Effect of incremental exercise on airway and systemic inflammation in patients with COPD

Airway and systemic inflammation are features of chronic obstructive pulmonary disease (COPD), and there is growing interest in clarifying the inflammatory processes. Strenuous exercise induces an intensified systemic inflammatory response in patients with COPD, but no study has investigated the airway inflammatory and anti-inflammatory responses to exercise. Twenty steroid-na?ve, ex-smokers with diagnosed COPD (forced expired volume in 1 s = 66 ± 12%) underwent baseline collection of venous blood and induced sputum followed by an incremental exercise test to symptom limitation 48 h later. Additional venous blood samples were collected following exercise at 0, 2, and 24 h, while induced sputum was collected 2 and 24 h after exercise. Sputum and blood samples were analyzed for differential cell count, CD4(+) and CD8(+) T lymphocytes (serum only), interleukin (IL)-6, IL-8, IL-10, chemokine (C-C motif) ligand 5 (CCL5), and high sensitivity C-reactive protein (serum only). There was an increase in the number of sputum eosinophils (cells/gram, P = 0.012) and a reduction in sputum IL-6 (P = 0.01) 24 h postexercise. Sputum IL-8 and CCL5 were also persistently decreased after exercise (P = 0.0098 and P = 0.0012, respectively), but sputum IL-10 did not change. There was a decrease in serum eosinophils 2 h after exercise (P = 0.0014) and a reduction in serum CCL5 immediately following and 2 h postexercise (P < 0.0001). Both serum eosinophils and CCL5 returned to baseline levels within 24 h. An acute bout of exercise resulted in a significant increase in the number of sputum eosinophils, which may be mediated by serum CCL5. However, there was also a reduction in sputum proinflammatory cytokines, suggesting some anti-inflammatory effect of exercise in the lungs of steroid-na?ve patients with COPD.     

Inflammatory response in induced sputum mononuclear cells from patients with acute exacerbation of asthma

Examination of sputum provides a direct method to investigate airway inflammation non-invasively in particular Th1 (IL-2, IFN-gamma) and Th2 (IL-4, IL-10) cytokine production. IL-2, IL-4, IL-10 and IFN-gamma cytokine were studied in induced sputum mononuclear cells of asthmatic patients. Sputum induction was performed on 10 patients and 10 normal controls. Basal and mitogen-stimulated cytokine production was determined in induced sputum T-cell culture. Supernatants were collected and assayed not only with specific ELISA but also with polymerase chain reaction (PCR) techniques. Data showed a significantly higher production of IL-10 by both the ELISA and the RT-PCR techniques in asthmatic patients compared with sputum mononuclear cells from healthy controls. IL-4 production was detected at a low level using the ELISA method in asthmatic patients. The RT-PCR analysis detected a significantly IL-4-mRNA expression in all asthmatic patients, compared with controls. Results of IL-10 and IL-4 mRNA expression were reproducible. We did not find any alteration in the expression of the type 1 derived cytokines (IL-2 and IFN-gamma) in asthmatic patients or in healthy controls. Our study showed a tendency of induced sputum mononuclear cells to express a Th2-like cytokine pattern in acute exacerbation of asthmatic patients, where IL-10 and IL-4 are synthesized in larger amounts. The combination of sputum induction as a non-invasive tool to explore the lung and the identification of disease-associated cytokine expression and of specific cytokine mRNA should help elucidate mechanisms of the immunologically mediated inflammatory responses in asthma.     

High mobility group protein B1 (HMGB1) in Asthma: comparison of patients with chronic obstructive pulmonary disease and healthy controls

