Maria Cristina Scarpa

Exhaled Nitric Oxide and Breath Condensate pH in Asthmatic Reactions Induced by Isocyanates

BACKGROUND We investigated the usefulness of measurements of fractional exhaled nitric oxide (FeNO) and pH of exhaled breath condensate (EBC) for monitoring airway response after specific inhalation challenges with isocyanates in sensitized subjects. METHODS Lung function (FEV(1)), FeNO, and pH in argon-deaerated EBC were measured before and at intervals up to 30 days after a specific inhalation challenge in 15 subjects with isocyanate asthma, in 24 not sensitized control subjects exposed to isocyanates, and in 3 nonasthmatic subjects with rhinitis induced by isocyanate. Induced sputum was collected before and 24 h after isocyanate exposure. RESULTS Isocyanate-induced asthmatic reactions were associated with a rise in sputum eosinophil levels at 24 h (p < 0.01), and an increase in FeNO at 24 h (p < 0.05) and 48 h (p < 0.005), whereas FeNO level did not vary with isocyanate exposure in subjects with rhinitis and in control subjects. FeNO changes at 24 h positively correlated with corresponding sputum eosinophil changes (rho = 0.66, p < 0.001). A rise in pH was observed in the afternoon samples of EBC, irrespective of the occurrence of isocyanate-induced asthmatic reactions. CONCLUSIONS We demonstrated that isocyanate-induced asthmatic reactions are associated with a consistent delayed increase in FeNO but not with the acidification of EBC.

Ceramide Expression and Cell Homeostasis in Chronic Obstructive Pulmonary Disease

Background: Increased expression of ceramide has been detected in emphysema. Ceramide promotes autophagy and apoptosis, which concur with cellular homeostasis. Objectives: To determine whether ceramide expression is associated with the development of chronic obstructive pulmonary disease (COPD) and with altered cellular homeostasis in lung parenchyma. Methods: We studied 10 subjects with severe COPD, 13 with mild/moderate COPD, 11 with idiopathic pulmonary fibrosis (IPF), 12 non-COPD smokers, and 11 nonsmoking controls. The immunoreactivity for ceramide along with markers of autophagy (LC3B), apoptosis (cleaved caspase-3), and cell proliferation (MIB1) was quantified in alveolar walls. Results: Ceramide expression was increased in COPD patients compared with control smokers and was related to the impairment of gas exchange but not to the degree of airflow limitation. In COPD, an important activation of apoptosis and autophagy pathways was observed, particularly in patients with severe disease, that was not counterbalanced by cell proliferation. Upregulation of ceramide was observed even in subjects with IPF in whom activation of apoptosis and autophagy was negligible and cell proliferation was instead the most prominent feature. Conclusions: Ceramide expression, which is increased in COPD and even more so in IPF, appears to be neither specific nor related to COPD severity, probably representing a broader marker of lung damage. In contrast, apoptosis and autophagy are characteristics of the COPD pathology, particularly in its most severe stage.

Mucosal immune environment in colonic carcinogenesis: CD80 expression is associated to oxidative DNA damage and TLR4-NFκB signalling.

