 7 days at considerably lower doses than the EAR.5,18,26 Moreover, these data suggest that higher
previously studied. levels of HFA-MDI formulation steroids are necessary to
This study has demonstrated that once-a-day treatment inhibit IgE-mediated early responses to inhaled allergen,
with 40 mg or 80 mg ciclesonide for 7 days is indeed such as mast cell degranulation, compared with lower
efficacious in this model of allergen-induced bronchocon- levels of steroids that appear to suppress the late response
striction and airway inflammation. Furthermore, we were effectively, likely through inhibition of proinflammatory
able to determine the lowest effective dose of ciclesonide cytokine gene expression.27
for attenuating all allergen-induced responses investigated Ciclesonide HFA-MDI 200 mg 4 times daily adminis-
in this study. In contrast with the 40 mg dose, ciclesonide tered in a previous study28 did not affect urine cortisol
at 80 mg per day attenuated the allergen-induced early levels after 4 weeks treatment, which suggests that the
airway response, the sustained fall in FEV1 measured 80 mg dose in the current study would have a very low
24 hours postchallenge, and the allergen-induced accu- potential for systemic activity. We did not measure
mulation of eosinophils into the airways. Whether 80 mg systemic safety markers, because no effect would have
represents a plateau beyond which there is no further been expected. It is unknown, however, whether sys-
attenuation of allergen-induced responses is unknown. temic activity in compartments such as the circulation
Compared with the aforementioned study of 800 mg and/or bone marrow is an unidentified yet important site
ciclesonide DPI BID resulting in approximately 51% of action of inhaled steroids. There is certainly evidence
attenuation in the late fall in FEV1,19 80 mg in the current showing that steroids provide anti-inflammatory effects
AUGUST 2005
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 morning immediately after waking, and approximately
systemic activity with low-dose ciclesonide treatment 1 hour preallergen challenge. Hence, the drug was present
supports the notion that steroid regulation of allergen- at the highest possible levels during challenges. Whether
induced bone marrow responses is likely through sup- this degree of efficacy would have been observed had drug
pression of proinflammatory mediators generated in administration and allergen challenge been separated by a
airways that subsequently control the bone marrow, longer time interval is unknown, but is important to
rather than the belief that steroids have a direct effect consider, because the protective effects of inhaled steroids
on cells in the bone marrow.30 Because new generation against allergen-induced early responses, airway eosino-
steroids are preferred as a result of their low systemic philia, and allergen-induced airway hyperresponsiveness
effects, this question of whether some systemic activity is are partially or completely lost as early as 12 hours after
also required will become an important issue that needs discontinuation of therapy.18
to be addressed. Direct comparisons between various ICS were not
There was a numerical, though insignificant (P > .025), performed in this study, largely because of the difficulties
dose-response for low doses of ciclesonide during the late associated with 4-way crossover studies. Although a direct
response, which is believed to be one of the most sensitive comparison would need to be performed to indicate the
variables used to demonstrate a dose-response.16 Surprisingly, relative potency of ciclesonide, the dose of 80 mg appears
these data demonstrate dose-responses to measurements to be similar to proven effective doses of other inhaled
of airway obstruction and inflammation at 24 hours steroids; 50 ug mometasone furoate BID has been shown
postchallenge. This is an unexpected finding, indicating to attenuate significantly the EAR, LAR, and 24-hour
these variables may be worthy of evaluation in subsequent postallergen sputum eosinophils.16 This study has pro-
trials evaluating dose-responses. vided new information regarding the minimally effective
Although results from this study suggest that 80 mg doses of inhaled ciclesonide for inhibition of allergen-
ciclesonide may be the minimally effective dose for induced airway responses and the apparent local anti-
protection against the EAR and serum ECP levels, it is inflammatory effects on the airways. Further evaluation of
noteworthy that there was a significant effect of both ciclesonide will be required to address whether these low
80 mg and 40 mg ciclesonide on the LAR and percentage doses are clinically effective.
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 We thank Dr A. Widmann for logistic support of this study and
the study, ciclesonide was always administered in the Mrs C. Veltman for help in patient recruitment. We also thank Tara
Strinich, Irene Babirad, and Tracy Rerecich for help in sample Institutes of Health, National Heart Lung and Blood Institute; 2002.
preparation and enumeration. NIH publication number 02-3659.
16. 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.
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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

treatment
Louis Venter, MSc,c Stefan Leichtl, PhD,d Christine Schmid-Wirlitsch, PhD,d

Dirk Bredenbröker, 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 pulmonary diseases, such as asthma. (J Allergy Clin Immunol
increasing incidence worldwide. Roflumilast is an oral, once- 2005;116:292-8.)
daily inhibitor of phosphodiesterase type 4 that prevents the
breakdown of cyclic adenosine monophosphate levels, leading Key words: Asthma, roflumilast, phosphodiesterase type 4, allergen
to inhibition of proinflammatory signaling. provocation, inflammation, late phase
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. Asthma is a worldwide public health concern that has
Methods: Twenty-three patients with mild asthma with an been increasing in prevalence, particularly in developed
FEV1 of 70% of predicted value or greater were enrolled in countries.1,2 In the United States approximately 31 million
a randomized, double-blind, placebo-controlled, 3-period persons have been given a diagnosis of asthma.3 Further-
crossover study. Patients participated in 3 treatment periods more, the economic burden of asthma (eg, prescription
(7-10 days) separated by washout periods (2-5 weeks). drugs, hospitalization, and loss of productivity) has also
Patients received 250 mg of oral roflumilast, 500 mg of
increased over the past 20 years, with the economic
roflumilast, or placebo once daily. Allergen challenge was
performed at the end of each treatment period, followed by
costs associated with asthma estimated to exceed those
FEV1 measurements over the ensuing 24 hours. of tuberculosis and HIV-AIDS combined.4
Results: Late asthmatic reactions (LARs) were reduced by 27% Asthma is characterized by chronic inflammation and
(P = .0110) and 43% (P = .0009) in patients treated with 250 airway hyperresponsiveness (AHR), leading to recurrent
and 500 mg of roflumilast, respectively, versus placebo. episodes of wheezing, breathlessness, chest tightness, and
Roflumilast, 250 and 500 mg, also attenuated early asthmatic coughing.5 A primary goal of asthma therapy is to achieve
reactions by 25% (P = .0038) and 28% (P = .0046), although not and maintain control of clinical symptoms by improving
to the same extent as LAR attenuation. Roflumilast was well lung function and reducing AHR. In addition, reducing
tolerated. No serious adverse events or discontinuations caused the frequency of asthmatic exacerbations and improving
by adverse events were reported.
health-related quality of life are important therapeutic
Conclusion: Once-daily oral roflumilast modestly attenuated
early asthmatic reactions and, to a greater extent, LARs to
goals. There are several therapeutic options for long-term
allergen in patients with mild allergic asthma. Pronounced maintenance (ie, controllers) and symptom relief (ie,
suppression of late responses in an allergen challenge model relievers) available to asthmatic patients. Commonly pre-
suggests that roflumilast might have anti-inflammatory activity, scribed fixed-dose combination therapies based on inhaled
which could provide clinical efficacy in chronic inflammatory 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
From athe Department of Internal Medicine, University of Stellenbosch, Cape asthma in all patients. Long-term use of ICSs can also
Town; bMonash Centre for Inflammatory Diseases, Monash University and potentially cause serious systemic side effects,6 and poor
Medical Centre, Melbourne; cALTANA Madaus (Pty) Ltd, Rivonia; and compliance is an additional concern.7 Furthermore, a small
d
ALTANA Pharma AG, Konstanz. number of patients are unresponsive to ICS therapy and
Disclosure of potential conflict of interest: Drs Bredenbröker, Schmid-
Wirlitsch, Leichtl, and Venter are employees of ALTANA Pharma, and
require alternative therapeutic options.8 Novel therapies
Dr Bardin has served as a consultant to ALTANA Pharma and received that increase the natural anti-inflammatory response in
research support from GlaxoSmithKline, AstraZeneca, Schering Plough, inflammatory target cells have the potential to meet the
and Boehringer-Ingelheim. The other authors have no conflict of interest current unmet medical need for asthmatic patients.
to disclose.
Inhaled allergen challenge in patients with mild allergic
Received for publication August 26, 2004; revised April 8, 2005; accepted for
publication April 18, 2005. asthma results in an early asthmatic reaction (EAR),
Available online June 1, 2005. followed by a late-phase response (the late asthmatic
Reprint requests: Philip G. Bardin, FRACP, PhD, Monash Medical Centre, reaction [LAR]). The EAR is a consequence of the
246 Clayton Rd, Clayton 3168, Melbourne, Australia. E-mail: p.bardin@ activation and degranulation of cells expressing allergen-
southernhealth.org.au.
0091-6749/$30.00
specific IgE. Mediators are released that induce nerve
Ó 2005 American Academy of Allergy, Asthma and Immunology stimulation, mucus hypersecretion, vasodilation, and
doi:10.1016/j.jaci.2005.04.023 microvascular leakage.
292
J ALLERGY CLIN IMMUNOL van Schalkwyk et al 293
predicted value or greater, were between 18 and 50 years of age, and

Abbreviations used were not receiving treatment with asthma medications other than
AHR: Airway hyperresponsiveness short-acting bronchodilators for symptom relief. Inclusion criteria
AUC: Area under the curve also required a positive allergen skin prick test response and
cAMP: Cyclic adenosine monophosphate hyperresponsiveness to methacholine, with a provocative concentra-

COPD: Chronic obstructive pulmonary disease tion resulting in a 20% decrease in FEV1 (PC20FEV1) of 16 mg/mL or
EAR: Early asthmatic reaction less. All patients provided informed written consent, and the Human
ICS: Inhaled corticosteroid Research Ethics Committee of the University of Stellenbosch (Cape
treatment
LAR: Late asthmatic reaction Town, South Africa) approved the study.
PC20FEV1: Provocative concentration resulting in a
20% decrease in FEV1 Study design and measurements
PDE4: Phosphodiesterase type 4
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).
The LAR is believed to reflect mechanisms of asthmatic Randomization was performed after the first washout period at the
inflammation because in this response activated airway first treatment visit. Study medication was taken between 7 AM and
cells release cytokines and chemokines locally and into the 10 AM daily.
At the first baseline visit, FEV1 was measured with a mobile
circulation, thus stimulating the release of inflammatory
spirometer (Vitalograph, Hamburg, Germany), and AHR to meth-
leukocytes, particularly eosinophils and their precursors,
acholine challenge was assessed for each patient. At the second
from the bone marrow into the circulation. Inflammatory baseline visit, allergen challenge was done to determine a provoca-
cells in the peripheral blood are then recruited into the tive concentration causing an early response to allergen (FEV1 25%
inflamed airways, where they augment airway inflamma- decrease) and late response (FEV1 15% decrease), as detailed
tion and increase AHR. below. During the baseline visits, patients were assessed for adher-
Roflumilast (3-cyclo-propylmethoxy-4-difluorome- ence to inclusion and exclusion criteria. In eligible patients methacho-
thoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) is an oral, line challenge was performed on the first treatment visit, and allergen
once-daily phosphodiesterase type 4 (PDE4) inhibitor in challenge was performed on the second treatment visit (ie, after
clinical development as long-term maintenance therapy taking study medication for 7-10 days). The FEV1 measurements
were done over the ensuing 24 hours at 5, 10, 15, 30, 45, and
for chronic obstructive pulmonary disease (COPD) and
60 minutes; subsequently at 1-hour intervals for the next 11 hours; and
asthma. Phosphodiesterases hydrolyze the second mes-
at 4.5, 5.5, and 24 hours after allergen challenge. At the conclusion of
senger cyclic adenosine monophosphate (cAMP) to the 24-hour period after allergen challenge, methacholine challenge
5#-adenosine monophosphate, rendering it inactive. The was repeated.
PDE4 isozyme has localized activity in the lung, and Allergen and methacholine challenges were performed according
PDE4 inhibitors, such as roflumilast, block cAMP hy- to the bronchial provocation technique described by Chai et al,12 as
drolysis in the airways. The inflammatory response is previously published by Bardin et al.13 The challenge tests were
highly sensitive to levels of cAMP, and by preventing the performed by using a Spira Elektra breath-actuated inhalation
breakdown of cAMP, PDE4 inhibitors are associated with dosimeter (SPIRA OY; Hämeenlinna, Finland). The duration of
a natural anti-inflammatory activity. As a second messen- 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
ger, cAMP blocks proliferation and chemotaxis of inflam-
inspiratory flow of 0.6 to 0.8 L/s. Provocation began with 5 breaths of
matory cells (eg, lymphocytes), inhibits proinflammatory
saline inhalation, each lasting 5 seconds, followed by holding the
cell activity (eg, phagocytosis and respiratory burst), and breath for 2 seconds (from functional residual capacity to total lung
suppresses the release of inflammatory and cytotoxic capacity). Challenges were performed only if baseline FEV1 was 70%
mediators (eg, TNF-a) in the lungs.9 In previous in vitro of predicted value or greater, if it was within 12% of the initial value
and in vivo studies, roflumilast decreased inflammatory measured at baseline, and if, after saline inhalation, the decrease in
cell infiltration into the airways, total protein levels, and FEV1 was 10% or less. At initial assessment, a decrease in FEV1 of
TNF-a release.10 Thus by maintaining levels of cAMP 25% or greater from the postsaline value within the first 2 hours after
in key airway inflammatory cells, roflumilast acts as a the challenge defined the EAR, whereas the LAR was characterized
steroid-free anti-inflammatory agent that might have util- 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
ity in diseases such as asthma and COPD.11
hours after challenge) and with a typical gradual deterioration in
This proof-of-concept study investigated the effect of
FEV1.
repeat doses of roflumilast (250 and 500 mg) on allergen- Each patient was challenged with a single allergen identified on
induced asthmatic responses and AHR in patients with the basis of reactivity during the skin prick test. The allergens used in
mild allergic asthma. 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
METHODS always started with the lowest allergen concentration, and FEV1 was
recorded 5, 10, and 15 minutes after inhalation. If the decrease in
Patients FEV1 was less than 10% of the postsaline FEV1, a 10-fold higher
Patients were included in the study if they had a history of allergen concentration was administered; if the decrease was 10% to
wheezing consistent with mild asthma, had an FEV1 of 70% of 15%, a 5-fold higher concentration was administered; and if the
294 van Schalkwyk et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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 corresponding to the primary variable, LAR, and on the AUC from
characteristics 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
Parameter Patients (n = 23) 3-period crossover design for both the LAR and EAR. Geometric
Median age, y (range) 28 (20-44) means and 95% CIs were calculated for the differences between
Mean height 6 SD, cm 168 6 10.7 population means. Point estimates and 95% CIs were calculated for
Mean weight 6 SD, kg 76 6 19.3 the maximum decrease analysis. Percentage reductions in the EAR
Sex distribution, n (%) and LAR were calculated for the differences between population
Female 12 (52) least-squares means for the per-protocol population. Other secondary
Male 11 (48) end points were analyzed in a descriptive manner with geometric
Smoking habits, n (%) means, and 95% CIs were calculated where appropriate.
Never smoked 19 (83)
Exsmokers 3 (13)
RESULTS
Current smokers 1 (4)
Mean FEV1 6 SD, L 3.17 6 0.82 Patients
Mean FEV1 6 SD, % predicted 89 6 10 Patient demographics and characteristics at baseline are
Mean PC20FEV1 6 SD, mg/mL 4.09 6 3.79
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 per-
decrease in FEV1 was greater than 15% to 25%, the same allergen protocol population varied among the different efficacy
concentration was administered a second time. The concentration of analyses because of missing or invalid data. Safety data
allergen causing a decrease in postsaline FEV1 of greater than 25% are reported for the intent-to-treat population that was
was used in each patient for allergen provocation on subsequent study actually exposed to each treatment. The numbers of
days. If needed, patients received single doses of inhaled beclome- patients exposed to 500 mg of roflumilast, 250 mg of
thasone or budesonide (800 mg) for stabilization of their condition
roflumilast, and placebo were 23, 21, and 22, respectively.
after allergen challenge.
Three patients were exsmokers, and 1 patient was a current
For methacholine challenge, if the decrease in postsaline FEV1
was 10% or less, 5 breaths of the lowest methacholine concentration smoker. Median age was 28 years. At baseline, the mean
(0.03 mg/mL) were inhaled. Doubling concentrations of methacho- FEV1 was 3.17 L (SD, 0.82), 89% of the predicted value.
line were then administered until either FEV1 decreased to 20% or
greater of the postsaline value or the highest available methacholine
Lung function and response to
concentration (16 mg/mL) was reached. The PC20FEV1 was calcu- allergen challenge
lated by means of linear interpolation of the FEV1 dose-response Patients treated with 250 and 500 mg of roflumilast
curve.13 experienced a significant attenuation of the LAR after
Clinical laboratory evaluations, electrocardiography, vital signs allergen challenge compared with those treated with
measurement, and physical examinations were performed at the
placebo. Treatment with 250 mg of roflumilast led to a
beginning and end of the study. Adverse events were assessed
throughout the study.
27% reduction in the LAR by means of AUC analysis
(P = .0110) compared with placebo, whereas 500 mg of
Statistical methods roflumilast led to a 43% (P = .0009) reduction compared
The primary efficacy variable of this study was inhibition of the with placebo (Table II). Therefore roflumilast-induced
LAR. Attenuation of the EAR and AHR were secondary end points. attenuation of the LAR showed a dose-related trend.
Pairwise tests were used to compare the effects of the 3 treatments on Significant attenuation of the LAR compared with placebo
the area under the curve (AUC) from 2 to 12 hours of the FEV1, was maintained for 12 hours after allergen challenge
TABLE II. Treatment differences in the LAR and EAR with AUC analysis
LAR AUC2-12h EAR AUC0-2h

Point estimate Point estimate
Roflumilast dose Patients,* n (95% CI) P value Reduction, % (95% CI) P value Reduction, %

Roflumilast, 250 21 20.148 .0110 27 20.146 .0038 25
mg, vs placeboà (20.272 to 20.024) (20.248 to 20.044)
Roflumilast, 500 mg, 19 20.243 .0009 43 20.179 .0046 28
treatment
vs placeboà (20.382 to 20.104) (20.307 to 20.050)
Roflumilast, 500 mg, 19 20.084 .1113 21 20.015 .3851 3
vs roflumilast, 250 mgà (20.223 to 0.056) (20.118 to 0.089)
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 treatment period, headache was reported by 4 (18%) of 22
resulted in a modest yet statistically significant inhibition patients, 6 (29%) of 21 patients, and 8 (35%) of 23 patients
of the maximum decrease in FEV1 during the LAR of 17% treated with placebo, 250 mg of roflumilast, and 500 mg of
(P = .0321) and 33% (P = .0002), respectively, compared roflumilast, respectively. The majority of headaches
with placebo (Table III). Both measures of lung function reported during the treatment period (73%) were consid-
(AUC and maximum decrease) demonstrated that roflu- ered by the investigator to be unlikely or not related to
milast inhibits bronchoconstriction associated with anti- study medication. The 6 adverse events associated with the
gen challenge. digestive tract were considered by the investigator to be
On the basis of the analysis of the AUC of FEV1, likely related to study medication, but no adverse events
patients treated with 250 and 500 mg of roflumilast also were judged definitely related. There were no clinically
experienced a statistically significant attenuation in the relevant changes in vital signs, electrocardiographic
EAR compared with placebo. Treatment with 250 mg of results, or clinical laboratory parameters.
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 DISCUSSION
were no statistically significant differences between the
250- and 500-mg roflumilast doses with respect to LAR Asthma is characterized by chronic inflammation of the
and EAR attenuation (Table II). There was a modest airways that might be responsive to treatment with PDE4
reduction of 14% in the maximum decrease in FEV1 during inhibitors. We have assessed the benefits of the PDE4
the EAR for both 250 and 500 mg of roflumilast compared inhibitor roflumilast in patients with mild asthma using an
with placebo (Table III). allergen challenge model. Roflumilast had only a modest
AHR was only slightly modified by roflumilast. During effect on the EAR but demonstrated a more pronounced
roflumilast treatment, the PC20FEV1 ratio was 1.0 or effect on the LAR. This suggests a potential anti-inflam-
greater, indicating that airway responsiveness did not matory role for roflumilast because it is believed that
increase despite the preceding allergen challenge (1.03 allergen-induced LARs are linked to an influx of inflam-
for 250 mg of roflumilast and 1.11 for 500 mg of matory cells and mediators associated with an inflamma-
roflumilast). The PC20FEV1 ratio was less than 1.0 tory response.14-16
(0.87) in patients treated with placebo, which reflects an In several in vivo and in vitro animal models, roflumi-
increase in AHR (Table IV). There was an apparent trend last has demonstrated multiple anti-inflammatory effects,
in doubling doses between roflumilast and placebo treat- including inhibition of inflammatory cell infiltration and
ment, but the difference between treatments did not reach reduction of TNF-a release in the lungs.11 In a previous
statistical significance. study in patients with exercise-induced asthma, 500 mg of
roflumilast reduced the decrease in FEV1 after exercise
Safety challenge by 41% and the median TNF-a levels by 21%
Roflumilast was well tolerated at both dose levels tested. versus placebo.17 Exercise-induced bronchoconstriction is
No serious adverse events or discontinuations because of regarded as an asthmatic airway reaction to nonspecific
adverse events occurred during the study. Most adverse stimuli.18,19 As a common characteristic of asthma, exer-
events were mild to moderate in intensity and were related cise-induced bronchoconstriction is an appropriate model
to the digestive tract (eg, diarrhea and gastrointestinal in which to study the efficacy of roflumilast; however,
disorder) or the nervous system (eg, headache). Headache alternative models are needed to elucidate the mechanism
was the most common adverse event. During baseline, of action of roflumilast in patients with asthma. The
6 (26%) of 23 patients reported headaches. During the allergen challenge model (particularly the LAR) is a
296 van Schalkwyk et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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 EAR
Point estimate Point estimate
Roflumilast dose (95% CI) P value Reduction, % (95% CI) P value Reduction, %
Roflumilast, 250 mg, 4.712 (0.427 to 8.997) .0321 17 4.015 (0.082 to 7.948) .0457 14
vs placeboà
vs placeboà
vs roflumilast 250 mgà
*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 tory actions, roflumilast might confer earlier benefits
airway inflammation and has previously been used to compared with corticosteroids and more comprehensive
assess the influence of anti-inflammatory medications on anti-inflammatory benefits compared with a cysteinyl
the EAR and LAR.20 The significant inflammatory com- leukotriene receptor antagonist.
ponent of the LAR has been attributed to an influx of The objective of this study was to evaluate the effect of
activated eosinophils and the release of cytokines and roflumilast by assessing its effect on the EAR and LAR in
chemokines that lead to the recruitment of additional patients with allergen-induced asthma. Roflumilast at
inflammatory cells, such as basophils, lymphocytes, and daily doses of 250 mg and 500 mg significantly dimini-
monocytes. In a similar challenge model montelukast and shed the LAR, confirming attenuation of allergen-induced
budesonide demonstrated a partial reduction of inflam- responses to single-dose administration of roflumilast
mation, as judged by changes in sputum inflammatory found in early studies.22 Roflumilast attenuation of the
cells; however, budesonide did not significantly reduce the LAR showed a dose-related trend: compared with pla-
EAR.21 As a targeted PDE4 inhibitor with anti-inflamma- cebo, 250 mg of roflumilast resulted in a mean attenuation
TABLE IV. PC20FEV1 ratios* by dose groupy airway muscle tone. Indeed, previous studies have shown
Placebo Roflumilast, Roflumilast,
that roflumilast does not have direct bronchodilatory
(n = 15) 250 mg (n = 18) 500 mg (n = 18) effects.29
Side effects have restricted the use of PDE inhibitors in
Mean PC20 0.87 1.03 1.11
asthma.20 Because the PDE4 inhibitors are more targeted,

FEV1 ratio
95% CI 0.55 to 1.36 0.59 to 1.81 0.67 to 1.85
they also tend to produce fewer adverse effects, such as
Mean doubling 20.20 0.04 0.15 nausea and headache. Roflumilast was well tolerated in
treatment
factor this study. Most side effects were mild to moderate in
95% CI 20.85 to 0.45 20.77 to 0.86 20.59 to 0.89 intensity. Headache, diarrhea, and mild nausea were the
most common adverse events and appeared to be dose
*Ratio of second visit of treatment period versus first visit of treatment period. dependent. All were transient and did not result in treat-
This table summarizes PC20FEV1 data for the per-protocol population.
ment discontinuation. In the current study the frequency of
headache is likely to be affected by the considerable
of 27%, whereas treatment with 500 mg of roflumilast proportion of patients who reported headache at baseline
compared with placebo resulted in a mean attenuation of (26%) and the high incidence of headaches in the placebo
43%. There was a greater degree of protection against group (18%). The incidence of adverse events in this study,
decreases in FEV1 during the LAR than the EAR, notably headache, was higher than that reported by studies
supported by both AUC and maximum decrease analyses. of the safety and efficacy of roflumilast conducted in larger
There was a more modest 14% reduction of the EAR with populations.30,31
250 and 500 mg of roflumilast, as determined by means of In conclusion, the current study demonstrates that
maximum decrease analysis; however, these data support roflumilast, a targeted PDE4 inhibitor, inhibits the LAR,
the AUC analysis, which is a more comprehensive exam- which might result from the anti-inflammatory activity
ination of the entire EAR and LAR. Roflumilast also has a of roflumilast. Further clinical studies with surrogate
modest effect on the EAR, which is mediated principally markers of inflammation (eg, induced sputum) are needed
by mast cell degranulation. Corticosteroids do not have a to confirm the anti-inflammatory effects of roflumilast.
significant effect on the EAR, which is likely because of Additionally, these data suggest that roflumilast shows
their lack of effect on mast cell degranulation.21,23 The promise as an oral, once-daily, steroid-free treatment for
early effect of roflumilast on the EAR might suggest a asthma. Additional studies to determine the clinical
different onset of anti-inflammatory effect than is achieved benefits of roflumilast in asthma and COPD are needed.
with corticosteroids. Although modest, the reduction of
the EAR by roflumilast might result from mast cell We thank our patients who participated in the study. The nursing
stabilization, mediator release, or both. Roflumilast had support of Dot Steyn and Wendy Lee is acknowledged, and we thank
more pronounced effects on the LAR, and the higher dose Marianne Koopman for her expert measurements of pulmonary
reduced responses by almost half. Because of the inflam- function.
matory processes underpinning the LAR, PDE4 inhibitors
might be particularly suitable to prevent cellular recruit-
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11. Bundschuh DS, Eltze M, Barsig J, Wollin L, Hatzelmann A, Beume R. In roflumilast on allergen challenge in asthmatics after a single dose
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active PDE4 inhibitor. J Pharmacol Exp Ther 2001;297:280-90. 23. Palmqvist M, Bruce C, Sjostrand M, Arvidsson P, Lotvall J. Differential
12. Chai H, Farr RS, Froehlich LA, Mathison DA, McLean JA, Rosenthal effects of fluticasone and montelukast on allergen-induced asthma.
RR, et al. Standardization of bronchial inhalation challenge procedures. Allergy 2005;60:65-70.
J Allergy Clin Immunol 1975;56:323-7. 24. Kidney JC, Boulet LP, Hargreave FE, Deschesnes F, Swystun VA,
13. Bardin PG, Dorward MA, Lampe FC, Franke B, Holgate ST. Effect of O’Byrne PM, et al. Evaluation of single-dose inhaled corticosteroid
selective phosphodiesterase 3 inhibition on the early and late asthmatic activity with an allergen challenge model. J Allergy Clin Immunol 1997;
responses to inhaled allergen. Br J Clin Pharmacol 1998;45:387-91. 100:65-70.
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14. Rossi GA, Crimi E, Lantero S, Gianiorio P, Oddera S, Crimi P, et al. 25. Larsen BB, Nielsen LP, Engelstatter R, Steinijans V, Dahl R. Effect of
Late-phase asthmatic reaction to inhaled allergen is associated with early ciclesonide on allergen challenge in subjects with bronchial asthma.
recruitment of eosinophils in the airways. Am Rev Respir Dis 1991;144: Allergy 2003;58:207-12.
379-83. 26. Pizzichini MMM, Kidney JC, Wong BJO, Morris MM, Efthimiadis A,
15. Niimi A, Amitani R, Yamada K, Tanaka K, Kuze F. Late respiratory Dolovich J, et al. Effect of salmeterol compared with beclomethasone on
response and associated eosinophilic inflammation induced by repeated allergen-induced asthmatic and inflammatory responses. Eur Respir J
exposure to toluene diisocyanate in guinea pigs. J Allergy Clin Immunol 1996;9:449-55.
1996;97:1308-19. 27. Weersink EJ, Aalbers R, Koeter GH, Kauffman HF, De Monchy JG,
16. Larsen GL, Wilson MC, Clark RA, Behrens BL. The inflammatory Postma DS. Partial inhibition of the early and late asthmatic response by a
reaction in the airways in an animal model of the late asthmatic response. single dose of salmeterol. Am J Respir Crit Care Med 1994;150:1262-7.
Fed Proc 1987;46:105-12. 28. Dente FL, Bacci E, Bartoli ML, Cianchetti S, Di Franco A, Giannini D,
17. Timmer W, Leclerc V, Birraux G, Neuhäuser M, Hatzelmann A, Bethke T, et al. One week treatment with salmeterol does not prevent early and late
et al. The new phosphodiesterase 4 inhibitor roflumilast is efficacious asthmatic responses and sputum eosinophilia induced by allergen
in exercise-induced asthma and leads to suppression of LPS-stimulated challenge in asthmatics. Pulm Pharmacol Ther 2004;17:147-53.
TNF-a ex vivo. J Clin Pharmacol 2002;42:297-303. 29. Engelstätter R, Wingertzahn M, Schmid-Wirlitsch C, Leichtl S,
18. Hofstra WB, Sont JK, Sterk PJ, Neijens HJ, Kuethe MC, Duiverman EJ. Bredenbröker D, Wurst W. Roflumilast, an oral, once-daily phosphodi-
Sample size estimation in studies monitoring exercise-induced broncho- esterase 4 (PDE4) inhibitor, does not exhibit bronchodilatory activity.
constriction in asthmatic children. Thorax 1997;52:739-41. Presented at: 2004 ACAAI Meeting; November 12-17, 2004; Boston,
19. Cockcroft DW. Nonallergic airway responsiveness. J Allergy Clin Mass.
Immunol 1988;81:111-9. 30. Leichtl S, Schmid-Wirlitsch C, Bredenbröker D, Rathgeb F, Wurst W.
20. Torphy TJ. Phosphodiesterase isozymes: molecular targets for novel Roflumilast, a new, orally active, selective phosphodiesterase 4 inhibitor,
antiasthma agents. Am J Respir Crit Care Med 1998;157:351-70. is effective in the treatment of asthma [abstract]. Eur Respir J 2002;
21. Leigh R, Vethanayagam D, Yoshida M, Watson RM, Rerecich T, Inman 20(suppl 38):303s.
MD, et al. Effects of montelukast and budesonide on airway responses 31. Izquierdo JL, Bateman ED, Villasante C, Schmid-Wirlitsch C,
and airway inflammation in asthma. Am J Respir Crit Care Med 2002; Bredenbröker D, Wurst W. Long-term efficacy and safety over one
166:1212-7. year of once-daily roflumilast, a new, orally active, selective, phospho-
22. Nell H, Louw C, Leichtl S, Rathgeb F, Neuhäuser M, Bardin PG. Acute diesterase 4 inhibitor, in asthma [abstract]. Am J Respir Crit Care Med
anti-inflammatory effect of the novel phosphodiesterase 4 inhibitor 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 J ALLERGY CLIN IMMUNOL
AUGUST 2005
11. Bundschuh DS, Eltze M, Barsig J, Wollin L, Hatzelmann A, Beume R. In roflumilast on allergen challenge in asthmatics after a single dose
vivo efficacy in airway disease models of roflumilast, a novel orally [abstract]. Am J Respir Crit Care Med 2000;161(suppl):A200.
active PDE4 inhibitor. J Pharmacol Exp Ther 2001;297:280-90. 23. Palmqvist M, Bruce C, Sjostrand M, Arvidsson P, Lotvall J. Differential
12. Chai H, Farr RS, Froehlich LA, Mathison DA, McLean JA, Rosenthal effects of fluticasone and montelukast on allergen-induced asthma.
RR, et al. Standardization of bronchial inhalation challenge procedures. Allergy 2005;60:65-70.
J Allergy Clin Immunol 1975;56:323-7. 24. Kidney JC, Boulet LP, Hargreave FE, Deschesnes F, Swystun VA,
13. Bardin PG, Dorward MA, Lampe FC, Franke B, Holgate ST. Effect of O’Byrne PM, et al. Evaluation of single-dose inhaled corticosteroid
selective phosphodiesterase 3 inhibition on the early and late asthmatic activity with an allergen challenge model. J Allergy Clin Immunol 1997;
responses to inhaled allergen. Br J Clin Pharmacol 1998;45:387-91. 100:65-70.
treatment
14. Rossi GA, Crimi E, Lantero S, Gianiorio P, Oddera S, Crimi P, et al. 25. Larsen BB, Nielsen LP, Engelstatter R, Steinijans V, Dahl R. Effect of
Late-phase asthmatic reaction to inhaled allergen is associated with early ciclesonide on allergen challenge in subjects with bronchial asthma.
recruitment of eosinophils in the airways. Am Rev Respir Dis 1991;144: Allergy 2003;58:207-12.
379-83. 26. Pizzichini MMM, Kidney JC, Wong BJO, Morris MM, Efthimiadis A,
15. Niimi A, Amitani R, Yamada K, Tanaka K, Kuze F. Late respiratory Dolovich J, et al. Effect of salmeterol compared with beclomethasone on
response and associated eosinophilic inflammation induced by repeated allergen-induced asthmatic and inflammatory responses. Eur Respir J
exposure to toluene diisocyanate in guinea pigs. J Allergy Clin Immunol 1996;9:449-55.
1996;97:1308-19. 27. Weersink EJ, Aalbers R, Koeter GH, Kauffman HF, De Monchy JG,
16. Larsen GL, Wilson MC, Clark RA, Behrens BL. The inflammatory Postma DS. Partial inhibition of the early and late asthmatic response by a
reaction in the airways in an animal model of the late asthmatic response. single dose of salmeterol. Am J Respir Crit Care Med 1994;150:1262-7.
Fed Proc 1987;46:105-12. 28. Dente FL, Bacci E, Bartoli ML, Cianchetti S, Di Franco A, Giannini D,
17. Timmer W, Leclerc V, Birraux G, Neuhäuser M, Hatzelmann A, Bethke T, et al. One week treatment with salmeterol does not prevent early and late
et al. The new phosphodiesterase 4 inhibitor roflumilast is efficacious asthmatic responses and sputum eosinophilia induced by allergen
in exercise-induced asthma and leads to suppression of LPS-stimulated challenge in asthmatics. Pulm Pharmacol Ther 2004;17:147-53.
TNF-a ex vivo. J Clin Pharmacol 2002;42:297-303. 29. Engelstätter R, Wingertzahn M, Schmid-Wirlitsch C, Leichtl S,
18. Hofstra WB, Sont JK, Sterk PJ, Neijens HJ, Kuethe MC, Duiverman EJ. Bredenbröker D, Wurst W. Roflumilast, an oral, once-daily phosphodi-
Sample size estimation in studies monitoring exercise-induced broncho- esterase 4 (PDE4) inhibitor, does not exhibit bronchodilatory activity.
constriction in asthmatic children. Thorax 1997;52:739-41. Presented at: 2004 ACAAI Meeting; November 12-17, 2004; Boston,
19. Cockcroft DW. Nonallergic airway responsiveness. J Allergy Clin Mass.
Immunol 1988;81:111-9. 30. Leichtl S, Schmid-Wirlitsch C, Bredenbröker D, Rathgeb F, Wurst W.
20. Torphy TJ. Phosphodiesterase isozymes: molecular targets for novel Roflumilast, a new, orally active, selective phosphodiesterase 4 inhibitor,
antiasthma agents. Am J Respir Crit Care Med 1998;157:351-70. is effective in the treatment of asthma [abstract]. Eur Respir J 2002;
21. Leigh R, Vethanayagam D, Yoshida M, Watson RM, Rerecich T, Inman 20(suppl 38):303s.
MD, et al. Effects of montelukast and budesonide on airway responses 31. Izquierdo JL, Bateman ED, Villasante C, Schmid-Wirlitsch C,
and airway inflammation in asthma. Am J Respir Crit Care Med 2002; Bredenbröker D, Wurst W. Long-term efficacy and safety over one
166:1212-7. year of once-daily roflumilast, a new, orally active, selective, phospho-
22. Nell H, Louw C, Leichtl S, Rathgeb F, Neuhäuser M, Bardin PG. Acute diesterase 4 inhibitor, in asthma [abstract]. Am J Respir Crit Care Med
anti-inflammatory effect of the novel phosphodiesterase 4 inhibitor 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.
Duration of postviral airway
hyperresponsiveness in children
with asthma: Effect of atopy

Paraskevi Xepapadaki, MD, PhD,* Nikolaos G. Papadopoulos, MD, PhD,* Apostolos
treatment
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 Abbreviations used
is an important risk factor for asthma persistence. AHR: Airway hyperresponsiveness
Objective: We sought to evaluate whether atopy is a risk NW: Nasal wash
factor for prolonged AHR after upper respiratory tract SPT: Skin prick test
infections (URIs). URI: Upper respiratory tract infection
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 Asthma is characterized by abnormalities in lung
first reported cold, a nasal wash specimen was obtained. function, variable airway obstruction, and airway hyper-
Methacholine provocation was performed 10 days and 5, 7, 9, responsiveness (AHR)1; among others, a significant cor-
and 11 weeks later. In case a new cold developed, the relation exists between the degree of AHR and both
provocation schedule was followed from the beginning. clinical severity and medication needs for asthma.2 A
Results: Viruses were detected in 17 (68%) of 25 patients
number of stimuli, including respiratory viruses, are able
during their first cold, with rhinovirus being most commonly
identified (82%). AHR increased significantly 10 days after the
to induce AHR.3 Increased AHR to histamine in healthy
URI, equally in both groups (P = .67), and remained so up to subjects after upper respiratory tract infections (URIs)
the fifth week. Duration of AHR in subjects experiencing a lasting up to 6 weeks was observed more than 20 years
single URI ranged from 5 to 11 weeks, without a significant ago.4 Human experimental infections with human rhino-
difference between groups. In the duration of the study, atopic viruses confirmed that increased AHR to nonspecific
children experienced more colds and asthma exacerbations stimuli is observed for up to 4 weeks after a viral infection
than nonatopic children. Thus for duration of AHR, in allergic subjects.5-7 Furthermore, AHR after viral
significant prolongation was noted in the atopic group when infections has been documented in animal models of
assessed cumulatively. paramyxovirus, respiratory syncytial virus, and influenza
Conclusion: In asthmatic children the duration of AHR after
virus infections.8-10 Most asthma exacerbations in chil-
a single natural cold is 5 to 11 weeks. However, an increased
rate of symptomatic cold and asthma episodes in atopic children
dren are associated with URIs, attributed in their majority
is associated with considerable cumulative prolongation of AHR, to rhinoviruses.11,12 Virus-induced AHR is increased in
which might help explain the role of atopy as a risk factor for atopic individuals compared with in healthy control
asthma persistence. (J Allergy Clin Immunol 2005;116: subjects.5,13 Moreover, atopy is one of the strongest risk
299-304.) factors for the development and persistence of asthma,
especially in childhood.14 More than 60% of asthmatic
Key words: Asthma, airway hyperresponsiveness, atopy 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
From the Allergy Unit, 2nd Pediatric Clinic, University of Athens.
There are suggestions that genes responsible for atopy and
*Drs Xepapadaki and Papadopoulos contributed equally to this work. AHR act together to develop the full asthma phenotype.17
Disclosure of potential conflict of interest: N. Papadopoulos has received Furthermore, it has been proposed that the defective
grants–research support from GlaxoSmithKline and AstraZeneca. epithelial repair cycle, which is characteristic of asthma
P. Saxoni-Papageorgiou has received grants–research support from
and strongly correlates to AHR, is amplified by exposure
Novartis and Schering-Plough. All other authors—none disclosed.
Received for publication November 11, 2004; revised March 28, 2005; to TH2 cytokines.18 Nevertheless, our current understand-
accepted for publication April 4, 2005. ing of the mechanisms connecting atopy, AHR, and
Available online May 24, 2005. asthma is still incomplete. The classical model of sensi-
Reprint requests: Paraskevi Xepapadaki, MD, PhD, UPC Research tization and exposure to particular allergens19 can only
Laboratories, 13, Levadias 11527, Goudi, Greece. E-mail: panvik@hol.gr.
0091-6749/$30.00
partly explain the epidemiologic observations. More
Ó 2005 American Academy of Allergy, Asthma and Immunology recently, we and others have shown that the response of
doi:10.1016/j.jaci.2005.04.007 atopic asthmatic individuals to rhinovirus infection is
299
300 Xepapadaki et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
defective, with a suboptimal TH1 response and a shift Statistical analysis

toward a TH2 phenotype.20,21 Such a response might lead Comparisons of baseline characteristics were performed with the
to incomplete viral clearance and inflammation persis- Fisher exact test for categoric variables and t tests for continuous
tence, potentially perpetuating AHR.22 variables. A common cold and an asthma exacerbation were defined
On the basis of the above, we hypothesized that atopy as an increase of the relevant symptom score totals over the personal
could be a risk factor for prolongation of AHR after median value for 2 days or more. The duration of each episode was
respiratory viral infections. The aim of this study was to defined as the time from symptom initiation to return to the median
treatment
prospectively evaluate the duration of AHR in children value. Severity of each was defined as the maximum symptom score.
The Wilcoxon signed-rank test and the Mann-Whitney U test were
with asthma after naturally occurring respiratory infections
used to compare independent and paired outcome variables, respec-
and assess the role of atopy as a risk factor in this respect. tively. The time to PC20 restoration was displayed in Kaplan-Mayer
curves compared with the log-rank test. P values of less than .05 were
METHODS considered significant.
Patients
Twenty-five children (16 boys), 7 to 12 years of age, with RESULTS
intermittent asthma and a history of disease exacerbations attributable Baseline characteristics
to URI only, were recruited for the study. Asthma diagnosis was
Table I shows the demographic, socioeconomic, and
based on history, 12% or greater improvement in FEV1 after
bronchodilation, and AHR (PC20 16 mg/mL). Classification of disease characteristics of the 2 groups, as obtained at
asthma severity was performed according to the Global Initiative for baseline. Atopic asthmatic children had statistically sig-
Asthma.23 Atopy was evaluated by using skin prick tests (SPTs) to a nificantly higher levels of total IgE (P = .04). A trend
panel of 18 locally relevant allergens performed outside the pollen toward more cases of positive family history of atopy was
season. Exclusion criteria were a history of seasonal or allergen- also noted in the atopic group (P = .08). In respect to all
driven symptoms, current or previous use of specific immunotherapy, remaining characteristics, the groups were homogeneous,
use of inhaled steroids within the previous 2 months, recent (<2 with no significant differences when statistically com-
months) URI, and chronic conditions potentially affecting airway pared. Baseline spirometric values did not differ between
responsiveness.24 All parents provided written consent, and the study
the 2 groups (Table I).
was approved by the hospital’s ethics committee.
Detection of respiratory viruses with PCR
Study design
All NW specimens obtained at baseline were negative
A prospective case-control design with 9 months’ follow-up
for the presence of respiratory viruses. PCR revealed the
(September-June) was used. Clinical evaluation, IgE determination,
SPT, and methacholine bronchial provocation were performed at presence of a virus in 17 (68%) of 25 patients during their
baseline. Questionnaires and a nasal wash (NW) specimen were also subsequent first URI. The most commonly identified virus
obtained. Patients were grouped according to the presence of atopy was rhinovirus (14/17 [82%]). Adenovirus was found in 4
(with n = 13). Children (or their parents) were asked to prospectively (23%) of 17 positive samples, whereas one child had both
complete diary cards with upper and lower respiratory tract rhinovirus and adenovirus. Virus identification rates did
symptoms.11 During the first reported URI, judged either subjectively not differ significantly between groups (nonatopic, 58%;
or by means of increased daily symptom scores (4), an NW was atopic, 77%; P = .34).
performed. Methacholine provocation was performed 10 days, and
5, 7, 9, and 11 weeks later. In case a new cold developed within this Duration of AHR
time period, the above provocation schedule was followed from All subjects completed the study. Methacholine re-
the beginning. Bronchodilators were used as relievers; in case sponsiveness at baseline was slightly, but not signifi-
of persistence or deterioration of asthma symptoms, subjects were
cantly, higher in atopic individuals (time = 0, atopic
instructed to receive systemic steroids, although these were not
needed on any occasion. 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
Methacholine provocation natural cold within the study period. AHR increased
Methacholine provocation was performed with the 2-minute significantly 10 days after the first reported URI, equally
breathing dosing protocol.25 The aerosols were generated with a in both groups (time = 10th day, atopic PC20 = 2.3 6 1.1
nebulizer output of 0.13 mL/min (Air Dynaval Taema). The proce- mg/mL, nonatopic PC20 = 2.6 6 0.9 mg/mL; P = .002 in
dure was discontinued if FEV1 decreased 20% or greater from comparison with their respective baseline values in both
baseline values or when a 16 mg/mL concentration had been cases and P = .67 between groups). This increase re-
administered.26 Spirometry was performed according to the American mained statistically significant up to the fifth week after
Thoracic Society guidelines.27
the onset of the cold, progressively decreasing, although
Virus detection still without statistically significant differences, between
the groups (time = fifth week, atopic PC20 = 3.7 6 1.3
RT-PCR was used for detection of viral RNA in NW samples, as
previously described.28 PCR reactions were performed for rhinovi- mg/mL, nonatopic PC20 = 4.7 6 1.3 mg/mL; P = .005
ruses; enteroviruses; respiratory syncytial virus; coronaviruses OC43 and P = .021, respectively, in comparison with baseline
and 229E; influenza viruses A and B; parainfluenza viruses 1, 2, and P = .29 between groups).
and 3; adenoviruses; Chlamydia pneumoniae; and Mycoplasma The above analysis was performed in subjects with
pneumoniae.29,30 no additional URI during that period (time = 0,
J ALLERGY CLIN IMMUNOL Xepapadaki et al 301
TABLE I. Patient characteristics: Comparison of baseline

characteristics between atopic and nonatopic asthmatic
children included in the study
Baseline Nonatopic Atopic

characteristics (n = 12) (n = 13) P value

Age (y) 10.4 6 1.8 10.3 6 1.2 NS
Sex (boys) 50% 77% NS
treatment
IgE (mg/dL) 78.9 6 14.5 339.4 6 117.1 .04
Residence (urban) 66% 54% NS
Density 1.5 1.2 NS
(persons per room)
Pet ownership 12% 13% NS
Exposure to tobacco 75% 62% NS
smoke
School type (public) 83% 85% NS
Paternal education 73% 69% NS
level (secondary
education)
Maternal education 58% 54% NS
level (secondary
education)
Father or mother with 60% 85% .08
atopy
Colds in the previous 3.5 3.6 NS
year (n)
Asthma exacerbations 3.3 3.3 NS
in the previous year (n)
Emergency visits for 2.3 2.7 NS
asthma in the previous
year (n) FIG 1. Linear regression model of AHR changes from the 10th day
Hospitalizations for 0.1 0 NS to the seventh week after a single URI in atopic (A) and nonatopic
asthma in the previous (B) children with intermittent virus-induced asthma. A significant
year (n) and time-dependent AHR decrease is observed in both cases. The
FVC (% mean) 6 SD 92.9 6 11.3 88.5 6 9.3 NS predicted time for AHR to return to baseline did not differ between
the groups.
FEV1 (% mean) 6 SD 81.4 6 6.7 78.2 6 7.4 NS
FVC, Forced vital capacity.

whereas only 9 (67%) of 13 of the atopic children returned
to their baseline PC20 value by day 200 (P = .0068),
n = 25; time = 10 days, n = 24; time = 5 weeks, n = 23). showing that in assessing the natural history of the disease,
After the fifth week, several children experienced addi- increased airway responsiveness, possibly caused by
tional colds, and therefore provocations were rescheduled repetitive colds, is considerably more prolonged in atopic
according to the study design; comparisons were not done asthmatic children and might in fact last more than
on fixed time points. Eleven weeks after the initial URI, 6 months.
10 children (3 atopic and 7 nonatopic children) had no
further cold, and their methacholine responsiveness had Common cold and asthma exacerbation
returned to their respective baseline value. The mean characteristics
duration of AHR in these subjects was 7.0 6 2.0 weeks Atopic children had generally more disease episodes
(median, 5 weeks; range, 5-11 weeks) for the atopic than nonatopic children (Fig 3). The average number of
children and 7.3 6 1.0 weeks (median, 7 weeks; range, colds that each subject experienced during the whole study
5-11 weeks) for the nonatopic children. In addition, using period was marginally higher in the atopic group (atopic
a simple linear regression model based on AHR values group, 4.6 6 0.4; nonatopic group, 3.5 6 0.4; P = .06).
from the 10th day on and including all subjects with a The average duration of each cold did not differ between
single URI, the predicted time for AHR to return to its groups (atopic group, 10.4 6 1.8 days; nonatopic group,
respective baseline value was 5.6 to 8.9 weeks for the 9.4 6 1.3 days; P = .81), as was the case for cold severity
atopic children and 6.7 to 10.2 weeks for the nonatopic (atopic group, 2.2 6 0.2; nonatopic group, 2.9 6 0.3;
children (Fig 1). P = .12).
However, important differences, as shown in Fig 2, Atopic asthmatic children experienced significantly
were observed when the cumulative duration of AHR was more exacerbations during the study period (5.1 6 0.6
assessed prospectively in the complete cohort. All 12 vs 3.2 6 0.3 of the nonatopic children, P = .01). There
nonatopic asthmatic children returned to their baseline were no differences in the duration or severity of asthma
PC20 values by day 120, after the first reported URI, exacerbations between the groups (duration: atopic group,
AUGUST 2005
TABLE II. Seasonal distribution of asthma exacerbation

characteristics in atopic and nonatopic asthmatic children
Number Duration (d) Severity (index)
Nonatopic
Winter 1.50 (0.55) 10.17 (15.21) 1.43 (0.55)

Spring 2.00 (1.26) 4.67 (1.63) 1.10 (0.33)
P value .52 .79 .29
treatment
Atopic
Winter 2.67 (1.50) 9.25 (4.80) 1.52 (0.53)
Spring 1.42 (0.67) 7.08 (4.89) 1.31 (0.35)
P value .02 .15 .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 DISCUSSION
difference is significant (P = .0068, survival analysis).
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-to-
moderate colds and very mild, if any, asthma exacerba-
FIG 3. Upper respiratory tract symptom exacerbations, colds, and tions.32 Furthermore, fixed time points for up to 8 weeks
asthma exacerbations experienced during a 9-month period in after infection have been used for outcome evaluation.7,33
atopic (open bars) and nonatopic (filled bars) children with inter- In this respect these studies excluded, to a considerable
mittent asthma. *P < .05, #P = .06.
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
8.5 6 0.9 days; nonatopic group, 11.4 6 2.3 days; our study, when the duration of AHR was assessed after a
P = .65; severity: atopic group, 4.4 6 0.8; nonatopic single infection, either in the subgroup of subjects who did
group, 4.6 6 0.9; P = .60). not have additional infection for up to 11 weeks, or by
No statistically significant differences were found in means of a simple regression model, it was 7 weeks on
either the duration or severity of symptom scores between average, ranging from 5 to 11 weeks. This time span is
patients with positive and negative virus identification still longer than previously reported in human subjects
(data not shown). (10 days to 6 weeks)4,34 and comparable with time spans
All atopic children had positive SPT responses to reported studies prospectively evaluating postviral AHR
common pollen allergens (with a wheal of 3 mm).31 in experimental animals (2-22 weeks).8,10,35,36 Neverthe-
One atopic child also presented with a positive SPT less, when AHR was assessed in the long term after
response to mites. To address the question of whether multiple colds, a considerable proportion of atopic sub-
prolonged AHR in atopic children might be affected by jects remained hyperresponsive for considerably longer,
exposure to allergens to which they were sensitized, we in some cases more than 6 months. This is consistent with
compared the characteristics of asthma exacerbations the notion that additional, naturally occurring colds might
during the pollen season (March-June) and the cold season further prolong AHR, as recently shown in an experimen-
(October-January). There were no differences in any such tal animal setting,37 and might prove important in consid-
characteristics within the nonatopic group (Table II). ering the natural history of the disease, as well as
However, within the atopic group, the number of asthma management of such patients. Therefore although our
exacerbations was significantly higher during the cold hypothesis that atopy might lead to postviral AHR
season, suggesting that exposure to allergens could not prolongation was shown not to be directly true because
account for additional disease burden in these patients there was no difference in AHR duration after a single
(Table II). infection, the total duration of AHR was in fact
J ALLERGY CLIN IMMUNOL Xepapadaki et al 303
significantly longer in atopic than nonatopic children and thelial cells derived from atopic subjects are more suscep-
that was associated with an increased number of colds, as tible to ex vivo rhinovirus infection than cells from healthy
well as asthma episodes. control subjects.51
Patient characteristics did not differ between the groups A weakness of the present study is that PCR viral
at baseline; it cannot be excluded, however, that subclin- identification was performed only for the first reported

ical inflammation might have been greater in atopic cold and not for subsequent colds. Nevertheless, it is
subjects, which were slightly, but not significantly, more without doubt that respiratory viral infections are the
treatment
hyperresponsive at baseline. cause of the common cold, whereas the association of
Several studies have shown that viral infection induces colds with subsequent asthma exacerbations is also well
greater changes in nonspecific AHR in patients with established.11,12
respiratory allergy than in healthy control subjects.5,7,38,39 The degree of airway responsiveness is indicative of
Accordingly, it has been assumed that after a viral asthma severity and counts as an indirect marker of airway
infection, the response to allergen exposure might be inflammation.2,52 In this respect prolongation of virus-
exaggerated, a notion elegantly confirmed by using induced AHR might well reflect persistent airway inflam-
segmental bronchial provocations with allergen.40,41 mation after multiple insults. Perpetuation of subclinical
Furthermore, clinical studies have shown synergy be- airway inflammation could have a substantial effect on the
tween viral infection and allergen exposure in sensitized risk of asthma persistence, relapse later in life, or both,
individuals.42,43 Nevertheless, other studies have shown providing a possible mechanism for the well-established
that allergen exposure does not necessarily augment virus- role of atopy as a major risk factor for the persistence of
mediated responses.44,45 This might also be the case in the asthma and AHR from childhood to adulthood.53,54
atopic population of our study, who, even though they In conclusion, the duration of AHR in children with
were sensitized to pollen allergens, had more symptoms intermittent virus-induced asthma ranges from 5 to 11
during the fall and winter months rather than during the weeks after a single infection but might be considerably
pollen season. This was not completely unexpected prolonged with repeated infections. Atopic subjects pre-
because all our subjects were selected for a postinfectious sent with more symptomatic colds and thus cumulatively
asthma phenotype during the 2 previous years, apparently have significantly more prolonged AHR. Prolongation of
reporting symptoms only after colds and not after allergen virus-induced AHR in atopic subjects might help explain
exposure. Certainly, the possibility that unidentified the well-established role of atopy as a risk factor for
allergens might have contributed to some exacerbations asthma persistence.
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
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Mechanisms of asthma and allergic inflammation
Dissecting asthma using focused transgenic

modeling and functional genomics
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
Mechanisms of asthma and

allergic inflammation
Background: Asthma functional genomics studies are aspects of asthma. (J Allergy Clin Immunol 2005;116:
challenging because it is difficult to relate gene expression 305-11.)
changes to specific disease mechanisms or pathophysiologic
features. Use of simplified model systems might help to address Key words: Asthma, IL-13, calcium-activated chloride channel,
this problem. One such model is the IL-13/Epi (IL-13–over- trefoil factor, 15-lipoxygenase, intelectin
expressing 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 Asthma results from a complex interplay between
epithelial cell. These mice develop airway hyperreactivity and genetic and environmental factors.1 Microarray studies
mucus overproduction but not airway inflammation. of lung tissue from subjects with asthma and from animals
Objective: To identify how effects of IL-13 on airway epithelial with experimental allergic asthma typically reveal hun-
cells contribute to gene expression changes in murine asthma dreds of genes that are differentially expressed in com-
models and determine whether similar changes are seen in parison with normal lung.2-4 However, it has been difficult
people with asthma.
to relate gene expression changes to specific mediators,
Methods: We analyzed gene expression in ovalbumin allergic
cell types, or pathophysiologic features.
mice, IL-13–overexpressing mice, and IL-13/Epi mice with
microarrays. We analyzed the expression of human orthologues Models that focus on one particular mediator or cell
of genes identified in the mouse studies in airway epithelial cells type or on specific pathophysiologic features might help
from subjects with asthma and control subjects. overcome this obstacle. We developed a transgenic mouse
Results: In comparison with the other 2 models, IL-13/Epi mice model to analyze effects of a single cytokine, IL-13, on
had a remarkably small subset of gene expression changes. a single cell type, the nonciliated airway epithelial cell.5
Human orthologues of some genes identified as increased in the IL-13 expression is increased in the airways of subjects
mouse models were more highly expressed in airway epithelial with asthma,6,7 and IL-13 is necessary and sufficient for
cells from subjects with asthma than in controls. These the development of experimental asthma.8-10 IL-13 activates
included calcium-activated chloride channel 1, 15-lipoxygenase,
the signaling molecule signal transducer and activator of
trefoil factor 2, and intelectin.
transcription factor 6 (STAT6) in many cell types.11
Conclusion: The combination of focused transgenic models,
DNA microarray analyses, and translational studies provides a Overexpression of IL-13 in the airways of mice with
powerful approach for analyzing the contributions of specific normal STAT6 expression (tg–IL-13 mice) causes mucus
mediators and cell types and for focusing attention on a limited overproduction, airway hyperreactivity, inflammation,
number of genes associated with specific pathophysiologic 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
From athe Department of Medicine, Allergy-Immunology Division,
Northwestern University Feinberg School of Medicine, Chicago; and bthe in nonciliated airway epithelial cells. These IL-13/Epi
Department of Medicine, cthe Lung Biology Center, dthe Division of mice developed mucus overproduction and airway hyper-
Pulmonary and Critical Care Medicine, ethe Cardiovascular Research reactivity but not inflammation, fibrosis, or emphysema.5
Institute, and fthe Program in Immunology, University of California San Here we apply a genomics-based approach to identify
Francisco School of Medicine.
Supported by National Institute of Health grants HL56835 and HL72301
gene expression changes in the IL-13/Epi focused model,
and by the UCSF Sandler Center for Basic Research in Asthma. the tg–IL-13 model, and a conventional allergic asthma
Disclosure of potential conflict of interest: D. A. Kuperman, none disclosed. model. Inclusion of the focused model allowed us to
C. A. Lewis, none disclosed. P. G. Woodruff, none disclosed. M. W. pinpoint a remarkably small number of gene expression
Rodriguez, none disclosed. Y. H. Yang, none disclosed. G. M. Dolganov,
changes that were consistently associated with airway
none disclosed. J. V. Fahy, none disclosed. D. J. Erle, none disclosed.
Received for publication January 6, 2005; revised February 28, 2005; accepted hyperreactivity and mucus production. Furthermore, we
for publication March 9, 2005. used these results to guide translational studies that show
Available online May 2, 2005. the relevance of some of these gene expression changes in
Reprint requests: David J. Erle, MD, UCSF Box 2922, San Francisco, CA people with asthma. These experiments demonstrate that
94143-2922. E-mail: erle@itsa.ucsf.edu.
0091-6749/$30.00
focused transgenic models combined with microarrays
Ó 2005 American Academy of Allergy, Asthma and Immunology can lead to an improved understanding of the pathogenesis
doi:10.1016/j.jaci.2005.03.024 of complex diseases such as asthma.
305
306 Kuperman et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
Microarray analysis
Abbreviations used Lung gene expression was analyzed by hybridizing Cy5-labeled
GAPD: Glyceraldehyde-3-phosphate dehydrogenase cDNA from mouse lungs (5 mice per group, each hybridized
IL-13/Epi: IL-13–overexpressing transgenic mice with separately) along with Cy3-labeled reference lung cDNA pooled
STAT6 expression limited to epithelial cells from wild-type mice. Tracheal perfusate samples were analyzed
tg–IL-13: IL-13–overexpressing transgenic mice similarly, except that amplified cRNA from each mouse was com-
STAT6: Signal transducer and activator of transcription pared with an amplified cRNA reference pool made by using tracheal
factor 6 perfusate samples from wild-type mice. DNA microarrays used
UCSF: University of California San Francisco in these experiments were produced by using the Operon Bio-
technologies (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://
METHODS www.ncbi.nlm.nih.gov/geo, accession number GSE1438).

Mice We used an approach that allowed us to estimate differential gene
The University of California San Francisco (UCSF) Committee on expression between the various groups we studied on the basis of
Animal Research approved the use of mice for these experiments. linear models, as previously described.14,15 To determine whether
Care and use of animals complied with the United States Public Health there were significant differences in gene expression between groups,
Service’s Policy on Humane Care and Use of Laboratory Animals by we calculated the odds ratio (probability of being differentially
Awardee Institutions (#3400-01). Three groups of transgenic mice expressed/probability of not being differentially expressed). When
were used in these experiments: (1) CC10-IL-131, Stat61/2 mice (tg– the log2 of the odds ratios (known as the B-statistic) was greater than
IL-13 mice); (2) CC10–IL-131 Stat62/2 mice; and (3) CC10–IL-131, 0, we classified the gene as differentially expressed.16,17 Hierarchical
Stat62/2, CC10-hStat61 mice (IL-13/Epi mice). CC10 refers to the clustering, a method for grouping together genes with similar
Clara cell specific promoter used to express IL-13 and human STAT6 expression patterns, was performed by using Acuity 4.0 software
(hSTAT6).12 The development and characterization of these trans- (Axon Instruments, Union City, Calif). In addition, genes were
genic mice have been previously described.5 Mice used here were classified on the basis of expression patterns to determine whether
backcrossed 5 times onto the Balb/c genetic background. tg–IL-13 they were increased relative to controls in 1, 2, or all 3 of the
mice have an intact Stat6 gene and IL-13–driven activation of STAT6 experimental groups (ovalbumin, tg–IL-13, and IL-13/Epi) as fol-
in a wide range of cells in the lung, resulting in airway inflammation, lows. Seven pseudogene vectors were created to represent genes that
mucus overproduction, airway hyperreactivity, subepithelial fibrosis, were increased only in 1 of the 3 models ([0,0,1]; [0,1,0]; and [1,0,0]),
and emphysema. IL-131 Stat62/2 mice (used as negative controls) genes increased equally in 2 models ([0,1,1]; [1,0,1]; [1,1,0]), and
lack STAT6 and did not develop any detectable IL-13–induced lung genes increased equally in all 3 models ([1,1,1]). Each differentially
pathology. IL-13/Epi mice also lack mouse STAT6 but express expressed gene was assigned a vector in 3-dimensional space
human STAT6 selectively in airway epithelial cells. hSTAT6 is according to the median log fold-change gene expression values
functional in mice, and epithelial-restricted activation of Stat6 by determined for that gene in the 3 experimental groups. All vectors
IL-13 induced airway hyperreactivity and mucus production without were scaled to unit length, and each gene was matched to the closest
airway inflammation, subepithelial fibrosis, or emphysema. Wild-type pseudogene, as determined by Euclidean distance.
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.
Human subjects
These studies were approved by the UCSF Committee on Human
Antigen sensitization and challenge Research and conducted in compliance with the Declaration of
Mice were sensitized by intraperitoneal administration of 50 mg Helsinki principles. Written informed consent was obtained from all
grade V ovalbumin mixed with adjuvant (10 mg aluminum potassium subjects. All subjects were adult nonsmokers (,10 pack-year total
sulfate) 3 times at weekly intervals. Control mice received adjuvant smoking history with last cigarette .1 year before the study). Medical
alone. Beginning 1 week after the last injection, mice were challenged histories were obtained, physical examinations were performed,
3 times by intranasal administration of ovalbumin (1 mg in 50 mL symptom questionnaires were collected, and spirometry was per-
PBS) at daily intervals. Control mice were challenged with PBS formed as described.18 Airway reactivity was measured by deter-
alone. Tissues were harvested for isolation of RNA 24 hours after the mining the PC20.19 The 28 healthy control subjects had no history of
last challenge. lung disease and were not hyperreactive (PC20 . 16 mg/mL). All 30
subjects with asthma had a previous physician diagnosis of asthma,
Isolation and labeling of RNA from mice were hyperreactive (PC20 , 8 mg/mL), and used only short-acting
Whole-lung RNAs were purified by using Trizol (Invitrogen, inhaled b-adrenergic–agonist medications for therapy. Individuals
Carlsbad, Calif). Integrity of all RNA samples used in this study was with an asthma exacerbation or respiratory infection within the
confirmed with a model 2100 bioanalyzer (Agilent Technologies, Inc, previous 6 weeks or significant medical problems other than asthma
Palo Alto, Calif). Cy3-labeled and Cy5-labeled lung cDNAs were and those using inhaled or systemic corticosteroids or leukotriene
prepared as described.13 To obtain samples enriched for airway antagonists were excluded.
epithelial cell RNA, the superior portion of the trachea was
cannulated, and the trachea and proximal major bronchi were excised Bronchial epithelial brushings
from the thorax and slowly perfused with 0.35 mL lysis buffer Bronchoscopy was performed, and bronchial brushings were
(RNeasy kit; Qiagen Inc, Valencia, Calif). RNA was isolated from obtained randomly from right or left lower lobe bronchial segments
perfusate according to the manufacturer’s instructions. Because the by using 4 disposable cytology brushes. The brushes were gently
amount of RNA obtained from tracheal perfusates was only ~1 mg, a vortexed in sterile saline. Cells from all brushes were pooled, yielding
T7 RNA polymerase-based method13 was used to prepare Cy3- a single sample for each subject. An aliquot was removed for
labeled and Cy5-labeled amplified cRNAs for array hybridizations. cytocentrifugation, stained with Diff-Quik (Baxter, McGraw Park,
J ALLERGY CLIN IMMUNOL Kuperman et al 307

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 FIG 2. Gene expression patterns. Grouping revealed genes with
Air-liquid interface cultures were established by using published 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)
protocols.20 Primary normal human bronchial epithelial cells (lot
all 3 models. The number of genes (left) and representative genes
3F1191; Cambrex Bio Science, Baltimore, Md) were seeded onto
(right) is shown for each group. Phenotypic attributes of each
12-mm–diameter Corning Transwells (Corning, NY) containing model are shown at the bottom. AHR, Airway hyperreactivity.
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
RESULTS
modified Eagle media to bronchial epithelial growth media) for Microarray analysis of gene expression in
8 days. After the differentiation period, IL-13 (0-10 ng/mL) was mouse asthma models
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 We used DNA microarrays to analyze gene expression
RNeasy kit. 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
Analysis of gene expression by real-time PCR
was composed of mice with transgenic overexpression of
Primers and a probe for mouse genes (see Table E1 in the Journal’s IL-13 in the lung (tg–IL-13 model); these mice had an
Online Repository at www.mosby.com/jaci) and human genes (see
intact gene for STAT6, a key signaling molecule required
Table E2 in the Online Repository at www.mosby.com/jaci) were
designed by using Primer Express software (Perkin Elmer, Boston,
for IL-13 activity. The third model was composed of IL-13
Mass). First-strand cDNA synthesis and PCR were performed by transgenic mice with STAT6 expression limited to non-
using ABI Prizm 7700 or 7900 Sequence Detection Systems (Applied ciliated airway epithelial cells (IL-13/Epi model). In
Biosystems, Foster City, Calif). For mouse lung and cultured human whole-lung samples, 805 gene transcripts were differen-
airway epithelial cell studies, cycle thresholds for each gene were tially expressed (increased or decreased) in at least 1 model
normalized to those of glyceraldehyde-3-phosphate dehydrogenase (Fig 1). Of these, 509 genes were increased or decreased
(GAPD). For human bronchial brushing samples, a 2-step PCR by 2-fold in at least 1 model. There were 583 gene
approach21 was used, and transcript copy numbers were normalized expression changes in the ovalbumin model and 351
on the basis of the geometric mean expression values of 3 house- changes in the tg–IL-13 model. In contrast, only 18
keeping genes (GAPD, elongation factor 1a1, and cyclophilin A) as
changes were seen in the IL-13/Epi focused transgenic
described.22 To identify genes that were differentially expressed
between subjects with asthma and normal subjects, we performed a
model. Comparison of the focused transgenic model to the
Wilcoxon rank-sum test and calculated the corresponding adjusted other models allowed for the identification of a small
P value by using the Westfall and Young23 maxT algorithm subset of gene expression changes that are attributable to a
implemented in Bioconductor’s multtest package (Bioconductor; specific mechanism and also are relevant to pathogenesis
open source software for bioinformatics, www.bioconductor.org).24 in complex systems.
AUGUST 2005
TABLE I. Genes induced by allergen and by direct effects of IL-13 on airway epithelial cells*
Fold-change by arrays Fold-change by PCR

Symbol Description Ova tg–IL-13 IL-13/Epi Ova tg–IL-13 IL-13/Epi
Clca3 Chloride channel calcium activated 3 78.3 59.8 28.2 820.3 1530.7 1448.2
Slc26a4 Solute carrier family 26, member 4 9.9 29.8 20.0 18.7 40.1 10.3
Retnla Resistin-like a 4.3 23.8 23.7 ND ND ND
Itln Intelectin 7.6 10.8 19.2 9.5 73.8 146.2
Nadsyn NAD synthetase 1 17.2 12.5 6.4 0.7 0.5 0.8
Atp2a1 Ca11 transporting ATPase 15.3 8.0 3.9 0.4 0.1 0.9
AMCase Acidic mammalian chitinase 2.5 12.3 8.6 3.7 7.4 4.4
Muc5ac Mucin 5, subtypes A and C 12.1 6.8 3.8 33.5 23.4 7.8
Agr2 Anterior gradient 2 3.8 7.7 4.3 11.1 21.6 10.2
Tff1 Trefoil factor 1 3.3 6.4 5.7 52.6 592.2 224.7

Reg3g Regenerating islet-derived 3g 6.4 4.1 3.2 ND ND ND
Pigr Polymeric immunoglobulin receptor 2.5 8.4 2.3 1.5 12.3 2.2
Muc5b Mucin 5, subtype B 4.0 5.1 2.8 3.6 7.4 13.2
D630002J15Rik RIKEN cDNA D630002J15 gene 2.8 4.3 2.0 ND ND ND
Scin Scinderin 2.8 2.9 3.0 21.9 21.8 34.5
Alox15 15-lipoxygenase 3.9 1.9 2.0 5.2 1.6 2.8
H2-Q7 Histocompatibility 2, Q region locus 7 2.4 2.1 2.1 ND ND ND
1110001D15Rik RIKEN cDNA 1110001D15 gene 2.1 2.1 2.4 ND ND ND
Tff2 Trefoil factor 2 4.5 4.5 1.8 3.5 8.6 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 of gene transcripts is consistent with the idea that
Control Subjects
effects of IL-13 on these cells are important in allergic
subjects with asthma P inflammatory responses.8,9 A total of 73 transcripts were
increased in the tg–IL-13 model but not increased (or
Number 28 30 —
increased to a much lesser extent) in the other 2 models
Age 36 6 8 38 6 13 NS
Sex 16 F/12 M 18 F/12 M NS
(Fig 2, C). These represent genes that are induced by
PC20 (mg/mL) 60.8 6 1.4 0.9 6 1.3 ,.0001 prolonged high-level overexpression of IL-13 but not by
FEV1/forced vital 80.6 6 1.4 71.1 6 1.4 ,.0001 acute allergen challenge and may be involved in the
capacity (%) pathogenesis of subepithelial fibrosis and emphysema,
FEV1 (% predicted) 106.5 6 13.2 85.2 6 13.9 ,.0001 pathologic features present in the tg–IL-13 model but not
the other 2 models.
*Values represent means 6 SDs.
We were especially interested in identifying genes that
were increased in all 3 models. Analysis of whole-lung
To aid in interpretation, we grouped transcripts accord- samples revealed 35 genes that had similar fold increases
ing to the magnitude of change in expression across the in all models (Fig 2, D). Because some epithelial gene
3 models. Transcripts were grouped by determining whether expression changes might have been undetectable in
the expression changes were seen in 1, 2, or all 3 of whole lung samples, we also analyzed gene expression
the models (see Table E3 in the Online Repository at in tracheal perfusate samples enriched for airway epithe-
www.mosby.com/jaci). There were 17 transcripts with at lial cell RNA. In these samples, we detected 276 genes
least 2-fold increases in the IL-13/Epi model but only 3 with altered expression (increased or decreased) in at least
transcripts with at least 2-fold decreases; therefore, we 1 model (see Table E4 in the Online Repository at
focused our subsequent analyses on increased genes. A www.mosby.com/jaci). Of these, 107 genes were changed
total of 239 transcripts were increased in the ovalbumin by 2-fold. There were 43 genes changed in the ovalbu-
model but not induced (or induced to a much lesser extent) min model and 239 in the tg–IL-13 model. There were 76
in the tg–IL-13 or IL-13/Epi models (Fig 2, A). These genes changed in the IL-13/Epi model, but only 22 of
changes are likely largely attributable to allergen-induced these were changed by 2-fold or more. Some genes had
lymphocyte activation and to production of mediators similar expression changes in both analyses, but many
other than IL-13. There were 247 genes increased simi- other changes were identified only in lung samples or only
larly in the ovalbumin and tg–IL-13 models but not in the in tracheal samples. In part, this reflects the fact that the
IL-13/Epi model (Fig 2, B). These allergen-induced genes tracheal sample was enriched for large airway epithelial
are apparently increased because of IL-13 effects on cells cells, whereas the whole-lung sample contained smaller
other than nonciliated airway epithelial cells, and the large airway epithelial cells as well as many other cell types. In
TABLE III. Gene expression in airway epithelial cells from control subjects and subjects with asthma
Median gene copy number

Fold
Symbol Description Difference* Control Asthmatic Adjusted P
4 4
Clca1 Chloride channel calcium activated 1 216.5 0.06 3 10 8.21 3 10 .0001
Itln Intelectin 3.4 1.73 3 105 6.12 3 105 .0002
Alox15 15-lipoxygenase 1.3 2.28 3 106 3.27 3 106 .06
Tff2 Trefoil factor 2 2.7 0.55 3 104 1.00 3 104 .06
Muc5ac Mucin 5, subtypes A and C 1.5 1.67 3 107 2.07 3 107 NS
Tff1 Trefoil factor 1 1.4 1.47 3 105 2.17 3 105 NS
Agr2 Anterior gradient 2 1.1 0.90 3 107 1.04 3 107 NS

Slc26a4 Solute carrier family 26, member 4 0.8 3.72 3 104 3.35 3 104 NS
Scin Scinderin 1.0 4.70 3 105 4.99 3 105 NS
Muc5b Mucin 5, subtype B 25.0 1.84 3 106 0.42 3 106 .0001
AMCase Acidic mammalian chitinase — NDà ND —
*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 tra-
cheal 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 FIG 3. Intelectin (Itln) expression in human airway epithelial cells.
expression of this gene. A, Intelectin gene expression by airway epithelial cells from control
subjects and subjects with asthma. Medians and interquartile
PCR validation of gene expression changes ranges are shown. B, IL-13 treatment of cultured human airway
in mouse models epithelial cells induced increased expression of intelectin tran-
scripts.
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 IL-13 induces intelectin expression in cultured
transcripts listed in Table I did not have obvious human human airway epithelial cells
orthologues. Eleven of the remaining 14 transcripts were One of the transcripts substantially increased in airway
increased by at least 2-fold in both ovalbumin allergic and epithelial cells from subjects with asthma was intelectin, a
IL-13/Epi mice by PCR (Table I). Our mouse modeling pattern recognition molecule that has not been previously
approach therefore resulted in the selection of 11 genes implicated in asthma. IL-13 treatment of cultured primary
that could be studied in people with asthma. human bronchial epithelial cells led to a large increase in
intelectin transcripts (Fig 3, B), demonstrating that IL-13
Airway epithelial gene expression in human
directly increases intelectin expression and that other cell
subjects with and without asthma
types are not required.
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 DISCUSSION
substantial and highly significant increases in the expres-
sion of the calcium activated chloride channel Clca1 We combined focused transgenic modeling, functional
(orthologue of murine Clca3) and intelectin in subjects genomics, and translational studies in human subjects to
with asthma (Table III and Fig 3, A). There were smaller help understand important aspects of the complex patho-
increases in expression of 15-lipoxygenase and trefoil genesis of asthma. As expected, microarray analysis of
factor 2 in subjects with asthma. One transcript, mucin 5b, an allergic model of asthma revealed hundreds of gene
was less abundant in subjects with asthma, and other expression changes. Analysis of the tg–IL-13 model,
transcripts examined were not significantly different. where IL-13 acts on many different cell types to produce
We were unable to consistently detect acidic mammalian extensive lung pathology, also showed hundreds of
chitinase transcripts in either group by using 2 different changes, many of which were similar to those seen in
sets of PCR primers and probes. the allergic model. By using the data from these 2 models,
AUGUST 2005
it would have been extremely challenging to determine We did not find significant increases in expression of
how particular gene expression changes relate to specific the other human homologues that we examined. To some
disease mechanisms or to select a reasonable number of extent, this likely reflects difficulties inherent in detecting
candidate genes for further study. By analyzing a third gene expression changes in limited samples (epithelial
model (IL-13/Epi) that focused on the effects of a single brushings) taken from human subjects with stable mild
mediator, IL-13, on a single cell type, the airway epithelial to moderate disease, as opposed to whole-lung analysis
cell, we were able to generate a dramatically shorter list of of mice with homogeneous genetic backgrounds housed
differentially expressed genes. These expression changes in a controlled environment and subjected to a potent
were associated with airway hyperreactivity and mucus disease-inducing stimulus. One transcript, Muc5b, was
overproduction, which are features of all 3 models, but not significantly increased in each of the mouse models, but
with inflammation, fibrosis, or emphysema, which are expression of both the transcript and the protein is de-
absent in the IL-13/Epi model. creased in people with asthma (Woodruff et al, unpub-
Our approach was designed to reduce the complexity lished data). Differences in Muc5b expression might relate
inherent in microarray studies of disease pathogenesis. to the fact that human Muc5b is expressed predominantly
Other microarray studies have reduced complexity by in submucosal glands.35 These glands are abundant in
analyzing homogeneous populations of cultured cells or human airways but are absent in the mouse, with the
cells isolated from tissues by laser capture microscopy or exception of a single gland in the trachea. This is an
flow-cytometric sorting. Although those approaches can example of the limits of mouse modeling. Our PCR assay
be useful, an important advantage of the focused trans- was unable consistently to detect the transcript for another
genic model approach that we describe here is that it orthologue, AMCase, but a recent report demonstrated
isolates the effects that a single mediator exerts on a single that airway epithelial production of AMCase is also
cell type in vivo. increased in subjects with asthma.36 Including transla-
We used translational studies to determine whether the tional studies helped to clarify the relevance of the animal
gene expression changes first identified by using mouse models to human disease and allowed us to draw novel
models were also seen in human disease. Four of the inferences about the activity of a specific mechanism in
orthologues identified in the models (the chloride channel human disease.
Clca1, intelectin, 15-lipoxygenase, and trefoil factor 2) We conclude that the combination of focused trans-
were increased in airway epithelial cells from people with genic models, DNA microarray analyses, and translational
asthma. In another study involving a subset of the human studies provides a powerful approach for analyzing the
subjects used for this study, we found that expression of contributions of specific mediators and cell types and for
Clca1 transcripts and protein was increased in airway focusing attention on a limited number of genes associated
epithelial cells from subjects with asthma (Woodruff et al, with specific pathophysiologic aspects of complex dis-
unpublished data). Previous reports suggest that induction eases like asthma.
of the mClca3/hClca1 chloride channel in airway epithe-
lial cells is important for mucus production.25-27 15- We thank Dean Sheppard and Andrea Barczak for their advice and
Lipoxygenase produces 15S-hydroxyeicosatetraenoic acid, Xiaozhu Huang, Louis Nguyenvu, Michael Salazar, and the staffs of
which has been reported to trigger mucus secretion in dogs,28 the Sandler Center Animal Physiology and Microscopy Core and the
promote contraction of human airway smooth muscle,29 UCSF National Heart, Lung, and Blood Institute Shared Microarray
and potentiate increases in allergen-induced early Facility for technical assistance.
asthmatic responses in human beings.30 Trefoil factor
2 was shown to be increased in an allergic mouse model
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phosphatidylinositol 3-kinase regulation. Am J Physiol Lung Cell Mol 35. Sharma P, Dudus L, Nielsen PA, Clausen H, Yankaskas JR, Hollings-
Physiol 2003;285:L730-9. worth MA, et al. MUC5B and MUC7 are differentially expressed in
21. Dolganov GM, Woodruff PG, Novikov AA, Zhang Y, Ferrando RE, mucous and serous cells of submucosal glands in human bronchial
Szubin R, et al. A novel method of gene transcript profiling in airway airways. Am J Respir Cell Mol Biol 1998;19:30-7.
biopsy homogenates reveals increased expression of a Na1-K1-Cl- 36. Zhu Z, Zheng T, Homer RJ, Kim YK, Chen NY, Cohn L, et al. Acidic
cotransporter (NKCC1) in asthmatic subjects. Genome Res 2001;11: mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway
1473-83. activation. Science 2004;304:1678-82.
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
Background: Adenosine 5 monophosphate (AMP) has been

shown to cause bronchoconstriction and a sensation of chest Abbreviations used
tightness when inhaled by asthmatic subjects. This response is AMP: Adenosine 5# monophosphate
attenuated after repeated inhalation of bradykinin, suggesting CGRP: Calcitonin gene related peptide
that AMP may act in part by the release of neuropeptides. NEP: Neutral endopeptidase
Objective: This study examined neuropeptide release in the NKA: Neurokinin A
human airway after endobronchial AMP challenge. NKB: Neurokinin B
Methods: Endobronchial AMP challenge was performed in NPK: Neuropeptide K
20 subjects and tachykinin levels were measured after PC20 AMP: Provocative concentration of AMP causing a
endobronchial AMP challenge and after placebo endobronchial 20% fall in FEV1
challenge with saline. SP: Substance P
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 Atopic asthmatic and non-asthmatic subjects cough and
(P=.37). bronchoconstrict in response to stimuli such as inhaled
Conclusion: This study demonstrates that endobronchial AMP AMP and sulphur dioxide; however, asthmatics tend to
challenge causes tachykinin release in the human airway respond to lower concentrations.6 Cough and chest tight-
in vivo. (J Allergy Clin Immunol 2005;116:312-7.) ness are both common symptoms in asthma and are related
to stimulation of sensory nerves.6
Key words: Tachykinin, neuropeptide, adenosine, endobronchial Evidence exists that while AMP acts mainly via primed
challenge
mast cells, the agent also stimulates vagal nerves. Pre-
treatment with ipratropium (an anti-cholinergic agent) has
Adenosine is a naturally occurring purine nucleoside a bronchoprotective effect on the response to AMP,
that functions as a constituent of nucleic acid, as an suggesting activation of cholinergic nerves in the response
intracellular and autocoid mediator.1 Elevated levels have to AMP.7,8 Inhalation of AMP and bradykinin cause a
been found in bronchoalveolar lavage fluid of asthmatic greater sensation of chest tightness than does inhalation of
subjects as compared to normal subjects, suggesting that methacholine, for the same degree of bronchoconstriction,
adenosine may be a mediator in asthma.2 Inhalation of suggesting that the former acts on sensory pathways.9
adenosine monophosphate (AMP), which is rapidly Repeated inhalation of bradykinin attenuates the response
dephosphorylated to adenosine in vivo,3 causes broncho- to inhaled AMP suggesting that both of these agents act in
constriction in atopic asthmatic and non-asthmatic sub- part via liberation of neuropeptides from sensory nerves.10
jects but not in non-atopic, non-asthmatic subjects.4 The Hong et al11 have shown that pulmonary C fibers (the
related nucleoside guanosine has no effect, suggesting that nerve fibers containing neuropeptides) in the rat are
this is a specific receptor mediated effect.5 activated after right atrial injection of adenosine, impli-
cating these nerves in the response to AMP in this model.
The purpose of this study was to examine neuropeptide
From aRespiratory Research Group, School of Medicine, Queen’s University release in vivo in the human airway after endobronchial
of Belfast and Departments of bClinical Biochemistry and Metabolic AMP challenge.
Medicine and cMedicine, Queens University, Belfast.
Funding: Northern Ireland Chest Heart and Stroke Association.
Received for publication November 18, 2004; revised March 10, 2005;
METHODS
accepted for publication March 28, 2005.
Available online June 17, 2005. Subjects
Reprint requests: Liam G. Heaney, MD, MRCP, Regional Respiratory Centre,
Level 8, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, UK.
Ethical approval was granted by the Research Ethics Committee
BT9 7AB. E-mail: l.heaney@qub.ac.uk. of the Queen’s University of Belfast. All subjects gave written
0091-6749/$30.00 informed consent. All subjects were non-smokers and had not
Ó 2005 American Academy of Allergy, Asthma and Immunology received any anti-histamines or inhaled or oral steroids in the preceding
doi:10.1016/j.jaci.2005.03.034 six months. Asthmatics were recruited if they (1) had a prior clinical
312
J ALLERGY CLIN IMMUNOL Crummy et al 313
diagnosis of asthma and a history of intermittent shortness of breath TABLE I. Visual analogue reaction for grading of response
or wheeze, (2) were atopic (reacted to at least one allergen on skin after endobronchial challenge
prick testing), and (3) had FEV1 greater than 60% predicted. All other
subjects had no symptoms suggestive of asthma; atopic non-asthmatic Analogue
subjects had at least one positive skin prick test as aforementioned. score Reaction observed
All subjects attended on two occasions. At the screening visit, 0 No reaction
informed consent was obtained and clinical assessment, skin prick 1 Subject coughs after instillation of AMP
testing, and AMP inhalation challenge were performed. At the (no coughing after saline challenge)
subsequent visit (at least 72 hours after screening visit), bronchos- 2 Subject coughs/immediate bronchial pallor
copy and endobronchial AMP challenge were performed. then hyperemia/increased mucus secretion
after instillation of AMP
Skin prick testing 3 Bronchoconstriction observed after instillation

Skin prick testing was performed using a standardized puncture of AMP
technique,12 using allergen preparations of house dust mite, cat, and
dog protein and grass pollen (Dome-Hollister-Stier, Epernon Cedex, Adapted from Polosa et al.1
France). A positive reaction was taken as a wheal size of 3 mm or
administered endobronchial dose being 400 mg/mL. There was
more.
a time lapse of 3 minutes after each concentration given to observe
for any visual reaction using the analogue outlined in Table I. The
Inhalational challenging endobronchial challenge was terminated either when there was a
Spirometry was performed according to American Thoracic visible reaction to AMP, when the maximum concentration of
Society Guidelines13 using a Vitalograph spirometer (Buckingham, AMP had been administered or when it was necessary to terminate
UK). AMP (Sigma-Aldrich Ltd., Poole, UK) was freshly prepared in the challenge for reasons of patient comfort. Three minutes after the
0.9% saline in doubling concentrations ranging from 0.391 mg/mL to final concentration of adenosine had been administered, a further
400 mg/mL. The AMP provocation test was performed using the two- bronchial wash of 20 mL of saline was performed and aspirated
minute tidal breathing method of Cockcroft et al14 using a Medix after a minimum dwell time. Subjects remained under observation for
Turbonebuliser (Leicestershire, UK) with an output of 0.13 mL/min. a period of at least two hours after the procedure.
PC20 AMP was calculated by linear interpolation.
Processing of samples
Bronchoscopy A total cell count was measured using a modified Neubauer
AMP was freshly made up on the morning of the bronchoscopy in hemocytometer and was expressed as the number of cells 3105/mL
0.9% saline from a stock solution of 400 mg/mL. At bronchoscopy, of BAL. Cell viability was assessed by Trypan blue exclusion
subjects were given intravenous Midazolam (up to 14 mg) to achieve staining. Viable cells are expressed as a percentage of total cell
mild sedation and the hypopharynx was anaesthetised using 4% numbers. Samples were centrifuged at 200 3 g for 10 minutes at 4°C
lignocaine spray. Vocal cord and tracheal anaesthesia was achieved to separate any debris and added to a protease inhibitor cocktail
using 4 mL of 4% lignocaine introduced trans-cricoidally. Oxygen (see Appendix) and stored at 270°C for subsequent analysis.
was routinely applied at 2 L/min via nasal cannulae. Heart rate,
ECG, and oxygen saturations were monitored throughout the Neuropeptide measurement
procedure. The bronchoscope (240 IT Olympus Optical Co. Ltd. NKA was measured using radioimmunoassay, utilizing a
Tokyo, Japan) was introduced orally and 2-mL aliquots of 2% N-terminal specific anti-serum that was raised in guinea pigs to
lignocaine were used as necessary to anesthetize the airways below synthetic human NKA (Amersham Bioscience UK Ltd product
the carina to suppress coughing. number IM168, Buckinghamshire, UK). It cross-reacts fully with
The site of the subsequent endobronchial challenge was rando- NKB and NPK but less than 0.1% with SP. The detection limit for the
mized prior to bronchoscopy. Subjects were randomly assigned a assay is 2 ng/L.
number, which determined the site of the active challenge to either CGRP immunoreactivity was measured using a commercial
the right middle or upper lobes, and randomization was constrained CGRP human radioimmunoassay (RIA) kit (catalogue number
to achieve balance. The placebo challenge was automatically RIK009, Peninsula Laboratories, San Carlos, Calif). This antibody
assigned to the opposite site from the active challenge. is a rabbit anti-human CGRP peptide (II) antibody. The label was
125
The bronchoscope was initially wedged in a segmental orifice of I-Tyr0-CGRP (catalog number Y6011). The limit of detection for
the site randomized for the placebo challenge and a baseline bronchial this assay is 2 ng/L and the antibody cross-reacts 100% with human
wash with 20 mL of saline was performed and aspirated back after CGRP (II), human CGRP, and rat CGRP. It cross-reacts <0.001%
minimum dwell time. A placebo challenge of 5 mL of saline was with rat calcitonin C-terminal adjacent peptide and less than 0.02%
administered to the same segment and the segment closely observed with insulin, glucagon, somatostatin, SP, vasoactive intestinal pep-
for any visible reaction. After 3 minutes, a second bronchial wash tide, and gastrin releasing peptide.
using 20 mL of saline was performed and aspirated back after Substance P (SP) was measured using a commercially available
minimal dwell time. ELISA (catolog number DE1400, R&D Systems, Minneapolis,
The active (AMP) challenge was then performed in the other site. Minn). It shows no significant cross-reactivity with NKA, neurokinin
Again a baseline bronchial wash was performed using 20 mL saline B (NKB), and neuropeptide K (NPK). The limit of detection of this
and immediately aspirated back under gentle suction. Then the active assay is 8 pg/mL.
challenge with 5 mL AMP was performed. The initial AMP con- For radioimmunoassays, lavage fluid was extracted using a
centration administered was one tenth that which caused a 20% fall in previously validated technique.16 In brief, cleared plasma and
FEV1 on the prior inhalational challenge or if the subject had been traysolol were added to equal volume of lavage fluid followed by
unresponsive to adenosine one tenth of the maximum concentration precipitation of large molecular weight proteins in 60% alcohol and
during the inhalation challenge (400 mg/mL). Up to two subsequent the sample centrifuged (30 min, 3 1500 g). Thiomersal was added to
AMP doses were given at quadrupling concentrations, the maximum the supernatant. This was then decanted, the extract was evaporated to
314 Crummy et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
TABLE II. Demographic details of subjects
Normals (N) Atopics (AT) Asthmatics (AS)
Number 7 6 7
Age (y), Median (IQR) 21 (21-24) 21 (21-24) 26 (21-30)
Sex (n = male) 4 3 2
FEV1% predicted, Mean (SD) 114.0 (8.4) 99.8 (14.2) 90.0 (14.8)
FVC% predicted, Mean (SD) 97.7 (11.0) 99.2 (15.4) 95.6 (16.4)
PC20 AMP (mg/mL), Median (IQR) 640.0 413.9 (144.8-640.0) 3.6 (2.3-7.9)
Maximum dose of endobronchial AMP (mg/mL), Median (range) 160.0 (40.0-400.0) 40.0 (10.0-160.0) 1.3 (0.15-10.0)
Number of aliquots of endobronchial AMP, Median (range) 2 (1.0-3.0) 11 (1.0-2.0) 11 (1.0-3.0)
dryness and the sample assayed. For SP ELISA, samples were Sep- 12.5 mg/mL, 326 mOsm; 40 mg/mL, 403 mOsm; 100
pakked (C18 Sep-pak; Waters, Milford, Mass) and eluted using a mg/mL, 567 mOsm; 400 mg/mL, 1292 mOsm.
60:40 solution of acetontirile and 1% trifluoro-acetic acid, dried down Endobronchial responses to AMP challenge are shown
and reconstituted in buffer prior to assay. Using these extraction and
in Figure 1. Instillation of endobronchial AMP led to
assay methods, peptide recovery was >90%. We have previously
immediate coughing in all subjects after administration of
characterised NKA, SP, and CGRP immunoreactivity in bronchoal-
veolar lavage fluid using HPLC, confirming it to be target peptide.17 adenosine, which was not seen after instillation of the
saline placebo challenge. However, there was no evidence
Measurement of osmolality of of generalised lung involvement (eg, wheeze, hypoxia)
AMP solutions in response to the endobronchial AMP challenge in any
subject.
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
As immediate coughing was observed in all groups,
(Advanced Micro-osmometer 3300; Advanced Instruments Inc, for the purposes of analysis all subjects were considered
Pomona, Calif). together. There was a significant increase in median (IQR)
NKA and substance P levels [10.0 (5.0–15.0) vs. 20.0
Statistical analysis (10.6–25.0) pg/mL, P < .01 and 227. 8 (176.9–278.6) vs.
All statistical analysis was performed using SPSS version 11.0 318.1 (190.6–422.9) pg/mL, P < .01] respectively when
(Statistical Package for Social Sciences, Chicago, Ill). All data were post-saline and post-AMP levels were compared (Fig 2).
tested for normality of distribution using Shapiro-Wilk W tests. There was no significant change in CGRP levels (P=.37,
Where the data were skewed values are quoted as median and inter- Fig 2). There was no significant difference between
quartile range (IQR), unless otherwise stated. For non-parametric groups in NKA, CGRP, or substance P levels at baseline
data, comparisons were made between paired samples using the or post-challenge.
Wilcoxon analysis and for unpaired samples using the Mann- There was no correlation between the change (post-
Whitney U test. Comparison between more than two groups were
AMP–post-saline) in NKA and substance P after AMP
performed using Kruskall-Wallis analysis. Post hoc multiple com-
challenging (r = 0.27, P=.25) or between the change
parisons were then performed to demonstrate the underlying statis-
tical differences (Stats Direct, Cambridge, UK). Non–parametric in CGRP and substance P (r = 0.25, P=.34). There was
correlations were calculated using Spearman’s rank correlations. no significant correlation between the change in NKA or
Subjects who did not achieve at least a 20% drop in FEV1 after substance P and the PC20 AMP for the group as a whole
inhaling 400 mg/mL of AMP were given an assigned PC20 value of (r = 0.09, P=.71, r = 0.26, P=.31 respectively). There was
640 mg/mL for statistical analysis. P values <.05 were regarded as no difference between the changes in NKA observed
statistically significant. between those who bronchoconstricted after endobron-
chial challenge (visual reaction 3) and those who did not
RESULTS (P=.30).
A total of 24 subjects were recruited. Three subjects

(1 normal, 1 atopic, and 1 asthmatic) did not complete DISCUSSION
the endobronchial challenge. Twenty-one subjects com-
pleted the endobronchial challenge protocol (1 sample This study provides the first evidence of in vivo
was inadequate for processing). The demographic details tachykinin release after a chemical airway challenge in
for the remaining 20 subjects (7 normals, 6 atopic humans. During the study it was observed that many of the
non-asthmatics, and 7 atopic asthmatics) are shown in subjects who were sedated and who did not cough during
Table II. There was no difference between the groups in the placebo saline challenge or baseline lavage at the
terms of age or baseline spirometry. active challenge site, started to cough immediately after
Measurements of osmolality of saline and AMP solu- the instillation of endobronchial AMP. In three cases this
tions showed the following: 0.9% saline, 281 mOsm; coughing induced by AMP was believed to be distressing
0.39 mg/mL, 282 mOsm; 3.125 mg/mL, 297 mOsm; enough to necessitate termination of the procedure.

