早期戒烟大鼠肺病理及炎性介质变化

目的观察早期戒烟后大鼠肺组织病理及炎性介质表达变化规律。方法选用Wistar雄性大鼠80只,随机分为对照组及早期戒烟后0天、1周、2周、4周、6周、8周、12周组。采用酶联免疫吸附方法测定各组大鼠血清中IL-8的蛋白质含量,S-P免疫组化学方法检测肺组织NF-κB p65的表达,并光镜下观察HE染色切片、对大鼠气道炎症进行病理学评分。结果早期戒烟组大鼠可见气道上皮细胞纤毛发生粘连、倒伏,上皮细胞空泡变形、坏死、增生,炎症细胞浸润;其血清IL-8浓度、肺组织NF-κB的表达及气道炎症病理评分在戒烟后各时相点较未吸烟对照组明显升高,有统计学意义（P〈0.05）。早期戒烟组大鼠血清IL-8的浓度、肺组织NF-κB的表达及肺组织病理炎症评分在戒烟后略有上升、且在戒烟后8周达到高峰,但随后在戒烟12周时可见IL-8的浓度有下降趋势,肺组织病理炎症反应有所减轻。结论早期戒烟大鼠在戒烟早期虽可见炎症反应略有加重,但随戒烟时间延长,仍可见炎症反应有所减轻。因此,提倡及早且坚持戒烟。     

Transitions in Smoking Behaviour and the Design of Cessation Schemes

The intake of nicotine by smoking cigarettes is modelled by a dynamical system of differential equations. The variables are the internal level of nicotine and the level of craving. The model is based on the dynamics of neural receptors and the way they enhance craving. Lighting of a cigarette is parametrised by a time-dependent Poisson process. The nicotine intake rate is assumed to be proportional with the parameter of this stochastic process. The effect of craving is damped by a control mechanism in which awareness of the risks of smoking and societal measures play a role. Fluctuations in this damping may cause transitions from smoking to non-smoking and vice versa. With the use of Monte Carlo simulation the effect of abrupt and gradual cessation therapies are evaluated. Combination of the two in a mixed scheme yields a therapy with a duration that can be set at wish.     

Patterns of MiRNA Expression in Arctic Charr Development

Micro-RNAs (miRNAs) are now recognized as a major class of developmental regulators. Sequences of many miRNAs are highly conserved, yet they often exhibit temporal and spatial heterogeneity in expression among species and have been proposed as an important reservoir for adaptive evolution and divergence. With this in mind we studied miRNA expression during embryonic development of offspring from two contrasting morphs of the highly polymorphic salmonid Arctic charr (Salvelinus alpinus), a small benthic morph from Lake Thingvallavatn (SB) and an aquaculture stock (AC). These morphs differ extensively in morphology and adult body size. We established offspring groups of the two morphs and sampled at several time points during development. Four time points (3 embryonic and one just before first feeding) were selected for high-throughput small-RNA sequencing. We identified a total of 326 conserved and 427 novel miRNA candidates in Arctic charr, of which 51 conserved and 6 novel miRNA candidates were differentially expressed among developmental stages. Furthermore, 53 known and 19 novel miRNAs showed significantly different levels of expression in the two contrasting morphs. Hierarchical clustering of the 53 conserved miRNAs revealed that the expression differences are confined to the embryonic stages, where miRNAs such as sal-miR-130, 30, 451, 133, 26 and 199a were highly expressed in AC, whereas sal-miR-146, 183, 206 and 196a were highly expressed in SB embryos. The majority of these miRNAs have previously been found to be involved in key developmental processes in other species such as development of brain and sensory epithelia, skeletogenesis and myogenesis. Four of the novel miRNA candidates were only detected in either AC or SB. miRNA candidates identified in this study will be combined with available mRNA expression data to identify potential targets and involvement in developmental regulation.     

