Jae-Jun Song

Effect of diesel exhaust particles on human middle ear epithelial cells

OBJECTIVEnIn the present study, we investigate whether diesel exhaust particles (DEPs) cause cytotoxicity and induce inflammation or increase the expression of mucin in immortalized human middle ear epithelial cell lines (HMEECs). Several publications have shown an association between traffic-related air pollutants and otitis media. Additionally, DEP have been shown to cause inflammation and an allergic response in the airways.nnnMETHODSnCell viability following DEP treatment was investigated in HMEECs using the MTT assay. We measured the expression of the inflammatory cytokines TNF-α and COX-2 and the mucin genes MUC5AC and MUC5B using semiquantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.nnnRESULTSnCell viability tests showed that exposure to more than 80 μg/mL of DEP caused a decrease in cell viability. DEP exposure also increased the expression of MUC5AC, but did not induce the expression of MUC5B in HMEECs.nnnCONCLUSIONnDEP decreased cell viability, induced an inflammatory response, and increased mucin gene expression in HMEECs. These findings support the hypothesis that environmental diesel exposure is a risk factor for otitis media.

Tracheal reconstruction by mesenchymal stem cells with small intestine submucosa in rabbits

AIMnThe increasing number of newborns requiring intubation and artificial ventilation in the sophisticated premature and intensive care units of recent years has been followed by a concomitant increase in the number of children who develop tracheal stenosis as a sequela of prolonged intubation, with a consequent increasing need for tracheal surgical repair. The aim of this study was to evaluate tracheal reconstruction by monolayered autologous mesenchymal stem cells (MSCs) with small intestine submucosa (SIS) in a rabbit model.nnnMETHODSnTwelve male rabbits were randomly divided into three groups: rabbits with tracheal defects without reconstruction (untreated group, n=4), rabbits with tracheal defects given porcine small intestine submucosa graft (SIS group, n=4), and rabbits with tracheal defects that underwent transplantation of monolayered mesenchymal stem cells on SIS (SIS+MSC group, n=4). Histological and endoscopic analyses were performed by hematoxylin-eosin staining (H&E), Prussian blue staining and endoscopy.nnnRESULTSnTracheal stenosis in the SIS+MSC group was minimal, compared to the untreated group and SIS group. Specimens obtained from the untreated and SIS groups showed severe infiltration of inflammatory cells and granulation tissue formation into the trachea. In the SIS+MSC group, however, minimal infiltration of the inflammatory cells and granulation tissue formation were observed. Twelve weeks following the operation, regeneration of pseudostratified columnar epithelium was confirmed by H&E staining with minimal inflammatory cell infiltration in the SIS+MSC group. Moreover, Prussian blue staining clearly demonstrated the presence of labeled MSCs in the regenerated tissue of SIS+MSC group.nnnCONCLUSIONSnThese results demonstrate that tracheal reconstruction by MSCs with SIS is effective in rabbits with tracheal defects with minimal mortality and morbidity, which appears to be a promising strategy in the treatment of tracheal defects.

Protective effect of NecroX, a novel necroptosis inhibitor, on gentamicin-induced ototoxicity

INTRODUCTIONnNecroX is a novel necrosis and necroptosis inhibitor that shows scavenger activity against mitochondrial reactive oxygen species (ROS) and cytoprotective activity against various insults. These findings raise the possibility of its protective effect in ototoxicity. This study was performed to investigate the protective effect of NecroX on gentamicin (GM)-induced hair cell loss in neonatal mouse cochlea cultures.nnnMATERIALS AND METHODSnThe protective effects of NecroX were measured by phalloidin staining of cultures from postnatal day 2-3 mice with GM-induced hair cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect apoptosis. The radical-scavenging activity of NecroX was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.nnnRESULTSnNecroX showed a significant and concentration-dependent protective effect against GM-induced hair cell loss, and hair cells retained their stereocilia well. NecroX decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. Additionally, NecroX showed direct radical scavenging activity in the DPPH assay.nnnCONCLUSIONSnIn this study, we demonstrated the protective effect of NecroX on GM-induced hair cell loss in neonatal cochlea cultures, and suggest that it may be of therapeutic use in the treatment of drug-induced ototoxicity.

