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Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Course 514.020 Immunopathological Lab Course
Basic knowledge in allergology
Eva Untersmayr-ElsenhuberDepartment of Pathophysiologyand Allergy ResearchMedical University Vienna
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Schedule for Nov. 16 and 17DAY 3: Nov. 16, 2010 Meeting point09:00 – 10:00 Introduction in allergology and
presentation of a case studyPD Dr. Eva Untersmayr‐Elsenhuber
Library at 4Q
10:00‐10:30 Basic techniques in allergology: SDS‐PAGE and ImmunoblotDr. Susanne Diesner & Mrs. Cornelia Schultz
Lab, end of corridor 4Q
10:30 – 10:45 Break10:45 – 12:30 Hands‐on in the lab: SDS‐PAGE
Dr. Diesner & Mrs. SchultzLab, end of corridor 4Q
DAY 4: Nov. 17, 2010 Meeting point09:00 – 09:30 Characteristics of food allergens
PD Dr. Eva Untersmayr‐ElsenhuberLibrary at 4Q
09:30‐10:30 Allergen purification via HPLCMrs. Cornelia Schultz
Christian Doppler Lab
10:30 – 10:45 Break10:45 – 12:30 Hands‐on in the lab: Immunoblot
Dr. Diesner & Mrs. SchultzLab, end of corridor 4Q
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
detailed case history
• Reactions timely related with exposition
• No symptoms if trigger is avoided
• Family history regarding atopic disorders
• Known allergies
• Diet diary
Diagosis of allergy
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
serum‐IgE
anti‐IgE
allergen
• Total IgE: PRIST > 100 kU/L• Specific IgE: CAP• Component-resolved diagnosis• Histamin release tests• Skin prick tests with extracts• Prick-to-prick tests with fresh food
Serological and skin testing
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Mean serum concentration:IgE: 0.02-0.5 mg/mlIgG: 8-16 mg/ml
Percentage of total Ig:IgE: 0.002 %IgG: 80 %
Serum half life time: 2 days
Peak IgE levels occure 4-6 weeks after peak of pollenseason
Total IgE >1000ng/mLmajor diagnostic criteria forallergic bronchopulmonaryaspergillosis
IgE antibodiesIgE IgG
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Diseases with elevated IgE levels
• Atopic diseases• Parasitic infections (eg.
Strongyloidiasis, ascariasis, schistosomiasis)
• Nonparasitic infections (eg. EBV, CMV, HIV, M. tuberculosis)
• Inflammatory disease (eg. Kimura disease, Churg-Stauss vasculitis, Kawasaki disease)
• Malignancies (eg. Hodgkin lymphoma, IgE myeloma)
• Cutaneous diseases (eg. Bullouspemphigoid)
• Cystic fibrosis• Nephrotic syndrome• Primary immunodeficiency
diseases (eg. Hyper-IgEsyndrome, Wiskott-Aldrich syndrome, Omenn syndrome, immune dysregulation, X-linkedinheritance, atypical DiGeorgesyndrome
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Ig switch: - transcription through upstream constant switch region- DNA cleavage of ssDNA at transcription site- DNA repair: recombine VDJ domain + new C domain
Immunoglobulin class switch
IgM expressing B-cell IgG1 expressing B-cell
2 signals for IgE switch1) IL4 or IL13 via STAT6:
activates transcriptionat Sε
2) CD40L (T-cells) andCD40 (B-cells):activates DNA switchrecombination
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
High affinity IgE receptor FcεRITetrameric FcεRIαβγ2
According to: Kraft S, Kinet JP. Nature Immunol 2007
αγ2 β
mast cells, basophils
αβ γ2
Antigen independent
effects
Increased cell survival
Antigen dependenteffects
Mediator release
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Trimeric FcεRIαγ2
According to: Kraft S, Kinet JP. Nature Immunol 2007
α
γ2
MHC class II
monocytes, macrophages, dendritic cells, Langerhans cells, eosinophils
Mediator release
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
ligand:receptor = 1:1 high affinity (1010 M-1) due to low dissociation rate, each binding site has lower intrinsic affinity
1 FcεRIα chain with 2 asymmetric IgE interaction sites:
- 4 solvent-exposed tryptophans large hydrophobic surface- C-C’ loop in the receptor D2 domain
bind dimeric, symmetric IgE Fc (Cε3)
FcεRI and IgE interaction
Garman SC et al. Nature 2000Wan T et al. Nature Immunol 2002
Metzger H. Immunol Rev 1992
Gould HJ et al. Nat Immunol Rev 2008
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Glycoprotein, 45 kDa,single chainhomology to C-type(Ca2+-dependent) lectins IgE and CD21 bind tolectin domain
lower affinity for IgE (107 M-1)
B-cells, activated T-cells, monocytes, macrophages, eosinophils, Langerhans cells, intestinal epithelial cells, platelets
Low affinity IgE receptor FcεRII/CD23
Mossalayi MD et al. EMBO J 1992
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
CD23 regulates IgE synthesis
Down-regulation of IgE synthesis
Co-crosslinking of mCD23 and mIgE by allergen-IgEcomplex Inhibition of B-cell proliferation and IgE production
