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Objectives. Air pollution is an increasing global concern, and its effect on allergic inflammation has attracted the attention of many researchers. Particulate matter (PM) is a major component of ambient air pollution, and heavy metals are the primary toxic constituents of PM. As previous studies on the impact of air pollutants on allergic inflammation did not adequately mimic real-world atmospheric exposure, we developed an experimental model to investigate the effects of aerosolized air pollutants on nasal epithelial cells and fibroblasts. Methods. We collected particulate matter 2.5 (PM2.5) samples from ambient 24-hour air samples obtained in Seoul from August 2020 to August 2022, and then conducted component analysis for metallic constituents. Primary nasal epithelial cells and nasal fibroblasts, obtained and cultured from the turbinate tissues of human participants, were treated with PM2.5. The associations of heavy metals identified from the component analysis with cytokine expression were investigated. A three-dimensional (3D)-hybrid culture model, consisting of co-culture of an air-liquid interface and nasal fibroblast spheroids, was constructed to observe the impact of aerosolized air pollutants. Results. Among the heavy metals, Si was the predominant component of PM2.5, and Zn showed the highest correlation with the concentration of PM2.5 in Seoul. PM2.5, Zn, and Si increased the production of epithelial cell-derived cytokines, and PM2.5 and Zn exhibited similar trends with one another. Exposure of the 3D-hybrid model to aerosolized PM2.5 and Zn resulted in elevated periostin, alpha-smooth muscle actin, and fibronectin expression in fibroblast spheroids, and those without an epithelial barrier exhibited a similar increase in periostin expression. Conclusion. Ambient air pollutants in the form of aerosols increase the expression of allergic inflammatory cytokines in both nasal epithelial cells and fibroblasts. Regulations on air pollution will help reduce the global burden of allergic diseases in the future.

BackgroundChronic rhinosinusitis (CRS) is an inflammatory disease affecting more than 10% of the global adult population. It is classified into Th1, Th2, and Th17 endotypes and eosinophilic and non-eosinophilic types. Th2-based inflammation and eosinophilic CRS (ECRS) are associated with tissue remodeling and fibrinolytic system impairment.ObjectiveTo elucidate the role of eosinophils in inducing fibrin deposition in CRS nasal polyp tissues and explore potential regulatory mechanisms.MethodsWe analyzed the expression of genes related to the serpin family and fibrinolytic system using Gene Expression Omnibus and Next-generation sequencing data. Differentially expression genes (DEGs) analysis was used to compare control and nasal polyp tissues, followed by KEGG and Gene ontology (GO) analysis. We measured the expression and correlation of plasminogen activator-1 (PAI-1), tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and urokinase plasminogen activator surface receptor (u-PAR) in CRS tissues, and evaluated the effect of eosinophils on the fibrinolytic system using a cytokine array and co-culture.ResultsNasal polyp tissues showed upregulated PAI-1, u-PA, and u-PAR expression and downregulated t-PA expression. Fibrinolytic system-related genes positively correlated with Th2 cytokines, except for t-PA. Eosinophil-derived Chitinase-3-like protein 1 (CHI3L1) increased PAI-1 expression and decreased t-PA levels in fibroblasts and epithelial cells. The inhibition of CHI3L1 suppresses these alterations.ConclusionCHI3L1 contributes to fibrin deposition by impairing the fibrinolytic system during nasal polyp formation. The regulation of CHI3L1 expression may inhibit fibrin deposition and edema in ECRS, presenting a potential treatment for this condition.

Abstract Many hangover cure products containing natural ingredients that are also effective against alcohol-related liver damage or improve liver function have recently become available. In addition to curing liver damage, antioxidants, anti-inflammatory agents, and blood ethanol reduction aids are emerging as relief targets that reduce hangover symptoms. We investigated the ameliorating effect of WON-21 herbal medicinal products by studying the mixing ratio of oriental medicine concept with respect to antioxidant potential, anti-inflammation, and aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) enzyme activities. WON-21 and its components exerted antioxidant and anti-inflammatory effects. Rutin, taxifolin, and quercetin showed superior antioxidant effects compared to the other components. WON-12 effectively reduced iNOS and COX-2 in LPS-stimulated macrophages. Quercetin and apigenin were 2 compounds effective for the inhibition of iNOS and COX-2. WON-21 and quercetin also significantly increased the activities of ALDH and ADH enzymes in a concentration-dependent manner.

Th2 inflammation and epithelial-mesenchymal transition (EMT) play crucial roles in the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to investigate the hypothesis that MMP-12, produced by M2 macrophages, induces EMT in nasal epithelial cells, thereby contributing to airway inflammation and remodeling in CRSwNP. The expression levels of MMP-12 were measured by RT-PCR in CRS nasal mucosa and THP-1 cells. mRNA and protein levels of E-cadherin, vimentin, α-SMA, and fibronectin were determined using RT-PCR, western blotting, and immunofluorescence staining in primary nasal epithelial cells and air-liquid interface culture. The expression of MMP-12 was significantly increased in CRSwNP and M2-like THP-1 cells. In co-culture with primary nasal epithelial cells and M2-like THP-1 cells, E-cadherin expression was inhibited, and fibronectin, vimentin, and α-SMA expression were increased. MMP-12 decreased E-cadherin but induced fibronectin, vimentin, and α-SMA mRNA and protein expression in primary nasal epithelial cells and air-liquid interface culture. MMP408, an MMP-12 inhibitor, inhibited EMT-related factors. These findings suggest that MMP-12 expression in M2 macrophages induces EMT in nasal epithelial cells and may contribute to the pathogenesis of CRSwNP.

