Qualitative and Quantitative Analysis of the Major Bioactive Components of Juniperus chinensis L. Using LC-QTOF-MS and LC-MSMS and Investigation of Antibacterial Activity against Pathogenic Bacteria.

Plants in the genus Juniperus have been reported to produce a variety of chemical components, such as coumarins, flavonoids, lignans, sterols, and terpenoids. Here, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were applied to qualitatively and quantitatively analyze the major bioactive components in an ethanolic crude extract from the leaves of Juniperus chinensis L., which grows naturally in Korea. In addition, the antibacterial activity of the crude extract against pathogenic bacteria was investigated. Using LC-QTOF-MS analysis, we identified ten compounds, of which six were confirmed to be flavonoid and lignan-based components as the major bioactive components, i.e., isoquercetin, quercetin-3-O-α-l-rhamnoside, hinokiflavone, amentoflavone, podocarpusflavone A, and matairesinoside. Among them, a quantitative analysis performed using LC-MS/MS revealed that the levels of quercetin-3-O-α-l-rhamnoside and amentoflavone in the crude extract were 203.78 and 69.84 mg/g, respectively. Furthermore, the crude extract exhibited potential antibacterial activity against 10 pathogenic bacteria, with the highest antibacterial activity detected against Bordetella pertussis. Thus, further studies of the leaf extract of J. chinensis L. must be carried out to correlate the compounds present in the extract with the antibacterial activity and elucidate the mechanisms of action of this extract against bacteria.

Identification and Comparison of Bioactive Components of Two Dryopteris sp. Extract Using LC-QTOF-MS.

Dryopteris sp. is known for its various pharmacological effects and is used as a traditional medicine in Asia. The present study investigated the chemical composition and antimicrobial activity of Dryopteris sp. distributed in Korea. The chemical compounds in the ethanolic extracts of Dryopteris lacera and Dryopteris bissetiana were investigated by ultra-high performance liquid chromatography-quadrupole time-of-flight-mass spectrometry analysis and identified by exploring the UNIFI traditional medicine library. Flavonoids such as juglanin, 6-hydroxyluteolin 7-O-laminaribioside, peltatoside, kaempferitrin, hyperoside, and astragalin were identified in both D. lacera and D. bissetiana. Neochlorogenic acid was identified as a caffeoylquinic acid in D. bissetiana. Both extracts of D. lacera and D. bissetiana exhibited antibacterial activity against Gram-positive pathogens, Staphylococcus aureus and Streptococcus mutans. The minimum inhibitory concentration of D. bissetiana against S. aureus was less than 625 ppm. The antibacterial activity was attributed to the identified phenolic compounds, juglanin, 6-hydroxyluteolin 7-O-laminaribioside, kaempferitrin, astragalin, and neochlorogenic acid. Therefore, D. lacera and D. bissetiana can be used as Gram-positive selective antibiotics for further investigation.

Bacillus velezensis TSA32-1 as a Promising Agent for Biocontrol of Plant Pathogenic Fungi.

The use of synthetic fungicides has caused major problems such as soil and water pollution and negatively affects non-target species. Microbial biocontrol agents are needed for crop disease management to reduce agrochemical use. Bacillus and related genera produce secondary metabolites with agricultural applications, such as the pathogen-control agent Bacillus velezensis. We isolated B. velezensis TSA32-1 from soil and identified its characteristics by sequencing its 16S rRNA. B. velezensis TSA32-1 showed enzyme activity and antimicrobial effects against phytopathogenic fungi by inhibiting the growth of Fusarium graminearum, F. fujikuroi, Alternatia alternate, and Diaporthe actinidiae. Additionally, B. velezensis TSA32-1 protected diseases in corn and pepper seeds caused by F. graminearum and Pythium ultimum. The complete genome of B. velezensis TSA32-1 was 4.05 Mb with a G+C content of 46.3 mol % and possessed the bacillaene biosynthesis cluster, a polyketide that inhibits protein biosynthesis. We also detected a surfactin synthesis cluster, known as non-ribosomal peptide synthetases, which biosynthesizes the antibacterial substance lipopeptide. Surfactin, and fengycin family compounds, secondary metabolites known as key factors in biological control, also detected B. velezensis TSA32-1 which shows potential as a biocontrol agent for controlling plant pathogens in agriculture.

