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1. Supplementary data on the characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, which confers herbicide tolerance, and on the compositional assessment of field grown MST-FGØ72-2 soybean expressing HPPD W336

Author

Rozemarijn Dreesen, Annabelle Capt, Regina Oberdoerfer, Isabelle Coats, and Kenneth Edward Pallett

Subjects
Computer applications to medicine. Medical informatics, R858-859.7, Science (General), Q1-390
Abstract

Supplementary data are provided which are supportive to the research article entitled “Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean” (Dreesen et al., 2018) [1]. The conducted supplementary analyses include the characterization of additional Escherichia coli-produced HPPD W336 protein batches used as a surrogate in HPPD W336 safety studies, the assessment of potential glycosylation and monitoring of stability in simulated intestinal fluid and during heating of the HPPD W336 protein. Furthermore, data are provided on conducted field trials and subsequent compositional analysis in MST-FGØ72-2 soybean grain of compounds related to the tyrosine degradation pathway and the metabolism of homogentisate.

Published
2018
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2. Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure

Author

Jaap Akkerdaas, Muriel Totis, Brian Barnett, Erin Bell, Tom Davis, Thomas Edrington, Kevin Glenn, Gerson Graser, Rod Herman, Andre Knulst, Gregory Ladics, Scott McClain, Lars K. Poulsen, Rakesh Ranjan, Jean-Baptiste Rascle, Hector Serrano, Dave Speijer, Rong Wang, Lucilia Pereira Mouriès, Annabelle Capt, and Ronald van Ree

Subjects
Immunologic diseases. Allergy, RC581-607
Abstract

Abstract Background Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. Aim To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. Methods Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2–2.5–4.0) and pepsin-to-protein ratio (PPR: 10–1–0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. Results At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. Conclusions Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair.

Published
2018
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3. Abstracts from the Food Allergy and Anaphylaxis Meeting 2016

