Direct generation of functional dopaminergic neurons from mouse and human fibroblasts.

Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

DFL23448, A Novel Transient Receptor Potential Melastin 8-Selective Ion Channel Antagonist, Modifies Bladder Function and Reduces Bladder Overactivity in Awake Rats.

The transient receptor potential melastin 8 ion channel (TRPM8) is implicated in bladder sensing but limited information on TRPM8 antagonists in bladder overactivity is available. This study characterizes a new TRPM8-selective antagonist (DFL23448 [5-(2-ethyl-2H-tetrazol-5-yl)-2-(3-fluorophenyl)-1,3-thiazol-4-ol]) and evaluates it in cold-induced behavioral tests and tests on bladder function and experimental bladder overactivity in vivo in rats. DFL23448 displayed IC50 values of 10 and 21 nM in hTRPM8 human embryonic kidney 293 cells activated by Cooling Agent 10 or cold, but it had limited activity (IC50 > 10 µM) at transient receptor potential vanilloids TRPV1, TRPA1, or TRPV4 or at various G protein-coupled receptors. In rats, DFL23448 administered intravenously or orally had a half-life of 37 minutes or 4.9 hours, respectively. DLF23448 (10 mg/kg i.v.) reduced icilin-induced "wet dog-like" shakes in rats. Intravesical DFL23448 (10 mg/l), but not vehicle, increased micturition intervals, micturition volume, and bladder capacity. During bladder overactivity by intravesical prostaglandin E2 (PGE2), vehicle controls exhibited reductions in micturition intervals, micturition volumes, and bladder capacity by 37%-39%, whereas the same parameters only decreased by 12%-15% (P < 0.05-0.01 versus vehicle) in DFL23448-treated rats. In vehicle-treated rats, but not in DFL23448-treated rats, intravesical PGE2 increased bladder pressures. Intravenous DFL23448 at 10 mg/kg, but not 1 mg/kg DFL23448 or vehicle, increased micturition intervals, micturition volumes, and bladder capacity. During bladder overactivity by intravesical PGE2, micturition intervals, micturition volumes, and bladder capacity decreased in vehicle- and 1 mg/kg DFL23448-treated rats, but not in 10 mg/kg DFL23448-treated rats. Bladder pressures increased less in rats treated with DFL23448 10 mg/kg than in vehicle- or 1 mg/kg DFL23448-treated rats. DFL23448 (10 mg/kg i.v.), but not vehicle, prevented cold stress-induced bladder overactivity. Our results support a role for bladder TRPM8-mediated signals in experimental bladder overactivity.

Ouabain Contributes to Kidney Damage in a Rat Model of Renal Ischemia-Reperfusion Injury.

Warm renal ischemia performed during partial nephrectomy has been found to be associated with kidney disease. Since endogenous ouabain (EO) is a neuro-endocrine hormone involved in renal damage, we evaluated the role of EO in renal ischemia-reperfusion injury (IRI). We measured plasma and renal EO variations and markers of glomerular and tubular damage (nephrin, KIM-1, Kidney-Injury-Molecule-1, α1 Na-K ATPase) and the protective effect of the ouabain inhibitor, rostafuroxin. We studied five groups of rats: (1) normal; (2) infused for eight weeks with ouabain (30 µg/kg/day, OHR) or (3) saline; (4) ouabain; or (5) saline-infused rats orally treated with 100 µg/kg/day rostafuroxin for four weeks. In group 1, 2-3 h after IRI, EO increased in ischemic kidneys while decreased in plasma. Nephrin progressively decreased and KIM-1 mRNA increased starting from 24 h. Ouabain infusion (group 2) increased blood pressure (from 111.7 to 153.4 mmHg) and ouabain levels in plasma and kidneys. In OHR ischemic kidneys at 120 h from IRI, nephrin, and KIM-1 changes were greater than those detected in the controls infused with saline (group 3). All these changes were blunted by rostafuroxin treatment (groups 4 and 5). These findings support the role of EO in IRI and suggest that rostafuroxin pre-treatment of patients before partial nephrectomy with warm ischemia may reduce IRI, particularly in those with high EO.

The long noncoding RNA SUMO1P3 as urinary biomarker for monitoring bladder cancer progression.