FIG 1. The frequency of endobronchial response to AMP challenge in normals, atopics, and asthmatics.
The immediacy of the cough after AMP challenge those of non-asthmatics, the asthmatics in this study were
suggests that the cough is mediated by afferent sensory mild both in terms of airway hyperresponsiveness and
nerves. It has previously been shown that AMP acts on treatment requirements and may not reflect differences
vagal nerves.7,8,9 The co-existent increase in NKA and that may occur with more significant asthma.
substance P suggest the mechanism may involve stimu- There was no correlation between the changes in NKA,
lation of sensory C fibres with antidromic release of CGRP, or substance P. This is perhaps not surprising in
tachykinins. the case of NKA, as this neuropeptide may be located
Stimulation of sensory C fibers and release of neuro- independently of the other two peptides.22 However, it is
peptides have previously been implicated in cough. It is surprising that there was no correlation between the
known that inhalation of capsaicin acting through the changes seen in substance P and CGRP because they are
VR-1 receptor causes cough.18,19 Capsaicin also liberates often co-localized in the same neurones. There are a
neuropeptides from sensory nerves and after repeated number of possible explanations for this discrepancy.
inhalation the cough diminishes, possibly due to depletion While neuropeptides are co-localized the exact amounts
of neuropeptides and suggesting propagation of cough present may vary depending on the location of the tissue.23
by neuropeptides in this model. There is evidence, in There may also be differential paths of degradation.
animal models, that AMP can act directly on pulmonary Substance P and NKA are primarily degraded in the
C fibers11; however, the exact receptor involved seems to airways by the enzyme neutral endopeptidase (NEP).
vary between species. In rats it was found that adenosine However, the exact degradation pathway of CGRP has
potentiated the response of pulmonary C fibers to chem- not yet been elucidated. It has been shown that CGRP is
ical stimuli and that this response was attenuated by pre- subject to degradation by tryptase while the tachykinins
treatment with an A1 receptor antagonist20; while in NKA and substance P are not.24 Most of the subjects in
guinea pig lung A2 agonists decreased the release of SP this study had tryptase release after endobronchial AMP
from pulmonary C fibers.21 Thus, it is possible in this challenging,15 which may explain why there was no
study that AMP was acting directly on nerve endings to difference in CGRP levels after AMP challenge and also
cause cough via a central mechanism rather than directly why there is no relationship between CGRP and SP levels.
by neuropeptide release, although this potential mecha- In addition, it may be that differential release accounts for
nism has not been well studied in the human airway. the patterns of neuropeptide release observed. The relative
The coughing after endobronchial AMP challenge and amount of neuropeptide released may be dependent on
subsequent release of tachykinins was observed across all the frequency with which the nerve is stimulated as well as
groups of subjects. While it has been suggested that the the quantity of each peptide in the nerve ending.25 How-
nerves of asthmatics may be more sensitive to stimuli than ever, the precise mechanisms remain to be established.
316 Crummy et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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 neuro-
peptide release31 and mannitol challenge in asthmatics
causes cough that is independent of bronchoconstric-
tion.32 Osmolality of the adenosine concentrations dem-
onstrated that the challenges used in normal subjects were
relatively hyperosmolar compared to normal saline/
plasma; however, this was not the case for the concen-
trations 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 asth-
matic 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 mecha-
nisms 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 endobron-
chial AMP challenge causes immediate cough and sig-
nificant 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.
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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
Background: Psychologic stress has major effects on many

organs and cellular systems. The hypothalamic-pituitary- Abbreviations used
adrenal axis, corticotropin-releasing factor (CRF), mast cells, a-Helical CRF: a-Helical CRF–(9-41)
and nerves have all been shown to be involved in intestinal CRF: Corticotropin-releasing factor
epithelial responses to stress. There has been little information Isc: Short-circuit current
in the literature on stress and the lung. PD: Potential difference
Objective: To investigate Wistar rat tracheal epithelial SP: Substance P
responses to acute water avoidance stress (1 hour). WAS: Water avoidance stress
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 was statistically evident for as long as 5 to 7 weeks.3
evidence for tracheal mast cell activation and degranulation Indeed, it has become increasingly evident in recent years
was found after stress. Stress and CRF injection both enhanced that the effects of acute and chronic stress on physiologic
responses to substance P, but these effects were not inhibited by function can be important in the initiation, development,
a-helical CRF. and/or perpetuation of many human diseases.4 The intes-
Conclusion: The data suggest that acute stress affects tracheal
tine has long been thought of as a target for stress. The role
epithelium and sensitizes it to enhanced responses to substance P,
of stress as a determinant in intestinal disease is well
partly through mast cell activation. Many but not all of these
effects are mediated by CRF. These results offer the possibility established both experimentally and clinically5-7 and may
that stress may be involved in inflammatory diseases of the lung well be relevant to the study of asthma.8 Irritable bowel
such as asthma. (J Allergy Clin Immunol 2005;116:318-24.) syndrome, as in asthma, is characterized by smooth
muscle hyperreactivity.9 The major prospective determi-
Key words: Mast cell, hypothalamic-pituitary-adrenal axis, short- nant for irritable bowel syndrome after acute infectious
circuit current, corticotropin-releasing factor, substance P diarrhea is accompanying poor psychosocial circumstan-
ces.10 Activation of mast cells colocalized with enteric
The role of stress in asthma is a controversial subject, nerves11,12 is a significant hallmark of disease. Finally,
which is reflected by the literature on this subject.1 A experimental acute and chronic stress causes intestinal
recent investigation of college students clearly showed hypersecretion, permeability, mucin release, and smooth
that stress associated with examinations enhanced several muscle hyperreactivity.5-7,13-16 These effects seem to be
outcome measures such as sputum eosinophils, eosino- mediated peripherally by neuropeptides such as substance
phil-derived neurotoxin levels, and IL-5. The authors P and neurotensin, as well as by the autonomic nervous
concluded that stress could act as a cofactor with inhaled system, and centrally by corticotrophin-releasing factor
antigen to enhance inflammation and asthma severity.2 (CRF). Despite this evidence for stress effects on the
Asthma exacerbations in children increase significantly intestine, little attention has been paid experimentally to
within 1 to 2 days of a major stressful event. This effect the possible effects of stress on lung function,17 although
the effect of stress on the development and expression of
From aDivision of Foods, National Institute of Health Sciences, Tokyo; bthe
atopy has begun to receive attention.18 This report
Department of Dermatology, Gunma University Graduate School of represents one of the first such attempts. In it we show
Medicine, Maebashi; and cthe Departments of Medicine and Pathology that acute stress, mediated in part through the action of
and Molecular Medicine, McMaster University and the Brain-Body CRF, has a stimulatory effect on ion secretion by rat
Institute, St Joseph’s Healthcare, Hamilton.
tracheal epithelium, and is accompanied by evidence for
Dr Akiyama and Dr Amano contributed equally to this work.
Supported by the Medical Research Council of Canada and St Joseph’s mast cell activation and degranulation. At the same time,
Hospital Foundation, Hamilton, Ontario, Canada. both stress and CRF prepare (sensitize) tissues for en-
Received for publication November 22, 2004; revised March 21, 2005; hanced subsequent responses to substance P (SP). These
accepted for publication March 28, 2005. studies may have important implications for the role of
Available online May 24, 2005.
Reprint requests: Hiroshi Akiyama, PhD, Division of Foods, National Institute
stress in lung diseases such as asthma.
of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501
Japan. E-mail: akiyama@nihs.go.jp.
METHODS
0091-6749/$30.00
Ó 2005 American Academy of Allergy, Asthma and Immunology Details of the methods can be found in the Journal’s Online
doi:10.1016/j.jaci.2005.03.040 Repository (www.mosby.com/jaci).
318
J ALLERGY CLIN IMMUNOL Akiyama, Amano, and Bienenstock 319
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.
Stress model
Water avoidance stress (WAS) was chosen as the model of stress.

This model appears to have similar effects and modes of action
FIG 1. Effect of WAS on tracheal baseline Isc. Rats were subjected
insofar as intestinal responses and the hypothalamic-pituitary-adrenal to WAS or sham stress for 1 hour or injected intraperitoneally with
axis are concerned.5,19 Stress sessions were performed between 1000 CRF (50 mg/kg) or a-helical CRF (250 mg/kg) 30 minutes before
and 1300 hours to minimize diurnal variations in response. 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)
and then washed 3 times in the same buffer. Samples were postfixed
(a-helical CRF), a CRF 1 and 2 antagonist (Peninsula Lab, Belmont,
in 2% osmium tetroxide for 1 hour, dehydrated in graded ethanol,
Calif), were dissolved in PBS according to the manufacturer’s
and embedded in Spurr resin. The sections were photographed at a
instructions, aliquoted, and kept frozen at 270°C until used. The
magnification of 30003 in a transmission electron microscope
neutral endopeptidase inhibitor, phosphoramidon, was obtained from
(JEOL 1200 EX, Tokyo, Japan). At least 20 mast cells from each
Sigma Chemical Co (St Louis, Mo) and dissolved in PBS. SP (Peninsula
rat were analyzed by 2 investigators who were blind to the
Lab) was dissolved in PBS, aliquoted, and stored at 0°C before use. All
experiments. Interobserver error did not vary by more than 3%.
drugs were used fresh. Immediately before the experiments, the drugs
Activation of mast cells was defined by the presence of granules with
were diluted in PBS to the appropriate concentrations.
altered or absent electron-dense content.
Ussing chamber experiments Fecal pellet output
Trachea. The trachea was removed, immersed in 37°C oxygen- The number of fecal pellets expelled by each rat per hour was
ated Krebs buffer, opened along the anterior margin, and immediately used as an indirect measure of colonic motility, as previously
mounted in Ussing chambers. Ussing chambers measure current described.13,20
across living tissue. In the case of tracheal tissue, the current
measured reflects chloride ion secretion by the epithelium. Tissue Experimental design and pharmacological
was mounted in an Ussing chamber and the spontaneous potential treatments
difference (PD) clamped at 0 volts (WP Instruments automated volt-
age clamp; WP Instruments, Nacro Scientific, Mississauga, Ontario, A pilot study showed that colonic epithelial abnormalities were
Canada). The injected current, the short-circuit current (Isc in mA/ maximal at 1 hour after initiation of WAS. The rats were injected
cm2), required to maintain 0 PD, was continuously measured and intraperitoneally with CRF (50 mg/kg) 1 hour before mounting the
indicates net active ion transport. At intervals, the PD across the tissue tissue. Others had previously determined that this dose was maximal
was measured to allow calculation of tissue ion conduction (indicates in intestinal Isc responses.5 The dose (250 mg/kg) of a-helical CRF
net passive ion flux across the preparation) using the Ohm law and the has been shown to block intestinal responses to WAS effectively5 and
PD and Isc values. Baseline values for Isc, an indicator of ion was injected intraperitoneally 30 minutes before the WAS protocol.
secretion, and G, an indicator of ion permeability, were measured 15
minutes after mounting the tissues, and then every 5 minutes for 40
Measurement of serum corticosterone
minutes. The tissue was considered equilibrated after 15 minutes. Levels were determined by the HPLC method of Wong et al21 with
Abnormal baseline values of G > 50 millisiemens/cm2 were consid- some minor modifications.
ered damaged and were excluded.
Colon. Tissue was treated as described elsewhere.5 Statistics
The data were expressed as means 6 SEMs. A P value of less
Tracheal responses to SP in vitro than .05 was considered significant. Differences between the values were
Substance P in amounts of 10 mL to a final concentration of tested by using the Student t test or the Scheffe method after ANOVA.22
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. RESULTS
The Isc was calculated as the difference between the baseline Isc Corticosterone levels
value and the maximum value within 10 seconds of addition of SP. In
all experiments, phosphoramidon (final concentration: 1025 mol/L) For corticosterone levels, see Fig E1 in the Online
was added to the chamber buffer 10 minutes before adding SP. Repository (www.mosby.com/jaci). The results from the
HPLC standards were linear over a dose range of 10 to
Electron microscopy 1000 ng/mL carbon-treated serum. Control values in
The tissue was fixed in 2% glutaraldehyde in 0.1 mol/L sodium normal rats were 102.6 6 19.5. It was evident that the
cacodylate buffer (pH 7.4) for 2 hours at room temperature, washed, sham animals were undergoing mild stress, because their
left overnight at 4°C in 0.2 mol/L sodium cacodylate buffer, pH 7.4, levels were 270 6 56.1, which differed significantly from
320 Akiyama, Amano, and Bienenstock J ALLERGY CLIN IMMUNOL
AUGUST 2005
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 physiology. The time point of 1 hour was chosen to
(785.1 6 121.8) differed significantly both from sham examine the effect of CRF on the trachea because the
(P < .01) and control (P < .01) groups. The responses to an stress exposure lasted for 1 hour. We saw no difference
intraperitoneal injection of CRF (50 mg/kg) in PBS 1 hour at 2 or 4 hours from that seen at 1 hour. There was
before mounting the tissues in Ussing chambers were no significant difference between any of the groups
912.6 6 201.8, which again differed significantly from in conductance values (see Table E1 in the Online
control (P < .01). The injection of a-helical CRF before Repository at www.mosby.com/jaci). Pretreatment with
the stress or CRF protocol reduced the levels to approx- a-helical CRF reduced the Isc seen in stress to the levels
imately the same as those of the sham (234.2 6 29.4). This of control and sham (P < 0.05). The effects of a-helical
level, however, differed significantly from the control CRF (42.8 6 6.4) alone did not differ significantly
values (P < .05). between control (43.5 6 1.9) and sham (41.8 6 3.4)
groups. No changes in conductance were seen as a result
Defecation during stress and response of treatment with a-helical CRF in any of the groups
to a-helical CRF examined.
See Fig E2 in the Online Repository (www.mosby.com/
jaci). Measurement of the number of fecal pellets per hour Effects of stress and CRF on SP-induced
corresponded to values expected in controls (0.5 6 0.3), tracheal epithelial Isc
sham (1.1 6 1.0), or stress (6.4 6 0.7) and those animals We wished to establish whether stress sensitized
pretreated with a-helical CRF (3.8 6 1.0). The number tissues for responses to other agonists such as SP. SP
seen in the stress group differed significantly from both effects on the generation of tracheal epithelial Isc have
control and sham groups (P < .01), whereas the animals been shown by others to be effective only if delivered on
pretreated with a-helical CRF had a reduced but not the luminal side.23-25 We confirmed these results (Fig 2)
significantly reduced number of pellets. and established that the effect of SP at a concentration
of 1026 mol/L was optimal on tissue equilibrated for
Effects of stress, CRF, and a-helical CRF 15 minutes. No differences were seen with or without the
on colonic epithelial physiology addition of phosphoramidon. As seen in Table I, both
See Fig E3 in the Online Repository (www.mosby.com/ stress (P < .01) and sham stress (P < .05) significantly
jaci). Baseline Isc responses 15 minutes after mounting increased the response to standardized amounts of SP
were approximately the same in control (30.7 6 6.3) and compared with controls. Similarly, CRF injection (50 mg/
sham groups (37.7 6 6.1). WAS significantly increased kg) intraperitoneally 1 hour before the experiment sig-
Isc relative to sham (P < .05) and control groups (P < .01). nificantly increased responsiveness of the tissue to SP
Intraperitoneal injection of CRF (25 mg/kg in saline) (P < .01). No changes in conductance were seen at any
confirmed that this produced significant increases in Isc time in these experiments.
responses. a-Helical CRF had no effect on colonic Isc5
and reduced the effects of stress to baseline.5 Effects of a-helical CRF on stress
and CRF effects
Effects of stress, CRF, and a-helical CRF Results of experiments with a-helical CRF are shown
on tracheal epithelial physiology in Fig 3. a-Helical CRF, which in earlier experiments at
Stress caused increases in Isc compared with both sham this concentration (250 mg/kg) was effective in reducing
(P < .05) and control groups (P < .01; Fig 1). The effect of stress effects, had no effect on the sensitization induced
intraperitoneal injection of CRF (50 mg/kg) 1 hour before by stress for subsequent SP responses. Surprisingly, the
the tissue was mounted in the Ussing chamber was control experiment of injection of a-helical CRF by itself
significant compared with sham (P < .01) and control sensitized the trachea equally as well as stress or CRF so
(P < .01) groups. This concentration has been shown that there were no significant differences compared with
by others5 to have maximal effects on colon epithelial control between the stress, CRF, a-helical CRF plus
TABLE I. Effect of stress and intraperitoneal CRF

injection on tracheal responses to SPy
DIsc (mA/cm2)
Control 47.9 6 4.4

Sham 105.1 6 21.7*
Stress 107.1 6 6.4**
CRF 129.5 6 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.
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, and a-helical CRF groups. Pretreatment with stress for 1 hour or injected intraperitoneally with CRF (50 mg/kg).
One hour after the initiation of stress, 90 minutes after a-helical
a-helical CRF before injection with CRF did not reduce
CRF, or 1 hour after CRF injection alone, tracheas were mounted
the enhanced response to SP. In fact, the response of this in Ussing chambers. SP (1026 mol/L) was added to the luminal
group showed a significant enhancement over CRF itself side of the chamber at 50 minutes after mounting. Bars represent
(P < .05), but not over the a-helical CRF alone. means 6 SEMs; **P < .01, *P < .05 vs control; P < .05 vs CRF.
Effects of stress on mast cell morphology

peripheral systemic administration.5-7,37,38 These effects
Acute stress caused mast cell activation (Table II) and
are mostly mediated through mast cell activation, because
in some cases degranulation (Fig 4). There were signifi-
pharmacological inhibition of mast cell degranulation
cant differences between control and stressed animals
prevents many of the consequences of acute stress,5,6,32
(P < .05) in the trachea as well as in the colon (P < .01).
and mast cell deficient rats fail to exhibit these find-
Nevertheless, some loss of density of granule contents
ings.14,15 Mast cell–nerve interactions and communica-
was also found in both colon and trachea of control
tion11,39,40 have been shown to be involved in the
animals. If this criterion is used to establish activation,
generation of a variety of intestinal and lung physiologic
an average of 22% of total mast cell granules in various
responses to antigen.16,25,39-41 These interactions35 appear
normal tissues, such as skin, ileum, heart, and cranial
to be crucial for the generation of many of the functional
dura mater,26-30 show signs of activation under normal
intestinal effects of stress, because SP antagonists6 block
physiologic conditions.
these effects. They are also inhibited by cholinergic,
adrenergic, and autonomic ganglion blockade through
DISCUSSION the use of a variety of well defined pharmacologic
agents.5
To the best of our knowledge, this is the first report of At no time did we see any increase in conductance, a
the effects of psychological stress on lung epithelial reflection of the integrity of the tracheal epithelium,
physiology. We have shown that in Wistar rats, acute whereas this is invariably seen as a stress response in the
water avoidance stress caused increased tracheal epithelial intestine.5,34 This probably reflects a general difference
Isc activity, and that this was accompanied by morpho- between trachea and intestine, because antigen exposure
logic evidence of mast cell activation and degranulation, of tracheal tissue from sensitized rats, despite causing
was mimicked by systemic CRF injection, and could be large increases in Isc25 and mast cell degranulation,21
largely reversed by a CRF receptor competitive inhibitor, never showed increased conductance.25,30
a-helical CRF. Stress effects are complex end results of a range
There are many reports now that have focused on of interacting signals between a variety of systems and
the effects of different forms of acute or chronic psy- cells of different types. Indeed, the results obtained with
chological stress on various intestinal physiologic param- a-helical CRF inhibition of stress on corticosterone levels
eters.6,7,14,15,20,31,32 These include experimental results, are instructive, because the levels of corticosterone were
obtained mostly16 (but not exclusively) with animal tis- reduced only to those found in sham animals, and still
sues, and include effects on fluid or ion secretion and differed significantly from those of controls. This suggests
absorption, permeability,5,15,23,33,34 motility,6,13,20 and that factors in addition to CRF and its pathway are
pain perception.32 Tissue responses have been shown to operative in the production of corticosterone and not
vary according to the experimental animal, strain, and inhibited by this antagonist of CRF receptors 1 and 2.42,43
tissue source, ie, stomach,35 jejunum,15,31,33 ileum,36 and Indeed, behavioral changes apparently mediated by CRF
colon.5,6,13,32 1 receptors can still be seen in transgenic CRF knockout
Thus, a significant body of literature now exists that mice.44 Furthermore, stress-enhanced inflammation in a
suggests acute stress effects in the intestine are largely hapten sensitization model of colitis was shown not to be
mediated by CRF,5,6,37,38 because many of the physio- caused by CRF.45
logical consequences can be reproduced equally either Corticotropin-releasing factor is known to have both
by intracerebral injection of CRF6,7,32,38 or through its proinflammatory29,46 and anti-inflammatory47-50 effects.
AUGUST 2005
TABLE II. Mast cell activation during stressy
Mast cell
activation
Total granular change % Total
Tissue Condition granules Total low densityz activation§
Trachea Control 2631 600 22.5 6 2.0

Stress 3234 1163 35.3 6 2.9*
Colon Control 2728 619 22.0 6 2.0
Stress 2352 940 41.0 6 1.7**
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 degran-
ulation 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 FIG 4. Representative electron micrographs of mast cells in rat
(Table II; Fig 4), it appears likely that directly or indirectly, tracheal epithelium from control (A) and stress (B) rats. Tracheas
CRF causes tracheal mast cell degranulation. Mast cell were fixed 15 minutes after mounting in Ussing chambers. Mast
degranulation is one of the first effects of allergen inha- cells from control groups showed some signs of activation. Mast
cells from stress groups were more activated, as indicated by loss
lation. Stress mediated lung mast cell degranulation is of granule density (arrows point to such granules in B). Magnifi-
another mechanism for enhancement of the consequence cation: 30003.
of allergen inhalation in the sensitized host and would
further promote airways inflammation. Although rat tra-
cheal and lung mast cells are a mixture of mucosal and a-helical CRF inhibitor but also that the stress-induced
connective tissue types,54 both seem to respond to stress enhancement of the effect of SP was not inhibited at all by
and CRF, as evidenced by the fact that injection of CRF the a-helical CRF inhibitor. Further complexity to inter-
into rat skin caused degranulation of primarily connective pretation was added by the observation that the a-helical
tissue–type mast cells,29 and stress caused elevations of CRF inhibitor alone enhanced the SP effect but notably
rat mast cell protease II,6 a specific marker of mucosal had no effect itself on Isc generation either in the colon or
mast cells. trachea. Last, a-helical CRF did not reduce the activity of
Our results also showed that both acute stress and the CRF in this model but actually enhanced it, confirming its
peripheral systemic injection of CRF enhanced the action activity independent of CRF. We can conclude that
of SP on epithelial cell secretion. Joachim et al55 recently although stress effects on Isc may be mediated largely
showed that stressed mice exhibited increased bronchial by CRF, its effects on enhancement of SP may be through
hyperreactivity and that this and the increase in airways additional and different mechanisms.
inflammation was caused by SP. Our experimental results Several possible explanations may be entertained to
were anticipated in view of the data of McAlexander and explain these unexpected findings. First, a-helical CRF
Undem,56 who showed that CRF caused enhancement of has weak intrinsic activity37,42 in several different sys-
tachykinin-induced contractions of guinea pig tracheo- tems.43,59 It might have influenced SP receptor number or
bronchial smooth muscle. However, our results could not affinity or have agonist activity on new, as yet undiscov-
be predicted, because there are now several reports ered CRF receptors expressed in target tissue. That the
showing that CRF can have a protective effect.47-50 stress effect itself is, however, not wholly dependent on
These results range from experiments involving antigen- CRF is further evidenced by our observations that corti-
induced plasma extravasation and that induced by SP costerone levels after stress in a-helical CRF pretreated
or vagal stimulation, all the way to stress-induced worsen- animals were not reduced to levels seen in controls, but
ing of a hapten model of colitis48 both in hypo Lewis rather only to those seen in sham animals.
(LEW/N) and hyper (Fischer, 344/N) CRF responders to The biological significance and relevance of these
stress.57,58 Interpretation of the role of CRF in the experiments to asthma lie in the primary observation
modulation of stress effects is complicated by our own that acute stress can cause moderate effects on tracheal
results, which clearly indicated not only that effects on function. It seems reasonable to argue teleologically that it
stress-induced Isc increases were inhibited totally by the would be detrimental to the host if hypersecretion by the
tracheal epithelium occurred in response to acute stress, 11. Stead RH, Tomioka M, Quinonez G, Simon GT, Felten SY, Bienenstock J.
Intestinal mucosal mast cells in normal and nematode-infected rat
whereas intestinal hypersecretion would not be deleterious
intestines are in intimate contact with peptidergic nerves. Proc Natl Acad
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Allergen-induced substance P synthesis in
large-diameter sensory neurons innervating
the lungs
Benjamas Chuaychoo, MD, Dawn D. Hunter, PhD, Allen C. Myers, PhD,
Marian Kollarik, MD, PhD, and Bradley J. Undem, PhD Baltimore, Md

Background: Tachykinins such as substance P are localized in Key words: Allergic inflammation, phenotypic switch, neurogenic
unmyelinated slow-conducting C fibers that can be activated by inflammation, substance P, nodose ganglion, sensory nerve, vagus
noxious stimuli and tissue inflammation. Substance P is seldom nerve
expressed in fast-conducting large-diameter (A-fiber) vagal
sensory neurons. We have previously found that allergic
inflammation causes a phenotypic change in tachykinergic In both the somatosensory and visceral-sensory system,
innervation of the trachea such that the production of sensory neuropeptides, exemplified by the tachykinin
substance P is induced in large-diameter sensory neurons substance P (SP), are commonly found stored in the
projecting mechanosensitive A fibers to the trachea. peripheral and central terminals of unmyelinated sensory
Objective: To evaluate whether allergic inflammation also
C fibers.1-3 Inflammatory reactions in the rat paw have
induces substance P synthesis in large-diameter sensory
stretch-receptor neurons innervating guinea pig lungs, and to
been found to evoke a phenotypic switch in the nature of
investigate potential mechanisms by which this may occur. tachykinergic innervation.4,5 After a local inflammatory
Methods: Sensitized guinea pigs were exposed to allergen reaction in the paw, the preprotachykinin gene is induced
(ovalbumin) aerosol. One day later, immunohistochemical de novo in large-diameter, low-threshold, touch-sensitive
analysis was performed on vagal sensory neurons that had been Ab fibers.4 Simply touching the paw can then lead to
retrogradely labeled from the lungs. neuropeptide release in the spinal cord that, in theory,
Results: Ovalbumin inhalation caused a significant increase could lead to an increase in neurotransmission to the
in substance P expression in large-diameter neurofilament- extent that this innocuous touch stimulus is interpreted
positive nodose ganglion neurons that innervate the lungs as a noxious pain-producing stimulus (ie, an allodynia).
(P < .05). This effect was decreased by ipsilateral vagotomy.
A similar inflammation-induced phenotypic switch can
Exposing isolated nodose ganglia to the sensitizing antigen,
ovalbumin, also significantly increased substance P expression
occur in vagal sensory innervation of the airways. In the
compared with control. respiratory tract of most mammals, including guinea pigs
Conclusion: Allergic inflammation induces substance P and human beings, SP-containing fibers are located mainly
synthesis in large-diameter (A-fiber) nodose ganglion neurons in the upper airways, trachea, and main bronchi.1,6 There
innervating guinea pig lungs. This could contribute to the are relatively few SP containing fibers in the lungs. In
hyperreflexia seen in allergic airway disease. The full healthy guinea pig trachea and large bronchi, tachykinins
expression of this phenotypic switch in vagus nodose are localized nearly exclusively in nociceptive C fibers
ganglion neurons requires intact vagus nerve, but if allergen derived from small-diameter cell bodies in jugular sensory
reached the systemic circulation in sufficient quantities, it ganglia.2,3 One day after allergic inflammation in the lungs,
could also affect substance P synthesis by local activation of
SP production is increased in the lungs and in vagal sensory
vagal ganglionic mast cells. (J Allergy Clin Immunol
2005;116:325-31.)
neurons.7 On further analysis, it was determined that part of
the increase in SP production occurred de novo in large-
diameter 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 mechanosen-
From the Johns Hopkins University School of Medicine and Bloomberg
School of Public Health.
sors apparently designed to evoke cough on activation.10
Supported by the Heart, Lung and Blood Institute of the National Institutes of They differ from classically described intrapulmonary
Health (Bethesda, Md) and scholarship funding to Dr Chuaychoo from the stretch-sensitive fibers in neurophysiology, activation
Faculty of Medicine, Siriraj Hospital, Mahidol University (Bangkok, profile, and distribution in the lungs.10 The intrapulmonary
Thailand).
stretch-sensitive Ab fibers are further subclassified as
Received for publication February 13, 2005; revised March 30, 2005; accepted
for publication April 4, 2005. either rapidly adapting receptors (RARs) or slowly adapt-
Available online June 1, 2005. ing receptors (SARs). Whether the stretch-sensitive fibers
Reprint requests: Bradley J. Undem, PhD, Johns Hopkins Asthma and Allergy in the lungs can be induced by inflammation to produce
Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: neuropeptides in a fashion similar the tracheal touch-
bundem@jhmi.edu.
0091-6749/$30.00
sensitive fibers is unknown.
Ó 2005 American Academy of Allergy, Asthma and Immunology There is also little known about the mechanism by
doi:10.1016/j.jaci.2005.04.005 which airway inflammation leads to phenotypic changes
325
326 Chuaychoo et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
consecutive increasing concentrations of ovalbumin (0.01%, 0.03%,

Abbreviations used 0.1% ovalbumin diluted in saline), with 10 minutes of exposure for
NGF: Nerve growth factor each concentration to the maximum dose unless the animal developed
RAR: Rapidly adapting receptor signs of allergic response (gasping; rapid, shallow breathing; or
SAR: Slowly adapting receptor coughing), whereupon it was removed to breathe ambient air. After
SP: Substance P 24 hours, the animals were killed with CO2 inhalation and
Triton-BSA-PBS: PBS containing 0.3 % TritonX-100 and exsanguinated. We found no difference in the overresponse of the
1% BSA animal to ovalbumin between actively and passively sensitized
Trk: Tyrosine kinase receptor 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).
of the neurons located in the remote vagal sensory ganglia Identification of nonretrogradely labeled
(nodose and jugular ganglia situated near the base of the large-diameter nodose neurons
brain). A logical scenario is that the allergic reaction leads We previously reported that allergen challenge induced a pheno-
to the release of certain neurotrophic factors such as nerve typic switch to SP production in large-diameter nodose ganglion
growth factor (NGF) that interact with tyrosine kinase neurons (>25 mm diameter) that project nerve fibers specifically to
receptor (Trk) A on the terminals. Activation of neuro- the trachea.8 To evaluate whether allergic inflammation induces a
trophin Trk receptors at nerve terminals can signal events similar phenotypic switch in large-diameter neurons innervating
to the cell body via internalization and transport of the beyond the trachea, we first evaluated all nodose ganglion neurons
activated receptors along the nerve axon.11 However, with diameters larger than 25 mm for SP immunoreactivity in control
animals and after antigen challenge. Nodose ganglia were removed
there are alternative mechanisms to consider. The vagal
and fixed in 4% formaldehyde (fixative) for 2 hours at 4°C, rinsed
sensory ganglia that contain the cell bodies of vagal 3 times in 0.1 M PBS (pH 7.4) and then cryoprotected with 18%
sensory neurons are enriched with mast cells. In sensitized sucrose in PBS for 18 to 24 hours. Serial cryostat sections of the
animals, antigen can activate these ganglionic mast cells, nodose ganglia (12 mm thickness) were mounted on 4 consecutive
leading to local mediator release and change in neuronal slides, such that the first slide had sections 1, 5, 9., the second 2, 6,
excitability.12 It is possible, therefore, that the inhaled 10, and so forth, and the alternate slides were used for the analysis.
allergen is not triggering tachykinin synthesis from within The sections were dried at room temperature for 30 minutes and
the lungs, but rather, allergen is systemically absorbed and rinsed with water and PBS and incubated with blocking solution
interacts with mast cells locally within the vagal sensory containing 1% BSA, 10% goat serum, and 0.5% Tween 20 in PBS at
ganglia. room temperature for 1 hour. The slides were then processed for
double immunofluorescence staining with a mixture of rabbit poly-
In the current study, experiments were performed to
clonal antibody to SP (1 mg/mL; Peninsula Laboratories Inc, San
address 2 hypotheses. First, allergic inflammation can Carlos, Calif) and mouse mAb to 160 kd neurofilament protein (13
lead to induction of tachykinin synthesis not only in mg/mL; Chemicon, Temecula, Calif) diluted in PBS containing 0.3 %
tracheal Ad neurons but also in neurons that project Ab TritonX-100 and 1% BSA (Triton-BSA-PBS; Sigma Chemical Co, St
stretch-sensitive fibers to the lungs. Second, the mecha- Louis, Mo) for 24 hours at 4°C. The sections were washed 3 times
nism for allergen-induced SP production in bronchopul- with Triton-BSA-PBS and covered with a mixture of goat antirabbit
monary A-fiber nodose neurons requires intact vagal fluorescein (20 mg/mL diluted in Triton-BSA-PBS; Vector
nerve fibers. 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
METHODS with higher pH PBS (pH 8.6), then coverslipped with antifade
glycerol (Fluoromount, Molecular Probes, Eugene, Ore). Slides were
All experiments were approved by the Johns Hopkins Animal
examined under epifluorescence (Olympus DX60 microscope;
Care and Use Committee. For active sensitization, male Hartley
Olympus Corp, Melville, NY) by using appropriate filter combina-
guinea pigs (Hilltop, Scottsdale, Pa) weighing 100 to 300 g were
tions for fluorescein (excitation filter 450-480 nm; barrier filter 500-
immunized by intraperitoneal injection (10 mg/kg) of ovalbumin
515 nm) and Texas red (excitation filter 510-550 nm; barrier filter
(10 mg/mL) dissolved in saline on day 1, day 3, and day 5. On day 21
570-590 nm). The 160-kd neurofilament protein has been reported to
after the final sensitization, the actively sensitized animals were killed
be a selective marker for myelinated neurons,2,3 most of which are
by CO2 asphyxiation and exsanguinated. The blood was collected,
large-diameter neurons (>25 mm and mean diameter ;40 mm3,8);
and serum containing ovalbumin-specific IgG1 was isolated. For
only large-diameter, neurofilament-positive, nodose ganglion neu-
passive sensitization to ovalbumin, male Hartley guinea pigs
rons were evaluated for the expression of SP. Negative controls
weighing 100 to 300 g were sensitized by intraperitoneal injection
included slides treated with only secondary antibodies and slides in
of serum (2 mL/kg) containing IgG1 that was collected from guinea
which available commercially obtained isotype controls were used as
pigs actively sensitized to ovalbumin13 and challenged 1 day after
the primary antibodies for rabbit and mouse (Vector Lab). These
sensitization. Control guinea pigs were not injected with serum but
resulted in categorically negative staining.
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
Identification of lung-projecting
used in the vagotomy studies as a means to decrease the intra-animal large-diameter neurons
variation in the allergen response. In the in vivo studies, sensitized Nodose ganglion neurons were retrogradely labeled from the lung
(active or passive) and unsensitized (control) animals were exposed with True Blue (Chemicon) a week before sensitization and allergen
to aerosolized antigen in a Plexiglas chamber (volume, 8 L) with challenge. True Blue is fluorescent dye that is taken up by the nerve
J ALLERGY CLIN IMMUNOL Chuaychoo et al 327
terminals and travels in membranous vesicles back to the cell soma by neurons to determine whether antigen induced the expression of
retrograde axoplasmic flow. Accordingly, it is extensively used in SP ex vivo.
neurobiology to retrogradely backfill neurons. Guinea pigs were
anesthetized with intramuscular injection of ketamine (50 mg/kg) and Vagotomy studies
xylazine (2.5 mg/kg) and received two 1-mL (3%) transdermal
The animals were anesthetized as described, and 25 mL 5% Fast
injections of True Blue dye (in PBS containing 1% dimethyl
Blue dye (Sigma Chemical Co, St Louis, Mo) was instilled into the
sulfoxide; Molecular Probes) into the right lung using a 5-mL
trachea as describe previously.14 A week later, the animals were
Hamilton syringe. One week after the retrograde labeling, the animals
divided into unsensitized (control) and sensitized groups. In the
were exposed to aerosolized antigen as described.
sensitized group, the animals were passively sensitized with intra-
Twenty-four hours after antigen challenge, the animals were killed
peritoneal injection of ovalbumin-sensitized serum as described. A
by intraperitoneal injection of an overdose of sodium pentobarbital
day after the sensitization, under general anesthesia as described, the
(150 mg/kg) and perfused via the ascending aorta with a rinsing

vagus nerve was unilaterally ligated caudal to the nodose ganglion
solution containing procaine (100 mg/mL) and heparin (10,000 IU/L)
with suture in 2 places approximately 1 cm apart, and the section of
in PBS followed by fixative. Intrathoracic organs (lung, trachea,
vagus nerve between sutures was removed. On the following day, the
esophagus, heart, and surrounding soft tissues) and nodose ganglia
animals were challenged with ovalbumin inhalation as described.
were removed and put into fixative for 2 hours at 4°C, then rinsed
Unsensitized (control) animals had a similar unilateral vagotomy and
3 times in PBS. Lungs were cut in sections of 1 to 2 mm to identify the
were challenged with ovalbumin. Twenty-four hours after the antigen
dye distribution in parenchyma, blood vessels, and airways. Trachea
challenge, the animals were killed with CO2 inhalation and
and esophagus were examined inside and outside the lumens. Ganglia
exsanguinated. The nodose ganglia were removed bilaterally and
from animals were used only if the dye was delimited primarily to the
fixed for double immunofluorescence staining with antibodies to
lung parenchyma and distal airways, with no dye diffusion into the
neurofilament (160 kd) and SP as described in the nonlabeled large-
large airways or other extrapulmonary sites.
diameter nodose neuron study. Only Fast Blue–labeled neurons that
These nodose ganglia from the allergen challenged animals were
were neurofilament-positive were evaluated. For vagotomy, Fast
prepared and sectioned as described. The alternate slides were
Blue was used; no differences in labeling were noted between Fast
examined, and all True Blue–labeled neurons were photographed
Blue and True Blue, and the latter was used for the lung labeling
to identify labeled cells in case some labeled neurons faded after
procedure described.
immunostaining. The sections were double stained as described with
the exception of using a rat mAb to SP (5 mg/mL diluted in Triton-
BSA-PBS; Chemicon) with the mouse mAb to 160-kd neurofilament Statistical analysis
(13 mg/mL diluted in Triton-BSA-PBS; Chemicon) for 24 hours at The data were analyzed by using the Student paired or nonpaired
4°C as primary antibodies, followed by a mixture of goat antirat t tests. Data were expressed as means 6 SEMs. P < .05 was considered
antibody labeled with Alexa Fluor 594 (20 mg/ mL diluted in Triton- significant.
BSA-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
RESULTS
Blue was visualized with an UV filter (excitation filter 330-385 nm; Nonretrogradely labeled large-diameter
barrier filter 400-420 nm). Only True Blue–labeled neurons with nodose neuron study
neurofilament-positive immunofluorescence were evaluated to de-
termine whether allergic inflammation induces SP production. We We have previously made exhaustive studies of cell diam-
tested both SP antibodies in the same ganglion sections and found eters and neurofilament staining in vagal sensory neurons
there was no difference in staining (not shown). projecting to the respiratory tract.3,9 The cell diameters
of neurofilament-negative neurons form a unimodal pop-
ulation with a mean diameter of about 20 mm. The cell
Isolated ganglia studies
diameters of neurofilament-positive neurons also form a
The animals were actively sensitized with intraperitoneal injec- unimodal distribution with an average diameter of 43 6 8
tions of ovalbumin as described. Three weeks after the sensitization,
(n = 713) in one study and 40 6 3 (n = 307) in another
the animals were killed by CO2 inhalation and exsanguinated. Nodose
study. In the current study, we evaluated 781 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 ganglion neurons that were <25 mm in diameter and
bicarbonate solution (composed of NaCl, 118 mM; KCl, 5.4 mM; found that >99% were neurofilament-negative, whereas
NaH2PO4, 1.0 mM; MgSO4, 1.2 mM; CaCl2, 1.9 mM; NaHCO3, 25.0 among the 792 neurons with diameters >30 mM, all were
mM; dextrose, 11.1 mM; and gassed with 95% O2, 5% CO2, pH 7.4). neurofilament-positive. On the basis of these studies, and
Each individual nodose ganglion was incubated (37 °C) in 2 mL in accordance with conclusions drawn from more direct
Krebs bicarbonate solution containing antibiotics (penicillin/strepto- studies in the somatosensory system,15 we conclude that
mycin, 100 U/mL) and continuously oxygenated (95% O2, 5% CO2). the large diameter neurofilament-positive population of
The solution was changed every 15 minutes 4 times to wash and neurons project myelinated A fibers to peripheral tissues.
equilibrate the tissues. The nodose ganglia from the same animal were
In unsensitized (control) animals, we found that only 78
divided into a control and an antigen challenge group. In the antigen
of 2034 large neurons were SP-immunoreactive (average
challenge group, ovalbumin (10 mg/mL) was added to the solution.
The ganglia were incubated for various periods (0, 4, 8, 12, 18 hours) of 3.8% 6 0.6%; n = 4 animals). In ovalbumin-sensitized
at 37 °C and then removed from the test tubes and fixed and processed animals, the number of large-diameter neurons positive for
for double immunofluorescence staining for SP and neurofilament as SP was substantially higher, 712 of 1939 (average of
described in the nonlabeled large-diameter nodose neuron study. We 36.7% 6 3.1%; n = 4 animals; P < .01; compared with
evaluated all large-diameter, neurofilament-positive nodose ganglion unsensitized animals, Fig 1, C; part of these data was
AUGUST 2005
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,

FIG 1. SP immunoreactivity (SP-IR) in nodose ganglion neurons
12 h and n 5 8 for 18 h). At 18 hours after OVA treatment, the
1 day after the antigen challenge with ovalbumin (OVA) inhalation
percentage of SP immunoreactivity (SP-IR) is significantly in-
in (A) unsensitized (control) and (B) sensitized guinea pigs. Repre-
creased in large-diameter (>25 mm) neurofilament (NF)–positive
sentative photomicrographs of lung neurons labeled with True
neurons (16% 6 2%; n = 8) compared with the control (2% 6 0.8%;
Blue (TB) dye with double immunofluorescence staining of SP
n = 6). Data are presented as means 6 SEMs. *P < .01.
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 lung- animals degranulate and release inflammatory mediators
labeled (n = 8, unsensitized; n = 9, sensitized). Note, tracheal la-
beled neuron data taken from our previous study.8 Data are
on antigen challenge.12 We therefore next addressed the
presented as the means 6 SEMs. *P < .01 between sensitized question whether a local allergic reaction within the
and unsensitized animals). nodose ganglion is sufficient to induce SP expression in
large-diameter neurofilament-positive neurons.
The nodose ganglia were isolated from actively sensi-
tized animals and exposed to 10 mg/mL ovalbumin or
obtained from our previous study8 and is included here for vehicle (Krebs bicarbonate solution) ex vivo. Ovalbumin
comparison purposes). effectively induced SP synthesis in neurofilament-positive
neurons beginning at between 8 and 12 hours of exposure.
Lung-labeling experiment After 18 hours of ovalbumin treatment, approximately
To address more specifically the hypothesis that SP 16 % of neurofilament-positive neurons were SP-positive
synthesis could be induced in intrapulmonary A-fiber (n = 8), compared with ;2% in the vehicle treated ganglia
neurons, we used a retrograde labeling strategy. The (n = 6; P < .01; Fig 2).
retrograde tracer True Blue was injected into right lung.
One week after the injection, both actively sensitized and Vagotomy studies
unsensitized (control) groups were challenged with in- The data from the isolated ganglia studies suggest that if
haled ovalbumin. The animals were killed 1 day after inhaled ovalbumin can reach the vagal sensory ganglia in
antigen challenge, and the distribution of the dye was sensitized animals, it may be capable of acting locally to
examined in the thoracic tissues. Thirty-two animals were induce SP synthesis in neurofilament-positive neurons. To
used for this experiment. In 17 animals, we confirmed that address this question further, in vivo experiments were
the dye was delimited primarily to the lung parenchyma conducted in passively sensitized (or control) animals with
and distal airways, with no dye diffusion into the large unilateral vagotomy performed before ovalbumin inhala-
airways or extrapulmonary sites. Fifteen animals were tion. The induction of SP immunoreactivity in the nodose
excluded from further analysis as a result of diffusion of ganglion neurons was compared between the vagotomy
the dye to the large airways and/or cardiac tissue. side and contralateral side with intact vagus. If the
Among the successfully labeled animals, 9 animals allergen-inducing signal for SP synthesis travels to the
were unsensitized (controls) and 8 were sensitized. All cell body via the nerve fiber, then the induction will be
animals were challenged with ovalbumin, and 1 day later, noted only in the ganglia associated with an intact vagus
the neurons were evaluated for SP immunoreactivity. nerve. In these studies, either the right or left vagus nerve
In control animals, an average of 18% 6 4% of the was severed in a given animal. The data obtained were the
large-diameter neurofilament-positive neurons were SP- same whether the right or left ganglia was studied, and
immunoreactive. In sensitized animals challenged with therefore, the results were pooled.
ovalbumin, 30% 6 6% of large-diameter neurofilament- Fast Blue dye (25 mL) was instilled into cervical trachea
positive neurons were SP-positive (P < .05 compared with 1 week before the antigen challenge. This procedure
control animals; Fig 1, C). labeled both extrapulmonary and intrapulmonary compart-
ments. All animals were then challenged with aerosolized
Isolated ganglia studies ovalbumin. Twenty-four hours after the antigen challenge,
We have previously noted that mast cells located within the animals were killed, and labeled neurofilament-positive
the nodose ganglia isolated from ovalbumin-sensitized neurons were evaluated for the SP immunoreactivity.
Vagus nerve intact side. In unsensitized animals,

3% 6 1% (n = 4) of neurofilament-positive neurons ex-
pressed SP in nodose ganglia of an intact vagus nerve. As
expected, in sensitized animals, the percentage of neuro-
filament-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 SP-
positive in nodose ganglia on the side in which the vagus
nerve was cut. Comparing these results with those ob-
FIG 3. Histogram demonstrating the percentage of SP immunore-
served in ganglia obtained from the side with an intact

activity (SP-IR) in Fast Blue–labeled neurofilament (NF)–positive
vagus (3% 6 1%) suggests that severing the vagus per se
nodose neurons in intact versus severed vagus nerves 1 day after
signals the induction of tachykinin synthesis in a subset of the ovalbumin inhalation. Data are presented as means 6 SEMs;
neurofilament-positive nodose neurons. In sensitized and n = 4 per group. *P .05.
challenged animals, there was a significant (P < .05) but
slight increase in the percentage of the neurofilament- sensitive A fibers to the trachea and lungs, respec-
positive neurons that were SP-positive in nodose ganglia tively.3,10,16 Fischer et al7 were first to publish that allergic
on the side of the vagotomy (Fig 3). However, this inflammation in guinea pig airways is associated with an
apparent ovalbumin effect in ganglia ipsilateral to the increase in preprotachykinin gene transcription and SP
vagotomy (1.8-fold increase) was significantly less than synthesis in nodose ganglion neurons. Subsequently, we
that observed on the vagus intact side (9.7-fold increase). showed that this could occur in large-diameter neurofila-
ment-positive neurons that projected low-threshold touch-
sensitive Ad fibers to the trachea.8 Further investigation
DISCUSSION indicated that tracheal nodose Ad fibers in the guinea pig
represent a unique type of sensory nerve that evokes cough
The results support the hypothesis that allergic inflam- on activation.10
mation in guinea pig lungs can lead to a switch in sensory In the current study, we designed experiments to
SP innervation such that, in addition to sensory C fibers, determine the effects of allergic inflammation in the lung
SP is found in cough-causing A fibers in the trachea8 and on SP content of neurons that project A fibers to intra-
in stretch-sensitive A fibers in the lungs (current study). pulmonary structures (ie, RAR/SAR population). We
In addition, the results are consistent with the hypothesis injected small volumes of dye directly into the lung
that this phenotypic switch in tachykinergic innervation parenchyma and subsequently evaluated retrogradely
requires signals that, at least in part, reach the cell bodies in labeled neurofilament-positive neurons. In the allergen-
the distant sensory ganglia via the vagal nerve fibers. challenged animals, about 30% of the lung-specific
In healthy mammals, most neuronal tachykinins (SP neurofilament-positive neurons expressed SP immunore-
and neurokinin A) in the mammalian respiratory tract are activity. This value is similar to that observed in tracheal-
found in sensory C fibers.1,2 The vagal C-fiber neurons in labeled neurons. We have previously reported that under
the guinea pig respiratory tract are derived from cell control conditions, very few (1% to 3%) of neurofilament-
bodies situated in the jugular and nodose ganglia.2,3,16 The positive neurons in the nodose ganglia express SP immu-
jugular C fibers, found in extrapulmonary airways and noreactivity.8 Likewise, Kummer et al2 have reported that
intrapulmonary bronchi, are more likely to contain SP than nodose neurons labeled from healthy guinea pig lungs are
the nodose C fibers located within the lungs.16 Regardless essentially all SP-negative. We were therefore surprised
of the type of C fiber, these nerves are typically not thought by our finding that 18% of the neurofilament-positive
to be activated in healthy animals under normal circum- neurons labeled from the lung were SP-positive in the
stances. Rather, they are recruited to action by noxious control animals. We suspect that this may be a result of
stimuli or pathological conditions such as tissue inflam- some inflammation caused by our intraparenchymal
mation.17 On activation of a tachykinin-containing nerve, injections of the dye itself. In any event, the results
the tachykinins are released into synapses in the central support the hypothesis that allergic inflammation can lead
nervous system, where they serve to evoke pain in the to induction of tachykinin synthesis in stretch-sensitive A
somatosensory system4,18 and augment cough19,20 and fibers within the lungs.
parasympathetic reflexes in the respiratory system.21-24 In our previous electrophysiological analysis of nodose
When released from the peripheral nerve terminals via A fibers projecting to the guinea pig lungs, we found that
axon reflexes, the tachykinins may cause or contribute to they represented a rather homogenous population of fast-
local inflammatory reactions.25-27 conducting stretch-sensitive nerves.16 All of the fibers had
Substance P is seldom expressed in large-diameter conduction velocities in the range of 9 to 25 m/s and could
(A-fiber) sensory neurons innervating the trachea or be segregated on the basis of their adaptation to prolonged
lungs.2,3,28 The large-diameter neurons innervating the lung inflation into either RAR or SAR phenotypes. The
guinea pig respiratory system are located mainly in the finding that 30% of nodose A-fiber neurons express SP
nodose ganglia and project touch-sensitive and stretch- after allergen challenge indicates that SP production can
AUGUST 2005
be induced in some vagal stretch-sensitive nerves. This Also supporting the hypothesis that the site of initiation of
raises the possibility that SP could be released from their the phenotypic switch is within the lung is the finding that
central terminals in the brain stem during breathing, the effect of allergic inflammation on SP induction in
independently of noxious stimuli. This would be analo- large-diameter neurons can be mimicked with respiratory
gous to the phenotypic switch described in touch-sensitive tract viral infection.9 The virus infection in this model is
Ab fibers of the somatosensory system, in which such thought not to extend beyond the airway epithelium. The
an effect has been suggested to underlie sensations of small ovalbumin-induced effect observed in nodose
allodynia.4 neurons ipsilateral to the vagotomy may have been a
How allergic inflammation causes increased SP immu- result of the fact that the vagus was cut caudal to the point
noreactivity in nodose neurons is not known. By using a where the superior laryngeal nerve meets the vagus. Thus,
similar model system, Fischer et al7 provided direct the pathway remained intact for that population of A fibers
evidence that allergen-induced increase in tachykinin that project to the larynx and pharynx via the superior
synthesis occurs at the level of preprotachykinin gene laryngeal nerve.

transcription in the cell nucleus. We considered 2 path- The question remains as to the nature of the chemical
ways by which inhaled allergen can influence gene mediators capable of being released after allergic inflam-
transcription in the cell bodies located at a distant vagal mation that can lead to long-distance nuclear signaling via
sensory ganglion. First, the allergen could reach the the vagal axons. On the basis of the literature, it seems
systemic circulation and activate mast cells within the likely that a neurotrophin molecule such as NGF may be
ganglion, causing a local effect on the neighboring causally involved.31 Neurotrophins and their Trk recep-
neurons. Second, the allergic inflammation within the tors are, in fact, designed to signal gene transcriptional
airway wall could influence the nerve terminals in a events from interactions occurring at distant terminals.11
manner that sends long-distance signals to the cell nucleus In addition, NGF is associated with allergic reactions,32
via the afferent nerve fiber. and we have previously reported that microinjection of
When the nodose ganglia isolated from sensitized NGF into the tracheal wall leads to SP production in large-
guinea pig are subsequently exposed to the sensitizing diameter neurofilament-positive nodose neurons innervat-
antigen, ganglionic mast cells degranulate, mast cell– ing the trachea.33 Similar results have also been observed
associated mediators are released, and the electrical in the mouse.34
excitability of resident neurons is altered.12 In the current
study, we found that antigen administered to the nodose We thank Ms Holly K. Rohde for her technical assistance.
ganglia ex vivo caused a ;10-fold increase in the percent-
age of SP-expressing neurofilament-positive neurons.
This effect is presumably secondary to the activation of
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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
Background: (R)- and (S)-Enantiomers of albuterol likely Key words: b2-adrenergic agonist enantiomers, airways, mucus,
exert differential effects in patients with asthma. The edema, inflammation, hyperresponsiveness
(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 Adrenergic receptors are composed of a- and b-
augments bronchospasm and has proinflammatory receptors that bind endogenous catecholamines, such as
actions. epinephrine. Although 3 subtypes of b-adrenergic recep-
Objective: The study aim was to determine whether the tors exist, smooth muscle relaxation producing vasodila-
(S)-enantiomer, in contrast to the (R)-enantiomer, has tion and bronchodilation is mediated by the b2-receptor.
adverse effects on allergic airway inflammation and Short-acting b2-adrenergic receptor agonists rapidly
hyperresponsiveness in a mouse asthma model. induce bronchodilation in patients with asthma and are
Methods: Mice sensitized to ovalbumin (OVA) intraperitoneally used for relief of acute symptoms, prevention of exercise-
on days 0 and 14 were challenged with OVA intranasally on
induced asthma, and management of acute severe asthma.
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
Racemic albuterol contains equal concentrations
(1 mg kg21 d21 administered by means of a miniosmotic (50:50) of the (R)- and (S)-enantiomers (ie, enantiomers
pump from days 13-36) on airway inflammation and that are nonsuperimposable mirror images).1 The (R)-
hyperreactivity was determined. enantiomer of albuterol binds to b2-adrenergic receptors
Results: In OVA-sensitized/OVA-challenged mice, (R)-albuterol with nearly 100-fold greater affinity than the (S)-enanti-
significantly reduced the influx of eosinophils into the omer, suggesting that the (S)-enantiomer does not act
bronchoalveolar lavage fluid and airway tissue. (R)-Albuterol through b-adrenergic receptor activation.1 Whereas the
also significantly decreased airway goblet cell hyperplasia and (R)-enantiomer of albuterol (levalbuterol) exerts the
mucus occlusion and levels of IL-4 in bronchoalveolar lavage bronchodilating properties of albuterol, the (S)-enantio-
fluid and OVA-specific IgE in plasma. Although (S)-albuterol
mer has adverse effects, including augmentation of bron-
significantly reduced airway eosinophil infiltration, goblet
cell hyperplasia, and mucus occlusion, it increased airway
chospasm and proinflammatory activities.2-5 In murine
edema and responsiveness to methacholine in OVA-sensitized/ mast cells (S)-albuterol increases IgE-induced histamine
OVA-challenged mice. Allergen-induced airway edema and IL-4 production, whereas the (R)-enantiomer lacks
and pulmonary mechanics were unaffected by these effects.2 Anti-inflammatory effects of (R)-albuterol,
(R)-albuterol. such as inhibition of T-cell proliferation, might be negated
Conclusion: Both (R)- and (S)-enantiomers of albuterol reduce by the presence of the (S)-enantiomer.3
airway eosinophil trafficking and mucus hypersecretion in a Differential effects of the enantiomers might result
mouse model of asthma. However, (S)-albuterol increases from differences in pharmacokinetics.1 The initial step in
allergen-induced airway edema and hyperresponsiveness. the metabolism of the (S)- and (R)-enantiomers is sulfate
These adverse effects of the (S)-enantiomer on lung function
conjugation, a stereospecific process in human airway
might limit the clinical efficacy of racemic albuterol. (J Allergy
Clin Immunol 2005;116:332-40.)
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
From the Departments of Medicine and Pathology, University of Washington. binding to lung tissue.
Supported by National Institutes of Health grants AI04989 and HL073722
and by a grant from Sepracor Inc.
In this study we characterized the effects of the
Disclosure of potential conflict of interest: E. Chi and E. R. Banerjee—none (R)- and (S)-enantiomers of albuterol on allergic airway
disclosed. W. R. Henderson, Jr, receives grants–research support from inflammation and hyperresponsiveness in a mouse asthma
Sepracor, Inc. model that mimics key features of human asthma.8
Received for publication August 4, 2004; revised April 1, 2005; accepted for
Although prior studies in guinea pigs and human subjects
publication April 12, 2005.
Available online June 1, 2005. have demonstrated that the (S)-enantiomer of albuterol can
Reprint requests: William R. Henderson, Jr, MD, Department of Medicine, induce airway hyperreactivity, there are no prior studies
Center for Allergy and Inflammation, Box 358050, University of examining the effect of (S)-albuterol versus (R)-albuterol
Washington, 815 Mercer Street, Seattle, WA 98109. E-mail: joangb@ on both airway hyperresponsiveness and the TH2
u.washington.edu.
0091-6749/$30.00
phenotype (ie, allergen-induced airway eosinophil traf-
Ó 2005 American Academy of Allergy, Asthma and Immunology ficking, mucus metaplasia, edema, and TH2 cytokine
doi:10.1016/j.jaci.2005.04.013 release) in an in vivo asthma model. We report that both
332
J ALLERGY CLIN IMMUNOL Henderson, Banerjee, and Chi 333
independent of functional residual capacity, tidal volume, and

Abbreviations used respiratory rate.
AP-1: Activator protein 1
BAL: Bronchoalveolar lavage Light microscopy-morphometry
OVA: Ovalbumin After pulmonary function testing, bronchoalveolar lavage (BAL)
Penh: Enhanced pause 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
(R)- and (S)-enantiomers reduce allergen-induced airway randomly selected and examined in a blinded manner. Sections were
eosinophil and mucus gland hyperplasia. However, only stained with hematoxylin and eosin, the total inflammatory cell

(S)-albuterol increases airway edema and responsiveness infiltrate was assessed on a semiquantitative scale (0-41), the number
to methacholine, effects that would decrease the thera- of eosinophils per unit of airway area (2200 mm2) was determined by
peutic efficacy of racemic albuterol. using a point-counting system (Image-Pro Plus point-counting sys-
tem software, Version 1.2 for Windows; Media Cybernetics, Silver
Spring, Md),12 and interstitial and perivascular airway edema were
METHODS assessed.13,14 Airway goblet cells (as a percentage of total airway
cells) were identified by means of Alcian blue staining,12 and the
Study protocol degree of mucus plugging of the airways (0.5 mm to 0.8 mm in
All animal use procedures were approved by the University of diameter) with the percentage occlusion of the airway diameter was
Washington Animal Care Committee. Female BALB/c mice (6-8 classified on a 0 to 41 scale on the basis of the following criteria:
weeks of age; The Jackson Laboratory, Bar Harbor, Me) received an 0, no mucus; 1, approximately 10% occlusion; 2, approximately 30%
intraperitoneal injection of 100 mg of ovalbumin (OVA; 0.2 mL of occlusion; 3, approximately 50% occlusion; 4, greater than approx-
500 mg/mL) complexed with alum on days 0 and 14 (Fig 1). Mice imately 80% occlusion.12
were anesthetized with 0.2 mL of ketamine (6.5 mg/mL)/xylazine
(0.44 mg/mL) in normal saline administered intraperitoneally before Cytokine assays
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 IL-4, IL-5, IL-10, GM-CSF, TNF-a, IL-2, and IFN-g were
normal saline with alum administered intraperitoneally on days 0 and assayed in BAL fluid with Bio-Plex Mouse Cytokine assays (Bio-
14 and 0.4 mL of saline without alum administered intranasally on Rad Laboratories, Hercules, Calif) that are bead-based multiplex
days 14, 25, and 35. In both the saline- and OVA-treated groups, sandwich immunoassays with a limit of detection of less than 10 pg/
miniosmotic pumps (200 mL, Alzet Model 2004; Durect Corp, mL. IL-13 was assayed in BAL fluid with a mouse IL-13 immuno-
Cupertino, Calif) containing either (R)- or (S)-albuterol (1 mg kg21 assay (Quantikine M; R&D Systems, Minneapolis, Minn), with a
d21, 6 mL/d delivery administration) were inserted subcutaneously limit of detection of less than 1.5 pg/mL.
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 OVA-specific IgE assay
compounds by local capillaries results in systemic administration. OVA-specific IgE was assayed by modification of the method
Each study group consisted of 4 to 6 animals. The 1 mg kg21 d21 of Iio et al.15 Nunc 96-well flat-bottom plates (Nalge Nunc
dose of albuterol enantiomer infusion was selected on the basis International, Rochester, NY) were coated with 50 mg/mL OVA in
of prior work by Sartori et al,9 demonstrating that continuous release 13 PBS overnight at room temperature, washed 3 times with
of racemic albuterol (2 mg kg21 d21) subcutaneously by means of 13 PBS plus 0.05% Tween-20 (wash buffer), blocked with 3% BSA
miniosmotic pump produced steady-state, high-plasma levels of in 13 PBS for 1 hour at room temperature, and washed 4 times with
albuterol (1025 M) in mice. 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
Pulmonary function testing 4 times with wash buffer, and blotted dry by inverting over paper
In vivo airway responsiveness to methacholine was determined towels. One hundred microliters (1:100 in 13 PBS) of biotin-
on day 36 in conscious, freely moving, spontaneously breathing mice conjugated rat anti-mouse IgE mAb (clone R35-72; BD Biosciences,
by using whole-body plethysmography (Model PLY 3211; Buxco San Diego, Calif) was added to each well and incubated overnight at
Electronics Inc, Sharon, Conn), as described by Hamelmann et al.10 4°C and then washed 4 times with wash buffer and blotted dry. Then
Mice were challenged with aerosolized saline or increasing doses 100 mL per well (1:1000 in 13 PBS) streptavidin-horseradish
(2 and 10 mg/mL) of methacholine generated by an ultrasonic peroxidase–conjugated secondary antibody (BD Biosciences) was
nebulizer (DeVilbiss Health Care, Inc, Somerset, Pa) for 2 minutes. added, and samples were incubated at 37°C for 90 minutes, then
The degree of bronchoconstriction was expressed as enhanced pause washed 4 times with wash buffer, and blotted dry. One hundred
(Penh), a calculated dimensionless value that correlates with mea- microliters of substrate solution (ie, 1 tablet of 2,2#-azinobis [3-
surement of airway resistance, impedance, and intrapleural pres- ethylbenzthiazoline-sulfonic acid, ABTS; Sigma Chemical Co, St
sure.9-11 Penh readings were taken and averaged for 4 minutes Louis, Mo] dissolved in 100 mL of 0.05 M phosphate-citrate buffer,
after each nebulization challenge. Penh is calculated as follows: pH 5.0, and 25 mL of 30% H2O2) was added per well, and color was
Penh ¼ ½ðTe =Tr 21Þ3ðPEF=PIFÞ, where Te is expiration time, Tr is developed for 30 minutes at room temperature. OD 405 nm was
relaxation time, PEF is peak expiratory flow, and PIF is peak measured by using an AD 340C absorbance detector (Beckman
inspiratory flow 3 0.67 coefficient. The time for the box pressure Coulter Inc, Fullerton, Calif). For the IgE standard curve, a sandwich
to change from a maximum to a user-defined percentage of the ELISA was used in which in separate assay plates biotin-conjugated
maximum represents the relaxation time. The Tr measurement begins rat anti-mouse IgE mAb (clone R35-72, BD Biosciences) was used
at the maximum box pressure and ends at 40%. Because Penh is the to coat the wells, and instead of plasma samples, known concen-
ratio of measurements obtained during the same breath, it is mainly trations of purified anti-mouse IgE (clone C38-2, BD Biosciences)
334 Henderson, Banerjee, and Chi J ALLERGY CLIN IMMUNOL
AUGUST 2005
FIG 1. Study protocol. i.p., Intraperitoneal; i.n., intranasal

were incubated; assays were run as described above. The standard mice (Fig 2, A). Airway goblet cells increased to 38.0% of
curve was constructed by using a linear regression analysis of the total airway cells in OVA-treated mice compared with
absorbances against serial dilutions of known concentrations of 0.4% in saline control mice (P = .0001, OVA vs saline;
mouse IgE. Pooled mouse plasma from OVA-sensitized/OVA-
Fig 4, A). The mucus occlusion of the airway diameter
challenged mice was used as a positive control.
morphometric score increased 15-fold in the OVA-treated
Statistical analysis mice compared with control mice (P < .0001, OVA vs
saline; Fig 4, B). Allergen-induced goblet cell hyperplasia
The data are reported as the means 6 SE of the combined
and mucus occlusion of airway diameter were inhibited by
experiments. Differences were analyzed for significance (P < .05) by
means of ANOVA with the protected least-significant-difference
both the (R)-enantiomer (Fig 2, C) and (S)-enantiomer
method (Statview II; Abacus Concepts, Berkeley, Calif). (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)
RESULTS and airway mucus occlusion by 41.4% (P = .0042,
R-albuterol/OVA vs OVA; Fig 4, B). (S)-Albuterol
Effect of (R)- and (S)-enantiomers of albuterol reduced goblet cell hyperplasia by 44.8% (P = .0095,
on allergen-induced airway inflammation S-albuterol/OVA vs OVA; Fig 4, A) and mucus occlusion
Airway infiltration by eosinophils. A marked infiltra- of the airways by 35.7% (P = .0088, S-albuterol/OVA
tion of inflammatory cells that were predominantly vs OVA; Fig 4, B).
eosinophils around the airways and pulmonary blood Airway edema. Airway edema was observed in the
vessels was observed in the lung interstitium of OVA- lungs of OVA-treated mice (Fig 2, B) compared with
treated mice (Fig 2, B) compared with that seen in saline- that seen in saline-treated control mice (Fig 2, A).
treated control mice (Fig 2, A) on day 36, 24 hours after the (S)-Albuterol markedly increased airway edema in OVA-
last intranasal OVA or saline challenge. By means of treated mice (Fig 2, D vs B). In contrast, (R)-albuterol had
morphometric analysis (Fig 3, A and B) of the histologic no effect on allergen-induced edema in the airways of
sections (Fig 2, C and D vs B), administration of (R)- OVA-treated mice (Fig 2, C).
and (S)-albuterol by means of miniosmotic pumps Cytokine release. Significant levels (P < .05) of IL-4,
(1 mg kg21 d21 dose from days 13-36) significantly IL-5, IL-13, and GM-CSF were found in the BAL fluid of
decreased the influx of total inflammatory cells (P = OVA-treated mice compared with levels in the saline
.0035, R-albuterol/OVA vs OVA; P = .0226, S-albuterol/ control group (Fig 5). The increased levels of IL-4 in
OVA vs OVA; Fig 3, A) and eosinophils (P = .021, R- OVA-treated mice were reduced 70.5% and 52.2% by
albuterol/OVA vs OVA; P = .008; S-albuterol/OVA vs (R)-albuterol and (S)-albuterol, respectively; the reduction
OVA; Fig 3, B) into the lung interstitium. Compared with was statistically significant only for the (R)-enantiomer
the saline group (Fig 3, C), OVA-sensitized/OVA-chal- (P = .043, R-albuterol/OVA vs OVA; Fig 5). There was
lenged mice exhibited a marked increase in BAL fluid no significant effect of either the (R)- or (S)-enantiomer of
eosinophils to 2.5 6 0.5 3 105 eosinophils/mL (P < albuterol on the increased BAL fluid levels of IL-5, IL-13,
.0001, OVA vs saline; Fig 3, C), which represented 41.8% and GM-CSF in the OVA-treated animals. The levels of
of total BAL fluid cells. In OVA-sensitized/OVA-chal- IL-10, TNF-a, IL-2, and IFN-g were not significantly
lenged mice, treatment with (R)-albuterol significantly increased in the BAL fluid of OVA-treated mice compared
inhibited the influx of eosinophils into BAL fluid by with levels seen in saline-treated control mice; neither
40.6% (P = .0043, R-albuterol/OVA vs OVA; Fig 3, C). enantiomer affected the levels of these cytokines in OVA-
In contrast, (S)-albuterol had no significant effect on treated animals (Fig 5).
eosinophil influx into the BAL fluid of OVA-treated mice OVA-specific IgE. Plasma OVA-specific IgE was
(Fig 3, C). absent in saline-treated control mice and present in
Airway mucus hypersecretion. Hyperplasia of airway OVA-treated control mice (Fig 6). (R)-Albuterol and
goblet cells and hypersecretion of mucus were observed in (S)-albuterol reduced OVA-specific IgE levels in OVA-
OVA-treated mice (Fig 2, B) compared with in control treated mice; the reduction was statistically significant

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), OVA-
treated 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 had been challenged with 3 intranasal doses of OVA, a
vs OVA; Fig 6). protocol that we have previously shown to induce airway
inflammation and mucus hypersecretion but that is sub-
Effect of (S)- and (R)-enantiomers of
optimal for inducing airway hyperresponsiveness.16,17
albuterol on allergen-induced airway This protocol was used because an augmenting effect
hyperresponsiveness of (S)-albuterol on airway hyperresponsiveness could
Pulmonary mechanics were assessed in response to have been masked in our mouse asthma model protocol,
aerosolized methacholine by means of noninvasive in vivo in which bronchial hyperresponsiveness is achieved
plethysmography on day 36, 24 hours after the last through administration of 4 intranasal doses of OVA
intranasal OVA challenge. The OVA-sensitized mice in mice sensitized by 2 intraperitoneal OVA doses.16,17
AUGUST 2005
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 had no effect on Penh (percentage of air) values of
bronchial responsiveness to methacholine (Fig 7). In the saline control group. At 0, 2, and 10 mg/mL metha-
contrast, (R)-albuterol did not alter airway responsiveness choline doses, non–OVA-sensitized/OVA-challenged
to methacholine in OVA-sensitized/OVA-challenged mice administered (R)-albuterol had 106.4%, 95.7%,
mice (Fig 7). and 101.1% Penh (percentage of air) values of the saline
control animals.
Effect of (R)- and (S)-enantiomers of albuterol
in non–OVA-sensitized/OVA-challenged mice
Miniosmotic pumps containing either (R)- or (S)- DISCUSSION
albuterol were placed subcutaneously in saline control
animals for a 24-day treatment period before pulmonary In this mouse model of asthma, we found both over-
function testing and assessment of lung histopathology lapping and distinct actions of the (S)- and (R)-enan-
(Fig 8) to examine the effect of the albuterol enantiomers tiomers of albuterol on key features of allergen-induced
independently of a modification of the allergic inflamma- airway inflammation and responsiveness to methacholine.
tory response. (R)-Albuterol significantly reduced the following features
Lung morphology. The (S)-enantiomer of albuterol did of allergen-induced airway inflammation: BAL fluid
not induce airway edema independently of an allergic levels of IL-4 and eosinophils, airway eosinophil infiltra-
response. No airway edema or inflammation was seen tion, goblet cell hyperplasia, and mucus occlusion and
in saline-treated mice administered either (S)-albuterol circulating levels of OVA-specific IgE. Although (S)-
(Fig 8, B) or (R)-albuterol (Fig 8, C). albuterol also decreased airway tissue eosinophilia, goblet
Pulmonary mechanics. The (S)-enantiomer of albuterol cell hyperplasia, and mucus plugging of the airways, no
did not affect airway reactivity in non–OVA-sensitized/ significant effect on the influx of eosinophils into the BAL
OVA-challenged mice. At 0, 2, and 10 mg/mL methacho- fluid was observed. (S)-Albuterol, but not (R)-albuterol,
line challenge doses, Penh (percentage of air) values had adverse effects on airway function by increasing
were, respectively, 105.1%, 92.3%, and 100.0% of the airway edema and hyperresponsiveness in OVA-treated
values of saline control animals. Similarly, (R)-albuterol mice.