Author

Guillaume Pouessel, Claire Claverie, Julien Labreuche, Jean-Marie Renaudin, Aimée Dorkenoo, Mireille Eb, Anne Moneret-Vautrin, Antoine Deschildre, Stephane Leteurtre, Linus Grabenhenrich, Margitta Worm, Sabine Dölle, Kathrin Scherer, Isidor Hutteger, Morten Christensen, Carsten Bindslev-Jensen, Charlotte Mortz, Esben Eller, Henrik Fomsgaard Kjaer, Leonor Carneiro-Leão, Jenny Badas, Alice Coimbra, Dikla Pivko Levy, Moshe Ben-Shoshan, Ayelet Rimon, Shira Benor, Nicolette J. T. Arends, Nikki Edelbroek, Hans de Groot, Joyce A. M. Emons, H. Kim A. Brand, Dirk Verhoeven, Leonieke N. van Veen, Nicolette W. de Jong, Geunwoong Noh, Eun Ha Jang, Mariona Pascal, Olga Dominguez, Mònica Piquer, Montserrat Alvaro, Rosa Jimenez-Feijoo, Jaime Lozano, Adriana Machinena, Maria del Mar Folqué, Maria Teresa Giner, Ana María Plaza, Paul Turner, Nandinee Patel, Marta Vazquez-Ortiz, Sarah Lindsley, Lucy Walker, Simon Rosenberg, Adriano Mari, Claudia Alessandri, Ivana Giangrieco, Lisa Tuppo, Chiara Rafaiani, Georg Mitterer, Michela Ciancamerla, Rosetta Ferrara, Maria Livia Bernardi, Danila Zennaro, Maurizio Tamburrini, Maria Antonetta Ciardiello, Christian Harwanegg, Antonio Fernandez, Regina Selb, Philippe Egenmann, Michelle Epstein, Karin Hoffmann-Sommergruber, Frits Koning, Martinus Lovik, E. N. Clare Mills, Javier Moreno, Henk van Loveren, Jean-Michel Wal, Susanne Diesner, Cornelia Bergmayr, Barbara Pfitzner, Vera Elisabeth Assmann, Philipp Starkl, David Endesfelder, Thomas Eiwegger, Zsolt Szepfalusi, Heinz Fehrenbach, Erika Jensen-Jarolim, Anton Hartmann, Isabella Pali-Schöll, Eva Untersmayr, Soren Wille, Peter Meyer, Caroline Klingebiel, Jonas Lidholm, Angelica Ehrenberg, Jonas Östling, Isabelle Cleach, Jean-Louis Mège, Joana Vitte, Roberta Aina, Pawel Dubiela, Sabine Pfeifer, Merima Bublin, Christian Radauer, Piotr Humeniuk, Stefan Kabasser, Riccardo Asero, Gador Bogas, Francisca Gomez, Paloma Campo, Maria Salas, Inmaculada Doña, Esther Barrionuevo, Maria Auxiliadora Guerrero, Cristobalina Mayorga, Ana Prieto, Domingo Barber, Maria Jose Torres, Annette Jamin, Andrea Wangorsch, Barbara Ballmer, Stefan Vieths, Stephan Scheurer, Danijela Apostolovic, Jelena Mihailovic, Maja Krstic, Maria Starkhammar, Tanja Cirkovic Velickovic, Carl Hamsten, Marianne van Hage, Francine C. van Erp, Edward F. Knol, Hannah M. Kansen, Bo Pontoppidan, Yolanda Meijer, Cornelis K. van der Ent, André C. Knulst, Rebekah Sayers, Helen Brown, Adnan Custovic, Angela Simpson, Claire Mills, Juliane Schulz, Network for Online Registration of Anaphylaxis (NORA), Jaap Akkerdaas, Muriel Totis, Annabelle Capt, Corinne Herouet-Guicheney, Ronald van Ree, Tushar Banerjee, Antima Banerjee, Mathilde Claude, Grégory Bouchaud, Roberta Lupi, Laure Castan, Olivier Tranquet, Sandra Denery-Papini, Marie Bodinier, Chantal Brossard, Rosella De Poi, Elisa Gritti, Emiliano De Dominicis, Bert Popping, Patrizia Polverino de Laureto, Kati Palosuo, Anna Kaarina Kukkonen, Anna Pelkonen, Mika Mäkelä, Nanju Alice Lee, Johanna Rost, Sridevi Muralidharan, Dianne Campbell, Sam Mehr, Catherine Nock, Joseph Baumert, Steve Taylor, Carla Mastrorilli, Salvatore Tripodi, Carlo Caffarelli, Serena Perna, Andrea Di Rienzo Businco, Ifigenia Sfika, Arianna Dondi, Annamaria Bianchi, Carlotta Povesi Dascola, Giampaolo Ricci, Francesca Cipriani, Nunzia Maiello, Michele Miraglia del Giudice, Tullio Frediani, Simone Frediani, Francesco Macrì, Chiara Pistoletti, Iride Dello Iacono, Maria Francesca Patria, Elena Varin, Diego Peroni, Pasquale Comberiati, Loredana Chini, Viviana Moschese, Sandra Lucarelli, Roberto Bernardini, Giuseppe Pingitore, Umberto Pelosi, Roberta Olcese, Matteo Moretti, Anastasia Cirisano, Diego Faggian, Alessandro Travaglini, Mario Plebani, Maria Carmen Verga, Mauro Calvani, Paolo Giordani, Paolo Maria Matricardi, Noe Ontiveros, Francisco Cabrera-Chavez, Julie Galand, Etienne Beaudouin, The Anaphylaxis Working Group of the French Allergology SocietyThe Anaphylaxis Working Group of the French Allergology Society, Florence Pineau, Shinobu Sakai, Kayoko Matsunaga, Reiko Teshima, Colette Larré, Sandra Denery, Sebastian Tschirner, Valérie Trendelenburg, Gabriele Schulz, Bodo Niggemann, Kirsten Beyer, Youcef Bouferkas, Younes Belabbas, Djamel Saidi, Omar Kheroua, Kamel Eddine El Mecherfi, Malika Guendouz, Abir Haddi, Hanane Kaddouri, Luis Amaral, Ana Pereira, Susana Rodrigues, Mareen Datema, Laurian Jongejan, Michael Clausen, Andre Knulst, Nikolaos Papadopoulos, Marek Kowalski, Frédéric de Blay, Aeilko Zwinderman, Karin Hoffman-Sommergruber, Barbara Ballmer-Weber, Montserrat Fernandez-Rivas, Shan Deng, Jia Yin, Charlotte Eisenmann, Maria Nassiri, Rabea Reinert, Johanna P. M. van der Valk, Roy Gerth van Wijk, Yvonne Vergouwe, Ewout W. Steyerberg, Marit Reitsma, Harry J. Wichers, Huub F. J. Savelkoul, Berber Vlieg-Boerstra, Anthony E. J. Dubois, Fabrícia Carolino, Ana Rodolfo, Josefina Cernadas, Dasha Roa-Medellín, Ana Rodriguez-Fernandez, Joaquín Navarro, Vicente Albendiz, María Luisa Baeza, Sonsoles Intente-Herrero, Andrea Mikkelsen, Kirsten Mehlig, Lauren Lissner, Linda Verrill, Stefano Luccioli, Jolanda van Bilsen, Frieke Kuper, André Wolterbeek, Tanja Rouhani Rankouhi, Lars Verschuren, Hilde Cnossen, Prescilla Jeurink, Johan Garssen, Léon Knippels, Jossie Garthoff, Geert Houben, Winfried Leeman, M. Eleonore Pettersson, Afke M. M. Schins, Gerard H. Koppelman, Boudewjin J. Kollen, Svitlana Zubchenko, Sarah Kuntz, Pablo Mérida, Montserrat Álvaro, Monica Piquer, Carmen Riggioni, Juan Heber Castellanos, Rosa Jimenez, Melanie Cap, Elodie Drumez, Stéphanie Lejeune, Caroline Thumerelle, Clémence Mordacq, Véronique Nève, Sonia Ricò, Margherita Varini, Rita Nocerino, Linda Cosenza, Antonio Amoroso, Margherita Di Costanzo, Carmen Di Scala, Giorgio Bedogni, Roberto Berni Canani, Paul J. Turner, Paloma Poza-Guedes, Ruperto González-Pérez, Inmaculada Sánchez-Machín, Victor Matheu-Delgado, Erik Wambre, Anne-Sofie Ballegaard, Charlotte Madsen, Juliane Gregersen, Katrine Lindholm Bøgh, Philippe Aubert, Michel Neunlist, Antoine Magnan, Daniel Lozano-Ojalvo, Alba Pablos-Tanarro, Leticia Pérez-Rodríguez, Elena Molina, Rosina López-Fandiño, Akila Rekima, Patricia Macchiaverni, Mathilde Turfkruyer, Sebastien Holvoet, Lénaïck Dupuis, Nour Baiz, Isabella Annesi-Maesano, Annick Mercenier, Sophie Nutten, Valérie Verhasselt, Ines Mrakovcic-Sutic, Srdan Banac, Ivana Sutic, Zdenka Baricev-Novakovic, Ingrid Sutic, Valentino Pavisic, Rosa Muñoz-Cano, Teodoríkez Jiménez-Rodríguez, Daniel Corbacho, Jordi Roca-Ferrer, Joan Bartra, Aleksandar Bulog, Vladimir Micovic, Lidia Markiewicz, Agata Szymkiewicz, Anna Szyc, Barbara Wróblewska, Bryan M. Harvey, Lucien F. Harthoorn, A. Wesley Burks, Georgios Rentzos, Anna-Lena Bramstång Björk, Ulf Bengtsson, Colin Barber, Chrystyna Kalicinsky, Christine Breynaert, Lieve Coorevits, Cornelia Jansen, Erna Van Hoeyveld, Kristin Verbeke, Anne-Marie Kochuyt, Rik Schrijvers, Diana Deleanu, Adriana Muntean, Maria Konstantakopoulou, Maria Pasioti, Anastasia Papadopoulou, Anna Iliopoulou, Nikolaos Mikos, Evangelia Kompoti, Eunice Dias de Castro, Borja Bartalomé, Kok Loong Ue, Elizabeth Griffiths, Stephen Till, Kate Grimshaw, Graham Roberts, Anna Selby, Indre Butiene, Jose Ignacio Larco, Ruta Dubakiene, Ana Fiandor, Alessandro Fiocchi, Nikos Papadopoulos, Sigurveig Sigurdardottir, Aline Sprikkelman, Anne-Fleur Schoemaker, Paraskevi Xepapadaki, Thomas Keil, Zizi Cojocariu, Beatriz Secades Barbado, Vasti Iancu, Esozia Arroabarren, Marta Goñi Esarte, Miren Arteaga, Mayra Coutinho Andrade, Denise Borges, Jorge Kalil, Pedro Giavina Bianchi, Rosana Camara Agondi, Rinkesh Kumar Gupta, Akanksha Sharma, Kriti Gupta, Mukul Das, Premendra Dwivedi, Rusudan Karseladze, Liana Jorjoliani, Lali Saginadze, Mariam Tskhakaia, Katia Basello, Gabriele Piuri, Attilio Francesco Speciani, Michela Carola Speciani, Carla Camerotto, Francesco Zinno, Olga Pakholchuk, Svitlana Nedelska, Stefano Pattini, Maria Teresa Costantino, Silvia Peveri, Danilo Villalta, Eleonora Savi, Andrea Costanzi, Vera A. Revyakina, Marina A. Kiseleva, Elena D. Kuvshinova, Inna A. Larkova, Anton A. Shekhetov, Diana Silva, André Moreira, José Plácido, Hanneke van der Kleij, Esther van Twuijver, Robbert Sutorius, Pieter-Jan de Kam, Jenny van Odijk, Helen Lindqvist, Elin Lustig, Amyra Ali Azamar Jácome, Karla Leversia Borjas Aguilar, Miguel García Domínguez, David Alejandro Mendoza Hernández, Cristiano Caruso, Cono Casale, Gian Lodovico Rapaccini, Antonino Romano, Italo De Vitis, Renata