biology of basophils & basophil activation test
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Picture from www.blog.bryanmjones.com, access November 2015
Biology of Basophils & Basophil Activation Test
Suda Sibunruang, M.D.
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Outline • Development and morphology
• Functional and Phenotypic Markers
• Mediators and effector functions
• Role in health and diseases
• Basophil activation test
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Pictures from www.nobelprize.org & http://intranet.tdmu.edu.ua, access November 2015
Paul Ehrlich (1854-1915)
1879
Identified by staining with basic dyes
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Access from www.commons.wikimedia.org, November 2015
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Schroeder JT. Immunological Reviews 2011;242:144–60 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
IL-3 affects essentially every aspect of basophil biology including development and maturation, survival, acute or late effects on
mediator release, and direct stimulation of IL-13 production
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Siracusa MC. et. al. J Allergy Clin Immunol 2013;132:789-801
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Siracusa MC. et. al. J Allergy Clin Immunol 2013;132:789-801
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Picture from http://static.wikidoc.org & Siracusa MC. et. al. Immunology and Cell Biology 2010;88:275–84
CD34+ myeloid hematopoietic progenitors
IL - 3
IL – 5 IL-3, GM-CSF
SCF
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Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
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Stone KD., et. al. J Allergy Clin Immunol 2010;125:S73-80 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
Picture from http://hdimagelib.com, access November 2015
Storage granule contents
• Major preformed mediator is histamine
• Histamine complexes with proteoglycans, most notably chondroitin sulfate, as opposed to heparin sulfate in the mast cell, and dissociates after exocytosis by ion exchange and changes in pH • Contain less heparin than mast cell • Tryptase in basophil are thought to be much lower than in mast cells; however, there can be variability Clearly caution in using tryptase as the sole specific marker for identifying the presence of mast cells
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Access from www.en.wikipedia.org & www.commons.wikimedia.org, November 2015
Morphology
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Schroeder JT. Immunological Reviews 2011;242:144–60
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Stone KD., et. al. J Allergy Clin Immunol 2010;125:S73-80 Kawakami T. and Galli SJ. Nature Reviews Immunology 2002;2:773-86
Differ more than alike
Basophils • Segmented nucleus • Larger & fewer granules
Mast cell
Basophil
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Siracusa MC. et. al. Immunology and Cell Biology 2010;88:275–84
Functional and Phenotypic Markers
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Siracusa MC. et. al. Immunology and Cell Biology 2010;88:275–84
• Immunoglobulin Fc receptors FcεRI, FcγRIIb • Cytokine receptors IL-3R,IL-5R, GM-CSFR • Chemokine receptors CCR2, CCR3 • Complement receptors CD11b, CD11c, CD35, CD88 • PG receptors CRTH2 • TLRs
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Basophil Activation
• Immunoglobulin E dependent pathway
• Immunoglobulin E independent pathway
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Karasuyama H. et. al. Nature Reviews Immunology 2009;9:9-13 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
Human basophils may express between 5,000 and 1 million FcεRI sites per cell Estimated 200 IgE-receptor cross-links needed to initiate mediator release from basophils
Immunoglobulin E dependent pathway
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Moon TC. et. al. Front Immunol 2014;5:1-18 Spencer LA. et. al. Front Immunol 2014;5:496
IgE dependent pathway
Mast cell
Piecemeal degranulation: vesicular transport of small packets of materials from secretory granules to cell surface
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Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408 Hausmann OV. et. al. Immunol Allergy Clin N Am 2009;29:555–66
Basophil activation markers: CD63 & CD203c
glycoprotein expressed in membranes of cytoplasmic granules
CD203c is a glycosylated transmembrane molecule Basophils express low level of CD203c, which is increased with basophil activation and may correspond to piecemeal degranulation
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Siracusa MC. et. al. Immunology and Cell Biology 2010;88:275–84
Mediators - Preformed mediators - Newly syn. Leukotrienes & prostaglandins - Cytokines & chemokines Significant source of IL-4 and IL-13 vital to pathogenesis of allergic disease
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Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