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 function. Sympathetic (ie, adrenergic), parasympathetic
(R)-albuterol inhibited infiltration of eosinophils into (ie, cholinergic), and sensory-efferent (ie, tachykinin-
airway tissue, but only (R)-albuterol significantly reduced mediated) pathways regulate mucus secretion from airway
eosinophil influx into BAL fluid. Although a correlation epithelial goblet cells and submucosal glands.20 In human
between BAL fluid levels of IL-5 and eosinophilia is airways the cholinergic response is predominant and is
typically seen, there was no reduction in the increased IL-5 mediated by muscarinic M3-receptors on the mucus secre-
levels of OVA-treated mice administered either albuterol tory cells.20 In ovine tracheal epithelial cells (R)-albuterol
enantiomer. Short- and long-acting b2-adrenergic agonists increased ciliary beat frequency, whereas (S)-albuterol
might facilitate eosinophil apoptosis, thereby reducing had no significant effect.21 Increased mucociliary clear-
airway eosinophilia.18 Albuterol (0.1-10 mM) in vitro dose ance rates by b2-adrenergic agonists have been reported in
dependently decreases colony numbers and increases some patients with asthma.22 The TH2 cytokines IL-4 and
apoptosis of eosinophil progenitor cells from the blood IL-13 have potent effects on mucus secretion.23,24
of patients with asthma.19 In contrast, racemic albuterol, Administration of each cytokine independently stimulates
(R)-albuterol, and (S)-albuterol do not affect apoptosis of airway mucus accumulation in mice.24 MUC5AC gene
antigen-specific human T-cell lines.3 expression and BAL fluid mucus protein release are
An unexpected finding of this study was the reduction increased in IL-4 transgenic mice.23 In addition, inhibition
in airway goblet cell hyperplasia and mucus hypersecre- of IL-4 by administration of soluble IL-4 receptor reduces
tion by both the (R)- and (S)-enantiomers of albuterol. airway mucus hypersecretion and inflammatory cell traf-
Limited data exist regarding the effect of (R)- and ficking to the lungs in OVA-treated mice.25 We found that
(S)-enantiomers of albuterol on airway mucus gland the increased BAL fluid levels of IL-4, but not IL-13,
AUGUST 2005
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 bronchoconstric-

tion 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 mice, suggesting a proinflammatory effect unique to
(R)-albuterol, with a trend toward IL-4 reduction by (S)-albuterol. This effect of (S)-albuterol was not observed
(S)-albuterol. Thus the albuterol enantiomers might mod- in nonsensitized/nonchallenged mice. In 2 sheep models
ulate allergen-induced airway inflammation and mucus of altered lung fluid balance, the effect of aerosolized
hypersecretion through IL-4, rather than IL-5 or IL-13, racemic albuterol and its (R)- and (S)-enantiomers on lung
signaling. We have recently demonstrated a similar dis- epithelial permeability has been examined.29 Pretreatment
cordance between IL-4 and IL-5/IL-13 in mediation of with (S)-albuterol increased the level of albumin in the
allergen-induced airway eosinophilia and mucus hyper- epithelial lining fluid in sheep receiving histamine to
secretion.14 In a mouse asthma model the selective increase lung permeability. This effect was not observed
redox effector factor 1 inhibitor PNRI-299, which inhibits after pretreatment with either (R)-albuterol or its racemate.
the transcription factor activator protein-1 (AP-1), sig- In sheep receiving an increase in left atrial pressure to
nificantly decreased airway eosinophil infiltration and increase hydrostatic forces, only (S)-albuterol increased
mucus occlusion and lung gene expression of IL-4 but not lung water volume. Thus in the presence of altered
IL-5 or IL-13.14 During T-cell activation, a complex lung fluid balance, (S)-albuterol, but not (R)-albuterol,
interaction between nuclear factor of activated T cells and might increase lung epithelial permeability. Our finding
AP-1 is necessary for inducible expression of IL-4.26 In that (S)-albuterol increases allergen-induced interstitial
contrast, transcriptional regulation of IL-5 and IL-13 edema indicates a potential adverse effect of this enanti-
might be independent of AP-1 binding.27 The induction omer in patients with asthma.
of goblet cell hyperplasia and eosinophilia by IL-4 in triple (S)-Albuterol significantly increased bronchial re-
IL-5/IL-9/IL-13–knockout mice further demonstrates the sponsiveness to methacholine challenge in OVA-sensi-
key role IL-4 exerts in the development of the TH2 tized/OVA-challenged mice, an adverse effect on airway
phenotype.28 In our studies the reduction in IL-4 in function not shared by (R)-albuterol. In nonsensitized/
OVA-treated mice administered (R)-albuterol correlated nonchallenged mice (S)-albuterol did not independently
with a decrease in circulating OVA-specific IgE. affect the airway response to methacholine. We used
We found that (S)-albuterol, but not (R)-albuterol, whole-body plethysmography to assess airway hyperre-
augmented the interstitial edema observed in OVA-treated activity to methacholine in the study groups. Although

FIG 8. Effect of (R)- and (S)-albuterol enantiomers on airway histology in non–OVA-sensitized/non–OVA-
challenged 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 for a 10-day period increased bronchial hyperresponsive-
Penh as an indirect measure of pulmonary mechanics,30-32 ness to both histamine and OVA.5 Chronic capsaicin
Penh values correlate well with airway resistance mea- treatment prevented the (R,S)- and (S)-albuterol–induced
sured directly in anesthetized, tracheotomized, and bronchial hyperresponsiveness in this model to indicate
mechanically ventilated mice.10,11 A strong correlation the importance of capsaicin-sensitive sensory nerves in
also exists between Penh values and the intensity of the (S)-albuterol-mediated development of airway hyperre-
allergen-induced airway eosinophil infiltration in the sponsiveness.5 Recent studies by Agrawal et al4 indicate
mouse asthma model.33 that (S)-albuterol activates proconstrictor and proinflam-
(S)-Albuterol might mediate its augmenting effect on matory pathways in human bronchial smooth muscle
bronchial hyperresponsiveness through several modes of cells. (S)-Albuterol significantly increased the expression
action, including parasympathetic and sympathetic path- and activity of Gia-1 protein and reduced Gs protein in
ways. In OVA-sensitized guinea pigs continuous expo- these cells.4 (S)-Albuterol also increased the intracellular
sure to (R,S)- and (S)-albuterol, but not (R)-albuterol, free calcium concentration in the bronchial smooth
AUGUST 2005
muscle cells after methacholine stimulation.4 These in C57BL/6 mice. Am J Physiol Lung Cell Mol Physiol 2001;280:
L363-8.
proconstrictor effects of (S)-albuterol were accompanied
12. Henderson WR Jr, Tang L-O, Chu S-J, Tsao S-M, Chiang GKS, Jones F,
by stimulation of phosphatidylinositol 3#-OH-kinase et al. A role for cysteinyl leukotrienes in airway remodeling in a mouse
and nuclear factor kB proinflammatory pathways in the asthma model. Am J Respir Crit Care Med 2002;165:108-16.
smooth muscle cells. (R)-Albuterol induced the opposite 13. Oh SW, Chong IP, Dong Keun L, Jones F, Chiang GKS, Kim HO, et al.
effects on Gia-1, Gs, and intracellular free calcium con- Tryptase inhibition blocks airway inflammation in a mouse asthma
model. J Immunol 2002;168:1992-2000.
centration in the bronchial smooth muscle cells, which 14. Nguyen C, Teo J-L, Matsuda A, Eguchi M, Chi E, Henderson WR Jr,
indicates separate mechanisms of action of the enan- et al. Chemogenomic identification of Ref-1/AP-1 as a novel therapeutic
tiomers.4 target for asthma. Proc Natl Acad Sci U S A 2003;100:1169-73.
In summary, the actions of the (R)- and (S)-enantiomers 15. 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
of albuterol in the lungs of allergen-sensitized/allergen-
eosinophilic recruitment into airways in BALB/c mice. Int Arch Allergy

challenged mice are complex. Although both enantiomers
Immunol 2002;127:217-25.
reduce mucus hypersecretion and trafficking of eosino- 16. Henderson WR Jr, Lewis DB, Albert RK, Zhang Y, Lamm WJE, Chiang
phils to the lungs after allergen challenge, only the GKS, et al. The importance of leukotrienes in airway inflammation in a
(S)-enantiomer induces airway edema and hyperrespon- mouse model of asthma. J Exp Med 1996;184:1483-94.
17. Zhang Y, Lamm WJE, Albert RK, Chi EY, Henderson WR Jr, Lewis
siveness to methacholine. Additional studies are needed DB. Influence of the route of allergen administration and genetic
to delineate the specific effects of the (R)- and (S)- background on the murine allergic pulmonary response. Am J Respir
enantiomers of albuterol on airway inflammation and Crit Care Med 1997;155:661-9.
hyperresponsiveness in patients with asthma. 18. Johnson M. Effects of b2-agonists on resident and infiltrating inflam-
matory cells. J Allergy Clin Immunol 2002;110(suppl):S282-90.
19. Wang CH, Lin HC, Lin CH, Yu CT, Liu SL, Huang KH, et al. Effect of
We thank Gertrude Chiang, Falaah Jones, and Ying-tzang Tien theophylline and specific phosphodiesterase IV inhibition on prolifera-
for excellent technical assistance and Rachel Norris for typing this tion and apoptosis of progenitor cells in bronchial asthma. Br J
manuscript. Pharmacol 2003;138:1147-55.
20. Rogers DF. Pharmacological regulation of the neuronal control of airway
mucus secretion. Curr Opin Pharmacol 2002;2:249-55.
21. Frohock JI, Wijkstrom-Frei C, Salathe M. Effects of albuterol enan-
tiomers on ciliary beat frequency in ovine tracheal epithelial cells. J Appl
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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
Background: Allergy skin testing guides developing avoidance that technicians are sufficiently trained on the correct use of
plans and writing an immunotherapy prescription. The goal that device. (J Allergy Clin Immunol 2005;116:341-6.)
for the allergist is to apply allergen skin testing to the
appropriate patient population by using a device that Key words: Skin testing, device, performance, variability, pain
minimizes both false-negative and false-positive findings while
Rhinitis, sinusitis, and

minimizing patient discomfort. New skin testing devices
The US Joint Council of Allergy, Asthma and Immu-
ocular diseases
continue to be developed with a trend toward production of
multiheaded devices. Data on the performance of these devices nology1 and the European Academy of Allergology and
in a head-to-head prospective fashion are limited. Clinical Immunology2 recommend percutaneous testing
Objective: Our goal was to study 8 commonly used devices to as the primary test for diagnosis of IgE mediated allergic
compare their performance in a head-to-head fashion. disease. Skin testing is also the preferred method for
Methods: In a prospective, double-blind fashion, the selecting allergens to be included in immunotherapy.3
performance of 8 skin test devices was evaluated. Devices Given this, the findings on the initial skin test panel are
were tested with histamine and saline on both the arms and very important clinical data. If a particular device is too
back of each subject. Devices were rotated over 4 testing sensitive (resulting in false-positive findings), the patient
sessions, at least a week apart, so each device was tested in
may receive an antigen that is not required to achieve
each anatomic testing location. Performance elements
clinical benefit. On the other hand, a high false-negative
examined included wheal, flare, pain, sensitivity, specificity,
and intradevice variability. rate for a particular device will result in a patient not
Results: We found significant differences in all areas of receiving a needed antigen while undergoing immuno-
device performance among all devices examined. Multiheaded therapy. The goal for the allergist is to perform allergen
devices also demonstrated significant intradevice variability skin testing in an appropriate patient population by using
and were more painful than single devices. Furthermore, a device that minimizes both false-negative and false-
multiheaded devices had larger reactions on the back, whereas positive findings. In addition, it is desirable to use a device
single devices had larger reactions on the arms. that results in minimal patient discomfort. Previous studies
Conclusion: Statistically significant differences exist among comparing devices for skin prick (ie, prick and puncture)
all devices tested. Providers should consider this data when
testing have revealed significant differences in the size
choosing a device that suits their practice setting and ensure
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
From athe Department of Allergy and Immunology and bthe Department of
appeared to result from the degree of trauma imparted
Clinical Investigations, Walter Reed Army Medical Center, Silver Spring;
c
private practice, Fort Lauderdale; and dthe Asthma and Allergic Disease to the skin by the device, an interpretation that was
Center, University of Virginia Health System. reinforced by the fact that those producing larger wheals
Supported by the Walter Reed Army Medical Center, Department of Clinical also caused more patient discomfort.6
Investigations. New skin devices continue to be developed, with a
Disclosure of potential conflict of interest: L. Borish has consultant arrange-
ments with PDL, Syngenta, and Sepracor. No other relevant conflicts of
current trend toward devices that allow for application
interest to disclose. of several antigens simultaneously, referred to as multi-
The opinions or assertions contained herein are the private views of the authors headed. This may limit technician time and increase
and are not to be construed as official or as reflecting the views of the efficiency. In addition, multiheaded devices have increas-
Department of the Army or the Department of Defense.
ing popularity in children, in whom the acceptance of a
Received for publication September 3, 2004; revised March 28, 2005; accepted
for publication March 28, 2005. few multiple test devices tends to be better than many
Available online May 16, 2005. individually applied devices. In a recent letter to the editor,
Reprint requests: Warner W. Carr, MD, Division of Experimental Nelson et al7 compared 3 new multidevices with previ-
Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant ously reviewed devices. In this communication, signifi-
Ave, Silver Spring, MD 20910. E-mail: wmcarr@comcast.net.
0091-6749/$30.00
cant differences were noted with the smallpox needle on
Ó 2005 American Academy of Allergy, Asthma and Immunology the back and the Greer Track (Greer Labs, Lenoir, NC) on
doi:10.1016/j.jaci.2005.03.035 the arm. Given this, we reviewed 4 devices that allow
341
342 Carr et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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 specif-
ically intended to determine sensitivity, specificity, var-
iability, and pain. With these results, providers will be
able to determine which device is best suited for their FIG 1. The left image illustrates the 4 test zones of the back, and the
practices. 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.
METHODS
Study design
ocular diseases
device (measured on a scale from 0-10). On the basis of this scale, a

The study was a prospective, double-blind clinical trial and was
reviewed and approved by the Walter Reed Army Medical Center level of 1 to 2 is considered minimal pain. The greatest reported pain
Clinical Investigation Committee and the Human Use Committee. All was recorded for that particular test site and session. Pain was
subjects enrolled into the study voluntarily agreed to participate and recorded within seconds of application of the skin test device to
minimize the influence of histamine on pain perception. Results were
gave written informed consent. Each subject underwent testing in 4
sessions, with each at least 1 week apart. Each device was tested both recorded for pain sensation on the arm and on the back.
on the arm and the back, with histamine (10 mg/mL; Hollister-Stier, Subjects
Spokane, Wash) and glycerol-saline (Hollister-Stier) during each
session. During the course of 4 sessions, the locations on the arm and Male or female subjects age 18 to 70 years, with or without
back were rotated to ensure each device was tested on the upper and allergies, were eligible for the study. Subjects were excluded if they
lower arm and upper and lower back. Therefore, each session yielded had dermatographism, severe atopic dermatitis, or asthma, or were
4 test sites per device: 2 histamine tests (1 back and 1 arm) and 2 taking antidepressants. Antihistamines were withheld for 1 week
glycerol-saline tests (1 back and 1 arm). Fig 1 illustrates the back and before testing. Leukotriene antagonists and H2 antagonists were
left arm test regions for one test session. Over the course of the study, withheld for 72 hours before testing.
sites were rotated to give an equal number of tests in all areas to offset
Devices
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 Four single-headed devices and 4 multiheaded devices were
arms, with an even distribution between left and right. All heads of a tested. Single headed devices included the Greer Pick (Greer Labs),
multiheaded device were tested with histamine at the histamine Accuset (ALK-Abelló, Inc, Round Rock, Tex), Sharptest (Panatrex,
site, and all heads were tested with saline at the saline site. At the Inc, Placentia, Calif), and Quintip (Hollister-Stier). Multiheaded
conclusion of the fourth session, a mean result was determined for devices tested were the Quintest (Hollister-Stier), Quantitest
each head of the multiheaded devices, and from this, intradevice (Panatrex, Inc), Greer Track, and Multi-Test II (Lincoln Diag-
variability was determined. Single device test sites were spaced at nostics, Inc, Decatur, Ill; Fig 2).
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 Skin testing
examined, this resulted in 132 individual pricks per subject per All testing was performed first on the arms, and once results were
session. The total number of individual pricks over the course of obtained and recorded, testing proceeded on the back. The wheal and
the study for each subject was 528. With 13 subjects completing flare results were recorded at 15 minutes by obtaining the longest
the study, this yielded 6864 individual prick sites for examination. orthogonal diameters. Mean diameters were used for statistical
Before each session, antihistamines were withheld for at least 1 week, analyses. Pain was recorded immediately after application of each
and H2 antagonists and leukotriene antagonists were withheld for skin test device. Positive test solution consisted of 10 mg/mL
72 hours. histamine (Hollister-Stier), with standard glycerol saline (Hollister-
To maintain objectivity, the technician who performed all of the Stier) used as a negative solution.
tests was blind to the contents of the test solution, either histamine
or saline. A second technician who was not in the room during Statistical analysis
application of each device recorded the results. This technician was Results were analyzed by using repeated-measures ANOVA, with
blind to the device used as well as to the solution used. Before the the within-subject factors body site (upper arm, lower arm, upper
study was initiated, a representative of the manufacturer trained the back, lower back) and device. Thirteen subjects were needed to power
technician who performed the skin tests on each device. This step was the study adequately to detect a minimum difference of 2 mm
taken to achieve the best possible results by using the manufacturer’s between each device. When calculating sensitivity and specificity, a
recommended skin testing technique. true-positive result was considered a histamine wheal of 3 mm or
Pain assessment was performed by using the Wong-Baker FACES greater, and a true-negative result was a glycerol-saline wheal less
pain rating scale8 immediately after application of each skin test than 3 mm. A result was considered false-negative if a histamine
J ALLERGY CLIN IMMUNOL Carr et al 343

ocular diseases
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 32.2 years (range, 22-57), and 7 subjects had a history of
if the glycerol-saline site was 3 mm or greater. Results are presented atopy.
as the means 6 SDs, and for multiheaded devices, the average of all
heads was used in the calculation of sensitivity and specificity. Interdevice comparisons
Sensitivity and specificity of each device are presented as proportions
with 95% CIs, and devices were compared by using the Fisher exact Histamine and saline reactions are presented in Table I.
test (2-tailed). Sensitivity was calculated by dividing true-positive Controlling for site (arm vs back), there was a significant
results by the sum of true-positive and false-negative results. difference in histamine wheal size among all devices in
Specificity was calculated by dividing true-negative results by the each device group (P < .008 for all comparisons), except
sum of true-negative and false-positive results. for no significant difference between the Accuset and the
When multiheaded devices were analyzed, intradevice variability Quintip (P = .28) and the Multi-Test II and Quantitest
was described by using the coefficients of variation (CVs; presented (P = .27). The largest reactions to histamine base were
as medians with the interquartile range) for each device. For each found with 2 single devices, Sharptest and Greer Pick.
multiheaded device, the wheal produced by each head was compared
There were no significant differences in saline wheal
by using repeated-measures ANOVA.
reactions. In addition, all mean histamine flares were
Pain scores were compared among devices by using the Wilcoxon
signed-rank test: median pain scores were presented as well as the greater than 10 mm, and mean saline flares were below
proportion of pain scores above a value of 2 (representing mild pain 5 mm. Table II gives the number of results that exceeded
on the Wong-Baker FACES pain rating scale). For interdevice the limits for positive and negative reactions set for
comparisons of pain, wheal, and flare size within the single-headed histamine and saline. For histamine wheal reactions, the
or multiheaded groups, there are 6 possible pairwise analyses. Using Greer Pick gave the lowest number of false-negative
a Bonferroni correction of the overall experimental P value of .05, results (2/208 or 0.96%); the range for single devices was
a P value of .008 (.05/6) or less is considered significant. 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.
RESULTS Single devices demonstrated a high degree of repro-
ducibility, with CVs ranging from 0.22 to 0.37 (Table I).
Twenty subjects were recruited for the study, and The CV reported in Table I for the multiheaded devices
7 subjects did not complete because of pregnancy (1) represents a CV of the mean of all heads. For intradevice
and military operational requirements (6). Eight men variability, or differences between each head of a multi-
and 5 women completed the study. The mean age was headed device, see Table III.
AUGUST 2005
TABLE I. Outcome measures for 8 devices*
Histamine wheal, Histamine flare, Saline wheal, Saline flare, Sensitivity % Specificity %
mean 6 SD mean 6 SD CV mean 6 SD mean 6 SD (95% CI) (95% CI)
Single devices
Sharptest 7.1 6 1.7 31.6 6 8.4 0.22 0.03 6 0.3 3.2 6 2.8 97 (91-991) 99 (94-991)
Greer Pick 6.6 6 1.8 33.3 6 9.5 0.37 0.0 6 0.0 2.7 6 2.4 98 (93-991) 100 (97-100)
Accuset 5.1 6 1.9 24.3 6 10.7 0.34 0.1 6 0.5 1.5 6 2.4 92 (85-97) 98 (93-991)
Quintip 4.8 6 1.7 22.6 6 9.3 0.36 0.0 6 0.0 1.1 6 2.6 95 (89-99) 100 (97-100)
Multidevices
Multi-Test II 5.9 6 1.3 26.0 6 5.7 0.23 0.02 6 0.2 3.3 6 1.5 93 (91-95) 99 (98-991)
Quantitest 5.7 6 1.6 25.6 6 7.3 0.34 0.01 6 1.6 3.2 6 1.8 89 (86-91) 99 (98-991)
Quintest 4.3 6 1.4 19.9 6 7.8 0.25 0.0 6 0.0 0.8 6 1.5 86 (82-89) 100 (99-100)
Greer Track 3.2 6 1.3 16.5 6 6.4 0.42 0.012 6 0.1 3.4 6 1.4 56 (52-60) 99 (98-991)
*Values for wheal and flare expressed in millimeters.
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
ocular diseases
Histamine wheal, mm Histamine flare, mm Saline wheal, mm Saline flare, mm

Total test <3 Range <10 Range >3 Range >10 Range
Single devices
Sharptest 208 3 0-10 1 9-60 0 0-3 1 0-15
Greer Pick 208 2 0-12 2 7-75 0 0 0 0-10
Accuset 208 8 0-8 11 0-50 1 0-4 1 0-12
Quintip 208 5 0-10 9 0-40 0 0 2 0-15
Multidevices
Multi-Test II 1664 57 0-12 42 0-50 4 0-4 4 0-20
Quantitest 1664 94 0-11 85 0-62 1 0-5 3 0-32
Quintest 1040 73 0-11 93 0-50 0 0 1 0-15
Greer Track 1664 366 0-11 361 0-47 2 0-5 1 0-22
TABLE III. Intradevice variability for multiheaded Arm versus back comparisons
devices expressed as CV
There was a significant difference in histamine wheal
Histamine wheal,* CVy (interquartile sizes between the arms and backs for all devices (P <
Multidevices mean 6 SD range) .0005; Fig 3). Histamine wheals for all single devices were
Multi-Test II 5.9 6 1.3 0.20 (0.14-0.44) significantly larger on the arms (P < .05 for all compar-
Quantitest 5.7 6 1.6 0.23 (0.14-0.50) isons), and wheals for all multiheaded devices (except the
Quintest 4.3 6 1.4 0.25 (0.18-0.59) Quintest) were larger on the back (P < .0013). The
GreerTrack 3.2 6 1.3 0.93 (0.70-1.39) Quintest device was larger on the back, but this difference
did not reach statistical significance (P = .17). There was
*Values expressed in millimeters.
CV median (interquartile range), intradevice variability. no significant difference between upper and lower arm.
There also was no significant difference between upper
and lower back.
Multidevices: intradevice variability
Sensitivity and specificity Intradevice variability reactions are presented in Table
The results of device sensitivity and specificity are III. Analyzing the multiheaded devices for intradevice
listed in Table I. All single devices and the Multi-Test II variability, there were significant differences in the wheal
had sensitivities >90%, and there was no significant sizes between the various heads for each device (P < .009
difference in sensitivity among the single devices and for all devices). Fig 4 illustrates the intradevice variability
the Multi-Test II. The Multi-Test II was more sensitive for the Greer Track. With the 8-headed devices (Greer
compared with all other multiheaded devices (P < .002). Track, Multi-Test II, and Quantitest), the greatest degree
The Quintest was less sensitive than the Greer Pick, of variability was found comparing the interior heads (S2,
Sharptest, and Multi-Test II. The Greer Track was less S3, S6, S7) with the corner heads (S1, S4, S5, S8) for all of
sensitive than all other devices (P < .0005). them.
J ALLERGY CLIN IMMUNOL Carr et al 345
FIG 3. Mean histamine wheal size in millimeters for all devices.
TABLE IV. Pain outcome measures for 8 devices
Pain %*
Mean pain Median pain (95% CI)
Single devices

Sharptest 1.17 1 13% (7-22)
ocular diseases
Greer Pick 0.88 1 5% (1-11)
Accuset 0.94 1 9% (4-16)
Quintip 1.0 1 7% (2-14)
Multidevices
FIG 4. Mean intradevice variability of the Greer Track. Sites are Multi-Test II 1.62 1 26% (17-36)
labeled S1 through S8, with sites S1, S4, S5, and S8 representing Quantitest 1.74 1 26% (17-36)
the corners and S2, S3, S6, and S7 representing the interior heads. Quintest 1.45 1 17% (10-26)
Greer Track 2.04 2 34% (23-43)
*Percentage of values above 2 on the Wong-Baker FACES pain rating

The greatest degree of intradevice variability was found scale (percentage of values interpreted as greater than mild pain).
within the Greer Track.
Pain
Median pain scores for all of the devices was 1.0, except are statistically significant differences among virtually all
for the Greer Track, with a median of 2.0 (Table IV). devices tested. One device that stands out with the lowest
Reports of pain were considered minor, with only 1 pain performance in all areas is the Greer Track. This device
rating reported above 6 on a scale from 0 to 10 on the was the most painful, had the smallest mean histamine
Wong-Baker FACES pain rating scale.8 The highest pain wheals and flares, was the least sensitive, and had the
rating was for the Greer Track (34% of pain scores above greatest degree of intradevice variability. These statistical
2), and the minimum pain reported was for the Greer Pick findings of performance may very well equate to clinically
(5% of pain scores above 2). All single devices were significant differences in performance. Excluding the
significantly less painful than the multiheaded devices Greer Track, it is unknown whether the statistical differ-
(P < .0005). Comparing the single devices, Sharptest pain ences among the remaining 7 devices will equate to
scores were significantly higher than Greer Pick (P < clinically significant differences in performance. Of the
.0005) and Accuset (P = .001). For the multidevices, remaining 7 devices, all had mean histamine wheals
Greer Track scores were significantly higher than all other greater than 3 mm and mean histamine flares greater
devices (P < .0005 for all comparisons), and the Quantitest than 10 mm, with sensitivities from 86% to 97%. In
was more painful than the Quintest (P = .001). In addition, addition, all of the remaining 7 devices had specificities
pain was negatively associated with sensitivity of 98% or greater. Therefore, each individual provider
(r = 20.77; P = .027), because the devices with greater should determine which device is best for that provider’s
sensitivity had lower pain scores. For the Greer Track practice. Keep in mind that these studies were performed
multidevice with 56% sensitivity, 34% of pain scores under the best of circumstances, with all tests conducted
were above 2. For the Greer Pick single device with by 1 technician who was certified by a representative of
98% sensitivity, only 5% of pain scores were above 2. 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
DISCUSSION device. In addition, these findings may not be directly
applicable to allergen skin testing, because we looked only
We have concluded a head-to-head prospective com- at histamine and glycerol-saline responses. A separate
parative study of 8 skin test devices and found that there study may be required to compare devices for this purpose.
AUGUST 2005
When choosing a skin test device, a few points are In contrast with previous studies, we did not find a clear
worth consideration. First, in our study, single devices had relationship between pain and wheal size.6 In fact, the
larger reactions on the arm, and multidevices had larger device that resulted in the greatest degree of pain had the
reactions on the back. Historically, it has been thought smallest mean histamine wheal size. Again, we think it is
that the back has been more reactive than the arms, and up to the individual practitioner to consider these differ-
although our multiheaded device data concur with this, ences when using a given device in practice.
our single-headed device data do not. There are several Finally, when comparing sensitivity and specificity,
possibilities for this observation, including intraoperator there are few differences among devices, with 2 excep-
variability, inadvertent operator bias, or a true difference. tions. The Greer Track was less sensitive than all other
With regard to the difference between single and multidevices, and the Quintest was less sensitive than the Greer
headed devices, we think that this difference is related to Pick, Sharptest, and Multi-Test II. With the 6 remaining
the back being a flatter surface; therefore, better contact is devices, there was no significant difference among
made with all of the test sites on a multiheaded device. The sensitivities. In addition, we found no significant differ-
arms are technically more challenging when placing a ences in specificity among devices. Overall, we found a
multiheaded device, given natural curvatures. To com- very low false-positive rate in all devices when using the
pensate for the curvatures and to ensure contact with all manufacturer’s recommended skin testing technique.
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. CONCLUSION
However, we think that this rocking motion is responsible

ocular diseases
for the differences noted between individual test sites We have completed a prospective, head-to-head com-
within a given multiheaded device, or intradevice varia- parison of the performance of 8 skin test devices. This
bility (Fig 4). With this rocking motion, more pressure is study was performed under the best of clinical circum-
exerted on the skin from the corner test sites. It is important stances, with 1 technician, trained by a representative of
to note that single devices also have differences in the the manufacturer, who performed all skin testing, and
recommended technique of application. The Quintip and another technician who read all of the results. We have
Sharptest use a simple downward perpendicular pressure, found significant differences among all devices tested.
and both of these devices have a depth control feature. Whether this equates to clinical differences is yet to be
Manufacturer-recommended techniques for the Greer Pick determined. Overall, skin testing is associated with min-
and Accuset are slightly more complicated. The skin imal pain, and individual providers should choose a skin
surface is penetrated at an angle, and then a flick, prick- test device on the basis of their own practice setting.
not-puncture technique is used. Neither of these last 2 As new devices are being produced, this study suggests
devices has a depth control feature. Given the tech- the need for continued evaluation of these devices in a
nique and lack of depth control, the Greer Pick and prospective, nonbiased fashion.
Accuset may result in greater intertechnician variability
if care is not taken to control for these features.
REFERENCES
However, with correct technique, and by using 1 tech-
1. Li JT, Lockey RF, Bernstein IL, Portnoy JM, Nicklas RA. Allergen
nician, all single devices had sensitivities greater than immunotherapy: a practice parameter. Ann Allergy Asthma Immunol
90% while maintaining specificities of 98% or greater. 2003;90(suppl):1-40.
Another observation from our study is that skin testing 2. Position paper: immunotherapy. (EAACI) The European Academy of
is not a painful procedure on average. The mean pain Allergology and Clinical Immunology. Allergy 1993;48(suppl 14):7-35.
3. Spector SL, Nicklas RA. Practice parameters for the diagnosis and
scores for all devices ranged from 0.88 to 2.04. Using the
treatment of asthma. J Allergy Clin Immunol 1995;96:707-870.
Wong-Baker FACES pain rating scale,8 this is considered 4. Adinoff AD, Rosloniec DM, McCall LI, Nelson HS. A comparison of six
mild pain. The degree of pain was significantly associated epicutaneous devices in the performance of immediate hypersensitivity
with the type of device used, with the multiheaded devices skin testing. J Allergy Clin Immunol 1989;84:168-74.
more painful than the single devices. However, when 5. Nelson HS, Rosloniec DM, McCall LI, Ikle D. Comparative performance
of five commercial prick skin test devices. J Allergy Clin Immunol 1993;
making this comparison, it is noteworthy that with a 92:750-6.
minimal increase in pain, as many as 8 times more tests are 6. Nelson HS, Lahr J, Buchmeier A, McCormick D. Evaluation of devices
applied. Therefore, with a pain score of 0.88, the Greer for skin prick testing. J Allergy Clin Immunol 1998;101:153-6.
Pick applied 1 skin test, and with a pain score of 1.62, the 7. Nelson HS, Kolehmainen C, Lahr J, Murphy J, Buchmeier A. A com-
parison of multiheaded devices for allergy skin testing. J Allergy Clin
Multi-Test II applied 8 skin tests. It is unclear whether
Immunol 2004;113:1218-9.
these observed statistical differences in pain would equate 8. Wong-Baker FACES Pain Rating Scale. In: Wong DL, Hockenberry-
to significant clinical differences, because all pain was Eaton M, Wilson D, Winkelstein ML, Schwartz P. Wong’s essentials of
considered mild. 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
Jürgen Reisinger, MSc,a Friedrich Horak, MD,a Gabrielle Pauli, MD,e
Marianne van Hage, MD,d Oliver Cromwell, PhD,f Franz König,c
Rudolf Valenta, MD,b and Verena Niederberger, MDa Vienna, Austria,
Stockholm, Sweden, Strasbourg, France, and Reinbek, Germany
Background: We have performed a double-blind, placebo- Conclusion: Our data demonstrate that vaccination with
controlled injection immunotherapy study with genetically genetically modified allergens induces IgG antibody responses
modified derivatives of the major birch pollen allergen, against the corresponding natural allergen not only in serum
Bet v 1 (Bet v 1–trimer, Bet v 1–fragments). but also in mucosal fluids, where they may protect against

Objective: To investigate whether vaccination with genetically allergen-induced inflammation. (J Allergy Clin Immunol
modified allergens induces allergen-specific antibodies in nasal 2005;116:347-54.)
ocular diseases
secretions and to study whether these antibodies affect nasal
allergen sensitivity. Key words: Allergen-specific immunotherapy, IgG antibodies,
Methods: A randomly picked subgroup of patients (n = 23; genetically modified allergens, nasal provocation
placebo, n = 10; trimer, n = 10; fragments, n = 3) was subjected
to an extensive analysis of serum samples and nasal lavage
In the last 15 years, considerable progress has been
fluids and to nasal provocation testing. Bet v 1–specific IgG1-4
made in the field of molecular allergen characterization.
and IgA antibodies were determined in serum samples
obtained before and after vaccination, after the birch pollen The repertoire of the disease-eliciting allergens has been
season, and 1 year after start of vaccination as well as in nasal recreated in the form of recombinant allergens for the most
lavage fluids obtained after the birch pollen season and 1 year common allergen sources and can be used for diagnostic
after start of vaccination by ELISA. Nasal sensitivity to tests that allow dissection of the reactivity profiles of
natural, birch pollen–derived Bet v 1 was determined by active patients down to a molecular level.1,2 In attempts to reduce
anterior rhinomanometry after the birch pollen season and IgE-mediated side effects during allergen-specific immu-
1 year after start of vaccination. notherapy, several research groups have used genetic
Results: Vaccination with genetically modified Bet v 1 engineering and peptide chemistry to develop allergen
derivatives, but not with placebo, induced Bet v 1–specific
derivatives with reduced allergenic activity.3-7 By using
IgG1, IgG2, and IgG4, and low IgA antibodies in serum, which
genetically engineered derivatives of the major birch
also appeared in nasal secretions, but no IgG3 antibodies. The
levels of therapy-induced Bet v 1–specific IgG4 antibodies in pollen allergen, Bet v 1, we have conducted a first double-
nasal secretions were significantly (P < .05) associated with blind, placebo-controlled immunotherapy study in pa-
reduced nasal sensitivity to natural, birch pollen–derived tients allergic to birch pollen.8 One group of patients was
Bet v 1 as objectively determined by controlled nasal treated with a mixture of 2 recombinant Bet v 1 fragments
provocation experiments. that exhibited strongly reduced IgE reactivity and aller-
genic activity; a second group received a recombinant
Bet v 1 trimer, also characterized by reduced allergenic
From athe Department of Otorhinolaryngology, and bthe Department of activity; and the third group was treated with aluminium
Pathophysiology, Center for Physiology and Pathophysiology, Vienna hydroxide alone, which was used as adjuvant for the
General Hospital, and cthe Department of Medical Statistics, Medical subcutaneous injections.8-12 The analysis of the immuno-
University of Vienna; dthe Department of Medicine, Clinical Immunology logical mechanisms showed that vaccination with the
and Allergy Unit, Karolinska Institutet and Hospital, Stockholm; eService
de Pneumologie, Hôpitaux Universitaires de Strasbourg; and fAllergopharma
genetically modified Bet v 1 derivatives induced de novo
Joachim-Ganzer KG, Reinbek. serum IgG antibodies that recognized the Bet v 1–wild-
Supported by grants F01811 and F01815 of the Austrian Science Fund and by type allergen and inhibited Bet v 1–induced histamine
a research grant from Biomay, Vienna. release from basophils.8 Furthermore, we obtained evi-
dence that boosts of the IgE memory responses caused by
for publication April 1, 2005.
Available online June 1, 2005. seasonal allergen exposure were reduced in actively
Reprint requests: Verena Niederberger, MD, Department of Otorhinolaryn- vaccinated patients.8
gology, Vienna General Hospital, AKH, Medical University of Vienna, In this sub-study, we asked whether vaccination with
Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: Verena. the genetically engineered allergens also leads to an
Niederberger@meduniwien.ac.at.
0091-6749/$30.00
induction of allergen-specific antibodies in the nasal
Ó 2005 American Academy of Allergy, Asthma and Immunology mucosa, the main site of allergic inflammation, and if
doi:10.1016/j.jaci.2005.04.003 so, whether such antibodies are associated with reduced
347
348 Reisinger et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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.
Cumulative injected
Pat.-Nr. Age Sex Sensitized to Symptoms Treatment Nr. of injections dose (mg)
2 33 m b, g r, c trimer 10 265
8 39 m b r, c, a trimer 9 185
15 42 m b r, c, a trimer 8 165
18 36 f b r, c, a trimer 10 169
26 27 f b, g, w r, a trimer 8 165
33 51 f b, a r, c, a trimer 8 163
34 28 m b, g, w, a r, c, a trimer 8 165
41 45 m b r, c, a trimer 9 245
44 37 m b r, c trimer 8 69
49 33 m b r, c trimer 8 165
36 45 f b, w, a r, c, a, d fragments 9 103
42 51 m b, g, w, a r, c, a fragments 7 85
64 36 m b, g r, c fragments 9 245
3 28 f b, g, w r, c placebo 8 0
5 38 m b, w r, c, a placebo 8 0
ocular diseases
6 23 m b, g, a r, c placebo 8 0
28 26 f b r, c placebo 9 0
37 30 m b, g, w, a, m r, c placebo 9 0
51 55 f b, w, a r, c placebo 9 0
56 36 m b r, c placebo 8 0
57 40 f b r, c, a placebo 8 0
58 43 m b, g, w, a r, c, a placebo 9 0
61 35 m b, g r, c, a placebo 9 0
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 in-
depth analysis of serum and nasal lavage fluids regarding
allergen-specific antibodies and extensive nasal provoca-
tion in a subgroup of 23 subjects recruited at random from FIG 1. Experimental protocol. Patients received a single course of
patients participating in the immunotherapy trial. 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.
METHODS
Recombinant allergens and vaccine (Stockholm, Strasbourg, Vienna). Patients were included if they had a
formulation positive result with skin prick test and ImmunoCAP (3.5 kU/L;
Pharmacia, Uppsala, Sweden) for Bet v 1 and natural birch pollen
Recombinant major birch pollen allergen, Bet v 1,13 mimicking extract and had a history of moderate to severe seasonal allergic
natural birch pollen–derived Bet v 1 was obtained from Biomay rhinoconjunctivitis attributable to birch pollen. Subcutaneous injec-
(Vienna, Austria). Recombinant Bet v 1 fragments and trimer were tions of aluminium hydroxide–adsorbed allergen derivatives con-
expressed in Escherichia coli and purified as described.9,10 Alumin- taining increasing doses (1-80 mg) of recombinant Bet v 1 derivatives
ium hydroxide adsorbates containing 100 mg protein/mL adsorbate (Bet v 1–trimer, Bet v 1–fragments) or placebo were administered
trimer or aluminium hydroxide alone (placebo) were formulated as at intervals of 1 to 2 weeks as a preseasonal treatment. After reaching
described14 following Good Manufacturing Practice guidelines. the maximal dose, the treatment was continued at 4-week intervals
until before the flowering season. Clinical outcome was monitored
Vaccination of patients allergic to birch pollen
by questionnaires, skin prick tests, and nasal provocation tests. The
with Bet v 1 derivatives or placebo study was approved by the local ethical committees, and written
The immunotherapy study was conducted as a placebo-controlled, informed consent was obtained from each patient.
double-blind, randomized vaccination trial over a period of 12 In a substudy, nasal lavage fluids were collected in 23 randomly
months with 1 preseasonal treatment course in 3 study centers picked patients from a total of 71 at the Vienna center. Serum samples
J ALLERGY CLIN IMMUNOL Reisinger et al 349

ocular diseases
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 the end piece of a narrow plastic tube. The respective side of the nose
tests were analyzed after deblinding of the immunotherapy study was slowly filled and emptied 5 times with 5 mL prewarmed 0.9%
(Table I). 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
Experimental protocol
2000), after treatment (February/March 2001), after the birch pollen
The sequence of serum sampling, nasal washings, and nasal season (May 2001), and in autumn/winter 2001. Nasal lavage fluids
provocation tests is illustrated in Fig 1. and serum samples were stored at 220°C until analysis.
Collection of nasal lavage fluids Nasal provocation tests
and serum samples Nasal provocation tests were performed with natural birch pollen
Nasal lavage fluids were obtained from 23 patients (treated with extract containing defined Bet v 1 concentrations before treatment
Bet v 1 fragments, n = 3; Bet v 1 trimer, n = 10; placebo, n = 10) (November/December 2000), after the birch pollen season (May
after the birch pollen season (May 2001) and 1 year after beginning of 2001), and after 1 year (October 2001). Allergen solutions were
the study (autumn/winter 2001). freshly prepared from the standardized lyophilized birch pollen
To obtain nasal secretions, patients sat with their head bent extract, were kept at 4°C between tests, and were discarded after
forward, and 1 nostril was sealed with a foam plastic plug encircling 48 hours.
AUGUST 2005
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 determi-
nations); 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
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
FIG 3. Association of vaccination-induced nasal Bet v 1–specific were considered statistically significant.
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
RESULTS
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 Demographic, clinical, and immunological
ocular diseases
with Bet v 1–fragments; white spots, placebo treatment.

characterization of patients allergic
to birch pollen
Thirteen men and 10 women with a mean age of
The baseline levels of nasal parameters (nasal flow and resistance) 38 years were analyzed. Nine of these patients were aller-
were established by active anterior rhinomanometry (Allergopharma gic exclusively to birch pollen, whereas the other 14 pa-
Rhinomanometer; Allergopharma, Reinbek, Germany) without any tients also had allergic symptoms to other allergen sources.
interaction and 10 minutes after application of 0.9% sodium chloride. The patients were sensitized exclusively to the major
Thereafter, the patient received increasing doses of birch pollen birch pollen allergen, Bet v 1, within birch pollen. All
extract solution containing 0.0064 mg/mL, 0.064 mg/mL, 0.64 mg/ patients had allergic rhinitis, all but 1 also had allergic
mL, 6.4 mg/mL, and 64 mg/mL Bet v 1, or placebo. Approximately
conjunctivitis, and 13 had mild seasonal allergic asthma.
0.05 mL (1 pump action) of allergen solutions was administered into
both nostrils by using a metered dose pump. During application of test On an average, patients had received 8.52 injections
solutions, patients had to hold their breath in full inspiration to avoid (placebo = 8.50, range = 8-9; treatment = 8.54, range = 7-10).
bronchial provocation. Changes in nasal parameters were determined Actively treated patients had received a cumulative
15 and 20 minutes after allergen application. The nasal flow and injected dose of 164.4 mg recombinant protein on average
resistance values with saline were used as a reference for calculations (range = 69-265 mg).
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; Vaccination with recombinant Bet v 1
sneezing: 3-53 = 1 score, >53 = 2 scores; systemic symptoms— derivatives induces Bet v 1–specific
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
IgG1, IgG2, and IgG4 but not IgG3 and
scores). The test was regarded as positive if a 40% or more reduction IgA antibodies in nasal secretions
of nasal air flow was obtained or if a symptom score of 3 was Levels of IgG1-4 subclass and IgA antibodies specific
exceeded. If the test was negative after 15 and 20 minutes, the testing for Bet v 1 were measured in nasal secretions obtained in
procedure was repeated with a 10-fold dose increase. When the test May (after the birch pollen season) and in October 2001
became positive, the maximum allergen concentration tolerated by
(1 year after the beginning of immunotherapy). In May
the patient was recorded, and the provocation test was concluded.
2001, IgG1 levels to Bet v 1 were significantly higher in
Reduction of nasal sensitivity to birch pollen extract in nasal
provocation experiments was expressed as change in tolerated nasal lavage fluids from actively treated patients than in
concentration steps. nasal lavage fluids from patients who had received
placebo (P < .05; Fig 2, A, left panel). We also found
Measurement of antibodies specific for higher Bet v 1–specific IgG2 and IgG4 levels in nasal
wild-type Bet v 1, Bet v 1–fragments, secretions from actively treated patients compared with
and Bet v 1–trimer by ELISA the placebo group, but these differences did not reach
significance (IgG2, Fig 2, B, left panel; IgG4, Fig 2, C, left
Serum IgG1, IgG2, IgG3, and IgG4 subclass antibodies (serum
dilution, 1:50) specific for recombinant (r) Bet v 1, Bet v 1–fragments,
panel). In October 2001, 1 year after treatment, antibody
and Bet v 1–trimer were measured as described.15 Bet v 1–specific concentrations declined in the nasal lavage samples,
serum IgA antibodies (serum dilution, 1:50) were detected by using indicating that vaccination-induced elevations of Bet v 1–
an alkaline phosphatase–conjugated mouse monoclonal antihuman specific IgG levels in nasal secretions show a kinetic
IgA antibody (G20-359; dilution, 1:1000; BD Pharmingen, San similar to that of serum antibody levels (Fig 2, A-C, left
FIG 4. Vaccination with Bet v 1 derivatives (trimer, fragments) induces IgG1 (A) and IgG4 (B) antibodies to wild-
type 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.

ocular diseases
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 between the cumulative injected dose of the derivatives
IgG3 (data not shown) and IgA (Fig 2, D, left panel) levels. and IgG1 (May 2001) in nasal lavage fluids (Fig 3).
Furthermore, we noted no relevant difference between
May 2001 and October 2001 measurements of nasal IgG3 Bet v 1–specific nasal antibodies mirror
and IgA levels. serum antibody responses
The levels of Bet v 1–specific IgG1 in nasal secretions Sera had been collected at several time points (Fig 1;
were dependent on the dose of genetically modified before therapy, autumn 2000; after therapy, spring 2001;
allergens injected during immunotherapy. A statistically after the birch pollen season, May 2001; and 1 year after
significant correlation (r = 0.446; P < .05) was found the beginning of the study, autumn 2001).
AUGUST 2005
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
ocular diseases
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 either the nasal IgG2, IgG3, and IgA levels and the results
Bet v 1–specific serum IgG1 (Fig 2, A, right panel), IgG2 from the May nasal provocation tests, or the October nasal
(Fig 2, B, right panel), and IgG4 (Fig 2, C, right panel) antibody levels and the outcomes of the October nasal
antibody levels than placebo-treated patients in February provocation tests. None of the patients reacted to saline in
2001 and May 2001 (all P values <.05). No relevant nasal provocation tests.
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 DISCUSSION
specific for intact Bet v 1 as well as for Bet v 1 derivatives
(Fig 4). We have recently described that injection immunother-
Significant correlations were found between serum apy with genetically modified derivatives (ie, rBet v 1
and nasal lavage fluid IgG1 (r = 0.572; P < .01; Fig 5, A), fragments, rBet v 1 trimer) of the major birch pollen
IgG2 (r = 0.498; P < .05; Fig 5, B), and IgG4 (r = 0.623; allergen, Bet v 1, induces serum IgG antibody responses
P < .01; Fig 5, C) antibody levels in May and between recognizing the wild-type allergen, which inhibit allergen
IgG4 levels in October 2001 (r = 0.461; P < .05; Fig 5, D). induced histamine release from basophils in vitro.8 In this
No such correlations were found concerning IgA levels substudy, we demonstrate that IgG antibodies appear also
(data not shown). in nasal secretions and that their presence is associated
with reduced allergen-specific nasal sensitivity.
Vaccination-induced Bet v 1–specific IgG
Treatment of patients with genetically modified ver-
antibody levels are associated with reduced sions of Bet v 1 induced the generation of antibodies, not
nasal sensitivity to Bet v 1 only against the Bet v 1–fragments and Bet v 1–trimer, but
All 23 patients completed the first (November/ also against wild-type Bet v 1 allergen in serum and nasal
December 2000) and third (October 2001) provocation lavage fluids. This finding can be explained by the fact that
test, but 2 trimer-treated patients and 1 placebo-treated both types of genetically engineered Bet v 1 derivatives
patient did not undergo the second (May 2001) provoca- were found to induce IgG antibodies in animals. These
tion. Therapy-induced Bet v 1–specific IgG4 antibody IgG antibodies recognized natural, pollen-derived Bet v 1
levels (May 2001) in nasal secretions were significantly and inhibited allergic patients’ IgE binding to Bet v 1.9,17
correlated with a reduced nasal sensitivity to birch pollen– Because the therapy-induced responses to wild-type
derived Bet v 1 allergen (r = 0.590; P < .01; Fig 6, B). Bet v 1 were as strong as (ie, trimer) and sometimes
Furthermore, there is a weak correlation between nasal even stronger than to the derivatives (ie, fragments), it
IgG1 levels and reduced nasal sensitivity (r = 0.425), but appears that most of the IgG response induced by vacci-
this did not achieve statistical significance (P = .062; Fig nation with the derivatives is directed against the wild-
6, A). By contrast, there were no associations between type allergen. The composition of the Bet v 1–specific
antibodies in nasal lavage fluids mirrored that of the serum 5. Ferreira F, Wallner M, Breiteneder H, Hartl A, Thalhammer J, Ebner C.
Genetic engineering of allergens: future therapeutic products. Int Arch
antibodies regarding subclasses and kinetics. In serum as
Allergy Immunol 2002;128:171-8.
well as in nasal lavage fluids, Bet v 1–specific IgG1, IgG4, 6. Vrtala S, Focke-Tejkl M, Swoboda I, Kraft D, Valenta R. Strategies for
and IgG2, low IgA, and no IgG3 responses were induced converting allergens into hypoallergenic vaccine candidates. Methods
by vaccination, and the kinetics of antibody levels were 2004;32:313-20.
similar in serum and nasal lavage fluids. Bet v 1–specific 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
IgG levels were high after vaccination (May) and declined retaining human T lymphocyte reactivity. J Immunol 2004;173:5872-9.
almost to baseline by October of the same year. The latter 8. Niederberger V, Horak F, Vrtala S, Spitzauer S, Krauth MT, Valent P,
observations and previous studies18,19 on allergen-specific et al. Development of a novel allergy vaccine: genetically engineered
antibody reactivities in mucosal fluids support the as- allergens prevent progression of allergic disease. Proc Nat Acad Sci
U S A 2004;101(suppl 2):14677-82.
sumption that the nasal allergen-specific IgG may result
9. Vrtala S, Hirtenlehner K, Vangelista L, Pastore A, Eichler HG, Sperr
from transudation. WR, et al. Conversion of the major birch pollen allergen, Bet v 1 into
The importance of the vaccine-induced Bet v 1–specific two nonanaphylactic T cell epitope-containing fragments: candidates for
IgG antibodies is indicated by the fact that their concen- a novel form of specific immunotherapy. J Clin Invest 1997;99:1673-81.
trations were associated with a reduction of nasal allergen 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
sensitivity and by the observation that this protective pollen allergen Bet v 1. FASEB J 2001;15:2045-7. (Express article
effect disappeared after their decline in October. We and Available at: www.fasebj.org/cgi/reprint/00-0767fjev1).
others have found that the vaccine-induced IgG antibodies 11. Pauli G, Purohit A, Oster JP, De Blay F, Vrtala S, Niederberger V, et al.
inhibited allergen-induced degranulation of basophils20-23 Comparison of genetically engineered hypoallergenic rBet v 1 derivatives

with rBet v 1 wild-type by skin prick and intradermal testing: results
in vitro and hence assume also that the nasal Bet v 1–
ocular diseases
obtained in a French population. Clin Exp Allergy 2000;30:1076-84.
specific IgG may act as blocking antibodies that mask IgE 12. van Hage-Hamsten M, Kronqvist M, Zetterstrom O, Johansson E,
epitopes of Bet v 1 and thus prevent mast cell degranu- Niederberger V, Vrtala S, et al. Skin test evaluation of genetically
lation in the submucosal tissues. In addition, it is possible engineered hypoallergenic derivatives of the major birch pollen allergen,
that the Bet v 1–specific IgG acts as protective shield 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
preventing the intrusion of allergen into the submucosa. In pollen season. J Allergy Clin Immunol 1999;104:969-77.
fact, several recent studies point to the importance of 13. Ferreira FD, Hoffmann-Sommergruber K, Breiteneder H, Pettenburger
allergen-specific IgG antibodies for the success of immu- K, Ebner C, Sommergruber W. Purification and characterization of
notherapy.20,24 For example, it has been demonstrated that recombinant Bet v 1, the major birch pollen allergen: immunological
equivalence to natural Bet v 1. J Biol Chem 1993;268:19574-80.
allergen-specific IgG can inhibit IgE-mediated allergen
14. Mahler V, Vrtala S, Kuss O, Diepgen TL, Suck R, Cromwell O, et al.
presentation to T cells.25 Vaccines for birch pollen allergy based on genetically-engineered
The nasal mucosa is one of the most important sites for hypoallergenic derivatives of the major birch pollen allergen, Bet v 1.
allergic inflammation, and there is also increasing evi- Clin Exp Allergy 2004;34:510-2.
dence that local IgE production in the nasal mucosa plays 15. Vrtala S, Susani M, Sperr WR, Valent P, Laffer S, Dolecek C, et al.
Immunologic characterization of purified recombinant timothy grass
an important role in systemic IgE responses.26 In this pollen (Phleum pratense) allergens (Phl p 1, Phl p 2, Phl p 5). J Allergy
context, it should be noted that patients vaccinated with Clin Immunol 1996;97:781-7.
the genetically modified Bet v 1 derivatives had exhibited 16. Denepoux S, Eibensteiner PB, Steinberger P, Vrtala S, Visco V,
lower boosts of systemic IgE production after seasonal Weyer A, et al. Molecular characterization of human IgG monoclonal
birch pollen exposure than the placebo-treated group.8 It is antibodies specific for the major birch pollen allergen Bet v 1: anti-
allergen IgG can enhance the anaphylactic reaction. FEBS Lett 2000;
hence tempting to speculate that vaccine-induced nasal 465:39-46.
IgG antibodies may inhibit the boosting of IgE memory 17. Vrtala S, Akdis CA, Budak F, Akdis M, Blaser K, Kraft D, et al. T cell
cells in the nasal mucosa by allergen contact in vaccinated epitope-containing hypoallergenic recombinant fragments of the major
patients. birch pollen allergen, Bet v 1, induce blocking antibodies. J Immunol
2000;165:6653-9.
Should further studies provide evidence for the impor- 18. Hoffmann-Sommergruber K, Ferreira ED, Ebner C, Barisani T,
tance of allergen-specific nasal IgG antibody responses Korninger L, Kraft D, et al. Detection of allergen-specific IgE in tears
for the success of immunotherapy, it will be possible to of grass pollen-allergic patients with allergic rhinoconjunctivitis. Clin
develop more effective protocols for allergen-specific Exp Allergy 1996;26:79-87.
19. Aghayan-Ugurluoglu R, Ball T, Vrtala S, Schweiger C, Kraft D,
immunotherapy.
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.
REFERENCES 20. Flicker S, Valenta R. Renaissance of the blocking antibody concept in
1. Valenta R, Kraft D. From allergen structure to new forms of allergen- type I allergy. Int Arch Allergy Immunol 2003;132:13-24.
specific immunotherapy. Curr Opin Immunol 2002;14:718-27. 21. Mothes N, Heinzkill M, Drachenberg KJ, Sperr WR, Krauth MT, Majlesi
2. Hiller R, Laffer S, Harwanegg C, Huber M, Schmidt WM, Twardosz A, Y, et al. Allergen-specific immunotherapy with a monophosphoryl lipid
et al. Microarrayed allergen molecules: diagnostic gatekeepers for allergy A-adjuvanted vaccine: reduced seasonally boosted IgE production and
treatment. FASEB J 2002;16:414-6. inhibition of basophil histamine release by therapy-induced blocking
3. Valenta R. The future of antigen-specific immunotherapy of allergy. Nat antibodies. Clin Exp Allergy 2003;33:1-11.
Rev Immunol 2002;2:446-53. 22. Ball T, Sperr WR, Valent P, Lidholm J, Spitzauer S, Ebner C, et al.
4. Singh MB, de Weerd N, Bhalla PL. Genetically engineered plant Induction of antibody responses to new B cell epitopes indicates
allergens with reduced anaphylactic activity. Int Arch Allergy Immunol vaccination character of allergen immunotherapy. Eur J Immunol 1999;
1999;119:75-85. 29:2026-36.
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23. Clinton PM, Kemeny DM, Youlten LJ, Lessof MH. Histamine release from 25. Wachholz PA, Kristensen Soni N, Till SJ, Durham SR. Inhibition of
peripheral blood leukocytes with purified bee venom allergens: effect of allergen-IgE binding to B cells by IgG antibodies after grass pollen
hyperimmune beekeeper plasma. Int Arch Allergy Immunol 1989;89:43-8. immunotherapy. J Allergy Clin Immunol 2003;112:915-22.
24. Wachholz PA, Durham SR. Mechanisms of immunotherapy: IgG 26. Durham SR, Gould HJ, Hamid QA. Local IgE production in nasal
revisited. Curr Opin Allergy Clin Immunol 2004;4:313-8. allergy. Int Arch Allergy Immunol 1997;113:128-30.
ocular diseases
AUGUST 2005
23. Clinton PM, Kemeny DM, Youlten LJ, Lessof MH. Histamine release from 25. Wachholz PA, Kristensen Soni N, Till SJ, Durham SR. Inhibition of
peripheral blood leukocytes with purified bee venom allergens: effect of allergen-IgE binding to B cells by IgG antibodies after grass pollen
hyperimmune beekeeper plasma. Int Arch Allergy Immunol 1989;89:43-8. immunotherapy. J Allergy Clin Immunol 2003;112:915-22.
24. Wachholz PA, Durham SR. Mechanisms of immunotherapy: IgG 26. Durham SR, Gould HJ, Hamid QA. Local IgE production in nasal
revisited. Curr Opin Allergy Clin Immunol 2004;4:313-8. allergy. Int Arch Allergy Immunol 1997;113:128-30.
ocular diseases
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
Background: The pharmacokinetics and pharmacodynamics Key words: H1-antihistamine, levocetirizine, pharmacokinetics,
of medications may differ between children and adults, pharmacodynamics, wheal, flare, allergic rhinitis, urticaria, children
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 The pharmacokinetics (absorption, distribution, metab-
not yet been studied prospectively in school-age children. olism, and excretion) and pharmacodynamics of many
Objectives: This study was performed to investigate medications, including those used in the treatment of
levocetirizine pharmacokinetic disposition and pharma- allergic diseases, have not been optimally investigated in

codynamics in relation to skin reactivity to histamine
the pediatric population.1 In the absence of such clinical
ocular diseases
in children aged 6 to 11 years.
Methods: Blood samples were obtained at predose baseline
pharmacology data, drug doses and dose intervals have to
and at defined intervals up to and including 28 hours after be extrapolated from those recommended for adults, and
a 5-mg levocetirizine dose. Concurrently, epicutaneous tests the dose and dose interval selected may not be optimally
with histamine phosphate, 1 mg/mL, were performed. efficacious or safe in children. Indeed, many drug regu-
Wheals and flares were traced at 10 minutes, and the areas latory agencies now mandate clinical pharmacology stud-
were measured with a computerized digitizing system. ies in the pediatric population.2
Results: In children aged 8.6 6 0.4 years (6 SEM), the peak More than 40 H1-antihistamines are used in the treat-
levocetirizine concentration was 450 6 37 ng/mL, and the time ment of allergic rhinitis, urticaria, and other diseases.3
at which peak concentrations occurred was 1.2 6 0.2 hours. Most of the orally administered H1-antihistamines are
The terminal elimination half-life was 5.7 6 0.2 hours, the
available in dosage formulations suitable for administra-
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
tion to children and even to infants; however, only 11 of
areas, the wheals and flares produced by histamine the 40 H1-antihistamines have been studied prospectively
phosphate were significantly decreased from 1 to 28 hours, in children with regard to their pharmacokinetics and
inclusive (P < .05). Mean maximum inhibition of wheals pharmacodynamics.4-23 These studies have generally been
and flares occurred from 2 to 10 hours (97% 6 1%) and conducted after administration of a single dose,5-10,12-20
from 2 to 24 hours (93% 6 1%), respectively. but 3 studies have been performed at steady state,11,12,20
Conclusions: Levocetirizine had an onset of action within and in a few studies, a population pharmacokinetic
1 hour and provided significant peripheral antihistaminic design21-23 has been used. The clinical pharmacology of
activity for 28 hours after a single dose. Once-daily dosing a few of the first-generation H1-antihistamines, such as
may be optimal in children aged 6 to 11 years, as it is in
chlorpheniramine, brompheniramine, diphenhydramine,
adults. (J Allergy Clin Immunol 2005;116:355-61.)
and hydroxyzine, was investigated after they had been
used in children for several decades. In contrast, the
pharmacokinetics and pharmacodynamics of the second-
generation H1-antihistamines cetirizine, fexofenadine,
ebastine, loratadine, levocetirizine, and mizolastine have
From athe Department of Pediatrics and Child Health, Department of been investigated in the pediatric population relatively
Immunology, Canadian Institutes of Health Research National Training early in drug development.
Program in Allergy and Asthma, Faculty of Medicine, and bthe Faculty of
Pharmacy, Department of Pediatrics and Child Health, Faculty of Medicine,
In the present study our objective was to characterize
The University of Manitoba. the pharmacokinetics and pharmacodynamics of the
Supported by an Institutional Grant from UCB Pharma, Inc (Belgium) to the new H1-antihistamine levocetirizine in children aged 6
University of Manitoba and the Health Sciences Centre. to 11 years. Levocetirizine24-29 (Fig 1) is the active R-
Disclosure of potential conflict of interest: Grants and research support from
enantiomer of the racemate cetirizine. It is highly selective
UCB Pharma.
Received for publication December 22, 2004; revised April 4, 2005; accepted for the human histamine H1-receptor, at which it has
for publication April 11, 2005. twice the binding affinity of cetirizine. Levocetirizine
Available online June 1, 2005. has conformational stability and is not converted to
Reprint requests: F. Estelle R. Simons, MD, FRCPC, 820 Sherbrook St, dextrocetirizine, the S-enantiomer, which has 30-fold
Winnipeg, Manitoba, Canada R3A 1R9. E-mail: lmcniven@hsc.mb.ca.
0091-6749/$30.00
less binding affinity than cetirizine at the H1-receptor.
Ó 2005 American Academy of Allergy, Asthma and Immunology Levocetirizine is minimally metabolized; during the
doi:10.1016/j.jaci.2005.04.010 week after administration of a single oral 14C-labeled dose
355
356 Simons and Simons J ALLERGY CLIN IMMUNOL
AUGUST 2005
26, and 28 hours afterward. The first 1 mL of blood was discarded.

Abbreviations used After each sample was obtained, the catheter was rinsed with 1.5 mL
EC50: Plasma concentration producing 50% of Emax of 0.9% saline. Blood samples were centrifuged at room temperature
Emax: Maximum effect attributable to medication 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 investi-
to adults, 85.4% and 12.9% of the drug can be recovered gator who performed epicutaneous tests with histamine phosphate,
unchanged in urine and feces, respectively.26 Like enan- 1 mg/mL, on the volar surfaces of the forearms by using sterilized
tiomers of other medications, levocetirizine is considered disposable straight needles (Coates & Clark, Greer, SC) and the prick-
to be a new chemical entity, and as such, its pharmaco- through drop technique. All skin tests were performed in duplicate.
kinetics, pharmacodynamics, efficacy, and safety need 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.
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 Analytic methods
also have peripheral H1-antihistaminic activity lasting at Levocetirizine concentrations were determined in plasma samples
least 24 hours after a single dose. 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

ocular diseases
precision were better than 10% throughout the range. The lower limit
METHODS of quantification for the assay was 12 ng/mL.
To test the hypothesis stated above, we performed a prospective, Wheal-and-flare circumferences were traced with a pen at
open-label, single-dose study of levocetirizine involving objective 10 minutes and transferred to paper by using transparent tape. The
pharmacokinetic and pharmacodynamic measurements. Approval tracings were scanned, and the areas were calculated with Sigma-
for levocetirizine administration was obtained through a New Drug Scan (Jandel Scientific, San Rafael, Calif). By using this system, with
Submission to Health Canada. The study protocol was approved wheal-and-flare sizes ranging from 0.05 to 5.0 cm2 and a sample size
by the University of Manitoba Research Ethics Board on the Use of of 14 children, differences of 20% could be detected with a 95% level
Human Subjects in Research. Before study entry, written assent was of confidence.
obtained from each child, and written informed consent was obtained
from the parent or parents of each child. Data analysis
Selection of participants Pharmacokinetics. The pharmacokinetic parameters were calcu-
lated by using the noncompartmental analysis approach. The elimina-
Children were eligible to participate if they were 6 to 11 years of tion rate constant (Ke) was calculated from the plasma levocetirizine
age, weighed 20 to 40 kg, and had mild allergic rhinitis. They were concentration (C) versus time (t) data measured after Cmax had
excluded if they had any recent acute illness or any other health occurred, within 0.5 to 2 hours after dosing, by using equation 1:
problem except for mild intermittent or persistent asthma or if they
required any oral medication, including any oral H1-antihistamines, C ¼ C°e-Ket
in the week before study entry or during the study. The only medi-
cations permitted before and during the study were as follows: low- where C° is the plasma concentration extrapolated to zero time after
dose (100 mg) intranasal glucocorticoids for rhinitis and low-dose application of equation 1 by using WIN-NONLIN (Scientific
(250 mg) inhaled glucocorticoids and as-needed inhaled albuterol Consulting, Apex, NC). The elimination half-life (t1/2) was calcu-
for asthma. lated by using equation 2:
Study outline t1=2 ¼ ln 2=Ke

During a preliminary visit to the Manitoba Institute of Child Health Although levocetirizine appears to be well absorbed, there is no
Pediatric Allergy Laboratory, the children were assessed for their intravenous formulation, and therefore the absolute bioavailability
ability to meet the inclusion criteria of the study. Medical history was (F) is unknown. Total body clearance (Cl) and apparent volume of
obtained, and physical examination, complete blood count, urinalysis, distribution (Vd) were calculated as Cl/F and Vd/F, as shown in
and assessment of hepatic and renal function were performed. The equation 3:
children were given the opportunity to become familiar with the test
procedures. Cl=F ¼ AUC=Dose
In addition to the medication restrictions noted previously, before
the levocetirizine dose and for 28 hours afterward, study participants and equation 4:
refrained from ingesting methylxanthine-containing substances (eg, Cl=F
cola, chocolate, or cocoa). After an 8- to 10-hour overnight fast, at Vd=F ¼
Ke
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 where AUC is the area under the plasma levocetirizine concentration
dosing, only clear juice or water was permitted. versus time curve from time zero to 28 hours.
EMLA local anesthetic cream (Astra, Mississauga, ON, Canada) Pharmacodynamics. The pharmacodynamic parameters maxi-
was applied to potential venipuncture sites. An indwelling intrave- mum effect attributable to medication (Emax) and plasma concen-
nous catheter (Critikon, Tampa, FL) was inserted, and 2.5-mL blood tration producing 50% of Emax (EC50) were calculated by using
samples were obtained before dosing and at 0.5, 1, 2, 3, 4, 6, 8, 10, 24, WIN-NONLIN (Scientific Consulting) and equation 5:
J ALLERGY CLIN IMMUNOL Simons and Simons 357

ocular diseases
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.
Emax C TABLE I. Demographics

E¼
EC50 1C
N = 14* (9 boys)
where E is the clinical effect, percentage suppression of histamine- Age: 8.6 6 0.4 y
induced wheal or flare, and C is the levocetirizine plasma concen- Weight: 30.4 6 2.2 kg
tration at which E occurs.30 Height: 132.5 6 3.3 cm
Statistical analysis. Absolute wheal-and-flare areas over time Body mass index: 16.9 6 0.6
were analyzed by using 1-way ANOVA, with subject and time as Levocetirizine dose: 0.18 6 0.01 mg/kg
variates, analysis of covariance with predose wheal-or-flare areas as
the covariates, and the Tukey and Bonferroni multiple-range tests. All values are presented as means 6 SEM.
Differences were considered to be significant at P values of less than *At the time of the study, 4 children were using an intranasal
glucocorticoid for mild persistent allergic rhinitis, and 2 were using an
or equal to .05.
orally inhaled glucocorticoid for mild persistent asthma.
RESULTS
0.82 6 0.05 mL/min/kg, and the mean apparent volume of
The 14 Caucasian children (9 boys) with mild allergic distribution was 0.4 6 0.02 L/kg. The mean residence
rhinitis enrolled in the study had a mean (6 SEM) age of time was 6.8 6 0.3 hours.
8.6 6 0.4 years, a mean weight of 30.4 6 2.2 kg, a Pharmacodynamic data were available on all 14 chil-
mean height of 132.5 6 3.3 cm, and a mean body mass dren. Wheal-and-flare areas after testing with histamine
index of 16.9 6 0.6. They received a single 5-mg phosphate, 1 mg/mL, are shown in Fig 3. Compared
levocetirizine dose, which was equivalent to a mean dose with predose values, the wheals were significantly sup-
of 0.18 6 0.01 mg/kg (Table I). Complete pharmacoki- pressed (P < .05) from 1 to 28 hours, inclusive, with
netic data were available on only 13 of the 14 children the mean maximum suppression of 97% 6 1% occurring
because of missing blood samples in 1 child. The mean from 2 to 10 hours. Compared with predose values, the
plasma levocetirizine concentration versus time plot is flares were significantly suppressed (P < .05) from 1 to 28
shown in Fig 2. The pharmacokinetic parameters, in- hours, inclusive, with the mean maximum suppression of
cluding elimination rate constants, area under the plasma 93% 6 1% occurring from 2 to 24 hours. The relationships
concentration versus time curve, oral clearance, and between the plasma levocetirizine concentrations and
apparent volume of distribution, were calculated by percentage suppression of wheal-and-flare responses
using noncompartmental analysis. compared with predose values versus time are shown in
The mean maximum plasma levocetirizine concen- Fig 4.
tration of 450 6 37 ng/mL occurred at a mean time of Pharmacodynamic analysis resulted in calculation of an
1.2 6 0.2 hours (Table II). The mean terminal elimination EC50 estimate of 16.1 6 2.2 ng/mL and an Emax estimate
half-life was 5.7 6 0.2 hours, the mean area under the of 104.3% 6 2.6% for wheal suppression, and an EC50 of
plasma levocetirizine concentration versus time plot was 1.4 6 0.1 ng/mL and an Emax of 94.5% 6 0.4% for flare
3549 6 342 ng/mL/h, the mean oral clearance rate was suppression.
AUGUST 2005
FIG 2. Plasma levocetirizine concentration (mean 1 SEM) versus time plot after ingestion of levocetirizine,
5 mg.
ocular diseases
TABLE II. Levocetirizine pharmacokinetics about 1 hour. In the absence of an intravenous levocetir-
izine formulation, true bioavailability cannot be deter-
Cmax (ng/mL) 450 6 37 mined. On the basis of the mean levocetirizine terminal
tmax (h) 1.2 6 0.2
elimination half-life of 5.7 hours that was found, dosing
t1/2 (h) 5.7 6 0.2
AUC (ng/mL/h) 3549 6 342
every 24 hours on a regular basis would be expected to
Cl/F (mL/min/kg) 0.82 6 0.05 lead to minimal or no levocetirizine accumulation in
Vd/F (L/kg) 0.4 6 0.02 plasma. On the basis of significant wheal-and-flare sup-
pression from 1 to 28 hours after dosing, levocetirizine
Values are presented as means 6 SEM. would be expected to have significant H1-antihistaminic
Cmax, Maximum plasma concentration; tmax, time of maximum plasma activity throughout the dosing interval.
concentration; t1/2, terminal elimination half-life; AUC, area under the
plasma concentration versus time plot; Cl, oral clearance; F, oral
In pharmacokinetic and pharmacodynamic studies of
bioavailability; Vd, apparent volume of distribution. H1-antihistamines, although outcome measures such as
blood tests and skin tests are highly objective, they are
inherently invasive, and the studies therefore present
There were no serious adverse effects. Two children unique challenges in children.2,4 Study designs do not
experienced some sneezing, nasal congestion, and dis- usually involve a placebo control,7-20 not only because of
charge, and 1 child had intermittent coughing. The nasal ethical constraints and parental concerns about the use of
symptoms were attributed to allergic rhinitis, and the placebo, but also because a potent H1-antihistamine
cough was attributed to asthma; that is, the respiratory suppresses wheals and flares by up to 100%,5,6,12,13 thus
symptoms were considered to be due to underlying making it difficult to maintain double-masked observa-
allergic diseases and to be unrelated to administration of tions and measurements.
the study drug. Two hours after dosing, one child had The objective, standardized, histamine-induced wheal-
nausea that was relieved by eating, and 23 hours after and-flare bioassay is useful for studying the onset, amount,
dosing, another child had a sore stomach that was relieved and duration of activity of H1-antihistamines. Skin tests
by eating. These gastrointestinal symptoms were attrib- with histamine relate to the suppression of wheals, a
uted either to overnight fasting or to anxiety about test primary symptom and sign in urticaria, and flares, which
procedures and were considered to be probably unrelated are caused by an axon reflex and are thus related to
to the study drug. One child was more tired than usual 6 histamine indirectly rather than directly. Whether skin test
hours after the dose of the study drug, and 2 children were suppression correlates with events in the airways remains
more tired than usual 12 hours after dosing. Although this controversial31; however, it is noteworthy that in allergy
fatigue might have been due to the intensity of the practice worldwide, skin tests with allergen are performed
procedures during the first 12 to 13 hours of the study, it in lieu of nasal and bronchial allergen challenges to
was considered to be possibly related to the study drug. ascertain the relevance of allergens to allergic rhinitis
and asthma symptoms, and in the clinical setting cutane-
ous responses are assumed to reflect airways responses.
DISCUSSION The pharmacokinetics and pharmacodynamics of lev-
ocetirizine, reported here in children aged 6 to 11 years,
In this study levocetirizine appeared to be well differ slightly from those reported previously in adults
absorbed, with peak plasma concentrations occurring at with a mean age of 35 6 2 years and a mean weight of

ocular diseases
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.
AUGUST 2005
67.3 6 2.3 kg, in whom the time of maximum plasma 5. Simons FER, Johnston L, Simons KJ. Clinical pharmacology of the
H1-receptor antagonists cetirizine and loratadine in children. Pediatr
concentration is 0.73 6 0.7 hours, the terminal elimination
Allergy Immunol 2000;11:116-9.
half-life value is 7.8 6 0.3 hours, the clearance rate is 6. Simons FER, Semus MJ, Goritz SS, Simons KJ. H1-antihistaminic
0.62 6 0.02 mL/min/kg, and the apparent volume of activity of cetirizine and fexofenadine in allergic children. Pediatr
distribution is 0.41 6 0.02 L/kg.24,25 As noted previously, Allergy Immunol 2003;111:1244-8.
after administration of radioactively labeled levocetirizine 7. Simons FER, Luciuk GH, Simons KJ. Pharmacokinetics and efficacy of
chlorpheniramine in children. J Allergy Clin Immunol 1982;69:376-81.
to adults, 85.4% of the drug is eliminated unchanged in the 8. Simons FER, Simons KJ, Becker AB, Haydey RP. Pharmacokinetics and
urine, and 12.9% is eliminated unchanged in the feces antipruritic effects of hydroxyzine in children with atopic dermatitis.
within 1 week.26 The duration of action of a single J Pediatr 1984;104:123-7.
levocetirizine dose is greater than 24 hours in adults.27 9. Simons KJ, Watson WTA, Martin TJ, Chen XY, Simons FER. Diphen-
hydramine: pharmacokinetics and pharmacodynamics in elderly adults,
The pharmacokinetics and pharmacodynamics of lev-
young adults, and children. J Clin Pharmacol 1990;30:665-71.
ocetirizine reported here in children aged 6 to 11 years 10. Simons FER, Roberts JR, Gu X, Kapur S, Simons KJ. The clinical
also differ from those reported previously in very young pharmacology of brompheniramine in children. J Allergy Clin Immunol
children. In a prospective study in children with a mean 1999;103:223-6.
age of 20.7 6 3.7 months and a mean weight of 11.6 6 11. Schmidt-Redemann B, Brenneisen P, Schmidt-Redemann W, Gonda S.
The determination of pharmacokinetic parameters of ketotifen in steady
1.8 kg, the time of maximum plasma concentration was state in young children. Int J Clin Pharmacol Ther Toxicol 1986;24:496-8.
1 hour, the terminal elimination half-life was 4.1 6 0.67 12. Watson WTA, Simons KJ, Chen XY, Simons FER. Cetirizine: a phar-
hours, the clearance was 1.05 6 0.10 mL/min/kg, and the macokinetic and pharmacodynamic evaluation in children with seasonal
apparent volume of distribution was 0.37 6 0.06 L/kg.20 allergic rhinitis. J Allergy Clin Immunol 1989;84:457-64.
13. Simons FER, Watson WTA, Simons KJ. Pharmacokinetics and pharma-
Rapid elimination of levocetirizine was also found in a
ocular diseases
codynamics of ebastine in children. J Pediatr 1993;122:641-6.

population pharmacokinetic study in which cetirizine was 14. Simons FER, Bergman JN, Watson WTA, Simons KJ. The clinical
given to 343 children aged 14 to 46 months, and timed pharmacology of fexofenadine in children. J Allergy Clin Immunol 1996;
sparse blood samples were obtained at steady state 98:1062-4.
for measurement of plasma levocetirizine (the active 15. Lin CC, Radwanski E, Affrime MB, Cayen MN. Pharmacokinetics of
loratadine in pediatric subjects. Am J Ther 1995;2:504-8.
enantiomer or eutomer) and dextrocetirizine (the inactive 16. Salmun LM, Herron JM, Banfield C, Padhi D, Lorber R, Affrime MB.
enantiomer or distomer) values.22,23 The population phar- The pharmacokinetics, electrocardiographic effects, and tolerability of
macokinetic model used predicted that with increasing loratadine syrup in children aged 2 to 5 years. Clin Ther 2000;22:613-21.
body weight, levocetirizine oral clearance would increase 17. Desager JP, Dab I, Horsmans Y, Harvengt C. A pharmacokinetic
evaluation of the second-generation H1-receptor antagonist cetirizine in
by 0.044 L/h/kg, and levocetirizine volume of distribution
very young children. Clin Pharmacol Ther 1993;53:431-5.
would increase by 0.639 L/kg. Taken together, the results 18. Pariente-Khayat A, Rey E, Dubois MC, Vauzelle-Kervroedan F, Pons G,
of these 2 studies indicate that in very young children, D’Athis P, et al. Pharmacokinetics of cetirizine in 2- to 6-year-old
compared with older children and adults, higher levoce- children. Int J Clin Pharmacol Ther 1995;33:340-4.
tirizine doses may be needed on a milligram per kilogram 19. Spicak V, Dab I, Hulhoven R, Desager J-P, Klanova M, de Longueville
M, et al. Pharmacokinetics and pharmacodynamics of cetirizine in infants
basis, and twice-daily dosing may be required. and toddlers. Clin Pharmacol Ther 1997;61:325-30.
Development of organ function and many of the 20. Cranswick NE, Fuchs M, Turzikova J. Efficacy, safety and pharmaco-
maturational changes affecting pharmacokinetic disposi- kinetics of levocetirizine in allergic children age 1-2 years. Clin
tion of drugs is ongoing throughout infancy and child- Pharmacol 2004;75:P46.
21. Mentré F, Dubruc C, Thénot J-P. Population pharmacokinetic analysis
hood, but elimination through the renal route is largely
and optimization of the experimental design for mizolastine solution in
completed by age 4 to 5 years.1 This study provides a children. J Pharmacokinet Pharmacodyn 2001;28:299-319.
rationale for administration of levocetirizine, 5 mg, once 22. Hussein Z, Pitsiu M, Majid O, Aarons L, de Longueville M, Stockis A,
daily in children aged 6 to 11 years, as in adults, with the et al. Retrospective population pharmacokinetics of levocetirizine in
expectation of prompt onset of action and significant long- atopic children receiving cetirizine: the ETAC study. Br J Clin
Pharmacol 2005;59:28-37.
lasting antihistaminic activity at the H1-receptor. 23. Simons FER, on behalf of the ETAC Study Group. Population pharma-
cokinetics of levocetirizine in very young children: the pediatricians’
We thank Dr Marc de Longueville and Dr Eugene Baltes for their perspective. Pediatr Allergy Immunol 2005;16:97-103.
24. Baltes E, Coupez R, Giezek H, Voss G, Meyerhoff C, Strolin Benedetti M.
collaboration. We also thank Dr Nestor Cisneros; Gail Bonin, RN; and
Absorption and disposition of levocetirizine, the eutomer of cetirizine,
especially Sandra S. Goritz, RN, for their contributions to this study.
administered alone or as cetirizine to healthy volunteers. Fundam Clin
Pharmacol 2001;15:269-77.
25. Tillement JP, Testa B, Bree F. Compared pharmacological character-
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JS, Kauffman RE. Developmental pharmacology—drug disposition, 26. Benedetti MS, Plisnier M, Kaise J, Maier L, Baltes E, Arendt C, et al.
action and therapy in infants and children. N Engl J Med 2003;349: Absorption, distribution, metabolism and excretion of [14C]levocetiri-
1157-67. zine, the R enantiomer of cetirizine, in healthy volunteers. Eur J Clin
2. Steinbrook R. Testing medications in children. N Engl J Med 2002;347: Pharmacol 2001;57:571-82.
1462-70. 27. Devalia JL, De Vos C, Hanotte F, Baltes E. A randomized, double-blind,
3. Simons FER. Advances in H1-antihistamines. N Engl J Med 2004;351: crossover comparison among cetirizine, levocetirizine, and ucb 28557 on
2203-17. histamine-induced cutaneous responses in healthy adult volunteers.
4. Simons FER. H1-antihistamines in children. In: Simons FER, editor. Allergy 2001;56:50-7.
Histamine and H1-antihistamines in allergic disease. 2nd Ed. New York: 28. Bachert C, Bousquet J, Canonica GW, Durham SR, Klimek L, Mullol J,
Marcel Dekker, Inc; 2002. p. 437-64. et al. Levocetirizine improves quality of life and reduces costs in
long-term management of persistent allergic rhinitis. J Allergy Clin 30. Holford NHG, Sheiner LB. Understanding the dose-effect relationship:
Immunol 2004;114:838-44. clinical application of pharmacokinetic-pharmacodynamic models. Clin
29. Potter PC, for the Pediatric Levocetirizine Study Group. Efficacy and Pharmacokinet 1981;6:429-53.
safety of levocetirizine on symptoms and health-related quality of life of 31. Simons FER. Antihistamines. In: Adkinson NF Jr, Yunginger JW, Busse
children with perennial allergic rhinitis. Ann Allergy Asthma Immunol. WW, Bochner BS, Holgate ST, Simons FER, editors. Middleton’s allergy:
In press 2005. principles and practice. 6th ed. St Louis: Mosby, Inc; 2003. p. 834-69.

