G. Feil

In vitro myogenic differentiation of human bone marrow-derived mesenchymal stem cells as a potential treatment for urethral sphincter muscle repair.

To explore a new treatment strategy for urinary incontinence, human bone marrow mesenchymal stem cells (MSC) of the first in vitro passage were exposed to 5‐azacytidine (AZA) to induce myogenic differentiation, and cultured for a total of six passages. Expression of stem cell surface antigens and intracellular α‐actin was examined by flow cytometry at the end of each passage and compared to that of native MSC (not exposed to AZA) cultured in parallel. To analyze differentiation into striated muscle, expression of the transcription factor MyoD1 and myosin heavy chain (MyHC) was examined by RT‐PCR. Both native and AZA‐exposed MSC of all passages were negative for the progenitor/endothelial antigen CD34, leukocytic CD45, and endothelial/monocytic CD31. In contrast, the MSC markers CD73, CD90, CD105, and intracellular actin were detected in both groups of MSC throughout the culture period. After an initial increase, the expression level of MSC antigens decreased over time particularly in AZA‐exposed MSC. Expression of smooth muscle α‐actin also declined, but was greater in AZA‐exposed MSC throughout the culture period. Varying percentages of MSC cultures expressed MyoD1 and MyHC mRNA. In late passages, AZA‐exposed MSC tended to be more frequently positive than native MSC. In pilot experiments, transplantation of MSC into the bladder neck tissue of athymic rats was feasible; long‐term analyses are pending. We conclude that independent of AZA exposure, MSC express smooth and striated muscle antigens. Treatment with AZA slightly increases myogenic differentiation, but may not be necessary in future studies of MSC as a treatment modality for urinary incontinence.

In Vitro Investigations of Tissue-Engineered Multilayered Urothelium Established from Bladder Washings

OBJECTIVE Human urothelial cells (HUCs) are commonly isolated from native urothelium requiring open or endoscopic surgery. The aim of this study was to raise primary monolayer cultures of HUCs from bladder washings, to generate multilayered urothelial sheets in vitro, to characterise the sheets immunologically, and to prove their viability. METHODS Irrigation fluids were taken from 29 adult patients. Isolated cells were cultured in serum-free keratinocyte medium. Confluent monolayer cultures were stratified, and evolved cell sheets were harvested after 10-16 d. Pancytokeratins and cytokeratin 20 (CK20) in the stratified cultures and the detached sheets were immunologically detected. To exclude the presence of mesenchymal cells, antibodies against fibroblast surface antigen and smooth muscle alpha-actin were used. In addition, expression of p63 and uroplakin III was investigated. The viability of the detached cell sheets was proven by establishing explant cultures of small sheet sections. RESULTS Confluent primary HUC cultures were established in 55.2% of the collected bladder washings between days 15-20. Multilayered urothelium developed in 62.5% of the monolayers. Histology revealed stratified cell layers similar to native urothelium. Both stratified cultures and detached sheets stained 100% positive for pancytokeratins and partially for CK20, indicating differentiation into superficial cells. No positive staining was observed with the mesenchymal markers used. p63 was expressed partially. Uroplakin III expression was not observed. Cell sheet viability was confirmed by rapid cell outgrowth in explant cultures. CONCLUSIONS Isolation of HUCs from bladder washings is a minimally invasive approach to establish primary urothelial cultures for creating autologous multilayered urothelial sheets.

Nuclear matrix protein-22: a prospective evaluation in a population at risk for bladder cancer. Results from the UroScreen study

Study Type – Diagnostic (non‐consecutive cohort without consistently applied reference standard)

