Browsing by Author "Avery, Anne, committee member"
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Item Open Access Biologic and biochemical features of prion pathogenesis(Colorado State University. Libraries, 2016) Hoover, Clare Elizabeth, author; Hoover, Edward A., advisor; Zabel, Mark D., advisor; Avery, Anne, committee member; Tjalkens, Ronald, committee memberPrions are the causative agents of a group of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies. Prions are unique in that disease is initiated when the normal prion protein (PrPC) undergoes a conformational change and propagates through a process of templated conversion to an infectious, misfolded, isoform (PrPRES, PrPCWD, or PrPSc) which can assemble into oligomers and amyloid fibrils. Disease is associated with prion accumulation in the central nervous system, causing the pathologic lesions of neurodegeneration, white matter spongiosis, and a reactive astrogliosis. Previous work has demonstrated the process of prion propagation and disease pathogenesis can be influenced by conversion cofactors, inhibitors, and biologic systems. Heat shock proteins have been shown to protect against the toxic disease effects of denatured and aggregated proteins in several models of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and spinocerebellar ataxia. In this dissertation, I investigated if heat shock protein 72 (HSP72) expression in neurons could protect against prion disease-associated pathology through a cell culture and mouse model of murine-adapted scrapie strain RML. In contrast to the role in other neurodegenerative diseases, HSP72 did not alter the prion disease course or amount of prion conversion in either disease model. Chronic wasting disease (CWD) is a naturally occurring, horizontally transmitted prion disease affecting wild and captive cervid populations that is rapidly expanding into new states and countries. Studies investigating the distribution of PrPCWD during early subclinical CWD infection have detected prions in the oropharyngeal lymphoid tissues as early as 1.5 months; however, the complete tissue distribution of PrPCWD immediately following prion exposure and the chronological progression of prion tissue accumulation remains unknown. Here, I show prions initially accumulate in the oropharyngeal lymphoid tissues following mucosal exposure and rapidly disseminate to all systemic lymphoid tissues prior to neuroinvasion. These findings will help better understand the early pathogenesis of CWD prior to clinical disease and potentially identify therapeutic targets. Prion disease diagnosis relies on demonstration of the misfolded isoform by immunodetection, amyloid seeding assays, or animal bioassays, all assays which may require separate sample preparations precluding examination by multiple tests. To address this limitation, I developed a new technique to detect PrPCWD amyloid seeding in fixed paraffin-embedded (FPE) tissues by real-time quaking induced conversion (RT-QuIC). FPE RT-QuIC proved to be more sensitive than IHC for prion detection and the use of RT-QuIC amyloid formation kinetics yielded a semi-quantitative estimate of the prion burden in samples without the cost and time of animal bioassays. The normal cellular prion protein resides in cell membrane lipid rafts, which has been shown to be a site of pathogenic conversion. Previous in vitro assays have highlighted the ability of lipids to promote prion formation but knowledge is limited regarding the capacity of lipids to inhibit prion formation. Here, I show endogenous polar brain lipids directly inhibit prion amyloid formation in RT-QuIC in a dose-dependent manner. This work is the first to identify an inhibitory role of lipids and suggests the prion conversion process is influenced by a balance of pro-conversion and inhibitory molecules.Item Open Access Changes in autoreactive B cell lifestyle early in development of autoimmunity(Colorado State University. Libraries, 2017) Smith, Mia J., author; Dow, Steven, advisor; Cambier, John, advisor; Avery, Anne, committee member; Webb, Craig, committee memberType 1 diabetes (T1D) is an autoimmune disorder characterized by destruction of the pancreatic beta cells, leading to decreased production of insulin and hyperglycemia. Although environmental factors contribute, genetic factors are likely the primary determinants of risk. With recent advances in GWAS studies, hundreds of risk-conferring alleles have been discovered for T1D. For most cases the exact mechanisms by which these genes and their gene products contribute to development of autoimmunity remains to be elucidated. However, given that T1D requires the activation of autoantigen-specific T and B cells that are normally silenced by immune tolerance, it is likely a combination of HLA and non-HLA alleles act in concert to undermine normal tolerance mechanisms, allowing activation of these autoreactive cells. Although T cells are the primary effectors of beta cell destruction in T1D, autoreactive B cells are thought to act primarily as antigen presenting cells. In a healthy