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Important considerations in the choice of future sustainable protein sources for human application are tolerance, nutritional quality, and potential health benefits. We evaluated, in a double-blind cross-over intervention trial, tolerance, nutritional quality, and potential health effects of two sustainable protein sources. Thirty-six apparently healthy older adults (age 62.3 ± 7.2yrs, BMI 25 ± 3 kg/m2) received 40 g/day bovine-plasma protein (BP), corn protein (CP) or, as a benchmark, whey protein (WP) for one week with a washout period of one week in-between. In 12 participants, we also determined postprandial amino acid (PAA) uptake kinetics upon consumption of 20 g BP, CP, or WP. Changes in self-reported gastrointestinal complaints and intestinal permeability assessed using a multi-sugar acetylsalicylic acid test did not differ between the interventions. Clear differences in PAA responses were observed after consumption of the different proteins, but clear essential amino acid responses were observed for all proteins. BP consumption resulted in a small but significant increase in blood pressure outcomes, and CP consumption resulted in a small but significant decrease in insulin levels when compared to the other interventions. In conclusion, alternative protein concentrates and isolates studied here can be consumed in relative high quantities without experiencing unwanted GI complaints or gut barrier dysfunction and they can be a good source of essential amino acids. The rise in blood pressure observed during the BP intervention, potentially linked to the elevated salt content of the BP, constitutes a potential health issue. Future studies with longer intervention periods might however be recommended.

Abstract Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.

Abstract Food security is under increased pressure due to the ever-growing world population. To tackle this, alternative protein sources need to be evaluated for nutritional value, which requires information on digesta peptide composition in comparison to established protein sources and coupling to biological parameters. Here, a combined experimental and computational approach is presented, which compared seventeen protein sources with cow’s whey protein concentrate (WPC) as the benchmark. In vitro digestion of proteins was followed by proteomics analysis and statistical model-based clustering. Information on digesta peptide composition resulted in 3 cluster groups, primarily driven by the peptide overlap with the benchmark protein WPC. Functional protein data was then incorporated in the computational model after evaluating the effects of eighteen protein digests on intestinal barrier integrity, viability, brush border enzyme activity, and immune parameters using a bioengineered intestine as microphysiological gut system. This resulted in 6 cluster groups. Biological clustering was driven by viability, brush border enzyme activity, and significant differences in immune parameters. Finally, a combination of proteomic and biological efficacy data resulted in 5 clusters groups, driven by a combination of digesta peptide composition and biological effects. The key finding of our holistic approach is that protein source (animal, plant or alternative derived) is not a driving force behind the delivery of bioactive peptides and their biological efficacy.

We studied several sustainable alternative protein concentrates and a whey protein concentrate as reference, to determine their protein composition, digestibility and quality using the harmonized INFOGEST static protocol. The proteins concentrates were analyzed to determine their conversion factor, degree of hydrolysis, true ileal digestibility, in vitro digestible indispensable amino acid score (IVDIAAS) and total absorbable amino acids and total essential amino acids. The results revealed that whey, blood plasma concentrate and yeast protein concentrate have a high mean true ileal indispensable amino acid in vitro digestibility (91.1–85.8%), closely followed by corn, pea, potato and proteins derived from lesser meal worms (ranging between 82.5 and 77.9%). Mycoprotein gave a much lower score in the mass balance, so that its other results could not be interpreted as being reliable. Whey, potato, blood plasma and yeast protein concentrates ranked highest in IVDIAAS (between 119 and 97.2), followed by lesser meal worm and pea (between 73.8 and 57.8) with corn protein concentrate having the lowest IVDIAAS due to underrepresentation of lysine. The method and data presented in this paper can form a start for further applying the INFOGEST in vitro digestion protocol to evaluate protein quality.

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

Allergen recognition and processing by antigen presenting cells is essential for the sensitization step of food allergy. Macrophages and dendritic cells are both phagocytic antigen presenting cells and play important roles in innate immune responses and signaling between the innate and adaptive immune system. To obtain a model system with a homogeneous genetic background, we derived macrophages and dendritic cells from THP-1 monocytes. The difference between macrophages and dendritic cells was clearly shown by differences in their transcription response (microarray) and protein expression levels. Their resemblance to primary cells was analyzed by comparison to properties as described in literature. The uptake of β-lactoglobulin after wet-heating (60°C in solution) by THP-1 derived macrophages was earlier reported to be significantly increased. To analyse the subsequent immune response, we incubated THP-1 derived macrophages and dendritic cells with native and differently processed β-lactoglobulin and determined the transcription and cytokine expression levels of the cells. A stronger transcriptional response was found in macrophages than in dendritic cells, while severely structurally modified β-lactoglobulin induced a more limited transcriptional response, especially when compared to native and limitedly modified β-lactoglobulin. These results show that processing is relevant for the transcriptional response toward β-lactoglobulin of innate immune cells.