High mobility group protein B1 (HMGB1) has been implicated as an important mediator in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). However, the expression of HMGB1 in plasma and sputum of patients with asthma and COPD across disease severity needs to be defined. The objective of the study was to examine the induced sputum and plasma concentrations of HMGB1 in COPD and asthmatic patients to determine differences in HMGB1 levels between these diseases and their relationship with airway obstruction and inflammatory patterns. A total of 147 participants were enrolled in this study. The participants included 34 control subjects, 61 patients with persistent asthma (according to the Global Initiative for Asthma [GINA] guidelines) and 47 patients with stable COPD (stratified by Global Initiative for Chronic Obstructive Lung Disease [GOLD] status). Spirometry was performed before sputum induction. HMGB1 levels in induced sputum and plasma were determined by enzyme-linked immunosorbent assay. Sputum and plasma concentrations of HMGB1 in patients with asthma and COPD were significantly higher than concentrations in control subjects and were significantly negatively correlated with forced expiratory volume in 1 s (FEV(1)), FEV(1) (% predicted) in all 147 participants. The levels of HMGB1 in induced sputum of COPD patients were significantly higher than those of asthma patients and healthy controls (P < 0.001). This difference was present even after adjusting for sex, age, smoking status, daily dose of inhaled corticosteroids and disease severity. There were no significant differences in HMGB1 levels between patients with eosinophilic and noneosinophilic asthma. HMGB1 levels in asthmatic and COPD patients were positively correlated with neutrophil counts and percentage of neutrophils. In multivariate analysis, the two diseases (asthma and COPD) and disease severity were independent predictors of sputum HMGB1, but not smoking, age or use of inhaled corticosteroids. In conclusion, these data support a potential role for HMGB1 as a biomarker and diagnostic tool for the differential diagnosis of asthma and COPD. The importance of this observation on asthma and COPD mechanisms and outcomes should be further investigated in large prospective studies.     

诱导痰黏蛋白5AC对支气管哮喘和慢性阻塞性肺疾病的鉴别价值

摘要 目的：对支气管哮喘（BA）患者和慢性阻塞性肺疾病（COPD）患者诱导痰黏蛋白5AC（MUC5AC）、炎症细胞和炎症因子水平进行比较以及相关性分析,评估MUC5AC鉴别BA和COPD的价值。方法：选取2018年9月至2019年9月来海南医学院第一附属医院就诊的BA稳定期患者（BA组）60例,同期COPD稳定期（COPD组）患者60例。诱导痰法采样并处理痰液,检测诱导痰MUC5AC、炎症细胞中性粒细胞（Neu）、巨噬细胞（Mac）、嗜酸性粒细胞（Eos）、淋巴细胞（Lym）、炎症因子血管内皮生长因子（VEGF）、细胞间黏附分子-1（ICAM-1）、白介素13（IL-13）和白介素17（IL-17）水平,通过Pearson相关分析对MUC5AC水平与Neu、Mac、Eos、Lym、VEGF、ICAM-1、IL-13、IL-17的关系进行分析。此外,采用受试者工作特征(ROC)曲线分析MUC5AC、炎症细胞及炎症因子鉴别诊断BA及COPD的效能。结果：COPD组诱导痰MUC5AC水平高于BA组,差异有统计学意义（P＜0.05）;COPD组诱导痰Mac和Eos水平均低于BA组,COPD组诱导痰Neu水平高于BA组,差异均有统计学意义（P＜0.05）;COPD组诱导痰VEGF、ICAM-1、IL-13和IL-17水平均低于BA组,差异均有统计学意义（P＜0.05）;Pearson相关分析结果显示,诱导痰MUC5AC与炎症细胞Mac、Eos和炎症因子VEGF、ICAM-1、IL-13和IL-17呈负相关（P＜0.05）,与炎症细胞Neu呈正相关（P＜0.05）。经ROC曲线分析可得：诱导痰MUC5AC鉴别BA和COPD的曲线下面积、灵敏度、特异度以及约登指数均高于炎症细胞Neu、Mac、Eos以及炎症因子VEGF、ICAM-1、IL-13、IL-17。结论：COPD患者诱导痰MUC5AC水平高于BA患者,MUC5AC与炎症细胞和炎症因子有关,MUC5AC的检测有助于鉴别BA或COPD,其有望作为临床BA或COPD的监测指标和治疗靶点。     

Sputum neutrophils as a biomarker in COPD: findings from the ECLIPSE study

Introduction

The percentage of neutrophils in sputum are increased in COPD patients, and may therefore be a biomarker of airway inflammation. We studied the relationships between sputum neutrophils and FEV₁, health status, exacerbation rates, systemic inflammation and emphysema, and long term variability at 1 year.

Methods

Sputum samples were obtained from 488 COPD patients within the ECLIPSE cohort. 359 samples were obtained at baseline, and 297 after 1 year. 168 subjects provided samples at both visits. Serum interleukin-6 (IL-6), IL-8, surfactant protein D and C-reactive protein levels were measured by immunoassays. Low-dose CT scans evaluated emphysema.

