ImPARAS | - Proceedings · 2018. 12. 19. · Roberto Berni Canani . University of Naples 'Federico II', Italy . Dr.Roberto Berni Canani is internationally recognized as one of the

4th ImpARAS conference June 19-21, 2018

Portici, Italy

Page 1

Proceedings of the

4th International ImpARAS Conference

This conference is organized by COST Action FA1402 ImpARAS Improving Allergy Risk Assessment Strategy for new food proteins

www.imparas.eu


Portici, Italy

Page 2

Be aware that the personal information mentioned in this Conference book may be used for COST Action FA1402 (ImpARAS) purposes only

Sponsors & organizers


Portici, Italy

Page 3

Scope and welcome address As chair of COST Action ImpARAS FA1402, it is a real pleasure to welcome you at our fourth and last ImpARAS conference in Portici in Italy. ImpARAS, Improving Allergenicity Risk Assessment Strategy (for novel and modified proteins), is an European network that aims to build an interdisciplinary European network of scientists with a broad range of expertise to discuss, with an out-of-the-box view, new ideas and more predictive models and approaches to improve the current allergenicity risk assessment strategy. ImpARAS is helping to develop an improved allergenicity risk assessment strategy for novel proteins, by adding more predictive tools to the current risk assessment strategy, and accelerate the introduction of novel protein (sources) onto the market, to mitigate the concern of consumers around novel or genetically modified protein (products) and to advice policy makers on the safety of novel protein (products). The networks focusses on different topics:

• Physical/chemical properties of proteins impacting allergenicity, including effect of processing, matrix effect, glycosylation, lipid binding, digestion, bioinformatics, protein purifications and analysis and others.

• In vitro methods to predict sensitization to food allergy, including epithelial transport of proteins, DC-T cell interactions, activation innate and adaptive immune system and others.

• In vivo methods to predict sensitization to food allergens, including mouse, rat or other models to measure effect on the immune system and others.

• Allergenicity Risk assessment, including current status, examples and applications, visions and others.

ImpARAS is a COST Action that is supported for 4 years (December 2014 – December 2018) by COST (European Cooperation in Science and Technology). More over 240 scientist from Industry, Universities, knowledge centers and regulatory bodies from 32 countries are united in ImpARAS. The network is active through a range of networking tools, such as meetings, workshops, conferences, training schools, and exchange of staff between partners also called short-term scientific missions (STSMs). ImpARAS is open to researchers from universities, public and private research institutions, as well as to NGOs, industry and SMEs. So you are welcome to join our network! You can find more information on our ImpARAS website; www.ImpARAS.eu or become member of our LinkedIn group. I would like to take this opportunity to thank the local organizers for all their efforts to make this conference a success and to welcome you in the wonderful atmosphere of Italy. Also thanks to the scientific committee that made its best to offer you such an exciting and interesting program. As our COST Action comes to an end, we can be proud of what we have achieved within our four Working Groups. I hope that you will enjoy the conference, make new contacts and start new opportunities with colleagues from other countries. Of course I also hope to meet you again during our last Working Group meeting in October 2018, held in Nantes, France.


Portici, Italy

Page 4

Summary Scientific and organizing committees ..................................................................................................... 5

Scientific Program ................................................................................................................................... 7

CVs invited speakers ............................................................................................................................. 10

List of Oral presentations ...................................................................................................................... 16

List of Poster presentations .................................................................................................................. 18

List of Flash presentations .................................................................................................................... 20

Abstracts Oral, Poster & Flash presentations ....................................................................................... 21

Participants ........................................................................................................................................... 64

Local Information .................................................................................................................................. 66


Portici, Italy

Page 5

Scientific and organizing committees Scientific committee

René Crevel RENE CREVEL Consulting Limited, UK, (Vice Chair)

Kitty Verhoeckx TNO, The Netherlands, (Chair)

Gabriel Mazzucchelli University of Liege, Belgium

Karin Hoffmann-Sommergruber Medical University of Vienna, Austria

Edyta Sienkiewcz-Szlapka University of Warmia and Mazury, Poland

Erwin Roggen 3rsmc, Denmark

Katrine Lindholm-Bøgh National Food Institute, Technical University of Denmark Denmark

Liam O’Mahony Swiss Institute of Allergy and Asthma Research, Switzerland

Ben Remington TNO, The Netherlands

Anne Constable Nestlé Research Centre, Switzerland

Paola Roncada Istituto Sperimentale Italiano Lazzaro Spallanzani, Italy


Portici, Italy

Page 6

Organizing committee

Paolo Masi CAISIAL Italy

Pasquale Ferranti University of Naples Italy

Annalisa Romano University of Naples Italy

Gianfranco Mamone University of Naples Italy

Kitty Verhoeckx TNO, The Netherlands

Marloes van der Wal-Bellaart TNO, The Netherlands

Astrid Kruizinga TNO, The Netherlands


Portici, Italy

Page 7

Scientific Program Day 1 – June 19th

12:30-14:00 Registration 14:10-14:20 Opening, welcome and practicalities by Pasquale Ferranti, University of Naples,

Matteo Lorito, Director of the Department of Agricultural Sciences and Paolo Masi, Director of the CAISIAL

14:20-14:30 Presentation of COST Action FA1402 ImpARAS Kitty Verhoeckx, Chair of the Action, TNO, The Netherlands 14:30-15:15 Keynote lecture Cecilia Berin “Immune profiling of pediatric food allergic cohorts” 15:15-15:35 Angelika Tscheppe “Contribution of conformational and linear IgE epitopes to ARA

H2-specific IgE-binding - in vitro and in vivo studies” 15:35-15:55 Mónica Martínez-Blanco “Egg protein induce adjuvant-independent oral sensitization

and the mixture of egg white and egg yolk enhances T cell responses in BALB/C mice”

15:55-16:25 Coffee Break 16:25-16:45 Clélia Villemin “Acid-hydrolyzed gliadins strengthen food allergies through early

sensitization” 16:45-17:05 Anne-Sofie Ravn Ballegaard “Protein-chemical features of five different wheat

products affect the sensitising capacity through the skin”

17:05-18:00 Flash presentations of ESRs on STSM ( 5 min + 1 min questions) Simona Bavaro - Autoclaving process to reduce almond allergenicity: impact on the peptidic pattern analyzed by after

simulated digestion and ex vivo intestinal epithelium passage Ying Deng - Effects of Structural Modifications on Immunogenicity of Food Proteins

Shu-Hua Liu - Characterisation of peanut allergens and possible post-translational modifications (PTMs)

Marija Perusko - New insights into allergenic relationship between red meat and cow’s milk

Ivana Prodic - Digestomics of walnut and its nsLTPs allergens reveals their ultimate resistance to gastric digestion Julia Klueber - Functional potency testing of allergenic and non-allergenic tropomyosins using RBL cell mediator

release as a potential tool in food allergen risk assessment

Eszter Schall - Purification of different LTPs and investigation of their behaviour in an in vitro gastrointestinal

digestion experiment Sahar Kazemi - Sensitising potential of gluten products via intact, damaged and inflamed skin

Angelika Tscheppe - Influence of conformational and linear IgE epitopes on Ara h 2-specific IgE-binding in a mouse model of

peanut allergy 19:00-21:00 Dinner


Portici, Italy

Page 8

Day 2 - Wednesday June 20th 09:00-10:30 Working group 1-2-3-4 meeting

10:30-11:00 Coffee Break 11:00-11:45 Keynote lecture: Roberto Berni Canani “Gut microbiome as potential target for

food allergy treatment” 11:45-12:05 Ivan Dimitrov “AllerScreener: a bioinformatic tool for sensitization and cross-

reactivity assessment of Proteins” 12:05-12:35 Richard Goodman “Understanding Real Risks and Risk Management for Food Allergy

and Celiac Disease” 12:35-13:35 Lunch During lunch there will be a presentation by our sponsor Annette J. Sauer, R-

Biopharm AG, Germany “Best practices in using food allergen testing”

13:35-14:20 Keynote lecture: Paul Turner “Determinants of severity: predicting life-threatening anaphylaxis reactions”

14:20-14:40 Mauro Marengo “Managing allergenic risks at the industrial level: egg proteins

residues in pasta” 14:40-16:00 Poster session 16:00-16:30 Coffee Break 16:30-16:50 Joost Smit “Receptor-mediated transfer, uptake and degradation of allergens

towards and inside dendritic cells” 16:50-17:10 Harry Wichers “Epitopes in sensitisation and in elicitation: the impact of food

processing” 17:10-17:30 Daniel Lozano-Ojalvo “PDL2+ CD11b+ dermal dendritic cells capture topical antigen

through hair follicles to prima LAP+ regulatory T cells” 17:30-17:50 Stephane Hazebrouck “Mapping of conformational IgE-binding epitopes of peanut

allergen Ara h 2 with chimeric 2S-albumins” 18:00-19:00 MC meeting 20:00-22:00 Conference Dinner


Portici, Italy

Page 9

Day 3 - Thursday June 21st

09:00-10.00 Keynote lecture: Lucien Harthoorn “Seeing the unseen": a perspective on building scientific evidence in non-IgE mediated food allergy”

10:00-10:45 Achievements and update from the working groups

Gabriel Mazzucchelli, Erwin Roggen, Katrine Lindholm Bᴓgh, Anne Constable 10:45-11:15 Coffee Break 11:15-11:35 Cláudia Raposo “Modulation of fish allergenicity towards the production of a low

allergen farmed fish using Creatine enriched diets” 11:35-11:55 Ana Pilar Tobajas “Effect of technological treatments on immunoreactivity and

allergenicity of the allergenic protein Pru p 3 from peach” 11:55-12:15 Caterina Villa “Milk proteins in food products as affected by thermal processing

assessed by immunochemical and DNA-based methods” 12:15-12:35 Hulya I. Buyukkestelli “Deactivation of allergens in some foods using ultrasound” 12:35-13:00 Closing of the conference


Portici, Italy

Page 10

CVs invited speakers


Portici, Italy

Page 11

Cecilia Berin

Icahn School of Medicine, U.S.A. Cecilia Berin is an Associate Professor of Pediatrics and Member of the PRIISM Immunology Institute at the Icahn School of Medicine at Mount Sinai. Her laboratory focuses on immune mechanisms of allergy and tolerance to foods. The laboratory uses state of the art immune profiling approaches, including mass cytometry, multi-parameter flow cytometry, and single cell sequencing to uncover the immune basis underlying phenotypic heterogeneity of food allergy.

The laboratory develops approaches for immune profiling for NIH-sponsored clinical trials, including immunotherapy and allergy prevention trials geared toward young pediatric cohorts. In addition to human immunology, the Berin Laboratory uses mouse models of food allergy to study immune mechanisms of food allergy in tissues, with emphasis on the immune link between the skin and gastrointestinal tract. Dr. Berin’s research is funded by the National Institutes of Health.

.

Lecture: “Immune profiling of pediatric food allergic cohorts” Day 1- Tuesday June 18th 14:30 - 15:15


Portici, Italy

Page 12

Roberto Berni Canani

University of Naples 'Federico II', Italy

Dr.Roberto Berni Canani is internationally recognized as one of the leading researchers in the area of pediatric food allergy, gastroenterology, and nutrition. He received his MD and PhD degrees from University of Naples “Federico II”, Naples, Italy. He is board certified in Pediatrics. Chief of the Pediatric Allergy Program and founder of the Congenital Food-induced Diarrheal Disorders Website (www.congenitaldiarrhealdisorders.net). A website dedicated to the investigation and the care of congenital diarrheal disorders.

He is author of more than 300 publications among scientific paper on international journals, chapters of books and reviews. His publications are widely quoted (collectively more than >9000 citations as at March 2018). Of these 30 papers have been cited >100 times. H-index score 44. Total impact factor score: >900. Associate editor of World Journal of Gastroenterology, Journal of Pediatric Gastroenterology and Nutrition, Journal of Gastroenterology and Hepatology Research, Allergies, International Journal of Autoimmune Disease & Therapy. He is member of many national scientific societies (Italian Society of Pediatrics, Italian Society of Pediatric Gastroenterology Hepatology and Nutrition, Italian Society of Pediatric Allergy and Immunology, Italian Society of Infectious Diseases, Italian Society of Pediatric Research). Vice-president of the Italian Society of Pediatric Gastroenterology Hepatology and Nutrition (SIGENP) (2008-2012) and actual chief of the food-induced diseases working group (from 2014).He is member of the European Academy of Allergy and Clinical Immunology (EAACI) and of the European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN). He has obtained several scientific prizes for the research activity. Member of the Expert Panel (from 2011) and of the Dietetic products, Nutrition and Allergies Panel of the European Food Safety Authority (EFSA) (2012-2015).

From 2013 he is enclosed in the list of Top 100 Italian Scientists of VIA-Academy (http://www.topitalianscientists.org/Top_italian_scientists_VIA-Academy.aspx). Key contributions: Dr. R. Berni Canani has developed an interdisciplinary research team and a highly integrated research clinical program to better understand the pathogenic mechanisms of food-induced diseases and provide the best care for children affected by these disorders. The Dr. Berni Canani laboratory is mainly focused on the study of basic aspects of human nutrition and selected food-induced diseases: immunonutrition, food allergy, congenital defects of digestion and transport of nutrients and electrolytes, obesity-related non-alcoholic fatty liver disease and dyslipidemia, aiming to move disease biology from the laboratory to clinical practice. The main study aim of the program is focused on the

Lecture: “Gut microbiome as potential target for food allergy treatment” Day 2- Wednesday June 19th 11:00 - 11:45

http://www.topitalianscientists.org/Top_italian_scientists_VIA-Academy.aspx


Portici, Italy

Page 13

identification of best immune-nutritional strategies to induce immune tolerance in children affected by food allergy, to limits autoimmunity and infectious diseases. A bench to bedside approach is used to investigate pathophysiological basis of pediatric nutrition and food-induced diseases including animal models, peripheral blood cells culture from children, epigenetics biomarkers involved in IgE- and in non-IgE-mediated food allergy, gut microbiota composition analysis using 16sRNA technology, short chain fatty acids and essential fatty acids metabolism. His studies on gut microbiome, immune tolerance and immunonutrition have already been translated into novel concepts and lead to a paradigm shift in the pathogenesis and treatment of food allergy and of other pediatric diseases.


Portici, Italy

Page 14

Paul Turner

Imperial College London, England

Dr Paul Turner is MRC Clinician Scientist and Clinical Senior Lecturer in Paediatric Allergy & Immunology within the MRC & Asthma UK Centre in Allergic Mechanisms at Imperial College London. He is also a Clinical Associate Professor at the University of Sydney, Australia. In 2012 he was awarded a UK Medical Research Council Fellowship, to assess mechanisms of acute allergic reactions to peanut, and how this may be modulated during oral immunotherapy. His research centres on the pathophysiology of severe allergic reactions to food, and how this impacts upon allergen risk management and the use of precautionary allergen labelling.

Paul also is a Clinical Trials Specialist at Public Health England, and has led a number of multicentre studies assessing the safety and efficacy of the intranasal influenza vaccine. Paul is an Honorary Consultant at a number of London teaching hospitals, including St Mary's, the Royal Free and the Royal Brompton.

