Heterogeneous spontaneous and interleukin-4-induced nitric oxide production by human monocytes.

The generation of nitric oxide by human monocytes has long been a subject of controversy because of the difficulty of rationalizing this production. In this work we evaluated the capacity of human monocytes to produce nitric oxide (NO) as measured by nitrite (NO2-) release. Resting unstimulated monocytes (2 x 10(6) cells/ml) were found to produce significant amounts of NO2- after 8 to 12 days in culture. This production appeared to be highly heterogeneous. Indeed, approximately, 75% of monocytes from the different donors produced up to 10 microM NO2- and were considered low producers; the last 25% produced higher amounts of NO2- (from 10 to 110 microM) and were considered high producers. In any case the spontaneous production of NO2- by monocytes was overcome in the presence of 1 mM N omega-monomethyl-L-arginine (LNMMA). This inhibitory effect was reversed in the presence of an excess of L-arginine (5 mM), indicating that this process is effectively dependent on L-arginine metabolism. Because interleukin-4 (IL-4) is considered an important NO-regulatory cytokine, its regulatory effect on this spontaneous production of NO was also evaluated. In the presence of a defined dose of IL-4 (1 to 100 ng/ml) the spontaneous production of the high-producing population of monocytes was abrogated, whereas IL-4 stimulated the production by the low-producing population of monocytes, which was suppressed in the presence of LNMMA. The present data indicate that NO production by human monocytes is heterogeneous and that IL-4 can be a potent inducer or inhibitor of this production, suggesting a variability in the activation state of these cells.

Airway remodelling in the pathogenesis of asthma.

The inflammatory and remodelling processes that underlie asthma result from a highly complex interaction between various cell types. Apart from inflammatory cells, such as eosinophils, activated T cells, mast cells and macrophages, structural tissue cells such as epithelial cells, fibroblasts and smooth muscle cells can also play an important effector role through the release of a variety of mediators, cytokines and chemokines. This results in an acute inflammatory response that is characterized by vascular leakage, mucus hypersecretion, epithelial shedding and widespread airway narrowing. At the same time, through the release of mediators, cytokines, chemokines and growth factors, epithelial and mesenchymal cells cause persistence of the inflammatory infiltrate and induce structural changes in the airway wall, such as increased thickness of the basement membrane, increased collagen deposition, changes in bronchial microcirculation, and smooth muscle hypertrophy and hyperplasia. The end result of airway inflammation and remodelling is an increased thickness of the airway wall, leading to a reduced baseline airway calibre and exaggerated airway narrowing.

The expression of basic fibroblast growth factor (bFGF) in tumor-associated stromal cells and vessels is inversely correlated with non-small cell lung cancer progression.

Tumor progression results from complex interactions between tumor and tumor-associated host tissue. Basic fibroblast growth factor (bFGF), via activation of its receptor, FGFR-1, has been postulated to be an important inducer of host stromal response and angiogenesis. To assess the putative role of tumor-associated stromal cells and vessels in tumor progression, we studied non-small cell lung cancer (NSCLC) from 84 patients, including 51 squamous cell carcinomas and 33 nonsquamous cell carcinomas, by immunohistochemical detection. bFGF and FGFR-1 immunoreactivity was observed in tumor and in tumor-associated stromal cells and vessels. The expression of bFGF and FGFR-1 in stromal cells was higher in squamous than in non-squamous cell carcinomas (respectively, P = .007 and P = .0004). We found that bFGF and FGFR-1 expression in tumor and tumor-associated stromal cells and vessels was directly correlated with host stromal response, as assessed by intratumoral extension of stroma, but not with angiogenic response, as assessed by microvessel count. Although FGFR-1 expression of tumor cells was directly correlated with T-stage (P = .03), bFGF expressions of tumor-associated stromal cells and vessels were inversely correlated with lymph node metastasis (respectively, P = .0001 and P = .0002) and advanced pathological stage (respectively, P = .03 and P = .01). These findings suggest that bFGF might mediate host stromal response in NSCLC and that the expression of bFGF in tumor-associated stromal cells and vessels might have an inhibitory role in NSCLC progression.

Effect of age upon airway obstruction and reversibility in adult patients with asthma.