individual, autoreactive B cells are normally silenced by one of three mechanisms: receptor editing, clonal deletion, or anergy. In this work I determined B cells bearing antigen receptors with high affinity for insulin are found only in the anergic B cell compartment, termed BND, of healthy individuals. Importantly, these cells leave this compartment in a proportion of first-degree relatives (FDRs), and in all autoantibody positive pre-diabetics and new onset diabetics. We posited people at risk for development of T1D carry autoimmune risk alleles that impair proper silencing of autoreactive B cells by anergy, allowing these cells to become activated and contribute to disease. In order to test this, I analyzed the HLA class II alleles and over 50 high risk non-HLA alleles in BND sufficient and deficient FDRs. I found loss of anergic insulin-binding B cells (IBCs) in FDRs was associated with the high risk T1D HLA alleles and polymorphisms in the high risk non-HLA loci, INS, PTPN2, PTPN22, and IKZF3. The associations of loss of B cell anergy with these particular risk alleles suggest insulin-reactive T cells and changes in negative regulation of B cell signaling contribute to the unstable anergic phenotype observed in autoimmune patients. In our T1D studies, we found loss of anergic IBCs was correlated with loss of the entire anergic B cell population, irrespective of their specificity, suggesting loss of B cell anergy could be a common phenomenon in other autoimmune diseases. In addition, many risk alleles for T1D are shared among other autoimmune diseases, including HLA and PTPN22, suggesting B cell anergy could be compromised in other autoimmune disorders in which similar contributing risk alleles are at play. Hence, I also analyzed the frequency and phenotype of thyroglobulin (Tg) and thyroid peroxidase (TPO) binding B cells, as well as total B cells, in early onset and long standing autoimmune thyroid disease (AITD) patients compared to healthy controls. Similar to studies in T1D, early onset AITD patients had a significant decrease in anergic Tg and TPO-binding B cells that was correlated with a decrease in total anergic B cells. Furthermore, loss of anergic Tg-binding B cells was inversely correlated with Tg autoantibodies and Tg-binding B cells expressed high levels of the activation marker CD86. These findings suggest activation of high affinity thyroid reactive B cells that are normally silenced by anergy, likely leads to production of autoantibodies. In order to further elucidate the possible contribution a breakdown in anergy of autoreactive B cells has in development of autoimmunity, I studied the phenotype and functional status of IBCs in diabetes susceptible (NOD) and diabetes resistant (C57BL/6) mice transgenic for the 125Tg heavy chain. This transgene increases the frequency of peripheral IBCs to a level that is easily detectable (~0.5-2% of total splenic B cells depending on the strain) [33]. In these mice, I found that high affinity IBCs were phenotypically and functionally anergic in C57BL/6 mice, but the equivalent in NOD appeared activated and functionally responsive, accumulated in the pancreas, and expressed insulin peptides in association with MHC II on their cell surface. Accumulation of these B cells in the pancreas correlated with retention and activation of insulin-reactive CD4 T cells. Hence, these mouse studies nicely summarize what I hypothesize occurs in autoimmune humans; namely, anergy is impaired in autoreactive B cells, likely due to genetic risk alleles, which allows them to become activated and provide critical antigen presenting function to cognate antigen-reactive T cells. These studies are significant in that they are the first studies to identify a breach in B cell anergy occurs early in development in multiple autoimmune disorders in humans, which is likely driven by a combination of autoimmune risk alleles that alter thresholds for B cell activation, enabling them to become activated and participate in disease through antigen presentation and autoantibody production. Furthermore, these studies highlight the utility of loss of B cell anergy as a possible biomarker for increased risk for development of autoimmune disorders.Item Open Access Chronic wasting disease strain diversity, distribution and transmission(Colorado State University. Libraries, 2021) Wagner, Kaitlyn, author; Zabel, Mark, advisor; Avery, Anne, committee member; MacNeill, Amy, committee member; Moreno, Julie, committee memberChronic wasting disease (CWD) is an invariably fatal prion disease affecting captive and free-ranging cervids, including white-tailed deer, mule deer, moose, elk and reindeer. Since the initial discovery of the disease in the 1960's, CWD has spread across the US and Canada, South Korea, and, most recently, Europe. While some outbreaks of CWD were caused by transport of infected animals from endemic regions, the origin of CWD in other epizootics is unclear and not all outbreaks have been characterized. Previous studies have shown that there are multiple strains of CWD; however, the