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Clinical Immunology Revision Notes Lecture 1- Hypersensitivity INAPPROPRIATE IMMUNE RESPONSES CAN LEAD TO TISSUE DAMAGE     

Immunologically primed individual Further contact with the antigen Secondary boosting of the immune response Reaction may be excessive, leading to tissue damage Coombs and Gell defined four types

Coombs and Gell classification of four types of sensitivity reaction.

TYPE 1: ANAPHYLACTIC HYPERSENSITIVITY  Type 1 allergic disease / atopic disease  Hereditary predisposition to produce IgE against common environmental antigens (allergens)  Some of the commonest causes of ill-health TYPE 1 ALLERGIC REACTIONS ARE DUE TO ACTIVATION OF TH2 CELLS AND THE OVERPRODUCTION OF IgE ANTIBODIES  Reason for IgE overproduction is unclear  Increasing evidence for lack of exposure to bacteria that stimulate TH1 in early life favours TH2 phenotype  Hygiene hypothesis - lower level of allergic disease in rural communities

T cell differentiation during human immune responses

TYPE 1 ALLERGIC REACTIONS ARE DUE TO ACTIVATION OF TH2 CELLS AND THE OVERPRODUCTION OF IgE ANTIBODIES          

Limited number of allergens deposited in low doses on mucosal epithelium or skin Particularly efficient way of activating TH2 cells and inducing IgE responses Allergic individuals have larger numbers of allergen-specific TH2 cells in their blood These cells produce more IL-4 per cell than in normal people Allergens are processed by Langerhans’ or other APCs Cleaved into small peptides Presented to uncommitted TH0 cells In an allergic response these differentiate into TH2 lymphocytes These release cytokines that inhibit TH1 cell activation and upregulate IgE production TH2 derived cytokines, particularly IL-2 and IL-13, together with the B-cell surface molecule CD40, induce isotype (class) switching in newly generated IgM bearing Bcells from µ to ℰ resulting in IgE production

THERE IS A STRONG GENETIC COMPONENT TO ALLERGIC DISEASES     

Genetic susceptibility not clearly defined High levels of IgE in certain allergic families HLA linkage to allergen sensitivity, e.g. ragwort and dust mites Genetic locus regulating IgE identified on C5 (also IL-4, 5, and 9) Polymorphism in IL-9 gene associated with asthma

CLUSTERING OF IgE RECEPTORS ON MAST CELLS THROUGH CROSS-LINKING TRIGGERS ANAPHYLAXIS  Mast cells display high affinity receptors for the Cℰ2:Cℰ3 region of IgE (as do circulating basophils)  Cross-linking of these receptor-bound antibodies by multivalent antigen leads to receptor aggregation and triggers release of inflammatory mediators  Intracellular mechanism: breakdown of PI to IP3  generation of DAG  increase of cytoplasmic free Ca2+  i.e. cascade  causes granules to fuse with plasma membrane and synthesis of lipid mediators from arachidonic acid (leukotriene and prostaglandin synthesis)

MAST CELL AND BASOPHIL MEDIATORS  Mast cell degranulation is the major initiating event  Release of preformed mediators: histamine, heparin, tryptase, neutral protease, various eosinophil and neutrophil chemotactic factors   histamine ➔ immediate symptoms of bronchoconstriction, vasodilation, mucus secretion, oedema  can be reproduced by skin test ➔ weal and flare reaction

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mast cell activation also releases newly formed lipid mediators: leukotrienes (LTB4, LTC4 and LTD4) prostaglandin D2 (PGD2) platelet activating factor (PAF) all potent bronchoconstrictors

LATE PHASE ALLERGIC REACTION         

mast cells and basophils also produce proinflammatory cytokines: tumour necrosis factor (TNF) IL-1, IL-4, IL-5 12h+ late phase reaction cellular infiltrate of CD4+ cells, monocytes and eosinophils these cells release TH2-type cytokines (IL-4, IL-5) results in further inflammation at site of allergen late reaction resembles delayed hypersensitivity reaction because of infiltration of T-cells distinguished by presence of eosinophils and TH2 cells

