abstract - oegaioegai.org/oegai/2-pdf/abstractbuch_oegai_2014.pdfbioscience s ehealthcare r 3 m2...

Abstract Book

ÖGAI Annual MeetingSalzburg

2014

Nov. 6 - 8, 2014 Große Universitätsaula am Max Reinhardt Platz im Festspielbezirk

AustrianSociety for Allergology andImmunology

Sponsors & Exhibitor Map 2Welcome & Committee 3General Information 4Programme at a glance 5 - 6Programm: Klinischer Allergietag 7Invited Speakers/ Vortragende 8 - 9Exhibitors 10 - 11Abstracts: Titles and Authors 12 - 15Abstracts: Keynote Lectures 16 - 23Abstracts: Klinischer Allergietag 24 - 29Abstracts: Oral and Poster Sessions 30-146

PartnersandExhibitors

supportingprogrammes

ExhibitorMap

FWF SFB F46: Towards prevention & therapy of allergy(Coordinator: Prof. Rudolf Valenta)

FWF PhD Program W1248: Molecular, Cellular and Clinical Allergology(MCCA) (Coordinator: Prof. Winfried Pickl)

FWF PhD Program W1213: Immunity in Cancer and Allergy(ICA)(Coordinator: Prof. Josef Thalhamer)

FWF PhD Program W1253: Host response in opportunistic infections(HOROS) (Coordinator: Prof. Reinhard Würzner)

FWF PhD Program W1212: Inflammation and Immunity (IAI) (Coordinator: Prof. Maria Sibilia)

Universität Salzburg Schwerpunkt Biowissenschaften und Gesundheit(Coordinator: Prof. Josef Thalhamer)

Austrian Society for Allergology and ImmunologyJahrestagung 2014, Salzburg, November 6-8, 2014

Partners and Exhibitors

Exhibitors

Seite 2

Große Universitätsaula

(Vortragssaal)

FOYER, 1. OG

Stiegenhaus Furtwängler-park

Max-Reinhardt-Platz

Steckdose

ÖGAI Jahrestagung 2014Universität, Foyer 1. ObergeschoßAussteller Standplan

Bencard

Allergy

ALK

Abe

lloEu

bio

&Pr

epot

ech

Stallergenes

eBiosciencean Affymetrix company

9 m2

Meda

Pharma

BD

Biosciences

Bayer

Healthcare

3 m2

MiltenyiBiotec GmbH

ShireHAL

Allergy

Presentation:Alraune

MenzlMT

3 m2

Mabtech

AB4 m2

Biozym


Welcome and Committee

Committee

Dear friends and colleagues,

On behalf of the Local Organising Committee and the Austrian Society for Allergology and Immunology (ÖGAI) I would like to warmly welcome you to Salzburg for the ÖGAI 2014 Annual Meeting. The congress venue, the Große Aula, is one of the most beautiful and prestigious rooms of the University of Salzburg. This is where Wolfgang Amadeus Mozart’s opera Apollo et Hyacinthus (K. 38), composed at the age of eleven, was given its first performance on 13 May 1767.

The meeting’s scientific program covers basic and clinical immunology, infectious diseases, immune therapies, as well as basic and clinical allergology. These topics will be addressed by outstanding keynote speakers during plenary lectures and by young scientists, who will have the opportunity to present and discuss their work in oral and poster presentation sessions. I would like to give a special thanks to all the speakers for their participation and willingness to share their knowledge, results, and ideas. I extend my thanks to all the sponsors and exhibitors for their invaluable contribution and support for the meeting.

I hope this meeting will provide you with a stimulating, enjoyable and inspiring experience in a magical place filled with the sounds of beautiful music.

Fatima Ferreirafor the organizing committee

Organizing Committee

Fatima FERREIRA-BRIZA(President)

Albert DUSCHLJosef THALHAMERMichael WALLNERRichard WEISSJutta HOREJS-HÖCKGabriele GADERMAIER

Organizing Office

Karin MAYR-NESTELBACHERDNA-CONSULT SciencetainmentTorsten KLADEDoris STAMPFER

Scientific CommitteeThomas HAWRANEKParacelsus Medizinische Privatuniversität SalzburgBarbara BOHLEMedizinische Universität WienWinfried PICKLMedizinische Universität WienStefan WÖHRLFloridsdorfer Allergie Zentrum Werner ABERERMedizinische Universität GrazNorbert REIDERMedizinische Universität Innsbruck Doris WILFLINGSEDERMedizinische Universität Innsbruck

Publishing Info Content: Organizing Committee / Organizing OfficeAbstracts: The authors are responsible for the content of the abstractsDesign & Organisation: Karin Mayr-Nestelbacher • DNA-CONSULT SciencetainmentPrint: Huttegger Druck Salzburg Liability is excluded for all printing errors and omissions

ÖGAI • Austrian Society for Allergology and ImmunologyOffice: Veronika MaierhoferInstitut für Immunologie • Lazarettgasse 191090 Vienna • Austria • Tel.: 43(0)660/4977161 Fax: 431-40160-933201 • [email protected]

Seite 3

ContactPublishingInfo

Welcome


General Information

RegistrationThe registration desk will be located at the entrance of the „Große Universitätsaula am Max-Reinhardt-Platz im Festspielbezirk“ during the following hours:Thursday, Nov. 6th. 07:00 - 17:00Friday, Nov. 7th. 08:00 - 17:00Saturday, Nov. 8th 08.00 - 14:00

Contact Registration Desk0043 676 7774565 • Nestelbacher

Opening hours • MeetingThursday, Nov. 6th. 07:00 - 17:00Assembly and Prizes 17:00 - 18:30Welcome Reception 18:30 - 20:00Friday, Nov. 7th. 08:00 - 17:00

Saturday, Nov. 8th 08.00 - 14:00Farewell Gulasch 13:15 - 14:00

Opening hours • ExhibitorsCommercial exhibition will be on display in the Foyer during the following hours:Thursday, Nov. 6th. 11:00 - 17:00Welcome Reception 18:30 - 20:00 Friday, Nov. 7th. 10:30 - 17:00Saturday, Nov. 8th 08.00 - 11:30Farewell Gulasch 13:15 - 14:00

AddressGroße Universitätsaula am Max-Reinhardt-Platzim Festspielbezirk • 5020 Salzburg

ParkingPlease use the Mönchsberggarage for parking. For a reduction of the ticket price please bring the ticket to the registration desk - it will be punched there.

Registration feesThe registration fee for participants includes:• admission to the annual meeting and the exhibition • digital meeting documentation • daily coffee• lunch on Thursday and Friday is not included; lunch for sale• free WLAN• Welcome Reception• Farewell Gulasch on Saturday

Name badgesOur participants are asked to wear their name badges all the time. The badge is essential for admission to all scientific sessions, exhibits and it is the entrance ticket to the welcome reception.

WLANWLAN is provided for free for participants and exhibitors. Please fetch the login data at the registration desk.

Scientific SessionsAll talks will be held in the Aula of the congress-venue.

Oral PresentationsPlease provide the data of your presentation at the info desk until 9.00 am on the day of your presentation. Poster PresentationsThe scientific posters will be presented in two Poster Sessions on Thursday and on Friday. Posters will be on display in the entrance foyer (ground floor) and should be placed on the assigned board on Thursday, Nov. 6th at 11:00 and removed not before Friday, Nov. 7th after 17:00. Please also note the schedule of poster sessions in the programme. Pins will be provided. Your poster number is printed in the list pages 12 - 15.

Digital AbstractbookAll abstracts of this Meeting are already available as a free PDF-Download. You find it at www.oegai.org

Meeting Attending ConfirmationIf you need a confirmation please come to the registration desk.

DFP ConfirmationYou will get the confirmation for DFP points at the registration desk.

General AssemblyWe cordially invite all ÖGAI members to the geral assembly on Thursday, Nov. 6th at 17:00 in the Aula.

Welcome ReceptionWe and the exhibitors invite you to the Welcome Reception on Thursday, Nov. 6th at 18:30 in the Kahn-Foyer on the 1st floor of the Universitätsaula.

Science ChatYoung scientists are invited for a „fireside chat“ with some of our international speakers. Scientists will share with young investigators some of what they have experienced and learned in their life in science: Thursday, Nov. 6th. at 20:00(duration: 1 hour) The meeting point will be announced at the registration desk.

After the Science Chat we offer a

Pub CrawlJoin us and „investigate“ the „Salzburger Beisl-Szene“. Please ask for infos at the registration desk.

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General Information


Programme at a glance

Chair Opening:Winfried PicklVienna

Chairs Session 1:Josef ThalhamerSalzburg

Karin Hoffmann-SommergruberVienna

Chairs Session 2:ReinhardWürznerInnsbruck

DorisWilflingsederInnsbruck

Chairs Session 3:Rudolf Valenta Vienna

GabrieleGadermaier Salzburg

Chairs Poster 1.1:Magnus Wickman SwedenEva Untersmayr-Elsenhuber, Vienna

Chairs Poster 1.2:Hannes Stockinger Vienna Iris Gratz, Salzburg

Chairs Poster 1.3:Sandra Scheiblhofer SalzburgMartin HimlySalzburg

Thursday: November 6th, 2014

07:00 - 08:55 Registration 08:55 - 09:00 Opening 09:00 - 09:30 Opening Keynote Lecture: Christian Münz (Switzerland) “Autophagy-mediated antigen presentation” Invited by FWF PhD Program W1248: Molecular, Cellular and Clinical Allergology (MCCA) (Coordinator: Winfried F. Pickl)

09:30 - 11:00 Session 109:30 - 10:00 Keynote Lecture 1: Magnus Wickman (Sweden) „Childhood-to-adolescence evolution of allergen-specific IgE antibodies“ Invited by FWF PhD Program W1213: Immunity in Cancer and Allergy (ICA) (Coordinator: Josef Thalhamer)

10:00- 11:00 Oral Presentations 1 Thomas EIWEGGER: Engineering hypoallergenic variants of the peanut allergens Ara h 2 and Ara h 6 Heidi A. MÜLLER: The major cow milk allergen, Bos d 5, is able to bind siderophore bound iron and influences T-helper cells according to its binding state Teresa STEMESEDER: Why I? IgE profiling and lifestyle analysis of 501 Austrian school children Eva WOLLMANN: Natural clinical tolerance to peanut in African patients is caused by poor allergenic activity of peanut IgE Victoria GARIB: Differences in molecular sensitization profiles towards animal allergens revealed by allergen micro-array in two generation of patients with respiratory allergy Christina BANNERT: Preventive sublingual immunotherapy in preschool children: first evidence for safety and pro-tolerogenic effects

11:00 - 11:30 Coffee Break (sponsored by MEDA Pharma)

11:30 - 13:00 Session 2 11:30 - 12:00 Keynote Lecture 2: Teunis Geijtenbeek (The Netherlands) ”C-type lectins in infection and immunity” Invited by FWF PhD Program W1253: “Host response in opportunistic infections (HOROS)” (Coordinator: Reinhard Würzner)

12:00 - 13:00 Oral Presentations 2 Gerald WIRNSBERGER: Jagunal-homolog 1 is a critical regulator of neutrophil function in fungal host defense Daniela GALLERANO: Mapping of IgG, IgG-subclass, IgA and IgM reactivity profiles in African and European HIV-infected individuals with an HIV-1 clade C proteome-based array Leonhard X. HEINZ: Identification of a lipid-modifying enzyme as novel regulator of innate immune responses A. WAGNER: Age-related differences of humoral and cellular immune responses to primary Japanese Encephalitis vaccination Barbara KLEIN: The impact of an acute allergic inflammation on neurogenesis and microglia in the hippocampal dentate gyrus

13:00 - 14:00 Lunch break

14:00 - 15:30 Session 3 14:00 - 14:30 Keynote Lecture 3: Jean Bousquet (France) „MeDALL: Mechanisms of the Development of ALLergy“ Invited by FWF SFB F46: Towards prevention and therapy of allergy (Coordinator: Rudolf Valenta)

14:30 - 15:30 Oral Presentations 3 Claudia KITZMÜLLER: Innate responses and antigen-presenting capacity of oral epithelial cells Barbara GEPP: Phosphorylation of CREB is upregulated in keratinocytes after treatment with the major birch pollen allergen, Bet v 1, and birch pollen lipids Raffaela CAMPANA: Epicutaneous application of recombinant Bet v 1 and Bet v 1 derivatives induces allergen-specific IgG and T cell responses Guido A. GUALDONI: Azithromycin inhibits IL-1 beta secretion of innate immune cells by specificly inhibiting the NLRP3 inflammasome Anastasia MESHCHERYAKOVA: Patient-specific immunological imprint within complex ovarian cancer tissues

15:30 - 17:00 1st Poster Session with coffee (sponsored by MEDA Pharma) Poster <34> to <82> Presentations 1.1 Allergenic Molecules and Epidemiology, 1.2 Infection and Immunity, 1.3 Development and Therapy of Allergies and other Immune Diseases

17:00 ÖGAI general assembly, awards ceremony medals, prizes and welcome reception



Chairs Session 6:Jutta Horejs-Höck, Salzburg

Fatima FerreiraSalzburg

Chairs Poster 2.1:Fatima FerreiraSalzburgDirk StrunkSalzburg

Chairs Poster 2.2:Stephen GalliU.S.A. Zsolt SzépfalusiVienna

Chairs Poster 2.3:Claude-Agnès Reynaud, France Gerhard Zlabinger Vienna

Chairs Closing:Ursula Wieder-mann-SchmidtVienna

Michael Wallner Salzburg

Chairs Session 4:Patrizia StoitznerInnsbruck

Richard Weiss Salzburg

Chairs Session 5:Albert DuschlSalzburg

Barbara Bohle Vienna

Friday: November 7th, 2014

08:00 - 09:00 Registration08:55 - 09:00 Opening

09:00 - 10:30 Session 409:00 - 09:30 Keynote Lecture 4: Claude-Agnès Reynaud (France) “Diversity of human memory subsets” Invited by FWF PhD Program W1213: Immunity in Cancer and Allergy (ICA) (Coordinator: Josef Thalhamer)

09:30 - 10:30 Oral Presentations 4 Yoan MACHADO: Carbohydrate coupling as a tool for modulation of the allergenicity and immunological properties of the major birch pollen allergen Bet v 1.0101 Iris K. GRATZ: Controlling the balance between effector and regulatory T cells in peripheral tissues Mariana VÁZQUEZ-STRAUSS: Dual Role of NSAIDs: overcoming melanoma resistance and enhancing cytotoxic potential of innate immune cells Elisabeth GLITZNER: Specific roles for dendritic cell subsets during initiation and progression of psoriasis Daniela ORTNER: The role of Langerhans cells and natural killer cells in cancer immune surveillance

10:30 - 11:00 Coffee Break (sponsored by Sony DADC)

11:00 - 12:30 Session 511:00 - 11:30 Keynote Lecture 5: Stephen Galli (USA) „Testing the toxin hypothesis of allergy: Roles of mast cells and IgE in innate and acquired resistance to venoms” Invited by FWF SFB F46: Towards prevention and therapy of allergy (Coordinator: Rudolf Valenta)

11:30 - 12:30 Oral Presentations 5 Nazanin SAMADI: Characterization of the CD8+ T cell response to allergens Martín R. CANDIA: Does signal one strength influence the phenotype and function of human allergen-specific T lymphocytes? Anna ONDRACEK: Nitrated Beta-Lactoglobulin enhances the anaphylactic response in a murine food allergy model Regina M. SELB: The trimeric complex of recombinant CD23 together with allergen and allergen-specific IgE Marion STEGER: Complement opsonization of A.fumigatus modifies dendritic cell maturation and up-take Kuan-Wei CHEN: Failure of specific immunotherapy (SIT) with house dust mite (HDM) allergen extracts due to lack of HDM allergens capable of inducing specific IgG responses in the therapeutic extracts

12:30 - 13:30 Lunch break

13:30 - 15:00 Session 6 13:30 - 14:00 Keynote Lecture 6: Alberto Mantovani (Italy) “Molecular functions and regulation circuits of inflammatory cytokines” InvitedbyFWFPhDProgramW1212:Inflammationand Immunity (IAI) (Coordinator: Maria Sibilia)

14:00 - 15:00 Oral Presentations 6 Michaela MITTERMEIR: Signaling capacities of the IL-31 receptor complex Madhura MODAK: Bidirectional polarization of T cell function via CD43 L. PANGRAZZI: Memory T cells and plasma cells niches in human bone marrow Alexander ZWIRZITZ: The urokinase receptor as a positive regulator of T cell activation Liisa ANDERSEN: Molecular analysis of MAZR function in CD4+ T cells Julia MARSCHALLINGER: Structural and functional rejuvenation of the aged brain by an approved anti-asthmatic drug

15:00 - 17:00 2nd Poster Session with coffee (sponsored by Sony DADC) Poster <83> to <115> Presentations 2.1 Cellular Immunology, 2.2 Allergens and Effector Mechanisms, 2.3 Cytokines and Signal Transduction 17:00 - 17:30 Closing Keynote Lecture: Maria Yazdanbakhsh (The Netherlands) “Helminths and allergies: the IgE trick“ Invited by FWF PhD Program W1248: Molecular, Cellular and Clinical Allergology (MCCA) (Coordinator: Winfried F. Pickl)17:30 - 18:00 Poster and oral presentations prize awards

Seite 7


Programmüberblick Klinischer Allergietag



KlinischerAllergietagin Deutsch

Vorsitzende:Michael StudnickaSalzburg

Stefan Wöhrl Wien

Vorsitzende:ThomasHawranekSalzburg

PetraZieglmayerWien

Samstag, 8. November 2014

8:00 - 9:00 Registrierung

9:00 - 9:15 Begrüßung

9:15 - 9:50 „Nussknacker-Suite“: Fälle aus dem allergologischen Alltag: Univ. Doz. Dr. Wolfgang Hemmer War es die Hirse? Und war es wirklich der Weizen? - Zwei simple, aber doch lehrreiche rezente Fälle Ass. Dr. med. Isabella Pohl Frau mit Ausschlag Doz. Dr. Stefan Wöhrl Copaxone-Allergie in 1 ¾ Patienten OA Dr. Thomas Hawranek (Penicillin-Allergie – einmal anders)

9:50 - 10:25 Prof. Dr. med. Dr. phil. Johannes Ring (München)

Risiken der Allergiezunahme:

Was wir aus den Ost-West–Studien nach der

deutschen Wiedervereinigung gelernt haben.

10:25 - 11:00 Prof. Dr. med. Knut Brockow (München)

Analgetikaintoleranz: Braucht wirklich jeder

Patient einen Provokationstest?

11:00 - 11:30 Kaffeepause (gesponsert von MEDA Pharma)

11:30 - 12:05 Prof. Dr. med. Margitta Worm (Berlin)

Volkskrankheit Anaphylaxie: Werden unsere

Patienten ausreichend versorgt?

12:05 - 12:40 Privatdoz. Dr. med. Jörg Kleine-Tebbe (Berlin)

Wie evident ist die Rekombinanten-basierte

Diagnostik in der Praxis?

12:40 - 13:15 Univ. Prof. Dr. med. Georg Stingl (Wien)

Biologika – ein modernes Allheilmittel?

„Farewell-Gulasch“ und Bier (gesponsert von Sony DADC)

Alle Sprecher des Klinischen Allergietages sind eingeladen vomSchwerpunkt Biowissenschaften und Gesundheit

der Universität Salzburg (Koordinator: Josef Thalhamer)


Invited Speakers

Jean Bousquet, born in 1946, is Full Professor of Pulmonary Medicine at Montpellier University, France. For 13 years, he was the Director of the Inserm (Institut de la Santé de et de la Recherche Médicale) laboratory “Immunopa-thology of Asthma”. He is Vice-President of the European Union-funded network of excellence GA2LEN (Global Allergy and Asthma European Network). He was granted as coordinator of the large scale Framework Programme 7 (EU) Inte-grated Project MeDALL (Mechanisms of the Development of Allergy). He has chaired GINA (Global Initiative for Asthma), National Heart, Lung and Blood Institute (NIH) and was the founder of ARIA (Allergic Rhinitis and its Impact on Asthma), in collaboration with the World Health Organization (WHO). Jean Bousquet was Chairman of the WHO Global Alliance against Chronic Respira-tory Diseases (GARD 2005-2013). He has been the editor-in-chief of Allergy (second ranking journal in the field, 2003-2009, IF: 6.2). His H factor is 90.

Stephen J. Galli is the chair of the Department of Pathology, the Mary Hewitt Loveless, MD Professor, and a professor of pathology and of microbiology and immunology at Stanford University School of Medicine, and, since 2009, the Co-Director of the Stanford Center for Genomics and Personalized Medicine. His research focuses on the development and function of mast cells and basophils and the development of new animal models for studying the roles of these cells in health and disease. Steve’s work has been recognized by Scientific Achieve-ment Awards from the International Association of Allergy & Clinical Immu-nology (1997) and the World Allergy Organization (2011), the Rous-Whipple Award of the American Society for Investigative Pathology (2014), and by his election to various honorary societies, including the Collegium Internationale Allergologicum, the Institute of Medicine of the US National Academies and the Accademia Nazionale dei Lincei (National Academy of the Lynxes) in Rome.

Teunis B.H. Geijtenbeek is Professor of Cellular and Molecular Immunology at the Academic Medical Center, University of Amsterdam, Amsterdam. He studies the role of C-type lectin receptors in pathogen interactions and how these receptors shape adaptive immune responses. Different pathogens such as HIV-1 and M. tuberculosis subvert the function of C-type lectin receptors to infect the host. C-type lectin receptor also interact with allergens and can be important for aberrant immune responses observed during allergy.

Alberto Mantovani is Professor of Pathology at the University of Milan and Scientific Director of the Istituto Clinico Humanitas. He has contributed to the advancement of knowledge in the field of Immunology formulating new para-digms and identifying new molecules and functions. As of spring 2014 he has had over 60.000 citations and has an H-index of 123 (Scopus).

Claude-Agnès Reynaud is director of research at Institut Necker-Enfants Malades, Paris Descartes Medical school, and head of the group “Develop-ment of the immune system”. She has a long-standing interest in molecular mechanisms of Ig gene repertoire formation, and more recently extended the focus of her studies to the functional diversity of B-cell memory/effector subsets, both in the mouse and in humans.

Christian Münz is professor and co-director of the Institute of Experimental Immunology at the University of Zürich, Switzerland. His lab studies primarily the immune control of the persistent human tumor virus Epstein Barr virus and the involvement of macroautophagy in antigen processing for MHC pre-sentation. After obtaining his PhD from the University of Tübingen, Germany, and his postdoctoral training at the Rockefeller University, New York, USA, where he became assistant professor, he returned to Europe in 2008 to his current position.

Magnus Wickman is a board certified paediatrician and paediatric allergologist with certified training in epidemiology at Karolinska Institutet, Stockholm, Swe-den. His work unites population based medicine/epidemiology, clinical medi-cine in particular food allergy, molecular allergology, and population genetics. Since1994 until 2014 he has been PI for the Swedish population based birth cohort BAMSE which started in 1994 and enrolled 4000 new born babies.Seite 8

Vortragende am Klinischen Allergietag, 8.11.2014


Invited Speakers


Invited Speakers / Vortragende

Maria Yazdanbakhsh is Head of Parasitology at Leiden University Medical Cen-ter, The Netherlands, and leads the section “Leiden Immunoparasitology Group” that works on the immune response to parasites, focusing on immune modula-tion. Her speciality has been on helminth infections and TH2 responses.

Prof. Dr. Knut Brockow, Dermatologe und Allergologe aus der Klinik und Poliklinik für Dermatologie und Venerologie am Biederstein, Technische Univer-sität München, interessiert an Arzneimittelreaktionen, Anaphylaxie und Mast-zellerkrankungen wie Mastozytose, bis 2013 über 6 Jahre Präsident der Abtei-lung “Arzneimittelallergie” der EAACI und beteiligt an vielen Positionspapieren und Empfehlungen zur Arzneimittelüberempfindlichkeit.

Priv.-Doz. Dr. Jörg Kleine-Tebbe, seit 2002 mit Dr. Gerald Hanf u. Dr. Ju-liane Ackermann im Allergie- und Asthma-Zentrum Westend (AAZW) in Berlin, Gemeinschaftspraxis für Allergologie, Dermatologie, Innere Medizin und Um-weltmedizin. Besondere Interessen betreffen die Diagnostik und Therapie von Soforttyp-Allergien und IgE-assoziierten Erkrankungen.

Prof. Dr. med. Dr. phil. Johannes Ring ist Director emeritus Haut und Al-lergieklinik Biederstein, TU München, die er von 1995 – 2014 leitete. Vorstand-smitglied und Präsident verschiedener allergologischer und dermatologischer Gesellschaften, u. a. Deutsche Gesellschaft für Allergologie und klinische Im-munologie (DGAKI), European Society for Dermatological Research (ESDR), Collegium Internationale Allergologicum (CIA), European Academy of Derma-tology and Venerology (EADV).Seine Forschungsschwerpunkte umfassen: Entzündliche Hauterkrankungen, Neurodermitis, Arzneimittel- und Nahrungsmittel-Allergie, Immuntherapie, Al-lergie und Umwelt. Zur Zeit ist Prof. Dr. med. Dr. phil. Johannes Ring Chief-Editor des Journal European Academy Dermatology Venerology (JEADV).

Univ. Prof. Dr. med. Stingl has contributed greatly to our understanding of the skin as an immune organ under physiologic and pathologic (e.g. psoria-sis, atopic dermatitis, syphilis, HIV-infection) conditions. His major accomplish-ments include the discovery of Langerhans cells as immunocytes and of the in-digenous T cell population of rodent skin. In the recent years, his research group has elucidated the mode of action of several immunomodulatory drugs. He is a member of the board of several scientific societies and organizations, advisory panels, scientific journals and committees, including honorary membership of the two leading societies of dermatological research, i.e. The Society for Inves-tigative Dermatology and the European Society for Dermatological Research. He was recently elected a Foreign Associate of the Institute of Medicine of the US National Academies and holds a membership in the Austrian and German National Academy of Sciences.

Prof. Dr. med. Margitta Worm wurde 2003 auf die Universitätsprofessur mit dem Schwerpunkt Immunmodulation allergischer Erkrankungen an der Chari-téUniversitätsmedizin Berlin berufen. Aktuell leitet sie die Hochschulambulanz der Klinik für Dermatologie, Venerologie und Allergologie und die klinisch ex-perimentelle Allergologie in Immunologie des Allergie-Centrums der Charité. Zu ihren Forschungsschwerpunkten gehören die atopische Dermatitis, Mech-anismen der Anaphylaxie, Nahrungsmittelallergie, Immunmodulation aller-gischer Erkrankungen und die Immunologie von B-Zellen. 2006 hat sie das deutschsprachige Anaphylaxie-Register initiiert, wo 106 allergologische Zentren in Deutschland, Österreich und der Schweiz angeschlossen sind. Prof. Dr. med. Worm ist Vorstandsmitglied der Deutschen Gesellschaft für klinische Immunolo-gie und Allergologie sowie Mitglied in einschlägigen Fachgesellschaften (BDG, DDG, DGAKI, EAACI) und führt regelmäßig Reviewertätigkeiten für nationale und internationale Journale durch.

Seite 9

Vortragende der “Nussknacker-Suite”:

OA Dr. Thomas Hawranek • Univ.-Doz. Dr. Wolfgang HemmerAss. Dr. med. Isabelle Pohl • Priv.- Doz. Mag. Dr. Stefan Wöhrl

Austrian Society for Allergology and ImmunologyJahrestagung 2014, Salzburg, November 6-8, 2014 Exhibitors

ALK-AbellóAllergie-Service GmbHBäckermühlweg 59, 4030 Linz, Austriawww.alk.net/atwww.allergie-plattform.at

Als führender Anbieter von hochqua-litativen Produkten zur spezifischen Diagnose bzw. kausalen Immunthera-pie der Typ-I Allergien und als kom-petenter Partner von ÄrztInnen und ApothekerInnen ist es unser zentrales Anliegen, die Versorgung allergie-kranker Menschen durch exzellente Produkte, Ausbildung und Öffentlich-keitsarbeit zu verbessern und so zu mehr Gesundheit und Lebensqualität beizutragen. Als kompetenter Partner allergologisch tätiger Fachärzte in Ös-terreich entwickeln und vertreiben wir von ALK-Abelló Allergentherapeutika zur ursächlichen Behandlung von All-ergischer Rhinitis und Konjunktivitis, allergischem Asthma bronchiale und allergischen Reaktionen auf Bienen- und Wespenstiche. Neben den sub-kutanen Darreichungsformen stehen sublinguale Präparate zur Verfügung. Im Jänner 2007 führten wir als erstes Unternehmen weltweit die kausale Tablettenimmuntherapie gegen eine Gräserpollenallergie ein. Die Produkt-palette umfasst weiterhin Allergie-diagnostika zur Pricktestung, Intra-kutantestung sowie zur nasalen und konjunktivalen Provokation. Wir von ALK-Abelló Österreich betreiben in Ko-operation mit der ALK-Abelló Zentrale in Dänemark und mit ALK Deutschland klinische Forschung auf höchstem Ni-veau, um die Therapie allergiekranker Menschen zu verbessern. Wir forschen ebenfalls im Bereich rekombinanter Allergene und Adjunvantien, um die Möglichkeiten der kausalen Allergiet-herapie zu verbreitern.

Bayer Austria Ges.m.b.H.Herbststraße 6-10, 1160 Wienwww. bayerhealthcare.at

Bayer HealthCare erforscht, entwi-ckelt, produziert und vertreibt innova-tive Produkte, um die Gesundheit von Mensch und Tier weltweit zu verbes-sern.Zu seinen Divisionen zählen neben Pharmaceuticals, Consumer Care, Animal Health und Medical Care.Bayer HealthCare ist mit seinen zahl-reichen klinischen Studien in den un-terschiedlichsten Indikationsgebieten ein stark vertretenes forschendes Pharmaunternehmen in Österreich. Darüber hinaus ist das Unternehmen in Österreich mit Marketing und Ver-trieb befasst.

Mehr unter www.bayerhealthcare.at

BD Life Sciences – BiosciencesBecton Dickinson Austria GmbHConcorde Business Park 1/E/1/72320 Schwechat, ÖsterreichTel.: +43 1 [email protected]

Biosciences bedient den gesamten Bereich der biologischen und medi-zinischen Forschung und bietet die größte Auswahl an innovativen Gerä-ten und Reagenzien für die zelluläre Analyse an.Ein breites Spektrum an Analyzern und Sortern für die Durchflusszytome-trie, verbunden mit kompetenter Be-ratung und Service, ermöglichen die passgenaue Lösung.für Ihr Labor. Unser Produktangebot und unsere Kompetenz machen uns zum interes-santen Partner für Forschungs- und Routinelabors, für die Industrie sowie für die wachsende Zahl von bio- und gentechnologischen Einrichtungen.

Für die klinische Durchflusszytome-trie stehen gebrauchsfertige, CE-IVD zertifizierte Lösungen im Bereich der Immunphänotypisierung von Lympho-zyten, Leukämie- und Lymphomdia-gnostik, Stammzellenumeration sowie für die Restzellbestimmung in Blutprodukten zur VerfügungUnsere Lösungen zur Automatisierung der Prozesse ermöglichen eine effizi-entere Abarbeitung, die hochwertigen Reagenzien erlauben eine präzisere Diagnostik für bessere klinische Ent-scheidungen.

Bencard Allergie GmbHLerchenfelder Straße 13/6/42,1070 Wien, www.bencard.at

Bencard Allergie arbeitet seit 80 Jah-ren an Lösungen für die spezifischen Immuntherapie. Es ist uns gelungen, mit neuen Testverfahren und Pro-dukten die Qualität der Versorgung allergischer Patienten entscheidend zu verbessern. 1999 konnten wir unser Ziel mit der Einführung unseres Pro-duktes POLLINEX® Quattro realisie-ren. 2004 wurden wir dafür mit dem MMW-Arzneimittelpreis ausgezeichnet, eine Auszeichnung für die spezifische Immuntherapie und speziell für POL-LINEX® Quattro. Zahlreiche klinische Studien unterstreichen die Effektivität dieses Konzeptes. Wir sind stolz, in enger Zusammenarbeit mit unseren Kunden die Zukunft der spezifischen Immuntherapie aktiv zu gestalten

Biozym Scientific GmbHBiozym Scientific GmbH, Postfach,D-31833 Hessisch Oldendorf, Deutsch-

land, Tel:+49 5152-9020,[email protected] Biotech Trading GmbH, Wehli-strasse 27b/1/9, A-1200 Wien, Öster-reich, Tel:+43 1 3340156-0www.biozym.com, [email protected]

Seit unserer Firmengründung in Ha-meln 1986 verstehen wir uns als ei-genständige und leistungsorientierte Vertriebs- und Serviceorganisation für unseren Zielmarkt „ Life Science „ in Deutschland, Österreich und weiteren europäischen Ländern. Unser Produktportfolio umfasst hoch-leistungsfähige Gerätesysteme, Con-sumables und Feinchemikalien. Darü-ber hinaus werden speziell angepasste Servicedienstleistungen angeboten. Unsere molekularbiologischen Schwerpunkte sehen wir in Anwen-dungsbereichen PCR-Technologie, Next-Generation Sequencing und in vitro Transkription, sowie Identifizie-rung und Aufreinigung molekularbio-logischer Substanzen.In Österreich bieten wir exklusiv für den Bereich Immunologie und Zellbi-ologie Produkte der Firma Biolegend an. Der Vertrieb unserer Produkte er-folgt über ein hoch motiviertes Au-ßendienstteam aus erfahrenen Mo-lekularbiologen und Biochemikern in Zusammenarbeit mit produktgrup-penorientierten Produktmanagern.

eBioscience, an Affymetrix companyCampus Vienna Biocenter 2, 1030 Wien, Austria, Tel.: +43 1 796 40 40 305,[email protected]

eBioscience, an Affymetrix company ist ein führender Anbieter im Bereich der Durchflusszytometrie und bie-tet eine breite Palette an Antikörpern und Fluorochromen für die Forschung im Bereich Life Science. Ein weiterer Schwerpunkt im Portfolio von eBi-oscience sind klassische, sowie Bead-based Multiplex Immunoassays für die Quantifizierung von Zytokinen, Wachstumsfaktoren und weiteren lös-lichen Proteinen. Neben Assays für die Quantifizierung von Proteinen werden auch Reagenzien für die Detektion von RNA angeboten.Das Unternehmen entwickelt jedes Jahr über 800 innovative Produkte für die Forschung in Immunologie, Onko-logie, Zellbiologie und Stammzellenbi-ologie.

EubioTel.: 0043-1-895 01 45, Schwender-gasse 17, 1150 Vienna, Austriawww.eubio.at, [email protected]


Seite 10

Austrian Society for Allergology and ImmunologyJahrestagung 2014, Salzburg, November 6-8, 2014 Exhibitors

We are a privately owned company in Vienna, Austria founded by Andreas Köck in 1995. Our main goal is to pro-vide the Austrian research community with state-of-the-art Life Science re-agents. At the moment our database and online ordering system contains ~ 500.000 products.Andreas Köck combines more than 30 years of research experience at the Medical University of Vienna, in the USA and the Netherlands with several years of experience in marketing and business development.We are the distributing company for the best original manufacturers of life science products.

HAL AllergyHandelsgesellschaft mbHJohnstrasse 4, A-1150 WienTel:+43 (0) 1 / 985 98 80E-Mail: [email protected]: www.hal-allergy.com

HAL Allergy ist einer der führenden All-ergenhersteller in Europa mit mehr als 50 Jahren Erfahrung auf diesem Ge-biet..Im Jahr 1959 begann HAL Aller-gy in einem kleinen Labor im Zentrum von Haarlem in den Niederlanden. Mit dem Ausbau des Unternehmens zu einem mittelständischen Pharmaun-ternehmen wurden die Aktivitäten in ein größeres Gebäude in Haarlem ver-legt.Im Jahr 2009 unternahm das mitt-lerweile 50 Jahre alte Unternehmen einen weiteren Schritt und zog um.Die Architektur des Gebäudes veränderte sich damit von einem Haus aus den fünfziger und sechziger Jahren in eine hochmoderne Produktionsstätte:In Bezug auf die Technologie wurden modernste Anlagen installiert und das Labor ist jetzt mit fortschrittlicher La-borausrüstung ausgestattet.Hierdurch kann HAL Allergy seinen zukünftigen Wachstumsanforderungen gerecht werden und seine Mission weiter ver-folgen, die Lebensqualität von Aller-gikern mit Hilfe biopharmazeutischer Produkte für die ursächliche Behand-lung zu verbessern.

Mabtech ABBox 1233, 131 28 Nacka Strand, Swe-den, Tel: +46 8 716 27 00,[email protected]

Mabtech is a Swedish Biotech company that produces high quality monoclonal antibodies and kits for ELISA and ELI-Spot. We have led the development of the FluoroSpot assay, which enables simultaneous analysis of secreted ana-lytes at the single cell level making it particularly useful when studying po-lyfunctional T cells or when the supply

of cells is limited. It can also be used to separately define and enumerate B cells secreting antibodies of different isotypes in the same well.

Meda Pharma GmbhGuglgasse 15, 1110 Wien,[email protected], www.meda.at

Meda ist ein global agierendes, füh-rendes Spezialitäten Pharmaunter-nehmen, das kostengünstige und zugleich am medizinischen Bedarf ori-entierte Arzneimittel anbietet. Durch Einführungen, Weiterentwicklung und Zukauf innovativer Produkte im Be-reich Allergie und Dermatologie ver-sucht Meda ständig den Bedürfnissen der Patienten gerecht zu werden.Meda ist eine Niederlassung der Meda AB., Schweden. Meda ist seit 2005 in Österreich erfolgreich tätig und setzt seine Produktschwerpunkte neben den Bereichen Allergologie und Dermato-logie vor allem bei Atemwegserkran-kungen, Schmerztherapie, Prävention und Behandlung von Knochenbruch-krankheiten/Osteoporose, Mineral-stoffwechsel sowie im Fachgebiet der Gynäkologie. Im rezeptfreien Apothekenbereich bietet Meda bewährte Präparate wie CB12, Naloc, Endwarts, Ce-Limo plus, Kamillosan, Travelgum sowie Magnofit direkt an.Mehr Informationen erhalten Sie un-ter www.meda.at

Ferdinand MenzlMedizintechnik GmbHA-1220 Wien, Donaufelderstrasse 199, Tel: 01 / 2558960-0www.menzl.com, [email protected]

Ferdinand Menzl Medizintechnik wurde vor über 25 Jahren als auf Reparaturen und Wartung von medizintechnischen Geräten spezialisierter Gewerbebe-trieb in Wien gegründet. Im Laufe der Jahre wurden Handelsbetriebe an-gegliedert, die sich mit dem Vertrieb medizintechnischer Geräte zur The-rapie und Diagnose von Atemwegs-erkrankungen befassen sowie auf die Vermeidung und Sanierung von Aller-genen spezialisiert sind. Regelmässige Teilnahmen an Fortbildungsveranstal-tungen und Kongressen halten die Mitarbeiter auf dem neuesten Stand und machen sie zu Spezialisten in ih-ren Tätigkeitsbereichen.

Miltenyi Biotec GmbHFriedrich-Ebert-Strasse 68, 51429 Bergisch Gladbach, Germany,Phone: [email protected]

Miltenyi Biotec is a global provider of products and services that advance

biomedical research and cellular the-rapy. Since 1989, we have developed innovative andreliable technologies for scientists and clinicians around the world. Our integrated portfolio of tools covers techniques of sample preparati-on, cellseparation, flow cytometry, cell culture, molecular analysis, and pre-clinical imaging. Our expertise covers research areas like immunology, stem cell biology, neuroscience, and cancer, and clinical research areas that inclu-dehematology, graft engineering, as well as apheresis. Today, we are more than 1,300 employees in 22 countries – all dedicated to empowering scien-tific discovery and advancing cellular therapy.

Shire • Medikamente gegen seltene ErkrankungenKärntner Ring 5-7/7. StockA-1010 Wien, Tel. 01-205 [email protected], www.shire.at

Seltene Erkrankungen sind häufig ge-netisch bedingt, mit zunehmenden Komplikationen verbunden und en-den manchmal auch tödlich. Für viele dieser Erkrankungen gibt es derzeit keine oder nur eingeschränkte Thera-piemöglichkeiten. Shire gelang es bis heute Medikamente gegen vier sel-tene genetische Erkrankungen zu ent-wickeln und auf den Markt zu bringen: Morbus Hunter, Morbus Fabry, Morbus Gaucher und hereditäres Angioödem. Die Entwicklung von Therapien für di-ese seltenen Erkrankungen ist das Er-gebnis langjähriger Forschungsarbeit. Shire setzt sich mit großem Engage-ment für die Erforschung und Entwick-lung weiterer Therapien im Bereich der seltenen Erkrankungen ein.Shire. We enable people with life-altering conditions to lead better lives.

