Research Report 2015 Contents

Foreword ��5

Medical Theoretical Research Units

Biocenter Medical Biochemistry ��8 Neurobiochemistry ��12 Clinical Biochemistry ��14 Biological Chemistry ��16 Cell Biology ��18 Genomics and RNomics ��22 Molecular Biology ��24 Experimental Pathophysiology and Immunology ��28 Molecular Pathophysiology ��30 Developmental Immunology ��32 Bioinformatics ��34

Department of Physiology and Medical Physics Physiology ��36 Biomedical Physics ��40

Department of Medical Genetics, Molecular and Clinical Pharmacology Cell Genetics ��42 Genetic Epidemiology ��44 Human Genetics ��46 Biochemical Pharmacology ��50 Molecular and Cellular Pharmacology ��52

Department of Anatomy, Histology and Embryology Clinical and Functional Anatomy ��54 Neuroanatomy ��56 Histology and Embryology ��58

Department of Hygiene, Microbiology and Social Medicine Hygiene and Medical Microbiology ��60 Virology ��64 Social Medicine ��66

Institute of Pharmacology ��68

Department of Medical Statistics, Informatics and Health Economics Medical Statistics and Informatics ��70 Health Economics ��74

Department of Pathology General Pathology ��76

Institute of Legal Medicine ��78

Clinical Research Units

Center of Operative Medicine Visceral, Transplant and Thoracic Surgery ��82 Cardiac Surgery ��86 Vascular Surgery ��90 Plastic, Reconstructive and Aesthetic Surgery ��92 Trauma Surgery ��94 Urology ��96 Orthopeadic Surgery ��98 Anaesthesiology and Critical Care Medicine General and Surgical Critical Care Medicine �� 102

2 Research Report 2015 Medical University of Innsbruck Center of Internal Medicine Internal Medicine I �� 104 Internal Medicine II �� 108 Internal Medicine III ��112 Internal Medicine IV ��116 Internal Medicine V �� 120 Internal Medicine VI �� 124 Joint Institution for Emergency Medicine and Critical Care Medicine �� 128

Center of Psychiatry and Pychotherapy General and Social Psychiatry �� 132 Biological Psychiatry �� 134 Psychosomatic Medicine ��136 Medical Psychology �� 138 Child and Adolescent Psychiatry �� 140

Center of Neurology and Neurosurgery Neurology �� 142 Neurosurgery ��146

Center of Obstetrics and Gynecology Obstetrics and Gynecology �� 148 Gynecological Endocrinology and Reproductive Medicine �� 152

Center of Otorhinolaryngology and Hearing, Speech and Voice Disorders Otorhinolaryngology �� 154 Hearing, Speech and Voice Disorders ��156

Center of Diagnostic Radiology Diagnostic Radiology �� 158 Neuroradiology ��162

Center of Dentistry and CMF-Surgery Dental Prosthetics and Restorative Dentistry ��166 Orthodontics ��168 Cranio-Maxillo-Facial and Oral Surgery �� 170

Centre of Pediatrics Pediatrics I �� 174 Pediatrics II �� 178

Department of Nuclear Medicine �� 180

Department of Therapeutic Radiology and Oncology �� 184

Department of Dermatology and Venereology �� 188

Department of Ophthalmology and Optometry ��190

Women’s Health Center ��192

Institute for Neuroscience ��194

FWF–funded Programmes

Molecular Cell Biology and Oncology – DK MCBO ��198 Host Response in Opportunistic Infections – DK HOROS �� 200 Signal Processing in Neurons – DK SPIN �� 202 SFB-F44–Cell Signaling in Chronic CNS Disorders �� 204

Core Facility Net �� 207

Research Report 2015 Medical University of Innsbruck 3 4 Foreword

Dear readers,

it is a great pleasure for me to present the first This common project of the western Austrian research report of the Medical University of Innsbruck Universities and associates (Leopold Franzens Uni- (MUI), detailing our research activities in 2013–2014. versity, Innsbruck; Paris Lodron University, Salzburg; Mozarteum, Salzburg; Johannes Kepler University, Since its inception as an independent University in Linz; Universität für künstlerische und industrielle 2004, MUI in collaboration with the Tiroler Kranken Gestaltung, Linz) is financed by the FederalMinistry anstalten (TILAK GmbH, now Tirol Kliniken) has of Science, Research and Economy (BMWFW). Two made a deep commitment to fostering research of the primary intentions of the knowledge centre innovation and excellence with the aim of creating are to simplify the search for university based knowledge to advance health. cooperation partners and to facilitate the rapid launching of projects. The project resulted in the In the past years MUI has continued to built on its realisation of a research report and competence established research strengths namely genetics, map. epigenetics and genomics, as well as infectology, immunology and organ/tissue replacement, neuro The following pages present the profiles and sciences and oncology. With almost € 70 million results for each of the MUI’s clinical departments external research funding (2013/2014), including and institutes and underline the discoveries and three FWF funded doctoral programmes (HOROS, advances which have come about thanks to the SPIN, MCBO), two special research programmes enormous effort of our scientists. (SFB-021 “Cell Proliferation and Cell Death in Tumors” and SFB-F44 “Cell Signaling in chronic I would like to take this opportunity to thank all the CNS disorders”), 30 EU Projects and the K1 Project scientists for their contribution and their continuing Oncotyrol, the K Project VASCage and two Christian efforts in the name of our University. Doppler labs (approved in 2014), the MUI also has Furthermore I would like to thank all the people in- earned a well-deserved reputation among physi- volved in putting this report together and hope that cians and scientists as a place where faculty and you will enjoy reading our first research report. students can pursue creative and novel ideas in a collaborative and supportive environment.

The current research report was created as part of a Univ.-Prof.in Dr.in Christine Bandtlow project of the Knowledge Transfer Centre West (WTZ) . Vice Rector for Research and International Relations

Research Report 2015 Medical University of Innsbruck 5 6 Research Report 2015 Medical University of Innsbruck Medical Theoretical Research Units

Research Report 2015 Medical University of Innsbruck 7 Biocenter Medical Biochemistry

differentiation processes are essential for by binding of a positive regulatory sub growth, development and integrity of multi unit, the cyclin. Sequential activation and cellular organisms. Before cells commit to inactivation of specific CDKcomplexes divide, they are exposed to a flood of diverse is required for cell cycle progression. and sometimes conflicting signals aimed p21 (CDK-interacting protein, Cip1) p27 to regulate cell growth, differentiation, cell (Kinase inhibitory protein, Kip1) and p57 proliferation or cell fate. Multiple external (Kip2) constitute one out of two families as well as internal signals can impinge on of CDK inhibitors (CKI) that bind to CDKs the central cell cycle control machinery in and control their activity. Their expression, order to promote or block cell proliferation. localisation and modifications play a cen- All signals need to be properly processed tral role in regulating CDK kinase activity and integrated to maintain body and organ especially in G1-phase and the decision homeostasis. Incorrect signal interpreta- between proliferation and cell cycle exit. In tion, processing or integration can lead to addition to their canonical function in CDK hypo- or hyperproliferative disorders, in- kinase regulation, they can also exert CDK cluding diseases like cancer or anaemia. independent functions. For example, the CDK inhibitor p27 can regulate cell motility The decision to continue proliferation or to and cell migration, linking this tumour sup- Head of Division: exit from the cell cycle into quiescence is pressor protein not only to hyperprolifera- Univ.-Prof. Dr. Ludger Hengst usually made during a specific window of the tion but also to cancer metastasis. eukaryotic cell division cycle. The cell cycle Contact: can be subdivided into four phases. DNA Among others, we identified the CDK Biocenter, Innrain 80–82 replication during S-phase is separated by inhibitor protein p27Kip1. Its activity, 6020 Innsbruck gap phases G1 and G2 from the segregation localisation or stability can be regulated of the duplicated DNA and other cellular by diverse mitogen signalling pathways. [email protected] components in M-phase (mitosis and cy- We investigate how these pathways control Phone: +43 512 9003 70110 tokinesis). Cells can decide to withdraw Cip/Kip family protein expression, localiza- Fax: +43 512 9003 73130 from proliferation or to commit to another tion, modification, activity or function and https://www.i-med.ac.at/imcbc/ round of cell division until they progress study their physiological roles in normal medclinchemfolder/medclinchem.html over the restriction point, a specific point cells and cancer cells. in G1 phase (Fig. 1). Progression over the p27 regulates cell cycle progression over restriction point renders the cell cycle mito- the restriction point. Abundant p27 binds Keywords gen independent and committed to another and inactivates CDKs and can prevent cell round of cell division. proliferation. The CDK inhibitor protein Cell cycle, cell proliferation, signal trans becomes unstable upon cyclin / CDK2 duction, ribosom, phospho-dynamics We investigate molecular mechanisms activation, as cells traverse the restriction that link diverse signalling networks to the point and progress towards S-phase. A pos- Research Focus central cell cycle control machinery. At the itive feedback loop couples p27 ubiquitin- core of this machinery is a conserved family dependent degradation to CDK activation • Cell Cycle & Proliferation (Ludger Hengst) of protein kinases, called cyclin-dependent (Fig. 2). The molecular mechanism that can • Signal Transduction & Proliferation kinases (CDKs). CDKs become activated initiate this feedback loop in presence of (Wolfgang Doppler) • Phospho-Dynamics (Peter Gruber) • Ribosomal Proteins (Wolfgang Piendl) • Eco- & Nutritional Biochemistry (F. Überall)

General Facts

Research in the Division of Medical Biochemistry is focused on signalling pathways regulating cell proliferation, apoptosis and differentiation in mammalian tissues with special reference to malignant cells. In addition, RNA-protein interactions are investigated with a focus on protein translation and ribosome function.

Research Fig. 1: Overview of the mammalian cell cycle. Cent-ral CDK/cyclin complexes are indicated Cell Cycle and Proliferation next to the cell cycle position when they are active and the Cip/Kip CDK inhibitors are Ludger Hengst shown next to CDK/Cyclin complexes, which they bind. The green arrow indicates that p21 Precisely coordinated cell division and and p27 are not only inhibitors but also acti-vators of cyclin D / CDKs.

8 Research Report 2015 Medical University of Innsbruck Medical Biochemistry

Fig. 2: A feedback loop controls CDK2 activation at the restriction Fig. 3: Oncogenic tyrosine kinases like JAK2, Src or BCR-Abl can point. Active CDK2 triggers the degradation of its own inhibitor p27, phosphorylate p27. This leads to the activation of bound CDK2. The promoting the switch-like activation of CDK2 kinase at the restric- activated CDK2 can phosphorylate the bound inhibitor p27, leading tion point. to its degradation and strong CDK activation. abundant CDK inhibitors and thus inactive This involves the selective degradation response, STAT1 prevents or restricts the CDKs has long remained a puzzle. of the ubiquitin ligase subunit Skp2 and development of spontaneously formed tum- We observed that oncogenic tyrosine results from inhibition of protein geranyl ors. However, particularly at later stages of kinases including BCR-Abl, Src or JAK2 geranylation. tumor development, where the anti-tumor can activate p27-bound CDKs by directly We also identified novel p27 mRNA binding immune response is blunted by the tumor phosphorylating the inhibitor. This tyrosine proteins that regulate the IRES- and and immune cells are frequently subvert- phosphorylation ejects an inhibitory helix Cap-dependent translation of p27 and ed to facilitate the growth and survival of of p27 from the catalytic cleft of the CDK investigated the role of p27 in apoptosis. the tumor, STAT1 can contribute to tumor- and permits the p27-bound CDK complex Current Research Projects: promoting effects. to bind ATP and to phosphorylate sub- Function and regulation of CDK-inhibitory Ongoing Research: strates. Among these substrates is p27 proteins. We are investigating the role of STAT1 in itself. Phosphorylation of p27 by the bound Role of translational control for the decision the infiltration, differentiation and biolog- CDK generates a phosphodegron which between cell proliferation and withdrawal ical function of tumor-infiltrating immune can initiate the ubiquitin-proteasomal from the cell cycle. cells. Our focus is on HER2-positive breast degradation of the CDK inhibitor (Fig. 3). Regulation of cytokine receptor signalling. cancer. We are particularly interested on Using this mechanism, that can be abused changes in the composition and function by diverse oncogenic tyrosine kinases, STAT1 in Cancer of tumor infiltrating immune cells upon mitogen signals can inactivate and de Wolfgang Doppler treatment by chemotherapeutic agents, stabilise the inhibitor p27 and thereby Strengthening a productive anti-tumor which act on the tumor epithelium but also promote CDK activation and cell cycle pro- immune response as well as suppressing influence the anti-tumor immune response. gression. Additional mechanisms include tumor-promoting activities of immune cells Major Achievements: translational control or involve regulation by represent important therapeutic options in We could demonstrate anti-tumor as well the ubiquitin proteasome system. cancer treatment. For the rational design as tumor promoting properties of STAT1 in Ongoing Research: of appropriate strategies to achieve these spontaneously growing mammary tumors. Regulation of cell cycle progression through goals, a more refined knowledge of the They are promoted by two different sub- G1 phase by tyrosine kinases, translation- mechanisms regulating the recruitment, sets of immune cells, namely CD8+ T al control in and of the cell cycle; tempo- differentiation, expansion and function of cells and tumor associated macrophages ral and spatial regulation of Cdk-inhibitory tumor-infiltrating immune cells is manda- (TAMs). CD8+ T cells contribute to the anti- proteins during cell cycle progression and tory. In this context, we investigate the role neoplastic activity of chemotherapeutic in apoptosis, regulation of ubiquitin ligase of a key mediator of the action of inter- agents, i.e. doxorubicin and lapatinib, and activity in G1, molecular mechanism of ferons on cells of the innate and acquired statin-induced cell cycle arrest, cell cycle immune system, the signal transducer and control by erythorpoietin and its receptor activator of transcription 1 (STAT1). It acts EpoR. Mouse knock-in models of cancer as a transcription factor to induce the ex- development. pression of genes required for antigen Major Achievements: processing, maturation and recruitment We discovered a novel mechanism that of immune effector cells, and of genes re- triggers p27 degradation, CDK activation quired for the antiviral defense. STAT1 also and cell cycle progression and identified co-operates with the cellular machinery different oncogenic tyrosine kinases in- regulating proliferation and apoptosis. cluding Src, BCR-Abl or JAK2, which induce In cancer, STAT1 has been shown to fulfill p27 tyrosine phosphorylation. We identi opposite roles in either promoting or imped- fied novel mechanism that control the ing tumor development, depending on the Fig. 4: Proposed role of STAT1, IFN-gam- localisation of p27. Recently, we elucidated stage of tumor development and the parma and the CXCR3 – binding chemokines the molecular mechanisms that induces ticular type of tumor: As a mediator of the CXCL9, CXCL10 and CXCL11 in the anti p27 stabilisation in the presence of statins. interferon-dependent anti-tumor immune tumor activity of doxo-rubicin and lapatinib.

Research Report 2015 Medical University of Innsbruck 9 Biocenter

this is critically dependent on STAT1. By Current Research Projects: this means, STAT1 serves in the anti-tumor We are exploring the mechanisms by which response. STAT1 was also shown to be re- STAT1 contributes to the chemotherapy- quired for the regeneration of the B-cell induced anti-tumor immune response. In compartment after doxorubicin induced particular, we are investigating the role of bone-marrow toxicity by promoting the the STAT1 target genes CXCL9, CXCL10 development of early B-cell precursors in and CXCL11 in the recruitment and the bone marrow. It is thereby important differentiation of CD8+ T cells. for the recovery of the B-cell lineage after treatment with this anti-cancer drug. On the Phospho-Dynamics other hand, STAT1 is positively influencing Peter Gruber the infiltration of mammary tumors with PKC is a family of serine/threonine TAMs by transcriptionally inducing the ex- specific protein kinases. The different PKC pression of the macrophage growth factor isozymes play important roles in diverse CSF1. We could show that intense local pro- signal transduction pathways. PKCepsilon liferation of fully differentiated macrophages has been reported to be enriched at the Fig. 6: Ribosomal protein L1 from the ar- rather then low-pace recruitment of blood- growth cones of extending neurites in chaeon Sulf-olobus acidocaldarius in com- borne precursors drives the accumulation differentiating neural cells, to participate plex with 23S rRNA of TAMs, which themselves are promoting in nerve growth factor signaling as well as tumor growth. The tumor promoting effect in transmitter release and to participate in stronger protein-RNA interaction might of STAT1 via influencing differentiation and cell death and survival. Since so far only substantially contribute to the thermal tol- infiltration of TAMs was supported by the very few substrates of PKCepsilon have erance of ribosomes in thermophilic organ- results of a retrospective study. There we been identified with reasonable certainty, isms. Our investigations are focusing on the could show an association of STAT1 mRNA the identification of PKC-downstream identification and characterization of those levels with macrophage infiltration and bad targets represents the next major area of structural features of RNA-binding proteins prognosis in breast cancer. research in this field. Since PKCepsilon is that modulate the affinity for their specific an important sensitizing kinase in the CNS RNA binding site. In this context we deter- and an attractiv e drug target for treatment mined the crystal structures of L1-rRNA and of several diseases, especially against pain L1-mRNA complexes at high resolution (in (hyperalgesia), addiction and anxiety dis collaboration with our Russian partners). orders, the discovery of additional drug targets through identification of PKCepsilon Function of Ribosomal Protein L1 substrates and the elucidation of signaling L1 is a two-domain protein with N and C pathways involved, might help to find novel termini located in domain I. In close collab- points of attack for therapeutic inter oration with a Russian group we succeeded vention and to manifest the importance of in constructing a truncation mutant of PKCepsilon in the nervous system. L1 representing domain I by deletion of Major Achievements: the central part of L1 (= domain II). We • Generation of novel PKCepsilon/RACK2 could demonstrate that domain I alone is interaction inhibitors sufficient forspecific RNA binding, where- • (Patent application: ‘Novel inhibitors of as domain II stabilizes the L1-23S rRNA Protein Kinase C epsilon signaling’) complex. • Identification of novel PKCepsilon sub- Major Achievements: strates Solution of the structure of the L1 protu- Fig. 5: X-ray structure of the C2-like domain Future Goals: berance in the ribosome (with the Russian of PKCepsilon. The protein backbone is il- • Identification of novel PKCepsilon sub- collaborator); see Fig. 6. lustrated as cartoon, while the protein seg- strates and elucidation of signaling Construction of a truncated mutant of ribo- ment EAVSLKPT (RACK2 binding domain on pathways involved somal protein L1 and elucidation of its role PKCepsilon and isozyme specific inhibitory • Further improvement of the potency of in RNA binding peptide) is highlighted in the yellow box. PKCepsilon/RACK2 protein-protein inter- Control of ribosomal protein synthesis in The pharmacophore model is aligned with action inhibitors mesophilic and thermophilic archaea the structure of a novel small molecule in- As bacteria and eukarya, archaea have hibitor. RACK2 (receptor for activated C Ribosomal Proteins to coordinate the synthesis of about 60 kinase 2) represents a protein that by bind- Wolfgang Piendl ribosomal proteins with each other and ing to PKCepsilon defines its final sorting Interaction of Ribosomal Proteins with with three rRNAs. Research is focusing on in the cell. By using inhibitory peptides or rRNA and mRNA the MvaL1 operon (encoding ribosomal similarily structured other small molecules, We are investigating ribosomal protein L1 proteins L1, L10 and L12) and on the MvaL3 the trans-location and thus the function of from different (hyper)thermophilic archaea operon (encoding 5 ribosomal proteins) PKCepsilon is inhibited. These tools are em- and bacteria. They exhibit a 10 to 100 from mesophilic and thermophilic Methano ployed for the identification of novel PKCep- fold higher affinity to their specificbinding coccus species. As in bacteria, regulation of silon substrates by quantitative phosphop- sites on rRNA and mRNA compared to the operons takes place at the level of trans- roteomics. that of their mesophilic counterparts. This lation. The regulator protein MvaL1, and

10 Research Report 2015 Medical University of Innsbruck Medical Biochemistry

MvaL4, respectively, binds preferentially to • Extent research on bioactivities of essen- Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation. Vosper its binding site on the 23S rRNA, and, when tial oil contained VOC J, Masuccio A, Kullmann M, Ploner C, Geley S, Hengst L. in excess, binds with lower affinity to its ONCOTARGET. PubMed: 25605247 regulatory binding site on its mRNA (in the Eco– and Nutrional Biochemistry & Lapatinib and doxorubicin enhance the Stat1-dependent anti- tumor immune response. Hannesdottir L, Tymoszuk P, Parajuli case of MvaL1 a structural mimic of the 23S Nutrigenomics N, Wasmer M-H, Philipp S, Daschil N, Datta S, Koller J-B, Tripp rRNA binding site) and thus inhibits trans Florian Überall CH, Stoitzner P, Mueller-Holzner E, Wiegers GJ, Sexl V, Villunger A, Doppler W. EUROPEAN JOURNAL OF IMMUNOLOGY. 2013; lation of all cistrons of the operon. • Risk/benefit assessment of the impact of 43(10); 2718–2729. Future Goals: natural products on cells through the use In situ proliferation contributes to accumulation of tumor-asso- To define the translational step at which of cellular model systems ciated macrophages in spontaneous mammary tumors. Tymo- szuk P, Evens H, Marzola V, Wachowicz K, Wasmer M-H, Datta archaeal L1 inhibits its own synthesis • Identification of gene expressionS , Mueller-Holzner E, Fiegl H, Boeck G, van Rooijen N, Theurl I, To study the mechanism of MvaL4-mediated signatures and genome wide pathway and Doppler W. EUROPEAN JOURNAL OF IMMUNOLOGY. 2014; 44(8); autoregulation of its operon in Archaea network analysis 2247–2262. Replenishment of the B cell compartment after doxorubicin-in- • Cellular detox systems: duced hematopoietic toxicity is facilitated by STAT1. Datta S, VOC Bioactivity Assessment Phase I: Cytochrome P450 isoenzymes Parajuli N, Tymoszuk P, Ottina E, Parson W, Sgonc R, Villunger A, Doppler W. JOURNAL OF LEUKOCYTE BIOLOGY. 2014; 95(6); Johanna M. Gostner (Johannes Hochleitner); 853–866. So far, cellular reactions to single volatile Phase II: Keap1/Nrf2 signalling (Martina High STAT1 mRNA levels but not its tyrosine phosphorylation organic compounds (VOCs) have been in- Naschberger) and a detailed understand- are associated with macrophage infiltration and bad prognosis in breast cancer. Tymoszuk P, Charoentong P, Hackl H, Spilka R, vestigated in detail only sparsely, due to ing of cellular redox regulated pathways Mueller-Holzner E, Trajanoski Z, Obrist P, Revillion F, Peyrat JP, Fiegl the difficulties in the modelling of realistic • Development of new kitchen appliances H, Doppler W. BMC CANCER. 2014; 14(S); 257. exposure scenarios in vitro. In cooperation for healthier cooking (cooperation with Thienoquinolines as Novel Disruptors of the PKC epsilon/RACK2 Protein-Protein Interaction. Rechfeld F, Gruber P, Kirchmair J, with Bioenergy2020+, a novel exposure PHILIPS Austria GmbH) and of a small- Boehler M, Hauser N, Hechenberger G, Garczarczyk D, Lapa GB, incubator system has been developed that scale bio-sensor (cooperation with CTR) Preobrazhenskaya M N, Goekjian P, Langer T, Hofmann J. JOURNAL enables long-term exposures to VOCs over (all + Maria Lerchbaumer) OF MEDICINAL CHEMISTRY. 2014; 57(8); 3235–3246. Pathway-focused bioassays and transcriptome analysis contribute a broad concentration range. Exposures of Risk/benefit assessment of natural prod- to a better activity monitoring of complex herbal remedies. Klein air-liquid interphase cultures of lung cell ucts – such as volatile organic compounds A, Wrulich O A, Jenny M, Gruber P, Becker K, Fuchs D, Gostner JM, models were realized with formaldehyde. and phytochemicals – is an integral part Ueberall F. BMC GENOMICS. 2013; 14(S); 133. Immunoregulatory Impact of Food Antioxidants. Gostner J, Ciardi Although results cannot be extrapolated of bio-medicine and in the development of C, Becker K, Fuchs D, Sucher R. CURRENT PHARMACEUTICAL DE- directly to in vivo, this in vitro approach potential therapeutics. Therefore, we have SIGN. 2014; 20(6); 840–849. can contribute to risk-benefitassessments . developed suitable and reliable cellular Selected Funding Furthermore, when investigating other models to achieve a deep understanding of • EPO-Can – Gaining Sag on the Epotein’s Sage. L. Hengst. VOCs, tryptophan breakdown via enzyme the impact of natural products on cell biolo- EU-Projekt – EU-FP7. 157,930.40€ indoleamine 2,3-dioxygenase is frequently gy, with a primary focus on redox-regulated • Funktion der p27 Tyrosin Phosp horylierung durch BCR-Abl, JAK2 und FLT3 in der Tumorentstehung – FWF Einzelprojekt affected. pathways. The identification of gene expres- P 24031. L. Hengst. 342,412.00€ Major Achievements: sion signatures and genome wide pathway • DK MCBO Teilprojekt. Molecular Mecanism of the statin- induced cell cycle arrest. L. Hengst. FWF. 118,135€ • For the first time, a long term treatment and network analysis is at the core of our • Protein Kinase C Epsilon-induced phosphoproteome P. Gruber. of cells to gaseous formaldehyde was analyses. Thereby, our specific focus tack- FWF Einzelprojekt P 25491. 267,177.75€ • Volatile Öle. Herta Firmberg Programm. Johanna Gostner. FWF. realised les the cell’s own detoxification systems. T 703. 223,500€ • Kif6 as potential target in A549 cells In this regard, Phase I detoxification, regu- • PHYTORAF I: Isolation, quantification and bioactivity assessment of plant allelochemicals, F. Überall, FFG 834251, bmvit identified lated via cytochrome P450 isoenzymes is cluster “Intelligent technologies” 135,000€. Future Goals: investigated in depth, as well as Phase II, • VocOnCell: Design, realization and validation of a novel incubator system to expose cell cultures to volatile compounds, F. • Investigating formaldehyde as endo orchestrated by the Keap1/Nrf2 signalling Überall, FFG 834169, bridge project. 475,000€. genous signalling molecule pathway. We strive to translate the findings • TRENDS IN NUTRITION: Implementation of novel food processing technologies for healthy nutrition, F. Überall, FFG 840590 • Exploring tryptophan-kynurenine pathway of our research into applications of use for headquarter project of PHILIPS Austria. 679,300€ activation by VOC society and in this context we have taken • ABIOTIC STRESS IN EXTREMOPHILES: Characterization of abiotic stress factors of cyanobacteria – isolation of active part in the development of new kitchen ap- principles, Mit M. Ganzera, LFU. F. Überall, FWF Einzelprojekt pliances for healthier cooking as part of a P 24168. 171,003€. fruitful cooperation with PHILIPS Austria Collaborations GmbH. This project was initiated in 2013. • Joyce M Slingerland, University of Miami, USA Within this framework, the advancements • R. W. Kriwacki, St. Jude Hosp. of Sick Children, Memphis, USA thus obtained have been extrapolated into • Hartmut Halfter, Universität Münster, Germany • Pierre Roger, Bruxelles, Belgium the development of a small-scale biosensor • Stephen J. Elledge, Harvard, Boston, USA in accordance with CTR. • Joan Conaway, Stowers Institute Kansas City, USA • Drorit Neumann, Tel Aviv University, Israel • Terrance Lappin, Queens University Belfast, UK Selected Publications • Elizabeth M Jaffee, Johns Hopkins, Baltimore, USA • Johannes Kirchmair, University of Hamburg, Germany CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidi- • Gennady Lapa, Gause Institute of New Antibiotics, Moscow Fig. 7: shows green tea polyphenol (-) Epigal- rectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation • Johann Hofmann (senior advisor) locatechin-3-gallate in the active site cavity at the Restriction Point. Bisteau X, Paternot S, Colleoni B, Ecker K, • Prof. Dr. M. Garber, Institute of Protein Research, Russian Coulonval K, De Groote P, Declercq W, Hengst L, Roger PP. Academy of Sciences, Pushchino, Moscow Region, Russia of the human drug metabolizing enzyme Cy- PLOS GENETICS. 2013; 9(5); e1003546. • Ulrich-Merzenich G., “Omics”-technologies in phytomedicine, Universitätsklinikum Bonn, Germany tochrome P450 2D6. The genetic algorithm The p27-Skp2 axis mediates glucocorticoid-induced cell cycle • Moscat J., Protein kinase C signaling, Genome Research Insti- GOLD was used for docking the flexible arrest in T-lymphoma cells. Kullmann MK, Grubbauer C, Goetsch tute Cincinnati, USA K, Jaekel H, Podmirseg SR, Trockenbacher A, Ploner C, Cato ACB, • Tonissen K., Eskitis Inst. for Drug Discovery, Griffith Univ., AUS non-generic (-) EGCG in the binding site of Weiss C, Kofler R, Hengst L. • Schwarzentruber P., Pyrosequencing & microbiome, Micros- CYP2D6. CELL CYCLE. 2013; 12(16); 2625–2635. tech, Olten, CH

Research Report 2015 Medical University of Innsbruck 11 Biocenter Neurobiochemistry

PNS – both in the developing and mature receptor components p75NTR and NgR in nervous system – RTN4-A/Nogo, is the only normal and diseased brains. RTN member with a defined function in the adult brain. Nogo-A was originally identified Major Achievements: as a myelin-derived inhibitor of neurite out- • Identification of RyR2 as a novel interac- growth and has been implicated as a major tion partner of RTN1 in neurons factor preventing neuronal regeneration • Identification of VersicanV0/V1 as a spe- and compensatory sprouting in the adult cific NgR2 ligand that controls epidermal CNS. Over the past few years, considerable innervation progress has been made in our understanding of the structure-function relationship Future Goals: of Nogo-A, its axonal receptors, and the Characterization of the physiological func- intracellular signaling cascades mediating tion of RTN protein interactions in neurons inhibition of axon outgrowth. However its physiological significance as an intracellular Purine-mediated Neuroprotection protein of neurons is unknown. Gabriele Baier-Bitterlich In order to improve the functional recov ery Recent studies in our lab highlight novel and clinical outcome after a stroke, a major Head of Division: functions of RTN-4A/Nogo-A and other RTN focus of current stroke research is the Univ.-Prof.in Dr.in Christine Bandtlow isoforms as important intracellular regula- development of new neuroprotective and tors of axonal and dendritic morphogenesis neuroregenerative strategies that aim to Contact: in vitro and in vivo. Present aims are to un- rescue the stroke-affected, but still viable Biocenter, Innrain 80–82 ravel the molecular mechanisms that me- tissue (penumbra, see Fig. 2). The rapid 6020 Innsbruck diate these effects and to analyse proteins degradation of cellular ATP during hypoxia/ that specifically interact with neuronally ex- ischemia results in the production and [email protected] pressed RTN proteins. In addition, we use release of purine nucleosides. Growing Phone: +43 512 9003 70281 several knock-out mouse model systems evidence suggests that purine nucleosides Fax: +43 512 9003 73110 to explore and define the role of the Nogo might act as trophic factors in hypoxic rain. https://www.i-med.ac.at/ neurobiochemistry/index.html

Keywords

Neuroscience, neuronal growth, central nervous system, brain, cytoskeletal changes

Research Focus

• Characterization of myelin-associated neurite growth inhibitors and their cognate receptors in the central nervous system. • Signal transduction of neurite growth inhibitors in the brain with special emphasis on effects on cytoskeletal changes in neuronal growth cones.

Research

Neurobiochemistry Christine Bandtlow This laboratory is primarily interested in delineating the physiological functions of reticulon proteins (RTN) and their signaling molecules in the nervous system. Related to their association with the ER, RTN proteins have been suggested to play a role in the regulation of intracellular trafficking of proteins involved in exo- and endocytosis, but their precise cellular functions remain unknown. Although many RTN isoforms show distinct expression patterns in the CNS and Fig. 1: Signalling by Nogo

12 Research Report 2015 Medical University of Innsbruck Neurobiochemistry

Fig. 2: Even a short blockade of oxygen flow in brain results in a rapid decline of cellular TPA and a subsequent loss of neuronal function and viability. Tissue in the ischemic core is beyond therapeutic rescue, however the penumbral tissue is affected by ischemia but still viable and therefore the key target for neuroprotective interventions. In response to hypoxia/ischemia purine nucleosides are produced and released from cells, and growing evidence suggests that they might act as trophic and neuroprotective factors in the central- and peripheral nervous systems. (Thauerer et al, 2012, 2014). Cell death and neurite formation (Hoechst (blue) and Propidium iodide staining (red)).

Therefore, purine nucleoside/adenosine wide-field microscopes. Access to an inte- Selected Publications receptors and their signaling pathways are grated stereology microscope/software Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons. a key focus of neuroprotective strategies . system for neuron tracing (Neurolucida) is Kaya L, Meissner B, Riedl M C, Muik M, Schwarzer C, Ferraguti Previous results in our laboratory demon offered in collaboration with the Institute of F, Sarg B, Lindner H, Schweigreiter R, Knaus H-G, Romanin C, Bandtlow C E. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR strated the positive impact of purine nucleo Pharmacology. CELL RESEARCH. 2013; 1833: p. 1421–1433. sides on viability and neurite outgrowth Nogo-A couples with Apg-1 through interaction and co-ordinate (see microscopy picture in Fig. 2) in hypoxic In collaboration with the Leopold-Franzens- expression under hypoxic and oxidative stress. Kern F, Stanika R I, Sarg B, Offterdinger M, Hess D, Obermair G J, neuronal PC12 cells and primary rat cere- University Innsbruck a gated STED super Lindner H, Bandtlow C E, Hengst L, Schweigreiter R. bellar granule neurons. We could further resolution microscope was purchased co- BIOCHEMICAL JOURNAL. 2013; 455: p. 217–227. show that activation of p42/p44 mitogen- funded by the BMWF, which is operational Peripheral nerve regeneration and NGF-dependent neurite out- activated kinases (alias ERK1/2), hypoxia- since beginning of 2014. As a second growth of adult sensory neurons converge on STAT3 phosphorylation downstream of neuropoietic cytokine receptor gp130. inducible transcription factor-1 (HIF1-alpha) superresolution technique, STORM was Quarta S, Baeumer B E, Scherbakov N, Andratsch M, Rose-John S, and protein kinase C-related kinase (PKN1) evaluated on the iMIC and will soon be of- Dechant G, Bandtlow CE, Kress M. J Neurosci. 2014 Sep 24;34(39):13222-33. are crucial for purine nucleoside-mediated ficially offered. Both gSTED and STORM are Loss of Nogo receptor homolog NgR2 alters spine morphology of regeneration and/or survival of neurons. able to resolve small structures clearly be- CA1 neurons and emotionality in adult mice. These findingsresulted in further fund- low Abbe’s diffraction limit (250 to 300 nm). Borrie S C, Sartori S B, Lehmann J, Sah A, Singewald N, Bandtlow C E. Front Behav Neurosci. 2014 May 15;8:175. ing to investigate the MAPK/HIF1-alpha Nogo receptor homolog NgR2 expressed in sensory DRG neurons (B. Thauerer, T421-B18, TWF) and PKN1 Software support is offered from basic user controls epidermal innervation by interaction with Versican. (G. Baier-Bitterlich, P26002-B24) signal- training to complex calculations. We cur- Bäumer B E, Kurz A, Borrie S C, Sickinger S, Dours-Zimmermann M T, Zimmermann D R, Bandtlow C E. ing cascades in purine nucleosid-mediated rently support Fiji, CellProfiler andMATLAB . J Neurosci. 2014 Jan 29;34(5):1633-46. neuroprotection. A server-based deconvolution software Modulation of phenylalanine and tyrosine concentrations by package (Huygens Professional) enabling ischemia and guanosine in neuronal PC12 cells. Thauerer B, Geisler S, Fuchs D, Baier-Bitterlich G. Biooptics/Microscopy the improvement of the resolution of light PTERIDINES. 2013; 24: p. 245–250. Martin Offterdinger microscopic images, including widefield, Selected Funding The MUI Biooptics/microscopy facility, im- laser scanning confocal, spinning disk and • FWF, W1206: Doctoral College “Signal processing in neurons”, plemented in 2009 at the Division of Neu- also gSTED superresolution images is suc- Bandtlow Christine E robiochemistry, provides university-wide cessfully running already for sev eral years. • FWF, P26002-B24: “Analyse der Rolle der PRK1 in der Neuro protektion”, Baier-Bitterlich Gabriele access to advanced equipment, training, Challenging interactive 3D image analyses • FWF, T421-B18: “Mechanismus der HIF -1alpha Aktivierung”, education and expertise in light microsco- are done using Imaris. Thauerer Bettina py to all scientists on campus. We currently Collaborations offer assisted access to several research mi- Microscopy-related teaching is offered in • Paul Lingor, Univ. Göttingen, Germany croscopes and image processing software. several PhD programmes and prior to inde- • Dieter Zimmermann, Univ. Zurich, Switzerland pendent usage of any microscope all users • Michail Sitkovsky, Dana-Farber Cancer Institute, Boston, USA Presently, we are harboring two laser scan- receive an instrument-specific training. Core Facilities ning confocal microscopes (Leica SP5, For all scientific equipment within the core • Leica SP5 laser scanning confocal microscope • Till, iMIC-microscope, multifunctional microscope for live cell Zeiss, LSM510 Meta) and a multifunctional facility biooptics (microscopes), an im- imaging (TIRF, spinning disk, FRAP) microscope (Till, iMIC) for live cell imaging, proved on-line booking system has been • SP8 gSTED microscope • Zeiss LSM510Meta laser scanning confocal microscope which is equipped for TIRF, spinning disk, established in 2012 in order to ensure easy • two widefield microscopes and FRAP. Moreover, we are harboring two and fair access to all users. • Neurolucida (in collaboration with the Institute of Pharmacology)

Research Report 2015 Medical University of Innsbruck 13 Biocenter Clinical Biochemistry

Keywords Micro-Analysis Facility). Our main technologies are: LC/CE-ESI-MS , HPLC (e.g. RPC, Protein Analysis, PTM Identification, Mass HILIC, IEC, GPC), Capillary electrophoresis, Spectrometry, Phospho-proteomics, Pro- Phospho-proteomics, Chromatin immune- teome-wide Quantification, Capillary Elec- precipitation, Co-immunoprecipitation and trophoresis, HPLC, Method Development, proteome-wide quantification (e.g. SILAC, Epigenetics, Protein Microanalysis Facility iTRAQ, TMT, Dimethyl labeling).

Research Focus Research

• Development of multidimensional At present, we have two main research in- LC/CE-MS-based methods terests. The first focuses on the valuatione • PTM identification of various nuclear and of capillary electrophoresis-mass spec- extracellular proteins trometry (CE-MS) as an alternative pro- • Identification of novel phospho-histone teomics tool for nanoLC-MS. Up to now, binding proteins LC-MS is the commonly used technique for • Identification of histone-modification pat- the analysis of complex protein mixtures in terns at the nucleosomal level proteomics. The use of CE as a complemen- Vice Head of Division (interim): • Method development for top-down and tary separation method to reversed-phase ao. Univ.-Prof. Dr. Herbert Lindner middle-down proteomics using ETD- and HPLC has not yet become fully accepted HCD-fragmentation as an alternative. One of the reasons is the Contact: • Development of targeted protein absolute on-line interfacing of CE with MS that allows Biocenter, Innrain 80–82 quantification methods stable electrospray processes without com- 6020 Innsbruck promising the quality of separation or the General Facts detection sensitivity. A recently published [email protected] concept of a sheathless interface based Phone: +43 512 9003 70310 Our lab focuses on the development of on a separation capillary with a porous tip high-resolution methods for the separation acting as nanospray emitter combines low Head of Division (interim): and identification of post-translationally flow characteristics of CE with an integrat- Univ.-Prof. Dr. Ludger Hengst modified proteins in order to investigate ed ESI source, as a promising alternative their biological significance. A set of separa- to nanoLC-ESI-MS. We recently published [email protected] tion methods based on capillary electropho- the successful application of CE-MS for Phone: +43 512 9003 70110 resis (CE), reversed-phase chromatography, the analysis of moderately complex mix- hydrophilic interaction liquid chromatogra- tures consisting of distinctly acetylated, https://www.i-med.ac.at/imcbc/ phy (HILIC) and mass spectrometry (MS) phosphorylated, methylated and deamidat- clinbiochemfolder/clinbiochem.html was introduced in our lab. Now, as a re- ed proteins as well as of microsequence sult of a continuous development program variants differing only slightly in mass and over many years, our group offers a wide charge. Our present aim is to evaluate the range of analytical methods and servic- suitability of CE-MS for its use in identifica- es to support the work of other research tion and quantification of deamidation re- scientists in the University (see Protein lated products. Deamidation of asparaginyl © Anal Chem 2011: 83(19):7297–305 Fig. 1: The design of a sheathless interface for coupling capillary electrophoresis to mass spectrometry (Faserl et al. 2011: Anal. Chem. 83(19): 7297–305).

14 Research Report 2015 Medical University of Innsbruck Clinical Biochemistry

residues is one of the most frequent modifications in peptides and proteins, and in the majority of cases is a non-enzymatic reaction that takes place under physiological conditions in vitro in the course of isolation or storage, and in vivo during development and/or aging of cells. This posttranslational modification reaction often follows a

complex mechanism, producing a mixture arz Schw Schw . of aspartyl, succinimidyl and isoaspartyl . © S arz forms, with L-isoaspartyl dominating. Due to © S its biochemical importance, various analyt- Fig. 2: Nano-LC-MS system consisting of a Fig. 3: Nanospray Flex™ ion source (Thermo ical techniques for the characterization of Q Exactive™ Hybrid Quadrupole-Orbitrap Scientific™). deamidated peptides have been described, Mass Spectrometer (Thermo Scientific™) however it is still a challenging task to dis- and a nano-LC gradient system UltiMate tinguish between isoAsp, Asp and Asn resi- 3000 (Dionex). dues. Isoaspartyl residues can significantly affect protein structure and typically alter • MALDI TOF/TOF 4800 plus analyzer (AB Selected Publications biological activity and function of peptides Sciex) Comparing and combining capillary electrophoresis electrospray and proteins. • Procise 492 protein sequencer (Applied ionization mass spectrometry and nano-liquid chromatography electrospray ionization mass spectrometry for the characteriza- Biosystems) tion of post-translationally modified histones. Sarg B, Faserl K, Our second interest is directed to • Nano-LC gradient systems UltiMate 3000 Kremser L, Halfinger B, Sebastiano R, Lindner HH. MOL CELL PROTEOMICS. 2013; 12(9): p. 2640–56. quantitative proteomics, which is a powerful (Dionex) Histone H5-chromatin interactions in situ are strongly modulat- approach used for both discovery and • Probot microfraction collector (LC-Pack- ed by H5 C-terminal phosphorylation. Kostova NN, Srebreva L, targeted proteomic analyses to understand ings) for on-line MALDI target preparations Markov DV, Sarg B, Lindner HH, Rundquist I. CYTOMETRY A. 2013; 83: p. 273–9. global proteomic dynamics in a cell, tissue • Various capillary electrophoresis and MALDI-MS tissue imaging identification of biliverdin reductase B or organism. Most quantitative proteomic HPLC Systems overexpression in prostate cancer. Pallua JD, Schaefer G, Seifarth analyses entail the isotopic labeling of • Solar M6 dual Zeeman spectrometer C, Becker M, Meding S, Rauser S, Walch A, Handler M, Netzer M, Popovscaia M, Osl M, Baumgartner C, Lindner H, Kremser L, Sarg proteins or peptides, which can then be (Thermo Fisher Scientific) for trace ele- B, Bartsch G, Huck CW, Bonn GK, Klocker H. differentiated by mass spectrometry. ment analysis J PROTEOMICS. 2013; 91: p. 500–14. Relative quantitation methods (SILAC, Characterization of the Link between Ornithine, Arginine, Pol- yamine and Siderophore Metabolism in Aspergillus fumigatus. ICAT, ICPL and isobaric tags) are used to Provided Services: Beckmann N, Schafferer L, Schrettl M, Binder U, Talasz H, Lindner compare protein or peptide abundance • Comprehensive protein identification of H, Haas H. PLOS ONE. 2013; 18;8(6):e67426. between samples. Recently, we performed simple and complex protein digests from Quantitative proteomics using ultralow flow capillary electrophoresis-mass spectrometry. Faserl K, Kremser L, Müller M, Teis D, quantitative proteome analyses using gel bands, immunoprecipitations (IP), and Lindner HH. ANAL CHEM. 2015 May 5;87(9):4633–40. metabolically labelled yeast proteins. whole-cell/secreted/tissue digests by Samples were analyzed by CE- and LC- mass spectrometry Selected Funding ESI-MS and the characteristic features • Molecular mass determination of intact • Coupling capillary electrophoresis to mass spectrometry for protein and proteome analysis; Industrial Project with AB SCIEX of the two approaches and the number proteins or peptides • Investigation of in-vivo O-Glycosylation of the low abundance of identified and quantified peptides and • Localization and quantification of marker NT-proBNP by Affinity Proteomics Methods and Mass Spectrometry in blood plasma from patients with severe heart proteins were compared. Moreover, the post-translational modifications (phos- failure two MS data sets were mined for post- phorylation, acetylation, methylation, etc.) • Industrial Project with Roche Diagnostics GmbH Penzberg, Germany translationally modified peptides, e.g. • Mass spectrometry supported determina- • Determination of changes in histone acetylation and methyla- acetylated, phosphorylated, deamidated tion of protein complex structure by identi- tion of Histone H4 in peripheral blood mononuclear cells isolated from human panic disorder patients prior to – during – and and oxidized forms. fication of chemical cross-linked peptides following – therapy; SFBF44 “Cell signaling in chronic CNS • Enrichment of phosphopeptides by Im- disorders” Protein Micro-Analysis Facility mobilized Metal Affinity Chromatography Collaborations ao. Univ.-Prof. Dr. Herbert Lindner (IMAC, etc.) • Reinhard Dallinger, Institute of Zoology, LFU, Innsbruck, Austria The Protein Micro-Analysis Facility is • Quantitative Proteomics using isotope la- • Peter Ladurner, Institute of Zoology, LFU, Innsbruck, Austria established within the Division of Clinical beling strategies (e.g. SILAC, iTRAQ, TMT, • Nicolas Singewald, Institute of Pharmacy, LFU, Innsbruck, A • Margarethe Geiger, Depart. of Vascular Biology and Thrombosis Biochemistry and is dedicated to provide etc.) Research Centre of Physiology and Pharmacology, Vienna, A investigators with equipment, expertise • Lable free Quantification • J. Ausio, University of Victoria, Victoria, Canada • M. Freitas, Ohio State Univ. Medical Center, Columbus, USA and custom services for the detection, • Separation technologies including analyti- • N. Guzman, Princeton Biochemicals Inc., New Jersey, USA characterization and quantification of cal and semi-preparative liquid chromatog- • Pedro Suau, Universitat Autonoma de Barcelona, Spain • S. Krylov, Department of Chemistry and Centre for Research proteins and peptides on a recharge basis. raphy (e.g. ion exchange, rev erse-phase, on Biomolecular Interactions, York University, Toronto, Canada The facility maintains a suite of state of the normal phase) and capillary electropho- • D.D. Chen, Department of Chemistry, University of British Co- lumbia, Vancouver Canada art instrumentation including: resis • R. Cole, Johns Hopkins University School of Medicine, Balitimo- • Q Exactive Plus (Thermo Fisher Scientific) • Quantitative amino acid analysis re, USA hybrid FT mass spectrometer LTQ Orbitrap • Quantitative elemental analysis using XL ETD (Thermo Fisher Scientific) atomic absorption spectrometry (AAS) Core Facilities Protein Micro-Analysis Facility: • LTQ VELOS mass spectrometer (Thermo • Capillary electrophoresis-electrospray [email protected] Fisher Scientific) ionization-mass spectrometry (CESI-MS) https://www.i-med.ac.at/iftz/zentrale_gruppen/proteinfacility/

Research Report 2015 Medical University of Innsbruck 15 Biocenter Biological Chemistry

areas include pteridine and lipid metabolism incidence. NF1 patients have an increased as well as intracellular signal transduction risk of developing tumours, show a variety and its regulation, particularly in the of developmental defects and frequently context of small guanine nucleotide binding have learning disabilities. The tumour (G) proteins. suppressor gene NF1 encodes the giant protein neurofibromin (320 kDa) and is not Another focus is gaining an understanding functional in NF1 patients due to genetic of novel biotechnologies in the context of alterations. Our long term vision is to define biomedicine and how they create and at the the functional spectrum of neurofibromin same time serve emerging markets as well in as much detail as possible. Our research as to explore their ethical, social and cultural activities currently include determining the dimensions. Research and teaching in this structure of full length neurofibromin as ellw field has led to numerous cooperations (see as the definition of its interaction partners below) and the participation in the building (collaborations with Frank McCormick, of a local interinstitutional platform of UCSF, and Lukas A. Huber, Innsbruck). expertise on ethics in the context of health In addition, we explore the mechanism and medicine. of neurofibromin-mediated repression of MHCII protein expression (collaboration Head of Division: Our methods spectrum includes bio with Andreas von Deimling, Heidelberg). Univ.-Prof. Dr. Klaus Scheffzek chemical techniques such as FPLC/HPLC A major activity in the reporting for preparative protein purification and period comprised the implementation Contact: analysis, eukaryotic cell culture, various and finalization of our biomolecular Biocenter, Innrain 80–82 biophysical as well as bioanalytical methods crystallography platform that includes 6020 Innsbruck and biomolecular X-ray crystallography. a robot based crystallization screening laboratory (ArtRobbins/DunnLab, Rigaku) [email protected] We consider teaching a major responsibility with automated imaging system and an Phone: +43 512 9003 70330 in the education of young scientists and X-ray generator (Microstar, Bruker). Fax: +43 512 9003 73110 contribute to the respective activities https://www.i-med.ac.at/imcbc/ for students of the Medical as well as the Neuropsychoimmunology molecularcellbiologyfolder/ Leopold-Franzens University Innsbruck. Dietmar Fuchs molcellbiol.html Mood changes and depression are common Research in patients suffering from inflammatory disorders like virus infections, autoimmune Keywords Structural Biology and Mechanisms of syndromes, malignant tumour diseases the Neurofibromatosis ypeT 1 Protein and also in the overweight, but the patho Structural biology, biochemistry, molecular Klaus Scheffzek genesis of symptoms is still unclear. Several biology, clinical chemistry, protein purifica- We aim to understand the disease of our recent studies showed associations tion, high pressure liquid chromatography, mechanism of neurofibromatosis type-1 between neuropsychiatric deviations bioethics (NF1), a genetic disease with relatively high in patients and increased neopterin

Research Focus

• Structural biology of disease proteins with a special focus on neurofibromatosis type-1, an inherited disease with relatively high incidence but poorly understood pathogenic mechanisms. • The biochemistry and clinical chemistry of pteridine and tryptophan metabolism. Pteridines are structurally related to the vitamins folic acid and riboflavin but, in contrast to these, can be formed in the mammalian body. • Ethical, cultural and social issues concerning the use of innovative technologies in biomedicine.

General Facts

We are performing basic research on Fig. 1: Since our move to the CCB building we have been implementing a now running biomolecular systems that can impact on platform dedicated to biomolecular sample production, crystallization and structure human health and disease. Our research determination.

16 Research Report 2015 Medical University of Innsbruck Biological Chemistry

concentrations and tryptophan breakdown (Kyn/Trp) in the blood. Our most recent work sheds more light on this observation and its relevance for neuropsychoimmunology. We have also observed higher blood phenyl alanine levels and higher phenylalanine to tyrosine ratios in such patients and also in healthy elderly individuals. Especially in combination with the measurement of Kyn/Trp, Phe/Tyr determinations can support treatment decisions as to whether serotonergic or noradrenergic/adrenergic/ dopaminergic treatment options are more likely to be useful in the individual patient. Clinical and in vitro studies utilizing the model of freshly isolated peripheral blood mononuclear cells (PBMC) were performed, Open Access Communications © Nature and in the years 2013/14 more than 50 Fig. 2: Position of Sjögren-Larsson syndrome causing amino acid changes in the fatty papers derived from multiple collaborations aldheyde dehydrogenase structure. worldwide have been published by the group. The reference given below refers to a mutagenesis screens. We now proceed genetic/genomic services. PhD projects review article in which the general concept to the manipulation of the expression of deal with the ethics of reprogenetics in behind our research activities is described this enzyme in cultured cells and in model developing countries and with the definition and from which detailed references can be organisms in order to study its physiological of “race” in pharmacogenomics. extracted (Capuron et al., Curr Pharm Des role. Fatty aldehyde dehydrogenase, 2014;20:6048–57). the subsequent enzyme in ether lipid Selected Publications metabolism, causes a rare inherited disease Functional MHC Class II Is Upregulated in Neurofibromin- Alkylglycerol Monooxygenase, a Novel when its function is lost, the Sjögren Deficient Schwann Cells. Reuss DE, Mucha J, Holtkamp N, Mueller U, Berlien H-P, Mautner VF, Ehemann V, Platten M, Scheffzek K, von Ether-Lipid Cleaving Enzyme Larrson Syndrome. This is a severe disease Deimling A. JOURNAL OF INVESTIGATIVE DERMATOLOGY. 2013; Katrin Watschinger, Gabriele Werner- characterized by developmental delays 133: p. 1372–1375. Felmayer, Georg Golderer and and a skin phenotype called ichtyosis. Activated Immune System and Inflammation in Healthy Ageing: Relevance for Tryptophan and Neopterin Metabolism. Capuron L, Ernst R. Werner Although humans have more than a dozen Geisler S, Kurz K, Leblhuber F, Sperner-Unterweger B, Fuchs D. Building on the 30 years plus focus on different aldehyde dehydrogenases, they CURRENT PHARMACEUTICAL DESIGN. 2014; 20: p. 6048–6057. pteridine research in the institute, we are cannot compensate for a deficiency in fatty A gatekeeper helix determines the substrate specificity of Sjogren-Larsson Syndrome enzyme fatty aldehyde dehydroge- currently focusing on ether lipid metabolism. aldehyde dehydrogenase. We characterized nase. Keller MA, Zander U, Fuchs JE, Kreutz C, Watschinger K, The central enzyme for the degradation by biochemical and structural methods the Mueller T, Golderer G, Liedl KR, Ralser M, Krauetler B, Werner ER, of these compounds, alkylglycerol mono human fatty aldehyde dehydrogenase and Marquez JA. NATURE COMMUNICATIONS. 2014; 5: p. 4439. Genetics as Social Practice – Transdisciplinary Views on Science oxygenase, is dependent on the cofactor found that a special additional structural and Culture. Prainsack B, Schicktanz S, G Werner-Felmayer. ISBN tetrahydrobiopterin. Tetrahydrobiopterin, a feature of this enzyme, a gatekeeper helix, 978-1-4094-5549-3; Ashgate, Farnham (UK); January 2014. compound structurally related to the vita- is responsible for its specificity to fatty http://www.ashgate.com/isbn/9781409455493. mins folic acid and riboflavin, is synthesized aldehydes. Selected Funding in the animal body, and is required for five • Structural basis for the modulation of dendritic spine density by specific, crucial ydroxylationh reactions. In Bioethics the interaction of neurofibromin and the AAA-ATPase p97/vcp, FWF, MCBO graduate school, Klaus Scheffzek addition to alkylglycerol monooxygenase Gabriele Werner-Felmayer • Characterization of immunomodulatory effects of nanoparticles these include the conversion of phenyl Based upon interdisciplinary dialogues in vitro, Austrian Science Fund (FWF), Dietmar Fuchs • Role of alkyglycerol monooxygenase and tetrahydrobiopterin alanine to tyrosine, the first essential step with colleagues from philosophy, social and biosynthesis in adipocyte function. Autonomous Province of in the degradation of the essential amino political science, medical law, economics Bolzano/Bozen–South Tyrol (Division for the Promotion of Edu- cation, Universities and Research), Katrin Watschinger acid phenylalanine, the biosynthesis of the and health management, we focus on neurotransmitters dopamine, epinephrine epistemology and culture in biomedicine Collaborations and serotonin as well as the biosynthesis as well as its ethical dimensions. Special • Lucile Capuron, University of Bordeaux, France • Keith Channon, Jonathan Hodgkin, University of Oxford, United of the versatile messenger molecule nitric emphasis is placed upon the concepts Kingdom oxide. of hope and promise in genomics, • Frank McCormick, San Francisco, USA The alkylglycerol monooxygenase reaction medically assisted reproduction and • Andreas von Deimling, Heidlerberg, Germany • Magnus Gisslen, Lars Hagberg, Östra University Hospital, Goth- was first described in 1964, along with the stem cell research. Current projects deal enburg, Sweden subsequent conversion of its fatty aldehyde with international regulation of assisted • Harald Mangge, Eva Reininghaus, Medizinische Universität Graz, Austria product to the corresponding fatty acid by reproductive technologies, with third • Teo T Postolache, University of Baltimore, MD, USA fatty aldehyde dehydrogenase. It took until party cross-border reproductive care, with • Richard W Price, Institut of Neurology, San Francisco General Hospital, UCSF, USA 2010 when we managed to assign a sequence unintended traumatization of patients in • Markus Ralser, University of Cambridge, United Kingdom to the very labile integral membrane protein the context of medicalised reproduction, The bioethics branch collaborates with colleagues from numer- alkylglycerol monooxygenase, which could and with prevailing determinist views in the ous institutions at local, national and international levels (King’s College London, University Medicine Goettingen). It is the Aus- not be purified so far. We then explored the dynamic field of genetics/genomics that are trian partner of the International Network of the UNESCO Chair biochemistry of the enzyme by site directed particularly powerful in marketing personal in Bioethics (Haifa).

Research Report 2015 Medical University of Innsbruck 17 Biocenter Cell Biology

Signal Transduction and Proteomics mice to determine the biological roles of the Lukas Huber Lab complex in vivo, at the organism level. We would like to understand how specific cell fate decisions are made by using a pool As a result of an interaction proteomics of very similar kinases within MAPK signal- screen using TAP -MS (Tandem Affinity ling pathways. We are especially interested Purification, coupled to Mass Spectrometry) in the role of scaffold proteins in organizing we have identified several proteins signal transduction complexes and study interacting with the LAMTOR complex. The the spatial and temporal resolution of sig core interactome includes all members of naling mechanisms as well as their cyto- the LAMTOR complex, the RAG GTPases plasmic effectors and target genes. The aim (that mediate the translocation of mTORC1 of our research is to understand the phys- to endosomes/lysosomes), and SLC38A9 iological function of scaffold complexes in (a previously uncharacterized member of MAPK signaling at the molecular level in the solute carrier family 38, that we recently cells and at the level of the entire organism. identified as an integral component of the amino acid-sensing machinery that controls The LAMTOR complex – At the crossroad the activation of mTORC1 (Rebsamen et al., between signal transduction and endo Nature 2015). We are currently analysing Head of Division: somal biogenesis this comprehensive interactome data Univ.-Prof. Dr. Lukas A. Huber Over the last two decades we and others functionally with a special focus on the could show that the LAMTOR complex (late interplay between signaling and endosomal Contact: endosomal/lysosomal adaptor, MAPK and biogenesis (Fig. 1). Biocenter, Innrain 80–82 MTOR activator) is strictly recruited to the 6020 Innsbruck membrane of late endocytic compartments, The LAMTOR complex: disease models from where it actively influences MAPK, The use of animal models led to great [email protected] mTORC signaling and endosomal trafficking. progresses in understanding the role of the Phone: +43 512 9003 70170 The complex is involved in several endosomal adaptor Lamtor2 in endosomal/ Fax: +43 512 9003 73100 biological processes including immunity, lysosomal trafficking. In a first approach www.i-med.ac.at/cellbio early embryogenesis, tissue homeostasis, LAMTOR2 knockout mice were generated, cellular proliferation and migration. but severe defects during embryogenesis We use complementary molecular and resulted in no viable offspring (Teis et cellular biology methods, stretching from al., J Cell Biol. 2006). During this time, proteomics, to live and confocal microscopy, four patients, all siblings, suffering from electron microscopy, and structural biology, a primary immunodeficiency syndrome, Research Focus/Keywords in order to functionally characterize the due to a hypomorph LAMTOR2 allele, LAMTOR complex at the molecular and were identified and demanded for further • Signal Transduction and Proteomics cellular levels. In addition, we use model investigations of the immune functions • Cell Differentiation organisms such as yeast and knockout LAMTOR2 (Bohn et al., Nat Med. 2007). • Membrane Traffic and Signaling

General Facts

We study molecular mechanisms that control the organization and function of living cells. To address these fundamental questions, we use a combination of genetic model systems, state-of the-art microscopy and quantitative proteomics. Our division provides an international and dynamic research environment for Master & PhD students as well as PostDocs. We have numerous national and international collaborations with academic partners and biotech companies. We are embedded the international PhD program MCBO (Molecular Cell Biology and Oncology). We participate in and coordinate several EU projects.

Research

Three research groups are currently active at the division of Cell biology: Fig. 1: Core interactome of the LAMTOR complex.

18 Research Report 2015 Medical University of Innsbruck Cell Biology

decreased signaling, while cDC and pDC expansion was caused by boosted mTORC1 activation (Scheffleret al., 2014).

Microvillus inclusion disease – intra cellular trafficking and epithelial polarity Microvillus inclusion disease is an auto somal recessive enteropathy characterized by intractable diarrhea setting on within the first few weeks of life. The hallmarks of MVID are a lack of microvilli on the surface of villous enterocytes, occurrence of intracellular vacuoles lined by microvilli (microvillus inclusions), and the cytoplasmic accumulation of periodic acid-Schiff (PAS)- positive vesicles in enterocytes. Together with our collaborators from the Fig. 2: Primary LAMTOR2+/+ and LAMTOR2-/- macrophages infected with Salmonella Department of Pediatrics I, MUI, and the (green). After 24 h an increased bacteria load in LAMTOR2-/- macrophages was observed. Division of Histology and Embryology, MUI, Actin (red) and Hoechst (blue). Scale bars: 10 mm (Taub et. al., 2012). we were the first to identify mutations in MYO5B, encoding the unconventional type Vb myosin motor protein, in a first Therefore, we have established conditional partners and downstream targets involved cohort of nine MVID patients (Mueller et al., knockout mouse models to investigate the in phagolysosomal maturation underlying Nature Genetics 2008). In a follow-up study, role of LAMTOR2 in antigen presenting cells an efficient antimicrobial response. we described further 15 novel nonsense including macrophages and dendritic cells and missense mutations in MYO5B in 11 (DCs). The correct uptake and processing DCs are initiators of adaptive immunity unrelated MVID patients (Ruemmele et al., of pathogens and antigen presentation and unlike macrophages also able to prime Human Mutation 2010). in the context of the immune response naïve T cells. Investigation of a DC specific Further investigations have focused on the is strictly regulated by the endosomal/ knockout mouse revealed a crucial role of role of Myosin Vb and its interplay with lysosomal system. Using a mouse model LAMTOR2 for DC homeostasis. While soon Rab Small GTPases in the establishment where Lamtor2 was specifically depleted after birth the epidermal Langerhans cell of correct epithelial polarity by making use in macrophages, key players in the immune (LC) network is dispersed due to increased of a CaCo2 RNAi cell model (Thoeni et al., system, we have been able to demonstrate apoptosis and a proliferation defect (Sparber Traffic 2014). that LAMTOR2 is a host defense factor et al., 2014), the aging animals suffer from Recently, we have identified novel mutations against pathogens (Fig. 2) (Taub et al. J a massive expansion of conventional (cDCs) in the STX3 gene, causing a variant form of Cell Sci. 2012) and our findings correlate and plasmacytoid DCs (pDCs) cumulating MVID in patients negative for mutations in with the previously described immuno in a myeloid proliferation syndrome (MPD) MYO5B (Wiegerinck et al., Gastroenterolo deficiency syndrome. We are currently (Fig. 3). As cellular mechanism causing gy 2014). Syntaxin 3 is an apical t-SNARE following this line of results combining the those phenotypes a deregulation of late protein pivotal for polarized apical exo mouse genetic approach with a proteomic endosomal LAMTOR complex dependent cytosis and secretion in epithelial cells. approach aimed at deepening our MAPKinase and mTORC1 signaling were By using a CaCo2 cell model for epithelial/ understanding of the Lamtor2 interaction identified. Loss of LCs was related to a enterocyte polarity and state of the art genome-editing technologies we focus on the intracellular cascade and the proteins involved, which ensure correct polarized intracellular traffic in order to maintain proper epithelial polarity (Fig. 4).

Cell Differentiation Ilja Vietor Lab The interplay between cell proliferation and differentiation controls not only development but also regeneration and therefore its regulatory mechanisms are of interest as therapeutic targets. Based on our studies we predict that the transcriptional co-repressor TPA inducible Fig. 3: Dendritic cell infiltrate in a spleen Fig. 4: Polarized epithelial CaCo2 cyst show- sequence 7 (TIS7) is one of the players section of a DC specific LAMTOR2 knockout ing basolateral proteins (green), apical pro- affecting the cellular regeneration events. mouse model at the age of three months. teins (white and red) and nuclei (blue). TIS7 inducible by the mitogen TPA or red: CD11c, blue: hematoxylin. growth factors is differentially expressed

Research Report 2015 Medical University of Innsbruck 19 Biocenter

TIS7 WT

TIS7 SKMc15 dKO

Fig. 5: Muscle satellite cells grown under differentiation conditions. Immunofluorescence microscopy images depict: MF20-differentiated myoblasts marker protein (red), pICln-TIS7-interacting methylosome subunit protein (green), and DAPI (blue).

*** Fig. 6: Fused TIS7 wt skeletal muscle 45 40 cells grown under differentiation condi- +/+ +/+ 40 35 +/+ +/+ tions in culture. Raster electron scanning -/- -/- micrograph. 35 30 30 25 25 *** p<0,001 20 20 15 *** body fat (%) body -/- -/- 15

body weight gain (%) gain weight body 10 10

5 5

body fat tissuecontentbody (%) *** p<0,001 0 0 chow diet Fig. 8: Left:6 months TIS7 oldSKMc15 males; double n=5 knockout mice are significantly leaner. 6 months old male Fig. 7: Lack of fat vacuoles in the jejunum of mice; n= 6. Chow diet. Right: TIS7 SKMc15 double knockout mice gain significantly less TIS7 SKMc15 double knockout mice (right). weight upon feeding with high fat diet. 11 weeks male mice; n= 11; 3 weeks high fat diet. Oil red oil staining; magnification 40x. in various polarized cell types. We have polarized cell types. We have shown that the lung disease in cystic fibrosis. This lung shown that TIS7 represses transcription in TIS7 interacts with the SIN3 complex disease is the major cause of morbidity and an HDAC-dependent manner. In the TIS7- and regulates transcription in an HDAC- mortality in cystic fibrosis, an autosomal regulated downstream target genes we dependent manner. In the promoter region recessive disease caused by mutations in have identified a common regulatory motif, of TIS7-regulated downstream target genes CFTR. In cystic fibrosis, chronic infection a so-called transcription factor “module”. we have identified a common regulatory and dysregulated neutrophilic inflammation TIS7 expression increases during the motif C/EBPalpha-Sp1 transcription lead to progressive airway destruction. process of tissue regeneration following factor “module”. Furthermore, TIS7 has Neutrophils, but not macrophages, from a challenge like muscle crush damage or the ability to inhibit the Wnt signaling TIS7-deficient mice showed blunted intestinal resection. Our previous studies in an HDAC-dependent manner. TIS7 effector function. In vivo, TIS7 deficiency have shown that in TIS7 knockout mice expression increases during the process of caused delayed bacterial clearance from the differentiation and fusion potential of tissue regeneration following a challenge the airway, but also less inflammation and myoblasts is impaired. like muscle crush damage or intestinal disease. In humans, TIS7 polymorphisms resection. Our previous studies have shown were significantly associated with variation The interplay between cell proliferation that in TIS7 knockout mice the expression of in neutrophil effector function. These and differentiation controls not only myogenic regulatory proteins is deregulated data indicated that TIS7 modulates the development but also regeneration. and the differentiation and fusion potential pathogenesis of cystic fibrosis lung disease Regulation of these two mechanisms is of of muscle satellite cells is impaired. through the regulation of neutrophil effector interest because they represent possible function. These findings were published as a therapeutic targets. Based on our studies, Major Achievements: mutual collaboration in the journal Nature. we predict that the transcriptional co- The group of our collaborators around regulator TPA-inducible sequence 7 (TIS7) Prof. Chris Karp at the Cincinnati College A second member of a novel gene family, is one of the players affecting cellular of Medicine, USA, using TIS7 knockout SKMc15, is a protein which shares regeneration events. TIS7, induced by mice generated in our lab as a specific with TIS7 high homology at the amino the mitogen TPA or growth factors, is experimental animal model, identified TIS7 acid level. Therefore, our laboratory differentially expressed in several different to be the main modifier of the severity of generated SKMc15 single as well as TIS7

20 Research Report 2015 Medical University of Innsbruck Cell Biology

SKMc15 double knockout mice and now concentrates on the identification of the functional role of both genes and their protein products. Interestingly, the TIS7 SKMc15 double knockout mice have a prominent phenotype: they are significantly smaller and leaner and, most importantly, they are resistant against weight gain upon feeding with the high fat-diet. We are currently searching for the mechanism responsible for this phenotype on the molecular level.

Future Goals: • Identification of molecular mechanisms responsible for the smaller body size and the lack of body fat deposits in TIS7 SKMc15 double knockout mice. The long Fig. 9: Schematic representation of the MVB Pathway. term goal of this project is to be able to design strategies for intervention with possible signaling pathways. • Identification of TIS7-interacting proteins and analyses of their biological role. In this project we will concentrate on further characterization of interactions between TIS7 protein complex components, mainly on their in vivo interactions within the living cell. The main focus will be on regulatory mechanisms by which TIS7 modulates gene expression of muscle-specific Fig. 10: (A) 3D-Modeling of cryo-fixed cells. Fig. 11: (A) Fluorescence microscopy of liv- genes during myogenesis. MVBs (yellow), Intralumenal MVB vesicles ing yeast, vacuole (red), GFP-CPS (green). • Identification and detailed analysis of epi- (ILVs) (red), vacuole (blue). (B) Model of ES- (B) Graphical representation of quantitive genetic mechanisms of transcriptional CRT-III and Vps4 during ILV neck constric- proteomics. regulation affected by TIS7. tion.

Membrane Traffic and Signaling host cells, micro-vesicle formation at the Selected Publications David Teis Lab plasma membrane, plasma membrane The coordinated action of the MVB pathway and autophagy ensu- Cell growth and survival requires the selective repair, quality control of nuclear pore res cell survival during starvation. Müller M, Schmidt O, Angelova M, Faserl K, Weys S, Kremser L, Pfaffenwimmer T, Dalik T, Kraft C, degradation of cellular components. Failure complex assembly and nuclear envelop Trajanoski Z, Lindner H, Teis D. ELIFE. 2015, Apr 22;4:e07736. in cellular degradation systems result in reformation and sealing. SLC38A9 is a component of the lysosomal amino acid sensing severe defects in cell homeostasis, which in machinery that controls mTORC1. Rebsamen M, Pochini L, Stasyk T, de Araujo ME, Galluccio M, Kandasamy RK, Snijder B, Fauster A, turn can cause in a wide variety of diseases Just how the ESCRT machinery catalyzes Rudashevskaya EL, Bruckner M, Scorzoni S, Filipek PA, Huber KV, ranging from cancer to neurodegeneration. these membrane remodeling reactions is Bigenzahn JW, Heinz LX, Kraft C, Bennett KL, Indiveri C, Huber LA. We are particularly interested in the unclear. NATURE. 2015 Mar 26;519(7544):477–81. LAMTOR2 regulates dendritic cell homeostasis through FLT3-de- molecular mechanisms required for the One key question in our lab is how the pendent mTOR signalling. Scheffler JM, Sparber F, Tripp CH, selective degradation of integral membrane ESCRT machinery sculpts membranes and Herrmann C, Humenberger A, Blitz J, Romani N, Stoitzner P, Huber proteins. A key step for the selective how ESCRT mediated membrane remodel LA. Nat Commun. 2014 Oct 22;5:5138. degradation of membrane proteins occurs ing and scission is coordinated with cargo The late endosomal p14-MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration. Schiefermeier on endosomes, where the endosomal sorting (Fig. 10 B). N, Scheffler JM, de Araujo ME, Stasyk T, Yordanov T, Ebner HL, complexes required for transport (ESCRTs) Offterdinger M, Munck S, Hess MW, Wickström SA, Lange A, Wunderlich W, Fässler R, Teis D, Huber LA. bind to and sort ubiquitinated membrane Furthermore we would like to understand J Cell Biol. 2014 May 26;205(4):525–40. proteins via the multivesicular body (MVB) how the ESCRT dependent degradation Coordinated binding of Vps4 to ESCRT-III drives membrane neck pathway into the lumen of lysosomes for of membrane proteins contributes to cell constriction during MVB vesicle formation. Adell MA, Vogel GF, Pakdel M, Müller M, Lindner H, Hess MW, Teis D. degradation (Fig. 9). This process requires growth and survival, particularly given the J Cell Biol. 2014 Apr 14;205(1):33–49. a membrane remodeling reaction that buds key role of ESCRTs during developmental intraluminal MVB vesicles (ILVs) away from and disease. Selected Funding the cytoplasm and into the lumen of MVBs • FWF: Special research program SFB021 (LAH, DT) (Fig. 10 A). Topologically similar ESCRT To address these questions we use yeast • PhD Program MCBO (LAH, DT) • START-Prize (DT) dependent membrane budding reaction as the best suited model system combining • P18531-B12 (TV) are required in distinct cellular processes, genetics with quantitative proteomics, • P22350-B12 (IV) • P26682-B21 (LAH) including membrane scission at the end of biochemical methods and different imaging • EU: FP7 Optatio (LAH) cytokinesis, release of budding HIV from approaches (Fig. 11 A, B). • HFSP Career Development Award (DT)

Research Report 2015 Medical University of Innsbruck 21 Biocenter Genomics and RNomics

Therefore, we have characterized the small • Member of the 7th framework EU: ncRNA transcriptome, invoIved in the ncRNAPain differentiation of mouse embryonic stem (ES) cells into neural cells, by generating Core Facilities: specialized ribonucleo-protein particle • High-throughput sequencing: (RNP)-derived cDNA libraries. By high- Genome Seq Core throughput sequencing and transcriptional • Expression profiling: profiling we identified hundreds of novel Affymetrix Core Facility ncRNAs that are involved in ES cell differentiation. Based on these findings, Research we have generated a custom microarray chip that covers 1500 novel neuro-specific Role of ncRNAs in Neurodevelopmental ncRNAs. By this approach, we have analysed Disorders the differential expression of ncRNAs in Alexander Hüttenhofer mouse models for neurodegenerative Small non-protein-coding RNAs (ncRNAs) diseases such as Alzheimer’s disease, play important roles in the regulation of Parkinson’s disease and Epilepsy. gene expression and have been implicated A second research focus is the regulation in a number of diseases of the central Head of Division: of ribosomal translation by natural and nervous system (CNS). miRNAs represent Univ.-Prof. Dr. Alexander Hüttenhofer non-natural modifications introduceda well characterized class of small ncRNAs into functionally important regions of the for which numerous commercial tools (e.g. Contact: ribosome as well as those incorporated into qPCR panels, micro arrays) have been Biocenter, Innrain 80–82 the coding sequences of the mRNAs. developed in order to screen for their 6020 Innsbruck differential expression in human patients as Major Achievements: well as animal disease models. Indeed, by [email protected] • Coordination GEN-AU Programme: these approaches several miRNAs have been Phone: +43 512 9003 70250 ncRNAs: from identification to functional implicated in the etiology of neurological Fax: +43 512 9003 73100 characterization diseases. In addition to miRNAs there http://www.rnomics.at/ • Member of the 7th framework EU: SysKid also exists a large number of short ncRNA • PhD program participant: SPIN: species (sized about 18 – 200 nt), which are signal processing in neurons either poorly characterized or that belong • Member of SFB program: to other known classes of ncRNAs (i.e. Keywords Cell signaling in chronic CNS disorders snRNAs, snoRNAs, or piRNAs) for which no

Non-coding RNAs; RNPs, ribosomal RNA, RNA sequencing (RNAseq), ribosome, translation

Research Focus

In cells from all organisms two different types of RNA molecules are found: messenger RNAs (mRNAs), and the so-called “non-protein-coding RNAs” (ncRNAs). Many known ncRNAs, such as microRNAs, are involved in the regulation of gene expression. Our group focuses on the identification and functional characterization of regulatory non-coding RNAs in various model organisms. In particular, we are interested in the identification of ncRNAs regulating neural development and in the identification of ncRNAs involved in human diseases.

General Facts

Our group works on the identification and function of regulatory non-coding RNAs (ncRNAs) in various model organisms for Fig. 1: Two classes of RNA species are transcribed from genomes of all organisms: messen- which we have coined the term “Experimental ger RNAs (mRNAs) and non-coding RNAs (ncRNAs); ncRNAs are not translated into proteins RNomics”. In particular, we are interested in and many of them are able to regulate gene expression by regulating transcription or trans- ncRNAs involved in neurological diseases. lation of mRNAs and thus act a genetic switches.

22 Research Report 2015 Medical University of Innsbruck Genomics and RNomics

high-throughput tools have been developed disease patients and their comparison with Although more than 100 different types to perform expression profiling. Thus in this healthy controls we were able to identify have been identified, mRNAs were thought project, which is part of the SFB-F44 “Cell 51 differentially expressed ncRNAs in to be only rarely modified. signaling in chronic CNS disorders”, we have Alzheimer’s disease and could also show Whereas N6-methyladenosine (m6A) developed an unbiased and comprehensive that 60 % of the ncRNAs that are present was described already 4 decades ago microarray platform to profile the on our customized microarray exhibit and shown to be the most abundant expression of thousands of these novel expression signals above background in modification in eukaryotic mRNAs, the ncRNA species from mouse brain tissues. human tissue. In addition, we focused presence of 5-methylcytosine (m5C) and To date we have applied this customized on the biochemical characterization of pseudouridine (Ψ) in this class of RNA microarray, designated as neuro-ncRNA the novel ncRNA candidates using in situ was only identified recently. The influence chip, to selected mouse models for LTCC hybridization in order to define the cellular of any of these modificationson ribo activity and CNS disorders e.g. Alzheimer’s as well as subcellular localization patterns somal translation is largely unknown and disease and Multiple-system atrophy. of ncRNAs. has been mainly a matter of speculation. Thereby we discovered more than 100 Employing a cell free translation system, novel ncRNA candidates whose expression Identification of ncRNA Patterns we systematically investigate the effects was found to be de-regulated in comparison as Biomarkers for Pain and Inter- of single modified mRNA residues on the to wild type controls. In the Alzheimer Individual Variations fidelity and efficiency of translation. mouse model, we identified two snoRNAs, Alexander Hüttenhofer whose expression was deregulated prior to This project is part of the EU Project Modifications of the Ribosomal Decod- amyloid plaque formation. Interestingly, the “ncRNAPain” and aims to identify pain ing Site and their Impact on Translation presence of snoRNAs could be detected predisposing ncRNA patterns and to apply Matthias Erlacher in cerebral spine fluid samples in humans, them as biomarkers for pain and inter- Ribosomal decoding is an essential process thus potentially serving as early diagnostic individual variation. This aim will be achieved in every living cell. During protein synthesis makers for Alzheimer’s disease. In addition, by identifying altered expression patterns the 30S ribosomal subunit needs to we could show the applicability of our of ncRNAs/miRs in painful vs. non-painful accomplish binding and accurate decoding customized microarray to human post- diabetic neuropathies (dPNP), in complex of mRNAs. Through mutational studies mortem brain tissue of Alzheimer’s disease regional pain syndrome (CRPS) after trauma and the analysis of high-resolution crystal patients and healthy individuals; Through vs. patients after trauma without CRPS structures nucleotides G530, A1492 and the expression profiling of post mortem and in addition in painful and non-painful A1493 of the 16S ribosomal RNA have come human brain samples from Alzheimer’s nerve lesions (NL). An initial quality check into focus as important elements for the showed that ncRNAs levels can be robustly decoding process. In order to biochemically measured in white blood cells and in serum . investigate decoding in greater detail we A first analysis performed in the white applied an in vitro reconstitution approach. blood cells and serum of n=10 patients This approach allowed us to exchange or with painful and non-painful dPNP revealed eliminate single chemical groups on A1492 that ncRNA profiles canalmost perfectly and A1493 and to test their impact on differentiate between these two subgroups. translational efficiency and fidelity.

Modified RNA Nucleotides Regulate Selected Publications Ribosomal Translation Generation of a neuro-specific microarray reveals novel diffe- Matthias Erlacher, rential expressed noncoding RNAs in mouse models for neuro degenerative diseases. Gstir R, Schafferer S, Scheiderler M, Alexander Hüttenhofer Misslinger M, Griehl M, Dachil N, Humpel C, Obermair GJ, RNA modifications can be found in every Schmuckermair C, Striessnig J, Flucher BE, Hüttenhofer A. organism in all three domains of life. RNA. 2014; 20 (12): p. 1929–1943. Micro RNAs in nociceptive circuits as predictors of future clinical applications. Kress M, Hüttenhofer A, Landry M, Kuner R, Favereaux A, Greenberg D, Bednarik J, Heppenstall P, Kronenberg F, Malcangio M, Rittner H, Uçeyler N, Trajanoski Z, Mouritzen P, Birklein F, Sommer C, Soreq H. Front Mol Neurosci. 2013; 6: S 33.

Selected Funding SFB F44-11 – Cell signaling in chronic CNS disorders, FWF 7th framework EU: SysKid 7th framework EU: ncRNAPain

Collaborations • Joerg Vogel, MPI, Berlin, Germany • Ralph Bock, MPI Potsdam, Germany • Jürgen Brosius, University of Münster, Germany • Yuuchi Soeno, Nippon University, Tokyo, Japan • Norbert Polacek, University of Bern

Core Facilities Fig. 2: Non-natural modifications can be site-specifically incorporated into the 16S rRNA, to • Genome Seq Core determine their impact on decoding. • Affymetrix core facility

Research Report 2015 Medical University of Innsbruck 23 Biocenter Molecular Biology

expression regulation the high standard of research quality. • Posttranslational acetylation of regula- Staff of the Division of Molecular Biology also tory non-histone proteins contribute substantially to the curricular teaching activities at the MUI. Notably, the General Facts division chair Peter Loidl is Vice Rector for Academic Affairs at the university. He also The Division of Molecular Biology is home took a leading role in the establishment of to six independent research groups, whose the two new study directions in Molecular scientific interests range from the investiga- Medicine (Bachelor and Master studies). tion of diverse aspects of filamentous fungi Alexandra Lusser is coordinator, Hubertus physiology and metabolism, to the study of Haas is deputy coordinator of the PhD secretory lipocalins, to research into the na- Program “Regulation of Gene Expression” ture and significance of chromatin remode- and Bernhard Redl is coordinator of the ling mechanisms. Molecular Medicine Master program. A common long-term goal of all groups is Beyond that, all group leaders are teaching to explore the relationship of the diverse lectures, seminars and practical courses processes mentioned above with diagnos- in the curricula of human medicine, dental tics and treatment of human disease. In medicine, molecular medicine (bachelor Head of Division: this regard, the groups of Hubertus Haas, and master) and of the PhD curriculum. In Univ.-Prof. Dr. Peter Loidl Gerald Brosch/Stefan Grässle, and Flor- addition, most group leaders are involved entine Marx-Ladurner strive to elucidate in the teaching of Biology students at the Contact: pathogenicity determinants and potential Leopold Franzens University Innsbruck. Biocenter, Innrain 80–82 drug targets of filamentous fungi. Moreover, 6020 Innsbruck they examine the regulatory mechanisms of Research secondary metabolites (e.g. penicillin), se- [email protected] cretory proteins and components, such as Physiology, Gene Regulation and Phone: +43 512 9003 70201 antimicrobial proteins and iron-chelating si- Secondary Metabolism in Filamentous Fax: +43 512 9003 73100 derophores, both of which have potential in Fungi http://mol-biol.i-med.ac.at/ antifungal therapy and diagnosis. The Redl Gerald Brosch, Stefan Graessle, Huber- group investigates the mechanism of action tus Haas, Florentine Marx-Ladurner of lipocalins, which are secretory scaven- Fungi affect the life of mankind in positive ger proteins. Thus, lipocalins are important and negative ways. On the one hand, Keywords components of the innate immune system, fungi are major players in saprobic yet they might also be involved in allergic decomposition, they mutually interact Chromatin and epigenetics, histone mod- reactions. The Lusser group conducts stud- with plants (mycorrhiza), serve as food ifying enzymes, ATP-dependent chromatin ies of fundamental gene regulatory mecha- source (mushrooms) or in food production remodeling, RNA methylation, filamentous nisms involving the remodeling of chroma- (e.g. bread, cheese, alcohol), and produce fungi, iron metabolism, fungal infection, tin structure as well as posttranscriptional widely used primary (e.g. citric acid) and siderophores, lipocalins, antimicrobial modification of RNAs. One particular focus secondary metabolites (e.g. penicillin). On proteins, innate immunity and allergy is on understanding chromatin changes in the other hand, some fungi are pathogens the context of neurodegenerative diseases of plants (e.g. Fusarium spp.) and animals Research Focus and behavioral disorders. Finally, the Loidl (e.g. Aspergillus fumigatus), or spoil food group investigates histone modifying en- by contamination or toxin production (e.g. Physiology, gene regulation and second- zymes, in particular the role of acetylation aflatoxin). Therefore, fungi impact ecology, ary metabolism in filamentous fungi of regulatory non-histone proteins. biotechnology, medicine, agriculture and • Iron metabolism in filamentous fungi: Together, the researchers make use of a links to human disease wide array of experimental model systems • Functions of histone modifying enzymes including filamentous fungi Aspergillus( , in gene regulation, fungal physiology Penicillium, Achremonium), the fruit fly and as targets for novel antifungal sub- Drosophila melanogaster, and mammalian stances models such as different cell lines as well • Structure, mechanism of action and as knock-out mice. applicability of antimicrobial peptides/ The participation of members of the division proteins secreted by filamentous fungi in several intra- and extramural network activities such as the FWF -funded PhD Lipocalins and their involvement in programs “HOROS” and “MCBO”, the special innate immunity and allergy research network SFB “Cell signaling in chronic CNS disorder” as well as the EU-FP7 Epigenetics and epitranscriptomics: Marie Curie International Training Network Fig. 1: Cover Figure of Natural Product Re- • Biological roles of ATP-dependent chro- “Nucleosome4D”, the Infect-ERA network ports 31(10). “Fungal siderophore metabo- matin remodeling enzymes “AspMetNet” and the D-A-CH network on lism with a focus on Aspergillus fumigatus”. • RNA methylation and its impact on gene iron sensing in filamentous fungi attests to Haas H. 2014.

24 Research Report 2015 Medical University of Innsbruck Molecular Biology

food industry. The best-studied fungal mechanisms. Characterization of fungal organism is Saccharomyces cerevisiae. In iron uptake and storage, particularly the several respects, however, the physiology siderophore system. of this unicellular organism is not Regulatory and structural links of iron comparable to that of the more complex homeostatic mechanims and other filamentous fungi (e.g. iron metabolism, metabolic pathways, e.g. pH regulation, light regulation, secondary metabolism). ergosterol biosynthesis, hypoxia adaptation. Three research groups in the Division of First-time in vivo PET-imaging of fungal Molecular Biology address different aspects infections using 68Gallium-labelled sidero- of filamentous fungal physiology ranging phores from iron metabolism and its significance Future Goals: for pathogenesis (Haas), to chromatin- Detailed characterization of the iron Fig. 2: Production of penicillin (PN) in differ- linked mechanisms of gene expression homeostasis-maintaining mechanisms of ent Aspergillus HDAC-mutants. A bacterial control (Brosch/Graessle) and the nature filamentous fungi (in particular ofAspergilli ) growth inhibition assay plate with Kocuria and mechanisms of action of antimicrobial and applied medical and biotechnological rhizophila as indicator organism was used proteins produced by filamentous fungi exploitation of the knowledge gained. to quantify PN in the medium of wild type (Marx-Ladurner). (wt), ΔhdaA, ΔhosA, and two double mutant Functions of Histone Modifying strains after 24h, 48h, and 60h of growth. Iron Metabolism in Filamentous Fungi: Enzymes in Gene Regulation and The relative sizes of bacterial growth inhi- Links to Human Disease Fungal Physiology bition zones correspond to relative accumu- Hubertus Haas Gerald Brosch and Stefan Graessle lation of PN in the culture medium of the Our central research goal is to characterize In addition to distinct regulatory sequences fungus. Whereas the class 2 HDAC HdaA the fungal metabolism and to exploit in gene promoters, the readout of genetic has a repressing effect, the class 1 enzyme this knowledge for both improvement of information in eukaryotes is significantly HosA seems to be crucial for the production antifungal therapy and diagnosis of fungal controlled at the chromatin level. In addition of PN in Aspergillus nidulans. infections as well as improvement of the to ATP-dependent chromatin remodeling biotechnological potential of fungi. Current and the methylation of DNA on distinct research focus is the iron/siderophore cytidines, covalent posttranslational in the HDAC RpdA, which are essential for metabolism of Aspergilli. A. fumigatus is a modifications of histones have profound the viability of Aspergillus and thus might typical saprobic filamentous ascomycete structural and functional consequences serve as targets for future antifungal but also the most common airborne fungal for the transcription program of a cell. therapy. pathogen of humans. It causes allergic The main research focus of the lab lies on Identification of the HDAC HosA as a major and invasive disease depending on the elucidating the functional impact of histone regulator of secondary metabolites in immune status of the patient. Unsatisfying acetylation and histone/protein arginine filamentous fungi. diagnostic and therapeutic possibilities methylation on fungal physiology. We are Future Goals: are reflected in a high mortality rate. particularly interested in studying to what Characterization of RmtD substrates and A. fumigatus and its low-pathogenic relative extent histone modifying activities are elucidation of the biological role of RmtD. Aspergillus nidulans produce extracellular involved in fungal pathogenicity as well as Development of antifungal strategies tar- siderophores (triacetylfusarinine C) for iron to investigate their role in the regulation of geting the fungal-specific domains of RpdA. acquisition and intracellular siderophores secondary metabolite production. Elucidation of novel HosA-regulated small (ferricrocin) for storage and distribution To this end, we have generated Aspergillus bioactive molecules of Aspergillus. of iron. Siderophore biosynthesis is strains with individual or pairwise deletions regulated by two transcription factors, of all protein arginine methyltransferase Structure, Mechanism of Action and SreA and HapX. Siderophores are central (PRMT) genes and of the class I histone Applicability of Antimicrobial Peptides/ components of the fungal metabolism as deacetylases (HDACs) RpdA and HosA. Proteins Secreted by Filamentous Fungi they affect germination, sexual and asexual Using these tools along with specifically Florentine Marx-Ladurner reproduction, oxidative stress resistance engineered transgenes, we study their Filamentous fungi secrete a wide array of and virulence. Lack of siderophore impact on viability and metabolism of the different proteins into the external medium, biosynthesis renders A. fumigatus fungus. Moreover, we perform proteomic and which are used for diverse functions, such apathogenic. Consequently, the siderophore transcriptomic analyses to (i) characterize as nutrient assimilation, quorum sensing, system represents a novel attractive target novel substrates and (ii) to investigate the host invasion and colonization, etc. Apart for improvement of antifungal therapy and contribution of HDACs and PRMTs to the from some secreted enzymes, which have diagnosis of fungal infections. regulation of secondary metabolism as well been developed for a variety of commercial Additional research topics include light reg- as of stress response genes. uses (mainly for the fermentation industry), ulation, nitrogen metabolism, noncoding Major Achievements: only few extracellular proteins are well RNAs, secondary metabolism (e.g. ceph- Identification of a novel PRMT (RmtD) in characterized with respect to their alosporin biosynthesis by Achremonium filamentous fungi, which differs from other function as pathogenicity or cell signaling chrysogenum) and improvement of molecu- PRMTs in that it does not accept canonical factors. Our main scientific interest is to lar tools for the manipulation of fungi. substrates (histones, RNPs) but instead identify, isolate and further characterize Major Achievements: methylates three as yet unknown proteins. on the molecular, structural and functional Identification and characterization of Identification and functional characteriza- levels novel extracellular proteins with fungal iron-regulatory and iron-sensing tion of two fungal-specific protein domains antimicrobial activity from Penicillium

Research Report 2015 Medical University of Innsbruck 25 Biocenter

Fig. 3: Structure and function of the small, cationic and cysteine-rich Penicillium chrysogenum antifungal protein PAF. (A) PAF consists of 55 amino acids and exhibits five anti-parallelβ -strands (green arrows) that are connected by four loop regions (blue). The cysteines are marked in yellow and form three disulfide bonds that stabilize the protein. Superimposed on the scheme of the secondary structure are the hydrophobic (red) and hydrophilic (blue) patches exposed on the protein surface that are responsible for full protein activity. (B) PAF inhibits the growth of the human pathogen Aspergillus fumigatus. Fungal hyphae treated with 64 μM PAF show reduced growth and hyper-branching, a typical effect of PAF on the morphology of sensitive fungi. In the untreated control neither growth reduction nor changes in morphology can be detected. chrysogenum, Aspergillus nidulans and involved in a variety of physiological Epigenetics and Epitranscriptomics Aspergillus fumigatus. Antimicrobial processes including retinoid, fatty acid and proteins are promising candidates for the pheromone signaling, immunomodulation, Chromatin Remodelling and development of novel therapies applicable inflammation, detoxification, modulation of RNA Modifications in medicine as well as in agriculture growth and metabolism, tissue development, Alexandra Lusser and in the food industry to prevent and apoptosis, and even behavioral processes. Eukaryotic DNA is assembled into a treat microbial infections. Therefore, the Whereas the structural basis of lipocalin- nucleoprotein complex termed chromatin. detailed characterization of these proteins ligand binding is now well understood, there The basic repeating unit of chromatin is is of crucial importance and a prerequisite is a major lack of knowledge regarding the nucleosome, which consists of 147 bp for the development of new therapeutic the mechanisms by which lipocalins exert of DNA wrapped around an octamer of approaches and their successful application their biological effects. This is mainly the core histones H2A, H2B, H3 and H4. in the future. due to the fact that only limited data The way in which DNA is organized in Major Achievements: are available on lipocalin receptors and the chromatin allows for highly efficient First steps towards biotechnological lipocalin-receptor interactions, although compaction of the genetic material and utilization of antimicrobial proteins and it is well accepted that many, if not all, of provides additional levels of control to understanding their structure-function these proteins are able to bind to specific the regulation of nuclear processes such relationship. cell receptors. Our main research focus as transcription, replication, repair and Future Goals: is on the identification of cellular lipocalin recombination. We are interested to learn Identification of molecular targets for the receptors, characterization of the molecular how the establishment and maintenance development of new therapeutic drugs. mechanisms of receptor-ligand interaction Characterization of additional cellular and the biological processes beyond functions of antifungal proteins apart from receptor binding. In addition, we study novel their antimicrobial activity. Development functions of lipocalins in innate immmunity of new chimeric antifungal proteins with and allergy. enhanced activity and improved specificity. Major Achievements: Elucidation of the lipocalin allergen uptake Lipocalins and their Involvement in in dendritic cells Innate Immunity and Allergy Future Goals: Bernhard Redl Identification and characterization of novel We investigate structural and functional lipocalin receptors with a focus on human features of human lipocalins. The protein proteins. superfamily of lipocalins consists of • We will use a set of biochemical methods, small, mainly secretory proteins defined including in vivo crosslinking, affinity puri- on the basis of conserved amino acid fication and mass spectrometry analysis Fig. 4: Types of molecular recognition prop- sequence motifs and their common for identification of specific receptors for erties of lipocalins. (1) lipophilic ligands structure. Functionally, they are important the lipocalins ApoD and the major dog and illustrated as cargo. (2) soluble macro- extracellular carriers of lipophilic cat allergens Can f 1 and Fel d 4. molecule ligands such as proteins. (3) a compounds in vertebrates, invertebrates, • Isolation of receptors for human odorant- lipocalin-specific membrane receptor is re- plants and bacteria. There is increasing binding proteins (hOBP) by using phage- sponsible for cellular uptake of the lipoca- evidence that this group of proteins is display technology. lin-ligand complex.

26 Research Report 2015 Medical University of Innsbruck Molecular Biology

Selected Publications Fungal siderophore biosynthesis is partially localized in peroxisomes. Gruendlinger M, Yasmin S, Lechner BE, Geley S, Schrettl M, Hynes M, Haas H. MOLECULAR MICROBIOLOGY. 2013; 88: p. 862–875.

Regulation of Sulphur Assimilation Is Essential for Virulence and Affects Iron Homeostasis of the Human-Pathogenic Mould Asper- gillus fumigatus. Amich J, Schafferer L, Haas H, Krappmann S. PLOS PATHOGENS. 2013; 9: p. e1003573.

The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess. Gsaller F, Hortschansky P, Beattie SR, Klammer V, Tuppatsch K, Lechner BE, Rietzschel N, Werner ER, Vogan AA, Chung D, Muehlenhoff U, Kato M, Cramer RA, Brakhage AA, Haas H. EMBO JOURNAL. 2014; 33: p. 2261–2276.

Fungal siderophore metabolism with a focus on Aspergillus fumigatus. Haas H. NATURAL PRODUCT REPORTS. 2014; 31: p. 1266–1276.

An endogenous promoter for conditional gene expression in Acre- monium chrysogenum: The xylan and xylose inducible promoter xyl1(P). Blatzer M, Gsaller F, Abt B, Schrettl M, Specht T, Haas H. JOURNAL OF BIOTECHNOLOGY. 2014; 169: p. 82–86.

Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis. Winter M, Moser MA, Meunier D, Fischer C, Machat G, Mattes K, Lichtenberger BM, Brunmeir R, Weissmann S, Murko C, Humer C, Meischel T, Brosch G, Matthias P, Sibilia M, Seiser C. EMBO JOURNAL. 2013; 32: p. 3176–3191. Fig. 5: 3D modelling of the XIST lncRNA region A predicts a stacked helix conformation for Antifungal proteins: More than antimicrobials? Hegedues N, Marx repeat 8 (R8). The methylated cytosines in R8 (marked in red in inset) are facing the outer F. FUNGAL BIOLOGY REVIEWS. 2013; 26: p. 132–145. surface of the helix, which can explain the interference of cytosine methylation with PRC2 Long non-coding RNAs as targets for cytosine methylation. Amort T, Souliere MF, Wille A, Jia X, Fiegl H, Woerle H, Micura R, Lusser A. binding (Amort et. al., 2013). RNA BIOLOGY. 2013; 10: p. 1003–1009.

Copper(I)oxide surface modified cellulose fibers-Synthesis, char- of eukaryotic chromatin affects those Posttranslational Acetylation of acterization and antimicrobial properties. Emam HE, Manian AP, processes. We are approaching this question Siroka B, Duelli H, Merschak P, Redl B, Bechtold T. Regulatory Non-histone Proteins SURFACE & COATINGS TECHNOLOGY. 2014; 254: p. 344–351. by studying the molecular mechanisms and Peter Loidl Antibacterial activity of rifamycins for M. smegmatis with compar- biological context of chromatin assembly Histones are prominent substrates of ison of oxidation and binding to tear lipocalin. Staudinger T, Redl and remodeling processes. Major research posttranslational modifications, likeB, Glasgow BJ. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS. 2014; 1844: p. 750–758. questions in my lab are: acetylation, methylation, phosphorylation (i) The biochemical analysis of chromatin and others which all can cause structural Selected Funding assembly processes using in vitro assays and functional rearrangements in chromatin • In search of novel human lipocalin receptors, FWF, Bernhard Redl and single molecule techniques in and therefore represent essential elements • New antifungal strategies: structure and function of NFAP, collaboration with the C. Dekker lab at the of the complex epigenetic histone code. FWF ‑Meitner Program, Lazlo Galgóczi • Novel molecular mechanisms of iron sensing and homeostasis in TU Delft. (ii) The study of biological functions During the last years it became more filamentous fungi, FWF (D-A-CH), Hubertus Haas of the ATP-dependent chromatin remodeling and more clear that a huge number of • Characterization of heme metabolism in Aspergillus fumigatus, FWF, Hubertus Haas factor CHD1 in Drosophila and in the mouse non-histone proteins are substrates for • Host response in opportunistic infections, HOROS PhD Program, and (iii) the study of centromeric chromatin enzymes that were initially identified as FWF, Hubertus Haas • Systematic identification of antifungal drug targets by a metabol- assembly in Drosophila. In addition, we have histone-modifying enzymes: this holds true, ic network approach (AspMetNet), FWF (Infect-ERA), Hubertus recently become interested in exploring in particular, for histone acetyltransferases Haas • Hunting for new antifungal strategies: the antifungal protein PAF, cytosine methylation of poly(A)RNAs and (HATs) and HDACs. The focus of our research FWF, Florentine Marx-Ladurner to investigate its impact on gene regulation is the analysis of functional consequences • Structure and function of the antifungal proteins PAFB and NFAP, (“epitranscriptomics”). of acetylation of non-histone proteins, such OTKA-FWF bilateral funding, Florentine Marx-Ladurner • Cytosine methylation as a new mechanism to regulate long as the nucleolar transcription factors UBF non-coding RNAs, TWF/FWF, Alexandra Lusser Major Achievements: and PAF53 and the cell cycle regulatory • Study of centromeric chromatin assembly pathways in Drosoph- Characterization of the requirement of protein Rb2/p130. ila melanogaster, MCBO-PhD Program, FWF, Alexandra Lusser CHD1 for sperm chromatin reorganization Major Achievements: Collaborations and histone variant incorporation in vivo. Identification of UBF and PAF53 as Elaine Bignell, Imperial College, London, UK; Axel Brakhage, F. Involvement of CHD1 in Drosophila stress well as of Rb2/p130 as substrates for Schiller University Jena, Germany; Robert Cramer, Geisel School of Medicine at Dartmouth, USA; Michael J. Hynes, Univ. of Mel- response and in local immune response. posttranslational acetylation. bourne, Australia; Jean-Paul Latgé, Institut Pasteur, Paris, France; Identification of distinct cytosineDemonstration of cell-cycle dependence of Antonio DiPietro, Univ. Cordoba, Spain; William Nierman, George Washington Univ., Rockville, USA; gillian Turgeon, Cornell Univ., methylation on the long-noncoding RNAs Rb2/p130 acetylation and characterization Ithaca, USA; Cees Dekker, Nynke Dekker, Technical University XIST and HOTAIR and its impact on RNA- of the cross talk between Rb2/p130 Delft, Netherlands; Chin-Yan Lim, A*-STAR, Singapore; Dmitry Fyodorov, Albert Einstein College of Medicine, Bronx, USA; Gyula protein interaction. acetylation and cell cycle-dependent Batta, University of Debrecen, Hungary; László Galgóczi, Univer- Future Goals: phosphorylation. sity of Szeged, Hungary; Nick D. Read, University of Manchester, UK; José F. Marcos, IATA, Valencia, Spain; Nancy Keller, University Study of biological roles of CHD1 in Future Goals: of Wisconsin, Madison, USA; Manfred Jung, Albert-Ludwigs-Uni- Drosophila Study of the effects of mutations of versität Freiburg, Germany; Antonello Mai, Università degli Studi di Roma “La Sapienza”, Italy; Gianluca Sbardella, Università di Study the prevalence and physiological acetylatable lysines in Rb2/p130 on cell Salerno, Fisciano, Italy; Arne Skerra, TU Munich, Freising, Ger- significance of RNA base modifications cycle progression. many; Ben J. Glasgow, University of California, Los Angeles, USA.