Lecture: “Determinants of severity: predicting life-threatening anaphylaxis reactions”

Day 2- Wednesday 19th 13:35 - 14:20


Portici, Italy

Page 15

Lucien Harthoorn

Nutricia Research, The Netherlands

Lucien Harthoorn is Research Program Leader Paediatric Care - Allergy in Nutricia Advanced Medical Nutrition and leading a team of scientists at the Nutricia Research Innovation Centre, Utrecht, The Netherlands.

He and his team are responsible for the innovation pipeline and evidence generation which is from preclinical science and clinical studies to scientific publications and building collaborations with national and international research groups and institutions in the field of paediatric allergy.

Lucien has a MSc in Biomedical Sciences from the Free University Amsterdam, The Netherlands where he graduated in Pharmacology in 1997. He obtained his PhD in Biochemical Physiology and Endocrinology at Utrecht University, The Netherlands in 2001. Lucien was postdoctoral researcher in Neurosciences and Endocrinology with special emphasis on mechanisms of satiety and the development of obesity in Amsterdam, The Netherlands. He had a Research Scientist position in Food Intake Regulation at the Top Institute Food & Nutrition in Wageningen, The Netherlands, where he co-led a work package of the EU-Diogenes project (Diet-Obesity-Genes). Then, Lucien was Senior Scientist & Project Leader at Mead Johnson Nutrition based both at the European R&D office in The Netherlands and the Global R&D centre in Evansville, U.S.A. and has been participating in The International Life Sciences Institute (ILSI) and several Food Intake and Metabolic Programming platforms and working groups. In 2011 he started as Research Manager for the Advanced Medical Nutrition Division of Danone - Nutricia Research, leading projects with major universities and Contract Research Organisations.

Lucien is (co)authoring +40 publications in the field of Endocrinology, Physiology, Neurosciences, Immunology and Allergy and is (co)inventor of 6 patents. He is a guest lecturer in Pharmacology and Physiology.

Lecture: “’Seeing the unseen’: a perspective on building scientific evidence in non-IgE mediated food allergy”

Day 3- Thursday October 20th 09:00 - 10:00


Portici, Italy

Page 16

List of Oral presentations O01 Contribution of conformational and linear IgE epitopes to ARA H 2-specific IgE-binding – in

vitro and in vivo studies. Angelika Tscheppe, Medical University of Vienna, Austria

O02 Egg proteins induce adjuvant-independent oral sensitization and the mixture of egg white and egg yolk enhances T cell responses in BALB/C mice. Monica Martínez Blanco, CIAL CSIC, Spain

O03 Acid-hydrolyzed gliadins strengthen food allergies through early sensitization. Clélia Villemin, INRA, France

O04 Protein-chemical features of five different wheat products affect the sensitising capacity through the skin.

Anne-Sofie Ravn Ballegaard, DTU, Denmark

O05 AllerScreener: a bioinformatic tool for sensitization and cross-reactivity assessment of proteins

Ivan Dimitrov, Medical University of Sofia, Bulgaria

O06 Understanding Real Risks and Risk Management for Food Allergy and Celiac Disease. Richard Goodman, University of Nebraska-Lincoln, U.S.A.

O07 Immune cell profiles characteristic for food allergic patients with and without allergen-specific IgE by high-dimensional mass cytometry.

Unni C. Nygaard, Norwegian Insitute of Public Health, Norway

O08 Managing allergenic risks at the industrial level: egg protein residues in pasta. Mauro Marengo, University of Milan, Italy

O09 Receptor-mediated transfer, uptake and degradation of allergens towards and inside dendritic cells.

Joost Smit, Institute for Risk Assessment Sciences, Utrecht University, The Netherlands

O10 Epitopes in sensitisation and in elicitation: the impact of food processing. Harry Wichers, Wageningen UR Food & Biobased Research, The Netherlands

O11 PDL2+ CD11b+ Dermal dendritic cells capture topical antigen trough hair follicles to prime Lap+ regulatory T Cells.

Daniel Lozano-Ojalvo, CIAL, CSIC, U.S.A.

O12 Mapping of conformational IgE-binding epitopes of peanut allergen Ara h 2 with chimeric 2S-albumins.

Stéphane Hazebrouck, INRA (National Institute for Agronomic Research), France

O13 Modulation of fish allergenicity towards the production of a low allergen farmed fish using Creatine enriched diets..

Cláudia Raposo, University of Algarve, Portugal

O14 Effect of technological treatments on immunoreactivity and allergenicity of the allergenic protein Pru p 3 from peach

Ana Pilar Tobajas, University of Zaragoza, Spain

O15 Milk proteins in food products as affected by thermal processing assessed by immunochemical and DNA-based methods.

Caterina Villa, REQUIMTE-LAQV/FFUP, Portugal


Portici, Italy

Page 17

O16 Milk proteins in food products as affected by thermal processing assessed by immunochemical and DNA-based methods.

Hulya I. Buyukkestelli, Ege University, Turkey


Portici, Italy

Page 18

List of Poster presentations P01 Catalase as a novel IgE reactive protein from banana should be involved in component-

resolved allergy diagnosis. Françoise Nesic, Faculty of Chemistry, University of Belgrade, Serbia

P02 The hidden “digestome”: do the current analytical approaches provide comprehensive peptide inventory of a food digest?. Gianluca Picariello, Instituto di Scienze dell'alimentazione - Consiglio Nazionale delle Ricerche (CNR), Italy

P03 Modulation of fish allergenicity towards the production of a low allergen farmed fish using EDTA enriched diets. Denise Schrama, Universidade do Algarve, CCMAR, Portugal

P04 Use of in vitro digestive models for the prediction of antinutritional properties and allergenic risk of food proteins. Krisztina Takács, NARIC-FSRI, Hungary

P05 How deep do we really know the peanut digestome?. Gianfranco Mamone, Institute of Food Sciences CNR, Italy

P06 Allergenicity risk assessment of sunflower seed protein.. Jihana Achour, INRA, France

P07 Resolving the complexity of peanut allergome using 2DE gel based and gel free proteomic analysis. Luigia Di Stasio, University of Naples, Italy

P08 Detection and quantification of fish as a potential allergenic food: comparison of two real-time PCR approaches targeting the 16S rRNA gene. Isabel Mafra, REQUIMTE-LAQV, Faculty of Pharmacy, University of Porto, Portugal

P09 High Pressure Processing and Food Allergens. Merve Eda, Ege University, Turkey

P10 Proteomics and B-cell reactivity analysis of water-soluble proteins of oat in patients with celiac disease.

Daniel Sánchez, Czech Academy of Sciences, Czech Republic

P11 Technological Processes Effects on the Allergenicity of Food Proteins: Gaps and Future Needs. Birgül Hizlar, Ege University, Turkey

P12 Activated intestinal epithelial cells conditioned with 2’-Fucosyllactose and CpG ODN might instruct moDC to drive Th1 differentiation. Veronica Ayechu Muruzabal, Utrecht University, The Netherlands

P13 Fast amperometric immunoplatform for ovomucoid traces determination in fresh and baked foods. Sara Benedé Pérez, Complutense University of Madrid, Spain

P14 In germ-free mice the degree of allergic sensitization is strictly dependent on diet endotoxin content. Hana Kozakova, Czech Academy of Sciences, Czech Republic


Portici, Italy

Page 19

P15 Celiac disease: a rare cause of hyposideremic anemia in adults. Elena Gologan, "Grigore T. Popa" University of Medicine and Pharmacy, Romania

P16 Egg yolk modifies the response of intestinal cells and dendritic cells in vitro. Leticia Perez-Rodriguez, CIAL, CSIC, Spain

P17 Observation of the level of inflammatory bowel factors and intestinal microbiota composition in the context of cow's milk protein allergy occurrence in the north-eastern region of Poland. Barbara Wróblewska, Polish Academy of Sciences, Poland


Portici, Italy

Page 20

List of Flash presentations F01 Autoclaving process to reduce almond allergenicity: impact on the peptidic pattern analyzed

by after simulated digestion and ex vivo intestinal epithelium passage. Simona Bavaro, ISPA-CNR, Italy

F02 Effects of Structural Modifications on Immunogenicity of Food Proteins. Ying Deng, WFBR, The Netherlands

F03 Characterisation of peanut allergens and possible post-translational modifications (PTMs). Shu-Hua Liu, Medical University of Vienna, Austria

F04 New insights into allergenic relationship between red meat and cow’s milk. Marija Perusko, University of Belgrade, Serbia

F05 Digestomics of walnut and its nsLTPs allergens reveals their ultimate resistance to gastric digestion. Ivana Prodic, Chemical University of Belgrade, Serbia

F06 Functional potency testing of allergenic and non-allergenic tropomyosins using RBL cell mediator release as a potential tool in food allergen risk assessment. Julia Klueber, Luxembourg Institute of Health, Luxembourg

F07 Purification of different LTPs and investigation of their behaviour in an in vitro gastrointestinal digestion experiment. Eszter Schall, Budapest University of Technology and Economics, Hungary

F08 Sensitising potential of gluten products via intact, damaged and inflamed skin. Sahar Kazemi, Medical University of Vienna, Austria

F09 Influence of conformational and linear IgE epitopes on Ara h 2-specific IgE-binding in a mouse model of peanut allergy. Angelika Tscheppe, Medical University of Vienna, Austria


Portici, Italy

Page 21

Abstracts Oral, Poster & Flash presentations


Portici, Italy

Page 22

O01

Contibution of conformational and linear IgE epitopes and linear IgE epitopes to ara h2-

specific IgE binding – in vitro and in vivo studies

A. Tscheppe1, D. Palmberger2, C. Radauer1, L. S. Van Rijt3, M. Bublin1, C. Hafner4, W. Hemmer5, V. Mayr1, C. Palladino1, A. Logiantara3, R. Van Ree3, R. Grabherr2, H. Breiteneder1

1Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria 2Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria 3Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands 4Department of Dermatology, University Hospital St. Pölten, Karl Landsteiner University of Health Sciences, St. Pölten, Austria 5Floridsdorf Allergy Center, Vienna, Austria Introduction: Ara h 2 with reduced IgE-binding capacity is a promising candidate for allergen specific immunotherapy. Little is known about the role of conformational and linear IgE epitopes. Therefore, we aimed to define the patient-specific IgE epitope profiles of Ara h 2. Methods: An Ara h 2 mutant (mt) lacking most linear epitopes and the wild-type protein (wt) were expressed in the baculovirus-insect cell system. Purified allergens and the natural protein (n) were reduced and alkylated (red/alk). IgE-binding was tested by ELISA using sera of 54 Ara h 2 allergic children and adults. C3H/HeOUJ mice were orally sensitized with peanut extract and challenged intraperitoneally with the various proteins to determine their anaphylactogenic potencies. Results: Mt, wt and nAra h 2 showed the predicted masses and structures. Complete red/alk was verified. In a patient specific manner, IgE-binding was reduced up to 50% due to the loss of conformational epitopes by red/alk (p<0.001). Likewise, IgE-binding was reduced up to 70% when linear epitopes were mutated (p<0.001). Patients with high levels of Ara h 2 specific IgE tend to recognize more the linear epitopes (r=0.305, p=0.025), and patients with low levels of Ara h 2 specific IgE recognize more conformational epitopes (r=-0.5, p=0.0001). In vivo, mice reacted with anaphylaxis upon challenge with mt, wt and n but not with the red/alk proteins, indicating that the destruction of conformational epitopes is required for the production of a safe hypoallergen. Furthermore, the challenge did not affect the present Ara h 2 specific IgE or IgG1 response. Conclusions: The obtained results indicate that both epitope types are important for allergen specific IgE-binding in a patient specific manner. In contrast to the published literature, destruction of the 3D structure is required for safe hypoallergens.

Supported by the Austrian Science Fund doctoral program W1248-B30, the Medical University of Vienna and Cost Action FA1402 (ImPARAS).


Portici, Italy

Page 23

O02

Egg proteins induce adjuvant-independent oral sensitization and the mixture of egg white

and egg yolk enhances T cell responses in BALB/c

Daniel Lozano-Ojalvo, Leticia Pérez-Rodríguez, Mónica Martínez-Blanco, Elena Molina and Rosina López-Fandiño

Instituto de investigación en Ciencias de la Alimentación (CIAL). CSIC. SPAIN [email protected] Young scientist. ImpARAS COST Action student. Oral presentation. Topic WG3 Background. Egg is the second most frequent source of allergic reactions in children. Egg yolk (EY) amounts to one third in weight of a fresh whole egg, but its contribution to egg allergy has not been investigated in depth. This study assesses whether EY influences the sensitizing capacity of egg white (EW) in mice. Methods. BALB/c mice were exposed to EW, EY and their mixture, using a model of adjuvant-free orally-induced allergy. The phenotype and proportion of DC and T cell subsets was examined in the MLNs by flow cytometry. Expression of Th2-skewing factors by DCs was assessed by RT-qPCR and cytokine release by MLN cells stimulated ex vivo was analyzed. Results. Gavage with the egg preparations induced maturation of MLN DCs, as shown by the upregulation of CD80 and CD86 on MLN CD11c+ and CD11c+CD103+ cells compared to naïve mice. In addition, EW, EY and the mixture EW:EY upregulated the expression of OX40L, whereas the expression of Jagged-2 was only significantly enhanced in the MLN of mice administered EY and EW:EY. An increased percentage of activated Th2 cells was detected in the MLN of mice administered EY, and GATA3 was overexpressed in mice that received EW:EY. Following ex vivo stimulation, the mixture EW:EY released IL-4 and IL-5 from the MLN cells of mice administered EY and EW:EY, but EW did not trigger the production of Th2 cytokines by cells belonging to any of the experimental groups

Conclusions. The results obtained reveal that EY provides Th2-adjuvant stimuli to the immune system that may increase the susceptibility to develop egg allergy..


Portici, Italy

Page 24

O03

Acid-hydrolyzed gliadins strengthen food allergies through early sensitization.

Laure Castan†,1,2,3,4, Clélia Villemin†,1, Mathilde Claude1, Philippe Aubert4,5, Michel Neunlist4, Chantal Brossard1, Antoine Magnan2,3,4, Sandra Denery1, Grégory Bouchaud†,1 and Marie Bodinier†,1

1INRA, UR1268 BIA, rue de la Géraudière, BP 71627, F-44316 Nantes, France 2INSERM, UMR1087, Institut du thorax, Nantes, F-44000 France 3CNRS, UMR6291, Nantes, F-44000 France 4Université de Nantes, Nantes, F-44000 France 5INSERM UMR913, Institut des Maladies de l'Appareil Digestif (IMAD), Faculté de Médecine, Nantes, F-44000, France † These authors contributed equally to this work Background Food allergies result from a complex immune response involving both innate and adaptive immune cells. Major proteins of wheat flour, gliadins, appear to be important allergens, and their characteristics can influence the allergic response. Our study investigates the immune reaction when developing a food allergy to gliadins in native, deamidated or hydrolyzed forms. Method We analyzed the immune response after one or two intraperitoneal sensitizations and after oral challenge with each gliadin form. Results We demonstrate that deamidated gliadins induce a stronger allergic reaction compared with native gliadins. Moreover, deamidation induces an earlier increase in intestinal permeability associated with more pronounced Th2 polarization together with a decrease in regulatory immunosuppressive response. Conclusion Altogether, our data indicate that industrial processes such as deamidation or hydrolysis impact food allergenicity through immune modulation and help us to develop tools to determine how these processes can influence this reaction and encourage or decrease allergic reactions.