OBJECTIVE: In a cross-sectional study we evaluated the effect of aging (separately from that of duration of disease) on airway obstruction and reversibility by comparing two groups of non-smoker patients with asthma. METHODS: We compared two groups of patients: group A, which had 50 subjects (8 men and 42 women) aged 59.7+/-4.6 years (mean +/- SD), and group B, comprised of 51 subjects (19 men and 32 women) who were 35.7+/-7.4 years old. The groups were selected because of comparable baseline degree of obstruction (FEV1 % of predicted, 67.8+/-20.3 in group A; 73.0+/-19.6 in group B, NS) and duration of the disease (14.0+/-11.7 years vs 11.2+/-9.1, NS). Spirometric examination, with a bronchodilator test, was performed and subjects not reaching 85% of predicted were submitted to a 4-week course of inhaled steroids. RESULTS: Although a higher number of subjects from group B responded to the acute bronchodilator test (p < 0.001), the maximum response achievable with treatment (steroid or bronchodilator) (deltaFEV1 expressed as the percent of predicted) was not statistically different between groups (12.0+/-17.5 vs 16.0+/-23.9). The mean FEV1 attainable after treatment (deltaFEV1%PT) was significantly lower in the older group (p = 0.0006). Within groups, the baseline FEV1% did not correlate with age; it was inversely correlated with the duration of the disease (p < 0.03 and p < 0.01, respectively). In both groups deltaFEV1 was inversely related with the baseline FEV1, whereas FEV1%PT was correlated with the duration of the disease, with a slope nearly doubled in group B (p < 0.001). CONCLUSIONS: Both the process of aging and the prolonged exposure to disease effects are important factors in determining the functional characteristics of chronic asthma: In particular, aging is associated not only with a reduced acute responsiveness to bronchodilators, but also with a reduced slope of the duration-FEV1%PT relationship that suggests a slowing of the rate of loss of reversibility of uncertain biological meaning.

MUC1 (episialin) expression in non-small cell lung cancer is independent of EGFR and c-erbB-2 expression and correlates with poor survival in node positive patients.

AIM: To examine tumour samples immunohistochemically for MUC1 (episialin), epidermal growth factor receptor (EGFR), and c-erbB-2, since the disruption of the cell-cell adhesion system by MUC1 and the c-erbB oncoprotein family is known to be important in the development of metastasis in human cancers. METHODS: 93 tumour samples from patients with early stage non-small cell lung cancer treated with surgery alone were examined for episialin, EGFR, and c-erbB-2. RESULTS: Episialin depolarised expression did not correlate with any of the histopathological variables examined (T,N stage, grade, histology, Ki67 proliferation index). No correlation was observed between episialin and EGFR or c-erbB-2 expression. Survival analysis showed that episialin depolarised expression correlated with poor prognosis (p = 0.003), especially in squamous cell cases (p = 0.0003). Episialin expression defined a group of patients with poor prognosis in the node positive category (p = 0.003). In multivariate analysis episialin was the most significant independent prognostic factor (p = 0.007), followed by N stage (p = 0.04). CONCLUSIONS: Depolarised expression of episialin is associated with poor outcome in early stage non-small cell lung cancer. Despite the similar activity on the cadherin cell-cell adhesion system, the expression of episialin and c-erbB oncoproteins is likely to be activated within different pathogenic pathways.

Effects of gemcitabine on cell proliferation and apoptosis in non-small-cell lung cancer (NSCLC) cell lines.

We evaluated the antiproliferative and the proapoptotic ability of gemcitabine in three non-small-cell lung cancer (NSCLC) cell lines. NCI-H292 (mucoepidermoid carcinoma), NCI-CorL23 (large-cell carcinoma) and NCI-Colo699 (adenocarcinoma) cells were cultured with and without 0.5, 0.05 and 0.005 microM gemcitabine for 24, 48 and 72 h, respectively. Gemcitabine exerted a stronger and earlier antiproliferative and proapoptotic effect on H292 cells than on CorL23 or Colo699 cells. Fas receptor expression was increased in all three cell lines and was higher in Colo699 than in CorL23 cells. The incubation of NSCLC with anti-Fas agonistic monoclonal antibody (CH11) induced cell apoptosis in H292 cells, demonstrating that the Fas receptor was functionally active. Finally, gemcitabine and CH-11 exerted a synergistic effect on cell apoptosis in H292 cells. This study demonstrates that gemcitabine induces apoptosis in NSCLC and that this effect might be exerted by modulating functionally active Fas expression, and these effects of gemcitabine were stronger in H292 cells than in either CorL23 or Colo699 cells.