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Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
Basophils play a dual role in allergic inflammation, depending on antigen concentrations: At low concentrations of antigen, basophils may modulate immune responses of B cells, T cells, and eosinophils through production of IL-4 and IL-13 As they infiltrate lesion site, and concentration of antigen increases, basophils may take on more of an effector role by secreting two substances (i.e., histamine and LTC4)
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Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
IgE dependent & independent pathway
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Marone G. et. al. Trends Immunol 2001;22:229-32
Shed- or virus-bound-gp120 can interact with variable, heavy chain 3 (VH3)+ IgE bound to FcεRI+ cells of individuals at an early stage of infection
Induces IL-4 and IL-13 release and favours a shift towards a Th2 phenotype
HIV-1 transactivation (Tat) protein secreted by infected cells interacts with CCR3 receptor and is a potent chemoattractant
Extracellular Tat can also induce IL-4 and IL-13 release
Human FcεRI+ cells express CCR3 and CXCR4, which are co-receptors for several strains of HIV-1, suggesting that these cells could be infected by certain strains of HIV-1
High IgE levels often observed in some HIV-infected patients
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Schroeder JT. Adv Immunol 2009;101:123-61 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
Activation of TLR 2 and 4 can lead to production of BAFF and IL-13, indicating that microbiota might modulate immunity by interacting with basophils
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Chen K. and Cerutti A. Current Opinion in Immunology 2011;23:345–52
IgD immune complexes
Augment mucosal immune responses by promoting B and T cell activation, leukocyte recruitment and direct microbial killing
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Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
Basophil degranulation is restricted to signals induced by FcεRI crosslinking, C5a, and IL-3
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Siracusa MC. et. al. J Allergy Clin Immunol 2013;132:789-801
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Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma
Tschopp CM, et al. Blood 2006;108:2290-9 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
Basophils synthesize and release granzyme B in response to IL-3 stimulation The significance of this finding remains undetermined
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Schroeder JT. Adv Immunol 2009;101:123-61
Only cytokines reported to negatively regulate basophils are type 1 interferons which limit IL-3-induced cytokine production
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Abbas AK, et al. Cellular and Molecular immunology 2014 Eighth Edition
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Abbas AK, et al. Cellular and Molecular immunology 2014 Eighth Edition
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Picture from http://what-when-how.com, access November 2015
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Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
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Naclerio RM, et al. N Engl J Med 1985;313:65-70
Inflammatory mediators in late antigen-induced rhinitis
Obj: To investigate mechanisms responsible for late-phase response in patients with allergies Method: We measured four biochemical mediators (histamine, tosyl-L-arginine methyl ester [TAME]-esterase, kinin, and prostaglandin D2) in nasal secretions after nasal challenge with pollen antigen in 12 patients with allergy and 3 persons for control
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Kepley CL, et. al. Am J Resp Crit Care Med 2011;164:1053-8 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
Contribution of basophils to LPR is based on their presence in tissues within hours following experimental allergen challenge Late response is largely mediated by basophils
Immunohistochemical Detection of Human Basophils in Postmortem Cases of Fatal Asthma
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Dvorak HF, et al. J Exp Med 1970;132:558-82 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
Picture from www.twineagles.org, access November 2015
Delayed-type hypersensitivity
When given rhus toxoid (the active agent in poison ivy) or dinitrochlorobenzene in a patch test, sensitized subjects developed skin reactions that were characterized by a cellular infiltrate into dermis consisting of up to approximately 16% basophils by 3 - 6 days after application of antigen
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Siracusa MC. et. al. Immunology and Cell Biology 2010;88:275–84
Basophils in immune regulation Interact with T or B cell
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Karasuyama H. et. al. Nature Reviews Immunology 2009;9:9-13 Sokol CL, Medzhitov R. Mucosal Immunol 2010;3:129-37
