Allergy

Question 1

What is an allergic response?

Answer 1

• The cardinal features of an adaptive immune response are:
− Memory
− Specificity
− Discrimination between self and non-self
• These are helpful for protective immunity but in an allergic response, it is these 3 features that are driving the response
• An allergic response is an over-active specific response to harmless/innocuous antigens resulting in pathology.

• Innocuous antigens are those that do not usually promote an immune reaction in most individuals, and are referred to as allergens.
• Common allergens – food, medications, pollen, dust mites, latex, animal dander

Question 2

Describe the Gel and Coombs classification of reactions (Immune reactant, antigen, effector mechanism, example)

Answer 2

Type 1 (immediate):
IgE - Soluble antigen - Mast Cell activation - allergic rhinitis, asthma, systemic anaphylaxis

Type 2:
IgG, IgM - cell/matrix associated antigen - FcR+ cells (phagocytes, NK) - drug allergies, blood transfusion reactions, HDN.

Type 3:
IgG - soluble antigen - FcR+ cells, complement - serum sickness

Type 4 (delayed):
Th1 - soluble antigen - macrophage activation - contact dermatitis, tuberculin reaciton. 

Th2 - soluble antigen - eosinophil activation - chronic asthma and rhinitis

CTLs - cell associated antigen - cytotoxicity - contact dermatitis

Question 3

Why should you never get an allergic reaction the first time you encounter the antigen?

Answer 3

• All true allergic responses develop in two stages:
1. Induction/sensitization → this is the first exposure. The individual is ‘primed’
2. Elicitation → Subsequent exposure to the same allergen. The sensitized individual shows clinical manifestations.
• You should therefore never get an allergic reaction the first time you encounter the allergen – because the immune system needs to be primed to develop the T cell or Ab response.

Question 4

Why is an intolerance different to an allergy?

Answer 4

• With an intolerance, you don’t need the lag phase – you get a response the first time.
• With for eg) lactose intolerance, you need to ingest grams of the substance. With an allergen, once sensitized, you are hyperreactive – may only need to ingest mg or even ng amounts to elicit a response.

Question 5

Describe the 3 major forms of Type II hypersensitivity.

Answer 5

• Involves the production of IgG or IgM that react with cell/matrix associated antigens causing cell lysis or phagocytosis.

Blood transfusion reactions:
• Blood group O make antibodies to A and B
• Blood group A make antibodies to B
• Blood group B make antibodies to A
• Blood group AB don’t make antibodies to either type

These antibodies are usually IgM:
• If you get a wrong blood type, you make IgM which binds to the transfused RBC
• IgM is good at fixing complement
• Causes fever, chills, nausea, vomiting

Haemolytic disease of the newborn:
• If a Rh- mother has a Rh+ baby, she may be immunized against the Rh+ antigen during childbirth by haemorrhage etc, and produce IgG (sensitization)
• If the mother has another Rh+ baby, the anti-Rh IgG can cross the placenta and bind to the fetus’s RBCs
• This results in opsonisaiton of the cells, and their phagocytosis and destruction in the liver or spleen (elicitation).
• Results in an enlarged spleen and liver and toxicity due to bilirubin

Drug induced hypersensitivies:
• Although too small themselves to stimulate an immune response, some drugs or their metabolites can bind to red blood cells or platelets
• The drug-self protein complex can create a new antigen that appears foreign to the immune system
• These stimulate antibody production against the drug (sensitization)
• In subsequent administration of the drug, the antibodies bind and cause either complement mediates lysis or opsonisation through Fc receptors and subsequent phagocytosis (elicitation).

→ eg) Penicilin binds to RBCs, resulting in anaemia
→ eg) Quinidine binds to platelets, resulting in thrombocytopenia

• Symptoms usually resolve when the drug is withdrawn.

Question 6

Describe the features of Type III hypersensitivities.

Answer 6

• Caused by antibody-antigen immune complexes against soluble antigen, and subsequent deposition in small blood vessels.

Systemic disease (eg, infection such as malaria, or in response to serum administration, ie, therapeutic anti-venom):
• Serum sickness is the historic example of systemic disease
• Animal serum containing antitoxin antibodies was given as treatment for diphtheria or tetanus to neutralize the toxins secreted by the bacteria.
• Also used as anti-venom (ie, Abs against venom of snake-bite)
• Because the serum came from another speciies, stimulates a strong Ab response
− If the person is given a second dose od serum, the antibodies bound to the serum proteins forming immune complexes
− A second dose may not even be necessary for complex formation, as IgG antibody has a long halflife in serum → it may persist long enough to be the sensitizer and subsequent inducer
− These complexes are eposed in blood vessel walls, especially those of the kidney, skin and joints, where they fix complement leading to inflammatory responses.
• Symptoms include fever, rashes, arthritis and kidney malfunction.

Local disease (eg, after repeated inhalation of antigen):
• Pigeon fancies disease → caused by inhalation of pigeon antigens in dried faecal particles
• Farmers lung → caused by the inhalation of mould spores in hay

→ Type III HSs are more common in patients with immune disease, eg) SLE

Question 7

What is atopy and the atopic march?

Answer 7

The Atopic March:
• Atopy is the genetic tendency to develop the classic allergic diseases → atopic dermatitis, rhinitis and asthma.
− This is why people with allergic disease tend to have more than one
− Atopy involves the capacity to produce IgE in response to common environmental proteins such as house dustmite, grass pollen and food allergens.
• A clear progression from early-life atopic dermatitis to later rhinitis, food allergy and stham has been commonly observed and is termed the atopic march.

Question 8

What are the IgE mediated disease prevalences in developed countries?

