ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of HertfordshireHatfield, Hertfordshire, UK

For further volumes:http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols andmethodologies in the critically acclaimedMethods in Molecular Biology series. The series wasthe first to introduce the step-by-step protocols approach that has become the standard in allbiomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagentsneeded to complete the experiment, and followed by a detailed procedure that is supportedwith a helpful notes section offering tips and tricks of the trade as well as troubleshootingadvice. These hallmark features were introduced by series editor Dr. John Walker andconstitute the key ingredient in each and every volume of the Methods in Molecular Biologyseries. Tested and trusted, comprehensive and reliable, all protocols from the series areindexed in PubMed.

Basophils and Mast Cells

Methods and Protocols

Second Edition

Edited by

Bernhard F. Gibbs

Division of Experimental Allergology and Immunodermatology, Department of Human Medicine,School of Medicine and Health Sciences, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany

Franco H. Falcone

Institute for Parasitology, Justus Liebig University Giessen, Biomedizinisches ForschungszentrumSeltersberg (BFS), Giessen, Germany

EditorsBernhard F. GibbsDivision of Experimental Allergologyand ImmunodermatologyDepartment of Human MedicineSchool of Medicine and Health SciencesCarl von OssietzkyUniversity of OldenburgOldenburg, Germany

Franco H. FalconeInstitute for ParasitologyJustus Liebig University Giessen, BiomedizinischesForschungszentrum Seltersberg (BFS)Giessen, Germany

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-0716-0695-7 ISBN 978-1-0716-0696-4 (eBook)https://doi.org/10.1007/978-1-0716-0696-4

© Springer Science+Business Media, LLC, part of Springer Nature 2020This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,expressed or implied, with respect to the material contained herein or for any errors or omissions that may have beenmade. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of SpringerNature.The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Acknowledgements

The cover image shows a peripheral blood basophil immunostained with BB1 antibody,showing the presence of basogranulin (green) in the secretory granules (copyright Andrew FWalls and Akinori Mochizuki).

v

Preface to the Second Edition

The well-received first edition of our book, Basophils and Mast Cells: Methods and Protocols,highlights the need for detailed methods that focus on basophils and mast cells, especiallythe use of primary cells. This second edition builds on and extends the strengths of theprevious one. The large majority of chapters have been revisited and, where necessary,updated by their original authors. In addition to refining existing methods, the new editionalso contains newly developed methods provided by cutting-edge researchers in the field.

To accommodate the new chapters, we have expanded the structure of the book fromoriginally four into six parts: after an introduction to the basic biology of basophils and mastcells in Part I, Part II focuses on obtaining the cells by purification or culture of stem cellsprogenitors. Here, a new chapter has been added by Siebenhaar and colleagues, describingthe generation of peripheral CD34+ stem cell-derived mast cells (Chapter 5). Similarly,MacGlashan describes two protocols for the generation of basophils from CD34+ progeni-tors, starting with two different materials: the convenient (but more expensive) commercialsources of CD34+ progenitors, or leukapheresis pack. As an interesting aside, MacGlashanreminds us in his introduction to Chapter 6 that although basophils are normally consideredshort-lived cells, their residence time in circulation (with estimates ranging from 12 h to afew days) is more likely to be just the tip of the iceberg, as a much longer time is spent duringthe process leading to maturation in the bone marrow, before they are released intocirculation. Our understanding here is very patchy.

Part III of the book is now dedicated to basophil and mast cell lines. After the updatedsummary of the most commonly used mast cell and basophil lines by Passante (Chapter 10),three new chapters describe the use of humanized reporter systems. As a possible alternativeto BATs and MATs, described in the next part, which require speedy processing of bloodsamples and flow cytometry equipment, IgE reporter systems are also available. These can becombined with allergen arrays, as described by Kalli and coauthors in Chapter 12. Barwaryet al. (Chapter 13) describe a new, easy to measure humanized IgE reporter system. Here,the cells are preloaded with a red fluorescent protein targeted to the granules, which is thenreleased upon activation, and can be conveniently measured without the need for expensivereagents or long incubation times. Of course, other research arenas have also advanced sincethe publication of our first edition, such as biomedical nanotechnology, and we therefore feltthat it was important to include protocols tailored to the study of the interactions betweennano- or submicron-scale materials and basophils or mast cells, as in Chapter 14 by Alelwaniand her coauthors.

