Browsing by Subject "Cystic Fibrosis"

Now showing 1 - 8 of 8
  • Thumbnail Image
    Item
    Association of Genetic Variation and Exercise Capacity with Clinical Indicators of Disease in Cystic Fibrosis
    (2019-08) Zavala, Hanan
    Introduction Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), which results in ion dysregulation and mucous buildup, most notably in the lungs. Previously a childhood disease, advancement in treatment options has greatly improved clinical course leading to longer lifespans. This progress has created a need for more sensitive clinical measures and personalized medicine options. Exploring genetic modifiers of disease along with response to exercise would provide valuable and unique information, contributing to better, more personalized assessment and management of disease. Purpose The purpose of this dissertation is two-fold. The first aim was to explore the impact of genetic variation at amino acid 663 of the sodium channel epithelial 1 alpha gene (SCNN1A) on clinical features of CF. In Study One it was hypothesized that subjects with at least one copy of the gain-of-function variant T663 (AT/TT) would have poorer clinical outcomes than those homozygous for the wild-type variant A663 (AA). In studies Two and Three the primary aim was to examine the clinical value of the six-minute walk test (6MWT) and one-minute sit-to-stand test (1STS) in the management of CF. It was hypothesized that response to the 6MWT and 1STS would be strongly correlated and measures from each test would be associated with clinical outcomes of disease. Methods Thirty-five CF subjects were enrolled and all had at least one copy of the F508del mutation. Buccal swabs were collected and samples were analyzed for genetic variation at position 663 of the SCNN1A gene (AA, AT/TT). The 6MWT and 1STS were performed. Continuous monitoring of heart rate (HR) and peripheral blood oxygen saturation (SpO2) was taken during exercise testing. The desaturation-distance ratio (DDR) was calculated using SpO2 measures and six-minute walk distance (6MWD). Medical charts were reviewed for pulmonary function and indicators of disease status. Depending on distribution of data, independent Student’s t-test or Mann Whitney U test was used to compare means. Correlations were performed using Spearman test and Fisher’s exact test was used to analyze categorical variables. Cox regression was used to assess days to pulmonary exacerbation. All data are presented as mean ± standard deviation unless otherwise noted. Significance was set at 0.05. Results There were no statistically significant differences in clinical outcomes between the AA and AT/TT genotypes. Clinically relevant observations in regards to lung function over time and exercise performance were noted and warrant further research. Further, 6MWD and 1STS repetitions were significantly correlated but neither outcome correlated with measures of pulmonary function. However, DDR was significantly correlated with several measures of pulmonary function, suggesting it is a better indicator of lung function than 6MWD alone. Additionally, those who desaturated during the 1STS (change in SpO2 > 4% from rest) had significantly lower lung function compared to those who did not. Neither 6MWD nor 1STS repetitions was associated with pulmonary exacerbation during follow-up. Those who experienced a pulmonary exacerbation during follow-up had significantly greater DDR compared to those who did not have an exacerbation. Conclusions Variation at position 663 of the SCNN1A gene may modify pulmonary disease in patients with CF, though further research is needed. Additionally, the 6MWT and 1STS show promise in providing unique and meaningful information about CF disease and, used in conjunction with typical 6MWT outcomes, DDR may be a helpful tool in evaluating exercise capacity in patients with CF.