Answer 8

• Asthma 5-17% → depends on the country. Croatia has low incidence, Portugal has high. ~5 million being treated for asthma in the UK (1 in 12 adults, 1 in 11 children)
• Allergic rhinitis 10-20%
• Food allergy 1-2% adults, 5-8% children → Different in children and adults because children tend to grow out of food allergy more than asthma and rhinitis.
• Eczema 1 in 5 children, 1 in 12 adults → Again with eczema, 50% children will grow out of it
• Systemic anaphylaxis (50% due to food allergy, the rest is inset stings etc..) → 63-99 fatalities per year in the US. >50% are teenagers. But of the hospitalization cases, only 0-3% are fatal – so as long as you get to hospital, prognosis is good.

Question 9

Why is it problematic that a major affected population of IgE mediated disease sufferers are 16-25?

Answer 9

• Problematic because adolescents are programmed to undertake risky behavior
• When living at home, the environment would be regulated to reduce allergens – but when they move out, they are less likely to be as stringent
• Also peer pressure – friends all going out for food, you want to join in.

Question 10

What immune response does a type I hypersensitivity evoke, and what roles do the cytokines play?

Answer 10

• Type I hypersensitivity is a Th2 response
− The immune system is seeing the allergen as a parasite
− Th2 cytokines in Type I reactions:
− IL-4 → IgE antibody production. Drives the type I reaction
− IL-13 → Hypersecreton of mucin
− IL-5 → Activation and recruitment of eosinophils
− IL-4/IL-5 → Differentiation and activation of mast cells

Question 11

Describe the biphasic response of anaphylaxis

Answer 11

• An acute reaction occurs immediately after allergen exposure, followed by a late phase reaction several hours later
• Symptoms during the acute phase are due to release of pre-formed mediators, but the late phase response involves influx of inflammatory cells.
− Mast cell degranulation is an acute response → leads to immediate weal and flare
− Further production of cytokines can lead to the late phase response (8hrs later) due to eosinophilia and Th2 cell recruitment.
− Results in quite severe inflammation and can lead to remodeling of the airways → increased mucin, increased muscle proliferation leading to decreased lumen of the airways.
− Eventually get bronchial hyperreactivity – non-specific triggers such as cold air and excersie trigger the asthmatic response, not just antigen.

Question 12

Describe the local symptoms of a Type I hypersensitivity.

Answer 12

• Depends on the site of challenge

Local response:
• Skin → allergen crosses the skin barrier, resulting in activation of resident mast cells. Local histamine gives increased blood flow and permeability, giving the local wheal and flare – increased fluid, redness, swelling and rashes.
• Airways → inhalation of allergen results in mast cell activation in the airways. This gives bronchial smooth muscle contraction, giving decreased lumen of the airways. Increased mucous production, coughing, wheezing and sneezing aims to get rid of the allergen.
• GI tract → allergen crosses the intestinal epithelium and results in mast cell activation giving increased fluid and peristalsis. Leads to expulsion of GI tract contents in an effort to get rid of the allergen.

Question 13

Describe the systemic symptoms of type I hypersensitivity.

Answer 13

• If an allergen enters the blood stream
• Is much more severe
• Insect stings can cause it, as they inject into bloodstream as well as tissue
• Blood vessels → mast cell activation in the bloodstream results in systemic histamine release along with other factors including platelet activating factor.
− PAF acts on platelets to produce histamine, this acts back on the basophils, giving a positive feedback loop.
− Strong vasodilatory activity of histamine leads to anaphylactic shock due to loss of blood pressure and fluid in the lungs
− Eventually leads to unconsciousness and death.
• Anaphylaxis may begin with severe itching of the eyes or face, and within minutes, progress to more serious symptoms → swallowing and breathing difficulties, abdominal pain, cramps, vomiting, diarrhea, hives, angioedema.

Question 14

What are the allergens that cause anaphylaxis?

Answer 14

• Food generally the most common cause
− Nuts, shellfish, dairy, egg whites, sesame seeds
− Normally will just cause a local response, but if the allergen concentration is big enough it may leak into the blood stream.
• Wasp and bee stings also common cause
• Exercise can be a trigger if occurring after eating allergy-provoking food
• Pollens and other inhaled allergens rarely cause anaphylaxis

→ Can be a particular problem if you already have a condition such as asthma, as the shock affects the airways, which are already pre-disposed to aberrant vasoconstriction.

Question 15

Describe the specific features of food allergy.

Answer 15

• Loss of oral tolerance associated with diminished Treg response → exemplified by patients with IPEX syndrome (mutated Foxp3) → these people develop severe allergic disorders.
• The epithelium is also a key player in allergy development:
− ECs serve as a barrier against foreign proteins, and increased epithelial permeability is associated with antigen sensitization and allergy development.
− ECs produce cytokines that promote Th2 skewing by DCs → in murine models of asthma, IL-33 production by ECs after sensitization promoted interactions between DCs and T cells that augmented Th2 responses.
− Food allergy commonly associated with atopic dermatitis, implicating the skin as a possible route of entry for food allergen.
− Mutations in filaggrin, which encodes the epidermal filament-aggregating protein, is associated with decreased keratinocyte barrier function and increased risk of food allergy.

• Why specific food allergens trigger the response is unclear
• Although, some food Ags possess the potential to stimulate the innate immune response
− Eg, peanut allergen Ara h1 binds to DCs
− Milk sphingomyelin activations type 2 cytokine responses
• Changes in microbial flora associated with allergic sensitisaiton
− Mice with decreased commensal colonies exhibit increased food allergen sensitivity, high serum IgE and increased circulating basophils.

Question 16

Describe the main ways of diagnosis of type I hypersensitivity.

