ABSTRACT Asthma exemplifies a major medical concern and is a considerable cause of morbidity and mortality in Western society. Controversy still exists regarding the most effective mode and intensity of exercise training for asthmatics. Thus, the purpose of the study was to determine whether walking or jogging at 60% of age-predicted heart rate maximum can increase effort-dependent pulmonary function parameters in moderate, persistent asthmatics. Forty-four sedentary asthmatics were randomly assigned to either a non-exercising control (NE) group (n = 22) or an eight-week moderate-intensity aerobic exercise (AE) group (n = 22). Results indicated that the subjects in the AE training group significantly (p = 0.05) increased their forced vital capacity (FVC), forced expiratory volume in one second (FEV1), peak expiratory flow (PEF), maximal voluntary ventilation (MVV) and inspiratory vital capacity (IVC). The NE group did not exhibit any significant changes in any of the measured variables. Therefore, walking or jogging at 60% heart rate maximum for 30 minutes three times a week for eight weeks can effectively improve the effort-dependent pulmonary parameters in moderate, persistent asthmatics. This represents a strong argument to support the inclusion of this mode of aerobic training in the treatment of moderate, persistent asthma due to its effectiveness, inexpensiveness and lowrisk.

Lėtinė obstrukcinė plaučių liga (LOPL) - lėtai progresuojanti kvėpavimo takų obstrukcija, atsirandanti dėl nenormalaus uždegiminio plaučių atsako į žalingas su oru įkvepiamas daleles ar dujas. Tai liga, lemianti didelį sergamumą ir mirtingumą visame pasaulyje. Sergant LOPL labiausiai pažeidžiami apatiniai kvėpavimo takai, o taip pat periferiniame kraujyje nustatomas uždegimo žymenų koncentracijų padidėjimas. Pastarasis rodo, jog vyksta sisteminis uždegimas. Kvėpavimo takų sekretas yra biologinė terpė, kurioje esantys imuniniai žymenys atspindi vietinį uždegimą. Tiek vietinio, tiek ir sisteminio uždegiminio proceso metu pagrindinėmis ląstelėmis įvardijamos neutrofilai. LOPL yra daugiakomponentė liga ir siekiant suprasti šios ligos metu vykstančius įvairius patogeninius mechanizmus, būtina įvertinti vietinį ir sisteminį uždegimą jungiančias jungtis bei principus. Norint nustatyti vietinio ir sisteminio uždegimo sąsajas, svarbu įvertinti kvėpavimo takų sekreto tiesioginį poveikį kraujo neutrofilų funkcijoms. Šio darbo tikslas buvo įvertinti kraujo neutrofilų chemotaktinį ir fagocitinį aktyvumą bei reaktyvių deguonies formų (ROS) susidarymo ypatumus sergant LOPL. Kraujo neutrofilų chemotaksis tirtas aktyvinant neutrofilus klasikiniu chemotaksį skatinančiu veiksniu- interleukinu-8 (IL-8), indukuotais skrepliais bei indukuotais skrepliais papildytais skirtingomis IL-8 koncentracijomis (10, 30, 100 ng/ml). Fagocitinis kraujo neutrofilų aktyvumas bei ROS susidarymas buvo tirtas... [toliau žr. visą tekstą] / Chronic obstructive pulmonary disease (COPD) - slowly progressive airway obstruction resulting from abnormal inflammatory response to lung damaging particles of inhaled air or gas. It is a major cause of high morbidity and moratlity worldwide. Patients with COPD has lung injury, but determined increased blood levels of inflammatory markers shows and systemic inflammation. Neutrophils are the key inflammatory cells both in local and systemic inflammatory process. COPD is heterogeneous disease and in order to understand a variety of pathogenic mechanisms it is necessary to evaluate local and systemic inflammation combination characteristics and principles. It is also important to investigate if airway mucus influence blood neutrophils activity. The aim of this study was to evaluate both chemotactic and phagocytic activity and reactive oxygen species (ROS) production in peripheral blood neutrophils during COPD. Blood neutrophils chemotaxis was measured by activation of the interleukin-8 (IL-8), induced sputum and induced sputum and IL-8 different concentration (10, 30 and 100 ng/ml) combination. Phagocytic activity and ROS generation of blood neutrophils was measured after neutrophils activation with serum osponized/non-osponized S. aureus bacteria, induced sputum and induced sputum combination with S. aureus bacteria. The concentrations of 10, 30 and 100 bact./neutrophil S. aureus were used for all experiments. All features of neutrophils in vitro were analyzed by flow... [to full text]

