PROGRAMM 5. Symposium Urologische Forschung
5. Symposium Urologische Forschung der Deutschen Gesellschaft für Urologie CME Zellbiologie des Urogenitalsystems Entwicklung, Homöostase, Pathogenese PROGRAMM In Kooperation mit der Arbeitsgemeinschaft Uropathologie der Deutschen Gesellschaft für Pathologie 14. bis 16. November 2013 Aula im Hauptgebäude der JLU-Gießen Inhalt GRUßWORT .............................................................................................................................................3 HINWEISE ................................................................................................................................................5 ALLGEMEINE HINWEISE ................................................................................................................................................................. 5 LAGEPLAN ...................................................................................................................................................................................... 6 AUF-FORSCHUNGSPREISE ............................................................................................................................................................. 7 PUBLIKATION DER ABSTRACTS ....................................................................................................................................................... 7 CME-ZERTIFIZIERUNG ................................................................................................................................................................... 7 HINWEISE FÜR REFERENTEN & MODERATOREN ............................................................................................................................ 8 Vorträge .........................................................................................................................................................................................8 Poster..............................................................................................................................................................................................8 SCIENTIFIC PROGRAM.............................................................................................................................9 Thursday, 14.11.2013 .............................................................................................................................................................. 11 Friday, 15.11.2013 ................................................................................................................................................................... 13 Saturday, 16.11.2013............................................................................................................................................................... 20 SOCIAL PROGRAM ................................................................................................................................25 Begrüßungsabend...................................................................................................................................................................... 27 Experimenteller Abend ............................................................................................................................................................. 27 ABSTRACTS............................................................................................................................................29 ANHANG ...............................................................................................................................................87 AUF-PREISTRÄGER .......................................................................................................................................................................89 SPONSOREN .................................................................................................................................................................................91 AUSBLICK: 6. AUF-SYMPOSIUM 2014 IN HOMBURG/SAAR ......................................................................................................93 2 Grußwort Liebe Kolleginnen und Kollegen, wir freuen uns, Ihnen den Tagungsband für das nunmehr 5. Symposium der Arbeitsgruppe urologische Forschung (AuF) der Deutschen Gesellschaft für Urologie überreichen zu können. Diese Veranstaltung steht unter dem Leitthema „Zellbiologie des Urogenitalsystems - Entwicklung, Homöostase und Pathogenese“ und wird in guter Tradition gemeinsam mit der Arbeitsgemeinschaft Uropathologie durchgeführt. Die Funktion des Urogenitalsystems wird durch seine vielfältigen, oft hochspezialisierten und im Körper einzigartigen Zellen sichergestellt. Störungen in der Funktion, Interaktion und Homöstase dieser Zellen sind die Ursache vieler Erkrankungen, mit denen die Urologie befasst ist. Das Symposium soll daher die normale Funktion des Urogenitalsystems und seine Erkrankungen aus dem Blickwinkel der Zellbiologie beleuchten. Ziel ist es, die Fortschritte auf diesem Gebiet zu präsentieren und zu diskutieren, wie sie in der urologischen Forschung weitergeführt und in der klinischen Praxis aufgenommen werden können. Auch methodische Entwicklungen sollen nicht zu kurz kommen. Für das 5. AuF-Symposium konnten wir namhafte Wissenschaftler als Referenten gewinnen. Neben der Möglichkeit mit diesen ausgewiesenen Experten ins direkte Gespräch zu kommen, bieten wir insbesondere den urologischen Nachwuchswissenschaftlerinnen und Nachwuchswissenschaftlern mit moderierten Vortrags- und Postersitzungen geeignete Foren zur Präsentation ihrer eigenen Arbeiten. Wir freuen uns, auch diesmal wieder ehemalige Eisenberger-Stipendiaten begrüßen zu können, die über ihre Projekte sowie gemeinsam mit ihren Betreuern über Ablauf und Bedeutung ihrer Stipendien sprechen werden. Wir heißen Sie herzlich willkommen in Gießen und wünschen Ihnen einen angenehmen Aufenthalt! 3 4 Allgemeine Hinweise 5 Lageplan 3 5 1 4 2 11 6 AuF-Forschungspreise Die Arbeitsgruppe Urologische Forschung der DGU prämiert auf seinem Symposium 2013 wieder vier herausragende Vortrags- und Posterpräsentationen mit je 500 €. Die von der Deutschen Gesellschaft für Urologie e.V. gestifteten Forschungspreise werden in den beiden Kategorien jeweils an einen Mediziner und einen Naturwissenschaftler vergeben. Über die Preisvergabe entscheidet eine Jury: Prof. Dr. rer. nat. Gerhard Unteregger PD Dr. med. Frederik Roos Prof. Dr. med. Ruth Knüchel-Clarke Prof. Dr. med. Sven Perner Die Preisverleihung findet am Samstag, den 16.11.2013 im Rahmen der Abschlusssitzung um 12:45 Uhr statt. Publikation der Abstracts Alle Abstracts der präsentierten Beiträge werden in der Januar-Ausgabe 2013 der Zeitschrift „Der Urologe“ zitierfähig veröffentlicht. Die Abstacts der vergangenen drei AuF-Symposien können unter den folgenden InternetAdressen abgerufen werden: 1. Symposium: http://link.springer.com/article/10.1007/s00120-009-2165-3 2. Sympoisum: http://link.springer.com/article/10.1007/s00120-010-2485-3 3. Symposium: http://link.springer.com/article/10.1007/s00120-011-2785-2 4. Symposium: http://link.springer.com/article/10.1007/s00120-012-3077-1 CME-Zertifizierung Das 5. Symposium Urologische Forschung der DGU ist eine von der Akademie der Deutschen Urologen in Zusammenarbeit mit der Landesärztekammer Hessen mit 16 CME-Punkten zertifizierte und evaluierte Veranstaltung. Die gemäß den Fortbildungskriterien vergebenen CME-Punkte werden bundesweit von allen Landesärztekammern anerkannt. Die CME-Registrierung der Teilnehmer erfolgt täglich an den Empfangstischen des Symposiums. Bitte halten Sie dazu Ihre EFN-Nummer (Barcode-Aufkleber) bereit. Die Akademie übernimmt die Meldung der über EFN registrierten Teilnehmer an die einzelnen Landesärztekammern. 7 Hinweise für Referenten & Moderatoren VORTRÄGE Vorträge sind auf 7 Min. plus 3 Min. Diskussion begrenzt. Wir möchten Sie freundlich bitten, Ihre Redezeit strikt einzuhalten. Tagungssprache ist grundsätzlich Deutsch. Es kann aber wegen der internationalen Beteiligung auch gerne auf Englisch vorgetragen werden. Wir möchten Sie in jedem Fall darum bitten, Ihre Vortragsfolien in englischer Sprache zu erstellen. Ihnen steht im Hörsaal ein Windows-Laptop mit einer aktuellen Microsoft PowerPointVersion zur Verfügung. Bitte achten Sie auf Windows-Kompatibilität Ihrer ppt-Dateien, da wir kein individuelles Anschliessen von Apple-Laptops ermöglichen können. Die Medieannahme befindet sich im Tagungshörsaal. Reichen Sie bitte Ihre Präsentationsdatei möglichst eine halbe Stunde vor Beginn Ihrer Session dort ein. Sollte Ihr Vortrag Videosequenzen enthalten, achten Sie auch darauf, die entsprechenden Dateien zusätzlich zu Ihrer ppt-Datei abzugeben. POSTER Poster sind hochformatig bis DIN A0 zu erstellen. Die Präsentation der Poster erfolgt auf Deutsch oder wahlweise auf Englisch. Auf jeden Fall sollen die gedruckten Poster wegen der internationalen Beteiligung in englischer Sprache erstellt sein. Bitte hängen Sie Ihr Poster rechtzeitig in der Pause vor Ihrer Session auf und anschließend wieder ab. In den moderierten Posterpräsentationen erklären die Autoren - am Poster stehend - kurz und knapp ihre Arbeiten. Die Redezeit ist dabei auf 3 Min. plus 2 Min. Diskussion begrenzt. Unsere Moderatoren sind angewiesen, streng auf die Einhaltung der Redezeit zu achten und die Vortragenden zugunsten eines harmonischen Programmablaufs nötigenfalls zu unterbrechen. 8 SCIENTIFIC PROGRAM 9 10 THURSDAY, 14.11.2013 17:0017:20 Welcome Florian Wagenlehner & Wolfgang Schulz, Task Force Urological Research (AuF) Joybrato Mukherjee, President of the Justus-Liebig-University Giessen Wolfgang Weidner, Director of the Clinic of Urology, University of Giessen Jan Fichtner, President of the German Society of Urology Ruth Knüchel-Clarke, Chairwoman of the Working Group Uropathology 1st Symposium Focus: Tumour stem cells and circulating tumour cells Moderation: Bernd Wullich & Wolfgang Schulz 17:2017:50 V1.1 The molecular characterisation of disseminated cancer cells - Is it clinically relevant? Nikolas Stöcklein Dept. of Experimental Surgical Oncology, University of Duesseldorf V1.2 Connecting the machineries of asymmetric division and tumor suppression in stem cells Salvatore Pece Institute FIRC of Molecular Oncology, Milan, I Short lectures 17:5018:20 18:2019:30 V1.3 Targeting a cancer stem cell population in renal cell cancer using small molecule inhibitors Annika Fendler Clinic of Urology, Charité Berlin V1.4 Epigenetic silencing of ITIH5 is associated with bladder cancer progression and early relapse of pT1 high grade urothelial tumors Michael Rose Institute of Pathology, RWTH Aachen V1.5 Expression changes of long noncoding RNA HOTAIR have tissue specific effects on HOX gene expression and phenotype in urothelial carcinomas Michèle Hoffmann Clinic of Urology, University of Duesseldorf 11 V1.6 Prevalence and clinical features of HOXB13 mutation carriers in German prostate cancer patients Manuel Lüdeke Clinic of Urology, University of Ulm V1.7 Immunohistochemical study on invasive tumor cells of clear cell renal cell carcinoma Anja Rabien Clinic of Urology, Charité Berlin from 19:45 12 Get Together at Dach Café Ludwigsplatz 11, Giessen; http://dachcafe.com FRIDAY, 15.11.2013 Keynote Lecture Jenny Southgate Moderation: Wolfgang Weidner & Klaus Steger 08:3009:00 K1 Developmental aspects of the urinary tract Jenny Southgate Jack Birch Unit, Dept. of Biology, University of York, UK 2nd Symposium Focus: Andrology Moderation: Wolfgang Weidner & Klaus Steger 09:0009:30 V2.1 Role of epigenetic in reproduction Klaus Steger Clinic of Urology, University of Giessen V2.2 Testicular stem cells Stefan Schlatt Center of Reproduction Medicine und Andrology, University of Muenster Short lectures 09:3010:00 10:0010:30 V2.3 Transcriptional control of tubular lumen size control in normal and diseased kidneys Zeliha Yesmin Yurtdas Max Delbrueck Center for Molecular Medicine, Berlin V2.4 Influence of testosterone on the regulation of inflammatory responses in testicular and immune cells Monika Fijak Institute for Anatomy and Cell Biology, University of Giessen V2.5 New biomarkers to differentiate malignant germ cell tumours of the testis established by the SILAC-method in combination with high-resolution mass spectrometry Felix Bremmer Institute of Pathology, University of Goettingen 10:3011:00 Coffe Break / Poster Exhibition 13 Poster I 1st Poster Presentation Moderation: Walburgis Brenner, Jörg Ellinger & Helge Taubert 11:0012:15 Brenner 15 Posters Ellinger 14 P1.1 Transcript levels of Piwi-like 1-4 genes are associated with clinicopathological parameters in renal cell carcinomas Omar Al-Janabi Clinic of Urology, University of Erlangen P1.2 HIF-2α is highly expressed in papillary renal cell carcinoma type II: a potential reason for the worse prognosis? Carl Ludwig Behnes Institute of Pathology, University of Goettingen P1.3 Deregulation of the CSN-CRL pathway during urological tumorigenesis? Linda Gummlich Clinic of Surgery, Charité Berlin P1.4 Quantitative imaging of intratumoral heterogeneity in clear cell renal carcinoma Rouven Höfflin Dept. of Molecular Urooncology, University of Heidelberg P1.5 Prediction of second-line therapy response in metastatic renal cell carcinoma patients by blood plasma marker proteins Sebastian Hölters Clinic of Urology, University of Homburg/Saar P1.6 Targets and function of dysregulated microRNAs in clear cell renal cell cancer Julia Liep Clinic of Urology, Charité Berlin P1.7 Integrated microRNA and mRNA signature associated with the transition from the locally confined to the metastasized renal cell carcinoma Zofia Wotschofsky Clinic of Urology, Charité Berlin P1.8 Molecular cytogenetic differences between Collecting Duct Carcinomas and upper urinary tract urothelial carcinomas Volker Jung Clinic of Urology, University of Homburg/Saar Taubert 12:1513:15 P1.9 Novel antiangiogenic compounds with antitumor activity for innovative approaches in cisplatin resistant testicular germ cell cancer treatment Bianca Nitzsche Institute of Physiology, Charité Berlin P1.10 Epigenetic regulation of genes involved in epithelial mesenchymal transition in prostate cancer Ulrike Brandt Clinic of Urology, University of Giessen P1.11 Epigenetic intervention counteracts resistance towards temsirolimus in prostate cancer Jasmina Makarevic Clinic of Urology, University of Frankfurt a.M. P1.12 Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and proteomics expression data Regina Reszka Metanomics, Berlin P1.13 Cytostatic drug WIST-C overcomes docetaxel induced resistance Martina Rottach Clinic of Urology, University of Greifswald P1.14 Absolute quantification by qRT-PCR - a new and sensitive method to measure absolute amounts of miRNA in high risk prostate cancer tissue Maria Schubert Clinic of Urology, University of Wuerzburg P1.15 Evaluation of Patient Derived Molecular Neuroendocrine Signatures in PC3 using RNAseq data Jost von Hardenberg Clinic of Urology, University of Mannheim Lunch 15 3rd Symposium Focus: Immunology / Infectiology of the genito-urinary tract Moderation: Florian Wagenlehner & Hans Krause 13:1513:35 V3.1 Bacterial virulence factors Ulrich Dobrindt Institute for Hygiene, University of Muenster V3.2 Host-response in urogenital infections Hamid Hossain Institute of Medical Microbiology, University of Giessen V3.3 Interstitial cystitis Lars-Christian Horn Institute of Pathology, University of Leipzig V3.4 Immunologie of testes / epididymides Andreas Meinhardt Institute for Anatomy and Cell Biology, University of Giessen Short lectures 13:3513:55 13:5514:10 14:1014:25 14:2514:45 V3.5 Infection of the prostate caused by Propionibacterium acnes Behnham Sayanjali Max-Planck-Institute for Infection Biology, Berlin V3.6 Investigation of Escherichia coli urinary tract isolates with characteristics of small colony variants Johannes Putze Institute for Hygiene, University of Muenster 14:4515:15 16 Coffee Break / Poster Exhibition Poster II 2nd Poster Presentation Moderation: Susanne Füssel, Frederik Roos & Katharina Braun 15:1516:30 Füssel 15 Posters Roos P2.1 OASIS/CREB3L1 is downregulated in human bladder tumors and mediates suppression of cancer cell spreading and migration in vitro Laura Dierichs Institute of Pathology, RWTH Aachen P2.2 Responses of bladder cancer cells towards tyrosine-kinase inhibition by dovitinib (TKI-258) in relation to the eptithelial mesenchymal transition status Jörg Hänze Clinic of Urology, University of Marburg P2.3 HDAC inhibition suppresses bladder cancer cell adhesion to collagen under flow conditions Eva Juengel Clinic of Urology, University of Frankfurt a.M. P2.4 Invasion of urothelial carcinoma of the bladder: MMP7 is associated with increased cell invasion in urothelial cancer: evidence from functional models Daniel Knauf Clinic of Urology, University of Mannheim P2.5 Analysis of microRNA expression in urothelial carcinoma of the upper urinary tract Stephanie Kriebel Clinic of Urology, University of Bonn P2.6 Acute epididymitis induces alterations in sperm protein composition Adrian Pilatz Clinic of Urology, University of Giessen P2.7 Investigations on the localization and significance of the prolactin-inducible protein (PIP) in the male urogenital tract Srikanth Karnati Institute for Anatomy and Cell Biology, University of Giessen P2.8 Role of TET3 and 5-hmC in establishment of sperm epigenome and in production of fertile sperm Undraga Schagdarsurengin Clinic of Urology, Dept. of Molecular Andrology, University of Giessen 17 Braun 18 P2.9 Mixed testicular atrophy is related to atherosclerosis in the ApoE(-/-) / LDL receptor(-/-) double knockout mouse model: New data of the arterial supply of the tubuli Kai Steinfeld Clinic of Urology, University of Giessen P2.10 Polysialylation of NCAM correlates with onset and termination of seasonal spermatogenesis Sebastian Galuska Institute of Biochemistry, University of Giessen P2.11 Bacterial epididymitis induces fibrotic transformation and regional changes in the activin/follistatin ratio Vera Michel Institute for Anatomy and Cell Biology, University of Giessen P2.12 Necrosis is the dominant cell dead pathway in UPEC induced epididymoorchitis model and is responsible for structural and functional damage of rat testis Sudhanshu Bhushan Institute for Anatomy and Cell Biology, University of Giessen P2.13 Identification of mouse sperm glycoprofile to evaluate possible alterations after urogenital tract infection: possible implications for immune privilege and sperm function Ferhad Khosravi Institute for Anatomy and Cell Biology, University of Giessen P2.14 Uropathogenic E. coli inactivate host survival AKT signalling pathway in Sertoli cells Zhengguo Zhang Institute for Anatomy and Cell Biology, University of Giessen P2.15 Urethral brush cells are polymodal cholinergic chemosensors Klaus Deckmann Institute for Anatomy and Cell Biology, University of Giessen 4th Symposium Focus: Cellular biology of the lower urinary tract Moderation: Roman Nawroth & Karl-Dietrich Sievert 16:3016:50 V4.1 Cell-test systems for stem cell-based tissue engineering Sabine Neuss-Stein Institute of Pathology, RWTH Aachen V4.2 Sphinkter regeneration using MSCs – options and limitations Karl-Dietrich Sievert Clinic of Urology, University of Tuebingen V4.3 Afferent system: non-neuronal cholinergic system Wolfgang Kummer Institutr of Anatomy und Cell Biology, University of Giessen Short lectures 16:5017:10 17:1017:30 17:3018:00 V4.4 Cellular changes during BPS Christian Gratzke Clinic of Urology, LMU Muenchen V4.5 Expression of Aquaporin water channels by human urothelium: contribution to transurothelial permeability in vitro and modulation by osmolality Peter Rubenwolf Clinic of Urology, University of Mainz V4.6 3D-ultrastructure of the human urinary bladder revealed by FIB-SEM and 3View SEM Jochen Neuhaus Clinic of Urology, University of Leipzig Keynote Lecture Hans Lilja Moderation: Roman Nawroth & Karl-Dietrich Sievert 18:0018:30 K2 Relationship between the biology of PSA and related kallikrein-markers and prostate cancer in man and mice (models) Hans Lilja Memorial Sloan-Kettering Cancer Center, New York, NY, USA from 19:00 Experimental Evening at Museum Mathematikum Liebigstraße 8, Gießen; http://www.mathematikum.de 19 SATURDAY, 16.11.2013 EisenbergerSession Crosstalks Tutor & Fellow Moderation: Gerhard Unteregger & Max Burger 09:0009:30 E1.1 The Department of Clinical Laboratories, Surgery and Medicine at Memorial Sloan-Kettering Cancer Center as host for Eisenberger-fellows of the DGU Hans Lilja Memorial Sloan-Kettering Cancer Center, New York, NY, USA 09:3010:00 E1.2 MSP, PSA und hK2 in der Pathogenese des Prostatakarzinoms Katharina Braun Clinic of Urology, University of Bochum/Marienhospital Herne E2.1 The Department of Transplant and Infection Immunology as host for Eisenberger-fellows of the DGU Martina Sester Dept. of Transplantation and Infection Immunology, University of Homburg/Saar E2.2 T-regs in metastasised renal cell carcinoma Martin Janssen Clinic of Urology, University of Homburg/Saar 10:0010:30 20 Coffee Break / Poster Exhibition Poster III 3rd Poster Presentation Moderation: Peter Olbert, Sven Perner & Martin Janssen 10:3012:00 Olbert 15 Posters Perner P3.1 GDNF-family neurotrophic factors in urinary bladder and expression of their receptors (GFRα) in bladder sensory neurons Rajender Nandigama Institute for Anatomy and Cell Biology, University of Giessen P3.2 Treatment strategy dependent tissue injury produced by a new acoustic lens design for electromagnetic lithotripters Andreas Neisius Clinic of Urology, University of Mainz P3.3 Coming