Lung cancer is the leading cause of death from cancer in the world. First, we hypothesized that microRNA expression is altered in the bronchial epithelium of patients with lung cancer and that incorporating microRNA expression into an existing mRNA biomarker may improve its performance. Using bronchial brushings collected from current and former smokers, we profiled microRNA expression via small RNA sequencing for 347 patients with available mRNA data. We found that four microRNAs were under-expressed in cancer patients compared to controls (p<0.002, FDR<0.2). We explored the role of these microRNAs and their gene targets in cancer. In addition, we found that adding a microRNA feature to an existing 23-gene biomarker significantly improves its performance (AUC) in a test set (p<0.05). Next, we generalized the biomarker discovery process, and developed a visualization tool for biomarker selection. We built upon an existing biomarker discovery pipeline and created a web-based interface to visualize the performance of multiple predictors. The “visualization” component is the key to sorting through a thousand potential biomarkers, and developing clinically useful molecular predictors. Finally, we explored the molecular events leading to the development of COPD and ILD, two heterogeneous diseases with high mortality. We hypothesized that integrative genetic and expression networks can help identify drivers and elucidate mechanisms of genetic susceptibility. We utilized 262 lung tissue specimens profiled with microRNA sequencing, microarray gene expression and SNP chip genotyping. Next, we built condition specific integrative networks using a causality inference test for predicting SNP-microRNA-mRNA associations, where the microRNA is a predicted mediator of the SNP’s effect on gene expression. We identified the microRNAs predicted to affect the most genes within each network. Members of miR-34/449 family, known to promote airway differentiation by repressing the Notch pathway, were among the top ranked microRNAs in COPD and ILD networks, but not in the non-disease network. In addition, the miR-34/449 gene module was enriched among genes that increase in expression over time when airway basal cells are differentiated at an air-liquid interface and among genes that increase in expression with the airway wall thickening in patients with emphysema. / 2019-07-31T00:00:00Z

