支气管哮喘和慢性阻塞性肺疾病患者诱导痰中血管内皮生长因子水平的变化及意义

目的探讨血管内皮生长因子（VEGF）在支气管哮喘和慢性阻塞性肺疾病（COPD）发病中的作用。方法分别选择缓解期支气管哮喘患者（哮喘组）30例、稳定期COPD患者（COPD组）28例和健康志愿者（健康对照组）24例进行肺功能测定和用诱导痰检查方法对痰进行炎性细胞分类计数,并用EMSA法测定诱导痰上清液中VEGF水平。结果哮喘组诱导痰中嗜酸粒细胞数为0．9（0．4—1．4）×10^9/L,明显高于COPD组和健康对照组的0．1（0—0．2）×10^9/L、0．0（0～0．1）×10^9/L（P值均＜0．05）;COPD组诱导痰中中性粒细胞数为2．3（1．8～2．8）×10^9/L,明显高于哮喘组和健康对照组的1．1（0．2～1．9）×10^9/L、1．0（0．8～1．2）×10^9/L（P值均＜0．05）。哮喘组、COPD组和健康对照组诱导痰上清液中VEGF水平分别为（2．3±0．5）、（0．3±0．1）、（0．9±0．2）μg／L,三组间比较差异有统计学意义（P值均＜0．05）。哮喘组诱导痰上清液中VEGF水平与诱导痰中嗜酸粒细胞数呈正相关（r=0．62,P＜0．05）,与第1秒用力呼气容积（FEV。）占预计值百分比呈负相关（r=-0．56,P＜0．05）;COPD组诱导痰上清液中VEGF水平与FEV,占预计值百分比呈正相关（r=0．43,P＜0．05）,与诱导痰中中性粒细胞数无相关性（r=0．21,P＞0．05）。结论支气管哮喘患者诱导痰上清液中VEGF表达上调,VEGF可能参与了支气管哮喘的气道炎性反应过程;COPD患者诱导痰上清液中VEGF表达下降,VEGF可能参与了COPD的发病过程。     

外周血嗜酸性粒细胞升高对肝移植后急性排斥反应的诊断价值

目的 评价外周血嗜酸性粒细胞升高对肝移植后急性排斥反应(AR)的诊断价值.方法 回顾性分析肝移植受者的临床资料,将术后具有完整肝活检和肝活检当天或前1天血常规和肝功能等临床数据的101例受者纳入研究.根据肝活检结果将受者分为AR组和非急性排斥反应(NAR)组,比较两组嗜酸性粒细胞绝对值和百分率等指标的差异.比较白细胞分类与发生AR的相关性.采用ROC分析确定嗜酸性粒细胞绝对值和百分率的最佳截断值,并分析其诊断AR的敏感性和特异性等.结果 AR组嗜酸性粒细胞绝对值和百分率分别为(0.127±0.161)×109/L和(2.8±7.3)％,均显著高于NAR组的(0.071±0.100)×109/L和(1.0±1.1)％(P＜0.05) ;外周血嗜酸性粒细胞升高与AR的发生呈明显正相关(P＜0.05).ROC分析显示,嗜酸性粒细胞绝对值和百分率的最佳截断值分别为0.145×109/L和2.35％,此时约登指数最高(分别为0.17和0.185),诊断AR的敏感性和特异性分别为28.6％和88.4％、28.6％和89.9％.结论 肝移植后外周血嗜酸性粒细胞的升高与AR的发生有关,对诊断AR具有较高的特异性,但敏感性较低.     

难治性哮喘的病因初探及其防治措施

哮喘是由多种细胞(如嗜酸性粒细胞、肥大细胞、T细胞、中性粒细胞、气道上皮细胞等)和细胞组分参与的气道慢性炎症性疾病.这种慢性炎症导致气道反应性的增加,通常出现广泛多变的可逆性气流受限,并引起反复发作性的喘息、气急、胸闷或/和咳嗽等症状1.     

