Nitrites in induced sputum as a simple and cheap non-invasive marker of airway inflammation for asthmatic schoolchildren.

To determine if there are differences in the nitric oxide metabolites (nitrites) in sputum of patients with persistent asthma and healthy schoolchildren, we performed a case-control study in a tertiary care hospital in Arequipa, Perú. Nitrites in induced sputum samples were measured using the Griess assay in 30 persistent asthmatics (mean age of 10.1 yr) and 30 controls (mean age of 11.9 yr). The mean +/- s.d. of nitrites among asthmatics was significantly higher than the controls (16.30 +/- 8.6 vs. 10.25 +/- 4.68 nmol/ml, respectively, p = 0.001). Moreover, the nitrite level in the sputum in children with severe persistent asthma was higher than in the level found in the moderate and mild asthmatics (32.83 +/- 9.48 vs. 18.10 +/- 1.96 vs. 11.84 +/- 4.73 nmol/ml, respectively, p < 0.01 for linear trend). This study showed for the first time in children that asthmatics have significantly higher levels of nitrites in induced sputum than healthy controls and that the level of nitrite correlates with the severity of the asthma. Nitrite levels in sputum, a simple and cheap, non-invasive method, may be a good alternative to measure the severity of inflammation in asthmatic children.     

哮喘患儿诱导痰液中NO_3~-/NO_2~-含量的变化及其意义

探讨儿童哮喘诱导痰液中一氧化氮(NO)代谢终产物硝酸盐/亚硝酸盐(NO_3~-/NO_2~-)含量的变化及临床意义,采用3％高渗盐水雾化吸入诱导患儿咳痰,0.1％DTT和PBS缓冲液裂解痰液,应用硝酸酶还原法和荧光酶标法分别测定诱导痰液中No_3~-/NO_2~-和嗜酸性粒细胞阳离子蛋白(ECP)含量,便携式肺功能仪测定肺通气功能。结果表明,稳定期、轻度和中、重度发作期患儿诱导痰液中NO_3~-/NO_2~-浓度较对照组显著增高,P<0.05;ECP含量较对照组显著增高,P<0.05;不同时期哮喘患儿各组间诱导痰液中NO_3~-/NO_2~-含量和ECP均有显著性差异,P<0.05。哮喘患儿诱导痰液中NO_3~-/NO_2~-和ECP含量与1秒钟用力呼气容积占预计值百分比(FEV1.0％)呈显著负相关,r=0.512,P<0.01;而NO_3~-/NO_2~-含量与痰液中ECP浓度呈显著正相关r=0.607,P<0.01。提示NO参与了儿童哮喘的发生和发展,诱导痰液中NO_2~-/NO_2~-水平可反映气道炎症的变化,可用于儿童哮喘病情监测及指导药物治疗。     

诱导痰液与哮喘患儿气道炎症的关系探讨

目的探讨简便有效的诱导痰液及细胞分析方法,了解儿童哮喘发作期气道炎症的特点。方法运用高渗盐水雾化吸入诱导痰液的方法,收集３７例哮喘发作期患儿、４８例正常儿童（对照组）痰液。一部分痰液经二硫苏糖醇（ＤＴＴ）液化后,在血球计数板上行细胞总计数;另一部分痰液涂片经瑞氏、甲苯胺蓝染色行细胞分类计数。结果哮喘组８４％取痰成功,诱导过程中呼气峰流速（ＰＥＦ）未见下降（Ｐ＞００５）。痰液细胞总计数哮喘组为（１１２±８９）×１０９／Ｌ,对照组为（７１±６２）×１０９／Ｌ（Ｐ＜００５）。嗜酸性粒细胞、肥大细胞在哮喘发作期气道中明显升高（Ｐ＜００１）。结论诱导痰液分析、评判哮喘患儿气道炎症是一种安全、可靠的新研究方法;嗜酸性细胞、肥大细胞为哮喘发作的主要效应细胞。     

Eosinophil cationic protein in induced sputum as a marker of inflammation in asthmatic children

We evaluated the usefulness of eosinophil cationic protein (ECP) in induced sputum and in serum as markers of asthmatic inflammation in children. We measured ECP in serum and in total indticed sputum samples from 14 children (7-11 years) with newly detected asthma before and after treatment, and from ten healthy non atopic controls of the same age. The patients inhaled budesonide, 800 μg/m2/day for the first month and 400 μg/m2/day for the next 5 months, both divided into two doses, and nedocromil, 4 mg three times daily for the following 6 months. In both sputum and serum, ECP levels were higher in the patients than in the controls, but the difference was more distinct in sputum. Significant clinical improvement during the treatment was accompanied by a decrease in sputum ECP. whereas serum ECP did not change. The results suggest that induced sputum is useful as a non invasive source material for evaluating asthmatic inflammation in children, total sputum ECP being more sensitive than serum ECP for diagnosing and monitoring asthma.     