Answer 16

Detection of IgE:
• Specific serum IgE ex vivo (RAST/IMMUNOCAP) → the presence of IgE in serum is atopic (out of place). If this is the case, the individual is said to be atopic – predisposed to allergy. However, they may not show clinical symptoms.
• Skin prick test → the most common.
− A few ul allergen introduced to the epidermis using a lancet → can be either preparations of allergen extract, eg) pollen, cat; or in the case of certain foods such as fruit, fresh allergen is used.
− If a person is sensitized, they will give a wheal and flare reaction → allergen cross-linkes the FceR on skin mast cells, causing mast cell activation
− The wheal and flare subsides within an hour, but is replaced with a late phase reaction later.

Provocation challenge:
• Typically used for food allergy
• Detection of IgE tells us if the individual is pre-disposed to allergy, but the provocation challenge tells us if they do exhibit a response.
− Food allergy → double bind placebo controlled food challenge
− Rhinitis → nasal challenge
− Asthma → bronchial provocation test
• The purpose is to find out exactly what allergen the person is allergic too

Question 17

Describe avoidance and pharmacotherapy as maintenance of type I hypersensitivity.

Answer 17

Avoidance
• The main method of management – identification of offending allergen and avoidance

Pharmacotherapy
• Treatment of the symptoms:
− Anti-histmines
− Mast cell stabilisting compounds
− Topical and systemic corticosteroids to reduce inflammation
− Adrenaline (EpiPen) for systemic anaphylaxis → life saving option in case of accidental exposure
• All provide moderate or partial relief

Question 18

Describe desensitisation as an immunotherapy for type I hypersensitivity.

Answer 18

1. Desensitisation
   • Effective for allergic rhinitis (used for the last 100 years)
   • Repeat subcut. or sublingual applications of small amounts of allergen
   • Induces an altered immune response (IgG4/Tregs)
   • Disease modifying – you are altering the quality of the immune response (IgG4 mops up the allergen before it gets chance to bind to IgE. There is also negative feedback, if you are not longer stimulating IgE on mast cells they downregulate their receptors)
   • Possible adverse systemic effects
   • Also lengthy time required for therapy

Potential for food allergy? Subcut injection of food allergen quite dangerous.

Anagnostou et al, 2014: Study of Induction of Tolerance to Oral Peanut (STOP)
• Phase 2 randomised trial
• 99 Children aged 7-15 years with peanut allergy
• Daily immunotherapy (2mg, 5mg, 12.5mg, 25mg, 100mg….) peanut prptein administered as peanut flour
• Doses increased at 2 –weekly intervals
• Control group underwent peanut avoidance
• The primary outcome (tolerance of up to 1400mg peanut protein) was observed in 62% of the active group, and none of the control group in phase 1.
• In phase 2, 54% tolerated 1400mg challenge, and 91% tolerated daily 800mg dose.
• Quality of life scores improved
• Side effects mostly mild GI symptoms
• Study concluded that peanut oral immunotherapy should be performed

→ Main goal of densitisations is to desensitize to the point where they can tolerate accidental exposure, not necessarily so they can eat peanut at wil.
→ However, this is useful as it is the fear of whether food contains peanut that effects their quality of life.

Question 19

What is immunotherapy in terms of type I hypersensitivity responses?

Answer 19

training the immune system away from an IgE response – towards a less harmful IgG response, or even better, tolerance.

Question 20

Describe prevention as an immunotherapy to maintain type I hypersensitivity responses.

Answer 20

• Stopping the allergy before it starts
• One example from a Jewish community in isreal – had low incidence of peanut allergy, but when they moved to the UK, they suddenly had high incidence → so isn’t genetic, there is some environmental factor
− Noted that Bamba (popular snack) is peanut flour
− Theory that early exposure to peanut was a good thing

Du Toit et al: LEAP – Learning Early about Peanut Allergy
• Randomised trial of peanut consumption in infants at risk for peanut allergy
− Assigned 640 infants with severe eczema, egg allergy or both to consume or avoid peanuts until 60 months of age
− Infants were tested to see if they already had peanut allergy
− In the 530 who initially were negative for peanut allergy → prevalence of allergy at 60 months was 13.7% in the avoidance group, and 1.9% in the consumption group.
− In the participants who initially were positive for peanut allergy → prevalance of allergy at 60 months was 35% in the avoidance group, and 10.6% in the consumption group.
− Levels of peanut-specific IgG4 were higher in the consumption group
• Induces altered immune response (increased IgG4)
• Prevention and cure
• Low incidence of adverse effects

→ Important, because in 1998 we gave advice that patents should avoid giving their children peanut if they have eczema – but this is bad! As exposure seems to help prevent.

Question 21

Describe monoclonal antibody therapy as maintenance of type I hypersensitivities.

Answer 21

• Omalizumab is currently the only approved biologic for the treatment of allergic disease
− Approved for use for moderate/severe allergic asthma and chronic urticarial disease
− Binds circulating IgE and decreases levels by 99% → find in Western society as parasites not much of a problem, so we can handle decreased IgE
− Results in decreased mast cell degranulation
− Also results in downregulated IgE receptor expression
− Problem is that it is expensive → costs €1700 per patient per month. So not used that much

