Immuno: Hypersensitivity
what are the newly synthesized mediators of mast cells upon activation?
chemokines/cytokines eicosanoids - LTs, thromboxanes, PGs PAF
what is the hygiene hypothesis?
children exposed to fewer, less heavy infections than parents so they have a less experienced/educated immune system; less practice with real infections leads immune system to see where danger doesn't exist
what happens in the effector phase?
clinical manifestations due to inflammatory mediators released by mast cells and basophils
type II hypersensivity reaction Ab binds inappropriately to Ag on cell surface and ____ activated
complement
how is complement mediated type II reaction done? 1.) _______ on surface of cell leading to lysis 2.) ________ via C3b binding to Fc of Ab's and destruction by macrophages or neutrophils
complement fixation opsonization
what are the mechanisms of cytolysis/cytotoxicity in type II reaction? (3)
complement mediated ADCC antibody mediated cellular dysfunction
chronic inflammation in allergic asthma is characteristic due to the ....
constant infiltration of immune cells
what does serotonin do from mast cells
constrict smooth muscle, increased vascular permeability
what happens when histamine binds H1 receptor in smooth muscle?
constriction
clinical manifestations type IV
contact dermatitis
inflammatory mediators in allergic Asthma lead to __ of smooth muscle, ____ vasc permeability, ___ mucous secretions
contraction increase increase
_____ are used to treat secondary mediators of allergic rxns (inhaled)
corticosteroids
antigens in allergic contact dermatitis form ____ bonds with soluble molecules or skin proteins
covalent
contraction of smooth muscle of wall of stomach from mast cell degranulation causes...
cramping and vomiting
_____ of two IgE molecules on surface of mast cell activates the cell to immediately degranulate
crosslinking
type IV hypersensitivity reaction is also called...
delayed type hypersensitivity
mast cell degranulation causing contraction of smooth muscle in the intestines causes...
diarrhea
physiological consequences of IgE mediated mast cell degranulation depends on the ____ and ___ of allergen
dose route of entry
serum sickness is most often caused today by _________ and _______
drug induced such as large amounts of penicillin monoclonal therapy with non humanized monoclonals
nasal mucus drainage in allergic rhinitis due to...
eosinophils
mast cell degranulation results in the recruitment of...
eosinophils and basophils
anaphylaxis is treated with....
epinephrine
historically, serum sickness is due to treatment of infectious diseases with ______ to neutralize toxins
equine serum
what is a hypersensitivity reaction?
exaggerated normal adaptive immune response
what happens in the sensitization stage of DTH?
expand number of ag specific T cells (1-2 weeks)
eosinophils are activated via ___ stimuli resulting in release of toxic mediators and inflammatory molecules
external
Celiac disease (DTH) is an immune response to ___ products
gluten
clinical manifestations type II
hemolytic anemia
result of the drugs that go bad in type II reaction is ___
hemolytic anemia and thrombocytopenia
allergens have a high or low solubility to elute from particles
high
irritation of nose in allergic rhinitis is due to ___ release
histamine
what are the preformed mediators of mast cells
histamine serotonin enzymes TNFalpha
result of type IV reaction is dmg to...
host cells
what is the disease from in granulomatous hypersensitivity?
host continually trying to isolate and contain pathogen
what is type III hypersensitivity reaction?
immune complex mediated hypersensitivity
mechanism of injury of type III reaction: 1.) fixation of complement by _______ 2.) activation of ____ cascade 3.) release biologically active fragments ____ 4.) vasc permeability and recruitment of PMN that release proteases and can dmg glomerular basement membrane
immune complexes complement C3a C5a
in a type III reaction, what happens to immune complexes?
immune complexes of certain sizes deposit in tissues because there are too many
desensitization of someone with allergic reactions involves giving allergy shots with ___________ to hopefully ________
increasing amounts of allergen increase the amount of IgG so much that it rids the allergen before IgE has time to react
mast cell degranulation initiates ______ response
inflammatory
what causes the symptoms of a hypersensitivity I reaction?
inflammatory mediators released by activated mast cells
why is type IV reaction delayed?
involved activation proliferation and mobilization of antigen specific T cells
favorite sites for deposition of immune complexes?
kidneys, joints, heart, small vessels
what do the preformed components of eosinophils do?
kill directly
what is the most damaging part of allergic asthma: early response or late response?
late response
late response of allergic asthma is due to ______ and other mediators
leukotrienes
symptom of arthus reaction Type III is localized or systemic erythema and hard swelling?
localized
clinical manifestations of type 1 response are dependent on....
location of allergen
a low or high dose of allergens favors Th2 response -> CD4 T cells secreting IL4
low
allergens have a high or low molecular weight for diffusion from mucus and easily carried to APC
low molecular weight
neutrophils release ____ enzymes which can cause tissue damage from phagocytosis
lysosomal
________ is a toxic protein of eosinophils that poisons parasites, host cells, and induces mast cells to release histamine
major basic protein
all clinical manifestations of type 1 reaction are due to _______ at the site
mast cell degranulation
food allergies are due to...
mast cell degranulation causing local permeability of BVs
main effector cells of allergic reactions
mast cells
IgE made by B cells rapidly attaches to what cells?
mast cells and basophils
type I hypersensitivity is due to IgE degranulation of ______
mast cells, basophils, and eosinophils
50% of people make IgE to airborne antigens encountered on ___ surfaces
mucosal
if allergen inhaled or ingested, what mast cells activated?
mucosal
H2 receptors are located where?
mucosal cells
______ come in close contact with the Ab-Ag complexes via their Fc receptors and are activated
neutrophils
is the mast cell specific for antigen?
no
will you have a hypersensitivity reaction to the first time you see an allergen/antigen?
no
will a skin test determine is an allergen is the cause of asthma?
no, use PEFR
local edema in allergic rhinitis due to....
obstruction of airways
serum sickness is of limited duration because....
once Ag is gone, symptoms recede
N.B. Anaphylactoid Reactions are caused by .....
other substances that induce mast cell degranulation such as exercise, drugs, chemicals
immune response in the gut to food allergy probably evolved to successfully eradicate...
parasites
reactions to ____ can fall under all 4 types of hypersensitivities
penicillin
most common cause of anaphylactic shock?
penicillin or related drugs
how is CD8 mediated contact sensitivity accomplished?
