Downregulation of exosomal let-7d and miR-16 in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a degenerative interstitial lung disease with both a poor prognosis and quality of life once the diagnosis is made. In the last decade many features of the disease have been investigated to better understand the pathological steps that lead to the onset of the disease and, moreover, different types of biomarkers have been tested to find valid diagnostic, prognostic and therapy response predictive ones. In the complexity of IPF, microRNA (miRNAs) biomarker investigation seems to be promising. METHODS: We analysed the expression of five exosomal miRNAs supposed to have a role in the pathogenesis of the disease from serum of a group of IPF patients (n = 61) and we compared it with the expression of the same miRNAs in a group of healthy controls (n = 15). RESULTS: In the current study what emerged is let-7d down-regulation and, unexpectedly, miR-16 significant down-regulation. Moreover, through a cross-sectional analysis, a clustering of the expression of miR-16, miR-21 and miR-26a was found. CONCLUSIONS: These findings could help the individuation of previously unknown key players in the pathophysiology of IPF and, most interestingly, more specific targets for the development of effective medications.

Mitochondrial DNA alteration in obstructive sleep apnea.

BACKGROUND: Obstructive Sleep Apnea (OSAS) is a disease associated with the increase of cardiovascular risk and it is characterized by repeated episodes of Intermittent Hypoxia (IH) which inducing oxidative stress and systemic inflammation. Mitochondria are cell organelles involved in the respiratory that have their own DNA (MtDNA). The aim of this study was to investigate if the increase of oxidative stress in OSAS patients can induce also MtDNA alterations. METHODS: 46 OSAS patients (age 59.27 ± 11.38; BMI 30.84 ± 3.64; AHI 36.63 ± 24.18) were compared with 36 control subjects (age 54.42 ± 6.63; BMI 29.06 ± 4.7; AHI 3.8 ± 1.10). In blood cells Content of MtDNA and nuclear DNA (nDNA) was measured in OSAS patients by Real Time PCR. The ratio between MtDNA/nDNA was then calculated. Presence of oxidative stress was evaluated by levels of Reactive Oxygen Metabolites (ROMs), measured by diacron reactive oxygen metabolite test (d-ROM test). RESULTS: MtDNA/nDNA was higher in patients with OSAS than in the control group (150.94 ± 49.14 vs 128.96 ± 45.8; p = 0.04), the levels of ROMs were also higher in OSAS subjects (329.71 ± 70.17 vs 226 ± 36.76; p = 0.04) and they were positively correlated with MtDNA/nDNA (R = 0.5, p < 0.01). CONCLUSIONS: In OSAS patients there is a Mitochondrial DNA damage induced by the increase of oxidative stress. Intermittent hypoxia seems to be the main mechanism which leads to this process.

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.

Exhaled matrix metalloproteinase-9 in lung cancer.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) has been recognized in several types of tumor development and progression, including lung cancer, for its role in the degradation and remodeling of lung tissue. Furthermore, increased MMP-9 has been commonly described in the serum and airways of non-small cell lung cancer (NSCLC) patients. OBJECTIVE: The aim of this study was to investigate, for the first time, MMP-9 in the exhaled breath condensate (EBC) of NSCLC patients. PARTICIPANTS: We enrolled 40 NSCLC patients and 40 controls affected by transudative pleural effusion. MEASUREMENTS: MMP-9 concentrations were measured in the EBC, whole blood (WB), and pleural effusion (PE) of all the subjects under study using enzyme immunoassay (EIA) kits. RESULTS: MMP-9 levels were found to be significantly higher in EBC, WB, and PE of NSCLC patients compared with controls. A positive correlation was observed between MMP-9 in EBC, cigarettes smoked, and stage of cancer. CONCLUSION: Exhaled MMP-9 was elevated in NSCLC patients, especially during tumor progression, and could represent a suitable noninvasive marker in the diagnosis and monitoring of lung cancer.

Microsatellite alterations and cell-free DNA analysis: could they increase the cytology sensitivity in the diagnosis of malignant pleural effusion?