Portici, Italy

Page 25

O04

Protein-chemical features of five different wheat products affect the sensitising capacity through the skin

Anne-Sofie R. Ballegaard1, Cristian Piras2, Gabriel Mazzucchelli3, Clélia Villemin4, Véronique Solé-

Jamault4, Sandra Denery-Papini4, Charlotte B. Madsen1, Katrine L. Bøgh1 1National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark 2Department of Veterinary Medicine, University of Milan, Milan, Italy 3Laboratory of Mass Spectrometry - MolSys, Department of Chemistry, University of Liege, Liege, Belgium 4UR1268 BIA, INRA, Nantes, France Background: Allergic sensitisation to foods may occur in infancy without prior oral exposure to the offending food. This has led to the assumption that food allergy sensitisation may occur through alternative routes, such as the skin. Recently, concerns have been raised regarding the safety of personal care products containing hydrolysed wheat proteins due to allergic reactions. The aim of the study was to investigate the impact of protein-chemical features on the skin sensitising capacity of five different wheat products; an unmodified, an enzyme hydrolysed, and three different acid hydrolysed gluten products. Methods: Wheat products were characterised for size distribution profile, protein/peptide fingerprint and degree of deamidation. To study the sensitising capacity of the five wheat products a dose-response study was conducted in naïve Brown Norway rats. Products were applied on slightly damaged skin without use of adjuvant. Rats were subsequently given post-immunisations by oral gavage. The sensitising capacity and cross-reactivity were evaluated by means of different ELISAs, immunoblotting and ear swelling test. Results: The protein-chemical features varied greatly between the products. All five products were able to induce a specific antibody response and sensitise through the slightly damaged skin, in a dose-dependent manner, though differences were seen between the products. Evaluating the cross-reactivity between the products, profound differences could be observed which correspond to the protein-chemical characteristics. Acid hydrolysed gluten products behaved differently than the enzyme hydrolysed gluten product indicating newly formed epitopes after acid hydrolysis. The protein sensitisation pattern differed between products.

Conclusion: This study showed that all five gluten products were able to sensitise through slightly damaged skin in a dose-dependent manner, though the pattern of sensitisation depended on the degree of modification.


Portici, Italy

Page 26

O05

AllerScreener: a bioinformatic tool for sensitization and cross-reactivity assessment of

proteins

Ivan Dimitrov and Irini Doytchinova Faculty of Pharmacy, Medical University of Sofia, Bulgaria The initial sensitization to foods arises in the GI tract. Food allergens are digested there, absorbed and endocytosed by dendritic cells or other antigen presenting cells (APC). In APC, the antigen fragments are further processed into oligopeptides which bind to the MHC class II proteins and the complex is presented on the cell surface for recognition by Th2 cells. The recognition leads to activation of B cells which mature to IgE-producing plasma cells. Here we present AllerScreener – a tool for prediction of sensitization ability of proteins. AllerScreener performs in silico digestion of allergens into peptides and then assesses the allergenicity of each peptide by similarity search in a database of allergenic peptides. The database contains 261921 peptides, derived after in silico digestion of 2491 known allergens from 336 species and subsequently predicted to bind to MHC class II proteins. AllerScreener is able to recognize novel unknown allergenic proteins using only information of their primary structure. Moreover, AllerScreener is able to identify cross-reactive proteins from different species.

AllerScreener is freely available at: https://www.ddg-pharmfac.net/AllerScreener/


Portici, Italy

Page 27

O06

Understanding Real Risks and Risk Management for Food Allergy and Celiac Disease

Richard E. Goodman

University of Nebraska-Lincoln The known prevalence of food allergy (FA) and celiac disease (CD) was very low in the 1950s. Clinical improvements increased the rates of diagnosis through the 1980s as proteins causing FA and CD were identified. Diagnoses became more common during the 1980s and parents were warned to not feed their children peanuts until the age of four. Yet, the prevalence of allergy to peanut increased four-fold. Were we avoiding tolerance induction while reducing infectious disease? Now FA prevalence is 6-8% and CD 1.3%. Food practices have changed with increasingly complex mixtures, increased processing and cooking. Commercial bakeries are using highly processed glutens to increase the speed and reduce costs of bread production. While FA and CD consumers are individuals, homologous proteins from similar foods are the dominant elicitors (2S albumins, lipid transfer proteins, vicilins, glycinins, tropomyosins, parvalbumins, arginine kinase and glutens) from related sources. We are not able to accurately predict which new proteins will be allergens, or who will become allergic. Protein sequences of genetically engineered (GE) crops are searched against the AllergenOnline.org and the associated Celiac databases for identification of matches that are potential risks. However, we are not paying attention to new processed foods that mix high concentrations of allergen homologues, process and consume them in new ways. There are no cases of increased risks of FA or CD from GE crops. Thresholds of detection are getting smaller while thresholds of reactivity being ignored for highly allergenic foods. Are we being misled by data from tests of protein digestibility and immune responses in animal models? Now clinicians have the LEAP data on early introduction of peanuts and it seems avoidance increased sensitization and allergy in <20 years. This presentation will challenge some of the paradigms being used for “risk assessment”. We need to educate consumers, not scare them.


Portici, Italy

Page 28

O07

Immune cell profiles characteristic for food allergic patients with and without allergen-specific IgE by high-dimensional mass cytometry

Friedrike Sonnet1,2, Ellen Namork1, Kanutte Huuse3, Hubert Dirven1 and Unni C Nygaard1

1Department of toxicology and risk assessment, Norwegian Institute of Public Health 2Utrecht University 3Department of Cancer Immunology, Oslo University Hospital Fifty percent of reports to the Norwegian Food Allergy Register of adverse reactions to food do not have allergen-specific IgE in serum (sIgE negative). Yet the pathophysiology of non-IgE-mediated food allergy is poorly understood. New, broader insight into immunological mechanisms and development of new diagnostic biomarkers are therefore needed. This pilot study aimed to identify biomarkers for sIgE-negative food allergy by comparing high-dimensional immune cell profiles between patients with adverse reactions to food with sIgE or without sIgE, and healthy control subjects (n=8-11). PBMCs were stained with two antibody panels, a 34-marker surface panel and a 28-marker functional panel against surface and intracellular targets following PMA and ionomycin stimulation. After mass cytometric detection, the data were analyzed by manual gating/unsupervised clustering and biostatistical analyses. The majority of the observed differences was related to the activation state and cytokine production, and was common for the sIgE positive and sIgE negative food allergy groups but differed from the control group. In particular, a subpopulation of intermediate monocytes and a CD4+ CD294+ T-cell subpopulation showed significantly increased expression of various activation markers in both food allergy groups. After stimulation, both food allergy groups had a decreased abundance of polyfunctional CD4+ and CD8+ memory T cells. Characteristic for the IgE-negative food allergic patients were an increased CD25 expression in a naive CD4+ CD294+ T-cell population and a significantly decreased production of IFN-γ and TNF-α in subpopulations of CD8+ T cells and natural killer cells. In conclusion, the high-dimensional immune cell profiling performed gives comprehensive information of the immunological cell signatures for food allergic patients with or without sIgE and provides a rationale for further studies to identify immune cell patterns linked to and possible predicting food allergy in humans.


Portici, Italy

Page 29

O08

Managing allergenic risks at the industrial level: egg proteins residues in pasta

Marengo Mauro, Bonomi Francesco, Iametti Stefania DeFENS, University of Milan, Italy Background. As of today, limits for allergens content in food have not been defined. Therefore, each industry set up a proper self-control manual that contains recommendations and risk evaluation. Egg proteins represent one of the major food allergens. The same pasta-making industrial plants are often used for production of both egg-pasta and semolina pasta, that may represent a risk for sensitive people since residual egg proteins may be present in semolina pasta. In this context it is fundamental to define the amount of semolina pasta, produced in egg-pasta line plant, that should be discarded. In this study, we assessed the amount of egg proteins in pasta samples taken at various time after the change from egg-pasta to semolina pasta. To investigate the role of drying in protein extraction, we detected the egg proteins in the same pasta samples before and after drying. Methods. Two commercial sandwich ELISA kits – from R-Biopharm and Morinaga - were used for egg proteins extraction and detection in fresh and dried pasta samples prepared with semolina and water on a production line previously used for the production of egg-containing pasta. Results. Recovery of egg proteins from the pasta matrix was only possible by using a combination of choatropes and reductants in the extraction medium. Pasta drying step decreased the amount of material extracted from the pasta with both kits, with noticeable differences. Appropriate protocols allowed monitoring the progressive disappearance of egg proteins from the industrial pasta, at least down to levels in the 0.8-1.0 ppm. Conclusion. Under appropriate conditions, current protocols and commercial immunochemical kits proved suitable for monitoring the progressive disappearance of egg proteins when shifting production lines in an industrial pasta production plant. This makes it possible to define a possible self-control procedure.


Portici, Italy

Page 30

O09

Receptor-mediated transfer, uptake and degradation of allergens towards and inside dendritic cells.

Joost Smit1, Friederike Sonnet1, Stef Koppelman2, Dion Zijtveld1, Raymond Pieters1

1Immunotoxicology group, Institute for Risk Assessment Sciences, Utrecht University, the Netherlands. 2University of Nebraska, Nebraska, USA Although cellular uptake and intracellular degradation of proteins are crucial for the induction of adaptive immune responses, it is unknown how this occurs in immune cells that are critical during allergic sensitization, such as dendritic cells (DC). Moreover, industrial processing influences allergen structure, size and activity, which might affect their cellular uptake and degradation and hereby the development of allergy. In our studies, we determined whether we are able to measure uptake and degradation of allergens towards and inside DC and whether differences exist in handling of (processed) allergens due to above mentioned factors. In addition, we investigated which routes of uptake and which protein properties are responsible for the transfer of allergens to and the uptake and degradation by DC in the intestine. Using CACO-2 epithelial cells and mouse bone marrow-derived DC, we studied the transfer and cellular uptake and processing of the peanut proteins Ara h 1, 2, 3, and 6 and of native and glycosylated Bos d 5 (ß-lactoglobulin). Profound differences were observed in the transfer towards and uptake by DC between the different peanut proteins and after glycosylation of Bos d 5. In addition, differences between allergens and between native and processed allergen were observed in intracellular degradation and presentation by DC. Several pathways involved in the uptake ofallergens were discovered by specific antagonists and allergens were chemically deglycosylated, which showed the importance of sugar moieties on allergens. Together, we show that allergenicity of (peanut) proteins may be, at least partly, determined at the level of allergen uptake and breakdown inside the antigen presenting cell. This process was significantly affected by protein modification and mediated by the level of glycosylation of the allergen. These findings may be relevant to the risk evaluation of existing allergens but also of novel or modified proteins. This also illustrates the usefulness of in vitro, cell-based assays to examine initial processes of allergenic sensitization to proteins.


Portici, Italy

Page 31

O10

Epitopes in sensitisation and in elicitation: the impact of food processing

Harry Wichers Wageningen Food & Biobased Research, Bornse Weilanden 9, 6708WG Wageningen, The Netherlands [email protected] The rapidly increasing prevalence of various types of allergies is increasingly becoming acknowledged as real. Recently, EAACI forecasted that, by 2025, 50% of the European population will suffer from some kind of allergic health issue. Efforts to deal with this alarming scenario should therefore not only be directed towards ‘managing’ the problem, e.g. in countermeasures such as labelling and informing the general public, but also be directed towards attempts to prevent such conditions to develop in the first place, and/or to re-balance disturbances in immunity that underlie the development of allergies. Nearly all of the foods that we consume are heat-processed, e.g. to confer taste, to prolong shelf-life and/or to ascertain microbiological safety. Such heat processing affects the structure of proteins, including that of allergens, that are contained in such foods. In turn such structural modifications impact on the allergenicity of these proteins, both in initiation of possible allergic reactions and in their elicitation. A concise overview of the impact of processing-related protein modifications on their sensitising potential and their elicitation potential will be discussed for peanut and cow’s milk as examples. For both peanut and milk, processing-derived modification lead to modified immunogenicity and allergenicity. Different processing types appear to exert different effects, presenting perspectives to use food processing as a tool to moderate immunogenicity and allergenicity.


Portici, Italy

Page 32

O11 PDL2+ CD11b+ Dermal dendritic cell capture topical antigen through hair follicles to prime

LAP + regulatory T cells

Leticia Tordesillas1,2, Daniel Lozano-Ojalvo1,2, David Dunkin3, Lucie Mondoulet4, Judith Agudo2, Miriam Merad2, Hugh A Sampson1,2,4 and M Cecilia Berin1,2.

1Pediatric Allergy & Immunology, 2Immunology Institute, 3Pediatric Gastroenterology, Icahn School of Medicine at Mount Sinai, New York, US. 4DBV Technologies, Montrouge, France. [email protected] Young scientist. ImpARAS COST Action Member. Oral presentation. Topic WG3. Background. Epicutaneous immunotherapy has proved to be efficient in the induction of immune tolerance and a safe alternative treatment of food allergies. The aim of this work was to determine the immunological mechanisms involved in the skin handling of the allergen and the induction of LAP+ regulatory T cells (Tregs). Methods. We examined how ovalbumin (OVA) applied topically using Viaskin® patches was acquired by dendritic cell subsets in the skin and transferred to the draining lymph nodes. Murine models with BALB/c and C57BL/6 backgrounds were used to study cellular populations involved in the acquisition and transport of OVA. In addition, adoptive transfer experiments using DO11.10 or OT-II mice allowed the investigation of allergen presentation to CD4+ T cells. Finally, the influence of hair follicle in OVA acquisition was studied using hairless SKH1 mice. Results. Data showed that OVA applied topically was acquired and transported to skin-draining lymph nodes by Langerhans cells (LCs) and CD11b+ cDC2s, but not cDC1s. In addition, LCs and CD11b+ cDC2s expressing the marker PDL2 could prime CD4+ T cells to generate LAP+ Tregs. However, depletion of LCs using anti-CSF1R antibodies and a Langerin-DTR mouse model showed that LCs were neither required nor sufficient for T cell priming or tolerance induction. A central role for CD11b+ cDC2s in presentation of topical antigen was confirmed using CD11c-CRE-IRF4fl/fl mice that lack IRF4-dependent cDC2s. Dermal CD11b+ cDC2s acquired antigen through the hair follicle, shown by the abolishment of antigen delivery to the lymph nodes and induction of tolerance in hairless SKH1 mice.

Conclusion. This work indicates a key role for the hair follicle niche and CD11b+ cDC2s expressing PDL2 in antigen acquisition and generation of immune tolerance to food allergens.