Results

Sputum neutrophil % increased with GOLD stage. There was a weak association between % sputum neutrophils and FEV₁ % predicted (univariate r² = 0.025 and 0.094 at baseline and year 1 respectively, p < 0.05 after multivariate regression). Similar weak but significant associations were observed between neutrophil % and health status measured using the St Georges Respiratory Questionairre. There were no associations between neutrophils and exacerbation rates or emphysema. Associations between sputum neutrophils and systemic biomarkers were non-significant or similarly weak. The mean change over 1 year in neutrophil % was an increase of 3.5%.

Conclusions

Sputum neutrophil measurements in COPD are associated weakly with FEV₁ % predicted and health status. Sputum neutrophil measurements were dissociated from exacerbation rates, emphysema and systemic inflammation.     

Pneumocystis carinii pneumonia: detection of parasites in sputum and bronchoalveolar lavage fluid by monoclonal antibodies.

Diagnosis of pneumocystis pneumonia is based on identifying Pneumocystis carinii cytochemically in material from the lung. The silver methenamine staining methods most commonly used are technically difficult and lack specificity. The diagnostic value of immunocytological identification of the parasite was evaluated by using mouse monoclonal antibody 3F6, specific for human pneumocystis, to identify P carinii in bronchoalveolar lavage fluid and sputum by immunofluorescence and was compared with that of other variables. Bronchoalveolar lavage was performed on 25 patients positive for HIV antibody with clinically suspected pneumocystis pneumonia and 40 patients negative for HIV antibody who presented with interstitial disorders of the lung. Lavage fluid showed pneumocystis only in the patients positive for antibody, the parasite being detected in 19 by immunofluorescence and in 17 by a modified silver methenamine staining method. Chest x ray films obtained at the time of bronchoscopy showed interstitial or alveolar shadowing in 17 of the 19 patients, but clinical symptoms and the presence of antibodies to pneumocystis did not seem to be predictive. Sputum samples were collected during 43 episodes of clinically suspected pneumocystis pneumonia in patients positive for HIV antibody. Pneumocystis was detected consistently more commonly by immunofluorescence than the silver strain in sputum collected routinely and induced by inhalation of saline. In 17 patients bronchoalveolar lavage followed sputum collection, and the sensitivity of detection of pneumocystis in immunofluorescence in sputum compared with lavage fluid was 57% (8/14). Immunofluorescence was suitable for specimens fixed in ethanol and seemed highly specific and more sensitive than the standard cytochemical methods for identifying pneumocystis.     

细菌感染/定植诱导慢性阻塞性肺疾病患者痰β-防御素2表达

目的：初步探讨下呼吸道细菌感染/定植对慢性阻塞性肺疾病（COPD）患者痰β-防御素2（BD-2）表达水平的影响。方法：36例戒烟COPD患者和30名不吸烟/戒烟对照人员纳入研究。在COPD患者的急性加重期和稳定期分别采集自然痰进行普通细菌培养（简称痰培养）,并行细胞分类计数和BD-2浓度测定;采集对照人员诱导痰行细胞分类计数和BD-2浓度测定以资对照。结果：COPD患者急性加重期和稳定期痰培养阳性比例分别为30/36和14/36。COPD患者痰细胞总数和中性粒细胞比例、淋巴细胞比例高于对照组而巨噬细胞比例低于对照组;痰菌阳性和痰菌阴性的急性加重期COPD患者痰细胞总数、巨噬细胞比例、中性粒细胞比例分别为（6.0±0.9）×106/g、（23.6±3.9）%、（66.0±5.9）%和（3.1±0.5）×106/g、（34.3±4.3）%、（55.7±4.4）%,痰菌阳性和痰菌阴性的稳定期COPD患者痰细胞总数、巨噬细胞比例、中性粒细胞比例、淋巴细胞比例分别为（4.4±0.8）×106/g、（28.6±6.4）%、（64.0±7.2）%和（3.0±0.5）×106/g、（41.4±5.7）%、（45.4±5.1）%。COPD患者稳定期痰BD-2浓度[（211±98）ng/L]低于急性加重期[（300±83）ng/L]而高于对照组[（127±41）ng/L];痰菌阳性稳定期COPD患者痰BD-2浓度高于对照组而与急性加重期无统计学差异;痰菌阴性稳定期COPD患者痰BD-2浓度低于急性加重期而与对照组无统计学差异。结论：下呼吸道细菌感染/定植是COPD患者痰BD-2表达升高的重要诱导因素,也是COPD患者急性加重发作的重要原因。     

