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201. Die Expression muskarinerger und purinerger Rezeptoren in Urothelzellen und suburothelialen Myofibroblasten im Rahmen des Bladder Pain Syndrom/Interstitielle Zystitis (BPS/IC)

Author

Feige, Thomas, Neuhaus, Jochen, Stolzenburg, Jens Uwe, Gaunitz, Frank, and Medizinische Fakultät der Universität Leipzig

Subjects
IC, BPS/IC, interstitielle Zystitis, Bladder pain syndrome, ddc:610, Bladder pain syndrome
Abstract

Die Dysregulation von Neurotransmittersystemen spielt eine wesentliche Rolle für die Pathophysiologie des Bladder Pain Syndrom/Interstitielle Zystitis (BPS/IC). Diese Arbeit beschäftigt sich daher mit der veränderten Expression muskarinerger (M2, M3) und purinerger (P2X1, P2X2, P2X3) Rezeptoren auf Urothelzellen und suburothelialen Myofibroblasten der Harnblasenwand im Rahmen BPS/IC. Dazu wurde eine Gruppe von Patientinnen mit Verdacht auf BPS/IC (n=17) einer Kontrollgruppe (n=7) gegenübergestellt. Die Gewebeproben der Patientengruppe sind im Zuge der Basisdiagnostik (transurethrales Harnblasenmapping) bei klinischem Verdacht auf das Vorliegen eines BPS/IC entnommen worden. Das Gewebe der Kontrollgruppe entstammt makroskopisch unauffälligen Bereichen von Harnblasen, welche im Rahmen einer radikalen Zystektomie oder einer lateral erweiterten endopelvinen Resektion (LEER-OP) entnommen worden sind. Die semi-quantitative Analyse der Rezeptorexpression erfolgte mittels indirekter Immunfluoreszenz, die Auswertung erfolgte mittels konfokaler Laserscanningmikroskopie. Die Ergebnisse der Patientengruppe wurden mit denen der Kontrollgruppe verglichen. Im Rahmen dessen wurde auch ein individuelles Rezeptorprofil für jeden Patienten erstellt. Es zeigte sich eine Hochregulation von M2R, M3R und P2X1R auf Urothelzellen sowie eine Hochregulation von M2R, M3R, P2X1R und P2X2R auf suburothelialen Myofibroblasten in der BPS/IC-Gruppe im Vergleich zur Kontrollgruppe. Des Weiteren zeigten sich individuelle Unterschiede in den Rezeptorprofilen der Patienten. Die Ergebnisse werden vor dem Hintergrund einer möglichen Beteiligung der Regulation von muskarinergen und purinergen Rezeptoren an der Pathophysiologie des BPS/IC diskutiert. Ferner werden diagnostische und therapeutische Möglichkeiten einer erweiterten Mollekulardiagnostik diskutiert.:Bibliografische Angaben 4 Abkürzungsverzeichnis 6 1. Einleitung 8 1.1. Das Bladder Pain Syndrom 8 1.1.1. Definition 8 1.1.2. Epidemiologie 10 1.1.3. Ätiologie und Pathophysiologie 10 1.1.4. Diagnostik 13 1.1.5. Therapie 16 1.2. Anatomische und physiologische Grundlagen 20 1.2.1. Das Urothel 21 1.2.2. Lamina propria 22 1.2.3. Muskarinerge Acetylcholinrezeptoren 23 1.2.4. Purinerge Rezeptoren 27 1.3. Fragestellung und Zielsetzung 31 2. Material und Methoden 32 2.1. Material 32 2.2. Methoden 33 2.2.1. Immunhistologie 33 2.2.2. Konfokale Laserscanning Mikroskopie und Auswertung 33 3. Ergebnisse 37 3.1. Lichtmikroskopie 37 3.2. Laserscanningmikroskopie 40 3.2.1. Rezeptorexpression im Urothel 40 3.2.2. Statistischer Vergleich von Kontroll-und BPS/IC-Gruppe im Urothel 42 3.2.4. Statistischer Vergleich von Kontroll-und BPS/IC-Gruppe auf sMF 46 3.2.5. Rezeptorprofile von Urothelzellen und sMF 47 3.3. Zusammenfassung 52 4. Diskussion 53 4.1. Diagnostisches Dilemma 53 4.2. Rezeptorverteilung im Urothel 61 4.3. Rezeptorverteilung auf sMF 64 4.4. Diagnostische und therapeutische Möglichkeiten des Rezeptorprofiling 65 4.5. Methodik der vorliegenden Arbeit 69 4.7. Zusammenfassung 71 Anhang 73 Referenzen 75 Erklärung über die eigenständige Abfassung der Arbeit 84 Lebenslauf: 85 Danksagung: 86

Published
2016

202. HISTOLOGISCHE UNTERSUCHUNG ZUR BI- UND MULTIPOLAREN RADIOFREQUENZABLATION DER NIERE

Author

Blachut, Lisa, Stolzenburg, Jens-Uwe, Neuhaus, Jochen, Bechmann, Ingo, Gaunitz, Frank, and Medizinische Fakultät

Subjects
Nierenzellkarzinom, Radiofrequenzablation, RFA, RFA, kidney cancer, ddc:610
Abstract

Das Nierenzellkarzinom ist in Europa die 10. häufigste Tumorerkrankung [1]. Da das Prädi- lektionsalter bei 62 Jahren liegt [2] und die Inzidenz mit steigendem Alter zunimmt, geht die Erkrankung häufig mit einer Vielzahl an Komorbiditäten einher. Dies fordert eine Auseinan- dersetzung mit der Anwendung minimal invasiver Methoden, die das operative Risiko mini- mieren. Immernoch wird die Therapie durch radikale Nephrektomie favorisiert. Ziel der Stu- die war es deshalb Entscheidungshilfen für die Anwendung der Radiofrequenzablation (RFA) zu liefern. Daher untersuchten wir an Hausschweinen technische Einflussgrößen auf Behandlungszeit, Ablationsvolumen und Läsionsform bei bipolarer und multipolarer RFA im Vergleich. Dabei ergaben sich nach 3-D-Rekonstruktion größere Ablationsvolumina im mul- tipolaren Modus, wobei sich signifikante Formunterschiede in beiden Anwendungen heraus- kristallisierten. Bei multipolarer RFA ergaben sich größere Inhomogenitäten. Die Ablation kleinerer Tumorgrößen unter 2 cm erscheint mit bipolarer RFA sicher. Patienten mit Tumo- ren bis zu 3 cm profitieren von mutipolarer Radiofrequenzablation. Die Anwendung multi- polarer RFA bei Tumoren größer 3 cm gestaltet sich durch die inhomogene Ablation schwie- rig und bietet ein schwer kalkulierbares Risiko für die Patienten. Es konnte kein Vorteil durch Steigerung des Energietransfers innerhalb eines Modus nachgewiesen werden, da dies mit einer längeren Behandlungszeit einherging, jedoch nicht mit einer Zunahme der Läsiongröße korrelierte. Bei Patienten mit Niereninsuffizienz, Einzelnieren oder bilateralen Tumoren liegt ein Hauptaugenmerk der Therapie auf dem Erhalt der Nierenfunktion. Um eine mögliche Schädigung von gesundem Nierengewebe durch die RFA zu detektieren, färbten wir die Mar- kerproteine aktivierte Caspase3 und HSP70 immunohistochemisch an. Bei Ablation nahe des Nierenbeckenkelchsystems konnten beide Proteine sowohl im System des distalen Sammel- rohrs, als auch im Nierenmark gefunden werden. Hauptsächlich war das Auftreten jedoch auf die Randgebiete der zentralen Koagulationszone begrenzt. Die Radiofrequenzablation scheint daher ein sicheres Verfahren für die Therapie von Tumoren ≤ 3 cm zu sein.:1 Einführung und Fragestellungen 1 1.1 Einführung 1 1.2 Das Nierenzellkarzinom 2 1.2.1 Epidemiologie 2 1.2.2 Risikofaktoren 3 1.2.3 Klinik des Nierenzellkarzinoms 4 1.2.4 Diagnostik und Therapieverfahren bei Nierenzellkarzinomen 4 1.3 Radiofrequenzablation 7 1.3.1 Pathophysiologie und Technische Details der Radiofrequenzablation 7 1.3.2 Monopolare, bipolare und multipolare Radiofrequenzablation 8 1.3.3 Histologische Betrachtungen der Radiofrequenzablation 9 1.4 Theoretische Einführung in die Untersuchungsparameter 10 1.4.1 Apoptose und Enzyme der Apoptosekaskade 10 1.4.2 Heat Shock Proteine 12 1.5 Fragestellungen und Ziele der Studie 13 2 Publikationsmanuskript 14 3 Zusammenfassung und Schlussfolgerungen 23 4 Literaturverzeichnis 28

