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IntroductionSevere equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses.MethodsProteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses’ sera (n=5 per group).ResultsIg binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses’ sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics’ sera on four but higher binding with healthy horses’ sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses’ serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA.DiscussionAntigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.

Abstract Background The domestic mite Blomia tropicalis is a major source of allergens in tropical and subtropical regions. Despite its great medical importance, the allergome of this mite has not been sufficiently studied. Only 14 allergen groups have been identified in B. tropicalis thus far, even though early radioimmunoelectrophoresis techniques (27 uncharacterized allergen complexes) and comparative data based on 40 allergen groups officially recognized by the World Health Organization (WHO)/IUIS in domestic astigmatid mites suggest the presence of a large set of additional allergens. Methods Here, we employ a multiomics approach to assess the allergome of B. tropicalis using genomic and transcriptomic sequence data and perform highly sensitive protein abundance quantification. Findings Among the 14 known allergen groups, we confirmed 13 (one WHO/IUIS allergen, Blo t 19, was not found) and identified 16 potentially novel allergens based on sequence similarity. These data indicate that B. tropicalis shares 27 known/deduced allergen groups with pyroglyphid house dust mites (genus Dermatophagoides). Among these groups, five allergen‐encoding genes are highly expressed at the transcript level: Blo t 1, Blo t 5, Blo t 21 (known), Blo t 15, and Blo t 18 (predicted). However, at the protein level, a different set of most abundant allergens was found: Blo t 2, 10, 11, 20 and 21 (mite bodies) or Blo t 3, 4, 6 and predicted Blo t 13, 14 and 36 (mite feces). Interpretation We report the use of an integrated omics method to identify and predict an array of mite allergens and advanced, label‐free proteomics to determine allergen protein abundance. Our research identifies a large set of novel putative allergens and shows that the expression levels of allergen‐encoding genes may not be strictly correlated with the actual allergenic protein abundance in mite bodies.

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.

BackgroundAsthma is not well investigated in equatorial Africa and little is known about the disease-associated allergen molecules recognized by IgE from patients in this area. The aim was to study the molecular IgE sensitization profile of asthmatic children and young adults in a semi-rural area (Lambaréné) of an equatorial African country (Gabon), to identify the most important allergen molecules associated with allergic asthma in equatorial Africa.MethodsFifty-nine asthmatic patients, mainly children and few young adults, were studied by skin prick testing to Dermatophagoides pteronyssinus (Der p), D. farinae (Der f), cat, dog, cockroach, grass, Alternaria and peanut. Sera were obtained from a subset of 35 patients, 32 with positive and 3 with negative skin reaction to Der p and tested for IgE reactivity to 176 allergen molecules from different allergen sources by ImmunoCAP ISAC microarray technology and to seven recombinant Blomia tropicalis (Blo t) allergens by IgE dot blot assay.ResultsThirty-three of the 59 patients (56%) were sensitized to Der p and 23 of them (39%) were also sensitized to other allergen sources, whereas 9 patients (15%) were only sensitized to allergen sources other than Der p. IgE serology analyses (n=35) showed high IgE-binding frequencies to the Blo t allergens Blo t 5 (43%), Blo t 21 (43%) and Blo t 2 (40%), whereas the Der p allergens rDer p 2, rDer p 21 and rDer p 5 (34%, 29% and 26%) were less frequently recognized. Only few patients showed IgE reactivity to allergens from other allergen sources, except to allergens containing carbohydrate determinants (CCDs) or to wasp venom allergens (i.e., antigen 5).ConclusionOur results thus demonstrate that IgE sensitization to mite allergens is very prevalent in asthmatics in Equatorial Africa with B. tropicalis allergen molecules representing the most important ones associated with allergic asthma.

IgE-mediated allergy to birch pollen affects more than 100 million patients world-wide. Bet v 1, a 17 kDa protein is the major allergen in birch pollen responsible for allergic rhinoconjunctivitis and asthma in birch pollen allergic patients. Allergen-specific immunotherapy (AIT) based on therapeutic administration of Bet v 1-containing vaccines is an effective treatment for birch pollen allergy but no allergen-specific forms of prevention are available. We developed a mouse model for IgE sensitization to Bet v 1 based on subcutaneous injection of aluminum-hydroxide adsorbed recombinant Bet v 1 and performed a detailed characterization of the specificities of the IgE, IgG and CD4+ T cell responses in sensitized mice using seven synthetic peptides of 31-42 amino acids length which comprised the Bet v 1 sequence and the epitopes recognized by human CD4+ T cells. We then demonstrate that preventive systemic administration of a mix of synthetic non-allergenic Bet v 1 peptides to 3-4 week old mice significantly reduced allergic immune responses, including IgE, IgG, IgE-mediated basophil activation, CD4+ T cell and IL-4 responses to the complete Bet v 1 allergen but not to the unrelated major grass pollen allergen Phl p 5, without inducing Bet v 1-specific allergic sensitization or adaptive immunity. Our results thus demonstrate that early preventive administration of non-allergenic synthetic T cell epitope-containing allergen peptides could be a safe strategy for the prevention of allergen-specific IgE sensitization.

