6th International Symposium on Molecular Allergology (ISMA)

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

there is neither a diagnostic nor a treatment tool commercially available. Hence Polistes sp. venom extract is used for skin tests and immunotherapy of Brazilian wasp venom allergic patients. The proximity of physicians and researchers in our group allows the use of ELISA and western blot analysis as a diagnosis support tool. Polistes venom is devoided of cross-reactive carbohydrate determinants (CCDs) allowing CCD-free differential diagnosis of P. paulista allergy. The question regarding protection of Polybia allergic patients treated with Polistes venom extract in the immunotherapy remains unanswered. Materials and methods: Twenty patients with clinical history of anaphylaxis to wasp venom were included and tested by ImmunoCAP ® to Polistes(i4) and MUXF3 (o214). Skin prick (SPT) and intradermal test (ID) was performed using commercial Polistes venom extract. ELISA and western blot (WB) was made using commercial Polistes venom extract and Polybia venom extract produced by our group besides HRP ELISA for IgE anti-CCD detection. Results: Patients presented clinical manifestations of anaphylaxis with symptoms that included urticaria, angioedema, diarrhea, respiratory symptoms and loss of consciousness. SPT was negative and the allergy was confirmed by ID test. In the IgE WB, all patients were positive to Polybia and Polistes recognizing multiple IgE-reactive bands different in each venom extract. In spite of this in IgE ELISA where the magnitude of IgE response to Polybia is 7 times higher than to Polistes only 5 patients are positive for both venoms from those only 2 are positive for CCD. Results from ELISA, WB and CAP for Polistes are not coincident. Patients positive in CAP for Polistes are many times negative in ELISA. Conclusions: Polistes venom extract is not suitable for diagnosis and treatment of Polybia patients as the sensitization pattern showed some differences. Despite the already reported cross-reactivity with homologs, possible new allergens unique in each venom were observed presenting distinct molecular masses not yet described. Conflicting results using extract goes along with previously reported problems in standardization of extracts used for diagnosis and treatment. Recombinant production and characterization of the Polybia venom allergens would help to elucidate the diagnoses and allow proper therapy for P. paulista venom allergic patients. Background: A rising prevalence of peanut allergy has been observed within the past decades. Routine diagnostic measures applying aqueous extracts (e.g. for skin prick test or IgE-detection measures) can lead to false negative results due to a lack of potential lipophilic allergens. Oleosins, a class of oil body proteins, have been found to be triggers of severe allergic reactions to hazelnut and sesame. Since data for peanut oleosins are scarce, the aims of the study were the isolation, the molecular characterization and the assessment of the oleosin allergenicity. Moreover, we searched for a specific diagnostic approach to close the existing detection gap in the established peanut allergy diagnostics. Materials and methods: A comprehensive oleosin isolation procedure was established which comprises extraction and subsequent step by step purification of oil bodies along with preparative electrophoresis. Protein identification was achieved by N-terminal sequencing, peptide mass fingerprinting and homology search against databases.
The IgE-binding capacity of oleosins was evaluated in western blot experiments with sera of peanut-allergic, tolerant and non-allergic individuals. A flow cytometric basophil activation test was used for the diagnosis of oleosin-allergic patients. Results: Oleosins were isolated and purified from the complex lipophilic matrix of peanut. Mass spectrometry analysis identified all known eight peanut oleosins, ranging from 15.5 to 17.5 kDa as well as further oil body related proteins (steroleosins and caleosins). IgE-binding to purified oleosins was observed in 20 of 39 sera from peanut-allergic patients by means of immunoblotting. Thus, oleosins meet the criteria to be classified as new allergens and isoallergens according to the WHO/IUIS allergen nomenclature subcommittee, and were accepted and designated as Ara h 14 and Ara h 15 in May 2015. Positive immunoblot results were observed solely in patients suffering from severe allergic reactions. IgE-dependent basophil activation was induced in vitro in a dose-dependent manner in peanut-allergic patients, but not in controls. Conclusions: A novel strategy for the simultaneous isolation of the lipophilic peanut allergens oleosins was successfully established. The allergenicity of the distinct oleosins was illustrated by IgE-detection in immunoblot and confirmed by a functional cellular assay, the basophil activation test. Oleosins proved to be relevant allergens that may cause more severe allergic reactions. Keywords: Oleosins; Allergens; Isolation; Diagnosis Background: Oral allergy syndrome (OAS) is a common adverse reaction to the ingestion of plant foods in patients with pollen-related allergic rhinoconjunctivitis. There is limited epidemiological information on childhood OAS in regions with highly complex pollen exposure. We aimed the present study to investigate OAS, its risk factors and underlying IgE sensitization patterns in children living in a Mediterranean country. Materials and methods: This cross-sectional study assessed 1271 children (age 4-17 years) with pollen-related seasonal allergic rhinoconjunctivitis (SAR), enrolled by 16 Italian outpatient clinics. Data about OAS symptoms and their food triggers were acquired by a standardized questionnaire. Skin prick tests (SPTs) were performed with commercial pollen and food extracts. Specific IgE to the panallergens Phl p 12 (profilin), Bet v 1 (pathogenesis related protein class 10, PR-10) and Pru p 3 (non-specific lipid transfer protein, nsLTP) were tested by ImmunoCAP FEIA. Results: Oral allergy syndrome was observed in 300/1271 (23.6 %) children with a prevalence increasing from age 5 year (24 %) to age 16 year (37 %). Risk factors for OAS were a female gender (p < 0.001), Clin Transl Allergy 2016, 6(Suppl 2):38 maternal OAS (p < 0.001), living in the Northern Italy (p < 0.05), and passive exposure to tobacco smoke (p < 0.01). OAS was strongly associated with other SAR comorbidities: asthma (p < 0.001), anaphylaxis (p < 0.005), urticaria and/or angioedema (p < 0.001), atopic dermatitis (p < 0.001), and gastrointestinal symptoms (p < 0.001). IgE to one or more panallergens were found in 229/300 (76.3 %) of OAS affected children. Cucurbitaceae (melon and watermelon) were associated with IgE sensitization to Phl p 12; Rosaceae (apple, peach, pear) were strongly associated with IgE sensitization to Bet v 1; peanuts and tree nuts (hazelnut and walnut) were associated with IgE sensitization to Pru p 3. Kiwi's allergy was the most frequent cause of OAS in children not sensitized to any of the three examined panallergens. Conclusions: In a geographic area with complex pollen exposure, OAS is a highly frequent complication of childhood seasonal allergic rhinitis, even at preschool age. IgE sensitization to profilin, PR-10 and nsLTP explains most but not all of OAS childhood morbidity and kiwi allergy is the most important trigger. These results call for the inclusion of early diagnosis and OAS treatment in guidelines of pollen-related seasonal allergic rhinitis in childhood. Keywords: Oral allergy syndrome; Panallergens; Profilin; Lipid-transfer protein; PR-10 Symposium 6: Molecular AIT: which approaches will make it to market? Background: Amongst tree pollen allergies, birch is one of the main causes of winter and spring pollinosis in the temperate climate zone of the Northern hemisphere. This is caused by sensitization of patients towards Bet v 1, the major birch pollen allergen. Most of these patients not only suffer from rhinitis and asthma, but also develop adverse reactions towards various fruits, nuts, and vegetables-most frequently against apple and hazelnut. These reactions are limited to the oral cavity and triggered by food proteins structurally related to Bet v 1, as these allergens are also able to cross-link Bet v 1 specific IgE on mast cells and basophils. Therefore, we designed a hybrid molecule (MBC4) by assembling parts of Bet v 1 from birch, Mal d 1 from apple and Cor a 1 from hazelnut. For an increased safety profile, we introduced a mutation into the backbone of MBC4 to reduce its IgE binding capacity. Materials and methods: Parental allergens, as well as MBC4 were expressed in E. coli, purified to homogeneity and characterized physico-chemically. Sera from clinically diagnosed patients with birch pollen allergy and concomitant pollen-food syndrome towards apple and hazelnut were analyzed by IgE ELSIA and mediator release assay to determine the IgE binding capacity of MBC4. Additionally, we monitored the immunological behavior in vivo in a mouse model. Results: Significantly reduced IgE binding and allergenic effector function of MBC4 was observed by ELISA and mediator release assay, respectively. Analyses of patients´ sera revealed that the hybrid molecule showed a hypoallergenic factor of 10000 when compared to Bet v 1. Although IgE binding was reduced, MBC4 was able to induce crossreactive IgG antibodies to parental allergens in vivo, whereas murine IgE antibodies revealed a very limited cross-reactivity. Additionally, when splenocytes from mice where re-stimulated with either parental allergen or MBC4, a cross-reactive T cell response was observed in ELISpot assays. Conclusions: As MBC4 revealed reduced IgE binding and allergic function in mice and man, and further was able to induce a cross-reactive T cell response as well as cross-reactive IgG antibodies, it presents itself as a suitable vaccine candidate to treat birch pollen and associated food allergies towards apple and hazelnut. This work was supported by FWF L688 and ÖNB 12533 grants. Keywords: Birch pollen allergy; Oral allergy syndrome; Allergen immunotherapy; Hybrid molecule Clin Transl Allergy 2016, 6(Suppl 2):38 fruits, vegetables, grains, peanut, tree nuts and also in pollens. It is a common cause of food-induced anaphylaxis (FIA) in adults living in Mediterranean area. LTP has also been proposed as a main cause of food-dependent exercise-induced anaphylaxis (FDEIA). We aimed to describe clinical characteristics and allergen sensitization profiles in patients with FIA related to LTP. Materials and methods: Twenty eight patients were included [mean age 27.1 (SD ± 12.8) years, 29 % <18 years and 50 % male) with clinical history of FIA, whose allergological work-up confirmed sensitization to LTP. Patients were tested with a multiple plant-food and pollen panel and specific IgE to LTP allergens. LTP sensitization was assessed by in vivo (Pru p 3, LTP extract, Bial-Aristegui ® ) or by in vitro tests (specific IgE, ImmunoCAP / ISAC, ThermoFisher ® ). Results: Median age of first anaphylactic episode was 26.5  yrs; 46 % had asthma; 68 % were atopic and 54 % had pollinosis (mugwort, wall pellitory, plane tree, olive and cypress). Co-sensitization to profilins was found in 14 %. Overall in our center, LTP-induced anaphylaxis represents 15.7 % of all causes of FIA. Foods implicated in anaphylactic reactions were: Rosaceae fruits-36 % (peach and apple), tree nuts-29 % (walnut, cashew nut and hazelnut), seeds-25 % (sesame, sunflower seed and flaxseed) and other vegetables-21 % (peanut, green bean, goji berry, tomato and maize). In three cases the food implicated remained unidentified. Three patients had FDEIA (green bean, tree nuts, tomato and maize). Clinical manifestations were: mucocutaneous 100 %, respiratory 86 %, cardiovascular 25 % and gastrointestinal 21 %. In 75 % the reaction occurred within the first 30 minutes after food ingestion; 29 % had ≥3 FIA before etiologic diagnosis. Conclusions: LTP are important allergens of FIA in Portugal. LTP sensitization is responsible for a heterogeneous group of offending plantfoods, taxonomically unrelated, which can induce recurrent reactions. It is a useful risk marker in patients with severe reactions due to an unknown cause. The association of LTP-induced anaphylaxis with pollinosis is relevant in our country. The unpredictable clinical expression depends on the effect of cofactors such as exercise. The management of avoidance plans can be particularly challenging due to LTP be a widely cross-reacting allergens in plant-foods. Keywords: Lipid transfer proteins; Panallergens; Anaphylaxis; Food allergy; Food-dependent exercise-induced anaphylaxis Background: There are few reports of allergic reactions to exotic fruits belonging to the Sapindaceae or Anacardiaceae plant families. Materials and methods: We evaluated IgE sensitization profile in a 22-year-old woman with seasonal allergic rhinoconjunctivitis and history of food-dependent exercise-induced anaphylaxis to pistachio, anaphylaxis to lychee, mango and polyfloral bee polen dietary supplement. Skin prick testing (SPT) was performed with commercial extracts, prick-prick testing with edible foods, serum specific IgE levels were determined by immunoblot test systems and fluorescent enzyme immunoassay (FEIA). Oral challenge tests were not performed due to clear history and ethical reasons. Results: SPT revealed positive wheal reactions to Asteraceae pollen extracts of Ambrosia elatior (10 mm), Artemisia vulgaris (4 mm) and Helianthus annuus (5 mm). SPT with liquid extract of lychee (5 % purée of Litchi chinensis, fruit of the Sapindaceae family) was also positive (4 mm). Prick-to-prick tests with both Anacardiaceae native fruits were positive: mango (Mangifera indica) fruit pulp (5 mm) and pistachio (Pistacia vera) drupe seed (3 mm). Values of serum pollen-specific IgE by immunoblot were high for ragweed (79 kU/L, EAST class 5) and mugwort (4.6 kU/L, EAST class 3) pollen. Specific IgE to mango (<0.35 kU/L, FEIA class 0) and lychee (<0.35 kU/L, EAST class 0) fruits were not detected. Molecular allergy assessment revealed involvement of profilin in this particular phenotype of pollen-food allergy association, specific IgE to rBet v2 (3.2 kU/L, EAST class 2) being considered a profilin sensitization biomarker, homologous (hm) with mugwort Art v 4, ragweed Amb a 8, lychee Lit c 4 and mango Man i 3 profilins. IgE sensitization to other tested allergen components was not found (<0.35 kU/L): rBet v 6 (isoflavone reductase hm with lychee Lit c IFR), rBet v 1 (protein hm with mango Man i 14 kD), rAra h 1 (7S vicilin hm with pistachio Pis v 3), rAra h 2 (2S albumin hm with pistachio Pis v 1), rAra h 3 (11S globulin hm with pistachio Pis v 2 and Pis v 5) and plant cross-reactive carbohydrate determinant (CCD). Conclusions: In regions such as Southern Romania, where Asteraceae pollen sensitization is important, allergists must be aware of the potential role of cross-reactivity between mugwort and/or ragweed pollen and several exotic fruits, allergy risk of bee pollen supplements, and the role of in vivo and in vitro allergy testing, including component-resolved diagnosis.
Consent: Written informed consent was obtained from the patient for publication of this abstract and any accompanying images.