New biologics:
• New Anti-IgE’s → eg, Tigelizumab, Novartis)
− Antibodies also being made against an intracellular sequence M19, that specifically targets B-cell expressing IgE and not serum IgE
• Anti-CD23 (the receptor for IgE) → Lumiliximab, Biogen
• Anti-cytokine therapy
− The cytokines produced by Th2 response (IL-4, IL-5, IL-12) are key drivers of the inflammatory response
− These are increased in patients with food allergy
− Mutations in IL-4 receptor and IL-13 are associated with increased risk of food allergy
− Anti-IL4 receptor antibody Dupilumab successfully improved the clinical symptoms of patients with asthma or atopic dermatitis.
• TLR agonist, natural polymer and cell-based adjuvants:
− Combining allergen with Th1 promoting immune adjuvants
− Promotes DC antigen internalization and Th1 polarisation, restoring the Th1/Th2 balance
− These adjuvants mimic PAMPS that bind to TLRs
− eg) AIC is CpG DNA conjugated to Ragweed allergen
− Stimualtes Th1 resposne instead of Th2, as CpG DNA is viral
− Improved the clinical symptoms in patients with allergic rhinitis
− Because of the strong Th1 response, there are concerns that TLR agonists will reduce Tregs
− However, there is some evidence that Tregs work in concert with TLR agonists to suppress adverse Th2 responses.
− Other proposed adjuvants include fungal chitosan → treatment of mice with peanut allergen bound to chitosan reduced allergen specific IgE responses and promoted Th1
• Epithelial cell targets
− In addition to serving as a barrier, ECs contribute to the the pathology of food allergy by interaction with allergens and secreting TH2 promoting mediators
− ECs express CD23, that transports antigen-bound IgE across the epithelium to the underlying tissue
− CD23 expression is upregulated by IL-4
− CD23 is increased in people with food allergy
− Treatment with anti-CD23 prevents IgE transport

Question 22

What is the prevlance of atopic allergy around the world, and how has it changed?

Answer 22

• There is a rising incidence of atopic allergy.
− In the 1920’s and 30’s, less then 1 in 1000 had asthma
− This increased 10 fold in the 70’s
− In 2000, approaching 10% of people had asthma → so over 70 years, there has been 100 fold increase.
− The % of 9-12 year olds with a topic disease has been steadily increasing from the 60s up until around 2005, where it began to plateu.

• Prevalence of atopic disease around the world:
− Increase in prevalence has a distinct geographic distribution
− Atopic disease is highly prevalent in developed countries such as US, UK and Australia
− Russia, Africa and South East Asia have low incidences

Question 23

What are 6 possible drivers for increased prevalence of atopic allergy?

Answer 23

• Changes in diet → PUFAs – type 6 PUFA in margarine tends to be a risk factor for allergy. PUFA 3 found in oily fish is protective.
• Increased exposure to allergens → As we have got more affluent, have centrally heated houses and carpets, increasing house dustmites. However, doesn’t fit entirely – Scandinavian house don’t tend to have carpets etc.. but have high levels of allergy.
• Selective adjuvant properties of chemicals and pollutants → Powerful study conducted when the Berlin wall was erected. Similar gene pool across Germany, but in East Germany there were different regulations and higher levels of pollutant. These actually had lower levels of allergy, but higher bronchitis. The thalates (plasticizing agents) have also been implicated in increased risk.
• Changes in infection/pathogen microenvironment
• Changes in indoor air quality
• Other lifestyle changes → Increased use of antibiotcs, so we aren’t getting infection. People are now having fewer children, so less likely to interact with siblings.

Question 24

What is the hygiene hypothesis, and what was the initial evidence supporting this?

Answer 24

Strachan, 1989: Hay fever, hygiene and household size
• For eczema – if you were the oldest child, the number of younger brothers and sisters you had had no effect, you would develop eczema regardless
• If you were the youngest, there was a dose-dependent relationship – the more siblings you had, the less likely you were to develop
− The theory for it not affecting the older sibling is that eczema manifests in the first 6 months – if you are the oldest, you don’t have any siblings around you at this point.
• In hayfever, there was also a dose-dependent relationship if you were older

→ Strachan formulated the following hypothesis “infections in early childhood, transmitted by unhygienic contact with older siblings, might prevent the development of allergic illnesses.

Question 25

What further evidence supported the hygiene hypothesis?

Answer 25

Riedler et al, 2001:
• It is thought some enteric microbes may protect against allergy
• Looked at populations of 6-13 year olds in rural Austria, Germany and Switzerland and noted if they moved there Swiss farming lifestyle > no farm at all
• The Amish not only have more exposure to microbes in the farm environment, but they also have much larger family sizes, so have more sibling exposure.

Exposure to domestic animals may also confer protection:
• Pet ownership has been shown to be inversely associated with atopy and asthma in children and adults

Other evidence in support of the hygiene hypothesis:
• Early daycare attendance → decreased asthma
• Early use of antibiotics → increased allergic disease
• Italian military recruits → increased hep A antibody, decreased atopic syndrome
• Children in Guinnea-Bissau → BCG vaccination decreased allergy
• Japanese school children → DTC to tuberculin, decreased atopy
• Finnish children → adopic disease preceded by an imbalance in the gut microflora

Question 26

What is the immunology of the hygeine hypothesis?

Answer 26

• The neonatal immune system has a Th2 bias – this is because in the womb, the mother is Th2 so she doesn’t reject the fetus. If she were Th1, would get GvHD reaction to the fetus
• The ability to produce IL-12 (and ilicit a Th1 response to balance it out) is age dependent
• Th2 skew diminishes in non-allergic children during 2 years
• In allergic children, they do not develop the same ability to produce IL-12, so they maintain a Th2 skew.

Question 27

Describe the selectivity of hygeine effects.

Answer 27

Not all infections are created equal:
• Respiratory viral infections → not protective, cause wheezing. Respiratory syncytial virus/human rhinovirus correlated with asthma
• No evidence for protection resulting from measles virus
• No difference in prevalence in relation to MMR, VZV, CMV and HSV
• Therefore, seems that protection is specific to certain infections, and perhaps at particular times of life.
− One study found that croup and repeated ear infections were inversely associated with atopy, whereas bronchiolitis was positively associated with a asthma.