Basophils secrete IL-4 & TSLP, which induce differentiation of naive T cells into TH2 cells in cooperation with dendritic cells
TSLP is capable of inhibiting IL-12 production by DCs, thereby further promoting Th2 cell differentiation
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Voehringer D. Trends Parasitol 2009;25:551-6
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Gibbs BF. Clin Exp Med 2005;5:43–9
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Siracusa MC. et. al. J Allergy Clin Immunol 2013;132:789-801
Role in health and diseases
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Karasuyama H. et. al. Nature Reviews Immunology 2009;9:9-13
Roles of basophils in allergy
1) In sensitized animals, specific IgE are produced and captured by circulating basophils through FcεrI 2) When the same antigens enter skin, IgE-bearing basophils are recruited to lesion, possibly by chemokines released by phagocytic cells that have captured antigen, and are activated when Ag binds to IgE–FcεrI complex 3) Activated basophils secrete cytokines and other factors that in turn act on tissue-resident cells, such as fibroblasts, 4) Induce them to produce more chemokines 5) Inflammatory cells, such as eosinophils and neutrophils, are then recruited to skin lesion in response to chemokines that are released by tissue-resident cells , resulting in chronic allergic inflammation
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Role of basophils in allergic diseases
Important in many if not all allergic diseases:
• Anaphylaxis
• Allergic rhinitis
• Asthma
• Urticaria
• Food allergies
Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
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Role of basophils in other clinical conditions
• Infections
• Autoimmunity
• Malignancy
Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
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http://www.dailymail.co.uk/sciencetech/article-2957265/When-white-blood-cells-ATTACK-Amazing-video-captures-moment-immune-takes-parasitic-worm.html
Immune system takes down a worm
Access from http://i.dailymail.co.uk, November 2015 Caenorhabditis elegans
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Voehringer D. Trends Parasitol 2009;25:551-6 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
Long-held belief that basophils (and eosinophils) are involved in natural immunity to parasitic infections IL-4–secreting basophils are critical for expulsion of these worms
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Role of basophils in other clinical conditions
• Infections
• Autoimmunity
• Malignancy
Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
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Charles N and Rivera J. Curr Allergy Asthma Rep 2011;11:378–87 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
Correlations between elevated serum IgE autoantibodies, and activated basophils with severity of lupus nephritis
IgE anti-dsDNA/dsDNA immune complexes bind to basophils FcεRI, causing basophil activation with homing to lymphoid organs
Activated basophils produce IL-4 and promote Th2 adaptive immune response that enhances autoantibody production
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Schuerwegh JM, et al. PNAS. 2010;107:2586–91 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
Rheumatoid arthritis
Citrullinated proteins can activate basophils due to IgE anti-citrullinated protein
Basophil activation may contribute to pathogenesis of autoimmune diseases as diverse as: • Antiglomerular basement membrane disease • Membranous nephropathy • ANCA associated vasculitis
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Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408 Stone KD., et. al. J Allergy Clin Immunol 2010;125:S73-80
Kawakami T. and Galli SJ. Nature Reviews Immunology 2002;2:773-86
Indirect therapeutic modulation of basophil activation, such as omalizumab, will be extended to direct therapeutic targeting of basophils to inhibit activation and promotion of a pathogenic Th2 microenvironment
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Role of basophils in other clinical conditions
• Infections
• Autoimmunity
• Malignancy
Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
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Wimazal F. et. al. Cancer 2010;116:2372-81 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
Retrospective study of prognostic significance of eosinophils and basophils in 1008 patients with de novo MDS in Austria & German
Increased no. of basophils are common features of AML and accelerated phase of CML
Basophils, greater than 250/ul, was associated with reduced survival
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Nievergall E. et. al. Blood 2014;123:1218-28
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Hoffmann HJ. et. al. Allergy 2015;70:1393-405 Karasuyama H. et. al. Nature Reviews Immunology 2009;9:9-13