Introduction: Urothelial Bladder Cancer (BC) is the ninth most common cancer worldwide. It is classified into Non Muscle Invasive (NMIBC) and Muscle Invasive Bladder Cancer (MIBC), which are characterized by frequent recurrences and progression rate, respectively. The diagnosis and monitoring are obtained through invasive methods as cystoscopy and post-surgery biopsies. Thus, a panel of biomarkers able to discriminate BC based on grading or staging represents a significant step forward in the patients' workup. In this perspective, long non-coding RNAs (lncRNAs) are emerged as reliable candidates as potential biomarker given their specific and regulated expression. In the present work we propose two lncRNAs, the Small Ubiquitin Modifier 1 pseudogene 3 (SUMO1P3), a poorly characterized pseudogene, and the Urothelial Carcinoma Associated 1 (UCA1) as candidates to monitor the BC progression. Methods: This study was a retrospective trial enrolling NMIBC and MIBC patients undergoing surgical intervention: the expression of the lncRNA SUMO1P3 and UCA1 was evaluated in urine from 113 subjects (cases and controls). The receiver operating characteristic curve analysis was used to evaluate the performance of single or combined biomarkers in discriminating cases from controls. Results: SUMO1P3 and UCA1 expression in urine was able to significantly discriminate low grade NMIBC, healthy control and benign prostatic hyperplasia subjects versus high grade NMIBC and MIBC patients. We also demonstrated that miR-320a, which binds SUMO1P3, was reduced in high grade NMIBC and MIBC patients and the SUMO1P3/miR-320a ratio was used to differentiate cases versus controls, showing a statistically significant power. Finally, we provided an automated method of RNA extraction coupled to ddPCR analysis in a perspective of clinical application. Discussion: We have shown that the lncRNA SUMO1P3 is increased in urine from patients with high grade NMIBC and MIBC and that it is likely to be good candidate to predict bladder cancer progression if used alone or in combination with UCA1 or with miRNA320a.

Inhibition of phosphodiesterase 4 enhances clitoral and vaginal blood flow responses to dorsal clitoral nerve stimulation or PGE1 in anesthetized female rats.

INTRODUCTION.: Cyclic adenosine 3'5' monophosphate (cAMP) is produced by adenylate cyclase after activation by, e.g., vasoactive intestinal polypeptide or prostaglandin E1 (PGE1). The cAMP-degrading phosphodiesterase 4 (PDE4) is expressed in the vagina and clitoris, but no information is available on the functional role for PDE4-related signals in the female neurovascular genital response. AIM.: The aim of this study is to study the effect of inhibition of PDE4 with rolipram on nerve- and PGE1-induced vaginal and clitoral blood flow responses of rat. METHODS.: Measure of clitoral and vaginal blood flow and blood pressure in anesthetized rats during activation of the dorsal clitoral nerve (DCN) before and after intraperitoneal administration of rolipram or sildenafil (phosphodiesterase type 5 inhibitors [PDE5]) and nitro-L-arginine (L-NNA) (nitric oxide synthase inhibitor). Effect by topical administration of PGE1 on genital blood flow was also evaluated. MAIN OUTCOME MEASURE.: Blood flow was recorded as tissue perfusion units (TPU) by a Laser Doppler Flowmeter. Mean arterial blood pressure (MAP) was recorded (cmH2 O) in the carotid artery. Blood flow responses are expressed as TPU/MAP. Unpaired t-test and an analysis of variance were used. RESULTS.: Compared with control stimulations, rolipram (0.3 mg/kg) caused a twofold increase in peak blood flow (P < 0.05) and fourfold increase of the rate of clitoral blood flow during activation of the DCN (P < 0.05). Simultaneously, a twofold increase in peak blood flow and threefold increase in rate of blood flow were noted in the vagina (P < 0.05). Similar effects were noted for sildenafil (0.2 mg/kg) (P < 0.05). Inhibitory effects by L-NNA (60 mg/kg) on blood flow responses to DCN activation were significantly lower for rats treated with rolipram than with sildenafil (P < 0.05). PGE1-induced (10 µg) blood flow responses were significantly higher (P < 0.05) in rats treated with rolipram than with sildenafil. CONCLUSIONS.: These findings suggest that the cAMP/PDE4 system may be of similar functional importance as the nitric oxide/cyclic guanosine monophosphate/PDE5 pathway for neurovascular genital responses of the female rat.

Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism.

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (-50 ± 3%) and sphingosine 1-phosphate (S1P, -40 ± 4%), which ended up to reduction in cell motility (-46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

Mesenchymal stem cells expressing therapeutic genes induce autochthonous prostate tumour regression.

Mesenchymal stem cells (MSC) as vehicles of therapeutic genes represent a unique tool to activate drugs within a neoplastic mass due to their property to home and engraft into tumours. In particular, MSC expressing the cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) have been previously demonstrated to inhibit growth of subcutaneous prostate cancer xenografts thanks to their ability to convert the non-toxic 5-fluorocytosine into the antineoplastic 5-fluorouracil. Since both the immune system and the tumour microenvironment play a crucial role in directing cancer progression, in order to advance towards clinical applications, we tested the therapeutic potential of this approach on animal models that develop autochthonous prostate cancer and preserve an intact immune system. As cell vectors, we employed adipose-tissue and bone-marrow MSC. CD-MSC toxicity on murine prostate cancer cells and tumour tropism were verified in vitro and ex-vivo before starting the preclinical studies. Magnetic Resonance Imaging was utilised to follow orthotopic tumour progression. We demonstrated that intravenous injections of CD-MSC cells, followed by intraperitoneal administration of 5-fluorocytosine, caused tumour regression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which develops aggressive and spontaneous prostate cancer. These results add new insights to the therapeutic potential of specifically engineered MSC in prostate cancer disease.

Expression of fatty acid amide hydrolase (FAAH) in human, mouse, and rat urinary bladder and effects of FAAH inhibition on bladder function in awake rats.