外源性肺表面活性物质对呼吸机相关性肺损伤大鼠炎性反应的影响

目的 探讨外源性肺表面活性物质(PS)对呼吸机相关性肺损伤(VILI)大鼠炎性反应的影响.方法 成年雄性Wistar大鼠28只,体重310-388 g,采用随机数宁表法,将大鼠随机分为4组(n=7),对照组(C组)、VILI组、PS组和空气对照组(A组).采用高气道压机械通气(气道峰压40cmH2O,通气频率20次/min,PEEP 0)20 rin制备VILI模型.C组麻醉后即经股动脉放血处死.VILI 组于模型制备成功后放血处死.PS组和A组造模后采用自制吸痰管吸除气道内水肿液后经气道分别注入PS 100 mg/kg(50 mg/ml)和等容量空气,行机械通气(VT10 ml/kg,通气频率45次/min,PEEP 7.5cm H2O)120 min后放血处死.采集股动脉血样及气道内水肿掖,采用ELISA法测定血浆IL-6、IL-10、巨噬细胞炎性蛋白-2(MIP-2)和TNF-α浓度,采用Bradford蛋白浓度测定法测定气道内水肿液蛋白浓度.光镜下观察肺组织病理学及中性粒细胞数目.结果 4组血浆TNF-α浓度比较差异无统计学意义(P ＞0.05).与C组比较,VILI组血浆MIP-2、IL-10和IL-6浓度及中性粒细胞计数升高(P＜0.05),气道水肿液量和水肿液蛋白浓度比较差异无统计学意义(P＞0.05).与A组比较,PS组中性粒细胞计数减少(P＜0.05),气道水肿液量和水肿液蛋白浓度比较差异无统计学意义(P＞0.05),血浆MIP-2、IL-10和IL-6浓度差异无统计学意义(P＞0.05).VILI组、A组和PS组肺组织炎性损伤明显.结论 外源性PS治疗VILI大鼠可减少肺组织中性粒细胞募集,但不能抑制炎性细胞因子的释放.     

大剂量维生素C对哮喘小鼠肺部感染的影响

目的 观察维生素C对小鼠哮喘模型中肺部感染的作用.方法 用100 μg卵白蛋白诱导小鼠哮喘,每组小鼠腹腔内注射3 mg或5mg[溶于100μl磷酸盐缓冲液(PBS)]维生素C,或同体积的PBS,每组8只小鼠.第24天用气道阻力描计法测定气道阻力.小鼠第25天在深度麻醉下处死得到支气管肺泡灌洗液和肺组织,对肺部炎性细胞总数以及血管和支气管周围炎性细胞浸润层数进行计数.用酶联免疫吸附试验(ELISA)评估其对Th1/Th2平衡的影响.结果 在哮喘模型中,只注射PBS组中的气道阻力均明显高于正常对照组(P＜0.05).6.25、12.50、25.00 g/L维生素C治疗乙酰胆碱激发哮喘组与正常对照组气道阻力相似.而50g/L乙酰胆碱激发哮喘组中使用维生素C治疗明显高于正常对照组,但低于PBS处理组,维生素C可以降低呼吸道对乙酰胆碱的敏感性(P＜0.05).在实验组支气管肺泡灌洗液细胞中巨噬细胞和嗜酸细胞在PBS组及维生素C治疗组细胞中分别占33.6％和63.1％、20.2％和63.1％.维生素C可减少支气管肺泡灌洗液中炎性细胞的数量(P＜0.05),并可减少血管周围炎性细胞(5.0±2.9比6.4±1.5,P＜0.05)及细支气管周围炎性细胞(1.2±0.9比2.6±1.8,P＞0.05)浸润厚度.维生素C不能调节Th1/Th2之间的平衡(P＞0.05).结论 维生素C虽不能转换Th1/Th2的之间的平衡,但有明显抗炎作用.     

白细胞介素-15对小鼠胃癌的治疗作用

目的 建立小鼠胃癌模型,检测荷瘤状态对小鼠免疫细胞的影响,同时给予白细胞介素-15(IL-15)免疫基因治疗,以检测IL-15对胃癌的预防及治疗效果.方法 采用多因素联合攻击法诱导小鼠胃癌,同时采用IL-15表达质粒载体进行免疫基因治疗.20周后取胃标本行病理学检查,并取血及脾细胞检测T细胞亚群及CD4+ CD25+调节性T细胞(Treg).同时进行自然杀伤(NK)细胞的细胞毒性试验,通过统计学方法分析结果.结果 多因素联合攻击法诱导胃癌发生率为37.5％.诱癌组小鼠血清IL-15水平(9.20±2.92) ng/L明显降低(P＜0.05),且其脾NK细胞杀伤活性(216.91±117.80) U/L明显降低(P＜0.05).诱癌组小鼠外周血Treg水平(8.07±6.62)％明显升高(P＜0.05),而对照组小鼠脾细胞Treg水平(4.40±3.34)％明显降低(P＜0.05).各组小鼠之间外周血T细胞亚群变化差异无统计学意义(P＞0.05).诱癌组小鼠脾细胞CD3+T细胞水平(78.31±29.79)％明显升高(P＜0.05),而CD4+T细胞(19.98±5.77)％及CD8+T细胞水平(10.15±1.72)％明显降低(P＜0.05).结论 多因素联合攻击法为小鼠胃癌模型建立提供了良好的模型.小鼠荷瘤状态下免疫功能下降,通过IL-15免疫基因治疗可提高机免疫功能,起到抗肿瘤的效果.     

