Theses and Dissertations

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    Using gene expression and mutational profiling to characterize canine acute myeloid leukemia and assess their comparative features with human acute myeloid leukemia
    (Colorado State University. Libraries, 2023) Harris, Adam, author; Avery, Anne, advisor; Avery, Paul, committee member; Dow, Steven, committee member; Duval, Dawn, committee member
    Acute myeloid leukemia (AML) is an aggressive heterogenous hematopoietic neoplasm that afflicts both dogs and people. Over 10,000 individuals (about the seating capacity of Cameron basketball stadium at Duke University) in the United States succumb to AML-related deaths every year. Treatment options for AML have made little progress in the past few decades and prognosis for both human and canine AML (cAML) remains dismal. However, there are large ongoing multi-institutional studies devoted to advancing medical management for human AML (hAML) by providing targeted therapeutics to patients based on their molecular characteristics. A preclinical model for testing novel therapies could accelerate the development of better treatments in people. We hypothesize that cAML will have similar underlying molecular features as human AML and dogs could be a translational model for developing therapeutics focused on treating AML. The goals of this thesis were to assess the gene expression programs and mutational profiles of cAML and compare our findings with available human AML data. First, we established diagnostic criteria for defining cAML using flow cytometry. Next, we globally assessed normal hematopoiesis in dogs using single cell transcriptomics to generate a hematopoietic tree for defining the cellular composition of cAML. Additionally, we investigated the mRNA expression and genetic variants in cAML to ultimately compare the molecular features with pediatric and adult AML subtypes. We hope this work advances our knowledge of cAML molecular characteristics and adds further credentials to the dog as spontaneous model for human AML.
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    The role of vitamin A and mycobacterial lipids in the immunopathogenesis of Mycobacterium tuberculosis infections
    (Colorado State University. Libraries, 2023) Harris, Macallister C., author; Podell, Brendan, advisor; Zabel, Mark, committee member; Henao Tamayo, Marcela, committee member; Ryan, Elizabeth, committee member; Basaraba, Randall, committee member
    Until recently Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), was the deadliest and most ubiquitous infectious disease in the world, infecting an estimated 1.7 billion people as of 2018 and killing 1.6 million people in 2021. However, recently COVID-19 has displaced this disease as the most fatal disease, and in the process of doing so has disrupted integral structural apparatuses meant to monitor and treat TB in endemic countries. This degradation has led to an unprecedented rise in disease transmission with a 4.3% rise in infection, with 10.6 million new infections in 2021. Further compounding the problem of this wave of Mtb infections is the rise of multidrug resistant infections, accounting for 3.9% of all new infections and responsible for 14% of Mtb fatalities in 2017. With an upsurge in case incidence, faltering antibiotic regimens and a variably effective 100-year-old vaccine, there is a new push in understanding disease comorbidities and alternative treatments for TB. Although over 1 billion people are presumed to be infected with Mtb, though not all of these patients have clinical TB. In fact, the WHO estimates that 1/3 of infections results in latent disease, a non-clinical phase of the infection which can last in definitively or eventually progress into active, clinical apparent TB. Patient comorbidities have become an intense area of study for further defining the delineating factors that direct infected individuals into either active infection, latent infection, or overcoming the infection. Two defining comorbidities, HIV status and diabetes have been correlated to an increased risk for having progressive TB. HIV infections in itself has been demonstrated to cause a 20 to 30 fold increase risk in clinical manifestations of a Mtb infection, leading to 187,000 deaths in 2021. An equally prolific area of Mtb research lies in developing new, efficacious prophylactic and active infection treatment regimens. As previously mentioned, current first line drugs regimens, such as rifampicin and