Eleni Litsiou

CXCL13 production in B cells via Toll-like receptor/lymphotoxin receptor signaling is involved in lymphoid neogenesis in chronic obstructive pulmonary disease.

RATIONALE Little is known about what drives the appearance of lymphoid follicles (LFs), which may function as lymphoid organs in chronic obstructive pulmonary disease (COPD). In animal infection models, pulmonary LF formation requires expression of homeostatic chemokines by stromal cells and dendritic cells, partly via lymphotoxin. OBJECTIVES To study the role of homeostatic chemokines in LF formation in COPD and to identify mechanism(s) responsible for their production. METHODS Peripheral lung homeostatic chemokine and lymphotoxin expression were visualized by immunostainings and quantified by ELISA/quantitative reverse transcriptase-polymerase chain reaction in patients with COPD with and without LFs. Expression of lymphotoxin and homeostatic chemokine receptors was investigated by flow cytometry. Primary lung cell cultures, followed by ELISA/quantitative reverse transcriptase-polymerase chain reaction/flow cytometry, were performed to identify mechanisms of chemokine expression. Polycarbonate membrane filters were used to assess primary lung cell migration toward lung homogenates. MEASUREMENTS AND MAIN RESULTS LFs expressed the homeostatic chemokine CXCL13. Total CXCL13 levels correlated with LF density. Lung B cells of patients with COPD were important sources of CXCL13 and lymphotoxin and also expressed their receptors. Cigarette smoke extract, H2O2, and LPS exposure up-regulated B cell-derived CXCL13. The LPS-induced increase in CXCL13 was partly mediated via lymphotoxin. Notably, CXCL13 was required for efficient lung B-cell migration toward COPD lung homogenates and induced lung B cells to up-regulate lymphotoxin, which further promoted CXCL13 production, establishing a positive feedback loop. CONCLUSIONS LF formation in COPD may be driven by lung B cells via a CXCL13-dependent mechanism that involves toll-like receptor and lymphotoxin receptor signaling.

Inspiratory resistive breathing induces MMP-9 and MMP-12 expression in the lung

Inspiratory resistive breathing (IRB) is characterized by large negative intrathoracic pressures and was shown to induce pulmonary inflammation in previously healthy rats. Matrix metalloproteinases (MMP)-9 and -12 are induced by inflammation and mechanical stress in the lung. We hypothesized that IRB induces MMP-9 and -12 in the lung. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve, connected to an inspiratory resistance, with the tidal inspiratory tracheal pressure set at 50% of the maximum. Quietly breathing animals served as controls. After 3 and 6 h of IRB, respiratory mechanics were measured, bronchoalveolar lavage (BAL) was performed, lung injury score was estimated, and lung MMP-9 was estimated by zymography and ELISA. MMP-9 and MMP-12 immunohistochemistry was performed. Isolated normal alveolar macrophages were incubated with BAL from rats that underwent IRB. After 18 h, MMP-9 and -12 levels were measured in supernatants, and immunocytochemistry was performed. Macrophages were treated with IL-1β, IL-6, or TNF-α, and MMP-9 in supernatants was measured. After 6 h of IRB, leukocytes in BAL increased, and IL-1β and IL-6 levels were elevated. Elasticity and injury score were increased after 3 and 6 h of IRB. Lung MMP-9 levels increased after 6 h of IRB. MMP-9 and MMP-12 were detected in alveolar macrophages and epithelial (bronchial/alveolar) cells after 3 and 6 h of IRB. MMP-9 and MMP-12 were found in supernatants after treatment with 6 h of IRB BAL. Cytosolic immunostaining was detected after treatment with 3 and 6 h of IRB BAL. All cytokines induced MMP-9 in culture supernatants. In conclusion, IRB induces MMP-9 and -12 in the lung of previously healthy rats.

