3th Student Retreat Microbiology and Immunology PhD Program · 2015-04-23 · Microbiology and Immunology PhD Program of the Lifescience Zurich Graduate School. This year the retreat

3th Student Retreat

Microbiology and Immunology PhD Program

Ascona 18th – 20th September 2010

In September 2009, the 2nd MIM PhD Student Retreat took place at

Parpan, canton of Graubünden.

3th MIM PhD Student Retreat 2010

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The Organizing Committee 2010

Anna Katharina Kraus

Helge Frebel

Pascale Vonäsch

Sanja Mandaric

Antonia Fettelschloss

The MIM-Program Logo was created by Luzia Reutimann

m crobiology& mmunologyi

Welcome

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Dear Students, It is a great pleasure to welcome you to the 2010 Student Retreat of the Microbiology and Immunology PhD Program of the Lifescience Zurich Graduate School. This year the retreat will take place in the southern part of Switzerland, in Ascona located in the canton of Ticino. We will spend the time discussing about our research projects during the oral presentations and poster sessions. In addition, we are pleased to announce that several guest speakers from different fields followed our invitation to participate in this meeting. Apart from the scientific part, there will be plenty of time to get to know each other during these 3 days, especially at the party on Saturday evening and during the vineyard trip on Sunday. Our retreat provides a good opportunity for building contacts between the students of the MIM program, in particular for those students who have recently started their PhD. We would like to thank you for participating in this meeting, and wish you a great and pleasant time. See you in Ascona, The 2010 Organizing Committee


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Ascona www.ascona.ch Ascona is situated to 196 m above sea level on the northern shore of Lake Maggiore. Sheltered by the mountains of the Alps, Ascona enjoys an exceptional mild and sunny climate suitable for growth of magnolias and palms, as well as for wine production. There are plenty of opportunities for pleasant walks among gardens, along the shores of the lake and on the grape-covered slopes above the lake in friendly little villages such as Ronco. The glamour of the canton Ticino with its delicious cuisine, wine and stunning natural beauty has made this area especially attractive for tourists.

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Acknowledgments

We would like to thank the MIM PhD Program within the Life Science Zurich Graduate School for financing this event.

We express our gratitude to all invited speakers for joining us in the retreat and giving their talks.

Thanks to the organizers of the last retreat for providing us with useful information and their experience.

Many thanks to Olympia Stefani for the administrative support as well for help with the organization.


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confirmed the potential of lentivirus vectors that transcriptionally target transgene expression to DCs for antigen-specific tolerance induction in autoimmune diseases. This strategy completely prevented MOG induced EAE in mice. We will further investigate the mechanisms of vector mediated tolerance induction, in particular the involvement of regulatory T cells and cytokines. The strategy presented here is particularly promising for clinical applications and important for addressing fundamental questions in immunity and tolerance.

Ascona

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Contact Details Accommodation in Ascona Casa Moscia Via Moscia 89 CH-6612 Ascona

phone: +41 91 791 12 68 fax: +41 91 791 59 32 The Organizing Committee Helge 078 725 71 03 Anna 077 457 48 24 Pascale 079 790 32 93 Sanja 076 501 97 29 Antonia 078 697 86 25

3thMIM Student Retreat 2010

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Program Saturday, 18 September 2010

07.00 Departure from Zurich

10.30 Arrival in Ascona

11.30 – 11.50 Mounting of posters (odd numbers)

11.50-12.15 Opening remarks

12.15 – 13.15 Lunch

13.30 – 14.30 Lecture by Prof. Isabel Roditi, “One hundred years of sleeping sickness: sex, drugs and RNA"

14.30 – 16.00 Student talks ST 01-04

16.00 – 17.00 Coffee break + check-in + distribution of the keys

17.00 – 18.00 Student talks ST 05-07

18.30 – 20.00 Dinner

20.00 – 21.30 Poster session (odd numbers)

22.00 Party

Sunday, 19 September 2010

08.00 – 08.50 Breakfast

08.50 – 09.30 Student talks ST 08-09

09.30 – 10.30 Lecture by Prof. John McKinney “Individuality of Bacterial Responses to Antimicrobials“

10.30 – 11.00 Coffee break

11.00 – 12.00 Student talks ST 10-12

12.00 – 12.15 Change posters (even numbers)

12.15 – 13.15 Lunch

13.30 Afternoon trip-visit to vineyard

18.30 Dinner

19.40 – 20.00 Change of posters (even numbers)

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P 39

Viral vector-mediated transcriptional targeting of dendritic cells for antigen-specific tolerance induction in EAE/MS.

Bruna de Andrade Pereira, Mathias Ackermann, Cornel Fraefel and Christiane Dresch

Institute of Virology, University of Zurich, Zurich, Switzerland [email protected]

The cause of multiple sclerosis (MS) is unknown and the pathogenic processes leading to disease development is incompletely understood. Current knowledge supports a T cell mediated autoimmune pathogenesis targeting myelin components or myelin-producing cells. Immunization of susceptible animals with myelin antigens or transfer of myelin antigen-reactive T cells induces experimental autoimmune encephalomyelitis (EAE), an inflammatory disorder of the CNS which closely resembles MS. The aim of this project was to induce permanent, antigen-specific tolerance in EAE/MS. The strategy includes the ex vivo modification of autologous hematopoietic stem cells (HSC) with lentiviral vectors that express antigens involved in EAE/MS from a dendritic cell (DCs) specific promoter. After re-infusion, the modified HSC will give rise to all cells of the immune system including antigen expressing dendritic cells. As lentivirus vectors mediate the genomic integration of transgenes in HSC, there is a constant supply of antigen expressing “steady-state” DCs. We hypothesized that the stable antigen presentation by these cells in thymus and periphery in a non-inflammatory condition would tolerize self-reactive T cells and, therefore, prevent/revert EAE/MS disease. We demonstrated the effectiveness of this strategy for inducing myelin oligodendrocyte glycoprotein (MOG)-specific tolerance in an EAE model in mice. We show the efficient deletion of MOG specific T cells in chimeras that received HSC transduced with MOG-expressing lentivirus vector. Also, 0% of mice treated with these cells didn’t developed EAE upon induction (clinical score 0), while 100% of mice that received BM cells transduced with a GFP-expressing control lentivirus vector developed EAE. We also showed that Foxp3+ regulatory T cells were generated in healthy mice. Histological analysis of CNS revealed demyelination in both brain and spinal cord of diseased mice. We


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P 38

The role of IL10 in cytomegalovirus infection

Sanja Mandaric, Senta Walton and Annette Oxenius Institute for Microbiology, ETH Zurich [email protected]

During an immune response to virus, the host induces multiple proinflammatory immune mechanisms, trying to cope with the infection. However, development of such a response has to be tightly regulated since potential damage to the host would be counter-productive. IL-10 is an immunosuppressive cytokine that plays a potent regulatory role in host response to infection. It can inhibit expression of proinflammatory cytokines, modulate function of antigen-presenting cells and directly or undirectly suppress effector T cell response.

The overall impact of IL10 in chronic viral infections is complex and has not yet been fully understood. Cytomegaloviruses (CMVs) are species specific b-herpesviruses. Upon resolution of primary infection CMVs persist life-long in their respective host in the form of latency from which it can reactivate in the case of immunosuppressive conditions (transplant patients, AIDS patients). By infecting mice with mouse cytomegalovirus (MCMV) we are interested to elucidate the role of IL10 in acute and the chronic phase of herpesvirus infection, in particular to explain the effects of this cytokine on virus-specific T cell reposnse and consequently on viral clearance.

We showed that IL10 or IL10R deficient mice exhibit reduced levels of viral replication upon MCMV infection. In acute phase of infection, we detected increased virus-specific CD4 T cell responses in IL10-/- or IL10R-/- mice, whereas virus-specific CD8 T cell responses were not pronouncedly affected. Analysis of the phenotype and activation status of different dendritic cell types from IL10 -/- mice revealed enhancement in expression of costimulatory and MHC molecules. Current focus of the project is to determine the possible mechanism leading to increase of MCMV-specific CD4 T cell response and viral clearance in IL10-/- mice.

Program

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20.00 – 21.30 Poster session (even numbers)

Monday, 20 September 2010

08.00 – 09.40 Breakfast + check- out

09.40 – 10.40 Lecture by Prof. Kerry L. Tucker “How an American biochemist made his research career in Europe”

10.45 – 11.15 Coffee break

11.15 – 12.00 Student talks ST 13-14 12.15 – 13.15 Lunch

13.30 – 14.30 Lecture by Dr. Stephane Bieri “The future is in the genes - Plant Biotechnology at BASF”

14.30 – 15.00 Final words and awards, departure

18.30 Arrival in Zurich

Student Talks time-frame: 12 min talk, 3 min discussion

Saturday, 18 September 2010 ST 01 14.30 by Willem van de Ween ST 02 14.50 by Andrea Mekker ST 03 15.10 by Pietro Cippa ST 04 15.30 by Chrisitan Lange ST 05 17.00 by Wolfgang Kratky ST 06 17.20 by Kristina Kakalacheva ST 07 17.40 by Antonia Fettelschloss Sunday, 19 September 2010 ST 08 08.50 by Céline Robert ST 09 09.10 by Helge Frebel ST 10 11.00 by Pascale Vonäsch ST 11 11.20 by Anna Kraus ST 12 11.40 by Olmo Sonzogni


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Monday, 20 September 2010 ST 13 11.15 by Stephan Schwager ST 14 11.35 by Tina Herbst

Poster Abstracts

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P 37

Negative Regulation of the IFN- signaling by the Influenza Matrix Protein (M1)

Thomas Ludersdorfer, Bettina Oberle, Jovan Pavlovic Institute of medical Virology, UZH [email protected]

Complex organisms have evolved sophisticated mechanisms to

prevent and control infection by various pathogens or viruses. Among these mechanisms the interferon (IFN) system represents the first step of defense against viral infection in vertebrates. Secretion of IFNs prepares uninfected cells for combating incoming virus. Products of IFN-stimulated genes (ISGs) mediate an antiviral action. Influenza A is counteracting the IFN system by inhibiting the type I IFN production through the non-strucural viral protein 1 (NS1). Paradoxically the NS1 deletion mutant of influenza virus can still propagate in the host cell.

This study demonstrates an antagonistic activity of the M1-protein of Influenza A in the IFN-alpha/beta signaling pathway. An IFN-stimulated response element (ISRE)-promoter-driven reporter system was used to detect the negative regulation of the IFN signaling cascade by the M1-protein. This effect was independently confirmed by direct measurement of MxA levels in Influenza A infected cells. These data identified the IFN-alpha/beta signaling cascade as a target of the virus. The M1-protein did not exert an effect on IFN transcription, suggesting that in contrast to the Influenza NS1 protein, it interfered with the action rather than the production of IFN-alpha/beta.


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P 36 The impact of HIV-1 replication on the activation of CD8+ T cells with specificities for different antigens

Sonia Bastidas, Anna Haas, Huldrych Guenthard and Annette Oxenius

Institute for Microbiology, Zürich, ETH Zürich [email protected]

Continuous loss of CD4+ T lymphocytes and systemic immune

activation are hallmarks of chronic HIV-1 infection. In chronically HIV-1 infected individuals a state of chronic immune activation can be observed; which is characterized by an elevated expression of activation markers on T cells. It is suggested that apoptosis (activation induced cell death) of these abnormally activated cells is one of the main causes of T-cell decline. Interestingly, T cells with an activated phenotype are neither necessarily HIV-specific nor HIV-infected. Usually 1-2% of all circulating CD8+ T cells during chronic infection are HIV specific whereas the level of CD8+ T cells exhibiting activated phenotypes (as judged by CD38 and HLA-DR expression) is in the range of 60% indicating that immune activation is not restricted to HIV-specific CD8+ T cells. Several hypotheses have been forwarded to explain the mechanism of CD8+ T cell activation. It is believed that most of these cells are activated by nonspecific mechanisms, including cross-reactivity and cytokine-driven activation, a phenomenon referred to as bystander activation. The mechanisms underlying this phenomenon are not well defined. Additionally, little to nothing is known about the antigen-specificity of bystander activated CD4+ and CD8+ T-cells.

Pre-existing data of our group indicate that cessation of antiretroviral therapy (ART) specifically leads to activation of CD4+ T cells with specificities for persistent Herpes virus antigens in the absence of detectable reactivation of these members of the Herpes virus family. In this study we expanded our analysis to another cohort of patients to investigate whether these observations also apply for CD8+ T cell responses specific for persistent viral antigens such as EBV, HSV, CMV, Adenovirus and VZV during viral rebound. Based on our preliminary data we propose that this HIV-driven activation also occurs for CD8+ T cells specific for persistent antigens, which likely represent a large part of activated CD8+ T cells in untreated HIV infection and thereby significantly contribute to chronic immune activation.