STALLERGENESÖsterreich GmbH Mariahilfer Straße 103/1/26, 1060 Wien, Austria,Tel: +43 1 533 74 [email protected]

Stallergenes bietet einen ganzheit-lichen Ansatz bei allergiebedingten Er-krankungen und stellt Allergologen ein umfangreiches Produktspektrum von der Diagnostik bis zur spezifischen Immuntherapie zur Verfügung.Mit seinem einzigartigen Know-how hat sich das Unternehmen unermüd-lich der Innovation verschrieben. Stallergenes leistet damit einen wert-vollen Beitrag zur Entwicklung der spezifischen Immuntherapie und bie-tet Allergiepatienten immer leistungs-fähigere Lösungen.


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Seite 12

Oral Presentation 1

<1> Engineering hypoallergenic variants of the peanut allergens Ara h 2 and Ara h 6Thomas EIWEGGER (1)*, Merima BUBLIN(2)*, Klara SCHMIDTHALER(1), Maria KOSTADINOVA(2), Christian RADAUER(2), Christine HAFNER(3), Zsolt SZÉPFALUSI(1), Eva-Maria VARGA(4), Heimo BREITENEDER(2) *contributed equally

<2> The major cow milk allergen, Bos d 5, is able to bind siderophore bound iron and influences T-helper cells according to its binding stateHeidi A. Müller(1) , Luis. F. Pacios(2), Cristina Gomez-Casado(2), Hofstetter G.(1), Georg A. Roth(4), Josef Singer(5), Araceli Diaz-Perales(2), Erika Jensen-Jarolim(1,5), Roth-Walter F.(1)

<3> Why I? IgE profiling and lifestyle analysis of 501 Austrian school childrenTeresa STEMESEDER(1), Eva KLINGLMAYR(1), Bettina SCHWEIDLER(1), Lisa LUEFTENEGGER(2,3), Stephanie MOSER(4), Roland LANG(3), Martin HIMLY(1), Gertie J. OOSTINGH(2), Arne BATHKE(5), Joerg ZUMBACH(4), Thomas HAWRANEK(3), Gabriele GADERMAIER(1)

<4> Natural clinical tolerance to peanut in African patients is caused by poor allergenic activity of peanut IgEEva WOLLMANN(1), Carl HAMSTEN Carl(2,3), Elopy SIBANDA(4), Mary OCHOME(1), Margarethe FOCKE-TEJKL(1), Anna ASARNOJ(2,5), Annika ÖNELL(6), Gunnar LILJA(7), Daniela GALLERANO(1), Christian LUPINEK(1), Theresa THALHAMER(8), Richard WEISS(8), Josef THALHAMER(8), Magnus WICKMAN(7,9), Rudolf VALENTA(1), Marianne VAN HAGE(2)

<5> Differences in molecular sensitization profiles towards animal allergens revealed by allergen micro-array in two generation of patients with respiratory allergyVictoria GARIB, Eva WOLLMANN, Rudolf VALENTA

<6> Preventive sublingual immuno-therapy in preschool children: first evidence for safety and pro-tolerogenic effectsChristina BANNERT(1), Zsolt SZÉPFALUSI(1), Leila RONCERAY(1), Elisabeth MAYER(1), Michaela HASSLER(1), Eva WISSMANN(1), Eleonora DEHLINK(1), Saskia GRUBER(1), Alexandra GRAF(2), Christian LUPINEK(3), Rudolf VALENTA(3), Thomas EIWEGGER(1), Radvan URBANEK(1)

Oral Presentation 2

<7> Jagunal-homolog 1 is a critical regulator of neutrophil function in fungal host defenseGerald WIRNSBERGER(1), Florian ZWOLANEK(2), Johannes STADLMANN(1,3), Luigi TORTOLA(1), Shang Wan LIU(1), Thomas PERLOT(1), Päivi JAERVINNEN(4), Gerhard DUERNBERGER(1,3), Ivona KOZIERADZKI(1), Renu SARAO(1), Alba DE MARTINO(5), Kaan BOZTUG(6,7), Karl MECHTLER(1,3), Karl KUCHLER(2), Christoph KLEIN(4), Ulrich ELLING(1) and Josef M. PENNINGER(1)

<8> Mapping of IgG, IgG-subclass, IgA and IgM reactivity profiles in African and European HIV-infected individuals with an HIV-1 clade C proteome-based arrayDaniela GALLERANO(1), Portia NDLOVU(2), Ian MAKUPE(2), Margarete FOCKE-TEJKL(1), Kerstin FAULAND(3), Eva WOLLMANN(1), Elisabeth PUCHHAMMER-STÖCKL(4), Walter KELLER(3), Elopy N. SIBANDA(5) and Rudolf VALENTA(1)

<9> Identification of a lipid-modifying enzyme as novel regulator of innate immune responsesLeonhard X. HEINZ(1), Christoph L. BAUMANN(1), Marielle KÖBERLIN(1), Berend SNIJDER(1), Manuela BRUCKNER(1), Riem GAWISH(1), Omar SHARIF(1), Kumaran KANDASAMY(1), Jacques COLINGES(1), Keiryn L. BENNETT(1), Astrid FAUSTER(1), Guanghou SHUI(2), Sylvia KNAPP(1), Markus R. WENK(2) and Giulio SUPERTI-FURGA(1)

<10> Age-related differences of humoral and cellular immune responses to primary Japanese Encephalitis vaccinationA. WAGNER(1), E. GARNER-SPITZER(1), J. JASINSKA(1), M. PAULKE-KORINEK(1), M. HOFER(1), K. STIASNY(2), F. X. HEINZ(2), H. KOLLARITSCH(1), U. WIEDERMANN(1, 3)

<11> The impact of an acute allergic in-flammation on neurogenesis and micro-glia in the hippocampal dentate gyrusBarbara KLEIN(1,2), Richard WEISS(3), Sebastien COUILLARD-DESPRES(2,4), Josef THALHAMER(3), Ludwig AIGNER(1,2)

Oral Presentation 3

<12> Innate responses and antigen-presenting capacity of oral epithelial cellsClaudia KITZMÜLLER(1), Martina SCHUSCHNIG(1), and Barbara BOHLE(1)

<13> Phosphorylation of CREB is upregulated in keratinocytes after treatment with the major birch pollen allergen, Bet v 1, and birch pollen lipidsBarbara GEPP(1), Nina LENGGER(1), Christian RADAUER(1), Florian GRUBER(2), Michael MILDNER(2), Heimo BREITENEDER(1)

<14> Epicutaneous application of recombinant Bet v 1 and Bet v 1 derivatives induces allergen-specific IgG and T cell responses RAFFAELA CAMPANA(1), Katharina MORITZ(2), Angela NEUBAUER(3), Hans HUBER(3), Rainer HENNING(3), Katharina BLATT(4), Gregor HOERMANN(5), Tess M. BRODIE(6), Alexandra KAIDER(7), Peter VALENT(4), Federica SALLUSTO(6), Stefan WÖHRL(2), Rudolf VALENTA(1,*)

<15> Azithromycin inhibits IL-1 beta secretion of innate immune cells by specificly inhibiting the NLRP3 inflammasomeGuido A. GUALDONI(1), Tilman R. LINGSCHEID(2), Peter STEINBERGER(1), Gerhard J. ZLABINGER (1)

<16> Patient-specific immunological imprint within complex ovarian cancer tissuesAnastasia MESHCHERYAKOVA(1), Erika BAJNA(1), Susanne WÖHRER(1) ElfiSCHARF(1),MartinSVOBODA(1),Erika JENSEN-JAROLIM(1,2), Georg HEINZE(3), Peter BIRNER(4), Diana MECHTCHERIAKOVA(1)

Oral Presentation 4

<17> Carbohydrate coupling as a tool for modulation of the allergenicity and immunological properties of the major birch pollen allergen Bet v 1.0101MACHADO Yoan(1), MAYR Melissa(1), THALHAMER Theresa(1), HÖPFLINGER Veronika(1), SCHEIBLHOFER Sandra(1), THALHAMER Josef(1), WEISS Richard(1)

<18> Controlling the balance between effector and regulatory T cells in peripheral tissuesDouglas F. PINHEIRO(1), Megan M. MAURANO(1), Abul K. ABBAS(3), Iris K. GRATZ(1,2,4)

<19> Dual Role of NSAIDs: overcoming melanoma resistance and enhancing cytotoxic potential of innate immune cellsMariana VÁZQUEZ-STRAUSS, Georg STINGL

<20> Specific roles for dendritic cell subsets during initiation and progression of psoriasisElisabeth GLITZNER(1), Ana KOROSEC(1), Patrick M. BRUNNER(2), Barbara DROBITS(1), Nicole AMBERG(1), Helia B. SCHONTHALER(3), Tamara KOPP(2), Erwin F. WAGNER(3), Georg STINGL(2), Martin HOLCMANN(1), Maria (1)

<21> The role of Langerhans cells and natural killer cells in cancer immune surveillanceDaniela ORTNER(1), Christoph TRIPP(1), Nicole AMBERG(2), Maria (2), Björn E. CLAUSEN(3) and Patrizia STOITZNER(1)

Oral Presentation 5

<22> Characterization of the CD8+ T cell response to allergensNazanin SAMADI, Claudia KITZMÜLLER , Barbara BOHLE, Rene GEYEREGGER, Beatrice JAHN-SCHMID

<23> Does signal one strength influence the phenotype and function of human allergen-specific T lymphocytes?Martín R. CANDIA(1), Peter TAUBER(1), Alina NEUNKIRCHNER(1,2), Doris TRAPIN(1), Sandra ROSSKOPF(1), Peter STEINBERGER(1) and Winfried F. PICKL(1,2).

<24> Nitrated Beta-Lactoglobulin enhances the anaphylactic response in a murine food allergy modelSusanne C. DIESNER(1,2), Cornelia SCHULTZ(1), Chloé ACKAERT(3), Gertie J. OOSTINGH(4), Anna ONDRACEK(1), Caroline STREMNITZER(1), Denise HEIDEN(1), Josef SINGER(1), Franziska ROTH-WALTER(1,5), Judit FAZEKAS(1), Thomas EIWEGGER(2), Zsolt SZÉPFALUSI(2), Erika JENSEN-JAROLIM(1,5), Ernst-Hanno STUTZ(3), Albert DUSCHL(3) and Eva UNTERSMAYR(1)

<25> The trimeric complex of recombinant CD23 together with allergen and allergen-specific IgERegina M. SELB(1), Julia ECKL-DORNA(1), Christian LUPINEK(2), Birgit LINHART(2), Andrea TEUFELBERGER(3), Walter KELLER(3), Kenneth H. ROUX(4), Rudolf VALENTA(2) and Verena NIEDERBERGER(1)

Seite 13


Seite 13

<26> Complement opsonization of A.fumigatus modifies dendritic cell maturation and up-takeMarion STEGER(1), Emina JUKIC(1), Wilfried POSCH(1), Cornelia LASS-FLÖRL(1), Hubertus HAAS(2), Doris WILFLINGSEDER(1)

<27> Failure of specific immunotherapy (SIT) with house dust mite (HDM) allergen extracts due to lack of HDM allergens capable of inducing specific IgG responses in the therapeutic extractsKuan-Wei CHEN (1), René ZIEGLMAYER (2), Petra ZIEGLMAYER (2), Patrick LEMELL (2), Friedrich HORAK (2), Rudolf VALENTA (1), Susanne VRTALA (1)

Oral Presentation 6

<28> Signaling capacities of the IL-31 receptor complexMichaela MITTERMEIR, Elisabeth MAIER, Stefanie ESS, Albert DUSCHL, Jutta HOREJS-HOECK

<29> Bidirectional polarization of T cell function via CD43Madhura MODAK, Petra CEJKA, Petra WAIDHOFER-SÖLLNER, Sabrina JUTZ, Otto MAJDIC, Peter STEINBERGER, Gerhard ZLABINGER, Johannes STÖCKL

<30> Memory T cells and plasma cells niches in human bone marrowL. PANGRAZZI, B. JENEWEIN, J. LAIR, M. KRISMER, B. WEINBERGER, B. GRUBECK-LOEBENSTEIN

<31> The urokinase receptor as a positive regulator of T cell activationAlexander ZWIRZITZ(1), Karin PFISTERER(1), Vladimir LEKSA(2) and Hannes STOCKINGER(1)

<32> Molecular analysis of MAZR function in CD4+ T cellsLiisa ANDERSEN, Alexandra SCHEBESTA, Alexandra GÜLICH, Shinya SAKAGUCHI and Wilfried ELLMEIER

<33> Structural and functional rejuvenation of the aged brain by an approved anti-asthmatic drugJulia MARSCHALLINGER(1,2), Barbara KLEIN(1,2), Iris Schäffner (3,4), Sébastien COUILLARD-DESPRES(2,5), Claudia SCHMUCKERMAIR(6), D. Chichung Lie (3,4), Nicolas SINGEWALD (6), Ludwig AIGNER(1,2)

Poster Session 1.1

<34> Identification of linear and conformational epitopes of bovine alpha -lactalbuminYuan SHUILIN, Li XIN, Chen HONGBING, Gao JINYAN, Wu ZHIHUA, Yang ANSHU, Tong PING(1,2)

<35> In-print of the environment on the molecular sensitization profile towards pollen allergens revealed by allergen micro-array.Viktoriya GARIB (1), Eva WOLLMANN (1), Gulnara DJAMBEKOVA (2), Rudolf VALENTA

<36> Characterization of recombinant gamma-gliadin protein and its evaluation for diagnosis of wheat hypersensitivitiesBharani Srinivasan1, Margarete Focke-Tejkl1, 2, Milena Weber1, Sandra Pahr1, Alexandra Baar1, Michael Hertl3 , Raja Atreya4, Markus.F.Neurath4, Harald Vogelsang5, Wolf-Dietrich Huber6, Rudolf Valenta1, 2

<37> Construction of a phage display library from Escherichia coli to study IgE-reactive bacterial antigens.Sheron DZORO, Irene MITTERMANN, Rudolf VALENTA

<38> The immune response against the timothy grass pollen allergen Phl p 5 in non-allergic humans depending on different environmentsAlmedina ISAKOVIC, Christoph HILLEBRAND, Theresa THALHAMER, Sandra SCHEIBLHOFER, Josef THALHAMER, Richard WEISS

<39> Immunogenicity of the major peach allergen Pru p 3: Does protein conformation, physico-chemical properties and stability matter?Stephanie EICHHORN(1), Isabel PABLOS(1), Serge VERSTEEG(2), Laurian ZUIDMEER-JONGEJAN(2), Ronald VAN REE(2), Fatima FERREIRA(1), Gabriele GADERMAIER(1)

<40> ImmunoCAP cellulose displays cross-reactive carbohydrate epitopes and can cause false-positive test results in patients with anti-CCD IgE antibodiesWolfgang HEMMER(1), Stefan WÖHRL(1), Felix WANTKE(1), Friedrich ALTMANN(2)

<41> Investigation of the structural and immunological behavior of Art v 3 upon thermal treatmentSabrina WILDNER(1,2), Lorenz STOCK(2,3), Adriano MARI(4), Hanno STUTZ(2,3) and Gabriele GADERMAIER(1,2)

<42> Characterization and IgE epitope mapping of Tri a 37, a serological marker for severe wheat food allergySandra PAHR(1,2), Regina SELB(3), Claudia CONSTANTIN(1), Milena WEBER(1), Margit FOCKE-TEIJKL(1), Gerhard HOFER(4), Andela DORDIC(4), Walter KELLER(4), Nikolaos G. PAPADOPOULOS(5), Stavroula GIAVI(5), Mika MÄKELÄ(6), Anna PELKONEN(6), Verena NIEDERBERGER(3), Susanne VRTALA(1,2), Rudolf VALENTA(1)

<43> Analysis of interactions of the major birch pollen allergen Bet v 1 with naturally occurring and synthetic ligandsLorenz AGLAS(1), Claudia ASAM(1), Stefanie GILLES(2), Claudia TRAIDL-HOFFMANN(2), Fatima FERREIRA(1), Michael WALLNER(1)

<44> Structural and immunological study of allergenic defensin-like proteins from mugwort, ragweed and feverfewIsabel PABLOS(1), Stephanie EICHHORN(1), Peter BRIZA(1), Christof EBNER(2), Naveen ARORA(3), Stefan VIETHS(4), Gabriele GADERMAIER(1), Fatima FERREIRA(1)

<45> Production and characterization of recombinant Per a 1, a major allergen of Periplaneta americanaBianca KASTNER(1), Stephanie EICHHORN(1), Isabel PABLOS(1), Sabrina WILDNER(1), Ines FORSTENLEHNER(2), Stefan VIETHS(3), Naveen ARORA(4), Gabriele GADERMAIER(1), Fatima FERREIRA(1)

<46> Renaissance of a well-known contact-allergenTamar KINACIYAN

<47> Recognition of birch pollen aller-gen Bet v 1 by endolysosomal proteasesRegina FREIER and Hans BRANDSTETTER

<48> Investigation of indoor allergen exposure and IgE sensitizationBettina SCHWEIDLER(1), Teresa STEMESEDER(1), Eva KLINGLMAYR(1), Lisa LÜFTENEGGER(2,3), Stephanie MOSER(4), Roland LANG(2), Martin HIMLY(1), Gertie J. OOSTINGH(3), Arne BATHKE(5), Jörg ZUMBACH(4), Thomas HAWRANEK(2), Gabriele GADERMAIER(1)

<49> Proteomic characterization of the Blomia tropicalis allergen Blo t 2 Juan R URREGO(1), João PONTE(1), Michael WALLNER(2), Carina PINHEIRO(1), Neuza ALCANTâRA-NEVES(1), Peter BRIZA(2), Fátima FERREIRA(2)

<50> Stability and micro-heterogeneity of Cor a 14, the 2S albumin from hazelnutSabine PFEIFER(1), Pawel DUBIELA(1), Merima BUBLIN(1), Karin HUMMEL(2), Christine HAFNER(1,3), Karin HOFFMANN-SOMMERGRUBER(1)

Poster Session 1.2

<51> Biomarkers of Infallation in juvenile idiopathic arthritis (JIA)Juergen BRUNNER(1), Thomas GINER(1), Guenter WEISS(2), Dietmar FUCHS(3)

<52> The Toll-like receptor 4 agonist MRP8/14 protein complex (Calprotectin) in autoinflammation: Potential biomarker in chronic nonbacterial osteomyelitis – a case reportJürgen BRUNNER(1)

<53> Interleukin 1 blockade with cana-kinumab for Hyper IGD syndrome (HIDS)Jürgen BRUNNER(1), Elisabeth BINDER(1), Daniela KARALL(1), Johannes ZSCHOCKE(2), Christine FAUTH(2)

<54 Complement analysis in patients with Juvenile Idiopathic Arthritis (WIELISA, SC5b9-ELISA)T. GINER(1), L. HACKL(1), R. WÜRZNER(2), J. BRUNNER(1)

<55> Functional analyses of total complement activity in an ELISA-based kit which replaces haemolytic assays: a decade of experience and future perspectivesReinhard WÜRZNER(1), Tom Eirik MOLLNES(2), Francesco TEDESCO(3), Peter GARRED(4), Lennart TRUEDSSON(5), Malcolm W. TURNER(6), Moh R. DAHA(7), Robert B. SIM(8)

<56> Reactive oxygen species mediated regulation of linker for activation of T cells in autoimmunityFlorian FORSTER(1), Clara MARQUINA(1), Bernhard MALISSEN(2), Rikard HOLMDAHL(1)

<57> Serum markers of antiphospholipid syndrome detection and their diagnostic significance in the pediatric practiceAnna BATYROVA, Irina KONDRAKHINA, Olga KOZHEVNIKOVA, Anna GEVORKYAN

<58> Complement-opsonized HIV overcomes SAMHD1 restriction in DCsWilfried POSCH(1), Marion STEGER(1), Ulla KNACKMUSS(1), Felipe DIAZ-GRIFFERO(2), Cornelia LASS-FLÖRL(1), Arnaud MORIS(3), Oliver T. KEPPLER(4), Doris WILFLINGSEDER(1)

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<59> Triggering CD11b/c on DCs promotes immediate Th17 polarization during acute HIV-1 infectionWilfried POSCH(1), Andrea SCHROLL(2), Asier SAEZ-CIRION(3), Gianfranco PANCINO (3), Teunis GEIJTENBEEK(4), Cornelia LASS-FLÖRL(1), Günter WEISS(2), Doris WILFLINGSEDER(1)

<60> Evaluation of vaccine responsiveness against tick-borne encephalitis (TBE) and hepatitis A in allergic patients – differences to healthy controls?Erika GARNER-SPITZER(1), Michael HOFER(1), Reinhard JARISCH(2), Tamar KINACIYAN(3), Michael KUNDI(4), Ursula WIEDERMANN(1)

<61> SOD1 Protects From Type I Interferon-Driven Oxidative Damage in Viral HepatitisAnannya BHATTACHARYA*(1), Ahmed N. HEGAZY*(2,3,4), Nikolaus DEIGENDESCH**(5), Lindsay KOSACK**(1), Jovana CUPOVIC(6), Richard K. KANDASAMY(1), Andrea HILDEBRANDT(1), Doron MERKLER(7), Anja A. KÜHL(8), Christopher SCHLIEHE(1), Isabel PANSE(2,3), Bojan VILAGOS(1), Kseniya KHAMINA(1), Isabelle ARNOLD(4), Lukas FLATZ(6), Haifeng C. XU(9), Philipp A. LANG(9), Alan ADEREM(10), Giulio SUPERTI-FURGA(1), Jacques COLINGE(1), Burkhard LUDEWIG(6), Max LÖHNING(2,3),†, Andreas BERGTHALER(1),†

<62> Comparison of the immunogenicity and reactogenicity after subcutaneous (s.c.) or intra muscular (i.m.) vaccination with a tick-borne encephalitis vaccineStefan HOPF(1), Erika GARNER-SPITZER(1), Michael HOFER(1), Ingrid DEMEL(1), Michael KUNDI(2), Otfried KISTNER(3) and Ursula WIEDERMANN(1)

<63> STAT1 in myeloid cells is required to limit early replication and persistence of murine cytomegalovirus in vivoMario BIAGGIO(1), Caroline LASSNIG(1,2), Rita ROM(1), Astrid KRMPOTIC(3), Stipan JONJIĆ(3),BirgitSTROBL(1)andMathiasMÜLLER(1,2)

Poster Session 1.3

<64> T cell- independent boost of Memory IgE responses by B cell epitopesMeena NARAYANAN(1), Margarete FOCKE-TEJKL(2), Rudolf VALENTA(1,2), Birgit LINHART(1)

<65> House dust mite extract is a robust source of enzymes that impair epithelial barrier functionK. Oida (1,2), L. Einhorn (1,3), S. Vrtala (4,5), Y. Resch (4), L. Panakova (6), F. Roth-Walter (1), J. Fazekas (1,3), H. Matsuda (2), A. Tanaka (7), E. Jensen-Jarolim (1,3)

<66> Rise of total IgE levels upon omalizumab treatment is not due to activation of IgE+ memory B cellsJulia ECKL-DORNA (1), Renate FRÖSCHL (1), Christian LUPINEK (1), Renata KISS (1), Katharina MARTH (1), Raffaela CAMPANA (1), Katharina BLATT (1), Peter VALENT (1), Regina M. SELB (1), Andrea MAYER (1), Katharina GANGL (1), Irene STEINER (1), Philippe GEVAERT (2), Rudolf VALENTA (1), Verena NIEDERBERGER (1)

<67> Hypoxia contributes to melanoma phenotype change by affecting the response to vemurafenibDaniela PUCCIARELLI(1), Nina LENGGER(1),MartinaTAKÁČOVÁ(2),Heimo BREITENEDER(1), Silvia PASTOREKOVA(2), Christine HAFNER(1,3)

<68> Novel approach for combined treatment of birch pollen and associated food allergiesHeidi HOFER(1), Claudia ASAM(1), Michael HAUSER(1), Peter BRIZA(1), Christof EBNER(2), Fatima FERREIRA(1), Michael WALLNER(1)

<69> Allergen specific antibody responses in non-atopic humans. Implications for antigen uptake and dendritic cell polarizationChristoph HILLEBRAND, Almedina ISAKOVIC, Josef THALHAMER, Richard WEISS

<70> Monitoring the induction of blocking antibodies during birch pollen AITSara HUBER(1), Heidi HOFER(1), Roland LANG(2), Thomas HAWRANEK(2), Michèle RAUBER(3)(4), Fatima FERREIRA(1), Michael WALLNER(1)

<71> Codon harmonization enhances heterologous expression of Bet v 1 in E. coliMaria Alejandra PARIGIANI(1), Yoan MACHADOf(1), Sabrina WILDNER(1), Peter BRIZA(1), Lorenz AGLAS(1), Martin WOLF(1), Fátima FERREIRA (1), Michael WALLNER(1)

<72> Role of allergen-based component-divided diagnostics in choice of allergy therapy approach for children with pollinosisM.A SNOVSKAYA., L.S. NAMAZOVA-BARANOVA, O.V. KOZEVNIKOVA, A.K. GEVORKYAN

<73> Immunity towards neo-antigen in skin grafts: Mouse models for skin gene-therapySophie KITZMUELLER(1,2), Douglas PINHEIRO(1), Thomas KOCHER(2), Johann BAUER(2), Iris GRATZ(1,2,3)

<74> The immune response to laminin 5 in skin gene therapy in epidermolysis bullosaAna I. SANCHO(1,2), Maria M. KLICZNIK(1), Johann W. BAUER(2), Iris K. GRATZ(1,2,3)

<75> Neuraminidase-coated, allergen-loaded microparticles are a safe and efficient novel food allergy treatment optionSusanne C. DIESNER(1,2), Cornelia SCHULTZ(1), Xue-Yan WANG(3), Katharina BEITL(1), Franziska ROTH-WALTER(1,4), Denise HEIDEN(1), Anna ONDRACEK(1), Josef SINGER(1), Judit FAZEKAS(1), Caroline STREMNITZER(1), Thomas EIWEGGER(2), Zsolt SZÉPFALUSI(2), Isabella PALI-SCHÖLL(1,4), Erika JENSEN-JAROLIM(1,4), Franz GABOR(3) and Eva UNTERSMAYR(1)

<76> Development of a humanized mouse model to study the immune response to skin gene therapy in epidermolysis bullosa (EB)Maria M KLICZNIK(1), Eva MURAUER(2), Iris K GRATZ(1,2,3)

<77> Novel meta-analysis of gene expression in mouse allergic asthmaBerislav BOSNJAK*(1), Michela RIBA*(2), Jose Manuel GARCIA-MANTEIGA*(2), Michelle M. EPSTEIN*(1), Elia STUPKA*(2)

<78> 5- and 10-year post-relapse survival of pediatric and young adult sarcoma patients after a dendritic cell-based cancer immunotherapy.Friedrich ERHART(1), Alexander M. DOHNAL(1), Thomas FELZMANN(2), Philipp FUNOVICS(3), Leo KAGER(4,5), Volker WITT(4,5), Susanna LANG(6), Carmen VISUS(2)

<79> Can225IgG, a new canine anti-EGFR antibodyJudit FAZEKAS(1,2), Josef SINGER(1,2), Wei WANG(3), Marlene WEICHSELBAUMER(1), Alexander MADER(4), Miroslawa MATZ(1), Willibald STEINFELLNER(4), Yuri SOBANOV(1), Diana MECHTCHERIAKOVA(1), Michael WILLMANN(5), Edzard SPILLNER(6), Renate KUNERT(4), Erika JENSEN-JAROLIM(2,1).

<80> Neonatal colonization with recombinant lactic acid bacteria for prevention of poly-sensitizationPriya SARATE(1), Stefan HEINL(2), Hana KOZÁKOVA(3), Reingard GRABHERR(2), Irma SCHABUSSOVA(1), Ursula WIEDERMANN(1)

<81> Immune alterations after thymectomy in early childhoodManuela ZLAMY(1), Reinhold WÜRZNER(2), Walther PARSON(3), Heidemarie HOLZMANN(4), Verena JELLER(1), Martina PRELOG(5)

<82> Towards a non-allergenic peptide mix containing the T cell epitopes of relevant house dust mite allergensHuey-Jy HUANG(1), Mirela CURIN(1), Srinita BANERJEE(1), Kuan-Wei CHEN(1), Tetiana GARMATIUK(1), Yvonne RESCH(1), Raffaela CAMPANA(1), Margarete FOCKE-TEJKL(1), Rudolf VALENTA(1), Susanne VRTALA(1,3)

Poster Session 2.1

<83> The fibroblast network serves as guiding structure for directed monocyte migration in 3D synovial tissue culturesRuth BYRNE(1), Karolina VON DALWIGK(1), Günter STEINER1, Johannes HOLINKA2, Reinhard WINDHAGER2, Josef S. SMOLEN1, Hans KIENER1, Clemens SCHEINECKER(1)

<84> Selective activation of cannabinoid receptor 2 primes eosinophils for enhanced migratory responsivenessRobert FREI, Gerald PARZMAIR, Silke SCHRANZ, Akos HEINEMANN, Eva STURM

<85> Dendritic cells use IgE-mediated antigen cross-presentation to generate cytotoxic T cells in response to low dose soluble antigenBarbara PLATZER (1), Kutlu G. ELPEK (2), Viviana CREMASCO (2), Kristi BAKER (3), Cornelia SCHULTZ (1, 4), Eleonora DEHLINK (1, 5) Kai-Ting C. SHADE (6), Robert M. ANTHONY (6), Richard S. BLUMBERG (3), Shannon J. TURLEY (2) and Edda FIEBIGER (1)

<86> HAX1 deletion impairs BCR-internalization, leading to delayed apoptosis of mature B cellsGertrude ACHATZ-STRAUSSBERGER

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Seite 15

<87> EGFR is required in liver macrophages for IL-1-induced IL-6 production and hepatocellular carcinoma formationHanane LANAYA(1)*, Anuradha NATARAJAN(1)*, Karin KOMPOSCH(1)*, Liang LI(2), Nicole AMBERG(1), Stefanie K. WCULEK(1), Martina HAMMER(1), Rainer ZENZ(1), Markus PECK-RADOSAVLJEVIC(3), Wolfgang SIEGHART(3), Michael TRAUNER(3), Hongyang WANG(2), Maria SIBILIA(1)

<88> Inducible Neo-Antigen Expression in Steady State. Langerhans Cells mediates Immunological ToleranceHelen STRANDT(1), Veronika HÖPFLINGER(1), Peter HAMMERL(1), Daniel H. KAPLAN (2), Dagmar WIRTH(3), Josef THALHAMER(1), Angelika STOECKLINGER(1)

<89> Elucidating the effects of costimulation blockade on allo-reactive cytotoxic T cellsSilke SCHROM(1), Sarah AHMADI(1), Barbara DILLINGER(1), Wolfgang HOLTER(1,2), Alexander DOHNAL(1), Andreas HEITGER(1,2)

<90> The role of RGS16 in immune regulationBastien A. HUBER(1), Angela HALFMANN(1), Klara SOUKUP(1), Alexander M. DOHNAL(1)

<91> Migratory skin dendritic cells are involved in CD8+ T cell responses against the melanoma-associated antigen gp100David G. MAIRHOFER(1), Vincent FLACHER(2), Christoph H. TRIPP(1), Björn E. CLAUSEN(3), Suzie CHEN(4), Patrizia STOITZNER(1)

<92> T cells from Multiple Myeloma patients exhibit features of T-cell exhaustionClaudia ZELLE-RIESER(1), Shanmugapriya THANGAVADIVEL(1), Wolfgang WILLENBACHER (2), Rainer BIEDERMANN(3), Richard GREIL(1,4) and Karin JÖHRER(1)

<93> The role of Langerin-positive skin dendritic cells in chemical skin carcinogenesisChristoph H. TRIPP(1), Daniela ORTNER(1), Sandrine DUBRAC(1), David E. SCHLÖGL(1), Kerstin KOMENDA(1), Björn E. CLAUSEN(2) and Patrizia STOITZNER(1)

<94> Lowering T cell activation strength in vivo polarizes T cell differentiation towards a regulatory phenotypeDouglas F. PINHEIRO(1), Sophie SKITZMUELLER(1,3), Maria Magdalena KLICZNIK(1,3), Gertrude ACHATZ(1), Abul K.ABBAS(2), Iris GRATZ(1,3,4).

Poster Session 2.2

<95> Multi-sensitization to hymenoptera venomsKarine MARAFIGO DE AMICIS (1,2,6); Alexandra SAYURI WATANABE (2,3); Clovis Eduardo GALVAO (2,4); Daniele DANELLA FIGO (1,2); José Roberto APARECIDO DOS SANTOS-PINTO (2,5); Mario Sergio PALMA (2,5); Fábio FERNANDES MORATO CASTRO (1,2,3); Jorge KALIL (1,2,3,4); Fatima FERREIRA–BRIZA (6); Gabriele GADERMAIER (6); Keity SOUZA SANTOS (1,2);

<96> Beside Der p 1 and Der p 2, also other house dust mite allergens have high allergenic activityYvonne RESCH(1), Mira ŠILAR(2), Peter KOPAČ(2),MihaelaZIDARN(2),Kuan-Wei CHEN(1), Wayne THOMAS(3), Peter KOROŠEC(2), Mitja KOŠNIK(2), Rudolf VALENTA(1) and Susanne VRTALA(1,4)

<97> Expression of recombinant canine, feline and equine FcepsilonRI-alpha chains for improved IgE profiling in these speciesLukas EINHORN(1,2), Judit FAZEKAS(1,2), Martina MUHR(1,2), Alexandra SCHOOS(1,2), Lucia PANAKOVA(3), Krisztina MANZANO-SZALAI(1), Kumiko OIDA(1,4), Josef SINGER(2), Erika JENSEN-JAROLIM(1,2)

<98> High density IgE recognition of the major grass pollen allergen Phl p 1 revealed with single chain IgE antibody fragments obtained by combinatorial cloningChristoph MADRITSCH(1), Elisabeth GADERMAIER(1), Uwe RODER(2), Christian LUPINEK(1), Rudolf VALENTA(1) and Sabine FLICKER(1) <99> Generation of new cassette vectors for the targeted presentation of human-relevant antigens (allergens) in humanized mouse modelsBernhard KRATZER(1), Alina NEUNKIRCHNER(1,2), Winfried F. PICKL(1,2)

<100> Identification and characterization of natural adjuvants in birch pollenDagmar WERNER, Birgit NAGL, Barbara BOHLE

<101> The role of neutrophils in IgE-mediated allergyDominika POLAK, Birgit NAGL, Claudia KITZMÜLLER, Barbara BOHLE

<102> Cor a 8 displays reduced stability upon heat and digestion treatmentPawel DUBIELA(1), Sabine PFEIFER(1), Merima BUBLIN( 1, 2), Christine HAFNER(1), Karin HOFFMANN-SOMMERGRUBER(1)

Poster Session 2.3

<103> HIV opsonization modulates DC signalingUlla KNACKMUSS(1), Wilfried POSCH(1), Marion STEGER(1), Michael BLATZER(1), Kristian PFALLER(2), Cornelia LASS-FLÖRL(1), Doris WILFLINGSEDER(1)

<104> Interleukin-2/anti-interleukin-2 antibody complexes expand T regulatory cells and protect against allergen-induced airway hyperreactivityAlina NEUNKIRCHNER(1,2), Daniela WOJTA-STREMAYR(1,2), Klaus G. SCHMETTERER(2), Lukas MAGER(2) Victoria REICHL(1,2), Edward ROSLONIEC(3), Ronald NAUMANN(4), Gerhard DEKAN(5), Beatrice JAHN-SCHMID(6), Barbara BOHLE(1,6) and Winfried F. PICKL(1,2)

<105> Resveratrol intake enhances indolamine-2,3-dioxygenase activity in humansGuido A. GUALDONI(1), Katharina A. MAYER(1), Johanna M. GOSTNER(2), Dietmar FUCHS(3), Gerhard J. ZLABINGER(1)

<106> The CD58-CD2 axis is the primary costimulatory pathway in human CD28 negative CD8 T cellsJudith LEITNER(1), Dietmar HERNDLER-BRANDSTÄTTER(2), Beatrix GRUBECK-LOEBENSTEIN(2), Gerhard ZLABINGER(1), Peter STEINBERGER(1)

<107> Basophils from house dust mite-allergics show an altered TLR profile from birch pollen- or non-allergic individualsMarkus STEINER(1), Thomas HAWRANEK(2), Michael SCHNEIDER(3), Andrea HARRER(2), Jutta HOREJS-HOECK(1), Fatima FERREIRA(1), Martin HIMLY(1)

<108> The soluble cytoplasmic tail of CD45 (ct-CD45) induces quiescent anergy in human T cellsAlexander PUCK, Maria SEYERL, Stefan HOPF, Otto MAJDIC, GerhardZLABINGER, Judith LEITNER, Peter STEINBERGER, Johannes STÖCKL

<109> The anti-malarial drugs chloroquine and hydroxy-chloroquine show suppressive potency on purified human CD4+ T-cellsRalf SCHMIDT(1), Franz RATZINGER(1), Sabrina JUTZ(2), Peter STEINBERGER(2), Winfried F. PICKL(2) and Klaus G. SCHMETTERER(1)

<110> STAT3 governs granzyme B and IL-10 production in CD4+ T-cellsKlaus G. SCHMETTERER(1,2), Alina NEUNKIRCHNER(1), Daniela WOJTA-STREMAYR(1) Judith LEITNER(1), Peter STEINBERGER(1), and Winfried F. PICKL(1) <111> Endotoxin contaminations in recombinant proteins activate primary human CD1c+ dendritic cellsHarald SCHWARZ, Maria SCHMITTNER, Albert DUSCHL, Jutta HOREJS-HOECK

<112> Generation of a multi-parameter reporter T cell lineSabrina JUTZ(1), Sandra ROSSKOPF(1), Judith LEITNER(1), Klaus SCHMETTERER(2), Katharina GRABMEIER-PFISTERHAMMER(3), Peter STEINBERGER(1)

<113> Azithromycin suppresses CD4+ T-cell activation by direct modulation of mTOR activityFranz RATZINGER(1), Helmuth HASLACHER(1), Wolfgang POEPPL(2), Gregor HOERMANN(1), Johannes J KOVARIK(3), Sabrina JUTZ(4), Peter STEINBERGER(4), Heinz BURGMANN(2), Wolfgang F PICKL(4) and Klaus G SCHMETTERER(1)

<114> The impact of NOD1 on IL-10 signalingTheresa NEUPER, Albert DUSCHL and Jutta HOREJS-HOECK

<115> The tryptophan metabolite picolinic acid suppresses proliferation and metabolic activity of CD4+ T-cellsJohanna PRODINGER(1), Julia LOACKER(1), Ralf SCHMIDT(1) Franz RATZINGER(1), Sabrina JUTZ(2), Peter STEINBERGER(2), Gregor HÖRMANN(1), Winfried F. PICKL(2) and Klaus G. SCHMETTERER(1)

Sparkling Science Poster

<116> ALRAUNE - Allergy Research in Rural, Alpine and Urban NetworksDavid SCHWARZENBACHER(1)*, Julia ZLOEBL(1)*, Teresa STEMESEDER(2), Eva KLINGLMAYR(2), Edith OBERKOFLER(1), Gabriele GADERMAIER(2)

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Austrian Society for Allergology and Immunology, Nov 6 - 8, 2014, Salzburg

Invited by FWF PhD Program W1248: Molecular, Cellular and Clinical Allergology (MCCA) (Coordinator: Winfried F. Pickl)

Christian MÜNZ

Viral Immunobiology, Institute for Experimental Immunology, University of Zürich, Switzerland

Antigen preservation for presentation is a hallmark of potent antigen presenting cells. We could show that in human macrophages and dendritic cells, a subset of phagosomes gets coated with Atg8/LC3, a component of the molecular machinery of the cellular catabolic pathway macroautophagy, and maintains phagocytosed antigens for prolonged presentation on MHC class II molecules. These Atg8/LC3 positive phagosomes are formed around antigen with TLR2 agonists and require ROS production by NOX2 for their generation. A deficiency in the NOX2 dependent formation of these antigen storage phagosomes could contribute to compromise anti-fungal immune control in chronic granulomatous disease (CGD) patients. While, thus, a role of the macroautophagy machinery for extra- and intracellular antigen presentation on MHC class II molecules has been established, the role of this pathway for MHC class I presentation to CD8+ T cells remains largely unexplored. We could demonstrate that macroautophagy deficiency in dendritic cells leads to an enhanced CD8+ T cell priming during influenza A virus infection in vivo, resulting in decreased pathology. This increased CD8+ T cell stimulation is caused by elevated MHC class I surface levels in macroautophagy deficient cells. While MHC class I expression and antigen processing seems unaffected, MHC class I molecules are stabilized on the cell surface of macroautophagy deficient cells due to decreased internalization and degradation. These findings suggest that stimulation of macroautophagy to harness its pathogen degrading functions should be explored with caution, since it could compromise priming of CD8+ T cell responses.

Autophagy-mediated antigen presentation

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Magnus WICKMAN

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

Background: Allergic sensitisation is not necessary related to current or later onset of allergy-related diseases. The search for sensitisation patterns of allergen molecules early in life related to the trajectories of asthma, rhinitis and eczema is warranted. The aim of this study was to evaluate the role of IgE to microarrayed allergen molecules using longitudinal data of a large birth cohort in relation to onset, persistence and/or disappearance of allergy related diseases among children followed from 4 to 16 years.