Published
2014

203. ATP induced intracellular calcium response and purinergic signalling in cultured suburothelial myofibroblasts of the human bladder: ATP induced intracellular calcium response and purinergic signalling in cultured suburothelial myofibroblasts of thehuman bladder

Author

Cheng, Sheng, Neuhaus, Jochen, Stolzenburg, Jens-Uwe, Reichenbach, Andreas, and Universität Leipzig

Subjects
Urologische Klinik, Urologie, ATP, Calciumion, Zellkultur, Biomembran, Purinozeptor, urinary bladder physiology, urgency, myofibroblast, interstitial cell, cultured cells, ATP, Calcium Imaging, purinergic signalling, purinergic receptor, immunofluorescence, confocal laserscanning microscopy, CLSM, spontaneous calcium activity, ddc:610, Harnblase, Physiologie, Dranginkontinenz, Myofibroblasten, Interstitielle Zellen, Zellkultur, ATP, Calcium Imaging, purinerge Rezeptoren, purinerge Signalverarbeitung, konfokale Laserscanningmikroskopie, CLSM, spontane Kalziumaktivität
Abstract

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.:1. Introduction............................................................................ 1 1.1. Anatomy and histology of the human urinary bladder..................... 1 1.1.1. Anatomy of the human urinary bladder..................................... 1 1.1.2. Structure of the human urinary bladder wall............................... 2 1.2. Normal bladder function and bladder dysfunction.......................... 3 1.2.1 Normal bladder function......................................................... 3 1.2.2 Sensory aspect.................................................................... 4 1.2.3 Overactivity or hypersensitivity of bladder.................................. 5 1.3 The role of functional cell types and interaction in urinary bladder... 6 1.3.1 The role of urothelium.......................................................... 7 1.3.2Theroleofsuburotheliamyofibroblast...................................... 7 1.3.3Theroleofdetrusorsmoothmusclecells.................................. 9 1.3.4 Possible interactions in urinary bladder cell types........................ 10 1.4 ATP function and Purinergic signalling in bladder........................... 11 1.5 Spontaneous activity of bladder................................................... 13 2. Objective.................................................................................. 15 3. Material and methods............................................................... 16 3.1. Ethics Statement........................................................................ 16 3.2. Cell preparation.......................................................................... 16 3.3. Solutions and chemicals............................................................. 19 3.4. Intracellular calcium measurements............................................. 20 2.4.1. Preparing cells for Calcium Imaging.......................................... 20 2.4.2. Preparing workspace of calcium imaging................................... 20 2.4.3. Calcium imaging recording...................................................... 22 3.5 Data analysis with automated Fluorescence analysis..................... 22 3.6 Confocal Immunofluorescence.................................................... 25 3.7 Statistics................................................................................. 26 4. Results.................................................................................. 27 4.1 Spontaneous calcium activity of sMF........................................... 27 4.2 ATP effects on calcium response in sMF...................................... 27 4.3 Analysis of purinergic receptors involved.................................... 30 3.3.1 Agonist stimulation.............................................................. 30 3.3.2 Signal inhibition by specific antagonists................................... 31 4.4 Confocal immunofluorescence of purinergic receptors.................. 32 5. Discussion............................................................................. 34 5.1 Myofibroblast identification....................................................... 34 5.2 Spontaneous activity in the bladder............................................ 36 5.3 ATP modulated calcium activity in sMF....................................... 37 5.4 purinergic signalling in sMF........................................................ 39 6. Summary................................................................................ 42 7. References.............................................................................. 45 Declaration............................................................................. 50 Acknowledgements................................................................. 51

Published
2011

204. Verankerungselemente im urethralen Kontinenzapparat des Mannes

Author

Weißenfels, Paul, Stolzenburg, Jens-Uwe, Neuhaus, Jochen, Schwalenberg, Thilo, Löffler, Sabine, Hohenfellner, Markus, Wedel, Thilo, and Universität Leipzig

Subjects
ddc:610, Anatomie, Urologie, Beckenboden, Harnkontinenz, Harnröhrenverschluss, anatomy, urology, urinary continence, urethral closure mechanism
Abstract

Die Belastungsinkontinenz des Mannes ist eine häufige Komplikation der radikalen Prostatovesikulektomie. Abgewandelte Operationstechniken und Rekonstruktions-verfahren führen zu verbesserten postoperativen Kontinenzraten und weisen auf die Bedeutung einzelner Komponenten am urethralen Verschlussmechanismus hin. Im Vergleich zum weiblichen ist der männliche Harnkontinenzmechanismus nur ansatzweise verstanden. In der vorliegenden Arbeit werden die am urethralen Verschluss beteiligten Komponenten und ihre Verbindungen zu angrenzenden Strukturen makroskopisch und mikroskopisch analysiert und auf ihre funktionelle Bedeutung untersucht. Zehn konservierte Leichname wurden für Untersuchungen des männlichen Urogenitaltraktes genutzt, aus drei Becken wurden Organpakete für die Erstellung von mikroskopischen Schnittpräparaten entnommen. Die histologischen Untersuchungen konnten zeigen, dass die Form des Musculus sphincter urethrae externus im Transversalschnitt von der Schnitthöhe abhängig ist. Seine Fasern strahlen in die Faszie des Musculus levator ani ein. Auch die Fasern des Corpus perineale inserieren in die Faszie dieses Muskels. Anhand dieser Ergebnisse wird die zentrale Rolle des Musculus levator ani im männlichen Harnkontinenzapparat deutlich. Der eigentliche Verschlussdruck wird demnach durch den Musculus levator ani aufgebaut und durch das Corpus perineale von dorsal auf die Urethra übertragen. Die Aufgabe des Musculus sphincter urethrae externus wird nicht als primär aktiv, sondern als dynamisches Widerlager für die membranöse Urethra beschrieben. Außerdem geht die Arbeit auf die noch wenig erforschte Mediatorfunktion der glatten Muskulatur im männlichen Urogenitaltrakt ein. Diese Ergebnisse präzisieren die Vorstellung des Kontinenzmechanismus des Mannes als Zusammenspiel von unterschiedlichen Komponenten und geben Anlass, aktuelle Standardtechniken der Beckenchirurgie zu überdenken und den Fokus auf schonende bzw. rekonstruktive Operationsverfahren zu richten.:Inhaltsverzeichnis Seite Bibliographische Beschreibung……………………………….………........…………… 3 Inhaltsverzeichnis………………………………………………………….........………….. 4 Verzeichnis der Abkürzungen…………………………………….…….......…………… 7 Verzeichnis der Abbildungen……………………………………….…….......…………. 8 Verzeichnis der Tabellen…………………………………………….…………........……. 9 1 Einleitung……………………………………………………….………...........………. 10 1.1 Epidemiologie der Harninkontinenz………………………….…...…………… 10 1.2 Stellenwert der radikalen Prostatektomie im Spektrum der operativen Therapieverfahren zur Behandlung des Prostatakarzinoms…............... 11 1.3 Protektion und Rekonstruktion von Kontinenzstrukturen....…..... 13 1.4 Zielstellung der Arbeit……………………………………………………....…...... 15 2 Material und Methoden……………………………………………………........…. 16 2.1 Instrumentarium und Reagenzien…………………………………………….. 16 2.2 Körperspender…………………………………………………………….......…....... 16 2.3 Konservierung…………………………………………………………....….......…... 16 2.4 Makroskopische Darstellung………………………………………………....…... 17 2.5 Gewebeentnahme…………………………….................................…..... 19 2.6 Fixation und Einbettung…...................................………………….... 20 2.7 Färbung………………………………………………………….…………...........……. 21 2.8 Histologische Auswertung…………………………………....…………………... 22 3 Ergebnisse……………………………………………............………………………… 23 3.1 Makroskopie………………………………………………………..........…………….. 23 3.1.1 Musculus sphincter urethrae externus – ein fächerförmiger Muskel mit ausgeprägter Vaskularisierung......................................... 23 3.1.2 Corpus perineale…...........................................…………....... 26 3.1.3 Musculus levator ani – Bestandteil der ventralen Suspension des Harnkontinenzorgans………………………………………...............…………… 26 3.2 Mikroskopie……………………………………………………….........…………....... 28 3.2.1 Musculus sphincter urethrae externus und Corpus perineale inserieren im Musculus levator ani und lateralen Venenplexus…...….… 28 3.2.2 Form und Verlauf des Musculus sphincter urethrae externus........………........................................................................... 32 3.2.3 Glatte Muskulatur als dynamische Verankerung des Harnkontinenzorgans......................................................... 34 4 Diskussion…………………………………………...........………………………….... 37 4.1 Struktur und Form des Musculus sphincter urethrae externus…. 37 4.2 Verankerung des Musculus sphincter urethrae externus …......... 39 4.3 Kontroverse Ansichten zum Corpus perineale und seiner Verankerung….................................................................................. 41 4.4 Glattmuskuläre Komponenten im männlichen Kontinenzsystem.43 4.5 Vorstellungen zum männlichen Harnkontinenzmechanismus…… 44 4.7 Experimentelle Arbeiten zum Harnkontinenzorgan im Vergleich..48 4.8 Auswirkungen der Ergebnisse auf die Praxis…………………......……… 53 5 Zusammenfassung……………………………………………………........…….… 55 Anhang…………………………………………………………………............……………….. 59 Erklärung über die eigenständige Abfassung der Arbeit……….......………. 64 Lebenslauf…………………………………………………………………………...........….... 65 Danksagung………………………………………………………………...........……………. 66 Literaturverzeichnis………………………………………………........………………...... 67