Gene expression profile of early in vitro biofilms of Streptococcus pneumoniae

In this study, the gene expression profile of early in vitro Streptococcus pneumoniae biofilm with respect to planktonic cells in cDNA microarray analysis is reported. Microarray analysis with respect to planktonic cells was performed on total RNA extracted from biofilms grown in 24‐well microtiter plates. To validate the microarray results, real‐time RT‐PCR was performed on 13 differentially expressed genes and one constitutively expressed gene. The cDNA‐microarray analyses identified 89 genes that were significantly differentially expressed in biofilm and planktonic cells. Genes involved in isoprenoid biosynthesis, cell wall biosynthesis, translation and purine and pyrimidine nucleotide metabolic pathways were exclusively expressed in the biofilms, whereas transcription regulator genes were exclusively expressed in planktonic cells. The real‐time RT‐PCR results of 13 differentially regulated genes were completely in agreement with the microarray data. The exclusive up regulation in biofilms of genes involved in the mevalonate pathway, cell wall biosynthesis, translation and purine and pyrimidine nucleotide metabolic pathways suggests that expression of these genes may be required for initial biofilm formation, and growth and survival of bacteria in biofilms. The up regulation of related genes suggests that cells in biofilms may be under stress conditions and possibly actively involved in the protein synthesis required to adapt to a new environment.

Guggulsterone suppresses LPS induced inflammation of human middle ear epithelial cells (HMEEC)

OBJECTIVEnGuggulsterone is a bioactive constituent of resinous sap originating from the guggul tree, Commiphora mukul, which has been used over several thousands of years to treat various diseases, including atherosclerosis, rheumatism, and obesity. However, the effect of guggulsterone inflammatory reactions induced by lipopolysaccharide (LSP) is not known. The aim of this study was to evaluate the anti-inflammatory effect of guggulsterone on cultured human middle ear epithelial cells (HMEEC).nnnMETHODSnThe effect of guggulsterone on LPS-induced tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2) expression was evaluated in HMEEC by real-time reverse transcription polymerase chain reaction (RT-PCR). LPS-induced COX-2 production and degradation of the inhibitor kB-alpha (IkB-α) were determined by Western blot analysis.nnnRESULTSnGuggulsterone significantly inhibited LPS-induced upregulation of TNF-α and COX-2 in a dose-dependent manner. COX-2 protein production by LPS was significantly suppressed by the guggulsterone pretreatment. Furthermore, LPS-induced IkB-α degradation was suppressed by the guggulsterone pretreatment.nnnCONCLUSIONSnThese results show that the guggulsterone has inhibitory effect on TNF-α expression and COX-2 production and it may be mediated through its inhibition of nuclear factor-kB activation. Our findings provide an insight into the molecular mechanisms underlying the anti-inflammatory activities of guggulsterone in relationship to otitis media.

Microarray analysis of microRNA expression in LPS induced inflammation of human middle ear epithelial cells (HMEECs)

OBJECTIVESnThe primary aim of this study was to reveal the relationship between inflammatory response of human middle ear epithelial cell (HMEEC) and microRNA (miRNA).nnnMETHODSnIn experimental group, cells were treated with lipopolysaccharide (LPS) for 2 h. No LPS was treated in the control group. Total RNA was extractedand used for miRNA microarray analysis. The predicted targets of miRNA with significant change were obtained using miRBase. To assess the function of predicted target gene lists, we evaluated the frequency of specific gene ontology (GO) terms among the predicted target genes of the miRNA with significant change using DAVID (Database for Annotation, Visualization and Integrated Discovery).nnnRESULTSnAfter normalization, the number of the differentially expressed genes was 15. Among them, 5 miRNAs were up-regulated and 10 miRNAs were down-regulated in LPS group compared with control group. The most enriched GO terms in the predicted target genes of miRNA with increased expression were developmental process, response to biotic stimulus, acute inflammatory response, and regulation of cell growth. The most enriched GO terms in the predicted target genes of miRNA with decreased expression were developmental process, cell differentiation, endocytosis, cell communication, IκB kinase/NFκB cascade, complement activation, innate immune response and cell adhesion.nnnCONCLUSIONSnIn conclusion, we identified the differentially expressed miRNA in LPS induced acute inflammation of HMEECs whose expression profile may provide a useful clue for the understanding of pathophysiology of otitis media. Our work indicates that miRNA play important role in the pathogenesis of otitis media.