Part IV describes diagnostic applications. This part has also been expanded and updated.The new Chapter 16 by Ebo and coauthors now introduces BATs in more depth, inparticular with respect to their use in understanding drug hypersensitivity reactions. Twouseful flow cytometry techniques applied to basophils are presented by Elst and coauthors:in Chapter 17, a technique which, rather than detecting changes in surface expression ofactivation markers such as CD63 or CD203c, allows staining of exteriorized granularmaterials. The process takes advantage of the ability of fluorescently labeled avidin to bindto negatively charged proteoglycans contained in the granule matrix, which are exteriorizedduring degranulation; the method correlates well with CD63 upregulation [1]. InChapter 18, the same authors remind us that not all activation pathways for mast cells and

vii

basophils are mediated via crosslinking of the high affinity IgE receptor and describe aprotocol for the assessment of Mas-related G protein-coupled receptor (MRGPRX2)expression on the two cell types. The MRGPRX2 receptor is increasingly recognized asbeing involved in many systemic pseudo-allergic (or anaphylactoid) reactions to smallmolecules, such as basic secretagogues and several drugs [2]. Measuring cellular activationby flow cytometry is by no means limited to basophils. Similar techniques can be also appliedto mast cells: the new Chapter 19 by Bahri et al. describes a novel method for mast cellactivation tests (MATs), extending the instrumentarium available to researchers in our field.

Part V focuses on functional studies. The new Chapter 20 by Bechmann et al. should beof great interest to the reader planning to study gene expression patterns in basophils; asshown in this chapter, the method chosen to purify RNA has a strong bearing on the results(i.e., the quality and integrity of RNA) obtained—an important lesson in times where manylabs rely on “convenient” commercial kits for this kind of work.

A new chapter now enables to assess the role of basophils in lung disease. Althoughbasophils have been known for several decades to play an important role in asthma, and weredescribed in sputum by Kimura and coworkers in 1975 [3], studying their role in differentforms of asthmatic disease has been difficult due to the lack of appropriate protocols. Thetechnique described by Fux et al. in Chapter 23 will help laboratories interested in examin-ing the roles of basophils in different asthma phenotypes. Part VI closes with protocols forknockout and disease models.

In total, 11 new chapters have been added in this edition. We are confident that thesecond edition of our book will provide you with even more essential cutting-edge tools foryour research into the functions of mast cells and basophils. Given that these cells appear tobe involved in numerous diseases as well as regulating many physiological functions, there isa considerable unmet need to shed more light on these cell types. We hope that theseprotocols will be of great benefit and wish you every success in your research.

Oldenburg, Germany Bernhard F. GibbsGiessen, Germany Franco H. Falcone

References

1. Joulia R, Mailhol C, Valitutti S, et al (2017) Direct monitoring of basophil degranulation by usingavidin-based probes. J Allergy Clin Immunol 140:1159–1162.e6

2. McNeil BD, Pundir P, Meeker S, et al (2015) Identification of a mast-cell-specific receptor crucial forpseudo-allergic drug reactions. Nature 519:237–241

3. Kimura I, Tanizaki Y, Saito K, et al (1975) Appearance of basophils in the sputum of patients withbronchial asthma. Clin Allergy 5:95–8

viii Preface to the Second Edition

Preface to the First Edition

According to the latest epidemiological findings, it appears that allergic diseases will soon beaffecting over 40% of the population in both Europe and elsewhere. There is, therefore, anoverwhelming unmet need to unravel the immunological causes of allergic inflammationand to improve anti-allergic therapies. In recent years, it has come to light that mast cells andtheir blood-borne basophil counterparts not only contribute significantly to the symptomsof allergy but also play a role in supporting the underlying tendency of the immune systemto respond in a pro-allergic manner. This immunomodulatory function has particularly ledto a resurgence into basophil research and may one day finally uncover the biological role forthis enigmatic and rare cell type which so readily releases a number of major inflammatoryand immunomodulatory chemical mediators within very short periods of time. Moreover,the discovery that both mast cells and basophils potentially contribute to a variety ofdiseases, such as urticaria, cancer, and autoimmune diseases, as well as physiological func-tions, has increased the need for improved tools for researching these cells (see Chapter 1).