Cigarette smoking (CS) represents the main causative agent underlying development and progress of COPD. Recently, involvement of CS in the pathogenesis of COPDassociated muscle abnormalities is becoming increasingly evident. Nevertheless, involved triggers and underlying mechanisms remain largely unknown. This study was conceived in order to examine effects of cigarette smoke exposure on skeletal muscle morphology, vascular supply and function. For this purpose, we have specifically designed murine COPD/emphysema model and gastrocnemius muscle was examined, while in vitro experiments were conducted using murine C2C12 skeletal muscle myocytes. In addition to the mild emphysematous changes present in the lungs of CS-exposed mice, our results demonstrated evident signs of muscle atrophy reflected by decreased fiber cross-sectional area, profound fiber size variation and reduced body mass. Furthermore, we have observed impairment in terminal myogenesis and lower number of myonuclei in skeletal muscles of CS-exposed animals despite evident activation of muscle repair process. Additionally, our results demonstrate capillary rarefaction in skeletal muscles of CS-exposed animals which was associated with deregulation of hypoxia-angiogenesis signaling, reduced levels of angiogenic factors such as HIF1-α and VEGF and enhanced expression of VHL and its partner proteins PHD2 and Ube2D1. The results of our in-vitro experiments demonstrated that VHL and its ubiquitination machinery can be synergistically regulated by TNF and hypoxia consequentially impairing angiogenic potential of skeletal muscle myocytes. Finally, we have shown that CS elicits chronic ER stress in murine skeletal muscles which is associated with activation of ERAD and apoptotic pathways as mirrored by elevated expression of Usp19, caspase 12 and caspase 3 in skeletal muscles of CSexposed animals. Moreover, molecular and morphological alterations in CS-exposed mice resulted in impairment of muscle function as reflected by their impaired exercise capacity. Taken together, from our results it is evident that cigarette smoke exposure elicits set of morphological, vascular and functional changes highly resembling those observed in COPD. Additionally, CS induces wide range of molecular alterations and signaling pathway deregulations suggesting profound effects of cigarette smoke exposure on skeletal muscle cell homeostasis.

Pulmonary rehabilitation is a highly evidenced intervention used in the management of patients with chronic obstructive pulmonary disease. Both patients and healthcare professionals have anecdotally acknowledged motivation as a key element in a programme. It has been suggested by some authors that motivation should be a prerequisite to entry, yet there is no evidence to support this suggestion. The purpose of this study therefore, was to provide some theory about the role of motivation in pulmonary rehabilitation and to produce a measurement instrument to enable further quantitative study. Methods A qualitative, exploratory investigation using focus groups and face-to-face interviews with patients undergoing a pulmonary rehabilitation programme was undertaken to generate data around factors influencing motivation. Results were used to develop a 43 item self-report questionnaire. The questionnaire was administered to 77 patients before and after a pulmonary rehabilitation programme along with other health status measures. The questionnaire was tested for reliability and validity. Item reduction was performed using factor analysis. Results Motivation within the context of a PR programme was shown to consist of a number of psychological, social and circumstantial variables that fell into 3 broad dimensions: Essential motivation, external motivation and functional outcome. A key finding was that attending pulmonary rehabilitation had an enormous positive influence on the patients’ essential motivation. The questionnaire was reduced to 21 items and principal components analysis demonstrated 9 factors within the questionnaire. These were function, self-efficacy, effort, optimism, tenacity, self worth, isolation, ability and achievement. The questionnaire was named the Malvern pulmonary rehabilitation motivation questionnaire (MPMQ) for identification. The MPMQ was shown to be reliable with internal consistency, reproducibility on test-retest and sensitivity to change. Correlations were found between the MPMQ and health related quality of life, anxiety and depression, breathlessness, exercise capacity and hospital admissions during the previous 12 months. Motivation score was significantly lower in patients who dropped out of the programme and was significantly higher at the end than the start of a programme. Conclusion The MPMQ has been shown to be a reliable tool with sound evidence of validity that can be used to objectively assess patients’ motivation within the context of a pulmonary rehabilitation programme. These findings need to be supported with further evidence for the validity and reliability of the questionnaire. Further investigation of the association of MPMQ score and adherence in pulmonary rehabilitation is needed along with further exploration of the determinants of motivation. This would enable specialist staff to identify patients who are likely to have adherence problems and channel efforts into effective cognitive-behavioural interventions in the ongoing effort to establish the optimum pulmonary rehabilitation programme.

