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BackgroundPsoriasin is known to be expressed in diverse organs, where it exerts antimicrobial activity. Psoriasin is also involved in the local host defense mechanism against pathogens. We hypothesized that allergy-related T-helper cell type 2 (Th2) cytokines may regulate the expression of psoriasin.MethodsWe treated normal human nasal epithelial (NHNE) cells with IL-4 or IL-13. Using human nasal tissues, we compared the expression level of psoriasin. We performed real-time polymerase chain reaction and Western blot assays using NHNE cells. Immunohistochemical staining and Western blot assays were performed with human nasal tissues. Furthermore, we studied the antimicrobial activity of nasal secretions from normal and allergic rhinitis patients.ResultsIL-13 markedly down-regulated psoriasin expression at the gene and protein levels in NHNE cells, and it also decreased the amount of psoriasin protein that was secreted into the extracellular compartment in NHNE cells. IL-4 had no statistically significant effect. Results from immunohistochemical staining and Western blot assays showed that psoriasin expression was decreased in allergic rhinitis patients compared with control subjects. Nasal secretions of allergic rhinitis patients exhibited decreased antimicrobial activity compared with control subjects.ConclusionWe found that Th2 cytokines regulated psoriasin expression in NHNE cells, and psoriasin expression was decreased in allergic rhinitis patients compared with control subjects. The decreased expression of psoriasin may be related to the reduction in antimicrobial capacity of nasal secretions under allergic conditions, resulting in an increase in susceptibility to viruses or bacterial infections.

In this study, the far-red-emitting fluorescence probe 1, containing a rhodamine derivative and a hydrazide reactive group, was developed for peroxynitrite detection and imaging. This probe, which is cell permeable and shows high sensitivity and selectivity in fluorometric detection of peroxynitrite over other ROS/RNS, was successfully utilized to detect exogenous and endogenous peroxynitrite in HeLa and RAW 264.7 cells, respectively. More importantly, 1 can also be used to detect endogenous peroxynitrite generated in Pseudomonas aeruginosa (PAO1)-infected mouse bone marrow-derived neutrophils. We anticipate that the new probe will serve as a powerful molecular imaging tool in investigations of the role(s) played by peroxynitrite in a variety of physiological and pathological contexts.

Purpose To obtain validated clinical values suitable for developing a gustatory function test, including umami taste, in a Korean population. Materials and methods The investigation involved 297 participants with self-reported normal sense of taste and smell. Liquid solutions were used for the assessment of gustatory function. The test consisted of 30 taste solutions [six concentrations of five tastants (sweet, bitter, salty, sour, and umami)]. For evaluation of overall gustatory function, the number of detected or correctly recognized taste thresholds was combined to form a "taste score." Results Mean values of each detection and recognition threshold for the five tastes in men were consistently lower than those of women. The 10th percentile of taste score for recognition was used as the cut-off value for distinguishing normogeusia from hypogeusia. In subgroup analysis, total taste score from recognition thresholds revealed a significant negative correlation with age, indicating lower scores for increasing age. Taste score for non-smokers was significantly higher than that of smokers, in terms of detection and recognition of taste sensitivities. Conclusion This gustatory function test was easy to perform, affordable, and time-saving, with the capacity to self-produce and obtain reliable data. Gustatory function was more sensitive in young people, women, and non-smokers.

Study objectives Investigating the exact pathophysiology of obstructive sleep apnea syndrome (OSAS)-induced hearing loss is critical. We sought to verify the hypothesis that a correlation exists between mitochondrial dysfunction in inner ear hair cells and the auditory dysfunction induced by chronic intermittent hypoxia (CIH) in a murine model of sleep apnea. Methods C57BL/6J adult male mice were randomized to 4 weeks of CIH (n = 12) or normoxia (Sham) (n = 12). Hearing threshold was determined by auditory brainstem response. The activity of mitochondria was compared between CIH and Sham mice. Histological assessment and transmission electron microscopy were performed for assessing morphologic changes in mitochondria. The number of mtDNA copies as well as the levels of PGC1-α, Tfam, and VDAC (voltage-dependent anion channel) were determined in the hair cells of CIH mice. Results We observed that hearing ability in CIH mice was impaired and hair-cell mitochondria in CIH mice were fewer compared to that in Sham and also displayed an aberrant morphology. The mRNA levels of PGC-1α and Tfam were higher in the CIH group than in the Sham group. Moreover, the expression of VDAC was increased in the tectorial membrane, the basilar membrane, and especially in the inner hair cells of CIH mice. Conclusions This study using CIH mice as a model for OSAS provides evidence of an association between OSAS and auditory function alteration, as well as of mitochondria being part of the pathophysiology of hearing impairment. Further investigation is required to determine whether mitochondria could serve as a valid target for preventive or therapeutic purposes.

Study objective No studies have investigated sequential changes in the heart on magnetic resonance imaging (MRI), along with observation of functional lung phenotypes and genetics, over the duration of chronic intermittent hypoxia (CIH). We investigated chronological changes in heart and lung phenotypes after CIH using a mouse model to provide new insights into the pathophysiology of sleep apnea-induced cardiovascular disease. Methods C57BL/6J adult male mice were randomized to 4 or 8 weeks of CIH. Cardiac cine-MRI images were analyzed to assess functional parameters of right ventricle (RV). Histopathological features of myocytes and pulmonary vessels, as well as genes involved in the endothelin (ET) system, were investigated. Results Function of the RV reduced significantly at 4 weeks and continuously decreased following another 4 weeks of CIH, although the rate of decrease was attenuated. Notably, persistence of reduced ejection fraction and end-systole RV wall thickness (WT) and increases in the ET system of the lungs and blood strongly implied the development of pulmonary hypertension after 8 weeks of CIH. Conclusions RV dysfunction with reduced end-systole RV WT could be a late phenotype in long-standing CIH and possibly also in obstructive sleep apnea.

To evaluate the potential efficacy of steroid-soaked, absorbable calcium alginate nasal packing following endoscopic sinus surgery.Prospective, randomized, single-blinded, placebo-controlled trial.Twenty-two patients (44 nostrils) who had chronic rhinosinusitis with polyps underwent bilateral endoscopic sinus surgery. Only those with an intersinus difference in Lund-Mackay severity score of 1 or less were included. In each patient, one randomly selected nostril was packed with calcium alginate soaked with 2 mL of triamcinolone (40 mg/mL) (triamcinolone group), whereas the contralateral nostril received an identical packing soaked in 2 mL of normal saline (saline group). Two independent investigators blinded to the packing allocation scored the surgical field using the validated Perioperative Sinus Endoscopy (POSE) scores 1, 4, and 8 weeks after surgery.All 44 nostrils were analyzed; the Lund-Mackay scores did not differ significantly between the groups before surgery. Eight weeks after surgery, the total POSE scores were significantly lower in the triamcinolone group (P = .014). The POSE scoring parameters were then compared between groups, and the following variables were significantly different: middle turbinate synechiae with the lateral wall (P = .037), polypoid degeneration of the ethmoid cavity (P = .006), and sphenoid sinus severity (P = .036).This study demonstrated that steroid-soaked, absorbable nasal packing can be used to enhance wound healing after endoscopic sinus surgery and to prevent polypoid changes in the nasal mucosa.1b. Laryngoscope, 128:311-316, 2018.