One of the most problematic features of mast cells and basophils is their strikingdifferences in behavior and response to various stimulating agents and drugs dependingon their location and the species from which they are studied. This limits, to some extent,our ability to extrapolate findings from, for example, mouse to human, but also tissue totissue and the use of tumor mast cell and basophil lines. It is therefore imperative that bettertools for the isolation of these cells from primary tissues are forthcoming. This indeed hasbeen the case in recent years and we now present a book which, we feel, will significantly helpanyone conducting research into these cells from the most practical point of view: definitivelab-based protocols targeted to those who actually do the work!

Whether you are planning to purify basophils or mast cells from peripheral blood(Chapter 2) or tissues (Chapter 3), or want to culture them from precursors from buffycoat blood (Chapter 4) or bone marrow (Chapter 5), you will find well-tested protocols inthis book. Other chapters discuss the use of mast cells in organotypic skin models(Chapter 6), the difficulty of counting basophils (Chapter 7), how to stain mast cells andbasophils in tissues (Chapter 8), how to assess mediator release (Chapter 10), and signaltransduction (Chapter 15). Also discussed is the suitability of using mast cell or basophil-likecell lines as surrogates for primary mast cells and basophils (Chapter 9) and the use ofreporter cell lines (Chapter 13) for the detection of allergen-specific IgE in serum samples.Chapter 12 presents protocols which can be used to measure mast cell migration usingmicroscopy. Chapter 14 describes protocols that can be used for gene silencing of mast cellsand basophils, two cell types that are notoriously difficult to transfect.

Researchers more interested in ex vivo or in vivo analysis will find protocols for humanbasophil activation tests (BATs) in Chapter 11, for mast cell phenotyping (Chapter 16), orthe study of murine basophils by flow cytometry in Chapter 17. Chapter 18 closes within vivo models that can be used for the analysis of mast cell functions.

Our book is written by leading research scientists in the field of basophil and mast cellbiology. The impetus for this consortium of authors was given by the recently establishedEU/ESF-BMBS COSTAction BM1007 “Mast Cells and Basophils—Targets for InnovativeTherapies” which we would like to acknowledge for providing the ideal platform forscholarly collaboration in this field.

ix

We sincerely hope that this methodology book will provide you with all the necessarytools for your research into mast cells and basophils, and we wish you every success in thequest of shedding more light on these fascinating cell types.

Oldenburg, Germany Bernhard F. GibbsNottingham, UK Franco H. Falcone

x Preface to the First Edition

Contents

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vPreface to the Second Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiPreface to the First Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ixContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

PART I INTRODUCTION AND HISTORICAL BACKGROUND

1 Paradigm Shifts in Mast Cell and Basophil Biology and Function:An Emerging View of Immune Regulation in Health and Disease. . . . . . . . . . . . . 3Ana Olivera and Juan Rivera

PART II OBTAINING THE CELLS: PURIFICATION, CULTURE, AND COUNTING

2 Purification of Basophils from Peripheral Human Blood . . . . . . . . . . . . . . . . . . . . . 35Franco H. Falcone and Bernhard F. Gibbs

3 Mast Cell Purification Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Jasmine Farrington, Elizabeth P. Seward, and Peter T. Peachell

4 Generation of a Human Allergic Mast Cell Phenotypefrom CD133+ Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Hans Jurgen Hoffmann

5 Generation and Culture of Peripheral CD34+ Stem Cell-DerivedMast Cells (PSCMCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Yanyan Luo, Stefan Frischbutter, Jorg Scheffel, and Frank Siebenhaar

6 Modulating the Human Basophil Phenotype During Its Developmentand Maturation: Basophils Derived from In Vitro Cultures of CD34+

Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Donald MacGlashan Jr.

7 Generation of Mast Cells from Murine Stem Cell Progenitors . . . . . . . . . . . . . . . . 85Emily J. Swindle

8 Integration of the Human Dermal Mast Cell into the OrganotypicCo-culture Skin Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Jonghui Kim, Sven Guhl, Magda Babina, Torsten Zuberbier,and Metin Artuc

9 The Absolute Basophil Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109Elena Borzova

xi

PART III BASOPHIL/MAST CELL LINES

10 Mast Cell and Basophil Cell Lines: A Compendium . . . . . . . . . . . . . . . . . . . . . . . . . 127Egle Passante

11 Use of Humanized RBL Reporter Systems for the Detectionof Allergen-Specific IgE Sensitization in Human Serum . . . . . . . . . . . . . . . . . . . . . 145Daniel Wan, Xiaowei Wang, Ryosuke Nakamura,Marcos J. C. Alcocer, and Franco H. Falcone

