Ulrich Schwantes

Expression of nicotinic acetylcholine receptor subunit mRNA in mouse bladder afferent neurons

Nicotinic acetylcholine receptors (nAChR) influence bladder afferent activity and reflex sensitivity, and have been suggested as potential targets for treating detrusor overactivity. Mechanisms may include indirect effects, e.g. involving the urothelium, and direct action on nAChR expressed by afferent neurons. Here we determined the nAChR repertoire of bladder afferent neurons by retrograde neuronal tracing and laser-assisted microdissection/reverse transcriptase polymerase chain reaction (RT-PCR), and quantified retrogradely labelled nAChRα3-subunit-expressing neurons by immunohistochemistry in nAChR α3β4α5 cluster enhanced green fluorescent protein (eGFP) reporter mice. Bladder afferents distinctly expressed mRNAs encoding for nAChR-subunits α3, α6, α7, β2-4, and weakly α4. Based upon known combinatorial patterns of subunits, this predicts the expression of at least three basically different subunits of nAChR - α3(∗), α6(∗) and α7(∗) - and of additional combinations with β-subunits and α5. Bladder afferents were of all sizes, and their majority (69%; n=1367) were eGFP-nAChRα3 positive. Immunofluorescence revealed immunoreactivities to neurofilament 68 (NF68), transient receptor potential cation channel vanilloid 1 (TRPV1), substance P (SP) and calcitonin gene-related peptide (CGRP) in eGFP-nAChRα3-positive and -negative neurons. For each antigen, all possible combinations of colocalisation with eGFP-nAChRα3 were observed, with eGFP-nAChRα3-positive bladder neurons without additional immunoreactivity being most numerous, followed by triple-labelled neurons. In conclusion, more than one population of bladder afferent neurons expresses nAChR, indicating that peripheral nicotinic initiation and modulation of bladder reflexes might result, in addition to indirect effects, from the direct activation of sensory terminals. The expression of multiple nAChR subunits offers the potential of selectively addressing functional aspects and/or sensory neuron subpopulations.

Refractory overactive bladder: a common problem?

Introduction and hypothesisUnsatisfactory treatment outcome sometimes is described as frequently occurring in patients treated with first-line therapy for overactive bladder (OAB). The present article reviews the different circumstances which may result in failure to respond to lifestyle interventions, behavioral therapy, and/or antimuscarinic treatment.MethodsAn extensive literature search was conducted to identify relevant articles on pathophysiological, clinical, and pharmacological aspects of refractory OAB.ResultsMissing definition, unrealistic individual expectation of treatment outcomes, lack of communication between physician and patient as well as pathophysiological and pharmacological processes were identified as relevant for failure to respond to first-line OAB treatment. Increase of patient’s motivation to adhere to the prescribed treatment, critical examination of the patient in regard to the initial diagnosis, and individual adjustment of antimuscarinic therapy may be appropriate tools to improve treatment outcome in OAB patients.ConclusionsOverall, the incidence of refractory OAB seems to be overestimated. There are several approaches to improve therapy results.

In vitro susceptibility testing of yeasts to nystatin – low minimum inhibitory concentrations suggest no indication of in vitro resistance of Candida albicans, Candida species or non-Candida yeast species to nystatin

In total, 14 yeast strains originating from patients with dermatomycoses, and 7 control strains (isolates from strain collections and collaborative ring trials) were investigated regarding their in vitro susceptibility to the polyene antifungal agent nystatin. Testing was performed using a broth microdilution assay based on the standardized method of susceptibility testing of yeasts per EUCAST (The European Committee on Antimicrobial Susceptibility Testing). Minimum inhibitory concentrations (MIC) for nystatin were measured. The reading of the MIC values was performed by both visual examination, and spectrophotometric measuring after 24 and 48 hours’ incubation time at 36°C. The visual read-out of growth inhibition revealed MICs for nystatin in a range from 3.7 to 7.4 IU/mL (0.625 to 1.25 μg/mL) for all Candida species tested. One of the Candida (C.) albicans strains, and both strains of C. glabrata and C. tropicalis, showed low MIC values of 3.7 IU/mL (0.625 μg/mL). Geotrichum candidum and Trichosporon mucoides were also inhibited by nystatin. The control strains (C. albicans, C. glabrata, C. parapsilosis, C. krusei and C. tropicalis) confirmed the values which were found for the wild strains. The spectrophotometric measuring of the turbidimetry revealed slightly lower MIC values for Candida species. Spectrophotometric measurment of Geotrichum candidum and Trichosporon mucoides was unsuccessful or not possible; however, visual reading of the results was carried out effectively. Nystatin showed very good in vitro activity against these non-Candida yeast species. In conclusion, very good in vitro activity of nystatin against all tested yeast strains could be detected. The in vitro efficacy was independent of the origin of the strains, as both the wild strains isolated from patients in this study, and the control strains originating from strain collections, were inhibited. Correspondence to: Dr. Pietro Nenoff, Dermatology, Laboratory Medicine, Allergy & Andrology Specialist; Main Focus: Tropical and Travel Dermatology (DDA), Mölbiser Hauptstraße 8, 04571 Rötha/OT Mölbis, Germany; Tel: +4934347-50 323; Fax +49-34347-50 123; E-mail: pietro.nenoff@gmx.de Received: October 31, 2016; Accepted: November 17, 2016; Published: November 22, 2016 Introduction The common use of azole antifungal agents, in particular oral fluconazole, but also topical azoles, for the treatment of cutaneous and oral candidiasis, and possibly due to the use of azole fungicides in agricultural crop protection, has led to the emergence of azole and echinocandine resistance of clinical isolates of Candida (C.) albicans and other Candida species [1]. An alternative therapeutic approach is the use of topical polyene antifungals such as amphotericin B or nystatin. Only limited current data exists regarding Candida species which are resistant to polyene antifungals. Recently, a total of 201 clinical C. albicans isolates from Turkey investigated by Etest were found to be susceptible to amphotericin B [2]. A similar situation can be found for nystatin. However, in vitro susceptibility testing studies have shown a low percentage of Candida species isolated from HIV positive patients to be resistant to nystatin [3]. In this study, a total of 14 currently isolated yeast strains from dermatomycosis patients, and seven reference strains (controls), including isolates from the DSMZ strain collection (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and inter-laboratory ring tests of INSTAND e.V. (the German Society for Promoting Quality Assurance in Medical Laboratories, Düsseldorf, Germany), were tested for their in vitro susceptibility to the polyene antifungal agent nystatin. A broth microdilution assay which corresponded to the method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and allowed for determination of the minimal inhibitory concentration (MIC) of nystatin, was used for this purpose. Materials and methods