Real-Life Study for the Diagnosis of House Dust Mite Allergy - The Value of Recombinant Allergen-Based IgE Serology

Background: Dermatophagoides pteronyssinus is one of the most important perennial allergen sources worldwide. Molecular diagnostics using the commercially available major allergens (Der p 1 and Der p 2) in combination with Der p 10 do not detect house dust mite (HDM) sensitization in a number of cases when used alone. The objective was to evaluate the IgE reactivity profiles of these patients using an experimental immunoassay biochip. Methods: Sera of HDM-allergic patients (positive skin prick test, CAP class 1 for allergen extract, and positive intranasal provocation) were tested for IgE antibodies against Der p 1, Der p 2, and Der p 10 by ImmunoCAP fluorescence enzyme immunoassay. Negatively tested sera were examined by an experimental chip containing 13 microarrayed HDM allergens. Results: Of 97 patients tested, 16 showed negative results to Der p 1, Der p 2, and Der p 10. MeDALL chip evaluation revealed 5 patients mono-sensitized to Der p 23, and 11 patients were negative for all HDM MeDALL chip components. Seven sera were available for further testing, and 3 of them showed IgE reactivity to dot-blotted nDer p 1, and 2 reacted with high-molecular weight components (>100 kDa) in nitrocellulose-blotted HDM extract when tested with 1251-labeled anti-IgE in a RAST-based assay. The HDM extract-specific IgE levels of the 11 patients were <3.9 kU/I. Conclusions: Recombinant allergen-based IgE serology is of great value when conventional IgE diagnostics fails. Der p 23 is an important HDM allergen, especially when major allergens are negative. Therefore, it would be desirable to have Der p 23 commercially available. Further research concerning the prevalence and clinical significance of different HDM allergens is needed. (C) 2016 S. Karger AG, Base

Molecular profiling of allergen-specific antibody responses may enhance success of specific immunotherapy

Background: House dust mites (HDMs) are among the most important allergen sources containing many different allergenic molecules. Analysis of patients from a double-blind, placebo-controlled allergen-specific immunotherapy (AIT) study indicated that patients may benefit from AIT to different extents depending on their molecular sensitization profiles. Objective: Our aim was to investigate in a real-life setting whether stratification of patients with HDM allergy according to molecular analysis may enhance AIT success. Methods: Serum and nasal secretion samples from patients with HDM allergy (n = 24) (at baseline, 7, 15, 33, and 52 weeks) who had received 1 year of treatment with a well-defined subcutaneous AIT form (Alutard SQ 510) were tested for IgE and IgG reactivity to 15 microarrayed HDM allergen molecules with ImmunoCAP Immuno-solid-phase Allergen Chip technology. IgG subclass levels to allergens and peptides were determined by ELISA, and IgG blocking was assessed by basophil activation. In vitro parameters were related to reduction of symptoms determined by combined symptom medication score and visual analog scale score. Results: Alutard SQ 510 induced protective IgG mainly against Dermatophagoides pteronyssinus (Der p) 1 and Der p 2 and to a lesser extent to Der p 23, but not to the other important allergens such as Der p 5, Der p 7, and Der p 21, showing better clinical efficacy in patients sensitized only to Der p 1 and/or Der p 2 as compared with patients having additional IgE specificities. Conclusion: Stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT

Immunological Interactive Effects between Pollen Grains and Their Cytoplasmic Granules on Brown Norway Rats

International audienceBackgroundGrass pollen is one of the most important aeroallergen vectors in Europe. Under some meteorological factors, pollen grains can release pollen cytoplasmic granules (PCGs). PCGs induce allergic responses. Several studies have shown that during a period of thunderstorms the number of patients with asthma increases because of higher airborne concentrations of PCGs.ObjectiveThe aims of the study were to assess the allergenicity of interactive effects between pollen and PCGs and to compare it with allergenicity of Timothy grass pollen and PCGs in Brown Norway rats.MethodsRats were sensitized (day 0) and challenged (day 21) with pollen grains and/or PCGs. Four groups were studied: pollen-pollen (PP), PCGs-PCGs (GG), pollen-PCGs (PG), and PCGs-pollen (GP). Blood samples, bronchoalveolar lavage fluid, and bronchial lymph node were collected at day 25. IgE and IgG1 levels in sera were assessed by enzyme-linked immunosorbent assay. Alveolar cells, protein, and cytokine concentrations were quantified in bronchoalveolar lavage fluid. T-cell proliferation, in response to pollen or granules, was performed by lymph node assay.ResultsInteractive effects between pollen and PCGs increased IgE and IgG1 levels when compared with those of the negative control. These increases were lower than those of the PP group but similar to the levels obtained by the GG group. Whatever was used in the sensitization and/or challenge phase, PCGs increased lymphocyte and Rantes levels compared with those of the pollen group. The interactive effects increased IL-1α and IL-1β compared with those of the PP and GG groups.ConclusionsImmunologic interactive effects have been shown between pollen and PCGs. For humoral and cellular allergic responses, interactive effects between the 2 aeroallergenic sources used in this study seem to be influenced mainly by PCGs