BackgroundChronic rhinosinusitis (CRS) is an inflammatory disease affecting the sinuses or nose. Persistent inflammatory responses can lead to tissue remodeling, which is a pathological characteristics of CRS. Activation of fibroblasts in the nasal mucosal stroma, differentiation and collagen deposition, and subepithelial fibrosis have been associated with CRS.ObjectivesWe aimed to assess the inhibitory effects of doxycycline and deoxycholic acid-polyethyleneimine conjugate (DA3-Doxy) on myofibroblast differentiation and extracellular matrix (ECM) production in nasal fibroblasts stimulated with TGF-β1.MethodsTo enhance efficacy, we prepared DA3-Doxy using a conjugate of low-molecular-weight polyethyleneimine (PEI) (MW 1800) and deoxycholic acid (DA) and Doxy. The synthesis of the DA3-Doxy polymer was confirmed using nuclear magnetic resonance, and the critical micelle concentration required for cationic micelle formation through self-assembly was determined. Subsequently, the Doxy loading efficiency of DA3 was assessed. The cytotoxicity of Doxy, DA3, PEI, and DA-Doxy in nasal fibroblasts was evaluated using the WST-1 assay. The anti-tissue remodeling and anti-inflammatory effects of DA3-Doxy and DA3 were examined using real-time polymerase chain reaction (Real-time PCR), immunocytochemistry, western blot, and Sircol assay.ResultsBoth DA3 and DA3-Doxy exhibited cytotoxicity at 10 μg/ml in nasal fibroblasts. Doxy partially inhibited α-smooth muscle actin, collagen types I and III, and fibronectin. However, DA3-Doxy significantly inhibited α-SMA, collagen types I and III, and fibronectin at 5 μg/ml. DA3-Doxy also modulated TGF-β1-induced changes in the expression of MMP 1, 2, and 9. Nonetheless, TGF-β1-induced expression of MMP3 was further increased by DA3-Doxy. The expression of TIMP 1 and 2 was partially reduced with 5 μg/ml DA3-Doxy.ConclusionsAlthough initially developed for the delivery of genetic materials or drugs, DA3 exhibits inhibitory effects on myofibroblast differentiation and ECM production. Therefore, it holds therapeutic potential for CRS, and a synergistic effect can be expected when loaded with CRS treatment drugs.

Abstract Background Adipose tissue is known to serve as an abundant and readily accessible source of adipose-derived stem cells (ADSCs) as an alternative to bone marrow. Collagenase is one of the most widely used methods for the isolation of ADSCs from adipose tissue, but it takes a long time, and there are also debates about safety. We propose an ultrasonic cavitation-treated method that can significantly reduce time and avoid the problem of using xenogeneic enzymes in ADSCs isolation. Methods ADSCs were isolated from adipose tissue using the enzyme treatment method and the ultrasonic cavitation treatment method. Cell proliferation was measured using cell viability assay. The expression levels of the surface markers of ADSCs were estimated by real-time PCR. After, ADSCs were cultured in chondrogenic, osteogenic, or adipogenic differentiation medium; the differentiation potential of ADCSs was analyzed by Alcian blue, Alizarin Red S, Oil Red O, and real-time PCR. Results The cells treated with collagenase and ultrasound had similar cell yields and proliferation after isolation. The difference in the expression of surface markers of ADSCs was not statistically significant. ADSCs showed differentiation potential into adipocytes, osteocytes, and chondrocytes, and there was no difference between the enzyme treatment method and the ultrasonic cavitation treatment method. The yield of the ADSC increased in time- and intensity dependently. Conclusions Ultrasound certainly serves as a promising method in advancing ADSC isolation technology.

The devastating effects of COVID-19 have highlighted the importance of prophylactic and therapeutic strategies to combat respiratory diseases. Stimulator of interferon gene (STING) is an essential component of the host defense mechanisms against respiratory viral infections. Although the role of the cGAS/STING signaling axis in the innate immune response to DNA viruses has been thoroughly characterized, mounting evidence shows that it also plays a key role in the prevention of RNA virus infections. In this study, we investigated the role of STING activation during Influenza virus (IFV) infection. In both mouse bone marrow-derived macrophages and monocytic cell line THP-1 differentiated with PMA, we found that dimeric amidobenzimidazole (diABZI), a STING agonist, had substantial anti-IFV activity against multiple strains of IFV, including A/H1N1, A/H3N2, B/Yamagata, and B/Victoria. On the other hand, a pharmacological antagonist of STING (H-151) or the loss of STING in human macrophages leads to enhanced viral replication but suppressed IFN expression. Furthermore, diABZI was antiviral against IFV in primary air–liquid interface cultures of nasal epithelial cells. Our data suggest that STING agonists may serve as promising therapeutic antiviral agents to combat IFV.

Background Glycolytic reprogramming is a key feature of chronic inflammatory disease. Extracellular matrix (ECM) produced by myofibroblasts plays an important role in tissue remodeling of nasal mucosa in chronic rhinosinusitis (CRS). This study aimed to determine whether glycolytic reprogramming contributes to myofibroblast differentiation and ECM production in nasal fibroblasts. Methods Primary nasal fibroblasts were isolated from the nasal mucosa of patients with CRS. Glycolytic reprogramming was assessed by measuring the extracellular acidification and oxygen consumption rates in nasal fibroblast, with and without transforming growth factor beta 1 (TGF-β1) treatment. Expression of glycolytic enzymes and ECM components was measured by real-time polymerase chain reaction, western blotting, and immunocytochemical staining. Gene set enrichment analysis was performed using whole RNA-sequencing data of nasal mucosa of healthy donors and patients with CRS. Result Glycolysis of nasal fibroblasts stimulated with TGF-B1 was upregulated along with glycolytic enzymes. Hypoxia-inducing factor (HIF)-1α was a high-level regulator of glycolysis, and increased HIF-1α expression promoted glycolysis of nasal fibroblasts, and inhibition of HIF-1α down-regulated myofibroblasts differentiation and ECM production. Conclusion This study suggests that inhibition of the glycolytic enzyme and HIF-1α in nasal fibroblasts regulates myofibroblast differentiation and ECM generation associated with nasal mucosa remodeling.