Middle East Respiratory Syndrome Coronavirus Superspreading Event Involving 81 Persons, Korea 2015.

Since the first imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection was reported on May 20, 2015 in Korea, there have been 186 laboratory-confirmed cases of MERS-CoV infection with 36 fatalities. Ninety-seven percent (181/186) of the cases had exposure to the health care facilities. We are reporting a superspreading event that transmitted MERS-CoV to 81 persons at a hospital emergency room (ER) during the Korean outbreak in 2015. The index case was a 35-yr-old man who had vigorous coughing while staying at the ER for 58 hr. As in severe acute respiratory syndrome outbreaks, superspreading events can cause a large outbreak of MERS in healthcare facilities with severe consequences. All healthcare facilities should establish and implement infection prevention and control measure as well as triage policies and procedures for early detection and isolation of suspected MERS-CoV cases.

Association between occupational dust exposure and prognosis of idiopathic pulmonary fibrosis: a Korean national survey.

BACKGROUND: Previous studies have investigated the relationship between occupational and environmental agents and idiopathic pulmonary fibrosis (IPF). However, there have been few studies regarding the prognosis of patients with IPF according to patient occupation. METHODS: We investigated whether occupational dust exposure was associated with clinically decreased lung function and poor prognosis. The Korean Interstitial Lung Disease Research Group conducted a national survey to evaluate the clinical, physiologic, radiologic, and survival characteristics of patients with IPF. A total of 1,311 patients with IPF were stratified into five groups according to their occupation: (1) unemployed or homemakers (n = 628); (2) farmers, fishers, or ranchers (n = 230); (3) sales or service personnel (n = 131); (4) clerical or professional personnel (n = 151); and (5) specific dust-exposed workers (n = 171). RESULTS: The mean age of subjects at diagnosis, was 67.5 ± 9.7 years. Current smokers were 336 patients, 435 were exsmokers, and 456 were never smokers. Dust-exposed workers showed early onset of IPF (61.3 ± 8.6 years; P < .001) and a longer duration of symptoms at diagnosis (17.0 ± 28.2 months; P = .004). Aging (P = .001; hazard ratio [HR], 1.034; 95% CI, 1.014-1.054), FVC % predicted at diagnosis (P = .004; HR, 0.984; 95% CI, 0.974-0.995), and dust-exposure occupation (P = .033; HR, 1.813; 95% CI, 1.049-3.133) were associated with mortality. CONCLUSIONS: These findings indicate that occupational dust may be an aggravating factor associated with a poor prognosis in IPF.

Idiopathic pleuroparenchymal fibroelastosis presenting in recurrent pneumothorax: a case report.

Idiopathic pleuroparenchymal fibroelastosis (PPFE) is a rare, recently classified entity that consists of pleural and subjacent parenchymal fibrosis predominantly in the upper lungs. In an official American Thoracic Society/European Respiratory Society statement in 2013, this disease is introduced as a group of rare idiopathic interstitial pneumonias. We describe a case of a 76-year-old woman with cough and recurrent pneumothorax. She was admitted to our hospital with severe cough at first. High resolution computed tomography (HRCT) disclosed multifocal subpleural consolidations with reticular opacities in both lungs, primarily in the upper lobes, suggesting interstitial pneumonia. Rheumatoid lung was diagnosed initially through an elevated rheumatoid factor, HRCT and surgical biopsy at the right lower lobe. However, one month later, pneumothorax recurred. Surgical biopsy was performed at the right upper lobe at this time. The specimens revealed typical subpleural fibroelastosis. We report this as a first case of idiopathic PPFE in Korea after reviewing the symptoms, imaging and pathologic findings.

A multicenter phase II study of belotecan, a new camptothecin analogue, in elderly patients with previously untreated, extensive-stage small cell lung cancer.