Fungal immunomodulatory proteins (FIPs) are a group of proteins found in fungi, which are extensively studied for their immunomodulatory activity. Currently, more than 38 types of FIPs have been described. Based on their conserved structure and protein identity, FIPs can be classified into five subgroups: Fve-type FIPs (Pfam PF09259), Cerato-type FIPs (Pfam PF07249), PCP-like FIPs, TFP-like FIPs, and unclassified FIPs. Among the five subgroups, Fve-type FIPs are the most studied for their hemagglutinating, immunomodulating, and anti-cancer properties. In general, these small proteins consist of 110–125 amino acids, with a molecular weight of ~13 kDa. The other four subgroups are relatively less studied, but also show a noticeable influence on immune cells. In this review, we summarized the protein modifications, 3-dimensional structures and bioactivities of all types of FIPs. Moreover, structure-function relationship of FIPs has been discussed, including relationship between carbohydrate binding module and hemagglutination, correlation of oligomerization and cytokine induction, relevance of glycosylation and lymphocyte activation. This summary and discussion may help gain comprehensive understanding of FIPs' working mechanisms and scope future studies.

Allergy is becoming a rapidly increasing problem worldwide, and in vitro models are frequently used to study the mechanisms behind the different types of allergic response. The dendritic cell (DC)–T-cell model can be used to study sensitization. However, lipopolysaccharide (LPS) is often used to maturate the DCs, but it gives rise to a DC1 phenotype, whereas Th2-driven inflammatory diseases such as allergy are characterized by the involvement of the DC2 phenotype. Our aim was to create a DC2–T-cell human model (human moDC2s) to study in vitro sensitization and validate the model using polyunsaturated fatty acids (PUFAs) that were previously shown to have immunomodulatory properties. We found that the generated DC2s expressed OX40L and drove naive T-cells into IL-13 production of CD4+ effector T-cells. In line with in vivo findings, n−3 long-chain (LC)PUFA docosahexaenoic acid (DHA) effectively decreased the DC2's surface expression of OX40L, as well as the IL-12p40 and IL-23 cytokine production by DC2s and subsequently lowered IL-13 production by DC2-induced effector T-cells. Similar cytokine production effects were found with eicosapentaenoic acid (EPA) and arachidonic acid (AA), whereas linoleic acid (LA) increased OX40L surface expression and subsequent T-cell-derived IL-13/IFNγ ratios, suggesting an increased risk of allergy development. Altogether, these data show that human moDC2s are able to induce Th2-type IL-13 secretion by T-cell differentiated in the presence of these DC2s and that this model can be differentially modulated by PUFAs. These results are in line with previous in vivo studies using PUFAs, indicating that this model may be of use to predict in vivo outcomes.

Although an impact of processing on immunogenicity of food proteins has clearly been demonstrated, the underlying mechanisms are still unclear. We applied 3 different processing methods: wet heating (60 °C) and low- or high-temperature (50 °C or 130 °C, respectively) dry-heating in absence or presence of reducing sugars, to β-lactoglobulin (BLG), lysozyme and thyroglobulin, which represent dietary proteins with different pI or molecular weight. Uptake of the soluble fraction of the samples was tested in two types of, genetically homogeneous, antigen-presenting cells (macrophages and dendritic cells derived from THP-1 monocytes). This revealed a strong correlation between the uptake of the different protein samples by macrophages and dendritic cells, and confirmed the key role of hydrophobicity, over aggregation, in determining the uptake. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but not in cytokine production, by THP-1 macrophages.

For the determination of the binding of heated cow’s milk whey proteins such as β-lactoglobulin to the receptors expressed on immune cells, inhibition ELISA with the soluble form of the receptor for advanced glycation end products (sRAGE) and scavenger receptor class B (CD36) has been successfully used in the past. However, binding to heated and glycated caseins in this read-out system has not been tested. In this study, inhibition ELISA was applied to measure the binding of cow’s milk casein alone, as well as all milk proteins together, which underwent differential heat treatment, to sRAGE and CD36, and we compared those results to a dot blot read out. Moreover, binding to sRAGE and CD36 of differentially heated milk protein was measured before and after in vitro digestion. Casein showed binding to sRAGE and CD36, independent from the heat treatment, in ELISA, while the dot blot showed only binding to high-temperature-heated milk protein, indicating that the binding is not related to processing but to the physicochemical characteristics of the casein. This binding decreased after passage of casein through the intestinal phase.

