Sensitization profiles to purified plant food allergens among pediatric patients with allergy to banana.

Banana fruit allergy is well known, but neither immunoglobulin E recognition patterns to purified plant food allergens nor true prevalences of putative banana allergens have been established. This study aimed to characterize β-1,3-glucanase and thaumatin-like protein (TLP) as banana allergens, testing them, together with other plant food allergens, in 51 children with allergic reactions after banana ingestion and both positive specific IgE and skin prick test (SPT) to banana. Banana β-1,3-glucanase and TLP were isolated and characterized. Both banana allergens, together with kiwifruit TLP Act d 2, avocado class I chitinase Pers a 1, palm pollen profilin Pho d 2 and peach fruit lipid transfer protein (LTP) Pru p 3, were tested by in vitro and in vivo assays. Banana β-1,3-glucanase (Mus a 5) was glycosylated, whereas banana TLP (Mus a 4) was not, in contrast with its homologous kiwi allergen Act d 2. Specific IgE to both banana allergens, as well as to peach Pru p 3, was found in over 70% of sera from banana-allergic children, and Mus a 4 and Pru p 3 provoked positive SPT responses in 6 of the 12 tested patients, whereas Mus a 5 in only one of them. Both peptidic epitopes and cross-reactive carbohydrate determinants were involved in the IgE-binding to Mus a 5, whereas cross-reactivity between Mus a 4 and Act d 2 was only based on common IgE protein epitopes. Profilin Pho d 2 elicited a relevant proportion of positive responses on in vitro (41%) and in vivo (58%) tests. Therefore, Mus a 4 and LTP behave as major banana allergens in the study population, and profilin seems to be also a relevant allergen. Mus a 5 is an equivocal allergenic protein, showing high IgE-binding to its attached complex glycan, and low in vivo potency

The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.

Blocking the bioactivity of allergens is conceptually attractive as a small-molecule therapy for allergic diseases but has not been attempted previously. Group 1 allergens of house dust mites (HDM) are meaningful targets in this quest because they are globally prevalent and clinically important triggers of allergic asthma. Group 1 HDM allergens are cysteine peptidases whose proteolytic activity triggers essential steps in the allergy cascade. Using the HDM allergen Der p 1 as an archetype for structure-based drug discovery, we have identified a series of novel, reversible inhibitors. Potency and selectivity were manipulated by optimizing drug interactions with enzyme binding pockets, while variation of terminal groups conferred the physicochemical and pharmacokinetic attributes required for inhaled delivery. Studies in animals challenged with the gamut of HDM allergens showed an attenuation of allergic responses by targeting just a single component, namely, Der p 1. Our findings suggest that these inhibitors may be used as novel therapies for allergic asthma

Development of a Sandwich ELISA to Measure Exposure to Occupational Cow Hair Allergens

Background: Cow hair and dander are important inducers of occupational allergies in cattle-exposed farmers. To estimate allergen exposure in farming environments, a sensitive enzyme immunoassay was developed to measure cow hair allergens. Methods: A sandwich ELISA was developed using polyclonal rabbit antibodies against a mixture of hair extracts from different cattle breeds. To assess the specificity of the assay, extracts from other mammalian epithelia, mites, molds and grains were tested. To validate the new assay, cow hair allergens were measured in passive airborne dust samples from the stables and homes of farmers. Dust was collected with electrostatic dust fall collectors (EDCs). Results: The sandwich ELISA was found to be very sensitive (detection limit: 0.1 ng/ml) and highly reproducible, demonstrating intra-and interassay coefficients of variation of 4 and 10%, respectively. The assay showed no reactivity with mites, molds and grains, but some cross-reactivity with other mammalian epithelia, with the strongest reaction with goat. Using EDCs for dust sampling, high concentrations of bovine allergens were measured in cow stables (4,760-559,400 mu g/m(2)). In addition, bovine allergens were detected in all areas of cattle farmer dwellings. A large variation was found between individual samples (0.3-900 mu g/m(2)) and significantly higher values were discovered in changing rooms. Conclusion: The ELISA developed for the detection of cow hair proteins is a useful tool for allergen quantification in occupational and home environments. Based on its low detection limit, this test is sensitive enough to detect allergens in passive airborne dust. Copyright (C) 2011 S. Karger AG, Base

Component-resolved diagnosis of pollen allergy based on skin testing with profilin, polcalcin and lipid transfer protein pan-allergens