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Invited Speakers


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"One hundred years of sleeping sickness: sex, drugs and RNA" Prof. Isabel Roditi Institute of Cell Biology, University of Bern, Bern, Switzerland Isabel Roditi`s research focuses on the regulation and function of surface glycoproteins that govern survival and transmission of Trypanosoma brucei, the parasite that causes sleeping sickness, by its insect host, the tsetse fly.

Some of the deadliest parasites afflicting humans and their domestic animals are transmitted by insects. These include various sub-species of Trypanosoma brucei which cause sleeping sickness in humans and Nagana in ruminants in large areas of sub-Saharan Africa. These diseases would be even more widespread were it not for the fact that T. brucei is strictly reliant on the tsetse fly for its transmission between mammals. The insect is not a mere "flying syringe" that mechanically transfers parasites from one host to the next as it takes a blood meal. Instead, there is a complex interaction between the parasite and the tsetse as the trypanosomes differentiate, proliferate and migrate through different tissues before being transmitted to a new host. Prof. Roditi is interested in learning how trypanosomes sense their environment and how they regulate gene expression in order to adapt to different hosts, with particular emphasis on genes coding for stage-specific coat proteins.Trypanosomes typically employ multiple levels of post-transcriptional control. Using several approaches, including biochemical purification of RNA-binding complexes and genome-wide screens with RNAi libraries, prof. Roditi discovered several novel RNA-binding proteins that regulate stage-specific mRNAs. She is also interested in analysis of the function of parasite proteins in transmission by tsetse. Futhermore, her research also focuses on genome-wide screens for the identification of genes involved in drug-resistance and differentiation of the parasite.

Poster Abstracts

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PEDSV.15 will be stably transfected with wild-type and mutant EGFR and their susceptibility to HCMV infection will be assessed. EGFR may have a key role in HCMV entry into pEC.


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P 35 HCMV entry mechanisms into porcine endothelial cells Taveira A1, Ponroy N1, Schneider M2, Sinzger C3, Seebach JD4, Millard AL*1, Mueller NJ*1

*Contributed equally

1Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, Switzerland, 2Laboratory for Transplantation Immunology, Division of Internal Medicine, University Hospital Zurich, Zurich, Switzerland, 3Institute of Medical Virology and Epidemiology of Virus Diseases, University of Tuebingen, Tuebingen, Germany,4Service of Clinical Immunology and Allergology, Department of Internal Medicine, University Hospital and Medical Faculty, Geneva, Switzerland [email protected]

Transplantation has emerged as the best therapy to treat patients with end-stage organ failure. However, a major limitation is the increasing shortage of donor organs. Xenotransplantation using pig organs is a promising experimental approach to increase availability of organs. Clinical pig-to-human trials involving islet cells are currently performed. Infection/reactivation of human cytomegalovirus (HCMV) is the most important complications post-transplantation. We previously showed that HCMV is able to replicate into porcine cells in vitro. In order to prevent HCMV entry into porcine cells, we investigated its potential entry pathways in porcine versus human endothelial cells (EC).

Primary human EC, as well as porcine aortic (PEDSV15) and porcine microvascular bone-marrow derived (2A2) EC lines were analyzed for HCMV entry receptor expression. In parallel, cells were inoculated with the endotheliotropic (TB40/E) or the fibrotropic (TB40/F) HCMV strains. Viral replication was assessed through the expression of viral immediate early (IE) proteins by immunofluorescence and western blotting techniques.

All cell types expressed high levels of integrin 1. PDGFR and EGFR were detected on 2A2 and to a lower extend on human EC. Interestingly, EGFR was undetectable and PDGFR expression lower on PEDSV.15. Treatment of PEDSV.15 with EGF failed to activate the EGFR signaling pathway suggesting either the absence of EGFR or the presence of a nonfunctional EGFR on PEDSV.15. Shortly after infection both TB40/E- and to a higher extent TB40/F-infected 2A2 showed a strong expression of IE proteins. On the contrary, IE expression was reduced and delayed in PEDSV.15, correlating with the differences observed in entry receptor expression. To further study the role of EGFR in HCMV infection of pEC,

Invited Speakers

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Professor Isabel Roditi is co-director of the Institute of Cell Biology at the University of Berne in Switzerland. She received her Ph.D. in 1983 from the University of Cambridge, Wolfson College, and her D.Habil. in 1993 from the University of Berne. She did postdoctoral work with the Medical Research Council in Cambridge and later at the Institute for Genetics and Toxicology at Karlsruhe University in Germany. In 1993 she received the Helmut Horten Förderpreis, and, in 2001, she received the Cloetta Prize; in 2001, she was elected a member of the Swiss Academy of Medical Sciences.


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Individuality of Bacterial Responses to Antimicrobials Prof. John McKinney Laboratory of Bacteriology, Global Health Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Research in the group of John McKinney focuses on understanding the mechanisms underlying bacterial persistence in the context of host immunity and antimicrobial therapy, using M. tuberculosis as a model pathogen.

Bacteria behave as individuals. Mutation and genetic exchange are important drivers of bacterial individuation, but these events are relatively rare. At much higher frequencies, genetically identical cells display metastable variation in growth rate, response kinetics, stress resistance, and other quantifiable phenotypes. These between-cell differences arise from non-genetic sources, such as stochastic fluctuations in gene expression and asymmetric partitioning of components at cell division. Temporal variation at the single-cell level generates phenotypic diversity at the population level. This diversity is critical for bacterial persistence in changing environments because it ensures that some individuals will survive a potentially lethal stress that would otherwise extinguish the population. For example, the refractoriness of bacterial infections to antibiotic therapy has been ascribed to spontaneous variants ("persisters") that survive and regrow despite prolonged antibiotic exposure. The persisters are not antibiotic resistant mutants and it is unclear why they tolerate antibiotics that kill their genetically identical siblings.

Our studies focus on the mechanistic basis of the reversible persister switch in the clinically important microorganism Mycobacterium tuberculosis. We use automated time-lapse fluorescence microscopy in conjunction with microfluidic and microelectromechanical systems to analyze bacterial responses to antibiotics at the single-cell level. Our studies show that the conventional interpretation of the persister phenomenon – that persistence is due to pre-existing subpopulations of dormant cells – is incorrect. Instead, we find no correlation between the growth rates of individual cells and their probability of survival during antibiotic exposure. We also find that the survival probability of closely related sister cells is strongly and positively correlated. These observations suggest that the factors that determine whether a cell lives or dies are metastable within individual cell lineages. In the specific case of isoniazid, a frontline anti-tuberculosis drug, we find that the isoniazid-activating catalase is expressed in apparently random bursts that are strongly correlated with cell death.

Poster Abstracts

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P 34 The Molecular Basis for Host Recognition of the Commensal Microbe Lactobacillus reuteri

Patrycja Konieczna, Mario Ziegler, Ruth Ferstl, Liam O’Mahony

Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland

Background. Lactobacillus reuteri is a commensal microbe which has been previously demonstrated to attenuate the severity of inflammation in murine models of colitis and respiratory allergy. The mechanism underpinning this protective host immune response is thought to include the induction of T regulatory cells. However, the molecular basis for the induction of T regulatory cells by the commensal microbiota is unknown. Aim. To elucidate the molecular basis for host recognition of Lactobacillus reuteri. Methods. Monocyte derived dendritic cells and the monocytic cell line THP-1 were utilized as cell culture models. Cells were stimulated with different doses of bacteria and cytokine secretion was measured using luminex. Transcription factor activation was measured using flow cytometry and gene expression quantified using qRT-PCR. Pattern recognition receptors were blocked using specific antibodies or specific ODNs. Results. Lactobacillus reuteri stimulated dendritic cells secrete high levels of IL-10 and low levels of IL-12p70. In addition, the level of cytokine secretion was dependent on the dose of bacteria used. Induction of NF-kB, p38 MAPK and ERK was a late event (greater than 90 minutes) suggesting that phagocytosis and intracellular activation is important for cytokine induction. Neutralisation studies implicate cell surface c-type lectin receptors and intracellular toll-like receptors as important pattern recognition receptors for recognition of Lactobacillus reuteri. Conclusion. Lactobacillus reuteri induces secretion of the regulatory cytokine IL-10 by dendritic cells and activation of this cellular response requires both cell surface c-type lectin receptors and intracellular toll-like receptors.


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P 33 Lytic and latent antigen-specific T cell responses against EBV-mediated B cell transformation Antsiferova Olga, Christian Münz Laboratory of viral Immunobiology, Institute of Experimental Immunology, UniZH [email protected] Epstein-Bar virus is a ubiquitous human virus. Primary infection takes place in childhood usually in asymptomatic fashion and proliferation of latently infected B cells is kept under the control of T cells. However, delayed encounter with the virus leads to the syndrome of infectious mononucleosis (glandular fever), which is characterized by striking lymhocytosis with the massive expansion of a few dominant clones of EBV-specific CD8+ T cells. Afterwards, the patient attains a life-long control of the virus. However, immunosuppression and re-activation of the virus is implicated in the pathogenesis of several malignances of lymphoid (Burkitt's lymphoma, Hodgkin`s disease etc) and epithelial cell origin (nasopharengial carcinoma etc). The reconstitution of immunocompetent human immune system components in severely immune compromized mice allows us to study the immune control of EBV in vivo. As during chronic EBV infection in humans lytic and latent (tumorogenic) EBV antigens are expressed in reconstituted and infected mice. Both of these have been demonstrated to be targets of cytotoxic T cells against established transformed B cells in vitro. To study in more details the dependence of T cell control on these antigens, expressed by the transformed B cell, we use recombinant viruses either with the wild type EBV genome or deficient in BZLF1 – one if the immediate transactivators of the virus that is required for the initiation of the lytic viral cycle. These tools allow us to follow the latent transforming EBV infection in vitro and in vivo in absence or presence of lytic EBV replication. The study aims to characterize the B cell transformation by recombinant EBV and analyse the contribution of lytic EBV antigen specific T cell responses to protective immune control of EBV in vivo.

Invited Speakers

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We conclude that stochastic fluctuations in gene expression at the single-cell level can determine cell fate (death or persistence) in response to potentially lethal environmental perturbations. Professor John McKinney received his Ph.D. from The Rockefeller University (New York, NY) in 1994 for studies on cell cycle regulation in Saccharomyces cerevisiae in the laboratory of Fred Cross. From 1995 to 1998, he was a postdoctoral fellow in the laboratory of William Jacobs at the Albert Einstein College of Medicine (Bronx, NY), where he studied mechanisms of persistence in Mycobacterium tuberculosis. In 1999, he returned to Rockefeller University to establish his own laboratory as an Assistant (1999-2004) and then Associate (2004-2007) Professor. In July 2007, the lab relocated to the Global Health Institute in the School of Life Sciences at the École Polytechnique Fédérale de Lausanne (EPFL) in Switzerland, where John McKinney is Professor and Head of the Laboratory of Bacteriology.


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A bicontinental life in the laboratory: How an American biochemist made his research career in Europe Prof. Kerry L. Tucker Interdisciplinary Center for Neurosciences, University of Heidelberg, Heidelberg, Germany Prof. Kerry L. Tucker is a research group leader at the University of Heidelberg, Institute of Anatomy and Cell Biology. He accepted our invitation to talk about his scientific career and his unique way of becoming a group leader.

Dr. Kerry L. Tucker studied Biochemistry at Harvard College in Cambridge, Mass., where he worked in the laboratory of Prof. Tomas Kirchhausen at Harvard Medical School researching clathrin-associated proteins. This led to an Artium Baccalaureus from Harvard College in June 1990. The next seven years he spent pursuing a Ph.D. at the Massachusetts Institute of Technology in Cambridge, Mass., involving first a stay in the laboratory of Prof. Ethan Signer studying repeat-induced gene silencing in A. thaliana, followed by four years in the laboratory of Dr. Rudolf Jaenisch at the Whitehead Institute of Biomedical Research. Here Dr. Tucker made a genetic investigation into the anatomical origins of DNA methylation-mediated genomic imprinting, which led to a Ph.D. in Biology in June of 1997.

Dr. Tucker then switched to both a new continent and to the study of neuroscience, entering the laboratory of Dr. Yves-Alain Barde at the Max Planck Institute of Neuroscience in Munich, Germany. There he engineered a mouse line to express green fluorescent protein specifically in all neurons of the embryonic central and peripheral nervous systems, which allowed the visualization of the development of these structures. Dr. Tucker established his own laboratory at the Interdisciplinary Center for Neurosciences at the University of Heidelberg in the Institute of Anatomy and Cell Biology in Oct. 2003. There he researches the development of the embryonic and postnatal cerebral cortex. He holds courses for students of pharmacy and molecular biotechnology (IPMB) and teaches also anatomy to medical students.

Poster Abstracts

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P 32

Intracellular defenses control HIV-1 diversification

Gustavo Gers Huber1, Annette Audigé1, Viviana Simon2, Roberto F. Speck1*

1Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, Switzerland1 and 2Department of Medicine, Division of Infectious Diseases, and Department of Microbiology, Mount Sinai School of Medicine, Global Health and Emerging Pathogens Institute, New York, U.S.

