The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

11beta-hydroxysteroid dehydrogenases as regulators of pulmonary corticosterone during the granulomatous response to trehalose 6,6'-dimycolate

Protection against Mycobacterium tuberculosis requires development and maintenance of granulomatous lesions, a feature considered to be the pathological hallmark of Tuberculosis (TB) disease. Upon encountering Mtb or mycobacterial antigens, specifically trehalose 6,6'-dimycolate (TDM), a strong local pro-inflammatory response is initiated. Systemic production of anti-inflammatory glucocorticoids (GCs) is also induced. Emergence of these antagonists at the inflammatory foci is counterproductive to development of the granulomatous structure and detrimental to host protection against TB. Therefore, it was hypothesized that local enzymatic regulation of GCs occurs locally at the site of granulomatous inflammation. The experiments described here strongly suggest that 11β-hydroxysteroid dehydrogenases (11βHSDs) shuttle GCs between active and inert forms during the acute granulomatous response, supporting the net reduction of corticosterone. The patterns of GC and 11βHSD regulation were specific to the lung (the site of inflammation) and were not observed in other tissues. Furthermore, 11βHSD2, which decreases corticosterone concentrations, was not expressed in models of dysregulated granulomatous inflammation. These findings suggest that cellular exposure to local active GC concentrations is restricted via 11βHSDs as a mechanism to initiate and maintain granuloma formation. The information derived from the experiments outlined in this dissertation provides a better understanding of the events required for establishment and maintenance of the protective granulomatous response. As a practical consequence, exploiting 11βHSD2 modulation of GCs at the site of Mtb infection may lead to improvement of Tuberculosis treatment strategies.^

Adenosine induced pulmonary fibrosis and the contribution of the adenosine A2B receptor to the induction of osteopontin in a mouse model of pulmonary fibrosis

Pulmonary fibrosis is a devastating and lethal lung disease with no current cure. Research into cellular signaling pathways able to modulate aspects of pulmonary inflammation and fibrosis will aid in the development of effective therapies for its treatment. Our laboratory has generated a transgenic/knockout mouse with systemic elevations in adenosine due to the partial lack of its metabolic enzyme, adenosine deaminase (ADA). These mice spontaneously develop progressive lung inflammation and severe pulmonary fibrosis suggesting that aberrant adenosine signaling is influencing the development and/or progression of the disease in these animals. These mice also show marked increases in the pro-fibrotic mediator, osteopontin (OPN), which are reversed through ADA therapy that serves to lower lung adenosine levels and ameliorate aspects of the disease. OPN is known to be regulated by intracellular signaling pathways that can be accessed through adenosine receptors, particularly the low affinity A2BR receptor, suggesting that adenosine receptor signaling may be responsible for the induction of OPN in our model. In-vitro, adenosine and the broad spectrum adenosine receptor agonist, NECA, were able to induce a 2.5-fold increase in OPN transcripts in primary alveolar macrophages. This induction was blocked through antagonism of the A2BR receptor pharmacologically, and through the deletion of the receptor subtype in these cells genetically, supporting the hypothesis that the A2BR receptor was responsible for the induction of OPN in our model. These findings demonstrate for the first time that adenosine signaling is an important modulator of pulmonary fibrosis in ADA-deficient mice and that this is in part due to signaling through the A2BR receptor which leads to the induction of the pro-fibrotic molecule, otseopontin. ^

Joint effects of pulmonary function and aerobic power on survival in a cohort of healthy adults