Portici, Italy

Page 33

O12 Mapping of conformational IgE-binding epitopes of peanut allergen Ara h 2 with chimeric

2S-albumins

Stéphane Hazebrouck1, Blanche Guillon1, Evelyne Paty2, Karine Adel-Patient1 and Hervé Bernard1

1 UMR CEA-INRA Service de Pharmacologie et d’Immunoanalyse, Université Paris-Saclay, 91991 Gif-sur-Yvette, France and 2 Université Paris Descartes, APHP, Hôpital Necker Enfants Malades, 75743 Paris, France. BACKGROUND: 2S-albumins Ara h 2 and Ara h 6 are the most potent peanut allergens and specific IgE responses toward these proteins are good predictors of clinical reactivity. Contrary to linear IgE-binding epitopes which have been extensively studied, data on conformational epitopes are limited. We investigated these conformational epitopes by producing chimeric 2S-albumins. METHODS: Chimeric 2S-albumins were produced in E. coli by replacing Ara h 2 domains with the corresponding ones from Ara h 6. After purification and refolding, secondary and tertiary structures of the chimeric proteins were assessed by CD-spectroscopy and by competitive inhibition of IgE-binding to native 2S-albumins with sera exhibiting high cross-reactivity between Ara h 2 and Ara h 6. Allergenicity of chimeras was further investigated by IgE-binding measurements with sera from 11 peanut-allergic patients and by degranulation assay of RBL-SX 38 cells. RESULTS: All chimeric constructions exhibited a proper refolding since cross-reactive IgE antibodies to Ara h 2 and Ara h 6 recognized the chimera with similar affinity. Additionally, IgE-binding measurements performed with sera depleted of Ara h 6-specific IgE antibodies permitted to locate the non-cross-reactive conformational epitopes of Ara h 2. These epitopes were required to induce an efficient degranulation of mast cells passively sensitized with IgE antibodies from patients sensitized only to Ara h 2.

CONCLUSIONS: Conformational cross-reactive epitopes of Ara h 2 and Ara h 6 were not impacted in chimeric 2S-albumins. Although the profile of epitopes recognition remained highly patient specific, the major conformational epitopes specific to Ara h 2 were located in the central domain.


Portici, Italy

Page 34

O13 Modulation of fish allergenicity towards the production of a low allergen farmed fish using

Creatine enriched diets

Cláudia Raposo1, Denise Schrama1, Pedro Rodrigues1 1CCMAR, Universidade do Algarve, Campus de Gambelas, 8005 – 139 Faro, Portugal ([email protected]) Background: Fish, despite its high nutritional value and benefit in human diets, is one of the most common elicitors of food---allergic reactions, which are a global concern for the public health. The main allergen is β---parvalbumin (PV), a small, highly stable calcium---binding muscle protein, responsible for IgE---mediated anaphylactic reactions to fish. In fish--- allergic patients, clinical cross---reactivity between different fish species is highly common due to the highly conserved amino acid and structural identity between the homologs. As no immunotherapy is available, complete avoidance of the allergenic food is currently the only solution, thus the production of a hypoallergenic fish might be a valid alternative. This work aimed to modulate fish allergenicity using diets supplemented with creatine as an expression modulator of PV. Methods: Gilthead seabream were raised using standard feed for control fish and specifically designed diets, enriched with different concentrations of creatine (2%, 5% and 8%) to target allergenicity. Parvalbumin’s muscle concentration was addressed through ELISA. Proteomics was used to characterize the fish muscle proteome and the allergens’ expression. Fish quality and welfare were established with biochemical, texture and sensorial analysis. Results: Cortisol levels were significantly lower in fish fed with 8% creatine, comparing to control fish (p=0.0498). Fish quality traits showed no significant differences (p<0.05). Parvalbumin’s concentration levels showed no significant differences between treatments (p<0.05) using ELISA. Proteomics identified 127 spots with 7 being significantly different and involved in muscle contraction. Conclusion: Overall welfare of fish raised under creatine---diet seems not to be affected by the novel feeding conditions. Creatine supplementation seems to slightly affect the allergenic potential of Sparus aurata with a 20% reduction of parvalbumin expression in fish fed with creatine 2% enriched diets.


Portici, Italy

Page 35

O14

Effect of technological treatments on immunoreactivity and allergenicity of the allergenic

protein Pru p 3 from peach

Tobajas AP1, Catalán T1, Segura-Gil I1, Sánchez L1, Colás C2, Calvo, M., Pérez MD1* (1)Departamento de Producción Animal y Ciencia de los Alimentos. Facultad de Veterinaria. Instituto Agroalimentario de Aragón (IA2) (Universidad de Zaragoza-CITA), Zaragoza, España. (2)Servicio de Alergología. IIS-Aragón. Zaragoza, España. *[email protected] Pru p 3 is the major allergen in peach and is considered the responsible of systemic symptoms after peach ingestion. Severity of peach allergy is probably related to the high stability of Pru p 3 to food processing and proteolysis degradation. Because peaches are often consumed after processing to obtain juice, nectar or jam, the aim of this study was to investigate the effect of technological treatments on Pru p 3 in order to produce peach-based products able to decrease their potential allergenicity. To perform this study, Pru p 3 was purified from peel peach by chromatographic techniques. Pure protein was inoculated into rabbits to raise antibodies, which were used to develop a sandwich ELISA. The denaturation of Pru p 3 subjected to technological treatments was determined by measuring the loss of immunoreactivity using the developed ELISA. Furthermore, changes in allergenicity were determined by an inhibition assay with treated samples and sera from peach allergic patients using the ImmunoCAP allergen Pru p3 system. Results showed that heat treatments of peach extract at 85ºC and 95 ºC up to 40 min did not affect significantly reactivity of Pru p 3 with rabbit IgG or human IgE. Pressure treatment at 400, 500 and 600 MPa at 20ºC for 10 min did not denature Pru p 3 whereas a denaturation degree lower than 20% was observed at 600 MPa for 10 min at 50 ºC. However, pressure treatments did not change Pru p 3 IgE binding properties. Pru p 3 showed a high resistance to degradation by most proteases assayed. However, after incubation with an alkaline and an acid protease, the band of Pru p 3 observed by SDS-PAGE showed a large disappearance after 2 h. Likewise, those samples showed a marked decrease of Pru p 3 reactivity with IgG and IgE. These preliminary results show that treatment of Pru p 3 with certain proteases is effective in producing hypoallergenic peach extract. Pru p 3 is the major allergen in peach and is considered the responsible of systemic symptoms after peach ingestion. Severity of peach allergy is probably related to the high stability of Pru p 3 to food processing and proteolysis degradation. Because peaches are often consumed after processing to obtain juice, nectar or jam, the aim of this study was to investigate the effect of technological treatments on Pru p 3 in order to produce peach-based products able to decrease their potential allergenicity. To perform this study, Pru p 3 was purified from peel peach by chromatographic techniques. Pure protein was inoculated into rabbits to raise antibodies, which were used to develop a sandwich ELISA. The denaturation of Pru p 3 subjected to technological treatments was determined by measuring the loss of immunoreactivity using the developed ELISA. Furthermore, changes in allergenicity were determined by an inhibition assay with treated samples and sera from peach allergic patients using the ImmunoCAP allergen Pru p3 system. Results showed that heat treatments of peach extract at 85ºC and 95 ºC up to 40 min did not affect significantly reactivity of Pru p 3 with rabbit IgG or human IgE. Pressure treatment at 400, 500 and 600 MPa at 20ºC for 10 min did not denature Pru p 3 whereas a denaturation degree lower than 20% was observed at 600 MPa for 10 min at 50 ºC. However, pressure treatments did not change Pru p 3 IgE binding properties. Pru p 3 showed a high resistance to degradation by most proteases assayed. However, after incubation with an alkaline and an acid protease, the band of Pru p 3 observed by SDS-PAGE showed a large disappearance after 2 h. Likewise, those samples showed a marked decrease of Pru p 3 reactivity with IgG and IgE. These preliminary results show that treatment of Pru p 3 with certain proteases is effective in producing hypoallergenic peach extract.

mailto:[email protected]


Portici, Italy

Page 36

O15

Milk proteins in food products as affected by thermal processing assessed by

immunochemical and DNA-based methods

Caterina Villa1, Joana Costa1, M. Beatriz P. P. Oliveira1 and Isabel Mafra1* 1REQUIMTE-LAQV, Faculty of Pharmacy, University of Porto, Porto, Portugal. *[email protected] Background: Cow’s milk allergy is one of the most common food allergies in early childhood, which can persist through adult life. Milk proteins are present in uncounted food products, as ingredients (cheeses, yogurts) or as technological aids (sausages, ham), exposing allergic individuals to a constant threat. Food processing, such as heat treatments can alter the structure/stability of proteins, subsequently affecting their allergenicity [1]. This work intends to exploit different thermal treatments, in order to understand their impact on the allergenicity of milk proteins, as well as on the immunological and DNA-based methods used for milk detection in foods. Methods: Model mixtures containing known quantities (10-0.00001%) of cow’s milk protein concentrate (technological aids) in turkey meat were prepared and submitted to two processing methods, simulating the production of ham (67°C, 5 h) and sausages (121°C, 15 min). The effect of thermal processing on milk was evaluated by PCR-based methods targeting different mitochondrial genes (cytb, 12S rRNA and 16S rRNA) of cow and by immunoassays (SDS-PAGE and immunoblotting) using specific antibodies for milk proteins (e.g. anti-beta-lactoglobulin). Results: Preliminary results obtained by real-time PCR targeting the cytb gene showed a sensitivity of 0.01% of milk protein concentrate in turkey meat (raw) with optimal performance parameters. The thermal processing affected the detection of milk, enabling a relative sensitivity of 0.1%. Likewise, the profile of proteins of milk concentrate was severely altered by the thermal treatments, both in ham and sausage model mixtures. Conclusion: The thermal processing has a negative effect on the detection of milk proteins as assessed by both protein- and DNA-based methods. Currently, the effect of thermal processing on milk protein concentrate is being evaluated by PCR systems targeting different markers genes, as well as by immunoassays targeting different allergenic proteins (e.g. caseins). References: [1] Villa, C., Costa, J., Oliveira, M. B. P. P., Mafra, I. (2018). Bovine Milk Allergens: A Comprehensive Review. Comprehensive Reviews in Food Science and Food Safety, 17:137-164. Acknowledgments: This work was supported by FCT (Fundação para a Ciência e Tecnologia) through projects AlleRiskAssess–POCI-01-0145-FEDER-031720, UID/QUI/50006/2013–POCI/01/0145/FEDER/007265 with financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 and by the project NORTE-01-0145-FEDER-000011. Caterina Villa and Joana Costa are grateful to FCT grants (PD/BD/114576/2016 and SFRH/BPD/102404/2014, respectively) financed by POPH-QREN (subsidised by FSE and MCTES).



Portici, Italy

Page 37

O16

Deactivation of allergens in some foods using ultrasound

Sedef Nehir El, Hulya I. Buyukkestelli, Sibel Karakaya Ege University, Food Engineering Department, Nutrition Science, Bornova Izmir, Turkey Allergenic foods include foods from both plants and animals such as peanuts, tree nuts, wheat, soy, cow's milk, eggs, fish, and shellfish. Typically proteins known as allergens or antigens, in their presence immunological mechanisms can elicit acute response in sensitized/allergic individuals. Food allergy and sensitivity to immunoglobulin E (IgE)-mediated reactions are becoming more serious health issue in recent years with the number of novel food products and ingredients launched on the market by the industry. During processing, interactions between proteins and other ingredients can lead to structure and conformation modifications and induce allergenicity. Otherwise, some allergens show a resistance to process and in this point, effective process parameters or deactivation techniques to reduce their allergenicity are needed. A number of thermal and nonthermal food processing technology for reducing allergenicity have been studied on various allergenic foods. Ultrasound is an emerging technique in food industries, frequently used for homogenization, emulsification, meat tenderization (meat), and fruits and vegetables dehydration processes. Ultrasound uses high energy mechanical waves, cavitation bubbles are formed which induces cyclic generation and collapse of cavities followed by formation of localized region of high pressure and temperature surrounding these collapsed cavities which can bring about conformational changes to food proteins and thereby influence their allergic reactivity by such as altering allergen epitopes. Understanding of the mechanisms of deactivating food allergens in foods by ultrasound treatment may be a good approach for food allergy/sensitization for food industry. In this presentation the application of ultrasound for potential reduction of food allergenicity and some results of our project titled “effect of Maillard reaction on the allergenicity of proteins in chicken and shrimp, their protein isolates and hydrolyzates” will be highlighted.


Portici, Italy

Page 38

P01

Catalase as a novel IgE reactive protein from banana should be involved in component-resolved allergy diagnosis

Andrijana Nešić1*, Jasana Nikolić1*, Uta Jappe2,3, Marija Gavrović-Jankulović1

1Department of Biochemistry, Faculty of Chemistry University of Belgrade, Belgrade, Serbia 2Division of Clinical and Molecular Allergology, Research Center Borstel, Airway Research Center North, Member of the German Center for Lung Research, Borstel, Germany 3Interdisciplinary Allergy Outpatient Clinic, Department of Pneumology, University of Lubeck, Germany *equally contributing authors Objective: Diagnostic reagents based on food allergen extracts often lack sufficient sensitivity. The introduction of well characterized allergens in the component-resolved allergy diagnosis has become a promising strategy to achieve reliable food allergy diagnosis. Material and methods: Catalase from banana was detected as IgE reactive protein in immunoblot. To evaluate its diagnostic potential, enough amounts of a recombinant catalase homologue was produced in the procaryotic expression system. The protein was isolated and biochemically and immunochemically characterized. IgE reactivity of catalase was analysed in a group of thirteen persons with suspected allergy to banana. The study was performed with the approval of the Ethics Committee of the University of Lubeck, Germany. Result: The recombinant catalase was produced in E. coli as 57 kDa protein with an isoelectric point of 8.3. The recombinant enzyme was purified by a combination of immobilized metal affinity chromatography (IMAC) and ion exchange chromatography under native conditions. Catalase activity of the recombinant protein was confirmed in zymography test with hydrogen peroxide. The recombinant protein showed IgE reactivity in 7 out of 13 tested patients with suspected allergy to banana in immunoblot. Conclusions: Catalase from banana fruit is identified as a novel allergen, with proposed designation as Mus a 7. IgE reactive recombinant Mus a 7 was produced and should be included in a component-resolved allergy diagnosis. Acknowledgement: This study was supported by the Ministry of Education, Science, and Technological Development, Republic of Serbia (Grant no. 172049).


Portici, Italy

Page 39

P02

The hidden “digestome”: do the current analytical approaches provide comprehensive peptide inventory of a food digest?