Histamine is an important mediator of allergic reactions, and mucus hypersecretion is a major allergic symptom. However, the direct effect of histamine on mucus secretion from airway mucosal epithelia has not been clearly demonstrated. TMEM16A is a Ca2+-activated chloride channel, and it is closely related to fluid secretion in airway mucosal epithelia. We investigated whether histamine directly induces fluid secretion from epithelial cells or submucosal glands (SMG) and mechanisms related, therewith, in allergic airway diseases. In pig airway tissues from the nose or trachea, histamine was a potent secretagogue that directly induced strong responses. However, gland secretion from human nasal tissue was not induced by histamine, even in allergic rhinitis patients. Histamine type 1 receptor (H1R) and histamine type 2 receptor (H2R) were not noted in SMG by in situ hybridization. Cultured primary human nasal epithelial (NHE) cells were used for the measurement of short-circuit current changes with the Ussing chamber. Histamine-induced slight responses of anion secretions under normal conditions. The response was enhanced by IL-4 stimulation through TMEM16A, which might be related to fluid hypersecretion in allergic rhinitis. Pretreatment with IL-4 augmented the histamine response that was suppressed by a TMEM16A inhibitor. TMEM16A expression was enhanced by 24-h treatment of IL-4 in human nasal epithelial cells. The expression of TMEM16A was significantly elevated in an allergic rhinitis group, compared with a control group. We elucidated histamine-induced fluid secretions in synergy with IL-4 through TMEM16A in the human airway epithelium. In addition, we observed species differences between pigs and humans in terms of gland secretion of histamine.

The endolymphatic sac (ES) is a cystic structure of the inner ear connected to the cochlea and vestibule, which plays a role in regulating ion homeostasis in inner ear fluid. Disruption of ion homeostasis can cause inner ear disorders with hearing loss and dizziness, such as Meniere’s disease. Herein, we found, for the first time, functional evidence for the involvement of β1- and β2-adrenergic receptors in apical electrogenic ion transport by human ES epithelium by using electrophysiological/pharmacological and molecular biological methods, which were dependent on K+ and Cl− ion transport. The apical electrogenic transport was absent or very weak in ES epithelia of patients with Meniere’s disease. These results suggested that adrenergic stimulation via β1- and β2-adrenergic receptors in the human ES was involved in regulation of inner ear fluid ion homeostasis and impairment of this response could be a pathological mechanism of Meniere’s disease.

Objective Vitamin D has immune modulatory functions, and thus its relationship with allergic disease has been evaluated in a number of studies. However, no concrete link between serum IgE level and vitamin D has been established. Thus, the purpose of study was to confirm the association of serum vitamin D level with the serum IgE (total and Dematophagoides farinae specific IgE) in Korean adolescents. Methods A total of 365 subjects between 10 and 18 years of age were enrolled through the Korea National Health and Nutrition Examination Survey (KNHANES). Serum levels of vitamin D, total IgE, and Dermatophagoides farinae (Df) specific IgE were measured, and data for potential confounding variables were collected. Multivariate regression analyses were used to determine the independent effects of these variables. Results Levels of both total and Df specific IgE were positively correlated with serum concentration of vitamin D (Spearman's rho ( R ) = 0.126, p = 0.016; R = 0.152, p = 0.004, respectively). These results were confirmed by multivariate linear regression analysis after adjusting for sex, age, and body mass index (coefficient ( B ): 10.45 95% confidence interval (CI): 0.59–20.31; B : 1.06, 95% CI: 0.31–1.80, respectively). And, the association between serum vitamin D and IgE showed the different results depending on the presence or absence of Df sensitization. Conclusions Serum concentration of vitamin D was positively associated with levels of both total IgE and Df-specific IgE in Korean adolescents.

Objective The mouse is the most popular animal model in olfactory research. Behavior tests with odorants are essential for determining olfactory phenotype. To the best of our knowledge, the mouse olfactory behavior test has not been standardized, making the results vulnerable to inter-observer variation. We sought to develop a new mouse olfactory behavior test assessed by an automatic video tracking system with minimal inter-observer variation. Methods A video-tracking system was used to automatically track mouse behavior in standard breeding cages with C57BL/6N mice. We tested two odorants (peanut butter for the preference test, 2MB acid for the avoidance test) and distilled water (for a control). Mouse behavior was recorded for 3 min and analyzed. For the preference test, investigation time was measured. For the avoidance test, time spent in sectors away from the odorant zone was measured. To confirm our experimental settings, we also evaluated an anosmia mouse model prepared with intranasal administration of ZnSO4. Results All strains of mice showed reproducible behavior patterns of preference or avoidance for the odorants. The anosmia mouse model, as expected, failed to show an olfactory ability for preference or avoidance, and this was well-matched by histologic changes caused by the ZnSO4 treatment. The automatic video tracking system successfully tracked and automatically calculated mouse behavior with good reproducibility. Conclusion Our olfactory behavior test offers a simple and accurate method to evaluate olfactory function in mice. This test can be utilized as a possible standard method to search for features of olfactory phenotypes in mice.

Objectives/Hypothesis The soft palate is composed of several muscles. So far, these muscles have been analyzed separately and the whole structure of the soft palate has been described very vaguely. The purpose of this study was to describe the structure of soft palate collectively. Study Design Human cadaver dissection. Methods Anatomical dissection was performed on 20 Korean, formalin-fixed cadavers (13 men and 7 women). The heads were randomly assigned to either dissection groups, medial-to-lateral (10 heads) or posterior-to-anterior (10 heads). Results Fibro-fatty layer was located in the oral side of the soft palate and became thick and dense along the midline. The levator veli palatini was distributed on the nasal side of the soft palate. The average vertical distance from the top of palatopharyngeal arch to the levator veli palatini was 12.3 ± 5.2 mm. The palatopharyngeus was also spread out widely and filled the palatopharyngeal arch. The periosteum of the hard palate became the fascia of the soft palate, which was merged with the aponeurosis of the tensor veli palatini in the midline. The Passavant's ridge was located between the levator veli palatini and palatopharyngeus, inserting onto the aponeurosis. The palatoglossus was very thin and embedded in the palatoglossal arch. Conclusion The muscles of the soft palate are intermingled with each other in the fibro-fatty layer. Comprehensive anatomical knowledge is needed to reduce unnecessary damage and obtain good surgical results for obstructive sleep apnea. Level of Evidence N/A. Laryngoscope, 123:2900–2904, 2013

