Bartosz Dybowski

Urethral distension as a novel method to simulate sphincter insufficiency in the porcine animal model

Objectives:  To describe a novel animal model of intrinsic sphincter deficiency.

The Effect of Endoscopic Administration of Autologous Porcine Muscle-derived Cells Into the Urethral Sphincter

OBJECTIVE To verify the fate of autologous porcine myogenic cells after endoscopic administration into the urethral sphincter. METHODS This study was performed on pig animal models. The muscle-derived cells (MDCs) were isolated and identified. After the third passage, the 6 × 10(7) of PKH26 labeled cells were injected into the urethral sphincter using a urethrocystoscope. The urethras were collected after 28 days. To analyze the fate of injected cells, the PKH26 presence, the desmin expression, and the distribution of acetylcholine receptors were evaluated in the tissue sections. Moreover, the maximal urethral closure pressure (MUCP) was assessed in experimental and control groups at day 1 and day 28. RESULTS The isolated porcine MDCs expressed desmin and were able to differentiate into myotubes in vitro. At day 28 after the transplantation, the depots of PKH26-positive cells were observed in the muscular layer, but also in the submucosa. The staining for desmin revealed that cells located in the muscle layer were integrated with muscle fibers that possessed acetylcholine receptors. However, cells administered into nonmuscle tissue did not express desmin. Urethral pressure profilometry demonstrated no significant differences between MUCP in the transplanted group in comparison to the control group at day 28. CONCLUSION The present study demonstrates the successful endoscopic transplantation of myogenic cells into the urethral sphincter. The experiments indicated the key importance of precise cell administration in terms of their fate after the injection.

P27(Kip1) and Ki-67 expression analysis in transitional cell carcinoma of the bladder.

P27Kip1 protein is a cell cycle inhibitor which blocks the transition of cells from G1 to S phase, while Ki-67 is the most specific marker of proliferative activity. Both proteins are independent predictors of clinical outcome in various neoplasms. The aim of the study was to assess the prognostic value of p27Kip1 and Ki-67 expression in urothelial bladder tumours. P27Kip1 and Ki-67 expressions were evaluated immunohistochemically in archival samples of 45 superficial and 26 invasive transitional cell carcinomas obtained by a transurethral resection. In the patients with superficial tumours, disease-free survival (DFS) was positively influenced by good histological differentiation as well as by concurrent high p27Kip1 and low Ki-67 expression. Multivariate analysis has confirmed that tumour grade and p27Kip1/Ki-67 status were independent predictors of DFS (p=0.028 and p=0.029, respectively). P27Kip1 or Ki-67 expressions did not influence overall survival. We conclude that a variable combined of p27Kip1 and Ki-67 expressions is a better predictor of DFS in superficial bladder tumours than either protein alone.

Predicting stone composition before treatment – can it really drive clinical decisions?

Introduction Determination of stone composition is considered to be crucial for the choice of an optimal treatment algorithm. It is especially important for uric acid stones, which can be dissolved by oral chemolysis and for renal stones smaller than 2 cm, which can be treated with extracorporeal shockwave lithotripsy (ESWL). Material and methods This short review identifies the latest papers on radiological assessment of stone composition and presents a comprehensive evaluation of current scientific findings. Results Stone chemical composition is difficult to predict using standard CT imaging, however, attenuation index measured in Hounsfield units (HU) is related to ESWL outcome. Stone density >1000 HU can be considered predictive for ESWL failure. It seems that stone composition is meaningless in determining the outcome of ureterolithotripsy and percutaneous surgery. Alternative imaging techniques such as Dual–Energy CT or analysis of shape, density and homogeneity of stones on plain X–rays are used as promising methods of predicting stone composition and ESWL outcome. Conclusions New imaging techniques facilitate the identification of uric acid stones and ESWL–resistant stones. Therefore, they may help in selecting the best therapeutic option.