R. Cocco, Carolina Aranda, Marcia C. Mallozi, Jackeline F. Motta, Lilian Moraes, Antonio Pastorino, Nelson Rosario, Ekaterini Goudouris, Arnaldo Porto, Neusa F. Wandalsen, Emanuel Sarinho, Flavio Sano, Dirceu Solé, Constantinos Pitsios, Maria Petrodimopoulou, Ekaterini Papadopoulou, Maria Passioti, Meropi Kontogianni, Nino Adamia, Ekaterina Khaleva, Ana Prieto del Prado, George Du Toit, Edyta Krzych, Urszula Samolinska-Zawisza, Konrad Furmanczyk, Aneta Tomaszewska, Filip Raciborski, Agnieszka Lipiec, Piotr Samel-Kowalik, Artur Walkiewicz, Jacek Borowicz, Boleslaw Samolinski, Aimee Lou Nano, Marysia Recto, Maria Luisa Somoza, Natalia Blanca López, Diana Pérez Alzate, Francisco Javier Ruano, Maria Isabel Garcimartín, Elisa Haroun, Maria Vázquez de la Torre, Antonia Rojas, Montserrat López Onieva, Gabriela Canto, Alexandra Rodrigues, Andreia Forno, António Jorge Cabral, Rute Gonçalves, Ilya Vorozhko, Tatyana Sentsova, Olga Chernyak, Svetlana Denisova, Lidia Ilènko, Valery Muhortnich, Caroline Zimmermann, Alexander Rohrbach, Faisal R. Bakhsh, Kollen Boudewijn, Anne-Marie Oomkes-Pilon, Dorien Van Ginkle, Mira Šilar, Anja Jeverica, Tina Vesel, Tadej Avčin, Peter Korošec, Johanna van der Valk, Irene Berends, Nicolette Arends, Maurits van Maaren, Harry Wichers, Joyce Emons, Anthony Dubois, Nicolette de Jong, Oksana Matsyura, Lesya Besh, Chung-Hsiung Huang, Tong-Rong Jan, Gary Stiefel, Jean Tratt, Kerrie Kirk, Fabricia Carolino, Stefania Arasi, Lucia Caminiti, Giuseppe Crisafulli, Chiara Fiamingo, Jlenia Fresta, Giovanni Pajno, Ben Remington, Astrid Kruizinga, W. Marty Blom, Joost Westerhout, Sabina Bijlsma, Joe Baumert, Mark Blankestijn, Henny Otten, Rob Klemans, Anouska D. Michelsen-Huisman, Harmieke van Os-Medendorp, Astrid G. Kruizinga, Astrid Versluis, Gert van Duijn, H. Mary-Lene de Zeeuw-Brouwer, Jacqueline J. M. Castenmiller, Hub P. J. M. Noteborn, Geert F. Houben, Kristian Bravin, David Luyt, Bushra Javed, Phil Couch, Christopher Munro, Phil Padfield, Matt Sperrin, Aideen Byrne, Lizalet Oosthuizen, Carina Kelleher, Fiona Ward, Niamh Brosnan, Graham King, Eva Corbet, Josué Alejandro Huertas Guzmán, Montserrat Bosque García, Oscar Asensio, Laura Valdesoiro Navarrete, Helena Larramona, Xavier Domingo Miró, Katarzyna Pyrz, Moira Austin, Yanne Boloh, Philip Couch, Deirdre Galloway, Pilar Hernandez, Jonathan O’B. Hourihane, Fiona Kenna, Barbara Majkowska-Wojciechowska, Lynne Regent, Marina Themisb, Sabine Schnadt, Aida Semic-Jusufagic, Audrey Dunn Galvin, Tiina Kauppila, Mikael Kuitunen, Nikolaos A. Kitsioulis, Nikolaos Douladiris, Sofia Kostoudi, Ioanna Manolaraki, Dimitris Mitsias, Emmanouil Manousakis, Nikolaos G. Papadopoulos, Rebecca Knibb, Jennifer Hammond, Richard Cooke, Jaakko Yrjänä, Anna-Maija Hanni, Päivi Vähäsarja, Oona Mustonen, Teija Dunder, Petri Kulmala, Eva Lasa, Carmen D’Amelio, Sara Martínez, Alejandro Joral, Gabriel Gastaminza, Maria Jose Goikoetxea, David C. A. Candy, Marleen T. J. Van Ampting, Manon M. Oude Nijhuis, Assad M. Butt, Diego G. Peroni, Adam T. Fox, Jan Knol, Louise J. Michaelis, Ines Padua, Patricia Padrao, Pedro Moreira, Renata Barros, Hanan Sharif, Manzoor Ahmed, Nehad Gomaa, Joris Mens, Koen Smit, Frans Timmermans, Tomaž Poredoš, Anja Koren Jeverica, Marjeta Sedmak, Evgen Benedik, Meta Accetto, Mirjana Zupančič, Glauce Yonamine, Gustavo Soldateli, Bruna Aquilante, Antonio Carlos Pastorino, Cleonir Lui de Moraes Beck, Andrea Keiko Gushken, Mayra de Barros Dorna, Cristiane Nunes dos Santos, Ana Paula Moschione Castro, Abdulhadi Al-Qahtani, Rand Arnaout, Agha Rehan Khaliq, Rashid Amin, Farrukh Sheikh, Jorge Alvarez, Marta Anda, Miriam Palacios, Montserrat De Prada, Carmen Ponce, Bianca Balbino, Riccardo Sibilano, Thomas Marichal, Nicolas Gaudenzio, Hajime Karasuyama, Pierre Bruhns, Mindy Tsai, Laurent L. Reber, Stephen J. Galli, Ana Reis Ferreira, Josefina R. Cernadas, Aida del Campo García, Sara Pereiro Fernández, Nerea Sarmiento Carrera, Fernando Bandrés Sánchez-Cruz, José Ramón Fernández Lorenzo, Stephanie Claus, Claudia Pföhler, Franziska Ruëff, Regina Treudler, Mercedes Escarrer Jaume, Agustin Madroñero, Maria Teresa Guerra Perez, Juan Carlos Julia, Charlotte Hands Plovdiv, Lee Gethings, Jim Langridge, Karine Adel-Patient, Hervé Bernard, Ivona Barcievic-Jones, Raditsa Sokolova, Rumyana Yankova, Mariya Ivanovska, Marianna Murdjeva, Tatyana Popova, Svetlan Dermendzhiev, Martin Karjalainen, Ulrike Lehnigk, Duncan Brown, Julie C. Locklear, Julie Locklear, Ioana Maris, Jonathan Hourihane, Cristina Ornelas, Joana Caiado, Manuel Branco Ferreira, Manuel Pereira-Barbosa, Yolanda Puente, Juan Carlos Daza, Francisco Javier Monteseirin, Natalia Ukleja-Sokolowska, Ewa Gawronska-Ukleja, Magdalena Zbikowska-Gotz, Zbigniew Bartuzi, Lukasz Sokolowski, Aine Adams, Bernard Mahon, Karen English, Nelly Gourdon-Dubois, Laetitia Sellam, Bruno Pereira, Elodie Michaud, Khaled Messaoudi, Bertrand Evrard, Jean-Luc Fauquert, Francisca Palomares, Gador Gomez, Maria Jose Rodriguez, Luisa Galindo, Ana Molina, Lorella Paparo, Maurizio Mennini, Rosita Aitoro, Adam Wawrzeńczyk, Michał Przybyszewski, Anna Wawrzeńczyk, Hulya Ercan Sarıcoban, Meltem Ugras, Zerrin Yalvac, Bertine M. J. Flokstra-de Blok, J. L. van der Velde, Andrea Vereda, Clara Ippolito, Amaranta Traversa, Daniela Adriano, Daniela Manila Bianchi, Silvia Gallina, Lucia Decastelli, Melina Makatsori, Anne Miles, Sonja Posega Devetak, Iztok Devetak, Soraya Ainad Tabet, Jeanette Fisker Trandbohus, Pernille Winther, Hans-Jørgen Malling, Kirsten Skamstrup Hansen, Lene Heise Garvey, Chia-Chi Wang, Yin-Hua Cheng, Chun-Wei Tung, Mariola Dietrich, Ingo Marenholz, Birgit Kalb, Sarah Grosche, Katharina Blümchen, Rupert Schlags, Mareike Price, Sylke Rietz, Jorge Esparza-Gordillo, Susanne Lau, Young-Ae Lee, Ali Almontasheri, Mohammad Al Bahkali, Sahar Elshorbagi, Abdullah Alfhaid, Mashary Altamimi, Eman Madbouly, Hassan Al-Dhekri, Rand K. Arnaout, Maria Basagaña, Sira Miquel, Borja Bartolomé, Bettina Brix, Stefanie Rohwer, Sandra Brandhoff, Alena Berger, Waltraud Suer, Alf Weimann, Cristina Bueno, Laura Martín-Pedraza, Sara Abián, Pablo San Segundo-Acosta, Juan Carlos López-Rodríguez, Rodrigo Barderas, Eva Batanero, Javier Cuesta-Herranz, María Teresa Villalba, Magna Correia, Filipe Benito-Garcia, Cristina Arêde, Susana Piedade, Mário Morais-Almeida, James Hindley, Ross Yarham, Anna Kuklinska-Pijanka, David Gillick, Karine Patient, Martin D. Chapman, Katrine L. Bøgh, Ana Miranda, Eugénia Matos, Anna Sokolova, Huan Rao, Ivona Baricevic-Jones, Frances Smith, Wentong Xue, Helga Magnusdottir, Anna G. Vidarsdottir, Sigrun Lund, Anders Blom Jensen, Bjorn R. Ludviksson, Reyna Simon, Robert Elfont, Sean Bennett, Robert Voyksner, Maria de Lurdes Torre, Songül Yürek, Margaretha A. Faber, Annick Bastiaensen, Evelyne Mangodt, Athina van Gasse, Ine Decuyper, Vito Sabato, Margo M. Hagendorens, Chris H. Bridts, Luc S. De Clerck, Didier Ebo, Susanne Schwarz, Mandy Ziegert, Saskia Albroscheit, Christian Schwager, Skadi Kull, Jochen Behrends, Niels Röckendorf, Frauke Schocker, Andreas Frey, Arne Homann, Wolf-Meinhard Becker, Uta Jappe, Nesrine Zaabat, Sylvia Osscini, Chantal Agabriel, Benoît Sterling, Ania Carsin, Valérie Liabeuf, Monica Maćków, Alina Zbróg, Monica Bronkowska, Justine Courtois, Romy Gadisseur, Catherine Bertholet, Pierre Lukas, Etienne Cavalier, Philippe Delahaut, Birgit Quinting, Margareta Brandt Gertmo, Ewa Ternesten Hasseus, Vladyslava Barzylovych, Júlio Oliveira, Luis F. Ensina, Carolina S. Aranda, Leire Dopazo, Rebeca Lopez, Raquel Perez, Laura Santos-Diez, Agurtzane Bilbao, Juan Miguel Garcia, Ignacio García Núñez, María Ángeles Algaba Mármol, María José Barasona Villarejo, José Antonio Bácter Martos, Marina Suárez Vergara, José María Ignacio García, Agata Michalska, Grzegorz Sergiejko, Robert Zacniewski, Ileana-Maria Ghiordanescu, Cristina Deaconu, Mihaela Popescu, Roxana Silvia Bumbacea, Alkerta Ibranji, Elida Nikolla, Gjustina Loloci, Nanna Juel-Berg, Lau Fabricius Larsen, Lars Kjaergaard Poulsen, João Marcelino, Ricardo Prata, Ana Célia Costa, Fátima Duarte, Marta Neto, Jennifer Santos, Luís Câmara Pestana, Daniel Sampaio, Paola Minale, Paola Dignetti, Donatella Bignardi, Irena Nedelea, Florin-Dan Popescu, Mariana Vieru, Florin-Adrian Secureanu, Carmen Saviana Ganea, Miguel Vieira, José Pedro Moreira Silva, Timothy Watts, Sophia Watts, Marta Lomikovska, Marina Peredelskaya, Natalia Nenasheva, Ivana Filipovic, Zorica Zivkovic, Djordje Filipovic, Jennette Higgs, Amena Warner, and Carla Jones

