Role of accelerated aging in limb muscle wasting of patients with COPD.

Purpose: Skeletal muscle wasting is an independent predictor of health-related quality of life and survival in patients with COPD, but the complexity of molecular mechanisms associated with this process has not been fully elucidated. We aimed to determine whether an impaired ability to repair DNA damage contributes to muscle wasting and the accelerated aging phenotype in patients with COPD. Patients and methods: The levels of phosphorylated H2AX (γH2AX), a molecule that promotes DNA repair, were assessed in vastus lateralis biopsies from 10 COPD patients with low fat-free mass index (FFMI; COPDL), 10 with preserved FFMI and 10 age- and gender-matched healthy controls. A panel of selected markers for cellular aging processes (CDKN2A/p16ink4a, SIRT1, SIRT6, and telomere length) were also assessed. Markers of oxidative stress and cell damage and a panel of pro-inflammatory and anti-inflammatory cytokines were evaluated. Markers of muscle regeneration and apoptosis were also measured. Results: We observed a decrease in γH2AX expression in COPDL, which occurred in association with a tendency to increase in CDKN2A/p16ink4a, and a significant decrease in SIRT1 and SIRT6 protein levels. Cellular damage and muscle inflammatory markers were also increased in COPDL. Conclusion: These data are in keeping with an accelerated aging phenotype as a result of impaired DNA repair and dysregulation of cellular homeostasis in the muscle of COPDL. These data indicate cellular degeneration via stress-induced premature senescence and associated inflammatory responses abetted by the senescence-associated secretory phenotype and reflect an increased expression of markers of oxidative stress and inflammation.

2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with several extra-pulmonary effects of which skeletal muscle wasting is one of the most common and contributes to reduced quality of life, increased morbidity and mortality. The molecular mechanisms leading to muscle wasting are not fully understood. Proteomic analysis of human skeletal muscle is a useful approach for gaining insight into the molecular basis for normal and pathophysiological conditions. METHODS: To identify proteins involved in the process of muscle wasting in COPD, we searched differentially expressed proteins in the vastus lateralis of COPD patients with low fat free mass index (FFMI), as a surrogate of muscle mass (COPDL, n = 10) (FEV1 33 ± 4.3% predicted, FFMI 15 ± 0.2 Kg.m-2), in comparison to patients with COPD and normal FFMI (COPDN, n = 8) and a group of age, smoking history, and sex matched healthy controls (C, n = 9) using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, combined with mass spectrometry (MS). The effect of silencing DOT1L protein expression on markers of cell arrest was analyzed in skeletal muscle satellite cells (HSkMSCs) in vitro and assessed by qPCR and Western blotting. RESULTS: A subset of 7 proteins was differentially expressed in COPDL compared to both COPDN and C. We found an increased expression of proteins associated with muscle homeostasis and protection against oxidative stress, and a decreased expression of structural muscle proteins and proteins involved in myofibrillogenesis, cell proliferation, cell cycle arrest and energy production. Among these was a decreased expression of the histone methyltransferase DOT1L. In addition, silencing of the DOT1L gene in human skeletal muscle satellite cells in vitro was significantly related to up regulation of p21 WAF1/Cip1/CDKN1A, a marker of cell arrest and ageing. CONCLUSIONS: 2D-DIGE coupled with MS identified differences in the expression of several proteins in the wasted vastus lateralis that are relevant to the disease process. Down regulation of DOT1L in the vastus lateralis of COPDL patients may mediate the muscle wasting process through up regulation of markers of cell arrest and senescence.

Home monitoring of breathing rate in people with chronic obstructive pulmonary disease: observational study of feasibility, acceptability, and change after exacerbation.

