Protein Kinase D Is Dispensable for Development and Survival of Drosophila melanogaster.

Members of the Protein Kinase D (PKD) family are involved in numerous cellular processes in mammals, including cell survival after oxidative stress, polarized transport of Golgi vesicles, as well as cell migration and invasion. PKD proteins belong to the PKC/CAMK class of serine/threonine kinases, and transmit diacylglycerol-regulated signals. Whereas three PKD isoforms are known in mammals, Drosophila melanogaster contains a single PKD homolog. Previous analyses using overexpression and RNAi studies indicated likewise multi-facetted roles for Drosophila PKD, including the regulation of secretory transport and actin-cytoskeletal dynamics. Recently, involvement in growth regulation has been proposed based on the hypomorphic dPKDH allele. We have generated PKD null alleles that are homozygous viable without apparent phenotype. They largely match control flies regarding fertility, developmental timing and weight. Males, but not females, are slightly shorter lived and starvation sensitive. Furthermore, migration of pole cells in embryos and border cells in oocytes appears normal. PKD mutants tolerate heat, cold and osmotic stress like the control but are sensitive to oxidative stress, conforming to the described role for mammalian PKDs. A candidate screen to identify functionally redundant kinases uncovered genetic interactions of PKD with Pkcδ, sqa and Drak mutants, further supporting the role of PKD in oxidative stress response, and suggesting its involvement in starvation induced autophagy and regulation of cytoskeletal dynamics. Overall, PKD appears dispensable for fly development and survival presumably due to redundancy, but influences environmental responses.

Establishing and monitoring of urethral sphincter deficiency in a large animal model.

BACKGROUND: Different methods for induction and monitoring of urethral sphincter deficiency were explored in a large animal model. METHODS: Sphincter deficiency was established in female pigs by dilatation and cauterization, and amount and frequencies of voiding were monitored and explored by pad test. Sphincteric closure pressures were recorded prior to and immediately after treatment of each animal, and on day 21 by two techniques: standard urethral pressure profilometry (s-UPP) and high-definition urethral pressure profilometry (HD-UPP). Tissue samples of the urethrae were analyzed by histochemistry (AZAN- and Sirius Red staining) and by immunohistochemistry detecting desmin and fast-myosin to depict muscular tissues. RESULTS: After 3 weeks of observation animals treated by dilatation plus electrocautery presented with sphincter deficiency: measurements by both, s-UPP and HD-UPP demonstrated the maximal closure pressure reduced to baseline levels and a diminished area under the curve. Histological analyses documented, that dilatation yielded a pitted connective tissue and cauterization lead to muscle damage. Animals treated by either dilatation only or proximal injury only recovered within 3 weeks. By pad test no significant differences between untreated and treated animals or between the differently treated groups were recorded. CONCLUSION: Significant urethral sphincter deficiency can be induced in female pigs by a combination of urethral dilatation and distal electrocautery. Sphincter deficiency can be measured by standard and high-definition urethral pressure profilometry. It was maintained over 21 days after induction and correlated with visible changes in the tissue structure of the distal urethra.

Precise injection of human mesenchymal stromal cells in the urethral sphincter complex of Göttingen minipigs without unspecific bulking effects.

AIM: To investigate if injection of cells in the urethral sphincter complex causes unspecific bulking effects. METHODS: Human mesenchymal stromal cells were isolated, expanded, and characterized. For transurethral injection, cells were labeled with the fluorescent dye PKH26 and in magnetic resonance imaging associated experiments with superparamagnetic particles. Aliquots of cells in 250 µL solvent were injected under vision in the urethral sphincter of immuno-suppressed Göttingen minipigs. Sphincteric closure pressure was recorded by standard and high-definition urethral pressure profilometry prior to and after cell injection. The animals were sacrificed after surgery or after 3 weeks, 3, 6, or 12 months of follow-up. The localisation of the injected cells was explored by histochemistry. Sham-treated animals served as controls. RESULTS: PKH26-labeled cells survive injections in sphincter tissue samples by Williams cystoscopic injection needle well. In our animal study, the cellular depots were detected in the submucosa or in deeper zones of the sphincter, depending of the length of the injection needle (4-8 mm). Adverse effects associated with injection of cells or solvent such as a noteworthy bleeding, incontinence, or obstruction, were not recorded (n = 96 minipigs). However, a transient infiltration of macrophages was detected 3 weeks after cell injection. Changes in the urethral pressure profiles were not observed in cell-treated (n = 72) compared to sham-treated animals (n = 24). CONCLUSIONS: Injection of small aliquots of cells to investigate cell therapies in minipigs is a feasible and safe procedure, and it does not bias the intrinsic urethral wall pressure.

Collagen cell carriers seeded with human urothelial cells for urethral reconstructive surgery: first results in a xenograft minipig model.