Background:
We report the case of a 27-year-old man that started suffering from recurrent episodes of anaphylaxis, following the ingestion of meals containing mixed foods. The first crisis occurred when he was 20 years old, with generalized urticaria and angioedema, hypotension and blurred vision after ingestion of pizza. Six months later another episode occurred after ingestion of meatballs and spaghetti. One year later, the same symptoms occurred with mixed food. Interestingly, all the systemic episodes developed 30-60 minutes after exercise. Self-administered adrenaline was prescribed and the patient was advised not to exercise at least 2 hours after eating. Between the ages of 21 and 26 no more anaphylactic episodes were reported. This patient lost follow up. Two years later he was readmitted in our outpatient department after treatment in the emergency room for exercise induced anaphylaxis. He also has complains of intermittent respiratory symptoms predominantly in the spring, since the age of 26. Materials and methods: Skin prick tests (SPT) to commercial extracts of aeroallergens, foods allergens, Pru p 3 and profilin were carried out. Prick-to-prick tests (PP) and serum specific IgE determinations (sIgE) to airborne allergens and some plant foods were also performed, according to case history. Molecular diagnosis with ImunoCAP-ISAC and specific IgE determinations to some components were also performed. Oral provocation tests (OPT) were performed to some of the suspected foods. Results: Sensitisation to grass pollens, mugwort and multiple foods was demonstrated as shown in the Table 1 below. OPT to peach and pizza crust with exercise was negative. Conclusions: In this patient with recurrent food-dependent exercise anaphylaxis the culprit allergen seems to be a non-specificLTP, as sIgE to Pru p 3 and Tri a 14 were positive and sIgE to ω-5-gliadin negative. Non-specific LTPs have been associated with cofactor-enhanced fooddependent anaphylaxis in southern Europe. The variability of anaphylactic reactions occurred in this patient could be explained by the type of exercise, food with different wheat processing and the season of the year. Keywords: Anaphylaxis; Tri A 14; Wheat; Exercise Consent: Written informed consent was obtained from the patient for publication of this abstract and any accompanying images.
Background: Oral administration of iron salts for iron deficiency treatment is usually well tolerated. Adverse gastrointestinal side effects are the most common. Exanthematous eruptions or anaphylactic reactions following a parenteral iron dextran injection or intravenous sucrose preparations are very uncommon We report a case of anaphylactic reaction caused by intravenous administration of iron salts. We also present the results of the allergy study Materials and methods: Case Description A 71-years-old woman with a personal history of type 2 diabetes, dyslipidemia and iron deficiency anemia was referred to our department to be tested for a possible allergic reaction to iron salts. The patient reported that after starting the infusion immediately presents generalized micropapular urticarial reaction associated with intense feeling of heat and hypotension, so the infusion was stopped and treated with intramuscular methylprednisolone and dexchlorpheniramine. However she tolerate oral iron in ferrous form Results: Skin tests were performed with FERIV ® 20 mg/mL [sucrose and hydroxide iron (III)] and with excipients of the commercial formulations. Positive responses were obtained in intradermal tests with FERIV ® (1/1000 and 1/10,000). The excipients tested proved negative Basophil activation test (BAT) were performed with FERIV ® and FER-ROCUR 40 mg [40 mg Fe 3+ ] oral solution (tolerated by the patient). Positive responses were obtained in BAT with FERIV ® (concentrations: 10 −1 and 10 −2 ) and FERROCUR (concentration: 10 −2 ). The excipients tested proved negative. BAT performed with FERIV ® and FERROCUR in 2 healthy controls were also negative We could not perform an intradermal tests with FERROCUR because the patient had a multiple organ dysfunction syndrome and died Conclusions: Iron salts are seldom responsible for allergic reactions. A few cases of eruptive dermatosis have been reported However, very few studies examine mild anaphylactic reactions or severe anaphylactic reactions after intravenous administration.
The pathogenesis of anaphylactic reactions to iron dextran has not yet been elucidated. In most reports, these episodes occur after the first contact with the drug. This should support an anaphylactoid origin rather than a truly anaphylactic reaction. In our case, both the positive skin tests and the negative results in the control subjects tested corroborate the specificity of the response and demonstrate the existence of an immediate, possibly IgE-mediated hypersensitivity mechanism Keywords: Intravenous iron salts; Anaphylaxis; Basophil activation test Consent: Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. Background: Many IgE-mediated food allergies in adults are caused by cross-reactivity between aeroallergens and food allergens. Peanuts are not frequently involved in these pollen-food syndromes or associations. Materials and methods: From adult patients with pollen-induced allergic rhinitis referred to our allergy clinic in one year-period, screened for suggestive history of allergy to Arachis hypogaea legume, we found only one 34-year-old female with moderate-severe persistent allergic rhinoconjunctivitis, positive skin prick tests to five grass mix (6 mm wheal) and Artemisia vulgaris (5 mm wheal) pollen extracts, and convincing history of oral allergy syndrome to peanuts. Specific IgE sensitization pattern was assessed using multi-parameter immunoblot test system for defined partial allergen diagnosis. Results: Serum specific IgE levels were found increased for Thimothy grass pollen (99 kU/L) and mugwort pollen (2.5 kU/L). IgE sensitization to single purified allergen components revealed genuine sensitization to Pooideae grass pollen: Phl p 1 (55 kU/L) and Phl p 5 (17.5 kU/L). Serum IgE to cross-reactive carbohydrate determinant marker was absent (<0.35 kU/L), important in vitro assessment for excluding false positive IgE to peanuts in patients with grass pollen allergy. Specific IgE against plant cross-reactive allergen components, such as grass polcalcin (Phl p 7 crossreactive with weed Art v 5) and profilin (Phl p 12 cross-reactive with weed Art v 4), were not found. Low antibody serum titer of specific IgE to whole peanut extract was detected (0.7 kU/L). Serum specific IgE against peanut allergen components involved in early sensitization and systemic reactions, the storage proteins rAra h 1 (7S vicilin), rAra h 2 (2S albumin) and rAra h 3 (glycinin legumin), and the nonspecific lipid transfer protein (LTP) involved in oral and/or systemic reactions rAra h 9, were not detected (<0.35 kU/L). IgE sensitization to defensins Art v 1, Ara h 12, Ara h 13 may explain cross-reactivity. In addition, specific IgE to Ara h 5 (profilin), Ara h 8 (PR-10 Bet v 1-like protein), Ara h 10 and Ara h 11 (oleosins) were not available for component resolved diagnosis, but IgE to profilin biomarker rBet v 2 and PR-10 biomarker rBet v 1 were not detected (<0.35 kU/L). Conclusions: A multiplex immunoblot assessment of single purified allergen peanut components can be used to determine the risk of systemic reactions in selected patients with pollinosis and IgE sensitization to peanuts. Consent: Written informed consent was obtained from the patient for publication of this abstract and any accompanying images.  Background: A significant increase of IgG (especially IgG 4 ) anti-allergen antibodies is significantly associated with the success of SIT. However, in non-allergic subjects, the presence of IgG specific for allergens is well documented, and their role is controversial.

Materials and methods:
We studied the IgG anti-allergen antibodies in 142 allergic patients with food and/or respiratory allergy symptoms and 53 control subjects, clinically asymptomatic and free of IgE sensitization, all living in the Southwest of France. After having determined the IgE sensitization profile by means of the ImmunoCAP ISAC ™ , we re-incubated the biochips with anti-human IgG polyclonal antibodies (Fc-specific) coupled to the AlexaFluor 647. The raw fluorescence values were converted to arbitrary units of IgG by means of a calibration serum having IgG reactivity toward 5 allergen components (nGal d3, rBla g 2, nArt v 3, nBos d lactoferrin, nGal d 1). Results: We found that more than 80 % of individuals, allergic or not, have some IgG directed against egg white (nGal d1, d2), cow's milk (nBos d5, d8), or one component from olive tree pollen (rOle e 9). For 32 components, allergic patients have concentrations of specific IgG significantly higher than non-allergic individuals (p < 0.001). Among the allergen components derived from the same organism, some are able to induce more frequently an IgE-sensitization, while other components mainly induce an IgG response, regardless of the glycosylation of these components. Clin Transl Allergy 2016, 6(Suppl 2):38 Conclusions: Our results demonstrate that, in allergic patients, IgG sensitization is mostly directed against the same targets as that of IgE sensitization. This implies that, out of the context of SIT, IgG are most probably not able to protect allergic patients from IgE-mediated symptoms. In addition, we found that at least for some allergens (e.g. pollens from hazelnut, timothy-grass and olive tree), the selection of the immunoglobulin isotype during anti-allergen humoral responses is dependent on the allergen molecule itself ( Table 2).
Undiluted sera from 195 individuals were studied by using the Immu-noCAP ISAC ™ biochip. After having been used to measure specific IgE concentrations, the same biochips were then re-incubated with polyclonal anti-human IgG antibodies conjugated to alexa fluor 647. For the determination of specific IgG concentrations, the raw fluorescence data were converted to arbitrary IgG units by using calibration curves established from the IgG reactivity of a control serum against 5 components. Only allergen components for which IgG sensitization levels were significantly different between the sensitized/allergic and non-sensitized groups are shown. ( Background: The current paradigm claims that tolerance induced via regulatory T cells is the major mechanism to maintain a non-allergic status in healthy individuals. Here, we examine a new hypothesis, which postulates that also non-allergic individuals mount antigenspecific immune responses against environmental antigens and that different immune response types exist and maintain this healthy condition. Furthermore, we hypothesize that depending on the living environment, non-allergic immune responses can be different. Hence, we assessed the immune status of non-allergic people living in a farming environment, who are regularly exposed to the major grass pollen allergen Phl p 5 in the context of a diverse microbial environment (animal sheds, haylofts, harvesting activities) and non-allergic people living in an urban environment, who lack such microbial exposure and diversity. Materials and methods: Because of the low frequency of antigenspecific memory T cells in non-allergic donors, peripheral blood mononuclear cells were expanded antigen-specifically with rPhl p 5. After enrichment of antigen-specific memory T cells, proliferation markers, transcription factors, and cytokine secretion allowed identification of different T helper subsets like TH1, TH2, Treg, and TH17. Moreover, antigen-specific IgE, IgG1, IgG4, and IgA antibody levels in non-allergic humans were measured by ELISA.