closer to endoscopic “real time target identification” - Technical improvements of an experimental lithotripsy laser system with spec-tral realtime object analysis Arkadiusz Miernik Clinic of Urology, University of Freiburg P3.4 Overexpression of Drosha is associated with outcomes in patients with urothelial carcinomas Nadine Ratert Clinic of Urology, Charité Berlin P3.5 Alternative therapy strategies for the non-muscle invasive bladder cancer on the basis of carbon nanomaterials Christiane Rieger Clinic of Urology, University of Dresden P3.6 The function of Kruppel-like factor 4 (KLF4) as a transcriptional repressor of IGF2-promoter during prostate carcinogenesis Temuujin Dansranjavin Clinic of Urology, University of Giessen P3.7 Resveratrol and prostate a very complicated “dangerous liaison” Marina Di Domenico Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, I P3.8 Cardiolipin species composition differs in benign prostate epithelia and prostate cancer and may be associated with tumor progression Sebastian Fussek Clinic of Urology, University of Greifswald 21 Janssen 22 P3.9 MiR-205 is progressively down-regulated in lymph node metastasis but fails as a prognostic biomarker in high-risk prostate cancer Charis Kalogirou Clinic of Urology, University of Wuerzburg P3.10 Re-expression of an epigenetically repressed microRNA inhibits prostate cancer cell growth Nina Korzeniewski Dept. of Molecular Urooncology, University of Heidelberg P3.11 HMGB1 modulates immune responses in a cell-specific manner in the rat experimental autoimmune orchitis Ferial Aslani Institute for Anatomy and Cell Biology, University of Giessen P3.12 Epigenetic dysregulation of inflammatory factors in prostatitis and prostate cancer Siva Reddy Velagala Institute for Anatomy and Cell Biology, University of Giessen P3.13 Androgen receptor regulates CD4+CD25+Foxp3+ T cell differentiation by binding to foxp3 locus Magdalena Walecki Institute for Anatomy and Cell Biology, University of Giessen P3.14 Functional analysis of specific T cells in patients under BCG-therapy Julia Elsässer Dept. of Transplantation and Infection Immunology, University of Homburg/Saar P3.15 Urine protein profiling identified Alpha-1-microglobulin and Haptoglobin as biomarkers for early diagnosis of acute allograft rejection following kidney transplantation Beatrice Stubendorff Clinic of Urology, University of Homburg/Saar 5th Symposium Focus: Epithelial-mesenchymal transition / Tumour-stroma-interaction Moderation: Ruth Knüchel-Clarke & Kerstin Junker 12:0012:20 V5.1 Stromal regulation and metastasis Jonathan Sleeman Center of Biomedicine and Medical Technology, University of Heidelberg V5.2 Tumour-stroma-interactions Gerhard Unteregger Clinic of Urology, University of Homburg/Saar V5.3 2-Photon microscopy of protective immunity in vivo Stephan Halle Institute of Immunology, University of Hannover 12:2012:40 12:4013:00 13:0013:15 Awards Resumee Outlook 2014 from 13:15 Short Lunch Bernd Wullich, Chairman of AuF Florian Wagenlehner & Wolfgang Schulz, AuF Gerhard Unteregger & Frederik Roos, AuF 23 24 SOCIAL PROGRAM 25 26 BEGRÜßUNGSABEND Donnerstag, 14.11.2013, ab 19:45 Uhr im Restaurant „Dach Café“ In gewohnt legerer Atmosphäre möchten wir Sie am ersten Abend des Symposiums zu einem Get Together in der Gaststätte Dach Café in Gießen begrüßen. Der Kostenbeitrag für diese Veranstaltung beträgt 20 €, für Studenten 10 €. Kontakt: Restaurant Dach Café Ludwigsplatz 11, 35390 Gießen Tel. 0641 – 686 91 000 Wegbeschreibung siehe Lageplan auf S. 5 EXPERIMENTELLER ABEND Freitag, 15.11.2013, ab 19:00 Uhr im Museum „Mathematikum“ Zum Festabend laden wir Sie herzlich in das besondere Ambiente des Museum Mathematikum ein. Während der zwischenzeitlich angebotenen Führungen erleben Sie Mathematik zum Anfassen. Der Kostenbeitrag für diese Veranstaltung ist in den Tagungsgebühren enthalten. Kontakt: Museum Mathematikum Liebigstraße 8, 35390 Gießen Tel. 0641 – 969 79 70 Wegbeschreibung siehe Lageplan auf S. 5 27 28 ABSTRACTS 29 30 V1.3 Targeting a cancer stem cell population in renal cell cancer using small molecule inhibitors Fendler A1,2,3, Busch J2, Jung K2,3, Birchmeier W1 1 2 3 Department of Signal Transduction, Invasion and Metastasis of Epithelial Cells, Max Delbrück Center of Molecular Medicine, Berlin Department of Urology, Charité – University Hospital Berlin Berlin Institute of Urologic Research, Charité – University Hospital Berlin Tumors frequently exhibit extensive intratumoral heterogeneity and it has been hypothesized that cancers are hierarchically organized and sustained by cancer stem cells (CSC) that are able to self renew and recapitulate tumor heterogeneity. CSC’s have been linked to therapy resistance and the specific inhibition of the CSC population might help to develop novel and more efficient treatment strategies of cancer. Current therapies of metastatic renal cell carcinoma are not curative, highlighting the ongoing need for novel and more efficient strategies. Thus, we aimed to identify a cancer stem cell population in clear cell renal cell cancer (ccRCC) and to investigate the potential of small molecule inhibitors to specifically target the CSC population. To identify a population with CSC properties in ccRCC, primary renal cancer cells were isolated from patients undergoing nephrectomy. Fluorescence activated cell sorting (FACS) identified a rare population of CXCR4+/c-Met+/CD44+ cells in primary renal cancer cells. These cells displayed higher sphere formation in vitro and their frequency increased with higher tumor stage. Xenograft assays in immune deficient NSG mice support the idea of a tumor-initiating capacity of this population. To assess treatment strategies that specifically target the cancer stem cell population in ccRCC, small molecule inhibitors directed against c-Met (Crizotinib), CXCR4 (AMD 3100), Wnt/-Catenin (ICG001, XAV939, LF3), GSK3 (CHIR 99021), Notch (DAPT), and MAPK signaling (U0126) were tested on their effect on sphere formation and cell proliferation of adherent cultures. Additionally small molecule inhibitors that have been approved for patient therapy (Sorafenib, Rapamycin) were tested. Small molecule inhibitors showed mixed effect on the proliferation of adherent cells and sphere formation and we could distinguish different classed of inhibition: (i) inhibition of both spheroid formation and growth of adherent cells, (ii) inhibition of spheroid formation but no effect on growth of adherent cells, (iii) no influence on sphere formation but inhibition of adherent cell growth, (iv) no effect. In conclusion, we identified small molecule inhibitors that can specifically target a population with cancer stem-like properties in ccRCC, while others appear to only target more differentiated cells. Targeting the CSC population might have a beneficial effect in treatment of ccRCC in the future. Contact: [email protected] 31 V1.4 Epigenetic silencing of ITIH5 is associated with bladder cancer progression and early relapse of pT1 high grade urothelial tumors Rose M1, Gaisa NT1, Antony P1, Fiedler D1, Heidenreich A2, Otto W3, Denzinger S3, Bertz S4, Hartmann A4, Karl A5, Knüchel R1, Dahl E1 1 2 3 4 5 Molecular Oncology Group, Institute of Pathology, Medical Faculty of the RWTH Aachen University Department of Urology, Medical Faculty of the RWTH Aachen University Department of Urology, Caritas St. Josef Medical Centre, University of Regensburg Institute of Pathology, University Hospital Erlangen Department of Urology, LMU Munich Background: Invasive bladder cancer is known to cause unfavorable prognosis. Disease management of this urothelial cancer subtype is still poor due to lack of prognostic and predictive markers, and most of these patients die in consequence of metastatic disease. Accumulating indications have shown that inter- -trypsin inhibitor heavy chain 5 (ITIH5) is associated with tumor suppression, especially metastasis, in various cancers such as breast cancer. However, its putative role in bladder cancer is completely unknown. Therefore, we aimed to study ITIH5 expression as well as its prognostic and biological impact on human bladder cancers. Methods: Epigenetic ITIH5 gene regulation was studied in bladder cell lines and primary bladder tumors by real-time PCR, immunohistochemistry, methylation-specific PCR (MSP) and pyrosequencing. In-vitro chromatin remodeling was approached by 5-aza-2’-deoxycytidine/ trichostatin A (DAC/TSA) treatment of bladder cancer cell lines. Statistical evaluation of ITIH5 methylation and ITIH5 expression with patient characteristics and clinical outcome was performed using SPSS15.0. The biological function of ITIH5 was examined by re-expression of a full-length ITIH5 cDNA in the urothelial cancer cell line RT112 that served as in vitro model for proliferation, colony spreading and tumor cell migration studies. Results: Real-time analyses showed downregulation of ITIH5 mRNA in 61% (n=45) of urothelial cancer samples, particularly in muscle-invasive tumors (p<0.001). ITIH5 loss in bladder cancer was further pronounced on protein level. DNA methylation analysis verified tumor-specific ITIH5 promoter methylation in 50% of pTa low grade and 68% of invasive bladder tumors. This epigenetic promoter modification was tightly linked (p<0.001) with the inactivation of ITIH5 mRNA synthesis in bladder tumor samples. A direct correlation between ITIH5 DNA methylation and its expression was functionally confirmed by in vitro demethylation experiments. Moreover, pyrosequencing analysis revealed that ITIH5 hypermethylation frequency was closely associated with progressive bladder cancers. In light of that, a cohort (n=120) of clinically challenging pT1 high grade bladder tumors was analyzed for ITIH5 expression. We found an association between ITIH5 protein loss and unfavorable prognosis of bladder cancer patients (recurrence-free survival; hazard ratio: 4.35, p=0.048). Finally, stable ITIH5 re-expression in human RT112 bladder cancer cells mediated suppression of both cell migration and colony spreading. Conclusions: We provide for the first time evidence that ITIH5 loss by aberrant ITIH5 promoter methylation during bladder cancer development may promote bladder cancer progression. Moreover, ITIH5 protein might become a prognostic biomarker for relapse risk stratification in high grade bladder cancer patients. Contact: [email protected] 32 V1.5 Expression changes of long noncoding RNA HOTAIR have tissue specific effects on HOX gene expression and phenotype in urothelial carcinomas Heubach J, Niegisch G, Schulz WA, Hoffmann MJ Urologische Klinik, Heinrich-Heine-Universität, Medizinische Fakultät Objective: Tumor heterogeneity in urothelial carcinoma (UC) which complicates diagnosis and therapy may be caused by aberrant differentiation. In many cancers, aberrant differentiation may be a consequence of deregulated homeotic HOX gene expression. However, expression and regulation of HOX genes is poorly studied in UC. We have therefore determined the expression patterns of HOXC and HOXD genes and the long noncoding RNA (lncRNA) HOTAIR. We studied its function in UC as it has been reported to regulate posterior HOXD genes and induce a more aggressive phenotype in breast cancer. Materials and Methods: Expression of HOX genes and HOTAIR was determined by qRT-PCR in UC tissues and cell lines. HOTAIR expression was modulated by ectopic expression and siRNA in selected UC cell lines. Cell migration was analyzed by means of transwell Boyden chambers coated with Collagen IV. Results: HOXC5-6 and C11-13 genes were generally overexpressed in tumor tissues and many cell lines. Adjacent HOTAIR was strongly overexpressed in a subset of tumors and in 7/12 cell lines. We did not find the expected inverse correlation between HOTAIR and HOXD10, but rather a strong positive correlation between HOTAIR and HOXD12 (r=0.93). Neither ectopic expression nor siRNA knockdown of HOTAIR resulted in HOXD10 expression changes, except in a single cell line, 5637. Functional assays with cell lines stable transfected with HOTAIR revealed cell type dependent effects on cell proliferation and migration. Conclusion: Our data confirm that major changes in HOX expression take place in urothelial carcinoma contributing to altered differentiation. Although we also found HOTAIR to be often overexpressed in UC its effects differ from those described in other tumors demonstrating that effects of lncRNAs can be tissue or even cell type specific. Supported by Forschungskommission der Medizinischen Fakultät der Heinrich-Heine-Universität Düsseldorf Contact: [email protected] 33 V1.6 Prevalence and clinical features of HOXB13 mutation carriers in German prostate cancer patients Lüdeke M1,2, Xu J3, Zheng SL3, Sonntag P4, Schulwitz H4, Rinckleb AE1,2, Schrader AJ1, Schrader M1, Vogel W2, Hoegel J2, Herkommer K4, Maier C1,2 1 2 3 4 Department of Urology, University Hospital Ulm Institut of Human Genetics, University Hospital Ulm Center for Cancer Genomics, Wake Forest University School of Medicine, Winston-Salem, USA Department of Urology, Klinikum rechts der Isar, Technische Universität München Introduction: Recently, a germline missense mutation in the homeobox gene HOXB13 (G84E) has been proposed as high risk variant for prostate cancer (PrCa) susceptibility in a cohort of European Americans. In order to assess the relevance of the G84E mutation in the German population, we performed a case control study on familial and sporadic PrCa patients. Methods: The study cohort consisted of 379 unrelated familial and 367 sporadic PrCa cases, as well 1015 controls. Additionally, 646 affected relatives from familial cases were included for associations with clinical features (stratification by Gleason score 8, advanced tumor stage or PSA at diagnosis >20ng/ml). Genotyping of HOXB13 G84E was conducted in part by an iPLEX massarray and by TaqMan method. Results: Carriers of G84E were rare in controls (0.4 %) and showed increased frequencies in both sporadic (1.6 %) and familial PrCa cases (3.2 %). Estimated risks were OR = 4.2 (p = 0.026) and OR = 8.3 (p = 0.0003) respectively. The risk effect size increased with the number of affected individuals per pedigree: OR = 12.6 (p < 0.0001) for 3 or more, OR = 14.4 (p < 0.0001) for 4 or more affected. The strongest association with clinical features was observed between G84E and advanced tumor stage (OR = 9.2; p < 0.0001). Conclusion: The frequency of HOXB13 mutation carriers in our study cohort was comparable to the observations in European Americans, establishing HOXB13 as the first high risk gene for PrCa in the German population. The association between G84E mutations and advanced tumor stage may be of greater interest for clinical practice, but needs further validation. Also the penetrance of the HOXB13 G84E mutation should be investigated in further studies in order to elucidate its suitability as a genetic predictor for PrCa. Contact: [email protected] 34 V1.7 Immunohistochemical study on invasive tumor cells of clear cell renal cell carcinoma Rabien A , Blauhut S1, Ergün B1, Jung K1,2, Kilic E3 1 1 2 3 Department of Urology, Charite-Universitätsmedizin Berlin Berlin Institute for Urologic Research, Berlin Institute of Pathology, Charité – Universitätsmedizin Berlin Background: The molecular mechanisms that lead to invasion of renal cell carcinoma (RCC) are not clear until now. As about 30% of the patients have metastases even at the time of diagnosis and the five-year survival rate is just about 70%, new insights in early metastatic processes are needed to better understand the conditions for invasion and to find suitable biomarkers for renal cancer. We investigated the molecular characteristics of invasive tumor cells of the major tumor subtype of clear cell RCC, which were currently invading blood vessels or fat tissue in the environment of the tumor. Material and Methods: In a first approach nine cases of clear cell RCC with respective invasive properties were selected by an experienced pathologist (E.K.) using hematoxylin / eosin stained sections of formalin-fixed paraffin-embedded (FFPE) tissue. Immunohistochemical staining of FFPE tissue containing blood vessels or fat tissue with invading tumor cells was done using primary antibodies for molecules driving / inhibiting invasion (matrix metalloproteinases MMP-2, -9, -14, EMMPRIN, RECK), for markers of the epithelial / mesenchymal phenotype (cytokeratin-19, Ecadherin, N-cadherin, S100A4, ZEB1, beta-Catenin) and for stem cells (CD105, in progress: CD44, CMET, CXCR4). Results: Depending on the marker, staining of the invading tumor cells was heterogenous. Some single cells were positive for the epithelial marker cytokeratin-19. The epithelial E-Cadherin as well as the mesenchymal N-Cadherin congruently stained (often larger) areas of the tissue. S100A4 and ZEB1 showed nuclear staining in many cells which accumulated in distinct areas at the edge of the blood vessels. MMP-2 was expressed at the whole edges of blood vessels, but less in the invading cells. CD105 stained blood vessels and rarely single cells which need to be differentiated from microcapillaries. The MMP inhibitor RECK appeared in occasional granular spots spread in the tumor cells. Beta-Catenin as well as EMMPRIN were widely expressed at the plasma membrane of tumor cells with extremely rare beta-catenin staining in the cytoplasm. Staining of the tumor and invading tumor cells seemed to be equal. In contrary, MMP-9 and MMP-14 levels were higher at the edge of the tumor mass and in the invading tumor cells. Conclusions: Heterogenous expression of the majority of markers investigated points to different cell types within the population of invading tumor cells, apart from tumor-supporting cells. Epithelial-tomesenchymal transition does not seem to play a role in invading clear cell RCC, or it is a temporally short phenomenon which could not be recognized in these samples. Elevated expression of MMP-9 and MMP-14, however, could indicate an important regulation which will be further examined. Contact: [email protected] 35 V2.3 Transcriptional control of tubular lumen size control in normal and diseased kidneys Yurtdas Y1,2,3,*, Aue A1,*, Ruffert J1,*, Hinze C1,3,*, Kilic E4, Schmidt-Ott KM1,3 Max-Delbrueck Center for Molecular Medicine Department of Urology, Charité Universitätsmedizin Berlin 3 Department of Nephrology, Charité Universitätsmedizin Berlin 4 Department of Pathology, Charité Universitätsmedizin Berlin * equal contribution 1 2 Introduction: The establishment of lumen structure of defined size requires highly regulated genetic and cellular interactions for the maintenance of physiological function of the kidney. Under pathological conditions, perturbed lumen size formation can be found in cystic kidney diseases and also in renal tumors. The transcription factor Grainyhead-like2 (Grhl2) is an epithelium specific mammalian homolog of Drosophila grainyhead, which is specifically expressed in epithelial cells of the distal nephron and in the collecting duct of the kidney. Understanding the mechanisms governing renal lumen size regulation can provide useful insights into the pathogenesis of cystic kidney diseases and renal tumors. In this study, we show that Grhl2 regulates collecting duct lumen size and identify a zinc finger transcription factor Ovol2 as a novel relevant target gene downstream of Grhl2. Methods: Formalin fixed, paraffin embedded and frozen tissue specimens of healthy human kidney and mouse kidney sections were evaluated immunohistochemically. The role of Grhl2 in kidney development was investigated in vivo by the generation of conditional Grhl2 knockout mouse model. In parallel, in vitro analysis by using an inner medullary collecting duct cell line (IMCD-3) in 3D culture was performed to analyze the role of Grhl2 in epithelial morphogenesis on a cellular level. Grhl2 loss of function and gain of function experiments were carried out on IMCD-3 cells via lentiviral gene transfer and an integration of Grhl2 rescue construct, respectively. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) from IMDC-3 cells were performed to determine the individual target genes downstream of Grhl2. To test the significance of Ovol2 downstream of Grhl2, Ovol2 was overexpressed in Grhl2-deficient IMDC-3 cells. Results: Immunohistochemical analysis of kidney sections from both mice and humans showed that the expression of Grhl2 was restricted in the nuclei of collecting duct and distal tubule cells of the kidney. The measurement of collecting duct lumen area showed a remarkably reduced lumen size in vivo and cyst lumen area in 3D culture in vitro in the Grhl2 knockout and knockdown scenario, respectively. Furthermore, the new Grhl2 key targets were found via gene expression analysis and ChIP-seq experiments. We focused on Ovol2 as the target gene of Grhl2 in the establishment of lumen formation. Our analysis showed that the re-expression of Ovol2 in Grhl2 knockdown IMCD-3 cells partially rescued a lumen phenotype, compared to Grhl2 knockdown IMDC-3 cells. Conclusion: Our results reveal the role of Grhl2 and its target gene Ovol2 in the regulation of lumen size and show that Grhl2/Ovol2 axis crucially influences lumen size in collecting duct. For further studies, we aim to characterize the molecular and cellular function of Grhl2/Ovol2 cascade in kidney diseases. Contact: [email protected] 36 V2.4 Influence of testosterone on the regulation of inflammatory responses in testicular and immune cells Fijak M1, Sauber L-J1, Walecki M1, Eisel F1, Aslani F1, Wahle E1, Bhushan S1, Hackstein H2, Schuler G3, Meinhardt A1 1 2 3 Department of Anatomy and Cell Biology, Justus-Liebig-University Giessen Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University Giessen Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-LiebigUniversity Giessen Beside their spermatogenic function androgens also play also a role in the modulation of autoimmune disease and contribute to suppression of inflammatory/autoimmune response. Previously, we have shown that substitution of reduced testosterone levels in a rat model of chronic testicular inflammation - experimental autoimmune orchitis (EAO) - has led to a significant amelioration of disease characteristics by inhibition of inflammatory responses in the testes. This was documented by a strong decrease of elevated macrophage and CD4+T cells numbers in the interstitial space concomitant with significant increase of “anti-inflammatory“ regulatory T cells (CD4+CD25+Foxp3+) as well as significantly lower mRNA levels of TNF-, IL-6 and MCP-1 as compared to EAO controls. Anti-inflammatory effects of testosterone supplementation during chronic testicular inflammation prompted us to investigate a putative direct influence of androgens on the generation of regulatory T cells and immune response in Sertoli (SC) and peritubular cells (PTC), both important contributors to immunological balance in the testis. Collected conditioned media from isolated Leydig cells were used for cultivation of isolated splenic T cells. Differentiation of regulatory T cells was estimated by measurement of expression of Foxp3 transcription factor by FACS and secretion of IL-10 and TGF- by ELISA. In another approach, isolated primary SC and PTC were pre-incubated with different concentrations of testosterone before induction of inflammatory response by LPS. IL-6, IL-10, TNF- and MCP-1 mRNA expression was investigated by quantitative real-time RT-PCR. Testosterone in conditioned media collected from Leydig cells stimulated the expression of Foxp3 transcription factor in splenic CD4+ T cells as well secretion of IL-10 and TGF- by these cells. The specificity of testosterone effect was proven by addition of androgen antagonist flutamide. In SC and PTC inflammatory response induced by LPS was inhibited by pre-incubation with increasing concentrations of testosterone. Significant reduction of TNF- mRNA levels were achieved in the presence of 1000 nM and 100 nM of testosterone in SC and PTC, respectively. The anti-inflammatory effect of testosterone in SC was abolished by use of flutamide. In view of the high intratesticular testosterone levels under normal conditions and their decrease under inflammatory conditions, our data point to a role of androgens in the immune homeostasis of the testis. Androgen action could be mediated by direct effect on SC and PTC as well as on the de novo generation and functional differentiation of regulatory T cells. Contact: [email protected] 37 V2.5 New biomarkers to differentiate malignant germ cell tumours of the testis established by the SILAC-method (stable isotope labelling by/with amino acids in cell culture) in combination with high-resolution mass spectrometry Bremmer F1, Bohnenberger H1, Oellerich T2, Kueffer S1, Strauss A3, Urlaub H4, Serve H2, Radzun HJ1, Ströbel P1, Maatoug Y1 and Behnes CL1 1 2 3 4 Institute of Pathology, University Medical Center, Göttingen Department of Hematology/Oncology, Johann Wolfgang Goethe University, Frankfurt Clinic for Urology, University Medical Center, Göttingen Bioanalytical Mass Spectrometry, MPI Biophysical Chemistry, Göttingen Background: Malignant germ cell tumours of the testis are the most common malignant tumours in young men between 18 to 35 years. Differentiation of the histological subtypes is essential for the therapeutic management. Thus it is important to find new biomarkers for the various histological subtypes. Furthermore, biomarkers may help to understand pathophysiological processes in these tumour types. Methods: In this study we carried out quantitative proteomic studies using high-resolution mass spectrometry in combination with the SILAC method (stable isotope labeling by amino acid in cell culture). For this purpose, the two germ cell tumour cell lines NTERA-2 and TCAM-2 were cultured in the presence of amino acids of different mass and subsequently analyzed by mass spectrometry. The detected proteins were further investigated by western blot and immunohistochemical analysis. Results: The SILAC and high-resolution mass spectrometry of the investigated cell lines revealed a total number of 342 differential expressed proteins. After intensive research in literature- and protein-data bases we chose antibodies against Destrin, CD81, and PHF6 for western blot analysis. The results confirmed the findings of mass spectrometry analysis. We further verified these findings on immunohistochemical analysis of 248 formalin-fixed and paraffin-embedded testis tumour tissue samples (seminomas, embryonic carcinomas and mixed germ cell tumours)allowing to distinguish different germ cell tumours subtypes, especially seminomas and embryonic carcinomas. Conclusion: (I) High-resolution mass spectrometry in combination with the SILAC method is suitable to differentiate between different tumour cell lines on the protein level. (II) The results of SILAC measures are reproducible in western blot analysis. (III) The method is helpful to establish new biomarkers differentiating histological subtypes of tumours. (IV) The detected proteins can be applied for immunohistochemical analysis on formalin-fixed and paraffin-embedded tumour tissue samples especially to distinguish seminomas from embryonic carcinomas as shown in this study. Contact: [email protected] 38 P1.1 Transcript levels of Piwi-like 1-4 genes are associated with clinicopathological parameters in renal cell carcinomas Al-Janabi O1, Wach S1, Nolte E1, Weigelt K1, Rau TT2, Stöhr C2, Legal W1, Schick S3, Greither T4, Hartmann A2, Wullich B1, Taubert H1 1 2 3 4 Urologische Klinik, Universitätsklinikum Erlangen, Friedrich-Alexander University Erlangen Institute of Pathology, Universitätsklinikum Erlangen, Friedrich-Alexander University Erlangen Tumorcenter, Friedrich-Alexander University Erlangen Center for Reproductive Medicine and Andrology, Martin Luther University Halle Background: Piwi-like gene family members (Piwil 1-4) are considered as stem-cell associated genes/proteins. They are expressed predominantly in the germ line but with a re-expression in different tumors. The expression of Piwil 1-4 genes has not been studied and correlated with clinicopathological parameters in renal cell carcinomas (RCC) yet. Material and methods: Transcript levels of Piwil 1-4 were analyzed by quantitative real time PCR in 74 clear cell RCC (ccRCC) tissues and corresponding normal tissues. Results: A strong and significant correlation of transcript levels of Piwil 1, 2, and 4 but not with Piwil 3 could be detected in the tumor tissues and in the normal tissues (P < 0.001; Spearman’s rank test). Piwil 4 gene expression was significantly higher in ccRCC than in corresponding normal renal tissue (P < 0.001; Mann-Whitney U-test). When separating the ccRCC patient cohort according to the median of Piwil 1-4 expression in a low and a high expression group and according to age in younger (≤ 64 yrs) and older patients (>64yrs); the younger patients displayed significantly higher mRNA expression levels of Piwil 1 in comparison to the older patients (P = 0.01; Fisher’s exact test). Interestingly, the expression of Piwil 1 showed a left-right polarity in the normal tissues but not in the tumor tissues (P = 0.0005; Fisher’s exact test). Conclusion: Associations between the expression levels of the Piwi-like family members and clinicopathological parameters could be detected for ccRCC suggesting a potential role in diagnostics and tumorigenesis of ccRCC and in embryology at least for renal tissue. Contact: [email protected] 39 P1.2 HIF-2 is highly expressed in papillary renal cell carcinoma type II: a potential reason for the worse prognosis? Behnes CL1, Thelen P2, Strauss A2, Radzun H-J1 Ströbel P1, Bremmer F1 1 2 Department of Pathology, University of Göttingen, Göttingen Department of Urology, University of Göttingen, Göttingen Background: Hypoxia inducible factors (HIFs) are widely expressed in different cell types and human cancers. HIFs are heterodimeric complexes composed of an alpha subunit (HIF-1, HIF-2 and HIF3) and a beta subunit (HIF-1) and promote cancer progression and neoangiogenesis by inducing VEGF secretion. Whereas HIF-1 and its functions are well known especially in renal cell cancer, HIF2 has not been investigated so far. We analyzed the expression of HIF-1 and HIF-2 in comparison to the expression of VEGF and capillary density in papillary renal cell carcinoma (RCC), which represents a rare tumor and is divided, based on histological criteria, into two subtypes, of which type II papillary RCC shows a worse prognosis. Methods: In the present study the expression of HIF-2, HIF-1, VEGF, CD31 and Ki67 were examined in 42 papillary RCC of histological type I and 32 papillary RCC of histological type II (n = 74) by immunohistochemistry. To demonstrate significant differences or correlations the data were subsequently statistically analysed. Results: Subtype II papillary RCC showed a significant higher expression of HIF2, whereas papillary RCC subtype I demonstrated a significant higher expression of HIF-1. The VEGF expression, the capillary density (CD31), and the proliferation rate (Ki67) were significant higher in papillary RCC subtype II in comparison to subtype I. Further analysis showed a significant correlation between VEGF expression and capillary density only in papillary RCC subtype II. A correlation between HIF-2 and the proliferation rate could not be demonstrated in both subtypes of papillary RCC. Conclusion: Papillary RCC types II demonstrate a significant increased expression of HIF-2 and VEGF in comparison to papillary RCC type I, which shows a significant higher expression of HIF-1. The HIF-2 and VEGF could be the reason for the higher capillary density as well as for the worse prognosis of subtype II compared to subtype I. Contact: [email protected] 40 P1.3 Deregulation of the CSN-CRL pathway during urological tumorigenesis? Gummlich L1,2, Kilic E3, Jung J2,4, Dubiel W1 1 2 3 4 Department of General, Visceral, Vascular and Thoracic Surgery, Division of Molecular Biology, Charité – Universitätsmedizin Berlin Berlin Institut for Urological Research, BFIU, Charité – Universitätsmedizin Berlin Department of Pathology, Charité – Universitätsmedizin Berlin Department of Urology, Charité – Universitätsmedizin Berlin Urological cancers belong to the most frequently occurring tumors. Renal cell carcinoma (RCC), for example, accounts for approximately 2% of all cancers worldwide. Despite a lot of effort spent to personalize therapeutical approaches, a group of RCC patients still appears to be therapy-resistant. New applicable targets are desperately needed for the treatment of urological cancer. The COP9 signalosome (CSN)-cullin-RING ubiquitin (Ub)-ligase (CRL) pathway is a prominent segment of the Ub proteasome system (UPS). It specifically ubiquitinates regulatory proteins and is often deregulated in cancer. Altered expression of the CRL components called F-box proteins in RCC influences the presence of key tumor promoting proteins in urological tissues. The exact mechanism of how deregulated CRL components are integrated in urological tumorigenesis is however unknown. Recently, our group revealed a post-transcriptional fine-tuning of COP9 signalosome (CSN) biosynthesis regulated by the c-myc/Lin28b/Let-7 pathway. Interestingly, analysis of RCC patient samples revealed down regulated let-7 miRNAs depending on metastatic state. Based on these observations we think that the CSN-CRL pathway is an attractive subject for urological cancer research. CSN-CRL pathway components were immunohistochemically stained in a subset of RCC samples to determine their expression pattern in urological tumor tissue. Selected CSN-CRL pathway components are expressed partly cytosolic and in the nucleus of the tubule, ureter, as well as the tumor cells. CSN subunit expression levels varied only slightly, whereas CAND1 staining revealed a deregulation in the intensity in urological cancer tissue nuclei. CSN subunit 8 and CAND1 are currently further examined in a tissue microarray with an appropriate cohort focusing on nucleus staining patterns. Interestingly, no subunit of the CSN complex was expressed irregularly in 4 RCC cell lines, implying a deregulation of the whole complex rather than a single subunit. The CAND1Skp2-p27 axis was also observed deregulated in these cell lines. In 786-O cells Skp2 is overexpressed whereas p27 appears with an atypical double band in immunoblots. In order to study the interplay of particular CSN-CRL pathway components and the possible impact of the p27 double band during renal tumorigenesis further investigations are in progress. Contact: [email protected] 41 P1.4 Quantitative imaging of intratumoral heterogeneity in clear cell renal carcinoma Höfflin R , Lahrmann B2, Grabe N2, Roth W3, Hadaschik B4, Pahernik S4, Hohenfellner M4, Duensing S1,4 1 1 2 3 4 Section of Molecular Urooncology, University Hospital Heidelberg Hamamatsu Tissue Imaging and Analysis Center (TIGA), BIOQUANT, University of Heidelberg Department of Pathology, University Hospital Heidelberg Department of Urology, University Hospital Heidelberg Background: Recent results from next generation sequencing analyses show a high degree of intratumoral genomic heterogeneity in advanced clear cell renal cell carcinoma (ccRCC). Moreover, reconstruction of tumor phylogeny based on mutational data suggests a branched pattern of clonal evolution of tumor cell populations. The two most commonly mutated genes with therapeutic relevance in ccRCC are VHL and mTOR. It is possible to indirectly determine the mutational status of these genes by analyzing the abundance of their respective downstream targets or changes in protein phosphorylation using routine immunohistochemistry (IHC). This proof-of-concept study was designed to test whether intratumoral heterogeneity centered on the VHL and mTOR pathway can be analyzed on the protein level in a quantitative manner and exploited as prognostic biomarker and tool for clinical decision-making. Methods: Formalin-fixed, paraffin-embedded (FFPE) specimens from a total of 34 primary tumors and metastatic lesions were processed for routine IHC analysis and analyzed for markers of pVHL loss (upregulation of HIF1- and/or HIF2-), activation of mTOR (phospho-mTOR S2448, phospho-S6 ribosomal protein S235/236) and expression of the proliferation marker Ki-67. Stained sections were scanned using a Nanozoomer 2.0 HT Scansystem at 20x magnification. The quantitative evaluation was performed with Visiopharm Software using two different algorithms: 1. positive pixel count to quantify cytoplasmic staining for pTOR S2448 and pS6RP S235/236. 2. positive nuclear count to quantify staining for HIF1-, HIF2- and Ki-67. In order to compare identical regions, adjacent sections were aligned and virtually segmented into squares of 1 mm². After exclusion of nontumorous tissue, scores were computed for individual squares and later combined into 3D maps to reflect abundance of protein expressin in a given tumor area. Results: We were able to obtain quantitative protein expression data for all markers and tumors. We found that intratumoral heterogeneity of protein expression is extensive. One of our key findings is that an activation of the mTOR signaling pathway occurs preferentially in the tumor periphery/invasion front. In addition, we detected strong focal pTOR S2448 immunostaining in two out of ten small ccRCCs with synchronous distant metastases. These focal lesions were suggestive of epithelial-mesenchymal-transition (EMT) and are currently being further investigated. Based on quantitative IHC image analyses we were able to create 3D maps representing differences in protein expression for all tumors analyzed. Conclusion: We have established an image analysis method for IHC/brightfield microscopy which makes it possible to visualize intratumoral heterogeneity of biomarkers in 3D. As this method can be fully automated and is applicable to other biomarkers, it can potentially be integrated into routine pathology and clinical decision-making. Contact: [email protected] 42 P1.5 Prediction of second-line therapy response in metastatic renal cell carcinoma patients by blood plasma marker proteins Hölters S1, Bergmann L2, Grünwald V3,Keilholz U4, Ohlmann C1 ,Staehler M5, Schmerler D6, Junker K1 1 2 3 4 5 6 Clinic of Urology and Pediatric Urology, Saarland University Medical Center, Homburg Medical Clinic II,University Hospital Frankfurt Department of Haematology, Haemostaseology, Oncology and Stem Cell Transplantation, Hannover Medical School Medical Clinic, Charité - Universitätsmedizin Berlin Department of Urology, University Hospital Munich Department of Clinical Chemistry and Laboratory Medicine, University Hospital Jena Introduction & Objectives: For patients with metastatic renal cell carcinoma (mRCC) vascular endothelial growth factor (VEGF)-targeted therapy (e.g. Sunitinib or Sorafenib) represents a promising first-line therapy after tumournephrectomy. For patients who progress during first-line therapy, inhibitors of mammalian target of rapamycin (mTOR) (e.g. Temsirolimus or Everolimus) represent an option for second-line therapy. Within the framework of the MARC-2 clinical trial, the aim of our study is the identification and validation of marker proteins associated with second-line therapy (Everolimus) response. Material & Methods: We included blood plasma samples from 19 mRCC patients receiving Everolimus. Therapy response was defined as stable disease and partial remission. We analysed the samples by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDITOF MS) using CM10 and Q10 arrays. For the identification of potential marker proteins, 2dimensional gelelectrophoresis and matrix-assisted laser desorption/ioniziation time-of-flight mass spectrometry (MALDI-TOF MS) will be conducted. Results: We identified 20 mass peaks by SELDI-TOF MS with Q10 and 16 with CM10 chips for the discrimination between responders (defined as stable disease or partial remission after two month of therapy) and non-responders (defined as progressive disease after 2 month of therapy). With the Q10, but not with the CM10 chip, we obtained five peaks for the discrimination between both patient groups before the first drug application. These peaks could be useable to preselect patients for the second-line therapy with Everolimus. For therapy monitoring, we found seven mass peaks (four with Q10 and three with CM10 chips) after two weeks and 24 mass peaks (11 with Q10 and 13 with CM10 chips) after four weeks of therapy to differentiate between responders and non-responders. These mass peaks could be useful to estimate the therapy response for the patients. Conclusion/Outlook: Our preliminary results argue for the presence of blood plasma proteins which potentially predict therapy response to Everolimus before as well as during therapy. The confirmation and identification of the SELDI-TOF MS outcomes by 2-dimensional gel electrophoresis and MALDITOF MS is on-going as well as the screening of an extended sample set. Sponsored by: iOMEDICO AG Contact: [email protected] 43 P1.6 Targets and function of dysregulated microRNAs in clear cell renal cell cancer Liep J , Wotschofsky Z1,2, Jung K1,2 1,2 1 2 Department of Urology, Charité – Universitätsmedizin Berlin Berlin Institute for Urologic Research, Charité – Universitätsmedizin Berlin Background: MicroRNAs (miRNAs) play a pivotal role in various types of tumors. Literature data reveal also the role of miRNAs in carcinogenesis and tumor progression of clear cell renal cell cancer and its metastasis. In a previous study (doi:10.7150/ijbs.5106) using microarray analysis and RT-PCR techniques, we could identify a specific miRNA expression pattern with several dysregulated miRNAs whereby most of them were down-regulated. Epigenetic mechanism could be revealed to be a key player in this downregulation of some of the found miRNAs. The altered miRNA-profile together with predicted miRNA-target interactions affords a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression. The aim of our study was to identify targets and function of some selected miRNAs of our profiling (miR-127 and miR-145). Materials and Methods: We started with a comprehensive target research using different search machines (miRWalk and targetscan) to get a list of predicted targets for each miRNA. Based on literature research we chose some potential targets out of this list, which could be interesting for carcinogenesis. To test, if these potential targets are really regulated by the miRNAs, renal cell lines (786-O and ACHN) were transfected with the miRNAs. The impact of the miRNA overexpression towards the appropriate target expression on mRNA level by RT-qPCR was measured. Furthermore, we wanted to investigate the effect of the miRNAs by means of scratch assay. Results: By this approach, we could identify some potential targets of our miRNAs of interest. The two genes BIRC2 and TNFRSF10B were downregulated after increasing miR-145 concentration, and the polycomb group protein EZH2 seems to be regulated by miR-127. The functional analysis reveals that miR-127 seems to have an influence on migration of tumor cells. Conclusions: Our study identifies some new miRNA-target interactions. Furthermore, the functional analysis gives hint that the found miRNA-target interaction plays an important