12 Use of Humanized Fluorescent Reporter Cell Line RBLNFAT-DsRed for the Detection of Allergen-Specific IgE in PatientSera Using Allergen Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155Marina Kalli, Marcos J. C. Alcocer, Andrew J. Blok,and Franco H. Falcone

13 NPY-mRFP Rat Basophilic Leukemia (RBL) Reporter:A Novel, Fast Reporter of Basophil/Mast Cell Degranulation . . . . . . . . . . . . . . . . 163Nafal J. S. Barwary, Daniel Wan, and Franco H. Falcone

14 Use of Engineered Nanoparticles (ENPs) for the Studyof High-Affinity IgE FcεRI Receptor Engagement and Rat Basophilic Leukemia (RBL) Cell Degranulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171Walla Alelwani, Raed A. Alharbi, Daniel Wan, Driton Vllasaliu,Franco H. Falcone, and Snow Stolnik

PART IV DIAGNOSTIC APPLICATIONS

15 Flow Cytometric Allergy Diagnosis: Basophil Activation Techniques . . . . . . . . . . 183Chris H. Bridts, Vito Sabato, Christel Mertens,Margo M. Hagendorens, Luc S. De Clerck, and Didier G. Ebo

16 Basophil Activation Experiments in Immediate Drug Hypersensitivity:More Than a Diagnostic Aid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197Didier G. Ebo, Jessy Elst, Athina Van Gasse, Leander De Puysseleyr,Margaretha A. Faber, Margo M. Hagendorens, Lina Mayorga,Christel Mertens, Chris H. Bridts, Luc S. De Clerck, and Vito Sabato

17 Basophil Activation Techniques: Staining of ExteriorizedGranule Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213Jessy Elst, Vito Sabato, Margo M. Hagendorens, Michel van Houdt,Margaretha A. Faber, Chris H. Bridts, Didier G. Ebo,and Christel Mertens

18 Measurement and Functional Analysis of the Mas-Related GProtein-Coupled Receptor MRGPRX2 on Human Mast Cellsand Basophils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219Jessy Elst, Vito Sabato, Margo M. Hagendorens, Michel van Houdt,Margaretha A. Faber, Chris H. Bridts, Didier G. Ebo,and Christel Mertens

19 Mast Cell Activation Test (MAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227Rajia Bahri and Silvia Bulfone-Paus

xii Contents

PART V WHOLE CELLS: FUNCTIONAL STUDIES

20 Quantity and Quality of Basophil RNA Depend on the RNAExtraction Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241Victor G. Bechmann, Anette Teo Hansen, Lars K. Poulsen,and Bettina M. Jensen

21 Measuring Histamine and Cytokine Release from Basophilsand Mast Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247Bettina M. Jensen, Ewa A. Bartko, Katrine Baumann,and Per S. Skov

22 Detection of Mast Cells and Basophils by Immunohistochemistry . . . . . . . . . . . . 263Andrew F. Walls and Cornelia Amalinei

23 Detection of Basophils and Other Granulocytes in InducedSputum by Flow Cytometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281Lionel Rohner and Michaela Fux

24 Mast Cell Migration and Chemotaxis Assayed by Microscopy . . . . . . . . . . . . . . . . 293Monika Bambouskova, Zuzana Rubıkova, Lubica Draberova,Pavel Draber, and Petr Draber

25 Basophil Stimulation and Signaling Pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311Edward F. Knol and Bernhard F. Gibbs

26 Gene Silencing Approaches in Mast Cells and PrimaryHuman Basophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323Vadim V. Sumbayev and Bernhard F. Gibbs

27 Identification and Immunophenotypic Characterizationof Normal and Pathological Mast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331Jose Mario Morgado, Laura Sanchez-Munoz, Cristina Teod�osio,and Luis Marıa Escribano Mora

PART VI WHOLE ORGANISM: DISEASE AND KNOCKOUT MODELS

28 Mast Cell-Mediated Reactions In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357Stefan Frischbutter, Jorg Scheffel, and Frank Siebenhaar

