**2. Trigger factors, allergens and allergy testing in atopic dermatitis**

The rate of increase in prevalence in AD in recent years is too rapid to be accounted for by changes in population genetics. Therefore environmental factors are the most likely modulating influences. The two principal aspects that have attracted attention are pollution and microbes. The identification of relevant pollutants that might contribute to the expression of atopic phenotype is still confused. Interaction with environmental microbes may be important in the causation of AD in a number of ways. Early-life exposure may condition maturation of the immune system so that apparent dysregulation associated with IgE production and allergy formation does not occur. Significant differences have been observed in the prevalence of allergies between urban and rural population within one country and observations confirm greater allergy sensitization in first-born and a lower frequency in children from large families. Evidence suggests that microbes entering via fecal-oral route have a greater protective effect against the development of atopic disease than those entering via the respiratory route (Burns et al., 2004).

genetically determined primary defect probably affecting the skin barrier thus playing a key role in the development of AD (Burns et al., 2004). The integrity of the skin barrier involves several components, including regulation of proteolysis of corneodesmosomes, the lipid lamellae and generation of natural moisturizing factor (MNF) from the breakdown products of the structural protein filaggrin. An imbalance of these components makes the epidermal barrier more susceptible to irritants, which in turn can lead to a disturbance of skin barrier function allowing penetration of microbes and allergens into the epidermis and dermis interdependence (Reitamo et al., 2008). In a recent meta-analysis filaggrin gene defects were shown to increase the risk of developing allergic sensitization, AD, and allergic rhinitis. The presence of filaggrin gene mutations correlated strongly with disease severity and treatment failure, and also increased the risk of asthma in AD patients (Van den Oord & Sheikh, 2009). IgE is composed of two identical heavy and light chains, that form the constant Fc domain and the antigen-binding sites, through which the IgE molecule binds to its cell surface receptors. Raised serum IgE levels are most commonly seen in parasitic infections as a defensive response. IgE mediated hypersensitivity is largely regulated by T lymphocytes. AD is generally thought to be due to an imbalance between Th1 and Th2 cells, with a Th2 cell predominant immune profile in its active stage. Th2 cells produce interleukin (IL) -4, IL-5, IL-6 and IL-13, which induce IgE production and activation of eosinophils, producing the

In general various cells contribute to the underlying pathomechanism of AD including Langerhans cells (LC), macrophages, T cells, B cells, keratinocytes, endothelial cells, eosinophils and mast cells. These cells communicate with each other in Th2 predominant cytokines as well as chemokines, prostanoids, proteases and reactive oxygen species

A number of environmental factors can trigger and perpetuate the inflammatory skin cascade in AD including irritants, foods, aeroallergens, infection. All four classic Coombs' classification types of hypersensitivity (type I-IV) have been implicated in the pathophysiology of AD, including pseudo allergy. The hypersensitivity types can occur either alone or in combination in the AD individual. In the extrinsic type of AD high levels of specific IgE antibodies and raised total IgE levels in peripheral blood have been significantly associated with severity of dermatitis (Reitamo et al., 2008 & Burns et al., 2004).

The rate of increase in prevalence in AD in recent years is too rapid to be accounted for by changes in population genetics. Therefore environmental factors are the most likely modulating influences. The two principal aspects that have attracted attention are pollution and microbes. The identification of relevant pollutants that might contribute to the expression of atopic phenotype is still confused. Interaction with environmental microbes may be important in the causation of AD in a number of ways. Early-life exposure may condition maturation of the immune system so that apparent dysregulation associated with IgE production and allergy formation does not occur. Significant differences have been observed in the prevalence of allergies between urban and rural population within one country and observations confirm greater allergy sensitization in first-born and a lower frequency in children from large families. Evidence suggests that microbes entering via fecal-oral route have a greater protective effect against the development of atopic disease

products. High affinity IgE receptors play a crucial role in promoting this process.

**2. Trigger factors, allergens and allergy testing in atopic dermatitis** 

than those entering via the respiratory route (Burns et al., 2004).

acute signs and symptoms of AD.

AD has a Th2 cell predominant immune profile in its active stage. Th2 cells produce IL-4, IL-5, IL-6, IL-13, which induce IgE production and activation of eosinophils, producing the acute signs and symptoms of AD. Genetic factors, reduced bacterial stimulation in early infancy and a disruption in skin barrier and function are thought to contribute to the Th1-Th2 cell imbalance of skin in AD. Eczematous skin lesions evolve as a result of complex interactions between IgE bearing antigen presenting cells, T cell activation, mast cell degranulation, keratinocytes, eosinophils, and a combination of immediate and cellular immune response. A number of environmental factors have been reported to induce and perpetuate this inflammatory response of the skin including food and aeroallergens, microbes and irritants. All four classic types of hypersensitivity reactions have been implicated in the pathophysiology of AD including pseudo allergy. They can occur either alone or in combination in AD individuals (Reitamo et al., 2008 & Burns et al., 2004).