ocular diseases
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
Background: The mechanisms by which eosinophilic patients with CRS to secondary bacterial infections. (J Allergy
inflammation damages the epithelium and contributes to Clin Immunol 2005;116:362-9.)
recurrent acute exacerbations in chronic rhinosinusitis (CRS)
have not been fully elucidated. Key words: Eosinophils, chronic rhinosinusitis, mucus, degranula-
Objective: We tested the hypotheses that eosinophils deposit tion, major basic protein
toxic major basic protein (MBP) in the mucus and that MBP
reaches concentrations able to damage the sinonasal

epithelium. A recent survey by the National Center for Health
ocular diseases
Methods: Tissue specimens with mucus attached to the tissue Statistics reported that 14.2% (29.2 million patients) of
were carefully collected from 22 patients with CRS and the US adult population recalled a health professional’s
examined by using immunofluorescence staining for MBP. This diagnosis of sinusitis.1 Rhinosinusitis is now preferred to
immunofluorescence was digitally analyzed to determine the the previous term sinusitis ‘‘. because sinusitis is almost
area covered by MBP and the intensity of the staining always accompanied by concurrent nasal airway inflam-
(estimating MBP concentration). Levels of MBP in extracts
mation. .’’2 The economic effect of chronic rhinosinus-
from nasal mucus were quantitated by means of RIA.
itis (CRS) is huge; in the US the direct cost was estimated
Results: Heterogeneous eosinophilia was evident within tissue
and mucus specimens. All tissue specimens showed intact in 1996 at $5.6 billion per year, and the indirect cost was
eosinophils, but diffuse extracellular MBP deposition, as a estimated as more than 70 million lost activity days per
marker of eosinophil degranulation, was rare. In contrast, all year.3 Patients with CRS have long-term nasal congestion,
mucus specimens showed diffuse MBP throughout and thick mucus production, loss of sense of smell, and
abundant diffuse extracellular MBP deposition within clusters intermittent acute exacerbations secondary to bacterial
of eosinophils. Digitized analyses of MBP immunofluorescence infections; they also experience severe quality-of-life
revealed increased area coverage (P < .0001) in mucus impairment.2,4 As an additional burden, CRS lacks a
compared with that seen in tissue. Estimated concentrations plausible cause. To date, the US Food and Drug
of MBP within the clusters suggested toxic levels. MBP
Administration has not approved any drug or treatment
concentrations in mucus extract reached 11.7 mg/mL; MBP
for CRS; no medical intervention has ever been efficacious
was not detectable in healthy control subjects.
Conclusion: In patients with CRS, eosinophils form clusters in a controlled clinical trial.
in the mucus where they release MBP, which is diffusely CRS is an inflammatory disease of the nasal and
deposited on the epithelium, a process not observed in the paranasal mucosa with persistent symptoms for longer
tissue. Estimated MBP levels far exceed those needed to than 3 months; its ultimate end stage is inflammatory
damage epithelium from the luminal side and could predispose 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 gran-
ules contain several cytotoxic proteins,8 and eosinophil
From athe Department of Otorhinolaryngology–Head and Neck Surgery, cthe granule major basic protein (MBP) is directly toxic to
Department of Internal Medicine, Division of Allergic Diseases, and dthe extracellular microorganisms as well as host tissue,
Department of Biochemistry and Molecular Biology, Mayo Clinic
Rochester; bthe Department of Otorhinolaryngology, University at
including respiratory mucosa.9 CRS specimens show
Buffalo, The State University of New York; and ethe Departments of epithelial damage that is colocalized with MBP deposi-
Dermatology and Medicine, University of Utah, Salt Lake City. tion.6,7,10 In vitro, MBP directly damages respiratory
Supported by grants from the National Institutes of Health (AI 49235, and sinus epithelium in a time- and dose-dependent
AI 09728) and from the Mayo Foundation.
manner.11,12
Received for publication May 7, 2004; revised March 4, 2005; accepted for
publication March 31, 2005. Recent histologic analyses of CRS specimens sug-
Available online June 17, 2005. gested that intact eosinophils migrate from the tissue into
Reprint requests: Jens Uwe Ponikau, MD, Department of Otorhinolaryngol- the mucus to form distinct and characteristic clusters.13,14
ogy, Mayo Clinic Rochester, 200 First St SW, Rochester, MN 55905. Therefore we tested whether eosinophils release MBP in
E-mail: ponikau.jens@mayo.edu.
0091-6749/$30.00
the mucus, but not in the tissue, and whether MBP reaches
Ó 2005 American Academy of Allergy, Asthma and Immunology concentrations capable of damaging the sinonasal epithe-
doi:10.1016/j.jaci.2005.03.049 lium in patients with CRS.
362
J ALLERGY CLIN IMMUNOL Ponikau et al 363
The tissue and the attached mucus were each evaluated with the
Abbreviations used following scoring system, which was previously used to describe
CRS: Chronic rhinosinusitis eosinophil7 and neutrophil10 infiltration and degranulation. Because
MBP: Major basic protein of the heterogeneity of the eosinophilic and neutrophilic inflamma-
tion, 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/
METHODS scattered; 2, many present/abundant) for tissue and for attached
mucus using the criteria listed below:
Patient selection
d intact eosinophils or neutrophils;
The diagnostic guidelines and criteria for CRS were consistent d punctate staining (MBP or elastase within intact extracellular
with those adopted at the recent Rhinosinusitis Consensus granules); and
Conference.2 All patients had symptoms consistent with CRS for d diffuse staining (extracellular MBP or elastase not in granules).
longer than 3 months, inflamed mucosa on endoscopy, and a coronal
computed tomographic scan demonstrating mucosal thickening of In addition, eosinophils forming clusters within the mucus
greater than 5 mm in more than 2 sinuses. Retention cysts and cystic (eosinophilic mucin) were noted as present (1) or absent (2), and
fibrosis are differential diagnoses to CRS, and if these diseases were diffuse MBP staining within the clusters was noted as present (1) or
diagnosed, those patients were excluded from the study. Because absent (2). Neutrophil clusters and elastase deposition were evalu-
complete immunologic evaluations were not performed in all pa- ated similarly.
tients, we have not excluded patients (if any) with immunodeficien-

cies. With regard to noneosinophilic inflammatory sinusitis, we found
ocular diseases
eosinophilia in tissues from all patients of our otherwise unselected Digital analyses of MBP and elastase
patient population. Patients were not preselected for having eosino- immunofluorescence in tissue and mucus
philic CRS.
Computer analysis of sections stained for MBP or elastase by
means of immunofluorescence was performed to evaluate objectively
Histologic analyses of specimens the overall areas covered by MBP or elastase, as well as the intensities
For the histologic analyses, specimens were collected from 22 of the staining (as indirect markers for MBP or elastase concen-
consecutive patients with CRS undergoing endoscopic sinus surgery. trations).6 Briefly, we used a confocal microscope (LSM510
During surgical intervention, we used Blakesley surgical forceps to Confocal Microscope; Carl Zeiss, Inc, Oberkochen, Germany) to
carefully and gently collect the maximum amounts of tissue and survey and select both the least and most intense areas of MBP or
mucus, and we ensured that the mucus remained attached to the tissue, elastase staining in the tissue and the mucus. First, digital images
which was immediately fixed in formalin. Four specimens from the (512 3 512 pixels, 4003 magnification, 488-nm excitation wave-
ethmoid sinuses of healthy individuals (nonallergic and no asthma) length) of areas that showed maximal fluorescence staining (most
undergoing septoplasty procedures served as negative controls. The intense accumulation of either eosinophils or neutrophils or diffuse
Institutional Review Board of the Mayo Clinic approved the study. extracellular MBP or elastase deposition) were obtained. Second,
Paraffin-embedded tissue blocks with attached mucus were cut in digital images of the corresponding areas on the serial section, which
5-mm-thick serial sections, mounted on positively charged slides, and was stained with normal rabbit IgG, as the negative control, were
stained with the following: (1) hematoxylin and eosin; (2) antibody to recorded. Third, by using image-analysis software (KS400 Image
eosinophil MBP using rabbit antihuman MBP15-17; (3) antibody to Analysis System, Carl Zeiss, Inc), the threshold for each negative
neutrophil elastase10 using rabbit antihuman elastase (IgG fraction; control image was calibrated to a baseline value that showed no
Cortex Biochem, San Leandro, Calif); and (4) negative control for positive pixels. Fourth, this background threshold was used to analyze
MBP and elastase (normal rabbit IgG). All specimens were incubated the corresponding area on the MBP or elastase immunofluorescence-
in 10% normal goat serum to block nonspecific binding by the stained specimen. Any pixels recorded were quantitated as a percent-
second-stage antibody and in 1% chromotrope 2R to block non- age of an area (512 3 512 pixels) positive for MBP or elastase. The
specific binding of fluorescein dye to the eosinophils.6 Fluorescein image-analysis software then compared the different areas within the
isothiocyanate–conjugated goat anti-rabbit IgG was used as the specimen and determined the area with the highest percentage of
secondary antibody.15-17 MBP was chosen to assess eosinophil positive pixels; this percentage indirectly indicated the area of
infiltration and degranulation because it is the predominant eosino- maximal inflammatory eosinophilic or neutrophilic infiltrate (tissue)
phil granule protein. It accounts for roughly 50% of the total protein or maximal MBP or elastase deposition (mucus). A similar survey was
mass in the eosinophil granule,18 and the release of MBP is highly made to find the area in the MBP or elastase immunofluorescence
correlated with the release of the other granule proteins.19 Elastase, specimen with the least fluorescence in the tissue and in the mucus;
the predominant neutrophil granule protein, was studied to assess once located, these areas were compared with the respective
neutrophil infiltration and degranulation. corresponding areas in the negative control serial section and analyzed
as above.
Semiquantitative analysis of MBP and
elastase immunofluorescence in tissue
and mucus Electron microscopy and immunogold
Three examiners independently examined the entire specimen. To labeling for MBP
exclude artifacts from trauma caused through the removal of the We also used electron microscopy to investigate the morphology
specimen during surgery, only areas of untouched mucosa covered of eosinophils and eosinophil granules and to localize MBP in tissues
with mucus were evaluated. In contrast, areas with obvious tears or from 3 patients with CRS, as described earlier.20 After the primary
influx of red blood cells, indicating trauma with bleeding in the areas antibody, affinity-purified antibody to MBP, we used a secondary
touched by the forceps, were excluded. antibody conjugated to 15 nm colloidal gold particles.17
364 Ponikau et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
Quantitative analysis of MBP in mucus showing various amounts of punctate staining were sim-
Nasal mucus was collected by means of direct trap suctioning ilar between tissue and mucus. Diffuse MBP deposition in
from the nasal cavity (middle meatus region) and from one maxillary the mucus was abundant in all (22/22) CRS specimens but
sinus under endoscopic guidance with a sinus secretion collector was not observed in the tissue, except in one small area
(Xomed Surgical Products, Jacksonville, Fla) from 12 additional from 1 specimen (1/22). Intact neutrophils were evident in
patients with CRS who met the same diagnostic criteria as described both tissue (10/22) and mucus (14/22) specimens. In
above and from 9 healthy control subjects. The mass of each mucus contrast to the numerous areas showing diffuse MBP
specimen was obtained, and a 3-fold excess of normal saline (0.15 M deposition in the mucus of all 22 specimens (Fig 2, A),
NaCl) was added. After vigorous vortexing for 10 seconds (33), the
isolated areas of diffuse elastase deposition were noted in
mucus suspension was centrifuged, and the resulting supernatant
fluid was frozen at 270°C. (This vortexing step probably did not
the mucus of only 9 of 22 patients. The majority of
release the intracellular intragranular MBP, which requires more specimens showed a virtual absence of diffuse elastase in
severe conditions. Specifically, to extract MBP, eosinophils need to the mucus (Fig 2, B).
be incubated for 30 minutes at room temperature in 0.5% NP-40 Eosinophils forming clusters within the mucus (eosin-
[Sigma-Aldrich, St Louis, Mo] and 0.01 M HCl8). Finally, MBP ophilic mucin) were present in 22 of 22 specimens, and
levels in the mucus supernatant fluid were determined by means of diffuse deposition of MBP in and around these clusters
RIA, essentially as described earlier.21 was seen in 22 of 22 specimens. In contrast, neutrophil
cluster formation was observed in only 5 of 22 specimens,
Statistical analysis and elastase deposition in these clusters was observed in
only 4 of 22 specimens. None of the specimens from
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 healthy control subjects (0/4) were positive for intact
ocular diseases
.05 was considered significant. Data are presented as means (6 SD) eosinophils or neutrophils or punctate or diffuse staining
or medians (ranges). for MBP or elastase (results not shown).
Digital analyses of MBP and elastase

RESULTS immunofluorescence
Patient demographics In the tissue the maximum area positive for MBP
Patient demographics have been previously described.6 immunofluorescence staining had a median of 18.35%
Briefly, the mean age of the 22 patients with CRS was (range, 0.64% to 56.63%); by contrast, the maximum area
47 years (range, 16-86 years); 11 were women; the mean positive for elastase immunofluorescence was signifi-
number of sinus operations was 1.8 (range, 0-7); the mean cantly less (median, 3.11%; range, 0.05% to 46.2%;
duration of disease was 8.6 years (range, 2-27 years); the P < .002; Fig 3). In the mucus the maximum area positive
incidence of aspirin idiosyncrasy was 41%; 11 had for MBP immunofluorescence staining had a median of
increased serum levels of total IgE (>128 U/mL, 2 SDs 93.28% (range, 3.62% to 100%); by contrast, the maxi-
above the mean value of healthy adult control subjects); mum area positive for elastase immunofluorescence was
and 10 were considered allergic, as defined by a positive also significantly less (median, 29.30%; range, 0.10% to
skin prick test response to at least one allergen from a 85.42%; P < .001; Fig 3). Furthermore, the maximum area
panel of 16 common aeroallergens. The incidence of positive for MBP immunofluorescence in the mucus
physician-diagnosed asthma was 68% (15/22); the other (median, 93.28%) was significantly increased compared
7 patients underwent a methacholine challenge, and 5 had with the maximum mean area in tissue (median, 18.35%;
positive responses. P < .0001).
Eosinophil and neutrophil infiltration and Electron microscopy and immunogold

degranulation in tissue and mucus labeling for MBP
Although intact eosinophils were abundant in numer- In tissue from a patient with CRS, both an intact
ous areas in the tissue (Fig 1, A and B), the most striking eosinophil containing the characteristic electron-dense
observation was the abundance of diffuse MBP staining granule cores with MBP and extracellular granules are
(not in cells and not in granules) in the mucus compared visible (Fig 4, A). However, the immunogold MBP stain
with its absence in the tissue (Fig 1, A-D). Eosinophils shows MBP confined within the intact granules (Fig 4, B),
formed cell clusters in the mucus, and diffuse MBP corresponding with the punctate staining in MBP immu-
staining was observed within and around these clusters nofluorescence (see Fig 1, E). Diffuse MBP immunogold
(Fig 1, A-D and F). Punctate staining, indicating intact staining is not seen in the tissue (Fig 4, B), which is
extracellular eosinophil granules, was frequently observed consistent with the lack of diffuse MBP immunofluores-
in both tissue and mucus (Fig 1, E and F). Compared with cence staining seen in the tissue (see Fig 1, E).
MBP staining, only isolated areas in tissue and mucus
stained for neutrophil elastase (results not shown). Measurement of MBP in the mucus
As summarized in Fig 2 (top), tissue in all specimens As shown in Fig 5, the mean concentration of detectable
showed abundant intact eosinophils (22/22) compared MBP in mucus extracts from the maxillary sinuses in
with mucus (11/22). The numbers of patient specimens patients with CRS was 4.2 mg/mL (6 3.1 mg/mL; range,

ocular diseases
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, subepi-
thelial 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).
AUGUST 2005
ocular diseases
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 thelium; in contrast, our specimens were carefully col-
were also able to harvest sufficient mucus from the nasal lected to ensure that the mucus remained attached to the
cavity to detect a mean MBP concentration of 4.1 mg/mL harvested tissue. Thus we could document the extent,
(6 2.6 mg/mL; range, 0-8.0 mg/mL), which did not differ localization, and degranulation pattern of the heteroge-
significantly from the mean concentration in the maxillary neous eosinophilia and neutrophilia in both the tissue and
sinuses. In contrast, in mucus specimens from the nasal the mucus. Eosinophil, rather than neutrophil, inflamma-
cavities of the healthy control subjects, no MBP could be tion was predominant in both tissue and mucus. Striking
detected above the sensitivity of the assay (0.010 mg/mL). extracellular deposition of diffuse MBP (especially in
Thus even the lowest MBP concentration detected in clusters) was unique to the mucus in all 22 patients with
mucus from the maxillary sinus of a patient with CRS CRS and was not found in the tissue (with the exception of
(0.31 mg/mL) was at least 30-fold greater than that of one small area in 1 patient). In contrast, extracellular
healthy control subjects. 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
DISCUSSION this study could explain the sinonasal epithelial erosion
observed in patients with CRS.6 As seen in Fig 1, A and C,
The underlying eosinophilic inflammation is in- the outer layers of the epithelium are eroded away, but a
creasingly recognized to play an important role in the layer of basal epithelial cells still remains. This observa-
pathogenesis of CRS, and its association with epithelial tion, as well as the marked deposition of toxic eosinophil
damage has been suspected.6,7 How the eosinophil granule MBP in the mucus, suggests that the damage to
actually mediates the pathophysiology, such as damaging the epithelium occurs from the outside (luminal side). The
the epithelium, remains unclear. Earlier studies in patients damaged epithelium might provide an entry port for colo-
with CRS used tissue biopsy specimens without mucus7,10 nizing bacteria that are present in the sinuses of patients
and showed MBP deposition within damaged sinus epi- with CRS, as well as healthy control subjects.22,23 Thus

ocular diseases
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 analyses to compare the brightness of anti-MBP staining
MBP, in the mucus could be a crucial predisposing factor in confocal microscopic images. Overall, 17 of 22 speci-
for the secondary bacterial infections that likely mediate mens showed brighter immunofluorescence staining for
the acute exacerbations of CRS. In healthy control MBP in mucus compared with that seen in tissue; no
subjects, the absence of MBP in the mucus might also brightness differences were noted for intact cells in tissue,
explain the lack of epithelial damage and, consequently, blood vessels, or mucus. Thus we estimate that areas of dif-
the lack of bacterial infections, despite the presence of fuse MBP in the mucus might exceed 33 mg of MBP/mL.
bacteria.22,23 Taken together, these findings might explain Because MBP is toxic to and causes erosion of the
the complex clinical course of CRS with its occasional epithelium at concentrations less than 10 mg/mL,12 our
bacterial exacerbations; indeed, the numbers of infiltrat- MBP immunofluorescence results suggest that MBP
ing tissue neutrophils have been directly correlated to the reaches local concentrations in the mucus of patients
numbers of bacteria present.24 Overall, these exacerba- with CRS far exceeding those necessary to mediate
tions are superimposed on the persistent and underlying epithelial damage. In addition, inspissation (dessication)
eosinophilic inflammation uniformly seen in all patients. of mucus at mucosal surfaces in vivo might lead to a further
Although we used an RIA for MBP in extracts from increase in the local MBP concentration. We observed
mucus specimens, this procedure likely underestimated striking MBP deposition directly adjacent to the damaged
the actual local concentrations for the following reasons. epithelium (Fig 1, A-D).
First, we attempted to extract MBP from the thick mucus In the tissue, extracellular MBP is confined to intact
through vortexing in saline, and thus only the MBP that eosinophil granules (ie, punctate staining), as shown by
actually dissolved in the saline could be measured. The means of MBP immunofluorescence staining (Fig 1, B,
remaining mucus and probably a large amount of undis- D, and E) and by means of electron microscopy and
solved MBP had to be discarded. Second, the concentra- immunogold labeling for MBP (Fig 4). The apparent lack
tion of MBP was based on the entire volume of the mucus of subepithelial tissue damage, as assessed with hematox-
specimen; because portions of the mucus do not contain ylin-and-eosin staining of specimens from patients with
MBP (Fig 3), our results underestimate the local maximal CRS, suggests that intact granules containing MBP might
MBP concentrations. not have physiologic or damaging actions in the tissue.
To address this potential underestimation, we calcu- The accumulation of diffuse MBP immunofluorescence in
lated the approximate MBP concentration in an eosinophil the mucus (and not in the tissue) suggests that the cells
to be 33 mg/mL or 2.1 3 1023 M (8.98 3 1029 mg [mass found in the tissue are in transit toward their final target in
MBP/eosinophil]/2.68 3 10210 mL [volume/eosino- the mucus, with ongoing deposition of MBP (ie, the
phil]).8 Although the immunogenic epitopes might not cluster of eosinophils surrounded by the diffuse cloud of
be equally available for MBP in mucus compared with MBP). This pattern of cluster-forming eosinophils appears
MBP in the granules of intact cells, we used digital strikingly similar to the eosinophils’ role in their immune
AUGUST 2005
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
ocular diseases
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 eosinophils release cytotoxic MBP in the mucus, but not in
organisms, subsequently releasing granular proteins (in- the tissue, at concentrations likely exceeding those needed
cluding MBP) that destroy the parasite.25 to damage the epithelium in patients with CRS. Therefore
It is generally assumed that eosinophil cytolysis and one might need to take not only the tissue but also the
piecemeal degranulation are distinct mechanisms by mucus into account when trying to understand the patho-
which granules and, subsequently, granule proteins are physiology of CRS and probably other airway diseases. In
released in diseased airway tissue.26 Instead, we found that addition, this new understanding suggests a beneficial
effect in therapies that target primarily the underlying and 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
presumably damage-inflicting eosinophilic inflammation
Proc 1999;74:877-84.
instead of the secondary bacterial infection. 14. Braun H, Busina W, Freudenschuss K, Beham A, Stammberger H.
‘‘Eosinophilic fungal rhinosinusitis’’: a common disorder in Europe?
Laryngoscope 2003;113:264-9.
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1. Lethbridge-Cejku M, Schiller JS, Bernadel L. Summary health statistics specific staining of eosinophil granule major basic protein. J Immunol
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2. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas immunofluorescence of eosinophil granule major basic protein in lung
RA, et al. Rhinosinusitis: establishing definitions for clinical research tissues of patients with bronchial asthma. Lancet 1982;2:11-6.
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1999;103:408-14. Adkinson NF Jr, Bochner BS, Yunginger JW, Holgate ST, Busse WW,
4. Gliklich RE, Metson R. The health impact of chronic sinusitis in patients Simons FE, editors. Middleton’s allergy: principles and practice. 6th ed.
seeking otolaryngologic care. Otolaryngol Head Neck Surg 1995;113: Philadelphia: Mosby; 2003. p. 305-32.
104-9. 19. Ott NL, Gleich GJ, Peterson EA, Fujisawa T, Sur S, Leiferman KM.
5. Kaliner MA, Osguthorpe JD, Fireman P, Anon J, Georgitis J, Davis JL. Assessment of eosinophil and neutrophil participation in atopic derma-
Sinusitis: bench to bedside. Current findings, future directions. Otolar- titis: comparison with the IgE-mediated late-phase reaction. J Allergy
yngol Head Neck Surg 1997;116(suppl):S1-20. Clin Immunol 1994;94:120-8.

6. Ponikau JU, Sherris DA, Kephart GM, Kern EB, Gaffey TA, Tarara JE, 20. Popken-Harris P, Checkel J, Loegering D, Madden B, Springett M,
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et al. Features of airway remodeling and eosinophilic inflammation in Kephart G, et al. Regulation and processing of a precursor form of
chronic rhinosinusitis: is the histopathology similar to asthma? J Allergy eosinophil granule major basic protein (proMBP) in differentiating
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7. Harlin SL, Ansel DG, Lane SR, Myers J, Kephart GM, Gleich GJ. 21. Wagner JM, Bartemes K, Vernof KK, Dunnette S, Offord KP, Checkel
A clinical and pathologic study of chronic sinusitis: the role of the JL. Analysis of pregnancy-associated major basic protein levels through-
eosinophil. J Allergy Clin Immunol 1988;31:867-75. out gestation. Placenta 1993;14:671-81.
8. Abu-Ghazaleh RI, Dunnette SL, Loegering DA, Checkel JL, Kita H, 22. Kalcioglu MT, Durmaz B, Aktas E, Ozturano O, Durmaz R. Bacteriology
Thomas LL, et al. Eosinophil granule proteins in peripheral blood of chronic maxillary sinusitis and normal maxillary sinuses: using culture
granulocytes. J Leukoc Biol 1992;52:611-8. and multiplex polymerase chain reaction. Am J Rhinol 2003;17:143-7.
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Intranasal tolerance induction with
polypeptides derived from 3 noncross-
reactive 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
Background: Specific immunotherapy is less effective in mucosal polyvalent vaccine to prevent multiple sensitivities in
patients with multiple allergic sensitizations compared with atopic patients. (J Allergy Clin Immunol 2005;116:370-6.)
monosensitized patients.
Objective: We therefore established a mouse model of Key words: Animal model, type I allergy, polysensitization, mucosal
polysensitization to the major birch and timothy grass pollen tolerance, polypeptides, hybrid, IL-10, TGF-b
allergens to test whether allergic polysensitization can be
ocular diseases
prevented by multiple allergen application via the mucosal

route.
Methods: Female BALB/c mice were immunized About 25% of the population in industrialized countries
intraperitoneally with recombinant (r) Bet v 1, rPhl p 1, and has type I allergy, a genetically determined immunolog-
rPhl p 5. For intranasal tolerance induction, a mixture of the
ical disorder. In Europe, the most common seasonal
complete allergens was compared with allergen-derived
airborne allergens are derived from white birch (Betula
immunodominant peptides applied either as a mixture or as
a synthetic hybrid peptide composed of the T-cell epitopes verrucosa) and timothy grass (Phleum pratense).1 More
of the 3 allergens. than 90% of the patients allergic to birch pollen (BP) react
Results: Intranasal application of the mixture of the complete to the major allergen Bet v 1, and in patients allergic
allergen molecules did not prevent polysensitization to the same to grass pollen, Phl p 1 and Phl p 5 represent major
allergens. In contrast, pretreatment with a mixture of the allergens.2-4
immunodominant peptides or the hybrid peptide led to There is substantial clinical evidence that many patients
significantly reduced allergen-specific IgE responses in sera, with allergy are cosensitized to several unrelated airborne
IL-4 production in vitro, and suppressed airway inflammation. allergens. In this context, recent studies demonstrated that
TGF-b mRNA levels did not change, and IL-10 production was
BP allergy is frequently associated with sensitization to
significantly suppressed after the pretreatment. The fact that
other inhalant allergens, including grass pollen, and that
the reduction of IL-10 was not abrogated after IL-10 receptor
neutralization and that tolerance was not transferable with multiallergies often develop with increasing age.5,6
splenocytes indicates that the suppression of TH2 responses One of the most effective treatments for type I allergy,
in polysensitized mice might not be mediated by especially in young and monosensitized patients, is spe-
immunosuppressive cytokines. cific immunotherapy, performed by repeated subcutane-
Conclusion: Our study demonstrates that it is possible to ous injections of increasing amounts of allergen extracts.7
suppress allergic immune responses simultaneously to several In patients with multiple sensitivities, specific immuno-
clinical important allergens. Thus, mucosal coapplication of therapy is known to be of low efficacy and is associated
selected peptides/hybrid peptides could be the basis of a 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 indi-
From athe Department of Specific Prophylaxis and Tropical Medicine, Center viduals.
for Physiology and Pathophysiology, Medical University of Vienna; and
b
the Department of Clinical Chemistry and Molecular Diagnostics, Hospital
Such improvements could be achieved by the use of
of the Philipps University Marburg. recombinant allergens or allergen-derived peptides ac-
Supported by grants from the Austrian Science Fund (F01814, P14634-PAT, cording to the patient’s sensitization profile,9-11 instead
Y0784GEN, FWF T163). of natural allergen extracts containing a variety of
Received for publication July 9, 2004; revised March 10, 2005; accepted for
allergenic molecules. Recently the possibility of using
publication April 1, 2005.
Available online May 24, 2005. prophylactic allergy vaccines has been discussed.12
Reprint requests: Ursula Wiedermann, MD, PhD, Department of Specific Following the concept of vaccination against many
Prophylaxis and Tropical Medicine, Center for Physiology and Patho- infectious diseases, prophylactic treatment to most com-
physiology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 mon allergens could be a possible strategy for early
Vienna, Austria. E-mail: ursula.wiedermann@meduniwien.ac.at.
0091-6749/$30.00
allergy prevention. In addition, the change to a less
Ó 2005 American Academy of Allergy, Asthma and Immunology invasive route of administration, such as application of
doi:10.1016/j.jaci.2005.04.002 allergens via the mucosal surfaces, might even enhance
370
J ALLERGY CLIN IMMUNOL Hufnagl et al 371
Synthesis, purification, and characterization

Abbreviations used of peptides for intranasal pretreatment
aa: Amino acid Subsequent to the identification of the immunodominant regions
BP: Birch pollen of Bet v 1, Phl p 1, and Phl p 5 by epitope mapping, the respective
r: Recombinant peptides (see Results and Tables E1-E3 in the Journal’s Online
RBL: Rat basophil leukemia 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 hexafluoro-
phosphat 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 patient’s compliance to treatment. In several experi-
the peptides were purified to >90% purity by preparative HPLC
mental animal studies, it has been shown that mucosal (Pichem, Graz, Austria).
tolerance is highly effective in preventing allergic dis-
ease.13-17 A recent study in mice demonstrated that the Dose-finding and kinetic studies for
intranasal route of desensitization can be even more intranasal pretreatment
effective than the intradermal route.18 We previously Dose-finding experiments and kinetic studies with Bet v 1, Phl p 1,
demonstrated in a murine model of allergic sensitization and Phl p 5 proteins were performed by using 0.1 to 100 mg/antigen
to BP that mucosal administration of recombinant (r) Bet v 1 applied 3 times at 7-day intervals or every third day for 3 weeks. In
suppressed allergic sensitization and airway inflamma- line with previous studies in monosensitized mice, a treatment

tion in naive and in sensitized mice.16,17,19 In the current regimen with 10 mg applied 3 times every 7 days was chosen.16,17
ocular diseases
study, we established a murine model of polysensitization Dose-finding experiments with the immunodominant peptides (1-100
to the noncross-reactive major birch and grass pollen mg/peptide) revealed that low-dose application (ie, 5 mg/peptide) was
allergens Bet v 1, Phl p 1, and Phl p 5. By using this model, optimal for intranasal tolerance induction.
we tested whether it is possible to induce tolerance Polysensitization and intranasal pretreatment
simultaneously against these different pollen allergens
Polysensitization was performed by 3 intraperitoneal injections
by intranasal application of either the allergen proteins or
(days 22, 36, and 50) of a mixture of 5 mg rBet v 1, 5 mg rPhl p 1, and
the allergen-derived peptides composed of the immuno- 5 mg rPhl p 5 adsorbed to aluminium hydroxide (Al[OH]3; Serva,
dominant epitopes thereof. Our results show that intra- Heidelberg, Germany) at 14-day intervals (group 1). For intranasal
nasal application of polypeptides successfully prevented pretreatment, a mixture of the 3 allergens, Bet v 1, Phl p 1, and Phl p 5
allergic polysensitization. (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
METHODS volume of 30 mL (group 3). Intranasal pretreatment with the hybrid
Animals peptide was performed by using a concentration of 20 mg construct in
30 mL per application (group 4). Control mice were intranasally sham-
Female inbred 7-week-old BALB/c mice (n = 5 per group) were treated with 30 mL 0.9% NaCl before polysensitization (group 1).
obtained from Charles River (Sulzfeld, Germany). All experiments One week after the last intraperitoneal immunization, an aerosol
were approved by the Animal Experimentation Committee of the challenge with 1% wt/vol BP and Phleum extract was performed on
University of Vienna and the Federal Ministry of Education, Science 2 consecutive days, as previously described.17
and Culture.
Sampling
Recombinant allergens and natural Blood samples were taken before treatment and 2 days after the
aerosol challenge (day 60) by tail bleeding. After the bleeding, the
allergen extracts
mice were sacrificed, spleen cell suspensions were prepared, and
rBet v 1, rPhl p 1, and rPhl p5 were obtained from Biomay AG bronchoalveolar lavages were collected, as previously described.16,17
(Vienna, Austria). Birch pollen (B verrucosa) and timothy grass
pollen (P pratense) were purchased from Allergon (Välinge, Airway eosinophilia
Sweden), and extracts were prepared as previously described.16 Bronchoalveolar lavage samples were spun onto microscope
slides and stained with hematoxylin and eosin (Hemacolor; Merck,
Epitope mapping studies Darmstadt, Germany). The number of eosinophils was expressed
as percentage of the total counted cell number, as previously
For T-cell epitope mapping, a panel of 50 peptides of the Bet v 1 described.19
molecule (Cambridge Research Biochemicals Limited, Cambridge,
United Kingdom), 77 peptides of Phl p 1 (Cambridge Research
Detection of allergen-specific antibody
Biochemicals Limited), and 92 peptides of Phl p 5 (provided by
Dr Helmut Fiebig, Reinbek, Germany) were used. Spleen cell sus- levels in serum
pensions from Bet v 1, Phl p 1, and Phl p 5 immunized mice were Microtiter plates (Nunc, Roskilde, Denmark) were coated with
incubated with each of the dodecapeptides (2 mg/well), spanning the each of the recombinant allergens (5 mg/mL) before incubation with
whole amino acid (aa) sequence of the respective antigens. The sera. Rat antimouse IgG1, IgG2a, and IgE antibodies (1/500;
dodecapeptides overlapped by 9 residues. Proliferative responses Pharmingen, San Diego, Calif) were used, followed by peroxidase-
were measured according to a previous description.16 conjugated mouse antirat IgG antibodies (1/2000; Jackson Immuno
372 Hufnagl et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
TABLE I. Allergen-specific antibody levels, T-cell proliferation, and cytokine production in polysensitized mice
rBet v 1 rPhl p 1 rPhl p 5 BP Phleum
IgG1* 1.98 6 0.28 1.92 6 0.05 1.61 6 0.27 IFN-gà 1501.9 6 621.1 1307.9 6 372.7
IgE* 0.85 6 0.21 0.48 6 0.05 0.53 6 0.02 IL-4à 28.6 6 14.6 65.8 6 17.3
IgG2a* 0.51 6 0.35 0.31 6 0.12 0.75 6 0.37 IL-5à 51.7 6 42.52 246.7 6 105.1
Stimulation index 2.97 6 0.51 2.63 6 1.02 4.41 6 2.38
*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 Adoptive cell transfer
of baseline levels (0.058 6 0.025) from preimmune sera.
Pretreatment with the peptide mixture or the hybrid peptide was
Rat basophil leukemia cell mediator performed as described. Eight days after the last intranasal applica-
tion, donor spleen cells (1 3 107) were intravenously injected into
release assay naive recipients.17 Four hours after the cell transfer, the recipients
Rat basophil leukemia (RBL)-2H3 cells were incubated with sera were polysensitized as described, and immune responses were
ocular diseases
obtained from pretreated and polysensitized mice at dilutions of 1/30 evaluated 7 days after the last immunization.
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 Statistics
analyzed for b-hexosaminidase activity as previously described.21
Data are expressed as means 6 SEMs from 3 independent
Results are reported as percentages of total b-hexosaminidase
experiments. Differences between groups were tested by Kruskal-
released after addition of 1% Triton X-100 and are shown after
Wallis tests. Post hoc comparisons for all pairs of groups were
subtraction of baseline release levels (1.13 6 0.81) obtained with
performed applying Tukey-Kramer tests. P values below .05 were
preimmune sera.
considered significant. Pairwise comparison of sensitized versus
Lymphocyte proliferation and pretreated groups was performed by using the Mann-Whitney U test.
cytokine production
Proliferation of splenocytes (2 3 105 cells/well) after stimulation RESULTS
with recombinant allergens (2 mg/well) was measured as previously
described.16 Polysensitized mice displayed comparable
IFN-g, IL-4, IL-5, and IL-10 production was measured in spleen immune responses to each of the
cell suspensions incubated for 40 hours with BP (25 mg/well) or 3 allergens
Phleum extract (25 mg/well) as described.16 IL-5 was measured in
bronchoalveolar lavage fluids as previously described.17 For neutral- Immunization with rBet v 1, rPhl p 1, and rPhl p 5
ization of the IL-10 receptor in vitro pooled splenocytes were cultured induced comparably high IgG1, IgE, and IgG2a levels
in the presence of rat antimouse IL-10 receptor mAb (60 mg/mL; and strong lymphocyte proliferative responses to each of
BD Biosciences, Heidelberg, Germany) or control rat IgG1, the allergens (Table I). In addition, similar levels of IL-4,
k isotype antibody (BD Biosciences).22 Cytokine levels are shown IL-5, and IFN-g were detected after stimulation of spleen
in pg/mL after subtraction of baseline levels (IFN-g, 147.23 6 61.6 cell suspensions from polysensitized mice with BP and
pg/mL; IL-4/5, 4.8 6 4.1 pg/mL; IL-10, 27.75 6 23.3 pg/mL) of Phleum extract (Table I). Naive splenocytes stimulated
unstimulated cultures. with rBet v 1, rPhl p 1, rPhl p 5, BP, or Phleum extract did
Quantification of TGF-b mRNA expression not differ in their proliferative responses or in cytokine
by real-time RT-PCR levels from medium control levels (data not shown).
Total RNA was isolated from pooled spleen cell suspensions on Epitope mapping studies for characterization
the day of sacrifice by using RNeasy Minikit (Quiagen, Valencia,
of the immunodominant peptides of Bet v 1,
Calif), treated with DNase (Quiagen), and then reverse-transcribed
into cDNA by using random hexamers (GeneAmp RT-PCR kit; Phl p 1, and Phl p 5
Perkin Elmer, Boston, Mass). Gene expression was determined by In Bet v 1 immunized mice, 1 immunodominant T-cell
quantitative real-time PCR by using predesigned TaqMan Gene epitope, MGETLLRAVESY, was located at the C-termi-
Expression Assays according to the manufacturer’s protocol on an nus corresponding to the aa sequence position 139-150.23
ABI 7700 sequence detection system (Applied Biosystems, Foster One immunodominant region of Phl p 1, AGELELQF-
City, Calif). Amplification of the endogenous control 18S rRNA
RRVKCKY, corresponding to the aa sequence position
(Applied Biosystems) was performed to standardize the amount of
sample cDNA added, and relative quantitation was performed by 127-141, was identified, also described as a T-cell–
using the standard curve method (Applied Biosystems). The thermal reactive region in human beings.24 Concerning Phl p 5,
cycle conditions were 50°C for 2 minutes and 95°C for 10 minutes, 2 immunodominant regions, KVDAAFKVAATAANA
followed by 40 cycles of amplification at 95°C for 15 seconds and corresponding to the aa sequence 166-180 (peptide 1) and
60°C for 1 minute. TVATAPEVKYTVFETALK corresponding to the aa
sequence 226-243 (peptide 2), were identified. Compar-

ison 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 polysen-
sitized mice.
Synthesis of peptides for intranasal

tolerance induction
According to the epitope mapping studies, we
synthesized single peptides from Bet v 1 (aa: SKEM-
GETLLRAVESYLLAHSDE), Phl p 1 (aa: LRSAGEL-
ELQFRRVKCKYPEG), 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: MGETLLRAVESYAGELELQFRRVK-
CKYTVATAPEVKYTVFETALK; see Tables E1-E3 in

the Online Repository at www.mosby.com/jaci).
FIG 1. IgE-dependent allergen-specific basophil degranulation (RBL
ocular diseases
assay) (A), and IL-4 production (B) in vitro from protein mixture
Intranasal tolerance induction with the (hatched bars), peptide mixture (gray bars), and hybrid peptide–
peptide mixture and the hybrid peptide, pretreated mice (black bars) compared with polysensitized controls
but not with the protein mixture, (white bars). *P < .05, **P < .01, pretreated vs polysensitized.
suppressed TH2 immune responses and
airway inflammation in polysensitized mice degranulation remained unchanged in polysensitized
recipients (Fig 3, C).
Sera from peptide mixture as well as from hybrid
peptide–pretreated mice, but not from mice pretreated
with the complete proteins, induced significantly lower DISCUSSION
IgE-induced basophil degranulation in vitro than sera from
polysensitized mice (Fig 1, A). In accordance with this In the current study, we demonstrate that it is possible to
reduction, IL-4 levels were significantly suppressed in prevent the development of multiple sensitivities to birch
spleen cell cultures from mice pretreated with the peptide and grass pollen allergens by intranasal antigen applica-
mixture or the hybrid peptide. In contrast, IL-4 levels were tion. Successful tolerance induction was achieved by
enhanced or remained unchanged in the protein-pretreated intranasal administration of the immunodominant pepti-
group (Fig 1, B). Unlike the allergen-specific TH2 des of Bet v 1, Phl p 1, and Phl p 5, but not by the mixture
responses, IgG2a antibody production was not signifi- of the allergen proteins.
cantly changed by either of the pretreatments (Table II). It was recently shown that the dose of coadministered
Similarly, IFN-g levels in vitro remained unchanged antigens is most important for the establishment of a
except for a significant enhancement in BP-stimulated balanced TH2 immune response.25 Accordingly, in the
splenocytes from the hybrid peptide–pretreated mice current study, we demonstrated that immunization with
(Table II). the optimal doses of Bet v 1, Phl p 1, and Phl p 5 (ie, 5 mg
Airway inflammation, characterized by eosinophils and each) led to comparable humoral and cellular immune
IL-5 production in bronchoalveolar lavages, was signifi- responses to all 3 allergens/antigens (Table I). Moreover,
cantly reduced after pretreatment with the peptide mixture the immunodominant epitopes recognized by T cells from
or the hybrid peptide (Fig 2). polysensitized mice were identical to some of the T-cell
epitopes in patients allergic to birch and grass pol-
Polytolerance induction is not regulated len.24,26,27 Thus, our murine model of multiple allergen
by TGF-b or IL-10 sensitivity showed similar immunological characteristics
No significant changes in TGF-b mRNA expression to those of human pollinosis.
were detected after pretreatment with the peptide mixture From previous studies we know that rBet v 1 acts as a
or the hybrid peptide compared with polysensitized potent mucosal tolerogen in monosensitized mice.16,17,19
controls (Fig 3, A). IL-10 levels were significantly sup- However, when rBet v 1 was intranasally applied before
pressed in peptide mixture as well as hybrid peptide– polysensitization with the 3 allergens, the tolerizing
pretreated mice (Fig 3, B). Neutralization of IL-10 receptor effects of Bet v 1 toward itself were considerably im-
in vitro did not abrogate the suppression of IL-10 levels in paired.28 In addition, in polysensitized mice, mucosal
the pretreated groups (Table III). Moreover, adoptive cell pretreatment with Bet v 1 alone did not alter the immune
transfer experiments showed that tolerance was not trans- response toward the coapplied grass pollen allergens,
ferable by spleen cells, because IgE-induced basophil indicating that no bystander or linked suppression can be
AUGUST 2005
TABLE II. Allergen-specific IgG2a antibody levels and IFN-g production in vitro in polysensitized and pretreated mice
IgG2a (OD)* IFN-g (pg/mL)y

rBet v 1 rPhl p 1 rPhl p 5 BP Phleum
Polysensitized 0.51 6 0.35 0.31 6 0.12 0.75 6 0.37 1501.9 6 621.1 1307.9 6 372.7
Protein-tolerized 0.29 6 0.19 0.29 6 0.26 0.71 6 0.51 2160.7 6 752.1 1885.6 6 553.3
Peptide-tolerized 0.93 6 0.71 1.02 6 0.77 1.56 6 0.46 2305.7 6 798.5 1753.5 6 657.1
Hybrid-tolerized 1.25 6 1.03 1.07 6 0.82 1.38 6 0.61 3486.9 6 184.1** 1869.7 6 204.1
*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.
ocular diseases
FIG 2. Number of eosinophils (A) and IL-5 levels (B) in bronchoal-

veolar lavage fluids from polysensitized mice (white bars), from
peptide mixture–pretreated mice (gray bars), and from mice pre-
treated 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 FIG 3. A, TGF-b mRNA expression from peptide mixture (gray bar)
possible to reduce IgE-dependent basophil degranulation, and hybrid peptide–pretreated mice (black bar) shown as relative
allergen-specific antibody responses, or cytokine produc- values in comparison with polysensitized controls (white bar). B,
tion with the mixture of the proteins in polysensitized IL-10 production in vitro from polysensitized (white bars), peptide
mixture (gray bars), and hybrid peptide–pretreated (black bars)
mice. These results are in line with other studies showing mice. *P < .05, **P < .01, pretreated vs polysensitized. C, RBL assay
that the tolerogenic efficacy was impaired when more after adoptive transfer of spleen cells from peptide mixture (trans-
than 1 protein antigen was ingested via the nasal or oral fer, gray bars) or hybrid peptide–pretreated mice (transfer, black
route.30,31 bars) compared with polysensitized controls (white bars).
Several studies have demonstrated that allergen-
derived immunodominant peptides applied via mucosal Indeed, intranasal administration of either of the peptide
surfaces have a high tolerogenicity in naive and in primed mixture or the hybrid peptide led to a highly significant
animals.15,32,33 It was further shown that treatment of mice reduction of allergen-specific TH2 responses (Fig 1) and
with a peptide containing a single immunodominant airway inflammation (Fig 2). Other studies showing that
epitope led to inhibition of immune responses directed application of a panel of peptides suppressed TH2 immune
to the other epitopes on the natural allergen, a phenom- responses were directed only against single allergens,32,34
enon called intramolecular suppression.33 Therefore, to whereas this is the first report showing that peptide-
enhance the efficacy of tolerance induction in the poly- induced tolerance can be directed against 3—and probably
sensitized mice, we applied a mixture of the immunodo- more—different antigens/allergens.
minant peptides or a synthetic hybrid composed of the Our observation that the mixture of protein allergens
major T-cell epitopes of Bet v 1, Phl p 1, and Phl p 5. was unable to induce tolerance, whereas the polypeptides
TABLE III. IL-10 levels after neutralization of IL-10 nisms such as clonal anergy, described in a model of
receptor in vitro* peptide-induced tolerance,32 or cytokine-independent
Control Ab aIL-10 receptor Ab
mechanisms, such as cell-cell contact interaction via the
Notch signalling pathway40 or via cytotoxic T lympho-
BP Phleum BP Phleum
cyte-associated antigen 4 expression on antigen-specific
Poly-sens 241.82 940.66 283.94 1095.78 regulatory T cells,41 might rather play a role in our model
Peptide-tol 138.63 472.41 158.91 661.14 of polytolerance induction.
Hybrid-tol 165.84 630.72 283.74 790.85 In conclusion, this is the first report showing that
*Pooled splenocytes from polysensitized (poly-sens), peptide mixture
mucosal application of polypeptide constructs inhibited
(peptide-tol), or hybrid peptide (hybrid-tol) pretreated mice were stimulated polysensitization to several antigens/allergens. Thus,
with BP or Phleum extract in the presence of a neutralizing anti IL-10 these data could be the basis for the development of a
receptor antibody or an isotype control. IL-10 levels (pg/mL) in mucosal polyvalent allergy vaccine for primary preven-
supernatants were measured by ELISA.
tion of multiple sensitivities in atopic individuals. It
remains to be investigated whether polytolerance induc-
were able to do so, leads to the speculation that because of tion can be used for treatment of already established
the different conformation of the allergens, distinct antigen- multisensitivities.
presenting cells were targeted.35 It might further be
speculated that the reduced/nontolerogenic capacity of We thank Karin Baier for technical assistance, Dr Michael Kundi
the protein mixture is a result of an intermolecular competi-

for statistical analysis, and Dr Helmut Fiebig for providing the Phl p 5
tion among a higher number of epitopes for the accessi- peptides.
ocular diseases
bility 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 REFERENCES
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23. Bauer L, Bohle B, Jahn-Schmid B, Wiedermann U, Daser A, Renz H, 37. Smith TR, Larche M. Investigating T cell activation and tolerance in
et al. Modulation of the allergic immune response in BALB/c mice by vivo: peptide challenge in allergic asthmatics. Cytokine 2004;28:49-54.
subcutaneous injection of high doses of the dominant T cell epitope from 38. Southwood S, Sidney J, Kondo A, del Guercio MF, Appella E, Hoffman
the major birch pollen allergen Bet v 1. Clin Exp Immunol 1997;107: S, et al. Several common HLA-DR types share largely overlapping
536-41. peptide binding repertoires. J Immunol 1998;160:3363-73.
24. Schenk S, Breiteneder H, Susani M, Najafian N, Laffer S, Duchene M, 39. Bohle B, Radakovics A, Jahn-Schmid B, Hoffmann-Sommergruber K,
et al. T-cell epitopes of Phl p 1, major pollen allergen of timothy grass Fischer GF, Ebner C. Bet v 1, the major birch pollen allergen, initiates
(Phleum pratense): evidence for crossreacting and non-crossreacting sensitization to Api g 1, the major allergen in celery: evidence at the
T-cell epitopes within grass group I allergens. J Allergy Clin Immunol T cell level. Eur J Immunol 2003;33:3303-10.
1995;96:986-96. 40. Hoyne GF, Le Roux I, Corsin-Jimenez M, Tan K, Dunne J, Forsyth LM,
25. Von Garnier C, Astori M, Kettner A, Dufour N, Corradin G, Spertini F. et al. Serrate1-induced notch signalling regulates the decision between
In vivo kinetics of the immunoglobulin E response to allergen: bystander immunity and tolerance made by peripheral CD4(1) T cells. Int
effect of coimmunization and relationship with anaphylaxis. Clin Exp Immunol 2000;12:177-85.
Allergy 2002;32:401-10. 41. Fowler S, Powrie F. CTLA-4 expression on antigen-specific cells but not
26. Müller WD, Karamfilov T, Kahlert H, Stuwe HT, Fahlbusch B, IL-10 secretion is required for oral tolerance. Eur J Immunol 2002;32:
Cromwell O, et al. Mapping of T-cell epitopes of Phl p 5: evidence for 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
Background: Allergy skin tests were administered in the second Key words: Allergens, allergic sensitization, allergy skin test,
and third National Health and Nutrition Examination Surveys epidemiology, NHANES II, NHANES III, survey
(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 Over the last 2 or more decades, rates of asthma have
rates in NHANES III and identified predictors of one or more increased in the United States and worldwide, although
positive test responses. Comparisons with NHANES II were there is some evidence that asthma rates might have
also made. peaked.1-3 One of the most important risk factors for
Methods: In NHANES III, 10 allergens and 2 controls were asthma is sensitization to one or more allergens. The
occupational respiratory
tested in all subjects aged 6 to 19 years and a random half- National Center for Health Statistics included allergy
sample of subjects aged 20 to 59 years. A wheal-based definition
Environmental and
skin testing in the second and third National Health and
of a positive test response was used.
Nutrition Examination Surveys (NHANES II and III),
disorders
Results: In NHANES III, 54.3% of the population had positive
which were conducted from 1976 through 1980 and 1988
test responses to 1 or more allergens. Prevalences were 27.5%
for dust mite, 26.9% for perennial rye, 26.2% for short through 1994, respectively, to estimate and monitor the
ragweed, 26.1% for German cockroach, 18.1% for Bermuda prevalence of allergic sensitization in the United States.
grass, 17.0% for cat, 15.2% for Russian thistle, 13.2% for white Although skin test results from NHANES II have been
oak, 12.9% for Alternaria alternata, and 8.6% for peanut. published,4 a comprehensive summary of skin test results
Among those with positive test responses, the median number from NHANES III has not been published, nor has a
of positive responses was 3.0. Adjusted odds of a positive test comparison between NHANES II and III data been
response were higher for the following variables: age of 20 to 29 published. The primary objectives were to estimate rates
years, male sex, minority race, western region, old homes, and of positive skin test responses in NHANES III and to
lower serum cotinine levels. For the 6 allergens common to
identify predictors of a positive test response to 1 or more
NHANES II and III, prevalences were 2.1 to 5.5 times higher in
allergens. A secondary objective was to compare positive
NHANES III.
Conclusions: The majority of the US population represented in skin test response rates between NHANES II and III;
NHANES III was sensitized to 1 or more allergens. Whether however, methodological differences between the 2
the higher prevalences observed in NHANES III reflect true surveys, which this article describes in detail, provide
changes in prevalence or methodological differences between challenges for comparing and interpreting rate differences
the surveys cannot be determined with certainty. (J Allergy between the 2 surveys.
Clin Immunol 2005;116:377-83.)
METHODS
a
From the Laboratory of Respiratory Biology, Division of Intramural Research, NHANES II and III
National Institute of Environmental Health Sciences, National Institutes of
Health, Research Triangle Park, and bthe Division of Allergy, Immunology, NHANES II and III were two in a series of population-based
and Transplantation, National Institute of Allergy and Infectious Diseases, surveys conducted by the National Center for Health Statistics to
National Institutes of Health, Bethesda. determine the health and nutritional status of the US population. Both
This analysis of National Health and Nutrition Examination Survey (NHANES surveys used a complex design to sample the civilian, noninstitu-
III) data was funded by the National Institute of Environmental Health tionalized population. In NHANES II, questionnaires and medical
Sciences and the National Institute of Allergy and Infectious Diseases. examinations were administered to 20,322 individuals aged 6 months
Received for publication March 1, 2005; revised April 25, 2005; accepted for to 74 years, whereas in NHANES III, 31,311 individuals aged 2
publication May 12, 2005.
months to 90 years were interviewed and examined.
Reprint requests: Darryl C. Zeldin, MD, NIEHS/NIH, PO Box 12233, MD
Allergy skin testing in NHANES II and III
D2-01, Research Triangle Park, NC 27709. E-mail: zeldin@niehs.nih.gov.
0091-6749 Prick-puncture allergy skin testing was performed in NHANES II
doi:10.1016/j.jaci.2005.05.017 and III; however, there were important differences in age eligibility,
377
378 Arbes et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
TABLE I. Prevalences of positive skin test responses

Abbreviations used among the US population aged 6 to 59 years
NHANES II: The second National Health and Nutrition represented in NHANES III
Examination Survey
Allergen tested Percentage (SE)
NHANES III: The third National Health and Nutrition
Examination Survey Indoor allergens
Dust mite 27.5 (1.02)
German cockroach 26.1 (0.82)
Cat 17.0 (1.00)
At least one indoor allergen 43.0 (1.12)
medical exclusion criteria, number and types of allergens tested, Outdoor allergens
standardization of allergen extracts, and reading times for the Perennial rye 26.9 (0.88)
reactions. Overviews of the allergy skin test protocols for both Short ragweed 26.2 (1.03)
surveys are presented here; however, details of the protocols can be Bermuda grass 18.1 (0.81)
found elsewhere.5,6 Russian thistle 15.2 (0.92)
In NHANES II, prick-puncture allergy skin tests to 8 allergens White oak 13.2 (0.78)
(house dust, cat, dog, Alternaria alternata, mixed giant-short rag- Alternaria alternata 12.9 (0.69)
weed, oak, perennial ryegrass, and Bermuda grass) and 2 controls At least one outdoor allergen 40.0 (1.22)
(positive and negative) were administered to all subjects aged 6 to 74 Food allergen: peanut 8.6 (0.51)
years. Each of the allergens was commercially available and US Food At least one indoor or outdoor allergen 53.9 (1.02)
and Drug Administration licensed, but none was standardized. A At least one of any type 54.3 (1.00)
standardized extract is one for which a reference standard for potency
exists. The positive control was histamine phosphate, and the nega-
tive control was 50% glycerol saline. Subjects in 48 of the 64 primary
sampling units were tested with a histamine base concentration of positive and negative controls was at least 1 mm. For NHANES II
0.1 mg/mL, a less than optimal concentration, whereas the rest were results, measurements from the 20-minute reading were used.
tested with the optimal concentration of 1.0 mg/mL.7 Lengths and In NHANES II, 11,769 of the 16,204 subjects who were age
widths of wheals (raised area in the middle of the reaction) and flares eligible for skin testing were aged 6 to 59 years, and of those, 11,062
Environmental and
(reddish area around the wheal) were measured at 10 and 20 minutes. had a wheal-based result for the 6 allergens common to both surveys.
Subjects with a history of allergy to cats, dogs, or ragweed were not Of the 11,062 subjects, 7230 had a valid skin test panel, 3024 had an
disorders
initially tested for those allergens. At the 10-minute reading, if the invalid panel, and 808 were missing a positive control result. The
subject reacted to fewer than 3 of the remaining allergens, then dog, NHANES II analysis was limited to the 7230 subjects; however, a
cat, and ragweed were tested on the other arm. If 3 or more responses secondary analysis was conducted without regard to the valid panel
were positive, then only ragweed was tested on the other arm. criterion (n = 11,062).
In NHANES III, prick-puncture allergy skin tests to 10 allergens In NHANES III, there were 12,106 age-eligible subjects, and of
(Table I) and 2 controls (positive and negative) were administered to those, 10,863 participated in skin testing, 174 were excluded for
all subjects aged 6 to 19 years and a random half-sample of subjects medical reasons, and 1069 refused or were unavailable for testing. Of
aged 20 to 59 years. The positive control was histamine phosphate the 10,863 subjects, 10,841 had a result for all 10 allergens, and of
(concentration is not published), and the negative control was 50% those, 10,508 had a valid skin test panel, 332 had an invalid panel, and
glycerol saline.8 Only house dust mite, cat, and short ragweed 1 subject was missing a positive control result. The NHANES III
allergens were standardized (personal communication with Paul analysis was limited to the 10,508 subjects.
Turkeltaub, MD, December 2, 2004). Lengths and widths of wheals
and flares were measured after 15 minutes (6 5 minutes). Subjects Statistical analyses
were medically excluded from skin testing if they usually did not have Percentages (with SEs) of the population with positive skin test
trouble breathing in their chest or lungs but were having trouble responses were estimated among the populations aged 6 to 59 years
breathing at the time of the examination, although not from a cold; if represented by the surveys. Sociodemographic or medical examina-
they usually had trouble breathing in their chest or lungs and had tion variables were assessed as potential predictors of one or more
more trouble breathing at the time of the examination; if they had a positive skin test responses in NHANES III. The complete list can be
severe response to allergen skin testing previously; or if they had viewed in Table E1 in the Online Repository in the online version of
severe eczema or infection on both arms. this article at www.mosby.com/jaci. Potential predictors were eval-
For comparisons between NHANES II and III, prevalences of uated first with x2 statistics and then with multivariable logistic
positive skin test reactions in NHANES II were estimated for the regression by using a backward selection process. The process began
6 allergens and ages (6-59 years) common to both surveys. The with all potential predictors in the model and ended with variables at a
6 allergens were cat, ragweed (mixed giant and short in NHANES II P value of .050 or less. Education, rather than poverty/income ratio,
and short in NHANES III), perennial rye, oak (oak in NHANES II and was modeled as an indicator of socioeconomic status because the
white oak in NHANES III), Bermuda grass, and A alternata. latter had a large number of missing values, and serum cotinine level
was modeled in place of smoker in the home because serum cotinine
Definition of a positive skin test response level is a biomarker for tobacco smoke exposure. Two-way interac-
For our analyses of NHANES II and III skin test data, we tions between sex, age, and race-ethnicity were evaluated and
considered an allergen-specific skin test response positive if the skin adjusted for the other predictors in the model. Only interactions
test panel was valid and the difference between the mean of the significant at the .050 level were reported.
wheal’s length and width for the allergen-specific test and the Statistical analyses were conducted with SAS Version 9.1 (SAS
negative control was at least 3 mm. A skin test panel was considered Institute, Cary, NC) or SUDAAN Release 9.0 (RTI International,
valid if the difference between the mean wheal diameters of the Research Triangle Park, NC) software. All percentages and odds
J ALLERGY CLIN IMMUNOL Arbes et al 379
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)
Among the
Among population
the total with positive
Allergen type population test responses
Indoor only* 13.7 (0.55) 25.3 (1.13)

Outdoor only 9.7 (0.68) 17.9 (1.26)
Peanut only 0.3 (0.11) 0.6 (0.20)
Indoor and outdoor only 22.2 (1.10) 41.0 (1.57)
Indoor and peanut only 0.2 (0.06) 0.3 (0.11)
Outdoor and peanut only 1.2 (0.18) 2.3 (0.32)
FIG 1. Percentage of the US population aged 6 to 59 years Indoor, outdoor, and peanut 6.9 (0.50) 12.6 (0.88)
(NHANES III) by numbers of positive skin test responses. None 45.7 (1.00) –
Total 100.0 (0.00) 100.0 (0.00)
*House dust mite, cat, or German cockroach.

ratios reported in this article were weighted to represent population
Short ragweed, perennial rye, Alternaria alternata, Bermuda grass,
estimates, and all SEs were adjusted for the complex survey design. Russian thistle, or white oak.
Numbers of subjects reported in this article are unweighted.
RESULTS NHANES III: Predictors of 1 or more

NHANES III: Prevalences of positive positive test responses
skin test responses The independent predictors of 1 or more positive test
Environmental and
Table I shows the prevalences of positive skin test responses were sex, age, race-ethnicity, census region,
responses among the US population aged 6 to 59 years. home construction year, and serum cotinine level. The
disorders
More than half of the population had positive test distributions of these predictors in the US population and
responses to one or more allergens. The highest preva- their adjusted odds ratios are shown in Table III. The
lences were for dust mite, rye, ragweed, and cockroach, distributions of all tested predictors and their bivariate
and the lowest prevalence was for peanut. A positive test associations with each of the 10 allergen skin tests can be
response to at least 1 indoor allergen was slightly more found in Table E1 in the Online Repository in the online
common than a positive test response to at least 1 outdoor version of this article at www.mosby.com/jaci.
allergen (43.0% vs 40.0%), even though twice as many Age was bivariately associated with each allergen test
outdoor allergens were tested. (Table E1). The prevalence of 1 or more positive test
The percentage of the population with a positive test responses, as well as the adjusted odds ratio, increased
response decreased as the number of positive test re- from the first decade of age to the second, peaked in the
sponses increased from 1 to 10 (Fig 1). A solitary positive third decade, and then decreased through the sixth decade
skin test response was seen in 15.5% (SE = 0.48) of the (Table III).
total population and 28.7% (SE = 0.95) of the population For each allergen tested, male subjects were more likely
with positive test responses. The 2 most common solitary than female subjects to have positive test responses (Table
reactions were to cockroach (4.3% [SE = 0.42] of the total E1). The adjusted odds of having 1 or more positive test
population) and dust mite (4.2%, SE = 0.24). The preva- responses were 1.6 times greater in male subjects (Table
lences of a solitary reaction to the other allergens ranged III). The odds ratio for sex did not differ by age (P value
from 0.10% to 1.70% of the total population. The mean for sex-age interaction term = .518); however, it did
and median numbers of positive test responses among differ by race-ethnicity (P value for sex-race interaction
those with positive test responses were 3.5 (SE = 0.06) term = .027). With the sex-race interaction term in the
and 3.0, respectively. model (model not shown), the adjusted odds ratios com-
Table II shows how positive skin test responses— paring male subjects with female subjects were 1.6 (95%
classified as indoor, outdoor, and peanut—were distrib- CI, 1.3-2.0) for non-Hispanic whites, 1.4 (95% CI, 1.1-
uted among the total US population and among those with 1.8) for non-Hispanic blacks, 1.1 (95% CI, 0.9-1.4) for
positive test responses. Among those with positive test re- Mexican Americans, and 2.0 (95% CI, 1.1-3.8) for others.
sponses, 41% reacted to a combination of indoor and out- Race-ethnicity was bivariately associated with a posi-
door allergens (but had negative test responses to peanut). tive test response to 7 of the 10 allergens (Table E1).
A positive test response to peanut alone was quite rare Compared with non-Hispanic whites, the adjusted odds
(0.6%), as were positive test responses to indoor allergens of having 1 or more positive test responses were greater
and peanut (0.3%) and outdoor allergens and peanut for each of the other 3 race-ethnicity categories (Table
(2.3%). III). However, as mentioned in the previous paragraph,
AUGUST 2005
TABLE III. Prevalences and odds ratios for the indepen- pattern remained in the adjusted model for 1 or more
dent predictors of 1 or more positive skin test responses positive test responses (Table III).
among the US population aged 6 to 59 years represented
in NHANES III Comparisons between NHANES II and III
The prevalences in NHANES II for positive test
Adjusted* Wald F
Percentage odds ratio test,
responses to the 6 allergens and ages (6-59 years) common
Predictor (SE) (95% CI) P value 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,
Age (y)
6-9 45.6 (2.19) 1.0 (reference)
5.2% (SE = 0.49) for Bermuda grass, 4.5% (SE = 0.29)
10-19 55.5 (1.33) 1.7 (1.3-2.1) for A alternata, and 3.1% (SE = 0.32) for cat. The
20-29 60.0 (1.79) 2.1 (1.6-2.8) NHANES III prevalences for those 6 allergens were 2.1
30-39 56.5 (1.96) 1.8 (1.4-2.4) to 5.5 times higher (Table I), and the NHANES III
40-49 50.5 (2.71) 1.5 (1.1-2.0) population was much more likely to react to at least 1 of
50-59 49.1 (2.70) 1.4 (1.0-1.8) <.001 the 6 allergens (41.9% [SE = 1.23] vs 21.8% [SE = 0.94]).
Sex As shown in Fig 2, rates of positive test responses were
Female 49.2 (1.23) 1.0 (reference) consistently higher in NHANES III than in NHANES II at
Male 59.4 (1.21) 1.6 (1.4-1.8) <.001 each age group.
Race-ethnicity
For both surveys, the prevalences without the valid-
Non-Hispanic white 51.3 (1.17) 1.0 (reference)
Non-Hispanic black 62.0 (1.26) 1.6 (1.4-1.9)
panel criterion were systematically less, although only
Mexican American 57.1 (1.28) 1.2 (1.0-1.4) slightly less. For example, the rate for a positive test
Other 64.0 (2.85) 1.5 (1.2-2.0) <.001 response to 1 or more of the 6 allergens decreased from
Census region 41.9% to 41.4% in NHANES III and 21.8% to 19.6% in
South 50.8 (1.40) 1.0 (reference) NHANES II.
West 58.0 (1.51) 1.3 (1.1-1.6)
Northeast 57.9 (2.78) 1.2 (0.9-1.8)
Midwest 52.8 (2.57) 1.1 (0.9-1.5) .042 DISCUSSION

Environmental and
Year home constructed

1974 to present 53.1 (1.40) 1.0 (reference)
The main finding of this study was that 54.3% of the
disorders
1946-1973 52.1 (1.60) 0.9 (0.8-1.1)

Before 1946 59.4 (1.69) 1.3 (1.1-1.6) .002 population represented by NHANES III had 1 or more
Cotinine (ng/mL) positive skin test responses to 10 common allergens. With
0.035-0.100 56.9 (2.17) 1.0 (reference) the limited number of allergens tested, this might be an
0.100-10.00 55.4 (1.63) 0.9 (0.7-1.1) underestimation of the prevalence of allergic sensitization
10.00-1080.00 51.0 (1.48) 0.7 (0.5-0.9) .012 in the US population. On average, an individual with a
positive test response reacted to 3 to 4 allergens, and most
*Adjusted for each variable in the table.
with positive test responses reacted to a combination of
indoor and outdoor allergens as opposed to indoor,
there was a significant interaction between sex and outdoor, or peanut allergens alone. For each of the 6
race-ethnicity. Among female subjects, the adjusted allergens tested in both NHANES II and III, the preva-
odds ratios for race-ethnicity (with non-Hispanic whites lence of a positive test response was higher in NHANES
as the referent) were 1.8 (95% CI, 1.4-2.2) for non- III at each decade of age.
Hispanic blacks, 1.4 (95% CI, 1.2-2.7) for Mexican Even though we analyzed positive skin test response
Americans, and 1.4 (95% CI, 0.9-2.1) for others. Among rates for the allergens and ages common to both surveys,
male subjects, those adjusted odds ratios were 1.5 (95% there were differences between the surveys that could not
CI, 1.2-1.8), 1.0 (95% CI, 0.8-1.2), and 1.7 (95% CI, 1.2- be controlled, such as differences in medical exclusion
2.5), respectively. criteria, in reading times of the reactions, in the histamine
Census region was bivariately associated with tests to concentrations for the positive controls, and in the quality
the outdoor allergens ragweed, rye, grass, and thistle of the allergen extracts. It would seem doubtful that
(Table E1). For 1 or more positive test responses, the differences in medical exclusion criteria, reading times,
adjusted odds ratio was lowest for the south and highest and histamine concentrations would have contributed
for the west (Table III). significantly to the differences in rates because only a
Home construction year was bivariately associated with small percentage of subjects were excluded for medical
a positive test response to dust mite, cockroach, ragweed, reasons in each survey, reading times overlapped some-
and peanut (Table E1). The prevalence of one or more what, and results remained essentially the same irrespec-
positive test responses, as well as the adjusted odds ratio, tive of whether the histamine control was used in the
was greatest in the oldest homes (Table III). definition of a positive test response. One methodological
Serum cotinine levels were bivariately associated with difference that could potentially explain the differences in
4 of the 6 outdoor allergens (Table E1); however, for those positive skin test response rates is the potency of the
allergens, the lowest cotinine level was associated with the allergens used. Only the cat and ragweed allergens tested
highest prevalence of a positive test response. That same in NHANES III were standardized, and without standard-
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).
Environmental and
ization, it cannot be assumed that the potencies of 1 or more positive skin test responses, with rates peaking
disorders
the allergens were the same between surveys. In fact, at age 20 to 29 years. In cross-sectional studies it is often
unstandardized allergen extracts can vary greatly in their difficult to determine whether age effects are real or are
potencies.9 due to a cohort effect (ie, the effect of capturing a high-risk
The relative potencies of the allergens used in these 2 cohort at a point in time). However, the age-specific
surveys are unknown, and because of this, it cannot be comparisons between NHANES II and III (Fig 2) provide
stated with any certainty that the increases in positive skin strong evidence that the prevalence of allergic sensitiza-
test response rates observed between NHANES II and III tion truly peaks in the third decade of life. If the NHANES
were due to true increases in reactivity in the US popu- III finding had been due to a cohort effect, then NHANES
lation. However, we would like to present 2 arguments II rates would have peaked at a younger age.
that support true increases. The prevalence of 1 or more positive test responses was
First, potency between unstandardized allergens could higher among male than female subjects, and the preva-
be greater, less, or the same, and it would seem unlikely lence was higher for male subjects at each decade of life. In
that the potency would have been systematically greater the general population, male subjects have higher levels of
for all 6 of the NHANES III allergens. An example of the serum IgE than female subjects at any given age,13 but
variability one might expect between allergen prepara- whether sex influences sensitization primarily through a
tions can be found within NHANES II itself. Within genetic or an environmental pathway is not known. The
NHANES II, complete panels of allergens were purchased higher prevalence of allergic sensitization among male
from 2 different manufacturers, and subjects were tested subjects at any age is in contrast to the pattern seen with
with one panel or the other.7 In a comparison of positive asthma. For asthma, the prevalence is greater in male
skin test response rates between these 2 panels of aller- subjects during childhood but greater in female subjects
gens, Gergen and Turkeltaub7 found that one panel gave during the teenage and adult years.14 This contrast
higher rates for 3 allergens, lower rates for 1 allergen, and suggests that factors other than allergic sensitization are
similar rates for 4 allergens; however, none of the absolute responsible for the sex-related shift in asthma prevalence
differences was greater than 4.7%. Second, the increases observed at or near puberty.
seen between NHANES II and III are consistent with Compared with non-Hispanic whites, the odds of hav-
reports from other countries, such as Japan,10 the United ing 1 or more positive skin test responses were increased
Kingdom,11 and Denmark.12 for the other 3 race-ethnicity categories. For NHANES II,
In NHANES III, sex, race-ethnicity, age, census region, Gergen et al4 reported that the age-adjusted prevalence of
home construction date, and serum cotinine level were 1 or more positive test responses was higher in blacks than
independent predictors of 1 or more positive skin test whites; however, the difference was not statistically sig-
responses. Age was the strongest independent predictor of nificant. In a study of allergic sensitization among children
AUGUST 2005
in NHANES III, Stevenson et al15 argued that race or that the increases in positive skin test response rates
ethnicity differences in sensitization likely reflect differ- observed between NHANES II and III represent an
ences in environmental exposures rather than genetics. In increase in the reactivity of the US population, such an
finding race-ethnicity a strong predictor of positive test increase would be consistent with studies from other
responses to dust mite, cockroach, and A alternata, countries. In NHANES 2005-2006, total and allergen-
those authors reasoned that the association was most specific IgE levels are being measured in all subjects,
likely to be due to differences in housing and community along with levels of indoor allergens in their homes.
environments, which would lead to differences in allergen
exposures. We thank Drs Stephanie London and Donna Baird (Epidemiology
For census region and home construction date, the Branch, National Institute of Environmental Health Sciences) for
allergen-specific results suggest that these predictors providing helpful comments during the preparation of this
affect sensitization primarily through an environmental manuscript.
pathway. Census region was bivariately associated with
positive test responses to outdoor allergens only, which
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3. Verlato G, Corsico A, Villani S, Cerveri I, Migliore E, Accordini S, et al.
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survey of US housing, it was shown that older homes had Results of an Italian study. J Allergy Clin Immunol 2003;111:1232-8.
higher levels of dust mite, cockroach, and mouse allergens 4. Gergen PJ, Turkeltaub PC, Kovar MG. The prevalence of allergic skin
than newer homes.16,17 test reactivity to eight common aeroallergens in the U.S. population:
results from the second National Health and Nutrition Examination
Higher serum cotinine levels predicted lower preva-
Survey. J Allergy Clin Immunol 1987;80:669-79.

lences of 1 or more positive test responses. Active
Environmental and
5. National Health and Nutrition Examination Survey III. Training manual

smoking has been associated with increased serum levels for allergy component. Available at: http://www.cdc.gov/nchs/data/
of total IgE; however, the published literature on the nhanes/nhanes3/cdrom/nchs/manuals/train.pdf. Accessed November 9,
disorders
relationship between either active or passive smoking and 2004.