continuous spread and the unclear origin of several outbreaks warrant continued surveillance and further characterization of strain diversity. Moreover, studies implicating extraneural prions as more zoonotic motivated us to examine within-host prion strain diversity. The overarching goal of the work presented here was threefold: 1) address CWD strain differences between lymphoid and brain tissue from the same animal, 2) assess if there are any differences in CWD from either within or between contiguous and non-contiguous outbreaks and 3) address aspects of plant-vectored CWD transmission. The work presented here has important implications for understanding strain diversity within and between deer, as well as identifies samples that appear to be novel strains that warrant follow up assessment. Finally, we show how plants may be playing a role in vectoring infectious prions shortly after exposure. This research has important implications for our understanding of prion strain diversity and distribution as well as adds insight to plant-vectored prion transmission. First, we assessed differences between lymph node-derived and brain-derived prions from within the same animal to characterize strain differences within a single animal. To do this, we assessed isolates using biochemical techniques including electrophoretic mobility, glycoform ratio and conformational stability. Interestingly, we found that there were biochemical differences between lymph node and brain isolates, novel intermediate conformations of the prions in the brain (but not the lymph node) and increased variability in the lymph node-derived prions. Collectively, these results suggest that there are more diverse prion strains in the periphery and are distinct from neurological prions. The research discussed here advances our understanding of the differences between lymph node-derived and brain-derived prions. In addition to within-host strain comparisons, we also wanted to assess biochemical strain differences from naturally infected cervid species. Numerous studies have examined CWD strains upon passage into transgenic mouse models. For the purposes of our research, we wanted to examine CWD strains from the natural host for a number of reasons: 1) bioassay is expensive and time consuming, making strain characterization challenging, 2) research indicating that host factors other than PrPC may be influencing strain characteristics and 3) to determine if we could detect dramatic biochemical differences in strains, thereby providing an easier method to determine CWD strain prevalence in cervid populations without bioassay. Because the origin of CWD is unknown and some outbreaks of CWD have no clear exposure/connection to ongoing CWD outbreaks, this research would provide insight into the evolution and origin of CWD. Here, we show that there are some cases of CWD that present with novel biochemical characteristics that distinguish them from other CWD isolates. These instances suggest a new strain has emerged or that there is differential evolution in these subpopulations. Importantly, this work highlights that there is a lot more variability CWD biochemical characteristics than previously described. As a part of the strain typing project, two samples were received from captive white-tailed deer in Texas. These samples immediately proved to be a challenge to work with because they were behaving in an unusual way in our biochemical strain typing assays. In short, these isolates behaved in strange ways depending on the detergent class with which they were being digested. Because there was no known introduction of CWD to this captive herd, we were suspicious that we were seeing a novel strain of CWD. Isolates were passaged into cervid and human PrP mice. Upon passage, these isolates looked like classical CWD in Tg33 mice and, fortunately, don't appear to have any zoonotic transmission potential into human PrP mice. Importantly, this work highlights that CWD can present in a unique way in a cervid host but cause a classical-type disease in transgenic animals. Finally, we examined the role of plants to transmit CWD. Previous research implicated plants as having a possible role as a vector in prion transmission. We built upon this previous research by using CWD prions rather than hamster prions and a different plant model. The research presented here will show that plants are able to uptake prions shortly after exposure, but that these prions are no longer detected by 72 h. The work presented here implicates plants as potential CWD vectors in the short term.Item Embargo Comprehensive investigation of chronic enteropathy in dogs through a prospective clinical trial, immunoassays, and RNA-sequencing(Colorado State University. Libraries, 2024) Manchester, Alison C., author; Dow, Steven, advisor; Lappin, Michael R., advisor; Avery, Anne, committee member; Webb, Craig, committee memberChronic enteropathy is a common condition in dogs causing recurrent or persistent gastrointestinal clinical signs. Pathogenesis is thought to involve intestinal mucosal inflammatory