慢性阻塞性肺疾病患者支气管肺泡灌洗液细菌宏基因组DNA的制备

【目的】优化稳定期慢性阻塞性肺疾病(chronic obstructive pulmonary diseases,COPD)患者支气管肺泡灌洗液(bronchoalveolar lavage fluids,BALF)细菌宏基因组DNA的提取方法,以便于高效提取微量的细菌DNA进行后续的PCR反应和测序。【方法】取稳定期COPD患者的BALF 5mL,离心收集细胞。为了有效提取样品中革兰氏阳性菌的基因组,对QIAGEN的DNA提取试剂盒的操作步骤进行优化:加入裂解缓冲液ATL后首先运用研磨珠和多功能生物样品匀质器破碎菌壁,再加入蛋白酶K孵育,然后加入裂解缓冲液AL振荡混匀。无水乙醇沉淀DNA后,将全部溶液过柱,用洗液AW1和AW2各洗柱一次,最后加50μL洗脱液洗脱DNA。提取的DNA定量后,运用PCR方法检测样本中的细菌16S rDNA量,并按照测序要求构建DNA文库进一步验证。【结果】试剂盒优化法提取的BALF的DNA总量为467.5(135.0-1697.5)ng,明显高于按照传统酚-氯仿法提取的DNA总量95.0(0-612.5)ng,并且所提取的DNA可以很好的扩增细菌的16S rDNA以及构建DNA文库,改良后的扩增产物明显增多(P=0.002)。【结论】使用DNA提取试剂盒结合研磨珠和多功能生物样品匀质器破菌壁的方法能够更高效的提取BALF中微量的宏基因组DNA,为进一步的测序和菌群分析打下基础。     

DNA damage in peripheral blood of patients with chronic obstructive pulmonary disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a condition characterized by chronic airway inflammation and remodeling, lung parenchymal inflammation, and destruction resulting in expiratory airflow obstruction, hyperinflation of the lung with loss of elastic recoil, and impairment of gas exchange. Skeletal muscles in individuals with COPD generate free radicals at rest, and production increases during contractile activity. Overproduction of free radicals may result in oxidant-antioxidant imbalance in favor of oxidants. This study evaluated the levels of genetic damage in peripheral blood of patients with COPD using the cytokinesis-blocked micronucleus (CBMN) and the comet assays. The study was conducted with 25 patients with COPD and 25 controls matched for age and sex. Results of both comet and CBMN assays showed an increase in the level of DNA damage. In the group of patients with COPD, the mean frequency of binucleate cells with micronuclei was 6.72+/-3.02, and in the control group, 4.20+/-2.08 (p=0.00233). Mean comet value was 26.84+/-19.61 in patients with COPD and 7.25+/-7.57 in the control group (p=0.00004). The increased frequency of micronuclei in patients with COPD was primarily assigned to clastogenic events and DNA amplification because the frequency of nucleoplasmic bridges and buds was also increased. Oxidative stress in lung cells is a constant source of free radicals that damage genetic material of both lung and circulating cells.     

夏季老年慢性阻塞性肺疾病患者口咽菌群分析

分析夏季老年慢性阻塞性肺疾病（COPD）患者与健康人群的口咽菌群构成,旨在确定老年COPD患者与健康人群上呼吸道菌群间的差异。

选择2018年6—8月沈阳市沈阳医学院附属第二医院COPD患者29例和健康体检者25例,采集口咽拭子进行细菌16S rRNA高通量测序。通过菌群多样性分析、物种组成和物种差异分析,比较老年COPD患者与健康人群口咽部微生物的异同。

老年COPD患者口咽中菌群丰富度显著高于健康人群,物种多样性低于健康人群。在门水平上,老年COPD患者口咽菌群中拟杆菌门相对丰度降低,放线菌门相对丰度显著增高;在属水平上,老年COPD患者口咽菌群中罗氏菌属、放线菌属和劳特罗普氏菌属丰度均显著高于健康人群,奈瑟菌属和普雷沃菌属相对丰度降低,差异均具有统计学意义（P＜0.05）。

老年COPD患者口咽部正常菌群组成发生变化,机会致病菌如罗氏菌属、劳特罗普氏菌属比例增加,提示夏季老年COPD患者口咽菌群失调。

     

Interferon-gamma (r-IFN-gamma) induced activation of alveolar macrophages (AM) from anergic patients with chronic obstructive pulmonary disease (COPD).