isoniazid, are faltering against multidrug resistant Mtb. Additionally, the only widely available vaccine, the Bacillus Calmette-Guerin vaccine, has high efficacious against several forms of TB, like adolescent TB meningitis, but has variable to low efficacy in control of classic pulmonary TB. These factors are driving research of novel treatments and vaccines that utilize niche characteristics of Mtb, host immunity and overall TB disease pathogenesis to prevent clinical disease manifestation. This thesis centers at the intersection of these two areas of this immunologic research, Mtb comorbidities and investigating a novel branch of immunology, by investigating the immunopathogenesis of TB in relation to a newly recognized comorbidity, vitamin A deficiency, and examining the utility of harnessing a niche lipid-based branch of immunology, CD1 lipid restricted immunology, to combat TB disease progression. In chapter one, this thesis, stemming from the foundational epidemiological work of Dr. Megan Murray, endeavored to investigate the pathophysiology of vitamin A deficiency and TB in a highly translatable guinea pig model. This novel vitamin A animal model demonstrated that vitamin A deficiency leads to a dysregulated immune system with atypically granulomatous lesions, skewed inflammatory population and contradicting inflammatory gene profiles. These changes lead to progressive clinical disease with increased pulmonary bacterial burden and splenic lesion burdens. Additionally, this chapter was able to demonstrate the ability to partially alter the detrimental effects of vitamin A deficiency during the course of an active Mtb infection via reintroduction of a vitamin A sufficient diet. In chapter two, this thesis aimed to establish the kinetic expression of CD1 glycoproteins, the lipid antigen presenting molecule homologous to MHC I and the linchpin to lipid-restricted immunology, within tissues of infected guinea pigs. This work demonstrated CD1b upregulation during the early adaptive immunology phase of infection by profiling of CD1 glycoproteins via flow cytometric analysis, immunohistochemical tissue evaluation, and CD1 specific qPCR profiling. This study went further by not only establishing kinetic expression of this molecule but correlating that expression back to a functional cytolytic response driven by CD1-restricted T cells. Lastly, chapter three of this thesis aimed to build on the CD1 work in chapter two by assessing the role CD1 lipid restricted immunity plays in the disease pathogenesis of Mtb infections. To perform this work CD1 glycoprotein invivo modulation techniques were perfected via the use of CD1 ortholog specific synthetic antisense RNA, morpholinos, to down regulate CD1 protein translation. To stimulate CD1 expression upregulation, a pleotropic growth factor, FLT3-L, previously shown to upregulate CD1, was utilized. This chapter demonstrated that these compounds were able to alter CD1 expression in vivo when evaluated via flow cytometric analysis, immunohistochemically, and via altered qPCR profiles. Additionally, this chapter showed the ability to hamper the functional cytolytic activity of CD1-restricted T cells via CD1 down regulation. Although CD1 glycoprotein expression was altered, disease progression of Mtb in the guinea pig, as measured by lesion and bacterial burden, remained unaltered. Collective these aims serve as the beginning investigative studies into two unique and potential promising arenas of translational Mtb research. The discovery that vitamin A resupplementation mitigates vitamin A deficiency within chapter 1 has launched a host of different investigations examining the ability of this essential micronutrient to not only prevent clinical TB but to also serve as an immunologic boost to enhance the efficacy of a variety of Mtb treatment regimens. Chapters 2 and 3 reexamined CD1 immunity and the utility of the guinea pig for lipid immunologic work. Though much work has yet to be done in this niche branch of immunology, these chapters lay the ground work and provide the new tools for future studies into potential efficacious and translatable lipid-based vaccination schemes. Tuberculosis, caused by Mycobacterium tuberculosis, is a disease with global ramifications that requires innovative research to meet the new challenges on the horizon. This dissertation looked to meet those challenges by contributing new knowledge to two developing fields of immunologic research while also laying out the tools for future groundbreaking work.