La broncho-pneumopathie chronique obstructive (BPCO) est une maladie respiratoire grave, qui est le plus souvent secondaire à l’exposition à des substances toxiques inhalées. La fumée du tabac est le principal facteur de risque identifié mais certains milieux professionnels tels que les métiers agricoles sont associés à un risque accru de BPCO. Plusieurs études ont été menées ici avec pour objectifs (1) d’estimer la prévalence de BPCO dans plusieurs catégories de professions agricoles, (2) de créer un questionnaire de screening adapté au milieu agricole qui permettrait de pallier au sous-diagnostique de cette maladie en identifiant les sujets redevables d’un examen spirométrique et (3) d’étudier les facteurs deassociés à la BPCO en milieu agricole.La prévalence de la BPCO est environ deux fois plus importante en milieu agricole qu’en population générale. Néanmoins, cette prévalence est très variable selon la population d’étude (type d’activité, région d’exercice, caractéristiques de la ferme) et le critère de jugement utilisé (mesure pré- ou post-bronchodilatateur, critère GOLD ou LIN, prise en compte des symptômes), comme l’ont prouvé les résultats présentés ici.Une standardisation de la méthodologie pour les études épidémiologiques analysant le risque de BPCO en milieu agricole est donc nécessaire. Cette standardisation doit imposer un critère diagnostique robuste, une mesure de l’exposition la plus précise possible et l’inclusion d’un groupe de témoins sans exposition professionnelle. / Chronic obstructive pulmonary disease (COPD) is a severe disease that is in most cases secondary to the inhalation of noxious particles. Tobacco smoking is the main causal factor identified for this disease but some occupational exposures have also been associated with higher prevalence of COPD.The studies reported here aimed (1) to estimate the prevalence of COPD in the spectrum of agricultural works, (2) to develop a screening questionnaire appropriate to agricultural workers in order to identify subjects most likely to have COPD and (3) to study factors associated with COPD in farmers.The main findings are the following: prevalence of COPD is about twice higher in farmers than in the general population. However, this prevalence varies with study population (type of activity, region of exercise, characteristic of the farm) and the diagnostic criteria used (pre- or post-bronchodilator measure, criteria for airflow limitation, assessment of respiratory symptoms or not).It is concluded that standardization of the methodology used for epidemiologic investigations of COPD in farmers may be scheduled with (1) strong diagnostic criteria, (2) clear and robust measurement of occupational exposure and (3) inclusion of a control group of unexposed subjects.