PURPOSE: Belotecan is a new camptothecin analogue and a potent topoisomerase I inhibitor. The aim of this phase II study was to investigate the efficacy and toxicity of belotecan in previously untreated elderly patients with small cell lung cancer (SCLC). METHODS: A total of 26 patients, aged ≥65 years, with previously untreated, extensive-stage SCLC were enrolled in the study. Belotecan was administered by daily intravenous infusion at 0.5 mg/m(2)/day for 5 consecutive days every 3 weeks. RESULTS: The overall response rate and disease control rate of chemotherapy on an intention-to-treat basis were 35 and 54 %, respectively. The median overall survival was 6.4 months, and the median time to progression was 2.8 months. The most common toxicity was hematologic. Grade 3 or 4 neutropenia occurred in 80.8 % of patients, and grade 3 or 4 thrombocytopenia in 15.3 %. Non-hematologic toxic effects of grade 3 or 4 were uncommon. CONCLUSION: Belotecan had modest efficacy and well-tolerated toxicity in previously untreated, elderly SCLC patients. Single belotecan could be a promising treatment option, considering its lower toxicity in elderly patients who are unsuitable candidates for platinum plus etoposide chemotherapy.

Thrombin induces epithelial-mesenchymal transition via PAR-1, PKC, and ERK1/2 pathways in A549 cells.

Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts. The origins of myofibroblasts are resident fibroblasts, fibrocytes, and epithelial-mesenchymal transition (EMT). We investigated the effects of thrombin, an important mediator of interstitial lung fibrosis, on EMT in A549 human alveolar epithelial cells. We show that thrombin induced EMT and collagen I secretion through the activation of PAR-1, and PKC and ERK1/2 phosphorylation in A549 cells. These effects were largely prevented by a specific PAR-1 antagonist, short interfering RNA (siRNA) directed against PAR-1, or specific PKCα/ß, δ, and ε inhibitors. These data indicated that interaction with thrombin and alveolar epithelial cells might directly contribute to the pathogenesis of pulmonary fibrosis through EMT. Targeting PAR-1 on the pulmonary epithelium or specific inhibitors to PKCα/ß, δ, and ε might stop the fibrotic processes in human idiopathic pulmonary fibrosis by preventing thrombin-induced EMT.

Clinical Utility of Two Interferon-gamma Release Assays on Pleural Fluid for the Diagnosis of Tuberculous Pleurisy.

BACKGROUND: The release of interferon-gamma (IFN-γ) by T lymphocytes increases after rechallenge with Mycobacterium tuberculosis antigen, especially, at a localized site of tuberculosis (TB) infection. We aimed to compare the clincial efficacy of two commercial IFN-γ release assays from pleural fluid for the diagnosis in tuberculous pleurisy. METHODS: We performed T-SPOT.TB and QuantiFERON-TB Gold tests simultaneously on pleural fluid and peripheral blood samples from patients with pleural effusion, in South Korea, an area with intermediate TB burden. RESULTS: Thirty-six patients were enrolled prospectively, and tuberculous pleurisy was found in 21 patients. Both the numbers of IFN-γ secreting T cells and the concentration of IFN-γ were greater in the pleural tuberculous group, comparing with the non-tuberculous group. Moreover, in the tuberculous group, there was a significant difference in IFN-γ producing spot-forming cells using the T-SPOT.TB method between pleural fluid and peripheral blood. The receiver operating characteristic (ROC) curve, was the greatest for pleural fluid T-SPOT.TB test, followed by peripheral blood T-SPOT.TB test, peripheral blood QuantiFERON-TB Gold test, and pleural fluid QuantiFERON-TB Gold test (area under the ROC curve of 0.956, 0.890, 0.743, and 0.721, respectively). The T-SPOT.TB assay produced less indeterminate results than did QuantiFERON-TB Gold assay in both pleural fluid and peripheral blood. CONCLUSION: These findings suggest that the pleural fluid T-SPOT.TB test could be the most useful test among the IFN-γ release assays for diagnosing tuberculous pleurisy in an area with an intermediate prevalence of TB infection.