Mechanisms of Acid and Base Secretion by the Airway Epithelium

One of the main functions of the airway epithelium is to inactivate and remove infectious particles from inhaled air and thereby prevent infection of the distal lung. This function is achieved by mucociliary and cough clearance and by antimicrobial factors present in the airway surface liquid (ASL). There are indications that airway defenses are affected by the pH of the ASL and historically, acidification of the airway surfaces has been suggested as a measure of airway disease. However, even in health, the ASL is slightly acidic, and this acidity might be part of normal airway defense. Only recently research has focused on the mechanisms responsible for acid and base secretion into the ASL. Advances resulted from research into the airway disease associated with cystic fibrosis (CF) after it was found that the CFTR Cl⁻ channel conducts HCO ₃ ⁻ and, therefore, may contribute to ASL pH. However, the acidity of the ASL indicated parallel mechanisms for H⁺ secretion. Recent investigations identified several H⁺ transporters in the apical membrane of the airway epithelium. These include H⁺ channels and ATP-driven H⁺ pumps, including a non-gastric isoform of the H⁺-K⁺ ATPase and a vacuolar-type H⁺ ATPase. Current knowledge of acid and base transporters and their potential roles in airway mucosal pH regulation is reviewed here.     

Permeation of Therapeutic Drugs in Different Formulations across the Airway Epithelium In Vitro

Background

Pulmonary drug delivery is characterized by short onset times of the effects and an increased therapeutic ratio compared to oral drug delivery. This delivery route can be used for local as well as for systemic absorption applying drugs as single substance or as a fixed dose combination. Drugs can be delivered as nebulized aerosols or as dry powders. A screening system able to mimic delivery by the different devices might help to assess the drug effect in the different formulations and to identify potential interference between drugs in fixed dose combinations. The present study evaluates manual devices used in animal studies for their suitability for cellular studies.

Methods

Calu-3 cells were cultured submersed and in air-liquid interface culture and characterized regarding mucus production and transepithelial electrical resistance. The influence of pore size and material of the transwell membranes and of the duration of air-liquid interface culture was assessed. Compounds were applied in solution and as aerosols generated by MicroSprayer IA-1C Aerosolizer or by DP-4 Dry Powder Insufflator using fluorescein and rhodamine 123 as model compounds. Budesonide and formoterol, singly and in combination, served as examples for drugs relevant in pulmonary delivery.

Results and Conclusions

Membrane material and duration of air-liquid interface culture had no marked effect on mucus production and tightness of the cell monolayer. Co-application of budesonide and formoterol, applied in solution or as aerosol, increased permeation of formoterol across cells in air-liquid interface culture. Problems with the DP-4 Dry Powder Insufflator included compound-specific delivery rates and influence on the tightness of the cell monolayer. These problems were not encountered with the MicroSprayer IA-1C Aerosolizer. The combination of Calu-3 cells and manual aerosol generation devices appears suitable to identify interactions of drugs in fixed drug combination products on permeation.     

Smoking Cessation Induces Profound Changes in the Composition of the Intestinal Microbiota in Humans

Background

The human intestinal microbiota is a crucial factor in the pathogenesis of various diseases, such as metabolic syndrome or inflammatory bowel disease (IBD). Yet, knowledge about the role of environmental factors such as smoking (which is known to influence theses aforementioned disease states) on the complex microbial composition is sparse. We aimed to investigate the role of smoking cessation on intestinal microbial composition in 10 healthy smoking subjects undergoing controlled smoking cessation.

Methods

During the observational period of 9 weeks repetitive stool samples were collected. Based on abundance of 16S rRNA genes bacterial composition was analysed and compared to 10 control subjects (5 continuing smokers and 5 non-smokers) by means of Terminal Restriction Fragment Length Polymorphism analysis and high-throughput sequencing.

Results

Profound shifts in the microbial composition after smoking cessation were observed with an increase of Firmicutes and Actinobacteria and a lower proportion of Bacteroidetes and Proteobacteria on the phylum level. In addition, after smoking cessation there was an increase in microbial diversity.

Conclusions

These results indicate that smoking is an environmental factor modulating the composition of human gut microbiota. The observed changes after smoking cessation revealed to be similar to the previously reported differences in obese compared to lean humans and mice respectively, suggesting a potential pathogenetic link between weight gain and smoking cessation. In addition they give rise to a potential association of smoking status and the course of IBD.     