Gianluca Picariello*,1, Maristella De Cicco1, Gianfranco Mamone1, Pasquale Ferranti2

1Instituto di Scienze dell’Alimentazione – National Research Council (CNR), Avellino, ITALY 2Dipartimento di Agraria – Università di Napoli “Federico II”, Portici (Napoli), Italy *correspondence to: [email protected] Protein digestion is a multi-phasal event producing a heterogeneous mixture of peptides. In general, “digestomes” include small-/medium-sized oligopeptides as well as nearly intact proteins. The electrophoretic methods, most often exploited to assess digestibility of food proteins, are inadequate to provide information about the nature of the peptide digests. Peptide components of a food digestome can be identified by chromatography coupled to tandem mass spectrometry (MS/MS) sequencing and software-assisted matching of experimental with expected MS/MS spectra of peptides released from a set of possible proteins. The complexity of the digestomes renders unreal the eventuality of assigning hundreds-to-thousands individual peptides manually. Analyzing a gastroduodenal digest of whey proteins by HPLC coupled to high-resolution/high-sensitivity MS/MS (Orbitrap Q-Exactive, Thermo, USA), we sought to evaluate if the typical analytical strategies yield comprehensive peptide coverage of a food digestome. Ordinary proteomic/peptidomic workflows employ automated ion selection for fragmentation, typically excluding low-mass and singly charged species. Furthermore, matching score from bioinformatic algorithms increases proportionally with the peptide length, so introducing an intrinsic bias against small peptides. Therefore, many small-sized peptides – probably not involved in the pathogenesis of food allergies – escape identification in the untargeted analysis of a food digestome. Remarkably, the analysis of a HPLC-MS/MS run with different proteomic search engines generated significantly different peptide inventories, so underlying the necessity to refine the searching algorithms. Contrarily to the common belief that whey proteins are almost completely degraded by gastroduodenal proteases (β-lactoglobulin is partly only gastro-resistant), a multitude of low-intensity large-sized polypeptides as well as disulfide cross-linked hetero-oligomers derived from both -lactalbumin and β-lactoglobulin survived digestion, as confirmed after pre-fractionation of the digests. However, the large polypeptides remained unidentified, even when the digests were analyzed with different techniques, such as MALDI-TOF MS, before and after S-S reduction. These findings evidence that the current picture of the food digestomes might be partial, only representing an “iceberg tip” and rise relevant implications for the evaluation of stability/susceptibility of food proteins to digestion, in view of the allergenicity risk assessment. Overall, data herein highlight the demand of improved approaches for the comprehensive untargeted characterization of the food digestomes, aiming at the discovery of possible food-derived bioactive peptides. Keywords: Food peptide digestomes, HPLC-MS/MS, disulfide cross-linked peptides, protein digestibility, food allergy


Portici, Italy

Page 40

P03

Modulation of fish allergenicity towards the production of a low allergen farmed fish using EDTA enriched diets

Denise Schrama1, Cláudia Raposo1, Annette Kuehn2, Martine Morriset3 and Pedro Rodrigues1

1CCMAR, Universidade do Algarve, Campus de Gambelas, 8005 – 139 Faro, Portugal ([email protected]) 2Luxembourg Institute of Health, Department of Infection and Immunity, 29, rue Henri Koch, L-4354 Esch-sur-Alzette, Luxembourg 3National Unit of Immunology and Allergology, Centre Hospitalier de Luxembourg, Luxembourg Background: Common food allergies, type I hypersensitivities involving the production of food-specific IgE-antibodies, are a global concern for the public health. Despite of its high nutritional value and benefit in human diets, fish is an important elicitor of food-allergic reactions. β-parvalbumin, a small, highly conserved and stable, calcium-binding muscle protein is the main fish allergen. In fish-allergic patients, clinical cross-reactivity between different fish species develops often due to IgE cross-recognition of highly similar epitope regions. Currently, no therapeutic cure is available why the clinical management of fish allergies relies on a strict avoidance diet. Methods: Sparus aurata were raised for 90 days using standard and specifically designed diets, enriched with EDTA to target allergenicity. Proteomics was used to characterize fish protein/allergens and their muscle expression. IgE immunoblots using sera from patients (n=16) with clinical history of fish allergy were performed to address the allergenic potential. Fish quality and welfare were established with biochemical, texture and sensorial analysis. Results: Cortisol levels show no significant differences between conditions (p>0.05). IgE-immunoblots were positive for all patients using muscle extracts from control. Most IgE-test (13/16) were also positive for the fish samples produced using EDTA-diets while IgE-signals were weaker as compared to the control fish. Fish quality show significant differences in springiness and cohesiveness (p<0.05). Water holding capacity does not seem to be affected by 3% EDTA. Proteomics identified 14 significantly different spots being involved mainly in the function of the contractile apparatus. Conclusion: Supplementation with EDTA in fish diet of S. aurata seems to change muscle protein expression profiles and possibly, the allergenicity of this species. Overall welfare of fish raised under EDTA-diet seems not to be affected by the novel feeding conditions.


Portici, Italy

Page 41

P04

Use of in vitro digestive models for the prediction of antinutritional properties and allergenic risk of food proteins

Krisztina Takács, Éva Gelencsér

NARIC Food Science Research Institute, Department of Biology, Budapest, Hungary Simulated gastrointestinal digestion (SGID) is widely used in food and nutrition science, which results from the fact that human experiments are often costly, resource-intensive and ethically objectionable. Consequently, in vitro alternatives that may determine the bioavailability of dietary proteins or predict their antinutritive or allergenic properties may lead to knowledge based and safe innovations. The digestibility rate of the proteins effects on their bioactivity amongst others influenced by their structure and the biochemical processes undergoing during digestion. Proteins may retain or lose their allergenic activity, and may also change their antinutritive properties. Trypsin and chymotrypsin inhibitory activity of soya bean protease inhibitors were studied during digestion process (oral, gastric and intestinal phases) using SGID model. Purified inhibitors (KTI, BBI) and native and dielectric heat treated soya bean matrixes were used for the assessment. Digestion properties were studied also for a well characterized milk protein allergen, β-lactoglobulin. SDS-PAGE, native-PAGE or 2-DE methods were used for separating the proteins according to Mw. Trypsin and chymotrypsin inhibitor activities of the separated proteins were detected using enzyme reaction based colour identification. IgE reactivity was assessed by immunoblot using sensitized human sera. It was found that the antinutritive properties of soya bean inhibitors were affected by the food matrix and the heat treatments during digestion. However the inhibitory activity was stable at low pH in gastric phase they were fully eliminated in the intestinal phase. It was also observed that -lactoglobulin was resistant to digestion at low pH and degraded to small peptides at the intestinal phase which could lead to resistance of allergenic epitopes as well. It can be conclude that the resistance to digestion is an important factor in the prediction of further bioactivity of dietary proteins.


Portici, Italy

Page 42

P05

How deep do we really know the peanut digestome?

Luigia DI Stasio,a,b Gianluca Picariello,b Pasquale Ferranti,b Gianfranco Mamonea aInstitute of Food Sciences, CNR, via Roma 64, 83100 Avellino, Italy bDepartment of Agriculture, University of Naples Federico II, Via Università, Portici (Naples), Italy Stability to proteolytic degradation in the digestive tract is considered a general feature shared by most food allergens. In the last years, susceptibility of food allergens to proteolysis has been typically assayed by using single purified proteins. Such an approach could suffer from scarce relevance, since the matrix components might affect the accessibility of proteases to allergens, thereby contributing to the bioaccessibility and hence to the bioavailability of allergenic determinants (epitopes). Another important relevant aspect, barely addressed so far, is the analysis of peptide fragments arising from the proteolysis process. In fact, the majority of the studies aiming at assessing the digestion stability of allergens only monitored the degradation of allergens by SDS-PAGE, neglecting the release of immunologically active proteolytic peptides, which escape the electrophoretic detection. In a previous study, we determined the stability of the major peanut allergens directly in the natural matrix using an in vitro static model that simulates the gastrointestinal digestion including the oral, gastric, duodenal and intestinal phases. Immunoreactive, large-sized fragments of Ara h 2, Ara h 6 and Ara h 3 survived hydrolysis. Smaller IgE-binding resistant peptides mainly arising from Ara h 3 and Ara h 1 were identified as well. However, a significant amount of disulfide-linked polypeptides remained unassigned due to the drawback of analyzing the heterogeneous digestome of peanuts. In the present study, disulfide-linked polypeptides arising from gastrointestinal digestion of whole peanuts were enriched by size exclusion chromatography (SEC) and further identified by LC-high resolution-MS/MS before and after Cys-reduction. Disulfide cross-linked peptides mainly arose from N-terminal region of Ara h 3. A detailed mapping of the peanut resistant peptides including disulfide ones could improve the knowledge about the molecular pathogenic mechanism underlying food allergy to peanut. Keywords: disulfide-linked polypeptides; peanut; gastrointestinal digestion


Portici, Italy

Page 43

P06

Allergenicity risk assessment of sunflower seed protein

Jihana Achour, Blanche Guillon, Marine Guinot, Karine Adel-Patient, Hervé Bernard and Stéphane Hazebrouck

Service de Pharmacologie et Immunoanalyse, Laboratoire d’Immuno-Allergie Alimentaire, CEA, INRA, Université Paris-Saclay, F-91191 Gif-sur-Yvette, France Background: World population is growing and demand in food proteins is then constantly increasing. We thus need to identify new sustainable protein sources. In this regard, vegetable raw materials appear to be an interesting alternative for animal proteins because of its low environmental impact. Sunflower seeds (SFS) display high protein-content (16%) and are already consumed in some countries such as Spain. However, adverse immune-mediated reactions have been observed in allergic patients. These reactions have been mainly attributed to IgE responses against Hel a 3 (nsLTP) and/or SFA-8 (2S-albumin). Objectives: We aimed (1) to identify the main allergenic proteins of SFS and the related risk of cross-reactivity with other food constituents and (2) to develop a mouse model of allergenic sensitization to SFS protein. Methods: Using dehulled and defatted whole SFS extract, several fractions were prepared by using selective precipitation with ammonium sulfate and separation by RP-HPLC. Protein fractions were then characterized by 1D- and 2D-gel electrophoresis and by mass-spectrometry (MALDI-TOF), using approaches addressed in the 2nd ImpARAS training school. Allergenicity of SFS proteins have been tested in an experimental model of mice injected intraperitoneally with whole SFS extract. Results: Protein precipitation induced with 40% ammonium sulfate led to an efficient separation of low MW proteins, mainly 2S-albumins and LTPs, from high MW proteins, mainly 11S globulin subunits. Several 2S albumins and LTPs were then isolated and identified, including SFA-8 and Hel a 3. In parallel, mice sensitized intraperitoneally exhibited a dose-dependent response to whole SSP extract. Preliminary results showed that 2S albumins seemed to be more immunogenic than LTPs. We also identified 2S-albumins that were at least as immunogenic as SFA-8.

Conclusion: The development of mouse models is still ongoing and oral route of sensitization will be soon investigated. The fraction containing globulin 11S subunits will be further characterized and IgE-binding capacity of SFS proteins will be then evaluated with sera from patients allergic to SFS and/or to other food allergens.


Portici, Italy

Page 44

P07

Resolving the complexity of peanut allergome using 2DE gel based and gel free proteomic analysis

Luigia Di Stasio,a,b,* Salvatore De Caro,a Gianluca Picariello,a Maria Adalgisa Nicolai,b

Pasquale Ferranti,b Gianfranco Mamonea aInstitute of Food Sciences, CNR, via Roma 64, 83100 Avellino, Italy bDepartment of Agriculture, University of Naples Federico II, Via Università, Portici (Naples), Italy *Corresponding author: Luigia Di Stasio E-mail: [email protected] Peanuts are responsible of one among the most severe and life-long persistent food allergies, affecting nearly 1% of children and 0.6% of adults within the general population of Westernized countries. Because of the large use of peanut as a food ingredient and its involvement in allergenic reactions, the molecular structure of peanut proteins has been the subject of fervent investigation over the last decades. The knowledge about the composition of peanut allergens has been significantly advanced by the latest progresses of proteomic methods. In this study, the complexity of the peanut allergome proteins was resolved using complementarily 2-DE-based proteomics under both reducing and non-reducing conditions and gel-free shotgun proteomics. The combination of the two electrophoretic condition running methodologies was beneficial to the purpose of comprehensive coverage of the peanut proteome. 2-DE analysis enabled the detection of the major storage proteins and disclosed naturally occurring high molecular weight disulphide-linked multimers of Ara h 1 and Ara h 3 as well as high molecular weight aggregates of Ara h 1 stable under reducing conditions. The occurrence of these protein complexes reveals natural interactions between Ara(s) subunits with a possible involvement in the allergenic potential of peanut, still unknown. The shotgun proteomic analysis confirmed the identification of major peanut allergens and enabled the access to the "deep" proteome of peanut seeds, allowing the identification of 149 gene products, including minor allergens escaped 2-DE detection. These results provide insights into the global complexity of the peanut allergome.



Portici, Italy

Page 45

P08 Detection and quantification of fish as a potential allergenic food: comparison of two real-

time PCR approaches targeting the 16S rRNA gene

Telmo J. R. Fernandes1, Joana Costa1, M. Beatriz P. P. Oliveira1 and Isabel Mafra1* 1REQUIMTE-LAQV, Faculty of Pharmacy, University of Porto, Porto, Portugal. *E-mail: [email protected] Background: Fish is one of the most common allergenic foods that should be accurately labelled to protect the health of allergic consumers. Despite its compulsory labelling [1], the allergic individuals can inadvertently consume the offending foods due to mislabelling or cross-contamination during processing. This emphasises the need of verifying the labelling compliance, which should rely on highly sensitive, accurate, specific and fast methods to enable the detection of trace levels of allergens in foods. In the present work, the development of two real-time PCR approaches is proposed targeting the 16S rRNA mitochondrial region, as a universal marker for fish detection in foods at trace levels [2]. Methods: Model mixtures of fish in béchamel sauce were prepared in the range of 50- 0.0001% (w/w) for method development and blind samples (8.0, 4.0, 2.5 and 0.25%) for method validation. Two real-time PCR approaches based of the universal EvaGreen dye and TaqMan probes were compared in terms of assay performance, validation and applicability. Besides, several commercial samples (n=34) were analysed. Results: Both systems showed similar absolute sensitivities down to 0.01 pg of fish DNA and adequate real-time PCR performance parameters. The relative sensitivity of the probe system was down to 0.0001% (1 mg/kg) of fish in béchamel, with a dynamic range of 50-0.0001%, while the Evagreen system only reached 0.05%, after which the amplification was non-specific. The validation results with blind samples showed that both systems were precise in the tested range, but trueness, expressed as bias, was within ±25% only in the probe system because at the highest tested level (8%) with the EvaGreen system the error was high. Conclusion: The quantitative probe system provided the best results, being successfully validated in terms of precision and trueness, and further applied to verify labelling compliance of processed foods, suggesting a high level of mislabelling and/or fraudulent practices. References: [1] Regulation (EU) No 1169/2011 of 25 October 2011 on the provision of food information to consumers. Official Journal of the European Union, L304, 18–63. [2] Fernandes, T.J.R., Costa, J., Oliveira, M.B.P.P., Mafra, I. (2018). Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches, Food Chemistry, 245: 1034-1041. Acknowledgments: This work was supported by FCT (Fundação para a Ciência e Tecnologia) through projects AlleRiskAssess – POCI-01-0145-FEDER-031720 and UID/QUI/50006/2013 – POCI/01/0145/FEDER/007265 with financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 and by the project NORTE-01-0145-FEDER-000011. T.J.R. Fernandes and J. Costa are grateful to FCT grants (SFRH/BD/93711/2013 and SFRH/BPD/102404/2014, respectively) financed by POPH-QREN (subsidised by FSE and MCTES). The codfish species were kindly provided by Pascoal & Filhos SA.