measures basophil response to allergen cross-linking IgE
Basophil activation test (BAT)
Compared with determination of sIgE in serum, BAT reflects a functional response as basophil activation
can be induced by cross-linking of FceRI, which requires more than binding of sIgE to allergen
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Diagnostic evaluation
Determine clinically relevant allergen in addition to clinical history, skin prick test, and specific IgE
• Food
• Insect venom
• Drug allergy
• Chronic urticaria: Alternative for autologous serum skin test (ASST)
Basophils from healthy donors are challenged with patients’ serum
Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Sensitivity & specificity
• Drug allergy: Beta-lactams, Neuromuscular blocking
agent (NMBA), quinolones , radio contrast media, and pyrazolones
sensitivity 50%, specificity up to 93%
• Food allergy:
sensitivity 77-98%, specificity 75 – 100 %
• Hymenoptera venom allergy:
sensitivity 85-100%, specificity 83 – 100 %
Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Monitor patients
• Allergen immunotherapy
• Anti-IgE treatment
• Natural resolution of allergy
Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Hoffmann HJ. et. al. Allergy 2015;70:1393-405
Basophil reactivity is the dose (range) at which maximal response occurs Basophil sensitivity is the dose at which half of the maximal response occurs Concentration at which 50% of basophils respond = EC50 ‘CD-sens’ = 1/EC50 x 100
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Hoffmann HJ. et. al. Allergy 2015;70:1393-405
Basophil reactivity reflected severity Basophil sensitivity reflected threshold of response to allergen
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Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Advantages
• Fewer resources
• More reproducible than challenge test
• Less stressful for the patient
• Avoids severe allergic reactions
Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Nonresponders
• 6–17% of population
• Nonresponders can experience allergic symptoms and have positive SPT with relevant allergens
• Remain unresponsive to stimulation through FceRI under standard BAT conditions
Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Kepley CL, et. al. J Allergy Clin Immunol 1999;104:279-84
Differences in intracellular signaling pathway
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Considerations when taking a blood sample for BAT (1)
• Remember to…
Check absolute basophil counts of above
50 cells/µl within 24 hours prior to
performing BAT
Personal communication, Jettanong Klaewsongklam, M.D., 2015
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Considerations when taking a blood sample for BAT (2)
• Antihistamines do not influence BAT but systemic steroids and cyclosporin A should be avoided
• Blood samples should preferentially be taken within 1 yr of the most recent exposure to allergen
• It is possible to use blood samples within 24 h to document sensitization, but may lose reactivity
Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Considerations when taking a blood sample for BAT (3)
• Basophil function is mostly assessed in heparin-stabilized blood
Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Hoffmann HJ. et. al. Allergy 2015;70:1393-405
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Allergen concentration range
Benzylpenicillin 2 &0.4 mg/ml
Amoxicillin 1.25 & 0.25 mg/ml
PPL 0.25 & 0.05 mg/ml
MDM 0.25 & 0.05 mg/ml
Cephalosporins 1.25 & 0.25 mg/ml
ASA 1 & 0.2 mg/ml
Diclofenac 0.3 & 0.06 mg/ml
Naproxen 1 & 0.2 mg/ml
Paracetamol 1 & 0.2 mg/ml
Metamizol 2.5 & 0.25 mg/ml
Atracurium 0.5 &0.05 mg/ml
Vecuronium 2-0.5 mg/ml
Hoffmann HJ. et. al. Allergy 2015;70:1393-405
Negative test with a parent drug does thus not rule out that reacts to a metabolite of drug
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Summary (1)
• Basophils have long been seen as a surrogate with which to study the more elusive mast cell. This view, however, is no longer valid, because substantial developmental and physiologic evidence now shows that these two cell types have more differences than similarities
Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
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Summary (2)
• Once thought to do little other than to secrete histamine and LTC4, basophils are now well recognized to be the predominant cellular source of IL-4 and IL-13—perhaps the two most important cytokines with a role in the pathogenesis of allergic disease
Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
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Summary (3)
• This information, along with evidence that these cells infiltrate allergic lesions and are capable of responding to a wide variety of stimuli, has sparked a renewed interest in basophils and in their role in allergic inflammation and disease.
Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
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Thank you for your attention