Telehealth programs to promote early identification and timely self-management of acute exacerbations of chronic obstructive pulmonary diseases (AECOPDs) have yielded disappointing results, in part, because parameters monitored (symptoms, pulse oximetry, and spirometry) are weak predictors of exacerbations. PURPOSE: Breathing rate (BR) rises during AECOPD and may be a promising predictor. Devices suitable for home use to measure BR have recently become available, but their accuracy, acceptability, and ability to detect changes in people with COPD is not known. PATIENTS AND METHODS: We compared five BR monitors, which used different monitoring technologies, with a gold standard (Oxycon Mobile®; CareFusion®, a subsidiary of Becton Dickinson, San Diego, CA, USA). The monitors were validated in 21 stable COPD patients during a 57-min "activities of daily living protocol" in a laboratory setting. The two best performing monitors were then tested in a 14-day trial in a home setting in 23 stable COPD patients to determine patient acceptability and reliability of signal. Acceptability was explored in qualitative interviews. The better performing monitor was then given to 18 patients recruited during an AECOPD who wore the monitor to observe BR during the recovery phase of an AECOPD. RESULTS: While two monitors demonstrated acceptable accuracy compared with the gold standard, some participants found them intrusive particularly when ill with an exacerbation, limiting their potential utility in acute situations. A reduction in resting BR during the recovery from an AECOPD was observed in some, but not in all participants and there was considerable day-to-day individual variation. CONCLUSION: Resting BR shows some promise in identifying exacerbations; however, further prospective study to assess this is required.

Application of Mixed Effects Limits of Agreement in the Presence of Multiple Sources of Variability: Exemplar from the Comparison of Several Devices to Measure Respiratory Rate in COPD Patients.

INTRODUCTION: The Bland-Altman limits of agreement method is widely used to assess how well the measurements produced by two raters, devices or systems agree with each other. However, mixed effects versions of the method which take into account multiple sources of variability are less well described in the literature. We address the practical challenges of applying mixed effects limits of agreement to the comparison of several devices to measure respiratory rate in patients with chronic obstructive pulmonary disease (COPD). METHODS: Respiratory rate was measured in 21 people with a range of severity of COPD. Participants were asked to perform eleven different activities representative of daily life during a laboratory-based standardised protocol of 57 minutes. A mixed effects limits of agreement method was used to assess the agreement of five commercially available monitors (Camera, Photoplethysmography (PPG), Impedance, Accelerometer, and Chest-band) with the current gold standard device for measuring respiratory rate. RESULTS: Results produced using mixed effects limits of agreement were compared to results from a fixed effects method based on analysis of variance (ANOVA) and were found to be similar. The Accelerometer and Chest-band devices produced the narrowest limits of agreement (-8.63 to 4.27 and -9.99 to 6.80 respectively) with mean bias -2.18 and -1.60 breaths per minute. These devices also had the lowest within-participant and overall standard deviations (3.23 and 3.29 for Accelerometer and 4.17 and 4.28 for Chest-band respectively). CONCLUSIONS: The mixed effects limits of agreement analysis enabled us to answer the question of which devices showed the strongest agreement with the gold standard device with respect to measuring respiratory rates. In particular, the estimated within-participant and overall standard deviations of the differences, which are easily obtainable from the mixed effects model results, gave a clear indication that the Accelerometer and Chest-band devices performed best.

Circulating desmosine levels do not predict emphysema progression but are associated with cardiovascular risk and mortality in COPD.

Elastin degradation is a key feature of emphysema and may have a role in the pathogenesis of atherosclerosis associated with chronic obstructive pulmonary disease (COPD). Circulating desmosine is a specific biomarker of elastin degradation. We investigated the association between plasma desmosine (pDES) and emphysema severity/progression, coronary artery calcium score (CACS) and mortality.pDES was measured in 1177 COPD patients and 110 healthy control subjects from two independent cohorts. Emphysema was assessed on chest computed tomography scans. Aortic arterial stiffness was measured as the aortic-femoral pulse wave velocity.pDES was elevated in patients with cardiovascular disease (p<0.005) and correlated with age (rho=0.39, p<0.0005), CACS (rho=0.19, p<0.0005) modified Medical Research Council dyspnoea score (rho=0.15, p<0.0005), 6-min walking distance (rho=-0.17, p<0.0005) and body mass index, airflow obstruction, dyspnoea, exercise capacity index (rho=0.10, p<0.01), but not with emphysema, emphysema progression or forced expiratory volume in 1 s decline. pDES predicted all-cause mortality independently of several confounding factors (p<0.005). In an independent cohort of 186 patients with COPD and 110 control subjects, pDES levels were higher in COPD patients with cardiovascular disease and correlated with arterial stiffness (p<0.05).In COPD, excess elastin degradation relates to cardiovascular comorbidities, atherosclerosis, arterial stiffness, systemic inflammation and mortality, but not to emphysema or emphysema progression. pDES is a good biomarker of cardiovascular risk and mortality in COPD.