Results:
We could show that IFN-γ is the dominant cytokine after rPhl p 5 restimulation in non-allergic townspeople pointing to a TH1 biased immune response and we could also confirm this finding by staining of TH1 associated transcription factor T-bet. On the other hand, farmers displayed similar numbers of IL-10 and IFN-γ producing cells and as well a balanced expression of transcription factors FoxP3 and T-bet. Additionally, we detected significantly higher Phl p 5-specific IgG1 titers than IgG4 titers in both non-allergic groups. Interestingly, townspeople showed significantly increased Phl p 5-specific IgG4 titers and IgG seroconversion compared to famers.

Conclusions:
In summary, it can be stated that tolerance induction is not the only mechanism to maintain a non-allergic state but rather that diverse antigen-specific immune response types are common in non-allergic individuals. Multiple mechanisms of naturally acquired protection exist and depending on the living environment different immune response types can establish and maintain a healthy nonallergic status.

Background:
The major ragweed pollen allergen Amb a 1 has been classified as member of the pectate lyase allergen family. To date, five different Amb a 1 isoforms have been officially acknowledged by the IUIS allergen nomenclature subcommittee. Moreover, pectate lyases have been identified as major allergens within the pollen of mugwort, a weed species which together with ragweed belongs to the botanical order of Asteraceae, as well as within several trees belonging to the Cupressaceae order. Thus, we thought to investigate cross-reactivity pattern as well as sensitization profiles of Amb a 1 isoforms as well as related allergens from different sources. Materials and methods: Pectate lyase pollen allergens from short ragweed (Amb a 1), mugwort (Art v 6), cypress (Cup a 1), mountain cedar (Jun a 1), and Japanese cedar (Cry j 1) were purified from aqueous pollen extracts. Moreover, three Amb a 1 isoforms (Amb a 1.01, 02, and 03, respectively) were either purified from pollen extracts or produced as recombinant proteins in P. pastoris. The allergens were characterized physico-chemically and thereafter IgE binding was assayed by immunoblot, ELISA, cross-inhibition, and mediator release assays. Results: For cross-reactivity profiling of Amb a 1 with homologous pectate lyases we found that each of the four cohorts included in the study showed a distinct sensitization fingerprint, which reflected the natural allergen exposure of the patients. Moreover, we analyzed the IgE binding to different Amb a 1 isoforms using sera of Amb a 1 sensitized individuals from Central Europe. Within this cohort, we found that all three tested Amb a 1 isoforms were recognized by IgE to a similar extent. Conclusions: Our data suggests that according to sensitization profiles pectate lyase allergens can be clustered into four categories, which are Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1. Whereas cross-reactivity between Asteraceae and Cupressaceae allergens was limited, we found considerable cross-reactivity within each order.  Table 3). In the SPT, the average LTP wheal diameter in patients that presented allergic reaction to rosacea was 8.7 mm (OAS), 7 mm (OAS and Systemic Symptoms) and 8.7 mm (Systemic Symptoms). We confirmed a statistically significate (p < 0.01) association between LTP and Pollen sensitisations, namely Grass, Cynodon

Conclusions:
In the LTP sensitised patients, we verified a greater sensitization to latex and foods, which occurred with a significantly greater frequency associated to rosacea fresh fruits. The LTP SPT wheal diameter wasn't higher in patients with a more severe symptomatology; the gravity of reactions was similar between both LTP sensitised and nonsensitised patients. We also found a correlation between LTP and Pollen sensitisation (increase in the frequency of LTP sensitisation when the number of Pollen sensitisation is greater).

Materials and methods:
A randomized cohort of 501 Austrian pupils from three geographical regions (alpine, urban, rural) donated capillary blood samples which were analyzed for IgE sensitization to 112 single allergens using the ImmunoCAP ISAC. To examine the exposure to indoor allergens from house dust mites, cats, dogs and molds, house dust samples were analyzed using a multiplex array. Demographic data, self-reported health status including allergies and other lifestyle conditions were collected using a detailed questionnaire. Results: Fifty-seven percent of subjects declared to suffer from allergies including self-reported adverse reactions, while 21 % stated to have clinically confirmed allergies. IgE reactivity to any of the 112 molecules on the ISAC chip was observed in 53 % of subjects. Highest sensitizations were found to allergens from grass pollen (Phl p 1: 27 %), house dust mites (Der p 2/Der f 2: 18 %), tree pollen (Bet v 1: 16 %) and animal hair (Fel d 1: 14 %). The majority of subjects showed a complex sensitization profile, while exclusive mono-sensitizations to insect venom, mites or grass pollen were found in 35 % of sensitized subjects. IgE reactivity to pollen allergens is mainly driven by sensitization to grass pollen allergens in this region. Fel d 1 and Can f 1 were the most abundant allergens in the collected house dust samples. The sensitization rate to house dust mite was significantly higher in urban regions, whereas pupils in alpine regions were found to be less exposed to mite allergens which translated into lower IgE sensitization. Despite elevated levels of mite allergens found on farms, a decreased sensitization rate to Der p 2 was found in pupils living on farms compared to those living in flats. Lifestyle factors, such as smoking and cat ownership, as well as having allergic family members, showed a significant influence on the overall sensitization of individuals. Background: The aim of our study was to assess the sensitization patterns to perennial inhalant allergens by means of molecular diagnostic approach in the region of Pilsen, Czech Republic. Materials and methods: The microarray system ImmunoCAP ISAC has been used for specific IgE detection to 112 different molecules. Sera from 779 patients sensitized to at least one perennial inhaled allergen molecule were subject of analysis. These patients suffered from at least one of the following diagnoses: chronic rhinitis (73 %), bronchial asthma (41 %), atopic dermatitis (34 %), urticaria or angioedema (19 %) and/or anaphylaxis (11 % Isolated sensitizations to molecules derived from storage mites Lep d 2 and/or Blo t 5 without sensitization to other mite-derived molecules were observed only exceptionally (0.9 %). Similarly, sensitization to at least one cockroach specific molecule (Bla g 1, 2, 5) was very rare (in 1.0 %). Sensitization to a tropomyosin (Der p 10 and/or Bla g 7) was observed in 3.6 %. Co-sensitization of Der p 10 with other mitederived molecules was observed in 1.2 %. The frequency of sensitization to mold-derived molecules was 33.4 %, the most frequent being Alt a 1 (26.8 %). Sensitization to Aspergillus derived molecules (Asp f 1 and/or Asp f 3 and/or Asp f 6) was 6.9 %. Conclusions: Molecular diagnosis of allergy gives a more precise and comprehensive insight into perennial inhalant allergen sensitization patterns than extract-based testing, allowing for better understanding of the sensitization process and regional differences. The data presented may help to improve the diagnostic and treatment procedures, especially focusing the need to quantify the content of the most frequent sensitizing molecules in the diagnostic and therapeutic allergen extracts used in the respective region. Keywords: Sensitization; Epidemiology; Perennial inhalant allergens; Microarray; Molecular diagnostics Background: Recent studies have shown that the allergic IgE response to grass pollen starts years before disease onset as a weak monosensitization or oligosensitization phenomenon and increase in serum concentration and complexity through a "molecular spreading" process during preclinical and early clinical disease stages. We aimed this study to investigate whether the development of the IgE response to house dust mite allergen molecules follows similar rules. Materials and methods: We have taken advantage of the data of Multicenter Allergy Study, a prospective long-term observational birth cohort study, started in 1990 and recruiting 1314 infants born in 5 German cities. Blood samples were collected at 1, 2, 3, 5, 6, 7, 10, 13 and 20 years of age. Sera with IgE antibodies to an extract of Dermatophagoides pteronyssinus were further tested for the presence of IgE Background: Tropomyosin (Pen a 1) is a pan-allergen. The homology between tropomyosins from mites, crustaceans, molluscs, insects and nematodes is responsible for the cross-reactivity between them. Although Pen a 1 in vitro sensitization has been described, its in vivo sensitization has been poorly characterized. Objectives: Study the prevalence of Pen a 1 sensitization by skin prick test (SPT) and its relation with a precise history of crustacean and/or molluscs allergy, in a population of patients with respiratory allergy sensitized to mites. Materials and methods: Data regarding the 1030 patients followed during 9 months in an Allergy outpatient clinic were retrospectively analysed. Demographic and clinical information and the results of SPT-hospital standard panel for inhalants, shrimp extract and Pen a 1 (Leti ® ) were collected. Positive result defined as a wheal diameter ≥3 mm. Data analysis using STATA 13. Results: There were included 450 patients with respiratory allergy (56 % rhinitis, 8 % asthma, 35 % both). All patients had sensitization to mites and were skin tested with a shrimp and Pen a 1 extracts. All the comparisons were made between patients with or without Pen a 1 and shrimp extract sensitization. Pen a 1 SPT has higher specificity (96.67 %) and negative predictive value (41.67 %) in the confirmation of clinical crustacean and/or molluscs allergy. On the other hand, shrimp extract showed a higher sensitivity (53.33 %). With regard to the degree of concordance between shrimp and Pen a 1 sensitization, the percentage of agreement were 90 % (majority of discordant pairs: shrimp+/tropomyosin−), Cohen's kappa coefficient was 0.39 (moderate concordance). Conclusions: Although the percentage of Pen a 1 sensitized patients is small, these patients have higher cutaneous reactivity to mites (wheals with higher diameter). Pen a 1 sensitized patients have clinical history of crustacean and/or molluscs allergy more frequently than patients without Pen a 1 sensitization or with shrimp sensitization. The higher specificity of Pen a 1 SPT makes it an important tool for diagnostic confirmation of clinical allergy to crustacean and/or molluscs, in patients with respiratory allergy sensitized to mites.

