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Additional file 1: Table S1. Clinical and demographic data of 150 mugwort pollen-allergic individuals sIgE against mugwort extract (w6), Art v 1 (w231) and Art v 3(w233) determined by ImmunoCAP, ND, not determined. AS asthma; AR allergic rhinitis; C, conjunctivitis; E, eczema. I-1, I-2, I-3, I-4 indicates the patients serum used in ImmunoCAP inhibition assay belonging to four groups of different sensitization patterns (1, Art v 1 and Art v 3 positive; 2, Art v 1 positive, Art v 3 negative; 3, Art v 1 negative, Art v 3 positive; 4, Art v 1 and Art v 3 negative). The 82 patients reported in previously studies17, 20, 28 are indicated by an asterisk. Table S2. GenBank accession numbers for three allergen groups in seven Artemisia species.Table S3. Productivity of the three group allergens purified by specific mAb. Table S4. ImmunoCAP IgE characterization of serum pools from five areas. Figure S1. Six Artemisia species collected from China. Figure S2. SDS-PAGE of natural purified Art v 1, Art v 2 and Art v 3 homologous allergens from six Chinese Artemisia species. a, natural Art v 1 homologues purified by specific mAb A7-G4-E6; b, natural Art v 2 homologues purified by specific mAb C9-C1 shown in six different gels; c, natural Art v 3 homologues purified by specific mAb A2-B8. Figure S3. Mass spectra of natural purified Art v 1(a), Art v 2(b) and Art v 3(c) homologues. The peptides verified by LC–MS/MS are shown in red and highlighted. Figure S4. ELISA quantification of three allergen components in Artemisia spp. pollen. a, Chinese silver mugwort (A. argyi) pollen extract (ArE) in SDS gel and reaction to polyclonal antibodies (pAb) by Western blot; b, ELISA standard curve for Art v 1 allergen (mAb A7-G4-E6 and rabbit pAbs); c, ELISA standard curve for Art v 2 homologous allergen (mAb C9-C1 with rabbit pAbs); d, ELISA standard curve for Art v 3 homologous allergen with two mAbs (mAbs A2-B8 and A9-G10) with representative different isoforms. Figure S5..Inhibition of IgE binding to Art v 1 and Art v 3 with seven pollen extracts using two serum pools. a: inhibition ELISA coated with rArt v 1.0101, serum pool from Datong, Shanxi (nArt v 1: 11 kUA/l); b: inhibition ELISA coated with rArt v 1.0101, serum pool from Yantai, Shandong (nArt v 1: 6.51 kUA/l); c: inhibition ELISA coated with rArt v 3.0201, serum pool from Datong, Shanxi (CAP nArt v 3: 11.27 kUA/l); d: inhibition ELISA coated with rArt v 3.0201, serum pool from Yantai, Shandong (CAP nArt v 3: 9.65 kUA/l). Figure S6. Inhibition of sera pool from Shanxi with extract from different species at 100 μg/ml in ELISA coated with 10 μg/ml of different pollen extracts and mixture. 6-mix, mixture of six Artemisia spp. extract in the same proportions.

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd

BACKGROUND: Allergies arise from aberrant Th2 responses to allergens. The processes involved in the genesis of allergic sensitization remain elusive. Some allergens such as derived from house dust mites, have proteolytic activity which can induce oxidative stress in vivo. A reduced capacity of the host to control oxidative stress might prime for allergic sensitization. METHODS: Two different strains of mice were compared for their anti-oxidant and immune response to HDM. Protease activity of the HDM extract was reduced to investigate its role in oxidative stress induction in the airways and whether this induction could determine allergic sensitization and inflammation. The role of oxidative stress in allergic sensitization was also investigated in humans. An occupational cohort of animal workers was followed for development of sensitization to rodent urinary proteins. Levels of oxidative stress in serum and anti-oxidant responses by PBMCs were determined. RESULTS: Susceptibility to allergic sensitization to mite allergens in mice was highly dependent on host genetic background and was associated with oxidative stress in the lungs before allergen exposure and poor anti-oxidant response after allergen exposure. Reduction of mite protease activity limited its capacity to induce oxidative stress and allergic inflammation in mice. We showed that also in human subjects, oxidative stress before allergen exposure and poor anti-oxidant responses were associated with predisposition to occupational allergy. CONCLUSION: Our study indicates that oxidative stress condition before allergen exposure due to an inadequate anti-oxidant response primes for allergic Th2 responses. This article is protected by copyright. All rights reserved.