Research Report 2015 Medical University of Innsbruck 27 Biocenter Experimental Pathophysiology and Immunology

Theodor von der Wense rebuilt the institute, sulted in the development of an Hsp60- and acted as Ordinarius until 1973. He died based, orally tolerizing vaccine against in 1977 (http://de.wikipedia.org/wiki/ atherosclerosis. Theodor_von_der_Wense). The Immunology of Fibrosis Kurt Loewit, a descendant of the above Fibrosis is an important consequence of mentioned Moritz Loewit, took over as various pathological conditions ranging interim chief until 1975, when Georg Wick from tissue damage, over inflammation, was nominated. Wick – an immunologist with reactions against foreign body implants to long-term training in USA in the laboratory “spontaneous” fibrotic diseases, always of Witebsky, the founder of the concept of being associated with inflammatory autoimmunity – extended the institute to a immunologic processes. An impaired large unit of sometimes 50 collaborators function of regulatory T cells (Treg) within working in different fields, .i e. besides fibrotic tissues has been recently shown by immunology also in endocrinology and us. www.autoimmunity.at molecular biology. A large armamentarium of research as well as diagnostic methods Systemic Sclerosis – Roswitha Sgonc was implemented. Later, the institute was The group is interested primarily in the renamed as Institute of PATHOPHYSIOLOGY pathogenesis and therapy of systemic Head of Division (interim): (reflecting its teaching subject more sclerosis (SSc), which is studied in human Univ.-Prof. Dr. Lukas A. Huber properly), and finally divided into 3 smaller patients as well as in the spontaneous units (Divisions), i.e. avian model UCD-200/206, the only animal Contact: model that manifests the whole clinical, Biocenter, Innrain 80–82 • Experimental Pathophysiology & Immu- histopathological and serological spectrum 6020 Innsbruck nology (Georg Wick, later interimistically of human SSc. Thus, only the comparative Lukas A. Huber) study of UCD-200/206 chickens and [email protected] • Molecular Pathophysiology human SSc made it possible to identify Phone: +43 512 9003 70171 (Reinhard Kofler) microvascular endothelial cells as the Fax: +43 512 9003 73960 • Developmental Immunology primary target of the autoimmune attack. www2.i-med.ac.at/expatho/ (Andreas Villunger) After many years studying pathomechanisms index.html and genetic factors underlying the disease, All three joined the Biocenter, where they we are now focusing on the development of act independently with respect to their novel therapeutic approaches. There is an Keywords research interests, yet are still combined unmet need for an effective pro-angiogenic in their teaching duties in what is called therapy of ischemic lesions in patients with Autoimmunity, molecular endocrinology, BEREICH PATHOPHYSIOLOGIE. SSc. Vascular alterations in both human atherosclerosis, systemic sclerosis, fibrosis, and avian SSc predominantly affect the teaching Pathophysiology to medical, PhD Research microvasculature. Initially, endothelial cell (MCBO) and Molecular Medicine students apoptosis is induced by anti-endothelial cell Laboratory of Autoimmunity antibody-dependent cellular cytotoxicity Research Focus Georg Wick (ADCC) via the Fas/Fas ligand pathway. The Immunology of Atherosclerosis Intimal proliferation, occlusion of blood • The Immunology of Atherosclerosis. Heat This project of the last two decades vessels, and capillary rarefaction lead to shock protein 60, “danger signal” “attract- resulted in the formulation of a new decreased blood flow, a state of chronic ing” preexisting innate and adaptive anti- “Autoimmune Concept for the Development ischemia, and to clinical manifestations Hsp60 immunological reactions. of Atherosclerosis”, supported by solid such as fingertip ulcers and comb • The Immunology of Fibrosis, impaired data from in vitro and animal experiments lesions. Tissue hypoxia normally induces function of regulatory T cells (Treg). as well as from cross-sectional and angiogenesis, but in SSc vascular repair and • Pathogenesis and therapy of systemic prospective longitudinal studies in human angiogenesis seem to be strongly disturbed. sclerosis. cohorts. In essence, this concept states One of the key molecules in the induction that classical atherosclerosis risk factors of angiogenesis is vascular endothelial General Facts first act as endothelial stressors inducing growth factor (VEGF). In SSc chronic and the expression of a stress protein (heat uncontrolled over-expression of VEGF The former institute of EXPERIMENTAL shock protein 60 – Hsp60) which then acts results in chaotic vessels, and intractable PATHOLOGY was initiated in the late 19th as a “danger signal” and thus serves as a fingertip ulcers. Vice versa, VEGF is a potent century. Moritz Loewit, from the Institute of target for pre-existing innate and adaptive mediator of angiogenesis if it is available “Experimental Pathology” in Prague, moved anti-Hsp60 immunity. Our present research in a temporally and spatially controlled in 1887 to Innsbruck to become the first is focused on fashion. We have addressed this therapeutic professor here. Hermann Pfeiffer was chief a) the elucidation of the migratory pathways dilemma in SSc by a novel approach using from 1919–1921, Gustav Bayer followed of HSP60-reactive T-cells into the arterial a VEGF121 variant that covalently binds him in 1922 and led the institute until intima and to fibrin, and gets released on demand by 1938 when the NS regime forced him, an b) the continuation and extension of our EU cellular enzymatic activity, only as long intellectual Jew, to end his life. After WW2, FR7-funded project TOLERAGE that re- as needed. With this approach we mimic

28 Research Report 2015 Medical University of Innsbruck Experimental Pathophysiology and Immunology Sgonc . © R Fig. 1: Model for the development and pathophysiology of systemic Fig. 2: Adaptive immune resonse in fibrosis. sclerosis. nature, where longer VEGF isoforms are Future Goals: enterological S.”. He has written several bound to extracellular matrix components Elucidation of novel therapeutic strategies textbooks: “Pathophysiology – Molecular, until liberated in a tightly controlled manner in SSc. Cellular and Systemic Basis of Diseases”, by local enzymatic activity of cells invading Maudrich, Vienna, 2007, and “Molecules of the matrix. Using UCD-206 chickens, we Teaching Pathophysiology – Molecular Life and Mutations”, Karger, Basel, 2002. could show that cell-demanded release of Endocrinology – Siegfried Schwarz locally applied fibrin-bound VEGF121 leads The Laboratory of Molecular Endocrinology Selected Publications to the formation of morphologically normal has focussed on various hormone/neuro- The role of heat shock proteins in atherosclerosis. Wick G, Jakic blood vessels, and clinical improvement of transmitter binding proteins and receptors B, Buszko M, Wick MC, Grundtman C. Nat Rev Cardiol. 2014 Sep;11(9):516–29. Review. PMID: 25027488. early and late ischemic lesions. Over all, as well as their ligands. Pentraxin 3 (PTX3) plasma levels and carotid intima media thick- 79.3 % of the lesions treated with VEGF121- Key papers describe: ness progression in the general population. Baragetti A, Knoflach fibrin showed clinical improvement, whereas • Discovery of Sex Hormone Binding Glob- M, Cuccovillo I, Grigore L, Casula M, Garlaschelli K, Mantovani A, Wick G, Kiechl S, Willeit J, Bottazzi B, Catapano AL, Norata GD. 71.0 % of fibrin treated controls, and 93.1 % ulin (SHBG) in cerebrospinal fluid (CSF) Nutr Metab Cardiovasc Dis. 2014 May;24(5):518–23. PMID: of untreated lesions deteriorated. This was and interaction of SHBG with Danazol 24462365. accompanied by significantly increased (non-genomic actions of steroids) The immunology of fibrosis. Wick G, Grundtman C, Mayerl C, Wimpissinger TF, Feichtinger J, Zelger B, Sgonc R, Wolfram D. growth of stable microvessels, up-regulation • First demonstration of homocysteate as Annu Rev Immunol. 2013;31:107–35. Review. PMID: 23516981. of the pro-angiogenic VEGF receptor-2 an NMDA-selective excitatory agonist Chlamydia pneumoniae infection acts as an endothelial stressor (VEGFR-2) and its regulator TAL-1, and • First description of an epitope map of the with the potential to initiate the earliest heat shock protein increase of endogenous endothelial VEGF glycoprotein hormone hCG 60-dependent inflammatory stage of atherosclerosis. Kreutmayer S, Csordas A, Kern J, Maass V, Almanzar G, Offterdinger M, expression. This study suggests that cell- • Construction of epitope-selective Öllinger R, Maass M, Wick G. Cell Stress Chaperones. 2013 demanded release of VEGF121 from fibrin immunoassays for glycoprotein hormones May;18(3):259–68. PMID: 23192457. matrix induces controlled angiogenesis • Demonstration of different orientations of The BH3-only protein Bad is dispensable for TNF ‑mediated cell death. Ottina E, Sochalska M, Sgonc R, Villunger A. by differential regulation of VEGFR-1 and receptor-bound agonistic vs. antagonistic Cell Death Dis. 2015 Jan 22;6:e1611. doi: 10.1038/cd- VEGFR-2 expression shifting the balance hCG dis.2014.575. PMID: 25611386. towards the pro-angiogenic VEGFR-2, • Prediction of the 3D structure of the extra- Selected Funding and shows the potential of covalently cellular domain of the hCG receptor • Effects of VEGF121 modified fibrin on ischemic lesions conjugated VEGF ‑fibrin matrices for the • Characterization of an apoptotic activity in systemic sclerosis: a new therapeutic approach. FWF: therapy of ischemic lesions. within urinary hCG preparations towards P23230-B13, Roswitha Gruber-Sgonc • The role of Vascular Associated Lymphoid Tissue (VALT) in the Major Achievement: Kaposi’s sarcoma cells Development of Atherosclerosis-“Inside out or outside in”,TWF, Effective therapy of ischemic skin lesions in • Demonstration of the importance of vaso- Bojana Jakic an animal model of SSc. pressin in critically ill patients • Lokale Immunreaktionen bei der Entstehung der Atheroskle- rose [Das vaskulär assoziierte lymphoide Gewebe (VALT) bei Atherosklerose], OeNB, Georg Wick Siegfried Schwarz teaches Pathophysiolo- gy to Medical, PhD (MCBO) and Molecular Collaborations Medicine students in most of the modules Jeremy Saklatvala and Robin Wait, Kennedy Institute of Rheuma- tology, University of Oxford, UK (“Human Body”, “Regulation of Bodily Func- Oliver Distler, Center of Exp. Rheumatology, University Hospital tions”, “Endocrine S. (system)”, “Blood Zurich, Switzerland Andreas Zisch†, Department of Obstetrics, University Hospital S.” and venipuncture practicum, “Heart Zurich, Switzerland

arz & Vascular S.”, “Kidney”, “Lung”, “Neuro- Olov Ekwall, Department of Rheumatology, Göteborg, Sweden Susanne Kerje, Department of Medical Sciences, Uppsala Univer-

Schw logical S.”, “Embryology”, “Aging”, “Bone . sity, Sweden

Research Report 2015 Medical University of Innsbruck 29 Biocenter Molecular Pathophysiology

or cell death induction in leukaemia cells ed heterogeneity and complexity of this (Kofler) with the ultimate goal to apply response encompassing both protein- and this knowledge to improve therapy and microRNA-encoding genes. Although GC diagnosis of human diseases. may cause changes in protein lev els in the The Applied Bioinformatics Group (Rainer) absence of mRNA regulations, the trans supports our high-throughput analyses and lational efficiency of transcripts is not affect- the MUI-Expression Profiling Facility, which ed by GC. Concerning the transcriptional re- is also attached to the DMP. sponse to GC in vivo, it varies considerably Arno Helmberg coordinates our teaching in the different molecular subtypes of this obligations and serves as Vice Chairperson disease. Regarding the anti-leukaemic of the Senate of our University. effect, repression of mRNA for key regu- The research in our Division is supported by lators of G2/M transition was commonly grants from the FWF, the MCBO graduate observed in all ALL subtypes whereas we college and various other sources including failed to observe a common transcriptional the Cancer Aid Society and Tyrolean control of apoptosis genes. The data sug- Cancer Research Institute that is headed by gest that GC-induced cell death does not Reinhard Kofler. result from transcriptional regulation of the apoptotic machinery itself but might rather Head of Division: Research result from a widespread deregulation of Univ.-Prof. Dr. Reinhard Kofler gene expression. Leukaemia Apoptosis Contact: Reinhard Kofler Major Achievements: Biocenter, Innrain 80–82 Glucocorticoids (GC) trigger cell death and • Defining the GC-regulated transcriptome 6020 Innsbruck cell cycle arrest in certain lymphoid cells in children during systemic GC mono- and are therefore used for the therapy of therapy and numerous other biologically [email protected] lymphoid malignancies, most important- relevant lymphoid systems including Phone: +43 512 9003 70360 ly childhood acute lymphoblastic leukae- the first identification of GC-regulated Fax: +43 512 9003 73960 mia (ALL). We aim to understand the anti- microRNAs http://dmp.i-med.ac.at/index.php/ leukaemic effects of GC and the causes • Functional analyses of numerous GC for GC resistance to develop concepts for response genes improved therapies. • Delineating the genes responsible for Keywords To this end, we determined gene expres- GC-induced cell cycle arrest and providing sion profiles of ALL cells in children prior novel concepts explaining GC-induced cell Cell cycle, mitosis, apoptosis, childhood to, and in the course of GC treatment, us- death acute lymphoblastic leukaemia, gluco- ing whole genome expression profiling. By corticoid, expression profiling, functional inclusion of peripheral blood lymphocytes Future Goals: gene analysis, gene knock-out and knock- from GC-exposed non-leukemic donors, • Completion of our analyses of GC-induced down by RNAi, viral vector systems, cyclin ALL cell lines and mouse thymocytes, an cell death by novel bioinformatics tools dependent kinase essentially complete list of GC-regulated • Development of a fluorescence-activated candidate genes in clinical settings and cell sorting (FACS)-based assay to deter- Research Focus experimental systems was generated, al- mine cancer-killing activity in leukocytes lowing immediate analysis of any gene for for potential cell-based cancer therapy • Molecular mechanisms of the anti its potential significance to GC-induced leukaemic effects of glucocorticoids (GC) cell death or cell cycle arrest. In addition to Cell Cycle Control using gene expression profiling of children conventional gene expression profiling, we Stephan Geley with acute lymphoblastic leukaemia during performed alternative transcripts analysis, Cellular reproduction relies on the faithful GC treatment followed by bioinformatics GC receptor binding site definition (ChIP-on- copying and segregation of the genome and functional gene analyses. CHIP), translatome analyses, and microRNA during defined phases of the celldivision • Identification and functional analysis of profiling. cycle, which is controlled by cyclin- proteins required for faithful chromosome Our “Applied Bioinformatics Group” (see dependent kinases (CDK). CDK1 is required segregation during mitosis. below) performs integrative analyses of for entry into mitosis and formation of • Analysis of non-cell cycle related functions these data and generates lists of candidate the mitotic spindle, a microtubule-based of cyclin-dependent kinases and of mitotic genes and pathways that need to be func- apparatus required for chromosome ubiquitin ligase (APC/C) function during tionally tested for their potential biological segregation. The microtubule-based motor development. role. For this we have developed lentiviral proteins, dynein and kinesins, play important expression systems allowing efficient analy- roles in aligning chromosomes within the General Facts sis of many genes in multiple cell lines. mitotic spindle, allowing their equational segregation into daughter cells. Spindle The Division of Molecular Pathophysiology Current Results: formation, chromosome segregation and (DMP) aims at a better molecular under- Defining GC-regulated genes andexit from mitosis are carefully monitored standing of fundamental biological pro- transcripts in a variety of experimental by the spindle assembly checkpoint (SAC) cesses, like regulation of cell cycle (Geley) and clinical systems revealed an unexpect- to avoid aneuploidy, which can result in

30 Research Report 2015 Medical University of Innsbruck Molecular Pathophysiology

port for clients of the Expression Profiling Unit and perform data analyses of the microarray data sets.

Current Projects: Identification of genes facilitating glucocorticoid therapy response in children with acute lymphoblastic leukaemia using multiple regression analysis of microarray and clinical data

Major Achievements: We defined the transcriptional response of Fig. 1: Fzr1 is essential for vertebrate development. Zebrafish embryos were depleted from ALL cells to treatment with synthetic GCs Fzr1 by morpholino antisense technology and show severe developmental retardation. prednisolone and dexamethasone; investigated the role of GCs in whole genome translational gene regulation, developed a severe birth defects or cancer. The SAC Novel CDKs and their functions: method to determine chromosome copy controls the activity of the anaphase- CDK14,15 and 16,17,18 are a group of poor- number alterations based on whole genome promoting complex/cyclosome (APC/C) ly characterised CDKs that can all interact gene expression data, and improved ubiquitin ligase, which controls chromo- with and become activated by membrane pre-processing and differential splicing some segregation and exit from mitosis . bound cyclin Y. These kinases show overlap- analysis for Affymetrix Exon microarrays. After mitosis the APC/C remains activated ping expression patterns. In vertebrates, we by FZR1 to establish G1 phase, which is have defined CDK16 as being essential for required for cellular growth as well as cel- spermatogenesis and could show that cyc- lular differentiation. Only 4 of the known 26 lin Y binding is regulated by 14-3-3 proteins Selected Publications CDKs are involved in cell cycle regulation. and phosphorylation. CLP1 links tRNA metabolism to progressive motor-neuron loss. The other members are either involved in Hanada T, Weitzer S, Mair B, Bernreuther C, Wainger BJ, Ichida J, Hanada R, Orthofer M, Cronin SJ, Komnenovic V, Minis A, Sato F, transcription or have other, less well char- Major Achievements: Mimata H, Yoshimura A, Tamir I, Rainer J, Kofler R, Yaron A,Eggan acterised, functions. • FZR1 knock-out mice and zebrafish model KC, Woolf CJ, Glatzel M, Herbst R, Martinez J, Penninger JM. NATURE. 2013; 495: p. 474–480. system (in collaboration with P. Aanstad, The synthetic glucocorticoids prednisolone and dexamethasone Results: LFU, Innsbruck) regulate the same genes in acute lymphoblastic leukemia cells. Microtubule based motor proteins: • Role of chromokinesins in mitosis (in Bindreither D, Ecker S, Gschirr B, Kofler A, Kofler R, Rainer J. The chromokinesins KIF4A and KIF22(hKid) collaboration with H. Maiato, Porto) BMC GENOMICS. 2014; 15: p. 662. contribute to chromosome congression. • Regulation of CDK16 by CCNY Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces. hKid is the major polar ejection force that • Regulation of Spindly by lipidation Barisic M, Aguiar P, Geley S, Maiato H. Nat Cell Biol. 2014 counteracts dynein-dependent poleward • Development of conditional RNAi and Dec;16(12):1249–56. chromosome movements. The main motor gene knock-out systems Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis. Wandke C, Barisic responsible for metaphase plate formation M, Sigl R, Rauch V, Wolf F, Amaro AC, Tan CH, Pereira AJ, Kutay U, is the kinetochore motor Cenp-E, but hKid Future Goals: Maiato H, Meraldi P, Geley S. is required for the correct orientation of Define the role of Fzr1 in ciliogenesis, under- J Cell Biol. 2012 Sep 3;198(5):847–63. chromosome arms. Kif4A regulates chro- stand how farnesylation controls the func- Cyclin-dependent kinase 16/PCTAIRE kinase 1 is activated by cyclin Y and is essential for spermatogenesis. Mikolcevic P, Sigl mosome compaction and might regulate tion of Spindly at kinetochores, investigate R, Rauch V, Hess MW, Pfaller K, Barisic M, Pelliniemi LJ, Boesl M, kinetochore stiffness. Dynein is recruited to the role of KIF4A in chromosome compac- Geley S. Mol Cell Biol. 2012 Feb;32(4):868–79. the kinetochores via Spindly. Spindly needs tion, identify substrates of CCNY-CDK16. Selected Funding to be farnesylated for its localisation and • MCBO Graduate Programme (W1101-P13), FWF, Reinhard function at the kinetochore. Applied Bioinformatics Kofler Johannes Rainer • MCBO Graduate Programme (W1101-P06), FWF, Stephan Geley FZR1 function in vertebrate development: Our primary research focus is on whole- • RANSFOG EU FP6 LSHC-CT-2004-503438, Stephan Geley FZR1 is a substrate recruitment factor of genome gene expression analyses where Collaborations the ubiquitin ligase APC/C. It activates the we are particularly interested in the analysis • H. Kovar, R. Panzer, S. Strehl, St. Anna Kinderspital, Vienna APC/C during late mitosis and in G1-phase. of high-density microarray and high through- • J. Penninger, IMBA, Vienna Loss of Fzr1 function causes shortening of put sequencing data and the dev elopment • B. Meister, R. Crazzolara, Department of Pediatrics, MUI • T. Hunt,Cancer Research UK, London) G1-phase and CDK-activity dependent pre- of new as well as adaptation of existing • R. Fässler, MPI Martinsried, Munich mature entry into S-phase, which causes methods for their analysis. In this context • P. Meraldi, Univ. Geneva, Switzerland DNA damage and p53 responses that can we are analysing data sets generated in our • H. Maiato, Univ. Porto, Portugal • M. Morgan, Fred Hutchinson Cancer Research Center, Seattle, lead to senescence, cell cycle arrest or ap- leukaemia apoptosis group, to determine USA optosis. We have generated Fzr1 deficient the transcriptional effects of glucocorti- • F. Theis, Helmholtz-Zentrum München, Germany mice and zebrafish and, in addition to the coids in acute lymphoblastic leukaemia and • P. Aanstad and D. Meyer, LFU Innsbruck effects on cell cycle regulation, found that to delineate and understand the treatment Core Facilities Fzr1 is essential for vertebrate development response and resistance mechanisms in Expression Profiling Unit (EPU, Affymetrix Core Facility): by regulating ciliogenesis. such patients. In addition, we provide sup- http://biocenter.i-med.ac.at/expression-profiling-unit

Research Report 2015 Medical University of Innsbruck 31 Biocenter Developmental Immunology

Survival-promoting Bcl-2 family members, Bad Bid Bim i.e. Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1 share four common Bcl-2 homology domains (BH1-BH4). All of these proteins are critical Bax Bak for cell survival, since the loss of any one of them causes the premature cell death of certain cell types. Consistently, over Bcl-w Bcl-2 Bcl-x Mcl-1 A1 expression of Bcl-2 pro-survival molecules is associated with prolonged cell survival Bad Bmf Noxa and resistance to cytotoxic drugs in a number of model systems, but more important- Puma Bid Bim ly, also in tumor patients. The pro-apoptotic Bcl-2 family members Fig. 1: Liaisons in the Bcl2 family. The Bcl2 can be divided into two classes: the Bax- family can be categorized into three classes like proteins, i.e. Bax, Bak, Bok that contain of proteins that control mitochondrial cell three BH-domains (BH123 or multi-domain death by complex protein–protein inter pro-apoptotic Bcl-2 proteins) and the actions, indicated here. These interactions BH3-only proteins. The latter include Bim, are based on different affinities between Head of Division: Bid, Puma, Noxa, Bmf, Bad, Hrk and Bik individual family members and are in Univ.-Prof. Dr. Andreas Villunger that are unrelated in their sequence to fluenced by ariousv posttranslational each other or other Bcl-2 family members protein modifications. Contact: (except for the BH3-domain). Biocenter, Innrain 80–82 We utilise genetically modified model Ongoing Projects: 6020 Innsbruck systems to study the role of pro-survival BH3-only proteins in the regulation of B cell Bcl-2 family proteins and BH3-only proteins survival [email protected] in tumor and lymphocyte development. Lymphocyte development and function in Phone: +43 512 9003 70380 the absence of A1 Fax: +43 512 9003 73960 Caspase-2, Cell Cycle Control and the PIDD in caspase -2 and NF -kB activation www.apoptosis.at DNA-Damage Response Tumor suppression/promotion by Cells that have been exposed to DNA- Caspase -2 and its partners damaging influences aim to repair the in- Identification of Caspase -2 substrates flicted damage. However, when this attempt Research Focus/Keywords fails, cells usually activate an apoptotic Non-Coding RNAs in Hematopoiesis program to avoid the spread of cells with Sebastian Herzog • Apoptosis & Tumor Biology compromized genomes. The molecular In the last decade, our understanding of • Non-coding RNAs in Hematopoiesis basis of these life/death decisions is still the human genome and its regulation has • Glucocorticoids & Immunology not entirely clear. dramatically changed. Initially considered The p53-induced protein with a death as “junk”, it is now clear that the non- General Facts domain (PIDD) has been identified as a protein coding regions, which comprises gene activated in response to p53 upon about 98 % of the ~3·109 DNA bases, is Work of the different groups in this division DNA-damage. Together with the adapter extensively transcribed and gives rise to focuses on the development of the immune molecule RAIDD, PIDD has been implicated numerous non-coding RNAs. The function system with an emphasis on cell death in the activation of Caspase -2, an endo- of these non-coding RNAs, however, is often signalling, its cross-talk with the cell cycle peptidase implicated in apoptosis and cell unclear. machinery and the role of steroid hormones cycle control. PIDD has recently also been in the establishment of self-tolerance and implicated in DNA damage-induced NF -kB MicroRNAs in Early Lymphocyte Develop miRNA function. activation and cytokine release, promoting ment and Transformation the transcription of inflammatory genes by MicroRNAs (miRNAs) are small, non-coding Research forming a complex with the kinase RIP -1 RNAs that mediate posttranscriptional and Nemo. Caspase -2 is an ill-defined silencing of a predicted 60 % of protein- Apoptosis, Tumor Biology & Leukocyte protease that has been implicated in mul- coding genes in mammals. Since their Development tiple cellular responses including the one discovery, they have emerged as central Andreas Villunger triggered by deprivation of metabolites, mediators of many, if not all biological BH3- Only Proteins in Cell Death heat shock or DNA damage triggered cell processes. In our work, we aim to decipher and Disease cycle control. However, the contribution of how miRNAs regulate complex transcrip- Whether a cell continues to live in response caspase -2 to these responses is in many tional networks, focusing on lymphocyte to diverse forms of stress or undergoes cases still unclear (Fig. 2). development as a well established model apoptosis along the intrinsic cell death We are currently investigating the role of system. In particular, we want to elucidate signaling pathway is largely determined by these proteins in tumor suppression and aim the role of individual miRNAs under physio- the complex interplay between individual to identify Caspase 2-specific substrates in logical conditions as well as upon aberrant members of the Bcl-2 protein family that order to gain further insight into the func- expression, which mimics an oncogenic can either promote or prevent apoptosis. tions of this multi-protein complex. situation. To this end, we combine gain- and

32 Research Report 2015 Medical University of Innsbruck Developmental Immunology

Nucleus DR transcriptional programmes both in health T cells that migrate to the periphery (pos- DNA-damage and disease. Among the non-coding RNA itive selection). Glucocorticoid hormones species identified so far, the group of long (GC) have been suggested to influence ATM/ATR non-coding RNAs (lncRNAs), which are these processes, or example by inducing

Chk1 arbitrarily defined as the set of ncRNAs with apoptosis in developing T cells with the Heat-shock a length of more than 200 nucleotides, are thymus itself producing GCs! In addition,

p53 the least well understood. In a MUI-START- GC resistance of thymocytes against GC-in- BID funded project, we initially applied RNAseq duced apoptosis is associated with auto Caspase-2 to the challenge of defining the lympho- immune diseases. We focus therefore on PIDD- osome tBID cyte linRNA transcriptome. The expression the following questions: i) what is the mo- of most of the lincRNAs appears to be re- lecular background of thymocyte resistance Golgi stricted to certain developmental stages, to GC-induced apoptosis in animal models suggesting the have a specific functional of autoimmune diseases?, and ii) what fac- Mitochondrium role in the associated processes. However, tors determine sensitivity to GC-induced Amplification Loop? Amplification Caspase-9 in order to decipher the function of individu- apoptosis in immature vs. mature thymo- al lincRNAs, we now use shRNA knockdown cytes (Fig. 3)? Caspase-3 ER and CRISPR/Cas9-mediated genome modi- Apoptosis fication together with RNA pulldown. Selected Publications Deregulated cell death and lymphocyte homeostasis cause pre- Regulation of Immunity mature lethality in mice lacking the BH3-only proteins Bim and Bmf. Labi Verena, Woess Claudia, Tuzlak Selma, Erlacher Miriam, Fig. 2: Schematic model summarizing the Jan Wiegers Bouillet Philippe, Strasser Andreas, Tzankov Alexandar, Villunger most important apoptotic path-ways with a Impact of life span on regulatory T cell Andreas. BLOOD. 2014; 123: p. 2652–2662. suggested involvement of caspase-2 activity, maturation and function Minor cell-death defects but reduced tumor latency in mice lack- i.e. cell death in response to DNA-damage, Regulatory T cells (T ) expressing the ing the BH3-only proteins Bad and Bmf. Baumgartner F, Woess C, reg Pedit V, Tzankov A, Labi V, Villunger A. ER-stress, heat shock, and death receptor transcription factor Foxp3 play an essen- ONCOGENE. 2013; 32: p. 621–630. (DR) ligation. ATM, Ataxia telangiectasia tial role in maintaining immune homeo Loss of PIDD limits NF ‑KB activation and cytokine production but mutated; ATR, Ataxia telangiectasia and stasis and preventing autoimmunity. A not cell survival or transformation after DNA damage. Bock FJ, Rad3 related; PIDDosome, protein complex spontaneous loss of function-mutation in Krumschnabel G, Manzl C, Peintner L, Tanzer MC, Hermann-Kleiter N, Baier G, Llacuna L, Yelamos J, Villunger A. CELL DEATH AND consisting of PIDD, RAIDD and caspase-2; foxp3 in ‘scurfy’ mice leads to fulminant DIFFERENTIATION. 2013; 20: p. 546–557. TRAIL, tumor necrosis factor related apop- lymphoproliferation and multi-organ auto- Death of p53-defective cells triggered by forced mitotic entry tosis inducing ligand; FADD, Fas-associat- immunity. A more detailed knowledge of in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome. Manzl C, Fava LL, Krumschnabel G, ed death domain (modified according to G. the factors that affect Treg maturation and Peintner L, Tanzer MC, Soratroi C, Bock FJ, Schuler F, Luef B, Geley Krumschnabel et al., 2009) number in the thymus and Treg homeo S, Villunger A. CELL DEATH & DISEASE. 2013; 4: p. e942. stasis under either normal conditions or Haematopoietic stem cell survival and transplantation efficacy is loss-of-function approaches, both in vit- during the course of an immune response is limited by the BH3-only proteins Bim and Bmf. Labi V, Bertele D, Woess C, Tischner D, Bock FJ, Schwemmers S, Pahl HL, Geley S, ro as well as in vivo, with biochemical and essential for the development of potent and Kunze M, Niemeyer CM, Villunger A, Erlacher M. molecular techniques. more efficient therapeutics for autoimmune EMBO MOLECULAR MEDICINE. 2013; 5: p. 122–136. diseases. It is also currently unclear how Increased leukocyte survival and accelerated onset of lymphoma in the absence of MCL-1 S159-phosphorylation. Lindner LincRNAs in Hematopoiesis and Immune life span influences the capacity of regT to SE, Wissler M, Gründer A, Aumann K, Ottina E, Peintner L, Function suppress immunity. As a means to study Brauns-Schubert P, Preiss F, Herzog S, Borner C, Charvet C, Projects such as ENCODE have clear- maturation and function of Treg cells, we use Villunger A, Pahl HL, Maurer U. ly demonstrated that the non-coding foxp3GFP knock-in mice that coexpress GFP Oncogene. 2014 Oct 30;33(44):5221–4. part of the human genome is extensively under the control of the endogenous foxp3 Selected Funding transcribed and contributes significant- promoter. This allows convenient detection • The role of Checkpoint kinase 1 (Chk1) in normal hematopoie- ly to the orchestration and fine -tuning of and purification of Treg cells by flow cytome- sis and Myc-driven transformation. Schuler F., ÖAW try and it is possible to isolate nearly 100 % • Investigating the role of prosurvival Bcl-2 family member A1 in T cell mediated immunity, Tuzlak S., ÖAW pure Treg cells using this approach. • Neue Einsichten in die Bcl2 Familie, Villunger A., FWF • Die Rolle des PIDDosoms in der Tumorentstehung, Villunger A., CD4+ CD8+ Glucocorticoids and T Cell Development FWF Isotype FITC • Die Rolle von BH3 Proteinen in der B Zell Homöostase, Villunger Selection processes in the thymus ensure A., FWF + + CD4 CD8 • Glukokortikoide und regulatorische T-Zellen, Wiegers G.-J., FWF GR-FITC that mature peripheral T cells fulfill two essential criteria: activation by foreign pep- Collaborations + tides bound to (host) MHC molecules, but CD8 • Georg Häcker, Freiburg, DE Isotype tolerance to self-derived peptides present- • Andreas Strasser, WEHI, Melbourne, AUS CD8-CY FITC • Pascal Schneider, Lausanne, CH CD4-PE ed in the same context. To this end, thymo- • Alexander Egle, PMU, Salzburg, AT GR-FITC cytes that express T cell receptors (TCRs) • Veronika Sexl, Vienna, AT with high avidity for self antigen:MHC and • Jörg Hackermüller at Helmholtz Center for Environmental Rese- Fig. 3: Glucocorticoid receptor (GR) ex therefore are potentially autoreactive, arch (UFZ), Leipzig, DE • Falus A., Dept. of Genetics, Cell- and Immunobiology (earlier pression in thymocyte subsets. Thymo-cytes undergo apoptosis (negative selection). Dept. of Biology) at Semmelweis University, Budapest, HU were stained for CD4, CD8 and GR, washed In contrast, thymocytes expressing TCR • Reul J. M., Laboratories of Integrative Neuroscience and En- docrinology (LINE), University of Bristol, UK and mounted with Mowiol. Isotype control with moderate avidity for self antigen:MHC • Boyd R. L., Department of Immunology, Monash University, and GR stained thymo-cytes were mixed 1:1. are rescued and differentiate into mature Clayton, Victoria, Australia

Research Report 2015 Medical University of Innsbruck 33 Biocenter Bioinformatics

2. Cancer immunology. Our aim is to deci- clinical data. We aim to identify causative pher tumor-immune cell interaction using genes, prioritize candidates for experimen- a combined computational-experimental tal studies, and further characterize path- approach. Specifically, we are address- ways contributing to the pathophysiology of ing the question as to how is the immune diseases. system shapes the mutational spectrum of the tumor during progression. Cancer Immunology Most advanced solid tumors remain incur Bioinformatics Services able and are resistant to chemotherapeu- We provide bioinformatics services for tics and targeted therapies. A series of researchers at the Biocenter and at the recent studies reported baffling intratumor Innsbruck Medical University as well as heterogeneity which may contribute to this for external experimental collaborators. failure. While increasing attention is being A high-performance computational infra- paid to the mutational spectrum of various structure and a number of software tools cancers, little attention has been devot- are maintained and continuously adapted ed either to define the immunogenicity of to state-of-the-art software technology (see these mutations or to characterize the im- http://icbi.at). The software development mune responses they elicit. Identification of Head of Division: is directed towards specialized databases, nonsynonymous mutations processed and Univ.-Prof. DI Dr. Zlatko Trajanoski analytical pipelines, and web-services. We presented in an immunologically relevant also advise scientists on designing experi- manner will not only highlight the mech- Contact: ments and support analyses of high-dimen- anisms driving tumor progression but will Biocenter, Innrain 80–82 sional data sets including: also provide a rich source of novel immuno 6020 Innsbruck -- whole-genome/whole-exome data, therapeutic targets. Thus, it is of utmost -- RNA-Seq, importance to decipher the cross-talk be- [email protected] -- ChIP-Seq, tween the tumor and the immune system Phone: +43 512 9003 71401 -- microbiome, during tumor development. Our long-term Fax: +43 512 9003 73100 -- high-content microscopy data, and goal is to develop a mechanistic multi-scale http://icbi.at -- proteomics data

Research

Computational Genomics Keywords Recent advances in genome sequencing technologies are rapidly changing the re- Bioinformatics, computational genomics, search and routine work of biologists and next generation sequencing, gene expres- human geneticists. Due to the brisk decline sion, cancer, immunology, mathematical of costs per base pair, next-generation se- modeling, data integration quencing (NGS) is now affordable even for small-to-mid sized laboratories. Whole- Research Focus genome sequencing and whole-exome sequencing have proven to be valuable • Analyses of diverse functional genomics methods enabling discovery of the genetic data in the context of human diseases and causes of rare Mendelian disorders as well integration with clinicopathological infor- as of complex diseases. The current bottle mation. neck is not the sequencing of the DNA it- • Modeling of the interaction between self but lies in structuring the processes of tumor and the immune system data management and in the sophisticated computational analysis of the experimental General Facts data. In order to get meaningful biological results, each step of the analysis workflow The research activities at the Division of (as for example depicted in Fig. 1) needs to Bioinformatics are directed towards two be carefully considered, and appropriate major thrusts: specific tools need to be used for particular 1. Computational genomics. We compu- experimental setups. Furthermore, the chal- tationally explore diverse functional lenge of ‘next-generation biology/genetics’ genomics data in the context of human is to narrow down the list of candidate vari diseases. By analyzing high-dimensional ants and interpret the remaining variants. data sets we aim to identify and prioritize The major focus of our research activities is © Briefings in Bioinformatics (Oxford University Press) candidate genes and further character- to narrow down the genome search space Fig. 1: Basic workflow of whole-exome ize pathways contributing to the patho- by integrating and analyzing disparate data (whole-genome) sequencing projects for physiology of diseases. sources including various omics data and variant detecion

34 Research Report 2015 Medical University of Innsbruck Bioinformatics

tumor heterogeneity, clonal evolution and tumor progression, which can then be experimentally verified.

Selected Publications Characterization of the immunophenotypes and the antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy. Angelova M, Charoentong P, Hackl H, Fischer M, Snajder R, Krogsdam AM, Waldner MJ, BIndea G, Mlecnik B, Galon J, Trajanoski Z. GENOME BIOLOGY. 2015; 16:64. A survey of tools for variant analysis of next-generation genome sequencing data. Pabinger S, Dander A, Fischer M, Snajder R, Sperk M, Efremova M, Krabichler B, Speicher MR, Zschocke J, Trajanoski Z. BRIEFINGS IN BIOINFORMATICS. 2014; 15: p. 256–278. Co-expressed genes prepositioned in spatial neighborhoods stochastically associate with SC35 speckles and RNA polymerase II factories. Rieder D, Ploner C, Krogsdam A M, Stocker G, Fischer M, Scheideler M, Dani C, Amri E-Z, Mueller WG, McNally JG, Trajanoski Z. CELLULAR AND MOLECULAR LIFE SCIENCES. 2014; 71: p. 1741– Fig. 2: Spinchart of molecular phenotypes (3 inner circles) and immunophenotypes in 598 1759. colorectal cancer patients Spatio-temporal dynamics of intratumoral cells reveal the immune landscape in human cancer. Bindea G, Mlecnik B, Tosolini M, Kirilovsky A, Waldner M, Obenauf model and investigate the tumor-immune ymous mutations of these patients we were AC, Angel H, Frederiksen T, Lafontaine L, Berger A, Bruneval P, cell interaction in colorectal cancer and also able to predict the tumor-epitopes Fridman WH, Becker C, Speicher MR, Trajanoski Z, Galon Z. IMMUNITY. 2013; 39:p. 782–795. in a number of different solid tumor types (neo-antigens) and could show that those GPViz: dynamic visualization of genomic regions and variants af- including breast and lung adenocarcino- are rarely shared between patients, indi- fecting protein domains. ma. Towards this goal we are exploring the cating that therapeutic vaccination needs Snajder R, Trajanoski Z, Hackl H. BIOINFORMATICS. 2013; 29: p. 2195–2196. immunogenicity of the colorectal cancer to be personalized rather than provided for mutanome using deep mining of publicly a larger cohort. The most shared epitope Selected Funding available data sets and carrying out exper- candidates are shown in Fig. 3; the epitope • SFB Cell Proliferation and Cell Death in Tumors, FWF, Zlatko imental studies using cell and mouse mod- TEYKLVVVGAV generated by mutations in Trajanoski • DK MCBO, FWF, Zlatko Trajanoski els. By analyzing RNAseq data of more than the KRAS gene was shared by 28 colorectal • Bioinformatics Tyrol, Standortagentur Tirol , Zlatko Trajanoski 500 colorectal cancer patients we could cancer patients. Additionally, we are build- • Oncotyrol, Zlatko Trajanoski • APERIM, EU Horizon2020, Zlatko Trajanoski (coordinator) obtain the landscape of infiltrating immune ing mathematical models at various scales cell types within the different moclecular and performing simulations that will help Collaborations subtypes (see Fig. 2). From the non-synon- us to identify immune signals controlling Jerome Galon, INSERM U872, Paris, France

Fig. 3: Neo-antigens shared in at least 7 of the 598 colorectal cancer patients; more commonly shared epitopes are shown in a lager font (Wordle), the gene symbols of the corresponding mutated genes are shown in brackets.

Research Report 2015 Medical University of Innsbruck 35 Department of Physiology and Medical Physics Physiology

Keywords microscopy and electrophysiology. The eight research groups are partners in local, Neurophysiology, renal physiology, muscle national and international consortia and physiology, lung physiology, cell volume reg- are funded by the European Commission ulation, ion channels, toxicology, electro- (4 projects), the FWF (7 projects), the physiology, cell biology, nociception ÖNB and private foundations with funding totalling € 4.2 million. Research Focus Research Scientific research at the Division of Physiology, Innsbruck, focuses on basic and On the Trail of Chronic Pain preclinical translational experimental work Michaela Kress in the areas of neurophysiology and the Chronic pain syndromes which develop physiology of muscle and epithelial organs. after nerve damage, trauma, or surgery Current research projects include the are characterized by persistent and severe science of normal biomolecular functions pain. They induce anxiety and depression in healthy individuals including their organs and greatly impair patients’ quality of life. and component cells. An understanding of One in five Europeans suffer from chronic Head of Division: normal functions can lay the groundwork pain, many of them for more than two years, Univ.-Prof.in Dr.in Michaela Kress to explore the pathogenesis of common some even longer. Chronic pain therefore diseases like COPD, renal dysfunction constitutes not only a heavy burden for Contact: or chronic pain. The principal level of individual patients and their families, but Fritz-Pregl-Straße 3/1 research is at the level of cellular models, also for national health systems in Europe 6020 Innsbruck organs and systems, and employs an since treatment costs can take up between integrated interdisciplinary approach. 1.5 and 3 % of a country’s gross domestic [email protected] Significantopportunities for innovation are product (GDP) per year. Advancing scientific Phone: +43 512 9003 70801 expected to arise from ongoing projects research in this field is thus a societal Fax: +43 512 9003 73800 developing human iPSCs into humanised need and a crucial undertaking in order to www.i-med.ac.at/dpmp/physiologie/ model systems and microRNAs as novel facilitate improved patient care. The main biomarkers and druggable targets for research aim of the group is to understand chronic neuropathic pain disorders. the pathogenesis of neuropathic and neurogenic pain disorders. We tackle General Facts these challenges with an interdisciplinary approach and a wide spectrum of The major task of the Division of Physiology methods and model systems including is the study and teaching of human primary neuron cultures and neuronal cell physiology. Physiology aims to understand lines (Fig. 1). how organisms survive and function. This is a challenging subject dealing with physical and chemical factors that are responsible for the origin, development and progression of life. The study of physiology includes the understanding of the workings of a given cell and its interaction with the cellular environment, through to the complex interaction of different cell types in tissues and organs and the interactions of these organ systems which are critical for the maintenance of whole body homeostasis and life. By understanding the normal function of organisms and their parts, we can better understand the processes that occur when these systems go awry in disease states. Eight research groups are involved in cutting edge research in the fields of nociception, calcium signalling, cell membranes and renal and alveolar epithelial physiology We employ a wide range of models and techniques including Fig. 1: Undifferentiated (top left) and differ- cell culture, imaging, gene and protein entiated (top right) neuroblastoma cell and expression, calcium microfluorimetricGFP transfected sensory neuron (lower pan- measurements, high resolution live el, blue) and actin staining (purple).

36 Research Report 2015 Medical University of Innsbruck Physiology

1. Neuroimmune Interactions aims to decode these biological molecules’ We explore interactions between the role, which perform multiple vital roles in nervous system and the immune system our genetic make-up and in the generation that occur upon nerve injury. At present we of chronic pain syndromes. are mainly interested in proinflammatory cytokines (interleukin 6, LIF, OSM), and the Calcium Channels in Muscle and Brain effects they have on neuronal function and Bernhard E. Flucher, Gerald Obermair regeneration after nerve injury. We currently Voltage-gated calcium channels are focus on the regulation of ion channels key regulators of cellular functions in and neuron excitability (Fig. 2) by signals electrically excitable cells. They control the originating from immune cells including communication between nerve and muscle microglia and the neurons themselves cells, the force of muscle contraction, in a research project and a PhD project and are importantly involved in regulating both funded by the FWF. Neurons can be muscle growth and differentiation during The Journal of Neuroscience reprogrammed in vitro with viral vectors as development and in response to exercise. . shown in Fig. 3 and this technology is used In nerve cells they regulate a variety to explore the signalosome of cytokine of vitally important functions including receptors and of non-coding RNAs which neurotransmitter release, gene regulation, have recently emerged as a novel family of and neuronal plasticity. The importance of

cellular regulators. voltage-gated calcium channels is reflected © Society for Neuroscience by a range of disorders related to aberrant 2. Non-Coding RNAs calcium channel functions such as the Fig. 3: Composite image of immunofluo- As novel players, non-coding RNAs and muscle diseases myotonic dystrophy rescence-labelled cultured neurons from their suitability as disease biomarkers or and malignant hyperthermia as well as lethargic (CaV β4-null mutant) mice recon- treatment strategies are assessed. With neurological diseases including migraine, stituted with specific splice variants of the ncRNAPain, a new European research epilepsy, autism, ataxia, chronic pain, mood calcium channel β4 subunit. Expression pro- project sets out to further explore the disorders, and Parkinson’s and Alzheimer’s filing (heat map in the background) revealed biological mechanisms underlying chronic disease. Our research teams use state-of- that the nuclear β4e subunit (orange) regu- pain. Endowed with an overall funding the-art molecular genetics, molecular and lates expression of neuronal genes includ- budget of 6 million euros by the European cell biology, electrophysiology, histology, ing that of CaV2.1, its own primary channel Commission for four years, the project and high-resolution microscopy approaches partner in cerebellar synapses. focusses on non-coding RiboNucleic to study calcium channel functions in mus Acids (ncRNAs). M. Kress coordinates the cle and nerve cells: ncRNAPain consortium which together molecular identity and specific functions of with A. Hüttenhofer, Z. Trajanoski (both 1. Calcium Channels in the Neuro- several hitherto uncharacterised channel Biocenter), F. Kronenberg (Div. Gen. Muscular System isoforms. Structure-function studies Epidemiology) and 10 international partners In the past years we discovered the revealed the molecular determinants of the unique biophysical properties of the skeletal muscle calcium channel. Recently we demonstrated the importance of controlling calcium influx for muscle fiber type determination and how increased calcium influx leads to muscle disease.

2. Calcium Channels in the Brain In synapses presynaptic calcium channels control the release of neurotransmitter in response to action potentials and postsynaptic calcium channels are involved in mechanisms of synaptic plasticity. Ongoing projects are concerned with the assembly and composition of synaptic calcium channel complexes and their involvement in the development of the neuro-muscular junction. For example, at present we are addressing the roles of two specific constituents of brain calcium channels, both in basic cell biological mechanisms, such as synaptic transmission and synapse formation, as well as their involvement in neurological disorders such as epilepsy and Fig. 2: Voltage-gated sodium current traces and analysis Parkinson’s disease (Fig. 4).

Research Report 2015 Medical University of Innsbruck 37 Department of Physiology and Medical Physics

Fig. 4: Mechanism of cyclosporine A induced renal epithelial cell injury. Human RPTEC/TERT1 cells were cultured on microporous supports and treated for 14 days with 0, 5 and 15 µM CsA. Samples were harvested for transcriptomics, proteomics and metabolomics. The heat map depicts the activation of the ATF4 pathway with 15 µM CsA, but not with 5 µM at the indicated days (d).

Kidney Function and Mechanisms of derived stem cell cultures providing a fully as a major determinant in cell signalling, Kidney Diseases humanized, animal-derived component- gene expression and exocytosis of sur- Gerhard Gstraunthaler, Judith Lechner, free culture system to be used in stem cell factant and its extracellular transformation Paul Jennings technology and tissue engineering. into functional films. Research of the renal groups is focused on studying the physiology of proximal tubular 3. Gender Medicine Insulin Secretion and Cell Volume cells, which play a major role in the develop- As a new line of research, studies on sex Regulation ment of acute and chronic kidney diseases. specific differences in renal tubular cell Johannes Fürst physiology have been implemented (Sci- Diabetes mellitus occurs throughout the 1. Biomarkers of Renal Injury ence Fund of the Austrian Central Bank). world but is more common (especially We are investigating the molecular mecha- Periodical changes in renal tubular cell phys- type 2) in more developed countries. Its nism of chemical induced stress in cultured iology phased by the female hormone cycle prevalence is increasing rapidly and an human proximal tubular cells and we are have been detected, which are potentially estimated 750 million people will have also attempting to identify novel mechanis linked to the lower susceptibility of women diabetes by 2030. Although genetic tic biomarkers of proximal tubular injury to renal failure as compared with men. background and environmental (i.e. dietary) (EU projects PredictIV, Detective). Some of factors have been associated with diabetes the pathways frequently altered in chemical Respiratory Cell Physiology pathogenesis the underlying physiological induced stress are Nrf2 (oxidative stress), Thomas Haller mechanisms need to be explored in more p53 (DNA damage), HIF1alpha (hypoxia) Quantitative as well as qualitative perturba- detail. Our main interest is studying the and ATF4 (the unfolded protein response). tions in the pulmonary surfactant system of regulation of insulin secretion in pancreatic The delineation of these and other pathways different etiologies, including a disruption beta cells in vitro, with a focus on the have been achieved by applying omic tech- of the type II cell homeostasis (Fig. 6), are contribution of cell volume regulatory niques (transcriptomics, proteomics and increasingly considered as underlying caus- mechanisms. metabolomics) and often coincide with the es of a spectrum of idiopathic respiratory loss of differentiation markers. distress and interstitial lung diseases, some of which are associated with a significant 2. Improved Assay Systems morbidity and mortality. The research per- In order to develop and improve in vitro formed by the respiratory cell physiology systems for chemical safety assessment group focuses in particular on the regulation as alternatives to animal testing, several and mechanisms of surfactant secretion by human and animal renal tubular cell lines the type II pneumocytes, the significance were characterized in depth and human and the biophysical properties of surfactant induced pluripotent stem cell differentiation at the respiratory air-liquid interface, and on into renal lineages has been studied (EU IMI- alveolar epithelial cell physiology in general, Project StemBANCC, Fig. 5). Human platelet with a current emphasis on the refinement lysates were developed as substitute for fetal of organotypic cell culture systems (lung- bovine serum in cell culture media and have on-a-chip). In the past years, for example, Fig. 5: iPSCs differentiated into adipocyte been successfully applied to adult adipose- we identified the air-liquid phase boundary like cells

38 Research Report 2015 Medical University of Innsbruck Physiology 286:L210–20 . Physiol . Mol . Lung Cell Lung . Physiol . J . © Am

Fig. 6: Stretch-induced Ca2+-signalling and exocytosis in alveolar type II cells

Selected Publications • FWF, P24079: Synapses and disease in calcium channel al- pha2-delta subunit mouse models, Gerald Obermair Deletion of Interleukin-6 Signal Transducer gp130 in Small Sen- • FWF, P27031: The role of calcium channels in acetylcholine sory Neurons Attenuates Mechanonociception and Down-Regu- receptor pre-patterning during neuromuscular junction de- lates TRPA1 Expression. Malsch Philipp, Andratsch Manfred Vogl, velopment Bernhard E. Flucher Christian Link, Andrea S, Alzheimer Christian, Brierley Stuart M, • FWF, W1101: Doctoral College in “Molecular Cell Biology and Hughes Patrick A, Kress Michaela. Oncology” 4th funding period; Speaker: Bernhard E. Flucher JOURNAL OF NEUROSCIENCE. 2014; 34: p. 9845–9856. • FWF, SFB F4415: Importance of intra- and extracellular Cav1.3 Peripheral Nerve Regeneration and NGF ‑Dependent Neurite Out- modulators for synapse stability in normal and diseased striatal growth of Adult Sensory Neurons Converge on STAT3 Phospho- medium spiny neurons. SFB: Cell signalling in chronic CNS dis- rylation Downstream of Neuropoietic Cytokine Receptor gp130. orders, Gerald Obermair and Bernhard E. Flucher Quarta Serena, Baeumer Bastian E, Scherbakov Nadja, Andratsch • FWF, P20472: Cells at air-liquid interfaces, Thomas Haller Manfred, Rose John Stefan, Dechant Georg, Bandtlow Christine • D-150700-017-011: Stiftung ProCare, Gerhard Gstraunthaler E, Kress Michaela. JOURNAL OF NEUROSCIENCE. 2014; 34: p. 13222–13233. Collaborations Sphingosine-1-Phosphate-Induced Nociceptor Excitation and On- • Frederic Bois, University of Compiègne/INERIS, France going Pain Behavior in Mice and Humans Is Largely Mediated by • Zam Cader, University of Oxford, UK S1P3 Receptor. Camprubi-Robles M, Mair N, Andratsch M, Benetti • Laszlo Csernoch, University of Debrecen, Hungary C, Beroukas D, Rukwied R, Langeslag M, Proia RL, Schmelz M, • Norman P. Curthoys, Colorado State University, Ft. Collins, CO, Montiel AVF, Haberberger RV, Kress, M. USA JOURNAL OF NEUROSCIENCE. 2013; 33: p. 2582–2592. • Wolfgang Dekant, University of Würzburg, Germany • Valentina Di Biase, Medical University of Graz, Graz, Austria Differential Neuronal Targeting of a New and Two Known Calci- • Jutta Engel, Saarland University, Homburg, Germany um Channel beta(4) Subunit Splice Variants Correlates with Their • Veit Flockerzi, Saarland University, Homburg, Germany Regulation of Gene Expression. Etemad Solmaz, Obermair Gerald • Martin Heine, Leibniz Institute for Neurobiology, Magdeburg, J, Bindreither Daniel, Benedetti Ariane, Stanika Ruslan, Di Biase Germany Valentina, Burtscher Verena, Koschak Alexandra, Kofler Reinhard, • Paul Heppenstall, EMBL Monterotondo, Italy Geley Stephan, Wille Alexandra, Lusser Alexandra, Flockerzi Veit, • Jing Hu, University of Tübingen, Germany Flucher Bernhard E. • Rohini Kuner, University Heidelberg, Germany JOURNAL OF NEUROSCIENCE. 2014; 34: p. 1446–1461. • Marc Landry, University Bordeaux, France Stable incorporation versus dynamic exchange of beta subunits in • Amy Lee, University of Iowa, Iowa City, USA a native Ca2+ channel complex. Campiglio Marta, Di Biase Valenti- • Martin Leonard, Public Health England, UK na, Tuluc Petronel, Flucher Bernhard E. • Claude Libert, VIB, Belgium JOURNAL OF CELL SCIENCE. 2013; 126: p. 2092–2101. • Marzia Malcangio, Kings College London, UK • Josef Penninger, IMBA, Vienna, Austria SEURAT-1 liver gold reference compounds: a mechanism-based • J. Perez-Gil, Complutense University, Madrid, Spain review. Jennings Paul, Schwarz Michael, Landesmann Brigitte, • Markus Ritter, Paracelsus Medical University, Salzburg, Austria Maggioni Silvia, Goumenou Marina, Bower David, Leonard Martin • Claudia Sommer, University Würzburg, Germany O, Wiseman Jeffrey S. • Hermona Soreq, Hebrew University Jerusalem, Israel ARCHIVES OF TOXICOLOGY. 2014; 88: p. 2099–2133. • Bob van de Water, Leiden University, The Netherlands An overview of transcriptional regulation in response to toxicolog- • Vladimir Yarow-Yarovoy, University of California Davis, Davis ical insult. Jennings P, Limonciel A, Felice L, Leonard MO. CA, USA ARCHIVES OF TOXICOLOGY. 2013; 87: p. 49–72.

Selected Funding • EC-FP7, (No 602133) ncRNAPain: Non-coding RNAs for personalised pain medicine, Michaela Kress • EU 7th Framework, Predict-IV, Paul Jennings • EU 7th Framework, DETECTIVE, Paul Jennings • IMI-Projekt StemBANCC, Paul Jennings, Gerhard Gstraunthaler • FWF, P25345: S1P and post-operative pain, Michaela Kress • FWF, W1206: Doctoral College “Signal processing in neurons”, Michaela Kress • FWF, P23479: Expression and function of a new skeletal muscle calcium channel splice variant, Bernhard E. Flucher

Research Report 2015 Medical University of Innsbruck 39 Department of Physiology and Medical Physics Biomedical Physics

ple by their vibrational “fingerprint” by Monika Ritsch-Marte was recently elected means of the Raman effect (spontaneous Fellow of the Optical Society of America Raman or Coherent anti-Stokes Raman and elected into the Austrian Academy of Scattering = CARS). Sciences. Alexander Jesacher received the Highlights: non-scanning (wide-field) CARS prestigious Young Researcher Award of the microscopy, phase-sensitive CARS. Erlangen Graduate School in Advanced Op- • Solar UV radiation: optimisation of spec- tical Technology (SAOT). troradiometric instruments and development of analysis techniques for solar ra- Research diation spectra and aerosol optical depth. Highlights: the Austrian UV measurement Biomedical Optics network (UV-Index). Monika Ritsch-Marte and Stefan Bernet Holographic Optical Tweezers shape light General Facts into optical trapping patterns which can move or pull microscopic particles, such as The Division of Biomedical Physics pursues micro-organisms, micro-beads, living cells, application-oriented basic research pro- cell organelles, or DNA-strands, in a con- jects devoted to the development of novel trolled and contact-free way. Head of Division: physical methods and technologies in Recently the research group has strived o. Univ.-Prof. in Dr.in medicine or cell biology. Currently there at pushing the limits of trapping towards Monika Ritsch-Marte exist two Research Groups: the Biomedical increasingly large particles, including fast Optics Group and the UV-Radiation Group. swimming organisms (e.g. flagellates or Eu- Contact: The research is largely funded externally, glena species). The “macro-tweezers” sys- Müllerstraße 44 e.g. by FWF, ERC, and EU network grants. tem, a dual beam mirror trap with a large 6020 Innsbruck The Biomedical Optics Group has a high field of view, was established and used to visibility in the international Biophotonics catch the largest living specimens ever [email protected] community, in particular for contributions trapped by exclusively optical means. To Phone: +43 512 9003 70870 to Holographic Optical Tweezers and to increase the scope even further, an ultra Fax: +43 512 9003 73870 Synthetic Holographic Microscopy. The sonic standing wave was created which www.i-med.ac.at/dpmp/bmp research focus lies on wavefront shaping confines the particles to a plane in the with so-called Spatial Light Modulators middle of the probe chamber – enabling Keywords (SLMs), miniaturized liquid crystal displays computer-controlled manipulation of spec- with individually addressable micrometer- imens of interest by optical tweezers, e.g. Biophotonics, optical tweezers, nonlinear sized pixels. for uncontaminated sampling for PCR. microscopy, CARS microscopy, Raman The UV-Radiation group is interested in Moreover, in collaboration with a Biophys- microscopy, digital holographic microscopy, various aspects of solar UV radiation. They ics group in Amsterdam, acoustic trapping phase contrast, spatial light modulators, optimise spectroradiometric instruments was adapted for massively parallel probing UV measurements, solar UV radiation and develop analysis techniques to meas- of macromolecules in a lab-on-a-chip device ure solar radiation spectra and aerosol op- (cf. Fig. 1). Research Focus tical depth. They operate the Austrian UV monitoring network. Synthetic Holographic Microscopy employs • Optical micro-manipulation: contact-free SLM-based wavefrontntrol to enhance a mi- handling of microscopic particles (micro- Recent special recognitions and awards: croscope in specific ways: One can flexibly organisms, micro-beads, living cells, cell The Division of Biomedical Physics has emulate various contrast mechanisms (e.g. organelles, or DNA-strands) with laser light. hosted several international conferences in brightfield,darkfield or phase contrast), Highlights: trapping of the largest swim- the last few years, including the Trends in create multiplexed images (combining dif- ming micro-organisms ever trapped “all- Optical Manipulation conference series in ferent imaging settings in one recorded im- optically”; combined acoustic and optical Obergurgl. age), or steer and pattern the illumination in trapping of even larger specimens. • Holographic microscopy: wavefront shaping with miniaturized liquid crystal displays, so-called Spatial Light Modulators (SLMs) inside an optical microscope, to create novel types of microscopy techniques. Highlights: spiral phase contrast, halo-free Zernike phase contrast, multiplane imaging, single-shot quantitative differential interference contrast, lensless imaging Fig. 1: Acoustic Force Spectroscopy, developed in collaboration with the VU Amsterdam, uses through a scattering medium. an acoustic manipulation device that can exert forces from subpiconewtons to hundreds • Chemical (vibrational) imaging: label-free of piconewtons on thousands of biomolecules (such as single- or double-stranded DNA) in technique to visualize molecules in a sam- parallel in lab-on-a-chip devices (G. Sitters et al., Nature Methods 12, 47, 2015).

40 Research Report 2015 Medical University of Innsbruck Biomedical Physics

Fig. 2: Synthetic Holographic Microscopy: Multi-plane imaging by wavefront shaping with a spatial light modulator. Using RGB illumination and a colour camera, 21 planes of the sample (a spore) could be imaged simultaneously (A. Jesacher et al., Optics Express, 21, 11150, 2013). the microscope for structured illumination with the Institut Fresnel in Marseilles. This sitivity of the human skin to UV radiation . microscopy. allows one to retrieve also the spontaneous An example of these maps is shown in Last year the approach was used to develop Raman spectrum directly from the CARS Fig. 3, in which the measurement sites are a method for diffractive multi-plane imag spectrum, to gain complementary informa- marked. It gives the maximum UV-index on ing, i.e. the instantaneous optical imaging tion. As a second line of action the feasibil- 14.05.2015, clearly influenced yb cloudi- of the entire volume of a thick transparent ity and practicality of super-resolution in ness and topography. sample. With multicolor LED illumination CARS-microscopy is under investigation. and a colour camera, time-synchronous im- The capability for absolute UV measure- aging of 21 focal planes was demonstrated UV-Radiation ments is also applied for measurements successfully (cf. Fig. 2). Mario Blumthaler of artificial UV sources as, e.g. used in sun In the past two years the research group has beds in solaria. In cooperation with inter- Raman Microscopy provides information continued to optimise spectroradiometric national agencies (e.g. Commission Inter- on the chemical contents of a sample measurements of solar radiation using array nationale de l’Eclairage CIE) these data are without the need to introduce exogenous spectroradiometers. Precise determination interpreted in terms of harmful and poten- dyes. Spontaneous Raman illuminates the and correction of stray light is necessary for tially healthy effects. Measurements in con- sample with a laser and looks at the spec- any interpretation of biological effects of UV nection with workplace security and protec- trum of the inelastically scattered light. measurements because of the steep slope tion were also carried out. The Raman signal can be resonantly en- of the solar spectrum in the UVB wavelength hanced, by using two laser beams of differ- range, and the high sensitivity of biological Selected Publications ent colour: If the frequency difference of tissues at these wavelengths . As a result, Wide-field vibrational phase imaging in an extremely folded two laser beams matches a Raman-active the healthful effect of Vitamin D production box-CARS geometry. Berto P, Jesacher A, Roider C, Monneret S, Rigneault H, Ritsch-Marte M. OPTICS LETTERS. 2013; 38: p. 709– vibration of targeted molecules, a blue- and the negative effect of erythema can be 711 [Selected for the Virtual Journal for Biomedical Optics 8 (4), shifted anti-Stokes signal is generated by a quantified in dependenceon the environ- May 2013]. resonant four-photon-effect called Coher- mental conditions (day of the year, time Enhancing diffractive multi-plane microscopy using coloured illumination. Jesacher A, Roider C, Ritsch-Marte M. OPTICS EX- ent Anti-Stokes Raman Scattering (CARS). of the day, altitude, snow cov er, amount of PRESS. 2013; 21: p. 11150–11161 [Selected for the Virtual Jour- The combination of CARS with microscopic ozone, amount of aerosols). The absolute nal for Biomedical Optics 8 (6), June 2013]. imaging leads to CARS microscopy, which calibration of UV detectors in the laboratory Axial super-localisation using rotating point spread functions shaped by polarisation-dependent phase modulation. Roider C, enables fast recording of chemical maps is traceable to the German reference labo- Jesacher A, Bernet S, Ritsch-Marte M. OPTICS EXPRESS. 2014; on a micrometer scale. From a practical ratory Physikalisch-Technische Bundesan- 22: p. 4029–4037 [Selected for the Virtual Journal for Biomedical Optics 9 (4), April 2014] . point of view, CARS microscopy resembles stalt (PTB) and the outdoor measurements Lensless imaging through thin diffusive media. Harm W, Roider C, fluorescence microscopy, but without the are regularly compared with measurements Jesacher A, Bernet S, Ritsch-Marte M. OPTICS EXPRESS. 2014; need for fluorescent dyes. In the past a non- of the reference laboratory of the World 22: p. 22146–22156 [Selected for the Virtual Journal for Biomedi- cal Optics 9 (11), Nov. 2014]. scanning wide-field CARS microscope was Meteorological Organisation. Based on its Stray light correction of array spectroradiometers for solar UV developed in Innsbruck. capabilities for high quality UV measure- measurements. Nevas S, Gröbner J, Egli L, Blumthaler M. APPLIED In the past two years phase-sensitive meas- ments, the UV group is responsible for the OPTICS. 2014; 53: p. 4313–4319. urements of the CARS signal were record quality control and publication of the Aus- Selected Funding ed with a special camera, in collaboration trian UV monitoring network within a long • Coherently Advanced Tissue and Cell Holographic Imaging and term research grant of the Austrian Govern Trapping, ERC Advanced Investigator Grant P247024, Ritsch‑ mental Department for the Environment. Marte Monika, 2010–2015 • Advanced Wide-field CARS Microscopy, Austrian Science Fund FWF ‑Project P22085, Ritsch-Marte Monika, 2010–2013 At the moment, the results of 16 stations • Christian Doppler Laboratory for Microscopic and Spectro scopic Material Characterisation (MS-MACH), Christian Doppler in Austria and nearby Bavaria and Swit- Forschungsgesellschaft, (Coordinator: Assoc.Prof. D. Stifter, zerland are published in near real time Johannes Kepler Universität Linz), Ritsch-Marte Monika, 2010‑2017 (www.uv-index.at), together with a regional • Traceability for surface spectral solar UV radiation, EMRP map, showing the distribution of the level researcher grant, ENV03-REG1, Blumthaler Mario, 2011–2014 of UV exposure modulated by the actual Collaborations cloud cover, derived from actual pictures • Rigneault Hervé, Institut Fresnel, Marseille, France Fig. 3: The Div. for Biomedical Physics oper- of the Meteosat satellite. The data are pre- • Padgett Miles, University of Glasgow, UK ates the Austrian UV measurement network sented in units of the ‘UV-index’, which is an • Piestun Rafael, University of Colorado, Boulder, USA • Wuite Gijs, Vrije Universiteit Amsterdam, Amsterdam, NL generating the on-line UV-Index for Austria, internationally agreed quantity for harmful • Nevas Julian, Physikalisch-Technische Bundesanstalt, Berlin, DE which is updated every few minutes. UV exposure, taking into account the sen- • Gröbner Julian, World Radiation Center, Davos, Switzerland

Research Report 2015 Medical University of Innsbruck 41 Department of Medical Genetics, Molecular and Clinical Pharmacology Cell Genetics

immune-regulatory cytokines or displays Research targeted cytotoxicity, ultimately leading to recruitment of innate immune cell types Cell Genetics Team: Gottfried Baier, and the initiation of an effective immune Natascha Kleiter, Thomas Gruber, response. Nikolaus Thuille, Kerstin Siegmund, Christa Pfeifhofer-Obermair, Victoria In order to understand the physiology and Klepsch, Sebastian Peer et al. pathophysiology of T lymphocytes, it is necessary to decode the biochemical process- Due to its biological complexity cancer is es that integrate the signals received from still poorly understood. It has been shown antigens, cytokines, and integrins as well as both experimentally and epidemiologically inhibitory receptors. Our work aims to ex- that chronic inflammation can predispose plore and identify gene products of distinct individuals to cancer, and may additionally members of the AGC family of protein ser- represent an inseparable aspect of clinically ine/threonine kinases and their effector prevalent cancer entities. substrates that act as key players in me- Therefore, the decoding of both tumour diating proper T cell effector functions and and immune cell functions in cancer pro- in fine-tuning theimmune response. The gression will be of the utmost importance Head of Division: underlying goal of the work is to understand in combating this frequently incurable dis- Univ.-Prof. Dr. Gottfried Baier the selective functions of these proteins in ease. signal transduction pathways in lympho- My team and I were the first to reveal the Contact: cytes and to use this information to develop lymphocyte-intrinsic PKC/NR2F6/Cbl-b Peter Mayr Straße 1a strategies for manipulating the immune re- axis as an essential signalling node gov- 6020 Innsbruck sponse, either in order to promote immuno- erning the complex host-tumour inter- suppression in the context of autoimmune actions at the crossroads between inflam- [email protected] diseases, graft rejection and inflammatory mation and cancer. Phone: +43 512 9003 70514 responses or for augmentation as part of Modulation of this lymphocyte-intrinsic sig- Fax: +43 512 9003 73518 an innovative cancer immunotherapy-based nalling pathway renders CD4+ and CD8+ ef- http://www.baierlab.com/ approach. fector T cells capable of rejecting otherwise

Keywords

T lymphocyte signalling, T helper cell differentiation, T cell effector functions, autoimmunity, cancer immunity, innovative immunological therapy concepts

Research Focus

The Cell Genetics Team has expertise in signal transduction, mouse genetics, the differentiation of effector/memory T cells and the ability of these cells to alter adaptive immune responses. In particular, we have gained experience in investigating molecular signalling processes through the use of hypothesis-driven mechanistic studies and by utilising unbiased mass spectrometry based screens and next generation sequencing to characterise novel protein-protein and protein-DNA inter- actomes and transcriptomes.

General Facts

The function of mature T cells is to recognize and respond to foreign antigens by a complex activation process involving dif- Fig. 1: The major research topic of the group relates to the biochemical, molecular and func- ferentiation of the resting cell into a prolif- tional analysis of the signal transducing protein kinase network within the haematopoietic erating lymphoblast that actively secretes system.

42 Research Report 2015 Medical University of Innsbruck Cell Genetics

TUMOR IMMUNE EVASION

TUMOR DRAINING CD8+ LYMPH NODE Dendri'c cell

CD4+

IL-‐10 Type 2 Macrophage TGFβ Monocyte

MDSC

Type 1 Macrophage Dendri'c cell

CIRCULATING TUMOR CELLS IL-‐2 Cancer cell IFNγ

TUMOR MICROENVIRONMENT

MICROBIOTA THERAPY TUMOR IMMUNE SURVEILLANCE G. Baier and team, review MS in prepara'on Re-‐Educa'ng an eﬀec've an'-‐tumor immunity during cancer therapy

Fig. 2: Our research aims to elucidate the underlying inter- and intracellular mechanisms that shape the microenvironment at the tumour site either to support or to prevent tumour initiation, progression or tumour immune surveillance. lethal tumour burdens and their metasta- Selected Publications Fresser F, Leitges M, Soldani C, Viola A, Kaminski S, Baier G. ses in experimental murine cancer model Immunity. 2013 Jan 24;38(1):41–52. Orphan nuclear receptor NR2F6 acts as an essential gatekeeper systems in vivo. Mechanistically, Nr2f6 as of Th17 CD4+ T cell effector functions. Selected Funding well as Cblb knockout mice thereby dis- Hermann-Kleiter N, Baier G. CELL COMMUNICATION AND SIGNALING (CCS). 2014; 12: p. 38. play an immune contexture at the tumour • Systems Biology of T Cell Activation, Acronym: SYBILLA, plus BMfWF funding; http://www.sybilla-t-cell.de/; EC grant/FP7- The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis site that allows superior anti-tumour T cell HEALTH-Large-scale Integrating Project via natural killer cells. responses, increasing the survival rates of Paolino M, Choidas A, Wallner S, Pranjic B, Uribesalgo I, Loeser S, • Molecular mechanisms regulating tumor immunology; tumour-bearing mice. Jamieson AM, Langdon WY, Ikeda F, Fededa JP, Cronin SJ, Nitsch http://www.sfb021.at/, FWF R, Schultz-Fademrecht C, Eickhoff J, Menninger S, Unger A, Torka • T cell-intrinsic role of PKCα in canonical TGFβR signalling, FWF Since it has already been established that R, Gruber T, Hinterleitner R, Baier G, Wolf D, Ullrich A, Klebl BM, • Cbl-b inhibitory signalling pathways in cancer, FFG Bridge cancer-mediated immune evasion can lead Penninger JM. • TaNeDS research collaboration in cancer biology; Daiichi NATURE. 2014; 507: p. 508–512. to T cell dysfunction, our data strongly sug- Sankyo Ltd., Tokyo, Japan gest that T cell-based therapies could signif- Cbl-b mediates TGFß sensitivity by downregulating inhibitory SMAD7 in primary T cells. Collaborations icantly benefit from modulation of this novel Gruber T, Hinterleitner R, Hermann-Kleiter N, Meisel M, Kleiter I, NR2F6/Cbl-b inhibitory signalling pathway. Wang CM, Viola A, Pfeifhofer-Obermair C, Baier G. • Jürgen Wagner and Gerhard Zencke; Novartis Pharma, Basel, J Mol Cell Biol. 2013 Dec;5(6):358–68. Switzerland Such a strategy of intracellular NR2F6 • Michael Leitges, Biotechnology Centre of Oslo, University of and/or Cbl-b checkpoint-targeting will Engineering effective T-cell based antitumor immunity. Oslo, Norway Gruber T, Hinterleitner R, Pfeifhofer-Obermair C, Wolf D, Baier G. • Amnon Altman, LIAI, San Diego, USA improve the efficacy and broaden the appli- Oncoimmunology. 2013 Feb 1;2(2):e22893. cability of cancer immunotherapy regimens, • Steve Shaw, NIH, Bethesda, USA The Kinase PKC alpha Selectively Upregulates Interleukin-17A • Noah Isakov, Ben Gurion University of the Negev, Israel and thus has the potential in the future to during Th17 Cell Immune Responses. Meisel M, Hermann-Kleiter • Wallace Langdon, University of Western Australia, Perth, AUS lead to prolonged patient survival. N, Hinterleitner R, Gruber T, Wachowicz K, Pfeifhofer-Obermair C, • Arthur Kaser, Department of Gastroenterology, Cambridge, UK

Research Report 2015 Medical University of Innsbruck 43 Department of Medical Genetics, Molecular and Clinical Pharmacology Genetic Epidemiology

1. A protein chemistry and cell culture lab- renal disease apoA-IV is associated with all- oratory performs a variety of functional cause mortality and sudden cardiac death. and epidemiologic studies regarding Finally, afamin is a human plasma vitamin E– phenotypes related to lipoprotein metab- binding glycoprotein primarily expressed in olism and other metabolic phenotypes. the liver. After initial biochemical character- 2. A molecular-genetic lab performs se- isation we started first functional studies quencing and genotyping for various and demonstrated that transgenic mice projects, with a strong focus on mito- overexpressing afamin had increased body chondrial DNA as well as on targeted weight and serum concentrations of lipids evaluation of certain candidate genes. and glucose. In line with these results we 3. The computational & statistical genetics showed in three population-based studies lab focusses on statistics, epidemiology, including >5000 participants that afamin computer science, and bioinformatics is strongly associated with the prevalence and represents an important cross-link and development of metabolic syndrome between the various research groups. and its components. Further studies in patients with pregnancy complications and Besides these three pillars our institute polycystic ovary syndrome confirmed the includes the “Sequencing & Genotyping association between afamin and metabolic Head of Division: Core Facility” that offers Sanger-sequenc- syndrome-like phenotypes. Univ.-Prof. Dr. Florian Kronenberg ing, large scale genotyping projects and management of large epidemiological stud- Molecular-Genetic Lab Contact: ies. The output and success of our institute This lab is in charge of the DNA work related Schöpfstraße 41 is based on a constant dialogue between the to various genetic-epidemiological cohorts. 6020 Innsbruck disciplines in a problem-oriented and crit- It performs targeted sequencing and geno ical elucidation of the research questions. typing of complex regions with specially [email protected] designed assays. Measurement of relative Phone: +43 512 9003 70560 Research telomere length in several cohorts and its Fax: +43 512 9003 73561 association with cardiovascular disease, http://www3.i-med.ac.at/genepi/ Protein Chemistry Lab kidney disease, and cancer shed new light Lipoprotein(a) [Lp(a)] and its apolipo on this marker of aging. protein(a) isoforms, apolipoprotein A-IV Beside the nuclear genome, one of our and afamin belong to the three most inten- research topics relates to mitochondrial sively studied proteins in our lab. During DNA (mtDNA). Being strictly maternally in- Keywords recent years we measured these proteins herited without recombination, mtDNA is a in roughly 20,000 individuals from major powerful tool to reconstruct maternal relat- Genetic epidemiology, lipoprotein meta population-based studies from Europe and edness based on sequence polymorphisms. bolism, complex phenotypes, genome-wide the US. These measurements are the basis Mutations in mitochondrial genes have association studies, mitochondrial DNA, for genome-wide association studies (see been linked to several complex diseases. computational genetics, cloud computing, below). Therefore, mtDNA is targeted in medical-, risk factors, cardiovascular disease, bio- Lp(a) and its genetic determinants have population- and also forensic genetics. Our markers been one of the main topics since the group established a comprehensive work- foundation of this institute in 2004. We flow for mtDNA analysis comprising next Research Focus are associating these measurements with generation deep sequencing strategies as various diseases and disease-associated well as accurate data management. Our We aim to identify determinants of health parameters such as cardiovascular disease, open source software tools eCOMPAGT, and disease related to genetic variability, peripheral arterial disease, kidney disease HaploGrep and mtDNA-Server are contri- environmental components and biochemical and diabetes mellitus. Genetically deter- butions to an automated mtDNA data pro- parameters and to study their physiological mined apo(a) isoforms are the strongest ge- cessing. These pipelines in hand, we were or pathophysiological functions. Our pheno- netic risk factor for cardiovascular disease. able to evaluate phylogenetic aspects in types of interest are complex in nature due In a recent analysis in three independent the multi-ethnic population of Myanmar. to their interplay and are related to athero- populations including more than 6000 in- sclerosis, diabetes mellitus, metabolic syn- dividuals we showed a causal relationship drome, cancer, and associated intermediate between Lp(a) concentrations and peripher- phenotypes such as lipoprotein metabolism al arterial disease. and inflammation. A further apolipoprotein studied in our lab is apolipoprotein A-IV (apoA-IV). For this General Facts apolipoprotein we could show an association with cardiovascular disease. Further- Our Institute serves as a bridge between more, patients with kidney disease have basic and clinical research. We have three pronounced elevations of apoA-IV which different pillars that develop their strength are associated with progression of chronic by an interleaved collaboration. kidney disease. In patients with end-stage Fig. 1: Analysis of sequencing data

44 Research Report 2015 Medical University of Innsbruck Genetic Epidemiology

Division of Genetic Epidemiology

Director: Florian Kronenberg Administration: Anita Neuner

Protein Chemistry Lab Molecular-Genetic Lab Computational & Scientists: Scientists: Statistical Genetic Lab Hans Dieplinger Stefan Coassin Scientists: Bernhard Rupp Liane Fendt Lukas Forer Konrad Schmidt Federica Fazzini Salome Friedel Technical personnel: Anita Kloss-Brandstätter Barbara Kollerits Ramona Berberich Bernd Schöpf Claudia Lamina Linda Fineder * Barbara Fürnrohr * Sebastian Schönherr Melanie Harg * Julia Raschenberger * Hansi Weißensteiner Andrea Stöckl * Technical personnel: Eva Boes * Gertraud Erhart Christian Fuchsberger * Margot Haun Monika Summerer *

Sequencing & Genotyping Core Facility Several members of the GenEpi team are contributing to the Core Facility

Diploma and Master Students Alessandra Altamirano * Clemens Banas * Anja Laschkolnig * Johannes Linsenmeyer * Johannes Pohlhammer * Stephanie Stangl * Lena Vogl * * Former coworkers who successfully completed their projects. We very much appreciate Fig. 3: The Division of Genetic Epidemiology developed several their previous contributions open-source software solutions for local computing and remote computing. Currently three different services are provided within Fig. 2: Coworkers of the Division of Genetic Epidemiology Cloudgene, executed in a massively parallel way.

Furthermore, we are currently investigating tation Server, in which Cloudgene builds the Meisinger C, Rantner B, Stöckl D, Stadler M, Klein-Weigel P, Peters A, Fraedrich G, Kronenberg F. the role of mitochondrial mutations in pros- underlying architecture. From July 2014 to Atherosclerosis. 237:243–250, 2014. tate- and oral squamous cell carcinomas, August 2015 over 1 million genomes have Comparison and evaluation of cardiac biomarkers in patients elucidating whether somatic mutations are been successfully imputed using this free with intermittent claudication: Results from the CAVASIC Study. involved in disease development and the service. Besides the imputation service, two Kollerits B, Sturm G, Lamina C, Hammerer-Lercher A, Rantner B, Stadler M, Ziera T, Struck J, Klein-Weigel P, Fraedrich G, further prognosis of the disease. additional workflows (mtDNA NGS, GWAS) Kronenberg F. Clin. Chem. 59:692–702, 2013. have been integrated into Cloudgene and Computational & Statistical Genetic Lab provided to the community as free services. Selected Funding This lab is characterized by a profound In addition, we are involved in several large • SAXCESS “Stucture-function analysis of the human plasma gly- interplay between statistics, epidemiology, scale population studies (e.g. the German coprotein afamin, a potential drug target in the treatment of metabolic syndrome.”, EU (Marie Curie), Hans Dieplinger computer science and bioinformatics. In Chronic Kidney Disease Study, ncRNA-Pain • “ncRNAPain: Non-coding RNAs in neurogenic and neuropathic genome-wide association studies (GWAS) Study), in which we manage huge amounts pain mechanisms and their application for risk assessment, patient stratification and personalised pain medicine”, EU-FP7, we bring together the data from pheno of data and improve the overall quality of Florian Kronenberg typing done e.g. in the protein chemistry lab collecting phenotypic information. These ef- • “A genome-wide approach to evaluate the genetic basis of lipo protein(a)”, FWF, Claudia Lamina with the genotype data and we try to find forts resulted in two open source software • “Analysis of rare variants from sequencing data”, FWF (Schrö- hitherto unknown associations with genes. solutions: Askimed for managing pheno dinger Rückkehrpr.), Christian Fuchsberger Within the last ten years we were involved types and SNPflow for the standardized in several GWAS consortia. Our own main and automatic quality control of genotyping Collaborations focus is on studies with Lp(a), apoA-IV and data. • Gonçalo Abecasis, Center of Statistical Genetics, University of Michigan, Ann Arbor, USA afamin, but we are also involved in consortia • Enis Afgan, Ruđer Bošković Institute Zagreb, Croatia & Johns evaluating phenotypes such as adiponectin, Selected Publications Hopkins University, Baltimore, USA peripheral arterial disease, BMI and waist • Steven C. Hunt, Cardiovascular Genetics Division, University of Lipoprotein(a) concentrations, apolipoprotein(a) phenotypes Utah, Salt Lake City, USA circumference, metabolites, measures of and peripheral arterial disease in three independent cohorts. • Institute of Epidemiology, Helmholtz Zentrum München, kidney function and others. Using these Laschkolnig A, Kollerits B, Lamina C, Meisinger C, Rantner B, Neuherberg, Germany Stadler M, Peters A, Koenig W, Stöckl A, Dähnhardt D, Böger CA, • Günther Specht, Department of Database and Information Sys- methods resulted in a tenfold increase Krämer BK, Fraedrich G, Strauch K, Kronenberg F. tems; Institute of Computer Science, University of Innsbruck, Cardiovascular Research. 103:28–36, 2014. Innsbruck, Austria in the number of known gene-phenotype • Matthew Bowler, Synchrotron Diffraction, EMBL Grenoble, associations. Plasma concentrations of afamin are associated with the France One aim of this lab is to apply computer sci- prevalence and development of metabolic syndrome. Kronenberg F, Kollerits B, Kiechl S, Lamina C, Kedenko L, Meisinger C, Willeit Core Facilities ence to the field of molecular biology and J, Huth C, Wietzorrek G, Altmann ME, Thorand B, Melmer A, medical genetics. Since genetics turned Dähnhardt D, Santer P, Rathmann W, Paulweber B, Koenig W, The Sequencing & Genotyping Core Facility owns state-of-the-art into a big data science, we provide re- Peters A, Adham IM, Dieplinger H. equipment for Sanger sequencing, high throughput genotyping Circ. Cardiovasc. Genet. 7:822–829, 2014. and qPCR and fragment analysis. searchers ready-to-use analysis pipelines • Sequenom MassARRAY4 MALDI-TOF System: multiplex geno in cloud environments without the neces- SNPflow: a lightweight application for the processing, storing and typing and methylation analysis automatic quality checking of genotyping assays. Weissensteiner • QuantStudio 6 Flex System: large scale genotyping and qPCR sity to become acquainted with the under- H, Haun M, Schönherr S, Neuner M, Forer L, Specht G, • 3130xl and 3730s Systems: Sanger sequencing and fragment lying technical details. One of our efforts Kloss-Brandstätter A, Kronenberg F, Coassin S. analysis using either 16 (low throughput) and 48 capillaries PLoS One. 8: e59508, 2013. (high throughput) resulted in the novel data parallelization • Fragment Analyzer: automated fragment analysis for NGS platform Cloudgene. The success of this Correlation between a positive family risk score and peripheral library QC artery disease in one case-control and two population-based • 8 and 96 channel TECAN pipetting robots for large pipetting platform can be seen in the Michigan Impu- studies. Lamina C, Linsenmeyer J, Weissensteiner H, Kollerits B, jobs and automated sample normalization

Research Report 2015 Medical University of Innsbruck 45 Department of Medical Genetics, Molecular and Clinical Pharmacology Human Genetics

• New methods of genetic laboratory alterations lead to impaired mitochondrial diagnosis, mutation databases, molecular respiration and result in human patholo- genetic quality control gies, generally referred to as “mitochondrial diseases”. Most of the proteins of the res- General Facts piratory chain are encoded by nuclear DNA; however, 13 respiratory complex proteins The primary aim of the Division of Human (mRNAs), two ribosomal RNAs (rRNAs) and Genetics is clarifying the genetic determi a complete set of 22 transfer RNAs (tRNAs) nants of health and disease in humans, are encoded by mitochondrial DNA (mtD- with special focus on rare diseases that are NA), a circular genome inside mitochondria. inherited as monogenic traits, and on genetic variants that have a major impact on One essential step in mitochondrial main- human biology and substantial disease rele tenance is mitochondrial RNA (mtRNA) vance. This aim is achieved by combining transcript processing. Transcription of the comprehensive patient services and exper- mtDNA occurs on both DNA strands and tise in clinical genetics, molecular genetics produces long polycistronic transcripts of and cytogenetics with basic research. The mRNAs and rRNAs, usually interspersed institute includes the Centre for Medical with tRNAs. Mitochondrial protein transla- Head of Division: Genetics Innsbruck which provides medi- tion requires the correct processing of the Univ.-Prof. DDr. Johannes Zschocke cal genetic services for the entire Western polycistronic RNA molecules at the 5énd Austria with extensive outpatient clinics and and 3énd of the interspersed tRNAs to re- Contact: inpatient consultation in Innsbruck and sev- lease mature mRNAs, tRNAs, and rRNAs. Peter Mayr Straße 1/1 eral regional hospitals. The diagnostic labo- The initial processing step, the endonucle- 6020 Innsbruck ratories cover all relevant methods for DNA, olytic cleavage of precursor tRNAs at the RNA and chromosome analysis including 5énd, is performed by RNase P, an enzyme [email protected] classical cytogenetics, fluorescence-in-situ complex composed of three different pro- Phone: +43 512 9003 70500 hybridization (FISH), DNA-Array (molecular teins (MRPP1, HSD10, and MRPP3). Pro- Fax: +43 512 9003 73510 karyotyping), tumour cytogenetics, Sanger cessing at the 3énd is done by tRNase Z, www.humgen.at sequencing for a large number of individu- a single protein coded by the ELAC2 gene. al genes, massively parallel (“next generation”) sequencing – both panel and clinical We found that missense mutations in the exome – multiplex-ligation-dependent probe genes for HSD10 and MRPP3 resulted in re- amplification (MLPA), methylation and im- duced tRNA processing which lead to mito- Keywords printing analyses, fragment length typing chondrial damage due to impaired respira- and Southern Blot for microsatellite repeat tory complex formation. In HSD10 disease, Human genetics, molecular genetics, cyto analyses, and others. Due to the close link for instance, patients usually display a pro- genetics, mitochondrial genetics, transcript with the large basic research unit, interest- gressive neurodegenerative disease course analysis, tumour disposition syndromes, ing observations or unclarified cases may with loss of cognitive and motor function, genetic skin diseases, metabolic medicine, be directly transferred into further investi- epilepsy and progressive cardiomyopathy, cancer genetics gations on a research basis. The diagnostic and usually death in childhood. Impor- laboratories are equipped for a wide range tantly, tRNA processing is the first step in Research Focus of relevant cell biology techniques with a post-transcriptional mtRNA maturation special focus on DNA and RNA analyses which includes base modification, RNA sur- • Genetic causes of rare diseases, including: and the functional analysis of genetic alter- veillance, RNA packaging, and finally RNA –– Developmental disorders, intellectual ations. The division is dedicated to interdis- decay. It is estimated that approximately disability and dysmorphic syndromes ciplinary collaboration and is happy to carry 300 nuclear coded proteins are involved –– Inherited metabolic diseases out both diagnostic tests and research in- in mitochondrial gene expression and only –– Genetic skin diseases vestigations for a large number of hospital some of them are known to be involved in –– Genetic disease of the teeth and perio- centres in Innsbruck and elsewhere. mitochondrial disorders. However, there dontal tissue are many unsolved cases of mitochondrial • Genetic causes of tumours and tumour Research disease, and defining genetic causes and dispositions, including: patho-mechanisms remains a major chal- –– Inherited cancer disposition syndromes Mitochondrial RNA Maturation lenge. In our projects, we focus on novel –– Breast and ovarian cancer and its Diseases mutations which disrupt mtRNA biogenesis. –– Hamartomatous tumours Johannes Zschocke, Albert Amberger, –– Cytogenetics of haematological Andrea Deutschmann Major Achievements: Identification, clinical malignancies Mitochondria are the powerhouses of the characterization, and functional analysis • Transcription and transcript processing of cell. They have a central function in ener- of HSD10 disease; defining new defects mitochondrial DNA (mtDNA) gy production and play vital roles in several in mtRNA processing which contribute to • Transcription and transcript processing of cellular processes. Cellular energy is mainly mitochondrial disorders. nuclear genes, in particular splice mech- produced by the respiratory protein com- Future Goals: Developing diagnostic tools anisms plexes in mitochondria. Numerous genetic and establishing novel disease models to

46 Research Report 2015 Medical University of Innsbruck Human Genetics

Major Achievements: RNA-based assays for comprehensive characterization of mu- mtDNA transcription tations e.g. in the NF1 and PMS2 genes; substantial and ongoing contributions to polycistronic RNA the delineation of tumour spectrum and non-neoplastic features in CMMRD; lead de- 5´ velopment of clinical diagnostic criteria for the diagnosis CMMRD in a European consortium; identification of a prevalentBRCA1 mutation in the Tyrolean Zillertal and Lower MRPP1 HSD10 MRPP1 HSD10 endonucleolytic endonucleolytic pre-RNA Inn valleys which impacts on cancer inci- processing processing RNase P MRPP3 ELAC2 tRNase Z dence and patient care in this region. MRPP3 ELAC2 Future Goals: evaluation of ‘atypical’ splice mutations with the aim to deduce commonly applicable rules for the selection of variants likely to affect mRNA splicing for further mRNA analyses; development of strategies rRNA x2 mRNA x11 tRNA x22 RNA maturation for the detection of mutation-induced splice • modification • modification • modification alterations by NGS; delineation of the patho- • ribosome assembly • polyadenylation • aminoacylation genetic mechanisms, in particular second- Fig. 1: Schematic overview of mitochondrial RNA metabolism. The mitochondrial rRNAs, ary somatic mutations, underlying the de- mRNAs and tRNAs are transcribed from the L- and H-strands as polycistronic units that velopment of neoplastic and non-neoplastic undergo endonucleolytic processing by RNase P and tRNase Z. Following the liberation features in CMMRD patients; evaluation of of the individual mRNA, rRNA and tRNA transcripts, they undergo post-transcriptional clinical data on CMMRD patients in close modifications. Several nucleotides of rRNAs are modified necessary for proper mitochon- collaboration with the European consortium drial ribosome biogenesis. A poly(A) tail is added to mRNAs. Mitochondrial tRNAs undergo and with the ultimate goal to improve the extensive post-transcriptional nucleotide modification, in addition of being aminoacylated management of CMMRD patients with a cognate amino acid. Genetic Skin Diseases understand the remarkable heterogeneity now planning to transfer the experience and Hans Christian Hennies, Katja Eckl of human mitochondrial diseases caused by knowledge gained with RNA-based muta- The skin, the largest human organ, is a high- defects in mtRNA metabolism. tion analysis by Sanger sequencing into the ly structured, multi-layered organ that is in- massive parallel/next generation sequenc- dispensable for the protection of the organ- Cancer Genetics ing (NGS) era. ism from the environment. It is particularly Katharina Wimmer, Julia Vogt, exposed, together with lung and gut, and Johannes Zschocke A second aim of the research lab is the ge- prone to potentially life-threatening reac- This research group is strongly associated netic and clinical characterization of consti- tions of external agents. It exhibits not only with the molecular diagnostic lab of the tutive mismatch repair deficiency (CMMRD) a clearly defined morphology but is also institute that offers molecular genetic in- syndrome, a rare autosomal recessively characterized by a close interplay with the vestigations for a broad spectrum of cancer inherited cancer susceptibility syndrome immune system, which complements struc- susceptibility syndromes. caused by biallelic mutations in one of the tural features in the defence line against four DNA mismatch repair (MMR) genes, hostile intruders. One of the major aims of the research lab is MLH1, MSH2, MSH6 and PMS2. Biallelic the development and improvement of diag- PMS2 mutations for which we developed The dermatogenetics research group is nostic tools for the identification and classi- highly sensitive and reliable analysis ac- especially interested in the molecular fication of mutations that escape standard count for approximately 60 % of all cases characterization of rare skin diseases. We techniques. The lab developed RNA-based of this syndrome. CMMRD shows clinical have mainly focused on severe disorders assays that substantially increase mutation overlap with other cancer susceptibility of keratinization, including generalized detection rates in several tumour suppres- syndromes, in particular neurofibromatosis autosomal recessive congenital ichthyo- sor genes. These approaches can effectively type 1 (NF1), familial adenomatous polypo- ses (ARCI) and palmoplantar keratoder- uncover splice alterations caused by muta- sis and Lynch syndrome, and has only re- mas. Using homozygosity mapping and tions that either fully escape the detection cently been recognized as a distinct child- high-throughput sequencing techniques, of gDNA based assays or cannot be read- hood cancer susceptibility syndrome. As we have been analysing the mutation spec- ily classified as deleterious from the anal- such, there is still a lack of knowledge on trum in ARCI, which can be caused by mu- ysis of gDNA only. The evaluation of these the natural history of this syndrome. tations in more than nine different genes. ‘atypical’ splice mutations e.g. in NF1 also Using exome sequencing combined with allowed elucidating basic mechanism of A third cancer research project, in close linkage analysis, we have for the first time splice site definition and inactivation. RNA- collaboration with the University Hospital of identified mutations inCERS3 , the gene for based assays have also proved to be pivotal Gynaecology and Obstetrics, is aimed at the ceramide synthase 3, associated with ARCI to circumvent diagnostic obstacles that are molecular characterization of ovarian can- and revealed the importance of very long caused by the presence of pseudo-genes cer tissue by tumour-based massive parallel acyl chain ceramides for epidermal barri- of the mismatch repair gene PMS2. We are sequencing analyses. er function (Fig. 2).

Research Report 2015 Medical University of Innsbruck 47 Department of Medical Genetics, Molecular and Clinical Pharmacology

A major impediment to translational re- impeded by the epidermal barrier activity One of the research projects is aimed at search in rare skin diseases stems from the and difficulties in the bioavailability over the characterization of genetic alterations lack of suitable disease models. We have larger skin areas. We have therefore tested that cause aggressive periodontitis; this generated in vitro full-skin models with the efficacy ofrecombinant enzyme as a OeNB-funded project is led by Dr. Kapferer primary keratinocytes and fibroblasts. Skin drug for topical application in ARCI using who has recruited a Tyrolean 4-genera- models for congenital ichthyosis and other transglutaminase 1 deficient skin models. tion family with aggressive periodontitis keratinization disorders were made using The protein was transferred into the epider- in conjunction with clinical features of either patient cells or inactivation of single mis with the help of nanotransporters, and Ehlers-Danlos syndrome (representing genes through RNA interference. the epidermal barrier function was indeed an entity traditionally described as EDS Availability of patient keratinocytes, how effectively reconstituted after repeated type VIII. Aggressive periodontitis (in con- ever, is often very limited and their ability treatments of the ARCI skin model with en- trast to chronic periodontitis) is a rare in- to proliferate is restricted to few passages; zyme/transporter formulations. Based on flammatory disease leading to periodontal RNA interference, on the other hand, can these results we are confident that locally tissue destruction and, if untreated, tooth also interfere with other, unwanted path- substituted proteins are functional in the vi- loss at a young age. It is caused by a pertur- ways. We have therefore embarked on the able epidermis and can compensate for the bation of the homeostasis between the sub- use of induced pluripotent stem (iPS) defects of disease-associated genes. gingival microbiota and the host defences cells for skin modelling. To this end, patient in susceptible individuals. EDS VIII is a clini- fibroblasts are reprogrammed with stem- Ectodermal Diseases cally heterogeneous disorder characterized ness factors, and iPS cells are differentiated Johannes Zschocke, Anna Schossig, by the combination of periodontal disease to the epithelial fate and further to epider- Robert Gruber and variable connective tissue features. A mal keratinocytes (Fig. 3). These cells can The identification and characterization of limited number of patients and pedigrees be used for generating skin models, analys- rare genetic diseases affecting skin, skin with this condition have been described. ing specific therapeutics, and also system- appendages, and teeth entails collaborative The members of the Tyrolean family have atic investigations of potential drugs. projects with PD Dr. Ines Kapferer, Depart- been clinically and immunologically char- Current therapy for ARCI is still mostly symp- ment of Dentistry, and Prof. Dr. Matthias acterized, and we performed a parametric tomatic, and topical application of drugs is Schmuth, Department of Dermatology. linkage analysis and exome sequencing. At present a candidate gene in the chromosomal region 12p13 is under functional investigation. In transfected cells the effect of the mutation on the immunologic function and the connective tissue structure is tested.

Additional projects include the investigation of the genetic causes of rare dental diseases such as Kohlschütter-Tönz syndrome, a rare genetic disorder with amelogenesis im- perfecta and epilepsy, and radicular dentin dysplasia, characterized by abnormal dentin formation and early tooth loss due to abnormal development of tooth roots. Parametric linkage analysis in adequate families is followed by exome sequencing and functional characterization of candidate mutations.

Identification and characterization of inherited skin disorders is the research focus in the collaboration with the Department of Dermatology, with special attention on inherited disorders of keratinization. In a regular dermatology-genetics clinic, patients with rare inherited skin conditions receive comprehensive clinical care and diagnostic work-up involving, where appropriate, an NGS multi gene panel analysis developed Fig. 2: Congenital ichthyosis can be caused by ceramide synthase-3 deficiency (from Eckl et in our institute, covering genetic disorders al, J Invest Dermatol 2013 133: 2202–2211). A) Clinical presentation. B) Enzyme activity was of keratinization and a selection of connec- largely reduced in differentiated keratinocytes from the patient. Overexpression of mutant tive tissue disorders. Patients with specific CERS3 demonstrated almost complete loss of enzyme activity. C) Mutations in CERS3 led to phenotypes of unclear, presumably genetic loss of very long acyl chain ceramides in patient skin. Amounts of free esterifiedω -hydroxy- cause are offered further evaluation by link- lated ceramides with very long acyl chains were significantly reduced in lipid extracts from age analysis (where appropriate) and exome patient keratinocytes. sequencing as well as functional studies.

48 Research Report 2015 Medical University of Innsbruck Human Genetics

Fig. 3: Generation of epidermal keratinocytes by differentiation of induced pluripotent stem cells. A) Pluripotent stem cells were obtained by reprogramming of fibroblasts as shown by typical morphology, activity of alkaline phosphatase, and staining signals for Oct4 and Tra1-81 (left to right). B) Differentiation of induced pluripotent stem cells to the ectodermal fate showed synthesis of epithelial marker keratin 18, epidermal marker keratin 14, and ectodermal gatekeeper p63. C) Primary (first row) and iPS-derived keratinocytes were positive for epithelial markers CD104 (first and second row) and keratins 18, 14, and 5 (third row) in FACS analysis..

MR, Haag R, Hennies HC*, Schäfer-Korting M. • 3D melanocyte skin models; Max Planck Minerva Programme; Selected Publications EXP DERMATOL. 2014, 23:286–288. Katja Eckl. • Skin models for genodermatoses; FFG; Katja Eckl. Mutation or knock-down of 17beta-hydroxysteroid dehydro- Kohlschütter-Tönz Syndrome: Mutations in ROGDI and Evidence genase type 10 cause loss of MRPP1 and impaired processing of Genetic Heterogeneity. Tucci A, Kara E, Schossig A, Wolf NI, of mitochondrial heavy strand transcripts. Deutschmann AJ, Plagnol V, Fawcett K, Paisan-Ruiz C, Moore M, Hernandez D, Collaborations Amberger A, Zavadil C, Steinbeisser H, Mayr JA, Feichtinger RG, Musumeci S, Tennison M, Hennekam R, Palmeri S, Malandrini • Wyatt W. Yue, Structural Genomics Unit, University of Oxford, Oerum S, Yue WW, Zschocke J. A, Raskin S, Donnai D, Hennig C, Tzschach A, Hordijk R, Bast T, UK HUM MOL GENET. 2014, 23:3618–3628. Wimmer K, Lo CN, Shorvon S, Mefford H, Eichler EE, Hall R, Hayes I, Hardy J, Singleton A, Zschocke J, Houlden H. • William Newman, Centre for Genomic Medicine, University of Diagnostic criteria for constitutional mismatch repair deficien- HUMAN MUTATION. 2013, 34(2); 296–300. Manchester, UK cy syndrome: suggestions of the European consortium ‘care for • Johannes Mayr, SALK, Austria CMMRD’ (C4CMMRD). Wimmer K, Kratz CP, Vasen HF, Caron Selected Funding • Ute Moog, Institute of Human Genetics, Medical Center Heidel O, Colas C, Entz-Werle N, Gerdes AM, Goldberg Y, Ilencikova D, berg, Germany Muleris M, Duval A, Lavoine N, Ruiz-Ponte C, Slavc I, Burkhardt • Genetische und funktionelle Studien der molekularen Basis der • Agnès Bloch-Zupan, Faculty of Oral Medicine, University of B, Brugieres L. aggressiven Parodontitis, OeNB, Ines Kapferer-Seebacher, Anna Strasbourg J MED GENET. 2014, 51:355–65. Schossig • Ludwine Messiaen, University of Alabama at Birmingham, Bir- • Mitochondriale RNA-Prozessierung bei der HSD10-Krankheit Impaired ceramide synthesis causes autosomal recessive con mingham, AL, USA und anderen primären Mitochondriopathien, Stiftung Propter genital ichthyosis and reveals the importance of ceramide acyl • Christian Kratz, Department of Pediatric Hematology & Oncolo- Homines,Johannes Zschocke chain length for epidermal terminal differentiation. Eckl KM, gy, Hannover Medical School, Hannover, Germany • Unravelling orphan diseases: High-throughput genomics for Tidhar R, Thiele H, Oji V, Hausser I, Brodesser S, Preil ML, Önal- • Multiple Members of the European Consortium Care for CMM- rare skin diseases; OeNB; Hans Christian Hennies. Akan A, Stock F, Becker K, Casper R, Nürnberg G, Altmüller J, RD (C4CMMRD) Nürnberg P, Traupe H, Futerman AH, Hennies HC. • Topical application of proteins as a new therapy option for the treatment of severe, genetic skin diseases; DFG/FWF; Hans • Tony Futerman, Weizmann Institute of Science, Rehovot, Israel J INVEST DERMATOL. 2013, 133:2202–2211. Christian Hennies. • Sarah Hedtrich, Freie Universität, Berlin, Germany Increased cutaneous absorption reflects impaired barrier function • In vitro and in vivo models of congenital rare skin diseases for • Peter Nürnberg, Universität zu Köln, Köln, Germany of reconstructed skin models mimicking keratinisation disorders. molecular characterization and drug screening; ERA Net/E-Ra- • Eli Sprecher, Sourasky Medical Center and Sackler Faculty of Eckl KM, Weindl G, Ackermann K, Küchler S, Casper R, Radowski re; Hans Christian Hennies. Medicine, Tel Aviv, Israel

Research Report 2015 Medical University of Innsbruck 49 Department of Medical Genetics, Molecular and Clinical Pharmacology Biochemical Pharmacology

Based on this knowledge we intend to de- and β1a subunit that are crucial for this velop novel substances that may be spe- protein-protein signal transduction. Besides cifically applied in the modulation of the structure-functional domain mapping, our immune activity (stimulation or down-reg- research is devoted to investigate the role of ulation). the Ca2+ current through the skeletal muscle DHPR (Fig. 1), which is – in contrast to General Facts cardiac EC coupling – not (directly) involved in skeletal muscle EC coupling. To this aim Until his emeritation in 2009, Hartmut (supported by FWF grant P23229-B09) we Glossmann was director of the Division created a k. i. mouse model that lacks this of Biochemical Pharmacology. In October enigmatic DHPR inward current. Extensive 2009, Hans-Günther Knaus was appointed in vivo, ex vivo and in vitro experiments iden- interim director. tified this mysterious Ca2+ influx asvestigial The Division of Biochemical Pharmacology (paper in preparation). For the DHPR - RyR1 is substructured into two largely independ- protein-protein interaction in skeletal ent research groups, headed by Sandra muscle, precise membrane targeting is in- Santos-Sierra and Manfred Grabner. dispensable. It is required that four DHPRs congregate in a square formation opposite Vice Head of Division (interim): Research the four homo-domains of the RyR1; a con- ao. Univ.-Prof. Dr. Manfred Grabner stellation known as skeletal muscle tetrad. Manfred Grabner Contact: The basic mechanism of skeletal muscle Recent Major Achievements: Peter Mayr Straße 1 contraction is the release of Ca2+ from We investigated the role of the essential 2+ 6020 Innsbruck SR stores. Two distinct Ca channels, the DHPR β1a subunit for DHPR voltage sensing. 2+ voltage-gated L-type Ca channel or di In vitro expression of β1a/β3 chimeras in [email protected] hydropyridine receptor (DHPR) of the sur- β1-null zebrafish relaxed myotubes revealed Phone: +43 512 9003 70407 face membrane and the intracellular Ca2+ a pivotal role of the Src homology 3 (SH3) Fax: +43 512 9003 73440 release channel or ryanodine receptor domain and the C terminus of β1a in charge https://www.i-med.ac.at/ibp/ (RyR1) of the SR, play together in this com- movement restoration. Furthermore, substi- plex process called excitation-contraction tution of a P by A in the putative SH3-binding Head of Division (interim): (EC) coupling (see Fig. 1). Our research polyproline motif in the proximal C terminus Univ.-Prof. Dr. Hans-Günther Knaus focus is to reveal the structure-function of β1a (also of the cardiac/neuronal β2a 2+ relationship of this fascinating Ca channel and β4) fully obstructed α1S charge move- [email protected] crosstalk in skeletal muscle. We succeed- ment. Consequently, we postulate a model Phone: +43 512 9003 70440 ed to fine map domains in the DHPRα 1S according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage Keywords sensing in skeletal muscle (Fig. 2). Future Goals: In our new FWF grant Pharmacology, immunology, biochemistry, (P27392-B21) we want to explore molecular molecular biology, biophysics, physiology, structural biology, developmental biology, phylogeny

Research Focus

• The Grabner lab focusses on structural- functional studies of multiple components of the skeletal muscle excitation- contraction (EC) coupling machinery, using zebrafish and mouse as modelorganisms . Another focus of the Grabner lab is the role of the Ca2+-activated Cl- channel in skeletal muscle EC coupling. • Sandra Santos-Sierra: Pharmacological modulation of the innate immune Fig. 1: Sketch of the skeletal muscle EC coupling machinery depicting the main focus of our response: Our research interests center research. Using zebrafish and mouse model organisms we are trying to unravel the struc- on understanding the human innate ture-function relationship of different components (viz. DHPRα1S, β1a, CaCC, etc.) of the immune response in inflammatoryskeletal muscle EC coupling machinery. This additional in-depth comprehension can serve diseases, in particular phagocytes activity as the starting point for the development of a new generation of ion channel drugs with and signaling processes therein involved. enhanced selectivity.

50 Research Report 2015 Medical University of Innsbruck Biochemical Pharmacology

regions of the DHPR β1a subunit, essential for DHPR tetrad formation. In addition, our research also focuses on the role of the Ca2+-activated Cl- channel (CaCC; Fig. 1) in zebrafishskeletal muscle which is activated during EC coupling (MCBO, FWF W1101-B12). CaCC currents might play a role in shaping the muscle action potential.

Pharmacological Modulation of the Santos-Sierra (B)

Innate Immune Response . Sandra Santos-Sierra The innate immune system plays a crucial role not only in fighting infections, but also in numerous diseases and pathological conditions including cancer. Toll-like receptors (TLRs) are main components of this system. They recognize pathogens via ligation of pathogen associated molecular patterns (PAMPS), likewise they bind some host derived ligands resulting from tissue © ChemMedChem, Murgueitio et al (A), 2014, S damage (DAMPS). Thus, the central role Fig. 3: A. Small-molecules TLR2 antagonists. The inhibitory concentration lies in the low of TLRs in various inflammatory processes micromolar range and it was determined as the inhibition in TNFα production by human and in sepsis is well recognized. For this monocytes after TLR2 stimulation. B. Small-molecules TLR2 agonists. The compounds syn- reason, the discovery of substances with ergize with known TLR2 ligands (as shown in luciferase experiments) pointing to a possible modulatory activity on TLR signaling may allosteric binding site in the receptor. have important implications in the therapy of a broad spectrum of pathologies linked to inflammation. matory response and on the other hand, Toll-like receptor 2 (TLR2) recognizes bac- TLR2-agonists may be used in adjuvanticity terial di- and tri-acylated lipopeptides and in those settings in which the immune sys- also some host endogenous ligands re- tem shows unresponsiveness. leased after tissue damage (e.g. HMGB1, hyaluronan). However, up to date there has Selected Publications been a lack of synthetic TLR2 modulators. Domain cooperativity in the β1a subunit is essential for dihydro- We have developed several compounds, pyridine receptor voltage sensing in skeletal muscle. Dayal A, Bhat V, Franzini-Armstrong C, Grabner M. small-molecules, which are bona fide TLR2 PROC NATL ACAD SCI USA. 2013, 110(18):7488–7493. ligands. These were retrieved from a com- Prospective virtual screening in a sparse data scenario: design of bined in silico/in vitro screening for their small-molecule TLR2 antagonists. Murgueitio MS, Henneke P, Glossmann H, Santos-Sierra S* and potential to bind TLR2 in HEK293 cells Wolber G*.

. ChemMedChem. 2014 Apr;9(4):813–22. A

. overexpressing the receptor and bearing an S . U . NF B-dependent reporter construct. κ Selected Funding Two groups of compounds were selected • Structure-function link in the DHPR β1a subunit for tetrad Acad Sci Acad . and their mechanism of action is under char- formation and skeletal muscle motility (P27392-B21); FWF,

Natl Manfred Grabner / Coapplicant, Dr. Anamika Dayal . acterization: First, TLR2-antagonists which • Molecular Cell Biology and Oncology (W1101-B12); FWF, bind the TLR2/1 and TLR2/6 heterodimers Manfred Grabner. © Proc at the lipopeptide ligand (Pam3CSK4) bind- • Else Kroner Fresenius Stiftung, Sandra Sierra-Santos Fig. 2: Model of -induced restoration of EC β ing site, as indicated by molecular modeling Collaborations coupling parameters. Substitution of the 3 (Fig. 3 A); Second, TLR2-agonists whose β • Clara Franzini-Armstrong, Department of Cell and Developmen- SH3 domain together with the C-terminus binding mode is structurally not defined tal Biology, University of Pennsylvania, Philadelphia, U.S.A. by corresponding β1a sequence leads to full yet, as these compounds have synergistic • Francesco Zorzato, Dipartimento di Scienze Della Vita e Bio tecnologie, Universitá Ferrara, Ferrara, Italy restoration of β-induced charge movement activity with other TLR2 ligands (Fig. 3 B). • Werner Melzer, Institut für Angewandte Physiologie, Universität and thus proper EC coupling. According to The activity of the different compounds in Ulm, Ulm, Germany • Paul D. Allen, Molecular Biosciences, University of California at our model the β1a SH3 domain and C-ter- mouse and human immune cells has been Davis, U.S.A. minus (blue) bind intra-molecularly via tested and proof of their in vivo activity is • Isaac Pessah, College of Biological Sciences, University of California at Davis, U.S.A. the SH3-PXXP interaction (yellow dots) to under way. • Roger Bannister, School of Medicine, Division of Cardiology, adapt the accurate β conformation which In order to modulate TLR activity, small-mol- University of Colorado, U.S.A. • G. Wolber, Institute of Pharmacy, Free University of Berlin, in turn strongly induces (thick red arrows) ecules show better properties than natural Germany. the correct, fully functional conformation of TLR2 ligands (e.g. their synthesis is cheaper • P. Henneke, Center for Chronic Immunodeficiency, University Medical Center Freiburg, Germany. the DHPRα1S core region (4 cylinders), and and they can be purified to clinical grade). • J. Kirchmair, Center for Bioinformatics, University of Hamburg, thus restoring all parameters of skeletal Consequently, the novel TLR2-antagonists Germany. • K. Liedl and J. Fuchs, Faculty of Chemistry and Pharmacy, muscle EC coupling (modified from Dayal et may be applied in pathologies where TLR2 Leopold-Franzens-University Innsbruck, Austria. al, 2013, PNAS). over activation leads to an increased inflam- • F Lagler, Paracelsus Medical University Salzburg, Austria.

Research Report 2015 Medical University of Innsbruck 51 Department of Medical Genetics, Molecular and Clinical Pharmacology Molecular and Cellular Pharmacology

other Pharmacology units (Division for Bio- plays a key role in the perception of ther- chemical Pharmacology or the Institute of mal and inflammatory pain. Its molecular Pharmacology). Our unit employs a total of environment in dorsal root ganglia (DRG) 6 people. Besides the division head and a has however been largely unexplored. The part-time administrator, 2 research assis- channel complex from mouse DRG was tants and an animal care keeper are staff detergent-solubilized, isolated by immuno- members. The PhD student is employed precipitation using a panel of sequence- through a PhD program. Both research as- directed antibodies against the TRPV1 sistants and the division head are MDs and protein and mouse DRG membranes, and also have clinical duties, especially related subsequently analyzed by mass spectrom- to the institutional ethics committee. etry. A number of potential TRPV1 inter action partners were identified, amongst Research them cytoskeletal proteins, signal transduction molecules, and established ion Our lab is primarily interested in investigating channel subunits. Based on stringent spec- the composition, microenvironment and ificity criteria, the voltage-gated K+ channel distribution of various ion channels, in beta 2 subunit (Kvβ2), an accessory subunit particular several classes of potassium and of voltage-gated K+ channels, was identi- Head of Division: TRPV channels. All these channels appear to fied as being associated with native TRPV1 Univ.-Prof. Dr. Hans-Günther Knaus associate with other molecular components channels. Reverse co-immunoprecipita- in complexes, so-called ‘microdomains’. We tion and antibody co-staining experiments Contact: aim to establish the composition of these confirmed the TRPV1/Kvβ2 association. Peter Mayr Straße 1 ion channel complexes by use of various Patch-clamp experiments in the presence 6020 Innsbruck detergent solubilization protocols, immuno of Kvβ2 resulted in a significant increase precipitation experiments and sequencing of capsaicin-induced outward currents. [email protected] of isolated ion channel complexes through Biotinylation assays demonstrated that Phone: +43 512 9003 70440 mass spectrometry. Kvβ2 increased the cell surface expression Fax: +43 512 9003 73440 levels of TRPV1, which supports the electro- https://www.i-med.ac.at/molpharm/ Composition of the TRPV1 Microdomain physiological data. This project demonstrat- in the Peripheral Nervous System. ed the association of a Kvβ subunit with Hans-Günther Knaus the TRPV1 channel, and indicated that such The Transient Receptor Potential Vanilloid 1 interaction plays a role in the trafficking of Keywords (TRPV1, vanilloid receptor 1) ion channel TRPV1 to the plasma membrane.

Ion channels, protein biochemistry, anti bodies, proteomics, immunoprecipitation

Research Focus

Our research unit focusses on certain key aspects of voltage- and ligand-gated ion channels. We are interested in the subunit composition of several ion channel families as well as their cellular and subcellular distribution. In addition, our research aims to identify the respective ion channel nano domains through immunoprecipitation experiments as well as through high-sensitivity sequencing by mass spectrometry.

General Facts

Our research unit is quite small in terms of associated personnel, resources and associated lab space. We reside on the ground level of the Pharmacology/Genetics building in Peter-Mayr Strasse 1 in approx. 110m2 of lab- and office space. We have access to © Biochim Biophy Acta © Biochim Biophy a single animal holding room (mice) which is shared with the scientists of the Division Fig. 1: TRPV1 and Kvβ2 co-expression in sensory neurons: immunostaining of dissociated for Biochemical Pharmacology. The majority wildtype (left panels) and TRPV1-/- (right panels) sensory neurons with anti-TRPV1 and of equipment and facilities is shared with Kvβ2 antibodies.