Induction of B-cell apoptosis B-cell populationregulation
B-cell surface
Luo HY et al. J Immunol 1991Sherr E et al. J Immunol 1989
Hibbert RG et al. JEM 2005
From Hibbert RG et al. JEM 2005
Up-regulation of IgE synthesisCo-crosslinking ofmIgE and CD21 bytrimeric sCD23
Aubry JP et al. Nature 1992Hibbert RG et al. JEM 2005
IgE binding tomCD23 protectsmCD23 againstproteolysis andprevents formationof sCD23
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
high binding affinity and slow dissociation rates of IgE to the allergenIgE: 10-10 bis 10-11 MIgG: 10-7 bis 10-8 M
IgE – allergen interaction
Kim KE et al. Mol Immunol 1996Pierson L et al. J Immunol Meth 1998Jackola DR et al. Mol Immunol 2002Hantusch B et al. Immunol Lett 2005
IgE affinity to allergen among the highest biologicallyrelevant binding strength
IgE epitopes on allergens are mainly conformational
IgE to conformational rather than linear epitopescorrelate with tolerance development in milk and egg allergic children
Foote J et al. PNAS USA 1995
Xia L et al. Mol Immunol 2010Padavattan S et al. J Immunol 2009
Pedraza-Escalona M et al. Mol Immunol 2009
Järvinen KM et al. Allergy 2007Vila L et al. Clin Exp Allergy 2001
repeated epitope presentation on allergen surface required for crosslinking allergens are multimers Tan YW et al. J Biol Chem 2009
Schöll I et al. J Immunol 2005
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Sensitisation to allergens
Lymph nodesor mucosa
Galli SJ et al. Nature 2008
Airway Basophils, mastcells, NK T cells,…
Dendritic cell samplingEntrance through disrupted epitheliaProtease activity cleaving of epithelialtight junctions
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Early phase of allergy
Galli SJ et al. Nature 2008
Allergen-specific IgEcross-linkingFcεRI aggregationmast cell activation
Mediator release
BronchoconstrictionVasodilatationInceased vascularpermeability, increased mucusproduction
Transition to latephase: leukocyte recruitment
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Morphology of mast cell
http://www.bu.edu/histology/i/22602ooa.jpg
Tissue based inflammatory cellsRespond to signals of innate and adaptive immunity with immediate and delayed release ofinflammatory mediators
20 μm diameterOvoid or irregularly elongated cells with ovoid nucleusAbundant metachromatic cytoplasmatic granules (metachromatic stainingdue to sulfated proteoglycans)
cKIT (CD117) and FcεRI positiveOther cell-surface receptors depending on location and activation:FcγRIIa (in resting state) FcγRI (CD64) (in presence of IFN-γ)β2-adrenergic receptor, adenosine receptor A2B, prostaglandin E2 receptor,C3a, C5a-receptor, IL-3R, IL-4R, IL-5R, IL-9R, IL-10R, GM-CSFR, IFN- γR,CCR3, CCR5, CXCR2, CXCR4, nerve growth factor R, TLRs, …
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Mast cell maturation and tissue distribution
Hematop. Stem cell
Mast cellprogenitor
cKIT (CD 117) – SCF Ligand
Arise from CD34+ progenitors
Galli SJ et al. Nature 2008
Mucosalmast cell
Connectivetissuemast cell
Tryptase pos. In respiratoryand GI mucosaincreasedwithinflammation
Tryptase, mast cell-specific chymase pos. In connective tissue (dermis, submucosa of GI tract, heart, conjunctivae, perivascular)Small bowel of end-stage immunodeficiencies
Phenotype of maturemast cells depends on growth factor milieu
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
• Antigen-crosslinked surface-bound IgE
• Divalent Ab against IgE Fc region
• Anti-idiotypic Ab
• Anti- Fc receptor Ab
• Covalent cross-linked IgE
• Lectins
• Complement (C3a, C5a) throughC3aR, C5aR (CD88)
• Nerve growth factor
• IgG by FcγRI
• TLR ligands (eg. TLR3 dsDNA IFN-γ production in mast cells)
Mechanisms of mast cell activation
C3aC5a7
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Mast cell mediators
Granule-ass. preformed mediators• Histamine• Neutral proteases (tryptase in MCT,
tryptase, chymase, cathepsin G, carboxypeptidase in MCTC)
• Proteoglycans (heparin, chondroitinsulfates) neg. charged complexeswith histamine
• Chemotactic and activating factors(ECF-A, NCF)
Newly formed mediators• Lipoxygenase pathway products:
SRS-A (LTC4, LTD4), leukotrienes(LTB4)
• Cyclo-oxygenase products: prostaglandines and thromboxanes
• PAF
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Effector function of mediatorsChemo-attractants• NCF• ECF-A• LTB4
Activators• Histamine• PAF• Tryptase• Kininogenase
Spasmogens• Histamine• PGD2• LTC4• LTD4
preformed newly formed
Attractants ofNeutrophils, eosinohils, monocytes, basophils
Vasodil., Vascular permeability
Kinins Vasodil. edemaProteolyt. enzyme, activates C3Microthrombi
• Bronchial smooth muscle contraction
• Mucosal edema• Mucus secretion