AD is a common chronic skin condition associated with high morbidity and major public health implications. As prevention of disease is not yet a real option, reducing morbidity is main aim of treatment. Identifying the underlying pathophysiology of the individual's AD is very crucial. To date no standard test is available to diagnose AD. Sensitization to various allergens is a major part of triggering and perpetuating the inflammatory skin response in AD. Various tests have been developed to investigate the underlying type(s) of hypersensitivity reaction(s) involved in AD patients. None of the available tests so far have proven sensitive and specific enough to identify reliably relevance between clinical disease and test result. Precise understanding of these tests including their limitations together with accurate correlation of patient symptoms and signs are required in order to differentiate between allergy, intolerances and hypersensitivities, and achieve an appropriate clinical diagnosis (Robinson & Smart, 2008).

Allergy tests commonly used in practice include measurement of total levels of IgE and allergen specific IgE levels in serum (radio-allergosorbent test/RAST), skin prick testing (SPT) and atopy patch testing (APT). A different positive predictive cut off point exists for each allergen tested respectively and similar test results do not imply a similar clinical reaction to each allergen (Robinson & Smart, 2008).

SPT is performed to detect the presence of allergen specific IgE to foods, aeroallergens, antibiotics and latex. A drop of a solution containing the allergen is applied to the skin and a lancet used to prick the skin. The allergen binds to IgE on mast cells causing degranulation and the release of histamine. This manifests as a weal and flare reaction, the diameter of which can be measured. SPT is easy to perform and results are immediately available. It can trigger off allergic reactions including anaphylaxis. Various medications including antihistamines, H2-antagonists, tricyclic antidepressants and neuroleptics can interfere with SPT and need to be withheld prior to testing. A positive SPT result indicates only sensitization and does not always equate to clinically relevant allergy. It must be interpreted in the context of clinical history, clinical signs and allergen exposure. The size of the weal does not necessarily correlate with severity of clinical reaction (Robinson & Smart, 2008 & Goodwin, 2008). The RAST detects free allergen specific IgE in serum. It is less sensitive and specific than SPT and is particularly useful where SPT is contraindicated. Testing is available for a wide range of food and environmental allergens. The RAST results are not affected by prior drug use and can be performed in patients with widespread skin disease. The RAST can be reported semi quantitatively as a score or as quantitative measurement by using the CAP RAST technology. The process involves an allergy-impregnated disc being incubated with patient's serum. Allergen specific IgE, if present binds to allergen. The disc is

Trigger Factors, Allergens and Allergy Testing in Atopic Dermatitis 217

(HDM), dust mites, HDM feces, animal dander (cat and dog), molds and pollens. Sensitization to aeroallergens appears to be more prevalent in older children and adults, leading to aggravation of AD after bronchial inhalation. Allergen specific T- cells have been identified in affected skin of AD to grass pollen, birch pollen and HDM supporting the concept of a T-cell mediated specific immune response to inhalant allergens with clinical implications. Sensitization to inhalant allergens is thought to occur via skin as well as respiratory route. Allergens penetrate the disturbed epidermal barrier fix on the specific IgE present on LCs and activate them. The activated LCs migrate to a lymph node and present the allergen to T cells, provoking an eczematous reaction. The appearance of eczematous skin lesions is due to cytotoxic T-cell activity and not to the aeroallergen specific IgE. Repeated epicutaneous allergen application has been shown to elicit eczematous lesions in

AD individuals (Reitamo et al., 2008 & Goodwin, 2008).

Mite allergen activates LCs via IgE fixation Documented presence of HDM on skin

Induction of eczematous lesions following APT

Flare up of AD following allergen inhalation

Table 2. Routes of sensitization aeroallergens in AD

Induction of new eczematous skin lesions upon allergen inhalation

lacking the allergens to which they react (Reitamo et al., 2008& Burns et al., 2004).