Subjects
Immunologic diseases. Allergy, RC581-607
Published
2017
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4. Technical variability of 2D gel electrophoresis – Application to soybean allergens

Author

Catherine Pallen, Claire Friry-Santini, Corinne Herouet-Guicheney, and Annabelle Capt

Subjects
False positive rate, Soybean/allergen, Technical variability, Electrophoresis, Genetically modified, Toxicology. Poisons, RA1190-1270
Abstract

Two-dimensional gel electrophoresis (2-DE) technique is used as a performing technique to assess the variability of protein expression in crops, and especially soybean endogenous food allergens, which are a subset of proteins of interest for assessing whether genetically modified (GM) soybean has a different allergenic profile compared to its non-GM counterpart. On top of the biological variability of the 2-DE, which has already been studied by several laboratories, technical variability has to be evaluated. In this study, several sources of variability (number of gel replicates, protein extracts, study timings and operators) were assessed qualitatively and quantitatively on all detectable polypeptide spots as well as on food allergen spots. Results showed that the major source of variability was the number of gel replicates. Other sources were minor. This has a direct practical impact on the laboratory work as this supports the utilization of three or four gel replicates to get robust results. Furthermore, this implies that the study can be run over several days, and be performed by several trained operators, without impacting its reproducibility. Furthermore, 2-DE could detect a 2-fold change between two samples with an acceptable rate of false positives (below 7%). This level of sensitivity is acceptable in the context of safety assessment of GM soybean as the biological variability of proteins in soybean is higher than the technical variability shown in this study. Overall, the 2-DE technique is suitable for investigating endogenous food allergen variability between several soybean seeds, including GM and non-GM counterpart.