Human skin comprises the epidermis and dermis, which perform interactive functional activities with each other in order to maintain the skin’s tensile strength. In particular, the dermal layer is crucial for skin protection. However, skin aging destroys collagen and elastin fibers, causing wrinkles, pigments, and sagging. Skin aging-related factors, such as tumor necrosis factor-α (TNF-α), promote the generation of intercellular reactive oxygen species (ROS). These are known to stimulate the hypersecretion of matrix metalloproteinase-1 (MMP-1), which degrades collagen and inhibits collagen synthesis. In this study, as part of our ongoing discovery of natural products, we investigated potential natural products derived from ginkgo fruit (Ginkgo biloba fruit) with protective effects against TNF-α-induced skin aging. Phytochemical investigation of the MeOH extract of G. biloba fruits, aided by liquid chromatography–mass spectrometry, led to the isolation of 14 compounds (1–14) from the n-butanol-soluble fraction. These were structurally determined to be: (E)-coniferin (1), syringin (2), 4-hydroxybenzoic acid 4-O-β-D-glucopyranoside (3), vanillic acid 4-O-β-D-glucopyranoside (4), glucosyringic acid (5), (E)-ferulic acid 4-O-β-D-glucoside (6), (E)-sinapic acid 4-O-β-D-glucopyranoside (7), ginkgotoxin-5-glucoside (8), ginkgopanoside (9), (Z)-4-coumaric acid 4-O-β-D-glucopyranoside (10), (1′R,2′S,5′R,8′S,2′Z,4′E)-dihydrophaseic acid 3’-O-β-D-glucopyranoside (11), eucomic acid (12), rutin (13), and laricitrin 3-rutinoside (L3R) (14). Biological evaluation of the isolated compounds for their effects on intracellular ROS generation showed that, of these 14 compounds, L3R (14) inhibited TNF-α-stimulated ROS generation (p < 0.001 at 100 μM). Inhibition of ROS generation by L3R led to the suppression of MMP-1 secretion and protection against collagen degradation. The inhibitory effect of L3R was mediated by the inhibition of extracellular signal regulated kinase (ERK) phosphorylation. Furthermore, L3R diminished the secretion of pro-inflammatory cytokines interleukin 6 (IL-6) and interleukin 8 (IL-8). Based on these experimental results, L3R is a potential bioactive natural product that can be used to protect against skin damage, including aging, in cosmetics and pharmaceuticals.

The endemic and pandemic caused by respiratory virus infection are a major cause of mortality and morbidity globally. Thus, broadly effective antiviral drugs are needed to treat respiratory viral diseases. Small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (U-exo) have recently gained attention as a cell-free therapeutic strategy due to their potential for safety and efficacy. Anti-viral activities of U-exo to countermeasure respiratory virus-associated diseases are currently unknown. Here, we tested the antiviral activities of U-exo following influenza A/B virus (IFV) and human seasonal coronavirus (HCoV) infections in vitro. Cells were subject to IFV or HCoV infection followed by U-exo treatment. U-exo treatment significantly reduced IFV or HCoV replication and combined treatment with recombinant human interferon-alpha protein (IFN-α) exerted synergistically enhanced antiviral effects against IFV or HCoV. Interestingly, microRNA (miR)-125b, which is one of the most abundantly expressed small RNAs in U-exo, was found to suppress IFV replication possibly via the induction of IFN-stimulated genes (ISGs). Furthermore, U-exo markedly enhanced RNA virus-triggered IFN signaling and ISGs production. Similarly, human nasal epithelial cells cultured at the air-liquid interface (ALI) studies broadly effective anti-viral and anti-inflammatory activities of U-exo against IFV and HCoV, suggesting the potential role of U-exo as a promising intervention for respiratory virus-associated diseases.

Epithelial-to-mesenchymal transition (EM transition) is a process wherein epithelial cells lose their intrinsic characteristics and cell–cell junctions and differentiate into a mesenchymal phenotype. EM transition is an important feature of cancer invasion and metastasis. In this study, we aimed to investigate the inhibitory effect of gintonin (GT), an ingredient of ginseng, on EM transition using A549 cells. The proliferation of A549 cells was enhanced following treatment with 50, 75, and 100 μg/mL of GT. GT affected EM transition-induced gene and protein expression, specifically that of vimentin (Vim), N-cadherin (N-cad), zinc finger E-box-binding homeobox 1, and Twist in A549 cells. Furthermore, the transforming growth factor beta 1 (TGF-β1)-induced phosphorylation of Smad2 and Smad3 was suppressed by GT treatment. Immunofluorescence staining also showed that GT treatment decreased the TGF-β1-induced expression of Vim and N-cad in A549 cells. Therefore, GT may be used to suppress cancer cell metastasis via maintenance of the cell–cell junction’s integrity. However, further studies are required to pave the way for its translation into clinical application in cancer therapeutics.

BackgroundEosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis (CRS) and is a refractory or intractable disease. However, a reliable clinical marker or an effective treatment strategy has not yet been established. ECRS is accompanied by excessive eosinophil infiltration and Th2 inflammatory response, which is closely related to tissue remodeling in the upper airways.ObjectivesWe sought to investigate the effect of eosinophils on tissue remodeling in ECRS. The purpose of this study was to identify the effects of eosinophils on the expression of pro-inflammatory mediators and extracellular matrix (ECM) in nasal fibroblasts and the key mediators that stimulate them.MethodsButyric acid was used to differentiate EOL-1 cells into eosinophils. We co-cultured differentiated EOL-1 cells and fibroblasts to measure the expression of pro-inflammatory mediators and ECM in fibroblasts. Among the cytokines secreted from the differentiated EOL-1 cells, factors that induced tissue remodeling of fibroblasts were identified.ResultsTreatment with butyric acid (BA) differentiated EOL-1 cells into eosinophils. Differentiated EOL-1 cells induced fibroblasts to produce pro-inflammatory mediators, IL-6 and IL-8, and tissue remodeling factor, VEGF. It also induced myofibroblast differentiation and overexpression of ECM components. Differentiated EOL-1 cells overexpressed osteopontin (OPN), and recombinant OPN increased the expression of IL-6, IL-8, VEGF, and ECM components in nasal fibroblast. OPN was overexpressed in the nasal tissue of patients with ECRS and was associated with the severity of CRS.ConclusionsEosinophil-derived OPN stimulated nasal fibroblasts and contributed to inflammation and tissue remodeling in ECRS. Moreover, the expression level of OPN was proportional to the severity of ECRS. Therefore, OPN regulation is a potential treatment for ECRS.

Plant-derived phytochemicals are emerging as novel agents for protection against chronic disorders. Dangguisu-san is a herbal prescription to invigorate the blood and relieve pain. Among the numerous active constituents of Dangguisu-san, those expected to be effective at inhibiting platelet aggregation were predicted using a network pharmacological method, and their efficacy was experimentally demonstrated. All four identified chemical components, namely chrysoeriol, apigenin, luteolin, and sappanchalcone, suppressed the aggregation of platelets to a certain extent. However, we report, for the first time, that chrysoeriol acts as a strong inhibitor of platelet aggregation. Although additional in vivo studies are needed, among the complex constituents of herbal medicines, the components that exert an inhibitory effect on platelet aggregation were predicted using a network pharmacological method and experimentally confirmed with human platelets.

Nasal drug delivery is advantageous when compared with other routes of drug delivery as it avoids the hepatic first-pass effect, blood–brain barrier penetration, and compliance issues with parenteral administration. However, nasal administration also has some limitations, such as its low bioavailability due to metabolism on the mucosal surface, and irreversible damage to the nasal mucosa due to the ingredients added into the formula. Moreover, the method of nasal administration is not applicable to all drugs. The current review presents the nasal anatomy and mucosal environment for the nasal delivery of vaccines and drugs, as well as presents various methods for enhancing nasal absorption, and different drug carriers and delivery devices to improve nasal drug delivery. It also presents future prospects on the nasal drug delivery of vaccines and drugs.

Objectives Nasal packing after endoscopic sinus surgery is frequently used to control postoperative bleeding, enhance the wound healing process, and prevent lateralization of the middle turbinate, which causes insufficient ventilation. Many biodegradable materials have been developed to reduce pain and mucosal damage during packing removal. The purpose of this study was to compare the efficacy of Guardcel (Genewel Co.) middle meatal packing with a traditional nonabsorbable middle meatal packing, Merocel (Medtronic Xomed), on wound healing and patient satisfaction. Methods In this prospective, single-blind, randomized controlled study, we enrolled 32 consecutive patients (64 nostrils) undergoing bilateral endoscopic sinus surgery at Korea University Guro Hospital from February 2015 to August 2015. Guardcel and Merocel were inserted postoperatively into a randomly assigned side. Objective findings about bleeding, hemostasis, adhesion, and infection were evaluated with nasal endoscopy. Patients’ symptoms including pain and nasal obstruction were evaluated with a visual analog scale. Each evaluation was done at 2–3 days, 1 week, 2 weeks, and 4 weeks after surgery. Results At 2–3 days after endoscopic sinus surgery, the Guardcel side had a significantly less hemostasis time than the Merocel side (P=0.001). During this period, the pain during packing removal was significantly lower on the Guardcel-inserted side than the Merocel-inserted side (P=0.002). At two weeks after surgery, the adhesion score on the Guardcel side was significantly lower than that of the Merocel side (P=0.011). Other parameters during the study follow-up periods were not statistically significant. There were no severe adverse reactions. Conclusion Guardcel, a newly developed packing material, appeared to shorten the hemostasis time and reduce pain sensation at 2–3 days after surgery; it also prevented adhesion formation 2 weeks after surgery when compared with the control. Guardcel can be an effective and safe candidate to replace conventional packing materials after endoscopic sinus surgery.

