Elisabetta Pace

Cigarette smoke increases Toll-like receptor 4 and modifies lipopolysaccharide-mediated responses in airway epithelial cells.

Airway epithelium is emerging as a regulator of innate immune responses to a variety of insults including cigarette smoke. The main goal of this study was to explore the effects of cigarette smoke extracts (CSE) on Toll‐like receptor (TLR) expression and activation in a human bronchial epithelial cell line (16‐HBE). The CSE increased the expression of TLR4 and the lipopolysaccharide (LPS) binding, the nuclear factor‐κB (NF‐κB) activation, the release of interleukin‐8 (IL‐8) and the chemotactic activity toward neutrophils. It did not induce TLR2 expression or extracellular signal‐regulated signal kinase 1/2 (ERK1/2) activation. The LPS increased the expression of TLR4 and induced both NF‐κB and ERK1/2 activation. The combined exposure of 16‐HBE to CSE and LPS was associated with ERK activation rather than NF‐κB activation and with a further increase of IL‐8 release and of chemotactic activity toward neutrophils. Furthermore, CSE decreased the constitutive interferon‐inducible protein‐10 (IP‐10) release and counteracted the effect of LPS in inducing both the IP‐10 release and the chemotactic activity toward lymphocytes. In conclusion, cigarette smoke, by altering the expression and the activation of TLR4 via the preferential release of IL‐8, may contribute to the accumulation of neutrophils within the airways of smokers.

Leptin and leptin receptor expression in asthma.

BACKGROUND The adipokine leptin is a potential new mediator for bronchial epithelial homeostasis. Asthma is a chronic inflammatory disease characterized by airway remodeling that might affect disease chronicity and severity. TGF-beta is a tissue growth factor the dysregulation of which is associated with airway remodeling. OBJECTIVE We sought to determine whether a bronchial epithelial dysfunction of the leptin/leptin receptor pathway contributes to asthma pathogenesis and severity. METHODS We investigated in vitro the presence of leptin/leptin receptor on human bronchial epithelial cells. Then we studied the effect of TGF-beta and fluticasone propionate on leptin receptor expression. Finally, the role of leptin on TGF-beta release and cell proliferation was analyzed. Ex vivo we investigated the presence of leptin/leptin receptor in the epithelium of bronchial biopsy specimens from subjects with asthma of various severities and from healthy volunteers, and some features of airway remodeling, such as reticular basement membrane (RBM) thickness and TGF-beta expression in the epithelium, were assessed. RESULTS In vitro bronchial epithelial cells express leptin/leptin receptor. TGF-beta decreased and fluticasone propionate increased leptin receptor expression, and leptin decreased the spontaneous release of TGF-beta and increased cell proliferation. Ex vivo the bronchial epithelium of subjects with mild, uncontrolled, untreated asthma showed a decrease expression of leptin and its receptor and an increased RBM thickness and TGF-beta expression when compared with values seen in healthy volunteers. Furthermore, severe asthma was associated with a reduced expression of leptin and its receptor and an increased RBM thickness with unaltered TGF-beta expression. CONCLUSIONS Decreased expression of leptin/leptin receptor characterizes severe asthma and is associated with airway remodeling features.

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients.

Background Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms. Methods The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13). Results In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter. Conclusions This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.

TLR4 upregulation underpins airway neutrophilia in smokers with chronic obstructive pulmonary disease and acute respiratory failure

Activation of Toll-like receptors (TLR) seems to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Upon TLR activation the release of defensins, including human beta defensin 2 (HBD-2), may occur. In this study, we explored the innate responses in patients with respiratory failure, with and without COPD, requiring intubation and mechanical ventilation. Mini-bronchoalveolar lavage (mini-BAL) samples were collected from nonsmoker subjects without COPD (n = 10), smokers without COPD (n = 6), and smokers with COPD (n = 15). TLR4, TLR2, and HBD-2 expression was evaluated by immunocytochemistry; interleukin (IL)-8, IP-10, and HBD-2 concentrations were evaluated by enzyme-linked immunosorbent assay; chemotactic activity toward neutrophils and lymphocytes; and cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] and by flow cytometry with anti-TLR4 and with HBD-2 depleted and not depleted mini-BAL). COPD mini-BAL showed increased neutrophil numbers, reduced neutrophil apoptosis, increased TLR4 and HBD-2 expression, increased neutrophil chemotactic activity, reduced IP-10 concentrations, and reduced lymphocyte chemotactic activity compared with those in nonsmoker subjects without COPD. In the smokers without COPD the mini-BAL showed reduced TLR4 and HBD-2 expression, higher IP-10 concentrations, and higher chemotactic activity than in patients with COPD. The blocking of TLR4 activation and HBD-2 depletion increased neutrophil apoptosis. No differences were observed for TLR2 expression and IL-8 concentrations. This study strengthens the contribution of TLR4 to promoting airway neutrophilia in COPD.