EOSINOPHILS ARE PROMINENT IN ALLERGIC REACTIONS       

eosinophil accumulation is characteristic results from chemoattractants from mast cells eotaxin RANTES (Regulated upon Activation, Normal T-cell Expressed and Secreted) IL-5 - produced by TH2 cells and inhibits eosinophil apoptosis eosinophils release major basic protein and peroxidase destroys respiratory epithelium in asthmatics

ATOPIC ALLERGY – EXAMPLES - INHALED ALLERGENS    

Allergic rhinitis 10% population (grass pollen, animal dander, mite faeces, etc) hay fever most common symptoms within minutes

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Asthma chronic, reversible airflow limitation and inflammation of airways exposure to indoor allergens in infancy, regardless of FH, predisposes development influenced by multiple genetic and environmental factors

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involvement of mast cells, eosinophils, macrophages as mediator -secretors T-cells provide microenvironment to maintain inflammatory response repair-type response fibroblast growth factor (FGF) transforming growth factor - β (TGF-β) platelet-derived growth factor (PDGF) leads to: fibrosis hypertrophy of smooth muscle - narrows airways

ANAPHYLAXIS         

guinea pig model 1mg egg albumin - no obvious effect repeat 2-3 weeks later wheezes immediately, dies of asphyxia in minutes postmortem - intense constriction of bronchioles/bronchi, contraction of smooth muscle, dilation of capillaries humans - laryngeal oedema, hypotension, lower-airway obstruction: peanuts, shellfish, other food allergens, wasp/bee stings, drugs, e.g. penicillin in sensitised individuals, adrenaline (epinephrine) to counter anaphylaxis and urticaria, bee sting, generalised urticaria, hypotension, bronchospasm diffuse chronic urticaria, within minutes/hours, resolved within 12h

LATEX ALLERGY      

proteins in latex (sap of rubber tree) glove wearers acute reaction, rash, itching/redness of eyes, nasal symptoms, coughing, shortness of breath severe cases - anaphylactic shock aerosols in powdered gloves inhaled cross reaction with avocado and banana proteins

FOOD ALLERGY      

awareness? nuts, shellfish, milk, eggs, sulphiting agents sensitisation via breast milk mast cells in GI tract local reactions - diarrhoea, vomiting changes in gut permeability may allow allergen to enter body - systemic reactions

CLINICAL TESTS FOR ALLERGY Intradermal challenge Release of histamine and other mediators produces weal and flare reaction Max 30min, then subsides May be followed by late phase reaction, 24h, infiltration of eosinophils and T-cells - oedematous Alternatively measure allergen specific serum IgE by ELISA  Correlates well

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THERAPY  Chain of reaction provides several potential targets  Avoidance - not always possible MODULATION OF IMMUNOLOGIC RESPONSE  Desensitise by repeated subcutaneous injections of allergen  Activation of TH1-type cells, more IgG4, competition with IgE?  Xolair - monoclonal anti-IgE - block IgE binding to mast cell receptors MAST CELL STABILISATION  drugs - isoprenaline and sodium cromoglycate  make mast cells resistant to triggering  block chloride channel activity MEDIATOR ANTAGONISM     

Histamine H1-receptor antagonists β2 - agonists (salmeterol) - bronchodilators for 12h Theophylline - phosphodiesterase-inhibitor increases cAMP Leukotriene-receptor antagonists Anti - IL4 or anti - IL4 receptor

ATTACKING CHRONIC INFLAMMATION  Macrophage triggering  Corticosteroids  Suppress gene transcription for multiple inflammatory genes ACTION OF CORTICOSTEROIDS IN ALLERGIC DISEASE       