BACKGROUND Allergy diagnosis needs to be improved in patients suffering from pollen polysensitization due to the existence of possible confounding factors in this type of patients. OBJECTIVE To evaluate new diagnostic strategies by comparing skin responses to pan-allergens and conventional allergenic extracts with specific IgE (sIgE) to purified allergen molecules. METHODS One thousand three hundred and twenty-nine pollen-allergic patients were diagnosed by a combination of an in vitro method with a panel of 13 purified allergens, including major allergens and pan-allergens, using a high-capacity screening technology (ADVIA-Centaur®) and skin prick test (SPT) to pan-allergens and conventional extracts. RESULTS There was a high concordance (κ index) between in vitro (sIgE to major allergens) and in vivo (SPT to conventional extracts) methods in patients who were not sensitized to pan-allergens, but SPT with conventional extracts failed to diagnose patients with sensitization to pan-allergens. In patients who were simultaneously sensitized to polcalcins and profilins, there was a duplication both in the number of sensitizations to major allergens and in the years of disease evolution. There was a statistical association between sensitization to profilins and/or lipid transfer proteins and food allergy (P<0.0001). CONCLUSION The novel diagnostic strategy has proven to be a valuable tool in daily clinical practice. Introduction of routine SPT to pan-allergens is a simple and feasible way of improving diagnostic efficacy. Patients sensitized to pan-allergens should be tested by an adequate panel of allergenic molecules in order to identify the allergens that are responsible for the allergic disease

Component-Resolved in Vitro Diagnosis in Peach-Allergic Patients

BACKGROUND: The in vitro diagnosis of pollen-related food allergy presents low specifi city and reproducibility with many conventional extracts. This can be improved using natural purifi ed allergens, recombinant purifi ed allergens, or both. OBJECTIVE: We compared specifi c immunoglobulin (Ig) E determination (sIgE), the basophil activation test (BAT), the histamine release test (HRT), and the cellular allergen stimulation test (CAST) using natural and recombinant allergens in the diagnosis of peach allergy. METHODS: Thirty-two peach allergic patients were studied. Skin prick tests were performed with commercial peach and extract with Mal d 1, nPru p 3, and profi lin (nPho d 2). sIgE, BAT, CAST, and HRT were determined using rPru p 3, rMal d 3, rBet v 1, rMal d 1, and rMal d 4. RESULTS: Agreement between the techniques was good with all the allergens, except HRT with rMal d 1 and rMal d 4. With rPru p 3, sIgE, CAST, BAT, and HRT showed sensitivity values of 88%, 81%, 72%, and 69% and specifi city values of 100%, 93%, 97%, and 83%, respectively. In patients with systemic symptoms or contact urticaria, the values were 100%, 85%, 81%, and 81%. In patients with oral allergy syndrome, sensitivity to profi lins or homologues of Bet v 1 was detected in 100% of the cases by all the techniques, except by HRT with rMal d 1, which detected 66% of the cases. CONCLUSIONS: The use of single allergens in the in vitro diagnosis of peach allergy by specifi c IgE determination, BAT, and CAST offers high specifi city and sensitivity, with better results than the HRT

The minor house dust mite allergen Der p 13 is a fatty acid binding protein and an activator of a TLR2-mediated innate immune response

Background: The house dust mite (HDM) allergen Der p 13 could be a lipid-binding protein able to activate key innate signaling pathways in the initiation of the allergic response. We investigated the IgE reactivity of recombinant Der p 13 (rDer p 13), its lipid binding activities and its capacity to stimulate airway epithelium cells. Methods: Purified rDer p 13 was characterized by mass spectrometry, circular dichroism, fluorescence-based lipid binding assays and in-silico structural prediction. IgE binding activity and allergenic potential of Der p 13 were examined by ELISA, basophil degranulation assays and in-vitro airway epithelial cell activation assays. Results: Protein modeling and biophysical analysis indicated that Der p 13 adopts a β barrel structure with a predominately apolar pocket representing a potential binding site for hydrophobic ligands. Fluorescent lipid binding assays confirmed that the protein is highly selective for ligands and that it binds a fatty acid with a dissociation constant typical of lipid transporter proteins. The low IgE binding frequency (7%, n= 224) in Thai HDM-allergic patients as well as the limited propensity to activate basophil degranulation classifies Der p 13 as a minor HDM allergen. Nevertheless, the protein with its presumptively associated lipid(s) triggered the production of IL-8 and GM-CSF in respiratory epithelial cells through a TLR2-, MyD88-, NF-kB- and MAPK-dependent signaling pathway. Conclusions: Although a minor allergen, Der p 13 may, through its lipid binding capacity, play a role in the initiation of the HDM allergic response through TLR2 activation

A genomic analysis and transcriptomic atlas of gene expression in Psoroptes ovis reveals feeding- and stage-specific patterns of allergen expression