Published
2011

205. Ionizing radiation and photodynamic therapy lead to multimodal tumor cell death, synergistic cytotoxicity and immune cell invasion in human bladder cancer organoids.

Author

Reinhold A, Glasow A, Nürnberger S, Weimann A, Telemann L, Stolzenburg JU, Neuhaus J, and Berndt-Paetz M

Subjects
Humans, Cell Line, Tumor, Cell Death drug effects, Combined Modality Therapy, Apoptosis drug effects, Urinary Bladder Neoplasms drug therapy, Urinary Bladder Neoplasms pathology, Photochemotherapy methods, Photosensitizing Agents pharmacology, Organoids drug effects
Abstract

Background: Photodynamic therapy (PDT) and radiotherapy using ionizing radiation (IR) are promising options for organ-preserving treatment of bladder cancer (BCa). A combination therapy (IR+PDT) could be beneficial for BCa treatment., Purpose: For PDT, we used the near-infrared photosensitizer tetrahydroporphyrin-tetratosylate (THPTS) showing high therapeutic efficacy. Treatment responses were analyzed in BCa organoids., Methods: Organoids consisting of BCa cells lines, bladder fibroblasts and muscle cells were treated with IR (9 Gy) and/or PDT using THPTS (25, 50 μM; 20 J/cm 2 ). Cytotoxicity was determined by microscopy, cell-based assays and histology. The cell death mode was analyzed by applying specific inhibitors followed by immunofluorescence or qPCR analyses of cell death markers. A matrix-based co-culture model was used to study T cell migration into the environment of treated organoids., Results: PDT and/or IR resulted in concentration-dependent reduction of metabolic activity, organoid diameter and integrity. Higher cytotoxicity of IR+PDT vs. monotherapies was observed after 72 h. Non-malignant organoids showed no cytotoxic effects. While apoptosis, necroptosis and ferroptosis were clearly involved in cell death of T-24 cells, cytotoxicity in RT-112 cells was probably provoked by apoptosis, ferroptosis and pyroptosis. IR+PDT resulted in significant migration of Jurkat cells into ECM-embedded organoids within 3 days after treatment., Conclusion: Treatment with IR+PDT showed tumor-selective cytotoxicity with additive or synergistic effects in BCa organoids. Thereby, IR+PDT led to multimodal cell death depending on the cellular context. Migration of T cells into the organoid environment illustrates the immunogenic potential of IR+PDT. Therefore, it might be a promising approach for organ-preserving BCa treatment., Competing Interests: Declaration of competing Interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Published
2025
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206. Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization-A New Standardized Approach in Bladder Cancer Research.

Author

Berndt-Paetz M, Han S, Weimann A, Reinhold A, Nürnberger S, and Neuhaus J

Abstract

Three-dimensional tumor models have gained significant importance in bladder cancer (BCa) research. Organoids consisting of different cell types better mimic solid tumors in terms of 3D architecture, proliferation, cell-cell interaction and drug responses. We developed four organoids from human BCa cell lines with fibroblasts and smooth muscle cells of the bladder, aiming to find models for BCa research. The organoids were characterized in terms of cytokeratins, vimentin, α-actin and KI67 by immunoreactivity. Further, we studied ligand-dependent activation of the Wnt/β-catenin pathway and investigated the responses to anti-tumor therapies. The organoids mimicked the structure of an inverse bladder wall, with outside urothelial cells and a core of supportive cells. The cytokeratin staining patterns and proliferation rate were in conjunction with the origins of the BCa cells. RT-112 even showed stratification of the epithelium. Treatment with Wnt10B led to increased β-catenin (active) levels in high-grade organoids, but not in low-grade BCa cells. Doxorubicin treatment resulted in clearly reduced viability (10-30% vs. untreated). In contrast, the effectivity of radiotherapy depended on the proliferation status of BCa cells. In conclusion, cell-line-based organoids can form bladder-like structures and reproduce in vivo features such as urothelial differentiation and stratification. Thus, they can be useful tools for functional studies in BCa and anti-cancer drug development.

Published
2023
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207. 3D Tumor Models in Urology.

Author

Neuhaus J, Rabien A, Reinhold A, Koehler L, and Berndt-Paetz M

Subjects
Male, Humans, Cell Culture Techniques methods, Organoids pathology, Spheroids, Cellular pathology, Tumor Microenvironment, Urology, Urinary Bladder Neoplasms pathology, Carcinoma, Renal Cell pathology, Kidney Neoplasms pathology
Abstract

Three-dimensional tumor models have become established in both basic and clinical research. As multicellular systems consisting of tumor and tumor-associated cells, they can better represent tumor characteristics than monocellular 2D cultures. In this review, we highlight the potential applications of tumor spheroids and organoids in the field of urology. Further, we illustrate the generation and characteristics of standardized organoids as well as membrane-based 3D in vitro models in bladder cancer research. We discuss the technical aspects and review the initial successes of molecular analyses in the three major urologic tumor entities: urinary bladder carcinoma (BCa), prostate carcinoma (PCa), and renal cell carcinoma (RCC).

Published
2023
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209. circPDE5A regulates prostate cancer metastasis via controlling WTAP-dependent N6-methyladenisine methylation of EIF3C mRNA.