Background Allergic rhinitis (AR) and asthma are 2 entities of allergic airway diseases that frequently occur together, which is referred to as united airways . In contrast to this general concept, we hypothesized that innate immunity of the upper and lower airways is respectively distinctive, because the immunologic conditions of the nasal and lung mucosa as well as the functions of the immune cells within their epithelia are different. Objective We wanted to identify distinctive mechanisms of innate immunity in the nose and lung mucosa, which are responsible for house dust mite (HDM)–induced AR and allergic asthma (AA), respectively. Methods We constructed a mouse model of AR or AA induced by sensitization and consequent provocation with HDM extracts. Results HDM-derived β-glucans, rather than LPS, were proven to be essential to activating innate immunity in the nasal mucosa and triggering AR, which depended on Toll-like receptor 2 (TLR2), but not on TLR4; however, the LPS/TLR4 signaling axis, rather than β-glucans/TLR2, was critical to HDM-induced AA. These differences were attributed to the specific role of β-glucans and LPS in inducing the surface expression of TLR2 and TLR4 and their translocation to lipid rafts in nasal and bronchial epithelial cells, respectively. We also showed that dual oxidase 2–generated reactive oxygen species mediate both β-glucan–induced TLR2 activation and LPS-induced TLR4 activation. Conclusions We describe a novel finding of distinctive innate immunity of the nose and lungs, respectively, which trigger AR and AA, by showing the critical role of HDM-induced TLR activation via dual oxidase 2–mediated reactive oxygen species.

Anoctamin 1 (ANO1)/transmembrane protein 16A (TMEM16A) is a calcium-activated anion channel that may play a role in HCO 3 − secretion in epithelial cells. Here, we report that the anion selectivity of ANO1 is dynamically regulated by the Ca 2+ /calmodulin complex. Whole-cell current measurements in HEK 293T cells indicated that ANO1 becomes highly permeable to HCO 3 − at high [Ca 2+ ] i . Interestingly, this result was not observed in excised patches, indicating the involvement of cytosolic factors in this process. Further studies revealed that the direct association between ANO1 and calmodulin at high [Ca 2+ ] i is responsible for changes in anion permeability. Calmodulin physically interacted with ANO1 in a [Ca 2+ ] i -dependent manner, and addition of recombinant calmodulin to the cytosolic side of excised patches reversibly increased P HCO3 /P Cl . In addition, the high [Ca 2+ ] i -induced increase in HCO 3 − permeability was reproduced in mouse submandibular gland acinar cells, in which ANO1 plays a critical role in fluid secretion. These results indicate that the HCO 3 − permeability of ANO1 can be dynamically modulated and that ANO1 may play an important role in cellular HCO 3 − transport, especially in transepithelial HCO 3 − secretion.

Here, we studied the IFN-regulated innate immune response against influenza A virus (IAV) infection in the mouse lung and the therapeutic effect of IFN-λ2/3 in acute IAV lung infection. For viral infections, IAV (WS/33, H1N1, PR8 H1N1, H5N1) were inoculated into wild-type mice by intranasal delivery, and IAV mRNA level and viral titer were measured. To compare the antiviral effect of IFNs in vivo in the lung, neutralizing antibodies and recombinant IFNs were used. After intranasal inoculation of IAV into mice, viral infection peaked at 7 days postinfection, and the IAV titer also reached its peak at this time. We found that IFN-β and IFN-λ2/3 were preferentially induced after IAV infection and the IFN-λ2/3-mediated innate immune response was specifically required for the induction of IFN-stimulated genes (ISGs) transcription in the mouse respiratory tract. Neutralization of secreted IFN-λ2/3 aggravated acute IAV lung infection in mice with intact IFN-β induction; consistent with this finding, the transcription of ISGs was significantly reduced. Intranasal administration of IFN-λ2/3 significantly suppressed various strains of IAV infection, including WS/33 (H1N1), PR (H1N1), and H5N1 in the mouse lung, and was accompanied by greater up-regulation of ISGs. Taken together, our data indicate that the IFN-λ2/3-mediated innate immune response is necessary to protect the lungs from IAV infection, and intranasally delivered IFN-λ2/3 has the potential to be a useful therapeutic strategy for treating acute IAV lung infection.

Background Nasal secretions include cytokines and inflammatory mediators that are involved in the pathogenesis of upper airway inflammation. Objective We tried to find unknown biomolecules that are involved in the pathogenesis of chronic rhinosinusitis (CRS). Methods We collected nasal mucosal secretions from patients who were diagnosed as having CRS and who underwent endoscopic sinus surgery. A total of 63 patients who underwent nasal secretion collection were reviewed. Enzyme-linked immunosorbent assay was performed by using nasal lavage samples to evaluate which biomolecules were associated with the severity of inflammation based on the Lund-Mackay score. By using human nasal epithelial cells, we performed Western blot, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay to evaluate the secretory mechanism of heat shock protein (HSP) 70. Results We found that the level of interleukin 8 and HSP70 were significantly associated with the Lund-Mackay score and interleukin 17C, C-X-C motif chemokine 10, and HSP27 were not significantly associated. HSP70 was also significantly associated with the surgical outcome of the enrolled patients. Furthermore, we found that exposure to hypoxia and treatment of lipoteichoic acid induced the secretion of HSP70 but that lipopolysaccharide did not induce the secretion of HSP70 in human nasal epithelial cells. Conclusion Our findings indicated that HSP70 might play a role in the pathogenesis of CRS and the possibility of HSP70 as a biomolecule that represents the severity of CRS.

Objective This study aimed to re-assess the outcomes of different surgical methods for esthesioneuroblastoma (ENB) in a single institution, with emphasis on changes in surgical treatment with regard to endoscopic approaches in patients with ENB. Subjects and methods We retrospectively reviewed the data of 35 patients with ENB treated over the last 31 years. Results The 5-year overall and disease-free survival rates were 66.8% and 50.8%, respectively. Disease-free survival in the endoscopic surgery group was significantly higher compared to that of craniofacial surgery group (P = 0.035). In the endoscopic surgery group, nine of 10 patients did not exhibit local failure or regional recurrence over a mean followup period of 64.3 months, which was longer than the mean time to recurrence (22.0 months) observed in this study. Conclusion Given its significant survival outcomes and high rate of local control, endoscopic surgery could be preferred as a minimally invasive treatment with potentially low morbidity and possible oncological validity for the treatment of ENB.

High-mobility group box 1 (HMGB1) mediates various functions according to the location. We tried to investigate the role of HMGB1 in upper airway under hypoxic conditions. We cultured primary normal human nasal epithelium (NHNE) cells under hypoxic conditions and evaluated the movement of HMGB1 by western blotting, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) level was evaluated to estimate the translocation mechanism of HMGB1. The role of secreted HMGB1 was evaluated by ELISA assay. Furthermore, we collected human nasal mucosa samples and nasal lavage fluids from patients conditioned under hypoxic and non-hypoxic environment, and compared the expression of HMGB1 in human nasal mucosa samples by immunohistochemistry and the levels of HMGB1 in lavage fluids using ELISA assay. Hypoxia induced translocation of HMGB1 into the extracellular area and it was dependent on ROS produced by dual oxidase 2. Secreted HMGB1 was involved in the upregulation of interleukin (IL)-8. In human samples, HMGB1 was translocated from nucleus to the cytoplasm in hypoxic-conditioned nasal mucosa. HMGB1 was increased in nasal lavage samples of chronic rhinosinusitis patients, whose sinus mucosa was supposed to be hypoxic as compared with controls. We suggest that HMGB1 is secreted in hypoxic condition via ROS-dependent mechanism and secreted HMGB1 participates in IL-8 upregulation mediating inflammatory response.