Background: Psoroptic mange, caused by infestation with the ectoparasitic mite, Psoroptes ovis, is highly contagious, resulting in intense pruritus and represents a major welfare and economic concern for the livestock industry Worldwide. Control relies on injectable endectocides and organophosphate dips, but concerns over residues, environmental contamination, and the development of resistance threaten the sustainability of this approach, highlighting interest in alternative control methods. However, development of vaccines and identification of chemotherapeutic targets is hampered by the lack of P. ovis transcriptomic and genomic resources. Results: Building on the recent publication of the P. ovis draft genome, here we present a genomic analysis and transcriptomic atlas of gene expression in P. ovis revealing feeding- and stage-specific patterns of gene expression, including novel multigene families and allergens. Network-based clustering revealed 14 gene clusters demonstrating either single- or multi-stage specific gene expression patterns, with 3075 female-specific, 890 male-specific and 112, 217 and 526 transcripts showing larval, protonymph and tritonymph specific-expression, respectively. Detailed analysis of P. ovis allergens revealed stage-specific patterns of allergen gene expression, many of which were also enriched in "fed" mites and tritonymphs, highlighting an important feeding-related allergenicity in this developmental stage. Pair-wise analysis of differential expression between life-cycle stages identified patterns of sex-biased gene expression and also identified novel P. ovis multigene families including known allergens and novel genes with high levels of stage-specific expression. Conclusions: The genomic and transcriptomic atlas described here represents a unique resource for the acarid-research community, whilst the OrcAE platform makes this freely available, facilitating further community-led curation of the draft P. ovis genome

Applying the adverse outcome pathway (AOP) for food sensitization to support in vitro testing strategies

Background Before introducing proteins from new or alternative dietary sources into the market, a compressive risk assessment including food allergic sensitization should be carried out in order to ensure their safety. We have recently proposed the adverse outcome pathway (AOP) concept to structure the current mechanistic understanding of the molecular and cellular pathways evidenced to drive IgE-mediated food allergies. This AOP framework offers the biological context to collect and structure existing in vitro methods and to identify missing assays to evaluate sensitizing potential of food proteins. Scope and approach In this review, we provide a state-of-the-art overview of available in vitro approaches for assessing the sensitizing potential of food proteins, including their strengths and limitations. These approaches are structured by their potential to evaluate the molecular initiating and key events driving food sensitization. Key findings and conclusions The application of the AOP framework offers the opportunity to anchor existing testing methods to specific building blocks of the AOP for food sensitization. In general, in vitro methods evaluating mechanisms involved in the innate immune response are easier to address than assays addressing the adaptive immune response due to the low precursor frequency of allergen-specific T and B cells. Novel ex vivo culture strategies may have the potential to become useful tools for investigating the sensitizing potential of food proteins. When applied in the context of an integrated testing strategy, the described approaches may reduce, if not replace, current animal testing approaches

Long-term effects of allergen sensitization and exposure in adult asthma: a prospective study.

BACKGROUND: : We investigated the effects of sensitization and exposure to common domestic allergens on longitudinal changes in lung function and bronchial hyperresponsiveness. METHODS: : Subjects attended 2 visits that were 4 years apart. Skin prick testing was performed and household dust samples were collected for quantification of mite, dog, and cat allergens at baseline. Measurements of lung function, exhaled nitric oxide, and bronchial hyperresponsiveness were completed at both visits. RESULTS: : Dust samples were collected in 165 of the 200 subjects completing both visits. Mean length of follow-up was 47 months. Bronchial hyperresponsiveness, measured at both visits in 86 subjects, deteriorated in those exposed to high mite allergen levels compared with those not exposed [mean (95% CI) doubling dose change PD20 = -0.44 (-1.07 to 0.19) vs 0.82 (0.27 to 1.36)], but improved in those exposed to high dog allergen levels compared with those not exposed [1.10 (0.33 to 1.86) vs 0.10 (-0.39 to 0.58)]. The associations were significant in the multivariate models. Cat allergen exposure was not associated with any changes in lung function, exhaled nitric oxide, or bronchial hyperresponsiveness. CONCLUSIONS: : In a 4-year prospective cohort of persons with asthma, exposure to high levels of dust mite allergens at baseline was associated with a subsequent increase in bronchial hyperresponsiveness

Allergens, germs and asthma

Objective To explore asthma pathogenesis using data from upper and lower airways. Data Source English-language papers on human asthma and nasal polyp subjects from 1990 onwards. Study Selection High-quality studies in established journals. Results The recognition of its inflammatory nature led to a quantum leap in the understanding and treatment of asthma, with lives saved by inhaled corticosteroids. Further work at genetic, molecular, histological and clinical levels has shown that asthma is polymorphic and rarely involves isolated Th2 bronchial inflammation. Viral infections may act as an initiating event in children and adults, showing synergy with atopy. Chronic staphylococcal colonization of the mucosa may act as a promoter, as in atopic dermatitis. These two observations may be linked, with viruses providing an entry for bacteria into the mucosal epithelium. Conclusions Most asthma begins in the nose and involves allergy and infection: both viral and bacterial. The combination of atopy and infection suggests new possibilities for therapy