急性哮喘发作患儿诱导痰液炎性细胞、白细胞介素-6、-8变化的意义

目的 了解儿童哮喘急性发作时气道炎性特征及临床意义 方法 对34例急性发作期、24例稳定期哮喘患儿和15例正常儿童的诱导痰液进行炎性细胞计数和分类,并测定其中白细胞介素-6(IL-6)、IL-8水平,分析炎性细胞与上述细胞因子及最大呼气流速(PEF)的关系 结果 哮喘急性发作组总细胞数较正常对照组明显增高,急性发作组嗜酸性粒细胞(EOS)、中性粒细胞及单核细胞比例均高于正常对照组,淋巴细胞比例明显低于正常对照组,稳定组EOS比例明显高于正常对照组 EOS比例与PEF呈显著负相关,IL-8水平与中性粒细胞呈显著正相关,急性发作期IL-6水平与EOS呈显著正相关 结论 急性哮喘发作患儿气道炎症是由EOS、中性粒细胞及单核细胞参与的炎症,IL-6、IL-8可能是参与此过程的重要细胞因子 痰液EOS数与病情严重程度相关,可作为监测病情及疗效的指标之一 针对这些细胞因子的治疗具有潜在价值     

The relation between obesity and asthmatic airway inflammation

Epidemiologic studies suggest increased asthma prevalence in obese subjects. However, the relation between obesity and airway inflammation remains unclear. This cross-sectional study aims to investigate the relation between obesity indices and exhaled nitric oxide (ENO) and leukotriene B(4) (LTB(4)) in children with asthma. Asthmatic patients aged 7-18 yr old were recruited. Weight-for-height Z score was calculated from anthropometry. ENO was measured by online single-breath method using a chemiluminescence analyzer, whereas LTB(4) concentrations in exhaled breath condensate (EBC) were quantified using competitive enzyme immunoassay. Ninety-two asthmatics and 23 controls were recruited. The mean ENO and LTB(4) concentrations in EBC were higher in asthmatic patients (87 p.p.b. and 40.5 pg/ml) than controls (25 p.p.b. and 18.7 pg/ml) (p < 0.0001 for both). Obesity, as defined by weight >120% median weight-for-height, was not associated with any alteration in ENO or LTB(4) concentrations in patients with asthma. Besides, these inflammatory markers did not differ between asthmatics in the highest and lowest quartiles of weight-for-height Z score. On multivariate analysis, ENO showed significant correlation with age (beta = 0.511, p < 0.0001), peripheral blood eosinophil count (beta = 0.222, p = 0.019), plasma total IgE concentration (beta = 0.187, p = 0.050) and forced expiratory volume in 1-s (FEV(1); beta = -0.221, p = 0.014). None of the factors was associated with LTB(4) concentration in EBC. In conclusion, ENO and LTB(4) concentration in EBC are increased in childhood asthma. However, these inflammatory markers did not differ between obese and non-obese children with asthma.     

Surrogate markers of airway inflammation: inflammometry in paediatric respiratory medicine

Until recently, there has not been any practical way to assess airway inflammation non-invasively in paediatrics. Surrogate markers of airway inflammation are potentially of great importance in the diagnosis and monitoring of inflammatory airways disease in children. A large number of substances in blood, urine and exhaled air or induced sputum are currently under study to evaluate their possible usefulness as markers of airway inflammation. To be useful, a marker should be valid, preferably non-invasive, quick, reproducible, repeatable and cheap. In addition, markers should be studied in relation to their specific purpose because different markers may be useful for different types of airway inflammation. Few, if any, markers will fulfill all these requirements. Most research has focused on applications of markers in asthma, some data refer to cystic fibrosis, infections and ciliary dyskinesia. Of all surrogate markers, exhaled nitric oxide has been studied the most and seems to offer information that should be evaluated for its relevance to clinical practice. Before introducing markers of inflammation into daily practice, analysis of benefits and costs are needed. There is little doubt that 'inflammometry' will be a major step forward and will be useful in differentiating airways diseases and improving treatment.     