plant leaves touch skin such as poison ivy and substance comes in and binds covalently to surface proteins (hapten); activate CD8 T cells that will recognize the modified self-peptide:MHC I
PAF induces ______ to aggregate and release factors
platelets
____ mediators are stored in granules in the mast cells already
preformed
how does the human monoclonal Ab omalizumab prevent allergic reactions?
prevents IgE binding to mast cells
allergens can also be ______ to break down proteins
proteases
allergens are _______ so they can elicit a T cell response
proteins
what happens in the activation phase?
reexposure to antigen results in degranulation of mast cells and basophils
when dried up allergens contact the mucus, what happens to them
rehydrate and release antigenic proteins
in type III reaction, the mechanism of injury is the same or different in systemic or localized?
same
targets of type II and III are often _____ so will see more in autoimmunity and transplantation
self antigens
in a type II reaction, IgG or IgM is made against...
self protein
2 stages of DTH
sensitization elicitation
what are the phases of the allergic reaction
sensitization phase activation phase effector phase
clinical manifestations type III
serum sickness glomerulonephritis
only mast cells at _________ are activated by allergen
site of allergen introduction
how is CD4 mediated contact sensitivity done?
skin comes in contact with nickel in objects and small amount enters skin where you then get sensitized CD4 T cells; whenever you contact nickel again you get inflammatory reactino there
the eicosanoids lead to a slow or fast contraction? more or less potent than histamine?
slow more potent
what size of the immune complexes provide problems for clearing by phagocytes? why?
small or intermediate less avidly bound by Fc receptors on phagocytes so stick around longer
H1 receptors are on...
smooth muscle and endothelial cells
arthus reaction: subcutaneous type III is when an antigen is injected _______
subcutaneously
allergic asthma is due to activation of _______ in lower airways
submucosal mast cells
antibody mediated cellular dysfunction in type II reaction the antibodies bind to _______ that are critical for function of the cell
surface receptors
what is death due to in anaphylaxis?
swelling of airways and constriction of epiglottis
what are 5 IgE mediated allergic reactions?
systemic anaphylaxis acute urticaria (wheal and flare) allergic rhinitis (hay fever) allergic asthma food allergy
_______-- is basically the wheal and flare reaction, mast cell activation in the skin
urticaria (hives, welts)
clinical manifestations type I
urticaria, systemic anaphylaxis
what happens when histamine binds H1 receptor in endothelial cells?
vascular permeability increases causing drop in BP
allergic rhinitis mediators cause increased capillary permeability leading to localized _______
vasodilation
effect of histamine is dependent on...
where tissue reaction occurs
can antigens for type III reaction be exogenous? endogenous?
yes both
can DTH be beneficial
yes depending on antigen to give resistance
Systemic anaphylaxis
- "against protection", whole-body reaction that occurs when an *allergen enters bloodstream and activates mast cells associated w/ blood vessels* - Systemic degranulation of mast cells causes an increase of vascular permeability + constriction of smooth muscle - Anaphylactic shock occurs when large amounts of fluid leave the blood cause swelling and a drop in blood pressure. Multiple organs systems are deleteriously affected. Death is commonly caused by *asphyxiation due to airway constriction + swelling of epiglottis* - Most common causes are allergens that are *directly injected into bloodstream (drugs /insect venom)*, but also includes certain ingested allergens that are *rapidly absorbed in gut (peanuts, brazil nuts)*
Hypersensitivity Reactions
- "overreactions" of immune system to self (autoimmunity) or non-pathogenic environmental antigens (allergens) - a state of heightened reactivity to an antigen - results from a *sensitization phase (primary response*) and an *effector phase (secondary response)* - Thus, all allergic reactions are a *secondary response*
Type I (Immediate) Hypersensitivity
- *Immediate*: development of symptoms after allergen exposure (sec to min) - Primary cause of *anaphylaxis* where an allergen *enters bloodstream* and causes life-threatening complications - Immediate mechanism is though IgE-mediate mast cell activation - *normal immune mechanism for parasite clearance* - *Atopy* - genetic predisposition to produce more allergen-specific or more total IgE - (20% of pop) - In developed countries where helminth (worm parasite) infections are common, total IgE levels are much higher yet the rates of allergy are much lower!
Allergy
- *altered reactivity against an environmental antigen* that most people do not react against (versus self) - Type I hypersensitivity (rhinitis, asthma, food, venom) is the most prevalent form of allergic disease - However, not all allergies are type I hypersensitivities (*DTH, contact dermatitis = type IV*)
Basophils in immediate hypersensitivity
- *constituatively express Fc(e)R1* - unlike eosiniphils - must be recruited
Final note on mast cells
- *evolved to deal with parasites*! - IgE-deficiency greatly enhances *susceptibility to parasite infection* - express *TLRs and additional FcRs* that activate to promote neutrophil and T cell recruitment upon recognition of invading pathogen - important in both innate and adaptive immunity in mucosal and epithelial tissues - can be activated without IgE + degranulation!