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    A circuitous journey of virus characterization and surveillance in North and Central America
    (Colorado State University. Libraries, 2023) McMinn, Rebekah J., author; Ebel, Gregory D., advisor; Quackenbush, Sandra, committee member; Brault, Aaron, committee member; Neuwald, Jennifer, committee member
    The burden of ticks and the pathogens they carry is increasing worldwide. Powassan virus (POWV, Flaviviridae: Flavivirus), the only known North American tick-borne flavivirus, is of particular concern due to rising cases and the severe morbidity of human disease. In this dissertation we evaluated the recent emergence of POWV from a culmination of field (chapter 2), in vitro (chapter 3), and in-vivo (chapter 4) studies. In addition, we determined the applicability of a vector-enabled surveillance method (xenosurveillance) in Central America (chapter 5). We first used a genetic approach to evaluate the emergence of lineage II POWV, known as deer tick virus (DTV), in parts of North America where human cases occur. We detected DTV-positive ticks from eight of twenty locations in the northeastern United States with an average infection rate of 1.4%. High-depth whole genome sequencing of eighty-four new and archival POWV and DTV samples allowed us to assess geographic and temporal phylodynamics. We observed both stable infection in the northeastern United States and patterns of geographic dispersal within and between regions. Bayesian skyline analysis demonstrated DTV population expansion over the last fifty years. This is concordant with the documented expansion of Ixodes scapularis tick populations and suggests increasing risk of human exposure as the vector spreads. Finally, we isolated sixteen novel viruses in cell culture and demonstrated limited genetic change after passage, a valuable resource for future studies investigating this emerging virus. We then assessed in vitro phenotypes of POWV on human neuronal cells using 16 genetically diverse isolates obtained from a broad geographic and temporal range. We determined over a 10,000-fold range in peak viral titer and significantly decreased cell mortality for two Midwest DTV isolates, though no clear correlation between in vitro phenotype and geo-temporal characteristics could be made. We then performed whole genome sequencing of virus post neuronal cell passage to identify potential residues of interest. Again, no residues could be linked to phenotype, though several interesting residues with increased frequency post-neuronal cell culture were identified. Based on the significant in vitro diversity observed, we sought to assess pathogenesis and tick transmission phenotypes between isolates. We noted neurological disease in mice in both lineages of POWV, with potential low-virulence strains derived from coastal New York. Additionally, we observed an early neuroinvasion phenotype for a Midwest DTV isolate. The ability to infect I. scapularis ticks was determined by feeding on infected host mice (viremic) and through an artificial infection method. Surprisingly, infection rates in ticks via viremic or artificial infection remained consistent between all five isolates tested, resulting in 12-20% infection rate. Taken together, these data demonstrate potential genotype-independent ability to infect ticks and conversely, strain-dependent differences in pathogenesis. In chapter 5, we evaluated a vector-enabled surveillance method ('xenosurveillance') in rural Guatemala. Surveillance methods that permit rapid detection of circulating pathogens are desperately needed. Xenosurveillance is a novel surveillance approach that takes advantage of mosquito feeding behavior to identify blood-borne pathogens that may be circulating in human and animal hosts. This approach circumvents invasive blood sampling of individuals and results in an abundant sample source derived from both humans and animals. In this study, twenty households from two villages (Los Encuentros and Chiquirines) in rural, southwest Guatemala were enrolled and underwent weekly prospective surveillance for 16 weeks. When febrile illness was reported in a household, recently blood-fed mosquitoes were collected from within dwellings and blood samples taken from each member of the household. Mosquitoes were identified to species and blood sources identified by sequencing. Shotgun metagenomic sequencing was used to identify circulating viruses. Culex pipiens (60.9%) and Aedes aegypti (18.6%) were the most abundant mosquitoes collected. Bloodmeal sources were most commonly human (32.6%) and chicken (31.6%), with various other mammal and avian hosts detected. Several mosquito-specific viruses were detected, including Culex orthophasma virus. Human pathogens were not detected. While more intensive sampling may be needed to detect human pathogens, sampling mosquitoes that feed on humans and domestic animals may prove valuable for monitoring pathogens with zoonotic potential.