role in tumor progression and metastasis. These interesting findings are a good starting point for further analysis to investigate the detailed role of miRNAs in progression of clear cell renal cell cancer and its metastasis. Contact: [email protected] 44 P1.7 Integrated microRNA and mRNA signature associated with the transition from the locally confined to the metastasized renal cell carcinoma Wotschofsky Z1,2, Billaud J-N3, Jung M1,2, Meyer H-A1,4 1 2 3 4 Department of Urology, Charité – Universitätmedizin Berlin Berlin Institute for Urologic Research, Berlin Ingenuity Systems, Redwood City, USA Institute of Physiology, Charité – Universitätmedizin Berlin Background and Objective: MicroRNAs (miRNAs) are endogenous small non-coding RNAs that regulate gene expression by interfering translation or stability of target transcripts. One miRNA can interact with several hundred mRNAs, while one mRNA can be regulated by several miRNAs. This interplay between miRNA and their mRNA has been proposed as an important process in cancer development and progression. We have investigated molecular networks impacted by predicted mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma (ccRCC) diagnosed with or without metastasis. Material and Methods: miRNA and mRNA microarray expression profiles derived from primary clear cell renal cell carcinomas from patients with (in total 16 samples) or without diagnosed metastasis (in total 22 samples) were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of experimentally validated and predicted mRNA targets was used. By applying an expression pairing tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs and their target genes with opposite or same expression. The resulting identified interactions were revalidated by RT-qPCR in another cohort of RCC patients. The predicted miRNA-mRNA interactions were also tested by functional analyses using miRNA knock-down and over expression experiments in renal cancer cell lines. Results: Among the significantly differentially expressed miRNAs, we have identified 3 miRNAs (miR146a, miR-128a and miR-17-5p) that were upregulated in primary tumors from patients without metastasis and down regulated in primary tumors from patients with metastasis. We have further identified the mRNA targets which expression were inversely correlated to these 3 miRNAs, and have been previously experimentally demonstrated in cancer setting in humans. Specifically we showed that BRAC1, MCM10, CDKN3, UHRF1, IL8 were downregulated and targeted by miR-146a-5p. The relation between these identifies target genes and miRNA-146a was validated in cell culture experiments. Conclusions: We identified novel target genes of dysregulated miRNA which are involved in the transition from primary RCC without metastases into tumors generating distant metastasis. Contact: [email protected] 45 P1.8 Molecular cytogenetic differences between Collecting Duct Carcinomas and upper urinary tract urothelial carcinomas Jung V1,13, Becker F1,11,13, Parr M1,13, Hartmann A2,13, Füssel S3,13, Toma M4,13, Grobholz R5, Pflugmann T6, Wullich B7,13, Strauss A8,13, Behnes CL9,13, Otto W10,13, Stöckle M1,13, Junker K1,12,13 1 2 3 4 5 6 7 8 9 10 11 12 13 Department of Urology, University of the Saarland, Homburg/Saar Department of Pathology, University Hospital Erlangen Department of Urology, Technical University of Dresden Department of Pathology, Technical University of Dresden Department of Pathology, Kantonsspital Aarau, Aarau, CH Department of Urology, St.Franziskus Clinics, Mönchengladbach Department of Urology, University Hospital Erlangen Department of Urology, Georg-August University Göttingen Department of Pathology, Georg-August University Göttingen Department of Urology, Caritas Clinics St. Joseph, University Regensburg Urological Group Practice & Clinic Derouet/Pönicke/Becker, Boxberg Center, Neunkirchen Department of Urology, University Hospital Jena German Network of Renal Cell Tumors Collecting duct carcinoma (CDC) is a rare renal neoplasm that is associated with poor prognosis due to its highly aggressive course and limited response to immuno- or chemotherapy. Histologically, CDC is defined as a subtype of renal cell carcinomas, but in some cases, it is difficult to differentiate from urothelial carcinomas (UC). Therefore the aim of this study was to determine genetic alterations of CDC in comparison to that of urothelial carcinomas of the upper urinary tract (UUT-UC) to clarify the histological origin of this rare tumor entity. Twenty-nine CDC samples were obtained from seven different German centers and compared with twenty-six urothelial carcinomas of the upper urinary tract. Comparative genomic hybridization (CGH) was used to investigate the genetic composition of patients’ tumors and allowed the detection of losses and gains of DNA copy numbers throughout the entire genome. The clinical data were correlated with CGH results. CGH analysis of CDC revealed DNA aberrations in many chromosomes. DNA losses were more frequently observed than gains, while high-level amplifications were not detected. The mean frequency of CDC chromosomal aberrations (4.9/case) was slightly lower than that in UUT-UC (5.4/case). Recurrent CDC DNA losses occurred at 8p (n=9/29), 16p (9/29), 1p (n=7/29) and 9p (n=7/29), and gains occurred in 13q (n=9/29). In contrast to CDC, the most frequently detected UUTUC DNA aberration was a loss at 9q (n=13/26). DNA losses at 9q, 13q and 8q as well as gains at 8p showed significant variations in UUT-UC compared to CDC. There was no correlation between the patients’ clinical course and the presence or absence of these recurrent genetic alterations. CDCs are characterized by a different genetic pattern compared to UUT-UC. Regarding the published data on renal cell carcinoma, we conclude that CDC appears to be a unique entity among kidney carcinomas. Contact: [email protected] 46 P1.9 Novel antiangiogenic compounds with antitumor activity for innovative approaches in cisplatin resistant testicular germ cell cancer treatment Nitzsche B1,2, Pries A1, Schrader M3, Preissner R1, Honecker F4, Höpfner M1 1 2 3 4 Institute of Physiology, Charité – Universitätsmedizin Berlin Berlin Institute for Urologic Research, Charité – Universitätsmedizin Berlin Department of Urology, University of Ulm Department of Oncology and Hematology, Hubertus Wald Tumor Center - University Cancer Center Hamburg Objective: Effective treatment of testicular germ cell tumors (TGCT) resistant to conventional chemotherapy is still insufficient. As angiogenesis is essential for the development, growth and progression of tumors we hypothesised that targeting angiogenic growth factor receptor signalling pathways may be a promising approach for novel treatment of therapy resistant TGCTs. Design and method: Two recently identified antiangiogenic compounds, HP-2 and HP-14, blocking the VEGFR-2 and related signalling pathways of endothelial and VEGFR-2 expressing cancer cells were investigated for their suitability to inhibit the growth and vascularisation of normal and cisplatin-resistant testicular germ cell tumors (TGCT) in vitro and in vivo. Performing proliferation assays (crystal violet method), the antineoplastic effects of HP-2 and HP-14 alone or in combination with platinum compounds were evaluated in both platinum sensitive and –resistant testicular germ cell tumor cells (2102EP, 2102EP-R). For in vivo evaluations TGCT cells (2102EP, 2102EP-R, Tera-1, Tera-2) were inoculated onto the chorioallantoic membrane of fertilized chicken eggs (CAM assay). The developing tumors were treated with the HP-compounds and the inhibition of angiogenesis and tumor growth was documented. Results and conclusions: HP-2 and HP-14 effectively suppressed the growth of TGCTs, both in vitro and in vivo. We could show that the growth of testicular germ cell cancer cells, resistant to conventional platinum-based chemotherapy can be potently inhibited by the novel HP-compounds alone or in combination with cisplatin. Together, these data suggest that HP-2 and HP-14 may be interesting new drugs for targeted therapy of urologic cancers, particularly for those being resistant to the conventionally successful cisplatin-based interventions. Contact: [email protected] 47 P1.10 Epigenetic regulation of genes involved in epithelial mesenchymal transition in prostate cancer Brandt U1, Wagenlehner F1, Waliszewski P1, Steger K1, Weidner W1, Gattenloehner S2, Schagdarsurengin U1, Dansranjavin T1 1 2 Clinic of Urology, Pediatric Urology and Andrology; Justus-Liebig-University Giessen Institute of Pathology, Justus-Liebig-University Giessen Aim: Epithelial to mesenchymal transition (EMT) plays a pivotal role in molecular mechanisms of prostate cancer (PCa) metastasis. Hypoxia-inducible factor 1-alpha (Hif-1) is a key transcriptional factor controlling hypoxia induced EMT. In our study, we have analyzed the epigenetic regulation of EMT associated genes EPO, FBLN2, SLUG and SNAIL1 in PCa cell lines after stabilization of Hif-1 protein. Methods: The endogenous Hif-1 protein was stabilized and enriched by treatment with 1 mM HIF1 prolyl hydroxylase inhibitor dimethyloxaloylglycine (DMOG). We investigated the effect of Hif1-stabilisation and 5´-Aza-2´-deoxycitidine (Aza) 5 μM on relative expression of EPO, FBLN2, SLUG and SNAIL1 in prostate cancer cell lines LNCaP, Du145 and PC3 by qRT-PCR. The methylation of the promoter regions were analyzed by COBRA (combined bisulfit restriction analysis) and methylation specific PCR. Results: Untreated PCa cell lines exhibited no expression of all analyzed genes, except SLUG showing a weak relative expression (RE) in PC3 cells. The promoter regions of EPO, FBLN2, SLUG and SNAIL1 were partially methylated in Du145 and PC3 cells. In LNCaP cells all candidate genes, except SLUG, were unmethylated. The stabilization of Hif-1 protein with DMOG led to an overexpression of only EPO (RE 11) in LNCaP and of SNAIL1 (RE 10) in PC3 cells. Aza treatment resulted in the activation of EPO (RE 1,5), FBLN2 (RE 1,3), SLUG (RE 1,7) and SNAIL1 (RE 3,2) in LNCaP cells. In PC3 cells we observed the activation of FBLN2 (RE 4,3) and 5-fold upregulation of SLUG (from RE 0,6 up to RE 3,4). The combined treatment of Aza with DMOG resulted in the activation of SLUG (RE 1,8) in LNCaP cells and of SLUG (RE 3,3) and SNAIL1 (RE 0,6) in PC3 cells. Conclusion: These data provide evidence that the CpG-methylation status of genes regulating EMT modulates their inducibility by Hif-1. Therefore we suppose that the methylation status of Hif-1 target genes could be useful for characterization of EMT and of metastatic potential of PCa. Contact: [email protected] 48 P1.11 Epigenetic intervention counteracts resistance towards temsirolimus in prostate cancer Makarevic J, Tsaur I, Jüngel E, Haferkamp A, Blaheta R Department of Urology, University of Frankfurt Introduction: Specific blocking of the “mammalian target of rapamycin” (mTOR)-kinase by the mTORinhibitor temsirolimus (Torisel ®) have led to encouraging clinical results. However, chronic use of temsirolimus may lead to resistance, which counteracts the antitumoral effect of this drug. In this study, we evaluated growth and invasion of prostate cancer cells with acquired resistance to the mTOR-inhibitor temsirolimus. Further investigation was designed to determine whether additionally targeting histone deacetylase (HDAC) by an HDAC-inhibitor (valproic acid, VPA) might counteract undesired feedback mechanism caused by chronic use of temsirolimus. Material and Methods: Prostate cancer cells were either treated with temsirolimus over 12 months, starting at 1 nM and increasing stepwise to 5 μM (PC3TEM), or only treated with medium (PC3). Growth, proliferation, adhesion and invasion of PC3 versus PC3TEM were investigated. Expression of cell cycle regulating proteins, mTOR related intracellular signaling and the expression profile of alpha and beta integrin subtypes were also evaluated. Additionally, HDAC was blocked in PC3TEM cells and the consequences on invasive growth investigated. Results: PC3 TEM accumulated in the G2/M-phase, accompanied by cdk1, cdk2, cyclin B elevation and reduction of p21 and p27. The target proteins Akt, mTOR, rictor and p70S6k were strongly activated in PC3TEM compared to PC3 cells. Distinct modifications were also seen with respect to alpha and beta integrin expression. Additional application of VPA blocked growth and adhesion of PC3TEM cells, reverted integrin modulation and deactivated proteins of the mTOR signaling pathway. Discussion: Chronic use of the mTOR inhibitor temsirolimus causes prostate cancer cell resistance. HDAC-inhibition counteracts this process, possibly by cross-communicating with the mTOR signaling axis. Specific targeting of HDAC may, therefore, enhance the benefit of an mTOR-inhibitor based regimen. Contact: [email protected] 49 P1.12 Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and proteomics expression data Meyer H-A1, Kamlage B2, Reszka R3, SchatzP3, Stephan C1,4, Dietrich D5, Kristiansen G5, Jung K1,4 1 2 3 4 5 6 Institute of Physiology, Charité – Universitätsmedizin Berlin Department of Urology, Charité - Universitätsmedizin Berlin Metanomics GmbH, Berlin Metanomics Health GmbH, Berlin Berlin Institute for Urologic Research, Berlin Institute of Pathology, University Hospital of Bonn Background: Metabolite profiling research offers a deeper insight into biochemical changes in cancer metabolism. Moreover the integrated analysis of transcription, metabolomics and proteomics data can improve the understanding of the underlying biological processes. Material and Methods: A set of 254 metabolites was determined by gas chromatography/liquid chromatography-mass spectrometry in matched malignant and non-malignant prostatectomy samples from 95 prostate cancer (PCa) patients. Transcription profiling data were obtained from own micro array experiments (matched malignant and non-malignant prostatectomy samples from 15 PCa patients using Affymetrix U133 arrays) as well as public GEO expression data. Expression levels of selected proteins were determined by tissue micro array in 41 matched frozen tissue samples. The data were related to clinicopathological variables and disease recurrence in the follow-up. Transcription and metabolomics data were statistical analysed (ANOVA, Mann–Whitney U test) and significant regulated metabolites/genes/proteins were selected. Results: Significant regulated metabolites/genes between malignant and non-malignant samples were used for network analysis. Enriched pathways which are involved in PCa progression or recurrence such as carbohydrate and fatty acid metabolism were identified. The role of fatty acid metabolism in PCa was analysed in more detail. Several fatty acids such as cerebronic acid, 2hydroxybehenic acid, tricosanoic acid showed higher concentrations in malignant than in nonmalignant samples which correspond to the observed higher mRNA and protein expression level of fatty acid synthase (FASN) in PCa. In contrast to normal prostate tissue, where protein expression level of FASN was correlated to the level of measured metabolites we found in malignant samples a deregulation of the corresponding pathway. Conclusion: Our integrated analysis of transcription, metabolite and proteomics data confirm and extent the role of several biological pathways which are involved in PCa progression. Contact: [email protected] 50 P1.13 Cytostatic drug WIST-C overcomes docetaxel induced resistance Rottach M1, Peter T1, Mandelkow R1, Weiss M1, Walther R2, Burchardt M1, Stope MB1 1 2 Department of Urology, University Medicine Greifswald Department of Medical Biochemistry and Molecular Biology, University Medicine Greifswald Background: Induction of cytoprotective pathways during docetaxel treatment of advanced prostate cancer (PCa) frequently confers secondary resistance to therapy. The alternative taxane compound WIST-C given as second-line therapy restores pro-therapeutical effects followed by overcoming docetaxel-induced resistance mechanisms. The aim of this study was to identify and characterize WIST-C-mediated cytostatic mechanisms in PCa cells, particularly compared to docetaxel-induced molecular effects. Methods: Prostate cancer cell lines were treated with the taxane WIST-C and analyzed by proliferation assay (CASY TT cell analyser, Roche). Drug-dependent modulation of resistanceassociated heat shock proteins HSP27, HSP70, HSP90, as well as androgen receptor (AR) and prostate specific antigene (PSA) expression levels were monitored by Western blotting and quantitative RT-PCR. Furthermore, the induction and activation of apoptotic factors was assessed. Results: Prior to incubation experiments WIST-C IC50 concentrations were determined indicating the cytostatic efficacy of the drug. Analysis of putative factors of chemoresistance revealed decreased levels of HSP27 and AR accompanied by an attenuation of transcriptional activity of AR as shown by reduced levels of PSA mRNA. Moreover, WIST-C treatment led to an induction of the pro-apoptotic factor p53. Conclusion: These results indicate WIST-C efficacy counter-acting docetaxel-induced cytoprotective HSP27 machinery in PCa cells pointing to differential cellular responses to both taxanes in PCa cells. Contact: [email protected] 51 P1.14 Absolute quantification by qRT-PCR - a new and sensitive method to measure absolute amounts of miRNA in high risk prostate cancer tissue Schubert M1*, Kneitz B1, Spahn M2, Riedmiller H1, Kneitz S3 1 2 3 Department of Urology and Pediatric Urology, University Hospital, Wuerzburg; Comprehensive Cancer Center Mainfranken Department of Urology, Inselspital Bern, CH Department of Physiological Chemistry, University Wuerzburg Introduction: We previously showed miRNA-221 as potential prognostic biomarker whose downregulation is associated with clinical recurrence in high risk prostate cancer (PCa). To become a reliable biomarker expressional analyses from independent study centres are necessary for validation. The Δct-method (relative qRT-PCR) is widely used for this. Due to high error rates and only a relative quantification statement, comparison of PCR results of material from different study collectives is limited. Often statistical features (e.g. Z-Score) are necessary to adapt expression levels of different study groups to make results comparable. There is urgent need to develop a method that is: more exact in determination of expression levels, applicable in daily routine and allows for exact comparison of results. Based on these conditions we demonstrate the method of absolute miRNA-quantification. Material and Methods: A universal reference (miRXplore™) with predefined amounts of miRs was used and serial dilution performed for qRT-PCR. This allowed for determination of absolute amounts of miR (in attomol) within a sample. For validation of miR-221 we used the same collective (A: n=99) with clinical data as in our previous analyses. Absolute quantification for miR-221 and the housekeeper were performed, followed by Cox regression and Kaplan Meier (KM) analyses. Absolute quantification was performed in a second independent cohort (B: n=108) to compare expression levels. Results: The new method was well reproducible and revealed precise results. The expressional analysis of cohort A reached comparable significance levels as in relative qRT-PCR: KM estimates predicted sign. difference (p< 0.01) in groups with high and low miR-221 expression regarding clinical failure and cancer related death (CRD). Dichotomized miR-221 expression was multivariately sign. for prediction of CRD (p< 0.01; CI 0.004-0.3). Absolute quantification revealed approximated expression levels in both PCa cohorts. Conclusion: We demonstrate a new precise and reproducible method to quantitate absolute amounts of miRs. We confirm miR-221 as potential prognosticator in high risk PCa. By absolute quantification expressional analyses from different study centres are comparable more precisely. In the future this method might therefore be used for validation of potential biomarkers in independent study centres and prospective settings. *2011-2012: Supported by a Ferdinand Eisenberger grant of the Deutsche Gesellschaft für Urologie (German Society of Urology), grant ID ScM1/FE-11 Contact: [email protected] 52 P1.15 Evaluation of Patient Derived Molecular Neuroendocrine Signatures in PC3 using RNAseq data von Hardenberg J1,2, Kerr G1, Voloshanenko O1, Worst TS1,2, Michel MS2, Boutros M1 1 2 German Cancer Research Center (DKFZ), Div. Signaling and Functional Genomics, Heidelberg Department of Urology, University Medical Center Mannheim, University of Heidelberg Introduction: Neuroendocrine prostate cancer (NPCA) represents an aggressive subtype of prostate cancer. Apart from NCI-H660 there are no cell based model systems available to study this aggressive prostate cancer subtyp. Recently the established prostate cancer cell line PC3 was suggested to serve as a NPCA model system. In western blot analysis we observed the expression of chromogranin A (CGA) and neuron specific enolase (NSE) in PC3 but also in Du-145. This questioned the approach to characterize only according to two neuroendocrine markers. The aim of this study was to test if we could better estimate the NPCA signature in PC3 and other prostate cancer cell lines. We therefore evaluated patient derived molecular NPCA signatures in these cell lines. Methods: Western blot analysis were used to identify the protein expression of CGA and NSE in PC3, Du-145 and LNCaP. Subsequently, gene expression information from 17 prostate cancer cell lines was used to evaluate known molecular signatures of NPCA. Alignment