Methods: Questionnaire data and sera from follow-ups at 4, 8 and 16 years in a large population based birth cohort (BAMSE) were used. Sera were available from all three time points in 1699 children. A random sample of 786 of these children was used for IgE analyses to 140 allergen components using an allergen-chip based on ISAC technology developed in the MeDALL FP7-funded research program. Asthma, rhinitis and eczema (A, R and E) were defined from questionnaire data.

Results: IgE reactivity to the majority of all allergen molecules increased over time irrespective of current asthma, rhinitis or eczema. However, differences were seen in the evolution of IgE in particular for airborne allergens components compared to peanut allergen components. We sought to identify key allergen molecules as well as bystander molecules. At 4 years 5.2% were sensitized to 41 molecules with an absolute risk of 90% to have onset of asthma or rhinitis after 4 year. For eczema no such molecules could be identified. Specific allergy in relation to PR-10, cat and dog allergen molecules has been investigated. Conclusions: Analysis of IgE to microarrayed allergen components seems to be a promising tool in predicting onset and persistency of asthma or rhinitis during childhood, in order to find preventive treatment measures.

Childhood-to-adolescence evolution of allergen-specific IgE antibodies

Invited by FWF PhD Program W1213: Immunity in Cancer and Allergy (ICA) (Coordinator: Josef Thalhamer)

1st author = presenting author except when underlined

Abstract

Keynote Lecture

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Invited by FWF PhD Program W1253: “Host response in opportunistic infections (HOROS)” (Coordinator: Reinhard Würzner)

Teunis GEIJTENBEEK

Department of Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands. ([email protected])

Adaptive immune responses by dendritic cells (DCs) are controlled by pattern recognition receptors such as Toll-like receptors (TLRs) and C-type lectins. Similarly, innate sensing by these receptors is also involved in aberrant immune responses to allergens or pathogens. Therefore, it is important to understand how pathogens/allergens are sensed by DCs and what molecular mechanisms are triggered to initiate adaptive immunity. C-type lectins recognize specific carbohydrate structures expressed by pathogens and allergens. Our studies have shown that C-type lectins trigger signaling pathways that induce specific cytokines to dictate T cell differentiation. Recently, we have shown that innate signaling by C-type lectin DC-SIGN on DCs to fucose-expressing pathogens such as Schistosoma mansoni leads to the induction of specific T helper (Th) type 2 and follicular Th differentiation. I will discuss the molecular mechanisms involved in these processes. Furthermore, we have recently identified an important role for C-type lectins in sensing of allergens and the induction of Th2 responses. Thus, C-type lectins are crucial in tailoring immune responses to both pathogens and allergens, and I will discuss the different mechanisms underlying these processes.

C-type lectins in infection and immunity

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Invited by FWF SFB F46: Towards prevention and therapy of allergy(Coordinator: Rudolf Valenta)

Jean BOUSQUET

Hopital de Villeneuve, University of Montpellier, Montpellier, France

The origin of the epidemic of IgE-associated (allergic) diseases is unclear. MeDALL (Mechanisms of the Development of ALLergy), an FP7 European Union project (No. 264357), aims to generate novel knowledge on the mechanisms of initiation of allergy and to propose early diagnosis, prevention, and targets for therapy. A novel phenotype definition and an integrative translational approach are needed to understand how a network of molecular and environmental factors can lead to complex allergic diseases. A novel, stepwise, large-scale, and integrative approach will be led by a network of complementary experts in allergy, epidemiology, allergen biochemistry, immunology, molecular biology, epigenetics, functional genomics, bioinformatics, computational and systems biology. The following steps are proposed: (i) Identification of ‘classical’ and ‘novel’ phenotypes in existing birth cohorts; (ii) Building discovery of the relevant mechanisms in IgE-associated allergic diseases in existing longitudinal birth cohorts and Karelian children; (iii) Validation and redefinition of classical and novel phenotypes of IgE-associated allergic diseases; and (iv) Translational integration of systems biology outcomes into health care, including societal aspects. MeDALL will lead to: (i) A better understanding of allergic phenotypes, thus expanding current knowledge of the genomic and environmental determinants of allergic diseases in an integrative way; (ii) Novel diagnostic tools for the early diagnosis of allergy, targets for the development of novel treatment modalities, and prevention of allergic diseases; (iii) Improving the health of European citizens as well as increasing the competitiveness and boosting the innovative capacity of Europe, while addressing global health issues and ethical issues.MeDALL follows over 44,000 children assessed from birth to 4 yrs (22,000), 8 yrs (19,000) and after puberty (over 13,000) with a harmonized historical questionnaire for 199 questions using DataShaper. MeDALL provides a harmonized MeDALL-Core Questionnaire (MeDALL-CQ) used retrospectively in all cohorts and prospectively in 11 European birth cohorts. The harmonization of questions was accomplished in 4 steps: (i) collection of variables from 14 birth cohorts, (ii) consensus on questionnaire items, (iii) translation and back-translation of the harmonized English MeDALL-CQ into 8 other languages and (iv) implementation of the harmonized follow-up. Three harmonized MeDALL-CQs (2 for parents of children aged 4-9 and 14-18, 1 for adolescents aged 14-18) were developed. Eczema, rhinitis, and asthma often coexist (comorbidity) in children, but the proportion of comorbidity not attributable to either chance or the role of IgE sensitisation is unknown. We assessed these factors in children aged 4-8 years in a prospective study study from 12 ongoing European birth cohort studies using hypothesis-driven approaches. We assessed 16 147 children aged 4 years and 11 080 aged 8 years in cross-sectional analyses. Coexistence of eczema, rhinitis, and asthma in the same child is more common than expected by chance alone-both in the presence and absence of IgE sensitisation-suggesting that these diseases share causal mechanisms. Although IgE sensitisation is independently associated with excess comorbidity of eczema, rhinitis, and asthma, its presence accounted only for 38% of comorbidity, suggesting that IgE sensitisation can no longer be considered the dominant causal mechanism of comorbidity for these diseases. Similar results were observed using data-driven approaches and unsupervised statistical analyses.Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients’ antibody reactivity profiles towards each of the allergen molecules with only minute amounts of serum. The MeDALL allergen-chip has been developed for the specific and sensitive monitoring of IgE and IgG reactivity profiles towards more than 170 allergen molecules in sera collected in birth cohorts.Historical GWAS (23,000 children), prospective epigenetics (7,000), proteomics (1,000) and transcriptomics (500) are currently been analysed.

MeDALL: Mechanisms of the Development of ALLergy

Abstract

Keynote Lecture

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Invited by FWF PhD Program W1213: Immunity in Cancer and Allergy (ICA)(Coordinator: Josef Thalhamer)

Marc DESCATOIRE(1), Davide BAGNARA(1), Sandra WELLER(1), Deborah DUNN-WALTERS(2), Jean-Claude WEILL(1) and Claude-Agnès REYNAUD(1)

(1) Institut Necker-Enfants-Malades (INEM) INSERM U1151, Faculté de Médecine Paris Descartes, Site Broussais, Paris (France)(2) Department of Immunobiology, King’s College London School of Medicine (UK)

In mice, marginal zone B cells (MZB) represent a distinct B cell lineage that arises in the spleen where marginal zone precursor cells (MZP) differentiate under the control of the Notch2 pathway. In humans, the existence of such a lineage is still controversial. We proposed that, in humans, blood IgM+IgD+CD27+ B cells represent circulating splenic MZB cells with a differentiation pathway that could be conserved at least in part between mice and humans. We identified in human spleen from young children a putative MZP subset, characterized by its capacity to differentiate into MZB-like cells through Notch2 activation in vitro. It accounts on average for 5% of total splenic B cells in children and decreases throughout life. A transcriptomic analysis confirmed that MZP represent an intermediate differentiation stage between naive and MZB cells. A Notch2 induction signature predominated among genes discriminating MZP from naive B cells, a signature further developed among MZB cells. Altogether, our results suggest that the early development of human blood and splenic MZB cells, proceeds, as in the mouse, through a Notch2-dependent differentiation pathway, and strengthen the proposition that IgM+IgD+CD27+ B cells differ from germinal center-dependent IgM memory B cells generated during T-dependent responses. To further delineate MZ from IgM memory B cells, we performed high-throughput sequencing of different splenic B cell subsets, using IgD and CD27 to define three different populations with mutated Ig genes: IgD+CD27+ (“MZ”), IgD-CD27+ (“classical memory”, including cells with IgG, IgA and IgM isotypes - the latter ones being named “IgM-only”) and IgD-CD27- cells (double negative). We analyzed clones that share sequences between different subsets for their mutation frequency distribution, their CDR3 length, their VH family and JH gene usage, and compared these different characteristics with the bulk of sequences from their respective subset of origin, for which these parameters constitute a distinct signature. Clonal relationships between the IgM clones (originating from the MZ, M-only and double negative compartments) show that all sequences involved display the characteristics of IgM-only B cells, whatever their subset of origin. We therefore conclude that the clonal relationships shared between the various IgM subsets do not represent filiation between them, but rather correspond to a heterogeneous phenotype of the IgM-only population that concerns both IgD and CD27 expression, leading to a partial overlap with the MZ and double-negative gates. Clones shared between the MZ and the switched IgG and IgA compartment also show, for their IgM part, the mutation and repertoire characteristics of IgM-only cells and not of MZ B cells, reinforcing the conclusion that IgM-only are true memory B cells, and constitute the only subset showing precursor-product relationships with switched memory B cells. This analysis thus reinforces the notion that IgM memory and marginal zone B cells represent two different entities, with specific effector function in, respectively, T-dependent and T-independent responses.

Diversity of human memory subsetsMarginal zone B cells in humans: a distinct cell lineage differing from IgM memory B cells.

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Invited by FWF SFB F46: Towards prevention and therapy of allergy(Coordinator: Rudolf Valenta)

Stephen J. GALLI

Departments of Pathology and of Immunology and Microbiology, Stanford University, Stanford, California, USA. ([email protected])

It is well known that mast cells (MCs) and IgE antibodies are important effector elements in allergic disorders, and it is thought that they can contribute to host resistance to certain parasites. However, it has been unclear whether MCs or IgE have any other beneficial functions. In 1991, Margie Profet hypothesized that many allergens are derived from sources (such as nuts, seafood, or venoms) which either might (e.g., foods) or always (e.g., venoms) contain toxins (Profet, 1991). Profet also proposed that allergic reactions (manifested as immediately occurring symptoms such as coughing or diarrhea) evolved to allow the sensitized host to respond immediately to, and to neutralize and/or avoid, noxious substances which might be indicative of potentially life-threatening situations. However, until recently, Profet’s “toxin hypothesis” was largely ignored. In mammals, venoms provoke an innate inflammatory response and pathology reflecting the activities of the contained toxins. Venoms also can induce allergic sensitization and the development of specific IgE antibodies, which bind to FcepsilonRI on tissue mast cells (MCs) and blood basophils, priming them to release mediators of upon subsequent venom exposure. MCs also can be activated directly by certain venoms, in the absence of specific IgE, and work in mice indicates that innate functions of MCs, including degradation of venom toxins by MC-derived proteases, can enhance host resistance to the venoms of certain arthropods (including the honeybee) and reptiles (Metz et al, 2006; Schneider et al., 2007; Akahoshi, Song, et al., 2011). We found that mice injected with 1) amounts of honeybee venom similar to what could be delivered in 1-2 stings, or 2) sublethal amounts of Russell’s viper venom, developed specific Th2 responses which increased their survival after subsequent challenge with potentially lethal amounts of the venom (Marichal, Starkl et al., 2013). Our data indicate that IgE antibodies, FcepsilonRI, and FcepsilonRIalpha–bearing effector cells (probably MCs) contribute to such acquired resistance to venom. The evidence that IgE-dependent immune responses against venom can enhance survival in mice supports the hypothesis that one important function of IgE is to help to protect the host against noxious substances.

Testing the toxin hypothesis of allergy: Roles of mast cells and IgE in innate and acquired resistance to venoms

AcknowledgementsI thank all of the co-workers and collaborators who have contributed so importantly to the work reviewed in my presentation, and the NIH, USA and the Department of Pathology, Stanford University, for supporting of these studies.

Abstract

Keynote Lecture

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InvitedbyFWFPhDProgramW1212:InflammationandImmunity(IAI)(Coordinator: Maria Sibilia)

Alberto MANTOVANI

Humanitas Clinical and Research Center, University of Milan, 20089 Rozzano, Italy

Macrophages are key orchestrators of chronic inflammation. They respond to microenvironmental signals with polarized genetic and functional programmes. M1 macrophages which are classically activated by microbial products and interferon-γ, are potent effector cells which kill microorganisms and tumours . In contrast, M2 cells, tune inflammation and adaptive immunity; promote cell proliferation by producing growth factors and products of the arginase pathway (ornithine and polyamines); scavenge debris by expressing scavenger receptors; promote angiogenesis, tissue remodeling and repair. M1 and M2 cells represent simplified extremes of a continuum of functional states. Available information suggests that TAM are a prototypic M2 population. M2 polarization of phagocytes sets these cells in a tissue remodeling and repair mode and orchestrate the smouldering and polarized chronic inflammation associated to established neoplasia. Intrinsic metabolic features and orchestration of metabolism are key components of macrophage polarization and function. Recent studies have begun to address the central issue of the relationship between genetic events causing cancer and activation of protumour, smouldering, non resolving tumour-promoting inflammation. New vistas have emerged on molecules associated with M2 or M2-like polarization and its orchestration in cancer. Recently, proof-of-principle has been obtained that targeting TAM can be beneficial in human cancer.

ReferencesMantovani A, Allavena P, Sica A, Balkwill F Cancer-Related Inflammation. Nature 454: 436-444, 2008.Biswas S.K. and Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as paradigm. Nat Immunol 2010: 11, 889-896. Sica A and Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Invest. 2012: 122, 787-795.Biswas SK, Mantovani A. Orchestration of metabolism by macrophages. Cell Metab. 2012; 15(4): 432-7Germano G, Frapolli R, Belgiovine C, et al. Role of macrophage targeting in the antitumor activity of trabectedin. Cancer Cell 23: 249-262, 2013.Jaillon S, Moalli F, Ragnarsdottir B, et al. The humoral pattern recognition molecule PTX3 is a key component of innate immunity against urinary tract infection. Immunity 40: 621-632, 2014.

Molecular functions and regulation circuits of inflammatory cytokines

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Invited by FWF PhD Program W1248: Molecular, Cellular and Clinical Allergology (MCCA)(Coordinator: Winfried F. Pickl)

Maria YAZDANBAKHSH (1), Abena AMOAH (1,2), Firdaus HAMID (1,3), Daniel BOAKYE (2), Taniawati SUPALI (4), Ronald VAN REE (5)

(1) Leiden University Medical Center, Leiden, The Netherlands(2) Noguchi Memorial Institute for Medical Research, Accra, Ghana(3) Hassanudin University, Makassar, Indonesia(4) University of Indonesia, Jakarta, Indonesia(5) Academic Medical Center, Amsterdam, The Netherlands

Large geographical differences are seen in the prevalence of allergic disorders worldwide. In affluent countries and in urban centers of low income countries, allergic disorders are a serious problem whereas populations living in rural areas often appear to be protected. It is interesting that helminth infections which are strong inducers of Th2 responses are highly prevalent in rural areas of developing countries. This translates into high levels of total IgE antibodies as well as allergen specific antibodies that do not seem to lead to allergic disorders. There is evidence that IgE antibodies to helminthes can be cross reactive with allergens via the carbohydrate structures. These cross reactive antibodies have poor biological activity. The exact structures that the IgE antibodies are directed to are being studied in order to develop strategies that might allow the induction of IgE with little biological activity in terms of inducing basophil/mast cell degranulation. This might be an approach to prevent the development of allergic disorders.

Helminths and allergies: The IgE trick

Abstract

Closing

Keynote Lecture

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Eingeladen von: Schwerpunkt Biowissenschaften und Gesundheit der Universität Salzburg(Koordinator: Josef Thalhamer)

Johannes RING, Ursula Krämer, Roma Schmitz, Heidrun Behrendt

Director emeritus Haut und Allergieklinik Biederstein, TU München

Der überraschende Befund von niedrigeren Heuschnupfen- und Asthmaprävalenzraten in Ostdeutschland im Vergleich zu Westdeutschland zur Zeit des Mauerfalles und der deut-schen Wiedervereinigung gaben Anlass zu verschiedenen epidemiologischen Studien, die sämtlich in der Folge einen steilen Anstieg der Allergiehäufigkeit in Ostdeutschland zeigten. Nach ca. zehn Jahren hatten sich Ost und West in Deutschland allergologisch weitgehend „harmonisiert“. Dieses Phänomen soll als Modell betrachtet werden, um Ursachen für die „Allergie-Epidemie“ in der „westlichen“ Welt besser zu verstehen. Tatsächlich wurde bislang nie untersucht, ob sich ein solches Muster auch in allen vergleichenden Studien und für alle Manifestationen allergischer Erkrankungen nachweisen ließ. Es erscheint interessant, aus diesem „Experimentum dictaturae“ zu lernen, wodurch ein Volk in einem Land mit ähnlicher Ethnik, Kultur und klimatischer Kondition lediglich durch einen politisch bedingten unter-schiedlichen Lebensstil getrennt war,.

Insgesamt 14 Querschnittsstudien wurden einer Metaanalyse unterzogen, und die Verände-rungen der Ost-West-Verhältniszahlen in der Dekade nach der deutschen Wiedervereinigung verglichen.

Heuschnupfen als die typischste atopische Erkrankung zeigte die klarsten Unterschiede zur Zeit der Wiedervereinigung zwischen Ost- und Westdeutschland und die deutlichsten Kon-vergenzraten.

Es fand sich ein deutlicher Unterschied zwischen allergischen Atemwegserkrankungen (Heu-schnupfen, Asthma) und Hautmanifestation als atopisches Ekzem, welches in Ostdeutsch-land zur Zeit der Wiedervereinigung gleich häufig oder häufiger war, als die Atemwegsaller-gien.

Die Risikofaktoren für Atemwegsallergien (Ozon, Feinstaub, Verkehrsbelastung) unterschei-den sich deutlich von den klassischen Risikofaktoren für andere Atemwegserkrankungen wie z. B. Bronchitis (Schwefeldioxid, grober Schwebstaub, industrieller Smog). Häufig diskutierte Innenraumfaktoren wie Teppichboden, Bettzeug oder Haustierhaltung konnten für den An-stieg der Allergieprävalenz in Ostdeutschland nicht verantwortlich gemacht werden. Aller-dings fand sich als möglicher Faktor, der in allen Studien präsent war, Einzelraumheizung mit fossilen Brennstoffen sowie Krippenbesuch bzw. das Leben in einer Familie mit Geschwistern als Allergie-protektiv. Nicht in allen Studien untersucht, aber dennoch von Interesse: Wurm-infektionen schienen protektiv für Atemwegsallergien aber eher fördernd für atopisches Ekzem zu sein. In einer Studie fand sich ein protektiver Effekt der Keuchhustenimpfung, wel-che in Ostdeutschland vor der Wiedervereinigung in nahezu 100 % durchgeführt wurde, im Vergleich dazu nur bei 75 % der westdeutschen Kinder.

Die West-Ost-Unterschiede verschwanden bei Kindern, die nach der Wiedervereinigung ge-boren wurden, relativ schnell, blieben jedoch bei Erwachsenen-Studien noch länger erhalten.

Die gefundenen Auffälligkeiten stellen möglicherweise nur Marker von noch genauer zu identifizierenden Kausalfaktoren dar.

Literatur:Krämer U, Schmitz R, Ring J, Behrendt H. What can reunification of East and West Germany tell us about the cause of the allergy epidemic? Clin Exp Allergy, in press

Risiken der Allergiezunahme – Was wir aus den Ost-West-Studien nach der deutschen Wiedervereinigung gelernt haben

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Knut BROCKOW

Klinik und Poliklinik für Dermatologie und Allergologie am Biederstein, Technische Universität München, Biedersteiner Straße 29, 80802 München ([email protected])

Die häufigsten Auslöser von Arzneimittel-Unverträglichkeitsreaktionen sind möglicherweise Analgetika. Es wird eine durch Zyklooxygenasehemmung bedingte Reduktion von protektiven Prostaglandinen bzw. Vermehrung von Lipoxygenasestoffwechselprodukten angenommen. Bis zur allergologischen Klärung sollten alle in Frage kommenden Auslöser gemieden werden. Die allergologische Diagnostik von Überempfindlichkeitsreaktionen auf Arzneimittel wird in einer deutschsprachigen Leitlinie überarbeitet; daneben existieren Europäische Leitlinien. Alle empfehlen die Austestung betroffener Patienten zur Verhinderung erneuter Reaktionen. Zumeist werden die Auslöser einer Analgetika-Überempfindlichkeit durch Anamnese, Hauttest und In-vitro-Untersuchungen nicht sicher identifiziert, insofern sind zur Klärung Provokationstestungen notwendig. Diese Praxis wird oft nicht befolgt, da die personellen und methodischen Kapazitäten fehlen. Die allergologische Versorgung, ist anhand der DRGs nicht kostendeckend. Es gibt zu wenige allergologische Zentren, die bis zur Provokationstestung diagnostizieren. In dieser Siuation müssen häufig Kompromisse eingegangen werden. Bei mindestens dreimaliger Urtikaria nach Analgetikaeinnahme war die Wahrscheinlichkeit einer erneuten Reaktion > 95%. Bei eindeutigen Reaktionen sollte eine Austestung von Alternativanalgetika erfolgen, da hohe Kreuzreaktivitäten zwischen Cox1-hemmenden Analgetika bestehen. Cox2-Hemmer, wie Celecoxib werden von >95% aller Patienten vertragen, ebenso Opiatanalgetika und Paracetamol zumeist in niederigen Dosierungen ≤500mg. Das Risiko-/Nutzenverhältnis einer stationären Provokation mit diesen Ausweichanalgetika kann diskutiert werden.

Analgetikaintoleranz- braucht wirklich jeder Patient einen Provokationstest?


Abstract

Vortrag Klinischer

Allergietag

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Margitta WORM

Allergie-Centrum-Charité, Klinik für Dermatologie, Venerologie und Allergologie,Charité Universitätsmedizin Berlin

Die Anaphylaxie ist definiert als eine sich rasch entwickelnde Multiorganerkrankung, diepotentiell tödlich verlaufen kann. Nahrungsmittel, Insektengifte und Medikamente sind diehäufigsten Auslöser. Versorgungsaspekte der Anaphylaxie betreffen sowohl die Diagnostikals auch die Therapie, wobei hier das Akut- aber auch das Langzeitmanagementberücksichtigt werden müssen. Zur Diagnostik der Anaphylaxie allgemein muss ausversorgungsmedizinischer Sicht festgestellt werden, dass bestimmte Patientengruppen einallergologisches Zentrum zur diagnostischen Abklärung gar nicht erreichen. EineVerbesserung wäre hier durch eine Vernetzung von notärztlichen Zentren mitallergologischen Zentren zu erzielen.Die Akuttherapie anaphylaktischer Reaktionen umfasst die i.m. Applikation von Adrenalin mitHilfe eines Autoinjektors sowie Antihistaminika und Kortikosteroide. Das einzige Medikament,welches direkt den Symptomen einer schweren Anaphylaxie entgegenwirkt, ist Adrenalin.Daher wird es in allen derzeit verfügbaren nationalen und internationalen Leitlinien alsMedikament der ersten Wahl empfohlen. Die Daten aus dem Anaphylaxie Register, woPatienten mit schweren allergischen Reaktionen von allergologischen Zentren gemeldetwerden, zeigen jedoch, dass nur weniger als 20% der Patienten tatsächlich trotz desVorliegens einer schweren Reaktion mit Herzkreislauf- und/oder LungenbeteiligungAdrenalin erhalten. Eine detaillierte Analyse der Versorgung von Patienten nachSchweregrad ergibt, dass zwar Adrenalin bei Patienten in der allergischen Schocksituationhäufiger gegeben wird, jedoch der Einsatz beispielsweise in der Gruppe der Patienten mitrespiratorischen Symptomen und Hautsymptomen deutlich weniger häufig ist. Somit zeigendie Daten aus dem Anaphylaxie Register, dass die Akutversorgung der Patienten verbessertwerden muss. Dies kann durch verbesserte Aufklärungsmaßnahmen nicht nur für Patienten,sondern auch dem ärztlichen Personal erzielt werden. Weitere Studien, die belegen, dasseine frühzeitige Gabe von Adrenalin das Überleben und den Verlauf einer schwerenallergischen Reaktion verbessern kann, können dazu beitragen, dieses Ziel zu erreichen.Key words: Anaphylaxie, Notfallversorgung, AdrenalinReferenzen:Worm M, Moneret-Vautrin A, Scherer K, Lang R, Fernandez-Rivas M, Cardona V, Kowalski ML, Jutel M, Poziomkowska-Gesicka I, Papadopoulos NG, Beyer K, Mustakov T, Christoff G, Bilò MB, Muraro A, Hourihane JO, Grabenhenrich LB. First European data from the network of severe allergic reactions (NORA). Allergy. 2014 Oct;69(10):1397-404.Worm M, Eckermann O, Dölle S, Aberer W, Beyer K, Hawranek T, Hompes S, Koehli A, Mahler V, Nemat K, Niggemann B, Pföhler C, Rabe U, Reissig A, Rietschel E, Scherer K, Treudler R, Ruëff F.Triggers and treatment of anaphylaxis: an analysis of 4,000 cases from Germany, Austria andSwitzerland. Dtsch Arztebl Int. 2014 May 23;111(21):367-75.Grabenhenrich L, Hompes S, Gough H, Ruëff F, Scherer K, Pföhler C, Treudler R, Mahler V,Hawranek T, Nemat K, Koehli A, Keil T, Worm M. Implementation of anaphylaxis managementguidelines: a register-based study. PLoS One. 2012;7(5):e35778.

Volkskrankheit Anaphylaxie - werden unsere Patienten ausreichend versorgt?


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Tab. 1 Beispiele (Allergenquellen)

Höhere analytische Sensitivität

Größere analytische Spezifität/Selektivität

Erdnuss Ara h 10, Ara h 11 (Oleosine) Ara h 1, (7S-Globulin) Ara h 2 (2S-Albumin) Ara h 3, (11S-Globulin) Ara h 9 (LTP, Mittelmeerraum)

Soja Gly m 4 Glym 5 Gly m 6

Haselnuss Cor a 1 (Bet v 1-homolog) Cor a 14 (2S-Albumin), Cor a 9 (11S-Globulin) Cor a 8 (LTP, Mittelmeerraum)

Weizen Tri a 19 (Omega-5-Gliadin) Fleisch alpha-GAL alpha-GAL Bienengift Api m 1 Wespengift Ves v 5 Ves v 1, Ves v 5 Tab. 2 Pflanzenfamilien u. -spezies

Major-Allergene Minor-Allergene (Pollen-Panallergene)

Relevante Pollenpflanzen für Allergiesymptome in Deutschland

aus diversen Proteinfamilien

Profiline Polcalcine

Birkengewächse (Hasel, Erle, Birke, Hainbuche) u. Buchengewächse (Buche, Eiche)

Bet v 1 Bet v 2

Bet v 4

Ölbaumgewächse (Esche, Ölbaum)

Ole e 1 Ole e 2 Ole e 3

Süßgräser (incl. Roggen) Phl p 1, Phl p 5 Phl p 12

Phl p 7

Beifuß Art v 1 Art v 4 Art v 5 Ambrosia (Traubenkraut, Ragweed)

Amb a 1 Amb a 8 Amb a 10

Jörg KLEINE-TEBBE, Juliane ACKERMANN-SIMON, Gerald HANF

Allergie- u. Asthma-Zentrum Westend, Praxis Hanf, Ackermann & Kleine-Tebbe, Berlin

Allergenmoleküle (meist Proteine, seltener Kohlehydratepitope) eröffnen neue Möglichkeiten für die allergenspezifische Immunglobulin E (IgE)-Diagnostik.

Warum besitzt die Molekulare Allergiediagnostik Vorteile?

Folgende Gründe sprechen für den Einsatz von Einzelallergenen: 1. Erhöhte Testempfindlichkeit (analytische Sensitivität), besonders bei unterrepräsentierten oder fehlenden wichtigen Allergenen im Extrakt (Beispiele in Tab, 1, mittlere Spalte)2. Verbesserte Testselektivität (analytische Spezifität/Selektivität), besonders wenn das selektierte IgE-Repertoire gegen ein Allergen zusätzliche Aussagen zum potentiellen Risiko (Beispiele in Tab. 1, rechte Spalte), zur primären (genuinen) Sensibilisierung oder zur möglichen Kreuzreaktivität gestattet (Beispiele in Tab. 2, rechte Spalten).

Molekulare Allergologie in der Praxis: Zwischen Evidenz und anekdotischer ErfahrungWie evident ist die Rekombinanten-basierte Diagnostik in der Praxis?

Fortsetzung siehe S. 28f


Abstract

Vortrag Klinischer

Allergietag

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Zur Labordiagnostik bereits verfügbare Einzelallergene (fett gedruckt): Markerallergene (mittlere. Spalte) zur IgE-Diagnostik als Zeichen einer primären Sensibilisierung und Pollen-Panallergene (re. Spalten) als Hinweis auf und Basis für klinisch fraglich relevante Kreuzreaktionen zwischen sämtlichen Pollen(extrakt)spezies.

Wann kommt die Molekulare Allergiediagnostik in Frage?

Die richtige Indikation für den Einsatz von Einzelallergenen lässt sich nur 3. individuell (abhängig vom klinischen Kontext/Vorgeschichte) und 4. allergenspezifisch (abhängig von Allergenquelle und verfügbaren Einzelallergenen) und nicht pauschal begründen.

Wie wird die Molekulare Allergiediagnostik sinnvoll eingesetzt?

Folgende Beispiele bei Verdacht auf Nahrungsmittelallergie (NMA), Insektengiftallergie oder Sensibilisierung gegen Pollen-Panallergene illustrieren in Tab. 3 den Einsatz von Einzelallergenen zur allergenspezifischen IgE-Diagnostik.

Tab. 1 Beispiele (Allergenquellen)

Höhere analytische Sensitivität

Größere analytische Spezifität/Selektivität

Erdnuss Ara h 10, Ara h 11 (Oleosine) Ara h 1, (7S-Globulin) Ara h 2 (2S-Albumin) Ara h 3, (11S-Globulin) Ara h 9 (LTP, Mittelmeerraum)

Soja Gly m 4 Glym 5 Gly m 6

Haselnuss Cor a 1 (Bet v 1-homolog) Cor a 14 (2S-Albumin), Cor a 9 (11S-Globulin) Cor a 8 (LTP, Mittelmeerraum)

Weizen Tri a 19 (Omega-5-Gliadin) Fleisch alpha-GAL alpha-GAL Bienengift Api m 1 Wespengift Ves v 5 Ves v 1, Ves v 5 Tab. 2 Pflanzenfamilien u. -spezies

Major-Allergene Minor-Allergene (Pollen-Panallergene)

Relevante Pollenpflanzen für Allergiesymptome in Deutschland

aus diversen Proteinfamilien

Profiline Polcalcine

Birkengewächse (Hasel, Erle, Birke, Hainbuche) u. Buchengewächse (Buche, Eiche)

Bet v 1 Bet v 2

Bet v 4

Ölbaumgewächse (Esche, Ölbaum)

Ole e 1 Ole e 2 Ole e 3

Süßgräser (incl. Roggen) Phl p 1, Phl p 5 Phl p 12

Phl p 7

Beifuß Art v 1 Art v 4 Art v 5 Ambrosia (Traubenkraut, Ragweed)

Amb a 1 Amb a 8 Amb a 10

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Tab. 3 Klinisches Bild Allergologische

Verdachtsdiagnose Einzelallergene zur IgE-Diagnostik

Anaphylaxie nach Anstrengungen und Mahlzeit mit Weizenprodukt(en)

Anstrengungsabhängige Weizenallergie

Tri a 19 (Omega-5 Gliadin)

"Katzen-Schweinefleisch-Syndrom"

Allergie gegen tierische Serumalbumine

Fel d 2 oder Bos d 6

verzögerte Fleisch-Allergie (z.B. Urtikaria)

Sensibilisierung gegen -GAL -GAL (Thyreoglobulin)

Allergie z.B. auf Weintrauben, Heidelbeeren, Zitrusfrüchte

Sensibilisierung gegen Lipid-Transferproteine (LTP)

Pru p 3 (Pfirsich-LTP)

Orales Allergie-Syndrom (OAS) häufig auf Nüsse, Kern- und Steinobst etc., ggf. systemische Reaktionen auf Soja (nativ)

Sensibilisierung gegen Bet v 1-Homologe (PR 10 Proteine)

Bet v 1 und ggfs. Gly m 4 (Bet v 1-homolog)

OAS nach ungewöhnlichen pflanzlichen Nahrungsmitteln (Melone, Exoten wie Litschi, Zitrusfrüchte)

Sensibilisierung gegen Profiline

Pru p 4 (oder Bet v 4, Phl p 12, Hev b 8)

Doppelsensibilisierung gegen Bienen- und Wespengift

Serologische Kreuzreaktion durch gemeinsame Allergene in Bienen- u. Wespengift

Api m 1 Ves v 1 Ves v 5

Multiple Sensibilisierungen gegen Pollenextrakte (Baum-, Gräser- u. Kräuterpollen) fraglicher Relevanz

Serologische Kreuzreaktion durch Pollen-Panallergene (Profilin u./o. Polcalcin)

Bet v 2 oder Phl p 12 (Profilin)Bet v 4 oder Phl p 7 (Polcalcin)

5. Zur individuellen Vorhersage der klinischen Relevanz einer Sensibilisierung ist das IgE gegen Allergenmoleküle (z.B. ImmunoCAP, ThermoFisher, Freiburg) nur bedingt geeignet, da es sich um einen Sensibilisierungstest handelt. Bei Nahrungsmittelallergie lässt sich bestenfalls ein relatives Risiko aufgrund des IgE-Profils ermitteln, aber keine sichere Vorhersage zur (zukünftigen) Verträglichkeit treffen.

6. Die klinische Relevanz sämtlicher IgE-Befunde ist nur bei korrespondierenden Symptomen gegeben und muss individuell geprüft werden (Vorgeschichte, Symptomprotokoll, ggfs. Provokation mit der zugehörigen Allergenquelle). Somit ermittelt der behandelnde Arzt auch bei Verwendung von Einzelallergenen die klinische Relevanz der diagnostischen Ergebnisse, nicht der Test.

Weiterführende LiteraturCanonica GW, Ansotegui IJ, Pawankar R, Schmid-Grendelmeier P, van Hage M, Baena-Cagnani CE, et al. A WAO - ARIA - GA(2)LEN consensus document on molecular-based allergy diagnostics. The World Allergy Organization journal. 2013;6(1):17. PubMed PMID: 24090398. Pubmed Central PMCID: 3874689 (Open Access).Kleine-Tebbe J, Jappe U. Molekulare Allergiediagnostik: Entwicklung und die Bedeutung für die klinische Praxis. Allergologie. 2013;36:327-49.Kleine-Tebbe J, Jappe U. Molekulare Allergologie (Taschenbuch). Dustri-Verlag Dr. Karl Feistle, München-Deisenhofen 2014, ISBN 978-3-87185-491-0

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Contact / E-Mail:[email protected]

Thomas EIWEGGER (1)*, Merima BUBLIN(2)*, Klara SCHMIDTHALER(1), Maria KOSTADINOVA(2), Christian RADAUER(2), Christine HAFNER(3), Zsolt SZÉPFALUSI(1), Eva-Maria VARGA(4), Heimo BREITENEDER(2) *contributed equally

(1) Department of Paediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria(2) Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria(3) Karl Landsteiner Institute for Dermatological Research, St. Poelten, Austria (4) Department of Paediatrics, Respiratory and Allergic Disease Division, Medical University Graz, Austria

Background: Currently no allergen specific immunotherapy (IT) for peanut allergic individuals is available due to side effects. Modified allergens with a highly reduced IgE-binding capacity with preserved T cell epitopes are needed for IT.

Aim: We aimed to generate modified major peanut allergens Ara h 2 and Ara h 6 with reduced IgE binding, preserved T-cell epitopes and intact tertiary structure for treatment of peanut allergy.

Methods: The hypoallergens were engineered by swapping of neighbouring segments of 5-10 amino acids residues in surface exposed loops. Ara h 2 and Ara h 6 were purified from commercial roasted peanuts, and mutated Ara h 2 and Ara h 6 were produced in E. coli SHuffle T7 cells. IgE-binding (ELISA, Immunoblot), IgE cross-linking capacity (Basophil Activation Test), T-cell stimulatory capacity (3H-Thymidine incorporation assay, intracellular cytokine staining, multiplex ELISA) and whole mRNA expression in allergen specific T-cells were evaluated.

Results: Mutated Ara h 2 and Ara h 6 were fully capable in inducing allergen specific T-cell proliferation with mildly reduced Th2 cytokine induction. Basophil activation was reduced by 90-97%. Allergen specific T-cell gene array revealed minimal changes in the T-cell mRNA signature suggestive for keeping relevant T-cell epitopes and response mechanisms intact.Conclusions: Hypoallergenic peanut variants with significantly reduced IgE binding but intact T-cell activation may be an option for allergen-specific IT.

Engineering hypoallergenic variants of the peanut allergens Ara h 2 and Ara h 6

Acknowledgements

This work was funded by BRIDGE Grant 820127 from the Austrian Research Promotion Agency (FFG) and the Medical Scientific fund of the Mayor of the city of Vienna (no. 11013) and the SFP and PhD Program Molecular, Cellular and Clinical Allergy, MCCA (no w1248-B13)

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Heidi A. MÜLLER(1) , Luis. F. PACIOS(2), Cristina GOMEZ-CASADO(2), G. HOFSTETTER(1), Georg A. ROTH(3), Josef SINGER(4), Araceli DIAZ-PERALES(2), Erika JENSEN-JAROLIM(1,4), F. ROTH-WALTER(1)

(1) Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria(2) Biotechnology Department, Center for Plant Biotechnology and Genomics, Technical University of Madrid, Madrid, Spain(3) Department of Anesthesiology, General Intensive Care and Pain Medicine, Medical University of Vienna, Austria(4) Comparative Immunology and Oncology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria

Background: The major cow milk allergen Bos d 5 belongs to the lipocalin-family and as such is a promiscuous ligand transporter. In this study we investigated its ability to bind to iron-siderophore complexes and tested for immune-modulatory consequences.

Methods: Docking to Bos d 5 of catechol-based flavonoids (quercetin, myricetin, luteolin) selected as siderophores were performed using AutoDock Vina. Prussian blue staining of Bos d 5 carrying iron (holo-) or not (apo-form) were performed. Peripheral blood mononuclear cells (PBMCs) of 25 human subjects were activated and stimulated for 18h with apo- or holo-form. Subsequently, cells and supernatants were analysed by flow cytometry and their cytokine-content.

Results: Docking analysis of Bos d 5 revealed that it is able to bind iron via catechol-based flavonoids that act as siderophores. When incubated with PBMCs, only the apo-form led to an increase of CD4+positive cells and significantly elevated IL13 and IFNγ-levels. In contrast, holo-form decreased numbers of CD4 expressing cells and induced apoptosis.

Conclusion: Our data give evidence that Bos d 5 is capable of binding iron via siderophores. The apo-form promotes Th2 cells and inflammation, whereas the holo-form appears to be immunosuppressive.

The major cow milk allergen, Bos d 5, is able to bind siderophore bound iron and influences T-helper cells according to its binding state

Acknowledgements

This work was supported by the F4606-B19 of the Austrian Science Fund FWF. J. Singer was financially supported by CCHD W1205-B09 of the Austrian Science Fund, FWF. C. Gomez-Casado was financially supported with the grant BES-2010-034628of the FPI Programme from the Spanish Government (MICINN/MINECO).


1st author = presenting author except when underlined

Abstract 2

Oral Presentation

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Why I? IgE profiling and lifestyle analysis of 501 Austrian school childrenTeresa STEMESEDER(1), Eva KLINGLMAYR(1), Bettina SCHWEIDLER(1), Lisa LUEFTENEGGER(2,3), Stephanie MOSER(4), Roland LANG(3), Martin HIMLY(1), Gertie J. OOSTINGH(2), Arne BATHKE(5), Joerg ZUMBACH(4), Thomas HAWRANEK(3), Gabriele GADERMAIER(1)

(1) University of Salzburg, Department of Molecular Biology, Salzburg, Austria(2) Salzburg University of Applied Sciences, Biomedical Sciences, Salzburg, Austria(3) Paracelsus Medical University Salzburg, Department of Dermatology, Salzburg, Austria(4) University of Salzburg, School of Education, Salzburg, Austria(5) University of Salzburg, Department of Mathematics, Salzburg, Austria

This study aims to clarify reasons for the increasing numbers of patients suffering from allergic diseases by analyzing the IgE sensitization profiles and lifestyle of 501 Austrian school children aged 13-21 from different geographical regions.Capillary blood samples were obtained from all participants and analyzed using the ImmunoCAP ISAC. Demographic data, self-reported health status including allergies and other lifestyle conditions such as smoking and diet were surveyed in a questionnaire.Forty percent of subjects declared to suffer from allergies including self-reported adverse reactions. IgE-reactivity to any of the 112 molecules on the ISAC chip was observed in 53% of subjects. Highest sensitizations to inhalant allergens were found to grass pollen (36%), tree pollen (28%) and house dust mites (23%). No difference in overall IgE sensitization was found between alpine, urban and rural living areas whereas the sensitization rate to house dust mite was significantly higher in urban regions. Among smokers the sensitization rate of 76% was found to be significantly higher.Investigations of allergic and non-allergic subjects showed a very high sensitization rate and might allow identification of factors influencing the development of allergic diseases.