Altered metabolism has been implicated in the pathogenesis of inflammatory diseases. NADPH oxidase 4 (NOX4), a source of cellular superoxide anions, has multiple biological functions that may be of importance in inflammation and in the pathogenesis of human metabolic diseases, including diabetes. However, the mechanisms by which NOX4-dependent metabolic regulation affect the innate immune response remain unclear. Here we show that deficiency of NOX4 resulted in reduced expression of carnitine palmitoyltransferase 1A (CPT1A), which is a key mitochondrial enzyme in the fatty acid oxidation (FAO) pathway. The reduced FAO resulted in less activation of the nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome in human and mouse macrophages. In contrast, NOX4 deficiency did not inhibit the activation of the NLR family, CARD-domain-containing 4 (NLRC4), the NLRP1 or the absent in melanoma 2 (AIM2) inflammasomes. We also found that inhibition of FAO by etomoxir treatment suppressed NLRP3 inflammasome activation. Furthermore, Nox4-deficient mice showed substantial reduction in caspase-1 activation and in interleukin (IL)-1β and IL-18 production, and there was improved survival in a mouse model of NLRP3-mediated Streptococcus pneumoniae infection. The pharmacologic inhibition of NOX4 by either GKT137831, which is currently in phase 2 clinical trials, or VAS-2870 attenuated NLRP3 inflammasome activation. Our results suggest that NOX4-mediated FAO promotes NLRP3 inflammasome activation.

The Eastern Banded Structure (EBS) and Hydra~I halo overdensity are very nearby (d $\sim$ 10 kpc) objects discovered in SDSS data. Previous studies of the region have shown that EBS and Hydra I are spatially coincident, cold structures at the same distance, suggesting that Hydra I may be the EBS's progenitor. We combine new wide-field DECam imaging and MMT/Hectochelle spectroscopic observations of Hydra I with SDSS archival spectroscopic observations to quantify Hydra I's present-day chemodynamical properties, and to infer whether it originated as a star cluster or dwarf galaxy. While previous work using shallow SDSS imaging assumed a standard old, metal-poor stellar population, our deeper DECam imaging reveals that Hydra~I has a thin, well-defined main sequence turnoff of intermediate age ($\sim 5-6$ Gyr) and metallicity ([Fe/H] = $-0.9$ dex). We measure statistically significant spreads in both the iron and alpha-element abundances of $\sigma_{[Fe/H]} = 0.13 \pm 0.02$ dex and $\sigma_{[\alpha/{\rm Fe}]} = 0.09 \pm 0.03$ dex, respectively, and place upper limits on both the rotation and its proper motion. Hydra~I's intermediate age and [Fe/H] -- as well as its low [$\alpha$/Fe], apparent [Fe/H] spread, and present-day low luminosity -- suggest that its progenitor was a dwarf galaxy, which subsequently lost more than $99.99\%$ of its stellar mass., Comment: 17 pages, 14 figures, submitted to ApJ

Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.

Down syndrome is the most common genetic disorder and is characterized by three copies of chromosome 21. Regulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (distal part of chromosome 21), and its product functions as an endogenous inhibitor of calcineurin signaling. RCAN1 protein stability is regulated by several inflammatory signaling factors, though the underlying mechanisms remain incompletely understood. Here, we report that RCAN1 interacts with the inflammation-linked transcription factor, signal transducer and activator of transcription 2 (STAT2) in mammalian cells. STAT2 overexpression decreased levels of RCAN1 protein. Decreases in RCAN1 were blocked by a proteasome inhibitor, indicating that STAT2 regulates RCAN1 degradation via the ubiquitin-proteasome system. Co-immunoprecipitation/immunoblot analyses showed that STAT2 enhanced RCAN1 ubiquitination through the ubiquitin E3 ligase FBW7. This pathway appeared to be physiologically relevant, as treatment of cells with interferon-α reduced RCAN1 levels through the activation of STAT2 and FBW7. Together, these results suggest that STAT2 influences diverse cellular processes linked to RCAN1 by negatively affecting RCAN1 protein stability.

Airway mucosa is constantly exposed to various airborne microbes, and epithelial host defense requires a robust innate immunity. Recently, it has been suggested that NADPH oxidase (NOX) isozymes serve functional roles in toll-like receptor (TLR)-mediated innate immune responses. However, the molecular mechanism between TLR and NOX-mediated reactive oxygen species (ROS) production in human airway mucosa has been poorly understood.Here, we show that flagellin-induced ROS generation is dependent on dual oxidase 2 (DUOX2) activation, which is regulated by [Ca(2+)](i) mobilization in primary normal human nasal epithelial (NHNE) cells. Interestingly, we observed that silencing of DUOX2 expression in NHNE cells and nasal epithelium of Duox2 knockout mice failed to trigger mucin and MIP-2? production upon challenging flagellin.Our observation in this study reveals that flagellin-induced hydrogen peroxide (H(2)O(2)) generation is critical for TLR5-dependent innate immune responses, including IL-8 production and MUC5AC expression in the nasal epithelium. Furthermore, DUOX2-mediated H(2)O(2) generation activated by the flagellin-TLR5 axis might serve as a novel therapeutic target for infectious inflammation diseases in the airway tract.Taken together, we propose that DUOX2 plays pivotal roles in TLR5-dependent inflammatory response of nasal airway epithelium.

The symbiotic microbiota profoundly affect many aspects of host physiology; however, the molecular mechanisms underlying host-microbe cross-talk are largely unknown. Here, we show that the pyrroloquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) activity of a commensal bacterium, Acetobacter pomorum, modulates insulin/insulin-like growth factor signaling (IIS) in Drosophila to regulate host homeostatic programs controlling developmental rate, body size, energy metabolism, and intestinal stem cell activity. Germ-free animals monoassociated with PQQ-ADH mutant bacteria displayed severe deregulation of developmental and metabolic homeostasis. Importantly, these defects were reversed by enhancing host IIS or by supplementing the diet with acetic acid, the metabolic product of PQQ-ADH.