Clinical Benefits of 7 Years of Treatment with Omalizumab in Severe Uncontrolled Asthmatics

Rationale. Severe asthma is characterized by inadequate symptom control and by high rate of inflammation despite high doses of steroids. Omalizumab, a recombinant humanized monoclonal anti-IgE, provides a new therapeutic strategy in severe allergic asthma. Aims. This study was aimed to assess whether long-term treatment with omalizumab improved clinical control in severe asthmatics. Methods. We investigated omalizumab effects on asthma outcomes evaluating seven severe allergic asthmatic patients who were treated for 7 years with add-on omalizumab. Number of exacerbations, use of antibiotics, additional asthma medications (systemic steroids, nebulized steroids and bronchodilators), and spirometry were analyzed before and after omalizumab treatment. Results. Omalizumab was well tolerated by all the studied patients. It improved FEV1 and FEV1/FVC ratio and reduced symptom score, asthma exacerbations, use of antibiotics, and use of nebulized steroids, bronchodilators, and oral corticosteroids. These effects were evident after 4 years of treatment and more pronounced after 7 years of treatment. Conclusions. This study underlines the utility of a long-term treatment with omalizumab to improve asthma clinical outcomes in severe asthmatics.

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

Abstract 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 μM 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.

Variable p‐CREB expression depicts different asthma phenotypes

Background:  Chromatin modification may play a role in inflammatory gene regulation in asthma. Cyclic adenosine mono‐phosphate response element‐binding protein (CREB), with the specific co‐activator, the CREB‐binding protein (CBP), contributes to the acetylation of chromatin and to the transcription of pro‐inflammatory genes.

Fluticasone induces apoptosis in peripheral T-lymphocytes: a comparison between asthmatic and normal subjects

Apoptosis is an important mechanism allowing inflammation to be limited. Glucocorticoids are the most effective anti-inflammatory agents in asthma therapy and induce cell apoptosis. Since T-lymphocytes are critically involved in airway inflammation in asthma, the effects of fluticasone propionate (FP) on apoptosis in unstimulated and in interleukin (IL)-2 stimulated peripheral blood T-lymphocytes (PBTs) isolated from 14 normal and 19 mild-to-moderate asthmatic subjects were evaluated. Apoptosis was evaluated by: deoxyribonucleic acid (DNA) fragmentation electrophoresis, DNA content, annexin V binding, apoptosis related markers (Fas, B-cell lymphona leukaemia-2 (Bcl-2), Bax, and CD25), and by electron microscopy. FP induced apoptosis in unstimulated PBTs of normal and asthmatic subjects in a time-dependent fashion. In asthma, this effect was associated with a significant decrease of Bcl-2 expression, and with an increase of Bax/Bcl-2 ratio. In PBTs of asthmatics, FP also reduced Fas and CD25 expression. Moreover, in IL-2-stimulated PBTs from both asthmatics and normal subjects, FP was able to induce apoptosis and to reduce Bcl-2, Fas and CD25 expression, whereas negligible effects were detected on Bax expression. This study shows that the glucocorticosteroid, fluticasone, increases apoptosis and modulates expression of apoptosis-related markers in unstimulated and in interleukin-2 stimulated T-lymphocytes. This points towards a potential mechanism by which fluticasone exerts its anti-inflammatory effects.

Increased expression of IL‐19 in the epithelium of patients with chronic rhinosinusitis and nasal polyps

Chronic rhinosinusitis (CRS) is an inflammation of the nose and of the paranasal sinuses. The involvement of the respiratory epithelium in the mechanisms of CRS is poorly understood.

Effect of indomethacin on the kinetics of tumour necrosis factor alpha release and tumour necrosis factor alpha gene expression by human blood monocytes

In this investigation we have examined the effects of indomethacin, an inhibitor of the cyclooxygenase pathway of arachidonic acid, upon the kinetics of the release of tumour necrosis factor alpha (TNF) and of the expression of TNF gene by lipopolysaccharide (LPS)-stimulated human blood monocytes (BM). Following stimulation of BM with LPS, TNF was released within 2 h, reached peak values at 8 h and declined at subsequent time-points (24 and 48 h). Indomethacin (10(-5) M) slightly stimulated the production of TNF at 2, 4, and 8 h and prevented the decline of TNF observed at 24 and 48 h. This effect was related to the persistence of TNF synthesis, as demonstrated by kinetic evaluation of the expression of TNF gene performed by dot-blot analysis. The effects of indomethacin on TNF release and TNF gene expression were due to the inhibition of endogenous prostaglandin (PG)E2 production. In the absence of indomethacin, PGE2 release by the LPS-stimulated BM began concomitantly with the decline of TNF production by the same cells under the same stimulus. Indomethacin completely blocked PGE2 release at any time-point. Exogenous PGE2 suppressed the release of TNF and the expression of TNF gene in a dose-dependent fashion. Exogenous PGE2 completely reversed the effects of indomethacin on TNF production at 24 h. These findings suggest that indomethacin may significantly alter the kinetics of TNF release and TNF gene expression by LPS-stimulated BM. These effects are related, at least in part, to the inhibition of the production of endogenous PGE2, an important self-driven regulatory factor of the kinetics of TNF production.