6. National Center for Health Statistics (U.S.). Public use data tape
skin test response positivity is inconclusive.18,19 Chronic documentation: allergy skin testing: tape number 5309: National Health
tobacco smoke exposure can suppress the immune system and Nutrition Examination Survey, 1976-80. Hyattsville (MD): US
and impair host defenses,20 which could potentially lead to Department of Health and Human Services, Public Health Service,
lower sensitization rates; however, smoke avoidance National Center for Health Statistics; 1986.
7. Gergen PJ, Turkeltaub PC. National Center for Health Statistics (US).
among persons with allergies and asthma would also
Percutaneous immediate hypersensitivity to eight allergens, United
lead to lower rates. States, 1976-80. Washington (DC): US Department of Health and
Two potential predictors worth discussing that did not Human Services Public Health Service, National Center for Health
remain in the final prediction model were the presence Statistics; 1986.
of an indoor cat and the presence of an indoor dog. The 8. Plan and operation of the Third National Health and Nutrition Exam-
ination Survey, 1988-94. Series 1: programs and collection procedures.
role of pet exposure in the cause of allergic sensitization Vital Health Stat 1 1994:(32)1-407.
and disease is controversial. One limitation to this cross- 9. Gleich GJ, Leiferman KM, Jones RT, Hooton ML, Baer H. Analysis of
sectional analysis was the inability to assess the timing of the potency of extracts of June grass pollen by their inhibitory capacities
exposures and the development of allergic sensitization, in the radioallergosorbent test. J Allergy Clin Immunol 1976;58:31-8.
10. Nakagomi T, Itaya H, Tominaga T, Yamaki M, Hisamatsu S, Nakagomi
which could be an explanation for the lack of association
O. Is atopy increasing? Lancet 1994;343:121-2.
with indoor cat and dog. Interestingly, the presence of an 11. Sibbald B, Rink E, D’Souza M. Is the prevalence of atopy increasing?
indoor cat was not associated with a positive skin test Br J Gen Pract 1990;40:338-40.
response to cat allergen. One potential explanation for this 12. Linneberg A, Nielsen NH, Madsen F, Frolund L, Dirksen A, Jorgensen
null result could be the pervasiveness of cat allergen in US T. Increasing prevalence of specific IgE to aeroallergens in an adult
population: two cross-sectional surveys 8 years apart: the Copenhagen
homes. In the National Survey of Lead and Allergens in Allergy Study. J Allergy Clin Immunol 2000;106:247-52.
Housing, 99% of homes with an indoor cat and 56% of 13. Barbee RA, Halonen M, Lebowitz M, Burrows B. Distribution of IgE in
homes without an indoor cat had cat allergen levels that a community population sample: correlations with age, sex, and allergen
exceeded the proposed threshold for allergic sensitiza- skin test reactivity. J Allergy Clin Immunol 1981;68:106-11.
14. Asthma Prevalence, Health Care Use and Mortality, 2000-2001. Avail-
tion.21 In epidemiologic studies the more widespread an
able at: http://www.cdc.gov/nchs/products/pubs/pubd/hestats/asthma/
exposure is within a population, the more difficult it asthma.htm. Accessed April 29, 2003.
becomes to demonstrate its effects.22 Another potential 15. Stevenson LA, Gergen PJ, Hoover DR, Rosenstreich D, Mannino DM,
explanation could be cat avoidance among persons who Matte TD. Sociodemographic correlates of indoor allergen sensitivity
are sensitized to cats. 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,
In conclusion, the majority of the US population et al. House dust mite allergen in US beds: results from the First National
represented in NHANES III was sensitized to 1 or more Survey of Lead and Allergens in Housing. J Allergy Clin Immunol 2003;
allergens. Although it cannot be definitively concluded 111:408-14.
17. Cohn RD, Arbes SJ Jr, Yin M, Jaramillo R, Zeldin DC. National 20. Sopori ML, Kozak W. Immunomodulatory effects of cigarette smoke.
prevalence and exposure risk for mouse allergen in US households. J Neuroimmunol 1998;83:148-56.
J Allergy Clin Immunol 2004;113:1167-71. 21. Arbes SJ Jr, Cohn RD, Yin M, Muilenberg ML, Friedman W, Zeldin DC.
18. Burrows B, Halonen M, Lebowitz MD, Knudson RJ, Barbee RA. The Dog allergen (Can f 1) and cat allergen (Fel d 1) in US homes: results
relationship of serum immunoglobulin E, allergy skin tests, and smoking from the National Survey of Lead and Allergens in Housing. J Allergy
to respiratory disorders. J Allergy Clin Immunol 1982;70:199-204. Clin Immunol 2004;114:111-7.
19. Strachan DP, Cook DG. Health effects of passive smoking. 5. Parental 22. Rose G. Sick individuals and sick populations 1985. Bull World Health
smoking and allergic sensitisation in children. Thorax 1998;53:117-23. Organ 2001;79:990-6.
Environmental and
disorders
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
Background: Although endotoxin is known to increase

symptoms in allergic individuals, early exposure might Abbreviations used
decrease sensitization. Similarly, the presence of an animal in EU: Endotoxin units
the home has been associated with decreased sensitization to ICD: Ion-charging device
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 places, and houses without a cat, as well as in those with an
over 24 hours in homes with or without animals. The total animal.4-7 Despite (or because of) this high allergen
volume sampled was approximately 1000 m3, which provides exposure, children raised in a house with an animal are
quantities of allergen and endotoxin that can easily be less likely to become sensitized to animal allergens.8-12
measured with standard assays. One possible explanation for this paradoxical finding is
Results: The quantity of endotoxin ranged from less than 0.5 to that allergic families choose to avoid owning animals
more than 500 pg/m3, whereas cat and dog allergen ranged
because of the perceived risk of sensitization.10,13 We
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
have previously reported that high exposure to the cat
animal. However, airborne endotoxin levels were significantly allergen Fel d 1 induces a form of immune tolerance that is
lower in homes with a cat compared with homes with a dog allergen specific.9,14,15 Alternatively, it has been argued
Environmental and
(P < .001). In keeping with this, there was a significant that cats and other animals might increase agents such as
correlation between airborne Can f 1 and airborne endotoxin bacterial LPS (endotoxin) in the home.12 Endotoxin is
disorders
(r = 0.50, P < .01) but not between endotoxin and Fel d 1 known to favor a shift away from TH2 responses in mice
(r = 0.17, P = .27). and might have the same effect on children.16,17 In
Conclusions: The results demonstrate that endotoxin is present keeping with this, children raised in close contact with
in the air of almost all homes. Although higher levels were seen farm animals (ie, with high endotoxin exposure) have less
in homes with a dog, similar levels might be present in homes
allergic disease.18,19 However, published reports are not
with no animals. The results argue that the effects of cat
ownership cannot be explained by increased exposure to
consistent about the effects of pets on either floor or
endotoxin. (J Allergy Clin Immunol 2005;116:384-9.) airborne endotoxin levels.15,20,21
Measuring airborne allergen or endotoxin requires both
Key words: Airborne endotoxin, cats, dogs 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
Respiratory symptoms related to domestic animals are a the concentration in the air is low, low-volume samplers
significant health issue. However, in many studies the will require very sensitive assays and might still provide
prevalence of IgE specific for cats or dogs is lower than for inadequate samples.22,24 On the other hand, a high-
other major allergens, such as pollens or dust mites.1-3 volume collector may sample the air repeatedly and thus
This is not due to inadequate exposure because the major underestimate the airborne concentration, whereas collec-
allergens Fel d 1 and Can f 1 are found in schools, public tors 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
From the Asthma and Allergic Diseases Center, University of Virginia.
Supported by National Institutes of Health grants AI-20565 and AID/EHS
which the particles can be removed and analyzed. We have
grant P01-AI-50989. In addition, J.P.M received an unrestricted educational used this technique to measure airborne allergen levels in
grant from The Sharper Image. homes and airborne endotoxin levels in animal facili-
Disclosure of potential conflict of interest: None disclosed. ties.26,27 Those studies included validating the assay
Received for publication January 10, 2005; revised April 29, 2005; accepted
techniques and the collection efficiency of the device.
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, METHODS
Charlottesville, VA 22908-1355. E-mail: tap2z@virginia.edu.
Airborne sampling
0091-6749/$30.00
Ó 2005 American Academy of Allergy, Asthma and Immunology The machines used for airborne sampling (Ionic Breeze Quadras
doi:10.1016/j.jaci.2005.05.012 from The Sharper Image, San Francisco, Calif) cycle between 2
384
J ALLERGY CLIN IMMUNOL Platts-Mills et al 385
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.
distinct flow rates. For each device, we timed the periods P1 and P2 of there was a close correlation between samples for both endotoxin
Environmental and
each rate. By using a vanometer, the wind speed was measured at the (n = 56, r = 0.91) and Fel d 1 (n = 44, r = 0.93).
center of 114 squares, each with an area of 9 cm2, on a 2-dimensional
disorders
grid placed orthogonal to and directly in front of the machine. These Domestic sampling
speeds were averaged and multiplied by the measurement area to A total of 71 homes in Central Virginia were studied between
determine the flow rate at each speed, V1 and V2. The flow rate of November 2003 and May 2004 to collect dust and carry out air
each device was calculated as follows: ðP1 V1 1P2 V2 Þ=ðP1 1P2 Þ in sampling for 24 hours. Because seasonal variation has been reported
cubic meters per minute. The average flow rate of the 14 devices used for endotoxin, 43 homes were studied both in November-December
in this study was 1.48 6 0.09 m3/min.26,27 and April-May. A floor dust sample was also collected with a Hoover
A 20 L/min air-sampling pump and an ICD were run in parallel in handheld vacuum.
a room with artificially disturbed dust (by using a vacuum cleaner
without a filter) for 10-minute periods (n = 8) to determine the Animal room sampling
collection efficiency of the devices. The 20 L/min pump collected We sampled 20 mouse rooms from 4 different animal facilities
airborne particles by using the same prefilter as was used to clean the (vivariums) at the University of Virginia. Each room was sampled at
collection plates of the ICDs. All samples were extracted overnight in least twice. A variety of cage types are used, but for the purposes of
2 mL of PBS and assayed for endotoxin and Fel d 1. The flow rates of this study, we have distinguished only between open cages, which
the 2 devices and the amount of endotoxin and cat allergen measured have only a metal grill to prevent the animals from exiting the cage,
were used to determine the collection efficiency for ICDs. The mean and filter-topped cages of any configuration. The detailed methods
collection efficiency was 40.6% 6 9.0% for endotoxin and 51.7% 6 have been published elsewhere.27 All animals were used for research
12.0% for Fel d 1, which were not significantly different. For the studies that had been approved by the University of Virginia
purposes of comparison with other studies, an estimated sampling Institutional Animal Care and Use Committee.
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 Assays
sampling rate, we can convert values for the total quantities of Samples were assayed for Fel d 1 and Can f 1 by using 2-site mAb-
airborne endotoxin or allergen collected to airborne concentrations based ELISAs (Indoor Biotechnologies, Inc, Charlottesville, Va),
(Figs 1-3). which are sensitive to 1 ng/mL, and for endotoxin with the Limulus
All sampling used 2 ICDs in parallel running for 24 hours and Amoebocyte Lysate test QCL 1000, which is sensitive to 0.3
placed at least 6 feet apart and at least 4 feet from the wall. In each endotoxin units (EU)/mL (equivalent to 30 pg of endotoxin/mL;
case, the stainless-steel plates of the 2 ICDs were removed and Bio-Whittaker/Cambrex). Because extract freeze-thaw cycles are
cleaned with a series of 3 filters (Millipore prefilters, AP20, 35 mm; associated with a significant decrease in endotoxin concentration,
Millipore Corporation, Bedford, Mass) dampened with sterile water. samples were assayed for endotoxin immediately on extraction. The
Each filter was placed in a 3-mL syringe and extracted overnight at buffer used for extraction was used in each assay as a negative control
4°C. The 3 filters from the first ICD were extracted in 2 mL of 1% and was less than the level of detection in all cases.
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 Statistical analysis
measuring endotoxin. In preliminary experiments 2 ICDs were run in Because the exposure data had a log-normal distribution, all
parallel and both were assayed for either endotoxin or Fel d 1, and values were reported as geometric means with 95% CIs, and statistics
386 Platts-Mills et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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.
were performed on log-transformed data. Means were compared Presenting the results as endotoxin collected over 24
by using independent-sample t tests, whereas a least squares linear hours allows comparison between different homes and
regression was used to assess the seasonal variation in airborne with animal facilities sampled in the same way. The results
Environmental and
endotoxin, the correlation between airborne allergens and endotoxin,

show that endotoxin exposure in homes is lower than in
and the correlation between duplicate measurements for endotoxin
animal rooms where rats or mice are kept without a filter
disorders
and cat allergen. A P value of less than .05 was considered significant.
All statistical analyses were performed with SPSS 11 (SPSS Inc, top on the cage. However, in animal rooms where cage
Chicago, Ill). tops are present, which is the case for the majority of
animal facilities, the mean endotoxin airborne concentra-
tion was lower than that for homes (Table III).
RESULTS
Easily measurable concentrations of endotoxin and DISCUSSION

animal allergens were present in the floor dust samples.
There was a wide range of values, from 0.82 EU/mg to 660 In many studies the prevalence of IgE antibodies
EU/mg, and overall, there was no significant effect of specific for cat or dog allergen is lower than for other
animal ownership (Table I). There were large differences major allergens, such as pollens or dust mites.1-3 This is
in the concentration of cat and dog allergens, which was in not due to inadequate exposure because (1) the phenom-
keeping with the presence of animals. enon is more marked among children living in a house
The results for airborne endotoxin also showed no with a cat,8-12 (2) the allergens are present and airborne
overall difference between homes with animals and homes continuously, and (3) the major allergens Fel d 1 and Can f 1
without animals (Table II). The same data are presented as are found in schools, public places, and houses without
airborne concentrations (Fig 1). When the results were animals, as well as those with an animal. Our data
analyzed by the species of animal present, there were show that the presence of a cat in the home does not
highly significant differences. Homes with a cat or cats increase airborne endotoxin levels. Thus the allergen-
had significantly lower airborne endotoxin levels than specific tolerance to cat allergens cannot be attributed
homes with a dog or with both species (Fig 1). In 43 to increased endotoxin exposure in homes with a cat.
houses airborne sampling for endotoxin was carried out Inevitably, any sampling technique that collects a
twice, first in November-December and again in April- quantity of allergen or endotoxin that can be confidently
May (Fig 2). Overall, there was a good correlation measured will alter the particles present in the air. In
between the 2 measurements (r = 0.57, P < .001). In addition, it is well established that the airflow created by a
particular, the values in homes with a cat were consistently high-volume air filter can increase airborne allergen
lower (ie, <30 pg/m3). Comparing airborne dog allergen levels.23 Thus all methods of measuring airborne concen-
levels with airborne endotoxin levels showed a significant trations of allergen or endotoxin involve a compromise.
positive correlation (r = 0.56, P < .001; Fig 3, A). The The particle collector used here has several disadvantages
association was not significant between endotoxin and cat and some major advantages. The disadvantages are (1)
allergen (r = 0.13, P = .33; Fig 3, B). those associated with high-volume sampling and (2) that
Environmental and
disorders
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 successfully measured airborne endotoxin levels by using
estimate of the efficiency with which particles are col- very low-volume sampling (ie, 2 L/min).22,28 However,
lected. The advantages of the device are that (1) it is silent that required extrasensitive assays and extensive precau-
and therefore well accepted for use in any room, including tions to avoid endotoxin contamination. The collector
bedrooms; (2) sampling of particles off the stainless-steel used here has no electrical safety concerns. Most pumps
plates is simple and very consistent (this is not possible used for collecting airborne allergen are not approved as
with most devices designed to clean the air, including all domestic appliances and therefore should not be left in a
high-efficiency particulate air filters); and (3) it collects home without the presence of an investigator. The range of
particles from large volumes of air, but because of the results observed (ie, from <5 to >5000 EU/24 hours or
wide aperture, the velocity of air coming out is relatively <0.5 to >500 pg/m3) is such that small differences in the
low. estimated collection efficiency (ie, between 41% and
In preliminary experiments sampling air at 18 L/min for 52%) would not affect the interpretation of our results.
2 to 6 hours, we were not able to detect significant In an additional experiment (data not shown), airborne
endotoxin levels in most houses. Other groups have endotoxin and Fel d 1 levels were measured before and
388 Platts-Mills et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
TABLE I. Floor dust concentration of endotoxin and allergens*
N Endotoxin (EU/mg)y Fel d 1 (mg/g) Can f 1 (mg/g)
No animals 23 49.8 (29.9-82.9) 2.5 (1.3-4.7) 2.3 (1.4-4.0)

Animals 39 63.9 (47.1-86.8) 25.1 (7.9-79.9) 49.4 (17.2-142)
Cat(s) only 13 82.8 (45.3-151) 583 (201-1690) 0.65 (0.24-1.7)
Dog(s) only 15 49.9 (31.3-79.5) 0.52 (0.24-1.1) 405 (226-728)
Both species 11 66.1 (39.5-110.7) 121 (36.1-405) 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 Endotoxin (EU/24 h)y Fel d 1 (ng/24 h) Can f 1 (ng/24 h)
No animals 28 115 (61-217) 6.6 (4.9-8.8) 3.3 (2.7-4)

Animals 43 240 (155-372) 102 (64-162) 138 (87-219)
Cat(s) only 16 68 (44-105) 488 (361-660) 3.4 (2.3-4.9)
Dog(s) only 15 447 (250-800) 4.7 (3.6-6.1) 777 (566-1067)
Both species 12 588 (282-1230) 1076 (545-2126) 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).
TABLE III. Airborne endotoxin in homes and animal rooms present, it remains unclear whether inhaled endotoxin in
Environmental and
n EU/24 h (GM)
homes contributes either to sensitization or symptoms.
The concentrations reported here in homes are higher than
disorders
Homes without animals 28 115 (61-217) the concentrations reported to give rise to symptoms
Homes with animals 43 240 (155-372) among animal handlers.28 However, those studies used
Mouse rooms (open cages) 8 1930 (752–4940)*
low-volume sampling and reported a much smaller range
Mouse rooms (filter tops on cages) 20 47.8 (31.9-71.6)
of results than we have found here or in animal facilities.27
GM, Geometric mean. Furthermore, recent short-term challenge studies found
*Significantly higher than homes with or without animals (P < .001). that doses of less than 10,000 EU produced very little
Significantly lower than homes with animals (P < .001) or without immediate change in the lungs.30
animals (P = .024).
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.
after disturbance of dust in an experimental room by using Our data argue in favor of a completely different inter-
a domestic vacuum cleaner without a filter. The results pretation of the effects of cat ownership. In our studies on
suggested that 90% of both airborne endotoxin and cat 4 different cohorts, the effect of cat ownership has been cat
allergen levels fell within 10 minutes. The rapid falling specific. In particular, cat ownership has no effect on the
rates of both endotoxin and cat allergen suggest that the IgE antibody response to dust mite allergens.9,15 In
level of disturbance is as important as the concentration in addition, the immune response to Fel d 1 includes the
reservoirs of dust. The observation that endotoxin levels in IL-4–dependent isotype IgG4.9,10 Thus the nonallergic
the floor dust of houses with cats or dogs are similar, yet response to cat has the features of a modified TH2 response
the airborne levels are significantly different, might sug- and not the TH1 response that would be predicted if the
gest that dogs were a greater cause of disturbance. tolerant response was related to increased endotoxin
Differences in the quantity of endotoxin either airborne exposure.16-18
or in the floor dust of houses with a cat cannot explain why It is important to recognize that there are major differ-
the presence of a cat in the house is associated with a form ences in the ways that cats and dogs are kept, and our
of immune tolerance. However, this is not to say that results might be relevant to specific housing conditions. In
airborne endotoxin is irrelevant to the response. In mice New Zealand the floor dust levels of endotoxin in homes
the immune response to inhaled ovalbumin can be with or without cats (13.7 vs 17.4 EU/mg, not significant)
suppressed or changed by high exposure to endotoxin, were lower than the levels seen here.15 Thus we could be
but the IgE antibody response is enhanced by small looking at 2 different phenomena overlapping in different
quantities of endotoxin.16 We have found airborne endo- studies. The first effect is tolerance induced specifically by
toxin in almost all homes, and this might be sufficient to cat allergen exposure, whereas the second, a nonspecific
act as an adjuvant for responses to inhaled allergens.29 At effect of animal ownership, including dog ownership,
could reflect increased exposure to endotoxin or other events in development of the modified Th2 response to cat allergen.
J Immunol 2004;172:2763-72.
bacterial products.
15. 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
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exposure to cat or dog protect against later allergy development? Clin Stockwell JD, et al. Reducing exposure to laboratory animal allergens.
Exp Allergy 1999;29:611-7. Comp Med 2003;53:486-92.
9. Platts-Mills T, Vaughan J, Squillace S, Woodfolk J, Sporik R. Sensiti- 25. Swanson MC, Agarwal MK, Reed CE. An immunological approach to
zation, asthma, and a modified Th2 response in children exposed to cat indoor aeroallergen quantitation with a new volumetric air sampler:
allergen: a population-based cross-sectional study. Lancet 2001;357: studies with mite, roach, cat, mouse, and guinea pig antigens. J Allergy
752-6. Clin Immunol 1985;76:724-9.
10. Perzanowski MS, Ronmark E, Platts-Mills TA, Lundback B. Effect of 26. Custis NJ, Woodfolk JA, Vaughan JW, Platts-Mills TAE. Quantitive
cat and dog ownership on sensitization and development of asthma measurement of airborne allergens from dust mites, dogs, and cats using
among preteenage children. Am J Respir Crit Care Med 2002;166: an ion charging device. Clin Exp Allergy 2003;33:986-91.
696-702. 27. Platts-Mills J, Custis N, Kenney A, Tsay A, Chapman M, Feldman S,
11. Custovic A, Hallam CL, Simpson BM, Craven M, Simpson A, Wood- et al. The effects of cage design on airborne allergens and endotoxin in
cock A. Decreased prevalence of sensitization to cats with high exposure animal rooms: high-volume measurements with an ion-charging device.
to cat allergen. J Allergy Clin Immunol 2001;108:537-9. Contemp Top Lab Anim Sci 2005;44:12-6.
12. Ownby DR, Johnson CC, Peterson EL. Exposure to dogs and cats in the 28. Pacheco KA, McCammon C, Liu AH, Thorne PS, O’Neill M, Martyny J,
first year of life and risk of allergic sensitization at 6 to 7 years of age. et al. Airborne endotoxin predicts symptoms in non-mouse-sensitized
JAMA 2002;288:963-72. technicians and research scientists exposed to laboratory mice. Am J
13. Anyo G, Brunekreef B, de Meer G, Aarts F, Janssen NA, van Vliet P. Respir Crit Care Med 2003;167:983-90.
Early, current and past pet ownership: associations with sensitization, 29. Liu AH. Endotoxin exposure in allergy and asthma: reconciling a
bronchial responsiveness and allergic symptoms in school children. Clin paradox. J Allergy Clin Immunol 2002;109:379-92.
Exp Allergy 2002;32:361-6. 30. Alexis NE, Lay JC, Almond M, Peden DB. Inhalation of low-dose
14. Reefer AJ, Carneiro RM, Custis NJ, Platts-Mills TA, Sung SS, Hammer endotoxin favors local T(H)2 response and primes airway phagocytes
J, et al. A role for IL-10-mediated HLA-DR7-restricted T cell-dependent in vivo. J Allergy Clin Immunol 2004;114:1325-31.
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
Background: Mechanical injury to the skin by scratching is an

important feature of atopic dermatitis (AD). Abbreviations used
Objective: To investigate the role of COX-2 in allergic skin AD: Atopic dermatitis
inflammation elicited by epicutaneous (EC) sensitization via BAL: Bronchoalveolar lavage
introduction of ovalbumin through shaved tape-stripped skin. CysLT: Cysteinyl leukotriene
Methods: COX-2 mRNA was measured by quantitative PCR, DC: Dendritic cell
and COX-2 protein was measured by Western blotting. We EC: Epicutaneous
investigated the effect of administration of the COX-2 selective HPF: High-power field
inhibitor NS-398 during EC sensitization with ovalbumin in a PG: Prostaglandin
mouse model of AD characterized by eosinophil skin PPAR: Peroxisome proliferator-activated receptor
infiltration, elevated total and antigen specific IgE, and a WT: Wild type
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 conversion of arachidonic acid to prostaglandin (PG) G2
after ovalbumin sensitization. Infiltration by eosinophils and and PGH2. PGH2 is subsequently converted to a variety of
expression of IL-4 mRNA in ovalbumin-sensitized skin sites, eicosanoids that include PGE2, PGD2, PGF2a, PGI2, and
ovalbumin specific IgE and IgG1 antibody responses, and IL-4 thromboxane A2.1 The spectrum of prostaglandins pro-
secretion by splenocytes after ovalbumin stimulation were all duced depends on the downstream enzymatic machinery
significantly increased in EC mice that received NS-398. In expressed in a particular cell type. Prostaglandins have
contrast, ovalbumin specific IgG2a antibody response and both autocrine and paracrine effects. These are mediated
IFN-g secretion by splenocytes after ovalbumin stimulation
by an array of receptors, which are differentially expressed
were significantly decreased in these mice. COX-2–deficient
by various cell types. Two classes of prostaglandin
mice also exhibited an enhanced systemic TH2 response to
EC sensitization. receptors exist: the membrane G-coupled receptor class,
Conclusion: These results demonstrate that COX-2 limits the ie, E-prostanoid 1-4 receptors for PGE2; and the nuclear
TH2 response to EC sensitization and suggest that COX peroxisome proliferator-activated receptor (PPAR) class,
inhibitors may worsen allergic skin inflammation in patients ie, PPARa, PPARg, and PPARd, which acts as a tran-
Food allergy, dermatologic
diseases, and anaphylaxis
with AD. (J Allergy Clin Immunol 2005;116:390-6.) scription factor on ligand binding.2 Nonsteroidal anti-
inflammatory drugs inhibit COX, leading to a marked
Key words: Atopic dermatitis, allergic skin Inflammation, NS-398, decrease in prostaglandin synthesis and inflammation.3
COX-2, TH1, TH2 Two COX isoforms, COX-1 and COX-2, have been
identified and are encoded by distinct genes.4 COX-1 is
Prostaglandins are formed by the oxidative cyclization expressed in nearly all tissues under basal conditions,
of the central carbons within 20 carbon polyunsaturated suggesting that its major function is to generate prosta-
fatty acids. 5-COX is the key enzyme involved in the glandin 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
From the Division of Immunology, Children’s Hospital; and the Department include TGF-a, platelet-derived growth factor, epidermal
of Pediatrics, Harvard Medical School. growth factor, and fibroblast growth factor, all have been
Dr Laouini and Dr ElKhal contributed equally to the article. shown to induce COX-2 expression.6-9
Supported by National Institutes of Health grant AI-31541.
Prostaglandins have profound effects on the immune
Received for publication July 27, 2004; revised March 31, 2005; accepted for
publication March 31, 2005. response. A large body of data suggests that addition of
Available online June 1, 2005. PGE2 in vitro inhibits IL-12 production and promotes
Reprint requests: Raif S. Geha, MD, Enders 8, Division of Immunology, IL-10 production by antigen-presenting cells, inhibits the
Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. E-mail: production of TH1 cytokines, and promotes TH2 cell
raif.geha@childrens.harvard.edu.
0091-6749/$30.00
differentiation.10,11 Furthermore, PGE2 was shown to
Ó 2005 American Academy of Allergy, Asthma and Immunology enhance IL-4–driven isotype switching to IgE.12 Topical
doi:10.1016/j.jaci.2005.03.042 application of PGE2 suppresses the cutaneous immune
390
J ALLERGY CLIN IMMUNOL Laouini et al 391
response.13 The PPARg agonist 15-deoxy-d(12,14)-pros- peritoneally daily for the duration of the sensitization period. NS-398
taglandin J(2) also inhibits IL-12 production by macro- (25 mg/mL in dimethyl sulfoxide) was diluted in a 5% NaHCO3
phages14 and ameliorates experimental autoimmune solution before injection.
encephalomyelitis.15 In a model of allergic airway in- Histological analysis
flammation, COX inhibition by nonsteroidal anti-in-
Specimens were fixed in 10% buffered formalin and embedded in
flammatory drug increased IL-5 and IL-13 production
paraffin. Multiple 4-mm sections were stained with hematoxylin and
in bronchoalveolar lavage (BAL) fluid and airway eosin. Individual cell types were counted blinded in 15 to 20 high-
hyperresponsivenessAHR.16 Furthermore, lung inflam- power fields (HPFs) at 10003.
matory indices, which include BAL cells, proteins, and
IgE as well as lung inflammation as determined by Quantitative RT-PCR for COX enzyme
histopathology, were significantly increased in the ab- mRNA expression
sence of either COX-1 or COX-2.17 Five hundred milligrams of skin was homogenized by using a
COX-1 is basally expressed at low levels in skin.18 Skin Polytron RT-3000 (Kinematica AG, Brinkmann Instruments Inc) in
injury by UV light has been shown to induce COX-2 lysis buffer solution provided in the RNAqueous extraction kit
expression,19 whereas mechanical injury increases PGE2 (Ambion Inc, Austin, Tex). RT was performed by using transcriptor
in the skin.20 This may be mediated by IL-1 and TNF-a first-strand cDNA synthesis kit (Roche Diagnostic, Foster City,
released from keratinocytes, fibroblasts, and mast cells.21-23 Calif). PCR reactions were run on an ABI Prism 7700 (Applied
Biosystems, Foster City, Calif) sequence detection system platform.
Atopic dermatitis (AD) is an inflammatory skin disease
Taqman primers with 6-carboxyfluorescein-labeled probe were
that frequently occurs in subjects with personal or family
obtained from Applied Biosystems. The housekeeping gene b2-
history of atopic disease.24 Mechanical injury to the skin microglobulin was used as a control. The relative gene expression
by scratching is an important feature of AD. We have among the different samples was determined by using the method
developed a mouse model of allergic skin inflammation described by Pfaffl.27
elicited by epicutaneous (EC) sensitization with ovalbu-
min. This model displays many of the features of human Determination of COX-2 protein expression
AD, including a dermatitis characterized by dermal infil- and PGE2 levels in skin
tration of T cells and eosinophils and increased local Five hundred milligrams of skin was homogenized in 1 mL 0.1
expression of TH2 cytokines and by a systemic allergen mol/L PBS solution (pH = 7.4) containing 1 mmol/L EDTA, 0.1
specific TH2 response characterized by IgG1 and IgE mmol/L indomethacin, and a cocktail of protease inhibitors. Fifteen
antibodies and IL-4 secretion by splenocytes after in vitro microliters of this solution was used for Western blotting for COX-2,
stimulation with ovalbumin.25 We used this model to with a rabbit anti–COX-2 antiserum (Abcam Inc, Cambridge, Mass)
followed by horseradish peroxidase–conjugated donkey antirabbit
assess the role of COX-2 in allergic skin inflammation.
antibody (Amersham Bioscience, Temecula, Calif). The blots were
reprobed with mAb to actin (Chemicon International, Piscataway,
NJ) followed by horseradish peroxidase–conjugated sheep antimouse
METHODS antibody (Amersham Bioscience) for loading control. The rest of the
material was extracted as described,28 and the extract used to
Mice determine PGE2 concentration by ELISA (Cayman Chemicals, Ann
BALB/c mice were obtained from the Jackson Laboratory (Bar Arbor, Mich).
Harbor, Me). COX-2+/2 mice on C57BL6x129/SvlmJ background
Competitive RT-PCR evaluation of

and genetically matched controls were obtained from Taconic
(Germantown, NY). Homozygous COX-2–deficient mice were cytokine mRNA in skin
obtained by genotyping the offspring of COX-2+/2 parents, and, as Competitive RT-PCR evaluation of cytokine mRNA in skin was
previously described, were not fertile.26 All mice were kept in a performed as described previously.29 Skin biopsies were immediately
pathogen-free environment. All procedures performed on the mice frozen in dry ice. The samples were homogenized in Trizol (GIBCO
were in accordance with the Animal Care and Use Committee of the BRL, Carlsbad, Calif) by using a Polytron RT-3000. RNA extraction
Children’s Hospital. was performed following the manufacturer’s instructions. cDNA was
synthesized from 10 mg total RNA in a 40-mL reaction mix by using
EC sensitization Superscript II (GIBCO BRL). The primers used to amplify cDNA for
EC sensitization of female mice 4 to 6 weeks old was performed as b2-microglobulin, IL-4, and IFN-g and DNA amplification were as
described previously.25 Briefly, the skin of anesthetized mice was described previously.29 To quantify cytokine mRNA, a fixed amount
shaved and tape-stripped 6 times. Ovalbumin (grade V; Sigma of reverse-transcribed cellular mRNA was coamplified in the pres-
Chemical Co, St Louis, Mo) 100 mg in 100 mL normal saline, or ence of serial dilutions of a multispecific internal plasmid control
placebo (100 mL normal saline), was placed on a patch of sterile (pMUS3), which contains nucleotide sequences of multiple cyto-
gauze (1 cm 3 1 cm), which was secured to the skin with a transparent kines.30 Results were expressed as a ratio of cytokine cDNA to
bio-occlusive dressing (Tegaderm, Owens & Minor Inc, Franklin, b2-microglobulin cDNA. We have recently found that the results of
Mass). Each mouse had a total of three 1-week exposures to the patch competitive RT-PCR for determination of cytokine mRNA in skin
separated by 2-week intervals. On day 49, the mice were killed and compare favorably with those of quantitative RT-PCR. In 2 experi-
their tissues examined. ments 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
Treatment with COX-2 inhibitor skin IL-4 mRNA expression after ovalbumin sensitization was 4.9-
Mice were given 1 mg/kg of the selective COX-2 inhibitor NS-398 fold using competitive PCR compared with 4.3-fold using quantita-
(Biomol Research Laboratories, Inc, Plymouth Meeting, Pa) intra- tive RT-PCR.
392 Laouini et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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 sensitized skin sites compared with saline-sensitized

IgG1, IgG2a, and IgE antiovalbumin antibodies were determined skin sites or with unmanipulated skin (Fig 1, C). The
by ELISA following the procedures we previously described.25 increased COX-2 mRNA expression observed 8 hours
after stripping was associated with significantly increased
IL-4 and IFN-g synthesis by spleen cells level of the COX metabolite PGE2 (Fig 1, D). There was
Single cell suspensions of spleen cells were prepared and cultured no increase in PGE2 levels in either ovalbumin-sensitized
at 2 3 106/mL in 24-well plates in the presence of ovalbumin (50 or saline-sensitized skin sites.
mg/mL) as previously described.31 Supernatants were collected after
96 hours. IL-4 and IFN-g were determined by ELISA (Pharmingen, Eosinophil infiltration is increased in
San Diego, Calif). ovalbumin-sensitized skin sites of mice
Statistical analysis treated with COX-2 inhibitor
The nonparametric Mann-Whitney test was used to compare the There was no difference in the numbers of eosinophils
different mice groups. 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
RESULTS skin of control untreated BALB/c mice, consistent with
previous observations.25 There were significantly more
Tape stripping induces expression of eosinophils in ovalbumin-sensitized skin of mice treated
COX-2 mRNA in normal mouse skin with NS-398 than in ovalbumin-sensitized skin of un-
We used quantitative RT-PCR to examine the effect of treated controls (Fig 2, A). Ovalbumin sensitization caused
tape stripping on COX-2 mRNA expression in mouse an increase in skin mononuclear cells that was modestly
skin. Low levels of COX-2 mRNA were detectable in but significantly higher in mice treated with NS-398
uninjured skin. After tape stripping 6 times, COX-2 compared with untreated controls (Fig 2, B).
mRNA expression increased, with peak levels 8 hours
poststripping, and returning to normal 48 hours later (Fig IL-4 expression is increased in EC skin sites
1, A). In contrast, there was no detectable increase in the of mice treated with COX-2 inhibitor
levels of COX-1 mRNA levels in the skin after tape We used competitive PCR to measure cytokine mRNA
stripping. COX-2 and COX-1 mRNA levels in ovalbu- in skin. Low and comparable levels of IL-4 and IFN-g
min-sensitized skin sites did not significantly differ from mRNA were detected in saline sensitized skin from
those in saline-sensitized or unmanipulated skin sites (Fig untreated mice and mice treated with NS-398 (Fig 3).
1, B). Western blotting analysis demonstrated that COX-2 Consistent with previous results,25 expression of IL-4
protein expression in the skin increased 8 hours after tape mRNA, but not IFN-g mRNA, markedly increased in
stripping (Fig 1, C). In contrast, there was no detectable ovalbumin-sensitized skin sites of untreated control
increase in COX-2 protein expression in ovalbumin- BALB/c mice. IL-4 mRNA was significantly increased
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.
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
FIG 5. Serum levels of ovalbumin (OVA) specific IgG1 (A), IgE (B),
T cells.
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.
Treatment with COX-2 inhibitor enhances *P < .05.
antigen specific IgE and IgG1 antibody

responses to EC sensitization with ovalbumin
The TH2 cytokine IL-4 plays an important role in IFN-g after ovalbumin stimulation in vitro.31 Fig 4, B,
isotype switching to IgE and IgG1, whereas the TH1 shows that splenocytes from EC mice treated with NS-398
cytokine IFN-g plays an important role in isotype switch- secreted significantly higher amounts of IL-4, and signif-
ing to IgG2a.32 To investigate whether COX-2 inhibition icantly less IFN-g, than splenocytes of unsensitized,
enhanced the systemic TH2 response to EC sensitization untreated controls. These results suggest that COX
with ovalbumin, we measured total and ovalbumin spe- products normally limit the systemic TH2 response to
cific IgE and IgG1 in serum. Fig 4, A, shows that EC-introduced antigen and promote the systemic TH1
ovalbumin specific IgG1 and IgE levels were significantly response.
higher in mice treated with NS-398. Treatment with
NS-398 had no effect on the IgG2a antibody response. Increased systemic TH2 response and
These results suggest that COX products normally down- decreased systemic TH1 response in
regulate the systemic IgE and IgG1 antibody response to COX-2–deficient mice
EC-introduced antigen. NS-398 may have effects other than COX-2 enzyme
inhibition. To ascertain that the effect of NS-398 on the
COX-2 inhibition causes increased systemic TH response to EC sensitization was a result of COX-2
TH2 response and decreased systemic TH1 inhibition, we examined the response of COX-22/2
response to EC sensitization mice to EC immunization. Fig 5 shows that ovalbumin
We have previously shown that splenocytes from specific IgE levels were significantly higher and ovalbu-
BALB/c mice with EC with ovalbumin secrete IL-4, and min specific IgG2a levels were significantly lower in
AUGUST 2005
The increased infiltration by mononuclear cells ob-

served 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
FIG 6. Cytokine production by spleen cells from EC mice, COX-22/2 expression of TH2 selective chemokines by skin cells35
mice, and WT controls (n = 6 for each group). Columns and error and enhances TH2 cell infiltration. Increased secretion of
bars represent means 6 SEMs. *P < .05. SAL., Saline. 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.
COX-2–deficient mice than in wild-type (WT) controls. Treatment of mice with the COX-2 inhibitor NS-398
COX-2 deficiency had no detectable effect on the IgG1 promoted the TH2 systemic response to EC sensitization.
antibody response. Fig 6 shows that splenocytes from EC- This was evidenced by enhanced serum IgG1 and IgE
immunized COX-22/2 mice secreted significantly more antibody levels to ovalbumin mice (Fig 4, A) and increased
IL-4 and significantly less IFN-g than splenocytes of IL-4 secretion by splenocytes in response to stimulation
genetically matched WT controls. with ovalbumin (Fig 4, B). In contrast, IFN-g secretion
was significantly decreased (Fig 4, B). Studies with COX-
22/2 mice strongly supported the conclusion that the
DISCUSSION enhancing effect of NS-398 on the TH2 response to EC
immunization was a result of inhibition of COX-2 activity.
The results of this study suggest that COX-2 products After EC immunization, these mice mounted a signifi-
limit the systemic TH2 response and enhance the systemic cantly higher ovalbumin specific IgE antibody response
TH1 response to epicutaneously introduced antigen and and their splenocytes secreted significantly more IL-4 and
promote skin infiltration with eosinophils in a mouse significantly less IFN-g than splenocytes from WT
model of allergic dermatitis. controls (Figs 5 and 6).
Both COX-1 and COX-2 were expressed in unmanip- Taken together, our results suggest that COX-2 prod-
ulated mouse skin (Fig 1, A), consistent with previous ucts normally limit the development of TH2 cytokines and
reports on mouse and human skin.18,33,34 Mechanical promote the development of TH1 cytokines in response to
injury by tape stripping transiently upregulated COX-2 EC sensitization with antigen. The fact that there was no
mRNA but not COX-1 mRNA expression in mouse skin. detectable increase in COX-2 or COX-1 mRNA expres-
This finding is consistent with the observation that PGE2 sion, COX-2 protein expression, or PGE2 levels in oval-
levels increase in human skin after tape stripping,20 which bumin-sensitized skin compared with either saline
we confirmed in this mouse study (Fig 1, C). sensitized or unmanipulated skin (Fig 1, B-D) suggests
Keratinocytes, fibroblasts, mast cells, endothelial cells, that the effects of COX-2 inhibition may be exerted early
and tissue macrophages have all been reported to express in the sensitization phase of our model, which is depen-
COX-2 after activation.21-23 IL-1b and TNF-a are known dent on tape stripping, because we are unable to sensitize
inducers of COX-2 mRNA expression.6,7 A correlation the mice without it. However, we cannot rule out an effect
has been observed between levels of PGE2 and IL-1a in on later events via the inhibition of baseline COX-2
tape-stripped skin.20 activity in the skin.
The COX-2 selective inhibitor NS-398 enhanced der- Our findings of increased IL-4 response to EC sensiti-
mal infiltration with eosinophils in our model (Fig 2, A). zation with inhibition or lack of COX-2 is in agreement
Eosinophil infiltration of the skin in our model is depen- with previous findings that administration of the COX
dent on their expression of the eotaxin receptor CCR3,31 inhibitor indomethacin 2 days before and during intraper-
and that eotaxin expression is dependent on IL-4.29 itoneal immunization enhances mRNA expression and
Expression of mRNA for the TH2 cytokine IL-4, but not secretion of the TH2 cytokines IL-5 and IL-13 in the lung
for the TH1 cytokine IFN-g, in ovalbumin-sensitized skin after allergen challenge.16 Similarly, allergic lung inflam-
sites was significantly enhanced in NS-398–treated mice mation as evidenced by eosinophils in BAL fluid and lung
(Fig 3). This may have contributed to increased eosinophil histopathology and TH2 cytokine secretion are enhanced
skin infiltration. The TH2 cytokine IL-5 is also important in intraperitoneally immunized mice deficient in COX-1
for eosinophil infiltration of the skin in our model. or COX-2.17,36 In these studies, COX inhibition did not
Although we did not measure IL-5 expression, it is likely result in a significant increase in the IgE antibody response
that it was also enhanced in NS-398–treated mice and to intraperitoneal immunization, and TH cytokine secre-
contributed to their exaggerated skin eosinophilia, be- tion by splenic T cells was not examined.
cause IL-5 and IL-4 expression by TH2 cells is usually There is a plethora of evidence that the COX-2 product
concordant. PGE2, which is present in skin of patients with AD,37
inhibits the development of TH1 cells and promotes the 6. Raz A, Wyche A, Siegel N, Needleman P. Regulation of fibroblast
development of TH2 cells in vitro,10,38 although discrepant cyclooxygenase synthesis by interleukin-1. J Biol Chem 1988;263:
3022-8.
results have also been reported.39,40 This inhibitory effect 7. Diaz A, Chepenik KP, Korn JH, Reginato AM, Jimenez SA. Differential
is exerted in a large part at the level of the dendritic cells, regulation of cyclooxygenases 1 and 2 by interleukin-1 beta, tumor
because PGE2 is a potent inhibitor of IL-12 production by necrosis factor-alpha, and transforming growth factor-beta 1 in human
these cells10 and an inhibitor of IL-12b1 receptor and lung fibroblasts. Exp Cell Res 1998;241:222-9.
8. Saha D, Datta PK, Sheng H, Morrow JD, Wada M, Moses HL, et al.
IL-12b2 receptor expression.41 On the basis of these Synergistic induction of cyclooxygenase-2 by transforming growth
in vitro results, one would expect that decreased PGE2 factor-beta1 and epidermal growth factor inhibits apoptosis in epithelial
generation in the skin subsequent to inhibition or lack cells. Neoplasia 1999;1:508-17.
of COX-2 may promote the TH1 response and inhibit the 9. Goppelt-Struebe M, Rehm M, Schaefers HJ. Induction of cyclooxygen-
ase-2 by platelet-derived growth factor (PDGF) and its inhibition by
TH2 response to EC sensitization. In fact, the reverse was
dexamethasone are independent of NF-kappaB/IkappaB transcription
observed. However, PGE2 may not be the most abundant factors. Naunyn Schmiedebergs Arch Pharmacol 2000;361:636-45.
or most relevant prostaglandin generated in injured skin. 10. van der Pouw Kraan TC, Boeije LC, Smeenk RJ, Wijdenes J, Aarden
Langerhans cells generate high amounts of PGD2 but LA. Prostaglandin-E2 is a potent inhibitor of human interleukin 12
very little amounts of other prostaglandins.42 The prosta- production. J Exp Med 1995;181:775-9.
11. Kalinski P, Hilkens CM, Snijders A, Snijdewint FG, Kapsenberg ML.
noid PGI2 limits lung allergic inflammation,43 and mice IL-12-deficient dendritic cells, generated in the presence of prostaglandin
deficient in the PGI2 receptor mount an exaggerated TH2 E2, promote type 2 cytokine production in maturing human naive T
response.43,44 Further studies are needed to examine the helper cells. J Immunol 1997;159:28-35.
nature of prostaglandins that accumulate in the skin after 12. Roper RL, Brown DM, Phipps RP. Prostaglandin E2 promotes B
lymphocyte Ig isotype switching to IgE. J Immunol 1995;154:162-70.
mechanical injury and the roles of individual prostaglan-
13. Rheins LA, Barnes L, Amornsiripanitch S, Collins CE, Nordlund JJ.
dins in modulating the TH response to EC sensitization. Suppression of the cutaneous immune response following topical appli-
Inhibition or lack of COX-2 activity may result in cation of the prostaglandin PGE2. Cell Immunol 1987;106:33-42.
enhanced leukotriene synthesis because of both increased 14. Azuma Y, Shinohara M, Wang PL, Ohura K. 15-Deoxy-delta(12,14)-
availability of arachidonic acid substrate and release from prostaglandin J(2) inhibits IL-10 and IL-12 production by macrophages.
Biochem Biophys Res Commun 2001;283:344-6.
the inhibitory effect of prostaglandins on 5-lipooxyge- 15. Diab A, Deng C, Smith JD, Hussain RZ, Phanavanh B, Lovett-Racke
nase–activating protein expression.45 Increased amounts AE, et al. Peroxisome proliferator-activated receptor-gamma agonist
of leukotriene C4 in the skin may promote CC chemokine 15-deoxy-delta(12,14)-prostaglandin J(2) ameliorates experimental auto-
ligand 19-dependent mobilization of antigen bearing immune encephalomyelitis. J Immunol 2002;168:2508-15.
dendritic cells (DC) to lymph nodes,46 resulting in the 16. Peebles RS Jr, Dworski R, Collins RD, Jarzecka K, Mitchell DB,
Graham BS, et al. Cyclooxygenase inhibition increases interleukin 5 and
exaggeration of what is already a TH2-skewed response to interleukin 13 production and airway hyperresponsiveness in allergic
EC sensitization. Cysteinyl leukotrienes (cysLTs) may mice. Am J Respir Crit Care Med 2000;162:676-81.
promote the induction of TH2 responses by DCs, as 17. Gavett SH, Madison SL, Chulada PC, Scarborough PE, Qu W, Boyle JE,
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tion of DCs pulsed with antigen and cysLTs enhance 18. Abd-El-Aleem SA, Ferguson MW, Appleton I, Bhowmick A, McCollum
allergic inflammation compared with DCs pulsed with CN, Ireland GW. Expression of cyclooxygenase isoforms in normal
antigen alone.47 CysLTs also promote eosinophil loco- human skin and chronic venous ulcers. J Pathol 2001;195:616-23.
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21. Scholz K, Furstenberger G, Muller-Decker K, Marks F. Differential
increased in the skin of patients with AD,37 and COX-2 expression of prostaglandin-H synthase isoenzymes in normal and
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AD lesions.49 Our results clearly show that COX-2 22. Warnock LJ, Hunninghake GW. Multiple second messenger pathways
inhibition may exacerbate AD by promoting the systemic regulate IL-1 beta-induced expression of PGHS-2 mRNA in normal
human skin fibroblasts. J Cell Physiol 1995;163:172-8.
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23. Leong J, Hughes-Fulford M, Rakhlin N, Habib A, Maclouf J, Goldyne
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keratinocytes: association of COX-2 expression with human keratinocyte
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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
Background: It has been reported that patients with cholinergic

urticaria have a type 1 allergy to autologous sweat; however, Abbreviations used
the pathogenesis of that disorder has not been fully elucidated. CU: Cholinergic urticaria
Objective: We investigated the responsiveness to autologous ASST: Autologous serum skin test
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. Cholinergic urticaria (CU), which was first described
Histamine release from peripheral blood basophils induced
by Duke1 in 1924, is characterized by unique clinical
by autologous sweat was measured.
Results: Eleven of 17 patients with cholinergic urticaria showed
features: pinpoint sized, highly pruritic wheals with
positive reactions in skin tests with their own diluted sweat. surrounding erythema that occur after sweating during
Substantial amounts of sweat-induced histamine release from physical exercise, taking a bath, raising the body temper-
autologous basophils were observed in 10 of 17 patients. Eight ature, and emotional stress. In typical cases, this disorder
of 15 patients with cholinergic urticaria showed positive usually occurs in young adults. Occasionally, this disorder
reactions in the autologous serum skin tests. All 6 patients who is accompanied with angioedema and anaphylactic reac-
developed satellite wheals after the acetylcholine test showed tions.2,3
hypersensitivity to sweat. Further, patients whose eruptions The pathogenesis of CU has not yet been well clarified
were coincident with hair follicles showed positive responses to despite the fact that numerous investigators have de-
the skin test with autologous serum, whereas patients whose
scribed its clinical characteristics and possible pathogen-
eruptions were not coincident with hair follicles did not.
Conclusion: On the basis of these findings, we propose that
esis. In patients with CU, injection of acetylcholine
cholinergic urticaria should be classified into 2 distinct (mecholyl) into normal-appearing skin produces a wheal
subtypes. The first (nonfollicular) subtype shows strong positive and flare reaction, often surrounded by smaller satellite
reactions to autologous sweat and negative reactions to lesions that are similar to the skin symptoms of CU.4
autologous serum. The second (follicular) subtype shows Acetylcholine is thus believed to play a significant role in

weak reactions to autologous sweat and positive reactions to the development of the symptoms of CU. Another aspect
autologous serum. (J Allergy Clin Immunol 2005;116: of the pathogenesis of CU has focused on sweat itself on
397-402.) the basis of evidence that this unique eruption occurs after
sweating. Adachi et al5 found that 20 patients with CU
Key words: Cholinergic urticaria, sweat, autologous serum, skin showed immediate-type reactions after an intradermal
test, histamine release test, acetylcholine test skin test with autologous sweat. Kobayashi et al6 pre-
sumed that the leakage of sweat into the dermis because of
ductal occlusion at the superficial acrosyringium causes
From the Division of Dermatology, Department of Clinical Molecular CU. Kaplan et al7 and Sigler et al8 demonstrated plasma
Medicine, Kobe University Graduate School of Medicine.
Disclosure of potential conflict of interest: A. Fukunaga, none disclosed;
histamine elevations after exercise challenge of patients
T. Bito, none disclosed; K. Tsuru, none disclosed; A. Oohashi, none with CU.
disclosed; X. Yu, none disclosed; M. Ichihashi, none disclosed; C. Nishigori, We performed this study to clarify further the possi-
none disclosed; T. Horikawa, none disclosed. ble involvement of sweat-mediated and autoimmune-
Received for publication November 11, 2004; revised May 13, 2005; accepted
mediated mechanisms in CU and in its clinical features.
for publication May 17, 2005.
Available online July 15, 2005. Skin responsiveness was evaluated after the intracutane-
Reprint requests: Tatsuya Horikawa, MD, Division of Dermatology, ous injection of autologous sweat and serum. We assessed
Department of Clinical Molecular Medicine, Kobe University Graduate the correlation between the degrees of skin reactions and
School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, amounts of in vitro histamine released from basophils after
Japan. E-mail: thorikaw@med.kobe-u.ac.jp.
0091-6749/$30.00
stimulation with autologous sweat. We further analyzed
Ó 2005 American Academy of Allergy, Asthma and Immunology the relationship between the clinical symptoms of CU and
doi:10.1016/j.jaci.2005.05.024 the characteristics of these tests.
397
398 Fukunaga et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
METHODS medium was measured by an ELISA with a characteristic detection

profile.12
Subjects
Eighteen patients with CU were enrolled at the Dermatological Statistical analysis
Institute of Kobe University Hospital. CU was confirmed by the The statistical significance of differences was determined by using
development of numerous small wheals after exercise until sweat- the Student t test. Some data were analyzed by regression analysis
ing.9 All of the patients had no aquagenic urticaria. The character- by using the statistical package StatView J (Abacus Concepts Inc,
istics of patients with CU are described in Table I. Their age ranged Palo Alto, CA). A difference was considered statistically significant
between 15 and 31 years (mean, 21.8 years). Nine patients had a past at P < .05.
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 RESULTS
explanations. Relevant drugs such as histamine H1-receptor antag-
onists were withdrawn for at least 24 hours before the examination.
Skin tests for autologous sweat
All of the patients had never had systemic corticosteroids for at least Of 17 patients with CU, except for 1 patient who
3 months before the examination. showed mechanical urticaria in skin test for autologous
sweat, 11 (64.7%) showed positive reactions to their own
Materials 1/100 diluted sweat by measuring the diameter of wheals
Venous blood was taken into sterile glass tubes and allowed to clot (Table II). In contrast, all 10 healthy controls showed
at room temperature for 30 minutes. Serum was separated by cen- negative reactions to their own 1/100 diluted sweat,
trifugation at 500g for 20 minutes and passed through a 0.45-mm whereas a few healthy controls had positive reactions to
MILLEX HV membrane (Millipore, Molsheim, France). Sweat was their own 1/10 diluted sweat (data not shown). Sweat-
collected from each patient’s forearm after exercise. The sweat was induced wheals in the skin tests were significantly greater
sterilized after collection by using a 0.45-mm MILLEX HV mem- for patients with CU than for healthy control subjects
brane, and it was preserved at 280°C before use. Sweat samples were
(Fig 1, A).
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.
Sweat-induced histamine release
from basophils
Skin test technique We investigated the histamine release from basophils of
Samples of autologous diluted sweat (0.02 mL), autologous serum 17 patients with CU and of 10 healthy controls after
(0.05 mL), and 0.9% sterile saline (0.02 or 0.05 mL) were separately incubation with autologous sweat. Of 17 CU patients’
injected intradermally into the volar aspect of the forearm of each basophils, 10 (58.8%) showed positive responses (more
subjects when they were quiet and with no wheal. The diameters of than 5% histamine release) after incubation with 1/100
wheals and erythema were measured after 15 minutes. Reactions diluted autologous CU sweat, whereas none of the 10
were assessed as positive if the diameter of the wheal induced by healthy controls’ basophils did with 1/100 diluted autol-
sweat and serum was equal to or larger than 6 mm. The sterile saline-
ogous normal sweat. Four of the CU patients’ basophils
induced wheals of all subjects were below 4 mm and 2 mm when the
showed positive responses to 1/1000 diluted CU sweat.
amounts of 0.05 and 0.02 mL were injected, respectively.
The overall values of percent histamine release from
basophils of patients with CU were significantly larger

Local provocation test
than those from healthy control subjects (Fig 1, B).
Responses to acetylcholine chloride (Ovisot; Daiichi, Tokyo,
Japan) were evaluated. Acetylcholine (0.1 mL) was intradermally Correlation of skin tests for autologous sweat
injected at a concentration of 100 mg/mL diluted with saline. The
development of satellite wheals around the injection site was con-
with sweat-induced histamine release from
sidered as positive. Simultaneously, we checked sweating around basophils
the injection site by the iodine-starch technique, and all patients We examined whether sweat-induced histamine release
tested showed sweating by this test.10 All of the normal controls from CU basophils correlates with skin tests for autolo-
tested showed a significant number of tiny sweating points by this gous CU sweat in 16 patients. As shown in Fig 1, C,
method.
% histamine release correlated positively with the area in
wheal using skin tests on 1/100 diluted sweat. These
Basophil histamine release test
results indicate that the degree of percent histamine release
A histamine release test was performed in vitro by using for autologous sweat represents the responsiveness of skin
HRT (Shionogi, Osaka, Japan) as previously described.11 Venous tests for autologous sweat.
peripheral blood samples from patients with CU and normal healthy
controls, 20 mL, and antibasophil antibodies conjugated to magnetic Autologous serum skin tests
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 Of 15 patients with CU, 8 (53.3%) showed a positive
basophils in each well were then trapped with a chandelier-shaped response in the autologous serum skin test (ASST; Table
magnet and transferred to another microplate, where the basophils II). In contrast, all 6 healthy controls showed a negative
were stimulated at 37°C for 1 hour with autologous sweat, anti-IgE response for ASST. Most patients with CU who had a
antibody, and digitonin, respectively. Histamine released into the negative response for ASST tended to show a positive
J ALLERGY CLIN IMMUNOL Fukunaga et al 399
TABLE I. Clinical characteristics of patients with cholinergic urticaria
Patient Age (y) Sex History Accompanying symptoms Total IgE (IU/mL) IgE RAST
1 26 Female Atopic dermatitis None 919 Mite, Candida

2 25 Female Atopic dermatitis Asthma 5518 Cedar, orchard grass
3 22 Male None Cold urticaria 432 Wheat
4 20 Male None None 85 Not done
5 21 Male Atopic dermatitis None 4080 Mite, Candida, wheat
7 22 Female Allergic rhinitis Cold urticaria 1842 Mite, Candida
8 19 Male None None Not done Not done
10 26 Male None None Not done Not done
11 31 Male Allergic rhinitis None 194 Not done
12 19 Female Atopic dermatitis Cold urticaria, angioedema 342 Mite
13 24 Female Atopic dermatitis None 907 Mite
14 15 Male None Cold urticaria 1302 Mite, Candida, orchard grass
15 26 Female None None 144 Negative
16 18 Female Atopic dermatitis None 118 Mite
17 19 Male Urticaria Cold urticaria Not done Not done
18 17 Male Allergic rhinitis None 423 Not done
TABLE II. Details of results for skin tests, histamine release test, acetylcholine test, and clinical chracterization
% Histamine release
Autologous sweat skin test by sweat
Autologous Acetylcholine Characteristics
Patient (Erythema*) (Whealy) 1/100z 1/1000z serum skin test§ testk of eruption
1 31 3 16 20 3 14 100 36.6 Negative Not done Nonfollicular

2 25 3 20 10 3 10 89.6 23.6 Negative Positive Nonfollicular
3 11 3 10 9 3 8 0.5 0 Negative Positive Nonfollicular
4 23 3 20 8 3 8 7.3 0.4 Negative Positive Nonfollicular
5 11 3 10 11 3 10 21.4 6.5 Negative Positive Undetermined
6 10 3 10 7 3 7 Not done Not done Negative Positive Nonfollicular
7 24 3 23 10 3 8 40 3.9 Not done Positive Nonfollicular
8 25 3 22 9 3 9 48.8 0.2 Not done Not done Nonfollicular
9 20 3 15 7 3 6 54.8 6.7 Positive Not done Undetermined
10 15 3 13 6 3 5 63.2 4.6 Positive Not done Undetermined
11 030 0 3 0 10.6 1.3 Positive Not done Follicular
12 10 3 8 10 3 8 2.1 0.2 Positive Negative Follicular

13 939 0 3 0 1 0 Positive Negative Follicular
14 Mechanical urticaria 0.2 0 Not done Not done Follicular
15 030 0 3 0 0.4 0 Positive Negative Follicular
16 736 0 3 0 0 0 Positive Negative Nonfollicular
17 030 0 3 0 0.4 0.4 Positive Negative Follicular
18 737 4 3 4 1.1 0 Negative Negative 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 wheals around the central large wheal were seen only in
with autologous CU sweat. In contrast, a few patients with patients with CU.4 However, these satellite wheals seemed
a positive response for ASST tended to show hypersen- to develop only in a few patients with CU.13 We therefore
sitivity for sweat (Table II). examined whether the patients with CU showed satellite
wheals by acetylcholine injection. In this study, 6 (50%) of
Intradermal acetylcholine test the 12 patients with CU tested showed a positive response
After intradermal injections of relatively high concen- for acetylcholine (Table II). Almost all of the patients who
trations of cholinergic agents, the typical satellite pinpoint were checked for sweating by iodine-starch method
AUGUST 2005
those from patients who showed negative responses for

the acetylcholine test (Fig 2, B). In addition, certain
satellite wheals after the acetylcholine test were recogniz-
able as coincident with perspiration points when sweat-
ing points were detected by the starch-iodine method
(Fig 2, C).
Characterization of the clinical phenotype
When patients with CU developed wheals after exer-
cise, 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
We demonstrated that the majority of patients with CU

are highly sensitive to autologous sweat. The heteroge-
neous responses in skin tests with autologous sweat
suggest that patients with CU have various degrees of
hypersensitivity to sweat. We further observed that var-
ious amounts of histamine were detected in the medium
when basophils were incubated in the presence of autol-
ogous sweat, which suggests that autologous sweat itself
contains factors that can induce histamine release. The
amounts of histamine released from CU basophils corre-
lated 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
FIG 1. Differences in responses to sweat in patients with CU and release tests using autologous sweat correlate with the
normal controls. A, The areas of wheals induced by intradermal intracutaneous test. Previously, Adachi et al5 reported that
injection with autologous sweat. B, Values of the histamine release
all patients with CU examined showed immediate-type
from basophils stimulated with CU sweat or normal sweat. C, The
relationship of responsiveness of skin tests with CU sweat and CU skin reactions to intradermal tests with sweat at various
sweat-induced histamine release from CU basophils. 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
showed sweating after acetylcholine injection in our positive reaction to their own 1/100 diluted sweat. Certain
series, indicating that the absence of the satellite wheals patients who showed a negative response at 1/100 dilution
by the agent is not attributed to the dyshidrosis. Compared might have shown a positive response at higher concen-
with patients who showed a negative response for the trations (1/10 and higher dilutions). In other words, those
acetylcholine test, the areas of CU sweat-induced wheals patients with CU who showed positive skin responses in
in the skin tests were significantly greater in patients who this study might represent the presence of strong hyper-
showed satellite wheals for the acetylcholine test (Fig 2, sensitivity to sweat.
A). The values of CU sweat-induced histamine released Interestingly, Hide et al14 recently reported that patients
from basophils of patients who showed positive responses with atopic dermatitis show hypersensitivity to autologous
for the acetylcholine test were significantly greater than sweat antigens. They speculate that this phenomenon is
J ALLERGY CLIN IMMUNOL 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 sweat-
FIG 2. The relationship between responses to sweat and acetyl- induced histamine release from basophils in CU patients with
choline tests in patients with CU. A, The areas of wheals induced by nonfollicular or follicular wheals. C, A representative picture of
intradermal injection with CU sweat in patients with CUs with or wheals consisting of follicles.
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.
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
an IgE-mediated response, because histamine release was 2). This means that those with hypersensitivity to sweat
impaired by removal of IgE from patients’ basophils and tend to develop satellite wheals after stimulation with
myeloma IgE blocked the sensitization of basophils with acetylcholine, a sweat inducer. Moreover, we observed
the patient’s serum. It is of interest to note that patients that satellite wheals after the acetylcholine test were

with CU frequently have atopic dermatitis.5,15 Adachi coincident with perspiration points by the iodine-starch
et al5 have shown that leukocytes from a normal healthy method (Fig 2, C). These results are compatible with the
donor did release histamine on sweat challenging after idea that sweat leakage from sweat ducts induces small
being sensitized with a patient’s serum. Therefore, it wheals in certain patients with CU.
might be possible that, similar to atopic dermatitis, hyper- Circulating functional histamine-releasing autoanti-
sensitivity to sweat in patients with CU could be an bodies reactive against either the a-subunit of the high-
IgE-mediated response. affinity IgE receptor (FceRIa) or IgE have been identified
An attractive hypothesis for the pathomechanisms of in more than one third of patients with chronic idiopathic
CU is that sweat leaks from the sweat duct into the urticaria.17-22 The ASST is now recognized as a suitable
dermis.6,16 Several cases of CU have been described that screening test for such autoantibodies in such patients.4
are associated with hypohidrosis/anhidrosis.6,16 Occlu- However, it is still unclear whether the wheal-inducing
sion of the superficial acrosyringium might result in sweat factors in the patient’s sera in this study are these
leakage into the dermis in patients with CU and anhidro- autoantibodies, and this issue should be further clarified
sis.6 If those patients with CU are hypersensitive to sweat, in the future. Sabroe et al23 have reported that only 1 of 9
the leaking sweat possibly induces urticarial symptoms patients with CU had a positive ASST. In contrast, we
around the sweat ducts, resulting in small pinpoint wheals. showed here that 8 of 15 patients had a positive ASST. The
Commens and Greaves4 examined 12 patients with CU by discrepancy of the ratio of responsiveness in ASST be-
intradermal testing with methacholine and found that tween their findings and ours might be attributed to the
satellite wheals were induced in only 6 of them. It is not yet patient population; that is, the majority of the patients in
clear why only some patients with CU develop satellite their study might have had the nonfollicular type. So far, it
wheals after injection of cholinergic agents. We showed is unclear whether we might enroll more follicular-type
AUGUST 2005
patients than the usual population in CU. Therefore, this 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
issue should be further studied in the future.
Clin Immunol 1975;55:394-402.
We observed that certain patients with CU develop 8. Sigler RW, Levinson AL, Evans R III, Horakova Z, Kaplan AP.
wheals in association with hair follicles, whereas the other Evaluation of patients with cold and cholinergic urticaria. J Allergy
patients do not. This phenomenon is similar to that seen Clin Immunol 1979;63:35-8.
in aquagenic urticaria, in which follicular wheals develop 9. Kobza Black A, Lawlor F, Greaves MW. Consensus meeting on the
definition of physical urticarias and urticarial vasculitis. Clin Exp
after contact with water. We found that patients with non- Dermatol 1996;21:424-6.
follicular-type CU tend not only to show satellite wheals 10. Wada M, Takagaki T. A simple and accurate method for detecting the
after the acetylcholine test but also to have hypersensitiv- secretion of sweat. Tohoku J Exp Med 1948;49:284.
ity to sweat as determined by skin tests and by histamine 11. Adachi A, Fukunaga A, Hayashi K, Kunisada M, Horikawa T. Anaphy-
laxis to polyvinylpyrrolidone after vaginal application of povidone-
release tests (Table II). On the other hand, most of the
iodine. Contact Dermatitis 2003;48:133-6.
patients with follicular-type CU showed a positive reac- 12. Nishi H, Nishimura S, Higashiura M, Ikeya N, Ohta H, Tsuji T, et al.
tion to ASST and no satellite wheals by acetylcholine or A new method for histamine release from purified peripheral blood
hypersensitivity to sweat (Table II). On the basis of these basophils using monoclonal antibody-coated magnetic beads. J Immunol
findings, we strongly believe that CU should be classified Methods 2000;240:39-46.
13. Commens CA, Greaves MW. Tests to establish the diagnosis in
into 2 subtypes from the clinical and pathological aspects. cholinergic urticaria. Br J Dermatol 1978;98:47-51.
The relationship between CU and hair follicles should be 14. Hide M, Tanaka T, Yamamura Y, Koro O, Yamamoto S. IgE-mediated
examined further. hypersensitivity against human sweat antigen in patients with atopic
In summary, 2 subtypes were identified in patients with dermatitis. Acta Derm Venereol 2002;82:335-40.
15. Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI.
CU. The first subtype shows nonfollicular wheals, a
Fitzpatrick’s dermatology in general medicine. 6th ed. New York:
hypersensitivity to autologous sweat, satellite wheals in McGraw-Hill; 2003. p. 1132-3.
the acetylcholine test, and negative reactions to autolo- 16. Itakura E, Urabe K, Yasumoto S, Nakayama J, Furue M. Cholinergic
gous serum. The second subtype shows follicular wheals, urticaria associated with acquired generalized hypohidrosis: report of a
a very weak hypersensitivity to sweat, no satellite wheals 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.
in the acetylcholine test, and positive reactions to autol- Autoantibodies against the high-affinity IgE receptor as a cause of
ogous serum. Thus, we suggest that the pathogenesis of histamine release in chronic urticaria. N Engl J Med 1993;328:
CU involves hypersensitivity or autoimmunity to sweat. In 1599-604.
considering the pathogenesis of CU, the classification 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
presented here may be useful to this unique disorder.
affinity IgE receptor in chronic urticaria. J Invest Dermatol 1996;106:
1001-6.
REFERENCES 19. Fiebiger E, Maurer D, Holub H, Reininger B, Hartmann G, Woisetschl-
1. Duke WW. Urticaria caused specifically by the action of physical agents. ager M, et al. Serum IgG autoantibodies directed against the alpha
JAMA 1924;83:3-9. chain of Fc epsilon RI: a selective marker and pathogenetic factor for a
2. Kaplan AP, Natbony SF, Tawil AP, Fruchter L, Foster M. Exercise- distinct subset of chronic urticaria patients? J Clin Invest 1995;96:
induced anaphylaxis as a manifestation of cholinergic urticaria. J Allergy 2606-12.
Clin Immunol 1981;68:319-24. 20. Tong LJ, Balakrishnan G, Kochan JP, Kinet JP, Kaplan AP. Assessment
3. Lawrence CM, Jorizzo JL, Kobza-Black A, Coutts A, Greaves MW. of autoimmunity in patients with chronic urticaria. J Allergy Clin
Cholinergic urticaria with associated angio-oedema. Br J Dermatol 1981; Immunol 1997;99:461-5.
105:543-50. 21. Zweiman B, Valenzazo M, Atkins PC, Tanus T, Getsy JA. Character-
4. Commens CA, Greaves MW. Tests to establish the diagnosis in istics of histamine-releasing activity in the sera of patients with chronic
cholinergic urticaria. Br J Dermatol 1978;98:47-51. idiopathic urticaria. J Allergy Clin Immunol 1996;98:89-98.
5. Adachi J, Aoki T, Yamatodani A. Demonstration of sweat allergy in 22. Ferrer M, Kinet JP, Kaplan AP. Comparative studies of functional and
cholinergic urticaria. J Dermatol Sci 1994;7:142-9. binding assays for IgG anti-Fc(epsilon)RIalpha (alpha-subunit) in
6. Kobayashi H, Aiba S, Yamagishi T, Tanita M, Hara M, Saito H, et al. chronic urticaria. J Allergy Clin Immunol 1998;101:672-6.
Cholinergic urticaria, a new pathogenic concept: hypohidrosis due to 23. Sabroe RA, Grattan CEH, Francis DM, Barr RM, Black AK, Graves MW.
interference with the delivery of sweat to the skin surface. Dermatology The autologous serum skin test: a screening test for autoantibodies in
2001;204:173-8. 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 São José,c António Bugalho de Almeida, MD, PhD,d Alda Manique, MD,d
Leonor Bento, MD, PhD,e Cândido Pinto Ricardo, PhD,b,f and Maria Margarida
Oliveira, PhDb,g Lisboa, Oeiras, and Algés, Portugal
Background: The safety issues regarding foods derived from

genetically modified (GM) plants are central to their Abbreviations used
acceptance into the food supply. The potential allergenicity Bt: Bacillus thuringiensis
of proteins newly introduced in GM foods is a major safety EPSPS: 5-Enolpyruvylshikimate-3-phosphate synthase
concern. GM: Genetically modified
Objective: We sought to monitor, in potentially sensitive GMO: Genetically modified organism
human populations, the allergenicity effects of 5 GM PAT: Phosphinotricine acetyl transferase
materials obtained from sources with no allergenic potential RUR: Roundup Ready
and already under commercialization in the European Union. SPT: Skin prick test
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 Recombinant DNA technology or genetic engineering
with food and inhalant allergy and individuals with asthma- allows the transfer of single genes from one organism to
rhinitis. We have also tested IgE immunoblot reactivity of sera another, even if distantly related, a feat impossible through
from patients with food allergy to soya (Roundup Ready) and conventional plant breeding. As a result, a genetically
maize (MON810, Bt11, Bt176) samples, as well as to the pure
modified organism (GMO) will contain a modified or
transgenic proteins (CryIA[b] and CP4 5-enolpyruvylshikimate-
3-phosphate synthase).
additional trait encoded by the introduced gene or genes,
Results: None of the individuals undergoing tests reacted which generally results in additional proteins.
differentially to the transgenic and nontransgenic samples Potential benefits for world agriculture derived from
under study. None of the volunteers tested presented detectable GMOs could be enormous, including the possibility of
IgE antibodies against pure transgenic proteins. producing higher yields of more nutritious food in more
Conclusion: The transgenic products under testing seem to be sustainable regimens.1-5
safe in terms of allergenic potential. We propose postmarket With the development of the new modification tech-
testing as an important screening strategy for putative allergic niques, there is the increasing concern of emergence of
sensitization to proteins introduced in transgenic plants. new food allergies. An example of such a situation is the
(J Allergy Clin Immunol 2005;116:403-10.)
Brazil nut allergen (2S protein), which when overex-
pressed in soybean was found to retain its allergenicity
Key words: Transgenic food, allergenicity, immune response,

public health, food safety, recombinant DNA technology
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
From aInstituto Nacional de Saúde Dr Ricardo Jorge, Lisboa; bInstituto de
Tecnologia Quı́mica e Biológica/Instituto de Biologia Experimental e hypersensitivity reactions that can be sudden, severe, and
Tecnológica, Oeiras; cClı́nica Médica e de Diagnóstico Dr Joaquim life-threatening.8 The best estimates are that IgE-mediated
Chaves, Algés; dClı́nica Universitária de Pneumologia do Hospital de food allergies affect approximately 1% to 2% of the adult
Santa Maria, Lisboa; eDepartamento de Clı́nica Pediátrica do Hospital de population9,10; in children this value is estimated to be 2% to
Santa Maria, Lisboa; fInstituto Superior de Agronomia, Tapada da Ajuda,
Lisboa; and gDepartamento Biologia Vegetal, Faculdade de Ciências de
8%.11,12
Lisboa, Lisboa. Before market introduction, genetically modified (GM)
Supported by Fundacxão Calouste Gulbenkian, research project food products are subjected to extensive assessment of
SDH.SP.I.01.11 and by Comissão de Fomento da Investigacxão em potential effects to human health, including toxicity and
Cuidados de Saúde, research project no. 186/01.
potential allergenicity. When the gene source is an aller-
Received for publication January 11, 2005; revised March 22, 2005; accepted
for publication April 12, 2005. genic food, in vitro and clinical tests are available to assess
Available online June 1, 2005. the allergenicity of the transferred protein or proteins.
Reprint requests: Rita Batista, BSc, Instituto Nacional de Saúde Dr Ricardo However, most genes transferred through genetic engi-
Jorge, Av Padre Cruz, 1649-016 Lisboa, Portugal; E-mail: rbatista@ neering are obtained from organisms with no allergenic
itqb.unl.pt.
0091-6749/$30.00
history. In such cases the assessment of allergenicity
Ó 2005 American Academy of Allergy, Asthma and Immunology becomes more difficult to obtain because of the absence of
doi:10.1016/j.jaci.2005.04.014 valid methods and models.13-16
403
404 Batista et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
TABLE I. Transgenic flour products tested in SPTs and in IgE Immunoblot reactivity assays
Origin and Method of testing

Date of Responsible certification of and human
Material Characteristics commercialization company the material population studied
2% GM Insect resistance (CryIA[b] 1998 Syngenta Institute of Reference SPTs in a population

Bt11 maize gene) and ammonium Materials and of allergic children
glufosinate tolerance Measurements (27 individuals);
(PAT gene); 35S pro; certified IgE immunoblot
NOS 3# t reactivity assay with sera
from patients with food
allergy (57 individuals)
100% GM Insect resistance (CryIA[b] 1997 Syngenta SPTs in a population
Bt176 maize gene) and ammonium of allergic children
glufosinate tolerance (27 individuals);
(PAT gene); 35S pro; 35S t IgE immunoblot
reactivity assay with
sera from patients
with food allergy
(57 individuals)
National Service of
100% GM Ammonium glufosinate 1998 Bayer Crop SPTs in a population of
Plant Protection
T25 maize tolerance (PAT gene); Sciences patients with asthma-
(DGPC); not certified
35S pro; 35S t rhinitis (50 individuals)
100% GM Insect resistance (CryIA[b] 1998 Monsanto SPTs in a population
MON810 gene); 35S pro; NOS3# t with asthma-rhinitis
maize (50 individuals); IgE
immunoblot reactivity
assay with sera from
patients with food allergy
(24 of the 57 individuals)
5% GM Gliphosate resistance 1996 Monsanto Institute of Reference SPTs in a population of
RUR soya (CP4EPSPS gene); Materials and allergic children (27
35S pro; NOS 3# t 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 Proteccxão de Culturas.
In this study we have monitored the IgE response of food-derived products implied a consumption of GM soya and
allergy-sensitive populations to GM maize and soya maize.
products (Table I). The transgenes in maize and soya The food inquiry was performed on 106 healthy volunteers to find
were obtained from sources with no allergenic history and out which maize- and soya-derived products (from a list of 205 dif-
ferent products) they had already consumed. The population studied
approved for human consumption in the European Union.
included individuals with ages from 1 to 41 years, with an average of
The IgE response of the same individuals to nonmodified 12.4 years (48 male and 58 subjects).
products was also analyzed for comparison.
Transgenic quality of the noncertified
flour samples
METHODS In addition to the 3 noncertified transgenic products listed in
This study was evaluated and approved by the Research Ethic Table I, nontransgenic analogues were also tested as controls. For
Committees of the Hospital of Santa Maria and the National Institute the noncertified material (Table I), we have first confirmed the
of Health, Lisbon, Portugal. All individuals participating in this study transformation event and the absence of cross-contamination among
or their parents also provided informed consent. them.
For these analyses, DNA was isolated by using the cetyltrimethyl-
Food inquiry ammonium bromide method,17 with 3 replicas per sample. DNA
Because of the fact that IgE-mediated allergic reactions require quality and concentration were analyzed by means of agarose gel
prior exposure, resulting in sensitization, we have performed a food electrophoresis, and maize-specific amplifiable DNA was detected
inquiry to evaluate the consumption of soya and maize food-derived by using PCR amplification of a 226-bp sequence from the maize
products. Bearing in mind that since 1998 all the GM products invertase gene.18
under testing were approved for commercialization in the European The presence or absence of the 35S Cauliflower Mosaic Virus
Union (Table I), we assumed that consumption of maize and soya promoter in the transgenic (Table I) and control samples was checked
J ALLERGY CLIN IMMUNOL Batista et al 405
by using standard protocols for the amplification of a 195-bp DNA through color development. Ten milligrams of lyophilized samples
sequence.18 was diluted in the kit buffer provided, and 100 mL of each sample
Transformation event–specific PCR reactions were performed to (approximately 200 mg of total protein) was eluted along the strip.
verify the presence of MON810, T25, and Bt176 transgenic events.18 For protein quantification, the Bio-Rad protein assay (Bio-Rad
Different internal controls were always used to detect putative laboratories) was used, with turkey albumin (Merck) as a standard.
contaminations. In each case whole or digested (HaeIII or Hinf I)
PCR product size was compared with expected values.18 Skin testing of the 2 populations
Skin tests were performed in 2 human populations with positive
Preparation of protein extracts for histories of food allergy, inhalant allergy, or both, as well as a
human skin prick testing and positive SPT response for related allergens; one group was composed
IgE immunoblot reactivity of 27 children with food and inhalant allergy from the Paediatrics
Allergy Department of the Hospital of Santa Maria, and the other was
Maize and soya protein extracts were made by Laboratorios Leti,
composed of 50 patients with asthma-rhinitis from the University
SL (Madrid, Spain) according to approved pharmaceutical prepara-
Clinic of Pneumology from the Hospital of Santa Maria (see Tables
tive and safety procedures for the production of diagnostic skin prick
E1 and E2 in the Online Repository in the online version of this
test (SPT) materials. About 10 g of each of the maize and soya flour
article at www.mosby.com/jaci). The children were tested with the
samples was extracted for 16 hours in 1:20 (wt/vol) PBS (pH 7.4).
extracts of Bt176, Bt11, RUR, and nontransgenic analogues; for the
After centrifugation, the pellet was discarded, and the supernatant
asthma-rhinitis population, we used the extracts of MON810, T25,
was extensively dialyzed against bidistilled water. The extracts were
and nontransgenic analogues for testing (Table I).
centrifuged, filter sterilized, and freeze-dried. For human SPTs, the
Skin tests were performed by using the prick procedure,19 and
extracts were resuspended to 10 mg/mL maize or soya freeze-dried
results were read after 20 minutes. The results were classified as
material (approximately 2 mg of total protein/mL for MON810, Bt11,
positive when the larger diameter of the wheal exceeded 3 mm.
Bt176, and control samples; approximately 3 mg of total protein/mL
Histamine hydrochloride, 10 mg/mL (Leti), was used as a positive
for T25 and control samples; and approximately 3.5 mg of total
control, and Phenolate saline serum with glycerine (Leti) was used as
protein/mL for Roundup Ready [RUR] and control samples).
a negative control.
For the IgE immunoblot reactivity assay conducted with sera from
All the protein extracts were first tested on a control population of
patients with food allergy, we used an extract prepared with food to
20 nonallergic healthy individuals.
which the person undergoing the test was allergic as a positive control
extract. Four grams of food material was homogenized in liquid
Sera for the IgE immunoblot reactivity assay
nitrogen and precipitated with 20 mL of 10% trichloroacetic acid
(wt/vol) in cold acetone containing 20 mM dithiothreitol for 1 hour at Patient sera were provided by the Joaquim Chaves Clinic and were
220°C. The precipitate was collected by means of centrifugation obtained from 57 individuals who had a positive history of docu-
(15 minutes at 14,000g at 4°C), washed twice with 20 mM dithio- mented food allergy, as well as a positive value equal to or higher than
threitol in cold acetone, and allowed to dry completely. class 3 on specific UniCAP test (Pharmacia Diagnostics, Seixal,
Portugal; see Table E3 in the Online Repository in the online version
Quality of transgenic proteins in maize of this article at www.mosby.com/jaci). All 57 sera were first assayed
and soya extracts for reactivity against nontransgenic maize and soya by means of
specific IgE UniCAP testing. The sera were then tested for IgE
ELISA GMO Check Bt maize test kit (SDI Europe, London, immunoblot reactivity against Bt11, Bt176 maize, and RUR soya, as
United Kingdom) was used to evaluate the presence-absence of well as against nontransgenic analogues (Table I). MON810 maize
Bt CryIA(b) protein in the lyophilized extracts prepared by and its nontransgenic analogue, as well as pure CryIA(b) (Research
Laboratorios Leti. Ten milligrams of dry extract was resuspended Diagnostics, Inc) and CP4EPSPS (Monsanto Co), were used to test
in 200 mL of the kit extraction buffer provided, and all nonsoluble the IgE immunoblot reactivity of sera of the 24 more sensitive

material was removed by means of centrifugation (10 minutes at patients (Table I).
11,000g). The manufacturer’s instructions were followed thereafter,
using approximately 200 mg of total protein. SDS-PAGE and protein transfer to
To evaluate the presence or absence of CP4 5-enolpyruvylshiki- nitrocellulose membranes
mate-3-phosphate synthase (CP4EPSPS) protein in RUR soya and
nontransgenic analogues, the lyophilized materials were tested with Samples were diluted 1:2 in sample buffer (0.125 M Tris-HCl [pH
an ELISA GMO Check RUR Soya Grain test kit (Strategic Diag- 6.8], 4% SDS, 20% vol/vol glycerol, 0.2 M dithiothreitol, and 0.02%
nostics Inc). Five milligrams of dry extract was diluted in 200 mL of bromophenol blue) and boiled for 5 minutes before electrophoresis
kit extraction buffer. The nonsoluble material was removed by means in a 0.75-mm-thick 10% acrylamide gel with 4% stacking gel.20 After
of centrifugation (10 minutes at 11,000g). The manufacturer’s electrophoresis, the proteins were blotted onto hybond ECL nitro-
instructions were followed thereafter, using approximately 2.5 mg cellulose membranes (Amersham Biosciences, Carnaxide, Portugal)
of total protein. by means of wet transfer in 25 mM Tris, 192 mM glycine, 0.1% SDS,
Thirty micrograms of each sample was also run by means of and 20% methanol for 1 hour at 75V at room temperature.
SDS-PAGE and immunobloted with rabbit anti-Bt CryIA(b) poly-
IgE immunoblot reactivity assay of sera
clonal antibodies (RDI, Flanders, NJ) or goat anti-CP4EPSPS serum
(Monsanto Co, St Louis, Mo; see description below). from patients with food allergy
It was impossible to obtain commercial anti-phosphinotricine The detection of patient sera IgE reactivity was carried out after
acetyl transferase (anti-PAT) antibodies, and there is no commercially electrophoresis of 30 mg (60 mg/cm gel width) of MON810, Bt11,
available ELISA kit for PAT. We therefore decided to use the Trait LL Bt176, and RUR transgenic samples and nontransgenic analogues
corn grain test kit (Strategic Diagnostics Inc) to evaluate the presence and 25 ng (50 ng/cm gel width) of pure CryIA(b) and CP4EPSPS and
or absence of PAT in Bt176, Bt11, T25, and nontransgenic analogues. transfer to nitrocellulose membrane.
This kit uses PAT-specific antibodies coupled to a color reagent and Blots were blocked overnight at 4°C with PBS-T (58 mM
incorporated into strips, allowing the detection of PAT in an extract Na2HPO4, 17 mM NaH2PO4.H2O, 68 mM NaCl, and 0.2% Tween
AUGUST 2005
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 Probability of consumption of products
(l estimates) with transgenic protein
n 95% CI P = .235 95% CI
Total 106 39.3 35.2-43.4 0.999902 0.99974-0.99999

Sex
Male 48 34.8 29.2-40.4 0.999959 0.99895-0.99993
Female 58 43.0 37.1-48.2 0.999719 0.99983-0.99999
Age group (y)
<5 20 29.5 22.6-36.3 0.999024 0.99506-0.99980
5-10 56 41.1 35.2-46.9 0.999936 0.99974-0.99998
10-25 11 48.8 35.6-62.0 0.999990 0.99976-1.00000
25 19 38.9 27.5-50.3 0.999893 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
Tecnológica Good Laboratory Practices Microbiology laboratory data).
20) and 5% skimmed milk powder (or 3% BSA for patients with milk of one product with maize or soya having transgenic proteins), we can
allergy) and washed with PBS-T before incubation in serum diluted then calculate the probability of one individual having been in contact
1:10 in blocking solution for 1 hour and 30 minutes at room with transgenic proteins, which is 12e2lp .
temperature. After washing with PBS-T, the membranes were To estimate this probability, we used as l the mean number of
incubated for 1 hour at room temperature in alkaline phosphatase– consumed products with maize or soya obtained in the survey, and as
conjugated monoclonal anti-human IgE (Southern Biotechnology p the proportion of maize and soya products detected with transgenic
Associates, Birmingham, Ala) diluted 1:2000 in blocking solution, proteins calculated by using the Instituto de Biologia Experimental e
washed with PBS-T and assay buffer, and incubated for 5 minutes Tecnológica Good Laboratory Practices Microbiology laboratory
with CDP-Star solution with Nitro-Block II enhancer (Tropix data during the last 2 years.
Western-Star Immunodetection System).
Blots were observed after exposure (5 seconds-30 minutes) to a
high-performance chemiluminescence Hyperfilm ECL (Amersham RESULTS
Biosciences). Food inquiry
Immunoblot detection of Bt CryIA(b) All 106 individuals participating in this inquiry con-
and CP4EPSPS sumed some of the 205 products presented. The extreme
The procedure was identical to the one described for IgE
cases, with lower and higher numbers of consumed
immunobloting of patient’s sera, with the following differences. For products, were relative to a 1-year-old and 9-year-old
Bt CryIA(b), the first antibody incubation was performed in rabbit girl with 4 and 129 consumed products, respectively.
anti-Bt CryIA(b) polyclonal (Research Diagnostics, Inc) diluted The mean of consumed products with maize and soya
1:1400 in blocking solution, and the second antibody incubation was 39.3 (95% CI, 35.2-43.4), and the probability of
was performed in goat anti-rabbit IgG-AP conjugate (Tropix-Applied an individual having eaten GM food was near 100%
Biosystems, Porto, Portugal) diluted 1:2800 in blocking solution. For (Table II).
CP4EPSPS, the first antibody incubation was performed in goat anti-
CP4EPSPS serum (Monsanto Co) diluted 1:5000 in blocking solu- Transgenic quality of the noncertified
tion, and the second antibody incubation was performed in anti-goat flour samples
IgG-alkaline phosphatase conjugate (Sigma, Sintra, Portugal) diluted
1:2500 in blocking solution.
All 6 tested samples (Bt176, T25, MON810, and the
nontransgenic analogues) showed the expected bands
Statistical analysis when checking for amplifiable maize DNA (data not
To estimate the probability of one individual from the Portuguese shown), and only the 3 transgenic samples showed the
population having once been in contact with transgenic proteins expected amplicon of the 35S promoter (data not shown).
present in maize or soya foods, we used (1) the results from the food The final confirmation that all the samples tested were
inquiry and (2) the percentage data of maize and soya products with correctly labeled and that there was no cross-contamina-
detectable transgenic proteins provided by Instituto de Biologia tion among them was obtained from construct-specific
Experimental e Tecnológica Good Laboratory Practices Microbiol- PCR (Fig 1). As expected, the digestion of the obtained
ogy laboratory. This laboratory is one of the 2 national laboratories amplicons confirmed the accuracy of the specific PCR
responsible for food GMO detection.
(data not shown).
Assuming that the number of products with maize or soya
consumed by the population is a Poisson random variable with the Quality of transgenic proteins in maize
expected value l and that the probability of an individual having
consumed a product with transgenic proteins (provided he or she had
and soya extracts
consumed n products with maize or soya) is modeled by using As described in the Methods section, Laboratorios
binomial distributions21 (n = number of experiences, p = probability Leti protein extracts were tested for the presence of the
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.

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 Trait LL corn grain test kit. With this system, we have also
by using ELISA (data not shown) and Western blotting confirmed the absence of PAT in nontransgenic analogues
(Fig 2) in MON810, Bt11, and Bt176 extracts and (data not shown).
was absent from the nontransgenic control analogues.
CP4EPSPS was also detected by means of ELISA (data Allergenicity tests
not shown) and Western blotting (Fig 3) in RUR ex- Skin testing of the 2 populations. Only individuals
tract and was absent from the nontransgenic analogue. with maize sensitivity, soybean sensitivity, or both had
Both pure CryIA(b) and CP4EPSPS proteins were de- positive results against the protein extracts under testing;
tected with the respective specific antibodies (data not however, none of the volunteers reacted differentially to
shown). GM versus non-GM samples (Table III and Tables E1 and
In T25, Bt11, and Bt176 samples PAT protein was E2 in the Online Repository in the online version of this
detected in 200 mg of total protein solutions by using the article at www.mosby.com/jaci).
AUGUST 2005
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 methods of testing have been questioned.28 Considering
tests performed by using SPTs and IgE immunoblot that the past few decades have witnessed a significant
reactivity assays increase in IgE-mediated allergic diseases, the allergenic
potential of these novel foods is a major concern in public
IgE immunoblot
SPTs reactivity assay health.
The food inquiry performed in this study indicated that
No. of Positive No. of Positive
individuals responses individuals responses
the probability of an individual having eaten GM food was
tested (%) tested (%) near 100% (Table II). This value is probably underesti-
mated because each individual probably consumed each
GM protein
PAT 77 0 NT 2
product several times, which was not considered in statis-
CRY1A(b) 77 0 57 0 tical calculations.
CP4EPSPS 27 0 57 0 Also, it is possible that the first sensitization occurred
during breast-feeding in the individuals submitted to SPTs
NT, Not tested. 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
All the patients had wheals larger than 3 mm for the individuals participating in this study had already been
histamine, and none of them reacted against the negative in contact with the products tested.
control. The DNA and protein quality analysis performed in this
IgE Immunoblot reactivity assay of sera from study confirmed the quality of flour samples and maize
patients with food allergy. Two types of Western and soya protein extracts (Figs 1-3). In the Western assay
blotting results (Figs 4 and 5) were observed. In Fig 4 for the detection of CryIA(b) in Bt11, Bt176, MON810,
serum from an individual with octopus allergy of class 4 and nontransgenic control analogues (Fig 2), the multiple
on specific UniCAP testing reacted only against positive bands approximately equal to the CryIA(b) trypsin resis-
controls. In Fig 5, concerning an individual with peanut tant core observed are likely the products of endogenous
allergy of class 6 on a specific UniCAP test, positive grain proteases.30 Some of the protein is degraded further
signals were observed against the positive control but also to produce lower-molecular-weight bands, including a
against other maize and soya protein extracts. 30-kd product previously reported.30
None of the volunteers tested presented differential As already mentioned, we have performed this study on
signals against nontransgenic versus transgenic protein sensitive populations. The population submitted to SPTs
extracts (Table III). All 24 individuals tested against pure and immunoblot analyses was composed of individuals
transgenic proteins (CP4EPSPS and CryIAb) presented no with food allergy and inhalant allergy, many of them
detectable reactions against these controls. children. Children are more susceptible to food allergies
than adults. This higher susceptibility is probably the
result of immunologic immaturity and, to some extent,
DISCUSSION immaturity of the gut.31,32 In addition, children who have
preexisting food allergies are more likely to experience
Although absolute certainties regarding GM food risks allergic reactions to other foods introduced in their diets.
to health and the environment will hardly be obtained, The absence of detectable differences in IgE reactivity
reports regarding potential problems have raised public between GM maize and soya samples and the correspond-
concern. Some of the concerning issues include the ing wild-type samples obtained in this study is in accor-
putative toxicity-allergenicity of crops expressing foreign dance with some previously published results.33,34
proteins,22-25 although these fears have not been con- The appearance of nondifferential bands on some
firmed in some later studies,26,27 and the adequacy of the chemiluminescence films for maize and soya protein
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.