Sequence variation is central to the ability of HIV-1 to evade

immune responses and antiretroviral therapeutics. Over the past years, it has become clear that, if left unchecked, DNA/RNA editing enzymes of the APOBEC3 family are potent mutagens of retroviral genomes. The HIV protein Vif normally counteracts APOBEC3 action. However, proviruses with footprints of past deamination have been found in many HIV-1 infected patients suggesting that Vif inactivation occurs frequently in vivo. Moreover, HIV-1 strains with Vif alleles unable to completely counteract the activity of APOBEC3 enzymes have been identified suggesting that full APOBEC3 neutralization is not necessary for survival of HIV-1 as a population/quasi-species.

We hypothesize that innate intracellular inhibitors modulate viral evolution in vivo and, thereby, indirectly also determine the pathogenicity of HIV-1. Thus, the APOBEC cytidine deaminases, which likely evolved as a host defense against (endogenous) retroviruses, might have been usurped by HIV-1 and facilitate its escape from immunological and pharmacological inhibition. The accessory viral protein Vif is at the core of this viral /host interphase and we will test to what extend partially active Vif phenotypes shape viral diversity and are responsible for rapid emergence of specific drug resistant HIV-1 strains. Prohibitive costs have so far prevented testing these questions in monkey animal models but our well established humanized mouse model overcomes these limitations and provides a biologically highly relevant, cost efficient experimental model system.


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P 31 Identification of Candida albicans specific T cell epitopes Eva Bär, Salomé LeibundGut Institute of Microbiology, ETH Zurich [email protected]

The commensal fungus Candida albicans is of major clinical relevance causing several diseases like disseminated, vaginal or oropharyngeal candidiasis (OPC) particular in immunodeficient individuals. Protection from fungal disease is dependent on the innate and the adaptive part of the immune system. Dendritic cells senses microbes via pattern recognition receptors, elicits antimicrobial responses and trigger T cells. It is well established that CD4+ T cells play a central role in protecting the host against fungal infection. This is illustrated by the fact that OPC is an AIDS-defining illness. However, the mechanisms by which the CD4+ T cells confer protection remain largely unclear. Therefore, a better understanding of molecular and cellular events leading to T cell responses during fungal infection is necessary in order to develop new therapeutic and preventive strategies against fungal infections.

This project will focus on the identification of fungus-derived MHC class II peptides presented to Candida-specific CD4+ T cells in mice. Antigenic epitopes will be eluted from MHC class II molecules of infected antigen-presenting cells and sequenced by HPLC-MS/MS techniques. The identified peptides will be used in immunization strategies against C.albicans infections. In addition, the requirement of the epitope-containing fungal proteins with respect to Candida-pathogenicity and virulence will be evaluated. Because host defense mechanisms vary by anatomical site another aspect of the project will be to investigate how the distribution and selection of MHC class II epitopes differ between diverse C. albicans-associated diseases. Together, these experiments shall provide important new insights into the pathogenicity of C.albicans and the mechanisms of CD4+ T cell-mediated immune response to infection.

Invited Speakers

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“The future is in the genes - Plant Biotechnology at BASF”

Dr. Stéphane Bieri BASF Plant Science Company GmbH, Limburgerhof, Germany Dr. Stéphane Bieri comes from the field of plant biotechnology. He followed our invitation to talk about the social implications of genetic engineering of food.

Dr. Stéphane Bieri was trained as a molecular biologist at the University of Basel and holds a Ph.D. degree from ETH Zürich. He then moved to the Max Planck Institute for Plant Breeding Research in Cologne and later to the University of Zürich to work on how plants are able to withstand pathogens through resistance genes. Specifically, he worked on a receptor from barley that is unique to plants and allows to recognize the presence of pathogen derived molecules and initiates defence mechanisms. Such receptors often play a role in tolerance or immunity of crops against a variety of pathogens in the field.

In 2007 he joined BASF Plant Science, a research and technology platform that aggregates all plant biotechnology activities of BASF SE to work on fungal disease resistance in cereals. He is now responsible for the coordination of all greenhouse related work and biosafety officer at the site of Limburgerhof.


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Student Abstracts

Important notice: This is a closed and private student meeting. None of the data presented here or printed in this booklet are to be considered published.

Poster Abstracts

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P 30 Investigating the role of ARFs and ARLs during the later stages of encystation in Giardia lamblia.

Samuel Rout1, Carmen Faso1, Adrian B. Hehl1

1Institute of Parasitology, University of Zurich [email protected]

Giardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is a highly-reduced flagellated unicellular eukaryotic microorganism and is the most common causative agent for diarrhea. Giardia species have two major life cycle stages: - an infective trophozoite (binucleate) and dormant cyst (quadrinucleate). Transmission of the parasite to the host requires encapsulation of the trophozoite into an environmentally resistant cyst. This stage conversion is dependent on a functional and highly regulated secretory pathway where cyst wall material (CWM) is transported from the endoplasmic recticulum to the plasma membrane in secretory competent vesicles’- named encystation specific vesicles (ESVs). ESVs are non- steady state organelles and arise only upon induction of encystation and are analogous to Golgi bodies in higher eukaryotes.

Experiments using Giardia homologues of components of secretory pathway revealed the effect of Sar1, Rab1 and Arf1 GTPases (grouped in the small Rho GTPases) on correct cyst wall formation. In particular, expression of a dominant negative ADP ribosylation factor 1 (ARF1) lead to formation of naked cysts which lacked infectivity. We found 6 additional ARF and ARF like (ARLs) sequences in the Giardia database whose functions are yet unknown. By using tagged versions of these genes we are currently investigating their effect on encystation and their role in correct cyst formation.

It is known that ARF proteins recruit clathrin to form clathrin coated vescisles (CCVs) in higher eukaryotes. However no CCVs have been shown in Giardia so far. Nevertheless association of clathrin heavy chain (CLH) to late ESVs has already been described. Therefore the role of ARF and ARLs in clathrin recruitment and ESV maintenance remains unclear. We hypothesize that CLH lattices might be involved in ESV maintenance by preventing homotypic fusion between ESVs. Based on this, we predict that expression of CLH recruitment incompetent ARF mutant would lead to enlarged ESVs and an inhibition of cyst wall formation.


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P 29 Improvements in the modular antigen translocation (MAT) vaccine technology

Mattia Garbani, Reto Crameri

Swiss Institute for Allergy and Asthma Research, Davos University of Zurich [email protected]

Delivering pharmaceuticals inside a cell is one of the factors limiting their bioavailability, in order to overcome this problem many different methods have been established. One of those strategies is the use of cell penetrating peptides, which lead to the internalization of a carried protein. The Modular Antigen Translocation (MAT) Vaccine exploits this property to bring Invariant-Chain (li) linked recombinant antigens into cells: the molecule is intracellularly targeted by li to the endosome, where proteolytic digest, charging of MHC-II molecules and finally antigen presentation takes place. By the li-mediated targeting of the immunogenic protein directly to the MHC-II, the immunresponse can be improved.

The MAT vaccine (through the tat-derived peptide) is potentially internalized in every cell; this fact lead to great losses. The aim of this project is to create a new generation of MAT vaccines in which the tat peptide is substituted or supplemented by a newly discovered peptide, which specifically binds to dendritic cells. With this new approach we are aiming to specifically target antigen presenting DCs, potentially leading to further improved immunogenicity of the cargo. If direct targeting of DCs is proven to be successful, oral delivery of modified MAT vaccines can be investigated, which would drastically reduce costs and improve patients compliance by substituting injections.

Student Talks

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Student Talks


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ST 01 Identification and Characterization of Human Regulatory B cells Willem van de Veen1, Barbara Stanic1, Görkem Yaman2, Beate Rückert1, Deniz Akdis1, Cezmi Akdis1 and Mübeccel Akdis1

1 Swiss Institute of Allergy and Asthma Research (SIAF), Zürich University, Davos, CH 2 Microbiology Department, Yüzüncü Yil University, Van, TR [email protected]

B cells do not only produce antibodies but memory B cells that express certain cytokines can regulate antigen-specific T cell responses. Another B cell-related immune regulatory response is the induction of blocking, non-inflammatory IgG4 antibodies, which characterizes all high dose antigen tolerance models. The lack or loss of regulatory B cells has been demonstrated by exacerbated symptoms in experimental autoimmune encephalitis, chronic colitis, contact hypersensitivity, collagen-induced arthritis and non-obese diabetic mouse models. A regulatory B cell subset remains to be elucidated in humans. We hypothesize that if a B cell plays an anti-inflammatory role, the antibody isotype produced by this B cell after differentiation to a plasma cell should be anti-inflammatory. The aim of this study is to identify and characterize a human IL-10-producing B cell subset and to determine whether these cells differentiate into IgG4-secreting plasma cells. Treatment with a TLR9 agonist induced strong B cell proliferation and high levels of IL-10 production in purified peripheral- as well as tonsil-derived B cells. Such treatment led to production of IgG4 at the mRNA and protein level and this effect was strongly enhanced when cultures were supplemented with exogenous IL-10. Isolation of B cells that secrete IL-10 after TLR9 ligation showed that these cells produce higher levels of IgG4 than cells that do not secrete IL-10. Furthermore, IL-10-producing B cells suppressed antigen-specific T cell proliferation whereas B cells that did not produce IL-10 were unable to suppress such a response. In addition, B cells specific for the major bee venom allergen phospholipase A2 were purified from beekeepers that developed immunological tolerance towards bee venom. These cells expressed higher levels of IgG4 mRNA compared to other B cells. Taken together, these data demonstrate that human suppressive memory B cells exist that produce mainly IgG4 after differentiation into plasma cells.

Poster Abstracts

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P 28 Mechanisms of T cell help during viral infections Josh Crouse, Annette Oxenius Institute of Microbiology, ETH Zurich [email protected]

Certain pathogenic infections (such as influenza, LCMV, and VSV) can induce a strong primary CD8 T cell response even in the absence of CD4 T cell help, whereas other pathogenic infections (such as Adenovirus, HSV-1 and VV) strictly require the presence of CD4 T cell help to induce a primary CD8 T cell response. It is believed that the ability of infectious agents to directly activate antigen presenting cells via pattern recognition receptors allows for the circumvention of CD4 T cell help. However, the mechanisms that render particular viral infections T help dependent are largely unknown. It is possible that the cytokine environment elicited by certain viral infections can account for their ability to elicit T cell help independent CD8 T cell responses. In line with this, we have found that upon Vaccinia virus infection, IL-12 is induced in a T cell help dependent manner, whereas LCMV induces a potent Type-I interferon response, without the help of CD4+ T cells. The Type-I interferon response is mounted at the expense of IL-12 production. Based on these findings, we will compare LCMV infection, a prototype of a T cell help independent infection to Vaccinia virus infection in vivo with the aim to obtain detailed knowledge about the mechanisms which render certain viral infections T cell help dependent or independent, with a focus on the role of Type-I interferons in the survival of activated CD8+ T cells.


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P 27 HIV-1 quasispecies analysis by ultra-deep sequencing Francesca Di Giallonardo, Niko Beerenwinkel, Osvaldo Zargodi, Huldrych F. Günthard, Rémy Bruggmann, Marzanna Künzli-Gontarczyk, and Karin J. Metzner Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University Zurich ETH Zurich [email protected]

The diversity of human immunodeficiency viruses (HIV-1) within one infected person is high. In fact, the virus has a high mutation rate that leads to a large genetic heterogeneity. With normal Sanger sequencing, mutants present at ≥20-25% can be detected, however, it is known that some resistant viruses present at low frequencies (minorities) can cause virological failure of antiretroviral therapy (ART). Therefore new methods are needed to detect quasispecies which can lead to therapy failure.

We used the 454 sequencing technology from Roche to sequence the HIV-1 protease. This high-throughput DNA sequencing technology allows resolving the genetic variation in a sample by direct sequencing and generates a large number of individual reads from a population of interest. In a first experiment we wanted to test the detection limit of this method. For this approach, two different viral stocks were mixed in different amounts (1% to 99% and 0.1 % to 99.9%). To exclude potential amplification bias due to the experimental procedure and to estimate the recombination rate 5 different virus stocks were mixed in equal amounts, splitted into 6 different samples, processed individually throughout the complete protocol, and sequenced.

We found the detection limit to be approx. 1%, variants present at 0.1% could not be found. The six independent samples of the virus stock mixture revealed that the frequencies of each of the 5 mixed viruses was similar with two exceptions in 1/6 samples suggesting that PCR errors of preferential amplification are uncommon. However, the recombination rate was estimated to be up to 10%.

Our 454 sequencing protocol is suitable to detect viruses at low frequencies (≥1%) and repeatedly results in similar quantifications of virus mixtures. Thus, further optimization processes will focus on the reduction of the recombination rate.