Dietary non-digestible polysaccharides (NDPs) might promote intestinal health via immuno-modulation. Immunomodulatory effects of NDP are most likely brought about by antigen processing cells such as macrophages that populate the intestine, although the mechanisms are still poorly understood. We validated the in vitro model of M1 and M2 macrophages to mimic the intestinal inflammatory and tolerant macrophages using literature and microarray-derived gene markers. All these markers were used to characterise the macrophage phenotype following NDP stimulation. This identified an alternative subset, termed M(NDP), which commonly modulated a set of 126 genes, involved in migration, metabolic processes, cell cycle, and inflammatory immune function. This gene-based analysis for macrophage subsets provides an additional tool to characterise NDP bioactivity for their in vivo potential.

Abstract Background The introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP). Main body The proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell–cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential. Conclusion The application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs.

N-3 long chain polyunsaturated fatty acids (LCPUFAs) are considered to possess protective properties for human health by impacting on immunological reactions. An “inflammation-suppressive” effect appears to be the common denominator of the beneficial effects of most of these dietary components which may protect against the development of chronic immune disorders such as (food) allergy. LCPUFAs, especially n-3 LCPUFAs, have been shown to interact with both the sensitization as well as the effector phase in food allergy in pre-clinical models. In this review, we explore the anti-allergic properties of LCPUFAs by providing an overview of clinical, in vivo and in vitro studies. Furthermore, we discuss the susceptibility of LCPUFAs to lipid oxidation and possible strategies to support the efficacy of LCPUFAs in reducing the allergy risk by using additional components with anti-oxidative and anti-inflammatory capacities such as the flavonoid quercetin. Finally, we propose new strategies to prevent (food) allergy using combinations of LCPUFAs and additional nutrients in diets or supplements, and postulate to investigate the use of LCPUFAs in allergic symptom relief.

The immune system provides host protection to infection with pathogenic organisms, while at the same time providing tolerance upon exposure to harmless antigens. Thus, an impaired immune function is associated with increased susceptibility to infections with increased disease severity and thereby necessitating the therapeutic use of antibiotics. Livestock performance and feed efficiency, in addition to their health status, are dependent on the microbial load of their gut, the barrier function of the intestinal epithelium and the activity of the mucosal immune system, all of which can be modulated by dietary components. The majority of feeds that are consumed in pets and livestock have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars called Maillard reaction (MR). Maillard reaction products (MRPs) and advanced Maillard reaction products (AGEs) determine taste, smell, and color of many food products therefore the MR is highly relevant for the feed industry. MRPs interact with different types of immune receptors, including the receptor for advanced glycation end products (RAGE) and immunomodulatory potential of feed proteins can be modified by Maillard reaction. This MR has become an important concern since MRPs/AGEs have been shown to contribute to increasing prevalence of diet-related chronic inflammatory states in the gut with negative health consequences and performance. The immunomodulatory effects of dietary MRPs and AGEs in livestock and pet animals are far less well-described, but widely considered to be similar to the relevant concepts and mechanisms obtained in the human field. This review will highlight immunological mechanisms underlying initiation of the innate and adaptive immune responses by MRPs/AGEs present in animal feeds, which are currently not completely understood. Bridging this knowledge gap, and taking advantage of progress in the human field, will significantly improve nutritional quality of feed and increase the prevention of diet-mediated inflammation in animals.

Objective:In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation.Methods: Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed.Results: Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins (TJP) 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation.Conclusions: Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the distribution of claudin 7.

Human tyrosinase (TYR) is a glycoprotein that initiates the first two reactions in the melanin biosynthesis pathway. Mutations in its encoding gene cause Oculocutaneous Albinism type I (OCA1), the most severe form of albinism, which is a group of autosomal recessive disorders characterized by reduced or absent production of melanin in skin, hair and eyes. Despite extensive structural and characterization studies of its homologues in lower eukaryotic organisms, the catalytic mechanism of human TYR and the molecular basis of OCA1 are largely unknown. In this work, we have carried out a large-scale recombinant expression of TYR that has enabled us to obtain high yields of pure and active protein, required for crystallization trials and screening of skin whitening agents, which is highly demanded in the cosmetic industry. Addition of an N-terminal honeybee melittin signal peptide for secretion of the produced protein into the (protein-free) medium, as well as a cleavable His-tag at the C-terminus, was crucial for increasing the yield of pure protein. We have successfully crystallized two TYR variants, in both glycosylated and deglycosylated forms, showing preliminary X-ray diffraction patterns at 3.5 Å resolution. Hence, we have established an expression and purification protocol suitable for the crystal structure determination of human TYR, which will give unique atomic insight into the nature and conformation of the residues that shape the substrate binding pocket that will ultimately lead to efficient compound design.