贲门癌患者外周血T淋巴细胞亚群及细胞因子表达的变化

目的 观察贲门癌手术患者机体免疫状态的变化.方法 采用流式细胞术检测97例贲门癌患者T淋巴细胞亚群(Th1、Th2、CD3+、CD4+、CD8+细胞),采用双抗体夹心酶联免疫吸附试验( ELISA)检测血清中白细胞介素(IL)-2、干扰素(IFN)-γ、IL-4、IL-10、可溶性白细胞介素-2受体R(sIL-2R)和肿瘤坏死因子(TNF)-β水平.结果 贲门癌患者术前外周血Th1/Th2细胞比值(0.43±0.13)较正常对照组(0.58±0.12)降低(P＜0.05),其Th1细胞表达的IL-2、IFN-γ等细胞因子水平低于正常对照组;Th2细胞表达的IL-4、IL-10细胞因子水平高于正常对照组(P＜0.05).T细胞介导的细胞免疫及血清sIL-2R和TNF-β水平与肿瘤TNM分期相关,其特征为:CD3+、CD4+细胞减少(P＜0.05),而CD8+细胞增加(P＜ 0.01);CD4 +/CD8+细胞比值下降(P＜0.01),血清sIL-2R和TNF-β水平升高(p＜0.05) 贲门癌根治术后1个月CD3+、CD4+细胞回升,CD8+细胞回降,同时血清sIL-2R和TNF-β水平降低(P＜0.01).结论 贲门癌患者外周血Th1/Th2细胞比值降低,细胞因子表达失衡,患者存在免疫功能抑制.     

白细胞介素-6通过p38蛋白调控髓核细胞的增殖和迁移

目的 观察白细胞介素-6(IL-6)对髓核细胞增殖和迁移的调控作用.方法 体外分离培养大鼠髓核细胞,将其随机分为对照组、IL-6组、加压组、IL-6+加压组和IL-6+ p38蛋白抑制剂(SB203580)+加压组.给予相应处理后,应用细胞计数试剂盒-8(CCK-8)法评估髓核细胞增殖能力,应用划痕实验评估细胞迁移能力.结果 CCK-8法检测各组细胞吸光度值,未加压环境下,IL-6组吸光度值(1.08±0.02)较对照组(0.67±0.01)明显升高(P＜0.05);加压环境下,IL-6组吸光度值(1.51±0.06)也较对照组(0.88±0.04)明显升高(P＜0.05).细胞划痕实验中,未加压环境下,IL-6组迁移距离[(27.73±3.15)像素]较对照组[(12.90±4.77)像素]明显升高(P＜0.05);加压环境下,IL-6组迁移距离[(52.81±3.72)像素]也较对照组[(14.32±3.84)像素]明显升高(P＜0.05).加压环境下使用SB203580阻断p38蛋白后,吸光度值(0.81±0.04)和迁移距离[(33.82±3.72)像素]均较IL-6组[(1.51±0.06)、(52.81±3.72)像素]显著下降(P＜0.05).结论IL-6能通过p38蛋白通路促进髓核细胞的增殖和迁移.     