Mucus hypersecretion is a prominent feature of respiratory diseases, and MUC5B is a major airway mucin. Mucin gene expression can be affected by inflammatory mediators, including prostaglandin (PG) D(2,) an inflammatory mediator synthesized by hematopoietic PGD synthase (H-PGDS). PGD(2) binds to either D-prostanoid receptor (DP1) or chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). We investigated the mechanisms by which PGD(2) induces MUC5B gene expression in airway epithelial cells. Western blot analysis showed that H-PGDS was highly expressed in nasal polyps. Similar results were obtained for PGD(2) expression. In addition, we could clearly detect the expressions of both H-PGDS and DP1 in nasal epithelial cells but not CRTH2. We demonstrated that PGD(2) increased MUC5B gene expression in normal human nasal epithelial cells as well as in NCI-H292 cells in vitro. S5751, a DP1 antagonist, inhibited PGD(2)-induced MUC5B expression, whereas a CRTH2 antagonist (OC0459) did not. These data suggest that PGD(2) induced MUC5B expression via DP1. Pretreatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) blocked both PGD(2)-induced ERK mitogen-activated protein kinase (MAPK) activation and MUC5B expression. Proximity ligation assays showed direct interaction between RSK1 and cAMP response element-binding protein (CREB). Stimulation with PGD(2) caused an increase in intracellular cAMP levels, whereas intracellular Ca(2+) did not have such an effect. PGD(2)-induced MUC5B mRNA levels were regulated by CREB via direct interaction with two cAMP-response element sites (-921/-914 and -900/-893). Finally, we demonstrated that PGD(2) can induce MUC5B overproduction via ERK MAPK/RSK1/CREB signaling and that DP1 receptor may have suppressive effects in controlling MUC5B overproduction in the airway.

Objectives We investigated the bulging and dehiscence of neurovascular structures in the sphenoid sinus and their relationships to the pneumatization of the sphenoid sinus. Methods One hundred sagittally hemisected cadaveric heads were examined. The degree of pneumatization of the sphenoid sinus was determined. Bulging and dehiscence of the internal carotid artery (ICA), optic nerve, maxillary nerve, and vidian nerve were examined, and the distances between these structures and the anterior or superior wall of the sphenoid sinus were measured. Additionally, the degree of bony thickness over these structures was determined. Results The prevalences of bulging of the optic nerve, segments 1 and 3 of the ICA, and the maxillary and vidian nerves were 56%, 34%, 65%, 41%, and 52%, respectively. The greater the degree of pneumatization, the more frequently did the structures bulge into the sphenoid sinus. The optic nerve was found to be in close proximity to the anterior and superior walls of the sphenoid sinus. The bone over the surrounding structures was very thin, especially for the complete sellar type. Conclusions The prevalence of bulging of the optic nerve, the ICA, and the maxillary and vidian nerves increased in proportion to the degree of sphenoid sinus pneumatization.

Objectives/Hypothesis: The morphologic features of upper and lower lateral cartilage and septal cartilage of the cadaveric nose were analyzed to provide practical anatomical knowledge for Asian rhinoplasty. Study Design: Cadaveric dissection. Methods: A total of 21 Korean adult cadavers were dissected. External nasal morphology was observed, measured, photographed, and analyzed. Histologic features were observed with a light microscope in coronally-transected specimens stained with hematoxylin and eosin. Results: The lengths of the upper and lower lateral cartilage of Korean cadaveric noses were similar to those of white noses. The widths of the upper and lower lateral cartilage were substantially smaller in Korean cadaveric noses than in those of whites. Upper lateral cartilage include substantial transverse portions near the keystone area that should be preserved in component reduction rhinoplasty. The relationships between the upper lateral cartilage and the lower lateral cartilage were divided into four types. Type I, in which the upper lateral cartilage and lower lateral cartilage are interlocked to form a Z-shape, is the most common. The posterior portion of the septal cartilage, which is connected to the perpendicular plate of the ethmoid and vomer, is thickest. Conclusions: Cartilaginous structures of Asian noses were substantially different from those of whites in terms of their shape, size, thickness, and relationship with other structures. The data from surgical anatomical observations of the cartilaginous framework of Korean cadaveric noses provided in this report will provide valuable information for performing rhinoplasty on Asian patients. Laryngoscope, 2010

The specific molecular profiles of ovarian cancer interface zones (IZ), the region between tumors and normal tissues, were evaluated using a new method involving matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS). We analyzed three ovarian serous carcinomas using MALDI-IMS. Principal component analysis (PCA) was used to evaluate the quality of tissue spatial features based on MALDI-IMS, and for analysis of large data sets of MALDI-IMS. Two-dimensional gel electrophoresis and fluorescence microscopy were used to verify interface-specific proteins. Unique profiles were identified for the tumors, the normal zone, and the IZ. Through MALDI analysis, two interface-specific proteins, plastin 2 and peroxiredoxin 1 (PRDX 1), were identified as differentially regulated between zones. Fluorescence microscopy revealed high expression levels of plastin 2 and PRDX 1 along the IZ of ovarian tumors. This comparative proteomics study using tissue MALDI-IMS suggested that the IZ is different from the adjacent tumor and normal zones, and that plastin 2 and PRDX 1 may be interface markers specific to ovarian tumors.

Background The Down syndrome candidate region-1 gene ( DSCR1 , also known as RCAN1 ) is situated close to the Down Syndrome Critical Region (DSCR), which contains genes responsible for many features of Down syndrome. DSCR1 modulates calcineurin phosphatase activity, though its functional role is incompletely understood. Methods Here we investigated the role of DSCR1-1S isoform in IL-1 receptor (IL-1R)-mediated signaling by analyzing interaction between DSCR1-1S and the IL-1R pathway components Tollip, IRAK-1, and TRAF6. Results Co-immunoprecipitation analyses of HEK293 cells revealed that DSCR1-1S interacted with Tollip, an IRAK-1 inhibitor, leading to the dissociation of IRAK-1 from Tollip. Similarly, both DSCR1-1S and Tollip interacted with TRAF6, with DSCR1 reducing interaction between Tollip and TRAF6. DSCR1-1S also stimulated IL-1R-mediated signaling pathways, TAK1 activation, NF-κB transactivation, and IL-8 production, all downstream consequences of IL-1R activation. General significance Together, these results suggest that DSCR1-1S isoform positively modulates IL-1R-mediated signaling pathways by regulating Tollip/IRAK-1/TRAF6 complex formation.

EGb 761, extracted from Ginkgo biloba leaves, has been proven to induce caspase-3-dependent apoptosis in oral cavity cancer cells. Since EGb 761 is a composition of various components, it is important to identify which components are responsible for its anticancer effects to reduce the total dosage and to avoid toxicity. Therefore, the study aimed to determine the effective compounds of EGb 761 that induce apoptosis in oral cavity cancer cells and to identify whether caspase-3 was involved in apoptosis of oral cancer cells by EGb 761 components. The results of cell proliferation assays on oral cavity cancer cells showed that kaempferol and quercetin significantly inhibited cellular proliferation at a concentration of 40 microM. Flow cytometry showed that the antiproliferative effects of each component were due to increased apoptosis. Kaempferol and quercetin induced apoptosis in various oral cancer cell lines (SCC-1483, SCC-25 and SCC-QLL1) and showed cleavage of poly (ADP-ribose) polymerase (PARP). Caspase-3 activity assay revealed that induction of apoptosis by kaempferol and quercetin was caspase-3-dependent. In conclusion, the results suggest that kaempferol and quercetin, two components of EGb 761, effectively induce caspase-3-dependent apoptosis of oral cavity cancer cells and can be considered as possible anti-oral cavity cancer agents.