Abstract To investigate the potential role of vitamin D (1,25(OH)2D3) in preventing the development of nasal polyps, we examined the effect of vitamin D on myofibroblast differentiation and extracellular matrix (ECM) production in TGF-β1-induced nasal polyp-derived fibroblasts (NPDFs) and elucidated the mechanisms underlying its inhibitory effect. 1,25(OH)2D3 significantly reduced expression levels of α-SMA, a myofibroblast marker, and fibronectin, a representative ECM component, in a dose-dependent manner in TGF-β1-induced NPDFs. 1,25(OH)2D3 suppressed activated Smad2/3 in time-course. Up-regulation of α-SMA, fibronectin and phosphorylation of Smad2/3 by TGF-β1 was unaffected by 1,25(OH)2D3 in NPDFs after vitamin D receptor-specific siRNA transfection. We confirmed that the Smad2/3-specific inhibitor SIS3 inactivated Smad2/3 and reduced α-SMA and fibronectin expression. Furthermore, acetylation of histone H3 was compromised by 1,25(OH)2D3, leading to inhibition of collagen 1A1, collagen 1A2 and α-SMA gene expression. Treatment with 1,25(OH)2D3 also significantly suppressed TGF-β1-enhanced contractility and motility in a contraction assay and Transwell migration assay. Finally, 1,25(OH)2D3 had a similar effect in ex vivo organ cultures of nasal polyps. Taken together, our results suggest that 1,25(OH)2D3 might be an effective therapy for nasal polyps by reducing myofibroblast differentiation and ECM production mediated by Smad2/3-dependent TGF-β1 signaling pathways in NPDFs.

Abstract Chronic rhinosinusitis with nasal polyps (CRSwNP) is closely associated with tissue remodeling. Epithelial-to-mesenchymal transition (EMT), a process of tissue remodeling, can be a therapeutic target of CRSwNP. Glucocorticoids are a type of steroid hormone that is used primarily in medical therapy for patients with CRSwNP; however, their effects on EMT in the airway epithelium remain unknown. To investigate the effects of dexamethasone and fluticasone propionate, a class of glucocorticoids, on transforming growth factor-β1 (TGF-β1) -induced EMT, we used A549 cells, human primary nasal epithelial cells (hPNECs) and ex vivo organ culture of the inferior turbinate. TGF-β1 induced changes in cell morphology, suppressed the expression of E-cadherin and enhanced the expression of a-smooth muscle actin, vimentin and fibronectin in A549 cells. However, glucocorticoids inhibited EMT, migration and invasion enhancement by TGF-β1. We found that the induction of phosphorylated ERK, p38 and the activity of Snail and Slug transcription factors by TGF-β1 were suppressed by glucocorticoids. Glucocorticoids also had a similar effect in hPNECs and ex vivo organ cultures of the inferior turbinate. These findings suggest that glucocorticoids might be a useful therapy for preventing tissue remodeling by blocking the EMT initiated by TGF-β1-induced MAPK and Snail/Slug signaling pathways in CRSwNP.

We hypothesized that differences in the microbiome could be a cause of the substantial differences in the symptoms of and treatment options for adult and pediatric patients with chronic rhinosinusitis (CRS). First, we characterized the differences in the nasal microbiomes of pediatric and adult CRS patients. Swabs were obtained from 19 patients with chronic rhinosinusitis (9 children and 10 adults). The bacterial 16S rRNA gene was pyrosequenced to compare the microbiota of the middle meatus. No significant differences were found in species richness and alpha-diversity indices between the two groups. However, in the comparison of diversity between groups using the unweighted pair group method with arithmetic mean (UPGMA) clustering of microbiome taxonomic profiles, we observed a relatively clear separation between the adult and pediatric groups. Actinobacteria had a significantly higher relative abundance in the adult group than in the pediatric group at the phylum level. At the genus level, Corynebacterium showed significantly higher relative abundance in the adult group than in the pediatric group. This is a comparative study between the microbiomes of adult and pediatric CRS patients. We expect this study to be the first step in understanding the pathogenesis of CRS in adults and children using microbiome analysis.

Cigarette smoke exposure has been shown to be associated with chronic rhinosinusitis and tissue remodeling. The present study aimed to investigate the effects of cigarette smoke extract (CSE) on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) production in nasal fibroblasts and to determine the underlying molecular mechanisms. Primary nasal fibroblasts from six patients were isolated and cultured. After the exposure of fibroblasts to CSE, the expression levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured by real-time PCR, ELISA, and immunofluorescence staining. The enzymatic activities of MMP-2 and MMP-9 were measured by gelatin zymography. Reactive oxygen species (ROS) production was analyzed using dichloro-dihydro-fluorescein diacetate and Amplex Red assays. PI3K/Akt phosphorylation and NF-κB activation were determined by Western blotting and luciferase assay. CSE significantly increased MMP-2 expression and inhibited TIMP-2 expression but did not affect MMP-9 and TIMP-1 expression. Furthermore, CSE significantly induced ROS production. However, treatment with ROS scavengers, specific PI3K/Akt inhibitors, NF-κB inhibitor, and glucocorticosteroids significantly decreased MMP-2 expression and increased TIMP-2 expression. Our results suggest that steroids inhibit CSE-regulated MMP-2 and TIMP-2 production and activation through the ROS/ PI3K, Akt, and NF-κB signaling pathways in nasal fibroblasts. CSE may contribute to the pathogenesis of chronic rhinosinusitis by regulating MMP-2 and TIMP-2 expression.

(1) Background: Tissue remodeling and extracellular matrix (ECM) accumulation contribute to the development of chronic inflammatory diseases of the upper airway. Endoplasmic reticulum (ER) stress is considered to be the key signal for triggering tissue remodeling in pathological conditions. The present study aimed to investigate the role of ER-stress in TGF-β1-stimulated nasal fibroblasts and inferior turbinate organ cultures; (2) Methods: Fibroblasts and organ cultures were pretreated with 4-phenylbutyric acid (PBA) and stimulated with TGF-β1 or thapsigargin (TG). Expression of ER-stress markers, myofibroblast marker, and ECM components was measured by Western blotting and real-time PCR. Reactive oxygen species (ROS) were quantified using 2′,7′-dichlorofluorescein diacetate. Cell migration was evaluated using Transwell assays. Contractile activity was measured by collagen contraction assay; (3) Results: 4-PBA inhibited TGF-β1 or TG-induced ER-stress marker expression, phenotypic changes, and ECM. Pre-treatment with ROS scavengers inhibited the expression of TGF-β1-induced ER-stress markers. Migration and collagen contraction of TGF-β1 or TG-stimulated fibroblasts were ameliorated by 4-PBA treatment. These findings were confirmed in ex vivo organ cultures; (4) Conclusions: 4-PBA downregulates TGF-β1-induced ER-stress marker expression, migration, and collagen contraction via ROS in fibroblasts and organ cultures. These results suggest that ER-stress may play an important role in progression of chronic upper airway inflammatory diseases by aiding pathological tissue remodeling.