Cytotoxic type II hypersensitivity
- *hemolytic anemia + thrombocytopenia* - aka antibody-dependent cell-mediated cytotoxicity (*ADCC*) - classic example= *penicillin-modified erythrocytes* - Cellular destruction is mediated by *complement + FcR-bearing macrophages* (effector phase) - Symptoms include rashes, anemia, bleeding, myocarditis - No diagnostic skin tests - biopsy or a blood test for pre-existing Abs
The nature of allergens that stimulate IgE production
- *small, stable, soluble, enzymatically active proteins* - Are proteins - elicit Th2 responses following antigen presentation on MHC class II - Many have enzymatic activity - dust mites (protease), detergents (subtilisin), aspergillus (fungal proteinase), meat tenderizer (papain) - Tend to be of low molecular weight allowing them to readily diffuse across mucosa - Highly stable (persist for a long time in tissue) and soluble
Asthma
- 9.1% of children, type I + IV hypersensitivity 1) Allergic asthma: *immediate hypersensitivity* reaction that causes breathing difficulties: lower mucosal airway mast cells degranulate + cause airway smooth muscle to contract + swell due to fluid influx - *Chronic inflammation* is often a hallmark of allergic asthma and is characterized by a *constant leukocyte infiltration (Th2, eosinophils, and neutrophils)* 2) Chronic asthma: airways *obstructed frequently by mucus plugs*. cause is not always re-exposure to the sensitizing IgE-specific allergen. *Chemicals* (smoke) or pathogen products that generate Th2 cells can trigger mucus obstruction - In this scenario chronic asthma is *type IV* hypersensitivity (T cell-mediated)
Type II hypersensitivity in blood transfusions
- ABO blood groups = most important antigens to match for blood transfusions due to threat of inducing type II hypersensitivity - *A + B antigens similar to bacterial carbs*, so most individuals are already sensitized against A, B, or both - *Complement fixation* causes hemolysis (effector) - *Cross-match test*: mix donor RBC w/ host serum + look for hemolysis - Opposite mixing doesn't matter as donor antibody would not be in large enough quantities to trigger lysis of host RBS
IgE and allergens
- Allergens in the sensitization phase have a unique ability to promote IgE production - *Mucosal routes of contact and low dose exposure*, typical for many allergens, *favor IgE* production rather than IgG - *Th2 cells (IL-4, IL-5, and IL-13)* are responsible for promoting IgE class switch
Type II hypersensitivity: noncytotoxic autoimmune disorders
- Almost always *mediated by IgG* - no complement fixation! - Instead, Abs directed against a specific EC receptor or ligand - influence ligand binding, signal transduction etc - MG, Graves - Testing: examining blood for autoantibodies against target tissue or molecule
Type II hypersensitivity
- Antibody driven (primarily *IgG*, can include IgM) - Antigens include host (blood components, collagen/ECM) as well as foreign products (drug or pathogen) - that are capable of *modifying proteins on surface of host cells + form new antigens, part foreign particle/ part host particle*! - drug-induced anemia, autoimmune (Goodpasture's, Pemphigus, Grave's Disease), blood transfusion reactions - can be cytotoxic or non-cytotoxic - *not immediate*, but reactions can occur *within hours* of exposure.
Arthus vs wheal
- Arthus takes longer, different presentation
Type I hypersensitivity pharmacological treatment
- Asthma: *b2-adrenergic receptor agonists, inhaled corticosteroids, cromoyln (blocks degranulation), theophylline* 1. *Epinephrine* (adrenaline) - used to treat anaphylaxis, vasoconstrictor used to increase blood pressure and relax constricted bronchial smooth muscle 2. *Anti-histamine* - blocks binding of histamine to H1 receptors (not an antibody) on vascular endothelial and epithelial cells. Topically for hives, inhaled for rhinitis 3. *Corticosteroids* - general leukocyte inhibitor that is effective in combating inflammation. Topically for eczema, inhalation for asthma and rhinitis 4. *b-adrenergic agonists* - opposite of b-blockers used to treat CVD. Mimics effects of epinephrine
Celiac Disease
- Common for antigens found in western diets, including glutens and related proteins. Strong genetic predisposition - *Th1 cells* in sensitized individuals recognize these antigens as foreign and activate resident tissue macrophages in gut - Inflammation is induced causing *destruction of villi lining*, diarrhea, anemia, malabsorption of nutrients (iron) - not a direct lysis effect like type II - Can become chronic unless the antigen is removed from diet (now considered auto-immunity) - Patients with celiac disease also *have B cells that make IgG and IgA* to various gluten-related products and present to T cells. - *Autoantibody test followed by biopsy* to diagnose
Serum sickness
- Early treatment for *bacterial infection*: injection of *serum from horse immunized w/ same pathogen* Protective Abs in horse serum can clear bacterial infection, but also *cause type III hypersensitivity 7-10 days later* - Highly conc foreign Ag (horse proteins) form smaller IC w/ host IgG --> blood vessel deposition - Limited duration - will go away unless more antigen is injected - still used in rare instances today to make *anti-venoms* - can also occur with *mAb therapies* when a large amount of foreign protein (mAb) is injected
Early and late phase of type I hypersensitivity
- Early: 0-30 min *direct degranulation of mast cells* that happens in seconds - Late: 6-8 hours later when *eicosanoids, cytokines, and chemokines* are newly synthesized and produced by mast cells + eosinophils
Farmer's lung
- IC-mediated hypersensitivity can also be directed against *inhaled antigens (molds, plants, animals)* in people w/ constant exposure (farm workers) - Unique properties of these antigens as well as concentrated doses *causes IgG rather than IgE class switching* (as opposed to allergy!) - IC are deposited in alveoli where they stimulate inflammatory responses, *lung damage, and difficulties breathing (asthma-like)*
Type I hypersensitivity prevention
- IgE --> *IgG4* neutralizes antigen, doesn't bind Fc(epsilon) receptor or induce degranulation
Food allergies
- IgE made against only an extremely small # of potential antigens that are ingested (grains, nuts, milk and related products, egg, soy, legumes, shellfish) - Ingested food allergens are able *diffuse across epithelial barrier in gut + activate mucosal mast cells* - Symptoms include cramps, diarrhea, and vomiting that result from fluid increases and smooth muscle contractions (potent mechanism for *expulsion of parasites* in gut) - Food allergens can readily go systemic when absorbed into the bloodstream and cause anaphylaxis. Food allergens also migrate towards skin epithelium where mast cells activate + cause *urticaria (hives)*
Mast Cell (pic)
- IgE's cross link, multiple ones bind same allergen
Arthus reaction
- Key reaction for type III hypersensitivity - Experimentally, purified antigen is injected under skin. If Abs present, *erythema and induration (hardness) within 6-24 hours* - IC deposition reaction that occurs locally in the skin - Arthus reactions are also a rare complication of vaccines that contain *tetanus or diphtheria toxoids*
IgE
- Produced by long-lived plasma cells in lymph nodes, mucosal tissue (respiratory + GI) - Very little in circ, *rapidly binds FceR1-expressing cells (mast cell, basophil, eosinophil)* after production localizing IgE to mucosal and epithelial tissues (3-4 week half-life) - normal immune response = recognize + expel parasites (helminths)
Contact hypersensitivity (type IV)
- Reaction that results in *allergic contact dermatitis, resembles eczema* (type I) but mechanism is completely different - Contact hypersensitivities typically *employ CTL cells that destroy epidermal tissue @ site of contact* - CD4+ T helper cells promote CTL function --> skin inflammation - *Patch test* - in vivo testing on back by applying small chambers containing various known contact allergens. Results read on days 2-4 - Common fragrances, chemicals, venoms, metals, to determine cause of unexplained skin inflammation
Eosinophils!