PURPOSE: Urethral strictures are a common disease of the lower urinary tract in men. At present, the use of buccal mucosa is the method of choice for long or recurrent strictures. However, autologous tissue-engineered grafts are still under investigation for reconstructive urological surgery. The aim of this pilot study was to evaluate the use of human urothelial cells (HUC) seeded on bovine collagen type I-based cell carriers (CCC) in an animal model and to evaluate short-term outcome of the surgical procedure. METHODS: Four male Göttingen minipigs were used with immunosuppression (cyclosporine A) for this pilot xenograft study. HUC obtained from human benign ureteral tissue were stained by PKH26 and seeded on a collagen cell carrier (CCC). Seven weeks after urethral stricture induction and protective vesicostomy, cell-seeded CCC was implanted in the urethra with HUC luminal and antiluminal, respectively. After two weeks animals were euthanized, urethrography and histological assessment were performed. RESULTS: Surgery was technically feasible in all minipigs. Stricture was radiologically established 7 weeks after induction. CCC was visible after two weeks and showed good integration without signs of inflammation or rejection. In the final urethrography, no remaining stricture could be detected. Near porcine urothelium, PKH26-positive areas were found even if partially detached from CCC. Although diminished, immunofluorescence with pankeratin, CK20, E-cadherin and ZO-1 showed intact urothelium in several areas on and nearby CCC. CONCLUSION: Finally, this study demonstrates that the HUC-seeded CCC used as a xenograft in minipigs is technically feasible and shows promising results for further studies.

Assessing the reproducibility of high definition urethral pressure profilometry and its correlation with an air-charged system.

INTRODUCTION: Recently, a new urodynamic method for the assessment of stress urinary incontinence called high definition urethral pressure profilometry (HD-UPP) has been introduced. This method combines a novel microtip catheter with advanced signal processing to enable spatial data location and the reconstruction of a pressure image inside the urethra. In order to assess the reproducibility of HD-UPP data, we statistically evaluate HD-UPP datasets and compare them to data from a double balloon air-charged system. MATERIALS AND METHODS: Both catheters are used on sedated female minipigs. Data from the microtip catheter are processed through a signal reconstruction algorithm, urodynamic features are extracted, and compared to the air-charged system. Reproducibility of HD-UPP data is assessed by statistically evaluating consecutive, intra-individual datasets. RESULTS: HD-UPP delivers results in agreement with previous comparisons of microtip and air-charged systems. The average deviation of two consecutive, intra-individual pressure images is very low at 7 cm H2 O. CONCLUSIONS: HD-UPP provides physicians with detailed information on the pressure distribution inside the urethra. Through comparison with an air-charged catheter, it is shown that HD-UPP delivers results in agreement with previous studies on the comparison of microtip and air-charged catheters. It provides excellent reproducibility, as the difference between sequentially measured profiles from the same minipig is significantly lower than the one between profiles from different minipigs.

Signal processing in urodynamics: towards high definition urethral pressure profilometry.

BACKGROUND: Urethral pressure profilometry (UPP) is used in the diagnosis of stress urinary incontinence (SUI) which is a significant medical, social, and economic problem. Low spatial pressure resolution, common occurrence of artifacts, and uncertainties in data location limit the diagnostic value of UPP. To overcome these limitations, high definition urethral pressure profilometry (HD-UPP) combining enhanced UPP hardware and signal processing algorithms has been developed. In this work, we present the different signal processing steps in HD-UPP and show experimental results from female minipigs. METHODS: We use a special microtip catheter with high angular pressure resolution and an integrated inclination sensor. Signals from the catheter are filtered and time-correlated artifacts removed. A signal reconstruction algorithm processes pressure data into a detailed pressure image on the urethra's inside. Finally, the pressure distribution on the urethra's outside is calculated through deconvolution. A mathematical model of the urethra is contained in a point-spread-function (PSF) which is identified depending on geometric and material properties of the urethra. We additionally investigate the PSF's frequency response to determine the relevant frequency band for pressure information on the urinary sphincter. RESULTS: Experimental pressure data are spatially located and processed into high resolution pressure images. Artifacts are successfully removed from data without blurring other details. The pressure distribution on the urethra's outside is reconstructed and compared to the one on the inside. Finally, the pressure images are mapped onto the urethral geometry calculated from inclination and position data to provide an integrated image of pressure distribution, anatomical shape, and location. CONCLUSIONS: With its advanced sensing capabilities, the novel microtip catheter collects an unprecedented amount of urethral pressure data. Through sequential signal processing steps, physicians are provided with detailed information on the pressure distribution in and around the urethra. Therefore, HD-UPP overcomes many current limitations of conventional UPP and offers the opportunity to evaluate urethral structures, especially the sphincter, in context of the correct anatomical location. This could enable the development of focal therapy approaches in the treatment of SUI.

High definition urethral pressure profilometry: Evaluating a novel microtip catheter.