Inhibitory effect of receptor for advanced glycation end products (RAGE) on the TGF-ß-induced alveolar epithelial to mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is a lethal parenchymal lung disease characterized by myofibroblast proliferation. Alveolar epithelial cells (AECs) are thought to produce myofibroblasts through the epithelial to mesenchymal transition (EMT). Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation is associated with renal fibrosis during diabetes and liver fibrosis. RAGE is expressed at low basal levels in most adult tissues except the lung. In this study, we evaluated the interaction of ligand advanced glycation end products (AGE) with RAGE during the epithelial to myofibroblast transition in rat AECs. Our results indicate that AGE inhibited the TGF-ß-dependent alveolar EMT by increasing Smad7 expression, and that the effect was abolished by RAGE siRNA treatment. Thus, the induction of Smad7 by the AGE-RAGE interaction limits the development of pulmonary fibrosis by inhibiting TGF-ß-dependent signaling in AECs.

A multicenter phase II study of belotecan, a new camptothecin analogue, as a second-line therapy in patients with small cell lung cancer.

Belotecan (Camtobell, CKD602) is a new camptothecin derivative antitumor agent that belongs to the topoisomerase inhibitors. The aim of this phase II study was to evaluate the efficacy and safety of single agent belotecan as a second-line therapy in patients with small cell lung cancer (SCLC). Patients who were previously treated for SCLC were entered into the study. Belotecan was given by daily intravenous infusion for five consecutive days, every three weeks. Twenty-five patients were enrolled in this study. On an intention-to-treat basis, belotecan induced an overall response rate of 24%, a median overall survival of 9.9 months, a median time to progression of 2.2 months, and a 1-year survival rate of 38.3%. Grade 3/4 neutropenia developed in 88.0% of patients and grade 3/4 thrombocytopenia in 40.0%. Nonhematologic toxicity of grade 3 or 4 was low. The results suggest that belotecan is relatively active and well tolerated as a second-line agent in patients with SCLC.

Inhibitory effects of anti-immunoglobulin E antibodies on airway remodeling in a murine model of chronic asthma.

BACKGROUND: Airway remodeling is one of the cardinal features of asthma and is thought to play a pivotal role in refractory or persistent asthma. Immunoglobulin E (IgE) has a major effect on the pathogenesis of asthma. The aim of this study was to investigate the effects of anti-IgE antibody not only on airway inflammation and bronchial hyperresponsiveness, but also on airway remodeling in a murine model of chronic asthma. METHODS: The authors developed a mouse model of chronic asthma in which ovalbumin (OVA)-sensitized female BALB/c-mice were exposed to intranasal OVA administration twice a week for 3 months. Anti-IgE antibodies were administered intravenously starting on the 38th day and once a month thereafter for 3 months during the intranasal OVA challenge. RESULTS: Mice that were chronically exposed to OVA developed sustained eosinophilic airway inflammation and airway hyperresponsiveness (AHR) to methacholine and showed increased levels of collagen, hydroxyproline, and alpha-smooth muscle actin, as compared with control mice. Treatment with anti-IgE antibody inhibited the development of AHR, eosinophilic inflammation, and airway remodeling. Moreover, anti-IgE antibody treatment reduced the levels of interleukin (IL)-5 and IL-13 in the bronchoalveolar lavage fluids, although it did not affect the levels of IL-10, transforming growth factor-beta, and activin A. CONCLUSION: These results suggest that anti-IgE antibody treatment modulates the airway inflammation and remodeling associated with chronic allergen challenge. The inhibition of inflammation may be related to the regulation of Th2 cytokines. However, the mechanisms underlying the blocking of airway remodeling by anti-IgE antibody remain to be elucidated.

Inhibitory effect of CXC chemokine receptor 4 antagonist AMD3100 on bleomycin induced murine pulmonary fibrosis.