Rationale: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of deaths worldwide. Patients with COPD have episodes of aggravated symptoms called exacerbations caused by pathogens or pollution. Respiratory viruses are associated with a significant number of COPD exacerbations with the most common virus being the rhinovirus (RV). The mechanisms by which RVs trigger COPD exacerbations are still unclear. Using human whole lung tissue explants (WTE), a novel model of RV-induced COPD exacerbations is proposed. Methods: WTE from COPD patients and smokers were initially stimulated with TLR ligands that are known to activate the same receptors as RV: poly(I:C) for TLR3 and R848 for TLR7/8 activation. Pro-inflammatory cytokines and type I and III IFN gene expression was measured by ELISA and qRT-PCR, respectively. A neutralising antibody against TNFα, a corticosteroid, and a panel of inhibitors targeting TLR pathway (p38 MAPK, IKK-2 and IRAK1/4) was applied to the tissue from COPD patients to establish which signalling pathways are responsible for the inflammatory response and IFN release. Explants from COPD patients and smokers was also exposed to two RV serotypes, RV-16 and RV-1B, in order to compare findings with a clinically relevant stimulant. Results: Poly(I:C) and R848 caused a significant increase of protein and gene expression of pro-inflammatory cytokines (TNFα, CCL5 and IL-6). Type I and III IFN gene expression was also significantly increased. Using the two ligands together caused a synergistic release of TNFα and CCL5. Tissue from COPD patients released more pro-inflammatory cytokines and expressed less IFNβ when compared to smokers. TNFα neutralisation inhibited subsequent release of CCL5 and IL-6. Dexamethasone and p38 MAPK inhibitor decreased TLR3- and TLR7/8-induced pro-inflammatory response whereas IKK-2 and IRAK1/4 inhibition had little effect on cytokine release. Dexamethasone and IKK-2 showed limited effect on IFN gene expression whereas p38 MAPK inhibitor significantly decreased and IRAK1/4 inhibition enhanced IFN expression. RV-16 induced modest but significant pro-inflammatory response in lung tissue, whereas RV-1B did not induce cytokine release. Both serotypes induced type I and III IFN gene expression. Tissue from COPD patients showed a lower expression of IFNβ and IFNλ when compared to smokers. Conclusion: This tissue explant was responsive to both synthetic TLR ligands and RV. The release of pro-inflammatory cytokines in response to TLR stimulation was partially inhibited by steroid. p38 MAPK is involved in TLR-induced inflammation but it also further decreases the already impaired IFN gene expression in COPD tissue. The role of IKK-2 and IRAK1/4 in TLR-induced innate immune response remains unclear. This model is a valuable system to study the mechanisms underlying RV-induced COPD exacerbations and also to test new inhibitors in the whole tissue environment.

Introduction Chronic obstructive pulmonary disease (COPD) has emerged as a major policy focus for health systems throughout Western Europe. This reflects the increased prevalence, associated healthcare utilisation and costs of COPD, and the potential to substantially improve outcomes through achieving reductions in smoking. The aim of this PhD was to develop projections for the prevalence, healthcare costs and number of deaths in people with COPD in England and Scotland over a 20-year horizon (i.e. from 2011 to 2030). Methods I undertook a phased programme of work, which began with a systematic review of the published and unpublished literature to identify models that were suitable for estimating and/or projecting the prevalence and disease and economic burden from COPD. This involved searching Medline, Embase, CAB Abstracts, World Health Organization (WHO) Library and Information Services and WHO Regional Indexes, and Google over the time period 1980-2013. The models were then critically appraised for their quality of reporting. From these, I selected the Dutch Model developed by Erasmus University for generating projections. Suitable data sources from both England and Scotland were identified, sourced and carefully processed in order to run the modelling exercises. Rates of incidence and prevalence were calculated using English and Scottish healthcare datasets and population data were obtained from the Office for National Statistics (ONS) and the General Register Office for Scotland (GROS). Relative risks for all-cause mortality among people with COPD were calculated from the Clinical Practice