- Resting ones do not express FceR1, but *IL-5 activation induces FceR1* = *secondary responders in type I hypersens* - Presence is typically associated with *chronic allergic inflammation* as the actions of Th2 and mast cells eventually lead to their accumulation - Many of their products also act back on mast cells (trigger histamine release) and other eosinophils (activation and granule release) that is responsible for chronic inflammation
Type IV hypersensitivity
- T-cell mediated - *does not involve class switching, Ab production, or complement* - mediated by T helper (*CD4+*) cells - Commonly know as *delayed-type* (DTH) - Occurs *1-3 days after exposure* - Delayed b/c of time it takes to process + present antigen + activate T cells - Prominent example is *TB test* - *Contact hypersensitivity* - Typically involves haptens or metals that activate CD4 and CTLs (CD8) cells to destroy tissue - Sensitization is similar - antigen is presented on MHC II to CD4+ T but the antigen and environment are *favorable for development of Th1 (or Th17) responses*
Type I hypersensitivity and disease
- The effect of an allergen mainly depends on the tissue where the mast cell is activated
Summary: types 2-4
- Type II hypersensitivity is caused by IgG or IgM reactive against cellular surface or matrix antigens. Triggers complement fixation, ADCC, inflammation (also are non-cytotoxic mechanisms) - Type III hypersensitivity is caused by the deposition of immune complexes in blood vessels or other tissues. These activate complement, mast cells, and other leukocytes that causes hemorrhage (Arthus reaction) - Type IV hypersensitivity is T cell-mediated. Th1 cells release IFNg to activate macrophages and cause tissue destruction. CTLs are involved in many contact hypersensitivities.
Antigen factors influencing IC deposition
- Type III hypersensitivity generally occurs when antigen is present in excess (*concentrated doses or recurring exposures*) - During *early phase* of exposure, *antigen usually in excess* (Ab lacking) - larger antigens w/ multiple epitopes are able to cross-link Abs + efficiently fix complement, enabling IC clearance - *2 IgG molecules per complex* are needed to fix complement - Once deposited, ICs form aggregates that fix complement --> inflammation, hemorrhage, etc...
Type I hypersensitivity in skin - Eczema
- atopic dermatitis - chronic itchy skin rash, breakages and fluid discharge - Etiology is poorly understood, linked to people w/ *familial history of airway allergies* - Unlike airway allergies, *often disappears after childhood*
Eosinophils (pre-packaged granulocytes)
- bi lobed
Spirometry test to measure lung function
- breathing should be way reduced initially - recovers, then there's a late phase rxn
Penicillin
- can be 1,2,3 - pain in ankles indicates immune complex deposition (III) - think IgG - RAST to measure IgE Abs against penicillin
Allergic rhinitis (hay fever)
- common allergy that cause sneezing, coughing, and congestion to *inhaled allergens* such as pollen, grasses + pet dander - loosely *grouped with allergic sinusitis* - IgE activation on mast cells results in local edema that *constricts nasal airways* --> congestion + *nasal discharge containing eosinophils*
DTH effector phase (pic)
- different from 1-3 because of TH1 emphasis
Poison ivy
- hapten!
Eosinophils
- highly restricted
Type I hypersensitivity in skin - Urticaria
- hives or "nettle-rash" - raised and itchy swellings that are basically multiple wheal and flare reactions on skin (allergy test) - Primarily mediated by mast cells + histamine - same reaction can be observed w/ histamine injection alone - Primary causes are *insect bites+ food allergens* that have entered blood stream
Mast Cells (expresses IgE receptor)
- in mucosal and epithelial tissues (*enables rapid detection* and response to danger - pathogenic in type I hypersensitivity) - IgE binds mast cells via FceR1 in *homocytotropic manner* (binds cells of same host where it was created) IgE binding to *FceR1* is *strongest* of this type (*irreversible*) - IgE binds in absence of antigen, serves as an antigen-specific receptor similar to those on B and T cells - Difference is speed (rapid degranulation) and specificity (IgE molecules with different specificities exist on same mast cell)
Summary: Type I
- mediated by IgE bound to mast cells (and others) that recognizes normally harmless environmental antigens (allergens) - Sensitization phase occurs though induction of antigen-specific Th2 responses followed by interaction w/ B cells that results in class switching to IgE. Effector phase occurs when IgE bound to mast cells becomes cross-linked and causes degranulation - Early phase of response is directly attributable to mast cell degranulation; late phase = result of eosinophil, basophil, and Th2 recruitment and synthesis of new allergenic products
In vivo skin testing (allergies)
- shows allergy to ragweed
Type I hypersensitivity in skin - angioedema
- similar to hives but occurs in *deeper subcutaneous tissue* - Symptoms more representative of *systemic reaction* (food or drug)
Mast cell granules (early mediators)
- sm *contractions* widen blood vessels - sm *constrictions* cause bronchospasm
The sensitization phase (phase 1 for hypersensitivity)
- small Ag crosses mucosa, ingested by APC - T cell, B cell activated, IgE produced - individual is now sensitized!
Allergens table
- symptoms determined by route of ingestion!