data from RNA Sequencing (RNAseq) experiments from different labs were compared and integrated by estimating expression using a standardized workflow. Expression information of known basal, luminal and neuroendocrine genes was then used to profile these 17 cell lines. Additionally, recently identified molecular signatures of NPCA in patient samples were used for unsupervised clustering. Results: CGA was highly expressed in PC3 and Du-145 but not in LNCaP cells. The profiling of RNAseq expression data according to known prostate markers proofed the feasiblity of this approach. The NPCA cell line NCI-H660 grouped by itself. PC3 and Du-145 did not show high expressions of neuroendocrine markers in the RNAseq data. Using the patient derived NPCA signature NCI-H660 showed a unique expression profile not clustering with other cell lines. We could not observe a NPCA signature in other established cell lines. Conclusion: Identifying molecular signatures using RNAseq helps to more precisely characterize prostate cancer cell lines. A molecular NPCA signature could only be observed in the established neuroendocrine cell line NCI-H660 although PC3 and Du-145 showed expressions of CGA and NSE on the protein level. Contact: [email protected] 53 V3.6 Investigation of Escherichia coli urinary tract isolates with characteristics of small colony variants Putze J1, Greune L2, Schmidt MA2, Svanborg C3, Dobrindt U1 1 2 3 Institut für Hygiene, Universitätsklinikum Münster Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung - ZMBE, Münster Division of Microbiology, Immunology and Glycobiology - MIG, Lund University, Lund, S Introduction: Urinary tract infections (UTIs) are a worldwide occurring disease with an estimate of more than 10 million cases per year in Western Europe. The most prevalent causative agent of UTIs is Escherichia coli. Besides symptomatic UTI, E. coli also causes asymptomatic bacteriuria (ABU). ABU strains cause only mild or no symptoms during carriage. Bacterial small colony variants (SCVs) grow slow and form tiny colonies compared to other strains of the same species. The appearance of SCVs has mainly been described for Staphylococcus aureus and Pseudomonas aeruginosa. SCVs are known to cause chronic-persistent infections, being more resistant to antibiotic treatment, occurring intracellularly and may display several other virulence- associated traits. On the other hand, SCVs frequently exhibit deficiencies like auxotrophy and electron-transport-defects also accounting for the slow growth behavior. Material and Methods: We focused on traits of UPEC which contribute to persistent and recurrent infection and characterized two reisolates of ABU E. coli strain 83972 obtained from deliberately colonized patients showing an SCV and SCV-like phenotype, respectively as well as additional isolates with decreased growth ability from asymptomatic carriage. We analyzed growth kinetics as well as bacterial cell morphology. In addition, phenotypic features like curli and cellulose expression, biofilm formation and resistance against antibiotics were assessed. We compared the adhesion characteristics to human bladder epithelial cells and human kidney epithelial cells. Furthermore the invasion capacity, uptake and survival in non-professional and professional phagocytes were determined. As a key aspect we sequenced the reisolates of E. coli ABU strain 83972. Results: UPEC and ABU isolates with decreased growth rate from cases of extended bladder colonization differed in their phenotypic traits. SCV characteristics could not be determined for all slow-growing urinary isolates. The SCV and the SCV-like reisolates of ABU E. coli 83972 differ in their phenotypes from their progenitor 83972. Conclusion: Our results demonstrate that SCV formation is also one strategy of asymptomatically colonizing E. coli to adapt to adverse and changing growth conditions in the urinary tract. We discuss our findings against the background of bacterial traits which distinguish UTI and ABU strains or contribute to adaptation to prolonged urinary tract colonization. Contact: [email protected] 54 P2.1 OASIS/CREB3L1 is downregulated in human bladder tumors and mediates suppression of cancer cell spreading and migration in vitro Dierichs L1, Rose M1, Schubert C1, Gaisa NT1, Heer M1, Heidenreich A2, Knüchel R1, Dahl E1 1 2 Molecular Oncology Group, Institute of Pathology, Medical Faculty of the RWTH Aachen University Department of Urology, Medical Faculty of the RWTH Aachen University Background: Invasive bladder cancers are associated with an unfavorable clinical outcome. Understanding the underlying biological mechanisms of this cancer subtype may help to approach novel therapeutic strategies. Previously, DNA array expression analysis identified the bZIP transcription factor OASIS (old astrocyte specifically induced substance), also known as CREB3L1, to be downregulated during bladder cancer development. We furthermore showed that aberrant promoter methylation of OASIS in bladder cancer tumors is associated with invasive high grade tumors. In this study, we aimed to decipher the functional role of OASIS loss in bladder cancer by generating a bladder cancer cell model overexpressing OASIS. Furthermore, the putative OASIS target gene HTRA3 was analyzed in this cellular system. Methods: OASIS and HTRA3 mRNA expression was analyzed in a large cohort of bladder cancer samples (n=64), comprising carcinoma in situ (CIS), papillary and invasive bladder tumor samples by using real-time PCR. Normal urothelium tissues (n=14) served as control. Using immunohistochemistry OASIS protein expression was determined. In order to characterize a putative tumor suppressive role OASIS, we established a stable gain-of-function in vitro model using the invasive J82 urothelial cancer cell line. Based on this, we analyzed colony growth (colony formation assay) and tumor cell motility (wound healing assay) of both independent J82-mock and J82-OASIS clones. Correlation of the OASIS expression and HTRA3 expression was performed by calculating a Spearman correlation coefficient. Results: Real-time PCR analysis showed a downregulation of OASIS mRNA in bladder cancer tissues (Δ fold change: -3.8) that was also evident on protein level. OASIS re-expression in the invasive bladder cancer cell line J82 led to a significant (p<0.001) suppression of colony growth. Moreover, we functionally demonstrated a reduction of tumor cell migration in vitro that is potentially mediated by an OASIS-induced HTRA3 expression. A positive correlation of OASIS and HTRA3 expression was also confirmed in bladder cancer tissues (n=48) (Spearman test; r=0.6649, p<0.001). Conclusion: In the current study we provide for the first time evidence that OASIS expression may represses bladder cancer invasion. By identifying HTRA3 as a potential target gene of OASIS in invasive bladder cancer cells a putative impact of OASIS on TGF- signaling could be suggested but have to further analyzed in future studies. Contact: [email protected] 55 P2.2 Responses of bladder cancer cells towards tyrosine-kinase inhibition by dovitinib (TKI258) in relation to the eptithelial mesenchymal transition status Hänze J, Henrici M, Hegele A, Hofmann R, Olbert P Klinik für Urologie und Kinderurologie, Philipps Universität Marburg Dovitinib (TKI-258) is a receptor tyrosine kinase (RTK) inhibitor targeting fibroblast growth factor receptor (FGFR), and structurally related RTKs. Dovitinib is investigated as anticancer drug in various cancers including bladder cancer with aberrant RTK signalling. Here, we analyzed the dovitinib response in relation to the epithelial mesenchymal transition (EMT) status as a possible predictor of therapy response. EMT status was determined in human bladder cancer cell lines based on mesenchymal marker N-cadherin and epithelial marker E-cadherin. Dovitinib dependent responses were analyzed by determination of IC50 values in viability/proliferation dose response curves and by survival fractions in colony formation experiments. We observed significant correlations of these parameters with the EMT status. Thus, the EMT status in bladder cancer cells may be exploited to predict therapy responses towards dovitinib treatment. Contact: [email protected] 56 P2.3 HDAC inhibition suppresses bladder cancer cell adhesion to collagen under flow conditions Juengel E1, Santos SM2, Schneider T1, Makarevic J1, Hudak L1, Bartsch G1, Haferkamp A1, Blaheta RA1 1 2 Klinik für Urologie und Kinderurologie, Universitätsklinikum, Goethe-University, Frankfurt a.M. Institut für Klinische Pharmakologie, Universitätsklinikum, Goethe-University, Frankfurt a.M. Introduction: The influence of the histone deacetylase (HDAC)-inhibitor, valproic acid (VPA), on bladder cancer cell adhesion in vitro was investigated in this paper. Materials & Methods: TCCSUP and RT-112 bladder cancer cells were treated with VPA (0.5 or 1 mM) twice or thrice weekly for 14 days. Controls remained untreated. Tumour cell interaction with immobilized collagen was evaluated by a flow-based adhesion assay using a shear force of 2 or 4 dyne/cm2. The effects of VPA on the integrin adhesion receptors 3, 5, ß1, ß3 and ß4 were assessed by flow cytometry to determine integrin surface expression and by western blotting to determine the cytoplasmic integrin level. Results: VPA, 0.5 mM and 1 mM, significantly prevented binding of both RT-112 and TCCSUP cells to collagen, compared with the untreated controls. Adhesion was reduced to a higher extent when RT112 (subjected to 2 dyne/cm2) or TCCSUP (subjected to 2 or 4 dyne/cm2) tumour cells were treated with VPA three times a week, compared to the two times a week protocol. VPA caused a significant up-regulation of the integrin 3, 5, ß1, ß3 and ß4 subtypes on the TCCSUP cell surface membrane. In RT-112 cells, only integrin 5 was elevated on the cell surface following VPA exposure. Western blotting revealed an up-regulation of 3, 5, ß3 and ß4 integrins and down-regulation of the integrin ß1 protein by VPA in TCCSUP. VPA also up-regulated 5 and down-regulated ß1 integrin in RT-112 cells, but also reduced 3 and ß3 in TCCSUP. VPA exerted adhesion-blocking properties on bladder cancer cells under physiologic flow conditions. The effects were accompanied by distinct modifications of the integrin expression profile, which differ depending on the cell lines used. Conclusion: Application of VPA might be an innovative option to prevent bladder cancer dissemination. Contact: [email protected] 57 P2.4 Invasion of urothelial carcinoma of the bladder: MMP7 is associated with increased cell invasion in urothelial cancer: evidence from functional models Knauf D¹, Gorzelanny C², Erben P¹, Schneider SW², Steidler A¹, Bolenz C¹ ¹ Department of Urology, Mannheim Medical Center, University of Heidelberg, Mannheim ² Department of Experimental Dermatology, Mannheim Medical Center, University of Heidelberg, Mannheim Introduction: Matrix metalloproteinases (MMPs) play a crucial role in the lymphovascular invasion (LVI) of malignant cells and metastatic spread of cancer. Elevated levels of MMP7 have been reported to correlate positively with invasiveness of urothelial carcinoma (UC) cells and are associated with a poorer oncological outcome. Therefore we aimed to functional evaluate MMP7 using in vitro assays in UC. Material & Methods: MMP7 expression was characterized in human UC cells (UMUC-3, RT112, HT1197, T24/83 and RT4) and in a benign urothelial cell line (Urotsa) by quantitative RT-PCR and Western Blot. The invasive potency of the different cell types where determined using an in vitro invasion assay based on electrophysiological resistance breakdown across a MDCK-C7 monolayer. A coefficient close to zero reflects high invasive potency. MMP7 expression was modulated using RNAi interference before seeding in the invasion assay. Results: Urothelial T24/83, HT1197, RT112, RT4 and UroTsa cells expressed the inactive pro-form of MMP7, whereas the active form was only detected in HT1197 and RT112 possessing the highest invasive potency (coefficients of 0.073 and 0.076, respectively). Optimiced transfection of UC HT1197 cells with MMP7 siRNA inhibited MMP7 expression and reduced significantly the invasive behaviour in the invasion assay (p<0.005, Mann-Whitney U-test). Conclusions: Our results indicate an invasive potency of UC cells dependent on the expression of the active form of MMP7. Therapeutical targeting of MMP7 could be an option in the prevention of lymphovascular invasion and metastatic spread of UC cancer cells. Contact: [email protected] 58 P2.5 Analyse der microRNA Expression im Urothelkarzinom des oberen Harntrakts Kriebel S , Schmidt D1, Kristiansen G2, Müller SC1, Ellinger J1 1 1 2 Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum Bonn Institut für Pathologie, Universitätsklinikum Bonn Background: MicroRNAs play a major role in the cancerogenesis of multiple malignant tumours and are also relevant for the urothelial Carcinoma of the urinary bladder. So far, the microRNA expression of the urothelial carcinoma of the upper urinary tract (UUT-UC) has not been investigated. Methodology: RNA was extracted out of 47 UUT-UC samples as well as 36 corresponding ureter samples. The expression of the microRNAs was analyzed using quantitative Real-Time PCR. As target genes 11 microRNAs were selected, which had been described in previous studies as dysregulated in the urothelial cancer of the urinary bladder. A statistical analysis was carried out using the MannWhitney Test including a Bonfferoni correction for multiple testing (significance level p<0.0045). Results: MicroRNAs miR-21, miR-96, miR-135, miR-141, miR-182, miR-205, miR-429, miR-520b (all p<0,001) were significantly upregulated in UUT-UC; miR-10a (p=0,012), miR-200b (p=0,006) and miR1244 (p=0,600) were similary expressed as in the normal tissue. Especially miR-96 and miR-182 allowed a precise differentiation (area under curve >0.92) between the two groups. The microRNA miR-205 (p=0,002) was upregulated in the poorly differentiated UUT-UC (G3 vs. G2/G1). Conclusion: MicroRNA expression profiling allows a differentiation of normal urothelium and UUTUC; microRNAs (especially miR-96 and miR-182) could be used as non-invasive Biomarkers in urine or blood. There also exists a potential prognostic relevance for miR-205. Contact: [email protected] 59 P2.6 Acute epididymitis induces alterations in sperm protein composition Pilatz A , Karnati S2, Lochnit G3, Paradowska-Dogan A1, Lang T4, Schultheiss D5, Schuppe H-C1, Hossain H6, Baumgart-Vogt E2, Weidner W1, Wagenlehner F1 1 1 2 3 4 5 6 Klinik für Urologie, Kinderurologie und Andrologie, Justus-Liebig-Universität, Gießen Institut für Anatomie und Zellbiologie, AG Medizinische Zellbiologie, Justus-Liebig-Universität, Gießen Institut für Biochemie, Justus-Liebig-Universität, Gießen Institut für Anatomie und Zellbiologie, AG Reproduktionsbiologie, Justus-Liebig-Universität, Gießen Urologische Belegabteilung, Evangelischen Krankenhaus Mittelhessen, Gießen Institut für medizinische Mikrobiologie, Justus-Liebig-Universität, Gießen Introduction: Infection and inflammation in the urogenital tract are important etiological factors implicated in male infertility. Here, the acute epididymitis is the only ascending infection with direct impact on epididymis and testis. In spite of epididymitis occurring frequently in patients within reproductive years, the impact on fertility has not been systematically investigated. To date, it is unknown if urogenital infections cause changes in protein composition of sperm. Materials and Methods: Between 2010 and 2012, 8 patients with acute epididymitis, gave written informed consent and provided a semen sample three months after initial presentation and treatment according to the guidelines. As a control group, 10 healthy men were included in the study. Semen analysis was performed within 1 h of collection according to WHO 2010 recommendations. To select the motile/viable sperm for proteome analysis, direct swim-up technique was performed from 1 ml of total semen. Samples of patients and controls were separately pooled due to low protein content. Proteome analysis was performed by 2D electrophoresis and protein identification by MALDI-TOF mass spectroscopy. The sub-cellular localization and biological function of the identified proteins was determined using the information of the UniProt Knowledgebase. In addition representative sperm preparations were examined by immunofluorescence for the presence of the subunit of the mitochondrial ATP synthase (ATP5B). Results: Proteome analysis identified 35 proteins in sperm from epididymitis patients were downregulated, irrespective of subcellular localization and biological functions. According to the literature, 13 of these have been reported to be differentially expressed after capacitation, 10 showed an impaired expression in infertile males, and 13 proteins have been found in the epididymal fluid. Most of the proteins were part of the cytoskeleton, followed by the cytoplasm, nucleus and mitochondria. Most of the proteins were classified to the following categories: “organization and cell motility", "transcription, translation, and protein folding" and "energy and metabolism". Immunofluorescence analysis confirmed ATP5B is less abundant in epididymitis samples compared to controls. Conclusions: Even when normal semen parameters are observed in patients following epididymitis by conventional semen analysis, significant changes in the sperm protein composition occurs. These changes may be implicated as additional factors contributing to postinflammatory subfertility/infertility. Contact: [email protected] 60 P2.7 Investigations on the localization and significance of the prolactin-inducible protein (PIP) in the male urogenital tract Karnati S1, Xiao Y1, Janga H1, Jongik D2, Izykowsky N2, Schuppe H-C3, Baumgart-Vogt E1, Weidner W3, Wagenlehner F3, Pilatz A3 1 2 3 Institut für Anatomie und Zellbiologie, AG Medizinische Zellbiologie, Justus-Liebig-Universität, Gießen Institut für Pathologie, Medizinische Hochschule Hannover Klinik für Urologie, Kinderurologie und Andrologie, Justus-Liebig-Universität, Gießen Introduction: The prolactin-inducible protein (PIP) is a glycoprotein that is found in both the salivary glands and the ejaculate. The localization of PIP in the genital tract is contradictory in the literature. Several publications describe PIP as a biomarker for metastatic breast cancer, carcinoma of the prostate as well as for azoospermia. Further, proteomic studies performed in various diseases of the urogenital tract showed that PIP is differentially regulated; however, the exact biological function of this protein in the ejaculate (seminal plasma, sperm) is unknown. Materials und Methods: To investigate the localization of PIP in the urogenital tract, immunohistochemistry was performed in mouse and human tissues of testis, epididymis, seminal vesicles and prostate. In addition, human sperms were stained for PIP by immunofluorescence. Finally, with a commercial ELISA, the quantification of PIP in seminal plasma as well as in sonicated sperms purified by the swim-up technique was performed. A total of 95 seminal plasma samples were analyzed for PIP (all with complete semen analysis according to WHO 2010): 10 samples before and after vasectomy of identical men, 10 samples from patients with acute epididymitis and 65 samples from the andrological routine were used in this study. Results: In murine and human genital organs PIP was localized exclusively in the cytoplasm of epithelial cells of the seminal vesicle. The PIP concentration in seminal plasma in men before vasectomy was 480 ± 63 pg / ml and thus comparable to the situation after vasectomy with 458 ± 37 pg / ml (p = 0.2). Patients with completed antimicrobial treatment for acute epididymitis had significantly lower PIP levels with concentrations of 431 ± 68 pg / ml (p <0.01). Correlation analysis in 95 seminal samples revealed PIP concentrations to be significantly associated with the fructose concentrations (classic seminal vesicle marker) in semen (r = 0.3, p <0.01), while no significant correlations were evident with zinc (prostate marker) and alpha-glucosidase (epididymis marker) as well as the sperm parameters (concentration, motility, morphology). PIP was not detectable with ELISA in swim-up purified and sonicated sperm samples. Immunofluorescence for PIP on human sperms showed no staining, suggesting that PIP is not expressed in human sperms. Conclusions: PIP is exclusively expressed in the seminal vesicle of the urogenital system and constitutes a component of the seminal plasma. The observed down regulation of PIP in patients with acute epididymitis might suggest a possible accompanying inflammation of the seminal vesicles. Contact: [email protected] 61 P2.8 Role of TET3 and 5-hmC in establishment of sperm epigenome and in production of fertile sperm Dansranjavin T1, Deuker J1, Steger K1, Bergmann M2, Weidner W1, Spiess A3, Schorsch M4, Schagdarsurengin U1 1 2 3 4 Abteilung Molekulare Andrologie, Klinik und Poliklinik für Urologie, Kinderurologie und Andrologie, JLU Giessen Institut für Veterinär-Anatomie, -Histologie und Embryologie, JLU Giessen Abteilung für Andrologie, Universitätsklinikum Hamburg-Eppendorf Kinderwunschzentrum Wiesbaden Objectives: Ten eleven translocation dioxygenase 3 (TET3) promotes DNA-demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine and is, therefore, a key player of postfertilization chromatin remodeling in paternal pronucleus. The aim of our study was to analyze, whether TET3 and 5-hmC are involved in heterochromatization during spermiogenesis, and