Inhibition of phosphodiesterase activity, airway inflammation and hyperresponsiveness by PDE4 inhibitor and glucocorticoid in a murine model of allergic asthma

Phosphodiesterase 4 (PDE4) isozyme plays important roles in inflammatory and immunomodulatory cells. In this study, piclamilast, a selective PDE4 inhibitor, was used to investigate the role of PDE4 in respiratory function and inflammation in a murine asthma model. Sensitized mice were challenged with aerosolized ovalbumin for 7 days, piclamilast (1, 3 and 10 mg/kg) and dexamethasone (2 mg/kg) were orally administered once daily during the period of challenge. Twenty-four hours after the last challenge, airway hyperresponsiveness to methacholine was determined by whole-body plethysmography, airway inflammation and mucus secretion by histomorphometry, pulmonary cAMP-PDE activity by HPLC, cytokine levels in bronchoalveolar lavage fluid and their mRNA expression in lung by ELISA and RT-PCR, respectively. In control mice, significant induction of cAMP-PDE activity was parallel to the increases of hyperresponsiveness, inflammatory cells, cytokine levels, mRNA expression as well as goblet cell hyperplasia. However, piclamilast dose-dependently and significantly improved airway resistance and dynamic compliance, and the maximal effect was similar to that of dexamethasone. Piclamilast treatment dose-dependently and significantly prevented the increase in inflammatory cell number and goblet cell hyperplasia, as well as production of cytokines, including eotaxin, TNFalpha and IL-4. Piclamilast exerted a weaker inhibitory effect than dexamethasone on eosinophils and neutrophils, had no effect on lymphocyte accumulation. Moreover, piclamilast inhibited up-regulation of cAMP-PDE activity and cytokine mRNA expression; the maximal inhibition of cAMP-PDE was greater than that exerted by dexamethasone, and was similar to dexamethasone on cytokine mRNA expression. This study suggests that inhibition of PDE4 by piclamilast robustly improves the pulmonary function, airway inflammation and goblet cell hyperplasia in murine allergenic asthma.     

The epithelial cholinergic system of the airways

Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types-ciliated, basal and secretory-are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs.     

Theoretical and experimental evaluation of the effects of an argon gas mixture on the pressure drop through adult tracheobronchial airway replicas

Argon has the potential to be a novel inhaled therapeutic agent, owing to the neuroprotective and organoprotective properties demonstrated in preclinical studies. Before human trials are performed, an understanding of varying gas properties on airway resistance during inhalation is essential. This study predicts the effect of an 80% argon/20% oxygen gas mixture on the pressure drop through conducting airways, and by extension the airway resistance, and then verifies these predictions experimentally using 3-D printed adult tracheobronchial airway replicas.The predicted pressure drop was calculated using established analytical models of airway resistance, incorporating the change in viscosity and density of the 80% argon/20% oxygen mixture versus that of air. Predicted pressure drop for the argon mixture increased by approximately 29% compared to that for air. The experimental results were consistent with this prediction for inspiratory flows ranging from 15 to 90 slpm. These results indicate that established analytical models may be used to predict increases in conducting airway resistance for argon/oxygen mixtures, compared with air. Such predictions are valuable in predicting average patient response to breathing argon/oxygen mixtures, and in selecting or designing delivery systems for use in administration of argon/oxygen mixtures to critically ill or injured patients.     

Treatment of central type lung cancer by combined cryotherapy: Experiences of 47 patients