Genome-wide mRNA expression profiling in vastus lateralis of COPD patients with low and normal fat free mass index and healthy controls.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) has significant systemic effects beyond the lungs amongst which muscle wasting is a prominent contributor to exercise limitation and an independent predictor of morbidity and mortality. The molecular mechanisms leading to skeletal muscle dysfunction/wasting are not fully understood and are likely to be multi-factorial. The need to develop therapeutic strategies aimed at improving skeletal muscle dysfunction/wasting requires a better understanding of the molecular mechanisms responsible for these abnormalities. Microarrays are powerful tools that allow the investigation of the expression of thousands of genes, virtually the whole genome, simultaneously. We aim at identifying genes and molecular pathways involved in skeletal muscle wasting in COPD. METHODS: We assessed and compared the vastus lateralis transcriptome of COPD patients with low fat free mass index (FFMI) as a surrogate of muscle mass (COPDL) (FEV1 30 ± 3.6%pred, FFMI 15 ± 0.2 Kg.m(-2)) with patients with COPD and normal FFMI (COPDN) (FEV1 44 ± 5.8%pred, FFMI 19 ± 0.5 Kg.m(-2)) and a group of age and sex matched healthy controls (C) (FEV1 95 ± 3.9%pred, FFMI 20 ± 0.8 Kg.m(-2)) using Agilent Human Whole Genome 4x44K microarrays. The altered expression of several of these genes was confirmed by real time TaqMan PCR. Protein levels of P21 were assessed by immunoblotting. RESULTS: A subset of 42 genes was differentially expressed in COPDL in comparison to both COPDN and C (PFP < 0.05; -1.5 ≥ FC ≥ 1.5). The altered expression of several of these genes was confirmed by real time TaqMan PCR and correlated with different functional and structural muscle parameters. Five of these genes (CDKN1A, GADD45A, PMP22, BEX2, CGREF1, CYR61), were associated with cell cycle arrest and growth regulation and had been previously identified in studies relating muscle wasting and ageing. Protein levels of CDKN1A, a recognized marker of premature ageing/cell cycle arrest, were also found to be increased in COPDL. CONCLUSIONS: This study provides evidence of differentially expressed genes in peripheral muscle in COPD patients corresponding to relevant biological processes associated with skeletal muscle wasting and provides potential targets for future therapeutic interventions to prevent loss of muscle function and mass in COPD.

The PROactive innovative conceptual framework on physical activity.

Although physical activity is considered an important therapeutic target in chronic obstructive pulmonary disease (COPD), what "physical activity" means to COPD patients and how their perspective is best measured is poorly understood. We designed a conceptual framework, guiding the development and content validation of two patient reported outcome (PRO) instruments on physical activity (PROactive PRO instruments). 116 patients from four European countries with diverse demographics and COPD phenotypes participated in three consecutive qualitative studies (63% male, age mean±sd 66±9 years, 35% Global Initiative for Chronic Obstructive Lung Disease stage III-IV). 23 interviews and eight focus groups (n = 54) identified the main themes and candidate items of the framework. 39 cognitive debriefings allowed the clarity of the items and instructions to be optimised. Three themes emerged, i.e. impact of COPD on amount of physical activity, symptoms experienced during physical activity, and adaptations made to facilitate physical activity. The themes were similar irrespective of country, demographic or disease characteristics. Iterative rounds of appraisal and refinement of candidate items resulted in 30 items with a daily recall period and 34 items with a 7-day recall period. For the first time, our approach provides comprehensive insight on physical activity from the COPD patients' perspective. The PROactive PRO instruments' content validity represents the pivotal basis for empirically based item reduction and validation.