whether TET3 has an impact on male fertility status. Methods: TET3-expression in spermatogenesis was examined by in-situ hybridization and by immunohistochemistry assays on human and bovine testis sections exhibiting normal spermatogenesis. Presence of 5-hmC was analyzed by immunofluorescence. TET3-mRNA-level in spermatozoa was analyzed by qRT-PCR in ejaculates from healthy normozoospermic donors (n=28) and patients, who underwent intracytoplasmic sperm injection (ICSI, n=48) and in vitro fertilization (IVF, n=19). Post-fertilization TET3 expression was analyzed in bovine early embryos. Results: TET3-mRNA was detectable in human as well as in bovine testis in spermatocytes up to round spermatids, whereas TET3-protein was expressed in round up to elongated spermatids. We could detect the presence of 5-hmC in round up to elongating spermatids. Sperm of healthy donors and work-up sperm of IVF-patients showed the highest TET3-mRNA in comparison to ICSI-patients (p<0.001). Spermatozoa from younger men (≤35y.) exhibited significant higher TET3-mRNA in comparison to elders (p=0.016). Among patients, elevated TET3-mRNA associated significantly to higher progressive motility (>50% vs. ≤50%, p<0.001) and to pregnancy (pregnancy vs. no pregnancy, p=0.02). Conclusions: The present study reveals for the first time the function of TET3 in chromatin remodeling process during spermiogenesis. Moreover, we show that the level of TET3-mRNA in spermatozoa is associated to fertility parameters. Contact: [email protected] 62 P2.9 Mixed testicular atrophy is related to atherosclerosis in the ApoE(-/-) / LDL receptor(/-) double knockout mouse model: New data of the arterial supply of the tubuli Steinfeld K1, Middendorff R2, Kampschulte M3, Mietens A2, Langheinrich A3, Krombach GA3, Linn T4, Mühlfeld C5, Wudy S6, Hartmann M6, Pilatz A1, Paradowska-Dogan A1, Altinkilic B1, Bergmann M7, Weidner W1 1 2 3 4 5 6 7 Department of Urology, Pediatric Urology and Andrology, University of Giessen Department of Anatomy and Cell Biology, University of Giessen Department of Radiology, University of Giessen Department of Internal Medicine, University of Giessen Department of Functional and Applied Anatomy, Medical School Hannover Department of General Pediatrics and Neonatology, University of Giessen Department of Veterinary Anatomy, Histology and Embryology, University of Giessen Aims/Objectives: We wanted to investigate the possible relationship between atherosclerotic lesions and unexplained infertility in men using the mouse model mentioned below and clinical studies with special focus on the arterial supply of the tubuli. Material and Methods: The ApoE /LDL receptor double knockout (KO) mouse model is an ideal tool to investigate altheroslerosis related spermatogenetic alterations comparable to humans. In testes from KO- and wild-type mice at the age of 20, 40, 60 and 80 weeks testis volume and total vascular volume fraction were quantified by micro-CT. In semithin sections total length, volume and surface area of capillaries were estimated by newCAST. Spermatogenesis was analyzed by spermatogenetic scores. Sperm counts were quantified in the epididymis. Testosterone levels were determined in serum. Results: KO mice exhibit diminished testis and total vascular volume fraction, changes of total length (P=0.0001), volume and surface area (P=0.0046) of capillaries, mixed atrophy in various seminiferous tubules and a reduction of testosterone levels. Conclusions: Mixed testicular atrophy in KO mice is linked to reduced testis volume, vascular volume fraction and low testosterone serum levels, suggesting a direct relation between atherosclerosis and disturbed spermatogenesis. (DFG, KFO 181/2-Project 8) Contact: [email protected] 63 P2.10 Polysialylation of NCAM correlates with onset and termination of seasonal spermatogenesis Hänsch M1, Simon P1, Schön J2, Geyer R1, Middendorff R3, Müller K4, Galuska SP1 1 2 3 4 Institute of Biochemistry, Justus-Liebig-University, Giessen Berlin-Brandenburg School for Regenerative Therapies, (BSRT), Charité, Berlin Institute for Anatomy and Cell Biology, Justus-Liebig-University, Giessen Leibniz Institute for Zoo and Wildlife Research, Berlin Roe deer (Capreolus capreolus) are seasonal breeders and cyclic structural changes of roe bucks’ testis come along with a totally arrested (winter) and a highly activated spermatogenesis (summer). For this reason, roe buck represents an interesting model to study general mechanisms of initiation and termination of spermatogenesis. We investigated if polysialic acid (polySia) - a linear homopolymer of 2,8-linked sialic acids, which could act as a negative regulator of cell-cell adhesion might be involved in the activation and/or inactivation of spermatogenesis. To address this point, testis samples of adult male roe deer were collected after castration at different time point of the year (February, April, June, August, October, December). Intriguingly, we observed that polySia attached to the neural cell adhesion molecule NCAM was enhanced during the onset of spermatogenesis in April. In addition, polySia was highly expressed in December. Predominantly, polySia was detectable between Sertoli cells and spermatogonia in the basal regions of testicular tubules as well as in the adluminal part of Sertoli cells. Thus, polySia is expressed during key steps of the “on/off mechanisms” of seasonal spermatogenesis and might represent one mediator of the interaction as well as communication between Sertoli cells and spermatogonia. Contact: [email protected] 64 P2.11 Bacterial epididymitis induces fibrotic transformation and regional changes in the activin/follistatin ratio Michel V, Bhushan S, Middendorff R, Meinhardt A Institute for Anatomy and Cell Biology, Department of Reproduction Biology, Justus-Liebig-University Giessen Uropathogenic E.coli (UPEC) are identified in >80% of urinary tract infections, with 40% of acute epididymitis patients showing persistent impaired semen parameters despite antibiotic treatment. This clinical observation suggests a link between urogenital infection and male infertility. Activin A, a critical mediator of inflammation and fibrosis, increases dramatically during acute infection in many tissues, and inhibition of activin by its binding protein, follistatin, may reduce the severity of disease and damage. The aim of this study was to investigate the activin-to-follistatin ratio in epididymitis, and its potential correlation with epididymal inflammation and fibrosis. We established an acute experimental epididymitis mouse model by infection of C57BL/6 mice with the uropathogenic isolate UPEC CFT073. Sham-operated C57BL/6 mice injected with PBS served as the control group. Following 3 days of infection, preliminary immunohistochemical and qRT-PCR experiments indicated regional changes in the epididymal activin-follistatin ratio. Fibrosis development in the caput, corpus and cauda epididymis was visualized histologically by Masson-Goldner staining. Acute UPEC infection resulted in an increase in collagen fibers and severe fibrotic transformation of the epididymal tissue architecture particularly around tubule cross-sections. These findings point to a putative link between the fibrotic damage following UPEC-induced acute epididymitis and the activin-follistatin signalling axis. Future studies will investigate the chronic progression of epididymal fibrosis and elucidate the potential for targeting these pathways in the treatment and fertility preservation of epididymitis patients. Contact: [email protected] 65 P2.12 Necrosis is the dominant cell dead pathway in UPEC induced epididymo-orchitis model and is responsible for structural and functional damage of rat testis Bhushan S1, Lu Y1, Tchatalbachev S2, Marconi M3, Bergman M4, Weidner W3, Chakraborty T2, Meinhardt A1 1 2 3 4 Institute for Anatomy and Cell Biology, Depratment of Reproductive Biology, Giessen Institute for Medical Microbiology, Giessen Institute of Pediatric Urology and Andrology, Clinic and Policlinic of Urology, Giessen Institute for Veterinary Anatomy, Department of Histology, and Embryology, Giessen Introduction: Bacterial infections of the male genital tract result from ascending canalicular infections of the male excurrent ducts and thus could cause male infertility. Uropathogenic Escherichia coli (UPEC) is a relevant pathogen in urogenital tract infection. Method and materials: To explore how testicular defenses react against UPEC infection, epididymoorchitis model was established by injecting UPEC CFT073 into vas deference in close proximity to the epididymis. Mimicking an infection ascending from the urinary tract, in the testis UPEC bacteria were found exclusively in the interstitial space 7 days post infection. Tracer experiments revealed that the integrity of the blood-testis and blood epididymis barrier was intact 7 days post-infection indicating that evasion of bacteria occurs probably intracellularly. In the testis, UPEC infection resulted in impairment of spermatogenesis by germ cell loss, damage of testicular somatic cells, a decrease in sperm numbers and a significant increase in TUNEL (+) cells. To investigate potential mechanism of germ cell death, hall mark steps of apoptosis were investigated. Activation of caspase-8 (extrinsic apoptotic pathway), caspase-3/-6 (intrinsic apoptotic pathway), caspase-1 (pyroptosis pathway) and the presence of 180 bp DNA fragments, all of which serve as indicators of the classical apoptotic pathway and pyroptosis, were not observed in infected testis. Notably, electron microscopical examination revealed degenerative features of Sertoli cells (SC) in UPEC infected testis. Furthermore, the passive release of high mobility group protein B1 (HMGB1), as an indication of the necrosis, was observed in infected testis. Conclusion: In summary, the findings indicate that UPEC infection causes the induction of an organized self-destruction cascade termed programmed necrosis in the testis, which may ultimately be the major mechanism contributing to impairment. Contact: [email protected] 66 P2.13 Identification of mouse sperm glycoprofile to evaluate possible alterations after urogenital tract infection: possible implications for immune privilege and sperm function Khosravi F¹, Lang T¹, Mink W², Kühnhardt S², Galuska SP², Meinhardt A¹ ¹ Institute for Anatomy and Cell Biology, Justus-Liebig-University, Giessen ² Institute of Biochemistry, Justus-Liebig-University, Giessen Introduction: Uropathogenic E. coli (UPEC) are a common etiological cause of genito-urinary tract infections such as prostatitis, epididymitis or epididymo-orchitis affecting in total approximately 1015% of all infertile men. Eradication of pathogens occurs via antibiotic treatment, but in 50-60% of acute epididymitis, patient impairment of spermatogenesis remains. The sperm glycocalix is generated during spermatogenesis as well as during epididymal maturation and O-linked and Nlinked glycans are essential for fertility, e.g. induction of sperm acrosome reaction and sperm-zona pellucida binding and interaction. Changes in the glycoprofile may predispose damaged spermatozoa for recognition by immune cells. There are three types O- glycan transferases: Nacetylgalactosaminyltransferase, O-mannosyltransferase and O-fucosyltransferase are initiating enzymes for O-glycan biosynthesis. These enzymes have tissue and/or cell specific expression. Aim: We aimed to determine the N- and O-glycan profile of mouse sperm under normal and infectious conditions. We hypothesise that UPEC or secreted virulence factors may impair male reproductive potential by possible modification of sperm glycoproteins. Results: In first step expression levels of 21 different O-glycan transferases were analysed in the mouse epididymis. Then we have established chromatography systems for determination and quantification of monosaccharide moieties of sperm. This system is functional to evaluate sugar alternations during sperm maturation. Conclusions: Expression of O-glycan transferse genes in epididymis indicates that epididymis plays an important role in synthesis of sperm glycocalyx. Glycoprofile analysis of mouse sperm indicates the alterations in sperm glycocalyx during sperm maturation in epididymis. Outlook: We are trying to identify the O- and N-linked saccharides of sperm by mass spectrometry analysis. We plan to evaluate the alterations of sperm glycoprofile and expression level of glycan transferases in mouse epididymis after UPEC infection and their consequences in male infertility. Contact: [email protected] 67 P2.14 Uropathogenic E. coli inactivate host survival AKT signalling pathway in Sertoli cells Zhang Z , Bhushan S1, Tchatalbachev S2, Chakraborty T2, Meinhardt A1 1 1 2 Department of Anatomy and Cell Biology, Unit of Reproductive Biology, Justus-Liebig-University Giessen Department of Medical Microbiology, Justus-Liebig-University Giessen Uropathogenic Escherichia coli (UPEC) are the major cause of acute and chronic ascending bacterial genital tract infections in men which ultimately can result in impaired spermatogenesis. UPEC can modulate host survival pathways to attenuate host inflammatory responses and escape from host immune response. Here, we have shown that in isolated rat Sertoli cells (SC) UPEC virulence factor alpha-hemolysin can inactivate AKT, also known as Protein Kinase B (PKB), by dephosphorylation. The inactivation of AKT leads to activation of several downstream targets by dephosphorylation, one of which is FOXO (Forkhead box class of transcription factors). We have observed the activation of FOXO1 and FOXO3 by dephosphorylation at serine 256 and serine 253 in SC. Following dephosphorylation, FOXOs can remain in the nucleus and execute diverse cellular functions such as cell cycle arrest, apoptosis, reactive oxygen species detoxification and DNA repair. We have demonstrated that the FOXO localize in the nucleus consequently FOXO DNA-binding activity significantly increased after UPEC infection in SC. Although FOXO translocate to the nucleus with high DNA binding activity, we have not observed any significant change in the expression of cyclin D1 (a regulator of cell cycle progression), p27Kip1 and p15INK4B (both of which can inhibit cell cycle progression). Similarly we have not observed any visible change in the expression of SOD2 (Sodium dismutase, a detoxification enzyme of reactive oxygen species) and Catalase. The underlying mechanism of FOXO targeted gene silencing still remains unclear. Taken together these results suggest that UPEC can evade host immune cell response by manipulating host surviving signalling pathway. Moreover suppression of AKT signalling pathway may lead to damage of Sertoli cells which could impair spermatogenesis and germ cell death. Contact: [email protected] 68 P2.15 Urethral brush cells are polymodal cholinergic chemosensors Deckmann K1, Filipski K1, Krasteva-Christ G1, Rafiq A1, Althaus M2, Fronius M2, Bschleipfer T3, Kummer W1 1 2 3 Institute for Anatomy and Cell Biology, JLU Giessen Institute of Animal Physiology, JLU Giessen Department of Urology, JLU Giessen Epithelial cell with a tuft of stiff microvilli on the cell surface are named brush cells. They have been reported in the gastrointestinal tract and the respiratory tract. For the respiratory tract it is know that brush cell are chemosensors. Recently, we identified them in the urethra as well. They are cholinergic (expressing the acetylcholine synthesizing enzyme choline acetyltransferase=ChAT) and express the brush cell marker protein, villin, and components of the canonical taste transduction signaling cascade (-gustducin, PLC2, TRPM5). In this work, we investigated the functional similiarity of urethral brush cells to type II taste cells of the tongue described in literature. Type II taste cells detect bitter, sweet and umami gustatory stimuli via taste receptors of the Tas1R and Tas2R families and the canonical taste transduction signaling cascade to generate signals to activate afferent nerve fibers. We used an antibody directed against an extracellular domain of the cation channel TPRM5 to isolate the presumptive urethral brush cells for RT-PCR analysis. This revealed mRNA expression of several taste receptors. In whole cell patch-clamp recordings, the bitter substance denatonium benzoate caused activation of a delayed and long-lasting inward current in 15/21 cells. In CLSM recording of free intracellular calcium concentration ([Ca2+]i) we investigated the responses to various stimuli. Candidate chemoreceptor cells from two different strains of choline acetyltransferase (ChAT)-eGFP mice were identified by their eGFP fluorescence, and from wild-type mice by fluorescent (FITC) antibody labeling of TPRM5. 91% (43/47) of the cells responded to ATP (0.5 mM) with an increase in [Ca2+]i, 92% (45/49) to denatonium benzoate (25 mM; bitter compound), and 90% (38/42) to L-glutamate (25 mM; “umami”). The response to denatonium benzoate was dose-dependent (2.5-25 mM).The effect could be blocked by a specific TRPM5 blocker (TPPO; 0.25 mM) as well as by specific PLC 2 Inhibitor U73122 (10 μM). Interestingly, urethral brush cells act as polymodal sensors in contrast to unimodal type II taste cells. Like taste cells, they communicate to neighboring cells. We noted [Ca2+]i rise in response to denatonium in non-eGFP expressing cells when situated in the vicinity of ChAT-eGFP positive cells. In the presence of cholinergic blockers, denatonium evoked [Ca2+]i increase only in ChAT-eGFP cells but not in eGFP negative cells, demonstrating stimulus evoked cholinergic signaling between chemosensory and surrounding cells. We interpret this cell type as a sentinel at the entrance to the urogenital tract initiating protective mechanisms by acetylcholine release. Funding: LOEWE Schwerpunkt “Non-neuronale cholinerge Systeme” Contact: [email protected] 69 V4.5 Expression of Aquaporin water channels by human urothelium: contribution to transurothelial permeability in vitro and modulation by osmolality Rubenwolf P1,3, Georgopoulos NT2, Baker S3, Southgate J3 1 2 3 Department of Urology, Johannes Gutenberg University Medical School Mainz, Mainz Department of Chemical and Biological Sciences, School of Applied Sciences, University of Huddersfield, UK Jack Birch Unit for Molecular Carcinogenesis, University of York, UK Background: It is generally assumed that human urothelium is impermeable to water and to the constituents of the urine. However, recent evidence indicates that human urothelium expresses a network of water- and urea-transporting channels, the aquaporins, which may constitute a molecular basis for transurothelial water and solute transfer. This finding challenges the traditional concept of the impermeable urothelial barrier, as it suggests that urothelium may be able to mediate water and solute transport and thus modify the composition and final concentration of the urine. The aim of the study was to study the functional aspects of urothelial AQPs. Methods: In an in vitro model of human urothelium, we have investigated how changes in osmolality affect AQP expression and have examined the role of AQP channels in water and urea transport. The effect of exposing normal human urothelial cell cultures to osmotic stress was examined immunochemically. Barrier strength of differentiated cultures was assessed by measuring transepithelial electrical resistance (TER) and determining permeability coefficients for water and urea. Mercuric chloride was used as an AQP channel blocker. Results: AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium revealed a significant barrier function (mean TER 3862 Ω.cm²), with mean diffusive water and urea permeability coefficients PD of 6.3 and 2.4 cm/s, respectively. AQP blockade with mercuric chloride resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase in water and urea flux across an osmotic gradient. Conclusion: When considered in toto with the emerging evidence from other studies, our results support an active role for human urothelium in sensing and responding to hypertonic salt concentrations through alterations in AQP protein expression, with AQP channels as a potential mechanism for modifying urine composition. The contribution of these mechanisms to normal urinary physiology has yet to be realised, but may be of particular significance in disorders where the urothelial barrier is compromised, such as dysfunctional bladder syndromes and infectious or interstitial cystitis. Contact: [email protected] 70 V4.6 3D-ultrastructure of the human urinary bladder revealed by FIB-SEM and 3View SEM Neuhaus J1, Schröppel B2, Wolburg H3, Fallier-Becker P3, Dass M4, Zimmermann H4, Schwalenberg T1, Stolzenburg J-U1 1 2 3 4 Department of Urology, University of Leipzig Natural and Medical Sciences Institute at the University of Tuebingen Department of Pathology, University of Tuebingen Zeiss Microscopy Labs, Training, Application and Support Center (TASC), Munich Objective: Urinary bladder function depends on the structural integrity of its cellular components. Pathological conditions, e.g. bladder pain syndrome / interstitial cystitis (BPS/IC), are characterized by macroscopic, microscopic and ultrastructural alterations of the bladder wall, including suburothelial myofibroblasts. Ultrastructural features of those cells are well known. However, their three-dimensional appearance has not been addressed so far at ultrastructural level. We here present the first 3D-reconstruction of suburothelial myofibroblasts of the human bladder at ultrastructural level using two different electron microscopic approaches. Material and Methods: Bladder biopsies from two male patients