Lymphocytes apoptosis in patients with acute exacerbation of asthma

Asthma is characterized by airway inflammation, which can be now assessed by the analysis of induced sputum. Ten patients with asthma were investigated during acute exacerbation for the quantification of apoptosis, for Bcl-2 and Fas expression, in induced sputum lymphocytes. They were compared to 12 patients with chronic obstructive pulmonary disease (COPD), and 10 healthy controls. Spontaneous apoptosis was determined by staining nuclei with propidium iodide, and analyzed with a FACScan. Bcl-2 was measured by Western blotting, and results were obtained by densitometric scanning, done by the gel proanalyser. The investigation of Fas was performed using the streptavidin-biotin preroxidase-complex method. Patients with asthma and patients with COPD exhibited a significant increase of cellularity, percentage of neutrophils, eosinophils and lymphocytes when compared to healthy controls. Apoptosis in induced sputum mononuclear cells was found decreased in patients with asthma compared to COPD patients and healthy controls. The quantification of apoptosis was measured after exposure to anti-cytokine antibodies. Anti-TNF-alpha antibody blocked the apoptosis in both patients groups and healthy controls, suggesting that TNF-alpha acted as an inducer of apoptosis. Anti-IL-10 blocked apoptosis completely exclusively in patients with asthma. Bcl-2 expression was found to be increased in induced sputum mononuclear cells from patients with asthma, compared to healthy controls and patients with COPD. Expression of Fas could be detected in patients with asthma, at a lower level than COPD patients and healthy controls. Distinct mechanisms of apoptosis were found in patients with asthma and patients with COPD, characterized by different levels of Bcl-2 and Fas expression. Induction of apoptosis should be a beneficial process in allergic inflammation traduced in induced sputum mononuclear cells. The apoptosis process is assumed by two different mechanisms in asthma and COPD. Our findings indicated that in asthmatic patients, activated lymphocytes accumulate in the bronchi; because of their prolonged survival that maintains inflammation.     

Macrorheology of cystic fibrosis,chronic obstructive pulmonary disease & normal sputum

Background

Prior microrheologic assessments of selected, microlitre plugs of cystic fibrosis (CF) sputum suggest no intrinsic rheologic abnormality. However, such analyses may not be representative of CF sputum as a whole. We therefore reassessed this question using whole sputum macrorheology. Additionally, we wished to further explore the relationships between sputum rheology, inflammation and infection.

Methods

Dynamic oscillatory macrorheometry was performed on whole expectorated sputum from stable adults with CF (n = 18) and COPD (n = 12) and induced sputum from normal controls (n = 7). Concomitant sputum inflammatory mediator levels were measured in CF and COPD samples. Sputum collected from CF subjects (n = 6) at commencement and completion of intravenous antibiotic therapy for an infective exacerbation was also assessed.

Results

CF sputum neutrophil elastase activity (NE) was significantly related to degree of sputum purulence (p = 0.049) and correlated significantly with measures of sputum viscoelasticity (r = 0.696, p = 0.008 for storage modulus G'' at 9 Hz). There were significant differences in viscoelasticity between subject groups when samples were compared irrespective of appearance/degree of sputum purulence. However, the macrorheology of mucoid CF sputum did not differ from normal sputum (eg median (range) G'' at 9 Hz 2.25 (0.79, 3.26) vs 2.04 (1.4,4.6) Pa, p = 1). In contrast, mucoid COPD samples demonstrated significantly greater viscoelasticity (G'' at 9 Hz 4.5 (2.4, 23) Pa) than sputum from both CF (p = 0.048) & normal subjects (p = 0.009). Antibiotic therapy during exacerbations was associated with significant reductions in CF sputum viscoelasticity, with mean (SD) G'' at 9 Hz decreasing from 28.5 (11.5) Pa at commencement to 6.4 (4.6) Pa on day 7 (p = 0.01).

Conclusion

The macrorheologic properties of whole, mucoid CF sputum are not different from normal, confirming the results of prior microrheologic studies. Instead, CF sputum viscoelasticity is related to secondary infection, decreases with intravenous antibiotic therapy and correlates with inflammation. In contrast, COPD sputum demonstrates inherently greater viscoelasticity, providing a novel target for potential therapeutic interventions.     