Approximately 30% of the world population suffers from immunoglobulin-E (IgE)-mediated allergy. IgE-mediated allergy affects the respiratory tract, the skin and the gastrointestinal tract and may lead to life-threatening acute systemic manifestations such as anaphylactic shock. The symptoms of allergy are mediated by IgE-recognition of causative allergen molecules from different allergen sources. Today, molecular allergy diagnosis allows determining the disease-causing allergens to develop allergen-specific concepts for prevention and treatment of allergy. Allergen-specific preventive and therapeutic strategies include allergen avoidance, vaccination, and tolerance induction. The implementation of these preventive and therapeutic strategies requires a detailed knowledge of the relevant allergen molecules affecting a given population. China is the world´s most populous country with around 1.4 billion inhabitants and an estimated number of more than 400 million allergic patients. Research in allergy in China has dramatically increased in the last decade. We summarize in this review article what is known about the dominating allergen sources and allergen molecules in China and what further investigations could be performed to draw a molecular map of IgE sensitization for China as a basis for the implementation of systematic and rational allergen-specific preventive and therapeutic strategies to combat allergic diseases in this country.

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.

More than 30% of the world population suffers from allergy. Allergic individuals are characterized by the production of immunoglobulin E (IgE) antibodies against innocuous environmental allergens. Upon allergen recognition IgE mediates allergen-specific immediate and late-phase allergic inflammation in different organs. The identification of the disease-causing allergens by demonstrating the presence of allergen-specific IgE is the key to precision medicine in allergy because it allows tailoring different forms of prevention and treatment according to the sensitization profiles of individual allergic patients. More than 30 years ago molecular cloning started to accelerate the identification of the disease-causing allergen molecules and enabled their production as recombinant molecules. Based on recombinant allergen molecules, molecular allergy diagnosis was introduced into clinical practice and allowed dissecting the molecular sensitization profiles of allergic patients. In 2002 it was demonstrated that microarray technology allows assembling large numbers of allergen molecules on chips for the rapid serological testing of IgE sensitizations with small volumes of serum. Since then microarrayed allergens have revolutionized research and diagnosis in allergy, but several unmet needs remain. Here we show that detection of IgE- and IgG-reactivity to a panel of respiratory allergens microarrayed onto silicon elements is more sensitive than glass-based chips. We discuss the advantages of silicon-based allergen microarrays and how this technology will allow addressing hitherto unmet needs in microarray-based allergy diagnosis. Importantly, it described how the assembly of silicon microarray elements may create different microarray formats for suiting different diagnostic applications such as quick testing of single patients, medium scale testing and fully automated large scale testing.

A large number of allergens have been discovered but we know little about their potential to induce inflammation (allergenic activity) and symptoms. Nowadays, the clinical importance of allergens is determined by the frequency and intensity of their IgE antibody binding (allergenicity). This is a rather limited parameter considering the development of experimental allergology in the last 20 years and the criteria that support personalized medicine. Now it is known that some allergens, in addition to their IgE antibody binding properties, can induce inflammation through non IgE mediated pathways, which can increase their allergenic activity. There are several ways to evaluate the allergenic activity, among them the provocation tests, the demonstration of non-IgE mediated pathways of inflammation, case control studies of IgE-binding frequencies, and animal models of respiratory allergy. In this review we have explored the current status of basic and clinical research on allergenic activity of indoor allergens and confirm that, for most of them, this important property has not been investigated. However, during recent years important advances have been made in the field, and we conclude that for at least the following, allergenic activity has been demonstrated: Der p 1, Der p 2, Der p 5 and Blo t 5 from HDMs; Per a 10 from P. americana; Asp f 1, Asp f 2, Asp f 3, Asp f 4 and Asp f 6 from A. fumigatus; Mala s 8 and Mala s 13 from M. sympodialis; Alt a 1 from A. alternata; Pen c 13 from P. chrysogenum; Fel d 1 from cats; Can f 1, Can f 2, Can f 3, Can f 4 and Can f 5 from dogs; Mus m 1 from mice and Bos d 2 from cows. Defining the allergenic activity of other indoor IgE antibody binding molecules is necessary for a precision-medicine-oriented management of allergic diseases.

Abstract Background Exposure to the house dust mite Dermatophagoides pteronyssinus (D.p.) increases the risk for developing allergic diseases in humans and their best friends, the dogs. Here, we explored whether this allergenic mite via its enzymes may impact the cutaneous extracellular matrix (ECM), which critically determines epithelial barrier integrity both structurally and functionally. Methods Two extracts obtained from either dust-purified or cultured D.p. bodies were used in the present study. To assess the potential impact of D.p. on protein components of the ECM, proteolytic activity of the D.p. extracts were determined by casein and gelatin gel zymography, and their N-acetyl-β-hexosaminidase activity determined colorimetrically. In addition, IgE-dependent and innate degranulation potential of D.p. was examined in canine MPT-1 mast cells and neurite outgrowth assay using rat pheochromocytoma PC-12 cells. Results In gel zymography, both extracts digested the substrates casein and gelatin in a dose-dependent manner, especially at alkaline pH, and effective in a wide range of temperatures (30 °C−42 °C). In particular, a 25-kDa band corresponding to Der p 1, the major D.p. allergen for humans, was found enzymatically active in both casein and gelatin gels regardless of the presence of metal ions and of alkaline conditions. Besides protease activity, N-acetyl-β-hexosaminidase activity was detected in both extracts, suggesting that D.p. affects the cutaneous ECM through deteriorating both proteins and glycosaminoglycans. While both D.p. extracts induced IgE-dependent mast cell degranulation, much less innate effects on mast- and neuronal cells were observed. Conclusions Our data highlight that D.p. is a robust source of several distinct enzymes with protease- and N-acetyl-β-hexosaminidase activities. In alkaline milieu they can degrade components of the ECM. Therefore, D.p. may contribute to epithelial barrier disruption especially when the skin surface pH is elevated.