Student Talks

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ST 02 The role of persistent viral infection in immune senescence Andrea Mekker, Urs Karrer Division of Infectious Diseases an Hospital Epidemiology, Department of Internal Medicine, University Hospital of Zurich, Zurich, Switzerland [email protected]

Aging of the immune system, commonly termed immune senescence, is clinically associated with an increased frequency and severity of infectious diseases, cancer, chronic inflammatory disorders and autoimmunity. Immune senescence is also linked to age-associated accumulation of memory and effector T cells reducing the naïve T cell pool, which is necessary for further immune protection. Memory inflation (i.e. a pattern of longitudinal accumulation of certain epitope-specific CD8+ T cells) is a peculiarity of the T cell response against Cytomegalovirus (CMV) infections in humans and mice, and signifies an accelerated accumulation of memory T cells. To test the hypothesis whether and how persistent viral infections might propagate immune senescence we established a mouse model for infection enhanced immune senescence (IEIS). To this end, high dose MCMV-infected C57BL/6 mice were challenged with the recombinant Vaccinia-G2 (G2: LCMV-epitope) virus and the CD4 and CD8 T cell immune response to Vaccinia and LCMV were investigated. As hypothesized, the Vaccinia-specific T cell response in young, persistently MCMV infected mice resembled those of aged uninfected mice and was considerably decreased compared to young Vaccinia-G2 challenged mice, which were not MCMV infected. The Vaccinia T cell response was dramatically reduced in aged MCMV-infected mice, and was associated with a reduced viral clearance compared to age-matched non infected animals. Similar results were obtained for the LCMV specific CD8 T cell response, indicating a severe role of MCMV infection in propagating immune senescence.


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ST 03 Bcl-2 inhibition: a new concept to prevent solid allograft rejection Pietro Cippà, Thomas Fehr Institute of Physiology, University of Zürich [email protected] Introduction: Novel, highly effective, but less toxic immunosuppressive drugs are urgently needed to improve long- term outcome after solid organ transplantation. Considering the crucial role of apoptosis in the regulation of the adaptive immune response, pharmacological inhibition of Bcl-2 proteins - major regulators of the intrinsic apoptotic pathway - represents an innovative immunosuppressive concept. Here we tested for the first time the antagonist of anti-apoptotic Bcl-2 proteins ABT-737 as an inhibitor of allogeneic immune reactions in vitro and in vivo. Methods: In vitro, ABT-737 was tested in a mixed lymphocyte reaction model with BM3.3 transgenic responder cells (expressing on CD8 T cells a transgenic T cell receptor specific for the MHC class I molecule Kb) reacting against B6 stimulators. In vivo, skin graft rejection was studied in an MHC class I mismatched mouse model (bm1 to B6). To assess tissue selectivity of ABT-737 in vivo, we analyzed by immunohistochemistry the number of apoptotic cells in different organs after injection of ABT-737. Results: ABT-737 potently suppressed allogeneic immune reactions in vitro on every level of T cell effector function. A more detailed analysis of the immunosuppressive mechanism demonstrated, that ABT-737 selectively induced apoptosis in T cells reducing the alloreactive clone size, but did not impair the physiological functions of the remaining viable T cells. Moreover, the pro-apoptotic effect of ABT-737 in vivo was selective to lymphocytes, as shown by an increased number of apoptotic cells in the spleen, but not in the kidney or the liver. Eventually, ABT-737 significantly prolonged skin allograft survival in an MHC class I mismatched model. Conclusion: Bcl-2 inhibition represents a novel and promising strategy to suppress allogeneic immune reactions and to prevent rejection after solid organ transplantation. The high selectivity of ABT-737 for lymphocytes suggests a low toxicity profile of the novel immunosuppressive treatment.

Poster Abstracts

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P 26 Characterization and Manipulation of Innate Immune Responses in the Domestic Cat Céline Robert, Valentino Cattori, Marina Meli, Hans Lutz Clinical Laboratory, Vetsuisse Faculty, University of Zurich [email protected]

In the prevention of pandemics due to emerging viral diseases, the manipulation of innate immunity holds the advantage of inducing in the host rapid defence mechanisms against a broad range of pathogens. The domestic cat appears to be an ideal model to study innate immune responses, as it is an out-bred species naturally affected by many viruses that resemble in their biological properties those affecting humans. With the objective to extend knowledge on the innate immune mechanisms in the context of feline viral infection, several real-time PCR systems have been developed for this species, to detect expression of genes already known to play an important role in early immune responses to viruses in mice and humans. These novel tests enable to measure the implication of relevant markers of innate immunity such as Toll-like receptors 3, 7, 8 and 9, various cytokines including interferon alpha and omega, as well as the intracellular antiviral protein Mx. In a series of in vitro experiments using the cat as a model, early immune responses to inoculation of feline primary immune cells and common cell lines with various viruses, namely the feline immuno-deficiency-, leukemia-, herpes-, calici-, and coronaviruses, are characterized. Furthermore, the immunologic responses elicited in these cells with a synthetic CpG-containing molecule are associated with the potential of this well-known immune response modifier to create resistance in vitro to the mentioned feline viral infections. Altogether, this work sheds light on early immune mechanisms to a broad range of viruses, and serves as basis for future experiments aimed at the prevention of viral infections not only in felids, but in humans and other species as well.


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P 25 Regulation of macroautophagy during lytic infection of EBV

Heike Nowag, Christian Münz

Instiut of experimental Immunology, University Zurich [email protected]

Macroautophagy is a major degradational system of the cell, which, in contrast to the ubiquitin-proteasomal pathway, engulfs long-lived proteins and organelles in a characteristic double-membrane vesicle, the so-called autophagosome. Autophagosomes will then fuse with lysosomes in order to dedrade their cargo.

Since it is involved in cellular homeostasis macroautophagy is upregulated following stress response to starvation or infection by intracellular pathogens.

In order to avoid the degradation by macroautophagy, RNA and DNA virus can subvert the pathway for the benefit of the host or for their own benefit. For viruses, manipulating autophagy pathway can result in various effects, like an increased viral replication, or an enhanced apoptosis of their host. We were interested of the role of macroautophagy upon Epstein Barr virus (EBV) replication.

Epstein Barr virus is a y-herpesvirus, which contains classical viral latency features, with reversibility as a hallmark (lytic phase).

Little is known about the lytic phase of Epstein Barr virus (EBV) infection and macroautophagy pathway. We used an in vitro inducible model of EBV reactivation to address this question. By inducing the lytic phase in an EBV infected B cell line (AKBM) through B-cell receptor cross-linking, we found that macroautophagy was significantly upregulated upon induction of the lytic phase. To further address the question of who may benefit from this upregulation, EBV viral titer upon inhibition or enhancement of macroautophagy are now evaluated.

In parallel we are addressing the question of the molecular pathway involved in macroautophagy upregulation upon viral reactivation. This study will help us to understand if EBV benefits from macroautophagy upregulation.

Student Talks

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ST 04 Genetic diversity amongst canine papillomaviruses Christian E. Lange 1,2, Kurt Tobler 2 and Claude Favrot 1

1 Dermatology Department, Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland 2 Virology Institute, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland [email protected]

Papillomaviruses (PVs) are responsible for various skin diseases in humans and occur in a broad genetic diversity; to date almost 200 human PVs in five genera. PVs have also been found in various animal species and could be linked to a couple of skin diseases. Oral papillomas in dogs have been well characterized, but in recent years novel canine PVs (CPVs) have been discovered associated with various types of lesions, including in situ carcinomas and endophytic papillomas. To shed more light into the largely unexplored CPV field, we evaluated lesional and nonlesional skin and mucous membrane specimens using several different PV-PCR protocols as well as rolling circle amplification.

In lesional skin (endophytic papillomas and in situ carcinomas), three novel CPVs (CPVs 5, 6 and 7) were discovered, which after complete sequencing could be grouped into three proposed clades, yielding hints about the characteristics of putative further CPVs.

Many of the non lesional samples of the oral mucosa and several of interdigital skin were also deemed positive for PV DNA. This supports the hypothesis, that not only humans harbor PVs in their healthy skin but dogs as well. These findings support proposed parallels between PV induced epidermal diseases in humans and dogs and encourage further studies in that direction.


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ST 05 CD8+ T cell tolerance induced by inflammation-matured dendritic cells Wolfgang Kratky, Roman Spörri and Annette Oxenius Institute of Microbiology, ETH Zurich The interplay between CD8+ T cells and dendritic cells (DCs) plays a crucial role in the ability of the immune system to eliminate infected cells. Efficient priming of naïve T cells by DCs requires three signals: 1. the cognate antigen, 2. costimulation, and 3. immunomodulatory cytokines/etc. Only fully mature DCs which have recognized conserved microbial structures (PAMPs) via pattern recognition receptors (e.g. toll like receptors, TLRs) can provide all these signals. However, DCs can also be activated in trans by inflammatory mediators secreted by PAMP-triggered DCs, a process likely to happen frequently during the course of an infection, where a huge discrepancy between the number of infected vs. activated cells can be observed. The aim of this project is to investigate whether inflammation can substitute for direct pathogen recognition in CD8+ T cell priming. In particular we want to explore whether these trans-activated DC are able to prime fully functional CD8+ T cells. With these experiments we want to define the requirements for full DC- and consequently CD8+ T cell function in more detail. We could show that although DCs activated in trans acquire features that are prerequisites for T cell priming such as upregulation of costimulation and MHC, and migration to secondary lymphoid organs. Nevertheless, this is insufficient for an efficient CD8+ T cell response. Our data further indicates that that inflammation-matured DCs may rather induce CD8+ T cell tolerance or deletion. Here we describe a mechanism which strictly couples the recognition of the pathogen to the quality of the response, thereby preventing priming of CD8+ T cells of irrelevant or unwanted specificities in an inflammatory setting.

Poster Abstracts

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P 24 Protein quality control in the ER: The role of Pdi1/Htm1 complex Ivan Hang, Robert Gauss, Markus Aebi Institute of Microbiology, ETH Zurich [email protected]

Endoplasmic reticulum provides an optimal environment for maturation of secretory proteins. Upon translocation to the ER proteins acquire N-linked glycan attachment, whose structure is being modified to reflect their different folding states. In addition to protein structure, chaperones in the ER recognize this N-linked glycan, which provides information for their further processing.

With its ability to take part in protein folding by catalyzing creation and rearrangement of disulfide bonds Pdi1 is one of the major components of the ER folding machinery that is present in all eukaryotic organisms. In yeast cells, it can be found in complex with Htm1, which recognizes glycan structure on the protein surface and acts as a manosidase to generate the terminal signal, directing proteins to degradation pathways.

As a main tool in our research we will use Pdi1-1, the first viable Pdi1 point-mutant. Pdi1-1 is unable to interact with Htm1p and thus affects its manosidase function, which disrupts the clearance of unfolded proteins from the ER. On the other hand, this single point mutation doesn't interfere with Pdi1 essential functions. By characterizing Pdi1-1 and its relationship with Htm1p we will be able to understand the functionality of the complex even further, which plays the key role in yeast ER quality control.


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P 23 Quorum sensing regulates the putisolvin production through orphan receptor PsoR in Pseudomonas putida affecting swarming motility and Biofilm formation Cárcamo-Oyarce G., Lumjiaktase P. and Eberl L. Departament of Microbiology, Institute of Plant Biology, University of Zurich.

Bacteria can modulate their behavour by releasing and responding to the accumulation of signal molecules; this cell-to-cell communitacion process is called quorum sensing (QS). In Gram-negative bacteria, the most common signal molecule is an acylated homoserine lactone (AHL). The plant-beneficial rhizobacteria Pseudomonas putida utilizes the PpuI/R QS system, which modulates the biofilm formation and regulates the production of cyclic lipopeptides putisolvins I and II. The cyclic lipopeptides are biosurfactantes necessary for swarming motility and can affect the biofilm structure. The putisolvin synthetase gene cluster (pso) is composed by three genes, termed psoA, psoB and psoC. Interestingly, the psoR gene, which is located upstream of psoA, shows high similarity to LuxR-type regulatory genes and is required for the expression of the pso cluster. The aim of this study was to evaluate the role of PsoR in the QS-regulated synthesis of putisolvin in P. putida. Using psoR::gfp reporter, we found that the psoR expression was induced by addition of AHLs in ppuI mutant strain. Structural analysis of biofilm formation, preformed in flow chambers systems, indicated that psoR mutant strain formed characteristic tower-like microcolonies, similar to psoA and ppuI mutant strains, whereas the wild-type strain developed a homogenous biofilm. These results suggest that the QS system control putisolvin production through PsoR.