BACKGROUND: Cannabinoid receptor (CB)-mediated functions may be involved in the regulation of bladder function, but information on endocannabinoid signals during micturition is scarce. OBJECTIVE: Investigate the expression of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in human, rat, and mouse bladders and study the effects of inhibition of FAAH during urodynamics in awake rats. DESIGN, SETTING, AND PARTICIPANTS: Bladder tissue from humans, mice, and rats was used for measurements. Female Sprague-Dawley rats were administered the FAAH inhibitor oleoyl ethyl amide (OEtA) or vehicle intravenously (IV) or intravesically (IVES) with or without rimonabant (CB1 antagonist) or SR144528 (CB2 antagonist). MEASUREMENTS: Real-time transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and cystometry in awake rats. RESULTS AND LIMITATIONS: Messenger RNA and protein for FAAH was expressed in the mucosa of human, mouse, and rat urinary bladders. Immunoreactivities for FAAH and CB2 were codistributed in rat and human urothelium. IV OEtA (0.3mg/kg) to rats increased intercontraction intervals (ICIs), micturition volume (MV), bladder capacity (BC), and threshold pressure (TP) by 17±1%, 16±1%, 17±1%, and 19±5%, respectively (all p<0.05 vs baseline). IVES OEtA (1 and 10mg/l) in rats dose-dependently increased (p<0.05 vs baseline) ICI (19±2% and 35±5%), MV (15±3% and 32±4%), BC (16±2% and 34±4%), and TP (15±1%, 21±3%). SR144528 (IVES 5mg/l) abolished all effects of OEtA, whereas rimonabant only counteracted effects of OEtA on TP. CONCLUSIONS: Bladder mucosa of all species expressed FAAH. Rat and human urothelium coexpressed FAAH and CB2. The FAAH inhibitor OEtA altered urodynamic parameters that reflect sensory functions of micturition in rats. Suggesting a role for the endocannabinoid system in bladder mechanoafferent functions of rats, effects of IVES OEtA were abolished by an IVES CB2 antagonist and partly counteracted by an IVES CB1 antagonist.

Neoadjuvant short-term intensive intravesical mitomycin C regimen compared with weekly schedule for low-grade recurrent non-muscle-invasive bladder cancer: preliminary results of a randomised phase 2 study.

BACKGROUND: The schedule for intravesical chemotherapy administration has not been definitively established in patients with low-grade recurrent non-muscle-invasive bladder cancer (NMIBC). OBJECTIVE: To assess both the feasibility and the efficacy of a short-term intensive schedule of neoadjuvant intravesical chemotherapy in patients with recurrent NMIBC. DESIGN, SETTING, AND PARTICIPANTS: A randomised phase 2 clinical study included 54 patients with recurrent NMIBC who were submitted to neoadjuvant chemotherapy intravesical instillations according to two different timing schedules. The study was performed at a tertiary care referral centre. INTERVENTION: Intravesical mitomycin C (MMC) 40 mg/40 ml was administered according to a schedule of either one instillation per week for 6 wk (group 1) or three instillations per week for 2 wk (group 2) prior to transurethral resection (TUR). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Local and systemic toxicity were investigated using the US National Cancer Institute's (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v.4.0 questionnaire at each instillation and the SF-36 questionnaire at randomisation and before TUR. A video-recorded cystoscopy and TUR were performed within 14 d after treatment completion. RESULTS AND LIMITATIONS: Groups 1 and 2 each were assigned 27 cases. Two patients (7.4%) in group 2 could not complete the scheduled treatment because of severe lower urinary tract symptoms. No statistically significant difference in SF-36 domain score was documented pre- and post-treatment between groups. Likewise, no statistically significant difference in treatment-related toxicity according to the CTCAE v.4 questionnaire was registered. Twelve patients (44.4%) in group 1 and 19 patients (70.4%) in group 2 (p=0.054) had complete tumour response. The small number of patients included represents the main limitation of the study. CONCLUSIONS: The intensive short-term schedule of neoadjuvant chemotherapy is safe and without additional toxicity compared with the weekly regimen. The increased ablative effect may be explained by the improved adherence of the scheduled timing to the duplication rate of tumour cells.

Beta-lactam antibiotic offers neuroprotection in a spinal muscular atrophy model by multiple mechanisms.

Spinal muscular atrophy (SMA) is a devastating genetic motoneuron disease leading to infant death. No effective therapy is currently available. It has been suggested that ß-lactam antibiotics such as ceftriaxone may offer neuroprotection in motoneuron diseases. Here, we investigate the therapeutic effect of ceftriaxone in a murine model of SMA. Treated animals present a modest, but significant ameliorated neuromuscular phenotype and increased survival, which correlate with protection of neuromuscular units. Whole gene expression profiling in treated mice demonstrates modifications in several genes including those involved in RNA metabolism toward wild-type. The neuroprotective effect seems to be mediated by multiple mechanisms that encompass the increase of the glutamate transporter Glt1, the transcription factor Nrf2, as well as SMN protein. This study provides the first evidence of a potential positive effect of this class of molecules in SMA. Further investigation of analogs with increased and more specific therapeutic effects warrants the development of useful therapies for SMA.