Tiotropium bromide exerts anti-inflammatory effects during resistive breathing, an experimental model of severe airway obstruction

Introduction Resistive breathing (RB), a hallmark of obstructive airway diseases, is characterized by strenuous contractions of the inspiratory muscles that impose increased mechanical stress on the lung. RB is shown to induce pulmonary inflammation in previous healthy animals. Tiotropium bromide, an anticholinergic bronchodilator, is also shown to exert anti-inflammatory effects. The effect of tiotropium on RB-induced pulmonary inflammation is unknown. Methods Adult rats were anesthetized, tracheostomized and breathed spontaneously through a two-way non-rebreathing valve. Resistances were connected to the inspiratory and/or expiratory port, to produce inspiratory resistive breathing (IRB) of 40% or 50% Pi/Pi,max (40% and 50% IRB), expiratory resistive breathing (ERB) of 60% Pe/Pe,max (60% ERB) or combined resistive breathing (CRB) of both 40% Pi/Pi,max and 60% Pe/Pe,max (40%/60% CRB). Tiotropium aerosol was inhaled prior to RB. After 6 h of RB, mechanical parameters of the respiratory system were measured and bronchoalveolar lavage (BAL) was performed. IL-1β and IL-6 protein levels were measured in lung tissue. Lung injury was estimated histologically. Results In all, 40% and 50% IRB increased macrophage and neutrophil counts in BAL and raised IL-1β and IL-6 lung levels, tissue elasticity, BAL total protein levels and lung injury score. Tiotropium attenuated BAL neutrophil number, IL-1β, IL-6 levels and lung injury score increase at both 40% and 50% IRB. The increase in macrophage count and protein in BAL was only reversed at 40% IRB, while tissue elasticity was not affected. In all, 60% ERB raised BAL neutrophil count and total protein and reduced macrophage count. IL-1β and IL-6 levels and lung injury score were increased. Tiotropium attenuated these alterations, except for the decrease in macrophage count and the increase in total protein level. In all, 40%/60% CRB increased macrophage and neutrophil count in BAL, IL-1β and IL-6 levels, tissue elasticity, total protein in BAL and histological injury score. Tiotropium attenuated the aforementioned alterations. Conclusion Tiotropium inhalation attenuates RB-induced pulmonary inflammation.

The differential effects of inspiratory, expiratory, and combined resistive breathing on healthy lung

Combined resistive breathing (CRB) is the hallmark of obstructive airway disease pathophysiology. We have previously shown that severe inspiratory resistive breathing (IRB) induces acute lung injury in healthy rats. The role of expiratory resistance is unknown. The possibility of a load-dependent type of resistive breathing-induced lung injury also remains elusive. Our aim was to investigate the differential effects of IRB, expiratory resistive breathing (ERB), and CRB on healthy rat lung and establish the lowest loads required to induce injury. Anesthetized tracheostomized rats breathed through a two-way valve. Varying resistances were connected to the inspiratory, expiratory, or both ports, so that the peak inspiratory pressure (IRB) was 20%–40% or peak expiratory (ERB) was 40%–70% of maximum. CRB was assessed in inspiratory/expiratory pressures of 30%/50%, 40%/50%, and 40%/60% of maximum. Quietly breathing animals served as controls. At 6 hours, respiratory system mechanics were measured, and bronchoalveolar lavage was performed for measurement of cell and protein concentration. Lung tissue interleukin-6 and interleukin-1β levels were estimated, and a lung injury histological score was determined. ERB produced significant, load-independent neutrophilia, without mechanical or permeability derangements. IRB 30% was the lowest inspiratory load that provoked lung injury. CRB increased tissue elasticity, bronchoalveolar lavage total cell, macrophage and neutrophil counts, protein and cytokine levels, and lung injury score in a dose-dependent manner. In conclusion, CRB load dependently deranges mechanics, increases permeability, and induces inflammation in healthy rats. ERB is a putative inflammatory stimulus for the lung.

Physical characterization of the aerosol of an electronic cigarette: impact of refill liquids.

Abstract Electronic cigarettes are used to evaporate a mixture of solvents, nicotine and flavors. Liquid particles can be generated under these conditions due to evaporation/condensation. The objective of this work is to measure the physical characteristics of the aerosol emission of an e-cigarette using different refill liquids. The aerosol particle number and size are determined with a Scanning Mobility Particle Sizer. Seven liquids are used: propylene glycol (PG), glycerol (VG), a mixture 1:1 of PG/VG, the mixture with 2% or 5% of a commercial flavor, the mixture with 1.2% of nicotine and the mixture with 1.2% of nicotine and 2% of flavor. Particle concentrations of the aerosol emitted from the electronic cigarette are 300–3000 times higher than that of the ambient air. Propylene glycol emits several times more than glycerol. The addition of a flavor or nicotine has little effect on the emission of the total number emitted. The count median diameter of the electronic cigarette particles is 200–400 nm, depending on the liquid used. Count median diameter of emitted particles is affected by the liquid used.