Correlation of nitrites in breath condensates and lung function in asthmatic children

We aimed to evaluate the value of exhaled breath condensates in monitoring airway inflammation in childhood asthma before and after high altitude climate therapy.
Forty-eight asthmatic children on regular anti-asthma treatment with a normal FEV₁ and positive skin prick test for house dust mites were recruited. All children had been referred to an alpine clinic for high altitude climate therapy, because of persistent asthmatic symptoms despite use of daily anti-inflammatory treatment. Subjects were assessed on their arrival and before departure from the alpine clinic. Spirometry, bronchial provocation tests and measurements of nitrites in breath condensates were performed.
Median levels of nitrites were significantly higher before than after high altitude climate therapy (1.27 vs. 0.93 μ m ; p = 0.008). In addition, MEF₅₀ improved significantly (p < 0.0005). There was a significant correlation between nitrites in breath condensates and MEF₅₀ (r = −0.63, p < 0.0001), symptoms (r = 0.47, p = 0.0007) and airway hyper-reactivity (AHR) (r = −0.41, p = 0.004).
In summary, we found a reduction in nitrites in breath condensates after a high altitude climate therapy. Significant correlations were found between nitrites and MEF₅₀, AHR and symptoms. We conclude that the measurement of nitrites may be feasible to objectively assess airway inflammation in asthmatic children in order to detect ongoing inflammation in children with normal FEV₁ but persistent symptoms.     

Non‐invasive markers of airway inflammation and remodeling in childhood asthma

To evaluate the relationship between pro‐inflammatory and pro‐remodeling mediators and severity and control of asthma in children, the levels of IL‐8, MMP‐9, TIMP‐1 in induced sputum supernatants, the number of sputum eosinophils, as well as FeNO, were investigated in 35 asthmatic children, 12 with intermittent (IA) and 23 with moderate asthma (MA), and 9 controls (C). The patients with asthma were followed for 1 yr and sputum was obtained twice during the follow‐up. Biomarker levels were correlated with the number of exacerbations. We found that IL‐8, MMP‐9, TIMP‐1 and the numbers of eosinophils in induced sputum, as well as FeNO, were increased in children with IA and MA in comparison to C. The ongoing inflammation was confirmed by increased nuclear p65 NF‐κB subunit localization in sputum cells. In MA, FeNO measurements, sputum eosinophils and IL‐8 levels, positively correlated with the occurrence of disease exacerbations during a 1‐yr follow‐up. According to FeNO, sputum eosinophils and IL‐8 sputum concentrations, and the number of exacerbations, two distinct phenotypes of MA were identified. This study shows that the presence of bronchial inflammation is detectable in the airways of some IA, as well as in the airways of MA, despite the regular ICS treatment. This study also proposes the need to perform large prospective studies to confirm the importance of measuring specific biomarkers in induced sputum, concomitantly to FeNO analyses, to assess sub‐clinical airway inflammation and disease control in children with asthma.     

Effect of montelukast on exhaled NO in asthmatic children exposed to relevant allergens

The level of exhaled nitric oxide (FENO) is increased in house dust mite (HDM)-sensitized asthmatic children after exposure to HDM antigen, and inhaled steroids can prevent this increase. The aim of this study was to evaluate whether montelukast could prevent an increase in FENO levels in allergic asthmatic children after a brief period of exposure to relevant allergens. Sixteen children were evaluated at the residential house 'Istituto Pio XII' (Misurina, Bellunio, Italy) in the Italian Alps, a dust mite-free environment. FENO levels were evaluated before ( t ₀) and immediately after ( t ₁) the children were exposed to HDM allergens for 2 weeks in their homes at sea level. No significant difference in FENO was observed in the fluticasone-treated group of children after 2 weeks at sea level. In the group treated with montelukast, an increase in FENO was observed between t ₀ and t ₁, which failed to reach statistical significance. These preliminary data suggest that oral montelukast could be effective in preventing the relapse in airway inflammation in allergic asthmatic children who are occasionally exposed to relevant allergens for a short period of time.     

