Clinical research in cystic fibrosis (CF) requires study endpoints that are sensitive to airways disease, repeatable and non-invasive. Despite significant advances in the treatment of CF, lung function assessments continue to rely on the forced expiratory volume in 1 second (FEV1). Although simple to perform, it lacks sensitivity, is difficult for younger subjects, and changes over time. An alternative method of assessing lung physiology is to derive measures of ventilation heterogeneity from inert gas washout tests. In early lung disease, measures of gas mixing appear to be more sensitive than spirometry. In addition, since only tidal breathing is required, they are more physiological and are more straightforward for younger subjects. Widespread use has been impaired by the lack of a robust and cost effective gas analyser technology. The work presented in this thesis concerns the adaptation, validation and then use of a novel gas analyser (Innocor) in a clinical system for the performance of multiple breath washouts. Lung clearance index (LCI), a simple measure of ventilation heterogeneity, has been calculated from washouts in 52 adults with CF and 50 healthy controls. LCI was more sensitive to disease than FEV1 in CF, being elevated in 11 of the 12 CF patients with normal spirometry. In healthy subjects, LCI has been shown to be repeatable and reproducible, with a narrow range of normal that is stable over a wide age range. In a separate study of 19 patients, LCI has also been shown to improve with treatment of an exacerbation in CF. Correlation with changes in other biochemical (serum CRP, peripheral blood white cell count, sputum IL-8, sputum neutrophil) clinical (symptom score) or structural (computed tomography) markers was poor. Short term change in LCI has also been demonstrated in CF patients in response to chest physiotherapy, although there was considerable heterogeneity of response in terms of both LCI and volume of lung ventilated by tidal breathing (as measured by washout functional residual capacity). In addition to LCI, multiple breath phase III slope analysis has been performed on washouts of CF patients and healthy controls, and this has been compared to other measures of lung physiology. Proposed measures of convective and diffusive gas mixing have been shown to be unreliable in CF. These studies have also been the first to demonstrate multi-centre use of washout tests as endpoints. The technology described here offers the possibility of a simple and reliable system for performing multiple breath washouts, though at present it is not available commercially. The studies have added to the understanding of the utility and reliability of washout tests, as well as some of their limitations. It is hoped that in future LCI will be an important clinical endpoint in therapeutic intervention studies in CF, and that it will also offer new ways to follow changes in lung physiology in other diseases.

Small cell lung cancer (SCLC) is a highly aggressive malignancy with extreme mortality and morbidity. Although initially chemo- and radio-sensitive, almost inevitable recurrence and resistance occurs. SCLC patients often present with metastases, making surgery not feasible. Current therapies, rationally designed on underlying pathogenesis, produce in vitro results, however, these have failed to translate into satisfactory clinical outcomes. Recently, research into cancer stem cells (CSCs) has gained momentum and form an attractive target for novel therapies. Based on this concept, CSCs are the cause of neoplastic tissue development that are inherently resistant to chemotherapy, explaining why conventional therapies can shrink the tumour but are unable to eliminate the tumour completely, leading to eventual recurrence. Here I demonstrate that SCLC H345 and H69 cell lines contain a subset of cells expressing CD133, a known CSC marker. CD133+ SCLC sub-population maintained their stem cell-like phenotype over a prolonged period of culture, differentiated in appropriate conditions and expressed the embryonic stem cell marker Oct-4 indicating their stem-like phenotype. Additionally, these cells displayed augmented clonogenic efficacy, were chemoresistant and tumorigenic in vivo, distinct from the CD133- cells. Thus, the SCLC CD133 expressing cells fulfil most criteria of CSClike definition. The molecular mechanisms associated with CD133+ SCLC chemoresistance and growth is unknown. Up-regulated Akt activity, a known promoter of resistance with survival advantage, was observed in CD133+ SCLC cells. Likewise, these cells demonstrated elevated expression of Bcl-2, an anti-apoptotic protein compared to their negative counterpart explaining CD133+ cell chemoresistance phenotype. Additionally, CD133+ cells revealed greater expression of neuropeptide receptors, gastrin releasing peptide (GRP) and V1A receptors compared to the CD133- cells. Addition of exogenous GRP and arginine vasopressin (AVP) to CD133+ SCLC cells promoted their clonogenic growth in semi-solid medium, illustrating for the first time neuropeptide dependent growth of these cells. A novel peptide (peptide-1) was designed based on the known structure of the substance P analogues that have shown benefit in animal models and in early clinical trials. This compound inhibited the growth of SCLC cells in in vitro with improved potency and stability compared to previous analogues and reduced tumorigenicity in vivo. Interestingly, peptide-1 was more effective in CD133+ cells due to increased expression of neuropeptide receptors on these cells. In conclusion, my results show that SCLC cells retain a sub-population of cells that demonstrate CSC-like phenotype. Preferential activation of Akt and Bcl-2 survival pathways and enhanced expression of neuropeptide receptors contribute to CD133+ SCLC chemoresistance and growth. Therefore, it can be proposed that CD133+ cells are the possible cause of SCLC development, treatment resistance and disease recurrence. Despite being chemoresistant, CD133+ cells demonstrated sensitivity to peptide-1. The identification of such new analogue that demonstrates efficacy towards resistant CD133+ SCLC cells is a very exciting step forward in the identification of a potential new therapy for resistant disease.

