**3. The allergy tests in diagnosis of AEDS**

162 Atopic Dermatitis – Disease Etiology and Clinical Management

The alteration of the epidermal structure due to filaggrin mutation induces a significant reduction of the lipidic component, particularly of ceramide levels (11) that leads to the well known phenomenon of trans-epidermal water loss (TEWL) resulting in dry skin and itching, while the impaired skin barrier associated with filaggrin deficiency favours the penetration

Concerning allergens, it is conceivable that their facilitated access favours the sensitization process, especially for house dust mites, the protease activity of their major allergen being a further enhancing factor (13). Once entered, allergens are captured by dendritic cells (14) that activate an initially local Th2 but a later Th1 response along with a systemic Th2 response inducing the Ig isotype switching to IgE synthesis and the involvement of eosinophils (2). In this kind of response an important factor seems to be thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine expressed by barrier epithelial cells able to activate myeloid-derived dendritic cells, macrophages and mast cells (15). The ongoing inflammatory process is then sustained by the Th2-related cytokine such as IL-5, IL-13, TNF-alpha, IL-17, and IL-31, the latter being primarily expressed in skin-homing Th2 cells (16). An important clinical aspect of the allergen-caused AEDS is the frequent evolution of manifestations to respiratory symptoms such as asthma and rhinitis. This process has been defined as the "atopic march" (17). Also in this case, filaggrin null mutation were found to be associated with the development of asthma (18). However, more than one model of atopic march was reported, because in a significant number of children asthma precedes AEDS as the onset manifestation of the atopic disease (19). Of note, in subjects with AEDS and no filaggrin gene mutations, the cytokines IL-4 and IL-13 (typical of the Th2 profile) are able to inhibit the expression of filaggrin (20). This suggests that if filaggrin deficiency predisposes to atopy, atopy is also

Regarding microbes, the normal skin defence is based on integrity of *stratum corneum* and on immune response by neutrophils and macrophages by production of substances which kill the microbes and by phagocytosis. Numerous antimicrobial peptides produced by keratinocytes and belonging to the classes of beta-defensins and cathelicidins are able to disrupt or penetrate the microbe membrane thus protecting the skin from infections (21). It has been demonstrated that in patients with AEDS there is a deficiency in the expression of beta-defensins and cathelicidins that may account for the susceptibility to skin infection, especially from *Staphilococcus aureus* (22). *S. Aureus* has a major role in AEDS, as indicated by the following features: 1) a very high density of colony-forming units *S. aureus* per cm2 of inflamed atopic skin lesions (23); 2) the higher affinity for *S. aureus* of the atopic skin compared with nonatopic or psoriatic skin (24); 3) the reduction of *S. aureus* counts on atopic skin sites following effective topical treatment (25). In any form of AEDS, an additional pathophysiologic role is played by the cytokine IL-31 produced by keratinocytes, which exerts a potent pruritogenic effect (26). In fact, pruritus is associated with skin lesions caused by scratching or excessive washing and to consequent damage of keratinocytes and release of mediators, but also of autoantigens, and generation of autoantibodies (27). Moreover, autoreactivity phenomena also concur to pathogenesis of AEDS. In particular, manganese superoxide dismutase (MnSOD) of both human and foreign origin – especially from the long known *Malassezia spp* yeast (28) – may act as autoallergen. Specific IgE against human MnSOD correlating with the disease activity were detected in patients with AEDS, suggesting that human MnSOD with molecular mimicry with MnSOD from *Malassezia* may play a role, as showed by its

of foreign noxae, especially allergens and microbes (12).

likely to impair the filaggrin-dependent skin barrier.

When food allergens are the cause of AEDS, the commonly used allergy tests, such as skin prick tests and in vitro measurement of specific IgE antibodies, have a role in the diagnostic work-up (30). Concerning food-specific IgE measurement, an useful application was suggested by Sampson and Ho, who identified in a group of 196 children and adolescents with AEDS the food-specific IgE levels predicting a positive result of a double-blind, placebo-controlled food challenge. Such levels, showing a positive predictive value higher than 95%, corresponded to 6 kU/L for egg, 32 kU/L for milk, 15 kU/L for peanut, and 20 kU/L for fish (31). The same author later confirmed the utility of specific IgE concentrations in predicting symptomatic allergy also for other foods such as wheat and soy (32). However, the advances in the knowledge of pathophysiology of AEDS, and particularly the understanding that in this disease the mechanisms of delayed hypersensitivity prevail, suggested the need of new diagnostic tools. The atopy patch test (APT) was recently defined as an important tool in diagnosis of AEDS, because it seems to have a greater significance than skin prick test or RAST, which simply detect the presence of specific IgE antibodies. Thus, allergy tests assessing only the immediate IgE-mediated phase of the allergic response can only partially detect the operating mechanisms. Instead, there is notable evidence supporting the capacity of the APT to reproduce the pathophysiologic events of AEDS.

