Fernando de Miguel

Sustained beneficial effects of intraprostatic botulinum toxin type A on lower urinary tract symptoms and quality of life in men with benign prostatic hyperplasia

To present a comprehensive experience with intraprostatic botulinum toxin‐type A (BoNT‐A) injection in men with symptomatic benign prostatic hyperplasia (BPH) and to assess the efficacy on lower urinary tract symptoms (LUTS) and quality of life (QoL).

Stat3 enhances transactivation of steroid hormone receptors

BackgroundSteroid hormone receptors (SHRs) are members of the superfamily of ligand-activated transcription factors that regulate many biological processes. Co-regulators act as bridging molecules between the SHR and general transcription factors to enhance transactivation of target genes. Previous studies demonstrated that Stat3 is constitutively activated in prostate cancer and can enhance prostate specific antigen (PSA) expression and promote androgen independent growth. In this study, we investigate whether Stat3 can enhance steroid hormone receptors activation.MethodsCV-1 cells in which plasmids expressing androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR) or estrogen receptor (ER) were cotransfected with a constitutively active STAT3 mutant.ResultsStat3 stimulates the transcriptional activity of all four SHR tested, AR, GR, PR and ER, in a hormone-dependent manner. Stat3 acts in a synergistic fashion with other coactivators such as SRC-1, pCAF, CBP, and TIF-2 on the transcriptional activity of these SHR. In addition, Stat3 significantly enhanced the sensitivity of androgen receptor in response to androgen. STAT3 did not affect the specificity of AR for other steroid hormones other than androgen or binding of AR to other hormone responsive elements.ConclusionsThese findings suggest that Stat3 can enhance the transactivation of AR, GR, PR and ER, and activated Stat3 could have a role in the development or progression of a hypersensitive AR.

Green onions : Potential mechanism for hepatitis A contamination

The largest documented foodborne hepatitis A outbreak in U.S. history occurred in November 2003. The source of that outbreak was green onions from a farm in Mexico. Two biomarkers were used to determine ways in which hepatitis A virus (HAV) can contaminate onions. Fluorescent microspheres (1.0 to 10 microm) and HAV vaccine were placed on the soil and the surfaces of pot-grown onions and in the liquid medium of hydroponically cultivated onions. Reverse transcription PCR (RT-PCR) was used to identify HAV RNA. Microspheres were found on the outside and inside of the pot-grown onions for up to 60 days. RT-PCR revealed HAV RNA from the vaccine in well-washed green onions. In the hydroponically grown onions, microspheres were found throughout the onion after only 1 day. RT-PCR also revealed HAV RNA inside the hydroponically grown onions. Both biomarkers support the hypothesis that HAV can contaminate the inside of the growing onion and can be taken up intracellularly through the roots. Once inside, the particles are impossible to remove by cleaning.

Intravesical instillation of human urine after oral administration of trospium, tolterodine and oxybutynin in a rat model of detrusor overactivity

To study the effects of antimuscarinics excreted into human urine on normal bladder in a rat model of detrusor overactivity.

Functional and immunohistochemical characterization of CB1 and CB2 receptors in rat bladder.

OBJECTIVES To determined the localization of CB(1) and CB(2) receptors in rat bladder and investigate the effect of a mixed CB(1)/CB(2) receptor agonist, ajulemic acid (AJA), on chemically evoked release of the sensory neuropeptide calcitonin gene-related peptide (CGRP). METHODS Whole rat bladders were incubated in a series of tissue baths containing physiologic salt solution to measure baseline CGRP release by enzyme immunoassay. Capsaicin (30 nM) and adenosine triphosphate (10 muM) were used to provoke CGRP release in the presence or absence of AJA. Specificity of AJA for CB(1) and CB(2) receptors was determined using antagonists. Localization was determined by immunofluorescence for CB(1) and CB(2) receptors in fixed bladders. RESULTS Immunofluorescence showed the localization of CB(1) and CB(2) receptors in the bladder. Mean baseline CGRP release was 605 +/- 62 pg/g of bladder weight, and AJA had no effect on CGRP release. The addition of adenosine triphosphate/capsaicin significantly increased the CGRP release over baseline, by 44% (P < .05), and AJA application significantly decreased CGRP release, by 29% compared with controls (P < .05). The CB(1) and CB(2) antagonists AM 251 and AM 630, respectively, reversed the blunting effect of AJA on evoked CGRP release, resulting in an increase of 40% and 38% over baseline, respectively. CONCLUSIONS CB(1) and CB(2) receptors are localized in the urothelium of rat bladder, and application of AJA inhibits the evoked release of CGRP by acting on CB(1) and CB(2) receptors. These findings identify a potential new pathway for study in the evaluation and treatment of painful bladder syndrome/interstitial cystitis.