infiltrates, but histopathological evaluation does not predict treatment response, inform prognosis, or correlate with clinical remission. Many dogs may improve clinically with dietary intervention, but between 15 to 40% of dogs are refractory to all therapies. This negatively impacts quality of life for dogs and their families and can lead to euthanasia. Better understanding of the cellular and molecular differences between CE and health is necessary to improve outcomes for these dogs, and to enable use of the dog as a translational model for study of inflammatory intestinal conditions across species. The goal of this work was to critically evaluate the pathogenesis of CE in dogs through use of in vitro assays, a prospective clinical trial, and next-generation sequencing based approaches. Preliminary studies have highlighted an important role for intestinal bile acids in the pathogenesis of canine and human chronic enteropathies. Fecal bile acid populations differ between healthy dogs and dogs with CE. However, there has been little work to evaluate potential consequences of these metabolic shifts in dogs. We therefore investigated potential immunomodulatory roles of primary and secondary bile acids through in vitro experiments with canine macrophages. Both the primary bile acid cholic acid (CA) and the secondary bile acid lithocholic acid (LCA) influenced LPS-induced cytokine production via canine monocyte-derived macrophages similarly, with suppression of TNF-α secretion and enhancement of IL-10 secretion. Neither BA altered the expression of the BA receptor TGR5. Transcriptomic analysis revealed that CA activated inflammatory signaling pathways in macrophages involving type II interferon signaling and the aryl hydrocarbon receptor, whereas LCA activated pathways related to nitric oxide signaling and cell cycle regulation. Thus, we concluded that both primary and secondary BAs are active modulators of macrophage responses in dogs, with differential and shared effects evident with sequencing analysis. Diet is the most effective management strategy for dogs with CE, enabling two-thirds of patients to achieve clinical remission from their disease. Various dietary strategies may be beneficial. Nutritional formulae sourcing protein from amino acids have been used for the induction of remission in human Crohn's disease patients for decades. We conducted a prospective clinical trial involving exclusive feeding of the first diet sourcing protein from individual amino acids to 23 client-owned dogs with CE to determine its ability to induce clinical remission and begin to tease apart mechanisms of action. After 2 weeks of EL, 68% of dogs consuming the diet were classified as responders. At the conclusion of the 8 week feeding trial, 16/23 dogs (70%) were considered clinical responders. Feeding EL caused shifts in fecal bacterial communities, which differed between responders and non-responders, suggesting that diet's ability to modulate gut bacterial populations may predict its efficacy. Serum biomarker concentrations were unchanged throughout the study apart from serum alkaline phosphatase activity. Results of this study indicate that an amino acid based diet is another option to treat dogs with CE and implicates the intestinal microbiota in achievement of remission in these patients. Most studies comparing healthy and CE dogs completed to date have been limited in scope, evaluating individual or a small collection of biomarkers or cell types. This has hampered advancement of the understanding of CE pathogenesis in dogs. Ultimately, this results in generic treatment strategies for dogs and leaves a substantial proportion unable to achieve clinical remission from their disease. To this end, we applied next-generation transcriptomic sequencing to mRNA from duodenal biopsies from CE dogs and healthy beagle dogs. Results of this analysis highlighted important roles for epithelial cell gene signatures in differentiating CE tissues from healthy ones. Commonly implicated cytokines like TNF-α, IL-12, or IL-10 were not differentially expressed, but pathway analysis highlighted a potential role for upregulation of anti-viral pathways in CE dogs. This preliminary study underscores the power of RNA sequencing to provide a broad overview of cellular activities in tissues of interest, and question widely accepted theories regarding dysfunction present in the gut of dogs with CE. Single-cell RNA sequencing offers a high-resolution molecular technique enabling characterization of gene expression on an individual cell basis. This approach overcomes traditional barriers to disease investigation (e.g., species-specific reagents) and allows for definition of cell subtypes within heterogeneous samples. We thus employed single-cell RNA sequencing to catalog and compare the diversity of cells present in duodenal mucosal endoscopic biopsies from 3 healthy dogs and 4 dogs with CE. We identified populations of epithelial cells, T cells, myeloid cells, and plasma cells, with contributions from both the healthy and CIE samples. Neutrophils from CE