Course 514.020 of the Medical University Vienna Immunopath. Lab Course Basic knowledge in allergology Eva Untersmayr‐Elsenhuber Department of Pathophysiology and Allergy Research Course 514.020 Immunopathological Lab Course Basic knowledge in allergology Eva Untersmayr-Elsenhuber Department of Pathophysiology and Allergy Research Medical University Vienna
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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)
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
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)
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
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