Research Datalink and mortality data were obtained from the ONS and GROS. The Model was thus adjusted to apply to England and Scotland. I then travelled to the Netherlands to work with the developers of the Dutch Model and ran a baseline model and an array of sensitivity analyses with modified inputs to the Model. Finally, my Rotterdam colleagues calculated uncertainty intervals for some of the estimates using probabilistic analysis. Results Using the probabilistic means and uncertainty intervals, in England, the modelled prevalence of diagnosed COPD among males of all ages in 2011 was 1.8% (95% uncertainty interval 1.8-1.9) increasing to 2.0% (1.7-2.1) by 2030. In females, in England, the baseline estimate was 1.8% (1.7-1.8) in 2011 increasing to 2.4% (2.0-2.6) in 2030. In Scotland, the modelled prevalence among males was 1.9% (1.8-1.9) in 2011 and this was projected to stay the same at 1.9% (1.7-2.2) by 2030. In females in Scotland, the estimated prevalence was 2.2% (2.1- 2.3) in 2011 and was projected to increase to 2.5% (2.1-2.7) in 2030.Using the Model I estimated that overall in 2011 there were a total of 952,000 (941,000-966,000) people with diagnosed COPD in England and 106,000 (103,000-110,000) in Scotland and that these numbers would increase to 1,325,000 (1,117,000-1,408,000) in England in 2030 and 125,000 (113,000-136,000) in Scotland in 2030, respectively. The greatest increase in COPD was projected to be in females over 65 years of age in both countries. The total annual direct healthcare costs of COPD in England were projected to increase from £1.60 (95% uncertainty interval 1.18-2.5) billion in 2011 to £2.35 (1.85-3.08) billion in 2030. In Scotland, costs were projected to increase from £170 (128-268) million in 2011 to £210 (165-274) million in 2030. These costs were calculated in terms of 2011 costs without the application of any economic trends (i.e. no annual increase applied for inflation). The number of deaths among people with COPD in England was estimated to be 99,000 (93,000-129,000) in 2011, increasing to 129,000 (126,000-133,000) in 2030. In Scotland there were estimated to be 10,000 (9,000-12,000) deaths in 2011, increasing to 14,000 (13,000-15,000) in 2030. The Dutch Model demonstrated a 39% increase in the number of people with COPD in England and a 17% increase in Scotland between 2011 and 2030. It provided an estimate of a 30% increase in deaths among people with COPD in England and of a 43% increase in Scotland. Overall, there was a projected 46% increase in the direct healthcare costs required to care for people with COPD in England and a 23% increase in Scotland between 2011 and 2030. The reasons for these differences are largely due to higher COPD-related excess mortality in Scotland and to differences in the data used for populating the model in both countries. Conclusions There are likely to be substantial increases in the number of people with COPD, associated morbidity, direct healthcare costs and mortality in both England and Scotland over the next two decades. These increases in numbers will predominantly occur in females over 65 years of age and are likely to have substantial societal impact in terms of organising the health and social care for this frail population.

Le monoxyde d'azote (NO) est une molécule produite par l'ensemble des cellules des voies aériennes. Sa synthèse à partir de la L-arginine fait appel à un groupe d'enzymes : les NO synthases (NOS). Il existe trois isoformes de NOS exprimées par des cellules ayant des fonctions diverses conférant ainsi au NO produit une spécificité d'action dépendante de la cellule et de la « situation » ayant entraîné sa synthèse. Les travaux effectués dans le cadre de cette thèse explorent des rôles différents (bronchoréactivité, immunitaires) du NO produit par les voies aériennes ainsi que la mesure du NO dans le contexte de l'anesthésie – réanimation. L'objectif du premier travail était de « revisiter » la fonction régulatrice du tonus bronchique dans le cadre d'une pathologie respiratoire, l'objectif du second travail était méthodologique (étude des sources anatomiques du NO expiré chez le patient sous ventilation mécanique), et l'objectif du troisième travail était d'utiliser la mesure validée dans le second travail pour évaluer les fonctions immunes du NO en réanimation.Premier travail. La compétition entre les NO synthases et les arginases pour leur substrat commun, la L-arginine, pourrait être impliquée dans régulation de la réactivité et du remodelage bronchique chez le patient