Mucus overproduction and airway obstruction are common features in airway mucosal inflammation. The mechanism by which LPS induces MUC5AC overexpression, however, has not been fully explored. The aims of this study were twofold: first, to examine the ATP-dependent mechanism by which LPS induces MUC5AC gene expression, and second, to identify specific molecules that could suppress LPS-induced MUC5AC expression at a G-protein-coupled receptor level. Here, we suggest that LPS from Pseudomonas aeruginosa induces MUC5AC overproduction by both an ATP-dependent pathway and an ATP-independent pathway. In addition, we showed that Regulator of G-protein signaling (RGS) 4 plays as a suppressor for ATP-induced MUC5AC expression by interacting with G alpha q in a GTP-dependent manner in vivo. These results give additional insights into the molecular mechanism of negative regulation of mucin overproduction and enhance our understanding of mucus hypersecretion during airway mucosal inflammation.

According to our previous study, Ginkgo biloba extract (GBE) suppresses IL-1beta-induced MUC5AC gene expression in NCI-H292 cells via the ERK and p38 MAPK pathways. This study sought to identify which ingredients of GBE suppress IL-1beta-induced MUC5AC gene expression in NCI-H292 cells and to examine which MAPKs are related to MUC5AC gene suppression for each ingredient. After the cells were pretreated with each ingredient and treated with IL-1beta (10 ng/mL), MUC5AC mRNA expression was determined by RT-PCR and real-time PCR. The results showed that kaempferol (KP) and quercetin (QC) suppressed MUC5AC mRNA expression in a dose-dependent manner, both with significant inhibition starting from 40 microm (equal concentration to about a twelfth or thirteenth dose of GBE). MAPK proteins were determined by western blot analysis after pretreatment with KP, QC and GBE. All three suppressed the phosphorylation of ERK and p38 kinases. In conclusion, the data suggested that KP and QC, essential ingredients in GBE, may overcome the dose problem of GBE and play a valuable role, clinically, in controlling mucin hypersecretion in airway inflammation.

Background/Aim: Proteinase-activated receptor-2 (PAR2), which is activated by trypsin, is known to be associated with expression of adhesion molecule integrins. We previously demonstrated that Helicobacter pylori induced the expression of integrin α5 and β1 in human gastric epithelial cells. The present study aims to investigate whether H. pylori in a Korean isolate (HP99) induces the expression of PAR2, which mediates the expression of integrin α5 and β1 and thus cell adhesion to fibronectin in gastric epithelial AGS cells. Methods and Results: mRNA expressions of PAR2, trypsinogen 1 and 2, and integrin α5 and β1 were assessed by RT-PCR analysis while protein levels of PAR2, trypsin as well as integrin α5 and β1 were determined by Western blot analysis. The activity of trypsin in the medium was determined by fluorometric analysis. Cell adhesion assay was performed colorimetrically. H. pylori induced the expressions of PAR2 and integrin α5 and β1 of the cells. H. pylori increased mRNA expression of trypsinogen 1 and 2 as well as the level and activity of trypsin in the medium. H. pylori induced cell adhesion to fibronectin. H. pylori-induced expression of integrin α5 and β1 and adhesion of the cells to fibronectin were inhibited in the cells transfected with PAR2 antisense oligonucleotide or treated with a soybean trypsin inhibitor, anti-integrin α5 antibody or β1 antibody. Conclusion:H. pylori induces the expression of integrin α5 and β1 and adhesion of the cells to fibronectin through PAR2 which is induced and may be activated by trypsin in H. pylori-infected gastric epithelial cells. PAR2 may have an important role in gastric cell adhesion and possibly carcinogenesis associated with H. pylori.

The intracellular negatively regulatory mechanism which affects IL-1beta-induced MUC8 gene expression remains unclear. We found that SOCS3 overexpression suppressed IL-1beta-induced MUC8 gene expression in NCI-H292 cells, whereas silencing of SOCS3 restored IL-1beta-induced MUC8 gene expression. Sequentially activated ERK1/2, RSK1, and CREB by IL-1beta were not affected by SOCS3, indicating that SOCS3 has an independent mechanism of action. Using immunoprecipitaion and nano LC mass analysis, we found that SOCS3 bound NonO (non-POU-domain containing, octamer-binding domain protein) in the absence of IL-1beta, whereas IL-1beta treatment dissociated the direct binding of SOCS3 and NonO. A dominant-negative SOCS3 mutant (Y204F/Y221F) did not bind to NonO. Interestingly, SOCS3 overexpression dramatically suppressed MUC8 gene expression in cells transfected with wild-type or siRNA of NonO. Moreover, silencing of SOCS3 dramatically increased NonO-mediated MUC8 gene expression caused by IL-1beta compared to NonO overexpression alone, suggesting that SOCS3 acts as a suppressor by regulating the action of NonO.

Objectives/Hypothesis: Saline irrigation would be more effective for chronic sinusitis patients if it had bactericidal effects. Low concentrations of hypochlorous acid may be used as a nasal irrigation solution. First, we developed a 0.85% NaCl solution by adding NaCl to tap water (pH 7.0 and 8.4) and measuring the concentration of free chlorine and hypochlorous acid after giving the solution a short electrical impulse of 20 seconds. Then we investigated whether the derived hypochlorous acid had a toxic effect on human primary nasal epithelial cells, if and what effect it had on the expression of mucin genes, and, finally, if it had bactericidal, fungicidal, or virucidal effects. Study Design: In vitro biochemical experiment. Methods: We treated human primary nasal epithelial cells with 3.5 ppm of hypochlorous acid and then examined the cells for cytotoxicity. We also investigated the bactericidal, fungicidal, and virucidal effects by challenging the cells with the following microorganisms Aspergillus fumigatus,Haemophilus influenzae,Klebsiella pneumoniae,Rhizopus oryzae,Candida albicans, Methicillin-resistant Staphylococcus aureus,Streptococcus pneumoniae, and Streptococcus pyogenes. To study the virucidal effects of HOCl, we used the human influenza A virus to challenge the cells. Results: In the cytotoxicity assay and in the morphological examination, the cells did not show anytoxicity at 30 minute or 2 hours after treatment with HOCl. More than 99% of bactericidal or fungicidal activity was noted for all species, except for Candida albicans, in tap water at either pH 7.0 or 8.4. In addition, a 3.2-log10 reduction was achieved in cells challenged with the human influenza A virus. Conclusions: A low concentration HOCl solution can be used as an effective nasal irrigation solution.

Exogenous ATP activates purinoreceptors on the cell surface that regulate diverse cellular functions, including mucous cell secretion in the respiratory epithelium. In this study, ATP increased MUC5AC mRNA in primary human nasal epithelial cells and in NCI-H292 pulmonary adenocarcinoma cells in vitro. ATP-induced MUC5AC mRNA was mediated by phospholipase Cbeta3. A dominant-negative mutation in the PDZ binding domain of PLCbeta3 inhibited ATP-induced MUC5AC gene expression. ATP sequentially activated the phosphorylation of Akt, ERK1/2, p38, RSK1, and cAMP-responding element-binding protein (CREB) in a protein kinase C-independent manner. ATP-induced MUC5AC mRNA levels were regulated by CREB via direct interaction with c-Ets1 on the MUC5AC gene promoter (located -938 to -930). Effects of CREB and c-Ets1 were additive. Inhibition of either CREB or c-Ets1 inhibited ATP-induced MUC5AC gene expression. Stimulation with ATP caused the direct binding of CREB and c-Ets1 to the MUC5AC promoter, increasing the phosphorylation of c-Ets1. Chromatin immunoprecipitation assays demonstrated that in the presence of ATP, both c-Ets1 and CREB bound to the MUC5AC promoter. The effects of exogenous ATP on MUC5AC gene expression are mediated by a complex regulatory cascade controlling interactions between CREB and c-Ets1 that bind to a promoter element in the MUC5AC gene enhancing MUC5AC gene transcription. ATP-dependent activation of MUC5AC gene expression via CREB-c-Ets1 may contribute to mucous cell hypersecretion associated with common respiratory disorders.