Abstract Since mite allergens are the most relevant inducers of allergic diseases worldwide, resulting in significant morbidity and increased burden on health services, the International Collaboration in Asthma, Allergy and Immunology (iCAALL), formed by the American Academy of Allergy, Asthma and Immunology (AAAAI), the American College of Allergy, Asthma and Immunology (ACAAI), the European Academy of Allergy and Clinical Immunology (EAACI), and the World Allergy Organization (WAO), has proposed to issue an International Consensus (ICON) on the clinical consequences of mite hypersensitivity. The objectives of this document are to highlight aspects of mite biology that are clinically relevant, to update the current knowledge on mite allergens, routes of sensitization, the genetics of IgE responses to mites, the epidemiologic aspects of mite hypersensitivity, the clinical pictures induced by mites, the diagnosis, specific immunotherapeutic approaches, and prevention.

Table of contents ORAL ABSTRACTS Symposium 1: Biochemistry, structure and environment of the allergen: what makes a protein an allergen? O1 Two cell-membrane peptidases carrying galactose-alpha-1,3-galactose are implicated in delayed anaphylactic reactions upon pork kidney ingestion in patients with IgE-antibodies to alpha-Gal Christiane Hilger, Kyra Swiontek, Jörg Fischer, François Hentges, Christiane Lehners, Martine Morisset, Bernadette Eberlein, Tilo Biedermann, Markus Ollert O2 Structure solution of Pla l 1 suggests similar folding of Ole e 1-like family members but distinct immunological properties Sabrina Wildner, Teresa Stemeseder, Regina Freier, Peter Briza, Roland Lang, Eva Batanero, Mayte Villalba, Jonas Lidholm, Thomas Hawranek, Fatima Ferreira, Hans Brandstetter, Gabriele Gadermaier Symposium 2: New allergen molecules in the spotlight O3 Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed (Ambrosia artemisiifolia) Philippe Moingeon, Rachel Groeme, Julien Bouley, Véronique Bordas, Maxime Le Mignon, Laetitia Bussières, Aurélie Lautrette, Laurent Mascarell, Vincent Lombardi, Véronique Baron-Bodo, Henri Chabre, Thierry Batard, Emmanuel Nony O4 Production and characterization of polybia paulista recombinant antigen 5: a valuable diagnostic tool Karine Marafigo De Amicis, Alexandra Sayuri Watanabe, Daniele Danella Figo, José Roberto Aparecido Dos Santos-Pinto, Mario Sergio Palma, Fabio Fernandes Morato Castro, Jorge Kalil, Therese Wohlschlager, Peter Briza, Sabrina Wildner, Fatima Ferreira-Briza, Gabriele Gadermaier, Keity Souza Santos Symposium 3: Progress in molecular and cellular diagnosis O5 Basophil activation test with recombinant Pru p 3; identifying genuine peach allergic patients Margaretha Faber, Athina Van Gasse, Vito Sabato, Margo M. Hagendorens, Chris H. Bridts, Luc S. De Clerck, Araceli Diaz Perales, Didier Ebo O6 Nanofluidic technology enables rapid, near-patient quantification of allergen-specific IgE Petra Zavadakova, Aurélie Buchwalder, Fabien Rebeaud, Iwan Märki Symposium 4: Relevance of molecular diagnostics for intervention and treatment O7 Longitudinal analysis of Bet v 1-specific epitope repertoires during birch pollen immunotherapy Barbara Gepp, Nina Lengger, Christian Möbs, Wolfgang Pfützner, Christian Radauer, Barbara Bohle O8 A natural CCD-free tool: is polistes sp. venom suitable for polybia paulista diagnosis and therapy? Karine Marafigo De Amicis, Alexandra Sayuri Watanabe, Clovis Eduardo Galvao, Daniele Danella Figo, Jose Roberto Aparecido Santos-Pinto, Mario Sergio Palma, Fabio Fernandes Morato Castro, Jorge Kalil, Fatima Ferreira, Gabriele Gadermaier, Keity Souza Santos Symposium 5: The advent of molecular allergology in epidemiology O9 Peanut oleosins: from identification to diagnostic testing Christian Schwager, Skadi Kull, Frauke Schocker, Jochen Behrends, Wolf-Meinhard Becker, Uta Jappe O10 Endotypes of oral allergy syndrome in childhood: a molecular diagnostic approach Carla Mastrorilli, Salvatore Tripodi, Carlo Caffarelli, Riccardo Asero, Arianna Dondi, Giampaolo Ricci, Carlotta Povesi Dascola, Elisabetta Calamelli, Andrea Di Rienzo Businco, Annamaria Bianchi, Tullio Frediani, Carmen Verga, Iride Dello Iacono, Diego Peroni, Giuseppe Pingitore, Roberto Bernardini, Paolo Maria Matricardi Symposium 6: Molecular AIT: which approaches will make it to market? O11 Mbc4: an innovative molecule to tackle birch pollen and concomitant food allergies Heidi Hofer, Claudia Asam, Michael Hauser, Peter Briza, Martin Himly, Christof Ebner, Fatima Ferreira O12 Challenges and solutions associated with the production of recombinant Bet v 1 allergen as a therapeutic protein Emmanuel Nony, Maxime Le Mignon, Pierrick Lemoine, Karine Jain, Kathy Abiteboul, Monica Arvidsson, Sabina Rak, Philippe Moingeon Clinical Cases: Breakthroughs and headaches from CRD: interactive session CC1 Anaphylaxis caused by lipid transfer proteins: a complex clinical pattern syndrome Inês Mota, Filipe Benito Garcia, Angela Gaspar, Cristina Arêde, Susana Piedade, Graça Sampaio, Graça Pires, Luís Miguel Borrego, Cristina Santa-Marta, Mário Morais-Almeida CC2 IgE sensitization profile in a patient with asteraceae pollen-exotic fruits association Florin-Dan Popescu, Mariana Vieru, Florin-Adrian Secureanu CC3 Food-dependent: exercise induced anaphylaxis. Which component to blame? Rosa Anita Rodrigues Fernandes, Isabel Carrapatoso, Raquel Gomes, Celso Pereira, Ana Todo-Bom CC4 Anaphylaxis to intravenous iron preparations in a patient that tolerates oral administration María Cecilia Martín Fernández De Basoa, Javier Barrios Regio, Juan De Castro Cordova, Antón Fernández Ferreiro CC5 IgE sensitization pattern in an adult patient with oral allergy syndrome to peanuts and pollinosis from southern Romania Florin-Dan Popescu, Mariana Vieru, Florin-Adrian Secureanu CC6 Evidence of specific IgE to plant-derived cross-reactive carbohydrate determinant in a patient with delayed anaphylaxis to red meat Mariana Vieru, Florin-Dan Popescu, Florin-Adrian Secureanu POSTER PRESENTATIONS Poster Session 1: Molecular allergology and epidemiology P1 Atopic children produce stronger and more frequent IgG responses than non-atopic children: longitudinal data from the German MAS birth cohort Olympia Tsilochristou, Serena Perna, Alina Schwarz, Alexander Rohrbach, Antonio Cappella, Laura Hatzler, Carl-Peter Bauer, Ute Hoffmann, Johannes Forster, Fred Zepp, Antje Schuster, Raffael D’amelio, Ulrich Wahn, Thomas Keil, Susanne Lau, Paolo Maria Matricardi P2 The IgG sensitization profiles against 112 allergenic components support the absence of a protective role of IgG in allergic individuals, outside of the context of SIT Pol André Apoil, Claire Mailhol, Anne Broué-Chabbert, Agnès Juchet, Alain Didier, Elodie Carrer, Thomas Lanot, Antoine Blancher P3 The immune response against the timothy grass pollen allergen Phl p 5 in non-allergic humans Almedina Kurtaj, Christoph Hillebrand, Gerda Fichtinger, Martin Danzer, Christian Gabriel, Theresa Thalhamer, Sandra Scheiblhofer, Josef Thalhamer, Richard Weiss P4 Analyzing the cross-reactivity profile of the major ragweed allergen Amb a 1 Martin Wolf, Michael Hauser, Ulrike Pichler, Teresa Twaroch, Gabriele Gadermaier, Christof Ebner, Hidenori Yokoi, Toshiro Takai, Alain Didierlaurent, Adriano Mari, Peter Briza, Heidrun Behrendt, Angela Neubauer, Frank Stolz, Fátima Ferreira, Michael Wallner P5 LTP (Pru p 3) sensitisation in skin prick test: which means in clinical practice? Sara Carvalho, Tatiana Lourenço, Joana Cosme, Fátima Cabral Duarte, Amélia