Keywords: Tropomyosin; Skin prick test; Sensitization
Background: Sensitization to inhalant allergens is an increasing problem around the world as well as in Lithuania. The aim of this study was to evaluate the distribution patterns of allergy to inhalant allergens in the population of Lithuania. Materials and methods: A retrospective study was conducted in 2013-2014 year period in public institution "Centro Poliklinika" involving 664 patients of whom 49.5 % (n = 329) were male and 50.5 % (n = 335)-female. Patients with suspicion of allergy to inhalants were tested for allergen-specific immunoglobulin E (IgE) using OPTIGEN ® mix 36 and inhalant 36 panels (Hitachi Chemical Diagnostics, Inc. U.S.A). A patient was considered allergic when spIgE to an allergen was more than class 2. Results: In our study 31.0 % (n = 206) of participants were allergic to at least one inhalant allergen. 76.7 % (n = 158) of them were children, age median was 5.0 Background: Allergies due to the venoms of hymenoptera can cause severe systemic reactions. In spite of the progress of component-resolution in the last years, diagnosis as well as therapy of venom allergy is still challenging. Amongst others this is due to extensive cross-reactivity between different venoms. In this study the antigens 5 of 7 hymenoptera species were recombinantly produced, characterized in detail and their cross-reactivity analyzed. Materials and methods: The antigens 5 Ves v 5, Vesp c 5, Pol d 5, Pol a 5, Dol m 5, Sol i 3 and the potentially hypoallergenic Poly s 5 were recombinantly produced in insect cells. The resulting purified proteins were characterized by immunoblotting und structural models were generated. Moreover, sera of venom allergic patients were assessed for IgE cross-reactivity and additionally basophil activation tests were performed. Results: All antigens 5 were successfully produced in Sf9 insect cells. As expected from sequence alignments structural models reveal identical folding, although surface charges differ between the different molecules. However, due to the use of Sf9 cells as expression host all antigens 5 were devoid of carbohydrate-based cross-reactivity. The analysis of sera from Ves v 5-reactive and Polistes allergic patients revealed extensive cross-reactivity of all antigens 5, independent of glycosylation. Some sera showed distinct reactivity profiles with the diverse antigens 5 and others reacted with all of them. Additionally, basophil activation tests from Ves v 5-reactive patients could reveal the same cross-reactivity with all antigens 5. This indicates the presence of shared as well as of individual IgE epitopes. Conclusions: The comparative analysis of antigens 5 from 7 different hymenoptera species revealed extensive cross-reactivity between all allergens on protein level. This implicates that antigens 5 are inappropriate marker allergens for the diagnostic discrimination between antigen 5-containing venoms. Moreover, detailed analyses on a molecular level can contribute to elucidate the clinical impact in the observed cross-reactivity. Background: Venoms of hymenoptera are responsible of 1/3 of anaphylactic shocks and 17 allergens have been described in the IUIS data bank in various hymenoptera species including Apis, Vespa, Vespula, Dolichovespula and Polistes. In 2004 a new sub species of Vespa, Vespa velutina, the yellow-leg asian hornet, was introduced in south west of France and is now present in more than 75 % of the French territory quickly progressing towards north and east of France. These hornets are bee-killers and build their nest close to human houses. Thus they represent a risk factor for biodiversity as well as for human health because their stings result in a huge loco-regional or systemic reaction. Some anaphylactic shocks have been reported. Materials and methods: In order to unravel the proteins and allergenic repertoire of the venom of asian hornet, an immunoproteomic study was designed. Results: A protein extract from 20 venom sacs was performed and run in 1 or 2-dimensional (1 or 2D) gel electrophoresis. Western blotting using sera from patients diagnosed allergic to the venom of european hymenoptera (bees, yellow jackets or hornets) revealed numerous IgE reactive bands demonstrating that individuals, allergic to european hymenoptera venom, have IgE recognizing asian hornet venom proteins. Mass spectrometry analysis of picked protein spots in 2D experiments, followed by data bank searches, allowed the identification of allergens homologous to already known allergens in european hymenoptera venom (Vespa magnifica and affinis, Apis mellifera, Dolichovespula maculata, Vespula vulgaris and germanica and Polybia paulista) such as phospholipase A1, antigen 5, arginine kinase, hyaluronidase and dipeptidyl di peptidase IV. Conclusions: In conclusion the data show that some cross reactivities exist between European hymenoptera and asian hornet venom allergens. Whether specific unique allergens are present in the Vespa velutina venom is under investigation. The results will improve the diagnosis of allergy to hymenoptera venom and help in designing the appropriate extract for a specific immunotherapy of patient allergic to asian hornet. Keywords: Hymenoptera asian hornet Background: Glycosylation plays an important role in the recognition and uptake of allergens by antigen presenting cells (APCs) and the modulation of immune responses. In addition, cross-reactive carbohydrate determinants (CCDs) constitute epitopes for human IgE, although their recognition seems not to be associated with clinical symptoms. Thus, elucidation of carbohydrate structures and investigation of their biological function is essential, to understand immunological properties of natural allergens. Materials and methods: D. pteronyssinus and D. farinae extracts and natural house dust mite major allergens were assessed for the presence and identity of glycans by Periodate-Schiff staining, detection by c-type lectins, carbohydrate-specific antibodies and mass spectrometry. Results: Glycan structures were detected by Periodate-schiff staining to be present on high molecular weight proteins in mite extracts from both species. Binding of the lectins GNA, PNA and DSA to many of these glycan-carrying proteins indicates the presence of N-linked high mannose and O-linked glycans comprising a core GalGalNAc 1 . Investigation by a 1,3-fucose-specific antibody excluded the presence of the CCD 1,3-fucose in the investigated samples. Applying mass spectrometry, the presence of various N-linked high mannose structures linked to a core HexNAc 2 glycan was confirmed. Analyses of purified natural major group 1 and group 2 allergens showed that group 1 allergens from D. pteronyssinus and D. farinae can carry the N-glycans HexNAc 1 , HexNAc 2 and HexHexNAc in a predicted N-glycosylation consensus sequence. Furthermore, our analyses demonstrate that group 2 allergens from both species can carry at four homologous sites modifications by hexose monosaccharides and in addition at one of these residues a polyhexose. Conclusions: Our results reveal a complex glycosylation pattern of proteins in D. pteronyssinus and D. farinae extracts that presumably influences significantly the allergenic and immunogenic properties of house dust mite proteins. The immunological relevance of the individual identified carbohydrate modifications needs to be elucidated in detail in further studies. Our data could contribute to development of innovative approaches in allergen specific immunotherapy. Background: Core structures of N-glycans modified with α1,3-fucose and β1,2-xylose represent the most frequently recognized IgE epitope. The lack of specific antibodies and the complexity of N-glycosylations so far hampered molecular insights into this important interaction. Structural characteristics as well as fine specificity of antibodies against such carbohydrates remain largely unclear. Materials and methods: Core glycan-specific IgE and Fab antibodies were produced by baculovirus-mediated infection of insect cells or in mammalian cells. Functionality and affinity of resulting antibodies was shown by ELISA and SPR analyses. Fab antibodies were used for crystallisation and structure determination in complex with an epitope disaccharide. Fine specificity and potential cross-reactivity with carbohydrate antigens was assessed using glycan arrays. Results: Recombinant IgE and Fab antibodies exhibited pronounced immunoreactivities in ELISA and dissociation constants in the nanomolar range. Structural analyses of a Fab in complex with the disaccharide epitope surrogate provided evidence for establishment of the IgE epitope by several antibody residues interacting with the core fucose. Glycan arrays that include a variety of synthetic carbohydrates representing the most frequently identified binding epitopes proved the specificity of the interaction of core-glycan-specific antibodies. Conclusions: In summary, our data provide insights into the specificity of recognition of complex N-glycans in pathological conditions. Background: Many allergens are glycoproteins that carry one or several carbohydrates linked to the protein structure. Not only proteins but also carbohydrates can stimulate the production of IgE antibodies Clin Transl Allergy 2016, 6(Suppl 2):38 and be strong inducers of Th2 responses. Galactose-α-1,3-galactose (α-Gal) is a mammalian carbohydrate with significance in a novel type of food allergy. Patients with IgE against α-Gal report severe allergic symptoms 3 to 6 hours after red meat consumption. We investigated whether IgE from red meat allergic patients recognize other mammalian glycans than α-Gal or glycans from the plant kingdom and venoms of importance in allergy. Furthermore, we examined whether red meat allergic patients' IgE response is directed against the carbohydrate-protein complex or against the glycan part only. Materials and methods: Sera from 24 red meat allergic patients were analyzed on ImmunoCAP for IgE against α-Gal, MUXF3, nCup a 1, nArt v 1 and with Streptavidin ImmunoCAP for biotinylated-Gal-α1,3-Gal, biotinylated-Neu5Gc, as well as enzymatically deglycosylated biotinylated-Gal-α1,3-Gal. The absence of the α-Gal epitope after enzymatic deglycosylation was identified and verified by an anti-α-Gal monoclonal antibody, IgE from red meat allergic patients by IgEimmunoblot as well as α-Gal-IgE inhibition experiment. Results: We found that all 24 red meat allergic patients neither had an IgE antibody response against the other abundant mammalian glycan N-glycolylneuraminic acid nor against cross-reactive carbohydrate determinants from plant or venom sources (nCup a 1, nArt v 1 and MUXF3). Deglycosylation of an α-Gal containing protein, bovine thyroglobulin, significantly reduced the IgE response. Furthermore, molecular dynamic (MD) analysis was applied to explore the nature of interaction between nsLTPs and tested ligands. ptraj analysis was used to process MD trajectories and to provide information about RMS deviation from a reference structure, hydrogen bonding, timecorrelation functions and diffusional behavior. Results: Due to pre-incubation of Pru p 3 with lipids a concentration dependent reduction of ANS binding was observed. Pru p 3 incubated (1:1; 1:10) with lauric acid showed 19 and 66 % of ANS fluorescence reduction, respectively, compared with Pru p 3 without lipids. For oleic acid (1:1; 1:10) reduction was 7 and 77 %, respectively. For other tested lipids and nsLTPs, this reduction was not seen. Molecular dynamic analysis suggests changes in protein structure due to binding of certain ligands. Interaction of Pru p 3 with oleic acid moved the C-terminal loop out towards the surface of the molecule. This interaction is stabilized by hydrogen bonds with Arg32. Interestingly, the region affected by conformational changes is one that was shown to be one of the main epitopes of Pru p 3.

Conclusions:
In this study, we observed that Pru p 3 was able to bind lauric and oleic acid, while other lipids did not interact. Molecular dynamic simulation has proved differences in the binding capacity of Pru p 3 with the ligands that can lead to conformational changes. The allergen-lipid interaction may act as a potential danger signal during the allergic sensitization phase or increase allergenicity during the effector phase. However, this remains to be investigated. Keywords: Non-specific lipid transfer proteins; Protein-ligand interaction; 3D structure Background: Improving production yield and elevating quality of recombinantly produced molecules is a high priority for the biotechnological as well as for the clinical sector. This issue has been tackled from numerous angles, one of them being codon usage bias. Crucial information concerning the overall protein synthesis procedure is encoded by the codon usage frequency; and hence even single synonymous codon mutations can significantly affect gene expression levels along with protein folding and function. Suboptimal codon usage bias can significantly limit heterologous protein expression. "Codon harmonization" is a strategy that effectively minimizes codon usage disparities between native and heterologous host by closely matching their respective codon usage frequencies. In this study we investigated the effects of codon harmonization in the recombinant production of the allergen Bet v 1.0101. Materials and methods: Different batches of rBet v 1 and its harmonized version, Bet-Harm, were produced in parallel. Production yields were quantified by SDS-PAGE densitometry. All batches were physicochemically analyzed by mass spectrometry, amino acid analysis, circular dichroism, dynamic light scattering, Fourier transform infrared spectroscopy and ANS-binding assays. Immunological properties of the rBet v 1 and Bet-Harm batches were compared by endolysosomal degradation assays, ELISA and mediator release assays. Results: A significant increase in protein yield and solubility was observed for Bet-Harm compared to rBet v 1. Besides, the two proteins displayed no alterations in their secondary structure elements, their behavior in solution and their ligand-binding ability. Furthermore, no differences in the proteolytic susceptibility, IgE-binding and IgE crosslinking ability of rBet v 1 and Bet-Harm were observed. Conclusions: Codon harmonization is an effective approach towards increasing protein expression levels and should be considered as a potent strategy for overcoming protein production problems.

Materials and methods:
The goal of our study is to characterize and compare the structures, the stabilities and the flexibilities of allergenic PR-10 proteins from various sources (i.e. birch pollen and apple) by different NMR spectroscopic techniques. Results: We rationalize the different allergenic and immunological properties of Bet v 1 and Mal d 1 and obtain structural insight into the cross-reactivity of these two allergens. Our data provide a comprehensive picture of the interplay between structure, dynamics and function of different allergens from the PR-10 family. Conclusions: Among the factors that can contribute to the allergenic potential of proteins, we hypothesize that intrinsic dynamic and structural processes are not only critical but even essential for their immunologic properties. Background: Polcalcins are small calcium-binding pollen proteins (around 9 kDa). They have been identified in pollen from diverse plant families where they are considered minor allergens. Due to their sequence homology and conserved structure, they show a high crossreactivity. Polcalcins are not usually as much represented as other allergens in pollen extracts, for that reason their availability as purified proteins would represent an useful tool for diagnosis and/or immunotherapy purposes in allergic individuals. The objective of this study was to purify the native polcalcin from different pollen allergenic sources by developing a purification method based on affinity chromatography. Materials and methods: RNA from Chenopodium album pollen was obtained and used in RT-PCR with specific primers to obtain the polcalcin (Che a 3) cDNA. This cDNA was cloned in pQE31 vector and the protein expressed in Escherichia coli. The recombinant protein was purified by affinity chromatography and size exclusion filtration, characterized by immunoblot and sequencing and used to produce rabbit polyclonal antibodies. These antibodies anti-rChe a 3 were also purified and used to identify polcalcin in different pollen extracts by direct ELISA. An anti-rChe a 3 antibody-sepharose column was packed for the purification of polcalcin from C. album and Olea europaea extracts. Clin Transl Allergy 2016, 6(Suppl 2):38