atteint de BPCO. Le but du premier travail était d'évaluer la relation entre expression de cette balance enzymatique, et les effets pharmacologiques de l'inhibition des NOS et des arginases sur la réactivité bronchique ex vivo à l'acétylcholine de patients sans et avec une BPCO peu sévère. Pour cela, nous avons étudié les bronches de 22 patients. Des études immunohistochimiques nous ont permis de mettre en évidence une expression bronchique NOS-2 plus importante chez les patients BPCO comparée aux patients non BPCO. De plus, les études pharmacologiques réalisées en cuve à organe ont permis de mettre en évidence une tension bronchique de base plus importante chez les BPCO, corrélée à l'expression de la NOS-2 et au degré d'obstruction bronchique (VEMS). L'utilisation d'inhibiteur des NOS diminuait cette tension de base. Nous avons démontré ainsi qu'une augmentation de l'expression NOS-2 chez le BPCO était impliquée dans l'augmentation du tonus bronchique de base et dans l'obstruction bronchique.Second travail. Les variations de NO expiré après chirurgie cardiaque avec circulation extracorporelle (CEC) demeurent controversées. Le but de ce deuxième travail était de déterminer quelle source de NO expiré (bronchique ou alvéolaire) était modifiée après CEC, et d'étudier les effets de la ventilation mécanique pendant la CEC sur ces variations. Nous avons étudié ainsi 32 patients ventilés ou non durant une chirurgie cardiaque avec CEC. Nous avons observé une diminution significative du NO expiré d'origine bronchique après la CEC. Cette diminution n'était pas observée lors du maintien durant la CEC d'une ventilation avec pression expiratoire positive (PEEP). Ce travaille permettait de conclure que la diminution du NO bronchique après la CEC pouvait être liée à une occlusion des petites voies aériennes. Cette atteinte de petites voies aériennes était prévenue par la PEEP.Troisième travail. Enfin, dans ce troisième travail, nous avons émis l'hypothèse que la mesure du NO produit par les voies aériennes (NO expiré et nasal) pouvait constituer un marqueur prédictif de survenue d'infection nosocomiale (fonctions immunitaires du NO). Dans une étude observationnelle chez 45 patients de réanimation ventilés (15 patients ont développé une infection nosocomiale), le NO nasal était le seul marqueur significativement plus bas chez les patients développant une infection nosocomiale (le NO expiré, les dosages d'IL6 et d'IL10 ainsi que le score SOFA n'étaient pas différents entre les deux groupes). Un NO nasal inférieur à 148 ppb 72 heures après l'admission du patient, permettait de prédire la survenue d'une infection nosocomiale avec une sensibilité et une spécificité de 80% et de 70% respectivement et un odds ratio de 2.7. Le développement de ce bio marqueur simple à mesurer permettrait de mettre en place des stratégies préventives (immunonutrition avec de la L-arginine). / In the respiratory tract, NO is produced by a wide variety of cell types and is generated via oxidation of l-arginine that is catalyzed by the enzyme NO synthase (NOS). NOS exists in three distinct isoforms: neuronal NOS (NOS-1), inducible NOS (NOS-2), and endothelial NOS (NOS-3). NO derived from the constitutive isoforms of NOS (NOS-1 and NOS-3) and other NO-adduct molecules (nitrosothiols) have been shown to be modulators of bronchomotor tone. On the other hand, NO derived from NOS-2 seems to be a proinflammatory mediator with immunomodulatory effects. This thesis explores the physiological and pathophysiological role of endogenous nitric oxide in the airways, and the clinical aspects of monitoring nitric oxide in exhaled air of patients with respiratory disease.First Study: competition between nitric oxide synthases (NOSs) and arginases for their common substrate l-arginine could be involved in the regulation of cholinergic airway reactivity and subsequent airway remodeling. The aims of this study were to evaluate the relationships between the expression of this enzymatic balance and the effects of NOS and arginase inhibition on bronchoconstrictive response to acetylcholine of patients without and with early chronic obstructive pulmonary disease (COPD). Twenty-two human bronchi were investigated for immunohistochemistry and modulation of acetylcholine-induced airway constriction. Significantly increased expression of NOS2 in immunoblots of bronchial tissue and staining in smooth muscle cells was evidenced in patients with COPD compared with control subjects. Forced expiratory volume in 1 s (FEV1) and NOS2 expression were negatively