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Background House dust mite (HDM) allergens are major elicitors of allergic reactions worldwide. Objective Identification, characterization, and evaluation of diagnostic utility of a new important HDM allergen was performed. Methods A cDNA coding for a new Dermatophagoides pteronyssinus (Dp) allergen, Der p 37, was isolated from a Dp expression library with allergic patients' IgE antibodies. Recombinant Der p 37 (rDer p 37) expressed in Escherichia coli was purified, then characterized by mass spectrometry, circular dichroism, and IgE reactivity by ImmunoCAP ISAC technology with sera from 111 clinically defined HDM-allergic patients. The allergenic activity of rDer p 37 was studied by basophil activation and CD4+ T-cell responses by carboxyfluorescein diacetate succinimidyl ester dilution assays. Specific antibodies raised against rDer p 37 were used for the ultrastructural localization of Der p 37 in mites by immunogold transmission electron microscopy. Results Der p 37, a 26 kDa allergen with homology to chitin-binding proteins, is immunologically distinct from Der p 15, 18, and 23. It is located in the peritrophic membrane of fecal pellets. Der p 37 reacted with IgE antibodies from a third of HDM-allergic patients and induced specific basophil- and CD4+ T-cell activation. Der p 37 IgE–positive patients had significantly higher IgE levels to major HDM allergens, reacted with more HDM allergens, and had a higher risk (odds ratio = 3.1) of asthma compared to Der p 37–negative patients. Conclusions Der p 37, a new Dp allergen recognized by a third of HDM-allergic patients, may serve as a surrogate marker for severe HDM sensitization and asthma.

BACKGROUND: Confirmation of the clinical relevance of sensitisation is important for the diagnosis of allergic rhinitis. OBJECTIVE: To investigate the usefulness of an in vitro basophil activation test and component-resolved diagnosis in distinguishing between symptomatic allergic rhinitis patients and asymptomatic sensitization to house dust mites (HDMs). METHODS: Thirty-six subjects with a positive skin prick test (SPT) for HDM were divided into a symptomatic (n = 17) and an asymptomatic (n = 19) group on the basis of their clinical history and a nasal provocation test. A basophil CD63 response to in vitro stimulation with Dermatophagoides pteronyssinus whole allergen extract and the IgE reactivity profiles for Der p 1, 2, 4, 5, 7, 10, 11, 14, 15, 18, 21, 23 were evaluated. Serum IgE and IgG specific to D pteronyssinus whole allergen extract and total IgE were measured. RESULTS: There were no statistically significant differences in the levels of IgE (IgE levels were higher in symptomatic patients with P = 0.055) and IgG specific to D pteronyssinus and total IgE. Symptomatic patients showed a lower threshold for in vitro basophil activation (3.33 ng/mL vs 33.3 ng/mL), a higher area under the curve (AUC) of basophil activation (171 vs 127) (P = 0.017), a higher response to positive control with anti-FcεRI stimulation (97% vs 79%) (P < 0.001), a recognition of more HDM allergens (4 vs 2) and more frequent sensitization to rDer p 7 (P = 0.016) and rDer p 23 compared to asymptomatic subjects (P = 0.018). There was a positive correlation (r = 0.63; P < 0.001) between the number of recognized allergens and the AUC of basophil activation. CONCLUSION AND CLINICAL RELEVANCE: In the subjects studied, the differences in the basophil response to D pteronyssinus allergen extract, number of recognized HDM allergens and reactivity to rDer p 7 and rDer p 23 distinguish symptomatic from asymptomatic HDM sensitisation better than SPT or allergen extract-specific IgE. Information regarding the clinical relevance of sensitization is important for the prescription of allergen-specific immunotherapy.

BackgroundSeveral studies indicate that Der p 7 is an important and clinically relevant allergen of Dermatophagoides pteronyssinus which should be included in vaccines for treatment of house dust mite (HDM) allergy. Aim of this study was to characterize the IgE epitopes of Der p 7.MethodsRecombinant Der p 7 was expressed and purified, analyzed for fold by circular dichroism and tested for its allergenic activity by basophil activation. Seven overlapping, surface-exposed peptides (P1–P7) with a length of 27 to 37 amino acids, which spanned the Der p 7 sequence, were synthesized and tested for IgE reactivity and allergenic activity by basophil activation assay. Carrier-bound peptides were studied for their ability to induce allergen-specific IgG antibodies in rabbits. Peptide-specific antibodies were used to inhibit allergic patients` IgE binding to Der p 7 by ELISA for mapping of IgE epitopes.ResultsrDer p 7 showed high allergenic activity comparable with Der p 5, Der p 21, and Der p 23. None of the seven tested peptides showed any IgE reactivity or allergenic activity when tested with HDM- allergic patients indicating lack of sequential IgE epitopes on Der p 7. IgE inhibition experiments using anti-peptide specific IgGs and molecular modeling enabled us to identify discontinuous, conformational IgE epitopes of Der p 7.Conclusion and Clinical RelevanceIgE epitopes of Der p 7 belong to the conformational and discontinuous type whereas sequential Der p 7 peptides lack IgE reactivity. It should thus be possible to construct hypoallergenic vaccines for Der p 7 based on carrier-bound allergen peptides.