52 Research Report 2015 Medical University of Innsbruck Molecular and Cellular Pharmacology

Identification of Potential Novel Inter action Partners of the Sodium‑Activated Potassium Channels Slick and Slack in Mouse Brain and their Respective Distribution. Hans-Günther Knaus For this research we used a combined approach of (co)-immunoprecipitation studies, Western blot analysis, double immunofluorescence and mass spectrometric sequencing and immunohistochemical distribution studies, in order to investigate protein- protein interactions of the Slick and Slack channels and their respective distribution profiles. We found that Slick and Slack channels co-assemble into the same cellular complex. Double immunofluorescence

experiments revealed that Slick and Slack © Sandra Rizzi channels co-localize in distinct mouse brain Fig. 2: Model of the transmembrane topology of a Slick / Slack channel. regions. Moreover, we identified the small cytoplasmic protein beta-synuclein and the Major Achievements: an pharmacies on public expense by out- transmembrane protein 263 (TMEM 263) Complete panels of sequence-directed an- patients (2006–2014) from the Federation as novel interaction partners of both, native tibodies against a large number of different of Austrian Social Insurance Institutions Slick and Slack channels. In addition, the ion channel families were characterized. By (Hauptverband der Sozialversicherung- inactive dipeptidyl-peptidase (DPP 10) use of these antibodies, the microdomain sträger) are being analysed for optimiza- and the synapse associated protein 02 environments of some of these ion chan- tion/savings potential, prescription of ge- (SAP 102) were identified as constituents nels were established. nerics, demands for regulations and further of the native Slick channel complex in the Future Goal: To characterize some of these aspects. mouse brain. We were also able to establish novel interaction partners in terms of their Achievements: The results together with the immunohistochmical distribution pro- precise function in the respective ion chan- a thorough interpretation and a discussion files for Slick and Slack channels in mouse nel complex. of the pharmacology (including risk-benefit brain sections. In some brain regions, these evaluations) of the prescribed substances two potassium channel isoforms are clearly Pharmacoeconomics: Austrian Drug were provided to the Hauptverband as a segregated, while in other regions they Prescription Report scientific basis for further decision-making. show significant coexpression, at least at Georg Wietzorrek Future Goal: The Austrian Drug Prescrip- the light microscopical level. Data from all prescriptions filled in Austri- tion Report will be published as a textbook.

Selected Publications Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons. Dolga AM, de Andrade A, Meissner L, Knaus HG, Höllerhage M, Christophersen P, Zischka H, Plesnila N, Höglinger GU, Culmsee C. Cell Death Dis. 2014 Jan 16;5:e999. Identification of voltage-gated K(+) channel beta 2 (Kvβ2) subunit as a novel interaction partner of the pain transducer Transient Re- ceptor Potential Vanilloid 1 channel (TRPV1). Bavassano C, Marvaldi L, Langeslag M, Sarg B, Lindner H, Klimaschewski L, Kress M, Ferrer-Montiel A, Knaus HG. Biochim Biophys Acta. 2013: p. 3166–75. Palmitoylation of the β4-subunit regulates surface expression of large conductance calcium-activated potassium channel splice variants. Chen L, Bi D, Tian L, McClafferty H, Steeb F, Ruth P, Knaus HG, Shipston MJ. J Biol Chem. 2013; S.13136–44. Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons. Kaya L, Meissner B, Riedl MC, Muik M, Schwarzer C, Ferraguti F, Sarg B, Lindner H, Schweigreiter R, Knaus HG, Romanin C, Bandtlow CE. Biochim Biophys Acta. 2013; p. 1421–33.

Selected Funding PhD program SPIN B05, ZFW12060-05, Austrian Research Foun- dation FWF

Collaborations Peter Ruth, Pharmacology, Tuebingen, Germany Fig. 3: Public expenses on drugs and calculated savings potential by indications, 2012. Bernd Fakler, Physiology II, Freiburg, Germany

Research Report 2015 Medical University of Innsbruck 53 Department of Anatomy, Histology and Embryology Clinical and Functional Anatomy

in the development, exchange and imple- medical disciplines. Among these, there are mentation of clinical and clinically applied workshops and surgical courses organised anatomy used in research and teaching as a in cooperation with the corresponding clin- basic science with indirect and direct bene- ics of the Medical University of Innsbruck fits for patients. and also with international organizations The Division of Clinical and Functional Anat- and societies. omy is staffed by eleven scientific and ten administrative members. Research The Division operates laboratory facilities for histology and immunohistology, an ultra The research at the Division of Clinical and sound laboratory, and an extensive body Functional Anatomy is grouped around sev- donation program, both for educational and eral main topics: development, ultrasound, scientific purposes. inner ear, clinical and applied anatomy and The Division contributes much to the pre- medical anatomy education. clinical training of future medical practitioners. The broad spectrum of learning activ- Human Development ities includes lectures as well as practical The main focus within this research topic exercises. Therefore, also some scientific is the analysis of humans, whereas most Head of Division: efforts deal with medical education and the developmental research is conducted us- Univ.-Prof. Dr. Erich Brenner, MME body donation program. Further education- ing species such as rodents, birds or fish. al activities include regular courses for the We contributed to the development of the Contact: continuing medical education of different utero-vaginal anlagen, where we could Müllerstraße 59 6020 Innsbruck

[email protected] Phone: +43 512 9003 71121 Fax: +43 512 9003 73112 www.anatomie-innsbruck.at/49.html

Keywords

Clinical anatomy, functional anatomy, ultra- sonography, phlebology/lymphology, medical education, inner ear

Research Focus

• Clinical and Functional Anatomy in collaboration with several clinical departments (e.g. Traumatology, Visceral Surgery, Plas- tic and Reconstructive Surgery, Radiology, etc.) • Developmental Anatomy of the male urinary tract • Developmental Anatomy of inner ear • Ultrasound guided blockades of plexus and peripheral nerves

General Facts

Mors Auxilium Vitae – Death Aids Life. This saying, mounted on a door of the department, aptly characterizes the basic conceptual design and objectives of our group.

This means that anatomy is not considered 2014, 196(6): 390 © Annals of Anatomy a discipline decoupled from daily medical Fig. 1: Causes-of-death data from body donors. The data list the prevailing fatal diseases and practice, but the whole Division’s activity is external circumstances of the last 25 years (1988–2013) as stated in the death certificates focused on the welfare and health of peo- of body donors forwarded to us. The area of each circle is proportional to the absolute ple. The Division therefore sees its main role number of deaths.

54 Research Report 2015 Medical University of Innsbruck Clinical and Functional Anatomy ain Medicine 2014 39(1):20 2014; 355(2): 273 . © Cell Tissue Res © Regional Anesthesia and P Fig. 2: Graphic illustration of two human mono-polar or “ampu- Fig. 3: The popliteal-sciatic block. Model photograph. Scanning of the tated” SG cells with surrounding SGCs (red) together with results popliteal fossa (A). The popliteal artery (PA) is seen in the cross-sec- from immunohistochemistry (TrkB, tyrosine kinase B receptor; tional view (B). The tibial nerve (TIB) is localized between the PA and BDNF, brain-derived neurotrophic factor; nNOS, nitric-oxide the skin (B). The TIB is then followed by sliding the transducer ceph- synthase; GFL, GDNF family ligand; C-ret, GFL receptor; GFRal- alad until the common peroneal nerve (PEN) merges with the TIB. At pha, GDNF family receptor alpha; NTRN, neurturin; hSGNs, hu- this point of the sciatic nerve (SCN) bifurcation, the needle is inserted man spiral ganglion cells; SGCs, satellite glial cells; GDNF, gli- at a very shallow angle to the transducer from the lateral side of the al-cellline-derived neurotrophic factor; Cx30, connexin 30; Cx36, thigh and advanced in-plane in a lateral-to-medial direction with the connexin 36; P75NTR, P75 neurotrophin receptor). end point between the 2 nerves (AB). Magnetic resonance (MR) imaging in the sagittal plane depicts the distribution of the injectate in and above the popliteal fossa spreading both cephalad and caudad to surround the SCN. show that the epithelial differentiation fol- Inner Ear specifically, the development of a colo- lows a cephalad direction. In the hindgut, Research on this topic is performed in proctological skills laboratory or improving we demonstrated that the expression pat- close collaboration with Anneliese Schrott- courses in regional anaesthesia. tern of distinct HOX genes differs markedly Fischer and Rudolf Glückert from the ENT between animal models. The urethral devel- Department. Together, we investigated the opment was analysed with special empha- possibility of using silica nanoparticles as Selected Publications sis on gender-differences. Thus, we could vectors for drugs and genes. Furthermore, Anatomical-coloproctological skills lab. demonstrate a significantly closer bladder we found that sepsis leads to significant Aigner F, Resch T, Oberhuber R, Kronberger I, Hoermann R, Fritsch H, Pratschke J, Oberwalder M. outlet in male foetuses compared to fe- hearing impairment. This physiological al- EUROPEAN SURGERY. 2014; 46: p. 21–24. males, suggesting that the development of teration was linked to apoptosis in the sup- Development of an Innovative 3D Cell Culture System to Study Tu- the male inner genitals requires a cellular porting cells of the organ of Corti and to a mour – Stroma Interactions in Non-Small Cell Lung Cancer Cells. Amann A, Zwierzina M, Gamerith G, Bitsche M, Huber JM, Vogel stimulus by the urogenital sinus epithelium, disturbance of the synapses of the inner GF, Blumer M, Koeck S, Pechriggl EJ, Kelm JM, Hilbe W, Zwierzina and we could show that during the develop- hair cells. H. PLOS ONE. 2014; 9: p. e92511. ment of the female urogenital system the Ultrasound-Guided Single-Penetration Dual-Injection Block for primary incidence is the formation of the Clinical and Applied Anatomy Leg and Foot Surgery A Prospective, Randomized, Double-blind Study. urethra. This is followed by the establish- Research in clinical and applied anatomy is Borglum J, Johansen K, Christensen MD, Lenz K, Bendtsen TF, ment of the vagina, which clearly depends mainly driven by questions received from Tanggaard K, Christensen AF, Moriggl B, Jensen K. REGIONAL AN- on the proper differentiation of the urogeni- collaborating clinicians. ESTHESIA AND PAIN MEDICINE. 2014; 39: p. 18–25. Histomorphometric Evaluation of Ischemia-Reperfusion Injury tal system/urethra. Therefore, this topic is widespread and cov- and the Effect of Preservation Solutions Histidine-Tryptophan- ers sub-topics such as the scaphoid bone, Ketoglutarate and University of Wisconsin in Limb Transplanta- the posterolateral corner of the knee, intra- tion. Ultrasound Hautz T, Hickethier T, Blumer M J F, Bitsche M, Grahammer The primary focus of this line of research operative pelvic neuromonitoring and the J, Hermann M, Zelger B, Messner F, Pechriggl EJ, Krapf C, is to develop novel ultrasound-guided clin- detailed mapping of the nasal muscles. Kimelman M, Brandacher G, Lee WPA, Margreiter R, Pratschke J, Schneeberger S. ical techniques, such as the block of the TRANSPLANTATION. 2014; 98: p. 713–720. superior cervical ganglion, the assessment Medical Education A New Nasopharyngeal Dynamic Reference Frame Improves of different tissues by means of sonoelasto This topic comprises more general aspects, Accuracy in Navigated Skull Base Targets. Kral F, DiFranco M, Puschban J, Hoermann R, Riechelmann H, graphy and the comparison of different such as an analysis of the philosophy and Freysinger W. blocks for regional anaesthesia. ethics of anatomy teaching, but also more SURGICAL INNOVATION. 2014; 21: p. 283–289.

Research Report 2015 Medical University of Innsbruck 55 Department of Anatomy, Histology and Embryology Neuroanatomy

Keywords

Cellular neuroscience, peripheral axonal regeneration, glioma, fluorescence imaging, comparative neuroanatomy

Research Focus

• Axotomy-induced plasticity in peripheral neurons • Receptor tyrosine kinase trafficking in axonal regeneration and glial proliferation

Aims: Interference with receptor tyrosine kinase trafficking and signalling • to promote neurite outgrowth and axonal regeneration Head of Division: • to inhibit glioma proliferation and tumour Univ.-Prof. Dr. Lars Klimaschewski growth

Contact: The Fibroblast Growth Factor Receptor Müllerstraße 59 (FGFR1) and regulators of ERK signalling 6020 Innsbruck such as Sprouty proteins are in the focus of our studies. [email protected] Phone: +43 512 9003 71160 General Facts 22224 Fax: +43 512 9003 73112 . www.neuroanatomy.at The Division of Neuroanatomy at the Medical University of Innsbruck offers 1002/dneu lectures and seminars in functional as well . as comparative Neuroanatomy for MD and PhD students. Our research is focused on the morphological consequences of growth © Developmental Neurobiology DOI: 10 © Developmental

Fig. 2: Sprouty2 deficient sensory neurons Inhibition of RTK in culture reveal different axon outgrowth endocytosis: Axon branching patterns.

Enhanced recycling factor signalling and receptor tyrosine of RTKs: kinase trafficking. Applying mainly cellular Axonal elongation Early endosome methods combined with high-resolution Dendritic branching imaging, we are studying fundamental neurobiological phenomena such as axon Inhibition of outgrowth and glial proliferation. degradation of RTKs: Axonal and dendritic growth Research

Peripheral Axon Outgrowth and Nerve Regeneration In recent years the cellular basis for in sufficient or incorrect axonal regeneration and the consequent lack of functional Lysosome recovery has been unravelled in various Recycling endosome laboratories. Neurotrophic factors such as the neurotrophins and the FGFs are crucial- Fig. 1: Trafficking of RTKs in neurons. ly involved in stimulating neurite outgrowth

56 Research Report 2015 Medical University of Innsbruck Neuroanatomy

Fig. 3: Multicolor labeling of human glioma cells. U373 cells treated with nontargeting or Spry2-siRNA are shown 3 days after transfection. Early endosomes contain higher amounts of FGFR1 and its ligand FGF-2 in glioma cells with reduced Spry2 levels.

and re-myelination after nerve injury. Ongo- induces positive and negative signals. Pajere K, Hopf R, Schmidhammer R, Hausott B, Redl H, Nógrá- di A, Klimaschewski L. NEUROSCIENCE LETTERS. 2014, 566, ing research in many laboratories focusses RTKs are activated by ligand binding, auto 280–285. on three different aspects: phosphorylated and ubiquitinated, i.e. the Enhanced axon outgrowth and improved long-distance axon re- 1. The molecular mechanisms of neuronal small molecule ubiquitin is attached to lysine generation in Sprouty2 deficient mice. Marvaldi L, Thongrong survival in response to a lesion, residues at various sites on them. Following S, Kozłowska A, Frei A, Pritz C O, Bäumer B, Ronchi G, Geuna S, 2. the modification of neuronal gene ex- internalization the receptor is either re Hausott B, Klimaschewski L. DEVELOPMENTAL NEUROBIOLOGY. 2014, 75, 217–231 pression patterns required for axonal cycled or degraded in the endosomal/lyso regeneration and somal pathway. Negative receptor signalling Rho-independent stimulation of axon outgrowth and activation of the ERK and Akt signaling pathways by C3 transferase in sensory 3. the changes in the axonal environment, involves the coordinated action of ubiquitin neurons. Auer M, Schweigreiter R, Hausott B, Thongrong S, Höltje particularly within the distal part of the ligases such as c-Cbl, adaptor proteins M, Just I, Bandtlow C, Klimaschewski L. FRONTIERS IN CELLULAR NEUROSCIENCE. 2012, 6, 43 lesioned nerve. such as Grb2, negativ e feedback molecules Our laboratory is interested in the elucida- such as Sprouty, cytoplasmatic kinases Membrane turnover and receptor trafficking in regenerating axons. Hausott B, Klimaschewski L. EUROPEAN JOURNAL OF tion of the intrinsic mechanisms of neuronal and phosphoinositol metabolites. We are NEUROSCIENCE. 2015, in press. survival, neurite outgrowth and peripheral particularly interested in the trafficking of axon regeneration activated by stimulation fibroblast growth factor receptor type 1 Selected Funding of receptor tyrosine kinases, in particular and its regulation by Sprouty. FGFRs are Austrian Science Funds (within the PhD program ‘Signal Proces- FGFR1. abundant in the nervous system and display sing in Neurons’ SPIN, W1206-B05) several key roles in brain development and Receptor Tyrosine Kinase Trafficking disease. Collaborations and Signalling • Ludwig Boltzmann Institute for Traumatology, Vienna, Austria Extracellular signals activate receptor- Selected Publications • Department of Neurosciences, University of the Basque Coun- try, Spain triggered intracellular signal transduction C3 exoenzyme lacks effects on peripheral axon regeneration in • Department of Physiology, Universitat Autonoma de Barcelona, pathways. At the same time, receptor tyro vivo. Auer M, Allodi I, Mohammed B, Udina E, Neiss W, Navarro X, Spain Klimaschewski L. JOURNAL OF THE PERIPHERAL NERVOUS SYS- • Department of Clinical and Biological Sciences, University of sine kinases (RTKs) initiate a cascade of TEM. 2013, 18, 30–36. Torino, Italy events comprising ‘negative signalling’, • Department of Biochemistry, The Norwegian Radium Hospital, Sorting of FGF receptor type 1 in a human glioma cell line. Irschick Norway thereby decreasing the amplitude of R, Trost T, Karp G, Hausott B, Auer M, Claus P, Klimaschewski L. • Center for Anatomy, Hannover Medical School, Germany positive signals and modulating the level HISTOCHEMISTRY AND CELL BIOLOGY. 2013, 139, 135–148. • Institute for Anatomy, University Cologne, Germany • Center for Anatomy, University Berlin (Charite), Germany of growth factor-induced stimulation. Inhibition of calpains fails to improve regeneration through a • Department of Genetics and Bioengineering, Yeditepe Univer- Hence, the same receptor simultaneously peripheral nerve conduit. Hausner T, Marvaldi L, Mártone G, sity Istanbul, Turkey

Research Report 2015 Medical University of Innsbruck 57 Department of Anatomy, Histology and Embryology Histology and Embryology

–– Comparison of systemic and topical mammalian cell cultures, (biopsy) samples application of nanoparticles from patients and various model organisms –– Topical application of such as mice, yeast, flatworms and the fluorochrome-bearing nanoparticles to freshwater polyp Hydra. image early cancer Membrane traffic is studiedwith special emphasis on endo/lysosomal pathways Research and intracellular signalling (Adell et al. 2014; Schiefermeier et al. 2014). Clinical and Applied Histology Furthermore, we are interested in the and Cytology relationships between membrane traffic, Günter Klima cytoskeleton and loss of cellular polarity, as Long-term collaborations with Clinical presented by Microvillus Inclusion Disease, Departments of the Innsbruck University a fatal hereditary enteropathy affecting neo- Medical Center, i.e. Department of Visceral, nates (Fig. 1; Wiegerinck et al., 2014; Thoeni Transplant and Thoracic Surgery, Depart et al., 2014). Finally, we perform methodo- ment of Urology, Department of Plastic-, logical research on various organisms from Reconstructive- and Aesthetic Surgery, all kingdoms of life, aiming at the improve- Department of Anesthesiology and Critical ment and development of advanced spec- Head of Division: Care Medicine, Center of Internal Medicine, imen preparation and labelling procedures ao. Univ.-Prof. Dr. Günter Klima Department of Cranio-maxillofacial and for biomedical electron microscopy Oral Surgery. (Schmiedinger et al., 2013). Contact: Müllerstraße 59 Cellular Electron Microscopy Endothelial Biology Group 6020 Innsbruck Michael W. Hess Paul Debbage Our cell biological research concen- Nanomedicine aims to apply the advantages [email protected] trates on ultrastructural aspects of intra of nanoscale structured materials to en- Phone: +43 512 9003 71170 cellular membrane trafficking in eukary- hance the targeting efficiency and reduce Fax: +43 512 9003 73170 otic cells and tissues, performed in close the toxicity of drug delivery, and to improve https://www.i-med.ac.at/ahe/ collaboration with the groups of L. A. Huber monitoring of therapeutic progress. Since histologie-embryologie/ and D. Teis (Division of Cell Biology) as well 2004 our group has collaborated with other as T. Müller and A. Janecke (Department groups in the Medical University Innsbruck of Paediatrics I). Cryo-based (immuno-) and the Leopold-Franzens-University in Keywords electron microscopy and 3D-modelling Innsbruck, and with partners elsewhere in based on electron tomography are our Austria to carry out translational research in Histology, electron microscopy, ultra preferred approaches for investigating Nanomedicine. As a founder member of the structure research, membrane trafficking, endocytic pathways, nanomedicine, trans lational research, endothelial cells, nano particles, albumin, magnetic resonance imaging, fluorescence optical imaging, tissue barriers, systemic imaging, topical imaging, colon carcinoma

Research Focus

• Classical histological and cytological analyses through use of light- and scanning electron-microscopy with emphasis on clinically applied research topics (G. Klima) • Ultrastructural investigation of subcellular constituents (organelles, cytoskeleton) in the context of intact cells and tissues (M. Hess) • Imaging in cancer diagnosis (P. Debbage): –– Interactions of albumin-based nano particles with tissues and cells –– Magnetic Resonance Imaging by use of gadolinium-bearing albumin nano Fig. 1: Microvillus Inclusion Disease caused by homozygous truncating mutation in the particles apical t-SNARE protein syntaxin 3 (STX3). Electron micrograph from severely affected en- –– Fluorescence Optical Imaging by use of terocytes (small intestine epithelial cells) showing characteristic atrophy and local loss of fluorochrome-bearing albumin nano brush-border microvilli at the cell surface, associated with pathological accumulation of particles glycoprotein-rich, dark vermiform membrane vesicles (arrow) in the cell periphery.

58 Research Report 2015 Medical University of Innsbruck Histology and Embryology

tions, our group is now coordinating an EU ERA-Net Transcan project (“NanoE- FEct”) aimed at early diagnosis of colorectal carcinomas by nanoparticle-mediated fluorescence imaging (total funding volume slightly higher than € 1,000,000).

Future Goals: the translational research we presently carry out in the NanoEFEct project is aimed at early diagnosis of colon carcinomas, but the technology we are developing could also be applied to target any carcinomas arising in organs that can be accessed by endoscopy; these include other segments of the gastrointestinal tract, the genitourinary tract, the upper respiratory tract and the larger airways of the lungs. The technology can also be applied to target Fig. 2: at left, albumin in its native configuration as a heart shaped molecule. Each of the the earliest stages of inflammation, which several linkers attached to it bears a single gadolinium atom (shown as a gold ball) which underlies a range of diseases and is often generates contrast in MRI. This principle of albumin with a linker bearing a signal-emitter is associated with malignancies. In the 3–5 the basis of our translational research in the NanoEFEct project, but there the signal-emitter year term, we are also interested in learning is a fluorochrome. At right, the dimensions of the albumin molecule are shown; we use these to load the nanoparticles with drugs, aiming to plan quantitative aspects of the nanoparticles in the translational project. The image is to achieve local treatment of tumours that taken from our publication Stollenwerk et al. (2010) Histochem Cell Biol. 133(4):375–404. cannot be excised.

Austrian National Consortium “Nanohealth” This made us aware of how little we know Selected Publications we collaborated with teams in Vienna, of the potential tissue toxicity of nano Coordinated binding of Vps4 to ESCRT-III drives membrane neck Innsbruck and Graz, learning to design and particles bearing active substances, and constriction during MVB vesicle formation. Adell MAY, Vogel GF, Pakdel M, Mueller M, Lindner H, Hess MW, Teis D. synthesize protein-based nanoparticles of other potential toxicities not detected in JOURNAL OF CELL BIOLOGY. 2014; 205: p. 33–49. of high quality (Figs. 2, 3): mechanically cell-based assays. It prompted us to review The late endosomal p14-MP1 (LAMTOR2/3) complex regulates stable, of highly reproducible size, bearing the blood-tissue barriers which regulate the focal adhesion dynamics during cell migration. standard numbers of both gadolinium tags transfer of nanoscale objects across the Schiefermeier N, Scheffler JM, de Araujo MEG, Stasyk ,T Yordano v T, Ebner HL, Offterdinger M, Munck S, Hess MW, Wickstroem SA, and lectin targeting groups, and being non- vascular endothelial lining and thus control Lange A, Wunderlich W, Faessler R, Teis D, Huber LA. cytotoxic according to OECD guidelines. their distribution within the body. We found JOURNAL OF CELL BIOLOGY. 2014; 205: p. 525–540. However, after intravascular application that systemic application of nanoparticles Microvillus inclusion disease: loss of Myosin vb disrupts intra cellular traffic and cell polarity. Thoeni CE, Vogel GF, Tancevski they accumulated rapidly in the von Kupffer (e.g. by intravascular injection) faces I, Geley S, Lechner S, Pfaller K, Hess MW, Müller T, Janecke AR, macrophages and in the endothelial cells multiple barriers maintained by the body Avitzur Y, Muise A, Cutz E, Huber LA. TRAFFIC. 2014; 15: p. 22–42. lining the liver sinusoids, the gadolinium between the blood and the internal com- Loss of Syntaxin 3 Causes Variant Microvillus Inclusion Disease. remaining in the liver (and kidneys and partments of almost all tissues (Debbage Wiegerinck CL, Janecke AR, Schneeberger K, Vogel GF, van Haaften-Visser DY, Escher JC, Adam R, Thoeni CE, Pfaller K, spleen) in large amounts for at least 2 and Thurner, 2010, Pharmaceuticals 3 (11), Jordan AJ, Weis CA, Nijman IJ, Monroe GR, van Hasselt PM, Cutz weeks after application. 3371–3416). Once the nanoparticles have E, Klumperman J, Clevers H, Nieuwenhuis EES, Houwen RHJ, van Haaften G, Hess MW, Huber LA, Stapelbroek JM, Mueller T, reached their targets within a tissue, high Middendorp S. signalling and drug-release rates can be GASTROENTEROLOGY. 2014; 147: p. 65–68. obtained exclusively within that tissue. To Cryo-Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates. achieve that however, the nanoparticle re- Schmiedinger T, Vogel G F, Eiter Ol, Pfaller K, Kaufmann WA, Floerl quires 3–5 different targeting groups to A, Gutleben K, Schoenherr S, Witting B, Lechleitner TW, Ebner H-L, “navigate” the barriers between the blood Seppi T, Hess MW. TRAFFIC. 2013; 14: p. 886–894. and the tissue cells. This raises significant Proteomics of cancer stem cells. Skvortsov S, Debbage P, Skvortsova I. INT. JOURNAL OF RADIATION BIOLOGY. 2014. 90: regulatory hurdles. p. 653–658.

We therefore considered topical application Selected Funding of nanoparticles. In this case, the blood- NanoEFEct, EU ERA-NET Transcan, Paul Debbage tissue barriers exclude the nanoparticles Collaborations Fig. 3: A transmission electron microscope from the systemic circulation. Small • Institute of Cell Biology, Histology and Embryology, Medical image of negatively contrasted nanoparticles amounts of nanoparticles could therefore Univ. of Graz, AT (seen here as white spheroids). These be applied to achieve strong signalling, high • Centre for Medical Basic Research, Medical Univ. of Graz, AT high-quality particles do not aggregate, are local drug concentrations, and a minimum • Dept. of Pharmaceutical Technology, LFU Innsbruck, AT • Medical Clinic 1 – Gastroenterology, Pneumology and Endocri- highly uniform in size, and each contains of material transfer into the systemic nology, University Clinics Erlangen,Germany approximately 10 of the albumin molecules circulation, reducing side-effects potentially • Inst. for Materials and Chemistry, SINTEF, Trondheim, Norway shown in Fig. 2 (Abdelmoez, Thurner et al., • Faculty of Engineering. University of Porto, Portugal to extremely low levels. • CESAR (Central European Society for Anticancer Research – 2010, Histochem Cell Biol. 134(2), 171–196). As a consequence of these considera- EWIV), Vienna, AT

Research Report 2015 Medical University of Innsbruck 59 Department of Hygiene, Microbiology and Social Medicine Hygiene and Medical Microbiology

Keywords General Facts

Infectious diseases, hygiene, infection, Infectious diseases are turning into one of fungi, EHEC, HIV, dendritic cells, platelets, the most frequent causes of death in the complement, N-chlorotaurine world. Presently, bacteria and fungi are constantly developing resistance to antibiotics Research Focus and antimycotics, resulting in an increase of emerging pathogens spread worldwide. Un- Understanding Infections: derstanding the biological principles under- From Pathogenesis to Diagnosis lying the mechanisms by which infectious • The tasks of the Division of Hygiene and agents adapt, and undermining the defence Medical Microbiology (HMM) comprise re- mechanisms of a host is critical for fighting search, teaching, laboratory diagnosis of diseases. infectious diseases, environmental, hospi- HMM conducts basic and translational tal and technical hygiene. research into molecular mechanisms of • Scientific activitiescover fungal patho- pathogenesis of bacterial, viral, or fungal genicity and virulence factors, molecular infections and different strategies for their mechanisms of host pathogen-interaction prophylaxis and therapy. HMM’s mission is Head of Division: including the complement system, ba- to coordinate and strategically align transla- Univ.-Prof.in Dr.in Cornelia Lass-Flörl sic immunological research (interactions tional infection research with the aim of de- of dendritic cells/T-cells), antimicrobial veloping new diagnostic, preventative and Contact: agents (antimycotics and endogenous an- therapeutic methods for treating infectious Schöpfstraße 41 tiseptics), enterohemorrhagic E. coli and diseases. To achieve this, HMM has formed 6020 Innsbruck prevention of nosocomial infections. thematic translational units of scientists, • HMM seeks to prevent illness and death each dedicated to one specific pathogen or [email protected] from targeted infectious disease threats infectious disease. HMM is one of the larg- Phone: +43 512 9003 70703 through research and the translation of est microbiology diagnostic laboratories in Fax: +43 512 9003 73700 scientific information into real-world, Austria, with an average sample throughput www.i-med.ac.at/hygiene/ practical applications, policies, and solu- of 250 000 specimens per year. (HMM is tions (Fig. 1). associated to all major hospitals in Tyrol,

Fig. 1: Translation of in vitro to in vivo – overview of research questions targeted at HMM.

60 Research Report 2015 Medical University of Innsbruck Hygiene and Medical Microbiology

Research

Emerging Infections with a Focus on Enterohemorrhagic Escherichia Coli (EHEC)-Induced Hemolytic Uremic Syndrome (HUS) Experiences over the last decade clearly demonstrate the vulnerability of modern society due to emerging pathogens. Out- breaks and epidemics virtually affect all aspects of our lives, with constant threats to our health. A prompt health-care response is critical to prevent a rapid spread of infection. A thorough intensive knowledge of the pathogen, its reservoirs, its risk-factors as well as its associated broad-spectrum drugs is immensely important. Researchers (WG Orth-Höller & WG Würzner) investigate the interaction of EHEC virulence factors with the complement cascade and evaluate whether a transient complement blockade opens new therapeutic strategies. HMM will pursue immune modulation strategies focusing on the role of complement, and will Fig. 2: Identifying underlying networks contributing to infectious diseases. provide genetic factors potentially responsible for disease development (Fig. 4). seeking it in a key position in the diagnostic multifactorial, e.g. the advent of medical Exploiting Immune laboratory landscape in Austria.) progress, the successful application of im- Response to Infection munosuppression in transplanted patients, The human immune system is constantly ac- Our research group consists of 6 Associate and the use of immunomodulatory agents tive combating diseases. Researchers have Professors, 5 Post-Docs, several PhD- and for treating various diseases from cancer developed novel methods for assessing the bachelor students and 7 technical assis- to rheumatoid arthritis. Reducing the inci- immune response in molecular detail focus- tants, the diagnostic of 9 medical doctors, 3 dence relies on rapid and specific diagnosing on HIV-1 and opportunistic fungal path- Post-Docs, and 26 technical assistants. tics, effective antifungal drugs, novel immu- ogens. During acute and chronic phase of The mission of HMM is to bridge the gap be- notherapeutic strategies, and adherence to infection, dendritic cells (DC), macrophages tween basic and translational research into infection control and sterility practices. and platelets are of major interest. Various microbial pathogenesis (Fig. 2). aspects of opsonization (complement, anti- CD-Fungus deals with the following three body) are considered in all in vitro experi- main research questions: How to best find, mental set-ups to mimic the in vivo situation treat and prevent mucor mycosis? close to reality. One focus is to study the

Tackling these key questions needs 1. recognising MM as such 2. identification of the source and type of infection 3. identification of the pathogen Christian-Doppler-Laboratory 4. understanding the underlying patho- for Invasive Fungal Infections mechanisms In 2015 a “Christian-Doppler-Laboratory for 5. initiation of early targeted treatment and Invasive Fungal Infections” was set up. With- 6. providing a clean and safe hospital in the estimated 2 million fungal species on environment. earth, about 600 cause diseases in humans, and the most important are Candida, Asper- CD-Fungus attempts to unravel scientific gillus, Mucorales, and Cryptococcus. Fungal questions raised by implementing 3 mod- infections are increasing and are associat- ules which will ultimately advance our un- ed with excessive morbidity and mortality derstanding of fungal pathology, improve di- (Fig. 3). Over 300 million people are acutely agnosis and treatment of MM and enhance or chronically infected, leading to death, patients’ outcome and safety in terms of long term illness, and reduced work capac- prevention of nosocomial and hospital-as- Fig. 3: Invasive fungal lung infection display- ity. The reasons for emergence are likely sociated infections. ing hyphae (mucor species) and yeast cells.

Research Report 2015 Medical University of Innsbruck 61 Department of Hygiene, Microbiology and Social Medicine

HMM will support epidemiological, translational and clinical studies to improve the management of fungal diseases.

N-Chlorotaurine: Assessing of New Antiseptic Solutions and Antimicrobial Surfaces N-chlorotaurine, a long-lived oxidant produced by activated human leukocytes has been synthesized as sodium salt in our division and is under clinical investigation for local treatment of infections of multiple body regions, including sensitive ones (Fig. 6). Basic research assesses its microbial activity against biofilms and investigates the activity against emerging pathogens (WG Nagl). While antibiotics are frequently considered the first line of containment for nosocomial infections, there is increasing effort being devoted to prevent infections. Researchers at the division are screening surface materials that prevent bacteria, viral Fig. 4: Involvement of the complement system in the pathogenesis of EHEC-associated HUS. and fungal contamination and persistence on medical surfaces (WG Mayr-Lass-Flörl). impact of viral opsonization patterns on sig- ed to treatment failure is limited. Hence, Laboratory Diagnostics, Hospital and nalling pathways within DCs, on DC matu- researchers decipher the role of hypoxia Technical Hygiene ration, and on DC antigen presentation to in the onset of infections and various new The division HMM fulfils its tasks in detec- CD4+ and CD8+ T cells (WG Wilflingseder in vitro and in vivo models will be applied tion and identification of pathogens causing & WG Posch; Fig. 5). Another goal is to eval- (WG Binder-Lass-Flörl, WG Wilflingseder, infections. This covers bacteriology, para- uate the interaction of platelets and fungal WG Speth). HMM research intends to pur- sitology, mycobacteriology and mycology. pathogens. Such interplay might result in sue immune modulation and new treatment The diagnostic laboratories are certified either mutual platelet activation or inhibi- strategies to provide promising options for according to ISO 9001:2009. Special parts tion hence resulting in additive antifungal the development of novel and more effec- are controlled by external audits in accord- defence or excessive inflammation and tive antifungal therapies. This topic is also ance to §67 Austrian Medicines Law and thrombosis (WG Speth-Rambach). Another dealt with in the FWF –funded doctoral pro- FDA, Division of Manufacturing and Product project aims at elucidating fungal proteins gramme of excellence, HOROS, for HOst Quality. Within the sector of hospital and which allow pathogens to escape from Response in Opportunistic infectionS and technical hygiene (accredited according complement interactions, subsequently the Christian Doppler laboratory for inva- to ISO/IEC 17025 and ISO/IEC 17020) protecting the fungus from the destructive sive fungal infections. guidelines for the prevention of infectious action of an activated immune system (WG diseases are developed and controlled Würzner). This ground-breaking research is Therapy-Resistant Fungal Infections corresponding to the statutory pre-setting forming the basis for the development of A disturbing and rapid increase in infections for technical facilities (e.g. disinfection novel treatment strategies and eventually of caused by antimycotic-resistant fungal machines). vaccines. pathogens is a big public health concern presently in the medical field. Most severe Fungal Infections of the and fatal cases result from healthcare- Immunocompromised Patient associated fungal infections, which are Our aging population and the growing increasingly caused by Candida, Aspergil- prevalence of chronic diseases has forced lus and Mucorales. Hence, a major focus modern medicine to use aggressive can- is to investigate azole and echinocandin- cer therapies and organ or bone marrow resistance in yeasts and moulds and to transplantation, which result in or require discover new resistance mechanisms (WG immunosuppression. In immunocompro- Lackner-Lass-Flörl). Another main focus is mised patients, fungal pathogens, usually to identify the underlying mode of ampho- efficiently controlled by the immune sys- tericin B resistance in Aspergillus terreus. tem, can cause life-threatening diseases In this context, we evaluate mitochondria that may be difficult to treat with currently as crucial modulator of polyene resistance available anti-mycotics. While the degree (WG Wilflingseder-Blatzer-Jukic-Lass-Flörl). of immune alteration is a major contributor The mission of HMM is to bridge the trans- to fungal diseases in immunocompromised lational gap between basic research and Fig. 5: Three-dimensional image of dendritic patients, knowledge of other factors relat- the development of novel antifungal drugs. cells infected with HIV-1.

62 Research Report 2015 Medical University of Innsbruck Hygiene and Medical Microbiology

H2O Taurin H O - 2 2 e O2 + HOCl N-Chlortaurin NADPH2 Cl- Chlorierte AS/Peptide Dichloramine H+ MPO 2 O H O Monochloramin NADPH- 2 2 2 + Bakterium H EPO Monobromamin Oxidase- Dibromamine Bromierte AS/Peptide Komplex Br- Superoxid- HOBr N-Bromtaurin H2O Dismutase Taurin H2O

NADP++2 H+

Fig. 6: Synthesis of N-chlorotaurine by human granulocytes.

Selected Publications Selected Funding Collaborations

Bactericidal activity of N-chlorotaurine against biofilm forming FWF I 661-B09: Oxystress and human fungal pathogens: clinical Jacques Meis and colleagues, Canisius Wilhelmina Hospital and bacteria grown on metal discs. and applied aspects. 2011–2014 Radboud University Medical Centre, Nijmegen, The Netherlands Coraca-Huber DC, Ammann C, Fille M, Hausdorfer J, Nogler M, Sybren de Hoog, Fungal Biodiversity Centre, Utrecht, The Neth- Nagl M. FWF I-656-B09: Biomarker and antifungal resistance in Aspergil- erlands ANTIMICROB AGENTS CHEMOTHER. 2014;58:2235–2239. lus. 2011–2014 Maiken C. Arendrup, Staten Serum Institut, Copenhagen, Den- mark Position and numbers of FKS mutations in C. albicans selectively Axel Brackhage and colleagues, Hans Knöll Institut, Jena, Germa- FWF W1253-B24: HOROS Doctoral Programme of Excellence influence in vitro and in vivo susceptibility to echinocandin treat- ny “Wirtsabwehr bei opportunistischen Infektionen”. 2014–2018 ment. Ricardo Araujo and colleagues, Institute of Molecular Pathology Lackner M, Tscherner M, Schaller M, Kuchler M, Mair C, Sartori B, and Immunology of the University of Porto, Portugal FWF KLI459: Tolerability of inhaled N-chlorotaurine in humans – a Istel F, Cavling Arendrup M. Kevin Kavanagh, National University Ireland, Maynooth, Ireland phase I clinical study. 2015–2016 ANTIMICROB AGENTS CHEMOTHER. 2014;58:3626–3635. Mike Birch, f2G Manchester, UK Helge Karch and colleagues, University Münster, Münster, Ger- Shiga toxin 2 reduces complement inhibitor CD59 expression on FWF P22165-B13: DCs exposed to complement-opsonised HIV: A many human renal tubular epithelial and glomerular endothelial cells. key to better vaccines? 2010–2013 Simon Satchell, University of Bristol, Bristol, United Kingdom Ehrlenbach S, Rosales A, Posch W, Wilflingseder D, Hermann M, Asier Saez-Cirion, Unité de Régulation des Infections Rétrovirales, Brockmeyer J, Karch H, Satchell SC, Würzner R, Orth-Höller D. FWF P24598-B13: HIV infection and transmission close to reality. Institut Pasteur, France INFECT IMMUN. 2013;81:2678–2685. 2012–2016 Arnaud Moris, INSERM UMRS945, Infection et Immunité, UPMC, France Blocking Hsp70 enhances the efficiency of Amphotericin B treat- FWF W011010-21: DC-iphering complement- and Fc-recep- Teunis Geijtenbeek, Center of Infection and Immunity, Academic ment in resistant Aspergillus terreus strains. tor-mediated HIV-1 incorporation in and effects on DC function in Medical Center, Netherlands Blatzer M, Blum G, Jukic E, Posch W, Gruber P, Nagl M, Binder U, search for novel therapeutical targets. 2015–2019 Felipe Diaz-Griffero, Department of Microbiology and Immunolo- Maurer E, Sarg B, Lindner H, Lass-Flörl C, Wilflingseder .D gy, Albert Einstein College of Medicine, NY 10461, US ANTIMICROB AGENTS CHEMOTHER. 2015 [Epub ahead of print]. FWF P25389-B13: Deciphering the role of Th17 paradigm for viral Frank Ebel, Max-von-Pettenkofer-Institut, München, Deutschland infections. 2013–2016 Admar Verschoor, TU München, München, Deutschland Identification of spergillusA fumigatus surface components that Jean-Paul Latgé, Institut Pasteur, Paris, Frankreich mediate interaction of conidia and hyphae with human platelets. FWF P26117-B20: Relevance of platelets and complement for the Sven Krappmann, Universitätsklinikum Essen, Deutschland Rambach G, Blum G, Latgé JP, Fontaine T, Heinekamp T, Hagleitner pathogenesis of invasive fungal infections 2014–2016 Jürgen Löffler, Universitätsklinikum Würzburg, Deutschland M, Jeckström H, Weigel G, Würtinger P, Pfaller K, Krappmann S, Donald C. Sheppard, McGill University Montreal, Canada Löffler J, Lass-Flörl C, Speth .C CD-Labor für Invasive Pilzinfektionen. 2015–2022 FP7-PEOPLE- J INFECT DIS. 2015 [Epub ahead of print]. 2013-ITN: Immediate T-helper 17 polarization upon triggering CD11b/c on ITN (Marie Sklodowska-Curie actions). From omics to patient im- HIV-exposed dendritic cells. proving diagnostics of pathogenic yeasts. 2015–2019 Wilflingseder D, Schroll A, Hackl H, Gallasch R, Frampton ,D Lass- Flörl C, Pancino G, Saez-Cirion A, Lambotte O, Weiss L, Kellam P, Trajanoski Z, Geijtenbeek T, Weiss G, Posch W. J INFECT DIS. 2015 [Epub ahead of print].

Research Report 2015 Medical University of Innsbruck 63 Department of Hygiene, Microbiology and Social Medicine Virology

viruses, Dr. Janine Kimpel, vector vac- abrogated both limitations, by substituting cines, associated with D. v. Laer, as well as the VSV envelope protein G by the glyco- Dr. Zoltán Banki, complement and dendritic protein GP of the lymphocytic choriomen- cell vaccines, associated with H. Stoiber. ingitis virus, thus rendering a chimeric virus termed VSV-GP (Muik et al., Journal of Virol- Around 30 % of the employees work in the ogy, 2011). serological and virological diagnostics group, which services the university hospi- VSV-GP-based Viral Vector Vaccines tal Innsbruck (LKI), the regional hospitals Janine Kimpel and the medical practices in Tirol. Funding: FFG Bridge Using VSV-GP expressing the model antigen The division has developed an oncolytic ova, we could show that VSV-GPova can viral cancer vaccine as well as comple- boost the initially potent immune response ment enhanced therapeutic antibodies. To to ova further upon repeated applications. In drive these two developments into clinical contrast, as already described previously by application, two companies were founded, others, we found that other vector vaccine ViraTherapeutics GmbH (founder D. v. Laer) such as adenovirus and VSV wild-type did and Lysovac (founder H. Stoiber), re- not boost the immune response induced Head of Division: spectively. ViraTherapeutics has recently by the first application any further upon Univ.-Prof.in Dr.in secured an investment that will cover the additional injections (Tober et al., 2014). Dorothee Holm-von Laer development up to early clinical phase II VSV-GP is currently used as the basis for the trials. development of a prophylactic HIV vaccine. Contact: Peter Mayr Straße 4b The division collaborates with several inter- VSV-GP-based Oncolytic Cancer 6020 Innsbruck national groups and also with groups and Vaccines clinics in Innsbruck: Hematology and On- Guido Wollmann [email protected] cology (Gastl), Urology (Culig, Horninger), Funding: FWF, FFG Research Studio Austria, Phone: +43 512 9003 71701 Gynaecology (Fiegl, Mart), Dermatology ViraTherapeutics Fax: +43 512 9003 73701 (Romani) a.o. Viruses that selectively replicate in can- http://www3.i-med.ac.at/virologie/ cer cells and thereby lyse cancer tissue The division has established and is now in are called oncolytic viruses. It is becoming charge (Dr. Janine Kimpel) of the BSL2 and more and more clear, that the therapeu- Keywords BSL3 animal facilities of Innsbruck Medical tic effects observed are not only achieved University. by direct viral destruction of cancer cells Virology, diagnostic of viral diseases, innate but also by a strong anti-cancer immune immunity, virotherapy, cancer immunother- Research response. This is thought to be induced apy by the release of cancer antigens during VSV-GP: a Viral Vaccine Vector and oncolysis in the presence of the virus-in- Research Focus Oncolytic Cancer Vaccine duced inflammatory environment. VSV-GP Dorothee von Laer was found to be safe and therapeutically The major focus of the Division of Virology The vesicular stomatitis virus (VSV) is a neg- effective in a broad range of cancer types lies on the development of novel therapeu- ative strand RNA virus, with rapid replication in mouse models (Fig. 2, Muik et al., 2014). tics and vaccines. This research spans from and growth to high titres. It has proven to be In addition, we found strong synergism with elucidating the modes of action of novel a potent vaccine vector and oncolytic virus. a DC-based vaccine for melanoma in mice therapeutic strategies to the clinical transla- However the neurotoxicity of VSV at higher (co-supervised by Z. Banki). Currently clin- tion and development in the spin-off biotech doses and the rapid induction of neutraliz- ical grade virus production and toxicology companies of the division. Specific foci are: ing antibodies has limited the use of this tests are under way to prepare for first-in- virus in the clinic. The group of D. v. Laer man studies. • Virus-based oncolytic cancer vaccine strategies and their mode of action. • Viral vector-based vaccines primarily against HIV. • Complement-enhanced vaccines and therapeutic antibodies.

General Facts

© Study group of Prof. Dr. H. Stoiber The Division of Virology has two professors: Dorothee von Laer, the director, and Heri Fig. 1: Displacement of factor H (fH) to enhance complement-dependent cytotoxicity (CDC). bert Stoiber, the deputy director. (A) Binding of fH through short consensus repeats (SCR) 7 and 18-20 to the surface of tumor In addition, there are three junior group cells avoid CDC. (B) Addition of recombinant SCR7 or SCR18-20 removes fH from the cells leaders: Dr. Guido Wollmann, oncolytic and (C) makes them accessible to CDC.

64 Research Report 2015 Medical University of Innsbruck Virology © Cancer Research

Fig. 2: rVSV(GP)-treatment led to long-term survival of both xenogeneic and syngeneic CNS-tumour bearing mice. (A) N=3 U87-RFP orthotopic glioma-bearing NOD/SCID mice were treated i.v. with a single dose of 108 PFU rVSV(GP)-GFP at 10 days post-transplantation (dpt). Animals were sacrificed at 3 days post-injection and immunohistochemical analysis of coronal brain sections was performed with TO-PRO-3 iodide as nuclear counterstain (blue). A representative fluorescent micrograph is shown with an arrow indicating the area of progressing cellular disintegration. (B) Cohorts of n≥9 U87-RFP orthotopic glioma-bearing NOD/SCID mice were treated i.v. with either single or multiple doses of 108 PFU rVSV(GP)-GFP at 10 dpt or 10, 17 and 24 dpt, respectively. Control mice were injected with PBS. Animals were monitored for event-free survival over a period of 125 dpt.

HCV and Complement play a pathologic role in chronic viral infec- Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity, and enhance oncolytic potency. Heribert Stoiber tions like HIV and HCV (Chougnet, AIDS Muik A, Stubbert LJ, Jahedi RZ, Geiß Y, Kimpel J, Dold C, Tober R, Funding: FWF W1253 HOROS Res. Hum. Retroviruses, 2007; Manigold, Volk A, Klein S, Dietrich U, Yadollahi B, Falls T, Miletic H, Stojdl D, Bell J C, von Laer D. The hepatitis C virus (HCV) specifically in- Lancet Infect. Dis., 2007). We have found JOURNAL OF CANCER RESEARCH. 2014; 1;74(13): S 3567–3578. corporates CD59 but not other regulators that the expansion of FV-induced Tregs is VSV-GP: a potent viral vaccine vector that boosts the immune re- of complement activation (RCAs), such as impaired in mice deficient for complement sponse upon repeated applications. Tober R, Banki Z, Egerer L, Muik A, Behmüller S, Kreppel F, CD46 or CD55, into the viral envelope (Ejaz, C3, suggesting that the complement system Greczmiel U, Oxenius A, von Laer D, Kimpel J. PLoS One, 2012). The incorporated CD59 is involved in Treg responses. This exciting JOURNAL OF VIROLOGY. 2014; 88(9): S 4897–4907. protects HCV only partially against comple- finding is the basis for a new project, which Reduction of complement factor H binding to CLL cells improves the induction of rituximab-mediated complement-dependent cy- ment-mediated lysis. Thus an additional fac- aims to define key molecules involved in the totoxicity. tor/RCA must be involved in the protection interaction between complement and Tregs Hörl S, Bánki Z, Huber G, Ejaz A, Windisch D, Muellauer B, Willenbacher E, Steurer M, Stoiber H. of the virus against complement. We aim to during retroviral infections. LEUKEMIA. 2013; 27(11): S 2200–2208. characterize additional RCA(s) that protect Complement factor H-derived short consensus repeat 18-20 en- HCV against complement, which could be a Memory Inflation hanced complement-dependent cytotoxicity of ofatumumab on chronic lymphocytic leukemia cells. potential therapeutic target. Zoltán Banki Hörl S, Banki Z, Huber G, Ejaz A, Müllauer B, Willenbacher E, Funding: FWF J3484 Steurer M, Stoiber H. Complement-Enhanced Therapeutic In recent years, accumulation of specific HAEMATOLOGICA. 2013; 98(12): S 1939–1947. Low prevalence of HPV detection and genotyping in non-muscle Antibodies CD8+ memory T cells – termed memory in- invasive bladder cancer using single-step PCR followed by reverse Heribert Stoiber, Zoltán Banki flation – has appeared to be one of the most line blot. The antitumor activity of monoclonal anti- important aspects of cytomegalovirus im- Pichler R, Borena W, Schäfer G, Manzl C, Culig Z, List S, Neururer S, Von Laer D, Heidegger I, Klocker H, Horninger W, Steiner H, bodies for the treatment of different can- munobiology. Dr. Z. Banki studied this phe- Brunner A. cers is mediated mainly by antibody-de- nomenon during his 12-months at Oxford WOLRD JOURNAL OF UROLOGY. 2015; Epub ahead of print. pendent cellular cytotoxicity (ADCC) and University in the group of Paul Klenerman. Selected Funding complement-dependent cytotoxicity (CDC). He worked with a novel model of memory • A chimeric oncolytic rhabdovirus for the treatment of melano- Unfortunately, the efficacy of CDC is strong- inflation that is based on replication-defi- ma, FWF - P 25499-B13, Univ.-Prof. Dr. Dorothee von Laer • Evaluation of a semi-replication competent VSV system as HIV ly impaired due to the expression and acqui- cient adenovirus and characterized the role vaccine, FFG-Bridge, Univ.-Prof. Dr. Dorothee von Laer sition of regulators of complement activa- of latently infected non-hematopoietic cells • AWS Pre-Seed, Univ.-Prof. Dr. Dorothee von Laer tion (RCA) on tumor cells. A prominent RCA in memory inflation. • HOROS – Host response in opportunistic infections, FWF ‑W1253, Univ.-Prof. Dr. Heribert Stoiber in fluid phase is factor H (fH), which asw not investigated in this context so far. We Epidemiology of HPV Infection Collaborations found that abrogating fH function provides a Wegene Borena • J. Schmitz, B. Haynes; Harvard Medical School, Boston, USA • A. van den Pol; Yale University, New Haven, USA novel approach to improve the CDC efficacy The epidemiology of HPV infection is being • A. Oxenius; ETH Zürich, Zürich, Switzerland of therapeutic antibodies (Hörl, Leukemia, studied in Austria, where hardly any data • HP Kiem; Fred Hutchinson Cancer Research Center, Seattle, USA 2013; Hörl, Haematologica, 2013). are available so far, and in different patient • J. Bell; CICR – Centre for Innovative Cancer Research, Ottawa, groups (Pichler et al. 2015). USA • H. Miletic; University of Bergen, Bergen, Norway Complement and T Cell Responses in • L. Hansmann, S. Newrzela; Johann-Wolfgang-Goethe University, Retroviral Infections Selected Publications Frankfurt a. M., Germany • F. Kreppel; University Ulm, Ulm, Germany Heribert Stoiber, Zoltán Banki Type I interferons protect T cells against NK cell attack mediated • L. Lehmann; Ludwigs-Maximilian-Univeristy, Munich, Germany The implication of Tregs in viral infection by the activating receptor NCR1. Crouse J, Bedenikovic G, Wiesel M, Ibberson M, Xenarios I, Von was first described for mice persistently in- Laer D, Kalinke U, Vivier E, Jonjic S, Oxenius A. Facilities fected with Friend virus (FV), but Tregs also IMMUNITY. 2014; 19;40(6): p. 961–973. BSL-2 and BSL-3 mouse facility

Research Report 2015 Medical University of Innsbruck 65 Department of Hygiene, Microbiology and Social Medicine Social Medicine

General Facts threaten human health and well being. This main effect or direct effects model has a The division has a strong focus on teach- certain weakness, however. ing public health to medical students com- Human reactions to environmental condi- plemented by in-depth practica on a wide tions occur within an ecological context range of health related themes. that shapes their responses. One key ele- The current head of the division was the re- ment of such a contextualised research ori- sponsible module organiser (2.31: Humans, entation is that individual, social and other family, society and the environment) for the factors of the natural and built environment medical curriculum (6. to 7. Semester). can substantially alter the direct noise re- Members of the division are further involved sponse function (see Fig. 1). in teaching public health doctors in environmental health. Soundscape and Blood Pressure in The division supports governments in en- Schoolchildren vironmental health impact assessments at Peter Lercher larger scales and more focused health im- Most research on the effects of noise on pact assessments at the community and blood pressure in children has focused on smaller regional levels. a direct effects model using sound levels Head of Division (interim): One member of the division is supporting as indicator. We published such a well-cited ao. Univ.-Prof. Dr. Peter Lercher the regional government and communities analysis in 2001 (Evans et al.). A re-analysis in providing restorative options and facili- of the data set was conducted within the Contact: ties for handicapped persons (wheel chair EU-funded 7th Framework project ENNAH Sonnenburgstraße 16 routes, handbike-routes, barrier-free tourist and the COST-Soundscape project with a 6020 Innsbruck destinations etc.) contextual perspective on effect modification by dispositional (low birth weight) and [email protected] Research contextual (Housing, soundscape assess- Phone: +43 512 9003 71253 ment, chronic stress) factors. Fax: +43 512 9003 73250 Sound Exposure and Health: https://www.i-med.ac.at/ Our Research Perspective We found children with premature births sozialmedizin/en/ Empirical research on the health impacts and elevated chronic stress (i.e. overnight of environmental noise has focused mainly cortisol) more susceptible to adverse blood on the critical question of whether certain pressure responses to road traffic noise. intensities of sound exposure can harm or Furthermore, residence in a multi-dwelling

Keywords

Environmental and social epidemiology, Environmental health impact assessment, Combined effects, Transportation Noise & vibration & air pollution, Psychoacoustics, Soundscape research, Public health, Health related quality of life

Research Focus

• Integrated environmental health impact assessment • Cardiovascular effects of environmental noise exposure • Effects of environmental noise & air pollution on HRQoL • Air pollution, social factors and respiratory health • Dispositional factors, coping styles as effect modifiers in environmental health studies • Combined effects of noise, vibration and air pollution exposure on annoyance and health • Effects of quiet areas and other restorativ e residential neighbourhoods on health & Fig. 1: Block diagram outlining the soundscape perception process and its moderation by well-being context leading to direct and indirect human responses (Lercher & Schulte-Fortkamp 2013)..

66 Research Report 2015 Medical University of Innsbruck Social Medicine

5 10 15 health assessments with slightly different < 37 weeks ≥ 37 weeks instruments. In addition the noise exposure assessment was improved.

We found reported hypotension to be sig- 130 nificantly associated with rail and total

mmHg noise exposure and strongly modified by 125 weather sensitivity. Reported hypotension medication showed associations of similar 120 size with rail and total noise exposure – but without effect modification by weather sensitivity. This replication study showed also 115 that the noise effect varies significantly by sex, age and body mass index and is mod- 110 erated by adjacent main roads and related annoyance but not by highways. ≥ 60 dBA,Ldn 105 Stress, Meditation and Blood Pressure Systolic blood pressure , < 50 dBA,Ldn Harald Hörmann

© Journal of the Acoustical Society of America © Journal of the Acoustical (PhD in preparation) 5 10 15 20a-Dihydrocortisol,µg/8h Fig. 2: Systolic blood pressure by gestational age and urine cortisol, adjusted for BMI, sex, family history, education, house type, area quiet, sound*gestation unit, as well as perceived quietness of the Noise and hypotension in adults: Selected Publications area, did not modify the traffic noise im- A novel relationship? Association and moderation of self-reported hypotension with traffic noise exposure: A neglected relationship. Lercher Peter, pacts: rather, each factor had its own, inde- Peter Lercher Widmann Ulrich. NOISE HEALTH. 2013; 15(65): p. 205–216. pendent effect on resting blood pressure. Although earlier short-term experimental The ecological context of soundscapes for children’s blood pres- These results complement earlier findings studies showed not only increases but also sure. Lercher Peter, Evans Gary, Widmann Ulrich. JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA. 2013; 134(1): p. 773– (Lercher et al. 2002), where we showed decreases in blood pressure after noise 781. an effect modification in children with low exposure, this fact has been neglected in Hypotension and environmental noise: a replication study. birth-weight/premature birth on mental research during the past 25 years. We had Lercher Peter, Widmann Ulrich, Thudium Jürg. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH. health. posed questions about hypotension and 2014; 11(9): p. 8661–8688. hypotension medication in two surveys and Soundscape of European Cities and Landscapes–Harmonising. wanted to test whether hypotension is a Lercher Peter, Schulte-Fortkamp Brigitte. In AIA/DAGA CONFE- Highly weather sensitive potential health outcome of chronic noise RENCE, 40th Italian (AIA) Annual Conference on Acoustics and Moderate weather sensitive the 39th German Annual Conference on Acoustics (DAGA). 2013; exposure. p. 8–21. Not at all weather sensitive 0.8 In the first survey (N=1989), self-report- Collaborations • Gary W. Evans, Cornell University, Ithaca, USA ed hypotension was associated non-line- • Jürg Thudium, Oekoscience-Institute, CH-7000 Chur, Switzer- arly with noise exposure (P=0.044) in the land 0.6 • Ulrich Widmann, AUDI AG, I/ET, D-85045 Ingolstadt, Germany presence of a strong sex × age effect mo • Andre Fiebig, HEAD acoustics GmbH, D-52134 Herzogenrath, dification (P<0.0001). A further significant Germany • Dietrich Kühner, Independent noise consultant, D-51519 0.4 modification by noise was observed with re- Odenthal, Germany ported symptoms of exhaustion (P=0.03). • Helen Lin, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Weather sensitivity showed a significant • Dick Botteldooren, Acoustics Research Group, Ghent Universi- interaction with noise sensitivity (P=0.02) ty, B-9000 Ghent, Belgium 0.2 • Brigitte Schulte-Fortkamp, TU Berlin, Berlin, Germany and also a non-linear interaction with age • Michael Cik, TU-Graz, 8010 Graz, Austria Predicted probability: hypotension probability: Predicted (P=0.02). The results remained stable after • Mark Brink, Federal Office for the Environment & ETH Zurich, Switzerland © International Journal of Environmental Research and Public Health © International adjustment for variables known to be asso- • Terry Hartig, Uppsala University, Sweden 50 60 70 ciated with constitutional hypotension. Low blood pressure readings were not associat- Railway sound exposure level, dBA, Ldn ed (too small N).

Fig. 3: Predicted probability of reported hy- In the second survey we conducted a rep- potension past year with rail sound level lication analysis on two samples (N=800 exposure by weather sensitivity. Adjusted & N=567). The smaller sample was an for 16 potential confounders and interaction intensive study with anthropometric and terms blood pressure readings and more in depth

Research Report 2015 Medical University of Innsbruck 67 Institute of Pharmacology Pharmacology

and international societies to the promotion of the amygdala and their participation in of Pharmacology. This is done through the amygdala neural circuits processing senso- board functions (R. Fischer-Colbrie, Secre- ry stimuli. tary) in the Austrian Pharmacological So- Future Goals: Characterization of the long- ciety (APHAR) and the organization of the range connections of amygdala GABAergic Annual Meeting of the APHAR every 3 years neurons and their behaviourally-relevant in Innsbruck. plasticity. Furthermore, the Department of Pharma- cology provides independent drug and Neuropeptides in Fear and Anxiety therapeutic information to doctors through Ramon Tasan the “Pharmainformation” bulletin and con- The laboratory investigates the role of neu- tributes to a variety of public bodies (e.g. ropeptides in modulating emotional behav- Ethic Committee of the Medical University iours that are related to fear and hunger. of Innsbruck) involved in the evaluation of A further aim is to unravel the underlying drug safety and development. synaptic correlates of these emotional responses. Avoiding danger and finding food Research are two intimately associated, life-sustain- ing behaviors that are organized in survival Head of Institute: Neural Circuits Underlying Fear and circuits and strongly modulated by emo- Univ.-Prof. Dr. Francesco Ferraguti Anxiety – Francesco Ferraguti tions. Maladaptation within such survival The laboratory is primarily interested in un- circuits can induce dysregulated, pathologi- Contact: derstanding the neural mechanisms medical behavior, resulting in the development of Peter Mayr Straße 1a ating emotional information processing in feeding- or anxiety-disorders. Interestingly, 6020 Innsbruck the amygdala, and the role that classical neuropeptides are essential modulators of neurotransmitters have, e.g. glutamate and both, energy homeostasis and anxiety-relat- [email protected] GABA, on the acquisition, storage and inhi- ed behaviors. For instance, PP-fold peptides, Phone: +43 512 9003 71204 bition of fear memories. including neuropeptide Y (NPY), peptide YY Fax: +43 512 9003 73400 Although a large body of in vivo work has (PYY) and pancreatic polypeptide (PP) are www.i-med.ac.at/pharmakologie/ suggested that fear and extinction of fear released during states of hunger or acute are encoded by specific neuronal activity danger. While the anxiolytic and fear-reduc- patterns with characteristic temporal dy- ing properties of these neuropeptides are Keywords namics, neuroanatomical information on increasingly evident, a potential interaction the underlying neural networks activated of feeding and fear has not been elucidated Neuropeptides, metabotropic glutamate re- during a particular behavioural task is still so far. ceptors, opioid receptors, neuropeptide Y largely lacking. A first step in understanding Neuropeptides are highly enriched in the receptors, fear learning, anxiety disorders, these networks is the characterization of amygdala and hippocampus, two brain re- epileptogenesis the main cell types of the amygdala and the gions that are fundamentally involved in identification of their participation in intrin- controlling emotional behaviors. There, they Research Focus sic and extrinsic circuitries of this region. are considered to act as essential media- Our work in recent years involved primarily tors significantly shaping synaptic function- • Characterization of the neural networks the anatomical, pharmacological and phys- ing. Through a multidisciplinary approach, underlying physiological and pathological iological characterization of different GAB- which involved immunohistochemistry, fear/anxiety and identification of novel Aergic cell types of the basolateral complex neuronal tract tracing, ex vivo slice electro- treatment strategies. and of the intercalated cell masses of the physiology with pharmaco- and optogenetic • Etiology and novel treatments of temporal rodent amygdala. Currently, taking advan- approaches in different transgenic mouse lobe epilepsy. tage of recent developments in molecu- lines, we have demonstrated that several lar genetics, viral trans-synaptic tracing neuropeptides of the gut-brain axis are fun- General Facts and novel ultrastructural techniques (e.g. damentally involved in the modulation of SDS-digested freeze-fracture replica immu- The Department of Pharmacology, estab- nogold labelling), we investigate long-range lished in 1886, is a centre of excellence in connections between amygdala GABAergic Neuropharmacology, and uses a variety of neurons and cortical or subcortical brain cutting-edge experimental approaches to structures as well as structural synaptic address fundamental research questions plasticity of amygdala inhibitory networks. related to the identification of novel molec- Moreover, we examine the pharmacologi- ular targets and the development of new cal and anatomical bases of anxiety disor- therapeutic concepts for neuropsychiatric ders in models of Parkinson’s disease. In disorders. particular, we seek to determine whether The Department provides training in phar- dopamine-depletion of the amygdala elicits Fig. 1: Axonal projections (shown in blue) of macology to both medical undergraduate pathological anxiety in mice. a large intercalated neuron (shown in red) and graduate students. An additional task of Major Achievements: Identification of nov- of the amygdala located in the intermediate the Institute is to contribute within national el cell types of the intercalated cell masses capsule.

68 Research Report 2015 Medical University of Innsbruck Pharmacology

specific overexpression of tetanus toxin introduced by stereotactic injections of a respective viral vector into the subiculum or nucleus reuniens thalami. Tetanus toxin is then selectively expressed in GABA/soma- tostain, GABA/parvalbumin or glutamate/ calretinin neurons at the site of injection Fig. 2: EEG depth electrode recording from the ipsilateral hippocampus of an epileptic and impairs neurotransmitter release from mouse before and after KOR agonist treatment. these neurons. We are then monitoring EEG activity in these mice for one month and fear and fear extinction behaviour, an effect Major Achievements: Established that the probe development of epilepsy. So far, we that highly depends on the homeostatic sit- activation of KOR plays an important role in have demonstrated that selective silencing uation of the individual, emphasizing a mu- epileptogenesis. of GABA/parvalbumin neurons in the subic- tual interaction of survival circuits for fear Future Goals: Investigate the potential of ulum leads to spontaneous limbic seizures and hunger. G-protein biased KOR agonists in epilepsy. and highlighted the crucial role of these Major Achievements: Identification of neu- neurons in the manifestation of temporal ropeptides of the gut-brain axis that are also Neuronal Circuitries of the Subiculum lobe epilepsy. fundamentally involved in the modulation of in Epileptogenesis Major Achievements: Identification that fear and fear extinction behavior. Meinrad Drexel and Günther Sperk selective silencing of GABA/parvalbumin in- Future Goals: Characterization of peripher- The group currently investigates the role of terneurons in the subiculum leads to spon- al modulators of hunger and satiety which GABAergic interneurons of the subiculum taneous limbic seizures. could also affect fear learning, closing the in the generation of epileptic seizures. Ep- Future Goals: Investigate the role of GABA loop of the gut-brain axis in controlling emo- ileptic seizures are generated by abnormal, ergic interneurons of the subiculum in the tionally driven behaviors. excessive or synchronous neuronal activ- generation of epileptic seizures. ity. Malfunctioning of microcircuits of the Opioid Systems in Epilepsy and Emo- hippocampus, thalamus or cortex may be Selected Publications tional Control – Christoph Schwarzer causative. Neurophysiological information Large intercalated neurons of amygdala relay noxious sensory The laboratory investigates the role of the formed in the hippocampus is processed information. Bienvenu TCM, Busti D, Micklem BR, Mansouri M, Magill PJ, Ferraguti F, Capogna M. endogenous dynorphin/kappa opioid re- and transmitted to multiple brain areas. JOURNAL OF NEUROSCIENCE. 2015; 35:5. 2044–57. ceptor (KOR) system in epilepsy and epilep- Recently, we obtained evidence that mal- Sensory inputs to intercalated cells provide fear-learning modula- togenesis. Moreover, by gaining insight into functioning of the subiculum, the main out- ted inhibition to the basolateral amygdala. Asede D, Bosch D, Lüthi A, Ferraguti F, Ehrlich I. NEURON. 2015; 86:2. 541–54. the functional neuroanatomy of the dynor- put area of the hippocampus, is crucially Expression of GABA receptor subunits in the hippocampus and phin/KOR in emotional control, a further involved in the generation of epileptic sei- thalamus after experimental traumatic brain injury. aim is to minimize potential side-effects of zures in animal models of temporal lobe epi- Drexel M, Puhakka N, Kirchmair E, Hörtnagl H, Pitkänen A, Sperk KOR agonist treatment. lepsy. In particular GABA/somatostatin and G. NEUROPHARMACOLOGY. 2015; 88. 122–33 Epilepsy is one of the most frequent neuro- GABA/parvalbumin neurons targeting the Maternal inheritance of the Gnas cluster mutation Ex1A-T affects size, implicating NESP55 in growth. Eaton SA, Hough T, logical diseases, which presently cannot be dendritic trees and the somata of pyrami- Fischer-Colbrie R, Peters J. cured. A high number of patients are refrac- dal neurons, respectively, as well as afferent MAMMALIAN GENOME. 2013; 24:7–8. 276–85. tory to pharmacological treatment, render- glutamate/calretinin neurons originating in GPER1 (GPR30) knockout mice display reduced anxiety and altered stress response in a sex and paradigm dependent manner. ing surgical removal of parts of the brain as the nucleus reuniens thalami may be im- Kastenberger I, Schwarzer C. the ultimate solution. paired in their function. HORMONES AND BEHAVIOR. 2014; 66. 628–36. In recent years, we provided evidence, that We use transgenic mice that allow cell- Selected Funding the activation of KOR plays an important • Signaling in neurons, Austrian Science Fund (FWF) Doctoral role in epileptogenesis. Thus, dynorphin college Program no. W12060, F. Ferraguti & C. Schwarzer. deficient mice display faster progression • Cell signaling in chronic CNS disorders, FWF Spezialforschungs- bereich Program no. F44-17, F. Ferraguti. and more neurodegeneration in models of • NPY in adult neurogenesis and hippocampus-dependent fear epileptogenesis than wild-type animals. learning, FWF grant no. P25851, R. Tasan. • Gene therapy in temporal lobe epilepsy, FWF, grant no. I977, Application of a KOR agonist during epilep- C. Schwarzer. togenesis reduces neurodenegeration and • Neuronal circuitries of the subiculum in epileptogenesis, FWF grant no. P26680, G. Sperk. neurochemical alterations. On the other hand, activation of KOR is known to induce Collaborations dysphoria in humans. In male mice, endog- Prof. Aiba A., the University of Tokyo, Tokyo, Japan. Prof. Becker A., Dept. Neuropathology, University of Bonn, Bonn, Germany. enous dynorphin acting on KOR exerts anx- Prof. Beck-Sickinger A., University of Leipzig, Leipzig, Germany. iogenic effects. In female mice, anxiogenic Dr. Bonaventure P., Johnson & Johnson Pharmaceutical Research effects of dynorphin may depend on an in- & Development, USA. Prof. Capogna M., Oxford University, Oxford, UK. Prof. Colmers W., Dept. Pharmacology, University of Alberta, terplay with the estrogen system. Based on Edmonton, Canada. Dr. Ehrlich I., University of Tübingen, Tübin- these findings, we presently investigate the gen, Germany. Prof. Heilbronn R., Dept. Virology, Campus Ben- jamin Franklin, Charité – Medical School, Berlin , Germany. Prof. potential of G-protein biased KOR agonists Herzog H., Garvan Institute of Medical Research, Australia. Prof. in epilepsy. In order to do so, we apply 4 Lüthi A., Friedrich Miescher Institute, Basel, Switzerland. Prof. Pape H.C., Westfälische Wilhelms-Universität, Münster, Germany. channel in vivo EEG in the kainic acid model of temporal lobe epilepsy with behavioral Core Facilities testing. Fig. 3: Microcircuits of the subiculum. • Neurolucida (in collaboration with the Biooptics facility)

Research Report 2015 Medical University of Innsbruck 69 Department of Medical Statistics, Informatics and Health Economics Medical Statistics and Informatics

Biobanking and BioMolecular Resources Stata, SPSS, etc.) and data management Research Infrastructure (BBMRI.MUI) software (REDCap). Lalit Kaltenbach has developed an e-CRF system for the interna- BBMRI.MUI aims to establish a state-of-the- tional, multicentre LEVOREP trial. art biobanking infrastructure at the Medi- cal University of Innsbruck and to increase Other multi-centre trials with major partici close cooperation between and harmoniza- pation of our division are the EU-funded tion of local, national and international bi- Gannet53 randomized controlled trial, as obanks. well as other studies such as the PLATA, VITRIS, AFREEZE, ForaC and FIinTIC trials. General Facts The division with Lalit Kaltenbach, as re- The Division of Medical Statistics and sponsible IT manager, runs five Austria-wide Informatics provides a major contribution to registries: the HIR (heart failure) registry, the teaching of medical students. Besides the PTCA (percutane coronary intervention) offering obligatory lectures in semesters 1, registry, the IIK (invasive interventional car- 5 and 8, we focus on teaching diploma and diology) registry, the Ablation registry, and PhD students. Students who are working on the Parkinson registry. The Austrian socie- Head of Division (interim): their diploma and PhD theses are advised ties of Cardiology and Neurology are part- ao. Univ.-Prof. Dr. Hanno Ulmer on the use of appropriate statistical meth- ners in these projects. ods. Contact: The statistical consulting and the participa- Schöpfstraße 41/1 Additionally, we provide statistical consultation in these projects are fundamental for 6020 Innsbruck tions to all researchers of MUI with a focus the strong publication record of the divi- on clinical studies. We support clinical re- sion. In relation to the number of employ- [email protected] searchers in all aspects of statistical study ees, the division has a top rank within MUI Phone: +43 512 9003 70900 planning, protocol writing, applications for in recent years regarding total number of Fax: +43 512 9003 73922 ethical review, data management, statistical publications. In the years 2013 to 2014, a https://www.i-med.ac.at/msig/ analysis and publication. We provide exper- total of 76 original research papers were tise regarding the usage of statistical (SAS, published by researchers of the division

Females Males proportion cured median survival uncured proportion cured median survival uncured 0,0 0,0 0,0 0,0 Keywords pancreas 5% AML 8% 0.3 y. AML Medical statistics, biostatistics, statistical pancreas 10% 0.4 y. AML oesophagus 10% 0.5 y. pancreas ; stomach liver 11% 0.5 y. pancreas ; oesoph. methods, epidemiology, medical informat- lung 14% 0.6 y. lung ; CNS/brain lung 14% 0.6 y. lung ics, medical documentation, clinical trials, CNS/brain 17% 0.7 y. liver AML 15% 0.7 y. stomach 0.8 y. bladder CNS/brain 22% registries, risk prediction, prevention stomach 22% 0.9 y. liver ; CNS/brain liver 25% stomach 28% 1.1 y. rectum 1.1 y. kidney Research Focus 1.2 y. colon 1.2 y. bladder 1.4 y. cervix Cancer Epidemiology ovary 40% head/neck 39% bladder 42% 1.7 y. head/neck

In cancer epidemiology, we investigate met- 1.9 y. head/neck abolic and lifestyle factors as potential risk 2.0 y. ovary colon 51% 2.1 y. colon factors for cancer incidence and mortality. rectum 55% bladder 57% 2.2 y. rectum head/neck 56% rectum 58% colon 61% larynx 61% Statistical and Epidemiological Methods

2.7 y. larynx The focus of the division lies in the devel- kidney 72% opment and application of statistical and cervix 74% epidemiological methods for modelling complex associations in the framework of classification and regression analysis.

Medical Informatics and Documentation

100,0 4,0 100,0 4,0 Medical informatics, which is a multidis- © Cancer Causes and Control ciplinary research field, targets the use of Fig. 1: Estimated levels of the proportion cured (left, in %) compared to levels of the median information technology in order to improve survival time of the uncured (right, in years) in Tyrol during 2005–2009 by cancer site, health care. stratified by sex.

70 Research Report 2015 Medical University of Innsbruck Medical Statistics and Informatics

Pancreas, female Pancreas, male

100 100

90 90

80 80

70 70

60 60

50 50

40 40

30 30 relative survival (%) relative relative survival (%) relative 20 20

10 10

0 0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 years since diagnosis years since diagnosis

Rectum, female Rectum, male

100 100

90 90

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60 60 50 50 Fig. 2: Estimated relative survival, overall 40 40 (upper curve, gray) and of the uncured (low- 30 30 relative survival (%) relative survival (%) relative er curve, black), for pancreas and rectum 20 20

10 10 cancer according to duration since diagno-

0 0 sis in Tyrol during 2005–2009, stratified by 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 sex. © Cancer Causes and Control years since diagnosis years since diagnosis

(mostly co-authorships). In order to support nation data from about 570 000 individuals Using data from the Vorarlberg health ex- researchers with submitting their PhD and from Sweden, Norway and Austria. Meas- amination database (VHM&PP), we investi- habilitation theses, Joachim Masser devel- urements such as height, weight, blood gated the role of elevated gamma-glutamyl- oped the SCORE program. It enables the pressure, blood glucose, triglycerides and transferase in endometrial cancer survival. administration of the personal publication total cholesterol were recorded between This is, so far, the most recent publication record of MUI researchers, including impact 1972 and 2004. regarding gamma-glutamyltransferase, fin- factors, citations and journal rankings. Individuals in the database have been fol- ishing up a series of top publications in jour- lowed until their death, emigration or the nals such as Circulation, Arteriosclerosis, Sabrina Neururer (chair of the organizing end of follow-up, according to the principles thrombosis, and vascular biology, Cancer committee) and Hanno Ulmer (president of epidemiological cohort studies. To obtain research and International journal of can- of the society, chair of the scientific com- cause of death and cancer incidence infor- cer. mittee) organised the 2013 Conference of mation, the database was linked to cancer the Austro-Swiss Region of the International registries in each country. More recently, we have set up a case-con- Biometric Society. trol study to investigate the association of The division is a key partner in the Me-Can aluminium exposure and breast cancer. Research project. Susanne Strohmaier, Wegene Bore- Caroline Linhart is working on this study in na, and Michael Edlinger published papers cooperation with MUI scientists from the Cancer Epidemiology regarding the association of metabolic Department of Gynaecology, the Universi- Hanno Ulmer, Michael Edlinger factors with liver, gall-bladder and brain ty Hospital for Plastic, Reconstructive and In cancer epidemiology, we investigate cancer, as well as the associations of total Aesthetic Surgery and the Division of Clini- metabolic and lifestyle factors as potential cholesterol and triglycerides with cancer in- cal Biochemistry at MUI. risk factors for cancer incidence and mor- cidence and mortality. tality. Metabolic syndrome is a cluster of Statistical and Epidemiological Methods factors characterized by obesity, hyperten- Besides our international collaborations, we Hanno Ulmer sion, dyslipidemia and high blood glucose. cooperate locally with the TILAK Institute of Classification and regression trees are com- The prevalence of metabolic syndrome is Epidemiology (Willi Oberaigner). In 2014, monly applied for exploration and modelling rising worldwide. Individuals with metabolic a paper applying proportion cured models of complex data. They are able to handle syndrome have a higher risk of cardiovascu- to Tyrolean cancer data was published by strongly non-linear relationships with high lar diseases and diabetes, but less is known Michael Edlinger. Currently, he is also in- order interactions and different variable about the association with cancer. volved in a large clinical cohort study (CAR- types. DIIGAN) in association with the University We participated in the Me-Can project, con- Hospital for Internal Medicine III (Cardiolo- Thomas Grubinger developed a new R taining a large database with health exami- gy and Angiology) at MUI. package “evtree”, available from the Com-

Research Report 2015 Medical University of Innsbruck 71 Department of Medical Statistics, Informatics and Health Economics

prehensive R Archive Network at http:// integrate data repositories and to support alogue (Österreichischer Leistungskatalog) CRAN.R-project.org/package=evtree, pro- medical documentation. Formal, semanti- for procedure coding. A four-step approach viding evolutionary methods for learning cally enriched knowledge representation (comparative analysis, definition analysis, globally optimal classification and regres- by means of ontologies provides a powerful typological analysis, ontology implementa- sion trees. solution to facilitate semantic interoperabil- tion) for formalizing the Austrian procedure ity and knowledge sharing within the scope catalogue is proposed, which provides a More recently, this research group has set a of e-health, medical documentation, or bio novel, systematically developed, strong new focus on causal inference, applying me- banking. An ontology represents classes framework to semantically enrich proce- diation analysis to epidemiologic research of entities of the real world and focuses on dure classifications. problems. Josef Fritz is currently working the definition of concepts and relations be- on the contribution of cardiovascular risk tween them. They offer a good solution for Philipp Hofer is currently working on the factors to the gender gap in mortality from addressing the challenge of machine-read evaluation of ontologies in order to repre- coronary heart disease. able concepts in order to support health sent biosample collections and the usage care providers and researchers with their of ontologies for automated data integra- Medical Informatics and Documentation daily work. tion. This includes semantic equivalences Georg Göbel between different data model representa- In the division of Medical Statistics and In- Sabrina Neururer developed ontologized tions. In this context, we developed a novel formatics, we have a strong focus on the versions of classification systems for health approach that combines competency eval- use of semantic web technology in order to care, especially the Austrian procedure cat- uation and query expansion to assess the usefulness of existing ontologies for the biobanking domain.

Biobanking and BioMolecular Resources Research Infrastructure (BBMRI.MUI) Georg Göbel BBMRI.MUI is a part of the Austrian BBM- RI.at project, which aims to develop a biobanking infrastructure in Austria in order to increase cooperation and harmonization between biobanks. Since 2014, the local BBMRI.MUI research team, consisting of Georg Göbel, Sabrina Neururer, Philipp Hofer, Thomas Insam, Anette Zeilner and Helga Hauffe, has worked on establishing common guidelines for the collection of human biosamples and on data management in order to implement a university-wide state-of-the-art biobanking infrastructure at the Medical University of Innsbruck. Cur- rently, several independent decentralized collections of human biomaterials are located at different MUI divisions. Based on the approval of the local ethics committee, most of these separate sample collections are linked to pseudonymized, detailed clinical data. The project aims at integrating archived biospecimens with clinical and molecular data in a collaborative environment that emphasizes scientific insights, while ensuring security and compliance. As a preliminary result, a university-wide biobank registry based on international standards was implemented and it will be linked with the European-wide BBMRI registry. © Journal of Statistical Software © Journal of Statistical

In the future, these projects will go beyond the sample-centric workflows of conven- Fig. 3: Trees for customer targeting constructed by rpart (upper panel) and ctree (lower tional biobanks by integrating patient mate- panel). The target variable is the customer’s choice of buying the book. The variables rials, clinical, specimen, genetic and molec- used for splitting are the number of art books purchased previously (art), the number ular assay data, to deliver a holistic, unified of months since the first purchase (first), the frequency of previous purchases at the view, enabling researchers to facilitate data Bookbinder’s Book Club (freq), and the customer’s gender. exploration and hypotheses driven research

72 Research Report 2015 Medical University of Innsbruck Medical Statistics and Informatics

Selected Publications Selected Funding A prospective study on metabolic risk factors and gallbladder • BBMRI.MUI, Austrian BMWFW (GZ 10.470/0016-II/3/2013), cancer in the metabolic syndrome and cancer (Me-Can) collab- Georg Göbel orative study. Borena W, Edlinger M, Bjørge T, Häggström C, Lindkvist B, Nagel Collaborations G, Engeland A, Stocks T, Strohmaier S, Manjer J, Selmer R, Tretli S, Concin H, Hallmans G, Jonsson H, Stattin P, Ulmer H. • Odd Aalen, University of Oslo, Oslo, Norway PLoS One. 2014 Feb 21;9(2):e89368. • Tone BjØrge, Bergen University, Bergen, Norway • Larry Brant, National Institute on Aging, Baltimore MD, USA Site-specific proportion cured models applied to cancerregistry • Hans Concin, Arbeitskreis für Vorsorge- und Sozialmedizin, Bre- data. genz, Austria © Journal of Statistical Software © Journal of Statistical Edlinger M, Ulmer H, Cvancarova M, Oberaigner W. • John Danesh, University of Cambridge, Cambridge, UK Cancer Causes & Control. 2014 Mar;25(3):365–73. • Rick Grobbee, University Medical Centre Utrecht, Utrecht, the Netherlands evtree: Evolutionary Learning of Globally Optimal Classification • Leo Held, University of Zurich, Zurich, Switzerland and Regression Trees in R. • Cecily Kelleher, University College Dublin, Dublin, Ireland Fig. 4: Class distribution of the simulated Grubinger T, Zeileis A, Pfeiffer KP. • Yuan Lu, Harvard University, Cambridge MA, USA 4 × 4 chessboard problem with zero noise, JOURNAL OF STATISTICAL SOFTWARE. 2014; 16: p. 1–29. • Anna Lukanova, German Cancer Research Centre, Heidelberg, plotted on the (X1, X2)-plane. The two class- A concept of a MIABIS based register of biosample collections at Germany the Medical University of Innsbruck. • Jonas Manjer, Lund University, Malmö, Sweden es are indicated by black circles and gray Hofer P, Fiegl H, Angerer J, Mueller-Holzner E, Chamson M, • Gabriele Nagel, University of Ulm, Ulm, Germany, crosses, respectively. Klocker H, Steiner E, Hauffe H, Zschocke J, Goebel G. • Petra Peeters, Imperial College, London, UK STUDIES IN HEALTH TECHNOLOGY AND INFORMATICS. 2014; • Ruth Pfeiffer, National Cancer Institute, Bethesda MD, USA 205: p. 293–297. • Pär Stattin, Umeå University, Umeå, Sweden without extra programming or IT support. • Ewout Steyerberg, Erasmus MC University Medical Centre Rot- Total Serum Cholesterol and Cancer Incidence in the Metabolic terdam, Rotterdam, the Netherlands Multi-level user access control ensures that Syndrome and Cancer Project (Me-Can). • Reinhild Strauss, Medical University of Vienna, Vienna, Austria all collaborators can work effectively while Strohmaier S, Edlinger M, Manjer J, Stocks T, Bjorge T, Borena W, • Jaakko Tuomilehto, University of Helsinki, Helsinki, Finland Haggstrom C, Engeland A, Nagel G, Almquist M, Selmer R, Tretli S, • Christopher Wild, International Agency on Research of Cancer, ensuring compliance with patient consent Concin H, Hallmans G, Jonsson H, Stattin P, Ulmer H. Lyon, France and maintaining regulatory guidelines. PLoS One. 2013; 8: p. e54242. • Kurt Zatloukal, Medizinische Universität Graz, Austria

Fig. 5: Core concepts and relations of the Austrian procedure catalogue ontology. Blue concepts represent static information from the initial procedure catalogue.

Research Report 2015 Medical University of Innsbruck 73 Department of Medical Statistics, Informatics and Health Economics Health Economics

Research they intend to encourage efficient delivery and to discourage the provision of unnec- DRGs in Austria and Europe essary services, i.e. to overcome some of Conrad Kobel the drawbacks of more traditional hospital Payment mechanisms represent one of the reimbursement systems. A case payment fundamental building blocks of any health system that fulfils these hopes requires system, introducing powerful incentives carefully balanced incentives as well as for actors in the system and fierce tech- a methodologically sound system. Espe- nical design complexities. Inpatient case cially, DRGs need to accurately reflect the payments, mainly referred to as diagnosis resources and costs of treating a group of related groups (DRGs), are nowadays used similar patients. as a payment mechanism with ambitious Case-based hospital financing systems aims: they seek to reimburse providers fair- have been adopted in an increasingly large ly for the work they undertake. Moreover, number of countries around the world.

Head of Division (interim): ao. Univ.-Prof. Dr. Hanno Ulmer

Contact: Schöpfstraße 41/1 6020 Innsbruck

[email protected] Phone: +43 512 9003 70900 Fax: +43 512 9003 73922 https://www.i-med.ac.at/msig/

Keywords

Diagnosis related groups (DRGs), patient classification, cost analyses, length of stay analyses, hospital cost accounting, hospital quality, hospital efficiency, hospital performance, hospital payment

Research Focus

Development and improvement of hospital financing systems based on diagnosis related groups (DRGs)

General Facts

The Division of Health Economics has a long tradition regarding the development and improvement of the Austrian hospital financing system (Leistungsorientierte Krankenanstaltenfinanzierung, LKF). It was established in 2004 on the initiative © British Medical Journal of Professor Karl Peter Pfeiffer, following the former Ludwig-Boltzmann-Institute of Epidemiology and Health System Research. In 2009, the division joined the European Fig. 1: Number of DRGs and relative price range for three episodes of care in 11 countries. Union funded EuroDRG (diagnosis related The length of the bars indicates the range of the price index, which compares country spe- groups in Europe: towards efficiency and cific DRG weights (relative weights, tariffs, or scores) with the weight of an index DRG quality) project. Karl Peter Pfeiffer, Conrad (price index = 1) for the episode of care (into which a standard case without complications Kobel and Caroline Linhart were the core would be classified). The size of the circles represents the number of DRGs used to classify researchers in this project. patients.

74 Research Report 2015 Medical University of Innsbruck Health Economics

Fig. 2: Graphical illustration of the distribution of DRGs into MDCs and partitions in different systems. The columns represent the DRG systems. The wider a column, the higher the total number of groups of this DRG system is in comparison to the others. The rows represent the MDCs, labelled alternately on both sides of the figure. Medicare Severity (MS)-DRGs served as the reference for this comparison. The higher a cell, the higher the share of groups in this system’s MDC is. For example, the column representing the GHM system is more than three times wider than the column representing the AR-DRG system. In addition, the differently coloured parts within MDCs of every system show the distribution of cases into medical, surgical and other partitions.

They are built around patient classification and less complex cases, and a slightly high- Selected Funding systems, i.e. DRG systems, which classify er proportion of surgical DRGs. EuroDRG, Diagnosis related groups in Europe: towards efficiency hospital cases into DRGs on the basis of Our research focus was to compare simi- and quality, EC, Contract/Grant agreement number: 223300, Karl classification variables such as diagnoses, larities and differences across systems in Peter Pfeiffer treatments, and demographic characteris- Europe in comparison to the Austrian LKF Collaborations tics. DRGs condense the confusingly large system. We aim to provide the scientific • Andrew Street, Anne Mason, Padraic Ward, James Gaughan, number of different patients treated in foundation for the further development Silvio Daidone, Centre for Health Economics, University of York, hospitals into a manageable number of (a) of the LKF system, delivering empircal re- York, England clinically meaningful and (b) economically search results on the consequences of cer- • Mona Heurgren, Lisbeth Serdén, Mats Talbäck, National Board of Health and Welfare, Stockholm, Sweden homogenous groups. tain system characteristics. • Martine Bellanger, Josselin Thuilliez, National School of Public In all systems, DRGs are organized into Ma- Health, Paris, France jor Diagnostic Categories (MDCs) or similar • Siok Swan Tan, Leona Hakkaart-van Roijen, Ken Redekop, In- stitute for Health Policy & Management, Erasmus Universitair categories, and a distinction is made be- Medisch Centrum Rotterdam, The Netherlands tween surgical and medical cases, which Selected Publications • Francesc Cots, Pietro Chiarello, Xavier Salvador, Xavier Castells, Parc de Salut Mar, Barcelona, Spain are again separated into different partitions. Diagnosis related groups in Europe: moving towards transparency, • Zeynep Or, Thomas Renaud, Institute of Research and Informa- efficiency, and quality in hospitals? In addition, most systems attempt to distin- tion on Health Economics, Paris, France Busse R, Geissler A, Aaviksoo A, Cots F, Häkkinen U, Kobel C, • Maria Swiderek, Katarzyna Czach, Katarzyna Wiktorzak, Agata guish between patients with different levels Mateus C, Or Z, O’Reilly J, Serdén L, Street A, Tan SS, Quentin W. Szymczak, Katarzyna Klonowska, Paweł Sakowski, National He- BMJ. 2013;346:f3197. of complexity or severity by further subdi- alth Fund, Warsaw, Poland viding (splitting) DRGs. However, the spe- Coronary artery bypass grafts and diagnosis related groups: pa- • Unto Häkkinen, Mikko Peltola, Hanna Ratto, Kirsi Vitikainen, Na- cific design features of different countries’ tient classification and hospital reimbursement in 10 European tional Institute for Health and Welfare, Helsinki, Finland countries. • Jeni Bremner, Paul Giepmans, European Health Management systems as well as the dynamics of change Gaughan J, Kobel C. Health Econ Rev. 2014;4:4. Association, Brussels, Belgium remain relatively poorly understood. Refine- Why do patients having coronary artery bypass grafts have dif- • Reinhard Busse, Alexander Geissler, David Scheller-Kreinsen, ment of DRG systems over the past decade ferent costs or length of stay? An analysis across 10 European Wilm Quentin, Department of Health Care Management, Berlin countries. University of Technology, Germany or so has led to DRG systems with more Gaughan J, Kobel C, Linhart C, Mason A, Street A, Ward P, EuroDRG • Jacqueline O’Reilly, Brian McCarthy, Economic and Social Rese- DRGs, better distinction between complex group. Health Econ. 2013; Suppl 2:77–88. arch Institute Dublin, Ireland

Research Report 2015 Medical University of Innsbruck 75 Department of Pathology General Pathology

of oncology, surgery, radiology, nuclear as well as potential therapeutic targets. medicine, head & neck as well as cranio- Furthermore the function of the androgen maxillofacial surgery, dermatology and receptor and the role of stem cells, cyto other departments. kines and inflammation in tumor progression and treatment resistance are evaluated Research in close cooperation with the Department of Urology. Another field of interest is bladder Oncology cancer currently concentrating on bio Main topics are the diagnosis of rare tumor markers for risk, prognosis, resistance to entities, tumor biology and mechanisms BCG-therapy and the prevalence of HPV in- of treatment resistance, evaluation of bio- fection in superficial bladder cancer. markers to predict individual risk and prognosis, support diagnosis and assist in treat- In addition the biobank for frozen tissue ment allocation as well as identification of located at our division includes tissue potential therapeutic targets using molecu- samples from prostate cancer and other lar pathological methods. Current projects urological tumors (kidney cancer, bladder deal with the early detection of lung cancer, cancer). For research purposes, samples of the role of molecular pathology for therapy other malignancies, such as breast cancer, Head of Division (interim): in lung cancer patients and potential prog- are also stored there. ao. Univ.-Prof.in Dr.in Bettina Zelger nostic markers in pancreatic carcinoma. Hematopathology Contact: The research groups for uropathology, The main field of interest is the pathology Müllerstraße 44 hematopathology and cranio-maxillofacial of malignant lymphomas, focusing on the 6020 Innsbruck surgery represent a particular field of microenvironment and the role of auto interest at our division and are therefore phagy in multiple myeloma as part of [email protected] highlighted separately. the European Union Seventh Framework Phone: +43 512 9003 71301 Programme (FP7/2007–2013 OPTATIO Fax: +43 512 9003 73301 Uropathology under grant agreement no. 278570), the https://www.i-med.ac.at/pathologie/ The main topic in the field of uropathology is expression of PD1 and PDL1 in multiple prostate cancer focusing on tumor biology myeloma (Fig. 2) and the morphology, im- (Fig. 1), mechanisms of treatment resist- mune phenotype and molecular pathology ance, evaluation of biomarkers for diagno- (MYD88 and CXCR4 mutations) of lympho Keywords sis, risk prognosis and therapy allocation plasmacytic/plasmacytoid lymphomas.

Oncology, uropathology, hematopathology, gastrointestinal pathology, oropharyngeal pathology, endocrine pathology, pathology of infections, immunology, transplantation

Research Focus

• oncology • immunology and transplantation pathology

General Facts

The Division of General Pathology focusses on diagnostic clinical pathology and is responsible for the routine pathological diagnosis of biopsies and surgical specimens obtained from most of the Clinical Departments of the Medical University of Innsbruck, with an emphasis on oncology, especially of the lymphatic tissue, the urogenital tract and oropharyngeal tumors. A biobank consisting of formalin-fixed paraffin-embedded material is located at our division, thus making us an important connecting link between basic science and clinical research. This translational research is reflected by a close cooperation Fig. 1: Prostate cancer with loss of basal cells and expression of AMACR as well as a normal with clinicians and researchers in the fields prostate gland (double staining immunohistochemistry for p63 (brown) and AMACR (red); 40x)

76 Research Report 2015 Medical University of Innsbruck General Pathology

close cooperation with the Department of Endocrinology, Gastroenterology and Metabolic Diseases and the evaluation of new methods of detection of Mycoses from blood and tissue using PCR based methods and molecular imaging techniques (MALDI-MS) in cooperation with the Depart- ment of Hygiene, Microbiology and Social Medicine as well as the Institute of Forensic Medicine.

Neuropathology Fig. 2: Bone marrow with multiple myeloma The Division of Neuropathology is currently and expression of PDL1 (double staining im- vacant. Routine diagnosis of neurological munohistochemistry for MUM1 (brown) and diseases and specific tumors in tissue spec- PDL1 (red); 40x). imens of human brain are currently made in close cooperation with Prof. J. Hainfellner, Current projects also deal with aggres- Institute of Neurology, Medical University of sive B-cell-lymphomas including micro- Vienna (MUV). environment, potential prognostic bio- Latest research projects in cooperation markers such as C-MYC translocations, and with the Department of Neurology and therapeutic targets such as PD1 and PDL1. Neurosurgery focusing on the morphological and immunohistochemical evaluation of In addition, a wide number of clinical the infiltrative zone of brain metastases, the studies are provided with tumor samples consequences of anterior cervical discecto- from our bio-bank of paraffin-embedded my on patients with cervical herniation and tissue specimens. the expression of somatostatin receptor in meningioma are supervised at the Division Cranio-Maxillofacial Pathology of General Pathology. This project aims at the application and integration of clinical, molecular patholog- Selected Publications ical, molecular imaging (MALDI-IMS, FTIR ERG rearrangement prevalence in prostate cancer: higher imaging and µCT), bioinformatics and pro- frequency in young age and in low PSA prostate cancer. Schaefer G, Mosquera JM, Ramoner R, Park K, Romanel A, Steiner E, tein identification technologies to identify Horninger W, Bektic J, Ladurner-Rennau M, Rubin MA, Demichelis molecular signatures allowing the stratifi- F, Klocker H. DISTINCT PROSTATA CANCER AND PROSTATIC DISEASES. 2013;16:132–138. cation of patients who are susceptible to Clinical presentation of cutaneous adnexal tumors. Zelger B, curative treatment of oral squamous cell Kazakov DV, Zelger BG. PATHOLOGE. 2014;35(5):487–96. carcinoma. Patient samples that have been Loss of membranous expression of the intracellular domain of and are still being collected at the Depart- EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer. Fong D, Moser P, Kasal A, Seeber A, Gastl ment for Cranio-Maxillofacial and Oral Sur- G, Martowicz A, Wurm M, Mian C, Obrist P, Mazzoleni G, Spizzo G. gery and at the Department for Pathology HISTOPATHOLOGY. 2014;64(5):683–692. (biobank) will be systematically collected, Improved accuracy of discrimination between IgM Multiple Myeloma (MM) and Waldenstroem’s Macroglobulinaemia (WM) by dissected, and prepared to be accessible testing for MYD88 L265P mutations. Willenbacher W, Willenbacher for this study. E, Brunner A, Manzl C. BR J HAEMATOL. 2013;161(6):902–904. The immunology of fibrosis. Wick G, Grundtman C, Mayerl C, Wimpissinger TF, Feichtinger J, Zelger B, Sgonc R, Wolfram D. Infectiology, Immunology and ANNU REV IMMUNOL. 2013;31:107–135. Transplantation The main topics include composite allograft Selected Funding The spectrum of B-cell-neoplasias with plasmacytic/plasmacytoid transplantations, limb transplantation in differentiation in bone marrow: Diagnostic workup for rational animal experiments, hand transplantations treatment allocation; Medizinischer Forschungsfond Tirol (MFF); Dr. Ella Willenbacher (Hematology)/Ass. Prof. PD Dr. Andrea of patients at the Innsbruck Medical Univer- Brunner-Véber (Pathology) sity with a special emphasis on morphol- The Role of HPV infection regarding non-muscle invasive bladder ogy and standardized diagnosis, immune cancer; Medizinischer Forschungsfond Tirol (MFF); Dr. Renate phenotype of the inflammatory infiltrate Pichler (Urology), Ass. Prof. PD. Dr. Andrea Brunner Véber (Pa- thology) and therapeutic approaches resulting from Multidimensional Approach of Spectra and Morphology of Oral this knowledge. Also samples from human Squamous Cell Carcinoma; Land Tirol; Prof. Christian Huck and animal tissue (rat and mouse models) (Analytische Chemie, Leopold Franzens Universität)/ao. Univ.- Prof. Dr. Bettina Zelger (Pathology) will be stored for future research. Collaborations Further fields of ttentiona are the immu- • Prof. Dr. Martina Prelog, Department of Pediatrics, University of Würzburg, Würzburg, Germany nology and new therapeutic approaches • Prof. Hainfellner/Dr. A. Wöhrer- Klinisches Institut für Neuro in chronic inflammatory bowel disease in logie, AKH Wien, AT

Research Report 2015 Medical University of Innsbruck 77 Institute of Legal Medicine Legal Medicine

Research international police organizations and judi- cial systems (e.g. FBI) to generate forensic Forensic Molecular Biology evidence and to serve as a basis for internal Walther Parson recommendations.

International Mitochondrial DNA mtDNA Next Generation Sequencing Reference Laboratory Nuclear DNA (nDNA) analysis on the human EMPOP (European forensic mtDNA remains of the 43 Mexican students missing population database) resulted in only one identification that was In the past 20 years forensic molecular bi- reported in December 2014. The remaining ology has faced substantial technological 16 specimens did not contain enough intact developments. Short Tandem Repeat (STR) nDNA to allow further identification. Mito- loci have become the golden standard for chondrial DNA (mtDNA) analysis is often (human) identification in crime case, ID and applied in cases where nDNA fails to pro- paternity case work. Major developments vide results. But in this specific case quan- have been witnessed in the field of the uni- tification of mtDNA in the 16 unidentified parentally inherited genomes mitochondrial remains was also negative. The results sug- and Y-chromosomal DNA. Also, the applica- gest that the excessive heat has destroyed Head of Institute: tion of X-chromosomal markers has found the nuclear and mitochondrial DNA in the o. Univ.-Prof. Dr. Richard Scheithauer a vital niche in forensic DNA analysis. The remains at least to an extent, that the con- Innsbruck Institute of Legal Medicine took ventional methods applied so far cannot be Contact: responsibility in setting up the “EDNAP used for successful analysis. A novel tech- Müllerstraße 44 forensic mtDNA population database” nology termed “Massively Parallel Sequenc- 6020 Innsbruck (EMPOP), a new concept that addresses ing” (MPS) could serve as a useful tool to the necessary quality standards criteria for further investigate these remains. MPS has [email protected] data generation, analysis and transfer quali- a couple of advantages over conventional Phone: +43 512 9003 70600 ty control. Mitochondrial DNA (mtDNA) has DNA methods, including an increased suc- Fax: +43 512 9003 73600 the appealing characteristics of a multi- cess rate when analysing severely degraded www.gerichtsmedizin.at copy target molecule under strict maternal DNA. This technology is currently evaluated inheritance that makes it an informative for its application in forensics to identify marker for forensic, population and medical further remains. The Institute of Legal Med- genetic investigations. Research on mtDNA icine at the Medical University of Innsbruck, at the Institute of Legal Medicine Innsbruck has over three years of experience and is goes back to the mid-nineties, where basic therefore pioneering research with MPS protocols and population studies in the hu- technology. Experimental data on test sam- man field and species-specific identification ples demonstrate that the new sequencing methods were introduced. Reference mtD- method produces useful results. In a case, NA database is used by international labora- where conventional DNA analysis failed, ap- tories for forensic and population/medical plication of MPS technology would be a last genetic research. In addition it is used by attempt.

Keywords

Forensic medicine, human identification, mitochondrial DNA, EMPOP, Y-STR, pharmacogenetics, drug monitoring, spectrometry

Research Focus

• Analysis of Short Tandem Repeat (STR) loci, mitochondrial and Y-chromosomal DNA on archaeological samples. Develop- ment of molecular photo fitting for prediction of the geographical source as well as physical traits of an individual. • Qualitative and quantitative analysis of small bioorganic molecules. Common targets are drugs, pharmaceuticals, endo genous compounds, and metabolites Fig. 1: Eric Pokorak (Unit Chief, Mitochondrial DNA Unit, FBI) & Douglas Hares (Custodian thereof included in all kinds of biological of the US National DNA Database, FBI) visit the Institute of Legal Medicine (Prof. Richard samples (e.g. biofluids, cells, tissues). Scheithauer, Prof. Walther Parson) for collaboration on mtDNA interpretation.

78 Research Report 2015 Medical University of Innsbruck Legal Medicine

Fig. 2: Dipl-Ing.(FH) Kathrin Arnhard, Prof. Herbert Oberacher and Julia Steger at the lab of the core facility.

Forensic Archaeology edge algorithms and software tools for Studying the Reducing Potencies of Antioxidants with the Elec- trochemistry Inherently Present in Electrospray Ionization-Mass The institute has a long standing tradition small molecular identification via automat- Spectrometry. Plattner S, Erb R, Chervet J-P, Oberacher H. in being repeatedly consigned to handle in- ed interpretation of tandem mass spectral ANALYTICAL AND BIOANALYTICAL CHEMISTRY. 2014; 406: 213–224. ternational requests on DNA fingerprinting data. Developed and validated assays find Concept for estimating mitochondrial DNA haplogroups using a such as the DNA identification of the Asian applicability in the comprehensive analysis maximum likelihood approach (EMMA). Tsunami-victims, the remains of the Russian of complex biological samples as part of the Röck AW, Dür A, van Oven M, Parson W. FORENSIC SCI INT GENT. 2013; 7(6): 601–609. Tsar family or historical cases such as the service provided by the Core Facility Meta Comparison of morphological and molecular genetic sex-typing putative Mozart skull and the remains of bolomics. on mediaeval human skeletal remains. Friedrich Schiller. Recently, Walther Parson The mission of the Core Facility Metabolo- Bauer CM, Niederstatter H, McGlynn G, Stadler H, Parson W. FORENSIC SCI INT GENT. 2013; 7(6): 581–586. has been invited to join the King Richard III mics is to serve as an enabling resource for Getting the Whole Picture: Adding Patient-Reported Outcomes identification research group as an expert research and development programs at the to Adjuvant Endocrine Treatment Evaluation in Premenopausal in mitochondrial DNA analysis. In this spe- Medical University of Innsbruck. We aim to Breast Cancer Patients. Oberguggenberger A, Goebel G, Beer B, Oberacher H, Meraner V, cific case reference samples were availa- provide expertise and state-of-the-art tech- Sztankay M, Sperner-Unterweger B, Zeimet AG, Marth C, Hubalek ble from two living individuals: Michael Ib- nologies for the qualitative and quantitative M, Holzner B. BREAST JOURNAL. 2014; 20: 555–557. sen and Wendy Duldig, who are 19 and 21 analysis of small bioorganic molecules. Selected Funding generations removed from King Richard III, Common targets are drugs, pharmaceuti- • Maximizing mtDNA Testing Potential with the Generation of respectively. Using genetic and non-genetic cals, endogenous compounds, and metabo High-Quality mtGenome Reference Data, NIJ mtDNA WGS 2011-MU-MU-K402 evidence thus strongly supporting, beyond lites thereof included in all kinds of biolog- • European Forensic Genetics – Network of Excellence, EURO- reasonable doubt, that the skeleton is Rich- ical samples (e.g. biofluids, cells, tissues). FORGEN FP7-SEC-2011-285487 • Application of forensic DNA fingerprinting for the investiga- ard III, which represents the eldest human tion of archaeological and modern human migration patterns identification case known to date. in a geographically defined Alpine population, FWF, Volders P22880-B12 Selected Publications • Simulation of redox processes involving nucleic acids, FWF, Pro- Bioanalytical Mass Spectrometry Group DNA Commission of the International Society for Forensic jekt P 22526-B11, 2010–2013 and Core Facility Metabolomics Genetics: revised and extended guidelines for mitochondrial DNA typing. Collaborations Herbert Oberacher Parson W, Gusmao L, Hares DR, Irwin JA, Mayr WR, Morling N, • Institute of Mathematics, University Innsbruck, Innsbruck, Pokorak E, Prinz M, Salas A, Schneider P M, Parsons TJ. Our research is interdisciplinary, focusing Austria FORENSIC SCI INT GENT. 2014; 13: 134–142. on different aspects of bioanalytical chem- • Institut Geschichtswissenschaften / Europäische Ethnologie, Evaluation of next generation mtGenome sequencing using the University Innsbruck, Innsbruck, Austria istry and its application in metabolomics, Ion Torrent Personal Genome Machine (PGM). • Zentralinstitut für Bluttransfusion und Immunologische Abtei- forensic science, medical and pharmaceuti- Parson W, Strobl C, Huber G, Zimmermann B, Gomes SM, Souto L, lung, Tirol Kliniken, Innsbruck, Austria • Armed Forces DNA Identification Laboratory, Rockville, USA cal research, as well as pharmacogenetics. Fendt L, Delport R, Langit R, Wootton S, Lagace R, Irwin J. FORENSIC SCI INT GENT. 2013; 7(5): 543–549. • Bundeskriminalamt Wiesbaden, Wiesbaden, Germany The analytical methods, on which our • Institut für Veterinärpathologie, University Giessen, Giessen, Identification of the remains of King Richard III. Germany research is mainly focused, are high- King TE, Fortes GG, Balaresque P, Thomas MG, Balding D, Delser • Department of Internal Medicine, Innsbruck Medical University, resolution bioorganic mass spectrometry, PM, Neumann R, Parson W, Knapp M, Walsh S, Tonasso L, Holt J, Innsbruck, Austria Kayser M, Appleby J, Forster P, Ekserdjian D, Hofreiter M, Schurer • Department of Gynecology and Obstetrics, Innsbruck Medical separation techniques as well as different K. NAT COMMUN. 2014; 5: 5631. University, Innsbruck, Austria • Department of Neurology, Innsbruck Medical University, Inns- kinds sample preparation techniques, such Evaluation of the Sensitivity of the “Wiley Registry of Tandem bruck, Austria as extraction, polymerase chain reaction or Mass Spectral Data, MSforID” with the “NIST/NIH/EPA Mass Spectral Library”. electrochemistry. Furthermore, we spend Oberacher H, Whitley G, Berger B. Core Facilities much effort on the development of cutting- JOURNAL OF MASS SPECTROMETRY. 2013; 48: 487–496. • Metabolomics

Research Report 2015 Medical University of Innsbruck 79 80 Research Report 2015 Medical University of Innsbruck Clinical Research Units

Research Report 2015 Medical University of Innsbruck 81 Center of Operative Medicine Visceral, Transplant and Thoracic Surgery

Sepsis and basic scientists. In a “bed to bench Treatment of abdominal sepsis with open and back” approach: complex treatment abdomen treatment and negative pressure. regimes and clinical trials can be further enhanced by molecular in-depth analysis. General Facts Furthermore, the research line is The Department of Visceral, Transplant, supplemented by development of proof of and Thoracic Surgery maintains not only concept trials employing a large variety of an internationally established high volume micro-surgically most challenging organ transplant program with transplantation and limb transplantation models in small as of all solid organs (kidney, liver, pancreas, well as large animals. small bowel, cluster and in cooperation with the Department of Cardiac Surgery Together with Prof. Jakob Troppmair as head heart and lung), as well as vascularised of the DSL research laboratory, senior staff composite allografts, but also covers as a surgeons and/or senior surgical residents Central Hospital with tertiary patient care investigate infectious, oncological and the entire field of general, visceral and transplantation-related topics in cell culture, thoracic surgery in adults and children. small animal models and specimens from clinical trials in collaboration with regional Director: Translational research takes place at or international research colleagues. Within Univ.-Prof. Dr. Dietmar the Department of Visceral, Transplant, a project, group leaders usually mentor Öfner-Velano, MAS, MSc, F.A.C.S. and Thoracic Surgery with its associated diploma students, who hereby have the Daniel Swarovski Research Laboratory. unique opportunity to develop not only Contact: Work is proceeding along three main axes, basic science but also (micro)surgical skills. Anichstraße 35 which cover the fields of main interest in 6020 Innsbruck transplantation, surgical oncology and Research infectiology, namely ischemia-reperfusion [email protected] injury, sepsis and metastatic disease. In Oncology – Metastatic Disease Phone: +43 512 504 22600 parallel to patient care with an extended Priv.-Doz. Dr. Alexander Perathoner, Fax: +43 512 504 22602 quality assurance program and risk Ass.-Prof. Dr. Florian Augustin www.chirurgie-innsbruck.at management, the Daniel Swarovski Oncological science is one of the most Research Laboratory (DSL) represents important and dynamic areas of surgical a high-end research unit which creates science. The Department of Visceral, Keywords a perfect symbiosis between clinicians Transplant and Thoracic Surgery is able to

Metastatic surgery, ischemia-reperfusion injury, sepsis

Research Focus

Oncology – Metastatic Disease • Clinical studies on surgical oncology including both retrospective single center studies and prospective international studies, according to the affected organs • Clinical and experimental studies on peritoneal carcinomatosis and metastastic disease

Ischemia-Reperfusion Injury in Organ Transplantation One main focus of our research is to gain a better understanding of the pathophysio logical mechanisms underlying ischemia- reperfusion (I/R) injury. I/R injury represents a threat, to which all transplanted organs are subjected in the process of transplantation and which is known to crucially influence graft and patient long-term Fig. 1: Development of transplant vasculopathy. Aortic grafts taken from a tetrahydro survival. Identification of the mechanisms biopterin-treated (group VIII) or untreated donors (IV), subjected to 24h cold ischemia time involved would not only help to better (CIT), and reperfused for 4 weeks. H&E (left column) and Elastica van Gieson (right column) understand this process but also to identify staining. (a+b) untreated with cold ischemia time, (c+d) tetrahydrobiopterin-treated with new treatment targets. cold ischemia time, (e) intimal thickening (μm). Results are expressed as mean ± SEM.

