Peptidome profiling of induced sputum by mesoporous silica beads and MALDI-TOF MS for non-invasive biomarker discovery of chronic inflammatory lung diseases.

Induced sputum is recognized as being of increasing importance for the diagnosis and monitoring of chronic inflammatory lung diseases. The main purpose of this study is to provide a valid approach to better fractionate and characterize the still under-estimated low-molecular weight proteome of induced sputum by using mesoporous silica beads (MSBs) SPE coupled to MALDI-TOF MS. Sputum peptides were captured from both derivatized and non-derivatized MSBs and then profiled by MALDI-TOF MS. Depending on the chemical groups present on the mesoporous surface, complex peptide mixtures were extracted from induced sputum and converted into reproducible MALDI profiles. The number of peaks detected as a function of S/N was evaluated for each mesoporous surface. More than 400 peaks with an S/N>5 were obtained in comparison to 200 peaks detected without MSBs. Additionally, as a proof-of-principle, we investigated the ability of this platform to discriminate between the "sputome" of patients with asthma and chronic obstructive pulmonary disease, and between these groups and those of healthy control subjects. Six m/z peaks emerged as potential diagnostic peptidic patterns able to differentiate these inflammatory airway diseases in the sputome range. Human α-defensins (human neutrophil peptide (HNP)1, HNP2, HNP3) and three C-terminal amidated peptides, one of which is phosphorylated on serine, were identified by MALDI-TOF/TOF MS. These findings may contribute to defining a high-throughput screening MS-based platform for monitoring key peptidic-biomarkers for inflammatory and chronic respiratory diseases in induced sputum samples.

Exhaled matrix metalloproteinase-9 (MMP-9) in different biological phenotypes of asthma.

BACKGROUND AND OBJECTIVES: Airway remodeling is a main feature of asthma. Different biological phenotypes of severe asthma have been recently recognized by the ENFUMOSA study group and among these one is characterized by neutrophilic airway inflammation. Concentrations of MMP-9 in airways have been suggested as a marker to monitor airway remodeling in asthma. OBJECTIVE: The aim of the present study was to explore airway remodeling in different biological phenotypes of asthma by measuring MMP-9 in EBC and correlating these with other variables. METHODS: Sixty consecutive subjects with asthma and 20 healthy controls were enrolled in the study. Exhaled MMP-9, pH and NO levels and inflammatory cells in sputum were measured in all subjects enrolled. RESULTS: We observed an increase of exhaled MMP-9 in asthmatic subjects compared to controls. Higher exhaled MMP-9 concentrations were described in severe asthmatics compared to mild to moderate especially in those with neutrophilic airway inflammation. We further found a correlation between exhaled MMP-9 and percentage of neutrophils in sputum, FEV1, exhaled NO and pH. CONCLUSION: Our results seem to substantiate the feasibility of measuring exhaled MMP-9 in the breath of asthmatic patients. MMP-9 may be considered a proxy of the amount of the ongoing airway remodeling in asthma. MMP-9 has been shown to be differentially released in different phenotypes of asthma. The measure of exhaled MMP-9 could help to monitor the ongoing airway remodeling, recognize severe stages of asthma, and possibly help determine the appropriate choice of therapy.

Dyspnea perception in asthma: role of airways inflammation, age and emotional status.

OBJECTIVES: Dyspnea perception in asthmatics differs between subjects. Poor perception is usually associated with increased risk of asthma attack/exacerbation. The advanced stage of the disease and the presence of eosinophilic airways inflammation have been recently recognized as being responsible for poor dyspnea perception. However, few studies are available on this topic. DESIGN: The aim of this study was to analyse the influence of inflammatory pattern, age and affective status on dyspnea perception in asthmatic subjects. SUBJECTS AND INTERVENTIONS: Seventy-one consecutive asthmatic patients were recruited and underwent induced sputum, exhaled NO measurement and breath condensate collection. Perception of dyspnea was evaluated as a BORG-VAS/FEV(1) slope before and after the broncho-reversibility test and correlated with the stage of asthma, inflammatory markers, age and depression scale. RESULTS: Dyspnea perception decreases with the worsening of asthma, with the advance of age and of depression status. Furthermore, airways inflammation plays a key role in the decline of dyspnea perception as proved by the negative correlation observed between inflammatory cells in sputum, exhaled pH and NO and BORG-VAS/FEV(1) slope. CONCLUSIONS: The results of our study suggested that airways inflammation, depression status, advance age and severity of asthma influence dyspnea perception and suggest a straight control to identify and better manage poor preceptor asthmatics.