BACKGROUND Aedes aegypti and Dermatophagoides pteronyssinus contain important allergens including cross-reactive tropomyosins. However, the functional and clinical relevance of their cross-reactivity is still debated. OBJECTIVE To analyse the humoral and cellular cross-reactivity of recombinant Aed a 10.01, Aed a 10.02 and Der p 10. METHODS Sera from 15 Austrian house dust mite-allergic, Der p 10-sensitized individuals were tested for IgE reactivity to recombinant tropomyosins in ELISA, inhibition ELISA and basophil activation tests. BALB/c mice were immunized with Aed a 10.01 or Aed a 10.02, and their sera were assessed for reactivity to all tropomyosins. Splenocytes were stimulated with all tropomyosins and synthetic peptides representing the amino acid sequence of Aed a 10.01. RESULTS IgE antibodies of Der p 10-sensitized patients cross-reacted with both tropomyosins from A. aegypti. Aed a 10.01 was a more potent inhibitor of IgE binding to Der p 10 and a stronger activator of basophils sensitized with Der p 10-specific IgE than Aed a 10.02. Murine antibodies raised against Aed a 10.01 and Aed a 10.02 cross-reacted with Der p 10. Aed a 10.01-specific antibody showed stronger cross-reactivity with Der p 10 than Aed a 10.02-specific antibody. Splenocytes from both groups of mice proliferated similarly to all tropomyosins. Five cross-reactive T cell-activating regions were identified. CONCLUSION AND CLINICAL RELEVANCE Tropomyosins from D. pteronyssinus and A. aegypti show humoral and cellular cross-reactivity, involving 5 potential T cell-activating regions. The more pronounced cross-reactivity of Aed a 10.01 and Der p 10 matched the higher sequence similarity of both proteins.

BACKGROUND Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

Arginine kinase (AK) was first identified as an allergen in the Indian-meal moth and subsequently shown to occur as allergen in various invertebrates and shellfish. The cDNA coding for AK from the house dust mite (HDM) species Dermatophagoides pteronyssinus, Der p 20, has been isolated, but no recombinant Der p 20 (rDer p 20) allergen has been produced and characterized so far. We report the expression of Der p 20 as recombinant protein in Escherichia coli. rDer p 20 was purified and shown to be a monomeric, folded protein by size exclusion chromatography and circular dichroism spectroscopy, respectively. Using AK-specific antibodies, Der p 20 was found to occur mainly in HDM bodies, but not in fecal particles. Thirty percent of clinically well-characterized HDM allergic patients (n = 98) whose immunoglobulin E (IgE) reactivity profiles had been determined with an extensive panel of purified HDM allergens (Der f 1, 2; Der p 1, 2, 4, 5, 7, 10, 11, 14, 15, 18, 21, 23 and 37) showed IgE reactivity to Der p 20. IgE reactivity to Der p 20 was more frequently associated with lung symptoms. AKs were detected in several invertebrates with specific antibodies and Der p 20 showed IgE cross-reactivity with AK from shrimp (Litopenaeus vannamei). Thus, Der p 20 is a cross-reactive HDM allergen and may serve as a diagnostic marker for HDM-induced lung symptoms such as asthma.

The respiratory epithelium is a barrier against pathogens and allergens and a target for therapy in respiratory allergy, asthma and chronic obstructive pulmonary disease (COPD). We investigated barrier-damaging factors and protective factors by real-time measurement of respiratory cell barrier integrity. Barrier integrity to cigarette smoke extract (CSE), house dust mite (HDM) extract, interferon-γ (IFN-γ) or human rhinovirus (HRV) infection alone or in combination was assessed. Corticosteroids, lipopolysaccharide (LPS), and nasal mucus proteins were tested for their ability to prevent loss of barrier integrity. Real-time impedance-based measurement revealed different patterns of CSE-, HDM-, IFN-γ- and HRV-induced damage. When per se non-damaging concentrations of harmful factors were combined, a synergetic effect was observed only for CSE and HDM. Betamethasone prevented the damaging effect of HRV and CSE, but not damage caused by HDM or IFN-γ. Real-time impedance-based measurement of respiratory epithelial barrier function is useful to study factors, which are harmful or protective. The identification of a synergetic damaging effect of CSE and HDM as well as the finding that Betamethasone protects against HRV- and CSE-induced damage may be important for asthma and COPD.

PURPOSE OF REVIEW: Molecular allergology uses pure, mainly recombinant and structurally defined allergen molecules and allergen-derived epitopes to study mechanisms of IgE-associated allergy, to diagnose, and even predict the development of allergic manifestations and to treat and prevent IgE-associated allergies. Atopic dermatitis, a chronic inflammatory skin disease is almost always associated with IgE sensitization to allergens. However, also non-IgE-mediated pathomechanisms seem to be operative in atopic dermatitis and it is often difficult to identify the disease-causing allergens. Here we review recent work showing the usefulness of molecular allergology to study mechanisms of atopic dermatitis, for diagnosis and eventually for treatment and prevention of atopic dermatitis. RECENT FINDINGS: IgE sensitization to airborne, food-derived, microbial allergens, and autoallergens has been found to be associated with atopic dermatitis. Using defined allergen molecules and non-IgE-reactive allergen derivatives, evidence could be provided for the existence of IgE- and non-IgE-mediated mechanisms of inflammation in atopic dermatitis. Furthermore, effects of epicutaneous allergen administration on systemic allergen-specific immune responses have been studied. Multi-allergen tests containing micro-arrayed allergen molecules have been shown to be useful for the identification of culprit allergens in atopic dermatitis and may improve the management of atopic dermatitis by allergen-specific immunotherapy, allergen avoidance, and IgE-targeting therapies in a personalized medicine approach. SUMMARY: Molecular allergology allows for dissection of the pathomechanisms of atopic dermatitis, provides new forms of allergy diagnosis for identification of disease-causing allergens, and opens the door to new forms of management by allergen-specific and T cells-targeting or IgE-targeting interventions in a personalized medicine approach.