辅助性T细胞亚群分化在Ⅲ型前列腺炎免疫发病机制中的作用

目的 探讨前列腺液(EPS)中CD+4T细胞亚群辅助性T细胞(Th细胞)分化在Ⅲ型前列腺炎免疫发病机制中的作用. 方法门诊诊断前列腺炎患者76例,年龄18～47岁,平均32岁.患者均有慢性前列腺炎典型临床症状,病程均＞3个月.按美国国立卫生院分类方法分为ⅢA型组(47例)、ⅢB型组(29例).其中ⅢA型组根据炎症程度又分为ⅢAl组(轻度炎症26例)和ⅢA2组(重度炎症21例).另设健康对照组(16例),年龄19～45岁,平均31岁.采用双抗体夹心酶联免疫法检测EPS中Th1类细胞因子(IFN-γ)、Th2类细胞因子(IL-4)水平及Th1/Th2比值(IFN-γ/IL-4),比较各组问差异.结果 ⅢA、ⅢB组IFN-γ水平[(134.78±43.67),(109.82±30.09)pg/m1]与对照组[(60.63±15.16)pg/m1]比较,差异有统计学意义(P＜0.05),ⅢA组较ⅢB组升高更明显(P＜0.05);ⅢA组IL-4水平[(51.99±20.59)pg/m1]与对照组[(53.88±17.92)pg/m1]比较差异无统计学意义P＞0.05),ⅢB组IL-4水平[(76.40±17.99)pg/m1]明显上调(P＜0.05);ⅢA组IFN-γ/IL-4水平(2.94±1.12)明显高于对照组(1.20±0.48,P＜0.05),Ⅲ B组(1.49±0.48)无明显改变(P＞0.05).与对照组比较,Ⅲ A1组IL-4水平[(63.03±18.86)pg/m1]无明显变化(P＞0.05),而ⅢA2组水平[(30.20±13.16)pg/m1]显著下调(P＜0.05);ⅢA1组和m A2组IFN-γ水平均明显上调[(127.65±36.57),(143.49±50.76)pg/m1],P＜0.05),但2组间差异无统计学意义(P＞0.05);ⅢA2组IFN-γ/IL-4明显高于ⅢA1组(3.67±0.82 vs 2.34±0.97,P＜0.05).结论 ⅢA型前列腺炎Th1细胞分化占优势,Th1/Th2平衡向Th1漂移,以细胞免疫反应为主;Th细胞分化也参与了ⅢB型前列腺炎的发病,但Th1/Th2处于相对平衡状态.Th1的优势分化可能是导致前列腺局部炎症发展的原因之一.     

胰腺癌患者外周血CD4-CD8-T细胞及白细胞介素-2、-6、-7水平测定的研究

目的 检测60例胰腺癌患者及60例正常人外周血CD4-CD8-T细胞及白细胞介素(IL)-2、IL-6、IL-7水平,探讨其与胰腺癌的关系.方法 采用流式细胞仪检测60例胰腺癌患者外周血CD4 - CD8 -T细胞水平;酶联免疫吸附试验( ELISA)法检测60例胰腺癌患者外周血清IL-2、IL-6、IL-7水平,并与60名健康人群进行对照比较.结果 胰腺癌患者外周血CD4 - CD8 -T细胞占CD3+T细胞的比例为(3.43±0.88)％,低于正常人群(5.66±1.23)％(P＜0.05);胰腺癌患者外周血中IL-2水平为(2.00 ±0.42) ng/L,低于正常对照组(5.50 ±0.64) ng/L(P＜0.05);IL-6水平为(210.68±10.82) ng/L,高于正常对照组(1.77 ±0.22) ng/L(P＜0.05);IL-7水平为(1.89±0.32) ng/L,低于正常对照组(6.35 ±0.56) ng/L(P ＜0.05).结论 胰腺癌患者外周血中CD4-CD8-T细胞的比例降低,IL-2、IL-7水平降低及IL-6水平升高可能与胰腺癌的发生有关.     

Comparison of airway immunopathology of eosinophilic bronchitis and asthma

BACKGROUND: Eosinophilic bronchitis is a condition characterised by a corticosteroid responsive cough, sputum eosinophilia, and normal tests of variable airflow obstruction and airway responsiveness. We performed a detailed comparative immunopathological study to test the hypothesis that the different airway function in patients with eosinophilic bronchitis and asthma reflects differences in the nature of the lower airway inflammatory response. METHODS: Exhaled nitric oxide was measured and induced sputum, bronchoscopy, bronchial wash (BW), bronchoalveolar lavage (BAL), and bronchial biopsy were performed in 16 subjects with eosinophilic bronchitis, 15 with asthma, and 14 normal controls. RESULTS: Both eosinophilic bronchitis and asthma were characterised by an induced sputum, BW and BAL eosinophilia, an increased number of epithelial and subepithelial eosinophils, and increased reticular basement membrane thickness. The median concentration of exhaled nitric oxide was higher in those with eosinophilic bronchitis (12 ppb) or asthma (8.5 ppb) than normal controls (2 ppb) (95% CI of the difference 5 to 16, p<0.0001 and 2 to 11.3, p=0.004, respectively). There were no group differences in epithelial integrity or the number of subepithelial T lymphocytes, mast cells or macrophages. CONCLUSION: With the exception of our previously reported association of smooth muscle mast cell infiltration with asthma, the immunopathology of eosinophilic bronchitis and asthma are similar which suggests that eosinophilic airway inflammation, increased exhaled nitric oxide, and increased basement membrane thickening are regulated independently of airway hyperresponsiveness.     