Most patients with central type lung cancer (CTLC) are not candidates for surgery; systemic chemotherapy and external beam radiotherapy are the main treatments but have not greatly affected patient outcome. Combined percutaneous and endobronchial cryotherapy has been used successfully to treat CTLC; this study aimed to determine its feasibility and safety. Forty-seven patients with unresectable CTLC (22 endotracheal, 26 tracheal wall and 21 extratracheal tumors) underwent 69 sessions of combined percutaneous cryosurgery, endobronchial cryosurgery and airway stenting. The long diameter of all tumors was <5 cm. Biopsy showed non-small cell lung cancer (NSCLC) in 40 patients (medium or well differentiated in 20 cases, poorly differentiated in 20) and small cell lung cancer (SCLC) in seven. Within 3 days after treatment, ventilatory capacity and performance status had obviously increased and cough, signs of dyspnea, hemoptysis and atelectasis improved significantly, but symptoms of pneumothorax and pleural effusion emerged. After 2 weeks, all complications had disappeared completely, as had cough. Progression-free survival (PFS) for endotracheal tumors (8 ± 4 months) was shorter than that for tracheal wall (13 ± 6 months, P < 0.05) and extratracheal (14 ± 8 months, P < 0.01) tumors. The PFS of NSCLC (11 ± 5 months) was significantly longer than that of SCLC (4 ± 2 months, P < 0.0001). The PFS of medium or well differentiated CTLC (15 ± 8 months) was significantly longer than that of poorly differentiated CTLC (7 ± 3 months, P < 0.0001). In conclusion, combined cryotherapy is a safe and effective treatment for CTLC, with PFS largely influenced by tumor location and pathologic type.     

Tracheal submucosal gland serous cells stimulated in vitro with adrenergic and cholinergic agonists A morphometric study

Summary A morphometric analysis was made of alterations in serous cell structure induced by adrenergic and cholinergic agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following agonists: phenylephrine, terbutaline, or methacholine (all at 10^–5 M). Controls were incubated similarly in medium containing no drugs or medium containing both the agonist and an excess of the appropriate antagonist (phentolamine, propranolol or atropine, all at 10^–4 M).Electron microscopic observation and stereological analysis of the incubated samples revealed that the volume density of serous cell granules in controls (0.30 ± 0.02, mean ± SE, n = 4) was significantly reduced by phenylephrine (0.19 ± 0.03, n = 4) and methacholine (0.17 ± 0.01, n = 4), but not by terbutaline (0.27 ± 0.04, n = 4). The presence of antagonists in the medium prevented the observed changes (phenylephrine/phentolamine: 0.29 ± 0.03, n = 3 and methacholine/atropine: 0.33 ± 0.06, n = 3). In addition, the volume density of intracellular vacuoles in controls (0.02 ± 0.005, n = 4) was increased in response to methacholine stimulation (0.12 + 0.05, n = 4), but not in response to the other agonists. This effect was blocked by atropine (0.01 ± 0.00, n = 3).We conclude that serous-cell granules are discharged by both alpha-adrenergic and cholinergic, but not beta-adrenergic stimulation. In addition, cholinergic stimulation evokes the formation of intracellular vacuoles, a possible indication of active ion and water transport.     

平喘颗粒对慢性哮喘大鼠Notch通路相关因子的影响

摘要 目的：通过观测各组慢性哮喘大鼠Notch信号通路相关因子Notch2、Jagged1、HES-1的变化，以及肺组织气道重塑的改善状况，探讨平喘颗粒在哮喘的治疗方面所发挥的作用机制。方法：按随机法将60只大鼠均分至空白组、模型组、地塞米松组、平喘颗粒组中。除空白组外，剩余的3组大鼠经卵蛋白+氢氧化铝致敏，复制慢性哮喘大鼠模型，空白组用生理盐水代替致敏液。造模前各组予相应的药物灌胃处理。6周后造模完成，处死大鼠并取其肺组织进行HE染色，在光镜下观察病理变化。使用Real-time PCR法检测Notch2、Jagged1 mRNA，Western blot法检测HES-1蛋白的表达程度。结果：与空白组相比，其余各组肺组织均可观察到明显气道重塑改变，Notch2、Jagged1 mRNA及HES-1蛋白表达明显上升（P＜0.05）；与模型组相比，平喘颗粒和地塞米松可缓解气道重塑，各因子表达情况显著下降（P＜0.05），两组间比较则无明显差异（P＞0.05）。结论：Notch2、Jagged1、HES-1表达程度与气道重塑呈正相关，平喘颗粒通过降低Notch2、Jagged1、HES-1表达改善气道重塑，可能是其防治哮喘发生发展的机制。     

Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8⁺ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.     