Author

Ding L, Wang R, Zheng Q, Shen D, Wang H, Lu Z, Luo W, Xie H, Ren L, Jiang M, Yu C, Zhou Z, Lin Y, Lu H, Xue D, Su W, Xia L, Neuhaus J, Cheng S, and Li G

Subjects
Cell Line, Tumor, Gene Expression Regulation, Neoplastic, Humans, Male, Methylation, RNA, Messenger metabolism, Cell Cycle Proteins genetics, Eukaryotic Initiation Factor-3 genetics, Prostatic Neoplasms pathology, RNA Splicing Factors genetics, RNA, Circular genetics
Abstract

Background: Circular RNA (circRNA) is a novel class noncoding RNA (ncRNA) that plays a critical role in various cancers, including prostate cancer (PCa). However, the clinical significance, biological function, and molecular mechanisms of circRNAs in prostate cancer remain to be elucidated., Methods: A circRNA array was performed to identified the differentially expressed circRNAs. circPDE5A was identified as a novel circRNA which downregulated in clinical samples. Functionally, the in vitro and in vivo assays were applied to explore the role of circPDE5A in PCa metastasis. Mechanistically, the interaction between circPDE5A and WTAP was verified using RNA pulldown followed by mass spectrometry, RNA Immunoprecipitation (RIP) assays. m 6 A methylated RNA immunoprecipitation sequencing (MeRIP-seq) was then used to identified the downstream target of circPDE5A. Chromatin immunoprecipitation assay (ChIP) and dual-luciferase reporter assay were used to identified transcriptional factor which regulated circPDE5A expression., Results: circPDE5A was identified downregulated in PCa tissues compared to adjacent normal tissue and was negatively correlated with gleason score of PCa patients. circPDE5A inhibits PCa cells migration and invasion both in vitro and in vivo. circPDE5A blocks the WTAP-dependent N6-methyladenisine (m 6 A) methylation of eukaryotic translation initiation factor 3c (EIF3C) mRNA by forming the circPDE5A-WTAP complex, and finally disrupts the translation of EIF3C. Moreover, the circPDE5A-dependent decrease in EIF3C expression inactivates the MAPK pathway and then restrains PCa progression., Conclusions: Our findings demonstrate that FOXO4-mediated upregulation of circPDE5A controls PCa metastasis via the circPDE5A-WTAP-EIF3C-MAPK signaling pathway and could serve as a potential therapeutic targer for PCa., (© 2022. The Author(s).)

Published
2022
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210. [Interstitial cystitis: the latest findings on its aetiopathogenesis].

Author

Gonsior A and Neuhaus J

Subjects
Herpesvirus 4, Human, Humans, Inflammation, Cystitis, Interstitial etiology, Cystitis, Interstitial genetics, Epstein-Barr Virus Infections
Abstract

New findings provide progress in the understanding of the complicated aetiopathogenesis of interstitial cystitis/bladder pain syndrome (IC/BPS), whose causalities have only been deciphered in fragments so far. An increasingly complex network of pathomechanisms is emerging, in which the frequently mentioned mast cells and urothelial changes seem to be only a fragment of the pathological changes. The latest findings regarding a possible genetic and epigenetic predisposition are based on pedigree analyses, detection of single nucleotide polymorphisms and significant changes in differentially expressed genes. Multiple alterations can be detected at the molecular level. Platelet-activating factor, VEGF, corticotropin-releasing hormone and the inflammasome are important players in understanding the disease, but the pathomechanism underlying the "activation" of IC remains unclear. New starting points could be the detection of viruses (Epstein-Barr virus, BK polyomaviruses) or bacterial inflammation by pathogens that cannot be detected in standard cultures., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Published
2021
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211. Biomarkers in the Light of the Etiopathology of IC/BPS.

Author

Neuhaus J, Berndt-Paetz M, and Gonsior A

Abstract

In this review, we focused on putatively interesting biomarkers of interstitial cystitis/bladder pain syndrome (IC/BPS) in relation to the etiopathology of this disease. Since its etiopathology is still under discussion, the development of novel biomarkers is critical for the correct classification of the patients in order to open personalized treatment options, on the one hand, and to separate true IC/BPS from the numerous confusable diseases with comparable symptom spectra on the other hand. There is growing evidence supporting the notion that the classical or Hunner-type IC (HIC) and the non-Hunner-type IC (NHIC) are different diseases with different etiopathologies and different pathophysiology at the full-blown state. While genetic alterations indicate close relationship to allergic and autoimmune diseases, at present, the genetic origin of IC/BPS could be identified. Disturbed angiogenesis and impairment of the microvessels could be linked to altered humoral signaling cascades leading to enhanced VEGF levels which in turn could enhance leucocyte and mast cell invasion. Recurrent or chronic urinary tract infection has been speculated to promote IC/BPS. New findings show that occult virus infections occurred in most IC/BPS patients and that the urinary microbiome was altered, supporting the hypothesis of infections as major players in IC/BPS. Environmental and nutritional factors may also influence IC/BPS, at least at a late state (e.g., cigarette smoking can enhance IC/BPS symptoms). The damage of the urothelial barrier could possibly be the result of many different causality chains and mark the final state of IC/BPS, the causes of this development having been introduced years ago. We conclude that the etiopathology of IC/BPS is complex, involving regulatory mechanisms at various levels. However, using novel molecular biologic techniques promise more sophisticated analysis of this pathophysiological network, resulting in a constantly improvement of our understanding of IC/BPS and related diseases.

Published
2021
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212. Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells.

Author

Neuhaus J, Weimann A, and Berndt-Paetz M

Subjects
Cell Line, Humans, Protein Interaction Maps, Frizzled Receptors metabolism, Immunohistochemistry methods, Low Density Lipoprotein Receptor-Related Protein-6 metabolism, Proto-Oncogene Proteins metabolism, Receptor Tyrosine Kinase-like Orphan Receptors metabolism, Wnt Proteins metabolism, Wnt Signaling Pathway, Wnt-5a Protein metabolism
Abstract

The differential activation of Wnt pathways (canonical: Wnt/β-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca 2+ ) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate various downstream effectors. We established a proximity ligation assay (PLA) for the detection of endogenous FZD-co-receptor interactions and analyzed time-dependent Wnt pathway activation in cultured cells. Prostate cancer cells (PC-3) stimulated by Wnt ligands (Wnt5A, Wnt10B) were analyzed by Cy3-PLA for the co-localization of FZD6 and co-receptors (canonical: LRP6, non-canonical: ROR1) at the single-cell level. Downstream effector activation was assayed by immunocytochemistry. PLA allowed the specific (siRNA-verified) detection of FZD6-LRP6 and FZD6-ROR1 complexes as highly fluorescent spots. Incubation with Wnt10B led to increased FZD6-LRP6 interactions after 2 to 4 min and resulted in nuclear accumulation of β-catenin within 5 min. Wnt5A stimulation resulted in a higher number of FZD6-ROR1 complexes after 2 min. Elevated levels of phosphorylated myosin phosphatase target 1 suggested subsequent Wnt/PCP activation in PC-3. This is the first study demonstrating time-dependent interactions of endogenous Wnt (co-)receptors followed by rapid Wnt/β-catenin and Wnt/PCP activation in PC-3. In conclusion, the PLA could uncover novel signatures of Wnt receptor activation in mammalian cells and may provide new insights into involved signaling routes.

Published
2021
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213. Mechanosensitivity Is a Characteristic Feature of Cultured Suburothelial Interstitial Cells of the Human Bladder.

Author

Neuhaus J, Gonsior A, Cheng S, Stolzenburg JU, and Berger FP

Subjects
Aged, Cells, Cultured, Female, Humans, Male, Middle Aged, Urinary Bladder cytology, Calcium metabolism, Osmotic Pressure, Stress, Mechanical, Urinary Bladder, Overactive metabolism, Urinary Bladder, Overactive pathology, Urinary Bladder, Underactive metabolism, Urinary Bladder, Underactive pathology, Urothelium metabolism, Urothelium pathology
Abstract

Bladder dysfunction is characterized by urgency, frequency (pollakisuria, nocturia), and dysuria and may lead to urinary incontinence. Most of these symptoms can be attributed to disturbed bladder sensitivity. There is growing evidence that, besides the urothelium, suburothelial interstitial cells (suICs) are involved in bladder afferent signal processing. The massive expansion of the bladder during the filling phase implicates mechanical stress delivered to the whole bladder wall. Little is known about the reaction of suICs upon mechanical stress. Therefore, we investigated the effects of mechanical stimulation in cultured human suICs. We used fura-2 calcium imaging as a major physiological readout. We found spontaneous intracellular calcium activity in 75 % of the cultured suICs. Defined local pressure application via a glass micropipette led to local increased calcium activity in all stimulated suICs, spreading over the whole cell. A total of 51% of the neighboring cells in a radius of up to 100 µm from the stimulated cell showed an increased activity. Hypotonic ringer and shear stress also induced calcium transients. We found an 18-times increase in syncytial activity compared to unstimulated controls, resulting in an amplification of the primary calcium signal elicited in single cells by 50%. Our results speak in favor of a high sensitivity of suICs for mechanical stress and support the view of a functional syncytium between suICs, which can amplify and distribute local stimuli. Previous studies of connexin expression in the human bladder suggest that this mechanism could also be relevant in normal and pathological function of the bladder in vivo.