Spínola Santos, Ana Célia Costa, Manuel Pereira Barbosa P6 IgE profiles, allergen exposure and lifestyle of 501 Austrian pupils: investigation of influences on the development of allergic sensitizations Teresa Stemeseder, Eva Klinglmayr, Bettina Schweidler, Lisa Lueftenegger, Stephanie Moser, Patrick Doppler, Roland Lang, Martin Himly, Gertie J. Oostingh, Arne Bathke, Joerg Zumbach, Thomas Hawranek, Gabriele Gadermaier P7 Molecular profiles of sensitization to perennial inhalant allergens in a middle European region Petr Panzner, Martina Vachova, Tomas Vlas, Marek Maly P8 Evolution of the IgE response to house dust mite allergen molecules in childhood Daniela Posa, Serena Perna, Stephanie Hofmaier, Laura Hatzler, Alexander Rohrbach, Carl-Peter Bauer, Ute Hoffmann, Johannes Forster, Fred Zepp, Antje Schuster, Philippe Stock, Ulrich Wahn, Linus Grabenhenrich, Thomas Keil, Susanne Lau, Kuan-Wei Chen, Yvonne Resch, Susanne Vrtala, Rudolf Valenta, Paolo Maria Matricardi P9 Tropomyosin (Pen a1): to include or not to include in skin prick testing? Joana Cosme, Sara Carvalho, Tatiana Lourenço, Amélia Spínola Santos, Manuel Pereira Barbosa Immunoallergy Department - Hospital de Santa Maria – Centro Hospitalar Lisboa Norte, Lisbon, Portugal, Lisbon, Portugal; Immunoallergy Department - Hospital de Santa Maria – Centro Hospitalar Lisboa Norte, Lisbon, Portugal; Faculdade de Medicina de Lisboa, Lisbon, Portugal P10 Component-resolved IgE profiles in Georgian patients Tamar Abramidze, Nino Lomidze, Maia Gotua P11 Cross reactivity between food and pollen allergens in Lithuania according to spIgE evaluation Austeja Dapkeviciute, Ruta Einikyte, Jolita Norkuniene, Laima Skrickiene, Asta Miskiniene, Violeta Kvedariene P12 Distribution of inhalant allergy in the population of Lithuania Ruta Einikyte, Austeja Dapkeviciute, Jolita Norkuniene, Laima Skrickiene, Asta Miskiniene, Violeta Kvedariene Poster Session 2: Allergen molecules: identification, characterization, structure and function P13 Interference of antigen 5-based cross-reactivity in the diagnosis of hymenoptera venom allergy Maximilian Schiener, Bernadette Eberlein, Carmen Moreno-Aguilar, Gunilla Pietsch, Mareike Mc Intyre, Lea Schwarze, Dennis Rußkamp, Tilo Biedermann, Edzard Spillner, Ulf Darsow, Carsten Schmidt-Weber, Markus Ollert, Simon Blank P14 IgE cross-reactivity between European Hymenoptera and Asian hornet (Vespa velutina) venom allergens Cyril Longé, Andrea Brazdova, Jean-Louis Brunet, Claire Schwartz, Bruno Girodet, François Lavaud, Joelle Birnbaum, Nhân Pham Thi, Magalie Duchateau, Julia Chamot-Rooke, Laurence Guilloux, Marie-Ange Selva, Rémy Couderc, Hélène Sénéchal, Jean-Pierre Sutra, Pascal Poncet P15 Carbohydrate composition of house dust mite extracts and major group 1 and group 2 allergens Steffen Augustin, Linda Pump, Martin Wald, Thomas Eichhorn, Frank Fischer, Christoph Willers P16 Specificity of monoclonal antibodies against cross-reactive carbohydrate determinants Michaela Miehe, Melanie Plum, Sara Wolf, Frederic Jabs, Tim Raiber, Frank Bantleon, Henning Seismann, Thilo Jakob, Edzard Spillner P17 Red meat allergic patients have a selective IgE response to the a-Gal glycan Danijela Apostolovic, Anh Thu Tran, Sara Sanchez-Vidaurre, Tanja Cirkovic Velickovic, Maria Starkhammar, Carl Hamsten, Marianne Van Hage P18 Specificity of non-specific lipid transfer proteins and influence of the ligands on their three-dimensional structure Pawel Dubiela, Piotr Humeniuk, Sabine Pfeifer, Merima Bublin, Tomasz Borowski, Karin Hoffmann-Sommergruber P19 Real-time PCR analysis of Pru av 1 and Pru av 3 allergens Martie C.M. Verschuren, Shanna Bastiaan-Net, Defien Depoortere, Kay Foetisch, Stephan Scheurer, Harry J Wichers, Theo Noij P20 Specificity of anti-Pru av 1 antibodies for the detection of Pru av 1 isoallergens Martie C.M. Verschuren, Shanna Bastiaan-Net, Nikki M.E. Van Uden, Karel Vandenberghe, Kay Foetisch, Stephan Scheurer, Harry J. Wichers H.J., Theo H.M. Noij P21 Enhancing recombinant production yield of Bet v 1 through codon usage harmonization Anargyros Roulias, Maria Alejandra Parigiani, Heidi Hofer, Claudia Asam, Christof Ebner, Fátima Ferreira P22 Structural and dynamic insights into the world of PR-10 allergens Linda Ahammer, Sarina Grutsch, Martin Tollinger Poster Session 3: Allergen molecules: identification, characterization, structure and function P23 Purification of polcalcin from different pollen allergenic sources by antibody-affinity chromatography Raquel Moya, Mª Angeles López-Matas, Raquel Reyes, Jerónimo Carnés P24 Variations of wheat allergens in cultivars measured through a targeted quantitative mass spectrometry approach Colette Larré, Hélène Rogniaux, Roberta Lupi, Sandra Denery-Papini P25 Art v 1, Amb a 4 and Par h 1 defensin-like proteins share similar structural features but distinct immunological and allergenic properties Isabel Maria Pablos, Stephanie Eichhorn, Yoan Machado, Peter Briza, Christof Ebner, Jung-Won Park, Alain Didierlaurent, Naveen Arora, Stefan Vieths, Gabriele Gadermaier, Fatima Ferreira P26 Homogeneity or diversity of IgE-binding proteins in wheat dependant exercise induced anaphylaxis? Sandra Denery-Papini, Charlene Tanaka, Florence Pineau, Roberta Lupi, Martine Drouet, Etienne Beaudouin, Martine Morisset, Susan Altenbach P27 Deciphering the role of disulfide bonds and of repetitive epitopes in immunoglobulin E binding to wheat gliadins Sandra Denery-Papini, Hamza Mameri, Chantal Brossard, Roberta Lupi, Florence Pineau, Jean Charles Gaudin, Denise Anne Moneret-Vautrin, Etienne Beaudouin, Evelyne Paty, Martine Drouet, Olivier Tranquet, Colette Larré P28 Assessment of the allergenicity of soluble fractions from bread and durum wheats genotypes Roberta Lupi, Stefania Masci, Olivier Tranquet, Denise-Anne Moneret-Vautrin, Sandra Denery-Papini, Colette Larré P29 Isolation and characterization of Ara h 12 and Ara h 13: defensins, a novel class of peanut allergens Skadi Kull, Arnd Petersen, Marisa Böttger, Sandra Rennert, Wolf-Meinhard Becker, Susanne Krause, Martin Ernst, Thomas Gutsmann, Johann Bauer, Buko Lindner, Uta Jappe P30 Allergenicity attributes of different peanut market types Stef Koppelman, Shyamali Jayasena, Dion Luykx, Erik Schepens, Danijela Apostolovic, Govardus De Jong, Tom Isleib, Julie Nordlee, Joe Baumert, Steve Taylor, Soheila Maleki P31 The impact of peanut lipids on Ara h 1-induced immune responses in monocytes-derived dendritic cells Chiara Palladino, Barbara Gepp, Sofía Sirvent, Alba Angelina, Merima Bublin, Christian Radauer, Nina Lengger, Thomas Eiwegger, Oscar Palomares, Heimo Breiteneder P32 Compared allergenicity of native and thermally aggregated ovalbumin as large agglomerated particles Mathilde Claude, Roberta Lupi, Grégory Bouchaud, Marie Bodinier, Chantal Brossard, Sandra Denery-Papini P33 Simulation of the gastrointestinal digestion of the hazelnut allergens Cor a 9 and Cor a 11 by an in-vitro model and characterisation of peptidic products including epitopes by HPLC-MS/MS Robin Korte, Julia Bräcker, Jens Brockmeyer P34 Analysis of distribution of rice allergens in brown rice grain and allergenicity of the products containing rice bran Rie Satoh, Reiko Teshima Poster Session 4: Molecular approaches in AIT P35 Production of a recombinant hypoallergenic variant of the major peanut allergen Ara h 2 for allergen-specific immunotherapy Angelika Tscheppe, Dieter Palmberger, Merima Bublin, Christian Radauer, Chiara Palladino, Barbara Gepp, Nina Lengger, Reingard Grabherr, Heimo Breiteneder P36 