Student Talks

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ST 06 Epstein Barr Virus: Environmental Trigger and Therapeutic Target in Multiple Sclerosis Kristina Kakalacheva, Jan Lünemann Institute of Experimental Immunology, Univesity of Zurich [email protected] Predisposition to autoimmune diseases such as multiple sclerosis (MS) is genetically linked to certain MHC class II molecules and other immune modulators. However, genetic susceptibility is only one risk factor, and low concordance rates in monozygotic twins, as well as the geographical distribution of disease risk, suggest the involvement of environmental factors in the development of these diseases. Environmental factors such as infections have been implicated in the onset and promotion of autoimmunity. The most consistent data for a pathogenic role in the development of MS exist for the human -herpesvirus Epstein-Barr virus (EBV). Previous studies have shown that treatment-naïve children and adults with MS show selective expansions of T cells and increased humoral immune responses specific for the EBV-encoded nuclear antigen-1 (EBNA1), but not to other EBV- encoded or other virus-derived antigens. Moreover, expanded CD4+ T cells specific for EBNA1 recognized myelin antigens more frequently than other autoantigens that are not associated with MS. In our preliminary data, we demonstrate that increased immune responses to EBNA1 correlate with disease activity and predict disease progression in untreated MS patients. We would like to further investigate the mechanisms leading to changes in EBV-specific immunity in MS by addressing the following three scientific questions: (1) Are autoantigen-reactive EBNA1-specific T cells primed during symptomatic primary EBV infection? (2) Are EBV- and EBNA1-specific T cells detectable in the target organ and do they cross-recognize disease-associated autoantigens in patients with MS? (3) Can blockade of the host DNA binding site of EBNA1 protein regulate EBV infection and EBNA1-specific T cell immunity?

These studies will characterize mechanisms leading to deregulated host-EBV interactions in patients with MS and might open new therapeutic avenues to prevent the occurrence and to halt the progression of EBV-associated diseases.


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ST 07 Regulation of IL-1 surface expression and secretion Fettelschoss A, Kündig TM

Department of Dermatology, Zurich University Hospital

IL-1α and -β are important pro-inflammatory cytokines produced by myeloid cells of the immune system. Pro-IL-1 and - are produced upon Toll like receptor (TLR) stimulation via NFkB. An additional stimulus activating the inflammasome and caspase 1 is required for proteolytic cleavage and secretion of mature IL-1. The regulation of IL-1 remains unclear. IL-1 exists as a cell surface associated form and as a mature secreted form. Here we demonstrate that both forms of IL-1, the cell surface associated and the secreted form are biologically active. However, these two forms are differentially regulated.

Is a monocyte or dendritic cell stimulated by TLR, but receives no signal stimulating the inflammsome, the surface bound form of IL-1 is upregulated. Exclusively upon additional inflammasome activation, IL-1 will be secreted. These findings may partially explain, why certain inflammatory responses, i.e. those were a dendritic cell obtain only TLR signaling, are more dependent on IL-1 than others.

Poster Abstracts

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P 22 Biotoxicity of the fungal chimerolectin MOA: Therese Wohlschlager1, Alex Butschi2, Kathrin Zurfluh1, Sibylle von Esch2, Michael Hengartner2, Markus Aebi1, Markus Künzler1 1. Institute of Microbiology, ETH Zurich; 2. Institute of Molecular Biology, University of Zürich [email protected] Lectins are defined as proteins with at least one carbohydrate-binding domain, that are of non-immune origin, and do not modify the ligand enzymatically. In higher fungi (basidiomycetes and ascomycetes) many lectins have been identified, most of which are small in size, cytoplasmic, soluble and strongly up-regulated in fruiting bodies. Analogous to plants, fungal lectins are suggested to play a role in defense against predators and parasites, complementing secondary metabolites and cyclic peptides in the innate defense mechanisms.

Our research focuses on the identification and characterization of novel fungal lectins, as well as their carbohydrate-specific toxic effects towards target organisms. Here we describe the Marasmius oreades agglutinin (MOA) from the edible fairy ring mushroom with regards to structural, biochemical and functional aspects. Analogous to certain bacterial toxins MOA consists of a lectin domain combined with a domain of different biological activity.


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P 21 Discovering ERAD components in Saccharomyces cerevisiae Susanne Müller, Prof. M. Aebi, Dr. Robert Gauss Institute of Microbiology, ETH Zurich [email protected]

Saccharomyces cerevisiae has proven to be a good model organism for eukaryotic processes such as N-linked glycosylation of proteins. N-linked glycosylation takes place in the yeast endoplasmic reticulum (ER). It is a multistep process during which lipid-linked oligosaccharides (LLOs) are assembled in the cytoplasm and are transported to the ER lumen by the essential Rft1p flippase. In the ER, the LLOs are then linked to asparagine (N) residues on proteins.

ER quality control mechanisms supervise the correct folding and the integrity of the resulting glycoproteins. Unfolded and misfolded glycoproteins are recognised by modifications in their sugar chains and will be eliminated via ER-associated degradation (ERAD). The ERAD process in yeast is still under investigation, and therefore the list of known ERAD components might still not be complete.

The aim of this study is to discover previously undetected ERAD components in Saccharomyces cerevisiae.

Previous experiments have shown that the lethal knockout mutation in the Rft1p flippase can be compensated by mutations affecting ERAD components involved in membrane homeostasis. It has been proposed that incomplete membrane proteins, which would be degraded by ERAD under normal circumstances, can partially compensate for the loss of the Rft1 flippase by transferring LLOs from the cytoplasm to the ER lumen. We will employ this principle to conduct a synthetic viable screen of yeast double mutants. A collection of several thousand double mutants will be screened, each containing a deletion in RFT1 and and in one non-essential yeast ORF. We expect that strains with rft1 and a deletion in ERAD components will be viable since they are able to compensate for the loss of Rft1p. Promising ORFs will be selected and further characterised to elucidate their function in the ERAD model.

Student Talks

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ST 08 Induction of resistance to viral infections in the cat by stimulation of the innate immune system Céline Robert, Vera Rüegger, Valentino Cattori, Marina L. Meli and Hans Lutz Clinical Laboratory, Vetsuisse Faculty, University of Zürich, Switzerland

The mammalian innate immune system efficiently recognizes CpG DNA, which is abundant in various viral and bacterial genomes. CpG DNA is known to signal through Toll-like receptor 9 (TLR9) in immune cells and thus initiate rapid antiviral mechanisms including the secretion of type I interferon (IFN), as well as further induction by these cytokines of intracellular proteins capable of interfering with viral replication. These effects have already indicated prophylactic properties against viral diseases in mice; however they remain to be characterized in other species. The domestic cat appears to be an ideal model to study innate immune responses, as it is an out-bred species naturally affected by many viruses that resemble in their biological properties those affecting humans.

In a series of in vitro experiments using the cat as a model, the immunomodulatory and antiviral properties of a synthetic CpG molecule in feline cells were analyzed. Incubation with CpG DNA induced proliferation of feline peripheral blood mononuclear cells (PBMCs) and significantly increased the expression of IFN, IFNand IFNgenes in these cells by up to 1700, 3800 and 80-fold respectively. Furthermore, supernatants of PBMCs pulsed with CpG DNA for 24 hours could enhance the expression of the intracellular antiviral protein Mx in target PBMCs, FEA and CrFK cells. Most importantly, replication of feline retroviruses (FIV and FeLV) as well as feline enteropathogenic viruses (FCV, FHV and FCoV) could be significantly inhibited in target cells incubated with these supernatants prior to infection. Our observations not only confirm that synthetic CpG molecules can activate early immune responses and promote resistance of feline cells to a broad range of viruses, but also provide valuable information for the development of novel preventive methods against viral diseases in both veterinary and human medicine.


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ST 09 The imapct of the PD-1 deficiency during the early phase of chronic LCMV infection Helge Frebel, Annette Oxenius Institute of Microbiology, ETH Zurich [email protected]

Though the immune system is capable of clearing most types of viral infections, there are numerous viral pathogens that cause infections being slowly or never resolved and therefore leading to a protracted or chronic infection of the host. While the causes of the immune system’s inability to resolve distinct viral infections still are matter of intense investigation, it is known that endogenous immunoregulatory mechanisms can contribute to the failure of the immune system to control these infections. By infecting mice with the arenavirus LCMV (Lymphocytic choriomeningitis virus), a resolved or chronic infection can be induced, depending on the respective viral strain and the dose of injection. Chronic LCMV infection is marked by the functional exhaustion of CD8 and CD4 T cells which is characterized by a strong reduction of cytokine secretion (IL-2, TNF-α and IFN-γ), a reduced capability to proliferate and an impaired development of T cell memory. To date, it remains controversial, if the functional loss of virus-specific T cell immunity represents an immunoevasive mechanism employed by the pathogen or if T cell exhaustion develops as an endogenous mechanism to prevent self-inflicted immunopathology in the face of high viral loads.

Recent studies suggest the upregulation of the immunoinhibitory receptor programmed death 1 (PD-1) by virus-specific T cells to be intimately linked to their progressive exhaustion during chronic LCMV infection. Interestingly, the induction of a chronic LCMV infection in PD-1-/- mice is fatal within 7 days, implying this immunoinhibitory receptor to play a protective role and shedding light on a possibly protective function of T cell exhaustion.

This project aims at investigating the role of the PD-1/PD-L pathway during the early phase of chronic LCMV infection in terms of determining the impact of PD-1 deficiency on virus-directed immune responses and elucidating the immunological processes that lead to fatality.

Poster Abstracts

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P 20 Salmonella Typhimurium binds to HeLa cells via Fim-mediated reversible adhesion and irreversible TTSS-1 mediated docking

Saskia Kreibich

Institute of Microbiology, ETH Zurich [email protected]

The food-borne pathogen Salmonella enterica serovar Typhimurium (S.Tm) invades mammalian epithelial cells. This multi-step process comprises bacterial binding to the host cell, activation of the Salmonella type three secretion system-1 (T1), injection of effector proteins, triggering of host cell actin rearrangements and S.Tm entry. While the latter steps are well understood, much less is known about the initial binding step. Earlier work had implicated adhesins (but not T1) or T1 (but not other adhesins). Here, we have studied the Salmonella virulence factors mediating S.Tm binding to HeLa cells. Using an automated microscopy assay and isogenic S.Tm mutants we analyzed the role of T1 and of several known adhesins (Fim, Pef, Lpf, Agf, Shd) in host cell binding. In wild type S.Tm, host cell binding was mostly attributable to T1. However, in the absence of T1, Fim (but not Pef, Lpf, Agf, Shd) also mediated HeLa cell binding. Furthermore, in the absence of T1 and type I fimbriae (Fim) we still observed residual binding, pointing towards at least one additional, unidentified binding mechanism. Dissociation experiments established that T1-mediated binding was irreversible ('docking') while Fim-mediated binding was reversible ('reversible adhesion'). Finally, we show that non-invasive bacteria docking via T1 or adhering via Fim can efficiently invade HeLa cells, if actin rearrangements are triggered 'in trans' by a wild type S.Tm helper strain. Our data shows that binding to HeLa cells is mediated by at least two different mechanisms and that both can lead to invasion if actin rearrangements are triggered.


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P 19 Defining universal neutralizing epitopes of Influenza A virus hemagglutinin using phage display technology.

Arkadiusz Wyrzucki and Lars Hangartner

Institute of Medical Virology, University of Zurich [email protected]

Annual Influenza A epidemics account for higher morbidity than all other respiratory diseases taken together. Influenza viruses can tolerate changes in their antigenic structures and are therefore capable of escaping pre-existing immunity. Such changes result also in a dramatic increase of viral resistance to available drugs. Furthermore, vaccines protect only from one or few closely related strains, and therefore have to be annually newly formulated and applied. Thus, a novel, ‘one shot’ universal vaccine that provides immunity to wide range of Influenza A strains, including newly emerging strains, is highly desirable.

In this work, we aim at defining universal epitopes of hemagglutinin- the major target protein for neutralizing antibodies on the Influenza A virus surface. Hemagglutinin is responsible for virus entry into the cell by its interaction with sialylated receptors of the host. It has been previously showed that some individuals possess cross-neutralizing antibodies that bind to more than one of the sixteen hemagglutinin subtypes. Crystal structures revealed that Fab fragments of these antibodies interact with a hydrophobic portion on hemagglutinin stem region and block its pH-dependent conformational change necessary for viral-host membrane fusion. In our study we would like to extend the knowledge about universal hemagglutinin epitopes by building phage display Fab libraries based on the immune repertoire of such individuals. These libraries will be used in a modified selection process to identify individual Fabs that cross-react with different hemagglutinin variants. The most promising Fab candidates will then be used in chaperon mediated crystallography approach to reveal conserved epitopes within the hemagglutinin that can confer heterotypic and heterosubtypic protection. Ultimately, the data from structural studies will be used for design of a novel generation of immunogens that can elicit a broad spectrum of protection against seasonal and pandemic Influenza A viruses.