Chronic asthma
- tiny lumen! - chronic leuk infiltration around vessel
Serum sickness (pics)
- when large amounts of protein are injected (horse serums) - anti-venoms
allergic asthma reactions are what type of hypersensitivity reaction
1
Type II hypersensitivity: cytotoxic autoimmune disorders
1) *Goodpasture's disease* - Abs against *collagen* located in EC basement membrane, causes *glomerulonephritis* 2) *Pemphigus vulgaris* - antibodies against *cadherin* molecules of the epidermis, causes *skin blistering* 3) *Autoimmune thrombocytopenic purpura* - antibodies against *integrins* localized on the surface of platelets, leads to *excessive bleeding* 4) *Autoimmune hemolytic anemia* - antibodies against *Rh antigens*, causes anemia
Allergy in vitro tests
1) *RIST* - radioimmunosorbent test, used to *quantify total IgE* in serum. Not informative for verifying allergen type but *can identify atopic individuals* 2) *RAST* - radioallergosorbent test, used to identify presence of allergen-specific IgE in serum, ELISA. *Can identify causative allergens*
DTH effector phase
1) Antigen-specific CD4+ T helper cells (typically Th1) recognize their cognate foreign antigen and *release IFNg* + soluble mediators that recruit and activate additional inflammatory cells - Cytotoxicity occurs due to localized tissue inflammation - Vasodilation and increases in vascular permeability cause redness and swelling at site of insult 2) *Tuberculin response* - skin injection of TB can result in DTH response that can diagnosis existing infection or prior BCG vaccination (lung x-ray) 3) Insect venoms (non-immediate) can cause similar reactions; however, IgE-mediated insect sting hypersensitivities (type I) are more common
Etiology of Hypersensitivity
1) Genetic component - Polymorphisms risk factors for development of various hypersensitivities - One parent with allergies: 33%; both: 70% - *primary factor* but not only explanation 2) Hygiene Hypothesis - development of a healthy and functional immune system key in preventing aberrant immune function. -vaccinations, over use of antibiotics, clean and sterile households, lead to a poorly functioning immune system - we are exposed to fewer microbes with every generation - fewer allergies in kids on farms, w/ pets
Type IV hypersensitivity and autoimmunity
1) Insulin-dependent diabetes mellitus: *pancreatic b-cell antigens* 2) Rheumatoid arthritis: *synovial joint antigens* 3) Multiple sclerosis: *myelin sheath antigens*
Type I hypersensitivity - immunotherapy
1) Monoclonal antibodies - *Benralizumab* - blocks IL-5 receptor and depletes eosinophils (asthma) - *Omalizumab* - binds free IgE + IgE bound by B cells but doesn't bind IgE bound to FceR1 (asthma + urticaria) 2) *Pitrakinra* - human recombinant protein that acts as an antagonist to block shared subunit in IL-4 and IL-13 receptors
Type III hypersensitivity and autoimmunity
1) SLE - circulating *auto-IgG against nucleic acids* or surface antigens = systemic deposition leading to erythema (lupus = *wolf rash* on face) - Not just erythema; widespread inflammation, tissue destruction, and deposition in joints, kidneys.. - Destruction leads to the production of even more systemic antigens 2) Rheumatoid arthritis mediated by rheumatoid factor (IgG reactive against the Fc region of IgG) causes IC deposition in the joints and destruction
4 types hypersensitivity
1) Type I hypersensitivity - mediated by IgE and mast cells, basophils, and eosinophils 2) Type II hypersensitivity - mediated by *IgG (or IgM)* directed against cell surface and extracellular matrix antigens 3) Type III hypersensitivity - mediated by *Ag-bound IgG immune complexes* 4) Type IV hypersensitivity - mediated by T cells
Describe the pathogenesis of type 1 reactions
1) a sensitisation phase: occurs during the initial exposure to an antigen when the host develops an antigen-specific IgE response, which results in sensitisation of the host by the binding of the antigen-specific IgE to FCε receptors on the surface of mast cells 2) an effector phase: either through a second exposure or prolonged initial exposure to the IgE-specific antigen, there is cross-linking of 2 or more IgE molecules on the surface of the mast cells. This results in their activation and release of preformed and newly synthesised mediators. This phase can be limited to an acute inflammatory reaction (over a period of hours; characterised by responses associated with release of preformed vasoactive amines from mast cell and includes increased vascular permeability, smooth muscle contraction and influx of inflammatory cells) or can progress to a late-phase reaction (over a period of hours), or to a chronic reaction (persisting for days to years), both resulting from a more intense inflammatory cell infiltration (eosinophils, neutrophils, macrophages, T lymphocytes) and tissue damage.
Describe the stages of inflammatory reactions stimulated by CD4+ T cells
1) activation of CD4+ T cells (sensitisation stage): naïve CD4+ T cells recognise peptides displayed by dendritic cells and secrete IL2, which functions as an autocrine growth factors to stimulate proliferation of antigen-responsive T cells. At the time of T-cell activation, APCs produce IL12, which induces differentiation of CD4+ T cells to the Th1 subset. 2) responses of differentiated effector T cells (effector stage): upon prolonged or repeat exposure to an antigen, th1 cells secrete cytokines, mainly IFN-γ, activating macrophages (classically activated macrophages). Activated macrophages serve to eliminate the offending antigen; if the activation is sustained, continued inflammation and tissue injury result.
Type I hypersens in skin: Immediate versus late phase
1) initial urticaria 2) swelling, fluid accumulation (newly syntehsized LT + PG)
Allergic asthma
1) local mast cell degranulation - mucous production, increased permeability 2) amplification of inflammation, eosinophils
which hypersensitivity groups are antibody mediated?
1-3
Type ___ hypersensitivity reaction is binding of antibody directly to antigen on cell surface
2
type ___ hypersensitivity reaction is deposition of antigen antibody complexes
3
how many types of hypersensitivity reactions?
4
what is a late phase response?
6-8 hours after immediate rxn subsides and swelling due to leukotrienes, chemokines, and cytokines
What is atopy?
A genetic predisposition to synthesize inappropriate levels of IgE specific for external allergens
3 phases of systemic immune complex disease: 1.) ______ complexes form in circulation 2.) deposition of the ICs in various tissues 3.) ____ reactions
Ag-Ab inflammatory
What is a hypersensitivity reaction?
Altered reactivity to a specific antigen that results in pathologic reaction upon theexposure of a sensitised host to that specific antigen. All are characterised by sensitisation and effeftor phases. The sensitisation phase requires that the host must have had either a previous exposure or a prolonged exposure to the antigen so that he or she can develop an immune repsonse to the inciting agent. The pathology associated occurs in the effector phase and is most commonly manifested as an inflammatory reaction or as cell lysis.
What is an allergen?
An antigen that gives rise to an immediate hypersensitivity
Describe systemic type 1 hypersensitivity
Anaphylaxis This refers to an acute systemic hypersensitivity reaction to an antigen that is mediated by IgE and involved mast cells activation, resulting in a shock-like state often involving multiple organ systems. The clinical signs and pathology vary by species and often correlate to the primary shock organ in its most severe manifestation - death. The primary target tissues are blood vessels and smooth muscle. Fatal anaphylaxis may occur as a result of asphyxiation secondary to oedema of the upper airway, circulatory failure as a result of dilation of the splanchnic vascular bed or hypoxaemia as a result of sever bronchospasms. The specie most sensitive to the development of anaphylaxis is the guinea pig. The most common pathologic findings in most species are pulmonary oedema and emphysema, except for dogs, for which the major shock organ is the liver, and severe hepatic congestion and visceral haemorrhage are the most common finding. The types of antigens that can elicit a systemic anaphylactic reaction are diverse, but most commonly include drugs, vaccines, venom of stinging insects, and heterologous sera.