The Parasite Paradox
• Worldwide helminth infection and allergy do not overlap, but both are driven by Th2 responses
• You would therefore expect there to be more atopy where you have helminth infections
• Intestinal parasite infection therefore seems to be protective
• Prolonged treatment with anti-helminthics increases skin prick test reactivity and increases IgE responses
• In Venezuela and Vietnam, they have eradicated certain soil parasites, but this has now been followed by an increase in allergy.
• Reduced allergy in those with parasitic infection is not due to total absence of specific IgE – they still produce it, just don’t go on to display the clinical manifestations.
− 25% children had house dustmite IgE, only 15% displayed SPT reactivity

Question 28

Describe the “microbiome hypothesis:

Answer 28

Liu, 2015
• Not all infections are equal – some protective, some provocative
• Isnt just infections that are important, also the commensals → the microbiomes of healthy vs allergic people are different.
− Healthy lung – homogenous distribution. Same bacteria all over
− Allergic → different bacteria to the healthy people, and different bacteria in different plces
• Studies in GF mice have shown that exposure to the commensals are crticial for proper immune system development – they have reduced TRegs.
• GF mice had increased allergic airway inflammation compared to SPF mice
• Microbial exposures activate PRRs → leads to downstream suppression of Th2 expansion, and therefore Th2 mediates diseases.
• Early life exposure is critical → the maternal microbiome influences the neonate. Composition of maternal flora may be related to wheeze in finants.
• Animal models demonstrate cause and effect, eg) in a mouse model of asthma, can prevent it by giving lactobascillus
• Reduced intestinal biodiversity during infancy has been associated with increased allergy at school age

Question 29

What is the “old friends” hypothesis?

Answer 29

Rook and Burnett, 2005:
• Noted that along with an increase in allergy, has been an increase in autoimmune disease, eg) T1DM
• The old friends hypothesis states that we need to be able to have protective immunity against pathogens, but tolerance against the commensals.
• It goes on to state that the reason increases in atopic disease have changed is because our microbial history has changed.
• In Paleolithic times, we were hunter gatherers
− Lived in small groups
− Ate meat that we killed
− Gut microbes were environmental saprophytes (organisms picked up from soil and water)
• The first major epidemiological shift happened between Paleolithic and Neolithic times
− moved to large social groups → increased orofaecal circulation of organisms
− Started domesticating animals and living with them
− Rapid evolution of viruses such as measles, mumps and smallpox
− Got modified human and helminth cycles due to increased animal exposure
• We then moved to larger cities in the bronze age, and started to get disease epidemics (flu,plague)
• Then got the second major epidemiological shift to modern day society
− Most people live in cities or towns
− Even those that live in the country aren’t exposed to animals or the environentn the same
− Have clean water and food
− Using antibiotics
− Have a loss of the organsims that we used to pick up from mud, water, vegetation etc…
• The idea is that the helminthes and commensals we used to be exposed to, we ‘co-evolved with’ – so we developed tolerance to them
− With respect to helminthes, the inflammatory response needed to remove them may be too detrimental, so we tolerated them
− As they co-evolved with us, many helminthes secrete immunoregulatory factors that dampen down the immune response
− If we have high helminth burden, these are ‘old friends’ – they secrete the regulatory factors so we have high TReg levels, therefore high tolerance, therefore decreased atopic allergy.
− However, in modern society, we have less helminthes – therefore have less immunosuppression and instead have inappropriate reactions to allergens.
− Pathogens are not old friends, they don’t secrete regulatory factors, so we mount protective immunity against them.

Importance of Tregs:
• Importance of the immunoregulation can be seen with the absence of TRegs
• Scurfy mouse (Foxp3 KO) – die from lymphoproliferative disorders and IBD from inappropriate immune responses to intestinal antigen
• IPEX:
− Vary rare, only 14 documented cases worldwide
− Foxp3 mutant, so TRegs absent
− Early onset (infancy)
− Chronic dermatitis
− Neonatal T1DM
− Thyroiditis
− Eczema and food allergy

Question 30

What is the important of the route of exposure in terms of type I hypersensitivity?

Answer 30

• Exposure through the skin and lung epithelial → Th2 response
• Exposure through the GI tract → tolerising

• Skin exposure was first shown to evoke a Th2 response when studies tried to understand occult allergy → this is where parents were claiming their children were presenting with peanut allergy the first ime they had been given a peanut (when we know this biologically isn’t correct).

Lack G et al, 2003:
• Theorised that maybe there was maternal exposure to peanut, and the antigen subsequently crossing the placenta sensitized the child
• However, was shown there was no increased risk from nut intake during pregnancy
• However, a factor that was shown to be important was eczema → 44x more likely to have peanut allergy if they had severe eczema
• This brought up the idea of the atopic march
• Individuals that have eczema have been found to have a propensity to an overproduction of Th2 promoting cytokines, eg) TSLP and IL-33
• It has also been found that 50% of eczema patients have a defect in filaggrin → Mutations in filaggrin, which encodes the epidermal filament-aggregating protein, is associated with decreased keratinocyte barrier function
− Food allergic individuals have also been found to have these defects

• In terms of exposure through the GI tract

Fox et al, 2009:
• If you avoid peanuts as a child, but your household is eating peanuts, you get peanut exposure through the skin and you develop allergy
• However, if you ear peanut before 12 months, you can really inhibit the ability to produce allergy
− We also saw this before with the Israeli children when they ate Bamba
• So, early exposure is recommended
• The reason for this is because the gut is a site of high immune load
− Mucosal immunce cells good at inducing TReg production
− This is the default mechanism
− So, under physiological conditions, there allergen entering via the GI will encounter a very immunosuppressive environment.