Next-Generation Sequencing Analysis of MiRNA Expression in Control and FSHD Myogenesis

Emerging evidence has demonstrated that miRNA sequences can regulate skeletal myogenesis by controlling the process of myoblast proliferation and differentiation. However, at present a deep analysis of miRNA expression in control and FSHD myoblasts during differentiation has not yet been derived. To close this gap, we used a next-generation sequencing (NGS) approach applied to in vitro myogenesis. Furthermore, to minimize sample genetic heterogeneity and muscle-type specific patterns of gene expression, miRNA profiling from NGS data was filtered with FC≥4 (log₂FC≥2) and p-value<0.05, and its validation was derived by qRT-PCR on myoblasts from seven muscle districts. In particular, control myogenesis showed the modulation of 38 miRNAs, the majority of which (34 out 38) were up-regulated, including myomiRs (miR-1, -133a, -133b and -206). Approximately one third of the modulated miRNAs were not previously reported to be involved in muscle differentiation, and interestingly some of these (i.e. miR-874, -1290, -95 and -146a) were previously shown to regulate cell proliferation and differentiation. FSHD myogenesis evidenced a reduced number of modulated miRNAs than healthy muscle cells. The two processes shared nine miRNAs, including myomiRs, although with FC values lower in FSHD than in control cells. In addition, FSHD cells showed the modulation of six miRNAs (miR-1268, -1268b, -1908, 4258, -4508- and -4516) not evidenced in control cells and that therefore could be considered FSHD-specific, likewise three novel miRNAs that seem to be specifically expressed in FSHD myotubes. These data further clarify the impact of miRNA regulation during control myogenesis and strongly suggest that a complex dysregulation of miRNA expression characterizes FSHD, impairing two important features of myogenesis: cell cycle and muscle development. The derived miRNA profiling could represent a novel molecular signature for FSHD that includes diagnostic biomarkers and possibly therapeutic targets.     

人偏肺病毒在传代细胞和人呼吸道上皮细胞中分离和鉴定

人偏肺病毒（Human metapneumovirus,HMPV）是2001年鉴定出的新发呼吸道病毒,婴幼儿、老人和免疫抑制人群易感,引起上呼吸道和下呼吸道感染,目前尚无疫苗和特异性治疗方案。为获得北京地区HMPV临床流行毒株,本研究将经荧光定量PCR检测为HMPV阳性的鼻咽抽吸物样本分别接种LLC-MK2、Vero-E6和分化良好的人呼吸道上皮细胞（Human Airway Epithelium,HAE）,观察细胞病变、检测免疫荧光、电镜观察病毒形态、测定病毒滴度及分析复制特点,对分离获得的HMPV分离株进行鉴定。结果表明,HMPV感染LLC-MK2细胞可形成合胞体,但在Vero-E6中多呈单个细胞感染;经免疫荧光检测,HAE、LLC-MK2和Vero-E6细胞均可见绿色荧光;电镜结果可见病毒为近似球型的颗粒,有包膜和刺突,直径约在150nm～200nm之间;HMPV在HAE和LLC-MK2两种细胞上的复制特点基本相同。本研究成功建立了临床样本在LLC-MK2、Vero-E6和HAE分离培养HMPV的方法,分离并鉴定了HMPV临床分离株,为HMPV感染机制的研究奠定基础。     

Expression of Muscle-Specific MiRNA 206 in the Progression of Disease in a Murine SMA Model

Spinal muscular atrophy (SMA) is a severe neuromuscular disease, the most common in infancy, and the third one among young people under 18 years. The major pathological landmark of SMA is a selective degeneration of lower motor neurons, resulting in progressive skeletal muscle denervation, atrophy, and paralysis. Recently, it has been shown that specific or general changes in the activity of ribonucleoprotein containing micro RNAs (miRNAs) play a role in the development of SMA. Additionally miRNA-206 has been shown to be required for efficient regeneration of neuromuscular synapses after acute nerve injury in an ALS mouse model. Therefore, we correlated the morphology and the architecture of the neuromuscular junctions (NMJs) of quadriceps, a muscle affected in the early stage of the disease, with the expression levels of miRNA-206 in a mouse model of intermediate SMA (SMAII), one of the most frequently used experimental model. Our results showed a decrease in the percentage of type II fibers, an increase in atrophic muscle fibers and a remarkable accumulation of neurofilament (NF) in the pre-synaptic terminal of the NMJs in the quadriceps of SMAII mice. Furthermore, molecular investigation showed a direct link between miRNA-206-HDAC4-FGFBP1, and in particular, a strong up-regulation of this pathway in the late phase of the disease. We propose that miRNA-206 is activated as survival endogenous mechanism, although not sufficient to rescue the integrity of motor neurons. We speculate that early modulation of miRNA-206 expression might delay SMA neurodegenerative pathway and that miRNA-206 could be an innovative, still relatively unexplored, therapeutic target for SMA.     