Stable and temperature-responsive surfactant-free foamulsions with high oil-volume fraction.

There is more than foam: Optical microscopy images (with false colouring, see picture) depicting quick destabilization (within minutes) of foamulsions due to the coalescence of densely-packed oil droplets when heated at higher temperatures (65 °C).

Neuromuscular electrical stimulation prevents muscle function deterioration in exacerbated COPD: a pilot study.

PURPOSE: COPD is a condition with systemic effects of which peripheral muscle dysfunction is a prominent contributor to exercise limitation, health related quality of life (HRQoL) impairment, and is an independent predictor of morbidity and mortality. Pulmonary rehabilitation (PR) is a successful strategy to improve exercise tolerance and HRQoL through the improvement of muscle function in patients with stable COPD or early after severe exacerbations of COPD (SECOPD). However, muscle function further deteriorates during SECOPD before early PR programmes commence. We aimed to investigate the feasibility and efficacy of quadriceps neuromuscular electrical stimulation (NMES) applied during a SECOPD to prevent muscle function deterioration. METHODS: We have conducted a pilot study in eleven COPD patients (FEV(1) 41.3 ± 5.6 % pred) admitted to hospital with a SECOPD. We randomly allocated one leg to receive NMES (once a day for 14 days) with the other leg as a control (non-stimulated leg). We measured the change in quadriceps maximal voluntary contraction (ΔQMVC) as the main outcome. RESULTS: Mean quadriceps muscle strength decreased in control legs (ΔQMVC -2.9 ± 5.3 N, p = ns) but increased in the stimulated legs (ΔQMVC 19.2 ± 6.1 N, p < 0.01). The difference in ΔQMVC between groups was statistically significant (p < 0.05). The effect of NMES was directly related to the stimulation intensity (∑mA) applied throughout the 14 sessions (r = 0.76, p < 0.01). All patients tolerated NMES without any side effects. CONCLUSIONS: NMES is a feasible and effective treatment to prevent quadriceps muscle strength derangement during severe exacerbations of COPD and may be used to compliment early post-exacerbation pulmonary rehabilitation.

Straightforward preparation of organic colloidal particles by harnessing spontaneous non-covalent interactions of active molecules from natural origin.

We demonstrate a straightforward method to prepare organic colloidal particles based on the spontaneous molecular interactions between small molecular weight actives of natural origin. Representative reactive natural actives from three of the most researched classes of phytochemicals including berberine (isoquinoline alkaloid), tannic acid (polyphenol) and glycyrrhizin (olenane type saponin) were chosen for the study. Binding parameters (association constant, binding enthalpy and entropy) obtained from isothermal titration calorimetry indicated that berberine strongly interacted with tannic acid to form insoluble colloidal complex which could be stabilised in the presence of glycyrrhizin (due to its interaction with both berberine and tannic acid and also due to its amphiphilic nature). Working on this principle, the mutual interactions of these three natural actives were exploited to obtain stable spherical particles with a mean diameter of less than 100 nm (77 nm) simply by mixing the aqueous solutions of berberine:tannic acid:glycyrrhizin at molar ratio of 2:1:1. The involvement of aromatic chromophore (π-π*) system and charged N atom of berberine in the spontaneous interaction between berberine and tannic acid was confirmed from spectral analysis. X-ray diffraction study suggested formation of amorphous organic colloidal particles, and the spherical shape of colloidal particles was confirmed by transmission electron microscopy.

Quercetin loaded biopolymeric colloidal particles prepared by simultaneous precipitation of quercetin with hydrophobic protein in aqueous medium.

