Human muscle precursor cells overexpressing PGC-1α enhance early skeletal muscle tissue formation

Muscle precursor cells (MPCs) are activated satellite cells capable of muscle fiber reconstruction. Therefore, autologous MPC transplantation is envisioned for the treatment of muscle diseases. However, the density of MPCs, as well as their proliferation and differentiation potential gradually decline with age. The goal of this research was to genetically modify human MPCs (hMPCs) to overexpress the peroxisome proliferator-activated receptor gamma coactivator (PGC-1α), a key regulator of exercise-mediated adaptation, and thereby to enhance early skeletal muscle formation and quality. We were able to confirm the sustained myogenic phenotype of the genetically modified hMPCs. While maintaining their viability and proliferation potential, PGC-1α modified hMPCs showed an enhanced myofiber formation capacity in vitro. Engineered muscle tissues were harvested 1, 2 and 4 weeks after subcutaneous injection of cell-collagen suspensions and histological analysis confirmed the earlier myotube formation in PGC-1α modified samples, predominantly of slow twitch myofibers. Increased contractile protein levels were detected by Western Blot. In summary, by genetically modifying hMPCs to overexpress PGC-1α we were able to promote early muscle fiber formation in vitro and in vivo, with an initial switch to slow type myofibers. Therefore, overexpressing PGC-1α is novel strategy to further enhance skeletal muscle tissue engineering

Noninvasive PET Imaging and Tracking of Engineered Human Muscle Precursor Cells for Skeletal Muscle Tissue Engineering

Transplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible noninvasive tool to monitor cell survival, migration, and integration into the host tissue is still missing. METHODS: In this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by reverse transcriptase polymerase chain reaction, and infection efficiency was evaluated by fluorescent microscopy. The viability, proliferation, and differentiation capacity of the transduced cells, as well as their myogenic phenotype, were determined by flow cytometry analysis and fluorescent microscopy. (18)F-fallypride and (18)F-fluoromisonidazole, two well-established PET radioligands, were assessed for their potential to image engineered hMPCs in a mouse model and their uptakes were evaluated at different time points after cell inoculation in vivo. Biodistribution studies, autoradiography, and PET experiments were performed to determine the extent of signal specificity. To address feasibility for tracking hMPCs in an in vivo model, the safety of the adenoviral gene delivery was evaluated. Finally, the harvested tissues were histologically examined to determine whether survival of the transplanted cells was sustained at different time points. RESULTS: Adenoviral gene delivery was shown to be safe, with no detrimental effects on the primary human cells. The viability, proliferation, and differentiation capacity of the transduced cells were confirmed, and flow cytometry analysis and fluorescent microscopy showed that their myogenic phenotype was sustained. (18)F-fallypride and (18)F-fluoromisonidazole were successfully synthesized. Specific binding of (18)F-fallypride to hD2R hMPCs was demonstrated in vitro and in vivo. Furthermore, the (18)F-fluoromisonidazole signal was high at the early stages. Finally, sustained survival of the transplanted cells at different time points was confirmed histologically, with formation of muscle tissue at the site of injection. CONCLUSION: Our proposed use of a signaling-deficient hD2R as a potent reporter for in vivo hMPC PET tracking by (18)F-fallypride is a significant step toward potential noninvasive tracking of hD2R hMPCs and bioengineered muscle tissues in the clinic

A novel animal model for external anal sphincter insufficiency

Purpose: Reliable animal models are essential to evaluate future therapeutic options like cell-based therapies for external anal sphincter insufficiency. The goal of our study was to describe the most reliable model for external sphincter muscle insufficiency by comparing three different methods to create sphincter muscle damage. Methods: In an experimental animal study, female Lewis rats (200-250g) were randomly assigned to three treatment groups (n = 5, each group). The external sphincter muscle was weakened in the left dorsal quadrant by microsurgical excision, cryosurgery, or electrocoagulation by diathermy. Functional evaluation included in vivo measurements of resting pressure, spontaneous muscle contraction, and contraction in response to electrical stimulation of the afferent nerve at baseline and at 2, 4, and 6weeks after sphincter injury. Masson's trichrome staining and immunofluorescence for skeletal muscle markers was performed for morphological analysis. Results: Peak contraction after electrical stimulation was significantly decreased after sphincter injury in all groups. Contraction forces recovered partially after cryosurgery and electrocoagulation but not after microsurgical excision. Morphological analysis revealed an incomplete destruction of the external sphincter muscle in the cryosurgery and electrocoagulation groups compared to the microsurgery group. Conclusions: For the first time, three different models of external sphincter muscle insufficiency were directly compared. The animal model using microsurgical sphincter destruction offers the highest level of consistency regarding tissue damage and sphincter insufficiency, and therefore represents the most reliable model to evaluate future therapeutic options. In addition, this study represents a novel model to specifically test the external sphincter muscle function