CXC chemokine receptor 4 (CXCR4), which binds the stromal cell-derived factor-1 (SDF-1), has been shown to play a critical role in mobilizing the bone marrow (BM)-derived stem cells and inflammatory cells. We studied the effects of AMD3100, CXCR4 antagonist, on a murine bleomycin-induced pulmonary fibrosis model. Treatment of mice with AMD3100 in bleomycin-treated mice resulted in the decrease of SDF-1 in bronchoalveolar lavage (BAL) fluids at an early stage and was followed by the decrease of fibrocytes in the lung. AMD3100 treatment decreased the SDF-1 mRNA expression, fibrocyte numbers in the lung at an early stage (day 3) and CXCR4 expression at the later stage (day 7 and 21) after bleomycin injury. The collagen content and pulmonary fibrosis were significantly attenuated by AMD3100 treatment in later stage of bleomycin injury. AMD3100 treatment also decreased the murine mesenchymal and hematopoietic stem cell chemotaxis when either in the stimulation with bleomycin treated lung lysates or SDF-1 in vitro. In BM stem cell experiments, the phosphorylation of p38 MAPK which was induced by SDF-1 was significantly blocked by addition of AMD3100. Our data suggest that AMD3100 might be effective in preventing the pulmonary fibrosis by inhibiting the fibrocyte mobilization to the injured lung via blocking the SDF-1/CXCR4 axis.

Effects of elastase inhibitor on the epithelial cell apoptosis in bleomycin-induced pulmonary fibrosis.

Alveolar epithelial cell injury and apoptosis is consistent findings in human idiopathic pulmonary fibrosis (IPF). Epithelial cell apoptosis is known to be induced by leukocyte elastase in vitro. The authors hypothesized that synthetic neutrophil elastase inhibitor, sivelestat (ONO-5046), can inhibit the bleomycin-induced pulmonary fibrosis in rats by blocking the apoptotic pathways in epithelial cells. Adult rats were injected with intratracheal bleomycin. Sivelestat was given for 13 days intraperitoneally after bleomycin treatments. Similar experiments were carried out in which A549 cells, a human alveolar type II epithelial cell line, were treated with bleomycin or neutrophil elastase. In rats, sivelestat decreased neutrophil counts and the cytokine-induced neutrophil chemoattractant (CINC)-1 in the bronchoalveolar lavage (BAL) fluid of bleomycin-treated rats. Sivelestat also decreased the bleomycin-induced lung inflammatory cell apoptosis by decreasing caspase-3 and -9 activities. In A549 cells, sivelestat decreased the elastase-induced epithelial cell apoptosis but not the bleomycin-induced epithelial cell apoptosis. Similarly, sivelestat inhibited the elastase-induced cell death but not the bleomycin-induced cell death in MTT assays. Sivelestat also inhibited the elastase-induced caspase-3 and -9 activities and cytochrome c release from the mitochondria but did not inhibit the bleomycin-induced caspase activities in A549 cells. In conclusion, bleomycin caused the lung inflammatory cell apoptosis through the caspase-9 and -3 pathways in rats. Sivelestat inhibited pulmonary fibrosis by blocking these mitochondria-mediated apoptotic pathways in bleomycin-treated rats and in elastase-treated A549 cells. These findings suggest that sivelestat can suppress the bleomycin-induced pulmonary fibrosis by blocking neutrophil chemotaxis and by inhibiting the neutrophil elastase-induced lung cell apoptosis in rats.

Risk factors for acute respiratory distress syndrome during neutropenia recovery in patients with hematologic malignancies.

INTRODUCTION: Neutropenia recovery may be associated with deterioration in oxygenation and exacerbation of pre-existing pulmonary disease. However, risk factors for acute respiratory distress syndrome (ARDS) during neutropenia recovery in patients with hematologic malignancies have not been studied. METHODS: We studied critically ill patients with hematologic malignancies with the dual objectives of describing patients with ARDS during neutropenia recovery and identifying risk factors for ARDS during neutropenia recovery. A cohort of consecutive neutropenic patients with hematologic malignancies who were admitted to the intensive care unit (ICU) was studied. During a 6-year period, 71 patients recovered from neutropenia, of whom 38 (53.5%) developed ARDS during recovery. RESULTS: Compared with non-ARDS patients, patients who experienced ARDS during neutropenia recovery were more likely to have pneumonia, be admitted to the ICU for respiratory failure, and receive mechanical ventilator therapy. The in-ICU mortality was significantly different between the two groups (86.8% versus 51.5%, respectively, for patients who developed ARDS during neutropenia recovery versus those who did not during neutropenia recovery). In multivariate analysis, only occurrence of pneumonia during the neutropenic episode was associated with a marked increase in the risk of ARDS (odds ratio, 4.76). CONCLUSIONS: Patients with hematologic malignancies complicated by pneumonia during neutropenia are at increased risk for ARDS during neutropenia recovery.