ObjectivesTrichostatin A (TSA), an inhibitor of histone deacetylase, has been shown to play an important role in attenuating asthmatic inflammation. However, the effect of TSA in allergic rhinitis is not known. The aims of this study were to investigate the effect of TSA on allergic nasal inflammation and on the induction of regulatory T cells in a murine model of allergic rhinitis.MethodsBALB/c mice were sensitized intraperitoneally with ovalbumin (OVA) and then challenged intranasally with OVA. TSA (1 mg/kg) was given to the treatment group, and multiple parameters of allergic responses were evaluated to determine the effects of TSA on allergic rhinitis. Allergic nasal symptom scores, including frequency of rubbing and sneezing, were checked. Eosinophil infiltrations were stained with Chromotrope 2R, and the expression levels of OVA-specific IgE, T-helper 1 (Th1) cytokine (interferon-gamma [IFN-γ]), Th2 cytokines (interleukin [IL] 4 and IL-5) and Treg (Foxp3, IL-10, and transforming growth factor-beta [TGF-β]) were measured by quantitative reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay.ResultsTSA reduced the scores of allergic nasal symptoms and the amount of eosinophil infiltration into the nasal mucosa. TSA suppressed OVA-specific IgE levels and reduced expression of the IL-4 and IL-5. However, the expression of IFN-γ was unchanged in the treatment group. The levels of Foxp3, IL-10, and TGF-β were increased in pretreatment with TSA as compared to control group.ConclusionThis study shows that TSA induced antiallergic effects by decreasing eosinophilic infiltration and Th2 cytokines in a murine model of allergic rhinitis via regulation of Tregs. Thus, TSA may be considered a potentially therapeutic agent in treating allergic rhinitis.

BACKGROUND:In airway epithelium, thymus and activation-regulated chemokine (CCL17) and macrophage-derived chemokine (CCL22) are induced by defective epithelial barriers such as E-cadherin and attract the effector cells of Th2 immunity. However, the association between the epithelial barrier and CCL17 expression has not been studied in chronic rhinosinusitis with nasal polyp (CRSwNP). Thus, we aimed to evaluate the expression of CCL17 and its regulation by Th cytokines in nasal polyp (NP) epithelial cells. METHODS:The expression and distribution of CCL17, CCL22, E-cadherin and/or epidermal growth factor receptor (EGFR) were measured using real-time PCR, western blot, and immunohistochemistry and compared between normal ethmoid sinus epithelium and NP epithelium. In addition, the expression level of CCL17 was determined in cultured epithelial cells treated with IL-4, IL-5, IL-13, TNF-α, and IFN-γ. RESULTS:The expression of CCL17 was decreased in the NP epithelium compared to the epithelium of normal ethmoid sinus, whereas the expression of CCL22 was not decreased. E-cadherin was differentially distributed between the epithelium of normal ethmoid sinus and NP epithelium. EGFR was also decreased in NPs. Interestingly, the stimulation of cultured epithelial cells with Th2 cytokines, IL-4 and IL-5, resulted in an upregulation of CCL17 expression only in NP epithelial cells whereas the expression of CCL17 was increased in both normal epithelial cells and NP epithelial cells by Th1 cytokines. CONCLUSION:Our results suggest that the decreased expression of CCL17 in defective NP epithelium may be closely connected to NP pathogenesis and can be differentially regulated by cytokines in the NP epithelium of patients with CRSwNP.

ObjectivesCaffeic acids are known to have anti-oxidant, anti-inflammatory, immunomodulatory, and tissue reparative effects. The purposes of this study were to determine the effect of caffeic acid on transforming growth factor (TGF) β1-induced myofibroblast differentiation and collagen production, and to determine whether caffeic acid is involved in the antioxidant effect in nasal polyp-derived fibroblasts (NPDFs).MethodsNPDFs were pretreated with caffeic acid (1-10 µM) for 2 hours and stimulated with TGF-β1 (5 ng/mL) for 24 hours. The expression of α-smooth muscle actin (SMA), collagen types I and III, and Nox4 mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein was determined by actin ned by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the Sircol collagen dye-binding assay. The reactive oxygen species (ROS) generated by NPDFs were determined using 2',7'-dichlorfluorescein-diacetate. siNox4 was used to determine the effect of Nox4.ResultsThe expression of α-SMA and production of collagen were significantly increased following TGF-β1 treatment. In contrast, the level of expression of α-SMA and the level of production of collagen were decreased by pretreatment with caffeic acid. The activation of Nox4 and the subsequent production of ROS were also reduced by pretreatment with caffeic acid. The expression of α-SMA was prevented by inhibition of ROS generation with siNox4.ConclusionCaffeic acid may inhibit TGF-β1-induced differentiation of fibroblasts into myofibroblasts and collagen production by regulating ROS.

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF-β1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF-β1. We found that E-cadherin, vimentin, fibronectin, and α-SMA expression was increased in nasal polyps compared to inferior turbinates. TGF-β1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α-SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF-β1 on migration of A549 cells and suppressed TGF-β1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF-β1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF-β1 in upper airway chronic inflammatory disease such as CRS.