Dual-energy computed tomography for the differentiation of uric acid stones: ex vivo performance evaluation

We assessed the potential of dual-energy computed tomography (CT) for the differentiation between uric acid (UA)-containing and non-UA-containing urinary stones. Forty urinary stones of 16 different compositions in two sizes (<and≥5mm) were examined in an ex vivo model. Thirty stones consisted of pure calcium oxalate (whewellite or wheddellite), calcium phosphate (apatite, brushite, or vaterite), ammonium magnesium phosphate (struvite), UA, ammonium acid urate, ammonium phosphate, sodium hydrogen urate, or cystine, and ten stones were of mixed composition (UA-sodium hydrogen urate, whewellite-urate, wheddellite-urate, whewellite-brushite, or whewellite-brushite-struvite). Scans were performed using dual-source CT in a dual-energy mode with the tubes simultaneously operating at 80 and 140kV. Two readers analysed the data with respect to stone attenuation at each energy level. The stones were classified as UA- or non-UA-containing using manual attenuation measurements and software analysis results. Sensitivity, specificity, PPV, and NPV were calculated using crystallographic stone analysis as the gold standard. Twenty-six out of 40 stones (65%) contained no UA; 14 stones (35%) contained UA. When compared with UA-containing stones, the differences in attenuation values at 80 and 140kV were significantly (P<0.001) higher in stones containing no UA. The software automatically mapped 39/40 stones (98%). Only one (2%) 2mm UA-stone was missed. The software correctly classified all detected stones as UA- or non-UA-containing. The attenuation values of the missed stone were manually plotted into the analysis sheet which allowed for the correct classification of the stone (containing UA). Therefore, the sensitivity, specificity, PPV, and NPV for the detection of UA-containing stones was 100%. Ex vivo experience indicates that differentiation between UA- and non-UA-containing stones can be accurately performed using dual-source dual-energy C

Therapy decisions after diagnosis of prostate cancer in men with negative prostate MRI

Background: To investigate the clinical implications of magnetic resonance imaging (MRI) negative prostate cancer (PCa) in a cohort of men undergoing transperineal prostate biopsy. Methods: We included all men without prior diagnosis of PCa undergoing transperineal template saturation ± fusion-guided targeted biopsy of the prostate between November 2014 and March 2018. Before biopsy, all patients underwent MRI and biopsies were performed irrespective of imaging results. Baseline characteristics, imaging, biopsy results, and follow-up information were retrieved from the patient charts. Patients were classified as either MRI negative (Prostate Imaging Reporting and Data System [PIRADS] ≤ 2) or positive (PIRADS ≥ 3). ISUP grade group 1 was defined as clinically nonsignificant (nsPCa) and ≥2 as clinically significant PCa (csPCa). Primary outcome was the individual therapeutic decision after diagnosis of PCa stratified according to MRI visibility. Secondary outcomes were the sensitivity and specificity of MRI, and the urooncological outcomes after radical prostatectomy (RP). Results: From 515 patients undergoing prostate biopsy, 171 (33.2%) patients had a negative and 344 (66.8%) a positive MRI. Pathology review stratified for MRI negative and positive cases revealed nsPCa in 27 (15.8%) and 32 (9.3%) and csPCa in 26 (15.2%) and 194 (56.4%) of the patients, respectively. The rate of active treatment in the MRI negative was lower compared with the MRI positive cohort (12.3% vs. 53.2%; odd ratio [OR] = 0.12; p < 0.001). While men with negative MRI were more likely to undergo active surveillance (AS) than MRI positive patients (18.1% vs. 10.8%; OR = 1.84; p = 0.027), they rarely underwent RP (6.4% vs. 40.7%, OR = 0.10; p < 0.001). Logistic regression revealed that a negative MRI was independently protective for active treatment (OR = 0.32, p = 0.014). The specificity, sensitivity, negative, and positive predictive value of MRI for detection of csPCa were 49.2%, 88.2%, 56.4%, and 84.8%, respectively. The rate of adverse clinicopathological outcome features (pT3/4, ISUP ≥4, or prostate-specific antigen [PSA]-persistence) following RP was 4.7% for men with MRI negative compared to 17.4% for men with MRI positive PCa (OR = 3.1, p = 0.19). Conclusion: Only few men with MRI negative PCa need active cancer treatment at the time of diagnosis, while the majority opts for AS. Omitting prostate biopsies and performing a follow-up MRI may be a safe alternative to reduce the number of unnecessary interventions. Keywords: PIRADS; biopsy-naïve; imaging; invisible prostate cancer; transperineal biopsy; treatmen