Quercetin loaded biopolymeric colloidal particles were prepared by precipitating quercetin (water insoluble polyphenol) and zein (hydrophobic protein), simultaneously, by adding their hydro-alcoholic solution to aqueous solution in presence of sodium caseinate as an electrosteric stabiliser. The presence of protein resulted in altering the shape of quercetin precipitates from needle-like to spherical shape at higher zein proportions, as confirmed by transmission electron microscopy. The average particle size of zein:quercetin composite particles was below 200 nm (130-161 nm) with negative surface charge (-30 to -41 mV), as confirmed by dynamic light scattering and electrophoretic mobility data. Solid state characterisation (X-ray diffraction) and spectroscopic measurements (UV-Vis and IR spectroscopy) confirmed characteristic changes in quercetin due to the entrapment in the biopolymeric matrix of colloidal particles. Results from anti-oxidant study demonstrated the advantage of entrapping quercetin in the colloidal particles in terms of the chemical stability in the alkaline pH and against photodegradation under UV-light irradiation.

Cytotoxicity and induction of inflammation by pepsin in Acid in bronchial epithelial cells.

Introduction. Gastroesophageal reflux has been associated with chronic inflammatory diseases and may be a cause of airway remodelling. Aspiration of gastric fluids may cause damage to airway epithelial cells, not only because acidity is toxic to bronchial epithelial cells, but also since it contains digestive enzymes, such as pepsin. Aim. To study whether pepsin enhances cytotoxicity and inflammation in airway epithelial cells, and whether this is pH-dependent. Methods. Human bronchial epithelial cells were exposed to increasing pepsin concentrations in varying acidic milieus, and cell proliferation and cytokine release were assessed. Results. Cell survival was decreased by pepsin exposure depending on its concentration (F = 17.4) and pH level of the medium (F = 6.5) (both P < 0.01). Pepsin-induced interleukin-8 release was greater at lower pH (F = 5.1; P < 0.01). Interleukin-6 induction by pepsin was greater at pH 1.5 compared to pH 2.5 (mean difference 434%; P = 0.03). Conclusion. Pepsin is cytotoxic to bronchial epithelial cells and induces inflammation in addition to acid alone, dependent on the level of acidity. Future studies should assess whether chronic aspiration causes airway remodelling in chronic inflammatory lung diseases.

Vascular dysfunction in chronic obstructive pulmonary disease.

RATIONALE: Cardiovascular disease is a major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), which may in part be attributable to abnormalities of systemic vascular function. It is unclear whether such associations relate to the presence of COPD or prior smoking habit. OBJECTIVES: To undertake a comprehensive assessment of vascular function in patients with COPD and healthy control subjects matched for smoking history. METHODS: Eighteen men with COPD were compared with 17 healthy male control subjects matched for age and lifetime cigarette smoke exposure. Participants were free from clinically evident cardiovascular disease. MEASUREMENTS AND MAIN RESULTS: Pulse wave velocity and pulse wave analysis were measured via applanation tonometry at carotid, radial, and femoral arteries. Blood flow was measured in both forearms using venous occlusion plethysmography during intrabrachial infusion of endothelium-dependent vasodilators (bradykinin, 100-1,000 pmol/min; acetylcholine, 5-20 microg/min) and endothelium-independent vasodilators (sodium nitroprusside, 2-8 microg/min; verapamil, 10-100 microg/min). Tissue plasminogen activator (t-PA) was measured in venous plasma before and during bradykinin infusions. Patients with COPD have greater arterial stiffness (pulse wave velocity, 11 +/- 2 vs. 9 +/- 2 m/s; P = 0.003; augmentation index, 27 +/- 10 vs. 21 +/- 6%; P = 0.028), but there were no differences in endothelium-dependent and -independent vasomotor function or bradykinin-induced endothelial t-PA release (P > 0.05 for all). CONCLUSIONS: COPD is associated with increased arterial stiffness independent of cigarette smoke exposure. However, this abnormality is not explained by systemic endothelial dysfunction. Increased arterial stiffness may represent the mechanistic link between COPD and the increased risk for cardiovascular disease associated with this condition.