Describe antibody-mediated cellular dysfunction
Antibodies directed against cell surface receptors impair or dysregulate function without causing cell injury or inflammation. For example, in myasthenia gravis, antibodies reactive with acetylcholine receptors in the motor end plates of skeletal muscle block neuromuscular transmission and therefore cause muscle weakness.
What are the 4 steps involved in developing and producing a Type I hypersensitivity reaction?
Antigen activates Th2 and stimulates IgE class switching of B cells IgE is produced by plasma cells IgE binds to mast cells Re-exposure to allergen causes massive release of mediators
What is the etiology behind the tissue damage observed in Type III hypersensitivity?
Antigen-antibody IgG complexes activate complement and attract neutrophils to release lysosomal enzymes
What are the newly synthesised mediators?
Arachidonic acid metabolites, especially PGD2 and LTC4, D4, E4. Cytokines: IL4 (B lymphocyte activation and IgE synthesis), Il5 (chemotactic for eosinophils), Il6, TNFα (pathogenesis of shock during a systemic anaphylactic reaction). Platelet-activating factor (PAF) (increased vasodilation and vascular permeability, recruitment and activation of inflammatory cells)
refers to IgE mediated hypersensitivity
Atopy
_________ are used to treat allergic reactions for bronchodilation as a short acting, rescue therapy
B agonists
What type of lymphocytes are associated with Type I hypersensitivity?
B cells Th2
What are the 3 mechanisms of disease caused by Type II hypersensitivity?
Bind antigen and target for phagocytosis Complement and Fc-receptor mediated inflammation and tissue injury Interference of normal cellular function due to binding of physiologically important molecules of cellular receptors
Celiac disease: 1.) ______ cells react with gluten peptides in GALT activate tissue macrophages 2.) secretion of ______ cytokines 3.) continuous intake of gluten leads to....
CD4 inflammatory atrophy of SI villi
effector cells of DTH allergic contact dermatitis can be due to what cells
CD4 or CD8 T cells
contact dermatitis can also occur due to dmg directly from _________
CD8 T cells
enzymes from mast cells are used to remodel....
CT
if allergen is introduced through penetration (mm), what mast cells activated
CT
Describe how mast cell activation can occur?
Can occur through a number of IgE-mediated and non-IgE-mediated mechanisms: a. IgE-Mediated (type I hypersensitivity)= cross-linking of membrane-bound IgE by antigen b. Non-IgE-mediated (anaphylactoid reaction): cytokines (IL8), complement products (C3a, C5a), drugs (codein, morphine), physical stimuli (heat, cold, trauma).
What are the 2 methods of immunotherapy available to treat Type I hypersensitivity?
Desensitization ("allergy shots") Repeated injection of allergens to reduce IgE and produce IgG
Describe type 3 reactions
Due to the formation of insoluble antibody-antigen complexes. This results in activation of the complement system and the development of an inflammatory reaction at the sites of immune complex deposition.
_____ is produced by eosinophils and is a neurotoxin and helminthotoxin
ECP
What are the primary cells involved in a type 3 reaction?
FCR-bearing neutrophils and macrophages. Complement activation leads to the elaboration of factors that are chemotactic and attract neutrophils and macrophages to the site. These cells are activated and produce a number of proinflammatory cytokines. Early in the response, these cells release vasoactive amines that cause increased vascular permeability allowing the immune complexes to lodge within the vessel wall. Phagocytic cells are also stimulated to release their proteolytic enzymes and toxic free radicals, and these processes result in tissue and vascular damage. Antigen-antibody complexes form as a part of a normal immune response and usually facilitate the clearance of antigen by the phagocytic system without resulting in a type III hypersensitivity reaction.
IgE binds to ____ portion to the cells in the absence of antigen
FcR
allergen specific IgE binds to mast cells via what receptor
FcsigmaRI
anti-histamines such as benadryl block ______ receptors
H1
antihistamines are used to treat allergic reactions by binding to.....
H1 Receptors
histamine binds cells rapidly via ____ receptors
H1 and H2
What mediators are released during the degranulation observed in Type I hypersensitivity, and what is the function of each?
Histamine: increases vascular permeability, increased mucous secretion, smooth muscle contraction of bronchi Prostaglandin: contraction of respiratory smooth muscle, increased mucous secretion Leukotrienes: bronchial spasms
What is the principal macrophage-activating enzyme involved in Type IV hypersensitivity and what cell secretes it?
IFN-gamma, secereted by Th1
What induces activated B cells to switch to the IgE isotype?
IL-4
systemic Type III: Serum sickness occurs when foreign antigen is administered _______; symptoms occurs within 7-10 days due to deposition of ____ in tissues which corresponds with appearance of high affinity _____
IV ICs IgG
after exposure to allergen, what antibody is produced from IL-4 release?
IgE
antibody in type I reaction
IgE
in allergic rhinitis, airborne allergens reacting with ________ sensitized mast cells in nasal passages and conjunctiva
IgE
strength of type 1 response depends on amount of __
IgE
Mast cells and basophils have Fc receptors that bind ______; what is the receptor?
IgE FcsigmaRI
What causes degranulation observed in Type I hypersensitivity?
IgE binds to mast cells and basophils
mast cells and basophils are considered sensitized as long as....
IgE is bound to them
what happens in the sensitization phase?
IgE made to antigen and IgE binds mast cells
what is type I hypersensitivity reaction
IgE mediated hypersensitivity; immediate hypersensitivity, allergic
upon 1st exposure to allergen in type 1 response, endpoint is formation of _______
IgE plasma cells and allergen specific IgE
prerequisite for type I hypersensitivity reactions is that...
IgE produced upon first exposure to antigen
an immunological treatment for allergic reactions is to prevent production of IgE by shifting response from IgE to ________ - desensitization
IgG
antibody in type 3 reaction
IgG
if a person has circulating ____ specific for antigen, arthus reaction will develop
IgG
antibody in type II reaction
IgM, IgG
Describe localisation or generalisation of the immune complex deposition
Immune complex deposition may be localised to a tissue or generalised if the complexes are formed in circulation. Blood vessels, synovial membranes, glomeruli and the choroid plexus are particularly vulnerable to deposition of immune complexes. A majority of diseases involving type III hypersensitivity reaction are the result of persistent infections, autoimmune disease or inhalation of foreign antigens. Few examples are systemic lupus erythematosus, hypersensitivity pneumonia due to fungal spore inhalation, rheumatoid arthritis, equine infectious anaemia.