Pre-orchiectomy tumor marker levels should not be used for International Germ Cell Consensus Classification (IGCCCG) risk group assignment

PURPOSE To investigate whether the use of pre-orchiectomy instead of pre-chemotherapy tumor marker (TM) levels has an impact on the International Germ Cell Consensus Classification (IGCCCG) risk group assignment in patients with metastatic germ cell tumors (GCT). METHODS Demographic and clinical information of all patients treated for primary metastatic testicular non-seminomatous GCT in our tertiary care academic center were extracted from medical charts. IGCCCG risk group assignment was correctly performed with pre-chemotherapy marker levels and additionally with pre-orchiectomy marker levels. Agreement between pre-chemotherapy and pre-orchiectomy risk group assignments was assessed using Cohen's kappa. RESULTS Our cohort consisted of 83 patients. The use of pre-orchiectomy TMs resulted in an IGCCCG risk group upstaging in 12 patients (16%, 8 patients from good to intermediate risk and 4 patients from intermediate to poor risk) and a downstaging in 1 patient (1.2%, from intermediate- to good-risk). The agreement between pre-orchiectomy and pre-chemotherapy IGCCCG risk groups resulted in a Cohen's kappa of 0.888 (p < 0.001). CONCLUSIONS Using pre-orchiectomy TMs can result in incorrect IGCCCG risk group assignment, which in turn can impact on the clinical management and follow-up of patients with metastatic GCT. Thus, adherence to the IGCCCG standard using pre-chemotherapy TMs levels is recommended

Retroperitoneoscopic donor nephrectomy: donor outcome and complication rate in comparison with three different techniques

Four surgical techniques for living donor nephrectomy were analyzed retrospectively in terms of perioperative outcome and early complication rate. A total of 182 donor nephrectomies including 69 open (OLDN), 14 fully laparoscopic (LDN), 34 hand-assisted laparoscopic (HLDN) and 65 retroperitoneoscopic (RLDN) nephrectomies were analyzed. There was a significant difference in mean operating time (OPT) between the OLDN (160min) and RLDN (150min) as compared to the LDN (212min) and HLDN group (192min) (P<0.001). Mean warm ischemia time (WIT) was significantly shorter with OLDN (114s), RLDN (121s) and HLDN (128s) when compared to LDN (238s) (P<0.001). Major complication rate was comparable among the groups. Independent of the preferred technique, donor nephrectomy is associated with complication rates. RLDN is comparable to OLDN in terms of OPT, WIT. Learning endoscopic donor nephrectomy could be associated with a higher complication rat

Risk Factors and Treatment Outcomes of 1,375 Patients with Testicular Leydig Cell Tumors: Analysis of Published Case Series Data

AIMS Leydig cell tumors (LCTs) are rare but the most common non-germ cell testicular tumors. Only limited evidence exists for reliably differentiating between benign and malignant LCTs and for optimally managing the different types and stages of this rare disease. This review aims to synthesize the available evidence regarding the clinical presentation and clinicopathological characteristics associated with LCT malignancy and management. METHODS We analyzed published case series with LCTs patients. The association between clinicopathological variables and the presence of metastatic disease was assessed using regression analyses. RESULTS We included 357 reports, reviewing available data from 1,375 patients (median age: 34y). Testis-sparing surgery (TSS) was performed in 463 patients. Local recurrence after TSS occurred in 8 of 121 (7%) patients with available follow-up information. Metastases were found in 101 patients and were most often located in the retroperitoneal lymph nodes (60%), lungs (38%), and/or liver (29%). The multivariable models with or without multiple imputation predicting metastatic disease included older age, larger tumor size, the presence of any adverse factor (larger tumor diameter, necrosis, angiolymphatic invasion, pleomorphism, high mitotic index, atypia), and any protective factor (Reinke crystals, lipofuscin pigments, gynecomastia) with model AUCs of 0.93. Durable remission after resection of metastases or platinum-based chemotherapy was rarely seen. CONCLUSION Our risk tables using clinicopathological parameters can help identify patients harboring malignant tumors. These patients should undergo staging and either be followed or receive further treatment. In metastatic disease surgical and systemic treatment might result in disease control in in some patients