82 Research Report 2015 Medical University of Innsbruck Visceral, Transplant and Thoracic Surgery

offer complete surgical management of the whole spectrum of surgical malignancies from very frequent tumors such as colon cancer to very rare tumors such as peritoneal malignancies. Therefore, oncological research at the Department of Visceral, Transplant and Thoracic Surgery is also characterized by a broad field of variegated research topics according to the different affected organs (e.g. thyroid cancer, gastric cancer, lung cancer). Surgical science is typically dominated by clinical research: all patients with a malignant disease are registered in databases (e.g. Austrian HIPEC Registry) to allow periodic retrospective and prospective analyses. The ongoing surveillance of oncological patients also enables the Department to participate in important national (e.g. ABCSG 16/ SALSA Study and ABCSG 26/SOLE Study on extended endocrine therapy in breast Fig. 2: Capillary mesh of pancreata in dependence of donor treatment and donor genotype. cancer patients) and international studies Pancreata were taken from tetrahydrobiopterin-treated or untreated donors with the in- (e.g. international multicenter study on dicated genotypes, subjected to ischemia, and transplanted to wild type recipients of the surgical morbidity in lung cancer patients same background as the knockouts (n = 5 per group). The capillary mesh was stained by with VATS-lobectomy). The clinical research infusion with fluorescein labelled dextran and observed by confocal intravital fluorescence includes diagnostic tools (e.g. the diagnostic microscopy. A–D: nontransplanted organs of wild type, eNOS -/-, nNOS -/- and iNOS -/-. value of ultrasound in thyroid cancer) E–H: organs of untreated donors (wild type, eNOS -/-, nNOS -/- and iNOS -/-, respectively), as well as new treatment options (e.g. transplanted to wild type recipients, 4 h after reperfusion. I–L: organs of donors treated hyperthermic intraperitoneal chemotherapy with BH4 (wild type, eNOS -/-, nNOS -/- and iNOS -/-, respectively), transplanted to wild in patients with peritoneal carcinomatosis). type recipients, 4 h after reperfusion. © PLoS ONE. 9(11): e112570. doi:10.1371/journal.pone.0112570 Of course, experimental projects also play a very important role in oncological research at the Department of Visceral, Transplant at the Department of Visceral, Transplant Single center prospective analysis of and Thoracic Surgery: scientists in our own and Thoracic Surgery according to the cancer-associated cytokines in serum and or in cooperating laboratories (e.g. Daniel primary affected organs: peritoneal fluid of patients undergoing Swarovski Laboratory) work on various cytoreductive surgery (CRS) and projects, including studies on transcription Elastography predicts thyroid cancer: hyperthermic intraperitoneal chemotherapy factors (e.g. STAT-1 in gastric cancer and comparison of two methods (compression (HIPEC) for peritoneal surface malignancies colorectal cancer), adhesion molecules ultrasound elastography vs. acoustic as a potential tool for perioperative therapy (e.g. CD44v6 in patients with non small cell radiation force ultrasound elastography) monitoring. (Research Group Peritoneal lung cancer), cytokines (e.g. in peritoneal with respect to diagnostic value and Carcinomatosis) carcinomatosis) and numerous other feasibility. (Research Group Thyroid Cancer) proteins (e.g. lipocalin 2 in colon cancer Study on volatile organic compounds in non- patients). ABCSG Study 16/SALSA (Prospective, small cell lung cancer tumor tissue, aiming randomized, open-label, multi-center, to define tumor markers for monitoring of Given that the treatment of patients with phase-II-study evaluating the effect of a therapy and for early detection of recurrent metastatic disease has changed significantly secondary adjuvant endocrine therapy disease. (Research Group Lung Cancer) in the last years due to development of with anastrozole for 2 years vs. 5 years in multimodal therapies, the Department of patients with hormone-receptor-positive Ischemia-Reperfusion Injury Visceral, Transplant and Thoracic Surgery breast cancer after 5 years prior adjuvant ao. Univ.-Prof. Dr. Stefan Schneeberger, intends to establish this topic as a new endocrine therapy.) (Research Group Breast Ass.-Prof. Priv.-Doz. Dr. Manuel Maglione focus in surgical oncological science. The Cancer) Major advances in surgical techniques, anti aim of the Metastasis Research Group in biotic prophylaxes, preservation solutions the future will be to combine clinical and Nation wide survey in Austria: Is and immunosuppressive therapy, have experimental scientific projects in order laparoscopic adjustable gastric banding an elevated solid organ transplantation to the to improve understanding of metastatic underestimated risk factor for developing treatment of choice in patients suffering disease and treatment of patients with esophageal cancer? (Research Group from irreversible organ failure. metastases. Esophageal and Gastric Cancer) Currently, organ shortage as well as chronic The following list of different exemplary Expression of neutrophil gelatinase- allograft loss represent two major hurdles clinical and experimental studies displays associated lipocalin in colorectal cancer to further enhancing the success of solid the broad spectrum of oncological research (Research Group Colorectal Cancer) organ transplantation. For both, the limited

Research Report 2015 Medical University of Innsbruck 83 Center of Operative Medicine

tissue allotransplantation (VCA) has become a rapidly advancing field with more than 100 hand/forearm transplantations and 20 face transplantations carried out in transplant centres all over the world. We have recently established the first in-depth analysis of the tissue damage induced by I/R injury in rodent models. Employing electron microscopy, confocal microscopy and molecular analysis of tissue-infiltrating inflammatory cells and markers for tissue damage, we have identified the injury to individual tissues as well as the architecture of the graft. Further, novel solutions and techniques for rinsing and storage of the 0000000000000300 . tissue are currently being tested in order to enhance tissue conservation during the 1097/TP . process of transplantation and to allow for doi: 10

. prolongation of the ischemia time. Particular attention is paid towards preserving nerves and muscle, where the most significant damage has been established to occur: in Schwann cells and in subcellular components such as mitochondria. Building 2014 Oct 15;98(7):713-20 . on the experience from findings in organ transplantation, mechanisms proven to be relevant in preventing I/R injury may also

Fig. 3: A-D, TEM observations of the skeletal muscle and the sciatic nerve in saline-treated Another topic in our research unit is limbs on POD 10. Muscle (A, B): A, The ultrastructure of the muscle mitochondria (m) is well the analysis of the immunomodulatory preserved in both the controls and the 2-hr CI limbs, B, whereas in the 10-hr and 30-hr CI properties of tetrahydrobiopterin, a naturally group, a large fraction of the mitochondria is degenerated (asterisks). A and B: same magni- occurring essential co-factor structurally fication. Scale bar=0.5 µm. Nerve C and D: C, In the control, nerve fibers display a compact related to the vitamins folate and riboflavin. arrangement of their myelin lamellae around the axon. D, In all CI groups, however, either We could show in a pancreas transplantation vacuolization of the myelin sheath is seen or the myelin sheath has completely disappeared. model in mice that treating the donor C and D: same magnification. Scale bar=10 Km. µ-I, Confocal microscopy of the skeletal with exogenous tetrahydrobiopterin could muscle in saline-treated animals on POD 10. Staining of viable (green, SYTO 16) and avital effectively prevent lethal I/R injury in (red, PI) muscle cell nuclei was performed to further detect damage to skeletal muscle. E, the transplanted recipient. In addition Although only a slight loss of viable cell nuclei was observed in the anterior tibial muscle to its potent antioxidative properties, of 2 hr CI animals, compared to nontransplanted controls, viable muscle cell nuclei were tetrahydrobiopterin is also known as an significantly diminished in 10 hr (P=0.01) and 30 hr (P=0.003) CI animals. F and G, In essential co-factor for a set of 8 different nontransplanted controls and 2 hr CI animals, the majority of cell nuclei were vital (green). enzymes, including the three nitric oxide H and I, Prolonged CI (10 hr and 30 hr) led to a high number of avital cell nuclei in skeletal synthase (NOS) isoforms (neuronal, muscle, detected by PI staining (red). TEM, transmission-electron microscopy; CI, cold is- endothelial, and inducible). We have chemia; POD, postoperative days; PI, propidium iodide. identified neuronal nitric oxide synthase as a target for tetrahydrobiopterin treatment in the prevention of I/R injury. Whether potential use of available organs and Hence, prevention of I/R injury is probably the other tetrahydrobiopterin-dependent long-term graft failure, I/R injury plays an one of the most important goals in solid enzymes play immunomodulatory roles important role. organ transplantation. One of the long- will be the focus of future studies. Based standing points of interest of our research on these observations, current projects in Increasing both donor age and the incidence aims is to investigate ischemia/reperfusion this field focus on (1) prevention of severe of extended criteria donors (ECDs) provides injury following organ transplantation as well I/R injury in a brain death mouse model, us with organs which are more prone to as its long-term consequences. While the aiming to simulate the clinical situation of develop significant injury through I/R. As a phenotype of I/R injury has been quite well cadaveric organ donation; (2) the triggering consequence, and in order to avoid severe, established in solid organ transplantation, mechanisms of I/R injury in inducing irreversible I/R injury, these organs are no such detailed analysis is available for transplant vasculopathy and the potential more likely to be declined if a prolonged transplantation of vascularized composite role of tetrahydrobiopterin in preventing it, cold ischemia time is foreseen. Further, I/R tissue allografts such as the hand or the and (3) simvastatin, which is hypothesised to injury is known to crucially influence the face. While this field is relatively new, in prevent I/R injury by a tetrahydrobiopterin- development of chronic allograft rejection. the past 15 years vascularized composite mediated process.

84 Research Report 2015 Medical University of Innsbruck Visceral, Transplant and Thoracic Surgery

assays, RTqPCR, proteomics and gene chip Secondary Endpoints: 30 day hospital analysis, mediating the ultimate goal of mortality rate, ICU Stay , Hospital stay, eventually translating results into clinical VAC associated complications and overall reality. complication rate.

Research Focus Sepsis The study is approved by the local ethics Priv.-Doz. Dr. Reinhold Kafka-Ritsch committee and was initiated as a single The research on abdominal sepsis is center trial at the University Hospital integrated into the clinical routine of the Innsbruck, but is further planned to become department; the main focus is on develop- a multicenter trial with the surrounding ment of new strategies for the treatment of secondary hospitals. abdominal sepsis. The ongoing prospective randomized study is administered by our Fig. 4: (A) Expression of CD44v6 in a squa- study coordination office. mous cell carcinoma (SCC); tissue micro Selected Publications array (TMA) core overview, 80x magnifi- Abdominal sepsis with generalized peritoni Incisional hernia rate after open abdomen treatment with negative cation. (B) Expression of the receptor of tis is a life-threatening condition requiring pressure and delayed primary fascia closure. Brandl A, Laimer E, Perathoner A, Zitt M, Pratschke J, Kafka-Ritsch R. hyaluronic acid-mediated motility (RHAMM) immediate surgical intervention. Despite HERNIA. 2014; 18: p. 105–111. in a large-cell carcinoma (LCC); TMA core intervention, a high percentage of these Therapeutic Management and Outcome of Locoregional Recur- overview, 80x magnification. (C) Expression patients develop severe septic shock with rence After Curative Colorectal Cancer Therapy-a Single-Center Analysis. Kogler P, Kafka-Ritsch R, Sieb M, Sztankay A, Pratschke of CD44v6 in the above SCC case; 320x mag- multi-organ dysfunction. At the time of J, Zitt M. JOURNAL OF GASTROINTESTINAL SURGERY. 2014; 18: nification. (D) Expression of RHAMM in the emergency laparotomy, patient recovery is p. 2026–2033. above LCC case; 320x magnification. uncertain and stabilization of the patient in Up-regulation of Neutrophil Gelatinase-Associated Lipocalin in Colorectal Cancer Predicts Poor Patient Survival. Maier HT, Aigner the intensive care unit is recommended. We F, Trenkwalder B, Zitt M, Vallant N, Perathoner A, Margreiter C, A further topic in our research unit is the have developed a damage control concept Moser P, Pratschke J, Amberger A. protective role of hemeoxygenase-1 (HO‑1) using abdominal vacuum therapy to treat WORLD JOURNAL OF SURGERY. 2014; 38: p. 2160–2167. Crucial role for neuronal nitric oxide synthase in early microcir- and biliverdin. HO-1 is the rate-limiting patients’ abdominal sepsis. The primary aim culatory derangement and recipient survival following murine step in conversion of heme into biliverdin, of this concept is to enhance recovery and pancreas transplantation. Cardini B, Watschinger K, Hermann M, 2+ Obrist P, Oberhuber R, Brandacher G, Chuaiphichai S, Channon carbon monoxide and free iron (Fe ). Up- allow bowel reconstruction in a second-look KM, Pratschke J, Maglione M, Werner ER. regulation of HO-1 has been described operation. PLOS ONE. 2014; 9: p. e112570. during acute and chronic rejection as well Histomorphometric Evaluation of Ischemia-Reperfusion Injury and the Effect of Preservation Solutions Histidine-Tryptophan- as I/R injury, and has been considered To prevent retraction of the fascia and Ketoglutarate and University of Wisconsin in Limb Transplanta- to act as a cytoprotective enzyme. HO-1 enhance the possibility of direct closure tion. Hautz T, Hickethier T, Blumer MJF, Bitsche M, Grahammer heme degradation products act strongly of the abdominal wall, we combine use of J, Hermann M, Zelger B, Messner F, Pechriggl EJ, Krapf C, Kimelman M, Brandacher G, Lee WPA, Margreiter R, Pratschke J, anti-oxidatively, and this seems to be the negative-pressure therapy with dynamic Schneeberger S. crucial aspect in prevention of I/R injury sutures to the fascia. Moreover, our studies TRANSPLANTATION. 2014; 98: p. 713–720. by HO-1 induction. Hence, activating this aim to investigate the long-term outcome of Selected Funding pathway early after organ transplantation this patient population with special interest • RAF and BCL‑2 in the regulation of cell death under oxidative or even during organ recovery might in incisional hernia development. stress; Der Wissenschaftsfonds (FWF); MCBO ‑W01101; 2005– protect transplanted grafts from ischemia 2015; € 600,000; Univ.-Prof. Dr. Jakob Troppmair • Deutsche Forschungsgemeinaschaft (DFG); Auswirkung und reperfusion injury. Considering future At present we are performing a prospective Therapieversuche des Hirntodes in experimentellen und klinis- clinical applications, current projects aim randomized study on the surgical treatment chen Modellen; 2010–2014; € 399,398.06; Univ.-Prof. Dr. Katja Kotsch to target the HO-1 pathway in different of patients with colonic perforation and • Tiroler Zukunftsstiftung; MitoCom Tyrol; 2011–2014; small animal models by (1) application peritonitis, treating with a damage control € 169,943.50; ao.Univ.-Prof. Dr. Erich Gnaiger • Houskapreis B&C Privatstiftung; Houska-Preis 2011; MitoCom of nutraceuticals such as Ginseng and strategy and application of abdominal Netzwerk; 2012–2017; € 120,000; Resveratrol, which are known to induce vacuum therapy. ao.Univ.-Prof. Dr. Erich Gnaiger • Der Wissenschaftsfonds (FWF); Die Rolle von Lipocalin-2 als HO-1, and by (2) administration of the bile Marker und therapeutisches Target in der Nierentransplanta- pigment biliverdin, the degradation product The aim of the study is to demonstrate that tion; 2011–2014; € 165,350.20; ao. Univ.-Prof. Dr. Felix Aigner of heme, which is known to have important by using a damage control strategy with Collaborations anti-oxidative properties. topical negative pressure in patients with • Peter J. Friend, Rutger Ploegh, University of Oxford, Nuffield peritonitis caused by colonic perforation, a Department of Surgical Scieces, Oxford, UK Clearly, the backbone of the transplant faster recovery from sepsis and a higher rate • Keith M. Channon, University of Oxford, Division of Cardiovas- cular Medicine, Radcliffe Department of Medicine, Wellcome research unit consists of the various of colonic reconstruction can be achieved. Trust Centre forHuman Genetics, Oxford, UK established animal models, which closely Based on our published experience • Darius Mirza, University Hospitals Birmingham, Liver and Hepato-Pancreato-Biliary (HPB) Unit, Birmingham, UK resemble clinical everyday life. The with this damage control concept and • Gerald Brandacher, Andrew WP Lee, Johns Hopkins Medical experimental setting is completed by a reconstruction rate of 80 % in this life University, Department of Plastic Surgery, Baltimore, USA • University of Pittsburgh Medical Center (UPMC), Division of- morphological analysis, comprising intra threatening situation, we want to test this Plastic and Reconstructive Surgery, Department of Surgery, vital confocal fluorescence microscopy, concept in a prospective randomized trial. Pittsburgh, USA • Hans Schlitt, Universitätsklinikum Regensburg, Klinik und histopathology and immunohistochemistry, Primary Endpoint: Rate of patients having a Poliklinik für Chirurgie, Regensburg, GER electron microscopy, and also by reconstructed colon at discharge from the • Kurt Werner Schmid, Universitätsklinikum Essen, Institut für Pathologie, Essen, GER biomolecular methods including amongst hospital and at 6 months after emergency • Emmanuel Morelon, Hospices Civils de Lyon, Hospital Edouard others western blots, enzyme activity operation. Herriot – Transplantation, néphrologie et immunologie, Lyon, FR

Research Report 2015 Medical University of Innsbruck 85 Center of Operative Medicine Cardiac Surgery

Keywords allowing for a later application in diagnosis, prevention and treatment of patients. Tech- Myocardial infarction, hypoxia, angio niques cover areas of analytical chemistry, genesis, bypass graft biology, thoracic molecular and cellular biology, primary hu- aortic aneurysm, stem cells, pharmaco man cell culture, tissue and organ culture therapy, prevention, aging, risk factors studies, as well as patient-based studies.

Research Focus General Facts

The research strategy of the University Cli The University Clinic for Cardiac Surgery nic for Cardiac Surgery is split into two main aims to act as a modern cardiosurgical unit concepts and generally covers central as- with focus on surgical outcome, quality pects of cardiovascular surgery, medicine, control, and development of modern and biology. With application-oriented pro- surgical strategies combined with patient jects we seek i) to improve myocardial pro- well-being, and advanced training of staff. tection and regeneration after infarction, Permanent improvement in all areas of ii) to increase durability of coronary bypass activity of our department is our maxim. grafts, and iii) to develop new tests allowing Activities in our department include patient Director: for the early diagnosis of and screening for care, academic teaching, education, and Univ.-Prof. Dr. Michael Grimm thoracic aortic aneurysms in blood samples. research. Core expertises of our clinic are: Several of these studies are done in cooper- Contact: ation with companies. With basic science i) coronary bypass surgery with focus on Anichstraße 35 projects we seek to define fundamental mo- aggressive use of bilateral mammary arter- 6020 Innsbruck lecular and cellular pathophysiological pro- ies, and minimally invasive robotic coronary cesses leading to cardiovascular diseases, surgery [email protected] Phone: +43 512 504 22501 Fax: +43 512 504 22502 http://herzchirurgie.tirol-kliniken.at

Fig. 1: Molecular modelling and docking of leoligin (isolated from Edelweiss) with HMG-CoA-reductase suggests an unconventional binding mode. Alignment of bioactive conformations of statin molecules bound to HMG-CoA-reductase (A). Chemical function- alities shared by statins (B). Leoligin mapped to the common feature statin model. All but two chemical features (red spheres) were mapped by the ligand, suggesting similar protein-ligand interaction profiles to those of the currently used statins (C). Simvastatin bound to HMG-CoA-reductase in the PDB entry 1hw9 (D). The statin forms hydrophobic contacts, hydrogen bond acceptors and donors, and a charged interaction with Arg590. Suggest- ed binding orientation of leoligin within the HMG-CoA-reductase active site (E). Similar to simvastatin, hydrophobic contacts and hydrogen bond networks also including Arg590 are formed. Interaction types are color-coded: red – hydrogen bond acceptor, green – hydrogen bond donor, yellow – hydrophobic, red star – negative charge. Cooperation with Dr. Daniela Schuster, University of Innsbruck.

86 Research Report 2015 Medical University of Innsbruck Cardiac Surgery

ii) valve surgery is performed – 85 % of all solated procedures – with minimally invasive access without compromise in outcome quality (evaluated by contribution to international multicenter studies). The use of transcatheter valves (TAVI) is successfully implemented (in high–risk patients we have an overall mortality of 6 % – the calculated risk is 29 % (logistic EuroScore)).

The program on minimally invasive mitral valve repair via anterolateral thoracotomy has a very strong position in our unit. This program is not only the biggest in Austria, but also gives Innsbruck a strong inter Fig. 2: Histological state of a venous bypass Fig. 3: This electron microscopic image national reputation. Approximately 80 % of graft prior to implantation into the arterial shows events occuring on the endothelial all suitable mitral repair procedures are per- system (mouse model). We are currently surface of an atherosclerotic plaque in formed using this technique. About 95 % of running a study on the pathophysiology of the mouse aorta in reponse to cadmium these specific patients received successful vein graft intimal hyperplasia. First results exposure. A clear retraction of endothelial repair of the native valve, which is remarka- indicate that the current view on the genesis cells can be observed, which leads to the bly high. The annual surgical training course of intimal hyperplasia may be wrong – our opening of gaps between these cells, result- “Focus Valve” is held in Innsbruck and has results may open totally new treatment op- ing in a loss of endothelial barrier function. become the most important meeting on tions. A significant acceleration of plaque develop- advanced techniques in minimally invasive ment and rupture is the consequence. valve surgery in Europe, with surgeons from more than 30 countries attending. vii) Our research laboratory team is treatments as well as the development of iii) In advanced life support, Innsbruck has multidisciplinary and in close contact with novel treatment options. established a local network for mechanical the clinical team. Regular meetings allow circulatory support and in acute cardiogenic for a perfect communication and exchange Myocardial Protection and Regeneration shock ECMO has become the primary care of knowledge between the laboratory and The induction of regenerative processes for these patients. clinics. Our work is published in the top and angiogenesis after myocardial journals of the field, and our science has led infarction (MI) is the central goal of most iv) Being the second largest program in to company cooperation and consultation experimental approaches aiming to improve Austria, we also run a heart and lung trans- support to the Austrian Ministry of Health the treatment of MI patients. To achieve plantation program with excellent outcome and the World Health Organisation. this goal the Cardiac Surgery Research (1–year survival rates above 90 % in heart Laboratory follows three major strategies: and above 80 % in lung transplantation). Research i) pharmacological, ii) physical, and iii) bio Currently we offer various types of mechani- logical stimulation of regeneration. cal circulatory assist devices to our patients As a central part of the Department of Car- (HeartMate II, Heartware; for BiVentricular diac Surgery, the Cardiac Surgery Research Many years of research in all of the Support: Thoratec, Berlin Heart (pediatrics), Laboratory covers: scientific consulting and above areas have led to significant pre– and Total Artificial Heart (SynCardia)). analytics for clinical trials, scientific educa- clinical success. One major innovative tion of surgical assistants, PhD and medical approach, which was discovered and v) With our complete spectrum of child PhD projects, application–oriented research established in the Cardiac Surgery heart surgery, in recent years we have and basic research. Since 2007, the labo- Research Laboratory, is a classical bench– treated more children with congenital heart ratory has been headed by a biologist and to–bed side approach. The basis of this disease than ever before, and with excellent pathophysiologist (Ass-Prof. PD. Dr. David approach i) (team Bernhard) was laid results. In years 2008 to 2012 we carried Bernhard) (with a 3 year break during which by a cooperation with the Department of out 364 cases with cardio–pulmonary he headed the Cardiac Surgery Research Pharmacy of the University of Innsbruck, bypass and more than 200 without. The Laboratory of the Medical University of which set out to find compounds that complexity (defined yb Aristotle score) of Vienna), and includes three teams (i.e. work stimulate angiogenesis. 5–Methoxyleoligin, our cases is gradually increasing, with very groups Bernhard, Holfeld, and Bonaros). a compound isolated from the roots of low mortality. The goal of the Department of Cardiac Sur- Edelweiss, showed potent effects in vitro. gery is to mirror clinical projects and stud- In vivo the effects were even more surprising, vi) In the thoracic aortic aneurysm program ies by laboratory based projects and vice as the compound not only stimulated we offer a full spectrum of modern aortic versa, to cover the full spectrum of bench– angiogenesis, but also arteriogenesis surgery. Central to this are the weekly to–bed side and bed side–to–bench options. (formation of larger blood vessels), and aneurysm clinics for thoracic aortic rescued myocardial muscle mass in the aneurysm held by our unit. Here we see Application‑Oriented Science infarction area, altogether increasing about 500 patients a year, predominantly The central task in this field of research cardiac performance by + 21 %EF (ejection originating from Western Austria. is the improvement of current surgical fraction) in pre-clinical trials.

Research Report 2015 Medical University of Innsbruck 87 Center of Operative Medicine

Approach ii) (team Holfeld) applies shock veins is significantly lower e.g. compared waves to the infarcted area of the heart. to internal mammary arteries. However, Shock wave therapy improved cardiac due to its good availability the saphenous performance (left ventricular ejection vein will remain an important option fraction) by + 18 % EF (ejection fraction). in bypass surgery. In order to increase This effect is mediated by the induction saphenous vein patency the team of the of angiogenesis and other regenerative Cardiac Surgery Research Laboratory has processes. The major advantage of shock in the past conducted a study aiming to find waves, compared to all other kinds of compounds that reduce intimal hyperplasia treatments, and due to its focused mode of (the first pathobiological step leading to application, is that it is almost free of side loss of patency) in saphenous veins in organ effects; it is cheap, and easy to apply. culture. The compound found, Leoligin, not only inhibited intimal hyperplasia in vitro, Approach iii) (team Bonaros) seeks to but also potently increased patency in vivo stimulate myocardial regeneration by stem (pre–clinically), without toxicity. cell therapy. As studies in the past showed only a minor effect of stem cell therapy in Importantly, a single intra–operative human myocardial infarction, due to the application of the drug was sufficient to rapid disappearance of stem cells from the block intimal hyperplasia. Large animal infarction zone (hours), the core and focus models are currently running and the of this approach is to increase the duration laboratory is currently starting cooperation

of residence and survival of stem cells in with a company to develop an intra– © whiley-vch the infarction area. operative storage solution (medical device) for venous grafts, which allows for the Fig. 5: Cover of a book on cigarette smoke Importantly, the above strategies can also extracorporeal treatment of venous bypass toxicity, a core expertise of the research be combined. Approaches i) and ii) are grafts. This approach is protected by a team. Knowledge about cigarette smoke tox- protected by patents, large animal studies patent. The next goal is to apply this new icity is mainly used for disease prevention have been conducted for ii) and are planned drug in the form of drug eluting stents. issues. This expertise is also supplied to the in the near future for i) and iii). Toxicological Austrian Ministry of Health and the World studies for i) have been performed and Basic Science Health Organisation. Currently the team is did not show toxicity. The compound for Apart from the goal of science in general, investigating e-cigarette toxicity on the car- i) is now available in a synthetic form to aquire and gain knowledge, the basic diovascular system. (cooperation with the Technical University science projects of the Cardiac Surgery of Vienna). For ii) and iii), initial clinical trials Research Laboratory also aim to establish basis in cell biology. To do so, cell isolation have been performed. scientific based knowledge for future and culture methods were established, application–oriented studies and the and cell populations were expanded and Bypass Surgery (Team Bernhard) development of novel therapies. analyzed. The Vena saphena is used as blood vessel in about 50 % of all cases of bypass surgery. Thoracic Aortic Aneurysms One of the most significant findings, However, the patency of the saphenous Team Dumfarth/Schachner/Bernhard presented in the first ublishedp study of the As a laboratory–based mirror of the team, was that smooth muscle cells in the clinical Aortic Competence Center of the TAA aorta are aged cells, with significantly Departments of Cardiac Surgery, Vascular shortened telomeres, and with reduced Surgery and Radiology, the Cardiac Surgery metabolism, proliferation, and migration Research Laboratory has a major focus on rates. As these cells are generally difficult studies of the thoracic aortic aneurysm to expand, due to their aged phenotype, it is (TAA). The primary goal is to understand planned to set up a bio–bank, allowing us to the pathogenesis of the TAA. In the past continually acquire cells. many studies have been undertaken to address this issue. However, up to now, TAA Future research will include studies pathophysiology remains largely enigmatic. of smooth muscle cells, fibroblasts, endothelial cells, other cell types in the TAA A major problem in this field of research is vessel wall, and patient serum. Major areas the lack of proper in vivo models, as well as of study include: aging processes, and the of human samples showing early stages of correlation of cell culture findings with the diseases. Further, almost all studies in histo– and immunohistology, and evaluation Fig. 4: A histological section of Apo E mouse the past worked with total aortic wall tissue of patient data on progression and outcome. heart, stained with Masson Trichrome (protein and nucleic acid studies, as well as The goal: to reveal the driving forces in TAA Goldner. Animals were exposed to high cho- histo- and immunohistology). The Cardiac formation and progression and to use this lesterol for 12 weeks. Blue color indicates Surgery Research Laboratory is one of new knowledge for disease detection and fibrotic tissue/collagen, red is muscle, and the very few teams in the world to have to monitor progression (biomarker), and for brown-black indicates nuclei. expanded the study of TAA to include its prognosis and treatment.

88 Research Report 2015 Medical University of Innsbruck Cardiac Surgery

Search for Unknown Risk Factors for that occur after myocardial infarction. Collaborations Cardiovascular Diseases The intention is to define more precisely • Dr. Adam Cdsordas, Cardiology Zürich, Switzerland • Dr. Alexander Egger, Austria Drug Screening Institute, Inns- Team Bernhard the physiological response in the area bruck, Austria Based on solid estimates the reason (risk affected by myocardial infarction, and • Prof. Christian Hartinger, Oragnic Chemistry, Auckland Univer- sity, New Zealand factors) for about 25 – 50 % of cardiovascular to define adverse and beneficial effects. • Dr. P. Paulus, Department of Anesthesiology, Goethe-University diseases are unknown to date. In order to The major focus will be on mitochondrial Frankfurt, Germany • Prof. Hermann Stuppner, Institute of Pharmacy, University of define such unknown risk factors the team metabolism, a key factor in the redox state Innsbruck of the Cardiac Surgery Research Laboratory (mitochondria make up 1/3 of the total • Prof. Xing Li Wang, Texas Heart Institute, Houston, Texas, USA conducted studies in the past, and was able mass of a cardiomyocyte), the relevance to define two totally novel risk factors, which of inflammation (physiological intensity are relevant to the general population. In of inflammation, and pharmacological several studies we could show that even modulation), as well as differentiation and slightly increased levels of serum cadmium dedifferentiation processes occurring increase the risk for cardiovascular diseases after myocardial infarction. The first (early atherosclerosis). planned step is to identify the physiological processes and later to interfere with them Our team was the first to show this inter and to analyse the signaling transduction relation in a small human study (cooper pathways involved. The techniques used ation with the Department of Neurology, will involve high resolution respirometry, Medical University of Innsbruck). Further, and histological, immunohistological, and the pathobiology of cadmium–induced molecular biological techniques. The results atherosclerosis could largely be elucidated. may form the basis for novel therapeutic In recent years, large epidemiological strategies. Finally, it is important for us to studies (up to 10,000 individuals), as well mention that almost all of the above projects as prospective cohort studies, confirmed are not seen in an isolated manner but form our finding. Currently, two cadmium studies a unit. A good example for this statement is are ongoing. Ultimately, we hope that our the fact that cigarette smoking is the major results will contribute to a reduction in source of cadmium uptake by humans, acceptable exposure levels for humans. The and is one of only two well defined risk second risk factor that could be defined factors for the thoracic aortic aneurysm. is lead (Pb). Similarly to cadmium, slightly The connected scientific projects should increased serum levels of Pb also increase – in our view – connect molecules to cells, the risk for early atherosclerosis. to tissues, to organs, to the organism, and ultimately to the healing of patients. Pathophysiology of Cardiovascular Disease Risk Factors Team Bernhard Selected Publications Another major area of research during Bovine aortic arch: predictor of entry site and risk factor for the past years was analysis of the patho neurologic injury in acute type a dissection. Dumfarth J, Plaikner M, Krapf C, Bonaros N, Semsroth S, Rizzo JA, Fang H, Grimm M, biology of the cardiovascular disease risk Elefteriades JA, Schachner T. factor smoking. Despite its extremely high Ann Thorac Surg. 2014 Oct; 98(4):1339–46. doi: 10.1016/j.atho- racsur.2014.05.086. Epub 2014 Aug 20. relevance for diseases such as cancer, Low energy shock wave therapy induces angiogenesis in acute chronic obstructive pulmonary disease, hind-limb ischemia via VEGF receptor 2 phosphorylation. Holfeld and cardiovascular diseases, the patho J, Tepeköylü C, Blunder S, Lobenwein D, Kirchmair E, Dietl M, Kozaryn R, Lener D, Theurl M, Paulus P, Kirchmair R, Grimm M. physiological processes induced and PLoS One. 2014 Aug 5;9(8):e103982. doi: 10.1371/journal. aggravated by cigarette smoke were hardly pone.0103982. eCollection 2014. understood. The team of the Cardiac Epicardial shock-wave therapy improves ventricular function in a porcine model of ischaemic heart disease. Holfeld J, Zimpfer D, Surgery Research Laboratory investigated Albrecht-Schgoer K, Stojadinovic A, Paulus P, Dumfarth J, Thomas cigarette smoke–induced atherosclerosis in A, Lobenwein D, Tepeköylü C, Rosenhek R, Schaden W, Kirchmair R, Aharinejad S, Grimm M. a large number of studies. A published book, J Tissue Eng Regen Med. 2014 May 19. doi: 10.1002/term.1890 and recent invitations to contribute reviews [Epub ahead of print]. in cardiovascular top journals on this issue, Smoking and cardiovascular disease: mechanisms of endothelial dysfunction and early atherogenesis. Messner B, Bernhard D. highlight the significant contribution of Arterioscler Thromb Vasc Biol. 2014 Mar; 34(3):509–15. doi: the team. Currently, the team is analyzing 10.1161/ATVBAHA.113.300156. potential toxic effects of e-cigarettes. Shockwave therapy differentially stimulates endothelial cells: implications on the control of inflammation via toll-Like receptor 3. Holfeld J, Tepeköylü C, Kozaryn R, Urbschat A, Zacharowski K, Physiological Regeneration after Grimm M, Paulus P. Inflammation. 2014 Feb;37(1):65–70. doi: 10.1007/s10753- Myocardial Infarction 013-9712-1. Team Bonaros/Bernhard Impact of cold ischemia on mitochondrial function in porcine he- A major task for the future of the Cardiac arts and blood vessels. Wiedemann D, Schachner T, Bonaros N, Dorn M, Andreas M, Kocher A, Kuznetsov AV. Surgery Research Laboratory is to study Int J Mol Sci. 2013 Nov 7;14(11):22042–51. doi: 10.3390/ and elucidate physiological processes ijms141122042.

Research Report 2015 Medical University of Innsbruck 89 Center of Operative Medicine Vascular Surgery

Keywords Research

Peripheral arterial disease, cerebrovascular Carotid Artery Disease disease, aortic aneurysm, aortic dissection, We study the influence of timing on the vascular trauma, venous disease, arterial outcome of carotid endarterectomy and and venous thrombosis carotid artery stenting in symptomatic patients. Historically it was recommended Research Focus to postpone carotid surgery in symptomatic patients for at least four to six weeks after • Treatment of patients with symptoma the qualifying neurological event, to prevent tic stenosis of the internal carotid artery cerebral bleeding. However, results from (comparison of carotid artery stenting the recent literature indicate that carotid (CAS) and carotid endarterectomy (CEA), endarterectomy is most effective when investigation of the influence of timing on performed earlier, to be precise: within two the outcome after CEA and CAS); weeks. The rationale for the diminishing • Risk factor assessment in patients with benefit of surgery in the latter period is that peripheral arterial disease and investiga- the first days after an initial neurological tion of cardiovascular complication rates; event carry the highest risk of stroke Director: • Vascular trauma including early and long- recurrence due to plaque embolization. For Univ.-Prof. Dr. Gustav Fraedrich term outcome, functional analysis and over a decade now, we have been following quality of life; the question whether CEA can be performed Contact: • Aortic dissection and factors associated safely in the early days after stroke onset, Anichstraße 35 with prognosis and outcome; and conclude that CEA is safe and most 6020 Innsbruck • Perioperative anticoagulation manage- effective when carried out rapidly after the ment including novel oral inhibitors onset of symptoms. [email protected] (NOACs). Phone: +43 512 504 22587 Carotid artery stenting (CAS) appeared as Fax: +43 512 504 22559 General Facts an alternative treatment technique in the www.gefaesschirurgie-innsbruck.at early 1990s. So far the safety and efficacy At the Department of Vascular Surgery we of CAS in comparison with CEA has not are especially interested in atherosclerosis been proven in patients with symptomatic and its different clinical manifestations: stenosis of the internal carotid artery. In peripheral arterial disease, cerebrovascular addition, the influence of timing on the disease and aortic aneurysms. outcome of CAS is not fully understood. Using the pooled analysis of all three Another research focus is carotid artery disease, its optimal treatment and novel diagnostic techniques. For many years now we have participated in this field in international trials comparing carotid artery stenting and carotid endarterectomy.

In addition, we elaborate on the optimal treatment of aortic dissection and on the natural course of this degenerative disease.

Furthermore we focus on all forms of vascular trauma and their optimal therapy. We assess the functional capacity of patients following a vascular trauma and whether the outcome can be positively influenced.

In the last years a variety of new anti coagulants have appeared and we now eva luate these in daily clinical routine.

We are especially interested in testing these novel anticoagulants in perioperative management. We focus on advantages and possible dangers in surgical patients and attach special importance to surgeries in Fig. 1: Sonography and intraoperative view emergency situations. of a symptomatic carotid stenosis

90 Research Report 2015 Medical University of Innsbruck Vascular Surgery

European trials (Carotid Stenting Trialists’ repair of arterial and venous trauma: peri- patients with complicated dissections were Collaboration, CSTC) which compared CAS operative mortality and analysis of factors analysed with special attention to limb and CEA, we could demonstrate that CAS associated with early limb loss, early and revascularisation and to renal and visceral is especially harmful in the early days after long-term outcome including patency of malperfusion. Patients who were treated a qualifying neurological event. During the the repaired vessel, long-term functional with Stentgrafts were evaluated with respect first 7 days CAS carries a fourfold risk of analysis and quality of life. In our institution, to the consequences of coverage of the periprocedural complication compared to trauma mechanisms most frequently left subclavian artery, such as neurological CEA (Fig. 2). include blunt injuries, in contrast to other complications including stroke and spinal centres which have a majority of penetra cord ischemia, and ischemic complications Perspectives: The pooled analysis of the ting traumata (e.g. stab- and gunshot in of the left upper limb. In addition, we CSTC and CREST (Carotid Revascularization juries). In blunt injuries, vascular injuries are compared outcome parameters in patients Endarterectomy versus Stenting Trial) data regularly associated with bone and nerve with aortic dissection to data from patients concerning the influence of the timing injuries, which lead to a worse functional with thoracic aortic aneurysms and of treatment is ongoing. The results are outcome in the long term. traumatic aortic injuries. expected during for the next few weeks. This is particularly important in the upper Peripheral Arterial Disease (PAD), the limb. Our research group analysed factors Selected Publications CAVASIC Study associated with poor functional outcome, The risk of carotid artery stenting compared with carotid endarterectomy is greatest in patients treated within 7 days of The CAVASIC study (Cardiovascular risk by use of standardised questionnaires to symptoms. Rantner B, Goebel G, Bonati LH, Ringleb PA, Mas JL, factors in patients with intermittent report on long-term quality of life, limb Fraedrich G, Carotid Stenting Trialists’ Collaboration. claudication) was initiated in 2002 to function and cold intolerance, which is J Vasc Surg. 2013 Mar;57(3):619–626. carry out prospective investigation of another common finding in this setting. High-sensitivity cardiac troponin T in patients with intermittent claudication and its relation with cardiovascular events and all- patients with intermittent claudication and Furthermore, iatrogenic injuries were cause mortality—the CAVASIC Study. Pohlhammer J, Kronenberg F, to assess their cardiovascular morbidity analysed, with special interest in access Rantner B, Stadler M, Peric S, Hammerer-Lercher A, Klein-Weigel P, Fraedrich G, Kollerits B. and mortality rates. The follow-up site complications occurring during vascular Atherosclerosis. 2014 Dec;237(2):711–7. examinations were finished in 2011, and interventional procedures. Access site Outcome after interposition of vein grafts for arterial repair since then different analyses on cardio complications are uncommon in institutions of extremity injuries in civilians. Klocker J, Bertoldi A, Benda B, Pellegrini L, Gorny O, Fraedrich G. vascular risk factors have been completed; with large numbers of invasive vascular J Vasc Surg. 2014; 59(6): 1633–1637. once again we could demonstrate that procedures, however they represent an Risk factors for mortality and failure of conservative treatment af- patients with PAD carry a significant risk of important issue. We were interested in ter aortic type B dissection. Grommes J, Greiner A, Bendermacher B, Erlmeier M, Belau P, Kennes LN, Fraedrich G, Schurink GW, cardiovascular complications. The number strategies to reduce such complications, Jacobs MJ, Klocker J. of cardiovascular complications has not such as the use of local compression and J Thorac Cardiovasc Surg. 2014; 148(5): 2155–2160. significantly decreased over the last years the additional use of Vascular Closure Ischemia and functional status of the left arm and quality of life after left subclavian artery coverage during stent-grafting of tho- despite intensive medical treatment. Devices (VCDs), and in techniques and racic aortic pathologies. Klocker J, Koell A, Erlmeier M, Goebel G, Survival following a myocardial infarction, outcome of surgical repair of access site Jaschke W, Fraedrich G. J Vasc Surg. 2014; 60(1): 64–69. however, is much better; PAD patients most complications. frequently die from malignancy. Collaborations Aortic Dissection • Carotid Stenting Trialists’ Collaboration • Dr. Peter Klein-Weigel, Klinik für Angiologie, Vascular Trauma Multicentric analysis of data including Klinikum Berlin-Buch,Germany Institutional characteristics (diagnosis data patients with Type B Aortic Dissection • PD Dr. Jochen Grommes,Department of Vascular Surgery, RWTH Aachen/Maastricht registry back to 1989; big Trauma Center) aimed to evaluate risk factors for planning • Prof. Dr. A Greiner, Department of Vascular Surgery, Charité allowed analysis of various aspects of the of individual treatment. In addition, Berlin

Fig. 2: The risk of stroke or death after treatment of symptomatic carotid stenoses by endarterectomy (CEA) or stenting (CAS), adopted from Rantner et al (2013).

Research Report 2015 Medical University of Innsbruck 91 Center of Operative Medicine Plastic, Reconstructive and Aesthetic Surgery

ital Deformities/Reconstructive Surgery Keywords and Wound management/healing are supported by the research laboratory unit head- Fat, adipose derived stem cells, wound ed by C. Ploner. The laboratory staff com- healing, immune cells, transplantation, prises 2 clinical PhD students, one technical congenital deformities, thoracic wall assistant (BMA) and 2 clinical researchers. deformities, cytokines, leukocyte trafficking Research Research Focus The Impact of Fat Tissue on Wound • Physiology of fat tissue resident mesen Healing and Tumorigenesis chymal stem cells and adipocytes and Christian Ploner their impact on wound healing Despite great advances in tissue-engineering • Repair of congenital thoracic wall defor of the skin, impaired wound healing still mities and consequence on cardiopul remains one of the most serious problems in monary function and quality of life plastic surgery. We are primarily interested • Transplantation Immunity: Discrimination in defining mechanisms of (chronic) wound of rejection and inflammatory processes healing, especially delineating the role Director: in the skin of the fat tissue in this complex cellular Univ.-Prof. Dr. Gerhard Pierer interplay. Disdained as a dispensable tissue General Facts that is necessary for energy storage only, Contact: recent findings strongly suggested that fat Anichstraße 35 VISION tissue is a remarkable source of cytokines, 6020 Innsbruck The ISO-9001:2008 certified research unit metabolites and hormones. In addition, of the Plastic, Reconstructive and Aesthetic fat tissue harbors a high number of easily [email protected] Surgery was established in autumn 2011, accessible, undifferentiated adipose- Phone: +43 512 504 22731 enabling dedicated doctoral researchers derived stem cells (ADSC) that are actually Fax: +43 512 504 22735 to follow clinical goals based upon sound tested in clinical applications, including www.plastischechirurgieinnsbruck.at scientific principles. Our vision is to apply wound-healing approaches (Fig. 1). www.pci.or.at basic research techniques to address clinical challenges and translate these However, the mere transplantation of these findings into new therapeutic approaches. cells into wound beddings only marginally enhanced the healing process, and cells AIMS embedded in engineered matrices stayed 1. Improve the understanding of cellular entrapped and impacted wound healing by processes in wounds, especially de- secretory action rather than by proliferation fining the role of fat tissue or differentiation. Fat tissue has a great impact on physiological and psychological processes in Therefore, we initiated a project focusing the human body. We address the question on wound matrix controlled molecular proof how cellular and secretory components cesses affecting the regenerative potential of the fat tissue influence regeneration. of distinct cutaneous (keratinocytes, fibro- 2. Surgical correction of congenital tho- blasts) and subcutaneous cell types (adipo- racic wall deformities and the impact cytes, ADSC). One of the most important it has on physical and physiological matrix components for dermal wound heal- health ing is fibronectin, a high molecular weight We address how surgery of chest wall glycoprotein, which is recognized by spe- deformities impacts cardiopulmonal and cific cell surface proteins of the integrin psychological parameters and quality of family, namely integrin alpha 5 and alpha V. Head of Research Laboratory: life. In a first line of experiments, we delineated Christian Ploner, PhD 3. Transplantation immunology: the function of alpha 5 and V integrins in re- Discriminating early skin rejection generative cell types, especially ADSC and Contact: from skin inflammation basal keratinocytes. By applying lentiviral Anichstraße 35 The aim is to identify specific biomark- transduction technology we found that high 6020 Innsbruck ers for early detection of skin rejection expression of these integrins favors prolifer- in composite allotransplantation prior to ation and migration over differentiation and [email protected] histological findings. that this integrin-dependent phenotype is Phone: +43 512 504 82648 mainly mediated by the PI3K/AKT pathway. Fax: +43 512 504 22735 STRUCTURE www.plastischechirurgieinnsbruck.at Three research groups, equivalent to the 3 In a second study, we investigate how tumor www.pci.or.at major units of the Department, i.e. the units cells from distinct origin (multiple myeloma, for Breast/Limb/Nerve-Surgery, Congen- breast cancer, prostate cancer or ovarian

92 Research Report 2015 Medical University of Innsbruck Plastic, Reconstructive and Aesthetic Surgery

allografts treated with tacrolimus [n=10], ods of corrective thoracoplasty have been applying the multiplex analysis technology described. There is a significant challenge (LuminexTM). We found that levels of IL-4, in thoracoplasty surgery, which emphasizes TNF -α and IL-12p70 correlated best with aesthetic restoration of large deformities, early skin rejection, whereas TNF -α and but should cause only low morbidity. As a IL-12p70 were specific for muscle tissue result, there has been an increase in the rejection. Importantly, all identified mark- number of patients seeking surgical cor- ers preceded histological alterations. To rection. The aim of our prospective study is differentiate between early rejection and to evaluate the effect of the PE and PC dean inflammatory skin process, we applied a formity itself and whether there is a change contact hypersensitivity (CHS) and delayed of pulmonary function and quality of life in type hypersensitivity (DTH) model in Lewis patients after surgical repair. In the study, , in preparation, 2015 © Morandi et al. , in preparation, rats (Fig. 2). CHS and DTH result in skin irri- all patients will undergo preoperative and tation and swelling histologically compara- postoperative evaluation with Computed To- Fig. 1: Multipotency of isolated primary hu- ble to grade I-II rejection in our rat allotrans- mography CT scan, pulmonary function test man ADSC. In vitro expanded ADSC (A) were plant model. Based on a multivariate linear and cycle ergometry in an upright, and fur- subjected to adipogenic (B), chondrogenic discriminant analysis, IL-12p70 and TNF -α thermore, in a supine position, as well as a (C) or osteogenic (D) differentiation medi- were approved as specific biomarkers for transthoracic echocardiogram. Additionally, um for 2 weeks (adipogenesis) or 3 weeks early skin rejection. all patients will undergo a pre- and postop- (chondrogenesis, osteogenesis), respectivly. erative standardized questionnaire (includ- Paraformaldehyde fixed cells were stained ing quality of life, patient satisfaction and for lipid-containing droplets (OilRedO, B), physical activity) and will be examined by a sulfated proteoglycans (Alcian blue, C) or professional psychologist. We hypothesize calcium depositions (Alizarin Red, D). that our results will provide evidence that pulmonary function is related to the depth cancer) enslave proximal mesenchymal of the depression or protrusion and proba- stem cells and adipocytes. We are bly causal for functional deficits, and might especially interested in the mechanism by explain why repair of the defect can result which tumor cell secreted cytokines affect in improved pulmonary function, exercise adipocyte differentiation and delipidation at tolerance and quality of life. the molecular level. In first experiments, we found that, dependent on the tumor origin, adipocyte differentiation is enhanced Selected Publications or diminished by specific cytokines, Differentiation between acute skin rejection in allotransplantation suggesting a differential role of adipocytes and T-cell mediated skin inflammation based on gene expression analysis. Wolfram D, Morandi EM, Eberhart N, Hautz T, Hackl in the development of the primary tumor. H, Zelger B, Riede G, Wachter T, Dubrac S, Ploner C, Pierer G, Schneeberger S. Biomed Res Int. 2015;2015:259160. Cytokine Patterns in Acute Skin Rejec- Adipocyte-derived players in hematologic tumors: useful novel tion and Inflammatory Skin Processes targets? Jöhrer K, Ploner C, Thangavadivel S, Wuggenig P, Greil R. Dolores Wolfram Expert Opin Biol Ther. 2015 Jan;15(1):61–77. Epub 2014 Oct 11. Acute skin rejection in vascularized com- Clinical results and patient satisfaction after pectus excavatum repair using the MIRPE and MOVARPE technique in adults – 10 posite allotransplantation (VCA) is the ma- © Dolores Wolfram et al. 2015, Vascularized Composite Allotransplantation years experience. Del Frari B, Schwabegger AH. jor obstacle for wider adoption in clinical PLAST RECONSTR SURG. 2013;132(6):1591–602. practice. Clinical success and outcome of Fig. 2: Representative picture of (A) a trans- Insights from computational modeling in inflammation and acute rejection in limb transplantation. Wolfram D, Starzl R, Hackl H, VCA often depends on exact timing and planted limb showing clinically Grade II Barclay D, Hautz T, Zelger B, Brandacher G, Lee WP, Eberhart N, the extent of the given immunomodulatory rejection characterized by erythema and Vodovotz Y, Pratschke J, Pierer G, Schneeberger S. therapy. Traditionally, this decision is made swelling. (B) DTH reaction on the planta PLoS One. 2014 Jun 13;9(6). upon histological appraisals focusing on pedis on the right paw presenting with Development of a multipurpose GATEWAY-based lentiviral tetracy- cline-regulated conditional RNAi system (GLTR). Sigl R*, Ploner inflammatory markers that hardly allow dis- localized erythema and swelling and the C*, Shivalingaiah G, Kofler R, Geley .S crimination between skin rejection and an control footpad on the left side without any PLoS One. 2014 May 19;9(5). inflammatory skin process. inflammatory reaction. Selected Funding • Clinical Research (KLIF), Austrian Science Fund (FWF), Barbara Therefore, we initiated a study to establish Cardiopulmonary Function after Chest Del Frari new biomarkers that enable this discrimina- Wall Deformity Surgery • Driving plasma cells MADi: the role of Myeloma–Adipocyte interaction in the progression and drug resistance of multiple tion before the process is manifested in an Barbara Del Frari myeloma, Standortagentur Tirol, C. Ploner & K. Jöhrer inflammatory response. Pectus excavatum (PE) and carinatum (PC) are the most common types of congenital Collaborations To start with, we screened for the expres- anterior chest wall deformities. The deform- • Prof. Fiona Watt, Kings College, London, UK • Prof. Ivan Martin, Department of Biomedicine, University Hos- sion levels of 14 inflammatory mediators ities often present not only as an aesthetic pital Basel, CH in skin and muscle biopsies from synge- disturbance, but also in association with • Prof. W.P. Andrew Lee, Johns Hopkins University, Baltimore, USA • Prof. Gerhard Brandacher, Johns Hopkins University, Baltimore, neic grafts [n=10], allogeneic transplants obstructive pulmonary mechanics and ab- USA without immunosuppression [n=10] and normal cardiac physiology. Various meth- • Ravi Starzl, PhD, Carnegie Mellon University, Pittsburgh, USA

Research Report 2015 Medical University of Innsbruck 93 Center of Operative Medicine Trauma Surgery

documentation unit, a biomechanics labo- Research ratory, a morphology and cell biology laboratory and the Core facility Micro CT. This Clinical Studies and Documentation setting supports clinical research, as wells Unit as laboratory research on macroscopic, mi- Mariette Fasser, MSc croscopic and cellular level. In recent years the focus of clinical studies in trauma surgery has no longer been Clinical research in Trauma Surgery limited to medical device investigations: represents a special challenge, because of due to growing experience and know-how, its high throughput of in- and out-patients an expansion to interdisciplinary projects and its wide range of treatment modalities. and further aims became possible. One The main research fields are medical device example is the EU-funded DO-HEALTH studies and investigator-initiated trials study which is focused on Vitamin D which explore new treatment methods. supplementation, Omega 3 intake and Our study coordinators (“study nurses”) home exercise as prophylactic measures support clinical staff in the organization and aiding healthy aging (study goals: to reduce administration of clinical trials, ensuring a risk of falls, to improve cognitive impairment complete collection and archiving of data and cardiovascular improvement). The Director: and patient follow-ups according to the involvement of the department in this Univ.-Prof. Dr. Michael Blauth “Good Clinical Practice” guidelines and project was a new approach and opened legal requirements. The clinical study centre doors for similar highly representative Contact: unit was certified in 2011 by the Clinical epidemiological projects. However, the Anichstraße 35 Research Organization “AO Foundation”. main research focus continues to be on 6020 Innsbruck The main collaborators are the Clinical Trial musculoskeletal topics, with investigations Center of the Medical University Innsbruck of trauma related research questions e.g. [email protected] and the AO foundation/Trauma. fixation of distal radius fractures, studies Phone: +43 512 504 22821 on proximal humerus fractures with focus Fax: +43 512 504 22824 In the biomechanics laboratory material on osteoporotic bone and loss of fracture www.unfallchirurgie-innsbruck.at testing machines are available for in vitro reduction implant failure and steps to testing of soft and hard biological tissues, prevent fixation failure. with several custom-made test setups for various anatomical regions as well as joint simulators for the spine, shoulder and hand. These allow in vitro functional evaluation of interventional surgical procedures for the stabilisation or reconstruction of joints as well as of soft and hard tissue. Keywords Research projects have been carried out in collaboration with the Dept. of Anatomy Clinical studies, biomechanics, fracture and Embryology, Dept. of Neurosurgery, fixation, cell biology, osteoporosis, geriatric Dept. of Craniofacial Surgery and Dept. of patients Orthopaedic Surgery.

Research Focus The main focus of the morphological/cell biological laboratory lies on basic research Research in the Department of Trauma into osteoporosis and its underlying Surgery focuses on the evaluation, mechanisms and the resulting stem cell development and improvement of new differentiation defects, as well as on and existing treatments and therapies for research on intervertebral discs. To conduct traumatic and degenerative musculoskeletal these studies a fully equipped tissue diseases, not exclusively, however, with culture unit is at our disposal for adult Surgery of Orthopedic and Trauma © Archives focus on geriatric patients. stem cell differentiation experiments and Fig. 1: Follow-up X-ray 1 year after an un- investigations on intervertebral disc cells. stable proximal femur fracture was treated General Facts In addition histological, ultrastructural and with an augmented PFNA nail, showing a biochemical analysis can be carried out. well-healed fracture and no signs of osteo In the clinical routine setting the depart- Research projects have been carried out necrosis of the femoral head. ment is organised into teams specialised in collaboration with the Dept. of Anatomy, in anatomical regions. The clinical patient Histology and Embryology, Dept. of Biomechanics Laboratory based research, as well as the applied and Therapeutic Radiology and Oncology, Dept. Werner Schmölz, Assoc. Prof., PhD, basic research of all the clinical teams at of Plastic-, Reconstructive- and Aesthetic Dipl.-Ing (FH) the department, is supported by an infra- Surgery, as well as with several groups of Implant anchorage in spinal stabilisation structure consisting of a clinical study and the CCB. procedures in patients having reduced

94 Research Report 2015 Medical University of Innsbruck Trauma Surgery

bone quality still poses a challenge to the Morphology and Cell Biology Laboratory discs (IVD) after burst fracture of surgeons. Therefore, a new test setup was Hannes L. Ebner, PhD the cervical spine. Specimens were developed which allows the application To optimise future treatment of osteo studied macroscopically, histologically, of physiologic pedicle screw loading and porosis, the potential of aminobisphos- and ultrastructurally to define healthy is capable of reproducing the mechanism phonates to enhance the development of cells, balloon cells, and disc cell death of pedicle screw loosening seen in clinical bone-forming osteoblasts from progenitor (DCD). There was a positive correlation practice. Various augmentation techniques cells was evaluated. The aminobisphospho- between DCD and absorbed energy in and materials as well as screw designs to nates investigated significantly enhanced all compartments of bovine discs. Both enhance pedicle screw anchorage were in- osteoblast formation and thus provide species showed similar patterns of DCD vestigated. It could be shown that augmen- further insights into their possible mode of in the different compartments as well as tation significantly improves screw anchor- action in the treatment of osteoporosis. similarities in cell morphologies and in age and while for PMMA as augmentation matrix damage. material the technique is only of secondary Adipose-derived stromal cells (ASCs) importance, for other augmentation mate- are increasingly being used for ortho rials e.g. silicon, the enhancement strongly paedic-based tissue engineering, due to depends on the augmentation technique their ability readily to undergo osteogenic applied. To improve instrumentation for differentiation. We used in vitro and in vivo cervical and lumbar spinal fusion proce- approaches to evaluate the use of ASCs dures, experiments were conducted in vitro as a treatment strategy for age-related to investigate the primary and secondary osteoporosis. When differentiated in stiffness of various supplementary instru- conditioned culture media harvested from mentations. Based on the results, recom- osteoporotic patient-derived human ASCs, mendations on the type of instrumentations osteoporotic patient-derived human bone and their stiffness were given. marrow stromal cells showed a significant improvement in their osteogenic potential. After surgical treatment of cruciate liga- These findings support the use of ASCs as ment injuries, graft elongation and graft an autologous cell-based approach for the © European Spine Journal fixation affect post-surgical joint stability. treatment of osteoporosis. Fig. 3: 3D reconstruction of a section from a Therefore, currently established and re mesenchymal stem cell, based on an elec- cently developed new graft preparation The behaviour of bovine disc cells, and tron tomogram, showing a single, branched, techniques, as well as femoral and tibial changes in disc matrix following in vitro threadlike mitochondrion with cristae, graft-bone fixation techniques, were in compression, were tested to compare the marked here in yellow, and mitochondrial vestigated in experiments in vitro. It could findings to data on human intervertebral membrane, in red. be shown that a graft preparation technique which had recently been developed for less Selected Publications screws under cranio-caudal cyclic loading. Bostelmann R, Keiler A, Steiger HJ, Scholz A, Cornelius JF, Schmoelz W. invasive surgery resulted in an increased The influence of Local Bone Density on the Outcome of One hund- Eur Spine J. 2015 Mar 27. graft elongation after surgical treatment. red and Fifty Proximal Humeral Fractures Treated with a Locking Plate. Kralinger F, Blauth M, Goldhahn J, Käch K, Voigt C, Platz A, Biomechanical comparison of vertebral augmentation with silico- Hanson B. ne and PMMA cement and two filling grades. Schulte TL, Keiler A, J Bone Joint Surg Am. 2014 Jun 18;96(12):1026–1032. Riechelmann F, Lange T, Schmoelz W. Long-term result of augmented PFNA: a prospective multicener Eur Spine J. 2013 Dec;22(12):2695–701. trial. Kammerlander C, Doshi H, Gebhard F, Scola A, Meier C, A full list of the publications and the currently running projects is Linhart W, Garcia-Alonso M, Nistal J, Blauth M. listed on the departmental website: Arch Orthop Trauma Surg. 2014 Mar;134(3):343–9. www.unfallchirurgie-innsbruck.at Osteoanabolic effect of alendronate and zoledronate on bone marrow stromal cells (BMSCs) isolated from aged female osteo- Selected Funding porotic patients and its implications for their mode of action in the DO-HEALTH, EU- 7th framework, Http://www.do-health.eu treatment of age-related bone loss. Lindtner RA, Tiaden AN, Genelin K, Ebner HL, Manzl C, Klawitter M, Sitte I, von Rechenberg B, Blauth M, Richards PJ. Collaborations OSTEOPOROSIS INTERNATIONAL. 2014; 25: p. 1151–1161. Clinical Investigation unit: Therapeutic potential of adipose-derived stromal cells in age-re- • AO Foundation/Trauma lated osteoporosis. Mirsaidi A, Genelin K, Vetsch JR, Stanger S, Morphology and Cell Biology Laboratory: Theiss F, Lindtner RA, von Rechenberg B, Blauth M, Müller R, Kuhn • Prof. Dr. med. B. von Rechenberg, University of Zürich, Zürich, GA, Hofmann Boss S, Ebner HL, Richards PJ. Swisse BIOMATERIALS. 2014; 35(26):7326–35. • Prof. Dr. med. P. Pietschmann, MedUni Wien, Wien, Austria • Priv. Doz. Dr. P. J. Richards, University of Zürich, Zürich, Swisse Morphological similarities after compression trauma of bovi- • Priv. Doz. Dr. G. Krumschnabel, Oroboros Labs, Innsbruck, Aus- ne and human intervertebral discs: Do disc cells have a chance tria of surviving? Sitte I, Kathrein A, Klosterhuber M, Lindtner RA, Biomechanics Laboratory: Neururer SB, Rauch S, Kuhn V, Schmoelz W. • Prof. Dr. med. Tobias Schulte, University of Münster, Germany J Orthop Res. 2014; 32(9):1198–207. • Prof. Ralph Müller, ETH, Zürich, Swisse Biomechanical comparison of 2 anterior cruciate ligament graft • Priv-Doz. Dr. med Heiko Koller, Bad Wildungen, Germany preparation techniques for tibial fixation: adjustable-length loop • Priv. Doz Dr. med Stefan Freude, University of Tübingen, Ger- cortical button or interference screw. Mayr R, Heinrichs CH, many Fig. 2: In vitro test set-up used to apply Eichinger M, Coppola C, Schmoelz W, Attal R. • Dr. med. Richard Bostelmann, University of Düsseldorf, DE physiological loading to pedicle screws and Am J Sports Med. 2015 Jun;43(6):1380–5. • Dr. med Claudia Druschel, Charite University medicine, Berlin, Germany provoke screw loosening. Red arrows show Primary stiffness of a modified transforaminal lumbar interbody • Dr. Kanna Gnanalingham, Dept of Neurosurgery, Manchester, fusion cage with integrated screw fixation: cadaveric biomechani- UK the load application and the pivot axis to cal study. Keiler A, Schmoelz W, Erhart S, Gnanalingham K. allow a tilting motion of the screw in the Spine (Phila Pa 1976). 2014 Aug 1;39(17):E994-E1000. Core Facilities vertebral body. Effect of augmentation techniques on the failure of pedicle MicroCT

Research Report 2015 Medical University of Innsbruck 95 Center of Operative Medicine Urology

urological and neuro-urological outpatient linked to prostate cancer and is the primary clinics, two adult urological wards, and a therapeutic target in this malignancy. For pediatric ward. The Division of Experimental two decades, researchers of the Depart- Urology is integrated in the Department of ment of Urology have been contributing to Urology. A main focus for the Department worldwide efforts to elucidate the molecu- is the treatment of urological malignancies. lar mechanism of AR function and its role in progression and therapy resistance. In 1993, the European Prostate Center Innsbruck was founded in order to ensure Several mechanisms have been identified optimal patient care and clinical research and were the basis for the development of on prostate cancer and prostatic diseases . new AR targeting drugs that led to prolonged In addition to the diagnosis and treatment survival of patients. of prostate diseases, a prostate cancer- screening project called Tyrol Project was Current research is focused on the AR implemented in1993 to offer early detection transcriptome. This includes protein-coding and curative treatment of prostate cancer genes, such as AGR2, a cellular chaperone, for affected men. Reconstructive Urology which is a potential tumor marker, or covers surgical repair of the urogenital microRNA genes, such as the host genes Director: tract after trauma or for treatment of of miR22 and miR29a, two epigenetic reg- Univ.-Prof. Dr. Wolfgang Horninger incontinence, whereas neuro-urology treats ulators involved in invasion and apoptosis, patients with functional disorders of the respectively. Investigation of posttrans Contact: bladder and the sphincter. Pediatric Urology lational regulation of AR protein and activity Anichstraße 35 offers diagnoses and treatments for all uncovered a feed-back-loop regulation, 6020 Innsbruck congenital, as well as acquired genitourinary which is potentially targetable by the well- problems, from birth to adulthood. known diabetes drug, metformin (Fig. 1). [email protected] Phone: +43 50 504 24811 Research Fax: +43 50 504 24873 www.uro-innsbruck.at Prostate Cancer Screening Wolfgang Horninger More than 20 years after the introduction of prostate specific antigen (PSA) testing Keywords in clinical practice, early detection of prostate cancer is still a matter of debate. Some Prostate cancer and prostatic diseases, prostate cancer-screening studies, mainly urological tumors, cancer markers and conducted in Europe, showed a decrease screening, androgen receptor, cytokine & in prostate cancer mortality and a stage growth factors signaling, immunology & migration towards lower, potentially curable immunotherapy, urogenital tract, andrology prostate cancer stages at the time of diagnosis. Research Focus However, the same studies showed a con- • Prostate cancer treatment and therapy re- siderable number of overdiagnoses and sistance mechanisms overtreatment. Our own data (“Prostate • Androgen receptor signaling and its role in Cancer demonstration Project”, “Tyrol prostate cancer development, therapy and Study”) showed that 20 years after initiation therapy resistance of an area-wide early detection program, a • Tumor immunology and immunothera- 64 % decrease in prostate cancer mortality py with a focus on dendritic cells and was achieved. Overdiagnosis was seen in γδ-T-lymphocytes 16–20 % of all screened men. Therefore, the Fig. 1: Posttranslational feedback-regulation • Diagnostics and tumor markers for urolog- aim of our Prostate Cancer Unit is to identi- of androgen receptor. AR and its transcrip- ical tumors, in particular prostate cancer fy new, better markers for prostate cancer. tional activity is enhanced by the ribonu- Moreover, we try to optimize prostate can- clear protein complex, MID1/α4-PP2A via General Facts cer detection (mpMRI) and prostate can- stimulation of mRNA translation. On the The Innsbruck Department of Urology cer treatment to avoid overdiagnosis and other hand, AR inhibits MID1 expression. was founded in 1964 under the direction reduce the side effects of (over)-treatment. During therapeutic androgen deprivation of Hans Marberger. Georg Bartsch was or anti-androgen treatment, this fine-tuning director from 1988 to 2010 and since 2011, The Androgen Receptor – Key Regulator mechanism of AR activity is interrupted, the Urological Department is directed by in Prostate Cancer which may contribute to therapy resistance. Wolfgang Horninger. The Department covers Helmut Klocker Metformin, an anti-diabetic drug disrupts the entire diagnostic and therapeutic range The androgen receptor (AR), a hormone the MID1 protein complex and downregu- of urology, running five operating theaters, induced transcription factor, is intimately lates AR protein level.

96 Research Report 2015 Medical University of Innsbruck Urology

Cytokines and Growth Factors in stroma is thought to create an optimal Prostate Cancer microenvironment for tumor growth and Zoran Culig progression driven by “activated” stromal Researchers are primarily interested in the cells. Hence, the use of 3D co-culture regulation of cellular events by cytokines systems for in vitro cancer research is in castration therapy-resistant prostate a highly important issue when studying cancer. There is a particular focus on the molecular changes occurring in the interleukin-6, whose levels are up-regulated different cell types as well as for testing in prostate malignancy, and on endogenous novel therapies and drugs. Recently 3D regulators of cytokine signaling. In this prostate cancer organoid culture protocols context, it is especially interesting that were established. The studies have shown these molecules are implicated in the that the molecular expression pattern of cell acquisition of resistance to chemotherapy type specific markers is markedly altered in with docetaxel (Fig. 2). In order to improve 3D versus 2D cultures. In addition, androgen therapy in advanced prostate cancer, responsiveness, as well as drug responses, inhibition of growth factors and other are significantly changed in 3D epithelial- oncogenes up-regulated during prostate stromal co-culture organoids, suggesting cancer progression by androgen ablation a strong influence of fibroblasts on tumor therapy are investigated. Combination cell behavior (Fig. 3). Future goals will focus therapies will be developed in the future in on the interplay between epithelial cells and order to establish personalized therapies. fibroblasts using this 3D cell culture system This research work, performed in several with the aim of improving responsiveness to projects, received numerous international current therapies. recognitions for its contribution to the current understanding of prostate cancer Immunology and Immunotherapy of development and progression. Urological Tumors Martin Thurnher Fig. 4: Mevalonate metabolism. HMG-CoA re- The immunology/immunotherapy group ductase, the target of statins, catalyzes meva- has a research interest on how the immune lonate formation, which is further converted system reacts against growing tumors. Spe- into IPP and its isomer DMAPP. Sequential cifically, they are interested in the activation condensations form FPP and GGPP. Activat- of γδ-T-lymphocytes by intermediates of the ed FPP and GGPP are the building blocks for mevalonate pathway (Fig. 4). Since deregu- posttranslational protein prenylation. By in- lation of mevalonate metabolism can lead hibiting FPP synthase, nitrogen-containing to malignant transformation, γδ-T-cells also bisphosphonates induce depletion of down- play an important role in the immunosurveil- stream FPP and GGPP, and thus, inhibition lance of tumors, such as bladder cancer. Im- of prenylation, as well as the accumulation proved understanding of these interactions of IPP that is specifically recognized by cer- will foster the development of innovative tain γδ-T-lymphocytes. immunotherapies, as well as the establishment of prognostic markers and monitoring Selected Publications Fig. 2: SOCS-3 function in prostate cancer. technologies. http://urologielabor-innsbruck.tirol-kliniken.at/ SOCS-3 is a common negative regulator for page.cfm?vpath=publikationen-gesamtuebersicht/ publikationen--wissenschaft androgen and IL-6 pathways in prostate cancer. IL-6 may cause a pro-differentiation Selected Funding effect as evidenced by PSA increase. Sus- • Austrian Research Fund, FWF • K1 Center Oncotyrol tained activation of the JAK/STAT pathway • MUI Start Grant Fund is prevented by up-regulation of SOCS-3 • Tyrolean Research Fund • Tirolean Cancer Society (Krebshilfe Tirol) by IL-6. SOCS-3 is also a negative feedback • Research Fund of the Austrian National Bank regulator for androgen signaling in prostate • Medical Research Fund (MFF) cancer cells. • Astellas Pharma Investigator Driven Grant Collaborations Models for Prostate Cancer Research • Holger Sültmann, DKFZ Heidelberg, D Iris E. Eder-Neuwirt • Mark A. Rubin, Weill Cornell Medical College, New York, US • Michal R. Schweiger & Hans Lehrach, MPI for Molecular In-vitro prostate cancer research is mainly Genetics, Berlin, D conducted with immortalized cell lines, Fig. 3: LNCaP prostate cancer epithelial cells • William R.G. Watson, Conway Institute of the University College Dublin, IRE which lose relevant growth characteristics co-cultured with GFP-labeled cancer associ- • Francesca Demichelis, University of Trento, I when grown on a plastic surface. In addition, ated fibroblasts (CAF) at a ratio of 1:1 in 3D • Christian Fuchsberger & Johannes Rainer, EURAC Bolzano,I • Narisu Naris, NIH Bethesda, US the human prostate is composed not only Perfecta 96 well plates. Cells form irregular • Glen Kristiansen, University of Bonn, Bonn, D of epithelial cells, but also contains several stellate organoids within 4 days of 3D cul- • Normam, J. Maitland, University of York, UK • Andrew C.B. Cato, Karlsruhe Inst. of Technology, Karlsruhe, D types of stromal cells. In tumor tissue, the ture. CAFs appear as small green islands • Jan Bouchal, University of Olomouc, Olomouc, CR interplay between the epithelium and the within the organoid. • Philip A. Cole, Johns Hopkins University, Baltimore, US

Research Report 2015 Medical University of Innsbruck 97 Center of Operative Medicine Orthopeadic Surgery

Keywords electron microscopy (SEM) is available. Molecular identification of biofilms is under Orthopaedics, microbiology, medical micro development. Immunological research aims biology, bacteriology, molecular biology, to detect immune activation parameters as immunology, minimal-invasive surgery, med- signs of infection and monocyte differentia- ical engineering, biomedical engineering, tion and activation in co-evolution with mi- prosthetics crobial infection.

Research Focus Research

Clinical Research Clinical Research We perform applied studies mainly in the Bone Graft Substitutes in Posterior fields of spine, arthroplasty, knee, and pae- Lumbar Interbody Fusion diatric orthopaedics, with a special interest Thaler in infection, implant migration and postop- ß-tricalcium phosphate is widely used in erative car driving ability. We also host the cages in order to achieve bone fusion in office of the European Arthroplasty Register. posterior lumbar interbody fusion. In 45 discs CT assessment revealed inadequate Director: Experimental Orthopaedics fusion (non-union) in 17 levels (39 %). This Univ.-Prof. Dr. Martin Krismer The area of biomedical engineering focuses technique of PLIF using ß-TCP cannot be on allograft stability and bone harvesting. recommended. Contact: The area of Implant-related infections Anichstraße 35 develops studies on infections related to Brake Response Times after Various 6020 Innsbruck biofilm attachment. Surgeries Different Authors [email protected] General Facts In 43 patients, 6 weeks after unicom- Phone: +43 512 504 22691 partmental knee arthroplasty, brake Fax: +43 512 504 22701 Clinical Research response time reached preoperative values www.orthopaedie-innsbruck.at Additionally to a new VICON system in (Liebensteiner). In 12 patients, brake clinical use we perform gait and motion response times after anterior cervical analysis in our Biomechanics Lab (Dr. Haid) fusion were immediately ameliorated after and outdoor with a Lukotronic system. operation, but did not reach normal values after 4–6 weeks (Thaler). Studies on differ- Implant migration measurement based on ent knee and ankle braces as well as on EBRA was developed and is developing in herniated lumbar discs with paresis are cur- cooperation with the Unit of Geometry and rently running. CAD at Innsbruck University (Prof. Husty). Software and scanning equipment are Implant Registers available. Labek Data of implant registers of 5 countries Brake reaction time for car driving is showed a need for methodological improve- measured in an experimental setting with ment. Revision rate was approximately 10 % high accuracy. Postoperative measurements after 5 years, with a high rate of inlay frac- have been performed for various operations. tures which indicates potential for improvement of implants. In an analysis of world- Experimental Orthopaedics wide arthroplasty registers, data have been The unit of Experimental Orthopaedics is extracted with respect to reason for revision a research unit within the Department of surgery and pooled causes. Aseptic loosen- Orthopaedic Surgery, and is divided in two ing is responsible for 55 % of hip arthroplas- Head of Experimental Orthopaedics: major research areas. ty revisions, and for 30 % in the knee. Septic Univ.-Prof. Dr. Michael Nogler loosening was the cause for revision in 7.5 % The area of biomedical engineering in the hip and 15 % in the knee. Contact: (Dr. Putzer) focuses on biomechanical stud- Innrain 36 ies aiming to increase allograft stability AO Spine Injury Classification System 6020 Innsbruck in difficult revision cases and to improve Reinhold techniques and instruments for bone re- In this project of the AO Spine Classification [email protected] moval. In the area of implant-related infec- Group, five experienced spine trauma Phone: +43 512 9003 71691 tions (Dr. Coraça-Huber) different biofilm surgeons from various parts of the world Fax: +43 512 9003 73691 models are carried out using the main classified in a structured, iterative process www.orthopaedie-innsbruck.at strains associated with implant-related in- consecutive case with TL injuries. The fections. Antibiotic and antiseptic suscep- reasons for disagreements were examined tibility tests can be carried out. Scanning systematically during review meetings.

98 Research Report 2015 Medical University of Innsbruck Orthopeadic Surgery

a mature biofilm. The accumulation phase requires exopolysaccharide and/or protein- protein interactions. In mature biofilms, the accumulation or dispersal of cells is controlled by inter cell communication mediated by quorum sensing activation. Cells dispersed from a mature biofilm regain the physiological characteristics of planktonic cells and may disseminate causing acute infection. With this study we can obtain the molecular profile of the strains causing device-related infection. This knowledge can help us develop a preventive method against biofilm formation on implants and/or a treatment to remove already established biofilms.

Immunology of Biofilm Infection Ammann Here we study the interaction of the complement system with bacteria in nosocomial joint infections. Results achieved during this study will not only Fig. 1: S. epidermidis biofilm broaden the basic knowledge about complement in nosocomial joint infections In four successive sessions, the system Molecular Profile of Biofilms but also have clinical implications. This study was revised until consensus and sufficient Coraça-Huber not only increases our knowledge about reproducibility were achieved. Bacterial biofilm follows a life cycle. Bacterial interactions of bacteria causing nosocomial cells attach to a surface with proteins, infection with the innate immune system Gait Analysis after Ankle Arthrodesis adhesins and through other nonspecific but can ultimately lead to the development Versus Arthroplasty interactions with the substratum. Attached of a novel, indirect detection strategy Biedermann cells can develop into small clusters with helping reduce the number of false negative A gait analysis and clinical assessment have biofilm-like properties called microcolonies samples drawn from patients after joint been performed in 101 ankle arthroplasties and these can grow and accumulate into reconstruction surgery. and 40 ankle arthrodesis cases. Significant asymmetry in gait and reduced range of motion compared to normal remained after both procedures, although subjective outcome has been improved after both procedures. As hindfoot fusion improved postoperative function and pain more than arthroplasty, the implantation of current arthroplasty designs in large patient series may be questioned.

Experimental Orthopaedics Biofilm Formation and Antimicrobial Susceptibility Tests Coraça-Huber In this study we investigated whether biofilms growin vitro on metal discs and on microtiter plates.

The evaluation of the biofilms formed on different surfaces was assessed by comparing the antibiotic susceptibility of S. aureus and by examining the structure Fig. 2: Staphylococcal biofilm life cycle (FnBPs – fibronectin binding proteins; CltA – of S. aureus biofilms grown yb scanning fibrinogen-binding protein-clumping factor A; Cna – collagen binding protein; Bbp – bone electron microscopy (SEM). Also, several sialoprotein-binding protein; PNAG – poly-N-acetylglucosamine; PIA – polysaccharide inter biomaterials can be tested for biofilm cellular adhesion; Bap – biofilm associated protein; Bhp – Bap homologue protein; eDNA growth and efficacy tests for biomaterials – extracellular DNA; icaADBC – genes involved in the synthesis of PIA; bap gene – involved with antimicrobial properties. in the synthesis of Bap).

Research Report 2015 Medical University of Innsbruck 99 Center of Operative Medicine

Bone Tissue as Antibiotic Carrier Coraça-Huber, Putzer Bone grafts can either be used as large structural bone grafts from post-mortem donors or as bone chips from morselized femoral head donated by living patients undergoing total hip arthroplasty. Such bone chips are used to fill defects that require biomechanical stability, which can be achieved by compressing the chips into the defect site. Fresh frozen bone chips Fig. 3: Surgical Innovation contain the original osteoconductive and osteoinductive proteins, but can add the Selected Publications risk of local contaminations. Antibiotics Bactericidal Activity of N-Chlorotaurine against Biofilm-Forming Bacteria Grown on Metal Disks. Coraça-Huber DC, Ammann CG, delivered from an implanted biomaterial can Fille M, Hausdorfer J, Nogler M, Nagl M. be potentially used to prevent infections. ANTIMICROBIAL AGENTS CHEMOTHERAPY. 2014;58: p. 2235– Morselized bone allografts can be used 2239. Influence of Poly-N-Acetylglucosamine in the Extracellular Ma- as carriers by impregnating them with trix on N-Chlorotaurine Mediated Killing of Staphylococcus Epi- antibiotic solutions or by mixing them with dermidis. Ammann CG, Fille M, Hausdorfer J, Nogler M, Nagl M, Coraça-Huber DC. antibiotic powders. Also, biomechanical NEW MICROBIOLOGY. 2014; 37: p. 383–386. compression tests were carried out The Use of Time-of-Flight Camera for Navigating Robots in Com- for different preparation procedures to puter-Aided Surgery: Monitoring the Soft Tissue Envelope of Min- imally Invasive Hip Approach in a Cadaver Study. Putzer D, Klug S, study the mechanical effect of grain size Moctezuma JL, Nogler M. distribution, water and fat content and the SURGICAL INNOVATION. 2014; 21: p. 630–636. possibility of enhancing osteoconductive AO Spine Injury Classification System: a Revision Proposal for the Thoracic and Lumbar Spine. Reinhold M, Audige L, Schnake K, and osteoinductive properties of allograft Bellabarba C, Dai LY, Oner FC. by adding bioglass or platelet rich plasma. EUROPEAN SPINE JOURNAL. 2013; 22; p. 2184–2201. The Use of Beta-Tricalcium Phosphate and Bone Marrow Aspirate as a Bone Graft Substitute in Posterior Lumbar Interbody Fusion. Intelligent Antibiotic Carriers Thaler M, Lechner R, Gstöttner M, Kobel C, Bach C. Bone cements can be used as antibiotic EUROPEAN SPINE JOURNAL. 2013; 22: p. 1173–1182. carriers. Adding antibiotics in different concentrations is influencing the mechanical Collaborations • Heraeus Medical GmbH, Wehrheim, Germany properties, as well as the antibiotic release • BonAlive Biomaterials Ltd, Turku, Finland of the bone cement. Antibiotic loaded • Stryker Leibinger GmbH & Co. KG, Freiburg, Germany • Hochschule Offenburg, Offenburg, Germany cements as well as antibiotic loaded • Incocon GmbH, Attnang-Puchheim, Austria implants are a way to fight periprostetic • Innsbruck University, Unit of Geometry and CAD, (Prof. Husty). joint infections. This research is conducted Devices & Services in collaboration with Inocon GmbH and the • Zeiss Fluorescence Microscope Axio Lab.A1 Consiglio Nazionale di Ricerca di Parma. • Scanning Electron Microscope JSM-6010 InTouchScope • BactoSonic Ultrasonic Bath Bandelin • Formlabs Stereolithographic 3D Printer Computer Assisted Bone Removal • VICON Nexus 2.1.1 gait lab with 2 Amti OR6-7-1000 ground Procedures reaction force measurement plates • Lukotronic motion analysis for outdoor use In a collaboration project, an intraoperative • Brake reaction time measurement setting planning tool for bone removal procedures is being tested in surgical simulation scenarios. In a simulation study the maximum reachable depth of straight instruments inserted into the femoral canal was determined. Constraints for a simulated bone removal procedure in a femoral canal could be defined. In a cadaver study the usage of an optical digitizer was evaluated do define the soft tissue envelope of the surgical situs during hip arthroplasty. In almost every surgical procedure retraction of soft tissue is necessary. To control retraction forces and minimize soft tissue damages related to it, the possibility of using a semiactive robotic retractor holder was evaluated in a concept study.

100 Research Report 2015 Medical University of Innsbruck Orthopeadic Surgery

Research Report 2015 Medical University of Innsbruck 101 Center of Operative Medicine Anaesthesiology and Critical Care Medicine General and Surgical Critical Care Medicine

Keywords we feature regular EMS physician, airway management, transesophageal echo, and Anaesthesiology, critical care medicine, regional anaesthesia courses for in-house emergency medicine, pain medicine, and out of town participants, and organize palliative care, breathing gas, coagulation annual international mountain rescue, and emergency medicine conferences in Inns- Research Focus bruck. Individual schedules ensure work-life balance (i.e. 25 % of attendings and 10 % of Anaesthesiology, critical care medicine, residents are on various part time plans); emergency medicine, pain medicine, palli- transparent rotation assignments and leave ative care, cardiopulmonary resuscitation, of absence plan distribute fairness across airway management, vasodilatory shock, all parties. Forty percent of our attendings, posttraumatic shock, coagulation, hypo- and 49 % of our residents are female. We thermia, regional anaesthesia, transplan- have tenured 10 female colleagues to the tation, neuro- and obstetrical anaesthesia, Associate Professor level and 3 of them muscle relaxants, mountain rescue, micro- and one female clinical colleague achieved circulation, prediction models, breathing chair positions. One of our three chiefs of gas analysis. staff is female, and the female president Director: of the Austrian Society of Anesthesiology o. Univ.-Prof. Dr. Karl Lindner General Facts and Critical Care Medicine is based in our department. Prof. Linder has been the inter- Contact: The Department of Anaesthesiology and im director of “General and Surgical Critical Anichstraße 35 Critical Care Medicine employs 150 full- Care Medicine” since 2013. 6020 Innsbruck time equivalent physicians (about 60 residents), with additional nursing, secretar- Research [email protected] ial, technical, and research staff totalling Phone: +43 512 504 28503 about 450 employees for the entire depart- Our physicians provide extremely wide Fax: +43 512 504 28504 ment. Besides performing about 40,000 clinical services reaching from very basic www.anaesthesie-innsbruck.at anaesthesia cases per year in one of our clinical procedures (serving the Innsbruck 60 operating/diagnostic rooms, our depart- area as a county hospital) to complex tertiary ment is responsible for a general surgery, care of severely injured or sick patients, post-operative, transplantation, and trauma serving as a national referral medical center intensive care unit, six postanaesthesia care and also for Southern Tyrol in Italy. When units, anaesthesiology outpatient clinic, taking together this clinical backbone and medical and nursing student education, our scientific activities, we have substantial emergency medical service ground and potential to develop bench-to-bedside rotorwing unit, pain service, basic science treatments in any area of our specialty. research laboratory, and animal operating Continuous positive airway pressure room. This variety is very large even for a (CPAP) ventilation, arginine vasopressin university hospital, and allows a wide focus during cardiopulmonary resuscitation and in clinical care, teaching, and research. vasodilatory shock as well as fibrinogen Our department is one of the most popular during life-threatening haemorrhage are training institutions in Austria combining examples for current global implementation ambitious young residents and extreme- of novel treatment strategies that were ly experienced attendings. For example, developed in our department.

Fig. 1: Gerinnungsmanagment in der Inten- Fig. 2: Enceclopedia of Trauma: Chapter: Ad- sivmedizin. Fries D und Streif W. Spring- juncts to transfusion. Fries D. Springerver- erverlag. ISBN-13978-3-642-05003-9. lag. ISBN 978-3-642-29611-6.

102 Research Report 2015 Medical University of Innsbruck Anaesthesiology and Critical Care Medicine/General and Surgical Critical Care Medicine

Peter Mair et al. are studying the role of surgical procedures. Barbara Friesenecker Selected Publications snow avalanche-associated hypothermia et al. are studying end-of-life issues in the The exclusive use of coagulation factor concentrates enables and injuries. Corinna Velik Salchner and intensive care unit. Birgit Mair et al. analyse reversal of coagulopathy and decreases transfusion rates in patients with major blunt trauma. Innerhofer P, Westermann I, colleagues are studying coagulation and outcomes of helicopter emergency medicine Tauber H, Breitkopf R, Fries D, Kastenberger T, El Attal R, Strasak regional anesthesia aspects in critically system patients. Franz Wiedermann is A, Mittermayr M. INJURY. 2013;44:209–16. ill cardiovascular patients. Stephan studying the antiphospholipid syndrome. A review of the volatiles from the healthy human body. de Lacy Eschertzhuber and his team study critical Marc Kaufmann is responsible for the Costello B, Amann A, Al-Kateb H, Flynn C, Filipiak W, Khalid T, Osborne D, Ratcliffe NM. JOURNAL OF BREATH RESEARCH. care medicine aspects in transplantation helicopter emergency medical system 2014;8:014001. patients. Volker Wenzel and Karl-Heinz “Christophorus 1” in Innsbruck with a Pulmonary function after emergence on 100 % oxygen in patients Stadlbauer have recently completed special interest in mountain medicine. with chronic obstructive pulmonary disease: a randomized, controlled trial. Kleinsasser AT, Pircher I, Truebsbach S, Knotzer H, the “VITRIS.at” trial studying the role of Günter Luckner, Werner Pajk and Markus Loeckinger A, Treml B. ANESTHESIOLOGY. 2014;120:1146–51. vasopressin as on-top medication during Mittermayr have a special interest in Accidental hypothermia. Brown DJ, Brugger H, Boyd J, Paal P. life threatening posttraumatic shock. Pet paediatric anaesthesia and study the role of NEW ENGLAND JOURNAL OF MEDICINE. 2012;367:1930–8. ra Innerhofer, the current president of coagulation in cardiac surgery and complex European Resuscitation Council Vasopressor during Cardiopulmo- our professional society, is looking at cranial procedures. Karin Khünl-Brady nary Resuscitation Study Group. A comparison of vasopressin and epinephrine for out-of-hospital cardiopulmonary resuscitation. coagulation in posttraumatic haemorrhage et al. study the role of muscle relaxants. Wenzel V, Krismer AC, Arntz HR, Sitter H, Stadlbauer KH, Lindner and is conducting a randomized controlled Peter Paal and team assess accidental KH. NEW ENGLAND JOURNAL OF MEDICINE. 2004;350:105–13. clinical trial assessing coagulation factors hypothermia, especially in combination Fibrinogen concentrate in dilutional coagulopathy: a dose study in vs. fresh frozen plasma. Thomas Luger et with snow avalanches. Stefan Jochberger pigs. Martini Judith, Maisch Sonja, Pilshofer Lisa et al. TRANSFUSION. 2014; 54(1); 149–157. al. are leading the geriatric trauma center, studies the role of critical care medicine Management of postpartum hemorrhage (PPH). Algorithm of the the fastest growing section in traumatology, in developing countries. Wolfgang Lederer interdisciplinary D-A-CH consensus group PPH (Germany – Austria and study how to optimize anesthesia for is studying ethical aspects in emergency – Switzerland). Schlembach D, Moertl MG, Girard T et al. geriatric patients. Axel Kleinsasser et al. medicine. Judith Martini assesses the role ANAESTHESIST. 2014; 63(3); 234–242. assess pulmonary function postoperatively. of microcirculation and infusion solution Effects of Fibrinogen Concentrate After Shock/Resuscitation: A Comparison Between In Vivo Microvascular Clot Forma- Hans-Ulrich Strohmenger is our critical aspects in shock. Thomas Mitterlechner tion and Thromboelastometry. Martini Judith, Cabrales Pedro, incident analyst and works on making et al. study technical adjuncts to facilitate Fries Dietmar et al. ECRITICAL CARE MEDICINE. 2013; 41(11); clinical treatment safer. Arnulf Benzer and endotracheal intubation. Ruth Kröss studies E301-E308. The early use of fibrinogen, prothrombin complex concentrate, team is studying depth of anaesthesia in paediatric anaesthesia. Janett Kreutziger and recombinant-activated factor VIIa in massive bleeding. Fries neurosurgical patients. Günter Putz et al. and team are looking at prediction models D. TRANSFUSION. 2013; 53(1); 91s-95s. assesses the role of regional anaesthesia after trauma. For the sake of simplicity and in obstetric patients. Michael Baubin et readability, we have kept this list brief in Selected Funding al. studies quality issues in emergency order to give an overview, as the complete • Christophorus-Flugrettungsverein vom ÖAMTC und die Schweiz- erische Rettungsflugwacht, VITRIS-Project, Volker Wenzel medicine, which recently resulted in our list of ongoing projects would render • Suche nach antidotes für neue orale Antikoagulantien und emergency medicine system unit being the reading exhausting. Plättcheninhibitoren (NOAC Linie), OENB, Dietmar Fries best in the German resuscitation register Devices and Services analysis. Ingo Lorenz and colleagues Several of our colleagues are contributing 60 operating/diagnostic rooms, general surgery, post-operative, study critical care medicine. Dietmar experts in updating clinical guidelines transplantation, and trauma intensive care unit, six postanesthe- Fries and a large team analyse the role of such as in cardiopulmonary resuscitation, sia care units, anesthesiology outpatient clinic, medical and nursing student education, emergency medical service ground and fibrinogen after posttraumatic shock in a and coagulation management after severe rotorwing unit, pain service, basic science research laboratory, randomised controlled trial and after large hemorrhage. and animal operating room.

Research Report 2015 Medical University of Innsbruck 103 Center of Internal Medicine Internal Medicine I

Keywords • the cause and effects of intestinal inflammation particularly in connection with Gastroenterology, hepatology, endo diseases like Crohn’s disease and ulcera- crinology, metabolism, inflammation, tive colitis diabetes mellitus, microbiota, non-alcoholic • studies of the composition of the human fatty liver disease, nutrition, inflammatory microbiota and its effects on human bowel diseases health and different diseases • role and mechanisms of insulin resistance Research Focus in type 2 diabetes and non-alcoholic fatty liver disease with a focus on various diets The research focus of our department • bariatric surgery and metabolic/immuno- is translational research in the fields logical effects on the host of inflammation and metabolism and is • lipoprotein metabolism and atherosclero- represented by several research groups. The sis major objective of our research activities is to improve clinical management of patients General Facts with chronic inflammatory disorders, such The Department of Internal Medicine I has as Crohn’s disease, ulcerative colitis, type 2 its focus in the specific medical areas Director: diabetes or obesity. The major research of gastroenterology, endocrinology and Univ.-Prof. Dr. Herbert Tilg topics are: metabolism. Our division has about 40

Contact: Anichstraße 35 6020 Innsbruck

[email protected] Phone: +43 512 504 23539 Fax: +43 512 504 23538 http://inneremed1.tirol-kliniken.at . R . © Moschen A

Fig. 1: Adipose tissue: cellular components and molecules synthesised. Expansion of the adipose tissue during weight gain leads to the recruitment of macrophages through various signals, which might include chemokines synthesized by adipocytes, such as CC-chemokine ligand 2 (CCL2). These macrophages are found mainly around apoptotic adipocytes. Various mediators synthesized by adipocytes and resident macrophages might contribute to local and systemic inflammation. The overall adipocytokine-cytokine cocktail might favour a pro-inflammatory milieu. IL interleukin, TNF tumor-necrosis factor (from Tilg H., Moschen A.R., Nat Rev Immunol 2006, 6:772–83).

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employees and many members are involved both in clinical work and research. Our laboratories are perfectly equipped and our researchers are able to perform state-of- the art research in the field of cellular and molecular work. The common aim of our research activities is to increase knowledge in respective disease areas and to improve patient care. We have established many different national and international collaborations throughout the world and, especially in the field of gastroenterology, our department is internationally well established and well known.

Our research is funded by the Austrian Research Promotion Agency (FFG), Austrian Science Fund (FWF), European Union (FP7), Christian Doppler Research Association (CDG) and we have published our research in highly respected international Journals such as Nature, Nature Medicine, Nature Reviews of Immunology, Cell, New England Journal of Medicine, Proceedings National Academy of Sciences, Gastroenterology, . R

Gut, Hepatology and many others. .

Research © Moschen A Gastroenterology Intestinal inflammation has been the focus Fig. 2: The multiple parallel hits model. Lipotoxicity: (1) A liver loaded with lipids consisting of our research activities in the last two primarily of trigylcerides might reflect a benign process because trigylcerides might exert decades. One focus has been traditionally mostly protective effects. Furthermore, hyperleptinemia leads to oxidation of hepatic lipids, clinical research and our clinical research thereby also protecting this organ from lipotoxicity. When the capacity of peripheral and group has been involved in many important central organs of detoxifying “aggressive lipids” fails, lipotoxic attack of the liver might clinical studies in the last years which begin. Inflammation may precede steatosis in NASH. Gut-derived signals: Many signals led to major improvements in the clinical beyond endotoxin might affect hepatic steatosis and inflammation. Several pathways have management of patients with inflammatory been identified how the gut microbiota might influence host energy metabolism: (2) Absence bowel diseases (IBD) such as Crohn’s of the microbiota in germ-free mice correlates with increased activity of phosphorylated disease and ulcerative colitis. As prototypic AMPK in the liver and the muscle (not shown). (3) Some of the breakdown products of poly- examples, we have participated in the saccharides are metabolized to SCFAs. SCFAs such as propionate and acetate are ligands for seminal SONIC study published in the New the G protein-coupled receptors Gpr41 and Gpr43. Shortage of SCFAs might allow the evolu- England Journal of Medicine demonstrating tion of systemic inflammatory events. Such mechanisms elegantly combine diet, microbiota, the superior efficacy of anti-TNF agents in and the epithelial cell as “nutrient sensor”. (4) The microbiota decreases epithelial expres- combination with immunosuppressants in sion of fasting-induced adipocyte factor (Fiaf), which functions as a circulating lipoprotein the treatment of Crohn’s disease. We also lipase (LPL) inhibitor and therefore is an important regulator of peripheral fat storage. (5) contributed substantially to the success Several TLRs, such as TLR5 or TLR9, are not only able to affect microbiota but also to regu- of the clinical study programme with late metabolism, systemic inflammation, and insulin resistance, thus highlighting the role of vedolizumab demonstrating superior activity the innate immune system in metabolic inflammation as observed in NASH. (6) Various nu- of this agent especially in the treatment of trients such as trans fatty acids (TFAs), fructose or aryl hydrocarbon receptor (AhR) ligands ulcerative colitis. These studies have also such as 2,3,7,8-tetrachlorodibenzodioxin (TCDD) may directly lead to steatosis / liver in- been published in the New England Journal flammation. Adipose tissue-derived signals: Signals derived from the adipose tissue beyond of Medicine. toxic lipids might play a central role in NAFLD / NASH. (7) Here, adipocytokines such as adiponectin and leptin, certain proinflammatory cytokines such as TNFα or IL-6, and others Another major focus of our research (the death receptor Fas, PPARŲ) are of key relevance. The cytokine / adipocytokine milieu group has been preclinical models of IBD might be critical because ob / ob-adiponectin tg mice, although becoming severely obese, assessing various pathway mechanisms are not insulin-resistant. This suggests that in the hierarchy of processes soluble mediators of intestinal inflammation. Here, we could play the central role. Adipose-derived mediators might indeed affect target organs such identify in the past several years new as the liver, because JNK1 adipose-deficient mice are protected from diet-induced obesity, mechanisms contributing to intestinal and experiments have demonstrated that this effect is mediated mainly by IL-6 (a cytokine), inflammation, which were published in high which is of key importance in human obesity. (from Tilg H., Moschen A. R., Hepatology 2010; ranked Journals such as Cell or Journal of 52:1836–46)

Research Report 2015 Medical University of Innsbruck 105 Center of Internal Medicine

Experimental Medicine. Another major our research group are commonly invited Endocrinology and Metabolism research topic is the role of diet on immunity to the top international meetings in this Diet-induced obesity has become a huge and disease development. field. Importantly, we have also contributed socio-economic burden worldwide. Obesity intellectually substantially as we have is commonly associated with several Hepatology established disease models and hypotheses metabolic alterations, especially insulin Another focus of our research group is in this field of research. Our hypothesis resistance, type 2 diabetes, dyslipidemia non-alcoholic fatty liver disease (NAFLD). paper suggesting that NASH reflects a and, as a consequence, with strongly Inflammation has always been a major disease caused by multiple parallel hits increased cardiovascular morbidity and interest of our research group and we is highly cited (Tilg H. Hepatology 2010) mortality. Among others, high-fat and have published more than 20 years ago and internationally well respected. Several high-carbohydrate intake have been held a highly cited paper demonstrating the current studies are focussing on the role of responsible for increased incidences of important role of inflammatory cytokines innate immunity and microbiota in NAFLD. obesity and type 2 diabetes. in chronic liver diseases. Research in the For these studies we have established an last ten years has concentrated on the international network with highly respected While metabolic phenotypes of diet-induced role of innate immunity and microbiota in international leaders. In respect to our forms of obesity are quite similar, partly NAFLD and several key papers could be leadership in this field, ew have recently different pathophysiological mechanisms published in this field. Our contributions are also been involved in the development of seem responsible for glucose intolerance also reflected by the fact that members of European guidelines in NAFLD. and dyslipidemia. The overall aim of our

A C Paneth Basement Fig. 3: Dissecting Diet and Intestinal Im- Cruciferous vegetables cells membrane (broccoli, cabbage, and munity. Kiss et al.1 and Li et al.2 recently brussels sprouts) Stem cell reported that intestinal immune functions are dependent on dietary aryl hydrocarbon repector (AhR) ligands. Indole-3-carbinol is an AhR ligand found in cruciferous vegeta- Indolo[3,2−b]carbazole bles, such as broccoli and brussels sprouts. HNH O OH N Specialized After oral consumption, indole-3-carbinol is intraepithelial NH lymphocytes converted in the presence of gastric acid to high-affinity ligands such as indolo[3,2-b] 6-Formylindolo[3,2−b]carbazole Goblet Gastric cell carbazole or 6-formylindolo[3,2-b]carbazole acid Mucus (Panel A).

Antimicrobial AhR ligands activate chaperone-bound peptides AhRs that dimerize with the AhR nuclear translocator (Arnt) and regulate gene expression (Panel B). The studies showed that two cell types are Gastrointestinal critically dependent on dietary-derived AhR tract signals: specialized intraepithelial lympho- HNH O cytes (e.g. intraepithelial T-cell receptor Ų Dietary-derived cells) and CD4-RORŲt+ intestinal lymphoid NH AhR signaling Lumen cells with lymphoid tissue-inducing function (e.g. Peyer’s patches) (Panel C). B Specialized intraepithelial Mice lacking AhR signals (obtaining genet- lymphocytes and CD4−RORγt+ AhR ligand intestinal lymphoid cells ically or through dietary deprivation) lack specialized intraepithelial lymphocytes and intestinal lymphoid cells, which results

Cell membrane AhR in reduced epithelial turnover, reduced expression of antimicrobial peptides, an AhR Defensin Chaperone Chaperone altered microbiota, and increased susceptibility to intestinal inflammation (induced by Mucus Cytoplasm dextran sulfate sodium or in response to Cit- Goblet Arnt cell robacter rodentium infectin) (Panel C). The Arnt pathogenesis of enhanced inflammation in AhR Peyer’s Paneth Stem the mutant mice is incompletely understood AhR-regulated genes patches cells cell and probably involves defective interleu- Nucleus Arnt Tissue kin-22 production. AhR regeneration and repair .

R (Interleukin-22 is a cytokine that controls . DNA CD4−RORγt+ intestinal homeostasis and protects against AhRE intestinal lymphoid Interleukin-22 cells intestinal pathogens) (from Tilg H., NEJM

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work is to clearly define harmful effects of activator inhibitor 1, gallstone formation, Selected Publications different diets on relevant tissues or organs proprotein subtilisin/kexin type 9, non- Adipose tissue and liver expression of SIRT 1, 3 and 6 increase after extensive weight loss in morbid obesity. in glucose and lipid metabolism. In detail, alcoholic fatty liver disease and telomere Moschen AR, Wieser V, Gerner RR, Bichler A, Enrich B, Moser P, we currently investigate systemic and length. Ebenbichler CF, Kaser S, Tilg H. cellular effects of various carbohydrate- JOURNAL OF HEPATOLOGY. 2013; 59: p. 1315–22. Blockade of receptor activator of nuclear factor-ĸB (RANKL) or fat-enriched diets in a mouse model of Type 2 Diabetes and the Metabolic signaling improves hepatic insulin resistance and prevents de- diet-induced obesity. At the cellular level, Syndrome velopment of diabetes mellitus. Kiechl S, Wittmann J, Giaccari A, Knoflach M, Willeit P, Bozec A, Moschen AR, Muscogiuri G, Sorice our studies are focused on diet-specific Several studies were performed in order to GP, Kireva T, Summerer M, Wirtz S, Luther J, Mielenz D, Billmeier effects on adipose tissue, the liver, intestine identify risk factors for the development of U, Egger G, Mayr A, Oberhollenzer F, Kronenberg F, Orthofer M, Penninger JM, Meigs JB, Bonora E, Tilg H, Willeit J, Schett G. and skeletal muscle. In adipose tissue we type 2 diabetes mellitus and the metabolic NATURE MEDICINE. 2013: 19: p. 358–63. are especially interested in lipid droplet syndrome. ER stress transcription factor Xbp1 suppresses intestinal tumori- formation and adipogenesis. In particular, genesis and directs intestinal stem cells. Niederreiter L, Fritz TM, Adolph TE, Krismer AM, Offner FA, Tschurtschenthaler M, Flak MB, these studies aim to allow characterization Studies included the protective effects of Hosomi S, Tomczak MF, Kaneider NC, Sarcevic E, Kempster SL, of metabolically healthy and unhealthy uncoupling protein 2, the increased risk Raine T, Esser D, Rosentiel P, Kohno K, Iwawaki T, Tilg H, Blumberg RS, Kaser A. adipocytes. Additionally, our studies will for diabetes mellitus due to psychotrop- THE JOURNAL OF EXPERIMENTAL MEDICINE. 2013: 210: include investigations of the role of DPP‑IV ic and anti-epileptic drugs, parameters p. 2041–56 and apolipoprotein A5, which are both associated with increased insulin resist- IL-37 protects against obesity-induced inflammation and insulin well known candidate genes in metabolic ance, comparisons of different definitions resistance. Ballak DB, van Diepen JA, Moschen AR, Jansen HJ, Hijmans A, Groenhof GJ, Leenders F, Buflet P, Boekschoten MV, disease, in diet induced obesity and insulin of the metabolic syndrome as well as sta- Müller M, Kersten S, Li S, Kim S, Eini H, Lewig EC, Joosten LA, Tilg resistance. tistical analysis of different anthropometric H, Netea MG, Tack CJ, Dinarello CA, Stienstra R. surrogate-markers and their accuracy to NATURE COMMUNICATIONS. 2014: 5: p. 4711. Microbiota and diabetes: an evolving relationship. Tilg H, Another focus of our work is to more clearly correlate to cardiovascular risk factors as- Moschen AR. define the crosstalk between metabolically sociated with obesity. GUT 2014. 63: p. 1513–21. important tissues and organs: To do so we Selected Funding are currently studying the effects of various Diabetes Registry Tyrol (DRT) Establishment and development of new models of the metabolic diets on (adipo)cytokines, hormones and The DRT was established in 2005 in order syndrome as screening platform for phytodrugs, Austrian Rese- fatty acids which are all thought to play to register all patients with type 1 and arch Promotion Agenca (FFG), BRIDGE, € 109,000.00, 2013– a major role in the interaction between type 2 diabetes mellitus as well as women 2015; Univ.-Prof. Dr. Herbert Tilg Long-term effects of weight loss on atherosclerosis, Austrian metabolically important tissues. Results diagnosed with gestational diabetes mellitus Science Fund (FWF), € 177,000.00, 2014–2016; ao. Univ.-Prof. from these studies will hopefully allow new who attend the out-patient clinics in Tyrol. Dr. Christoph Ebenbichler insights into diet-specific contributions of Up to date, the DRT population comprises Body weight and cellular aging: effects of weight loss on the various tissues in pathophysiology of insulin approximately 7,500 patients and therefore telomere length, Austrian Science Fund (FWF), € 154,000.00, resistance and its harmful consequence on covers about 15 % of all prevalent diabetes 2014–2016; Priv.-Doz. Dr. Markus Laimer VASCage – Research Center of Excellence in Vascular Ageing, Aus- health. in Tyrol. A recent study investigated the trian Research Promotion Agency (FFG), COMET, € 360,000.00, association between diagnosis of type 2 2014–2018; Univ.-Prof. Dr. Herbert Tilg Metabolism, Diabetes and diabetes mellitus and certain types of HDL function and atherosclerosis: Studies in apolipoprotein E Knockout Rabbits, Austrian Science Fund (FWF), € 231,000.00, Atherosclerosis cancer in Tyrol. 2015–2018; ao. Univ.-Prof. Mag. Dr. Andreas Ritsch Our major research intentions are focused Christian Doppler Research Laboratory for metabolic crosstalk, on but not limited to bariatric surgery, Atherosclerosis Christian Doppler Research Association (CDG), € 770,000.00, type 1 and type 2 diabetes mellitus, Our research is focused on the implication 2015–2022; Assoz.-Prof. Priv.-Doz. Dr. Susanne Kaser atherosclerosis and its short and long of lipid and lipoprotein metabolism on the Collaborations term influence on metabolic homeostasis, development of cardiovascular diseases. • Charles A. Dinarello, Denver, Colorado, USA cardiovascular health, and telomere length. We are interested in the role of reverse • Georg Schett, Erlangen, Germany • Stefan Schreiber, Kiel, GermanyW cholesterol transport in this scenario. Our • Arthur Kaser, Cambridge, UK Bariatric Surgery research work encompasses basic research, • Fredrik Bäckhed, Gothenburg, Sweden • Patrice Cani, Brussels, Belgium Over the past decade, bariatric surgery was animal-based studies as well as clinical • Willem de Vos, Wageningen, The Netherlands one of our pronounced research topics. studies and is targeting the development of • Roberto Vettor, Verona, Italy We examined the short-, mid-, and long- new strategies for prevention and therapy term effects of pronounced and sustained of atherosclerotic diseases in humans. weight loss induced by bariatric surgery on metabolic homeostasis, cardiovascular Research Interests health and telomere attrition. Topics • Lipoprotein metabolism and atheroscle- examined in numerous studies on bariatric rosis surgery patients comprise phospholipid- • Reverse cholesterol transport with focus transfer-activity, HDL-C, markers of on High Density Lipoprotein (HDL), Cho- chronic inflammation, adipokines, visceral lesteryl Ester Transfer Protein (CETP) and adipose tissue, adipocyte fatty acid-binding Scavenger Receptor Class B Type I (SR-BI) protein, retinol-binding protein 4, markers • Transgenic rabbit models for atheroscle- of atherosclerosis, chemerin, matrix rosis metalloproteinase 7, interleukin 1 and 6, • Gene transfer (receptor mediated endo tumour necrosis factor alpha, plasminogen cytosis, viral vectors)

Research Report 2015 Medical University of Innsbruck 107 Center of Internal Medicine Internal Medicine II

Keywords • Treatment optimization in end stage liver disease and complications after liver Management of liver failure, chronic liver transplantation including recurrent and disease, cirrhosis and hepatocellular de novo malignancies, recurrent viral hep- carcinoma, treatment of chronic viral atitis and genetic risk factors for graft hepatitis, liver transplantation disease Treatment of portal hypertension and com- • Clinical trials with new therapeutic agents plications, endoscopic therapy of benign for liver and gut diseases. and malignant biliary and pancreatic disease, endoscopic diagnosis and treat- General Facts ment of benign and malignant mucosal disorders of the gastro-intestinal tract The department of internal medicine II links basic and clinical science in hepatology and Research Focus gastroenterology. It is structured into an inpatient ward, an outpatient center and the • Regulation of iron homeostasis with a spe- hepatology laboratory. This structure ena- cial emphasis on HFE and non-HFE hemo- bles us to carry out clinical research as well chromatosis. Functional characterisation as basic science. As a tertiary referential Director: of mutations in proteins involved in iron center for patients with gut and liver diseas- Univ.-Prof. Dr. Wolfgang Vogel metabolism. es and, in cooperation with the department

Contact: Anichstraße 35 6020 Innsbruck

[email protected] Phone: +43 512 504 23401 Fax: +43 512 504 24052 https://www.i-med.ac.at/patienten/ kliniken/innere_medizin_2.html

Fig. 1: Confocal microscopy (A) and iron export experiments (B) are part of functional characterization of ferroportin mutations. Confocal microscopy reveals prominent membrane fluorescence of wild-type and A77D, N144H, C326Y, A69T and D181V mutant GFP- ferroportin. Only ferroportin correctly localized to the plasma membrane is able to export iron from the cell. Wild-type, N144H, C326Y and A69T mutant ferroportin cause a more than 5-fold increase in iron export when compared to untransfected cells. No such increase can be observed in A77D and D181V mutant ferroportin overexpressing cells. These finding strongly support the concept that the apparent reduction in iron export function of D181V and A77D mutant ferroportin is caused by a defective pump mechanism and not by intracellular retention.