BACKGROUND: The exact diagnosis of malignant pleural effusions (PE) is difficult and often requires combined procedures, because the cytological examination of pleural fluid does not detect tumoral cells in 40% of malignant effusion cases. The aim of this study was to analyze microsatellite alterations (MA) in malignant PE and to determine their diagnostic value as an additional test to cytological examination. The increase in cell-free DNA levels was also evaluated as a signal of probable malignancy. METHODS: A total of 84 patients with PE were enrolled and underwent PE and whole blood and exhaled breath condensate analyses. Free DNA was measured by spectrophotometer analyses. DNA was extracted from all samples and analyzed for MA, using the microsatellite markers at chromosomes 3p, 12p, 5q, and 17p. RESULTS: The microsatellite analysis of PE exhibited a higher percentage of alterations in malignant PE than in benign PE. In addition to this, cell-free DNA in PE was seen to be significantly more elevated in malignant than in benign PE. The sensitivity of the sole cytology increased considerably when patients showed at least one MA or DNA>4 ng/µL in the PE. CONCLUSION: In conclusion, it was seen that the combination of the cytological examination with microsatellite analyses and cell-free DNA in pleural fluid could increase the sensitivity of the diagnosis in patients with PE who have a suspected malignancy, obviating the need for other invasive diagnostic procedures.

Could exhaled ferritin and SOD be used as markers for lung cancer and prognosis prediction purposes?

BACKGROUND: Today an increasing interest is being generated by the study of lung cancer markers in the exhaled breath condensate (EBC), precisely because this sample seems to lend itself to lung cancer early screening and follow-up. Indeed, ferritin and superoxide dismutase (SOD) have recently been recognized to play a role in lung cancerogenesis and patients' survival. The aim of this study was to evaluate the clinical value and the prognostic power of exhaled ferritin and exhaled SOD in patients with lung cancer. MATERIAL AND METHODS: Forty patients with nonsmall cell lung cancer (NSCLC) and 15 controls were enrolled in the study. All subjects under study underwent EBC collection and analysis of ferritin and SOD. A total of 36 patients were either given a follow-up of at least 25.5 months or followed up until death. RESULTS: Exhaled ferritin and SOD resulted as being higher in NSCLC than in controls and as being influenced by the stage of cancer. A pronounced survival difference was found in the presence of exhaled ferritin 300 ng/mL and exhaled SOD > 13.5 U/µL. CONCLUSIONS: In conclusion, although the results need to be confirmed on a larger and homogeneous population, we hypothesized that the notion of using the measurement of ferritin and SOD in the EBC could, if deemed feasible, have clinical implications in the monitoring of lung cancer and as an outcome predictor.

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.

Neutrophilic airways inflammation in lung cancer: the role of exhaled LTB-4 and IL-8.

BACKGROUND: Recent advances in lung cancer biology presuppose its inflammatory origin. In this regard, LTB-4 and IL-8 are recognized to play a crucial role in neutrophil recruitment into airways during lung cancer.Notwithstanding the intriguing hypothesis, the exact role of neutrophilic inflammation in tumour biology remains complex and not completely known.The aim of this study was to give our contribution in this field by investigating LTB-4 and IL-8 in the breath condensate of NSCLC patients and verifying their role in cancer development and progression. METHOD: We enrolled 50 NSCLC patients and 35 controls. LTB-4 and IL-8 concentrations were measured in the breath condensate and the blood of all the subjects under study using EIA kits. Thirty NSCLC patients and ten controls underwent induced sputum collection and analysis. RESULTS: LTB-4 and IL-8 resulted higher in breath condensate and the blood of NSCLC patients compared to controls. Significantly higher concentrations were found as the cancer stages progressed. A positive correlation was observed between exhaled IL-8 and LTB-4 and the percentage of neutrophils in the induced sputum. CONCLUSION: The high concentrations of exhaled LTB-4 and IL-8 showed the presence of a neutrophilic inflammation in the airways of NSCLC patients and gave a further support to the inflammatory signalling in lung cancer. These exhaled proteins could represent a suitable non-invasive marker in the diagnosis and monitoring of lung cancer.