Acknowledgements

The study was funded by Sparkling Science, a program of the Federal Ministry of Science, Research and Economy, Vienna, Austria.


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Eva WOLLMANN(1), Carl HAMSTEN Carl(2,3), Elopy SIBANDA(4), Mary OCHOME(1), Margarethe FOCKE-TEJKL(1), Anna ASARNOJ(2,5), Annika ÖNELL(6), Gunnar LILJA(7), Daniela GALLERANO(1), Christian LUPINEK(1), Theresa THALHAMER(8), Richard WEISS(8), Josef THALHAMER(8), Magnus WICKMAN(7,9), Rudolf VALENTA(1), Marianne VAN HAGE(2)

(1) Institute for Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna(2) Department of Medicine Solna, Clinical Immunology and Allergy Unit, Karolinska Institutet, and University Hospital, Stockholm, Sweden(3)CenterforInflammatoryDiseases,KarolinskaInstitutet,Stockholm,Sweden(4) University of Zimbabwe Medical School, Harare, Zimbabwe(5) Astrid Lindgren Children’s Hospital, Stockholm, Sweden(6)ThermofisherScientificImmunoDiagnostics,R&D,UppsalaSweden(7) Department of Pediatrics, Sachs’ Children’s Hospital, Stockholm, Sweden(8) Department of Molecular Biology, University of Salzburg and(9) Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

Peanut allergy may cause life-threatening allergic reactions. Here we investigated immunological patterns of clinical tolerance to peanut in peanut sensitized but asymptomatic patients from central Africa compared to Swedish peanut allergic vs. sensitized but asymptomatic patients.Sera from allergic patients (n=54) from Zimbabwe with IgE to peanut but without symptoms, and sera from peanut allergic (n=25) and sensitized but asymptomatic (n=25) patients from Sweden, were analyzed for total IgE and IgE, IgG and IgG4 reactivity towards Ara h 1-3, 6, 8, 9 using an allergen microarray. Allergenic activity was investigated by basophil activation assays. IgE to Ara h 2 peptide epitopes was analyzed.Fourty-six percent of the African and all peanut allergic Swedish patients showed IgE towards one of the highly allergenic peanut allergens (Ara h 1-3, 6, 9). However, 48% of the African patients had IgE to cross-reacting carbohydrates (CCD) with low allergenic activity and 60% of the Swedish asymptomatic patients had IgE against the PR-protein Ara h 8. Peanut IgE from both peanut asymptomatic patients groups showed very poor allergenic activity compared to IgE from peanut allergic patients. Asymptomatic patients almost completely lacked IgE to Ara h 2 peptide epitopes which were recognized by peanut allergic patients.Natural clinical tolerance to peanut in the African and Swedish patients could be explained by exclusive IgE to low allergenic peanut components e.g. such as profilins, CCD and/or PR-10 proteins and by poor allergenic activity of peanut-specific IgE.

Natural clinical tolerance to peanut in African patients is caused by poor allergenic activity of peanut IgE

Acknowledgements

Supported by the FWF-funded doctoral program IAI and SFB F46, the Swedish Research Council, the Stockholm County Council, the Swedish Heart-Lung Foundation, the Center for Inflammatory Diseases Karolinska Institutet, the Swedish Asthma and Allergy Association’s Research Foundation, the Swedish Cancer and Allergy Foundation, Konsul Th Bergs Foundation, the King Gustaf V 80th Birthday Foundation, the Hesselman Foundation, the Magnus Bergvall Foundation, Karolinska Institutet and the FP7-funded program MeDALL of the European Union.


Abstract 4

Oral Presentation

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Victoriya GARIB, Eva WOLLMANN, Rudolf VALENTA

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna

Background: The prevalence of allergic diseases has increased significantly over the past several decades. The exposure to animal allergens and IgE-sensitization to domestic inhalant allergens is a major risk factor for asthma. Objective: The aim of this study was to determine the IgE-sensitization profiles towards animal allergens in 2 generations of patients with respiratory allergy and their co-sensitizations with pollen allergens. Methods: Sera of 50 adult patients with allergic rhinitis and/or bronchial asthma (young adults; 20-29 years, n=20 and middle aged adults; 30-49 years, n=30) were analyzed using an allergen micro-array containing 112 different allergen molecules (ImmunoCAP ISAC; Thermo Scientific). All patients are native Uzbek from Tashkent metropolis.Results: Significant differences were found in the IgE sensitization patterns in the 2 groups: young patients show higher IgE sensitization rates towards animal allergens (75%) as compared to the elder group (30%). An increase of IgE reactivity from 23% in the middle-aged adults to 61% in younger patients against the uteroglobin rFel d 1 could be detected and mean rFel d 1 specific IgE ISU levels raised from 2.1 to 38.5. The younger generation also displayed specific IgE for the lipocalins rCan f 1, rFel d 4, and nMus m 1, and to the dog prostatic kallikrein rCan f 5, whereas no reactivity against rCan f 2 and rEqu c 1 was detectable. Also in this group IgE sensitization to animal serum albumins (nCan f 3, nEqu c 3, and nFel d 2) could be found. High co-sensitization rates to animal and the grass pollen allergens nCyn d 1 and rPhl p 1 were observed in the young group vs. more frequent co-sensitizations against animal and the weed pollen allergen nSal k 1 in the middle aged adults group. Significant differences were also found in the IgE sensitization patterns between the clinical groups: patients with bronchial asthma were more frequently co-sensitized to cat and dog allergens or cat and house dust mite allergens (28% vs. 10%, and 18% vs. 0% respectively) and showed higher mean IgE levels towards rFel d 1 (31.1 vs. 21.1 ISU-E) compared to the group with allergic rhinitis.Conclusion: In the young generation of patients with respiratory allergy higher rates of IgE sensitizations towards animal allergens were found compared to the elder patient group, most prevalent was the major cat allergen rFel d 1. Occurrence of IgE co-sensitization to unrelated allergens may increase the risk of asthma development and progression.

Differences in molecular sensitization profiles towards animal allergens revealed by allergen micro-array in two generation of patients with respiratory allergy

Acknowledgements

This study was supported by the Austrian Science Fund (FWF) and was performed in the framework of International Network of Universities for Molecular Allergology and Immunology.


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Christina BANNERT(1), Zsolt SZÉPFALUSI(1), Leila RONCERAY(1), Elisabeth MAYER(1), Michaela HASSLER(1), Eva WISSMANN(1), Eleonora DEHLINK(1), Saskia GRUBER(1), Alexandra GRAF(2), Christian LUPINEK(3), Rudolf VALENTA(3), Thomas EIWEGGER(1), Radvan URBANEK(1)

(1) Department of Pediatrics, Medical University of Vienna, Austria(2) Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna, Austria(3) Department of Pathophysiology and Allergy Research, Medical University of Vienna

Prevention of IgE sensitizations by allergen-specific immunotherapy has been described. Prospective data using a preventive approach in children is missing. We initiated a prospective pilot study investigating the safety, immunomodulatory and sensitization-preventive effect of sublingual immunotherapy (SLIT) in sensitized, clinically asymptomatic 2-5 year old children.In this double-blind, randomized, placebo-controlled study 31 mono/oligo-sensitized children to house dust mite or grass pollen were included. SLIT with the respective source (n=15) or placebo (n=16) was applied for 2 years. Skin prick testing and specific IgE and IgG measurements were recorded at baseline, 12 and 24 months. At the same time allergen-specific proliferation and Treg function was measured.Preventive SLIT in young children was safe. After 12 and 24 months of treatment the rate of allergen-specific sensitization was comparable in the verum and the placebo group. However, verum-treated patients displayed an up-regulation of allergen-specific IgG (p<0.05). IL10-dependent inhibition (p<0.05) was observed in vitro in the treatment group but not in the placebo group.We conclude that preventive SLIT is safe in 2-5 year old children and induces regulatory mechanisms involving allergen-specific IgG and IL10.

Preventive sublingual immunotherapy in preschool children: first evidence for safety and pro-tolerogenic effects


Abstract 6

Oral Presentation

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Gerald WIRNSBERGER(1), Florian ZWOLANEK(2), Johannes STADLMANN(1,3), Luigi TORTOLA(1), Shang Wan LIU(1), Thomas PERLOT(1), Päivi JAERVINNEN(4), Gerhard DUERNBERGER(1,3), Ivona KOZIERADZKI(1), Renu SARAO(1), Alba DE MARTINO(5), Kaan BOZTUG(6,7), Karl MECHTLER(1,3), Karl KUCHLER(2), Christoph KLEIN(4), Ulrich ELLING(1) and Josef M. PENNINGER(1)

(1) IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria(2) Medical University of Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, 1030 Vienna, Austria(3) IMP, Institute for Molecular Pathology, 1030, Vienna, Austria(4) Department of Pediatrics, Dr. Von Hauner Children’s Hospital, Ludwig-Maximilians University, Munich, Germany(5) CSF, Campus Science Support Facility, 1030 Vienna, Austria.(6) CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria(7) Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Austria

Neutrophils are key innate immune effector cells essential to fight bacterial and fungal pathogens. Here we report that mice carrying a hematopoietic lineage-specific deletion of Jagunal homolog 1 (Jagn1) cannot mount an efficient neutrophil-dependent immune response to the human fungal pathogen Candida albicans. Global glycobiome analysis revealed marked alterations in the glycosylation of proteins involved in cell adhesion and cytotoxicity of Jagn1-deficient neutrophils. Functional analysis confirmed marked defects in neutrophil migration in response to Candida albicans infection, impaired formation of cytotoxic granules, as well as defective MPO-release and killing of Candida albicans. GM-CSF treatment protected mutant mice from increased weight loss and accelerated mortality after Candida albicans challenge. Importantly, GM-CSF also restored the defective fungicidal activity of bone marrow cells from patients with JAGN1 mutations. These data directly identify Jagn1/JAGN1 as a novel regulator of neutrophil function in microbial pathogenesis and uncover a potential treatment option for human patients.

Jagunal-homolog 1 is a critical regulator of neutrophil function in fungal host defense

Acknowledgements

We thank all members of the Penninger laboratory for helpful discussions and technical support. We thank all members of the IMP-IMBA Biooptics service facility for assistance in cell sorting and image quantification. K.M. is supported by the European Community’s Seventh Framework Programme PRIME-XS (262067) and MeioSys (222883-2) and from the Austrian Science Fund SFB F3402, P24685-B24 and TRP 308-N15. K.K. was supported by a grant of the Medical University of Vienna, and in part by a grant from the FWF (FWF-P-25333-B08). C.K. is supported by the ERC, an EU ERARE program (NEUTRO-NET), the DFG Gottfried-Wilhelm-Leibniz program, DFG-SFB914 (Migration of Immune Cells) and the DZIF (German Center for Infection Research). J.M.P. is supported by grants from IMBA, the Austrian National Foundation, the Austrian Academy of Sciences, NEUTRO-NET, and an EU ERC Advanced Grant.


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Daniela GALLERANO(1), Portia NDLOVU(2), Ian MAKUPE(2), Margarete FOCKE-TEJKL(1), Kerstin FAULAND(3), Eva WOLLMANN(1), Elisabeth PUCHHAMMER-STÖCKL(4), Walter KELLER(3), Elopy N. SIBANDA(5) and Rudolf VALENTA(1)

(1) Division of Immunopathology, Department of Pathophysiology and Allergy Research, Medical University of Vienna, Austria(2) Gamma City Laboratory, Harare, Zimbabwe(3) Institute of Molecular Biosciences - Structural Biology, Karl Franzens University, Graz, Austria(4) Department of Virology, Medical University of Vienna, Austria(5) Asthma, Allergy and Immune Dysfunction Clinic, Parirenyatwa University Teaching Hospital, Harare, Zimbabwe

We studied epitope specificities of antibody responses in HIV-infected patients from Sub-Saharan Africa, where clade C is prevalent, and in patients from Europe, where infections are predominantly caused by clade B. A comprehensive set of HIV-1 clade C recombinant proteins and peptides derived from envelope proteins gp120 and gp41 was prepared, purified and used to measure IgG, IgG-subclass, IgA and IgM responses in the two populations and in African follow-up serum samples. Interestingly, African and European patients showed almost identical antibody reactivity profiles in terms of epitope specificity and involvement of IgG, IgG-subclass, IgA and IgM responses. Among gp120-derived peptides, the major epitopes were a peptide overlapping the V3 region and a C-terminal peptide, which were recognized by IgG1>IgG2=IgG4>IgG3, IgA>IgM and by the four IgG-subclasses, respectively. By contrast, gp41-derived-peptides were mainly recognized by IgG1 but not by the other IgG-subclasses, IgA or IgM. Among non-surface proteins, protease, reverse transcriptase+RNAseH, integrase, capsid and matrix proteins were the most frequently and strongly recognized antigens which showed broad IgG-subclass and IgA reactivity. Immune recognition of gp120 peptides and non-surface proteins involved all four IgG-subclasses and was indicative of a mixed Th1/Th2 immune response. Specificities and magnitudes of antibody responses in African patients were stable during disease and antiretroviral treatment, and persisted despite severe T cell loss. The HIV-1 clade C proteome-based test therefore allowed diagnosis and monitoring of antibody responses during the course of HIV-infections and assessment of specificity, isotype and subclasses.

Mapping of IgG, IgG-subclass, IgA and IgM reactivity profiles in African and European HIV-infected individuals with an HIV-1 clade C proteome-based array

Acknowledgements

This study was supported by a research grant from Biomay AG, Vienna, Austria.


Abstract 8

Oral Presentation

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Leonhard X. HEINZ(1), Christoph L. BAUMANN(1), Marielle KÖBERLIN(1), Berend SNIJDER(1), Manuela BRUCKNER(1), Riem GAWISH(1), Omar SHARIF(1), Kumaran KANDASAMY(1), Jacques COLINGES(1), Keiryn L. BENNETT(1), Astrid FAUSTER(1), Guanghou SHUI(2), Sylvia KNAPP(1), Markus R. WENK(2) and Giulio SUPERTI-FURGA(1)

(1) CeMM, Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria(2) Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore

Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and conspire to safeguard cellular homeostasis. Therefore, modulation of the cellular lipid landscape offers an excellent opportunity to globally adapt to environmental changes. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory processes that need to be tightly controlled. Here we report the identification of a lipid-modifying enzyme as novel GPI-anchored negative regulator of TLR signaling. Loss-of-function in macrophages led to a higher responsiveness upon TLR stimulation and knock-out mice manifested enhanced pro-inflammatory responses in TLR-dependent models, confirming our data in vivo. Revealing a role in lipid metabolism, knockdown of the enzyme profoundly changed both the global lipid composition and membrane fluidity. Lipidomics-based functional mapping of changes in lipid species abundance were predictive of its anti-inflammatory properties, mechanistically linking membrane lipid composition and innate immune signaling. Taken together, our results identify a membrane-modulating enzyme as novel regulator of innate immune signaling.

Identification of a lipid-modifying enzyme as novel regulator of innate immune responses

Acknowledgements

This work was funded by the Austrian Academy of Sciences and the European Research Council.

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Age-related differences of humoral and cellular immune responses to primary Japanese Encephalitis vaccination A. WAGNER(1), E. GARNER-SPITZER(1), J. JASINSKA(1), M. PAULKE-KORINEK(1), M. HOFER(1), K. STIASNY(2), F. X. HEINZ(2), H. KOLLARITSCH(1), U. WIEDERMANN(1, 3)

(1)InstituteofSpecificProphylaxisandTropicalMedicine,MedicalUniversityofVienna,Kinderspitalgasse 15, 1090 Vienna, Austria(2) Medical University of Vienna, Department of Virology, Vienna, Austria(3)DepartmentofRheumatology&InflammationResearch,InstituteofMedicine,UniversityofGöteborg, Sweden

Immunosenescence is associated with reduced B and T cell responses. It has been shown that booster vaccination is less effective in the elderly. However data on the efficacy of primary immunisation in the elderly is sparse. In a monocentric, open label, phase IV study we investigated humoral and cellular immune responses to primary Japanese Encephalitis (JE) vaccination in young (18-40 y) and elderly subjects (>60 y).All subjects (30/ group) received a primary course of an inactivated JE- vaccine. Neutralising antibody titers (NT) were determined in serum samples taken before vaccination, 1 and 5 weeks after the 2nd dose. PBMC´s were isolated prior to the 1st and 1 week after the 2nd dose for analysis of different T- and B-cell subsets and antigen restimulation.JE-NT´s were significantly lower in elderly compared to young participants. Furthermore 47% of the elderly were no/low-responders to JE-vaccination. Reduced humoral immune responses were associated with reduced cytokine production (e.g. IFN-g) in vitro and a significant increase of Treg cells in the elderly. Additionally, higher frequencies of late-differentiated effector and effector memory cells were detected in elderly. The majority of elderly subjects were seropositive for CMV, which correlated with reduced antibody


Abstract 10

Oral Presentation

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Barbara KLEIN(1,2), Richard WEISS(3), Sebastien COUILLARD-DESPRES(2,4), Josef THALHAMER(3), Ludwig AIGNER(1,2)

(1) Institute of Molecular Regenerative Medicine, Paracelsus Medical University Salzburg(2) Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University Salzburg(3) Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg(4) Institute of Experimental Neuroregeneration, Paracelsus Medical University Salzburg

Our aim was to investigate the effect of an acute allergic inflammation on the hippocampal neurogenic niche in C57BL/6 mice. For sensitization, we used the timothy grass pollen allergen Phl p 5 and aluminum hydroxide followed by intranasal challenges with Phl p 5 four days before the end of the experiment. Blood levels of Phl p 5-specific IgE, IgG1 and IgG2a confirmed sensitization. Immunohistochemical analyses of proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine (BrdU), injected four weeks before the end of the experiment, showed that the allergy group had higher cell proliferation in the hippocampal neurogenic niche compared to controls. Co-labelling of BrdU with NeuN (a marker for mature neurons) or glial fibrillary acidic protein (GFAP), showed that numbers of newly generated neurons (NeuN+) and GFAP+ astrocytes increased significantly in comparison to controls. In the same region, also more immature neurons expressing doublecortin (DCX) were found. Interestingly, fewer cells were positive for ionized calcium binding adaptor molecule-1 (Iba-1), a marker for microglia, and their soma was smaller. These results indicate that an acute allergic inflammation not only leads to altered microglial activity in the hippocampal dentate gyrus, but also affects neurogenesis.

The impact of an acute allergic inflammation on neurogenesis and microglia in the hippocampal dentate gyrus

Acknowledgements

This project is supported by the Research Fund of the Paracelsus Medical University Salzburg (PMU-FFF) as a RISE Project under grant agreement R-13/02/046-KLE.


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MACHADO Yoan(1), MAYR Melissa(1), THALHAMER Theresa(1), HÖPFLINGER Veronika(1), SCHEIBLHOFER Sandra(1), THALHAMER Josef(1), WEISS Richard(1)

(1) University of Salzburg, Department of Molecular Biology, Division of allergy and Immunology, Hellbrunnerstrasse 34, Salzburg

Due to its abundance of APCs, the skin represents an attractive target tissue for specific immunotherapy. Recently, we demonstrate the feasibility of efficiently targeting APCs through C-type lectin receptors using hypoallergenic neoglycoconjugates of papain with mannan. Here, a panel of polysaccharides, mannan, laminarin, glucomannan, inulin and dextran were efficiently coupled to Bet v 1.0101. Mice were immunized with these neoglycoconjugates via laser porated skin and the immunogenicity and cytokine profile elicited were measured. Allergenicity of Bet v 1.0101 glycoconjugates could be modulated by varying the sugar-to-protein ratio achieving an up to 1000-fold reduction in IgE cross linking capacity. In vitro uptake experiments using BMDCs revealed an increased uptake for Bet-Mannan neoglycoconjugates compared to Bet v 1.0101. Mice immunized with these neoglycocojugates showed high IgG1 and IgG2a antibodies. Bet-Mannan neoglycoconjugates shifted the immune response to a Th1/Th17 pattern evidenced by large amount of IL-17 and IFN-γ secreted by Bet v 1.0101 re-stimulated splenocytes isolated from immunized mice. These data suggest coupling carbohydrates to Bet v 1.0101 as an attractive approach for pollen allergen specific cutaneous immunotherapy.

Carbohydrate coupling as a tool for modulation of the allergenicity and immunological properties of the major birch pollen allergen Bet v 1.0101

Acknowledgements

This work was supported by the Austrian Science Fund (FWF Project # W1213).

Abstract 12

Oral Presentation

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Barbara GEPP(1), Nina LENGGER(1), Christian RADAUER(1), Florian GRUBER(2), Michael MILDNER(2), Heimo BREITENEDER(1)

(1) Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna (2) Department of Dermatology, Medical University of Vienna

Lipids accompanying or binding to allergens can be immunomodulating. Therefore, we analyzed the ability of the major birch pollen allergen, Bet v 1, to bind lipids from peanut, birch or grass pollen and tested the phosphorylation of the transcription factor CREB in primary keratinocytes after stimulation with Bet v 1 alone or Bet v 1 in combination with lipids. Total lipids from peanut, birch and grass pollen were extracted using chloroform/methanol. The binding of lipids to the hydrophobic cavity of Bet v 1 was verified by adding 1-anilinonaphthalene-8-sulfonic acid (ANS) and measuring the decrease of fluorescence at 484 nm. Phosphorylated CREB (p-CREB) levels were analyzed by Western Blot. After incubation of Bet v 1 with lipid extracts from both pollen species but not from peanut, a concentration dependent reduction of ANS binding was observed. Furthermore, Bet v 1 in combination with birch pollen lipids showed higher levels of p-CREB in keratinocytes compared with birch pollen lipids alone.The increased phosphorylation of CREB after treatment with Bet v 1 in combination with birch pollen lipids indicates that Bet v 1 may act as a transport vehicle able to release lipids to cell membranes and therefore providing a potential danger signal during the sensitization phase.

Phosphorylation of CREB is upregulated in keratinocytes after treatment with the major birch pollen allergen, Bet v 1, and birch pollen lipids

Acknowledgements

Supported by grant SFB-F4608 (HB) from the Austrian Science Fund.

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RAFFAELA CAMPANA(1), Katharina MORITZ(2), Angela NEUBAUER(3), Hans HUBER(3), Rainer HENNING(3), Katharina BLATT(4), Gregor HOERMANN(5), Tess M. BRODIE(6), Alexandra KAIDER(7), Peter VALENT(4), Federica SALLUSTO(6), Stefan WÖHRL(2), Rudolf VALENTA(1,*)

(1) Division of Immunopathology, Department of Pathophysiology, Center of Physiology and Pathophysiology, Vienna General Hospital (AKH), Medical University of Vienna, Austria; (2) Division of Immunology, Department of Dermatology, Allergy and Infectious Diseases, Vienna General Hospital (AKH), Medical University of Vienna, Austria; (3) Biomay AG, Vienna, Austria; (4) Division of Hematology and Hemostaseology, Department of Internal Medicine I, Vienna General Hospital (AKH), Medical University of Vienna, Austria; (5) Department of Laboratory Medicine, Vienna General Hospital (AKH), Medical University of Vienna, Austria; (6) Cellular Immunology Laboratory, Institute for Research in Biomedicine, Bellinzona, Switzerland; (7) Center for Medical Statistics, Informatics and Intelligent Systems, Section for Clinical Biometrics, Medical University of Vienna, Austria.

Allergen-specific immunotherapy (SIT) is the only allergen-specific and disease-modifying treatment for allergy but can induce side effects and suffers from inconvenient administration protocols. We have conducted a clinical trial using rBet v 1 and two hypoallergenic rBet v 1 fragments for epicutaneous administration (i.e., atopy patch testing) in 30 adult subjects (15 birch pollen allergic patients suffering from AD, 5 birch pollen-related RC patients, 5 allergic patients without birch pollen allergy and 5 non-allergic individuals). Blood samples were collected before and 6-8 weeks after application and used to compare allergen-specific IgE and IgG antibody levels, T cell and cytokine responses. Epicutaneous administration of rBet v 1 and rBet v 1 derivatives leads to a significant boosting of allergen-specific T cell proliferation and IgG production mainly in APT-positive birch pollen allergic patients, but not IgE production. In these patients significant increases in skin-homing CLA+ and CCR4+ T cells were observed after allergen administration. No systemic side effects were observed. Our results demonstrate that epicutaneous application of recombinant allergens boosts allergen-specific T cell and IgG antibody responses and thus may be considered as a possible route for SIT.

Epicutaneous application of recombinant Bet v 1 and Bet v 1 derivatives induces allergen-specific IgG and T cell responses

Acknowledgements

Supported by the Austrian Science Fund (FWF), Vienna, Austria.


Abstract 14

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Guido A. GUALDONI(1), Tilman R. LINGSCHEID(2), Peter STEINBERGER(1), Gerhard J. ZLABINGER (1)

(1) Institute for Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna(2) Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité -- Universitätsmedizin Berlin, Germany

Macrolides are a class of antibiotics widely used in the treatment of infectious diseases, azithromycin being one of the most prominent representatives. In addition to its anti-infective qualities, immune-modulating properties have been attributed to these substances, an effect which is being increasingly exploited in clinical medicine. However, the molecular impact on immune cell function remains poorly characterized. Investigating the cytokine-modulatory properties of azithromycin in human monocytes, we found a specific inhibition of IL-1 beta secretion upon LPS stimulation, whereas no significant effect was seen upon flagellin stimulation, thus indicating a specific effect on the NLRP3 inflammasome. Coherently, we found inhibition of caspase-1 cleavage whereas NF-kappaB signaling was unaffected. In contrast, other macrolides tested, did not alter cytokine production. Furthermore, our findings were confirmed in a murine endotoxin sepsis model, in which azithromycin treatment resulted in a specific IL-1 beta down-modulation and enhanced survival. Overall, we provide evidence of a novel facet of azithromycin’s immune-modulation which might help to better understand clinical observations and lead to novel applications of this well established drug.

Azithromycin inhibits IL-1 beta secretion of innate immune cells by specificly inhibiting the NLRP3 inflammasome

Acknowledgements

The authors thank Petra Waidhofer-Söllner for excellent technical assistance.


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Anastasia MESHCHERYAKOVA(1), Erika BAJNA(1), Susanne WÖHRER(1) ElfiSCHARF(1),MartinSVOBODA(1),ErikaJENSEN-JAROLIM(1,2),GeorgHEINZE(3),PeterBIRNER(4), Diana MECHTCHERIAKOVA(1)

(1) Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology & Immunology, Medical University of Vienna, Austria(2) Messerli Research Institute of the Medical University of Vienna, Veterinary University of Vienna and University of Vienna, Austria(3) Center for Medical Statistics, Informatics, and Intelligent Systems, Medical University of Vienna, Austria(4) Clinical Institute of Pathology, Medical University of Vienna, Austria

Ovarian cancer comprises a highly heterogeneous multifactorial disorder. Increasing attention is given to the role of local inflammation and tumor infiltrating immune cells in disease etiology. In the current study we asked whether patient-specific immunological imprint might be used as prognostic marker for estimation of disease development and clinical outcome. To get comprehensive overview, we examined large cohort of patients with primary ovarian cancer (n=67). Microscopy-based platform was used for automatic scanning and follow up quantitative analysis. Focus of the study was given to the CD45-positive leukocyte population, CD20-positive B lymphocytes and CD68-positive monocytes/macrophages. Unique advantage of this system provides opportunity to align the quantities of individual immune cell sub-populations with their spatial distribution accounting for tumor anatomy. Staining-derived datasets are used for alignment with clinical parameters and for building up of prognostic models, thus reflecting the individual impact of B-cell and macrophage lineages and their potential mutual interconnection. Such strategy represents an innovative approach toward patient’ stratification for prognostic monitoring and might open new directions for patient-orientated therapeutic interventions.Key words: Tumor immunology, patient-orientated immunological imprint

Patient-specific immunological imprint within complex ovarian cancer tissues

Acknowledgements

The work was supported by The Austrian Science Fund FWF P22441-B13 and P23228-B19


Abstract 16

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Innate responses and antigen-presenting capacity of oral epithelial cellsClaudia KITZMÜLLER(1), Martina SCHUSCHNIG(1), and Barbara BOHLE(1)

(1) Christian Doppler Laboratory for Immunomodulation, Department of Pathophysiology and Allergy Research, Medical University Vienna

Background: Epithelial cells of the oral cavity have important barrier functions and might be involved in the development of oral tolerance. Little is known about their possible role in the modulation of immune responses.

Aim: To analyse whether oral epithelial cells react to pathogen-associated molecular patterns (PAMPs) and up-regulate components of the antigen-processing and -presentation machinery under inflammatory or infectious conditions.

Methods: We analysed the expression of Toll-like receptors (TLR), NOD-like receptors (NLR) and C-type lectin receptors (CLR) by the buccal epithelial cell line HO-1-N-1, the sublingual epithelial cell line HO-1-u-1 as well as by primary human oral keratinocytes (HOK) using PCR and studied the functional response of receptor stimulation by IL-8 ELISA. We used Western Blot and flow cytometric analysis to check whether the cell lines up-regulate Cathepsin S and MHC class I and II molecules in response to direct stimulation with IFN-gamma and indirect stimulation by PAMPs.

Results: HO-1-N-1 expressed mRNA of TLR 1, 2, 4, 6 and NOD1 and increased IL-8 production after stimulation of TLR 2, 3, 4 and NOD1. HO-1-u-1 expressed mRNA of TLR 1, 3, 4, 5, 6, and NOD1 and secreted elevated levels of IL-8 after stimulation of TLR 3 and 5. HOK expressed mRNA of all the TLR and NLR analysed and reacted strongly to TLR 3 stimulation and less to a TLR 2 ligand. Direct stimulation with IFN-g led to an up-regulation of Cathepsin S and an increased expression of MHC class I and II molecules on the surface HO-1-N-1 and HOK. In ongoing experiments we found so far that HOK can increase the levels of MHC class I molecules also after stimulation of TLR 3.

Conclusion: Oral epithelial cells can be stimulated by TLR ligands. Depending on whether their origin is from buccal and sublingual regions they show differences to which stimuli they react to. Under inflammatory and infectious conditions oral epithelia could have antigen-presenting potential.

Acknowledgements

Supported by the Christian Doppler Research Association, BiomayAG, Austria, and the Austrian Science Fund, projects SFB F4610, W1212 and W1213


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Douglas F. PINHEIRO(1), Megan M. MAURANO(1), Abul K. ABBAS(3), Iris K. GRATZ(1,2,4)

(1) Department of Molecular Biology, University of Salzburg, Salzburg, Austria(2) Department of Dermatology, University of California San Francisco, San Francisco,CA 94143, USA(3) Department of Pathology, University of California San Francisco, San Francisco,CA 94143, USA(4) Division of Molecular Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria

Immune homeostasis in tissues is achieved by maintaining a delicate balance between pathogenic effector T cells (Teff) and protective regulatory T cells (Treg) specific for tissue-antigens (Ags). The factors responsible for maintaining this balance are largely unknown. Previous findings from our group suggest that tissues acquire the ability to attenuate autoimmune reactions upon repeated exposure to Ags. However, the mechanism by which this occurs is unknown.To study immune regulatory mechanisms in a T cell mediated autoimmune disease, we established two mouse models that feature tetracycline-inducible expression of chicken ovalbumin (Ova) in the epidermis. Expression of Ag in the skin elicits a T cell dependent inflammatory dermatitis. Using this model, we found that Ag persistence is a major determinant of the relative frequencies of Teff and Treg cells. Persistent Ag, a mimic of self Ag, lead to functional inactivation and loss of the Teff cells with preservation of Treg cells in the target tissue, both of which was associated with reduced ERK phosphorylation. Additionally, we found that the Ag dose determines early T cell differentiation decisions in vivo. These findings provide a framework for understanding how Treg and Teff cells respond to self-Ag in peripheral tissues.

Controlling the balance between effector and regulatory T cells in peripheral tissues

Acknowledgements

This work has been supported by grants to Iris K. Gratz from the NIH (AR064554), the Austrian Science Fund (FWF) (J2997-B13) and by the Dystrophic Epidermolysis Bullosa Research Association (DEBRA) International and DEBRA Austria.

Abstract 18

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Mariana VÁZQUEZ-STRAUSS, Georg STINGL

DIAID, Medical University of Vienna, Vienna, Austria.

As opposed to other solid carcinomas, melanoma is sometimes amenable to immunotherapy. In fact, we found that some, but not other, melanoma cell lines undergo lysis when treated with the soluble form of the lytic molecule TNF-related apoptosis-inducing ligand (TRAIL) and that the non-steroidal anti-inflammatory drug (NSAID) diclofenac can sensitize otherwise resistant cell lines to TRAIL-induced apoptosis. When we addressed the mechanisms underlying the latter phenomenon, we found that diclofenac induces upregulation of (i) the pro-apoptotic receptor TRAIL-R2 and (ii) pro-apoptotic molecules such as caspases 5 and 10, NOXA, CYCS, CHOP and XBP1 in melanoma cells. Surprisingly enough, Diclofenac proved to be equally effective as imiquimod in upregulating TRAIL expression on diverse leukocyte subpopulations (e.g. monocytes, mDCs, pDCs). In light of these results it appears that the anti-tumoral effects of diclofenac and probably also other NSAIDs are due to, at least, a bimodal action, i.e. by increasing the susceptibility towards lysis of the tumor cells as well as the tumoricidal potential of leucocytic effector cells.

Dual Role of NSAIDs: overcoming melanoma resistance and enhancing cytotoxic potential of innate immune cells.

Acknowledgements

FWF PhD Programm “Inflamation and Immunity”.

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Specific roles for dendritic cell subsets during initiation and progression of psoriasisElisabeth GLITZNER(1), Ana KOROSEC(1), Patrick M. BRUNNER(2), Barbara DROBITS(1), Nicole AMBERG(1), Helia B. SCHONTHALER(3), Tamara KOPP(2), Erwin F. WAGNER(3), Georg STINGL(2), Martin HOLCMANN(1), Maria SIBILIA(1)

(1) Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria(2) Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University of Vienna, Vienna, Austria(3) BBVA Foundation–CNIO Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

Several subtypes of dendritic cells (DCs) are found in skin lesions of psoriasis patients, but their involvement in disease pathogenesis is poorly understood. Here, we investigated a contribution of Langerhans cells (LCs) and plasmacytoid DCs (pDCs) in psoriasis. In both human psoriatic lesions and a psoriasis mouse model (DKO* mice), LC numbers are severely reduced, whereas pDC numbers are increased. Depletion of pDCs in DKO* mice prior to psoriasis induction resulted in a milder phenotype, whereas pDC depletion during established disease had no effect. In contrast, depletion of Langerin+ APCs before disease onset did not affect psoriasis development, whereas depletion from psoriatic DKO* mice aggravated disease symptoms. Using bone marrow (BM) chimeric mice, we found that disease aggravation was due to absence of LCs, but not other Langerin+ APCs, and that LC replenishment in psoriasis occurred from BM-derived precursors. LCs produced high levels of IL-10 in psoriatic mice, and LC depletion resulted in increased epidermal IL-23. In the absence of pDCs, IL-23 production was reduced, and blocking of IL-23R signaling greatly ameliorated disease symptoms. These results demonstrate that LCs exert an anti-inflammatory function during active psoriatic disease, while pDCs have an instigatory effect during disease initiation via IL-23 production.


Abstract 20

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The role of Langerhans cells and natural killer cells in cancer immune surveillanceDaniela ORTNER(1), Christoph TRIPP(1), Nicole AMBERG(2), Maria SIBILIA(2), Björn E. CLAUSEN(3) and Patrizia STOITZNER(1)

(1)Department of Dermatology and Venereology, Innsbruck Medical University, Innsbruck, Austria(2)Department of Medicine I, Institute for Cancer Research, Medical University of Vienna, Vienna, Austria(3) Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany

Non-melanoma skin cancers like basal cell carcinoma (BCC) are the most common skin tumors. These epithelial tumors develop in the basal layers of the epidermis due to UV exposure or chemical carcinogenesis. The aberrant activation of the hedgehog signaling pathway has been detected as one of the major genetic causes for BCC. A transgenic mouse model harboring an inducible activating allele of the hedgehog protein Smoothened (R26-SmoM2) develops skin alterations similar to BCC. We have attained first results using this mouse model showing that natural killer cells (NK) and Langerhans cells (LC) disappear from skin during tumorigenesis. Mice developing BCC were crossed with a mouse model that allows depletion of langerin-positive skin DC, which led to a higher tumor burden. These observations indicate that NK cells and LC are important for immunosurveillance early in tumorigenesis. Investigating the cytokine milieu in skin upon tumor progression showed an enhanced inflammatory and tumor-permissive environment with significantly increased IL-1β, TNF-α and TGF-β mRNA levels. Furthermore, tumor-bearing skin contained a huge population of CD11b+ Gr1+ cells, most likely myeloid derived suppressor cells. The interactions of these various immune cell types in the tumor situation need to be further investigated to understand how innate immune responses are altered during carcinogenesis. The elimination of NK cells and LC could be a potent tumor escape mechanism in BCC.


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Nazanin SAMADI, Claudia KITZMÜLLER , Barbara BOHLE, Rene GEYEREGGER, Beatrice JAHN-SCHMID

Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna

Allergy is the abnormal immune response against harmless antigens. Like any immune response, allergic reactions are mediated by both innate and adaptive immunity. T cells are part of adaptive immunity and play a main role in the development of type I allergy. The function of CD4+T cells in the pathogenesis and maintenance of allergic disorders have been broadly investigated while the role of CD8+T cells is poorly understood. Previous studies have demonstrated a controversial function of CD8+T cells in allergic disorders in human and murine models. The aim of this project is to identify and characterize allergen-specific CD8+ T cells in patients with different manifestations (rhinoconjunctivitis, atopic dermatitis and atopic bronchial asthma) of type I allergy. Different seasonal and perennial allergens will be studied. Allergen specific T cells from PBMCs will be expanded in vitro and these T cell lines and cloned T cells will be characterized for their phenotype and function. By now, we have optimized the expansion of allergen-activated CD8. Peripheral blood from patients were stained by proliferation dyes and stimulated with allergen. Proliferating cells were expanded by addition of growths factor/s in short time cultures and characterized. We could find significant CD8+T cell proliferation in all allergic diseases, but larger numbers of allergen-reactive CD8+T cells were found in T cell lines induced by cat hair and house dust mite extracts compared to pollen extracts from birch and grass. Further experiments are needed to identify the functionality of these allergen-reactive CD8+ T cells.

Characterization of the CD8+ T cell response to allergens

Acknowledgements

Supported by the Austrian Science Funds, project W1248.

Abstract 22

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Does signal one strength influence the phenotype and function of human allergen-specific T lymphocytes?Martín R. CANDIA(1), Peter TAUBER(1), Alina NEUNKIRCHNER(1,2), Doris TRAPIN(1), Sandra ROSSKOPF(1), Peter STEINBERGER(1) and Winfried F. PICKL(1,2).

(1) Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Christian Doppler Laboratory for Immunomodulation, Medical University of Vienna, Vienna, Austria.

TCR signal strength is a major relaying mechanism to control T cell differentiation and function. Since allergic sensitization but also late phase allergic reactions have been linked to altered CD4+ T cell function, we sought to investigate the contribution of TCR signal strength to the differentiation of allergen-specific CD4+ Th cells. We here used a recently developed Art v 1 TCR/HLA-DRB1*01:01 double transgenic system to define possible altered peptide ligands (APL) of the major mugwort pollen allergen, Art v 1(24-35). Furthermore, variation of the amount of antigenic peptides as well as targeted pharmacological inhibition of T cell signaling pathways was used to alter signal strength. For the quick and facile determination of the binding characteristics of APL a robust, flow cytometry-based assay for peptide binding to cell surface expressed HLA molecules was established. So far, two APL with differential T cell activating capabilities as determined by proliferation and cytokine secretion assays have been characterized. Moreover, some targeted drugs could be identified, which resulted in differential cytokine production in allergen-specific T cells. In summary, altering TCR signal strength might represent a useful way how to shape allergen-specific T cellular immune responses.

Acknowledgements

Supported by the Austrian Science Fund (FWF) project DK-W1248-B13 (as part of the PhD program Molecular, Cellular and Clinical Allergology, MCCA of the Medical University of Vienna), SFB F46909-B19 and Biomay AG.