The soluble tumor necrosis factor-alpha receptor suppresses airway inflammation in a murine model of acute asthma.

PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma. TNF-alpha blocking strategies are now being tried in asthma patients. This study investigated whether TNF-alpha blocking therapy inhibits airway inflammation and airway hyperresponsiveness (AHR) in a mouse model of asthma. We also evaluated the effect of TNF-alpha blocking therapy on cytokine production and adhesion molecule expression. MATERIALS AND METHODS: Ovalbumin (OVA) sensitized BALB/c female mice were exposed to intranasal OVA administration on days 31, 33, 35, and 37. Mice were treated intraperitoneally with soluble TNF-alpha receptor (sTNFR) during the OVA challenge. RESULTS: There were statistically significant decreases in the numbers of total cell and eosinophil in bronchoalveolar lavage fluid (BALF) in the sTNFR treated group compared with the OVA group. However, sTNFR-treatment did not significantly decrease AHR. Anti-inflammatory effect of sTNFR was accompanied with reduction of T helper 2 cytokine levels including interleukin (IL)-4, IL-5 and IL-13 in BALF and vascular cell adhesion molecule 1 expression in lung tissue. CONCLUSION: These results suggest that sTNFR treatment can suppress the airway inflammation via regulation of Th2 cytokine production and adhesion molecule expression in bronchial asthma.

Inflammatory and remodeling events in asthma with chronic exposure to house dust mites: a murine model.

Although animal models with ovalbumin have been used to study chronic asthma, there are difficulties in inducing recurrence as well as in maintaining chronic inflammation in this system. Using a murine model of house dust mite (HDM)-induced bronchial asthma, we examined the airway remodeling process in response to the chronic exposure to HDM. During the seventh and twelfth weeks of study, HDM were inhaled through the nose for three consecutive days and airway responsiveness was measured. Twenty-four hours later, bronchoalveolar lavage and histological examination were performed. The degree of overproduction of mucus, subepithelial fibrosis, and the thickness of the peribronchial smooth muscle in the experimental group was clearly increased compared to the control group. In addition, HDM-exposed mice demonstrated severe airway hyperreactivity to methacholine. In the bronchoalveolar lavage fluid, the number of total cells and eosinophils was increased; during the twelfth week, the number of neutrophils increased in the experimental group. With regard to changes in cytokines, the concentrations of IL-4, IL- 13, and transforming growth factor-beta (TGF-beta) were increased in the experimental group. The data suggest that eosinophils, IL-4, IL-13, and TGF-beta might play an important role in the airway remodeling process and that neutrophils may be involved with increased exposure time.

Time sequence of airway remodeling in a mouse model of chronic asthma: the relation with airway hyperresponsiveness.

During the course of establishing an animal model of chronic asthma, we tried to elucidate the time sequence of airway hyperresponsiveness (AHR), airway inflammation, airway remodeling, and associated cytokines. Seven-week-old female BALB/c mice were studied as a chronic asthma model using ovalbumin (OVA). After sensitization, mice were exposed twice weekly to aerosolized OVA, and were divided into three groups depending on the duration of 4 weeks, 8 weeks, and 12 weeks. At each time point, airway responsiveness, inflammatory cells, cytokines in bronchoalveolar lavage fluids (BALF), serum OVA-specific IgE, IgG1, IgG2a, and histological examination were carried out. AHR to methacholine, increased levels of OVA-specific IgG1 and IgG2a, and goblet cell hyperplasia were continuously sustained at each time point of weeks. In contrast, we observed a time-dependent decrease in serum OVA-specific IgE, BALF eosinophils, BALF cytokines such as IL-13, transforming growth factor-beta1, and a time-dependent increase in BALF promatrix metalloproteinase-9 and peribronchial fibrosis. In this OVA-induced chronic asthma model, we observed airway remodelings as well as various cytokines and inflammatory cells being involved in different time-dependent manners. However, increased airway fibrosis did not directly correlate with a further increase in airway hyperresponsiveness.