Roles of Peripheral and Central Nicotinic Receptors in the Micturition Reflex in Rats

PURPOSE We investigated the effects of nicotinic acetylcholine receptor activation in the bladder and central nervous system on the micturition reflex in urethane anesthetized rats. MATERIALS AND METHODS The effects of nicotinic acetylcholine receptor activation on bladder activity were examined during continuous infusion cystometrogram. Nicotine with or without the nicotinic acetylcholine receptor antagonist mecamylamine (Sigma Chemical Co., St. Louis, Missouri) was administered intravesically, intrathecally or intracerebroventricularly in normal or capsaicin pretreated rats. We also examined nicotine induced responses in dissociated bladder afferent neurons from L6 to S1 dorsal root ganglia that were sensitive to capsaicin using whole cell patch clamp recordings. RESULTS Intravesical nicotine (1 to 10 mM) significantly decreased intercontraction intervals in dose dependent fashion. This excitatory effect was abolished by co-application of mecamylamine (3 mM) as well as by capsaicin pretreatment. On patch clamp recordings 300 muM nicotine evoked rapid inward currents that were antagonized by mecamylamine in capsaicin sensitive bladder afferent neurons. Intrathecal and intracerebroventricular administration of nicotine (10 mug) decreased and increase intercontraction intervals, respectively. Each effect was antagonized by mecamylamine (50 mug) administered intrathecally and intracerebroventricularly. The spinal excitatory effect was significantly inhibited by the N-methyl-D-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (20 mug) given intrathecally or by capsaicin pretreatment, although the effects of capsaicin pretreatment were significantly smaller than those of (+)-MK-801 hydrogen maleate. CONCLUSIONS These results indicate that nicotinic acetylcholine receptor activation in capsaicin sensitive C-fiber afferents in the bladder can induce detrusor overactivity. In the central nervous system nicotinic acetylcholine receptor activation in the spinal cord and brain has an excitatory and an inhibitory effect on the micturition reflex, respectively. In addition, the nicotine induced spinal excitatory effect may be mediated by the activation of glutamatergic mechanisms.

Human muscle-derived cell injection in a rat model of stress urinary incontinence

We investigated the use of human muscle‐derived cells (hMDCs) for the treatment of stress urinary incontinence (SUI) in a nude rat model. hMDCs were isolated from adult skeletal muscle. Three groups of six animals consisting of controls, animals undergoing sciatic nerve transection (SNT) with periurethral sham‐injection, and SNT with hMDCs (1 × 106 cells/20 μl saline) were utilized. Leak point pressure (LPP) was measured 4 weeks following injection. Bilateral SNT resulted in a significantly lower LPP that was significantly higher following hMDCs than sham injection. The results demonstrate the efficacy of human muscle cell therapy alone in improving physiologic outcomes in an animal model of SUI. Muscle Nerve, 2007

Proteomic investigation on chronic bladder irritation in the rat.