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Clinical aspects of mast cell activation
Allergen dosageand entranceroute are decisivefor clinical reaction
MCTMCTCintravenoushigh dose
subcutaneouslow dose
inhalationlow dose
uptake withfood
Mast cell activation
Anaphylaxis Wheal andflare reaction
Allerg. rhinitisAsthma
Nausea, pain, vomiting diarreha
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Morphology of basophils
http://microanatomy.net/blood/basophil3.jpg
Share many features with mast cells: FcεRIsecretion of Th2 cytokinesmetachromic stainingrelease of histamine after activation
But distinct lineage
5-8 μm diameter, segmented condensed nucleus, little proliferative capacityRapid and potent expression of IL-4 and IL-13Express Cytokine receptors (IL-3R, IL-5R, GM-CSF receptor)
Chemokine receptors (CD11c, CD11c, CD35 and CD88)Ig receptors (FcεRI, FcγRIIb)TLR
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
http://focosi.altervista.org/blood-cell-development.jpg
Basophil development and activation• CD34+ progenitors• Differentiate and mature in bone marrow• Circulate in periphery, <1% peripheral
leukocytes• Differentiation driven by IL3• Express integrins and chemokine
receptors able to inflitrateinflammed tissue (skin in AD, airway of respiratory allergies)
Activation via IgE crosslinkingC3a, C5a, TLR2 and TLR4 IL-4, IL-13 secretionand potentiation of IgE activationIL-33 through ST2 receptor
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Mediators: preformed: Histamine, less heparin, low tryptase levelsnewly synthesized: LTC4, LTD4, LTE4, no PGD2 productioncytokines: IL-4, IL-13, GM-CSF source of early IL-4 for Th2 cell differentiation and amplification of IgE synthesis
Role in health and disease
Karasuyama H et al. Nat Rev Immunol 2009Min B. Nat Immunol 2008
Sullivan BM et al. Immunity 2009
Sullivan BM et al. Immunity 2009
• Physiological function remains unknown (host defense against parasites?)• Innate immunity (TLR2 expression)• Predominant source of IL-4 in allergen and helminth parasite activated
PBMCs• In late-phase allergic responses, found in increased numbers in lungs of
asthma patients dying of asthma
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Features of mast cells and basophils
OriginMaturationLifespanLocationSizeNucleusGranulesPeptido-glycansTryptasecontent
Mast cells Basophils
Hematopoietic stem cellConnective tissueMonthsTissue6-12 μmOval or roundSmaller and moreHeparin and chondroitinsulfatesHigh
Hematopoietic stem cellBone marrowDaysIntravascular circulation5-8 μmSegmentedLarger and fewerPredom. chondroitinsulfatesLow
Stone KD et al. JACI 2010
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Anaphylaxis= Serious allergic reaction with rapid onset, which can cause death
Anaphylaxis is unpredictable, can occur in anyone, anywhere at any timeUnderrecognized by patients and underdiagnosed by MDs
Incidence: doubled from 21/100.000 person/year (1980s) to 50/100.000 person/year (1990s)
with 70/100.000 person/year younger than19 years
Triggers: Food (peanut, treenut, shellfish, fish, milk, eggs)Medication (β-lactam antibiotics)Insect stings/bitesNatural latex rubberIgG-Ag complexesComplement and coagulation system activation
exercise, cold air or water, medication Simons FER. JACI 2009
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
http://www.bio.davidson.edu/courses/immunology/Students/spring2006/Witcher/figure%2012-11.jpg
Mechanisms
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Nomenclature of food adverse reactions
Food hypersensitivity
Food allergy Non-allergic foodhypersensitivity
IgE-mediatedfood allergy
Non-IgE mediatedfood allergy
immunologicallymediated response
Johansson SG et al. EAACI position paper 2001
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
IgE-mediated food allergy
Diesner SC. MD thesis MUW 2007
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
local: Oral allergy syndrome, angioedema, gastrointestinal pain, nausea, vomiting, diarrhea
Systemic: Urticaria, angioedema, eczema, rhinitis, asthma, anaphylaxis
Unclear correlation: Migraine, arthritis, fatigue, behavioral disorders
Symptoms of food allergy
Course 514.020 of the Medical University Vienna
Immunopath. Lab CourseBasic knowledge in allergology
Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research
Case study: Patient H. S.35 year old patient with known birch pollenallergy comes to the allergy outpatient clinicreporting increasing severeness of OAS upon food ingestion
Tiggering food: carrot, apple, hazelnut, peanut
CAP class birch: 4CAP class carrot: 2CAP class apple: 3CAP class hazelnut: 2CAP classpeanut: 4
Routes of sensitization for peanut allery?