Diagnosis of dog allergy in children : molecular assessment and refined characterization

Dog allergy is a common cause of rhinitis and asthma in children, yet the diagnosis is a clinical challenge. Allergic sensitization, i.e. the presence of serum IgE antibodies, to dog dander affect up to 30 % of all children and adolescents, but not all sensitized children display symptoms. The most important diagnostic tool, the detection of IgE antibodies to dog dander extracts in serum does not reveal which allergen molecule in the extract that gives rise to the allergic sensitization and symptoms. Through molecular allergy diagnostics it is now possible to detect allergic sensitization to specific allergen molecules from dog, but the clinical relevance of sensitization to the different dog allergen molecules is not yet clear. When our investigations were initiated in 2014, there were six recognized dog allergen molecules, Can f 1- Can f 6, of whom Can f 1, Can f 2, Can f 4 and Can f 6 belong to the lipocalin protein family. Can f 3 is the dog serum albumin, and Can f 5 is the male dog allergen prostatic kallikrein. The overall aim of this doctoral thesis was to improve diagnostics of dog allergy by identifying patterns of sensitization to dog allergen molecules associated with rhinitis and asthma in dog dander sensitized children and by exploring novel biomarkers and complementary diagnostic tests for dog allergy. In paper I, we found that a positive nasal provocation test with dog dander extract was associated with an increasing number of positive sensitizations to dog allergen molecules and with sensitization to allergens from the lipocalin protein family. When investigating the impact of the different allergens, we found that sensitization to Can f 3, Can f 4 and Can f 6 conferred an increased risk for a positive vs a negative nasal challenge. On the contrary, monosensitization to Can f 5 was associated with a negative nasal provocation test. In paper II, we showed that the basophil activation tests to allergen molecules, evaluated by the basophil allergen threshold sensitivity (CD-sens), were positive in a majority of the sensitized children with a positive, as well as in those with a negative nasal provocation test. However, the levels of CD-sens to dog dander and to Can f 1 were higher in children with a positive nasal provocation. The levels of IgG or IgG4 to the investigated allergens did not differ between sensitized children with a positive and a negative nasal provocation test, while sensitized children with a dog at home had higher levels of IgG4 to Can f 1 and Can f 5 and lower CD-sens to all investigated allergen molecules. In paper III, we performed nasal transcriptomic analysis in dog dander sensitized children and healthy controls. The most over-expressed gene in dog dander sensitized children was CST1, coding for Cystatin 1. CST1 expression was enhanced in a cluster of children with lower FEV1, increased bronchial hyperreactivity, pronounced eosinophilia and higher CD-sens to dog compared with other dog dander sensitized children. Finally, in paper IV, we showed that asthma in dog dander sensitized children was associated with multisensitization to furry animal allergen molecules and to lipocalins. Children with severe asthma had higher IgE levels to the dog lipocalins Can f 2, Can f 4 and Can f 6 than other dog dander sensitized children. Moreover, severe asthma was associated with symptoms of dog allergy evaluated by nasal provocation testing. In conclusion, we demonstrate that a detailed assessment using molecular allergy diagnostics may help clinicians to assess the impact of allergic sensitization on dog allergy and asthma morbidity. We found that multisensitization to dog allergens and sensitization to lipocalins is associated with dog allergy and that the analysis of CD sens, IgG4 antibodies and nasal gene expression may provide further information in the diagnosis of this common disease

Molecular Allergen-Specific IgE Recognition Profiles and Cumulative Specific IgE Levels Associated with Phenotypes of Cat Allergy

Cat allergy is a major trigger factor for respiratory reactions (asthma and rhinitis) in patients with immunoglobulin E (IgE) sensitization. In this study, we used a comprehensive panel of purified cat allergen molecules (rFel d 1, nFel d 2, rFel d 3, rFel d 4, rFel d 7, and rFel d 8) that were obtained by recombinant expression in Escherichia coli or by purification as natural proteins to study possible associations with different phenotypes of cat allergy (i.e., rhinitis, conjunctivitis, asthma, and dermatitis) by analyzing molecular IgE recognition profiles in a representative cohort of clinically well-characterized adult cat allergic subjects (n = 84). IgE levels specific to each of the allergen molecules and to natural cat allergen extract were quantified by ImmunoCAP measurements. Cumulative IgE levels specific to the cat allergen molecules correlated significantly with IgE levels specific to the cat allergen extract, indicating that the panel of allergen molecules resembled IgE epitopes of the natural allergen source. rFel d 1 represented the major cat allergen, which was recognized by 97.2% of cat allergic patients; however, rFel d 3, rFel d 4, and rFel d 7 each showed IgE reactivity in more than 50% of cat allergic patients, indicating the importance of additional allergens in cat allergy. Patients with cat-related skin symptoms showed a trend toward higher IgE levels and/or frequencies of sensitization to each of the tested allergen molecules compared with patients suffering only from rhinitis or asthma, while there were no such differences between patients with rhinitis and asthma. The IgE levels specific to allergen molecules, the IgE levels specific to cat allergen extract, and the IgE levels specific to rFel d 1 were significantly higher in patients with four different symptoms compared with patients with 1–2 symptoms. This difference was more pronounced for the sum of IgE levels specific to the allergen molecules and to cat extract than for IgE levels specific for rFel d 1 alone. Our study indicates that, in addition to rFel d 1, rFel d 3, rFel d 4, and rFel d 7 must be considered as important cat allergens. Furthermore, the cumulative sum of IgE levels specific to cat allergen molecules seems to be a biomarker for identifying patients with complex phenotypes of cat allergy. These findings are important for the diagnosis of IgE sensitization to cats and for the design of allergen-specific immunotherapies for the treatment and prevention of cat allergy