INTRODUCTION: Urethral pressure profilometry (UPP) is used in the diagnosis of stress urinary incontinence (SUI). SUI is a significant medical, social, and economic problem, affecting about 12.5% of the population. A novel microtip catheter was developed for UPP featuring an inclination sensor and higher angular resolution compared to systems in clinical use today. Therewith, the location of each measured pressure sample can be determined and the spatial pressure distribution inside the urethra reconstructed. In order to assess the performance and plausibility of data from the microtip catheter, we compare it to data from a double balloon air charged system. MATERIALS AND METHODS: Both catheters are used on sedated female minipigs. Data from the microtip catheter are processed through a signal reconstruction algorithm, plotted and compared against data from the air-charged catheter. RESULTS: The microtip catheter delivers results in agreement with previous comparisons of microtip and air-charged systems. It additionally provides a new level of detail in the reconstructed UPPs which may lead to new insights into the sphincter mechanism of minipigs. CONCLUSIONS: The ability of air-charged catheters to measure pressure circumferentially is widely considered a main advantage over microtip catheters. However, directional pressure readings can provide additional information on angular fluctuations in the urethral pressure distribution. It is shown that the novel microtip catheter in combination with a signal reconstruction algorithm delivers plausible data. It offers the opportunity to evaluate urethral structures, especially the sphincter, in context of the correct location within the anatomical location of the pelvic floor. Neurourol. Urodynam. 35:888-894, 2016. © 2015 Wiley Periodicals, Inc.

Eliminating pulse-induced artifacts in Urethral Pressure data.

Urethral Pressure Profilometry (UPP) is a tool in the diagnosis of urinary incontinence. The pressure profile along the urethra is measured by a special catheter in order to assess the contraction strength of the sphincter muscle. The use of microtip catheters with several pressure sensors and an integrated acceleration sensor enables signal reconstruction of the pressure distribution on the urethra's inside. Experimental data from minipigs exhibit artifact patterns in the pressure data. It is shown that these artifacts are caused by vascular pulsation in the sphincter structure. We therefore investigate different methods exploiting the time-correlation of the artifacts to eliminate pulse-induced artifacts in the pressure data without compromising the actual signal. Evaluation of these methods applied to experimental data conclude this work showing that both an Input-Model and Principal Component Analysis Decorrelation are effective at removing the artifacts.

A novel transgenic rat model for spinocerebellar ataxia type 17 recapitulates neuropathological changes and supplies in vivo imaging biomarkers.

Spinocerebellar ataxia 17 (SCA17) is an autosomal-dominant, late-onset neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat in the TATA-box-binding protein (TBP). To further investigate this devastating disease, we sought to create a first transgenic rat model for SCA17 that carries a full human cDNA fragment of the TBP gene with 64 CAA/CAG repeats (TBPQ64). In line with previous observations in mouse models for SCA17, TBPQ64 rats show a severe neurological phenotype including ataxia, impairment of postural reflexes, and hyperactivity in early stages followed by reduced activity, loss of body weight, and early death. Neuropathologically, the severe phenotype of SCA17 rats was associated with neuronal loss, particularly in the cerebellum. Degeneration of Purkinje, basket, and stellate cells, changes in the morphology of the dendrites, nuclear TBP-positive immunoreactivity, and axonal torpedos were readily found by light and electron microscopy. While some of these changes are well recapitulated in existing mouse models for SCA17, we provide evidence that some crucial characteristics of SCA17 are better mirrored in TBPQ64 rats. Thus, this SCA17 model represents a valuable tool to pursue experimentation and therapeutic approaches that may be difficult or impossible to perform with SCA17 transgenic mice. We show for the first time positron emission tomography (PET) and diffusion tensor imaging (DTI) data of a SCA animal model that replicate recent PET studies in human SCA17 patients. Our results also confirm that DTI are potentially useful correlates of neuropathological changes in TBPQ64 rats and raise hope that DTI imaging could provide a biomarker for SCA17 patients.

NKp80 defines and stimulates a reactive subset of CD8 T cells.

NKp80, an activating homodimeric C-type lectin-like receptor (CTLR), is expressed on essentially all human natural killer (NK) cells and stimulates their cytotoxicity and cytokine release. Recently, we demonstrated that the ligand for NKp80 is the myeloid-specific CTLR activation-induced C-type lectin (AICL), which is encoded in the natural killer gene complex (NKC) adjacent to NKp80. Here, we show that NKp80 also is expressed on a minor fraction of human CD8 T cells that exhibit a high responsiveness and an effector memory phenotype. Gene expression profiling and flow cytometric analyses revealed that this NKp80(+) T-cell subset is characterized by the coexpression of other NK receptors and increased levels of cytotoxic effector molecules and adhesion molecules mediating access to sites of inflammation. NKp80 ligation augmented CD3-stimulated degranulation and interferon (IFN)gamma secretion by effector memory T cells. Furthermore, engagement of NKp80 by AICL-expressing transfectants or macrophages markedly enhanced CD8 T-cell responses in alloreactive settings. Collectively, our data demonstrate that NKp80 is expressed on a highly responsive subset of effector memory CD8 T cells with an inflammatory NK-like phenotype and promotes T-cell responses toward AICL-expressing cells. Hence, NKp80 may enable effector memory CD8 T cells to interact functionally with cells of myeloid origin at sites of inflammation.