correlated. Pharmacological experiments demonstrated that resting tension was elevated in COPD compared with control subjects and was positively correlated with the expression of NOS2. The sole effect of the specific arginase inhibitor Nomega-hydroxy-nor-L-arginine was to decrease sensitivity in COPD patients, whereas NG-nitro-L-arginine methyl ester unexpectedly decreased resting tension because of a non-cGMP-dependent effect. In conclusion, an upregulation of NOS2 expression in COPD patients is involved in airway tone regulation and functional airflow limitation, whereas increased arginase activity is involved in airway sensitivity.Second Study: the change in exhaled NO after cardio-pulmonary bypass remains controversial. The aims were to determine whether exhaled NO sources (alveolar or bronchial) are modified after bypass, and whether mechanical ventilation (MV) settings during bypass modify exhaled NO changes. Thirty-two patients were divided into three groups: without MV during bypass and positive end-expiratory pressure (PEEP) (n=12), dead space MV without PEEP (n=10) and dead space MV with PEEP (n=10). Alveolar NO concentration and bronchial NO flux were calculated before and 1h after surgery using a two-compartment model of NO exchange developed in spontaneous breathing patients. Whereas a significant decrease in bronchial NO was found after bypass in the two groups without PEEP during bypass, this decrease was not observed in patients with dead space ventilation with PEEP. Alveolar NO was not significantly modified whatever the ventilation settings. In conclusion, the impairment of bronchial NO seemed related to airway closure since dead space mechanical ventilation with PEEP prevented its decrease.Third Study: the development of biomarkers able to predict the occurrence of nosocomial infection could help manage preventive strategies, especially in medical patients whose degree of acquired immunosuppression may be variable. We hypothesized that the NO fraction present in the airways (upper and lower) of critically ill patients under mechanical ventilation could constitute such a biomarker. We conducted an observational study in a medical intensive care unit. Forty-five patients (26 men; 72 [25th-75th percentiles] years [56-82]; Simplified Acute Physiology Score II, 63 [50-81], 14 infected) under mechanical ventilation (>3 days) underwent on day 1 and day 3 of their stay: nasal and exhaled (partitioned in bronchial and alveolar sources) bedside NO measurements, determination of urine NO end products and plasma cytokine (IL-6, IL-10) concentrations, and Sequential Organ Failure Assessment score calculation. Nosocomial infection incidence was recorded during the 15 subsequent days. Fifteen patients (33%) acquired a nosocomial infection. Nasal NO was the only marker significantly different between patients with and without subsequent infection (day 1, 52 ppb [20-142] vs. 134 [84-203], P = 0.038; day 3, 98 ppb [22-140] vs. 225 [89-288], P = 0.006, respectively). Nasal NO fraction 148 ppb or less at day 3 had an 80% sensitivity, a 70% specificity, and an odds ratio of 2.7 (95% confidence interval, 1.9-3.8) to predict acquisition of nosocomial infection. Nasal NO seems to be a relatively sensitive and specific biomarker of subsequent nosocomial infection acquisition.

Background: COPD is associated with an increased lung macrophage burden. Whilst lung macrophages may self-renew, recruitment of peripheral blood monocytes from the systemic circulation is considered to represent their principal means of replenishment. Through modulating expression of monocytic chemokines CCL2/CCL3 and their respective receptors (CCR2/CCR1+CCR5), IL-6 could play a key role in facilitating the recruitment of monocytes to the lungs of COPD patients. COPD is associated with enhanced pulmonary and systemic IL-6 levels; concentrations of the soluble IL-6 receptor sIL-6R may be an important determinant of IL-6 signalling in COPD. Trans-signalling through sIL-6R, IL-6 may facilitate recruitment of monocytes in COPD by influencing chemokine and chemokine receptor expression. Aims: 1) To compare levels of IL-6, sIL-6R, CCL2 and CCL3 in the plasma and sputum of COPD and controls. 2) To examine of the effects of IL-6 stimulation on monocyte chemokine receptor gene expression (CCR1, CCR2 and CCR5). 3) To compare subtypes (CD14++CD16-, CD14+CD16+, CD14-CD16++) and chemokine receptor expression (CCR1, CCR2, CCR5) of monocytes in COPD (paired stable & exacerbating) and controls. 