<p>Lung squamous cell carcinoma (LSCC) comprises ~30% of non-small cell lung cancers, and currently lacks effective targeted therapies. Previous immunohistochemical and microarray studies reported overexpression of Hedgehog (HH)-GLI signaling components in LSCC. However, they addressed neither the tumor heterogeneity nor the requirement for HH-GLI signaling. Here, we investigated the role of HH-GLI signaling in LSCC, and studied the therapeutic potential of HH-GLI pathway suppression. </p><p>Gene expression datasets of two independent LSCC patient cohorts were analyzed to study the activation of HH-GLI signaling. Four human LSCC cell lines were examined for HH-GLI signaling components. Cell proliferation and apoptosis were assayed in these cells after blocking the HH-GLI pathway by lentiviral-shRNA knockdown or small molecule inhibitors. Xenografts in immunodeficient mice were used to determine the <italic>in vivo<italic> efficacy of GLI inhibitor GANT61. </p><p>In both patient cohorts, we found that activation of HH-GLI signaling was significantly associated with the classical subtype of LSCC. <italic>GLI2<italic> expression level was significantly higher than <italic>GLI1<italic>, and displayed strong positive correlations with the prominent markers for the classical subtype (<italic>SOX2<italic>, <italic>TP63<italic> and <italic>PIK3CA<italic>) on chromosome 3q. In cell lines, genetic knockdown of SMO produced minor effects on cell survival, while GLI2 knockdown significantly reduced proliferation and induced extensive apoptosis. Consistently, the SMO inhibitor GDC-0449 resulted in limited cytotoxicity in LSCC cells, whereas the GLI inhibitor GANT61 was very effective. Importantly, GANT61 demonstrated specific <italic>in vivo<italic> anti-tumor activity in xenograft models of GLI-positive cell lines. </p><p>Taken together, we report SMO-independent regulation of GLI in LSCC, and demonstrate an important role for GLI2 in LSCC. Different from standard-of-care chemotherapy or small molecule inhibition of kinase signaling cascades, we present a novel and potent strategy to treat a subset of LSCC patients by targeting the GLI transcriptional network.</p> / Dissertation