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    Use of single-cell RNA sequencing and comparative immuno-oncology to gain insights into spontaneous canine cancers
    (Colorado State University. Libraries, 2023) Ammons, Dylan T., author; Dow, Steven, advisor; Avery, Anne, committee member; Thamm, Douglas, committee member; Basaraba, Randall, committee member
    Advances in human clinical medicine stem from discoveries and reports in model systems, therefore the use of biologically relevant models in essential for developing effective human therapeutics. Traditionally, small mammals, such as mice and rats, have been used to address basic science questions and they have contributed substantially to our understanding of biology. Despite widespread use and accessibility of rodent models, there is a growing awareness that findings in rodents frequently fail to translate to human medicine. In recent years, pet dogs have been proposed as an ideal model system to facilitate translational research. As such, the overarching themes of this dissertation are to (1) build upon the dog as a model by providing novel cell type transcriptomic references for immuno-oncology research and (2) investigate immunological correlates with treatment responses in clinical trials using dogs with spontaneously arising tumors. First, the introductory chapter discusses the dog as a model for human disease with a focus on the application in glioma and osteosarcoma (OS). The biological and molecular features of each tumor type are described, then current therapeutic approaches in dogs and human are discussed. After introducing the tumor types, two cell types, myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), are discussed in detail as they are key cell types throughout the dissertation. In the final section of the introduction, single-cell RNA (scRNA) sequencing, the technology foundational to the work presented here, is discussed in detail. In chapters 2 through 5 we focus on OS, a malignant tumor of the bone with minimal therapeutic options. In chapter 2 we generated a reference scRNA dataset of canine circulating leukocytes, then applied the dataset to investigate how the presence of a primary OS tumor impacts systemic immune cell transcriptomes. Through evaluation of 74,067 cells from 17 dogs (7 healthy, 10 OS) we identified relative increases in the abundances of polymorphonuclear (PMN-) and monocytic (M-) MDSCs and provided their transcriptomic signatures for further study. The reference aspect of the work constituents a comprehensive database with gene signatures for each of the 36 cell types identified in canine blood. This work provides key insights into OS induced changes to circulating immune cells while also providing a broadly applicable reference that can be applied to many different areas of canine research. In chapter 3 we generate another comprehensive database, this time focusing on characterizing the heterogeneity within canine OS tumors. Through analysis of 35,310 cells we identified exhausted T cells, mature regulatory dendritic cells (mregDCs), and 8 transcriptomically distinct macrophage/monocyte populations and provide their transcriptomic signatures. We used cell-cell interaction inference approaches to investigate active immune suppressive pathways in OS and found TAMs and mregDCs to be major contributors to T cell suppression. Lastly, we obtained an external human OS scRNA dataset to evaluate cell type homologies between dogs and human which suggested a high degree of similarities between the species. We hope the data generated in this chapter can be applied to enhance canine OS research and shed light on conserved immune suppressive pathways in OS. In chapter 4 we apply the datasets generated in chapters 2 and 3 to investigate how the tumor microenvironment (TME) impacts the transcriptional programs of infiltrating immune cells. To complete the analysis, we used data from circulating leukocytes of the 10 OS dogs in chapter 2 and the OS tumor-infiltrating immune cells identified in chapter 3. Through direct comparison of infiltrating and circulating immune cells we were able to confirm several tumor-induced changes reported in humans are also apparent in the dog. Key confirmatory findings in infiltrating immune cells included the upregulation of activation markers on T cells, increased relative abundance in exhausted T cells, and increased expression of immune suppressive molecules on myeloid cells. Overall, the analysis suggests overarching tumor-induced immunological changes are conserved between human and dogs. In chapter 5 we apply scRNA sequencing to investigate how a myeloid targeted combination therapeutic (losartan, ladarixin, and toceranib) impacts intratumoral and systemic immune responses. Analysis revealed broad immune cell depletion in the tumor and increases in circulating M-MDSCs in dogs receiving treatment. We identified modulation to multiple chemokine signaling axes which shed light on mechanisms associated with treatment-induced immune cell depletion. Finally, the analysis revealed profound impacts to tumor cells and fibroblasts, with treatment skewing transcriptomic profiles toward a hypoxic phenotype and increased insulin-like growth factor associated gene expression. Ultimately, this study represents the first insights into how any therapeutic modulates the OS tumor microenvironment at the single-cell level. Finally, in chapter 6 we conducted a canine glioma clinical trial to investigate the utility of another myeloid targeted therapy (vaccination, losartan, and propranolol). We observed treatment to induce partial tumor regression in 2 and stable disease in 6 of 10 dogs, for an overall clinical benefit rate of 80%. Through evaluation of antibody responses to vaccination, we identified a subset of patients to be immunological responders, which we found exhibited enhanced overall survival times relative to dogs that did not generate antibody responses. The findings from the clinical study suggest that myeloid targeted therapy for treatment of glioma may be a valuable approach that warrants further investigation in canine and human glioma patients. In conclusion, our work applying single-cell RNA sequencing resulted in the generation of valuable canine-specific cell type reference datasets and revealed key insights in osteosarcoma immunobiology. The work evaluating myeloid therapeutics in the setting of osteosarcoma and glioma provide mechanistic and clinical insight that can be applied to further study of the therapeutic approach. Overall, we hope the body of work presented here strengthens the foundation of the dog as a model for translational biomedical research.