Os nanocristais são partículas de fármacos cristalinos, com tamanho médio na faixa de submicrons, geralmente entre 200 e 500 nm, estabilizados por agentes estéricos ou eletrostáticos adsorvidos na superfície das partículas do fármaco. Sua dimensão reduzida proporciona propriedades especiais, como a adesividade às mucosas e o aumento de área superficial e da solubilidade de saturação, o que melhora significativamente a biodisponibilidade de fármacos pouco solúveis em água. Outra aplicação emergente dos nanocristais é na melhoria da entrega e da retenção de fármacos em tecidos e células tumorais. Estudos demonstraram que o flubendazol é um fármaco capaz de induzir a morte celular em tumores malignos e retardar o seu crescimento, por meio da alteração que provoca na estrutura dos microtúbulos e pela inibição da polimerização da tubulina. Foi demonstrada sua atividade antiproliferativa em linhagens de leucemia, mieloma, câncer intestinal, câncer de mama e neuroblastoma. O flubendazol é também um fármaco eficaz contra os helmintos, demonstrando atividade superior na eliminação dos vermes adultos, quando comparado com a dietilcarbamazina. Embora o flubendazol pareça ser uma molécula promissora, é um fármaco praticamente insolúvel em água (0,005 mg/mL). Para atingir o efeito terapêutico desejado, é necessário o desenvolvimento de uma formulação com melhores solubilidade e biodisponibilidade. Nesse sentido, o presente trabalho apresenta o preparo e a caracterização físico-química de nanocristais de flubendazol por meio da microfluidização. Foram realizados ensaios exploratórios para avaliar a performance de diferentes agentes estabilizantes nas suspensões: o polissorbato 80, o polaxamer 188 e o D-&#945; tocoferol polietilenoglicol 1.000 succinato (TPGS). A avaliação da distribuição do tamanho de partícula foi realizada por espalhamento de luz laser (LLS), espalhamento de luz dinâmica (DLS), análise de rastreamento de nanopartículas (NTA) e microscopia eletrônica de varredura (MEV). A utilização do TPGS favoreceu a obtenção de uma nanossuspensão com o menor diâmetro hidrodinâmico médio das partículas, de 253,9 &#177; 3,0 nm. Nos estudos exploratórios, também foram determinados os parâmetros ótimos de moagem do microfluidizador, sendo estabelecidos: 35.000 psi de pressão, temperatura do produto de 30°C (&#177; 5°C) e tempo de recirculação de 2 horas/100 gramas. Objetivando alcançar o menor diâmetro hidrodinâmico médio dos nanocristais, executou-se um planejamento estatístico no qual foi avaliada a influência da concentração de flubendazol (% p/p) e de TPGS (% p/p) na formulação. A análise revelou a significativa influência da concentração do TPGS na redução do tamanho de partícula e na estabilidade físico-química da nanossuspensão. Ensaios complementares de solubilidade demonstraram que o nanocristal proporcionou incremento na solubilidade de 2,3 e 3,2 e 5,2 vezes em HCl 0,1 N, tampão fosfato pH 6,8 e tampão fosfato salino pH 7,4, respectivamente. No ensaio de dissolução conduzido em HCl 0,1 N e 0,1% TPGS, observou-se significativo incremento, de 41% de fármaco dissolvido após 60 minutos, quando comparado com o flubendazol micronizado. As características do estado sólido do nanocristal foram avaliadas por meio de análise térmica (calorimetria exploratória diferencial e termogravimetria) e difratometria de raios X, não sendo observadas significativas alterações da estrutura cristalina. O presente trabalho também avaliou a efetividade dos nanocristais de flubendazol em tumores de pulmão, demonstrando sua expressiva capacidade de retardar o crescimento e diminuir o tamanho desses tumores em camundongos xenotransplantados. / Nanocrystals are drug particles with an average size in the sub-micron range, generally between 200 and 500 nm, stabilized by steric or electrostatic agents adsorbed on the surface of the drug particles. The reduced size provides special properties such as mucosal adhesiveness, increase in surface area and saturation solubility, which significantly improves the bioavailability of poorly water-soluble drugs. Another emerging application of nanocrystals is in the enhancement of drug delivery and retention in tumor tissues. Studies have shown that flubendazole is a drug capable of inducing cell death in malignant tumors and decelerating their growth, by altering the structure of the microtubules and inhibiting the tubulin polymerization. Antiproliferative activity has been demonstrated in leukemia, myeloma, intestinal cancer, breast cancer and neuroblastoma lines. In addition, flubendazole is also an effective drug against helminths, demonstrating superior activity in eliminating adult worms when compared to diethylcarbamazine. Although flubendazole appears to be a promising molecule, it is an insoluble drug in water (0.005 mg / mL). To achieve the desired therapeutic effect, it is necessary the development of a formulation with better solubility and bioavailability. In this context, the present research reports the physico-chemical preparation and characterization of flubendazole nanocrystals through microfluidization. Exploratory experiments were carried out to evaluate the performance of different stabilizing agents in formulations: polysorbate 80, polaxamer 188 and D-&#945; tocopherol polyethylene glycol 1000 succinate (TPGS). The determination of the particle size distribution determination was performed by laser light scattering (LLS), dynamic light scattering (DLS), nanoparticle scanning (NTA) and scanning electron microscopy (SEM). The use of TPGS favored the preparation of a nanosuspension with the lowest mean hydrodynamic size of the particles, of 253.9 &#177; 3 nm. In the exploratory studies, the optimum grinding parameters were also determined: 35,000 psi of microfluidizer pressure, product temperature of 30 ° C (&#177; 5 ° C) and recirculation time of 2 hours for each 100 grams of suspension. In order to reach the lowest average hydrodynamic diameter, a statistical design was applied in which the influence of flubendazole concentration (% w / w) and TPGS (% w / w) on the formulation was evaluated. The analysis revealed a significant influence of TPGS concentration on the particle size reduction and on the physicochemical stability of the nanosuspension. Complementary solubility tests showed that the nanocrystal provided an increase in solubility of 2.3, 3.2 and 5.2-fold in 0.1 N HCl, phosphate buffer pH 6.8 and phosphate buffer saline pH 7.4, respectively. In the dissolution test performed in 0.1 N HCl with 0.1% TPGS, a 41% increase of the drug dissolved after 60 minutes was achieved, when compared to micronized flubendazole. The solid-state characteristics of the nanocrystal were accessed through thermal analysis (differential scanning calorimetry and thermogravimetry) and X-ray diffraction and the results indicated that the crystal structure was not significantly altered. This research also evaluated the action of flubendazole nanocrystals in lung tumors, demonstrating expressive ability to retard growth and decrease the size of these tumors in xenotransplanted mice.