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.

Exhaled ERCC-1 and ERCC-2 microsatellite alterations in NSCLC patients.

BACKGROUND: The excision repair cross-complementation (ERCC) enzyme plays a rate-limiting role in nucleotide excision repair pathway. Microsatellite alterations (MAs) at the long arm of chromosome 19, where are located the ERCC genes, have recently been associated with non-small cell lung cancer (NSCLC) pathogenesis and reduced survival. The aim of the present study was to explore MAs at 19q in DNA from exhaled breath condensate (EBC) of NSCLC patients investigating their possible correlation with the smoking habit, with the biological behaviour of the tumour and their predictive survival power. METHODS: 34 NSCLC patients and 33 healthy controls (19 non-smokers and 14 smokers) were enrolled. All the subjects underwent 19q microsatellite analysis of their EBC. A total of 25 patients were either given a follow-up of at least 102 weeks, or were followed up until death. RESULTS: No MAs were found in EBC or WB in the healthy non-smokers, while 16% of exhaled MAs were found in healthy smokers and 25% of exhaled MAs in NSCLC patients. The number of MAs resulted related with tobacco consumption and with NSCLC patients' survival. CONCLUSIONS: In conclusion, the study of MAs at 19q resulted feasible in EBC-DNA. These genetic alterations are specific for lung cancer and predictive of survival in NSCLC patients. Our results suggest interesting clinical perspectives for the analysis of exhaled MAs at 19q in NSCLC patients.

Systemic and airway inflammation in sleep apnea and obesity: the role of ICAM-1 and IL-8.

The recurrent hypoxic stress that characterizes obstructive sleep apnea (OSA) seems to play a role in the increased adherence of neutrophils to endothelial cells as well as in the resulting migration of the former to the inflamed area. Intercellular adhesion molecule 1 (ICAM-1) and interleukin (IL)-8 are markers widely used in OSA studies to investigate inflammation. The aim of this study was to measure ICAM-1 and IL-8 levels in the breath condensate and in the plasma and inflammatory cells in the induced sputum of 12 obese OSA (OO) patients, 10 nonobese OSA (NOO) patients, 10 obese non-OSA (ONO) subjects, and 8 healthy subjects (HS) using a specific enzyme immunoassay (EIA) kit. A significant increase in both plasma and exhaled IL-8 and ICAM concentrations and percentage neutrophils was observed in the induced sputum of obese OSA patients, non-obese OSA patients, and obese non-OSA subjects compared with healthy subjects. However, although these inflammatory markers were found to follow an upward trend in obese OSA patients no difference was observed in both either non-obese OSA patients and obese non-OSA subjects. Finally, a significant positive correlation was found to occur among IL-8, ICAM-1, and sputum neutrophils, as well as across the apnea-hypopnoea index (AHI), TST 90%, body mass index (BMI), and neck circumference. The data obtained confirm the occurrence of an ICAM- and IL-8-mediated neutrophilic airway inflammation in both OSA and obese patients. The degree of inflammation, which seems to worsen in cases of comorbidity (OSA and obesity), is likely to be responsible for the increased risk of developing cardiovascular events observed in these subjects, and therefore, it deserves to be elucidated even more.

Cigarette smoke and increased COX-2 and survivin levels in exhaled breath condensate of lung cancer patients: how hot is the link?