Question 31

Describe the intervention strategies that tackle the hygeine hypothesis of type I hypersensitivity

Answer 31

• LEAP study → early gut vs skin exposure
• Helminth treatment
− After injection of Trichuris OVA, eggs hatch and colonise the colon but only for a short period of time, so the treatment has to be repeatred. Conversely however, the hookworm Necator administered percutaneously migrats through the vasculature and lungs to the SI, where it survives by feeding on blood from the mucosa, so it is long-lasting. In its natural state, this infection is a major public health problem as it causes GI problems.
− Studies on the relationship between helminth and atopy have generated mixed results, with both positive and negative results depending on species.
− Studies from helminth endemic areas suggested that certain helminth infections may protect against allergy and asthma, but a systemaic review concluded in general there was no overall protective effect. However, concurrent hookworm infection was protective. In contrast, Ascaris infection was associated with significantly increased risk of asthma → interesting given the fact both Necator and Ascaris pass through the lungs.
− Important to note that many helminth antigens crossreact with common allergens, and it may be that release of antigens from dying worms after anti-helminthic treatment cross-reacts with IgE and causes increased allergy → not the actual lack of helminthes themsvels.
• Probiotic interventions
− Many of the studies show a positive effect on eczema
− Looks like a mixture of probiotics is more effective than single
− However, not all studies were positive and nothing on wheezing, asthma or hayfever
− No current health advice to say taking probiotics may help, but likely to be in the future
• Prevention of the atopic march
− The idea is that if you can prevent the development of eczema, you can prevent the development of atopic disease (but this is very much a theory)
− To date, all interventions to try and prevent the atopic march using the following have not been successful:
− Cetirizine anti-histamine
− Topical calcineurin inhibitors
− Early moisturizing
− As we have seen, key features in the pathophysiology of allergic disease are barrier defects and abnormal immune resposnes.
− Observation that there is a role for transcutaneous sensitization to allergens lead to the study of aiming to prevent allergic disease through the creation of an artificial barrier on the skin by intensive moisturizing from birth.
− In one study, a significant effect of the use of daily emollient was found

Question 32

What are the problems with the hygeine hypotheis?

Answer 32

• It has now been established that the proportion of asthma attributable to atopy is usally less than 50%
• The original hygeiene hypothesis suggests that decreased microbial exposure would, through enhanced atpic immune response, increase the incidence of allergies and asthma → if true, then the protective effects would be more pronounced for atopic asthma. However, there is some evidence that nonatopic asthma may have increased more than atopic asthma.
• Asthma prevalence has begun to decline in children and adults in westernized countries, but it is unlikely these countries have become less clean and there is no evidence that family size has increased.
• The observed decline in asthma did not correspond with a decline in atopic eczema and food allergy, which continues to increase in prevalence
• A third anomaly is high asthma prevalence in Latin America, which appear unlikely to have lower infection rates then Spain and Portugal, which have lower asthma symptom prevalence.

Question 33

What are the general features of Type IV hypersensitivity

Answer 33

• AKA delayed hypersensitivity
• Does not involve antibody! Involves Th, Tc and macrophages
• Takes 24-72 hours to appear
• Differs from other hypersensitivis in that it is not always against harmless antigen, but is seen in many infections and can contribute to protection from pathogens
• Causes include: Posion Ivy, Nicken, Hapten Responses, reactive chemicals
• Accounts for 1% of all GP visits and 15% of occupational disaeses

Question 34

What are examples of contact allergens, and the clinical manifestations?

Answer 34

• Following repeated exposure to the chemical there is an eczematous reaction involving swelling, redness and severe itching which occurs 24-72 hours after contact with the allergen.
− It was found that chemical irritants were present in some brands of skin cream and cosmetics including Nivea and L’oreal cream, Piz Buin self tanning agents and baby wipes
− Preservative methyl isothiazolinone is routinely added to moisturisers – but told to remove it due to a spate of allergic reactions.
− PDD is found in hair dye and is a common contact allergen. This is okay for the consumer, as can just dye your hair a different colour, but if you were a hairdresser it would be an occupational allergy – they wouldn’t be able to work as a hairdresser anymore. So has socioeconomic effects.

Low molecular weight chemicals:
• Haptens are too small to trigger an immune response, so these chemicals must be protein reactive in order to make the protein/hapten complex that is seen by the immune system, so they tend to have unstable structures (ie, are electrophilic)
− PDD → the amine group is unstable and wants to live
− Dinitrochlorobenzene → chlorine group
− Methylisothiazolinone → heterocyclic ring with an N and S that want to open up
• They will bind to nucleophilic amino acids.
• Binding to the skin confers immunogenicity

Natural contact allergens
It isn’t just synthetic chemicals that cause contact dermatitis – natural substances cause it too
− Common one is nickel, found in jewelry → 34% Thai women allergic to nickel
− Believed to be so common because the exposure is intimiate, eg) you are making a hole in the skin for a piercing, then inserting the allergen into it
− Interestingly, if you have a brace made from nickel (oral exposure) – you are less likely to develop contact allergy. Makes sense from what we known about oral exposure being tolerising.
− Geraniol found in geraniums, roses and citrus peel is also common → bad for gardeners, florsts and bar tenders.

Question 35

Describe 3 proteins whos modification is relevant for allergic contact dermatitis

Answer 35

• Chemical protein modification by contact allergens is essential for their ability to induce skin inflammation
• Modifications may mimic or interfere with classic post-translational protein modifications, and thereby alter the function and localization of proteins and protein-protein interactions
• Up to now, 3 proteins have been identified whos modification is relevant for allergic contact dermatitis:
− KEAP1
− Cytosolic sensor protein that detects oxidative an electrophilic stress by oxidative or chemical modification of cysteines
− Results in the activation and nuclear translocation of Nfr2
− This activates the antioxidant phase II response
− Mice lacking Nfr2 have a much lower threshold for sensitization by contact allergens and increased contact hypersensitivity
− TRPA1
− Calcium influx channel of nociceptive neurons involved in pain, itching and inflammation
− Expressed on keratinocytes and endothelial cells
− its activation depends upon chemical modification of cysteine
− CHS by oxazolone are reduced in TRPA1 deficient mice
− TLR4
− Receptor for LPS
− Nickel and cobalt ions can bind to conserved histidines in human TLR4, inducing inflammatory signaling by NFkB

Question 36

Briefly describe what constitutes the induction and elicitation phase of allergic contact dermatitis.