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Susanne C. DIESNER(1,2), Cornelia SCHULTZ(1), Chloé ACKAERT(3), Gertie J. OOSTINGH(4), Anna ONDRACEK(1), Caroline STREMNITZER(1), Denise HEIDEN(1), Josef SINGER(1), Franziska ROTH-WALTER(1,5), Judit FAZEKAS(1), Thomas EIWEGGER(2), Zsolt SZÉPFALUSI(2), Erika JENSEN-JAROLIM(1,5), Ernst-Hanno STUTZ(3), Albert DUSCHL(3) and Eva UNTERSMAYR(1)

(1) Department of Pathophysiology and Allergy Research, Divison of Comparative Immunology and Oncology, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.(2) Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.(3) Department of Molecular Biology, University of Salzburg, Salzburg, Austria.(4) Biomedical Sciences, Salzburg University of Applied Sciences, Puch/Salzburg, Austria.(5) Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria.

We reported that nitrated food proteins have a reduced de novo sensitization capacity. Conversely, in vitro experiments showed an enhanced effector cell triggering capacity. Thus, we aimed to investigate the influence of nitration on the effector phase of food allergy.BALB/c mice were repeatedly immunized intraperitoneally with the milk allergen beta-lactoglobulin (b-LG) or the egg allergen ovomucoid (OVO) followed by two intragastric feedings. Thereafter, mice were systemically challenged with untreated, sham-nitrated or nitrated b-LG or OVO to evaluate their allergic response. The anaphylaxis marker mMCP1 was significantly elevated only in b-LG allergic animals challenged with nitrated b-LG compared to those challenged with untreated or sham-nitrated b-LG. This was confirmed by a significant drop of body temperature. Interestingly, this effect was not observed for OVO. Circular dichroism and SDS-PAGE analysis revealed an impact of nitration on the secondary structure exclusively for b-LG together with enhanced protein dimerization. Our data indicate that nitration differently affects the food allergens b-LG and OVO. In case of b-LG, nitration influenced the secondary structure and favoured dimerization causing an enhanced anaphylactic capacity in b-LG allergic animals.

Nitrated Beta-Lactoglobulin enhances the anaphylactic response in a murine food allergy model

Acknowledgements

Supported by the Austrian science fund project P21577-B11 and by the Wirtschaftkammerpreis 2014.


Abstract 24

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The trimeric complex of recombinant CD23 together with allergen and allergen-specific IgERegina M. SELB(1), Julia ECKL-DORNA(1), Christian LUPINEK(2), Birgit LINHART(2), Andrea TEUFELBERGER(3), Walter KELLER(3), Kenneth H. ROUX(4), Rudolf VALENTA(2) and Verena NIEDERBERGER(1)

(1)Department of Otorhinolaryngology, Medical University of Vienna, Vienna, Austria(2)Divison of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria(3)Institute of Molecular Biosciences, Karl Franzens University, Graz, Austria(4)Florida State University, Tallahassee, Florida, USA

CD23, the low affinity receptor for IgE, plays an important role in allergy. One major function is the IgE-facilitated allergen presentation to T cells and subsequent activation of allergen-specific T cells. Four CD23 protein versions were recombinantly expressed in SF9 insect cells. The characterisation via circular dichroism (CD) spectra and gel filtration showed folded, monomeric proteins. Binding of both monomeric human monoclonal and polyclonal serum IgE as well as IgE in complex with birch pollen allergen Bet v 1 to CD23 was demonstrated by ELISA and by surface plasmon resonance. We performed negative stain electron microscopy of the three molecules alone (i.e., CD23, monoclonal human IgE, Bet v 1) and after complex formation. After addition of Bet v 1 allergen (17 kDa) to monoclonal IgE (190 kDa) we observed an extension of one or both Fab arms of the antibody. Further addition of recombinant CD23 molecules (35 kDa) to the complex resulted in thickening of the antibody’s Fc structure in our pictures.In summary, we report the in vitro formation of a tri-molecular complex consisting of recombinant CD23, monoclonal allergen-specific IgE and Bet v 1 and take a first step towards the visualization of this complex by negative stain electron microscopy.

Acknowledgements

Supported by grants 4605, 4613 and in part by 4604 and P23350-B11 of the Austrian Science Fund (FWF).


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Marion STEGER(1), Emina JUKIC(1), Wilfried POSCH(1), Cornelia LASS-FLÖRL(1), Hubertus HAAS(2), Doris WILFLINGSEDER(1)

(1) Division of Hygiene and Medical Microbiology, Innsbruck Medical University(2) Division of Molecular Biology, Innsbruck Medical University, Innsbruck, Austria

In this study, interactions of dendritic cells (DCs) with differentially opsonized and non-opsonized Aspergillus fumigatus strains were investigated. Two different mutants were used, one lacking the pigment melanin (pksP mutant) and one the hydrophobin layer (rodA mutant). Opsonization pattern of the different strains, binding and internalization by DCs as well as cytokine secretion were studied. We demonstrated that melanin has the highest impact on the fungal virulence compared to the wildtype Aspergillus strain. Surprisingly, the rodlet layer showed a minor impact on complement (C) activation. C coating of conidia enhanced these processes compared to their non-opsonized counterparts independent on the fungal strain used. These data revealed, that melanin is one of the key effectors of masking C deposition and binding of conidia by DCs. Opsonization of swollen conidia favored the uptake and internalization and production of pro-inflammatory cytokines, resulting in a favorable TH1 immune response. These in vitro studies propose DCs or neutrophils in combination with complement opsonins as possible vaccines against invasive aspergillosis.

Complement opsonization of A.fumigatus modifies dendritic cell maturation and up-take

Abstract 26

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Failure of specific immunotherapy (SIT) with house dust mite (HDM) allergen extracts due to lack of HDM allergens capable of inducing specific IgG responses in the therapeutic extractsKuan-Wei CHEN (1), René ZIEGLMAYER (2), Petra ZIEGLMAYER (2), Patrick LEMELL (2), Friedrich HORAK (2), Rudolf VALENTA (1), Susanne VRTALA (1)

(1) Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria(2) Vienna Challenge Chamber, Allergy Center Vienna West, Austria

SIT with HDM allergen extracts is often not effective.We performed a retrospective blinded analysis of sera from hundred HDM allergic patients who had been treated with a HDM allergoid extract (n=52) or with placebo (n=48) for 51 weeks regarding IgE- and IgG-reactivity to a broad panel of micro-arrayed HDM allergens (nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 10, rDer p 11, rDer p 14, rDer p 15, rDer p 18, rDer p 21 and rDer p 23). Sera obtained before (week 0), during (week 23) and after SIT (week 51) were analyzed for allergen-specific IgE- and IgG-reactivity.Besides Der p 1 (77.9%) and Der p 2 (93.9%), patients frequently have IgE against Der p 4 (52.0%), Der p 5 (60.9%), Der p 7 (43.5%), Der p 21 (44.7%) and Der p 23 (78.8%). During the course of SIT, only Der p 1- and Der p 2-specific IgG levels increased significantly in the verum group whereas for all other tested allergens no induction of specific IgG antibodies was observed, indicating that the vaccine lacked these allergens in an immunologically active form. Interestingly, the assessment of clinical efficacy based on registration of the total nasal symptom scores (TNSS) of the patients determined by allergen exposure in an allergen exposure chamber before, during and after treatment showed that only patients sensitized exclusively to Der p 1 and/or Der p 2 showed a significant reduction of TNSS as compared to the placebo group, whereas patients sensitized also to other HDM allergens showed no significant improvement.Our results thus demonstrate that failure of SIT with HDM extracts can be due to lack of HDM allergens in therapeutic vaccines capable of inducing specific IgG responses.


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Michaela MITTERMEIR, Elisabeth MAIER, Stefanie ESS, Albert DUSCHL, Jutta HOREJS-HOECK

University of Salzburg, Department of Molecular Biology, Hellbrunnerstraße 34, 5020 Salzburg

Interleukin (IL-) 31 is a member of the IL-6 cytokine family. The human IL-31 receptor complex consists of OSMR-beta and the IL-31 receptor alpha (IL-31RA), of which five validated splice variants (two short and three long isoforms) are known. Compared to the short isoforms, the three long isoforms possess several tyrosine residues for STAT activation. To analyze the signaling capabilities of all IL-31RA isoforms, we cloned and transfected them into the human cell lines HeLa, HEK293 and Hep3B, all of which differ in their endogenous OSMR-beta expression. Reportergene-assays and Western Blotting in HeLa cells showed a dose-dependent STAT3 activation upon induction with IL-31. Similar experiments in the two other cell lines showed that co-expression of the second receptor chain OSMR-beta is essentially required for functional IL-31 receptor signaling. To identify the specific tyrosine residues responsible for signal transduction we introduced amino acid exchanges into the OSMR-beta chain. In addition, we identified the essential amino acids for JAK-binding and functional IL-31R signaling. Taken together, this study shows that long as well as short isoforms of human IL-31RA are capable of inducing STAT signaling. However direct interaction with OSMR-beta is required.

Signaling capacities of the IL-31 receptor complex

Acknowledgements

This study is supported by the Austrian Science Fund, FWF, grant number P23933.

Abstract 28

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Madhura MODAK, Petra CEJKA, Petra WAIDHOFER-SÖLLNER, Sabrina JUTZ, Otto MAJDIC, Peter STEINBERGER, Gerhard ZLABINGER, Johannes STÖCKL

Institute of Immunology, Medical University of Vienna, Vienna, Austria

CD43 is one of the abundant glycoproteins expressed on T cells. CD43 has been demonstrated to act as not only a potent co-receptor but also a negative regulator for T cell activation. To further investigate the role of CD43 in T cell activation and subsequent function, peripheral blood T cells were activated via two distinct CD43 epitopes recognized by monoclonal antibodies (mAbs) namely 6E5 and 10G7 along with TCR signaling. Both the CD43 mAbs were shown to be potent co-stimulators for T cell activation when cross-linked but they exert differential downstream effect upon ligation including differential activation of downstream signaling pathways, T cell cytokine production and also distinct effector function. T cells activated via 10G7 mAb were poorly restimulated and further acquired suppressive function compared to T cells activated either via 6E5 or via CD28. Thus, identifying a novel pathway to induce immune inhibitory T cells. Furthermore, inhibitory T cells do not directly act on responder T cells, but rather exhibit their effect via dendritic cells when added to allogenic mixed leukocyte reaction. Together our data suggests a unique role of CD43 in bidirectional polarization of T cells immunity, depending on its targeted epitope.

Bidirectional polarization of T cell function via CD43

Acknowledgements

The authors would like to thank Claus Wenhardt for his expert technical assistance


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Memory T cells and plasma cells niches in human bone marrowL. PANGRAZZI, B. JENEWEIN, J. LAIR, M. KRISMER, B. WEINBERGER, B. GRUBECK-LOEBENSTEIN

Institute for Biomedical Aging Research, University of Innsbruck

It is well understood that aging leads to a decline of immune function, a process known as immunosenescence. Latent infection with cytomegalovirus (CMV) may still aggravate the condition. For this reason it is very important to find new strategies to counteract the problem, in particular through the maintenance of immunological memory. During the last years, it has been demonstrated that memory T cells and plasma cells survive in bone marrow niches, well organized structures which support the homeostatic proliferation of these cells. Plasma cells home to CXCL-12+/-, CD4+ memory T cells to IL7+ collagen XI+/- and CD8+ memory T cells to IL-15+ bone marrow cells. CD4+ memory T cell and plasma cell niches have already been described in mice but not in humans and there is still relatively little information on CD8+ T cell niches in either species. Our group has demonstrated that, during aging, there is a decline of naïve and an accumulation of effector memory CD8+ T cells, which are maintained by IL-15 producing cells. Moreover, the numbers of plasma cells, which home to CXCL-12+ stromal cells, decrease in the bone marrow with aging. The aim of this study is to characterize the bone marrow niches responsible for the maintenance of CD8+, CD4+ memory T cells and plasma cells and to investigate how they are changing with aging and CMV infection. qPCR experiments revealed that IL-15 mRNA increased both with age and CMV infection, while IL-7 expression is reduced during aging but not affected by CMV. Immunofluorescence staining of tissue sections and FACS analysis of cell suspensions were performed to confirm results at the protein level. We conclude that changes in the bone marrow niches and chronic CMV infection may lead to decreased antibody responses in elderly.


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The urokinase receptor as a positive regulator of T cell activationAlexander ZWIRZITZ(1), Karin PFISTERER(1), Vladimir LEKSA(2) and Hannes STOCKINGER(1)

(1) Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna (2) Laboratory of Molecular Immunology, Institute of Molecular Biology, Academy of Sciences, Bratislava, Slovak Republic

T cell activation and migration of T cells are two central processes in adaptive immunity. We are interested in one particular molecule namely CD87 (urokinase receptor, uPAR), which seems to equip T cells with features important in both of them. CD87 is widely recognized as an important mediator of fibrinolysis and extracellular matrix degradation. Commonly it is hardly found on resting lymphocytes. However, it has already been shown that T cells start to express CD87 upon activation (Nykjaer et al 1994). Furthermore, there is evidence that CD87 is important in lymphocyte migration to sites of infection (Gyetko et al 2001). Yet, detailed mechanisms as well as CD87s role in other aspects apart from migration have so far not been investigated. We therefore examine CD87s function in T cell activation and migration in more detail. We observed that T cells overexpressing CD87 change their phenotype from suspension to adherent cells. In addition, these cells display an elevated response to T cell receptor stimulation as measured by calcium mobilization and IL-2 production. In our current experiments we want to clarify functional consequences of CD87 expression on T cells, dissect CD87s signaling pathways and elucidate the molecular mechanisms responsible for our observations.


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Liisa ANDERSEN, Alexandra SCHEBESTA, Alexandra GÜLICH, Shinya SAKAGUCHI and Wilfried ELLMEIER

Division of Immunobiology, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria

Transcriptional and epigenetic mechanisms play a key role in the regulation of T cell development and function. We identified the BTB zinc finger protein MAZR (also known as Patz1) as an important regulator of Cd8 gene expression in DN thymocytes and revealed its essential role in CD4/CD8 lineage choice of DP thymocytes. However, the role of MAZR in peripheral T cell development has not been elucidated so far. To comprehensively analyze the in vivo and in vitro role of MAZR in CD4+ T cells, we are employing conditional gene targeting approaches (using the Cd4-Cre deleter strain) as well as gain-of-function studies using retroviral-mediated overexpression strategies. Preliminary results suggest a role for MAZR in modulating Th17/Treg effector differentiation and function. Data from our ongoing experiments will be presented.

Molecular analysis of MAZR function in CD4+ T cells

Acknowledgements

Supported by FWF.


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Julia MARSCHALLINGER(1,2), Barbara KLEIN(1,2), Iris Schäffner (3,4), Sébastien COUILLARD-DESPRES(2,5), Claudia SCHMUCKERMAIR(6), D. Chichung Lie (3,4), Nicolas SINGEWALD (6), Ludwig AIGNER(1,2)

(1) Institute of Molecular Regenerative Medicine, Paracelsus Medical University, Salzburg, Austria(2) Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Salzburg, Paracelsus Medical University, Salzburg, Austria(3) Research Group Adult Neurogenesis and Neural Stem Cells, Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich-Neuherberg, Germany (4) Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany(5) Institute of Experimental Neuroregeneration, Paracelsus Medical University, Salzburg, Austria(6) Department of Pharmacology and Toxicology, Institute of Pharmacy and CMBI, Leopold-Franzens-University of Innsbruck, Innsbruck, Austria

Leukotrienes, mediators of inflammation, are well-studied in asthma and allergy, and leukotriene receptor antagonists such as the drug montelukast have been successfully developed to treat asthmatic patients. In the aged brain, elevated levels of leukotrienes might be involved in neuroinflammation and in age-related cognitive impairments, as well as in reduced adult neurogenesis, since we demonstrated that montelukast induced a significant increase in neuronal progenitor proliferation in vitro.Here, we tested if a 6-week oral montelukast (10 mg kg-1) treatment of young (4 months) and aged (20 months) rats has beneficial effects on cognition, and analyzed potential modes of action including neuroinflammation, blood-brain barrier integrity, neuronal activity, and hippocampal neurogenesis. To gain information on the role of the putative leukotriene receptor GPR17, we analyzed the impact of montelukast on adult FoxO deleted neurospheres, which show strongly reduced GPR17 expression.We showed that montelukast promotes neurogenesis, decreases neuroinflammation and restores blood-brain barrier specifically in aged rats. Most intriguingly, this already marketed anti-asthmatic drug, acting on leukotriene receptors, restored cognitive functions in aged rats.

Structural and functional rejuvenation of the aged brain by an approved anti-asthmatic drug

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Identification of linear and conformational epitopes of bovine alpha -lactalbumin

Yuan SHUILIN (1,2), Li XIN(1,2), Chen HONGBIN(1,2), Gao JINYAN(1,2), Wu ZHIHUA(1,2), Yang ANSHU(1,2), Tong PING(1,2)

(1) State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P.R. China(2) Department of life sciences and food engineering Nanchang University, Nanchang 330047, P.R. China

Background: Alpha-lactalbumin (ALA) is one of the major allergens in cow milk. However, the research on its epitopes was relatively limited, especially for conformational epitopes.

Methods: Patient’s sera specific for cow’s milk ALA were purified by affinity chromatography and then mimotopes against IgG and IgE was obtained by the biopanning of the Ph.D.-C7C and Ph.D.-12 phage display library, respectively. On the basis of mimotopes, continuous epitopes were defined using the mimic peptide frequency from Alignment UniProt and conformational epitopes were computed using Web service of Pepitope Server.

Results: IgE linear epitopes were located in AA41-46, AA55-60, AA62-72, AA74-76, AA85-90 and AA92-99,while position of IgG linear epitopes were AA37-46, AA52-54, AA56-59, AA63-72, AA74-76, AA81-90 and AA92-99, respectively. Five IgE and three IgG conformational epitopes were rendered with PyMOL.

Conclusions: It is first time to identify conformational epitopes of ALA. Some common residues appeared in linear and conformational epitopes , which provided a diagnostic tool giving information on the cow’s milk patients. This study indicated that many conformational epitopes were composed linear epitopes.


Acknowledgements

The work was supported by National High Technology Research and Development Program of China (863 Program, No. 2013AA102205), National Natural Science Foundation of China (No. 31260204 and 31301522)

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In-print of the environment on the molecular sensitization profile towards pollen allergens revealed by allergen micro-array.Viktoriya GARIB (1), Eva WOLLMANN (1), Gulnara DJAMBEKOVA (2), Rudolf VALENTA (1)

(1) Division of Immunopathology, Department of Pathophysiology and Allergy Research,Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna(2)SpecializedScientific-PracticalCenterforTherapyandMedicalRehabilitation(RSSPMCT&R),Tashkent, Uzbekistan

The green zones in Tashkent-city in Uzbekistan have been re-organized during the last 2 decades. Covering approximately 35% of the city´s surface, it includes vegetation commonly present in Central Asia such as saltwort and newly planted species like Bermuda grass. Determination of the molecular sensitization profiles of allergic patients is essential for the correct treatment with allergen specific immunotherapy but has not been established for allergic patients in the Central Asian area. The aim of this study was to determine the IgE-sensitization profile towards pollen allergens in patients with respiratory allergy from Tashkent in dynamic to new landscape. Sera of fifty adult patients with allergic rhinitis and/or asthma were analyzed using an allergen micro-array containing 112 different allergen molecules (ImmunoCAP ISAC; Thermo Scientific). 42/50 showed pollen associated allergic symptoms. We separated them into 3 groups according to their age (middle aged adults-1: MAA-1 (30-39 years), middle aged adults-2 MAA-2 (40-49 years), young adults YA (20-29 years)). We found that 78,7% of patients with respiratory allergy were polysensitized. The hierarchy of pollen sensitization is: nSal k 1 (54,7%) > nCyn d 1 (50%) > rPhl p 1 (38,1%) > nPla a2 (26,1%) > rPla l 1 (23,8%) > rPhl p 5 (21,4%). No IgE reactivity was observed towards rAln g 1, rBet v 1, rBet v 4, rCor a 1.001.01, rPar j 2, and rPhl p 11. The pattern of genuine sensitization “grass-weed-tree” appears with the highest frequency. A difference in the frequency of IgE sensitization could be detected in different age groups. A decrease of sensitization rates against nSal k 1 from 83,3% in the middle-aged adults to 30% in younger patients , and against rPla a 1 from 25% to 5% could be detected. On the other hand the number of young allergics with IgE against nCup a 1 is 4,8x greater than the number in the elder group with similar sensitization. Our data show the predominant role of nCyn d 1 in the Uzbek population as the most frequently detected allergen in all age groups. The results of the IgE profiling identified grass pollen and in particular Bermuda grass and salkwort as the most important pollen allergen sources in Tashkent and a variation in these results is observable upon implementation of environmental changes. These data show that the molecular sensitization profile towards pollen allergens in Central Asia is a consequence of the local flora and its alterations indicate the importance of allergen micro-array analysis for the selection of the correct immunotherapy treatment.


Acknowledgements

This study was supported by the Austrian Science Fund (FWF; DK: Inflammation and Immunity), American Austrian Foundation (AAF) and was performed within the framework of the International Network of Universities for Molecular Allergology and Immunology.

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Bharani Srinivasan1, Margarete Focke-Tejkl1, 2, Milena Weber1, Sandra Pahr1, Alexandra Baar1, Michael Hertl3 , Raja Atreya4, Markus.F.Neurath4, Harald Vogelsang5, Wolf-Dietrich Huber6, Rudolf Valenta1, 2

(1) Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Christian Doppler Laboratory for Allergy Research, Medical University of Vienna, Vienna, Austria(3) Departments of Dermatology and Allergology, Philipps University Marburg, Marburg, Germany(4) Medical Clinic 1, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany. (5) Department of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria(6) Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria

Celiac disease (CD) and dermatitis herpetiformis (DH) is caused by hypersensitive immune reaction to gliadins, but these proteins are poorly characterized. Our aim was to characterize gamma-gliadin (GG1) identified from CD-specific gliadin sub-fraction and to study its usefulness for diagnosis of wheat hypersensitivities and monitoring diet adherence. Methods: GG1 was recombinantly expressed and characterized biochemically and structurally. Immunological characterization was performed using CD, DH and control subject’s sera. IgA- and IgG-epitopes were identified using overlapping peptides. GG1 and peptide-specific antibodies were raised for tracing GG1 in cereals, wheat products and to study its resistance to digestion. Results: rGG1 is unfolded and oligomeric. IgA-reactivity to rGG1 is a specific marker for CD, DH and useful for monitoring diet adherence by CD patients. Peptide 4 from Pro/Gln-rich domain was the immunodominant epitope. GG1-related antigens were found in closely related but not in unrelated cereals. GG1 was detected in wheat products after baking and, the major IgA epitope containing region was resistant to digestion. Conclusions: rGG1 and its peptides may be useful for diagnosis, follow-up, understanding disease mechanism, prevention and therapy.

Characterization of recombinant gamma-gliadin protein and its evaluation for diagnosis of wheat hypersensitivities

Acknowledgements

This study was supported by the FWF-funded PhD program IAI and by the Medical University of Vienna and in part by a research grant from Phadia/Thermofisher Scientific, Uppsala, Sweden.


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Sheron DZORO, Irene MITTERMANN, Rudolf VALENTA

Department of Pathophysiology and Allergy Research,Medical University of Vienna, Vienna- Austria

We have found that approximately 30% of patients suffering from atopic dermatitis (AD) displayed IgE reactivity against a variety of antigens from E. coli. This finding is unusual because E. coli is known as a natural, tolerogenic constituent of the human microbiome, and exposure to bacteria has been reported to have anti-allergenic activity in various studies. Therefore as a first step, we have investigated by IgE inhibition experiments whether IgE reactivity to E. coli antigens is due to cross-reactivity with Staphyloccocus aureus or Malassezia sympodialis, which are frequent skin pathogens in AD. However, no evidence for relevant cross-reactivity was obtained. In order to identify genuine IgE-reactive components of E. coli, a commensal strain ATCC25922 was selected for investigation.

E. coli total genomic DNA was extracted and randomly fragmented by enzymatic restriction. Generated DNA fragments were ligated into a plasmid vector and plasmids with inserts were selected for amplification. Polymerase chain reaction was performed across the cloning region of the vector, to check nucleotide sequences of the inserts. Upon BLAST analysis, sequences of various constituents of E.coli were identified in the library, including transcription factors, chaperone and transporter proteins, isomerase enzymes and putative membrane proteins. The constructed library will now be screened by biopanning procedures, to identify IgE binding clones.

Construction of a phage display library from Escherichia coli to study IgE-reactive bacterial antigens.


Abstract 37

Poster Presentation

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The immune response against the timothy grass pollen allergen Phl p 5 in non-allergic humans depending on different environmentsAlmedina ISAKOVIC, Christoph HILLEBRAND, Theresa THALHAMER, Sandra SCHEIBLHOFER, Josef THALHAMER, Richard WEISS

University of Salzburg, Department of Molecular Biology, Division of Allergy and Immunology, Hellbrunnerstraße 34, 5020 Salzburg

The “hygiene hypothesis” states that a lack of exposure to microbial components during early childhood leads to a higher risk to develop allergic diseases, whereas a high diversity of microbes could be crucial for the establishment of a non-allergic immune system. In this study, we assess the immune status of non-allergic people living in a farming envorinment, who are regularly exposed to timothy grass pollen allergen in the context of a diverse microbial environment. And in addition non-allergic people living in an urban environment, who lack such microbial exposure. PBMCS from non-allergic donors are expanded antigen-specifically with Phl p 5. Expression of surface markers and transcription factors, as well as cytokine secretion allows identification of TH1, TH2, Treg and TH17 cells. Moreover, IgE, IgG1 and IgG4 antibody levels in non-allergic human plasma are measured. We found a high phenotypic diversity among non-allergic individuals, indicating that multiple mechanisms of naturally acquired protection exist. These established methods will allow to statistically assess the distribution of different T-cell subsets in the non-allergic immune system, depending on different environments and to find a potential link between exposures to microbes and the development of allergy.


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Stephanie EICHHORN(1), Isabel PABLOS(1), Serge VERSTEEG(2), Laurian ZUIDMEER-JONGEJAN(2), Ronald VAN REE(2), Fatima FERREIRA(1), Gabriele GADERMAIER(1)

(1) University of Salzburg, Department of Molecular Biology, Division of Allergy and Immunology, Salzburg, Austria(2) Department of Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands

The influence of molecular properties on adjuvant binding and the immunologic behavior was evaluated by comparing rPru p 3 C1 (C1), a potential hypoallergenic vaccine candidate against peach allergy and the wild-type rPru p 3 (WT).Analysis by circular dichroism spectroscopy, dynamic light scattering, and size-exclusion chromatography, showed a compact, alpha-helical fold for the WT protein whereas C1 adopted a loosened, random-coil conformation. Due to more accessible surface charges, binding of C1 to two different adjuvants (aluminum hydroxide, and aluminum phosphate) was enhanced compared to WT in in vitro experiments. In contrast to WT, C1 demonstrated a very high susceptibility to endolysosomal proteolysis. After immunizing Balb/c mice (n = 6) using adjuvant adsorbed proteins, the WT protein elicited a robust antibody response regardless of the formulation. Aluminum phosphate adsorbed C1 triggered an immune response in all mice (50% cross-reactive with WT), while aluminum hydroxide induced IgG in only one mouse.It could be demonstrated that adjuvants’ formulation, adsorption rate and endolysosomal stability influence the immunologic profile. These variables should be taken into account when designing and administering protein vaccines.

Immunogenicity of the major peach allergen Pru p 3: Does protein conformation, physico-chemical properties and stability matter?

Acknowledgements

The study was supported by the FAST project EU grant 201871.


Abstract 39

Poster Presentation

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Wolfgang HEMMER(1), Stefan WÖHRL(1), Felix WANTKE(1), Friedrich ALTMANN(2)

(1) Floridsdorf Allergy Center, Vienna, Austria, (2) Department of Chemistry, University of Natural Resources and Life Sciences, Vienna (BOKU), Austria

Rationale: CCDs in plant and insect venom extracts may cause false-positive in vitro test results. We noticed that some CCD-positive sera show multiple positive ImmunoCAP results even with CCD-free recombinant allergens.

Methods: IgE-binding to recombinant allergens and to allergen-free blank ImmunoCAPs (BIC) was compared in CCD-positive sera before and after CCD inhibition. Results: 35/52 (67%) CCD-positive sera (bromelain 1.01-59.6 kU/l) reacted with BIC ≥0.35 kU/l (0.35-4.22 kU/l). IgE-binding to BIC correlated with binding to bromelain (r=0.80) and was completely abolished by a CCD inhibitor. Binding to another five lots of BICs was lower but correlated strongly with the first lot (r≥0.94). Of 10 CCD-reactive sera (14.0-52.5 kU/l) tested on recombinant Phl p12, Fel d 1, Ara h 2, and Pru p 3, 8 were positive to all components (0.36-1.6 kU/l), 2/10 showed borderline results. Binding to components correlated with binding to bromelain (r=0.61) and BIC (r=0.97) and was completely blocked by CCDs. MS confirmed the presence of MMXF glycans in unprocessed and processed cellulose.

Conclusions: The ImmunoCAP cellulose allergen carrier contains varying traces of CCDs and can cause false-positive results to non-glycosylated allergens in patients with high levels of anti-CCD IgE antibodies.

ImmunoCAP cellulose displays cross-reactive carbohydrate epitopes and can cause false-positive test results in patients with anti-CCD IgE antibodies

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Investigation of the structural and immunological behavior of Art v 3 upon thermal treatmentSabrina WILDNER(1,2), Lorenz STOCK(2,3), Adriano MARI(4), Hanno STUTZ(2,3) and Gabriele GADERMAIER(1,2)

(1) University of Salzburg, Department of Molecular Biology, Division of Allergy and Immunology, Salzburg, Austria(2) Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, Salzburg, Austria(3) University of Salzburg, Department of Molecular Biology, Division of Chemistry and Bioanalytics, Salzburg, Austria(4) Associated Centers for Molecular Allergology, Rome, Italy

Artemisia vulgaris (mugwort) is an important elicitor of pollinosis in late summer and autumn. The structural and immunological behavior of Art v 3, the non-specific lipid transfer protein (LTP) upon thermal treatment was investigated. Recombinant Art v 3.0201 was produced in E. coli and purified using CEX. The molecule was heated up to 120 min at 95°C in acidic (pH 3.4) and neutral (pH 7.3) buffers. Circular dichroism showed high thermal stability at pH 3.4 while the alpha helical fold was lost upon 15 min heating at pH 7.3. Conformational changes were investigated by capillary electrophoresis hyphenated to time-of-flight mass spectrometer indicating a decay of disulfide bonds by beta elimination and formation of lanthionine(s). Native Art v 3 is monomeric presenting a hydrodynamic radius of 1.8 nm. Relaxing of the compact shape is observed upon heat treatment at pH 7.3 which is not attributed to protein aggregation as determined by SEC. Notably, IgE reactivity to Art v 3 was completely reduced upon heating at pH 7.3 but remained largely unaffected at pH 3.4 suggesting involvement of conformational epitopes. Susceptibility of Art v 3 to thermal treatment is highly dependent on ambient conditions and thus studies enrolling pollen and food derived LTPs should consider these facts.

Acknowledgements

The financial support by the Austrian Federal Ministry of Science, Research and Economy and the National Foundation of Research, Technology and Development is gratefully acknowledged.


Abstract 41

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Characterization and IgE epitope mapping of Tri a 37, a serological marker for severe wheat food allergySandra PAHR(1,2), Regina SELB(3), Claudia CONSTANTIN(1), Milena WEBER(1), Margit FOCKE-TEIJKL(1), Gerhard HOFER(4), Andela DORDIC(4), Walter KELLER(4), Nikolaos G. PAPADOPOULOS(5), Stavroula GIAVI(5), Mika MÄKELÄ(6), Anna PELKONEN(6), Verena NIEDERBERGER(3), Susanne VRTALA(1,2), Rudolf VALENTA(1)

(1) Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Christian Doppler Laboratory for the Development of Allergen Chips, Medical University of Vienna, Vienna, Austria(3) Department of ENT, Medical University of Vienna, Vienna, Austria(4) Institute of Molecular Biosciences, Karl-Franzens University Graz, Graz, Austria(5) Allergy Department, 2nd Pediatric Clinic, University of Athens, Athens, Greece(6) Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland

Wheat is an important staple food that can cause IgE-mediated food allergy. Recently, we identified Tri a 37 as a marker allergen for severe allergic reactions in wheat food allergic patients. The purpose of the present study was the recombinant expression of Tri a 37 in two different expression systems and their characterization regarding molecular, structural and immunological properties. Furthermore, we aimed to characterize relevant epitopes of Tri a 37.Recombinant Tri a 37 was expressed in a prokaryotic expression system using E.coli cells and in a eukaryotic expression system using baculovirus-infected insect cells, purified to homogeneity and characterized by means of SDS-PAGE, mass spectrometry, circular dichroism, chemical crosslinking and non-denaturing RAST-based binding assays. Five overlapping peptides were synthesized and used for epitope mapping. Tri a 37-specific rabbit antibodies were raised to perform inhibition experiments and to study its resistance to digestion. Tri a 37 expressed in the prokaryotic system was unfolded whereas using the eukaryotic expression system, we obtained an α-helically folded protein. In non-denaturing RAST-based experiments, both allergens showed comparable IgE-reactivity. IgE and IgG epitope mapping using synthetic peptides revealed that Tri a 37 contains sequential epitopes. Since major IgE epitopes of Tri a 37 are sequential epitopes, Tri a 37 is a true wheat food allergen that can be used for the ín-vitro diagnosis of severe wheat food allergy.


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Lorenz AGLAS(1), Claudia ASAM(1), Stefanie GILLES(2), Claudia TRAIDL-HOFFMANN(2), Fatima FERREIRA(1), Michael WALLNER(1)

(1) Department of Molecular Biology, University of Salzburg, Austria(2) Institute of Environmental Medicine, UNIKA-T, Klinikum rechts der Isar, Technische Universität München, Germany

Birch pollen allergy is one of the most prevalent allergies in Europe. More than 95% of birch pollen allergic patients are sensitized to Bet v 1, the major allergen of birch pollen. Structurally, Bet v 1 is a globular protein and has the ability to bind a variety of ligands within its hydrophobic cavity. The impact of ligand binding on the immunogenic and allergenic properties of Bet v 1.0101 (called Bet v 1 in the following) is still a matter of discussion. Therefore, we investigated the intrinsic ligand binding properties of recombinant Bet v 1.In this respect we used a selection of different ligands. Extrinsic fluorescence studies with ANS (8-anilinonaphthalene-1-sulfonic acid) were used to demonstrate the interaction of the protein with the ligands. Furthermore, the thermal and proteolytic stability was determined in the presence and absence of a ligand. Protein-ligand interactions were verified for each ligand. In terms of thermal and proteolytic stability, some ligands appeared to have a stabilizing effect, as observed for sodium deoxycholate, whereas others did not. Ligand binding appears to influence the structural properties of Bet v 1. It is possible that the stabilizing effect of a ligand may be a crucial characteristic of Bet v 1 during the sensitization process.

Analysis of interactions of the major birch pollen allergen Bet v 1 with naturally occurring and synthetic ligands


Abstract 43

Poster Presentation

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Structural and immunological study of allergenic defensin-like proteins from mugwort, ragweed and feverfewIsabel PABLOS(1), Stephanie EICHHORN(1), Yoan MACHADO(1),Peter BRIZA(1), Christof EBNER(2), Naveen ARORA(3), Stefan VIETHS(4), Gabriele GADERMAIER(1), Fatima FERREIRA(1)

(1)University of Salzburg, Department of Molecular Biology, Division of Allergy and Immunology, Salzburg, Austria(2)Allergy Clinic Reumannplatz, Vienna, Austria(3)CSIR-Institute of Genomic and Integrative Biology, Allergy and Immunology Section, Delhi, India(4)Paul-Ehrlich-Institut, Division of Allergology, Langen, Germany

Defensin-like proteins, Art v 1, Amb a 4 and Par h 1 are relevant allergens from mugwort, ragweed and feverfew pollen. Beyond sequence similarities and allergenicity, little is known about the structural and immunological features shared by these proteins. Thus, we aimed to produce the recombinant allergens for comprehensive physicochemical and immunological studies. For the first time the Par h 1 coding region including the signal peptide was identified from total RNA using a degenerated primer followed by 5’-RACE protocol. The recombinant proteins Art v 1.0101, Amb a 4.0101 and Par h 1 were expressed as non-fusion soluble proteins in E. coli Rosetta-gamiB pLysS. Purity of >95% was assessed by gel electrophoresis and mass spectrometry. Mass analysis confirmed protein identities. Circular dichroism and Fourier transform infrared spectroscopy analysis revealed similar foldings for Art v 1, Amb a 4 and Par h 1. The three allergens showed different stability to endolysosomal degradation. The analysis of weed sensitized patients’ sera from Austria displayed different sensitization profiles to Art v 1, Amb a 4 and Par h 1 with partial IgE cross-reactivity. These proteins will enable to study sensitization and IgE cross-reactivity of defensin-like allergens in large patients’ cohorts.

Acknowledgements

Funded by ERA New INDIGO project I1152.Immunity in Cancer and Allergy, PhD program-University of Salzburg.


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Production and characterization of recombinant Per a 1, a major allergen of Periplaneta americanaBianca KASTNER(1), Stephanie EICHHORN(1), Isabel PABLOS(1), Sabrina WILDNER(1), Ines FORSTENLEHNER(2), Stefan VIETHS(3), Naveen ARORA(4), Gabriele GADERMAIER(1), Fatima FERREIRA(1)

(1) University of Salzburg, Department of Molecular Biology, Division of Allergy and Immunology, Salzburg, Austria(2) University of Salzburg, Department of Molecular Biology, Division of Analytical Chemistry and Bioanalytics, Salzburg, Austria(3) Paul-Ehrlich-Institute, Division of Allergology, Langen, Germany(4) CSIR-Institute of Genomics and Integrative Biology, Allergy and Immunology Section, Delhi, India

The American cockroach Periplaneta americana is a major source of indoor allergens and frequently causes allergic reactions and asthma. Per a 1 can be found in the midgut and fecal excrements; however its function is not known yet. The aim of the study was the recombinant production, physico-chemical and immunological investigation of the allergen. The second tandem repeat of Per a 1.0103 (residues 197-378) was cloned in pHIS parallel 2 vector and expressed as soluble non-fusion protein in E. coli BL21 star. The protein was purified by anion exchange and size exclusion chromatography with a yield of ~9 mg/L. Purity of >95% was demonstrated by gel electrophoresis. The identity was confirmed by mass spectrometry and amino acid analysis. The CD depicted a curve typical for alpha helical proteins with a minimum at 222 nm. IgE binding was tested in ELISA using sera of 13 cockroach allergic patients. The purified protein will be used to evaluate IgE sensitization, cross-reactivity and clinical relevance in large patients’ cohorts from India, Europe and America.

Acknowledgements

The work was funded by the ERA New INDIGO project I1152.


Abstract 45

Poster Presentation

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Renaissance of a well-known contact-allergen

Tamar KINACIYAN

DIAID, Department of Dermatology, Medical University of Wien

Background and Objective: We report on two young ladies who came with massive eyelid eczema and conjunctivitis to our outpatient clinic. The history revealed in one patient that she had colored her eyebrows one day before the onset of eczema and the other colored her eyelashes. Methods: Patch tests with the European standard series and hairdresser materials were performed. In one patient additionally textile dyes were tested. Results: In the European standard series was p-Phenylendiamine (PPD) in both patients positive. In hairdresser materials, one patient reacted positive to p-Toluylendiamine, p-Aminophenol, 3-Apinophenol the second patient to p-Aminophenol, Hydroquinone and Dispersions orange.Conclusions: Re-evaluation of case history in both patients revealed contact dermatitis to henna-tattoo more than 10 years ago. The second patient developed scars from this contact dermatitis. Both had not yet consulted any allergist for these reactions and did not know that in all cases the causative substance was PPD. Thus, all henna-tattoo- or PPD-allergic patients should also be made aware of an allergic reaction to eyelash and eyebrow dyes.


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Regina FREIER and Hans BRANDSTETTER

Structural Biology Group, Department of Molecular Biology, University Salzburg

Proteolytic processing by endolysosomal proteases is a key event in the development of an allergic response. We investigate the idea that sensitizing allergens (e.g. Bet v 1a) are highly resistant, and can only be cleaved after endolysosomal proteases induce conformational changes. This hypothesis is supported by the identification of four initial Bet v 1a processing sites by cathepsin S (1), which are harbored within secondary structure elements, seemingly not accessible to proteolytic processing. In contrast, non-sensitizing structural homologues can easily be cleaved be many proteases. To study the conformational transition of allergens, crystallization of cathepsin S in complex with peptides corresponding to the four initial cleavage sites of Bet v 1a is intended. To prevent peptide cleavage we produced an active site dead mutant of cathepsin S. Interactions with Bet v 1a peptides in the active site should reveal which amino acids are important for the recognition. Additionally peptide conformations will allow to model the transition within the allergen. The accessibility of sensitizing and non-sensitizing allergens can be further studied by measuring binding kinetics with surface acoustic technology (SAW). The affinity of cathepsin S to intact and fragmented Bet v 1a will show if reordering precedes its processing by cathepsin S. Furthermore the comparison between sensitizing allergens and structural homologues might reveal the general importance of the recognition by proteases in allergenicity.