Summary A sinonasal inverted papilloma is a benign tumor which has been reported up to 4%. However, an inverted papilloma originating in the sphenoid sinus is even rarer, and a pediatric case has never before been reported. We report a case of pediatric inverted papilloma originating in the sphenoid sinus that presented with symptoms of sinusitis. Endoscopic sinus surgery was performed using a computer-aided image-guided system. The tumor was removed without any surgical complications or recurrence for 2 years.

Summary Introduction Olfactory neuroblastoma is a very aggressive tumour with high rates of locoregional recurrence and distant metastasis. Therefore, salvage therapy plays an important role in control of these neoplasms. In this study, we present our experience and treatment outcomes for salvage therapy in patients with recurrent olfactory neuroblastoma. Material and methods We retrospectively analysed 17 patients treated for advanced olfactory neuroblastoma during the last 15 years. Results The disease-free 5-year survival rate was 48% in all patients and 42% in the 17 patients with recurrence or distant metastasis. Salvage therapy was performed in 6 of 8 patients with recurrence or metastasis and proved successful in 50% of them. In the patients with locoregional recurrence, the success rate of salvage radiotherapy plus neck dissection was superior to salvage chemotherapy alone. In addition, 17% of the patients with distant metastasis after initial treatment died after salvage therapy. In 15% of patients with a clinical stage N0 at initial diagnosis, nodal recurrence developed and was successfully treated with salvage therapy. Conclusion Complete surgical resection, including craniofacial resection and postoperative radiotherapy without elective neck dissection, is the preferred approach in the treatment of advanced olfactory neuroblastoma. In locoregional recurrence, successful salvage therapy may include selective neck dissection and radiotherapy, but in cases of distant metastasis, the prognosis was poor.

Bony changes on CT imaging of inverted papilloma (IP) are useful for predicting tumor origin and recurrence sites. Because the lateral wall and floor of the sphenoid sinus are the most common origin and recurrence sites, the anterior wall of the sphenoid sinus should be opened as wide as the lateral wall and inferiorly to the level of the floor, especially in deeply pneumatized sphenoid sinuses.The incidence of isolated sphenoid IP is exceedingly low. So far, there have not been studies on the usual origin and recurrence sites of the sphenoid sinus. We sought to identify the sites of origin and recurrence and describe clinical characteristics, radiological features, and proper endoscopic management.We retrospectively reviewed medical records of patients with IP of the sphenoid sinus. Data collection included clinical presentations, origin and recurrent sites, radiological features, and surgical methods.Seven patients were identified. The most common symptoms were headache and nasal obstruction. Radiological findings showed that most tumors extended into the nasal cavity or ethmoid sinus combined frequently with erosion of the lateral wall or intersinus septum. We noted simultaneous attachment to multiple walls in five subjects, including both lateral wall and floor attachment in three subjects.

Objective Olfactory neuroblastomas are very aggressive tumors with a high locoregional recurrence rate and distant metastasis. Surgical treatment, including craniofacial resection, has been the main treatment modality, but treatment outcomes of concurrent chemoradiotherapy remain unclear. We present our experiences regarding the treatment outcome of patients with advanced olfactory neuroblastoma undergoing surgical treatment and concurrent chemoradiotherapy. Methods We retrospectively analyzed 16 patients treated for advanced olfactory neuroblastoma within the past 10 years. Results The disease-free 5-year survival rate of the patients ( n = 10) who underwent surgical treatment was 68%. The survival rate for patients ( n = 6) who received concurrent chemoradiotherapy was 42%. The difference in the survival rate was not statistically significant, and no difference in the recurrence rate between the two groups. In the group having surgical treatment, post-operative radiotherapy and salvage therapy were important to increase the survival rate. In the group having concurrent chemoradiotherapy, no patient experienced primary tumor progression. Conclusion Complete surgical resection, including craniofacial resection (CFR), and post-operative radiotherapy seem to be essential in the treatment of advanced olfactory neuroblastoma. However, concurrent chemoradiotherapy may be another primary treatment modality.

Epicatechin gallate (ECG) is the third major catechin component in green tea, but it shows strong biological activity in some aspects, including apoptosis, cell growth inhibition, and membrane transport system in various cells. We previously reported that ECG induces activating transcription factor 3 (ATF3), which is involved in pro-apoptosis in HCT-116 cells. In this report, we present a molecular mechanism by which ECG induces ATF3 expression at the transcriptional level. We found that Sp3 contributed to the basal expression of the ATF3 gene, whereas EGR-1 played an important role in ECG-induced ATF3 expression in HCT-116 cells, as assessed by EMSA and co-transfection experiments. These results suggested that EGR-1, a tumour suppressor protein, could substantiate ECG's role of ATF3 expression in human colorectal cancer cells. We also found that pro-oxidant activity of ECG contributed to ECG-induced ATF3 expression.

Objectives/Hypothesis: Although skin has been the most effective graft material for reconstructing the airway lumen, the use of squamous epithelium has many problems. If autologous airway squamous epithelium could differentiate into mucociliary epithelium after in vivo grafting, it could be an answer to these problems. In this study, we wanted to examine whether carrier-free nasal epithelial cell sheets composed of autologous squamous epithelium could be used as a substitute for skin in airway luminal reconstruction in three maxillectomy patients. Study Design: In vitro biochemical experiments with in vivo applications. Methods: We cultured nasal squamous epithelium from three maxillary cancer patients prior to maxillecotmy. These squamous cell sheets were grafted on the forearm free flap, and, after maxillectomy, the surgical defect was reconstructed with a prefabricated myocutaneous radial forearm free flap with the cultured nasal squamous epithelium. At 1 and 3 month intervals after the reconstructive surgery, the cultured cell grafted area was investigated with histologic phenotype, comparing the skin grafted area. Results: The autologous nasal squamous epithelial cell sheet differentiated into mucociliary epithelium without the crust or mucus stagnation that is usually observed in cases in which skin graft is used for airway reconstruction. Conclusions: We suggest that autologous cultured nasal squamous epithelium, which differentiates into mucociliary epithelium after in vivo grafting, can be used as a clinically relevant substitute for skin graft in airway luminal reconstruction.