Einteilung:Das Lungenkarzinom ist eine der häufigsten Krebstodesursachen der Welt. Die chirurgische Therapie mit onkologischer Resektion des Tumors bietet bessere Chancen für einen dauerhaften Therapieerfolg. Die Bedeutung der systematischen Lymphadenektomie im Sinne eines akkuraten Stagings und einer besseren Lokalkontrolle des Tumors ist unumstritten. In der Literatur wurden verschiedene LK-Befallsmuster bei den operierten NSCLC anhand verschiedener histomorphologischer Parameter beschrieben, um letztendlich eine passende Technik der Lymphadenektomie zu entwickeln. Patienten und Methoden: In unserer retrospektiven Studie wurde ein Kollektiv von 111 Patienten mit operierten nicht kleinzelligen Lungenkarzinome zwischen 2008 und 2013 untersucht. Das LK-Metastasierungsmuster wurde anhand verschiedener histomorphologischer Parameter untersucht. Ergebnisse: Eine zentrale Tumorlage, L1-Kategorie, sowie die zunehmende Tumorgröße zeigten eine signifikante Neigung zur LK-Metastasierung. Anhand der Tumorlokalisation im Lungenlappen ließ sich jedoch kein bestimmtes LK- Befallmuster sichern. Schlussfolgerung: Diese Daten unterstützen die Angaben der Literatur, in der eine systematische Lymphadenektomie unabhängig von den anderen Parametern weiter gefordert wird, um ein akkurates Staging zur erreichen und so eine optimale Therapie durchzuführen.fi

During fetal development, the lung is filled with fluid that is secreted by an active Cltransport promoting lung growth. The basolateral Na+,K+,2Cl- cotransporter (NKCC1) participates in Cl- secretion. The apical Cl- channels responsible for secretion are unknown but studies suggest an involvement of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is developmentally regulated with a high expression in early fetal development and a decline in late gestation. Perinatal lung transition is triggered by hormones that stimulate alveolar Na+ channels resulting in fluid absorption. Little is known on how hormones affect pulmonary Cl- channels. Since the rise of fetal cortisol levels correlates with the decrease in fetal CFTR expression, a causal relation may be assumed. The aim of this study was to analyze the influence of glucocorticoids on pulmonary Cl- channels. Alveolar cells from fetal and adult rats, A549 cells, bronchial Calu-3 and 16HBE14o- cells, and primary rat airway cells were studied with real-time quantitative PCR and Ussing chambers. In fetal and adult alveolar cells, glucocorticoids strongly reduced Cftr expression and channel activity, which was prevented by mifepristone. In bronchial and primary airway cells CFTR mRNA expression was also reduced, whereas channel activity was increased which was prevented by LY-294002 in Calu-3 cells. Therefore, glucocorticoids strongly reduce CFTR expression while their effect on CFTR activity depends on the physiological function of the cells. Another apical Cl- channel, anoctamin 1 showed a glucocorticoid-induced reduction of mRNA expression in alveolar cells and an increase in bronchial cells. Furthermore, voltage-gated chloride channel 5 and anoctamine 6 mRNA expression were increased in alveolar cells. NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells alike. The results demonstrate that glucocorticoids differentially modulate pulmonary Clchannels and are likely causing the decline of CFTR during late gestation in preparation for perinatal lung transition.