血管回声跟踪技术评价戒烟前后对动脉弹性影响的临床研究

目的:血管回声跟踪(echo-tracking,ET)技术探讨长期吸烟及戒烟对颈总动脉(common carotid artery,CCA)及肱动脉(brachial artery,BA)弹性的影响.方法:受试者为吸烟组(80例,男性,46±岁)和正常对照组(25例,男性,45±6岁),戒烟组在戒烟六个月后,根据戒烟情况将受试者分为成功戒烟组(23人)、部分戒烟组(25人)、戒烟失败组(32人),应用ET技术检测各吸烟组戒烟前及戒烟六个月后CCA及BA弹性(僵硬度β、弹性模量Ep、脉搏波传导速度PWVβ)的变化,同时检测每日吸烟量、吸烟指数、血脂、血糖、血压等各项指标,并与正常对照组比较.结果:(1)正常对照组与各吸烟组戒烟前及戒烟后血脂、血糖、血压、体重指数等指标比较均无统计学差异(P＞0.05).(2)各吸烟组戒烟前每日吸烟量、吸烟指数等指标比较均无统计学差异(P＞0.05).(3)各吸烟组戒烟前CCA及BA的β、Ep、PWVβ较正常对照组均明显增高,具有显著统计学差异(P＜0.001).(4)各吸烟组戒烟后,成功戒烟组CCA及BA的β、Ep、PWVβ与正常对照组无统计学差异(P＞0.05),成功戒烟组CCA及BA的β、Ep、PWVβ较戒烟失败组减低,具有统计学差异(P＜0.05,P＜0.01),部分戒烟组CCA及BA的β、Ep、PWVβ与正常对照组有统计学差异(P＜0.05).结论:吸烟引起CCA及BA弹性减低,成功戒烟可使CCA及BA弹性改善,BA改善效果更明显.     

Lung and Heart Diseases Are Better Predicted by Pack-Years than by Smoking Status or Duration of Smoking Cessation in HIV Patients

Background

The objective of this study was to assess the relationship of pack-years smoking and time since smoking cessation with risk of lung and heart disease.

Methods

We investigated the history of lung and heart disease in 903 HIV-infected patients who had undergone thoracic computed tomography (CT) imaging stratified by smoking history. Multimorbidity lung and heart disease (MLHD) was defined as the presence of ≥ 2 clinical or subclinical lung abnormalities and at least one heart abnormality.

Results

Among 903 patients, 23.7% had never smoked, 28.7% were former smokers and 47.6% were current smokers. Spirometry indicated chronic obstructive pulmonary disease in 11.4% of patients and MLHD was present in 53.6%. Age, male sex, greater pack-years smoking history and smoking cessation less than 5 years earlier vs. more than 10 years earlier (OR 2.59, 95% CI 1.27–5.29, p = 0.009) were independently associated with CT detected subclinical lung and heart disease. Pack-years smoking history was more strongly associated with MLHD than smoking status (p<0.001).

Conclusions

MLHD is common even among HIV-infected patients who never smoked and pack- years smoking history is a stronger predictor than current smoking status of MLHD. A detailed pack-years smoking history should be routinely obtained and smoking cessation strategies implemented.     

What Are the Major Determinants in the Success of Smoking Cessation: Results from the Health Examinees Study

Understanding mechanisms underlying smoking-related factors should be prioritized in establishing smoking prevention and cessation policy. The aim of this study was to identify factors significantly associated with smoking initiation and/or smoking cessation as well as the most important determinants of successful smoking cessation in a developed non-Western setting. Based on multiple logistic regression models, the odds ratios (ORs) for smoking initiation and cessation were estimated among males (N = 24,490) who had participated in the Health Examinees (HEXA) study. The Cox proportional hazards regression model was used to assess the association between selected predictors of smoking cessation and the likelihood of reaching this goal. Finally, Kaplan–Meier curves were constructed to illustrate the distribution of time from age at smoking initiation to age at smoking cessation. We found that the ORs for successfully quitting smoking increased with age, married status, educational achievement, having a non-manual job, drinking cessation and disease morbidity. Those exposed to secondhand smoking showed less likelihood of quitting smoking. A continual decrease in the ORs for successfully quitting smoking was observed according to increased smoking duration, smoking dose per day and lifetime tobacco exposure (p_trend <0.001). Among the selected predictors, lifetime tobacco exposure, educational attainment, alcohol drinking status and birth cohort were the major determinants in the success of smoking cessation. Our findings suggest that lifetime tobacco exposure, educational attainment, alcohol drinking status and birth cohort can determine success in smoking cessation. Public interventions promoting a smoke-free environment are needed to reinforce discouraging the initiation of, reducing, and quitting cigarette smoking.     