29 Identification of Murine Basophils by Flow Cytometry and Histology . . . . . . . . . 367Christian Schwartz and David Voehringer

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

Contents xiii

Contributors

MARCOS J. C. ALCOCER • School of Biosciences, University of Nottingham, Nottingham, UKWALLA ALELWANI • Department of Biochemistry, Faculty of Science, University of Jeddah,

Jeddah, Kingdom of Saudi ArabiaRAED A. ALHARBI • Department of Laboratory Medicine, Faculty of AppliedMedical Sciences,

Albaha University, Albaha, Kingdom of Saudi ArabiaCORNELIA AMALINEI • “Grigore T. Popa” University of Medicine and Pharmacy, Iasi,

Romania; Institute of Legal Medicine, Iasi, RomaniaMETIN ARTUC • Department of Dermatology and Allergy, Universit€atsmedizin Berlin,

Berlin, GermanyMAGDA BABINA • Department of Dermatology and Allergy, Universit€atsmedizin Berlin,

Berlin, GermanyRAJIA BAHRI • Lydia Becker Institute of Immunology and Inflammation, Division of

Musculoskeletal and Dermatological Sciences & Manchester Collaborative Centre forInflammation Research (MCCIR), School of Biological Sciences, Faculty of Biology,Medicine and Health, University of Manchester, Manchester, UK

MONIKA BAMBOUSKOVA • Laboratory of Signal Transduction, Institute of Molecular Geneticsof the Czech Academy of Sciences, Prague, Czech Republic; Department of Pathology andImmunology, Washington University in St. Louis, St. Louis, MO, USA

EWA A. BARTKO • Laboratory for Medical Allergology, Herlev-Gentofte Hospital, 2900Hellerup, Denmark

NAFAL J. S. BARWARY • Department of Biology, School of Science, University of Duhok, Duhok,Iraq

KATRINE BAUMANN • RefLab ApS, COBIS building, Copenhagen N, DenmarkVICTOR G. BECHMANN • Laboratory for Medical Allergology, Herlev-Gentofte Hospital,

Hellerup, DenmarkANDREW J. BLOK • Division of Molecular Therapeutics and Formulation, School of Pharmacy,

University of Nottingham, Nottingham, UKELENA BORZOVA • Department of Dermatology and Venereology, I.M. Sechenov First Moscow

State Medical University, Moscow, Russian FederationCHRIS H. BRIDTS • Department of Immunology, Allergology, Rheumatology, University of

Antwerp, Antwerp University Hospital, Antwerp, Belgium; Faculty of Medicine andHealth Science, Department of Immunology, Allergology, Rheumatology and the Infla-MedCentre of Excellence, University of Antwerp, Antwerp University Hospital, Antwerp,Belgium

SILVIA BULFONE-PAUS • Lydia Becker Institute of Immunology and Inflammation, Divisionof Musculoskeletal and Dermatological Sciences & Manchester Collaborative Centre forInflammation Research (MCCIR), School of Biological Sciences, Faculty of Biology,Medicine and Health, University of Manchester, Manchester, UK

LUC S. DE CLERCK • Department of Immunology, Allergology, Rheumatology, University ofAntwerp, Antwerp University Hospital, Antwerp, Belgium; Faculty of Medicine andHealth Science, Department of Immunology, Allergology, Rheumatology and the Infla-MedCentre of Excellence, University of Antwerp, Antwerp University Hospital, Antwerp,Belgium

xv

LEANDER DE PUYSSELEYR • Faculty of Medicine and Health Science, Department ofImmunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence,University of Antwerp, Antwerp University Hospital, Antwerp, Belgium

LUBICA DRABEROVA • Laboratory of Signal Transduction, Institute of Molecular Genetics ofthe Czech Academy of Sciences, Prague, Czech Republic

PAVEL DRABER • Laboratory of Biology of Cytoskeleton, Institute of Molecular Genetics of theCzech Academy of Sciences, Prague, Czech Republic

PETR DRABER • Laboratory of Signal Transduction, Institute of Molecular Genetics of theCzech Academy of Sciences, Prague, Czech Republic

DIDIER G. EBO • Department of Immunology and Allergology, AZ Jan Palfijn Gent, Ghent,Belgium; Faculty of Medicine andHealth Science, Department of Immunology, Allergology,Rheumatology and the Infla-Med Centre of Excellence, University of Antwerp, AntwerpUniversity Hospital, Antwerp, Belgium