aged 79 yrs and 83 yrs undergoing urothelial tumor transurothelial resection were included in this pilot study. The tissue was prepared for electron microscopy using either standard protocol or the enhanced heavy metal block staining protocol (Deerinck et al. 2010). We used a Crossbeam® Auriga 40, a Crossbeam® Auriga 60 (Zeiss), and a Sigma™VP 3View (Zeiss) to produce serial section electron microscopic image stacks of up to 1000 images. The minimum voxel resolution was 4.5 x 4.5 x 10 nm. Image stacks were pre-processed using ImageJ and 3D-reconstructions were done with Amira® 3D Image Analysis software (vsg, Düsseldorf, Germany). For comparison we did confocal immunofluorescence imaging using a LSM5 Pascal laserscanning microscope (Zeiss). Results: 3D-ultrastructural analysis of suburothelial myofibroblasts revealed the delicate membraneous structure of the cells, characterized by a network of cytoplasmic protrusions and spots of vesicle production, resembling exosomes, supported by CD9 confocal immunofluorescence. Interestingly, we could identify gap junctions between the protrusions of one single cell, i.e. establishing „short circuits“ within the cell. This feature was only revealed by careful examination of the three-dimensional structure of the cells. The sponge-like structure of the plasma membrane leads to an immense increase of the surface area. Suburothelial myofibroblasts were characterized by myofilaments, partial coverage by a basal lamina, fibronexus junctions and plasmalemmal attachment plaques. No classical adhesion junctions were found between those cells. Conclusions: We here show for the first time the delicate 3D-ultrastructure of suburothelial myofibroblasts in the human bladder. Quite surprising was the finding of gap junction formation between protrusions of the one and the same cell, the functional relevance of which has to be explored. Our results demonstrate the potency of 3D-ultrastructure analysis to gain further insights into the structure-function relationship of cells in the human bladder and might reveal new aspects of bladder pathology when extended to pathologically altered tissues in future studies. Contact: [email protected] 71 P3.1 GDNF-family neurotrophic factors in urinary bladder and expression of their receptors (GFR) in bladder sensory neurons Nandigama R1, Bschleipfer T2, Kummer W1, Nassenstein C1 1 Institute for Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany, 2 Department of Urology, Pediatric Urology and Andrology, Justus-Liebig-University, Giessen, Germany Bladder sensory C-fibre neurons express transient receptor potential (TRP) channels. TRPV1 plays a role in bladder pain and bladder hyperreflexia in inflammation. Neurotrophic factors like nerve growth factor (NGF) are usually associated with development of the peripheral nervous system. Recent studies, however, reported their role in inflammation dependent hypersensitivity of sensory neurons and their effects in TRP channel mediated excitability of sensory neurons. Although NGF has been associated in bladder inflammation, the role of glial cell line-derived neurotrophic factors (GDNF) has not been studied yet. Here we investigated expression of GDNF-family ligands (GFLs) in urinary bladder and of their receptors (GFR) in bladder sensory neurons. Quantitative real-time PCR showed expression of all GFLs in urinary bladder. Real-time PCR analysis revealed the following rank order of GFL expression: artemin (Ct value 29) > neurturin (Ct value 32) > GDNF (Ct value 36) > persephin (Ct value 38). RT-PCR analysis for receptors for GDNF-neurotrophic factors (GFR) showed expression of all GFR (1-4) and its co-receptor RET in L5-S2 DRG where cell bodies of bladder afferent neurons are located. Single-cell RT-PCR studies in TRPV1 positive isolated retrogradely labelled bladder sensory neurons showed expression of receptors GFR1 (receptor for GDNF; 1/7 cells), GFR3 (receptor for artemin; 6/7 cells) and its co-receptor RET (co-receptor for all GFLs; 6/7 cells), whereas expression of GFR2 (receptor for neurturin) was absent in all 7 single cells. Taken together, these data indicate that GDNF-family neurotrophic factors and their receptors (GFR) may play a role in bladder sensory pathways. Contact: [email protected] 72 P3.2 Treatment strategy dependent tissue injury produced by a new acoustic lens design for electromagnetic lithotripters Neisius A1,2, Smith N3, Kuntz NJ1, Schykowski T1, Astroza GM1, Lipkin ME1, Gustafson MR3, Simmons WN3, Preminger GM1, Zhong P1,3 1 2 3 Division of Urologic Surgery, Duke Comprehensive Kidney Stone Center, Durham, NC, USA University Medical Center Mainz, Department of Urology, Mainz Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA Purpose: The acoustic lens of a Siemens Modularis electromagnetic shock wave lithotripter has been further modified to reduce pre-focal cavitation while generating a pressure waveform and broad focal zone mimicking the Dornier HM3 electrohydraulic device. We sought to determine the threshold for the maximum acoustic energy which can be safely applied to a kidney under clinically relevant treatment protocols, and the dependency of tissue injury on pulse repetition frequency (PRF). Materials and Methods Tissue injury (TI) produced by the original and modified lenses in a swine model were first evaluated starting from the highest output level to determine the threshold energy for safe lithotripsy. Thereafter, TI was assessed under an effective acoustic pulse energy for the leading compressive wave (E+) of 44 mJ. A clinical protocol with a soft ramping scheme was used to deliver 3000 shock waves to each kidney using a PRF of 1.0 and 1.5 Hz, leading to a total accumulated energy of 112.84 J. Following lithotripsy, kidneys were perfused, harvested, dehydrated, cast in paraffin wax, and sectioned. Photographic images were taken every 120 μm and analyzed to determine the functional renal volume (FRV) damage. Results: Gross subcapsular hematomas were produced by both the original and modified lenses at E+ of 51 mJ. Using E+ of 44 mJ, the modified lens showed quantatively macroscopic tissue injury (subcapsular hematoma) in 1/6 renal units (17%) in the 1.5 Hz group. No macroscopic tissue injury was detected in the 1.0 Hz group (0/6 renal units). After processing of the digitalized images TI was detected in 0.432 % (±0.51%) of the FRV (with a maximum level of 1.324% in the kidney with the gross hematoma) in the 1.5 Hz group and in 0.009% (±0.015%) of the FRV in the 1 Hz group (p=0.025), respectively. Conclusions: This study demonstrates that the energy threshold for gross TI of the modified lens is comparable to the original lens. Our data further confirms that the initiation of TI depends on acoustic pulse energy, and the extent of TI depends on the total accumulated acoustic energy delivered to a kidney, as well as on PRF. Overall, our results suggest that a rational design of treatment strategies to minimize tissue injury in SWL is warranted. Supported by a Ferdinand Eisenberger grant of the Deutsche Gesellschaft für Urologie (German Society of Urology), grant ID NeA1/FE-11 Contact: [email protected] 73 P3.3 Coming closer to endoscopic “real time target identification” - Technical improvements of an experimental lithotripsy laser system with spec-tral real-time object analysis Miernik A1, Nuese C2, Knabe B2, Eilers Y2, Bolwien C2, Brandenburg A2, Lambrecht A2, Wetterauer U1, Schoenthaler M1 1 2 University Medical Centre Freiburg, Department of Urology, Freiburg, Fraunhofer Institute for Physical Measurement Techniques IPM, Freiburg Introduction: Photonic technologies are gaining increasing attention in different fields of medicine. Almost every application of laser energy induces a specimen spe-cific spectral response. Analysis of reflected spectra may be used for acquisition of additional diagnostic information. Future laser systems with integrated spectroscopic real-time analysis of targeted structures will provide a new therapeutic quality in dif-ferent fields of medicine. In urology, lasers are used for various applications such as the treatment of benign prostate hyperplasia or tumour ablation of the bladder. Concerning interventional uri-nary stone treatment, the Holmium-YAG laser is the gold standard for endoscopic disintegration. In this setting, a differentiation of organic and non-organic structures (tissue vs. stone vs. endoscope) might significantly increase safety and accuracy of the treatment. We hereby present constructional improvements of a previously de-scribed experimental laser system for endoscopic stone lithotripsy with real-time ob-ject identification. Methods: Pure samples of urinary stones of different compositions, native human caliculi, endoscope components and organic samples (porcine renal parenchyma, collecting system, blood) were analysed by an experimental fibre-coupled fluores-cence spectroscopy setup in a dry and a wet model. After determining the optimum laser excitation wavelength and acquisition of the spectral information with consecu-tive data analysis, an experimental setup including a photo diode controlled by a micro-computer was constructed to improve data processing. The system was evaluat-ed for its ability of real-time object identification. Results: With the experimental setup as described, it was possible to obtain specific spectra for all specimens. Mathematical post processing data revealed narrow band areas of the light spectrum required for discriminatory specimen analysis. Signal in-tensity levels were sufficient for a reliable discrimination of stone material, endoscope surfaces and organic samples. By optimizing the system, measurement (integration) times could be reduced significantly. Conclusions: Implementation of a computer-controlled photo diode into a fibre-coupled fluorescence spectroscopy laser system improved and accelerated data pro-cessing of acquired spectral signals significantly. This will improve the efficiency of real-time object analysis during endoscopic interventions. The hereby acquired data will promote the development of future medical laser systems with real time target identification. (Supported by the Ferdinand Eisenberger-Grand MiA1/FE-12) Contact: [email protected] 74 P3.4 Overexpression of Drosha is associated with outcomes in patients with urothelial carcinomas Ratert N1,2, Ecke T3, Jung K1,2, Erbersdobler A4, Kilic E5, Rabien A1 1 2 3 4 5 Department of Urology, Charite-Universitätsmedizin, Berlin Berlin Institute for Urologic Research, Berlin Department of Urology, Helios Klinikum Bad Saarow Institute of Pathology, Universität Rostock Institute of Pathology, Charité – Universitätsmedizin Berlin Background: It is already known that abnormal miRNA expression is associated with tumorigenesis. Aberrant microRNA (miRNA) expressions are also associated with different clinical outcomes in cancer patients. Consequently, it is postulated that proteins which are needed for miRNA maturation may also play a key role in tumor behavior. The biogenesis of miRNAs is a multistep process involving a couple of protein complexes. Briefly, miRNA genes are transcribed by RNA-Polymerase (RNAP)II/III into a long single or multiple primary miRNA (pri miRNA). Afterwards pri miRNA is processed by RNase III enzyme Drosha and its cofactor DiGeorge syndrome critical region gene 8 (DGCR8) into hairpin precursor miRNA (pre miRNA). In cytoplasm pre miRNA is converted by RNase III enzyme Dicer into double stranded miRNA duplex containing the mature as well as the complementary strand. Finally, mature miRNA is loaded onto the miRNA Induced Silencing Complex (miRISC). Argonaute proteins are the central components of miRISC and are needed for the repression of mRNA translation. The aim of this study was to investigate the expression of the miRNA-related proteins Argonaute1 (Ago1), Argonaute 2 (Ago2), and Drosha by immunohistochemical staining in bladder cancer and to evaluate the expression data in relation to clinicopathological parameters and as prognostic tools. Material and Methods: A total of 112 urothelial carcinomas and 30 normal tissue samples of the bladder collected by transurethral resection of the bladder (TURB) or cystectomy were used for the tissue microarray (TMA) study. The cases were not stratified in any way but selected in accordance to availability of tissue as well as of follow up data. Immunostaining was done with primary antibodies against Ago1, Ago2, and Drosha. Fast-Red TR/Naphthol AS-MX was used as chromogen. Negative controls were performed by staining with missing respective primary antibody. Immunostainings were evaluated as 0 (no immunoreactivity) or 1 (positive immunoreactivity). Results: Ago1, Ago2, and Drosha expression levels were up-regulated in malignant compared to normal bladder tissue samples. Performing separate Kaplan-Meier analysis for G1/G2 and G3 tumors, Drosha expression exhibited possible prognostic potential for G1/G2 (log rank test=10.875; P=0.001) but not for G3 tumors concerning overall survival. In order to avoid bias results for the prognostic analysis due to starting point of analysis we separated the patient collective in TURB and cystectomy treated patients. Drosha exhibited possible prognostic potential for TURB (log rank test=6.636; P=0.010) as well as for cystectomy patients (log rank test=4.139; P=0.042) with regard to the overall survival, whereas results for Ago1 and Ago2 remained insignificant. Conclusions: In this study, we investigated Ago1, Ago2, and Drosha expression in an immunohistochemical manner for the first time in bladder cancer. Drosha appears to be a potential prognosticator of overall survival time with respect to G1/G2 tumors. Contact: [email protected] 75 P3.5 Alternative therapy strategies for the non-muscle invasive bladder cancer on the basis of carbon nanomaterials Rieger C1, Kunze D1, Temme A2, Fuessel S1, Wirth MP1 1 2 Department of Urology, University of Technology, Dresden Department of Neurosurgery2, University of Technology, Dresden Introduction: An improvement of the intravesical treatment for recurrence and progression prophylaxis of the non-muscle invasive bladder cancer is needed. Therefore the use of siRNAs might represent a promising treatment option but also a multifunctional transporter for conventional chemotherapeutic (CT) and therapeutic nucleic acids (AS-ODN) for chemosensitization of the tumor cells will be an option. This novel carrier system is based on carbon nanotubes (CNTs) which can be functionalized for different purposes like increased mucoadhesive properties. To analyze such innovative cancer treatments suitable orthotopic BCa models are needed. Following instillation of human BCa cells labeled with a luciferase gene to the mouse bladder tumor growth can be monitored noninvasively in such in vivo models. Material and methods: UM-UC-3 BCa cells were stably transduced with the gene of the Fireflyluciferase. Different UM-UC-3LUC clones were then selected and characterized. Cells were transfected with siRNAs against Bcl-xL and survivin, either one siRNA alone (40 nM) or with combinations of two siRNAs (20 nM each). Cell counts, apoptosis induction as well as targets mRNA and protein levels were examined after 48h. Cells were also transfected with AS-ODNs against Bcl-xL (250nM). mRNA expression and cell counts were examined after 24h. The mucoadhesive properties of CNTs to pig or mouse bladder urothelium were assessed using Franz diffusion cells (Gauer Glas). Bladder tumor implantation was achieved by intravesical instillation of 2 x 106 tumor cells in nude mice. The bladder was pretreated with poly-L-lysine (0.1 mg/ml) or trypsin (0.5%). Results: Two UM-UC-3LUC clones were selected. Single and combined siRNA treatment strongly decreased the mRNA and protein expression of the targets in both UM-UC-3LUC clones. For example, siRNA treatment against survivin led to a 95% reduction of survivin mRNA level. Different Bcl-xL AS-ODNs were selected by mRNA structure prediction algorithms. BX2100 showed the strongest effect on UM-UC-3LUC cells. Different types of CNTs (varying in length and diameter) were tested in first ex vivo mucoadhesive studies on pig and mice bladders to show the attachment to urothelium. After inoculation of UM-UC-3LUC bladder cancer cells through a urethral catheter into the murine bladder an in vivo signal could be monitored using bioluminescence imaging. Conclusions: Two UM-UC-3LUC clones were successfully generated and characterized, which is an important requirement for the subsequent establishment of an orthotopic BCa model. The knockdown of antiapoptotic Bcl-xL by using siRNAs as well as AS-ODNs and the use of CNTs as multifunctional transporter represent a promising treatment option for BCa and will be further investigated in an in vivo BCa model. Funding provided by German Cancer Aid Contact: [email protected] 76 P3.6 The function of Kruppel-like factor 4 (KLF4) as a transcriptional repressor of IGF2promoter during prostate carcinogenesis Dansranjavin T1, Lammert A1, Wagenlehner F1, Waliszewski P1, Brandt U1, Steger K1, Weidner W1, Gattenloehner S2, Schagdarsurengin U1 1 2 Clinic of Urology, Pediatric Urology and Andrology; Justus-Liebig-University Giessen Institute of Pathology, Justus-Liebig-University Giessen Objective: KLF4 is a potent transcriptional repressor involved in stem cell maintenance and in tumorigenesis. The present study reveals KLF4-bonding in differentially methylated region of IGF2promoter 0 (IGF2-DMR0) and elucidates the role of KLF4 in the regulation of IGF2/H19-imprinting cluster in prostate carcinogenesis. Methods: PCa-cell lines (LnCAP and Du145), 57 PCa (prostate carcinoma) and 71 BPH (benign prostatic hyperplasia) were subjected to DNA-methylation analyses in IGF2-DMR0 and in imprinting control region (ICR) by bisulfite pyrosequencing, and to IGF2- and H19-mRNA-quantification by qRTPCR. Loss of imprinting (LOI) was determined by Apa I and Rsa I polymorphic sites within IGF2 and H19, respectively. KLF4-bonding on IGF2-DMR0 was analyzed by chromatin immunoprecipitation (ChIP). Results: KLF4 was significantly enriched in unmethylated IGF2-DMR0 of LnCAP and depleted in hypermethylated IGF2-DMR0 of Du145 cells (relative bond: 265 vs. 8). DNA-demethylation in Du145 with 5-azacytidine led to a 14-fold increase of KLF4-bond in IGF2-DMR0. In tissue specimen, DMR0hypermethylation was significantly associated to elevated IGF2-mRNA (p< 0.001) emphasizing the repressor role of KLF4 in prostate tumors. BPH exhibited more often IGF2-overexpression (33.8%) compared to PCa (15.8%) (p=0.025). In contrast to hypermethylation of IGF2-DMR0, ICRhypermethylation showed a significant association to H19- as well as to IGF2-LOI (p=0.005 and p=0.014, respectively). Summary: Our study demonstrates that detachment of the transcriptional repressor KLF4 from IGF2DMR0 by its hypermethylation contributes to IGF2-overexpression. IGF2-overexpression could be examined in BPH as well as in PCa. ICR-hypermethylation in PCa and BPH was correlated significantly to IGF2- and H19-LOI. Contact: [email protected] 77 P3.7 Resveratrol and prostate a very complicated “dangerous liaison” Di Domenico M1,6, Mancini FP2, Kisslinger A3, Iannelli P2, Iorio R1, Feola A1, Saar M4, Pierantoni GM5, Tramontano D5 1 2 3 4 5 6 Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, I Department of Sciences and Technologies, University of Sannio, Benevento, I Institute of Experimental Oncology and Endocrinology, CNR, Naples, I Department of Urology and Pediatric Urology, University of Saarland, Homburg/Saar Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Naples, I Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple University, Philadelphia, PA, USA Background: Several lines of evidence support a strong connection between nutrition and prostate cancer. One plausible link between diet and prostate cancer is oxidative stress which triggers a host of pro-carcinogenic processes. Dietary antioxidants have been demonstrated to promote protection against several human disorders and dietary chemoprevention is attracting increasing interest. Resveratrol, a phytoalexin from grapes, exerts a variety of biological protective effects that are antioxidative, pro-apoptotic/anti-cancer, and at least calorie-restrictive. P66Shc, a redox enzyme member of the Shc family, shows a striking convergence of activities with those of resveratrol. Therefore, we tested the effect of resveratrol on the activation of p66Shc in a prostate cell culture system. Methods The non-transformed human prostate cell line EPN and the EPN-PKM3 cells bearing PKM, a dead kinase mutant of the non-receptor kinase Pyk2, were used in this study. A soft-agar assay was performed on both cell lines and cell proliferation was analyzed in the presence of resveratrol by direct counting. Western blot analysis and immunoprecipitation served to study various molecular targets. Results: Resveratrol reduces cell proliferation of both cell lines and induces a dose- and timedependent increase of p66Shc Ser36 phosphorylation. Hereby the effect of resveratrol on p66Shc Ser36 phosphorylation is not ERK dependent. Conclusion: This is the first evidence linking resveratrol and p66Shc in the prostate and may provide insight into the mechanisms underlying the effect of resveratrol on prostate cell regulation. Key words: prostate cancer, resveratrol, p66shc Contact: [email protected] 78 P3.8 Cardiolipin species composition differs in benign prostate epithelia and prostate cancer and may be associated with tumor progression Fussek S¹, Evert K², Schild L³, Streitbörger A¹, Grossebrummel H¹, Pechoel M¹, Zimmermann U¹, Evert M², Burchardt M¹, Stope MB¹ ¹ Department of Urology, University Medicine Greifswald ² Department of Pathology, University Medicine Greifswald ³ Department of Pathobiochemistry, Otto-von-Guericke University of Magdeburg Background: Cardiolipin (CL) is a mitochondrial phospholipid serving an important role in membrane structure and mitochondrial function. The composition of CL acyl-chain residues has been found to be highly variable. Previously, fatty-acid residues of CL were shown to differ between tumor and nontumor cells in rat liver and brain cancer. Furthermore, a functional link between CL acyl-chain distribution and cell proliferation was reported in lymphocytes of patients with leukemia. In this study, the acyl-chain patterns of CLs in benign prostate epithelial cells and prostate cancer cells were investigated. Methods: CLs were extracted from established PCa cell lines (LNCaP, PC-3 and 22Rv1) and ten human prostate specimen by a modified Folch extraction procedure and analyzed using highperformance liquid chromatography tandem mass spectrometry (HPLC-MS / MS). Tissue purity was evaluated by two independent pathologists. Differences in CL expression were evaluated by SPSS software. Results: CL acyl-chain composition showed differences between benign prostate epithelial cells and prostate cancer cells. 