Browsing by Author "Basaraba, Randall, committee member"
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Item Open Access Bovine tuberculosis slaughter surveillance in the United States: assessment of its trace-back function 2001-2010(Colorado State University. Libraries, 2012) Mann, Heather, author; Olea-Popelka, Francisco, advisor; Orloski, Kathleen, committee member; Salman, Mo, committee member; Basaraba, Randall, committee memberThe detection of gross bovine tuberculosis (bovine TB) lesions in cattle at slaughter and the successful trace-back to the herd of origin is critical to the detection of infected herds and for the progress of the national bovine TB eradication program in the United States (U.S.). A national animal identification system to identify and trace individual animals is currently under development in the U.S.; however, it is not yet fully implemented. In order to quantify the impact slaughter surveillance and traceability of bovine TB infected cattle has on the eradication of bovine TB from the cattle population in the U.S., this study was conducted with the aim to determine the ability of the current bovine TB slaughter surveillance system to trace infected cattle back to the herd of origin. Data obtained for the period 2001-2010, in which 386 bovine lesions were confirmed as bovine TB in the U.S., were used for this study. The specific objectives for this study were 1) to review and document the available literature related to the history of bovine TB control in the U.S., focusing primarily on the current method of disease detection (slaughter surveillance) and the impediments to eradication in the U.S., 2) to quantify the number of successful trace-backs of bovine TB infected animals to their herd of origin during 2001-2010 3) to quantify the number of trace-backs that found at least one bovine TB infected ("affected") herd, and 4) determine if selected factors were associated with the probability of successfully tracing infected animals and finding infected herds. The results of this study indicates that the odds of successful trace-backs are 7.06 times greater for cattle with official identification than without official identification (OR 95% CI: 1.66, 29.93, p-value =0.008). Additionally, the odds of successful trace-back are 15.47 times greater for adult cattle compared to fed cattle (OR 95% CI: 4.47, 53.48, p-value<0.001). Thus, application of official ID on all classes of cattle would increase the probability of successfully tracing bovine TB cases back to a herd of origin; however, under the current system it will not ensure a complete success in tracing bovine TB infected cattle to the herd of origin. While adult cattle are currently more likely to be traced back than fed cattle, it is worth noting that the effort and time required to find the herd of origin for both adult and fed bovine TB cases can be substantial and is highly variable. The results of this study provide an important tool to aid U.S. officials in their decision making with respect to the evaluation and implementation of strategies for the national bovine TB control and eradication program.Item Open Access Correlation of HAS2-associated gene duplications with biological aggressiveness of mast cell tumors in Chinese Shar-Pei dogs(Colorado State University. Libraries, 2013) Garner, Alana Pavuk, author; Avery, Anne, advisor; Thamm, Douglas, committee member; Basaraba, Randall, committee memberCutaneous mucinosis in Shar-Pei dogs is the result of excessive dermal hyaluronan, a protein associated with angiogenesis and tumor cell motility in multiple human and canine neoplasms. Cutaneous mucinosis in Shar-Pei has been associated with gene duplications upstream of the hyaluronic acid synthase 2 gene (HAS2). The objective of this study was to evaluate the relationship between HAS2, cutaneous mucinosis, and features of mast cell tumor (MCT) aggressiveness in Shar-Pei dogs. Biopsies of cutaneous MCTs from 149 Shar-Pei and 100 non-Shar-Pei were graded according to two schemes for canine cutaneous MCTs. Biopsies of the Shar-Pei MCTs were also evaluated for degree of cutaneous mucinosis, depth of invasion, and microvessel density (MVD). Shar-Pei and non-Shar-Pei MCTs were evaluated via qPCR for relative copy number of the gene duplication upstream of HAS2. The proportion of grade III tumors was significantly higher in Shar-Pei than the general canine population (p=1.044e-11), with no difference in average age at diagnosis. Shar-Pei biopsies had significantly higher HAS2-associated gene segment duplications than non-Shar-Pei (p=1.128e-11), and copy number was significantly associated with the development of grade III tumors (p=0.0077), mitotic index > or = 7 (p=0.022), and tumoral MVD (p<0.05). Relative copy number was not significantly associated with the degree of cutaneous mucinosis or depth of invasion. Our data suggest a relationship between HAS2 gene duplications and features of MCT aggressiveness in Shar-Pei dogs.Item Open Access Feline foamy virus in domestic cats: use as a vaccine vector, characterization of infection, and association with other diseases(Colorado State University. Libraries, 2019) Ledesma-Feliciano, Carmen Denise, author; VandeWoude, Sue, advisor; Quimby, Jessica, committee member; Schountz, Tony, committee member; Basaraba, Randall, committee member; Frye, Melinda, committee memberFoamy viruses (FVs) are retroviruses from the Spumaretrovirinae subfamily. FVs are globally prevalent retroviruses with a unique molecular biology. FVs establish apparently apathogenic lifelong infections. Due to this, FVs are considered attractive vectors for vaccine and gene therapy development. Feline foamy virus (FFV) infects domestic cats and has widespread and high prevalence around the world. However, FFV has also been isolated from cats suffering from concurrent disease, including renal syndromes and other retroviral co- infections such as feline immunodeficiency virus (FIV). Much remains unknown about FFV infection and in vivo experimental infections are rare in the literature. To test FFV's use as a vaccine vector and understand the interaction between viral proteins and host antiviral restriction factors, we developed an infective chimeric vaccine containing lentiviral FIV vif replacing FFV bet. FFV Bet and FIV Vif counteract feline innate APOBEC3 (feA3) restriction factors through different mechanisms. FeA3 action on retroviral genomes lead to hypermutation and degradation of viral DNA. In vitro, we show that vif can replace bet to yield replication-competent chimeric viruses. We experimentally inoculated 12 domestic cats (n=4 per group in naïve, wild-type, and chimera-inoculated groups) with the FFV-Vif chimera and wild- type FFV in order to compare viral replication kinetics through PCR and specific antibody development through ELISA. Inoculation with the chimeric vector resulted in the development of a specific immune response against FFV Gag and Bet and FIV Vif proteins. In addition, we show that the domestic cat can be superinfected with different strains of FFV. The chimeric virus displayed attenuated infection in vivo, as provirus was not detected in PBMC for any chimera-only inoculated animals. Thus, Bet may have additional functions other than A3 antagonism required for successful in vivo infection. Our studies further exemplify how FV vaccine vectors are an attractive tool to counteract lentiviral infections and poses the possibility to induce immunity against other lentiviral antigens. In order to further characterize wild-type infection, we also collected blood, saliva, and urine over a 6-month time-period with a necropsy and tissue collection at the end of the study. Animals were monitored daily for clinical signs of disease and temperature and weight data were collected weekly. None of the cats showed clinical signs of infection and complete blood count and chemistry were unremarkable. However, we found significant differences in blood urea nitrogen, one of the markers used to assess renal function, when comparing infected versus control animals. All animals inoculated with wild-type virus showed a persistent proviremia (in PBMCs) and viral tissue tropism was primarily lymphoid with the exception of one cat that had an expanded tissue tropism to other lymphoid tissues and oral mucosa. This animal had altered viral kinetics compared to the rest of infected animals, in addition to a negative correlation between lymphocyte count and viral load. Histopathological analysis showed evidence of microscopic pathology in the kidneys, lung, and brain of infected animals. This same cat had an increase in urine protein at the time of highest PBMC proviremia. Additionally, transmission electron microscopy showed ultrastructural changes indicative of transient renal injury in the kidneys of infected animals. We additionally found electron dense structures in the cytoplasm of tubular epithelial of as of yet unknown origin. Due to the renal changes we saw in the experimental study and pathology reported in the literature, we conducted a survey of FFV in pet cats in the USA and Australia (AU) suffering from chronic kidney disease (CKD) and compared findings to age- and sex-matched controls without CKD. We found an association between CKD and FFV in males, and males in general are also at a significantly increased risk of FFV infection. We then assessed through an FFV serosurvey