Act primarily on delayed or chronic effects of allergen exposure and seen as anti-inflammatory Inhibit influx of eosinophils, basophils and lymphocytes into local sites Systemic administration - circulating eosinophils decrease rapidly because of margination Prevent eosinophil production in bone marrow Bind to a receptor that leads to inhibition of transcription of many cytokine genes Limited effect on leukotriene production and histamine release Downregulate T-cell activity - blocks contact and delayed hypersensitivity in skin tests

Lecture 2- Hypersensitivity Continued TYPE II: ANTIBODY-DEPENDENT CYTOTOXIC HYPERSENSITIVITY

 Abnormal antibody directed against cell or tissue (or abnormal cell - normal antibody)  May activate complement  Cause cell destruction by direct membrane damage

TYPE II: ANTIBODY-DEPENDENT CYTOTOXIC HYPERSENSITIVITY

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Alternatively, antibody may opsonise cells Removed by phagocytes C3b or antibody Also, ADCC (antibody-dependent cellular cytotoxicity) - extracellular non-phagocytic, non-sensitised leucocytes (neutrophils, macrophages, NK cells - large parasites)

TYPE II REACTIONS BETWEEN MEMBERS OF THE SAME SPECIES – ALLOIMMUNE  Transfusion reactions  ABO  Isohaemagglutinins (IgM - “natural antibodies”) - cross react with gut flora antigens TYPE II REACTIONS BETWEEN MEMBERS OF THE SAME SPECIES – ALLOIMMUNE  Rhesus incompatibility  RhD-negative mothers now treated prophylactically with avid anti-D IgG

TYPE II REACTIONS BETWEEN MEMBERS OF THE SAME SPECIES – ALLOIMMUNE     

Organ transplant rejection Hyperacute graft rejection mediated by preformed antibodies Performed by blood transfusion, failed transplant, multiple pregnancy Antibodies bind to endothelial cells in graft blood vessels resulting in thrombosis Not common - recipients checked for antibodies

AUTOIMMUNE TYPE II HYPERSENSITIVITY REACTIONS  Examples - autoimmune haemolytic anaemia, autoimmune thrombocytopenia (platelets), Hashimoto’s thyroiditis, Goodpasture’s syndrome

ANTI-RECEPTOR AUTOIMMUNE DISEASES  Myasthenia gravis - anti-acetylcholine receptor  Graves’ disease - anti-TSH receptor

TYPE II DRUG REACTIONS    

Drugs coupled to body components Hapten to antigen If IgE antibodies - anaphylactic response Type II - drug form antigenic complex with cell - causes lysis of cell, e.g. Sedormid (sedative)

TYPE III - IMMUNE COMPLEX-MEDIATED HYPERSENSITIVITY          

Excess of antigen over a protracted period Antibody plus antigen forms insoluble complex Give rise to acute inflammatory reactions Complement fixation releases C3a and C5a ➞ mast cell mediators release Increased vascular permeability Polymorphs attracted Attempt to phagocytose immune complexes Causes extracellular release of polymorph granular contents - “frustrated phagocytosis” Damage to local tissues Intensify inflammatory reaction

 renal sections of patients with (1) SLE (type III) and (2) Goodpasture’s syndrome (type II), stained with fluorescent anti-IgG (1) - “lumpy bumpy” deposits from complexes formed in the blood and deposited in the kidney (2) - even layer of staining on the glomerular basement membrane from anti-basement membrane IgG TYPE III - IMMUNE COMPLEX-MEDIATED HYPERSENSITIVITY    

Proportions of antibody and antigen important Ab xs - mild Ag xs - rapidly precipitated and localised Moderate to gross Ag xs - soluble complexes Soluble complexes bind (C3b to CR1 receptor) to erythrocytes, transported to fixed macrophages in the liver, safely inactivated

INFLAMMATORY LESIONS DUE TO LOCALLY FORMED COMPLEXES  Arthus reaction Intradermal  injection of soluble antigen into rabbits ➞ erythematous and oedematous reaction, peak 3 - 8h Interpulmonary Arthus reaction ➞ hypersensitivity pneumonitis, farmer’s lung - 6-8h after exposure to dust from mouldy hay  Pigeon-fancier’s disease - serum protein in dust from dried faeces  Rat handlers - serum protein in urine DISEASES RESULTING FROM CIRCULATING COMPLEXES      