samples exhibited a more inflammatory transcriptional program. T cells were broadly divided into non-resident and tissue resident subtypes, though minimal transcriptomic differences were appreciated within this class of cells. One subset of epithelial cells from CE dogs showed differential expression of a gene encoding a 2-pore potassium channel (KCNK16). Our results reveal a previously unappreciated cellular heterogeneity in canine duodenal mucosa and provides insights into molecular mechanisms underlying CE in dogs. The cell type gene signatures determined through this work will enable better understand the subtleties of canine intestinal physiology to allow more accessible interrogation of cellular activities in health and disease. The results of the studies described add further nuance and detail to understanding of the pathogenesis and management of canine CE. We have documented the power of transcriptomic analysis for differentiation of intestinal mucosal molecular programs in health and CE. Further investigation into intestinal bile acids, duodenal mucosal T cell subtypes and neutrophils, and intestinal epithelial cell activities are indicated.Item Open Access Effects of immunological targeting of two mosquito antigens and oral ingestion of anthelmintic drugs on the yellow fever mosquito, Aedes aegypti (Diptera: culicidae)(Colorado State University. Libraries, 2011) Deus, Kelsey Marie, author; Foy, Brian D., advisor; Avery, Anne, committee member; Black, William C., IV, committee member; Bowen, Richard A., committee memberAedes aegypti is one of the most important mosquito vectors of human arboviruses, including dengue viruses, chikungunya virus, and yellow fever virus. Human infection with these viruses constitutes an enormous global disease burden. Current control methods rely heavily on the use of insecticides, which are rapidly losing their utility due to the spread of insecticide resistance. Anti-vector vaccines and anthelmintic drugs with insecticidal properties have been proposed as novel means to decrease pathogen transmission by reducing the daily probability of mosquito survival. The aims of this dissertation research were to: evaluate the Ae. Aegypti mosquito lysosomal aspartic protease and the glutamate-gated chloride anion channel as potential mosquitocidal antigens, evaluate drugs frequently used in mass drug administration campaigns for their ability to induce a mosquitocidal effect when imbibed in a blood meal, to assess the variation in susceptibility of Ae. Aegypti strains to orally imbibed ivermectin, and finally to determine if resistance to ivermectin could be selected for in a genetically diverse laboratory strain of Ae. Aegypti . Despite the utilization of several immunization regimens, a specific mosquitocidal immune response against the Ae. Aegypti mosquito lysosomal aspartic protease could not be verified. In vitro experiments in which high titer glutamate-gated chloride anion channel serum was fed to mosquitoes failed to elicit a mosquitocidal response, suggesting that it is an unlikely mosquitocidal antigen. In vitro blood feeding experiments with several anthelmintic drugs revealed that high concentrations of macrocyclic lactones (including ivermectin, selamectin and moxidectin) were effective in reducing adult mosquito survival and that sublethal concentrations resulted in reduced fecundity and egg hatch rate. When imbibed in a blood meal, diethylcarbamazine, albendazole-sulfoxide and pyrantel pamoate, which are all currently used in human mass drug administration campaigns for controlling parasitic pathogens in humans, had no effect on adult mosquito survival. Significant differences in susceptibility to ivermectin, according to mosquito strain, were observed, with three permethrin-resistant strains of Ae. Aegypti being the most refractory to ivermectin, suggesting a possible permethrin-induced cross resistance mechanism to ivermectin. After subjecting a genetically diverse laboratory strain of Ae. Aegypti to three successive rounds of selection with orally imbibed ivermectin, no resistance to the drug was apparent. Although mass drug administration is unlikely to have any impact on the transmission of Ae. Aegypti vectored pathogens, Ae. Aegypti may prove to be a useful model for studying the effects of ivermectin in the mosquito, including studying potential resistance and cross-resistance mechanisms to anthelmintic drugs.Item Open Access 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 memberOsteosarcoma (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.Item Open Access Immune modulatory and antimicrobial properties of mesenchymal stromal cells delivered systemically(Colorado State University. Libraries, 2020) Johnson, Valerie, author; Dow, Steve, advisor; Zabel, Mark, advisor; Avery, Anne, committee member; Tjalkens, Ron, committee memberTo view the abstract, please see the full text of the document.Item Open Access Iron, hepcidin, and microcytosis in canine hepatocellular carcinoma(Colorado State University. Libraries, 2021) Polak, Klaudia