Published
2014
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5. Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure

Author

Muriel Totis, Rod A. Herman, Thomas Edrington, Gregory S. Ladics, Rakesh Ranjan, Rong Wang, Hector Serrano, Gerson Graser, André C. Knulst, Lucilia Pereira Mouriès, Lars K. Poulsen, Brian Barnett, Kevin C. Glenn, Erin Bell, Dave Speijer, Ronald van Ree, Jean Baptiste Rascle, Tom Davis, Jaap H. Akkerdaas, Scott McClain, Annabelle Capt, Experimental Immunology, APH - Personalized Medicine, APH - Global Health, AII - Inflammatory diseases, Medical Biochemistry, AGEM - Amsterdam Gastroenterology Endocrinology Metabolism, AII - Amsterdam institute for Infection and Immunity, and Ear, Nose and Throat

Subjects
0301 basic medicine, Pulmonary and Respiratory Medicine, Proteolysis, medicine.medical_treatment, Immunology, Genetically modified crops, medicine.disease_cause, 03 medical and health sciences, 0302 clinical medicine, Allergen, Pepsin, medicine, Immunology and Allergy, Antiserum, Protease, medicine.diagnostic_test, biology, business.industry, Research, RC581-607, 030104 developmental biology, 030228 respiratory system, Biochemistry, biology.protein, Immunologic diseases. Allergy, business, Digestion, Plant lipid transfer proteins
Abstract

Background Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. Aim To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. Methods Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2–2.5–4.0) and pepsin-to-protein ratio (PPR: 10–1–0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. Results At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. Conclusions Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair. Electronic supplementary material The online version of this article (10.1186/s13601-018-0216-9) contains supplementary material, which is available to authorized users.

Published
2018
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6. Supplementary data on the characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, which confers herbicide tolerance, and on the compositional assessment of field grown MST-FGØ72-2 soybean expressing HPPD W336

Author

Kenneth Edward Pallett, Isabelle Coats, Annabelle Capt, Rozemarijn Dreesen, and Regina Oberdoerfer

Subjects
Supplementary data, Multidisciplinary, Glycosylation, Chemistry, 010501 environmental sciences, lcsh:Computer applications to medicine. Medical informatics, 030226 pharmacology & pharmacy, 01 natural sciences, Intestinal fluid, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Biochemistry, Dioxygenase, Biochemistry, Genetics and Molecular Biology, lcsh:R858-859.7, Research article, Plant metabolism, lcsh:Science (General), 4-Hydroxyphenylpyruvate dioxygenase, 0105 earth and related environmental sciences, Homogentisate 1,2-dioxygenase, lcsh:Q1-390
Abstract

Supplementary data are provided which are supportive to the research article entitled “Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGO72-2 soybean” (Dreesen et al., 2018) [1] . The conducted supplementary analyses include the characterization of additional Escherichia coli-produced HPPD W336 protein batches used as a surrogate in HPPD W336 safety studies, the assessment of potential glycosylation and monitoring of stability in simulated intestinal fluid and during heating of the HPPD W336 protein. Furthermore, data are provided on conducted field trials and subsequent compositional analysis in MST-FGO72-2 soybean grain of compounds related to the tyrosine degradation pathway and the metabolism of homogentisate.

Published
2018

7. Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean

Author

Rozemarijn Dreesen, Regina Oberdoerfer, Kenneth Edward Pallett, Isabelle Coats, and Annabelle Capt

Subjects
0106 biological sciences, 0301 basic medicine, Pseudomonas fluorescens, Toxicology, 01 natural sciences, 4-Hydroxyphenylpyruvate Dioxygenase, 03 medical and health sciences, chemistry.chemical_compound, Amino Acids, Aromatic, Dioxygenase, Aromatic amino acids, Amino Acid Sequence, Homogentisate 1,2-dioxygenase, chemistry.chemical_classification, biology, Herbicides, Wild type, General Medicine, biology.organism_classification, Plants, Genetically Modified, Metabolic pathway, 030104 developmental biology, Enzyme, Phenotype, chemistry, Biochemistry, Tyrosine, Soybeans, 4-Hydroxyphenylpyruvate dioxygenase, 010606 plant biology & botany
Abstract

By transgenic expression technology, a modified 4-hydroxyphenylpyruvate dioxygenase enzyme (HPPD W336) originating from Pseudomonas fluorescens is expressed in MST-FGO72-2 soybean to confer tolerance to 4-benzoyl isoxazole and triketone type of herbicides. Characterization and safety assessment of HPPD W336 were performed. No relevant sequence homologies were found with known allergens or toxins. Although sequence identity to known toxins showed identity to HPPD proteins annotated as hemolysins, the absence of hemolytic activity of HPPD W336 was demonstrated in vitro. HPPD W336 degrades rapidly in simulated gastric fluid. The absence of toxicity and hemolytic potential of HPPD W336 was confirmed by in vivo studies. The substrate spectrum of HPPD W336 was compared with wild type HPPD proteins, demonstrating that its expression is unlikely to induce any metabolic shifts in soybean. The potential effect of expression of HPPD W336 on metabolic pathways related to tyrosine was investigated by comparing seed composition of MST-FGO72-2 soybean with non-genetically modified varieties, demonstrating that expression of HPPD W336 does not change aromatic amino acid, homogentisate and tocochromanol levels. In conclusion, HPPD W336 was demonstrated to be as safe as other food proteins. No adverse metabolic effects were identified related to HPPD W336 expression in MST-FGO72-2 soybean.

Published
2018

8. MOESM3 of Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure

Author

Akkerdaas, Jaap, Totis, Muriel, Barnett, Brian, Bell, Erin, Davis, Tom, Edrington, Thomas, Glenn, Kevin, Graser, Gerson, Herman, Rod, Knulst, Andre, Ladics, Gregory, McClain, Scott, Poulsen, Lars, Ranjan, Rakesh, Jean-Baptiste Rascle, Serrano, Hector, Speijer, Dave, Wang, Rong, Lucilia Pereira MourièS, Annabelle Capt, and Ree, Ronald

Abstract

Additional file 3: Fig. E3. SDS-PAGE and immunoblot is shown for both bovine collagen after duodenal digestion preceded by gastric digestion at pH 1.2/PPR 0.1. Under these conditions, bovine collagen is fully resistant to duodenal digestion.

Published
2018
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9. Abstracts from the Food Allergy and Anaphylaxis Meeting 2016