Objective measurements of physical fitness and pulmonary function are related individually to long-term survival, both in healthy people and in those who are ill. These factors are furthermore known to be related to one another physiologically in people with pulmonary disease, because advanced pulmonary disease causes ventilatory limitation to exercise. Healthy people do not have ventilatory limitation to exercise, but rather have ventilatory reserve. The relationship between pulmonary function and exercise performance in healthy people is minimal. Exercise performance has been shown to modify the effect of pulmonary function on mortality in people with chronic obstructive pulmonary disease, but the relationship between these factors in healthy people has not been studied and is not known. The purpose of this study is to quantify the joint effects of pulmonary function and exercise performance as these bear on mortality in a cohort of healthy adults. This investigation is an historical cohort study over 20 years of follow-up of 29,624 adults who had complete preventive medicine, spirometry and treadmill stress examinations at the Cooper Clinic in Dallas, Texas.^ In 20 years of follow-up, there were 738 evaluable deaths. Forced expiratory volume in one second (FEV$\sb1$) percent of predicted, treadmill time in minutes percent of predicted, age, gender, body mass index, baseline smoking status, serum glucose and serum total cholesterol were all significant, independent predictors of mortality risk. There were no frank interactions, although age had an important increasing effect on the risk associated with smoking when other covariates were controlled for in a proportional-hazards model. There was no confounding effect of exercise performance on pulmonary function. In agreement with the pertinent literature on independent effects, each unit increase in FEV$\sb1$ percent predicted was associated with about eight tenths of a percent reduction in adjusted mortality rate. The concept of physiologic reserve is useful in interpretation of the findings. Since pulmonary function does not limit exercise tolerance in healthy adults, it is reasonable to expect that exercise tolerance would not modify the effect of pulmonary function on mortality. Epidemiologic techniques are useful for elucidating physiological correlates of mortality risk. ^

Surfactants during upwelling events in the sea-surface microlayer and 1 m depth during METEOR cruise M117, Baltic Sea

Results from a study of surfactants (SAS) in the sea surface microlayer (SML) and underlying water (ULW) at different locations in the Baltic Sea. The total SAS concentrations were measured using phase-sensitive alternative current voltammetry with hanging mercury drop electrode (HMDE) in unfiltered samples. The concentrations of SAS were expressed as the equivalent concentration of nonanionic surfactants Triton-X-100. The enrichment factors (EF) of SAS were calculated as the ratio of concentration in SML to the corresponding ULW samples.

Noninvasive monitoring of serial changes in pulmonary vascular resistance and acute vasodilator testing using cardiac magnetic resonance

Objectives The study sought to evaluate the ability of cardiac magnetic resonance (CMR) to monitor acute and long-term changes in pulmonary vascular resistance (PVR) noninvasively. Background PVR monitoring during the follow-up of patients with pulmonary hypertension (PH) and the response to vasodilator testing require invasive right heart catheterization. Methods An experimental study in pigs was designed to evaluate the ability of CMR to monitor: 1) an acute increase in PVR generated by acute pulmonary embolization (n = 10); 2) serial changes in PVR in chronic PH (n = 22); and 3) changes in PVR during vasodilator testing in chronic PH (n = 10). CMR studies were performed with simultaneous hemodynamic assessment using a CMR-compatible Swan-Ganz catheter. Average flow velocity in the main pulmonary artery (PA) was quantified with phase contrast imaging. Pearson correlation and mixed model analysis were used to correlate changes in PVR with changes in CMR-quantified PA velocity. Additionally, PVR was estimated from CMR data (PA velocity and right ventricular ejection fraction) using a formula previously validated. Results Changes in PA velocity strongly and inversely correlated with acute increases in PVR induced by pulmonary embolization (r = –0.92), serial PVR fluctuations in chronic PH (r = –0.89), and acute reductions during vasodilator testing (r = –0.89, p ≤ 0.01 for all). CMR-estimated PVR showed adequate agreement with invasive PVR (mean bias –1.1 Wood units,; 95% confidence interval: –5.9 to 3.7) and changes in both indices correlated strongly (r = 0.86, p < 0.01). Conclusions CMR allows for noninvasive monitoring of acute and chronic changes in PVR in PH. This capability may be valuable in the evaluation and follow-up of patients with PH.