Mutagenesis of amino acids critical for calcium-binding leads to the generation of a hypoallergenic Phl p 7 variant Marianne Raith, Linda Sonnleitner, Doris Zach, Konrad Woroszylo, Margit Focke-Tejkl, Herbert Wank, Thorsten Graf, Annette Kuehn, Ines Swoboda P37 Are birch pollen allergen immunotherapy induced blocking antibodies protective for cross-reactive allergens? Claudia Asam, Sara Huber, Heidi Hofer, Roland Lang, Thomas Hawranek, Fátima Ferreira, Michael Wallner P38 High success of 58 subcutaneous immunotherapy for pets allergy in a polyallergic cohort of patients: a component resolved individually adapted treatment (CRIAT) Fabienne Gay-Crosier P39 Neutrophils are potential antigen presenting cells in IgE- mediated allergy Dominika Polak, Birgit Nagl, Claudia Kitzmüller, Barbara Bohle P40 Characterization of allergen-specific CD8+ T cells in type I allergy Nazanin Samadi, Claudia Kitzmüller, Rene Geyeregger, Barbara Bohle, Beatrice Jahn-Schmid Poster Session 5: Molecular and cellular diagnostic tests P41 Nanofluidic-based biosensors allow quantification of total circulating IgE from a drop of blood in 5 minutes Aurélie Buchwalder, Ariel Gomez, Fabien Rebeaud, Iwan Märki P42 Allergen microarray for the analysis of serum IgE binding profile and allergenic activity Jaana Haka, Liisa Hattara, Marika Heikkinen, Merja H Niemi, Juha Rouvinen, Petri Saviranta, Pekka Mattila, Kristiina Takkinen, Marja-Leena Laukkanen P43 Generation of a well-characterized panel of periplaneta americana allergens for component resolved diagnosis Stephanie Eichhorn, Isabel Pablos, Bianca Kastner, Bettina Schweidler, Sabrina Wildner, Peter Briza, Jung-Won Park, Naveen Arora, Stefan Vieths, Gabriele Gadermaier, Fatima Ferreira P44 Improved diagnostic sensitivity of recombinant Api m 1 and Ves v 5 in diagnosis of Hymenoptera venom allergy Mira Silar, Julij Selb, Rok Kogovsek, Mitja Kosnik, Peter Korosec P45 Added value of biomarkers of primary sensitization and cross-reactivity in patients with hymenoptera venom allergy Leticia Pestana, Alcinda Campos Melo, Ana Mendes, Maria Elisa Pedro, Manuel Pereira Barbosa, Maria Conceição Pereira Santos P46 Cosensitization to Alt a 1 and Act d 2: more than a fortuitous association? Françoise Bienvenu, Claire Goursaud, Lorna Garnier, Sandrine Jacquenet, Michaël Degaud, Sébastien Viel, Annick Barre, Pierre Rougé, Jacques Bienvenu, Joana Vitte P47 Molecular diagnosis for peanut allergy: ALFA method performs as well as established methods for Ara h 1, Ara h 2, Ara h 6, Ara h 9 and CCD Amel Bensalah, Isabelle Cleach, Laurent Mousseau, Chantal Agabriel, Valérie Liabeuf, Joëlle Birnbaum, Jean-Louis Mège, Joana Vitte P48 Evaluation of a food challenge service in relation to specific IgE to molecular components in children with suspected peanut allergy James Gardner, Minal Gandhi, Harsha Kariyawasam, Giuseppina Rotiroti P49 Component resolved diagnosis in cereal allergy Isabel Carrapatoso, Celso Pereira, Frederico Regateiro, Emília Faria, Ana Todo-Bom Poster Session 6: Molecular diagnosis in prevention and therapy P50 Pretreatment molecular sensitizations determine the sIgG4 induction during the updosing of SCIT and may be useful to identify clinically relevant additional sensitizations Johannes Martin Schmid, Ronald Dahl, Hans Juergen Hoffmann P51 Usefulness of recombinant latex allergens in immunotherapy’s decision and follow-up Inês Mota, Filipe Benito Garcia, Angela Gaspar, Mário Morais-Almeida P52 Omega-5-gliadin in the diagnosis of wheat-dependent anaphylaxis induced by ibuprofen but not by exercise Joana Cosme, Letícia Pestana, Amélia Spínola Santos, Manuel Pereira Barbosa P53 Food dependent exercise-induced anaphylaxis: a component-resolved and in vitro depletion approach to access IgE cross-reactivity Diana Silva, Teresa Vieira, Ana Maria Pereira, André Moreira, Luís Delgado P54 Olive pollen allergens: what are we missing? Sara Prates, Cátia Alves, Elena Finelli, Paula Leiria Pinto P55 Purified Alt a 1 extract in Alternaria alternata allergy diagnosis Bárbara Kong Cardoso, Cíntia Cruz, Filipa Semedo, Elza Tomaz, Filipe Inácio P56 Use of specific IgE Bos d8 (casein) to aid early introduction of dietary baked milk in children with cows’ milk allergy James Gardner, Santanu Maity, Giuseppina Rotiroti, Minal Gandhi P57 Molecular characterisation and immunoreactivity of a peanut ingredient for use in oral food challenges Ivona Baricevic-Jones, Justin T. Marsh, Phil E. Johnson, Anuradha Balasundaram, Anya-May Hope, Aafke Taekema, Angela Simpson, Aida Semic-Jusufagic, E.N. Clare Mills P58 Specific IgE to recombinant allergens of hazelnut and oral food challenge in children Gourdon Dubois Nelly, Sellam Laetitia, Pereira Bruno, Michaud Elodie, Messaoudi Khaled, Evrard Bertrand, Fauquert Jean-Luc Poster session 7/8: miscellaneous P59 What defines a protein as an allergen? A discussion of sources and sufficiency Richard E. Goodman P60 Cat allergy: relationship between clinical and molecular diagnostic María Cecilia Martín Fernández De Basoa, Antón Fernández Ferreiro, Elena Rodríguez Plata P61 Anaphylaxis to rabbit: the cat came in last Luis Amaral, Borja Bartolomé, Alice Coimbra, Jose L Placido P62 Dog allergy: relationship between clinical and molecular diagnostic María Cecilia Martín Fernández De Basoa, Antón Fernández Ferreiro, Elena Rodríguez Plata P63 Correlation of serum timothy grass-pollen specific IgE levels determined by two immunoblot test systems Mariana Vieru, Florin-Dan Popescu, Florin-Adrian Secureanu, Carmen Saviana Ganea P64 Development of oral food challenge formulations for diagnosis of fish allergy using powdered fish ingredients Carol Ann Costello, Ivona Baricevic-Jones, Martin Sorensen, Clare Mills, Adrian Rogers, Aage Otherhals P65 Fish and peanut allergens interact with plasma membranes of intestinal and bronchial epithelial cells and induce differential gene expression of cytokines and chemokines Tanja Kalic, Isabella Ellinger, Chiara Palladino, Barbara Gepp, Eva Waltl, Verena Niederberger-Leppin, Heimo Breiteneder P66 Interleukin 4 affects fat tissue metabolism and expression of pro-inflammatory factors in isolated rat adipocytes Dawid Szczepankiewicz, Ewa Pruszynska-Oszmalek, Marek Skrzypski, Krzysztof W. Nowak, Aleksandra Szczepankiewicz P67 Ozone induced airway hyperreactivity in PD-L2−/− mice model Gwang-Cheon Jang P68 Thymic stromal lymphopoietin (TSLP) and its receptor as targets for the development of anti-inflammatory inhibitory agents Iva Markovic, Andreas Borowski, Tina Vetter, Andreas Wohlmann, Michael Kuepper, Karlheinz Friedrich P69 The mononuclear phagocyte system in experimentally-induced allergic rhinitis Ibon Eguiluz Gracia, Anthony Bosco, Ralph Dollner, Guro Reinholt Melum, Anya C Jones, Maria Lexberg, Patrick G Holt, Espen Sønderaal Bækkevold, Frode Lars Jahnsen P70 Expression of histamine metabolizing enzymes is increased in allergic children Aleksandra Szczepankiewicz, Paulina Sobkowiak, Marta Rachel, Beata Narozna, Dorota Jenerowicz, Witold Swiatowy, Anna Breborowicz P71 Modifying the glycosylation of human IgE towards oligomannosidic structures does not affect its biological activity Melanie Plum, Sara Wolf, Frank Bantleon, Henning Seismann, Frederic Jabs, Michaela Miehe, Thilo Jakob, Edzard Spillner P72 Flying Labs: an educational initiative to transfer allergy research into high-school settings Michael Wallner, Heidi Hofer, Fatima Ferreira, Reinhard Nestelbacher P73 Clinical significance of antihistamines and Kujin, an anti-allergic Kampo medicine Hiroyuki Fukui