Comparison of inflammatory markers in induced and spontaneous sputum in a cohort of COPD patients

Background

Sputum induction is a non-invasive method for obtaining measurements of inflammation in the airways. Whether spontaneously sampled sputum can be a valid surrogate is unknown. The aim of this study was to compare levels of six inflammatory markers in sputum pairs consisting of induced and spontaneous sputum sampled on the same consultation either in a stable state or during exacerbations of chronic obstructive pulmonary disease (COPD).

Methods

433 COPD patients aged 40–76, Global initiative for chronic Obstructive Lung Disease (GOLD) stage II-IV were enrolled in 2006/07 and followed every six months for three years. 356 patients were followed for potential exacerbations. Interleukin-6, interleukin-8, interleukin-18, interferon gamma-inducible protein-10, monokine induced by gamma interferon and tumor necrosis factor-alpha (IL-6, IL-8, IL-18, IP-10, MIG and TNF-α) were measured by bead based multiplex immunoassay in 60 paired sputum samples from 45 patients. Albumin was measured by enzyme immunoassay, for concentration correction. Culturing for bacterial growth was performed on 24 samples. Bland-Altman plots were used to assess agreement. The paired non-parametric Wilcoxon signed-rank test, the non-parametric Spearman’s rank correlation test and Kruskal-Wallis test were used for statistical analyses. For all analyses, a p-value < 0.05 was considered significant.

Results

Agreement between the two measurements was generally low for all six markers. TNF-α was significantly higher in spontaneous sputum at exacerbations (p = 0.002) and trending higher at the steady state (p = 0.06). Correlation coefficients between the levels of markers in induced and spontaneous sputum varied between 0.58 (IL-18) to 0.83 (IP-10). In spontaneous sputum IL-18 and MIG were higher in ex-smokers (p < 0.05). The levels of all markers were higher in GOLD stage III & IV except for IL-6 in spontaneous sputum and IL-18 in induced sputum, compared with GOLD stage II, although not statistically significant. In spontaneous sputum the levels of IL-6 were significantly higher if Haemophilus influenzae (HI) was not cultured.

Conclusion

We observed a low agreement and significant differences in inflammatory markers between induced and spontaneous sputum, both at steady state and exacerbations. We recommend considering sampling method when reporting on inflammatory markers in sputum.     

Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis

Aim: The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF). Methods: Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT‐PCR‐DGGE and qPCR analysis of the bacterial and fungal communities was carried out. Results: Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae. Conclusions: The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4°C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.     

Sputum proteomics identifies elevated PIGR levels in smokers and mild-to-moderate COPD

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms leading to COPD and its progression are still poorly understood. In this study, induced sputum was analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis. The comparison of nonsmokers, smokers, and smokers with moderate COPD revealed 15 changed proteins with the majority, including polymeric immunoglobulin receptor (PIGR), being elevated in smokers and subjects with COPD. PIGR, which is involved in specific immune defense and inflammation, was further studied in sputum, lung tissue, and plasma by Western blot, immunohistochemistry/image analysis, and/or ELISA. Sputum PIGR was characterized as glycosylated secretory component (SC). Lung PIGR was significantly elevated in the bronchial and alveolar epithelium of smokers and further increased in the alveolar area in mild to moderate COPD. Plasma PIGR was elevated in smokers and smokers with COPD compared to nonsmokers with significant correlation to obstruction. In conclusion, new proteins in smoking-related chronic inflammation and COPD could be identified, with SC/PIGR being one of the most prominent not only in the lung but also in circulating blood.     

COPD phenotype description using principal components analysis

Background

Airway inflammation in COPD can be measured using biomarkers such as induced sputum and Fe_NO. This study set out to explore the heterogeneity of COPD using biomarkers of airway and systemic inflammation and pulmonary function by principal components analysis (PCA).

Subjects and Methods

In 127 COPD patients (mean FEV₁ 61%), pulmonary function, Fe_NO, plasma CRP and TNF-α, sputum differential cell counts and sputum IL8 (pg/ml) were measured. Principal components analysis as well as multivariate analysis was performed.

Results

PCA identified four main components (% variance): (1) sputum neutrophil cell count and supernatant IL8 and plasma TNF-α (20.2%), (2) Sputum eosinophils % and Fe_NO (18.2%), (3) Bronchodilator reversibility, FEV₁ and IC (15.1%) and (4) CRP (11.4%). These results were confirmed by linear regression multivariate analyses which showed strong associations between the variables within components 1 and 2.