Combinations of urine-based tumour markers in bladder cancer surveillance

Abstract Objective. The aim of the study was to investigate whether combinations of urine-based tumour markers including urinary cytology (Cytology or Cyt) increase the sensitivity in the detection of bladder cancer recurrence. Material and methods. Urinary cytology, NMP22, UroVysion (FISH) and ImmunoCyt™ (uCyt+) were determined in 221 patients during the follow-up of non-muscle-invasive transitional cell carcinoma (NMI TCC) before cystoscopy (n = 49) or with the suspicion of TCC recurrence before transurethral resection of the bladder (n = 173). For all markers alone as well as in all possible combinations (multimarker panels, MPs) sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were evaluated. MPs were considered positive if at least one marker was positive. Results. No malignancy was found in 108 patients, whereas recurrent TCC was confirmed in 113 patients. Sensitivity and specificity for Cytology were 84% and 62%, for NMP22 68% and 49%, for FISH 72% and 63%, and for uCyt+ 73% and 62%, respectively. The NPV was below 80% for all markers alone. Combinations of two and three markers increased the sensitivity as well as the NPV to over 90 and 80%, by reducing specificity to an average of 44% and 35%, respectively. The most sensitive combinations were NMP22, uCyt+ together with Cytology and FISH, and uCyt+ together with NMP22 (sensitivity for both combinations 98%). There was no further improvement when all four markers were combined. Conclusions. Combinations of tumour markers increased the sensitivity and NPV in the detection of recurrence of NMI TCC. A stepwise approach of tumour marker determination may be used to reduce the frequency of follow-up cystoscopies at a reasonable risk.

Clinical experience with survivin as a biomarker for urothelial bladder cancer

ObjectivesThis study was carried out as a prospective pilot study to evaluate the potential of survivin mRNA measurement in patients suspicious for urothelial bladder cancer (BC). Data were also analyzed for possible influences of secondary urological findings on survivin measurements.MethodsSurvivin was measured by an mRNA assay in voided urine samples of 50 patients with suspicion of new or recurrent BC prior to transurethral resection. Sample evaluation was possible in 49 cases. Histopathology revealed no malignancy in 17 (35%) and BC in 32 (65%) patients. Survivin mRNA was quantitated by real-time PCR from frozen cell pellets of centrifuged urine samples. A ROC analysis of the survivin data was performed.ResultsROC analysis identified the best cut-off level at 10,000 mRNA copies, resulting in a sensitivity of 53% and a specificity of 88%. Seven of the 20 pTa tumors (35%), all four pT1 (100%) and all four muscle-invasive tumors (100%) were detected. Of four patients with carcinoma in situ (Cis), 50% could be identified. Only two patients (4%) were assessed as false positive. Histologically confirmed cystitis and concomitant urological findings (inflammatory cells in urine, microhematuria and others) had no detectable influence on survivin measurements.ConclusionIn present group of patients, survivin was a reliable biomarker for high-grade urothelial BC (sensitivity 83%), but not for low grade (sensitivity 35%) urothelial BC with a high specificity (88%). No confounders influencing the results of survivin measurements could be identified.

The role of haematuria in bladder cancer screening among men with former occupational exposure to aromatic amines

Study Type – Diagnostic (validating cohort)

Schistosomiasis: a critical review.

Precise data on epidemiology, morbidity, post-treatment resolution, reinfection, and resurgence of schistosomiasis could be helpful in establishing purposeful treatment plans for the disease in endemic populations. Here we give a concise overview of recent publications on bilharziasis. A main emphasis is placed on studies on the prevalence of schistosomiasis, partly including long term surveillance of morbidity following treatment with praziquantel. As genito-urinary schistosomiasis may be a risk factor for the spread of HIV, the involvement of the reproductive tract has become another focus in research on the disease. A novel diagnostic tool, eosinophil cationic protein (ECP), is proposed to correlate with the degree of inflammation of the genito-urinary tract.

Comparison of cytology and nuclear matrix protein 22 for the detection and follow-up of bladder cancer.