Decreased peroxynitrite inhibitory activity in induced sputum in patients with bronchial asthma

BACKGROUND: The production of peroxynitrite, an extremely potent oxidant, is increased in inflammatory lung disease. It is therefore important to measure antioxidant activity against peroxynitrite in epithelial lining fluid to examine the physiological effects of peroxynitrite in the airways of patients with asthma. This study was designed to determine whether peroxynitrite inhibitory activity in induced sputum is correlated with clinical characteristics and airway inflammatory indices in asthmatic patients. METHODS: Inflammatory indices were measured in induced sputum from 25 patients with asthma and 12 normal control subjects. Peroxynitrite inhibitory activity was also measured by monitoring rhodamine formation in sputum samples. RESULTS: Peroxynitrite inhibitory activity in induced sputum was significantly lower in asthmatic patients (52.4 (24.5)%) than in normal control subjects (92.1 (3.9)%, p<0.0001). Its activity was significantly correlated with forced expiratory volume in 1 second (FEV(1)) % predicted (r=0.774, p<0.0001) and bronchial hyperreactivity to methacholine (r=0.464, p=0.023). There was a significant negative correlation between peroxynitrite inhibitory activity and the degree of eosinophilic airway inflammation (% eosinophils, r=-0.758, p<0.0001; eosinophil cationic protein, r=-0.780, p<0.0001). CONCLUSIONS: Decreased peroxynitrite inhibitory activity occurs in induced sputum of asthmatic patients. Since even in patients with stable asthma the airway lining fluid lacks peroxynitrite inhibitory activity, large amounts of peroxynitrite, which are further increased during an acute asthma attack, would not be completely inactivated and asthmatic airways might have markedly increased susceptibility to peroxynitrite induced airway injury.     

Differences in radiological/HRCT findings in eosinophilic bronchitis and asthma: implication for bronchial responsiveness

BACKGROUND: Airway hyperresponsiveness in asthmatics is considered to be one of the major consequences of airway inflammation and remodelling. Airway responsiveness is normal in patients with eosinophilic bronchitis (EB), despite eosinophilic inflammation of the airways comparable to that which occurs in asthmatics. Comparisons between asthma and EB should clarify the changes in airway morphology that are related specifically to AHR in asthmatics. METHODS: Eighteen asthmatic patients, 15 patients with EB, and 11 healthy subjects were recruited. Airway wall area percentage (WA%), centrilobular prominence, and air trapping were compared using thin slice section computed tomography. RESULTS: WA% was significantly greater in asthmatics than in patients with EB (72 (3.1)% v 54 (2.1)%, p = 0.032) and was similar in EB patients and controls (54 (2.1)% v 57 (1.8)%, p>0.05). Centrilobular prominence and air trapping were similar in EB patients and asthmatics and were significantly greater than in controls. CONCLUSION: WA% rather than air trapping or centrilobular prominence may be associated with the airway hyperresponsiveness that occurs in asthmatics but not in patients with EB.     

Cough receptor sensitivity to capsaicin does not change after allergen bronchoprovocation in allergic asthma