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 In this study we did not obtain any differential positive
cross-reactivity among various plant and animal pro- results, which allows us to conclude that the transgenic
teins.35,36 In the example presented in Fig 5, although products under testing seem to be safe regarding their
the patient tested had only documented peanut allergy allergenic potential. Although we succeeded in integrating
(class 6 on UniCAP test), it was shown that he also had a private clinic and a hospital in this study, it would be
IgE binding to other foods, such as almond (class 2), desirable to increase the size of the analyzed population
hazelnut (class 3), walnut (class 2), cashew (class 4), and eventually extend this work to other countries.
soybean (class 3), and maize (class 3). This fact justifies We also propose the development and use of clinical
the appearance of the nondifferential bands on maize and testing with specific IgE in the postmarketing surveillance
soya lanes. of foods produced through biotechnology. Positive test
Although IgE detection (either SPT or specific IgE) results should be followed by double-blind, placebo-
serves as a good indicator of sensitization but not neces- controlled food challenges under appropriate clinical
sarily of disease, in the clinical setting the absence of observation to identify true clinical reactions.38
detectable IgE was found to have excellent negative pre-
dictive accuracy indices and therefore might be very We gratefully acknowledge the National Service of Plant
useful in excluding the presence of immediate food hyper- Protection (DGPC) for providing BT176, T25, MON810, and
sensitivity.37 nontransgenic analogue maize samples; Laboratorios Leti for the
AUGUST 2005
preparation of maize and soya protein extracts; and Monsanto, derived products—qualitative nucleic acid based methods. Nov 2002.
especially Dr Richard Goodman, for providing the CP4EPSPS p. 23-66.
protein and the corresponding antiserum. Fernanda Spı́nola and 19. Sub-Committee on Skin Tests of the European Academy of Allergology
and Clinical Immunology. Skin tests used in type I allergy testing.
Cátia Peres are gratefully acknowledged for their advice regarding
Allergy 1989;44(suppl 10):1-59.
GMO detection. We also thank Margarida Santos, Helena Raquel,
20. Laemmli UK. Cleavage of structural proteins during the assembly of the
Madalena Martins, and Sara Silva for help in the preparation of the head of bacteriophage T4. Nature 1970;227:680-5.
food inquiry. Finally, we thank Phil Jackson for the final revision of 21. Bliss CI. Statistics in biology. Vol 1. New York: MacGraw-Hill; 1967.
the manuscript and Fundac xão Calouste Gulbenkian and Comissão de p. 558.
Fomento da Investigac xão em Cuidados de Saúde for funding. 22. Losey JE, Rayon LS, Carter ME. Transgenic pollen harms monarch
larvae. Nature 1999;395:214.
23. Ewen SWB, Pusztai A. Effect of diets containing genetically modified
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Advances in Asthma, Allergy, and Immunology Series 2005
Javier Chinen, MD, PhD,a and William T. Shearer, MD, PhDb Bethesda, Md,
and Houston, Tex
The authors selected articles published in the literature from

January 2004 through December 2004 that were relevant to Abbreviations used
the areas of basic and clinical immunology. Several articles APC: Antigen-presenting cell
explored the development of TH1 or TH2 response and the role CTL: Cytotoxic T cell
of the monocyte–T cell interaction. Others were articles CVID: Common variable immunodeficiency
describing the action of drugs commonly used in asthma to DGS: DiGeorge syndrome
inhibit cytokine responses and the anti-inflammatory role of DSS: Dextran sulfate sodium
nonimmune pulmonary cells present in the lung. Several HIGM: Hyper-IgM syndrome
reports show how dendritic cells are being developed as ICOS: Inducible costimulatory molecule
vehicles for DNA vaccines aimed at stimulating cellular IRD: Immune restoration disease
responses, an advance of great importance for HIV researchers NEMO: NF-kB essential modifier
working on vaccines, who are concerned about the different NK: Natural killer
ways HIV evades the immune response. Other publications PID: Primary immunodeficiency
described Toll-like receptors in diverse cells, including mast TCR: T-cell receptor
cells and CD41 T cells, for the recognition of viruses and TLR: Toll-like receptor
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. The areas of basic and clinical immunology continue to
Significant advances included the clinical prognosis in common develop at a fast pace, with numerous reports exploring
variable immunodeficiency for patients presenting with lung relatively new and old areas of these fields, such as the
pathology, the safety of live vaccines in partial DiGeorge biology of Toll-like receptors (TLRs) and the description
syndrome, the report of patients with complete DiGeorge
of primary immunodeficiencies (PIDs), respectively. The
syndrome with the presence of peripheral blood T cells, the
clinical spectrum of patients with NF-kB essential modifier
goal of this article is to review some of the significant
(NEMO) gene deficiency, the publication of a consensus progress in basic and clinical immunology published in
algorithm for the management of hereditary angioedema, and 2004, with focus on articles that the authors considered
the report of immune restoration syndrome in pediatric HIV of interest to the readers of The Journal of Allergy and
infection. (J Allergy Clin Immunol 2005;116:411-8.) Clinical Immunology.
Key words: Immunoregulation, HIV, immunodeficiency, innate

BASIC IMMUNOLOGY
immunity, complement
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 Regulation of the T-cell response:
Department of Allergy and Immunology, Texas Children’s Hospital, and TH1 versus TH2
the Departments of Pediatrics and Immunology, Baylor College of
Medicine, Houston. Wittmann et al1 investigated the cytokine secretion
The opinions expressed in this article do not necessarily represent the views of profile resulting from the interaction of monocytes derived
the National Human Genome Research Institute or the National Institutes of from peripheral blood and autologous CD41 T cells
Health.
isolated from inflammatory skin lesions induced by an
Disclosure of potential conflict of interest: None disclosed.
Received for publication May 4, 2005; accepted for publication May 6, 2005. allergen patch test. These activated T cells induced IL-12
Available online July 5, 2005. secretion by monocytes that were stimulated with IFN-g.
Reprint requests: Javier Chinen, MD, PhD, National Human Genome Research However, when the T cells were incubated with resting
Institute, 10 Center Drive, MSC 1611, Building 10/CRC Room 6-3340, monocytes, IL-12 secretion was not induced. In contrast,
Bethesda, MD 20892. E-mail: jchinen@mail.nih.gov.
0091-6749/$30.00
resting T cells did not inhibit IL-12 secretion in resting
Ó 2005 American Academy of Allergy, Asthma and Immunology monocytes. The authors further determined that this effect
doi:10.1016/j.jaci.2005.05.010 on IL-12 secretion was cell-contact specific and dependent
411
412 Chinen and Shearer J ALLERGY CLIN IMMUNOL
AUGUST 2005
TABLE I. Key advances in basic immunology blood T cells by fluticasone increases synergistically with
the addition of salmeterol. Increased apoptosis was asso-
1. Monocyte activation is necessary for induction of a TH1
response.
ciated with a more efficient caspase processing, increased
2. Salmeterol and fluticasone act synergistically to inhibit translocation of the glucocorticoid receptor, and reduction
secretion of inflammatory mediators in asthma. of the expression of phosphorylated IkBa. Salmeterol
3. A 10-kd protein from pulmonary Clara cells and IL-17F from alone did not produce apoptosis in the T cells.
bronchial epithelial cells are potent anti-inflammatory cytokines. A factor that affects the immune regulation of the
4. HLA alleles influence the generation of HIV CTL allergic response is the 10-kd protein secreted by
escape mutants and the risk of HIV transmission. the pulmonary Clara cells.6 This protein decreased the
5. Mast cells recognize viruses and are activated through expression of the TH2 cytokines IL-4, IL-5, and IL-13
Toll receptors. of ovalbumin-sensitized mouse splenocytes and of CD41
6. CD4 T cells can be directly activated through Toll receptors.
T cells that had been polarized into TH2 cells. This potent
7. Alternative splicing might be responsible for breaking immune
tolerance and the development of autoimmune disorders.
effect was dose dependent and associated with a reduction
of intracellular GATA-3, the transcription factor that
mediates TH2 response. This result underscores the
on the induction of T-bet expression in monocytes. T-bet dynamic interaction between immune cells and highly
is a signal transduction factor that is essential for the specialized lung cells in lung inflammation and asthma
development of the TH1 response. On the basis of these pathogenesis.
findings, the authors suggest that initial events in skin
atopic disease might involve the infiltration of activated DNA vaccines
T cells into the skin and interaction with resting antigen- DNA vaccination is being actively explored as an
presenting cells (APCs), resulting in absence of IL-12 and alternative for the treatment of allergic diseases. DNA-
development of a TH2 environment. The role of T-bet as based immunomodulation has been shown to switch the
the key protein for the induction of the TH1 response was immune response from a TH2- to a TH1-dominant re-
supported by the work of Lametschwandtner et al,2 who sponse in several mouse models.7 Klostermann et al8 used
induced T-bet expression in TH2 cells obtained from skin human dendritic cells transduced with an adenovirus
biopsy specimens of atopic individuals. TH2 cells ex- vector carrying the expression cassette for the grass pollen
pressing T-bet were able to secrete and express high levels Phl p 1 protein. When these cells were cocultured with
of IFN-g, TNF-a, IL-2, and IL-12, with a decrease of the T cells, they induced a TH1-like response, with prolifer-
expression levels of IL-4 and IL-5. In addition, they ation of the CD81 T-cell subtype, increased IFN-g
showed a reversion of chemokine expression profile from secretion, and less IL-4 and IL-5 secretion than when
TH2 to TH1. The immunologic events early in infancy that nontransduced dendritic cells were pulsed with the Phl p 1
can lead to atopic disease were investigated by Upham protein. A different delivery strategy for DNA vaccination
et al,3 who described that the HLA-DR expression in in allergy was shown by Ludwig-Portugall et al9 using a
monocytes obtained from cord blood stimulated with gene gun–mediated delivery in vivo. Although this
IFN-g correlated with IL-12 secretion induced by endo- method requires 100- to 1000-fold more DNA, it does not
toxin and had an inverted association with IL-13 secretion involve a viral vector. The authors used the gene gun to
induced by ovalbumin or dust mite. HLA-DR expression immunize mice percutaneously with a b-galactosidase–
in unstimulated monocytes was inversely associated with encoding plasmid and then sensitized the animal to
allergic disease at the 2-year follow up. These findings b-galactosidase protein. IgG2a was 10-fold increased,
suggest that early activation of APCs might decrease the and specific IgE was not detectable. In contrast, mice that
TH2 response and the risk of development of atopic were immunized intraperitoneally with b-galactosidase
disease. had high specific IgE levels. To increase the immunoge-
Two drugs commonly used in asthma, fluticasone and nicity of DNA vaccines, Jilek et al10 examined the use of
salmeterol, were shown to synergistically inhibit cytokine biodegradable microspheres as DNA carriers for prophy-
secretion and to influence the TH2 to TH1 balance.4 The laxis against anaphylaxis. They used microspheres made
drug combination inhibited the secretion of the TH1 of polylactidecoglycolide, a biodegradable material that
cytokines TNF-a and IFN-g by mitogen-stimulated is readily phagocytosed by dendritic cells.11 When mice
PBMCs from normal and asthmatic patients. In contrast, received subcutaneous polylactidecoglycolide micro-
the secretion of the TH2 cytokines IL-5 and IL-13 was spheres with DNA encoding phospholipase A, the bee
inhibited only in PBMC cultures from control subjects but venom major allergenic protein, and then were sensitized
not from asthmatic patients. The addition of a phospho- and challenged with a lethal dose of the phospholipase A
diesterase inhibitor to the combination suppressed IL-13 protein, anaphylaxis was prevented in 50 of 54 experi-
secretion in PBMCs from asthmatic patients, an effect that mental mice. Interestingly, the preventive effect was also
can be explained by the maintenance of high cyclic achieved in animals that received microspheres with
adenosine monophosphate levels. Pace et al5 were also nonspecific DNA. The authors also demonstrated similar
interested in the mechanism of action of the combination production of IgG2a, IL-4, IFN-g, and IL-10, suggesting
of fluticasone and salmeterol in T cells. These investiga- that polylactidecoglycolide microspheres drive APCs
tors found that the induction of apoptosis in peripheral toward the TH1 phenotype and could be considered for
J ALLERGY CLIN IMMUNOL Chinen and Shearer 413
therapeutic use in atopic patients. Another molecule in this Gag epitope. However, when this HIV strain was
studied was the CpG oligonucleotide, which was shown transmitted to an individual with different HLA alleles,
to drive TH1 cytokine expression in plasmacytoid den- this epitope reverted to the wild type. Supporting their
dritic dells obtained from patients with allergic rhinitis.12 finding, they demonstrated that a second mutation in the
When a CpG oligonucleotide was introduced to cocultures same epitope did not revert. The reasons why some
of dendritic cells and CD41 T cells, the cytokine secretion epitope mutations persist and others revert to the wild
profile changed from being predominantly composed of type are not clearly related to virus fitness, but certainly
IL-4 and IL-5 to being mostly IFN-a and TNF-a. This this is of concern for the design of vaccines targeting the
study suggests that mucosal dendritic cells can be con- anti-HIV CTL response. A related article by Dorak et al18
sidered as targets for DNA-based immunomodulation examined 125 couples who were initially HIV discordant
of the T-cell response. after which the spouse converted and 104 persistent HIV-
discordant couples. They found that the risk of HIV
Advances in cytokine research transmission to the spouse was 2-fold higher, independent
The production of IL-13 by human B lymphocytes and of viral load, if the couples shared one or both HLA-B
its role in IgE synthesis was investigated by Hajoui et al13 alleles than if they had different HLA-B alleles. This
using B cells isolated from the tonsils of healthy volun- suggests that HIV CTL escape mutants are transmitted
teers. They reported a 10-fold increase of IL-13 secretion more efficiently in a homogenous population and that they
when these cells were stimulated with anti-CD40 antibody will vary from population to population (Fig 1). Adding
and IL-4. When they added neutralizing anti-IL-13 anti- one more strategy for HIV immune evasion, Draenert
bodies, IgE levels decreased by 80%, and IgE transcripts et al19 reported mutations in the Gag protein outside a
decreased by 50%, suggesting that B-cell secretion of IgE particular epitope that altered a target site for protein
is regulated in part by IL-13 produced by the same B-cell processing in APCs and therefore cannot be presented,
population. Two articles published in the Journal focused making CTLs unable to lyse infected cells.
on the IL-17 family of inflammation proteins and the role Winchester et al20 investigated innate immunity in
of nonimmune cells in lung inflammation. Kawaguchi mother-to-child transmission of HIV, mediated by mater-
et al14 reported that a function of a newly identified IL-17F nal natural killer (NK) cells. The expression of HLA-
protein in primary bronchial epithelial cells was to induce B*4901 and B*5301 alleles inhibited mother-to-infant
GM-CSF secretion through activation of Raf-1/MEK- HIV transmission despite high maternal viral loads. They
ERK1/2, and therefore IL-17F participates in the patho- also bound the KIR30L1 NK receptor. The HLA-B*5001
physiology of allergic inflammation. A second article and B*3501 alleles, which differ from B*4901 and
examined the regulation of IL-17A in human airway B*5301 by only 5 amino acids, did not bind the
smooth cells obtained from patients undergoing lung KIR30L1 receptor and were associated with enhanced
surgery. IL-17A induced secretion of IL-6 after stimula- vertical transmission. The authors proposed that the
tion with TNF-a but not after stimulation with IL-1b. Of molecular basis of this observation involved maternal
note, there was no induction of other inflammation NK cell recognition by engagement of NK cell receptors
markers, such as intercellular adhesion molecule expres- with polymorphic ligands encoded by maternal HLA-B
sion or GM-CSF secretion.15 The expression of IL-10 and alleles. Moreover, they believe that the placenta is the
FoxP3, which phenotypically define regulatory T cells, site where protection against vertical HIV transmission
was compared in CD41 T cells obtained from patients occurs, mediated by interrelating adoptive and innate
with moderate and severe asthma and from healthy control immune recognition mechanisms.
subjects.16 This study found that FoxP3 mRNA expres- In the B-cell compartment, Moir et al21 found 42 genes
sion correlated with IL-10 mRNA expression, and it was upregulated in B cells from HIV-viremic patients com-
2-fold higher in asthmatic patients receiving steroids than pared with B cells from healthy control subjects. Most
in healthy control subjects or patients with mild asthma. of these genes were associated with the activation of the
In addition, it was shown that CD41CD251 T cells IFN-g pathway or with terminal differentiation of B cells. Basic and clinical immunology
expressed 11-fold more IL-10 and FoxP3 than total In addition, they showed that CD95 expression in B cells
CD41 T cells after being exposed to corticosteroids correlated with HIV viremia. This report is valuable for
in vitro. These results suggest that the anti-inflammatory the identification of genes involved in the mechanisms
effect of corticosteroids include the development of of B-cell dysfunction in HIV infection.
regulatory T cells secreting IL-10.
Innate immunity
HIV immunopathogenesis The role of TLRs in innate immunity continues to
The mechanisms by which HIV evades the immune expand and involve many different immune cells and
system are far from being completely elucidated. Leslie processes. Kulka et al22 added mast cells to the list of
et al17 studied the association of a specific cytotoxic T-cell effector cells participating in the recognition of viruses
(CTL) epitope in the HIV1 Gag protein and HLA alleles through TLRs. Mast cells had already been shown to
in an HIV-infected population. They found that HIV- respond to LPS and peptidoglycan through TLR-1, TLR-2,
infected individuals expressing the HLA alleles B57 and TLR-4, and TLR-6. The importance of mast cells on
B*5801 had selected for variants with a specific mutation shaping the innate immunity response to infection was
AUGUST 2005
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.
reviewed by Marshall and Jawdat.23 Mast cells are ation of colonic cells and were more susceptible to injury
not only activated by TLRs but also by complement caused by DSS or radiation. Similar mortality occurred
components, leading to the secretion of cytokines impor- when wild-type mice were deprived of commensal bacte-
tant for selective recruitment of effector cells. The expres- ria and then were treated with DSS. When these animals
sion of TLR-3 in mast cells derived from peripheral blood were given LPS, the animals were protected, suggesting
and 2 mast cell lines was newly reported, and their that TLR stimulation might mediate epithelial barrier
production of IFN-a and IFN-b in response to exposure repair. To provide a direct link between innate and
to dsDNA and to PolyI:C was also reported. A similar adaptive immunity, Gelman et al26 reported that mouse
response was obtained when mast cells were exposed CD41 T cells expressed TLR-3 and TLR-9 and responded
to UV light–inactivated influenza virus and to type 1 to CpG and polyI:C stimulation with increased survival
reovirus. and nuclear factor kB (NF-kB) activation. This observa-
Flo et al24 demonstrated that TLR-4 stimulation with tion might represent the response of the immune system
LPS in macrophages helped to control bacterial replication for infectious organisms that impair APCs by directly
by increasing levels of lipocalin 2 expression. This protein activating the adaptive immune cells.
inhibits iron uptake by Escherichia coli. Lipocalin 2
knockout mice became highly bacteremic after infection Immune mechanisms of drug allergy
Depta et al27 challenged the classical notion that
with E coli but not after infection with other bacteria less
dependent on iron. In the gut the existence of commensal haptens stimulate specific T cells only when they are
bacteria has prompted the question of how the gut controls covalently bound to proteins. The investigators trans-
inflammation, and it has been thought that inflammatory fected a mouse T-cell hybridoma to express a plasmid
bowel disease could be caused by inappropriate recogni- encoding a T-cell receptor (TCR) specific to sulfametox-
tion of antigens. Rakoff-Nahoum et al25 showed that mice azole. When these cells were exposed to the drug in the
deficient in TLRs have increased mortality than wild-type presence of fixed EBV-transformed B cells, they prolif-
mice after receiving dextran sulfate sodium (DSS) as a erated with a reactivity that was dependent on the level of
model for inflammation. These mice were deficient on the specific TCR expression. Increased TCR expression
MYD88, a signal transduction factor essential for sev- also correlated with cross-reactivity with other drugs that
eral TLR-mediated responses. The mice presented with share the sulfanilamide core structure but not with other
epithelial injury and severe colonic bleeding but not sulfonamides, like furosemide or celecoxib. Because fixed
leukocyte infiltration or bacterial overload. Previous B cells were used as APCs, these results showed that
administration of antibiotics did not modify the pathologic drugs can directly interact with TCRs and that there is no
changes. The TLR-deficient mice had increased prolifer- need for antigen processing to obtain T-cell reactivity to
sulfonamides. A study by Nassif et al28 examined the CLINICAL IMMUNOLOGY

phenotypes of lymphocytes obtained from 6 patients with
toxic epidermal necrolysis caused by hypersensitivity to Some key advances in clinical immunology are listed
a single drug, either cotrimoxazole and carbamazepine, in Table II.
tetrazepam, or piroxicam. These lymphocytes were 70%
to 90% CD81 CTLs and were specific for the offending Asthma and the immune response
drug in the cases of cotrimoxazole and carbamazepine but Hanania et al32 asked whether the corticosteroid ther-
not for tetrazepam or piroxicam. The authors believe that apy in patients with asthma affects the immune response to
the lack of specificity for the last 2 drugs can be explained influenza vaccine. Asthmatic subjects (n = 294) who were
by the uncertainty of the offending drug, but overall, the randomized to receive either placebo or inactivated influ-
data support the hypothesis that specific CTLs are respon- enza vaccine were divided in 2 groups, one that received
sible for toxic epidermal necrolysis pathology. medium- or high-dose inhaled corticosteroids and another
that received none or only low corticosteroid doses. The
Applications of the human genome sequence
serologic response to influenza serotype A was not
in immunology impaired with the use of corticosteroids, but the response
Ng et al29 took advantage of the completion of the to serotype B was slightly decreased, with a 2.1-fold
mapping and sequencing of the human genome to support increase of the titers compared with an increase of 2.5-fold
their hypothesis that alternative splicing of self-antigens in the group receiving no steroids or only low-dose
might play a role for the generation of autoantigens in steroids. Although actual protection against influenza
autoimmune disorders. Alternative splicing might disturb infection was not measured, the study places a word of
peripheral tolerance that has already been attained for the caution on the possible decreased immune response in
normal spliced protein. The authors randomly chose 45 asthmatic patients taking steroids.
self-proteins that have been implicated in autoimmunity
and compared their alternative splicing frequency with PIDs
9554 random proteins of the human genome. Forty-two A must-read article is the update in PIDs written by
percent of the random proteins present alternative splicing Notarangelo et al33 representing the International Union of
in contrast to 100% of the 45 proteins implicated in Immunological Societies Primary Immunodeficiency
autoimmunity. Eighty percent of these proteins might Diseases Classification Committee. The authors reviewed
undergo noncanonical alternative splicing, which was and classified PIDs reported up to their last meeting in
much higher than the 1% of randomly selected proteins. 2003. More than 100 PIDs have been defined and char-
More experimental data are needed to confirm this original acterized. Although rare, PIDs are diagnosed with more
hypothesis, which would provide insight into the patho- frequency and in more diverse ethnic groups. However,
genesis of autoimmunity disorders and novel therapy more efforts are needed for PID awareness in minority
development. In an example of high-yield gene search groups, as noted by Cunningham-Rundles et al,34 who
studies, Nakajima et al30 used a gene chip contain- looked in a database of over 120,000 inpatients of a
ing about 22,000 gene probes to compare transcripts general hospital for conditions suggestive of immuno-
expressed in CD41 cells, CD81 cells, basophils, eosino- deficiency. Fifty-nine patients were identified, and 17 of
phils, neutrophils, CD141 cells, and CD191 cells, focus- them had an undiagnosed PID. Eighty-six percent of these
ing on the expression of granulocyte-selective genes for previously undiagnosed patients with PIDs were African
ion channels. The authors found 17 novel transcripts from American or Hispanic.
51 with 5-fold greater expression than other leukocyte It is common to think that the de novo genetic defects of
lineages. Six of these 17 were eosinophil and basophil PIDs should occur during egg fertilization and embryo
specific. The authors reported the list of genes with formation. This idea was challenged by Holzelova et al.35
specific expression and stressed their importance for They investigated patients with the autoimmune lympho-
drug targets in allergic and inflammatory processes and proliferative syndrome but without identified mutations
their significance for drug development in allergic disease in the causative genes Fas, Fas L, Casp8, and Casp10.
and inflammation. Genetic variations influencing allergy The authors cleverly explored the double-negative T cells
were described by Hoffjan et al31 by screening 200 that accumulate in these patients. They identified Fas
children for 61 polymorphisms in 35 immunoregulatory mutations in these cells and subsequently in monocytes
genes. The polymorphisms were analyzed in regard to and CD341 cells, but the mutations were not present in
cytokine production and allergic sensitization, as well as mucosal cells or B cells or when total T cells were tested.
interaction between the polymorphisms. The authors The authors were able to demonstrate these somatic
found 5 associations that involved a reduced IL-13 mutations in 2 of 6 patients studied and showed that the
secretion, including polymorphisms in the genes for survival advantage conferred to a subset of lymphocytes
IL-13, TGF-b, IgE receptor, and nitric oxide. None of was enough to produce autoimmune lymphoproliferative
the genes were associated with atopic dermatitis. The syndrome.
authors concluded that variations in immunoregulatory Chinen and Puck36 reviewed the progress and current
genes might be risk factors for the development of allergic hurdles of gene therapy for PIDs. The French clinical trial
disease and childhood asthma. for X-linked severe combined immunodeficiency has now
AUGUST 2005
TABLE II. Key advances in clinical immunology inducible costimulatory molecule (ICOS) in their cohort
of patients with CVID and found mutations in the ICOS
1. The immune response to influenza vaccine in patients with
moderate-to-severe asthma taking inhaled or oral corticosteroids
gene in 2 of 9 families with autosomal-recessive CVID
is slightly decreased. and no mutations in the ICOS ligand gene. No polymor-
2. Somatic mutations in T-cell progenitors might cause ALPS. phic variants found in the ICOS gene sequence were
3. Four infants with XSCID were successfully treated with gene more common in patients with CVID than in the general
therapy in England. population. An additional 181 patients with sporadic
4. Patients with CVID with granulomatous lung disease, lymphoid CVID were examined, and no mutations were found.
hyperplasia, and lymphoid interstitial pneumonia have poor This report confirms that an ICOS gene defect might cause
survival prognosis. CVID, although it is responsible for only a minority of
5. Some patients with complete DGS might have detectable cases.
numbers of T cells in peripheral blood, although oligoclonal in
A comprehensive review of DGS or chromosome
nature and with poor function.
6. NEMO-deficient patients have increased susceptibility to pyo-
22q11.2 deletion syndrome by Sullivan43 emphasizes
genic and mycobacterial infections. Some of these patients might the spectrum of severity of each of the clinical findings
not have the ectodermal component of this syndrome. of this condition, including T-cell deficiency. A few cases
7. IRD occurs in pediatric HIV infection. might remain undiagnosed until the patient reached
childhood and receives live vaccines before immunologic
ALPS, Autoimmune lymphoproliferative syndrome; XSCID, X-linked severe status is assessed. Moylett et al44 studied a cohort of 53
combined immunodeficiency. 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 impor-
treated 11 infants, with successful T-cell restoration in 9 tant to note that this cohort of patients had only a mild-
of them; the other 2 had only partial reconstitution and to-moderate decrease of T cells. The recommendation on
subsequently received a conventional bone marrow trans- the use of live vaccines in this group of patients is still
plantation.37 In addition, Gaspar et al38 published their controversial, and a cautious approach is advised against
experience in 4 infants with X-linked severe combined live vaccines administration until more data are avail-
immunodeficiency in England who received gene therapy able. Markert et al45 reported an unusual presentation of
and achieved normal T-cell numbers. However, these 5 patients with DGS who had heart, parathyroid, and
successes were tempered with the occurrence of T-cell immune defects. In addition, they presented with rash
leukemia in 3 of the children from the French trial.37 These and lymphadenopathy. Although they had T cells and
malignancies were proved to be caused, at least in part, by some had mitogen proliferative responses, these T cells
insertion of the retroviral vector in oncogenes. New gene were oligoclonal and there was no evidence of thymus
vector designs are being investigated to reduce the risk of activity, as measured by the absence and low output of
cancer caused by insertional mutagenesis. naive cells. The authors concluded that the presence of
Gardulf et al39 reported their experience with the T cells did not necessarily mean the presence of thymus
subcutaneous self-administration of immunoglobulins activity and recommended that patients with DGS should
for patients with PID done at home, suggesting that the undergo an evaluation of thymus activity in addition to the
quality of life of these patients might increase with this assessment of T-cell numbers.
modality. Chinen and Shearer40 reviewed the pros and Two review articles by Etzioni and Ochs46 and Luo
cons of subcutaneous administration of immunoglobulins et al47 described and summarized the recent develop-
for immunodeficiency, which is being established as a ments on HIGM and the biology of the activation-induced
viable alternative to traditional intravenous infusions. cytosine deaminase, one of the proteins that, when miss-
Regarding specific PIDs, several advances have been ing, results in HIGM. Orange et al48 described 7 patients
made in common variable immunodeficiency (CVID), with anhydrotic ectodermal dysplasia with immuno-
hyper-IgM syndrome (HIGM), and DiGeorge syndrome deficiency caused by mutations in the NF-kB essential
(DGS). Bates et al41 investigated the clinical features of modifier (NEMO) gene. They demonstrated a particular
noninfectious pulmonary disease in patients with CVID. susceptibility to pyogenic and mycobacterial infections.
They found that 29 of 69 patients with CVID presented NEMO deficiency was initially described as a form of
with none of these abnormalities, 23 had respiratory HIGM but is currently classified as an innate immunity
symptoms but were radiologically normal, and 18 had defect.33 In a follow-up article, the authors described a
respiratory symptoms and radiologic diffuse abnormali- 16-year-old patient who had a mutation in the Ikb kinase
ties. Within this last group, those who had granulomatous portion of the NEMO gene, and although presenting with
lung disease, follicular bronchiolitis, lymphoid hyperpla- immunologic defects, the patient did not have the ecto-
sia, and lymphoid interstitial pneumonia (13/18 patients) dermal defect components of this condition.49 Niehues
had worse prognosis and survival than the other groups, et al50 reported a similar patient, although with a mutation
with a survival of 13.7 years since the time of diagnosis in exon 2 inducing a premature stop codon. These 2 cases
compared with 28.8 years in the other groups. In addition, underscore the variety of presentations of these rare
these patients also were at higher risk of lymphoprolifer- disorders and the need for continuing awareness for
ative disease. Salzer et al42 investigated the role of diagnosis.
Complement deficiency innate immunity. They are present in mast cells and CD41
Three highly recommended reference articles for the T cells for the recognition of viruses and bacteria, and they
management of complement deficiencies were published are involved in the control of iron metabolism that is
in 2004. One is a comprehensive review for the clinical essential for some bacteria species.
evaluation of complement deficiencies addressed for the Clinical research in immunologic diseases continues to
clinician and containing useful algorithms for the evalu- show remarkable progress. An updated classification for
ation of patients with suspected complement deficiency.51 PIDs is now available, with more than 100 genes identified
This article explains the genetics of these deficiencies, as responsible for these diseases. ICOS is known now to
clinical manifestations, and current therapeutic advances. be responsible for a minority of patients with CVID. With
The second article is a review of the history, genetics, the advances in the field of genetics, a second successful
clinical presentation, and current management of heredi- trial of gene therapy has been published, although 3 cases
tary angioedema caused by deficiency of C1 inhibitor, of leukemia have occurred in patients from the first trial.
written by Frank.52 In an effort to gain consensus in the Other advances were the definition of poor prognosis in
management of hereditary angioedema, an international CVID for patients presenting with specific inflammatory
conference was held in Ontario, Canada, in 2003 with the lung pathology, the estimation of safety for administration
participation of European and American researchers. of live vaccines in patients with partial DGS, the unusual
A summarizing consensus algorithm was drafted and presentation of complete DGS with the presence of host
published.53 Diagnostics and managements available T cells in peripheral blood, the presentation of the clinical
were reviewed, including appropriate use of tranexamic spectrum of patients with NEMO deficiency, the elabora-
acid, androgens, and C1 inhibitor concentrate. tion of a consensus algorithm for the management of
hereditary angioedema, and the description of immune
Immunorestoration syndrome in HIV infection restoration syndrome in HIV-infected children with pre-
Seven cases of immune restoration disease (IRD) were dominance of herpes zoster infections.
described in a cohort of 69 perinatally acquired HIV-
infected children.54 IRD presents as a severe inflammatory
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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
It has become evident that the gastrointestinal tract is

preferentially and profoundly depleted of CD41 T cells during Abbreviations used
acute HIV-1 infection. The enhanced susceptibility of GI: Gastrointestinal
gastrointestinal lymphoid tissue to HIV-1 is in part due to the SIV: Simian immunodeficiency virus
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 emphasized in the study of acute HIV-1 infection. Here we
a significant effect on HIV research in the near future. will summarize the recent findings suggesting the critical
(J Allergy Clin Immunol 2005;116:419-22.) role of the lymphoid system of the GI tract during acute
HIV and SIV infection.
Key words: Acute HIV-1, gastrointestinal tract, CD41 T cells
It has been well established that the GI tract harbors the
majority of the body’s complement of immune cells.1 GI
Acute infection is a critical time in the course of HIV-1 tract lymphocytes, placed in close proximity to the
infection. During this phase, the virus gains access to its external environment, are phenotypically distinct from
target cells, infects, replicates, disseminates, and simulta- peripheral blood lymphocytes; the majority of the intes-
neously establishes a pool of latently infected cells. As tinal lymphocytes (>90%) exhibit a memory phenotype.2
evidenced by the explosive growth of the epidemic in the In addition, because of constant exposure to a myriad of
last 2 decades, it is clear that HIV-1 is extremely adept food and microbial antigens, GI tract lymphocytes are
at accomplishing these tasks. Once established, untreated significantly more activated than peripheral blood lym-
HIV-1 infection culminates in profound immunodefi- phocytes.2 Furthermore, up to 70% of GI tract lympho-
ciency and death in the majority of individuals. Recent cytes express CCR5, a chemokine receptor that serves as
evidence derived from human subjects with acute HIV-1 an essential coreceptor for the entry of CCR5-tropic HIV-1
infection and macaques with acute simian immunode- into CD41 T cells.3 (In contrast, approximately 20% of
ficiency virus (SIV) infection suggests that the course of peripheral blood lymphocytes express CCR5.) Thus the
these infections might be determined during the acute vast population of activated memory CD41 T cells with
phase of infection. Because the virus entering a new host abundant expression of chemokine receptors provides
must negotiate a series of obstacles between entry, HIV-1 with an ideal environment to establish infection.
amplification, and dissemination, it is plausible that in- Unfortunately, the study of the human GI tract during
terventions made during acute HIV-1 infection have the acute HIV-1 infection is challenging. It is fraught with
potential to change the natural history of this disease. To difficulties in identifying individuals during acute infec-
date, much work has focused on understanding these tion and the added complexity of obtaining GI tract biopsy
events and have described changes exclusively within the specimens in the face of psychological and physical
peripheral blood. Until recently, mucosal sites, such as complications associated with acute HIV-1 infection.
the gastrointestinal (GI) tract, have been relatively under- 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
From aAaron Diamond AIDS Research Center and Rockefeller University, infection, at a time when little or no CD41 T-cell depletion
New York, and bBernhard-Nocht Institut fur Tropenmedizin, Hamburg. was evident in the peripheral blood.4,5 Furthermore,
Disclosure of potential conflict of interest: All authors—none disclosed.
intestinal CD41 T-cell depletion occurred regardless of
2005. whether viral inoculum was delivered intrarectally or
Available online July 5, 2005. intravenously.5 These studies were subsequently extended
Reprint requests: Saurabh Mehandru, MD, Aaron Diamond AIDS Research to demonstrate that GI tract lymphocyte depletion occurs
Center, The Rockefeller University, 455 First Ave, 7th Floor, New York, in all stages of SIV infection, including acute infection.4
NY 10016. E-mail: smehandr@adarc.org.
0091-6749/$30.00
Studies conducted during the 1990s indicated that intes-
Ó 2005 American Academy of Allergy, Asthma and Immunology tinal CD41 T-cell depletion might be an early feature of
doi:10.1016/j.jaci.2005.05.040 HIV-1 infection as well6 and that intestinal CD41 T-cell
419
420 Mehandru et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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.
depletion is more significant than depletion of CD41 T T-cell depletion was observed in the GI tract and was
cells in the peripheral blood.7 However, such studies were significantly greater than depletion in the peripheral blood
not conducted in patients identified during acute infection. (Fig 1). Since then, we have extended our studies to
Along with 2 other groups, we have recently demon- 26 subjects with acute and early HIV-1 infection and
strated that during acute HIV-1 infection, a preferential have observed far greater CD41 T-cell depletion in the
and profound depletion of CD41 T cells occurs within the intestines compared with in the peripheral blood (unpub-
GI tract.8-10 In the first of these studies, Guadalupe et al8 lished data). Having demonstrated a striking CD41 T-cell
described 2 individuals with acute HIV-1 infection in depletion within the GI tract, we have examined the
which significant CD41 T-cell depletion occurred within subsets of CD41 T cells in which this depletion was most
approximately 4 to 6 weeks of infection. Brenchley et al10 prominent. Because a majority of intestinal CD41 T cells
studied one individual with acute HIV-1 infection (in- express CCR53 and given that viruses during the early
fected for <1 month) and 4 individuals with early HIV-1 stages of HIV-1 infection are predominantly CCR5-
infection (duration of infection was 4-9 months). In all tropic,11 it was not surprising that Brenchley et al10 and
5 subjects, a preferential CD41 T-cell depletion was we9 both observed that GI tract lymphocyte depletion was
noted in the intestines. In fact, significant GI tract CD41 most significant among the CCR51 subsets of CD41 T
T-cell depletion was characteristic of all stages of HIV-1 cells. In addition, we have observed marked CD41 T-cell
infection.9,10 depletion in the effector sites (lamina propria) of the GI
Our initial focus was to describe changes within the GI tract, with relative sparing of the inductive sites (organized
tract of individuals with acute and early HIV-1 infection. lymphoid tissue). In contrast, however, HIV-1 RNA was
To this end, we studied 13 individuals identified during localized to the inductive sites. We believe that this
acute (n = 7) and early (n = 6) infection. Mean CD41 T- discrepancy is best explained by loss of target cells in
cell percentage in the GI tract was 15.7% 6 3.6% the effector compartment. We hypothesize that if we could
compared with a mean of 42.3% 6 14.7% in the blood examine a subject within the first 7 to 10 days of infection,
(P < .001). Thus in all of our subjects, profound CD41 viral RNA would be evident in the effector compartment
J ALLERGY CLIN IMMUNOL Mehandru et al 421
as well before the annihilation of the resident CD41 cells. subjects and SIV-infected macaques suggest an emerging
In a small cross-sectional cohort, we have observed that in model for the pathogenesis of HIV-1 infection. The large
contrast to the peripheral blood, restoration of CD41 T complement of resident memory cells in mucosal surfaces
cells in the GI compartment with highly active antiretro- get preferentially infected and fuel subsequent rounds
viral therapy is incomplete. We are currently examining of replication and infection. Therefore in acute stages
patients longitudinally to determine whether the timing of mucosal sites appear to propel the infection forward.
onset of antiretroviral therapy is associated with better GI What are the implications of these findings? A number
tract CD41 T-cell reconstitution, as has been suggested of significant issues emerge from these studies:
by Guadalupe et al8 in a limited cohort comprised of 2
1. By demonstrating that significant viral replication and
subjects.
immune depletion occurs at mucosal sites during
Recent data obtained from the SIV macaque model
acute infection, these findings provide compelling
provide startling insights into the degree of GI tract
evidence to the argument that mucosal sites should
CD41 T-cell infection and depletion as well. Studies
be the focus of further examination and should be
by Mattapallil et al12 and Li et al13 demonstrate rapid
considered in the monitoring of patients on therapy.
infection and destruction of GI tract memory CD41 T cells
2. These findings shatter the dogma that during acute
within days of infection with SIV. The study by Mattapallil
HIV-1 infection, there is little CD41 T-cell depletion
et al12 suggests that at peak viremia (day 10), as many as
in the body.
60% of GI tract memory CD41 T cells might be infected
3. These findings reinvigorate the debate regarding the
with SIV and that these cells are lost within 14 days of
timing of therapy in HIV-1 infection. Guidelines
infection. This results in profound immunodeficiency
recommending that treatment need not be initiated
that begins within days of infection, not months to years
until CD41 T-cell count decreases to less than 350
as was previously thought. The authors put forth the notion
cells/mm3 or until plasma viral load is more than
that memory CD41 T cells are killed by means of direct,
100,000 copies/mL14 should take into account these
virus-mediated destruction rather than bystander effects or
recent data demonstrating severe mucosal CD41
suppression of CD41 T-cell production.
T-cell depletion in acute and early HIV-1 infection.
Li et al13 showed, somewhat surprisingly, that GI tract
This said, however, it must be mentioned that the
cells infected initially with SIV have a nonactivated
clinical significance of mucosal immune depletion
(CD692CD252Ki672) phenotype. Peak infection of
remains uncertain. Redundancy in our immune system
memory CD41 T cells within the GI tract corresponded
might prevent long-term consequences, yet the pos-
to peak viremia, and depletion of GI tract CD41 T cells
sibility of early immune senescence exists, and the
coincided with a decrease in the peripheral viral load. Li
long-term consequences are not clear.
et al therefore suggested the concept of ‘‘substrate de-
4. These findings suggest a potential for the use of im-
pletion,’’ resulting in viral load reduction in the host. In
munomodulators, such as cyclosporine, during acute
contrast to Mattapallil et al,12 Li et al13 propose that CD41
HIV-1 infection with the goal of curtailing successive
T-cell depletion is caused by virus-triggered, Fas–Fas
rounds of viral infection and CD41 T-cell depletion
ligand–mediated apoptosis. It is likely that CD41 T-cell
within the GI tract.
destruction is multifactorial, caused by virus-induced
5. With regard to preventive efforts directed against
cytolysis, apoptosis, and the host’s own cytotoxic T-
HIV-1, recent data underscore the need to develop
lymphocyte, natural killer cell responses. Further work
strategies to protect mucosal surfaces from infection.
needs to be done to resolve this issue.
The use of microbicides and CCR5 blockers would
Concurrent studies in our laboratory have focused on
represent such interventions.
determining virologic and immunologic correlates of GI
6. Finally and perhaps most importantly, these findings
tract CD41 T-cell depletion. Our data (unpublished)
reemphasize the fact that mucosal immune responses
suggest that acute HIV-1 infection results in immunologic
must be targeted in the development of effective
activation and proliferation of GI tract CD41 T cells, Basic and clinical immunology
HIV-1 vaccines.
creating a local niche of viral replication. We have
observed a highly significant difference between the level The weight of current evidence places mucosal lym-
of CD41 T-cell infection in the GI tract and peripheral phoid tissue as pivotal in HIV-1 pathogenesis. These
blood. In addition, we have observed a striking difference findings are likely to have a significant effect on HIV
in HIV-1 viral RNA production within CD41 T cells research in the near future.
derived from the GI tract compared with from the periph-
eral blood. Thus we hypothesize that during acute infec-
tion, HIV-1 encounters a vast population of susceptible REFERENCES
cells within the GI tract and preferentially infects them. 1. Mowat AM. Anatomical basis of tolerance and immunity to intestinal
Viral infection results in immune activation and CD41 antigens. Nat Rev Immunol 2003;3:331-41.
T-cell proliferation, both of which augment viral produc- 2. Schieferdecker HL, Ullrich R, Hirseland H, Zeitz M. T cell differenti-
ation antigens on lymphocytes in the human intestinal lamina propria.
tion, setting up the next round of infection. The end re- J Immunol 1992;149:2816-22.
sult is profound CD41 T-cell depletion within the GI 3. Anton PA, Elliott J, Poles MA, McGowan IM, Matud J, Hultin LE, et al.
tract. Combined, the results from recent studies in human Enhanced levels of functional HIV-1 co-receptors on human mucosal
422 Mehandru et al J ALLERGY CLIN IMMUNOL
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T cells demonstrated using intestinal biopsy tissue. AIDS 2000;14: delay in restoration following highly active antiretroviral therapy. J Virol
1761-5. 2003;77:11708-17.
4. Smit-McBride Z, Mattapallil JJ, McChesney M, Ferrick D, Dandekar S. 9. Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan
Gastrointestinal T lymphocytes retain high potential for cytokine C, et al. Primary HIV-1 infection is associated with preferential depletion
responses but have severe CD4(1) T-cell depletion at all stages of of CD41 T lymphocytes from effector sites in the gastrointestinal tract.
simian immunodeficiency virus infection compared to peripheral lym- J Exp Med 2004;200:761-70.
phocytes. J Virol 1998;72:6646-56. 10. Brenchley JM, Schacker TW, Ruff LE, Price DA, Taylor JH, Beilman
5. Veazey RS, DeMaria M, Chalifoux LV, Shvetz DE, Pauley DR, Knight GJ, et al. CD41 T cell depletion during all stages of HIV disease occurs
HL, et al. Gastrointestinal tract as a major site of CD41 T cell depletion predominantly in the gastrointestinal tract. J Exp Med 2004;200:749-59.
and viral replication in SIV infection. Science 1998;280:427-31. 11. Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR. Change
6. Lim SG, Condez A, Lee CA, Johnson MA, Elia C, Poulter LW. Loss in coreceptor use coreceptor use correlates with disease progression in
of mucosal CD4 lymphocytes is an early feature of HIV infection. Clin HIV-1–infected individuals. J Exp Med 1997;185:621-8.
Exp Immunol 1993;92:448-54. 12. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M.
7. Schneider T, Jahn HU, Schmidt W, Riecken EO, Zeitz M, Ullrich R. Massive infection and loss of memory CD41 T cells in multiple tissues
Loss of CD4 T lymphocytes in patients infected with human immuno- during acute SIV infection. Nature 2005;434:1093-7.
deficiency virus type 1 is more pronounced in the duodenal mucosa than 13. Li Q, Duan L, Estes JD, Ma ZM, Rourke T, Wang Y, et al. Peak SIV
in the peripheral blood. Berlin Diarrhea/Wasting Syndrome Study replication in resting memory CD41 T cells depletes gut lamina propria
Group. Gut 1995;37:524-9. CD41 T cells. Nature 2005;434:1148-52.
8. Guadalupe M, Reay E, Sankaran S, Prindiville T, Flamm J, McNeil A, 14. Guidelines for the use of antiretroviral agents in HIV-infected adults
et al. Severe CD41 T-cell depletion in gut lymphoid tissue during and adolescents. Washington (DC): Department of health and Human
primary human immunodeficiency virus type 1 infection and substantial Services; 2005.
Editorial
Are you immunodeficient?
Francisco A. Bonilla, MD, PhD, and Raif S. Geha, MD Boston, Mass
Key words: Clinical immunology, primary immunodeficiency, in-

fectious diseases, genetics Abbreviation used
PI: Primary immunodeficiency
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 organism, the site of infection (localized vs disseminated),
to achieve immune function to provide efficient, self- the infection’s severity (degree of tissue or organ damage),
limited host defense against the biotic and abiotic the infection’s persistence or resistance to therapy, and the
environment while preserving tolerance to self.’’ The frequency of relapse or reinfection. In spite of the com-
challenge to practitioners is to translate this axiom into monality of these considerations early in the approach to
principles that answer specific clinical and academic the potentially immunodeficient patient, there are very few
needs. One essential message of Casanova et al is to data or agreement on where to best draw the dividing line
consider the susceptibility to infection limited to one or a between normal and abnormal along any of these dimen-
few pathogens and having Mendelian inheritance to be sions. It might also be important to consider that this line
within the spectrum of PI, regardless of the immunologic can be drawn differently under circumstances that differ
phenotype. They further argue that in light of this, with respect to, for example, the level of public hygiene,
academic and clinical needs will best be met by a the prevalence of particular pathogens, or the availability
classification system on the basis of clinical phenotype, of vaccinations. (Casanova et al,1 in fact, consider these
in contrast to the classic, or perhaps traditional, system on factors as masking the true prevalence of PI.) In addition,
the basis of immunologic phenotype.2,3 According to their Casanova et al consider the importance of Mendelian
scheme, Casanova et al1 distinguish conventional and (single gene) inheritance in a clinical definition. Perhaps
unconventional immunodeficiencies as those that do or this element could be generalized to any definable genetic
do not have clearly defined immunologic phenotypes, component to include interactions among mutations,
respectively. The authors consider these to be end points polymorphisms, or both that might determine a pheno-
of a spectrum rather than dichotomous. They further state type, as has been observed in some PI diseases.4 Whether
there has ‘‘never been a fully satisfactory classification of one is prepared to alter one’s conceptualization of immune
PID.’’ However, it is worth discussing whether any single deficiency to include unconventional forms, the matter of
system on the basis of clinical phenotype or immunologic definition requires further study from all sides (epidemi-
phenotype could ever be so. ology, immunology, and genetics) to provide a more solid
Regardless of how we might classify their diseases, framework for further discussion. Ultimately, the issue of
immunodeficient patients come to clinical attention pre- where to draw the line between normal and abnormal is
dominantly as a result of a predisposition to infection. This critical if the search for ‘‘currently unknown Mendelian
predisposition is manifested in one or more of the clinical primary immunodeficiencies’’1 is to set out with hope for
dimensions of infection: the inherent virulence of the meaningful discovery.
The criteria of normalcy in a system based on immu-
nologic phenotype relate to population distributions of
From the Division of Immunology, Children’s Hospital, and the Department
of Pediatrics, Harvard Medical School, Boston, Mass.
screening laboratory studies of immune function (eg,
Disclosure of potential conflict of interest: F. A. Bonilla has consultant serum immunoglobulin levels and specific antibody titers,
arrangements with Talecris Biotherapeutics and is on the speakers’ bureau peripheral blood lymphocyte subpopulations, in vivo or
for Accredo Therapeutics. R. Geha has no conflicts of interest to disclose. in vitro measures of T-cell function, assays of phagocyte
oxidative burst or adhesion, and complement function
2005.
Available online July 5, 2005. or serum component levels).2 This system affords con-
Reprint requests: Francisco A. Bonilla, MD, PhD, Children’s Hospital, venient statistical labels for normal and abnormal.
Immunology, Enders 809, 300 Longwood Ave, Boston, MA 02115. Unfortunately, the biologic and clinical correlates are
E-mail: francisco.bonilla@childrens.harvard.edu. also largely lacking here, as in the discussion of predis-
0091-6749/$30.00
position to infection above. This sometimes leads to an
Ó 2005 American Academy of Allergy, Asthma and Immunology important point raised by Casanova et al1 that certainly
doi:10.1016/j.jaci.2005.05.026 bears repeating: ‘‘patients with specific clinical infectious
423
424 Bonilla and Geha J ALLERGY CLIN IMMUNOL
AUGUST 2005
diseases but no overt immunological phenotype [are] TABLE I. Clinical-academic considerations or questions
being largely neglected.’’ in PI diseases and potential classification schemes to
However, this is not precisely a failure of a system or address them
classification based on immunologic phenotype, as much Biomedical aspects Basis for
as it reflects 3 distinct elements. First, one might choose of immunodeficiency classification of PI
simply not to categorize a limited infection predisposition
Infecting microbe(s) Microbial taxonomy
as a PI, one of the main points of Casanova et al1 (ie, if it is
Host with genetic Genetic catalog, mode
definable genetically, then we should). Second, we must lesion(s)-polymorphism(s) of inheritance
consider further what is meant by immunologic pheno- Altered immunopathogenesis Biochemical and cell
type. Casanova et al use the adjective ‘‘overt’’ to denote an of infection biologic mechanisms
abnormality detectable by the battery of screening tests Immune dysregulation Biochemical and cell
listed above. However, at some level, all genetically de- (atopy, autoimmunity, biologic mechanisms
finable PIs must have some immunologic phenotype; it is lymphoproliferation, malignancy)
the clinical phenotype that dictates the specific eval- Clinical syndrome of All clinical features
uation that will ultimately identify it (therefore we can immunodeficiency of the disease
agree on the value of the clinical classification here). And Diagnostic evaluation Immunologic phenotype
Therapy Anti-infective, immune
finally, it is clear that we have incomplete knowledge
reconstitution, response
regarding how the organism as a whole (including the to therapy
immune system) protects itself from infection. Only in Outcome Natural history, prognosis
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 it is truly ‘‘the least efficient physiological system at the
immune mechanisms. individual level’’1 and whether we might discover that a
In the final analysis, the matter of classification might large fraction of human subjects are immunodeficient
truly be secondary. Consider Table I, which shows a very depends on one’s perspective, as we have discussed, not-
simple scheme outlining the biologic and clinical elements withstanding the benefits of public hygiene, immuniza-
of PI and the classification systems that might be proposed tion, and antibiotics. This is apparent if one shifts focus
in response to or motivated by these different aspects of from the anthropocentric view and remembers that human
the interaction of a pathogen with a susceptible host. No pathogens and human beings evolve together.
system is superior in all cases, and some might even be At the bedside, the clinical immunologist’s interest
frankly cumbersome in some situations, but there is no is piqued by the constellation of history, symptoms, and
single system that optimally serves all needs. findings. Some version of the clinical classification of
We all work together toward the determination of which Casanova et al1 speak is foremost in our minds
complete sets or elements of a PI knowledge base, such while we determine the most efficient path toward
as the complete set of gene alterations that lead to defining the immunologic phenotype, making a definitive
susceptibility to infection, the complete set of clinical diagnosis, or both. Experience informs us that our ability
phenotypes of PIs, and the complete set of immunologic to define the immunologic phenotype with precision
phenotypes. These will not be conveniently ordered along depends utterly on the sophistication of the laboratory
1 or 2 dimensions that will be useful in every situation, methods available for study of the individual patient. We
as outlined above. What might serve best is a multiseg- note in passing that recent advances in molecular methods
mented database in which each segment orders the infor- can, in some instances, divorce the processes of defining
mation according to a distinct classification system and the immunologic phenotype from making a diagnosis in
every entry is linked to its entries in the other segments. the case of a PI that has already been defined at the
This type of organization is legion on the Internet (see, molecular level. For example, a 15-month-old boy pre-
for example, the National Center for Biotechnology sents with severe recurrent respiratory tract infections with
Information of the National Institutes of Health, Bethesda, encapsulated bacteria. We sequence his BTK gene and find
Maryland, at http://www.ncbi.nih.gov/, and the Institute a mutation or deletion consistent with X-linked agamma-
for Medical Technology Bioinformatics Group of the globulinemia. We have established a diagnosis without
University of Tampere, Tampere, Finland, at http:// knowing whether he is agammaglobulinemic or B lym-
bioinf.uta.fi/). phopenic. We do not advocate such an approach, however,
The adaptive immune system might be dispensable for because it perpetuates or even creates critical gaps in our
human development and survival in a germ-free environ- knowledge base.
ment.8 On the other hand, elements of innate immunity As we stated, the example applies only where the
interact with commensal flora and are required for normal molecular defect is known. The situation is different
function of some systems.9 In light of the interrelations of for the patient with a less well-understood form of PI.
immunity with other organ systems, the boundaries of the The level of laboratory sophistication required for the
immune system, as a whole, become less distinct. Whether definition of new forms of PI is an order of magnitude
J ALLERGY CLIN IMMUNOL Bonilla and Geha 425
beyond what suffices for diagnosis of known entities. 4. Foster CB, Lehrnbecher T, Mol F, Steinberg SM, Venzon DJ, Walsh TJ,
et al. Host defense molecule polymorphisms influence the risk for
Technologic advances might soon provide us with the
immune-mediated complications in chronic granulomatous disease.
ability to automate functional studies of pathogen-specific J Clin Invest 1998;102:2146-55.
immune responses and to link these to genomics-derived 5. Orange JS. Human natural killer cell deficiencies and susceptibility to
strategies to identify loci for study.10 Wherever they exist, infection. Microbes Infect 2002;4:1545-58.
these resources must be made available in some way to the 6. Orange JS, Levy O, Brodeur SR, Krzewski K, Roy RM, Niemela JE,
et al. Human nuclear factor kappa B essential modulator mutation can
larger community of clinical immunologists. The impor- result in immunodeficiency without ectodermal dysplasia. J Allergy Clin
tance of this cannot be overstated. We agree with Immunol 2004;114:650-6.
Casanova et al1 that the full spectrum of human suscep- 7. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, et al.
tibility to infection is largely waiting to be discovered. For Pyogenic bacterial infections in humans with IRAK-4 deficiency.
Science 2003;299:2076-9.
those with PI and those who study it, hope derives from a
8. Guerra IC, Shearer WT. Environmental control in management of
chance encounter with ‘‘a prepared mind,’’ timely recog- immunodeficient patients: experience with ‘‘David’’. Clin Immunol
nition, and the technology to repair it.11,12 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
REFERENCES intestinal homeostasis. Cell 2004;118:229-41.
1. Casanova J-L, Fieschi C, Bustamante J, Reichenbach J, Remus N, 10. Tian Q, Stepaniants SB, Mao M, Weng L, Feetham MC, Doyle MJ, et al.
von Bernuth H, et al. From idiopathic infectious diseases to novel primary Integrated genomic and proteomic analyses of gene expression in
immunodeficiencies. J Allergy Clin Immunol 2005;116:426-30. Mammalian cells. Mol Cell Proteomics 2004;3:960-9.
2. Bonilla FA, Bernstein IL, Khan DA, Ballas ZK, Chinen J, Frank MM, et al. 11. Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, et al.
Practice Parameter for the diagnosis and management of primary immu- Correction of ADA-SCID by stem cell gene therapy combined with
nodeficiency. Ann Allergy Asthma Immunol 2005;94(suppl):S1-63. nonmyeloablative conditioning. Science 2002;296:2410-3.
3. Notarangelo L, Casanova JL, Fischer A, Puck J, Rosen F, Seger R, et al. 12. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E,
Primary immunodeficiency diseases: an update. J Allergy Clin Immunol Nusbaum P, et al. Gene therapy of human severe combined immuno-
2004;114:677-87. deficiency (SCID)-X1 disease. Science 2000;288:669-72.

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 Créteil, France, and Frankfurt, Germany
Primary immunodeficiencies are typically seen as rare An immunologic definition and classification of pri-
monogenic conditions associated with detectable immunologic mary immunodeficiencies currently prevails and is ex-
abnormalities, resulting in a broad susceptibility to multiple pected to do so for the foreseeable future.1 Unfortunately,
and recurrent infections caused by weakly pathogenic and this has resulted in studies of otherwise healthy patients
more virulent microorganisms. By opposition to these
with specific infectious clinical diseases but no overt
conventional primary immunodeficiencies, we describe
nonconventional primary immunodeficiencies as Mendelian immunologic phenotype being largely neglected. The
conditions manifesting in otherwise healthy patients as a attention of most investigators and clinicians has remained
narrow susceptibility to infections, recurrent or otherwise, focused on the tip of the iceberg: those rare patients with a
caused by weakly pathogenic or more virulent microbes. noisy clinical phenotype (multiple, recurrent, and severe
Conventional primary immunodeficiencies are suspected on the infections) and a visible immunologic phenotype (defining
basis of a rare, striking, clinical phenotype and are defined on the primary immunodeficiency). The most striking exam-
the basis of an overt immunologic phenotype, often leading to ple of such conventional primary immunodeficiencies is
identification of the disease-causing gene. Nonconventional reticular dysgenesia, an exceedingly rare disorder associ-
primary immunodeficiencies are defined on the basis of a more ated with agranulocytosis and alymphocytosis, resulting in
common and less marked clinical phenotype, which remains
early-onset vulnerability to virtually all microorganisms
isolated until molecular cloning of the causal gene reveals a
hitherto undetected immunologic phenotype. Similar concepts and a rapidly fatal outcome in the absence of hematopoietic
can be applied to primary immunodeficiencies presenting other stem cell transplantation.2 Immunodeficiency is com-
clinical features, such as allergy and autoimmunity. monly ruled out in patients with a single severe infectious
Nonconventional primary immunodeficiencies thus expand disease (even if recurrent or life-threatening) and normal
the clinical boundaries of this group of inherited disorders routine immunologic workup (searching for signs of
considerably, suggesting that Mendelian primary inherited or acquired immunodeficiency). Self-contradic-
immunodeficiencies are more common in the general tory titles in the medical literature, such as ‘‘Fatal infection
population than previously thought and might affect children in an immunocompetent individual,’’ remain common.
with a single infectious, allergic, or autoimmune disease. Unusual infectious diseases are often described as idio-
(J Allergy Clin Immunol 2005;116:426-30.)
pathic, demonstrating caution and a desire to avoid the
direct incrimination of the patient’s genetic background.
Key words: Primary immunodeficiency, infectious diseases, idio-
pathic infections, inborn errors, Mendelian disorders, predisposi- However, some infections typically caused by weakly
tion to infection virulent (opportunist) microbes have been found to
be associated with a high frequency of familial forms,
parental consanguinity, or both, suggesting Mendelian
From aLaboratoire de Génétique Humaine des Maladies Infectieuses,
Université de Paris René Descartes-INSERM U550, Faculté de Médecine
predisposition. This group of nonconventional primary
immunodeficiencies is characterized by a very narrow
Necker, Paris; bUnité d’Immunologie et d’Hématologie Pédiatriques,

Hôpital Necker Enfants Malades, Paris; cService d’Immunologie spectrum of opportunistic infections limited to one micro-
Clinique, Hôpital Saint Louis, Paris; dKlinik für Kinderheilkunde, bial genus or species possibly, but not necessarily, recur-
Klinikum der J.W. Goethe Universität, Frankfurt; and eService de
rent in otherwise healthy patients with no detectable
Pédiatrie, Centre Hospitalier Intercommunal de Créteil, Créteil.
Our laboratory is supported in part by grants from the BNP-Paribas and immunologic abnormality on initial investigation.3 These
Schlumberger foundations, the Institut Universitaire de France, and the EU diseases do not fit easily into the classical classification of
grant QLK2-CT-2002-00846. primary immunodeficiencies.
Nonconventional primary immunodeficiencies include
for publication March 30, 2005.
Available online July 5, 2005. the syndromes of Mendelian susceptibility to mycobacte-
Reprint requests: Jean-Laurent Casanova, MD, PhD, Laboratoire de Génétique rial diseases (OMIM 209950,4 first described in 1951) in
Humaine des Maladies Infectieuses, Université de Paris René patients with mutations in the IL-12/23–IFN-g circuit
Descartes-INSERM U550, Faculté de Médecine Necker, Paris 75015, (first identified in 1996)5-8; recurrent invasive disease
France, EU. E-mail: casanova@necker.fr.
0091-6749/$30.00
caused by Neisseiria species in patients with mutations
Ó 2005 American Academy of Allergy, Asthma and Immunology affecting the terminal components of complement (C5 to
doi:10.1016/j.jaci.2005.03.053 C9) forming the membrane attack complex (first described
426
J ALLERGY CLIN IMMUNOL Casanova et al 427
in 1974)9,10; isolated chronic mucocutaneous candidiasis weakly virulent mycobacteria.20,27 Currently unexplained
(OMIM 114580, first described in 1969), which remains candidate infectious diseases include invasive pneumo-
unexplained genetically11,12; epidermodysplasia verruci- coccal disease18,19 and herpes simplex encephalitis,28,29
formis with disseminated warts caused by human papil- which have been diagnosed in at least a few patients with
lomaviruses belonging to group B1 (OMIM 226400, first conventional immunodeficiencies. Many life-threatening
described clinically in 1922 and subsequently shown to be infectious diseases might well turn out to result from the
a Mendelian trait [1939] conferring susceptibility to Mendelian inheritance of a specific predisposition, reflect-
papillomaviruses [1946-1966]) in patients with mutations ing a nonconventional primary immunodeficiency.
in EVER1 and EVER2 (first described in 2002)13,14; and Nonconventional primary immunodeficiencies are de-
X-linked lymphoproliferative syndrome caused by fined on clinical grounds, raising the issue of the classifi-
Epstein-Barr virus (OMIM 308240, first described in cation of primary immunodeficiencies. In fact, there has
1975) in patients with mutations in SAP (first described never been a fully satisfactory classification of primary
in 1998).15,16 Needless to say, the dichotomy between immunodeficiencies.1,30,31 This problem has become in-
conventional and nonconventional conditions is some- creasingly acute because of the explosion of knowledge
what artificial because there is really a continuum between in the field in the last 20 years, with at least 200 conditions
these 2 extremes.17 Patients with a recently described described clinically and more than 100 disease-causing
conventional primary immunodeficiency, IL-1 receptor- genes identified. Moreover, many more conventional and
associated kinase 4 deficiency, are particularly susceptible nonconventional primary immunodeficiencies are likely
to Streptococcus pneumoniae,18,19 and conversely, pa- to be identified in the near future. A genetic classification
tients with mutations in the IL-12–IFN-g axis are also was impossible in the early days before identification of the
susceptible to Salmonella species.20 In any event, neither disease-causing genes. Even today, genetic classification
the identification of a cellular phenotype nor that of the would be hindered by the lack of a well-defined temporal
causal gene suggested the existence of an underlying and spatial expression pattern for the disease-causing
primary immunodeficiency in patients with nonconven- genes, limiting our understanding of pathogenesis.
tional primary immunodeficiencies. Instead, primary im- Furthermore, different clinical syndromes might be caused
munodeficiency diagnosis was based on the relatively low by different mutations in the same gene, and the same
virulence of the microbe and the seemingly Mendelian syndrome might be caused by different genetic causes.
inheritance of predisposition to severe disease. Even if it were possible, a genetic classification would not
It would not be wise to limit the group of nonconven- actually be sufficient because phenotypes are obviously
tional primary immunodeficiencies to these 5 Mendelian more important than genotypes; the chief value of a
syndromes, to patients presenting unexplained infections genotype lies in its ability to account for a given pheno-
caused by weakly virulent opportunistic microorganisms, type. Accordingly, the McKusick catalog of human
or even to patients with recurrent infections caused by genetic disorders is merely a catalog and not a classifica-
more virulent pathogens. There is good reason to believe tion.4 Any classification system for academic and clinical
that other human conditions reflect currently unknown purposes must therefore be primarily phenotypic, although
Mendelian primary immunodeficiencies. First, genetic improvements in our understanding of the genetic basis
epidemiologic studies searching for familial forms and of primary immunodeficiencies might lead to changes
parental consanguinity have not been carried out for most in phenotypic classification. The phenotypic definition of
infectious, autoimmune, and allergic clinical syndromes. primary immunodeficiencies is clearly the necessary
Second, neither the absence of familial cases nor the starting point for phenotypic classification.
lack of consanguinity are sufficient to exclude Mendelian Historically, the identification of agammaglobulinemia
defects, and sporadic cases might reflect a genetic lesion, in 1952 by Ogden Bruton32 and the subsequent discovery
as illustrated by the first genetic lesion discovered in that its inheritance was X-linked and recessive33 was the
human subjects, trisomy 21, in patients with Down origin of current classifications of primary immunodefi-
syndrome.21 Third, the virulence of microorganisms is ciencies on the basis of a combination of immunologic Basic and clinical immunology
also a continuum, and many pathogenic microbes, such phenotypes and modes of inheritance.1,30,31,34-39 Primary
as Mycobacterium tuberculosis, are actually innocuous in immunodeficiencies are commonly classified into disor-
most human beings. A number of common infectious ders of T cells, B cells, phagocytes, and complement.
diseases are likely to reflect nonconventional primary They are then further classified according to the mode
immunodeficiencies in at least a fraction of patients. of inheritance and, when known, genetic cause. This
Consistent with this view, mycobacterial diseases caused method of classification poses a serious problem of de-
by weakly virulent BCG species were described as finition because it tightly links the concept of primary im-
idiopathic infections before the identification of defects munodeficiency with the observation of an immunologic
in the IL-12–IFN-g circuit.22,23 The identification of these phenotype. According to this view, even asymptomatic
defects has led to the recent description of 3 unrelated IgA-deficient individuals are immunodeficient, unlike,
families with a purely Mendelian form of predisposition to paradoxically, patients dying of infectious disease with-
bona fide tuberculosis,24-26 following on from the obser- out immunologic abnormality. Moreover, because many
vation that IL-12Rb1 deficiency had low penetrance for disease-causing genes are expressed in different cell types
the case-definition phenotype of clinical disease caused by in which mutant alleles might have different effects,
428 Casanova et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
clinical phenotypes, whether infectious, allergic, or auto- not operational yet because it awaits investigators in the
immune, are far from being consistent within each of the field to review all pathogens one by one, perhaps in a
4 cell type–based groups. What is the clinical similarity collaborative effort (eg, primary immunodeficiencies as-
between defects of C1 inhibitor and C9? Conversely, sociated with, for example, Pneumocystis species infection
X-linked agammaglobulinemia (primarily, but not exclu- or Toxoplasma species infection). This classification will
sively, a B-cell defect) clinically resembles HLA class I address the most relevant clinical question at the bedside
deficiency (often improperly classified as a T-cell defect). (Which immunodeficiency should the physician consider
This situation also results in paradoxical classifications, in a given infected patient?) and the most relevant immu-
with an immunologic phenotype attributed to one cell type nologic question at the bench (What role does a particular
but a genetic defect actually affecting another cell type. molecule play in immunity to infection in vivo?). Of
For example, CD40L deficiency is generally described as course, the genotype and immunologic phenotype are
a B-cell defect because of the hyper-IgM syndrome,40 but invaluable both clinically to tailor treatment options to
CD40L is expressed on T cells and is involved in the individual patients and immunologically to decipher the
interaction of T cells with both B cells and macrophages– molecular basis of immune responses.
dendritic cells. The focus on certain immunologic pheno- A corollary of this purely clinical definition and clas-
types, such as hyper-IgM syndrome, is misleading in sification of primary immunodeficiencies is that inborn
itself: there are more differences than common points Mendelian deficiencies of immunity are more common
between patients with mutations in CD40L (expressed than initially thought. Accordingly, newly described pri-
on T cells), AID (expressed in B cells), and NEMO mary immunodeficiencies, such as partial IFN-gR1 and
(expressed ubiquitously), all of which can result in hyper- signal transducer and activator of transcription 1 defi-
IgM syndrome. The current immunologic classification ciencies, have been shown to be transmitted as autosomal
of primary immunodeficiencies is thus imperfect both dominant traits in multiplex families, at odds with the
immunologically and clinically. classical view that primary immunodeficiencies are nec-
An ideal definition and classification of primary immu- essarily recessive traits because of their severity.43 Not all
nodeficiencies and inborn deficiencies should evidently severe infectious diseases will be found to reflect a
rely on clinical phenotype because this best reflects the Mendelian primary immunodeficiency or to be due to
physiologic effect of any deleterious genotype. Indeed, the inheritance of a major susceptibility gene, as seen in
immunodeficiencies in general, whether inherited or ac- leprosy with mutations in Parkin,44-46 because predispo-
quired, should be defined clinically, as opposed to immu- sition to infection might display truly polygenic determi-
nologically. Would anyone seriously suggest that it would nism. Nevertheless, it will be important in the future to
be better to define respiratory failure in terms of epithelial decipher the Mendelian genetic basis of infectious dis-
abnormalities rather than the physiologic consequences eases. Studies of autoimmune and allergic syndromes are
of insufficient oxygen inhalation? It is certainly useful to also likely to reveal novel Mendelian disorders. Once a
assess various parameters in the course of any organ clinical definition of immunodeficiency is accepted, pa-
failure, but the definition and monitoring of organ failure tients with infectious diseases, allergy, or autoimmunity
must be physiologic. Immunodeficiency is a failure to (in the broad sense of these terms, including angioedema,
achieve immune function to provide efficient, self-limited hemophagocytosis, and autoinflammation) should be
host defense against the biotic and abiotic environment considered as potential bearers of Mendelian primary
while preserving tolerance to self. Immunodeficiencies are immunodeficiencies. Accordingly, several primary im-
thus best defined in terms of the diverse forms of life- munodeficiencies were recently shown to present purely
threatening infections, allergies, or autoimmune reactions. as autoimmune,47 autoinflammatory,48 and hemophago-
The detection of an identifiable immunologic abnormality cytosis49 syndromes. Intriguingly, only one Mendelian
is less important and depends on the tools available to the disorder, C1 inhibitor deficiency, has thus far been found
investigator. Immunodeficiencies might also be consid- to be purely associated with autosomal-dominant angio-
ered in terms of whether they are inherited or acquired. edema, a syndrome related to (but possibly different from)
Most immunodeficiencies are actually idiosyncratic, re- allergy.50 Noninfectious immunologic diseases have only
flecting both nature (genetic background) and nurture (the recently emerged as a public health problem and do not
effect of the environment on the host). An ideal classifi- threaten mankind as acutely as infections. Records show
cation of primary immunodeficiencies should take this that life expectancy in Western Europe in the 18th century
into account, considering infectious syndromes one by one was about 25 years, whereas life expectancy is currently
(and possibly autoimmune and allergic syndromes as about 40 years in Sub-Saharan Africa, largely because
well). For example, primary immunodeficiencies associ- of the burden of infection.17,51 The current longer life
ated with mycobacterial41 or pneumococcal42 diseases are expectancy in developed countries primarily reflects
specifically associated with defects of interaction between recent developments in hygiene (preventing infection),
T-cells and phagocytes (involving in particular the IL-12/ vaccines (preventing disease), and antibiotics (preventing
23–IFN-g circuit and the respiratory burst) and bacterial a fatal outcome), rather than the intrinsic efficiency of our
sensing and opsonization (involving mucosal inflamma- immune system.51 Although the immune system serves
tion, complement, carbohydrate-specific antibodies, and well at the population level, ensuring the reproduction of
splenic macrophages), respectively. This classification is species, it is the least efficient physiologic system at the
J ALLERGY CLIN IMMUNOL Casanova et al 429
individual level. As indicated by medical and demo- 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: immu-
graphic data, most human subjects are immunodeficient
nological implications. Immunol Rev 2005;203:10-20.
and exposed to life-threatening infectious diseases.51 20. Fieschi C, Casanova JL. The role of interleukin-12 in human infectious
Many might carry a Mendelian primary immunodefi- diseases: only a faint signature. Eur J Immunol 2003;33:1461-4.
ciency, being thus perhaps the rule rather than the 21. Lejeune J, Gautier M, Turpin R. [Study of somatic chromosomes from
exception and paradoxically raising hope for scientists, 9 mongoloid children]. C R Hebd Seances Acad Sci 1959;248:1721-2.
22. Casanova JL, Jouanguy E, Lamhamedi S, Blanche S, Fischer A.
physicians, and patients. Immunological conditions of children with BCG disseminated infection.
Lancet 1995;346:581.
We thank Laurent Abel for critical reading of the manuscript and 23. Casanova JL, Blanche S, Emile JF, Jouanguy E, Lamhamedi S,
Altare F, et al. Idiopathic disseminated bacillus Calmette-Guerin
other members of the laboratory of Human Genetics of Infectious
infection: a French national retrospective study. Pediatrics 1996;98:
Diseases for helpful discussions. We also thank Gérard Orth for
774-8.
helpful discussions, and we thank 2 anonymous reviewers for their 24. Altare F, Ensser A, Breiman A, Reichenbach J, Baghdadi JE, Fischer A,
constructive criticisms. 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
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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
Background: Infant endotoxin exposure has been proposed

as a factor that might protect against allergy and the Abbreviations used
early childhood immune responses that increase the risk of EU: Endotoxin units
IgE production to allergens. OR: Odds ratio
Objective: Using a prospective study design, we tested the SI: Stimulation index
hypothesis that early-life endotoxin exposure is associated with TLR4: Toll-like receptor 4
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 Childhood allergic diseases, such as asthma and hay
age 2 to 3 years. fever, are increasing in prevalence, cause chronic ill
Results: We found that increased endotoxin levels were health, and are a substantial public health concern in
associated with decreased IL-13 levels in response to cockroach, developed countries.1,2 Measurable childhood sensitiza-
dust mite, and cat allergens, but not mitogen stimulation. tion to inhaled allergens occurs primarily after the age of
Endotoxin levels were not correlated with allergen- or mitogen- 3 years,3 but the immunologic underpinnings of allergic
induced IFN-g, TNF-a, or IL-10. Increased endotoxin levels disease and airway inflammation likely develop far earlier
were associated with decreased lymphocyte proliferation after
in life.4,5 Manifestation of an allergic phenotype likely
cockroach allergen stimulation. An inverse, although
results from the complex interplay of genetic, develop-
nonsignificant, association was also found between endotoxin
and proliferation to the other tested stimuli. mental, and environmental influences. Adaptive immune
Conclusion: Increased early-life exposure to household processes, including activation of helper T lymphocytes
endotoxin was associated with reduced allergen-induced and subsequent B-lymphocyte activation with IgE isotype
production of the TH2 cytokine IL-13 and reduced switching, underlie the process of allergic sensitization
lymphoproliferative responses at age 2 to 3 years in children in individuals genetically predisposed to development of
at risk for allergy and asthma. Early-life endotoxin-related allergic responses.6 Although still controversial,7,8 there
reduction of IL-13 production might represent one pathway is mounting evidence from animal models9-13 and the
through which increased endotoxin decreases the risk of epidemiologic literature14,15 suggesting that exposure to
allergic disease and allergy in later childhood. (J Allergy Clin
endotoxin, a potent activator of innate immunity, might
Immunol 2005;116:431-7.)
influence subsequent adaptive immune responses to aller-
gen. Furthermore, these studies suggest that the timing and
Key words: Endotoxin, lymphocyte proliferation, cytokine, child- Basic and clinical immunology
hood, allergy 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
From the Departments of aEpidemiology, bEnvironmental Health, and
e
Biostatistics, Harvard School of Public Health, and cThe Channing
might reduce the risk of allergy through its influence on
Laboratory, Department of Medicine, and dthe Department of Medicine, innate immunity and downstream T-cell and B-cell reg-
Brigham and Women’s Hospital and Harvard Medical School. ulation of cytokine and IgE expression.
Disclosure of potential conflict of interest: None disclosed. The immunologic pathway linking endotoxin exposure
Supported by NIEHS R01 ES-07036; NIEHS 2P30ES00002; NIH/NHLBI
to adaptive immunity and evidence for its effects on the
HL07427-23; and AI/EHS35786.
Received for publication December 10, 2004; revised March 28, 2005; development of allergic diseases have recently been
accepted for publication May 9, 2005. reviewed.7,16,17 Briefly, endotoxin is biologically active
Available online July 5, 2005. LPS, a primary component of the outer cell membrane of
Reprint requests: Diane R. Gold, MD, Channing Laboratory, 181 Longwood gram-negative bacteria.18,19 Even minute amounts of
Ave, Boston, MA 02115. E-mail: diane.gold@channing.harvard.edu
0091-6749/$30.00
endotoxin provoke innate immune responses in vitro and
Ó 2005 American Academy of Allergy, Asthma and Immunology in vivo.20 The nature of that response, which is only
doi:10.1016/j.jaci.2005.05.015 partially understood, might depend on the developmental
431
432 Abraham et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
stage of the organism (eg, infant or adult) and on the the parents for blood collection and longitudinal follow-up. The
sequence and mode of exposure to endotoxin and allergen. Brigham and Women’s Hospital Institutional Review Board ap-
Inhaled endotoxin activates the innate immune system proved the study protocol.
by binding transmembrane toll-like receptors (TLRs)
expressed on macrophages and dendritic cells21 through Environmental sampling and endotoxin
IL-12 signaling.22 Given the right timing of exposure and measurements
genotype, these endotoxin-activated antigen-presenting The home sampling protocol has been described previously.27 A
cells might stimulate production of IFN-g and other TH1 trained research assistant visited participants’ homes within 2 to 3
cytokines.23 Through production of IFN-g by TH1-biased months of the child’s birth. During these visits, conducted between
lymphocytes or through alternative pathways, endotoxin 1994 and 1996, detailed demographic, socioeconomic, parental
might downregulate TH2 cytokine (including IL-13) disease history, and home characteristics questionnaires were com-
secretion, IgE production, and consequent allergic dis- pleted, and standardized dust sampling was conducted in various
ease. Although studied extensively in animal models, few sites within the home, including the family room. Dust samples to be
prospective data are available on the effects of home LPS used for endotoxin assays were stored desiccated at 220°C until
extraction.
exposure on cytokine production in young children.
Endotoxin activity of dust samples was determined by using the
Using a prospective birth cohort with a parental history
kinetic Limulus amebocyte lysate assay with resistant-parallel-line
of allergy or asthma, we explored the hypothesis that estimation, as previously described.28-30 The Limulus amebocyte
early-life endotoxin exposure alters immune system re- lysate was supplied by BioWhittaker (Walkersville, Md). Reference
sponsiveness by examining the relationship between standard endotoxin was obtained from the United States
house dust endotoxin and PBMC responses to allergen Pharmacopoeia, Inc (Rockville, Md), and control standard endotoxin
stimulation. Specifically, we examined associations be- was supplied by Associates of Cape Cod (Woods Hole, Mass).
tween house dust endotoxin levels measured 2 to 3 months Results were reported in endotoxin units (EU) per milligram of dust
after the child’s birth and allergen- and mitogen-induced adjusted to account for lot-to-lot variation in Limulus amebocyte
cytokine and proliferative responses of mononuclear cells lysate sensitivity to house dust endotoxin and referenced to the
reference standard endotoxin EC5 and EC6 (US Pharmacopoeia, Inc;
isolated from peripheral blood sampled at age 2 to 3 years.
1 ng of EC5 and EC6 = 10 EU).30 Because of the prioritization
Previously, we had found that increased endotoxin levels
schema for assaying dust samples, the availability of endotoxin
in infancy were associated with decreased risk of eczema measurements was conditional on there being sufficient dust to first
in the first year of life, suggesting that endotoxin might assay for home allergens and fungi. As such, for 19 of the 115
have a protective effect against allergic disease and the subjects with biomarker outcome data, family room dust endotoxin
biologic pathways influencing the risk of allergy.24 We levels were unavailable.
hypothesized that endotoxin levels would be positively
correlated with levels of IFN-g, a TH1 cytokine, and PBMC responses
inversely associated with levels of the TH2 cytokine
At 2 to 3 years of age, blood sampling and analysis was conducted
IL-13, which can mediate isotype switching to IgE.25
in a subgroup of the study participants (n = 115). As previously
We further hypothesized that endotoxin levels would be described, selection of this subgroup was based on the home allergen
associated with decreased proliferative responses after levels measured during the initial home visit.31,32 The goal in
allergen stimulation. 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 Ficoll-
METHODS Hypaque centrifugation.33 Fresh cells were incubated in media;
media containing either 30 mg/mL cockroach allergen (Bla g 2), 30
Description of cohort mg/mL house dust mite allergen (Der f 1), or 1000 U/mL cat allergen
The Epidemiology of Home Allergens and Asthma study is a (Fel d 1); or media plus 10 mg/mL PHA. Optimal stimulant
longitudinal birth cohort study of environmental predictors of allergy concentrations used for the assay were determined in a prior dose-
and asthma development. A description of the recruitment of study response analysis.31
participants and study protocol has been previously published.26 In At 24 and 60 hours after the initiation of stimulation, supernatants
brief, 505 children from 499 families with a parental history of asthma were harvested, and cytokine concentrations were quantified by
or allergy were enrolled in a birth cohort study designed to examine means of ELISA (Endogen, Woburn, Mass). On the basis of prior
the effects of allergen exposure in early life on the development optimization for detection of cytokine levels, IL-10 and TNF-a were
of asthma. The Brigham and Women’s Hospital Human Research measured in the 24-hour samples, and IFN-g and IL-13 were
Committee approved the study. Informed consent was obtained from measured in the 60-hour samples. The lower limits of detection for
the parents for blood collection and longitudinal follow-up. Mothers cytokine assays were as follows: IFN-g, less than 2 pg/mL; IL-13,
in the greater Boston metropolitan area delivered at a large Boston less than 7 pg/mL; IL-10, less than 3 pg/mL; and TNF-a, less than 5
hospital were screened with the following questions: (1) Have you pg/mL. Because supernatant quantities were limited, cytokine assays
ever had asthma, hay fever, or allergies? (2) Has the biologic father were prioritized, with IFN-g levels being measured first. As a result,
of your child ever had asthma, hay fever, or allergies? Mothers fewer subjects have observations of IL-13, IL-10, and TNF-a
responding yes to either question were asked to complete a screening levels.32
questionnaire. Families were not approached if the index child was After incubation of PBMCs with allergen or mitogen for 72 hours,
premature, had a major congenital anomaly, or was in the neonatal 1 mCi of tritiated thymidine was added to each well. After incubation
intensive care unit or if the mother was less than 18 years old or could for an additional 8 hours, the cells were harvested, and tritiated
not speak English or Spanish. Informed consent was obtained from thymidine uptake was determined by means of b-counting.
J ALLERGY CLIN IMMUNOL Abraham et al 433
TABLE I. Characteristics of children in the cohort
Subjects with measurement Subjects with no measurement

of endotoxin and PBMC of both endotoxin and PBMC Total
Variable outcomes* (n = 96) outcomes (n = 402) (n = 498)
Sex, n
Male 60 (63%) 208 (52%) 268 (54%)
Female 36 (38%) 194 (48%) 230 (46%)
Race-ethnicity, n
White or Asian 79 (82%) 324 (81%) 403 (81%)
Other 17 (18%) 78 (19%) 95 (19%)
Income, n
<$50,000 27 (28%) 106 (26%) 133 (27%)
$50,000 68 (71%) 283 (70%) 351 (70%)
Family room Bla g (1 or 2), n
0.05 U/g 25 (26%) 84 (21%) 109 (22%)
<0.05 U/g 69 (72%) 291 (72%) 360 (72%)
Family room Der f 1, n
2 mg/g 43 (45%) 190 (47%) 233 (47%)
<2 mg/g 53 (55%) 206 (51%) 259 (52%)
Family room Fel d 1, n
1 mg/g 61 (64%) 234 (58%) 295 (59%)
<1 mg/g 34 (35%) 147 (37%) 181 (36%)
Cold before blood draw, n
Yes 24 (25%) – –
No 71 (75%) – –
*The total number varied according to specific outcomes, but 96 children had endotoxin and lymphocyte proliferation data.
Proliferation was quantified by calculating the stimulation index (SI) Our focus on the relation of endotoxin to IL-13 production
for each stimulant with the following formula: presented statistical challenges because of the skewed distribution of
the cytokine levels and because of the significant proportion of values
SI ¼ðMean valueof tritiatedthymidineuptakeforstimulatedsamplesÞ= below the limit of detection. First, we used nonparametric correlation
ðMean valueof tritiatedthymidineuptakeforunstimulatedsamplesÞ:31 analyses to relate continuous dust endotoxin with continuous cyto-
kine 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
IgE measurements sensitivity of our findings to the choice of number assigned to
Serum samples were assayed for total IgE and specific IgE the observations below the limit of detection. We then assessed the
antibodies to dust mite, cat, cockroach, and ovalbumin by using the relation between endotoxin and the odds of having measurable IL-13.
UNICAP System (Pharmacia, Uppsala, Sweden). IgE increase was Finally, in secondary analyses we tested for trends in the proportion
defined either as an IgE specific response (0.35 IU/mL) to at least of cytokine observations above the lower limit of detection when
one allergen or a total IgE level of greater than 60 IU/mL for age 2 to 3 grouped by tertile of endotoxin level.
years. 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
Statistical methods greater, Fel d 1 of 1 mg/g or greater, and Der f 1 of 2 mg/g or greater).31
All statistical analyses were conducted with SAS version 8.2 (SAS Reported household income was classified as being less than or
Institute Inc, Cary, NC). In primary analyses investigating lympho- greater than $50,000.
proliferative 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 RESULTS
improve the normality of residuals and facilitate interpretation of
model results. The relationships between log10-transformed SIs, Of the 505 children initially enrolled in the study, 7
log10-transformed endotoxin, and potential covariates were assessed were followed for less than 5 months in the first year of
by using ordinary least-squares univariate and multivariate regression life. Of the 498 with follow-ups, 115 had lymphocyte
models. The estimates for the relationship of the log of endotoxin proliferation measurements at age 2 to 3 years, of whom
to SI were used to calculate the percentage difference in SI for a 96 also had family room dust endotoxin measurements.
doubling of endotoxin. This enabled us to translate the estimates into
Compared with the 402 subjects without both lymphocyte
an easier-to-understand outcome that was scaled by a realistic (within
the range of our data) increase in endotoxin level. We also considered
proliferation and endotoxin data, the subsample with
SI as a dichotomous categorical by using cutoff points that have been both (n = 96) had a greater proportion of boys (Table I).
considered as positive PBMC responses to allergen or mitogen No other selection bias was detected. Endotoxin in the
stimuli in other studies (allergen responses, SI > 3; PHA responses, 96 family room dust samples had a geometric mean of 95
SI > 153).31 EU/mg (geometric SD, 1.8 EU/mg) and a median level of
AUGUST 2005
TABLE II. Distribution of endotoxin, IL-13, and PBMC lymphocyte proliferative response levels*
Variable Minimum Median 75th Percentile Maximum Detectable, n
Endotoxin (EU/mg), n = 96 19.3 101.2 138.4 518.8 96 (100%)