JESSY ELST • Faculty of Medicine and Health Science, Department of Immunology,Allergology, Rheumatology and the Infla-Med Centre of Excellence, University of Antwerp,Antwerp University Hospital, Antwerp, Belgium

MARGARETHA A. FABER • Faculty of Medicine and Health Science, Department ofImmunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence,University of Antwerp, Antwerp University Hospital, Antwerp, Belgium

FRANCO H. FALCONE • Institute for Parasitology, Justus Liebig University Giessen,Biomedizinisches Forschungszentrum Seltersberg (BFS), Giessen, Germany

JASMINE FARRINGTON • Department of Biomedical Science, University of Sheffield, WesternBank, Sheffield, UK

STEFAN FRISCHBUTTER • Department of Dermatology and Allergy, Charite—Universit€atsmedizin Berlin, Berlin, Germany

MICHAELA FUX • University Institute of Clinical Chemistry, University Hospital Bern,Inselspital, Bern, Switzerland

BERNHARD F. GIBBS • Division of Experimental Allergology and Immunodermatology,Department of Human Medicine, School of Medicine and Health Sciences, Carl vonOssietzky University of Oldenburg, Oldenburg, Germany

SVEN GUHL • Department of Dermatology and Allergy, Universit€atsmedizin Berlin, Berlin,Germany

MARGO M. HAGENDORENS • Faculty of Medicine and Health Science, Department ofImmunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence,University of Antwerp, Antwerp University Hospital, Antwerp, Belgium; Department ofPediatrics, University of Antwerp, Antwerp University Hospital, Antwerp, Belgium

ANETTE TEO HANSEN • Laboratory for Medical Allergology, Herlev-Gentofte Hospital,Hellerup, Denmark

HANS JURGEN HOFFMANN • Department of Respiratory Diseases and Allergy, AarhusUniversity Hospital and Department of Clinical Medicine, Aarhus University, Aarhus,Denmark

BETTINA M. JENSEN • Laboratory for Medical Allergology, Herlev-Gentofte Hospital, 2900Hellerup, Denmark

MARINA KALLI • Division of Molecular Therapeutics and Formulation, School of Pharmacy,University of Nottingham, Nottingham, UK; School of Biosciences, University ofNottingham, Nottingham, UK

JONGHUI KIM • Department of Dermatology and Allergy, Universit€atsmedizin Berlin,Berlin, Germany

xvi Contributors

EDWARD F. KNOL • Department of Immunology, University Medical Center Utrecht, Utrecht,The Netherlands

YANYAN LUO • Department of Dermatology and Allergy, Charite—Universit€atsmedizinBerlin, Berlin, Germany

DONALD MACGLASHAN JR • Asthma and Allergy Center, Johns Hopkins University,Baltimore, MD, USA

LINA MAYORGA • Allergy Research Group, Instituto de Investigaci�on Biomedica de Malaga-IBIMA and Allergy Unit, Hospital Regional Universitario de Malaga, ARADyAL,Malaga, Spain

CHRISTEL MERTENS • Department of Immunology, Allergology, Rheumatology, University ofAntwerp, Antwerp University Hospital, Antwerp, Belgium; Faculty of Medicine andHealth Science, Department of Immunology, Allergology, Rheumatology and the Infla-MedCentre of Excellence, University of Antwerp, Antwerp University Hospital, Antwerp,Belgium

LUIS MARIA ESCRIBANO MORA • Instituto de Estudios de Mastocitosis de Castilla La Mancha,Hospital Virgen del Valle, Toledo, Spain; Spanish Mastocytosis Network (REMA),Salamanca, Spain; Centro de Investigaci�on del Cancer, Salamanca, Spain

JOSE MARIO MORGADO • Instituto de Estudios de Mastocitosis de Castilla La Mancha,Hospital Virgen del Valle, Toledo, Spain; Spanish Mastocytosis Network (REMA),Salamanca, Spain; Cytognos S.L. Biotechnology, Salamanca, Spain

RYOSUKE NAKAMURA • Japan Agency for Medical Research and Development (AMED),Tokyo, Japan

ANA OLIVERA • Mast Cell Biology Section, Laboratory of Allergic Diseases, National Instituteof Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD,USA; Molecular Immunology Section, Laboratory of Molecular Immunogenetics, NationalInstitute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health,Bethesda, MD, USA