(1) Egger et al. (2011) Assessing Protein Immunogenicity with a Dentritic-Cell Line-Derived Endolysosomal Degradome In PLoS One

Recognition of birch pollen allergen Bet v 1 by endolysosomal proteases


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Bettina SCHWEIDLER(1), Teresa STEMESEDER(1), Eva KLINGLMAYR(1), Lisa LÜFTENEGGER(2,3), Stephanie MOSER(4), Roland LANG(2), Martin HIMLY(1), Gertie J. OOSTINGH(3), Arne BATHKE(5), Jörg ZUMBACH(4), Thomas HAWRANEK(2), Gabriele GADERMAIER(1)(1)University of Salzburg, Department of Molecular Biology, Salzburg, Austria(2)Paracelsus Medical University Salzburg, Department of Dermatology, Salzburg,Austria(3)Salzburg University of Applied Sciences, Biomedical Sciences, Salzburg, Austria(4)University of Salzburg, School of Education, Salzburg, Austria(5)University of Salzburg, Department of Mathematics, Salzburg, Austria

Allergic diseases were considerably increasing during the last decades; the reasons for this development are however not fully elucidated. The aim of our work is to assess the correlation between allergen exposition and IgE sensitization. Therefore, 501 school children (13-21 years) living in different geographic areas (urban, rural or alpine) from Austria participated in our study. Using a fluorescent multiplex array, exposition to indoor allergens from cat, dog, mites and molds were analyzed using house dust samples. IgE sensitizations to respective allergens were evaluated by ImmunoCap ISAC. The most prevalent allergens in the house dust were Fel d 1 and Can f 1, while Alt a 1 amounts were insignificant. Exposition to cat and dog allergens was higher in houses, while mite allergens were considerably elevated on farms. Students in alpine regions were less exposed to mites, which translated into lower IgE sensitization. Fel d 1 exposition was slightly higher in urban areas but did not lead to enhanced IgE development. Although exposed to equivalent Can f 1 amounts, subjects living in urban areas showed higher IgE sensitizations. We conclude that IgE sensitization is not generally associated with allergen exposition but is impacted by environmental and lifestyle factors.

Investigation of indoor allergen exposure and IgE sensitization

Acknowledgements

The study was funded by Sparkling Science, a program of the Federal Ministry of Science, Research and Economy, Vienna, Austria.


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Juan R URREGO(1), João PONTE(1), Michael WALLNER(2), Carina PINHEIRO(1), Neuza ALCANTâRA-NEVES(1), Peter BRIZA(2), Fátima FERREIRA(2)

(1) Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia, Brazil(2) Department of Molecular Biology, University of Salzburg, Austria

Background: Blomia tropicalis is an important source of allergens associated with allergic asthma in tropical and subtropical regions. However, the clinical importance of group 2 allergens from this mite species is still under debate. Until now only rare information of Blo t 2 is available, most of it obtained at the transcript level. Methods: Blomia tropicalis extracts were prepared from different breeds collected and cultured in Bahia, Brazil. Mite extracts were prepare using phosphate buffered saline and proteolytically digested with a set of distinct proteases. Peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS). Results: By MS analysis we were able to identify all isoforms of Blo t 2 at the proteome level and quantify their relative abundance. Moreover, we found for the first time that Blo t 2 is initiated by a signal peptide, which is processed during expression. The cleavage site determined by MS could be confirmed using the software SignalP 4.0.Conclusion: We successfully identified and quantified mature Blo t 2 isoforms at the protein level. This constitutes a crucial step for the selection of candidate molecules for recombinant production and immunologic characterization of this allergen.

Proteomic characterization of the Blomia tropicalis allergen Blo t 2

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Poster Presentation

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Sabine PFEIFER(1), Pawel DUBIELA(1), Merima BUBLIN(1), Karin HUMMEL(2), Christine HAFNER(1,3), Karin HOFFMANN-SOMMERGRUBER(1)

(1) Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria(2) VetCore Facility for Research, VetOmics, University of Veterinary Medicine Vienna, Austria(3) Karl Landsteiner Institute for Dermatological Research, St. Poelten, Austria

Allergens from nuts tend to induce severe allergic reactions in sensitive individuals. 2S albumins are water-soluble storage proteins sharing a characteristic structure and considered to be major allergens in many seeds and nuts. The aim of this study was to purify and characterize natural Cor a 14, the 2S albumin from hazelnut, and to investigate its allergenic activity.Cor a 14 was purified from raw hazelnuts, characterized and identified with a sequence-coverage of 100%. We estimated a molecular mass of ~12 kDa which is in accordance with the mature protein. Further, we could identify two different isoforms and showed microclipping at the C-terminus. CD spectra at room temperature showed the typical characteristics of 2S albumins and temperatures of more than 80°C were necessary to start unfolding of Cor a 14 demonstrating its high stability to heat treatment. In vitro digestion experiments, simulating gastric and duondenal conditions, revealed that Cor a 14 is completely resistant to proteolytic degradation. Native as well as heat-treated protein was recognized by sera from hazelnut allergic patients. However, reduction led to reduced IgE binding in more than 60% of tested sera.

Stability and micro-heterogeneity of Cor a 14, the 2S albumin from hazelnut

Acknowledgements

Supported by grants SFB F4603 and W1248 (Austrian Science Fund) to K. Hoffmann-Sommergruber and P. Dubiela, respectively.

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Juergen BRUNNER(1), Thomas GINER(1), Guenter WEISS(2), Dietmar FUCHS(3)

(1) Department of Pediatrics, Medical University Innsbruck, Austria (2) INTERNAL MEDICINE(3) Division of Biological Chemistry, Biocenter, , MEDICAL UNIVERSITY INNSBRUCK, Innsbruck, Austria

Background: Juvenile idiopathic arthritis (JIA) is a relevant autoimmune disease in children. T cells, B cells, and damage-associated molecular patterns (DAMPS) are involved in the pathogenesis of the disease. Biomarkers for JIA and its subtypes are not established. Pro-inflammatory pathways activate enzyme indoleamine 2,3-dioxygenase (IDO) which enhances tryptophan (Trp) conversion to kynurenine (Kyn). Thus, in conditions of chronic immune activation reduced Trp availability and production of Kyn and its down-stream metabolites may inhibit cell proliferation. In rheumatoid arthritis (RA) Trp concentrations are lower in patients than in controls and the Kyn/Trp ratios are higher and correlate with neopterin concentrations [1-3].

Objectives: To evaluate Tryptophan as Biomarker in JIA

Methods: In this study, Trp and Kyn metabolism was investigated in children with JIA and compared to serum neopterin concentrations. Fifty-four sera of 25 JIA patients and 10 samples of synovial fluid were examined with HPLC (Trp and Kyn) and Elisa (Neopterin, BRAHMS, Hennigsdorf, Germany). Eighteen sera from 18 children with non-inflammatory diseases were used as controls.

Results: Trp in the sera of patients was mean 57.2 + SD 19.0 µmol/L and Kyn was mean 2.40 + SD 0.81 µmol/L). Serum neopterin was 5.69 + SD 1.72 nmol/L. In the synovial fluid, neopterin was mean 10.5 + SD 7.41 nmol/L), Trp was 36.7 + SD 17.4 µmol/L and Kyn was 2.13 + SD 0.75 µmol/L). In control patients, neopterin was 6.93 + SD 3.10), Trp was 57.6 + SD 14.8) and Kyn was 2.60 + SD 1.60 µmol/L.

Conclusions: Serum Trp concentrations showed no relevant difference in JIA patients vs. controls. IDO activity reduces Trp primarily in the synovial fluid in JIA patients.

Biomarkers of Infallation in juvenile idiopathic arthritis (JIA)


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The Toll-like receptor 4 agonist MRP8/14 protein complex (Calprotectin) in autoinflammation: Potential biomarker in chronic nonbacterial osteomyelitis – a case report

Jürgen BRUNNER(1)

(1) Department of Pediatrics, Medical University Innsbruck, Austria

Background: The cytoplasmic S100 proteins derived from cells of myeloid origin. Calprotectin (MRP8/14 protein complex) might be a biomarker either for autoinflammation and autoimmunopathy. Since autoinflammatory diseases might be a diagnostic challenge calprotectin may be helpful in the diagnosis of autoinflammatory diseases. Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory, noninfectious disease. CNO describes a wide spectrum from a monofocal bone lesion to the chronic recurring multifocal osteomyelitis (CRMO). Laboratory and histopathological findings are nonspecific. In some patients systemic inflammatory signs such as elevated acute phase proteins cannot be found.

Objectives: To test the ability of Calprotectin (MRP8/14 protein complex) serum concentrations to monitor disease activity in patients with CNO.

Methods: Serum concentrations of Calprotectin (MRP8/14 protein complex) in a patient with CNO were determined by a sandwich ELISA. Results: Calprotectin (MRP8/14) level were raised heralding active disease when acute phase proteins (CrP, erythrocyte sedimentation rate). The calprotectin level was 7872,7 ng/ml (normal range 0-3000 ng/ml)

Conclusions: Calprotectin (MRP8/14) serum concentrations correlate closely with disease activity and may herald a flare before clinical manifestation. Therfore MRP8/14 serum concentrations are a biomarker indicating disease activity in CNO patients.


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Jürgen BRUNNER(1), Elisabeth BINDER(1), Daniela KARALL(1), Johannes ZSCHOCKE(2), Christine FAUTH(2)

(1) Department of Pediatrics, Medical University Innsbruck, Austria(2) Department of Human Genetics, Medical University Innsbruck, Austria

Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS; MIM# 260920) is a rare autosomal recessive autoinflammatory condition caused by mutations in the MVK gene, which encodes for mevalonate kinase. There is no standard treatment for HIDS.Case report:We report on a 2 year-old Austrian boy with recurrent episodes of fever, febrile seizures, arthralgias, and splenomegaly. Rash and abdominal pain were also seen occasionally. During attacks an acute-phase response was detected. Clinical and laboratory improvement was seen between attacks. These findings led to the tentative diagnosis of HIDS. Sequencing of the MVK gene showed a homozygous c.1129G>A (p.Val377Ile, also known as V377I) mutation in the child, while the healthy non-consanguineous parents were heterozygous. The mutation is known to be associated with HIDS. Therapy with nonsteroidal anti-inflammatory drugs during attacks had poor benefit. A further febrile episode resulted in a status epilepticus. Treatment with canakinumab was initiated and a final dose of 4 mg/kg every 4 weeks resulted in the disappearance of febrile attacks and a considerable improvement of patient´s quality of life during a 6-month follow-up period. The drug has been well tolerated, and no side effects were observed.Conclusion:Treatment with canakinumab is a therapeutical option for patients with HIDS.

Interleukin 1 blockade with canakinumab for Hyper IGD syndrome (HIDS)


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Complement analysis in patients with Juvenile Idiopathic Arthritis (WIELISA, SC5b9-ELISA)T. GINER(1), L. HACKL(1), R. WÜRZNER(2), J. BRUNNER(1)

(1) Department of Pediatrics, Medical University Innsbruck, Austria(2) Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Austria

The juvenile idiopathic arthritis is a well researched disease in the group of autoimmunopathies. Beside the deregulation of T-cells and cytokines also the complement system is involved in the pathogenesis of this group of diseases. This prospective longitudinal study investigated the contribution of the complement system in patients with juvenile idiopathic arthritis, using practicable ELISA techniques (Wieslab® screening kit; SC5b9 soluble terminal complement complex ELISA).Serum and plasma of the peripheral blood and the synovial fluid were investigated for the activity of the three complement pathways - classical (CP), mannose binding lectin (MBL), and the alternative pathway (AP) and total complement activity by measuring SC5b9. Results where compared to published reference controls and 18 children without activation of inflammation as an age matched control group.In total 57 samples of peripheral blood (PB) and 8 samples from synovial fluid (SF) from 28 children with JIA were investigated in a longitudinal observation during acute phase and remission. The screening of complement system showed debasement of the AP (8 of 10) and CP (7 of 10) in patients during acute phase (7 of 10). The SC5b9 measurement showed a significant (p<0.002) higher amount in plasma (3,6AU/ml in median) and serum (31,4AU/ml) during acute phase compared to the control group (serum - 7,72AU/ml and plasma – 1,25AU/ml in median). In conclusion the study confirmed, that the CP and AP of the complement system are main contributors in the pathogenesis of JIA. Because of significant elevation of SC5b9 in acute phase of JIA, complement blockade with Anti-C5 may be a therapeutically option in the future.


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Reinhard WÜRZNER(1), Tom Eirik MOLLNES(2), Francesco TEDESCO(3), Peter GARRED(4), Lennart TRUEDSSON(5), Malcolm W. TURNER(6), Moh R. DAHA(7), Robert B. SIM(8) (1)Division of Hygiene & Med. Microbiology, Innsbruck Medical University, Austria(2)Department of Immunology, Oslo University Hospital and University of Oslo, Norway(3)Department of Life Sciences, University of Trieste, Italy(4)Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, University of Copenhagen, Denmark(5)Department of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology, Lund University, Sweden(6)Immunobiology Unit, Institute of Child Health, University College London, UK(7)Leiden University Medical Centre, The Netherlands(8)Pharmacology Department, Oxford University, UK

An ELISA-based kit was introduced about a decade ago with the primary aim to screen for deficiencies in components of the complement (C) system (WIESLAB® Complement system Screen). The assay is based on specific and separate analysis of the classical, lectin and alternative pathway, respectively, using the common terminal end product C5b-9 as surrogate for hemolysis. This assay is particularly useful for detecting genetic C deficiencies and has helped to identify the cause of several severe and recurrent infections, renal diseases and systemic autoimmune diseases. Its use has confirmed the higher sensitivity and specificity compared to the “antique” hemolytic assays. In particular, this assay detects properdin deficiencies, which are not always detected in hemolytic assays. Its advantage is to assess the functionality of all three C pathways independently of biologically variable reagents. After ten years of clinical use, this assay has now extended its applications to quantitate the functionality of the C pathways in disease monitoring. New features of this assay include the use of different dilutions when testing C deficient sera, the assessment of C activation in HUS samples during acute phase or remission, and its usefulness to monitor patients undergoing C inhibitor therapy.

Functional analyses of total complement activity in an ELISA-based kit which replaces haemolytic assays: a decade of experience and future perspectives


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Reactive oxygen species mediated regulation of linker for activation of T cells in autoimmunityFlorian FORSTER(1), Clara MARQUINA(1), Bernhard MALISSEN(2), Rikard HOLMDAHL(1)

(1)MedicalInflammationResearch,MedicalBiochemistryandBiophysics,KarolinskaInstitutet,Stockholm, Sweden(2) Centre d’Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, Institut National de la Santé et de la Recherche Médicale, U631, Centre National de la RechercheScientifique,UMR6102,13288Marseille,France

Linker for activation of T cells (LAT) is a membrane adapter protein necessary for T cell development and signaling. In human arthritic synovial fluid lymphocytes, oxidative stress has been suggested to delocalize LAT into the cytosol, leading to unresponsive T cells. To test the consequences of oxidation on LAT, we created a mouse strain expressing LAT with cysteine to serine mutations at position 120/172. In vitro experiments revealed no differences in T cell activation, pointing to no cell intrinsic consequences of the mutation. In vivo we monitored a minor defect in the development of T cells at the transition from the double negative to the double positive state. In the periphery we show a reduced expression of CD62L. In addition we observe a higher inflammation in mice carrying the LAT mutation in a DTH model.To further investigate the consequences of LAT oxidation, we crossed the LAT C120/172S mice with mice showing defective NOX2 dependent ROS production due to a mutation in the Ncf1 gene. We tested the consequence of LAT mutation in ROS sufficient and deficient mice in the context of collagen induced arthritis. Surprisingly we didn’t see a difference in the ROS sufficient mouse, whereas we observed a protection of the LAT mutated mouse in the ROS deficient setting.


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Contact / E-Mail:[email protected], [email protected]

Anna BATYROVA, Irina KONDRAKHINA, Olga KOZHEVNIKOVA, Anna GEVORKYAN

FederalStateBudgetaryScientificInstitution“ScientificCentreofChildrenHealth”

Markers of antiphospholipid syndrome (APS) such as cardiolipin and betta-2 glycoprotein I IgM, IgG antibodies are often associated with increased risk of thrombus formation at various diseases. But it has been rare studied in pediatric practice. Aim: to estimate the cardiolipin and betta-2 glycoprotein I IgM, IgG levels diagnostic significance in children with suspicion of antiphospholipid syndrome (APS) from different departments. Methods: the sera of 105 children (2-16 years) were tested on ImmunoCAP.Results: 62.9% children from rheumatology, 17.1% - gastroenterology, 9.5% - neuropsychiatric, 8.6% - nephrology, 1, 9% - cardiology departments were included. The positive results of tests were revealed in one patient from gastroenterology department with nonspecific ulcerative colitis that showed high IgG to cardiolipin and betta-2 glycoprotein I levels, but not IgM. In rheumatology department 3 children with systemic lupus erythematosus - SLE (10,7% from 28) and 2 patients with juvenile rheumatoid arthritis (9.5% from 21) had at least one positive marker. Conclusions: these tests show very rare positive results in children even with clear determine diagnosis of SLE. It is necessary to use complex approach to establish APS and prevent thrombus formation.

Serum markers of antiphospholipid syndrome detection and their diagnostic significance in the pediatric practice

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Wilfried POSCH(1), Marion STEGER(1), Ulla KNACKMUSS(1), Felipe DIAZ-GRIFFERO(2), Cornelia LASS-FLÖRL(1), Arnaud MORIS(3), Oliver T. KEPPLER(4), DorisWILFLINGSEDER(1)

(1) Division of Hygiene and Medical Microbiology, Medical University of Innsbruck(2) Department of Microbiology and Immunology, Albert Einstein College of Medicine(3) INSERM, U1135, Center for Immunology and Microbial Infections - CIMI-Paris(4) Institute of Medical Virology, University of Frankfurt

DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1 and this non-permissiveness of DCs is suggested to go along with viral evasion. We here give insight into a substantial novel way of dendritic cell modulation at least during acute HIV-1 infection by triggering integrin receptor signaling. We found, that complement-opsonization of the virus is able to relieve SAMHD1 restriction in DCs, thereby initiating strong maturation and co-stimulatory capacity of the cells and stimulating efficient cellular and humoral antiviral immune responses. This newly described way of DC modulation by complement might be exploited to find novel therapeutic targets promoting DC immune functions against HIV.

Complement-opsonized HIV overcomes SAMHD1 restriction in DCs

Acknowledgements

Austrian Science Fund [FWF, P22165 and P24598 to DW, P25389 to WP] and the OeNB [project: 14875 to WP]

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Wilfried POSCH(1), Andrea SCHROLL(2), Asier SAEZ-CIRION(3), Gianfranco PANCINO (3), Teunis GEIJTENBEEK(4), Cornelia LASS-FLÖRL(1), Günter WEISS(2), Doris WILFLINGSEDER(1)

(1) Division of Hygiene and Medical Microbiology, Schöpfstrasse 41, Innsbruck, Austria(2) Department of Internal Medicine VI, Clinical Immunology and Infectious Diseases, Innsbruck Medical University, Anichstr. 35, Innsbruck, Austria (3) Unité de Régulation des Infections Rétrovirales, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris, France(4) Center for Experimental and Molecular Medicine, AMC, University of Amsterdam, Amsterdam, Netherlands

Early on in HIV-1 infection, gut Th17 cells are massively depleted leading eventually to compromised intestinal barrier function and excessive immune activation. Complement coating as predominantly found during the acute phase of infection was shown to act as an endogenous adjuvant for DC-mediated induction of virus-specific CTLs. Here, we show that DCs loaded with HIV-1 bearing high surface complement levels after incubation in plasma from HIV-infected individuals, secreted significantly higher concentrations of Th17-polarizing cytokines compared to DCs exposed to non-opsonized HIV-1. The enhanced Th17-polarizing capacity of in vitro generated and BDCA1+ DCs was linked to activation of ERK. Additionally, C3a produced from DCs exposed to complement-opsonized HIV was associated with the higher Th17 polarization. Our in vitro and ex vivo data, therefore, indicate that complement opsonization of HIV-1 strengthens DC-mediated anti-viral immune functions by simultaneously triggering Th17 expansion, intrinsic C3 formation and enhanced CTL responses via DC activation.

Triggering CD11b/c on DCs promotes immediate Th17 polarization during acute HIV-1 infection

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Evaluation of vaccine responsiveness against tick-borne encephalitis (TBE) and hepatitis A in allergic patients – differences to healthy controls?Erika GARNER-SPITZER(1), Michael HOFER(1), Reinhard JARISCH(2), Tamar KINACIYAN(3), Michael KUNDI(4), Ursula WIEDERMANN(1)

(1)InstituteforSpecificProphylaxisandTropicalMedicine,MedicalUniversityofVienna(2) Allergiezentrum Floridsdorf(3) Allergieambulanz der Universitätsklinik für Dermatologie, AKH, Medical University of Vienna(4) Institute for Public Health, Medical University Vienna

Type I allergies have increased drastically and afflict app. 30% of the western population. Allergic sensitization results in Th2 biased immune responses which eventually trigger allergic symptoms/diseases. Specific immunotherapy (SIT) is the only causative treatment, leading to activation of regulatory cells (IL10, TGF-ß) and a shift from Th2 toTh1 response. In a clinical trial we investigate whether responsiveness to routine vaccines is altered by allergy: allergic patients, either with SIT or symptomatically treated and healthy controls receive a booster vaccination against tick-borne encephalitis (TBE). To test primary responsiveness a subgroup is also vaccinated against hepatitis A.Immune response is evaluated with respect to specific Ab-titers, cytokine production of re-stimulated PBMC and quantification of naïve, memory, and regulatory subsets of B- and T-cells. Additionally, allergic- and SIT-status of participants are correlated with humoral/cellular immune responses. Preliminary data, namely Ab-titers and B- and T-cell subsets have been determined for the subgroup receiving both vaccines.The results of this study will clarify whether allergic sensitization/specific immunotherapy lead to impaired vaccine responsiveness and demand adaptations of vaccination schedules.


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SOD1 Protects From Type I Interferon-Driven Oxidative Damage in Viral HepatitisAnannya BHATTACHARYA*(1), Ahmed N. HEGAZY*(2,3,4), Nikolaus DEIGENDESCH**(5), Lindsay KOSACK**(1), Jovana CUPOVIC(6), Richard K. KANDASAMY(1), Andrea HILDEBRANDT(1), Doron MERKLER(7), Anja A. KÜHL(8), Christopher SCHLIEHE(1), Isabel PANSE(2,3), Bojan VILAGOS(1), Kseniya KHAMINA(1), Isabelle ARNOLD(4), Lukas FLATZ(6), Haifeng C. XU(9), Philipp A. LANG(9), Alan ADEREM(10), Giulio SUPERTI-FURGA(1), Jacques COLINGE(1), Burkhard LUDEWIG(6), Max LÖHNING(2,3),†, Andreas BERGTHALER(1),†

(1) CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Lazarettgasse 14 AKH BT25.3, Austria.(2) Experimental Immunology, Department of Rheumatology and Clinical Immunology, Charité–University Medicine Berlin, 10117 Berlin, Germany.(3) German Rheumatism Research Center (DRFZ), a Leibniz Institute, 10117 Berlin, Germany.(4)TranslationalGastroenterologyUnit,ExperimentalMedicineDivisionNuffieldDeptofClinicalMedicine,University of Oxford, John Radcliffe Hospital, Oxford, UK.(5) Max Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin, Germany.(6) Institute of Immunobiology, Cantonal Hospital St. Gallen, Rorschacherstrasse 95, 9007 St. Gallen, Switzerland.(7) Department of Pathology and Immunology, University of Geneva, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Geneva, Switzerland.(8) Department of Medicine I for Gastroenterology, Infectious Disease and Rheumatology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.(9) Department of Gastroenterology, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany.(10) Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, WA 98109-5219, USA.* These authors contributed equally to this work.** These authors contributed equally to this work.† These authors contributed equally to this work.

Tissue damage caused by viral hepatitis is a major cause of morbidity and mortality worldwide. It is driven by poorly understood processes involving interrelated immunological and metabolic events. Using a mouse model of viral hepatitis, we identified virus-induced early transcriptional changes in the redox pathways including downregulation of superoxide dismutase 1 (Sod1) in the liver. Sod1-/- mice exhibited increased inflammation and aggravated liver damage upon viral infection, which was ameliorated by antioxidant treatment. Thus, oxidative stress is a key mediator of virus-induced liver damage. Type I interferon (IFN-I) led to the downregulation of Sod1 in wildtype mice and caused oxidative liver damage in Sod1-/- mice in the absence of infection. Notably, blockade of IFN-I signaling protected against virus-induced liver damage. These results delineate a novel mechanism of innate immunity-driven immunopathology, linking IFN-I signaling with antioxidant host defense and infection-associated tissue damage.

Acknowledgements

A.Bh. is supported by a DOC fellowship of the Austrian Academy of Sciences. A.N.H. and R.K.K. were supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF 116-2012 and ALTF 314-2012). A.H. was supported by a stipend of the German Academic Exchange Service (DAAD). C.S. was supported by a fellowship within the Postdoc-Program of DAAD. G.S.-F. was supported by the ERC advanced grant i-FIVE. M.L. was supported by the German Federal Ministry of Education and Research (BMBF, T-Sys), the German Research Foundation (SFB618, TPC3; SFB650, TP28), and the Volkswagen Foundation (Lichtenberg program). A.B. was supported by the Austrian Academy of Sciences and by stand-alone grant #23991 of the Austrian Science Foundation (FWF).


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Comparison of the immunogenicity and reactogenicity after subcutaneous (s.c.) or intra muscular (i.m.) vaccination with a tick-borne encephalitis vaccineStefan HOPF(1), Erika GARNER-SPITZER(1), Michael HOFER(1), Ingrid DEMEL(1), Michael KUNDI(2), Otfried KISTNER(3) and Ursula WIEDERMANN(1)

(1)InstituteofSpecificProphylaxisandTropicalMedicine,CenterofPathophysiology,Infectiology & Immunology, Medical University Vienna, Austria (2) Institute of Environmental Health, Medical University of Vienna, Austria (3)ClinicalVirology&ScientificAffairs,BioScience,BaxterInnovationsGmbH,Vienna,Austria

Studies to determine the optimal vaccination route are being performed during the licensing studies for all vaccines evaluating immunogenicity and reactogenicity. However in various situations the vaccine might be applied subcutaneously. For this reason, the following study was performed to compare the immunogenicity and safety between s.c. and i.m. vaccination. Neutralizing antibodies, lymphocyte population distribution, cytokine production and the occurrence of side effects were analyzed and compared for both vaccination routes.Neutralizing TBE specific antibody titers and cytokine production upon antigen restimulation of isolated PBMCs did not significantly differ between the different vaccination groups. FACS analysis revealed only slight differences in naïve and memory lymphocyte populations between genders and vaccination routes, which however, did not have an obvious influence on vaccine specific humoral and cellular responses.Monitoring of side effects after vaccination showed a significant increase in local reactions after s.c. vaccine application compared to i.m. vaccination. Moreover women showed higher rates of side effects than men.The results show that the TBE vaccine can be applied either s.c. and i.m. leading to a comparable immune response. However, with respect to local side reactions the s.c. route seems to promote reactogenicity.


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Mario BIAGGIO(1), Caroline LASSNIG(1,2), Rita ROM(1), Astrid KRMPOTIC(3), Stipan JONJIĆ(3),BirgitSTROBL(1)andMathiasMÜLLER(1,2)

(1) Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria (2) University Center Biomodels Austria, University of Veterinary Medicine, Vienna, Austria(3) Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Croatia

Cytomegalovirus (CMV) infection is a frequent cause of congenital birth defects and can trigger severe life-threatening disease in immune suppressed individuals. Murine CMV closely resembles human CMV and is commonly used as model system to study acute and persistent viral infections. While NK and T cells play a predominant role in eliminating infected cells, the contribution of myeloid cells to the anti-MCMV defence is largely elusive. Signal transducer and activator of transcription 1 (STAT1) is a key transcription factor in signalling in response to all types of interferon and lack of STAT1 results in high susceptibility to bacterial and viral infections in humans and mice. Our laboratory has generated mice that specifically lack STAT1 in myeloid cells (Stat1ΔLysz), enabling us to study the importance of myeloid antiviral activity in vivo. Stat1ΔLysz mice showed an increased viral load and increased pathology in spleen and liver early after infection compared to littermate controls. Intriguingly, Stat1ΔLysz mice did not show decreased survival but a higher persistence of MCMV in salivary glands. We found similar activation of splenic and liver NK cells, whereas preliminary experiments show differences in CD8+ and CD4+ T cell activation. Current efforts are directed towards a more detailed characterization of anti-MCMV responses in distinct T cell subsets.

STAT1 in myeloid cells is required to limit early replication and persistence of murine cytomegalovirus in vivo

Acknowledgements

This work is funded by the FWF DK W1212 and the SFB F28 to BS and MM.

Abstract 63

Poster Presentation

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Meena NARAYANAN(1), Margarete FOCKE-TEJKL(2), Rudolf VALENTA(1,2), Birgit LINHART(1)

(1) Div.of Immunopathology, Dept.of Pathophysiology and Allergy Research, Center of Pathophysiology,Infectiology and Immunology, Medical University of Vienna, Austria(2)Christian Doppler Laboratory for Allergy Research, Medical University of Vienna, Austria

IgE-mediated allergy is characterized by the induction of allergen-specific Th2 response and production of allergen-specific IgE antibodies. Although many clinical studies have shown that IgE levels are boosted by repeated allergen contact, mechanisms underlying secondary IgE responses are not yet fully understood. Hence, aim was to study the molecular and cellular requirements for boosting allergen-specific IgE responses.We developed a mouse model based on hapten-carrier model, where 4 copies of a 31 amino acid peptide derived from Phl p 1, devoid of T cell epitopes recognized by BALB/c mice, was fused to a carrier, PreS and used for sensitization. Then,boosting was done with molecules containing peptide without or with an immunologically irrelevant carrier. For control purposes, boosting were also done with the same immunogen used for sensitization, carrier alone and PBS. Peptide-specific IgE responses determined by ELISA and RBL assays showed oligomeric peptides devoid of T cell epitopes could boost memory IgE responses. T cell responses studied by proliferation assays using mouse splenocytes failed to proliferate when stimulated with peptide, which attests the lack of allergen-specific T cell epitopes. Our results suggest B cell targeting approaches for established allergy.

T cell- independent boost of Memory IgE responses by B cell epitopes

Acknowledgements

Supported by grant P23350-B11 (FWF), Vienna, Austria.


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K. Oida (1,2), L. Einhorn (1,3), S. Vrtala (4,5), Y. Resch (4), L. Panakova (6), F. Roth-Walter (1), J. Fazekas (1,3), H. Matsuda (2), A. Tanaka (7), E. Jensen-Jarolim (1,3)

(1) Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Austria(2) Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Japan(3) Comparative Immunology and Oncology, Institute of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University Vienna, Austria(4) Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University Vienna, Austria(5) Christian Doppler Laboratory for the Development of Allergen Chips, Medical University Vienna, Austria(6) Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Austria(7) Laboratory of Comparative Animal Medicine, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Japan

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disorder. It is accepted that epithelial barrier dysfunction or disruption must be the initial event before sensitization. There is on the other hand a strong correlation between house dust mite (HDM) exposure and AD. HDMs are harmless per se, but are a source of allergens including such with enzymatic function.We investigated here the barrier disruption potential of HDM proteases by gel zymography in vitro using whole body extract from Dermatophagoides pteronyssinus. Regardless of the presence of Ca and Zn ions, and of a wide range of temperature, HDM extract degraded casein (as a readout for serine and cysteine protease activity) and gelatin gels (for collagenase activity). The HDM extract exhibited proteolytic activity at pH 5.0−9.0, thus by far exceeding the pH range of normal stratum corneum at pH 4.5−5.5.Exposure to HDM on a daily basis may play a crucial role in the onset of AD by impairing epithelial integrity via several enzymatic activities and by delivering allergens deep into the skin. This activity is independent on metal ions, temperature and effective in a wide pH range. The mechanism thus is robust and allows for penetration into the skin of even healthy individuals, depending on the dose.

House dust mite extract is a robust source of enzymes that impair epithelial barrier function

Contact / E-Mail:Presenting author: [email protected] author: [email protected]

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Rise of total IgE levels upon omalizumab treatment is not due to activation of IgE+ memory B cellsJulia ECKL-DORNA (1), Renate FRÖSCHL (1), Christian LUPINEK (1), Renata KISS (1), Katharina MARTH (1), Raffaela CAMPANA (1), Katharina BLATT (1), Peter VALENT (1), Regina M. SELB (1), Andrea MAYER (1), Katharina GANGL (1), Irene STEINER (1), Philippe GEVAERT (2), Rudolf VALENTA (1), Verena NIEDERBERGER (1)

(1) Medical University of Vienna, Spitalgasse 23, A-1090 Vienna, Austria AND (2) Upper Airway Research Laboratory (URL), Ghent University Hospital, Sint-Pietersnieuwstraat 25, Ghent, Belgium

Omalizumab targets free IgE and inhibits its binding to mast cells. Interestingly during omalizumab therapy an increase in total serum IgE levels has been observed. In this study we investigated whether the latter is caused by a prolonged half-life of IgE upon complex formation with omalizumab or by enhanced IgE production of IgE+ memory B cells upon crosslinking of their B cell receptor with omalizumab.Total and allergen-specific serum IgE were determined in patients before and after subcutaneous treatment with omalizumab (n=15) or placebo (n=5).Omalizumab treated patients showed a 2-6 fold increase of total IgE and a polyclonal rise in specific IgE. To investigate whether this rise could be due to enhanced IgE production by IgE+ memory B cells, we intranasally challenged patients (5/group) with omalizumab, placebo or Bet v 1 and measured total and allergen-specific IgE before and 8 weeks after the challenge. Intranasal omalizumab did not induce a change in total or allergen-specific serum IgE. A rise of Bet v 1-specific serum IgE was observed in Bet v 1 challenged patients as previously reported. Furthermore we tested the effect of omalizumab on IgE production by B cells in vitro. Omalizumab did not increase IL-4 and anti-CD40 induced IgE production in culture.In summary, we observed no effect of omalizumab on IgE production. Thus the total IgE increase is likely to be caused by complex formation of omalizumab with IgE in the blood, thereby prolonging its half-life.


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DanielaPUCCIARELLI(1),NinaLENGGER(1),MartinaTAKÁČOVÁ(2),HeimoBREITENEDER(1),Silvia PASTOREKOVA(2), Christine HAFNER(1,3)

(1) Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Institute of Virology, Department of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia(3) Karl Landsteiner Institute for Dermatological Research, St.Poelten, Austria

Melanoma is associated with an activating mutation in the gene encoding for BRAF. Vemurafenib is an inhibitor of the BRAF V600 mutation which arrests the cell-cycle and induces apoptosis in melanoma cells. One of the main factors which could affect the response to the drug is an inadequate supply of oxygen. We investigated the effects of hypoxia on melanoma cell cultures treated with vemurafenib. Three BRAF V600 mutant melanoma cell lines (M14, 518A2 and A375) were cultivated in hypoxia (2% pO2). Antiproliferative effects were evaluated in a real-time setting in the impedance-based x-CELLigence® system. In hypoxia, vemurafenib-treated M14 and 518A2 cells reduced cell growth (-40% and -38%) compared to normoxic, vemurafenib-treated cells. Surprisingly, hypoxic vemurafenib-treated A375 cells showed an enhanced cell proliferation rate (+34%) when compared to normoxic, vemurafenib-treated A375 cells. WB analyses of HIF1alpha and proteins involved in PI3K, JAK-STAT and MAPK pathways were performed. The expression of HIF1alpha was reduced in vemurafenib-treated M14 and 518A2 cells but not in A375 cells and was also detected in xenografted tumor samples. The results suggest that hypoxia alters the gene-expression pattern of proliferative melanoma cells in a cell-type specific manner.

Acknowledgements

This project is a part of the EU Marie Curie Initial Training Networks (ITN) Biomedical engineering for cancer and brain disease diagnosis and therapy development: EngCaBra. Project no. PITN-GA-2010-264417.


Hypoxia contributes to melanoma phenotype change by affecting the response to vemurafenib

Abstract 67

Poster Presentation

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Novel approach for combined treatment of birch pollen and associated food allergiesHeidi HOFER(1), Claudia ASAM(1), Michael HAUSER(1), Peter BRIZA(1), Christof EBNER(2), Fatima FERREIRA(1), Michael WALLNER(1)

(1) University of Salzburg, Department of Molecular Biology, Salzburg, Austria(2) Allergieambulatorium Reumannplatz, Vienna, Austria

Background: Birch pollen allergy is often accompanied by the oral allergy syndrome (OAS) towards fruits nuts and vegetables. It is triggered due to similar structure of the food allergens and the major birch pollen allergen Bet v 1. At present, only allergen immunotherapy (AIT) can meet the disease.

Methods: To address birch pollen and associated food allergies towards apple and hazelnut, we designed a hybrid protein (MBC4) comprising of T cell epitope containing stretches of all three allergens. In order to diminish IgE binding capacity a specific mutation was introduced. Humoral and cellular immune responses were screened to examine immunogenic and allergenic behavior of the hybrid molecule.

Results: The MBC4 molecule showed an altered folding verified by circular dichroism, and also significantly reduced IgE binding in comparison to parental allergens. In an in vivo mouse model we examined its immune regulatory behavior.

Conclusion: Reduced IgE binding of the MBC4 molecule reveals it as a hypoallergen, but even though the folding of the protein is disrupted, it remains immunogenic. Therefore, we think, that the novel hybrid protein seems to be a promising vaccine candidate for treatment of birch pollen and associated food allergies.

Key words: birch pollen allergy, oral allergy syndrome (OAS)


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Allergen specific antibody responses in non-atopic humans.

Implications for antigen uptake and dendritic cell polarizationChristoph HILLEBRAND, Almedina ISAKOVIC, Josef THALHAMER, Richard WEISS

University of Salzburg, Department of Molecular Biology, Division of Allergy and Immunology

In this study we investigated the humoral immune response in non-allergic donors from different environments and the impact of allergen specific IgG on antigen uptake and immune polarization. We found that donors living in a farming vs. urban environment differed concerning the percentages of IgG responders. Townspeople showed a higher rate of IgG antibody production than farmers. These differences in the seroconversion indicate that the environment and the amount of antigen exposure might have an influence on the immune response. Furthermore we showed that antigen uptake by CD1c+ myeloid dendritic cells in the presence of autologous plasma is highly dependent on IgG. We therefore established the methodology to measure gene expression of Fc receptors and immune polarizing genes in DCs that have taken up the antigen and their capacity to polarize T cell responses. Our preliminary results indicate that CD1c+ that have taken up allergen/IgG immune complexes highly differ in their gene expression and polarizing potential between individual donors. Taken together, we have established the methodology to investigate the impact of living conditions on IgG mediated DC polarization and the maintenance of T cell responses in non-allergic individuals.


Abstract 69

Poster Presentation

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Monitoring the induction of blocking antibodies during birch pollen AITSara HUBER(1), Heidi HOFER(1), Roland LANG(2), Thomas HAWRANEK(2), Michèle RAUBER(3)(4), Fatima FERREIRA(1), Michael WALLNER(1)

(1) Department of Molecular Biology, University of Salzburg, Salzburg, Austria(2) Department of Dermatology, Paracelsus Medical University Salzburg, Salzburg, Austria(3) Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria(4) Clinical and Experimental Allergology, Department of Dermatology and Allergology, Philipps University Marburg, Germany

Background: Bet v 1 represents the major allergen in birch pollen showing a sensitization rate of 95%. At present, allergen immunotherapy (AIT) represents the only curative approach to meet the disease. The induction of so called blocking antibodies denotes a hallmark of the treatment; however, treatment-induced allergen-specific IgG levels do not necessarily correlate with clinical success.

Methods: Five birch pollen-allergic patients were treated with conventional birch pollen AIT and sera were taken before treatment, after reaching the maintenance dose, and after 1 year follow-up. The humoral immune response during therapy was monitored by facilitated antigen binding (FAB) assays and ELISA.

Results: All patients showed an improvement of nasal provocation scores during AIT. Specific IgE levels were initially boosted; but declined during treatment. This was accompanied by the induction of allergen-specific IgG. All donors developed blocking antibodies during AIT as demonstrated by FAB assays.