BACKGROUND CD80 has been thought to play an active role in immunosurveillance as it has been found to be up-regulated in ulcerative colitis (UC) patients with dysplasia. The aim of the present study was to analyse early events in UC-related and non-inflammatory carcinogenesis with reference to CD80 expression to clarify what stimuli are involved in its up-regulation in these patients. PATIENTS AND METHODS Sixty-two patients affected with UC, UC with dysplasia, UC and cancer, colonic adenoma, or colonic cancer and 11 healthy subjects were enroled in our study. Tissue samples were taken from surgical specimens during colonic resection or during colonoscopy. Mucosal mRNA expression of Toll-like receptor-4 (TLR4) and nuclear factor-kappaB (NF-κB) was quantified with Real Time RT-PCR. TLR4, β-catenin and p53 expressions were analysed by immunohistochemistry. Mucosal levels of activated NF-κB were measured with immunometric assays while 8-Hydroxydeoxyguanosine (8-OHdG) levels were quantified by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Non-parametric tests were used for statistical analysis. RESULTS 8-OHdG mucosal levels were higher in the patients with UC + dysplasia with respect to those in the patients with UC only (p=0.03). CD80 mRNA mucosal levels were directly correlated with 8-OHdG mucosal levels (τ=0.26, p=0.04), TLR4 protein expression (τ=0.45, p<0.01) and NF-κB mRNA expression and activity (τ=0.24, p=0.02; τ=0.34, p=0.02, respectively). CD80 protein expression, instead, was directly correlated with 8-OHdG mucosal levels (τ=0.19, p=0.05) and inversely correlated with TLR4 mRNA expression (τ=-0.25, p=0.03). CONCLUSION Oxidative DNA damage peaked in UC-related dysplasia and was found to be directly correlated to CD80 expression. The direct correlation between TLR4 protein expression and CD80 mRNA and the indirect correlation between CD80 protein and TLR4 mRNA expressions give substance to the hypothesis that they play a role in immunosurveillance. No significant correlations between CD80 expression and p53 and β-catenin accumulation during oncogenesis were, instead, observed.

The role of non-invasive biomarkers in detecting acute respiratory effects of traffic-related air pollution.

The role of non‐invasive methods in the investigation of acute effects of traffic‐related air pollution is not clearly established. We evaluated the usefulness of non‐invasive biomarkers in detecting acute air pollution effects according to the age of participants, the disease status, their sensitivity compared with lung function tests and their specificity for a type of pollutant. Search terms lead to 535 titles, among them 128 had potentially relevant abstracts. Sixtynine full papers were reviewed, while 59 articles were excluded as they did not meet the selection criteria. Methods used to assess short‐term effects of air pollution included analysis of nasal lavage (NAL) for the upper airways, and induced sputum (IS), exhaled breath condensate (EBC) and exhaled nitric oxide (FeNO) for central and lower airways. There is strong evidence that FeNO evaluation is useful independently from subject age, while IS analysis is suitable almost for adults. Biomarker changes are generally observed upon pollutant exposure irrespective of the disease status of the participants. None of the biomarkers identified are specific for a type of pollutant exposure. Based on experimental exposure studies, there is moderate evidence that IS analysis is more sensitive than lung function tests, whereas this is not the case for biomarkers obtained by NAL or EBC. Cells and some cytokines (IL‐6, IL‐8 and myeloperoxidase) have been measured both in the upper respiratory tract (NAL) and in the lower airways (IS). Overall, the response to traffic exposure seems different in the two compartments. In conclusion, this survey of current literature displays the complexity of this research field, highlights the significance of short‐term studies on traffic pollution and gives important tips when planning studies to detect acute respiratory effects of air pollution in a non‐invasive way.

Mechanisms of Decrease in Fractional Exhaled Nitric Oxide During Acute Bronchoconstriction

BACKGROUND Fractional exhaled nitric oxide measured at expiratory flow of 50 mL/s (Feno50), a biomarker of airway inflammation, is affected by changes in airway caliber. Whether a lower Feno50 level during bronchoconstriction is only an artifact due to the strong flow dependence of this parameter is controversial. METHODS We aimed to evaluate the dynamics of airway and alveolar nitric oxide (NO) during acute bronchoconstriction induced by methacholine. Exhaled NO was measured at expiratory flows of 10, 50, 100, 150, and 250 mL/s before and after metacholine in 26 responders to methacholine and 37 nonresponders. Flow-independent parameters (airway wall NO flux, airway NO diffusing capacity, airway wall NO concentration, alveolar NO concentration) were calculated using a two-compartment model, and correction for NO axial back diffusion was applied. RESULTS Bronchoconstriction in responders was associated with a decrease in Feno50 (-28%, P < .0001), in airway wall NO flux (-34%, P < .0001), and in airway NO diffusing capacity (-15%, P < .05). In contrast, alveolar NO concentration was not affected by bronchoconstriction. Postmethacholine changes in Feno50 were more strictly related to the ventilation distribution, assessed by single-breath carbon monoxide uptake, than to larger airways caliber, assessed by FEV1. When bronchoconstriction was reversed by salbutamol, airway wall NO flux and airway NO diffusing capacity returned to values comparable to those measured premethacholine. CONCLUSIONS The changes in airway caliber induced by noninflammatory stimuli alter NO transport in the lung. The changes in NO dynamics are limited to conductive airways and are characterized by a reduction of NO flow to luminal space.