Answer 36

• Induction phase →
− allergen coming through the skin, draining to the lymph node, processed by APCs and presented to T cells. The DCs secrete cytokines that polarize the T cell response to Th1/Th17. Then get clonal expansion of allergen specific T cells which home back to the skin.
• Elicitation phase →
− The same allergen crosses the skin, and meets the allergen specific T cells.
− These T cells secrete:
− Chemokines → recruits macrophages
− Cytokines → IFNy activates the macrophages. GM-CSF stimulates monocyte production
− Cytotoxins → TNFa/b cause local tissue destruction and increase adhesion molecule expression

Question 37

How is LC migration regulated in allergic contact dermatitis.

Answer 37

• When you expose the skin to allergen, you get a loss of Langerhans cells and they become bigger – they are activated.
• This is accompanied by an accumulation of DCs in the lymph node
• However, only a proportion of the LCs will leave the skin at any one time – this is important, you don’t want to completely delete the population because you may get another insult.

Regulation of LC migration in man and mouse:
• LC biology is very similar in man and mouse
• Allergen stimulates the production of IL-1B by LCs → this acts in an autocrine manner to provide a signal for migration
• Secondly, it acts on neighbouring keratinocytes to produce TNFa, which acts back on the LCs to induce more IL-1
• The LC will then leave the skin and go to the draining lymph node

Question 38

What are the roles of the specific DC subsets in contact dermatitis, and how was this determined?

Answer 38

• As state, the LCs aren’t the only cell population in the skin, so how do we know these are the important ones?
• Use mouse transgenic models with either constitutive depletion of LCs, or inducible.

Inducible:
• DTR is the diphtheria toxin receptor
• As soon as you inject diphtheria toxin, you destroy the langerin+ cells → so at day 0, have no LCs and no Langerin+ cells
• However, after a week or so you have nearly have the dermal Langerin+ cells back, but no epidermal LCs
• So you have a model with either no Langerin+ cells at all, or some dermal but no epidermal.

Constitutive:
• huLangerin-DTA → at birth, have no epidermal LCS but have normal dermal Langerin+ cells.
• Batf3 → at birth, have no dermal Langerin+ cells but normal epidermal LCs

• The inducible system shows that without either type, you have a compromised CHS response
• However, dermal Langerin+ cells alone are sufficient to induce an allergic response
• In the constitutive system, you see an enhanced response in the absence of epidermal LCs → suggests these have a regulatory function serving to dampen immune responses.
• However, in the absence of dermal Langerin+ cells, epidermal LCs can maintain a CHS response, so in the absence of dermal Langerin+ DCs, epidermal LCs can perform their function

→ Tells you that both epidermal LCs and Langerin+ DCs can mediate the response.
→ This is known as redundancy – more than one way to get the same end point. This is because the immune sytem is important, so even if one part is compromised, you still get a response.

• However, it is likely that in the inact immune system, dermal Langerin+ DCs are the ones involved in the CHS response, and epidermal LCs play a regulatory role.
• It is only when part of the system is compromised that epidermal LCs become mediators of the allergic response.

This is highlighted in the kinetics of DC migration:
• It is the dermal Langerin+ DCs that arrive at the lymph node first
• These trigger the proliferation of CD8+ and CD4+ cells
• Later, the LCs arrive and these produce IL-10 to downregulate the immune response, because otherwise you would have proliferation occurring indefinitely.

This is true for potent contact allergens, however:
• The absence of epidermal LCs gives a compromised CHS response to low dose allergen, irrespective of whether dermal Langerin+ cells are present

Question 39

What are the LC interactions with T cells during contact dermatitis, and how are they activated to carry this out?

Answer 39

• They provide signal 1 – antigen in the context of MHC
• CD80 and CD86 interacting with CD28 on the T cell provide signal 2
• Soluble cytokine mediators can provide signal 3

However, in order for the DC to be activated, it has to sense antigen in the context of ‘danger’:
• These danger signals come from PAMPs during infection, or DAMPs resulting from trauma or stress to the tissue
• So, for a contact allergen to stimulate a productive T cell response, it must be considered ‘foreign’ in order to be recognized by TCR/MHC, and must also be able to upregulate the production of DAMPs
• They way it does this is 2-fold:
− Direct → direct activation of the PRR on the DC, eg) nickel ligation to TLR4. Also allergen may be an irritant which causes cell death and the release of DAMPs
− Indirect → eg) DNCB causes ECM breakdown, releasing hyaluronic acid, and this can act via TLR2
• It is postulated that the recognition of haptens (contact allergens) in the absence of danger signals leads to immunological tolerance, but in the presence of danger signals sensitization will be acquired

Question 40

What are complete and incomplete allergens in terms of contact hypersensitivity?

Answer 40

• Chemical allergens are commonly able to deliver both an antigenic stimulus and danger signals → complete allergens.
• Weaker allergens tend to be called incomplete allergens – they are immunogenic but do not cause danger signals themselves, they need to be seen in the context of danger signals ilicited by something else.
− We know this happens because we can enhance an allergic response by giving an allergen combined with an irritant (induces DAMP production)
− This is more like what we see in real life, we would usually get exposure to multiple chemicals as opposed to just one.

Question 41

What is the stimulation index in type IV hypersensitivities and what can it be used for?