Efficacy of simple short-term in vitro assays for predicting the potential of metal oxide nanoparticles to cause pulmonary inflammation.

BACKGROUND: There has been concern regarding risks from inhalation exposure to nanoparticles (NPs). The large number of particles requiring testing means that alternative approaches to animal testing are needed. OBJECTIVES: We set out to determine whether short-term in vitro assays that assess intrinsic oxidative stress potential and membrane-damaging potency of a panel of metal oxide NPs can be used to predict their inflammogenic potency. METHODS: For a panel of metal oxide NPs, we investigated intrinsic free radical generation, oxidative activity in an extracellular environment, cytotoxicity to lung epithelial cells, hemolysis, and inflammation potency in rat lungs. All exposures were carried out at equal surface area doses. RESULTS: Only nickel oxide (NiO) and alumina 2 caused significant lung inflammation when instilled into rat lungs at equal surface area, suggesting that these two had extra surface reactivity. We observed significant free radical generation with 4 of 13 metal oxides, only one of which was inflammogenic. Only 3 of 13 were significantly hemolytic, two of which were inflammogenic. CONCLUSIONS: Potency in generating free radicals in vitro did not predict inflammation, whereas alumina 2 had no free radical activity but was inflammogenic. The hemolysis assay was correct in predicting the proinflammatory potential of 12 of 13 of the particles examined. Using a battery of simple in vitro tests, it is possible to predict the inflammogenicity of metal oxide NPs, although some false-positive results are likely. More research using a larger panel is needed to confirm the efficacy and generality of this approach for metal oxide NPs.

Inflammatory lung secretions inhibit dendritic cell maturation and function via neutrophil elastase.

RATIONALE: Continuous episodes of infection are a feature of lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Lung antigen-presenting dendritic cells (DCs) sample inhaled antigen to initiate immune responses. Therefore, we hypothesized that inflammatory mediators, such as neutrophil elastase (NE) released into the lung, may be able to modulate their activity. OBJECTIVE: To determine whether sputum (from patients with COPD and those with CF) or NE can alter DC phenotype and function. METHOD: NE and sputum samples were incubated with immature or mature murine DCs (mDCs). DC phenotype and function were studied by fluorescence-activated cell sorter and Western Blot analysis, assessing their expression of costimulatory molecules and their ability to induce T cell proliferation. RESULTS: COPD/CF sputum samples and human NE downregulated the expression of CD40, CD80, and CD86 (but not major histocompatibility complex II) on DCs and inhibited LPS-induced DC maturation. This effect was partially (sputa) to significantly (NE) reversed by addition of recombinant secretory leukocyte protease inhibitor. Western Blot analysis showed that purified NE degraded CD86 in mDC lysates in a time- and dose-dependent fashion, and caused shedding of CD86 into the supernatants of mDC cultures. NE treatment also inhibited the antigen-presenting ability of mDCs, as measured by their ability to induce ovalbumin-specific D011.10-transgenic T-cell proliferation. CONCLUSIONS: Our data indicate that NE in lung inflammatory secretions of patients with COPD/CF may disable DCs and prevent them from mounting an adequate immune response. This may have implications for the infection-driven generation of disease exacerbations in these two pathologies.