Student Talks

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ST 10 Genome scale RNAi-screening reveals a role of the coatomer I complex in membrane localization of cholesterol, sphingolipids and Rho GTPases for SopE-mediated Salmonella Typhimurium host cell invasion Pascale Vonaesch, Benjamin Misselwitz, Sabrina Dilling and Wolf- Dietrich Hardt Institute of Microbiology, ETH Zurich [email protected]

Salmonella enterica subspecies I serovar Typhimurium are gram-negative, rod shaped facultative intracellular bacteria which cause severe gastroenteritis in human.

Virulence of Salmonella spp. depends on two pathogenicity islands (SPI-1 and SPI-2) encoding two type III secretion systems (TTSS) and different effector proteins which are injected trough these systems into the host cell. SPI-1 mediates the entry into and SPI-2 ensures survival of the bacteria in the host cell. With the help of four SPI-1 effector proteins (SopE, SopE2, SopB, SipA) Salmonella induces its entry into non- phagocytic, intestinal epithelial cells by acting on RhoGTPases and triggering the formation of actin- containing membranous protrusions ("membrane ruffles") leading to internalization of the bacteria. SopE acts as a guanyl nucleotide exchange factor (GEF) for Rac1, Cdc42 and maybe other RhoGTPases.

Up to date, it is not completely understood how SopE mediates invasion into epithelial cells. By genome-scale RNAi screening we identified 72 host cell proteins affecting SopE-mediated pathogen invasion. Follow-up assays assigned these "hits" to particular steps of the invasion process, i.e. docking, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella containing vacuole (SCV). Membrane ruffling was affected by known (Cdc42, Nap1 and the Arp2/3 complex) and by novel factors, i.e. Profilin 1, the sodium/potassium-transporting ATPase ATP1A1 and the E3 ligase Ring box protein 1 (Rbx1).

Knocking down the COPI-complex reduced effector injection and membrane ruffling. Most likely, both effects are attributable to mislocalization of cholesterol and sphingolipids into a large intracellular compartment. We speculate that the ruffling defect is explained by the concomitant recruitment of Rac1 and Cdc42 away from the cell membrane into a large intracellular compartment. Our findings suggest that the identified COPI-facilitated


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maintenance of cholesterol and sphingolipids in the cell membrane represents an unifying mechanism essential for host cell invasion of a wide range of pathogens.

Poster Abstracts

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P 18 Functional investigation of methanol dehydrogenase-like protein XoxF in Methylobacterium extorquens AM1: Sabrina Schmidt, Philipp Christen, Patrick Kiefer, Julia A. Vorholt


Methanol dehydrogenase-like protein XoxF of Methylobacterium extorquens AM1 exhibits a sequence identity of 50% to the catalytic subunit MxaF of periplasmic methanol dehydrogenase in the same organism. The latter has been characterized in detail, identified as a pyrroloquinoline quinone (PQQ)-dependent protein and shown to be essential for growth in the presence of methanol in this methylotrophic model bacterium. In contrast, the function of XoxF in M. extorquens AM1 has not yet been elucidated, and a phenotype remained to be described for a xoxF mutant. Here, we found that a xoxF mutant is less competitive than the wild type during colonization of the phyllosphere of Arabidopsis thaliana, indicating a function for XoxF during plant colonization. A comparison of the growth parameters of the M. extorquens AM1 xoxF mutant and the wild type during exponential growth revealed a reduced methanol uptake rate and a reduced growth rate for the xoxF mutant of about 30%. Experiments with cells starved for carbon revealed that methanol oxidation in the xoxF mutant occurs less rapidly compared to the wild type, especially in the first minutes after methanol addition. A distinct phenotype for the xoxF mutant was also observed when formate and CO2 production were measured after the addition of methanol or formaldehyde to starved cells. The wild type, but not the xoxF mutant, accumulated formate upon substrate addition and had a one-hour lag in CO2 production under the experimental conditions. Determination of the kinetic properties of the purified enzyme showed a conversion capacity for both formaldehyde and methanol. The results suggest that XoxF is involved in one-carbon metabolism in M. extorquens AM1.


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P 17 Adaptation of the HIV-specific B cell repertoire to viral escape Merle Schanz Institute of Medical Virology, University of Zürich [email protected]

The development of an effective human immunodeficiency virus 1 (HIV-1) vaccine is a major global health challenge. It is widely believed that a successful vaccine must induce neutralizing antibodies to prevent HIV infection. Indeed, passive administration of neutralizing antibodies has been shown to protect against HIV challenge in animal models.

Although most infected individuals develop a neutralizing antibody response, it usually cannot clear the infection because adaptation to the rapidly evolving escape variants of the virus lags behind. Understanding the mechanisms of protective antibody responses in infected individuals and especially the development of neutralizing antibodies to viral escape mutants is therefore of particular importance. The overall aim of this study is the investigation of how HIV specific B cell responses to viral escape mutants are induced by newly emerging virus quasispecies.

In initial experiments, methods for the isolation and characterization of HIV specific B cells were established. B cells isolated from HIV infected individuals in different disease stages were immortalized by Epstein Barr virus (EBV) transformation and their phenotype, function and antibody repertoire were analyzed. Furthermore, GFP labeled virions were used to identify and isolate HIV specific B cells from patients.

Student Talks

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ST 11 Donor CD40 is required for the IL-17 response to alloantigen Anna K Kraus, Pietro Cippa, Jin Chen, Rudolf P Wuethrich, Thomas Fehr Division of Nephrology, University Hospital Zurich, and Institute of Physiology, Univesity of Zurich [email protected]

Allo-reactive cytotoxic T lymphocytes (allo-CTLs) induce tubulointerstitial injury during kidney allograft rejection. CD40 is the only classical costimulatory molecule expressed on renal tubular epithelial cells (rTECs) under inflammatory conditions. Blockade of the CD40- and the CD28-pathways can induce long term allograft survival. However, it is still unclear whether CD40 blockade acts primarily on host or donor CD40. Moreover a role of CD40 on CD8 T cells for effective memory generation has been proposed. The aim of the study was to investigate the role of host and donor CD40 for direct T cell alloreactivity in vitro and in vivo.

Primary cultures of murine rTECs were prestimulated with IFN-β and IFN-γ to induce high surface expression of MHC and CD40. Responder T cells were restimulated in vitro with either autologous or allogenic fully MHC-missmatched splenocytes. Proliferation was measured by thymidine incorporation. Cytotoxicity was tested against rTECs from WT or CD40-/- mice using a 51Cr release assay. Cytokine analysis of the coculture supernatants was performed with standard ELISA assays. To assess the role of donor CD40 in vivo, fully MHC-missmatched skin grafts from WT and CD40-/- donors were performed with or without CD4 T cell depletion.

When CD8 and CD4 T cells were cocultured, the absence donor CD40 does reduce proliferation, IFN-γ production, and cytotoxicity. IL-17 production was completely abolished under these conditions. When CD8 T cells were stimulated without CD4 help, no cytotocicity was detected. Proliferation and IFN-γ production were overall reduced and even less, if stimulators lacked CD40. CD40expression on target cells or not did not have an influence on cytotoxicity.

We observed a prolongation of skin graft survival, if CD4 T cells were depleted, but the expression of CD40 on the donor skin did not have an


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impact on the time course of rejection. Kidney grafts from CD40-/- donor mice are currently performed.

No impact of CD40 expression on responder T cells in vitro could be detected. We will further investigate the role of reicipient CD40 for an effective generation of T cell memory.

The absence of donor CD40 during allostimulation in vitro reduces all effector functions of CTLs whereas the expression of CD40 on the target cells does not have an impact on cytotoxicity and skin graft rejection. IL-17 production of alloreactive CD4 T cells is dependent on donor CD40. Thus, donor CD40 blockade might be therapeutically useful to prevent kidney allograft rejection.

Poster Abstracts

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P 16 Influence of inflammation-induced changes on bacterial growth in the intestinal ecosystem Lisa Maier, Prof. Wolf-Dietrich Hardt Institute of Microbiology, ETH Zurich [email protected]

Gastrointestinal infections are a serious global health problem with 1.8 million victims per year according to the WHO. Severe bacterial diarrhea caused by Salmonella spp. infections mostly occurs in developing countries and is notably dangerous in young children and HIV patients. In addition to the innate and adaptive immune system, the commensal microflora populating the intestine at high density protects against those kinds of infections. This phenomenon is known as colonization resistance (CR). The enteric pathogen Salmonella enterica spp. I serovar Typhimurium (S. Tm) overcomes CR and out-competes most of the anaerobic commensal microflora by the induction of gut inflammation. Although it has been assumed that only pathogens can profit from gut inflammation, it was recently observed in our lab that certain commensal E. coli strains can also benefit from these conditions. Moreover, these E. coli strains (e.g. E. coli 8178) inhibited the pathogen S. Tm in a mouse model for acute Salmonella-induced colitis. Strikingly, E. coli 8178 was able to suppress intestinal growth of S. Tm more effectively than the current standard probiotic used for treatment of human Salmonellosis, E. coli Mutaflor (MF). The mechanisms of this inhibition as well as the molecular basis of inflammation-induced overgrowth in the gut are unclear.

The aim of this study is to identify and characterize genes of competitive E. coli (=competition factors) required for overgrowth and pathogen inhibition in the inflamed gut. In order to identify competition factors, a genetic screening method (transposon-mediated differential hybridization; TMDH) will be adapted.

These findings might help to improve probiotic E. coli MF for treatment and prophylaxis measures of human Salmonella infections and may lead to the design of therapeutic agents that specifically block or inhibit overgrowing E. coli in human inflammatory bowel disease.


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P 15 Functional characterisation of essential oligosaccharyltransferase subunits in Saccharomyces cerevisiae Jörg Breitling, Markus Aebi Institute of Microbiology, ETH Zurich [email protected]

N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is one of the most abundant posttranslational modifications. The transfer of the lipid-linked oligosaccharide precursors to specific asparagine residues of a nascent polypeptide chain is catalysed by the oligosaccharyltransferase (OTase). In higher eukaryotes the OTase is an oligomeric complex composed of up to eight subunits. Although the catalytic subunit has been identified the function of most of the other subunits of the OTase complex is still unclear. This study is aimed to elucidate the function of the essential OTase subunits OST1 and WBP1 in the yeast Saccharomyces cerevisiae. To this end a screening of a mutant library of OST1 and WBP1 was performed. Mutants of OST1 and WBP1 have been identified which impair growth of yeast cells in a dominant negative way and whose growth suppression effect is OTase dependent. The influence of these mutants on glycosylation of various target proteins and OTase complex formation is currently under investigation.

Student Talks

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ST 12 Influence of human cytomegalovirus infection on suppressor of cytokine signaling expression Olmo Sonzogni 1, Maddalena Ghielmetti 1, Lea Häberli 1, Anne-Laure Millard 1, Mårten Schneider 2, Roberto Speck 1, Regina Miller 1, Joerg D. Seebach 3, Marek Fischer 1, Nicolas J. Mueller 1 1Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Switzerland, 2Laboratory for Transplantation Immunology, 3Service of Clinical Immunology and Allergology, Department of Internal Medicine, University Hospital and Medical Faculty, Geneva Contact: [email protected]

Suppressor of cytokine signaling proteins (SOCS) are known to act as attenuators of inflammation. When a cytokine binds to its receptor, activation of the JAK/STAT pathway leads not only to the transcription of pro-inflammatory target genes like tumor necrosis factor α (TNFα) and interferon γ (IFNγ), but also to SOCS. It is well accepted that SOCS proteins then interfere directly with JAK and preventing phosphorylation of the cytoplasmic part of the receptor, or compete with STAT for the place on the phosphorylated residues. Some viruses like the herpes simplex virus 1 (HSV-1), are known to upregulate the expression of SOCS proteins, especially SOCS1 and SOCS3, and use these proteins to their advantage in their pathogenicity. The human cytomegalovirus (HCMV) belongs to the ß-herpes family virus and is close related to HSV-1. Infection of HCMV is usually asymptomatic but it is of major concern in immunocompromised patients. The aim of this study is to investigate the effect of HCMV infection on the expression of SOCS1 and SOCS3.

After infection of human endothelial cells (hEC), real time PCR analysis showed a 12-fold upregulation of SOCS1 mRNA expression 10 hours post infection (h p.i.) when compared to non-infected cells. In contrast, no modulation seemed to occur for SOCS3. As it was recently shown that HCMV can also productively infect porcine endothelial cells (pEC), we investigated the expression of SOCS1 and SOCS3 also in HCMV-infected pEC. In these cells, SOCS1 mRNA expression was 10 times higher 24h p.i than in non-infected cultures. In contrast SOCS3 mRNA expression again did not seem to be modulated by the infection.


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Investigations on the effect of HCMV infection on the protein levels of SOCS1 and SOCS 3 by western blot is currently ongoing, with specific antibodies against these proteins in both the human and the porcine settings. Further studies concerning the expression of SOCS1 and SOCS 3 in HCMV-infected EC will support a better understanding of SOCS-mediated control of inflammatory responses. Such findings could enable the development of novel approaches to reduce the damages due to HCMV infection in immunocompromised patients.