Background: House dust mites (HDM) are distributed almost worldwide and are the main elicitors of indoor allergies. Diagnosis of HDM allergy is mainly performed with natural allergen extracts which are often of insufficient quality. Objective: To comp are diagnostic tests based on specific IgE serology to natural HDM extracts (ImmunoCAP [d1] with tests based on recombinant HDM allergens (ImmunoCAP, ImmunoCAP ISAC, ELISA). Methods: Sera from 27 well-characterized HDM allergic patients were tested for IgE reactivity to the most important HDM allergen molecules (Der p 1, 2, 5, 7, 21 and 23) by ELISA, dot blot and ImmunoCAP ISAC microarray technology (allergen-chip containing 13 house dust mites allergens). Additionally, IgE-reactivity was tested to a mixture of the six most important HDM allergens using Streptavidin ImmunoCAP, and to natural house dust mites extract by ImmunoCAP (d1). Results: In each of the 27 house dust mites allergic patients IgE sensitization could be diagnosed with each of the serological tests. A strong positive correlation was found between IgE measurements performed with the extract-based ImmunoCAP and the ImmunoCAP containing a mix of the house dust mites allergen molecules (r = 0.9418, p

Background: IgE-reactivity to antigens from gram-positive and negative bacteria is common in patients suffering from respiratory and skin manifestations of allergy, but the routes and mechanisms of sensitisation are not fully understood. The analysis of the genome, transcriptome and microbiome of house dust mites has shown that Staphylococcus aureus and Escherichia coli species are abundant bacteria within the house dust mites microbiome. Therefore, our aim was to investigate if house dust mites are carriers of bacterial antigens leading to IgE sensitisation in patients suffering from atopic dermatitis. Methods: Plasma samples from atopic dermatitis patients (n = 179) were analysed for IgE-reactivity to a comprehensive panel of micro-arrayed house dust mites allergen molecules and to Staphylococcus aureus and Escherichia coli by IgE immunoblotting. Antibodies specific for Staphylococcus aureus and Escherichia coli antigens were tested for reactivity to nitrocellulose-blotted extract from purified house dust mites bodies and the IgE-reactive antigens were detected by IgE-immunoblot inhibition experiments. IgE antibodies directed to bacterial antigens in house dust mites were quantified by IgE ImmunoCAPTM inhibition experiments. Results: IgE-reactivity to bacterial antigens was significantly more frequent in atopic dermatitis patients sensitised to house dust mites than in atopic dermatitis patients without house dust mites sensitisation. Staphylococcus aureus and Escherichia coli antigens were detected in immune-blotted house dust mites extract and the presence of IgE-reactive antigens in house dust mites was demonstrated by qualitative and quantitative IgE inhibition experiments. Conclusions: House dust mites may serve as carriers of bacteria responsible for the induction of IgE sensitisation to microbial antigens.