IL-13 (pg/mg), n = 67
Bla g 2 <LD <LD 9.6 207.2 32 (48%)
Der f 1 <LD 8.6 30.5 397.3 47 (70%)
Fel d 1 <LD <LD 4.7 137.6 26 (39%)
PHA <LD 1425 2255 3812 64 (96%)
SI
Bla g 2 0.9 4.1 5.7 53.6 94 (100%)
Der f 1 0.4 3.3 5.9 32.5 95 (100%)
Fel d 1 0.3 2.2 4.4 27.5 96 (100%)
PHA 14.4 138.6 285.5 1205.1 96 (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 Association of house dust endotoxin and
proliferation and cytokine levels for this subsample were stimulation-induced cytokine production
similar to those previously demonstrated for the larger By using rank-based correlations, increasing house dust
sample (Table II).31,32
endotoxin levels measured early in life were inversely
We found no confounders of the association of endo- correlated with allergen-induced IL-13 but not IL-13
toxin with either the lymphoproliferative response or the induced by PHA (Table IV). For a doubling of endotoxin
cytokine production by stimulated PBMCs after consid-
levels, children had significantly reduced odds of having
ering living room allergen level, report of dog or cat, race- measurable-detectable allergen-induced IL-13 levels in
ethnicity, household income, or cold before blood draw response to all 3 allergens, with no association between
(data not shown). As previously reported for the larger
endotoxin- and PHA-induced IL-13 (Table V). With
cohort, cockroach allergen (Bla g 1 or 2), cat allergen increasing tertiles of endotoxin, a smaller percentage of
(Fel d 1), and dust mite allergen (Der f 1) levels measured children had measurable allergen-induced IL-13 levels
in family room dust samples were not significantly (above the limits of detection), although this trend was
associated with home endotoxin levels.34
only statistically significant for the response to Der f 1 (see
Fig E1 in the Online Repository in the online version of
Association of house dust endotoxin and SIs this article at www.mosby.com/jaci). We did not observe
We observed a consistent association between increased correlations between allergen- or mitogen-induced levels
dust endotoxin measured in the first months of a baby’s of IFN-g, TNF-a, and IL-10 in the supernatants of the
life and decreased SIs at age 2 to 3 years, although this isolated PBMCs and endotoxin (data not shown). We
association was only significant for the response to cock- found no evidence of an association between PHA-
roach allergen (Table III). Despite the absence of measur- induced IL-13 and endotoxin.
able confounders, to adjust for potential independent
influences on the lymphoproliferative responses, we ad-
justed for indicators of age at the time of blood draw, report DISCUSSION
of a cold in the week before blood draw, household in-
come level, and house dust allergen levels in the allergen- In this prospective cohort study of young children with
specific proliferation models in addition to reporting the a family history of allergic disease, we observed that
univariate models.31 The multivariate-adjusted relation- increased levels of family room dust endotoxin in infancy
ships between endotoxin and the SIs ranged from 24% to were associated with decreased allergen-induced IL-13
217%, being strongest and statistically significant for the production by mononuclear cells isolated from peri-
SI after cockroach allergen stimulation. When dichoto- pheral blood at age 2 to 3 years. We also found that
mized at previously defined cutoff points,31 positive endotoxin levels were associated with a downregulation
cockroach-induced lymphocyte proliferative responses of allergen- and mitogen-stimulated lymphocyte prolifera-
(SI > 3) were also inversely associated with endotoxin tion. Through the interactions of its Lipid A moiety with
levels (odds ratio [OR] of Bla g 2 SI 3 for a doubling in receptors such as TLR4, endotoxin is hypothesized to
family room endotoxin level: OR, 0.53; 95% CI, 0.27- modulate the innate immunity pathway and, through those
1.049; P = .07). Although the estimates for Der f 1 SI pathways, to influence adaptive immunity16,17 with re-
were negative, the analyses using SI as a dichotomous duced production of cytokines, such as IL-13, as seen in
outcome were not supportive of a significant association our study. This in turn might result in subsequent reduc-
of endotoxin with a lower odds of Der f 1 SI of greater tion of IgE levels, allergy, and allergic disease. In our
than 3 (P = .9) or Fel d 1 SI of greater than 3 (P = .7). cohort at this age, we have previously demonstrated an
TABLE III. Percentage change in lymphocyte proliferative response (SI) for a doubling of family room dust
endotoxin levels
Univariate* Multivariatey
Stimulus % Difference 95% CI % Difference 95% CI
Cockroach allergen (Bla g 2) 215 227 to 21 217 228 to 24

Dust mite allergen (Der f 1) 213 230 to 8 215 231 to 5
Cat allergen (Fel d 1) 25 221 to 15 24 221 to 16
PHA 211 228 to 10 29 227 to 12
*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.
association between lower IL-13 levels and reduced risk of proportions of IFN-g–producing CD4 T cells.15 In this
allergy/increased IgE levels.32 Increased endotoxin levels study the investigators did not evaluate cytokine secretion
were also associated with reduced risk of allergy, but this of lymphocyte proliferative response of antigen- or mito-
association was not statistically significant. A doubling of gen-stimulated PBMCs.
endotoxin in family room dust was associated with an OR Although this is, to the best of our knowledge, the first
of 0.71 (95% CI, 0.39-1.31) for increased IgE levels, as report of a prospective association of endotoxin with
determined by positive RAST results or high total IgE diminished IL-13 production in children, some animal
levels among 97 children with data on endotoxin in early models have demonstrated similar associations. However,
life and IgE levels at age 2 to 3 years. In a previous re- in animal models endotoxin exposure with TLR4 agonists
port from this study, we demonstrated that higher infant has been shown to both diminish and enhance IFN-g
endotoxin levels were associated with a significantly production, IL-13 production, and allergic responses.12
lower risk of eczema in the first year of life.24 The data The directionality and nature of the immune and allergic
we present in this article support the hypothesis that the responses appears to be dependent on many factors,
association of increased home endotoxin exposure with including the type of model, dose of allergen or TLR4
protection against childhood allergic disease might be agonist, timing of allergen or TLR4 agonist, use or not of
mediated in part through reduced TH2 cytokine expression adjuvant, and type of species or murine strain.9,11,39-42 In
in early life. one murine model prenatal exposure to LPS resulted in
Researchers in Europe have observed an association increased neonatal IFN-g secretion and decreased neona-
between a farming lifestyle and lower prevalence of al- tal IL-13 production after OVA exposure, with no protec-
lergic disease in children and hypothesized that increased tion against airway responsiveness.43 In another murine
exposure to endotoxin in infancy might be responsible model, Velasco et al12 showed that pulmonary adminis-
for this association through an early-life influence on tration of Lipid A before allergen sensitization decreased
innate immune development and its relation to adaptive eosinophilia, bronchoalveolar lavage fluid IL-13 levels,
immunity, cytokine production, and IgE production.14,35,36 serum IgE levels, airway hyperresponsiveness, and the
In a cross-sectional study of German, Austrian, and Swiss number of CD41 cells in the lung. Lipid A administration
children ages 6 to 13 years, endotoxin measured in the dust during allergen challenge diminished eosinophilia. In
of children’s mattresses was inversely associated with contrast, Delayre-Orthez et al42 showed that LPS during
asthma, atopic sensitization, and hay fever.37 Increased challenge enhanced allergen-induced eosinophilia but did
bed dust endotoxin levels were associated with decreased not enhance allergen-induced IgE or airway hyperrespon-
LPS-induced production of IFN-g, IL-10, IL-12, and siveness. A recent study compared the effects of Lipid A in
TNF-a. The researchers interpreted these findings as a terms of maturity of the immune system, species, dose,
global downregulation of the immune response. In con- and timing by examination of human cord and adult
trast, we found a specific endotoxin-related downregula- PBMCs and murine cells in vitro and in vivo.13 Lipid A
tion of a TH2 cytokine, IL-13, which can mediate isotype induced primarily a time- and dose-independent produc-
switching to IgE25,38 without an influence on IFN-g, tion of IFN-g.
IL-10, or TNF-a production. Our study contrasts with the In both human and animal models, the effects of
multicenter European study in many ways. We stimulated environmental endotoxin exposure, are likely to be de-
our PBMCs with allergen, our study was prospective, our pendent on the dose and timing of exposure and on the
cohort was predisposed to allergy by virtue of family developmental stage and genotype.44 These factors might
history, and our endotoxin levels, measured in infancy, explain seemingly contradictory epidemiologic findings of
were relatively low. In a small US cross-sectional study of endotoxin effects on allergic disease.7,16 As an irritant, in
infants with repeated wheeze, allergen-sensitized children those with or without asthma, endotoxin might increase
came from homes with higher endotoxin levels, and higher the severity of symptoms.31,45 Previously, we found that
home endotoxin levels were correlated with increased endotoxin was associated with increased risk of wheeze
AUGUST 2005
TABLE IV. Correlations between allergen- and mitogen- TABLE V. Relative odds of detectable versus nondetect-
induced cytokine levels and family room dust endotoxin able allergen- and mitogen-induced IL-13 for a doubling
levels* of family room endotoxin levels
Cytokine Correlation Cytokine Stimulus Obs. OR 95% CI

Stimulus (pg/mL) Obs. coefficient P value
IL-13
Bla g 2 IFN-g 80 20.05 .66 Bla g 2 67 0.36 0.16-0.81
IL-13 67 20.31 .01 Der f 1 67 0.27 0.11-0.70
IL-10 64 0.11 .38 Fel d 1 67 0.44 0.20-0.94
TNF-a 50 20.01 .94 PHA 67 0.89 0.19-4.17
Der f 1 IFN-g 85 0.01 .91
IL-13 67 20.27 .02 Obs., Number of observations.
IL-10 69 20.08 .51
TNF-a 50 20.09 .54
Fel d 1 IFN-g 80 0.06 .57 exclude the role of chance in determining the observed
IL-13 67 20.21 .08 association between endotoxin and proliferation after
IL-10 64 20.02 .89 stimulation with the other allergens and the mitogen.
TNF-a 50 20.03 .83
However, the consistently negative associations suggest
PHA IFN-g 85 0.08 .46
IL-13 67 0.12 .34
that this is not a chance observation. Although confound-
IL-10 69 20.05 .67 ing bias by unmeasured factors is always a possibility, we
TNF-a 50 20.14 .35 found no confounding by other measured exposures
(including dog) in our analyses evaluating the association
Obs., Number of observations. of endotoxin with detectable IL-13, and we adjusted for
*Rank-based (Spearman) correlation coefficients. 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
and repeated wheeze in the first year of life in this cohort; exposure that occurs within the first year of life. Our single
this might be secondary to its irritant effects.29 In the measure of endotoxin in the home is likely to represent this
siblings of the subjects in this cohort, the association of true exposure with error. Because of the prospective study
endotoxin with wheeze decreased over time, and as the design, differences between the observed measurements
child grew older, earlier-life endotoxin appears to have a of endotoxin and the subjects’ true endotoxin exposure
null or even protective effect on wheeze risk, perhaps are most likely nondifferential with respect to PBMC re-
because the wheeze was more related to allergic inflam- sponses. This measurement error would most likely have
mation.46 This is consistent with the hypothesis that attenuated the exposure effect estimates. A final limitation
endotoxin might increase the risk of irritant wheeze at is that we did not evaluate the allergens that we used to
the same time as decreasing the risk of allergy and its stimulate the PBMCs for the possible presence of endo-
pulmonary and extrapulmonary manifestations. In their toxin. Production of IL-13 is likely to be less influenced by
studies of children growing up on farms, Braun-Fahrlander trace LPS than production of cytokines involved in innate
et al37 have found consistent protective effects of endo- immunity, such as TNF-a. The possibility remains, how-
toxin on allergy but an increased risk of wheeze with ever, that increased home allergen levels in infancy are
increasing endotoxin levels among those without allergy. associated with reduced IL-13 production not only in
We had limited power to fully elucidate pathways response to allergens but also in response to a combination
leading from endotoxin exposure to cytokine production, of allergen and LPS.
early allergy, and subsequent allergic disease expression. In conclusion, in a cohort of children at risk of allergy
In this group of young children, when allergy, particularly and asthma, we conducted a prospective study on the re-
allergy to inhaled allergens, is just developing, we found lation of home endotoxin levels in infancy to subsequent
that increased levels of endotoxin were associated with a allergen-stimulated lymphocyte proliferative responses
lower risk of high IgE levels but that association was not and cytokine production. Our data suggest that increased
statistically significant. This might be due to small num- early-life endotoxin exposure might be associated with a
bers (low power) because we were only able to measure reduction in subsequent allergen-stimulated lymphocyte
both endotoxin and cytokines on a subset of the cohort proliferation and IL-13 secretion. Endotoxin-induced
or due to the age of the children because the allergic reduction in IL-13 secretion might be one early-life step
phenotype is not fully evolved by age 2 to 3 years. It is in the pathway to endotoxin-associated reduction in
quite possible that a skewing toward TH2 cytokine allergy and allergic disease.
production is an earlier step in the pathway toward full
expression of the allergic phenotype. The associations be-
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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 Näsman, Ph Lic,d Marita Troye Blomberg, PhD,e and
Gunnar Lilja, MD, PhDa Stockholm, Sweden
Background: There is indirect evidence that an increased by CMV coinfection. (J Allergy Clin Immunol 2005;116:
infectious burden is associated with a decreased prevalence 438-44.)
of IgE-mediated allergy during childhood.
Objective: To determine whether there is a relation between the Key words: Childhood, CMV, EBV, IgE, infections, sensitization,
serostatus of 13 different viruses and parentally reported serology, viral infections
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. The increasing prevalence of allergic disease has
Methods: A total of 246 infants were followed prospectively to become a major health problem in the industrialized parts
2 years of age with clinical examinations, skin prick test, and of the world.1 Epidemiological studies have shown that
specific IgE analyses and through analysis of seropositivity various markers of increased burden of infections are
against adenovirus, influenza, parainfluenza, respiratory associated with a decreased prevalence of allergy and
syncytial virus, CMV, EBV, herpes simplex virus, human asthma during childhood.2,3 Viral infections have been
herpesvirus 6, and varicella-zoster virus. implicated in influencing IgE-mediated sensitization, but
Results: There was some evidence that IgE sensitization (24%) their exact role remains controversial.3,4 It has been
tended to be more common among children who were sugested that many viruses influence the differentiation
seropositive against few compared with children who were
of T cells, thus causing an imbalance between TH1 and
seropositive against many viruses, but this was not statistically
significant, and there was no consistent trend across the groups. TH2 immune responses.5 Cytomegalovirus (CMV) and
IgE sensitization was statistically significantly less prevalent at Epstein-Barr virus (EBV) are persistent viral infections, as
2 years of age among infants who were seropositive against demonstrated by frequent presence of the virus in saliva
EBV but not other viruses (adjusted odds ratio, 0.34; 95% CI, and urine of healthy individuals,6 and may influence the
0.14-0.86). The interaction of seropositivity against both immune system with respect to the development of
CMV and EBV antibodies indicated a further reduction in the allergy, as recently proposed by Sidorchuk et al.7
risk for IgE sensitization (adjusted odds ratio for interaction, The aim of this study was therefore to elucidate the
0.10; 95% CI, 0.01-0.92), indicating effect modification interplay between CMV and EBV in relation to IgE
associated with seropositivity against CMV. sensitization among children at 24 months of age. The
Conclusion: Our results indicate that acquisition of EBV
study was also designed to investigate the association of
infection during the first 2 years of life is associated with a
reduced risk of IgE sensitization, and this effect is enhanced IgE sensitization with infectious burden measured as
seroprevalence against 13 different viruses, including
CMV, EBV, and respiratory syncytial virus (RSV), as
From athe Department of Pediatrics, Sachs’ Children’s Hospital, and bthe well as parentally reported infections.
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
METHODS
Clinical Research Centre, Örebro University Hospital; dthe Royal Institute

of Technology; and ethe Department of Immunology, Stockholm Subjects
University.
Families who where expecting a child were asked by the midwife
Disclosure of potential conflict of interest: None to disclose.
Supported by the Swedish Asthma and Allergy Association, Consul Th C at the maternity ward whether they were interested in participating in
Berg’s Foundation, the Samariten Foundation, Mjölkdroppen, the Vårdal the study. Only parents who reported they had a history of allergy in
Foundation, the Heart and Lung Foundation, GlaxoSmithKline, Brio AB, the mother, in both parents, or in neither parent were eligible. The
and the Karolinska Institute. Pharmacia Diagnostics AB supplied reagents parents provided a blood sample and underwent skin prick tests
for plasma IgE analyses. (SPTs). Only parents whose SPT results confirmed their positive or
Received for publication October 25, 2004; revised April 20, 2005; accepted negative history of respiratory allergy to pollen and/or furred pets
for publication April 21, 2005. were invited to continue. When evaluated at 24 months of age, 246
children (126 boys and 120 girls) born to the selected parents
Reprint requests: Caroline Nilsson, MD, Department of Pediatrics, Sachs’
participated in the study. One hundred two children had 2 allergic
Children’s Hospital, Stockholm South Hospital, S-118 83 Stockholm,
Sweden. E-mail: caroline.nilsson@sodersjukhuset.se. parents, 75 children had an allergic mother, and 69 children had no
0091-6749/$30.00 parental history of allergy. All infants were born full-term (>35 weeks
Ó 2005 American Academy of Allergy, Asthma and Immunology of gestation) at hospitals in Stockholm and had birth weights within
doi:10.1016/j.jaci.2005.04.027 the normal range (data not shown). The socioeconomic status of the
438
J ALLERGY CLIN IMMUNOL Nilsson et al 439
plasma with Pharmacia CAP-FEIA (Pharmacia-Upjohn, Uppsala,

Abbreviations used Sweden). A positive test was defined as an IgE antibody level 0.35
EBV: Epstein-Barr virus kilo Units allergen-specific antibodies (kUA) per liter.
CMV: Cytomegalovirus
HHV6: Human herpesvirus 6 Classification of the children
HSV: Herpes simplex virus In accordance with Johansson et al,8 the child was classified as
kUA: Kilo Units allergen-specific antibodies IgE-sensitized if at least 1 SPT was positive (3 mm) and/or if spe-
RSV: Respiratory syncytial virus cific IgE against at least 1 of the selected allergens was 0.35kUA/L.
SPT: Skin prick test To optimize the classification in IgE-sensitized and non–IgE-
VZV: Varicella-zoster virus sensitized children, the results of the in vivo and in vitro analysis
were combined.
Viral infections/serological methods

families was estimated through the father’s occupation grouped ac-
cording to the classification used by Statistics Sweden. Demographic The serostatus against 13 viruses was investigated: respiratory
data are presented in Table I. tract infections, including adenovirus, influenza (A/H1, A/H3, and
The study was approved by the Human Ethics Committee at influenza B), parainfluenza (types 1, 2, 3), and RSV; and herpesvirus,
Huddinge University Hospital, Stockholm (Dnr 75/97, 113/97), and including CMV, EBV, herpes simplex virus (HSV), human herpes-
the parents provided informed consent. virus 6 (HHV6), and varicella-zoster virus (VZV).
IgG against the EBV capsid antigen and HHV6 was determined
Clinical evaluation according to previously published immunofluorescence assays.9,10
A specific fluorescence in dilution of 1/20 was regarded as a sign of
The children were followed from birth to 2 years of age and were
seropositivity.
clinically evaluated at ages 6, 12, 18, and 24 months by 1 pediatrician
For HSV, CMV, and VZV IgG ELISA with purified nuclear
(C. N.).
antigens from the respective viruses cultivated in human fetal lung
fibroblasts were used.11,12 The cutoff for seropositivity was an
Skin prick testing
absorbance of >0.2 at a dilution of 1/100.
Skin prick tests against food and inhalant allergens were IgG antibodies against influenza A/H1, A/H3, and influenza B
performed among the children at 24 months of age according to the were determined with ELISAs by using recombinant influenza
manufacturer’s recommendation (ALK, Copenhagen, Denmark). antigens.13 IgG antibodies against parainfluenza (serotypes 1, 2, 3),
The SPT included food allergens: egg white (Soluprick weight to RSV, and adenoviruses were measured by ELISA. The viruses were
volume ratio, 1/100), cod (Soluprick 1/20), peanut, (Soluprick 1/20), cultured to full cytopathogenic effect either in human fetal lung
cow’s milk (3% fat, standard milk), and soybean protein (Soja Semp; fibroblasts (RSV, adenovirus) or MA 104 cells (monkey kidney cells,
Semper AB, Stockholm, Sweden). SPTs were also performed for parainfluenza) and prepared mainly by ultracentrifugation and son-
inhalant allergens: cat, dog, Dermatophagoides farinae, birch, and ification of clarified supernatants. For adenovirus, sonicated, infected
timothy (Soluprick 10 Histamine Equivalent Prick test). All parents cells were used. Preparations from the respective cell lines were used
were skin prick tested against the same inhalant allergens as the as control antigen in the assays using cell culture antigen. Optical
children but also against horse, rabbit, and mugwort. Histamine densities above 0.3 after subtraction of control antigen activity were
chloride (10 mg/mL) was the positive control and the allergen diluent regarded as a sign of past infection for the respiratory viruses.
the negative control. The SPT was considered positive if the wheal
diameter was 3 mm after 15 minutes. Statistics
Descriptive statistics were used to characterize the data. x2
Parents’ report of infections Analysis and the Student t test (2-tailed) were used for comparison
During the observation period, the parents were asked to record of IgE-sensitized and non–IgE-sensitized children where appropriate.
every infection that their child had in a structured diary. This included The number of serologically verified infections was normally
symptoms—runny nose, cough, vomiting, diarrhea, fever—and a distributed (Fig 1), and the parentally reported infections were close
doctor’s diagnosis where relevant. The diary consisted of sets of to normally distributed, and they were divided into quarters defined
structured questions, 1 set for each illness. The parents filled in the by quartiles by using the statistical program SPSS 11.0 for Windows
form every time the child was ill, marking the correct squares with an (SPSS Inc, Chicago, Ill). There was variation in the size of the groups Basic and clinical immunology
X and recording the date of the illness. Parents confirmed the events as a result of characteristics of the distribution.
recorded in the diary when they visited the outpatient clinic with the Odds ratios and 95% CIs were calculated for the development of
child at ages 6, 12, 18, and 24 months. In an attempt to record missed IgE sensitization. Data were adjusted for background variables by
illnesses, at each visit the parents were asked, ‘‘Has your child had using multiple logistic regression analysis. Adjustments were made
any illness since your last visit?’’ If additional illnesses were for sex, parental allergy (none, single-heredity, or double-heredity),
mentioned, they were added to the diary. maternal age, parental smoking, furred pets at home, months of birth,
older siblings, duration of breast-feeding, socioeconomic status,
Blood sampling parentally reported infections, and seropositivity against viruses. All
Venous blood samples were collected when the children were 24 of the measures were modeled as series of binary dummy variables.
months old. Plasma was separated by centrifugation and stored at The interaction of seropositivity for CMV and EBV was inves-
270°C pending analysis. tigated by using logistic regression, with adjustment for the main
effects.
Specific IgE P values <.05 were considered statistically significant. The data
Circulating IgE antibodies against cow’s milk, egg white, peanut, were analyzed by using Stata 7.0 (Stata Corp, College Station, Tex),
cod fish, soy bean, wheat, cat dander, dog dander, birch pollen, SPSS 11.0 for Windows, and the SAS System for Windows release
timothy pollen, and Dermatophagoides farinae were determined in 8.02 (SAS Institute, Cary, NC).
440 Nilsson et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
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
Whole cohort IgE-sensitized Nonsensitized OR; 95% CI ORadj; 95% CI
n (%) 246 59 (24.0) 187 (76.0)

Sex
Boy, n (%) 126 (51.2) 34 (27.0) 92 (73.0) 1 1
Girl, n (%) 120 (48.8) 25 (20.8) 95 (79.2) 0.71; 0.39-1.28 0.65; 0.32-1.31
Heredity
Nonheredity; n (%) 69 (28.0) 11 (15.9) 58 (84.1) 1 1
Double-heredity; n (%) 102 (41.5) 32 (31.4) 70 (68.6) 2.41; 1.12-5.20 3.79; 1.50-9.60
Maternal heredity; n (%) 75 (30.5) 16 (21.3) 59 (78.7) 1.43; 0.61-3.34 2.29; 0.83-6.31
Maternal age at delivery
Maternal age, 31.4 (21-44) 31.5 (22-44) 31.3 (21-43)
mean (range)
21-30 y, n (%) 103 (41.9) 22 (21.6) 81 (78.4) 1 1
31-44 y, n (%) 143 (58.1) 37 (25.9) 106 (74.1) 1.29; 0.70-2.35 1.42; 0.67-3.01
Month of birth
Born April-September, n (%) 148 (60.2) 27 (18.2) 121 (81.8) 1 1
Born October-March, n (%) 98 (39.8) 32 (32.7) 66 (67.3) 2.17; 1.20-3.93 2.23; 1.10-4.52
Exposure
Exclusive breast-feeding
0 mo, n (%) 13 (5.3) 2 (15.4) 11 (84.6) 0.61; 0.13-2.89 0.91; 0.13-6.60
0.5-3.9 mo, n (%) 34 (13.8) 10 (29.4) 24 (70.6) 1.40; 0.62-3.19 3.11; 1.10-8.85
4-5 mo, n (%)* 166 (67.5) 38 (22.9) 128 (77.1) 1 1
5.1-10 mo, n (%) 33 (13.4) 9 (27.3) 24 (72.7) 1.26; 0.54-2.95 1.21; 0.45-3.21
Mothers smoking, n (%) 10 (4.1) 1 (1.7) 9 (4.8) 0.34; 0.04-2.75 0.32; 0.03-3.19
Fathers smoking, n (%) 19 (7.7) 7 (11.9) 12 (6.4) 1.96; 0.73-5.24 2.24; 0.68-7.34
Furred pets at home, n (%) 45 (18.3) 9 (15.3) 36 (19.3) 0.76; 0.34-1.68 0.74; 0.28-1.94
Number of older siblings
0, n (%) 135 (54.9) 34 (25.2) 101 (74.8) 1 1
1, n (%) 81 (32.9) 24 (29.6) 57 (70.4) 1.25; 0.68-2.31 1.35; 0.65-2.80
>2, n (%) 30 (12.2) 1 (3.3) 29 (96.7) 0.10; 0.01-0.78 0.07; 0.01-0.65
Socioeconomic status
High, n (%) 141 (57.3) 34 (24.1) 107 (75.9) 1 1
Medium, n (%) 39 (15.9) 9 (23.1) 30 (76.9) 0.94; 0.41-2.19 0.97; 0.36-2.64
Low, n (%) 41 (16.7) 10 (24.4) 31 (75.6) 1.02; 0.45-2.28 1.35; 0.49-3.75
Studying, n (%) 11 (4.5) 4 (36.4) 7 (63.6) 1.80; 0.50-6.52 3.39; 0.67-17.06
Not specified, n (%) 14 (5.7) 2 (14.3) 12 (85.7) 0.52; 0.11-2.46 0.52; 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 However, the nonsensitized children were statistically

significantly more likely to have been born during the
IgE sensitization
summer than sensitized children and more frequently had
The children had an average age of 24.1 months (range, more than 1 sibling. The sensitized children were statis-
22-29) at the 24-month evaluation. Fifty-nine (24%) tically significantly more likely to have 2 atopic parents.
children were classified as IgE-sensitized. The in vitro The statistically significant association with short duration
test (allergen-specific IgE in plasma) was positive in 52 of breast-feeding was observed only in the adjusted
(22%) infants, whereas 35 (14%) infants had at least analyses, suggesting a complex set of associations or a
1 positive SPT against the selected food and inhalant chance finding.
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%; IgE sensitization and the parental report
dog, 4.9%; wheat, 4.1%; cat, 3.7%; birch, 3.7%; soy, of infections
2.0%; fish, 1.2%; timothy, 0.8%; and mite, 0.8%. The median number of parental reported infections was
There were no statistically significant differences in 13 (range, 4-24) during the first 24 months of life. For
sex, having a furred pet at home, or having smoking evaluation, the infants were divided into 4 groups defined
parents between IgE-sensitized and nonsensitized children by quartiles: 4 to 9, 10 to 13, 14 to 16, and 17 to 24
(Table I). reported infections. The association with IgE sensitization
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 dif-
ference between the groups, and the trend across the
groups was not consistent (Table II).
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 FIG 1. Frequency of seropositivity against the 13 selected viruses at
against at least 1 of the viruses studied, and 1 child had 24 months of age in IgE sensitized* (n = 59) and nonsensitized
detectable IgG against all 13. There were no significant (n = 187) infants. *At least 1 positive SPT 3 mm and/or at least
associations between the number of parentally reported 1 specific IgE against the selected allergens 0.35 kUA/L.
infections and the number of viruses identified through
serology (data not shown).
The mean number of viruses identified through serol- IgE sensitization associated with seropositivity against
ogy was 5 (Fig 1). The children were divided into 4 groups EBV among children seronegative against CMV was 0.75
defined by quartiles. Fifty-five children had antibodies (95% CI, 0.30-1.91) but was reduced to 0.07 (95% CI,
against 0 to 3 viruses, 95 children against 4 to 5 viruses, 42 0.01-0.57) among children who were seropositive against
against 6 viruses, and 54 against 7 to 13 viruses, respec- both viruses. Interaction testing revealed a statistically
tively. There was some suggestion that children with significant synergistic effect (effect modification), pro-
fewer antibodies were more often sensitized than children ducing an odds ratio for the interaction of both viruses
with antibodies against many viruses, but this result was with IgE sensitization of 0.10 (95% CI, 0.01-0.92) after
not statistical significant (Table II), and the pattern did not adjustment for the main effects.
show a consistent trend across the groups. We also observed an association with sensitization
among subjects who were seropositive against CMV and
IgE sensitization and seropositivity seronegative against EBV. Among those seronegative
against EBV (n = 182), 26 of 71 were sensitized among
to individual viruses
those infected with CMV, compared with 25 of 111 who
Respiratory viruses. The association between pres- were not infected with CMV. This produces an odds ratio
ence of antibodies against the individual viruses and of 1.99 (95% CI, 1.03-3.83), suggesting a modest in-
IgE sensitization is presented in Table II. There were creased risk associated with CMV infection in subjects
no statistically significant associations between IgE who are seronegative for EBV.
sensitization and seropositivity against the 8 airborne The serostatus against the other herpesviruses was not
viruses—adenovirus, influenza (A/H1, A/H3, and B), significantly associated with IgE sensitivity.
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 DISCUSSION
IgE-sensitized.
Herpesviruses. Seropositivity against the viruses The hypothesis that infectious diseases during early
belonging to the herpesvirus family—CMV, EBV, HSV, childhood may have a protective role against the devel-
HHV6 and VZV—and IgE sensitization are also presented opment of allergy, the hygiene hypothesis, was raised in
in Table II. Seronegativity against EBV was statistically the late 1980s.5
significantly associated with IgE sensitization. Sixty-four In the current study, we did not find strong evidence of
children (26%) were seropositive against EBV. Among an association between IgE sensitization and the number
these, 8 (12 %) were sensitized. This was significantly less of previous infections indicated either by parental report
than in the EBV seronegative group (odds ratio, 0.37; 95% or by seropositivity. However, IgE sensitization was less
CI, 0.16-0.82). The association remained statistically prevalent at 2 years of age among infants who were
significant after adjustment for all of the potential con- seropositive against EBV. The combination of having
founding factors (Table II). both CMV and EBV antibodies was more strongly
Cytomegalovirus IgG antibodies were detectable in 96 negatively associated with sensitization than would be
(39%) children. Among these, 27 (28%) were IgE-sensi- predicted by the individual associations of EBV or CMV
tized. There were no statistically significant differences in antibodies alone, indicative of an interactive effect.
the numbers of seropositivity or seronegativity against The prevalence of EBV antibodies in our study is
CMV when comparing the IgE sensitized and nonsensi- comparable with that of other industrialized countries.14
tized infants. However, seropositivity for EBV was more Previous studies of EBV are contradictory in relation to
negatively associated with sensitization among subjects development of atopy. Increased levels of antibodies
who were also seropositive for CMV. The odds ratio for against EBV were found in children 5 to 18 years old
AUGUST 2005
TABLE II. Seropositivity against the investigated viruses and parentally reported infections in IgE-sensitized and
nonsensitized children
Whole cohort IgE-sensitized Nonsensitized OR; 95% CI ORadj; 95% CI
n 246 59 187
Parainfluenza 1, n (%) 70 (28.5) 16 (27.1) 54 (28.9) 0.92; 0.48-1.76 0.93; 0.46-1.86
Parainfluenza 2, n (%) 30 (12.2) 7 (11.9) 23 (12.3) 0.96; 0.39-2.36 1.14; 0.44-2.99
Parainfluenza 3, n (%) 166 (67.5) 42 (71.2) 124 (66.3) 1.26; 0.66-2.38 1.13; 0.58-2.21
Influenza Panama, n (%) 47 (19.1) 13 (22.0) 34 (18.2) 1.27; 0.62-2.61 1.32; 0.62-2.82
Influenza Texas, n (%) 30 (12.2) 6 (10.2) 24 (12.8) 0.76; 0.30-1.97 0.63; 0.23-1.76
Influenza Beijing, n (%) 92 (37.4) 16 (27.1) 76 (43.8) 0.54; 0.28-1.03 0.52; 0.26-1.01
Adenovirus, n (%) 200 (81.2) 46 (78.0) 154 (82.4) 0.76; 0.37-1.56 0.77; 0.36-1.63
RSV, n (%) 154 (62.6) 33 (55.9) 121 (64.7) 0.69; 0.38-1.26 0.71; 0.37-1.36
HHV6, n (%) 207 (84.2) 47 (79.7) 160 (85.6) 0.66; 0.31-1.40 0.66; 0.30-1.46
HSV, n (%) 24 (9.8) 6 (10.2) 18 (9.6) 1.06; 0.40-2.81 1.10; 0.40-3.02
CMV, n (%) 96 (39.0) 27 (45.8) 69 (36.9) 1.44; 0.80-2.60 1.20; 0.64-2.27
VZV, n (%) 45 (18.3) 8 (13.6) 37 (19.8) 0.64; 0.28-1.45 0.60; 0.25-1.42
EBV, n (%) 64 (26.0) 8 (13.6) 56 (29.9) 0.37; 0.16-0.82 0.34; 0.14-0.86
Frequency of seropositivity
0-3 viruses, n (%) 55 (22.4) 16 (27.1) 39 (20.9) 1.57; 0.71-3.50 1.53; 0.59-3.97
4-5 viruses, n (%) 95 (38.6) 25 (42.4) 70 (37.4) 1.15; 0.47-2.84 0.78; 0.28-2.21
6 viruses, n (%) 42 (17.1) 7 (11.9) 35 (18.7) 1.02; 0.42-2.51 0.99; 0.35-2.80
7-13 viruses, n (%) 54 (22.0) 11 (18.6) 43 (23.0) 1 1
Parentally reported infections
4-9 infections, n (%) 54 (22) 17 (28.8) 37 (19.8) 1.48; 0.64-3.46 1.40; 0.50-3.95
10-13 infections, n (%) 75 (30.5) 13 (22.0) 62 (33.2) 0.68; 0.29-1.61 0.57; 0.21-1.54
14-16 infections, n (%) 62 (25.2) 16 (27.1) 46 (24.6) 1.12; 0.48-2.61 1.11; 0.40-3.08
17-24 infections, n (%) 55 (22.3) 13 (22.0) 42 (22.5) 1 1
OR, Odds ratio; ORadj, adjusted odds ratio.
with clinical signs of atopy compared with their nonatopic Several plausible explanations are possible, including the
counterparts.15 However, Calvani et al14 reported a higher idea that IL-10 homologues present in the viruses might
prevalence of atopy among EBV seronegative children in downregulate the antigen processing/presentation capac-
the age group 0 to 6 years, corroborating our results. ity of dendritic cells/macrophages and thereby switch off
A recent Swedish study (BAMSE) failed to demonstrate the host T-cell system, similar to the downregulation
that the EBV serostatus in 4-year-old children correlated observed for T regulatory cells.22,23 Alternately, both
with IgE sensitization.16 In developing countries, where EBV and CMV can polyclonally activate B cells to
EBV asymptomatically infects the majority of children produce antibodies with many different specificities and
before 3 years of age,17 the prevalence of atopy has been thereby hinder the capacity of allergens to cross-link the
reported to be lower than in industrialized countries.18 In B-cell receptor as seen for helmintic infections.24 Thus,
combination with our findings, these observations might these data and our results provide further support for the
indicate an age-dependent role of EBV, rather than EBV hypothesis that specific characteristics of EBV and/or
infection per se, in relation to IgE sensitization. CMV infection, rather than infection per se, might influ-
Cytomegalovirus becomes persistent after primary in- ence the risk of IgE sensitization.
fection and seems to induce a TH1 cytokines response.19 Interestingly, RSV infection, according to serology,
CMV is frequently transmitted from mothers to infants was not associated with IgE sensitization at 24 months of
during pregnancy, at delivery, or via breast milk.20 The age. Previous studies have suggested that hospitalization
few studies published on the relation between CMV and because of RSV infection is linked with atopy and
allergies are inconclusive.21 In the Swedish BAMSE induction of IgE synthesis.25 Discrepancies between dif-
study, no association was found between CMV seropos- ferent studies indicate that the vulnerability to RSV might
itivity and IgE sensitization in 4-year-old children. be associated with a propensity for asthma and allergy, and
However, among children with seropositivity against not that RSV per se causes asthma. Again, the character-
CMV and seronegativity against EBV, there was a pos- istics of infection may be important, and identification
itive association with sensitization to food allergens.7 This through hospitalization suggests more severe acute infec-
led us to test the interaction of seropositivity against CMV tion than the majority of children in our study would have
and EBV in relation to IgE sensitization. This indicated had. Blanco-Quiros et al26 reported that infants who de-
effect modification such that the negative association of veloped severe RSV bronchiolitis had low levels of IL-12,
EBV seropositivity with sensitization was further en- a strong TH1 inducer, in cord blood. We have recently
hanced in children who were also seropositive for CMV. published similar data showing low levels of IL-12 in cord
The mechanism for this putative interaction is unknown. blood of IgE sensitized infants at 24 months of age.27
These observations might indicate an interaction among confer greater protection, such as EBV infection in the
cytokines, RSV, and sensitization during early infancy. age-defined window of susceptibility with possible mod-
There was some indication that the numbers of sero- ification through interaction with CMV. Development of
logically verified viral infections were inversely correlated more precise measures of patterns of acute infection and
to IgE sensitization, but there was no consistent trend their immunological sequel will assist in our understand-
across the groups and no statistically significant associa- ing of how early in life viral infections are implicated in
tion. The number of parentally reported infections and the etiology of IgE sensitization.
seropositivity against the selected viruses did not corre-
late. This is not entirely surprising, because many viruses, We thank the families who participated in the study. We also thank
eg, rhinovirus and corona virus, which are proposed to be Anna Stina Ander, Johan Berggren, Jeanette Harrysson, Lena
the major causes of upper respiratory infections in infants, Jägdahl, and Monica Nordlund for assistance.
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
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tive. The strength of our study is that the atopic status of 1259-60.
the parents was characterized, and information on infec- 6. Grillner L, Strangert K. A prospective molecular epidemiological study
tions was collected prospectively by using diaries. of cytomegalovirus infections in two day care centers in Sweden: no
evidence for horizontal transmission within the centers. J Infect Dis
Importantly, objective serological measurements against 1988;157:1080-3.
antiviral antibodies were made, and the results were 7. Sidorchuk A, Wickman M, Pershagen G, Lagarde F, Linde A. Cyto-
adjusted for factors that might bias the evaluation. megalovirus infection and development of allergic diseases in early
However, we recognize some limitations. The study childhood: interaction with EBV infection? J Allergy Clin Immunol
2004;114:1434-40.
population was selected and not population-based.
8. Johansson SG, Hourihane JO, Bousquet J, Bruijnzeel-Koomen C,
Because we selected children with different family histo- Dreborg S, Haahtela T, et al. A revised nomenclature for allergy: an
ries of allergy, we believe that the group of children in our EAACI position statement from the EAACI nomenclature task force.
study is fairly comparable with children in the general Allergy 2001;56:813-24.
population. Furthermore, the exact sensitivity for sero- 9. Linde A, Fridell E, Dahl H, Andersson J, Biberfeld P, Wahren B. Effect
of primary Epstein-Barr virus infection on human herpesvirus 6,
positivity in some of the assays used to evaluate the cytomegalovirus, and measles virus immunoglobulin G titers. J Clin
respiratory infections is not fully known because they Microbiol 1990;28:211-5.
have been used mainly to detect ongoing infections, so the 10. Linde A, Andersson J, Lundgren G, Wahren B. Subclass reactivity to
rate of seropositivity is somewhat underestimated, but this Epstein- Barr virus capsid antigen in primary and reactivated EBV
infections. J Med Virol 1987;21:109-21.
should not introduce bias. The optimal approach would
11. Sundqvist VA, Linde A, Wahren B. Virus-specific immunoglobulin G
have been to perform neutralization assays, but serum subclasses in herpes simplex and varicella-zoster virus infections. J Clin
samples from infants are inevitably insufficient for tests Microbiol 1984;20:94-8.
using low serum dilutions. In contrast, the sensitivity and 12. Sundqvist VA, Wahren B. An interchangeable ELISA for cytomegalo-
specificity for seropositivity of the assays used for the virus antigen and antibody. J Virol Methods 1981;2:301-12.
13. Stepanova L, Naykhin A, Kolmskog C, Jonson G, Barantceva I,
herpesvirus infections have been proven reliable, as best Bichurina M, et al. The humoral response to live and inactivated
demonstrated by the correlation between initial serostatus influenza vaccines administered alone and in combination to young
and clinical outcome found in transplant recipients.31 It adults and elderly. J Clin Virol 2002;24:193-201.
could also be argued that analyzing 13 different viruses 14. Calvani M, Alessandri C, Paolone G, Rosengard L, Di Caro A,
De Franco D. Correlation between Epstein Barr virus antibodies, serum
could produce some associations with IgE sensitization by
IgE and atopic disease. Pediatr Allergy Immunol 1997;8:91-6.
chance. However, because an association specifically with 15. Strannegard IL, Strannegard O. Epstein-Barr virus antibodies in children
EBV was evaluated as a result of an a priori hypothesis with atopic disease. Int Arch Allergy Appl Immunol 1981;64:314-9.
based on the results of previous studies,14 the associations 16. Sidorchuk A, Lagarde F, Pershagen G, Wickman M, Linde A. Epstein-
reported for EBV are unlikely to be caused by chance. Barr virus infection is not associated with development of allergy in
children. Pediatr Infect Dis J 2003;22:642-7.
In summary, our results indicate that an EBV infection 17. Cohen JI. Epstein-Barr virus infection. N Engl J Med 2000;343:481-92.
during the first 2 years of life is associated with a reduced 18. Addo Yobo EO, Custovic A, Taggart SC, Asafo-Agyei AP,
risk of IgE sensitization. Some patterns of infection may Woodcock A. Exercise induced bronchospasm in Ghana: differences
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in prevalence between urban and rural schoolchildren. Thorax 1997; 25. Schauer U, Hoffjan S, Bittscheidt J, Kochling A, Hemmis S, Bongartz S,
52:161-5. et al. RSV bronchiolitis and risk of wheeze and allergic sensitisation in
19. Rentenaar RJ, Gamadia LE, van DerHoek N, van Diepen FN, Boom R, the first year of life. Eur Respir J 2002;20:1277-83.
Weel JF, et al. Development of virus-specific CD4(1) T cells during 26. Blanco-Quiros A, Gonzalez H, Arranz E, Lapena S. Decreased
primary cytomegalovirus infection. J Clin Invest 2000;105:541-8. interleukin-12 levels in umbilical cord blood in children who developed
20. Ahlfors K, Ivarsson SA, Harris S. Report on a long-term study of acute bronchiolitis. Pediatr Pulmonol 1999;28:175-80.
maternal and congenital cytomegalovirus infection in Sweden: review of 27. Nilsson C, Larsson A-K, Söderlund A, Gabrielsson S, Troye Blomberg
prospective studies available in the literature. Scand J Infect Dis 1999;31: M, Lilja G. Low numbers of IL-12-producing cord blood mononuclear
443-57. cells and immunoglobulin E sensitization in early childhood. Clin Exp
21. Wu CA, Puddington L, Whiteley HE, Yiamouyiannis CA, Schramm Allergy 2004;34:373-80.
CM, Mohammadu F, et al. Murine cytomegalovirus infection alters 28. Johnston SL. Natural and experimental rhinovirus infections of the
Th1/Th2 cytokine expression, decreases airway eosinophilia, and lower respiratory tract [review]. Am J Respir Crit Care Med 1995;152:
enhances mucus production in allergic airway disease. J Immunol S46-52.
2001;167:2798-807. 29. Celedon JC, Wright RJ, Litonjua AA, Sredl D, Ryan L, Weiss ST, et al.
22. Salek-Ardakani S, Arrand JR, Mackett M. Epstein-Barr virus encoded Day care attendance in early life, maternal history of asthma, and asthma
interleukin-10 inhibits HLA-class I, ICAM-1, and B7 expression on at the age of 6 years. Am J Respir Crit Care Med 2003;167:1239-43.
human monocytes: implications for immune evasion by EBV. Virology 30. Lau S, Nickel R, Niggemann B, Gruber C, Sommerfeld C, Illi S, et al.
2002;304:342-51. The development of childhood asthma: lessons from the German
23. Raftery MJ, Wieland D, Gronewald S, Kraus AA, Giese T, Schonrich G. Multicentre Allergy Study (MAS). Paediatr Respir Rev 2002;3:265-72.
Shaping phenotype, function, and survival of dendritic cells by cyto- 31. Ljungman P, Aschan J, Lewensohn-Fuchs I, Carlens S, Larsson K,
megalovirus-encoded IL-10. J Immunol 2004;173:3383-91. Lonnqvist B, et al. Results of different strategies for reducing cytomeg-
24. Smits HH, Hartgers FC, Yazdanbakhsh M. Helminth infections: protec- alovirus-associated mortality in allogeneic stem cell transplant recipients.
tion from atopic disorders. Curr Allergy Asthma Rep 2005;5:42-50. Transplantation 1998;66:1330-4.
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
Background: IgE antibody-producing B cells are enriched in

the nasal mucosa in patients with allergic rhinitis because of Abbreviations used
local class switching to IgE. The expressed IgE VH genes also CDR: Complementarity-determining region
undergo somatic hypermutation in situ to generate clonal FWR: Framework region
families. The antigenic driving force behind these events is R/S: Replacement/silent mutation ratio
unknown.
Objective: To examine the possible involvement of a super-
antigen 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.
IgE and its receptors are central to allergic disease,
Methods: We extracted mRNA from the nasal biopsy specimens manifested in different target organs, including the nose
of 13 patients and 4 nonallergic control subjects and PBMCs (allergic rhinitis), the lung (allergic asthma), the skin
from 7 allergic patients. IgE and IgA VH regions were RT-PCR (atopic dermatitis), and the gut (allergic gastroenteritis).
amplified, and the DNA sequences were compared with those IgE binds to effector and antigen-presenting cells bearing
of control subjects. We constructed a molecular model of VH5 IgE receptors (FceRI, CD23, or both) in mucosal tissues
to locate amino acids of interest. associated with the target organs mediating the allergic
Results: We observed a significantly increased frequency of response. In addition, IgE-mediated allergen presentation
IgE and IgA VH5 transcripts in the nasal mucosa of the to T helper cells might lead to renewed IgE antibody
allergic patients compared with the normal PBMC repertoire.
synthesis, epitope spreading, and exacerbation of allergic
Within IgE and IgA VH5 sequences in the nasal mucosa, the
distribution of replacement amino acids was skewed toward the
sensitivity. A large number of genetic and environmental
immunoglobulin framework regions. Three of 4 nonintrinsic risk factors for allergy have been identified, providing
hotspots of mutation identified in the VH5 sequences were in broad insight into the pathogenesis of allergic disease. The
framework region 1. The hotspots and a conserved VH5-specific factors that determine the susceptible target organ in
framework residue form a tight cluster on the surface of VH5. different individuals exposed to the same allergens are,
Conclusion: Our results provide evidence for the activity of however, unknown. We suggest that these factors might
a superantigen in the nasal mucosa in patients with allergic be localized in the target organ and might include super-
rhinitis. (J Allergy Clin Immunol 2005;116:445-52.) antigens.
Snow and coworkers1-5 have observed a bias in the
Key words: Human, allergic rhinitis, VH5, superantigen, B lympho- repertoire of IgE heavy-chain variable (IgE VH) regions in
cyte, mucosa
asthma, which exhibited the hallmarks of a superantigen.
There are 51 VH genes grouped into 7 gene families (VH1-
VH7), varying in size from 22 members in VH3 to 1 or
From aThe Randall Division of Cell and Molecular Biophysics, King’s College 2 members in VH5, VH6, and VH7.6 Each VH region has
London; bUpper Respiratory Medicine, National Heart and Lung Institute, 3 framework regions (FWR1-FWR3) alternating with
Imperial College, London; and cthe Department of Respiratory Medicine
and Allergy, Guy’s Hospital, London.
3 complementarity-determining regions (CDR1-CDR3).
*Dr Coker is currently affiliated with Clare Hall Laboratories, Cancer Research The FWRs determine the structural framework for the
UK, South Mimms, Hertfordshire, United Kingdom. antigen-binding sites of the CDRs. The CDR sequences
Supported by the Clinical Research Committee Royal Brompton and Harefield are inherently more prone to somatic hypermutation
Hospitals NHS Trust. HAC and HEH were supported by BBSRC PhD
during affinity maturation of antibodies in the immune
studentships, and HG and SRD were supported by project grants from
Asthma UK (grant no. 03/055) and the MRC (grant no. G0200485). response, whereas the FWR sequences are relatively
Received for publication August 25, 2004; revised March 23, 2005; accepted conserved.7 In peripheral blood B cells of healthy indi-
for publication April 22, 2005. viduals, the proportion of expressed VH regions from
Available online June 17, 2005. different VH families generally reflects the size of the
Reprints of this article are not available from the authors.
0091-6749/$30.00
family.8 In asthma, however, Snow and coworkers found
Ó 2005 American Academy of Allergy, Asthma and Immunology an overabundant use of the minor VH5 family in IgE in the
doi:10.1016/j.jaci.2005.04.032 blood, lung mucosa, and spleen of asthmatic patients1-3
445
446 Coker et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
and in the blood of the majority of 6 asthmatic patients.5 agreement with previous authors,9,19 we were unable to use healthy
Overabundant use of VH5 was also reported in the blood of subjects as a control group for IgE because PCR amplification of IgE
3 patients with atopic dermatitis9 but not in another study was only successful in the allergic subjects. Instead, we recruited 4
nonallergic subjects (AA2, MTS3, KB5, and SK6) and used biopsy
of 2 patients with atopic dermatitis.10 By contrast, in
specimens from 2 of the allergic patients (TL25 and CA30) plus 2
the blood of 2 subjects with peanut allergy, Snow and
additional patients with multiple allergies (JC1 and IB4) to examine
coworkers observed an overabundant use of VH1, sug- VH use in IgA.
gesting the activity of a peanut-associated superantigen.11 Volunteers were recruited from the Royal Brompton Hospital
B-cell superantigens, similar to T-cell superantigens, Allergy Clinic or by advertisement in the local press to donate a nasal
act by binding to immunoglobulin FWRs, leading to biopsy specimen. None had received immunotherapy, and any
clonal amplification of all members of the family. Because medication was discontinued at least 2 weeks before nasal biopsy.
the number of CDRs in the B-cell repertoire (millions) Biopsies were performed at the Royal Brompton Hospital, London,
is vastly greater than the number of VH families (7), United Kingdom, and processed as described previously.20 All such
this results in biased use of the selected family.12 work had the approval of the local ethics committee and the patients’
written informed consent. Blood samples were taken from 7 of the 16
Two previously identified B-cell superantigens,
patients who also donated nasal biopsy specimens (CD6, JB7, CM10,
Staphylococcus aureus protein A and HIV gp120, selec-
HD14, SO16, HD17, and AP19). PBMCs, including B cells, were
tively expand B cells expressing VH3.12-14 The interaction isolated from these samples, as described previously.19 The tissue
of protein A with the FWR of VH3 can be seen in the sample from the inferior turbinate resulted from operations performed
crystal structure of a complex with the VH3 Fab fragment at Guy’s Hospital, London, United Kingdom, with the approval of the
of an IgM antibody.15 Superantigen-selected B cells might Guy’s Research Ethics Committee and also with the patient’s written
also exhibit a skewed distribution of amino acid substitu- informed consent.
tions 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 Amplification and analysis of
a further criterion for CDR- versus FWR-oriented selec- VH region sequences
tion; the sequences WRC and WA, which occur more Total RNA was extracted from both the nasal mucosa and PBMC
frequently in the CDRs, are recognized as intrinsic samples, cDNA was produced, and VH-Ce sequences were PCR
hotspots of mutation.17 These 2 features of superantigen amplified by using the proofreading Pfu DNA polymerase (Promega,
selection of B cells were also linked to the VH5 over- Madison, Wis) from IgE-positive B cells. The VH-Ce PCR products
abundance in asthma and atopic dermatitis,2,9 which is were then cloned and sequenced. This entire procedure has been
described in detail previously.19 VH-Ca sequences were amplified
consistent with the influence of a superantigen and in
from cDNA in the same way as VH-Ce sequences, replacing the
contrast to the lack of such influence observed in VH5 Ce-specific primers with nested primers specific for Ca: Ca1, 5#-
B cells from normal spleen.18 TTTCGCTCCAGGTCACAC-3#; Ca2, 5#-GGGAAGAAGCCCT-
In a previous study of the expressed VH regions in GGACCAGGC-3#. The annealing temperature for the second round
allergic rhinitis, we presented evidence of local clonal of PCR was adjusted from 65°C to 69°C. Assignment of the VH genes
expansion, somatic hypermutation, and class switching in and their somatic mutations was carried out according to their
the nasal mucosa in patients with allergic rhinitis.19 In this homology with the germline sequences detailed on the VBase
study we present a detailed analysis of IgE and IgA VH database (www.mrc-cpe.cam.ac.uk).
family use in the nasal mucosa in patients with allergic Only unique sequences were included in the analysis, meaning
rhinitis. 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
METHODS B cells, each unique mutation isolated from the family members
was included in the mutational analysis.
Samples from patients with allergic rhinitis Statistical significance was determined by the use of x2 analysis
and nonallergic control subjects with the Yates correction for continuity (generating a more con-
Male and female donors with allergic rhinitis aged between 18 and servative calculation of significance when smaller data values are
55 years were recruited for this study. The allergic status of the donors used).
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 Molecular modeling of VH5 antibody structure
multiple allergies (CD6, JB7, CM10, HD14, SO16, AP19, SJ24, Because no VH5 antibody crystal structure is available, a model
TL25, CA30, and SLT1) who were allergic to grass and also allergens was generated for the VH5-51 germline sequence on the basis of the
such as animal dander or house dust mite, to which they could be known structure of the most closely matched antibody (PDB code:
perennially exposed. One tissue sample (HD17) originated from a 1CGS). The heavy-chain CDR1 and CDR2 (H1 and H2) loop lengths
patient with only grass pollen allergy. The samples were taken were identical in both antibody sequences, and the heavy-chain
throughout the year, with the patient with only grass pollen allergy CDR3 (H3) loop and the light chain (VL) domain structures were
undergoing biopsy within the grass pollen season. Of the 11 nasal taken directly from 1CGS to produce a complete Fv model. No steric
mucosa samples analyzed, 10 were biopsy specimens taken from the clashes were detected after substitution of the VH5-51 sequence. The
inferior turbinate, and one (SLT1) consisted of a piece of an inferior model was generated by using HOMOLOGY and displayed with
turbinate removed by surgery to alleviate nasal obstruction. In INSIGHT II (Accelrys, Cambridge, United Kingdom).
J ALLERGY CLIN IMMUNOL 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
RESULTS These data were taken from sequences pooled from

multiple patients. The occurrence of VH5 sequences
IgE VH gene use
across the PBMC samples appeared to be evenly distrib-
We examined a total of 62 in-frame VH region se- uted (with the 4 VH5 sequences occurring in 4 of the 7
quences derived from the nasal mucosa of the 11 patients different PBMC samples). This was in contrast to the
with allergic rhinitis* and 50 VH region sequences from distribution of VH5 sequences in the samples from the
the PBMCs of 7 of these patients. There was a clear nasal mucosa, in which the 18 VH5 sequences were
difference in the VH gene use observed in IgE B cells from isolated from only 5 of the 11 patients. There was a
the nasal mucosa of the allergic patients compared with particularly high level of VH5 use in the nasal mucosa of
that expected on the basis of the normal genomic PBMC patient CA30, in whom 11 of 18 sequences were from
repertoire21 and also compared with that observed in IgE- unrelated B cells expressing VH5 IgE-positive sequences.
positive PBMCs from the allergic patients (Fig 1).21 However, even when this patient was excluded from the
There was a highly significant decrease in the use of analysis, the increased VH5 IgE use (14%) in the allergic
VH3 by IgE-positive B cells in the allergic nasal mucosa nasal mucosa compared with that expected on the basis of
(34%) compared with that expected on the basis of the the normal PBMC repertoire was still highly significant
normal repertoire in PBMCs (55%) observed by previous (P < .005, x2 analysis), although the significance between
workers (P < .005, x2 analysis).21 This was not, however, the allergic nasal mucosa and allergic PBMCs was lost
significant when compared with that observed in the (P > .1, x2 analysis).
allergic PBMCs (50%; P > .1, x2 analysis).
There was also a highly significant increase in the use Hotspots of mutation in IgE VH5 sequences
of VH5 in the nasal mucosa (29%) compared with that from the allergic nasal mucosa Basic and clinical immunology
observed by previous researchers in normal PBMCs All of the sequences isolated from nasal biopsy spec-
(2.9%; P < .005, x2 analysis). The increased use of VH5 imens exhibited evidence of somatic hypermutation.
in the allergic nasal mucosa was significant when com- Somatic mutations evident within 17 distinct VH5 se-
pared with the 8% VH5 use observed in the allergic IgE- quences were compared with those from 19 randomly
positive PBMCs (P < .025, x2 analysis). There was no chosen non-VH5 sequences to determine whether there
significant difference in the use of VH5 in the allergic was evidence to suggest that the overuse of VH5 in the
PBMCs compared with that expected on the basis allergic nasal mucosa might have been a consequence of
of the normal PBMC repertoire (8% vs 2.9%; P > .1, B-cell selection (eg, by the presence of nonintrinsic
x2 analysis). hotspots of mutation).
The percentage variability of each codon was used to
identify apparent hotspots of somatic hypermutation
*Nucleotide sequences submitted to Genbank, accession numbers AY640472–
AY640533.
(Fig 2). The percentage mutation (including both silent
Nucleotide sequences submitted to Genbank, accession numbers AY640534– and replacement mutations) and the percentage mutation
AY640583. at each nucleotide were also analyzed (data not shown),
AUGUST 2005
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.
although neither identified any further hotspots of muta- TABLE I. The direction of somatic mutation at hotspots
tion to that evident from Fig 2. in VH IgE sequences from the allergic nasal mucosa
Of the hotspots of mutation identified in Fig 2, those at suggests the presence of 4 nonintrinsic hotspots in
Met 40 and Tyr 56 in the VH5 sequences and at codons VH5 sequences
52a, 56, and 84 in the non-VH5 sequences, when examined Hotspot From To O* Ey
in more detail, were disregarded because the mutations
were spread evenly across the 3 nucleotide positions of the VH 5
codon and had very different effects on the amino acid. Lys 23(1) AAG CAG Gln 6 2 Nonintrinsic
GAG Glu 2 4
The apparent hotspots at codon 52b and also codon 82a in
TAG Stop 0 2
the non-VH5 sequences were also disregarded because Gly 24(2) GGT GAT Asp 0 2 Nonintrinsic
codon 52b was mutated in both of only 2 sequences that GCT Ala 5 0
used this polymorphic codon, and the mutations at codon GTT Val 1 4
82a were divided between 3 Asn and 3 Ser residues, such Ser 28(3) AGC AGA Arg 3 1
that there were insufficient mutations of each for analysis. AGG Arg 2 2
The exact nature of the remaining apparent hotspots of AGT Ser 4 6
mutation was examined in detail to determine whether the Thr 30(2) ACC AAC Asn 1 1 Nonintrinsic
direction of mutation at each codon reflected the accepted AGC Ser 4 1
trends of somatic hypermutation, as defined by previous ATC Ile 2 5
Ser 31(2) AGC AAC Asn 7 7
authors.22 Those mutations that conformed to such a
ACC Thr 5 4
profile were defined as intrinsic, whereas those that clearly ATC Ile 0 1
differed were defined as nonintrinsic and therefore likely Ser 31(3) AGC AGA Arg 1 1
to have been selected in response to antigen (Table I).22 AGG Arg 0 1
The only hotspot apparent in the non-VH5 sequences AGT Ser 6 5
was a commonly identified hotspot, Ser 31,22 which was Tyr 32(3) TAC TAA Stop 0 0
classified as intrinsic. In contrast, although 9 of the 13 TAG Stop 0 1
remaining hotspots identified in the VH5 sequences also TAT Tyr 4 3
appeared to be intrinsic in nature, importantly, 4 apparent Tyr 52(2) TAT TCT Ser 1 1 Nonintrinsic
nonintrinsic hotspots were also identified. This is consis- TGT Cys 1 4
TTT Phe 5 2
tent with the non-VH5 IgE molecules having been targeted
Asp 53(3) GAT GAA Glu 4 2
toward a wide range of antigens, whereas the identification GAC Asp 2 4
of nonintrinsic hotspots of mutation in the VH5 sequences GAG Glu 1 1
suggests that the VH5 IgE molecules were targeted toward Ser 57 (3) AGC AGA Arg 0 1
a limited number of antigens. The 4 nonintrinsic mutations AGG Arg 0 1
evident in the VH5 sequences were present at Lys 23(1), AGT Ser 7 5
Gly 24(2), and Thr 30(2), each in FWR1, and also at Ala 71(2) GCC GAC Asp 0 1
Tyr 52(2), which was found in CDR2. According to GGC Gly 0 1
Kabat’s numbering, CDR1 incorporates codons 31 GTC Val 6 4
through 35, and CDR2 incorporates codons 50 through Ser 76(2) AGC AAC Asn 6 4
ACC Thr 1 2
65. FWR1 incorporates codons 1 through 30, FWR2
ATC Ile 0 1
incorporates codons 36 through 49, and FWR3 incorpo- Met 89(3) ATG ATA Ile 4 3
rates codons 66 through 95. ATC Ile 0 2
ATT Ile 1 0
Non-VH5
Analysis of replacement/silent mutation Ser 31(2) AGC AAC Asn 5 3
ratio values ACC Thr 0 2 Basic and clinical immunology
Because 3 of the 4 apparently nonintrinsic hotspots of ATC Ile 0 0
mutation in the VH5 sequences were unconventionally
*O denotes the observed number of somatic mutations.
present in the framework region, further analysis of the E denotes the expected direction of mutation on the basis of previous
ratio of replacement to silent mutations (R/S values) in the research.22
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 tions were present in FWR1, analysis of the R/S values of
difference between the CDR and the FWR (CDR, 3.46; the individual FWRs in the VH5 group of sequences was
FWR, 1.67; P < .025, x2 analysis). In stark contrast, there also carried out. FWR1 exhibited an R/S value of 2.16,
was no significant difference between the R/S values in the FWR2 exhibited an R/S value of 1.06, and FWR3
CDR and the FWR of the VH5 group of sequences (CDR, exhibited an R/S value of 2.61, suggesting that although
2.38; FWR, 2.06; P > .1, x2 analysis). FWR2 was conventionally conserved in the VH5 se-
Because the VH5 FWR R/S value was higher than quences, FWR1 and FWR3 contributed to the unusual
expected and also because 3 of the 4 nonintrinsic muta- overall FWR R/S value observed in the VH5 sequences.
AUGUST 2005
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 CA30A 0 0 0 0 11 0 0 11
CA30B 0 0 1 3 0 0 0 4
IgA CA30A 1 0 8 1 2 0 0 12
CA30B 2 0 6 1 0 1 0 10
IgA VH gene use

If a superantigen was involved in the biased use of FIG 3. Location of nonintrinsic hotspots in the 3-dimensional
VH5, this might be apparent in isotypes other than IgE. structure. Hotspots Lys23, Gly24 and Thr30 (FWR1), and Tyr52
We first examined this possibility by using 2 adjacent (CDR2) in VH5 are shown in yellow on a homology model of VH5-51.
biopsy specimens (CA30A and CA30B) from patient Exposed hydrophobic residue Ile75 is shown in green. CDRs are
indicated, encompassing both Kabat and Chothia24 definitions. The
CA30. remaining VH and VL FWRs are shown in dark and light blue.
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 Location of nonintrinsic hotspots in the
pattern, with 2 of 12 IgA VH sequences in biopsy structure of IgE VH5
specimen A but none of 10 in biopsy specimen B being The 4 nonintrinsic hotspots occurred at Lys 23(1),
VH5. Combining IgE and IgA results, 57% (13/37) of Gly 24(2), Thr 30(2), and Tyr 52(2). Of these, 3 were
sequences in biopsy specimen A were VH5, whereas 0% unusually situated within FWR1, with only Tyr 52(2) in
(0/14) of sequences in biopsy specimen B was VH5. None CDR2. Therefore it would seem likely that the putative
of 13 VH5 sequences in CA30 were related to any of the superantigen has contact with FWR1 and CDR2, although
others, suggesting polyclonal activation of B cells ex- the R/S values imply that FWR3 might also be involved.
pressing VH5 sequences locally in the region of biopsy The B-cell superantigen protein A has been shown to bind
specimen A. The results for CA30 demonstrate the linkage similarly to VH3, interacting with FWR1, FWR3, and
between VH5 bias in IgE and IgA and suggest that a B-cell CDR2.12,13,15,23
superantigen might influence the selection of VH in a local Because no crystal structure of a VH5 is available, we
manner. constructed a model on the basis of the known structure of
The FWR and CDR R/S values for IgA VH5 and non- the most closely matched antibody. There are 2 VH5
VH5 were determined and compared. R/S values for genes, VH5-51 and VH5-a, but only the former is ex-
IgA non-VH5 sequences in CA30 were comparable with pressed in the majority of the population. Fig 324 shows
those of IgE non-VH5 sequences (CDR, 4; FWR, 1.4) Lys 23, Gly 24, and Thr 30 in FWR1 and Tyr 52 in CDR2
and thus indicative of normal antigen selection. However, in our model of VH5-51.
as observed for IgE VH5, the FWR and CDR R/S values When the locations of the 4 nonintrinsic hotspots are
for IgA VH5 sequences were similar (CDR, 4; FWR, identified within this model, it is immediately apparent
4.3). that they are clustered together at the edge of the conven-
On analysis of 73 IgA VH sequencesà in 2 of the biopsy tional antigen-binding site, as defined by the 6 CDRs. Lys
specimens from the original cohort (TL25 and CA30) plus 23 and Gly 24 are adjacent to CDR1, and Thr 30 lies at the
2 additional biopsy specimens (JC1 and IB4) from allergic very boundary between FWR1 and CDR1; Tyr 52 lies at
patients, we observed VH5 to be significantly higher than the boundary between FWR2 and CDR2. Also shown in
in normal PBMCs (14% vs 2.9%; P < .005, x2 analysis).21 Fig 3 is residue Ile 75, a hydrophobic surface residue in an
The greater abundance of IgA-expressing compared with unusually exposed location but conserved in the vast
IgE-expressing B cells in the nasal mucosa allowed us to majority of VH5 sequences. In fact, all VH2, VH3, VH4,
determine the IgA VH use in the nasal mucosa of healthy and VH6 germline sequences have lysine at this position,
nonallergic subjects. Of 55 sequences§ from 4 nonallergic whereas it is isoleucine only in VH5. This cluster of
donors, we found a 9% frequency of VH5, which is also residues might thus define at least a part of the site at which
significantly different from that seen in normal PBMCs a putative superantigen might interact with IgE VH5.
(P <.05, x2 analysis).21
DISCUSSION
àNucleotide sequences submitted to Genbank, accession numbers AY971069-
AY971142
In our previous study of somatic hypermutation in VH
§Nucleotide sequences submitted to Genbank, accession numbers AY971012- sequences in IgE-expressing B cells in the nasal mucosa of
AY971068 patients with allergic rhinitis, we observed that 2 of a total
of 3 clonal families were derived from the VH5 family, and antibodies that recognize the carbohydrate I/i red blood
we detected an IgA VH5 clone related to one of the cell antigen are restricted to the VH4-34 gene, and residues
families.19 This hinted at a biased use of VH5 in the tissue 23-25 in FWR1 are implicated in binding.25 In addition,
similar to that found in asthma and atopic dermatitis.1-5,9 the crystal structure of the complex between a rheumatoid
We have therefore analyzed VH sequences from a large factor antibody and its autoantigen, IgG Fc, involved
cohort of patients with allergic rhinitis to test this hypoth- residues at the FWR1/CDR1 and FWR2/CDR2 bound-
esis. aries, enabling the CDR to remain available for conven-
Our results have revealed a significant bias toward VH5, tional antigen binding.26,27
even when one biopsy (CA30), which exhibited the most Remarkably, exactly the same features that we have
extreme bias, was excluded. A similar trend in the PBMCs observed in IgE VH5 in allergic rhinitis were previously
from a subgroup of these patients did not reach statistical reported in asthma.1-5 For example, VH5 represented 29%
significance. The greater bias in tissue suggests that a local and 33% of the IgE sequences in this allergic rhinitis study
event caused the VH5 bias. The similar trend in PBMCs and a previous asthma study,2 respectively. Exactly the
might result from the displacement of VH5-expressing same nonintrinsic hotspots, Lys 23(1), Gly 24(2), Thr
B cells from the tissue and dilution in the pool of normal 30(2), and Tyr 52(2), were observed in asthma1 as in this
B cells in the circulation. IgA VH sequences also exhibited study of allergic rhinitis but differing from those in atopic
a significant but less extreme bias toward VH5 compared dermatitis (Gly 35 and Thr 95).22 The similarities between
with that seen in IgE sequences. The demonstration of allergic rhinitis and asthma might not be surprising, given
VH5 bias in both IgE and IgA and colocalization in the that the upper and lower airways are physically contiguous
nasal mucosa support the superantigen hypothesis. The and exposed to some of the same aeroallergens, but are
putative superantigen might bind to the subset of B cells important, given the differing susceptibility of some
expressing VH5, leading to selective proliferation or individuals to asthma and others to rhinitis and that
rescue from apoptosis. The smaller bias in IgA VH5 might many individuals have both conditions. It might be
be due to the TH2 environment of the nasal mucosa, which fortuitous that the same minor VH family appears to be
favors class switching to IgE in rapidly dividing B cells. overexpressed in both allergic rhinitis–asthma and (at least
We observed an even smaller IgA VH5 bias in the normal in one of the 2 studies) atopic dermatitis. The selection of
nasal mucosa, which might hint that the bias precedes the different FWR mutations in the IgE VH5 in atopic
development of allergy. Allergen-specific B cells might be dermatitis, however, might point to the action of different
further selected from this population. superantigens.
Although there was a significant IgE VH5 bias in the 62 What might be the putative B-cell superantigen asso-
sequences analyzed, these sequences came from a minor- ciated with allergic rhinitis? About 37% of the population
ity (5/11) of the patient population. This must not be taken carries Staphylococcus aureus in the nasal mucosa.28
to indicate that a superantigen is likely to have been acting Certain S aureus enterotoxins are characterized as T-cell
in only the 5 patients, however. We have demonstrated superantigens that stimulate B-cell proliferation, IgE
previously19 and again here, exemplified by the compar- synthesis, or both.29-31 Some of these or others might act
ison of biopsy specimens A and B from patient CA30, that as B-cell superantigens. If a B-cell superantigen binds as
there are not necessarily any identical sequences or even we suspect to FWR1 of VH5, an allergen might still bind
clonal relationships between cells in adjacent 1- to 2-mm3 to the CDR on the immunoglobulin. This could have
biopsy specimens. These biopsy specimens (10-20 mg) important consequences for the activation of B cells
are less than 1% of the inferior turbinates (average weight, expressing VH5 and also the mast cells and dendritic cells
approximately 2 g). Thus sequences derived from a single that capture IgE VH5 in the tissue.
biopsy specimen are not necessarily representative of the
tissues in individual patients. Our VH5 bias is based on We thank Drs Rebecca Beavil, Pooja Takhar, Lyn Smurthwaite,
sampling a population of patients. The absence of VH5 and Graham Dunn (King’s College London) for helpful discussions
sequences in 6 of 11 of the biopsy specimens does not and practical advice.
imply that there are none elsewhere in the tissue.
Superantigens acting in other regions of the tissue might
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Antibody responses against galactocerebroside
are potential stage-specific biomarkers in
multiple sclerosis
Til Menge, MD,a Patrice H. Lalive, MD,a Hans-Christian von Büdingen, MD,a,b
Bruce Cree, MD, PhD,a Stephen L. Hauser, MD,a and Claude P. Genain, MDa
San Francisco, Calif, and Zürich, Switzerland
Background: Galactocerebroside, the major glycolipid of Key words: Galactocerebroside, myelin antigens, autoantibody,
central nervous system myelin, is a known target for multiple sclerosis, experimental allergic encephalomyelitis
pathogenic demyelinating antibody responses in experimental
allergic encephalomyelitis (EAE), the animal model of multiple
sclerosis (MS).
Objective: To address the importance of anti-galactocerebroside Multiple sclerosis (MS) is a chronic immune-mediated
(a-GalC) antibodies in MS and to evaluate them as biomarkers inflammatory demyelinating disease of the central nervous
of disease. system (CNS) characterized by heterogeneity in clinical
Methods: a-GalC IgGs were quantified from sera of patients presentation and underlying pathological mechanisms.1
with MS and in marmoset EAE by a new immunosorbent assay. There is currently no easy paraclinical marker to diagnose
Results: We report a significant difference in serum a-GalC MS subtypes and predict disease course accurately with-
IgG titers between patients with relapsing-remitting (RR)–MS
out lengthy periods of clinical follow-up.
and healthy controls (HCs; P < .001). The frequencies of
Several myelin autoantigens may serve as targets for
a-GalC antibody-positive subjects (a-GalC titers $ mean
HC titers 1 3 SD) are also significantly elevated in RR-MS the autoaggressive attack in MS—for example, myelin
compared with HC (40% vs 0%; P = .0033). Immunoaffinity protein myelin/oligodendrocyte glycoprotein (MOG), ex-
purified a-GalC IgGs from human serum bind to cultured pressed on the outermost lamellae of the myelin sheath
human oligodendrocytes, indicating that the ELISA detects and thus readily accessible to the immune machinery;
a biologically relevant epitope. Corroborating these findings, and a major CNS myelin glycolipid, galactocerebroside
a-GalC antibody responses in marmoset EAE were similarly (GalC), which accounts for 32% of the myelin lipid
found to be specifically associated with the RR forms and not content. Both MOG and galactocerebroside are highly
the peracute or progressive forms, in contrast with other encephalogenic in various models of experimental auto-
anti-myelin antibodies (P = .0256).
immune encephalomyelitis (EAE), the prototypic animal
Conclusion: (1) a-GalC antibodies appear MS-specific and
model for MS.2-4 Furthermore, passive antibody transfers
are not found in healthy subjects, unlike antibodies against
myelin proteins; (2) when present, a-GalC antibodies identify in myelin basic protein (MBP)–primed animals5-9 and
mostly RR-MS and may be an indicator of ongoing disease in vitro models have demonstrated the demyelinating
activity. This novel assay is a suitable and valuable method to properties of anti-galactocerebroside (a-GalC) and
increase accuracy of diagnosis and disease staging in MS. a-MOG antibodies.10-13 Antibody responses against these
(J Allergy Clin Immunol 2005;116:453-9.) myelin targets are thus factors that potentially regulate
From athe Department of Neurology, University of California San Francisco; none disclosed. C. Genain: has done consulting work for Aventis
and bthe Neurologische Klinik der Universität Zürich. Pharmaceuticals; named as the main inventor on a patent application
Supported by grants from the National Institutes of Health (NS4678-01 to ‘‘Methods to diagnose and prognose multiple sclerosis,’’ filed by University
Dr Genain and AI43073-11 to Dr Hauser), the National Multiple Sclerosis of California San Francisco, which includes data from this work; received
Society (RG3370-A-3 and 3438-A-7 to Dr Genain), the Cure MS Now fund, grants/support from National Institutes of Health (NS4678-01), National
the Lunardi Supermarkets, Inc, the Nancy Davis Center Without Walls, and Multiple Sclerosis Society (RG3370-A-3 and 3438-A-7); research contract
Aventis Pharmaceuticals. Dr Menge and Dr Lalive are postdoctoral research with Aventis Pharmaceuticals; donations from the Cure MS Now
fellows of the National Multiple Sclerosis Society. Foundation and the Lunardi Supermarkets, Inc; employed by University
Disclosure of potential conflict of interest: T. Menge: named as inventor on of California San Francisco; on the speakers’ bureau for Biogenidec, Teva
patent application ‘‘Methods to diagnose and prognose multiple sclerosis,’’ Pharmaceuticals, Serono, Inc.
filed by University of California San Francisco, which includes data from Received for publication January 7, 2005; revised March 9, 2005; accepted for
this work; received postdoctoral fellowship of the National Multiple publication March 11, 2005.
Sclerosis Society (FG 1476-A-1); employed by University of California Available online May 16, 2005.
San Francisco. P. H. Lalive: named as inventor on patent application Reprint requests: Claude Genain, MD, Department of Neurology,
‘‘Methods to diagnose and prognose multiple sclerosis,’’ filed by University Neuroimmunological Laboratories, C-440, University of California San
of California San Francisco, which includes data from this work; received Francisco, 513 Parnassus Ave, San Francisco CA 94143-0114. E-mail:
postdoctoral fellowship of the National Multiple Sclerosis Society (FG claudeg@itsa.ucsf.edu.
1476-A-1); received grant/support from Swiss National Foundation 0091-6749/$30.00
(PBGEB-102918); employed by University of California San Francisco. Ó 2005 American Academy of Allergy, Asthma and Immunology
H.-C. von Büdingen: none disclosed. B. Cree: none disclosed. S. L. Hauser: doi:10.1016/j.jaci.2005.03.023
453
454 Menge et al J ALLERGY CLIN IMMUNOL
AUGUST 2005
Animals
Abbreviations used Callithrix jacchus marmosets were cared for in accordance with
AM: Acute monophasic the guidelines of the Institutional Animal Care and Usage Committee.
CIS: Clinically isolated syndrome EAE was induced by immunization with 100 mg human white matter
CNS: Central nervous system homogenate as described.22 Plasma samples were obtained from
EAE: Experimental allergic encephalomyelitis EDTA-anticoagulated blood at baseline and at intervals of 2 to 4
GalC: Galactocerebroside weeks and stored at 240°C. The animals were scored every other day
a-GalC: Anti-galactocerebroside for the development of clinical signs and disability using a previously
HC: Healthy control published scale.22
HR: Hazard ratio
MBP: Myelin basic protein a-GalC ELISA
MOG: Myelin/oligodendrocyte glycoprotein Bovine brain–derived galactocerebroside (Matreya, Pleasant Gap,
MRI: Magnetic resonance imaging Pa) was dissolved in chloroform-methanol (2:1). For coating,
MS: Multiple sclerosis galactocerebroside was air-dried, stepwise resuspended in 65°C
PP: Primary-progressive hot ethanol (50% vol/vol) at a final concentration of 50 ug/mL,
rMOG: Recombinant rat myelin/oligodendrocyte with 100 uL added to wells of Polysorb 96-well microtiter plates
glycoprotein (extracellular domain) (Nunc, Rochester, NY), and incubated uncovered overnight at room
RR: Relapsing-remitting temperature (RT) for solvent evaporation. Plates were washed with
RT: Room temperature double-distilled H2O and blocked with 1% BSA (A7030; Sigma, St
SP: Secondary-progressive 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.
disease phenotype expression in the context of established After washing, specific antibody binding was detected by an alkaline
phosphate–labeled goat-anti-human IgG (A9544; Sigma) or by a
CNS inflammation.
horseradish peroxidase–conjugated rabbit-anti-monkey IgG (A2054;
The pathogenic involvement of anti-myelin antibodies
Sigma), diluted in ELISA buffer and incubated for 1 hour at RT. For
in human MS is less well established, because antibody human sera, binding was detected by reading the OD at 405 nm in a
titers against the myelin proteins do not unequivocally microplate reader (SpectraMax; Molecular Devices, Sunnyvale,
differ between control populations and patients with Calif) after incubation with paranitrophenyl phosphate (Moss,
MS.14-19 However, regardless of pathogenicity, anti- Pasadena, Md) for 30 minutes in the dark at RT. The marmoset
myelin antibodies have recently been proposed as pre- assay was developed with 3,3#,5,5#-tetramethylbenzidine (Pierce,
dictive disease markers.20 Rockford, Ill) for 15 minutes at RT and the OD read at 450 nm
Here, we examined whether a-GalC antibodies could wavelength.
serve as disease markers in MS. We demonstrate for the For specificity and sensitivity controls, a polyclonal rabbit-
anti-bovine galactocerebroside antiserum (G9152; Sigma) was used
first time that significantly elevated titers of a-GalC
and antibody binding detected by a horseradish peroxidase–labeled
antibodies are specifically found in relapsing-remitting goat-anti-rabbit IgG (A0545; Sigma). Quenching experiments were
(RR)–MS, and not in early or progressive forms of the performed by overnight pre-incubation with solubilized galactocer-
disease. In strong support of our clinical observations, ebroside; galactocerebroside was air-dried and resuspended in 65°C
longitudinal assessment of galactocerebroside reactivity hot ethanol at 200 ug/mL and further diluted in ELISA buffer to a final
during the course of relapsing EAE in marmosets indicates concentration of 2 ug/mL.
that appearance of antibodies against galactocerebroside is
delayed with respect to disease onset. 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
METHODS
and blocking with 3% BSA in PBS plus .05% Tween for 1 hour at
Patients and controls 37°C, marmoset samples were incubated for 1 hour at 37°C and
Sixty-five consecutive patients seen in our MS center, 51 meeting diluted 1:100 in 3% BSA in PBS plus .05% Tween. Antibody binding
the diagnostic criteria for clinically definite MS,21 were recruited for was detected by a peroxidase-labeled rabbit-anti-monkey IgG for
this study: 20 with RR-MS, 15 secondary-progressive (SP)–MS, and 1 hour at 37°C.
16 primary-progressive (PP)–MS (Table I). In addition, 14 patients
had a clinically isolated syndrome (CIS), ie, a single clinical attack Statistical analysis
suggestive of CNS demyelination. Twenty volunteers served as To express the results of the galactocerebroside assay, a signal-
healthy controls (HCs). Both untreated patients and patients treated to-background binding ratio was calculated as the ratio of OD
with IFN-b and glatiramer acetate were included in this study, but (signal) over OD (background). Positive controls, ie, a human sample
those treated with glucocorticoids within 3 months or on immuno- with strong binding signal, and negative controls, ie, ELISA buffer
suppressive therapy within 6 months of phlebotomy were excluded. only, omitting serum, were included on each plate. For human
Blood was drawn by venipuncture and clotted serum stored at samples, samples above the mean binding ratio 1 3 SD for the HC
240°C. Informed consent was obtained from the patients and HCs, group were considered positive. In the marmoset assay, samples were
and the study was conducted in accordance with Institutional Review considered positive for a binding ratio above 3 with ODGalC >0.1 and
Board approval. greater than 3-fold the baseline (unimmunized) sample. Statistical
J ALLERGY CLIN IMMUNOL Menge et al 455
TABLE I. Characteristics of patients with MS and HCs
Variable HC CIS RR-MS SP-MS PP-MS
N 20 14 20 15 16
Sex
Female 10 9 15 9 10
Male 10 5 5 6 6
Age (y)
Median 51.0 36.0* 43.0 45.0 51.0
Range 28-71 23-50 25-61 37-60 40-65
Disease duration
(mo)
Median NA NA 120 120 48
Range NA NA 9-266 24-384 9-216
Expanded Disability
Status Scale
Median NA 1.5 2.0à 5.5 4.5
SD NA 0.9 1.5 1.5 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).
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
analysis was conducted by using STATA 7.0 (StataCorp LP, College subgroup. Solid lines (—) denote mean binding ratios; dashed line
Station, Tex) and GraphPad Prism 3.0 (GraphPad Software, San (--) denotes threshold of detection (mean binding ratio of HC 1 3 SD
Diego, Calif). Categorical variables were compared by using the x2 (see B). B, Frequencies of a-GalC IgG seropositivity in human sera.
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 a titer of 1:12,800. Preincubation of the rabbit antiserum
analysis was used to assess time-dependent variables. Because the with galactocerebroside solubilized in ELISA buffer
binding ratios are not normally distributed, the binding ratio was (maximal solubility concentration, 2 ug/mL in aqueous
transformed by using an inverse ratio to generate a normal distribu- buffer) led to an 85% reduction in signal, proving spec-
tion for parametric analysis. ificity of the assay. A mAb reactive against MOG (8.18-
Antibody affinity purification C5) did not react with the coated galactocerebroside,
confirming the purity of the antigen. In serial dilutions of
Human serum was diluted in 10 mmol/L sodium phosphate buffer,
total IgG purified on protein G from either the human
pH 7.0 (SP buffer), and IgG was purified over a protein G column
(HiTrap HP; Amersham, Piscataway, NJ). Bound IgG was eluted positive control or pooled immune C jacchus sera, the
with 100 mmol/L glycine-HCl, pH 2.7, and dialyzed against the threshold of detection was 6.25 ug IgG per well. The
sodium phosphate buffer. For immunoaffinity purification of a-GalC interplate and intraplate coefficients of variation were 15%
antibodies, galactocerebroside was dissolved at 5.0 mg/mL in 65°C and 4%, respectively.
hot methanol and hydrophobically bound to a FF-octyl column
(HiTrap; Amersham) as previously described.24 The IgG fraction was Detection of a-GalC IgG in patients with MS
applied to this column and bound IgG eluted and dialyzed into PBS as
Quantitatively, significant differences in a-GalC
described.
antibody titers were found between HC and RR-MS
Immunohistochemistry (P < .001) as well as between patients with CIS and
The human oligodendrocytoma cell line HOG (kind gift of RR-MS (P < .05; ANOVA with Bonferroni correction for
Dr Glyn Dawson), known to express galactocerebroside,25 was multiple comparisons; Fig 1, A). There was a trend
grown in monolayers. Cells were trypsinized and plated at a density suggesting a difference for the antibody titers between
of 20,000 cells/well onto chamber glass slides (Nunc); fixed in ice- SP-MS and HC (P = .092). In contrast, there were no
cold methanol; blocked with 2% BSA and 2% FBS in PBS; and significant differences for a-GalC reactivity among the
stained with human serum (1:50), rabbit antiserum (1:50), or 1006- HC, CIS, and PP-MS subgroups. Even if the 2 patients
GalC (30 ug/mL), respectively, diluted in 1% BSA-PBS for 1 hour at
with the highest binding ratios in the RR-MS group were
RT and developed with fluorescein isothiocyanate–labeled anti-IgG
secondary antibodies (F3512 for human, F9887 for rabbit; Sigma).
excluded from the calculations, the difference in the
Control slides omitting the first antibodies were included. reciprocal binding ratio compared with the HC group
remained highly significant (P < .01).
RESULTS The threshold for positivity was 3.23 and is indicated in
Fig 1, A (dashed line; see Methods). The frequencies of
Validation of the a-GalC assay patients with RR-MS identified as a-GalC antibody–
The assay was validated by a rabbit antiserum reactive positive by this analysis were significantly higher com-
to bovine galactocerebroside, with reactivity detectable to pared with HC (40% vs 0%; P = .0033; Fisher exact test
AUGUST 2005
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 including the preclinical animals (Table II). In contrast,
comparisons). Again, there was a trend observed for a-GalC antibodies were detected only in animals with
a-GalC antibody positivity in SP-MS compared with HC RR-EAE, and not during the first attack of AM-EAE, even
(26.7% vs 0%; P = .026; not significant after correction in the severely affected animals or in animals displaying a
for multiple independent comparisons). Other pairwise progressive course (Table II). However, this could have
comparisons were not significant (Fig 1, B). resulted from the overall shorter observation period for
these animals (median, 28 and 60 days postimmunization
Immunoaffinity purification of a-GalC IgG vs 70 days postimmunization for RR-EAE; Table II).
and immunohistochemistry The a-GalC antibody response appeared significantly
To assess the specificity and biological relevance of the later compared with antibody responses against the myelin
ELISA assay, serum of 1 patient demonstrating a high protein rMOG and MBP in RR-EAE: median time lapse
a-GalC response (#1006) was subjected to immunoaffin- between immunization and appearance, 70 days for
ity purification of a-GalC IgG. From 50 mL serum, 190 ug a-GalC vs 45 days for a-rMOG and 27 days for a-MBP
a-GalC IgG (1006-GalC) was extracted by a custom- (P = .0256; log rank test for equivalence of survival
made galactocerebroside column. 1006-GalC reacted in functions). A Cox proportional hazard model showed
the ELISA with a detection limit 62.5 ng specific IgG per that the hazard ratios (HRs) for a-rMOG and a-MBP
well (0.625 ug/mL), and the signal could be quenched by antibody responses were significantly different from the
soluble galactocerebroside (45% signal reduction). These HR for a-GalC (HR a-rMOG = 5.56, P = .013; HR
galactocerebroside-purified IgGs showed staining of the a-MBP = 12.76, P = .001; Fig 3), indicating that a-GalC
human oligodendrocytoma cell line HOG identical to the antibodies occurred most distant from immunizations and
control rabbit a-GalC specific antiserum (Fig 2, A and B). thus onset of EAE in these animals.
These results unequivocally show that galactocerebroside
specific antibodies purified from human serum are cell-
surface binding on oligodendrocytes, and indicate that the DISCUSSION
newly implemented ELISA assay system likely detects
biologically relevant antibodies. We present here a reproducible solid-phase assay for
detection of galactocerebroside-specific IgG in human
Detection of a-GalC IgG responses in sera. These specific IgG were purified by means of a
marmoset EAE galactocerebroside immunoaffinity chromatography col-
Sequential sera of 20 animals immunized with human umn and were shown to retain the ability to bind to a
white matter homogenate were studied. Because of the galactocerebroside epitope expressed on human oligoden-
outbred nature of the animals, the clinical course of EAE is drocytes and in vitro by ELISA. The assays previously
not uniform: 9 animals displayed a RR-EAE disease described to measure such antibodies in MS18,19,26 iden-
course, and 2 animals did not remit during attacks but tified differences between controls and MS for cerebro-
progressively worsened over time (similar to a PP course). spinal fluid, but not serum, even with undiluted serum in a
Six animals were euthanized at onset of the first attack, solid-phase radioimmunoassay.18 The most likely expla-
termed acute monophasic (AM), and 2 of these had a nation for the differences we find between HC and MS
peracute disease course rapidly progressing to a score of 4. is the stratification for MS subgroups, which was not
An additional 3 animals were euthanized before the onset examined in previous studies.18,19 Indeed, comparing all
of clinical disease, at the time when pleocytosis was our 65 patients with MS as 1 group with HC showed no
present in the cerebrospinal fluid, demonstrating presence significant difference in the frequency of antibody-posi-
of CNS inflammation. Clinical information is summarized tive patients.
in Table II. The current a-GalC IgG assay is performed in serum at
Antibodies against rMOG and MBP were detected in all dilutions of 1:40 and above, which is considerably easier
but 1 of the animals regardless of their disease course, to access than cerebrospinal fluid and can be repeated
TABLE II. Characteristics of C jacchus marmoset EAE and antibody status
Disease Clinical onset Sacrifice Maximal clinical a-GalC First day a-rMOG First day a-MBP First day
Animal course (day PI) (day PI)y score (day PI)y IgG detected (PI)z IgG detected (PI)z IgG detected (PI)z
326-91 RR 14 120 4 (120) 1 23 1 63 1 23