OBJECTIVES Interstitial cystitis (IC) is a painful bladder syndrome associated with urinary frequency and urgency. Elusive cause of IC makes its diagnosis only possible by exclusion in many cases. In this study, we used proteomics for identifying disease-associated proteins in a rat model of chronic bladder irritation. METHODS Chronic irritation of the rat bladder was caused by a brief (90 seconds) intravesical instillation of 0.2 mL of 0.4 N HCl. Whole bladders were collected at different time points after treatment, snap frozen, and nuclear and cytosolic protein extracts were obtained. Samples were resolved in standard 2-dimensional (2D) gels stained with an improved Coomasie stain or by differential gel electrophoresis (DIGE). Differentially expressed spots were excised and identified by MALDI-TOF MS/MS. Histologic and Western blot analyses were also performed. RESULTS Bladder morphology and histologic appearance of bladder sections after HCl treatment reflected hemorrhage, edema, epithelial denudation, detrusor mastocytosis, and eosinophilia. Proteomic analysis of irritated rat bladder revealed marked overexpression of 4 nuclear proteins and marked underexpression of 1 nuclear protein compared with normal rat bladders. Among these proteins, inflammation-associated calgranulin A (over) and smooth muscle protein-22/transgelin (under) showed opposed expression patterns after bladder irritation. CONCLUSIONS Presence of mast cells and eosinophils and overexpression of calgranulin A confirm the inflammatory component of HCl-irritated bladder. Altered expression of nuclear proteins is of particular interest because of their possible role as a prognostic marker in inflammatory bladder disorders. However, more studies are needed before clinical application of these findings can be established.

In Vitro and In Vivo Effect of Lidocaine on Rat Muscle-Derived Cells for Treatment of Stress Urinary Incontinence

OBJECTIVES Lidocaine cytotoxicity has been reported in some cell types, which could affect its use as a local anesthetic in cell-based therapy. We evaluated the in vitro and in vivo effect of lidocaine on rat muscle-derived progenitor cells (MDCs). METHODS MDCs were isolated from rat skeletal muscle and purified using the preplate technique. For in vitro tests, the MDCs underwent either 2 hours of, or continuous, exposure to lidocaine (50 microM-5 mM). After 72 hours of incubation, cell viability was measured using the methylthiazololetetrazolium assay. For the in vivo tests, periurethral injection of either phosphate-buffered saline, MDCs (1 x 10(6) cells/20 microL), or 2% lidocaine plus MDCs was performed in pudendal nerve-transected rats. The leak point pressure (LPP) was measured at 4 weeks after the injection. RESULTS Lidocaine concentrations of < or = 500 microM had no effect on MDCs with continuous exposure. MDCs in 1 mM lidocaine showed decreased survival and no MDCs in 5 mM lidocaine survived. With a 2-hour exposure, only MDCs in the 5-mM lidocaine group showed decreased survival. Rats with nerve transection and phosphate-buffered saline injection showed significantly lower LPPs than the controls. The LPP was restored to a significantly greater level after MDCs only or lidocaine plus MDC injection. No statistically significant difference in LPP restoration was found between the MDC-only and lidocaine plus MDC injections. CONCLUSIONS Cytotoxicity to lidocaine was minimal at a physiologic concentration in vitro. The functional recovery of LPP by MDC treatment was not affected by lidocaine preinfiltration. Taken together, our data indicate that lidocaine can be applied as a local anesthetic in periurethral MDC injection without decreasing the efficacy of the therapy.

Gene Therapy for Neurogenic Erectile Dysfunction

Neurogenic erectile dysfunction (ED) is one of major causes of ED and is often difficult to treat. In this review, we report significant improvements of ED caused by diabetes mellitus (DM) or cavernous nerve injury by using a gene therapy approach in rat models of ED. Herpes simplex virus (HSV) vectors were used to deliver neurotrophic factors, such as neurotrophin 3 (NT3), glial cell line‐derived neurotrophic factor (GDNF) and neurturin (NTN), into cavernous nerves. The retrograde transport of HSV vector‐mediated neurotrophic factors to pelvic ganglion neurons occurred after the administration of the vector around the cavernous nerves. HSV vector administration around the cavernous nerves can improve DM‐induced ED and promote the recovery of erectile function after cavernous nerve injury. The HSV vector‐mediated delivery of neurotrophic factors could be applicable for the treatment of neurogenic ED. NT3, GDNF, and NTN would be the possible factors that could be used for this gene therapy application.