Published
2020
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214. Comparative study of the organisation and phenotypes of bladder interstitial cells in human, mouse and rat.

Author

Gevaert T, Neuhaus J, Vanstreels E, Daelemans D, Everaerts W, Der Aa FV, Timmermans JP, Roskams T, Steiner C, Pintelon I, and De Ridder D

Subjects
Animals, Female, Humans, Immunohistochemistry, Male, Mice, Microscopy, Electron, Rats, Rats, Sprague-Dawley, Interstitial Cells of Cajal ultrastructure, Mucous Membrane ultrastructure, Myocytes, Smooth Muscle ultrastructure, Urinary Bladder anatomy & histology
Abstract

With most research on interstitial cells (IC) in the bladder being conducted on animal models, it remains unclear whether all structural and functional data on IC from animal models can be translated to the human context. This prompted us to compare the structural and immunohistochemical properties of IC in bladders from mouse, rat and human. Tissue samples were obtained from the bladder dome and subsequently processed for immunohistochemistry and electron microscopy. The ultrastructural properties of IC were compared by means of electron microscopy and IC were additionally characterized with single/double immunohistochemistry/immunofluorescence. Our results reveal a similar organization of the IC network in the upper lamina propria (ULP), the deep lamina propria (DLP) and the detrusor muscle in human, rat and mouse bladders. Furthermore, despite several similarities in IC phenotypes, we also found several obvious inter-species differences in IC, especially in the ULP. Most remarkably in this respect, ULP IC in human bladder predominantly displayed a myoid phenotype with abundant presence of contractile micro-filaments, while those in rat and mouse bladders showed a fibroblast phenotype. In conclusion, the organization of ULP IC, DLP IC and detrusor IC is comparable in human, rat and mouse bladders, although several obvious inter-species differences in IC phenotypes were found. The present data show that translating research data on IC in laboratory animals to the human setting should be carried out with caution.

Published
2017
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215. Nuclear magnetic resonance spectroscopy as a new approach for improvement of early diagnosis and risk stratification of prostate cancer.

Author

Yang B, Liao GQ, Wen XF, Chen WH, Cheng S, Stolzenburg JU, Ganzer R, and Neuhaus J

Subjects
Biomarkers, Tumor blood, Biomarkers, Tumor urine, China, Humans, Male, Neoplasms blood, Neoplasms diagnostic imaging, Neoplasms urine, Prostatic Neoplasms blood, Prostatic Neoplasms urine, Reproducibility of Results, Risk Assessment, Early Detection of Cancer methods, Magnetic Resonance Spectroscopy, Metabolomics, Prostatic Neoplasms diagnostic imaging
Abstract

Prostate cancer (PCa) is the second most common male cancer worldwide and the fifth leading cause of death from cancer in men. Early detection and risk stratification is the most effective way to improve the survival of PCa patients. Current PCa biomarkers lack sufficient sensitivity and specificity to cancer. Metabolite biomarkers are evolving as a new diagnostic tool. This review is aimed to evaluate the potential of metabolite biomarkers for early detection, risk assessment, and monitoring of PCa. Of the 154 identified publications, 27 and 38 were original papers on urine and serum metabolomics, respectively. Nuclear magnetic resonance (NMR) is a promising method for measuring concentrations of metabolites in complex samples with good reproducibility, high sensitivity, and simple sample processing. Especially urine-based NMR metabolomics has the potential to be a cost-efficient method for the early detection of PCa, risk stratification, and monitoring treatment efficacy.

Published
2017
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216. Tetrahydroporphyrin-tetratosylat (THPTS): A near-infrared photosensitizer for targeted and efficient photodynamic therapy (PDT) of human bladder carcinoma. An in vitro study.

Author

Berndt-Paetz M, Weimann A, Sieger N, Schastak S, Riyad YM, Griebel J, Arthanareeswaran VKA, Stolzenburg JU, and Neuhaus J

Subjects
Apoptosis radiation effects, Cell Line, Tumor, Dose-Response Relationship, Drug, Dose-Response Relationship, Radiation, Humans, Radiation Dosage, Treatment Outcome, Apoptosis drug effects, Infrared Rays therapeutic use, Photochemotherapy methods, Photosensitizing Agents administration & dosage, Urinary Bladder Neoplasms drug therapy, Urinary Bladder Neoplasms pathology
Abstract

Background: Efficacy of PDT in muscle-invasive bladder cancer is hampered by low tissue penetration of most photosensitizers by short excitation wavelength. THPTS is excitable at near-infrared (760nm) allowing tissue penetration up to 15mm. We examined the cellular effects of THPTS-PDT in human bladder cancer cells., Material and Methods: We used four human transitional carcinoma cell lines, epithelial bladder progenitors (HBLAK) and bladder smooth muscle cells (HBSMC). We used flow cytometry to examine pharmacokinetics of THPTS, confocal laser scanning microscopy to analyze subcellular localization and production of reactive oxidative species (ROS), examined cytotoxicity and cell death pathways (qRT-PCR)., Results: Total uptake varied between cell lines and was significantly high in HBLAK and HBSMC. Lysosomal localization was mainly seen in cancer cells and HBLAK, while THPTS was distributed throughout the cytoplasm in HBSMC. Significant ROS production was detected 30min after THPTS-PDT. Growth arrest occurred within 4h and resulted in apoptotic and necrotic cytotoxicity after 24h. Cytotoxicity was dose-dependent and specifically high in cancer cells and HBLAK and significantly low in HBSMC., Conclusion: THPTS-PDT induces cellular mechanisms leading to cellular growth arrest, apoptosis and necrosis in human bladder cancer cells. These effects are only partly dependent on the total amount of THPTS uptake and rather dependent on its subcellular compartmentalization. HBSMC are hardly affected by THPTS-PDT confirming tumor specificity and safety. THPTS is a promising new photosensitizer with the unique advantage of deep tissue penetration allowing the treatment of solid tumors and warranting further animal studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Published
2017
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217. Anatomical study of pelvic nerves in relation to seminal vesicles, prostate and urethral sphincter: immunohistochemical staining, computerized planimetry and 3-dimensional reconstruction.

Author

Ganzer R, Stolzenburg JU, Neuhaus J, Weber F, Fuchshofer R, Burger M, and Bründl J

Subjects
Cadaver, Humans, Imaging, Three-Dimensional, Immunohistochemistry, Male, Middle Aged, Staining and Labeling, Pelvis innervation, Prostate innervation, Seminal Vesicles innervation, Urethra innervation
Abstract

Purpose: Studies of male pelvic neuroanatomy are mandatory to improve functional outcome after radical prostatectomy. We performed a topographical investigation of nerves on the course from the seminal vesicles along the prostate toward the striated urethral sphincter., Materials and Methods: Serial whole mount sections (1 mm intervals) of pelvic blocks of human adult male autopsy cadavers were investigated after immunohistochemical nerve staining. Computerized nerve quantification and planimetry of the total nerve surface area were performed within defined regions (ventral, ventrolateral, dorsolateral and dorsal) at the levels of the seminal vesicles and prostate, and at the striated urethral sphincter. The distance between the seminal vesicles and the nerves was measured. For improved topographical understanding 3-dimensional reconstructions were created. Differences between 3 independent variables were tested with the nonparametric Kruskal-Wallis test., Results: We studied a total of 969 whole mount sections of 5 cadavers. Nerves were arranged in a vertical plate lateral to the seminal vesicles. Mean ± SD distance to the seminal vesicles was 1.68 ± 0.84, 1.50 ± 0.12 and 1.76 ± 0.37 mm at the tip, middle and base, respectively. Periprostatic nerves were mainly found dorsolaterally. At the striated urethral sphincter 38.9% of nerves had shifted to the dorsal region. The total nerve surface area decreased significantly from the seminal vesicle tip (50.2 mm(2)) to the striated urethral sphincter level (13.3 mm(2)) (p = 0.0004)., Conclusions: Our findings underline that during nerve sparing prostatectomy nerve damage might occur during mobilization of the entire seminal vesicles, apical dissection and posterior reconstruction of the rhabdosphincter. Nerve planimetry revealed that 75% of the nerves from the seminal vesicles do not reach the striated urethral sphincter level and seem to innervate structures other than the corpora cavernosa., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Published
2015
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218. A global proteome approach in uric acid stimulated human aortic endothelial cells revealed regulation of multiple major cellular pathways.