Airway cell patterns in patients suffering from COPD and OSAS (Overlap Syndrome).

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) and chronic obstructive pulmonary disease (COPD) are two diseases that often coexist within an individual. This coexistence is known as Overlap Syndrome (OS). Both diseases are characterized by local and systemic inflammations, but no studies to date have investigated local airway inflammation in patients suffering from Overlap Syndrome. METHODS: We performed a Berlin Questionnaire to evaluate the presence of the principal OSAS symptoms, a pulmonary function test, and then a nocturnal oximetry and polysomnography in 72 patients that were divided into five groups: OS (n = 18), COPD (n = 15), OSAS (n = 16), 12 obese without OSAS or COPD, and one control group of 11 normal subjects. All patients underwent sputum induction and the analysis of cell patterns were evaluated in all groups. The relationship with the degree of obesity, airway obstruction and OSAS severity was also evaluated. RESULTS: The percentage of neutrophils in induced sputum was higher in OS (74.33% ± 14.8), COPD (63.33% ± 13.22) and OSAS (60.69% ± 17.6) subjects compared with control groups of obese (43.5% ± 17.49) and normal weight (32.04% ± 12.26). No difference was found among Overlap, COPD, and OSAS patients (p = 0.56). A negative correlation was found between PaO(2) and percentage of airway neutrophils (r = -0.29, p < 0.05); similarly, no correlations arose between BMI, FEV(1) or ODI. CONCLUSION: Patients suffering from Overlap Syndrome present a high percentage of neutrophils in induced sputum like patients affected by COPD or OSAS alone. Our result suggests that airway inflammations is always involved in all of these diseases, even though probably sustained by different mechanisms.

Validity and reproducibility of morphologic analysis of nasal secretions obtained using ultrasonic nebulization of hypertonic solution.

BACKGROUND: Collection of nasal secretions is important for the evaluation of upper airways inflammation in many nasal disorders. OBJECTIVE: To study the validity and reproducibility of nasal secretion cellularity induced by nebulization of hypertonic solution in patients with allergic rhinitis (AR), patients with nonallergic rhinitis with eosinophilia syndrome (NARES), and control subjects. METHODS: Sixty-eight individuals (29 with AR [mean +/- SD age, 33.3 +/- 16.9 years], 23 with NARES [mean +/- SD age, 46.4 +/- 16.6 years], and 16 controls [mean +/- SD age, 42.1 +/- 15.1 years]) underwent ultrasonic nebulization of hypertonic (4.5%) saline solution on 2 different occasions to study the validity and reproducibility of total and differential cell counts of nasal secretions. RESULTS: The mean +/- SD percentage of eosinophils was significantly higher in samples from patients with AR (20.8% +/- 23.1%) and NARES (18.7% +/- 22.8%) than in samples from controls (0.6% +/- 0.6%; P < .001 for both). There was a significant correlation between 2 samples of nasal secretions obtained on 2 different occasions for percentages of macrophages, neutrophils, eosinophils, and epithelial cells. CONCLUSIONS: The analysis of nasal secretions obtained using ultrasonic nebulization of hypertonic solution can distinguish patients with AR and NARES from controls. The reproducibility of this technique is good for macrophages, neutrophils, eosinophils, and epithelial cells. This method could be used to detect nasal airway inflammation in clinical settings.