Conclusion

COPD is a multi dimensional disease. Unrelated components of disease were identified, including neutrophilic airway inflammation which was associated with systemic inflammation, and sputum eosinophils which were related to increased Fe_NO. We confirm dissociation between airway inflammation and lung function in this cohort of patients.     

Compartment differences of inflammatory activity in chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is associated with local and systemic inflammation. The knowledge of interaction and co-variation of the inflammatory responses in different compartments is meagre.

Method

Healthy controls (n = 23), smokers with (n = 28) and without (n = 29) COPD performed spirometry and dental examinations. Saliva, induced sputum, bronchoalveolar lavage (BAL) fluid and serum were collected. Inflammatory markers were assessed in all compartments using ELISA, flow cytometry and RT-PCR.

Results

Negative correlations between lung function and saliva IL-8 and matrix metalloproteinase-9 (MMP-9) were found in smokers with COPD. IL-8 and MMP-9 in saliva correlated positively with periodontal disease as assessed by gingival bleeding in non-smokers.Tumor necrosis factor-α (TNF-α) in saliva, serum and TNF-α mRNA expression on macrophages in BAL-fluid were lower in smokers than in non-smokers. There were positive correlations between soluble TNF-α receptor 1 (sTNFR1) and soluble TNF-α receptor 2 (sTNFR2) in sputum, BAL-fluid and serum in all groups. Sputum interleukin-8 (IL-8) or interleukin-6 (IL-6) was positively correlated with sTNFR1 or sTNFR2 in non-smokers and with sTNFR2 in COPD.

Conclusion

Saliva which is convenient to collect and analyse, may be suitable for biomarker assessment of disease activity in COPD. An attenuated TNF-α expression was demonstrated by both protein and mRNA analyses in different compartments suggesting that TNF-α response is altered in moderate and severe COPD. Shedding of TNFR1 or TNFR2 is similarly regulated irrespective of airflow limitation.     

Increased interleukin (IL)-8 and decreased IL-17 production in chronic obstructive pulmonary disease (COPD) provoked by cigarette smoke

Recently, involvement of IL-17 in development of COPD has been noticed. Unlike IL-8, the role of IL-17 in COPD remains controversial. In order to further understand mechanisms in cigarette smoke (CS) induced COPD, we investigated IL-17 and IL-8 levels in different stages of COPD patients, and time courses of IL-17 and IL-8 release in CS induced COPD rats. A total of 73 elderly patients with COPD and 31 healthy volunteers were recruited in the study. IL-17 and IL-8 levels in the sputum and plasma were measured, and number of differential cells was counted. A newly developed CS induced rat COPD model was employed to study time courses of IL-17 and IL-8 release and nucleated cell accumulation. The results showed that IL-8 levels in the sputum of severe COPD patients were elevated by 16.5-fold, but IL-17 levels were reduced by 4.8-fold. While IL-8 correlated with neutrophils, IL-17 correlated with monocytes and lymphocytes. Similarly, level of IL-8 was increased, but IL-17 was decreased in the bronchoalveolar lavage fluid (BALF) of CS rats. Time course study showed that increased IL-8 production in the BALF initiated at 6 weeks, but decreased IL-17 production started at 10 weeks following CS exposure. In conclusion, increased IL-8 level in COPD patients appears mainly secreted from neutrophils and macrophages, whereas decreased IL-17 level seems resulted from reduced number of monocytes or damaged epithelial cells. Increased IL-8 (a proinflammatory cytokine) secretion and decreased IL-17 (a protective cytokine of airways) release can both contribute to development of COPD.     