Monitoring and mapping the presence and/or intensity of an environmental hazard through space, is an essential part of public health surveillance. Radon, a naturally occurring radioactive carcinogenic gas, is an environmental hazard that is both the greatest source of natural radiation exposure in human populations and the second leading cause of lung cancer worldwide. Concentrations of radon can accumulate in an indoor setting, and, though there is no safe concentration, various guideline values from different countries, organizations and regions provide differing threshold concentrations that are often used to delineate geographic areas at higher risk. Radon maps demarcate geographic areas more prone to higher concentrations but can underestimate or overestimate indoor radon risk depending on the concentration threshold used. The goals of this thesis are to map indoor radon risk in the province of British Columbia, identify areas more prone to higher concentrations and their associations with different radon concentration thresholds and lung cancer mortality trends. The first analysis was concerned with developing a data-driven method to predict and map ordinal classes of indoor radon vulnerability at aggregated spatial units. Spatially referenced indoor radon concentration data were used to define low, medium and high classes of radon vulnerability, which were then linked to regional environmental and housing data derived from existing geospatial datasets. A balanced random forests algorithm was used to model environmental predictors of indoor radon vulnerability and predict values for un-sampled locations. A model was generated and evaluated using accuracy, precision, and kappa statistics. We investigated the influence of predictor variables through variable importance and partial dependence plots. The model performed 34% better than a random classifier. Increased probabilities of high vulnerability were found to be associated with cold and dry winters, close proximity to major river systems, and fluvioglacial and colluvial soil parent materials. The Kootenays and Columbia-Shuswap regions were most at risk. We built upon the first analysis by assessing the difference between temporal trends in lung cancer mortality associated with areas of differing predicted radon risk. We assessed multiple scenarios of risk by using eight different radon concentration thresholds, ranging from 50 to 600 Bq m-3, to define low and high radon vulnerability. We then examined how the following parameters changed with the use of a different concentration threshold: the classification accuracy of each radon vulnerability model, the geographic characterizations of high risk, the population within high risk areas and the differences in lung cancer mortality trends between high and low vulnerability stratified by sex and smoking prevalence. We found the classification accuracy of the model improved as the threshold concentrations decreased and the area classified as high vulnerability increased. The majority of the population were found to live in areas of lower vulnerability regardless of the threshold value. Thresholds as low as 50 Bq m-3 were associated with higher lung cancer mortality trends, even in areas with relatively low smoking prevalence. Lung cancer mortality trends were increasing through time for women, while decreasing for men. We suggest a reference level as low as 50 Bq m-3 is justified for the province. / Graduate

We have studied gas flow and particle deposition in a realistic three-dimensional model of the bronchial tree, extending from the trachea to the segmental bronchi (7th airway generation for the most distal ones) using Computational Fluid Dynamics (CFD). The model is based on the morphometrical data of Horsfield et al. [J. Appl. Physiol., 31: 207-217, 1971] and on bronchoscopic and CT images, which give the spatial 3D-orientation of the curved ducts. It incorporates realistic angles of successive branching planes. Steady inspiratory flow varying between 50cm³/s and 500cm³/s was simulated as well as deposition of spherical aerosol particles (1 to 7 m diameter, 1g/cm³ density). Flow simulations indicated non-fully developed flows in the branches because of their relative short lengths. Velocity flow profiles in the segmental bronchi, taken one diameter downstream the bifurcation, were distorted compared with the flow in a simple curved tube, and wide patterns of secondary flow fields were observed. Both were due to the asymmetrical 3D configuration of the bifurcating network. Viscous pressure drop in the model was compared with results obtained by Pedley et al. [Respir Physiol, 9: 387-405, 1970], which are shown to be a good first approximation. Particle deposition increased with particle size and was minimal for approximately 200cm³/s inspiratory flow but it was highly heterogeneous for branches of the same generation.

No description available.