轻-中度哮喘患儿气道炎症类型与病情、糖皮质激素疗效的关系

目的探讨轻-中度支气管哮喘患儿初始治疗前气道炎症类型与病情及吸入糖皮质激素治疗反应的关系。方法以87例轻-中度哮喘患儿作为研究对象,在糖皮质激素吸入治疗(ICS)前进行痰液诱导及诱导痰细胞学分析,酶联免疫荧光法测定痰液嗜酸粒细胞阳离子蛋白(ECP)、ELISA法检测痰液IL-8、TGF-β1,儿童肺功能仪检测基础肺功能和小气道通气指标、乙酰甲胆碱(Mch)支气管激发试验测定气道高反应性(AHR)。20例健康体检儿童作为对照组,应用SPSS13.0软件进行统计学分析。结果87例轻-中度哮喘患儿根据诱导痰液EOS%分为嗜酸性粒细胞哮喘组(EA)64例,非嗜酸性粒细胞哮喘组(NEA)23例,EA与NEA组ICS治疗前痰液细胞学构成比、诱导痰上清液ECP、IL-8差异有统计学意义(P<0.05);两组间FEV1%预测值(FEV1%pred)、PEF%pred、中-重度AHR%、小气道阻塞(%)、痰液TGF-β1水平等指标差异有统计学意义(P<0.05)。ICS治疗4周后EA组基础肺功能指标、气道高反应性、小气道通气功能明显改善,而NEA组改善不明显。多元逐步回归分析结果表明初治痰液EOS%、FEV1%预测值、痰液TG...     

Vascular endothelial growth factor overexpression in induced sputum of children with bronchial asthma

Vascular endothelial growth factor (VEGF) induces angiogenesis and increases vascular permeability participating in narrowing of the airway lumen that follows lung injury. We sought to investigate the expression of VEGF in induced sputum during and after recovery from acute episodes of bronchial asthma in children. Eighteen asthmatic children with acute attacks of varying severity were subjected to VEGF estimation by an enzymatic immunoassay in induced sputum. They were followed up till complete remission of symptoms and signs and were then retested. VEGF was also estimated in sputum induced from age 34 and sex-matched healthy children enrolled as a control group. The sputum VEGF levels during acute asthma [median = 71 ng/ml; mean (s.d.) = 114.6 (121.8) ng/ml] were significantly higher than the levels estimated during remission [median = 50 ng/ml; mean (s.d.) = 45.7 (24.2) ng/ml] and both were higher than the corresponding levels of the control group [median = 36 ng/ml; mean (s.d.) = 31.3 (17.2) ng/ml]. VEGF levels during asthmatic episodes correlated positively to the recovery levels (r = 0.6, p = 0.009). The patients' VEGF expression did not vary with asthma severity, serum total IgE concentration, peripheral blood eosinophil count, or erythrocyte sedimentation rate of patients. Children on corticosteroids inhalation therapy at enrollment had sputum VEGF levels that were comparable to those on other therapies. The increased expression of sputum VEGF in asthmatic children reinforces the concept that it might have a pathogenetic role in bronchial asthma and may represent a biomarker of airway inflammation.     

Exhaled breath condensate eicosanoids and sputum eosinophils in asthmatic children: A pilot study

Cysteinyl leukotrienes (cys-LTs), LTB₄ and 8-isoprostane are increased in the exhaled breath condensate (EBC) from asthmatic patients. The aim of this study was to investigate whether the measurement of cys-LTs, LTB₄ and 8-isoprostane in EBC can reflect the level of airway inflammation assessed by induced sputum in asthmatic children sensitized to house dust mite (HDM) during natural avoidance of HDM allergens. Twelve children were evaluated at the time of admission (T0) and after 3 months of stay (T1) at the Istituto Pio XII (Misurina, Italian Dolomites 1756  m). Sputum eosinophil percentage and measurement of cys-LTs, LTB₄ and 8-isoprostanes in the breath condensate at T0 and T1 were evaluated. Eosinophil percentage in induced sputum was 8.5 ± 1.1% at T0 and 3.5 ± 0.4% at T1 (p = 0.011). Neutrophil percentage in sputum was 1.1 ± 0.5% at T0 and 1.5 ± 1.0% at T1 (ns). Cys-LTs mean level was 14.24 ± 4.53 pg/ml at T0 and 4.65 ± 0.68 pg/ml at T1 (p = 0.0125). LTB₄ level was 2.36 ± 0.19 pg/ml at T0 and 2.41 ± 0.23 pg/ml at T1 (ns). 8-Isoprostane level reduced from 17.47 ± 3.18 pg/ml at T0 to 7.36 ± 3.26 pg/ml at T1 (p = 0.003). This study show that exhaled cys-LTs and 8-isoprostane, as well as eosinophil percentage in induced sputum, are reduced after allergen avoidance in asthmatic children suggesting a potential application of EBC for the non-invasive evaluation of airway inflammation in asthma in allergic asthmatic children.     