Few studies with a limited number of patients have provided indications that cashew-allergic patients may experience severe allergic reactions to minimal amounts of cashew nut. The objectives of this multicentre study were to assess the clinical relevance of cashew nut sensitisation, to study the clinical reaction patterns in double-blind placebo-controlled food challenge tests and to establish the amount of cashew nuts that can elicit an allergic reaction.A total of 179 children were included (median age 9.0 years; range 2-17 years) with cashew nut sensitisation and a clinical history of reactions to cashew nuts or unknown exposure. Sensitised children who could tolerate cashew nuts were excluded. The study included three clinical visits and a telephone consultation. During the first visit, the medical history was evaluated, physical examinations were conducted, blood samples were drawn and skin prick tests were performed. The children underwent a double-blind placebo-controlled food challenge test with cashew nut during the second and third visits. The study showed that 137 (76.5%) of the sensitised children suspected of allergy to cashew nut had a positive double-blind placebo-controlled food challenge test, with 46% (63) manifesting subjective symptoms to the lowest dose of 1 mg cashew nut protein and 11% (15) developing objective symptoms to the lowest dose. Children most frequently had gastro-intestinal symptoms, followed by oral allergy and skin symptoms. A total of 36% (49/137) of the children experienced an anaphylactic reaction and 6% (8/137) of the children were treated with epinephrine.This prospective study demonstrated a strikingly high percentage of clinical reactions to cashew nut in this third line population. Severe allergic reactions, including anaphylaxis requiring epinephrine, were observed. These reactions were to minimal amounts of cashew nut, demonstrated the high potency of this allergens.www.ncbi.nlm.nih.gov/pubmed NTR3572.

An a-glucan was isolated from the culinary medicinal mushroom A. bisporus by hot water extraction, ethanol precipitation and DEAE-cellulose chromatography. The resulting material showed a single HMW peak excluded from a Sephadex G50 column that could completely be degraded by α-amylase treatment. After heating in 1% SDS a small additional peak of low MW eluted from the G50 column. The monosaccharide composition of the main peak was evaluated by HPLC, and was found to consist of a majority of glucose (97.6%), and a minor proportion of galactose (2.4%). Methylation analysis and degradation by a-amylase indicated the presence of an a-glucan with a main chain consisting of (1®4)-linked units, substituted at O-6 by α-D-glucopyranose single-units in the relation 1:8. Mono- (13C-, 1H-NMR) and bidimensional [1H (obs.),13C-HSQC] spectroscopy analysis confirmed the a-configuration of the Glcp residues by low frequency resonances of C-1 at d 100.6, 100.2, and 98.8 ppm and H-1 high field ones at d 5.06, 5.11, and 4.74 ppm. The DEPT-13C-NMR allowed assigning the non-substituted and O-substituted –CH2 signals at d 60.3/60.8 and 66.2 ppm, respectively. Other assignments were attributed to C-2, C-3, C-4, C-5 and C-6 of the non-reducing ends at d 71.8; 72.8; 70.0; 71.3 and 60.3/60.8 ppm, respectively. The minor proportion of galactose that was demonstrated was probably derived from a complex between the a-glucan and a low molecular weight galactan.

More understanding of the risk-benefit effect of the glycoalkaloid tomatine is required to be able to estimate the role it might play in our diet. In this work, we focused on effects towards intestinal epithelial cells based on a Caco-2 model in order to analyze the influence on the cell monolayer integrity and on the expression levels of genes involved in cholesterol/sterol biosynthesis (LDLR), lipid metabolism (NR2F2), glucose and amino acid uptake (SGLT1, PAT1), cell cycle (PCNA, CDKN1A), apoptosis (CASP-3, BMF, KLF6), tight junctions (CLDN4, OCLN2) and cytokine-mediated signaling (IL-8, IL1β, TSLP, TNF-α). Furthermore, since the bioactivity of the compound might vary in the presence of a food matrix and following digestion, the influence of both pure tomatine and in vitro digested tomatine with and without tomato fruit matrix was studied. The obtained results suggested that concentrations

BACKGROUND:Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. METHODOLOGY/PRINCIPAL FINDINGS:Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. CONCLUSIONS/SIGNIFICANCE:Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.