11 of 19 analyzed CL species differed statistically significant. Remarkably, among the benign samples the same CL pattern was found while the prostate cancer specimen showed a high variability. Moreover all three prostate cancer cell lines presented significant different CL fatty-acid residues contents. Conclusion: This study demonstrated the different acyl-chain composition of CL in benign prostate epithelial and prostate cancer cells. The differences of CL patterns within prostate cancer specimen and the cell culture cells suggest a role in prostate cancer progression. Contact: [email protected] 79 P3.9 MiR-205 is progressively down-regulated in lymph node metastasis but fails as a prognostic biomarker in high-risk prostate cancer Kalogirou C1, Spahn M1,2, Krebs M1,Joniau S3, Lerut E4, Burger M1, Scholz C-J5, Kneitz S6, Riedmiller H1, Kneitz B1 1 2 3 4 5 6 Klinik und Poliklinik für Urologie und Kinderurologie, Universtitätsklinik Würzburg Universitätsklinik für Urologie, Inselspital Bern, CH Universitätsklinik für Urologie, Leuven, B Universitätsklinik für Urologie, Leuven, B Interdisziplinäres Zentrum für klinische Forschungs, Universität Würzburg Biozentrum Würzburg, Institut für Physiologische Chemie I, Würzburg Abstract: The treatment of high-risk prostate cancer (HRPCa) is a tremendous challenge for urooncologists. The identification of predictive moleculobiological markers allowing risk assessment of lymph-node metastasis and systemic progression is essential to establish effective treatment. In the current study we investigate the prognostic potential of miR-205 in HRPCa study- and validation cohorts setting defined clinical endpoints for both. We demonstrate miR-205 to be significantly down-regulated in over 70 % of the HRPCa samples analysed and that reconstitution of miR-205 causes inhibition of proliferation and invasiveness in prostate cancer (PCa) cell lines. Additionally, miR-205 is increasingly down-regulated in lymph-node metastases compared to the primary tumour indicating that miR-205 plays a role in migration of PCa cells from the original location into extraprostatic tissue. Nevertheless down-regulation of miR-205 in primary PCa was not correlated to the synchronous presence of metastasis and failed to predict the outcome for HRPCa patients. Moreover, we found a tendency for miR-205 up-regulation to correlate with an adverse outcome of PCa patients suggesting a pivotal role of miR-205 in tumourigenesis. Overall we showed that miR205 is involved in the development and metastasis of PCa, but failed to work as a useful clinical biomarker in HRPCa. These findings might have implications for the use of miR-205 as a prognostic or therapeutic target in HRPCa. Keywords: high-risk prostate cancer; microRNA; miR-205; prognosis; biomarker Contact: [email protected] 80 P3.10 Re-expression of an epigenetically repressed microRNA inhibits prostate cancer cell growth Korzeniewski N1, Hohenfellner M2, Duensing S1,2 1 2 Section of Molecular Urooncology, University Hospital Heidelberg Department of Urology, University Hospital Heidelberg Background: The cause of prostate cancer is largely unknown, although compelling evidence exists that extensive chromatin remodeling, which also affects microRNA (miRNA) loci, occurs at early stages of prostate carcinogenesis. MiRNAs are small non-coding RNA molecules that play a crucial role in a plethora of regulatory cellular processes by controlling mRNA stability and protein expression. There is mounting evidence that miRNAs also play an important role in prostate cancer development and progression. Determining changes in the miRNA profile that occur following prostate carcinogenesis may reveal novel targeting of signaling pathways important for either the initiation of carcinogenesis or the continued propagation of prostate tumor cells. Here, we surveyed the expression of miRNAs after DNA hypomethylation in order to determine which miRNAs are affected by methylation-driven chromatin modification in prostate cancer cells. Methods: Hormone-sensitive LNCaP cells were treated for seven days with the DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) which is known to promote re-expression of tumor suppressor genes through promoter hypomethylation compared to control treated LNCaP cells. A miRNA array consisting of approximately 1400 capture probes (miRCURY; Exiqon) was then used to identify dysregulation of miRNAs in treated versus untreated LNCaP cells. Results: We identified a novel miRNA, mir-555, to be re-expressed following 5-aza-dC treatment of LNCaP cells and found that ectopic expression of this miRNA negatively affects cell cycle progression. We further discovered that the cell cycle arrest phenotype observed following ectopic miR-555 expression is dependent on AR expression. We next performed an oligonucleotide gene expression microarray analysis on the same 5-aza-dC treated samples and found that miR-555 potentially targets several genes important for mitotic progression, suggesting new targets for novel therapeutic interventions. Conclusion: Taken together, our results lend additional support to the notion that miRNAs play an important role in prostate cancer. We show that miRNAs are dynamically regulated during chromatin remodeling processes and we are currently utilizing these results to identify mitotic signaling pathways that could be novel therapeutic targets to prevent prostate cancer cell progression. Contact: [email protected] 81 P3.11 HMGB1 modulates immune responses in a cell-specific manner in the rat experimental autoimmune orchitis Aslani F1, Meinhardt A1, Elsässer H-P2, Schuppe H-C3, Bhushan S1, Fijak M1 1 2 3 Department of Anatomy and Cell Biology, Justus-Liebig-University, Giessen Institute of Cytobiology and Cytopathology, Philipps-Universität, Marburg Department of Urology, Pediatric Urology and Andrology, Justus-Liebig University of Giessen High mobility group box protein 1 (HMGB1), the non-histone chromosomal protein, plays an important role in onset and chronification of autoimmune diseases once released from the nuclei. In this study, we analyzed how HMGB1 can regulate inflammatory reactions in vivo in a rat model of experimental autoimmune orchitis (EAO) and in vitro in primary testicular cells. HMGB1 was translocated from the nuclei in EAO testis and in the testis of infertile men with leukocytic infiltrates. HMGB1 levels in EAO testis were elevated at late chronic phase of disease as compared to early proinflammatory cytokines such as IL-6 and TNF-. We found that testicular somatic cells show a cellspecific expression profile of HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE). The highly sensitive and specific proximity ligation assay was used to analyze HMGB1 receptor binding in testicular cells. HMGB1-TLR4 binding was dominant in testicular macrophages. However, Sertoli and peritubular cells showed higher levels of HMGB1-RAGE interaction. In support, HMGB1 triggered RAGE-dependent ERK1/2 MAPK and CREB activation in Sertoli and peritubular cells, whilst in testicular macrophages HMGB1 induced TLR4-signaling as evidenced by p38 MAPK and p65 NF-B phosphorylation which stimulated an increase in mRNA levels of TNF- and IL-6. Recent data shows that RAGE induced ERK activation leads to enhanced autophagy levels. In line with these data, extracellular HMGB1 triggered formation of LC3 positive punctae and double membrane autophagosomes in Sertoli cells. Increased autophagy levels in Sertoli cells might explain how these cells survive the inflammatory environment in EAO testis. Considering HMGB1’s late phase of action, inhibition of HMGB1 may be a putative target for therapeutic intervention in treatment of chronic testicular inflammation. Contact: [email protected] 82 P3.12 Epigenetic dysregulation of inflammatory factors in prostatitis and prostate cancer Velagala SR1, Seifer P1, Dansranjavin T1, Steger K1, Weidner W2, Wagenlehner F2, Schagdarsurengin U1 1 2 Dept. of Molecular Andrology, JLU Giessen Clinic of Urology, Pediatric Urology and Andrology, UKGM, Standort Giessen Objective: Recently, a new hypothesis has been proposed for prostate carcinogenesis that exposure to environmental factors such as infectious agents and dietary carcinogens, and hormonal imbalances lead to injury of the prostate and to the development of chronic inflammation and regenerative ‘risk factor’ lesions, referred to as proliferative inflammatory atrophy (PIA). This preliminary study aimed to analyze the epigenetic status of different inflammatory factors in prostate tumors in order to reveal the link between prostate inflammation and carcinogenesis. Material and Methods: Three prostate cancer cell lines PC-3, DU145 and LnCAP, and primary matching tissue samples from 14 PCa (prostate carcinoma) and 14 BPH (benign prostatic hyperplasia) were subjected to epigenetic studies, including CpG-promoter methylation analyses by COBRA (combined bisulfite restriction analysis) and bisulfite sequencing, 5-aza-CdR-treatment and qRTPCR. Additionally, we analyzed the mRNA of cytokines in ejaculate (spermatozoa) and in urine samples before and after prostate massage from 5 patients with prostatitis-syndrome (chronic pelvic pain syndrome, n=4; asymptomatic inflammatory prostatitis, n=1). Results: Based on available literature 44 inflammatory factors could be selected as involved in prostatitis and/or prostate cancer. In-silico analyses revealed that 12/44 have CpG-island promoters (C3, CCL5, CXCL5, CXCL12, ERß, HIF1, IL4, IL6, IL11, IL12A, IL12B, IL13 and NGF), among which 6 (IL6, CCL5, C3, IL4, IL13 and CXCL12) exhibited in PCa cell lines hypermethylated promoters and down regulated expression, respectively. Four latter genes could be reexpressed via 5aza-CdR-treatment. Analyses on primary tumor probes revealed that cytokines, especially IL4, IL13 and CXCL12, are frequently hypermethylated in PCa as well as in BPH. Interestingly, in comparison to IL4 and IL13, whose mRNA was frequently down regulated in the majority of PCa and BPH, CXCL12 exhibited an intense mRNA-expression in PCa and BPH. Studies on urine samples from prostatitis patients revealed that mRNA level of cytokines in first and exprimate urine is nearly the same, except some cases, where a prostate massage led to a massive (up to 5-fold) increase of cytokine-mRNA in urine (e.g. IL4, IL13 and CXCL12). Remarkably, spermatozoa from prostatitis patients, especially from asymptomatic inflammatory prostatitis, exhibited high levels of cytokine-mRNA (e.g. IL6, IL13 and CCL5). Conclusion: Our study provides indications that cytokines, particularly IL4, IL13 and CXCL12, are frequently epigenetically affected in prostate tumors and that these cytokines are present in high levels in the urine of prostatitis patients. Therefore, we suggest that these cytokines might play a crucial role in leading prostatitis to prostate tumor. Contact: [email protected] 83 P3.13 Androgen receptor regulates CD4+CD25+Foxp3+ T cell differentiation by binding to foxp3 locus Walecki M1, Eisel F1, Hackstein H2, Baal N2, Steger K3, Paradowska-Dogan A3, Meinhardt A1, Fijak M1 1 2 3 Department of Anatomy and Cell Biology, Giessen Institute for Clinical Immunology and Transfusion Medicine, Giessen Department of Urology, Pediatric Urology and Adrology, Giessen Regulatory T cells (Tregs) are able to inhibit proliferation and cytokine production in the effector T cells and play a major role in immune responses and prevention of development of autoimmune diseases. The experimental autoimmune orchitis (EAO) in the rodent is a model of chronic testicular inflammation leading to infertility. Our previous results have shown supplementation of EAO rats as well as rat splenic T cells in vitro with testosterone causes differentiation of Tregs characterized by increased expression of the transcription factor Foxp3. Foxp3 is a master regulator in the function of Treg cells and its expression is under epigenetic control. Many common transcription factors are involved in Foxp3 regulation as well as in the methylation status by binding the foxp3 locus. Peripheral naïve CD4+CD25-Foxp3- T cells can differentiate into Tregs by stimulation and induction of Foxp3 by several factors and interaction proteins. In the present study, we aim to investigate the effect of androgens on the expression, regulation and methylation status of human Foxp3 and putative interaction with androgen receptor (AR). We analyzed the effect of androgens on the differentiation of human male and female Treg cells. Our results showed that the Foxp3 expression significantly increased in T cells from human with lower testosterone level. We found an interaction of androgen receptor with foxp3 locus in isolated primary human CD4+CD25+CD127- regulatory T cells from male and female peripheral blood after the stimulation with androgens. No binding was detectable in CD4+CD25-CD127+ T cells (Foxp3 negative). The AR-foxp3 interaction changes the acetylation status of histones within the binding region but not the DNA methylation of tested CpG within foxp3 gene. Contact: [email protected] 84 P3.14 Functional analyses of specific T cells in patients under BCG-therapy Elsäßer J , Janssen M1,2, Becker F3, Suttmann H4, Ohlmann C-H1, Schmitt K5, Sester U2,6, Stöckle M1, Sester M2 1,2 1 2 3 4 5 6 Department of Urology, Saarland University, Homburg Department of Transplant and Infection Immunology, Institute of Virology, Saarland University, Homburg Boxberg Centre, Urological Group and Clinic Derout/Pönicke/Becker, Neunkirchen Urologicum Hamburg Department of Pathology, Saarland University, Homburg Department of Internal Medicine IV, Saarland University, Homburg Introduction: Immunotherapy with Bacille Calmette Guérin (BCG) is the standard-therapy for prevention of progress and relapse of non-muscle invasive urothelial carcinoma. However, knowledge on the induction and functionality of systemic T cell immunity is limited. Method: In 18 bladder cancer patients, blood was drawn longitudinally before each instillation during the induction course. Multiparameter flow cytometry is applied to quantify and characterize specific T cells from whole blood using intracellular cytokine staining after stimulation with purified protein derivative (PPD). PPD is used as a commercially available mycobacterial antigen preparation for tuberculin skin test and it allows detection of therapy-induced immunity due to its cross reactivity towards BCG. 54 age-matched healthy subjects served as controls. Results: Before the first instillation, 9/18 patients showed a pre-existing immunity toward PPD, which could be caused by BCG-vaccination or prior contact to mycobacteria. However, baseline levels of IFN- producing PPD-specific T cells were comparable to controls and showed a significant increase after completion of the 2nd instillation (p<0.0001). Systemic immunity was induced in all patients, although the increase was less pronounced in patients with pre-existing immunity. Consistent with repeated antigen-challenge, PPD-specific cytokine-profiling after instillation revealed a lower percentage of multifunctional IFN-γ/IL-2 double-positive T cells compared to healthy controls (p=0.0003). Of note, unlike in patients with pre-existing immunity, cytokine profiles in patients with primary immunity was shifted towards IL-2 single producing T cells (35.9% vs. 13.4%, p=0.02). Conclusion: Interestingly we found out, that patients under BCG-therapy shows similar cytokine profiles as patients with active tuberculosis. Decreased functionality is a typical feature of specific immunity in both patients with active TB and BCG therapy. Considering this in the context of tuberculosis diagnosing, among patients with active infection, the percentage of IL2 single positive cells may allow distinction between patients with primary infection and cases with boosted immunity after prior contact. Contact: [email protected] 85 P3.15 Urine protein profiling identified Alpha-1-microglobulin and Haptoglobin as biomarkers for early diagnosis of acute allograft rejection following kidney transplantation Stubendorff B1,2, Finke S1, Walter M1, Kniemeyer O3, von Eggeling F4, Gruschwitz T1, Steiner T1,5, Ott U6,7, Wolf G6,7, Wunderlich H1,8, Junker K1,2 1 2 3 4 5 6 7 8 Department of Urology, University Hospital, Jena Clinic of Urology and Pediatric Urology, Saarland University Medical Center, Homburg/Saar Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute and Integrated Research and Treatment Center – Center for Sepsis Control and Care (CSCC), University Hospital, Jena Institute of Human Genetics, Core Unit Chip Application (CUCA), University Hospital, Jena Department of Urology, Helios Hospital Erfurt KfH Kuratorium für Dialyse und Nierentransplantation e.V., KfH Nierenzentrum, Jena Department of Internal Medicine III, University Hospital, Jena Clinic of Urology and Pediatric Urology, St. Georg Hospital, Eisenach Introduction: Early diagnosis of acute allograft rejection and effective immunosuppressive therapy lead to improvements in graft survival following kidney transplantation. However, there are no noninvasive diagnostic parameters available that enable early and reliable detection of acute rejection. The objective of our study was to determine whether acute allograft rejection can be predicted at an early postoperative stage and to identify potential biomarkers for clinical practice. Materials and Methods: Urine samples of 116 kidney recipients were included. Rejection was proven by biopsy (n=58) and stable transplant function was monitored for at least 2 years (n=58). Postoperative urine samples were collected between 3rd and 10th day following transplantation. Urinary protein profiles were analyzed by SELDI-TOF-MS. Multiplex-Fluorescence-2DE and Peptide Mass Fingerprinting were used for identification of candidate proteins. Urinary concentration of candidate proteins was validated by ELISA. Results: A protein signature including 4 masses differentiated acute rejection from stable transplant patients at the postoperative stage with 73% sensitivity and 88% specificity. Alpha-1-microglobulin (A1MG) and Haptoglobin (Hp) were identified as putative biomarkers for acute rejection. Protein levels were significantly higher in postoperative urine samples from patients with upcoming rejection than in stable transplant patients (A1MG: 29.13 μg/ml vs. 22.06 μg/ml, p=0.001; Hp: 628.34 ng/ml vs. 248.57 ng/ml, p=0.003). The combination of both proteins enabled the diagnosis of early rejection with 85% sensitivity and 80% specificity. Conclusion: The specific urine protein pattern enables the prediction of early acute allograft rejection already few days after kidney transplantation. A1MG and Hp appear to be reliable rejection biomarkers. Further analyses have to show if early diagnosis is possible for patients with allograft rejection that occurs several month after transplantation by analyzing changes in urine protein pattern in regular intervals. Contact: [email protected] 86 ANHANG 87 88 AuF-Preisträger 2009: Dipl.-Biol. Annika Schäfer (verh. Fendler), Berlin Dr. med. Matthias Saar, Homburg/Saar 2010: Dr. rer. nat. Natalie Sampson, Innsbruck Dr. med. Friedemann Zengerling, Ulm 2011: Dipl.-Biol. Elke Nolte, Erlangen Dipl.-Biol. Elke Schneider, Mainz Rudi Ascherl & Dr. rer. nat. Kerstin Boll, Leipzig Dr. med. Matthias Heck, München 2012: PD Dr. med. Carsten Stephan, Berlin Dr. rer. nat. Bettina Schlick, Innsbruck Dr. med. Carl Ludwig Behnes, Göttingen Dr. rer. nat. Nina Korzeniewski, Heidelberg 89 90 Wir bedanken uns für die finanzielle Unterstützung bei unseren Förderern und Sponsoren: 91 Die DGU bedankt sich bei den genannten Firmen für die nachstehend aufgeführten Beträge als Gegenleistung für Werbezwecke/Standkosten: Astellas 10.000 € Farco-Pharma 7.500 € Pierre-Fabre 1.500 € Janssen 1.000 € Pfizer 1.000 € Takeda 1.000 € Apogepha 1.000 € Bionorica 1.000 € GlaxoSmithKline 800 € Lilly 700 € Bayer 500 € AMS 500 € Serag-Wiessner 500 € Ausblick: 5. AuF-Symposium 2013 in Gießen 92 6. Symposium CME Urologische Forschung der Deutschen Gesellschaft für Urologie Interdisziplinäre Forschung in der Urologie: Mehrwert durch Vernetzung Homburg 2014 In Kooperation mit der Arbeitsgemeinschaft Uropathologie der Deutschen Gesellschaft für Pathologie 13. bis 15. November 2014 Kulturzentrum Saalbau Homburg/Saar http://auf-symposium.dgu.de
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