无创性检查对哮喘长期缓解患儿气道炎症的评价

目的探索常用无创性检查对哮喘长期缓解的评价。方法选择45例哮喘患儿,分发作组、短期缓解组、长期缓解组;另设对照组12例。检测肺功能、乙酰甲胆碱(Mch)支气管激发试验、诱导痰液计数EOS%、酶联免疫荧光法测定痰液及血清嗜酸性细胞阳离子蛋白(ECP)、ELISA法检测痰液及血清IL-5。结果①46.15%长期缓解期患儿FEF75<80%预计值,高于对照组(P<0.05);FEF25为(106.3±14.8)%,高于发作组(P<0.05)。②长期缓解患儿气道高反应性(AHR)与对照组差异无统计学意义(P>0.05)。③缓解期患儿痰液EOS%,血清ECP,痰液、血清IL-5与对照组比较差异均无统计学意义(P>0.05);痰液ECP高于对照组(P<0.05)。④各小气道通气指标与Mch激发试验、痰液EOS%、ECP、IL-5呈负相关(P均<0.05)。⑤小气道阻塞哮喘患儿AHR重于小气道功能正常者(P<0.05),小气道阻塞患儿各小气道指标与痰IL-5呈负相关(P<0.05)。⑥Mch激发试验与痰液、血清ECP,痰液IL-5呈正相关(P均<0.05)。⑦两组缓解期患儿肺功能,AHR,痰液EOS%、ECP、IL-5,血清ECP、IL-5差异均无统计学意义(P均>0.05)。⑧上述指标对哮喘长期缓解无预测价值(P>0.05)。结论长期缓解哮喘患儿存在一定程度小气道功能异常及气道炎症,痰液ECP是反映气道炎症更敏感的指标。研究所选取的无创性检查对评价哮喘缓解均有一定指导作用,但对哮喘长期缓解无预测价值。     

哮喘患儿支气管诱导痰液中P物质含量变化的临床研究

目的　研究支气管哮喘患儿急性发作期、缓解期支气管诱导痰液中P物质含量变化及临床意义。方法　用高渗盐水超声雾化吸入法留取研究组及对照组儿童支气管诱导痰液。其中哮喘急性发作期组 34例 (3d内无静脉或口服激素史 ) ;哮喘缓解期组 30例 ;健康对照组 2 2例。用放射免疫方法检测各组支气管诱导痰中P物质含量。结果　哮喘患儿急性发作期支气管诱导痰中P物质含量明显高于缓解期及正常对照组 ,差异显著 ,P均 <0 0 1。缓解期组与正常对照组比较 ,差异无显著性 ,P >0 0 5。结论　哮喘患儿急性发作期支气管诱导痰中P物质含量显著增高 ,缓解期降低 ,接近正常水平 ;提示支气管诱导痰中P物质含量与儿童哮喘的发作及缓解关系密切 ,可做为一种临床监测指标。     

哮喘小鼠气道IL-6、信号转导和转录激活因子3的表达与活化及其与气道炎症的关系

目的研究气道IL-6、信号转导和转录激活因子3(STAT3)在哮喘小鼠气道中的表达与活化,及其与气道炎症的关系。方法 30只Balb/c小鼠随机分为正常对照组、哮喘未干预组和AG490干预组,每组10只。建立小鼠哮喘模型,苏木精-伊红(HE)染色观察肺组织病理改变,收集支气管肺泡灌洗液(BALF),进行炎性细胞分类计数,ELISA检测BALF中IL-4、IL-5、IL-6水平,免疫组织化学染色检测各组肺组织中STAT3表达,蛋白免疫印迹法检测肺组织STAT3和p-STAT3水平。应用SPSS13.0软件进行统计学分析。结果 HE染色显示哮喘未干预组可见明显气道炎症改变,AG490干预组炎症改变较轻;AG490干预组BALF中细胞总数、嗜酸粒细胞(EOS)以及IL-4、IL-5水平均低于哮喘未干预组(P<0.05)。哮喘未干预组气道STAT3表达水平、肺组织STAT3和p-STAT3水平明显高于对照组(P<0.05),AG490干预组肺组织p-STAT3水平明显低于哮喘未干预组(P<0.05)。哮喘小鼠气道STAT3表达水平及肺组织p-STAT3水平分别与BALF中EOS计数、IL-4、IL-5、IL-6水平等...