Nitric oxide induces MUC5AC mucin in respiratory epithelial cells through PKC and ERK dependent pathways.

BACKGROUND: Nitric oxide (NO) is generally increased during inflammatory airway diseases. This increased NO stimulates the secretion of mucin from the goblet cell and submucosal glands but the mechanism is still unknown precisely. In this study, we investigated potential signaling pathways involving protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the NO-induced MUC5AC mucin gene and protein expression in A549 cells. METHODS: Nitric oxide was donated to the A549 cells by NOR-1. MUC5AC mucin levels were assayed by enzyme-linked immunosorbent assay (ELISA). MUC5AC promoter activity was determined by measuring luciferase activity after the lysing the transfected cells. Activation of PKC isoforms were measured by assessing the distribution of the enzyme between cytosolic and membrane fractions using immunoblotting. Immunoblotting experiments using a monoclonal antibody specific to PKC isoforms were performed in the cytosol and membrane fractions from A549 cells. Western blot analysis for pERK and p38 were performed using the corresponding antibodies from the cell lysates after donating NO to the A549 cells by NOR-1. RESULTS: The transcriptional activity of MUC5AC promoter was maximal at the concentration of 0.1 mM NOR-1 for 1 hour incubation in transfected A549 cells. (+/-)-(E)-methyl-2-((E)-hydroxyimino)-5-nitro-6-methoxy-3-hexenamide (NOR-1) markedly displaced the protein kinase C (PKC)alpha and PKCdelta from the cytosol to the membrane. Furthermore, the PKC-alpha,betainhibitors, GO6976 (10 nM) and PKCdelta inhibitors, rottlerin (4 muM) inhibited the NOR-1 induced migration of PKCalpha and PKCdelta respectively. NOR-1 also markedly increased the MUC5AC promoter activity and mRNA expression, mucin synthesis and ERK1/2 phosphorylation. The PKC inhibitors also inhibited the NOR-1 induced MUC5AC mRNA and MUC5AC protein synthesis by inhibiting the activation of PKCalpha and PKCdelta with ERK1/2 pathways. CONCLUSION: Exogenous NO induced the MUC5AC mucin gene and protein through the PKCalpha and PKCdelta-ERK pathways in A549 cells. Inhibition of PKC attenuated NO-mediated MUC5AC mucin synthesis. In view of this findings, PKC inhibitors might be useful in the treatment of bronchial asthma and chronic bronchitis patients where NO and mucus are increased in the bronchial airways.

Co-administration of vaccination with DNA encoding T cell epitope on the Der p and BCG inhibited airway remodeling in a murine model of chronic asthma.

Therapeutic modalities of airway remodeling in asthma have proved to be unsuccessful regarding reversing the previously established chronic airway changes. Recently, the potential of plasmid DNA to inhibit the Th2 immune response has been demonstrated in animal models of asthma. Bacillus Calmette-Guerin (BCG) immunization also induced immunomodulation, which appeared to be reliant on the properties of the interferon-gamma that was produced. Mice were immunized with house dust mite extract (HDM). At the 3 week point, we injected BCG subcutaneously into mice on three successive weeks. One week after the BCG injection, we immunized mice with the DNA plasmid encoding for murine T-cell epitope on Dermatophagoide pteronyssinus 2 thrice weekly. At 9 weeks after immunization, we measured airway responsiveness. Twenty four hours later, we performed bronchoalveolar lavage and histological examinations. Co-administration of DNA vaccination and BCG resulted in a partial suppression of the overproduction of goblet cells and the thickness of the peribronchial smooth muscle in ongoing allergic responses. In the bronchoalveolar lavage fluid, the number of total cells and eosinophils was reduced, and regarding the change of cytokines, the concentration of IL-4 was also decreased, but interferon-gamma was increased in the co-administration group, opposed to the asthma group. These results suggest that co-administration of vaccination with the DNA encoding T-cell epitope and BCG are effective regarding ongoing allergic response and might constitute an ideal method for combating allergic disease in the future.