Portici, Italy

Page 46

P09

High Pressure Processing and Food Allergens

Merve Eda Eker, Sibel Karakaya Ege University, Department of Food Engineering Bornova İzmir, Turkey The ubiquitous presence of food allergens in diet have prompted increased awareness of food allergies warrants undertaking appropriate preventive measures to protect sensitive consumers from unwanted exposure to offending food allergens. Since foods/food ingredients are often subjected to variety of processing conditions, alterations in immunodominant epitopes may potentially affect protein allergenic properties by destroying existing epitopes or generating new ones. Thermal and non-thermal treatments may have different effects on epitopes. High pressure processing (HPP), one of the non-thermal food processes, is becoming attractive for the food industry especially its ability to inactivate pathogens in room temperature and retain sensory characteristics and nutritional quality of foods. HPP based on pressure execution in the range of 100-800 MPA can affect secondary, tertiary and quaternary structures of protein and leads to conformational changes. Studies revealed that these changes alter the binding capacity between epitope and IgE. As a result IgE and epitopes cannot attach each other so allergen protein immunoreactivity decrease. This review will focus on the removal of food allergens through HPP including the application of HPP technology for promoting the inhibition of allergen activity and for assisting allergen removal. Keywords: non-thermal treatment, high pressure processing, food allergen Young Scientist and MSc. Student: Merve Eda EKER (< 8 year experience) Topic: WG1 Type of presentation: Oral Presentation ImpARAS COST Action member: Sibel Karakaya, Sedef Nehir El


Portici, Italy

Page 47

P10

Proteomic and B-cell reactivity analysis of water-soluble proteins of oat in patients with celiac disease

1Daniel Sánchez, 2Iva Hoffmanová, 3Stanislava Štěpánová Honzová, 1Věra Hábová, 1Helena

Tlaskalová-Hogenová 1Laboratory of Cellular and Molecular Immunology, Institute of Microbiology v.v.i., Czech Academy of Sciences, Prague, Czech Republic 2Second Department of Internal Medicine, Third Faculty of Medicine, Charles University in Prague and University Hospital Královské Vinohrady, Prague, Czech Republic 3synlab czech Ltd., Prague, Czech Republic Properties and clinical significance of non-prolamin cereal proteins targeted by immune system of celiac patients are still matter of contention. The oat belongs to the nutrients-rich crop, however, it is not generally accepted as the alternative cereal for celiac disease patients. The oat is primary gluten-free and its avenins are considered as non-toxic for a number of celiac disease patients on a gluten-free diet. Despite this fact, the bias of oat toxicity restricted its use for preparation of gluten-free products. Seeming presence of toxic prolamins in oat flour can usually originate from contamination by wheat flour (gluten) during harvesting, and food processing. Nevertheless, non-gluten proteins – wheat alpha-amylase inhibitors, have been recently considered as potent activators of innate immune responses and possible targets of B-cell responses, both in active celiac disease patients and those on a gluten-free diet. Interestingly, the alpha-amylase inhibitor of oat was identified as a target for antibodies in patients suffering from celiac disease. In our study, we focused on the proteome analysis of hydrosoluble proteins extracted from oat kernel from different oat varieties grown in different geographical and agricultural conditions in the Czech Republic. Indeed, we found slightly differences between proteome composition of oat, which depends on the variety and the growing conditions. The analysis of IgA and IgG antibodies reactivity of patients with active celiac disease as well as those on a gluten-free diet revealed the specificity of these antibodies for several oat proteins between 40 – 80 kDa, in contrast to healthy controls. The identification of these proteins is in progress; it will enable us to define immunodominant compounds of oat proteome, and also possible the cross-reactivity of patients’ antibodies. Simultaneously, we will quantify the levels of the specific antibodies using ELISA. Finally, the clinical significance of detected antibodies against oat proteins will be assessed. Acknowledgements: The work was supported by projects 13-14608S of the Czech Science Foundation, TH03010019 of Technology Agency of the Czech Republic and Institutional Research Concept RVO: 61388971


Portici, Italy

Page 48

P11

Technological Processes Effects on the Allergenicity of Food Proteins:

Gaps and Future Needs

Birgül Hızlar, Sedef Nehir El, Sibel Karakaya

Ege University, Food Engineering Department, Nutrition Science, Bornova İzmir, Turkey Food allergy is a specific immune adverse response to digestion, inhalation, and contact with food allergen protein. Recently studies on allergenic reactions to foods and prevention of allergenic reactions have been intensified. The most important reason is that a certain proportion of the population is suffering from adverse health consequences as a result of the consumption of particular foods or food ingredients. Milk, egg, soy, crustacean/shellfish, fish, tree nut, peanut and wheat named major allergenic foods responsible for 90% of allergic reactions in worldwide. The allergenic activity of a food may decrease, remain unchanged, or even increase by food processing. Complex structure of food makes it difficult to predict the effects of processes on the allergen protein. The same food process may have different effects on the Ig-E binding capacity, digestibility and conformational structure of allergen proteins; this results in alteration of allergenicity of proteins. For this reasons, more studies are required both in vitro and in vivo. Although the effects of different treatments (thermal and non-thermal) on the IgE-binding capacity of several allergens have been investigated, less information is available on the effects of processing on clinical reactivity. There are gaps related with studies about the effects of food processing on allergen proteins and current literature data is insufficient. This review summarizes the effect of various technological processes on allergenicity of major food allergens and presents gaps in the literature. Keywords: Food allergen, food processing, allergenicity, epitope Young Scientist and PhD Student: Birgül Hızlar (< 8 year experience) Topic: WG1 Type of presentation: Oral Presentation ImpARAS COST Action member: Sibel Karakaya, Sedef Nehir El


Portici, Italy

Page 49

P12 Activated intestinal epithelial cells conditioned with 2’-Fucosyllactose and CpG ODN might

instruct moDC to drive Th1 differentiation

Veronica Ayechu-Muruzabal1, Atanaska I. Kostadinova1,2, Saskia A. Overbeek1,2, Bernd Stahl2, Johan Garssen1,2, Belinda van’t Land2,3 and Linette E.M. Willemsen1

1Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands. 2Nutricia Research, Utrecht, The Netherlands. 3Department of Paediatric Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands. In the current study, the immunomodulatory capacities of 2’-Fucosyllactose (2’FL), one of the most abundant human milk oligosaccharide, were compared to short chain galacto-and long chain fructo-oligosaccharides 9:1 (GF), which previously showed to promote Th1 and regulatory type immune polarization in the presence of CpG in an in vitro co-culture model. Additionally, the ability of 2’FL-exposed intestinal epithelial cells (IEC) to instruct immature monocyte derived dendritic cell (moDC) function was evaluated. HT-29 cell line, co-cultured with anti-CD3/CD28 activated peripheral blood mononuclear cells (PBMC), was apically exposed to 2’FL, GF or 2’FL/GF mixture (0.25, 0.5, or 1.0 w/v%) either or not combined with CpG ODN M362 (0.5 uM). IEC were washed and co-cultured with moDC. moDC were then used in an allogeneic assay where their capacity to induce naï ve CD4+ T-cell differentiation was evaluated. In presence of CpG, GF as well as 2’FL and 2’FL/GF enhanced IFN-gamma and IL-10 secretion of activated PBMC co-cultured with IEC compared to CpG alone (p<0.05), while IL-13 and IL-5 remained low. IEC-derived galectin-3, TGF-beta1 (both p<0.001), galectin-9 and galectin-4 (both p<0.05) of CpG-exposed cells was further increased by GF, 2’FL and/or 2’FL/GF compared to CpG alone. Only moDC co-cultured with activated IEC conditioned with 2’FL and CpG increased IFN-gamma secretion by CD4+ T-cells (p<0.05). These data imply that, similar to GF, exposure of IEC to 2’FL alone or 2’FL/GF combined with CpG polarize the immune response towards a Th1 and regulatory type. IEC-derived galectins might be involved in the immunomodulatory effects. moDC exposed to 2’FL and CpG-conditioned IEC instructed Th1 differentiation, suggesting that 2’FL can shape the adaptive immune response by affecting IEC function.


Portici, Italy

Page 50

P13

Fast amperometric immunoplatform for ovomucoid traces determination in fresh and baked foods

S. Benedéa, V. Ruiz-Valdepeñas Montielb, E. Povedanob, M. Villalbaa, L. Matac, P. Galán-Maloc, R.M.

Torrente-Rodríguezb, E. Vargasb, A.J. Reviejob, S. Campuzanob, J.M. Pingarrónb aDepartamento de Bioquímica y Biología Molecular I. Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain bDepartamento de Química Analítica, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain cZEULAB, S.L., Bari, 25, 50197 Zaragoza, Spain BACKGROUND: Under the current regulations, eggs should be mentioned in the ingredient list on the food product label. However, traces of egg residues can be unexpectedly present in some food products. Highly sensitive detection methods for egg are strongly demanded for regulatory agencies to protect the allergic population. Our objective was to develop the first electrochemical immunosensor for the highly sensitive determination of egg residues by means of ovomucoid (OM) determination, a very resistant protein against food processing. METHODS: The approach involved the selective capture of sandwich immunocomplexes formed by capture antibody-target protein allergen-HRP-labeled detector antibody onto carboxylic acid-functionalized magnetic beads. The resulting magnetic bioconjugates were captured on the surface of disposable screen-printed carbon electrodes to perform amperometric detection at 0.20 V vs. the silver pseudo-reference electrode in the presence of hydroquinone and H2O2. RESULTS: The electrochemical immunoplatform showed a wide range of linearity (0.3-25 ng/mL), a limit of detection of 0.1 ng/mL, which is much lower than those claimed for commercial ELISA kits, and a great selectivity against other white egg allergenic proteins. The developed immunosensor was successfully employed for the simple and accurate determination of trace amounts of OM in unprocessed (eggs and wheat flour) and baked (bread) food samples.

CONCLUSION: The amperometric immunosensor demonstrates successful applicability to carry out the determination of OM in raw and baked foods using simple and rapid protocols. The high sensitivity achieved makes the immunosensor very attractive for applicability in inspection programs to monitor the effectiveness of cleaning processes from production units in the food industry, thereby protecting both producers and consumers against unintentional ingestion of eggs and eggs-related products.


Portici, Italy

Page 51

P14

In germ-free mice the degree of allergic sensitization is strictly dependent on diet

endotoxin content

Hana Kozakova1, Martin Schwarzer1,2, Dagmar Srutkova1, Petra Hermanova1, Francois Leulier2, Irma Schabussova3

[email protected] 1Laboratory of Gnotobiology, Inst. of Microbiology of the Czech Academy of Sciences, v. v. i., Novy Hradek, Czech Republic, 2Inst. de Génomique Fonctionnelle de Lyon, Ecole Normale Supérieure de Lyon, Lyon, France, 3Inst. of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria Background: Type I allergy gnotobiotic mouse model of was established for assessment the microbiota role in the development of allergic sensitization. However, the role of bacterial residues in diet in this setting is poorly understood. The aim of our study was to compare the effect of bacterial contamination in sterile diet on the level of allergic sensitization to the main component of birch pollen Bet v 1 in germ-free mice (GF). Sterile grain-based diets ST1 and R03 were tested for the level of bacterial contamination and their immune contribution. Methods: We fed GF BALB/c mice by gamma-irradiated mouse nutritionally adequate diets ST1 and R03 at least two generations. LPS concentrations were determinate by the fluorescent PyroGen Recombinant Factor C Assay. Two-m-old GF females were subcutaneously sensitized on days 1, 14, and 28 with Bet v 1 with Alum as adjuvant. The seventh days after last immunization mice were killed. Humoral and cellular immune responses were evaluated by ELISA. Results: ST1 diet contained higher amount of bacterial DNA, approximately ten times more endotoxin, and induced higher, TLR4-dependent, cytokine production in dendritic cells compared to R03 diet. In a GF mouse model of sensitization to the major birch pollen allergen Bet v 1, feeding on ST1 was associated with decreased production of allergen-specific IgE and IgG1 antibodies in sera in comparison to R03. Furthermore, reduced levels of allergen-specific and ConA-induced cytokines IL-4, IL-5 and IL-13 accompanied by increased levels of IFN-γ were detected in splenocytes cultures of these mice.

Conclusion: We showed that not only intestinal microbiota influence the development of allergic responses in experimental settings, but also bacterial fragments in the sterile diet may have a profound effect on level of allergy sensitization under germ-free conditions.



Portici, Italy

Page 52

P15

Celiac disease: a rare cause of hyposideremic anemia in adults

Elena Gologan

"Grigore T. Popa" University of Medicine and Pharmacy, Lasi, Romania Background. Celiac disease (CD) is an immunoalergic disorder triggered by the ingestion of gluten containing food in susceptible persons. CD is a rare cause of anemia in adults. The diagnosis is difficult if the doctor does not consider this possible cause. Method. 55 adults with chronic hyposideremic anemia, no diarrhea and normal findings at upper digestive endoscopy, colonoscopy and small bowel X-ray examination were investigated in the University Hospital “St. Spiridon”, Institute of Gastroenterology and Hepatology, Iasi, Romania between January 2012 and June 2016 reffered by haematologist. Two categories of investigations done were: duodenal biopsies and specific immunological tests for CD (antigliadin, antiendomisium and anti tisular transglutaminase antibodies type IgG and IgA). Results. 55 adults with chronic hyposideremic anemia (age between 18 and 44 years, mean age 23.5) were investigated by duodenal biopsies and specific immunological tests for CD. 29 of them, had both category of investigations conclusive, 11 of the rest had Marsh 1 duodenal atrophy but negative immunological tests and only one had positive immunological tests with normal duodenal histology. A gluten free diet (GFD) was indicated for all the patients as a treatment option considering the pathophysiology of celiac disease involving both innate and adaptative immune response to gluten diet. The therapeutic test (consisting in remission of anemia) was positive in 28 of 29 cases (96.5%) from the first group (the non-responding one was set on corticotherapy with a good response), in only 3 cases from the second group (27.3%) and the single patient of the third group; totally in 32 cases (78%). Conclusions. A hyposideremic anemia in young adults even with no diarrhea and normal endoscopic findings may hide a celiac disease, the confirmation requiring immunology and pathological investigations. If suspected, response at GFD confirms the diagnostic.