Author

Oberbach A, Neuhaus J, Jehmlich N, Schlichting N, Heinrich M, Kullnick Y, Mohr FW, Kugler J, Baumann S, Völker U, and Adams V

Subjects
Aorta drug effects, Cells, Cultured, Endothelial Cells drug effects, Endothelium, Vascular cytology, Endothelium, Vascular drug effects, Endothelium, Vascular physiology, Humans, Signal Transduction drug effects, Aorta cytology, Aorta physiology, Endothelial Cells physiology, Proteome physiology, Signal Transduction physiology, Uric Acid pharmacology
Abstract

Background: Uric acid (UA) has been identified as one major risk factor for cardiovascular diseases. Lowering of serum UA levels improves endothelial function. The present study investigates for the first time concentration-dependent effects of UA on human aortic endothelial cells (HAEC) and the cellular pathways involved in global proteomic analysis., Methods: The concentration dependent effects of UA on HAEC were investigated by nanoLC-MS/MS and ingenuity pathway analysis to reveal putative cellular pathways. For verification of the identified pathways the abundance or activity of key proteins was measured using ELISA or Western blotting. NO production was quantified by confocal laser microscopy., Results: We identified ubiquitin-proteasome system (UPS) and eIF4 signaling as the major pathways regulated by UA. K-means clustering analysis revealed 11 additional pathways, of which NO, superoxide signaling and hypoxia were further analyzed. A complex regulatory network was detected demonstrating that 500μmol/L UA, which is well above the concentration regarded as pathological in clinical settings, led to diminishing of NO bioavailability. In addition a UA-dependent downregulation of eIF4, an upregulation of UPS and an increase in HIF-1α were detected., Conclusions: Here we show for the first time, that increasing UA levels activate different sets of proteins representing specific cellular pathways important for endothelial function. This indicates that UA may alter far more pathways in HAEC than previously assumed. This regulation occurs in a complex manner depending on UA concentration. Further studies in knockout and overexpression models of the identified proteins are necessary to prove the correlation with endothelial dysfunction., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Published
2014
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219. Sleeve gastrectomy reduces xanthine oxidase and uric acid in a rat model of morbid obesity.

Author

Oberbach A, Neuhaus J, Schlichting N, Kugler J, Baumann S, and Till H

Subjects
Animals, Blood Glucose metabolism, Disease Models, Animal, Gastrointestinal Hormones metabolism, Male, Obesity, Morbid etiology, Rats, Rats, Sprague-Dawley, Diet, High-Fat, Gastrectomy, Obesity, Morbid metabolism, Obesity, Morbid surgery, Uric Acid metabolism, Xanthine Oxidase metabolism
Abstract

Background: Serum uric acid (sUA) plays a major role in the development of morbidities associated with obesity, especially cardiovascular diseases. Within the purine pathway, xanthine oxidase (XOD) represents the key enzyme. The aim of this study was to investigate the dynamics of sUA and XOD following sleeve gastrectomy (SG) in a rat model of high-fat-diet (HFD) induced obesity., Patients: Over a period of 11 weeks, 30 rats received a HFD, and 10 rats received a low fat diet (LFD). Thereafter, 10 randomly selected HFD rats and 10 LFD rats were sacrificed. The remaining 20 HFD rats were randomly assigned to either SG or sham operation (SH) and studied 14 days postoperatively., Methods: The white adipose tissues (WAT) from visceral (intestinal and retroperitoneal) and inguinal (subcutaneous) depots were collected. sUA and urine UA (uUA) were measured by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Abundance and activity of XOD was investigated in the liver, colon, adipose tissue, and skeletal muscle by enzyme-linked immunosorbent assay (ELISA)., Results: HFD led to significant weight gain, elevated sUA levels, increased WAT and increase of XOD activity. Fourteen days postoperatively, SG rats showed a significant decrease of weight and adipose tissue, improved glucose metabolism, and changes of gut hormones. The sUA and uUA levels were significantly decreased following SG. Furthermore, XOD activity was significantly down-regulated in WAT., Conclusion: HFD induces elevated sUA levels by gain of WAT and increase of XOD activity. Following SG, the reduction of WAT as the major source of XOD and the lowering of XOD activity are the basis for the decrease of sUA., (Copyright © 2014 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

Published
2014
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220. Bariatric surgery in severely obese adolescents improves major comorbidities including hyperuricemia.

Author

Oberbach A, Neuhaus J, Inge T, Kirsch K, Schlichting N, Blüher S, Kullnick Y, Kugler J, Baumann S, and Till H

Subjects
Adolescent, Biomarkers blood, Case-Control Studies, Child, Comorbidity, Female, Gastric Bypass, Gastroplasty, Humans, Hyperuricemia blood, Hyperuricemia complications, Laparoscopy, Male, Obesity, Morbid complications, Pediatric Obesity complications, Pilot Projects, Weight Loss, Young Adult, Bariatric Surgery methods, Hyperuricemia epidemiology, Obesity, Morbid blood, Obesity, Morbid surgery, Pediatric Obesity blood, Pediatric Obesity surgery, Uric Acid blood
Abstract

Objective: Serum uric acid (sUA) is believed to contribute to the pathogenesis of metabolic comorbidities like hypertension, insulin-resistance (IR) and endothelial dysfunction (EDF) in obese children. The present pilot study investigated the association between sUA concentrations and loss of body weight following laparoscopic sleeve gastrectomy (LSG) or laparoscopic Roux-en-Y-gastric bypass (RYGB) in severely obese adolescents., Materials/methods: 10 severely obese adolescents underwent either LSG (n=5) or RYGB (n=5). 17 normal weight, healthy, age- and gender-matched adolescents served as a normal weight peer group (NWPG). Pre- and 12 months postoperatively, sUA and relevant metabolic parameters (glucose homeostasis, transaminases, lipids) were compared., Results: Preoperatively, sUA was significantly elevated in patients with severe obesity compared to NWPG. Twelve months after LSG and RYGB, a significant decrease in sUA, BMI, CVD risk factors, hepatic transaminases, and HOMA-IR was observed. Reduction in SDS-BMI significantly correlated with changes in sUA., Conclusions: sUA levels and metabolic comorbidities improved following bariatric surgery in severely obese adolescents. The impact of changes in sUA on long-term clinical complications of childhood obesity deserves further study., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Published
2014
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221. Intravesical treatments of bladder pain syndrome/interstitial cystitis.

Author

Neuhaus J and Schwalenberg T

Subjects
Administration, Intravesical, Botulinum Toxins, Type A administration & dosage, Cystitis, Interstitial diagnosis, Cystitis, Interstitial pathology, Dimethyl Sulfoxide administration & dosage, Humans, Neuromuscular Agents administration & dosage, Cystitis, Interstitial drug therapy
Abstract

Bladder pain syndrome/interstitial cystitis (BPS/IC) is a disabling chronic condition that affects up to 7% of women in the USA. In men, BPS/IC seems to be less common, but might be underestimated because it can be confused with chronic prostatitis. The aetiology and pathophysiology of BPS/IC are not well understood. Consequently, diagnosis and treatment is challenging and most therapies used to date are off-label. These therapies include bladder instillation with dimethyl sulfoxide (DMSO) and BCG, as well as hyperbaric oxygen therapy. Overall, botulinum neurotoxin A injection, intravesical sodium hyaluronate instillation and DMSO instillation seem to be the best-performing treatments, with response rates of 79%, 76% and 75%, respectively, and can be used effectively as second-line or third-line therapies for BPS/IC. However, additional high-quality randomized controlled trials are necessary to improve the available data.

Published
2012
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222. Individual receptor profiling as a novel tool to support diagnosis of bladder pain syndrome/interstitial cystitis (BPS/IC).