Mites belong to the most potent allergen sources and are considered a perennial allergen. Thirteen mite species have been identified in house dust so far, the two commonest mites in homes worldwide include *Dermatophagoides pteronyssimus* and *Dermatophagoides farinae.* Beds and overstuffed furniture are main foci for breeding dust mites. Airborne level of mite allergen during sleep in bedroom is tenfold higher than that found in living rooms of same house during daily activities. Mite allergens persist for months after death of mite. Exposure to high doses of mite allergen in early infancy has been associated with higher incidence of developing eczema. Efforts to create dust mite free environment yielded significant

The respiratory route in the induction of new skin lesions may be relevant in a subset of patients with concomitant allergic asthma. Early studies reported that inhalation of house dust or pollen extract can provoke exacerbations of AD. Skin lesions have also been shown to develop in AD individuals after placebo-controlled bronchial provocation of HDM, cat allergen or tree pollen. Aggravation of eczema is more pronounced in AD individuals with concomitant allergic asthma than in patients suffering from AD alone. Two possible explanations have been offered for this phenomenon. Allergen exposed in airways enters the circulation and is transported to skin activating there the inflammatory cascade. Another possibility is allergen induced airway inflammation causes release of mediators from inflammatory cells in skin already primed for AD. Substantial clinical improvement has been reported to occur when sensitized AD individuals are exposed to environments

**Transcutaneous route** 

**Respiratory route** 

then incubated with radio-labeled anti IgE and radioactivity measured to give level of specific IgE present to particular allergen. Positive RAST results indicate the presence of IgE to an allergen or cross reacting allergen, and are difficult to interpret in the presence of high levels of total IgE (>1000kU/L). Results therefore must always be interpreted in the context of clinical history (Robinson & Smart, 2008 & Goodwin, 2008).

APT is epicutaneous application of inhalant and food allergens on unaffected skin of AD individuals eliciting a delayed, type IV hypersensitivity reaction. It was traditionally developed to investigate association between AD and allergy to aeroallergens and proved in that context to have a higher specificity when compared to SPT and RAST. More recently the APT has been used to supplement the SPT in the diagnosis of food allergy, in an attempt to identify delayed reactions to food products. Reproducibility of APT results raises unsolved issues and is to date poor for most food allergens and good for inhalant allergens. Variables that can strongly influence results are allergen concentration, skin site and devices used for allergen application as well as reading time and/or criteria for defining positive reactions. These variables seem to be specific for each allergen (Lipozencić & Wolf, 2010).


#### Table 1. Factors affecting reproducibility of APT

Patch testing (PT) is an epicutaneous and the gold standard test in the diagnosis of contact allergic dermatitis (CAD). It is not a routine investigation for the diagnosis of AD, but ideal to rule out contact allergy (e.g. to topical medication) in the presence of AD. Recent evidence suggests that CAD is as common in AD individuals as in the general population and PT is recommended in AD patients with recalcitrant disease, hand eczema and in children (Fonacier & Aquino, 2010).

Oral food challenges are the "gold standard" for the diagnosis of food allergy. They can be open, single or double blinded, or placebo controlled. A range of vehicles can be used to disguise food (liquids or solid). Tolerance of a serving size of a food is generally considered evidence of lack of reactivity. In AD oral food challenges should be performed in a doubleblind placebo-controlled manner (Robinson & Smart, 2008).

Negative RAST, SPT and APT results have a high negative predictive value and are fairly reliable methods of excluding allergy. Positive results however require careful consideration and correlation with clinical history and clinical presentation (Robinson & Smart, 2008 & Goodwin, 2008).

#### **2.1 Aeroallergens**

Clinical manifestations of allergy to aeroallergens involve allergic rhinitis, asthma and exacerbation of AD. Patients with atopic predisposition can express one or all of these clinical manifestations. Filaggrin gene defects have been shown to increase the risk of developing allergic sensitization, AD, and allergic rhinitis (Van den Oord & Sheikh, 2009). A variety of inhalants have been implicated in exacerbations of AD including house dust mite (HDM), dust mites, HDM feces, animal dander (cat and dog), molds and pollens. Sensitization to aeroallergens appears to be more prevalent in older children and adults, leading to aggravation of AD after bronchial inhalation. Allergen specific T- cells have been identified in affected skin of AD to grass pollen, birch pollen and HDM supporting the concept of a T-cell mediated specific immune response to inhalant allergens with clinical implications. Sensitization to inhalant allergens is thought to occur via skin as well as respiratory route. Allergens penetrate the disturbed epidermal barrier fix on the specific IgE present on LCs and activate them. The activated LCs migrate to a lymph node and present the allergen to T cells, provoking an eczematous reaction. The appearance of eczematous skin lesions is due to cytotoxic T-cell activity and not to the aeroallergen specific IgE. Repeated epicutaneous allergen application has been shown to elicit eczematous lesions in AD individuals (Reitamo et al., 2008 & Goodwin, 2008).