Serum sickness Injection of relatively large doses of foreign serum Horse serum with anti-snake venom Now mouse monoclonal antibodies Serum sickness 8 days after injection Deposition of soluble complex in small blood vessels

DEPOSITION OF IMMUNE COMPLEXES AT OTHER SITES  Favoured sites - skin, joints, kidney TYPE IV: CELL-MEDIATED (DELAYED-TYPE) HYPERSENSITIVITY  Exaggerated interaction between antigen and the normal cell-mediated immune mechanisms  T-cell reactions may be autoreactive or against microorganisms, transplants, or fixed antigen on the skin (chemicals in contact dermatitis)  Following earlier priming, memory T-cells recognise antigen with class II MHC on antigen presenting cell  Stimulated into proliferation  Release proinflammatory cytokines - mediate hypersensitivity response (attracting macrophages and cytotoxic T-cells)  Sensitisation phase ➞ elicitation phase

 Mantoux reaction  Tuberculin into skin of sensitised individual (CMI)  24-48h erythematous reaction, lymphocytes and macrophages dominate TISSUE DAMAGE PRODUCED BY TYPE IV REACTIONS            

Infections Intracellular bacteria ➞ cavitation and caseation in human tuberculosis Granulomatous skin lesions in leprosy Chronic damage from macrophage accumulation and activation Chronic granuloma (walling-off), giant cells, fibroblasts Skin rash of smallpox and measles Contact dermatitis Chemicals in contact with skin forming new antigens Picryl chlorides, chromates, poison ivy, nickel jewellery Treated by suppressing T-cell function - corticosteroids Insect bites Organ-specific autoimmune disease, e.g. type 1 diabetes ➞ cell -mediated hypersensitivity is the major engine for tissue destruction

Lecture 3- Allergic Disease and the Role of the Laboratory in Diagnosis

ALLERGIC DISEASE

 Atopy (without place)- a (familial) predisposition toward the development of IgE to common environmental antigens  Most commonly manifested as allergic rhinitis but also as bronchial asthma, atopic dermatitis, or food allergy  Not everyone that has an allergy is atopic SENSITISATION AND ALLERGY SYMPTOMS Allergic sensitisation (atopy) and allergic symptoms are not synonymous. Only a proportion of patients who are skin prick positive to allergens will exhibit symptoms. The development of disease symptoms therefore depends on a variety of factors. Amongst adults born before 1970, recorded sensitisation rates increase over time, with increasing prevalence rates of allergic disease in each subsequent generation.  This increase will probably continue in the future,  Therefore, it is important that data on childhood sensitisation is collected, and that sensitised people are monitored to examine what happens in terms of disease in later life

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INTERNATIONAL COMPARISONS  The increase in allergy and atopy in the United Kingdom has been mirrored in many other developed countries in Western Europe, the United States, Canada, Australia and New Zealand.  However, developing countries in Africa and the Middle East still report a relatively low prevalence of allergy.  Prevalence of asthma symptoms in children - highest prevalence’s reported from centres in the United Kingdom, New Zealand, Australia and the Republic of Ireland,  Followed by most centres in North, Central, and South America.  The lowest prevalence’s were from centres in Eastern Europe, Indonesia, Greece, China, Taiwan, India, and Ethiopia.  Comparisons of the United Kingdom (which exhibited a high rate of asthma) and Albania (with a low rate of asthma) showed that the degree of allergic sensitisation in these two countries was similar  Highlighting the role that the environment must play in allergy disease development.  Marked differences in bowel flora have been found between genetically similar allergic and nonallergic infants living in Sweden (an area with a high prevalence of allergic conditions) and Estonia (an area with a low prevalence)  Before the unification of Germany, poorer children in the East exhibited lower prevalences of allergic disease than those in the West.  But following unification, and the changes in environment that resulted from this, the frequencies of hay fever and atopic sensitisation in these children rose significantly  Support for the allergy-protective effect of the traditional lifestyle has also come from studies on Steiner school children who follow an anthroposophic lifestyle.  This lifestyle involves the minimal use of medications, delayed vaccinations, a lower use of antibiotics and paracetamol, and a diet consisting of organic or fermenting vegetables.  Children following this way of life were shown to have a much lower rate of IgE sensitisation, asthma, hay fever and eczema than children attending non-Steiner schools in the same area. TYPES OF ALLERGIC DISEASE        