Results:
The RT-PCR allowed the obtaining of a 261 pb cDNA which sequence corresponded to Che a 3. A highly purified 10 kDa-protein was obtained after its expression and purification in two steps and its identity confirmed as Che a 3. Polcalcin was detected by ELISA in eight pollen extracts including Cynodon dactylon, C. album, Phleum pratense, O. europaea, Alnus glutinosa, Anthoxantum odoratum, Parietaria judaica, and Betula alba. Extracts from O. europaea and C. album were selected for the purification of native polcalcin. Purified Che a 3 and Ole e 3 were obtained with a high degree of purity (>95 %). Ole e 3 was identified by mass-spectrometry. Conclusions: Affinity chromatography based on specific antibodies is an efficient Materials and methods for the purification of native polcalcin from different allergenic sources. Background: Wheat is an important part of the daily diet of millions of people; however, this staple food is also responsible for food allergies. Food allergy has become a major health issue in developed countries, therefore there is an urgent need to develop analytical methods able to detect and quantify with a good sensitivity and reliability some specific allergens in complex food matrices. Wheat genus presents species with different ploidy levels, among which: diploid species with genome A (T. monococcum, einkorn); tetraploid with genomes A and B (durum wheat, Emmer); and hexaploid with genomes A, B and D (spelt, bread wheat). The bread wheat currently cultivated (T. aestivum) is hexaploid (genome ABD). It originated through spontaneous hybridizations between A, B and D genome of ancestral species and has been widely bred. In the case of prolamins, there is experimental evidence for a natural variation in the degree of biological activity between cereal genus (oat, rye, barley and wheat) as well as in the genus Triticum. T. monococcum, is an ancient species which has received renewed attention in the context of low impact and sustainable agriculture and because of its potential nutritional qualities. In addition, it was shown to exhibit a lower IgE-binding capacity compared to hexaploid species. We present a targeted MS/MS approach to compare the relative abundance of the major recognized wheat allergens in the salt-soluble (albumin/globulin) fraction of wheat grains. Twelve allergens were quantified in seven wheat varieties, selected from three Triticum species: species: T. aestivum (bread wheat), T. durum (durum wheat), and T. monococcum. Materials and methods: The allergens were monitored from one or two proteotypic peptides and their relative abundance was deduced from the intensity of one fragment measured in MS/MS. Results: Whereas the abundance of some of the targeted allergens was quite stable across the genotypes, others like alpha-amylase inhibitors showed clear differences according to the wheat species, in accordance with the results of earlier functional studies. Conclusions: This study enriches the scarce knowledge available on allergens content in wheat genotypes, and brings new perspectives for food safety and plant breeding. Background: Defensin-like proteins such as Art v 1, the major allergen from mugwort (Artemisia vulgaris), Amb a 4 from ragweed (Ambrosia artemisiifolia) and Par h 1 from feverfew (Parthenium hysterophorus) are important triggers of allergy. They consist of an N-terminal globular domain and a C-terminal proline-rich extended tail. To investigate the structural and immunological features of these proteins, Art v 1, Amb a 4 and Par h 1 were expressed in E. coli and purified to homogeneity. Materials and methods: Proteins were characterized using mass spectrometry, dynamic light scattering, circular dichroism and Fourier transform infrared spectroscopy. For immunological studies the proteins were subjected to endolysosomal degradation and in vitro antigen uptake with BMDCs. IgE recognition and cross-inhibition experiments were assessed by ELISA using patients' sera from Austria (n = 40), Canada (n = 38) and Korea (n = 27). The allergenic activity was studied by mediator release assay. Results: The identity of purified proteins was confirmed by mass spectrometry. The three proteins were monomeric as determined by dynamic light scattering. Fourier transform infrared spectroscopy revealed comparable content of α-helices and β-sheets, indicating similar foldings for the three allergens. Despite of their secondary structural similarity, the uptake kinetics were markedly different, with Amb a 4 being more efficiently internalized by BMDCs, followed by Art v 1 and Par h 1. The defensin domain of Art v 1 was more stable to endolysosomal proteolysis compare to Amb a 4 and Par h 1, pointing to possible differences in their immunogenicity. All three allergens triggered IgE-mediated basophil degranulation ranging from 15 to 40 %, demonstrating their allergenicity. The IgE reactivity of Art v 1 was significantly higher in Austrian and Korean cohorts compared with Amb a 4 and Par h 1, while in the Canadian cohort all allergens showed similar reactivity. Additionally, in cross-reactivity studies, Amb a 4 and Par h 1 showed a higher cross-reactivity between them compared to Art v 1, which in some cases was unable to inhibit the response against Amb a 4 or Par h 1. This data suggests the presence of shared-common IgE epitopes while others are allergen-specific. Wheat is a complex mixture of proteins; within a single cultivar, more than 100 constituents can be observed, among which, gliadins and glutenins present a high polymorphism and large sequence homologies. Two-dimensional immunoblot analysis after extraction of total proteins is a powerful technique to observe the diversity of IgE-binding in wheat flour, preserving the natural polymorphism, while eliminating cross-contamination issues. Materials and methods: Sera obtained from adult patients diagnosed with WDEIA were analyzed by immunoblot after two-dimensional separation of total proteins from the US wheat cultivar Butte 86. The availability of a proteomic map from this cultivar in which most of the major flour proteins were identified by tandem mass spectrometry makes it possible to determine the specific flour proteins that react with patients IgE. Comparison was carried out with ELISA results. Results: As expected, most of the sera (70 %) reacted strongly to omega-5 gliadins in ELISA and detected multiple corresponding 2-DE spots. This reactivity was often accompanied in immunoblot by weak reactivity to certain LMW-GS and alpha-gliadins which may correspond to cross-reactions, but that did not always correlate with ELISA results. The majority of patients with no IgE reactivity to omega-5 gliadins reacted to wheat LTP (about 20 % of cases). A few sera exhibited very different and diverse profiles: strong reactivity to all gliadins and LMW-GS, predominant binding to alpha-gliadins, specific binding to HMW-GS or specific binding to salt-soluble proteins including alphaamylase inhibitors and peroxidases. Conclusions: These data suggest that multiple proteins may be involved in WDEIA, even though some of these reactions occur as cross-reactions to the major allergens omega-5 gliadins. Wheat lacking omega-5 gliadins would result in decreased allergenic potential but would not be suitable for individuals already diagnosed with WDEIA. Background: The prolamin superfamily comprises the largest number of allergenic plant proteins. The members possess a conserved skeleton of 8 cysteine residues that are connected by intramolecular disulfide bonds. The sulfur-rich prolamins of wheat, alpha-gliadins, gamma-gliadins and low molecular weight glutenin subunits differ by their organization into two structural domains: the C-terminal (Ct-) domain, which include the cysteine skeleton, and the N-terminal (Nt-) domain, which contains repeating motifs. Wheat gliadins are frequently involved in food allergy to wheat and occasionally involved in baker's asthma. The present study aimed to decipher the molecular basis of α-and γ-gliadin allergenicity. Materials and methods: Different forms (natural/recombinant; native/reduced-alkylated) and fragments (Nt-and Ct-) of α-and γ-gliadins were produced to study the contribution of their structural domains and disulfide bonds to IgE binding. Their secondary structure was determined using synchrotron radiation circular dichroism (SRCD); sera from patients with food allergies to wheat or baker's asthma were used to analyze IgE binding and triggering potential on RBL cells. Results: The secondary structures of natural and recombinant proteins were slightly different. Compared with natural gliadins, recombinant proteins retained IgE binding but with reduced reactivity. When the proteins were reduced/alkylated, no significant effect was observed on the secondary structures of α-and γ-gliadin while significant decrease of IgE binding for both natural and recombinant gliadins was observed. Thus, reduction may have an indirect but drastic effect on the global folding of the proteins. Evaluation of the triggering potential of α-gliadin led to similar conclusions. IgE binding to peptides and recombinant domains of gliadins indicated the presence of epitopic regions in both domains. Conclusions: In Conclusions, the α-and γ-gliadins in their native form should be preferentially used for IgE reactivity detection. As for other allergens of the prolamin superfamily, formation of disulfide bonds appears to be of importance for IgE binding to a and g-gliadins. Disulfide bonds in the Ct-domain most likely play a role in generating conformational epitopes within this domain or which are formed by a combination of residues from both domains. Keywords: Food allergy; Wheat allergens; Epitopes The published version of this abstract can be found at [1].

Variations of wheat allergens in cultivars measured through a targeted quantitative mass spectrometry approach
Background: Although the uptake of the major peanut allergen Ara h 1 by dendritic cells (DCs) has been described, it is still not clear whether the allergen is able to sensitize by itself or whether there are other molecules involved. Evidence of the role of small molecules in the allergic sensitization, such as lipids, directly bound as ligands by the allergen or present in the allergen source, is emerging. Peanuts contain a significant amount of lipids. Therefore, we aimed to assess whether peanut lipids can bind to Ara h 1, and whether they can modulate the allergen-induced response in monocyte-derived dendritic cells (MoDCs). Materials and methods: Ara h 1 was purified from commercially available roasted peanuts by standard chromatographic methods. Extraction of total lipids from peanut was performed using the chloroform/ methanol method. The lipid concentration was determined by the sulfo-phospho-vanillin method. Lipid binding to Ara h 1 was evaluated by 1-anilinonaphthalene-8-sulfonic acid (ANS) displacement assay. ANS was used at 5 µM, and peanut lipids were added to Ara h 1 at 5, 50, and 100 µM. Fluorescence was measured at 484 nm. Monocytes were purified from PBMCs of 10 non-peanut allergic individuals and differentiated for 6 days with IL-4 and GM-CSF. The resulting MoDCs were treated with 10 µg/ml peanut lipids alone or in combination with 10 µg/ml Ara h 1. TNF-α levels in the cell culture supernatant were measured by ELISA. Results: Ara h 1 incubated with peanut lipids at the highest concentration, showed a reduction of ANS fluorescence by 30 % compared with Ara h 1 incubated with ANS alone. In MoDCs from 5 of 10 nonpeanut allergic individuals Ara h 1 increased the TNF-α production. Interestingly, peanut lipids reduced TNF-a levels in response to Ara h 1 stimulation by 65 %. In the other 5 donors, Ara h 1 did not elicit any TNF-α response, but peanut lipids in combination with Ara h 1 showed a non-significant increase of TNF-α levels. Conclusions: We observed that lipids extracted from peanut were able to bind to Ara h 1. Furthermore, peanut lipids diminish the Ara h 1-induced TNF-α response in MoDCs of non-allergic individuals. These data indicate that peanut lipids do play a role in the modulation of the immune response to Ara h 1. Background: Several studies reported data, sometimes contradictory, about the effect of heat-treatment of egg on allergy using mice or sera form egg allergic patients; but the possible impact of aggregation was not considered. Aggregation is an irreversible modification of proteins by formation of new intermolecular bonds. Aggregates of various morphologies may be generated depending on physico-chemical conditions. Ovalbumin, a major allergen of egg white, is prone to aggregate upon thermal processing. This study compares allergenicity of native and aggregated ovalbumin as large agglomerated particles on parameters from both phases of allergic reaction: sensitization and elicitation. Materials and methods: An ovalbumin solution (pH5-0.8 M) was extensively heated (80 °C for 6 h) to generate large agglomerated aggregates. A murine model of allergy was used to evaluate influence of ovalbumin structure on Ig production upon sensitization by intraperitoneal route. Using sera from mice sensitized to native or aggregated ovalbumin, Ig-binding and degranulation capacities of native and aggregated ovalbumin were measured by ELISA and with RBL cells.

