A Method to Isolate Pericytes From the Mouse Urinary Bladder for the Study of Diabetic Bladder Dysfunction / 대한배뇨장애요실금학회지

Purpose@#Pericytes surround the endothelial cells in microvessels and play a distinct role in controlling vascular permeability and maturation. The loss of pericyte function is known to be associated with diabetic retinopathy and erectile dysfunction. This study aimed to establish a technique for the isolation of pericytes from the mouse urinary bladder and an in vitro model that mimics in vivo diabetic bladder dysfunction. @*Methods@#To avoid contamination with epithelial cells, the urothelial layer was meticulously removed from the underlying submucosa and detrusor muscle layer. The tissues were cut into multiple pieces, and the fragmented tissues were settled by gravity into collagen I-coated culture plates. The cells were cultured under normal-glucose (5 mmol/L) or high-glucose (30 mmol/L) conditions, and tube formation, cell proliferation, and TUNEL assays were performed. We also performed hydroethidine staining to measure superoxide anion production. @*Results@#We successfully isolated high-purity pericytes from the mouse urinary bladder. The cells were positively stained for platelet-derived growth factor receptor-β and NG2 and negatively stained for smooth muscle cell markers (desmin and myosin) and an endothelial cell marker (CD31). The number of tubes formed and the number of proliferating cells were significantly lower when the pericytes were exposed to high-glucose conditions compared with normal-glucose conditions. In addition, there were significant increases in superoxide anion production and the number of apoptotic cells when the pericytes were cultured under high-glucose conditions. @*Conclusions@#To the best of our knowledge, this is the first study to isolate and culture pericytes from the mouse urinary bladder. Our model would be a useful tool for screening the efficacy of therapeutic candidates targeting pericyte function in diabetic bladder dysfunction and exploring the functional role of specific targets at the cellular level.

A Method to Isolate Pericytes From the Mouse Urinary Bladder for the Study of Diabetic Bladder Dysfunction / 대한배뇨장애요실금학회지

Purpose@#Pericytes surround the endothelial cells in microvessels and play a distinct role in controlling vascular permeability and maturation. The loss of pericyte function is known to be associated with diabetic retinopathy and erectile dysfunction. This study aimed to establish a technique for the isolation of pericytes from the mouse urinary bladder and an in vitro model that mimics in vivo diabetic bladder dysfunction. @*Methods@#To avoid contamination with epithelial cells, the urothelial layer was meticulously removed from the underlying submucosa and detrusor muscle layer. The tissues were cut into multiple pieces, and the fragmented tissues were settled by gravity into collagen I-coated culture plates. The cells were cultured under normal-glucose (5 mmol/L) or high-glucose (30 mmol/L) conditions, and tube formation, cell proliferation, and TUNEL assays were performed. We also performed hydroethidine staining to measure superoxide anion production. @*Results@#We successfully isolated high-purity pericytes from the mouse urinary bladder. The cells were positively stained for platelet-derived growth factor receptor-β and NG2 and negatively stained for smooth muscle cell markers (desmin and myosin) and an endothelial cell marker (CD31). The number of tubes formed and the number of proliferating cells were significantly lower when the pericytes were exposed to high-glucose conditions compared with normal-glucose conditions. In addition, there were significant increases in superoxide anion production and the number of apoptotic cells when the pericytes were cultured under high-glucose conditions. @*Conclusions@#To the best of our knowledge, this is the first study to isolate and culture pericytes from the mouse urinary bladder. Our model would be a useful tool for screening the efficacy of therapeutic candidates targeting pericyte function in diabetic bladder dysfunction and exploring the functional role of specific targets at the cellular level.

A Simple and Nonenzymatic Method to Isolate Human Corpus Cavernosum Endothelial Cells and Pericytes for the Study of Erectile Dysfunction

Species Distribution and Antifungal Susceptibility of Yeasts Isolated from Ear Specimens / 대한임상미생물학회지

BACKGROUND: Candida auris was first isolated from the ears of Japanese and Korean patients. However, the prevalence of yeast isolates from ear cultures and their antifungal susceptibility profiles in these nations remain unclear.METHODS: We assessed yeast isolates recovered from ear cultures from a university hospital in Korea over a 4-year period from January 2014 to December 2017. Species identification was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and/or sequence analysis. Antifungal minimal inhibitory concentrations (MICs) were determined using the broth microdilution method of the Clinical and Laboratory Standards Institute.RESULTS: Among 81 non-duplicate isolates from ear cultures, Cadida parapsilosis was the most frequently detected yeast species (34.6%), followed by C. auris (28.4%), Candida metapsilosis (9.9%), Candida orthopsilosis (8.6%), Candida albicans (7.4%), and others (11.1%). The MICs of the isolates were 0.125 to > 64 µg/mL, ≤0.03 to 4 µg/mL, 0.25 to 1 µg/mL, 0.125 to 1 µg/mL, and ≤0.03 to 2 µg/mL for fluconazole, voriconazole, amphotericin B, caspofungin, and micafungin, respectively. Of the 81 isolates, 44.4% (36/81) showed decreased susceptibility to fluconazole (MIC ≥4 µg/mL). Of the 23 C. auris isolates, 19 (82.6%) had a fluconazole MIC of ≥32 µg/mL. None of the isolates showed resistance to amphotericin B or echinocandins. Most of these patients suffered from chronic otitis media (84%).CONCLUSION: Candida parapsilosis complex and C. auris were the yeast species identified most frequently from ear cultures and they exhibited a high rate of fluconazole non-susceptibility, particularly C. auris.

Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque

PURPOSE: Epigenetic modifications, such as histone acetylation/deacetylation and DNA methylation, play a crucial role in the pathogenesis of inflammatory disorders and fibrotic diseases. The aim of this study was to study the differential gene expression of histone deacetylases (HDACs) in fibroblasts isolated from plaque tissue of Peyronie's disease (PD) or normal tunica albuginea (TA) and to examine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of HDAC7 in fibroblasts derived from human PD plaque. MATERIALS AND METHODS: For differential gene expression study, we performed reverse-transcriptase polymerase chain reaction for HDAC isoforms (1–11) in fibroblasts isolated from PD plaque or normal TA. Fibroblasts isolated from PD plaque were pretreated with HDAC7 siRNA (100 pmol) and then stimulated with transforming growth factor-β1 (TGF-β1, 10 ng/mL). Protein was extracted from treated fibroblasts for Western blotting. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 and myofibroblastic differentiation. RESULTS: The mRNA expression of HDAC2, 3, 4, 5, 7, 8, 10, and 11 was higher in fibroblasts isolated from PD plaque than in fibroblasts isolated from normal TA tissue. Knockdown of HDAC7 in PD fibroblasts inhibited TGF-β1-induced nuclear shuttle of Smad2 and Smad3, transdifferentiation of fibroblasts into myofibroblasts, and abrogated TGF-β1-induced production of extracellular matrix protein. CONCLUSIONS: These findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetic therapy for PD.

Comparison of the Bruker Biotyper and VITEK MS Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Systems Using a Formic Acid Extraction Method to Identify Common and Uncommon Yeast Isolates

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows rapid and accurate identification of clinical yeast isolates. In-tube formic acid/acetonitrile (FA/ACN) extraction is recommended prior to the analysis with MALDI Biotyper, but the direct on-plate FA extraction is simpler. We compared the Biotyper with the VITEK MS for the identification of various clinically relevant yeast species, focusing on the use of the FA extraction method. METHODS: We analyzed 309 clinical isolates of 42 yeast species (four common Candida species, Cryptococcus neoformans, and 37 uncommon yeast species) using the Biotyper and VITEK MS systems. FA extraction was used initially for all isolates. If ‘no identification' result was obtained following the initial FA extraction, these samples were then retested by using FA (both systems, additive FA) or FA/ACN (Biotyper only, additive FA/ACN) extraction. These results were compared with those obtained by sequence-based identification. RESULTS: Both systems correctly identified all 158 isolates of the four common Candida species after the initial FA extraction. The Biotyper correctly identified 8.7%, 30.4%, and 100% of 23 C. neoformans isolates after performing initial FA, additive FA, and FA/ACN extractions, respectively, while VITEK MS identified all C. neoformans isolates after the initial FA extraction. Both systems had comparable identification rates of 37 uncommon yeast species (128 isolates), following the initial FA (Biotyper, 74.2%; VITEK MS, 73.4%) or additive FA (Biotyper, 82.0%; VITEK MS, 73.4%). CONCLUSIONS: The identification rate of most common and uncommon yeast isolates is comparable between simple FA extraction/Biotyper method and VITEK MS methods, but FA/ACN extraction is necessary for C. neoformans identification by Biotyper.

Galangin Suppresses Pro-Inflammatory Gene Expression in Polyinosinic-Polycytidylic Acid-Stimulated Microglial Cells