The Mutual Interactions between Mesenchymal Stem Cells and Myoblasts in an Autologous Co-Culture Model

Both myoblasts and mesenchymal stem cells (MSC) take part in the muscle tissue regeneration and have been used as experimental cellular therapy in muscular disorders treatment. It is possible that co-transplantation approach could improve the efficacy of this treatment. However, the relations between those two cell types are not clearly defined. The aim of this study was to determine the reciprocal interactions between myoblasts and MSC in vitro in terms of the features important for the muscle regeneration process. Primary caprine muscle-derived cells (MDC) and bone marrow-derived MSC were analysed in autologous settings. We found that MSC contribute to myotubes formation by fusion with MDC when co-cultured directly, but do not acquire myogenic phenotype if exposed to MDC-derived soluble factors only. Experiments with exposure to hydrogen peroxide showed that MSC are significantly more resistant to oxidative stress than MDC, but a direct co-culture with MSC does not diminish the cytotoxic effect of H2O2 on MDC. Cell migration assay demonstrated that MSC possess significantly greater migration ability than MDC which is further enhanced by MDC-derived soluble factors, whereas the opposite effect was not found. MSC-derived soluble factors significantly enhanced the proliferation of MDC, whereas MDC inhibited the division rate of MSC. To conclude, presented results suggest that myogenic precursors and MSC support each other during muscle regeneration and therefore myoblasts-MSC co-transplantation could be an attractive approach in the treatment of muscular disorders.

The Anatomy of Caprine Female Urethra and Characteristics of Muscle and Bone Marrow Derived Caprine Cells for Autologous Cell Therapy Testing.

Cell therapy is emerging as an alternative treatment of stress urinary incontinence. However, many aspects of the procedure require further optimization. A large animal model is needed to reliably test cell delivery methods. In this study, we aim to determine suitability of the goat as an experimental animal for testing intraurethral autologous cell transplantation in terms of urethral anatomy and cell culture parameters. The experiments were performed in 12 mature/aged female goats. Isolated caprine muscle derived cells (MDC) were myogenic in vitro and mesenchymal stem cells (MSC) population was able to differentiate into adipo‐, osteo‐ and chondrogenic lineages. The median yield of cells after 3 weeks of culture amounted 47 × 10(6) for MDC and 37 × 10(6) for MSC. Urethral pressure profile measurements revealed the mean functional urethral length of 3.75 ± 0.7 cm. The mean maximal urethral closure pressure amounted 63.5 ± 5.9 cmH2O and the mean functional area was 123.3 ± 19.4 cm*cmH2O. The omega‐ shaped striated urethral sphincter was well developed in the middle and distal third of the urethra and its mean thickness on cross section was 2.3 mm. In the proximal part of the urethra only loosely arranged smooth muscle fibers were identified. To conclude, presented data demonstrate that caprine MDC and MSC can be expanded in vitro in a repeatable manner even when mature or aged animals are cell donors. Results suggest that female caprine urethra has similar parameters to those reported in human and therefore the goat can be an appropriate experimental animal for testing intraurethral cell transplantation. Anat Rec, 00:000–000, 2016.

Pressure-flow nomogram for women with lower urinary tract symptoms

Introduction Results of urodynamic studies performed in female patients are often difficult to interpret. The objective of the study was to develop a nomogram that would help in diagnosing functional bladder outlet obstruction (BOO) in neurologically intact women with any kind of lower urinary tract symptoms. Material and methods From the urodynamic database adult women were chosen with maximal flow rate (Qmax) ≤ 12 ml/s in a pressure-flow study. Four criteria were used to identify a group of patients suspected of BOO: thickened bladder wall, presence of bladder diverticula, subjective improvement on α-blockers and improvement of voiding symptoms on any form of treatment. The line separating high and low pressure zones on the pressure-flow chart was established according to the position of patients who met at least one of them. Results Sixty-seven patientswere investigated. Twenty-one women met at least one of the specified criteria. They had significantly higher voiding pressures (p det(Qmax) 35 cm H2O vs. 16.5 cm H2O; p = 0.002). A new nomogram with one separating line (p det(Qmax) = 1.5 × Q max+ 10) was proposed. The difference in the distribution of women fulfilling the criteria between high pressure zone and low pressure zone was highly significant (19/35 vs. 2/32; p < 0.0001). Sensitivity, specificity, positive and negative predictive values of our nomogram in identifying patients suspected of BOO was 90.5%, 65.2%, 54.3% and 94% respectively. Conclusions The new nomogram can be considered a screening test which efficiently excludes obstruction among women with low Q max in a pressure-flow study.