Conclusion: We did not find a correlation of either IgG or IgE titers with clinical data collected throughout the study. In FAB assays we could measure the capacity of AIT-induced IgG to block IgE/allergen complex formation, which correlated well with clinical success of the treatment.Key Words: AIT, blocking antibodies


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Maria Alejandra PARIGIANI(1), Yoan MACHADOf(1), Sabrina WILDNER(1), Peter BRIZA(1), Lorenz AGLAS(1), Martin WOLF(1), Fátima FERREIRA (1), Michael WALLNER(1)

(1) Department of Molecular Biology, University of Salzburg, Austria

Background: Codons are not used with equal frequencies across species. Synonymous codons were demonstrated to be critical in shaping cellular processes as they affect protein expression, structure and/or function. Disparities in codon usage between different organisms are a common reason for failure in heterologous gene expression often leading to misfolded or insoluble products. Therefore, the DNA sequence of the major birch pollen allergen Bet v 1 was modified to resemble the codon usage of E. coli.Methods: Several batches of Bet v 1 and its codon harmonized homologue Bet v 1 Harm were produced in E. coli and purified to homogeneity. All batches were extensively characterized with an array of physico-chemical methods to detect possible effects of codon harmonization on protein folding. Results: Harmonization of the Bet v 1 resulted in a significantly increased protein yield compared to wild-type Bet v 1. However, no differences in the primary or secondary structure of the protein were observed. Conclusion: Codon harmonization of the allergen Bet v 1 was shown to increase the heterologous expression of soluble protein whereas no influence on the allergen-fold was observed. Thus, the method may facilitate industrial scale production of allergenic proteins.

Codon harmonization enhances heterologous expression of Bet v 1 in E. coli

Abstract 71

Poster Presentation

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M.A SNOVSKAYA., L.S. NAMAZOVA-BARANOVA, O.V. KOZEVNIKOVA, A.K. GEVORKYAN

FederalStateBudgetaryScientificInstitution“ScientificCentreofChildrenHealth”

RTSS were count and sIgE levels to birch, alder, hazelnut, oak pollen allergens, rBetv1, rBetv2, rBetv4, rBetv6 were estimated with ImmunoCAP in 62 children (age 5-10 years) with pollinosis. Then 32 patients got two courses of sublingual ASIT with birch pollen (I group), 30 children had only symptomatic treatment (II group). Results: All patients had high sIgE levels to pollen allergens and rBetv1 and RTSS >11. sIgE to minor allergens were in 7 children (Ib group), other patients had no such antibodies (Ia group).After the first ASIT 12 children (Ia) had symptoms reduction and 11 from these had decrease of sIgE to all pollen allergens. No positive results were observed in Ib group. In II group 4 patients had symptoms and sIgE levels reduction.After the second ASIT 21 patients (Ia) had symptoms reducing (RTSS < 9,3). 19 patients also showed sIgE levels decreasing. Group Ib had no significant change. II group patients had clinical improvement in 5 cases.So effectiveness of ASIT was 34.7% and 67.3% for first and second course respectively.Conclusion: ASIT efficacy is depend on course therapy numbers. Two course of the therapy lead to clinical improvement in more than 65% patients. Application of diagnostic approach with minor allergens provides the accuracy of ASIT prescription.

Role of allergen-based component-divided diagnostics in choice of allergy therapy approach for children with pollinosis

Acknowledgements

The work performed within the agreement № 14.607.21.0017 with Ministry of Education and Science of Russia (unique identifier RFMEFI60714X0017)


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Sophie KITZMUELLER(1,2), Douglas PINHEIRO(1), Thomas KOCHER(2), Johann BAUER(2), Iris GRATZ(1,2,3)

(1) Department of Molecular Biology, University of Salzburg, Salzburg, Austria(2) Division of Molecular Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria(3) Department of Dermatology, University of California San Francisco, San Francisco, CA, USA

Epidermolysis bullosa (EB) is a blistering disease, caused by mutations of anchoring proteins in the skin. Replacing defective genes by ex vivo gene therapy and subsequent transplantation of skin grafts is among the most promising approaches for treating EB. However, graft acceptance may be complicated by the risk of an immune response against the neo-antigen expressed in the graft. Therefore one critical aspect for the success of this treatment is the induction and maintenance of tolerance towards the neo-antigen. In a polyclonal system mimicking the transplantation of a corrected skin graft in a patient with a null mutation we could show that the immune response against an epidermal neo-antigen leads to rejection of the grafted tissue. To analyse the underlying mechanisms we also established a monoclonal grafting model. In this model we transfer antigen (Ovalbumin) restricted CD4+ T-cells (DO11.10 TCR-tg) into mice which receive grafts expressing Ovalbumin as a neo-antigen and follow the antigen-specific responses in vivo. Analyzing skin grafts showed an impact of CD4+ T-cells to graft rejection. Our hypothesis that antigen-specific Treg cells can attenuate this process will serve as a basis for future immunomodulatory approaches to prevent the loss of transplanted skin grafts.

Immunity towards neo-antigen in skin grafts: Mouse models for skin gene-therapy

Acknowledgements

We thank Debra Austria (EB Immunologie) and the University of Salzburg for funding this project.


Abstract 73

Poster Presentation

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Ana I. SANCHO(1,2), Maria M. KLICZNIK(1), Johann W. BAUER(2), Iris K. GRATZ(1,2,3)

(1) Department of Molecular Biology, University of Salzburg, Salzburg, Austria(2) Division of Experimental Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria(3) Department of Dermatology, University of California San Francisco-School of Medicine, San Francisco, CA, USA

Background: Junctional epidermolysis bullosa (JEB), a blistering skin disease, can be caused by mutations in the gene coding for the skin protein laminin 5 (LAM5). No cure is available but reports suggest ex vivo genetically corrected protein transfer as a therapeutic option. As the newly introduced gene could induce adverse immune reactivity, especially in protein-deficient patients, we assess immune responses to LAM5 after ex vivo gene therapy of JEB.

Methods: Proliferation of LAM5-stimulated PBMCs, isolated from a JEB patient, was assessed before and after grafting. Moreover, LAM5-specific cytokine and antibody levels were determined by either ELISA or flow cytometry.

Results: The results showed no significant LAM5-specific cell proliferation before or after grafting. IL6 and IFN-γ were detected in patient’s serum consistent with the presence of wounds and infections. IL4, IL5 and IL17 were not present. No antigen-specific cytokines and no antibody titers were observed. We also established a method to follow immune responses in skin.

Conclusions: These results seem to indicate lack of antigen-specific immune responses after restoring LAM5 expression and protein functionality, suggesting ex vivo gene therapy of JEB as a safe therapeutic approach in patients that lack pre-existing immune reactivity.

The immune response to laminin 5 in skin gene therapy in epidermolysis bullosa

Acknowledgements

This work was supported by DEBRA Austria (“EB Immunologie”) and the University of Salzburg.


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Susanne C. DIESNER(1,2), Cornelia SCHULTZ(1), Xue-Yan WANG(3), Katharina BEITL(1), Franziska ROTH-WALTER(1,4), Denise HEIDEN(1), Anna ONDRACEK(1), Josef SINGER(1), Judit FAZEKAS(1), Caroline STREMNITZER(1), Thomas EIWEGGER(2), Zsolt SZÉPFALUSI(2), Isabella PALI-SCHÖLL(1,4), Erika JENSEN-JAROLIM(1,4), Franz GABOR(3) and Eva UNTERSMAYR(1)

(1) Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Department of Paediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria(3) Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna, Austria(4) Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria

Allergen-loaded PLGA-microparticles (MPs) coated with Neuraminidase (NA) from Vibrio cholera were shown to increase the intestinal uptake via M-cells. Here we aimed to test the safety and therapeutic efficiency of orally applied NA-coated MPs in naïve and food allergic mice.To evaluate the safety of six oral administration cycles of ovalbumin (OVA)-loaded MPs, allergic mice received uncoated or NA-coated MPs or were left untreated. While previously naïve animals developed significantly elevated OVA-specific serum IgA levels but no cytokine changes, we found significantly elevated IL-10 and IFN-gamma levels in allergic mice. To reveal the therapeutic potential of MPs, mice were sensitized with OVA via the oral route and received six oral immunotherapy cycles using OVA-loaded, uncoated or NA-coated MPs. As controls, mice were treated with OVA ig, were left untreated, or naïve. Treatment with NA-coated MPs induced an earlier recovery from anaphylaxis indicated by measurement of core body temperature. Significantly elevated levels of total intestinal IgA and IL-10 production of stimulated splenocytes were found in mice treated with NA-MPs compared to controls.Oral immunotherapy with NA-coated, allergen-loaded MPs represents a safe and efficient food allergy treatment in mice.

Neuraminidase-coated, allergen-loaded microparticles are a safe and efficient novel food allergy treatment option

Acknowledgements

Supported by the Austrian science fund project P21884. EJJ is supported by the Austrian science fund project SFB F4606-B19, CST and JF by W1205-B09 (CCHD).

Abstract 75

Poster Presentation

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Maria M KLICZNIK(1), Eva MURAUER(2), Iris K GRATZ(1,2,3)

(1) Department of Molecular Biology, University of Salzburg, Salzburg, Austria(2) Division of Experimental Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria(3) Department of Dermatology, University of California San Francisco-School of Medicine, San Francisco, CA, USA

The skin blistering disease, dystrophic epidermolysis bullosa (DEB), is caused by mutations in the gene coding for the skin protein Type VII collagen (C7). All therapeutic approaches aim at introducing the correct skin protein to EB patients. Most patients are deficient in C7 and will therefore recognize the therapeutic full-length protein as a neo-antigen. The immune reaction against the neo-antigen will lead to skin inflammation and clearance of C7 protein, which will diminish or destroy the success of any treatment approach.Methods: We are developing a humanized EB mouse model to study and manipulate human immune responses. In this model, we graft human skin and autologous PBMCs to NOD-scid IL2rγnull (NSG) mice. The reaction of human T cells to skin antigen will then be followed in vivo in the recipient mice.Results: Efficiency of the engraftment was analyzed by subtyping immune cells using lineage cell markers for T, B and myeloid cells. Additionally, we analyzed cytokine production of CD4+ and CD8+ T cells. The results showed that CD4+ and CD8+ T cells could stably engraft, although CD8+ cell engraftment was favored compared to CD4+ cells. T cells produced high levels of IFN-γ, which was consistent with the onset of graft versus host disease. While most myeloid cell types engrafted with good efficiency, no stable B cell population was established.Conclusions: Using this model we can follow the engraftment of hPBMCs and measure inflammatory responses mounted against skin antigens.

Development of a humanized mouse model to study the immune response to skin gene therapy in epidermolysis bullosa (EB)

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Berislav BOSNJAK*(1), Michela RIBA*(2), Jose Manuel GARCIA-MANTEIGA*(2), Michelle M. EPSTEIN*(1), Elia STUPKA*(2)

*contributed equally to this work

(1) Medical University of Vienna, Department of Dermatology, DIAID, Experimental Allergy, Währinger Gürtel 18-20, 1090 Vienna, Austria(2)CenterforTranslationalGenomicsandBioinformatics,SanRaffaeleScientificInstitute,ViaOlgettina 58, 20132 Milano, Italy

Individual microarray analyses in experimental allergic asthma have identified a large number of potential regulators and pathways. Relevant genes and pathways underlying disease pathogenesis may emerge from the integration of these datasets. We selected 6 publically available datasets for meta-analysis on the basis of the microarray platform and in vivo experimental protocol. Our strategy was to combine a top down pathway-centered and a bottom up gene-centered analyses. The first approach consisted of selecting asthma-specific pathways from a combination of ‘enriched biological terms’ in each individual study. In second approach, we combined differentially expressed genes from individual datasets to create a gene list. From 22,690 genes and total of 131 samples, we obtained a core-network of 903 interconnected genes unraveling known (STAT1, RELA) and previously unknown hubs (PPARA, HCLS1), as well as disease-related pathways, such as T-cell and B-cell mediated immunity, phagocytosis and circadian rhythm. To validate the analysis, we confirmed the expression changes of 42 genes in lungs from mice with allergic asthma. The power of this strategy is the possibility of uncovering key molecules underlying disease pathogenesis utilizing datasets derived from disparate experiments.

Novel meta-analysis of gene expression in mouse allergic asthma

Abstract 77

Poster Presentation

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Friedrich ERHART(1), Alexander M. DOHNAL(1), Thomas FELZMANN(2), Philipp FUNOVICS(3), Leo KAGER(4,5), Volker WITT(4,5), Susanna LANG(6), Carmen VISUS(2)

(1) CCRI, St. Anna Kinderkrebsforschung, Vienna, Austria(2) Activartis Biotech GmbH, Vienna, Austria(3) Department of Orthopaedic Surgery of the Medical University of Vienna, Austria(4) Department of Pediatric and Adolescent Medicine of the Medical University of Vienna, Austria(5) St. Anna Children’ Hospital, Vienna, Austria(6) Department of Pathology of the Medical University of Vienna

Cancer immunotherapy (CIT) based on dendritic cells (DC) is gradually developing into a more mature therapy option. Our approach involves the microbial danger molecule lipopolysaccharide in connection with interferon-gamma.In the years 2000-2008 we recruited 29 children, adolescents and adults suffering from sarcomas (48% osteosarcoma, 52% other) that had recurrent and/or metastatic disease and were thus eligible for an experimental therapy (phase I clinical trial). At the time of DC-CIT, 62% were in 2nd or higher complete surgical remission (CR) and 34% in progress; 3% had stable disease. After DC-CIT, 41% received further multimodal treatment.In summer 2014 we tracked the outcome of all 29 participants. We registered an overall 5-year survival rate of 31% and 10-year rate of 22% after advent of experimental treatment. For osteosarcoma alone, we observed respective rates of 36% and 17%. The rate of 5-year survivors with later additional multimodal therapy is 67%. For any efficacy evaluation, further clinical studies with a randomized, controlled design will be necessary.To explore factors associated with long-term survival, we investigated 9 vaccine variables and 74 host immune system variables. A significant association with survival was registered for helper T cell markers.

5- and 10-year post-relapse survival of pediatric and young adult sarcoma patients after a dendritic cell-based cancer immunotherapy.

Acknowledgements

We would like to thank Gerda Ricken, René Reitermaier, Simone Klingenbrunner and Katrin Fischhuber for excellent technical assistance.

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Judit FAZEKAS(1,2), Josef SINGER(1,2), Wei WANG(3), Marlene WEICHSELBAUMER(1), Alexander MADER(4), Miroslawa MATZ(1), Willibald STEINFELLNER(4), Yuri SOBANOV(1), Diana MECHTCHERIAKOVA(1), Michael WILLMANN(5), Edzard SPILLNER(6), Renate KUNERT(4), Erika JENSEN-JAROLIM(2,1).

(1) Comparative Immunology and Oncology, Institute of Pathophysiology and Allergy Research, Medical University of Vienna(2) Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna(3) Department of Immunology, Capital Medical University, Beijing, P. R. China(4) Department of Biotechnology, VIBT – BOKU – University of Natural Resources and Life Sciences, Vienna(5) Department for Companion Animals and Horses, Clinic for Internal Medicine Small Animals, University of Veterinary Medicine Vienna, Austria(6) Immunological Engineering, Department of Engineering, Aarhus University, Denmark

Although cancer is among the most common causes of death in aged dogs, no passive immunotherapy has been established yet.The human and canine Epidermal Growth Factor Receptors (EGFR) show striking homology as well as similar biological functions. Thus, we generated a canine anti-EGFR antibody (Can225IgG) in order to introduce passive immunotherapy also for veterinary patients.The antibody was produced in CHO DUKX-B11 cells and purified via Protein G affinity chromatography. CD-spectroscopy, immunoassays and cell proliferation assays provided information about assembly, folding, specificity, inhibitory functions and effector-cell mediated tumor-cell killing.We obtained around 120 mg of purified, correctly folded and assembled antibody. Furthermore, we could demonstrate that signal depletion by Can225IgG was sufficient to reduce proliferation and viability of tumor cells. Moreover, co-incubation of canine breast cancer cells with canine monocytes and Can225IgG led to significantly increased phagocytosis of tumor cells.In conclusion we report the first recombinant canine anti-EGFR antibody with the perspective of a clinical trial in dogs. In addition, we propose the use of better-suited model organisms for therapeutic drug studies, speeding up clinical trials for the benefit of both human and canine patients.

Can225IgG, a new canine anti-EGFR antibody

Acknowledgements

Supported by grants P 23398_B11 and DK W1205-B09 (CCHD) of the Austrian Science Fund, by Biomed Int R+D, Vienna, and by RotePfote - Cancer Research for pets. M. Weichselbaumer was a recipient of a scholarship of the University of Veterinary Medicine Vienna.

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Priya SARATE(1), Stefan HEINL(2), Hana KOZÁKOVA(3), Reingard GRABHERR(2), Irma SCHABUSSOVA(1), Ursula WIEDERMANN(1)

(1)InstituteofSpecificProphylaxisandTropicalMedicine,CenterforPathophysiology,Immunology and Infectiology, Medical University of Vienna, Vienna, Austria(2) (c/o CD Laboratory for Genetically Engineered Lactic Acid Bacteria Department of Biotechnology University of Natural Resources and Life Sciences VIBT, Vienna, Austria(3) Laboratory of Immunology and Gnotobiology, Institute of Microbiology of Academy of Sciences of Czech republic,V.V.I., Novy Hradek, Czech Republic

It is well recognized that allergic individuals are at risk to develop multiple allergies and such poly-sensitized individuals are difficult to treat by conventional therapeutic measures. We have recently established mouse models of poly-sensitization and demonstrated that allergic poly-sensitization can be suppressed by mucosal treatment with novel allergen chimers in adult mice. With respect to neonatal interventions we previously showed that colonization with recombinant probiotic strains expressing the allergen Bet v 1 successfully prevents allergic responses. In order to investigate whether the concept of neonatal colonization with recombinant probiotic bacteria could be used for prevention of allergic multi-sensitivities, we now have constructed a recombinant Lactobacillus plantarum, constitutively expressing a birch (Bet v 1) and grass pollen chimer (Phl p 1 and Phl p 5). Next, neonatal colonization will be performed in order to study the effects and interactions of this recombinant lactobacillus strain with the host immune system.

Neonatal colonization with recombinant lactic acid bacteria for prevention of poly-sensitization

Acknowledgements

FWF funding (MCCA program)

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Manuela ZLAMY(1), Reinhold WÜRZNER(2), Walther PARSON(3), Heidemarie HOLZMANN(4), Verena JELLER(1), Martina PRELOG(5)

(1) Department of PediatricsI, Medical University Innsbruck, Austria(2) Division of Hygiene and Microbiology, Medical University Innsbruck, Austria(3) Institute of Legal Medicine, Medical University Innsbruck, Innsbruck, Austria(4) Clinical Institute of Virology, Medical University of Vienna, Austria(5) University Children’s Hospital, University of Würzburg, Germany

The aging of the immune system (immunosenescence) starts soon after birth with thymus involution and continues throughout life. T cell immunosenescence is characterized by a decreased number of naïve T cells, a compensatory autoproliferation of peripheral memory T cells, an oligoclonal expansion of T cell receptor (TCR) distribution, and an expansion of perpetuated CD8+ T cells due to chronic viral infections (e.g. CMV). Consequently a low antibody response to vaccinations and neo-antigens, increased infection rates with associated morbidity and mortality rates as well as an increased incidence of autoimmune disorders is described. Former studies assumed that thymectomy in early childhood leads to a premature immunosenescence, mimicking changes of the aged immune system. Our study was aimed to investigate whether thymectomy in early childhood may serve as a model for premature immunosenescence. Therefore changes in the peripheral T cell subpopulations were measured in thymectomized patients and age-matched controls. Naïve and memory T cells, intestinal derived naïve T cells (CD103+), IL-7 receptor (CD127)-expressing T cells and TCR diversity were analyzed. The influence of serum IL-7 concentrations and latent CMV infection on specific T cell subpopulations was measured. To determine the influence of thymectomy on the immune response to neo antigens (tick-borne-encephalitis-virus vaccination and mumps-measles-rubella-vaccination) a clinical study was conducted. Our study revealed lower percentages of naïve T cells, especially within the CD8+ T cell pool. We found higher proportions of intestinal derived T cells in thymectomized patients compared to controls and a peripheral proliferation of pre-existing memory T cells. IL-7 values in thymectomized patients were slightly higher compared to controls. CMV seropositivity predicted a more monoclonal TCR distribution in thymectomized patients. Thymectomized children showed a delayed immune response to neo-antigens but a normal immune response to revaccination with a live-attenuated vaccine. Our study demonstrated alterations in the T cell immune system reminiscent of the immunological changes found in elderly. Thymectomized children represent a unique group to study the long-term consequences of suboptimal or lacking thymus output. It remains to be evaluated, if vaccine-specific IgG antibody concentrations of thymectomized children vanish earlier in later life than in controls, and if immunosenescence parameters accelerate with advancing age making thymectomized patients prone for inflammatory or degenerative diseases of the elderly.

Immune alterations after thymectomy in early childhood

Abstract 81

Poster Presentation

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Huey-Jy HUANG(1), Mirela CURIN(1), Srinita BANERJEE(1), Kuan-Wei CHEN(1), Tetiana GARMATIUK(1), Yvonne RESCH(1), Raffaela CAMPANA(1), Margarete FOCKE-TEJKL(1), Rudolf VALENTA(1), Susanne VRTALA(1,3)

(1) Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria(3) 3Christian Doppler Laboratory for the Development of Allergen Chips, Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria

Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 are the most important house dust mite (HDM) allergens. The aim of this study was to define a mix of non-allergenic T cell epitope-containing peptide mix of these allergens for tolerance induction. According to the amino acid sequences of these allergens we were able to synthesize 36 overlapping peptides covering the complete sequences of the 6 HDM allergens on a peptide synthesizer. The peptides could be purified in large amounts. They lacked secondary structure but most of them remained soluble in physiological buffers. Testing of the peptides for IgE reactivity with sera from HDM allergic patients showecker testing of the peptides for their ability to stimulate T cells was performed with PBMCs from HDM allergic patients using a CFSE dilution-based assay showing the presence of relevant HDM T cell epitopes in the peptides. Our results indicate that it may be possible to identify a hypoallergenic T cell epitope-containing peptide mix of the clinically relevant HDM allergens for tolerance induction.

Towards a non-allergenic peptide mix containing the T cell epitopes of relevant house dust mite allergens


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Ruth BYRNE(1), Karolina VON DALWIGK(1), Günter STEINER1, Johannes HOLINKA2, Reinhard WINDHAGER2, Josef S. SMOLEN1, Hans KIENER1, Clemens SCHEINECKER(1)

(1) Division of Rheumatology(2) Department of Orthopedics, Medical University of Vienna, VIENNA, Austria

The synovial lining tissue consists of fibroblast-like synoviocytes (FLS) and monocyte-derived macrophage-like synoviocytes (MLS) within a self-built meshwork of dense extracellular matrix (ECM) components. FLS are thought to direct ECM synthesis, assembly and degradation. Whether FLS themselves or the ECM network serve as guiding structures for MLS migration is incompletely understood.We studied the dynamics of synovial tissue modeling as well as MLS migratory behavior using a 3D synovial tissue in vitro model. Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. CD14+ monocytes (MO) were isolated from peripheral blood. FLS and MO were labeled with fluorescent membrane dyes and cultured in spherical extracellular matrix micromasses with an average size of 1.5 mm for up to two weeks. Second harmonic generation was used for the visualization of collagen fibers (ECM). Cell migration was monitored in individual micromasses by real-time confocal/multi-photon microscopy.The formation of a synovial lining-like layer on the outside of the 3D cell culture takes 2 – 3 days. The first signs of collagen fibers co-localized with FLS clusters and appear as early as day 1. They increase density alongside the establishment of the lining layer. The majority of MO was found to be in close contact with the FLS network with low tendency for migration. A minor fraction of MO displayed directed cell movement with an impressive maximum speed of up to 15 mcm/min. MO migration occurred in intimate contact with FLS but did not necessarily follow FLS network boundaries, but rather the cell cultures surface. However, for sitting as well as migrating MO contact with FLS seems to be more important than contact with ECM.The 3D synovial tissue culture system allows for monitoring and analyzing the dynamics of synovial lining modeling. Both, FLS and MO appear to cooperate in the organization of the synovial lining tissue with subtle migration patterns of MO in relation to the organized synovial lining architecture. Ongoing experiments address molecular mechanism(s) of MO – FLS interaction in order to identify potential targets for future therapeutic intervention in arthritis.

The fibroblast network serves as guiding structure for directed monocyte migration in 3D synovial tissue cultures

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Selective activation of cannabinoid receptor 2 primes eosinophils for enhanced migratory responsivenessRobert FREI, Gerald PARZMAIR, Silke SCHRANZ, Akos HEINEMANN, Eva STURM

Inst. of Experimental and Clinical Pharmacology, Medical University of Graz;Universitätsplatz 4, 8010 Graz, Austria

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) has been proposed to elicit eosinophil migration in a CB2 receptor/Gi/o-dependent manner. However, it was claimed recently that besides CB2 activation, 2-AG metabolites and the 15-lipoxygenase pathway are involved in the regulation of eosinophil migration induced by 2-AG. Here we explored the direct contribution of specific CB2 receptor activation to eosinophil effector function. We observed that the selective CB2 agonist JWH-133 potently enhanced chemoattractant-induced eosinophil shape change, chemotaxis, CD11b expression and adhesion. Moreover, selective CB2 stimulation evoked an increase in intracellular Ca2+ and mediated the activation of MAPK-kinase 1/2 and Rho-associated protein kinase via a pertussis toxin-insensitive G-protein. These data indicate that direct CB2 receptor activation primes eosinophils for an enhanced migratory responsiveness towards proinflammatory agents. Given its abundant expression in eosinophils and other inflammatory cells, antagonism of the CB2 receptor may be of therapeutic relevance in allergic inflammation and other eosinophilia-associated disorders.

Acknowledgements

We thank Birgit Brodacz and Iris Red for their skilled technical assistance.


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Barbara PLATZER (1), Kutlu G. ELPEK (2), Viviana CREMASCO (2), Kristi BAKER (3), Cornelia SCHULTZ (1, 4), Eleonora DEHLINK (1, 5) Kai-Ting C. SHADE (6), Robert M. ANTHONY (6), Richard S. BLUMBERG (3), Shannon J. TURLEY (2) and Edda FIEBIGER (1)

(1) Division of Gastroenterology and Nutrition, Boston Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA(2) Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA(3) Division of Gastroenterology, Brigham and Women’s Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA(4) Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria(5) Department of Pediatrics and Adolescent Medicine, Division of Pediatric Pulmology, Allergology and Endocrinology, Medical University of Vienna, 1090 Vienna, Austria(6)CenterforImmunologyandInflammatoryDiseases,MassachusettsGeneralHospital,Harvard Medical School, Boston, MA 02129, USA

Epidemiologic studies discovered an inverse association between IgE-mediated allergies and cancers. Mechanistic details as to how IgE contributes to tumordefense remain poorly understood. Using a humanized mouse model that mirrors expression of Fc-epsilon-RI, the high affinity IgE receptor, on dendritic cells (DCs) as seen in humans at steady state in the absence of inflammation, we demonstrate a novel IgE-mediated cross-presentation pathway. This cross-presentation pathway is executed by the CD8-negative DC subset in an IgE-dependent manner and is particularly potent in inducing cytotoxic T cells in response to low-dose soluble antigen. Interestingly, co-administration of danger signals and IL-12 are dispensable for efficient IgE-mediated cross-presentation. In classical tumor vaccination experiments with the B6-melanoma model, DCs that are loaded ex vivo with tumor antigen through IgE/Fc-epsilon-RI prevent tumor growth more potently than DCs loaded without IgE. In summary, our study delineates a novel cross-presentation pathway for soluble antigens that relies on the DC-bound IgE pool. Furthermore, our findings point to IgE-mediated cross-presentation as a target pathway to improved DC-based tumor vaccination strategies as currently developed for cancer therapy in humans.

Dendritic cells use IgE-mediated antigen cross-presentation to generate cytotoxic T cells in response to low dose soluble antigen

Acknowledgements

This work was supported by grants from the National Institutes of Health: K01DK093597 (to B.P.), DK53056 (to R.S.B.), 5R01 DK074500-08, 2P01AI045757-15, R21 CA182598-01 (to S.J.T.), T32 CA 070083-15 (to V.C.) and AI075037 (to E.F.) and by the Harvard Digestive Diseases Center Grant P30DK034854.

Abstract 85

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HAX1 deletion impairs BCR-internalization, leading to delayed apoptosis of mature B cellsGertrude ACHATZ-STRAUSSBERGER

Department of Molecular Biology, Division of Allergy and Immunology, University of Salzburg

HAX1 was identified in our group as an IgE-tail interacting protein. To determine the impact on IgE receptor mediated signaling we deleted HAX1 in mice, which caused a severe reduction in the number of lymphocytes in spleen, with an almost 70% reduction of B220+ cells. To explain the reduced cellularity of Hax1-/- mice and as HAX1 was thought to play a protective role in apoptotic processes, we investigated the general viability for bone marrow B progenitor cells and splenic B cells. The general survival of Hax1-/- bone marrow cells was inconspicuous, whereas Hax1-/- splenocytes showed elevated survival. By focusing on B220 cells, we showed decreased apoptosis of IgM stimulated splenic B cells, which by trend was also visible for B220 bone marrow cells. This, as we suggest, is reasoned by an impaired internalization of the BCR from Hax1-/- splenic B cells after IgM crosslinking. We strongly suggest an important role for HAX1 in BCR mediated internalization events, as we showed a tight interaction to the cytoplasmic domains of IgG1, IgG2a and IgE and identified KVKWI(V)F as the putative binding motif at the cytoplasmic domains. IgE B cells are considered to be prone for apoptosis. As the main function of HAX1 was found in the inhibition of apoptosis, we further want to investigate the impact on the apoptosis prone phenotype of mIgE B cells.


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Hanane LANAYA(1)*, Anuradha NATARAJAN(1)*, Karin KOMPOSCH(1)*, Liang LI(2), Nicole AMBERG(1), Stefanie K. WCULEK(1), Martina HAMMER(1), Rainer ZENZ(1), Markus PECK-RADOSAVLJEVIC(3), Wolfgang SIEGHART(3), Michael TRAUNER(3), Hongyang WANG(2), Maria SIBILIA(1)

(1) Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, 1090 Vienna, Austria.(2) National Center for Liver Cancer, International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, 225 Changhai Road, Shanghai 200438, China. (3) Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Division of Gastroenterology and Hepatology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. *These authors contributed equally to this work.

Hepatocellular carcinoma (HCC) is the sixth most frequent cancer with limited treatment options and poor prognosis. Tumorigenesis has been linked with macrophage-mediated chronic inflammation and diverse signaling pathways including the Epidermal Growth Factor Receptor (EGFR) pathway. The precise role of EGFR in HCC is unknown, and EGFR inhibitors have shown disappointing results in clinical trials likely due to the lack of biomarkers allowing patient stratification. Here we discover that EGFR is expressed in liver macrophages in both human HCC and in a mouse HCC model. Mice lacking EGFR in macrophages show impaired hepatocarcinogenesis, whereas mice lacking EGFR in hepatocytes unexpectedly develop more HCC due to increased hepatocyte damage and compensatory proliferation. The presence of EGFR-positive liver macrophages in HCC patients is associated with poor survival. Mechanistically, following IL-1 stimulation, EGFR is required in liver macrophages to transcriptionally induce IL-6, which triggers hepatocyte proliferation and HCC. This study highlights the complexity of EGFR signaling in HCC and demonstrates a new tumor-promoting mechanism for EGFR in non-tumor cells, which could lead to more effective precision medicine strategies.

EGFR is required in liver macrophages for IL-1-induced IL-6 production and hepatocellular carcinoma formation

Abstract 87

Poster Presentation

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Inducible Neo-Antigen Expression in Steady State Langerhans Cells mediates Immunological ToleranceHelen STRANDT(1), Veronika HÖPFLINGER(1), Peter HAMMERL(1), Daniel H. KAPLAN (2), Dagmar WIRTH(3), Josef THALHAMER(1), Angelika STOECKLINGER(1)

(1) Department of Molecular Biology, University of Salzburg, Austria(2) Department of Dermatology, Center for Immunology, University of Minnesota, Minneapolis, USA(3) Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research, Braunschweig, Germany

Epidermal Langerhans cells (LC) are highly efficient antigen presenting cells. However, their function for immune activation or tolerance induction is still not completely solved. To address the principal biological role of LC in vivo, we have developed transgenic mouse models which enable de novo antigen expression specifically in LC by Tamoxifen- (TAM) mediated activation of Cre recombinase. Thus, in these mice neo antigens such as Ovalbumin (OVA), E. coli beta-Galactosidase (bGal) or others are presented to the immune system selectively by LC. We found that antigen expression in resting LC did not induce antibody production however, it was sufficient for initial activation of OT-1 cells and, moreover, endogenous cytotoxic T cells. Interestingly, when we looked at later time points, CTL responses were suppressed, even after antigen challenge with the respective gene gun (GG) vaccine. We found regulatory T cells (Treg) highly relevant for immunosuppression, since depletion of CD25+ Treg cells, almost completely restored CTL responses to that of GG-immunized wild type mice. These data suggest that antigen presentation by resting LC results in T- and B cell tolerance which cannot be broken by a subsequent inflammatory antigen challenge.


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Silke SCHROM(1), Sarah AHMADI(1), Barbara DILLINGER(1), Wolfgang HOLTER(1,2), Alexander DOHNAL(1), Andreas HEITGER(1,2)

(1) CCRI, St. Anna Children’s Cancer Research Institute, Vienna, Austria(2) Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Austria

T cells (TCs), playing a key role in immune reconstitution after hematopoietic stem cell transplantation (HSCT) and guiding allo-immune reactions against malignant but also normal host tissues (GvHD) require costimulatory signals for full activation. By interfering with the CD28 pathway, anergy, i.e. antigen-specific non-responsiveness, is induced. The tolerizing effect of CD28 blockade via CTLA4-Ig has been described for the CD4+ TC population. We hypothesize that costimulation blockade (CB) using CTLA4-Ig or an anti-CD28 antibody also affects CD8+ cytotoxic TCs.

We performed mixed leukocyte reactions with Balb/c CD3+ TCs stimulated by C57BL/6 dendritic cells. In the presence of CB proliferation of CD8+ TCs was inhibited by ~65%, the degranulation marker CD107a, intracellular and secreted levels of GzmB and IFN-g were reduced with no difference using either CTLA4-Ig or anti-CD28. These data suggest that CB during allostimulation impairs the expansion and/or function of murine CD8+ TCs. We plan to perform cytotoxic T lymphocyte killing assays using the mouse lymphoma cell line EL4 as target cells and further elucidate whether CB directly affects CD8+ TCs or via control by CD4+ TCs. Understanding the effects of CB on cytotoxic T cells will be important for further HSCT research.

Elucidating the effects of costimulation blockade on allo-reactive cytotoxic T cells


Abstract 89

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Bastien A. HUBER(1), Angela HALFMANN(1), Klara SOUKUP(1), Alexander M. DOHNAL(1)

(1) Children’s Cancer Research Institute Vienna – St. Anna Kinderkrebsforschung Wien

Dendritic Cells (DCs) are important in immune regulation to prevent excessive effector T cell functions after lipopolysaccharide (LPS) encounter. Based on human data from monocyte-derived DCs we detected a potential target to drive DCs into their regulatory phenotype, the regulator of G-protein signaling protein 16 (RGS16). Except its mechanical function as a GTPase turning off G-protein coupled receptor signaling, there is not much known about RGS16 in DCs. RGS16 protein is not only present in DCs, we also found it in the supernatant of LPS-stimulated bone-marrow-derived DCs from mice. Furthermore, DCs silenced for RGS16 expression were able to induce higher proliferation in CD8+ T-cell populations compared to control DCs. As we are interested in immune-regulatory mechanisms in the tumor microenvironment we inoculated B16F10 melanoma into wild-type mice. In tumor-resident DCs mRNA expression was 2-fold lower than in the respective splenic DCs. However in the tumor cells, RGS16 expression was almost 10-fold higher. Further investigation will determine the role of RGS16 in immune regulation and its impact in the tumor microenvironment.

The role of RGS16 in immune regulation

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Migratory skin dendritic cells are involved in CD8+ T cell responses against the melanoma-associated antigen gp100David G. MAIRHOFER(1), Vincent FLACHER(2), Christoph H. TRIPP(1), Björn E. CLAUSEN(3), Suzie CHEN(4), Patrizia STOITZNER(1)

(1) Department of Dermatology and Venereology, Innsbruck Medical University, Innsbruck, Austria(2) Laboratory of Immunopathology and Therapeutic Chemistry, Institute de Biologie Moléculaire et Cellulaire, Strasbourg, France(3) Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany(4) Susan Lehman Cullman Laboratory for Cancer Research, Rutgers University, Piscataway, New Jersey, USA

Ectopic expression of the neuronal receptor, metabobotropic glutamate receptor 1 (Grm1), in melanocytes has been described to be involved in melanoma pathogenesis in human. The overexpression of Grm1 in melanocytes of the transgenic (tg)(Grm1)EPv mouse strain leads to spontaneous melanoma development. In this study we characterized the role of skin DC subsets in cross-priming of CD8+T cells in tumor immunity. Melanoma growth disturbed the DC network, especially Langerin+dermal DC (dDC) were reduced in skin and lymph nodes. Functionally this DC subset seemed to be impaired due to lower expression of CD40 and IL-15Rα in tumor mice. In order to highlight the importance of migratory DC in cross-presentation of endogenous tumor-associated gp100 antigen, we crossed tg(Grm1)EPv mice to LangerinEGFP and Langerin-DTR mice to allow sorting or depletion of various skin DC subsets. All sorted migratory skin DC subsets were able to cross-present endogenous gp100 antigen as determined by production of IFN-gamma and Granzyme B by gp100-specific CD8+T cells from transgenic pmel-1 mice. The depletion of Langerin+dDC and LC in vivo led to a reduction in proliferation and differentiation of adoptively transferred gp100-specific CD8+T cells. This effect could be restored when Langerin+dDC repopulated the skin. Our results demonstrate that skin DC, especially Langerin+dDC, are able to cross-prime CD8+T cell responses against a tumor-associated antigen, thereby they would be suitable targets for a immunotherapy against melanoma.


Abstract 91

Poster Presentation

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Claudia ZELLE-RIESER(1), Shanmugapriya THANGAVADIVEL(1), Wolfgang WILLENBACHER (2), Rainer BIEDERMANN(3), Richard GREIL(1,4) and Karin JÖHRER(1)

(1) Tyrolean Cancer Research Institute, Innsbruck, Austria(2) Department of Internal Medicine V, Innsbruck Medical University, Innsbruck, Austria(3) Department of Orthopedic Surgery, Innsbruck University Medical Center, Austria(4) Laboratory for Immunological and Molecular Cancer Research, IIIrd Medical Department, Paracelsus Medical University Salzburg, Salzburg, Austria

Immune evasion is a prerequisite for the establishment of cancers and has been delineated in several entities in detail. Recent studies suggest that immunological alterations involving the T-cell compartment are responsible for immune deficiencies. However, data in multiple myeloma are largely missing. Here, we investigated the phenotype and function of bone marrow and peripheral T cells of myeloma patients and healthy, age-matched donors. We found an upregulation of T cells expressing the molecules CTLA-4 (CD152), CD57 (human natural killer-1), 2B4 (natural killer cell receptor 2B4, CD244) and PD1 (programmed death receptor-1, CD279), which were previously associated with T-cell exhaustion and T-cell senescence. Functional studies confirmed decreased proliferative capacity of bone marrow CD8+ T-cells of multiple myeloma patients and downregulation of Tbet after T-cell activation, confirming the exhausted status of the CD8+ T-cells. Our results suggest that restoring the functional activity of bone marrow T-cells could enhance the efficacy of current immunotherapeutic strategies in multiple myeloma patients.

T cells from Multiple Myeloma patients exhibit features of T-cell exhaustion

Acknowledgements

This project was supported by a grant of the Austrian Cancer Society/Tirol and the EU-grant OPTATIO (FP2007-2013, no. 278570)


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Christoph H. TRIPP(1), Daniela ORTNER(1), Sandrine DUBRAC(1), David E. SCHLÖGL(1), Kerstin KOMENDA(1), Björn E. CLAUSEN(2) and Patrizia STOITZNER(1)

(1) Department of Dermatology and Venereology, Innsbruck Medical University, Innsbruck, Austria (2) Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany

The exposure to chemicals can cause carcinogenesis in the skin leading to the development of squamous cell carcinoma or basal cell carcinoma. Due to their location, skin dendritic cells (DC) are the first antigen presenting cells encountering transformed cells. In this project we wanted to understand the innate mechanisms occurring in chemical carcinogenesis in the skin. Langerin+ DC/Langerhans cells (LC) proved to be important for early events during carcinogenesis, because tumor formation was accelerated after application of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) on murine skin lacking these two cell types. In particular, we detected more gamma-H2AX-expressing keratinocytes, indicative of DNA damage in DMBA-treated skin devoid of Langerin+ DC/LC. The clearance of damaged and transformed skin cells is mediated by innate cells, such as NK cells. Application of DMBA led to an accumulation of NK cells in the skin and this process was dependent on the presence of Langerin+ DC/LC. Moreover, in vivo depletion of NK cells accelerated tumor development in a similar way as in the absence of Langerin+ DC/LC. Our findings demonstrate an important link between skin DC and innate effector cells in the clearance of transformed cells preceding carcinogenesis.

key words: weed pollen allergens

The role of Langerin-positive skin dendritic cells in chemical skin carcinogenesis


Abstract 93

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Douglas F. PINHEIRO(1), Sophie SKITZMUELLER(1,3), Maria Magdalena KLICZNIK(1,3), Gertrude ACHATZ(1), Abul K.ABBAS(2), Iris GRATZ(1,3,4).