whether FFV was associated with FIV and causing potentiation of FIV disease in two cohorts of naturally FIV-infected cats. One of the groups consisted of cats living in 1-2 cat households that did not experience much FIV-related morbidity and mortality, while the second group of cats housed in a large multicat household suffered from severe clinical symptoms and mortality. We hypothesized the reason for this discrepancy could be an increase in FFV/FIV co-infection rate in the group of cats with higher morbidity and mortality. We found that FFV is associated with FIV in these groups and that males are also at an increased risk for FFV infection. Finally, we conducted an in vitro co- infection study to assess potentiated infection as determined by more rapid development of cytopathic effects (CPE) and higher viral titers in the supernatant. GFox cells were inoculated with FFV and FIV in single, and dual simultaneous and staggered inoculations. A p26 ELISA was used to determine amount of FIV reactivity in the cells, while a chemiluminescent β- galactosidase assay was used to detect amount of β -gal produced in FeFAB FFV reporter cells. The in vitro assays showed increased permissivity of either virus following an initial infection of the other virus, showing these two retroviruses can accelerate and potentiate a secondary infection regardless of which virus infected initially. Overall, we have demonstrated the suitability of FFV as a vaccine vector candidate. Additionally, we have documented that FFV may cause subclinical alterations that in certain cohorts of domestic cats, may contribute to disease development in chronic cases. Finally, we showed that FFV interacts with another retrovirus and could potentially affect FIV-related disease. More studies should focus on the effects of FFV in chronic infections in addition to the effect of FFV on co-morbidities in a chronic timeline.Item Open Access Glyme-synthesized nanomaterials(Colorado State University. Libraries, 2021) Armstrong, James, author; Ackerson, Christopher J., advisor; Prieto, Amy, committee member; Kennan, Alan, committee member; Basaraba, Randall, committee memberNanomaterials include materials with at least one dimension in the nanometer range. These materials include nanoparticles, quantum dots, thin films, self-assembled materials, supramolecular materials and more. Nanoscience is an intriguing field for cutting edge research for energy, biology, medicine, optical and other applications. Coinage-metal (Au, Ag, Cu) nanomaterials are particularly of interest for the stability of nanoparticles synthesized with these metals. These metals can also be utilized to produce supramolecular assemblies, e.g. Hydrogels. In particular, this dissertation will cover four projects involving coinage metal nanomaterials. Chapter 2 discusses the ligand-exchange of a gold-thiolate nanocluster synthesized in diglyme, while chapters 3-5 investigate a unique supramolecular assembly of coinage-metal thiolates using glymes as antisolvent. Chapter 3 explores the underlying makeup of these amorphous assemblies, while chapters 4 and 5 investigate the application of this supramolecular assembly for additive manufacturing applications and antimicrobial applications, respectively. All of these products are linked through the synthesis and characterization of nanomaterials, which require the use of glymes (1,2-dme, diglyme, triglyme, etc.) as a necessary synthetic solvent or antisolvent. Nanoclusters are small, atomically precise nanoparticles with a metal core and a passivating layer of organic ligands. Coinage metal nanoclusters are studied for their stability, especially gold nanoclusters, allowing for long-term studies of properties and applications, as well as post-synthetic modifications. Precise control over ligand shell composition, particularly of mixed ligand layers is desired for control over nanocluster functionality. Supramolecular materials build bulk properties through noncovalent interactions. Self-assembled supramolecular materials utilize small molecules which assemble into larger secondary and tertiary structures. These materials are of interest for a broad range of applications like additive manufacturing and biological applications. The motivation behind this work was to explore nanomaterials which results from a glyme based synthesis. Gold nanocluster synthesis in diglyme is found to produce a stable gold-thiolate nanocluster with a single glyme ligand. The precision of a single-unique ligand could lead to further enhancements in nanocluster functionality in the future. Addition of glyme to a coinage-metal thiolate solution results in the rapid precipitation of a rigid supramolecular