In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models

Introduction Despite spectacular progress in cellular transplantology, there are still many concerns about the fate of transplanted cells. More preclinical studies are needed, especially on large animal models, to bridge the translational gap between basic research and the clinic. Herein, we propose a novel approach in analysis of cell transplantation effects in large animals explants using in vivo imaging system (IVIS®) or similar equipment. Material and methods In the in vitro experiment cells labeled with fluorescent membrane dyes: DID (far red) or PKH26 (orange) were visualized with IVIS®. The correlation between the fluorescence signal and cell number with or without addition of minced muscle tissue was calculated. In the ex vivo study urethras obtained from goats after intraurethral cells (n = 9) or PBS (n = 4) injections were divided into 0.5 cm cross-slices and analyzed by using IVIS®. Automatic algorithm followed or not by manual setup was used to separate specific dye signal from tissue autofluorescence. The results were verified by systematic microscopic analysis of standard 10 μm specimens prepared from slices before and after immunohistochemical staining. Comparison of obtained data was performed using diagnostic test function. Results Fluorescence signal strength in IVIS® was directly proportional to the number of cells regardless of the dye used and detectable for minimum 0.25x106 of cells. DID-derived signal was much less affected by the background signal in comparison to PKH26 in in vitro test. Using the IVIS® to scan explants in defined arrangement resulted in precise localization of DID but not PKH26 positive spots. Microscopic analysis of histological specimens confirmed the specificity (89%) and sensitivity (80%) of IVIS® assessment relative to DID dye. The procedure enabled successful immunohistochemical staining of specimens derived from analyzed slices. Conclusions The IVIS® system under appropriate conditions of visualization and analysis can be used as a method for ex vivo evaluation of cell transplantation effects. Presented protocol allows for evaluation of cell delivery precision rate, enables semi-quantitative assessment of signal, preselects material for further analysis without interfering with the tissue properties. Far red dyes are appropriate fluorophores to cell labeling for this application.

Optimum anesthesia for reliable urethral pressure profilometry in female dogs and goats.

To investigate the effects of propofol and isoflurane on urethral pressure profilometry of female dogs and goats, and to identify the method of anesthesia that least influences urethral pressure profilometry and to assess its reproducibility.

Impact of stage and comorbidities on five-year survival after radical cystectomy in Poland: single centre experience.

Introduction Long-term outcomes of patients treated for invasive bladder cancer in Poland are poorly documented in the literature. Impact of various clinical parameters on their survival is even less well studied. Radical cystectomy is a major surgery, so the patients’ condition can be equally important as cancer stage. The aim of the study was to assess 5-year overall survival (OS) after cystectomy and impact of comorbidity on OS in a single Polish academic centre. Material and methods Clinical data of all patients who underwent cystectomy in years 2004-2006 for urothelial cancer were retrospectively reviewed. Survival status was determined at least 5 years after surgery. Pathological variables, comorbidities, surgery delay and complications were evaluated as potential predictors of OS. Kaplan-Meier estimates of the survival function as well as Cox proportional hazards models were utilized. Results Thirty-day, 1-year and 5-year OS for 63 patients was 98.4%, 58.7% and 31.7%, respectively. None of the investigated parameters were significantly related to five-year OS. However, a composite parameter consisting of stage, diabetes status and postoperative course was found as a significant predictor. Five-year OS in 16 patients with pT1-2 and without diabetes and without post-operative complications was higher than in the remaining 47 patients (56% vs. 23%; P = 0.02). Conclusions Five-year OS in our group was lower than in most published international series but concordant with a previous Polish report. Improvement in survival after radical cystectomy may be expected when early diagnosis will be accompanied by optimal care of patients with diabetes mellitus and avoidance of postoperative complications.