Pott's puffy tumor is an infrequent entity characterized by a subperiosteal abscess associated with frontal bone osteomyelitis. It has become rare due to the development of antibiotics and is usually seen as a complication of frontal sinusitis. Although Pott's puffy tumor is more commonly described in children, it should also be included in the differential diagnosis of swelling on the forehead in adults. Once the diagnosis is suspected, appropriate imaging should be performed to evaluate the possible complications. The treatment of Pott's puffy tumor combines medical and surgical approaches in order to prevent further complications. The goal of surgery is to drain the sinus and to excise the infected bone if necessary. The endoscopic endonasal approach is a safe and effective alternative to the external approach. This report describes the case of a 25-year-old man with Pott's puffy tumor resulting from frontal sinusitis.

ObjectivesThe Korean version of the Sniffin' stick (KVSS) test is widely used in Korea to evaluate olfactory function. However, its validity and reliability have not been studied well. In this study, the authors administered the KVSS and the T%T olfactometer test to evaluate olfactory function and to establish relationships between these two test measures.MethodsTwo hundred and eleven patients participated in this prospective randomized study. One hundred and nine patients with no olfactory symptoms and 102 patients with decreased olfaction participated. All participants were underwent KVSS II and T&T olfactometer testing.ResultsThe mean recognition threshold of the T&T olfactometer was -1.8±0.9 for patients with normal olfaction and 4.0±2.6 for patients with decreased olfaction. The mean Threshold-Discrimination-Identification score of the KVSS II was 30.0±3.8 for patients with normal olfaction and 15.9±7.1 for patients with decreased olfaction. Correlation coefficient between the two tests was significantly high (rs=-0.725, P

BACKGROUND:Diesel exhaust particles (DEP) are a major source of air pollution. Nasal fibroblasts are known to produce various cytokines and chemokines. The aim of this study was to evaluate DEP-induced cytokines and chemokines in nasal fibroblasts and to identify the signaling pathway involved. METHODS:A cytokine and chemokine array performed after stimulation of nasal fibroblasts with DEP revealed that levels of IL-6 and IL-8 were increased most significantly among various cytokines and chemokines. RT-PCR and ELISA were used to determine the mRNA and protein expression levels of IL-6 and IL-8. Signaling pathways of p-38, Akt, and NF-κB were analyzed by western blotting, luciferase assay, and ELISA. Organ cultures of nasal interior turbinate were also developed to demonstrate the ex vivo effect of DEP on the expression of IL-6 and IL-8 and the associated signaling pathway. RESULTS:DEP increased the expressions of IL-6 and IL-8 in nasal fibroblasts at mRNA and protein levels. DEP induced phosphorylation of p38, Akt, and NF-κB, whereas inhibitors of p38, Akt, and NF-κB blocked these phophorylations and the expressions of IL-6 and IL-8. These findings were also observed in ex vivo organ culture of nasal inferior turbinate. CONCLUSIONS:DEP induces expression of IL-6 and IL-8 via p38, Akt, and NF-κB signaling pathways in nasal fibroblasts. This finding suggests that air pollution might induce or aggravate allergic rhinitis or chronic rhinosinusitis.

Tissue remodeling is believed to cause recalcitrant chronic rhinosinusitis (CRS). Epithelial-mesenchymal transition (EMT) is a novel clinical therapeutic target in many chronic airway diseases related with tissue remodeling. The aim of this study was to investigate the effect of trichostatin A (TSA) on transforming growth factor (TGF)-β1-induced EMT in airway epithelium and nasal tissue.A549 cells, primary nasal epithelial cells (PNECs), or inferior nasal turbinate organ culture were exposed to TSA prior to stimulation with TGF-β1. Expression levels of E-cadherin, vimentin, fibronectin, α-smooth muscle actin (SMA), histone deacetylase 2 (HDAC2), and HDAC4 were determined by western blotting and/or immunofluorescent staining. Hyperacetylation of histone H2 and H4 by TSA was measured by western blotting. After siHDAC transfection, the effects of HDAC2 and HDAC4 silencing on expression of E-cadherin, vimentin, fibronectin, α-SMA, HDAC2, and HDAC4 in TGF-β1-induced A549 were determined by RT-PCR and/or western blotting. We assessed the change in migration capacity of A549 cells by using cell migration assay and transwell invasion assay.TGF-β1 altered mRNA and protein expression levels of EMT markers including E-cadherin, vimentin, fibronectin, α-SMA, slug, and snail in A549 cells. Inhibition and silencing of HDAC2 and HDAC4 by TSA and siRNA enhanced TGF-β1-induced EMT in A549 cells. TSA blocked the effect of TGF-β1 on the migratory ability of A549 cells. In experiments using PNECs and inferior turbinate organ cultures, TSA suppressed expression of EMT markers induced by TGF-β1.We showed that EMT is induced by TGF-β1 in airway epithelial cells and nasal tissue via activation of HDAC2 and HDAC4, and that inhibition of HDAC2 and HDAC4 by TSA reduces TGF-β1-induced EMT. This observation indicates that histone deacetylase inhibitors such as TSA could be potential candidates for treatment of recalcitrant CRS related with tissue remodeling.