Forty-six anergic patients (37 males and 9 females, age range 55-79 yr) were selected from ninety-one patients suffering from COPD due to frequent exacerbations and impaired delayed cutaneous reactivity (43.9%). The phenotype of circulating lymphocytes, their proliferative response to a panel of polyclonal T-cell activators and the candidacidal activity (CA) of circulating PMNs (polymorphonuclear cells) were measured. In 13 patients presenting a defective CA of circulating PMNs, the in vitro response of alveolar macrophage CA to r-IFN-gamma was also determined. We found: 1) a significant reduction in the CL response to PHA in COPD patients vs controls; 2) a low PMN-CA in 23 (57%) COPD patients; 3) a non-significant difference in phenotype analysis in patients and controls; 4) lower CA of AMs in COPD patients than in controls; 5) restoration in vitro of CA by r-IFN-gamma in the group of anergic COPD patients presenting depressed CA. We conclude that a defective cell-mediated immunity could be the basis of the enhanced susceptibility to infectious exacerbations in many COPD patients and that, in vitro, it could be reversed by r-IFN-gamma treatment.     

Blood and sputum protein biomarkers for chronic obstructive pulmonary disease (COPD)

ABSTRACT

Introduction: Chronic obstructive pulmonary disease (COPD) is a heterogeneous set of disorders, characterized by airflow limitation, and reduced lung function. Despite increasing knowledge regarding its pathophysiology, there has been limited advancement in therapeutics and the current treatment strategy is symptom management and prevention of exacerbations.

Areas covered: Biomarkers represent important tools for the implementation of precision medicine. As fundamental molecules of all living processes, proteins could provide crucial information about how genes interact with the environment. Proteomics studies could act as important tools in identifying reliable biomarkers to enable a more precise therapeutic approach. In this review, we will explore the most promising blood and sputum protein biomarkers in COPD that have been consistently reported in the literature.

Expert commentary: Given the complexity of COPD, no single protein biomarker has been able to improve the outcomes of COPD patients. According to preliminary studies, precision medicine in COPD will likely require a combination of different proteins in a biomarker panel for clinical translation. With advancements in current mass spectrometry techniques, an enhancement in the identification of new biomarkers will be observed, and improvements in sequence database search can fill in potential gaps between biomarker discovery and patient care.     

Outcome measures in chronic obstructive pulmonary disease (COPD): strengths and limitations

Current methods for assessing clinical outcomes in COPD mainly rely on physiological tests combined with the use of questionnaires. The present review considers commonly used outcome measures such as lung function, health status, exercise capacity and physical activity, dyspnoea, exacerbations, the multi-dimensional BODE score, and mortality. Based on current published data, we provide a concise overview of the principles, strengths and weaknesses, and discuss open questions related to each methodology. Reviewed is the current set of markers for measuring clinically relevant outcomes with particular emphasis on their limitations and opportunities that should be recognized when assessing and interpreting their use in clinical trials of COPD.     

Role of epithelial mesenchymal transition (EMT) in chronic obstructive pulmonary disease (COPD)

Small airway fibrosis is the main contributor to physiological airway dysfunction in COPD. One potential mechanism contributing to small airway fibrosis is epithelial mesenchymal transition (EMT). When associated with angiogenesis (so called EMT-Type-3) it may well also be the link with the development of cancer, which is closely associated with COPD and predominantly in large airways. In a recent study published in Respiratory Research, Qin Wang and colleagues investigated the role of urokinase plasminogen activator receptor (uPAR) in EMT in small airway epithelium of COPD patients. However, there are some issues with the paper which we wish to comment on.     