Background: IgE-associated allergic sensitization to house dust mite (HDM) allergens is associated with a variety of allergic symptoms such as rhinitis, conjunctivitis, dermatitis and asthma. Allergen exposure chambers allow the precise assessment of clinical symptoms specifically induced with HDMs. Objective: To investigate if an association between serologic IgE and HDM-induced symptoms such as asthma, rhinitis and conjunctivitis can be established. Methods: The clinical symptoms of 91 HDM allergic patients were assessed in the Vienna Challenge Chamber during a controlled 6-hour exposure to HDM allergens. The symptoms assessed were total nasal symptom score, total ocular symptom score and asthma symptom score. Sera from the 91 patients were tested for IgE reactivity to 13 Dermatophagoides pteronyssinus allergens using allergen microarray technology (ImmunoCAP ISAC chip technology) and allergen-specific IgE levels to Dermatophagoides pteronyssinus (Der p) allergen extract were quantified by ImmunoCAP measurements. Results: Patients with high Der p-specific IgE levels measured by ImmunoCAP (> 10 kU/L) had higher IgE-levels to all important HDM allergens than patients with low IgE levels ( 6, mean value of four hours) also had higher asthma symptom score and total ocular symptom score, as well as higher IgE-levels to micro-arrayed Der p: 2, 5, 7, 21 and 23 compared to patients with low total nasal symptom score ( 4) and in patients with high Total Ocular Symptom Score (> 2) compared to those with low symptom scores. Conclusions: Our study indicates that IgE-reactivity profiles to micro-arrayed HDM allergens but not allergen-specific IgE levels to HDM extracts are associated with allergic symptoms to HDM exposure.