(1) Department of Molecular Biology, University of Salzburg, Salzburg, Austria(2) Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA(3) Division of Molecular Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria(4) Department of Dermatology, University of California San Francisco, San Francisco, CA 94143, USA

Immune homeostasis is governed by a fine balance of pathogenic effector T cells (Teff) and suppressive regulatory T cells (Treg). Here we aim to define the impact of tissue-antigen expression levels on T cell activation strength and its downstream effects on T cell differentiation. We use two Tetracycline(Tet)-inducible models for expression of ovalbumin (Ova) in skin. Keratin 5 promoter (K5) is expressed in the basal cell layer and involucrin (INV) in the upper layers of the epidermis. In these models we can follow adoptively transferred naïve Ova specific T CD4+ cells (DO11.10) in vivo. We find that K5-Ova-epxression leads to differentiation of both, Teff and Treg cells, while INV completely blocks Treg differentiation. Consequently, K5 results in self-resolving skin inflammation while INV leads to fatal disease. The latter can be reversed by in vivo Treg generation prior to Ova-induction. We found that INV leads to higher expression levels, which we can titrate by dilution of Tet. This reduces proliferation and production of effector cytokines and restores Treg cell differentiation. Taken together, this suggests that antigen load and initial TCR signal strength can be crucial in the decision of Teff vesus Treg differentiation with remarkable consequences on clinical outcome.key words: weed pollen allergens

Lowering T cell activation strength in vivo polarizes T cell differentiation towards a regulatory phenotype

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Karine MARAFIGO DE AMICIS (1,2,6); Alexandra SAYURI WATANABE (2,3); Clovis Eduardo GALVAO (2,4); Daniele DANELLA FIGO (1,2); José Roberto APARECIDO DOS SANTOS-PINTO (2,5); Mario Sergio PALMA (2,5); Fábio FERNANDES MORATO CASTRO (1,2,3); Jorge KALIL (1,2,3,4); Fatima FERREIRA–BRIZA (6); Gabriele GADERMAIER (6); Keity SOUZA SANTOS (1,2);

(1) Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, School of Medicine, University of Sao Paulo, Sao Paulo, Brazil(2) Institute for Investigation in Immunology (iii), INCT, Sao Paulo, Brazil(3) Division of Allergy, Clinics Hospital of School of Medicine, University of Sao Paulo, Sao Paulo, Brazil(4) Laboratory of Immunology, Heart Institute (InCor), LIM19, University of Sao Paulo, School of Medicine, Sao Paulo, Brazil(5) Institute of Biosciences of Rio Claro, University of Sao Paulo State (UNESP), Rio Claro, Brazil(6) Department of Molecular Biology, Division of Allergy and Immunology, University of Salzburg, Salzburg, Austria

Double sensitization to both, Apis mellifera and Vespula ssp. venom is common in up to 59% of the European hymenoptera venom allergic patients. In Brazil, Polistes sp. and Polybia paulista pose the major risk. Reports about double sensitization involving Apis mellifera and Solenopsis are rare, and there is nothing described about multi-sensitization to insects.Nine patients with anaphylactic reactions and sensitization to honeybee, wasp and fire ant were tested by ImmunoCap, skin prick test (SPT), dot blot (DB) and western blotting (WB) using commercial extracts of Apis mellifera, Polistes sp. and Solenopsis as well as Polybia paulista venom extract produced by our group.All patients were positive to four venoms in DB and WB. Four patients presented ImmunoCAP <0.35 kUA/l for one or two venoms. In WB, the patients recognized multiple IgE-reactive bands in each venom extract. Apart from homologous molecules, new allergens unique for specific venoms were identified presenting distinct molecular masses not present in the other sources.This is the first report of multi-sensitization to four hymenoptera insect venoms. Next steps are to produce recombinant species-specific allergens and evaluate them by component-resolved diagnostics.

Multi-sensitization to hymenoptera venoms

Acknowledgements

For the financial support of Fundação de Amparo à Pesquisa (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) from Brazil.

Abstract 95

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Beside Der p 1 and Der p 2, also other house dust mite allergens have high allergenic activityYvonneRESCH(1),MiraŠILAR(2),PeterKOPAČ(2),MihaelaZIDARN(2),Kuan-WeiCHEN(1),Wayne THOMAS(3), Peter KOROŠEC(2), Mitja KOŠNIK(2), Rudolf VALENTA(1) and Susanne VRTALA(1,4)

(1) Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria; (2) University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia;(3) Telethon Institute for Child Health Research, University of Western Australia, Perth, Australia;(4) Christian Doppler Laboratory for the Development of Allergen Chips, Medical University of Vienna, Austria

Introduction: Der p 1 and Der p 2, the two major allergens in house dust mites (HDMs), have been extensively studied, however, little is known about their allergenic activity in comparison to other HDM allergens.

Methods: Seven important HDM allergens (Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Der p 21 and Der p 23) were studied in regard to their IgE-binding frequency, the levels of IgE directed to the allergens as determined by Streptavidin ImmunoCAPs and their allergenic activity in CD63 based basophil activation tests, using sera from 30 clinically well-characterized HDM-allergic Slovenian patients.

Results: Although Der p 1 and Der p 2 were the most frequently recognized allergens (90%) and bound the highest levels of allergen-specific IgE (mean: 8.59 kU/L and 7.73 kU/L respectively) in the tested patients, they were not the most potent allergens in regard to basophil activation. In fact, the newly identified major allergen Der p 23 (IgE-binding frequency: 83%) induced the strongest basophil activation in sensitized patients, although the allergen-specific IgE levels were low (mean: 2.61 kU/L). Interestingly, Der p 5, a mid-tier allergen (IgE-binding frequency: 37%) showed the third highest IgE levels (mean: 4.18 kU/L) and the second strongest basophil activation.

Conclusion: Der p 1 and Der p 2 were described as the most important allergens in HDM allergy, however, other allergens additionally contribute strongly to the allergic symptoms in sensitized patients.


Acknowledgements

This work was supported by grants F4602 and F4605 of the Austrian Science Fund (FWF), Thermofisher, Uppsala, Sweden and the Christian Doppler Research Association, Austria.

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Expression of recombinant canine, feline and equine FcepsilonRI-alpha chains for improved IgE profiling in these speciesLukas EINHORN(1,2), Judit FAZEKAS(1,2), Martina MUHR(1,2), Alexandra SCHOOS(1,2), Lucia PANAKOVA(3), Krisztina MANZANO-SZALAI(1), Kumiko OIDA(1,4), Josef SINGER(2), Erika JENSEN-JAROLIM(1,2)

(1) Comparative Medicine, Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Austria(2) Comparative Immunology and Oncology, Institute for Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology andImmunology, Medical University of Vienna, Austria(3)… Clinical Department of Small Animal Internal Medicine, University of Veterinary Medicine Vienna, Austria(4)… Laboratory of Veterinary Molecular Pathology and Therapeutics, Tokyo University of Agriculture and Technology, Japan

IgE-based diagnosis has an overall lower clinical impact in veterinary medicine, possibly due to a lack of high-quality IgE detection reagents for diverse species. Therefore, we aimed to produce recombinant canine, feline and equine alpha chains of FcepsilonRI-alpha for high-affinity IgE detection in dogs, cats and horses to improve the specificity and spectrum of diagnostic IgE tests in animals. Combined with a custom SV40_Neo mammalian expression vector the Flag-tagged FcepsilonRI-alpha fusion proteins of canine, feline and equine were expressed in CHO-DUKX B11 cells. 384 clones of each species were selected according their productivity and quality by ELISA and Immunoblot and purified via anti-FLAG M2 affinity gel. The purified recombinant products were correctly folded as determined by CD-spectroscopy. Immunoblot and ELISA assays verified integrity of the recombinant alpha chains. The recombinant proteins detected allergen-specific serum IgE of the relevant species, but also showed crossreactivity to other species including humans. We here accomplished the expression of canine, feline and equine alpha chains for IgE detection in these species. These tools will improve IgE-diagnosis in veterinary allergology and comparison of sensitization profiles in humans and their animals.

Acknowledgements

Sources of funding: Austrian Science Fund project SFB F4606-B19 and FWF grant P23398-B11. L. E. was supported by the MCCA-PhD program W 1248-B13, J.S. and J.F. by W1205-B09.

Contact / E-Mail (of first author):[email protected]

Abstract 97

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Christoph MADRITSCH(1), Elisabeth GADERMAIER(1), Uwe RODER(2), Christian LUPINEK(1), Rudolf VALENTA(1) and Sabine FLICKER(1)

(1) Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology & Immunology, Medical University of Vienna,(2) GE Healthcare Europe GmbH, Freiburg, Germany

The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens with a molecular weight of approximately 25-30kDa. Group 1 allergens are recognized by more than 95% of grass pollen allergic patients. We investigated the IgE recognition of Phl p 1 using allergen-specific IgE-derived single chain antibody fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollen allergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1-derived peptides the binding sites of two ScFvs were mapped to the N-terminus of the allergen. In surface plasmon resonance experiments (SPR) they showed comparable high affinity binding to Phl p 1 as a human IgE-derived complete antibody recognizing the allergens’ C-terminus. In a set of SPR experiments simultaneous allergen recognition of all three binders was demonstrated. Even in the presence of the three binders, allergic patients’ polyclonal IgE reacted with Phl p 1 indicating high density IgE recognition of the Phl p 1 allergen. Our results show that multiple IgE antibodies can bind with high density to Phl p 1, which may explain the high allergenic activity and sensitizing capacity of this allergen.

High density IgE recognition of the major grass pollen allergen Phl p 1 revealed with single chain IgE antibody fragments obtained by combinatorial cloning

Acknowledgements

Funding Sources: This study was supported by grants F4607 and F4605 of the Austrian Science Fund (FWF).


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Generation of new cassette vectors for the targeted presentation of human-relevant antigens (allergens) in humanized mouse modelsBernhard KRATZER(1), Alina NEUNKIRCHNER(1,2), Winfried F. PICKL(1,2)

(1) Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Christian Doppler Laboratory for Immunomodulation, Medical University of Vienna, Vienna, Austria.

Allergies affect more than 25% of individuals in our population. However, experimental animals able to present the immunodominant peptides of human-relevant aeroallergens are scarce, making the investigation of novel strategies for allergy treatment or prevention difficult. To overcome these limitations, we created a pair of novel cassette vectors for tissue-specific expression of HLA-DR molecules in mice. The peptide binding domains of HLA-DRA*01:01 and HLA-DRB1*07:01 were chimerized to the corresponding constant domains of H-2 IEd alpha and beta. HLA sequences were flanked by unique Not I and Cla I restriction enzyme sites to enable rapid, directed exchange for other allelic sequences. HLA-DRA and –DRB1 variable sequences are flanked by ≥ 6kb of 5’- and 3’-genomic sequences, to conserve unique promoter/enhancer elements. Functional evaluation of cassette-encoded transgenes revealed MHC class II transactivator-dependent HLA expression, which was functionally confirmed in proliferation assays using allergen-specific T cells. The novel HLA::IE cassette vectors will contribute to the quick and facile creation of a collection of HLA transgenic mice, to better understand the mechanisms of immune-mediated diseases such as allergies and to evaluate novel immunotherapeutic concepts.

Acknowledgements

Supported by the Austrian Science Fund (FWF) project DK-W1248-B13 (as part of the PhD program Molecular, Cellular and Clinical Allergology, MCCA of the Medical University of Vienna), SFB F46909-B19 and Biomay AG.


Abstract 99

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Identification and characterization of natural adjuvants in birch pollenDagmar WERNER, Birgit NAGL, Barbara BOHLE


IgE-mediated allergy is a hypersensitivity reaction of the immune system to normally harmless antigens which is due to an aberrant Th2-dominated immune response. 25% of urban population suffer from allergic disorders and 1/4 reacts to birch pollen(BP). The major BP allergen Betv1 is recognized by 95% of birch pollen-allergic patients, whereas less than 50% react to the minor allergens Betv2, 3, 4, 6 and 7. By expanding BP-specific T-cell clones(TCCs) from the peripheral blood of BP-allergic patients we found BP extract-specific TCCs that were not specific for any of the known allergens in BP. So far, 17 BP extract-specific TCCs could be expanded. After stimulation with BP, signature cytokines for Th2 and Th1 were measured in supernatants of these TCCs. 59% belonged to the Th0, 35% to the Th1 and only 1% to the Th2 subset. This led to the assumption that there are distinct proteins in BP which induce a Th0/1-like response rather than a Th2-like reaction. The aim of this study is to identify and characterize those proteins. BP proteins were separated with size exclusion chromatography. BP extract-specific TCCs will be tested with the individual fractions. In parallel, the fractions will be subjected to endo-lysosomal degradation assays to draw conclusions on their immunogenicity. It has been demonstrated that proteins with higher resistance to endo-lysosomal proteolysis are more immunogenic. After identification and isolation of candidate proteins they will be analyzed in more detail. These Th0/1 subset supporting BP proteins may represent natural adjuvants which could be supplemented to vaccines for immunotherapy of BP allergy.

Acknowledgements

Supported by the Austrian Science Fund project W1212 and SFB F4610, CD Laboratory for Immunmodulation and Biomay AG


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Dominika POLAK, Birgit NAGL, Claudia KITZMÜLLER, Barbara BOHLE


Although large numbers of neutrophils are present in late-phase reactions, their role in allergic disorders is not well understood. These professional phagocytes can be activated to express MHCclassII molecules by stimulation with certain cytokines, chemokines and bacterial factors, such as GM-CSF, TNF-alfa, IL-8, IFN-gamma and LPS. Some studies have even shown that murine neutrophils are able to process and present antigens to CD4+T-cells. The aim is to assess whether human neutrophils act as antigen-presenting cells for allergen-specific T-cells. The isolation of pure neutrophils from human blood was established and they were assessed for the expression of MHCclassII and co-stimulatory molecules under different conditions by flow cytometry. Surface binding, internalization and intracellular degradation of fluorescence-labelled Bet v 1 by neutrophils were compared with monocytes. Endolysosomal proteases were isolated from neutrophils and monocytes. Finally, neutrophils and monocytes were used as antigen presenting cells for Bet v 1-specific T-cell lines. So far, we found that the mixture of IL-3,GM-CSF and IFN-gamma enhanced the expression of HLAclassII on neutrophils. Neutrophils internalized Betv1 with a different kinetic than monocytes. Endolysosomal extracts of neutrophils were produced and will now be used for degradation assays of Betv1. The resulting peptides will be analysed by mass spectrometry. In first experiments with Betv1-specific T-cell lines neutrophils plus Betv1 induced T-cell proliferation.Our data provide evidence that neutrophils can activate allergen-specific T-cells. Further experiments are on the way.

The role of neutrophils in IgE-mediated allergy

Acknowledgements

Supported by the Austrian Science Funds, project W1248 and SFB F4610.


Abstract 101

Poster Presentation

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Pawel DUBIELA(1), Sabine PFEIFER(1), Merima BUBLIN( 1, 2), Christine HAFNER(1), Karin HOFFMANN-SOMMERGRUBER(1)

(1) Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria; 2Karl Landsteiner Institute for Dermatological Research, St. Poelten, Austria

Background and Aim: So far, 10 hazelnut allergens have been identified, among those the ns lipid transfer protein, Cor a 8. The aim of the study is to purify and characterize the physicochemical and allergenic properties of Cor a 8. Methods: nCor a 8 was purified from raw hazelnuts by precipitation and chromatographical steps. The protein was identified by N-terminal sequencing and mass spectrometry. Immunological and physicochemical experiments were performed. Results: Mass spectrometry analysis provided 9.475 kDa for purified nCor a 8 which corresponds to the theoretical mass of 9.468 kDa (database access nr: 9QATH2). The intact N-terminus was verified by Edman-degradation. In stability assays purified Cor a 8 displayed reduced stability against enzymatic and heating treatment as compared to Pru p 3. Three out of six sera had IgE predominantly recognizing linear epitopes as compared to 3 sera which had IgE specific for conformational epitopes. In RBL assays Cor a 8 induced mediator release. Upon addition of hazelnut lipids this response was remarkably increased.Conclusions: Cor a 8 was purified from extract, displaying the physicochemical properties different to Pru p 3. In cellular assays the allergenic activity of Cor a 8 was considerably increased when adding hazelnut lipids, thus showing the impact of food matrix on the allergenicity of a single food protein.

Cor a 8 displays reduced stability upon heat and digestion treatment

Acknowledgements

Supported by grants SFB F4603 and W1248 (Austrian Science Fund) to K. Hoffmann-Sommergruber and P. Dubiela, respectively.

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Ulla KNACKMUSS(1), Wilfried POSCH(1), Marion STEGER(1), Michael BLATZER(1), Kristian PFALLER(2), Cornelia LASS-FLÖRL(1), Doris WILFLINGSEDER(1)

(1) Division of Hygiene and Medical Microbiology, Schöpfstrasse 41, Innsbruck, Austria, (2) Division of Embryology and Histology, Innsbruck Medical University, Müllerstrasse 59, Innsbruck, Austria

The immune response to viral infections comprises complex interplays between the virus and the immune system and aims in eradication of the pathogen with least damage to the host. Dendritic cells (DCs), which are essential for the generation of a protective antiviral immune response, are exploited by the viruses to evade innate and adaptive immunity. When HIV enters the body, virus becomes immediately opsonized with complement fragments which should help to eradicate the virions by the innate immune system. We earlier showed that differential opsonization of HIV significantly modulates antigen capture and presentation by DCs (Wilflingseder et al, 2007; Posch et al, 2012). We are now interested in investigating the initial signaling events in DCs upon exposure using differentially opsonized HIV in more detail to identify novel therapeutical targets. We here found that significantly higher amounts of complement-opsonized HIV (HIV-C), which induces a potent specific CD8+ T cell response via DCs, bound to DCs and HIV-C was able to overcome innate DC restriction in contrast to non-opsonized HIV (HIV). Additionally, HIV-C signaled via Rac1 and caused prolonged phosphorylation of ERK1/2 and JNK/SAPK, while HIV activated RhoA and did not induce MAPKs in DCs. Our data demonstrate the importance to distinguish the initial cellular events after attachment/internalization of differentially opsonized HIV and might provide a promising novel strategy to tackle HIV-1 infection.

HIV opsonization modulates DC signaling

Acknowledgements

We would like to thank our technician Karolin Lechleitner and the FWF (P24598 to DW, P25389 to WP) and the ÖNB (Project 14875).

Abstract 103

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Alina NEUNKIRCHNER(1,2), Daniela WOJTA-STREMAYR(1,2), Klaus G. SCHMETTERER(2), Lukas MAGER(2) Victoria REICHL(1,2), Edward ROSLONIEC(3), Ronald NAUMANN(4), Gerhard DEKAN(5), Beatrice JAHN-SCHMID(6), Barbara BOHLE(1,6) and Winfried F. PICKL(1,2)

(1) Christian Doppler Laboratory for Immunomodulation, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(3) Veterans Affairs Medical Center, Memphis, TN, USA(4) Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany(5) Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria(6) Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria

T-cells play a key role in the development and maintenance of allergic diseases and thus represent a promising target for therapeutic interventions. Evidence from SIT studies suggest that regulatory T-cells (Treg) might counterbalance the Th2-biased immune response. We here created double trangenic (tg) allergy mice expressing a human TCR specific for the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 and HLA-DR1 to study the response to the human-relevant aero-allergen in vivo. After allergen-specific sensitization, only double tg allergy mice, but not single tg or wt control mice showed enhanced airway-hyperreactivity (AHR) upon challenge, which was associated with airway and lung inflammation. Significantly, treatment of allergy mice with three daily i.p. injections of IL-2/anti-IL-2 mAb complexes induced a 6-fold increase of CD25+Foxp3+ Treg among CD3+CD4+T-cells in peripheral blood (17.9±6.2% compared to 3.1±1.4%). Furthermore, expansion of Treg by IL-2/anti-IL-2 mAb complexes before sensitization prevented mice from allergen-induced AHR upon challenge and significantly reduced Art v 1-specific serum IgE, when compared to sham-treated mice. In summary, expansion of Treg by IL-2/anti-IL-2 mAb complexes seems to be a promising strategy to interfere with allergic diseases.

Interleukin-2/anti-interleukin-2 antibody complexes expand T regulatory cells and protect against allergen-induced airway hyperreactivity

Acknowledgements

Support: Austrian Science Fund SFB-F4609-B19 & Christian Doppler-Research Association and Biomay AG

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Guido A. GUALDONI(1), Katharina A. MAYER(1), Johanna M. GOSTNER(2), Dietmar FUCHS(3), Gerhard J. ZLABINGER(1)

(1) Institute for Immunology; Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna(2) Division of Medical Biochemistry, Medical University of Innsrbuck(3) Division of Biological Chemistry, Medical University of Innsbruck

Resveratrol is a polyphenol compound found in a variety of nutrients and has been shown to have immunomodulating, anti-cancerogenic and cardioprotective effects. The regulation of the rate limiting enzyme of inflammatory tryptophan metabolism, indolamine-2,3-dioxygenase (IDO), has been proposed to be involved in resveratrol’s biological effects. These observations, however, rely on in vitro findings and animal studies. In order to analyze resveratrol’s IDO-modulation in humans, we performed a pilot study including 10 healthy volunteers with oral administration of 5g resveratrol (resveratrol n=8, placebo n=2). Plasma levels of tryptophan and kynurenine were analyzed to assess IDO activity. After resveratrol application, tryptophan levels dropped markedly within 2.5h (p<0.001) and 5h (p<0.001). Kynurenine levels were slightly elevated at 2.5h which resulted in an 1.33 and 1.30 fold increase in the kynurenine/tryptophan ratio at 2.5h (p<0.01) and 5h (p<0.01), respectively. The present study provides evidence for a profound modulation of tryptophan metabolism after oral resveratrol intake. Since IDO has been shown to play a crucial role in immunity, cancer development and regulation of vascular tone, the modulation of this enzyme might be involved in resveratrol’s biological effects.

Resveratrol intake enhances indolamine-2,3-dioxygenase activity in humans

Acknowledgements

The authors thank Dr. Michael Wolzt for support in the performance of the study.


Abstract 105

Poster Presentation

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The CD58-CD2 axis is the primary costimulatory pathway in human CD28 negative CD8 T cellsJudith LEITNER(1), Dietmar HERNDLER-BRANDSTÄTTER(2), Beatrix GRUBECK-LOEBENSTEIN(2), Gerhard ZLABINGER(1), Peter STEINBERGER(1)

(1) Institute for Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Austrian Academy of Sciences, Institute for Biomedical Aging Research, Immunology Division, Innsbruck, Austria

Aging and repeated antigen encounter eventually leads to a state known as immune senescence. Loss of CD28, the most potent costimulatory receptor on human T cells, is generally regarded to be a main predictor of biological aging of the human immune system. Besides CD28 there are numerous additional receptors that costimulate T cell activation. Little is known on the role of these costimulatory ligands in the function of CD28- T cells. We have examined such alternative pathways regarding their functional role in CD28-CD8+ T cells. We activated CD28-CD8+ T cells from elderly individuals in the presence of the most important costimulatory ligands CD58, 41BBL, ICOSL, CD166, CD70, OX40L, GITRL, CD54. Our study showed that most costimulatory molecules have low capacity to activate CD28- T cells, whereas the engagement of the CD2 molecule by its ligand CD58 strongly costimulated proliferation, cytokine production and effector function in this T cell subset. CD58 is broadly expressed on antigen presenting cells including dendritic cells. Blocking CD58 mAb greatly reduced the response of human CD28-CD8+ T cells to allogeneic DC as well as to viral antigens. Our results clearly identify the CD58/CD2 axis as the primary costimulatory pathway for CD8 T cells that lack CD28.

Acknowledgements

Supported by a grant from the ONB12731 to PS and Theodor Körner Fond to JL.


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Markus STEINER(1), Thomas HAWRANEK(2), Michael SCHNEIDER(3), Andrea HARRER(2), Jutta HOREJS-HOECK(1), Fatima FERREIRA(1), Martin HIMLY(1)

(1) University of Salzburg, Department of Molecular Biology, Salzburg, Austria(2) Paracelsus Medical University, Salzburg, Austria(3) Bühlmann Laboratories AG, Schönenbuch, Switzerland

Human basophils represent well-known allergic effector cells. However, an involvement of basophils in innate immunity regulated by TLRs has been hypothesized and TLR signaling has been postulated to influence the IgE-mediated pathway. We thus asked the question whether basophils derived from allergic patients differ from basophils of non-allergic donors regarding TLR stimulation. Basophils of birch pollen-, mite-, and non-allergic donors were purified via density centrifugation and negative magnetic cell sorting. TLR-1, 2, 4, and 6 expression levels were analyzed by flow cytometry. Up-regulation of CD203c and cytokine release of basophils after incubation with ligands for TLR-1/2, 2/6, and 4 were investigated using a multiplex assay for IL-4, 6, 8, 23, IFN-gamma, MCP-1, and TNF-alpha. Basophils of allergic and non-allergic donors expressed similar amounts of TLR-1 and 2, whereas TLR-4 seemed to be less prominent in mite-allergic donors. Surprisingly, IL-8 release of basophils of mite-allergic donors was higher upon TLR-4 stimulation. In contrast, TLR stimulation did not alter CD203c surface expression and only minor differences in IL-4 release were observed in all groups. In conclusion, these results point towards a possible link between allergy and TLR-signaling.

Basophils from house dust mite-allergics show an altered TLR profile from birch pollen- or non-allergic individuals

Acknowledgements

The study was funded by grant P18820-B13 of the Austrian Science Funds (FWF).

Abstract 107

Poster Presentation

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Alexander PUCK, Maria SEYERL, Stefan HOPF, Otto MAJDIC, GerhardZLABINGER, Judith LEITNER, Peter STEINBERGER, Johannes STÖCKL

Institute of Immunology, Center for Pathophysioloy, Infectiology and Immunology,Medical University of Vienna, Vienna, Austria.

The cytoplasmic tail of CD45 (ct-CD45) is proteolytically cleaved and released uponactivation of human phagocytes. The soluble ct-CD45 was found to act on T cells as aninhibitory, cytokine-like factor that reduces T cell proliferation. In this study, weaimed to elucidate the molecular mechanisms acting within T cells, upon ct-CD45binding. Here, we demonstrate that ct-CD45 induces a novel form of anergy in humanperipheral blood T cells. Ct-CD45 inhibited the function (proliferation, cytokineproduction) of human T cells and rendered the cells hyporesponsive to restimulation,which was reversible by exogenous IL-2 or IL-7. However, microarray analysis did notindicate induction of any classical anergy-associated genes. Instead, we foundinduction of Schlafen family member 12 (SLFN12) and of Krueppel-like factor 2 (KLF2).When we analyzed the expression patterns of cell cycle regulatory factors, we foundinhibition in the induction of cyclin D1 while other cyclins were unaltered. Insummary, ct-CD45 triggers an anergy program in T cells, which is reversible byexogenous IL-2, acting independently of classical anergy factors. From our data, theinhibition of cyclin D1 suggests a cell cycle arrest in the early G1 phase, thusmaking it distinct from canonical T cell anergy.

The soluble cytoplasmic tail of CD45 (ct-CD45) induces quiescent anergy in human T cells

Acknowledgements

We acknowledge Claus Wenhardt, Margarethe Merio, Petra Waidhofer-Söllner, Petra Cejkaand Jens Gerwien for their expert technical assistance.


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(1) Department of Laboratory Medicine, Medical University of Vienna, Austria(2) Institute of Immunology, Medical University of Vienna, Austria

Ralf SCHMIDT(1), Franz RATZINGER(1), Sabrina JUTZ(2), Peter STEINBERGER(2), Winfried F. PICKL(2) and Klaus G. SCHMETTERER(1)

The anti-malarial drugs chloroquine and hydroxy-chloroquine have immunomodulatory potency which is therapeutically harnessed in rheumatic diseases. However, the exact mechanisms of action on immune cells have not been fully described. Consequently, we assessed the influence of chloroquine on highly purified human CD4+ T-cells. T-cells were pre-incubated with chloroquine at concentrations of up to 10 µM which leads to intracellular accumulation of drug levels comparable to oral therapy in rheumatic diseases. Following activation with anti-CD3/anti-CD28 agonistic antibodies, chloroquine treated T-cells were suppressed in proliferation as well as secretion of cytokines. These effects were due to specific inhibition of activation since no decrease in cellular viability could be detected. In restimulation experiments, we determined that the effects of chloroquine were reversible after removal of the substance. Using Jurkat reporter cell lines expressing GFP under control of either the NFAT, NF-kB or AP-1 promoter, we observed that chloroquine specifically inhibited AP-1 promoter activity. In conclusion, we describe immunosuppressive functions of chloroquine on purified CD4+ T-cells and propose that inhibition of AP-1 activity is the main molecular mechanism of this effect.

The anti-malarial drugs chloroquine and hydroxy-chloroquine show suppressive potency on purified human CD4+ T-cells

Abstract 109

Poster Presentation

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Klaus G. SCHMETTERER(1,2), Alina NEUNKIRCHNER(1), Daniela WOJTA-STREMAYR(1) Judith LEITNER(1), Peter STEINBERGER(1), and Winfried F. PICKL(1)

(1) Institute of Immunology, Medical University of Vienna, Austria(2) Department of Laboratory Medicine, Medical University of Vienna, Austria

STAT3 is a key integrator of signals transmitted from cell surface immune receptors. To assess the effects of STAT3 activation in human T-cells, a constitutively active mutant of STAT3, i.e. STAT3C, was cloned into an IRES-GFP harboring retroviral vector. STAT3C-transgenic CD4+ T-cells showed clear-cut transgene expression and phenotypically displayed an effector T-cell surface phenotype. Besides, a significant up-regulation of intracellular granzyme B levels could be observed. STAT3C+ T-cells were hypo-responsive following stimulation. They further displayed a distinct cytokine secretion profile including a significant increase in IL-10 and a reduction of IL-2, IFN-gamma and IL-13 secretion. Of note, STAT3C+ T-cells activated via CD3/CD2 significantly suppressed effector T-cell proliferation. Mechanistically, suppression was dependent on the release of granzyme B and concomitant apoptosis of effector T-cells. Our observations are further substantiated by the finding that peripheral blood Tr1 cells reveal activation-induced hyperphosphorylation of STAT3. Furthermore, exposure of Tr1 cells to a STAT3 inhibitor could partially revert their hyporesponsiveness. This indicates a clear-cut relation between activation of STAT3 and the acquisition of a tolerogenic program.

STAT3 governs granzyme B and IL-10 production in CD4+ T-cells

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Harald SCHWARZ, Maria SCHMITTNER, Albert DUSCHL, Jutta HOREJS-HOECK

University of Salzburg, Department of Molecular Biology, Hellbrunner Str. 34, A-5020 Salzburg

Commercially available recombinant proteins are often produced in E. coli. Most suppliers guarantee less than 1 endotoxin unit (EU) of contamination in their products. We analysed several recombinant proteins for their endotoxin content and found endotoxin contaminations in the same range as stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have immunomodulatory effects, we stimulated different human immune cells with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002 – 2 ng/ml). We show that primary human CD1c+ dendritic cells especially can be activated by minimal amounts of LPS. In more detail, 20 pg/ml LPS are sufficient to significantly up-regulate the expression of IL-6 and IL-8, as well as the surface activation markers CD40, CD80 and CD86. Notably, we found that the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated to high CD14 expression levels observed in CD1c+ DCs that have been cultured in medium for 24 hours. To avoid the generation of erroneous data, we suggest different endotoxin detection assays to thoroughly screen recombinant proteins for endotoxin contamination when working with immune cells that are highly sensitive to LPS.

Endotoxin contaminations in recombinant proteins activate primary human CD1c+ dendritic cells

Acknowledgements

We thank Andrei Medvedev and Douglas Golenbock for providing the TLR4, MD-2 and CD14 expression vectors.


Abstract 111

Poster Presentation

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Sabrina JUTZ(1), Sandra ROSSKOPF(1), Judith LEITNER(1), Klaus SCHMETTERER(2), Katharina GRABMEIER-PFISTERHAMMER(3), Peter STEINBERGER(1)

(1) Division of Immune Receptors and T cell activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria(2) Department of Laboratory Medicine, Medical University of Vienna, Austria(3) Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria

Activation of T cells occurs via a complex signaling network. The transcription factors AP-1, NFAT and NF-kappaB play a central role in T cell activation and are required for cytokine expression, T cell differentiation and proliferation. The aim of this work is to establish a reporter T cell line enabling measurement of the activation of those three transcription factors simultaneously. Three reporter constructs were generated with response elements for the respective transcription factors and three different fluorescent proteins which do not overlap in their spectra. To measure AP-1, NFAT and NF-kappaB activation, mCherry, eGFP and CFP were used as reporter genes, respectively. The Jurkat cell line E6 was transduced with these constructs and a stable single cell clone was established. Cocultivation of reporter cells with T cell stimulator cells that trigger the TCR-complex led to an induction of AP-1, NFAT and NF-kappaB. The use of T cell stimulator cells bearing CD58 or CD80 further increased reporter gene expression. Moreover, this system can also be used to analyze inhibitory pathways e.g. PD1/PDL1. In conclusion, our multi-parameter T cell line is a powerful tool to measure the activation of the main signaling pathways in T cell activation.

Generation of a multi-parameter reporter T cell line

Acknowledgements

Supported by FWF grant 21964-B20

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Franz RATZINGER(1), Helmuth HASLACHER(1), Wolfgang POEPPL(2), Gregor HOERMANN(1), Johannes J KOVARIK(3), Sabrina JUTZ(4), Peter STEINBERGER(4), Heinz BURGMANN(2), Wolfgang F PICKL(4) and Klaus G SCHMETTERER(1)

(1) Department of Laboratory Medicine, Medical University of Vienna, Austria(2) Division of Infectious Diseases and Tropical Medicine, Department of Medicine I, Medical University of Vienna, Austria(3) Clinical Division of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, Austria(4) Institute of Immunology, Medical University of Vienna, Austria

Advanced macrolides, such as azithromycin (AZM) or clarithromycin (CLM), are antibiotics with immunomodulatory properties. Here we have sought to evaluate their in vitro influence on the activation of CD4+ T-cells.Isolated CD4+ T-cells were stimulated with agonistic anti-CD3/anti-CD28 monoclonal antibodies in the presence of different macrolides. Cell proliferation, cytokine secretion of supernatants and cell viability was assessed. Intracellular signaling pathways were evaluated using reporter cell lines, FACS analysis, immunoblotting and in in vitro kinase assays.AZM inhibited cell proliferation rate and cytokine secretion of CD4+ T-cells in a dose-dependent manner. At therapeutic concentrations, no reduction of cell viability was observed. In contrast, CLM showed only minor effects on T-cell function. Analysis of molecular signaling pathways revealed that treatment with AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target mTOR. In vitro kinase studies using recombinant mTOR showed that AZM inhibited mTOR activity. In contrast to rapamycin, this effect was independent of FKBP12.We show for the first time that AZM acts as immunosuppressive agent on CD4+ T-cells at therapeutically relevant concentrations by inhibition of mTOR activity.

Azithromycin suppresses CD4+ T-cell activation by direct modulation of mTOR activity

Acknowledgements

The study was conducted in cooperation with the MedUni Wien Biobank facility. The project was supported by grants of the Austrian Society for Laboratory Medicine and Clinical Chemistry (ÖGLMKC); the Medical Scientific Fund of the Mayor of the City of Vienna [Grant 13040BGM, 14041BGM]; and the Austrian Science Fund [Grant SFB F4609]. None of the founder had influence on any phase of this project.


Abstract 113

Poster Presentation

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Theresa NEUPER, Albert DUSCHL and Jutta HOREJS-HOECK

University of Salzburg, Department of Molecular Biology, Salzburg, Austria

NOD1 was originally found to recognize the bacterial cell wall component iE-DAP. However, in this study we show that NOD1 plays a novel role as intrinsic regulator of IL-10 signaling. We demonstrate that silencing of NOD1 in human moDCs causes a significant increase in IL-10 mRNA expression in response to IL-10, whereas the expression of CD86 was largely suppressed. As IL-10 treated moDCs are an accepted model of tolerogenic DCs, we analysed the role of NOD1 in these cells. We show that tolerogenic DCs lacking NOD1 produce lower levels of IL-6 and IL-12. Moreover, in co-culture experiments tolerogenic DCs deficient for NOD1 are more potent to induce IL-10 secretion by naïve CD4+ T cells, whereas IFN-gamma release is unaffected. These findings suggest that NOD1 may counter-regulate the tolerogenic potential of IL-10 stimulated DCs. Analysis of regulatory proteins, which may be involved in NOD1-mediated modulation of IL-10 signaling revealed that specifically SOCS2 mRNA expression was clearly decreased in moDCs lacking NOD1. Moreover, silencing of SOCS2 in moDCs does have similar effects on IL-10 targets as a lack of NOD1. Taken together, this study identifies NOD1 as a novel player in modulating IL-10 signaling and suggests that SOCS2 may be involved in this process.

The impact of NOD1 on IL-10 signaling

Acknowledgements

This project is supported by the Austrian Science Fund, FWF, grant number W1213.

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Johanna PRODINGER(1), Julia LOACKER(1), Ralf SCHMIDT(1) Franz RATZINGER(1), Sabrina JUTZ(2), Peter STEINBERGER(2), Gregor HÖRMANN(1), Winfried F. PICKL(2) and Klaus G. SCHMETTERER(1)

(1) Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria(2) Institute of Immunology, Medical University of Vienna, Vienna, Austria

Tryptophan metabolites such as kynurenine (KYN), 3-hydroxyanthranilic acid (3-OH-AA) and picolinic acid (PA) are key mediators of immunosuppression by cells expressing the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). However, studies on the influence of tryptophan metabolites on CD4+ T-cells have so far only focused on the effects of KYN and 3-OH-AA. Consequently, we assessed the influence of PA on cell viability, proliferation, cellular metabolism, cytokine secretion and production and up-regulation of surface markers following in vitro activation with agonistic anti-CD3/anti-CD28 antibodies. In contrast to KYN and 3-OH-AA, exposure of T-cells with PA did not affect cell viability, while proliferation and metabolic activity was suppressed in a dose-dependent manner. T-cell functions requiring protein synthesis, such as cytokine secretion and cell surface marker up-regulation, were not or only weakly inhibited by PA. Restimulation experiments revealed that PA-exposure induced a state of IL-2 resistant anergy. Phosphorylation of c-Myc at Ser62, was strongly reduced in PA-exposed T-cells while more upstream signaling molecules were not affected. In conclusion, PA mediates inhibition of cell cycle and metabolic activity while leaving protein production intact.

The tryptophan metabolite picolinic acid suppresses proliferation and metabolic activity of CD4+ T-cells

Abstract 115

Poster Presentation

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ALRAUNE - Allergy Research in Rural, Alpine and Urban Networks David SCHWARZENBACHER(1)*, Julia ZLOEBL(1)*, Teresa STEMESEDER(2), Eva KLINGLMAYR(2), Edith OBERKOFLER(1), Gabriele GADERMAIER(2)

(1) HBLA Ursprung, Elixhausen, Austria (2) University of Salzburg, Department of Molecular Biology, Salzburg, Austria

Students of HBLA Ursprung and BG Tamsweg work with scientists in an interdisciplinary project to find possible correlations between the exposition to indoor allergens, different lifestyle habits including physical exercise or diet, demographic data and the risk to develop an allergic disease. Blood from 501 school children living in different geographic regions were analyzed for IgE sensitization using ImmunoCAP ISAC microarrays. All participants collected a dust sample from their home to investigate the concentration of various indoor allergens. In order to evaluate the lifestyle, a questionnaire was created by scientists in co-operation with students. Students of HBLA Ursprung were peer-to-peer teaching general aspects of allergic diseases when visiting the schools. The project also focuses on transferring science to public. Presentations and experiments for events in science communication like “Lange Nacht der Forschung” or “Science Day” were prepared and the team will take part in a “Science Slam”. Preliminary results showed a very high IgE sensitization rate of 53% among all participants with grass pollen, house dust mites and birch pollen as major allergen source and statistical analysis will enable identification of factors influencing allergic diseases.

Acknowledgements

We thank our project partners from the University of Salzburg, University of Applied Sciences Salzburg, Paracelsus Medical University Salzburg and University of Education Salzburg. The study was funded by Sparkling Science, a program of the Federal Ministry of Science, Research and Economy, Vienna, Austria.

Contact / E-Mail:[email protected]; [email protected]

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Austrian Society for Allergology and Immunology, Nov 6 - 8, 2014, Salzburg1st author = presenting author except when underlined

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• Analyze FFPE, cell and blood lysates, total RNA

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