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of transplant surgery, as one of the three liver transplant centers in Austria, we have access to a large number of interesting patients with a wide spectrum of diseases. Furthermore our laboratory provides viral hepatitis diagnostics and genetic testing for HFE haemochromatosis and other genetic causes of iron disorders for primary and secondary care centers in western Austria.

The hepatology laboratory is a well-equipped laboratory carrying out clinical routine and basic research work. Full equipment for molecular biology work including genotyping, PCR, cloning and vector production is available. Furthermore, the laboratory has a sterile tissue culture hood and incubator for cell culture experiments, centrifuges, chromatography, ELISA reader and blotting equipment for biochemical experiments and a mass spectrometer. The close collaboration between our laboratory and the clinical wards is the key to translate findings made in basic research into clinical studies and to further investigate genetics and disease pathways in patients recruited from our inpatient or outpatient ward.

Research

Regulation of Iron Metabolism, HFE and Non-HFE Hemochromatosis The hepatology laboratory (Head Prof. Fig. 2: Proposed molecular mechanism of alpha-1-antitrypsin’s effect on hepcidin expres- Dr. Heinz Zoller) is primarily interested in sion via matriptase-2 inhibition: Hemojuvelin (HJV) is a membrane-bound coreceptor for the regulation of iron homeostasis with the heterodimeric BMP Type I receptor and BMP Type II receptor complex and is essen- a special emphasis on HFE and non-HFE tial for BMP-6-induced activation of SMAD proteins which increase hepcidin expression. hemochromatosis. Hemochromatosis is The serine protease matriptase-2 (MT-2) cleaves HJV and subsequently inhibits hepcidin the most common genetic and metabolic expression. Alpha-1-antitrypsin (A1AT) medicated inhibition of MT-2 thus stimulates hep- liver disease in adults and an important cidin expression via reduced hemojuvelin cleavage. differential diagnosis in patients with chronic liver disease. Although a majority of patients with hemochromatosis are beyond the sole classification of novel and causes a lethal neurological disease. A homozygous for the C282Y polymorphism mutations. focus of our working group is the functional of the HFE gene, non-HFE associated characterisation of novel mutations in the hemochromatosis variants like ferroportin Hepcidin regulation is a further research ceruloplasmin gene and the development disease or aceruloplasminemia are topic in our lab where we have shown that of new therapeutic strategies for this increasingly recognized to contribute to the hepcidin is induced by the acute-phase devastating disease. overall burden of iron overload disorders. protein alpha-1-antitrypsin, which is not primarily associated with iron metabolism. For this aim, we have established a colony Recent studies of our workgroup have Full exome sequencing in a family that of CP -knockout mice and are conducting investigated the disease mechanisms presented with severe alpha-1-antitrypsin experiments for viral gene transfer of the of novel mutations in the only known deficiency and liver iron overload at our intact CP -gene to establish a gene therapy mammalian iron exporter ferroportin. We outpatient center revealed the presence for this otherwise untreatable disease. have shown that some mutations cause a of heterozygous hepcidin mutations in Apart from this basic science work we loss of function while others cause a dose- affected individuals. Further in vitro studies have initiated a European registry to collect dependent hepcidin resistance. Hepcidin showed that alpha-1 antitrypsin regulates structured information on the clinical is known as the “iron hormone” and is the hepcidin expression via hemojuvelin and presentation, biochemistry, radiology, family main regulator of iron homeostasis. The matriptase-2. history, genetics and histology of patients results from our studies further indicate Aceruloplasminemia is another genetic with non-HFE hemochromatosis, ferroportin that intact iron export might be necessary disease causing liver iron overload but disease and aceruloplasminemia. This will for hepcidin-induced downregulation of also iron deposition in the central nervous help us determine the incidence, prognosis, ferroportin and therefore have implications system, which cannot be treated so far treatment strategies and complications

Research Report 2015 Medical University of Innsbruck 109 Center of Internal Medicine

of these rare syndromes and to identify major problem in hepatology. The second with Dr. Henninger from the department patients eligible for future clinical studies. research focus of our department is thereof radiology we have shown that MRI is a fore optimisation of patient care in this pop- valuable tool for non-invasive quantification Clinical Research Liver Disease and ulation and the improvement of long-term of liver iron and the degree of steatosis. This Liver Transplantation survival and quality of life in patients who will help us reduce the need of liver biopsies Chronic liver diseases can result in end undergo liver transplantation. The access to and gives us the possibility to follow-up stage liver disease or hepatocellular car- a large number of patients with chronic liver patients non-invasively, for example during cinoma necessitating liver transplantation disease and liver transplant recipients via clinical trials in non-alcoholic fatty liver dise as the only curative treatment. Although our inpatient and outpatient center enables ase. Recently, we were able to show that liver chronic hepatitis C, a formerly frequent us to carry out clinical research in the pre- transplantation is an excellent treatment cause of chronic liver disease, will de- and post-transplant phase. modality in patients with acute-on-chronic crease as a consequence of effective direct liver failure, but only a minority of affected antiviral agents, chronic or even end stage Magnetic resonance imaging (MRI) is an patients will undergo liver transplantation liver disease caused by alcoholic or non- important tool in the evaluation of patients as the mortality on the waiting list is high. alcoholic fatty liver disease, hemochroma- with chronic liver diseases. In several re This has further implications for organ tosis and metabolic diseases will remain a search projects conducted in collaboration allocation, which, at the moment, does not allow high urgency status for patients with chronic liver disease. 05.11.2008 – pre treatment 22.04.2009 – 2 months Deferasirox If patients successfully undergo liver transplantation, careful follow-up is a requirement to achieve good long-term survival rates. Recurrence of the underlying 750 disease is a common problem, especially in patients transplanted for fatty liver disease or hepatocellular carcinoma. We have identified rs738409-G in the PNPLA3

22.07.2009 – 5 months R2* 12.01.2011 – 10 months gene as a risk factor for hepatic triglyceride Deferasirox Deferiprone accumulation and graft steatosis after transplantation. Furthermore we were able to show that patients with hepatocellular 0 carcinoma outside classic transplant criteria can undergo transplantation without increased risk of recurrence depending on their response to neoadjuvant therapies. These observations can lead to a more tailor-made post-transplant surveillance adjusted to the individual risk factors of transplant recipients. 05.11.2008 – pre treatment 22.04.2009 – 2 months Deferasirox The future goal of our research unit is to further improve pre- and post-transplant care in patients with liver disease and to translate findings from basic research into clinical research and finally into clinical routine.

Clinical Trials in Inflammatory Bowel Disease and Chronic Liver Diseases 12.01.2011 – 10 months Deferiprone As a tertiary referral center for gut and liver diseases the department of internal medicine II has a large number of patients potentially available for clinical trials. This enables us to collaborate with other academic centers or pharmaceutic companies in multicenter trials and ensures our patients access to novel therapies not yet available outside clinical studies.

Fig. 3: Non-invasive liver and brain iron quantification in a patient with aceruloplasminemia. We conduct clinical trials in inflammatory Therapy with oral iron chelators caused a rapid normalization of liver iron concentration bowel disease, viral hepatitis, biliary but did not affect brain iron deposition or neurological symptoms. diseases, hepatocellular carcinoma or non-

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alcoholic liver disease. To ascertain the high quality requirements of good clinical practice our department employs two study coordinators, Mag. Toaba and Mag. Zotter.

Selected Publications Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor. Praschberger Roman, Schranz Melanie, Griffiths William JH, Baumgartner Nadja, Hermann Martin, Lomas David J, Pietrangelo Antonello, Cox Timothy M, Vogel Wolfgang, Zoller Heinz. BIOCHIMICA ET BIOPHYSICA ACTA. 2014; 1842(9): 1406–1412. Patatin-like phospholipase domain-containing protein 3 rs738409-G in recipients of liver transplants is a risk factor for graft steatosis. Finkenstedt Armin, Auer Claudia, Glodny Bern- hard, Posch Ursula, Steitzer Hansjoerg, Lanzer Gerhard, Pratsch- ke Johann, Biebl Mathias , Steurer Michael, Graziadei Ivo W, Vogel Wolfgang, Zoller Heinz. CLINICAL GASTROENTEROLOGY AND HE- PATOLOGY. 2013; 11(12): S.1667–1672.

Acute-on-chronic liver failure: excellent outcomes after liver transplantation but high mortality on the wait list. Finkenstedt Armin, Nachbaur Karin, Zoller Heinz, Joannidis Michael, Pratschke Johann, Graziadei Ivo W, Vogel Wolfgang. LIVER TRANSPLANTATION. 2013; 19(8): S. 879–886.

MNGIE Syndrome: Liver Cirrhosis Should Be Ruled Out Prior to Bone Marrow Transplantation. Finkenstedt Armin, Schranz Melanie, Bösch Sylvia, Karall Daniela, Scholl-Bürgi Sabine, Ensinger Christian, Drach Mathias, Mayr Johannes A, Janecke Andreas R, Vogel Wolfgang, Nachbaur David, Zoller Heinz. JIMD REPORTS. 2013; 10: S.41–44.

The ART of decision making: retreatment with transarterial che- moembolization in patients with hepatocellular carcinoma. W Sieghart, M Pinter, F Hucke, I Graziadei, M Schöniger-Hekele, C Müller, W Vogel, M Trauner, M Peck-Radosavljevic M. Hepatology. 2013; 57(6):2261–73.

A single determination of C-reactive protein at the time of diagnosis predicts long term outcome of patients with hepatocellular carcinoma. W Sieghart, F Hucke, M Pinter, I Graziadei , W Vogel, C Müller, H Heinzl, M Trauner, M Peck-Radosavljevic. Hepatology 2013. 57(6):2224–34.

The ART-strategy: sequential assessment of the ART score predicts outcome of patients with hepatocellular carcinoma re-treated with TACE. F Hucke, W Sieghart, M Pinter, I Graziadei, W Vogel, C Müller, H Heinzl, F Waneck, M Trauner, M Peck-Radosavljevic. J Hepatol. 2014; 60(1):118–26. How to STATE suitability and START transarterial chemoemboliza- tion in patients with intermediate stage hepatocellular carcinoma. F Hucke, M Pinter, I Graziadei, S Bota, W Vogel, C Müller, H Heinzl, F Waneck, M Trauner, M Peck-Radosavljevic, W Sieghart W. J Hepatol. 2014 Dec; 61(6):1287–96.

Collaborations • M. Peck-Radosavljevic, Vienna Medical University • MARC CREUS, Department of Chemistry, University of Basel, Basel, Switzerland • RANIA BAKRY, Department of Analytical Chemistry & Radi- ochemistry, Leopold Franzens University of Innsbruck, Inns- bruck, Austria • WILLIAM J. H. GRIFFITHS and TIMOTHY M. COX, Department of Medicine, Cambridge University Hospitals, NHS Foundation Trust, Cambridge, England • ANTONELLO PIETRANGELO, Center for Hemochromatosis, Uni- versity Hospital of Modena, Modena, Italy • MAYKA SANCHEZ-FERNANDEZ, Department of Genetics and Epigenetics, Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Barcelona, Spain

Devices • Maldi-TOF mass spectrometer • 2D high pressure liquid chromatography • Fast protein liquid chromatography • French Press • Lyophylisation • CCD-Camera/Imager • Genetic sequencing for different genes of iron regulation • Full exome sequencing (in collaboration) • Proteomics/Functional characterisation • HFE genotyping, HBV and HCV genotyping and quantification

Research Report 2015 Medical University of Innsbruck 111 Center of Internal Medicine Internal Medicine III

reduce the progression of diseased hearts degradation by blocking the protease DPP- to chronic heart failure, once lost, replace- IV/CD26. This strategy preserved SDF ‑1 af- ment of functional myocardium is very lim- ter MI, enhanced the migration of CXCR4+ ited. Therefore, therapies to augment and blood derived progenitors, improved car- preserve myocardial tissue are warranted. diac function, and decreased mortality in mice (Fig. 1). The same treatment combined 1. Cardiovascular Regeneration and Aging, with cell cycle activation in cardiomyocytes 2. Cardiac Imaging and Translational was even capable of enhancing myocardi- Research, and al regeneration after MI. Based on these 3. Clinical Studies. promising findings, we initiated the first clinical trial analysing the effect of G-CSF The focus of our experimental approach and a DPP-IV inhibitor treatment on cardiac in column 1 is the development of novel function after acute MI (SITAGRAMI-TRIAL; strategies for cardiac regeneration and EudraCT number: 2007-003941-34). neovascularization. Our specific aim is to understand the cell recruitment of progeni- However, SDF ‑1-CXCR4-mediated cell tor cells to the ischemic heart and vessels, recruitment and repair mechanisms are as well as the process of regeneration still barely understood. Therefore, we are Director: through transition from the neonatal stage currently working to expand our knowledge Univ.-Prof. Dr. to adulthood with the intention to develop about SDF ‑1/CXCR4 based cardiac repair Wolfgang-Michael Franz new regenerative therapies. Furthermore, utilizing novel CXCR4-EGFP reporter mice we are interested in the process of athero and tissue specific SDF ‑1 knock-out mice to Contact: sclerosis and angiogenesis. Another im- enhance the recruitment of progenitor cells Anichstraße 35 portant aspect of our experimental work to the heart. Our preliminary data suggest 6020 Innsbruck is to elucidate the process of aging. Since that inhibition of prolyl hydroxylase may patients suffering from premature aging be a promising target for HIF ‑1a mediated [email protected] (progeria) develop severe cardiovascular SDF ‑1 activation to increase stem cell Phone: +43 512 504 25613 morbidities like stroke, myocardial infarc- homing and myocardial repair. Fax: +43 512 504 25622 tion, and severe atherosclerosis, we are Future Goals: http://inneremed3.tirol-kliniken.at/ currently using this model to investigate • Lineage tracing of regenerative cell popu- new targets which can inhibit these aging lations in the neonatal and adult heart to processes. In our second column, we aim develop new therapies for severe heart Keywords to develop novel imaging protocols for TAVI failure. patients or to look for novel biomarkers with • Elucidating the role of aging-related en- cardiac regeneration, neovascularization, MRI based protocols. zymes in the development and progres- stem cells, SDF ‑1, CXCR4, DPPIV, transla- sion of heart failure. tional research, MRI, MSCT, clinical studies We are also focusing on translational research that combines basic research with II. Aging Related Splicing Variants in Research Focus clinical data. Additionally, our research unit Patients with Cardiomyopathy takes part in multinational projects and Ass.-Prof. M. M. Zaruba, S. K. Ghadge The focus of our cardiovascular research clinical trials that are depicted in column 3. (PhD), M. Messner (PhD student) group is based on 3 complementing Defined mutations in the human lamin columns to develop novel therapies for car- Research A gene or in enzymes processing the diac and vascular regeneration as well as important nuclear membrane protein LMNA the implementation of new imaging meth- Column 1: Cardiovascular Regeneration (e.g. Zmpste24) are causally involved in ods and translational research to the clinic: and Aging premature aging syndromes like progeria. Column 1: Cardiac Regeneration and I. Therapeutic Targeting of CXCR4+ Patients suffering from progeria develop Aging: The aim is to develop novel therapies Cell Populations in the Ischemic Heart severe cardiovascular morbidities like to build new heart tissue and vessels, Ass.-Prof. M. M. Zaruba, S. K. Ghadge stroke, myocardial infarction, and severe Column 2: The major goal is to utilize novel (PhD), M. Messner (PhD student) atherosclerosis leading to early death. imaging technologies like MSCT or MRT to In previous work, we showed that stem We are currently using this model to monitor and improve clinical therapies, cell mobilization with Granulocyte colony- investigate new targets that can inhibit Column 3: Monitoring of international stimulating factor (G-CSF) and Parathyroid this aging process. In the current projects phase 2–3 Clinical studies. hormone (PTH) was associated with im- we investigate whether premature aging proved cardiac function after MI. However, enzymes, like LMNA, play a role in the General Facts a major drawback is the inactivation of the development of heart failure. important homing factor, stromal derived Heart Diseases are the main reason for factor 1 (SDF ‑1) by activated proteases, like III. Neonatal MI Mouse Model to Study mortality and morbidity in the Western DPP-IV after myocardial infarction. There- Cardiac Regeneration world. Is-chemic cardiomyopathy following fore, we developed a novel dual medical Dr. B. Haubner (PhD), myocardial infarction (MI) is the most preva therapy based on mobilization of progenitor T. Schütz (PhD student) lent. Although new medical therapies can cells with G-CSF and inhibition of SDF -1 Cardiac regeneration is one of the prime

112 Research Report 2015 Medical University of Innsbruck Internal Medicine III

like vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (FGF), secretoneurin and catestatin induce proliferation and chemotaxis of endothelial cells and protect them from apoptosis. A therapeutic application was tested in vivo in several animal models by our group. In the so-called hind limb ischemia model, the application of a secretoneurin- expressing plasmid or catestatin as peptide was associated with a dramatic reduction of ischemia-related tissue defects and a significant improvement in limb reperfusion.

In the experimental myocardial infarction model, treatment with secretoneurin significantly improved cardiac parameters like left ventricular ejection fraction and reduced infarct size. These effects were induced by interaction with several tyrosine kinase receptors like VEGF ‑receptor 2 or mem- © modified according to Zaruba et al. , Expert Opin Biol Ther 2010 bers of the FGF ‑receptor family. Recently, Fig. 1: Targeting of CXCR4+ cells with DPP-IV/CD26 inhibition plus G-CSF treatment. After we could demonstrate effectiveness and acute myocardial ischemia, G-CSF administration mobilizes CXCR4+ progenitor cells from safety of secretoneurin gene therapy also BM. Thereupon, CD34+CXCR4+ cells circulate in enhanced numbers to the injured heart. in animal models with impaired vascular In the myocardium, CXCR4+ cells interact with functionally intact SDF-1. Pharmacological response like the Apo E -/- mouse. inhibition of DPPIV/CD26 prevents the degradation of intact SDF-1. Thus, stabilization of Future Goals: SDF-1 improves homing of mobilized stem cells. This strategy yielded enhanced neovas- • Development of tissue-specific gene ther- cularization, reduced cardiomyocyte apoptosis and finally improved cardiac function and apeutic vectors, survival by attenuating the development of ischemic cardiomyopathy. • clarify the molecular mechanism of secretoneurin and catestatin induced actions visions in cardiovascular therapeutics. transcriptome during the first week of on vascular cells in more detail. Accumulating experimental evidence hint at life using RNA-Seq (Fig. 2). Interesting a modest annual cardiomyocyte turnover in candidate genes will be identified using V. Macrovascular Protection and the adult mammalian heart that is obviously bioinformatics and consecutively targeted Regeneration not sufficient to replace millions of lost in vitro and in vivo to test the potential Dr. Christoph Brenner and Friedericke cardiomyocytes upon myocardial injury. influence on cardiac regeneration. Remm (Vet. med. PhD student) Newts and zebrafish are well-knownin vivo Future Goals: Identification of novel Disturbances of the vascular endothelial animal models for cardiac regeneration regulators of cardiac regeneration using integrity can occur during the process of but the evolutionary distance to humans the above described mouse neonatal atherosclerosis and its interventional therapy limits their translational potential. In 2011, myocardial infarction model in combination in diseased arteries. De‑endothelialized experiments by Porrello et al. reported a with our transcriptome database. The areas of the vascular wall can lead to local short window after birth of complete cardiac long-term aim of our group is to stimulate thrombus formation and thus can impair regeneration following apical resection in the cardiac regeneration in adult mammalian blood flow with resulting ischemic events. neonatal mouse. Stimulated by their work hearts by studying in vivo neonatal cardiac Although endogenous endothelium has we independently established a neonatal regeneration as an experimental model. remarkable capabilities for self-renewal, the mouse model of left anterior descending time span between injury and endothelial artery (LAD) ligation and proved excellent IV. Novel Strategies for Improved recovery is precarious. Therefore, we have recovery of murine neonatal hearts after Angiogenesis recently established a therapeutic regimen clinically relevant myocardial infarction ao. Univ.-Prof. Rudolph Kirchmair and to accelerate endothelial regeneration. (MI). We demonstrated that the heart of Dr. Markus Theurl After arterial denudation, attached platelets, neonatal mice regenerates after massive Our research focuses on endothelial cell activated endothelial and smooth muscle MI within one week after LAD ligation. biology and angiogenesis. Over the last cells secrete the cytokine SDF1 that can Importantly, this regenerative potential is years our laboratory identified the neuro bind to the CXCR4 receptor of circulating lost within seven days after birth. Therefore, peptides catestatin and secretoneurin as progenitor cells (ciPCs). Subsequently, we are fascinated by the question: What are novel angiogenic factors. These peptides these cells are recruited to the injured the transcriptional changes within the first derive from the precursor molecules vascular wall. After migration into the seven days that transform a mammalian secretogranin II or chromogranin A and diseased tissue, ciPCs can accelerate heart from a regenerative to a scarring are found mostly in the neuroendocrine endothelial regeneration mainly by secretion state? In order to answer this question, we system but are also expressed in the of paracrine factors. By pharmacological set out to analyse the time course changes ischemic skeletal muscle and myocardium. inhibition of SDF1 cleavage, e.g. by using of the mouse cardiac coding and noncoding Similar to prominent angiogenic factors the DPP4-inhibitor Sitagliptin, we were able

Research Report 2015 Medical University of Innsbruck 113 Center of Internal Medicine

to boost ciPC-recruitment and vascular re- delay after acute myocardial infarction is a European Union funded multicenter Re- endothelialization (Fig. 3). Future projects after primary-PCI using late-enhancement- search and Development project conducted of our group will now investigate the impact MRI. The group also utilizes this technique at six hospitals in the European Union bring- of pharmacological DPP4 inhibition on the to investigate novel biomarkers like ing together a pan-European consortium of development of atherosclerosis. copeptin with respect to the correlations of leading companies, universities, hospitals infarct size, as well as acute inflammatory and healthcare suppliers. VI. Experimental Ischemia/ processes after an acute myocarditis. The Reperfusion in Vivo Mouse Model group could show a good correlation with IV. Sleep apnea syndrome and cardiac re- Doz. Bernhard Metzler an impaired microvascular perfusion. Actual synchronization therapy in patients with The group investigates cardioprotective projects include the evaluation of pulse- conventional pacemakers. The study is mechanisms to reduce ischemia/reperfu- wave-velocity measurements of the aorta in funded by the OeNB Anniversary Fund. sion injury after myocardial infarction. In patients after acute MI. W. Dichtl particular, we are interested in signal transduction cascades PI3K and migfilin. III. Lab-2-Go: V. TAKINSULA: pilot study to evaluate Prof. J. Mair, S. Plangger, B. Will, ventral venous/hormonal changes in Column 2: Cardiac Imaging and K. Wegscheidler TakuTsubo cardiomyopathy. W. Dichtl Translational Research The Lab2Go project, co-funded by the Euro- I. New Cardiac Imaging Appraoches pean Commission, is a study to determine VI. P-Select in Study. with Multislice Computed Tomography the value of a point-of-care testing system P. Marschang, R. Kirchmair (MSCT) for measuring cardiac Troponin-I (cTnI) at The purpose of the study is to evaluate the ao Prof. G. Feuchtner, the patients’ bedside as an aid in the di- correlation of atherosclerotic plaque volume ao Prof. G. Friedrich, F. Plank agnosis of patients with the indications of and intima-media-thickness with soluble The aim of the group is to develop new a Myocardial Infarction (MI). A blood sam- p-selectin as a potential new biomarker for protocols for cardiac imaging utilizing multiple can be taken and tested by the doc- atherosclerosis. The study is funded by the slice computed tomography (MSCT). tor, nurse or paramedic to provide a cTnI OeNB Anniversary Fund. In particular, the following research topics measurement during clinical assessment, are on the agenda: rather than having to wait for laboratory re- VII. The Role of FGF23 and Klotho in • evaluation of TAVI patients with MSCT, sults. The aim is to have a solution available Chronic Heart Failure particularly with respect to calcification at the end of the project which meets the G. Pölzl, S. K. Ghadge, M. Messner, and their impact on the TAVI procedure. targeted system specifications, is accept- M. M. Zaruba • evaluation of the diagnostic value of MSCT ed by the intended users and is ready for The project comprises clinical studies in patients suffering from chest pain in co- clinical validation. The Point-of-Care sys- in a large heart failure cohort on the operation with EU sponsored multi-center tem that will be demonstrated is referred predictive role of FGF23 and sKlotho, as studies (DISCHARGE). to as the Minicare system – an innovative well as molecular, immunohistological, and • value of MSCT to relate coronary morphol- biophotonics based technology that em- immunofluorescence in endomyocardial ogy and size to the prediction of hemo ploys a surface sensitive optical detection biopsies from myopathic hearts. dynamic relevance. technique to determine a biomarker con- • MSCT based measurement of fractional centration in blood. The system is designed VIII. LevoRep Study flow reserve (FFR) to evaluate the hemo- to enable various biomarkers to be meas- G. Pölzl dynamic significance of coronary artery ured in whole blood within 10 minutes at The LevoRep study is a multicenter stenosis and the impact on further thera- lab-quality precision. The Lab2Go project randomised trial to test the efficacy and peutic approaches. • evaluation of coronary segments after Bioresorbable Vascular Scaffold (BVS) implantation: Possibilities of non invasive assessment of BVS. • development and composition of an Aus- trian TAVI registry with data related to the pre-peri-post interventional results together with the department of cardiac surgery. • development of general guidelines for MSCTA based appraisal of coronary arteries and protocols.

II. Development of New Heart-MRI Protocols and Biomarkers Doz. Bernhard Metzler, © Haubner et al. Aging, 2012 Ass.-Prof. G. Klug, S. Reinstadler, Fig. 2: Complete cardiac regeneration 28 days following myocardial infarction in a neonatal H. Feistritzer mouse heart (P1.5). Regenerative potential is lost within the first week after birth (P7.5). The focus of the group is to investigate the Transcriptional analyses between neonatal and 7 day old mouse hearts are used to unravel correlation of infarct size with the patient the enigma of bona fide in vivo cardiac regeneration.

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tigate the effects of POL6326 (CXCR4 antagonist) as a stem cell mobilizing agent, on cardiac function and infarct size and on safety and tolerability, in patients with reperfused ST-Elevation Myocardial Infarction (STEMI). PI: MM Zaruba.

Registries: • GABI_R registry German-Austrian registry to evaluate of short and long-term safety and success of the ABSORBTM Everolimus-coated bio resorbable scaffolds in patients with coronary artery disease. G. Friesdrich • Austrian TAVI registry (long-term follow-up) Source Registry. G. Friedrich • Austrian Heart Failure Registry. G. Pölzl • Acute Coronary Syndrome – STEMI pilot registry. J. Dörler • EORP Atrial Fibrillation Ablation Long-term Registry. M. Stühlinger

Selected Publications © Christoph Brenner Pluripotent-stem-cell-derived epicardial cells: a step toward artifi- Fig. 3: Schematic effect of Gliptin therapy. Circulating CXCR4+ progenitor cells can home to cial cardiac tissue. Brenner C, Franz WM. Cell Stem Cell. 2014 Nov 6;15(5):533–4. doi: 10.1016. sites of endothelial injury via the SDF1/CXCR4 pathway. SDF1 that is secreted by attached First treatment of a child suffering from severe ischemic cardi- platelets and activated smooth muscle and surrounding endothelial cells can bind to the omyopathy with G-CSF and sitagliptin. Zaruba MM, Hiergeist L, CXCR4-receptor and thus facilitate recruitment of progenitor cells into the injured vascular Mechea A, Kozlik-Feldmann R, Theisen D, Netz H, Franz WM. Int J Cardiol. 2013 Dec 10;170(2):e41–2. Epub 2013 Oct 26. wall. The dipeptidyl peptidase 4 (DPP-4) inactivates SDF1 by cleavage of two N-terminal Left ventricular global function index: Relation with infarct char- amino acids. Pharmacological inhibition of DPP-4 (Gliptin therapy) leads to an enhanced acteristics and left ventricular ejection fraction after ST-segment recruitment of progenitor cells. These progenitors can mediate an accelerated endotheli- elevation myocardial infarction. Reinstadler S, Klug G, Feistritzer HJ, Mayr A, Kofler M, Aschauer A, Schocke M, Müller S, Franz WM, al regeneration mainly by paracrine effects. (ECM = extracellular matrix, SM = smooth Metzler B. Int J Cardiology. 2014; 175: 579–81. Epub ahead of muscle cell, EC = endothelial cell, P = platelet). print 2014 Jun 10. Short-term inhibition of DPP-4 enhances endothelial regeneration after acute arterial injury via enhanced recruitment of circulating safety of pulsed infusions of levosimendan intravenous (IV) ferric carboxymaltose on progenitor cells. Brenner C, Kränkel N, Kühlenthal S, Israel L, in outpatients with advanced heart failure. exercise capacity in patients with chronic Remm F, Fischer C, Herbach N, Speer T, Grabmaier U, Laskowski A, Gross L, Theiss H, Wanke R, Landmesser U, Franz WM. It is an international study organized heart failure and iron deficiency. Int J Cardiol. 2014 Nov 15;177(1):266–75. and conducted at the Medical University PI: G. Pölzl Long-term predictive value of copeptin after STEMI: a cardiac Innsbruck. The study was started in 2009 • RELAX-AHF ‑EU (phase 3) magnetic resonance study. Reinstadler SJ, Klug G, Feistritzer HJ, Mair J, Schocke M, Franz FM, Metzler B. Int J Cardiology. 2014, and finalized in 2013. A biomarker sub- Sponsor: Novartis Pharma GmbH 172:359–360. Epub ahead of print 2014 Jan 16. study, in cooperation with the Medical Aim: This is a multinational, multicenter, University Würzburg, Germany (S. Störk, S. randomized, open-label study to confirm Selected Funding • MesP1 dependent signalling pathways during the formation of Frantz), is still ongoing. and expand the efficacy, safety and toler- common cardiovascular progenitors in vivo and in vitro. Deut- ability evidence of 48 hours intravenous sche Forschungsgemeinschaft (DFG). W. M. Franz • Elucidating the fate of CXCR4+ cell populations in the ischemic Column 3: Clinical Studies infusion of serelaxin (30 micrograms/ heart. Deutsche Forschungsgemeinschaft (DFG). M. Zaruba Our clinical study team supports the kg/day) when added to Standard of Care • Catestatin zur Behandlung der kardialen Ischämie. Fonds zur clinic with conducting national and (SoC) in patients admitted to hospital for Förderung der wissenschaftlichen Forschung (FWF). M. Theurl • Lab2Go project. EU-FP7 programm. J. Mair international multi-centre trials to answer Acute Heart Failure (AHF). • Anlaysis of the protective effects of pharmacological DPP-4-in- important issues in current cardiology. The PI: G. Pölzl hibition on endothelial regeneration and atherosclerosis. Else Kröner-Fresenius-Stiftung. Christoph Brenner following clinical trials are currently under • EINSTEIN-CHOICE (phase 3) • Schlafapnoe und kardiale Resynchronisation bei Patienten mit investigation: Sponsor: Bayer bisher konventioneller Schrittmachertherapie. OeNB Anniver- sary Fund. W. Dichtl • Global Leaders (phase 3) Aim: This is a multicenter, randomized, • P–Selectin Studie. OeNB Anniversary Fund. P. Marschang Sponsor: AstraZeneca double-blind, event-driven, superiority Aim: To determine if there is a better medi- study for efficacy. Patients with confirmed Collaborations • Michael Bader, Max-Delbrück-Center for Molecular Medicine cation strategy to prevent blood from clot- symptomatic DVT (Deep Vein Thrombosis) (MDC), Robert-Rössle-Str. 10, 13125 Berlin, Germany ting and at the same time minimizing the or PE (Pulmonary embolism) who complet- • Loren Field, PhD, Riley Heart Research Center, Indiana Universi- ty School of Medicine, Indianapolis, USA number of complications. ed 6 or 12 months of treatment of antico- • Ulf Landmesser, Department of Cardiology, Charité – Universi- PI: G. Friedrich agulation are eligible for this trial. tätsmedizin Berlin, Germany • Rüdiger Wanke, Institute of Veterinary Pathology, Ludwig-Maxi- • FER-CARS-05 Study (phase 4) PI: P. Marschang milians-University, Munich, Germany Sponsor: Daiichi Sankyo Inc. • CATCH-AMI (Phase 2) • Univ.-Prof. Dr. O. Müller, University of Heidelberg, Germany • Sushil Mahata, PhD, University of California, San Diego, USA Aim: to determine, relative to placebo, Sponsor: Polyphor Ltd. • Dieter Zimmermann, Univ. Zurich, Switzerland the effect of iron repletion therapy using Aim: The purpose of this study is to inves- • Michail Sitkovsky, Dana-Farber Cancer Institute, Boston, USA

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Keywords inpatient facilities and a state-of-the-art unit for extra-corporal therapy (haemo- as Chronic kidney disease, pathophysiology, well as peritoneal dialysis, plasmapheresis, systems biology, stratified/personalized liver support therapy, immunoadsorption) medicine, epidemiology, autoimmune allow service to a large clinical population disease, haemodialysis, cardiovascular and this background drives our translational mortality, renal transplantation research efforts. The laboratories of the Department hold a clinical routine as well as Research Focus a molecular biology (including microarray facility) and cell culture unit. Our clinical In order to better characterize patho- trial core unit manages investigator driven physiologically complex phenotypes such projects as well as the participation in large as chronic kidney disease, we apply modern multicentre clinical trials and a large bio- epidemiology and “omics”-techniques in banking effort. The common denominator conjunction with state-of-the-art systems of the Department’s research activity is the biology approaches to derive predictive area of personalized medicine in the various biomarkers to implement innovative, aspects of Nephrology and we collaborate stratified/personalized treatments. This with multiple academic and industry Director: translational research focus is supported partners in national and/or EU funded Univ.-Prof. Dr. Gert Mayer by experimental and clinical studies in projects (EMERGENTEC biodevelopment selected populations and by strong national Vienna, AbbVie, TEVA, University of Vienna, Contact: and international collaborations. University of Groningen, STENO Diabetes Anichstraße 35 Center, University of Glasgow, Semmelweis 6020 Innsbruck General Facts University, University of Silesia, University Erlangen, Charite Berlin, Stanford University, [email protected] The Department is the tertiary referral etc.). During our recent activities, we Phone: +43 512 504 25855 centre for patients with renal disease (native focused primarily on the application of Fax: +43 512 504 25857 kidney, renal replacement therapy, kidney systems-biology techniques within the FP -7 www.nephrologie-innsbruck.at transplantation) and hypertension for the funded project SysKid (www.syskid.eu), to Western region of Austria and Southern which our Department was one of the main Tirol. Specialised outpatient, as well as contributors.

Fig. 1: Alterations of proximal tubular cell (PTC) phenotype and gene expression in response to tubular injury.

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Fig. 2: Integrative systems biology analysis of renal gene expression data shows differential regulation of hypoxia and angiogenesis pathways in stable and progressive kidney diseases.

Research is able to stimulate acute inflammation tubular cells from patients with proteinuric via its synergistic effects with other pro- nephropathies revealed significant Basic Research Activities inflammatory cytokines early after injury, transcriptional de-regulation of pro-fibrotic but may attenuate chronic inflammation and but also tubulo-protective (e.g. BMP-7) Cellular and Molecular Nephrology fibrogenesis at later time points. Additional mechanisms. When compared to patients Univ.-Prof. Dr. Herbert Schramek gene silencing approaches identified some with stable renal disease, patients with Renal fibrosis is the final, common pathway of the intracellular signalling pathways progressive renal failure showed attenuated leading to nephron loss. The patho involved, for example STAT3 as an OSM- tubular VEGF ‑A expression despite a strong physiological mechanisms include tubular induced signalling molecule involved in hypoxia signal (see Fig. 2). These gene cell injury, infiltration of inflammatory both inhibition of CTGF expression and expression profiles also helped to define cells, accumulation of (myo)-fibroblasts, super-induction of CCL2 mRNA expression new, clinically relevant biomarkers. and rarefaction of the peritubular micro in human PTCs. Future goals include vasculature. Of all the cell types involved, the functional analysis of novel genes of Univ. Doz. Dr. Michael Rudnicki, a member proximal tubular epithelial cells (PTCs) interest involved in the regulation of PTC of our group, has recently started to focus on play a central role. The laboratory is phenotype during PTC injury, inflammation simultaneous analysis of renal miRNA and predominantly interested in this specific and progressive renal fibrosis. mRNA profiles. His work revealed regulatory cell type during tubulointerstitial injury and networks, in which specific miRNAs activate fibrogenesis. Utilizing pro-inflammatory Translational Research Activities entire signal transduction pathways such as and pro-fibrotic ligands, such as IL-1β, inflammation or apoptosis. TNF ‑α, and TGF ‑β1, we not only investigate Transcriptional Profiling and Systems novel molecular mechanisms, which are Biology Application in Chronic Renal Recently, transcriptomics data were com associated with cellular injury leading to an Disease – Univ.-Prof. Dr. Gert Mayer plemented by proteomic, metabolomic and activated, dedifferentiated PTC phenotype, Several years ago, we established (in genomic profiles in the large, multinational but also those which induce cell protection collaboration with the University of EU FP-7 funded project SysKid (Systems or repair. Distinct human PTC lines have Stanford, California) microarray technology Biology towards Novel Chronic Kidney been used to investigate expression and to study whole organ and, via application Disease; www.syskid.eu). In collaboration regulation of several pro-inflammatory of laser capture micro-dissection, renal with EMERGENTEC biodevelopment and and pro-fibrotic genes including CCL2 and compartment specific differential mRNA the Medical Universities of Vienna and CTGF, respectively (Fig. 1). Interestingly and miRNA expression in human and animal Groningen we developed a proprietary we discovered that oncostatin M (OSM) tissue. When compared to normal controls systems biology-derived molecular model

Research Report 2015 Medical University of Innsbruck 117 Center of Internal Medicine

of renal disease in type II diabetes and Additionally, he is exploring alternative in a multicenter national study we identified identified molecular processes associated dialysis modalities, such as electro-osmosis factors associated with risk of peritonitis. with progressive renal function loss. in collaboration with Prof. Thomas Bechtold Interestingly, oral active vitamin D therapy Biomarkers associated with these pathways (Research Institute of Textile Chemistry and was associated with decreased incidence were discovered and validated in large Textile Physics, University of Innsbruck). and improved survival. patient cohorts. Currently, we are working Finally, he also collaborates with the to match the disease specific molecular Department of Experimental Urology in In collaboration with the Department of profiles with drug mode of action molecular the field of cytokine signaling and prostate Nephrology, Ospedali Riuniti di Bergamo, profiles to gain access to targeted cancer. Italy, we examined the effect of an anti CD20 therapy (Fig. 3). antibody in frequently relapsing nephrotic Another area of interest is the effect of age- In order to validate our “in silico” derived diseases in children and in adults. Based ing on transcriptional profiles in the kidney, hypotheses on predictive biomarkers, we on data obtained in a prospective study and an area of special relevance to the field of are also leading several large-scale, national a review, we were able to show that this renal transplantation. Ass. Prof. DDr. Hannes and multinational prospective cohort studies approach is a valuable therapeutic option. Neuwirt, another team member, is working (e.g. The Austrian Dialysis and Transplant In collaboration with the Department for on biomarkers that predict long-term graft Registry; PROVALID, a study of over 4,000 Pediatrics, we treat and recruit patients function and is also interested in the role patients with type 2 diabetes in 5 European with rare diseases, in particular Fabry’s of the complement system in kidney trans- countries; TOPVAS, a project including over disease, into a European multicenter study plant models. 240 patients after renal transplantation in on enzyme replacement therapy. Austria). The data collected there form the In this context, we have recently published basis for outcomes and health economics Regarding the excessive cardiovascular a 3-biomarker-panel that was able to research on a European level. mortality in patients with end-stage renal predict renal function on top of serum disease, we are interested in the possible creatinine. We are currently establishing Clinical Research Activities detrimental effect of positive calcium mass a biobank of serum, urine and renal tissue balance during haemodialysis. We propose of zero-hour biopsies (taken at the time Targeted Therapy in Renal Disease that a disturbance in the rapidly accessible of transplantation) in renal transplant Doz. Dr. Rudnicki (PD, anti CD20, Mb. bone calcium pool in chronic renal disease recipients and a corresponding prospective Fabry), Dr. Markus Pirklbauer (cardio- diminishes the capacity to buffer a calcium registry in order to establish and validate vascular mortality on hemodialysis); load, thereby contributing to vascular biomarkers of renal function in this cohort. Dr. Andreas Kronbichler (autoimmune calcification. Based on an individual In the same framework, Dr. Neuwirt is also diseases) OENB grant (“Impact of the exchangeable working on the role of the complement Peritonitis is the most serious complication calcium pool on cardiovascular risk in system in chronic allograft nephropathy in in patients on peritoneal dialysis (PD). In an chronic hemodialysis patients”, Dr. Markus kidney transplant models in vitro. analysis of PD patients treated locally and Pirklbauer) a novel assessment approach

Fig. 3: Interference of a drug mechanism of action molecular model (left) with the diabetic nephropathy phenotype molecular model (right). Interfering processes on the drug (blue) and phenotype side (red) are indicated.

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for calcium mass balance and calcium experimental setting to a) increase the buffer capacity has been established in understanding of these diseases, b) bring hemodialysis patients. To elucidate patho forward this understanding to clinical utility physiological mechanisms underlying and c) translate this knowledge into design the high cardiovascular mortality in of clinical trials with the aim to individualize/ hemodialysis patients, we evaluate the personalize treatment measures. These association of our calcium kinetic data with effort are driven forwards via a collaboration clinical outcome and attempt to identify with Dr. David Jayne (Cambridge University novel biomarkers by using ELISA-based Hospitals, UK). serum analysis. Based on these new insights in calcium regulatory mechanisms, it is our Selected Publications aim to individualize hemodialysis treatment From molecular signatures to predictive biomarkers: modeling disease pathophysiology and drug mechanism of action. Heinzel by using novel approaches for calcium mass A, Perco P, Mayer G, Oberbauer R, Lukas A, Mayer B. balance assessment in order to improve Front Cell Dev Biol. 2014; 2:37. cardiovascular prognosis. This innovative A 3-biomarker-panel predicts renal outcome in patients with proteinuric renal diseases. Neuwirt H, Perco P, Kainz A, Mühlberger I, clinical project is performed in collaboration Leierer J, Braniff SJ, Mayer B, Mayer G, Rudnicki M. with national (Central Institute for Clinical BMC Med Genomics. 2014; 7:75. Chemistry and Laboratory Medicine, Tacrolimus increases Nox4 expression in human renal fibroblasts Innsbruck, Head: Prof. Griesmacher) and and induces fibrosis-related genes by aberrant TGF ‑beta receptor signalling. Kern G, Mair SM, Noppert SJ, Jennings P, Schramek H, international (PD Dr. med. Andreas Pasch, Rudnicki M, Mueller GA, Mayer G, Koppelstaetter C. University Hospital for Nephrology and PLoS ONE. 2014; 9: e96377. Hypertension, Inselspital Bern, Switzerland) SOCS-3 is downregulated in progressive CKD patients and regulates proliferation in human renal proximal tubule cells in a partners. The kidney is often affected by STAT1/3 independent manner. Neuwirt H, Eder IE, Puhr M, autoimmune disorders. Rudnicki M. Lab Invest. 2013; 93:123–34. Renal involvement in autoimmune connective tissue diseases. Kronbichler A, Mayer G. BMC Med. 2013; 11:95. Our research aim is to delineate patho genetic steps that lead to the onset of auto- Selected Funding immunity, predicting treatment failure and • SKID – Systems biology towards novel chronic kidney disease diagnosis and treatment; EU - FP7 (€ 1,882,583) Gert Mayer relapse, in particular in nephrotic syndrome • PROVALID – Multi-centre study regarding the cumulative (membranous nephropathy, minimal change incidence of renal outcomes in patients with type II diabetes in different European countries; AbbVie research fund disease and focal segmental glomeruloscle- (US$ 4,200,000) Gert Mayer rosis), anti-neutrophil cytoplasm antibody • TOPVAS – The Transplant Outcome Prediction Validation Study; TEVA research grant fund (€ 732,000) Gert Mayer (ANCA)-associated vasculitis and system- • Hämoelektroosmose; Austria Wirtschaftsservice Ges.m.b.H. ic lupus erythematosus. We have worked (€ 150,000) Hannes Neuwirt • Der labile Kalziumpool und seine Bedeutung für das kardio- on several projects including rituximab’s vaskuläre Risiko in Hämodialysepatienten; OENB (€ 150,000) efficacy in minimal change disease and fo- Markus Pirklbauer cal segmental glomerulosclerosis and more recently in ANCA-associated vasculitis. In Collaborations • Bernd Mayer, EMERGENTEC biodevelopment GmbH; Vienna, the latter entity, we recently published a Austria review summarizing factors that predispose • Rainer Oberbauer, Division of Nephrology and Dialysis; Medical University Vienna patients towards infections. In the future, • Harald Mischak, Mosaiques Diagnostics GmbH; Hannover, Ger- we want to decipher risk factors leading to many • Peter Rossing, STENO Diabetes Center; Gentofte, Denmark infections in rituximab treated patients and • Johannes Mann, Universität Erlangen; Erlangen, Germany in larger ‘general’ cohorts. • Dick de Zeeuw, Hiddo Lambers Heerspink, Academisch Zieken- huis; Groningen, The Netherlands • Andrzej Wiecek, Slaski Uniwersytet Medyczny Katowicach; Kat- Moreover, we will study renal involvement in towice, Poland • Laszlo Rosivall, Semmelweis University; Budapest, Hungary the context of ANCA-associated vasculitis • Patrick Mark, University of Glasgow; Glasgow, UK within the Diagnostic and Classification • Kitty Jager, Academisch Medisch Centrum bij de Universiteit van Amsterdam; Amsterdam, The Netherlands Criteria in Vasculitis Study (DCVAS). • Mariano Rodriguez, Universidad de Cordoba; Cordoba, Spain With the help of modern laboratory • Timothy Meyer, Stanford University School of Medicine; Cali- fornia, USA approaches (‘omics’), we aim to improve • David Jayne, Vasculitis and Lupus Clinic, Cambridge University patient care substantially by identifying a Hospitals; United Kingdom • Annette Bruchfeld, Division of Renal Medicine, Karolinska Insti- panel of peptides/proteins indicative of tutet; Stockholm, Sweden disease relapse in proteinase 3-positive • Luis Quintana, Servicio de Nefrología y Trasplante Renal, Uni- versidad de Barcelona; Barcelona, Spain vasculitis using SWATH-mass spectrometry • Daiki Nakagomi, Department of Allergy and Clinical Immunolo- and working on the nasal microbiome gy, Chiba University; Chiba, Japan • Thomas Neumann, Department of Internal Medicine III, Jena (metagenomics) in patients with University Hospital; Jena, Germany granulomatosis with polyangiitis (GPA, • Jae Il Shin, Department of Pediatric Nephrology, Yonsei Univer- sity College of Medicine, Severance Children’s Hospital; Seoul, Wegener’s). Korea • Piero Ruggenenti, Department of Nephrology, Ospedali Riuniti di Bergamo/Italy This clearly highlights our future goal • Andreas Pasch, University Hospital for Nephrology and Hyper- to combine clinical findings with an tension, Inselspital Bern; Bern, Schweiz

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Keywords General Facts

Personalized cancer medicine, geriatric Within the Medical University Department oncology, ageing, tumor immunology, of Internal Medicine UCIM5 comprises a angiogenesis, myeloma, EpCAM 50 bed-hospital including 20-bed stem cell transplation unit, an outpatient clinic and Research Focus day hospital, an oncology trial center (OTC), laboratory services, translational research The University Clinic for Internal Medi facilities and a FACS sorting core facility. cine 5– Hematology & Oncology – (UCIM5) represents a core member of the Compre In general, UCIM5 provides state-of-the- hensive Cancer Center Innsbruck (CCCI) art clinical care primarily for patients with and shares its goals and research with all hematological malignancies and solid the partner institutions of the CCCI. Primary tumors. Solid tumors include primarily goals of CCCI are to improve the clinical cancers of the lung, GI tract and sarcomas. care and outcome of cancer patients by Clinical care includes diagnostic services developing and conducting state-of-the art such as cytomorphology, immuno clinical trials and translational research. phenotyping and molecular genetics. Director: UCIM5 conducts translational and clinical Approximately 15 % of the patients are Univ.-Prof. Dr. Günther Gastl research and participates in national and currently entered into clinical trials. international clinical trial programs. The Annually approximately 50 clinical trial Contact: Clinic is particularly well-placed to readily protocols are carried out by the OTC; our Anichstraße 35 translate basic research advances into clinical trial portfolio includes the following: 6020 Innsbruck clinical progress. Approximately 50 % • clinical studies for the establishment and of the clinical trial program is confined improvement of diagnostic techniques [email protected] to phase III trials. Clinical research also (e.g. minimal residual disease assess- Phone: +43 512 504 24003 includes retrospective and prospective ment by multicolor flow cytometry, molec Fax: +43 512 504 25615 observational studies. For this purpose, ular tumor profiling by next generation www.haematologie-onkologie.at national web-based registries for chronic sequencing), myeloid leukemia and multiple myeloma • phase I-VI clinical trials for the develop- have been established and are being ment of investigational new anticancer coordinated by our Clinic. Moreover, drugs or drug combinations and the opti- UCIM5 participates and serves as a country mization of existing anticancer treatments coordinator for the European MDS Registry • the evaluation and validation of prognostic of the European Leukemia Net (ELN). scores (e.g. for geriatric cancer patients), UCIM5 has been a founding institution of including the integration of geriatric the K1 Center ONCOTYROL (Competence assessment for elderly cancer patients Center for Personalized Cancer Medicine) to adjust treatment algorithms, improve and has been receiving major funding by clinical outcome, ameliorate toxicities and the Austrian Agency for Advancement of improve cost-effectiveness. Research (FFG) for promoting strategies to improve personalized cancer care and UCIM5 is an academic teaching hospital foster precision oncology. and forms an integral part of the University

Fig. 1: Real time confocal imaging of EpCAM processing and signalling in human cells. Cleavage of EpCAM results in the generation of an extracellular shedded domain (EpEX) and an intracellular part (EpICD) translocating to the nucleus and inducing Wnt signalling.

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Hospital Innsbruck and the Medical Translational Cancer Research tumor types and to predict responsiveness University Innsbruck. UCIM5 participates UCIM5 houses 4 translational research of EpCAM+ carcinomas for the anti-EpCAM in teaching & training activities for medical groups. Forward and reverse tanslational antibody treatment. Carcinoma cells in graduate students and postdoctoral fellows, research benefits from the clinical trial general and in particular cancer stem and offers a clinical PhD program for Clinical program, hospital- and population-based cells process EpCAM glycoprotein by cell Cancer Research. registries, in-house biobanks, diagnostic fa- surface-associated proteases into a soluble cilities and modern laboratory technologies. extracellular fraction (EpEX) and a short Clinical research, disease management intracellular signaling peptide (EpICD). and continouos medical education are Experimental & Clinical Oncogenomics; Shuttling of EpICD into the cell nucleus structured into 8 separate clinical programs Leaders: Gerold Untergasser, Ph.D. stimulates Wnt signalling, c-myc expression headed by physician scientists: Michael Steurer, M.D. and tumor cell proliferation. Assays for Gilbert Spizzo, M.D. EpCAM expression in tumor specimens and Clinical Programs & Program Heads The experimental oncogenomics group the analysis of soluble EpEX in blood and • Acute leukemias & stem cell works on the detection of tumor-derived malignant effusions have been developed transplantation: David Nachbaur, M.D circulating DNA (ctDNA) in peripheral by this group. • Indolent lymphomas: blood, liquor and malignant effusions. Michael Steurer, M.D. The functional importance of genetic The Clinical Oncogenomics group is • Agressive lymphoma: abnormalities is evaluated in tissue culture integrated into the Molecular Cancer Wolfgang Willenbacher, M.D. systems and murine tumor models. Diagnostics Center and focusses on • Myeloma & other plasma cell dyscrasias: As an example, overexpression of the the genomic profiling of solid and liquid Eberhard Gunsilius, M.D transmembrane adhesion molecule EpCAM malignancies. Recently, a public-private- • Myelodysplastic syndromes, geriatric on epithelial cancer cells was found to partnership with Caris Life Sciences hematooncology, anemia in the elderly: indicate poor prognosis for various solid (Phoenix, Arizona) has been set up for a Reinhard Stauder, M.D., M.Sc. • Myeloproliferative syndromes: Stefan Schmidt, M.D. • Gastrointestinal tumors: Wolfgang Eisterer, M.D. • Head/neck & thoracic cancers: Georg Pall, M.D • Breast & genitourinary cancers: Christoph Leitner, M.D. • Koagulopathies: Clemens Feistritzer, M.D. • Palliative Medicine & Supportive Care: Walpurga Weyrer, M.D.

Research

Clinical Research: Oncology Trial Center (OTC) Clinical Research is organized by the OTC. The OTC staff includes administrative personnel study nurses and clinical investigators. For the planning and performance of academic trials the OTC collaborates with the Clinical Trial Competence Center at the Medical University Innsbruck. The clinical trial portfolio within the time-period 2013/2014 is summarized below.

Tumor Entities / No. of Ongoing Clinical Trials (Phase I-II / III-IV) CLL 09 (03/06) Lymphoma (non CLL) 13 (03/10) Myeloma 04 (01/03) Acute leukemias 12 (05/07) Myeloproliferative neoplasis 09 (06/03) MDS 05 (01/04) GI-Cancers 23 (10/13) Fig. 2: The chaperone protein GRP78 can be overexpressed in various malignancies and re- Lung Cancers 10 (02/08) leased into the tumor microenvironment, thereby mediating resistance to anticancer drugs Sarcomas 03 (02/01) and angiogenic inhibitors.

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molecular profiling program to identify notherapeutic clinical program. Immune cells expressing a variety of activating druggable molecular tumor targets and to biomarker research includes monitoring of and inhibitory killer-cell immunoglobulin- offer cancer patients customized therapies circulating and tumor-associated immune line receptors (KIR) have been reported to based on their individual tumor profiles. As cells in various maligancies. By multidimen- mediate a GvT effect without GvHD. Thus, a next step, an in-house Next Generation sional flow cytometry, expression of CD62L the clinical success of allogeneic SCT relies Sequencing (NGS) facility will be established was found to be decreased on T cells of on a small window which may be predicted to complement routine cancer diagnostics CML patients and to predict response to by the mismatch of one or several mHags by histopathology, immune phenotyping, TKI treatment. and/or on the KIR ligand status of the host cytogenetics and PCR-based molecular Another area of immune biomarker re- as well as the donors’ activating KIRs. This genetics. search is the analysis of immune check- research group expanded HA-1-specific point antigen expression and the assesse- CD8+ T cells from the patient’s peripheral Tumor Immunology ment of tumor-infiltrating immune cells in blood following donor lymphocyte infusion Leaders: Sieghart Sopper, Ph.D. hematological malignancies (e.g. myeloma) concomitantly with the disappearance of Brigitte Kircher, Ph.D. and solid tumors. leukemic cells and identified among HLA- Major research topics of this group are the identical patient/donor pairs the mHag enhancement of T cell mediated antitumor Allogeneic hematopoietic stem cell HA-1 as a valid target for the GvL effect. immunity by modifying T-cell signalling and transplantation (HSCT) represents a Further research is warranted to get a more the search for clinically applicable immune curative approach for hematologic thorough understanding of the molecular biomarkers to predict responsiveness for malignancies based on the induction targets and mechanisms of the anti-mHag- immuno-oncological interventions. In this of graft-versus-tumor (GvT) reactivity. specific T-cell response and the importance respect inhibition of Cbl-b signalling by However, its effectiveness can be limited of NK cell activity after HSCT. siRNA in T cells was found to markedly im- by the often severe toxicity of graft-versus- prove T-cell mediated antitumor immuni- host disease (GvHD). GvT activity and GvHD Tumor Cell Biology ty in murine tumor models. Together with share many similar pathways in cellular Leader: Heinz Zwíerzina, M.D. Dr. Penninger’s group (IMP, Vienna) and immunity. Both effects are mediated This research group searches for druggable Dr. Baier’s group (Molecular Immunology, primarily by donor T-cell recognition of molecular targets of cancer stem cells e.g. Medical University Innsbruck) this strategy minor histocompatibility antigens (mHag). in ovarian cancer, and has established an in is currently translated being into an immu- On the other hand, natural killer (NK) vitro 3D-tumor cell culture system including stromal fibroblasts and immune cells for anticancer drug screening. By differential gene expression analysis, the guanosine exchange factor VAV3 has been identified as potential stem cell specific target in epithelial ovarian cancer. Expression of a truncated variant of VAV3 was found to be associated with refractoriness to platinum- based therapies in a cohort of ovarian cancer patients. A patent has been filed on the use of VAV3 splice variants as a predictive biomarker for platinum-based chemotherapy in ovarian cancer.

For cancer drug screening the group has established a 3D-cell culture system (“hanging drops” method) to generate multicellular tumor spheroids. This cell culture method allows incorporation of immunocompetent cells and study in depth of tumor-immune cell intercations. Recently, this cell culture technique has been adapted for primary tumor cells isolated form fresh tumor biopsies or maligant effusions.

Tumor Angiogenesis Leader: Eberhard Gunsilius, M.D. The tumor angiogenesis group investigates predictive biomarkers for antiangiogenic therapies and screens natural and synthetic compounds for their anti-angiogenic activity in-vivo using the chorion allantois membrane assay. Chemoresistant carci Fig. 3: 3D-composite tumor & stromal cell culture for in vitro drug screening. noma cells were found to translocate

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Translational Implementation of Genetic ecules. Pircher A, Wellbrock J, Fiedler W, Heidegger I, Gunsilius E, Hilbe W. Oncology. 2014;86(1):46–52. Evidence in the management of MDS (TRIAGE–MDS) and the IWG–MDS Selected Funding (International Working Group – MDS). Monitoring GRP78 mediated resistance to treatment with novel drugs by specific ELISA: ONCOTYROL/FFG, Eberhard Gunsilius, The interplay of molecular aberrations, M.D.; 480,000.00 € assessment status and anemia are analyzed Microtissue engineering – innovative 3D coculture model for ex and compared with reference populations vivo drug efficacy testing & biomarker identification in NSCLC; ONCOTYROL/FFG, Heinz Zwierzina, M.D.; 425,111.00 € to elucidate mechanisms of ageing both in Austrian CML Registry – Predictive markers for the response, out- frail and in elderly cancer patients. These come and cost effectiveness: ONCOTYROL/FFG, Steafn Schmidt, analyses form the basis for individualized M.D.; 530,667.00 € © shutterstock treatment algorithms and establish the OPTATIO (Optimizing Targets & Therapeutics in high risk and refractory Multiple Myeloma): EU-FP7 Program, Wolfgang Willen- Fig. 4: Individualized treatment algorithms relevance of assessment scores including bacher, M.D. (coord.); 510,700.00 € are essential in elderly patients quality of life and functional capacities as Translational implementation of genetic evidence in the manage- patient-reported outcomes (PROs). ment of MDS (TRIAGE-MDS): EU-Horizon 2020; Reinhard Stauder, M.D., M.Sc.;190,000.00 € the endoplasmatic reticulum chaperone protein GRP78 from the cytoplasm onto Collaborations their cell membrane and to release this Selected Publications • Competence Center ONCOTYROL, Innsbruck, Austria • Arbeitsgemeinschaft Tumortherapie, Salzburg, Austria chaperone protein under stress conditions Reduction of complement factor H binding to CLL cells improves • Austrian Breast and Colon Cancer Study Group (ABCSG), Vien- the induction of rituximab-mediated complement-dependent cy- na, Austria into the tumor microenvironment. Soluble totoxicity. Hörl S, Bánki Z, Huber G, Ejaz A, Windisch D, Muellauer • MDS Net, Duesseldorf, Germany GRP78 can activate pro-survival signaling B, Willenbacher E, Steurer M, Stoiber H. • Central European Society for Anticancer Research (CESAR), Leukemia. 2013; 27(11): 2200–8. in tumor cells (intrinsic tumor resistance) Vienna, Austria and protect endothelial cells of the tumor EpCAM overexpression prolongs proliferative capacity of primary • Schweizerische Arbeitsgemeinschaft für klinische human breast epithelial cells and supports hyperplastic growth. Krebsforschung (SAKK), Bern, Switzerland vasculature against anti-angiogenic drugs Martowicz A, Rainer J, Lelong J, Spizzo G, Gastl G, Untergasser G. • Arbeitsgemeinschaft Internistische Onkologie (AIO), Berlin, (extrinsic tumor resistance). Mol Cancer. 2013; 12:56. doi: 10.1186/1476-4598-12-56. Germany • Vesalius Research Center, Leuven, Belgium Rituximab plus subcutaneous cladribine in patients with extranod- • European MDS Registry of the European Leukemia Net (ELN), T. al marginal zone B-cell lymphoma of mucosa-associated lymphoid de Witte, Nijmegen, The Netherlands This, GRP78 expression and release in the tissue: a phase II study by the Arbeitsgemeinschaft Medikamen- • Jansen, Joop , PhD; Radboud University Medical Center, Nijme- tose Tumortherapie. Troch M, Kiesewetter B, Willenbacher W, gen, The Netherlands tumor tissue can predict resistance against Willenbacher E, Zebisch A, Linkesch W, Fridik M, Müllauer L, Greil • EHA (Europ Hematology Association) SWG Elderly Task Force various anticancer drugs and angiogenesis R, Raderer M. Haematologica. 2013; 98: 264–68. in Hematology, D. Bron, Institut Jules Bordet, Brussels, Belgium inhibitors. Currently studies are ongoing to The role of the e3 ligase cbl-B in murine dendritic cells. Wallner • CALGB, Chicago, USA elucidate the underlying molecular mech- S1, Lutz-Nicoladoni C, Tripp CH, Gastl G, Baier G, Penninger JM, • Caris Life Sciences, Phoenix, USA Stoitzner P, Wolf D. PLoS One. 2013; 8(6): e65178. • IWG-MDS (International Working Group – MDS), Peter Green- berg MD, Stanford, USA anisms of GRP78-induced drug resistance PPT and VES-13 in elderly cancer patients: evaluation in mul- • International Society of Geriatric Oncology (SIOG), L. Balducci, and to measure soluble GRP-78 in tissue tidimensional geriatric assessment and prediction of survival. Lee Moffit Comprehensive Cancer Centre, Tampa, FL, USA Augschöll J, Kemmler J, Hamaker M, Stauder R. J Geriatr Oncol. • ASH (Am Soc Hematology) – Hematology and Aging Special In- and peripheral blood by a sensitive ELISA. 2014; 5: 415–421 terest Group, H. Klepin MD, Comprehensive Cancer Center of Development of an innovative 3D cell culture system to study Wake Forest University, NC, USA Biology of Ageing and Personalised tumour—stroma interactions in non-small cell lung cancer cells. Treatment in Elderly Cancer Patients Amann A, Zwierzina M, Gamerith G, Bitsche M, Huber JM, Vogel Core Facilities GF, Blumer M, Koeck S, Pechriggl EJ, Kelm JM, Hilbe W, Zwierzina • FACS Sorting Core Facility (Cell sorting, multidimensional flow Leader: Reinhard Stauder M.D., M.Sc. H. PLoS One. 2014; 9(3):e92511. cytometry) At present, elderly people (>70 yrs) Prevalence and possible causes of anemia in the elderly: a constitute about 50 % of cancer patients. cross-sectional analysis of a large European university hospital Devices & Services cohort. Bach V, Schruckmayer G, Sam I, Kemmler G, Stauder R. • Instrumentations: FACSAria Cell Sorter, AutoMACS Cell Sep- Despite this huge number, evidence-based Clinical Interventions in Aging. 2014; 9:1187–1196. arator, FACSCalibur Cell Analyzer, MagPix Biomarker Analyzer recommendations and guidelines are rare. The side population of ovarian cancer cells defines a heteroge- • Molecular Cancer Diagnostics Network (histomorphology, flow neous compartment exhibiting stem cell characteristics. Boesch cytometry, cytogenetics, PCR, NGS) The outcome of a given patient is influenced M, Zeimet AG, Reimer D, Schmidt S, Gastl G, Parson W, Spoeck F, by the biology of the tumor (“seed”) as well Hatina J, Wolf D, Sopper S. as by that of the patient (“soil”). To integrate Oncotarget. 2014; 30;5(16):7027–39. Expression of EpCAM(MF) and EpCAM(MT) variants in human aspects of individual patient, geriatric carcinomas. Fong D, Seeber A, Terracciano L, Kasal A, Mazzoleni assessment (GA) has been established and G, Lehne F, Gastl G, Spizzo G. J Clin Pathol. 2014;67(5): 408–14. implemented at UCIM5 for elderly cancer The relevance of a geriatric assessment for elderly patients with a haematological malignancy – a systematic review. Hamaker ME, patients. Thus, different dimensions in Prins MC, Stauder R. Leuk. Res. 2014; 38(3):275–83. a given patient including comorbidities, The G8 screening tool detects relevant geriatric impairments and performance status, functional activities, predicts survival in elderly patients with a haematological malig- mood (depression, quality of life), social nancy. Hamaker ME, Mitrovic M, Stauder R. Ann. Hematol. 2014; support and nutritional status are assessed. 93(6): 1031–40. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Paolino M, Choidas A, Wallner S, Pranjic The impact of restrictions due to these B, Uribesalgo I, Loeser S, Jamieson AM, Langdon WY, Ikeda F, Fededa JP, Cronin SJ, Nitsch R, Schultz-Fademrecht C, Eickhoff J, different dimensions on clinical outcome Menninger S, Unger A, Torka R, Gruber T, Hinterleitner R, Baier G, and therapy toxicity are being analyzed and Wolf D, Ullrich A, Klebl BM, Penninger JM. assessment based treatment algorithms Nature. 2014;507(7493):508–12. Neoadjuvant chemo-immunotherapy modifies CD4(+)CD25(+) developed in cooperation with SIOG, regulatory T cells (Treg) in non-small cell lung cancer (NSCLC) EHA and ASH. Molecular aberrations are patients. Pircher A, Gamerith G, Amann A, Reinold S, Popper H, Gächter A, Pall G, Wöll E, Jamnig H, Gastl G, Wolf AM, Hilbe W, Wolf characterized in myeloid neoplasms by D. Lung Cancer. 2014; 85(1):81–7. NGS in cooperation with EU-MDS (J Jansen, New antiangiogenic strategies beyond inhibition of vascular en- Nijmegen, NL; EU-Horizon 2020 Project: dothelial growth factor with special focus on axon guidance mol-

Research Report 2015 Medical University of Innsbruck 123 Center of Internal Medicine Internal Medicine VI

Keywords wards, three outpatients’ clinics with a focus on infectious disease/immunology Internal medicine, infectious diseases, and tropical medicine, rheumatology and immunology, rheumatology, pneumology, pneumology respectively. Apart from routine host-pathogen interaction, metal and lipid investigations of internal medicine, we also metabolism, immune mediated diseases, perform laboratory diagnostics of infectious immune deficiency, tropical medicine diseases, tropical infections, auto-immune disorders and immunodeficiency disorders Research Focus as well as functional pulmonary analyses along with bronchoscopy. A study center Based on the broad clinical expertise in our co-ordinates the clinical studies at our department the research at our institution institution. covers many different aspects of basic research and clinically relevant topics in the Several research groups investigate areas of infectious diseases, immunology, relevant topics in our fields of interest as rheumatology and pneumology, both at detailed below making use of up to date the level of laboratory based science and laboratory technologies of biochemistry, clinical research. The work includes clinical molecular biology, cell biology, immunology, Director: studies with a major aim being to translate microbiology and genetics both in vitro and Univ.-Prof. Dr. Günter Weiss the results of our scientific investigations at in vivo. Our laboratories are well equipped the bench to the bedside for the benefit of with modern infrastructure including high Contact: our patients. throughput PCR, a FACS analyser and an Anichstraße 35 in vivo fluorescence imager. A major goal 6020 Innsbruck General Facts of our institution is to provide high quality education in clinics and science, to provide [email protected] Apart from all aspects of general internal an excellent environment for international Phone: +43 504 23251 medicine our institution has a focus and competitive research in the laboratory Fax: +43 504 23317 core expertise in infectious disease, clinical and at the bedside, and to inspire medical http://inneremed6.tirol-kliniken.at immunology, rheumatology and pneumology doctors to combine research with their and has established itself as a reference clinical occupation and to critically evaluate centre for Western Austria in some of these the clinical practice and the currently medical fields. As there is significant clinical available information. and scientific overlap between these medical disciplines the combined expertise Research at our department creates a positive synergy and gain of knowledge for the optimized Laboratory for Infectious Diseases and treatment of patients and in performing Immunology clinical and laboratory based research. Our The laboratory is interested in host-pathogen department consists of three in patient interactions, the role of innate resistance © Journal of experiemntal medicine

Fig. 1: Nitric oxide (NO) produced by NO-synthase (Nos2) inhibits central metabolic pathways in Salmonella directly and also activates ferroportin (Fpn1)-mediated iron export via Nrf2. The subsequent reduction of intracellular iron levels restricts the availability of iron to intracellular microbes and enhances TNF-α and IL-12 production.

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Macrophage after RBC uptake Monocyte after RBC uptake Granulocyte after RBC uptake No RBC uptake in B-Cells

Fig. 2: Phagocytosis of red blood cells (RBC) by different immune cells genes in neutrophils and macrophages in 2013) and generated mammalian systems involved macrophages. We realized that host control of infection and orchestration with deletions of specific genes being of during stress erythrophagocytosis locally of innate and adaptive immune responses, importance for the delivery of metals/ proliferating macrophages are supported by and how metabolic alterations (metals, metabolites or establishment of protective on demand-recruited macrophages that are lipids, hormones, peptides) of the host or host immune responses. highly specialized for iron handling which microbial environment affects the course enables them to avoid iron mediated toxic of infections or auto-immune diseases. We In an attempt to identify regulatory path- tissue damage. aim to identify new targets for the treatment ways of iron homeostasis and erythro- of infections either by compromising host/ poiesis during hypoxia we uncovered a Immunometabolism microbial metabolism or through favourably new mechanism by which platelet derived Atherosclerosis is still the leading cause affecting host immune responses. growth factor-BB inhibits the expression of of death in industrialized countries, and the iron regulatory hormone hepcidin thus novel therapies that can lower low-density A second major focus of this laboratory is securing a sufficient supply of iron for the lipoprotein cholesterol (LDL-C) are needed. iron homeostasis in health and disease. synthesis of red blood cells (Sonnweber et Any approach promoting the transport of We have studied pathways leading to iron al., Gut 2014). excess cholesterol from atherosclerotic loading (genetic and disease related) plaque macrophages back to the liver via or absolute/functional iron deficiency Using a well-established animal model of plasma high-density lipoprotein (HDL) for along with its sequels such as anaemia ACD we identified serum hepcidin levels biliary and final faecal excretion is expected of chronic disease (ACD). Further, we to be a good predictive marker for the to prevent atherosclerosis, a mechanistic examine new therapeutic avenues to treat response to erythropoiesis stimulating concept called reverse cholesterol trans hemochromatosis or anaemia of chronic agents (ESA). We could further demonstrate port. We have recently reported that diseases by targeting the expression of that pharmacological inhibition of hepcidin arachidonic acid (AA)-derived bioactive iron regulatory hormones, modulating formation improves the therapeutic efficacy lipid mediators including leukotrienes and transcellular iron trafficking or affecting of ESAs, which may favour a reduction lipoxins critically influence whole body iron regulated immune effector pathways; of ESA dosages, costs and side effects cholesterol homeostasis in mammals the latter being based on our longstanding (Theurl et al., Haematologica 2014). We (Demetz et al., Cell Metabol 2014). We research on regulatory interactions between also used our mammalian model of ACD to discovered that pharmacological and iron and immune function and their role in explore and investigate new hepcidin and genetic alteration of AA metabolism aimed infections, auto-immunity and cancer. ferroportin modifying drugs to successfully at increasing lipoxin levels in the liver treat this type anaemia based on our promotes reverse cholesterol transport In 2013 we identified a novel pathway by previous proof of principle studies (Theurl and leads to increased clearance of which macrophages combat resistance et al., Blood 2009 and 2011), and some of LDL-C (Fig. 3). to infection with intracellular bacteria. We these drugs are currently already in phase I found that these cells increase transcellular and II clinical trials. In another project, we discovered that iron export via nitric oxide mediated fibrates, drugs used to lower plasma lipids, induction of the central iron export protein Over the last two years we also became ameliorate the survival rate of septic mice by ferroportin. This reduces the availability of interested in stress and inflammation positively influencing neutrophil migration, the growth factor iron for microbes while induced erythrophaghocytosis (Fig. 2), a which represent a major constituent of at the same time increases antimicrobial condition often seen in ACD. We realized innate immunity (Tancevski et al., EMBO Mol immune effector functions (Nairz et al., J Exp that this process is much more complex Med 2014). Taken together, our research at Med 2013; Fig. 1). To study microbe specific than described in textbooks and uncovered the intersection between lipid metabolism metabolic and immunological host pathogen a previously unknown mechanism of and immunology will help to pave the way responses at the cellular and systemic how erythrophagocytosis is regulated not only for the development of novel lipid- level we have established various infection (Theurl et al., in submission). Using different lowering drugs, but may also serve as an models including S. typhimurium, C. pneu genetically modified reporter mice ew are innovative treatment approach in sepsis. moniae, L. monocytogenes, S. aureus and currently exploring the location of stress E. coli (Bellmann-Weiler, Immunobiology erythrophagocytosis and the ontogeny of

Research Report 2015 Medical University of Innsbruck 125 Center of Internal Medicine

In the lab we work on pathophysiological mechanisms, especially in large vessel arteritides (including abdominal aortic aneurysm) and spondyloarthritis (Dejaco et al., Ann Rheum Dis 2014). Specifically ew focus on a mouse model for spondyloarthritis and pro-inflammatory cytotoxic T-cells, which use alternate stimulatory pathways since they lack the co-stimulatory molecule CD28, show signs of early aging and are considered as long- lived with reduced apoptosis.

Among our major achievements are the initialisation of the new International Criteria for Behçet’s Disease, establishment of NKG2D as part of a co-stimulatory pathway for CD4+CD28- T cells in giant cell arteritis and polymyalgia rheumatica and

Furthermore we have extended patient Fig. 3: Regulation of Cholesterol Metabolism by Lipid Mediators cohorts for clinical and pathophysiological Arachidonic acid (AA) is converted to prostaglandins (PGs) by cyclooxygenase (COX) en- studies and pathophysiological studies zymes. Aspirin inhibits the production of PGs and results in substrate diversion of AA to in spondyloarthritis and large vessel lipoxygenase (LOX) pathways in the liver and also promotes the generation of 15-epimeric arteritides. Hyperuricaemia is a frequent lipoxins. Arachidonate 5-lipoxygenase (Alox5) is involved in the biosynthesis of leukotriene clinical condition eventually leading to gout B4 (LTB4) and the lipoxins, LXA4 and LXB4. Aspirin treatment enhances reverse choles- (Fig. 4). While local urate deposition results terol transport in part by increasing the production of Alox5-derived mediators and subse- in activation of the immune system via the quent bile acid secretion through ATP-binding cassette subfamily b member 11 (Abcb11). NALP3 inflammasome, hyperurecemia These protective actions are recapitulated by administration of stable analogs of LXB4 (i.e. also causes systemic metabolic effects, 5-R/S-methyl LXB4). Adapted from: Spite M., Cell Metab. 2014 Dec 2;20(6):935–7 and increased circulating uric acid levels are a known risk factor for coronary heart

Biomarker Research, Diagnostics and clinical studies are undertaken to evaluate Biobanking the usefulness and effectivity of clinical These studies focus on the identification practices and therapy standards. of diagnostic and prognostic parameters in infectious diseases including sepsis Biobanking has been systematically and auto-immune disorders. The idea is to performed at our institution for many years identify new biomarkers or combinations and is of central importance for ongoing thereof to enable rapid and pathogen and future clinical studies and research in specific diagnosis of infectious diseases infectious, immunological, rheumatological in order to differentiate between viral and pulmonary as well as in rare diseases. and bacterial infections, and to assist Rheumatological research clinicians in the differentiation between an exacerbation of an auto-immune disease Our clinical rheumatological research and infectious complication in subjects with is focused on the evaluation of a new systemic inflammatory disorders or in an referral tool for primary care physicians immune-compromised host. Such markers and specialists to our outpatient unit, include small molecules derived from innate diagnostic issues (with specific impact on immune cells, metabolic products such as sonography), validation of new classification lipids, amino acid degradation compounds, criteria (e.g. for polymyalgia rheumatica anti-microbial peptides and volatile gases. and Behcet’s disease) and outcome of Along this line we aim to improve the quality rheumatic diseases (e.g. by supporting of state of the art diagnostics for infectious the international evaluation of the new diseases and auto-immune disorders along ASAS-health index). Our current work with establishment of clinically needed on a structured disease-specific clinical Fig. 4: Severe form of gout and identification diagnostic tests for rare diseases and database together with a clinical biobank of urate deposition by dual energy computer immuno-compromised patients. In addition, will further support translational research. tomography

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Fig. 5: Enhancing and suppressing effects of endothelin-1 and the endothelin-receptor blocker bosentan on outmigration of Langerhans cells from murine epidermal skin explants (left). Live cell imaging confirming ET-1 induced calcium mobilisation (right). disease. In a fruitful collaboration with BMPR2, ALK-2 and endoglin, analysis of the Ludwiczek S, Talasz H, Brandacher G Moser PL, Muckenthaler MU, Fang FC, Bogdan C, Weiss G. the clinic of radiology this association has iron status in these patients and improved J Exp. Med. 2013, 210: 855–873. been investigated by the combination of imaging with MRI studies are ongoing Hypoxia induced downregulation of Hepcidin is mediated by plate- state of the art ultrasound and computer projects. Among the more rare diseases, let derived growth factor BB. Sonnweber T, Nachbaur D, Schroll A, Nairz M, Seifert M, Demetz E, Haschka D, Mitterstiller AM, tomography (DECT) imaging along with patients with sarcoidosis, idiopathic lung Kleinsasser A, Burtscher M, Trübsbach S, Murphy AT, Wroblewski investigations into the effects of immune- fibrosis, CF ‑ and non-CF bronchiectasis V, Witcher DR, Mleczko-Sanecka K, Vecchi C, Muckenthaler MU, Pietrangelo A, Theurl I, Weiss G. modulatory drugs on the course of gout in as well as polycystic lung diseases are Gut. 2014; 63: 1951–59. vitro and in vivo. registered. The latter includes pulmonary Fibrates ameliorate the course of bacterial sepsis by promot- An ongoing clinical study currently Langerhans cell histiocytosis, a disease ing neutrophil rectruitment via CXCR2. Tancevski I, Nairz M, Duwensee K, Auer K, Schroll A, Heim C, Feistritzer C, Hoefer investigates the prevalence and nature of that may also result in PAH. We already J, Gerner RR, Moschen AR, Heller I, Pallweber P, Li X, Theurl M, anaemia in systemic rheumatic diseases, tested the presence of PAH drug targets in Demetz E, Wolf AM, Wolf D, Eller P, Ritsch A, Weiss G. EMBO Mol Med. 2014 Apr 22;6(6):810–20. its impact on disease activity, morbidity and the inflammatory cells of these patients and mortality along with its alteration by disease concluded that blockade of endothelin -1 Selected Funding modifying drugs. signalling might prevent outmigration of • European Union 7th Framework, Project: Eurocalin (Günter Langerhans cells (Fig. 5). Weiss) • FWF ‑ TRP-188: Role of Iron for Atherosclerosis (Günter Weiss) Rare Pulmonary Diseases • FWF ‑23853-MED: Thyreomimetika und Statine: Einfluß auf Re- The orphan lung diseases comprise many versen Cholesterintransport & Atherosklerose (Ivan Tancevski) • FWF ‑24749-MED: Hepcidin in Diagnose und Therapie ver- disorders, and assessment as well as Selected Publications schiedener Anämieformen (Igor Theurl) • FWF ‑ 25338: Toll like receptors and innate immunity in axial treatment of affected patients is a great NKG2D stimulated T-cell autoreactivity in giant cell arteritis spondyloarthritis: A TH1-prone mouse model and human stud- and polymyalgia rheumatica. Dejaco C, Duftner C, Al-Massad J, challenge, because of the lack of adequate ies, Austrian Research Foundation (Michael Schirmer) Wagner AD, Park JK, Fessler J, Aigelsreiter A, Hafner F, Vega S, guidelines. Therefore, present research is Sterlacci W, Grubeck-Loebenstein B, Tzankov A, Ness T, Boiardi L, focused on a systematic approach to these Salvarani C, Schirmer M. Collaborations ANN RHEUM DIS. 2013 Nov;72(11):1852–9. diseases along with translational research • Prof. Dr. Christian Bogdan, Univ. of Erlangen, Germany The Arachidonic Acid Metabolome Serves as a Conserved Reg- • Doz. Dr. Christian Datz, Obernsdorf, Austria projects. Patients with pulmonary rare ulator of Cholesterol Metabolism. Demetz E, Schroll A, Auer K, • Prof. Dr. Thomas Decker,M Perutz Laboratory, Vienna diseases are registered within a database Heim C, Patsch JR, Eller P, Theurl M, Theurl I, Theurl M, Seifert M, • Prof. Dr. Ferric Fang, Univ of Washington, Seattle, USA Lener D, Stanzl U, Haschka D, Asshoff M, Dichtl S, Nairz M, Huber • Prof. Dr. Matthias Hentze, EMBL, Heidelberg, Germany and a biobank with biological specimens E, Stadlinger M, Moschen AR, Li X, Pallweber P, Scharnagl H, Sto- • Prof. Dr. Martina Muckenthaler, Univ. of Heidelberg, Germany will be established. Patients with pulmonary jakovic T, März W, Kleber ME, Garlaschelli K, Uboldi P, Catapano • Prof. Dr. Sylvia Knapp, CeMM, Vienna AL, Stellaard F, Rudling M, Kuba K, Imai Y, Arita M, Schuetz JD, • Prof. Dr. Klaus Schuemann, Technical Univ of Munich, Germany arterial hypertension (PAH) represent Pramstaller PP, Tietge UJ, Trauner M, Norata GD, Claudel T, Hicks • Prof. Dr. Phil Swirski, Harvard Medical School, Boston, USA the major cohort of patients and efforts AA, Weiss G, Tancevski I. • Prof. Dr. Eric Mattheson, MAYO Clinic and Foundation, Roches- CELL METABOLISM. 2014 Nov 4;20(5):787–98. ter (MN), USA for improved screening within high risk • Prof. Dr. Carlo Salvarani, Arcispedale S.Maria Nuova, Reggio Nitric oxide-mediated regulation of ferroportin-1 controls mac- Emilia, Italy populations (e.g. patients with connective rophage iron homeostasis and immune function in Salmonella • Prof. Dr. Fereydoun Davatchi, University of Teheran, Iran tissue disease), mutational analysis for infection. Nairz M, Schleicher U, Schroll A, Sonnweber T, Theurl I, • Prof. Dr. Christos C. Zouboulis, Klinikum Dessau, Germany

Research Report 2015 Medical University of Innsbruck 127 Center of Internal Medicine Joint Institution for Emergency Medicine and Critical Care Medicine

Keywords medical emergency room including a short stay (maximum 24 hours) ward located in Acute kidney injury, sepsis, cardio pul- the Medizinzentrum Anichstrasse (MZA). monary resuscitation, biomarkers, micro Administratively, the unit is affiliated to the vesicles, secretoneurin, chronobiology, Department of Internal Medicine I (Director: plasma pharmacokinetics, target-site phar- Prof. Dr. Herbert Tilg). macokinetics, pharmacodynamics The unit is involved in several clinical mul Research Focus ticentre trials investigating early diagnosis and treatment of acute kidney injury, • Applied clinical as well bench-to-bedside treatment of severe infections and sepsis research covering several aspects of as well as antimicrobial pharmacokinetics. critical illness with special emphasis on Complementary in vitro models are used to acute kidney injury (AKI), sepsis, cardio- investigate inflammatory mechanisms of pulmonary resuscitation (CPR) renal injury. • Definition and clinical validation of biomarkers for diagnosis and prognosis of The research unit comprises the laboratory AKI and CPR of Inflammation Research (U-1-015) hosting Director: • Identification and characterisation of two research groups: the Intensive Care Univ.-Prof. Dr. Michael Joannidis microvesicles in severe sepsis Medicine group (Viktoria Haller, Ulrich • Chronobiology Harler, Julia Hasslacher, Georg Lehner) Contact: • Intensive care specific pharmacodynam- led by Michael Joannidis and the Clinical Anichstraße 35 ics and pharmacokinetics Pharmacokinetics group (Rene Welte) led 6020 Innsbruck by Romuald Bellmann. General Facts [email protected] Internal collaboration partners include all Phone: +43 512 504 24181 The Joint Institution for Emergency Medi- University Clinics (I-VI) of the Department Fax: +43 512 504 24199 cine and Critical Care Medicine was estab- of Internal Medicine, the Neurological www.i-med.ac.at/notfallmedizin/ lished in December 2012. Intensive Care Unit and the Surgical/ Clinically, it was designed as a core facility Trauma Intensive Care Unit as well as for the Department of Internal Medicine the Division of Hygiene and Medical providing a high level of Intensive Care Microbiology, the Division of Molecular and and Emergency Medicine. It comprises Cellular Pharmacology and the Department a level three intensive care unit and the of Medical Psychology.

Fig. 1: ROC analysis for prediction of poor neurological outcome at 72 h after CPR. SN secretoneurin, NSE neuron-specific enolase.

128 Research Report 2015 Medical University of Innsbruck Medical Emergency & Intensive Care Unit

Fig. 2: Characterisation of microvesivles in septic shock applying high-sensitivity flow cytometry (0.3 µM resolution).

Research biomarkers for AKI and prediction was Microvesicles in Severe Sepsis and independent of the aetiology of AKI or Septic Shock Intensive Care Medicine the underlying disease. Additionally Severe sepsis has a worldwide annual inci- Michael Joannidis to the clinical approach, function and dence of around 3/1000 inhabitants and a This group is involved in biomarker research release kinetics of several biomarkers mortality rate > 50 % when proceeding to in critical illness with a major interest in are characterised in a renal epithelial- septic shock. This syndrome is frequent- acute kidney injury and cardiopulmonary endothelial co-culture system developed ly complicated by devastating coagulation resuscitation. A second major focus is by our laboratory to simulate interstitial disturbances leading to disseminated intra- investigation of microvesicles in sepsis. inflammation. vascular coagulation (DIC). Microvesicles Chronobiology is the third field of interest. (MV) are capable of mediating pleiotropic inflammatory signals during sepsis and Acute Kidney Injury (AKI) Hypoxic Brain Damage after Cardio may play a key role in the propagation of AKI in the critically ill is associated with pulmonary Resuscitation (CPR) thrombin generation via phosphatidylser- significant mortality and long term morbidity Cardiac arrest is one of the major causes of ine exposure as well as in the initiation of including end stage renal disease. Based on death in cardiovascular disease, frequently blood coagulation by specific epitopes a prior project (OeNB Anniversary Fund, associated with long-term neurological such as tissue factor. In a project funded project 12094) we analysed conventional deficits in case of survival. Neuron specific- by the OeNB Anniversary Fund (project criteria for AKI, i.e. dynamic changes enolase (NSE), the recommended biomarker 13861), we investigated the hypothesis in serum creatinine and urinary output for outcome prediction since 2006, has that increased levels of endothelial derived significantly triggering the development been questioned recently because of being MV reflect endothelial dysfunction which of the current AKI criteria (KDIGO Clinical influenced by therapeutic ypothermiah as is considered a key element in the patho- Practice Guideline for Acute Kidney Injury, well as haemolysis frequently occurring physiology of sepsis. Developing a specific 2012, http://kdigo.org/home/guidelines/ after CPR. Secretoneurin (SN), a 33 amino high-sensitivity flow cytometry approach acute-kidney-injury/). acid neuropeptide is specifically expressed enabled us to measure previously undetect- in endocrine, neuroendocrine and neuronal able smaller MV until 0.3 µm (Fig. 2). We Furthermore, several renal tubular proteins tissues with a wide spread distribution in found that increased levels of MV detected were evaluated as biomarkers to achieve an the brain. SN exerts a variety of biological in patients with septic shock predominant- earlier and more specific diagnosis of AKI functions like induction of angiogenesis ly originate from circulating cells indicating aiming at improved prevention strategies. and is markedly upregulated by hypoxia. excessive leukocyte and platelet activation Participating in two large prospective For the first time we could demonstrate, in especially in lethal septic shock. cohort trials, cellular arrest markers, tissue a large prospective cohort of 150 patients inhibitor of metalloproteinsases 2 (TIMP -2) admitted to our ICU after successful CPR, Only single patients exhibited markedly and insulin-like growth factor-binding that SN enables outcome prediction within increased amounts of endothelial derived protein 7 (IGFBP7) were investigated. Their the first 24 hours, significantly earlier than MV. Moreover, our findings indicate that presumed mechanism of action is depicted NSE. CD31+/CD41- (i.e. platelet endothelial cell in Fig. 3. The SAPPHIRE and OPAL studies adhesion molecule positive and integrin demonstrated diagnosis of moderate or SN was not influenced by therapeutic alpha-IIb negative) MV, formerly frequently severe AKI (KDIGO stage 2 or 3) at least hypothermia. Combining SN and NSE in the considered to be mostly endothelium twelve hours earlier than conventional first 72 hours after CPR resulted in an UCA derived, might originate predominantly biomarkers. The achieved AUC > 0.8 was of 0.881 (95 % CI: 0.815-0.946) to predict from leukocytes. These results triggered higher than that of previously investigated poor neurological outcome (Fig. 1). a subsequent project funded by the OeNB

Research Report 2015 Medical University of Innsbruck 129 Center of Internal Medicine

in which we currently address the role of demonstrate significant effects of 24 hours • Definition of biomarker panels for neuro- distinct MV subtypes during DIC trying on call duty on the neuroendocrine system logic outcome prediction after CPR to identify the specific stimuli that cause leading to increased noradrenaline levels as • Investigating specific effects of MV sub- their release. To achieve this goal we well as significantly reduced concentration types on distinct proteolytic processes of establish coagulation assays that allow us ability, however, concealed by an individual the coagulation system in DIC to investigate the interactions between overestimation of performance. MV, the endothelium and the coagulation Clinical Pharmacokinetics system in a translational approach. This Major Achievements: Romuald Bellmann system will be capable of reflecting the key • Clinical validation cell cycle arrest pro- Severe infections are a common reason role of the activated endothelium during teins as biomarkers for AKI for critical illness. Adequate antimicrobial sepsis and DIC. • First time identification of SN as early pre- chemotherapy is crucial for the clinical dictor for severe hypoxic brain damage outcome. Sub-therapeutic antimicrobial Chronobiology Research after CPR dosage results in poor response and Continuous disruption of wake-sleep cycle • Establishment of high-sensitivity flow cy- may promote the emergence of resistant and chronic sleep deprivation have been tometric MV analysis and characterisation microorganisms. On the other hand, implicated to contribute to cardiovascular of circulating MV as being mainly platelet critically ill patients are at an enhanced risk disease as well as malignancy among other and leucocyte derived in septic shock of drug toxicity. Absorption, distribution, health issues. Investigating the effects of metabolism and elimination of drugs can be physicians’ nightshift we previously had Future Directions: altered by critical illness depending on the described significant cardiac arrhythmias as • Investigating long term outcome predic- state of disease, type of organ failure and well increased sympathetic activity during on tion of AKI by cell cycle arrest proteins and required treatment modality. An increasing call duties. In a consecutive study we could markers of fibrosis number of patients present with profound

Fig. 3: Proposed mechanistic involvement of the novel biomarkers in AKI: initial tubular cells sustain injury by various insults. In response to DNA and possibly other forms of damage, IGFBP7 and TIMP-2 are expressed in the tubular cells. IGFBP7 directly increases the expression of p53 and p21 and TIMP-2 stimulates p27 expression. These effects are conducted in an autocrine and paracrine manner via IGFBP7 and TIMP-2 receptors. The p proteins in turn, block the effect of the cyclin-dependent protein kinase complexes (CyclD-CDK4 and CyclE-CDK2) on the cell cycle promotion, thereby resulting in G1 cell cycle arrest for short periods of time presumably to avoid cells with possible damage from dividing. AKI, acute kidney injury; IGFBP7, insulin-like growth factor-binding protein 7; TIMP-2, tissue inhibitor of metalloproteinases-2. Ref. Kashani K et al, Critical Care 2013 Feb 6;17(1):R25.