Depolarized expression of episialin (EMA, MUC1) in lung adenocarcinoma is associated with tumor progression.

Carcinoma cell detachment is an important step in tumor progression and metastasis. Episialin (EMA), if expressed throughout the entire cell surface, may inhibit cell-cell and cell-matrix adhesion. We investigated whether the cellular distribution of episialin in non-small cell lung cancer (NSCLC) is associated with tumor progression. We evaluated the expression of episialin by immunohistochemical staining, in surgical specimens from 122 adenocarcinomas and 99 squamous cell carcinomas. Episialin was present in most NSCLC, with a higher percentage of immunoreactive neoplastic cells in adenocarcinoma than in squamous cell carcinoma (p = 0.0001). In adenocarcinoma the depolarized pattern was significantly associated with nodal metastasis (p = 0.005) and with advanced stage (p = 0.007). In conclusion, nodal metastasis and advanced pathological stage in adenocarcinoma are associated with a depolarized cellular distribution of episialin, suggesting a possible involvement of the molecule in cancer metastasis.

Safety and cellular assessment of bronchial brushing in airway diseases.

Bronchial brushing is a useful method for morphological and functional studies of bronchial epithelial cells (BECs) in various diseases. This technique has been found to be generally safe, but its safety in asthma and chronic bronchitis has not been fully assessed. The purpose of this study was to determine 1, whether bronchial brushing is a safe method in asthmatic and chronic bronchitis patients of differing severity and 2, to characterize the BECs obtained in terms of number, viability and purity. We evaluated 25 asthmatics of variable severity, 19 chronic bronchitis patients and 26 normal volunteers. Bronchoscopy and bronchial brushing were performed in a standardized manner by the same investigator. Safety was assessed by clinical follow-up of all subjects; continuous monitoring of arterial oxygen saturation during the procedure with a digital oximeter was carried out in a subsample of subjects. No complications were observed clinically during the procedure. There was a minimal fall in arterial oxygen saturation without a significant difference between the three groups of subjects. A consistent number of BECs was recovered and their viability, assessed by the trypan blue exclusion test, in asthmatics and chronic bronchitis patients was significantly lower than in controls (P < 0.05). Bronchial brushing is well tolerated and may be a valuable method of obtaining BECs in asthmatic and chronic bronchitis patients.

Airway inflammation in patients affected by obstructive sleep apnea syndrome.

Obstructive sleep apnea syndrome (OSAS) has been shown to be associated to upper airway inflammation. The object of the present study was to establish the presence of bronchial inflammation in OSAS subjects. In 16 subjects affected by OSAS, and in 14 healthy volunteers, airway inflammation was detected by the cellular analysis of the induced sputum. OSAS patients, as compared to control subjects, showed a higher percentage of neutrophils (66.7+/-18.9 vs. 25.8+/-15.6) (P<0.001) and a lower percentage of macrophages (29.4+/-18.4 vs. 70.8+/-15.3) (P<0.001). The percentage of eosinophils and lymphocytes were not significantly different in the two groups. OSAS subjects show bronchial inflammation characterized by a significant increase in neutrophils.

Growth factors in asthma.

Asthma is a chronic inflammatory disease of the airways, with associated repair processes. Both inflammatory and repair processes appear to be strictly related, and can lead to several histopathological alterations of the bronchial mucosa, such as the shedding of epithelium and increased thickness of the basement membrane. The integrity as well as the alterations of the bronchial structure are the consequence of several biological events, such as cell proliferation and death, cell activation and inhibition, and extracellular matrix (ECM) production and degradation. These events are critically regulated by polypeptides called growth factors (GFs), which are able, functioning in an autocrine and paracrine fashion, to affect and modulate cell functions and ECM turnover. Although the importance of GFs has been widely demonstrated in other pulmonary conditions, such as lung fibrotic diseases, their possible involvement in the pathogenesis of inflammatory and postinflammatory processes in asthma is still not completely clear. The aim of the present review was to discuss the biological evidence concerning the role of several growth factors, such as transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), granulocyte/macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF) and endothelin, in asthma and chronic bronchitis.