In biopsy-based studies, a Th2 cytokine pattern was found 24 hours after APT, but a shift to a Th1 pattern, as occurs in chronic AEDS skin lesions, was noted after 48 hours (33, 34). A more frequent positivity to APT was reported in patients with allergen-specific lymphocyte proliferation and expression of activation markers on peripheral blood T-cells following *in vitro* stimulation with house dust mite, cat or grass pollen allergens, than in patients without lymphocyte proliferation (35). Application of the APT to skin of subjects with AEDS was followed by an influx of inflammatory dendritic epidermal cells (36). A significant increase of TEWL was reported in the site of the APT application, both after 48 and 72 hours, compared with the control skin site (37). By immunohistochemical analysis, the presence of IgE on Langerhans cells was demonstrated in positive APT reactions to *Dermatophagoides* in patients with mite-associated AEDS (38).

Clinically, patients with a diagnosis of intrinsic AEDS because of negative IgE tests actually had a positive APT for dust mites (39). This aspect is of particular interest, because AEDS patients with negative SPT and IgE measurement in serum should be defined as nonatopic unless APT is performed. A number of studies evaluated how common such patients are, with different observations. In one study the rate of positive APT in nonatopic patients was 23% (40), while in another study comparing AEDS patients with extrinsic and intrinsic forms, the rate of positive APT was 47.4% and 66.6%, respectively (41). In a European multicenter study, which included 314 patients with AEDS, the frequency of clear-cut positive APT reactions ranged from 39% with dust mites to 9% with celery. A notable observation from the study was that positive APT in face of all SPT and sIgE testing negative was found in 7% of the patients, whereas a positive APT without SPT or sIgE for the respective allergen was seen in 17% of the patients (42). This

Atopic Dermatitis: From Pathophysiology to Diagnostic Approach 165

reaction initiated by immunologic mechanisms" (50) and includes all known mechanisms,

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as a replacement for atopy.

**4. References** 

S480.

lead the authors to conclude that, as no gold standard for aeroallergen provocation in AEDS exists, the relevance of aeroallergens for AEDS may be evaluated by APT in addition to SPT and sIgE. Moreover, in children with respiratory symptoms an exclusive positivity to APT with dust mites was observed (43). On the other hand, it was reported that in 63 children with mite-induced asthma and rhinitis, all with positive SPT and sIgE in serum, 16 (25%) were positive to mite APT too, indicating that delayed hypersensitivity reactions were involved (44).

These observations lead us to investigate the possible factors underlying the positive result of APT in subjects with respiratory symptoms. In our first study, conducted on 297 children (45), we could demonstrate that in patients with asthma or rhinitis a positive APT to dust mite was strongly associated with the presence of current or past AEDS. Instead, most subjects with respiratory disease but a negative history for AEDS had a positive SPT. Multivariate analysis showed that there was a high probability of a positive APT result in patients with AEDS (odds ratio 17.4), in patients with AEDS and respiratory disease (odds ratio 21.9), and in patients with past AEDS and respiratory disease (odds ratio 22.8). These observations were confirmed in a study on a large population of 465 children aged 0.4 to 17.6 years. They were divided into four groups: group A, current AEDS (40 patients); group B, current AEDS with respiratory symptoms (156 patients); group C, past AEDS with respiratory symptoms (203 patients); and the control group, respiratory symptoms with no history of AEDS (66 patients). The APT was significantly more frequently positive in groups with current AEDS (groups A and B) or past AEDS (group C) than in the control group, while SPT and RAST were significantly more frequently positive in the control group (46). Such significant differences in response to APT in patients with diverse clinical expressions suggest that distinctive immunologic mechanisms lie beneath the different manifestations of hypersensitivity to dust mites. It seems conceivable that in subjects with a negative history for AEDS sensitization occurs by respiratory route and leads to the development of a Th2 pattern of response with ongoing production of specific IgE and consequent positive SPT and in vitro IgE tests. By contrast, in the case mite allergens enter through the skin, as it occurs in exposure to common indoor concentrations of the major allergen Der p 1 (47), such entering being facilitated by its proteolytic activity and in the presence of a filaggrin-dependent skin barrier dysfunction, a different chain of events is likely to take place. This is ultimately revealed by positive APT and negative SPT and *in vitro* IgE tests.

The recent observations on the diagnostic significance of APT in patients with different clinical expressions of the disease highlighted the importance of delayed hypersensitivity in AEDS. This brings into question the role of simple IgE sensitization in AEDS and also the appropriateness of the term atopic when applied to AEDS. In fact, current evidence shows that up to two thirds of patients with AEDS are not atopic, therefore even to continue using the term atopic dermatitis is to be considered problematic (48, 49). In fact, the definition of atopy as "a personal or familial tendency to produce IgE antibodies in response to low doses of allergens" (50) seems not to be appropriate for AEDS, as many patients show a positive result to APT but not to IgE tests. At the same time, the definition atopy patch test seems unfounded, because the test does not reveal atopy, i.e. a type I hypersensitivity, but a type IV hypersensitivity according to Gell and Coombs classification (51). A unifying solution should be the use, in both cases, of the term allergy, which is defined as "a hypersensitivity reaction initiated by immunologic mechanisms" (50) and includes all known mechanisms, as a replacement for atopy.