BACKGROUND: The relationship between cough receptor sensitivity and eosinophilic inflammation of the airway in patients with asthma remains unclear. METHODS: Eighteen patients with asthma sensitised to house dust mite (HDM) were enrolled in a randomised parallel group study. Patients with asthma whose main symptom was cough were not enrolled in the study. Half the patients were randomly assigned to inhale saline and the other half to inhale HDM allergen. Cough receptor sensitivity to capsaicin, airway responsiveness to histamine, and sputum eosinophils analysed with hypertonic saline inhalation were investigated before and 24 hours after saline or HDM allergen bronchoprovocation. RESULTS: Patients inhaling saline showed no significant changes in sputum eosinophils (from 7.87% (95% confidence interval (CI) 5.08 to 12.19) to 8.60% (95% CI 3.03 to 14.18); p=0.97), airway responsiveness to histamine (from 726.68 micro g/ml (95% CI 251.90 to 2096.36) to 773.01 micro g/ml (95% CI 251.36 to 2377.23); p=0.96), or capsaicin sensitivity (from 7.23 micro M (95% CI 2.45 to 21.31) to 7.24 micro M (95% CI 2.46 to 21.31); p=0.96). Early asthmatic response was induced in all patients, and late asthmatic response was observed in six of nine patients inhaling HDM allergen. Although there were significant increases in sputum eosinophils (from 9.83% (95% CI 6.78 to 14.27) to 21.00% (95% CI 13.85 to 28.15); p<0.01) and airway responsiveness to histamine (from 784.16 micro g/ml (95% CI 318.24 to 1932.24) to 377.81 micro g/ml (95% CI 118.43 to 1205.24); p<0.05) 24 hours after HDM allergen inhalation compared with baseline levels, capsaicin sensitivity did not change significantly (from 5.75 micro M (95% CI 1.91 to 17.30) to 6.20 micro M (95% CI 2.21 to 17.38); p=0.77). CONCLUSIONS: These findings suggest that cough receptor sensitivity to capsaicin is not associated with eosinophilic inflammation of the airway in patients with allergic asthma whose main symptoms are wheezing and dyspnoea but not cough.     

Effects of inhaled tumour necrosis factor alpha in subjects with mild asthma

BACKGROUND: Inhaled tumour necrosis factor alpha (TNF alpha) has previously been shown to induce airway neutrophilia and increased airway reactivity in normal subjects. It was hypothesised that a similar challenge would increase airway reactivity in those with mild asthma, but that the inflammatory profile may differ. METHODS: Ten mild asthmatic subjects were recruited on the basis of clinical asthma and either a sensitivity to methacholine within the range defined for asthma or a 20% improvement in forced expiratory volume (FEV(1)) after 200 micro g salbutamol. Subjects inhaled either vehicle control or 60 ng recombinant human (rh)TNF alpha and were studied at baseline, 6, 24, and 48 hours later. Variables included spirometric parameters, methacholine provocative concentration causing a 20% fall in FEV(1) (PC(20)), induced sputum differential cell count, relative sputum level of mRNA of interleukins (IL)-4, IL-5, IL-9, IL-14, IL-15 and TNF alpha, and the exhaled gaseous markers of inflammation, nitric oxide and carbon monoxide. RESULTS: PC(20) showed an increase in sensitivity after TNF alpha compared with control (p<0.01). The mean percentage of neutrophils increased at 24-48 hours (24 hour control: 1.1 (95% CI 0.4 to 2.7) v 9.2 (95% CI 3.5 to 14.9), p<0.05), and there was also a rise in eosinophils (p=0.05). Relative levels of sputum mRNA suggested a rise in expression of TNF alpha, IL-14, and IL-15, but no change in IL-4 and IL-5. Spirometric parameters and exhaled gases showed no significant change. CONCLUSION: The increase in airway responsiveness and sputum inflammatory cell influx in response to rhTNF alpha indicates that TNF alpha may contribute to the airway inflammation that characterises asthma.     

Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients

BACKGROUND: Asthma is a chronic inflammatory disorder of the airways driven by T cell activation. Th2 cells and their cytokines are thought to play a role in the pathophysiology of allergic as well as non-allergic asthma. METHODS: Airway cells were obtained by sputum induction from 15 healthy and 39 asthmatic individuals and the airway T cell cytokine profiles (interleukin (IL)-4, IL-5, IL-13, IL-10 and interferon (IFN)-gamma) at the mRNA level were studied by real time RT-PCR. RESULTS: Asthma patients had increased expression of IL-5 (p = 0.001) and IL-13 (p = 0.03) mRNA in sputum compared with non-asthmatic controls. IL-4 mRNA and IFN-gamma mRNA were detectable in the sputum of 44% and 21% of patients, respectively, but not in controls. Sputum IL-10 mRNA levels did not differ significantly between patients and controls. Sputum mRNA expression levels of IL-4, IL-5, and IL-13 were significantly correlated with the percentage of eosinophils and were higher in subjects with allergic asthma than in those with non-allergic asthma (p = 0.03, p = 0.02 and p = 0.0002, respectively); they did not differ between mild asthmatic subjects and those with moderate to severe asthma. In contrast, IFN-gamma mRNA expression was higher in non-allergic than in allergic patients (p = 0.04) and higher in patients with moderate to severe asthma than in those with mild asthma (p<0.01). Sputum IL-5 mRNA levels (but not the other cytokine mRNA levels) were also correlated with exhaled nitric oxide (eNO) and with bronchial hyperreactivity expressed as the histamine concentration resulting in a 20% decrease in forced expiratory volume in 1 second. CONCLUSION: Real time RT-PCR analysis of mRNA in induced sputum confirms a predominance of Th2 cytokines in both allergic and non-allergic asthma. IL-5 levels reflect eosinophil infiltration as well as eNO levels and hyperreactivity, and levels of the Th1 cytokine IFN-gamma indicate asthma severity. The technique is a promising tool for use in further studies of asthma severity and disease activity.     