Effects of Inhalation of Thermal Water on Exhaled Breath Condensate in Chronic Obstructive Pulmonary Disease

Background: Inhalation of thermal water (TW) is traditionally used as part of the treatment of chronic obstructive pulmonary disease (COPD), but its benefit and mechanisms are controversial. We previously observed a reduced proportion of neutrophils in induced sputum after treatment with TW. Objectives: The aim of this study was to determine whether inhalation of TW in COPD patients is associated with biochemical changes of airway lining fluid, including a reduction in the neutrophil chemoattractant leukotriene B4 (LTB4). Methods: Thirteen COPD patients were randomly assigned to receive a 2-week course of TW and normal saline inhalation in a cross-over, single-blind study design. Exhaled breath condensate (EBC) was collected before and after treatments. LTB4 concentrations in EBC were determined by ELISA, and EBC pH was measured before and after argon deaeration. Results: No significant differences in LTB4 concentrations in EBC were detected with either treatment. A significant decrease in pH of non-deaerated EBC was observed after a standard course of TW (median 7.45, interquartile range 6.93–7.66, vs. median 6.99, interquartile range 6.57–7.19; p = 0.05), which disappeared after argon deaeration. Conclusions: There is no evidence that TW treatment affects LTB4 concentration in EBC. The results of EBC pH measurements suggest that TW inhalation induces an imbalance of volatile components of the buffer system in airway lining fluid.

Exhaled nitric oxide dynamics in asthmatic reactions induced by diisocyanates.

Isocyanate‐induced asthmatic reactions are associated with delayed increase in fractional exhaled nitric oxide measured at expiratory flow of 50 mL/s (FeNO50), a biomarker of airway inflammation. The time course of FeNO increase is compatible with the activation of NO synthase, but the origin of NO production in the lung is undetermined.

Cluster analysis of occupational asthma caused by isocyanates

Asthma due to isocyanates is a heterogeneous disease, which can be separated in 3 distinct phenotypes by unsupervised cluster analysis. We established that the causing agent is not sufficient to explain the heterogeneity of OA. Differences across clusters suggest that individual susceptibility and/or intensity of exposure are implicated.

Mucosal Immune Environment in Colonic Carcinogenesis: T-Cell Activation in Ulcerative Colitis and Dysplasia

(TMAs). Slides were immunostained for TLR9, α-SMA and CK, then digitalized and analyzed semiquantitatively by using a digital microscope software. Results: Significant elevation of cfDNA levels were found in CRC compared to that in healthy blood samples (p<0,05), but healthy and adenoma samples did not show major difference. In tissue samples increasing cytoplasmatic TLR9 expression was found during colorectal ADCS. The percentage of TLR9+ cells was significantly higher in CRC epithelium (68,25±24,33%) and also in adenoma (70,34±2,49%) than in healthy (32,92±8,314%) samples. The proportion of intraepithelial α-SMA+/CK+ cells did not show substantial difference between healthy (1,94±0,69%) and adenoma (1,62±0,78%) samples. However, significantly elevated (p<0,01) α-SMA+/CK+ (3,34±1,01%) cell number was observed in CRC compared with those found in the other two groups during ADCS. Conclusion: The number of intraepithelial α-SMA+/CK+ cells elevated parallel with the cfDNA level during ADCS, which may refer to the potential role of cfDNA in EMyT via TLR9 activation. Although the TLR9 level is already elevated in colorectal adenoma, the cfDNA level was sufficient only in carcinoma samples to lead to TLR9 activation.