130 Research Report 2015 Medical University of Innsbruck Medical Emergency & Intensive Care Unit

immunological dysfunction facilitating Selected Publications infections by opportunistic pathogens such Discovery and validation of cell cycle arrest biomarkers in hu- as invasive fungal infections associated man acute kidney injury. Kashani Kianoush, Al-Khafaji Ali, Ardiles Thomas, Artigas Antonio, Bagshaw Sean M, Bell Max, Bihorac with mortality rate exceeding 50 %. Azra, Birkhahn Robert, Cely Cynthia M, Chawla Lakhmir S, Optimum dosage of newly developed broad Davison Danielle L, Feldkamp Thorsten, Forni Lui G, Gong Michelle spectrum azoles and echinocandins guided Ng, Gunnerson Kyle J, Haase Michael, Hackett James, Honore Patrick M, Hoste Eric AJ, Joannes-Boyau Olivier, Joannidis Michael, by pharmacokinetic and pharmacodynamic Kim Patrick, Koyner Jay L, Laskowitz Daniel T, Lissauer Matthew considerations should contribute to E, Marx Gernot, McCullough Peter A, Mullaney Scott, Ostermann Marlies, Rimmele Thomas, Shapiro Nathan I, Shaw Andrew D, Shi improved outcome. Jing, Sprague Amy M, Vincent Jean-Louis, Vinsonneau Christophe, Wagner Ludwig, Walker Michael G, Wilkerson R. Gentry, Zacharowski Kai, Kellum John A. Therefore we assessed pharmacokinetics Critical Care. 2013 Feb 6;17(1):R25. of caspofungin, an echinocandin antifungal Derivation and validation of cutoffs for clinical use of cell cycle agent, in critically ill patients requiring arrest biomarkers. Hoste Eric AJ, McCullough Peter A, Kashani, continuous renal replacement therapy Kianoush, Chawla Lakhmir S, Joannidis Michael, Shaw Andrew D, Feldkamp Thorsten, Uettwiller-Geiger Denise L., McCarthy Paul, (CRRT) and in a control group not requiring Shi Jing, Walker Michael G, Kellum John A, Sapphire Investigators. CRRT. Caspofungin clearance by CRRT was NEPHROLOGY DIALYSIS TRANSPLANTATION. 2014; 29: p. 2054– very low and plasma pharmacokinetics 2061. almost unaffected by CRRT and Secretoneurin as a marker for hypoxic brain injury after cardiopulmonary resuscitation. Hasslacher Julia, Lehner Georg Franz, comparable to that in healthy volunteers. Harler Ulrich, Beer Ronny, Ulmer Hanno, Kirchmair Rudolf, This study proved that caspofungin can Fischer-Colbrie Reiner, Bellmann Romuald, Dunzendorfer Stefan, Joannidis Michael. be administered at standard dosage INTENSIVE CARE MEDICINE. 2014; 40: p. 1518–1527. to critically ill patients independent of Effects of 24 h working on-call on psychoneuroendocrine and requirement of CRRT. Since most infections oculomotor function: A randomized cross-over trial. Ernst Flori- occur in tissue rather than in the blood an, Rauchenzauner Markus, Zoller Heinz, Griesmacher Andrea, Hammerer-Lercher Angelika, Carpenter Roger, Schuessler stream, target-site pharmacokinetics might Gerhard, Joannidis Michael. be even more relevant for clinical outcome PSYCHONEUROENDOCRINOLOGY. 2014; 47: p. 221–231. than plasma pharmacokinetics. Therefore, Pharmacokinetics of Caspofungin in Critically Ill Patients on biliary AMB pharmacokinetics in patients Continuous Renal Replacement Therapy. Weiler Stefan, Seger Christoph, Pfisterer Hartwig, Stienecke Eva, Stippler Florian, treated with lipid-formulated AMB and Welte Rene, Joannidis Michael, Griesmacher Andrea, Bellmann biliary AMB pharmacodynamics by in vitro Romuald. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. 2013; 57: and ex vivo-simulations was investigated in p. 4053–4057. another project. Selected Funding Biliary AMB concentrations were lower Interaction between microvesicles, endothelium and the coagu- and displayed a slower rise and decline lation system in sepsis and DIC, OeNB Anniversary Fund (project 15708), Michael Joannidis than plasma levels. Fungal growth and AMB activity were impaired by bile. Thus, Collaborations treatment of fungal cholangitis with lipid- • Univ.-Prof. Dr. Thomas Staudinger, Intensive Care Unit, Internal formulated AMB is not supported by our Medicine I, Medical University Vienna, Vienna, Austria • Professor Stefan Kluge, Department of Intensive Care Medi- data. cine, Hamburg Eppendorf, Hamburg, Germany • Prim. Univ.-Prof. Dr. Christian Wiedermann, Department of In- Major Achievements: ternal Medicine, Central Hospital of Bolzano, Bolzano, Italy • John Kellum, MD, FCCM, FACP, University of Pittsburgh School Determination of pharmacokinetics of of Medicine, Pittsburg, PA, USA caspofungin and target site kinetics of lipid- • Ravindra L. Mehta, MD, UCSD Medical Centre, San Diego, CA, USA formulated AMB in the critically ill. • Univ.-Prof. Dr. Jaroslav Sterba, Department of Pediatric Oncol- ogy, University Hospital Brno and Masaryk University, Brno, Future Goals: Czech Republic • Dr. Piotr Smuszkiewicz, Department of Anesthesiology, Inten- Plasma and target-site pharmacokinetics sive Therapy and Pain Treatment, University Hospital Przybysze- and pharmacodynamics of other antifun- wskiego, Poznan, Poland • Univ.-Prof. Dr. Markus Müller, Priv. Doz. Dr. Markus Zeitlinger, gals and of trimethoprim sulfonamide com- Universitätsklinik für Klinische Pharmakologie, AKH, Wien, binations. Austria

Research Report 2015 Medical University of Innsbruck 131 Center of Psychiatry and Pychotherapy General and Social Psychiatry

departments of the Center of Psychiatry, long-standing field of research in the Psychotherapy and Psychosomatics. The Department. The latency between the latter is especially pertinent with respect suicidal impulse and the ensuing action, as to mutual interests shared with the well as relationships to previous physician Departments of Biological Psychiatry and contacts and hospitalization, have been Psychosomatics. Preclinical research is analysed by this group. Biological variables mostly of a translational nature and extends such as oxytocin and cholesterol were also and complements the clinical programmes. studied in suicide attempters and controls. Within the broad spectrum of research The work led by Dr. Deisenhammer’s group efforts, a few highlights will be briefly has also led to regional suicide prevention summarized in the following. measures, including attaching safety nets to a big highway bridge. Research Experimental Alzheimer Research Selected references, funding sources and Christian Humpel collaborations are briefly presented in the In close collaboration with the clinical Appendix. researchers, research in the Psychiatric Laboratory of Experimental Alzheimer’s Director (interim): Age Related Psychiatric Disorders disease focusses on investigating the Univ.-Prof. Dr. W. Wolfgang Imrich Blasko, Michaela Defrancesco development of beta-amyloid plaques in Fleischhacker This research group has its main base in Alzheimer’s disease and their association the Memory Clinic of the Department. In with brain blood vessels. Furthermore, Contact: addition to providing clinical service for the the migration of blood cells (monocytes Anichstraße 35 catchment area, specific areas of scientific and platelets) into the Alzheimer brain is 6020 Innsbruck interest include the neuropsychological and explored. Another research focus is to find anatomical underpinnings of mild cognitive and establish novel biomarkers in blood [email protected] impairment (MCI) and Alzheimer dementia. to diagnose Alzheimer’s disease and other Phone: +43 512 504 23669 In this context, a strong emphasis is given forms of dementia. In addition to peripheral Fax: +43 512 504 25267 to predicting the conversion risk from MCI markers, this lab also scientifically http://psychiatrie.tirol-kliniken.at/ to dementia. To this end, next to findings evaluates routinely acquired CSF samples from well-established neuropsychologi- from patients referred either from the cal tests, anatomical imaging findings and Memory Clinic or from the inpatient unit of Keywords both peripheral and central biomarkers are the Department. explored. The Departments of Neuroradi- Addiction, alzheimer, beta-amyloid plaques, ology and the Laboratory for Experimental Experimental Psychiatry Unit cocaine, conditioned place preference, Alzheimer Research are important collabo- Alois Saria, Gerald Zernig dementia, depression, diagnosis, drug de- rating partners. Further to that, Dr. Blasko’s The Experimental Psychiatry Unit is one pendence, ECT, electroconvulsive therapy, research group collaborates with the Vien- of the host labs for the international PhD health related behaviour, magnetic reso- na Transdanube Aging Study (VITA) spear- program “Signal Processing in Neurons”. nance imaging, mild cognitive impairment, headed by Dr. Peter Fischer. This is a large Research at the lab focusses on the platelets, social interaction, substance use scale population-based epidemiological mechanisms of reward and the mode of disorder, suicide, tau, therapeutic drug study evaluating the association of various action of psychoactive drugs. In addition, monitoring, unipolar, vessels physical, cognitive and social activities with the lab offers clinical service for psychiatric the risk for dementia. Driving ability of the patients, i.e. Therapeutic Drug Monitoring Research Focus elderly is a research topic studied in collab- (TDM). In this context, blood levels of over 30 oration with Ilsemarie Kurzthaler, who leads antidepressant or antipsychotic drugs are Age related psychiatric disorders, the Department’s traffic safety in psychiatry determined daily for the University Hospital behavioral and clinical psychology, neuronal group. Innsbruck and for additional hospitals and signal processing, preclinical substance physicians in Austria and Northern Italy, use research, suicide, therapeutic drug Affective Disorders and Suicide by use of liquid chromatography-tandem monitoring, treatment of affective disorders Armand Hausmann, mass spectrometry. TDM results are also Eberhard Deisenhammer exploited for addressing various research General Facts These two research groups, again, base questions, and the lab is the core laboratory their work on the clinical services provided for drug monitoring in two large European The Department of General and Social by the relevant in- and outpatient units of the multi-center studies (OPTiMiZE and Psychiatry has a strong focus on clinical Department. Established and experimental EULAST). research related to the services provided treatments for depression, ranging from by the in- and outpatient units of the phase II clinical trials to light therapy and The Experimental Psychiatry Unit is also a Department, which are located on the electroconvulsive therapy, are explored. partner in the Human Brain Project, one of campus of the Medical University Innsbruck. the two flagship research projects funded This allows for close collaboration with Depression is associated with a consider by the European Commission, involving other medical disciplines and the other able suicide risk, the latter being another over 100 partner universities in Europe

132 Research Report 2015 Medical University of Innsbruck General and Social Psychiatry

Sphingomyelin SM(d18:1/18:0) is significantly enhanced in cerebrospinal fluid samples. Koal T, Klavins K, Seppi D, Kemmler G, Humpel C. Journal of Alzheimer’s Disease. 2015; 44: 1193–1201. Platelets in the Alzheimers disease brain:do they play a role in cerebral amyloid angiopathy? Kniewallner KM, Ehrlich D, Kiefer A, Marksteiner J, Humpel C. Current Neurovascular Research. 2015;12: 4–14. Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor. Prast JM, Schardl A, Sartori SB, Singewald N, Saria A, Zernig G. Front Behav Neurosci. 2014 Dec 22;8:441. doi: 10.3389/fnbeh.2014.00441. eCollection 2014. Reacquisition of cocaine conditioned place preference and its inhibition by previous social interaction preferentially affect D1-medium spiny neurons in the accumbens corridor. Prast JM, Schardl A, Schwarzer C, Dechant G, Saria A, Zernig G. Front Behav Neurosci. 2014 Sep 24;8:317. doi: 10.3389/fnbeh.2014.00317. eCollection 2014. Perinatal use of aripiprazole: plasma levels, placental transfer, and child outcome in 3 new cases. Windhager E, Kim SW, Saria A, Zauner K, Amminger PG, Klier CM. J Clin Psychopharmacol. 2014 Oct;34(5):637–41. doi: 10.1097/ JCP.0000000000000171. Whole-exome sequencing identifies a polymorphism in the BMP5 gene associated with SSRI treatment response in major depression.Tammiste A, Jiang T, Fischer K, Mägi R, Krjutškov K, Pettai K, Esko T, Li Y, Tansey KE, Carroll LS, Uher R, McGuffin P, Võsa U, Tšernikova N, Saria A, Ng PC, Eller T, Vasar V, Nutt DJ, Maron E, Wang J, Metspalu A. J Psychopharmacol. 2013 Oct;27(10):915–20. Use of benzodiazepines in Alzheimer’s disease: a systematic review of literature. Defrancesco M, Marksteiner J, Fleischhacker WW, Blasko. Fig. 1: This picture shows a plaque in an Alzheimer mouse. The plaque (blue) is surrounded Int J Neuropsychopharmacol. 2015 May 19;18(10). by reactive astrocytes (red), most of them express a calcium channel (yellow). Green fluo- Changes in white matter integrity before conversion from rescence correspond to calcium-channel alpha1 subunit like-immunoreactivity (published mild cognitive impairment to Alzheimer’s disease. Defrances- co M, Egger K, Marksteiner J, Esterhammer R, Hinterhuber H, in Journal of Alzheimers disease). Deisenhammer EA, Schocke M. PLoS one. 2014 Aug 25;9(8). and some outside Europe. Alois Saria leads of stigma-management-programs and Bright versus dim ambient light affects subjective well-being but not serotonin-related biological factors. Stemer B, Melmer A, the “Education Program” of this project cognitive training programs in psychiatric Fuchs D, Ebenbichler C, Kemmler G, Deisenhammer EA. to coordinate education and training of patients. Psychiatry Res. 2015 Jun 27. a large number of PhD students in this Oxytocin plasma levels in psychiatric patients with and without recent suicide attempt. Deisenhammer EA, Hofer S, Schwitzer O, multidisciplinary project. Health psychology aims to evaluate our nic- Defrancesco M, Kemmler G, Wildt L, Hinterhuber H. otine cessation program and focusses on Psychiatry Res. 2012 Nov 30;200(1):59–62. Addiction Research, Preclinical aspects of body image and body modifica- The duration of the suicidal process: how muich time is left for intervention between consideration and accomplishement of a Gerald Zernig tion (e.g. in blind people). suicide attempt? Deisenhammer EA, Ing CM, Strauss R, Kemmler Impaired social interaction is a hallmark G, Hinterhuber H, Weiss EM. J Clin Psychiatry. 2009 Jan;70(1). symptom of many psychiatric diseases. Selected Publications This also pertains to substance use Leisure time activities and cognitive functioning in middle Euro- Selected Funding pean population-based study. Blasko I, Jungwirth S, Kemmler G, disorders. Dr. Zernig’s group studies Weissgram S, Tragl TH, Fischer P. Group Humpel: the neural bases of substance-induced European Geriatric Medicine. Volume 5, Issue 3, June 2014, Pages • Austrian Science Funds P24734-B24 200–20. • Austrian Science Funds P24541-B24 social interaction medications, using • Austrian Science Funds Sonderforschungsbereich SFB F44 The Child is Father of the Man.Review von relevanten Studien zur cocaine as a prototypical drug of abuse, Epidemiologie in der Kinder- und Jugendpsychiatrie. Fuchs M, Experimental Psychiatry Unit in an experimental model. In addition to Bösch A, Hausmann A, Steiner H. • Austrian Science Fund W1206 – Graduate School: Signal Pro- Z Kinder-Jugendpsychiatr Psychother. 2013;41(1):45–57. cessing in Neurons (PI: Gerald Zernig) behavioural modifications on the impact of • Austrian Science Fund P 23824-B18: Neurobiology of Social In- potentially beneficial interventions, such as Die Behandlung der Agitation beim psychiatrischen Notfall. teraction as Alternative for Drugs of Abuse (PI: Rana El Rawas/ Kasper S, Baranyi A, Eisenburger P, Erfurth A, Ertl M, Frey R, Alois Saria) for instance environmental enrichment or Hausmann A, Kapfhammer HP, Psota G, Rados C, Roitner-Vitzthum • Austrian Science Fund P 27852-B21: Does social interaction paired housing, changes in neurocircuitry E, Sachs GM, Winkler D. have an anti-stress effect? (PI: Rana El Rawas) CliniCum neuropsy. Sonderausgabe November 2013: 1–15. • European Commission FP7-ICT-2013-FET-F: Human Brain Pro- and neuroreceptor/neurotransmitter level Facial affect recognition in symptomatically remitted patients ject (PI Alois Saria) are investigated. with schizophrenia and bipolar disorder. Yalcin-Siedentopf N, Hoertnagl CM, Biedermann F, Baumgartner S, Deisenhammer EA, Collaborations Hausmann A, Kaufmann A, Kemmler G, Mühlbacher M, Rauch AS, Group Humpel Fleischhacker WW, Hofer A. Behavioral and Clinical Psychology Seven publications resulted from collaborations within and out- Schizophr Res. 2014;152(2–3):440–445. Verena Günther side the MUI. External collaborators were Affective prosody perception in symptomatically remitted • Prim. Prof. Josef Marksteiner (Psychiatry, Landekrankenhaus Behavioral and Clinical Psychology patients with schizophrenia and bipolar disorder. Hoertna- Hall/Tirol), focusses primarily on cognitive/behavioral gl C, Yalcin-Siedentopf N, Baumgartner S, Biedermann F, • PD Dr. Walter Kaufmann (IST, Klosterneuburg) and Deisenhammer EA, Hausmann A, Kaufmann A, Kemmler G, • Prof. Ingrid Strömberg (Umea, Sweden). aspects of chronically ill-patients (e.g. body Muehlbacher M, Rauch AS, Fleischhacker WW, Hofer A. Experimental Psychiatry Unit Schizophrenia Res. 2014;158(1–3):100–104. image in patients with an insulin pump, • Henry Markram (coordinator, Human Brain Project), EPFL Lau- psychological aspects of patients with an Vascular pathology of 20-month-old hypercholesterolemia mice in sanne, Switzerland comparison to triple-transgenic and APPSwDI Alzheimer’s disease • Anu Tammiste, Estonian Genome Center, University of Tartu, implantable cardioverter defibrillator...) and mouse models. Hohsfield LA, Daschil ,N Orädd G, Strömberg I, Estonia on the conceptualization and evaluation Humpel C. Molecular Cellullar Neuroscience. 2014, 63: 83–95. • Claudia Klier, Medical University Vienna, Austria

Research Report 2015 Medical University of Innsbruck 133 Center of Psychiatry and Pychotherapy Biological Psychiatry

through both investigator initiated grants three months. Using a pharmacokinetic as well as classical industry sponsored simulation program, we calculate the ratio phase II and phase III trials. In addition, of observed versus expected plasma levels international collaborators include Keio of the prescribed NGA and investigate its University in Tokyo, the University of Bergen relationship with changes in psychopatho- in Norway and Zucker Hillside Hospital logical symptoms. In addition, the relation- in New York as well as local collaborators ship between attitudes toward drug therapy within the Department and also other as an indirect indicator of adherence and medical disciplines of the Medical University NGA plasma levels is studied. Innsbruck. Psychooncology research also has strong international connections Cognition including among others the European Both neuro- and social (affective) cognition Organisation for Research and Treatment in present another focus of this research Cancer (EORTC) which also partially funds group. More recently the investigation of quality of life and patient related outcomes social cognition in symptomatically remitted research. patients suffering from serious mental illness has received much attention. To Research this end, a number of studies investigating Director: facial emotion recognition and affective Univ.-Prof. Dr. W. Wolfgang Selected references, funding sources and prosody as well as Emotional Intelligence Fleischhacker collaborations are briefly presented in the are conducted in patients suffering from Appendix. schizophrenia or bipolar disorder, their Contact: healthy siblings as well as healthy control Anichstraße 35 Schizophrenia Research subjects, to identify social cognitive 6020 Innsbruck Wolfgang Fleischhacker, Alex Hofer deficits as potential trait markers for these disorders. [email protected] Clinical Psychopharmacology Phone: +43 512 504 23669 Past and ongoing studies focus on These studies also explore the potential role Fax: +43 512 504 25267 antipsychotics, ranging from early drug of cognitive impairment on various levels as http://psychiatrie.tirol-kliniken.at/ development in phase II clinical trials all the an endophenotype for schizophrenia and/or way to large-scale international pragmatic bipolar disorder. effectiveness studies. The underlying Keywords theme is always enhancing treatment Resilience options for patients with schizophrenia. The Three ongoing studies investigate resilience Addiction, alcohol, antipsychotics, bipolar European First Episode in Schizophrenia and its biological correlates in patients with disorder, bone mineral density, cognition, Trial (EUFEST) was the first large-scale schizophrenia and bipolar disorder with a cognitive-behavioural therapy, compliance, independent comparative first episode focus on religion and culture. The primary depot, patient related outcomes, prosody, study worldwide, evaluating treatment aim of these studies is to investigate patient recognition, psychooncology, quality outcomes in close to 400 patients in 14 transcultural differences in resilience across of life, resilience, schizophrenia, stigma, European countries and Israel. In its wake, patients from two different geographical substance abuse disorder, therapeutic drug Optimization of Treatment and Management regions, Austria and Japan, that have monitoring, transcultural psychiatry of Schizophrenia in Europe (OPTiMiSE) FP-7 different religious and cultural backgrounds funded program and the European long- (i.e. Christianity and Buddhism). Research Focus acting in Schizophrenia Trial (EULAST) pursue related research questions with Another study investigates the degree alcoholism, drug safety, psychopharma- advanced methodology. The group is also and quality of resilience as well as its cology, schizophrenia, substance abuse involved in global efforts to improve clinical correlates (e.g. hope, self-esteem, social disorder trial design in order to ease the translation support) across 200 female and 200 male from rigorous randomized controlled students from local universities. Using General Facts clinical trial standards into every day 3 T-MRI and fMRI and focusing on sex clinical practice. In this context, the issues differences, we will subsequently examine Embedded into the clinical services of of treatment attitudes, compliance and drug potential structural and functional cerebral the center, the Department of Biological safety have been given particular emphasis, differences in subjects with a high degree of Psychiatry’s research groups have a long a prospective therapeutic drug monitoring resilience compared to subjects with a low standing tradition of dealing with various program in schizophrenia patients treated degree of resilience. topics hovering around schizophrenia. with new generation antipsychotics being These are supported by a number of one example of these efforts. Psychooncology, Quality of Life, international collaborators and funded Outcomes Research by grants from the European Union, the Patients with schizophrenia starting mono Bernhard Holzner Austrian Science Foundation, the European therapy with a new-generation antipsy- In close collaboration with the Department Group for Research in Schizophrenia and chotic (NGA) in a naturalistic treatment of Psychosomatics, this group studies the pharmaceutical industry, the latter setting are prospectively followed up for psychological outcomes in cancer patients,

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applying late breaking statistical and Selected Publications The EORTC emotional functioning computerized adaptive test: phases I-III of a cross-cultural item bank development. Gamper technical methodology. Health related Sibutramine in the treatment of antipsychotic-induced weight EM, Groenvold M, Petersen MA, Young T, Constantini A, Aaronson quality of life is the main outcome variable gain: a pilot study in patients with schizophrenia. Biedermann N, Giesinger JM, Meraner V, Kemmler G, Holzner B, EORTC Quality Falko, Eltanaihi-Furtmüller Nadja, Huber Regina, Kemmler Georg, of Life Group. Psychooncology. 2014 Apr;23(4):397–403. and hand held computer based self- Ebenbichler Christoph, Lechleitner Monika, Fleischhacker W The Computer-based Health Evaluation Software (CHES): a soft- assessments analysed by item response Wolfgang, Hofer Alex. Int Clin Psychopharmacol. 2014; 29: p. 181–184. ware for electronic patient-reported outcome monitoring. Holzner theory are used to develop computer- B, Giesinger JM, Pinggera J, Zugal S, Schöpf F, Oberguggenberger Risk of violence of inpatients with severe mental illness – do pa- AS, Gamper EM, Zabernigg A, Weber B, Rumpold G. adapted questionnaires, adding a detailed tients with schizophrenia pose harm to others? Edlinger Monika, BMC Med Inform Decis Mak. 2012 Nov 9;12:126. subjective (patient-related outcome) Rauch Anna-Sophia, Kemmler Georg, Yalcin-Siedentopf Nursen, Fleischhacker W Wolfgang, Hofer Alex. Getting the whole picture: adding patient-reported outcomes assessment of the psychological health Psychiatry Res. 2014; 219: p. 450–456. to adjuvant endocrine treatment evaluation in premenopausal breast cancer patients. Oberguggenberger A, Goebel G, Beer B, status of cancer patients. Some of this Affective prosody perception in symptomatically remitted patients Oberacher H, Meraner V, Sztankay M, Sperner-Unterweger B, work is done under the aegis of EORTC with schizophrenia and bipolar disorder. Hoertnagl Christine M, Zeimet AG, Marth C, Hubalek M, Holzner B. Yalcin-Siedentopf Nursen, Baumgartner Susanne, Biedermann Brest J. 2014 Sept-Oct;20(5):555–7. and with a support of the oncology units Falko, Deisenhammer Eberhard A, Hausmann Armand, Kaufmann in the Departments of Hematology and Alexandra, Kemmler Georg, Muehlbacher Moritz, Rauch Anna- Reduced olfactory sensitivity, discrimination and identification in Sophia, Fleischhacker W Wolfgang, Hofer Alex. patients with alcohol dependence. CI Rupp, M Kurz, G Kemmler, D Gynecology of the MUI. Schizophr Res. 2014; 158: p. 100–104. Mair, A Hausmann, H Hinterhuber, WW Fleischhacker. Alcoholism Clinical and Experimental Research. 2003, 27, 432– Measuring adherence to medication in schizophrenia: the rela- 439. Substance use Disorder, Clinical tionship between attitudes toward drug therapy and plasma levels of new-generation antipsychotics. Yalcin-Siedentopf Nursen, Low bone mineral density and impaired bone metabolism in young Sergei Mechtcheriakov - Claudia Rupp Wartelsteiner Fabienne, Kaufmann Alexandra, Biedermann alcoholic patients without liver cirrhosis: a cross-sectional study. P Malik, RW Gasser, G Kemmler, R Moncayo, G Finkenstedt, M Falko, Edlinger Monika, Kemmler Georg, Rettenbacher Maria A 27 bed alcohol rehabilitation inpatient Kurz, WW Fleischhacker. A, Widschwendter Christian G, Zernig Gerald, Fleischhacker W Alcoholism Clinical and Experimental Research. 2009, 33(2):375– unit as well as a large outpatient clinic for Wolfgang, Hofer Alex. 81. patients suffering from substance-related Int J Neuropsychopharmacol. 2014 Dec 7; 18(5). pii: pyu091. doi: 10.1093/ijnp/pyu091. and addictive disorders from the illegal Selected Funding Comments on ‘Cognitive remediation improves memory and psy- spectrum are the base for research in this chosocial functioning in first-episode psychiatric out-patients’. • Improving usability of the EORTC questionnaires in daily clinical clinical field. Much emphasis is devoted Stürz K, Günther V. Psychol. Med. 2014; 44(3): 671. practice by elaborated QoL result presentation in CHES-EORTC, EORTC Quality of Life Group, 2012–2014 to neuropsychological deficits in patients The impact of abdominoplasty after massive weight loss: A quali- • Web-based patients reported outcome monitoring in cancer pa- suffering from chronic alcoholism and their tative study. Stürz K, Piza H, Kinzl JF. tients (WEB-PROM), Austrian National Bank (ÖNB), 2011–2014 Annals of Plastic Surgery. 2013; 71(5): 547–549. impact on the development and sustenance • Determination of European utility weights for a cancer-specific Effectiveness of antipsychotic drugs in first-episode schizophre- preference-based quality of life measure derived from the EO- of alcoholism as well as their relevance with nia and schizophreniform disorder: an open randomised clin- RTC QLQ-C30, EORTC, 2014–2016 respect to treatment outcomes. Individually ical trial. RS Kahn, WW Fleischhacker, H Boter, M Davidson, Y • Validation and scoring of the EORTC CAT measures, FWF, Vergouwe, IPM Keet, MD Gheorghe, JK Rybakowski, S Galderisi, J 2014–2017 tailored cognitive training measures are Libiger, M Hummer, S Dollfus, JJ López-Ibor, LG Hranov, W Gaebel, • “Moodinflame” (Early diagnosis, treatment and prevention of employed to support structured treatment J Peuskens, N Lindefors, A Riecher-Rössler, DE Grobbee, for the mood disorders targeting the activated inflammatory response EUFEST study group. system), EU, finished 2013 programs. Lancet. 2008. 371: 1085–97. • Optimization of Treatment and Management of Schizophrenia in Europe (OPTiMiSE) The Optimisation of Treatment and Management of Schizophrenia • Alex Hofer, FWF Projektnummer: KLI-366 The effect of chronic alcohol intake on in Europe (OPTiMiSE) trial: Rationale for its methodology and a • Berndhard Holzner, FWF Projektnummer: P 26930 Einzel review of the effectiveness of switching antipsychotics. S Leucht, projekte bone metabolism is another area of interest I Winter-van Rossum, S Heres, C Arango, WW Fleischhacker, • Johannes Giesinger, FWF Projektnummer: J 3353 Schrödinger- in this research group. In collaboration B Glenthøj, Marion Leboyer, FM Leweke, S Lewis, P McGuire, A Programm Meyer-Lindenberg, D Rujescu, S Kapur, RS Kahn and IE Sommer. with the Department of Endocrinology Schizophrenia Bulletin. 2015 May;41(3):549–58. Collaborations the influence of alcohol on bone mineral Treatment adherence in schizophrenia: A patient-level meta-analysis of combined CATIE and EUFEST studies. P Czobor, RA Van • Henning Flechtner, Magdeburg University Hospital density and various biomarkers involved in • Susanne Singer, Leipzig University Hospital (Stepped care pro- Dorn, L Citrome, RS Kahn, WW Fleischhacker, J Volavka. ject) it is explored. European Neuropsychopharmacology. 2015 Aug;25(8):1158–66. • Fabio Efficace, GIMEMA Group, Roma, Italy Aripiprazole once-monthly for treatment of schizophrenia: a dou- • Hiroyuki Uchida, Keio University School of Medicine, Depart- With respect to illegal drugs, patients ble-blind, randomised, non-inferiority study. WW Fleischhacker, ment of Neuropsychiatry, Tokyo, Japan R Sanchez, PP Perry, N Jin, T Peters-Strickland, BR Johnson, RA • Roger Pycha, Hospital of Brunico, Department of Psychiatry, in methadone and opiate substitution Baker, A Eramo, RD McQuade, WH Carson, D Walling, JM Kane. Brunico, Italy programs constitute a large group of British Journal of Psychiatry. 2014, 205:135–144. • European Alliance against Depression • pro mente tirol interest. Currently, preferences for the Schizophrenia – Time to Commit to Policy Change. WW • University of Bergen, Norway different drugs available in substitution Fleischhacker, C Arango, P Arteel, TRE Barnes, W Carpenter, K • University Medical Center Utrecht, Netherlands Duckworth, S Galderisi, L Halpern, M Knapp, SR Marder, M Moller, • Zucker Hillside Hospital, New York programs and the relevance of subjective N Sartorius, P Woodruff. Schizophrenia Bulletin. 40 (3): 165–194, • European Group for Research in Schizophrenia (EGRIS) attitude are studied. 2014. • Keio University, Tokyo, Japan

Research Report 2015 Medical University of Innsbruck 135 Center of Psychiatry and Pychotherapy Psychosomatic Medicine

Keywords Research

Psychoimmunology, stress, depression, eat- Research in Psychoimmunology ing disorders, patient-reported outcome Priv.-Doz. Dr. Katharina Hüffner Psychoimmunology provides the opportunity Research Focus to study the interacting biological systems that might contribute to psychosomatic • Interdisciplinary psychosomatic research: and somatopsychic co-morbidities. In this using the field of “psychoimmunology” to context chronic stress conditions are used study psychosomatic and somatopsychic as a research paradigm for the investigation co-morbidities of individual stress reactions and related • Research in eating disorders changes in neurotransmitter-pathways and • Research in patient-reported outcome immune activation.

General Facts Several immunological mechanisms might link chronic somatic disease such as Research in the field of psychosomatic breast cancer and psychological distress. medicine: One possibility is that inflammatory Director (interim): • Is concerned with the complex interac- stimuli can influence the neurotransmitter Univ.-Prof.in Dr.in Barbara tions between physical, mental and social pathways known to be important in the Sperner-Unterweger conditions that can contribute to psycho- pathogenesis of depression. Specifically, somatic and somatopsychic comorbidities the serotonin pathway can be influenced Contact: • Is characterized by interdisciplinary ap- by cytokines which enhance the activity Anichstraße 35 proaches of the indole amine 2,3-dioxygenase (IDO) 6020 Innsbruck –– In co-operation with the Psychiatric/ or stress hormones which can activate Psychotherapeutic Consultation-Liaison tryptophan 2,3-dioxygenase (TDO). Both barbara.sperner-unterweger Service enzymes are involved in the break-down of @i-med.ac.at –– In co-operation with other clinical units tryptophan (TRP) to kynureine (KYN). Phone: +43 512 504 23691 outside the department of Psychiatry Fax: +43 512 504 24778 and Psychotherapy The resulting TRP depletion results in lower http://psychiatrie.tirol-kliniken.at/ levels of serotonin (5-HT) which could One major issue encountered during routine in turn increase the risk for depressive clinical work as well as in research is the symptoms (Fig. 1). In addition to influencing subjective judgement of individual patients the serotonergic neurotransmitter pathway, with regard to their mental and physical inflammatory stimuli can also affect well-being. We consider this as “basic catecholamine synthesis via the enzyme knowledge” essential for the evaluation of co-factor, tetrahydrobiopterin (BH4) or therapeutic concepts. cytokines may reduce the availability of

Fig. 1: Graphical depiction of the amino acid derived neurotransmitter biosynthesis pathways analyzed in our current studies. Changes induced by inflammation are indicated with an arrow.

136 Research Report 2015 Medical University of Innsbruck Psychosomatic Medicine

Fig. 2: Patient-reported outcome profile of an individual patient’s quality of life trajectory

BH4, which is necessary for the conversion obese patients who undergo bariatric Pathological eating and body dissatisfaction in middle-aged and older women. Mangweth-Matzek Barbara, Hoek Hans W, Pope of phenylalanine ((PHE) to tyrosine/TYR) by surgery. We also perform epidemiological Harrison G. phenylalanine hydroxylase (PAH). Tyrosine studies, e.g. middle aged males; and Jr. CURRENT OPINION PSYCHIATRY. 2014; 27(6): 431–435. hydroxylase which converts TYR into L-3, between female and male fitness-centre- Getting the whole picture: adding patient-reported outcomes to adjuvant endocrine treatment evaluation in premenopausal 4-dihydroxyphenylalanine (Levodopa) is sports activists with regard to eating breast cancer patients. Oberguggenberger Anne, Goebel Georg, also dependent on BH4. PHE/TYR ratio is behaviour, quality of life, and mood. Beer Beate, Oberacher Herbert, Meraner Verena, Sztankay Monika, Sperner-Unterweger Barbara, Zeimet Alan G, Marth used as an estimate of PAH activity (Fig. 2). Christian, Hubalek Michael, Holzner Bernhard. Patient-Reported Outcome THE BREAST JOURNAL. 2014; 20(5): 555–557. Notably, also tryptophan hydroxylase, Univ.-Doz. Dr. Bernhard Holzner Selected Funding an enzyme that converts TRP to 5-HT, is A major research focus of our group • “Moodinflame” (Early diagnosis, treatment and prevention of dependent on BH4. Currently, we study in 2013 and 2014 was the evaluation mood disorders targeting the activated inflammatory response healthy probands as well as patients with of medical treatments and the impact system), EU, finished 2013 • Platelet function as biomarker of depression – Can acute men- depression and depressive adjustment of chronic diseases from a patient’s tal stress increase the diagnostic value? Österreichische Natio- disorder with and without chronic somatic perspective. We have conducted a number nalbank, 01.01.2013 – 31.12.2015 • Sexual health during endocrine-treatment for breast cancer, disorders (e.g. cancer). These projects are of patient-reported outcome (PRO) studies Austrian Cancer Aid, 2013–2015 being carried out together with various in cancer patients investigating clinical as cooperation partners within the Medical well as methodological research questions. Collaborations University Innsbruck such as the Division Currently, we are extending our PRO • Harrison G. Pope, MD; Biological Psychiatry Laboratory, McLean Hospital/Harvard Medical School, Boston, USA of Biological Chemistry at the Biocenter research activities beyond oncology and • Hans W. Hoek, MD; Parnassia Psychiatric Institute, The Hague/ and the Department of Gynaecology and are planning and conducting studies in Department of Psychiatry, Groningen University, The Nether- lands / Department of Epidemiology, Columbia University, New Obstetrics and are supported by the EU grant further medical fields in addition to those York, USA “moodinflame” (Early diagnosis, treatment performed in- and outpatient groups from • Dr. K.M. Giesinger; Kantonsspital St. Gallen (Dept. of Orthope- dia), St. Gallen, Schweiz and prevention of mood disorders targeting the Division of Psychosomatic Medicine. • Univ.-Prof. Dr. Henning Flechtner; Magdeburg University Hos- the activated inflammatory response pital • Univ.-Prof. Dr. Susanne Singer; Leipzig University Hospital (Step- system – finished 2013) and an ongoing Selected Publications ped care project) • Prof. Dr. Fabio Efficace, GIMEMA Group, Roma, Italy ÖNB grant (Platelet function as a biomarker The menopausal transition—a possible window of vulnerability for depression – Can acute mental stress for eating pathology. Mangweth-Matzek Barbara, Hoek Hans W, Rupp Claudia, Kemmler Georg, Pope Harrison G-Jr, Kinzl Johannes. increase the diagnostic value?, 01.01.2013 INTERNATIONAL JOURNAL OF EATING DISORDER. 2013; 46(6): –31.12.2015). 609–616.

Psychometric evaluation of the EORTC computerized adaptive Research in Eating Disorders test (CAT) fatigue item pool. Petersen Morten Aa, Giesinger Univ.-Prof. Mag. Dr. Barabara Mangweth- Johannes M, Holzner Bernhard, Arraras Juan I, Conroy Thierry, Gamper Eva-Maria, King Madeleine T, Verdonck-de Leeuw Irma W, Matzek Young Teresa, Groenvold Mogens. Present research is focused on various QUALITY OF LIFE RESEARCH. 2013; 22(9): 2443–2454. aspects of clinical eating disorders: We Bioprofiling of plateletes in medicated patients with depression. currently study a) addictive symptoms in Hüfner Katharina, Kandler Christina, Koudouovoh-Tripp Pia, Egeter Jonas, Hochstrasser Tanja, Stemer Bettina, Malik Peter, Giesinger anorexic and bulimic patients, b) cultural Johannes, Humpel Christian, Sperner-Unterweger Barbara. differences and traumatic childhood in JOURNAL OF AFFECTIV DISORDERS. 2014; 172: 81–88.

Research Report 2015 Medical University of Innsbruck 137 Center of Psychiatry and Pychotherapy Medical Psychology

Keywords represents a psychological center of excellence that is dedicated to the psycho- Bio-psycho-social aspects of diseases, social evaluation of different transplant can- doctor-patient-relationship, psychotherapy didates, particularly of living kidney donors and recipients as well as other candidates Research Focus undergoing reconstructive or solid organ transplantation. • Psychoneuroimmunology: investigation of psychosomatic/psychoneuroimmunologi- The research group Cognitive Neuro cal complexity science cooperates locally and internation- • Patient reported outcomes ally. The facility “Lab for Cognitive Neuro- • Quality of life, health psychology, well sciences Innsbruck” is fully equipped with being, psychometric assessment, ques- electroencephalography (EEG), functional tionnaire development, positive psychol- near-infrared spectroscopy (fNIRS), and ogy behavioural measuring methods suitable • Transplant psychology: psychosocial eval- for studies in infants, children, adults, and uation, treatment protocols patients. • Cognitive Neuroscience: neuronal pro- Director: cessing for cognitive processes e.g. lan- The working group “Psychotraumatology o. Univ.-Prof. Dr. Gerhard Schüßler guage and Trauma Therapy” examines the • Psychotraumatology and Trauma Therapy effects of specific trauma-therapeutic Contact: • Psychotherapy research concerning treatment in patients with complex post- Schöpfstraße 23a emotions, diagnosis, efficacy and delivery traumatic stress disorders (cPTSD) and 6020 Innsbruck in patients with dissociative disorders. In General Facts this project the group works together with Gerhard.Schüß[email protected] European research groups. Phone: +43 512 504 27707 The Psychoneuroimmunology Laborato- Fax: +43 512 585418 ry run by ao.Univ.-Prof. DDr. Christian Schu- A broad training of medical students in http://psychiatrie.tirol-kliniken.at/ bert was founded in 1995. Currently, there doctor-patient-relationship and com- is strong collaborative activity with the munication is one major task. A psycho Division of Biological Chemistry, Biocentre, therapeutic inpatient and mainly outpatient Innsbruck Medical University, Innsbruck, clinic gives the opportunity for research in Austria (ao.Univ.-Prof. Dr. Dietmar Fuchs). this clinical field. In 2014, a highly competitive grant from the Austrian Science The Center for Advanced Psychology in Foundation (P 27228-G22) was received Plastic and Transplant Surgery (CAPPTS) to focus on the health and wellbeing of

Fig. 1: Simultaneous assessment of electrophysiological and vascular signals by means of the electroencephalography and the functional near-infrared spectroscopy.

138 Research Report 2015 Medical University of Innsbruck Medical Psychology

Fig. 2: Negative Incidents with Cortisol. Fig. 3: Positive Incidents with Neopterin.

medical students and practitioners. In par- (fNIRS), both adoptable also simultaneously. focus was on the health consequences of ticular health professionals in the medical severe stress in childhood, epigenetics, and field are prone to urnoutb . This research Health Psychology brain development after traumatic stress in project focuses on identifying key factors Stefan Höfer childhood. to promote health and wellbeing throughout Research focuses on two key areas: the medical curricula. The unique approach the fist key area is the development of Selected Publications is to investigate environmental (work- and international patient reported outcome OPD-2 – Operationalisierte Psychodynamische Diagnostik: Das Manual für Diagnostik und Therapieplanung. (Ed.; u.a. G. organisational psychology) as well as per- measures in different areas (i.e. heart Schüßler), Hans Huber. Arbeitskreis OPD. 2014. sonal factors (health psychology, positive disease) and different languages world- The first step is the hardest – emotion recognition in patients psychology) together to determine path- wide (i.e. Swedish, Danish, Norwegian). The with somatoform disorders. Beck Thomas, Breuss Margit, Kumnig Martin, Schüßler Gerhard. ways to health and wellbeing throughout application of health psychological theories ZEITSCHRIFT FÜR PSYCHOSOMATISCHE MEDIZIN UND PSYCHO- the professional life. to enhance the quality of life and wellbeing THERAPIE. 2013; 59: p. 385–390. of patients and the general population is the Psychoneuroimmunologie des Lebenslaufs: Einfluss von Stress second key area. Recently together with the in der Kindheit auf Immunfunktionsstörung und entzündliche Er- Research krankung im weiteren Leben. Schubert Christian. European Health Psychological Society and PSYCHOTHER PSYCHOSOM MED PSYCHOL. 2014; 64: p. 171– Psychoneuroimmunology the Federation of Austrian Associations of 180. Psychologists Prof. Höfer hosted the 28th Is there a link between physical activity and alcohol use?. Kopp Christian Schubert Martin, Burtscher Martin, Kopp-Wilfling Prisca, Ruedl Gerhard, Stress system dynamics during “life as it Conference of European Health Psychology Kumnig Martin, Ledochowski Larissa, Rumpold Gerhard. is lived”: integrative single-case studies on Society with over 800 participants. SUBST USE MISUSE. 2015; 50: p. 546–51. healthy women and women with LE, breast [Patterns of dysfunctional parenting styles and psychological disturbances in offspring]. Kumnig Martin, Höfer Stefan, Alex- cancer, depression et al. Psychotraumatology and andra Huber, Messner Carmen, Renn Daniela, Mestel Robert, Trauma Therapy Klingelhöfer Jürgen, Kopp Martin, Dören Stephan, Schüßler Gerhard, Rumpold Gerhard. Transplantation Psychology Astrid Lampe PSYCHOSOMATISCHE MEDIZIN UND PSYCHOTHERAPIE. 2013; Martin Kumnig In addition to the main task of trauma 59: p. 356–368. Psychological assessments are crucial therapy, the effects of training measures An overview of psychosocial assessment procedures in reconstructive hand transplantation: The past, present, and future for the evaluation and optimization of the to sensitize medical professionals for in psychosocial assessemnt of patients undergoing for recon- suitability of transplant patients, consider- domestic violence are reviewed and possible structive hand transplantation. Kumnig Martin, Jowsey-Gregoire Sheila G, Morenao Elisa, Brandacher Gerald, Azari Kodi, Rumpold ing solid organ or vascularized composite negative consequences of traumatic events Gerhard. allotransplantation (VCA). Psychological are surveyed regarding the prevalence for TRANSPLANT INTERNATIONAL. 2013; 27: p. 417–427. assessment is mandatory for living kidney illnesses. Within the project “The experience Electrophysiological evidence for modulation of lexical processing after repetitive exposure to foreign phonotactic rules. Rossi donation. and the stress processing of executives in Sonja, Hartmüller Tobias, Vignotto Micol, Obrig Hellmuth. emergency organisations” two measure BRAIN AND LANGUAGE. 2013; 127: p. 404–414. Lab for Cognitive Neurosciences packages have been developed; on one Selected Funding Sonja Rossi hand a self-management tool for executives • Optimizing patient/doctor interaction at the breast cancer The research group “Cognitive Neuro which helps the affected executives to unit at the LKH/Tirol, Land Tirol Qualitätsförderungsprogramm science” focuses primarily on neuronal efficiently adjust their stress load, on the 2011–2013, Christian Schubert • Language learning in monolingual and bilingual infants, 2013– processing mechanisms during first and other hand a guide for psychosocial experts 2015 Marie Curie Intra-European Felloship awarded by the Eu- second language acquisition in infancy and for the professional support of stress- ropean Commission, Sonja Rossi • Austrian Science Foundation 2014–2017, Stefan Höfer adulthood. Neuronal markers are assessed loaded executives. The annual meeting by means of the electroencephalography of the Society for Psychotraumatology Collaborations (EEG) and the near-infrared spectroscopy (DeGPT) 2015 took place in Innsbruck. The • Multiple international cooperations, s.a.

Research Report 2015 Medical University of Innsbruck 139 Center of Psychiatry and Pychotherapy Child and Adolescent Psychiatry

Keywords ual factors and how they contribute to the development of this complex and painful Child and adolescent psychiatry, child and condition. The aim of our research is to as- adolescent psychology, clinical psychiatry, sess attachment representations in adoles- psychopathology, psychotherapy, personal- cent patients with Anorexia Nervosa or Bu- ity disorders, eating disorders, attachment limia Nervosa. We will analyze attachment research, internet addiction, cyber-mobbing themes concerning parent-child relationships and experiences of separation, abuse Research Focus and loss in the attachment narratives of our adolescent ED patients. Assessment and Course of Personality Disorders in Adolescence Additionally, we are interested in the links The diagnosis of personality disorders between attachment representations, (PD) in adolescence has long been symptom severity, comorbid disorders and controversially discussed in the research personality pathology. One of the most literature. Major concerns include the challenging directions taken by attachment validity and stability of diagnosing PD, the researchers focuses on the influence of heterogeneity of the psychopathological attachment representations on the course Director: profile and stigmatisation in that age group. and outcome of mental disorders. To Univ.-Prof.in Dr.in Kathrin Sevecke date, there is no study investigating that Even though studies on temperament and influence in adolescent patients with ED. Contact: personality provide evidence that the main Thus, we want to examine the influence of Anichstraße 35 ingredients of adult PD are present during attachment representations on the outcome 6020 Innsbruck puberty, only very little is known about the and also, for the first time, analyse how far course of these disorders in adolescents. attachment can change after treatment. [email protected] Therefore, the purpose of this study is to ex- Phone: +43 512 504 23670 amine underlying dimensions (e.g. attach- General Facts Fax: +43 512 504 23650 ment patterns, emotion regulation, integra- https://www.i-med.ac.at/patienten/ tion level of mental structure) mediating the New research unit with a special focus ukl_kinder_jugendpsychiatrie.html course and outcome of PD in adolescents. on personality pathology and attachment Furthermore, we are interested in examin- research in child and adolescent psychiatry ing possible links between attachment representations, symptom severity, comorbid Collaborations inside MUI: disorders, course and outcome in adolescent patients with PD. Neuropsychoimmunology in Adolescent Patients with Eating Disorders Attachment and Eating Disorders in ao.Univ.-Prof. Dr. Dietmar Fuchs, Section for Adolescence Biological Chemistry, Biocenter Innsbruck The emerging body of attachment research Neural correlates and personality pathology in patients with eating disorders (ED) pro- in adolescents with eating disorders (Univ.- vides a promising insight into the interplay Prof. Dr. Elke Gizewski, Department of Radi- between environmental, social and individ- ology, Medical University of Innsbruck)

Dr. Astrid Bock, Dr. Manuela Gander, Dr. Martin Fuchs, Postdoc Researcher Postdoc Researcher Specialist for Child and Adolescent Psychiatry

140 Research Report 2015 Medical University of Innsbruck Child and Adolescent Psychiatry

Research

Assessment and Course of Personality Assessing Trauma in Children and Disorders in Adolescence Adolescents Prof. Dr. Kathrin Sevecke, Univ.-Prof. Dr. Barbara Juen, Dr. Manuela Gander, Dr. Astrid Bock Prof. Dr. Kathrin Sevecke The purpose of this study is to examine This study aims to improve the assess- underlying dimensions (e.g. attachment ment of trauma and analyse consequences patterns, emotion regulation, integration of traumatic experiences in children and level of mental structure) mediating the adolescents. course and outcome of PD in adolescents.

Furthermore, we are interested in exam- Selected Publications ining possible links between attachment Autismus oder „Psychopathy“? Roberz J, Lehmkuhl G, Sevecke K representations, symptom severity, co Forensische Psychiatrie, Psychologie, Kriminologie. 2013, 7:282– morbid disorders, course and outcome in 289. adolescent patients with PD. Tierquälerei als Symptom von Callous-Unemotional Traits bei in- haftierten Jungen und Mädchen. Sevecke K, Franke S, Krischer M Kindheit und Entwicklung. 2013, 22(3):165–173. Attachment and Eating Disorders in Schulabsentismus bei Kindern und Jugendlichen – Beilage zum Adolescence Fachjournal „neuropsychiatrie – vereinigt mit psychiatrie und psy- Prof. Dr. Kathrin Sevecke, chotherapie“. Sevecke K; 2014.

Dr. Manuela Gander, Dr. Astrid Bock Grenzfall ADHS – Diagnose und Differentialdiagnose im Volks The aim of our research is to assess schulalter: A Review. Bonatti E, Küng A, Sevecke K. attachment representations in adolescent Zeitschrift Neurologie Psychiatrie. Jatros 2014 (12–14). patients with Anorexia Nervosa and Bulimia Das dimensional-kategoriale Hybridmodell für Persönlichkeits störungen im DSM-5 aus jugendpsychiatrischer Perspektive – Nervosa and analyse their influence on the Kritik und Ausblick. Sevecke K, Schmeck K, Krischer M. Zeitschrift outcome. für Kinder- und Jugendpsychiatrie und Psychotherapie. 2014, 42 (4), 279–283.

Neuropsychoimmunology in Adolescent Persönlichkeitspathologie und Psychopathie bei verhaltensauf fälligen, delinquenten Jugendlichen. Sevecke K, Maya K, Krischer Patients with Eating Disorders und Gerd Lehmkuhl. Prof. Dr. Dietmar Fuchs, Prof. Dr. Kathrin Buch: Kriminologie-Jugendkriminalrecht-Strafvollzug. Heraus- geber Frank Neubacher, Michael Kubink. Band 59 Drucker & Sevecke Humblot. Berlin 2014. To examine relevant neurotransmitters in Borderline-Störungen und selbstverletzendes Verhalten. Sevecke plasma samples of patients with Anorexia K, Krischer M, In G Lehmkuhl, F Poustka, M Holtmann & H Steiner. Nervosa. Praxishandbuch Kinder- und Jugendpsychatrie. Hogrefe, ISBN: 978-3-8017-2538-9 (2014).

Neural Correlates and Personality Die Psychoptahy-Checkliste für Jugendliche. Sevecke K, Krischer Pathology in Adolescents with Eating M. Hogrefe. 2014. Disorders Attachment classification, psychophysiology and frontal EEG asymmetry across the lifespan. Gander M & Buchheim A. Prof. Dr. Elke Gizewski, Frontiers in Human Neuroscience. 9 (79): 1–16. Prof. Dr. Kathrin Sevecke Eating disorders in adolescence: Attachment issues from a de- To investigate neural correlates of adoles- velopmental perspective. Gander M, Sevecke K & Buchheim A. cents with eating disorders while watching Frontiers in Psychology. 2015;6: 1–12. visual food cues in an fMRI environment. Collaborations

Emotional and Structural Indicators • Univ.-Prof. Dr. Anna Buchheim, Institute of Psychology at the University of Innsbruck, Austria of Psychopathological Development in • Univ.-Prof. Dr. Barbara Juen, Institute of Psychology at the Adolescence University of Innsbruck, Austria • Univ.-Prof. Dr. Svenja Taubner, Institute of Psychology at the Dr. Astrid Bock Alpen-Adria University of Klagenfurt, Austria The aim of this study is to examine factors • Dr. Florian Juen, KBO Child Center Munich, Germany • Prof. Dr. med. Dipl. Psych. Klaus Schmeck, KJPK Basel, Switzer like affect regulation and emotion recogni- land tion that might contribute to the early onset • PD Dr. Maya Krischer, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Cologne, of psychopathology in adolescence. Germany

Internet-Addiction and Cyber-Mobbing in Psychiatric Patients Dr. Martin Fuchs, Prof. Dr. Kathrin Sevecke The major aim of this study is to analyse the prevalence rates of internet-addiction and cyber-mobbing experiences in psychiatric in- and outpatients.

Research Report 2015 Medical University of Innsbruck 141 Center of Neurology and Neurosurgery Neurology

surgery, neurooncology, epilepsy surgery and deep brain stimulation for movement disorders. Equally close collaborations exist with the Departments of Neuroradiology and Vascular Surgery and Cardiology in the field of stroke medicine. Research collaborations in the field of neuroimaging are centered upon the Department of Neuroradiology and Nuclear Medicine. Dedicated research infrastructures within the clinical department include the Division of Neurobiology (animal modelling of neurodegeneration), the neurological research laboratory (focus on biomarker research and neuroimmunology), as well as the recently established computational neuroscience unit for the development of novel image analysis algorithms. Director: o. Univ.-Prof. Dr. Werner Poewe Research © Lancet Neurol Contact: Research Group Stroke Anichstraße 35 Fig. 1: Thrombolysis administration in the Group leaders: 6020 Innsbruck nine countries of Tyrol in 2010 (A) and Univ.-Prof. Johann Willeit 2013 (B). Univ.-Prof. Stefan Kiechl, [email protected] Priv.-Doz. Michael Knoflach Phone: +43 512 504 23850 General Facts Research priorities of the neurovascular Fax: +43 512 504 23852 The Department of Neurology at Innsbruck research group include acute and post- www.i-med.ac.at/neurologie Medical University has a total of 114 in- acute stroke care, cardiovascular risk patient beds including a stroke unit (8 beds), prediction as well as cardiovascular ageing. a neurocritical care unit (10 fully-ventilated Based on the internationally well-respected beds, 6 post-immediate care beds), an Bruneck Study, a population based Keywords epilepsy monitoring unit (4 beds) and a longitudinal cardiovascular health study dedicated sleep laboratory (6 beds). The that has been running for 25 years, state of Stroke research, multiple sclerosis (MS), department acts as a referral center for the the art biomarker research is conducted in neurocritical care, parkinson’s disease (PD), entire spectrum of neurological diseases an international cooperation with a focus on multiple system atrophy (MSA), REM sleep encompassing the entire area of the state of the lipidome and the role of micro RNAs in behavior disorder, RBD, RLS, computational Tyrol and neighbouring areas. Furthermore, atherogenesis. The neurovascular research neuroscience the department receives referrals on an group participates in in large international Austria-wide scale when second opinions research and meta-analysis consortia Research Focus and specialized diagnostic and therapeutic (steering/writing committee: FSC, ERFC, procedures are required. The large number LSC, NPSC, IMT-PROG, GBD, various GWA The clinical Department of Neurology at of patients received into the department studies). the Medical University of Innsbruck has an each year (more than 6,000 in-patient internationally established research focus admissions per year, close to 40,000 out- The evaluation of the effect of the in the fields of epidemiology and pathophys patient contacts per year) results in a optimization of the Tyrol wide stroke iology of ischemic stroke, neurocritical care significant clinical routine workload. Key treatment pathway received broad inter (including infectious diseases of the ner collaborations in the field of clinical routine national acclaim (Fig. 1). Risk prediction vous system), Parkinson’s Disease, MSA include joint programs with the Department after TIA as well as impact of differences and degenerative movement disorders. of Neurosurgery, in particular vascular in stroke unit management on treatment In addition, the department is the only academic institution in Austria with an internationally recognized and certified sleep laboratory with a focus on research into the field of sleep-related movement disorders. The Division of Neurobiology is focused on animal modelling of neurodegeneration with an emphasis on multiple system atrophy. A recently established professorship for computational science is concerned with establishing novel image analysis Fig. 2: Involvement of microglia (CD11b) in the clearance of α-synuclein (hAS) in a model of algorithms for MRI datasets. MSA with accumulation of AS in oligodendroglia (CNP).

142 Research Report 2015 Medical University of Innsbruck Neurology

recognized clinical research programme focusing on degenerative movement disorders including Parkinson’s disease and related syndromes. Major contributions have been made in the field of clinical trials in PD, rating scale development for PD and MSA, natural history and imaging studies in parkinsonian syndromes and other movement disorders, with key papers in major journals such as Neurology, Brain or Lancet Neurology.

The research group is involved in multiple international research consortia and networks and between 2013 and 2014 Fig. 3: Three-dimensionally reconstructed human brain based on a T1-weighted MRI se- has authored and co-authored a total of 86 quence using FreeSurfer brain-analyzing tool. The silver-coloured image represents the cal- papers in peer-reviewed journals. Recent culated surface model of a human cerebral cortex with its detailed gyrification. Colour-cod- clinical research projects have focused ed cortical labels of both hemispheres reflecting various anatomical regions are illustrated on the epidemiology, genetics and natural in the left image. Data can be used to investigate regional and tissue specific neuroanatom- history of PD, MSA and other movement ical variations. disorders including HD and ataxias, on the validation of imaging and biomarker studies for PD, MSA, ataxias and related disorders, on the development, clinimetrics delays are addressed as part of the Austrian Research Group Neurocritical Care and and validation of new rating scales for PD, Stroke Unit Collaboration and Registry. Infectious Diseases of the Nervous MSA and ataxias and on clinical trials in PD Different approaches to post stroke care are System and related movement disorders. Present currently tested in a large scale randomized Group leaders: aims are to validate biomarkers for early disease management project (Stroke Card). Univ.-Prof. Erich Schmutzhard*, diagnosis and progression of PD, MSA and Johann Willeit and Stefan Kiechl head the Priv.-Doz. Ronny Beer, other movement disorders inlcuding HD ‘Research Center of Excellence in Vascular Priv.-Doz. Raimund Helbok* and ataxias as well as to introduce clinical Ageing – Tyrol’ which has its headquarters in The study group focuses both on clinical trials of novel therapies for these disorders Innsbruck (4.3 Mio €) (Fig. 1). and translational research. Invasive multi- including an immunisation trial against modal neuromonitoring, covering structural alpha-synuclein. Major achievements to Research Group Multiple Sclerosis and (imaging), metabolic (microdialysis, pbtiO2, date include the identification of risk factors Neuroimmunology CBF), functional monitoring (electrocor- and prodromal markers for the development Group leaders: ticography – COSBID) and the clinical as- of Parkinson’s disease and a natural history Univ.-Prof. Thomas Berger, pects of neurocritical care (blood pressure study of multiple system atrophy. Future Univ.-Prof. Florian Deisenhammer, management, nosocomial infections, venti- goals of the department encompass a Univ.-Prof. Markus Reindl lator associated pneumonia etc.) are major better characterization of premotor PD and Grounded in long-lasting and excellent foci in the departments research. Murine clinical trials on novel therapeutics for PD, research collaborations the main research aneurysmatic subarachnoid haemorrhage MSA and other movement disorders. topics are related to pathomechanisms, and murine cerebral malaria are the basis *state funded position (TirolKliniken) with clinical and therapeutic aspects of for translational research. Beyond that, inactive research grants in MUI autoimmune CNS and PNS disorders: fectious diseases of the nervous system multiple sclerosis (MS), antibody-associated and tropical neurology (cerebral malaria, Research Group Sleep Medicine neurological disease (neuromyelitis helminthic diseases of the brain, epilepsy in Group leader: optica [NMO], acute disseminated tropical countries) are a major focus of this Univ.-Prof. Birgit Högl, encephalomyelitis or NMDAR-encephalitis) research group. Univ.-Doz. Birgit Frauscher and immune neuropathies. Thus, scientific *state funded position (TirolKliniken) with The Sleep Disorders Research Group and foci are the role of human autoantibodies active research grants in MUI Sleep Disorders Clinic at the Department directed against different CNS tissue of Neurology have a highly modernized antigens (mainly MOG, AQP4 and Research Group Movement Disorders clinical sleep laboratory and a large sleep NMDA-R) and neutralizing antibodies in the Group leaders: disorders outpatient clinic. The unit monitoring of various MS treatments. These o. Univ.-Prof. Werner Poewe, serves the whole spectrum of clinical research activities are complementary Priv.-Doz. Sylvia Bösch*, sleep medicine including insomnia, sleep to and translatable to our neurological Univ.-Prof. Christoph Scherfler, breathing disorders, narcolepsy and laboratory diagnostics. In addition, Univ.-Prof. Klaus Seppi, other hypersomnia syndromes, circadian extensive databases for MS and NMO have Univ.-Prof. Gregor Wenning disorders, parasomnias (such as sleep been established, which are of paramount Movement disorder groups at the walking and REM sleep behavior disorder) importance for past and current national Department of Neurology and their partners and other sleep disorders, namely restless and international research activities. at MUI have established an internationally legs syndrome (RLS).

Research Report 2015 Medical University of Innsbruck 143 Center of Neurology and Neurosurgery

made important contributions regarding P, Langley S, Santer P, Rungger G, Willeit J, Kiechl S, Mayr M (2014); Bruneck Study. Circulation 129:1821–31. clinical presentation, diagnostic tools and pathogenesis of MSA. We are especially 4.Blockade of receptor activator of nuclear factor-kB (RANKL) signaling improves hepatic insulin resistance and prevents de- interested in α-synuclein mediated velopment of diabetes mellitus. Kiechl S, Wittmann J, Giaccari A, oligodendroglial pathology that includes Knoflach M, Willeit P, Bozec A, Moschen A, Muscogiuri G, Sorice GP, Kireva T, Summerer M, Wirtz S, Luther J, Mielenz D, Billmeier protein misfolding and aggregation as well U, Egger G, Mayr A, Oberhollenzer F, Kronenberg F, Orthofer M, as cell-to-cell propagation. We identified Penninger J, Meigs JB, Bonora E, Tilg H, Willeit J, Schett G (2013); fundamental interactions between MSA Nature Medicine. 19:358–63. pathology and mitochondrial or proteolytic 5. Circulating microRNAs as novel biomerkers for platelet activation. Willeit P, Zampetaki A, Dudek K, Kaudewitz D, King A, Kirkby stress. The latter contribute to the specific NS, Crosby-Nwaobi R, Prokopi M, Drozdov I, Langley S, Sivaprasad neuronal vulnerability in MSA and represent S, Markus HS, Mitchell JA, Warner T, Kiechl S, Mayr M (2013); powerful interventional targets. We were Circ Res 112:595–600. the first to demonstrate that the toll-like Research Group Multiple Sclerosis and Neuroimmunology 1. Overlapping demyelinating syndromes and anti-N-methyl-D-as- receptor 4 (TLR4) promotes α-synuclein partate receptor encephalitis. Titulaer MJ, Hoeftberger R, Iizuka T, clearance by microglia (Fig. 2). Leypoldt F, McCracken L, Cellucci T, Benson LA, Shu H, Irioka T, Hirano M, Singh G, Cobo C, Alvaro K, Kenichi Morales PS, Wirtz More recently our work also involved pre- PW, Yamamoto T, Reindl M, Rosenfeld MR, Graus F, Saiz A, Dalmau clinical screening of candidate neuroprotec- J. Ann Neurol. 2014; 75: p. 411–428.

tive and neuroregenerative strategies that 2. Guidelines for uniform reporting of body fluid biomarker are currently being evaluated for safety and studies in neurologic disorders. Gnanapavan S, Hegen H, Khalil M, Hemmer B, Franciotta D, Hughes S, Hintzen R, Jeromin A, efficacy in controlled clinical trials Havrdova E, Tumani H, Bertolotto A, Comabella M, Frederiksen J, Alvarez-Cermeno JC, Villar L, Galimberti D, Myhr KM, Dujmovic I, Fazekas F, Ionete C, Menge T, Kuhle J, Keir G, Deisenhammer F, Computational Neuroscience: Teunissen C, Giovannoni G. Group leader: Neurology. 2014; 83: p. 1210–1216.

Univ.-Prof. Christoph Scherfler 3. Antigen-Specific oleranceT by Autologous Myelin Peptide-Cou- The research unit for Computational pled Cells: A Phase 1 Trial in Multiple Sclerosis. Lutterotti A, Yousef S, Sputtek A, Stuerner KH, Stellmann JP, Breiden P, Fig. 4: SINBAR Study Group / Department of Neuroscience was established at the Reinhardt S, Schulze C, Bester M, Heesen C, Schippling S, Miller Neurology. Department of Neurology in October 2014 SD, Sospedra M, Martin R. to accommodate and support existing Science Translational Medicine. 2013; 5. research groups in the field of MRI and PET 4. The spectrum of MOG autoantibody-associated demyelinating For specific clinical questions regular and image analysis. The unit is tightly intertwined diseases. Reindl M, Di Pauli F, Rostasy K, Berger T. Nature Reviews Neurology. 2013; 9: p. 455–461. expanded polysomnography are performed with the Department of Neuroradiology and (expanded refers to, among others, addition- has broad access to the MRI core facility, Research Group Neurocritical Care and Infectious Diseases of the Nervous System: al EMG or EEG channels, transcutaneous equipped with a dedicated 3 Tesla research 1. Higher brain extracellular potassium is associated with brain capnography, etc.) as well as multiple sleep tomograph. Members of the laboratory are metabolic distress and poor outcome after aneurysmal subarachnoid hemorrhage. Antunes AP, Schiefecker AJ, Beer R, Pfausler B, latency tests, maintenance of wakefulness developing and exploring mathematical Sohm F, Fischer M, Dietmann A, Lackner P, Hackl WO, Ndayisaba tests, actigraphy, pupillographic sleepiness models of structural and functional image JP, Thomé C, Schmutzhard E, Helbok R. Crit Care. 2014 Jun 11;18(3):R119. test, DLMO etc. The Sleep Research Group analysis that will be translated into routine and Sleep Disorders Clinic have three board neurological and radiological assessment of 2. Addressing neurologic needs in sub-Saharan Africa: An opportunity for multisociety cooperation. Bower JH, Diop AG, Gouider certified expert neurologists/sleep spe- patients with CNS disorders. Due to a long- R, Schmutzhard E. cialists and one board certified sleep tech- lasting expertise in the field of movement Neurology. 2014 Sep 23;83(13):1207–9. nician, and additional highly trained staff. disorders the main focus of our research is in 3. Participants in the International Multidisciplinary Consensus The RLS outpatient clinic has been recog- the area of neurodegenerative parkinsonian Conference on Multimodality Monitoring. Intracranial pressure and cerebral perfusion pressure monitoring in non-TBI patients: nized as a Willis-Ekbom disease quality care disorders and has recently been extended special considerations. Helbok R, Olson DM, Le Roux PD, Vespa P. Center by the Willis-Ekbom disease/RLS to neurodegenerative dementias, sleep Neurocrit Care. 2014 Dec;21 Suppl 2:S85–94. foundation. In addition, the sleep research disorders and intracranial hemorrhages. 4. The speed of ultraearly hematoma growth in acute intracere- group at the Department of Neurology is bral hemorrhage. Sato S, Arima H, Hirakawa Y, Heeley E, Delcourt C, Beer R, Li Y, Zhang J, Jüettler E, Wang J, Lavados PM, Robinson entertaining a long-standing collaboration Selected Publications T, Lindley RI, Chalmers J, Anderson CS, INTERACT Investigators. with the sleep group at Barcelona Universi- Neurology 2014; 83:2232–2238. Research Group Stroke: ty (SINBAR Study Group) which has resulted 1. Thrombolysis and clinical outcome in patients with stroke af- 5. Nitric oxide synthase inhibition with the antipterin VAS203 im- in more than 10 high-level publications over ter implementation of the Tyrol Stroke Pathway: a retrospective proves outcome in moderate and severe traumatic brain injury: a observational study. Willeit J, Geley T, Schöch J, Rinner H, Tür A, placebo-controlled randomized Phase IIa trial (NOSTRA). Stover the past few years (Fig. 3). Kreuzer H, Thiemann N, Knoflach M, Toell T, Pechlaner R, Willeit JF, Belli A, Boret H, Bulters D, Sahuquillo J, Schmutzhard E, Zavala K, Klingler N, Praxmarer S, Baubin M, Beck G, Berek K, Dengg C, E, Ungerstedt U, Schinzel R, Tegtmeier F, NOSTRA Investigators. Engelhardt K, Erlacher T, Fluckinger T, Grander W, Grossmann J, J Neurotrauma 2014; 31:1599–606. Division of Neurobiology Kathrein H, Kaiser N, Matosevic B, Matzak H, Mayr M, Perfler R, Group leaders: Poewe W, Rauter A, Schoenherr G, Schoenherr HR, Schinnerl A, 6. A longitudinal study on nodding syndrome—a new African ep- Spiss H, Thurner T, Vergeiner G, Werner P, Wöll E, Willeit P, Kiechl ilepsy disorder. Winkler AS, Wallner B, Friedrich K, Pfausler B, Univ.-Prof. Gregor K. Wenning, S. (2015); Unterberger I, Matuja W, Jilek-Aall L, Schmutzhard E. Priv.-Doz. Nadia Stefanova Lancet Neurol. 14:48–56. Epilepsia. 2014; 55:86–93.

The research programme at the Division of 2. Prediction of cardiovascular disease risk with lipoprotein(a): Research Group Movement Disorders Neurobiology focuses on multiple system prospective 15-year outcomes. Willeit P, Kiechl S, Kronenberg F, 1. Correlation of dopaminergic terminal dysfunction and micro- Witztum JL, Santer P, Mayr M, Xu Q, Mayr A, Willeit J, Tsimikas structural abnormalities of the basal ganglia and the olfacto- atrophy (MSA), a neurodegenerative S (2014); ry tract in Parkinson’s disease. Scherfler C1, Esterhammer R, disorder associated with autonomic In the Bruneck Study. JACC 2014;64:851–60. Nocker M, Mahlknecht P, Stockner H, Warwitz B, Spielberger S, Pinter B, Donnemiller E, Decristoforo C, Virgolini I, Schocke M, failure, ataxia and parkinsonism. Over 3. Lipidomics profiling and cardiovascular disease risk in the Poewe W, Seppi K. the last decade our research group has prospecitive population-based. Stegemann C, Pechlaner R, Willeit Brain. 2013 Oct;136(Pt 10):3028–37.

144 Research Report 2015 Medical University of Innsbruck Neurology

2. Substantia nigra hyperechogenicity as a marker for Parkin- Research Group Multiple Sclerosis and Neuroimmunology: of Sao Paulo and the University of Chicago. son’s disease: a population-based study. Mahlknecht P, Seppi K, • 2013: HSRM Project, Austrian Federal Ministery of Science Stockner H, Nocker M, Scherfler C, Kiechl ,S Willeit J, Schmidauer “BIG-WIG MS” (Markus Reindl and Thomas Berger) The strong international network and collaborations are also vis- C, Gasperi A, Rungger G, Poewe W. • 2013: #I916-B13, Austrian Science Foundation (FWF) ERANET ualized through the pubmed publication track report of the Sleep Neurodegener Dis. 2013;12(4):212–8. ERARE “Eugene Devic European Network (EDEN)” (Markus Research Group, which comprises more than 100 international Reindl) collaborative studies, for the most part published in high ranking 3. Changing the research criteria for Parkinson’s disease: obsta- • 2007: #DK W1206, Austrian Science Foundation (FWF) “Doc- peer-reviewed international journals. These publications include cles and opportunities. Berg D, Lang AE, Postuma RB, Maetzler W, toral program SPIN”, Re-evaluation 2013 (Markus Reindl, dep- results from bicentric, oligocentric or multicentric studies, aca- Deuschl G, Gasser T, Siderowf A, Schapira AH, Oertel W, Obeso JA, uty speaker and PI) demic research projects, and guideline papers. Olanow CW, Poewe W, STern M. Lancet Neurol. 2013;12(5):514–24. Research Group Neurocritical Care and Infectious Diseases Research Group Multiple Sclerosis and Neuroimmunology: of the Nervous System: • Hans Lassmann, Monika Bradl, Romana Höftberger, Fritz 4. European Multiple System Atrophy Study Group. The natural • “Novel Biomarkers for DCI after SAH: Microparticles Revisit- Leutmezer and Alexander Zimprich: Medical University of Vi- history of multiple system atrophy: a prospective European co- ed”, FWF( Austrian Science Fund (Project KLI 375-B00), Ronny enna, Vienna, Austria hort study. Wenning GK, Geser F, Krismer F, Seppi K, Dürr S, Bösch Beer S, Köllensperger M, Goebel G, Pfeiffer KP, Barone P, Pellecchia • Michael Khalil, Christian Enzinger and Franz Fazekas: Medical University of Graz, Graz, Austria MT, Quinn P, Koukouni V, Fowler CJ, Schrag A, Mathias CJ, Giladi Research Group Movement disorders: N, Gurevich T, Dupont E, Ostergaard K, Nilsson CF, Widner H, • D. Berg: MJFF – Prospektive validation of risk markers for the • Kevin Rostasy: Childrens Hospital Datteln, Datteln, Germany Oertel W, Eggert KM, Albanese A, del Sorbo F, Tolosa E, Cardozo development of PD. Biomarkers 2007 • Edgar Meinl: LMU Munich, Munich, Germany A, Deuschl G, Hellriegel H, Klockgether T, Dodel R, Sampaio C, • C. Goetz. MJFF – Validation of Dyskinesia Rating Scales. • Bernhard Hemmer: TU Munich, Munich, Germany Coelho M, Djaldetti R, Melamed E, Gasser T, Kamm C, Meco G, EudraCT no. 2009-017968-17. 2009 • Sven Jarius and Brigitte Wildemann: University of Heidelberg, Colosimo C, Rascol O, Meissner WG, Tison F, Poewe W. • CHRUL (Coordinating Center Lille, France) – FAIR-PARK II – Heidelberg, Germany Lancet Neurol. 2013;12(3):264–74. HORIZON 2020– Research agreement no. 633190. 2015 • Orban Aktas and Hanspeter Hartung: University of Düsseldorf, Düsseldorf, Germany 5. Movement disorders: new insights into disease mechanisms Research Group Sleep Medicine: • Christine Stadelmann and Wolfgang Brück: Univ. of Göttingen, and treatment. Poewe W, Mahlknecht P. • I 21 20 B 27 01/2015-01/2018: Safer Screening for RBD, Göttingen, Germany Lancet Neurol. 2014; 13(1):9–11. funding from FWF Bilateral Austria/Argentina. Project Leader: • Andreas Lutterotti, Mireia Sospreda, Jan Lünemann and Roland Birgit Högl Martin: University of Zürich, Zürich, Switzerland Research Group Sleep Medicine: • 11/2012-10/2016: RLS-Iron: Investigation of iron metab- • L. Kappos: University of Basel, Basel, Switzerland 1. A Prospective Video-Polysomnographic Analysis of Movements olism in patients with idiopathic RLS, funding from the Gov- • Romain Marignier: University of Lyon, Lyon, France during Physiological Sleep in 100 Healthy Sleepers. Stefani A, ernment of Tyrol, translational research fund. Global project • Albert Saiz, Frances Graus, Josep Dalmau and M. Comabella: Gabelia D, Mitterling T, Poewe, W, Högl B, Frauscher B. Leader Birgit Högl University of Barcelona, Barcelona, Spain SLEEP MEDICINE. Sleep, 2014 • KLI 236 (former KLI 112) 2012–2016: Motor activity during sleep in health and disease Funded by FWF, Project Leader Bir- • Paddy Waters, Jackie Palace, Isabel Leite and Angela Vincent: 2. Sleep and Respiration in 100 Healthy Caucasian Sleepers – A git Frauscher. formally transferred to Gregor Wenning in 2012 University of Oxford, Oxford, UK Polysomnographic Study According to American Academy of • 2007–2011: Motor characterization of augmentation in rest- • Anne Fogdell-Hahn: Karolinska Institute, Stockholm, Sweden Sleep Medicine Standards. Mitterling T, Högl B, Schonwald V, less legs syndrome, funding from the Austrian Nationalbank, • Eva Havrdova: University of Prague, Prague, Czech Republic Hackner H, Gabelia D, Frauscher B. Anniversary Funds. 12594 Project Leader Birgit Högl • Jeffrey Bennett: University of Denver, Denver, Colorado SLEEP. 2014 38(6):867–875. • Douglas Sato, Tatsuro Misu and Kazuo Fujihara: Tokohu Univer- Division of Neurobiology sity, Sendai, Japan 3. Validation of an integrated software for the detection of rapid • Progression of microglial activation in a mouse model of MSA. eye movement sleep behavior disorder. Frauscher B, Gabelia D, FWF Doctoral Programme SPIN DK W1206-08 (Nadia Stefano- Biermayr M, Stefani A, Hackner T, Mitterling T, Poewe W, Högl B. Computational Neuroscience: va); SLEEP. 2014; 37: p. 1663–1671. • Neuroimage Wien Innsbruck Graz (WING) • Cardiovascular phenotyping of a transgenic mouse model of • EU Horizon 2020, FAIRPARK II multiple system atrophy, MUI-Start 2014-05-005 (Daniela 4. Quantitative assessment of isolated rapid eye movement (REM) • PRODEM AUSTRIA Kuzdas-Wood) sleep without atonia without clinical REM sleep behavior disorder: clinical and research implications. Sasai-Sakuma T, Frauscher B, Computational Neuroscience Mitterling T, Ehrmann L, Gabelia D, Brandauer E, Inoue Y, Poewe Miscellaneous • Hochschulraumstrukturmittel–Projekt: Neuroimage Wien W, Högl B. Innsbruck Graz (WING) SLEEP MEDICINE. 2014; 15: p. 1009–1015. Neurological Research Laboratory: The main goals of the Neurological Research laboratory (headed 5. Motor events during healthy sleep: a quantitative polysomno- Collaborations by Univ.-Prof. Markus Reindl) are clinically orientated neurosci- graphic study. Frauscher B, Gabelia D, Mitterling T, Biermayr M, ence, teaching and the development of novel methods for labo- Bregler D, Ehrmann L, Ulmer H, Högl B. ratory testing in neurology. In 2013 the Neurological Research Research Group Neurocritical Care and Infectious Diseases SLEEP. 2014; 37: p. 763–73, 773A-773B. Laboratory moved, together with the Institute of Neuroscience of the Nervous System: and the Neurosurgery Research Laboratory to Innrain 66, 2nd and • Craig Anderson, The George Institute for Global Health, Syd- rd Division of Neurobiology – key papers: 3 floor. In July 2003 and again in 2006, 2009 and 2012 the Neu- ney, Australia 1. Multiple-system atrophy. Fanciulli A, Wenning GK (2015); rological Research and Routine Laboratories were certified for • John Stover, Zürich, Switzerland: NOSTRA trialists N Engl J Med. 372:249–263. the International Standard EN ISO 9001:2008 • Peter Kremsner: Tübingen, Germany and Lambarene, Gabun: SMAC trialists 2. Toll-like receptor 4 is required for alpha-synuclein depend- • Peter Le Roux: Wynnewood, PA, USA, International Multidisci- ent activation of microglia and astroglia. Fellner L, Irschick R, plinary Consensus Conference on Multimodality Neuro-Mon- Schanda K, Reindl M, Klimaschewski L, Poewe W, Wenning GK, itoring. Stefanova N (2013); • JH Zhang: Loma Linda, CA, USA Glia. 61:349–360. • Sarah Gabriel, Cystinet-project, Antwerp, Belgium • Nino Stocchetti, Milano, Italy: SyNAPSE trialists 3. Systemic proteasome inhibition triggers neurodegeneration in a transgenic mouse model expressing human alpha-synuclein Research Group Sleep Medicine: under oligodendrocyte promoter: implications for multiple sys- • Oscar Gershanik, University of Buenos AIres, Argentina tem atrophy. Stefanova N, Kaufmann WA, Humpel C, Poewe W, • SINBAR, Sleep Innsbruck Barcelona Wenning GK (2012); • Joan Santamaria and Alex Iranzo, Hospital Clinic of Barcelona, Acta Neuropathol. 124(1):51–65. Barcelona, Spain • Claudia Trenkwalder, Paracelsus-Elena Clinic, Kassel, and Uni- 4. Toll-like receptor 4 promotes alpha-synuclein clearance and versity of Goettingen Germany survival of nigral dopaminergic neurons. Stefanova N, Fellner L, • University of Barcelona, Harvard University, Stanford Univer- Reindl M, Masliah E, Poewe W, Wenning GK (2011); sity, Johns Hopkins University, the Rush Medical Center, and Am J Pathol. 179:954–963. other universities in Japan, Latin America etc. 5. Multiple system atrophy: a primary oligodendrogliopathy. The strong international imbedding and connections of the sleep Wenning GK, Stefanova N, Jellinger KA, Poewe W, Schlossmacher research group is also apparent from the fact, that the group MG (2008); leader holds several current EC positions in international groups, Ann Neurol. 64:239–246. e.g. the International RLS study group IRLSSG, and the Interna- tional RBD study group (current secretary in both groups) and is Selected Funding the current chair of the medical advisory board of the RLS/WED foundation. Research Group Stroke: In 2013 and 2014 funding was also derived from the FWF (Transla- International guests, students, specialists and visiting professors tional Research Project TRP 188-B12, 360 K€). The neurovascular visited the Sleep Group came from Israel, Georgia, Argentina, research group participates in in large international research and Brazil, The Netherlands, Germany, Finland, Thailand, Japan, etc. meta-analysis consortia (steering/writing committee: FSC, ERFC, Birgit Högl, the Head of unit has in turn been invited as a Visiting LSC, NPSC, IMT-PROG, GBD, various GWA studies). Professor to Harvard Medical School, the Federal State University

Research Report 2015 Medical University of Innsbruck 145 Center of Neurology and Neurosurgery Neurosurgery

Keywords present in gliomas, whereas metastatic disease of solid cancers was believed to Neuro-oncology, cerebrovascular, neuroin- be circumscript lesions in the brain. The tensive care, neuromonitoring, neurotrau- surrounding tissues of metastatic disease ma, spine surgery, spinal implants, disc de- and primary gliomas are currently under generation, regenerative medicine investigation with 31-Phosphorus-MR- Spectroscopy and precise biopsies are Research Focus taken from these regions to demonstrate that infiltrative behavior in brain tumors • Characterization of myelin-associated (metastasis and gliomas) can be found by neurite growth inhibitors and their cognate state-of-the-art MR-imaging. The borders receptors in the central nervous system. and surroundings of resected metastases • Signal transduction of neurite growth in- are examined immunohistochemically. hibitors in the brain with special emphasis Operative techniques are currently under on effects on cytoskeletal changes in neu- investigation, as in cases of gliomas, ronal growth cones. fluorescence guided resections and intraoperative imaging with CT is evaluated General Facts through posthoc image rendering with Director: prototypic algorithms. The aim of this Univ.-Prof. Dr. Claudius Thomé The Department of Neurosurgery actively collaborative project with industrial partners hosts investigator-driven academic studies is to predict the extent of tumor resection Contact: and is participating in multinational trials in through intraoperative CT-imaging. Anichstraße 35 different fields of our daily practice. Clinical 6020 Innsbruck research is paralleled with experimental Selected Trials: work in our laboratory. We pursue the idea • Elastic Fusion: intraoperative CT is ren- [email protected] of leading innovative concepts from “bench dered by prototypic algorithms to gen- Phone: +43 512 504 27452 to bedside”, exemplarily shown in the erate a “virtual” MRI. Extent of resection Fax: +43 512 504 27453 case of chondrocyte cell transplantation is evaluated by neuroradiology (proof of http://neurochirurgie.tirol-kliniken.at after surgical treatment of lumbar disc principle) herniations. Besides ongoing research • 31P-MR-Spectroscopy: several tumor en- focusing on tumor cell infiltration into tities undergo 31P-MR-Spectroscopy to healthy brain tissue, we implemented determine chemical shift imaging based different pre- and postoperative routines profiles of tumor tissue to determine the “functional status” of • Biopsy: regions of high phosphorus con- patients after treatment of intracranial centration (high ATP-levels) are precisely pathologies. Our main clinical research examined through a targeted biopsy to focuses are divided into three (interacting) correlate P-concentration with histologic groups. patterns

Research Vascular/Neurotrauma/ Intensive Care In addition to prospective analysis Tumor of treatment results of vascular The neuro-oncology program of our pathologies (aneurysms, arteriovenous department is focusing on two main fields malformations and stroke) and the ongoing of interest, “infiltration” and “optimization improvements in technical standards and of functional outcomes” of patients after operative techniques, the use of hypoxic brain tumor surgery. Infiltration is always preconditioning and serum biomarkers

Fig. 2: Intraoperative fluorescence of a ma- Fig. 1: Multimodal treatment plan for the re- lignant brain tumor to delineate the lesion section of a solid brain tumor. from healthy brain tissue.

146 Research Report 2015 Medical University of Innsbruck Neurosurgery

are investigated to beneficially influence devices to reduce recurrences (Barricaid) IVD tissue tropism have not been identified patient treatment. The Department of and have conducted experimental and characterized. The use of AAV-mediated Neurosurgery is participating in several studies to elucidate the benefits of gene therapy for human intervertebral disc international registries and is in close regenerative approaches. Clinical studies research has not yet been investigated. collaboration with the Department of on corpectomies and instrumentation Neurology. Invasive multimodal monitoring procedures are supplemented with Selected Publications of brain oxygenation, metabolism and biomechanical studies to determine the Effectiveness of posterior decompression techniques compared with conventional laminectomy for lumbar stenosis. Overdevest blood flow, in combination with intracranial stability and durability of spinal implants GM, Jacobs W, Vleggeert-Lankamp C, Thomé C, Gunzburg R, Peul pressure is performed to evaluate the use needed for surgical stabilization of the W. Cochrane Database Syst Rev. 2015 Mar 11;3:CD010036. of these invasive techniques on patient human spine in close collaboration with the Imbalanced protein expression patterns of anabolic, catabolic, anti-catabolic and inflammatory cytokines in degenerative outcome after severe head trauma and Department of Trauma Surgery. cervical disc cells: new indications for gene therapeutic treat- subarachnoid hemorrhage. Researchers ments of cervical disc diseases. Mern DS, Beierfuß A, Fontana J, Thomé C, Hegewald AA. of the Department are currently abroad Selected Trials: PLoS One. 2014 May 7;9(5):e96870. in joined projects with the Mayo Clinic in • N-DISC: autologous chondrocyte cells to Enhancing tissue repair in annulus fibrosus defects of the interver- Rochester, Minnesota and in Loma Linda, be reinjected in the patient’s interverte- tebral disc: analysis of a bio-integrative annulus implant in an in-vivo ovine model. Hegewald AA, Medved F, Feng D, Tsagogiorgas C, California. bral disc several weeks after surgery to Beierfuß A, Schindler GA, Trunk M, Kaps C, Mern DS, Thomé C. prevent ongoing degeneration J Tissue Eng Regen Med. 2015 Apr;9(4):405–14. Severe head injury is a common • Barricaid: a composite material annulus Perfusion characteristics of Moyamoya disease: an anatomically and clinically oriented analysis and comparison. Schubert GA, medical condition at our department. closure device is implanted during stan Czabanka M, Seiz M, Horn P, Vajkoczy P, Thomé C. In addition to multimodal monitoring dard surgery for lumbar disc herniation Stroke. 2014 Jan;45(1):101–6. and pathophysiological investigations, a • DynorFuse: a multinational trial to investi- Distance to the neurooncological center: a negative prognostic factor in patients with glioblastoma multiforme. An epidemiologi- monocentric study was launched in close gate the effectiveness of rigid vs. dynamic cal study. Kerschbaumer J, Freyschlag CF, Bauer R, Obwegeser AA, collaboration with the Departments of fusion techniques in patients with spinal Schubert GA, Thomé C, Seiz M. Neuroradiology and Neurology to determine stenosis and mild signs of instability Anticancer Res. 2012 Dec;32(12):5515–9. the use and interpretation of early MRI data • ForaC: a multicenter randomized trial Selected Funding and their correlation to serum biomarkers on anterior versus posterior approach- Diverse Industry sponsored and academic clinical trials of brain injury. Furthermore, treatment es for cervical foraminal stenosis with of chronic subdural hematomas, a widely radiculopathy Collaborations underestimated disease predominantly Numerous research collaborations with institutions in Austria and neighboring countries in the fields of neurooncology (i.e. Vienna, arising in the elderly, was analyzed based on The experimental focus of the spine group Regensburg, Heidelberg), cerebrovascular neurosurgery (i.e. a monocentric randomized protocol. is biological treatment approaches for Aachen, Düsseldorf, Berlin) and regenerative medicine (i.e. Berlin, Mannheim). The multicenter clinical trials involve numerous part- intervertebral disc degeneration (IVD). IVD, ners throughout Europe. Researchers are currently staying at the Selected Trials: often accompanied by inflammatory and Mayo Clinic, Rochester, MS/USA and in Loma Linda, CA/USA within the scope of ongoing cooperations. • RIPAT: hypoxic preconditioning is applied patho-immunological processes, has been (randomized) to patients before elective described as structural failures of disc treatment of aneurysms. Serum biomark- tissues. Current treatment approaches are ers and neuropsychological outcomes are restricted to symptomatic therapies and do recorded pre- and postoperatively not address the option of biological repair • TIBI: multimodal invasive neuromonitor- of the discs. Intervertebral disc cells play ing in patients with severe head trauma a central role in the maintenance of discs in combination with early MRI data and by coordinating the expression of anabolic, serum biomarkers of neuronal injury catabolic, anti-catabolic and inflammatory cytokines affecting the extracellular Spine matrix. Our electronic database search has In accordance with our experimental identified several target genes that could research of spinal pathologies, our spine have a significant impact on disc matrix group is leading ongoing regenerative anabolism and catabolism. studies such as the N-DISC trial. Human (autologous) chondrocytes are cultivated By combinatorial relative mass value from herniated disc material and reinjected evaluations of the identified target proteins 3 months after standard surgery. This is in degenerative lumbar and cervical discs, the first clinical trial also investigating we have ascertained imbalanced protein prophylactic treatment of degenerated expression patterns of certain anabolic, discs. Another main (clinical) research catabolic, anti-catabolic and inflammatory interest is based on minimally invasive cytokines. Our progressive characterization surgery using state-of-the-art neuro- of the target genes gives us the opportunity navigation systems and imaging technology. to develop new therapeutic approaches. Recurrent pathologies (i.e. re-herniation Currently, we are establishing an adeno- of lumbar discs) might be reduced by associated virus (AAV) based gene closure of substance defects in the annulus therapeutic system as a new biological fibrosus. We collaborate with industrial treatment approach for degenerative disc partners to approve the use of closure diseases. So far, AAV serotypes with human

Research Report 2015 Medical University of Innsbruck 147 Center of Obstetrics and Gynecology Obstetrics and Gynecology

advanced and innovative therapies in this Research field, as well as to contribute to research in order to improve therapy options. The Clinical Trials: Gynaecologic Oncology AGO Studienzentrale is not only responsible Christian Marth for trial conduct within the department, A selection of the most important trials is but also for the coordination of AGO shown below: trials in other Austrian sites (i.e. manages A number of gynecological cancer trials applications to ethics committees and (surgical and therapeutic trials) are being other competent authorities etc.). Further conducted to assess efficacy and safety of more, the department has a trial office different types of therapies. for breast cancer trials with several study Selected trials are described below. nurses dedicated to support the patients in • The antiangiogenic treatment of advanced trials at the department. epithelial ovarian, primary peritoneal or fallopian tube cancer with AMG386 or pla- The pre-clinical studies are conducted cebo was evaluated in the first-line setting mainly in the Laboratory of Clinical with carboplatin and paclitaxel (AGO 30 Biochemistry (Head: Heidi Fiegl) and in the Trinova 3) and in the recurrent partially Morphological Laboratory (Head: Elisabeth platinum-sensitive or -resistant setting Director: Müller-Holzner until her death in July 2014; with pegylated liposomal doxorubicin Univ.-Prof. Dr. Christian Marth Afschin Soleiman since July 2014). Both (PLD) (AGO 28 Trinova 2) as Phase III ran- laboratories are certified according to domized double-blind trials to determine Contact: ISO 9001:2008 and house the bio bank whether AMG386 is superior to placebo in Anichstraße 35 of this department, which contains FFPE either setting. 6020 Innsbruck tissue samples from nearly 6,500 patients, • In the randomized open-label AGO 39 fresh frozen tissue samples from over Ovar 2.21 trial, two standard-of-care [email protected] 1,000 patients, ascites samples from over chemotherapies (Carboplatin+Gemcit- Phone: +43 512 504 23051 1,500 patients and serum samples (pre- abine vs. Carboplatin+PLD) in combi- Fax: +43 512 504 23055 therapeutic samples and samples drawn nation with antiangiogenic treatment https://www.i-med.ac.at/patienten/ during the follow-up period from over 3,000 (Bevacizumab) are compared to measure ukl_gynaekologie_geburtshilfe.html patients). efficacy in patients with first recurrence of platinum-sensitive epithelial ovarian, pri- Biobanking has been performed in the mary peritoneal or fallopian tube cancer. department since the 1980s and optimized • The Phase III randomized double-blind Keywords over the years. The serum-, tissue- and AGO 40 NOVA trial evaluates the efficacy tumour data applications are registered in of the PARP inhibitor Niraparib compared Clinical studies, gynecologic oncology, the Data Processing Registry of the Austrian to placebo as maintenance therapy in senology, biobanking translational research, Data Protection Commission. The clinical patients with platinum-sensitive ovarian biomarker-identification and personal data are stored separately in cancer, evaluated in a cohort of patients a clinical database, which is also registered with germline BRCA mutation and high- Research Focus in the Data Processing Registry. grade serous or high-grade predominant-

• Clinical and pre-clinical studies in women specific cancer in regard to prevention, diagnostics, therapy and follow-up care • Translational research • Collection of biological samples (tissues, serum, ascites, …,3) • Biomarker identification in breast and gynecological malignancies • Special interest in understanding the development of women specific cancer • Pregnancy Research

General Facts

The clinical trials conducted in the department are coordinated by AGO Studienzentrale as trial office of the non- profit organization GOA Austria (Association of gynecologic oncology in Austria) since 2002. The main aim is to enable Fig. 1: Univariate survival analyses according to L1CAM expression in 1021 patients with gynecologic cancer patients to receive FIGO stage I, type I endometrial cancers. A) Disease-free survival. B) Overall survival.

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ly serous histology but without germline dicates the need for adjuvant treatment. for more effective treatment strategies BRCA mutation. The aim is to assess This molecule might serve as a treatment to improve the dismal survival in these whether maintenance with Niraparib will target for the fully humanized anti-L1CAM patients, the GANNET53 trial (Ganetespib in extend the progression-free survival (PFS) antibody currently under development for metastatic, p53 mutant, platinum-resistant in these patients. clinical use. ovarian cancer) was initiated. GANNET53 is a Europe-wide, multi-centre Phase I Major Achievements: Major Achievements: and randomized Phase II clinical trial, Inhibition of Angiopoietins 1 and 2 with Identification of miR-21-3p as a positive targeting mutant p53, via an innovative new Trebananib in women with recurrent L1CAM regulator in several human carcino- Hsp90 (heat shock protein 90) inhibitor in epithelial ovarian cancer (EOC) provided a mas and miR-34a as a L1CAM suppressor platinum-resistant EOC patients. clinically meaningful prolongation in PFS. in endometrial carcinoma. Establishment of a standardized staining protocol for L1CAM The consortium consists of clinical trial Clinical Trials: Breast Cancer on formalin-fixed, paraffin-embedded tis- groups in gynecological oncology, university Christian Marth sues using automated platforms. centres as well as noted p53 scientists In the following, a selection of the most im- and three innovative enterprises. We aim portant clinical trials is shown: Future Goals: to substantially improve overall survival • The department is currently successfully Validation of the clinical usefulness of (OS) in EOC patients with metastatic type II participating in a Phase III trial (Belle 3) L1CAM in a prospective randomized trial. platinum-resistant tumours. On the molec which is investigating BKM120 with Ful- ular level, we aim to identify the tumours vestrant in HR+/HER2- breast cancer Identification and Targeting of Ovarian mutational status in all GANNET53 study (BC) patients, who progressed on or after Cancer Stem Cells patients. mTOR inhibitor based treatment. We are at Alain Zeimet and Daniel Reimer the moment the worldwide top recruiter in In general the majority of patients with Biomarker Identification in this trial. FIGO stage III/IV EOC initially responds to Gynaecologic Cancer • Treatment options for triple-negative BC initial surgery and standard chemotherapy Nicole Concin patients are still limited. Therefore, clinical but ultimately undergo relapse, probably A selection of the most important projects trials are of special interest in this setting. due to the survival of small populations of is shown: Two promising trials in this setting are cur- cells with tumour-repopulating potential, • The gamma-glutamyltransferase (GGT) rently being conducted in our department. also termed cancer stem cells (CSC). It is modulates crucial redox-sensitive func- These are the tnAcity (Gemcitabine/Car- a hallmark of CSC to survive anti-cancer tions in the cell. It has also been shown boplatin) and the placebo controlled, rand- treatment, which primarily target rapidly that GGT plays a role in tumour progres- omized Olympia Trial with the PARP -inhib- dividing tumour cells. They can give rise sion, invasion and drug resistance. There- itor Olaparib. to recurrences of tumours that are usually fore, the significance of preoperative • We are also involved in innovative chemo- more chemo-resistant and aggressive. Thus, serum GGT levels as a prognostic marker therapy trials. For example, the Phase II it would be desirable to develop additional is evaluated in gynecological cancers. AGMT-MBC 6 Trial evaluates Capecit- therapeutic modalities that could target • Circulating tumour cells (CTCs) have been abine in combination with Bedamustine in ovarian CSC to complement conventional demonstrated to have prognostic value in women with pre-treated locally advanced treatment options, ultimately enabling several tumour types. Recently identified or metastatic Her2-negative BC; or the complete eradication or at least long term new molecular markers for the CTC-detec- Kathrine Trial investigates the anti-body growth arrest of the disease. tion are analysed in blood of EOC patients drug conjugate Trastuzumab Emtansine in to evaluate their diagnostic usefulness. patients with HER2-positive primary BC. • In an Oncotyrol funded project, EOC cell lines were screened for putative CSC to Major Achievements: L1CAM Expression identify specific markers and to select tar- Identification of an association of elevated Alain Zeimet gets suitable for in vivo CSC depletion or GGT levels with poor survival rates among L1CAM is a 200 to 220k Dmembrane glyco- inhibition strategies. endometrial cancer and EOC patients. protein of the immunoglobulin superfamily and is crucially involved in neurogenesis. Major Achievements: Identification of HIF1 alpha as an indepen Moreover, L1CAM is expressed in a variety The side population of ovarian cancer cells dent prognostic factor for OS in advanced of tumours, where its presence is associat- defines a heterogeneous compartment primary EOC. ed with poor clinical outcome. exhibiting stem cell characteristics. • In a retrospective, international multicen- Impact of Aluminium on Breast Cancer tre cohort study, the L1CAM expression GANNET53 (EU Project) Nicole Concin was analysed by immunohistochemistry Nicole Concin There is a need for research clarifying re- in 1021 endometrial cancer specimens. Epithelial ovarian cancer (EOC) is the cently raised issues relating underarm an- L1CAM has been shown to be the best most lethal gynecological malignancy. tiperspirants containing aluminium salts prognostic factor in Fédération Interna- The predominance of aggressive type II with BC. Therefore we established the most tionale de Gynécologie et d’Obstétrique tumours, which are characterized by a high comprehensive study in biological samples (FIGO) stage I, type I endometrial can- frequency of p53 mutations, and primary of BC patients and controls. cers and shows clear superiority over the or acquired resistance to platinum-based standardly used multifactor risk score. chemotherapy profoundly contribute to the L1CAM expression in type I cancers in- high mortality rate. Addressing this need

Research Report 2015 Medical University of Innsbruck 149 Center of Obstetrics and Gynecology

Parallel Mechanisms between Embryo- DNA Demethylation Agents etiological factor for the development and Carcinogenesis Heidi Fiegl of cervical cancer. A subgroup of the 12 Nicole Concin Anaesthetic management of cancer HPV-types, referred to as high-risk HPVs, The aim of this study is to analyse the surgery may influence tumour recurrence. is associated with cervical carcinoma spatiotemporal distribution of relevant Previous results in our laboratory showed in situ and invasive cancers. High-risk biomarkers expressed in mesenchymal that at clinically relevant concentrations, HPV persistence is associated with the and epithelial structures within the Lidocaine, a prototype amide-type local increased expression of the high-risk HPV developing female secondary sexual anaesthetic, demethylates DNA of BC cell E7 oncoproteins, the major transforming organs and to identify diagnostically and/or lines in vitro. This modification of epigenetic proteins of such viruses, which is highly therapeutically markers. information may be of therapeutic relevance specific for the induction of cervical in the perioperative period, because a carcinogenesis. decrease in methylation can reactivate • In collaboration with the Institute for Bio Biomarker Identification in Cancer tumour suppressor genes and inhibit medical Aging Research, a project was Heidi Fiegl tumour growth. initiated to establish a novel ELISA assay A selection of the most important projects for the detection and quantification of E7 is shown below: Major Achievements: oncoproteins of high-risk HPV types for • Despite years of intensive study and Demonstrates that Ropivacaine exert the detection of cervical precancers and substantial progress in identifying new demethylating effects on specific BC cell cancers. Additionally, a validation of the biomarkers and creating new therapies lines. clinical performance diagnostic kit with for BC, this disease remains the most cervical smears from 2000 patients was common cause of cancer-related death Development and Validation of conducted. in women. The aim of an Oncotyrol Pan-High-Risk E7 ELISA funded project was the improvement of Andreas Widschwendter Analysis of Afamin in Serum Samples recurrence-risk prediction in BC patients Persistent infections by high-risk human Michael Hubalek based on the analyses of the epigenome, papillomaviruses (HPV) are the main Afamin is a human glycoprotein with an genome, transcriptome and metabolome in blood samples from BC patients. In a large-scale blood specimen collection in breast cancer centres in Austria (Innsbruck, Salzburg, and Vienna) and South-Tyrol (Brixen and Meran) blood samples from over 2,000 BC patients have been collected so far. • A central aim of preventive oncology is the identification of patients with a heritable predisposition to cancer. Identification of common founder mutations in certain ethnicities allows integration of genetic testing strategies in the routine care of all cancer patients. In collaboration with the Division of Human Genetics, we analysed specimens from breast and ovarian cancer patients treated in our Department and identified a TyroleanBRCA1 founder mutation. • Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. In an international collaboration, we analysed the functional role of epigenetic factors in endometrial cancer development. Fig. 2: Scientific principle of the GANNET53 trial. (Li et al, Cell Death& Diff 18:1904-13, 2011; Major Achievements: Li et al, Mol Cancer Res 9:577–88, 2011). Destabilisation of mutant p53 protein by inhibi- Identification of a highly prevalentBRCA1 tion of the Hsp90 chaperone causes subsequent degradation by MDM2 or CHIP E3 ligases: founder mutation in the Lower Inn Valley Stable complex formation with Hsp90 causes aberrant stabilisation of mutp53 in cancer that correlates to a local increase in the cells. MDM2 and CHIP, which in principle are capable of degrading mutp53, are unable to breast and ovarian cancer risks. degrade mutp53 as long as it is protected by the complex (‘caging’). Stabilised mutp53 exerts oncogenic gain-of-function (GOF). Acute depletion of mutp53 in tumour cells is strongly Identificationof HAND2 DNA methylation cytotoxic in all tested mutp53 solid cancer cell types tested (ovarian, breast, colon, and as a biomarker for early detection of prostate). Small molecule inhibitors of the Hsp90 ATPase (such as the highly potent sec- endometrial cancer and as a predictor of ond generation Hsp90inhibitor Ganetespib, or the weaker first generation 17AAG + SAHA) treatment response. acutely deplete mutp53, which is strongly cytotoxic in mutp53 harbouring tumour cells.

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apparent molecular weight of 87 kDa with pregnancy to assess the influence of preg- Selected Publications specific binding properties for vitamin E. nancy-associated conditions/ diseases. Anti-angiopoietin therapy with trebananib for recurrent ovarian Comparative proteomics has identified cancer (TRINOVA-1): a randomised, multicentre, double-blind, Afamin as a potential biomarker for ovarian Major Achievements: placebo-controlled phase 3 trial. Monk BJ, Poveda A, Vergote I, Raspagliesi F, Fujiwara K, Bae DS, Oaknin A, Ray-Coquard I, cancer. Identification of HE4 as a biomarker to Provencher DM, Karlan BY, Lhommé C, Richardson G, Rincón DG, • In collaboration with the Division of detect recurrent endometrial cancer Coleman RL, Herzog TJ, Marth C, Brize A, Fabbro M, Redondo A, Genetic Epidemiology has shown that Afa- in patients undergoing routine clinical Bamias A, Tassoudji M, Navale L, Warner DJ, Oza AM. LANCET ONCOLOGY. 2014;15:799–808. min serum concentrations were measured surveillance. in women with uncomplicated pregnan- Role of miR-34a as a suppressor of L1CAM in endometrial carcinoma. Schirmer U, Doberstein K, Rupp AK, Bretz NP, Wuttig cies in a retrospective cohort study at dif- Future Goals: D, Kiefel H, Breunig C, Fiegl H, Müller-Holzner E, Zeillinger R, ferent gestational ages and a prospective Initiation of a prospective analysis of HE4 in Schuster E, Zeimet AG, Sültmann H, Altevogt P. observational study in the first, second endometrial cancer patients in the setting ONCOTARGET. 2014;5;462–472. and third trimester. of a randomized control trial. Suppression of acetylpolyamine oxidase by selected AP-1 mem- • In a second project, Afamin levels were bers regulates DNp73 abundance: mechanistic insights for overcoming DNp73-mediated resistance to chemotherapeutic drugs. measured in serum samples of BC patients Pregnancy Research Bunjobpol W, Dulloo I, Igarashi K, Concin N, Matsuo K, Sabapathy and patients with benign breast diseases. Several different Members of the K. CELL DEATH AND DIFFERENTIATION. 2014; 21:1240–1249. Department Major Achievements: Selections of the most important studies L1CAM in Early-Stage Type I Endometrial Cancer: Results of a Identification of linearly increased of Afamin are shown: Large Multicenter Evaluation. Zeimet AG, Reimer D, Huszar M, Winterhoff B, Puistola U, Azim SA, Müller-Holzner E, Ben-Arie A, levels in maternal serum during pregnancy. van Kempen LC, Petru E, Jahn S, Geels YP, Massuger LF, Amant Working group of Irene Mutz-Dehbalaie F, Polterauer S, Lappi-Blanco E, Bulten J, Meuter A, Tanouye S, Oppelt P, Stroh-Weigert M, Reinthaller A, Mariani A, Hackl W, Association between Body Mass Index and Matthias Scheier Netzer M, Schirmer U, Vergote I, Altevogt P, Marth C, Fogel M. and Anastrozole Plasma Levels In industrialized countries a growing number JOURNAL OF THE NATIONAL CANCER INSTITUTE. 2013;105; Michael Hubalek of women delay reproduction until later 1142–1150. The efficacy of adjuvant endocrine treat- in life. The impact of advanced maternal Role of DNA Methylation and Epigenetic Silencing of HAND2 in ment with aromatase inhibitors, inhibiting age on the rate of perinatal mortality was Endometrial Cancer Development. Jones A, Teschendorff AE, Li Q, Hayward JD, Kannan A, Mould T, West J, Zikan M, Cibula D, the conversion of androgens to oestrogen in analysed in a retrospective cohort study in Fiegl H, Lee SH, Wik E, Hadwin R, Arora R, Lemech C, Turunen adipose tissue, might depend on the overall the state of Tyrol, Austria. H, Pakarinen P, Jacobs IJ, Salvesen HB, Bagchi MK, Bagchi IC, Widschwendter M. volume of adipose tissue. In collaboration PLOS MEDICINE. 2013;10; e1001551. with the Department of Psychiatry and Psy- Working group of Angela Ramoni chotherapy, the interaction between body The incidence of abnormally invasive Selected Funding mass index (BMI) and Anastrozole treat- placentation is rising. Massive, potentially ment as well as estrogenic was analysed. life-threatening blood loss during delivery GANNET53 European Union Seventh Framework Program (FP7) is planned for 5.5 years with a funding of 6 million Euros is the most feared complication. Prenatal (Prof. Dr. Nicole Concin). Major Achievements: diagnosis via ultrasound, followed by Identification of positive association be- appropriate peripartum management of this Collaborations tween BMI and Anastrozole plasma levels. condition, may help reduce morbidity and • Prof. Dr. Robert Zeillinger, Medical University Vienna, Vienna, mortality. In a study four cases of placenta Austria Human Epididymis Protein 4 (HE4) percreta and the chosen form of treatment • Prof. Dr. Ignace Vergote, Katholieke Universiteit Leuven, Leu- ven, Belgien Levels in Serum of Endometrial Cancer were described. • Dr. Neda Slade, Ruđer Bošković Institute, Zagreb, Croatia Patients and Pregnant Women • Priv.-Doz. Dr. Roland Reitsamer, Paracelsus Medical University, Salzburg, Austria Irene Mutz-Dehbalaie Working group of Susanne Jerabek- • Primar Dr. Arthur Scherer, Medical Services Hospital, Bressa- Human epididymis protein 4 (HE4) is a Klestil and Matthias Scheier none, Italy secretory protein that is overexpressed The incidence of fetal portosystemic • Primar Dr. Herbert Heidegger, Medical Services Hospital, Mer- an, Italy in patients with serous and endometrioid anastomoses is unknown, and it is presumed • Prof. Dr. Andreas Obermair, University of Queensland, Brisbane, epithelial ovarian and uterine cancers. HE4 that many cases remain undetected. Australia • Prof. Dr. Martin Oehler, University of Adelaide, Adelaide, Aus- has proven utility as a serum biomarker in However, portosystemic anastomoses are tralia EOC and is approved as an aid in monitoring associated with fetal growth restriction • Prof. Dr. Martin Widschwendter, University College London, Lon- recurrence or progressive disease in due to a diminished oxygen supply to don, United Kingdom • Dr. Heinrich Roehder, Biodesix, Colorado, USA EOC patients. Recently, our Department hepatocytes and hence, downregulation of • Dr. Peter Schulz-Knappe, Protagen AG, Dortmund, Germany identified HE4 as an independent prognostic liver function. In a study three cases and the Collaborations in Clinical Trials with (inter)national research marker in endometrial cancer patients. used form of treatment were described. groups: • In collaboration with the University of • AGO Austria – Arbeitsgemeinschaft Gynäkologische Onkologie • ENGOT – European Network for Gynecological Oncological Trial Queensland, HE4 was analysed in sequen- Major Achievements: groups (Cooperation with 19 trial groups) tial serum samples from endometrial can- Identification of an increased risk for • GCIG – Gynecologic Cancer InterGroup (Cooperation with 25 cer patients to analyse the HE4 kinetics stillbirth in women older than 40 years and international trial groups) • ABCSG – Austrian Breast & Colorectal Cancer Study Group between the time point of diagnosis and description of obesity and poor antenatal the time point of recurrence or freedom care as important amendable risk factors. International collaboration with trial groups outside of EN- GOT, GCIG or ABCSG trials: from recurrence. Recommendation for a detailed examination • Prof. Dr. Jalid Sehouli, Charité Berlin, Germany • In another project HE4 serum levels were of the fetal liver using color flow mapping to • Nord-Ostdeutsche Gesellschaft für Gynäkologische Onkologie evaluated in comparison to CA 125 (can- exclude portocaval anastomoses in cases (NOGGO e.V.) • EORTC – European Organization for Research and Treatment of cer antigen 125) levels in the course of of unexplained growth restriction. Cancer

Research Report 2015 Medical University of Innsbruck 151 Center of Obstetrics and Gynecology Gynecological Endocrinology and Reproductive Medicine

with the Department of Gynecology and Physiology and Pathology of the Obstetrics and the Morphologic Laboratory Menstrual Cycle and Early Pregnancy of this department, the central laboratory of Bettina Boettcher, Christoph Brezinka, the LKI, General Pathology Division, Division Valeria Colesselli, Agung Dewanto, of Clinical Biochemistry, Departments Katharina Feil, Verena Porto, Beata of Genetics and Genetic Epidemiology, Seeber, Ludwig Wildt (alphabetical order) Department of Neuroradiology, Depart The main research interests concern the ments of Neurology, Department of Nuclear regulation of the menstrual cycle, the Medicine, Division of Endocrinology of assessment of ovarian reserve and the the Department of Internal Medicine, large area of menstrual cycle dependent Divisions of Endocrinology and Metabolism (katamenial) disorders such as dysmenor- of the Department of Paediatrics and the rhea, endometriosis/adenomyosis and, as Department of Otolaryngology. additional examples, katamenial epilepsy and premenstrual syndrome. With regard to Research the pathophysiology of menstrual disorders, our main interest is in hyperandrogenemic Fertility Preservation disorders (PCO-syndrome). Katharina Winkler-Crepaz, Susanne Director: Hofer, Sarrah Ayuandari, Wolfgang Physiological Changes During the Univ.-Prof. Dr. Ludwig Wildt Biasio and Ludwig Wildt Menstrual Cycle One side effect of many of the commonly It has been known for a long time that women Contact: used chemotherapies, as well as pelvic ir- hyperventilate during the luteal phase of Anichstraße 35 radiation, is its deleterious effect on the go- the menstrual cycle and during pregnancy. 6020 Innsbruck nads. In women this can lead to premature We could show that hyperventilation starts menopause with its related diseases and already during the beginning of the rise of [email protected] sterility. In addition, ovarian tumours, chro- serum estradiol indicating final maturation Phone: +43 512 504 23275/23276 mosomal disorders such as Turner Mosaic of the preovulatory follicle and the start of Fax: +43 512 504 23277 or fragile X Premutation can lead to a pre- the fertile phase of the menstrual cycle. www.kinderwunsch-zentrum.at mature loss of ovarian function and fertility. Thus, determination of the decline of endexspiratory pCO2 pressure may be used One possible strategy to preserve female for determining the fertile phase of the cycle. Keywords fertility is the cryopreservation of ovarian After developing a highly sensitive device tissue prior to the gonadotoxic treatment. for easy determination of endexspiratory Endocrinology, reproductive biology and Once the patient is cured, ovarian function pCO2, studies are initiated to evaluate the medicine, fertility preservation, endo can be restored by autotransplantation of usefulness of this method in the context metriosis, PCOS, early pregnancy, menstrual the tissue. The Department of Gynecologi- of natural family planning. Moreover, our cycle dependent disorders, uterine fibroids cal Endocrinology and Reproductive Medi- findings indicate that some symptoms of gender identity disorders. cine was the first center to perform ovarian the premenstrual syndrome may be caused tissue cryopreservation in Austria. Oncolog- by excessive hyperventilation during the Research Focus ic patients from all over Austria, South Tyrol luteal phase of the cycle. and Southern Germany were referred to The main research topics of the Depart- Innsbruck. In order to improve the success Adenomyosis and Endometriosis ment of Gynecological Endocrinology and rate of cryopreservation and transplanta- Adenomyosis and Endometriosis are Reproductive Medicine are physiology and tion, xenotransplantation is an important common disorders of human females pathology of ovarian function, fertility pres- approach to assess the quality of ovarian during the middle and late reproductive ervation, polycystic ovarian syndrome, ade- tissue prior to transplantation, as well as to period of life. They are characterized by nomyosis/endometriosis, early pregnancy investigate the mechanism behind follicular dysmenorrhea, pelvic pain and infertility. We and recurrent pregnancy loss, uterine fi- development after transplantation. developed a new model of the pathogenesis broids, cycle dependent disorders, contra- of endometriosis/adenomyosis based on ception and disorders of gender identity. The fertility preservation unit of our clinic morphological and molecular biology data is primarily interested in studying follicu- called TIAR (Tissue injury and repair). This General Facts lar growth and functionality as well as to concept views adenomyosis and endo analyse molecular mechanisms involved metriosis as the consequence of constant All our research projects are performed in in follicular growth initiation and follicu- autotraumatisation of the uterus caused by intense cooperation between the research lar loss. This is done by in vitro assays, by exaggerated uterine contractions followed laboratory, the clinical units and the immunohistochemistry and by transplanta- by local injury, induction of prostaglandin IVF -laboratory of the Department. This tion and stimulation experiments in SCID- synthesis and aromatase activity and in allows translation of experimental medicine mice. Furthermore, we aim to optimize the turn a further estrogen-induced increase in to direct patient care, for example, in the existing fertility preservation techniques by the force of contractions. Pharmacological case of ovarian tissue cryopreservation developing specific stimulation and preser studies now underway are the logical in patients facing loss of fertility due to vation protocols. The concept followed at consequence of this concept, examining malignant disease. We collaborate closely our Department is shown in Fig. 1. the effects of aromatase inhibitors and

152 Research Report 2015 Medical University of Innsbruck Gynecological Endocrinology and Reproductive Medicine ©Archived Gynecolgy and Obstertics ©Archived Fig. 1: The Innsbruck protocol for fertility preservation in female Fig. 2: TIAR-Tissue injury and Repair cancer patients. novel prostaglandin antagonists on pain in on the efficacy and safety of progestins registered participants. He is a fellow of patients suffering from endometriosis. Pain and aromatase inhibitor intravaginal ring the American Institute of Ultrasound in perception, which may also be profoundly in endometriosis and assessment of safety Medicine and a Seminarleiter of ÖGUM. altered in endometriosis, is examined and efficacy of Vilaprisan in patients with Major Achievements: Publication of the using functional MRT in a model of pelvic uterine fibroids are currently performed. “Truffle” study in 2014 and 2015, getting pain. Along a similar line, the induction of the 2017 ISUOG conference to Austria. pain fiber growth in endometriotic lesions Gender Identity Disorders by neurotrophins and their receptors are The Department is a center for treatment Selected Publications examined using quantitative immuno of female to male (FtM) and male to female Novel dynamic culture system to support initiation of primordi- histochemistry and molecular biology. (MtF) transsexuals. So far, our interests al follicle growth in prepubertal mouse ovaries. Winkler-Crepaz K, Nederegger V, Ayuandari S, Rosenfellner D, Zervomanolakis I, Further studies examined some metabolic were mainly to study the effects of andro Hofer S, Wildt L, Ziehr SC. Fertil Steril. 2014 Sep;102(3):864–870. disturbances in endometriosis as indicated gens or estrogens, respectively on hormonal A first-in-human study of PDC31 (prostaglandin F2α receptor in- by Afamin serum levels. and metabolic parameters in transsexual hibitor) in primary dysmenorrhea. Böttcher B, Laterza RM, Wildt L, Seufert RJ, Buhling KJ, Singer CF, Hill W, Griffin P, Jilma B, Schulz M, patients. Now a functional MRT-study has Smith RP. Hum Reprod. 2014 Nov;29(11):2465–73. Polycystic Ovarian Syndrome been initiated which will examine the effect Metformin induces a prompt decrease in LH-stimulated Polycystic ovarian syndrome is the most of visual erotic stimuli on the activity of testosterone response in women with PCOS independent of its insulin-sensitizing effects. Kurzthaler D, Hadziomerovic-Pekic D, frequent endocrine disorder of women of specific brain regions in FtM transgender Wildt L, Seeber BE. Reprod Biol Endocrinol. 2014 Oct 11;12. early to mid reproductive life associated patients. Adenomyosis and endometriosis. Re-visiting their association and with signs of androgenization, irregular further insights into the mechanisms of auto-traumatisation. An MRI study. Leyendecker G, Bilgiycildirim A, Inacker M, Stalf T, Hup- cycles and infertility. It is frequently, Ultrasound in Gynecological Endo pert P, Mall G, Boettcher B, Wildt L. although not always associated with insulin crinology and Reproductive Medicine Arch Gynecol Obstet. 2015 Apr;291(4):917–32. resistance and symptoms of metabolic syn- Christoph Brezinka TRUFFLE study group. 2 year neurodevelopmental and intermedi- drome. In our studies we have focused on Ultrasound has become a central part of ate perinatal outcomes in infants with very preterm fetal growth restriction (TRUFFLE): a randomised trial. Lees CC, Marlow N, van diagnostic aspects of PCOS (highly sensitive diagnosis in all questions related to fertility Wassenaer-Leemhuis A, Arabin B, Bilardo CM, Brezinka C, Calvert assays for androgens in serum, diagnosis and early pregnancy but also to meno S, Derks JB, Diemert A, Duvekot JJ, Ferrazzi E, Frusca T, Gan- zevoort W, Hecher K, Martinelli P, Ostermayer E, Papageorghiou of insulin resistance and the diagnosis of pause, endometriosis and genital neo AT, Schlembach D, Schneider KT, Thilaganathan B, Todros T, Valca- heterozygous forms of C-21 Hydroxylase plasia. With state of the art equipment that monico A, Visser GH, Wolf H. Lancet. 2015 May 30;385(9983):2162–72. deficiency. is constantly being updated, the main effort is on training of medical staff of the depart Selected Funding Early Pregnancy Failure ment particularly in the use of 3D and 4D • Tyrolean Research Foundation (TWF), project number GZ: Early pregnancy failure (e.g. missed abor ultrasound, proper documentation with UNI-0404-1097, Dr. K. Winkler-Crepaz • Oesterreichische NAtionalbank, Anniversary Fund (OeNB), tion) has traditionally been treated by an online system and a constant quality project number 14641, Dr. K. Winkler-Crepaz dilatation and curettage. This is a surgical review process. Outside the department procedure and may have adverse effects there is strong activity in ultrasound Collaborations on the function of the endometrium with training courses and publication of • Fertiprotect Network: Germany, Switzerland and Austria • C. A. Schreiber, Univ. of Pennsylvania, Penn Medicine, USA respect to future pregnancies. We tried guidelines and manuals. Christoph Brezinka • S. Mechsner, Charitè Berlin, Germany to optimize the medical management of is a board member of the International • T. Woodruff, Northwestern University and the Oncofertility Consortium, USA women with early pregnancy failure. Women Society for Ultrasound in Obstetrics and • G Leyendecker, Kinderwunschzentrum Darmstadt, Germany are primarily being offered successful yet Gynecology ISUOG and member of the non-invasive treatment options for early mis ISUOG ultrasound safety committee, the Devices & Services carriage. Positive feedback through patient ultrasound education committee. He is • Ovarian tissue cryobank • CL-863 Freeze Control, Cryologic (automated freezer) questionnaires has confirmed the safety organizing the 2017 ISUOG conference • Tecan Reader, Genius Pro, Fluorescence/Luminescence/Ab- and acceptability of this treatment. Studies in Vienna which is expected to have 3000 sorbance Reader

Research Report 2015 Medical University of Innsbruck 153 Center of Otorhinolaryngology and Hearing, Speech and Voice Disorders Otorhinolaryngology

Laboratory by Univ.-Prof. Dr. Annelies is unknown and most likely dominating the Schrott-Fischer. These three major research application accuracy. This initial feasibility units are in close cooperation with the study aims at providing first data for ULE clinician scientists enabling translational with a research navigation system. Active research projects with clinical impact in optical navigation was done in CT-images of three major focuses of Otorhinolaryngology. a plastic skull, an anatomic specimen (both Furthermore the Department is in close with implanted fiducials), and a volunteer cooperation with the local industry, with anatomical landmarks exclusively. Each developing, for instance a laryngeal object was registered ten times with 3, 5, pacemaker or a vestibular implant. Further 7, and 9 registration points. Measurements clinical research focuses are focused on were taken at 10 (anatomic specimen and clinical oncology, rhonchopathy and clinical volunteer) and 11 targets (plastic skull). The aspects of hearing implants. active NDI Polaris system was used under ideal working conditions (tracking accuracy Research 0.23 mm root-mean-square, RMS; probe tip calibration was 0.18 mm RMS). Variances Molecular Biology and of tracking along the principal directions Oncology Laboratory were measured as 0.18 mm2, 0.32 mm2, Director: PD. Dr. Joszef Dudas and 0.42 mm2. ULE was calculated from Univ.-Prof. Dr. Herbert Riechelmann Cell cycle association and hypoxia regulation predicted application accuracy with of excision repair cross complementation isotropic and anisotropic models and from Contact: group 1 protein (ERCC1) in tumor cells of experimental variances, respectively. Anichstraße 35 head and neck cancer. 6020 Innsbruck The ULE was determined from the variances Excision repair cross complementation as 0.45 mm (plastic skull), 0.60 mm [email protected] group 1 (ERCC1) protein is considered (anatomic specimen), and 4.96 mm Phone: +43 512 504 23141 a prognostic marker in head and neck (volunteer). The predicted application Fax: +43 512 504 23144 cancer. In this study, clinical response to accuracy did not yield consistent values for http://hno.tirol-kliniken.at/ mitomycin C (MMC) or cisplatin (CDDP) the ULE. Quantitative data of application based radiochemotherapy (RCT) was as- accuracy could be tested against prediction sessed in 106 head and neck squamous cell models with iso- and anisotropic noise carcinoma (HNSCC) patients and compared models and revealed some discrepancies. with cell nuclear immunoreactivity (in % of positive nuclei) of the mouse monoclonal Keywords (clone: 8F1) ERCC1-antibody in tumor tissue samples. Drug delivery to the inner ear, inner ear morphology, accuracy of computer aided The same immunostaining was performed surgery, head & neck neoplasm, epithelial- in 26 normal mucosa samples. While mesenchymal transition ERCC1-immunoreactivity did not differ in normal pharyngeal mucosa and treatment Research Focus responders, non-responders revealed significantly lower (p=0.0064) ERCC1 • The role of Nanoparticles in drug delivery representation. Simultaneous in vitro stud- to the inner ear ies revealed that under hypoxic conditions, • Error analyses for computer assisted ERCC1-gene expression significantly de- surgery creased, whereas ERCC1+ cells might • Targeted therapy for head and neck squa- represent radiosensitive cells, which are mous cell carcinoma preferably in G2-phase of cell cycle, and © Tumor Biol. 2014 are non-hypoxic. (Fig. 1, Dudas et al., Tumor Fig. 1: Twenty-six normal mucosa samples General Facts Biol. 2014) and 106 HNSCC tumor tissue samples have been stained with 8 F1 ERCC1 antibody, and The major aim of the Department 4D Visualization Laboratory the percentage of stained cells in tumor of Otorhinolaryngology is to provide Univ.-Prof. Dr. Wolfgang Freysinger cell nests or in high power fields of normal independent basic and clinical research in Quantitative error analysis for computer mucosa has been evaluated. There was no the field of Otorhinolaryngology to optimize assisted navigation: A feasibility study. significant difference between the stain- patient treatment. The basic research is The benefitof computer-assisted navigation ing representation of normal mucosa and provided by three independent laboratories: depends on the registration process, at therapy responder HNSCC, but the therapy 1) the Molecular Biology and Oncology which patient features are correlated to nonresponder HNSCC patients have shown Laboratory by PD. Dr. Joszef Dudas; 2) the some preoperative imagery. The operator- significantly lower (p=0.0064) ERCC1 rep- 4D Visualization Laboratory by Univ.-Prof. induced uncertainty in localizing patient resentation compared to the normal mucosa Dr. Wolfgang Freysinger; 3) the Inner ear features—the user localization error (ULE)— and responder patients.