Author

Georgios Rentzos, Esben Eller, Annick Mercenier, Magna Correia, Pedro Moreira, Linda Verrill, Mário Morais-Almeida, Frauke Schocker, Carsten Bindslev-Jensen, Ulrike Lehnigk, Andreas Frey, Lars Verschuren, Anna-Lena Bramstång Björk, Lene Heise Garvey, Elena Molina, Katrine L. Bøgh, J. L. van der Velde, Montserrat Alvaro, Phil Padfield, Niamh Brosnan, Nelly Gourdon-Dubois, Christian Harwanegg, Manuel Branco Ferreira, Songül Yürek, Amyra Ali Azamar Jácome, Rashid Amin, José Ramón Fernández Lorenzo, Kirsten Beyer, Florin-Adrian Secureanu, Charlotte Bernhard Madsen, Julie Galand, Claudia Pföhler, Rabea Reinert, Heinz Fehrenbach, Anton Hartmann, Maria Salas, Arne Homann, Rand Arnaout, Sarah Lindsley, Nerea Sarmiento Carrera, Patrícia Padrão, Berber Vlieg-Boerstra, Alkerta Ibranji, Benoît Sterling, Maria Auxiliadora Guerrero, Maria L. Baeza, Maurizio Tamburrini, Eva Untersmayr, Tanja Cirkovic Velickovic, Farrukh Sheikh, Sarah Kuntz, Sara Martínez, Kriti Gupta, Maria de Lurdes Torre, Yvonne Vergouwe, Ross Yarham, Faisal R. Bakhsh, Marta Vazquez-Ortiz, João Marcelino, Tullio Frediani, Ricardo Prata, Anna Kaarina Kukkonen, Gustavo Soldateli, Ines Mrakovčić-Šutić, Elodie Drumez, Cristina Ornelas, Maria Vazquez de La Torre, Renata Barros, Agata Szymkiewicz, Aneta Tomaszewska, Stefanie Rohwer, Charlotte Eisenmann, Adriana Muntean, Matteo Moretti, Johanna P. M. van der Valk, Birgit Quinting, Stefan Kabasser, David Gillick, Michał Przybyszewski, Grzegorz Sergiejko, Antonio Jorge Cabral, Alba Pablos-Tanarro, Robert Elfont, Marit Reitsma, Roxana Silvia Bumbăcea, Nelson Rosario, Maria Livia Bernardi, Cristiane N. Santos, Christian Radauer, Sandra Denery-Papini, Geert F. Houben, Nicolette W. de Jong, Marta Anda, Alexander Rohrbach, Teodorikez Wilfox Jimenez-Rodriguez, María Teresa Villalba, Karine Patient, Harmieke van Os-Medendorp, Kathrin Scherer, Marta Goñi Esarte, Svetlan Dermendzhiev, Jossie Garthoff, Michelle M. Epstein, Catherine Bertholet, Bruna Pultrini Aquilante, Didier G. Ebo, Roberta Aina, Jorge Kalil, Petri Kulmala, Lars K. Poulsen, Barbara Ballmer, H. Mary-Lene de Zeeuw-Brouwer, Karin Hoffman-Sommergruber, Winfried Leeman, Anne Miles, Nehad Gomaa, Maria Teresa Costantino, Evelyne Mangodt, Maria Konstantakopoulou, Rosa Jimenez, Attilio Francesco Speciani, Helga Magnusdottir, Inmaculada Sánchez-Machín, H. Kaddouri, Raquel Perez, Astrid Versluis, Anthony E. J. Dubois, Anja Koren Jeverica, Zdenka Barićev-Novaković, Joost Westerhout, A.M. Plaza, Júlio Oliveira, Pierre Lukas, Oksana Matsyura, Giuseppe Pingitore, Mira Silar, Huub F. J. Savelkoul, Urszula Samolinska-Zawisza, Chantal Brossard, Ana Reis Ferreira, Reiko Teshima, Serena Perna, Mariola Dietrich, Dirk Verhoeven, Fiona Kenna, Paloma Poza-Guedes, Cristobalina Mayorga, Pilar Hernandez, Roberto Bernardini, Roberto Berni Canani, Maria Francesca Patria, Mariana Vieru, Julie Locklear, Esther van Twuijver, Marina A. Kiseleva, H. Kim A. Brand, Gabriele Schulz, Ileana-Maria Ghiordanescu, Nikolaos G. Papadopoulos, Danijela Apostolovic, Maria del Mar Folqué, Eva Lasa, Mohammad Al Bahkali, Arianna Dondi, Sofia Kostoudi, Simon Rosenberg, Véronique Nève, Rumyana Yankova, Barbara Ballmer-Weber, Maria Jose Goikoetxea, María José Barasona Villarejo, Teija Dunder, Tina Vesel, Elisa Gritti, Marianne van Hage, Laure Castan, Etienne Beaudouin, Margherita Di Costanzo, Natalia Ukleja-Sokołowska, Matthew Sperrin, Paul Turner, Saskia Albroscheit, Rebeca Lopez, Sonja Posega Devetak, Francisca Palomares, Laetitia Sellam, Jennifer Hammond, Astrid G. Kruizinga, Konrad Furmanczyk, Patricia Macchiaverni, Olga Pakholchuk, I V Vorozhko, Ania Carsin, Aleksandar Bulog, Joris Mens, Bianca Balbino, James P. Hindley, Jaap H. Akkerdaas, Jonathan Hourihane, Hilde Cnossen, Tatyana A Popova, Rupert Schlags, Natalia Nenasheva, A. Ehrenberg, Sigurveig T. Sigurdardottir, Anna Iliopoulou, Clara Ippolito, Piotr Samel-Kowalik, Paloma Campo, Gador Bogas, Constantinos Pitsios, Sarah Grosche, Karla Leversia Borjas Aguilar, Amena Warner, Birgit Kalb, Lénaïck Dupuis, Harry J. Wichers, Diana Silva, Gabriele Piuri, Linus Grabenhenrich, Emanuel Sarinho, M. Eleonore Pettersson, Rodrigo Barderas, Gary Stiefel, E. N. Clare Mills, Lali Saginadze, Ruta Dubakiene, Francesca Cipriani, Mindy Tsai, Marina Themisb, Michela Ciancamerla, Rebecca Knibb, Ruperto González-Pérez, Marina Peredelskaya, André C. Knulst, Irene Berends, S N Denisova, Montserrat Fernandez-Rivas, Paolo Maria Matricardi, Daniel Sampaio, Giorgio Bedogni, Sira Miquel, Koen Smit, Stefania Arasi, Sylvia Osscini, Justine Courtois, Cristina Deaconu, Srdan Banac, Kirsten Hansen, Jennette Higgs, Charlotte Hands Plovdiv, Carlotta Povesi Dascola, Noe Ontiveros, Rik Schrijvers, Carla Mastrorilli, Maria Passioti, Sebastian Tschirner, Bjorn R. Ludviksson, Joana Caiado, Daniel Corbacho, Sophie Nutten, Agustin Madroñero, Marina Suárez Vergara, Boudewjin J. Kollen, Catherine J Nock, Johanna Rost, Agnieszka Lipiec, Skadi Kull, Geert Houben, Carlo Caffarelli, Jordi Roca-Ferrer, Bolesław Samoliński, Isabelle Cleach, Colette Larré, Aideen Byrne, Corinne Herouet-Guicheney, Jonas Lidholm, Vera Assmann, W. Marty Blom, Adriano Mari, Adam T. Fox, Giuseppe Crisafulli, Afke M. M. Schins, Anastasia Papadopoulou, Flávio Sano, Richard Cooke, Franziska Ruëff, L Jorjoliani, Rebekah Sayers, Anne-Marie Kochuyt, Borja Bartalomé, Anne Moneret-Vautrin, Juan Carlos Julia, Jelena Mihailovic, Katarzyna Pyrz, Kollen Boudewijn, Margherita Varini, Paolo Giordani, Claudia Alessandri, Johanna van der Valk, Antoine Deschildre, Mariam Tskhakaia, Rosina López-Fandiño, Omar Kheroua, Nicolette J. T. Arends, Alina Zbróg, Chung-Hsiung Huang, Andrea Wangorsch, Ulf Bengtsson, Jim Langridge, Dasha Roa-Medellín, Jose Ignacio Larco, Bert Popping, Ana Prieto del Prado, José Antonio Bácter Martos, Rita Nocerino, Sophia Watts, Elisa Haroun, Danilo Villalta, Lee A. Gethings, María Ángeles Algaba Mármol, Ewa Gawrońska-Ukleja, Cornelia Jansen, André Moreira, V A Revyakina, Robert Zacniewski, Xavier Domingo Miró, Francesco Macrì, Mayra de Barros Dorna, Akanksha Sharma, Stephen J. Till, Ali Almontasheri, Chrystyna Kalicinsky, Robbert Sutorius, Akila Rekima, Mark A. Blankestijn, Nicolette Arends, Chiara Pistoletti, Merima Bublin, Manuel Pereira-Barbosa, Reyna Simon, Martin Karjalainen, Sam Mehr, Tushar Banerjee, Carmen Riggioni, Bertrand Evrard, Antonio Carlos Pastorino, Lidia Ilènko, Nikolaos Douladiris, Miguel Vieira, Erik Wambre, Francisco Cabrera-Chavez, Nikos G. Papadopoulos, Piotr Humeniuk, Gert van Duijn, Francesco Zinno, Young-Ae Lee, Lisa Tuppo, Hans de Groot, Léon M.J. Knippels, Pawel Dubiela, Henrik Fomsgaard Kjaer, Andrea Vereda, Djamel Saidi, Kok Loong Ue, Henk Van Loveren, Maria Jose Rodriguez, Marta Lomikovska, Elodie Michaud, Josefina Cernadas, Carmen Ponce, Marysia Recto, Frances Smith, Hassan Al-Dhekri, Shinobu Sakai, Thomas Eiwegger, Ana Rodolfo, Diego Peroni, Nikki Edelbroek, Waltraud Suer, Jenny van Odijk, Irena Nedelea, Borja Bartolomé, Lauren Lissner, Marjeta Sedmak, Annette Jamin, Italo De Vitis, Bo Pontoppidan, Annabelle Capt, Diego G. Peroni, Susana Rodrigues, Juan Carlos López-Rodríguez, David Endesfelder, Lesya Besh, Danila Zennaro, Charlotte G. Mortz, Kati Palosuo, María José Torres, Esozia Arroabarren, Lynne Regent, Laura Valdesoiro Navarrete, Ana Molina, Agha Rehan Khaliq, Phil Couch, Ana Miranda, Thomas Marichal, Riccardo Asero, Denise Borges, Dikla Pivko Levy, Miriam Palacios, Mireille Eb, Stephen J. Galli, Karin Hoffmann-Sommergruber, Anna Gudrun Vidarsdottir, Ifigenia Sfika, André Wolterbeek, Mauro Calvani, Edward F. Knol, Joyce A. M. Emons, Anne-Marie Oomkes-Pilon, Montserrat Bosque García, Daniel Lozano-Ojalvo, Filip Raciborski, Deirdre Galloway, Nanna Juel-Berg, Margareta Brandt Gertmo, Cornelia Bergmayr, Rob Klemans, Juliane Gregersen, Yolanda Meijer, Bettina Brix, Susanne Lau, Karine Adel-Patient, Hanneke van der Kleij, Mareike Price, Jean-Louis Mege, Lizalet Oosthuizen, Agurtzane Bilbao, Indre Butiene, Antonino Romano, Colin Barber, Rosana Camara Agondi, Nour Baïz, Soraya Ainad Tabet, Peter Korošec, Laurian Jongejan, Francine C. van Erp, José Pedro Moreira Silva, Nandinee Patel, Jaime Lozano, Prescilla V. Jeurink, Artur Walkiewicz, Bryan M. Harvey, Tiina J. Kauppila, Aida Semic-Jusufagic, Marianna Murdjeva, Miren Arteaga, Y. Bouferkas, Geunwoong Noh, Henny G. Otten, Sabine Dölle, Christopher Munro, O Dominguez, Gerard H. Koppelman, Leonieke N. van Veen, Vasti Iancu, Georg Mitterer, Patrizia Polverino de Laureto, Juliane Schulz, Ewa Ternesten Hasseus, Caroline Zimmermann, Ivona Barcievic-Jones, Shira Benor, Susanne Schwarz, Eun Ha Jang, Josefina Rodrigues Cernadas, Tadej Avcin, Joseph L. Baumert, Pernille Winther, Stéphane Leteurtre, Gabriela Canto, Louise J Michaelis, Jorge Alvarez, Gabriel Gastaminza, Michela Carola Speciani, Rute Gonçalves, Leire Dopazo, Evgen Benedik, Susanne C. Diesner, Luc S. De Clerck, A. Wesley Burks, Maurits S. van Maaren, Salvatore Tripodi, Philippe Aubert, Kamel Eddine El Mecherfi, Aline B. Sprikkelman, Ana Prieto, Margitta Worm, Edyta Krzych, Maja Krstic, Iride Dello Iacono, Melanie Cap, Alf Weimann, Maria Nassiri, Niels Röckendorf, Vladyslava Barzylovych, Marcia C. Mallozi, Pierre Bruhns, Jeanette Fisker Trandbohus, Ekaterini Papadopoulou, Vicente Albendiz, Timothy Watts, Uta Jappe, Javier Moreno, Maria Carmen Verga, Beatriz Secades Barbado, Carmen Di Scala, Roy Gerth van Wijk, Vladimir Mićović, Esther Barrionuevo, Giampaolo Ricci, Luisa Galindo, Ana Paula Beltran Moschione Castro, Oona Mustonen, Jia Yin, Lucia Caminiti, Jorge Esparza-Gordillo, N Adamia, Anna-Maija Hanni, Romy Gadisseur, Stefano Luccioli, Regina Treudler, Rosetta Ferrara, M. Guendouz, Jaakko Yrjänä, Philipp Starkl, Premendra D. Dwivedi, Javier Cuesta-Herranz, Johan Garssen, Ekaterini Goudouris, Sridevi Muralidharan, Kate Grimshaw, Carolina Sanchez Aranda, Ioana Maris, Manzoor Ahmed, Hajime Karasuyama, Stephanie Claus, Chantal Agabriel, Karen English, Dorien Van Ginkle, Eleonora Savi, Loredana Chini, Ine I. Decuyper, Sabine Schnadt, Valérie Trendelenburg, Jean-Luc Fauquert, Maurizio Mennini, Nikolaos Mikos, Ana Célia Costa, Steve L. Taylor, A. A. Schoemaker, Sara Abián, Margo M. Hagendorens, Andrea Di Rienzo Businco, Melina Makatsori, Eugénia Matos, Lucy Walker, Nikolaos A. Kitsioulis, David Alejandro Mendoza Hernández, Maria Starkhammar, Djordje Filipovic, Aine Adams, Mukul Das, Sonsoles Intente-Herrero, Natalia Blanca López, Marek L. Kowalski, Diana Deleanu, Bernard P. Mahon, Jean-Michel Wal, Lucia Decastelli, Mihaela Popescu, Aimee Lou Nano, Eva Batanero, Tong-Rong Jan, Yolanda Puente, Jacek Borowicz, Aimée Dorkenoo, J. Östling, Mashary Altamimi, Michel Neunlist, Zerrin Yalvaç, Sonia Ricò, Wentong Xue, Linda Cosenza, David C. A. Candy, Robert D. Voyksner, Montserrat De Prada, Abdulhadi Al-Qahtani, Sébastien Holvoet, Wolf-Meinhard Becker, Meropi D. Kontogianni, Bruno Pereira, F. Pineau, Eva Corbet, Kirsten Mehlig, Rosella De Poi, Jolanda H. M. van Bilsen, José Luís Plácido, Hans-Jørgen Malling, Chia-Chi Wang, Philip Couch, Kerrie Kirk, Agata Michalska, Sylke Rietz, Mariya Ivanovska, Victor Matheu-Delgado, Carl Hamsten, Francisca Gómez, Neusa Falbo Wandalsen, Mika J. Mäkelä, Tatyana Sentsova, Kristian Bravin, Philippe Delahaut, Hervé Bernard, Leonor Carneiro-Leão, Michele Miraglia del Giudice, Elena D. Kuvshinova, Jochen Behrends, Caroline Klingebiel, Meta Accetto, Claire Mills, Mariona Pascal, Miguel García Domínguez, Huan Rao, Carmen Saviana Ganea, Umberto Pelosi, Stefano Pattini, Bodo Niggemann, Annamaria Bianchi, Laura Martín-Pedraza, Anna Selby, Cristina Bueno, Stefan Vieths, Luis Felipe Ensina, Florin-Dan Popescu, Antonio Fernandez, Soren Wille, Erna Van Hoeyveld, Carina Kelleher, Athina L. Van Gasse, Anders Blom Jensen, Zorica Zivkovic, Moshe Ben-Shoshan, Sara Pereiro Fernández, Ronald van Ree, Antima Banerjee, Pablo Merida, Mandy Ziegert, Barbara Wróblewska, Morten Christensen, Gador Gomez, Pablo San Segundo-Acosta, Khaled Messaoudi, Anne-Sofie Ravn Ballegaard, Mikael Kuitunen, David Luyt, Vito Sabato, Nesrine Zaabat, Svitlana Zubchenko, Julien Labreuche, Elin Lustig, Katharina Blumchen, Guillaume Pouessel, Ana Fiandor, Stéphanie Lejeune, Paola Dignetti, Helen Brown, Anastasia Cirisano, Evangelia Kompoti, Anna Sokolova, Mercedes Escarrer Jaume, Shan Deng, Dirceu Solé, Chiara Fiamingo, Alessandro Travaglini, Nicolas Gaudenzio, Zbigniew Bartuzi, Antonia Rojas, Roberta Olcese, Nanju Alice Lee, Sandra Brandhoff, Lucien F. Harthoorn, Anna Maria Szyc, Andrea Costanzi, Sabine Pfeifer, Emiliano De Dominicis, Chiara Rafaiani, Yin-Hua Cheng, Elida Nikolla, Ignacio García Núñez, Diego Faggian, Lieve Coorevits, Ayelet Rimon, Erika Jensen-Jarolim, Jacqueline J.M. Castenmiller, Kristin Verbeke, Sean Bennett, Paraskevi Xepapadaki, Glauce Hiromi Yonamine, Anna Wawrzeńczyk, Rusudan Karseladze, Emmanouil Manousakis, Fiona Ward, Ivana Filipovic, Marie Bodinier, Viviana Moschese, Pedro Giavina Bianchi, Alice Coimbra, Alena Berger, Anton A. Shekhetov, Monica Maćków, Philippe Egenmann, Oscar Asensio, Lidia Hanna Markiewicz, Chun-Wei Tung, Zsolt Szépfalusi, Jan Knol, Frits Koning, Carolina S. Aranda, Annick Bastiaensen, Giovanni Battista Pajno, Leticia Pérez-Rodríguez, Ewout W. Steyerberg, Riccardo Sibilano, Valérie Verhasselt, Roberta Lupi, Lukasz Sokolowski, Adam Wawrzeńczyk, Dimitris I. Mitsias, Andreia Forno, Antoine Magnan, Christian Schwager, Ioanna Manolaraki, Alessandro Fiocchi, Tanja Rouhani Rankouhi, Carla Jones, Ana Pereira, Hannah M. Kansen, Michael Clausen, D. Bignardi, Assad M. Butt, Julie C. Locklear, Katrine Lindholm Bøgh, Valery Muhortnich, María Teresa Giner, Juan Miguel Garcia, Maria Luisa Somoza, Raditsa Sokolova, Maria Pasioti, Mirjana Zupancic, Joana Vitte, Duncan Brown, Arnaldo Porto, M. Turfkruyer, Lau Fabricius Larsen, Filipe Benito-Garcia, Mayra Coutinho Andrade, Meltem Ugras, Ingrid Sutic, Rand K. Arnaout, Etienne Cavalier, Marta Neto, Grégory Bouchaud, Elena Varin, Bushra Javed, Carla Camerotto, Monica Bronkowska, Cristiano Caruso, Luís Amaral, Jackeline F. Motta, Sahar Elshorbagi, Cornelis K. van der Ent, Alexandra Rodrigues, Silvia Peveri, Juan Heber Castellanos, Muriel Totis, Joan Bartra, Gjustina Loloci, Ivana Giangrieco, Ekaterina Khaleva, Joaquín Navarro, Kayoko Matsunaga, Jlenia Fresta, Jonathan O'b Hourihane, Sabina Bijlsma, Ana Rodriguez-Fernandez, Rosita Aitoro, Daniela Manila Bianchi, Rosa Jimenez-Feijoo, Mario Plebani, Marleen T. J. Van Ampting, Anthony E.J. Dubois, Clémence Mordacq, Simone Frediani, Martin Chapman, Helena Larramona, Fabrícia Carolino, Yanne Boloh, Ivona Baricevic-Jones, Mathilde Claude, Caroline Thumerelle, Päivi Vähäsarja, Mareen R. Datema, Abdullah Alfhaid, Diana Perez Alzate, Laura Santos-Diez, Graham King, Maria Teresa Guerra Perez, Jean-Marie Renaudin, Frieke Kuper, Josué Alejandro Huertas Guzmán, Harry Wichers, Thomas Keil, A. Haddi, Jennifer Santos, Regina Selb, Aida del Campo García, Maria Basagaña, Valentino Pavišić, Angela Simpson, Chris H. Bridts, Susana Piedade, Silvia Gallina, Isabella Pali-Schöll, Inês Pádua, Margaretha A. Faber, Frédéric de Blay, Luís Câmara Pestana, Ben Remington, Anna S. Pelkonen, Sandra Lucarelli, Ivana Šutić, Olivier Tranquet, Montserrat López Onieva, Antonio Amoroso, Paola Minale, Katia Basello, George Du Toit, Daniela Adriano, Adnan Custovic, Zbikowska-Gotz M, Domingo Barber, Inmaculada Doña, Christine Breynaert, Hanan Sharif, Manon M. Oude Nijhuis, Sandra Denery, Bertine M. J. Flokstra-de Blok, Rinkesh Kumar Gupta, Pieter-Jan de Kam, Dianne E. Campbell, Carmen M. D'Amelio, Nunzia Maiello, Ingo Marenholz, José María Ignacio García, Helen Lindqvist, Lilian Moraes, Cleonir Lui de Moraes Beck, Eunice Dias de Castro, Cono Casale, Barbara Majkowska-Wojciechowska, Maria Petrodimopoulou, Andrea Mikkelsen, Hub P. J. M. Noteborn, Hulya Ercan Saricoban, Moira Austin, Martinus Løvik, Graham Roberts, Isabella Annesi-Maesano, Aeilko H. Zwinderman, Sigrun H. Lund, Anouska D. Michelsen-Huisman, Fernando Bandrés Sánchez-Cruz, Francisco Javier Ruano, Valérie Liabeuf, Eman Madbouly, Pasquale Comberiati, Maria Isabel Garcimartin, Stephan Scheurer, Inna A. Larkova, Jean Tratt, Renata Rodrigues Cocco, Younes Belabbas, Lorella Paparo, Elizabeth Griffiths, Gian Lodovico Rapaccini, Audrey Dunn Galvin, Zizi Cojocariu, Isidor Hutteger, Claire Claverie, S.M. Nedelska, Anna Kuklinska-Pijanka, Jenny Badas, Mónica Piquer, Iztok Devetak, A.K.F. Gushken, Frans Timmermans, Adriana Machinena, Francisco Javier Monteseirin, Amaranta Traversa, Fátima Cabral Duarte, Tomaž Poredoš, Peter Meyer, Cristina Arêde, Rosa Muñoz-Cano, Barbara Pfitzner, Alejandro Joral, Juan Carlos Daza, Laurent L. Reber, Olga Chernyak, and Maria Antonetta Ciardiello