BACKGROUND: Allergies arise from aberrant Th2 responses to allergens. The processes involved in the genesis of allergic sensitization remain elusive. Some allergens such as derived from house dust mites, have proteolytic activity which can induce oxidative stress in vivo. A reduced capacity of the host to control oxidative stress might prime for allergic sensitization. METHODS: Two different strains of mice were compared for their anti-oxidant and immune response to HDM. Protease activity of the HDM extract was reduced to investigate its role in oxidative stress induction in the airways and whether this induction could determine allergic sensitization and inflammation. The role of oxidative stress in allergic sensitization was also investigated in humans. An occupational cohort of animal workers was followed for development of sensitization to rodent urinary proteins. Levels of oxidative stress in serum and anti-oxidant responses by PBMCs were determined. RESULTS: Susceptibility to allergic sensitization to mite allergens in mice was highly dependent on host genetic background and was associated with oxidative stress in the lungs before allergen exposure and poor anti-oxidant response after allergen exposure. Reduction of mite protease activity limited its capacity to induce oxidative stress and allergic inflammation in mice. We showed that also in human subjects, oxidative stress before allergen exposure and poor anti-oxidant responses were associated with predisposition to occupational allergy. CONCLUSION: Our study indicates that oxidative stress condition before allergen exposure due to an inadequate anti-oxidant response primes for allergic Th2 responses. This article is protected by copyright. All rights reserved.

BACKGROUND: Oil body-associated allergens such as oleosins have been reported for important allergenic foods such as peanut, sesame and hazelnut. Here we investigate whether oil body associated proteins (OAPs) are linked with specific clinical phenotypes and whether they are represented in skin prick test (SPT) reagents. METHODS: A hazelnut OAP fraction was characterized by mass-spectrometry (MS) to identify its major constituents. Polyclonal rabbit antibodies were generated against hazelnut OAPs. The presence of OAPs in commercially available hazelnut SPTs was studied by immunoblot and spiking experiments. OAP-specific IgE antibodies were measured in sera from patients with a convincing history of hazelnut allergy by RAST (n = 91), immunoblot (n = 22) and basophil histamine release (BHR; n = 14). RESULTS: Hazelnut OAPs were analysed by MS and found to be dominated by oleosins at 14 and 17 kDa, and a 27 kDa band containing oleosin dimers and unidentified protein. In 36/91 sera specific IgE against hazelnut OAPs was detected, and confirmed to be biologically active by BHR (n = 14). The majority (21/22) recognized the oleosin bands at 17 kDa on immunoblot, of which 11 exclusively. These OAP-specific IgE responses dominated by oleosin were associated with systemic reactions to hazelnut (OR 4.24; p = 0.015) and negative SPT (2 6.3, p = 0.012). Immunoblot analysis using OAP-specific rabbit antiserum demonstrated that commercial SPT reagents are virtually devoid of OAPs, sometimes (3/9) resulting in false-negative SPT. Spiking of SPT reagents with OAP restored serum IgE binding of these false-negative patients on immunoblot at mainly 17 kDa. CONCLUSION: Hazelnut allergens found in oil bodies dominated by oleosin are associated with more severe systemic reactions and negative SPT. Defatted diagnostic extracts are virtually devoid of these allergens, resulting in poor sensitivity for detection of IgE antibodies against these clinically relevant molecules.

RATIONALE: Hazelnut allergy ranks among the most frequently observed food allergies. Clinical symptoms range from the oral allergy syndrome to life threatening anaphylaxis. Diagnosis of hazelnut allergy partially relies on in vivo testing by means of skin prick testing (SPT). The aim of this study was to characterize hazelnut SPT extracts both in vitro and in vivo. METHODS: Hazelnut SPT extracts were investigated for protein concentration and composition. The major hazelnut allergen Cor a 1, lipid transfer protein (LTP) and thaumatin-like-protein (TLP) were monitored by competitive RIA and immunoblotting. SPT extracts (n = 6) were analyzed for skin reactivity and the correlation between the SPT extract protein concentration and the mean skin reactivity (HEIC) was determined in a group of hazelnut-allergic patients (n = 30). For one SPT extract, the threshold level for Cor a 1 was determined in Cor a 1-monosensitized patients (n = 5). RESULTS: Protein concentrations ranged from 0.2-14 mg/ml. Although some proteins were present in most extracts (bands at 10, 22-28, 32 and around 48 kDa), clear differences in composition were observed (both intra- and inter-variability). The concentration of the major hazelnut allergen Cor a 1 differed up to a factor 50 (0.6-32 micrograms/ml). LTP was virtually absent in 3/9 SPT extracts and variable quantities of TLP were detected by immunoblotting. Some patients (6/30) had a false-negative SPT with 3/6 SPT extracts. There was a clear correlation between the protein concentration and the mean HEIC (RPearson = 0.87). The threshold level for Cor a 1 was +/- 3.2 ng/ml as assessed with one of the products investigated. CONCLUSIONS: Heterogeneous protein concentration/composition of SPT extracts results in variable skin test responses. The absence of potentially severe allergens like LTP may lead to false-negative SPT results that jeopardize a patient's safety. From these results it can be concluded that there is a strong need for standardization of products for SPT