Author

Neuhaus J, Schulte-Baukloh H, Stolzenburg JU, Speroni di Fenizio P, Horn LC, Rüffert H, Hartenstein S, Burger M, Schulze M, and Schwalenberg T

Subjects
Aged, Biopsy, Cystitis, Interstitial metabolism, Female, Fluorescent Antibody Technique, Humans, Middle Aged, Muscle, Smooth metabolism, Muscle, Smooth pathology, Pain genetics, Pain metabolism, Pain pathology, Real-Time Polymerase Chain Reaction, Receptor, Muscarinic M2 genetics, Receptor, Muscarinic M2 metabolism, Receptor, Muscarinic M3 genetics, Receptor, Muscarinic M3 metabolism, Receptors, Histamine H1 genetics, Receptors, Histamine H1 metabolism, Receptors, Histamine H2 genetics, Receptors, Histamine H2 metabolism, Receptors, Purinergic P2X1 genetics, Receptors, Purinergic P2X1 metabolism, Receptors, Purinergic P2X2 genetics, Receptors, Purinergic P2X2 metabolism, Receptors, Purinergic P2X3 genetics, Receptors, Purinergic P2X3 metabolism, Syndrome, Transcriptome, Urinary Bladder metabolism, Urinary Bladder pathology, Urinary Bladder Neoplasms metabolism, Urinary Bladder, Overactive diagnosis, Urinary Bladder, Overactive genetics, Urinary Bladder, Overactive metabolism, Cystitis, Interstitial genetics, Cystitis, Interstitial pathology, Genetic Testing methods, Urinary Bladder Neoplasms genetics, Urinary Bladder Neoplasms pathology
Abstract

Purpose: Dysregulation of neurotransmitter receptors may contribute to bladder overactivity (OAB) symptoms. To address the question whether specific receptor expression patterns are associated with bladder pain syndrome/interstitial cystitis (BPS/IC), we examined the expression of muscarinic, purinergic and histamine receptors in the detrusor., Methods: Detrusor receptor expression was investigated in bladder biopsies of female BPS/IC patients (n = 44; age 60.64 ± 13.78, mean ± SD) and carcinoma patients (n = 11; age 58.91 ± 12.72) undergoing cystectomy. Protein expression of muscarinic (M2, M3), purinergic (P2X1-3) and histamine receptors (H1, H2) was analysed by confocal immunofluorescence, and gene expression was quantified by real-time polymerase chain reaction (qPCR)., Results: M2, P2X1, P2X2 and H1 receptor immunoreactivity (-IR) was significantly enhanced in BPS/IC compared to the control group, while there was no difference for M3-, P2X3- and H2-IR. We calculated a score, which separated BPS/IC from control patients with an AUC of 89.46%, showing 84.09% sensitivity and 90.91% specificity. Patients had a 9.25 times enhanced calculated risk for BPS/IC. In addition, two patient subgroups (M2 > M3 and M3 > M2) were observed, which differed in associated purinergic and histamine receptor expression., Conclusions: M2, P2X1, P2X2 and H1 were significantly upregulated in BPS/IC patients, and H2 was occasionally highly overexpressed. There was no significant correlation between receptor protein and gene expression, implying posttranslational mechanisms being responsible for the altered receptor expressions. On the basis of individual receptor profiles, upregulated receptors could be targeted by monotherapy or combination therapy with already approved receptor inhibitors, thereby promoting tailored therapy for patients suffering from BPS/IC-like symptoms.

Published
2012
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223. Anatomical description of the periprostatic nerves in the male rhesus monkey (Macaca mulatta).

Author

Ganzer R, Neuhaus J, Gratzke C, Blana A, Wieland WF, and Stolzenburg JU

Subjects
Animals, Autonomic Pathways physiology, Macaca mulatta physiology, Male, Models, Animal, Penile Erection physiology, S100 Proteins metabolism, Seminal Vesicles innervation, Autonomic Pathways anatomy & histology, Macaca mulatta anatomy & histology, Prostate innervation
Abstract

Objectives: Recent publications have revealed a variable course of the periprostatic nerves in humans. It is unclear to what extent nerves outside the dorsolateral region of the prostate are involved in the physiology of erectile function. As functional studies in humans are limited by ethical aspects investigations in animal models could provide further insight. The intention of this study was to give a detailed description of the topographical anatomy of autonomic nerves along the seminal vesicles and the prostate in male rhesus monkeys (Macaca mulatta) to investigate its suitability as an animal model for future physiological studies., Methods: Wholemount serial sections of pelvic organ blocks of ten male rhesus monkeys were investigated. Autonomic nerves were stained with an antibody against S100., Results: Autonomic nerves were dispersed along the dorsolateral to the ventrolateral aspect of the capsule of the prostate within a layer of connective tissue. There was no accumulation of vessels and nerves in the dorsolateral position of the prostate. The prostate is located dorsally to the urethra and does not encircle it. No adjacent nerves were found in the cranial two-thirds of the seminal vesicles., Conclusions: The male rhesus monkey is limited suitable as an animal model for studies on the periprostatic nerves provided the following differences to humans are considered: the special topography of the prostate, the nerve course along the seminal vesicles and the missing nerve accumulation dorsolaterally to the prostate.

Published
2011
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224. TGF-beta1 inhibits Cx43 expression and formation of functional syncytia in cultured smooth muscle cells from human detrusor.

Author

Neuhaus J, Heinrich M, Schwalenberg T, and Stolzenburg JU

Subjects
Aged, Aorta drug effects, Aorta physiology, Cells, Cultured, Connexin 43 drug effects, Down-Regulation drug effects, Female, Gap Junctions drug effects, Gap Junctions physiology, Gene Expression Regulation drug effects, Humans, Inflammation genetics, Male, Muscle, Smooth cytology, Muscle, Smooth drug effects, Muscle, Smooth physiology, Polymerase Chain Reaction, RNA, Messenger genetics, RNA, Neoplasm genetics, Transcription, Genetic, Urinary Bladder drug effects, Urinary Bladder physiology, Urinary Bladder physiopathology, Urinary Bladder Neoplasms genetics, Connexin 43 genetics, Transforming Growth Factor beta1 pharmacology, Urinary Bladder Neoplasms pathology, Urinary Bladder Neoplasms surgery
Abstract

Background: Human detrusor smooth muscle cells (hBSMCs) are coupled by connexin 43 (Cx43)-positive gap junctions to form functional syncytia. Gap junctional communication likely is necessary for synchronised detrusor contractions and is supposed to be altered in voiding disturbances. Other authors have shown that the pleiotropic cytokine TGF-beta1 upregulates Cx43 expression in human aortic smooth muscle cells., Objective: In this study, we examined the TGF-beta1 effects on Cx43 expression in cultured hBSMCs., Design, Setting, and Participants: hBSMC cultures, established from patients undergoing cystectomy, were treated with recombinant human TGF-beta1., Measurements: Cx43 expression was then examined by Western blotting, real-time PCR, and immunocytochemistry. Dye-injection experiments were used to study the size of functional syncytia., Results and Limitations: Dye-coupling experiments revealed stable formation of functional syncytia in passaged cell cultures (P1-P4). Stimulation with TGF-beta1 led to significant reduction of Cx43 immunoreactivity and coupling. Cx43 protein expression was significantly downregulated and Cx43 mRNA was only 30% of the control level. Interestingly, low phosphorylation species of Cx43 were particularly affected., Conclusions: Our experiments demonstrated a significant down regulation of connexin 43 by TGF-beta1 in cultured hBSMCs. These findings support the view that TGF-beta1 is involved in the pathophysiology of urinary bladder dysfunction.

Published
2009
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225. Histamine receptors in human detrusor smooth muscle cells: physiological properties and immunohistochemical representation of subtypes.

Author

Neuhaus J, Weimann A, Stolzenburg JU, Dawood W, Schwalenberg T, and Dorschner W

Subjects
Calcium metabolism, Carbachol pharmacology, Cells, Cultured, Female, Fura-2 pharmacology, Histamine pharmacology, Histamine Agonists pharmacology, Humans, Immunohistochemistry, Male, Receptors, Histamine classification, Myocytes, Smooth Muscle metabolism, Receptors, Histamine metabolism, Urinary Bladder cytology
Abstract

The potent inflammatory mediator histamine is released from activated mast cells in interstitial cystitis (IC). Here, we report on the histamine receptor subtypes involved in the intracellular calcium response of cultured smooth muscle cells (cSMC). Fura-2 was used to monitor the calcium response in cSMC, cultured from human detrusor biopsies. The distribution of histamine receptor subtypes was addressed by immunocytochemistry in situ and in vitro. Histamine stimulated a maximum of 92% of the cells (n=335), being more effective than carbachol (70%, n=920). HTMT (H1R-agonist), dimaprit (H2R) and MTH (H3R) lead to significant lower numbers of reacting cells (60, 48 and 54%). Histamine receptor immunoreactivity (H1R, H2R, H3R, H4R) was found in situ and in vitro. Histamine-induced calcium increase is mediated by distinct histamine receptors. Thus, pre-therapeutic evaluation of histamine receptor expression in IC patients may help to optimize therapy by using a patient-specific cocktail of subtype-specific histamine receptor antagonists.