No description available.

Lung function depends on the expansion and contraction of lung tissue during the respiratory cycle. The measurement of regional pulmonary function is of great interest and importance since many lung diseases can cause changes in biomechanical or material properties. It is also significant to study the radiation-induced changes in pulmonary function following radiation therapy. In this thesis, we propose a technique that uses four-dimensional (3D+time) CT imaging (4DCT), 3D non-rigid image registration to estimate regional lung function. Lung images reconstructed at different inflation levels are analyzed for dynamic lung function development during a breath cycle. We demonstrate local pulmonary function can be reproducibly measured using 4DCT in human subjects prior to RT. The image registration accuracy is validated using semi-automatic anatomic landmark picking system. The major contributions of this thesis include: 1) demonstrating the robustness and reproducibility of regional pulmonary function measurement using 4DCT in both sheep and human subjects, 2) developing approaches to improve the measurement reproducibility by dynamic lung volume matching and Jacobian normalization, 3) development and comparison four cubic metrics for reproducibility analysis, 4) research on time-varying lung ventilation in different breathing phases in both sheep and human subjects. Our contributions in this thesis are useful for diagnosis and assessment of lung diseases, useful for qualifying radiation induced changes in pulmonary function in irradiated and non-irradiated lung tissue.

The main function of the respiratory system is gas exchange. Since many disease or injury conditions can cause biomechanical or material property changes that can alter lung function, there is a great interest in measuring regional lung function and mechanics. In this thesis, we present a technique that uses multiple respiratory-gated CT images of the lung acquired at different levels of inflation with both breath-hold static scans and retrospectively reconstructed 4D dynamic scans, along with non-rigid 3D image registration, to make local estimates of lung tissue function and mechanics. We validate our technique using anatomical landmarks and functional Xe-CT estimated specific ventilation. The major contributions of this thesis include: 1) developing the registration derived regional expansion estimation approach in breath-hold static scans and dynamic 4DCT scans, 2) developing a method to quantify lobar sliding from image registration derived displacement field, 3) developing a method for measurement of radiation-induced pulmonary function change following a course of radiation therapy, 4) developing and validating different ventilation measures in 4DCT. The ability of our technique to estimate regional lung mechanics and function as a surrogate of the Xe-CT ventilation imaging for the entire lung from quickly and easily obtained respiratory-gated images, is a significant contribution to functional lung imaging because of the potential increase in resolution, and large reductions in imaging time, radiation, and contrast agent exposure. Our technique may be useful to detect and follow the progression of lung disease such as COPD, may be useful as a planning tool during RT planning, may be useful for tracking the progression of toxicity to nearby normal tissue during RT, and can be used to evaluate the effectiveness of a treatment post-therapy.

Lung segmentation is an important first step for quantitative lung CT image analysis and computer aided diagnosis. However, accurate and automated lung CT image segmentation may be made difficult by the presence of the abnormalities. Since many lung diseases change tissue density, resulting in intensity changes in CT image data, intensity-only segmentation algorithms will not work for most pathological lung cases. This thesis presents two automatic algorithms for pathological lung segmentation. One is based on the geodesic active contour, another method uses graph search driven by a cost function combining the intensity, gradient, boundary smoothness, and the rib information. The methods were tested on several 3D thorax CT data sets with lung disease. Given the manual segmentation result as gold standard, we validate our methods by comparing our automatic segmentation results with Hu's method. Sensitivity, specificity, and Hausdorff distance were calculated to evaluate the methods.