  • Thumbnail Image
    Item
    Bacterial communities associated with chronic rhinosinusitis and the impact of mucin degradation on Staphylococcus aureus physiology
    (2020-08) Lucas, Sarah
    Chronic rhinosinusitis (CRS) is a heterogeneous disease of the paranasal sinuses. Anatomic variation, genetic mutation, viral infection, and allergic response, are all considered to play etiological roles in CRS. Contributors to pathogenesis include immune dysfunction, impaired mucociliary clearance, increased mucus production and stasis, and bacterial virulence mechanisms, however their importance and order of occurrence is not well understood. Microbiological surveys have implicated Staphylococcus aureus as one of the most frequent bacteria in the CRS sinuses. Paradoxically, S. aureus asymptomatically colonizes the nasal passages of ~50% of healthy adults. This work aimed to further describe the diversity of bacterial communities in the paranasal sinuses of patients with CRS using genomic and bioinformatic methods, and reveal new insights into the balance between S. aureus commensalism and pathogenesis. CRS is particularly prevalent in people with the genetic disorder cystic fibrosis (CF). Bacterial infection of the upper airways is thought to be a focus for pulmonary infection, which accounts for the majority of morbidity and mortality in this population. Using 16S rRNA gene sequencing, we described highly similar bacterial communities between sinus mucus and lung sputum in CF patients. CF patients exhibited high interindividual variation, and several instances where a canonical CF pathogen (e.g. S. aureus, Pseudomonas aeruginosa) was seen in the sinus mucus, but not sputum, suggesting this is a location of initial pathogen colonization and adaptation. Further investigation of the bacterial communities in sinus mucus revealed that diversity of the bacterial community was significantly greater in communities with S. aureus as the dominant organism. In contrast, Pseudomonas dominated communities were less diverse, and associated with both increased patient age, and decreased lung function, though not significantly. These results contribute to a growing body of research suggesting a temporal progression of bacterial diversity in the CF airways towards a community characterized by P. aeruginosa dominance. We further hypothesized that the presence of other bacterial taxa could have differential effects on S. aureus physiology in the non-CF CRS. Using 16S rRNA gene sequencing, the bacterial communities were characterized in CRS and non-CRS patient sinus mucus accessed through endoscopic sinus surgery. Compared to the bacterial communities observed in non-CRS mucus, CRS was characterized by an increased proportional abundance of facultative and obligate anaerobic bacterial genera including Streptococcus, Prevotella, and Fusobacterium, and a decrease in Actinobacteria compared. Guided by taxonomy, we computationally predicted the metagenomic content of CRS-associated communities, which suggested an increased functional capacity for the degradation of mucin-glycoproteins - a highly abundant constituent of mucus responsible for its many biological and physical characteristics. Using anaerobic enrichment of CRS sinus mucus bacterial communities on mucins in a minimal medium, we demonstrated a mucin-degradation phenotype, and further described the bacterial communities to include anaerobic genera such as Streptococcus, Prevotella, Veillonella, and Fusobacterium. This work shows that mucins are not efficiently used as a growth substrate by S. aureus, however, mucin degradation and fermentation by CRS-derived bacterial consortia produced metabolites that could augment S. aureus growth. Furthermore, transcriptomic analysis suggests mucin degradation and fermentation products affect S. aureus metabolic state and virulence potential. This work presents high-resolution molecular characterization of the bacterial communities associated with CRS, demonstrates mucin-degradation as a bacterial phenotype carried out by these communities, and suggests a role for mucin-degradation in supporting the growth and virulence of S. aureus.
  • Thumbnail Image
    Item
    Bacterial Mucin Degradation in the Cystic Fibrosis Airways: A Potential Regulator of P. aeruginosa Pathogenicity.
    (2023-05) Villarreal, Alex
    Cystic fibrosis (CF) is characterized by impaired mucociliary clearance, leading to mucus accumulation and chronic bacterial respiratory infections. Pseudomonas aeruginosa (PA) is a canonical pathogen in these infections and is found to nutritionally benefit from cross-feeding interactions with co-colonizing mucin-degrading bacterial genera. However, the impact of these cross-feeding interactions on the regulation of PA virulence remains unclear. Implementing a reductionist experimental approach, this thesis aims to investigate the proposed cross-feeding model to identify key exchanged metabolites and elucidate mechanisms underlying PA virulence. To successfully isolate and analyze variables of the multifaceted cross-feeding model, several specialized experimental tools and techniques were developed and described in Chapter 2. These tools were employed in Chapter 3 to fully characterize the growth profiles of several individual representative mucin-degrading bacterial species and investigate the effects of their secondary metabolites on PA physiology. Chapter 4 builds upon this by characterizing complex mucin-degrading bacterial communities enriched from clinical CF sputum samples, and again investigating their effects on PA physiology in vitro. These data establish validated methods for the field of CF research, expand our understanding of PA physiology, and pave the path for future development of novel targeted CF therapeutic strategies.