Baicalin, a Chinese herbal medicine, has anti-fibrotic and anti-inflammatory effects. The aims of present study were to investigate the effects of baicalin on the myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction of interleukin (IL)-1β-stimulated nasal fibroblasts and to determine the molecular mechanism of baicalin in nasal fibroblasts.Nasal fibroblasts were isolated from the inferior turbinate of patients. Baicalin was used to treat IL-1β-stimulated nasal fibroblasts. To evaluate cytotoxicity, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. The expression levels of α-smooth muscle actin (SMA), fibronectin, phospho-mitogen-activated protein kinase (p-MAPK), p-Akt, p-p50, p-p65, and p-IκBα were measured by western blotting, reverse transcription-polymerase chain reaction (RT-PCR),or immunofluorescence staining. Fibroblast migration was analyzed with scratch assays and transwell migration assays. Total collagen was evaluated with the Sircol collagen assay. Contractile activity was measured with a collagen gel contraction assay.Baicalin (0-50 μM) had no significant cytotoxic effects in nasal fibroblasts. The expression of α-SMA and fibronectin were significantly down-regulated in baicalin-treated nasal fibroblasts. Migration, collagen production, and contraction of IL-1β-stimulated nasal fibroblasts were significantly inhibited by baicalin treatment. Baicalin also significantly down-regulated p-MAPK, p-Akt, p-p50, p-p65, and p-IκBα in IL-1β-stimulated nasal fibroblasts.We showed that baicalin down-regulated myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction via the MAPK and Akt/ NF-κB pathways in IL-1β-stimulated nasal fibroblasts.

Sclerosing polycyctic adenosis (SPA) is a rare lesion of unknown etiology morphologically resembling fibrocystic changes of the breast. To date, approximately 41 cases of SPA have been reported. Most cases of SPA have originated in the parotid and submandibular glands, with a few cases of intra-oral minor salivary gland origin. This is the first reported case of sclerosing polycystic adenosis of nasal minor salivary gland origin. The differential diagnosis of SPA includes polycystic disease, sclerosing sialadenitis, and benign and malignant glandular neoplasias. Although atypia ranging from mild dysplasia to carcinoma in situ can occur in some cases, SPA has a favorable outcome. It is important to be familiar with SPA to avoid aggresive treatment that results from a misdiagnosis. We present a case of a 49-year-old man who had 1-year history of right nasal obstruction.

Respiratory epithelial adenomatoid hamartoma (REAH) is a rare benign lesion in nasal cavity. We report two cases of REAH of the nasal cavity arising from nasal septum. The etiology of REAH is unknown although inflammation may induce gland proliferation observed in hamartomas. One of our cases was associated with nasal polyposis. REAH is a self-limiting disease, so it is important to differentiate REAH from other pathologic process, including inverted papilloma and low-grade adenocarcinoma. The treatment of choice is complete excision through a conservative approach.

Airway submucosal glands contribute to innate immunity and protect the lungs by secreting mucus, which is required for mucociliary clearance and which also contains antimicrobial, anti-inflammatory, anti-proteolytic and anti-oxidant proteins. We stimulated glands in tracheal trimmings from three lung donors and collected droplets of uncontaminated mucus as they formed at the gland orifices under an oil layer. We analyzed the mucus using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis identified 5486 peptides and 441 proteins from across the 3 samples (269-319 proteins per subject). We focused on 269 proteins common to at least 2 0f 3 subjects, of which 102 (38%) had protective or innate immunity functions. While many of these have long been known to play such roles, for many others their cellular protective functions have only recently been appreciated in addition to their well-studied biologic functions (e.g. annexins, apolipoproteins, gelsolin, hemoglobin, histones, keratins, and lumican). A minority of the identified proteins are known to be secreted via conventional exocytosis, suggesting that glandular secretion occurs via multiple mechanisms. Two of the observed protective proteins, major vault protein and prohibitin, have not been observed in fluid from human epithelial cultures or in fluid from nasal or bronchoalveolar lavage. Further proteomic analysis of pure gland mucus may help clarify how healthy airways maintain a sterile environment.

To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a β-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

Nasal polyposis is characterized by persistent inflammation and remodeling in sinonasal mucosa. Toll-like receptors (TLRs) play a role in the innate immune response to microbes in the sinonasal cavity. The aim of this study was to evaluate whether nasal polyp-derived fibroblasts (NPDFs) and organ-cultured nasal polyps can synthesize pro-inflammatory cytokines and matrix metalloproteinases (MMPs) after exposure to lipopolysaccharide (LPS), a TLR4 agonist. NPDFs and organ-cultured nasal polyps were isolated from nasal polyps of 8 patients and exposed to LPS. The mRNA and protein expression levels of TLRs, cytokines, and MMPs were determined using a gene expression microarray, real-time RT-PCR, western blot analysis, enzyme-linked immunosorbent assay, and immunofluorescence staining. The enzymatic activities of MMPs were analyzed using collagen or gelatin zymography. The protein expression level of MMP-1 increased in nasal polyp tissues compared to inferior turbinate tissues. LPS induced mRNA expression of TLR4, IL-6, IL-8, and MMP-1 and activated MAPK signaling in NPDFs. LPS promoted the release of interleukin (IL)-6 through extracellular signal-related kinase (ERK) and IL-8 through ERK and c-Jun N-terminal kinases (JNK). Production of IL-6 and IL-8 was induced by PI3K/Akt signaling in LPS-stimulated NPDFs. LPS increased the transcript and protein expression levels of MMP-1 and induced collagenase activity of MMP-1 via ERK and p38, but did not induce gelatinase activity of MMP-2 and MMP-9. LPS from Rhodobacter sphaeroides (LPS-RS) inhibited the stimulatory effects of LPS in NPDFs as well as in organ culture of nasal polyp. LPS triggers immune response via TLR 4 and activates MAPK and PI3K/Akt signaling pathway, which is involved in remodeling of nasal polyps.

To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (~50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ~0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.