Cystic fibrosis (CF) is an inherited disease leading to disrupted function of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. CF affects many organ systems including the pancreas, liver, intestine, sweat glands, and gallbladder. The leading cause of morbidity and mortality, however, is lung disease. A porcine model of CF was developed, and over time it develops lung disease that recapitulates many of the characteristics observed in humans with CF including airway remodeling, mucus accumulation, infection, and inflammation. At birth, and despite the absence of inflammation and infection, the CF pig airways exhibit a host of abnormalities including tracheal cartilage ring defects, abnormal appearing smooth muscle bundles, reduced trachea diameter, and reduced mainstem bronchi diameter. The primary objectives of this study were to construct an experimental method that allowed for the attainment of airway size information at multiple inflation pressures, to assess the extent of airway narrowing in the newborn CF porcine lung at 20 cmH2O, to determine the tracheal lobe volume for CF and non-CF, and to perform morphologic assessment of the parenchymal airspaces for CF and non-CF newborn pigs. Micro-computed tomography (micro-CT) was selected as the primary analysis tool. The volumetric, high resolution data sets of micro-CT provided a means to virtually track airways through the three dimensional space of the lung, and to image airways as small as 250 microns in diameter. Due to experimental constraints, only one lobe was analyzed: the tracheal lobe; it is the porcine equivalent of the human right upper lobe. Each excised tracheal lobe was cannulated and micro-CT scanned five times. Each lobe was scanned at multiple inflation pressures ranging from 0 to 20 cmH2O. The airways were segmented with a custom designed, substantially-automated computer algorithm. Quantitative analysis of airway size was done with the Pulmonary Workstation 2 software package. At a pressure of 20 cmH2O, the CF airway narrowing was most pronounced in the large airways of the tracheal lobe, and the percent difference in airway cross sectional area between CF and non-CF lessened for airways of smaller size. The volume of the newborn CF pig tracheal lobe was approximately twenty percent smaller than non-CF, but no differences were observed in tracheal lobe airspace histology between the groups. Airway size deviations at birth imply developmental abnormalities in utero that are dependent upon CFTR function. Additionally, the observation that reduced airway caliber exists only in relatively large airways suggests a time-dependent role of CFTR on airway development, as the large airways develop before the small ones in utero. These findings may provide insight to the early pathogenesis of CF lung disease.

Lung airway epithelium is the first line of defense against inhaled particulates such as cigarette smoke. Subepithelial dendritic cells (DC) survey the airway epithelial lining and represent the link between innate and adaptive immune response. No study has investigated the effect of cigarette smoke on DC in the presence of epithelial cells (EC). The purpose of this 4x2 factorial design study was to co-culture normal human bronchial epithelial (NHBE) cells with monocyte-derived dendritic cells (MDDC) and examine the effect of cigarette smoke condensate (CSC) and poly I:C stimulation (TLR3 ligand that mimics viral infection) on MDDC homotypic clustering, phagocytosis ability, surface marker expression, DC cytokine response, T cell (TC) proliferation and TC cytokine response. Experiments were performed with MDDC and TC derived from four individual donors. Two planned comparisons (DMSO vehicle control compared to CSC high dose in both the no poly I:C and poly I:C stimulated groups) were analyzed. In MDDC stimulated with CSC, there was a significant increase in homotypic clustering, reduced phagocytosis, increased CD54, increased CD83 and CD86 maturation marker expression. Although no significant changes were observed in MDDC cytokine production, IL10 exhibited a trend to increase with CSC exposure but failed to reach statistical significance. Despite evidence that CSC exposed MDDC are maturing, there was no increase in TC proliferation; however in poly I:C stimulated co-cultures, CSC exposure increased IL2, IL5, IL10 and IL13 expression while non-stimulated co-cultures exhibited an increase in IL10 following CSC exposure. This study indicates that cigarette smoke has the ability to decrease phagocytosis ability of DC and increases co-stimulatory maturation markers without inducing TC proliferation and potentiating a Th2 environment. These findings suggest that DC of smokers may be less likely to mount a normal immune response to invading pathogens thus providing evidence for smoker susceptibility to infection and how DC of smokers may respond to viral infection. In addition, these findings are particularly important in patients with allergic airway disease since increasing Th2 cytokines would increase the risk for disease exacerbation.