Portici, Italy

Page 53

P16 EGG YOLK MODIFIES THE RESPONSE OF INTESTINAL CELLS AND DENDRITIC CELLS IN VITRO

Mónica Martínez-Blanco, Daniel Lozano-Ojalvo, Leticia Pérez-Rodríguez, Elena Molina and Rosina

López-Fandiño Instituto de investigación en Ciencias de la Alimentación (CIAL). CSIC. SPAIN [email protected] Young scientist. ImpARAS COST Action student. Oral presentation. Topic WG2 Background. Eggs present allergens both in egg white (EW) and yolk (EY). EW proteins are considered to exhibit a higher allergenic potential. However, the main components of EY are lipids, that stand among the extrinsic factors that modify the allergenic properties of proteins. We aimed to investigate whether EY acts as a immunostimulating substance in vitro that promotes Th2 responses. Methods. Cocultures of Caco-2 cells and human PBMCs and cocultures of murine bone marrow-derived DCs (BM-DCs) and CD4+ T cells were conducted to examine epithelial and DC functions following stimulation with EW, EY and their mixture. Gene expression and flow cytometry analyses were carried out. Results. IL-33 was significantly upregulated following stimulation of Caco-2 cells with EY and with the mixture of EW:EY, but no changes were found when cells were exposed to EW alone. Upon incubation with BM-DCs, the egg preparations did not change the number of CD11c+ cells or the expression of the costimulatory molecules CD80 and CD86. Similarly, we did not observe changes in the expression of DC genes that modulate T cell differentiation, such as OX40L, Jagged-2 or IL-12p40, or in the secretion of the proinflammatory cytokines TNF-α or IL-6. However, subsequent cultivation of BM-DCs which had been stimulated with EW:EY with CD4+ T cells from mice intraperitoneally sensitized to EW distinctly induced the proliferation of Th2 cells.

Conclusion. EY promoted the release of IL-33 from intestinal epithelial cells, which interacts with DCs triggering Th2 responses on CD4+ T cells. The lack of a phenotypically detectable activation in BM-DCs in vitro cultured with EY was likely due to the absence of a IL-33 stimuli. However, there might be additional mechanisms through which EY favors Th2 polarization during DC presentation of allergens to specific T cells, and thus, influence the sensitization process



Portici, Italy

Page 54

P17

Observation of the level of inflammatory bowel factors and intestinal microbiota composition in the context of cow's milk protein allergy occurrence in the north-eastern

region of Poland

A. Szyc1, L. Markiewicz1, M. Zakrzewska2, E. Romaszko3, B. Wróblewska1 1 Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland 2 Non-Public Health Care Clinic of Allergy “ALLERGICA”, Olsztyn, Poland 3 Non-Public Health Care “ATARAX”, Olsztyn, Poland The World Allergy Organization statistics highlight that 11–26 million people in the European Union and 220–520 million world-wide suffer from food allergies. Patient reports of cow’s milk allergy (CMA) range between 1–17·5%, 1–13·5%, and 1–4% in preschoolers, children 5 to 16 years of age and adults, respectively. Patients suffering from CMA are often recommended to avoid the intake of dairy products. Anti-allergy steroids and antihistamine drugs may be also applied as an medical treatment to relieve symptoms. The course of allergy and used protocols can influence on the structure of intestinal microbiota and the level of inflammatory factors in the gut. The study evaluates the impact of cow’s milk protein allergy (CMPA) and applied methods of treatment on levels of inflammatory bowel factors and intestinal microbiota composition in group of 20 participants (1–14 years) with early-onset CMPA and 20 healthy peers living in the north-eastern region of Poland. Participants were diagnosed of CMPA using for the determination of antibodies/cytokines serum content standardized immunoassay tests and prick tests and /or oral challenge carried out by collaborating medical units. CMPA was confirmed if total IgE was increased and specific sIgE to CMP allergens exceeded <0.7kUA/L. The concentration of inflammatory bowel factors (calprotectin, α-1-antitripsin- α1AT) in feces was determined using ELISA tests. Bacterial quantity and biodiversity was determined using qPCR and PCR-DGGE technic with H’ Shannon-Wiener index calculation. 50% of the study population had implemented an elimination diet that completely excluded the consumption of milk and dairy products. 12% indicated alleviation of symptoms as a result of this procedure. Other people observed a exacerbation of symptoms despite the diet. Participants with CMPA and following an restrictive elimination diet and/or subjected to oral challenge as a diagnostic method are more prone to showing increased content of inflammatory factors: calprotectin (p = 0.041), and α-1-antitrypsin (p = 0.009).in comparison to healthy pers. The level of these markers, although increased, precluded the co-occurrence of other inflammatory bowel diseases. In the group of CMPA, significantly decreased quantity of the Lactobacillus and Bifidobacterium genus (p = 0.047) and increased number of E. coli (p <0.01) and yeast (p = 0.0007) were observed in comparison to healthy peers. Biodiversity of bacteria determined on the basis of H', indicated statistically significant differences in quality profiles between healthy individuals and the CMPA group, mainly within the Lactobacillus population. (H '-2.08 vs. 1.59, p <0.01) and Clostridium leptum (H' 3.26 vs. 2.53, p <0.05). Among mentioned procedures only elimination diet influenced the biodiversity if microbiota however this protocol is essential and its neglect is associated with allergic condition deterioration.


Portici, Italy

Page 55

F01

Autoclaving process to reduce almond allergenicity: impact on the peptidic pattern analyzed by after simulated digestion and ex vivo intestinal epithelium passage.

Simona L. Bavaro a*, Roberta Lupi b,c, Sandra Denery b, Colette Larre b and Linda Monaci a .

aInstitute of Sciences of Food Production, National Research Council of Italy (ISPA-CNR), Via Amendola 122/O, Bari (IT) bINRA, UR 1268 Biopolymers Interactions Assemblies, 44316 Nantes, France cUniversity of Tuscia, Department of Agricultural and Forestry Science, Via S. Camillo de Lellis s.n.c., 01100 Viterbo, Italy

Almond seeds are highly versatile and can be consumed singly as a seed or enter a large number of food products. On the other hands almonds belonging to the tree nuts are also allergens capable of triggering allergic reactions in sensitive individuals and European legislation requires the mandatory labeling whenever introduced into the foods. Due to their inclusion in many different products they can enter the diet of consumers raising some concern in allergic consumers [1]. To reduce this risk, several technological strategies have been devised to modify or remove allergens from foods. In this communication we studied the effect of high temperature and pressure on almond protein pattern and also investigated the resistance of immunogenic peptides produced during simulated digestion. Their capacity, to cross the intestinal epithelium and induce an allergic response in sensitive individuals was also examined. To this aim, raw and processed almond seeds underwent simulated digestion experiments and then were submitted to ex-vivo experiments on Balb/C mice by using Ussing chambers, in order to assess the impact on intestinal epithelium permeability and their passage through the epithelium. Ex-vivo experiments conducted on the jejunum of Balb/C mice showed a significant difference (P <0.05) in the intestinal permeability of the pre-hydrated autoclaved almonds compared to the raw and to the only autoclaved seeds. By contrast, no significant changes in tissue permeability were observed between controls (including or not enzymes) and raw and autoclaved almond digests after 10 minutes. In conclusion, the preliminarily results obtained in this work indicate that the type of treatment, has an impact on the digestion of almonds with an influence on the final protein pattern. Moreover, significant differences in intestinal absorption of digested samples were also highlighted in pre-hydrated autoclaved almonds compared to raw and autoclave products. Additional repetitions are needed to consolidate these preliminary results,further studies will be performed in order identify the immunogenic peptides capable of triggering the allergic response.

1) Costa, J., Mafra, I., Carrapatoso, I., Oliveira, M.B.P.P., Almond Allergens: Molecular Characterization, Detection, and Clinical Relevance. 2012


Portici, Italy

Page 56

F02

Effects of Structural Modifications on Immunogenicity of Food Proteins

Ying Deng1,2, Coen Govers1, Harry J. Wichers1,2

1Food Biobased Research, Wageningen UR, the Netherlands 2Food Chemistry, Wageningen UR, the Netherlands Contact: [email protected]

Protein misfolding occurs occasionally in human metabolism. Such events can trigger the innate immune system, initiate chronic immune activation and improper immune response. These are etiologically involved in e.g. neuro-inflammatory disorders like Alzheimer’s disease and Parkinson disease. Prevention of aggregation and processing-related misfolding, e.g. β-sheet structure induction, is also relevant in pharmaceutical protein preparation and may lead to unwanted immune responses towards such proteins. For instance, glycation of ovalbumin enhanced its uptake by dendritic cells and its immunogenicity compared to the native protein [1, 2]. Glycation of peanut protein is involved in the development of allergy, and aggregation of milk proteins affects their intestinal uptake and shifts this from epithelial uptake to uptake via Peyer’s Patches. The significance of food protein structure modifications as a result of processing is relatively underexposed. Heating, shearing and pH change are important parameters for possible structural changes in proteins, leading to misfolding, hydrophobicity changes, aggregation and glycation as a consequence of Maillard reaction. As a major allergen in cow’s milk, β-lactoglobulin (BLG) is used as an example protein to study the connection between structure change and immune system stimulation. Moreover, other two proteins-lysozyme from hen egg white and thyroglobulin from bovine with distinct-different molecular weight and pI from BLG are also tested to see if the treatment-derived changes is universal. In our model, treatment condition gives a much strong influence than different saccharide regarding the cellular uptake by THP-1 macrophage. Physico-chemical and biochemical properties of protein after processing with different methods differs correspondingly. Particle size, assembled form, hydrophobicity, and receptor binding capability are dominate parameters which determine its endocytosis extent. The result helps in defining the essential factor of biological molecules in APC’s recognition mechanism. Key words: Glycation, Aggregation, THP-1, Immunogenicity, Endocytosis References: 1. Ilchmann, A., et al., Glycation of a food allergen by the Maillard reaction enhances its T-cell

immunogenicity: Role of macrophage scavenger receptor class A type I and II. Journal of Allergy and Clinical Immunology, 2010. 125(1): p. 175-183.

2. Hilmenyuk, T., et al., Effects of glycation of the model food allergen ovalbumin on antigen uptake and presentation by human dendritic cells. Immunology, 2010. 129(3): p. 437-445.



Portici, Italy

Page 57

F03

Characterisation of peanut allergens and possible post-translational modifications (PTMs)

Shu-Hua Liu1, Jelena Mihailovic2, Katarina Smiljanic2, Michelle M. Epstein1,

Tanja Cirkovic Velickovic2,3,4,5

1 Department of Dermatology, Medical University of Vienna, Vienna, Austria 2 Faculty of Chemistry, CoE for Molecular Food Sciences, University of Belgrade, Belgrade, Serbia 3 Faculty of Chemistry, Department of Biochemistry, University of Belgrade, Belgrade, Serbia 4 Ghent University Global Campus, Incheon, Korea 5 Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Background Peanut allergy is the most common type of food allergy causing severe reactions or even fatal anaphylaxis in sensitised individuals. The major peanut allergens are Ara h 1, Ara h 2, Ara h 3, and Ara h 6 which cause the most severe responses. Their molecular properties have been characterised but possible post-translational modifications (PTMs) that might explain their severe allergenicity are not well understood. The goal of this study was to utilize a combination of nanoLC-Mass Spectrometry (MS)/MS methods and PEAKS Studio 8.0 (Bioinformatics Solutions Inc., Ontario, Canada) program to evaluate PTMs in the major peanut allergens. Method Acquired MS data of purified peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6 were analysed and identified via hybridized databases obtained from UniProt (www.uniprot.org). More than 1200 reviewed (Swiss-Prot) and unreviewed (TrEMBL) entries from peanut were combined with common MS contaminants, the Repository of Adventitious Proteins (cRAP), to create a hybridized database. We then focused on Ara h 2 (Conglutin-7) and Ara h 6 (Conglutin) because of their propensity to cause severe anaphylactic reactions. Epitopes found in the Immune Epitope Database (www.iedb.org) were analysed for possible PTMs by matching PEAKS PTM results with mapped positions of epitope sequences. Results We identified 37 proteins from the purified peanut allergens. There were 33 peanut proteins and 4 contaminants originating from human keratin and pig trypsin. Ara h 2 had 242 epitopes, 29 potential PTMs and 4 mutations. Eight of the epitopes had up to 8 possible PTMs. Several relevant PTMs were discovered, including tryptophan oxidation to oxolactone in position 25, sulfonation of N-terminus of cysteine in position 116 and oxidation of methionine in position 50 and 125. Notably, all had either a “NNQRCMCEALQ” or “QQIMENQSD” motif, which are linked to Th2 cytokines and T cell proliferation. We observed 8 epitopes, 9 likely PTMs and no mutations for Ara h 6 and half of the epitopes had possible PTMs and a maximum of 4 PTMs was found on one epitope. Conclusion The analysis of relevant peanut allergens by nanoLC-MS/MS methods and PEAKS Studio 8.0 program revealed several PTMs, which might have important ramifications due to their influence on allergenicity and digestibility resulting from modification properties by trypsin and other food protein enzymes. These data suggest that PTMs on certain peanut epitopes could be involved in the pathogenesis of severe food allergy to peanuts.


Portici, Italy

Page 58

F04

New insights into allergenic relationship between meat and cow’s milk

Marija Perusko1, Danijela Apostolovic2, Tanja Cirkovic Velickovic3,4,5, Marianne van Hage2

1Faculty of Chemistry - Innovation Center d.o.o., Belgrade, Serbia 2Department of Medicine, Solna, Immunology and Allergy Unit, Karolinska Institutet and University Hospital, Stockholm, Sweden 3Center of Excellence for Molecular Food Sciences & Department of Biochemistry, University of Belgrade - Faculty of Chemistry, Belgrade, Serbia 4Ghent University Global Campus, Yeonsu-gu, Incheon, South Korea 5Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium BACKGROUND Red meat allergy is a novel form of food allergy with severe delayed allergic reactions where IgE antibodies are directed against the carbohydrate galactose-α-1,3-galactose (α-Gal) epitope. The α-Gal epitope is abundantly expressed on glycolipids and glycoproteins from non-primate mammals. Many red meat allergic patients report allergic reactions after drinking cow’s milk. We investigated molecular basics of IgE reactivity to milk proteins among red meat allergic patients.

METHODS Milk proteins were tested for the presence of α-Gal epitope and for the efficiency to bind IgE from red meat allergic patients’ sera using methods of immunoblotting and inhibition ELISA. Raw milk and commercially available milk preparations were compared for the IgE binding in inhibition ELISA. Additionally, capacity of milk proteins to activate basophils of red meat allergic patient was assed.

RESULTS Bovine gamma globulin (BGG) reacted positively with anti-α-Gal mAb in immunoblot, while all the other tested proteins (α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin) were negative for the presence of this epitope. BGG was also shown to bind IgE antibodies from a pool of red meat allergic patients, and the binding was almost completely abrogated by thyroglobulin, a glycoprotein rich in α-Gal epitope. Additionally, ELISA experiment showed that BGG exerts a dose-dependent inhibition of red meat allergic patients’ IgE binding to α-Gal. Inhibition ELISA with raw milk and commercially available milk preparations showed that raw milk was a more potent inhibitor of the IgE binding than the commercially available milks. Importantly, BGG and milk successfully activated basophils of red meat allergic patient.

CONCLUSIONS In this study we identified BGG as a glycoprotein carrier of the α-Gal epitope in milk that bound IgE antibodies and furthermore activated basophils of red meat allergic patients. The results highlight the importance of milk as a clinically relevant allergen source among the red meat allergic population.