The relationship between telomere length and mortality in chronic obstructive pulmonary disease (COPD)

Some have suggested that chronic obstructive pulmonary disease (COPD) is a disease of accelerated aging. Aging is characterized by shortening of telomeres. The relationship of telomere length to important clinical outcomes such as mortality, disease progression and cancer in COPD is unknown. Using quantitative polymerase chain reaction (qPCR), we measured telomere length of peripheral leukocytes in 4,271 subjects with mild to moderate COPD who participated in the Lung Health Study (LHS). The subjects were followed for approximately 7.5 years during which time their vital status, FEV₁ and smoking status were ascertained. Using multiple regression methods, we determined the relationship of telomere length to cancer and total mortality in these subjects. We also measured telomere length in healthy “mid-life” volunteers and patients with more severe COPD. The LHS subjects had significantly shorter telomeres than those of healthy “mid-life” volunteers (p<.001). Compared to individuals in the 4^th quartile of relative telomere length (i.e. longest telomere group), the remaining participants had significantly higher risk of cancer mortality (Hazard ratio, HR, 1.48; p = 0.0324) and total mortality (HR, 1.29; p = 0.0425). Smoking status did not make a significant difference in peripheral blood cells telomere length. In conclusion, COPD patients have short leukocyte telomeres, which are in turn associated increased risk of total and cancer mortality. Accelerated aging is of particular relevance to cancer mortality in COPD.     

Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility

Cigarette smoking is the major environmental risk factor for chronic obstructive pulmonary disease (COPD). Genome-wide association studies have provided compelling associations for three loci with COPD. In this study, we aimed to estimate direct, i.e., independent from smoking, and indirect effects of those loci on COPD development using mediation analysis. We included a total of 3,424 COPD cases and 1,872 unaffected controls with data on two smoking-related phenotypes: lifetime average smoking intensity and cumulative exposure to tobacco smoke (pack years). Our analysis revealed that effects of two linked variants (rs1051730 and rs8034191) in the AGPHD1/CHRNA3 cluster on COPD development are significantly, yet not entirely, mediated by the smoking-related phenotypes. Approximately 30 % of the total effect of variants in the AGPHD1/CHRNA3 cluster on COPD development was mediated by pack years. Simultaneous analysis of modestly (r ² = 0.21) linked markers in CHRNA3 and IREB2 revealed that an even larger (~42 %) proportion of the total effect of the CHRNA3 locus on COPD was mediated by pack years after adjustment for an IREB2 single nucleotide polymorphism. This study confirms the existence of direct effects of the AGPHD1/CHRNA3, IREB2, FAM13A and HHIP loci on COPD development. While the association of the AGPHD1/CHRNA3 locus with COPD is significantly mediated by smoking-related phenotypes, IREB2 appears to affect COPD independently of smoking.     

Implication of Interleukin (IL)-18 in the pathogenesis of chronic obstructive pulmonary disease (COPD)

Interleukin (IL)-18 is a pro-inflammatory cytokine that was firstly described as an interferon (IFN)-γ-inducing factor. Similar to IL-1β, IL-18 is synthesized as an inactive precursor requiring processing by caspase-1 into an active cytokine. The platform for activating caspase-1 is known as the inflammasome, a multiple protein complex. Macrophages and dendritic cells are the primary sources for the release of active IL-18, whereas the inactive precursor remains in the intracellular compartment of mesenchymal cells. Finally, the IL-18 precursor is released from dying cells and processed extracellularly.IL-18 has crucial host defense and antitumor activities, and gene therapy to increase IL-18 levels in tissues protects experimental animals from infection and tumor growth and metastasis. Moreover, multiple studies in experimental animal models have shown that IL-18 over-expression results to emphysematous lesions in mice. The published data prompt to the hypothesis that IL-18 induces a broad spectrum of COPD-like inflammatory and remodeling responses in the murine lung and also induces a mixed type 1, type 2, and type 17 cytokine responses. The majority of studies identify IL-18 as a potential target for future COPD therapeutics to limit both the destructive and remodeling processes occurring in COPD lungs.     