Subjects
Pulmonary and Respiratory Medicine, Allergy, medicine.medical_specialty, 030504 nursing, business.industry, Immunology, RC581-607, medicine.disease, Meeting Abstracts, 03 medical and health sciences, 0302 clinical medicine, Food allergy, 030225 pediatrics, Family medicine, medicine, Immunology and Allergy, Immunologic diseases. Allergy, 0305 other medical science, business, Anaphylaxis
Published
2017
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10. Technical variability of 2D gel electrophoresis – Application to soybean allergens

Author

Claire Friry-Santini, Corinne Herouet-Guicheney, Annabelle Capt, and Catherine Pallen

Subjects
Electrophoresis, Health, Toxicology and Mutagenesis, Context (language use), macromolecular substances, Toxicology, Genetically modified, Soybean/allergen, Article, lcsh:RA1190-1270, False positive paradox, False positive rate, Polyacrylamide gel electrophoresis, GM, genetically modified, lcsh:Toxicology. Poisons, Gel electrophoresis, Two-dimensional gel electrophoresis, Spots, Chemistry, business.industry, 2-DE, two-dimensional gel electrophoresis, food and beverages, IPG, immobilized pH gradient, Genetically modified organism, Biotechnology, Technical variability, IEF, isoelectric focusing, CV, coefficient of variation expressed as percentage, SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, business, Biological variability
Abstract

Highlights • 2-DE is robust and suitable for comparing the GM soybean with its non-GM counterpart; its technical variability is lower than the biological variability. • Main source of variability is the gels so 3–4 gel replicates should be used. • Other sources of variability are minor, which gives some experimental flexibility, i.e. study can be run over several days, several operators., Two-dimensional gel electrophoresis (2-DE) technique is used as a performing technique to assess the variability of protein expression in crops, and especially soybean endogenous food allergens, which are a subset of proteins of interest for assessing whether genetically modified (GM) soybean has a different allergenic profile compared to its non-GM counterpart. On top of the biological variability of the 2-DE, which has already been studied by several laboratories, technical variability has to be evaluated. In this study, several sources of variability (number of gel replicates, protein extracts, study timings and operators) were assessed qualitatively and quantitatively on all detectable polypeptide spots as well as on food allergen spots. Results showed that the major source of variability was the number of gel replicates. Other sources were minor. This has a direct practical impact on the laboratory work as this supports the utilization of three or four gel replicates to get robust results. Furthermore, this implies that the study can be run over several days, and be performed by several trained operators, without impacting its reproducibility. Furthermore, 2-DE could detect a 2-fold change between two samples with an acceptable rate of false positives (below 7%). This level of sensitivity is acceptable in the context of safety assessment of GM soybean as the biological variability of proteins in soybean is higher than the technical variability shown in this study. Overall, the 2-DE technique is suitable for investigating endogenous food allergen variability between several soybean seeds, including GM and non-GM counterpart.

Published
2014

11. Cloning of laminin γ2 cDNA and chromosome mapping of the genes for the dog adhesion ligand laminin 5

Author

Guerrino Meneguzzi, Annabelle Capt, Jean-Paul Ortonne, Richard Guyon, Flavia Spirito, and Catherine André

Subjects
Keratinocytes, DNA, Complementary, Molecular Sequence Data, Biophysics, Biology, Ligands, Biochemistry, law.invention, Extracellular matrix, Dogs, Species Specificity, Sequence Analysis, Protein, law, Laminin, Complementary DNA, Animals, Humans, Amino Acid Sequence, Cloning, Molecular, Molecular Biology, Gene, Conserved Sequence, Cloning, Genome, Sequence Homology, Amino Acid, Gene Expression Profiling, Nucleic acid sequence, Chromosome Mapping, Cell Biology, Ligand (biochemistry), Molecular biology, Recombinant DNA, biology.protein, Cell Adhesion Molecules, Sequence Alignment
Abstract

Overexpression of the gamma2 chain of laminin-5 has been linked to tumor invasion and an unfavorable prognostic value, but the role of this adhesion molecule in cancer progression remains unclear. Because dog models of human cancers provide the opportunity of clarifying the relation between laminin-5 and tumor malignancy we have isolated and characterized the cDNA of dog gamma2 chain. Comparative analysis of the nucleotide sequence revealed high identity between the dog and the human gamma2, including the intermolecular molecule binding sites and the regulatory promoter sequences. Moreover, expression of a recombinant human gamma2 chain in dog keratinocytes results in assembly and secretion of hybrid laminin-5 molecules, which underscore the functional relevance of the gamma2 conserved domains. We have also determined the syntenic location of the dog laminin-5 loci on CFA7. Our study provides a basis for therapeutical approaches of epithelial cancers of gamma2 using dogs as large animal models.

Published
2003
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12. L'épidermolyse Bulleuse Jonctionnelle du Braque allemand : un modèle canin spontané de l'Épidermolyse Bulleuse Jonctionnelle de l'homme

Author

Guerrino Meneguzzi, Annabelle Capt, Éric Guaguère, and Flavia Spirito

Subjects
integumentary system, General Veterinary, gene therapy, junctional epidermolysis bullosa, dermatosis, dog, German shorthaired pointer, épidermolyse, bulleuse jonctionnelle, dermatose, thérapie génique, Braque allemand, chien
Abstract

Junctional Epidermolysis Bullosa in the german shorthaired pointer : a spontaneous model for Junctional Epidermolysis Bullosa in man A Junctional Epidermolysis Bullosa (JEB), described in the German shorthaired pointer, is a remarkable spontaneous canine model for Junctional Epidermolysis Bullosa in man. In the German shorthaired pointer, JEB is caused by a homozygous substitution of the 1514C-T nucleotide. This variation in sequence results in a non-conservative change of 14 amino acids (505T – I) in domain I of laminin-5 α3. This non-conservative substitution modifies the hydrophobicity profile of the peptidic fragment, and therefore alters the stability of the binding between the α3 chain and the β3γ2 heterotrimer, which could result in a breakdown of the muted polypeptide. A fraction of muted α3 could be incorporated in laminin-5, modifying its function and hindering the extracellular cleavage of the γ2 chain. Genetic complementation studies were carried out. An MMLV retrovirus expressing the whole cDNA, muted and wild, of the α3 chain was developed to be transducted in the keratinocytes of JEB dogs (KJEB). This phenotypic transversion was successful and brought back a3 chain secretion, and thus the production of functional laminin-5 molecules. In addition, the adhesion capabilities of these transducted KJEB in cultures also returned. Finally, the reconstitution of canine JEB epithelia expressing a hybrid laminin-5 is now possible, and is currently used in studies on the fate of these ex vivo grafts in JEB dogs., Une Epidermolyse Bulleuse Jonctionnelle (EBJ) est décrite chez le Braque allemand et constitue un remarquable modèle canin de l’épidermolyse bulleuse jonctionnelle de l’Homme. Chez le Braque allemand, cette maladie est causée par une substitution homozygote du nucléotide 1514 C-T chez les chiens EBJ. Cette variation de séquence induit un changement non conservatif de 14 acides aminés (505T – I) dans le domaine I de la laminine α3. Cette substitution non conservative modifie le profil d’hydrophobicité du fragment peptidique et altère donc la stabilité d’association de la chaîne α3 avec l’hétérotrimère β3γ2. Ceci pourrait aboutir à une dégradation du polypeptide muté. Une fraction d’α3 mutée pourrait s’incorporer dans la laminine 5, en altérer le fonctionnement et entraver le clivage extra-cellulaire de la chaîne γ2. Des expériences de complémentation génétique ont donc été réalisées. Pour cela, un rétrovirus MMLV exprimant l’ADNc entier muté et sauvage de la chaîne α3 a été construit pour être transduit dans les kératinocytes des chiens EBJ (KEBJ). Cette transversion phénotypique a été réussie et a permis aux KEBJ de sécréter de nouveau la chaîne α3 et ainsi, de produire des molécules de laminine 5 fonctionnelles. Par ailleurs, ces KEBJ transduits ont montré aussi la restauration de leur capacité d’adhésion en culture. Enfin, la reconstitution d’épithéliums canins EBJ exprimant une laminine 5 hybride est désormais possible et est actuellement à l’origine d’études sur le devenir de ces greffes ex vivo chez des chiens EBJ., Guaguère Eric, Capt Annabelle, Spirito Flavia, Meneguzzi Guerrino. L'épidermolyse Bulleuse Jonctionnelle du Braque allemand : un modèle canin spontané de l'Épidermolyse Bulleuse Jonctionnelle de l'homme. In: Bulletin de l'Académie Vétérinaire de France tome 156 n°2, 2003. pp. 47-51.