Published
2006
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226. Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells.

Author

Neuhaus J, Oberbach A, Schwalenberg T, and Stolzenburg JU

Subjects
Carbachol pharmacology, Cells, Cultured, Cystectomy, Histamine analogs & derivatives, Humans, Male, Receptors, Histamine drug effects, Receptors, Histamine metabolism, Receptors, Histamine H1 drug effects, Receptors, Histamine H1 metabolism, Receptors, Histamine H2 drug effects, Receptors, Histamine H2 metabolism, Receptors, Histamine H3 drug effects, Receptors, Histamine H3 metabolism, Urinary Bladder metabolism, Urinary Bladder Neoplasms, Calcium metabolism, Histamine pharmacology, Histamine Agonists pharmacology, Myocytes, Smooth Muscle metabolism, Urinary Bladder cytology
Abstract

Objectives: To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists., Methods: Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry., Results: H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells., Conclusions: Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.

Published
2006
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227. Histomorphology of canine urethral sphincter systems, including three-dimensional reconstruction and magnetic resonance imaging.

Author

Stolzenburg JU, Neuhaus J, Liatsikos EN, Schwalenberg T, Ludewig E, and Ganzer R

Subjects
Animals, Female, Male, Dogs anatomy & histology, Imaging, Three-Dimensional, Magnetic Resonance Imaging, Urethra anatomy & histology
Abstract

Objectives: To present a detailed anatomic description and comparison of the smooth and striated urethral sphincter in male and female dogs. We performed a thorough histologic evaluation, three-dimensional reconstruction, and magnetic resonance imaging of the lower urinary tract of male and female dogs., Methods: The lower urinary tract anatomy was investigated in 16 male and 18 female dogs by serial sectioning, including immunohistochemical staining and three-dimensional reconstruction. Magnetic resonance imaging performed in 5 male and 5 female dogs before histologic investigation helped to demonstrate the anatomy in vivo., Results: A urethral sphincter muscle in both sexes existed without muscular connection to the pelvic floor. It ran circularly and consisted of an inner smooth and outer striated muscular part. In the female dog, the striated muscle encircled the urethra and vagina in the caudal third of the membranous urethra (musculus urethrovaginalis). A urinary diaphragm (diaphragma urogenitale) could not be found histologically or by magnetic resonance imaging., Conclusions: The dog is a suitable animal model for investigations of the urethral sphincter. In the female dog, attention should be given to the special topography of the musculus urethrovaginalis.

Published
2006
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228. Does the extraperitoneal laparoscopic approach improve the outcome of radical prostatectomy?

Author

Stolzenburg JU, Truss MC, Bekos A, Do M, Rabenalt R, Stief CG, Hoznek A, Abbou CC, Neuhaus J, and Dorschner W

Subjects
Humans, Intraoperative Complications, Male, Postoperative Complications, Prostatic Neoplasms surgery, Treatment Outcome, Laparoscopy adverse effects, Laparoscopy methods, Peritoneum surgery, Prostatectomy adverse effects
Abstract

Laparoscopic radical prostatectomy (LRPE) became the operative procedure of choice for patients with clinically localized prostate cancer in selected urologic centers around the world. Principal advantages are the minimal invasive nature of the procedure, a superior visualization of the operative field because of the magnification of the optical system, an exact and watertight anastomosis, the possibility of early catheter removal, and a potentially reduced amount of blood loss. Recent data show that oncologic outcome is not compromised by the minimal invasive nature of the procedure. However, a major drawback of LRPE is the transperitoneal route of access to the extraperitoneal organ of the prostate. Therefore, principal disadvantages of LRPE are potential intraperitoneal complications. Endoscopic extraperitoneal radical prostatectomy is a further advancement of minimal invasive surgery because it overcomes the limitations of LRPE by the strictly extraperitoneal route of access, combining the advantages of minimal invasive surgery with the advantages of an extraperitoneal procedure. This article reviews the literature on minimally invasive (laparoscopic and endoscopic-extraperitoneal) radical prostatectomy.

Published
2004
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229. Smooth muscle cells from human urinary bladder express connexin 43 in vivo and in vitro.

Author

Neuhaus J, Weimann A, Stolzenburg JU, Wolburg H, Horn LC, and Dorschner W

Subjects
Aged, Blotting, Western, Female, Gap Junctions pathology, Gap Junctions physiology, Humans, In Vitro Techniques, Male, Middle Aged, Muscle, Smooth pathology, Myocytes, Smooth Muscle pathology, Urinary Bladder pathology, Urinary Bladder Diseases pathology, Connexin 43 analysis, Connexin 43 physiology, Muscle, Smooth chemistry, Muscle, Smooth physiopathology, Myocytes, Smooth Muscle chemistry, Myocytes, Smooth Muscle physiology, Urinary Bladder chemistry, Urinary Bladder physiopathology, Urinary Bladder Diseases physiopathology
Abstract

Smooth muscle cells (SMC) of the human urinary bladder are believed to be electrically coupled. However, it has not been successfully demonstrated the nature of the underlying cell-cell communication. Here, we used Western blot technique to demonstrate the gap junction protein connexin 43 (Cx 43) in human bladder musculature and in smooth muscle cell cultures. We found expression of Cx 43 in all samples of the detrusor (Mdv, n=6) and in five of six samples of the internal sphincter (Mv). Cx 43 expression was less in cell cultures, 60% showing Cx 43 expression. Iontophoretic application of the gap junction permeable fluorescent dye lucifer yellow revealed a mean of 2.6+/-2.2 (mean+/-SD, n=8) coupled cells in cultured smooth muscle cells. Our findings support the notion that Cx 43 is constitutively expressed in human bladder SMC in vivo. Thus, the function of the bladder is likely to be influenced by the organisation of smooth muscle cells into functional syncytia.

Published
2002
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230. Detrusor smooth muscle cells of the guinea-pig are functionally coupled via gap junctions in situ and in cell culture.

Author

Neuhaus J, Wolburg H, Hermsdorf T, Stolzenburg JU, and Dorschner W

Subjects
Animals, Biomarkers, Calcium metabolism, Cells, Cultured, Connexin 43 analysis, Electrophysiology, Fura-2, Guinea Pigs, In Vitro Techniques, Male, Muscle, Smooth cytology, Muscle, Smooth metabolism, Signal Transduction, Urinary Bladder cytology, Urinary Bladder metabolism, Gap Junctions ultrastructure, Muscle, Smooth physiology, Urinary Bladder physiology
Abstract

Intercellular communication between smooth muscle cells is crucial for contractile behaviour in normal and pathologically altered urinary bladder. Since the study of coupling is difficult in situ, we established cell cultures of bladder smooth muscle cells to analyse coupling mechanisms. Microinjection of Lucifer yellow demonstrated syncytia composed of only a few to several dozen cells. Electron-microscopic examination of freeze-fracture specimens and ultrathin sections revealed that the dye-coupling was based on typical gap junction formation between the cultured smooth muscle cells. Furthermore, we were able to demonstrate gap junctions within the tissue fragments from which the primary cultures were grown. By Western blotting, we found connexin-43-positive protein bands both in native tissue probes from the guinea-pig urinary bladder and in smooth muscle cell cultures. Extracellular electrical stimulation of single cells evoked calcium transients, as visualized by fura-2 ratiofluorimetry. Calcium waves propagated throughout the syncytia with a declining amplitude, showing that the calcium signal was not regenerative. Therefore, the calcium signal was probably transmitted by a diffusible factor. These findings correlated well with the dye-coupling that we found between detrusor smooth muscle cells in situ. The use of smooth muscle cell cultures therefore seems to be a feasible approach for studying coupling behaviour in vitro.

Published
2002
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