Gait mechanics in patients with chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is characterized by the frequent association of disease outside the lung. The objective of this study was to determine the presence of biomechanical gait abnormalities in COPD patients compared to healthy controls while well rested and without rest.

Methods

Patients with COPD (N = 17) and aged-matched, healthy controls (N = 21) walked at their self-selected pace down a 10-meter walkway while biomechanical gait variables were collected. A one-minute rest was given between each of the five collected trials to prevent tiredness (REST condition). Patients with COPD then walked at a self-selected pace on a treadmill until the onset of self-reported breathlessness or leg tiredness. Subjects immediately underwent gait analysis with no rest between each of the five collected trials (NO REST condition). Statistical models with and without covariates age, gender, and smoking history were used.

Results

After adjusting for covariates, COPD patients demonstrated more ankle power absorption in mid-stance (P = 0.006) than controls during both conditions. Both groups during NO REST demonstrated increased gait speed (P = 0.04), stride length (P = 0.03), and peak hip flexion (P = 0.04) with decreased plantarflexion moment (P = 0.04) and increased knee power absorption (P = 0.04) as compared to REST. A significant interaction revealed that peak ankle dorsiflexion moment was maintained from REST to NO REST for COPD but increased for controls (P < 0.01). Stratifying by disease severity did not alter these findings, except that step width decreased in NO REST as compared to REST (P = 0.01). Standardized effect sizes of significant effects varied from 0.5 to 0.98.

Conclusions

Patients with COPD appear to demonstrate biomechanical gait changes at the ankle as compared to healthy controls. This was seen not only in increased peak ankle power absorption during no rest but was also demonstrated by a lack of increase in peak ankle dorsiflexion moment from the REST to the NO REST condition as compared to the healthy controls. Furthermore, a wider step width has been associated with fall risk and this could account for the increased incidence of falls in patients with COPD.     

Specific IgA and metalloproteinase activity in bronchial secretions from stable chronic obstructive pulmonary disease patients colonized by Haemophilus influenzae

Background

Haemophilus influenzae is the most common colonizing bacteria of the bronchial tree in chronic obstructive pulmonary disease (COPD), and positive cultures for this potentially pathogenic microorganism (PPM) has been associated with local inflammation changes that may influence the relationships between H. influenzae and the bronchial mucosa.

Methods

A cross-sectional analysis of stable COPD patients enrolled in the Phenotype and Course of Chronic Obstructive Pulmonary Disease (PAC-COPD) Study, focusing on bronchial colonization by H. influenzae, was performed. Specific IgA against the PPM was measured by optical density, and metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) using ELISA in sputum samples. Levels in patients colonized by H. influenzae and non-colonized patients were compared.

Results

Sputum supernatant for the measurement of specific IgA against H. influenzae was available from 54 stable COPD patients, who showed levels of specific IgA significantly lower in colonized (n=21) than in non-colonized patients (n=33) (15 [4-37] versus 31 [10-75], p=0.033, Mann-Whitney U test). Proenzyme MMP-9 was measured in 44 patients, and it was higher in colonized (n=12, 1903 [1488-6699] ng/ml) than in non-colonized patients (n=32, 639 [373-972] ng/ml) (p<0.001, Mann-Whitney U test). Active form of MMP-9 was also higher in colonized (126 [25-277] ng/ml) than in non-colonized patients (39 [14-68] ng/ml) (p=0.021, Mann-Whitney U test), and the molar ratio between proenzyme MMP-9 and TIMP-1 was above 1 (2.1 [0.1-12.5]) in colonized patients, significantly higher than the ratio found in non-colonized patients (0.2 [0.08-0.5]) (p=0.030, Mann-Whitney U test).