  • Thumbnail Image
    Item
    CFTR Genotype, Not Circulating Catecholamines, Influence Cardiovascular Function in Cystic Fibrosis
    (2015-07) Bisch, Alexander
    Objective: Cystic fibrosis (CF) is a genetic disease that elicits affects throughout the body and is characterized by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) with direct, well-documented, pulmonary function consequences. It has been shown a single dose of a Beta-agonist increases cardiac output (Q) and stroke volume (SV) and decreases systemic vascular resistance (SVR) in healthy subjects. This effect is attenuated in CF subjects; however, it is unknown if this decreased cardiovascular response to an inhaled drug is due to inherent cardiovascular deficits from CFTR mutation, receptor desensitization from prolonged Beta- agonist use, or inhibited drug delivery to the blood stream due to mucus buildup in the lungs. This study sought to determine the effects of endogenous epinephrine (EPI) and norepinephrine (NE) on cardiovascular function in CF, and to evaluate cardiovascular function according to CFTR mutation [Delta]F508 Ins/DEL). Methods: Eleven CF subjects and 27 healthy control participants completed a cycle ergometery test with measures of Q, SV, SVR, and HR along with plasma measures of EPI and NE. We compared subjects by variables of cardiovascular function relative to EPI and NE, and also based on genetic variants of [Delta]F508 Ins/DEL. Results: CF subjects demonstrated significantly lower Q and SV at 50% of peak exercise and peak exercise than healthy subjects, and a higher SVR at rest, 50% of peak, and peak exercise. Additionally, CF subjects also demonstrated significantly lower Q and SV relative to NE at rest, however there were no differences in HR relative to NE or SVR relative to EPI. When SV was stratified for CFTR mutation type, there were significant differences at rest, 50% of peak exercise, and there was a trend towards significance at peak exercise. Subjects with a double deletion of the [Delta]F508 had lower SV when compared to single deletion subjects. There were moderate and significant correlations found between EPI and SV and EPI and Q, but not in EPI and SVR when the study population was evaluated as a whole. Within the healthy group, there were significant correlations between EPI and SV, and EPI and Q, but none between EPI and SVR. Only correlations between EPI and Q were seen in the CF group. Conclusion: These results demonstrate that CF subjects have lower cardiovascular function parameters. Further, these results suggest that this impairment in cardiovascular function is likely the result of impairment in CFTR function due to CFTR genotype differences of the [Delta]F508, rather than receptor desensitization or inhibited drug delivery.
  • Thumbnail Image
    Item
    Colonoscopic Screening Shows Increased Early Incidence and Progression of Adenomas in Cystic Fibrosis
    (2017-01) Niccum, David
    Background. Colorectal cancer is an emerging problem in cystic fibrosis (CF). The goal of this study was to evaluate adenoma detection by systematic colonoscopic screening and surveillance. Methods. We analyzed prospectively collected results of colonoscopies initiated at age 40 years from 88 CF patients at a single Cystic Fibrosis Center. We also reviewed results of diagnostic colonoscopies from 27 patients aged 30-39 years performed during the same time period at the Center. Results. The incidence of polyp detection increased markedly after age 40 in CF patients. Greater than 50% were found to have adenomatous polyps; approximately 25% had advanced adenomas as defined by size and/or histopathology; 3% were found to have colon cancer. Multivariate analysis demonstrated specific risk factors for adenoma formation and progression. Conclusions. Early screening and more frequent surveillance should be considered in patients with CF due to early incidence and progression of adenomas in this patient population.
  • Thumbnail Image
    Item
    Development of tools for genome engineering in swine.