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

Background

BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells.

Results

The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells.

Conclusions

Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.     

p38 MAPK通路在脂多糖致呼吸道上皮损伤中的作用

脂多糖（Lipopolysaccharide,LPS）是革兰阴性杆菌细胞壁的主要组成成分,也是一种很强的炎症反应和氧化应激诱导剂。呼吸道上皮是机体防御外界细菌、病毒、香烟烟雾等生物和化学因素损伤的天然屏障,在维持呼吸道局部微环境稳态中可发挥重要作用,也是吸入性药物治疗的主要靶细胞。呼吸道上皮结构完整性缺陷或功能紊乱还参与了哮喘、慢性阻塞性肺疾病等多种肺部疾病的发生和发展。LPS可引起呼吸道上皮损伤,但其具体的分子机制目前尚不清楚。p38丝裂原活化蛋白激酶（P38mitogen-activated protein kinase,p38 MAPK）作为MAPK家族四个亚家族成员之一,包含四个成员：p38α、p38β、p38γ和p38δ,可通过经典和非经典的p38 MAPK信号通路激活方式及通过激酶活性无关的功能参与调控炎症反应、细胞生长、细胞分化和细胞死亡等多种病理生理过程。本文就p38 MAPK信号通路在LPS致呼吸道上皮损伤中的作用做一综述。     

Clinical and computed tomographic predictors of chronic bronchitis in COPD: a cross sectional analysis of the COPDGene study

Background

Chronic bronchitis (CB) has been related to poor outcomes in Chronic Obstructive Pulmonary Disease (COPD). From a clinical standpoint, we have shown that subjects with CB in a group with moderate to severe airflow obstruction were younger, more likely to be current smokers, male, Caucasian, had worse health related quality of life, more dyspnea, and increased exacerbation history compared to those without CB. We sought to further refine our clinical characterization of chronic bronchitics in a larger cohort and analyze the CT correlates of CB in COPD subjects. We hypothesized that COPD patients with CB would have thicker airways and a greater history of smoking, acute bronchitis, allergic rhinitis, and occupational exposures compared to those without CB.

Methods

We divided 2703 GOLD 1–4 subjects in the Genetic Epidemiology of COPD (COPDGene®) Study into two groups based on symptoms: chronic bronchitis (CB+, n = 663, 24.5%) and no chronic bronchitis (CB-, n = 2040, 75.5%). Subjects underwent extensive clinical characterization, and quantitative CT analysis to calculate mean wall area percent (WA%) of 6 segmental airways was performed using VIDA PW2 (http://www.vidadiagnostics.com). Square roots of the wall areas of bronchi with internal perimeters 10 mm and 15 mm (Pi10 and Pi15, respectively), % emphysema, %gas trapping, were calculated using 3D Slicer (http://www.slicer.org).

Results

There were no differences in % emphysema (11.4 ± 12.0 vs. 12.0 ± 12.6%, p = 0.347) or % gas trapping (35.3 ± 21.2 vs. 36.3 ± 20.6%, p = 0.272) between groups. Mean segmental WA% (63.0 ± 3.2 vs. 62.0 ± 3.1%, p < 0.0001), Pi10 (3.72 ± 0.15 vs. 3.69 ± 0.14 mm, p < 0.0001), and Pi15 (5.24 ± 0.22 vs. 5.17 ± 0.20, p < 0.0001) were greater in the CB + group. Greater percentages of gastroesophageal reflux, allergic rhinitis, histories of asthma and acute bronchitis, exposures to dusts and occupational exposures, and current smokers were seen in the CB + group. In multivariate binomial logistic regression, male gender, Caucasian race, a lower FEV₁%, allergic rhinitis, history of acute bronchitis, current smoking, and increased airway wall thickness increased odds for having CB.

Conclusions

Histories of asthma, allergic rhinitis, acute bronchitis, current smoking, a lower FEV₁%, Caucasian race, male gender, and increased airway wall thickness are associated with CB. These data provide clinical and radiologic correlations to the clinical phenotype of CB.