Conclusions

Clinically stable COPD patients colonized by H. influenzae had lower levels of specific IgA against the microorganism and higher values of the active form of MMP-9 in their sputum supernatant than non-colonized patients. Bronchial colonization by H. influenzae may cause structural changes in the extracellular matrix through a defective defense and the production of active metalloproteinases.     

beta-Adrenoceptor-mediated thermogenesis and lipolysis in patients with chronic obstructive pulmonary disease

The present study investigated whether development or maintenance of a relatively increased fat mass in normal-weight patients with chronic obstructive pulmonary disease (COPD), despite periods of weight loss, may be related to impaired beta-adrenoceptor-mediated responses in lipid utilization and thermogenesis. Nine COPD patients and nine healthy controls (body mass index: 23.0 +/- 1.3 vs. 23.8 +/- 0.6 kg/m2, not significant; fat mass: 19.0 +/- 2.1 vs. 11.9 +/- 1.5 kg, P < 0.01) received consecutive 30-min infusions of 6, 12, and 24 ng x kg fat free mass(-1) x min(-1) isoproterenol. During beta-adrenergic stimulation, nonesterified fatty acid levels increased significantly less in COPD patients (P < 0.001). Respiratory exchange ratio decreased similarly in both groups, indicating a similar change in the rate of lipid to carbohydrate oxidation. Energy expenditure increased similarly in both groups during beta-adrenergic stimulation. However, because plasma isoproterenol concentrations were significantly higher in COPD patients, thermogenesis related to isoproterenol concentration was significantly reduced in this group (P < 0.05). In conclusion, beta-adrenoceptor-mediated lipolysis and thermogenesis are impaired in COPD patients. This may play a role in the development or maintenance of their relatively increased fat mass.     

Cytogenetic damage and ras p21 oncoprotein levels from patients with chronic obstructive pulmonary disease (COPD), untreated lung cancer and healthy controls

Human population has been continually exposed to benzene which is present in our environment as an essential component of petroleum. p-Benzoquinone (p-BQ) is one of the benzene metabolites and is thought to be an ultimate toxic or carcinogenic substance. For molecular analysis of carcinogen-induced mutations in mouse cells, we constructed a new shuttle vector plasmid pNY200 that has supF gene as a target of the mutations and replicates in mouse and in Escherichia coli cells. In p-BQ-treated pNY200 propagated in mouse cells, base substitutions were induced predominantly at G:C sites, and the major mutation was G:C-->A:T transition. Many tandem base substitutions were also induced at CC:GG sequences. By a postlabeling analysis and a polymerase stop assay, we confirmed that p-BQ adducts formed in DNA and mutation sites roughly correspond to the sites where the adducts were formed. Comparing data of pNY200 in mouse cells with those of the similar shuttle vector plasmid pMY189 in human cells should be important for extrapolation of data from mouse to human, because carcinogenicity of chemicals is tested in mice.     

Proteomic study of human bronchoalveolar lavage fluids from smokers with chronic obstructive pulmonary disease by combining surface-enhanced laser desorption/ionization-mass spectrometry profiling with mass spectrometric protein identification

Bronchoalveolar lavage fluid (BALF) is an important diagnostic source to investigate cellular and molecular changes in the course of lung disorders. The pattern of soluble proteins in BALF obtained from patients at different stages of respiratory disorders may provide deeper insights in the molecular mechanisms of the disease. We used surface-enhanced laser desorption/ionization mass spectrometry (MS) for differential protein display combined with reversed-phase chromatography and subsequent matrix-assisted laser desorption/ionization-MS or nanoliquid chromatography MS/MS analysis for protein identification to compare the protein pattern of BALF samples obtained from ten smokers suffering from chronic obstructive pulmonary disease (COPD), eight clinically asymptomatic smokers, and eight nonsmokers without pulmonary disease. In this context, we were able to identify small proteins and peptides, either differentially expressed or secreted in the course of COPD or in a direct response to cigarette smoke. The concentrations of neutrophil defensins 1 and 2, S100A8 (calgranulin A), and S100A9 (calgranulin B) were elevated in BALFs of smokers with COPD when compared to asymptomatic smokers. Increased concentrations in S100A8 (Calgranulin A), salivary proline-rich peptide P-C, and lysozyme C were detected in BALFs of asymptomatic smokers when compared to nonsmokers, whereas salivary proline-rich peptide P-D and Clara cell phospholipid-binding protein (CC10) were reduced in their concentration. The identified proteins and peptides might be useful in the future as diagnostic markers for smoke-induced lung irritations and COPD.     