Success rates are correlated mainly to completion of a smoking cessation program

Smoking cessation without professional help is achieved only in 3-5%. Smoking cessation clinics that combine behavioral and pharmaceutical support increase abstinence rates after 6 months from 35% to 55%, depending also from the health professional9s experience. In our smoking cessation clinic (SCC) in Evaggelismos hospital we enrolled 250 smokers the last 20 months. These smokers received behavioral support in groups of 5-6, pharmaceutical treatment (varenicline or NRTs) and educational material as is the usual practice of the clinic. The smokers are given 5 weekly appointments that last 1 hour each. Motivation and Confidence to quit were measured using a visual Analogue Scale (VAS) from 0-10. 0 corresponds to none motivation and self-confidence. Fangestrom Nicotine Dependence Score was also measured. The profile of our smokers was highly addicted (FNDT≥8), motivated (VASm: 6) but without self-confidence (VASsc: 3) or known health problems related to smoking (70%). 75% used varenicline, 10% used NRTs and 15% did not accept or tolerate pharmaceutical treatment. Our success rate at 3m was 58% and 45% at 1year. What is interesting is that the vast percentage (85, 7%) of smokers that failed to quit were the ones that were unable to follow the program (36% of total) mainly (85%) because the morning appointments conflicted their work. We conclude that a SC program that combines intense psychological support and pharmaceutical treatment leads to high SC rates in 1y. It appears that adherence to the program is of the most important factors that relate with success rates. We propose that either evening SCC should be available or abstinence from work due to participation to a SCC program should be justified.

The importance of early COPD diagnosis during a smoking cessation program

Background Many patients with mild–moderate COPD (chronic obstructive pulmonary disease), are asymptomatic. Since expressed symptoms are usually mild and mostly attributed to age, they are often underestimated [1,2]. Thus early COPD patients usually remain undiagnosed [3]. The aim of the study is to evaluate the rates of undiagnosed COPD cases in early stages of the disease (stage I and II according to GOLD classification), [4] in our smoking cessation program and to assess the effectiveness of COPD diagnosis as a motivational tool for quitting smoking.

Smoking cessation changes basic metabolism, body weight, leptin and insulin levels, adipose tissue percentage, index of insulin resistance and index of insulin secretion

Background It is known that nicotine increases energy expenditure, basic metabolism and has appetite-suppressing properties, which likely explains why smokers tend to have lower body weight than non-smokers and why smoking cessation is followed by weight gain up to 12 kgr (7 kgr in median). Thus smoking cessation often has the disadvantage of increasing the risk of metabolic syndrome; fact that both decreases the benefits of smoking cessation and can discourage smokers (mostly women) from quitting. On the top of that, weight gain has been found to increase the risk of relapse, particularly among normalweight or underweight women who report chronic dieting, but also among men. Varenicline is a nicotiniccholinergic receptor partial agonist and therefore should share nicotine’s properties. Thus we believe that during its intake for three months in the process of smoking cessation basic metabolism will be sustained in levels comparable to these while smoking. During this period ex-smokers are also protected from withdrawal syndromes and thus capable of following psychological and physical activity counselling in order to sustain stable body weight. Quitting smoking is a process of changing life habits and is extremely difficult due to the addictive properties of nicotine. Post cessation weight gain decreases the benefits and leads to increased relaption rates. Varenicline improves succession and decrease relaption rates. Materials and methods During the three months period of varenicline intake, we enrolled 15 smokers that visited our smoking cessation clinic and succeeded in quitting smoking. Eight used varenicline. Basic metabolism and body weight were measured before (BSC) and one month after smoking cessation (ASC).

Analysis of acceptance of the smoking ban in Greece.

Background Exposure to secondhand smoke, according to the World Health Organization is responsible for 603,000 deaths in 2004. The ban on smoking in public places in Greece was legislated but not implemented, as the adequate and effective measures were not taken. This study aims to determine the degree of acceptance of the last anti-smoking law and the parameters associated with the acceptance or not of the smoking ban.