Philippe Prince

Metalloproteinase-9 in Induced Sputum Correlates with the Severity of the Late Allergen-Induced Asthmatic Response

Background: Asthma is characterized by airway inflammation and remodeling in which matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP) play an important role. Allergen exposure activates the inflammatory/repair process in sensitized subjects. Induced-sputum analysis is a non-invasive method that allows the assessment of changes in inflammatory and remodeling mediators implicated in asthma. Objectives: To evaluate the changes in MMP-9 and its principal inhibitor (TIMP-1) in sputum and plasma of mild allergic asthmatic subjects after whole-lung allergen challenge. Methods: Induced sputum and blood samples were obtained at baseline, and 6 and 24 h after challenge. MMP-9 and TIMP-1 levels in sputum and plasma were measured by ELISA. Results: Allergen challenge increased the percentage of sputum eosinophils and MMP-9 levels 6 and 24 h after the challenge compared to baseline levels, but TIMP-1 levels did not vary significantly. A significant correlation was observed between MMP-9 levels at 6 h and the maximum percent fall in FEV1 during the late response. Throughout the study, MMP-9 levels correlated significantly with the number of neutrophils in sputum. Conclusions: This study shows that analysis of induced sputum is a useful tool to study the variations in MMP-9 and TIMP-1 levels following allergen challenge, therefore allowing to evaluate their role in allergen-induced airway damage and repair.

Expiratory flows and airway inflammation in elderly asthmatic patients

Asthma in the elderly is often underrecognized and suboptimally treated, resulting in an increased morbidity and mortality. The characteristics of asthma-related bronchitis and its optimal treatment remain to be determined in this population. We aimed to compare lung function and airway inflammation in elderly and younger asthmatic subjects. Data from two induced sputum databases were analyzed in three groups of asthmatic subjects (18-30 y, n = 136; 31-59 y, n = 385; 60-72 y, n = 172) and one group of healthy elderly subjects (60-89 y, n = 16). Expiratory flows and induced sputum cell counts were analyzed. Airway obstruction was more marked in elderly asthmatics compared with healthy elderly or younger asthmatic subjects (p < 0.01). An increase in sputum neutrophils and a decrease in macrophages and lymphocytes were observed in elderly asthmatics (p < 0.0001). Neutrophil percentages significantly increased with asthma severity in the young and the middle-aged groups, while they remained similar in elderly asthmatics regardless of asthma severity (p < 0.05). Neutrophil percentages weakly correlated with the dose of ICS in all asthmatics (r = 0.17, p < 0.0001). Age and dose of ICS were independent predictors of neutrophil percentage in asthmatic subjects in a regression model (R(2) = 0.12). Asthma in the elderly is associated with a more marked airway obstruction and sputum neutrophilia. Both age and the dose of corticosteroids need to be considered in the interpretation of the clinical relevance of sputum neutrophil count.

Airway Inflammatory Responses Following Exposure to Occupational Agents

BACKGROUND Airway inflammatory responses to specific inhalation challenges (SICs) with low-molecular-weight (LMW) and high-molecular-weight (HMW) agents have not been studied thoroughly. We assessed the changes in airway inflammatory cells following SIC in sensitized workers, and looked at the influence of various factors on the pattern of inflammatory responses to SIC. METHODS Induced sputum analysis was performed in workers sensitized to LMW (n = 41) or HMW agents (n = 41) after a control day and after a positive SIC. Cell counts were compared with lung function and various clinical parameters. RESULTS In the LMW group, eosinophils were increased following late asthmatic responses (median [interquartile range], 0.02 [0.04] × 10(6) cells/g vs 0.30 [0.80] × 10(6) cells/g and 1.0% [3.5] vs 8.9% [8.0], P < .05), as were neutrophil numbers (0.8 [1.3] × 10(6) cells/g vs 2.3 [5.4] × 10(6) cells/g, P = .04). In the HMW group, eosinophil percentages increased both after early (1.0% [2.2] vs 5.5% [14.5], P = .003) and dual asthmatic responses (4.5% [3.7] vs 15.0% [13.7], P = .02). In the LMW group, the increases in neutrophils were higher in current smokers than in ex-smokers or nonsmokers. The length of exposure to the agent, tobacco use, and baseline percentage of eosinophils were independent predictors of the change in eosinophils, whereas age and baseline neutrophil percentage were predictors of the change in neutrophils. CONCLUSIONS This study confirms that eosinophils and neutrophils are increased after SIC, whatever the causal agent. The type of agent is not predictive of the inflammatory response to SIC. Smoking is associated with a more neutrophilic response after SIC with an LMW agent.

Benefits of low-dose inhaled fluticasone on airway response and inflammation in mild asthma

RATIONALE Current guidelines suggest that asthma should be controlled with the lowest dose of maintenance medication required. OBJECTIVES To evaluate the effects of a low dose of inhaled corticosteroid compared to a placebo, on airway inflammation and responsiveness in patients with mild symptomatic asthma. METHODS In this randomized double-blind, placebo-controlled, parallel group study, we looked at the influence of inhaled fluticasone propionate 250 microg/day for 3 months followed by 100 microg/day for 9 months on airway inflammation and methacholine responsiveness in non-smoking subjects with mild allergic asthma. Subjects were evaluated at baseline and 3, 6, 9 and 12 months after treatments; a 2-week evaluation of respiratory symptoms and peak expiratory flow measurements was done before each visit. RESULTS Fifty-seven subjects completed the 3-month study period. Airway responsiveness, expressed as the PC20 methacholine, increased by 0.27 and 1.14 doubling concentrations, respectively, in placebo-treated (n=33) and in fluticasone-treated (n=24) asthmatic subjects (p=0.03). An additional improvement in PC20 up to 2.16 doubling concentrations was observed in the fluticasone-treated group during the 9-month lower-dose treatment (p=0.0004, end of low-dose period compared with placebo). Sputum eosinophil counts decreased after 3 months of fluticasone 250 microg/day compared with placebo (p<0.0001) and remained in the normal range during the 9-month lower-dose treatment. Respiratory symptoms and peak expiratory flows did not change significantly throughout the study in both groups. CONCLUSION In mild asthma, keeping a regular minimal dose of ICS after asthma control has been achieved, may lead to a further reduction in airway responsiveness and keep sputum eosinophil count within the normal range.