Up-regulation of blood arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease

Context and objective: Plasma arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease (COPD) were investigated. Methods: Plasma was extracted and free fatty acids (FFAs) were separated using column chromatography and measured by fluorescence. Plasma 20:4 levels and its percentage relative to total FFA levels (%20:4) were measured in COPD (n = 18) and control (n = 20) subjects. Results and conclusions: FFA levels were lower in COPD compared with normals. However, there was a significant increase in %20:4 levels in COPD patients (GOLD stage I/II 0.9 ± 0.4%; GOLD stage III/IV 1.1 ± 0.1%) compared with control subjects (0.6 ± 0.1, p < 0.05). %20:4 is a potential biomarker for COPD.     

Up-regulation of blood arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease

Context and objective: Plasma arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease (COPD) were investigated.

Methods: Plasma was extracted and free fatty acids (FFAs) were separated using column chromatography and measured by fluorescence. Plasma 20:4 levels and its percentage relative to total FFA levels (%20:4) were measured in COPD (n = 18) and control (n = 20) subjects.

Results and conclusions: FFA levels were lower in COPD compared with normals. However, there was a significant increase in %20:4 levels in COPD patients (GOLD stage I/II 0.9 ± 0.4%; GOLD stage III/IV 1.1 ± 0.1%) compared with control subjects (0.6 ± 0.1, p < 0.05). %20:4 is a potential biomarker for COPD.     

Depressed urokinase activity in bronchoalveolar lavage fluid from patients with sarcoidosis,silicosis or idiopathic pulmonary Rbrosis: relationship to disease severity

Intraalveolar fibrinolysis, is regulated by the concerted actions of plasmin, plasminogen activators (PAs), and their specific inhibitors (PAIs). This event is considered as a critical step in the pathogenesis of pulmonary fibrosis. The aim of this study was to evaluate whether local PA activity can be held as a marker of fibrosis in chronic interstitial lung disorders (ILD). Changes in both PA activity and PA-related proteins (urokinase-type PA (uPA), tissue-type PA (tPA), PAI-1 and PAI-2) were assessed in bronchoalveolar fluid (BALF) of 60 subjects: 18 healthy controls, 18 non-fibrotic sarcoidosis patients, 16 patients with idiopathic pulmonary fibrosis (IPF) and eight silicotic patients with established fibrosis. We observed a significant decrease of BALF PA activity in the three groups of patients as compared with controls. Reduction in BALF PA activity was compatible with lower uPA protein levels associated, especially in IPF patients, with an increased occurrence of PAI-1 and PAI-2 antigens. Soluble tPA antigen was never detected either in control subjects or in patients. Most importantly, the reduction in BALF PA activity and uPA protein levels was found to be most severe in patients with advanced fibrotic disease, namely IPF, while moderate and only weak alterations were found in silicosis and non-fibrotic sarcoidosis, respectively. In addition, significant positive correlations were found between BALF PA activity and functional impairment as assessed by TLC % and DL_CO%. Finally, the reduction in uPA and PA activity levels observed in BALF from sarcoidosis patients was found to be proportional to the degree of BAL lymphocytosis. These findings indicate that an intense reduction in BALF PA activity is associated with severe stages of the parenchymal disease, possibly reflecting the degree of the fibrotic process.