Markers of acute airway inflammation.

In asthma, acute inflammation induced by allergen challenge in allergic patients leads to an early and a late phase reaction. Immunoglobulin E (IgE)-mediated cell activation plays a crucial role in acute inflammation and leads to the release of proinflammatory mediators, such as histamine, eicosanoids, platelet-activating factor, oxygen-free radicals, neuropeptides and cytokines. These mediators are capable of inducing constriction of airways smooth muscle, increased secretion of mucus, and vasodilatation, as well as several important biological events, including modulation of the expression of adhesion molecules in endothelial and epithelial cells, and of inflammatory cell recruitment. Clinically, acute airway inflammation is characterized by a bronchoconstrictive response with acute symptoms, including wheezing and dyspnoea which can be reversed by short-acting bronchodilators, among which beta 2-agonists play a major role.

[Mechanisms of bronchial hyperreactivity. Role of the epithelium]. / Mécanismes de l'hyperréactivité bronchique. Rôle de l'épithélium.

Bronchial epithelial cells are present all along the airways. They play a role of barrier and protect the airways against inhaled pollutants. However it is known to day that they could play an active role, through synthesis and release of mediators which modulate the airway muscles, the epithelium permeability and the vascular tone. Shedding of the epithelium is a constant feature of the asthmatic airways as can be observed directly on the bronchial biopsies and indirectly through and increased number of epithelial cells in the bronchoalveolar lavage fluid. The epithelial shedding is well correlated to the bronchial hyperreactivity. In vitro, bronchial epithelial cells release higher quantities of 15 HETE, endothelin and PGE2 in asthmatics than in normal control subjects. They express HLA-DR and ICAM-1 in higher quantities in asthmatic patients. Bronchial epithelial cells might play an important role in inflammatory lesions which are observed in asthmatic patients. They could be a good target for new therapeutic approaches in asthma.

[Role of glucocorticosteroid receptors in bronchial asthma]. / Ruolo del recettore dei glucocorticosteroidi nell'asma bronchiale.

Glucocorticoids (GC) represent the cornerstone anti-inflammatory treatment of chronic asthma. A small proportion of asthmatics develop a severe form of the disease and require a chronic long-term treatment with oral GC. These patients, ascribed as GC dependent asthmatics, present an ongoing inflammation of the airways. GC dependent asthma should be differentiated from GC resistant asthma. GC resistant asthmatics are defined as patients whose baseline pre-bronchodilation FEV1 of less than 70-80% predicted improves by less than 15% following 1-2 weeks of 40 mg prednisolone daily. The effects of GC are mediated by the GC receptor (GR) alpha. By a process called trans-activation they increase the transcription of genes involved in either beneficial processes or certain side effects. Through trans-repression, they inhibit the transcription factors, including nuclear factor kappa B (NF-kappa B), thereby decreasing the expression of many genes encoding inflammatory mediators. In addition to GR alpha, an isoform deficient in hormone binding has been isolated in humans and termed GR beta, which functions as a dominant negative inhibitor of GR alpha. However, to act as such, GR beta has to be more abundant than GR alpha, and conflicting data have been obtained concerning the relative levels of the two isoforms in cell lines and freshly isolated cells. It seems however that overexpression of GR beta may play a role in GC-resistant asthmatics, whereas in GC-dependent asthmatics, a predominant GR alpha expression has been consistently found. Thus the persistence of inflammation in GC-dependent asthma does not seem to be associated with an overexpression of GR beta but with a dysfunction of the trans-repression or trans-activation processes mediating the anti-inflammatory effects of GR alpha.

Beclomethasone/formoterol vs fluticasone/salmeterol inhaled combination in moderate to severe asthma.