4) To compare the migratory ability of monocytes from COPD and controls. 5) To compare numbers of marginated CX3CR1+ monocytes in the pulmonary microvasculature and proliferation status (Ki67 positivity) of alveolar macrophages in COPD and controls. Methods: 1) MSD soluble marker analysis was performed on plasma and sputum supernatant. 2) Monocytes underwent stimulation with IL-6 and sIL-6R; chemokine receptor expression was determined by quantitative PCR. 3) Flow cytometry was performed on whole blood to determine monocyte subtype and chemokine receptor expression. 4) Monocyte migration towards sputum supernatant was assessed using a transwell system incorporating fluorescence based detection of DNA from migrated cells. 5) Immunofluorescence and immunohistochemistry was performed on lung tissue (obtained from patients undergoing surgical resection of lung carcinoma) to identify marginated (CX3CR1+CD14+, CX3CR1+CD16+) monocytes and proliferating alveolar macrophages (Ki67) respectively. Results and Conclusion: Levels of sIL-6R were increased in the lungs and systemic circulation of COPD patients implying potential for enhanced IL-6 trans-signalling: monocytes cultured in the presence of IL-6+sIL-6R upregulated expression of the CCR5 gene. A greater proportion of circulating COPD CD14++CD16- and CD14+CD16+ monocytes were demonstrated to express CCR5 compared to controls indicating that CCR5 ligands may have an important influence over monocyte migration in COPD. Levels of CCR5 ligand CCL3 were significantly elevated in COPD sputum supernatant; IL-6 levels were positively associated with CCL3 indicating that IL-6 trans-signalling may mediate lung chemokine expression. Nevertheless, COPD monocytes demonstrated impaired migration towards sputum supernatant and reduced margination to pulmonary microvessels. Despite this, the number of alveolar macrophages in COPD was increased; however this was not likely to be related to self-replication owing to low alveolar macrophage Ki67 expression.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic, inflammatory condition, characterised by airflow limitation. The use of glucocorticoids (GC) as an anti-inflammatory treatment in COPD has limited clinical benefits, and as such, new treatments are needed. Identifying key pathways involved in the inflammatory response in COPD may enable the development of novel treatments. The aims of this thesis were to examine the steroid sensitivity of an in vitro mixed sputum culture cell model, comparing COPD cells to smoking and non-smoking controls, examine expression of the intracellular signalling molecule p38 Mitogen Activated Protein Kinase (MAPK) in COPD lungs compared with controls, examine the GC and p38 MAPK inhibitor and dual therapy sensitivity of a bronchial epithelial cell line and finally, to understand the mechanisms by which a p38 MAPK inhibitor in combination with a GC synergistically inhibit pro-inflammatory mediator production in a bronchial epithelial cell line. Dexamethasone inhibits mixed sputum cell pro-inflammatory mediator release, with no differences in sensitivity observed between COPD and control cells. Isolated sputum neutrophils demonstrate modest sensitivity to dexamethasone, which is in contrast to blood neutrophils. There are increased numbers of cells positive for activated p38 MAPK in COPD lungs compared with controls, specifically localised to follicular B and CD8+ T cells, bronchial epithelial cells and alveolar and sputum macrophages. Lung and sputum neutrophils are devoid of activated p38 MAPK, and a pharmacological p38 MAPK inhibitor has no effect on pro-inflammatory mediator production from these cells. This is in contrast to blood neutrophils, whereby p38 MAPK activation can be induced following LPS stimulation and in vitro cell culture, and pro-inflammatory mediator release is inhibited by a p38 MAPK inhibitor. Dexamethasone and birb 796 inhibit stimulated pro-inflammatory mediator release from a bronchial epithelial cell line in a dose-dependent manner. Sensitivity to either drug is dependent on stimuli and the pro-inflammatory mediator analysed. There is additive and synergistic inhibition of pro-inflammatory mediator production when combination therapy comprising dexamethasone and birb 796 is used compared with either drug alone. This may be due to Birb 796 enhancing dexamethasone-mediated nuclear translocation of the glucocorticoid receptor, which may enhance the GC-mediated anti-inflammatory effects. Combination therapy may therefore be a useful therapeutic in the treatment of COPD.