EGLE PASSANTE • Sanofi Genzyme, Berkshire, UKPETER T. PEACHELL • Department of Infection, Immunity and Cardiovascular Disease, The

Medical School, University of Sheffield, Sheffield, UKLARS K. POULSEN • Laboratory for Medical Allergology, Herlev-Gentofte Hospital, Hellerup,

DenmarkJUAN RIVERA • Molecular Immunology Section, Laboratory of Molecular Immunogenetics,

National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutesof Health, Bethesda, MD, USA

LIONEL ROHNER • University Institute of Clinical Chemistry, University Hospital Bern,Inselspital, Bern, Switzerland

ZUZANA RUBIKOVA • Laboratory of Biology of Cytoskeleton, Institute of Molecular Genetics ofthe Czech Academy of Sciences, Prague, Czech Republic

VITO SABATO • Department of Immunology and Allergology, AZ Jan Palfijn Gent, Ghent,Belgium; Faculty of Medicine andHealth Science, Department of Immunology, Allergology,Rheumatology and the Infla-Med Centre of Excellence, University of Antwerp, AntwerpUniversity Hospital, Antwerp, Belgium; Department of Immunology and Allergology, AZJan Palfijn Gent, Ghent, Belgium

LAURA SANCHEZ-MUNOZ • Instituto de Estudios de Mastocitosis de Castilla La Mancha,Hospital Virgen del Valle, Toledo, Spain; Spanish Mastocytosis Network (REMA),Salamanca, Spain

Contributors xvii

JORG SCHEFFEL • Department of Dermatology and Allergy, Charite—Universit€atsmedizinBerlin, Berlin, Germany

CHRISTIAN SCHWARTZ • Institute of Clinical Microbiology, Immunology and Hygiene,University Hospital Erlangen, Erlangen, Germany

ELIZABETH P. SEWARD • Department of Biomedical Science, University of Sheffield, WesternBank, Sheffield, UK

FRANK SIEBENHAAR • Department of Dermatology and Allergy, Charite—Universit€atsmedizin Berlin, Berlin, Germany

PER S. SKOV • RefLab ApS, COBIS building, Copenhagen N, DenmarkSNOW STOLNIK • Division of Molecular Therapeutics and Formulation, School of Pharmacy,

University of Nottingham, Nottingham, UKVADIM V. SUMBAYEV • Medway School of Pharmacy, University of Kent, Chatham Maritime,

UKEMILY J. SWINDLE • School of Clinical and Experimental Sciences (MPT 810, Rm LF73, SAB,

Level F), Faculty of Medicine, University of Southampton, University HospitalSouthampton, Southampton, UK

CRISTINA TEODOSIO • Spanish Mastocytosis Network (REMA), Salamanca, Spain; Centro deInvestigaci�on del Cancer, Salamanca, Spain

ATHINA VAN GASSE • Faculty of Medicine and Health Science, Department of Immunology,Allergology, Rheumatology and the Infla-Med Centre of Excellence, University of Antwerp,Antwerp University Hospital, Antwerp, Belgium; Faculty of Medicine and Health Science,Department of Pediatrics, University of Antwerp, Antwerp University Hospital, Antwerp,Belgium

MICHEL VAN HOUDT • Faculty of Medicine and Health Science, Department of Immunology,Allergology, Rheumatology and the Infla-Med Centre of Excellence, University of Antwerp,Antwerp University Hospital, Antwerp, Belgium

DRITON VLLASALIU • Institute of Pharmaceutical Science, King’s College London, London,UK

DAVID VOEHRINGER • Infektionsbiologische Abteilung, Universit€atsklinikum Erlangen,Erlangen, Germany

ANDREW F. WALLS • Southampton General Hospital, University of Southampton,Southampton, UK

DANIEL WAN • University of Nottingham, School of Pharmacy, Nottingham, UK; MerckGroup, Glasgow, Scotland, UK

XIAOWEI WANG • School of Pharmacy, University of Nottingham, Nottingham, UK; School ofBiosciences, University of Nottingham, Nottingham, UK

TORSTEN ZUBERBIER • Department of Dermatology and Allergy, Universit€atsmedizin Berlin,Berlin, Germany

xviii Contributors