#### **Transcutaneous route**

216 Atopic Dermatitis – Disease Etiology and Clinical Management

then incubated with radio-labeled anti IgE and radioactivity measured to give level of specific IgE present to particular allergen. Positive RAST results indicate the presence of IgE to an allergen or cross reacting allergen, and are difficult to interpret in the presence of high levels of total IgE (>1000kU/L). Results therefore must always be interpreted in the context

APT is epicutaneous application of inhalant and food allergens on unaffected skin of AD individuals eliciting a delayed, type IV hypersensitivity reaction. It was traditionally developed to investigate association between AD and allergy to aeroallergens and proved in that context to have a higher specificity when compared to SPT and RAST. More recently the APT has been used to supplement the SPT in the diagnosis of food allergy, in an attempt to identify delayed reactions to food products. Reproducibility of APT results raises unsolved issues and is to date poor for most food allergens and good for inhalant allergens. Variables that can strongly influence results are allergen concentration, skin site and devices used for allergen application as well as reading time and/or criteria for defining positive reactions.

Patch testing (PT) is an epicutaneous and the gold standard test in the diagnosis of contact allergic dermatitis (CAD). It is not a routine investigation for the diagnosis of AD, but ideal to rule out contact allergy (e.g. to topical medication) in the presence of AD. Recent evidence suggests that CAD is as common in AD individuals as in the general population and PT is recommended in AD patients with recalcitrant disease, hand eczema and in children

Oral food challenges are the "gold standard" for the diagnosis of food allergy. They can be open, single or double blinded, or placebo controlled. A range of vehicles can be used to disguise food (liquids or solid). Tolerance of a serving size of a food is generally considered evidence of lack of reactivity. In AD oral food challenges should be performed in a double-

Negative RAST, SPT and APT results have a high negative predictive value and are fairly reliable methods of excluding allergy. Positive results however require careful consideration and correlation with clinical history and clinical presentation (Robinson & Smart, 2008 &

Clinical manifestations of allergy to aeroallergens involve allergic rhinitis, asthma and exacerbation of AD. Patients with atopic predisposition can express one or all of these clinical manifestations. Filaggrin gene defects have been shown to increase the risk of developing allergic sensitization, AD, and allergic rhinitis (Van den Oord & Sheikh, 2009). A variety of inhalants have been implicated in exacerbations of AD including house dust mite

These variables seem to be specific for each allergen (Lipozencić & Wolf, 2010).

of clinical history (Robinson & Smart, 2008 & Goodwin, 2008).

• Allergen concentration not standardized

Table 1. Factors affecting reproducibility of APT

• Differences in skin sites and pre-treatment of skin • Differences in reading time and duration of patch testing

blind placebo-controlled manner (Robinson & Smart, 2008).

• Different vehicles used

(Fonacier & Aquino, 2010).

Goodwin, 2008).

**2.1 Aeroallergens** 

Mite allergen activates LCs via IgE fixation Documented presence of HDM on skin Induction of eczematous lesions following APT

#### **Respiratory route**

Flare up of AD following allergen inhalation Induction of new eczematous skin lesions upon allergen inhalation

#### Table 2. Routes of sensitization aeroallergens in AD

The respiratory route in the induction of new skin lesions may be relevant in a subset of patients with concomitant allergic asthma. Early studies reported that inhalation of house dust or pollen extract can provoke exacerbations of AD. Skin lesions have also been shown to develop in AD individuals after placebo-controlled bronchial provocation of HDM, cat allergen or tree pollen. Aggravation of eczema is more pronounced in AD individuals with concomitant allergic asthma than in patients suffering from AD alone. Two possible explanations have been offered for this phenomenon. Allergen exposed in airways enters the circulation and is transported to skin activating there the inflammatory cascade. Another possibility is allergen induced airway inflammation causes release of mediators from inflammatory cells in skin already primed for AD. Substantial clinical improvement has been reported to occur when sensitized AD individuals are exposed to environments lacking the allergens to which they react (Reitamo et al., 2008& Burns et al., 2004).