Describe type 4 hypersensitivity
Immune complex hypersensitivity. This reaction is also known as cell-mediated hypersensitivity because it is the result of the interaction of T lymphocytes and the specific antigen to which they have been sensitised. The resulting immune response is usually mediated by the release of soluble cytokines from CD4+ lymphocytes, which act through mediator cells (mainly macrophages) to produce chronic inflammatory reactions. Because these responses are dependent on sensitised T lymphocytes and require 24 to 48 hours to develop, they are also referred as delayed-type hypersensivity (DTH).
Describe type 3 hypersensitivity
Immune complex hypersensitivity. This reaction occurs through the formation of antigen-antibody complexes that activate complement and result in tissue damage. The cell or tissue injury is similar to type II hypersensitivity reaction, although the underlying pathogenesis is different. Antigen-antibody complexes produce tissue damage mainly by eliciting inflammation at the sites of deposition. The pathologic reaction is usually initiated when antigen combines with antibody in the circulation, creating immune complexes that typically deposit in different tissues. Less frequently, the complexes may be formed at sites where antigen has been "planted" previously (called in situ immune complexes).
Describe localised type 1 hypersensitivity
In this case, the clinical signs and pathological findings are restricted to a specific tissue or organ. Localised reactions most commonly occur at epithelial surfaces such as skin, respiratory tract and gastrointestinal tract. Few examples: - allergic dermatitis, mostly in small animals (e.g. food allergy, flea bites...) - allergic rhinitis, mostly in ruminants (e.g. grass and weed pollens, mold spores)
eosinophils secrete ___ which causes degranulation of mast cells and basophils
MBP
Describe mast cells
Mast cells are a heterogeneous population of bone-marrow granulated cells. They can be divided into mucosal and connective tissue subpopulations and are primarily found adjacent to blood vessels and nerves where their mediators have their greatest influence.
What is responsible for the delay associated with Type IV hypersensitivity?
Migration of macrophages and T cells to the site of foreign antigens
What are the 4 drugs used to treat Type I hypersensitivity?
NSAIDs Antihistamines Steroids Theophylline or epinephrine
Describe type 2 reactions
Occurs when IgG or IgM is directed against either an altered self-protein or a foreign antigen bound to a tissue or a cell. The results can lead to either destruction of the tissue or cell by ADCC, complement-mediated lysis, or altered cellular function without evidence of tissue or cell damage.
What are the three basic antibody-mediated mechanisms that result in hypersensitivity
Opsonisation and phagocytosis, complement and Fc receptor-mediated inflammation and antibody-mediated cellular dysfunction
Describe opsonisation and phagocytosis
Phagocytosis is largely responsible for depletion of cells coated with antibodies. Cells opsonised by IgG antibodies are recognised by phagocyte Fc receptors. In addition, when IgM or IgG antibodies are deposited on the surfaces of cells, they may activate the complement system by the classical pathway. Complement activation generates by-products, mainly C3b and C4b, which are deposited on the surfaces of the cells and recognised by phagocytes that express receptors for these proteins. The net result is phagocytosis of the opsonised cells and their destruction. Complement activation on cells also leads to the formation of the membrane attack complex, which disrupts membrane integrity and cause osmotic lysis of the cells.
Idiopathic thrombocytopenic purpura (ITP) is caused by an antibody to what?
Platelets
What are the categories of mediators released by the mast cells?
Preformed/primary and newly synthesised/secondary
Type II reactions can be caused by good drugs that go bad that modify ___ or ___
RBCs platelets
Describe type 4 reactions
Result of activation of sensitised T lymphocytes to a specific antigen. The resulting immune response if either mediated by direct cytotoxicity or by the release of cytokines that act primarily through macrophages.
Describe type 1 reactions
Result of an IgE response directed against an environmental or exogenous antigen. The result is the release of vasoactive mediators from IgE-sensitised mast cells, and these mediators produce an acute inflammatory response.
What is the etiology of the tissue damage observed in Type IV hypersensitivity?
Sensitized T cells encounter antigen and release cytokines leading to macrophage activation
What are the preformed mediators?
Stored in cytoplasmic granules. Includes vasoactive amines (histamine, serotonin, adenosine); chemotactic factors for eosinophils and neutrophils; enzymes; proteoglycans (heparin, chondroitin sulfates)
What are the types of type 1 hyppersensitivities?
Systemic and localised
DTH is T or B cell mediated
T
what is type IV hypersensitivity reaction?
T cell mediated hypersensitivity - DELAYED 1-3 days after antigen
__________ are also recruited to sensitize the host against offending antigen; okay if its a parasite, not okay if its cat crap
T, B, and macrophageS
once activated, there is secretion of proinflammatory cytokines by Ag specific ____ cells
TH1
contact dermatitis dmg (DTH) is due to inflammatory response and tissue destruction due to _____ cells and ___
TH1 macrophages
what Th cells are activated in a DTH sensitizied individuals?
TH1 or TH17
IgE production is thought to be dependent on what Th cell due to secretion of what cytokine
Th2 IL-4
What factors determine whether a host will develop a type 1 hypersensitivity reaction?
The factors that determine whether a host will develop a type I hypersensitivity reaction are complex. The genetic makeup of the host and the dose and route of antigen exposure are most important. These factors influence whether the individual will have Th1 or Th2 response. The development of an IgE-secreting B lymphocyte from an immature B lymphocyte depends on activated CD4+ lymphocytes of the Th2 type. The cytokines that define a Th2-lymphocyte response have important roles in regulating the cells involved in a type 1 hypersensitivity reaction: IL3, IL4 and IL10 influence mast cell production; IL4 is involved in isotype switching to IgE; IL3 and IL5 influence eosinophil maturation and activation. The major cytokine that defines a Th1 response, IFNγ, inhibits the Th2 response. Thus an animal that develops predominantly a Th2 response to a particular antigen would be more likely to develop a type I hypersensitivity reaction as compared with one that develops predominantly a Th1 response. In humans, additional genetic influences can be linked to the human leukocyte antigen (HLA)-linked immune response genes. These genes appear to control allergen-specific IgE responses. The association of specific class I MHC molecules with an increased susceptibility to atopy (genetic predisposition to develop localised type I hypersensitivity reactions to innocuous antigens) in the dog has been proposed.