Bcl-2 expression in sputum eosinophils in patients with acute asthma

BACKGROUND: Eosinophils play a pivotal role in asthmatic airway inflammation. Apoptosis is thought to be critically relevant in promoting the clearance of inflammatory cells and the resolution of inflammation. Bcl-2 inhibits apoptosis in cellular systems. A study was undertaken to determine whether bcl-2 expression in sputum reflects the clinical severity of patients with asthma. The relationship between bcl-2 expression in sputum eosinophils and eosinophil activation markers such as interleukin (IL)-5 and eosinophil cationic protein (ECP) levels in sputum supernatant was evaluated. METHODS: Sputum was obtained from 18 patients with asthma. Fresh expectorated sputum separated from saliva was treated with an equal volume of dithiothreitol 0.1%, cytospun for cell differentials and bcl-2 stain, and the supernatant was collected for biochemical assay. Bcl-2+ eosinophils were stained using immunocytochemistry, ECP was measured by fluoroimmunoassay, and IL-5 was detected by sandwich enzyme linked immunosorbant assay. RESULTS: Twelve patients with severe or life threatening asthma had more bcl-2+ eosinophils (mean difference 46.8% (95% CI 27.0 to 66.6), p<0.01) and a higher ECP level (p<0.01) in the sputum than those with mild to moderate asthma (n = 6). IL-5 was frequently detected in patients with severe or life threatening asthma (11/12 versus 1/6, p<0.01). There was a significant positive correlation between bcl-2+ eosinophils and ECP levels (r = 0.61, p<0.01) and between bcl-2+ eosinophils and IL-5 levels (r = 0.83, p<0.01). There was a significant negative correlation between bcl-2+ eosinophils and FEV(1)/FVC (r = -0.54, p<0.05). CONCLUSION: The increased expression of bcl-2 in eosinophils from sputum of subjects with severe asthma suggests that bcl-2 may prolong survival and decrease apoptosis of airway eosinophils in asthma.     

Evidence for different subgroups of difficult asthma in children

BACKGROUND: Children with difficult asthma experience frequent symptoms despite treatment with high dose inhaled steroids. Persistent symptoms may result from persistent airway inflammation which can be monitored by measuring exhaled nitric oxide (NO). This study aimed to assess the role of airway inflammation, using NO as a surrogate, in children with difficult asthma and to investigate the response to oral prednisolone. METHODS: NO was measured in 23 children (mean age 11.7 years) with difficult asthma, before and after 2 weeks of treatment with oral prednisolone. The clinical response was assessed by spirometric tests, peak flow, bronchodilator use, and symptoms. Adherence to treatment was assessed by measuring serum prednisolone and cortisol concentrations. NO was measured in 55 healthy children to establish a normal range. RESULTS: NO concentrations were higher in asthmatic patients than in controls (geometric mean 11.2 v 5.3 ppb, p<0.01). Using grouped data, the concentration of NO fell following prednisolone (11.2 v 7.5 ppb, p<0.01) accompanied by an improvement in morning peak flow (p<0.05). The baseline NO concentration was raised (>12.5 ppb) in nine asthmatic patients and remained high after prednisolone in five. Thirteen had normal levels of NO (<12.5 ppb) before and after prednisolone. Thirteen asthmatic patients remained symptomatic following prednisolone; NO levels were raised on both occasions in five of these and were normal in seven. CONCLUSIONS: As a group, the asthmatic subjects demonstrated evidence of airway inflammation which responded to prednisolone. At least two subgroups of patients were identified: one with persistently raised NO levels despite treatment with oral prednisolone indicating ongoing steroid insensitive inflammation, and another with normal levels of NO. Both subgroups included patients with persistent symptoms, which suggests that different patterns of difficult asthma in children exist.     