Answer 41

• Following the 3 signals, you get clonal expansion of allergen specific T cells →both memory and effector
• You can measure this proliferation as a stimulation index
− The SI = the proliferation in the test chemical treated group divided by the control group
− An SI over 3 is a positive response
− DNCB is a very potent allergen, get a strong proliferation above 3-fold even at 5% concentration
− Skin irritants like PABA don’t stimulate strong proliferation even at concentrations of 10% and above
• So, T cell proliferation distinguishes between an allergen and an irritant
− Irritants cause the danger signal, but don’t cause immune recognition

Question 42

How do all the different cells come together to shape the immune response in contact dermatitis?

Answer 42

• Cytokine profile of high IFNy, IL-12 and IL-17 (low IL-4, IL-5 and IL-10) is indicative of a Th1 response
• The contact allergen is being mis-recognised as a virus or fungi
• We must also note that CD8+ T cells can differentiate into different subsets too:
− Tc1 → IFNy
− Tc2 → IL4, IL5
− Tc17 → IL-17
• This means the cytokine profile could be from CD4 or CD8+ T cells
• Can use flow cytometry to identify the type of cell:
− The CD8+ T cells secrete quite low levels of IL-17, and although increases a little after contact with allergen, it is still less than 1%
− With the CD4 ells, these are much better able to produce IL-17 – have 10% in the controls, incrasing to 30% after DNCB activation
− So it is inducing a CD4+ response

→ Langerhans cells are good at inducing IL-17 secreting cells.

So, to put it all together:
• Dermal Langerin+ DCs migrate first – these activate IFNy producing CD4+ and CD8+ cells
• Then we have a delayed influx of Langerhans cells that stimulate the activation of Th17 cells as well as downregulting the immune response to prevent an exaggerated response.
− The priming of naïve T cells seems to be paralleled by the induction of contact allergen specific TRegs that fail to prevent allergic contact dermatitis in the case of strong allergens, but may prevent it being caused by weaker allergens.
− In the elicitation phase, these TRegs help to downregulate the immune response – in their absence, it is much more prolonged
− The ratio between effector/memory T cells and TRegs seems to be controlled by the potency of the contact allergen

Question 43

Why is there a need for in vitro assays for contact allergen identification?

Answer 43

• Replacement of the murine local lymph node assay is a pressing issue, and animal testing is prohibited for the cosmetics industry
• Several in vitro assays that cover aspects of the sensitization phase, ranging from peptide reactivity to T cell priming have been developed
• May allow for complete replacement of animal testing.

Question 44

How is allergic contact dermatitis diagnosed?

Answer 44

Patch testing:
• Put patch on, leave on for 72 hours – cant shower
• Go back and have dermatologist read what you are allergic too

Question 45

How is allergic contact dermatitis treated?

Answer 45

• Identificaiton of allergy and avoidance
• Mid to high potency topical corticosteroids
• Extensive (>20%) hypersensitivity may warrant systemic steroids

Potential Therapy: From Parasite to Peptide
• We have discussed already how ‘old friends’ that have co-evolved with us can produce immunoregulatory molecules
− ES-62 is produced by a rodent nematode
− Known to cause immunomodulatory effects
− However, people don’t tend to want to be injected with parasite protein, so a group has cleaved it into small peptides and used this to identify the bit that has the activity
− Proven quite successful in terms of inhibiting allergic contact dermatitis.

Question 46

What is the role of IgE in chemical respiratory allergy?

Answer 46

• Highly diagnostic if present
• Relatively strong associated for some chemicals, eg) acid anhydrides and platinum salts
• However, other chemicals have an association of

Question 47

What is the shape of the immune response in chemical respiratory allergy?

Answer 47

We believe that respiratory allergy is a Th2 mediated response.
• IL-4 → IgE production
• IL-13 → hypersecretion of mucin
• IL-5 → activation and recruitment of eosinophils
• IL-4/IL-5 → differentiation and activation of mast cells

• Can prove this with experimental models in BALB/c mice:
− Graphs show contact allergens are activating type 1 cytokines, but respiratory allergens type 2 cytokines
• As with the contact allergens, this could be coming from Th2 cells or Tc2 cells
− The evidence suggests that respiratory allergens are activating Th2 cells
− It has been reported recently that in patients with occupation asthma, the IFN gene promoter is hypermethylated, giving reduced potential for IFN expression. This is consistent with preferential Th2 responses.

Why do contact allergens activate Th1, but respiratory Th2?

Role of DCs in the development of the T cell phenotype:
• In vitro work has suggested that DCs can develop down a DC1 or DC2 pathway depending on the context in which they experience the danger signals or antigen.
− Certain bacterial products such as CpG, RNA or IFNa that drive them down the DC1 pathway
− Products associated with parasites or histamine drive them down the DC2 pathway
− Once driven down a pathway, they will produce the cytokines that give signal 3 to drive Th1 or Th2 respectively.

Question 48

What is the LC migratory pathway like in chemical respiratory allergen as opposed to contact dermatitis?

Answer 48

• 4 hours after exposure, contact allergen is inducing migration
• It isn’t until 17 hours later that you see LC migration after the respiratory allergen
• Was subsequently shown that with the respiratory allergen, within half an hour of exposure, there was an upregulation of IL-10
− This IL-10 serves to restrain the LCs within the epidermis, stopping them leaving the skin quickly
− This can be reversed with anti-IL10
− There is no effect of IL-10 with DNCB
• Believed that this is because if you delay migration of LCs, you get Langerhan cell exhaustion – if they have been activated and maintained in the skin, they cant continue to produce the cytokines
− They downregulate their ability to produce IL-12, the key cytokine for Th1
− So the delayed LCs go down the Th2 pathway