通用PCR法在痰标本真菌检测中的临床应用价值分析

目的:探讨通用PCR法在痰标本真菌检测中的临床应用价值。方法:选择2015年9月至2017年9月本院收治的免疫力低下患者178例作为研究资料,各收集痰标本2份,其中一份用于PCR扩增,另一份经培养后若观察到真菌或可疑菌落,则提取后在此进行PCR扩增测序。比较3种方法对真菌的检出情况、真菌阳性率,与组织病理学结果进行比较,分析3种方法的诊断效能。结果:178份痰标本经平板培养可观察到107份真菌生长,其余71份未见真菌生长。挑取真菌及可疑菌落行PCR结果显示阳性、阴性分别115、63份。对痰标本直接PCR结果显示,阳性、阴性分别124、54份。PCR扩增产物测序结果显示大多是白色念珠菌感染,仅3份是曲霉菌感染。内对照扩增验证结果显示有1份阴性标本存在扩增抑制现象。3种方法的真菌阳性率:直接PCR法痰培养后PCR法痰培养,但组间无显著差异(P0.05)。3种方法诊断效能总体趋势:直接PCR法痰培养后PCR法痰培养,其中直接PCR法与痰培养后PCR法的特异度、准确度均显著高于痰培养法(P0.05),直接PCR法的敏感度显著高于痰培养法(P0.05)。结论:通用PCR法在痰标本真菌检测中的临床应用价值较高,直接PCR具有操作简单、快速等优势。     

Repeatability of and Relationship between Potential COPD Biomarkers in Bronchoalveolar Lavage,Bronchial Biopsies,Serum, and Induced Sputum

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, primarily affecting the airways. Stable biomarkers characterizing the inflammatory phenotype of the disease, relevant for disease activity and suited to predict disease progression are needed to monitor the efficacy and safety of drug interventions. We therefore analyzed a large panel of markers in bronchoalveolar lavage, bronchial biopsies, serum and induced sputum of 23 healthy smokers and 24 smoking COPD patients (GOLD II) matched for age and gender. Sample collection was performed twice within a period of 6 weeks. Assays for over 100 different markers were validated for the respective matrices prior to analysis. In our study, we found 51 markers with a sufficient repeatability (intraclass correlation coefficient >0.6), most of these in serum. Differences between groups were observed for markers from all compartments, which extends (von-Willebrand-factor) and confirms (e.g. C-reactive-protein, interleukin-6) previous findings. No correlations between lung and serum markers were observed, including A1AT. Airway inflammation defined by sputum neutrophils showed only a moderate repeatability. This could be improved, when a combination of neutrophils and four sputum fluid phase markers was used to define the inflammatory phenotype.In summary, our study provides comprehensive information on the repeatability and interrelationship of pulmonary and systemic COPD-related markers. These results are relevant for ongoing large clinical trials and future COPD research. While serum markers can discriminate between smokers with and without COPD, they do not seem to sufficiently reflect the disease-associated inflammatory processes within the airways.     

Toll-Like Receptor (TLR2 and TLR4) Polymorphisms and Chronic Obstructive Pulmonary Disease

Toll-like receptors (TLRs) participate in the defence against bacterial infections that are common in patients with Chronic Obstructive Pulmonary Disease (COPD). We studied all tagging SNPs in TLR2 and TLR4 and their associations with the level and change over time of both FEV(1) and sputum inflammatory cells in moderate-to-severe COPD. Nine TLR2 SNPs and 17 TLR4 SNPs were genotyped in 110 COPD patients. Associations of SNPs with lung function and inflammatory cells in induced sputum were analyzed cross-sectionally with linear regression and longitudinally with linear mixed-effect models. Two SNPs in TLR2 (rs1898830 and rs11938228) were associated with a lower level of FEV(1) and accelerated decline of FEV(1) and higher numbers of sputum inflammatory cells. None of the TLR4 SNPs was associated with FEV(1) level. Eleven out of 17 SNPs were associated with FEV(1) decline, including rs12377632 and rs10759931, which were additionally associated with higher numbers of sputum inflammatory cells at baseline and with increase over time. This is the first longitudinal study showing that tagging SNPs in TLR2 and TLR4 are associated with the level and decline of lung function as well as with inflammatory cell numbers in induced sputum in COPD patients, suggesting a role in the severity and progression of COPD.     

Sputum cytology in the diagnosis of pulmonary paracoccidioidomycosis

The presence of Paracoccidioides brasiliensis was determined in sputum samples from 50 patients with paracoccidioidomycosis using four different techniques: (a) cell-block preparations stained with silver methenamine, (b) direct microbiologie examination, (c) smears stained with Shorr, and (d) smears stained with silver methenamine. Overall, cell-block preparations and smears stained with silver methenamine proved to be the most sensitive techniques, followed by smears stained with Shorr and direct microbiologic examination in decreasing order of sensitivity. Sputum cytology tended to be less positive in patients with interstitial pulmonary lesions as determined by chest X-ray than in patients with alveolar lesions. In addition to its high sensitivity, cell-block preparation technique allows storage of blocks and slides for further studies.