Activity-Dependent Modulation of Odorant Receptor Gene Expression in the Mouse Olfactory Epithelium

Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.     

A Cost-Utility Analysis of Lung Cancer Screening and the Additional Benefits of Incorporating Smoking Cessation Interventions

Background

A 2011 report from the National Lung Screening Trial indicates that three annual low-dose computed tomography (LDCT) screenings for lung cancer reduced lung cancer mortality by 20% compared to chest X-ray among older individuals at high risk for lung cancer. Discussion has shifted from clinical proof to financial feasibility. The goal of this study was to determine whether LDCT screening for lung cancer in a commercially-insured population (aged 50–64) at high risk for lung cancer is cost-effective and to quantify the additional benefits of incorporating smoking cessation interventions in a lung cancer screening program.

Methods and Findings

The current study builds upon a previous simulation model to estimate the cost-utility of annual, repeated LDCT screenings over 15 years in a high risk hypothetical cohort of 18 million adults between age 50 and 64 with 30+ pack-years of smoking history. In the base case, the lung cancer screening intervention cost $27.8 billion over 15 years and yielded 985,284 quality-adjusted life years (QALYs) gained for a cost-utility ratio of $28,240 per QALY gained. Adding smoking cessation to these annual screenings resulted in increases in both the costs and QALYs saved, reflected in cost-utility ratios ranging from $16,198 per QALY gained to $23,185 per QALY gained. Annual LDCT lung cancer screening in this high risk population remained cost-effective across all sensitivity analyses.

Conclusions

The findings of this study indicate that repeat annual lung cancer screening in a high risk cohort of adults aged 50–64 is highly cost-effective. Offering smoking cessation interventions with the annual screening program improved the cost-effectiveness of lung cancer screening between 20% and 45%. The cost-utility ratios estimated in this study were in line with other accepted cancer screening interventions and support inclusion of annual LDCT screening for lung cancer in a high risk population in clinical recommendations.     

Cost Effectiveness of Free Access to Smoking Cessation Treatment in France Considering the Economic Burden of Smoking-Related Diseases

Context

In France more than 70,000 deaths from diseases related to smoking are recorded each year, and since 2005 prevalence of tobacco has increased. Providing free access to smoking cessation treatment would reduce this burden. The aim of our study was to estimate the incremental cost-effectiveness ratios (ICER) of providing free access to cessation treatment taking into account the cost offsets associated with the reduction of the three main diseases related to smoking: lung cancer, chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD). To measure the financial impact of such a measure we also conducted a probabilistic budget impact analysis.

Methods and Findings

We performed a cost-effectiveness analysis using a Markov state-transition model that compared free access to cessation treatment to the existing coverage of €50 provided by the French statutory health insurance, taking into account the cost offsets among current French smokers aged 15–75 years. Our results were expressed by the incremental cost-effectiveness ratio in 2009 Euros per life year gained (LYG) at the lifetime horizon. We estimated a base case scenario and carried out a Monte Carlo sensitivity analysis to account for uncertainty. Assuming a participation rate of 7.3%, the ICER value for free access to cessation treatment was €3,868 per LYG in the base case. The variation of parameters provided a range of ICER values from -€736 to €15,715 per LYG. In 99% of cases, the ICER for full coverage was lower than €11,187 per LYG. The probabilistic budget impact analysis showed that the potential cost saving for lung cancer, COPD and CVD ranges from €15 million to €215 million at the five-year horizon for an initial cessation treatment cost of €125 million to €421 million.

Conclusion

The results suggest that providing medical support to smokers in their attempts to quit is very cost-effective and may even result in cost savings.     

特异性miRNA调节机理研究进展

MicroRNA(miRNA)是一类新鉴定的非蛋白质编码小RNA,它们在多种生物学过程中发挥重要作用。最新研究表明许多miRNA表达受RNA编辑、差别加工和组织特异性增强子调节而呈现时间和空间特异性,而且基于miRNA调节设计分子药物的前景很好。深入理解miRNA调节的机理有助于揭示一些疾病的发病机理,发现干预治疗的新分子靶标,以及最终建立有效的基因疗法。因此,本文将对miRNA调节机理的最新研究进行综述。