assembly. The resultant metallogel exhibits properties unique from similar materials without the use of glyme in synthesis. The metallogel is composed of oligomers reminiscent of nanoparticle precursors; as such, metallogel-nanoparticle composites are readily synthesized.Item Open Access Molecular characterization of canine peripheral T-cell lymphoma(Colorado State University. Libraries, 2020) Harris, Lauren, author; Avery, Anne, advisor; Avery, Paul, committee member; Basaraba, Randall, committee member; Bailey, Susan, committee memberTo view the abstract, please see the full text of the document.Item Open Access Molecular characterization of novel transcription of antisense toxin-antitoxin RNA in regulating Mycobacterium tuberculosis(Colorado State University. Libraries, 2020) Dawson, Clinton C., author; Slayden, Richard A., advisor; Basaraba, Randall, committee member; Belisle, John, committee member; Karkhoff-Schweizer, RoxAnn, committee member; Tjalkens, Ronald, committee memberDespite more than seventy years of available anti-tuberculosis (TB) treatments, Mycobacterium tuberculosis (Mtb) remains the deadliest human pathogen. Novel short-course therapies are needed that effectively treat latent TB infection (LTBI), which is like a major source for new infections. However, the molecular determinants of LTBI, including a large repertoire of regulators encoded by Mtb that mediate survival, are largely uncharacterized. Gene expression studies have implicated numerous regulators and particularly toxin-antitoxin (TA) systems in Mtb pathogenesis. Whole genome sequencing (i.e. WGS) studies have linked the massive genomic expansion of TA systems along with other pathogen-specific gene families to the emergence of TB-causing mycobacteria. In addition, a multitude of TA systems show genotypic differences that distinguish between ancient and modern lineages of Mtb. These predominantly include lineage-specific changes in amino acids, altering antitoxin DNA-binding, and nucleotides, generating new promoters. These mutations have led to an overrepresentation of differentially expressed Mtb TA genes responsible for mediating epigenetic changes that are associated with gains in virulence of modern lineages. Thus, the work presented in this dissertation begins to define the novel co-regulation of TA systems that underlie Mtb pathogenesis. Unraveling of more complex regulation of Mtb TA systems will provide keen insights into the phenotypic changes responsible for Mtb survival and persistence in vivo. This will ultimately help to streamline research and development of novel antibiotics as well as host directed immunotherapies against hard-to-treat tubercle bacilli, effectively shortening the duration of TB treatment. TA systems are ubiquitous among bacteria, especially pathogens, and increasingly found to be essential for adaptation to host immune defenses and in vivo drug pressures, resulting in the development of persistent or chronic infections. Phylogenomics comparisons have revealed that Mtb encodes a significantly expanded repertoire of TA systems that are solely conserved by tubercle bacilli, including homologous ParDE/RelBE systems like RelBE2 (i.e. Rv2865-Rv2866). Herein, we report a novel antisense (as)RNA, we call asRelE2, which is uniquely encoded by Mtb and involved in differentially post-transcriptionally regulating relE2 mRNA levels as part of the response to host-associated stress such as low pH in a cAMP-dependent manner. This dynamic regulation of the tripartite relBE2/asrelE2 TA locus appear to be essential for long-term survival under acidic stress in vitro. In addition, the overexpression of relE2 is found to mediate phenotypic development of a persistent state in Mtb associated with increasing tolerance towards frontline anti-TB drugs isoniazid (Inh) and rifampicin (Rif). In mice, asRelE2 acts in differentially regulating bi-cistronic relB2 and relE2 mRNA levels in a host tissue-specific manner dependent upon the downstream effector functions of interferon gamma (i.e. IFN-γ) in murine TB. Specifically, relE2 and relB2 mRNA levels are found to steadily increase in lungs and in spleens, respectively, in the development of the chronic phase of Mtb infection. To our knowledge, this is the first time a Mtb TA system has been shown to be co-regulated by an asRNA antitoxin. Furthermore, this is linked with the development of the adaptive host immune response to Mtb, demonstrating that the post-transcriptional regulation of TA systems is an important mechanism, coordinating the epigenetic changes that are a hallmark of Mtb persistence and pathogenesis.Item Open Access Pharmacological characterization of losartan as a CCR2 antagonist and