OBJECTIVES This study was designed to determine the clinical usefulness of the nuclear matrix METHODS 84 patients suffering from bladder cancer or suspected bladder cancer, 25 patients with benign urological lesions and 60 healthy controls participated in a prospective study. Freshly voided spot urine samples were taken for cytological examination and determination of NMP 22 levels by enzyme-linked immunoassay. RESULTS The sensitivity of the NMP 22 test according to the tumor grading was (results of cytology in brackets): G1 25.0% (20.0%); G2 68.2% (59.1%), and G3 100.0% (66.7); overall sensitivity was 62.5% (45.0%). The sensitivity for superficial bladder cancer was 46.7% (36.7%) and for invasive bladder cancer 90.0% (70.0%). The specificity was 65.9% (88.9%). CONCLUSIONS NMP 22 is a reliable tool for detecting invasive bladder cancer. Results for the frequently occurring low grade superficial bladder cancer are as poor as those obtained with cytology. In addition benign lesions such as urolithiasis or urinary tract infection lead to false-positive results. Therefore cystoscopy has to be performed when trying to detect and follow-up bladder cancer.

Correlation of bFGF expression in renal cell cancer with clinical and histopathological features by tissue microarray analysis and measurement of serum levels

The prognostic value of bFGF for surgically treated renal cell cancer (RCC) patients was evaluated by immunohistochemistry (IHC) and the tissue microarray technique (TMA). Additionally, preoperative serum bFGF levels were correlated to tumour stage and the presence of metastases at initial diagnosis. Serum levels of bFGF were measured by ELISA in 39 healthy volunteers, in 37 patients with benign urologic diseases and in 74 RCC patients, 26 of whom revealed lymph node or distant metastases. bFGF expression as detected by IHC was investigated in 777 tissue cores from 259 different RCC patients [median follow-up: 138 (36–240) months]. Eighty eight patients died from tumour progression. For each patient, the TMA slides contained a tissue core from the primary tumour, its invasion front and the normal renal parenchyma. bFGF serum levels were higher in RCC patients vs healthy volunteers (P<0.01) and vs patients with benign urologic diseases (P<0.01). Metastasized patients revealed higher bFGF serum levels than organ-confined specimens (P<0.01). As detected by IHC only increased bFGF expression in the invasion front tissue correlated with the patients’ long-term survival (log rank test) (P=0.03). In multivariate analysis regional LN metastases (P<0.01), the histological grading (P<0.01), and an increased bFGF expression in the invasion front (P=0.04) independently predicted the patients’ clinical prognosis. Not the expression of bFGF in the primary tumour but in its invasion front reflects the aggressiveness of RCC, hereby indicating a different biological potential within both areas. The value of bFGF serum levels as indicators of systemic tumour dissemination remains to be determined.

Tissue Engineering der Harnröhre und des Harnleiters

ZusammenfassungErworbene oder angeborene Störungen von Harnröhre und Harnleiter erfordern häufig geeignetes Gewebe zur Rekonstruktion der entsprechenden Defekte. Verschiedene Biomaterialien wurden erfolgreich zur Wiederherstellung von Ureter und Urethra in Tiermodellen und mittlerweile auch klinisch eingesetzt. Azelluläre Matrizes beispielsweise können, wenn sie im Empfängerorgan implantiert werden, dort als Grundgerüst für eine „natürliche“ Geweberegeneration dienen. Resorbierbare Gewebe können auch als Träger zur Transplantation von Zellen zur Harnröhren- bzw. Harnleiterrekonstruktion verwendet werden. Eines der Hauptprobleme hierbei ist, Zellen des Urogenitaltrakts in ausreichender Menge in Primärkulturen zu züchten. Möglicherweise kann die Stammzellforschung in der Zukunft dazu beitragen, dieses spezifische Problem zu lösen.AbstractCongenital or acquired disorders of the urethra or ureter often require adequate tissue transfer for reconstruction. A variety of biomaterials have proved to be useful in the reconstruction of the urethra or ureter in animal models and meanwhile even clinically. Innovative tissues such as acellular matrices can be placed in the host and function as a scaffold to allow the natural process of tissue regeneration. Biodegradable scaffolds can also be used as cell transplantation vehicles for the reconstruction of urethral or ureteral tissue. One of the limitations of cell-based tissue engineering techniques however is the difficulty of growing genitourinary-associated cells in large quantities in primary cultures. It can be speculated that stem cell research might help to overcome this specific problem in the future.