106-90 RR 16 112 3 (96) 1 80 1 53 1 25
191-92 RR 16 112 2 (20) 1 81* 1 53* 1 27*
378-85 RR 16 112 2 (40) 1 26* 1 26* 1 56*
U062-02 RR 43 97 3 (84) 1 96 1 36 1 36
185-99 RR 7 86 2 (56) 1 70 1 15 1 28
U050-01 RR 21 86 2 (57) 1 70 1 45 1 15
U057-02 RR 32 82 1.5 (73) 1 62 1 62 1 18
U050-00 RR 21 78 3 (38) 1 78 1 29 1 29
Median 6 SD RR 21 6 11 98 6 16 2.0 6 0.8 70 6 25 42 6 17 29 6 12
(97 6 16)
127-93 CP 16 68 4 (56) 2 2 1 54* 1 26*
U052-01 CP 21 52 3 (40) 2 2 1 36 1 18
U025-00 AM 21 61 2 (57) 2 2 1 42 1 28
U023-00 AM 16 31 2 (28) 2 2 1 28 1 28
273-93 AM 12 28 3.5 (24) 2 2 1 28* 1 28*
274-93 AM 16 28 2 (20) 2 2 1 30* 1 30*
U021-99 AM 18 23 1.5 (23) 2 2 1 20 1 20
346-92 AM 17 22 3 (22) 2 2 1 23 1 23
Median 6 SD AM 16 6 3 28 6 15 2.0 6 0.8 28 6 8 28 6 4
(23 6 14)
U053-01 Preclinical NA 38 0 2 2 1 35 1 35
U061-02 Preclinical NA 31 0 2 2 2 2 1 31
U030-00 Preclinical NA 23 0 2 2 1 13 1 20
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.
multiple times. Most significant, serum a-GalC are spe-

cific 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 differ-
ences 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.
FIG 3. Time course of a-GalC and a-myelin protein IgG responses in
These observations imply that a-GalC antibodies can
immunized C jacchus. Serum dilutions, 1:100. (.) denotes onset of
help stratify different MS subgroups, namely RR-MS, a clinical signs; (-;-) denotes a-MBP positivity; (-*-) denotes a-rMOG
novel finding with high clinical relevance. Patients with positivity; (-n-) denotes a-GalC positivity. Significant levels for
CIS by definition have had 1 apparent clinical attack, median onset postimmunization (pi) of antibody positivity were
whereas patients with RR-MS are characterized by disease determined by a Cox proportional hazard model.
dissemination in time and space. A high proportion of CIS
who present with brain magnetic resonance imaging of established MS, their detection in patients with early
(MRI) abnormalities will proceed to develop RR- MS and CIS could potentially help correct and achieve an
MS,27,28 and indeed, for many of those patients, subclin- earlier diagnosis of definite MS than with conventional
ical MS or minor attacks may have been present for a criteria. The anti-myelin protein antibodies, on the other
considerable period. Thus it can be envisioned that hand, have recently been described as potential predictors
detection of a-GalC antibodies may permit staging of of early conversion in patients with CIS.20
MS forms according to time from the first demyelinating Critical for interpretation of our clinical findings in the
event. Because these antibodies appear to be characteristic absence of longitudinal measurements in human MS was
AUGUST 2005
the study of chronic relapsing marmoset EAE, which best investigation, in combination with MRI. In line with other
approximates MS complex pathophysiology. Specifically, recent reports on humoral immunity in MS and EAE,20,37
we found that the a-GalC responses occurred distinctly these novel findings continue to underscore the value of
after disease onset only in animals with RR forms of EAE a-myelin antibody assessment—both protein and glyco-
(Table II; Fig 3). This was in contrast to the a-rMOG and lipid—as biomarkers that will be used in the near future for
a-MBP responses that occurred in all animals tested, in MS diagnostics, staging, and prognosis.
some cases even before clinical onset. These findings are
in line with results from 2 other EAE models,29,30 in which We thank Salomon Martinez for expert animal work; Jerry
a-GalC antibodies were also present in the early chronic Hernandez, Drew Dover, and Kevin Morgan for help analyzing the
stage of guinea pig EAE29 and occurred after the clinical samples, and the clinical coordinators and neurologists at the
onset and after the development of a-MBP antibodies.30 University of California San Francisco MS Center for sample
Reactivity against rMOG was not tested in either of these collection.
studies.
Although the pathophysiological explanation for the
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Perilesional GM-CSF therapy of a chronic leg phagocytosis, GM-CSF stimulates the proliferation of
ulcer in a patient with common variable keratinocytes and the differentiation of myofibroblasts.1-3
immunodeficiency GM-CSF also increases wound tensile strength.4 The role
of bacterial infections in chronic wounds is not well
To the Editor: established. However, GM-CSF might prove useful in
Impaired wound healing characterizes multiple immu- chronically infected wounds. Previous studies have dem-
nodeficiency states, including common variable immuno- onstrated that GM-CSF enhances phagocyte killing of
deficiency (CVID). Chronic, nonhealing wounds often several microorganisms.5,6 In addition, GM-CSF was
ensue, with a considerable associated toll of pain, disfig- shown in animal studies to promote the healing of infected
urement, disability, and increased medical costs. These wounds.7 Interestingly, GM-CSF appears to be effective
chronic wounds are notoriously difficult to treat, often in chronic venous stasis ulcers, which one would not
prompting debridement and skin grafting. expect to be associated with decreased immune defense.
Here we report a patient with CVID with chronic leg Da Costa et al8 administered perilesional placebo or GM-
ulcers who responded to perilesional GM-CSF therapy. CSF in 200-mg or 400-mg doses. The healing rates were
The patient was a 68-year-old white man with CVID 57%, 61%, and 19% for the low-dose GM-CSF, high-dose
who had been on intravenous immunoglobulin replace- GM-CSF, and placebo, respectively. GM-CSF was also
ment for 15 years. He also had diabetes mellitus (type 2), used in a patient with CVID with bilateral leg ulcers
was on chronic steroid therapy for several years (for caused by dermatofibromas, with a dramatic response.9
steroid-dependent asthma), and more recently was on Our patient illustrates that in addition to venous stasis
methotrexate for chronic inflammatory myositis. In May ulcers and dermatofibroma-associated ulcers, perilesional
2002, he developed a right leg ulcer that did not heal and GM-CSF can be very effective in the treatment of infected
required skin grafting. leg ulcers in patients with CVID.
In January 2004, he developed another ulcer over his
Ammar Z. Hatab, MDa
right leg, after minor trauma, in an area distinct from the Deanna McDanel, PharmD, BCPSb
previous ulcer. The wound continued to progress despite Zuhair K. Ballas, MDa,c
debridement and local care, reaching a maximum size a
Division of Allergy/Immunology
of 7 cm 3 5 cm (Fig 1, A). Bacterial culture from the Department of Internal Medicine
ulcer (March 2004) grew Pseudomonas aeruginosa and C42/E-13, GH
methicillin-resistant Staphylococcus aureus. Standard Carver College of Medicine
therapy, including local care and topical and systemic anti- University of Iowa
b
biotics, was ineffective. GM-CSF treatment (sargramostim, Department of Pharmaceutical Care
University of Iowa Hospitals and Clinics and the
Leukine; Immunex Corp, Seattle, Wash) was begun in
College of Pharmacy
March 2004. He received 125 mg in 0.25 mL subcutane-
University of Iowa
ously at each of 4 sites distributed evenly around the c
Iowa City VA Medical Center
healthy edge of the ulcer (3, 6, 9 and 12 o’clock). This was Iowa City, Iowa
repeated weekly for 4 weeks.
The ulcer started improving a few days after the first REFERENCES
injection. After 4 weeks, the patient was instructed to wash 1. Groves RW, Schmidt-Lucke JA. Recombinant human GM-CSF in the treat-
the ulcer with GM-CSF (3 mL saline containing 15 mg ment of poorly healing wounds. Adv Skin Wound Care 2000;13:107-12.
GM-CSF) twice daily for another 4 weeks (Fig 1, B). The 2. Hancock GE, Kaplan G, Cohn ZA. Keratinocyte growth regulation by the
products of immune cells. J Exp Med 1988;168:1395-402.
ulcer healed completely in September 2004 (Fig 1, C). The
3. Gabbiani G. Modulation of fibroblastic cytoskeletal features during wound
patient reported no side effects from GM-CSF treatment. It healing and fibrosis. Pathol Res Pract 1994;190:851-3.
is worth noting that his methotrexate dose was actually 4. Jyung RW, Wu L, Pierce GF, Mustoe TA. Granulocyte-macrophage
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thelial cells. The resultant effect is to lay down connective 7. Robson M, Kucukcelebi A, Carp SS, Hayward PG, Hui PS, Cowan WT,
tissue and restore tissue architecture. During maturation, et al. Effects of granulocyte-macrophage colony-stimulating factor on
the wound contracts and gains tensile strength. 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,
GM-CSF has been shown to influence all phases of double-blind, placebo-controlled, dose-ranging study of granulocyte-
wound healing. In addition to its well characterized effects macrophage colony stimulating factor in patients with chronic venous
on neutrophil and monocyte proliferation, migration, and leg ulcers. Wound Repair Regen 1999;7:17-25.
460
J ALLERGY CLIN IMMUNOL Letters to the Editor 461
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.
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.

doi:10.1016/j.jaci.2005.04.008
Asthma caused by cyanoacrylate used in a

leisure activity
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 ortho- FIG 1. Results of SIC.
pedics).1 These agents are well known to cause occu-
pational 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 Occupational asthma caused by acrylates has been
been only one case reported of their causing respiratory described often, but in this case exposure took place only
symptoms out of the workplace.5 on occasion during a leisure activity; it is relevant to high-
We report bronchial asthma caused by cyanoacrylate light that in this nonatopic subject specific reactivity to the
in a 55-year-old man whose hobby was making miniature sensitizer persisted even 1 year after cessation of exposure.
planes, an activity that required the use of a cyanoacrylate This case underlines the sensitization strength of acrylates,
adhesive paste. This exsmoker had never experienced proved as well by the fact that very intermittent exposure
asthmatic or rhinitis symptoms. A year before being seen was enough to trigger bronchial asthma. This can also
at the clinic, he reported acute dyspnea during a weekend, reasonably explain why the subject was cured after
which is when he normally worked on his model planes; stopping exposure.
this episode required emergency care, followed by a short Dr Mona-Rita Yacoub is a postdoctoral fellow supported by
course of oral and inhaled corticosteroid therapy. After Asthma in the Workplace (Canadian Institutes of Health Research,
this occasion, he stopped practicing his hobby and did not Canadian Lung Association, Institut de recherche Robert-Sauvé en
require medication, except for short-acting bronchodilator santé et sécurité du travail du Québec).
occasionally when his respiratory symptoms were exac-
We thank L. Schubert for reviewing the manuscript.
erbated by physical exercise, cold temperature, and heavy
Mona-Rita Yacoub, MD
smells.
Catherine Lemière, MD, MSc
The results of skin prick tests to common aeroallergens Jean-Luc Malo, MD
were negative; spirometry showed an FEV1 of 2.9 L Department of Chest Medicine
(100% of predicted value), a forced vital capacity of 3.5 L Sacré-Coeur Hospital
(100% of predicted value), and an FEV1/forced vital 5400 West Gouin Blvd
capacity ratio of 83% (normal). Methacholine bronchial Montreal, Quebec, Canada H4J 1C5
responsiveness was normal (PC20 5 128 mg/mL; normal
value >16 mg/mL in our laboratory). The subject under-
REFERENCES
went a specific inhalation challenge (SIC) according to
1. Piirilä P, Kanerva L, Keskinen H, Estlander T, Hytönen M, Tuppurainen M.
a standardized procedure.6 Results are shown in Fig 1. On
Occupational respiratory hypersensitivity caused by preparations contain-
a control day, the patient was exposed to diluent paint ing acrylates in dental personnel. Clin Exp Allergy 1998;28:1404-11.
by means of nebulization for 30 minutes. Spirometry, 2. Quirce S, Baeza ML, Tornero P, Blasco A, Barranco R, Sastre J. Occupa-
methacholine testing, and induced sputum performed after tional asthma caused by exposure to cyanoacrylate. Allergy 2001;56:446-9.
diluent exposure produced normal results. On 2 subsequent 3. Weytjens K, Cartier A, Lemiere C, Malo JL. Occupational asthma to
diacrylate. Allergy 1999;54:289-90.
days, exposure to cyanoacrylate was carried out by asking 4. Kopferschmit-Kubler MC, Stenger R, Blaumeiser M, Eveilleau C,
the patient to mimic his leisure activity in a challenge Bessot JC, Pauli G. Asthma, rhinitis and urticaria following occupational
room, spreading cyanoacrylate glue on a piece of card- exposure to cyanoacrylate glues. Rev Mal Respir 1996;13:305-7.
board for progressively longer periods of time (totals of 5. Kopp SK, McKay RT, Moller DR, Cassedy K, Brooks SM. Asthma and
rhinitis due to ethylcyanoacrylate instant glue. Ann Intern Med 1985;102:

4 and 30 minutes of exposure on the 2 days). The test
613-5.
revealed a typical early late response. Induced sputum 6. Cartier A, Bernstein IL, Burge PS, Cohn JR, Fabbri LM, Hargreave FE,
performed before and after SIC demonstrated pronounced et al. Guidelines for bronchoprovocation on the investigation of occupa-
eosinophlia after the cyanoacrylate challenge: eosinophil tional asthma. J Allergy Clin Immunol 1989;84(suppl):823-9.
counts switched from 0.5% before SIC to 63% at the end Available online June 1, 2005.
of the last day of exposure. 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.

doi:10.1016/j.jaci.2005.04.008
Asthma caused by cyanoacrylate used in a

leisure activity
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 ortho- FIG 1. Results of SIC.
pedics).1 These agents are well known to cause occu-
pational 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 Occupational asthma caused by acrylates has been
been only one case reported of their causing respiratory described often, but in this case exposure took place only
symptoms out of the workplace.5 on occasion during a leisure activity; it is relevant to high-
We report bronchial asthma caused by cyanoacrylate light that in this nonatopic subject specific reactivity to the
in a 55-year-old man whose hobby was making miniature sensitizer persisted even 1 year after cessation of exposure.
planes, an activity that required the use of a cyanoacrylate This case underlines the sensitization strength of acrylates,
adhesive paste. This exsmoker had never experienced proved as well by the fact that very intermittent exposure
asthmatic or rhinitis symptoms. A year before being seen was enough to trigger bronchial asthma. This can also
at the clinic, he reported acute dyspnea during a weekend, reasonably explain why the subject was cured after
which is when he normally worked on his model planes; stopping exposure.
this episode required emergency care, followed by a short Dr Mona-Rita Yacoub is a postdoctoral fellow supported by
course of oral and inhaled corticosteroid therapy. After Asthma in the Workplace (Canadian Institutes of Health Research,
this occasion, he stopped practicing his hobby and did not Canadian Lung Association, Institut de recherche Robert-Sauvé en
require medication, except for short-acting bronchodilator santé et sécurité du travail du Québec).
occasionally when his respiratory symptoms were exac-
We thank L. Schubert for reviewing the manuscript.
erbated by physical exercise, cold temperature, and heavy
Mona-Rita Yacoub, MD
smells.
Catherine Lemière, MD, MSc
The results of skin prick tests to common aeroallergens Jean-Luc Malo, MD
were negative; spirometry showed an FEV1 of 2.9 L Department of Chest Medicine
(100% of predicted value), a forced vital capacity of 3.5 L Sacré-Coeur Hospital
(100% of predicted value), and an FEV1/forced vital 5400 West Gouin Blvd
capacity ratio of 83% (normal). Methacholine bronchial Montreal, Quebec, Canada H4J 1C5
responsiveness was normal (PC20 5 128 mg/mL; normal
value >16 mg/mL in our laboratory). The subject under-
REFERENCES
went a specific inhalation challenge (SIC) according to
1. Piirilä P, Kanerva L, Keskinen H, Estlander T, Hytönen M, Tuppurainen M.
a standardized procedure.6 Results are shown in Fig 1. On
Occupational respiratory hypersensitivity caused by preparations contain-
a control day, the patient was exposed to diluent paint ing acrylates in dental personnel. Clin Exp Allergy 1998;28:1404-11.
by means of nebulization for 30 minutes. Spirometry, 2. Quirce S, Baeza ML, Tornero P, Blasco A, Barranco R, Sastre J. Occupa-
methacholine testing, and induced sputum performed after tional asthma caused by exposure to cyanoacrylate. Allergy 2001;56:446-9.
diluent exposure produced normal results. On 2 subsequent 3. Weytjens K, Cartier A, Lemiere C, Malo JL. Occupational asthma to
diacrylate. Allergy 1999;54:289-90.
days, exposure to cyanoacrylate was carried out by asking 4. Kopferschmit-Kubler MC, Stenger R, Blaumeiser M, Eveilleau C,
the patient to mimic his leisure activity in a challenge Bessot JC, Pauli G. Asthma, rhinitis and urticaria following occupational
room, spreading cyanoacrylate glue on a piece of card- exposure to cyanoacrylate glues. Rev Mal Respir 1996;13:305-7.
board for progressively longer periods of time (totals of 5. Kopp SK, McKay RT, Moller DR, Cassedy K, Brooks SM. Asthma and
rhinitis due to ethylcyanoacrylate instant glue. Ann Intern Med 1985;102:

4 and 30 minutes of exposure on the 2 days). The test
613-5.
revealed a typical early late response. Induced sputum 6. Cartier A, Bernstein IL, Burge PS, Cohn JR, Fabbri LM, Hargreave FE,
performed before and after SIC demonstrated pronounced et al. Guidelines for bronchoprovocation on the investigation of occupa-
eosinophlia after the cyanoacrylate challenge: eosinophil tional asthma. J Allergy Clin Immunol 1989;84(suppl):823-9.
counts switched from 0.5% before SIC to 63% at the end Available online June 1, 2005.
of the last day of exposure. doi:10.1016/j.jaci.2005.04.015
Correspondence
Cystic fibrosis gene mutations and somnolence, and fatigue, and it was noted that nasal
chronic rhinosinusitis congestion and other rhinitis symptoms follow a circadian
rhythm, being more severe at night and early in the
To the Editor: morning. Chronotherapy, the timed dosing of rhinitis
In their review of the definition, pathophysiology, medications to manage optimally the diurnal variation in
treatment of rhinosinusitis by Meltzer et al,1 there is no nasal congestion and other rhinitis symptoms, was dis-
discussion of the association between mutations in the cussed, as well as the advantages and disadvantages of
cystic fibrosis transmembrane conductance regulator each medication class in relation to nighttime rhinitis
(CFTR) gene and chronic rhinosinusitis.2 Most of these symptoms; however, readers must draw their own con-
patients do not meet diagnostic criteria for cystic fibrosis clusions regarding the most effective medication class.
because their sweat chloride is not elevated above 60 Three large, double-blind, randomized, placebo-con-
mmol/L and they do not have evidence of 2 disease- trolled trials of montelukast 10 mg (MON), the only
causing CFTR gene mutations. However, some of the leukotriene receptor antagonist approved in the United
patients with CFTR gene mutations and chronic rhinosi- States for seasonal allergic rhinitis, were discussed in the
nusitis reported by Wang et al2 did have other features of supplement.2-4 Compared with placebo, MON adminis-
cystic fibrosis, such as infertility and infection with tered once daily at bedtime significantly reduced the
Pseudomonas aeruginosa. Identification of CFTR gene nighttime symptom scores and peripheral blood eosin-
mutations in patient with chronic rhinosinusitis may ophil counts in all 3 trials.
potentially be of clinical importance in the future, because However, in contrast with the 3 large trials cited for
pharmacologic therapies to overcome defective CFTR montelukast, the intranasal corticosteroid trials discussed
function are under development.3 varied considerably in size, scope, and design. The sup-
Clement L. Ren, MD plement did not include a discussion of a recent large,
Division of Pediatric Pulmonology and Allergy randomized, double-blind, double-dummy, parallel-
Room 4-3236
group trial in 705 subjects with seasonal allergic rhinitis
601 Elmwood Avenue
University of Rochester that compared the effectiveness of a 15-day course of
Rochester, NY 14642 intranasal fluticasone propionate aqueous nasal spray
200 mg (FPANS) with MON, both administered once
Editor’s note: This Correspondence has no accompanying reply. The daily in the evening.5 The results of this study showed
authors of the Meltzer article chose not to reply, saying that the that FPANS was consistently superior to MON for all
correspondence provides interesting information and is worthy of daytime and nighttime symptoms, including nasal con-
publication.
gestion. The nighttime symptoms (difficulty going to
sleep, nighttime awakenings, nasal congestion on awak-
REFERENCES
ening) and scoring in this trial were the same as in the
1. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas RA. 3 montelukast trials cited. In addition, a randomized,
Rhinosinusitis: establishing definitions for clinical research and patient
care. J Allergy Clin Immunol 2004;114:S156-212.
double-blind, double-dummy, placebo-controlled, paral-
2. Wang X, Moylan B, Leopold DA, Kim J, Rubenstein RC, Togias A, lel-group trial in 62 subjects with seasonal allergic
et al. Mutation in the gene responsible for cystic fibrosis and predis- rhinitis compared subjects treated with FPANS, MON,
position to chronic rhinosinusitis in the general population. JAMA 2000; MON 1 loratadine 10 mg (LOR), or placebo throughout
284:1814-9.
the grass pollen season.6 Subjects assessed their rhinitis
3. Zeitlin PL. Novel pharmacologic therapies for cystic fibrosis. J Clin Invest
1999;103:447-52. symptoms during the study and also underwent nasal
biopsy before and during the season for evaluation
Available online May 16, 2005. of local eosinophilic inflammation. Both MON and
doi:10.1016/j.jaci.2005.03.032
MON 1 LOR were less effective than FPANS for con-
trol of daytime and nighttime symptoms, including nasal
Leukotriene receptor antagonists are not blockage, and for reduction of pollen-induced nasal
as effective as intranasal corticosteroids eosinophilic inflammation on biopsy.
for managing nighttime symptoms of Furthermore, when evaluating nighttime rhinitis treat-
allergic rhinitis ment options, another important consideration is that many
patients with rhinitis have mixed rhinitis, which may in-
To the Editor: clude a combination of seasonal allergic, perennial allergic,
I wish to comment on the supplement published in the or perennial nonallergic rhinitis. Whereas MON is currently
November 2004 issue titled ‘‘Allergic Rhinitis After indicated only for relief of seasonal allergic rhinitis, FPANS
Hours: The Relevance and Consequences of Nighttime is indicated for the management of nasal symptoms of all
Symptoms.’’1 Nasal congestion associated with allergic 3 of these types of rhinitis.
rhinitis was identified as an important risk factor for I agree with the premise that therapy aimed at reducing
sleep-disordered breathing, sleep fragmentation, daytime nighttime nasal congestion is paramount for improving
463
Correspondence
Cystic fibrosis gene mutations and somnolence, and fatigue, and it was noted that nasal
chronic rhinosinusitis congestion and other rhinitis symptoms follow a circadian
rhythm, being more severe at night and early in the
To the Editor: morning. Chronotherapy, the timed dosing of rhinitis
In their review of the definition, pathophysiology, medications to manage optimally the diurnal variation in
treatment of rhinosinusitis by Meltzer et al,1 there is no nasal congestion and other rhinitis symptoms, was dis-
discussion of the association between mutations in the cussed, as well as the advantages and disadvantages of
cystic fibrosis transmembrane conductance regulator each medication class in relation to nighttime rhinitis
(CFTR) gene and chronic rhinosinusitis.2 Most of these symptoms; however, readers must draw their own con-
patients do not meet diagnostic criteria for cystic fibrosis clusions regarding the most effective medication class.
because their sweat chloride is not elevated above 60 Three large, double-blind, randomized, placebo-con-
mmol/L and they do not have evidence of 2 disease- trolled trials of montelukast 10 mg (MON), the only
causing CFTR gene mutations. However, some of the leukotriene receptor antagonist approved in the United
patients with CFTR gene mutations and chronic rhinosi- States for seasonal allergic rhinitis, were discussed in the
nusitis reported by Wang et al2 did have other features of supplement.2-4 Compared with placebo, MON adminis-
cystic fibrosis, such as infertility and infection with tered once daily at bedtime significantly reduced the
Pseudomonas aeruginosa. Identification of CFTR gene nighttime symptom scores and peripheral blood eosin-
mutations in patient with chronic rhinosinusitis may ophil counts in all 3 trials.
potentially be of clinical importance in the future, because However, in contrast with the 3 large trials cited for
pharmacologic therapies to overcome defective CFTR montelukast, the intranasal corticosteroid trials discussed
function are under development.3 varied considerably in size, scope, and design. The sup-
Clement L. Ren, MD plement did not include a discussion of a recent large,
Division of Pediatric Pulmonology and Allergy randomized, double-blind, double-dummy, parallel-
Room 4-3236
group trial in 705 subjects with seasonal allergic rhinitis
601 Elmwood Avenue
University of Rochester that compared the effectiveness of a 15-day course of
Rochester, NY 14642 intranasal fluticasone propionate aqueous nasal spray
200 mg (FPANS) with MON, both administered once
Editor’s note: This Correspondence has no accompanying reply. The daily in the evening.5 The results of this study showed
authors of the Meltzer article chose not to reply, saying that the that FPANS was consistently superior to MON for all
correspondence provides interesting information and is worthy of daytime and nighttime symptoms, including nasal con-
publication.
gestion. The nighttime symptoms (difficulty going to
sleep, nighttime awakenings, nasal congestion on awak-
REFERENCES
ening) and scoring in this trial were the same as in the
1. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas RA. 3 montelukast trials cited. In addition, a randomized,
Rhinosinusitis: establishing definitions for clinical research and patient
care. J Allergy Clin Immunol 2004;114:S156-212.
double-blind, double-dummy, placebo-controlled, paral-
2. Wang X, Moylan B, Leopold DA, Kim J, Rubenstein RC, Togias A, lel-group trial in 62 subjects with seasonal allergic
et al. Mutation in the gene responsible for cystic fibrosis and predis- rhinitis compared subjects treated with FPANS, MON,
position to chronic rhinosinusitis in the general population. JAMA 2000; MON 1 loratadine 10 mg (LOR), or placebo throughout
284:1814-9.
the grass pollen season.6 Subjects assessed their rhinitis
3. Zeitlin PL. Novel pharmacologic therapies for cystic fibrosis. J Clin Invest
1999;103:447-52. symptoms during the study and also underwent nasal
biopsy before and during the season for evaluation
Available online May 16, 2005. of local eosinophilic inflammation. Both MON and
doi:10.1016/j.jaci.2005.03.032
MON 1 LOR were less effective than FPANS for con-
trol of daytime and nighttime symptoms, including nasal
Leukotriene receptor antagonists are not blockage, and for reduction of pollen-induced nasal
as effective as intranasal corticosteroids eosinophilic inflammation on biopsy.
for managing nighttime symptoms of Furthermore, when evaluating nighttime rhinitis treat-
allergic rhinitis ment options, another important consideration is that many
patients with rhinitis have mixed rhinitis, which may in-
To the Editor: clude a combination of seasonal allergic, perennial allergic,
I wish to comment on the supplement published in the or perennial nonallergic rhinitis. Whereas MON is currently
November 2004 issue titled ‘‘Allergic Rhinitis After indicated only for relief of seasonal allergic rhinitis, FPANS
Hours: The Relevance and Consequences of Nighttime is indicated for the management of nasal symptoms of all
Symptoms.’’1 Nasal congestion associated with allergic 3 of these types of rhinitis.
rhinitis was identified as an important risk factor for I agree with the premise that therapy aimed at reducing
sleep-disordered breathing, sleep fragmentation, daytime 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 No prospective controlled study of IFA WBE treatment
class of medication (ie, intranasal steroids) should be efficacy or prospective study of the natural history of IFA
considered as initial therapy for relief of nighttime rhinitis allergy has been published. Retrospective studies have
symptoms. selection bias, and the natural history of allergy can vary
Robert A. Nathan, MD enormously between species. Large prospective studies
University of Colorado Health Sciences Center have found reaction rates on re-exposure to range from
2709 North Tejon 70% for the jack jumper ant through 50% for the honeybee
Colorado Springs, CO 80907 and to 25% for the yellow jacket.2,3 Individuals allergic to
Disclosure of potential conflict of interest: Dr Nathan receives IFA who react to multiple simultaneous stings (as often
grants/research support from Abbott, Altana, Aventis, AstraZeneca, occurs) may experience few reactions when exposed to
Bayer, Berlex, Boehringer Ingelheim, Bristol-Myers Squibb, CIBA smaller doses of venom.
Geigy, Dura, Forest, GlaxoSmithKline, Immunex, Janssen, Parke- Our randomized, double-blind, placebo-controlled trial
Davis, Pfizer, 3-M Pharmaceuticals, Proctor & Gamble, Roberts, of venom immunotherapy (VIT) provides a model that
Sandoz, Sanofi, Schering/Key, Sepracor, Sterling, Tap Pharm, could be used to assess IFA WBE.3 How did we justify our
Wallace, and Wyeth; is a consultant/scientific advisor for AMGEN, study, and why did 2 respected university ethics commit-
Altana, AstraZeneca, Aventis, Genentech, GlaxoSmithKline, Merck, tees approve? First, large studies that have demonstrated
Novartis, Pfizer, Schering/Key, Sepracor, and Viropharm; and is on
the safety of sting challenges after applying health and age
the speakers’ bureau for AstraZeneca, Aventis, Genentech/Novartis,
exclusion criteria included a total of 238 patients with
GlaxoSmithKline, Pfizer, and Schering/Key.
severe (Mueller grade IV) allergy. Second, the efficacy
Editor’s note: This Correspondence has no accompanying reply. data from 2 controlled trials of VIT to prevent honey bee
and vespid sting anaphylaxis are suboptimal by contem-
porary standards. One allocated treatment according to
REFERENCES patient choice, with outcomes determined by reactions
1. Meltzer EO, editor. Allergic rhinitis after hours: the relevance and conse-
occurring outside hospital that were unobserved by the
quence of nighttime symptoms. J Allergy Clin Immunol 2004;114:S133-53. investigators. The other was single-blind and stratified by
2. Philip G, Malmstrom K, Hampel FC, Weinstein SF, LaForce CF, Ratner using factors that do not influence reaction risk. Finally,
PH, et al. Montelukast for treating seasonal allergic rhinitis: a randomized, the efficacy of immunotherapy varies between species,
double-blind, placebo-controlled trial performed in the spring. Clin Exp
and we could not be sure of the efficacy of jack jumper ant
Allergy 2002;32:1020-8.
3. Nayak AS, Philip G, Lu S, Malice M-P, Reiss TF. Efficacy and toler- VIT.
ability of montelukast alone or in combination with loratadine in seasonal Without double-blinding, investigators can be misled
allergic rhinitis: a multicenter, randomized, double-blind, placebo- by personal bias and subjective features such as itch, mild
controlled trial performed in the fall. Ann Allergy Asthma Immunol flushing, breathlessness, anxiety, and hypotension asso-
2002;88:592-600.
4. van Adelsberg J, Phillip G, LaForce CF, Weinstein SF, Menten J, Malice
ciated with bradycardia and anxiety. It is notable that we
M-P, et al. Randomized controlled trial evaluating the clinical benefit of gave epinephrine to a patient who appeared to have a
montelukast for treating spring seasonal allergic rhinitis. Ann Allergy moderate reaction to an injection that was later revealed
Asthma Immunol 2003;90:214-22. to be placebo.3
5. Ratner PH, Howland WC, Arastu R, Philpot EE, Klein KC, Baidoo CA,
The inclusion of a blind placebo group is the only way
et al. Fluticasone propionate aqueous nasal spray provided greater im-
provement in daytime and nighttime nasal symptoms of seasonal allergic to be certain that a sting challenge adequately tests treat-
rhinitis compared with montelukast. Ann Allergy Asthma Immunol 2003; ment efficacy. This is an important ethical issue; we have
90:536-42. observed the death of a man who believed he was pro-
6. Pullerits T, Praks L, Ristioja V, Lotvall J. Comparison of a nasal tected by ant WBE, reinforcing the risks of a flawed
glucocorticoid, antileukotriene, and a combination of antileukotriene and
antihistamine in the treatment of seasonal allergic rhinitis. J Allergy Clin
evidence base. As explained, the exposure to a small
Immunol 2002;109:949-55. number of IFA in a sting challenge may be insufficient
to provoke anaphylaxis in many people with allergy.
doi:10.1016/j.jaci.2005.03.045 Furthermore, insect handling procedures may lead to a
depletion of venom as assessed at the time of venom sac
dissection.3
Efficacy of ant venom immunotherapy and Imported fire ant WBE may be an effective treatment,
whole body extracts because extracts contain venom proteins and quality con-
trol methods have been developed. However, uncertainties
To the Editor:
remain with regard to the natural history of IFA allergy
Golden1 presents a useful review of insect venom and whether the dose of venom delivered by IFA WBE
immunotherapy, but we disagree with his conclusion
extracts is sufficient to confer protection.
that imported fire ant (IFA) whole body extract (WBE)
Simon G. A. Brown, MBBS, PhD, FACEMa
has been proven efficacious. Golden1 stresses the need Robert J. Heddle, MBBS, PhD, FRACP, FRCPAb
to understand the natural history of sting allergy and Michael D. Wiese, BPharm, MClinPharmc
the importance of controlled studies. We add to this the Konrad E. Blackman, MBBS, FACEMc
need for prospective design, adequate randomization, and a
University of Western Australia
double-blinding. Fremantle Hospital
AUGUST 2005
sleep and quality of life, but also that the most effective No prospective controlled study of IFA WBE treatment
class of medication (ie, intranasal steroids) should be efficacy or prospective study of the natural history of IFA
considered as initial therapy for relief of nighttime rhinitis allergy has been published. Retrospective studies have
symptoms. selection bias, and the natural history of allergy can vary
Robert A. Nathan, MD enormously between species. Large prospective studies
University of Colorado Health Sciences Center have found reaction rates on re-exposure to range from
2709 North Tejon 70% for the jack jumper ant through 50% for the honeybee
Colorado Springs, CO 80907 and to 25% for the yellow jacket.2,3 Individuals allergic to
Disclosure of potential conflict of interest: Dr Nathan receives IFA who react to multiple simultaneous stings (as often
grants/research support from Abbott, Altana, Aventis, AstraZeneca, occurs) may experience few reactions when exposed to
Bayer, Berlex, Boehringer Ingelheim, Bristol-Myers Squibb, CIBA smaller doses of venom.
Geigy, Dura, Forest, GlaxoSmithKline, Immunex, Janssen, Parke- Our randomized, double-blind, placebo-controlled trial
Davis, Pfizer, 3-M Pharmaceuticals, Proctor & Gamble, Roberts, of venom immunotherapy (VIT) provides a model that
Sandoz, Sanofi, Schering/Key, Sepracor, Sterling, Tap Pharm, could be used to assess IFA WBE.3 How did we justify our
Wallace, and Wyeth; is a consultant/scientific advisor for AMGEN, study, and why did 2 respected university ethics commit-
Altana, AstraZeneca, Aventis, Genentech, GlaxoSmithKline, Merck, tees approve? First, large studies that have demonstrated
Novartis, Pfizer, Schering/Key, Sepracor, and Viropharm; and is on
the safety of sting challenges after applying health and age
the speakers’ bureau for AstraZeneca, Aventis, Genentech/Novartis,
exclusion criteria included a total of 238 patients with
GlaxoSmithKline, Pfizer, and Schering/Key.
severe (Mueller grade IV) allergy. Second, the efficacy
Editor’s note: This Correspondence has no accompanying reply. data from 2 controlled trials of VIT to prevent honey bee
and vespid sting anaphylaxis are suboptimal by contem-
porary standards. One allocated treatment according to
REFERENCES patient choice, with outcomes determined by reactions
1. Meltzer EO, editor. Allergic rhinitis after hours: the relevance and conse-
occurring outside hospital that were unobserved by the
quence of nighttime symptoms. J Allergy Clin Immunol 2004;114:S133-53. investigators. The other was single-blind and stratified by
2. Philip G, Malmstrom K, Hampel FC, Weinstein SF, LaForce CF, Ratner using factors that do not influence reaction risk. Finally,
PH, et al. Montelukast for treating seasonal allergic rhinitis: a randomized, the efficacy of immunotherapy varies between species,
double-blind, placebo-controlled trial performed in the spring. Clin Exp
and we could not be sure of the efficacy of jack jumper ant
Allergy 2002;32:1020-8.
3. Nayak AS, Philip G, Lu S, Malice M-P, Reiss TF. Efficacy and toler- VIT.
ability of montelukast alone or in combination with loratadine in seasonal Without double-blinding, investigators can be misled
allergic rhinitis: a multicenter, randomized, double-blind, placebo- by personal bias and subjective features such as itch, mild
controlled trial performed in the fall. Ann Allergy Asthma Immunol flushing, breathlessness, anxiety, and hypotension asso-
2002;88:592-600.
4. van Adelsberg J, Phillip G, LaForce CF, Weinstein SF, Menten J, Malice
ciated with bradycardia and anxiety. It is notable that we
M-P, et al. Randomized controlled trial evaluating the clinical benefit of gave epinephrine to a patient who appeared to have a
montelukast for treating spring seasonal allergic rhinitis. Ann Allergy moderate reaction to an injection that was later revealed
Asthma Immunol 2003;90:214-22. to be placebo.3
5. Ratner PH, Howland WC, Arastu R, Philpot EE, Klein KC, Baidoo CA,
The inclusion of a blind placebo group is the only way
et al. Fluticasone propionate aqueous nasal spray provided greater im-
provement in daytime and nighttime nasal symptoms of seasonal allergic to be certain that a sting challenge adequately tests treat-
rhinitis compared with montelukast. Ann Allergy Asthma Immunol 2003; ment efficacy. This is an important ethical issue; we have
90:536-42. observed the death of a man who believed he was pro-
6. Pullerits T, Praks L, Ristioja V, Lotvall J. Comparison of a nasal tected by ant WBE, reinforcing the risks of a flawed
glucocorticoid, antileukotriene, and a combination of antileukotriene and
antihistamine in the treatment of seasonal allergic rhinitis. J Allergy Clin
evidence base. As explained, the exposure to a small
Immunol 2002;109:949-55. number of IFA in a sting challenge may be insufficient
to provoke anaphylaxis in many people with allergy.
doi:10.1016/j.jaci.2005.03.045 Furthermore, insect handling procedures may lead to a
depletion of venom as assessed at the time of venom sac
dissection.3
Efficacy of ant venom immunotherapy and Imported fire ant WBE may be an effective treatment,
whole body extracts because extracts contain venom proteins and quality con-
trol methods have been developed. However, uncertainties
To the Editor:
remain with regard to the natural history of IFA allergy
Golden1 presents a useful review of insect venom and whether the dose of venom delivered by IFA WBE
immunotherapy, but we disagree with his conclusion
extracts is sufficient to confer protection.
that imported fire ant (IFA) whole body extract (WBE)
Simon G. A. Brown, MBBS, PhD, FACEMa
has been proven efficacious. Golden1 stresses the need Robert J. Heddle, MBBS, PhD, FRACP, FRCPAb
to understand the natural history of sting allergy and Michael D. Wiese, BPharm, MClinPharmc
the importance of controlled studies. We add to this the Konrad E. Blackman, MBBS, FACEMc
need for prospective design, adequate randomization, and a
University of Western Australia
double-blinding. Fremantle Hospital
J ALLERGY CLIN IMMUNOL Correspondence 465
Alma Street contain sufficient venom allergens to have acceptable,

Fremantle, WA 6160, Australia albeit suboptimal, efficacy. Also, unlike the winged
b
Department of Respiratory Medicine Hymenoptera WBE, there are relatively few reports of
Flinders Medical Centre
treatment failure with IFA WBE, and no fatalities. Brown
Bedford Park, Australia
c et al1 mention a fatal reaction but not the species of WBE
Royal Hobart Hospital
Hobart, Australia used for treatment or the dose, schedule, and duration
of treatment. However, our experience with the winged
Hymenoptera WBEs ‘‘demonstrates the value of a com-
plete understanding of the natural history of the disease in
REFERENCES
determining the efficacy and indications for treatment and
1. Golden DB. Insect sting allergy and venom immunotherapy: a model and the importance of clinical trials.’’2 As Brown et al1 point
a mystery. J Allergy Clin Immunol 2005;115:439-47.
2. Brown SGA, Franks RW, Baldo BA, Heddle RJ. Prevalence, severity, and
out, prospective design and blind treatment are also critical
natural history of jack jumper ant venom allergy in Tasmania. J Allergy to the strength of the evidence.
Clin Immunol 2003;111:187-92. The lack of IFA venom products for diagnosis and
3. Brown SGA, Wiese MD, Blackman KE, Heddle RJ. Ant venom immu- immunotherapy in the United States is a continuing gap in
notherapy: a double-blind, placebo-controlled, crossover trial. Lancet
our repertoire. It is not the ethics but the economics of
2003;361:1001-6.
clinical trials that has deterred the performance of double-
Available online June 17, 2005. blind, placebo-controlled clinical trials of IFA WBE and
doi:10.1016/j.jaci.2005.04.025
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
Reply of the Insect Committee of the American Academy of
Allergy, Asthma and Immunology, who have now re-
To the Editor: solved to explore the development of a controlled trial
I appreciate the comments of Brown et al.1 It was clearly of IFA WBE immunotherapy. (Nelson, personal com-
only by unintended oversight that the exemplary work of munication, March 2005). When the efficacy of current
the authors was not cited in my review that focused on treatment has not been proven up to current standards
treatment strategies in the United States.2 Their work on and the risk of treatment failure is a life-threatening
the natural history of ant venom allergy and their con- reaction, a controlled trial is clearly justified. Our
trolled trial of ant venom immunotherapy are a model to thanks to Brown et al1 for exposing this important
which we should aspire.3,4 In contrast, there has been no issue.
controlled trial of imported fire ant (IFA) whole body David B. K. Golden, MD
extract (WBE) immunotherapy, and the natural history of Johns Hopkins Asthma and Allergy Center
IFA allergy is unknown. 5501 Hopkins Bayview Blvd
I am dismayed that my statements were construed to Baltimore, MD 21224
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 REFERENCES
diagnostic accuracy.5-8 For this reason, I stated, ‘‘For fire 1. Brown SGA, Heddle RJ, Wiese MD, Blackman KE. Efficacy of ant
ant allergy, venom is the most accurate diagnostic material, venom immunotherapy and whole body extracts. J Allergy Clin Immunol
but WBE have shown adequate diagnostic sensitivity.’’ 2005;116:464-5.
2. Golden DBK. Insect sting allergy and venom immunotherapy: a model
My statement that ‘‘Fire ant immunotherapy is performed
and a mystery. J Allergy Clin Immunol 2005;115:439-47.
with WBE that contains sufficient venom allergens to 3. Brown SG, Franks RW, Baldo BA, Heddle RJ. Prevalence, severity and
provide reasonable clinical protection’’ was based on the natural history of jack jumper ant venom allergy in Tasmania. J Allergy
reported content of venom allergens in IFA WBE. I did Clin Immunol 2003;111:187-92.
not state that WBE was as potent as venom.8 The report 4. Brown SG, Wiese MD, Blackman KE, Heddle RJ. Ant venom immu-
notherapy: a double-blind placebo-controlled crossover trial. Lancet
of clinical efficacy of IFA WBE by Freeman et al9 had 2003;361:1001-6.
the strengths of prospective sting challenge (instead of 5. Strom GB, Boswell MD, Jacobs RL. In vivo and in vitro comparison of
retrospective field sting reports) and a limited (but fire ant venom and fire ant whole body extract. J Allergy Clin Immunol
highly significant) control group. Still, Freeman et al9 1983;72:46-53.
6. Paull BR, Coghlan TH, Vinson SB. Fire ant venom hypersensitivity,
concluded that ‘‘A controlled prospective trial of WBE
I: comparison of fire ant venom and whole body extract in the diagnosis
versus placebo is needed.to help define the natural of fire ant allergy. J Allergy Clin Immunol 1983;71:448-53.
history of IFA hypersensitivity.’’ Of recent interest are 7. Butcher BT, deShazo RD, Ortiz AA, Reed MA. Superiority of
reports of rush immunotherapy with IFA WBE to prevent Solenopsis invicta venom to whole body extract in RAST for diagnosis
systemic and large local reactions, but these too were of imported fire ant allergy. Int Arch Allergy Appl Immunol 1988;85:
458-61.
uncontrolled.10,11 8. Hoffman DR, Jacobson RS, Schmidt M, Smith AM. Allergens in
Together, the in vivo and in vitro evidence led to the Hymenoptera venoms, XXIII: venom content of imported fire ant whole
belief that unlike the other Hymenoptera, IFA WBE does body extracts. Ann Allergy 1991;66:29-31.
J ALLERGY CLIN IMMUNOL Correspondence 465
Alma Street contain sufficient venom allergens to have acceptable,

Fremantle, WA 6160, Australia albeit suboptimal, efficacy. Also, unlike the winged
b
Department of Respiratory Medicine Hymenoptera WBE, there are relatively few reports of
Flinders Medical Centre
treatment failure with IFA WBE, and no fatalities. Brown
Bedford Park, Australia
c et al1 mention a fatal reaction but not the species of WBE
Royal Hobart Hospital
Hobart, Australia used for treatment or the dose, schedule, and duration
of treatment. However, our experience with the winged
Hymenoptera WBEs ‘‘demonstrates the value of a com-
plete understanding of the natural history of the disease in
REFERENCES
determining the efficacy and indications for treatment and
1. Golden DB. Insect sting allergy and venom immunotherapy: a model and the importance of clinical trials.’’2 As Brown et al1 point
a mystery. J Allergy Clin Immunol 2005;115:439-47.
2. Brown SGA, Franks RW, Baldo BA, Heddle RJ. Prevalence, severity, and
out, prospective design and blind treatment are also critical
natural history of jack jumper ant venom allergy in Tasmania. J Allergy to the strength of the evidence.
Clin Immunol 2003;111:187-92. The lack of IFA venom products for diagnosis and
3. Brown SGA, Wiese MD, Blackman KE, Heddle RJ. Ant venom immu- immunotherapy in the United States is a continuing gap in
notherapy: a double-blind, placebo-controlled, crossover trial. Lancet
our repertoire. It is not the ethics but the economics of
2003;361:1001-6.
clinical trials that has deterred the performance of double-
Available online June 17, 2005. blind, placebo-controlled clinical trials of IFA WBE and
doi:10.1016/j.jaci.2005.04.025
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
Reply of the Insect Committee of the American Academy of
Allergy, Asthma and Immunology, who have now re-
To the Editor: solved to explore the development of a controlled trial
I appreciate the comments of Brown et al.1 It was clearly of IFA WBE immunotherapy. (Nelson, personal com-
only by unintended oversight that the exemplary work of munication, March 2005). When the efficacy of current
the authors was not cited in my review that focused on treatment has not been proven up to current standards
treatment strategies in the United States.2 Their work on and the risk of treatment failure is a life-threatening
the natural history of ant venom allergy and their con- reaction, a controlled trial is clearly justified. Our
trolled trial of ant venom immunotherapy are a model to thanks to Brown et al1 for exposing this important
which we should aspire.3,4 In contrast, there has been no issue.
controlled trial of imported fire ant (IFA) whole body David B. K. Golden, MD
extract (WBE) immunotherapy, and the natural history of Johns Hopkins Asthma and Allergy Center
IFA allergy is unknown. 5501 Hopkins Bayview Blvd
I am dismayed that my statements were construed to Baltimore, MD 21224
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 REFERENCES
diagnostic accuracy.5-8 For this reason, I stated, ‘‘For fire 1. Brown SGA, Heddle RJ, Wiese MD, Blackman KE. Efficacy of ant
ant allergy, venom is the most accurate diagnostic material, venom immunotherapy and whole body extracts. J Allergy Clin Immunol
but WBE have shown adequate diagnostic sensitivity.’’ 2005;116:464-5.
2. Golden DBK. Insect sting allergy and venom immunotherapy: a model
My statement that ‘‘Fire ant immunotherapy is performed
and a mystery. J Allergy Clin Immunol 2005;115:439-47.
with WBE that contains sufficient venom allergens to 3. Brown SG, Franks RW, Baldo BA, Heddle RJ. Prevalence, severity and
provide reasonable clinical protection’’ was based on the natural history of jack jumper ant venom allergy in Tasmania. J Allergy
reported content of venom allergens in IFA WBE. I did Clin Immunol 2003;111:187-92.
not state that WBE was as potent as venom.8 The report 4. Brown SG, Wiese MD, Blackman KE, Heddle RJ. Ant venom immu-
notherapy: a double-blind placebo-controlled crossover trial. Lancet
of clinical efficacy of IFA WBE by Freeman et al9 had 2003;361:1001-6.
the strengths of prospective sting challenge (instead of 5. Strom GB, Boswell MD, Jacobs RL. In vivo and in vitro comparison of
retrospective field sting reports) and a limited (but fire ant venom and fire ant whole body extract. J Allergy Clin Immunol
highly significant) control group. Still, Freeman et al9 1983;72:46-53.
6. Paull BR, Coghlan TH, Vinson SB. Fire ant venom hypersensitivity,
concluded that ‘‘A controlled prospective trial of WBE
I: comparison of fire ant venom and whole body extract in the diagnosis
versus placebo is needed.to help define the natural of fire ant allergy. J Allergy Clin Immunol 1983;71:448-53.
history of IFA hypersensitivity.’’ Of recent interest are 7. Butcher BT, deShazo RD, Ortiz AA, Reed MA. Superiority of
reports of rush immunotherapy with IFA WBE to prevent Solenopsis invicta venom to whole body extract in RAST for diagnosis
systemic and large local reactions, but these too were of imported fire ant allergy. Int Arch Allergy Appl Immunol 1988;85:
458-61.
uncontrolled.10,11 8. Hoffman DR, Jacobson RS, Schmidt M, Smith AM. Allergens in
Together, the in vivo and in vitro evidence led to the Hymenoptera venoms, XXIII: venom content of imported fire ant whole
belief that unlike the other Hymenoptera, IFA WBE does body extracts. Ann Allergy 1991;66:29-31.
AUGUST 2005
9. Freeman TM, Hyghlander R, Ortiz A, Martin ME. Imported fire ant 11. Walker R, Jacobs J, Tankersly M, Hagan L, Freeman T. Rush immu-
immunotherapy: effectiveness of whole body extracts. J Allergy Clin notherapy for the prevention of large local reactions secondary to
Immunol 1992;90:210-5. imported fire ant stings [abstract]. J Allergy Clin Immunol 1999;103:
10. Tankersley MS, Walker RL, Butler WK, Hagan LL, Napoli DC, Freeman S180.
TM. Safety and efficacy of an imported fire ant rush immunotherapy
protocol with and without prophylactic treatment. J Allergy Clin Available online June 17, 2005.
Immunol 2002;109:556-62. 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 immunopathol-
ogy 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 dis-

covered family of receptors involved in the innate
recognition of pathogens. TLRs have much homology
467
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
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
molecular patterns (PAMPs). Some PAMPs are bac-

terial 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,
FIG 1. Ten distinct TLRs exist in human subjects, recognizing
and cell recruitment. Because of their strong immu-
many PAMPs. TLRs can associate as heterodimers changing
their ligand specificity. nostimulatory capacities, many PAMPs are currently
studied as potential treatment agents for allergic
From Meakins-Christie Laboratories, Department of Pathology and
diseases.
Medicine, McGill University, Montreal, Canada. Because the TLRs are part of the innate immune
Received for publication April 21, 2005; accepted for publication system, they are not modified during an immune
April 22, 2005. response and are passed on to the progeny with little
Available online June 17, 2005.
Reprint requests: Qutayba Hamid, MD, PhD, McGill University, genetic change. This has prompted genetic studies to
Meakins-Christie Laboratory, 3626 St Urbain St, Montreal, Canada determine whether specific single nucleotide polymor-
H2X 2P2. E-mail: qutayba.hamid@mcgill.ca. phisms in the TLR genes are associated with atopy.
A recent study has suggested a polymorphism in
0091-6749/$30.00
Ó 2005 American Academy of Allergy, Asthma and Immunology TLR2 and TLR4 in Europeans to be associated with
doi:10.1016/j.jaci.2005.04.034 decreased atopy, dependent on PAMP exposure
J ALLERGY CLIN IMMUNOL August 2005

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.
(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
468 hypothesis, which states that lack of a ‘‘pathogenic
pressure’’ (increased hygiene) in early childhood
results in an imbalanced immune system hypersensi-
tive 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 allergen-
induced 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 immu-
notherapy has been shown to provide potential clinical
benefits. In a mouse model of allergy, physical linking
of CpG DNA to ragweed protein inhibited IgE
FIG 4. Detection of bromodeoxyuridine-positive proliferating expression, inhibited IL-5 expression, and promoted
cells colocalized with CD3-positive cells in explants of nasal mu-
IFN-g expression.5 Physical linking of the CpG DNA
cosa of children. The explants were stimulated with LPS (0.1 mg/
mL) for 2 hours (A) and 24 hours (B). Ten percent colocalization to the ragweed protein enhanced the effects, suggest-
was seen at 2 hours, and 70% colocalization was seen at 24 hours.3 ing that cells reacting to the allergen also have TLR9
August 2005 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.
469
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 FIG 7. Horseradish peroxidase immunocytochemistry for MBP-
positive cells in sections of patients receiving the ragweed CpG
DNA vaccine for immunotherapy (A) or placebo (B).
activated by the CpG DNA (Fig 6). Ragweed CpG REFERENCES

DNA injections in human clinical trials has been 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
shown to inhibit allergen-induced IL-4 mRNA ex- European farmers. J Allergy Clin Immunol 2004;113:482-8.
pression, IL-5 mRNA expression, and MBP-positive 2. Noguchi E, Nishimura F, Fukai H, Kim J, Ichikawa K, Shibasaki M,
cell numbers in the nasal mucosa of allergic patients et al. An association study of asthma and total serum immunoglobin
(Fig 7) and to reduce chest and nasal symptom scores.6 E levels for Toll-like receptor polymorphisms in a Japanese popu-
lation. Clin Exp Allergy 2004;34:177-83.
The compound can also increase the number of TLR9- 3. Tulic MK, Manoukian JJ, Eidelman DH, Hamid Q. T-cell prolifer-
positive cells in the nasal mucosa (Fig 8). As the role of ation induced by local application of LPS in the nasal mucosa of
PAMPs and TLRs is clarified in atopy and TLRs are nonatopic children. J Allergy Clin Immunol 2002;110:771-6.
4. Tulic MK, Fiset PO, Manoukian JJ, Frenkiel S, Lavigne F, Eidelman
better characterized, development of new therapies to
DH, et al. Role of toll-like receptor 4 in protection by bacterial
both prevent atopic diseases and treat existing disease lipopolysaccharide in the nasal mucosa of atopic children but not
will be possible. adults. Lancet 2004;363:1689-97.

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).
5. Tighe H, Takabayashi K, Schwartz D, Van Nest G, Tuck S, Eiden JJ,

et al. Conjugation of immunostimulatory DNA to the short ragweed
470 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 Fig 1 shows a 7-year-old Latin American boy with
of natural killer cells presenting as severe NK cell CAEBV. Typical of this condition is the
presence of bullous and ulcerative skin lesions after
skin reaction to mosquito bites exposure to mosquito bites. In addition, patients
Susan E. Pacheco, MD, Stephen M. Gottschalk, MD, develop high fever, lymphadenopathy in draining
Mary V. Gresik, MD, Megan K. Dishop, MD, nodes, and marked hepatosplenomegaly. Bullous
Takayuki Okmaura, MD, and Theron G.
McCormick, MD, Guest Editors
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.
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: Tgmccorm@texaschildrenshospital.org.
0091-6749/$30.00
doi:10.1016/j.jaci.2005.04.044 FIG 2.

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).
5. Tighe H, Takabayashi K, Schwartz D, Van Nest G, Tuck S, Eiden JJ,

et al. Conjugation of immunostimulatory DNA to the short ragweed
470 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 Fig 1 shows a 7-year-old Latin American boy with
of natural killer cells presenting as severe NK cell CAEBV. Typical of this condition is the
presence of bullous and ulcerative skin lesions after
skin reaction to mosquito bites exposure to mosquito bites. In addition, patients
Susan E. Pacheco, MD, Stephen M. Gottschalk, MD, develop high fever, lymphadenopathy in draining
Mary V. Gresik, MD, Megan K. Dishop, MD, nodes, and marked hepatosplenomegaly. Bullous
Takayuki Okmaura, MD, and Theron G.
McCormick, MD, Guest Editors
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.
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: Tgmccorm@texaschildrenshospital.org.
0091-6749/$30.00
doi:10.1016/j.jaci.2005.04.044 FIG 2.

FIG 3.
FIG 6.
Skin biopsy from a bullous lesion revealed subep-

idermal bullae with a dense dermal infiltrate of eosin-
ophils, 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 ‘‘hy-
persensitivity reaction.’’ However, this condition does
not meet criteria for an immunologic allergic or
hypersensitivity reaction to mosquitoes on laboratory
471
or clinical grounds.
FIG 4.
lesions develop within 24 hours after mosquito expo-

sure 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

Cell markers CD32, CD561, and/or CD161
Receptor for infection Unknown

Main transforming protein Unknown
Mosquito bite reactions Often present
Target population First 2 decades of life
Associated malignancies Hemophagocytic
lymphohistiocytosis,
NK cell leukemia,
NK cell lymphoma
FIG 5.

By current parameters, patients with NK cell symptomatic care, the optimal treatment option is bone
CAEBV disease seem to have normal immunity marrow transplantation.5
before development of the disease. An immunologic
evaluation in this patient revealed normal lymphocyte REFERENCES
proliferation to antigen and mitogens and functional 1. Miyazato H, Nakasuka S, Dong Z, Takakuwa T, Oka K, Hanamoto
antibodies to polysaccharide and protein antigens. H, et al. NK-cell related neoplasms in Osaka, Japan. Am J Hematol
Skin biopsy specimens from patients with NK cell 2004;76:230-5.
2. Tokura Y, Ishihara S, Tagawa S, Naoshiro S, Ohshima K, Takigawa M.
CAEBV related to mosquito bites are significant for Hypersensitivity to mosquito bites as the primary clinical manifestation
an inflammatory reaction composed primarily of of a juvenile type of Epstein-Barr virus-associated natural killer cell
NK cells (CD561CD3–) expressing EBV DNA by leukemia/lymphoma. J Am Acad Dermatol 2001;45:569-78.
in situ hybridization (Fig 7). 3. Ohga S, Nomura A, Takada H, Hara T. Immunological aspects of
Epstein-Barr virus infection. Crit Rev Oncol Hematol 2002;44:203-15.
PBMCs from affected patients often demonstrate 4. Kimura H, Hoshino Y, Kanegane H, Tsuge I, Okamura T, Kawa K,
30% to 70% NK cells, most infected with monoclonal et al. Clinical and virologic characteristics of chronic active
or oligoclonal EBV. In addition, EBV DNA PCR Epstein-Barr virus infection. Blood 2001;98:280-6.
5. Fujii N, Takenaka K, Hiraki A, Maeda Y, Ikeda K, Shingawa K, et al.
levels from PBMCs are significantly elevated, with
Allogeneic peripheral blood stem cell transplantation for the treat-
mean levels of 1042 copies/mg.4 A table distinguishing ment of chronic active Epstein-Barr virus infection. Bone Marrow
NK cell CAEBV is provided (Table I). Aside from Transplant 2000;26:805-8.
472

Articles of note. . . Beyond
Adverse events after influenza immunization in
Our
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
Pages
Burton Zweiman, MD, & Marc E. Rothenberg, MD, PhD, Editors
adverse effects of such immunization. This study
reviewed records of the Vaccine Adverse Event
Reporting System (VAERS), a passive surveillance findings will help stimulate a continued search for an
system begun by the US Food and Drug Adminis- effective, safe RSV vaccine.
tration and the Centers for Disease Control and Pre- (Falsey et al. N Engl J Med 2005;352:1749-59.)
vention in 1990. Since 1990, there were 166 reports
of adverse events (AEs) following receipt of the TFV Anaphylactic reaction to lupin flour
alone or along with other vaccines in children less Flour made from ground-up lupin beans is being
than 2 years old. These AEs have generally been quite used increasingly as a wheat flour substitute in
mild, (fever, transient rash). Seizures were the most com- some European countries. This case report described
mon serious AE, reported in 28 cases. Most of the a severe anaphylactic reaction in a 25-year-old woman
seizures occurred along with fever with onset within shortly after ingestion of a meal containing chicken,
2 days after immunization. No sequelae were re- fried potatoes, and onion rings with recovery after
ported. Although there is probably some underreport- intensive therapy. There was a past history of transient
ing of AE in the voluntary reporting in the VAERS, asthma at age 15 years and a prior anaphylactic
these findings suggest that TFV immunization is reaction to peanuts. It was found that the breading on
generally very safe and well tolerated by young the onion rings contained lupin flour. Subsequent skin
children. tests to peanuts and a crude extract of lupin flour were
(McMahon et al. Pediatrics 2005;115:453-9.) strongly positive. There is probably a 20% to 40%
cross-reacting homology between lupin and one of
Respiratory syncytial virus infection in elderly the allergens in peanuts. Lupin flour allergy has been
and high-risk adults reported mainly in European patients known to be
Respiratory syncytial virus (RSV) infection, exten- allergic to other legumes, particularly peanut, soy, or
sively investigated in children, is increasingly recog- pea. Indeed, reactions to lupin are one of the most
nized as a cause of illness in adults. This study common types of food-induced anaphylaxis in France.
prospectively investigated all respiratory illnesses in This report should be kept in mind, because lupin
cohorts of (1) healthy elderly patients (65 years of age beans and lupin flour are now becoming available for
or older), (2) high-risk adults (those with chronic heart, consumption in the United States.
lung, or airways disease), and (3) patients hospitalized (Radcliffe et al. Lancet 2005;365:1360.)
with acute cardiopulmonary conditions. RSV infec-
tions occurred annually in 3% to 7% of healthy elderly A new mechanism of nonatopic asthma elicited by
patients and in 4% to 10% of high-risk adults. The immunoglobulin free light chains
frequency of RSV infection was at least as great as that A significant proportion of asthmatic individuals are
of influenza A in these populations. In the hospitalized nonatopic, yet the mechanism by which an asthma
patients, RSV infection and influenza A resulted in exacerbation is triggered in these individuals is not
similar lengths of stay, rates of use of intensive care known. In this report, the investigators extended their
(15% and 12%, respectively), and mortality (8% and prior observation that antigen-specific immunoglobu-
7%, respectively). RSV infection accounted for 10.6% lin free light chains (LCs) mediate mast cell–depen-
of hospitalizations for pneumonia, 11.4% of hospital- dent hypersensitivity by examining the role of LCs in a
izations for chronic obstructive pulmonary disease, murine model of nonatopic asthma. In particular, they
and 7.2% of hospitalizations for asthma. The authors use a LC antagonist, the 9-mer F991, and abrogate the
concluded that RSV infection is an important illness development of airway hyperresponsiveness and pul-
in elderly and high-risk adults, with a disease burden monary infiltration. Using mast cell–deficient mice,
similar to that of nonpandemic influenza A in a they show that the role of LCs is dependent on mast
population in which the prevalence of immunization cells. Finally, they demonstrate that asthmatic indi-
for influenza is great. One would hope that these viduals (both atopic and nonatopic) have elevated sera
J ALLERGY CLIN IMMUNOL August 2005 Page 473

levels of jLC (but not kLC) compared with control mice, DT is only active on CD11c+ cells (primarily
individuals. These results substantiate a new mecha- DCs and macrophages). Indeed, administration of DT
nism that might be involved in triggering allergic, but to the lungs of these transgenic mice depleted CD11c+
not IgE-mediated, asthma and suggest that inhibition DCs and alveolar macrophages. Notably, DT abol-
of LC-mediated mast cell activation might be thera- ished the characteristic features of asthma, including
peutically useful. eosinophilic inflammation, goblet cell hyperplasia,
(Kraneveld et al. Proc Natl Acad Sci U S A 2005;102: and bronchial hyperreactivity. Furthermore, in the
1578-83.) absence of CD11c+ cells, TH2 cells did not produce
IL-4, IL-5, and IL-13 in response to OVA aerosol.
Suppressive effects of prostaglandin E receptor subtype Importantly, in CD11c-depleted mice, eosinophilic
EP3—the mechanism of aspirin sensitivity? inflammation and TH2 cytokine secretion were re-
Prostaglandins (PGs), including PGD2 and PGE2, are stored by adoptive transfer of CD11c+ DCs, but not
produced during allergic responses, yet PG synthesis by transfer of alveolar macrophages. These findings
inhibitors (eg, aspirin) are generally ineffective for identify lung DCs as key pro-inflammatory cells that
asthma. Inasmuch as PGD2 is a potent proinflamma- are necessary and sufficient for TH2 cell stimulation
tory mediator and smooth muscle constrictor, this during ongoing lung inflammation.
suggests that PGE2 might have an important regula- (van Rijt et al. J Exp Med 2005;201:981-91.)
tory (or protective) role in allergy. To address this
possibility, the investigators subjected mice with Daily versus as-needed inhaled corticosteroid treatment
specific deficiencies in each of the 4 PGE2 receptors of mild, persistent asthma
(EP1 through EP4) to an OVA-induced model of Most current national guidelines recommend daily use
asthma. Notably, mice deficient in EP3 had a marked of controller medications, such as an inhaled cortico-
increase in multiple aspects of asthma (including steroid (ICS), in the treatment of persistent asthma
inflammation and TH2 cytokine production), whereas (PA), even of mild degree. This randomized, double-
mice deficient in the other receptors were comparable blind study investigated whether treatment with daily
to wild-type mice. On the basis of these results sug- ICS (budesonide 200 micrograms bid), or daily
gesting a suppressive role for EP3 signaling, the leukotriene antagonist (zafirlukast 20 mg bid) was
investigators examined the impact of an EP3-selective more effective than daily placebo with as-needed ICS
agonist on the development of asthma. Indeed, the EP3 use in the treatment of mild PA in 199 adults. After
agonist inhibited airway inflammation, TH2 cytokine 1 year of treatment, there were no significant differ-
production, and bronchoconstriction, even when it ences in the major asthma outcome (increases in
was administered 3 hours after antigen challenge. average morning peak expiratory flow) among the 3
In addition, a significant number of allergen-induced treatment groups. The frequency of acute asthma
genes were inhibited by the EP3 agonist. Taken exacerbations requiring corticosteroid therapy was
together, these results call attention to the anti-allergic also not significantly different in the 3 groups. There
role of PGE2 and its EP3 receptor, providing a new were greater improvements in patients using ICS daily
paradigm for therapeutic intervention. Furthermore, than in placebo-treated individuals in prebroncho-
the results provide a possible explanation for aspirin- dilator FEV1 (P = .005), PC20 bronchial reactivity
sensitive asthma by suggesting that such individuals (P < .001), asthma control score (P < .001), sputum
might preferentially require the protective effects of eosinophils (P = .007), and number of symptom-free
the EP3 pathway (and are thus sensitive to aspirin). days (P = .03). However, the postbronchodilator FEV1
(Kunikata et al. Nat Immunol 2005;6:524-31.) and quality of life scores were not significantly
different between those treated daily with ICS and
Dendritic cells are critical for experimental asthma those receiving placebo. There were no differences in
Dendritic cells (DCs) have been shown to have an any asthma outcome scores between those treated with
important role in sensitization to inhaled allergens, but daily zafirlukast and those receiving placebo. These
their function in ongoing TH2 cell–mediated lung findings indicate that daily ICS treatment improves
inflammation is currently unknown. Using an OVA- some manifestations of mild PA in adults. However,
induced murine asthma model, the investigators show the authors concluded that daily ICS might not be
that airway DCs acquire a mature phenotype and needed in such asthmatic individuals; they can instead
interact with CD4 T cells within the lung tissue. To be treated with short intermittent courses of inhaled
study whether the DCs contributed to inflammation, or oral corticosteroids taken when asthma symptoms
they subsequently depleted the DCs from the airways worsen significantly. It is still uncertain whether these
using CD11c-diphtheria toxin (DT) receptor trans- findings can be extended to mild PA in children.
genic mice during the OVA aerosol challenge. In these (Boushey et al. N Engl J Med 2005;352:1519-28.)
Page 474 August 2005 J ALLERGY CLIN IMMUNOL

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T HE J OURNAL OF

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

The editors’ choice 239

Reviews and feature articles

Current reviews of allergy and clinical immunology

Y This month’s theme: Infection and immunity

About the cover

Ó 2005 American Academy of Allergy, Asthma and Immunology

J ALLERGY CLIN IMMUNOL August 2005 5A

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

The editors’ choice 239

Reviews and feature articles

Current reviews of allergy and clinical immunology

Y This month’s theme: Infection and immunity

About the cover

Ó 2005 American Academy of Allergy, Asthma and Immunology

J ALLERGY CLIN IMMUNOL August 2005 5A

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

The editors’ choice 239

Reviews and feature articles

Current reviews of allergy and clinical immunology

Y This month’s theme: Infection and immunity

About the cover

Ó 2005 American Academy of Allergy, Asthma and Immunology

J ALLERGY CLIN IMMUNOL August 2005 5A

Continuing Medical Education examination: Innate immune responses 250

Y Molecular mechanisms in allergy and clinical immunology

Continuing Medical Education examination: EBV the prototypical

Asthma diagnosis and treatment

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

J ALLERGY CLIN IMMUNOL August 2005 7A

Continuing Medical Education examination: Innate immune responses 250

Y Molecular mechanisms in allergy and clinical immunology

Continuing Medical Education examination: EBV the prototypical

Asthma diagnosis and treatment

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

J ALLERGY CLIN IMMUNOL August 2005 7A

Continuing Medical Education examination: Innate immune responses 250

Y Molecular mechanisms in allergy and clinical immunology

Continuing Medical Education examination: EBV the prototypical

Asthma diagnosis and treatment

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

J ALLERGY CLIN IMMUNOL August 2005 7A

Roflumilast, an oral, once-daily phosphodiesterase 4 inhibitor, attenuates 292

Duration of postviral airway hyperresponsiveness in children with 299

Mechanisms of asthma and allergic inflammation

Endobronchial adenosine monophosphate challenge causes tachykinin 312

Allergen-induced substance P synthesis in large-diameter sensory neurons 325

Continued on page 11A

Differential effects of (S)- and (R)-enantiomers of albuterol in a mouse 332

Rhinitis, sinusitis, and ocular diseases

Allergen-specific nasal IgG antibodies induced by vaccination with genetically 347

Levocetirizine: Pharmacokinetics and pharmacodynamics in children 355

Striking deposition of toxic eosinophil major basic protein in mucus: 362

Environmental and occupational respiratory disorders

Airborne endotoxin in homes with domestic animals: Implications for 384

Continued on page 13A

Food allergy, dermatologic diseases, and anaphylaxis

Basic and clinical immunology

Advances in Asthma, Allergy, and Immunology Series 2005

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

New York, NY, and Hamburg, Germany

Y David L. Perkins, MD, and Diane R. Gold, MD, Boston, Mass

Relationship to FIT: TPD/Preceptor Research Mentor ____JACI Editor