A Kit to Facilitate and Standardize the Processing of Sputum for Measurements of Airway Inflammation

BACKGROUND The use of inflammometry has been shown to be effective for managing asthma. However, sputum processing can be time consuming. Furthermore, methods of sputum processing can vary among facilities. To help with standardization and to simplify the procedure for laboratory staff, a novel, commercially available processing device (Accufilter, Cellometrics Inc, Canada) has been developed. OBJECTIVE To assess the validity of the Accufilter device and kit for recovery of treated specimens, and for quantitative sputum inflammatory cell counts by comparing intrasample measurements with those using the same procedure without the Accufilter device and kit. METHODS The present study was a wet laboratory comparison of induced sputum cell counts obtained from sputum processed with versus without the device and kit. Comparisons of each sputum specimen were performed by the same technologist in random order. RESULTS A total of 39 samples were processed using both the standard method and the Accufilter device. The intraclass correlation coefficients were high for the weight of the filtrate, and for eosinophil and neutrophil differential counts. CONCLUSION A good degree of agreement of results was apparent when the two methods were compared. The differences noted between both methods were minimal and did not modify clinical interpretation. The use of the Accufilter device and kit can be used in place of the standard method for sputum quantitative analysis, especially in centres with large sample loads.

Comparative clinical and airway inflammatory features of asthmatics with or without polyps.

BACKGROUND Nasal polyposis (NP) is associated with a more severe and steroid-resistant asthma. OBJECTIVE To compare clinical and airway inflammatory features of asthmatics with or without NP. METHODS Two groups of asthmatic patients were studied: group 1; n=39, with NP; group 2; n=40, without NP. Asthma control was assessed according to the Asthma Control Scoring System (ACSS). Expiratory flows, induced sputum, and blood eosinophils were also measured. RESULTS ACSS score was lower (poorer control) in group 1 (meanA+-SEM = 73A+-3%) compared with group 2 (82A+-2%, p=0.01). FEV1 (mean of predicted value A+- SEM) was 81A+-3 for group 1 and 96A+-3 for group 2 (p=0.001), and the FEV1/FVC ratio was lower in group 1 (70A+-2%) compared with group 2 (76A+-1%, p=0.01). Blood and induced sputum eosinophils, as well as fibronectin and eosinophil cationic protein levels, were higher in group 1. CONCLUSION Asthmatic subjects with NP have increased airway obstruction, increased inflammatory cells and reduced asthma control compared to those without NP. This may suggest a contribution of nasal polyps to the severity of asthma or a common susceptibility to develop upper and lower airways mucosal inflammation.

A fast, simple, and inexpensive method to collect exhaled breath condensate for pH determination

BACKGROUND Exhaled breath condensate (EBC) analysis is a noninvasive method for assessing lower airway inflammation. Various methods of collecting EBC have been described. However, they are often time-consuming or involve expensive equipment. OBJECTIVE To evaluate the efficiency and repeatability of a simple, fast, and inexpensive method of EBC collection for pH determination. METHODS Twenty-four mild asthmatic patients, 18 moderate-to-severe asthmatic patients, and 26 controls were asked to slowly exhale for 45 seconds into a -80 degrees C cooled metal cylinder covered with protective rubber and attached to a piece of tubing. The EBC was collected using a syringes plunger. The groups were compared regarding EBC pH. Reproducibility tests were also performed. Induced sputum samples were obtained for inflammatory cell counts. RESULTS We obtained approximately 50 microL of EBC for pH determination. Mild asthmatic patients had lower mean +/- SD pH values than controls (5.97 +/- 0.48 vs 6.36 +/- 0.34; P = .008), and corticosteroid-treated, moderate-to-severe asthmatic patients had mean +/- SD pH values similar to controls (6.23 +/- 0.38; P > .05). Mean +/- SD sputum eosinophil percentages were higher in both asthmatic groups than in controls (3.42% +/- 5.37% and 4.14% +/- 4.98% vs 0.04% +/- 0.12%; P < .001) and were not correlated with pH values in all groups. The mean intraday coefficient of variation for the method was 4.8% (range, 0.9%-8.8%). No correlation was found in all groups between sputum neutrophils and pH. CONCLUSIONS We developed a useful device for collecting EBC for pH evaluation that could provide an alternative to other methods when pH is the main variable evaluated.

Effects of protease inhibitors on mediator preservation in the supernatant of induced sputum.

Abstract Addition of protease inhibitors in induced sputum samples may help improve the recovery of mediators. The effect of protease inhibitors, in induced sputum, on the measurement of fibronectin, VEGF, IL‐5, IL‐6, and IL‐8 was assessed. Protease inhibitors were added to sputum supernatant of atopic asthmatic subjects. Mediators were measured by ELISA or EIA. No differences were found in VEGF, IL‐5, and IL‐8 levels between protease inhibitors studied. Concentrations of IL‐6 and fibronectin were higher when using, respectively, the commercial cocktail, and aprotinin. Protease inhibitors, if added, should be carefully chosen at the beginning of each study, to optimize the results.