Results:
We showed that heat-aggregation of ovalbumin as large particles enhanced IgG production and promoted a shift toward IgG 2a production in mice (pro-Th 1 profile) compared to native ovalbumin (pro-Th 2 profile). Moreover, the IgG repertory differed i.e. aggregated ovalbumin generated antibodies able to bind both native and aggregated ovalbumin. Large agglomerated aggregates displayed lower IgE-binding and RBL activation capacities. Conclusions: This work illustrates links between structure of ovalbumin and allergenicity potential on parameters from both sensitization and elicitation phases of the allergic reaction. It would appear that large agglomerated aggregates of OVA increased sensitization by producing more IgG and decreased elicitation. Keywords: Egg allergy; Food processing; Aggregation; Basophil activation Clin Transl Allergy 2016, 6(Suppl 2):38 Background: Allergic reactions against certain foods are triggered by epitopic regions in the allergen's amino acid sequence that show the ability to bind IgE. For an allergic reaction to occur, the passage of these epitopes through the intestinal mucosa in an immunologically intact form is required (not considering oral allergies that tend to show far less severe symptoms). Epitopes have been identified for many major tree nut allergens known to be able to trigger anaphylaxis. Still very little is known about their molecular fate during the gastro-intestinal passage and the IgE-binding potential of the digestive products. Materials and methods: A standardised multistage in-vitro model (Minekus et al. 2014), simulating the three phases of human digestion (oral, gastral, intestinal), was applied to ground hazelnut kernels. The time-dependent course of the in-vitro digestion processes was analysed by SDS-PAGE. A software assisted HPLC-MS/MS proteomics approach on a high resolution orbitrap mass spectrometer was used to elucidate the structure of peptidic degradation products and assess their relative concentrations over time. Results: Degradation was observed for both major allergens from hazelnut kernels, Cor a 9 and Cor a 11, in the gastral and intestinal phases accompanied by the formation of low-molecular products. While Cor a 11 showed higher stability against digestion both molecules were largely degraded after 120 min in the gastral and 10 min in the intestinal phase. The molecular structures of several peptidic products were identified by mass spectrometry. Time dependent formation and degradation kinetics of these peptides indicate that the gastrointestinal digestion of the intact allergens follows a multi-stage mechanism based on consecutive enzymatic activity resulting in a broad variety of peptidic structures. While the concentration of the intact allergens decreased rapidly during the digestion processes, several of the peptidic degradation products overlap with previously identified linear epitopes. Conclusions: Human digestion significantly affects the molecular structure of allergens by gastral and intestinal degradation processes. The major hazelnut allergens Cor a 9 and Cor a 11 are shown to be largely degraded before they reach the intestinal lumina whereas particular parts of the protein sequence resist digestion. The stability of specific linear epitope against gastrointestinal degradation therefore appears to play a crucial role for their allergenic potential. Keywords: Hazelnut allergy; In-vitro digestion; High resolution mass spectrometry Background: Rice allergy is a problem in both, Asian and Western countries. Several rice proteins have already been identified as causative agents of rice allergy. Since the first case of occupational contact urticaria was reported in a housewife and was due to the handling of rice bran in the form of rice bran pickles, it is necessary to understand the allergenicity of whole brown rice, not only polished rice but also rice bran, and to consider the sensitization by percutaneous exposure. In this study, we analysed the distribution of rice allergens in brown rice grain and the allergenicity in health food and cosmetics containing rice bran. Materials and methods: Brown rice (Oryza sativa L. cv koshihikari), polished rice, and rice bran were powdered and suspended in 1 M NaCl containing protease inhibitors or Laemmli sample buffer and proteins were extracted. For the health foods and cosmetics containing rice bran, samples were suspended in phosphate-buffered saline (PBS) and proteins were extracted. The extracted protein solutions were then centrifuged and filtered. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and analysed using western blotting with rabbit polyclonal antibody specific to two rice allergens, 19 kDa globulin and 52 kDa globulin.

Results:
The results of western blot analysis revealed that the 19 kDa globulin was hardly detected in the rice bran but was detected in the brown rice and the polished rice. On the other hand, 52 kDa globulin was detected in the rice bran and the brown rice, and was hardly detected in the polished rice. Regarding the proteins from the health foods and cosmetics, some of them showed clear protein bands and the others hardly showed the bands. Furthermore, 19 and/or 52 kDa globulins were detected in some products because the western blot analysis used the rice allergen specific antibody. Conclusions: 19 and 52 kDa globulins are localized mainly in the inner and outer parts of the rice grains, respectively. The finding that some health foods and cosmetics with rice bran contain 19 and 52 kDa globulins indicates that the patients with rice allergy need to be careful about using these products. Keywords: Rice; Rice bran; Globulin; Health foods; Cosmetics Background: Pollen from Fagales trees, especially birch pollen, is the main cause of early seasonal allergy in the temperate climate zone of the Northern hemisphere. Birch pollen allergic patients frequently develop allergies also towards various fruits, vegetables and nuts belonging to the botanical families of Rosaceae, Apiaceae and Fabaceae, respectively. Common symptoms in birch allergic patients include the oral allergy syndrome against apple and hazelnut. Allergen immunotherapy (AIT) is currently the only strategy to effectively cure allergies and the success of the treatment is influenced by the development of specific blocking antibodies. The aim of this study was to investigate whether blocking antibodies induced by successful birch pollen AIT using conventional allergen extracts would inhibit IgE binding to Bet v 1 related pollen as well as food allergens. Materials and methods: Therefore, a panel of 8 Fagales pollen allergens (Aln g 1.0101 from alder, Bet v 1.0101 from birch, Car b 1.0109 from hornbeam, Cas s 1.0101 from chestnut, Cor a 1.0104 from hazelnut, Fag s 1.0101 from beech, Ost c 1.0101 from hop-hornbeam and Que a 1.0301 from oak) as well as two related food allergens (Mal d 1.0108 from apple and Cor a 1.0401 from hazelnut) were recombinantly produced in E. coli, purified to homogeneity and analyzed physico-chemically. Serum samples from 5 different birch pollen allergic patients were collected before, after reaching maintenance dose and after 1-year after initiation of AIT with birch pollen extracts. Treatment efficacy was analyzed by nasal provocation tests. Facilitated antigen binding (FAB) assays were performed to determine the blocking activity of AIT induced IgG. Results: All five patients included in the study showed an improvement of nasal provocation scores during AIT. Moreover, all patients developed Bet v 1 specific blocking antibodies. However, FAB assays revealed that not all donors developed blocking antibodies against the whole panel of Bet v 1 related allergens. Conclusions: Therefore, we believe that successful birch pollen AIT cannot always ameliorate allergic symptoms towards related allergens emphasizing the need for improved treatment strategies. Keywords: Allergy; Fagales pollen; Facilitated antigen binding assay Background: Allergy to pets is a public health problem [1][2][3]. Geneva is a multiple complex community [4] where the use of component resolved diagnostic (CRD) is unavoidable [5]. Conclusions: Pets habits as food habits have changed in most urban European areas: they share frequently human life and are indoor allergens. It is shown here that in a multicomplex community like Geneva, most of patients consulting an allergy setting are highly sensitized to Feld1 and have another pets minor allergen's sensitization associated to Feld1. Once allergic to pets, >90 % of the patients are co-allergic to pollens and food. Immunotherapy being the only treatment to cure the allergic disease [6,7], improving schedule for individual patient should be an option. Component resolved diagnostic routine used in this setting is able to improve the SIT efficacy from 68 to 91 %, with an average (median) score of improvement for pets allergy by SIT of 84 %. ASR were recorded for 12 % of patients. SIT for pets is highly efficient and secure despite polysensitization and polyallergy. As for Canf1 for dog allergy [8], Feld1 alone seems not sufficient for diagnosis and consecutively treating cat allergic patient. Background: Neutrophils are present in large numbers in allergic latephase reactions. However, it is not yet clear whether they contribute to allergic inflammation. These professional phagocytes might present the allergen to allergen-specific T cells since they express MHC class II molecules upon stimulation with certain cytokines, chemokines and bacterial factors, such as GM-CSF, TNF-a, IL-8, IFN-g and LPS, respectively. In fact, murine neutrophils have been shown to process and present antigens to CD4 + T-cells. Our aim is to assess whether human neutrophils act as antigen-presenting cells for allergen-specific T-cells. Materials and methods: Neutrophils isolated from the peripheral blood of allergic donors were cultured under different conditions and analyzed for the expression of MHC class II, CD40, CD80, and CD86, by flow cytometry. Surface binding, internalization and intracellular degradation of fluorescence-labelled Bet v 1 by neutrophils were compared with monocytes. Microsomal proteases were isolated from both cell types and incubated with Bet v 1. The resulting proteolytic fragments were sequenced using mass spectrometry. Finally, neutrophils and monocytes were cocultured with Bet v 1-specific T-cell cultures generated from birch-pollen allergic donors in the presence or absence of Bet v 1 and proliferative responses of T-cells were assessed. Results: A cocktail of IL-3, GM-CSF and IFN-g enhanced the expression of HLA class II and CD80 on neutrophils. Neutrophils effectively internalized Bet v 1 and their uptake and endolysosomal degradation of the allergen was faster than by monocytes. In addition, neutrophils processed longer peptides of Bet v 1 than monocytes. Neutrophils pulsed with Bet v 1 induced proliferation in Bet v 1-specific T-cells specific for different epitopes distributed over its entire amino acid sequence. However, monocytes were the more potent antigen-presenting cells. Conclusions: Our data provide evidence that neutrophils may serve as antigen-presenting cells for allergen specific T-cells and thereby, play a role in the late phase reaction of IgE-mediated allergy. Background: Allergy is an IgE-mediated hypersensitivity reaction against harmless antigens. Like any immune response, allergic reactions are mediated by both innate and adaptive immunity. T cells are part of adaptive immunity and play a main role in the maintenance of IgE-mediated allergy. The function of CD4 + T cells in the pathophysiology of allergic disorders has been extensively investigated while the role of CD8 + T cells is still poorly understood and controversial. The aim of this project is to characterize allergen-specific CD8 + T cells in patients with different allergic manifestations (rhinoconjunctivitis, atopic dermatitis and atopic bronchial asthma). Materials and methods: Different seasonal (birch pollen and grass pollen) and perennial allergens (cat dander and house dust mite) were included. PBMCs from allergic patients were stained with the proliferation dye efluor 670 and incubated with allergen. Proliferating CD3 + CD8 + cells were then assessed for the expression of differentiation markers (CD27, CD28, CD45RO, CXCR3, CRTh2, PD-1), homing factors (CCR4, CD62L, CD29b), intracellular cytokines (IL-4, IL-5, IL-13, IL-17, IL-22 and IFN-γ, TNF-α), and cytotoxic proteins (granzyme B and perforin) by flow cytometry and compared to non-proliferating CD8 + T cells. Results: We found significant numbers of proliferating CD8 + T cells in all allergic manifestations, however, largest numbers were detected upon stimulation with house dust mite & grass pollen extracts. Furthermore, we observed a higher production of IL-4, granzyme B and perforin in proliferating than non-proliferating CD8 + T cells. In addition, a significantly higher expression of CD27, CD45RO, CD62L, and CD29b was detected in allergen-reactive CD8 + T cells. Conclusions: So far, we could confirm the existence of allergen-reactive IL-4 + CD8 + T cells in different allergic manifestations. These cells produce cytotoxic proteins and will be further characterized in future experiments. Clin Transl Allergy 2016, 6(Suppl 2):38 Background: In a world where information is instantly available, the need to provide rapid, near-patient, quantitative diagnosis is becoming an essential requirement. In response to this demand we have developed biosensors with a nanometric-size reaction chamber that enhances molecular interactions and thereby reduces incubation times of immunoassay from hours to seconds. The first application available for this system quantifies total circulating IgE from as little as 50 ul of blood in 5 min. The patient's sample is diluted with a solution containing fluorescently-labelled detection biomolecules that specifically bind to IgE, thus forming fluorescent molecular complexes. Through capillarity, the solution enters the biosensor where complexed IgE are captured on the targeted area. Fluorescence is then measured by a miniaturized microscope in the abioSCOPE reading unit, a novel and versatile in vitro diagnostic platform. Materials and methods: Matrix commutability, test linearity, effect of potential interfering substances and a comparison study have been performed to determine key analytical performance characteristics of the test. Results: The test is compatible with capillary blood or with serum or plasma samples. It has an assay measurable range from 10 to 400 kU/l of IgE. High levels of nonIgE immunoglobulins, icteric, hemolytic or lipemic samples do not interfere with test results. A comparison study between total IgE tested in the abioSCOPE against an industry gold standard (ImmunoCAP Total IgE ® in Phadia 250) showed an excellent agreement between the two systems, with values of sensitivity, specificity and accuracy of 88.7, 87.5 and 88.2 %, respectively. Conclusions: Thanks to this miniaturized biosensor and the abio-SCOPE, non-invasive quantification of protein at the point-of-care is performed in 5 minutes from a drop of whole blood collected from the patient's fingertip, whereas time-to-results using laboratory-based instruments usually takes hours. Validation of this technology allows the development of allergen-specific IgE tests to aid allergists in making precise and comprehensive allergy diagnoses. Background: More than 25 % of the population suffer from IgE-mediated allergic reactions and the number of allergic people is increasing. Consequently, there is increasing demand for strategies targeting early diagnosis, prevention and treatment of allergic sensitisation. Allergy diagnosis is traditionally carried out using allergen extracts in provocative or serological tests. By using component-resolved diagnostics, however, it is possible to identify the specific disease-eliciting allergen and, thus, to introduce more personalised and effective immunotherapy treatments for sensitised individuals. Materials and methods: A simple and rapid Array-in-Well (AiW) platform provides simultaneous IgE reactivity profiling for multiple allergens using only minute amounts of serum. Results: To establish a proof-of-concept, we printed purified and MS validated recombinant or native allergens representing relevant respiratory allergen sources, such as dog, horse and birch pollen allergens as an array into wells of 96-well microtiter plate. The IgE profiling against these allergens was carried out using serum samples collected from allergic donors. The binding of IgEs present in serum samples to the total of nine allergens present in a single well was measured and subsequently detected by fluorescently labelled human IgE-specific secondary antibodies. Fluorescence imaging of the microarrays was used to quantify the fluorescence intensity in each spot, thus resulting in a quantitative profile of IgEs present in the sample.
The allergenic activity of recombinant allergens in solution was measured by a competitive immunoassay on the above established AiW platform. Two recombinant allergens, wild-type allergen vs. natural hypoallergen, with different allergenic properties were compared by a competitive assay. The binding of serum IgEs to immobilised allergens was inhibited by the increasing amounts of the soluble allergens. Conclusions: The developed allergen microarray provides a promising, simple and cost-effective tool for simultaneous analysis of allergy-associated IgE antibodies enabling also the biological activity measurement of the allergens by the competitive immunoassay. The assay has apparent potential for high-throughput IgE profiling in the large patient cohorts targeting wide variety of allergens as well as the analysis of the allergenic activity of allergens.