Pulmonary Artery-Vein Segmentation in Real and Synthetic CT Images = Segmentación Arteria-Vena Pulmonares en Imágenes de CT Reales y Sintéticas

La tomografía axial computerizada (TAC) es la modalidad de imagen médica preferente para el estudio de enfermedades pulmonares y el análisis de su vasculatura. La segmentación general de vasos en pulmón ha sido abordada en profundidad a lo largo de los últimos años por la comunidad científica que trabaja en el campo de procesamiento de imagen; sin embargo, la diferenciación entre irrigaciones arterial y venosa es aún un problema abierto. De hecho, la separación automática de arterias y venas está considerado como uno de los grandes retos futuros del procesamiento de imágenes biomédicas. La segmentación arteria-vena (AV) permitiría el estudio de ambas irrigaciones por separado, lo cual tendría importantes consecuencias en diferentes escenarios médicos y múltiples enfermedades pulmonares o estados patológicos. Características como la densidad, geometría, topología y tamaño de los vasos sanguíneos podrían ser analizados en enfermedades que conllevan remodelación de la vasculatura pulmonar, haciendo incluso posible el descubrimiento de nuevos biomarcadores específicos que aún hoy en dípermanecen ocultos. Esta diferenciación entre arterias y venas también podría ayudar a la mejora y el desarrollo de métodos de procesamiento de las distintas estructuras pulmonares. Sin embargo, el estudio del efecto de las enfermedades en los árboles arterial y venoso ha sido inviable hasta ahora a pesar de su indudable utilidad. La extrema complejidad de los árboles vasculares del pulmón hace inabordable una separación manual de ambas estructuras en un tiempo realista, fomentando aún más la necesidad de diseñar herramientas automáticas o semiautomáticas para tal objetivo. Pero la ausencia de casos correctamente segmentados y etiquetados conlleva múltiples limitaciones en el desarrollo de sistemas de separación AV, en los cuales son necesarias imágenes de referencia tanto para entrenar como para validar los algoritmos. Por ello, el diseño de imágenes sintéticas de TAC pulmonar podría superar estas dificultades ofreciendo la posibilidad de acceso a una base de datos de casos pseudoreales bajo un entorno restringido y controlado donde cada parte de la imagen (incluyendo arterias y venas) está unívocamente diferenciada. En esta Tesis Doctoral abordamos ambos problemas, los cuales están fuertemente interrelacionados. Primero se describe el diseño de una estrategia para generar, automáticamente, fantomas computacionales de TAC de pulmón en humanos. Partiendo de conocimientos a priori, tanto biológicos como de características de imagen de CT, acerca de la topología y relación entre las distintas estructuras pulmonares, el sistema desarrollado es capaz de generar vías aéreas, arterias y venas pulmonares sintéticas usando métodos de crecimiento iterativo, que posteriormente se unen para formar un pulmón simulado con características realistas. Estos casos sintéticos, junto a imágenes reales de TAC sin contraste, han sido usados en el desarrollo de un método completamente automático de segmentación/separación AV. La estrategia comprende una primera extracción genérica de vasos pulmonares usando partículas espacio-escala, y una posterior clasificación AV de tales partículas mediante el uso de Graph-Cuts (GC) basados en la similitud con arteria o vena (obtenida con algoritmos de aprendizaje automático) y la inclusión de información de conectividad entre partículas. La validación de los fantomas pulmonares se ha llevado a cabo mediante inspección visual y medidas cuantitativas relacionadas con las distribuciones de intensidad, dispersión de estructuras y relación entre arterias y vías aéreas, los cuales muestran una buena correspondencia entre los pulmones reales y los generados sintéticamente. La evaluación del algoritmo de segmentación AV está basada en distintas estrategias de comprobación de la exactitud en la clasificación de vasos, las cuales revelan una adecuada diferenciación entre arterias y venas tanto en los casos reales como en los sintéticos, abriendo así un amplio abanico de posibilidades en el estudio clínico de enfermedades cardiopulmonares y en el desarrollo de metodologías y nuevos algoritmos para el análisis de imágenes pulmonares. ABSTRACT Computed tomography (CT) is the reference image modality for the study of lung diseases and pulmonary vasculature. Lung vessel segmentation has been widely explored by the biomedical image processing community, however, differentiation of arterial from venous irrigations is still an open problem. Indeed, automatic separation of arterial and venous trees has been considered during last years as one of the main future challenges in the field. Artery-Vein (AV) segmentation would be useful in different medical scenarios and multiple pulmonary diseases or pathological states, allowing the study of arterial and venous irrigations separately. Features such as density, geometry, topology and size of vessels could be analyzed in diseases that imply vasculature remodeling, making even possible the discovery of new specific biomarkers that remain hidden nowadays. Differentiation between arteries and veins could also enhance or improve methods processing pulmonary structures. Nevertheless, AV segmentation has been unfeasible until now in clinical routine despite its objective usefulness. The huge complexity of pulmonary vascular trees makes a manual segmentation of both structures unfeasible in realistic time, encouraging the design of automatic or semiautomatic tools to perform the task. However, this lack of proper labeled cases seriously limits in the development of AV segmentation systems, where reference standards are necessary in both algorithm training and validation stages. For that reason, the design of synthetic CT images of the lung could overcome these difficulties by providing a database of pseudorealistic cases in a constrained and controlled scenario where each part of the image (including arteries and veins) is differentiated unequivocally. In this Ph.D. Thesis we address both interrelated problems. First, the design of a complete framework to automatically generate computational CT phantoms of the human lung is described. Starting from biological and imagebased knowledge about the topology and relationships between structures, the system is able to generate synthetic pulmonary arteries, veins, and airways using iterative growth methods that can be merged into a final simulated lung with realistic features. These synthetic cases, together with labeled real CT datasets, have been used as reference for the development of a fully automatic pulmonary AV segmentation/separation method. The approach comprises a vessel extraction stage using scale-space particles and their posterior artery-vein classification using Graph-Cuts (GC) based on arterial/venous similarity scores obtained with a Machine Learning (ML) pre-classification step and particle connectivity information. Validation of pulmonary phantoms from visual examination and quantitative measurements of intensity distributions, dispersion of structures and relationships between pulmonary air and blood flow systems, show good correspondence between real and synthetic lungs. The evaluation of the Artery-Vein (AV) segmentation algorithm, based on different strategies to assess the accuracy of vessel particles classification, reveal accurate differentiation between arteries and vein in both real and synthetic cases that open a huge range of possibilities in the clinical study of cardiopulmonary diseases and the development of methodological approaches for the analysis of pulmonary images.