BACKGROUND:The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated. OBJECTIVE:To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level. METHODS:Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments. RESULTS:Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients. CONCLUSION:Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.

Recently, a protein homologous to glutathione-S-transferases (GST) was detected in prominent amounts in birch pollen by proteomic profiling. As members of the GST family are relevant allergens in mites, cockroach and fungi we investigated the allergenic relevance of GST from birch (bGST).bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. Sera from 217 birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The mediator-releasing activity of bGST was analysed with IgE-loaded rat basophil leukaemia cells (RBL) expressing human FcεRI. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses to either protein were assessed. IgE-cross-reactivity between bGST with GST from house dust mite, Der p 8, was studied with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen were assessed in water, simulated lung fluid, 0.9% NaCl and PBS. Eluted proteins were quantified by ELISA and analysed by immunoblotting.Only 13% of 217 birch pollen-allergic patients showed IgE-reactivity to bGST. In RBL assays bGST induced mediator release. Immunization of mice with bGST induced specific IgE and a Th2-dominated cellular immune response comparably to immunization with Bet v 1. bGST did not cross-react with Der p 8. In contrast to Bet v 1, only low amounts of bGST were released from pollen grains upon incubation in water and the different physiological solutions.Although bGST is abundant in birch pollen, immunogenic in mice and able to induce mediator release from effector cells passively loaded with specific IgE, it is a minor allergen for birch pollen-allergic patients. We refer this discrepancy to its limited release from hydrated pollen. Hence, bGST is an example demonstrating that allergenicity depends mainly on rapid elution from airborne particles.

Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptide were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37) and in the eukaryotic system (BvTri a 37) as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR) and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN). Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of sequential IgE epitopes indicates that sensitization to alpha-purothionin occurs via the gut. Both allergens can be used for in-vitro diagnosis of wheat food allergy. The induction of blocking IgG antibodies suggests the usefulness for immunotherapy.