Galangin (3,5,7-trihydroxyflavone) is a polyphenolic compound abundant in honey and medicinal herbs, such as Alpinia officinarum. In this study, we investigated the anti-inflammatory effects of galangin under in vitro and in vivo neuroinflammatory conditions caused by polyinosinic-polycytidylic acid (poly(I:C)), a viral mimic dsRNA analog. Galangin suppressed the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines in poly(I:C)-stimulated BV2 microglia. On the other hand, galangin enhanced anti-inflammatory interleukin (IL)-10 production. Galangin also suppressed the expression of pro-inflammatory markers in poly(I:C)-injected mouse brains. Further mechanistic studies showed that galangin inhibited poly(I:C)-induced nuclear factor (NF)-κB activity and phosphorylation of Akt without affecting MAP kinases. Interestingly, galangin increased the expression and transcriptional activity of peroxisome proliferator-activated receptor (PPAR)-γ, known to play an anti-inflammatory role. To investigate whether PPAR-γ is involved in the anti-inflammatory function of galangin, BV2 cells were pre-treated with PPAR-γ antagonist before treatment of galangin. We found that PPAR-γ antagonist significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-α and IL-6 in poly(I:C)-stimulated microglia. In conclusion, our data suggest that PI3K/Akt, NF-κB, and PPAR-γ play a pivotal role in mediating the anti-inflammatory effects of galangin in poly(I:C)-stimulated microglia.

Galangin Suppresses Pro-Inflammatory Gene Expression in Polyinosinic-Polycytidylic Acid-Stimulated Microglial Cells

Galangin (3,5,7-trihydroxyflavone) is a polyphenolic compound abundant in honey and medicinal herbs, such as Alpinia officinarum. In this study, we investigated the anti-inflammatory effects of galangin under in vitro and in vivo neuroinflammatory conditions caused by polyinosinic-polycytidylic acid (poly(I:C)), a viral mimic dsRNA analog. Galangin suppressed the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines in poly(I:C)-stimulated BV2 microglia. On the other hand, galangin enhanced anti-inflammatory interleukin (IL)-10 production. Galangin also suppressed the expression of pro-inflammatory markers in poly(I:C)-injected mouse brains. Further mechanistic studies showed that galangin inhibited poly(I:C)-induced nuclear factor (NF)-κB activity and phosphorylation of Akt without affecting MAP kinases. Interestingly, galangin increased the expression and transcriptional activity of peroxisome proliferator-activated receptor (PPAR)-γ, known to play an anti-inflammatory role. To investigate whether PPAR-γ is involved in the anti-inflammatory function of galangin, BV2 cells were pre-treated with PPAR-γ antagonist before treatment of galangin. We found that PPAR-γ antagonist significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-α and IL-6 in poly(I:C)-stimulated microglia. In conclusion, our data suggest that PI3K/Akt, NF-κB, and PPAR-γ play a pivotal role in mediating the anti-inflammatory effects of galangin in poly(I:C)-stimulated microglia.

Optimizing in vivo gene transfer into mouse corpus cavernosum by use of surface electroporation

PURPOSE: Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum. MATERIALS AND METHODS: Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 microg/40 microL), different electroporation settings (5-50 V, 8-16 pulses with a duration of 40-100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue. RESULTS: Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis. CONCLUSIONS: We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.

Molecular Identification and Amphotericin B Susceptibility Testing of Clinical Isolates of Aspergillus From 11 Hospitals in Korea

BACKGROUND: We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. METHODS: A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and beta-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. RESULTS: ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by beta-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of > or =2 microg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was < or =75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). CONCLUSIONS: Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance.

Juxtacortical Spots on Fluid-Attenuated Inversion Recovery Images in Cryptogenic Transient Ischemic Attack

BACKGROUND AND PURPOSE: Juxtacortical spots are detected frequently on fluid-attenuated inversion recovery (FLAIR) images, but have not been extensively researched in patients with transient ischemic attack (TIA). We hypothesized that juxtacortical spots on FLAIR images are partly associated with right-to-left shunt (RLS) in TIA without clear etiology. The possibility of an association between the presence of RLS and juxtacortical spots on FLAIR images in patients with TIA without clear etiology was investigated, and the imaging findings of patients with and without RLS were compared. METHODS: This was a retrospective study of TIA patients who visited our tertiary stroke center consecutively within 72 hours of TIA onset. Cryptogenic TIA was defined as no clear etiology despite a routine diagnostic workup. The presence of RLS was examined by transcranial Doppler with an agitated saline test or transesophageal echocardiography. Juxtacortical spots were defined as small and round hyperintensities in the juxtacortex on FLAIR images, excluding white-matter hyperintensities. RESULTS: Of the 132 patients with cryptogenic TIA examined for this study, 70 (53.0%) had RLS. Juxtacortical spots on FLAIR images were detected more frequently in patients with RLS than in those without. The independent factors for the presence of juxtacortical spots were RLS [odds ratio (OR)=3.802, 95% confidence interval (95% CI)=1.74-8.2; p=0.001] and age (OR=1.058, 95% CI=1.01-1.10; p=0.004) by multivariate analysis. The number of juxtacortical spots was significantly higher among patients with a moderate-to-large RLS than in those with a small or no RLS. CONCLUSIONS: The findings of the present study demonstrate a significant association between the presence of RLS and the occurrence of juxtacortical spots on FLAIR images in patients with cryptogenic TIA.