Glucocorticoids are steroid hormones that have potent anti-inflammatory actions. Endogenous glucocorticoid action is modulated by 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyses the interconversion of active glucocorticoids (cortisol, corticosterone) and intrinsically inert forms (cortisone, 11-dehydrocorticosterone). There are 2 isozymes; 11β-HSD type 1 regenerates active glucocorticoids in vivo whereas 11β-HSD type 2 inactivates glucocorticoids. Although 11β-HSD1 is highly expressed in the lung, its role there has been little explored. In this study, the expression and localization of 11β-HSD1 mRNA in lung was confirmed by in situ hybridization. Immunohistochemical staining of mouse lung localized 11β-HSD1 to the cytoplasm of fusiform cells in alveolar walls, in a multivesicular pattern characteristic of interstitial fibroblasts. A lung fibrosis model of inflammation was used to test the role and regulation of 11β-HSD1. The results suggest that levels of 11β-HSD1 mRNA and enzyme were not changed during bleomycin-induced lung inflammation. However, 11β-HSD1-deficient mice showed a more severe inflammatory response than congenic wild-type controls, with greater inflammatory cell infiltration into the lung, and increased levels of HO-1 and iNOS mRNA 14 days following bleomycin installation into lung. Picrosirius red staining of lung sections suggested more collagen deposition in 11β-HSD1-deficient mice than in wild-type controls during the course of the lung inflammatory response. Moreover, whereas naïve 11β-HSD1-deficient mice had significantly lower collagen content in lung (84% of WT levels, p<0.05). 28d after bleomycin there was no significant difference between genotypes (KO having 94% of WT levels, p=0.42) confirming more collagen production in 11β-HSD1-deficient mice following bleomycin. Fibroblasts are critical in the regulation of inflammatory responses and are essential in the model of bleomycin-induced lung injury. Lung fibroblasts may have a different transcriptional regulation of 11β-HSD1 compared to other tissues. In the majority of tissues, 11β-HSD1 can be transcribed from 2 promoters; the P1 promoter is the main promoter used in lung, with other tissues mainly using the P2 promoter. To address the relevance of the P1 promoter in lung and to identify the cell type using the P1 promoter, mouse lungs were collagenase-digested to isolate primary fibroblast and epithelial cells. Isolated lung fibroblasts highly expressed 11β-HSD1, predominantly from the P1 promoter. During passage, primary lung fibroblasts switched promoter usage from P1 to P2. In fibroblast primary culture, treatment with TGF-β for 72h markedly decreased 11β-HSD1 expression to 38% of untreated levels, an effect which was reversed by SB431542, a TGF-β receptor antagonist. Whilst TGF-β reduced levels of mRNA initiating at the P2 promoter, initiation from the P1 promoter was completely repressed. Treatment with TGF-β receptor antagonist increased levels of P1-initiated 11β-HSD1 mRNA by 6.6-fold compared to untreated cells. These data suggest that the switch in 11β-HSD1 promoter usage may be regulated by TGF-β during an inflammatory response. Furthermore, as the P1 and P2 promoters are differentially regulated (e.g. by C/EBPβ, a cytokine-responsive transcription factor), the promoter switch may place 11β-HSD1 under a different transcriptional regulation during inflammation. Taken together, these results suggest that 11β-HSD1 deficiency worsens lung inflammation and results in greater lung fibrosis. Therefore, amplification of intracellular glucocorticoids levels, by 11β-HSD1, may represent an important mechanism to limit the inflammatory response and shape fibroblast function, limiting subsequent collagen production and fibrosis.

Acute Lung Injury, characterised clinically as the Acute Respiratory Distress Syndrome is a catastrophic response to a range of pulmonary and non-pulmonary insults. Despite much work the key mechanisms involved in generating the exaggerated immune response that results in lung injury are not completely understood. Hypoxia-inducible factor-1 has been shown to be a key transcription factor in the myeloid cell response to inflammatory signals. The aims of this thesis were to develop a model of acute lung injury and to study the role of Hif-1 in the generation of lung injury in this model. A model of direct pulmonary injury as a result of intratracheal instillation of endotoxin is described. Using this model the role of myeloid cell Hif-1α was characterised using a myeloid cell specific conditional knockout system. The injury in Hif-1α deficient mice was quantitatively similar to the injury seen in wild type animals over a range of time points. However, the quality of the injury, assessed by a measure of nitric oxide mediated damage was reduced. The in vivo data were supported by in vitro studies using a murine macrophage cell line which showed that manipulation of the cellular oxygen tension in the presence of endotoxin alters the ability of the cell to generate nitric oxide. Furthermore, pharmacological manipulation of cellular Hif-1 levels by Dimethyloxallyl Glycine (DMOG) in the macrophage cell increased the generation of nitric oxide in response to endotoxin by altering the expression of a number of the isoforms of Nitric Oxide Synthase. In a final set of experiments the response to intratracheal endotoxin was modulated in mice by the concurrent administration of DMOG. As expected the qualitative response to endotoxin was similar but the NO mediated damage was enhanced in the animals administered DMOG. Manipulation of Hif-1 may have a role in the therapy of lung injury by altering the characteristics of the response.