UNLABELLED: One of the most current intriguing hypotheses on lung cancerogenesis envisages a role for inflammation as a possible trigger of both epithelial-mesenchymal transition and cancer development. Cigarette smoke has been suggested to be the main factor underlying the inflammation of the airways described in lung cancer patients. Cycloxygenase and survivin, a COX-2 dependent factor of apoptosis resistance, seem to play a key role in this regard. PURPOSE: The aim of this study was to study COX-2 and survivin in the airways of lung cancer patients and in those of a group of smokers in a view to increasing our understanding of the link between smoking, airway inflammation and lung cancer. PATIENTS AND METHODS: 70 NSCLC patients (28 smokers, 26 ex-smokers and 16 non-smokers) and 30 healthy subjects (20 smokers and 10 non-smokers) were enrolled in the study. Both COX-2 and survivin concentrations were measured in the exhaled breath condensates of all the subjects under study using EIA kits. RESULTS: Higher levels of exhaled survivin and COX-2 were found in NSCLC patients compared to healthy smokers and non-smokers. These levels were observed to be significantly elevated in smokers (patients with lung cancer and healthy) and ex-smokers compared to non-smokers and exhibited a positive correlation with the number of cigarettes smoked expressed as pack/year. A correlation was also found between exhaled COX-2 and survivin and the progression of cancer. CONCLUSIONS: We support the hypothesis that cigarette smoke be strongly connected to the inflammation of the airways observed in lung cancer patients. On the basis of the results obtained the use of exhaled breath condensate COX-2 and survivin levels could be suggested as two potential markers within an early non-invasive screening of populations of smokers at risk of lung cancer.

Prognostic value of exhaled microsatellite alterations at 3p in NSCLC patients.

UNLABELLED: Our research group has recently been able to demonstrate and validate the possibility of studying of 3p microsatellite alterations (MAs) in the DNA extracted from the exhaled breath condensate (EBC) of healthy smokers and of subjects with non-small cell lung cancer (NSCLC). In light of the interest that has recently been aroused in the novel molecular staging protocol of lung cancer, the evaluation of the prognostic power of the genetic alterations involved in lung cancerogenesis, including 3p microsatellite alterations could be of clinical interest. PURPOSE: The aim of this study was to investigate the outcome predictive power of exhaled 3p microsatellite alterations in lung cancer patients. PATIENTS AND METHODS: Seventy-one NSCLC patients were enrolled in the study. All the subjects under study had already undergone a 3p microsatellite analysis of their EBC. A total of 56 patients were either given a follow-up of at least 102 weeks, or were followed up until death. RESULTS: The number of 3p microsatellite alterations found in the exhaled breath condensate DNA exhibits a remarkable correlation with patients' survival. D3S2338 and D3S1289 account for the microsatellites with the highest positive prognostic power; loss of heterozygosis (LOH) D3S1289 has a negative prognostic value in adenocarcinoma while microsatellite instability (MI) and LOH D3S2338 influence survival in squamous cell carcinoma; and, independently of NSCLC stage, D3S1289 is associated with poor prognosis. CONCLUSIONS: In conclusion, 3p MAs in the DNA of exhaled breath condensate is strongly associated with NSCLC patients' survival. Our results suggest that it is possible to use the study of EBC MAs as an outcome predictor for lung cancer patients.

IL-2, TNF-alpha, and leptin: local versus systemic concentrations in NSCLC patients.

One recent line of cancer research shows increasing interest for biological factor such as IL-2, TNF-alpha, and leptin, which have been found to participate in the development and progression of non-small cell lung cancer (NSCLC). The aim of this study was to measure IL-2, TNF-alpha, and leptin concentrations in the airways and in the systemic circle of patients with NSCLC, investigating the role of these factors in the lung tumors. We enrolled 32 patients (17 men, 71 +/- 7 years) with a histological diagnosis of NSCLC and 20 healthy ex-smoker controls, negative for computed tomography of the chest (14 men, 69 +/- 8 years). IL-2, TNF-alpha, and leptin levels were measured in the serum, the urine, the bronchoalveolar lavage, the induced sputum, and exhaled breath condensate (EBC) of patients enrolled by means of a specific enzyme immunoassay kit. Higher concentrations of IL-2, TNF-alpha and leptin were found in NSCLC patients than in controls (p < 0.0001). A statistically significant increase of IL-2, TNF-alpha, and leptin concentrations was observed in patients from stage I to stage III of NSCLC. These findings suggest that IL-2, TNF-alpha, and the leptin play an important role in the cancerogenesis of NSCLC. Their measure in the EBC could be proposed as noninvasive markers for an early detection of NSCLC and in the follow-up of this tumor.