Airway epithelial cells regulate fluid and ion absorption and secretion to maintain pH and volume of airway surface liquid (ASL) (Smith & Welsh 1992, Van Scott et al. 1995, Coakley et al. 2003, Hunt 2006). The nasal epithelium, as part of the airway system, is known to actively secrete mucin and determine the electrolyte composition of nasal secretions. Disruption of these processes can lead to altered composition of nasal secretions and impairment of mucociliary clearance, common features of various nasal and paranasal diseases (Tos & Mogensen 1984). However, little is known about the mechanisms of fluid and electrolyte secretion and absorption in the human nasal epithelium. The anions HCO3− and Cl− are major constituents of cellular secretion and also affect ASL pH (Coakley & Boucher 2001). Various anion transporters, such as the anion exchangers (AEs), cystic fibrosis transmembrane conductance regulator (CFTR), sodium bicarbonate cotransporter (NBC) and calcium activated chloride channel (CaCC) are expressed in the human airway (Jacquot et al. 1993, Inglis et al. 2002, Mall et al. 2003, Paradiso et al. 2003). AEs translocate monovalent anions such as Cl− and HCO3− across the plasma membranes. Recent studies have identified two unrelated multigene families of AEs: the SLC4 and SLC26 transporters (Mount & Romero 2004, Alper 2006). The SLC4 transporter family of 10 genes includes three types of HCO3− transporters: AE (exchanges Cl− and HCO3−), NBC (transports Na+ and HCO3−) and Na+-driven Cl−–HCO3− exchangers (NDCBE). Four genes of this SLC4 family, SLC4A1, -A2, -A3 and SLC4A9, were reported as Cl−–HCO3− exchangers and these isoforms were named AE1, AE2, AE3 (bAE3 and cAE3) and AE4, respectively (Romero 2005). The SLC26 family consists of 10 genes (SLC26A1 to -A9 and -A11) encoding AEs that transport various anions including SO42−, Cl−, I−, OH− and HCO3− with variable specificity (Mount & Romero 2004). Among various isoforms, only AE2, bAE3 and SLC26A9 have been shown by RT-PCR to be expressed in human tracheobronchial tree and lung cancer cell lines (Al-Bazzaz et al. 2001, Lohi et al. 2002). Moreover, the possible effect of mucociliary differentiation on the expression of AE has not been reported in cultured airway epithelia. Recently, functional characterization of AEs has been investigated with pharmacological methods in various human cells and tissue including distal colon (Taylor et al. 2001) and pancreatic duct cell lines (Cheng et al. 1998). However, there are only a few reports on the function of AEs in the human airway, including the nasal epithelia (Loffing et al. 2000, Inglis et al. 2002, Mall et al. 2003). Therefore, the aim of this study was to investigate the regulation of mRNA expression of AEs in accordance with mucociliary differentiation, and the functional expression of AEs in both luminal and basolateral membranes in cultured normal human nasal epithelial (NHNE) cells. To this purpose, we first induced mucociliary differentiation of cultured NHNE cells and then confirmed differentiation with histological and molecular characterization. Next, we identified different types of AE isoforms present and any changes with respect to mucociliary differentiation in cultured NHNE cells. Finally, we examined the functional activity of AEs in the luminal and basolateral membranes of polarized monolayers of cultured NHNE cells.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

The purpose of our study was to establish a system for culturing normal human conjunctival epithelial (NHCE) cells under serum-free culture conditions without compromising their ability to differentiate into a mucous epithelium. To this end, small pieces of normal conjunctiva were biopsied from patients undergoing cataract surgery. Obtained NHCE cells were cultured in bronchial epithelial growth medium (BEGM) under serum free culture conditions and passage 3 cells were air-lifted. Cultured NHCE cells displayed typical epithelial morphology. Expression of cytokeratin 19 and conjunctival epithelial specific carbohydrate residue were detected. Air-lifted NHCE cells demonstrated an increase in stratification and differentiation into goblet cells up to 3weeks under air-liquid interface (ALI) culture condition. NHCE cells expressed MUC1, MUC4, MUC16, and MUC5AC mRNA, and MUC5AC production and secretion increased in a time dependent manner after culture under ALI conditions. Exposure of cells to proinflammatory cytokines (TNF-alpha or IFN-gamma) resulted in upregulation of MUC1, MUC4, MUC16, and MUC5AC gene expression. In conclusion, differentiated NHCE cells showed features of a multi-layered conjunctival epithelium, including goblet cells, and retained functional characteristics similar to those seen in vivo. This cell culture system can better facilitate investigation of conjunctival epithelial cell biology and goblet cell differentiation.

Squamous cell carcinoma in the lung originates from bronchial epithelial cells that acquire increasingly abnormal phenotypes. Currently, no known biomarkers are clinically efficient for the early detection of premalignant lesions and lung cancer. We sought to identify secreted molecules produced from squamous bronchial epithelial cells cultured with organotypic culture methods. We analyzed protein expression patterns in the apical surface fluid (ASF) from aberrantly differentiated squamous metaplastic normal human tracheobronchial epithelial (NHTBE) and mucous NHTBE cells. Comparative two-dimensional PAGE analysis revealed 174 unique proteins in the ASF of squamous NHTBE cells compared with normal mucociliary differentiated NHTBE cells. Among them, 64 well-separated protein spots were identified by liquid chromatography-tandem mass spectrometry, revealing 22 different proteins in the ASF from squamous NHTBE cells. Expression of six of these proteins [SCC antigen 1 (SCCA1), SCC antigen 2 (SCCA2), S100A8, S100A9, Annexin I, and Annexin II] in the squamous NHTBE cells was further confirmed with immunoblot analysis. Notably, SCCA1 and SCCA2 were verified as being expressed in squamous metaplastic NHTBE cells but not in normal mucous NHTBE or normal bronchial epithelium. Moreover, SCCA1 and SCCA2 expression increased in in vitro lung carcinogenesis model cell lines with increasing malignancy. In summary, we identified proteins that are uniquely secreted from squamous metaplastic primary human bronchial epithelial cells cultured by the organotypic air-liquid interface method. These ASF proteins may be used to detect abnormal lesions in the lung without collecting invasive biopsy specimens. [Cancer Res 2007;67(14):6565–73]

Stem cell factor (SCF) has important roles in the proliferation and differentiation of hematopoietic stem cells. The complex of c-Kit and its ligand SCF induce hematopoiesis, melanogenesis, and gametogenesis. However, the mechanism by which SCF induces cell proliferation in the human megakaryoblastic leukemia cell line, MO7e, and the signaling molecules involved, especially in downstream signaling of c-Kit, remain unclear. Here, we show that pharmacological inhibition of the PI3K pathway inhibits SCF/c-Kit signaling and cell proliferation. In addition, we find that the Shc/PDK1/PKC/Akt/c-raf signaling cascade is essential for SCF/c-Kit signal pathway. Our results also suggest that ERK5 is activated and translocated to the nucleus, activating CREB and STAT3. Interestingly, chrysin shuts down the SCF/c-Kit complex-induced signaling cascade. Taken together, these studies give additional insight into the molecular mechanism of SCF/c-Kit-induced cell proliferation and its inverse agonist, chrysin. Finally, these findings enhance our understanding of MO7e cell proliferation.