During wintertime temperature inversion episodes the concentrations of particulate air pollution, also defined as particulate matter (PM), in Utah’s Cache Valley have often been highest in the nation, with concentrations surpassing more populated and industrial areas. This has attracted much local and national attention to the area and its pollution. The Cache Valley has recently been declared to be in non-attainment of provisions of Federal law bringing to bear Federal regulatory attention as well. While there is epidemiological evidence indicating that PM is detrimental to public health, there is much less information indicating by which biological and molecular mechanisms PM can exert harm. This study was undertaken to better understand the mechanisms by which ambient PM collected in the Cache Valley can be harmful to human lung cells. Cache Valley PM was found to be mildly cytotoxic only at concentrations that were much greater than physiologically achievable, and such concentrations were difficult to obtain with the limited amounts of captured ambient PM. The limited cytotoxicity was despite apparent PM-induced pro-apoptotic signaling such as caspase-3 upregulation, and activation of caspase-12 and calpain. Cache Valley PM was found to be stressful to cells, triggering endoplasmic reticulum stress and the unfolded protein response. Cache Valley PM was also found to be inflammogenic leading to activation of pro-inflammatory transcription factors, increases in the release of pro-inflammatory cytokines and chemokines, as well as the upregulation of the activating receptors of these cytokines. The proinflammatory effects and absence of apoptosis, despite pro-apoptotic signaling of the Cache Valley PM on human lung cells appeared to stem from increased activation of the central pro-growth protein Akt with subsequent inactivation of the tumor suppressor P-TEN. These findings have indicated novel mechanisms of PM-related cellular stress and inflammation contributing needed information on what may be underlying mechanisms of PM associcated illnesses.

Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality as well as delayed clearance of the virus. Efforts to improve influenza infection outcomes in neonates typically center on prevention, although current vaccines fall short of complete protection and can only be administered in humans after 6 months of life. We propose that as the neonatal immune system responds differently than the adult immune system, interventions that are efficacious or tolerable in adults cannot be guaranteed to perform the same in neonates. T cell vaccines that target conserved influenza virus epitopes have been proposed for conferring protection to multiple influenza virus strains, but if T cell vaccines will be used in infants and adults, neonates must be able to respond to the same T cell antigens as adults. Mouse pups responded to influenza virus peptide PA224-233 but not NP366-374 during influenza virus infection in contrast to the codominant adult response. Mice infected as pups also generated diminished T cell memory compared to mice infected as adults and displayed skewed immunodominance during secondary infection. Adult bone marrow derived dendritic cells (BMDCs) improved viral clearance when loaded with influenza virus and promoted NP366-374-specific CD8+ T cell responses in infected pups. BMDC peptide vaccination could stimulate PA224-233-specific but not NP366-374-specific CD8+ T cell responses in pups, but, PA224-233 vaccination offered no protection to pups during lethal infection. These data suggest that altered immunodominance must be considered when stimulating CD8+ T cell responses in adults and neonates. Immaturity and active suppression of immune responses are both factors in neonatal vulnerability to disease. Specifically, active suppression of neonatal immunity by regulatory T cells (Tregs) has been proposed as a driving factor in diminished neonatal immunity, but removing these cells can compromise viral defense or increase deleterious inflammation. Mice that lacked Tregs displayed compromised anti-influenza antibody responses and decreased lymph node responses during influenza virus infection. A high proportion of pup Tregs also expressed Gata3. Transgenic pups with a Treg specific Gata3 knockout displayed an increase in Tbet expression in both conventional and regulatory T cells and an increase in IFNγ producing CD4+ T cells in the lungs during infection. These data suggest that Tregs are required for effective humoral responses to influenza virus and that Gata3 expression influences Treg suppressive function in neonates.