Portici, Italy

Page 59

F05 Digestomics of walnut and its nsLTPs allergens reveals their ultimate resistance to gastric

digestion Ivana Prodic1*, Pawel Dubiela2, Jelena Mihailovic1, Dragana Stanic-Vucinic1, Katarina Smiljanic1, Karin

Hoffmann-Sommergruber 2, Tanja Cirkovic Velickovic1,3,4

1University of Belgrade – Faculty of Chemistry, Center of Excellence for Molecular Food Sciences & Department of Biochemistry, 2 Dept. of Pathophysiology and Allergy Research, Medical University of Vienna, 3 Ghent University Global Campus, Incheon, Korea, 4 Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium *Corresponding author: E-mail address: [email protected] Background: Sensitization to non-specific lipid transfer protein (ns-LTPs) in plant foods is regarded as a risk factor for generalized allergic reactions. Stability to gastric digestion represents very important parameter of food proteins allergenicity. Usually studies of digestion were carried out on purified proteins, but has never been examined the influence of different food matrices on different allergens. Allergens from the nsLTP family are known to share a characteristic structure which is highly resistant to proteolysis, and therefore, IgE cross-reactivity of nsLTPs needs to be investigated in the environment such as complex food matrix. Objective: The aim of this research project is to reveal how proteins are digested (by Minekus protocol) within the natural food matrix and possible consequences on their allergenicity, with the special focus on ns-LTP. Methods: Pure nsLTPs from walnut were labelled with Alexa 633 and added to whole grain of grounded raw walnuts, incubated with human α-amylase, and pepsin, therefore mimicking the effects of oral and gastric digestion, in total duration of 2h. Proteins extracted from the mixture were analyzed by one-dimensional (1D) and two-dimensional SDS-PAGe, and respective 1D and 2D immunoblots. Results: Most proteins from pepsin digested walnut sample were more resistant to digestion according to 1D SDS PAGE. Pepsin digested raw walnut sample with nsLTP were assessed by 2D PAGE to compare profiles of the digested and control sample (no pepsin added). 2D SDSPAGE of digested and control walnut samples showed almost identical profiles, especially in the context of fluorescently labelled nsLTP allergens. These results demonstrate substantial resistance of nsLTP allergens to gastric digestion since they remained mostly intact after 2 h of gastric (pepsin) digestion.

Conclusion: Further research is needed to be able to grade stability/resistance of selected food allergens to gastric digestion as a consequence of food matrix modulating effects. We propose that certain combinations of foods and allergens could provide additional protection or on the contrary ease the digestion, by comparing trends between control and digested samples and between different digested combinations as well.



Portici, Italy

Page 60

F06

Functional potency testing of allergenic and non-allergenic tropomyosins using RBL cell mediator release as a potential tool in food allergen risk assessment.

Julia Klueber1, Françoise Codreanu-Morel2, Stefanie Randow3, Joana Costa4, Thorsten Graf1, Tanja Scheuermann1, Markus Ollert1,5, Kitty Verhoeckx6,7, Karin Hoffmann-Sommergruber8,

Martine Morisset2, Thomas Holzhauser3, Annette Kuehn1 1. Luxembourg Institute of Health, Department of Infection and Immunity, Esch-sur-Alzette, Luxembourg 2. National Unit of Immunology and Allergology, Centre Hospitalier de Luxembourg, Luxembourg, Luxembourg 3. Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany 4. REQUIMTE-LAQV/Faculdade de Farmácia da Universidade do Porto, Porto, Portugal 5. Department of Dermatology and Allergy Center, Odense Research Center for Anaphylaxis, University of Southern Denmark, Odense, Denmark 6. Department of Dermatology/Allergology, University Medical Centre Utrecht (UMCU), The Netherlands 7. TNO, Zeist, The Netherlands 8. Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria Background Crustaceans are one of the most common elicitors in food allergic reactions. Allergenic tropomyosins can be found in invertebrates, such as crustaceans and other arthropods such as insects. Homologs from vertebrates are known as non-allergenic proteins, an observation which is not well understood. The aim of this study was to compare the allergenic potency of various tropomyosins by mediator release experiments using rat basophil leukemia cells (RBL) and finally, to correlate these data to basophil activation tests (BAT). Method RBL-2H3 cells, having the IgE-binding α-chain of the human FcεR1 receptor, were passively sensitized with serum samples from 22 study participants (20 shrimp-allergic patients, 2 atopic controls) and stimulated with serial dilutions of eight tropomyosins (natural, recombinant) from different species (crustacean, mealworm, chicken) and one extract (crustacean), in order to characterize their allergenic potency. BAT results were available previously (7/20 patients). Results A positive RBL-cell test result was obtained in 5 out of 7 shrimp-allergic patients which had been previously tested in BAT (6/7 positive). For these patients, similar in-vitro allergenic potency of shrimp versus chicken tropomyosins was found in both BAT and RBL assay. One patient was negative in both tests, while another patient was borderline positive in BAT and negative in RBL assay. In addition, 13 shrimp-allergic patients have been tested in further RBL assays. Amongst the positive samples (9/13), shrimp and mealworm tropomyosins induced a comparable positive release at low concentrations, while chicken homologs showed a positive result only at approximately 100 to 1,000-fold higher concentration. Conclusions Tropomyosins from shrimp and chicken vary by their in-vitro allergenic potency by several orders of magnitude. Results from BAT and RBL assay were found to be highly similar. Such a protein pair, crustacean and chicken tropomyosin, might be beneficial as calibrators of functional allergenicity assessment assays on the basis of suitable human specimen.


Portici, Italy

Page 61

F07

Purification of different LTPs and investigation of their behaviour in an in vitro gastrointestinal digestion experiment

Eszter Schall1, Sándor Tömösközi1, María Garrido Arandia2, Araceli Díaz Perales2

1Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest, Hungary 2Centre for Plant Biotechnology and Genomics (UPM-INIA), Campus de Montegancedo, Madrid, Spain Background Lipid transfer proteins (LTP) are widely investigated because many plant-derived LTPs cause respiratory and food allergy. The ability of allergenic activity can be sought not only in the structure but may also affect the other components surrounding the allergenic protein. The aim of the work to produce and purify recombinant allergenic and non-allergenic LTPs and prepare protein extract from plants surrounding their natural matrix. A simulated gastrointestinal digestion process was performed to compare changes in LTPs by digestion. Methods Three different Pichia pastoris yeast clones were used to produce Tri a 14 (wheat allergen), Pru p 3 (peach allergen) and LTP 2 recombinant proteins. LTP 2 has a similar structure than Tr a 14 and Pru p 3 but it does not show any allergenic activity. After the expression, the proteins were purified with gel filtration chromatography and RP-HPLC. To get the wheat and peach protein extracts, wheat bran and peach peel were defatted with acetone and proteins were extracted with phosphate-buffered saline. Finally, an in vitro digestion experiment was performed and SDS-PAGE was used to compare the changes in the recombinant and natural form of allergens and non-allergenic LTP 2. Results During my work, I purified three recombinant LTPs and prepared protein extracts from peach and wheat. Investigation of the molecular size of the proteins after the simulated digestion by gastric and duodenal enzymes, the gel electrophoresis did not show any change. Conclusion The investigated LTPs show resistance to in vitro digestion but there is a need to involve more sensitive methods (mass spectrometry) to understand the processes during digestion.


Portici, Italy

Page 62

F08

Sensitising potential of gluten products via intact, damaged and inflamed skin

Sahar Kazemi1, Jeppe Madura Larsen2, Charlotte Bernhard Madsen2, Michelle Epstein1, Katrine Lindholm Bøgh2

1Department of Dermatology, Medical University of Vienna, Vienna, Austria 2National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark

Background: Allergic reactions to foods have been reported to occur after the first known ingestion. This might indicate that allergic sensitisation may occur through an alternative route other than the gastrointestinal tract. This phenomenon has, in particular, been observed among children with atopic dermatitis and has led to the hypothesis that allergic sensitisation to food might occur through the skin, potentiated by skin barrier defects and inflammation. Furthermore, there is more evidence that the skin route is important for allergic sensitisation as there have been reports where food derived ingredients in personal care products caused an allergy. For example, various cosmetics and personal care products contain wheat proteins and wheat protein derivatives for their emulsifying and foaming properties. The aim of this study was to establish an Altromin Brown Norway rat model of atopic dermatitis and irritant contact dermatitis to determine whether sensitisation to proteins occurs through the skin, especially during active inflammation.

Methods: Wheat tolerant Brown Norway rats were used to compare the sensitising capacity of proteins through inflamed, slightly damaged and intact non-inflamed skin. The rats were treated with titrated concentrations of the vitamin D-analogue, MC903 and Sodium lauryl sulfate (SLS) topically three times per week for 2 weeks. They were then administered a topical solution of MC903 or SLS mixed with either acid-hydrolysed wheat or PBS for five weeks. Sera and skin samples were analysed and the sensitisation was assessed by in-house wheat-specific IgG1 and IgE assays and skin inflammation was evaluated on skin sections stained with Hematoxylin and Eosin, Periodic acid–Schiff and Toluidine blue.

Results & conclusion: In this study, we observed that topical administration of low-dose SLS and MC903 and wheat treatment was unable to induce inflammation or irritation, though there was a minor degree of hyperkeratosis and hyperplasia and low wheat-specific IgE titers. High doses of SLS and MC903 in addition to wheat proteins induced severe skin inflammation and irritation that appeared histologically similar to atopic- and irritant contact dermatitis in patients. In conclusion, topical administration of high-dose SLS and MC903 induces a clinically relevant experimental model of atopic dermatitis and irritant contact dermatitis in rats and demonstrates that inflamed, irritated skin may be important for allergic sensitisation to food allergens.


Portici, Italy

Page 63

F09

See O1 (Angelika Tscheppe).


Portici, Italy

Page 64

Participants First name Name E-mail Country Jihana Achour [email protected] France Veronica Ayechu Muruzabal [email protected] The Netherlands Anne-Sofie Ravn Ballegaard [email protected] Denmark Simona Lucia Bavaro [email protected] Italy Sara Benedé Pérez [email protected] Spain Cecilia Berin [email protected] U.S.A. Roberto Berni Canani [email protected] Italy Marie Bodinier [email protected] France Gregory Bouchaud [email protected] France Laure Castan [email protected] France Anne Constable [email protected] Switzerland Joana Costa [email protected] Portugal René Crevel [email protected] United Kingdom Giovanna de Donato [email protected] Italy LUIGIA DI STASIO [email protected] Italy Araceli Diaz Perales [email protected] Spain Ivan Dimitrov [email protected] Bulgaria Merve Eda [email protected] Turkey Kamel El Mecherfi [email protected] France Michelle Epstein [email protected] Austria Christiane Kruse Fæste [email protected] Norway Pasquale Ferranti [email protected] Italy Giuseppina Garro [email protected] Italy Marija Gavrovic-Jankulovic [email protected] Serbia Éva Mária Gelencsér [email protected] Hungary Elena Gologan [email protected] Romania Richard Goodman [email protected] U.S.A. Lucien Harthoorn [email protected] The Netherlands Stephane Hazebrouck [email protected] France Birgül Hizlar [email protected] Turkey Karin Hoffmann-Sommergruber [email protected] Austria Hulya I. Buyukkestelli [email protected] Turkey Stefania Iametti [email protected] Italy Sahar Kazemi [email protected] Austria Julia Klueber [email protected] Luxembourg Hana Kozakova [email protected] Czech Republic Astrid Kruizinga [email protected] The Netherlands Colette Larre [email protected] France Katrine Lindholm Bogh [email protected] Denmark Shu-Hua Liu [email protected] Austria Daniel Lozano-Ojalvo [email protected] Spain Charlotte Bermhard Madsen [email protected] Denmark Isabel Mafra [email protected] Portugal Gianfranco Mamone [email protected] Italy Mauro Marengo [email protected] Italy Lidia Markiewicz [email protected] Poland Mónica Martínez-Blanco [email protected] Spain Stefania Masci [email protected] Italy Paolo Masi [email protected] Italy Rosalba Mauriello [email protected] Italy Gabriel Mazzucchelli [email protected] Belgium



















































Portici, Italy

Page 65

First name Name E-mail Country Elena Molina [email protected] Spain Laura Molinaro [email protected] Italy Sedef Nehir El [email protected] Turkey Adrijana Nesic [email protected] Serbia Maria Adalgisa Nicolai [email protected] Italy Leticia Pérez Rodríguez [email protected] Spain Marija Perusko [email protected] Serbia Gianluca Picariello [email protected] Italy Fabiana Pizzlongo [email protected] Italy Ivana Prodic [email protected] Serbia Cláudia Raposo [email protected] Portugal Pedro Rodrigues [email protected] Portugal Ana Rodrigues Costa [email protected] Portugal Erwin L Roggen [email protected] Denmark Annalisa Romano [email protected] Italy Daniel Sánchez [email protected] Czech Republic Anette Sauer [email protected] Germany Eszter Schall [email protected] Hungary Denise Schrama [email protected] Portugal Edyta Sienkiewicz-Szłapka [email protected] Poland Joost Smit [email protected] The Netherlands Mark Smits [email protected] Netherlands Samantha Strand [email protected] United Kingdom Thomas Stroheker [email protected] Switzerland Ana Pilar Tobajas [email protected] Spain Olivier Tranquet [email protected] France Angelika Tscheppe [email protected] Austria Paul Turner [email protected] United Kingdom Jolanda van Bilsen [email protected] The Netherlands Marloes van der Wal [email protected] The Netherlands Emilia Vassilopoulou [email protected] Cyprus Kitty Verhoeckx [email protected] The Netherlands Caterina Villa [email protected] Portugal Clélia Villemin [email protected] France Harry Wichers [email protected] The Netherlands Barbara Wróblewska [email protected] Poland Deng Ying [email protected] The Netherlands Petra Zrimšek [email protected] Slovenia

Be aware that the personal information mentioned in this Conference book may be used for COST Action FA1402 (ImpARAS) purposes only



mailto:[email protected];

































Portici, Italy

Page 66

Local Information Conference will be held at:

Dipartimento di Agraria, Università di Napoli, Royal Palace of Portici Via Università 100

80055, Portici (NA), Italy

How to reach the meeting venue:

By car: Highway Napoli-Salerno (direction Reggio Calabria). Exit: Portici-Ercolano. From Airport Capodichino: Take a taxi or bus "Alibus", Direction Naples - Piazza Garibaldi - Central Station then take train as illustrated below From Napoli train Central Station: • Circumvesuviana train line (Napoli Central station floor -1). Direction Sorrento or Direction

Poggiomarino, stop Portici Via Libertà. • Metropolitan train line (Napoli Central station) direction Salerno, stop Portici-Ercolano.

More information: Prof. Pasquale Ferranti, [email protected] tel. +39(0)812539359 Dr. Annalisa Romano, [email protected] tel. +39(0)812539458

Others Portici is at walking distance from the Herculaneum archeological excavations. Pompei and other archeological sites are connected by local train and are quite close. Also Sorrento, Sorrento Coast, Salerno Coast (Amalfi, Positano) are connected directly with local trains every 30 min.



Documents

ImPARAS | - Proceedings · 2018. 12. 19. · Roberto Berni Canani . University of Naples 'Federico II', Italy . Dr.Roberto Berni Canani is internationally recognized as one of the