Nrf2 is essential for protection against acute pulmonary injury in mice

Nrf2 is a member of the “cap ‘n’ collar” family of transcription factors. These transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a wide range of tissues, many of which are sites of expression for phase 2 detoxification genes. Nrf2−/− mice are viable and have a normal phenotype under normal laboratory conditions. The NF-E2 binding site is a subset of the antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant response elements are regulatory sequences found on promoters of several phase 2 detoxification genes that are inducible by xenobiotics and antioxidants. We report here that Nrf2−/− mice are extremely susceptible to the administration of the antioxidant butylated hydroxytoluene. With doses of butylated hydroxytoluene that are tolerated by wild-type mice, the Nrf2−/− mice succumb from acute respiratory distress syndrome. Gene expression studies show that the expression of several detoxification enzymes is altered in the Nrf2−/− mice. The Nrf2−/− mice may prove to be a good in vivo model for toxicological studies. As oxidative damage causes DNA breakage, these mice may also be useful for testing carcinogenic agents.

Multi-component assessment of chronic obstructive pulmonary disease : an evaluation of the ADO and DOSE indices and the global obstructive lung disease categories in international primary care data sets

Funding The International Primary Care Respiratory Group (IPCRG) provided funding for this research project as an UNLOCK group study for which the funding was obtained through an unrestricted grant by Novartis AG, Basel, Switzerland. The latter funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Database access for the OPCRD was provided by the Respiratory Effectiveness Group (REG) and Research in Real Life; the OPCRD statistical analysis was funded by REG. The Bocholtz Study was funded by PICASSO for COPD, an initiative of Boehringer Ingelheim, Pfizer and the Caphri Research Institute, Maastricht University, The Netherlands.

Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention

This study was supported by the UK Natural Environment Research Council (NE/H019456/1) to CJvdG, by the Wellcome Trust (WT 098051) to AWW and JP for sequencing costs, and by The Anna Trust (KB2008) to KDB. AWW and The Rowett Institute of Nutrition and Health, University of Aberdeen, receive core funding support from the Scottish Government Rural and Environmental Science and Analysis Service (RESAS). We thank Paul Scott, Richard Rance and the Wellcome Trust Sanger Institute’s sequencing team for generating 16S rRNA gene sequence data.

Use of intrinsic modes in biology: Examples of indicial response of pulmonary blood pressure to ± step hypoxia

Recently, a new method to analyze biological nonstationary stochastic variables has been presented. The method is especially suitable to analyze the variation of one biological variable with respect to changes of another variable. Here, it is illustrated by the change of the pulmonary blood pressure in response to a step change of oxygen concentration in the gas that an animal breathes. The pressure signal is resolved into the sum of a set of oscillatory intrinsic mode functions, which have zero “local mean,” and a final nonoscillatory mode. With this device, we obtain a set of “mean trends,” each of which represents a “mean” in a definitive sense, and together they represent the mean trend systematically with different degrees of oscillatory content. Correspondingly, the oscillatory content of the signal about any mean trend can be represented by a set of partial sums of intrinsic mode functions. When the concept of “indicial response function” is used to describe the change of one variable in response to a step change of another variable, we now have a set of indicial response functions of the mean trends and another set of indicial response functions to describe the energy or intensity of oscillations about each mean trend. Each of these can be represented by an analytic function whose coefficients can be determined by a least-squares curve-fitting procedure. In this way, experimental results are stated sharply by analytic functions.

Phenotypic switching in the human pathogenic fungus Cryptococcus neoformans is associated with changes in virulence and pulmonary inflammatory response in rodents

High-frequency reversible changes in colony morphology were observed in three strains of Cryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as 10−3. Mice infected with colony type W died faster than those infected with other colony types. The rat inflammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inflammation with caseous necrosis for infection with type C to minimal inflammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains of C. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate that C. neoformans undergoes phenotypic switching and that this process can affect virulence and host inflammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.

Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis

The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin β6 subunit (β6−/−), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

Nonlinear indicial response of complex nonstationary oscillations as pulmonary hypertension responding to step hypoxia

This paper is devoted to the quantization of the degree of nonlinearity of the relationship between two biological variables when one of the variables is a complex nonstationary oscillatory signal. An example of the situation is the indicial responses of pulmonary blood pressure (P) to step changes of oxygen tension (ΔpO2) in the breathing gas. For a step change of ΔpO2 beginning at time t1, the pulmonary blood pressure is a nonlinear function of time and ΔpO2, which can be written as P(t-t1 | ΔpO2). An effective method does not exist to examine the nonlinear function P(t-t1 | ΔpO2). A systematic approach is proposed here. The definitions of mean trends and oscillations about the means are the keys. With these keys a practical method of calculation is devised. We fit the mean trends of blood pressure with analytic functions of time, whose nonlinearity with respect to the oxygen level is clarified here. The associated oscillations about the mean can be transformed into Hilbert spectrum. An integration of the square of the Hilbert spectrum over frequency yields a measure of oscillatory energy, which is also a function of time, whose mean trends can be expressed by analytic functions. The degree of nonlinearity of the oscillatory energy with respect to the oxygen level also is clarified here. Theoretical extension of the experimental nonlinear indicial functions to arbitrary history of hypoxia is proposed. Application of the results to tissue remodeling and tissue engineering of blood vessels is discussed.