Hay fever Perennial rhinitis Asthma Urticaria Eczema Contact dermatitis Food allergy Allergy to insect stings

 Drug hypersensitivity  Anaphylaxis  Occupational allergic disorders  Occupational asthma – inflammation of airways secondary to dust, gases, fumes, vapours produced in the workplace  Chemicals and plastics – azo-dyes in textile industry  Wood/vegetable dust – flour in bakers •Biologic enzymes – trypsin in plastic EXAMPLES OF ALLERGIC REACTIONS AND ASSOCIATED SYMPTOMS      

Skin •Allergic eczema •Urticaria (hives) Eye/nose •Rhino-conjunctivitis •Sinusitis Oral •Oral allergy syndrome Respiratory •Asthma •Gastro-intestinal Diarrhoea •Vomiting •Colic Systemic •Anaphylaxis

RHINITIS  Allergic rhinitis is the most common immunological disorder  Heterogeneous group of nasal disorders causing nasal congestion, rhinorrhea, nasal itching, sneezing, often have conjunctivitis  Allergens include pollen, mould, house-dust mites, cockroach residue, animal dander  Small molecular weight airborne chemicals can act as haptens  Seasonal or perennial FOOD ALLERGY 6% of young children – cow’s milk, egg, peanut 4% of adults – shellfish, peanut, tree nut, fish Atopic family history is a risk factor Within minutes, urticaria and or angioedema, pruritis Contact urticaria may occur when food is handled e.g. oral allergy syndrome Chronic asthma and rhinitis not generally due to just food allergy but respiratory symptoms may accompany systemic food reactions  Anaphylaxis may occur

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ANAPHYLAXIS  Severe, life-threatening, systemic hypersensitivity reaction involving difficulty in breathing, circulatory collapse or both  3-20 per 100, 000 per year  Causes 20 deaths per year in UK  IgE and non-IgE-mediated  Most common causes – food (nuts, fish, shellfish), drugs, general anaesthetic agents (muscle relaxants), insect stings, latex  Food-induced takes longer to develop than drug- or insect-induced  Symptoms most commonly begin in the skin and mucous membranes TREATMENT    

Avoidance Anti-histamines Leukotriene inhibitors Inhibitors of mast cell activation e.g. Nedocromil sodium

Corticosteroids – inhaled, topical β2-Adrenoceptor agonists for asthma α-Adrenoceptor agonists are used to relieve nasal congestion Immunotherapy - subcutaneous or sublingual Uses allergen extracts of pollen, cat dander or HDM Recommended for the treatment of allergic rhinitis, venom hypersensitivity, some drug allergies and mild bronchial asthma  Induces immunological tolerance and the induction of blocking IgG4 antibodies through repeated exposure to allergen

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ALLERGEN  Grass pollen Weed pollens Tree pollens Moulds House dust mites Epidermis and animal proteins Food of animal origin Food of plant origin Venoms Parasites Drugs Occupational allergens  The following are key to allergenicity: Route of exposure >15μm – stops at nose, causes rhinitis 515μm – stops in bronchi, causes asthma  90% of individuals OTHER ASSAYS     

Other assay methods for the detection of specific IgE antibodies: Chemiluminescent assay by Hitachi Chemical Diagnostics (MAST: multiple thread allergosorbant test) Chemiluminescent assay by DPC Immunlite Chemiluminescent assay...