BACKGROUND: Recommended treatment for moderate to severe asthma is the combination of an inhaled corticosteroid and a long-acting beta(2)-agonist. The present study was designed to compare a new fixed combination of extrafine beclomethasone and formoterol, with the fixed combination fluticasone and salmeterol. METHODS: This was a phase III, multinational, multicentre, double-blind, randomized, two-arm parallel groups, controlled study. After a 2-week run-in period, 228 patients with moderate to severe asthma were randomized to a 12-week treatment with either beclomethasone 100 microg plus formoterol 6 microg or fluticasone 125 microg plus salmeterol 25 microg, both delivered two inhalations b.i.d. via a pressurized metered dose inhaler. RESULTS: The analysis of noninferiority on the primary outcome, morning peak expiratory flow in the last 2 weeks of treatment, showed no difference between groups (difference -3.32 l/min; 95% CI -17.92 to 11.28). A significant improvement from baseline in lung function, symptom score and rescue medication use was observed in both groups at all time points. Beclomethasone plus formoterol combination showed a significantly faster onset of bronchodilation when compared with fluticasone plus salmeterol with the difference maintained for up to 1 h postdosing. No differences were observed between treatments in the rate of asthma exacerbations, frequency of adverse events and overnight urinary cortisol/creatinine ratio. CONCLUSIONS: The new combination of extrafine beclomethasone plus formoterol is not inferior to the marketed combination of fluticasone and salmeterol in terms of efficacy and tolerability, with the advantage of a faster onset of bronchodilation. ( ClinicalTrials.gov number, NCT00394368).

Beclomethasone/formoterol versus budesonide/formoterol combination therapy in asthma.

The present study was designed to compare the fixed combination of beclomethasone and formoterol in a hydrofluoroalkane Modulite (Chiesi Farmaceutici, Parma, Italy) pressurised metered-dose inhaler (pMDI), with a combination of budesonide and formoterol administered via a Turbuhaler (AstraZeneca, Lund, Sweden) dry powder inhaler (DPI). This was a phase III, multinational, multicentre, double-blind, double-dummy, randomised, two-arm parallel groups, controlled study design. After a 2-week run-in period, 219 patients with moderate-to-severe asthma were randomised to a 12-week treatment with beclomethasone 200 microg plus formoterol 12 microg b.i.d. delivered via a pMDI or budesonide 400 microg plus formoterol 12 microg b.i.d. delivered via a DPI. The analysis of noninferiority on primary outcome, morning peak expiratory flow in the last 2 weeks of treatment, showed no difference between groups. A statistically significant improvement from baseline in lung function, symptoms and rescue medication use was observed in both groups at all time-points. No differences were observed between treatments in either rate of asthma exacerbations or frequency of adverse events. The new fixed combination of beclomethasone and formoterol in hydrofluoroalkane Modulite pressurised metered-dose inhaler is equivalent to the marketed combination of budesonide and formoterol in terms of efficacy and tolerability profile.

Near-fatal asthma phenotype in the ENFUMOSA Cohort.

BACKGROUND: Near-fatal asthma (NFA) is characterized by severe asthma attacks usually requiring intensive care unit admission. This phenotype of asthma has been studied mainly in acute conditions. METHODS: The aim of our study was to compare the clinical, functional and inflammatory characteristics of NFA patients with mild to severe asthmatics in stable conditions. We recruited 155 asthmatic patients from five centres of the European Network for Understanding Mechanisms of Severe Asthma: 67 patients with mild-to-moderate asthma controlled by low/medium doses of inhaled corticosteroids; 64 with severe asthma that, despite treatment with high doses of inhaled corticosteroids, long-acting beta2-agonists and for 1/3 also with regular oral corticosteroids, had at least one asthma exacerbation in the previous year; 24 with an NFA episode in the previous 5 years in the absence of inclusion criteria for the previous groups. All the patients were examined in stable conditions. RESULTS: NFA patients were taking less corticosteroids and were less compliant to prescribed asthma medications than the other two groups of patients. Lung function, blood gases, atopic status, sputum and blood inflammatory cell count of NFA patients were similar to mild-to-moderate, but not severe, asthmatic patients. CONCLUSIONS: In stable conditions patients with an NFA attack in the previous 5 years cannot be distinguished from patients with mild-to-moderate asthma, while they are different from severe asthmatics both in terms of lung function and of airway inflammation. The risk factor that characterizes this group of patients is reduced usage of prophylactic corticosteroids.