Pathological features and inhaled corticosteroid response of eosinophilic and non-eosinophilic asthma

Background

Non‐eosinophilic asthma is a potentially important clinicopathological phenotype since there is evidence that it responds poorly to inhaled corticosteroid therapy. However, little is known about the underlying airway immunopathology and there are no data from placebo‐controlled studies examining the effect of inhaled corticosteroids.

Methods

Airway immunopathology was investigated using induced sputum, bronchial biopsies, bronchial wash and bronchoalveolar lavage in 12 patients with symptomatic eosinophilic asthma, 11 patients with non‐eosinophilic asthma and 10 healthy controls. The patients with non‐eosinophilic asthma and 6 different patients with eosinophilic asthma entered a randomised, double‐blind, placebo‐controlled crossover study in which the effects of inhaled mometasone 400 μg once daily for 8 weeks on airway responsiveness and asthma quality of life were investigated.

Results

Patients with non‐eosinophilic asthma had absence of eosinophils in the mucosa (median 4.4 cells/mm² vs 23 cells/mm² in eosinophilic asthma and 0 cells/mm² in normal controls; p = 0.03) and normal subepithelial layer thickness (5.8 μm vs 10.3 μm in eosinophilic asthma and 5.1 μm in controls, p = 0.002). Non‐eosinophilic and eosinophilic asthma groups had increased mast cell numbers in the airway smooth muscle compared with normal controls (9 vs 8 vs 0 cells/mm², p = 0.016). Compared with placebo, 8 weeks of treatment with inhaled mometasone led to less improvement in methacholine PC₂₀ (0.5 vs 5.5 doubling concentrations, 95% CI of difference 1.1 to 9.1; p = 0.018) and asthma quality of life (0.2 vs 1.0 points, 95% CI of difference 0.27 to 1.43; p = 0.008).

Conclusions

Non‐eosinophilic asthma represents a pathologically distinct disease phenotype which is characterised by the absence of airway eosinophilia, normal subepithelial layer thickness and a poor short‐term response to treatment with inhaled corticosteroids.Clinicians have long regarded asthma as a heterogeneous disease,^1,2 although detailed clinicopathological studies have tended to emphasise the similarities in the underlying airway pathology and disordered function between patients.^3,4 The development of safe non‐invasive induced sputum techniques has provided the opportunity to study airway inflammation in a diverse range of patients. Using this technique, we and a number of other groups have identified a subset of adults who have clear physiological evidence of asthma but no induced sputum evidence of eosinophilic airway inflammation.^5,6,7 This asthma phenotype is potentially clinically important since several uncontrolled studies have suggested that it is associated with a poor short‐term and longer‐term response to inhaled corticosteroid.^5,8,9Non‐eosinophilic asthma is present in 53% of patients presenting to an adult respiratory clinic with symptomatic asthma.⁹ Other investigators have reported the absence of a sputum eosinophilia in up to 50% of patients with refractory asthma,⁹ patients studied during an asthma exacerbation¹⁰ and patients taking high doses of inhaled corticosteroids.⁶ In a recent longitudinal study of patients with severe asthma, the absence of sputum eosinophils has been reported to be a stable feature in a number of patients observed over 12 months;¹¹ another study showed that it was present in untreated symptomatic patients as well as those receiving inhaled corticosteroid therapy.⁹ These observations suggest that, in some patients at least, non‐eosinophilic asthma is a stable phenotype that is not solely explained by the effects of corticosteroid therapy.Several studies have noted that an airway neutrophilia is often present in patients with non‐eosinophilic asthma, and Wenzel et al⁷ reported a predominantly neutrophilic airway inflammatory response with an absence of eosinophils and normal basement membrane thickness in a subgroup of patients with refractory asthma from whom bronchial biopsy specimens were taken. These findings support the concept that non‐eosinophilic asthma is a pathologically distinct entity, although the extent to which these findings reflect the effects of treatment remains unclear.The aim of this study was to compare the immunopathology of eosinophilic and non‐eosinophilic asthma with normal controls in patients with symptomatic asthma who were not treated with inhaled corticosteroids. We also set out to compare the response to 8 weeks of treatment with the inhaled corticosteroid mometasone in a prospective randomised, double‐blind, placebo‐controlled crossover trial in patients with non‐eosinophilic asthma and in a subgroup with eosinophilic asthma.