Describe the inflammatory response
The immediate response is characterised by increased blood flow, increased vascular permeability and smooth muscle spasm. As the reaction progresses, additional leukocytes are recruited and they sustain the inflammatory response and cause cell damage. One of these leukocytes, eosinophils, is particularly important. Eosinophils are recruited to the sites of type I hypersensitivity reactions by chemokines, such as eotaxin and IL5. They release: - components of their granules (e.g. eosinophilic major basic protein, that is toxic not only to parasites but also normal cells and therefore contribute to the epithelial cells damage associated with chronic type I reactions) - lipid mediators (e.g. PAF, leukotrienes) - cytokines
Describe DTH mediated by CD4+ lymphocytes
The prototypical reaction is the localised tuberculin response, which is produced by the intracutaneous injection of purified protein derivative (PPD, also called tuberculin), a protein-containing antigen of the tubercle bacillus. In a previously sensitised individual, reddening and induration of the site appear in 8 to 12 hours, reach a peak in 24 to 72 hours and slowly subside.
Describe the tuberculin reaction
The quantity of antigen limits the extent of the inflammatory response, and resolution of the inflammation generally occurs in 5 to 7 days. This is in contrast to chronic infections with persistent intracellular organisms or poorly degradable intracellular antigens that develop into a granulomatous inflammation. Another example of type IV hypersensitivity reaction is contact allergy in man and other animals. In this disease there is topical sensitisation to an allergen and re-exposure leads to an inappropriate inflammatory response at the cutaneous site of challenge.
What factors determine whether a type 3 reaction will occur?
The relationship of the antibody response to the quantity of antigen: when antibody is in great excess of antigen, the antigen-antibody complexes formed are large and insoluble, and easily removed by the phagocytic system. The most pathogenic complexes are of small or intermediate size (formed in slight antigen excess), which bind to phagocytic cells with less avidity and circulate longer. The activity of the phagocytic system: in some instances immune complex hypersensitivity may be the result of the normal phagocytic system being overwhelmed.
Describe type 2 hypersensitivty/cytotoxic hypersensitivity
This type of hypersensitivity most often occurs as the result of the development of antibodies directed against antigens on the surface of a cell or in a tissue, with the result that the cell or tissue is destroyed. Antigen may be either endogenous (normal cell or tissue protein) or exogenous (e.g. a drug or microbial protein adsorbed to the cell). In some instances, the antigen may be a cell surface receptor and the antibody may activate or block the activation of the cell rather than cause the cytotoxicity (this is the pathogenesis of many immune-mediated and autoimmune diseases).; The largest group of cytotoxic hypersensitivity reactions involves the haematologic disorders with antibodies directed against antigens present on the surface of red blood cells or platelets. Type II hypersensitivity reactions most frequently involve IgM and IgG and occur within hours after exposure in a sensitised host.
When would antibody-mediated cell destruction occur?
Transfusion reactions. Haemolytic disease of the newborn (neonatal isoerythrolysis) in which there is an antigenic difference between the mother and the foetus. Autoimmune haemolytic anaemia or thrombocytopaenia. Certain drug reactions, in which a drug acts as a "hapten" by attaching to plasma membrane proteins of red cells
What are the types of hypersensitivity reactions?
Type 1 (intermediate), type 2 (cytotoxic), type 3 (immune complex) and type 4 (delayed-type)
What are the general characteristics of each type of hypersensitivity?
Type I: immediate hypersensitivity Type II: Antibody-mediated Type III: Immune complex Type IV: Delayed (T-cell mediated)
ABO incompatibility is an example of what type of hypersensitivity?
Type II
Grave's disease is an example of what type of hypersensitivity?
Type II
Myasthena gravus us an example of what type of hypersensitivity?
Type II
Rheumatoid arthritis is an example of what type of hypersensitivity?
Type III
Contact dermatitis is an example of what type of hypersensitivity?
Type IV
Type I diabetes mellitus is an example of what type of hypersensitivity?
Type IV
Describe complement and Fcreceptor-mediated inflammation
When antibodies deposit in fixed tissues, such as basement membranes and extracellular matrix, the resultant injury is due to inflammation. The deposited antibodies activate complement, generating by-products, including chemotactic agents, which direct the migration of polymorphonuclear leukocytes and monocytes, and anaphylotoxins which increase vascular permeability. This results in the production of other substances that damage tissues. Antibody-mediated inflammation is the mechanism responsible for tissue injury in some forms of glomerulonephritis or pemphigus disease.
what happens in the elicitation stage of DTH?
activated cells mediate recruitment of innate immune cells which leads to clinical manifestations
how is the FcsigmaRI-IgE receptor different from B and T cell receptors?
activation of mast cell is immediate very rapid and strong response to any antigen the person is sensitized all mast cells in area will degranulate one cell can have antigen receptors for more than one specificity
TNF alpha from mast cells is used to increase expression of ________ promoting leukocyte traffic from blood to site
adhesion molecules
an antigen that induces an allergic response is called an...
allergen
anaphylaxis is caused by...
allergens in blood that cause widespread mast cell activation
if allergen affects the conjunctiva of the eye in type 1 reaction, what is it called
allergic conjunctivitis
most common type I hypersensitivity reaction
allergic rhinitis
synthesis and slower response of cytokines, LTs, and PGD of eosinophils results in...
amplification of the response
________ - activation of mast cells deeper in subcutaneous tissue
angiodema
what is type II hypersensitivity reaction
antibody mediated hypersensitivity - IgG/IgM, cytolytic or cytotoxic
after the proinflammatory cytokines are released, there is recruitment and activation of ________ from TH17
antigen non specific inflammatory cells
granulomatous hypersensitivity is a DTH that is from.....
antigen persists and their is accumulation of macrophages leading to clusters of cells to form granulomas
where do mast cells usually reside?
around BVs, lining of gut, lungs
_______ is more prolonged allergic reaction in skin from inflammatory skin lesions from mast cells or bad inflammatory response
atopic dermatitis/eczema
antibody mediated cellular dysfunction in type II reactions is characteristic in ___ diseases such as myasthenia gravis and type II diabetes
autoimmune
what are three common examples of type II reaction ?
autoimmune diseases where antibodies are made against cell surface or matrix antigens incompatible blood transfusions hyperacute rejection
______ also take part in type I hypersensitivity reactions with mast cells
basophils
in a sensitized patient with allergic asthma, there is immediate constriction of ____
bronchi
PAF is the most potent cause of ___ and ___
bronchoconstriction vasodilation cause shock like syndromes