154 Research Report 2015 Medical University of Innsbruck Otorhinolaryngology

This could potentially be due to the facts antibodies. Bioefficacy of drug release asw Selected Publications that navigation and one prediction model tested with rolipram loaded LNCs to prevent Nanoparticle mediated drug delivery of rolipram to tyrosine kinase B positive cells in the inner ear with targeting peptides wrongly assume isotropic noise (tracking cisplatin-induced apoptosis. We established and agonistic antibodies. Glueckert R, Pritz CO, Roy S, Dudas J, is anisotropic), while the anisotropic noise different cell culture models with SH‑SY‑5Y Schrott-Fischer A. prediction model assumes an anisotropic and inner ear derived cell lines and used Front Aging Neurosci. 2015 May 19;7:71. Cell cycle association and hypoxia regulation of excision repair registration strategy (registration is neonatal and adult mouse explants. Uptake cross complementation group 1 protein (ERCC1) in tumor cells isotropic in typical navigation systems). and trafficking asw evaluated with FACS of head and neck cancer. Dudás J, Schartinger VH, Romani A, Schweigl G, Kordsmeyer K, Marta PI, Url C, Kral F, Riechelmann H. and confocal as well as transmission Tumour Biol. 2014 Aug;35(8):7807–19. The ULE data are presumably the first electron microscopy. Plain NPs show some Quantitative error analysis for computer assisted navigation: a quantitative values for the precision of selectivity in uptake related to the in vitro feasibility study. Güler Ö, Perwög M, Kral F, Schwarm F, Bárdosi ZR, Göbel G, Freysinger W. localizing anatomical landmarks and system properties, carrier material, and NP Med Phys. 2013 Feb;40(2):021910. implanted fiducials. Submillimetric size. localization is possible for implanted screws; Selected Funding anatomic landmarks are not suitable for Some peptide ligands provide enhanced • Echtzeit Resektionskontrolle bei navigierten Operationen an der lateralen Schädelbasis, ÖNB, approved 12 /2014, 100,000.- high-precision clinical navigation (Fig. 2) targeted uptake to neuronal cells but failed • CIGuide, FFG, approved 2014, 420,000.- (Güler et al. J. Med. Phys. 2013). to show this in cell cultures. Agonistic • 3D Rekonstruktion des Innenohrs, VAMEL, K-Regio, Land Tirol 197,810.- antibodies linked to silica NPs showed TrkB • Ion channels in human neurons, MEDEL, 218,000.- Inner Ear Biology activation and enhanced binding to inner • BDNF – TrcB signaling in Head and Neck Cancer; FWF, 112,623.- Univ.-Prof. Dr. Annelies Schrott-Fischer ear derived cells. Rolipram loaded LNCs Collaborations Nanoparticle mediated drug delivery of proved as effective carriers to prevent • TU München mit Informatik, Prof. Nassir NABAB rolipram to tyrosine kinase B positive cells cisplatin-induced apoptosis. • Universität Bern, HNO, Prof. Marco Caversaccio in the inner ear with targeting peptides and • Brigahm & Womens Hospital, Harvard, Boston, MA, USA, Prof. Ron KIKINIS agonistic antibodies. Most NPs with targeting ligands showed • Mechatronik, MCI, Innsbruck, DI Dr. Andres MEHRLE Brainlab, limited effects to enhance uptake. NP München • iSYS, Kitzbühel, CEO Dr. Michael VOGELE Systemic pharmacotherapies have aggregation and unspecific binding may • ACMIT, Wr. Neustadt, CEO Dr. Gernot Kronreif limitation due to blood-labyrinth barrier, change uptake mechanisms and impair • LSTMH, Liverpool, UK, • Semmelweis University Budapest, Inst. Pathology and Experi- so local delivery via the round window endocytosis by an overload of NPs. This mental Cancer Research Budapest, Hungary membrane opens a path for effective may affect survival signaling. NPs with • Department of Surgical and Molecular Pathology, National Insti- tute of Oncology, Budapest, Hungary treatment. Multifunctional nanoparticle antibodies activate survival signaling and • Department of Otorhinolaryngology, University Lübeck, Ger- (NP)-mediated cell specific drug delivery show effective binding to TrkB positive cells many • Cochlear Signaling and Tissue Engineering Laboratory, Labora- may enhance efficacy and reduce side but needs further optimization for specific tory Director, USA Josef M. Miller, Ph.D., effects. Different NPs with ligands to target internalization. • http://www.khri.med.umich.edu/research/miller_lab/index. php TrkB receptor were tested. Distribution, • Auditory Anatomy Laboratory,., Laboratory Director, USA Rich- uptake mechanisms, trafficking, and Bioefficiacy of rolipram release confirms ard Altschuler, Ph.D • http://www.khri.med.umich.edu/research/altschuler_lab/ bioefficacy of drug release of rolipram LNCs as encouraging vectors for drug index.php loaded NPs were evaluated. We tested lipid delivery of lipophilic agents to the inner • Universität Uppsala, Schweden, Helge Rask-Andersen http://www.medfarm.uu.se/ based nanocapsules (LNCs), Quantum Dot, ear with ideal release characteristics • Department für Anatomie, Histologie und Embryologie, Sektion silica NPs with surface modification by independent of endocytosis. für Neuroanatomie , MUI, Lars Klimaschewski, Barbara Hausott • Department für Anatomie, Histologie und Embryologie Sektion peptides mimicking TrkB or TrkB activating (Glückert et al. Front Aging Neurosci. 2015) für Klinisch-Funktionelle Anatomie, Brenner Erich, Elisabeth Pechriggl • Department für Anatomie, Histologie und Embryologie , Sektion Fig. 2: Rolipram-loaded lipid nanocapsules für Histologie und Embryologie, Kristian Pfaller (LNCs) partially rescue cisplatin-induced ap- • Veterinärmedizinische Universität, VetCore Facility for Re- search Imaging Unit, Stephan Handschuh optosis in postnatal mouse cochlea cultures • UMIT Hall Institut für Biomedizinische Informatik, Division für (P24). Spiral ganglion neurons (SGN) treat- Biomedizinische Bildanalyse, Karl Fritscher, Rainer Schubert • UMIT Hall, Institut für Elektrotechnik und Biomedizinische Tech- ed with cisplatin and rolipram-loaded LNCs nik,Christian Baumgartner exhibit less cleaved caspase 3 (CC3-red • MedEl Innsbruck ,Ingeborg Hochmair, Carolyn Garnham, Claude Jolly staining) immune reactivity (A) than con- • Frank Rattay, Computational Neuroscience and Biomedical trols exclusively treated with cisplatin (D). Engineering, Institute for Analysis and Scientific Computing, Vienna University of Technology, Austria In the spiral ligament (SL) cisplatin induces • University of Tampere, Finland, Ilmari Pyykko increased CC3 reactivity (J) while additional • University of Angers, France, Saulnier Patrick, Guillaume Bastiat • University of Southampton, UK, Tracey Newman treatment with rolipram-loaded LNCs re- • The Bionics Institute, East Melbourne, Australia, Andrew K. Wise duced the CC3-reacitivty (G). (A,D,G,J) CC3 • Inserm U 254 Neurobiologie de l’Audition, Montpellier, France, Eybalin Michel immunolabeling; (B,E,H,K) phalloidine labeling and DAPI staining; (C,F,I,L) bright field. Devices Scale bar 50 μm. • TissueFaxs from TissueGnostics 2015 . © Front Aging Neurosci

Research Report 2015 Medical University of Innsbruck 155 Center of Otorhinolaryngology and Hearing, Speech and Voice Disorders Hearing, Speech and Voice Disorders

Keywords Faculty in 1968. This was the first chair in these specialities in the German speaking Aural effects of noise, hearing disorders, countries. Today, the HSV is Austria’s hearing rehabilitation, sound localisation, largest institution in the fields of Audiology, sound localisation with hearing implants, Pediatric Audiology and Phoniatrics. It speech understanding with hearing im- offers a full range of clinical services for plants, tinnitus, voice disorders the diagnosis and treatment of disorders of hearing, speech, language, swallowing Research Focus and of childhood learning problems. In the 1990s, the Department was significantly • Assessing the auditory performance (e.g. involved in implementing Universal Newborn speech understanding, sound source Hearing Screening in Austria. localisation) of patients with hearing implants including cochlea and middle ear Equipped with an anechoic chamber and implants. tools for high-precision acoustic meas- • Assessing the auditory performance of urements, the Department is frequently normal hearing people whose hearing is involved in collaborations with commercial impaired by external factors like helmets firms (including producers of hearing im- Director: or caps. plants and hearing aids) who seek to test o. Univ.-Prof. Dr. Patrick Zorowka • Developing objective methods for hearing or evaluate their recently developed tech- implant fitting in children (e.g. Stapedius nology with patients or test persons under Contact: reflex measurement). suitable acoustic conditions. Anichstraße 35 • Analysing the effects of noise on hearing 6020 Innsbruck health through epidemiological studies of Research tinnitus and hearing complaints in adoles- [email protected] cents Hearing Loss in Cystic Fibrosis (CF) Phone: +43 512 504 23218 Markus Rungger Fax: +43 512 504 23217 General Facts In collaboration with the CF Unit of the www.hss-innsbruck.at Department of Pediatrics III of Innsbruck The Department HSV was founded in 1974, Medical University. after a “Chair in Audiology and Phoniatrics” Patients with Cystic Fibrosis (CF) and chro was established at Innsbruck Medical nic airway colonisation with Pseudomonas

Fig. 1: The anechoic chamber is a special room for high-precision psycho-acoustic measure ments. The background noise level in the chamber is less than –10 dB Hearing Level at all frequencies between 16 and 20,000 Hz. Loudspeaker boxes are mounted in a horizontal plane for testing a person’s sound source localization skills.

156 Research Report 2015 Medical University of Innsbruck Hearing, Speech and Voice Disorders

aeruginosa receive regular aminoglycoside them uses empirical data for the purpose of fitting based on ESRT are currently being therapy against their infection. To monitor falsification of assumptions and hypotheses evaluated and possibilities of extending the the ototoxic side-effects of the treatment, (as would be logically correct). Moreover, clinical applicability of the procedure are the patients undergo hearing tests (audio many theories suffer from conceptual being investigated. grams) before and after aminoglycoside uncertainty; others make wrong predictions application. In this research project, and again others have implications which High-Speed Visualization of Vocal audiograms of the CF patients are are inconsistent with other well-established Cord Vibration Anomalies retrospectively analysed and changes of theories. This research project undertakes Thomas Wöllner their hearing thresholds are statistically a logical and ontological analysis of models High-speed cameras (>4000 images/ linked to parameters of their disease, of Tinnitus pathogenesis. Its primary goal second) allow for a detailed and, to some including genotype of the mutation, is to reveal logical and ontological errors degree, real-time visualization of vocal cumulative aminoglycoside dose, type of included in them and, if possible, to eliminate cord vibrations. With the help of software aminoglycoside application, pulmonary them and revise the model assumptions. the recorded images can be analysed and function, pancreas insufficiency, and In addition, it will be demonstrated that a vocal cord movements which are irregular others. In detail, it is analysed which of lot of empirical research could be saved if, or functionally abnormal can be identified. these parameters is most predictive for a prior to empirical investigation, conceptual To detect irregularities which are clinically hearing threshold deterioration and which uncertainty and logical ambivalence of relevant, the software needs a decision frequencies of the hearing threshold are theories would be remedied. algorithm including criteria for recognizing most vulnerable to the above parameters. some movement patterns as normal Aural Effects of Noise and others as abnormal. This research Sound Localisation in Viktor Weichbold project attempts to define such criteria by Unilateral Deafness Loss of hearing sensitivity is a long-known correlating abnormal movement patterns Josef Seebacher effect of continuous exposure to loud noise. with clinical parameters of dysphonic Correct localization of sound sources In recent years it has become clear that voices. in a 3D environment requires binaural tinnitus and hyperacusis are also due to hearing. In patients with unilateral deafness inner ear damage from acoustic energy. To Selected Publications sound source localisation is impaired; induce tinnitus or hyperacusis, sound levels Effect of Wearing a Ski Helmet on Perception and Localization of Sounds. Ruedl G, Kopp M, Burtscher M, Zorowka P, Weichbold V, as a consequence their rate of correct need not be so high as to induce a hearing Stephan K, Koci V, Seebacher J. localisations is low. Restoring their hearing loss (i.e. >85 dB Sound Pressure Level). INT J SPORTS MED. 2014, 35(8): p. 645–650. with a hearing implant (cochlea or middle Rather, impulse noise is presumed to be the Evaluation of a minimally invasive surgical fixation technique for young children with the Concerto Pin cochlear implant system. ear implant) is supposed to improve their detrimental factor in these pathologies. This Schnabl J, Wolf-Magele A, Pok SM, Url C, Zorowka P, Sprinzl G. EUR localisation skills, however, research has study assesses the prevalence of tinnitus ARCH OTORHINOLARYNGOL. 2014 Mar 23. [Epub ahead of print] shown that the results remain below and hyperacusis in adolescents who play an Results of hearing screenings in 14-to 15-year old adolescents. Weichbold V, Holzer A, Newesely G, Zorowka P, Stephan K. expectations: despite restored bilateral instrument in a band and who are regularly HNO. 2013, 61(1): p. 25–29. hearing the patients perform worse than exposed to different sorts of noise. It Safety of intralesional cidofovir in patients with recurrent respira- normally hearing persons. This research is expected that adolescent musicians tory papillomatosis: an international retrospective study on 635 RRP patients. Tjon Pian Gi RE, Ilmarinen T, van den Heuvel ER, project investigates why unilaterally deaf who are frequently exposed to impulse Aaltonen LM, Andersen J, Brunings JW, Chirila M, Dietz A, Ferran persons with a hearing implant in the deaf noise (e.g. drummers) have more hearing Vilà F, Friedrich G, de Gier HH, Golusinski W, Graupp M, Hantzakos A, Horcasitas R, Jackowska J, Koelmel JC, Lawson G, Lindner F, side localize worse than normally hearing complaints than others who are exposed to Remacle M, Sittel C, Weichbold V, Wierzbicka M, Dikkers FG. persons. The hypothesis under investigation more stationary noise. EUR ARCH OTORHINOLARYNGOL. 2013, 270(5): p. 1679–1687. is that the processing of the acoustic signal Collaborations in the speech processor is so fast that the Stapedius Reflex in Cochlear • Fa. Grassmayr Glockengießerei, Innsbruck, Österreich interaural time differences (which sounds Implant (CI) Patients • SCOTT Sports SA, Givisiez, Schweiz normally have) are nearly annihilated. As Kurt Stephan these differences are an important cue Various studies (incl. from our research Devices and Services for the auditory system in determining group) have shown that the stapedius reflex • Medical outpatient unit • Audiology (incl. Anechoic Chamber) the direction where a sound comes from, can be elicited by electrical stimulation via a • Pediatric Audiology their diminishment may be the cause cochlear implant in deaf ears. The electrical • Logopedic unit for the deterioration of the localisation stapedius reflex threshold (ESRT) which can performance. If this hypothesis is true, a be detected by an objective test procedure slight deceleration of the signal processing was found to correlate with the upper in the speech processor should improve the limit of electrical stimulation (maximum localisation performance. comfortable loudness), a quantity which is most important for the fitting of CI speech Conceptual modelling of processors. Based on this correlation an Tinnitus pathogenesis objective fitting procedure was developed Viktor Weichbold which is routinely applied at the HSV There exists a huge mass of theories Department for the CI fitting of children attempting to explain the pathogenesis of and of patients who cannot verbally report Tinnitus. While most of them integrate data their loudness sensation when their hearing from empirical research in order to confirm is stimulated by a CI. Long-term stability their assumptions and hypotheses, none of of ESRT and clinical application of CI

Research Report 2015 Medical University of Innsbruck 157 Center of Diagnostic Radiology Diagnostic Radiology

• Digital imaging/PACS/post processing of scanning of extremities of patients, mostly imaging data for the evaluation of bone density (Osteo- • Clinical trials in oncology porosis). Our research projects are mostly clinically orientated. We focus on the rapid General Facts translation of research results into clinical practice. Also, most research projects are For clinical research, the Department is interdisciplinary. The Department of Radiol- equipped with state of the art imaging ogy and the Department of Neuroradiology equipment including 7 CT scanners (1 Dual have a close cooperation regard-ing train- source/three 64 row/one 32 row/two 16 ing, patient care and research. row), 3 MRI scanners (3T and 1.5T), 1 PET- CT (in cooperation with the Department of Research Nuclear Medicine), 3 angio suites (1 biplane) and 15 high end ultrasound systems. One Atherosclerotic Burden and its CT with a sliding gantry operating in an Relevance in Case of Different Diseases OR and an imaging suite is dedicated to and Treatment Strategies. stereotaxy and CT guided procedures. The Bernhard Glodny, Johannes Petersen Department operates completely digitally Cardiovascular and cerebrovascular seque- Director: using a comprehensive imaging archive that lae of atherosclerotic disease are one of the o. Univ.-Prof. Dr. Werner Jaschke was installed in the year 1999. leading causes of morbidity and mortality in There are 66 staff members including 30 humans, and concerns many different fields Contact: residents (radiologists in training). of medicine. Atherosclerosis can be detect- Anichstraße 35 ed using different modalities of diagnostic 6020 Innsbruck The Section of Experimental Radiology imaging. Moreover, treatments are planned is staffed with 6 phycisists with different using clinical imaging, and cardiovascular [email protected] areas of interest such as MRI, MRS, image risk profiles can be compiled individual- Phone: +43 512 504 22761 data processing,radiation protection and ly. Computed tomography can be used to Fax: +43 512 504 22758 computer applications. quantify the “atherosclerotic burden” of all http://radiologie.tirol-kliniken.at vascular territories in an objective and re- The Department houses research facilities producible manner. Relationships between for animals (Small Animals Research Lab). oral health and health in general have been High resolution RF coils for MR imaging suspected since many years. One of the of animals are available as well as access first observations to be made in this area to PET imaging. Large animals can be im- of study was that oral and general health is Keywords aged on 1 of the clinical CT scanners (32 impacted similarly by certain behavioural, rows/Siemens). The Core Facility MicroCT social and environmental factors. In the late Radiology, interventional radiology, MRI, CT, is equipped with 2 MicroCT scanners (vi- eighties, and the nineties of the last centu- angiography, contrast media, ultrasound, vaCT40/Scanco Medical and XtremeCT ry, a causal relationship between marginal PET-CT, robotics, interventional oncology, II/Scanco Medical) which are operated in periodontitis, and atherosclerosis was es- radiation protection, digital imaging, PACS, cooperation with the Department of Trau- tablished. microCT ma Surgery. Both microCT scanners can be used for the high resolution imaging of small This fitted perfectly with the concept of Research Focus animals (ranging from mice to rabbits). The atherosclerosis as an inflammatory disease. XtremeCTII is also used for high resolution The aim of the present studies is to adopt the • ultrasound (elastography, musculoskel- ettal ultrasound, ultrasound of peripheral nerves, ultrasound guided interventions) • multiparametric imaging of prostate cancer • MRI (quantification of fat/iron; cardiac MRI; spectrosopy of muscle/myocardium; musculo skeletal MRI including stress MRI of the hip and knee, MR mammography) • Cardiac CT • Dual Energy CT • Emergency Radiology, especially trauma • Sports injury • Interventional Radiology (endovascular/ Fig. 1: A typical CAP lesion of a tooth (14) in a semi-coronal reconstruction of a CT. The tooth oncology) shows an endodontic filling (a), a CAP lesion of a tooth (46) in a semi-sagittal reconstruction • Real time dose monitoring of patients of a CT (b), and a semicoronal reconstruction in the region 46 (c), showing the methods of • Diagnosis and treatment of HCC measurement of the distance between the crown and the alveolar ridge (double arrow) and • Percutaneous stereotactic RFA of measurement of the height of the bone (white line).

158 Research Report 2015 Medical University of Innsbruck Diagnostic Radiology

Non-Invasive Cardiac/Cardiovascular b) Diffusionweighted MRI of peripherial Imaging nerves (with M. Reinhold, Orth. Surgery) Gudrun Feuchtner Value of diffusion-weighted magnetic CT: 10 Multicenter trials resonance imaging for the diagnosis and MRI:Pulse wave velocity (PWV) is the treatment of patients with lumbar nerve proposed gold-standard for the assessment root entrapment syndromes. of aortic elastic properties. It is the aim of Fig. 2: 65-year-old woman with an invasive this research project to use MR based pulse c) Diffusion tensor imaging of the median ductal carcinoma showing inhomogeneous wave velocity imaging to assess aortic nerve in carpal tunnel syndrome. breast tissue at 9 o’clock as demonstrated by stiffness as a biomarker of myocardial wall PI: A. Klauser conventional mammography (a, b). Low-en- stress. ergy CESM images (c) are equivalent to d) Assessment of tumour microcirculation conventional mammography, while spectral Experimental Radiology by dynamic magnetic resonance imaging imaging shows a contrast enhancing mass. Wolfgang Recheis (DMRI): Tumour microcirculation is an Image processing and analysis including important biomarker for diagnosis, therapy theory of atherosclerosis as inflammatory Rapid Prototyping based on radiological outcome prediction and therapy monitoring. disease in order to ascertain whether caries data represent core interests and tasks of In our study group DMRI is applied for these may be causative for atherosclerosis as well, the work group “Experimental Radiology”. purposes to prostate carcinoma, rectal especially in cases in which the disease has These projects include multidimensional carcinoma, glioblastomas, etc. crossed the enamel. Consequent infections visualization, quantification of disease of the dental pulp and apical periodontitis patterns based on texture analysis, shape e) MR Molecular Imaging using Nanoparti may represent additional causes for analysis and others (see Fig. 3). Moreover, cles. Recent developments in nanotechnolo atherosclerosis. Dental caries (Fig. 1) turned our new core facility micro-CT allows for gy provide a wide spectrum of nano sized out to be an independent risk factor for a the depiction of structures in µm scale in all material for various applications, including higher atherosclerotic burden of the aorta. three spatial dimensions. tumour targeting and molecular imaging. Moreover, dental fillings showed an inverse The main task of our work in this field is to effect, and were found to be an independent Morphological and MR-Imaging implement MR measurement techniques protective factor for aortic atherosclerotic Benjamin Henninger, Christian Kremser to facilitate the preclinical characterization burden (Fig. 2). Chronic apical periodontitis Morphological and functional MR-imaging and testing of such materials from varying also emerged as a risk factor for higher in all organ systems deveolopment of novel research groups. Main contact: C. Kremser atherosclerotic burden. MR-imaging applications and MR sequences Examples of research projects Imaging of Prostate Cancer Imaging of Breast Cancer Image fusion and image guided biopsy Martin Daniaux, Tobias De Zordo a) MRI for the Evaluation of Diffuse Liver of the prostate Example: Dual-energy contrast-enhanced Disease: Evaluation of different MRI- Multimodal imaging of the GU-tract mammography methods (relaxometry, chemical-shift Friedrich Aigner, Daniel Junker Dual-energy contrast-enhanced mammog- imaging, multi-echo approach, screening PSA is commonly used in screening for raphy is one of the latest developments in dixon) for the determination of diffuse liver prostatic cancer. Patients with high PSA breast care. Imaging with contrast agents in disease (fat, iron or combined diease); levels frequently undergo systematic breast cancer has already been described influence of iron on the evaluation of liver prostatic biopsies. However, PSA is not in previous magnetic resonance imaging fat (see Fig. 4). a reliable indicator for prostate cancer and computed tomography studies. How- ever, high costs, limited availability—or high radiation dose—led to the development of contrast-enhanced spectral mammography (CESM). Our most recent research evaluated this novel technique and was supported by GE Health Care (literature below). The research team focussing on the diagnosis of breast cancer has a vast experience with all of the imaging modalities currently used for evaluating the breast. In case of a suspicious imaging finding ew perform fine needle aspiration and/or percutaneous biopsies using stereotaxy or ultrasound guidance. Our unit serves as the largest Fig. 3: Left: Application of micro-computed tomography to microstructure studies of the me- screening and assessment centre of the na- dicinal fungus Hericium coralloides. Pallua JD, Kuhn V, Pallua AF, Pfaller K, Pallua AK, tional breast-screening programme in Tirol. Recheis W, Pöder R., Mycologia. 2015 Jan-Feb;107(1):227–38. arrow) and of measurement of Approximately 10 000 mammograms and the height of the bone (white line) breast ultrasound studies are performed Right: 3D Visualization of the round window (pink), the scala tympani (light yellow) and the each year making our unit the biggest in length (measuring points= yellowish brown) of a sheep cochlea from micro CT datasets, Austria. which were used for round window area, cochlea length and scala tympani measurement.

Research Report 2015 Medical University of Innsbruck 159 Center of Diagnostic Radiology

and systematic prostate biopsies are nerves and ultrasound guided nerve root invasive and suffer from a rather high infiltration and pain therapy. One of the false negative rate. Thus, there exists the most recent publications illustrates our need for improving non invasive methods work: for detecting prostate cancer in its early The axillary nerve (AN) is frequently injured stages. Our group has dealt with imaging during shoulder trauma and imaging is of the prostate using ultrasound and required to define the site and extent of multiparametric MRI for nearly 2 decades. nerve injury. However, the AN has a rather Also, we used ultrasound since the early complex course through several soft tissue 90s for performing ultrasound guided compartments of the shoulder and axilla. biopsies. Recently, image fusion became Therefore, imaging of the nerve with MRI available. We use ultrasound/MRI-fusion and sonography is troublesome. Thus for guiding biopsies and avoiding “blind” detection and sonographic assessment systematic biospies of the prostate. This requires a thorough knowledge of local approach improves the detection of cancer topography. in large prostates, in the anterior portion of the prostate and in the inner gland. Also, Our investigation is aimed at defining reli- reporting imaging results using the PI-RADS able anatomical landmarks for AN-sonog- classification helps to avoid unnecessary raphy in 5 volunteers and later validating biopsies. A low PI-RADS classification is a the proposed sonographic examination Fig. 4: Above: 43year old patient with very reliable indicator for the absence of protocol in 10 unselected patients. With suspected diffuse liver disease. The prostate cancer. Our results indicate, that strict adherence to the proposed exam- screening dixon sequence (work in pro- patients with a high PI-RADS classification ination algorithm, sonography of the AN gress package 718B, Siemens Healthcare) should undergo an image guided biopsy was feasible in all volunteers and patients. can provide a fast diagnosis of the predo- which has a much higher true positive rate Furthermore, sonographic findings correlat- minant pathologic liver deposition – in this than “blind” biopsies (see Fig. 5). ed nicely with the gold standard “surgical case it shows a fatty liver. Below: Automated exploration” concerning the severity and two-point dixon screening for the evaluati- Musculosklettal Imaging topography of neural impairment. on of hepatic steatosis and siderosis: com- Andrea S. Klauser et al. parison with R2*-relaxometry and chemical • Sonography of Carpaltunnel: definition of Based on our study results we propose our shift-based sequences. cut off values algorithm for AN-sonography as the first- • Sonoelastography of epicondylitis: accu- line imaging tool for the assessment of racy compared to histology axillary nerve trauma. (PIC) • Sonoelastography of plantar fasciitis: accuracy compared to histology Interventional Oncology • Sonoelastography of achilles tendon: ac- Reto Bale curacy compared to histology • Image guided tumor ablation • US guided injections in CTS: Sonoelasto- • Stereotaxy graphic appearance • Robotics • MR-Tractography (DTI, ADI) in median • Targeting nerves in healty voluntees and CTS pa- • Interventional Oncology tients: comparison to so-nography • US guided injection in Sacroiliac joints of Stereotactic Ablation of Liver Tumors: Fig. 5: A 67-year old patient with an anterior children: to prove feasability Radiofrequency ablation (RFA) allows for located Gleason score 7b prostate cancer in • DECT in gout: comparsion to US, findings local curative tumour treatment by inducing the transitional zone (PSA 5.69 ng/ml), encir- in extraarticular regions coagulation necrosis with a high-frequency cled on the whole-mount step-section slide • Hip Traction MRI (FIG) alternating current. The major limiting factor (a): The carcinoma (arrows) shows low sig- of percutaneous ablation methods is the nal on T2-weighted images with ill-defined Ultrasound tumour size, requiring multiple overlapping margins (b), diffusion restriction on diffu- Hannes Gruber; Alexander Loizides ablation zones. 3D-planning allowing sion-weighted imaging (c, d), and hyperper- Research Focus of the Research Unit: for the simultaneous display of multiple fusion on dynamic contrast-enhanced MRI • Peripheral nerve sonography trajectories and increased accuracy is (e: red circle on perfusion map) with a focal • Sonographic evaluation of soft tissue required. In addition, the virtual 3D plan has plateau curve (f: red curve). An area with hy- massesv to be precisely transferred into the patient. perplastic nodules appears unsuspicious in • Ultrasound guided injections in the spine Our team has developed frameless T2-weighted images (b), diffusion-weighted • Sonography of the musculoskeletal sys- stereotactic aiming devices and imaging (c, d), but shows hyperperfusion on tem immobilization devices for precise dynamic contrast-enhanced MRI (e: green • Contrast enhanced sonography punctures in different body regions. circle on perfusion map) with a focal wash- Stereotaxy enables highly accurate ablation out curve (f: green curve), which is even The Section of surgical ultrasound is a probe positioning in liver tumors. In 2001 the more pathological (PI-RADS 5) than the per- leader in the development of ultrasound worldwide first stereotactic radiofrequency fusion of the carcinoma. techniques for evaluation of peripheral ablation (SRFA) of a liver tumor was

160 Research Report 2015 Medical University of Innsbruck Diagnostic Radiology

performed in Innsbruck. In the meantime MD, Putzer D, Seiz M, Recheis W, Jacobs AH, Stockhammer G, Hutterer M. more than 600 patients were successfully PLoS One. 2014 Apr 23;9(4):e95830. doi: 10.1371/journal. treated at our department. Recently a pone.0095830. eCollection 2014. database was established and connected Diffusion-weighted magnetic resonance imaging for the diagnosis of patients with lumbar nerve root entrapment syndromes: results to the hospital information system (HIS). All from a pilot study. Reinhold M, Ederer C, Henninger B, Eberwein the relevant information about every patient, A, Kremser C. Eur Spine J. 2015 Feb;24(2):319–26. doi: 10.1007/ s00586-014-3602-6 Fig. 6: Carpal tunnel syndrome: diagnosis by treatment and follow-up examinations is Pretreatment Evaluation of Microcirculation by Dynamic Con- means of median nerve elasticity—improved continuously collected. Local recurrence trast-Enhanced Magnetic Resonance Imaging Predicts Surviv- diagnostic accuracy of US with sonoelasto- rate, complications, long-term survival etc. al in Primary Rectal Cancer Patients. DeVries AF, Piringer G, Kremser C, Judmaier W, Saely CH, Lukas P, Ofner D. Int J Radiat graphy (Miyamoto et al., 2014). can now easily be calculated. Currently the Oncol Biol Phys. 2014 Dec 1;90(5):1161–7. doi: 10.1016/j. efficacy and long-term survival after SRFA ijrobp.2014.07.042. is evaluated for different tumor entities. Tumor targeting and imaging with dual-peptide conjugated multifunctional liposomal nanoparticles. Rangger C, Helbok A, In addition, a major topic of research is Sosabowski J, Kremser C, Koehler G, Prassl R, Andreae F, Virgolini the implementation of robotic devices for IJ, von Guggen-berg E, Decristoforo C. Int J Nanomedicine. 2013;8:4659–71. interventional procedures. Comparison of real-time elas-tography and multiparametric MRI for prostate cancer detection: a whole-mount step-section analy- Theragnostics/Image Guided Tumor sis. Junker D, Schäfer G, Kobel C, Kremser C, Bektic J, Jaschke W, Aigner F. AJR Am J Roentgenol. 2014; 202: W263–9. Therapy (Mitigate Project) Achilles tendon assessed with sonoelastography: histologic MITIGATE stands for Closed-loop Molecular agreement. Klauser AS, Miyamoto H, Tamegger M, Faschingbauer Environment for Minimally Invasive Treat- R, Moriggl B, Klima G, Feuchtner GM, Kastlunger M, Jaschke WR. Radiology. 2013 Jun;267(3):837–42. ment of Patients with Metastatic Gastroin- Ultrasound-guided versus computed tomography-controlled peri- testinal Stromal Tumours. radicular injections in the middle and lower cervical spine: a prospective randomized clinical trial. Obernauer J, Galiano K, Gruber H, Bale R, Obwegeser AA, Schatzer R, Loizides A. Gastrointestinal stromal tumour (GIST) Eur Spine J. 2013 Nov;22(11):2532–7. is a rare disease and frequently affects Laparoscopic liver packing to protect surrounding organs dur- Fig. 7: The 2 US scans a (longitudinal and young patients and often results in a ing thermal ablation. Schullian P, Weiss H, Klaus A, Widmann G, Kranewitter C, Mittermair C, Margreiter R, Bale R. axial) show the sonographic situation at the short life expectancy of less than 3 years. Minim Invasive Ther Allied Technol. 2014 Oct;23(5):294–301. “leaving” of the AN from the plexus. The 2 Currently there is only one class of effective US scans b (longitudinal and axial) show medications for systemic GIST therapy and Selected Fundings the sonographic situation at the “middle often the tumours develop drug resistance • K-REGIO Projekt Cardiospect, Land Tirol, Wirtschaftsförderung, ao. Univ.-Prof. Dr. Michael Schocke segment” and the 2 US scans c (longitudinal after a few years. The MITIGATE consortium • MITIGATE #602306, EU FP7, o. Univ.-Prof. Dr. Werner Jaschke and axial) show the sonographic situation at representing three European universities, • RLS-Iron, Land Tirol, Translationales Research Programm, ao. Univ.-Prof. Dr. Michael Schocke the “distal segment”. The white arrowheads three research organisations and four SMEs • DISCHARGE #603266; EU FP7; ao. Univ.-Prof. Dr. Gudrun indicate the AN, the white arrow indicates will pursue the ultimate goal of developing Feuchtner • Einsatz genetischer Algorithmen zur Erstellung realer, farbko- the posterior fascicle of the plexus, (*) in- new protocols and guidelines to effectively dierter 3D-Modelle zur gleichzeitigen Darstellung von Hämody- dicates the axillary artery, (^) indicate the diagnose and treat patients with metastatic namik und Morphologie in zerebralen arterio-venösen Malfor- mationen; Autonome Provinz Bozen, PD Dr. Wolfgang Recheis cortex of the humeral head, (Δ) indicate the GIST resistant to current treatment. • Oralkarzinom WIF ‑273-01-00015/014-0034, Land Tirol, Trans- subscapular muscle, the rings (0) indicate lationales Research Programm, PD Dr. Wolfgang Recheis the teres minor muscle, (~) indicates the MITIGATE is co-funded by the European Collaborations long head of the triceps muscle and the 3 Community’s Seventh Framework Pro- • iSYS Medizintechnik GmbH bowed, white arrows indicate the 3 terminal gramme (FP7/2007–2013) under grant • Siemens Healthcare Österreich branches of the AN forming within the ax- agreement no 602306 and will run from • Ergospect GmbH illary gap. October 2013–2017 (Fig. 9). Core Facilites • Micro CT Selected Publications • Neuroimaging Research The association of chronic apical periodontitis and endodontic therapy with atherosclerosis. Petersen J, Glaßl EM, Nasseri P, Crismani A, Luger AK, Schoenherr E, Bertl K, Glodny B. Clin Oral Investig. 2014 Sep;18(7):1813–23. doi: 10.1007/ s00784-013-1156-3. Epub 2013 Dec 12. Dual-energy contrast-enhanced spectral mammography (CESM). Daniaux M1, De Zordo T, Santner W, Amort B, Koppelstätter F, Jaschke W, Dromain C, Oberaigner W, Hubalek M, Marth C. Arch Gynecol Obstet. 2015 Mar 27. [Epub ahead of print]

Incremental prognostic utility of coronary CT angiography for asymptomatic patients based upon extent and severity of coronary artery calcium: results from the COronary CT Angiography EvaluatioN For Clinical Outcomes InteRnational Multicenter (CONFIRM). Cho I, Chang HJ, Ó Hartaigh B, Shin S, Sung JM, Lin FY, Achenbach S, Heo R, Berman DS, Budoff MJ, Callister TQ, Al-Mallah MH, Cademartiri F, Chinnaiyan K, Chow BJ, Dunning AM, DeLago A, Villines TC, Hadamitzky M, Hausleiter J, Leipsic J, Shaw LJ, Kaufmann PA, Cury RC, Feuchtner G, Kim YJ, Maffei E, Raff G, Pontone G, Andreini D, Min JK. Study.Eur Heart J. 2015 Feb 21;36(8):501–8. doi: 10.1093/eu- rheartj/ehu358. Epub 2014 Sep 8. An intra-individual comparison of MRI, [18F]-PET and [18F]-FLT Fig. 8: Stereotactic Ablation of Liver Tumors. PET in patients with high-grade gliomas. Nowosielski M, DiFranco Fig. 9: Stromal Tumours.

Research Report 2015 Medical University of Innsbruck 161 Center of Diagnostic Radiology Neuroradiology

some projects arise in cooperation with (including multicenter studies, e.g. SITS the Department of Radiology, and yet oth- open, ACTS II). The senior physicians lead ers arise from research experiences in the the younger colleagues with interest in cerebral processing of pain with emphasis neuroradiology and the PhD students of on gender differences. Beside these more the Department in all aspects of clinical experimental projects, Neuroradiology also studies, and cooperate with clinical carries out clinical studies addressing neu- partners within the MUI (mainly Neurology, rovascular disease (Fig. 1), brain tumors Neurosurgery, Psychiatry, Child and Youth and brain development. The Department Psychiatry, Neuropediatrics, Radiology, but of Neuroradiology administrates and leads also Gynecology, ENT, Nuclear Medicine, the Core Facility Neuroimaging Research Cardiology, Neonatology, Radiation Therapy, (CF -NIR). Orthodontics and others).

General Facts The more experimental research is still developing. Up to now, Neuroradiology has Structure of the Research Unit, 2 “Laufbahnstellen” (Tenure Track positions) Aims and Clinical Routine: with Ass. Profs. who lead their own research The Department of Neuroradiology was groups: “Diffusion Tensor Imaging (DTI) of Director: established in 2012 and is therefore spine and nerves” and “Multimodal imaging Univ.-Prof.in Dr.in Elke R. Gizewski still under development. Together with with focus on MR-Spectroscopy”. Within the the Department of General Radiology, latter group, one PhD student began studies Contact: the Neuroradiology is involved in the in 2015 (ÖNB grant) with focus on 31P MR- Anichstraße 35 radiographer and physician educational Spectroscopy (MRS) in cerebral diseases; 6020 Innsbruck programs, but also in research and clinical MRS and multimodal imaging represent a routine activities. Furthermore, the two significant focus of the Department (Fig. 2). [email protected] departments have a long-standing PhD Phone: +43 512 504 27095 program, and they also started a clinical One research focus of the Head of the Fax: +43 512 504 27096 PhD program in 2013. department is the cerebral processing of http://radiologie.tirol-kliniken.at pain. She was co-PI of two DFG-funded Neuroradiology is a department with a large research projects dealing with visceral pain clinical workload, which includes diagnostic imaging: “Extinction learning” and “Placebo and interventional neuroradiology for all modulation of visceral pain processing”, neuro-cases both of pediatric and of adult and is now translating that experience to patients. One focus of research is clinically local research (Fig. 3, and project listed related imaging and intervention studies below). The extensive experience with high

Keywords

High-field MRI, MR-spectroscopy, fMRI, DTI, VBM, multimodal imaging, dual-energy-CT, interventional neuroradiology

Research Focus

Neuroradiological research is to a large extent connected to and driven by its clinical partners (Neuro-Focus at the MUI, e.g. neurodegenerative and neuroimmunolog- ical disorders, epilepsy, sleep medicine, degenerative spine diseases, brain tumors, neurovascular diseases, psychiatric diseases…). Besides those main partners, Neuro- radiology is also involved in the projects of many other departments (clinical and theoretical) at the MUI, MCI and LFU. Fig. 1: Endovascular therapy of cerebral aneurysms is more and more important in clinical Projects generated within the Department of routine. The first row shows a recent development in stents which is here evaluated in clin- Neuroradiology itself mostly focus on tech- ical studies nical developments (dose reduction/dual The second row shows an example of endovascular stroke therapy with thrombektomy us- energy CT, MRI-sequence developments, ing one of the recently developed stent retrievers (arrows). Here, the Neuroradiology (in fMRI/VBM, 1H and 31P MR-Spectroscopy); cooperation with Neurology) is part of a big mulicenter study.

162 Research Report 2015 Medical University of Innsbruck Neuroradiology

field MRI (3 T and 7 T (now in cooperation with the Erwin L Hahn Institute in Essen, with the Excellence Centre of High Field MRI at the Medical University Vienna, and with the Imaging Unit of the DKFZ in Heidelberg)), both structural and fMRI (multimodal) also represent a further focus on brain processing related to cognitive and emotional processes, particularly with respect to possible gender differences. The fMRI group has a second focus on psychiatric research (resilience, affective disorders, eating disorders), and also on critical emotional situations arising in cardiology.

The Department of Neuroradiology has also a considerable number of collaborations outside the MUI.

Core Facility Neuroimaging Research (CF ‑NIR) The main modality of this CF is the BMWF ‑funded 3 Tesla-MRI-system, which establishes a core facility for MR-based neuroimaging research at the MUI. The Fig. 2: Multimodal imaging in tumor patients. Multicystic WHO grade IV glioma on the right 3T MRI was installed in 2011 and started hemisphere with edema in T2 weighted images (A, arrow), contrast-enhancement (B, ar- work exclusively for research use in 2012. row), restricted diffusion (C, arrow) and increased perfusion (D, arrow) of the solid parts. The CF ‑NIR is centrally administered by the 31P MRS spectrum, displaying the high energy metabolites phosphocreatine (PCr, arrow) Head of the Department of Neuroradiology, and ATP (E), the arrow is pointing at the relevant voxel. who leads an interdisciplinary Steering Board. The technical equipment is support- Research high impact in clinical care and therapy. ed by one physicist (since 2014) and an There are several relevant pelvic pain assistant radiographer. The Team “Neuro This section lists only those research types, including primary and secondary radiology” provides support to all associated projects which are mainly led by dysmenorrhea. To date, there is no scientists in technical and post-processing Neuroradiology (NR). Further collaborations, study that addresses interoceptive pain questions. Furthermore, the core facility de- mainly those involving the CF ‑NIR, have thresholds, subjective perception of pain, velops and introduces new MR sequences undertaken many additional projects, and and cerebral processing of such stimuli and technical equipment, such as improved yet other projects work with many further in patients with dysmenorrhea. The rectal coils. Above all, the Neuroimaging plat- radiological techniques. barostat distension model, which is well form offers opportunities to bring different established in the Essen laboratory and groups together and to transfer knowledge, Attachment and Cerebral Processing has now been transferred to Innsbruck, and it provides a setting for communication led by Prof. Dr. Buchheim and is a clinically relevant, valid and reliable and cooperation. Prof. Dr. Gizewski, in cooperation with interoceptive pain model. This paradigm One important recent development with the Dr. Labisch and Prof. Viviani is now being used in a pilot study on CF ‑NIR is the Neuroimage WING, which is Attachment is a core function in healthy dysmenorrhea patients, with the first a grant (Hochschulraumstrukturmittel) sup human life, but it is vulnerable in patients results showing typical activation in “pain porting an imaging platform at the Medical with psychiatric diseases. Well known tools matrix” areas (Fig. 3). Universities Innsbruck, Graz and Vienna. such as the “Adult Attachment Projective Neuroimage WING (WienINnsbruckGraz) Picture System” are available; however, this Cerebral Processing of Food Stimuli in is led by MUI (Department of Neurology, imaging system is not optimal for use in Young Anorectic Patients in Respect to Univ.-Prof. Dr. Christoph Scherfler: “Com fMRI experiments. The goal of this study is Personality Disorders and Gender putational Neuroimaging”, in cooperation to evaluate a new imaging suite we created, led by Prof. Gizewski, Prof. Sevecke, in co with the Department of Neuroradiology) which is especially adapted for fMRI in operation with NR: Dr. Steiger, Child and and was set up to collect and analyse healthy volunteers in a 3T MRI setting. Youth Psychiatry: Dr. Fuchs data from different sites and from pooled Some earlier studies have revealed alter patient populations. This will lead to Cerebral Processes of Enteroceptive ations in cerebral processing in adult higher efficiency and synergies in research Pain in Patients with Dysmenorrhoea anorectic patients. However, since they projects and also to a know-how transfer. led by Prof. Gizewski; NR: Dr. Siedentopf, were based on longstanding disease, Multiple sclerosis, movement disorders Dr. Steiger in cooperation with Prof. Wildt, their results could not give clear answers and dementias were defined as the starting Dr. Böttcher, and Prof. Elsenbruch (Essen) on how those functional and structural projects. Pelvic pain is an important symptom having differences developed in contrast to healthy

Research Report 2015 Medical University of Innsbruck 163 Center of Diagnostic Radiology

volunteers. We have therefore established metabolism in preterm children, using MRS. established imaging methods (see above) the application of these stimuli to young Additionally, fMRI and psychological tests and with clinical data that are routinely col- patients and will correlate the measured will be applied to obtain data from grown-up lected in the Department of Neurology. brain parameters with psycho-social data. former preterm children. 31P MRS in Healthy Volunteers and Resilience: Neuroimaging of Gender 31P MRS in Cerebral Gliomas and Brain Trauma Patients Differences in Healthy Subjects Metastases led by Ass.-Prof. Grams; led by Prof. Hofer and Prof. Gizewski in led by Ass.-Prof. Grams; NR: Dr. Walchhofer, Dr. Steiger in cooper cooperation with NR: Dr. Siedentopf NR: Dr. Walchhofer and Dr. Steiger in ation Prof. Thomé, Dr. Petr, Resilience represents the capacity of coop. with Prof. Thomé, Dr. Kerschbaumer, and Dr. Pinggera some individuals to remain healthy or Dr. Freyschlag, Prof. Stockhammer and 31P MRS is being performed in patients recover easily from adverse events, Dr. Nowosielski, Prof. Nevinny-Stickel with severe traumatic brain injury during despite marked negative circumstances By using MR spectroscopy of phosphorus the acute, sub-acute and chronic stages. and risk factors, whereas others under compounds (31P MRS) it is possible to Trauma influence on energy metabolism comparable conditions are particularly detect various metabolites of energy and on reorganization processes will be vulnerable to disorders and illness. Few metabolism and of membrane turnover. investigated and the results correlated studies have examined the structural 31P MRS is being applied in patients with with established imaging parameters (see correlates of resilience, and they involved cerebral gliomas and metastases in order above) and with clinical parameters. mostly subjects under risk circumstances to investigate tumour heterogeneity and the or suffering post-traumatic stress disorder effects of therapy not only on the tumorous DTI of Spinal Cord (PTSD). The Neuroradiology is involved area but on the healthy brain hemisphere led by Ass.-Prof. Dr. Cartes-Zumelzu; in this study firstly by analyzing a cross- as well. The resulting data will be correlated Radiology: Dr. Kremser in cooperation sectional survey to investigate resilience in with results obtained from established with Prof. Thomé, Prof. Feuchner, healthy volunteers, with a primary focus on methods such as 1H MR spectroscopy, Prof. Granata, PD Dr. Broessner and potential gender differences, and secondly MR perfusion and MR diffusion-weighted Siemens Medical Imaging by addressing the cerebral representation imaging, as well as with clinical, histological DTI is well established for analysis of white of resilience in the same individuals, with and PET parameters. matter and brain structure. However, emphasis on gender specificities. this method might also be helpful in 31P MRS in Stroke Patients spinal imaging, especially in degenerative MRI and MRS Parameters in Cerebral led by Ass.-Prof. Grams; diseases. However, it offers many Development of Preterm Infants NR: Dr. Walchhofer and Dr. Steiger in challenges. The sequence used is influenced led by Dr. Djurdjevic in cooperation with cooperation with Prof. Willeit by many anatomical structures which cause Prof. Kiechl-Kohlendorf, Prof. Gizewski and Dr. Knoflach artefacts, and it also has limited resolution. and Prof. Buchheim 31P MRS is being applied in patients with This project successfully optimized the Up to now, some studies have revealed acute, subacute and chronic ischemic sequence and then started the study of the structural parameters in preterm children stroke to gain further insights into the en- first patients showing degenerative cervical that indicate an unfavourable clinical ergy metabolism and reorganization mecha- changes with narrowing of the spinal canal outcome (e.g. the Innsbruck NEOBRAIN nisms of infarcted brain and surrounding ar- (Fig. 4). study). These first results led to formulate eas during the acute stage, and to monitor further hypotheses and to develop a study subacute and chronic changes. The results Dual Energy CT in Stroke Patients addressing not only structure but also will be correlated with those obtained from led by Ass.-Prof. Grams; NR: Dr. Kurz in co operation Prof. Poewe, Ass.-Prof. Glodny, Prof. Willeit, Dr. Knoflach, and Prof. Ortler Dual energy computed tomography (DECT) can distinguish up to three different materials or tissues. Various projects are investigating the differentiation of blood- brain-barrier disruption, haemorrhage, thromboembolic material, and infarcted and healthy brain, and are correlating them with conventional CT.

Dual Energy CT for Artefact Reduction in Patients with Cranial and Spinal Implants led by Ass.-Prof. Grams; NR: Prof. Gizewski, Dr. Kurz, in coopera tion with Ass.-Prof. Glodny, Prof. Ortler, Prof. Crismani Fig. 3: Activation of insular and DLPFC in the contrast pain versus no-pain in patients with DECT also offers the opportunity to reduce dysmenorrhea. These areas are understood today as main parts of pain processing and beam-hardening artefacts from metal areas involved in altered signalling in patients with chronic pain syndromes. implants by extrapolation of monochromatic

164 Research Report 2015 Medical University of Innsbruck Neuroradiology

Fig. 4: A shows an optimized DTI sequence from a healthy volunteer. The anisotropy can be calculated without significant artefacts and the tracts can be visualized in both sagittal and transverse orientation with high resolution. 4B shows two patients with degenerative spine disease and visualizes tract disturbances.

(MC) series. This method is being applied in Chemelli-Steingruber I. Eur J Radiol. 2013 Nov;82(11):1989–95. patients with cerebral clips, dentogenic and Correlation of dopaminergic terminal dysfunction and microstruc- tural abnormalities of the basal ganglia and the olfactory tract spinal implants. The aim of these studies in Parkinson’s disease. Scherfler ,C Esterhammer R, Nocker M, is to evaluate the presence of artefacts Mahlknecht P, Stockner H, Warwitz B, Spielberger S, Pinter B, Donnemiller E, Decristoforo C, Virgolini I, Schocke M, Poewe W, and to assess the surrounding tissue, in Seppi K. BRAIN. 2013; 136(S): 3028–3037. comparison to conventional computed Experimental human endotoxemia enhances brain activity dur- tomography. ing social cognition. Kullmann JS, Grigoleit JS, Wolf OT, Engler H, Oberbeck R, Elsenbruch S, Forsting M, Schedlowski M, Gizewski ER. Methods of Quantifying Supra-Aortal SOC COGN AFFECT NEUROSCI. 2014; 9(6): 786–793. and Intracranial Artery Calcifications Placebo analgesia in patients with functional and organic abdominal pain: a fMRI study in IBS, UC and healthy volunteers. Schmid J, led by Ass.-Prof. Grams; Langhorst J, Gaß F, Theysohn N, Benson S, Engler H, Gizewski ER, NR: Dr. Steinkohl, in cooperation Ass .-Prof. Forsting M, Elsenbruch S. GUT. 2015; 64(3): 418–427. PD Glodny, PD Dr. Beer, PD Dr. Helbok, Residual thrombembolic material in cerebral arteries after endovascular stroke therapy can be identified by dual-energy com- Prof. Ortler and Dr. Julia Kerschbaum puted tomography. Astrid E. Grams, Michael Knoflach, Rafael A method developed in our department to Rehwald, Johann Willeit, Martin Sojer, Elke R Gizewski, Bernhard quantify aortal calcification is being applied Glodny. AJNR. e-pub May 2015. to examine the supra-aortal and intracranial Selected Funding arteries. The amount of calcification will be • Characterization of brain metabolism in ischemic stroke with correlated with the incidence of intracranial MR spectroscopy of phosphorous compounds, ÖNB, Dr. Grams • As Co-investigator: Myocardial MRS correlates of cardiac sym- aneurysms or cerebral vasospasm after a pathetic Denervation in PD, FWF, Prof. Wenning and: Clinical subarachnoid haemorrhage. Neuroimaging / Neuroimage WING, BMWFS, Prof. Scherfler Collaborations Selected Publications • Austria: NEUROIMAGE WING (BMWFW grand: pooled MRI data Variability of clinical functional MR imaging results: a multi- collection and analysis Med. Universities Innsbruck, Graz, Wien center study. Wurnig MC, Rath J, Klinger N, Höllinger I, Geissler (Neurology and Neuroradiology, 7T MRI), MCI Innsbruck A, Fischmeister FP, Aichhorn M, Foki T, Kronbichler M, Nickel J, • Germany: University Hospital Essen (Medical Psychology & Siedentopf C, Staffen W, Verius M, Golaszewski S, Koppelstätter Behavioural Immunobiology, Forensic Psychiatry, Neurology, F, Knosp E, Auff E, Felber S, Seitz RJ, Beisteiner R. RADIOLOGY. Neurosurgery, Radiology), Erwin L. Hahn Institut Essen, DKFZ 2013; 268(2): 521–531. Heidelberg/MR-Imaging, Justus-Liebig University Giessen Cerebellar dysfunction in a family harboring the PSEN1 muta- (Neurology, Neuroradiology, Neuropediatrics), University Mar- tion co-segregating with a Cathepsin D variant p.A58V. Ehling burg (Neurology), LMU Munich and Technical University Dres- R, Noskova L, Stranecky V, Hartmannova H, Pristoupilova A, den (Neuroradiology), University Hamburg (Neuroradiology), Hodanova K, Benke T, Kovacs GG, Stroebel, T, Niedermueller U, Goethe University Frankfurt (Neuroradiology), University Tübin- Wagner M, Nachbauer W, Janecke A, Budka H, Boesch S, Kmoch gen (Psychosomatic Medicine) S. NEUROL SCIENC. 2013; 326(1–2): 75–82. • Switzerland: Hirslanden Clinic Zürich, Neuroradiology Enhancement patterns in the fibro cellular tissue in different kinds of plaques of the internal carotid artery. Rantner B, Core Facilities Sojer M, Kremser C, Cartes-Zumelzu F, Fraedrich G, Jaschke W, Neuroimaging Research Core Facility (3T MRI)

Research Report 2015 Medical University of Innsbruck 165 Center of Dentistry and CMF-Surgery Dental Prosthetics and Restorative Dentistry

dentition to biologic health, function, aes- Our data show that the main reason for thetics and comfort. This is accomplished failure is predominantly due to ceramic by using operative, periodontal dentistry fracture. Nonvital teeth significantly show techniques, and prosthodontics, integrated higher risk of failure (p<.0001). There is biomaterials and occlusion. 80 % of the cur- a 2.3-times greater risk of failure associ- riculum is provided by staff members of our ated with existing parafunction (bruxism, department, delivering information through p=.0045). lectures and clinical oversight of patient care 40 hours a week. We also serve a lead- Major Achievements: ing position in the preparation and analysis All-ceramic restorations offer a predictable of the MEDAT-Z test for all public dental and successful restoration with an estimat- schools in Austria. Regarding the high com- ed survival probability of 93.5 % over 10 petence in educational dentistry, our staff years and 78.5 % at 20 years. Significantly members are routinely consulted as exter- increased failure rates are associated with nal experts in international accreditations bruxism and nonvital teeth. of dental curricula. Future Goals: The department has access to well- Clinical trials on long term survival and Director: equipped research laboratories for dental effects of ceramic materials in implant Univ.-Prof.in DDr.in Ingrid Grunert materials. These laboratories are routinely prosthodontics used by the faculty and the under-graduate Contact: students. Although the department has Dental Material Sciences Anichstraße 35 been involved in many areas of prosthetic René Steiner, Herbert Dumfahrt 6020 Innsbruck and restorative dental materials research, Among the clinical researches, the in vitro they have been especially recognized for evaluation of dental materials represents [email protected] their leadership in the area of ceramics over the main research focus of our faculty. Phone: +43 512 504 27159 the past twenty years. Early involvement Series of experiments are performed on Fax: +43 512 504 27157 with these materials and techniques has the physical material properties like sur- www.zmk-innsbruck.at not only been beneficial in clinical experi- face roughness, adhesion stability, abrasion ence and research studies, but also in many resistance and thermostability. The present clinical publications. in vitro evaluation is focused on the ability Keywords of various ceramic polishing kits to mimic Research glazed dental ceramic surface. Clinically, all-ceramics, silicate-ceramic, CAD/CAM, chairside adjustments of ceramic resto- dental implant, dental materials, dentures, Longevity of Ceramic Restorations rations involve roughening of the ceramic periodontal diseases, rare diseases, Ulrike Beier, Herbert Dumfahrt surface by diamond polishing rotary instru- Silicate ceramic restorations are widely ments and subsequent layered to restore Research Focus used for veneers, inlays, onlays and crowns surface smoothness. The resulting surface in dentistry (Fig. 1a, 1b). Long-term data roughness has been shown to decrease Traditionally, the research of the depart- are of crucial importance to optimize clini- flexural strength, which may compromise ment addresses issues on dental material cal practice. Our present focus lies on the the long-term prognosis of the restoration. sciences, with a special focus on clinical evaluation of clinical quality, success rate and in vitro analyses of dental ceramics, as and estimated survival rate of silicate glass- Rough surfaces may have an abrasive effect well as dentures. ceramic restorations over a 20-year period. on antagonistic and adjacent teeth and lead The department further focuses on rare dis- Aesthetic match, porcelain surface, margin- to an increased adhesion of bacteria. Pol- eases with oral phenotypes, and especially al discoloration and integrity are evaluated ishing ceramic restorations may be done by on genetical and molecular basis of aggres- following modified California Dental Asso- using polishing kits, disks, or diamond pol- sive periodontal diseases. ciation/Ryge criteria during clinical exami- ishing paste materials. On the basis of the nation. Number of restoration failures and findings, none of the commercially available General Facts reasons for failures are recorded. ceramic polishing kits could create an ini-

The Department of Prosthodontics and Operative Dentistry is part of the Depart ment for Dental, Oral and Maxillo-facial Medicine. The Department of Prosthodontic and Operative Dentistry provides an impor tant role in the University School of Dental Medicine by establishing foundational concepts of dentistry to the pre-doctoral dental students. Our primary responsibility lies in the education and training of pre-doctoral students in restorative aspects of damaged Fig. 1: ceramic restoration of an abraded dentition (A) before, and (B) after treatment

166 Research Report 2015 Medical University of Innsbruck Dental Prosthodontics and Restorative Dentistry

tially smoother surface than glazed ceram- features for success in implantology. The sinterpreted as aggressive periodontitis. ic. The addition of a polishing step with di- aim of our present studies is the radio- 2. (2) Kohlschütter-Tönz syndrome is a rare amond polishing paste is recommended to graphical evaluation of marginal bone level genetic disorder with epilepsy, psych- achieve a significant improvement in ceram- changes around different titanium implant omotor regression, and severe enamel ic surface smoothness. The cost of ceramic systems after several years in function. defects (Fig. 2). polishing kits should not be considered as Machined-surface implants and rough- 3. (3) In a turkish family with consanguin- an indicator of performance. surface implants were early loaded with ous parents, heterotaxy-syndrome with individual bar retained overdentures. Our aberrant tooth shapes and root resorp- CAD-CAM-Fabricated Dentures data show that there is a significant differ- tions is inherited in an autosomal-reces- Patricia Steinmaßl, Ingrid Grunert ence (p<0.003) between the two implant sive mode. In all these families, we per- CAD/CAM manufacturing of dentures pre- systems at the baseline-measurements form genetical and medical analyses and sents an innovative option for fabricating (0.36mm machined vs. 0.71mm rough-sur- characterize the dental defects clinically dentures with a reduced amount of dentist face) and a highly significant difference for and histologically. visits and promises to provide dentures the annual bone loss (0.041mm machined with better surface properties, which could and 0.12mm rough-surface p<0.001)). promote denture hygiene. Denture hygiene is crucial for maintenance not only of oral, Both of the implant systems are clinically but also of general health, as microorgan- satisfying. Nevertheless, the machined- isms colonising the denture surfaces have surface-group shows a better radiological been shown to be correlated with severe performance than the rough-surface one. complications such as pneumonia, a common cause of death among elderly people. Future Goals: An appropriate denture fit is important for • Clinical trials with different implant sys- Fig. 2: Kohlschütter-Tönz syndrome is a rare denture retention. Only well stabilised den- tems in single tooth gaps genetic disorder with epilepsy, psychomo- tures enable good nutritional intake and • Clinical trials on patient satisfaction tor regression, and a severe enamel defect speech, and both these factors are very im- with yellow or brownish discoloration of the portant for the patient’s comfort and quality Hereditary Dental and Periodontal teeth. of life. Until now, hardly any preclinical and Disorders no clinical data are available on this topic. Ines Kapferer-Seebacher Selected Publications Periodontitis is an inflammatory disease Adjusting dental ceramics: An in vitro evaluation of the abil- To evaluate the material-scientific properties, linked to complex polymicrobial infection. ity of various ceramic polishing kits to mimic glazed dental ceramic surface. Steiner René, Beier Ulrike S, Heiss-Kisielewsky we analyse conventionally manufactured as In susceptible individuals it leads to perio- Irene, Engelmeier Robert, Dumfahrt Herbert, Dhima Mathilda. well as CAD/CAM manufactured dentures, dontal tissue destruction by the perturbing JOURNAL OF PROSTHETIC DENTISTRY. 2015; doi: 10.1016/j. prosdent.2014.12.007. [Epub ahead of print] comparing them in vitro with regard to the homeostasis between the subgingival A 7-year prospective radiographic evaluation of marginal bone surface roughness and porosity, measuring microbiota and the host defenses. Perio- level around two different implant systems: a randomized clinical them both initially, and then again after dontal research of our department com- trial. Burtscher Doris, Norer Burghard, Dalla Torre Daniel, Beier Ulrike S, Schubert K, Grunert Ingrid. CLINICAL ORAL IMPLANTS thermocycling and brushing them; we also bines genetical, molecular and microbio- RESEARCH. 2014; doi: 10.1111/clr.12444. [Epub ahead of print] measure the release of PMMA-monomer logical analyses of aggressive periodontitis. Longevity of silicate ceramic restorations. Beier Ulrike S, Dumfahrt from them. To estimate patient comfort, we In collaboration with the Division of Human Herbert. QUINTESSENCE INTERNATIONAL. 2014;45(8):637–644. measure denture fit, weight and thickness, Genetics, a Tyrolian 4-generation family with Randomized controlled trial: lip piercing: the impact of material on microbiological findings. Kapferer Ines, Beier Ulrike as well as stress resistance. A clinical study Ehlers-Danlos syndrome type VIII (EDS VIII) S, Jank Siegfried, Persson Rutger. PEDIATRIC DENTISTRY. assessing patient comfort, practicability is under investigation. EDS VIII is a clinically 2013;35(1):E23–28. and cost-effectiveness is planned. heterogeneous disorder associated primar- Instant dentin hypersensitivity relief of a single topical application of an in-office desensitizing paste containing 8% arginine ily with aggressive periodontal disease, and and calcium carbonate: a split-mouth, randomized-controlled The study design has been awarded the variable connective tissue features. Only a study. Kapferer Ines, Pflug Claudia, Kisielewsky Irene, Giesinger “ODV-Wissenschaftspreis des ZIV 2015” few patients and pedigrees with this condi- Johannes, Beier Ulrike S, Dumfahrt Herbert. ACTA ODONTOLOGI- CA SCANDINAVICA. 2013;71(3–4):994–999. and is currently under review for funding tion have been described. The members of by the Tiroler Wissenschaftsfonds and MUI the Tyrolian family have been characterized National and Selected Funding start. clinically and immunologically, and linkage • Genetic and Functional Studies to Identify the Molecular Basis of Aggressive Periodontitis, Jubilee Fund of the Austrian Natio- analysis and exome sequencing have been nal Bank, Kapferer-Seebacher Ines Implantology performed. At present a candidate gene in Doris Burtscher, Ingrid Grunert the chromosomal region 12p13 is under Collaborations Not only partially but also fully edentulous functional investigation. • Bernhard Gottlieb University Clinic of Dentistry, Vienna, Austria • Division of Prosthodontics, Restorative Dentistry, Periodontolo- patients benefit from the successful osseo gy and Implantology, Medical University Graz, Austria integration of dental implants, which are a Furthermore, several families with rare • International master for craniomandibular dysfunctions and musculoskeletal medicine, Fortbildungsinstitut ZÄT-INFO major advance in clinical dental treatment. diseases and oral phenotypes are currently • Mayo Clinic, Minnesota, USA Nowadays dental implants are available in also under investigation. For example: different materials, such as titanium-based 1. Radicular dentine dysplasia is a rare in- alloys or zirconia, each with various implant herited disorder characterized by abnor- surface conditions. In all cases, mainte- mal dentine formation, which results in nance of osseointegration and of a steady abnormal tooth roots and tooth loss at state in marginal bone level are imperative an early age; therefore, it is often mis-

Research Report 2015 Medical University of Innsbruck 167 Center of Dentistry and CMF-Surgery Orthodontics

Keywords

3D low budget printer, malocclusion, angle class II, BioBiteCorrector®, miniscrew

Research Focus

Orthodontic mini-implants, 3D low budget printer, tooth surface texture after adhesive removal, class II appliance Fig. 1: Model printed by Makerbot Replica- General Facts tor 2

Orthodontics is a special field of dentistry that focuses on the prevention and correction of malocclusion of teeth and jaws. Orthodontics is performed using removable and fixed appliances. Director: Univ.-Prof. Dr. Adriano Crismani Research

Contact: 3-D Low Budget Printer Anichstraße 35, MZA The aim of the study was to evaluate the 6020 Innsbruck benefits and drawbacks of 3D dentalmo del printing compared to standard dental Fig. 2: Model printed by Formlabs Form 1 [email protected] plaster casts. Two different 3D low budget Phone: +43 512 504 27198 printers were tested in the study. The Fax: +43 512 504 27199 advantages of 3D-printed casts compared The Application of the BioBiteCorrector® www.zmk-innsbruck.at to conventionally made plaster models are: in Orthodontics: Skeletal vs. lower weight and easier transport, high Dentoalveolar Changes resistance to abrasion, low risk of fracture, The BioBiteCorrector® (BBC) is an ease of access: reprinting models at any orthodontic device that is fixed on the time, no need for storage space. archwires of the multi-bracket appliance to treat class II malocclusions with no need 12 plaster casts were scanned with of compliance. It is composed of two triple the structured light scanner S600 ARTI telescopes, whereas the protrusion of the (Zirkonzahn GmbH, Gais, Italy). The lower jaw depends on the length of these data obtained were used to recreate telescopes. corresponding models with two low budget 3D-printers: Formlabs Form 1 (Formlabs Inc. The aim of this study was to determine Somerville, USA) and Makerbot Replicator 2 whether the effects of the BioBiteCorrector® (MakerBot® Industries, Brooklyn, USA). are also skeletal or have to be attributed to a dentoalveolar compensation. Formlabs Form 1 generates models with the stereolithography (SLA) technique, shaping For the purpose of a pilot study, lateral build part’s layer using an ultraviolet laser cephalometric radiographs of 10 patients and light-curing resin. The Makerbot were analysed. The first radiograph asw Replicator 2 prints models with the fused taken on the day that the BBC was fixed (T1), filament fabrication (FFF) technology, the second one was taken after the removal extruding thermoplastics from a nozzle, of the whole multi-bracket appliance (T2). layer by layer. The printed copies were then The patients had an average age of 14.9 rescanned with the structured light scanner. years and wore the BBC 6.5 months on average. The scans of plaster casts and corresponding printing copies were finally digitally The results show a significant change in compared using the GOMInspect software the position of the pogonion as well as the (GOM-Gesellschaft für Optische Messtech- lower incisal edge. nik mbH, Braunschweig, Germany) and the data were evaluated. The sagittal length of the mandible also increased significantly, which is indicative 3D-dental models can be a reliable alterna- of the skeletal impact of the treatment, in tive to conventional casts. addition to the dent alveolar effect.

168 Research Report 2015 Medical University of Innsbruck Orthodontics

and low levels of cortical bone formation at Selected Publications the head of the miniscrew may be a reason How Precise is the Production of Models by Low Budget 3D Printers? A Pilot Study. B Paal, M Bert, J Gröger, W Recheis, AG for higher failure rates. Bone morphogenetic Crismani. Inf Orthod Kieferorthopädie. 2014; 46;261–266. protein-2 (BMP-2) functionalized implant Is the Temperature Development During Debonding Identical for surfaces have previously been explored all Rotating Dental Instruments?. R Biedermann, K Winter, AG to enhance overall osseointegration and Crismani. Inf Orthod Kieferorthopädie. 2014; 46; 235–240. The Application of the BioBiteCorrector® in Orthodontics: Skel- induce cortex-bearing bone formation at ettal vs. Dentoalveolar Changes. J Schmid, E Pasin, T Magg, AC supraalveolar peri-implant defects. The aim Crismani. Inf Orthod Kieferorthopädie. 2014; 46;267–270. of this study was to investigate the effect Fig. 3: BioBiteCorrector® of a BMP-2 functionalized implant surface Collaborations • Wolfgang Recheis,Department of Radiology, Medical University on the stability and cortical-level bone-to- Innsbruck, Austria implant contact ratio (BICR) of orthodontic • Michael Rasse, Department of Oral and Maxillofacial Surgery, Medical University Innsbruck, Austria miniscrews. • Volker Kuhn, Department of Traumatology, Medical University 36 miniscrews (length: 6 mm; diameter: Innsbruck, Austria • Michael Bertl, Department of Orthodontics, Medical University 1.5 mm) were placed in 3 swines. Half Vienna, Austria of the miniscrews were coated with nano-crystalline diamond (NCD) and functionalized with BMP-2. Upon insertion, stability was evaluated by measuring resonance frequency (RF) with an Osstell ISQ wireless probe. An animal was sacrificed after 2, 4 and 12 weeks and resonance frequency analysis (RFA) was performed again. Recovered miniscrews and surrounding bone were scanned by micro-CT before sectioning for bright field microscopy. BICR was then evaluated in 2D on histological samples and in 3D on micro-CT scans at both the cortical (head/ neck) and trabecular (thread) level of the Fig. 4: cephalometric before inserting Bio- miniscrews. Stability, as expressed by BiteCorrector® an increase in RFA results, significantly increased for BMP-2 functionalized miniscrews after 4 and 12 weeks.

The neck/tread ratio of the 2D and 3D BICR showed significantly higher cortical level bone-to-implant contact of BMP-2 functionalized miniscrews at those time-points. There was a positive correlation between higher RFA results and cortical BICR. These results suggest an increase in stability of BMP-2 functionalized miniscrews as a result of higher BICR at the cortical bone level.

Fig. 5: cephalometric after inserting BioBite- Corrector®

BMP-2 Functionalization of Orthodontic Miniscrews Increases Stability and Cor- tical Level Bone-to-Implant Dontact: a Histomorphometric and Micro-CT Evaluation Sufficient cortical bone thickness has been shown to critically effect miniscrew stability Fig. 6: Micro CT of miniscrew

Research Report 2015 Medical University of Innsbruck 169 Center of Dentistry and CMF-Surgery Cranio-Maxillo-Facial and Oral Surgery

Keywords General Facts

Cranio-maxillofacial and oral surgery, im- The consequences of Radiation Therapy in plants, biological surfaces, osseointegra- Head and Neck Tumour Patients is a major tion, wound healing, tissue engineering, focus of the Department of Cranio-Maxillo- reconstructive medicine, nano technology facial and Oral Surgery since all aspects of wound healing including cellular behaviour, Research Focus blood flow and stem cell activation are influenced by radiation. • Reversing impaired healing of irradiat- Using smart implants as a new sensing ed bone through the use of immobilized technology allows us to gain an insight into growth factors on nanostructured osteo- bone healing based on serial analysis of synthetic Material impedance spectroscopic examinations be- • Smart Implants – Monitoring of osseous fore radiographic or histologic changes are healing and bone remodelling in vivo. detectable. • VascuBone – Development of a tool box for tailor-made angio-inductive or Vascu- Reconstructive Medicine: larized Bone implants Reconstructive facial surgery is one focus Director: Univ.-Prof. DDr. Michael Rasse

Contact: Anichstraße 35 6020 Innsbruck

[email protected] Phone: +43 512 504 24373 Fax: +43 512 504 24371 www.zmk-innsbruck.at

Fig. 1: Histological evaluation of the bone implant contact ratio (BICR) at the osteosynthesis screws. (A) Histological overview of the locking screw in bone subjected to toluidin blue O staining. (B) Detailed view of the osteosynthesis screw highlighting the bone contact areas (arrows). (C) BICR after 1, 2, 4, and 8 weeks of the 3 groups. BMP-2 immobilized on nano-crystalline diamond (green) resulted in an initial increase of BICR in the irradiated bone. Despite this initial increase the BICR after 8 weeks was lower compared with unirradiated bone (p ¼ .08) (blue).

170 Research Report 2015 Medical University of Innsbruck Cranio-Maxillo-Facial and Oral Surgery

of the Department of Cranio-Maxillofacial of 6 weeks and spectroscopic analysis of and Oral Surgery. After ablative tumour the sensor signals were archived for later surgery or resection of osteonecrotic and analyses. After 6 weeks the bone defects infected bone, free tissue transplants are together with the biosensor was explanted microvascularly anastomosed for facial re- and examined by means of micro-CT and habilitation. Research is focused on the de- histology. Serial analysis of the impedance velopment of minimally invasive or artificial spectroscopic examinations was performed transplants. by firstly fitting the data gathered during defect healing to a Cole-Cole equation. To achieve this goal the Department is part Thereafter the results were compiled, of the FP-7 framework project “Vascubone” and the spectra thus obtained revealed together with a further 14 partners. The de- gross changes in material densities during partment is a leader in the field of preclini- healing. Terminal analyses, by means of cal trials, provides the technology for hard micro-CT as well as histology, demonstrated tissue histology and immohistochemistry incomplete yet ongoing osseous healing as and plans and conducts animal trials. the defect was filled with both connective tissue as well as with newly formed R. Gassner, C. Zsifkovits, F. Kloss , R. Stigler, bone. Furthermore, no obvious signs of V. Offermanns, J. Laimer, M. Rasse inflammation were observed indicating that Department of Cranio-Maxillofacial and Fig. 2: Implantation of the biosensor and ra- the implanted biosensor is biocompatible. Oral Surgery, Medical University of Inns- diological control. (A, B) Creation of a full Our results on dielectric spectroscopy bruck, Innsbruck, Austria thickness calvarial defect; (C) Fixed bio- may serve as a potential method for close sensor; (D) Postoperative impedance meas- continuous monitoring of bone wound J. Steinkuehler urement; (E) Radiological control of sensor healing in craniomaxillofacial and oral Max Planck Institute for Colloids and Inter position in two planes. surgery in the future (Fig. 2). faces, TU Berlin, Berlin, Germany overcome shortcomings of bone healing 3. VascuBone M. Ertl, P. Ertl and osseointegration we used bioactive The FP7 Project Vascubone deals with the Department of Health and Environment, Na- BMP‑2 on nano-crystalline DIAMOND (NCD) development of an artificial vascularized nosystems, Austrian Institute of Technology, coated implants based on nanotechnology bone transplant for the reconstruction Vienna, Austria and physisorption which is an approach of large facial defects. This is achieved that has been patented (Steinmueller- through the use of a construct consisting of W. Kapferer, G. Lepperdinger Nethl D, Inventors: Steinmueller-Nethl D, a vascular bed, modified bone replacement Department of Biomedical Aging Research, Steinmueller D; Bonn G, Huck C, Najam- material and mesenchymal progenitor University of Innsbruck, Innsbruck, Austria Ul-Haq M, Rainer M, Stecher G; Kloss F, cells. The vascular bed is engineered by Gassner R. Biological Surfaces. Patent decellularicing a porcine gut segment with Doris Steinmüller Number: EP1824528 (Fig. 1). its supplying vascular bed and reseeding the vessels with endothelial progenitor cells Research 2. Smart Implants: Sensors as Technol- from the future recipient. ogy Asset 1. Bioactive Surfaces in Cranio-Maxillo- Radiology remains the gold standard to show Several animal experiments were Facial and Oral Surgery bone wound healing and osseointegration performed in order to improve the common Implants have revolutionized patient care in of implants despite the fact that it provides bone replacement material beta-tricalcium- all fields of medicine and dentistry. Titani- only a snap shot of the dynamic bone growth um has evolved as the leading raw material and regeneration process. Even histology when treatment of bone and cartilage dis- provides only a glimpse of the activity or eases/degeneration as well as tooth loss inactivity of osteoblasts, osteoclasts and necessitates osseointegration of individual- osteocytes in bone. ized tissue replacement options. The goal of this research was to test a This is mainly due to its bioinert properties. novel sensing technology that is based on Titanium implants in particular provide bioimpedance and which in due course may excellent results in healthy young and allow the monitoring of the osseous healing adult patients. But the success rate of any processes in an uninterrupted manner. A implant is hampered if the implant site sub-critical size defect with full thickness suffers from poor bone wound healing. Due was created in a rabbit calvaria that was to conditions affecting bone turnover and sufficiently large to accomodate the Fig. 3: Picture shows the mandibular con- homeostasis such as osteoporosis, age, biosensor which was mounted on a titanium tinuity defect bridged with reconstruction radiation therapy, bisphosphonate intake, mesh and inserted into the defect. The plates and the microvascular anastomo- infection, severe trauma or other pathology- mesh was then fixed to the adjacent bone sed artificial transplant. The magnification related bone changes, osseous healing at with micro-screws. Measurements were shows the circulation of the transplant after the implant site is frequently limited. To performed every 3 or 4 days during a period anastomosis (DDr. Stigler).

Research Report 2015 Medical University of Innsbruck 171 Center of Dentistry and CMF-Surgery

phosphate. The functionality of the beta TCP surface with nanocrystalline diamonds was tested in animal models. The diamond particles by themselves modified the wettability and cell adhesion properties of the surface and were further shown to bind covalently or via physisorption different growth factors such as BMP-2 and Angiopoietin-1. The new knowledge thus acquired on functionalized biomaterials and cell behaviour led to the application of such an artificial transplant for reconstructing a Fig. 4: Schematic illustration and histology (toluidine blue staining) of merged slides with mandibular continuity defect in sheep. The representatives of each group. The 1500 x 1000 mm2 box marked scheme (a, left) indicates Horizon2020 Proposal “VascuReGenTis” the standard area used to evaluate new bone formation. The area of new bone formation in- has been submitted in order to test this side this box was measured for all samples and used to calculate the percentage of de novo technique in clinical trials (Fig. 3). bone synthesis with respect to the total reference area. The side of the reference box facing the implant surface was used to evaluate the percentage of direct bone-to-implant contact 4. Bone Regenerating Effect of Stron- with respect to the total length of the reference. Minimal bone formation observed in the tium Functionalized Implant Surfaces Grade 4 Ti (a, right). 6.7 at.% Ti-Sr-O with a 60 minute pre-wash (b, left) and no pre-wash (b The functionalization of implant surfaces right) showed increased bone formation. A higher amount of bone apposition could be ob- has gained increased attention in the served for the 8.9 at.% Ti-Sr-O with a 60 minute pre-wash (c, left) and a 20 minute pre-wash last decade due to research into implant (c, right). The implant bodies are not shown to scale as these had a diameter of 1100 μm. dentistry. A lot of different approaches Scale bar is 1000 μm. Data and references cited in Offermanns et al. 2014. directed towards enhanced bone healing have been investigated over the past couple of years and they always attempt to Selected Publications Medical University of Innsbruck / Robert Stigler (Frank Kloss), Robert Gassner, Michael Rasse achieve rapid osseointegration of titanium Consortium with 15 participating institutions / universities implants. Since strontium (Sr) is known for BMP -2 immobilized on nanocrystalline diamond-coated titanium screws; demonstration of osteoinductive properties in irradiated “Strontium functionalized titanium implants”, Danish National Ad- vanced Technology Foundation, Frank Kloss/Vincent Offermanns its anabolic and anti-catabolic effects on bone. Kloss F, Singh A, Hächl O, Rentenberger J, Auberger T, Kraft bone, research has been focused on this A, Klima G, Mitterlechner T, Steinmüller-Nethl D, Lethaus B, Rasse M, Lepperdinger G, Gassner R (2013). Collaborations alkaline earth metal and its potential impact Head Neck 35: 235–241, 2013. (Journal of the Sciences and • Interdisciplinary Nanoscience Center (iNANO), Aarhus Univer- on osseointegration. Specialties of the) Head and Neck. doi: 10.1002/hed.22958. sity, Denmark • Danish Technological Institute (DTI), Aarhus, Denmark Bone marrow T cells from the femur are similar to iliac crest • Danish National Advanced Technology Foundation, Copen The objective of our studies was to derived cells in old age and represent a useful tool for studying hagen, Denmark the aged immune system. Pritz Theresa, Landgraf-Rauf Katja, • Elos Medtech Pinol A/S, Gørløse, Denmark investigate the performance of Ti implants Herndler-Brandstetter D, Rauf R, Lair J, Gassner R, Weinberger • Department of Engineering Sciences, Applied Materials Sci- with a Sr functionalized titanium coating Birgit, Krismer M, Grubeck-Loebenstein Beatrix (2013). ence, University of Uppsala, Sweden Immunity & Ageing. 2013 10:17 doi:10.1186/1742-4933-10-17. (Ti-Sr-O), which exhibits a continuous release of strontium, with respect to Systemic impact molds mesenchymal stromal/stem cell aging. Reitinger S, Schimke M, Klepsch S, de Sneeuw S, Yani SL, Gassner osseointegration.The examined Ti-Sr-O R, Ertl P, Lepperdinger G (2015, April 8). coatings, prepared from a magnetron co- Transfus Apher Sci / pii: S1473-0502(15)00072-5. doi:10.1016/j. transci.2015.04.008. sputtering process, differed from each other in coating thickness, Sr content and Accelerated bone ingrowth by local delivery of strontium from surface functionalized titanium implants. Andersen OZ, Offermanns Sr release characteristics and the observed V, Sillassen M, Almtoft KP, Andersen IH, Sorensen S, Jeppesen CS, increase in new bone formation was found Kraft DC, Bøttiger J, Rasse M, Kloss F, Foss M. Biomaterials. 2013;34:5883-90. to correlate with the amount of Sr released in vitro. The results indicate that sputtered Enhanced osseointegration of endosseous implants by predictable sustained release properties of strontium. Offermanns Ti-Sr-O coatings, showing sustained release V, Andersen OZ, Falkensammer G, Andersen IH, Almtoft KP, of Sr, accelerate osseointegration in healthy Sorensen S, Sillassen M, Jeppesen CS, Rasse M, Foss M, Kloss F. and osteoporotic bone plus in comparison J Biomed Mater Res B Appl Biomater. 2014;25:33279. to established surfaces and may thus have Selected Funding an impact on practical applications for Jubilee Fund of the Austrian National Bank (# 12246) and Synthes, medical implants. Salzburg, Austria (titanium reconstruction plates and screws): Title: Reversing impaired healing of irradiated bone by immobilized growth factors on nanostructured osteosynthesis material Major Achievements Include: The produc- Function: Principal Investigator: Robert Gassner (70 %); Co-Inves- tion of implant surfaces with predictable Sr tigator: Frank Kloss und Günter Lepperdinger (30 %) Tyrolean Future Fund – Translational Research Project release properties; Verification of beneficial Title: ‘Smart Implants – Monitoring of osseous healing and bone effects of Sr functionalized surfaces in vivo remodelling in vivo’. Function: Principal Investigator: Günter Lepperdinger / Bio- medical Aging Research , University of Innsbruck (33 %), Robert Future Goals: The evaluation of mechanical Gassner / CMF Surgery Innsbruck (33 %) und Peter Ertl / Austrian anchorage via push-out tests; Implementa- Research Center Seibersdorf (33 %) EU project FP7-HEALTH project: VascuBone – Development of tion of Sr functionalized implant surfaces in a tool box for tailor-made angio-inductive or Vascularized Bone orthopedic and dental implantology implants

172 Research Report 2015 Medical University of Innsbruck Cranio-Maxillo-Facial and Oral Surgery

Research Report 2015 Medical University of Innsbruck 173 Centre of Pediatrics Pediatrics I

• Haemolytic uraemic syndrome in integrated into interdisciplinary wards • Role of ACE2 in ARDS at the Children’s Centre. Interdisciplinary • mTOR inhibition in patients with neuro- means that the surgeons will in future blastoma and acute lymphatic leukaemia share the care of these children with the • Genetic characterization of rare chan paediatricians. Close cooperation is already nelopathies well established with our Departments of Pediatrics 2 (Neonatology) and 3 (Pediatric General Facts Cardiology, Pulmunology, Allergology and Cystic fibrosis). As part of the Medical University, the Children’s Hospital has a unique position It is our understanding that clinical psychol in providing the highest level of paediatric ogy is an integral part of clincal paediatrics care in western Austria. Simultaneously, and therefore we closely cooperate with the it represents an institution for scientific Department of Paediatric and Adolescent research as well as for the teaching of Psychiatry. We will place a special emphasis medical students. The training of physicians, on the participation of the Department of as well as their scientific training which is Human Genetics in the diagnosis and follow a prerequisite for scientific careers, is a up of rare diseases. The same applies to Director: priority at our hospital. This also includes imaging techniques in the field of diagnos- Univ.-Prof. Dr. Gerhard Gaedicke continued training of clinical specialists, tics. Paediatric Radiology, ultrasound, CT physicians, as well as nursing staff and scans and magnetic resonance tomography Contact: other medical professionals working with will be covered by the Department of Radi- Anichstraße 35 children and adolescents. ology in the Children’s Hospital. Overall, the 6020 Innsbruck intention is that in the future the specialists A great challenge will be the move into will come to the child in order to avoid cum- [email protected] the second construction block of the new bersome transport of sick children within Phone: +43 512 504 23501 building in 2015. The Paediatric Intensive the entire campus. Fax: +43 512 504 25450 Care Unit (PICU) will be extended and http://kinderzentrum.tirol-kliniken.at will take over the existing emergency Research department, which will be completed by an admission and observation ward Molecular Genetics of with eight beds. The general outpatient Congenital Diarrhoea Keywords clinic, as well as the special outpatient Assoc. Prof. Dr. Andreas Janecke, clinic, will be extended by a day clinic Assoc. Prof. Dr. Thomas Müller, Molecular genetics of rare childhood with the same number of beds thereby Assoc. PD Dr. Peter Heinz-Erian, diseases, cell biology, cancer biology, drug avoiding hospitalization, especially of Dr. Georg Vogel, discovery, biomarkers, inherited metabolic children with chronic diseases. Paediatric Assoc. Prof. Dr. Michael W. Hess, disorders, metabolomics, breath gas anal- surgery as well as all of the departments Univ.-Prof. Dr. Lukas Huber ysis, paediatric gastroenterology and hepa- of specialized surgery, which have been In 2014, our Microvillus inclusion disease tology, inflammatory bowel disease, paedi- until now treating children in different Research Group identified that loss of atric haematology and oncology, paediatric paediatric surgical wards within their Syntaxin 3 causes a variant form of the haemostaseology, diabetes in childhood, Departments, will in future have their beds congenital enteropathy microvillus inclusion epidemiology, national diabetes registry and international registry comparison

Research Focus

• Genetics of rare diseases, metabolomics • Genetic predisposition to chronic diarrhea, disruption of intestinal epithelial polarity • Cell death and metabolism in childhood malignancies; FOXO transcription factors, XIAP, Survivin • Biomarkers for autoimmune diseases • Genetic and clinical characterization of inherited metabolic diseases • Paediatric diabetes: epidemiology, national registry, long term complications, genetics of neonatal diabetes • Paediatric endocrinology: clinical and genetic studies on thyroid, adrenal glands and bone disorders

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disease. Understanding the pathogenesis of A clinical trial of gene modulation in ALL repositioning and in tight cooperations these disorders can significantly advance patients treated with glucorticoids has ena- with pharmaceutical institutes and the our knowledge of intestinal epithelial cell bled us to obtain significant information on pharmaceutical industry. Closely affiliated polarity. the details of leukemia biology and is cur- with both the division of Paediatric Oncology rently directed towards the exploration of and Haematology at the Medical University Paediatric Haematology/Oncology factors that affect chemo-sensitivity. of Innsbruck and the Tyrolean Cancer Members: Assoc. Prof. Dr. Roman Research Institute, our research extends Crazzolara, Dr. Gabriele Kropshofer, In addition we continue to explore disrupt- from the laboratory to the patient using the Assoc.-Prof. Dr. Bernhard Meister, ed signalling pathways in rare paediatric on- basic tools of molecular and cell biology, Univ.-Prof. Dr. Gerhard Gaedicke cology and haematology patients. Recently, genomics, genetic epidemiology, human and The Paediatric Oncology Lab is focused we identified deletion of KINDLIN-3 as an cell imaging technology and cell therapy. on identifying transcription factors that egress factor for osteoblasts, contributing Integrated in 5 local and 18 international regulate normal hematopoietic and leu to osteopetrosis in LAD-III patients. The clinical trials, the group’s bench-to-bedside kemic cell biology. The strategy of targeting characterization of IC1 deletion in a patient approach has enabled us to achieve greater these protective growth-activating mecha with Beckwith-Wiedemann syndrome has translation of research from biomedical nisms holds significant promise with regard demonstrated its role in elevated IGF ‑2 discovery into new prevention strategies, to the development of new therapeutic expression and the origin of Wilms tumours diagnostics, prognostics and therapies. options. Our recent data suggest that dis- in affected patients. ruption of the signalling molecules, mTOR Inherited Metabolic Diseases/Rare and p38MAPK, will enhance the efficacy Cancer Biology Diseases of current therapeutic agents. An ongoing Assoc. Prof. Dr. Michael Ausserlechner Assoc. Prof. Dr. Daniela Karall, clinical trial of the mTOR inhibitor Rapamy- Our research team investigates how PD Dr. Sabine Scholl-Bürgi cin in patients with relapsed neuroblastoma cell death and metabolism in childhood In recent years, our research has focused provides a source of patient samples which malignancies is controlled by FOXO on identification of new disorders (e.g. we will collect by our lab in order to further transcription factors and by Inhibitor of CoQ4, mitochondrial fission and fusion, elucidate the biology of ALL and its respon- Apoptosis proteins (XIAP and Survivin). SPENCD, PIGQ) and characterization of se(s) to mTOR inhibition. Further studies In a translational approach, we develop known disorders (e.g. LCHAD deficiency, into the signal transduction pathways inhib- strategies to target these death regulators GLUT1-deficiency syndrome, PIGA, FBXL4, ited by natural compounds are ongoing. with small compounds discovered by drug ALG8-CDG).

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Neuropaediatric Diseases PD Dr. Edda Haberlandt, Dr. Matthias Baumann We focussed our research on collaborations to describe rare channelopathies of neurological diseases (Ehlers Danlos, KCNQ2-, GABRG2-, GRIN2A-Mutation and Ring Chro- mosome 18).

Paediatric Diabetes Research Group Assoc. Prof. Dr. Sabine Hofer, Assoc. Prof. Dr. Elisabeth Steichen, Assoc. Prof. Dr. Daniela Baumgartner Sabine Hofer is a member of the scientific committee of the Austrian/German Diabetes registry DPV and is coordinating molecular basis of so-called ambiguous between bench and bedside. Our paediatric scientific research questions formed primary sexual characteristics. Furthermore, nephrology unit has become a European through the database. The main focus we evaluate treatment options for children, Reference Centre for HUS in childhood. since 2013 is the international comparison adolescents and adults who are born with Every 2 years we organize together with between diabetes management in children this condition and perform an assessment Prof. Würzner an international HUS meeting, and adolescents with the US. This group of health-related quality of life issues. This which is very highly esteemed by the large, focused on the complications screening study also encompasses individuals whose worldwide distributed HUS research family. of children with diabetes in testing and pattern of secondary sexual development establishing an early non-invasive method at a later stage differs from common Paediatric Intensive Care Unit (PICU) to measure aortic distensibility and expectations. As a centre participating in Dr. Uwe Klingkowski, stiffness – this work is ongoing. Another the German “DPV-Hypothyreose” study Assoc. Prof. Dr. Nikolaus Neu topic is devoted to rare neonatal forms of group for quality assurance in paediatric Our group of investigators (N. Neu, Dept. of diabetes and hyperinsulinism. endocrinology, longitudinal data of patients Pediatrics, B. Treml and A. Kleinsasser, Dept. with congenital primary hypothyroidism are of Anesthesiology) focuses on ACE2, a new Paediatric Endocrinology collected and analysed with regard to the principal enzyme in the renin-angiotensin- Dr. Klaus Kapelari, long-term follow-up of patients and in order aldosterone system. This enzyme might play Assoc. Prof. Dr. Elisabeth Steichen, to assess treatment within the framework a crucial role in the development of acute Assoc. Prof. PD Dr. Sabine Hofer of current guidelines. respiratory distress syndrome (ARDS). The Paediatric Endocrinology group We were able to demonstrate that ACE2 offers expertise in all areas of paediatric Paediatric Nephrology improves oxygenation and pulmonary blood endocrinology including disorders of Assoc. Prof. Dr. Siegfried Waldegger, flow in an experimental model system. Our growth, puberty, sex differentiation, Dr. Magdalena Riedl, Dr. Thomas Giner, findings provide the basis for clinical trials glucose metabolism, bone and mineral Dr. Alejandra Rosales that are currently underway. metabolism, the pituitary/hypothalamus, Haemolytic-uraemic syndrome (HUS) is the thyroid gland, the adrenal gland, and the a severe disease, leading to transient or Rheumatology and Paediatric gonads. The group’s main research focus chronic renal insufficiency. A bacterial toxin Infectious Diseases encompasses clinical and genetic studies produced by E. coli causes the classic form PD Dr. MMag. Jürgen Brunner, on the thyroid and adrenal glands and bone of the disease. These bacteria produce Dr. Manuela Zlamy PhD, disorders. Shiga-toxin and cause a gastrointestinal Assoc. Prof. Dr. Michaela Sailer-Höck, tract infection. Atypical forms of HUS are Mag. Ulrike Binder PhD, Normal values of thyroid function were induced by an overshooting activity of the Dr. Thomas Giner established by retrospective analysis of complement system. The exact registration The main research area in paediatric a big cohort of children and adolescents of the typical HUS as well as the investigation rheumatology is the development of showing and it revealed that hormone of the molecular mechanism causing the biomarkers for autoimmune diseases in levels change markedly during childhood. dysregulation of the complement system is infancy and adolescence. The complement Molecular investigation of patients with a primary research focus of our paediatric system, representing a component of association of congenital hypothyroidism, nephrology group. innate immunity, has been considered choreoathetosis, and pulmonary symptoms to be insignificant in the pathogenesis at the Institute of Experimental Pediatric We are closely working together with the of autoimmune diseases. The first Endocrinology (Charité Berlin) led to department of microbiology (Prof. Würzner) results of our work suggest an extremely the identification of mutations in the and the department of paediatric nephrology high turnover of complement in some transcription factor NKX2-1, which plays at the Hostpital for Sick Children at Toronto, paediatric autoimmune pathologies an important role in the development and Canada. With these collaborators we are and autoinflammatory diseases. The function of thyroid, basal ganglia, and the developing new methods for analyzing the complement system might be a potential lung. In collaboration with the German complement system. This helps to improve biomarker for monitoring autoimmune network “Netzwerk DSD” we are working the diagnosis of this rare but important diseases and may herald subclinical towards a better understanding of the disease and helps to bridge the gap inflammation.

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Selected Publications • Prof. Dr. Holger Prokisch, mitoNET (Network for diagnosis and therapy in mitochondrial diseases), Helmholtz Institute, BIRC5/Survivin enhances aerobic glycolysis and drug resist- München, Germany ance by altered regulation of the mitochondrial fusion/ fission • Prof. Dr. Stefan Kölker, EIMD (European Network for Intoxicati- machinery. Hagenbuchner J, Kuznetsov AV, Obexer P, on Type Metabolic Disorders), Medical University of Heidelberg, Ausserlechner MJ. ONCOGENE. 2013; 32(40); 4748–4757. Germany Loss of dermatan sulfate epimerase (DSE) function results in • Assoc. Prof. Dr. Martina Huemer, EHOD (European Network for Homocystinurias and Remethylation Defects), University musculocontractural Ehlers Danlos syndrome. Mueller Thomas, Children’s Hospital Zürich, Switzerland Mizumoto Shuji, Suresh Indrajit, Komatsu Yoshie, Vodopiutz Julia, • Prof. Dr. Thomas Obladen, iNTD (International Neurotransmitter Dundar Munis, Straub Volker, Lingenhel Arno, Melmer Andreas, Disease Network), Medical University of Heidelberg, Germany Lechner Silvia, Zschocke Johannes, Sugahara Kazuyuki, Janecke • Prof. Dr. Matthias R. Baumgartner, MMA-PA (methlymalonic Andreas R. and propionic acidemias) guideline group, University Children’s HUMAN MOLECULAR GENETICS. 2013; 22(18); 3761–3772. Hospital Zürich, Switzerland Tracking of Metabolic Control from Childhood to Young Adult- • Prof. Dr. Johannes Häberle, UCD (urea cycle disorders) guide- hood in Type 1 Diabetes. Hofer Sabine E, Raile Klemens, line group, University Children’s Hospital Zürich, Switzerland Froehlich-Reiterer Elke, Kapellen Thomas, Dost Axel, Rosenbauer • Prof. Dr. Ron Wevers, Dr. Dirk J. Lefeber, University Children’s Joachim, Grulich-Henn Juergen, Holl Reinhard W. Hospital Nijmegen, The Netherlands Austrian German Diabet Patienten V; German Competence Net- • Assoc. Prof. Dr. Dorothea Möslinger, for the Austrian Metabolic work Diabet. JOURNAL OF PEDIATRICS. 2014; 165(5); 956-U393. Group, University Children’s Hospital Vienna • Univ.-Prof. Dr. med. Dirk Föll, Department of Paediatric, Rheu- Loss of Syntaxin 3 Causes Variant Microvillus Inclusion Disease. matology and Immunology, University Children’s Hospital Wiegerinck Caroline L*, Janecke Andreas R*, Schneeberger Münster, Germany Kerstin*, Vogel Georg F*, van Haaften-Visser Desiree Y, Escher • Dr. Marco Gattorno, IRCCS Institute G. Gaslini, Pediatrics, Rheu- Johanna C, Adam Ruediger, Thoeni Cornelia E, Pfaller Kristian, matology, Clinical Immunology, Genua, Italy Jordan Alexander J, Weis Cleo-Aron, Nijman Isaac J, Monroe Glen R, van Hasselt Peter M, Cutz Ernest, Klumperman Judith, Clevers Hans, Nieuwenhuis Edward ES, Houwen Roderick HJ, van Haaften Gijs, Hess, Michael W#, Huber Lukas A#, Stapelbroek Janneke M#, Mueller Thomas#, Middendorp Sabine#. GASTROENTEROLOGY. 2014; 147(1); 65-U142. *shared first authors; #shared senior and corresponding authors COQ4 mutations cause a broad spectrum of mitochondrial disorders associated with CoQ10 deficiency. Brea-Calvo G*, Haack TB* , Karall D*, Ohtake A, Invernizzi F, Carrozzo R, Kremer L, Dusi S, Fauth C, Scholl-Bürgi S, Graf E, Ahting U, Resta N, Laforgia N, Verrigni D, Okazaki Y, Kohda M, Martinelli D, Freisinger P, Strom TM, Meitinger T, Lamperti C, Lacson A, Navas P, Mayr JA, Bertini E, Murayama K, Zeviani M, Prokisch H, Ghezzi D. AM J HUM GENET. 2015; 96: 309–317. *shared first authors Iron supplementation associated with loss of phenotype in autosomal dominant hypophosphatemic rickets. Kapelari K, Köhle J, Kotzot D, Högler W. J CLIN ENDOCRINOL METAB. 2015 Jul 17:jc20152391. [Epub ahead of print] PubMed PMID: 26186302. Selected Funding • Wirksamkeit neu identifizierter Krebsmedikamentein vivo PRIZE (Austrian Wirtschaftsservice) Project; Principal investigator: M.J. Ausserlechner • Effect of GUCY2C mutations on NA+/H+ exchanger 3 (NHE3) regulation in classic congenital sodium diarrhea (cCSD). Else Kröner-Fresenius-Stiftung; 2013_A230; Principal investigator: T. Müller • Translationale Forschung bei angeborenen Durchfallerkrankun- gen; OeNB Jubiläumsfonds Nr. 15627; Principal investigator: A.R. Janecke

Collaborations The research was supported by a grant from the Österreichische Forschungsföderungsgesellschaft and was performed in collaboration with • M. Schuster, Apeiron Biologics, and J. Penninger*, Institiute of Molecular Biotechnology of the Austrian Academy of Science, Vienna. • Prof. Dr. Tomas Obsil, Biophysical Chemistry, Charles University Prague, Prague, Czech Republic • Dr. Veronica Obsilova, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic • Prof. Dr. Jan Vesely, Organic Chemistry, Charles University Prague, Prague, Czech Republic • Prof. Dr. Consolato Sergi, Institute of Pathology, University of Alberta, Edmonton, Canada • Prof. Dr. Ralf Rieker, Institut of Pathology, University of Erlan- gen, Germany • Dr. Giampietro Viola, Department of Pediatrics, University of Padova, Italy • Prof. Dr. Allan Kaasik, Department of Pharmacology, University of Tartu, Estonia • Prof. Dr. Gerhard Wolber, Institute of Pharmaceutical Chemistry, FU-Berlin, Germany • Prof. Dr. Judith Rollinger, Pharmakognosie/Pharmazeutische Biologie, Universität Wien, Wien • Dr. Suse Benseler, Childhood Arthritis and Rheumatology Research Alliance (CARRA), Pediatric Rheumatology European Society: BRAIN WORKS; Toronto, Canada • Prof. Dr. Hans Clevers, Hubrecht Institute, Utrecht, The Nether- lands • Prof. Dr. Wolfgang Sperl, Assoc. Prof. Dr. Johannes A. Mayr, Mitocenter, University Children’s Hospital Salzburg

Research Report 2015 Medical University of Innsbruck 177 Centre of Pediatrics Pediatrics II

General Facts centres and with the local neuroscience and cardiovascular science group. The Department of Neonatology at the Medical University of Innsbruck is a Research perinatal centre offering the highest level of care. It provides care for all very preterm Neonatal Neuroscience – Clinical and and critically ill neonates in Tyrol and offers Experimental Research Groups a standardized follow-up programme until Neonatal brain injury is a major cause of these children reach school-age. infant mortality and morbidity and thus a problem of great global and national Researchers in the Department of concern. In industrialized Western Neonatology focus on both clinical and countries, the most common cause of basic science (www.neonatal-research.at), neonatal brain injury is prematurity. During with the aim of improving survival and long- the last years, improvements in neonatal term outcome of neonates. intensive care medicine have decreased preterm infant mortality. However, infants Clinical research includes the characteri- born prematurely remain at high risk of zation of neurodevelopmental and cardio neurodevelopmental delay and of lifelong Director: vascular outcome of very preterm infants handicap. To date, causal therapeutic Univ.-Prof.in Dr.in Ursula until school-age and the definition of risk strategies for neonatal brain injury are not Kiechl-Kohlendorfer predictors for adverse outcome. This en- available. Clinical management is based compasses multimodal monitoring of the on an optimization of perinatal care and Contact: neonatal brain (aEEG, MRI), and the eval- supportive measures and on identifying Anichstraße 35 uation of the role of nutrition/growth and infants at high risk for adverse neonatal 6020 Innsbruck research on the optimization of perinatal outcome. resuscitation. The department also focuses [email protected] on risk factors for and prevention of sudden Clinical Research Projects: Phone: +43 512 504 27307 infant death syndrome (SIDS). The basic Elke Griesmaier-Falkner, Vera Neubauer, Fax: +43 512 504 27308 research programme is dedicated to iden- Anna Posod, Ulrike Pupp Peglow, http://kinderzentrum.tirol-kliniken.at tifying mechanisms of neuroprotection in Ursula Kiechl-Kohlendorfer perinatal brain injury models and aims at assessing new therapeutic strategies. In Neuromonitoring of Preterm Infants addition, the department participates in a The amplitude-integrated electroencepha- world-wide quality improvement collabora- logram (aEEG) constitutes a practicable and tive – the Vermont Oxford Network – with fast method to address specific neurologi- the aim of following key neonatal outcomes cal risks by continuously monitoring elec- Keywords and thereby continuously improving patient trocortical activity during development. The care. There are close national and interna- research group is particularly interested in Preterm infants, neonatal neuroscience, tional collaborations with other perinatal the potential use of aEEG in preterm infants developmental outcome, cardiovascular risk, sudden infant death syndrome, neuroprotection, sigma-1 receptor ligands, mitochondrial metabolism, FOXO3, apoptosis

Research Focus

• Characterization of risk predictors for adverse outcome of preterm infants • Monitoring of the preterm brain (aEEG, MRI) • Investigation of effects of prematurity, neonatal growth and feeding practices (focus on human milk) on cardiovascular risk factors and neurodevelopmental outcome • Development of substances for neuroprotection and treatment of perinatal brain injury • Role of anti-apoptotic substances in mitochondrial metabolism • Impact of FOXO3 gene expression on cell death and stress resistance in neuronal cells Fig. 1: Aspects of neonatal brain injury

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for the evaluation of brain maturation and neonatal and adult brain injury. However, FOXO3 on cell death regulation and function, as well as its relation to long-term they finally failed to live up to researchers’ stress resistance in neuronal cells. FOXO neurodevelopmental outcome. In addition, expectations in clinical trials of adult brain transcription factors are central regulators magnetic resonance imaging is performed injury and showed unwanted side effects of cell death but also promote longevity, in non-sedated very preterm infants at term by triggering apoptotic neurodegeneration since they protect stem cells from oxidative age with the research focus on the evalua- in the undamaged developing brain, calling stress. The research group has been tion of brain morphology, injury pattern and for cautious use of these agents in the focusing on the pro- and anti-apoptotic maturation. vulnerable preterm organism. Of interest, functions of the transcription factor for DM it is well known that in addition to FOXO3 in neuronal cells. FOXO3-triggered Follow-up of Very Preterm Infants its NMDA antagonistic effect, it also is a apoptosis induced by cellular stress involves Children born preterm are at risk for sigma-1 receptor ligand. Therefore, in the a biphasic reactive oxygen species (ROS) neurodevelopmental delay or disorder. last years, the role of the sigma-1 receptor accumulation at the mitochondria due to Therefore, regular follow-up visits are and its ligands in perinatal brain injury uncoupling of mitochondrial respiration important not only to provide support for were further investigated and exogenous through the BH3-only protein Bim. Recent these children and their parents but also for sigma-1 receptor agonists (PRE-084 and studies in our lab identified C10orf10/ research purposes and for quality control of PPBP) were shown to have protective DEPP as a direct transcriptional target of neonatal intensive care. potential in neonatal brain injury. The focus FOXO3 which localizes to peroxisomes at present lies on the therapeutic potential and mitochondria. DEPP impairs cellular Experimental Research Projects: of endogenous sigma-1 receptor ligands, ROS detoxification and thereby sensitizes Neuroprotection such as dehydroepiandrosterone (DHEA) neuronal cells to ROS-induced cell death. Elke Griesmaier-Falkner, and its sulfate ester (DHEAS), which have Future Goals: Identification and charac- Karina Wegleiter, Anna Posod been shown to be strong neuroprotectants terization of FOXO3-interacting drugs that The neonatal neuroscience research in adult peripheral and central nervous inhibit the function of FOXO3 in neuronal laboratory evaluates the potential of system pathology. cells. substances to protect the preterm brain. In the second and third trimester of pregnancy, Neuronal Metabolism Selected Publications as well as after birth, the brain rapidly Judith Hagenbuchner Patent ductus arteriosus, low platelets, cyclooxygenase inhib- increases in size, shape and complexity. Mitochondria are at the centre of cellular itors, and intraventricular hemorrhage in very low birth weight preterm infants. Brunner B, Hoeck M, Schermer E, Streif W, During this period, the developing brain is pathways such as oxidative phosphorylation Kiechl-Kohlendorfer U. The Journal of Pediatrics. 2013; particularly vulnerable to insults, such as (ATP generation), the TCA cycle, glucose 163: p. 23–28. hypoxia-ischemia, free radical formation, metabolism and oxidation of fatty acids. In vivo and in vitro evaluation of the effect of PRE-084 on inflammation-sensitized hyperoxia-induced developing brain injury. inflammation and excitotoxicity mediated Alterations of mitochondrial metabolism Posod A, Krepp Y, Stock K, Urbanek M, Kiechl-Kohlendorfer U, by excess N-methyl-D-aspartate (NMDA) can therefore contribute to cell death as Griesmaier E. JOURNAL OF NEUROSCIENCE RESEARCH. 2013; Aug 23. doi: 10.1002/jnr.23271. receptor activation. We are specially well as to its prevention. The research group The sigma-1 receptor agonist 4-phenyl-1-(4-phenylbutyl) pip- interested in substances that have focusses on the role of the anti-apoptotic eridine (PPBP) protects against newborn excitotoxic brain in- already been tested and proven to show protein BIRC5/Survivin and its role in jury by stabilizing the mitochondrial membrane potential in vitro and inhibiting microglial activation in vivo. Wegleiter K, neuroprotective potential in animal models mitochondrial metabolism. Our recent data Hermann, M, Posod A, Wechselberger K, Stanika RI, Obermair GJ, of adult brain injury. As both white and grey revealed that Survivin leads to recruitment Kiechl-Kohlendorfer U, Urbanek M, Griesmaier E. matter structures in the developing brain of the main fission protein DNM1L/ EXERIMENTAL NEUROLOGY. 2014; 261: p. 501–509. The common antitussive agent dextromethorphan protects are known to be particularly susceptible, Drp1 and thus causes mitochondrial against hyperoxia-induced cell death in established in vivo and in these substances are tested for their fragmentation, which shifts neuroblastoma vitro models of neonatal brain injury. Posod A, Pinzer, K, Urbanek neuroprotective potential in pre-myelinating cells from oxidative phosphorylation to M, Wegleiter K, Keller M, Kiechl-Kohlendorfer U, Griesmaier E. NEUROSCIENCE. 2014; 274: p. 260–272. (pre- and immature) oligodendroglial and aerobic glycolysis (Warburg effect) as BIRC5/Survivin enhances aerobic glycolysis and drug resistance neuronal cell types. Furthermore three main energy source (Sanofi Prize of the by altered regulation of the mitochondrial fusion/fission machin- in vivo models have been established, Medical University in 2014). This metabolic ery. Hagenbuchner J, Kuznetsov AV, Obexer P, Ausserlechner MJ. ONCOGENE. 2013; 32: p. 4748–4757. mimicking key aspects of neonatal brain shift results in increased resistance to injury (excitotoxic, hyperoxic and hypoxic- intrinsic cell death signalling, and also to Selected Funding ischemic brain injury models). dependency of Survivin-expressing cells Early vascular ageing (EVA), part of the excellence initiative (Com- petence Centers for Excellent Technologies – COMET) of the Using an established neonatal mouse model on glycolysis. This allows us to kill Survivin Austrian Research Promotion Agency FFG: “Research Center of of excitotoxic brain damage, it could be expressing cells effectively by use of Excellence in Vascular Ageing – Tyrol, VASCage” (K-Project Nr. 843536) funded by the BMVIT, BMWFW, the Wirtschaftsagentur shown that the substance dextrometorphan glycolysis-inhibitors in vitro and in vivo. Wien and the Standortagentur Tirol; 1.1 Mio. Euro (DM) significantly reduced brain injury by reducing cell death. DM is a low-affinity FOXO3 and its Target Genes on Cell Collaborations NMDA receptor antagonist with anti- Death and Stress Resistance in • Allen Kaasik, Department of Pharmacology University of Tartu, Estonia inflammatory properties and is widely and Neuronal Cells – Petra Obexer • Christiane Richter‑Landsberg, Carl von Ossietzky University, safely used as an antitussive in both adults The pathophysiology of preterm brain Oldenburg, Germany • Jan Lewerenz, Department of Neurology, University Hospital and children. In the hyperoxia-mediated damage is multifactorial and phases of Ulm, Germany brain injury model DM also protected hypoxia and ischemia are known to play an • Martin Lee, Prolacta Bioscience, Monrovia, CA • Mechthild Vennemann, Inst. of Legal Medicine, Univ. of Müns- against cell death in vivo and in vitro. important role. Since FOXO transcription ter, Münster, Germany, These were promising results and during factors are activated by different cellular • Moon R, Goldberg Center for Community Pediatric Health, Chil- dren’s National Medical Center, Washington, US, and Blair PS, the last years, NMDA antagonists have stresses, our lab is primarily interested University of Britstol, UK (International Society for the Preven- been favoured as therapeutic agents in in the impact of the transcription factor tion of Infant Death)

Research Report 2015 Medical University of Innsbruck 179 Department of Nuclear Medicine Nuclear Medicine

Keywords well-funded and internationally respected preclinical Research & Development Unit of Peptide receptor radionuclide therapy, high quality. This group consists of several peptide ligand radionuclide therapy, radiochemists/pharmacists, medical phys- radioiodine refractory thyroid cancer, icists and PhD-students. Their work results hormone refractory prostate cancer, in the construction of radiotracers using dif- neuroendocrine tumours ferent modal systems including a variety of radiolabelled peptide analogues such as for Research Focus somatostatin, vasoactive intestinal peptide (VIP), CCK-2/gastrin, or prostata-specific The Department of Nuclear Medicine is membrane antigen (PSMA) ligand for spe- best known for the work with radiolabelled cific tumour targeting. Other important de- peptides, both for diagnostic and velopments are based on Arg-Gly-Asp (RGD) therapeutic purposes, a theme that we for imaging of angiogenesis in tumour le- have systematically explored over the sions, or on hepatic binding protein imaging last 2 decades. We identify and develop with galactosylated albumin for functional a variety of radiopharmaceuticals for liver reserve estimation. Radiopharmaceu- different targets for clinical use. Our goal ticals are produced at clinical grade in our Director: is to engineer more effective ligands/ dedicated laboratories for use in SPECT/CT Univ.-Prof.in Dr.in Irene J. Virgolini peptides/antibodies – “theranostics” – for or PET/CT studies. individualized treatment. Contact: About 20 whole body PET/CT studies are Anichstraße 35 General Facts performed daily in our PET-center. Patients 6020 Innsbruck prior evaluated by dosimetry following The Department of Nuclear Medicine accel- SPECT/CT studies are treated at our [email protected] erates translation of preclinical radiophar- Nuclear Medicine Therapy Unit with high Phone: +43 512 504 22651 maceutical research development (focus dose theranostics. Fax: +43 512 504 22659 on radiolabelled peptides) into clinical ap- www.nuklearmedizin-innsbruck.com plications towards imaging of biomarkers Radioiodine ablation therapy of thyroid used for cancer treatment (70 % of clinical cancer remnants, peptide receptor radio routine), treatment of neurological impair- nuclide therapy (PRRT) of neuroendocrine ment (20 % of clinical routine) or cardiac tumour patients and peptide ligand disease (10 % of clinical routine). The struc- radionuclide therapy (PLRT) of prostate ture of the Department of Nuclear Medicine cancer patients are our most important is based on a very creative, productive, therapy tools.