Poster Abstracts

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P 14 Metabolic characterization of the facultative methylotroph Methylobacterium extorquens AM1 on a C2 substrate

Kathrin Schneider, Remi Peyraud, Philipp Christen, Nathanael Delmotte, Patrick Kiefer, Stephane Massou, Jean-Charles Portais, Julia Vorholt


Methylobacetrium extorquens AM1 represents an important model

organism to study microbial growth on reduced one-carbon (C1) compounds; the genus colonizes the plant phyllosphere, where methanol but also other carbon substrates serve as carbon an energy source. For understanding in situ growth conditions in phyllosphere it is essential to have knowledge of metabolic characteristics on substrates other than C1. Many multi-carbon sources such as fatty acids, terpenes, waxes, and alcohols might be available on plants and are metabolized via the C2 intermediate acetyl-CoA. The first step in understanding how these substrates are metabolized is to characterize the metabolism for M. extorquens AM1 on acetate. In addition, acetate itself is a carbon source available in natural habitats; it is an end product in fermentation processes during degradation of complex compounds. To show which pathways are operating during growth on substrates metabolized via acetyl-CoA, we chose acetate as model compound. The elucidation of the network topology for growth on acetate for the isocitrate-lyase negative organism was performed by metabolome analysis and 13-C labeling experiments. 13C-based NMR flux analysis was used to elucidate the contribution of pathways and reveals overlap and differences in network operation in central metabolism relative to growth in the presence of methanol as sole carbon and energy source. Proteomic data were used to underline the operation of the proposed metabolic network.


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P 13 HIV-1 entry and its inhibition Axel Mann, Alexandra Trkola Institute of Medical Virology, University of Zurich [email protected]

Virus entry into target cells is the first step in the viral life-cycle and hence an important checkpoint for interference with viral infection by the host immune defense, vaccines and pharmaceutical agents.

Human Immunodeficiency Virus 1 (HIV-1) entry is mediated by the viral envelope glyco-proteins gp41 and gp120 and is initiated through the interaction of gp120 with the target cell CD4 receptor. A following cascade of conformational changes within the envelope glyco-proteins, involving engagement of a co-receptor, leads to fusion of the viral and cellular membranes and productive infection.

The overall aim of this thesis project is to dissect the modes by which antibodies and antiviral agents can inhibit viral entry. To this end a more detailed understanding of the cellular and viral moieties involved in viral entry is required, in order to gain new insights into the viral entry mechanism itself and to define new epitopes for neutralizing antibodies and entry inhibitors. Critical components that determine the efficacy of viral entry, both within the viral particle as well as properties of the target cell, are studied in various in vitro infection systems.

With the aim of gaining a more detailed concept of virus surface glycoprotein function, DARPin binding proteins specific for the viral envelope proteins are selected using ribosome display. Identified binders will be used to map epitopes on surface glyco-proteins that are critical for virus infectivity and are thus targets for future vaccines and drugs.

Student Talks

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ST 13 Drosophila melanogaster: a new infection host for assessing the pathogenicity of Burkholderia cepacia complex (Bcc) strains Stephan Schwager, Susanne Uehlinger and Leo Eberl

Department of Microbiology, Institute of Plant Biology, University of Zurich, Zurich, Switzerland Contact: Stephan Schwager, [email protected]

Species of the Burkholderia cepacia complex (Bcc) can cause disease in plants, and can establish opportunistic infection diseases in animals or in humans affected by immunodeficiency, chronically granulomatose or cystic fibrosis. However the mechanisms for virulence factors are poorly understood.

Recent advances in virulence factor identification have increased the demand for assessment of the pathogenic potential of Burkholderia strains, and hence there is a need for appropriate infection models. In previous studies different infection hosts, including mammals, nematodes, insects and plants, have been used. In this study we evaluated the fruit fly Drosophila melanogaster as a model host for studying Burkholderia sp. infection. The fly pricking model, intended to represent an acute infection, involves pricking flies with a needle inoculated with bacterial culture.

We compared the pathogenicity of Bcc strains in the fly model with that in two other infection models, namely Caenorhabditis elegans and Galleria mellonella. We also tested 27 transposon mutants, which were found to be attenuated in the C. elegans infection model, in the D. melanogaster pricking model. We found that several mutants strongly attenuated in the C. elegans model were also less virulent in the D. melanogaster pricking model. Mutants strongly attenuated in the G. mellonella model were generally attenuated in the D. melanogaster pricking model too.

The D. melanogaster model was found to give similar results to at least one of the two established models (C. elegans and G. mellonella) for each strain tested, which shows the potential of this model for the study of B. cepacia infection. The new D. melanogaster model is a very rapid, simple and efficient tool to test the pathogenicity of the Bcc.


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ST 14 Antibody-Mediated Control of Helminth-Induced Basophilia Tina Herbst†, Moria Prati†, Ben Marsland# and Nicola Harris*§ † Microbiology, Institute of Biology, Swiss Federal Institute of Technology, ETH, Zürich, # Service de pneumologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, §Swiss Vaccine Research, Global Health Institute, École Polytechnique Fédèrale de Lausanne, Switzerland.

Basophils represent a small subset of granulocytes and represent 0.01% to 0.3% of the white blood cells in a healthy individual. Basophils act in inflammatory responses and are known to play a role in both parasite infections and allergies. More recently basophils were reported to play an important role as antigen presenting cells capable to provoke Th2 immune response, and as effector cells important for protective immunity against helminth parasites. We now demonstrate an essential role for antibodies in promoting increased basophil production following infection with the murine helminth Heligmosoides polygyrus. Our data shows that helminth-induced basophilia requires the production of isotype-switched antibodies, most likely IgG or IgE. These antibodies promote basophil production in the bone marrow and are therefore essential for helminth-induced increases in blood and tissue basophil numbers. In contrast, Th2 cell differentiation and IL-3 production were not altered by the absence of isotype class switch recombination. These findings demonstrate that helminth-induced basophilia requires CD4+ T cell-dependent antibody production but occurs independently of CD4+ T cell cytokine production.

Poster Abstracts

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glucose2-glycans are present at different steps of synthesis and processing of oligosaccharides in the ER, it is important to specify in which step malectin is acting. Therefore, we want to determine and analyze binding partners of human malectin in the ER with co-immunoprecipitation and mass spectrometry.


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P 12 Protein Homeostasis Control in the Endoplasmic Reticulum Claudia Otto, Robert Gauss, Markus Aebi Institute of Microbiology, ETH Zurich [email protected]

Proteins that are either secreted or populate cellular membranes transit through the secretory pathway. The endoplasmic reticulum (ER) is the entry port to this pathway. Most proteins synthesized in the ER are glycoproteins that carry N-linked glycans. These glycans are co-translationally attached to an asparagine residue in the sequon asparagine-x-serine/threonine (x≠proline) and have an important role in quality control of the protein.

The precursor oligosaccharide is assembled on dolichol-pyrophosphate and transferred en block by oligosaccharyltransferase to nascent protein when the peptide chain enters the ER through the translocon complex. Once this N-glycan is transferred to the protein, it is immediately processed by different glucosidases and mannosidases in the ER lumen. The sequential removal of glucoses and mannoses generates defined intermediate structures that signal the folding state of the protein. The ER lumen contains several molecular chaperones, folding enzymes and lectins that can read the different glycan signals and appropriately assist folding of newly synthesized proteins. Taken together, these factors are known as ER quality control (ERQC). Proteins that pass the criteria of ERQC are transported to their site of action through the secretory pathway. If proteins fail to obtain their native folding state or if single subunits of multimeric complexes cannot assemble in a specific time frame, they are degraded via the ER-associated protein degradation (ERAD) pathway to maintain protein homeostasis in the ER.

In order to evaluate how the ER regulates its protein homeostasis we want to quantify and analyze the general turnover of ER proteins by a pulse chase - SILAC approach combined with multiple reaction monitoring (MRM). This will give us insights into the general flux of proteins through the ER and how the flux is altered under stress conditions. It will also give information about the half-life of ER chaperones. Recently, a novel ER-lectin named malectin was identified that specifically binds to glycans containing two glucose residues. To date, its function is completely unknown. As

Poster Abstracts

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Poster Abstracts


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P 01 Control of Bronchial Epithelial Cell Tight Junctions by Regulatory T cells and IL-35 Kerstin Wanke, Maciek Chalubinski, Paulina Wawrzyniak, Cezmi A.Akdis Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos [email protected]

In allergic asthma the nature of the interplay between the epithelium and resident T-cells is of great importance. Bronchial epithelial cells on the one hand represent a barrier that confers protectionand on the other hand play akey role in asthma associated lung inflammation. Factors that contribute to loss of integrity of this barrier may contribute to both, resultion of inflammation by trainage of inflammatory cells and inflammatory mediators and increase of inflammation by leakines for environmental factors, pro-inflammatory mediators and allergens. Responsible for the integrity of epithelial cells are the tight junction complexes. They form very close cell-cell contacts and control cell integrity, cell polarity and paracellular flow of molecules and cells. We intend to investigate the control of tight junctions by regulatory T cells (Tregs) and its cytokines. In previous findings we could show that the recently described T regulatory cell associated cytokine IL-35 reduced transepithelial resistance (TER) and led to stratifications of adjacent complexes, whereas neither IL-10 nor TGFbeta affected tight junctional complexes in air-liqud interface cultures (ALI) of human bronchial epithelial cells.

Here, we report that T regulatory cells were also able to reduce TER in ALI in a cell-cell contact independent manner. While reducing TER, mRNA levels of tight junctional proteins stayed stable. The findings for IL-35 and T regulatory cells were confirmed via FACS and confocal microscopy, which demonstrated stable protein expression of tight junction proteins accompanied by opening of tight junctions an therefore increasing epithelial permeability. An increase in mRNA levels of tight junction proteins after incubation with either IL-35 or Tregswas observed in cocultures with subconfluent monolayers of epithelial cells. We are currently focusing on mice models of asthma and Treg cell adoptive transfer models to further demonstrate the in vivo relevance of the findings.

Poster Abstracts

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P 11 Controlled displacement of cells by FluidFM technology Philipp Stiefel, Julia Vorholt Institute of Microbiology, ETH Zurich [email protected]

The Fluidic Force Microscope (FluidFM) is a novel instrument developed in the Laboratory of Biosensors and Bioelectronics of ETH Zurich. It combines the precise force-controlled positioning of an atomic force microscope (AFM) with nanofluidics. The AFM cantilever is converted into a syringe containing a microchannel through which fluids can flow from or to the tip. Different designs of the cantilever have been engineered to address specific experiments.

In the presented work cantilevers with a blunt aperture were used to spatially manipulate different types of cells (mammalian cells, yeast and bacteria). Thereby, for example yeast cells were moved to specific positions under liquid or from liquid onto solid agar. The force feedback, which is achieved by a laser pointing on the cantilever tip, allows a very gentle positioning onto the cell to protect it from being damaged. Additionally, during the manipulation procedure the force exerted to the cantilever can be recorded, which for example allows us to determine the force used to detach an adherent mammalian cell.

These results demonstrate that the FluidFM is a useful instrument (i) to pick up a selected cell and place it to a specific location and (ii) to precisely measure adhesion forces.


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P 10 Harnessing the Power of the Micro Deepa Mohanan, TM. Kündig, P. Johansen Department of Dermatology, University Hospital Zurich [email protected]

Allergen-specific immunotherapy (SIT) has been around for almost a hundred years. However, it’s only recently that we have obtained a better understanding of the immunological actions of SIT, which is effective by modulating T-cell responses and the inducing allergen neutralising IgG antibodies. There are several problems associated with conventional subcutaneous SIT and these include, duration of treatment, severe side effects, low clinical efficacy of conventional SIT for food allergens and some pollens, and the route of administration. Thus, there is an increasing interest in improving SIT vaccines. Microspheres are novel pharmaceutical dosage forms that through their particulate nature, act either directly by targeting the cells of the immune systems or indirectly by acting as a depot for the entrapped allergen. Thus the aim of this study is to improve SIT by harnessing the power of microparticles and to characterise the mechanisms by which they are able to do so. We will first setup the murine asthmatic model and then treat these animals using the conventional treatment model. Once we have established that, we will then establish a treatment protocol using antigen-loaded microspheres in asthmatic mice followed by further refinement studies of this treatment which includes surface modification of particles and administration routes. Finally, to better understand the mechanisms by which microparticles are enhancing SIT, we will then look at in vivo trafficking of these particles from the site of administration as well as the crucial roles different antigen presenting cells (APCs) by using conditional deletion of certain subsets of APCs.

Poster Abstracts

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In conclusion, we provide first evidence for a key role of T-regulatory cells in controlling bronchial epithelial cell integrity most likeyl via the recently described cytokine IL-35.