The effect of smoking on the transcriptional regulation of lung inflammation in patients with chronic obstructive pulmonary disease.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is believed to result from an abnormal inflammatory response in the lungs to noxious particles and gases usually found in cigarette smoke. OBJECTIVES: In this study, the molecular mechanisms for the enhanced proinflammatory cytokine gene transcription in COPD were investigated. METHODS: Lung tissue was examined from 56 subjects undergoing resection for peripheral lung tumors as follows: current smokers with (n = 14) and without COPD (n = 17), ex-smokers with COPD (n = 13), and nonsmokers (n = 12). The levels of inhibitor kappaB-alpha (IkappaB-alpha), histone deacetylase 2 (HDAC2), acetylated (ac-) histone H3 and H4, the transcription factor nuclear factor-kappaB (NF-kappaB), proinflammatory cytokine messenger RNA, and 8-isoprostane were measured. MEASUREMENTS AND MAIN RESULTS: IkappaB-alpha levels were significantly decreased in healthy smokers and current and ex-smoking patients with COPD when compared with nonsmokers (p < 0.001), with an associated increase in NF-kappaB DNA binding in current smokers (p < 0.05). An increase in acetylated histone 4 (ac-H4; p < 0.01) was found in current smokers. Conversely, ex-smokers with COPD showed an increase in ac-H3 (p < 0.05). Decreased levels of cytoplasmic, but not nuclear, HDAC2 protein levels were detected. From the cytokine profiles, no significant differences were detected; however, interleukin-12p40 expression correlated with ac-H4 in current smokers with COPD (p < 0.01). CONCLUSION: These data propose a role for modification of nucleosomal structure in inflammatory cytokine gene transcription in response to smoking. The imbalance between histone deacetylation and acetylation in favor of acetylation may contribute to the enhanced inflammation in smokers susceptible to the development of COPD.

Regulation of LPS-mediated inflammation in vivo and in vitro by the thiol antioxidant Nacystelyn.

Increased levels of proinflammatory cytokines are present in bronchoalveolar lavage fluid in various lung diseases. Redox-sensitive transcription factors such as NF-kappaB regulate gene transcription for these cytokines. We therefore studied the effect of a new thiol antioxidant compound, Nacystelyn (NAL), on IL-8 regulation in a human macrophage-derived cell line (THP-1). LPS (10 microg/ml) increased IL-8 release compared with control levels. This LPS activation was inhibited by coincubation with NAL (1 and 5 mM). Pretreatment with cycloheximide or okadaic acid, protein synthesis, and serine/threonine phosphatase inhibitors, respectively, did not modify inhibition of IL-8 release caused by NAL. NF-kappaB and C/EBP DNA binding were increased after LPS treatment compared with control, an effect inhibited by cotreatment with NAL. Activator protein (AP)-1 DNA binding was unaffected. The enhanced neutrophil chemotaxis produced by conditioned media from LPS-treated cells was inhibited when cells were cotreated with NAL. The selectivity of NAL inhibition upon IL-8 expression was studied. LPS-treated THP-1 cells also had higher levels of TNF-alpha, transforming growth factor (TGF)-beta1 and -3, MIP-1alpha and -beta, and RANTES gene expression. However, only LPS-induced IL-8 and TGF-beta1 expressions were inhibited by NAL. An anti-inflammatory effect of NAL was confirmed in vivo as shown by a reduction in LPS-induced neutrophil recruitment to the lungs following instillation of NAL into the lungs. Our studies demonstrate that NAL has anti-inflammatory properties in vitro and in vivo, may therefore have a therapeutic role in lung inflammation, and has the advantage over other antioxidant agents in that it may be administrated by inhalation.

Pulmonary and systemic effects of short-term inhalation exposure to ultrafine carbon black particles.