pre-clinical and pharmacodynamic assessment as a potential anti-metastatic therapy(Colorado State University. Libraries, 2017) Regan, Daniel P., author; Dow, Steven, advisor; Schenkel, Alan, committee member; Thamm, Douglas, committee member; Slansky, Jill, committee member; Basaraba, Randall, committee memberTo view the abstract, please see the full text of the document.Item Open Access The C3HeB/FeJ mice as a novel preclinical mouse model for Mycobacterium tuberculosis: an analysis of the host pathogenesis and the in vivo environment of the necrotic granuloma(Colorado State University. Libraries, 2014) Driver, Emily R., author; Lenaerts, Anne, advisor; Basaraba, Randall, committee member; Crick, Dean, committee member; Gustafson, Daniel, committee memberMycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), infects and kills millions each year. Actively infected patients exhibit a heterogeneity of disease lesions in the lungs, which make it difficult for current treatment regimens to effectively cure. Efforts are underway to develop novel pre-clinical drugs, and this requires a thorough re-evaluation of the current animal models used to test these formulations. A new mouse model using C3HeB/FeJ mice, deficient at the sst-1 locus develops a similar heterogeneity of pulmonary lesions to human disease when aerosol infected with MTB. The studies presented here have found the C3HeB/FeJ mice develop 3 types of lesions: type I) necrotic granulomas delineated with a collagen rim, type II) necrotic alveolitis, and type III) cellular inflammation. Type I lesions, consist of an accumulation of amphophilic cellular debris admixed with numerous extracellular bacilli. At the periphery of the necrotic core on the inside of the collagen rim is a cuff with foamy macrophages of variable size, which contain very large numbers of intracellular bacilli. Type II lesions, the alveolar septal walls remain mostly intact, while the alveolar spaces are filled with high numbers of dying neutrophils, intact cells, extracellular bacteria and debris, creating a honeycomb-like necrosis, which is only seen at very late stage in immunocompetent mouse models. Type III lesions, are disorganized and have a mix of lymphocytes, macrophages (epitheloid and foamy), and a small number of neutrophils with low numbers of intracellular bacilli. The necrotic encapsulated type I granuloma creates an environment that promotes hypoxia, creates a physical barrier, and causes physiological changes to the various bacterial populations. The Kramnik mouse model has higher populations of resistant bacteria when drug treated, such as isoniazid (INH), rifampin (RIF), and pyrazinamide (PZA). These drugs are less effective when administered as monotherapies in C3HeB/FeJ mice. The adaptations the MTB undertake in the core of the type I granuloma while under stress not only make drug treatment less effective, but also make standard acid-fast staining techniques, such as auramine-rhodamine less efficient at detecting bacilli. While, Sybr Gold, a novel acid-fast stain that binds to nucleic acid, is capable of staining bacilli throughout infection and in all lesion types. The discrepancy in staining ability of the two acid-fast stains is indicative of the physical target for auramine-rhodamine no longer being present or available for staining in the bacilli in the core of the necrotic granuloma. The knowledge on histopathology then leads us further to modify the granuloma in the Kramnik mouse model. Firstly, using collagen disrupters we aimed to improve treatment efficacy. Secondly, by using clinical TB strains the goal was to advance disease to potentiate cavitary disease and gain a greater understanding of strain relevance to treatment. Thirdly, by taking advantage of the structural similarity of the necrotic granuloma and solid cancerous tumors allow for the translation of therapy technologies, such as liposomal nanoparticles to be exploited for treatment of MTB. Preliminary findings suggest that the use nanomedicines in TB therapy may be an effective method of drastically reducing treatment as well as potential issues. The C3HeB/FeJ mouse strain is a highly relevant disease model that can be used for determining the efficacy of novel pre-clinical drugs and drug regimens, to gain a better understanding of disease pathogenesis, to understand the specific immunological events of disease, and to explore alternatives to standard therapy.Item Open Access 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 memberUntil 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.Item Embargo 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 memberAdvances 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.