Published
2003
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13. Investigation of endogenous soybean food allergens by using a 2-dimensional gel electrophoresis approach

Author

Corinne Herouet-Guicheney, David Rouquié, Annabelle Capt, Vaithilingam Sekar, and William H. Eby

Subjects
Food Safety, Context (language use), Genetically modified crops, Biology, Toxicology, medicine.disease_cause, chemistry.chemical_compound, Allergen, immune system diseases, Genotype, medicine, Humans, Electrophoresis, Gel, Two-Dimensional, Food science, Gel electrophoresis, Two-dimensional gel electrophoresis, business.industry, fungi, food and beverages, General Medicine, respiratory system, Allergens, Plants, Genetically Modified, respiratory tract diseases, Biotechnology, Genetically modified organism, chemistry, Glyphosate, Seeds, Soybean Proteins, Soybeans, business, Food Hypersensitivity
Abstract

As part of the safety assessment of genetically modified (GM) soybean, 2-dimensional gel electrophoresis analyses were performed with the isoxaflutole and glyphosate tolerant soybean FG72, its non-GM near-isogenic counterpart (Jack) and three commercial non-GM soybean lines. The objective was to compare the known endogenous human food allergens in seeds in the five different soybean lines in order to evaluate any potential unintended effect(s) of the genetic modification. In total, 37 protein spots representing five well known soybean food allergen groups were quantified in each genotype. Qualitatively, all the allergenic proteins were detected in the different genetic backgrounds. Quantitatively, among 37 protein spots, the levels of accumulation of three allergens were slightly lower in the GM soybean than in the non-GM counterparts. Specifically, while the levels of two of these three allergens fell within the normal range of variation observed in the four non-GM varieties, the level of the third allergen was slightly below the normal range. Overall, there was no significant increase in the level of allergens in FG72 soybean seeds. Therefore, the FG72 soybean can be considered as safe as its non-GM counterpart with regards to endogenous allergenicity. Additional research is needed to evaluate the biological variability in the levels of endogenous soybean allergens and the correlation between level of allergens and allergenic potential in order to improve the interpretation of these data in the safety assessment of GM soybean context.

Published
2010

14. Comparison of the human skin grafted onto nude mouse model with in vivo and in vitro models in the prediction of percutaneous penetration of three lipophilic pesticides

Author

Anne-Pascale Luzy, D. J. Esdaile, Annabelle Capt, and O. Blanck

Subjects
Male, Insecticides, Skin Absorption, Mice, Nude, Human skin, Absorption (skin), Pharmacology, Toxicology, Risk Assessment, Cypermethrin, chemistry.chemical_compound, Mice, Nude mouse, In vivo, Pyrethrins, Stratum corneum, medicine, Animals, Humans, Rats, Wistar, integumentary system, biology, General Medicine, Skin Transplantation, Pesticide, biology.organism_classification, In vitro, Rats, medicine.anatomical_structure, chemistry, Models, Animal, Biophysics, Malathion, Female, Hexachlorocyclohexane
Abstract

The evaluation of the degree of percutaneous penetration of agrochemicals is a key part of risk assessment for operators. The availability of suitable and predictive experimental models is crucial, in particular in the case of lipophilic compounds which persist in the stratum corneum (SC). Regulatory models (rat in vivo, human and rat in vitro) and the innovative human skin grafted onto nude mice (HuSki) model were compared for their ability to predict the human skin absorption. Radiolabelled malathion, lindane and cypermethrin (4 μg/cm2) were topically applied to each model. The % of applied dose absorbed and that present in skin and SC were evaluated at 24 h. Additionally, the absorption profile of cypermethrin was evaluated in the in vivo rat and HuSki models for up to 11 days. We found that the human in vitro and HuSki models closely predicted the human skin absorption at 24 h, while rat models overestimated the human skin absorption. Furthermore, our experiments with cypermethrin indicated that evaluation of % percutaneous absorption over extended periods of time was feasible with the HuSki model. In our studies the HuSki model overcame the limitations of the regulatory models and is promising to realistically refine the dermal absorption assessment of topically applied chemicals.

Published
2006

15. Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy

Author

Flavia Spirito, Fernando Larcher, Annabelle Capt, Olivier Danos, Marcela Del Rio, Eric Guaguere, and Guerrino Meneguzzi

Subjects
Keratinocytes, Genetic enhancement, Transgene, Biophysics, Biochemistry, Viral vector, Extracellular matrix, Mice, Dogs, Laminin, medicine, Animals, Molecular Biology, Cells, Cultured, Skin, integumentary system, biology, Cell Biology, Genetic Therapy, medicine.disease, Recombinant Proteins, Disease Models, Animal, medicine.anatomical_structure, Treatment Outcome, Immunology, Gene Targeting, biology.protein, Epidermolysis bullosa, Keratinocyte, Epidermolysis Bullosa, Junctional epidermolysis bullosa (veterinary medicine), Immunocompetence
Abstract

Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (alpha3beta3gamma2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the alpha3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials.

Published
2005

16. FC-27 A spontaneous dog model for in vivo gene therapy of junctional epidermolysis bullosa

Author

M. Del Rio, Flavia Spirito, Guerrino Meneguzzi, Eric Guaguere, and Annabelle Capt

Subjects
Basement membrane, Pathology, medicine.medical_specialty, integumentary system, General Veterinary, biology, Transgene, Genetic enhancement, Molecular biology, Extracellular matrix, medicine.anatomical_structure, Immune system, In vivo, Laminin, medicine, biology.protein, Junctional epidermolysis bullosa (veterinary medicine)
Abstract

Junctional epidermolysis bullosa (JEB) is a mechanobullous skin disease associated with mutations in the basement membrane components of the dermo-epidermal junction. This condition is a suitable prototype for proving the feasibility of a gene therapy approach to genodermatoses. As successful genetic treatment requires immunocompetent animal models to study the possible host immune response to the transgene, we have characterized a breed of short-haired pointers suffering from a mild form of JEB. These animals exhibit skin blistering and erosions following minor trauma. Immunohistochemical analysis of the skin biopsies and immunoprecipitation of spent medium from cultured JEB keratinocytes showed reduced expression and secretion of the α3 chain of laminin 5 (α3, β3, γ2), the major adhesion ligand of basal keratinocytes. The search for genetic mutations detected a homozygous insertional mutation (4818 + 207 ins6.5 kb) in intron 35 of the lama3 gene. Consequently, keratinocytes from dogs with JEB secrete reduced amounts of wild-type laminin 5, and adhesive properties of the keratinocytes are compromised. Retroviral transfer of wild-type dog α3 cDNA into JEB keratinocytes enhanced secretion of laminin 5 in the extracellular matrix and restored the adhesion, differentiation and proliferative capacity of the transduced cells. Transplantable fibrin-based skin equivalents made with transduced JEB keratinocytes and grafted onto SCID mice generated normal cohesive and stratified epithelia and showed stable localized deposition of laminin 5 at the dermo-epidermal junction. Our results form the basis for preclinical assays of gene therapy on a unique immunocompetent animal model for an inherited skin disease. Funding: DEBRA (UK) Foundation, Association Francaise Contre les Myopathies.

Published
2004
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17. Inherited Junctional Epidermolysis Bullosa in the German Pointer: Establishment of a Large Animal Model

Author

Annabelle Capt, Eric Guaguere, Jean-Paul Ortonne, Guerrino Meneguzzi, Flavia Spirito, and Anne Spadafora

Subjects
Keratinocytes, skin, Biopsy, LINE, Dermatology, laminin α3, Biology, Gene delivery, Biochemistry, Blister, Dogs, Laminin, Intronic Mutation, medicine, Animals, RNA, Messenger, Molecular Biology, Gene, Messenger RNA, Base Sequence, integumentary system, Intron, Genodermatosis, cell adhesion, Exons, Cell Biology, medicine.disease, Molecular biology, Introns, Pedigree, Disease Models, Animal, Intercellular Junctions, biology.protein, Epidermolysis Bullosa, Cell Adhesion Molecules, Junctional epidermolysis bullosa (veterinary medicine), genodermatosis
Abstract

Junctional epidermolysis bullosa (JEB) is a genodermatosis suitable for gene therapy because conventional treatments are ineffective. Here, we elucidate the genetic basis of mild JEB in a breed of dogs that display all the clinical traits observed in JEB patients. The condition is associated with reduced expression of laminin 5 caused by a homozygous insertion (4818+207ins6.5 kb) of repetitive satellite DNA within intron 35 of the gene (lama3) for the laminin alpha3 chain. The intronic mutation interferes with maturation of the alpha3 pre-messenger RNA resulting in the coexpression of a transcript with a 227 nucleotide insertion and a wild-type mRNA that encodes scant amounts of the alpha3 polypeptide. Our results show that the amino acid sequence and structure of the canine and human alpha3 chain are highly conserved and that the reduced expression of laminin 5 affects the adhesion and clonogenic potential of the JEB keratinocytes. These JEB dogs provide the opportunity to perform gene delivery in a naturally occurring genodermatosis and to evaluate host tolerance to recombinant laminin 5.

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