Subpollen particles are rich carriers of major short ragweed allergens and NADH dehydrogenases: quantitative proteomic and allergomic study

Background: Short ragweed (Ambrosia artemisiifolia) allergies affect more than 36 million people annually. Ragweed pollen grains release subpollen particles (SPP) of respirable size upon hydration or a change in air electrical conditions. The aim of this study was to characterize the proteomes and allergomes of short ragweed SPP and total pollen protein extract (TOT), and compare their effects with those of standard aqueous pollen protein extract (APE) using sera from short ragweed pollen-sensitized patients. Methods: Quantitative 2D gel-based and shotgun proteomics, 1D and 2D immunoblotting, and quantitative ELISA were applied. Novel SPP extraction and preparation protocols enabled appropriate sample preparation and further downstream analysis by quantitative proteomics. Results: The SPP fraction contained the highest proportion (94%) of the allergome, with the largest quantities of the minor Amb a 4 and major Amb a 1 allergens, and as unique, NADH dehydrogenases. APE was the richest in Amb a 6, Amb a 5 and Amb a 3, and TOT fraction was the richest in the Amb a 8 allergens (89% and 83% of allergome, respectively). Allergenic potency correlated well among the three fractions tested, with 1D immunoblots demonstrating a slight predominance of IgE reactivity to SPP compared to TOT and APE. However, the strongest IgE binding in ELISA was noted against APE. New allergenic candidates, phosphoglycerate mutase and phosphoglucomutase, were identified in all the three pollen fractions. Enolase, UTP-glucose-1-phosphate uridylyltransferase and polygalacturonase were observed in SPP and TOT fractions as novel allergens of the short ragweed pollen, as previously described. Conclusion and Clinical Relevance: We demonstrated that the complete major (Amb a 1 and 11) and almost all minor (Amb a 3, 4, 5, 6, 8 and 9) short ragweed pollen allergen repertoire as well as NADH oxidases are present in SPP, highlighting an important role for SPP in allergic sensitization to short ragweed.This is the peer‐reviewed version of the article: Smiljanic, K.; Apostolovic, D.; Trifunovic, S.; Ognjenovic, J.; Perusko, M.; Mihajlovic, L.; Burazer, L.; Hage, M. van; Velickovic, T. C. Subpollen Particles Are Rich Carriers of Major Short Ragweed Allergens and NADH Dehydrogenases: Quantitative Proteomic and Allergomic Study. Clinical & Experimental Allergy 2017, 47 (6), 815–828.[https://doi.org/10.1111/cea.12874

Identification of Critical Amino Acids in an Immunodominant IgE Epitope of Pen c 13, a Major Allergen from Penicillium citrinum

Background: Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13. Methodology/Principal Findings: Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A 148 –E 166) and S22 (A 243 –K 274) were recognized by sera from 90 % and 100 % of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T 261 –K 274), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity

Challenges for Allergy Diagnosis in Regions with Complex Pollen Exposures

Over the past few decades, significant scientific progress has influenced clinical allergy practice. The biological standardization of extracts was followed by the massive identification and characterization of new allergens and their progressive use as diagnostic tools including allergen micro arrays that facilitate the simultaneous testing of more than 100 allergen components. Specific diagnosis is the basis of allergy practice and is always aiming to select the best therapeutic or avoidance intervention. As a consequence, redundant or irrelevant information might be adding unnecessary cost and complexity to daily clinical practice. A rational use of the different diagnostic alternatives would allow a significant improvement in the diagnosis and treatment of allergic patients, especially for those residing in complex pollen exposure areas

Non-IgE-reactive allergen peptides deteriorate the skin barrier in house dust mite-sensitized atopic dermatitis patients

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by type 2 cytokine-driven skin inflammation and epithelial barrier dysfunction. The latter is believed to allow the increased penetration of chemicals, toxins, and allergens into the skin. House dust mite allergens, particularly Der p 2, are important triggers in sensitized individuals with AD; the precise actions of these allergens in epithelial biology remain, however, incompletely understood. In this study, we compared the effects of the protein allergen Der p 2 and a mix of non-IgE-reactive Der p 2 peptides on skin cells using patch tests in AD patients and healthy participants. We then analyzed mRNA expression profiles of keratinocytes by single-cell RNA-sequencing. We report that existing barrier deficiencies in the non-lesional skin of AD patients allow deep penetration of Der p 2 and its peptides, leading to local microinflammation. Der p 2 protein specifically upregulated genes involved in the innate immune system, stress, and danger signals in suprabasal KC. Der p 2 peptides further downregulated skin barrier genes, in particular the expression of genes involved in cell–matrix and cell–cell adhesion. Peptides also induced genes involved in hyperproliferation and caused disturbances in keratinocyte differentiation. Furthermore, inflammasome-relevant genes and IL18 were overexpressed, while KRT1 was downregulated. Our data suggest that Der p 2 peptides contribute to AD initiation and exacerbation by augmenting hallmark features of AD, such as skin inflammation, barrier disruption, and hyperplasia of keratinocytes

Biochemical, Biophysical and IgE-Epitope Characterization of the Wheat Food Allergen, Tri a 37

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