Mites belong to the most potent allergen sources and are considered a perennial allergen. Thirteen mite species have been identified in house dust so far, the two commonest mites in homes worldwide include *Dermatophagoides pteronyssimus* and *Dermatophagoides farinae.* Beds and overstuffed furniture are main foci for breeding dust mites. Airborne level of mite allergen during sleep in bedroom is tenfold higher than that found in living rooms of same house during daily activities. Mite allergens persist for months after death of mite. Exposure to high doses of mite allergen in early infancy has been associated with higher incidence of developing eczema. Efforts to create dust mite free environment yielded significant

Trigger Factors, Allergens and Allergy Testing in Atopic Dermatitis 219

aeroallergens, are the main tests available for investigation. Using these tests in combination and in consideration of clinical history and signs is likely to yield a higher clinically relevant result. Negative results of above investigations are fairly reliable methods of excluding allergy.

The prevalence of AD and food allergies (FA) appears to have increased in recent years in many western countries. In this context it is important to define the terms. FA and food hypersensitivity (FH) indicate an adverse clinical reaction to food due to interaction of food proteins with one or more immune mechanisms. Food intolerance (FI) is the result of non-

immunological reactions to foods and food additives (Reitamo et al., 2008).

• Food allergy involving other immunological mechanisms ( type IV)

• Food allergy due to IgE mediated mechanism (Coombs' classification, type I)

• Non-allergic food intolerance (pharmacological, metabolic, or toxic reaction to food) • Food aversion (often non-specific symptoms, unconfirmed by blinded food

FA has the greatest incidence in infancy and early childhood. Around 8% of children are thought to develop adverse reactions to food, most of them within the first year of life. The prevalence of FA varies between regions and is influenced by culture and genetic factors. The spectrum of food allergens has remained relatively unchanged despite the rise of FA incidence. Seven food items account for 90% of FA in children. These include cow's milk, egg, peanuts, soybeans, wheat, fish, and tree nuts. By three years of age the majority of children will have developed tolerance to these food items with the exception of peanuts/ tree nuts and seafood. In adults, IgE mediated FA is rare. The main food allergens in adults

Infants and children Adults

In general high-protein food allergens are considered to be more allergenic. Most food allergens are water-soluble glycoproteins that are particularly resistant to food processing,

Peanut Tree nuts Fish Shellfish

**2.2 Food allergens** 

challenge)

Table 5. Types of adverse reactions to foods

are peanuts, tree nuts, fish and shellfish (Kim, 2008).

Cow's milk Egg Peanut Tree nuts Soy Wheat Fish

Table 6. Major food allergens in children and adults

cooking and digestion.

improvement in clinical symptoms although not complete remission. AD patients not sensitized or not exposed to mites benefited as much from exposure to mite free environment as those with mite sensitization and/or exposure.


Table 3. Factors implicating aeroallergens underlying pathomechanism of AD

Many AD patients are highly sensitized to aeroallergens and experience precipitation of AD upon epicutaneous or bronchial allergen exposure. RAST and SPT help to verify or exclude the presence of IgE mediated hypersensitivity, by measurement of allergen specific IgE in serum and evaluation of IgE bound on mast cells. Neither test procedure considers those AD patients characterized by cell-mediated sensitization. The APT was established as a provocation test for a subgroup of AD patients to study the induction of eczema by aeroallergens after 24-72 hours. It is similar to nasal or bronchial provocation test in allergic rhinitis and asthma. Allergen specific IgE is not obligatory for a positive APT reaction. APT has shown a higher specificity towards inhalants (69-92%) when compared to SPT (44-53%) and serum specific IgE (42-64%), suggesting a more appropriate screening role for SPT and RAST in this context. APT does not replace the classical methods of diagnosis of IgEmediated allergy. APT are usually negative in individuals with respiratory allergy and healthy subjects pointing to AD specific triggering mechanisms required to produce APT positivity. Much effort has been undertaken to standardize APT procedures. The protocol of the European task Force on Atopic Dermatitis (ETFAD) provides a standardized APT technique (Ronchetti et al., 2008).

Despite above impressive data on APT as a diagnostic tool, final scientific proof for the relevance of aeroallergens as identified by positive APT, for clinical manifestation of AD is still missing. The question on how well APT can identify AD patients who would benefit from allergen avoidance remains to be answered.


#### Table 4. ETFAD consensus on APT indications

In summary sensitization to inhalant allergens is a major contributing factor to the pathogenesis and perpetuation of AD. It is found more commonly in older children and later life of AD individuals and is demonstrated by presence of aeroallergen specific IgE and positive SPT to aeroallergens. Bronchial inhalation challenge, RAST, SPT, and APT to aeroallergens, are the main tests available for investigation. Using these tests in combination and in consideration of clinical history and signs is likely to yield a higher clinically relevant result. Negative results of above investigations are fairly reliable methods of excluding allergy.