Fig. 1: Structure of the 89Zr-fusarinine-C (FSC)-complex and 89Zr-FSC-(RGD)3 in a mouse model targeting the receptor-positive tumour (red arrow), wheras the receptor-negative tumour shows no accumulation of the tracer (blue arrow)

180 Research Report 2015 Medical University of Innsbruck Nuclear Medicine

Research

Preclinical Research Activities – Research & Development Unit The research activities are focussed on preclinical research dedicated to the optimization and improvement of radiolabelling procedures for established radiopharmaceuticals, the in-house preparation of new radiopharmaceuticals for clinical studies, as well as the preclinical development of new radioligands for molecular imaging and therapeutic purposes.

Different research projects illustrate the activities in this field. The FWF project P25899-B23 “Novel 68Ga/89Zr-chelators for targeted biomolecules in PET” (project leader: Prof. Clemens Decristoforo) explores novel 02/2015 04/2015 opportunities to prepare highly specific radiolabelled biomolecules based on 68Ga and 89Zr, two radionuclides which have Fig. 2: Scintigraphy of a patient with metastasised prostate cancer before (A) and under (B) gained high attention for PET-application therapy with high-dose 177Lu-PSMA ligand (2 x 6 GBq). The images demonstrate significant in the last years. By starting from a cyclic uptake of the radiopharmaceutical by disseminated bone metastases responding to therapy. siderophore (fusarinine C, FSC) we have developed strategies to prepare new multimeric ligands for targeted molecular targeting CCK 2-receptors on different Gly-Trp-Met-Asp-Phe-NH2) for the diagnosis imaging for oncological applications. The human tumours, including medullary thyroid of MTC is transferred from bench to bedside. trimers 68Ga-FSC-(RGD)3 and [89Zr]FSC- carcinoma (MTC), small cell lung cancer, So far, a GMP-conform kit formulation for (RGD)3 with specific binding to alpha-v- insulinomas, carcinoids, gastrointestinal radiolabelling with In-111 was developed beta3 integrin showed a more than 3-fold stromal (GIST) tumours. Based on our initial and an Investigational Medicinal Product increased tumour uptake when compared studies on 68Ga-labelled GSA-derivatives for Dossier (IMPD) and all the documents for to a monomeric form of the peptide. Also non-invasive imaging of the functional liver the clinical trial application were elaborated. mono-, di- and trimeric FSC-minigastrin reserve using PET we developed alternative The authorization by the national authorities conjugates targeting CCK2 receptors compounds with improved metabolic as well as the EU is currently ongoing, labelled with 68Ga and 89Zr showed excellent stability and compared the new 68Ga-NOTA- suggesting a possible start of the clinical tumour uptake in respective mouse models. GSA in cooperation with the Department trial by mid-2015. (Fig. 1) of Preclinical Imaging and Radiopharmacy of the University Hospital Tübingen Prostate Cancer Theranostics Within the co-ordinated research project with our lead structure 68Ga-DTPA-GSA. New receptorial radiotracers binding (CRP) F22052 of the International Atomic Despite successful improvement of the to PSMA with significantly increased Energy Agency (IAEA) “Development metabolic stability imaging performance expression on prostate cancer (PC) cells and Preclinical Evaluation of Therapeutic was comparable. Both compounds have been proposed for PET-imaging. Radiopharmaceuticals Based on Lu- showed high uptake in the target organ The 68Ga-PSMA ligand HBED-CC proved 177 and Y-90 Labeled Monoclonal and rapid elimination from the body in the its feasibility to detect PC relapses and Antibodies and Peptides” (project leader: corresponding rat model. Based on this metastases with high sensitivity. Our Priv.‑Doz. Dr. Elisabeth von Guggenberg), in promising data, at the moment, an initial clinical studies demonstrated the great collaboration with other participants from clinical study with the 68Ga-DTPA-GSA is potential of 68Ga-PSMA PET/CT in patients Argentina, Brazil, Hungary, India, Iran, Italy, being prepared. with biochemical relapse. Furthermore, Macedonia, Poland, Saudi Arabia, Syria based on the high level expression of and Turkey we have preclinically evaluated Clinical Research Activities PSMA on PC cells we have started to treat two promising peptide derivatives for Within the ERA-NET Transcan call (FWF patients with metastasised disease with targeted radionuclide therapy. Specifically, project I1224-B19, project leader: Prof. high dose 177Lu-PSMA ligand. Initial results a bombesin analogue was evaluated Clemens Decristoforo) “Phase I clinical demonstrate high tumour control ability of for targeting gastrin releasing peptide trial using a novel CCK-2/gastrin receptor- this radiopharmaceutical with significant (GRP) receptors, a target molecule over- localizing radiolabelled peptide probe for implication on future PC therapy protocols expressed on a variety of human cancer personalized diagnosis and therapy of (Fig. 2). cells, including prostate, breast, lung, patients with progressive or metastatic and pancreatic cancer. Furthermore, a MTC” a new promising cholecystokinin-2- Thyroid Research Activities radiolabelled cyclic minigastrin analogue receptor targeting peptide CP04 (DOTA- Our recent thyroid research activities developed by our group was evaluated for DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr- concentrated on the therapy options of

Research Report 2015 Medical University of Innsbruck 181 Department of Nuclear Medicine

radioiodine-refractory thyroid cancer. of a thyroid cancer-specific quality of life Virgolini IJ. European Journal of Nuclear Medicine and Molecular Several potential kinase inhibitors have assessment tool, which currently is close Imaging. 2013 Feb; 40(3):364–372. [(68)Ga]NS(3)-RGD and [(68)Ga] Oxo-DO3A-RGD for imaging α(v) clinically been used with, however, a rather to the finalizing phase III. Results will be β(3) integrin expression: synthesis, evaluation, and comparison. broad range of side effects. Radiolabelled published in 2016. The department was Knetsch PA, Petrik M, Rangger C, Seidel G, Pietzsch HJ, Virgolini I, somatostatin analogues have clinically also part of the EORTC Quality of Life Decristoforo C, Haubner R. Nuclear Medicine and Biology. 2013 Jan;40(1):65–72. been implemented for treatment by our Group project on the development of a group. The diagnosis and therapy of the computer-adaptive test for role functioning Selected Funding MTC-subtype is addressed by the ERA-NET – the manuscript has been submitted for • Phase I clinical trial using a novel CCK-2/gastrin receptor-locali- Transcan project as well as by the CRP publication. zing radiolabelled peptide probe for personalized diagnosis and therapy of patients with progressive or metastatic medullary F22052 of the IAEA (see above). thyroid carcinoma. Project leader: Prof. Clemens Decristoforo; In 2014, a study on the weekly web- ERA-NET: GRANT-T-MTC FWF Project I 1224-B19/€ 219,703.- based tele-monitoring of self-reported • Novel 68Ga/89Zr-chelators for targeted biomolecules in PET. MITIGATE • Project leader: Prof. Clemens Decristoforo. In this concerted action several potential bone pain and quality of life in patients • FWF Project P 25899-B23 / € 238,011.- imaging diagnostics are currently evaluated. with metastasised prostate cancer has • MITIGATE: Closed-loop Molecular Environment for Minimally Invasive Treatment of Patients with Metastatic Gastrointestinal In WP7 the Department of Nuclear Medicine been initiated. Patients currently are Stromal Tumours. will perform the first diagnosticin vivo study consecutively included into the study. • WP7 Leader: Prof. Clemens Decristoforo in patients with GIST tumours, possibly By the end of 2014 a study on the effect • European Union FP7 under grant no 602306 / € 341,400.- starting this year. of music on the anxiety levels of patients Collaborations during nuclear therapy has been finalised. Clinical Cooperations: 68Ga-NODAGA-RGD for Imaging Hepato- The aim of the study was to offer music as • Prof. Dr. Richard Baum, Zentralklinik Bad Berka, Bad Berka, cellular Cancer (HCC) a method of relaxation and distraction to Germany • Prof. Dr. John Buscombe, Cambridge University Hospitals, Cam- In an initial study, together with the cancer patients who have to be isolated bridge, UK Department of Internal Medicine II, we for nuclear therapy and can only be offered • Prof. Pietro Muto, Ospedali dei Colli, Monaldi, Italia 68 • Prof. Annibale Versari, Az. Osp. Arcispedale S. Maria Nuova, studied the feasibility of Ga-NODAGA-RGD limited support during that period. Data are Reggio Emilia, Italia to image the hepatocellular carcinoma with currently being analysed. • Prof. Stefano Fanti, Policlinico S. Orsola-Malpighi, Bologna, Italia PET. Additionally, tolerability, biodistribution • Dr. Lisa Bodei, IEO Istituto Europeo di Oncologia, Milano, Italia • Dr. Chiara Maria Grana, IEO Istituto Europeo di Oncologia, Mi- and elimination from the body, in vivo lano, Italia stability, and radiation burden for the Selected Publications • Prof. Helmut Maecke, University Hospital Freiburg, Freiburg, Germany patient was studied in this Phase I/II study. Therapie des Patienten mit Radioiod-refraktärem, differenzier- • Prof. Georgios S Limouris, Athens University Medical Faculty, Ten patients with HCC were included. The tem Schilddrüsenkarzinom. Ein Konsensusstatement. Lindner C, Athens, Greece Dierneder J, Pall G, Pirich C, Hoffmann M, Raderer M, Becherer • Prof. Dr. Hans-Jürgen Wester, Technical University Munich, Gar- A, Niederle B, Lipp R, Lind P, Gallowitsch H, Romeder F, Virgolini I. compound was well tolerated with no ching, Germany Nuklearmedizin. 2014 Nov 25;54(1). observed side effects observed. Further • Prof. Dr. Thomas Beyer, Medical University Vienna, Vienna, Austria data collection and analysis is ongoing. (18)F ‑glyco-RGD peptides for PET imaging of integrin expression: efficient radiosynthesis by click chemistry and modulation of bi- • Prof. Dr. Marcus Hacker, Medical University Vienna, Vienna, odistribution by glycosylation. Maschauer S, Haubner R, Kuwert Austria Quality of Life T, Prante O. • Prof. Jean-Noël Talbot, AP-HP & Université P&M Curie, Paris, Molecular Pharmaceutics. 2014 Feb 3;11(2):505–15. France Patients treated with radiopharmaceuticals • Prof. David R. Vera, University of California, San Diego, United Tumor targeting and imaging with dual-peptide conjugated States are usually already at an advanced stage multifunctional liposomal nanoparticles. Rangger C, Helbok A, • Prof. Stanley J. Goldsmith, Weill Cornell Medical College, New of disease and due to regulations of Sosabowski J, Kremser C, Koehler G, Prassl R, Andreae F, Virgolini York, United States IJ, von Guggenberg E, Decristoforo C. radiation safety they have to stay isolated International Journal of Nanomedicine. 2013;8:4659–4671. • Prof. Ajit Shinto, Kovai Medical Centre and Hospital, Coimba- for the period of radioactive treatment. This tore, India Nuclear medicine 2013: from status quo to status go. Beyer T, • Prof. Anthony Goh, Singapore General Hospital, Singapore causes an additional level of anxiety for Hacker M, Schubiger A, Virgolini I, Wester HJ. • Prof. Emerita Andres-Barrenechea, St. Luke’s Medical Center, the patients. To support the wellbeing and European Journal of Nuclear Medicine and Molecular Imaging. Quezon City, Philippines 2013 Dec; 40(12):1794–1796. • Prof. Harvey Turner, University of Western Australia, Murdoch, well-feeling of the patients we have initiated A retrospective comparison between 68Ga-DOTA-TOC PET/CT and Australia translational projects integrating not only 18F ‑DOPA PET/CT in patients with extra-adrenal paraganglioma. • Prof. Mike Sathekge, University of Pretoria, Pretoria, South psychooncologists but also theologists, Kroiss A, Putzer D, Frech A, Decristoforo C, Uprimny C, Gasser Africa RW, Shulkin BL, Url C, Widmann G, Prommegger R, Sprinzl GM, • Prof. Alicja Hubalewska, Jagiellonian University, Kraków, Poland psychologists, nutritionists for the patients’ Fraedrich G, Virgolini IJ. • Prof. N. Ozlem Küçük, Ankara University, Ankara, Turkey European Journal of Nuclear Medicine and Molecular Imaging. • Prof. Levent Kabasakal, Istanbul University, Istanbul, Turkey supportive care. 2013 Dec; 40(12):1800–1808. Cooperations with Major Research Projects: Development of 68Ga-labelled DTPA galactosyl human serum al- • Peter Laverman, University Medical Center Nijmegen, The bumin for liver function imaging. Haubner R, Vera DR, Farshchi- Currently, the routine application of quality Netherlands Heydari S, Helbok A, Rangger C, Putzer D, Virgolini IJ. • Milos Petrik, PhD., Palacky University Olomouc, Czech Republic of life assessment is intensively investigated European Journal of Nuclear Medicine and Molecular Imaging. and fostered by respective institutions 2013 Aug;40(8):1245–1255. • Vladimir Tolmachev, Uppsala University, Sweden • Jagiellonian University Medical College, Krakow, Poland such as the EORTC Quality of Life Group or Design and Evaluation of Novel Radiolabelled VIP Derivatives • Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy the ISOQOL. At the department a routine for Tumour Targeting. Rangger C, Helbok A, Ocak M, Radolf T, • IRRP, NCSR “Demokritos”, Molecular Radiopharmacy, Athens, Andreae F, Virgolini IJ, von Guggenberg E, Decristoforo C. Greece computer-based quality of life assessment Anticancer Research. 2013 Apr;33(4):1537–1546. • National Centre For Nuclear Research, Radioisotope, Centre program has been implemented in 2011 and (68)Ga-DOTA-TOC uptake in neuroendocrine tumour and healthy POLATOM, Otwock, Poland is currently being evaluated. First results tissue: differentiation of physiological uptake and pathological • University Medical Centre Ljubljana, Ljubljana, Slovenia processes in PET/CT. Kroiss A, Putzer D, Decristoforo C, Uprimny • University Medical Center Mannheim, Germany providing new “real-life” information on C, Warwitz B, Nilica B, Gabriel M, Kendler D, Waitz D, Widmann • Universita degli Studi di Torino Unito, Torino, Italy thyroid cancer patients’ quality of life over G, Virgolini IJ. • Advanced Accelerator Applications, Saint-Genis-Pouilly, France European Journal of Nuclear Medicine and Molecular Imaging. • Universidad de la Republica Facultad de Ciencias, Montevideo, the course of treatment and follow-up have 2013 Apr; 40(4):514–523. Uruguay already been published. The department is Somatostatin receptor PET in neuroendocrine tumours: (68) • National Institute for Physics and Nuclear Engineering, Bucha- Ga-DOTA (0),Tyr (3)-octreotide versus (68)Ga-DOTA (0)-lanreotide. rest-Magurele, Romania furthermore involved in the EORTC Quality Putzer D, Kroiss A, Waitz D, Gabriel M, Traub-Weidinger T, Uprimny • All India Institute of Medical Sciences, New Delhi, India of Life Group project on the development C, von Guggenberg E, Decristoforo C, Warwitz B, Widmann G, • Nuclear Medicine, S. Orsola Hospital, Bologna, Italy

182 Research Report 2015 Medical University of Innsbruck Nuclear Medicine

Research Report 2015 Medical University of Innsbruck 183 Department of Therapeutic Radiology and Oncology Therapeutic Radiology & Oncology

electro-spinning of nano-fibrous cell cul- Equipment is available to perform flow ture substrates, developing cell-to-elec- cytometry analyses, long-term live- trode interfaces at micron-scales cell imaging (by light and fluorescence techniques), proteomics, metabolomics, General Facts intracellular ROS-quantification, advanced cell and tissue culturing (in-house Peter Lukas has headed the Department fabrication of nano-fiber scaffolds, 2D- and of Therapeutic Radiology and Oncology 3D-perfusion culture models), impedance (ROI) at the Medical University of Innsbruck and TEER-methods to assess tissue integrity, (MUI) since 1993. He has introduced cell-migration tracking, as well as up-take several improved treatment techniques studies and subcellular localization of nano- in radiotherapeutic routines, nowadays particles by scanning and transmission globally accepted as standard treatment electron microscopy. The second protocols. Besides his clinical experience, associated laboratory is the Laboratory for Peter Lukas has strongly promoted basic Experimental and Translational Research on and translational research in the field of Radiation Oncology (EXTRO-Lab), headed radiobiology at his department by Ira Skvortsova. This laboratory was established in 2006. Director: The Dept. of Therapeutic Radiology and o. Univ.-Prof. DI Dr. Peter Lukas Oncology (MUI) comprises eight units Research tasked with performing therapeutic as Contact: well as experimental irradiation (five linear Peter Lukas Anichstraße 35 accelerators to generate photon beams of The main Project in 2013/2014 was SEM- 6020 Innsbruck energies up to 20 MeV; two Brachytherapy PER: Secondary Malignoma – Prospective units, and a conventional x-ray-device up to Evaluation of the Radiotherapeutic dose dis- [email protected] 200 keV. Further Information: tribution as the cause for induction, funded Phone: +43 512 504 22800 www3.i-med.ac.at/strahlentherapie/de/ by Oncotyrol. Fax: +43 512 504 22869 start.php http://www3.i-med.ac.at/ Project partners are: strahlentherapie/ The first associated Laboratory of • University for Health Sciences, Radiobiology (headed by Thomas Seppi) • Medical Informatics and Technology utilizes experienced staff personnel (UMIT), Hall i. T. / Department of Biomed- Keywords (4 VPs) skilled in a broad spectrum of cell ical Computer Science and Mechatronics; biology as well as in nano-technological • Tiroler Landeskrankenanstalten (TILAK); Radiotherapy, oncology, radiosurgery, methodologies. • ELEKTA Oncology Systems. radiation biology, radiation protection, medical physics, medical biotechnology, medical molecular biology, cell biology, nanobiotechnology

Research Focus

Major research topics are: • Radio-Sensitizing in Radiation Oncology: Many of the new systemic substances used in Oncology have a radiosensitizing (RSP) or radioprotective (RPP) potential. Often little is known about these properties before going into clinical use because investigation in this concern is not part of the EU guidelines for marketing authorisa- tion. For Radiooncologists the knowledge of RSP or RPP is essential for the planning of any form of combined treatments. 8, 2013)

• High Precision Radiotherapy: Our Institu- . tion is famous worldwide for the development of Patient Fixation Devices for high precision radiotherapy • Radiobiology: Impact of low doses for the

induction of secondary malignoma; cell of Vol © TRAFFIC (Cover culture modelling, nano-particle applica- Fig. 1: Glioblastoma cells grown on functionalised electrospun gelatine-nanofibres (SEM- tions in vitro, modelling of micro-fluidics, micrograph; 2000x).

184 Research Report 2015 Medical University of Innsbruck Therapeutic Radiology and Oncology

Objectives: Based on the development of novel linear accelerator models, new radiation treatment techniques such as IMRT (Intensity Modulated Radiation Therapy), IGRT (Image Guided Radiation Therapy und VMAT (Volumetric Modulated Arc Therapy) have become applicable. According to the EU–directive 97/43, these techniques allow the theoretically best possible radiation dose distribution to preserve the normal tissue with the highest possible therapeutic effect on the underlying tumour. Note that these effects only hold for high doses. A negative site effect of using the aforementioned techniques is that considerably large volumes of the body of the patient will get “contaminated” through the use of small or minimal doses.

Well known radiobiologists such as Trott and Tubiana claim that radiation treatment with minimal doses is the reason for the induction of secondary malignancies Fig. 2: Radiation-induced apoptosis in 3D-tissues of glioblastoma cells co-cultured with which often occur several decades after fibroblasts (SEM-micrograph; 1000x). the particular treatment. This and the steep dose gradient in conventionally used techniques might explain the observation Thomas Seppi that nowadays only a small fraction of The associated Laboratory of Radiobiology tigate the potential of inducing drug release patients treated by radiotherapy go on to conducted several projects in the field of by gamma-ray/proton dilation as a trigger develop secondary malignancies, despite nano-technology and tissue engineering modality. NPs made of heavy metals, such the prediction of a higher incidence. funded by the Austrian Nano-Initiative (FFG) as gold, may enhance the efficacy of cancer or by other peer reviewed governmental radiotherapy by increasing the local absorp- Appropriate data sets to prove or disprove grants. The team, headed by Thomas Seppi, tion of photon as well as proton radiation. this theory are currently lacking . From 2012 is experienced in radiobiology, analytical onwards radiation therapy techniques such chemistry, nano-coatings in biomedical SPIOs can be detected by magnetic reso- as IMRT and VMAT will be available in the applications, laser-optical cell analyses, nance imaging, and thus, could be coupled Department of Therapeutic Radiology and electron-microscopy protocols, designing to gold to enable easily applicable cancer Oncology, at Medical University Innsbruck and prototyping of advanced cell culture theranostics. In particular, the core magnet- providing the best high dose conformity but models, as well as in molecular biology and ic properties of NPs will allow the identifica- simultaneously limiting the contaminating toxicology of cancer cells. tion of their bio-distribution in tumour tis- dose received by larger areas of healthy sues by MRT, whereas gold coating of NPs is tissue within which the target volume is Since 1998, 13 doctoral and 11 diploma used to increase photon energy deposition located. The objective of this project is to theses have been supervised in the fields of during radiotherapy of cancer. Investiga- develop the platform and framework to analytical chemistry, radiobiology, molecular tions to study the effects of concomitant perform a long term (range ~ 20 years) cell biology, and cancer research. Since NP-treatment in comparison to convention- study based on the preconditions given by then, scientific personnel and equipment al RT alone are performed in vitro by using this new radiotherapy technology. has mainly been financed yb granted in-house fabricated advanced cell culture fellowships. Currently, the research group is models specifically developed to maintain In order to tackle the problem, we plan engaged in projects related to nano-particle 3D-tumour cell constructs. For this pur- to develop a technology for providing a applications in vitro, modelling of micro- pose, funded research is focussed on the platform to collect and archive data from fluidics, electro-spinning of nano-fibrous cell micro-fluidic and extracellular requirements patients who were treated by these special culture substrates, as well as in developing of three-dimensionally arranged tumour cell techniques and to perform temporary cell-to-electrode interfaces (impedance and aggregates maintained in vitro. intermediate evaluations. Ongoing Projects TEER-analyses) at micron-scales. are concerned with the membership of A main objective of ongoing projects – per- Therefore, a special emphasis is placed by the permanent Quality Assurance Panel formed in collaboration with several local the research group on the study of the con- Radiooncology of the German Hodgkin and international partners – is to synthesize tribution of functionalised bio-surfaces in Lymphoma Group and the membership in advanced nanoparticles (NPs) composed enhancing cell adhesion and in protracting the Oncology Advisary Board of the Austrian of a coated super-paramagnetic iron oxide culture protocols in order to enable long- Ministry of Health with the mission to create core (SPIOs), to accommodate chemothera- term treatment investigations of fractionat- a national cancer programme peutics on the surface of NPs, and to inves- ed radiotherapy in vitro.

Research Report 2015 Medical University of Innsbruck 185 Department of Therapeutic Radiology and Oncology

Ira-Ida Skvortsova The research programme of the EXTRO-Lab FaDu-IRR and SCC25-IRR, which survived transition (EMT), stem cell differentiation, is dedicated to the development of novel after repeated exposure of the parental and blood vessel development. All of the biomarkers and therapeutic targets to pre- FaDu and SCC25 cells to ionizing radiation proteins identified using the proteomics dict and improve radiation tumour response (10 Gy, 16 MV X-rays) in an Electa Precise approach had abundant relationships with and individualize cancer therapy. In order to Linear Accelerator (Elekta Oncology Rac1 protein. Furthermore, Rac1 is closely reach this endpoint of the programme, it is Systems, Crawley, UK). The cells received linked to the intracellular pathways that are necessary to know more about the molec- a total dose of 100 Gy. The newly obtained predicted to be activated in IRR HNSCC ular background of primary and secondary IRR cells retained their radiation resistance cells (Fig. 3). (acquired) radiation resistance. even after 3 years of passaging. Exponentially growing HNSCC carcinoma Therefore a proteomics based approach is Additionally, the IRR cells possess not only cells were analysed for expression of ErbB widely used to identify the most commonly radiation resistance, but are also insensitive family members using RayBio® Human altered pathways in carcinoma cells to cisplatin, docetaxel, and EGFR blockers. EGFR Phosphorylation Antibody Array 1 exhibiting defined radiation responses. These facts have a profound clinical impact: Kit (RayBiotech, Inc, Norcross, GA, USA). Proteome-related experiments are being HNSCC relapsing after radiotherapy could Integrated density values (IDVs) were nor- conducted in our lab and also in collaboration also be resistant to chemo- and targeted malized against the signal intensities of with an OncoProteomics Laboratory in therapeutics, hence the arsenal of agents positive controls after background correc- Amsterdam (The Netherlands). Additionally available to treat HNSCC recurrences would tion. Columns represent the mean value Dr. Skvortsova has developed a series be very restricted. including standard deviation obtained from of collaborations with t leading cancer three independent experiments (*p < 0.05; translational researchers worldwide (USA, The software PathwayStudio 10.3 (Elsevier **p < 0.01; ***p < 0.001) (Fig. 4) Spain, Germany, Netherlands, Belgium, B.V., Amsterdam, The Netherlands) was Greece, Israel, Japan, Slovakia). used to analyze proteins showing differing Differences in the migration of parental expression in treatment-resistant IRR cells and IRR HNSCC cells, and Rac1 inhibitor- Since CSCs are refractory to conventional and in treatment-sensitive parental HNSCC treated cells (which induces a repression therapeutic approaches, it would be useful cells, in order to determine their common in cell migration), were determined using a to establish a preclinical in vitro model of targets (cell processes). In addition to our QCMTM 24-well colorimetric cell migration treatment resistance, in order to elucidate results reported in 2011, which illustrated assay (Merck Millipore, Darmstadt, the intracellular molecular mechanisms of involvement of proteins in cell motility, Germany), following the manufacturer’s CSC insensitivity to anti-cancer therapy. In- migration, invasion, adhesion and neoplasm instructions. HNSCC cells harvested in the deed, we have recently generated two radi- metastasis, further cell processes were appropriate serum-free quenching medium ation-resistant HNSCC cell lines (IRR cells): also identified: epithelial-to-mesenchymal were placed in the upper insert with an 8-µm

186 Research Report 2015 Medical University of Innsbruck Therapeutic Radiology and Oncology

pore size polycarbonate membrane. The lower chamber contained culture medium with a chemoattractant (10 % FCS). Plates were incubated for 24 hours at 37°C in a 5 % CO2 humidified atmosphere. HNSCC cells that migrated through the membrane were stained and then subsequently extracted using extraction buffer. The optical densities of dye extracts were read at 560nm using a microplate reader (Bio-Rad Microplate Reader 680, Bio-Rad Laboratories GmbH, Munich, Germany). **p < 0.01; ***p < 0.001. (Fig. 5)

Selected Publications Nucleophilic cross-linked, dextran coated iron oxide nanoparticles as basis for molecular imaging: synthesis, characterization, visualization and comparison with previous product. Borny R, Lechleitner TW, Schmiedinger T, Hermann M, Tessadri R, Redhammer G, Neumüller J, Kerjaschki D, Berzaczy G, Erman G, Popovic M, Lammer J, Funovics M. CONTRAST MEDIA MOL IMAGING. 2014; 10: S.18–27. Cryo-immunoelectron microscopy of adherent cells improved by the use of electrospun cell culture substrates. Schmiedinger T, 2014, 1(8): 513–522

Vogel GF, Eiter O, Pfaller K, Kaufmann WA, Flörl A, Gutleben K, . Schönherr S, Witting B, Lechleitner TW, Ebner HL, Seppi T, Hess MW. TRAFFIC. 2013; 8: p. 886–93.

Proteomics of Cancer Stem Cells. Skvortsov S, Debbage P, © Oncoscience Skvortsova I. Int J Radiat Biol.2014 Aug;90(8):653–8. Crosstalk between DNA repair and cancer stem cell (CSC) associ- Fig. 4: Receptor status (EGFR and ErbB2, ErbB3) in radio-resistant HNSCC cells ated intracellular pathways. Sergej Skvortsov, Paul Debbage, Peter Lukas, Ira Skvortsova. Seminars in Cancer Biology 06/2014; 31. DOI:10.1016/j.semcancer.2014.06.002. Rac1 as a multifunctional therapeutic target to prevent and combat cancer metastasis. Arnold CR, Abdelmoez A, Thurner G, Debbage P, Lukas P, Skvortsov S, Skvortsova I. Oncoscience. 2014, 1(8): 513–522.

Selected Funding • SEMPER: SEcondary Malignoma – Prospective Evaluation of the Radiotherapeutic dose distribution as the cause for induction, Oncotyrol, Lukas P. (400,000 €) • NanoDisc-Biomedical application of nano-surfaces on CD-for- mates, FFG-818050, Seppi T, Lechleitner T (588,700 €) • i-Scaff – Intelligent scaffolds of electro-spun nano-fibres in advanced cell culture models, TZS-Translational Research Pro- gram, Seppi T, Schmiedinger T (294,000 €) • Multiwell-Discs with reference microstructures, FFG-833467, Schmiedinger T (117,000 €)

Collaborations • Fachhochschule Vorarlberg, Feldkirch, Austria • Unit of Hydraulic Engineering, University of InnsbruckI; Inns- bruck, • Institute of Physics, Academy of Sciences Prague, Prague, Czech Republic • Università del Sacro Cuore, Rome, Italy • Department of Engineering, University of Trento, Trento, Italy • OncoProteomics Laboratory in Amsterdam (The Netherlands) • Anna Dubrovska, OncoRay – National Center for Radiation Re- search in Oncology • Medical Faculty Dresden Carl Gustav Carus, TU Dresden Fet- scherstr. 74 / PF4101307 Dresden, Germany • Connie R. Jimenez, OncoProteomics Laboratory, Dept. Medical Oncology, VUmc-Cancer Center Amsterdam, Room CCA 1-60, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands • Emilie Varin, Translational Research Officer, EORTC, Avenue .E Mounier 83/11, Brussels, Belgium • Takashi Imai, Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Ins- titute of Radiological Sciences, 4-9-1 Anagawa, Inage, Chiba 263-8555 Japan • Silvia Pastorekova, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 84505 Bratislava, Slovakia • MedAustron, Wiener Neustadt, Austria • University for Health Sciences, Medical Informatics and Tech- nology (UMIT), Hall i.T. / Department of Biomedical Computer Science and Mechatronics Fig. 5: Effects of Rac1 inhibitor on HNSCC cell migration © Oncoscience. 2014, 1(8): 513–522

Research Report 2015 Medical University of Innsbruck 187 Department of Dermatology and Venereology Dermatology, Venereology and Allergology & Experimental Dermatology Research Unit

Keywords immunobiology of the different types of skin dendritic cells, with emphasis Dendritic cells, genodermatoses, histio on epidermal Langerhans cells. The cytoses, HIV, immunity, lupus, melanoma, immunogenic function of Langerhans cells parasitoses, psoriasis, skin has been investigated in the context of skin cancer (melanoma and squamous Research Focus cell / basal cell carcinoma). Hereby, several different mouse tumor models, including • Dendritic cells spontaneously arising melanoma, have • Epidermal biology been used to determine the phenotype • Cutaneous immune system, autoimmunity of tumor infiltrating dendritic cells and • Infectious diseases of the skin, HIV/AIDS effector cells, such as T cells and natural • Photomedicine killer T cells. The occurrence and function of • Dermatohistopathology myeloid suppressor cells and their influence • Clinical trials on the growth and metastasizing potential of tumors was studied. Importantly, mouse General Facts models, in which defined subsets of skin dendritic cells can be depleted, have been Director: In the dermatology department, basic employed to clarify the importance of these Univ.-Prof. Dr. Matthias Schmuth research and patient care are intimately cells for the development, surveillance and interconnected with each other. This is ulti- ultimately therapy of tumors. Based on these Contact: mately of advantage to our patients, who to findings ew currently attempt to develop Anichstraße 35 benefit from new diagnostic and therapeu- novel alternative immunotherapies which 6020 Innsbruck tic approaches early in development. can be tested for their efficacy in mouse tumor models and eventually translated into [email protected] Research applications for human medicine. Phone: +43 512 504 24801 Fax: +43 512 504 23002 Differential Effects of Allergens and Research with Special Emphasis on the http://dermatologie.tirol-kliniken.at Non-Allergenic Antigens on Human Clinical Application of Dendritic Cells Dendritic Cells for Immunotherapy Christine Heufler-Tiefenthaler, Nikolaus Romani, Patrizia Stoitzner Norbert Reider Earlier research from our department has led Patients suffering from type I, IgE-mediated to the development of a widely used method allergies constitute an important clientele to generate large numbers of dendritic of our department. Dendritic cells initiate cells from monocytes of the human blood. and regulate virtually all immune reactions These dendritic cells have been used as a in the body, including the undesired allergic powerful adjuvant to generate anti-tumor hypersensitivities. It is still unclear why immune responses. In principle, dendritic chemically closely related molecules can cells from the blood of patients are grown be either allergenic or non-allergenic. We in vitro, “loaded” with tumor antigens work on identifying the molecular basis (peptides), and re-infused into the patients for these differential responses in human in order to elicit a potent cytotoxic anti- dendritic cells exposed to non-allergenic tumor T cell response. This approach has and allergenic lipocalins, the most frequent proven successful in several mouse models, group of animal derived respiratory and responses, though not curative, were allergens. Differential T cell responses and broad gene expression analyses of dendritic cells have been performed by microarray. Candidate molecules were identified, and Head of Experimental Dermatology: their intracellular trafficking and processing Univ.-Prof. Dr. Nikolaus by dendritic cells are now being studied Romani in detail. The expected results will add to our knowledge of the fundamental biology Contact: of dendritic cells and may help to better Anichstraße 35 understand the development of allergies to 6020 Innsbruck respiratory antigens. Fig. 1: Network of Langerhans cells in an [email protected] Immunological Studies on Dendritic epidermal sheet specimen. Approximately Phone: +43 512 504 28559 Cells of the Skin with Emphasis on 700 Langerhans cells reside in one square Fax: +43 512 504 23002 Immunosurveillance against Cancer millimeter of epidermis. These cells are di- Patrizia Stoitzner rectly targeted in situ by antibody-coupled The main topic of our research is the vaccines.

188 Research Report 2015 Medical University of Innsbruck Dermatology, Venereolgy and Allergology/Experimental Dermatology Research Unit

recorded clinical and immune parameters on chronic inflammatory skin disease are integrated with information about (psoriasis), allergies, skin cancer, HIV and response to therapy and thus provide a rich genetic skin diseases. Although most of scientific resource with which to address our current trials are pharma-initiated, the questions of both disease mechanisms Department also supports the planning and and therapeutic strategies. Recent work realization of investigator-initiated trials. has demonstrated serologic markers of The unit works in close collaboration with malignancy in patients with autoimmune the Coordination Center for Clinical Tri- disease. als (KKS) and the Comprehensive Cancer Center. Infectious Diseases of the Skin Robert Zangerle, Mario Sarcletti, Martin Selected Publications Gisinger, Maria Kitchen-Hosp, Reinhard Treatment modification in HIV-Infected individuals starting antiretroviral therapy between 2011 and 2014. Rappold M, Rieger Höpfl A, Steuer A, Geit M, Sarcletti M, Haas B, Taylor N, Kanatschnig M, This research program addresses questions Leierer G, Ledergerber B, Zangerle R. of HIV epidemiology and response to J Int AIDS Soc. 2014; 17:19768S. Murine Langerin+ dermal dendritic cells prime CD8+ T cells while Fig. 2: Electron microscopic visualisation of therapy using the large national OEHIVKOS Langerhans cells induce cross-tolerance. Flacher V, Tripp CH, lamellar bodies and secreted lipid contents cohort that was initiated, and is led by Mairhofer DG, Steinman RM, Stoitzner P, Idoyaga J, Romani N. in the intercellular space at the stratum researchers from the Innsbruck Department EMBO Mol Med. 6:1191–20, 2014. The late endosomal adaptor molecule p14 (LAMTOR2) represents granulosum – stratum corneum junction in of Dermatology. The cohort is increasingly a novel regulator of Langerhans cell homeostasis. Sparber F, the outer epidermis. linked to European and international Scheffler JM, Amberg N, rippT CH, Heib V, Hermann M, Zahner SP, Clausen BE, Reizis B, Huber LA, Stoitzner P, Romani N; collaborative efforts, e.g. the Antiretroviral Blood. 123:217–27, 2014. observed in clinical trials, including our Therapy Cohort Collaboration (ART-CC), Human skin dendritic cells can be targeted in situ by intrader- own patients. We started investigating the the Collaboration of Observational HIV mal injection of antibodies against lectin receptors. Stoitzner P, Schaffenrath S, Tripp CH, Reider D, del Frari B, Djedovic G, Ebner direct targeting of dendritic cells in the skin Epidemiological Research Europe (COHERE) S, Romani. Exp Dermatol. 23:909–915, 2014. with antibody-antigen conjugates (anti- in EuroCoord, the CASCADE Collaboration Eosinophilic leukocytoclastic vasculitis – a spectrum ranging from DEC-205, anti-langerin), by intradermal in EuroCoord, and EuroSIDA in EuroCoord. Wells’ syndrome to Churg-Strauss syndrome? Ratzinger G, Zankl J, Eisendle K, Zelger B. and epicutaneous application in a human Additional research addresses sexually Eur J Dermatol. 24:603–10, 2014. skin explant model. Different dendritic cell transmitted infections (STI) and skin Detection of Ro/SS-A antibodies in lupus erythematosus: what subsets were targeted differently. Based disorders caused by viruses (HPV), as well does it mean for the dermatologist? Böckle BC, Stanarevic G, Sepp on these findings ew are currently studying as by various parasites and fungi. NT. J Am Acad Dermatol. 68:385–94, 2013. In human monocyte derived dendritic cells SOCS1 interacting the T cell stimulatory functions of skin with CYTIP induces the degradation of CYTIP by the proteasome. dendritic cells in this model. We are testing Photomedicine Grabher D, Hofer S, Ortner D, Heufler .C PLoS One. 8:e57538, the hypothesis that antibody-conjugated Gudrun Ratzinger, Cornelia Gattringer 2013. Nail psoriasis masqueraded by secondary infection with Rhodotor- antigen elicits massively augmented T cell This research program addresses the ula mucilaginosa. Martini K, Müller H, Huemer HP, Höpfl .R responses. Such immunization, for instance effects of UV-irradiation as a causative Mycoses. 56:690–2, 2013. against neoantigens in cancer, could prove factor of photodermatoses, as well as the highly useful in tandem with immune therapeutic effects of UV-irradiation on Selected Funding • The role of PXR in epidermal homeostasis and immuni- checkpoint therapies. common inflammatory skin diseases and ty relevance to non-melanoma skin cancer; FWF P21449; skin cancer. Ongoing trials investigate the 2009–2014;Dubrac; € 308,414 • The role of the p14-MP1-MAP kinase scaffold complex in Epidermal Biology and Genodermatoses effects of phototherapy on the autoimmune Langerhans cell biologyof skin dendritic cells in cancer and Sandrine Dubrac, Robert Gruber, skin disorder scleroderma and on cutaneous infection. FWF P23548 ;2011–2015; Romani; € 301,744 • Development of immunotherapeutic strategies against skin Verena Moosbrugger-Martinz, T-cell lymphoma. cancer; MCBO project part 15; 2011–2014; Stoitzner; € 63,800 Matthias Schmuth • Einfluss onv Lipocalinrezeptoren auf dendritischen Zellen auf die Entwicklung von Allergie; ÖNB 15069; 2012–2015; Heufler; This research group focuses on the Dermatohistopathology € 100,000 biological processes that regulate the Bernhard Zelger • Dendritische Zellen: Brücke zwischen angeborener und erworbener Immunität bei Krebs; FWF P27001; 2014–2017; interplay between skin barrier function Dermatohistopathology research in Stoitzner; € 332,876 and cutaneous inflammation. Ongoing Innsbruck is renowned for innovative projects address the genetic causes, as concepts describing a variety of Collaborations well as epidermal structure and function morphologic discoveries in many skin • Universitäts Hautklinik, Wien (Profs. Georg Stingl, Adelheid Elbe-Bürger, Erwin Tschachler) in genodermatoses; i.e. disorders of disorders, with special emphasis on soft • Universitäts Hautklinik, Erlangen (Prof. Gerold Schuler) cornification and atopic dermatitis, as well tissue tumors and cutaneous vasculitis. • Laboratory of Cellular Physiology & Immunology, The Rocke feller University, NY (Prof. Michel Nussenzweig) as the role of nuclear hormone receptors Current collaborative research projects • Stanford University (Assoc. Prof. Juliana Idoyaga) (PPAR, LXR, PXR) in regulating inflammation include studies characterizing inflammation • The Malaghan Institute for Medical Research, Wellington, NZ (Prof. Franca Ronchese) and epidermal differentiation. and rejection in limb transplantation. • Erasmus Universität, Rotterdam und Johannes Gutenberg Uni- versität Mainz (Prof. Björn Clausen) • Université de la Mediterranée, Marseille (Prof. Bernard Cutaneous Immune System, Clinical Trials Malissen) Autoimmunity Norbert Reider, Gudrun Ratzinger, • University of California at San Francisco (Profs. Peter Elias, Ken Feingold) Norbert Sepp, Barbara Böckle, Robert Zangerle, Van Anh Nguyen, • Université de Strasbourg, Frankreich (Dr. Vincent Flacher) Gudrun Ratzinger Georg Weinlich, Matthias Schmuth • Universität Zürich, Schweiz (Prof. Christian Münz) This group has established registries of The department’s clinical trial unit carries autoimmune skin disorders. Carefully out numerous phase I-III clinical trials

Research Report 2015 Medical University of Innsbruck 189 Department of Ophthalmology and Optometry Ophthalmology and Optometry

funding from the “Fonds zur Förderung der proteolytically processed which results in wissenschaftlichen Forschung” (FWF). the generation of peptides, in particular Furthermore, there is cooperation with of SN, secretolytin, PE-11, WE-14, GE-25, several other institutes at the Medical catestatin, pancreostatin, vasostatin and University Innsbruck, especially with serpinin. The distribution of nerve fibres for Reiner Fischer-Colbrie from the Institute these peptides will be explored including of Pharmacology, Christian Humpel from evaluation of the origin of the nerve fibres. the Laboratory of Psychiatry, Heidi Fiegl from the Laboratory of Gynaecology and Ophthalmooncology Rudolf Kirchmair from the Department Nikolaos Bechrakis, Heidi Fiegl, Josef of Internal Medicine. The laboratory at Troger the Department of Ophthalmology is well Several tumours are characterized by equipped. The fact that there are standard abnormal DNA-methylation. This might methods well established in the laboratory also be the case for intraocular tumours and that cooperation exists with eminent such as uveal melanoma or retinoblastoma. scientists guarantees a very high quality of Therefore, the methylation pattern will the research in this Department. be explored in these tumours and will be compared with “normal” choroidal tissues. Director: Research Consequently, abnormal DNA methylation Univ.-Prof. Dr. Nikolaos Bechrakis can indeed be found and as a next step it Neuropeptides in the Eye should be determined whether specific Contact: Josef Troger, Yvonne Nowosielski, methylation patterns correlate with the Anichstraße 35 Nikolaos Bechrakis prognosis of the tumours. This topic is 6020 Innsbruck The aims of this topic are concentrated on currently being pursued. three items: 1) it should be investigated [email protected] whether certain neuropeptides contribute Ocular Neovascularization and Effects Phone: +43 512 504 23721 to pathologic neovascularisations in the of Therapeutic Angiogenesis Inhibition Fax: +43 512 504 23722 eye. The peptides of interest are substance Claus Zehetner, Nikolaos Bechrakis, http://augenklinik.tirol-kliniken.at/ P (SP), neuropeptide Y (NPY), secretoneurin Gerhard Kieselbach, Martina Kralinger (SN) and catestatin because each of Neovascular age-related macular degener these molecules exert a very pronounced ation (AMD) is the leading cause of visual proangiogenic activity. The diseases of loss in ageing western populations. The interest are wet age-related macular current standard of care involves intravitreal Keywords degeneration (ARMD), proliferative diabetic administration of monoclonal antibody- retinopathy, central retinal vein occlusion based therapies directed against vascular Neuropeptides, ophthalmooncology, anti- and retinopathy of prematurity since endothelial growth factor (VEGF). VEGF is VEGF therapy each of these diseases are characterized a multifunctional cytokine that regulates by the development of abnormal antiapoptotic pathways of endothelial cells Research Focus neovascularizations. 2) To find out whether in adult vasculature. Systemic VEGF acts as certain neuropeptides act endogenously a vascular protective factor and is essential The research at the Department of neuroprotectively on ganglion cells in the for maintaining the integrity and the anti- Ophthalmology in Innsbruck is focused on retina. Several neuropeptides are known to thrombogenic as well as anti-inflammatory three topics: have a very strong neuroprotective potency. properties of the endothelium. Our study • Neuropeptide research in the eye This is known from PACAP, VIP (and the group could demonstrate that intraocular • Investigations on tumours in the eye VIP-related proteins ADNF and ADNP) application of VEGF inhibitors can induce a • Anti-VEGF therapy especially in wet ARMD but also from SN, SP, NKA and serpinin and diabetic macular edema and since these peptides are present in the retina and are in close contact with General Facts ganglion cells, they might indeed act endogenously neuroprotectively. If this The main scientific aim at the Department could be confirmed, this ouldw have far- of Ophthalmology in Innsbruck is to perform reaching consequences in the therapy of basic research on the topics listed above. diseases such as glaucoma. The aim must For this purpose, a laboratory is available be consequently to develop eye drops that where most of the methods are established maintain or accelerate the neuroprotective which are necessary to make investigations mechanisms in the retina, which represents on the three topics, in particular cell culture a complete novel approach in the therapy of with migration and proliferation assays, this disease. 3) To investigate the presence histochemistry (immunofluorescence), and distribution of chromogranin-derived Fig. 1: Catestatin-immunoreactive nerve ELISAs, radioimmunoassays and RP-HPLC. neuropeptides. There are three main fibers in the stroma of the ciliary body (ar- There are two medical technicians and one granins: chromogranin (Cg) A, CgB and rows) and in the stroma of the ciliary pro- Ph.D. candidate who has been employed by secretogranin (Sg) II. These proteins are cesses (arrowheads).

190 Research Report 2015 Medical University of Innsbruck Ophthalmology and Optometry

Acta Ophthalmologica 2015

(pg/ml) with significantly different equilibrium significant reduction of systemic VEGF. Our 300 dissociation constants (K ). Data on study results are ofD translational relevance binding affinities demonstrated that afliberceptin regards binds to VEGF-A clinical withsafety an. Sustained approximatelyreduction 100-fold of systemic higher VEGF binding in the general 250 affinitycirculation than that is of an ranibizumab inadvertent or off-target bevacizumabeffect that and might it thereby increase neutralises the incidence of VEGF-Acardiovascular with greater anti-VEGF potency. class effects. 200 Accordingly,The selective it was therapeutic found that interference far with lowerfactors levels of of aflibercept the proangiogenic are required signalling for similarcircuit anti-VEGF for the efficacytreatment in neo-of pathologic vascularisationneovascularization models (Holash is likely et al.to result in 150 2002; Rudgecompensatory et al. 2007). increases of other factors Systemicinvolved exposure in this after process intravitreal. This could injectioninduce of anconv anti-VEGFerse regulatory agent effects is that another key factor that determines its 100 might weaken the therapeutic efficacy effect onof plasmaantiangiogenic VEGF concentrations. drugs. Our study group Concentrations of plasma VEGF Bioavailabilityfound a issignificant one of the systemic principal upregulation pharmacokineticof the proangiogenic properties ofcytokine drugs placental 50 and definedgrowth asfactor the quantity (PlGF) ofafter active intravitreal drug thatadministration reaches systemic of circulation.VEGF inhibitors. The engineeredSecondary molecularalterations design of proangiogenic of † MDD afliberceptfactors is ancould important potentially determinant promote an escape 0 Baseline 7 Days* 4 Weeks* Baseline 7 Days 4 Weeks Baseline of its highfrom bioavailability. angiogenesis Afliberceptinhibition and is may be Aflibercept Ranibizumab Control a novelresponsible type of multi-component for the decreased cyto- therapeutic kine trap,efficacy which or employs persistence the fusion of the of neovascular Fig. 1. Plasma levels of vascular endothelial growth factor (VEGF) before and after intravitreal Fig. 2: Plasma levels of vascular endothelial growth factor (VEGF) before and after intraparts- fromtissue endogenousin patients undergoing VEGF recep- antiangiogenic injection of anti-VEGF therapeutics in patients with exudative age-related macular degeneration. tors. The molecule consists of the Ig vitreal injection of anti-VEGF therapeutics in patients with exudative age-related macular therapy. Identification of factors that confer Systemic VEGF concentration was significantly decreased by aflibercept after 7 days and this domain of human VEGFR1 and the Ig degeneration. Systemic VEGF concentration was significantly decreased by aflibercept after antiangiogenic drug resistance would reduction persisted throughout 4 weeks. No significant effects were seen with ranibizumab. domain of human VEGFR2 fused with 7 days*Statistically and this significant reduction differences persisted are indicatedthroughout by an 4 weeks. asterisk. †NoThe significant minimum detectable effects dose were seen enable development of the next generation the constant region (Fc) of human with(MDD) ranibizumab. of VEGF defined*Statistically by the manufacturer significant was differences 9 pg/ml. All are measurement indicated outliers by an are asterisk. below †The of drugs for more effective treatment of the MDD. IgG1. Ranibizumab is a recombinant minimum detectable dose (MDD) of VEGF defined by the manufacturer was 9 pg/ml.humanised All ocular monoclonal vasculopathies IgG1. kappa- measurementat the 1-month outliers sampling are below point. the Con-MDD. described significant differences in the isotype antibody Fab fragment with a versely, the systemic levels of VEGF systemic concentration-time profiles molecularSelected weight Publications of about 48 kD, for after intravitreal ranibizumab after intravitreal injections of afliber- whichCatestatin-like the recombinant immunoreactivity 1gG1 in antibodythe rat eye. Gramlich Oliver W, Lorenz Katrin, Grus Franz H, Kriechbaum remained constant without suppression cept and ranibizumab. For rani- bevacizumabMaren, Ehrlich served Daniela, as Humpel the parent Christian, mol-Fischer-Colbrie Reiner, relative to baseline. bizumab, no accumulation could be ecule. ThereBechrakis has Nikolaos been E, Troger some Josef controversy. In a study by our group, we dem- detected during the loading dose of aboutNEUR theOPEPTIDES ability. 2014; ofbevacizumab 48: p. 7–13. to onstrated a significant reduction of three intravitreal injections, whereas penetrateSystemic the Upregula retination dueof PDGF to ‑B in being Patients aWith Neovascular AMD. systemic VEGF after intravitreal systemic accumulation was observed largerZehetner fullsize Claus, IgG1 Kirchmair (149 Rudolf kD)., Neururer Accord- Sabrina B, Kralinger administration of bevacizumab. In the for aflibercept. Correspondingly, the ingly, ranibizumabMartina T, Bechrakis was Nikolaos developed E, Kieselbach as Gerhard a F. INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE. 2014; 55: present study, a similar off-target effect systemic exposure increases in patients cleavedp. Fab337–344 lacking. an Fc region as the was observed in all patients treated treated with aflibercept and it has been smallerReduced-Fluence size was thought Photodynamic to enhance Therapy Combined its with Ranibi- with aflibercept. In both studies, no shown to be more than 10-fold greater diffusionzumab from for Nonproliferative the vitreous Macular intoTelangiectasia the Type 2. significant effects on plasma VEGF compared to a similar treatment regime retinaZehetner and Claus, choroid Haas Gertrud, (Ferrara Treiblmayr et Bernhard, al. Kieselbach Gerhard F, Kralinger Martina T. levels could be detected after intravi- with ranibizumab. The measured con- 2006).OPHTHALMOLOGICA Contrary to. 2013; this 229: p. 195–202 original. treal ranibizumab application (Zehet- centration of aflibercept was sufficient hypothesis,Plasma levels immunohistochemistryof vascular endothelial growth factor before and ner et al. 2013). The data of our to reach levels well above the IC50 for studiesafter and intravitreal findings injection of systemic of bevacizumab, VEGF ranibizumab and pe- current study for aflibercept are in line VEGF (Avery et al. 2014). blockadegaptanib after in patients intravitreal with age-related injection macular of degeneration, and in patients with diabetic macular oedema. with the recent findings of Wang et al. Aflibercept is the highest-affinity bevacizumabZehetner Claus, proved Kirchmair Rudolf the, Huber effective Stefan, Kralinger Martina (2014) who describe significantly sup- VEGF blocker described to date. The penetrationT, Kieselbach of Gerhard the F. larger molecule pressed systemic VEGF levels 1 month characteristics of an anti-VEGF agent throughBRITISH all retinalJOURNAL OF layers OPHTHALMOLOG and theY. cho-2013; 97: p. 454–459. Correlation of vascular endothelial growth factor plasma levels after intravitreal aflibercept injection are defined by differences in important roid, despiteand glycemic its control bigger in patients molecule with diabetic size retinopathy. and no significant effects for rani- physicochemical properties of the (HeiduschkaZehetner Claus, et al. Kirchmair 2007). Rudolf The, Kralinger ease Martina, of Kieselbach bizumab. respective molecules. Distinct binding penetratingGerhard. the retina and passing into ACTA OPHTHALMOLOGICA. 2013; 91: p. e470-e473. Pharmacokinetics might provide a kinetics of aflibercept and ranibizumab the systemic blood circulation across rationale for these systemic effects on are factors that help explain the varied the blood-retinaSelected Funding barrier is thought to VEGF plasma levels. Anti-VEGF effects on plasma VEGF levels. The be mediated by the presence of FcRn Fig. 3: Plasma levels of PlGF before and after intravitreal injection of anti-VEGF therapeutics VEGF and neuropeptides in experimental choroidal neovasculari- drugs are very potent agents with IC50 three intravitreally injected VEGF receptorszation, (Kim FWF, Nikolaos et al. Bechrakis 2009). in pa(inhibitorytients with concentration) neovascular AMD. values In in those the treatedinhibitors with thataflibercept, are currently PlGF levels used significant in -Aflibercept and bevacizumab both ly subnanomolarincreased after 7 range. days, and Avery this increase et al. persistedclinical practice throughout bind human4 weeks. VEGF-A No significantshare theCollaborations structural element of an Fc effects were seen in the ranibizumab and bevacizumab cohort. Statistically significant dif- Oliver Gramlich, Katrin Lorenz, Franz Grus, Maren Kriechbaum: Experimental Ophthalmology, Department of Ophthalmology, ferences (***P < 0.001, **P < 0.01). †The MDD of PlGF defined by the manufacturer was University Medical Center, Johannes Gutenberg-University Mainz, 12 pg/mL. All measurement outliers are below the MDD Germany e157

Research Report 2015 Medical University of Innsbruck 191 Women’s Health Center Women’s Health Center

of activity outside of the hospital, including included in the programme. We have also outpatient clinics and talks for women’s taught Gender Medicine at the annual organisations. The other part of the Gender advanced training week for the past 10 Medicine Unit is a research institute. One years and this year we intend to implement main topic is the implementation of Gender an official diploma for Gender Medicine. We Medicine into the curricula of all health are currently developing the programme for professions. At the Medical University of the courses, which will start next year. The Innsbruck we provide courses in human course will run for four terms: medicine, dental medicine and molecular medicine. Gender Medicine is included in We started with the Medical University, but the compulsory curriculum and also in the our aim is to implement Gender Medicine compulsory examinations. Moreover it is in the curricula of all health professionals; compulsory in the PhD-programmes. The for instance we also teach at the criteria for venia docendi also include a Fachhochschule Gesundheit (for midwives compulsory Gender Medicine course (SOS and all technical-medical professions) and Lehre). The main aim of this course is to the school of nurses (AZW). In this context teach participants how they can implement we also conduct research on medical Gender Medicine into all of their research students’ attitude towards gender and Director: programmes, not only in the case of clinical gender medicine. Medical students, but Univ.-Prof.in Dr.in topics but also in basic research. We try to also students from allied health professions Margarethe Hochleitner motivate our students to implement aspects have been studied with regard to their of gender specific medicine into their PhD- gender sensitivity in medicine. Contact: theses and to encourage them to produce Innrain 66/1st floor abstracts with a focus on gender issues Our research work also focuses on 6020 Innsbruck for submission to national or international migration. We perform a lot of surveys congresses as a minimum requirement. with our migrant medical students, [email protected] Thusfar we have had approximately 30 predominantly those of Turkish origin and Phone: +43 512 504 25710 posters concerned with gender topics also with our Turkish patients. Fax: +43 512 504 25719 accepted for presentation at national or http://fgz.i-med.ac.at/ international conferences. Another focus of our research is on sex and violence. We developed a questionnaire for Gender Medicine is also a (key) component recording patient histories that has been of the post-graduate training programmes approved by the Ethical Commission for Keywords of the doctor’s association in Austria. The our patients at the Women’s Health Clinic Gender Medicine ring lecture series is and by the midwives. The aim is for our Gender medicine, women’s health, lifelong learning, prevention, affirmative action for women, work-life-balance

Research Focus

• How to get Gender Medicine into the Medical Curricula? • How to get Gender Medicine into the Clinic? • Gender Medicine and Prevention, Cardiol- ogy • Women’s Empowerment, Women’s Careers, Work-Life-Balance • Diversity, Migration • How to deal with Sex and Violence in a Medical Setting

General Facts

The Gender Medicine Unit includes a Women’s Health Clinic focusing on all aspects of Women’s Health issues. It is a routing station within the University Clinics. There is a special focus on migrant women. The Women’s Health Clinic consists of an ambulance and a ward, but there is also a lot Ring Lecture Series Gender Medicine

192 Research Report 2015 Medical University of Innsbruck Women’s Health Center

Ring Lecture Series Gender Medicine

paper/form/questionnaire to be utilized in ascertain their study situation, problems performed in order to improve the working everyday clinical practice. and barriers that they may encounter and situation for women. Currently female and also their perception of sex and gender. male doctors in other countries are studied Additionally we study women’s careers in Furthermore, we analyse sex specific with regard to their career aspirations. medicine. In this context several issues differences within these groups. There is These studies will inform us about Austrian- have been focused upon, such as work- also a PhD-student and a post-doc working specific and medicine-specific challenges life balance, social and gender inequality on this topic. in women’s careers. There is also a PhD- and also the challenge of working in a student and a post-doc working on this male-dominated environment. Women Sex and Violence topic. still face disadvantages when it comes to We developed a questionnaire for patients climbing the career ladder and acheiving on the topic of sex and violence, which we higher ranks in academia. Currently, we have already used at our Women’s Health Selected Publications are also focusing on these issues in other Clinic. Currently, we work with the midwives How Do We Get Gender Medicine Into Medical Education? countries (Turkey, Israel) in order to study and their patients. The next cohort will be Hochleitner Margarethe, Nachtschatt, Ulrike, Siller, Heidi. HEALTH CARE FOR WOMEN INTERNATIONAL. 2013; 34: 3–13. the differences and similarities in women’s the male patients. Our aim is to develop a Female and Male Physicians in Academic Medicine: Is Work-Li- careers in medicine from an international questionnaire for all of the patients at all fe-Balance Still an Issue? perspective. We work on a lot of Women’s of the clinics that is accepted not only by Siller Heidi, Bader Angelika, Waldenberger-Steidl Barbara, Hochleitner Margarethe issues and are very pleased to have patients but also by the care providers, i.e. IN: Thege Britta, Popescu-Willigmann Silvester, Pioch Roswitha, incorporated Gender aspects into a wide doctors. There is also a PhD-student and a Badri-Höher Sabah. Paths to Career and Success for Women in Science. Springer range of research topics, encompassing post-doc working on this topic. Verlag: 2014. both basic and clinically applied science. RichterInnenstudie im OLG Sprengel Innsbruck Medical Students’ Attitude towards Siller Heidi, Voithofer Caroline, Hochleitner Margarethe Research Gender Medicine IN: Barta Heinz, Ganner Michael, Voithofer Caroline. Rechtstatsa- chenforschung heute: Tagungsband. 2013, Innsbruck university Medical students and students from press: 2014. Implementation of Gender Medicine allied health professions have been asked Progress of Gender Medicine in Europe. Siller Heidi, Hochleitner into the Curricula about their perceptions of the subjects Margarethe. Following the completion of the EuGiM of gender sensitivity and the gender role HEALTH CARE FOR WOMEN INTERNATIONAL (in press). project on Lifelong Learning, which ideology of doctors and patients. This Collaborations developed Gender Medicine curricula for research has identified gaps in knowledge/ • Univ.-Prof.in Dr.in Vera Regitz-Zagrosek, Charité, GiM, Berlin/ implementation into master studies and understanding that need to be filled in order Germany summer schools there is now a follow-up to improve students’ gender sensitivity in • Univ.-Prof.in Dr.in Karin Schenck-Gustafsson, Karolinska, Stock- holm/Sweden project called EuGenMed “Roadmap for a medicine. There is also a PhD-student and • Assoc.-Prof.in Dr.in Petra Verdonk, Amsterdam Medical Centre, gender-sensitive approach to health care a post-doc working on this topic. Amsterdam/Netherlands research and practice in Europe”. The • Univ.-Prof.in Dr.in Ineke Klinge, Maastricht University, Maastricht/ Netherlands “Innsbruck Model” of integration of sex and Women’s Careers in Medicine • Univ.-Prof.in Dr.in Alexandra Kautzky-Willer, MUW, Internal Medi- gender in the different curricula was invited Quantitative and qualitative data have been cine, Vienna/Austria • Univ.-Ass.in Dr.in Caroline Voithofer, Leopold Franzens as example of best practice. obtained in order to gather information • Universität, Zivilrecht, Innsbruck/Austria on the challenges experienced by women • Priv.-Doz.in Dr.in Susanne Perkhofer, FH gesundheit, Innsbruck/ Austria Migration wishing to progress in their respective • Dr.in Waltraud Buchberger MSc, AZW, Innsbruck/Austria We started a lot of questionnaires with careers. Additionally, a review of the • ao. Univ.-Prof.in Dr.in Barbara Juen, Leopold Franzens University, Institute for Psychology, Innsbruck/Austria Austrian, German and Turkish medical Austrian literature about women’s careers in • Prof.in Dr.in Patricia Davidson, Johns Hopkins University, Dean of students. The questionnaire seeks to medicine, pitfalls and positive aspects was the School of Nursing, Baltimore/USA

Research Report 2015 Medical University of Innsbruck 193 Institute for Neuroscience Neuroscience

General Facts A classical example of neuronal plasticity is the switch from noradrenergic to The Institute for Neuroscience is located cholinergic neurotransmission, which on the 3rd floor of the building at Innrain 66 occurs in differentiated postganglionic in close proximity to the laboratories of sympathetic neurons under the influence Psychiatry, Neurology and Neurosurgery. of target-derived signals. We identified the Members of the Institute participate in genome organizer Special AT-rich sequence the FWF ‑funded networks SFB-F44 and binding protein 2 (Satb2) as an acutely up- DK 106 SPIN. The Institute offers a modern regulated target gene of neurokine/p38 infrastructure and state-of-the-art research MAPK signaling in sympathetic neurons equipment. Laboratories include a stem undergoing trans-differentiation. cell laboratory licensed for biosafety level 2 work dedicated to the generation and Our gain-and loss-of-function studies differentiation of human iPSCs. Procedures revealed that Satb2 is both necessary have been implemented to generate, and sufficient to trigger the sympathetic steadily maintain and differentiate human neurotransmitter switch. We reasoned that iPSC-derived cell lines. Separate laboratory modulation of Satb2 and consequently rooms are dedicated to work with nucleic chromatin architecture by neurotrophic Director: acids and proteins. In addition, the institute factors might serve as a novel pathway Univ.-Prof. Dr. Georg Dechant supports a primary cell culture and animal involved in the long-term adaptive processes transplantation laboratory. Rooms are underlying higher brain functions. Contact: fully equipped with stereomicroscopes, Innrain 66/G3 epifluorescence and confocal microscopes In support of this hypothesis, recent results 6020 Innsbruck and a specialized room is dedicated to in our laboratory showed that Satb2 is histology, sectioning and immunostaining. induced by plasticity-mediating extracellular [email protected] signals such as BDNF or Ca2+-influx through Phone: +43 512 504 27297 Research L-type voltage-gated calcium channels in Fax: +43 512 504 27390 CNS neurons. The analysis of a conditional www.i-med.ac.at/neuroscience Neuronal Plasticity Group mutant lacking Satb2 in the adult forebrain Dr. Galina Apostolova (generated by our group), demonstrated that Satb2 is required for synaptic plasticity and Modulation of Higher-Order Chroma- long-term memory formation. In addition, tin Architecture – Implications for we found that Satb2 interacts with genome Neuronal Plasticity organizing proteins of the inner nuclear membrane and regulates the geometry of Keywords Complex behaviors such as learning and neuronal nuclei in the hippocampus in vivo. memory depend on changes in gene Our findings give us grounds to hypothesize Neuronal plasticity, neurotrophic factors, expression and subsequent long-lasting that a Satb2-containing DNA-protein cell signaling, transgenic animal models, adaptations in synaptic strength and complex determines both the nuclear induced pluripotent stem cells, neuronal structure. Our current research interests shape and chromosomal conformations in differentiation, human models of neurolog- are focused on the mechanisms of neuronal neurons downstream of L-VGCC and BDNF ical diseases plasticity, with special emphasis on the role signaling, thereby integrating plasticity- of chromatin conformation regulation in mediating extracellular signals into changes Research Focus adaptive gene transcription. in the transcriptome.

Our laboratory studies how nerve cells can be programmed and re-programmed depending on neural activity and neurotrophic growth factors. In transgenic mouse models we investigate activity- dependent mechanisms of learning and memory that depend on plastic chromatin reorganization in the cell nucleus. Another focus is to generate neurons from stem cells. We are developing protocols to differentiate human stem cells obtained with the “induced pluripotent stem cell” (iPSC) technology into specific neuronal populations. Based on these protocols we have established cellular models of human Fig. 1: Immunostaining for Satb2 in sagital mouse brain sections reveals strong Satb2 ex- neurological diseases. pression in the cortex and CA1 area of the hippocamus.

194 Research Report 2015 Medical University of Innsbruck Neuroscience

Project 2. Modeling Spinocerebellar A Ataxia Type 6 with Patient-Derived iPSCs B Spinocerebellar Ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease associated with CACNA1A gene, coding for the alpha 1 A subunit of P/Q type voltage-gated calcium channel CaV2.1. SCA6 mutation consists of CAG repeats leading to a short expansion of a polyglu- tamine stretch located in the cytoplasmic C-terminal tail of the channel protein. Cur- rently, the pathogenic mechanisms remain elusive, and no therapy is known. We aim to investigate the effect of the SCA6 mutation on CaV2.1 channel protein functionality directly in human neurons. We generated iPSC lines from SCA6 patients Fig. 2: Human neurons grown from pluripotent stem cells derived from a patient with and differentiated them in neurons. We are Friedreich Ataxia (A) and from a patient with Spinocerebellar Ataxia type 6 (B). using confocal microscopy to investigate Stainings for Peripherin (A, red), Frataxin (A, green) and CAV2.1 (B, red), the subcellular localization of CaV2.1 (scale bar 10um). channel protein and neuronal excitability, calcium currents and synaptic transmission in SCA6 neurons. Our further aims are Our future goals are to provide evidence which are subjected to differentiation into to use our validated strategy to develop that Satb2-dependent rearrangements of neurons. The disease-relevant neurons drug screening assays, and to extend it to the nuclear architecture and/or changes are interrogated in order to disclose the the analysis of other monogenic diseases in the epigenetic profiles are necessary molecular mechanisms causing and/or associated with mutations in the CACNA1A for higher cognitive functions and that driving the particular disease, especially gene. dysfunction of these mechanisms leads to for the known monogenic diseases. learning and memory deficits. We also aim Selected Publications to study whether cognitive deficits inherent Ongoing Projects: Alternative generation of CNS neural stem cells and PNS deriv- to normal aging and neuropsychiatric atives from neural crest derived peripheral stem cells. Weber M, Project 1. Modeling Friedreich Ataxia Apostolova G, Widera D, Mittelbronn M, Dechant G, Kaltschmidt diseases are caused by alterations in Satb2 with Patient-Derived iPSCs B, Rohrer H. Stem Cells. 2015 Feb;33(2):574–88. doi: 10.1002/ expression or function. Friedreich Ataxia (FRDA) is an autosomal stem.1880. recessive neurodegenerative disease Reacquisition of cocaine conditioned place preference and its inhibition by previous social interaction preferentially affect Stem Cell Group caused by an elongated intronic GAA repeat D1-medium spiny neurons in the accumbens corridor. Prast JM, Priv. Doc. Dr. med. univ. Roxana Nat in the gene encoding the mitochondrial Schardl A, Schwarzer C, Dechant G, Saria A, Zernig G. Front Behav Neurosci. 2014 Sep 24;8:317. doi: 10.3389/fnbeh.2014.00317. The recent availability of the human protein frataxin. Peripheral sensory neurons eCollection 2014. pluripotent stem cells (PSC) reprogrammed are the most susceptible cells for FRDA Peripheral nerve regeneration and NGF ‑dependent neurite from adult somatic cells provides a unique pathophysiology. Animal models of FRDA outgrowth of sensory neurons depends on STAT3 phosphorylation downstream of the neuropoietic cytokine receptor gp130. opportunity to investigate the mechanisms reproduced GAA repeat expansion, frataxin Quarta S, Baeumer BE, Scherbakov N, Andratsch M, Rose- of human nervous system development and deficiency, mitochondrial alterationsJohn MS, Dechant G, Bandtlow CE, Kress M. J. Neurosci. Sep 24;34(39):13222–33. doi: 10.1523/JNEUROSCI.1209-13.2014. its disorders. There are currently 2 major and neurodegeneration observed in the Induced pluripotent stem cells from Friedriech ataxia patients fail areas of interest: human disease, but the central questions to upregulate frataxin during in vitro differentiation to peripheral concerning FRDA pathophysiology sensory neurons. Eigentler A, Boesch S, Schneider R, Dechant G, Nat R. Stem Cells Dev. 2013 15:3271–82. 1. Explore the molecular mechanisms that remained elusive: why are specific neuronal Human pluripotent stem cells modeling neurodegenerative dis- regulate PSC conversion into specific populations particularly susceptible in eases. Nat R, Eigentler A, Dechant G. In “Pluripotent Stem Cells / neural progenitor populations, their FRDA and when during ontogeny does the Book 2”, ISBN 980-953-307-463-9 Editors InTech. neuronal subtype specification and pathology manifest itself in susceptible Selected Funding functional maturation. We apply to neurons? To address these questions, we • 2013–2016: FWF DK W1206 “Signal Processong in Neurons” mouse and human PSCs different neural have generated patient iPSC lines and Dechant induction, patterning and specification differentiated them to peripheral sensory • 2013–2016: FWF Stand-alone project P25014-B24 “Role of genome organizer Satb2 in adult brain function” Apostolova protocols, based on established models neurons (Eigentler et al.2013). Functional • 2012–2015: FWF SFB-F44 “Cell Signaling in Chronik CNS Dis- of nervous system development. characteristics are compared during in orders”/MUI associated project, Nat, Dechant • 2015–2019: FWF SFB-F44 “Cell Signaling in Chronik CNS Dis- vitro maturation of control and FRDA iPSC- orders” Apostolova, Dechant 2. Modeling human neurological disorders derived sensory neurons. Furthermore, • 2014–2016: FWF Standalone Project Nr P 26886-B19, “Mode- ling Friedreich Ataxia with patient iPSC-derived neurons”, Nat with iPSC derivatives in order to we analyse whether the frataxin deficit understand the molecular mechanisms affects the development of peripheral Collaborations of diseases in pathologically-relevant sensory neurons and their circuitries • Roland Foisner, Medical University Vienna; Vienna, Austria • Marin Korte; TU Braunschweig, Braunschweig; Germany phenotypes. We reprogram patient- after transplantation of human sensory • Nicolas Singewald; Innsbruck University, Innsbruck; Austria derived somatic cells into iPSC lines, precursors in chicken embryos. • Jörg Striessnig,Innsbruck University, Innsbruck; Austria

Research Report 2015 Medical University of Innsbruck 195 196 Research Report 2015 Medical University of Innsbruck FWF–funded Programmes

Doctoral Programmes (DK): DK Molecular Cell Biology and Oncology – MCBO DK Host Response in Opportunistic Infections – HOROS DK Signal Processing in Neurons – SPIN

Special Research Programmes (SFB): SFB-F44: Cell signaling in chronic CNS disorders

Research Report 2015 Medical University of Innsbruck 197 FWF ‑funded Programmes Molecular Cell Biology and Oncology – DK MCBO

at Austrian universities. Programs complementary and transferrable skills are initiated by consortia of leading required to perform front-line research. scientists and selected through a It is the main goal of MCBO to teach stringent international evaluation its students strategies that allow them process. Programs are regularly to efficiently and successfully study reviewed and can be extended up to a features of a particular molecule or total of 12 years. a specific signalling process at the subcellular or single-cell level, as well General Facts as in the context of an entire organism.

Molecular Cell Biology and Oncology MCBO offers a comprehensive system (MCBO) is an excellence PhD program of lectures and laboratory courses . at the Medical University of Innsbruck Peer-reviewed research projects, funded by the FWF (Austrian Science dedicated supervision by three- Fund), with participation of the member thesis committees and a lively University of Innsbruck. The goal of seminar program create a stimulating Speaker: the MCBO doctoral program is to equip research environment, conducive to ao. Univ.-Prof. Dr. young researchers with the knowledge, the successful completion of the PhD. Bernhard E. Flucher skills and attitudes necessary to excel in an independent scientific career in MCBO Facts Contact: basic and applied bio-medical sciences. Biocenter, Innrain 80–82 • Established in 2005 6020 Innsbruck Training • 45 students enrolled • 62 students graduated [email protected] Research training within MCBO is • Students form 18 nations and Phone: +43 512 9003 70818 designed to prepare students for solving 3 continents www.mcbo.at basic research questions and to teach • More than 180 publications in-depth knowledge of cell biology with • Competitive recruitment the ultimate goal of creating the basis • Courses and lectures in English Keywords for the development of novel treatments • Three to four years thesis research to fight prevalent human diseases. To • International symposia and meetings Doctoral research training, Molecular achieve this goal, MCBO is dedicated to • 6 months stays abroad at prestigious Cell Biology, providing its students with a multitude Universities like the University of Austrian Science Fund (FWF) W1101 of state-of-the-art methodological California (San Francisco), skills, important basic knowledge in the John Hopkins University (Baltimore), Focus – Research Training field of cancer cell biology and tumor Karolinska-Institut (Stockholm), immunology, as well as with a set of and many more. • State-of-the-art PhD training in molecular cell biology and oncology Research Training • Benchmark training standards: competitive recruitment, training opportunities, international exchange

Research Topics

• Cell Cycle • Cell Death • Cytoplasmic signaling • Calcium channels • Tumor-Immunology

FWF DK Program

The DK-Program by the Austrian Science Fund supports structured PhD programs at centers of excellence

198 Research Report 2015 Medical University of Innsbruck MCBO Team at mid-term retreat

MCBO Faculty Former Members

Baier Gottfried, Univ.-Prof. Dr. rer. nat. Gastl, Günther, Univ.-Prof. Dr. med. univ. Division of Translational Cell Genetics, MUI Department of Internal Medicine, MUI Culig Zoran, ao. Univ.-Prof. Dr. med. univ. Klocker, Helmut, ao. Univ.-Prof. Dr. rer. nat. Department of Urology, MUI Department of Urology, MUI Flucher Bernhard, ao. Univ.-Prof. Dr. phil. Kofler, Reinhard, Univ.-Prof. Dr. med. univ. Division of Physiology, MUI Division Molecular Pathophysiology, MUI Geley Stephan, ao. Univ.-Prof. Dr. med. univ. Troppmair Jakob, Univ.-Prof. Mag. Dr. rer. nat. Division of Molecular Pathophysiology, MUI Department of Visceral-, Transplant-and Thoracic Surgery, MUI Grabner Manfred, ao. Univ.-Prof. Dr. phil. Division of Biochemical Pharmacology, MUI Hengst Ludger, Univ.-Prof. Dr. rer. nat. Division of Medical Biochemistry, MUI Huber Lukas, Univ.-Prof. Dr. med. univ. Division of Cell Biology, MUI Funding (2005 – 2017): Kleiter Natascha, Priv.-Doz. Dr. rer. nat. Austrian Science Fund (FWF): € 10,037,000 Division of Translational Cell Genetics, MUI Medical University Innsbruck: € 3,365,000 University Innsbruck (LFU): € 250,000 Lusser Alexandra, ao. Univ.-Prof. Dr. rer. nat. Total: € 13,652,000 Division of Molecular Biology, MUI Scheffzek Klaus, Univ.-Prof. Dr. rer. Nat. Division of Biological Chemistry, MUI Stoitzner Patrizia, Assoz. Prof. Dr. rer. nat. Department of Dermatology and Venereology, MUI Striessnig Jörg, Univ.-Prof. Dr. med. univ. Division of Pharmacology and Toxicology, LFU Teis David, Assoc. Prof. Dr. rer. nat Division of Cell Biology, MUI Trajanoski Zlatko, Univ.-Prof. Dipl.-Ing. Dr. techn. Division of Bioinformatics, MUI Villunger Andreas, Univ.-Prof. Mag. Dr. rer. nat. Division of Developmental Immunology, MUI Wilflingseder Doris, Priv.-Doz. Mag. Dr. rer. nat. Division of Hygiene and Medical Microbiology, MUI MCBO Students

Research Report 2015 Medical University of Innsbruck 199 FWF ‑funded Programmes Host Response in Opportunistic Infections – DK HOROS

subjects. Six of the seven members annual retreat”, and the possibility to of the consortium work at the Medical offer a professorship to a distinguished University of Innsbruck (MUI), one is researcher from abroad. Thus, HOROS heading the Institute of Biomedical strengthens the scientific environment Aging Research (IBA), now part of of the research campus Innsbruck that Leopold Franzens University (LFU) of attracts not only the best students, Innsbruck. It is envisaged to strengthen but also distinguished scientists to the the cooperation between both local campus. universities in the coming periods. Four In October 2014 six HOROS and two members of the consortium are medical HOROS-associate students started doctors – one works at bedside and 3 their projects. Another four students mainly preclinically – three are natural started in March 2015. scientists. All 7 contribute to various aspects of host-pathogen interaction, Funding comprising inherited and acquired immunity. On the infection side fungal, FWF DK W1253-B24, € 2,200,000 Speaker: bacteriological or virological models ao. Univ.-Prof. DDr. are in use. Collaborations Reinhard Würzner From Basic Research to • Peter Zipfel, Univ. Jena, Germany Contact: Clinical Implementation • Peter Garred, Univ. Copenhagen, Schöpfstrasse 41 Denmark 6020 Innsbruck HOROS is carried out by scientists • David Denning, Univ. Manchester, and physicians working in clinical Great Britain [email protected] departments or “pre-clinical” research • Beate Kehrel, Univ. Münster, Germany Phone: +43 512 9003 70707 institutes of both universities, thus • Jürgen Löffler, Univ. Würzburg, Fax: +43 512 9003 73700 providing an important translational Germany www.horos.at link between basic research and clinical • Axel Brakhage, Univ. Jena, Germany application. MUI is very interested • Ioav Cabantchik, Univ. Jerusalem, to have a broad and excellent DK on Israel “Infection, Immunity & Transplanta tion”, • Ferric Fang, Univ. Washington/ Keywords one of its major officially stated research Seattle; USA topics. A strong liaison with industrial • Andreas Radbruch, DRFZ Berlin, Host response, opportunistic partners has been established for Germany infections supplementary funding. HOROS fosters • Guiseppe del Giudice, Novartis, immunity, transplantation, even closer collaborations between Siena, Italy biogerontology research groups and the strategic • Stefan G. Tullius, Univ. Boston, USA added values lie in an attractive • Ondrej Viklicky, Univ. Prague, Czech Research Focus educational curriculum, more coherent Replublic and practical than previously. HOROS • Thomas Pietschmann, Scientists and physicians of the provides perfect means to finance Univ. Hannover, Germany Innsbruck campus, working in the research stays of PhD students in • Alexandra Trkola, Univ. Zurich, related fields of Infection,Immunity, collaborators’ laboratories, a “HOROS Switzerland Transplantation and/or Biogerontology, have decided to join forces and have created a structured and multi disciplinary research and training programme of excellence (DK). Their intention is to cooperatively and synergistically investigate genetic and environmental parameters, which destroy the immune homeostasis during host-pathogen interaction, thus leading to opportunistic infections, seldom developing in healthy, but Ao. Univ.-Prof. Dr. med. Reinhard Würzner, Ph.D (Speaker HOROS, right) and quite often in immuno-compromised ao. Univ.-Prof. Mag. Dr. Markus Reindl (Clinical Department of Neurology, SPIN).

200 Research Report 2015 Medical University of Innsbruck HOROS Faculty Members and students

HOROS Principal Investigators ao. Univ.-Prof. DDr. Reinhard Würzner phasis on the diagnosis, prevention and therapy of invasive HOROS-Speaker infections and antifungal drug resistance. Identification of factor H binding complement evasion molecules in fungi Univ.-Prof. Dr. rer. nat. Katja Kotsch: Reinhard Würzner is an expert in complement evasion strategies, Influence of donor and recipient in particular of bacteria and fungi, but also of complement age on the outcome in SOT related kidney diseases. He is the coordinating deputy head Katja Kotsch is working at the Dept. of Visceral, Transplant of all MUI doctoral programmes and also heading the related and Thoracic Surgery and was recently appointed as Professor doctoral programme “Infectious Diseases”. for Experimental Transplantation Immunology. Her research interests are related to the diagnosis and therapy of acute and Univ.-Prof. Dr. med. Beatrix Grubeck-Loebenstein chronic rejections in solid organ transplantation by defining Deputy-Speaker risk factors of allograft survival including the increasing age of The role of the human bone marrow for the transplant donors and recipients. In addition she is interested in regulation of immune responses in old age the impact of infections in this transplantation setting. Beatrix Grubeck-Loebenstein is directing the Institute for Biomedical Aging Research (IBA) which is now part of LFU. ao. Univ.-Prof. Dr. rer. nat. Heribert Stoiber: She is a leading scientist in biogerontology and in particular in Mechanisms for the specific acquisition of immunology of old age and has inaugurated the related doctoral complement regulator proteins by HCV programme “Aging”. Her contribution to HOROS will bridge Heribert Stoiber is specialized in medical microbiology and medical and science faculties of both universities. virology and deputy head of the Division for Virology. His research focuses on the interplay of viruses with the com- ao. Univ.-Prof. Mag. Dr. rer. nat. Hubertus Haas: plement system, in particular with evasion mechanisms viruses Siderophore-mediated diagnosis of fungal infections apply to circumvent the lytic action of the human complement Hubertus Haas is working at the Biocenter. He is a basic system. scientist involved in fungal diseases and in particular interested in the iron homeostasis of the fungus and the role of iron as Univ.-Prof. Dr. med. Günter Weiss: virulence factor. One of his targets are siderophores which allow Role of NRAMP-1 in the control of host resistance the fungus to acquire iron in hostile environments. against infection with intracellular bacteria Günter Weiss is professor of clinical immunology and infec- Univ.-Prof. Dr. med. Cornelia Lass-Flörl: tious diseases and is directing the Department of Internal Establishment of a human lung tissue Medicine VI. His research interest focuses on disorders of iron model to study fungal infections homeostasis and host-pathogen interaction with a special em- Cornelia Lass-Flörl is directing the Division of Hygiene and phasis on regulatory interactions between iron homeostasis, Medical Microbiology. natural resistance genes and immune function in various infec- Her research focuses on fungal infections with a special em- tions. He is the coordinator of CIIT at IMU.

Research Report 2015 Medical University of Innsbruck 201 FWF ‑funded Programmes Signal Processing in Neurons – DK SPIN

was set up to allow multidisciplinary TEACHING interaction at the interface of molecular and clinical neuroscience. Our aim is to The main goal of SPIN is to equip reach a new level of understanding of students with the practical and theo- the fundamental integrative processes retical knowledge they need in order to that govern the signaling within and actively contribute to future scientific between nerve cells under normal and advances. In order to obtain a PhD at pathological conditions. our institution, students must carry out an experimental study and complete The SPIN program has identified three the courses in the PhD curriculum. broader areas of research: • molecular/cellular neuroscience Monitoring and Mentoring • neuronal physiology and SPIN students work under the tutelage pathophysiology of a supervising professor and a board • behavioral neuroscience of advisors, the “Thesis Steering Committee” Speaker: We have initiated a variety of integrated Univ.-Prof. Dr. Georg Dechant PhD projects that combine all three Thesis Steering Committee (TSC) levels, which proves to be an ideal A thesis steering committee is Contact: learning and training environment for assembled for each student. The TSC Institute for Neuroscience our students. consists, in addition to the supervisor, Innrain 66, 6020 Innsbruck of two experienced researchers. During What is SPIN? the process of preparing the thesis, the [email protected] steering committee is to evaluate and Phone: +43 512 504 27297 The SPIN doctoral program is an supervise the progress of the PhD work http://www.neurospin.at/ initiative of the Medical University of in regular and structured meetings with Innsbruck (MUI) and the University of the student. Innsbruck (LFUI). It was established in Research Focus September 2007 with the support of Progress Report the Austrian Science Fund (FWF) and Each week, one student in the SPIN Neurological and psychiatric disorders offers interdisciplinary postgraduate network presents their progress with are one of the greatest threats to public training in translational Neuroscience their research to other students and health. Research across a wide range for excellent Austrian and international faculty members and thus marks the of sectors and disciplines is needed to students. It combines the expertise in central communication platform for all bring about the changes that people Neurosciences across departments, SPIN members. with such disorders need. making it currently the only doctoral In precisely this spirit, we use an college in Austria with an exclusive EXTRAS integrative, crossover approach. SPIN focus on Neuroscience. Students also benefit from a number

202 Research Report 2015 Medical University of Innsbruck SPIN Faculty Members and students of additional offers within the SPIN SPIN Principal Investigators | Members on Campus program. These include annual retreats Univ.-Prof. Dr. Christine Bandtlow in attractive locations in and around Division of Neurobiochemisty Austria, stays abroad to maintain old and make new contacts in their Univ.-Prof. Dr. Georg Dechant respective field, academic (soft) Institute for Neuroscience skills training workshops, as well as Univ.-Prof. Dr. Francesco Ferraguti collaborations and joint events with Department of Pharmacology our neighboring Graduate School of Systemic Neurosciences (GSN) LMU Univ.-Prof. Dr. Lars Klimaschewski Munich. Division of Neuroanatomy Univ.-Prof. Dr. med. Michaela Kress SPIN in Numbers Department of Physiology and Biomedical Physics • PIs: 10 ao. Univ.-Prof. Dr. Markus Reindl • Departments: 8 Clinical Department of Neurology • Current students: 22 (14 female, 8 male) ao. Univ.-Prof. Dr. Christoph Schwarzer • Alumni: 25 Department of Pharmacology (15 female, 10 male) ao. Univ.-Prof. Dr. Nicolas Singewald • Nationalities: 15 Department of Pharmacology and Toxicology (LFUI) ao. Univ.-Prof. Dr. Nadia Stefanova FUNDING Department of Neurology, Division of Neurobiology FWF: € 4,899,980.50 (until the end of 2016) ao. Univ.-Prof. Dr. med. Gerald Zernig MUI: € 2,448,773.50 (until the end of 2015) Experimental Psychiatry Unit

Research Report 2015 Medical University of Innsbruck 203 FWF ‑funded Programmes SFB-F44 – Cell Signaling in Chronic CNS Disorders

for the pathophy siology of Parkinsonian associated with progressive multisystem disorders (Parkinson’s disease, Multiple neurodegeneration. Our group will provide System Atrophy), Alzheimer’s disease and detailed characterization of the functional abnormal fear and anxiety. phenotype of a transgenic mouse model with targeted overexpression of α-syn in Members and Projects MUI oligodendrocytes as an important readout Importance of Intra- and Extracellular for preclinical drug screening for MSA. To Cav1.3 Modulators for Synapse Stability identify underlying pathogenic mechanisms in Normal and Diseased Striatal Medium and candidate targets for future therapies Spiny Neurons we will focus on the putative bilateral inter Gerald Obermair, Bernhard E. Flucher actions between epigenetic factors, and Division of Physiology α-syn aggregation and propagation in MSA In brain dendritic spines are small postsyn- models. The outcomes are likely to critically aptic membrane protrusions on neuronal enhance our insights into the pathogenesis dendrites involved in excitatory synaptic and progression of MSA. The results will transmission and synaptic plasticity. Neu- have immediate relevance for interventional ronal L-type calcium channels are locat- target discovery which in turn will promote ed in dendritic spines and contribute to future clinical trial activities in MSA patients. Coordinator : the local concentration of the ubiquitous Univ.-Prof. Dr. Jörg Striessnig (LFU) second messenger calcium. Thereby calci- Identification of Regulatory ncRNAs in um channels integrate synaptic signals, ef- Chronic CNS Disorders Contact: fect changes in spine morphology and the Alexander Hüttenhofer Institute of Pharmacy synaptic structure and contribute to basic Division for Genomics and RNomics Biocenter, Innrain 80–82, Innsbruck neuronal functions including learning and This project aims to identify regulatory University of Innsbruck (LFU) memory formation. Neurological diseases ncRNAs involved in neuronal development are often accompanied by synaptic adap- and chronic CNS disorders. Using special [email protected] tations including altered form and function probes and techniques recently developed Phone: +43 512 507 58801 of dendritic spines. For example, a specific in this lab ncRNAs that are regulated in dis- Fax: +43 512 507 58899 loss of dendritic spines of striatal neurons ease and may therefore contribute to signal- http://www.uibk.ac.at/pharmazie/ has previously been shown to be involved in ing pathways involved in neurodegeneration pharmakologie/sfb-f44/ the pathology of Parkinson’s disease (PD). will be identified and characterized. Existing Interestingly a loss of dendritic spines in the expertise will also be used to probe for reg- striatum may also underlie the development ulatory ncRNAs participating in L-type Ca2+ General Facts of L-DOPA-induced dyskinesia, the major channel-mediated plasticity and nuclear sig- debilitating side effect of the common treat- naling in various neurons. Chronic diseases of the central nervous ment for PD. In our project of the first SFB system (CNS), such as fear/anxiety dis funding period we have identified a specific Function of Special AT-rich Sequence- orders and neurodegenerative diseases role of a specific L-type calcium channel and Binding Protein 2 (SatB2) in Aging and occur with high and increasing prevalence. its interaction with postsynaptic proteins in Neuronal Pathophysiology Since molecular disease mechanisms are regulating the stability of dendritic spines. Georg Dechant, Galina Apostolova not fully understood, current drug therapies Building on this important result, we now Institute for Neuroscience are often unsatisfactory. The development of test in the ongoing project whether and how Complex behaviors such as learning and novel and improved therapeutic strategies this proposed mechanism contributes to the memory depend on changes in gene ex- requires the identification of innovative tar- etiology of PD and other neuronal diseas- pression and subsequent long-lasting adap- gets for therapeutic intervention. Therefore es. To this end we are employing high- and tations in synaptic strength and structure. competent laboratories at the two Innsbruck super-resolution fluorescence microscopy Current research interests of this group universities joined their complementary ex- and state-of-the-art electrophysiology. Our are focused on the mechanisms of neuro- pertise to comprehensively study signaling results will contribute to the understanding nal plasticity, with special emphasis on the pathways that bear such potential. The ma- of synaptic adaptations during neurological role of chromatin conformation regulation in jor research focus is on L-type calcium chan- disorders and probe the therapeutic po- adaptive gene transcription. nels (LTCCs) and epigenetic modulators, in tential of targeting the identified synaptic A classic example of neuronal plasticity is particular histone deacetylases (HDACs). mechanisms. the switch from noradrenergic to cholinergic These pathways appear to participate in the neurotransmission, which occurs in differ- etiology of several neurological and neu- Alpha-synuclein – a Pathogenic Trigger entiated postganglionic sympathetic neuropsychiatric disorders. Moreover, recent and Interventional Target in Multiple rons under the influence of target-derived preliminary findings from our consortium System Atrophy signals. We identified the genome organizer suggest that they can be (patho-) physiolog- N. Stefanova, G. Wenning Special AT-rich sequence binding protein 2 ically linked. Department of Neurology (Satb2) as an acutely up-regulated target Strong local expertise is bundled to study Multiple system atrophy (MSA) is a dis- gene of neurokine/p38 MAPK signaling in Ca2+-mediated, epigenetic and non-coding tinctive neurodegenerative disorder char- sympathetic neurons undergoing trans-dif- RNA (ncRNA)-mediated regulatory mecha- acterized by oligodendroglial cytoplasmic ferentiation. Gain-and loss-of-function stud- nisms to disclose the role of these pathways inclusions of fibrillarα -synuclein (α-syn) and ies of this group revealed that Satb2 is both

204 Research Report 2015 Medical University of Innsbruck necessary and sufficient to trigger the sym- fear extinction training may facilitate extinc- and context-independent manner is expect- pathetic neurotransmitter switch. Therefore tion learning and the concurrent plasticity. ed to foster the rational development of nov- modulation of Satb2 and consequently chro- These hypotheses will be experimentally el cognitive enhancers which may be used matin architecture by neurotrophic factors addressed by means of a multidisciplinary as augmenting CBT adjuncts to treat anxiety might serve as a novel pathway involved in approach combining optogenetics, viral disorders more effectively. the long-term adaptive processes underly- monotransynaptic tracing and novel ultra ing higher brain functions. structural techniques. A mouse model for Role of Cav1.2 and Cav1.3 Calcium- In support of this hypothesis, recent results non-motor symptoms of early PD, lacking Channels for Parkinson’s Disease and in our laboratory showed that Satb2 is in- motor impairments, will also be established Neuropsychiatrc Disorders duced by plasticity-mediating extracellular and characterized. Therefore, this project Jörg Striessnig signals such as BDNF or Ca2+-influx through will complement other investigations within Department of Pharmacology and L-type voltage-gated calcium channels in this SFB on aberrant signaling mechanisms Toxicology, University of Innsbruck CNS neurons. The analysis of a conditional leading to selective neurodegeneration (e.g. L-type Ca2+ channels (LTCCs) have recent- mutant lacking Satb2 in the adult forebrain PD), altered neural plasticity and abnormal ly emerged as novel drug targets for the (generated by our group), demonstrated that fear memory processing. treatment of Parkinson’s disease (PD) with Satb2 is required for synaptic plasticity and already licensed or new channel blockers. long-term memory formation. In addition, Members University of Innsbruck (LFU) The concentrations of available drugs re- we found that Satb2 interacts with genome Epigenetic Mechanisms in Aberrant quired for effective block, the LTCC channel organizing proteins of the inner nuclear Memory Regulation isoforms in involved in PD pathophysiology membrane and regulates the geometry of Nicolas Singewald and the mechanisms of neuroprotection are neuronal nuclei in the hippocampus in vivo. Department of Pharmacology and still not known. This group recently identi- Therefore Satb2-containing DNA-protein Toxicology, University of Innsbruck fied human mutations that strongly point to complex may determine both the nuclear Effective long-term treatment for fear and a crucial role of brain LTTC gain of function shape and chromosomal conformations in anxiety-related disorders is a continuing (including the so-called Cav1.3 subtype) for neurons downstream of L-VGCC and BDNF challenge. One emerging treatment strate- the pathophysiology of psychiatric diseas- signaling, thereby integrating plasticity- gy is combining exposure-based cognitive es (particularly autism spectrum disorders, mediating extracellular signals into changes behavioural therapy (CBT) with cognitive ASD). This suggests a pathogenic and thus in the transcriptome. enhancers. Key results from the first SFB also potentially therapeutic role of brain Future goals are to provide evidence that funding period (FP) support the utility of LTCCs beyond PD. New tools and assays will Satb2-dependent rearrangements of the this approach for long-term fear inhibition. be developed to determine if LTCCs in the nuclear architecture and/or changes in the Specifically, ew provide evidence that brain are blocked as effectively as Cav1.2 epigenetic profiles are necessary for higher histone deacetylase (HDAC) inhibitors and channels in the cardiovascular system. This cognitive functions and that dysfunction of facilitating dopaminergic signaling act as will allow predictions if already licensed these mechanisms leads to learning and cognitive enhancing strategies to rescue drugs can be used for neuroprotection in memory deficits. Another aim is to study aberrant fear extinction consolidation in PD or the therapy of selected ASD patients whether cognitive deficits inherent to nor- S1 (129S1/SvImJ) mice. Additional find- with Cav1.3 mutations. Moreover, suitable mal aging and neuropsychiatric diseases ings indicate that non-coding RNAs, includ- mouse models will be established that will are caused by alterations in Satb2 expres- ing – microRNAs (miRNAs), and Cav1.3 allow us to study the functional conse- sion or function. channel-mediated signaling – may be addi- quences of human ASD mutations in differ- tional promising targets to exploit for novel ent tissues, in particular the brain. Finally Dopamine Regulation of Amygdala pro-cognitive properties supporting extinc- we ask the question if knockout of Cav1.3 Inhibitory Circuits: Relevance for tion memory formation. Building on results channels or chronic inhibition of these chan- Pathological Fear Structures obtained in the 1st FP, we now aim to elucidate nels leads to compensatory upregulation of Francesco Ferraguti the how and where in the brain HDAC inhibi- other ion channels that could counteract Institute or Pharmacology tion, enhancement of dopaminergic signal- their pharmacological action. These highly Parkinson’s disease (PD) is classically con- ing, interference with non-coding RNAs- or translational questions will be addressed sidered as a movement disorder resulting Cav1.3 mediated-signalling can augment in collaboration with other members of the from the loss of dopaminergic (DAergic) fear extinction to form a persistent and con- consortium. Our work has immediate rele- neurons. However, a number of non-motor text-independent fear inhibitory memory. In vance for better understanding of Ca2+-de- symptoms, including pathological fear and addition, we aim to improve the tolerability pendent human disease mechanisms and anxiety, predate the emergence of motor im- of exposure based therapy by combining ongoing drug discovery in industry. pairment. PD could then be seen as a multi the therapeutic actions of non-sedative dimensional disease. Dopamine exerts a anxiolytic drugs which do not impair ex- Members from other Universities pivotal role in the regulation of fear respons- tinction learning and appropriate cognitive Birgit Liss, University of Ulm es most likely by affecting GABAergic trans- enhancers. Finally, this project started to Ludwig Aigner, Paracelcus Medizinische mission within the amygdaloid complex. We identify potential epigenetic biomarkers in Privatuniversität, see homepage postulate that pathological fear in early PD blood cells that can be associated with the results from altered associative plasticity in sensitivity to and the extent of the therapeu- Former Members (1st Funding Period) the basolateral amygdala (BLA) mostly de- tic effect of exposure therapy in anxiety dis- Christian Humpel, Josef Marksteiner pendent on the reduced function of DA on order patients. Revealing mechanisms via Dept. for General and Social Psychiatry, MUI specific local interneurons. In addition, en- which rescue of impaired fear extinction can Alexandra Lusser hanced phasic DAergic transmission during be achieved in a better tolerated, persistent Division of Molecular Biology, MUI

206 Research Report 2015 Medical University of Innsbruck Core Facility Net www.corefacilitynet.org

What is the Core Facility Net? Benefits Core Facilities at Medical University of Innsbruck The inter-university core facility network Work togehther with experts has produced Austria’s largest platform for Many experts, who are willing to cooperate, Protein Micro-Analysis Facility the exchange of life science technology have given their commitment to the ao. Univ.-Prof. Dr. Herbert Lindner services and expertise. network. They provide a short summary [email protected] about their expertise – try searching for a Tel.: +43 512 9003 70310 The three Medical Universities of Graz, special expertise and get a list of experts! For more details see page 15 Innsbruck and Vienna, as well as the Austrian Institute of Technology (AIT) and Know about high-end infrastructure Sequencing and Genotyping Facility the University of Veterinary Medicine Vienna Available infrastructure is represented and Univ.-Prof. Dr. Florian Kronenberg are participating in the setup of this “shared described on the web portal, including infor- [email protected] technology space”. They are linking up mation like User Manuals, experts related Tel.: +43 512 9003 70560 existing technologies worth approximately to this special device and other information. For more details see page 45 30 million € and the expert know-how of With help of the integrated search function- 80 specialists from the four core areas of ality it’s possible to find it quickly. Innsbruck Flow Cytometry Unit OMICS-technologies, imaging techniques, Priv.-Doz. Dr. Sieghart Sopper biocomputing and functional biomodels. Geo Information [email protected] Look out for experts by searching the map Tel.: +43 512 504 82596 In addition to the research infrastructure – a full Google Map Integration is available For more details see page 120 network, pan-university, post-graduate therefore. Every content, like experts, further education and training will be devices or even documents/publications Biooptics provided, especially to young researchers, in can be Geo-tagged. Priv.-Doz. Dr. Martin Offterdinger order to initiate and encourage networking. [email protected] In the future, additional partner institutions Calendar Tel.: +43 512 9003 70287 will be integrated into the network. The At the portal all events provided by the For more details see page 13 technical realisation of the project was members could be found in one place. To carried out in cooperation with the FAW help finding the relevant events for you, use Micro CT GmbH; the research infrastructure platform the integrated filter functionality – filter by Dipl.-Ing. Dr. Volker Kuhn is open to the public since July 2014. site or by tags, or just search for it. [email protected] The total cost of the project is more than Tel.: + 43 512 504 80857 520,000 € and has been approved for Document Management For more details see page 94 152,000 € of financing as a part of a federal Take advantage from the built-in document fund for infrastructure in higher education management system, including history and Neuroimaging Research Core Facility by the former Federal Ministry of Science versioning, working together on the same Univ.-Prof. Dr. Elke R. Gizewski and Research. document and more. [email protected] Tel.: +43 512 504 24202 For more details see page 163

Metabolomics o. Univ.-Prof. Dr. Richard Scheithauer [email protected] Tel.: + 43 512 9003 70600 Assoz. Prof. Dr. Herbert Oberacher Tel.: +43 512 9003 70639 [email protected] For more details see page 79

Genome Seq Core Expression Profiling Univ.-Prof. Dr. Alexander Hüttenhofer [email protected] Tel.: +43 512 9003 70250 For more details see page 22

Research Report 2015 Medical University of Innsbruck 207 Imprint Research Report 2015 of the Medical University of Innsbruck A publication of the Medical University of Innsbruck; editors and layout: Mag. Maria Magdalena Mair, Damla Celikel, Nadine Nössing; Servicecenter Forschung, Schöpfstraße 45, A-6020 Innsbruck language editors: Prof. Dr. Paul Debbage, Mag. pharm. Gudrun Thurner PhD, Jonathan Vosper PhD, James Wood PhD, Augustine Boima images: Medical University of Innsbruck, ao. Univ.‑Prof. Dr. Siegfried Schwarz, Florian Lechner, Shutterstock pictures of buildings: MUI/Franz Oss (3), Siegfried Schwarz (1), MUI/Christof Lackner (1), Tirol Kliniken/Gerhard Berger (1) cover layout: David Bullock; cover photos: MUI/Florian Lechner (3); printed by Alpina Druck, Austria The head of the research unit is responsible for the content and pictures of the relevant contribution. Typing and printing errors reserved.

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