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    Establishing canine osteosarcoma as a solid tumor model for the evaluation of B7-H3 CAR T cell therapy
    (Colorado State University. Libraries, 2023) Cao, Jennifer, author; Dow, Steven, advisor; Avery, Anne, committee member; Schenkel, Alan, committee member; Thamm, Douglas, committee member
    Osteosarcoma (OS) is a highly aggressive primary bone cancer that mainly affects children and young adults. OS is the third most common childhood cancer, after lymphoma and brain tumors. Major advances in the 1980's in neoadjuvant chemotherapy has increased 5-year survival rates in OS from 30% to 70%. Unfortunately, for patients that do not respond to standard therapy or that have metastatic disease the 5-year survival rate is still 20% with no major improvements in the last 4 decades. Approximately 15-20% of patients have metastatic lesions at the time of diagnosis and 25-30% of all OS patients will develop metastatic disease. For this subset of patients advances in treatment options are desperately needed. OS also occurs in high rates in large breed dogs with an estimated 10,000 cases in dogs per year in the United States compared to 1,000 cases per year in humans. The dog has been a well-established translational model for OS due to the similar clinical presentation, cell origin, histological features, and disease progression between canine and human OS. Development of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment for advanced and relapsed B-cell lymphomas and leukemias. CAR T cell targeting of B cell marker CD19 has shown up to 90% complete remission in patients with advanced B cell leukemia. However, efforts to expand CAR T cell therapy to solid tumor types have not seen the same clinical success as with blood cancers. Major barriers unique to solid tumor CAR T cell therapy are A) selection of tumor associated antigen target, B) CAR T cell trafficking to tumor sites from the circulation and C) immune suppressor cells within the tumor microenvironment (TME). To develop more effective CAR T cell therapies against solid tumor, we utilized canine OS as a translational animal model. To establish canine osteosarcoma as a platform for evaluating B7-H3 CAR T cell therapy, first we validated B7-H3 as an antigen target in canine OS. We found differential expression of B7-H3 with high levels of B7-H3 expression on OS cell lines and FFPE biopsies, whereas normal canine tissues were B7-H3 negative or low. Next, we optimized generation of canine B7-H3 CAR T cells from whole blood isolated from tumor bearing dogs to maximize both T cell expansion and CAR transduction efficiency. We also found that the addition of cytokines IL-7 and IL-15 minimize CAR T cell exhaustion due to ex vivo activation and expansion. We next determined that canine B7-H3 CAR T cells exerted antigen specific killing and cytokine activity against B7-H3+ canine OS cell lines. To address issues with CAR T cell trafficking we evaluated the addition of chemokine receptor CXCR2 to B7-H3 CAR T cells. To assess the utility of the B7-H3-CXCR2 CAR we determined that canine OS cell lines secreted high levels of ligand chemokine CXCL8 at baseline. To further evaluate functionality, we evaluated the two CAR constructs in a mouse xenograft model of canine OS. We found that the B7-H3-CXCR2 CAR construct had significantly greater anti-tumoral activity than the single B7-H3 CAR construct in inhibiting tumor growth and achieving complete tumor elimination. Studies were also designed to determine if modifying the TME with combination drugs losartan and propranolol improved CAR T cell activity. This is based on recent successful studies with losartan and propranolol in dogs with OS and glioma. We found that combination losartan and propranolol decreased the population of mouse CD11b+Ly6Chigh tumor associated macrophages (TAMs) to xenografted canine OS tumors. In vitro assays showed that immune suppressive macrophages enhance B7-H3-CXCR2 CAR T cell function when co-cultured, likely through CAR activation by macrophage B7-H3 expression. Collectively, the results from these studies pave the way for assessing B7-H3-CXCR2 CAR T cells in dogs with metastatic OS. Clinical outcomes in spontaneous OS in dogs will likely give more clinically relevant results serving as a platform for evaluating new CAR T cell therapies and combination therapies with TME modification, radiation, or checkpoint blockade. Success of this work can provide a new adoptive cell immunotherapy treatment option to patients both canine and human with metastatic osteosarcoma. Additionally, B7-H3-CXCR2 CAR T cells can be applied to other B7-H3 positive CXCL8 secreting tumor types.