Background:
In tropic and sub-tropic regions American cockroach (Periplaneta americana) is a major source of indoor allergens, frequently causing allergic reactions and asthma. From this source no single major allergen is able to predict patients' sensitization and therefore serve as a marker allergen in allergy diagnosis. As a result there is a need for component resolved diagnosis (CRD) in American cockroach allergy to determine sensitization patterns and point out treatment strategies. Within the ERA-Net New Indigo project GENALL (Genetically engineered allergens for component-resolved diagnosis and immunotherapy of airway allergies) a set of well-characterized recombinant Periplaneta americana allergens will be produced. Materials and methods: Per a 1.0103 (residues 197-378), Per a 2.0101, and the C-terminal domain of Per a 3.0101 (residues 426-675), were recombinantly produced in E. coli and purified to homogeneity. Proteins were physico-chemically characterized by mass spectrometry (MS), amino acid analysis (AAA) and circular dichroism spectroscopy (CD). The IgE binding capacity of the proteins was determined by ELISA experiments using sera of cockroach allergic patients from India (n = 25) and South Korea (n = 10). Results: In MS the primary sequence of the proteins was confirmed. With AAA the expected amino acid distribution was obtained and the exact protein concentrations were determined. Spectra obtained from CD revealed partially intact secondary structural elements. The ability of the three allergens to bind IgE was confirmed by ELISA. While for Per a 2 and Per a 3 sensitization frequencies ranged from 40 to 60 %, Per a 1 appeared to be a minor allergen in the tested patients' cohorts, showing reactivity with only one of the tested sera. Conclusions: Recombinantly produced Per a 1, Per a 2 and Per a 3 were physico-chemically characterized, and their IgE binding capacity was shown by ELISA. In further studies, all ten described Periplaneta americana allergens will be used for testing large patients' cohorts from Europe, Asia and America in order to evaluate the sensitization patterns. Cross-reactivity studies between American cockroach allergens and their homologues from German cockroach (Blattella germanica) might help to elucidate the primary sensitizer. Background: The ISAC â microarray explores sensitization to 112 molecular allergens. The analysis of such a broad panel is useful for the detection of overlooked associations. We report cosensitization to Alt a 1 (Alternaria alternata acidic glycoprotein) and Act d 2 (kiwi thaumatin-like protein TLP), the only TLP tested by ISAC â . Objectives: -to study the frequency of Alt a 1 and Act d 2 cosensitization.
-to try and understand its mechanism Materials and methods: Data from 807 ISAC â 112 microarrays (Thermo Fisher) were collected: 221 patients from Lyon, Central France (62 % males, mean age: 12 years) 586 patients from Marseille, Southern France (51 % males, mean age: 13 years) Results: In both centers, more than 80 % of patients sensitized to Act d 2 were also sensitized to Alt a 1 (Lyon 82 %, Marseille 86 %). Conversely, only 35 % of Alt a 1 sensitized patients from Lyon were also sensitized to Act d 2, but the percentage reached 61 % in Marseille patients. The median level of Alt a 1 specific IgE (sIgE) was significantly higher in cosensitized patients than in Alt a 1 monosensitized patients: 5.4 ISU versus 1.99 for Lyon (p < 0.01) and 17.3 ISU versus 3.7 for Marseille (p < 0.0001). Importantly, cosensitization to Alt a 1 and other kiwi allergens present on the microarray (Act d 1, Act d 5, Act d 8) was infrequent. Clinical data were not available for all patients. Act d 2 sensitization was not always clinically relevant with respect to kiwi fruit. Most patients cosensitized to Alt a 1 and Act d 2 displayed respiratory symptoms. Discussion: Gomez-Casado C. (FEBS Letters 2014) demonstrated that: • Alt a 1 is co-localized with Act d 2 in kiwi • electrostatic interactions exist between Act d 2 and Alt a 1 • Alt a 1 behaves as a competitive inhibitor of the TLP.
Moreover, Act d 2 spotted on ISAC â is a natural purified allergen (as opposed to Alt a 1 which is a recombinant one). Therefore, Act d 2 might be contaminated by Alt a 1 during the purification process which could induce recognition of Act d 2 spots by Alt a 1 sIgE. Alternatively, true cross-reactivity between Act d 2 and Alt a 1 cannot be excluded at this stage. Conclusions: Considering its frequency, Alt a1/Act d 2 cosensitization observed in 2 centers is unlikely to be incidental. The mechanism underlying this finding needs further investigation, including the search for (i) Alt a 1 association with other TLP, (ii) structurally similar epitopes, and (iii) clinical relevance of Act d 2 sensitization. Keywords: Act D 2; Alt A 1; Kiwi; Alternaria; Thaumatin-like-protein

Background:
The aim of this study was to ascertain if negative specific IgE to peanut component Ara-h2 is able to predict peanut tolerance whilst positive specific IgE to Peanut Ara-h2 is more likely associated with peanut allergy in a population of cosmopolitan children with suspected peanut allergy, resident in the central and Greater London region and attending a tertiary level allergy clinic.

Materials and methods:
The study is a retrospective evaluation of peanut challenge cases in relation to peanut challenge outcomes, results of peanut skin prick test and peanut specific IgE and specific IgE for peanut molecular components (Ara-h2 and Ara-h8). In addition children with a recent (within 12 months) history of peanut reaction for whom specific IgE for peanut molecular component was available were also included in the analysis. Results: The analysis of our data suggests that specific IgE to Ara h 2 is a more specific test for predicting outcome of a peanut food challenge when compared to peanut specific IgE, with specificities ranging from 85 to 95 %, depending on the cohort examined. These similar values across cohorts are reflected in this study data. In our peanut allergic subjects the median value for Ara h2 was 6.7 and 0.09 kua/l in the peanut tolerant group. This was found to be statistically significant For each method, sIgE results were converted in 7 classes (0 to 6) based on the intensity of the measured signal. Ideal concordance was defined as two same-class results, good concordance as adjacent class results, poor concordance as a difference of 2 classes, and no concordance when results differed by more than 2 classes Clin Transl Allergy 2016, 6(Suppl 2):38 (p = <0.0001). The AUC for was found to be 0.91 and therefore significant in predicating peanut challenge outcome. Conclusions: Different cut off points for SPT and specific IgE have been presented in a number of studies, but so far either a low sensitivity or low specificity has reduced the reliability as a replacement for oral challenges and thereby the clinical impact. The usage of componentresolved diagnostics have been reported as more useful compared to whole peanut protein specific IgE. In line with other studies we found that Ara h 2 was the best predictor of peanut challenge outcomes.
Whilst the results of the study do not reduce the need for challenging patients, the need for food challenges may be reduced by combing the results of Ara H2 IgE and clinical history. Background: Component resolved diagnosis (CRD) provides a step forward in identifying trigger allergens and assessing the risk of severe reactions in food allergy. This study aims to characterize, over a oneyear period, the clinical and molecular profiles of patients with immediate food reactions to cereals, followed in a Food Allergy outpatient consultation. Materials and methods: Ten patients (8 males) with an average age of 42 ± 12 years were included. We recorded the first food/drink containing cereals associated with clinical manifestations of food allergy and screened potential co-factors involved in the reactions. Our investigation included the evaluation of symptoms after ingestion of other foods and related to inhalant allergens. Skin prick tests (SPT) to cereal flours, to inhalant allergens and to Pru p 3 were performed. We carried out specific IgE (sIgE) determinations to cereal flours and to some components: Gluten, Tri a 19, Tri a 14, Pru p 3 and Pru p 4. We performed additional SPT, prick-to-prick tests and sIgE in selected cases, depending on clinical history. Results: Two patients described symptoms after drinking beer and other two after ingesting pizza. One patient referred wheezing after eating corn or bread when exercising. The remaining patients developed food reactions after eating meals containing mixed foods that always included cereals, particularly wheat. Nine patients had systemic reactions and severe anaphylaxis occurred in six of them. In the four patients with positive sIgE to Tri a 19, anaphylaxis only occurred associated with exercise. However, two of them had recurrent urticaria while treated with low doses of aspirin. Four patients had sIgE to Tri a 14. Two of them described systemic reactions with variable severity and even anaphylaxis when co-factors such as exercise, alcohol or drug intake were present. One patient was only sensitised to gluten. All the patients sensitised to Tri a 14 had sIgE to Pru p 3. Besides cereal allergy, most of the patients had other food allergies. Seven patients had respiratory allergies related to inhalant allergens. Conclusions: Cereal allergy diagnosis is a clinical challenge. Sensitisation to Tri a 19 seems to be directly related to wheat-dependent exercise induced anaphylaxis, but for those positive to Tri a 14 other different components and co-factors may be crucial for clinical severity. CRD improved the accuracy of cereal allergy diagnosis, particularly wheat allergy. Results: Case report of 36 years-old man with allergic (mites) rhinosinusitis, referred to our Immunoallergy department for an episode of generalized urticaria, itching, dyspnea and hypotension that was preceded, 30 minutes earlier, by the ingestion of wheat bread with butter followed by one ibuprofen tablet. By that time, he was in the eighth day of antibiotherapy with amoxicillin/clavulanic acid and in third day of ibuprofen-600 mg every 12hours, for a dental infection. The patient was admitted in the emergency department and the symptoms disappeared after administration of intramuscular adrenaline, intravenous antihistamines and corticosteroids. He refers no relation of this event with physical activity and had no previous complaints with wheat ingestion with or without associated exercise. SPT showed positive results to dust mite, shrimp, tropomyosin and wheat (wheal 4 mm) but negative to gliadin extract. Prick-prick reaction to wheat flour was positive (wheal 8 mm). The levels of serum total IgE was >2000 UI/mL and sIgE to wheat, gluten and rTri a 19 (ω-5-gliadin) were 4.67, 7.7 and 21.60 KUA/L respectively. Using the ImmunoCAP ISAC (Thermo Fisher Scientific, Sweden) there were determined: rTri a 19 (ω-5-gliadin) of 9.2 ISU-E and nFag e 2, rTri a 14 and nTri a aA_TI all negatives. Exercise challenge tests were performed without and with prior wheat ingestion were both negative. There were also performed an oral provocation test with meloxicam without wheat that was negative. The diagnosis of wheat-dependent anaphylaxis induced by NSAIDs was made and the patient was prescribed with an epinephrine auto-injector 0.3 mg.

P49 Component resolved diagnosis in cereal allergy
Recommendations to avoid taking NSAIDs 3 h before and after wheat ingestion were made. Conclusions: NSAIDs are common triggers of WDEIA but its role as cofactor of wheat-dependent anaphylaxis is not well studied. We present a case of wheat-dependent anaphylaxis induced by NSAIDs where the determination of rTri a 19 (ω-5-gliadin) was an important toll in the diagnosis. All individuals performed diagnostic work-up with skin prick tests (SPTs) and specific IgE (sIgE) for baseline characterization and LTP sensitization confirmation. For in vitro immune-depletion procedure, duplicate samples of each patient serum were pre-incubated with the suspected native allergen (peach, walnut or wheat, respectively) in a solid phase (ImmunoCAP ® ). The eluted serum, containing unbound IgE, was collected and samples were re-tested and compared using ImmunoCAP ISAC ® . Results: The first patient showed LTP sensitization to Prup-3, Cor-a-8, Jug-r-3, Arah-9; after pre-incubation with peach there was 100 % depletion of sIgE to all LTPs. The second patient had SPTs and sIgE positive to Rosaceaes, non Rosaceae fruits and nuts, including mango, orange and walnuts; ISAC detected LTP sensitization (Prup-3, Cor-a-8, Jug-r-3, Arah-9); walnut depleted sIgE by 60 % to Ara-h-9, 67 % to Prup-3 and Pla-a-3, 75 % to Artv3, 88 % to Jugr-3 and 100 % to Cor-a-8. The patient with previous baker-asthma, SPTs and sIgE were positive to wheat, although ω-5-gliadin was negative; ISAC showed sensitization to Prup-3, Jug-r-3, Arah-9, Tri-a-14; wheat immunodepletion occurred by 100 % only to Tri-a-14.