Mucosal expression of ENaC and AQP in experimental otitis media induced by Eustachian tube obstruction

OBJECTIVEnWe investigated the expression of the epithelial sodium channel (ENaC) and aquaporins (AQPs) in the middle ear mucosa of a rat model of otitis media with effusion caused by surgical obstruction of the Eustachian tube.nnnMETHODSnSixty-four rats were randomly assigned to either undergo unilateral Eustachian tube obstruction (groups 1, 2, and 3) or to undergo no procedure (control group). Bony Eustachian tubes were approached through ventral incisions and obstructed with electrocautery. On days 14, 28, and 56, the ears were evaluated, and the rats were sacrificed for otoscopic evaluation and real-time RT-PCR. Immunohistochemistry was done for ENaC-alpha and AQP-1.nnnRESULTSnThe level of ENaC-alpha expression decreased 0.28- and 0.73-fold at 2 and 4 weeks, respectively, but increased 1.48-fold at 8 weeks (p<0.05). The change in ENaC-beta expression at 2 weeks was insignificant. However, the level of ENaC-beta expression increased 3.17- and 7.85-fold at 4 and 8 weeks, respectively (p<0.05). The level of ENaC-gamma expression increased 1.51-, 4.82- and 14.79-fold at 2, 4 and 8 weeks, respectively (p<0.05). The level of AQP-1 expression decreased 0.10- and 0.04-fold at 4 and 8 weeks, respectively (p<0.05). The change in AQP4 expression at 4 and 8 weeks was insignificant (p>0.05). The pattern of immunoreactivity of ENaC-alpha and AQP-1 was similar with that of gene expression.nnnCONCLUSIONnThe experimental methods provoked reproducible otitis media with effusion. This model is well suited for studies of middle ear homeostasis during disease pathogenesis. Middle ear mucosa homeostasis is altered significantly by ETO, and the subunits of AQP proteins show a characteristic expression pattern over time.

Protective role of NecroX-5 against neomycin-induced hair cell damage in zebrafish

AbstractNecroX-5, one of the derivatives of NecroX series compounds, is a mitochondrial reactive oxygen species and reactive nitrogen species scavenger that inhibits cell death against various kinds of oxidative stresses. The objective of the present study was to evaluate the effects of NecroX-5 on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five days post-fertilization, zebrafish larvae were exposed to 125xa0μM neomycin and one of the following NecroX-5 concentrations for 1xa0h: 10, 25, 50, and 75xa0μM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy (nxa0=xa010). The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Ultrastructural changes were evaluated using scanning electron microscopy. NecroX-5 decreased neomycin-induced hair cell loss in the neuromasts (NecroX-5 50xa0μM: 13.4xa0±xa02.0 cells, 125xa0μM neomycin only: 8.1xa0±xa01.2 cells; nxa0=xa010, Pxa0<xa00.05) and decreased the TUNEL reaction. The ultrastructural analysis showed that the structures of mitochondria and hair cells within the neuromasts were preserved in zebrafish exposed to 125xa0μM neomycin and 50xa0μM NecroX-5. NecroX-5 decreased apoptosis and mitochondrial damage. In conclusion, NecroX-5 attenuated neomycin-induced hair cell loss in zebrafish.n

Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers.

In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4–6xa0bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1xa0pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5u2009±u20091.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohens kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.

Expression of ENaC in LPS-induced inflammation of middle ear mucosa

Abstract Conclusion: The expression of all three subunits of the epithelial sodium channel (ENaC) decreased after the lipopolysaccharide (LPS) injection and normalized as the fluid collection resolved. This implies that fluid collection in acute otitis media may be caused by the inhibition of ENaC function. Objectives: We investigated the expression levels of subunits of ENaC after an injection of LPS into the middle ear cavity of rats. Methods: Seventy-two Sprague-Dawley rats were assigned randomly into the five groups that received an LPS injection and the control group. A transtympanic injection of LPS (1 mg/ml) was done and rats were sacrificed 6, 12, 24, 72, and 120 h after the procedure. Real-time RT-PCR was carried out for ENaC-α, -β, -γ, and immunohistochemistry was performed for ENaC-α and -β. Results: The level of ENaC-α expression decreased 0.50-fold and 0.55-fold at 6 and 12 h, respectively (p < 0.05), but it normalized at 24 h. It increased 3.64-fold at 72 h and 3.24-fold at 120 h (p < 0.05). At 6 and 12 h after the LPS injection into the middle ear cavity, inflammation was induced and ENaC-α immunoreactivity decreased. At 24 h, ENaC-α immunoreactivity was normalized, then at 72 and 120 h after the injection it was increased.