While environmental particles are associated with mortality and morbidity related to pulmonary and cardiovascular (CV) disease, the mechanisms involved in CV health effects are not known. Changes in systemic clotting factors have been associated with pulmonary inflammation. We hypothesized that inhaled ultrafine particles result in an inflammatory response which may stimulate systemic clotting factor release. Adult male Wistar rats were exposed to either fine or ultrafine carbon black (CB) for 7 h. The attained total suspended particle concentrations were 1.66 mg/m(3) for ultrafine CB and 1.40 mg/m(3) for fine CB. Particle concentration of ultrafine particles was more than 10 times greater than that of fine particles and the count median aerodynamic diameter averaged 114 nm for the ultrafine and 268 nm for the fine carbon particles. Data were collected immediately, 16 and 48 h following exposure. Only ultrafine CB caused an increase in total bronchoalveolar lavage (BAL) leukocytes, whereas both fine (2-fold) and ultrafine (4-fold) carbon particles caused an increase in BAL neutrophils at 16 h postexposure. Exposure to the ultrafine, but not fine, carbon was also associated with significant increases in the total numbers of blood leukocytes. Plasma fibrinogen, factor VII and von Willebrand factor (vWF) were unaffected by particle treatments as was plasma Trolox equivalent antioxidant status (TEAC). Macrophage inflammatory protein-2 mRNA was significantly increased in BAL cells 48 h following exposure to ultrafine CB. The data show that there is a small but consistent significant proinflammatory effect of this exposure to ultrafine particles that is greater than the effect of the same exposure to fine CB.

Nacystelyn inhibits oxidant-mediated interleukin-8 expression and NF-kappaB nuclear binding in alveolar epithelial cells.

Nacystelyn (NAL), a recently developed lysine salt of N-acetyl-L-cytokine (NAC) has mucolytic and antioxidant properties. In this study, we investigated the effect of NAL upon oxidant-mediated interleukin (IL)-8 release and the activation of the redox-sensitive transcription factors AP-1, NF-kappaB, and C/EBP in a human alveolar epithelial cell line (A549). NAL (5 mM) enhanced intracellular glutathione (GSH) after 4 h and abolished H(2)O(2)-induced IL-8 release from A549 cells. This was associated with inhibition of NF-kappaB and C/EBP DNA-binding, measured by the Electrophoretic Mobility Shift Assay (EMSA). NAL also abolished the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyl transferase (CAT) reporter system, transfected into A549 cells. Supernatants obtained from H(2)O(2)-treated A549 cells induced chemotaxis of polymorphonuclear neutrophils, which could be inhibited by co-incubation with NAL. These data indicate that NAL may be used to modulate pro-inflammatory process by inhibiting cytokine release in the lungs and thus has therapeutic potential in inflammatory lung diseases.

Potential role of IL-8, platelet-activating factor and TNF-alpha in the sequestration of neutrophils in the lung: effects on neutrophil deformability, adhesion receptor expression, and chemotaxis.

The microvasculature of the normal lung contains a pool of sequestered neutrophils, which is markedly enhanced in acute lung inflammation. Lung neutrophil sequestration is determined by the cells' deformability and adhesivity to capillary endothelium, and is a pre-requisite for emigration into the airspaces. We assessed the effect of several pro-inflammatory mediators associated with acute lung inflammation on these factors. Platelet-activating factor, IL-8 and formyl-Met-Leu-Phe (fMLP) induced a marked, but transient reduction in neutrophil deformability. Also, increased surface expression of the beta(2)-integrin and CD11b, and shedding of L-selectin (CD62L) was observed for these stimuli. TNF-alpha in contrast caused a small decrease in cell deformability only after 30 min, and shedding of L-selectin, but no change in CD11b levels. However, TNF-alpha-pretreatment markedly enhanced the fMLP response for cell deformability, CD11b expression and CD62L loss. Moreover, all pre-treatments were found to induce chemokinesis, and all except fMLP, enhanced fMLP-directed chemotaxis. We were able to demonstrate, using specific TNF-alpha receptor antagonists, that the TNF-alpha-induced changes in chemotaxis were mediated through the 55-kDa receptor. Also, inhibitors of the mitogen activated protein (MAP) kinase signaling pathway showed that the p38 MAP kinase pathway was involved for fMLP-directed chemotaxis of TNF-pretreated neutrophils, although activation of the extracellular signal-regulated kinase (ERK) pathway was also seen. These data demonstrate the differential role of pro-inflammatory mediators in controlling neutrophil sequestration and migration, which may orchestrate the severity of the inflammatory response in such respiratory diseases as chronic obstructive pulmonary disease and asthma.