    (2009-09) Carlson, Daniel Fred
    Heightened interest in relevant models for human disease, in the production of transgenic livestock for biomedical applications, and new and pending releases of genome sequences for rat, cow, and pig have increased the need for improved methods for germline transgenesis. Transpositional transgenesis (TnT) offers an efficient and precise mechanism for genome integration of transgene DNA that avoids incorporation of CG-rich vector DNA and multi-copy transgene concatemerization that can lead to suppression of gene expression and transgene instability. We have developed and tested a transposon toolbox (including Sleeping Beauty, Tol2, piggyBac, and Passport) in pig cells. We have also demonstrated the activity of Cre and Flp recombinases in cultured pig cells and have implemented that technology for regulated activation of gene expression in swine. The application of transposon and recombinase technologies significantly enhances both the means and possible complexity of pig genetic modification. The use of enhanced cis and trans components of the Sleeping Beauty (SB) transposon system for animal transgenesis by pronuclear injection (PNI) resulted in tremendous improvement in the creation of transgenic laboratory mice, rats, and pig embryos, increasing both the frequency of transgenic founders, and the number of transgenes per founder, overall elevating the number of potential transgenic lines by 10-20-fold. Genetic modification of pigs by tandem pig transgenesis and cloning also benefits from TnT, resulting in multiple independent insertions per cellular clone which permits the assessment of several alleles upon segregation from a single founder. Finally, towards the development of a porcine model of cystic fibrosis (CF), we’ve created a mouse phenocopy of CF based on RNA interference using the SB transposon system. Building on insights gained in CFTRRNAi mouse, we’ve developed tightly regulated CFTRRNAi alleles in pigs to circumvent the lethal neonatal meconium ileus that confounds the CFTR-knockout model, hopefully simplifying investigation of postnatal CFTR deficiency in a large animal. Given its simplicity, versatility and high efficiency, TnT represents a compelling non-viral approach to modifying the porcine germline.
  • Thumbnail Image
    Item
    Investigating The Role Of Cystic Fibrosis Transmembrane Conductance Regulator On Oxidative Stress In Colorectal Cancer
    (2020-05) Singhania, Mekhla
    Colorectal cancer (CRC) is the third leading cause of cancer-related deaths among both men and women in the United States, with an estimated 53,200 deaths in 2020 (Siegel, Miller, & Jemal, 2020). Our lab identified Cystic Fibrosis Transmembrane Regulator (CFTR) as a CRC tumor suppressor gene in mice and humans. However, the mechanism by which CFTR acts as a tumor suppressor gene in CRC is unknown. To identify potential pathways, we compared gene expression in CFTR-low expressing CRC vs. CFTR high expressing tumors in several publicly available databases using gene set enrichment analysis. These analyses revealed that a common subset of genes is responsive in CFTR-low expressing tumors and in HIF1α-high tumors. HIF1α protein is induced by oxidative stress and is known to play a protective role in the oxidative stress pathway. To investigate the significance of this correlation, I hypothesized that downregulation of CFTR protects against oxidative stress. To test this hypothesis, I compared cell viability in human Caco-2 CRC cell lines: one in which CFTR has been knocked out by CRISPR-Cas9 modification (CFTR-KO) vs. matched parental controls (CFTR-WT). Each cell line was treated with menadione to induce oxidative stress. When cell viability was measured using an MTT assay, a trend was observed, where CFTR-KO cells were less affected by menadione than CFTR-WT. This suggests that CFTR deficiency may help colon cancer cells to survive better in an oxidative stress environment. To determine if CFTR deficiency promotes this survival of CRC cells by change in ROS levels, ROS detection assays were carried out to compare ROS levels between CFTR WT and CFTR KO cell lines. We observed a trend where CFTR-WT cells had higher ROS levels than CFTR-KO in this oxidative stress environment. This suggests that CFTR deficiency maybe promoting survival of CRC cells in an oxidative stress environment by reducing ROS levels. This work will help to better understand the role of CFTR as a tumor suppressor gene in CRC.
  • Thumbnail Image
    Item
    Newborn Screening for Cystic Fibrosis
    (2008-11-24) Fulmer, Calie
    Cystic fibrosis is a genetic, life-long disease of the lungs and digestive tract with many complications. Early diagnosis of cystic fibrosis through neonatal screening improves nutrition, improves growth, and healthier lungs.