Conclusions:
In-vitro immunodepletion may be an useful assay to guide the diagnostic study, deciding which allergens to test, and also eviction recommendations in patients with FDEIA sensitized to a panallergen like LTP. Clin Transl Allergy 2016, 6(Suppl 2):38 and ISAC (ImmunoCAP-ISAC) we observed an excellent concordance between the two tests for grass but not for olive pollen. The aim of this study was to characterize the pattern of sensitization to molecular allergens in patients with positive SPT to olive and pollen polysensitization.

Materials and methods:
We did a retrospective analysis on a sample of 42 patients with asthma and/or rhinitis, with positive SPT to olive and at least one more pollen, who had specific IgE detection by ISAC.
We analized the results of ISAC, focusing on olive pollen specific allergens (Ole e 1, Ole e 7 and Ole e 9) and crossreactive allergens. Background: Alternaria alternata (Aa) has been considered a relevant allergen in airways atopic disease and specific immunotherapy may be indicated. Alt a 1 is the major allergen and immunotherapy extracts standardization is based on it. On the other hand sensitization to minor allergens may compromise the efficacy and/or the safety of the treatment. Our aim was to estimate the prevalence of Aa sensitization among atopic patients in Portugal, as well as compare the use of a total Aa extract to a purified Alt a 1 extract in the diagnosis.

Materials and methods:
We performed skin prick tests in 102 consecutive patients referred to our outpatient allergy clinic using our standard battery of aeroallergen extracts and also included Aa total and Alt a 1 Diater ® extracts. Papules with medium diameter 3 mm or more were considered as positive tests. We characterized Aa sensitive patients and analysed differences between Aa total and Alt a 1 results. Background: Casein (Bos d8) is identified as major allergen in cow's milk and in high levels often indicates allergy to both fresh and baked milk. It may be used to predict and aid the management of acute cow's milk allergy in children, with low levels indicating a tolerance to baked milk products Casein is a heat stable protein and the proteins remain stable even after 120 mins boiling at 100 °C. This is unlike α-lactalbumin which disappears after 30 mins, β-lactoglobulin after 15 mins and Lactoferrin after 10 mins. A large study suggested that 75 % of children with a recent diagnosis of cow's milk protein allergy tolerated baked milk. Tolerance of baked milk is a good indicator for outgrowing allergy and with the addition of baked milk into the diet appears to accelerate tolerance. Although there is a paucity of published evidence to support the practice, home reintroduction of baked milk products has become routine practice through experience in allergy services in the UK. There are limited cutoff levels for casein published in literature. We have audited the use of casein spIgE in managing children with cows' milk allergy in our UK based population. Materials and methods: Casein spIgE testing was incorporated into our evaluation of children with cow's milk allergy. Supervised baked milk challenges were performed on selected children using 'malted milk' biscuits.

Results
• A total of 98 tests were requested over a period of 7 years.
• 42 children with confirmed cows' milk allergy were challenged.
• 29 of them had eczema.
• SPT was performed to fresh and cows' milk reagent (Diagenics) • 41 children passed and successfully introduced baked milk and remain symptom free with regular exposure. • 9 of these children have outgrown their milk allergy and tolerate fresh milk. • See Tables 5 and 6 of results Conclusions: Low levels of casein specific IgE give a good indicator of outcome to challenge and aids early selection of patients for baked milk challenges. This is especially important in clinical settings with no on site intensive care where careful challenge risk stratification is important. Early inclusion of dietary baked milk accelerates the resolution of cows' milk allergy and widens the choice of foods for these children.
The child with casein of 29.8 passed their in-hospital food challenge. When baked milk was introduced at home, she became symptomatic and currently remains milk free. Discarding this result we get the following results Background: Although double-blind placebo-controlled food challenge (DBPCFC) is established as the current "gold standard" for food allergy diagnosis, the criteria by which food challenge ingredients should be evaluated for suitability have not been standardised. Furthermore, the amount of allergic material in food challenges should be comparable to commonly consumed materials. We aimed to characterise the peanut flour in a DBPCFC with respect to peanut allergen content and IgE reactivity to determine suitability for use. Materials and methods: Roasted peanut flour (four batches) and raw peanut flour were used. Sera from peanut allergic individuals were obtained from the Manchester Allergy and Respiratory Tissue Bank. Peanut flours were characterised using 2-dimensional (2D) SDS-PAGE, immunoblots, IgE immunoblots, IgE ELISA and mass spectrometry. The peanut flour-incurred challenge meal was used to determine its reactivity with sera from allergic individuals. Results: Peanut flour was characterised by immunoblotting to show the presence of the major allergens (Ara h 1, Ara h 2, Ara h 6 and Ara h 3). Protein aggregation was observed in all the immunoblots as a consequence of roasting (most prominent was aggregation of Ara h 1 and Ara h 3 acidic subunit). Allergens were profiled using untargeted mass spectrometry. IgE immunoblots and immunoassay using sera from allergic patients confirmed consistent patterns of reactivity of peanut flour between batches. The IgE immunoblots performed with extracts of the peanut-incurred dessert challenge meals demonstrated little or no difference in the reactivity compared to the roasted peanut flour itself. Conclusions: Variation between batches of peanut flour with regards to peanut allergen profiles and IgE reactivity was minimal. IgE reactivity of peanut was maintained after being incurred into the chocolate dessert. These data show the suitability of the peanut flour as an active ingredient in oral food challenges. Background: Reports that proteins are allergens are increasingly common in peer-reviewed journals, open access articles of varied review qualities or by automated computer annotation. The impact of reporting a protein as an allergen can have significant consequences related to diagnostic accuracy and food risk assessment processes. But there is no standard definition accepted for proof of allergenicity. How should we define allergens? Who should define a protein as an allergen? The intent of this discussion is to consider sources of information and definitions focuses on allergens causing IgE mediated hypersensitivities. Materials and methods: Allergens are defined primarily based on IgE binding tests with proteins purified from allergenic sources, proteins produced as clones from allergenic sources or simply by computer sequence comparisons to those of known allergens. While clinical responses to the pure protein could prove that a protein is an allergen, such tests are never conducted due to ethical considerations or lack of appropriately identified, voluntary allergic subjects. Publications typically report predicted (cDNA) translated protein sequences or partially characterized determined amino acid sequences. Subjects are often declared as allergic or sensitized with little or no clinical symptoms reported and without background information related to responses to other allergenic sources. This brief review is intended to stimulate researcher's efforts. Results: Sources defining allergens have different criteria and purposes. The WHO/IUIS database provides a nomenclature system with a short-hand name for allergenic proteins (genus, species and number), typically prior to publication, based on assurances from the submitter. Clin Transl Allergy 2016, 6(Suppl 2):38

P57
Criteria are that a minimal number of allergic subjects (typically 5), produce IgE that binds to the protein. The AllergenOnline.org database expert panel reviews published information of IgE binding to the taxonomic-protein group with a minimal requirement of specific IgE binding with preference for demonstration of allergenic activity (skin prick test, basophil activation) for risk assessment. Sequence sources (NCBI/UniProt) are increasingly auto-annotated based on keywords or low identity matches. Cross-reactive carbohydrate determinants can skew results

Conclusions:
The burden of proof is on you, the allergen researcher, to clearly report objective characteristics and results! Background: Allergy to cat is a frequent cause of rhinitis, asthma or contact urticaria. The prevalence of sensitization to different cat allergens is not well known but it seems that different patterns of sensitization to cat allergens in patients with cat allergy can help us to predict the severity and persistence of symptoms.

Materials and methods:
We select 39 sensitized patients to cat (51 % female, median age 28 years). Specific IgE measurement to cat allergens Fel d1, Fel d2 and Fel d4 was performed by microarray ISAC ® (ThermoFisher Scientific, Sweden), a value >0.3 ISU was considered as positive. Skin prick test was performed with an ALK-Abelló extract (Denmark). Association of Specific IgE measurements and presence and type of rhinitis or asthma was studied . Statistical analysis was performed using Fisher test and chisquared test. Results: 95 % of patients had specific IgE to Fel d1, 10 % to Fel d2, and 10 % to Fel d4. 82 % were monosensitized to Fel d1, 3 % to Fel d2 and 3 % to Fel d4. 75 % of sensitized patients to Fel d4 were also sensitized to Fel d1. Fel d2 was associated with severity of rhinitis and asthma (p < 0.01, p < 0.01, respectively). Fel d4 was associated with presence of asthma symptoms (p < 0.04). Direct contact with cats was associated both to persistence and severity of rhinitis (p < 0.01, p < 0.01, respectively). A positive skin prick test to cat was associated with rhinitis and asthma symptoms (p < 0.01, p < 0.01, respectively). Background: Although rabbits are common domestic pets, severe allergic reactions to rabbits at home are rarely reported. There are some cases of mild to moderate allergic rhinitis, conjunctivitis, pruritus and/or asthma in laboratory animal caretakers with frequent exposure. Moreover, few studies have evaluated the allergenic relationship between rabbits and other furry animals. We present the case of a 52 year-old woman, who presented with anaphylaxis minutes after inhalant exposure to rabbits in September 2013. She had a history of asthma and rhinitis. She worked as a cook since 1987 and some of her tasks involved, occasionally, preparing rabbit for meals, including skinning. Materials and methods: Skin prick tests were positive to house dust mites and cat epithelium. Prick-to-prick test was positive to raw rabbit meat. ImmunoCAP ® test revealed high values of total IgE (1072 kU/L) as well as specific IgE to rabbit epithelium (28.2 kU/L) and rabbit meat (14.4 kU/L). Sodium dodecye sulfate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting with extracts from rabbit (meat, epithelium, urine) and cat (epithelium, albumin) was performed.
Results: Two IgE-binding bands with molecular masses of 50-60 kDa were identified. SDS-PAGE immunoblotting-inhibition was carried out using cat epithelium extract as solid phase. All rabbit extracts assayed (meat, epithelium, urine) were able to induce a total IgE binding inhibition as well as a sample of cat albumin. Conclusions: Sensitization to cat albumin and rabbit proteins (50-60 kDa) were detected, which were probably different isoforms of rabbit albumin. These results suggest that rabbit was probably the primary sensitizer, contrary to what was expected and highlight the importance of molecular diagnosis. Background: Allergy to dog is a frequent cause of rhinitis, asthma or contact urticaria. The prevalence of sensitization to different dog allergens is not well known but it seems that different patterns of sensitization to dog allergens in patients with dog allergy can help us to predict the severity and persistence of symptoms.

Materials and methods:
We select 41 sensitized patients to dog (61 % female, median age 31 years). Specific IgE measurement to dog allergens Can f1, Can f2, Can f3 and Can f5 was performed by microarray ISAC ® (ThermoFisher Scientific, Sweden), a value >0.3 ISU was considered as positive. Skin prick test was performed with an ALK-Abelló extract (Denmark). Association of Specific IgE measurements and presence and type of rhinitis or asthma was studied. Statistical analysis was performed using Fisher test and chisquared test. Results: 44 % of patients had specific IgE to Can f1, 17 % to Can f2, 12 % to Can f3, and 66 % to Can f5. 20 % were monosensitized to Can f 1, 2 % to Can f2, 5 % to Can f3 and 44 % to Can f5. Can f1 was associated with persistent rhinitis (p 0.01), Can f3 with severity of rhinitis and asthma (p < 0.01, p 0.01, respectively), and Can f5 to both persistence and severity of rhinitis (p 0.02, p < 0.01, respectively). Sensitization to several allergens in patients (1, 2, 3 or 4) was associated with persistent asthma or rhinitis (p 0.02, p 0.01, respectively), and with moderate severity (p 0.04). Direct contact with dogs was associated with both, persistency and severity of rhinitis (p 0.02, p 0.03, respectively). The wheal diameters of skin test with commercial extract of dog were smaller in patients monosensitized to Can f 5.