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P 02 Reactions of plants to bacterial volatiles: from love to hatred * D. Blom, * C. Fabbri, ** E. Connor, ** F. Schiestl, *** T. Boller, * L. Eberl and * L. Weisskopf * Institute of Plant Biology, University of Zurich ** Institute of Systematic Botany, University of Zurich *** Institute of Plant Biology, University of Basel

Increasing evidence indicates that bacteria can interact with plants through the emission of volatile compounds. Here, we report the highly contrasting effects of volatiles from 45 rhizosphere strains on the model plant Arabidopsis thaliana. Plants and bacteria were grown for three weeks in a compartmental Petri dish assay. Effects of bacteria ranged from over 5-fold plant growth promotion (Pandoraea norimbergersis) to plant killing (Chromobacterium violaceum) and were, for each strain tested, strongly dependent on culture medium and bacterial density. This led us to investigate the effect of quorum-sensing on the volatile-mediated impact of bacteria on plants. Disrupting quorum-sensing in the plant growth promoting strain Burkholderia pyrocinia did not lead to reduced positive effects. In contrast, the deleterious impact of Chromobacterium violaceum and Pseudomonas aeruginosa was significantly lower in the respective quorum-sensing mutants. Further experiments using mutants unable to produce hydrogen cyanide (HCN) and external chemical supply of HCN to growing plants established this compound as the main candidate for the observed plant growth inhibition by bacterial volatiles. Cyanogenesis was found to be regulated by quorum-sensing in C. violaceum and P. aeruginosa, suggesting that the reduced virulence of quorum-sensing mutants on A. thaliana is caused by the loss of the ability to produce HCN.

Poster Abstracts

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P 09 Induction Kinetics of the Cell Wall Stress Stimulon of Staphylococcus aureus Using Different Antibiotics V. Dengler, R. Heusser, B. Berger-Bächi and N. McCallum Institute of Medical Microbiology, University of Zurich [email protected]

The emergence of methicillin resistant S. aureus (MRSA) that are able to acquire multiple resistance determinants severely restricts antibiotic therapy options. Antibiotic resistance is affected by several different global regulators of virulence factors, metabolism and stress responses. In S. aureus exposure to cell wall stress, such as cell wall active antibiotics, induces a set of gene known as the cell wall stress stimulon (CWSS). Deletion of VraRS, the two component system that regulates the CWSS gene expression, leads to decreased resistance to diverse cell wall antibiotics indicating a role of the stress response in various resistance phenotypes. To measure induction of the cell wall stress response we have constructed promoter-reporter (lacZ and luciferase) gene fusions to three members of the stimulon, sas016, sa0908 and tcaA. We analysed induction kinetics of the stress response using antibiotics that target different enzymatic steps or precursor molecules of cell wall synthesis. Maximal induction levels ranged from 3- to 65-fold when cells were stressed with minimal inhibitory concentrations (MIC) of antibiotics. Patterns of induction stimulated by the different types of antibiotics and the concentration dependency of induction also varied greatly. There appeared to be no direct correlation between antibiotic targets or their bactericidal efficiency with levels of induction of the protective cell wall stress response. Inactivation of VraR decreased resistance levels to CWSS-inducing antibiotics in varying degrees, with MIC values dropping from 2- to 16-fold. There seemed to be no links between the strength of CWSS induction by antibiotics and the impact of the CWSS on their respective resistance phenotype.


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P 08 The role of IL-17 in the early phase of OPC Kerstin Weidner, Salomé LeibundGut Institute of Microbiology, ETH Zurich

The fungus Candida albicans lives as part of the normal microflora in healthy individuals without triggering any harmful effects but it can cause severe forms of candidiasis in immunocompromised individuals such as HIV-positive patients. Immune-protective mechanisms have long been thought to be Th1-mediated. However, more recently IL-17 was recognized as one of the most critical factors protecting from Candidiasis. Whereas the source of IL-17 was first described as being a novel subset of T helper cells, Th17 cells, it is now clear that different innate cell populations can also secrete IL-17. Because C.albicans is cleared rapidly after primary pathogen-exposure in our mouse model of oropharyngeal Candidiasis (OPC), we suspect that the protective IL-17 is rather of an innate source than T-cell derived. The aim of this project is to unravel the detailed role of IL-17 in host protection from oropharyngeal Candidiasis. For this, the kinetics of IL-17 production in the infected organs will first be established on both the transcriptional and translational level. Second, the cellular source of IL-17 at different time points shall be identified with the help of IL-17 reporter mice. Correlating those results with the invasion of different types of immune cells into the infected tissues, a model for emergence and effects of IL17 during the early phase of an OPC shall be established.

Poster Abstracts

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P 03 Role of T cells and iNKT cells in Pseudomonas aeruginosa lung infection: Pascal Ziltener, Annette Oxenius Institute of Microbiology, ETH Zurich [email protected]

T cells and iNKT cells are innate immune cells localized preferentially at peripheral sites such as the lung mucosa. Pseudomonas aeruginosa is an opportunistic pathogen that causes severe pneumonia in immunocompromised people such as patients with cystic fibrosis. However, the involvement of T cells and iNKT cells in host defense against P. aeruginosa lung infection is poorly defined. We examined survival, the bacterial burden, cellular infiltration and cytokine responses in mice infected intranasally with P. aeruginosa. Survival was reduced TCR-/- mice, and bacterial clearance was delayed in both TCR-/- mice and J18-/- mice compared to WT. IL-1, TNF and IL-6 were increased in the lungs and BALF of TCR-/- mice, while J18-/- mice had reduced IL-1 in the lungs and reduced TNF and IFN in the BALF, suggesting an anti-inflammatory and a pro-inflammatory role for T cells and iNKT cells 24 hours post infection, respectively.


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P 04 Modelling the whole scope of antiretroviral therapy in humanized NOG mice Marc Nischang1, Stefan Baenziger1, Ursula Hofer1, Erika Schlaepfer1, Stephan Regenass2, Guenter Kraus3, Roger Sutmuller3, Roberto Speck1

1Division of Infectious Diseases, 2Institute of Clinical Immunology, University Hospital Zurich, Switzerland, and 3Tibotec BVBA, Mechelen, Belgium

Even though established antiretroviral treatment are very potent in inhibiting HIV replication, many open questions and unsolved problems remain− What is the best treatment strategy to prevent transmissions? Is it possible to eradicate the virus completely? One step towards resolving these issues is the establishment of a simple and robust small animal model.

We evaluated the potential of modelling suppressive treatment, pre- and post-exposure prophylaxis in humanized mice. Newborn NOG mice were transplanted with human cord blood hematopoietic stem cells and these humanized mice then were challenged with HIV YU-2 by intraperitoneal injection of 106 TCID50. Four weeks later we verified infection by measuring plasma viral load and started a triple ART using food pellets containing antiretrovirals. After six weeks of treatment 14 of 21 mice showed viral loads below detection limits, and in most of the other animals’ viral loads were reduced by several logs. In contrast all control mice showed an increase in HIV RNA viral load over time. For the PrEP scheme mice received a single application of a novel long acting drug at different time points before HIV challenge. The PEP scheme consisted of treatment with the same drug at different time points after challenge, continued by weekly applications for 30 days. In both schemes viral load was analysed 30 days after stop of treatment. 3 of 5 (PrEP) and 7 of 7 (PEP) untreated control animals showed viral replication. In the PrEP setup animals receiving therapy one day before HIV exposure were completely protected from infection (0 of 5 animals infected). PEP was effective early after challenge. 0 of 7 animals were infected if the drug was applied immediately (+0d), at later time points the protective effect gradually declined. Pharmacokinetic analysis in the humanized mice indicated that the antiretroviral drugs were present at sufficient concentrations.

In conclusion, humanized NOG mice are a valuable tool to investigate antiretroviral therapy strategies. By suppressing viral replication in the humanized mice, we now have a model to study HIV latency and

Poster Abstracts

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reveal new therapeutic targets in T cell-mediated inflammatory CNS diseases such as MS.


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P 07 Autophagy-mediated antigen presentation in central nervous system autoimmunity: Christina Sina, Jan Lünemann Institute of Experimental Immunology, University Hospital Zurich [email protected]

The emergence, activation and expansion of CD4+ autoreactive T cells are considered crucial events in the development of multiple sclerosis (MS), a common autoimmune disease of the central nervous system (CNS). The mechanisms mediating autoreactive CD4+ T cell activation as well as relevant autoantigens towards which autoreactive CD4+ T cells are directed in MS are incompletely characterized.

Previous studies could demonstrate constitutive autophagosome formation in MHC class II-positive cells, including dendritic, B cells and epithelial cells. In these cells, autophagosomes continuously fused with multivesicular MHC class II-loading compartments and targeting of antigen to autophagosomes via fusion to the autophagosome-associated protein Atg8/LC3 led to strongly enhanced MHC class II presentation to CD4+ T cells. These data show that macroautophagy constitutively and efficiently delivers cytosolic proteins for MHC class II presentation. Notably, inflammatory stimuli such as proinflammatory cytokines and TLR agonists upregulate macroautophagy in antigen-presenting cells suggesting that macroautophagy-mediated antigen processing could particularly enhance immune responses within inflamed tissues.

We hypothesize that macroautophagy-dependent presentation of autoantigens facilitates the activation of autoreactive T cells within the CNS. We will monitor macroautophagy in reporter mice during the course of experimental autoimmune encephalomyelitis (EAE), an antigen-driven CD4+ T cell mediated animal model of MS. We will next use conditional gene targeting in mice to investigate whether macroautophagy in professional antigen presenting cells influences the development and/or progression of EAE. In order to relate these findings to human disease, we will address whether cerebrospinal fluid-infiltrating CD4+ T cells in MS patients preferentially recognize substrates of macroautophagy loaded onto MHC class II molecules.

These studies are expected to provide insights into an as yet unexplored potential mechanism of autoimmune CNS inflammation and may

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possible eradication approaches. Furthermore, long-acting anti-HIV compounds may be a valuable addition to the existing ART.


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P 05 Characterization of C.albicans specific T helper cell responses: André Gladiator, Salomé LeibundGut Institute of Microbiology, ETH Zurich [email protected]

Fungal infections gained increasing clinical relevance during the last decades. Among the causative agents of such infections is the ubiquitous fungus Candida albicans. As a commensal it colonizes skin and mucosal surfaces. Healthy individuals develop stable interactions with the fungus but in the course of immunodeficiency (e.g. AIDS or immunosuppressive treatment related to organ transplantation) it can convert to an opportunistic pathogen and may cause different forms of candidasis as well as lethal systemic infections.

Immune responses to fungal infections are complex and incompletely understood as they strongly depend on both the innate and adaptive branch of the immune system.As evidenced from HIV-infected individuals, CD4+ T cells play a particularly important role for protection of disease. Therefore this project aims at characterizing C. albicans-specific T-cells with regard to their activation and expansion properties, their effector functions as well as their protective capacity. Furthermore the project aims at cloning the T-cell receptor (TCR) of these C. albicans-specific T-cells in order produce a transgenic mouse that harbors T cells only expressing the C. albicans-specific TCR. This will constitute a powerful tool to analyze monoclonal T-cell responses in detail and additionally facilitate the search for so far unknown C. albicans-derived immunodominant T cell epitopes.

Finally the results from this project shall increase the understanding of immune responses to fungal infections and may all together offer valuable insights for the development of new anti-fungal therapy.

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P 06 Identification of Plant-Probiotic Bacteria in the Phyllosphere Gerd Innerebner, Claudia Knief, Julia A. Vorholt Institute of Microbiology, ETH Zurich [email protected]

Aerial plant surfaces are colonized by diverse microorganisms called epiphytes. Among these inhabitants of the phyllosphere, certain groups of bacteria have been systematically found in association with plants. Recently, we identified members of the Alphaproteobacteria, namely Methylobacterium spp. and Sphingomonas spp., to be highly abundant on healthy field-grown clover, soybean, and Arabidopsis thaliana plants. However, their function in the leaf-surface ecosystem is largely unknown and we hypothesized a possible role as “gate keeper” bacteria to protect plants against pathogens. In order to investigate this, we set up in-vivo competition experiments using plant-surface isolates as biocontrol strains, Pseudomonas syringae pv. tomato (Pst) as pathogen, and Arabidopsis thaliana as model plant. We could show that some of the commensal bacteria tested indeed protect plants when infected with P. syringae and other pathogens by keeping Pseudomonas cell numbers low and by delaying disease symptoms. Additionally, we observed plant growth-promoting activity of these epiphytic bacteria in germination assays with tobacco seedlings. Future experiments are aiming towards a more detailed understanding of the interactions between plants, beneficial bacteria, and pathogens.

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3th Student Retreat Microbiology and Immunology PhD Program · 2015-04-23 · Microbiology and Immunology PhD Program of the Lifescience Zurich Graduate School. This year the retreat