Zofia, author; Olver, Christine, advisor; Avery, Anne, committee member; Santangelo, Kelly, committee member; Shropshire, Sarah, committee memberHepatocellular carcinoma (HCC) is the most common primary liver tumor found in dogs. There is evidence that iron dysregulation is associated with HCC pathogenesis in both humans and dogs. Anemia and thrombocytosis were common hematologic abnormalities detected in about half of dogs with massive HCC, and microcytosis was present in approximately 31% of dogs in one study. Additionally, humans with hereditary hemochromatosis have an increased risk of HCC. The liver is the major organ site for iron storage and metabolism containing numerous iron regulatory proteins which may play an important role in canine HCC. Since microcytosis is associated with iron restricted erythropoiesis, our first objective was to determine whether neoplastic hepatocytes exhibit differential expression of iron regulatory genes as well as hepatic iron stores in normocytic versus microcytic HCC cases in an initial pilot study. Next, we aimed to quantify and compare expressions of a larger set of iron regulatory and human HCC-related genes among canine HCC tumor tissue, adjacent peritumoral liver tissue, non-specific reactive hepatitis liver tissue from non-HCC dogs, and normal liver tissue, as well as to quantify and compare estimated hepatic iron stores. We hypothesized that canine HCC tumor tissue exhibits iron overloading and higher expression of hepcidin and its upstream regulators (IL-6 and BMP6), which would promote intracellular iron availability for neoplastic hepatocyte proliferation. We also hypothesized that microcytic HCC cases would exhibit higher expression levels of hepcidin in tumor tissue compared to tumors from normocytic dogs. Additionally, we explored associations between clinical parameters and RNA levels of iron regulatory genes as well as estimated hepatocellular iron stores in both HCC tumor and the adjacent, peritumoral tissues. We expected to find gene expression patterns in canine HCC tumor tissue related to abnormal regulation of iron metabolism and other pathways similar to what has been described in human malignancies. Cases were selected from a database search for canine HCC and included if complete pre-operative blood work was available and there was adequate formalin-fixed paraffin-embedded (FFPE) tissue for RNA isolation for all cases. Hematologic and clinical parameters were recorded and used for correlation studies. All liver sections were reviewed by a board-certified veterinary anatomic pathologist. RNA was isolated from FFPE blocks and NanoString nCounter platform was used to quantify RNA counts for selected genes. Sections were stained with Perls Prussian Blue stain and hepatocytic iron stores were estimated using NIS-Elements software. Contrary to our hypotheses, all canine HCC tumors had markedly decreased expression of hepcidin (HAMP) and depletion of hepatocellular iron stores. Other iron-related genes down-regulated in canine HCC tumor tissue included TfR2 (an upstream regulator of hepcidin), STEAP2, LTF, HMOX1, CYBRD1and SFXN5. Tumor tissue overexpressed TfR1, STEAP3, and LCN2. No significant differences in RNA levels or iron stores were found between tumors of microcytic and normocytic HCC cases, but the adjacent peritumoral tissue was markedly iron loaded and exhibited negative correlation between hepcidin RNA levels and mean cell volume (MCV) as well as serum iron. Microcytic HCC cases were associated with noteworthy clinical findings such as increased ALT, lower HCT and serum iron, and histologically more poorly differentiated tumors. Differential expression of genes involved in Wnt signaling and ferroptosis was observed in canine HCC tumor versus the adjacent peritumoral liver tissue.Item Open Access Metformin: a tool to better understand T cell mediated protection against Mycobacterium tuberculosis(Colorado State University. Libraries, 2020) Haugen Frenkel, Jessica D., author; Basaraba, Randall J., advisor; Obregón-Henao, Andrés, committee member; Podell, Brendan K., committee member; Chicco, Adam, committee member; Avery, Anne, committee memberTo view the abstract, please see the full text of the document.Item Open Access Mucosal and systemic immune correlates of protection against feline enteric coronavirus infection(Colorado State University. Libraries, 2019) Pearson, Morgan, author; Dean, Gregg, advisor; Schountz, Tony, committee member; Webb, Craig, committee member; Avery, Anne, committee memberFeline infectious peritonitis (FIP) is a disease with high mortality that results from a mutation in the genome of the relatively harmless and ubiquitous feline coronavirus (FCoV) (Licitra, Millet et al. 2013). FIP causes a deadly effusive and/or granulomatous disease in cats (Kipar, May et al. 2005). Because FIP is always fatal, our aim is to aid with the development of a vaccine against the parent virus FCoV. The goal of this study is to complete a comprehensive assessment of the mucosal immune response associated with FCoV infection and clearance. Previous research has shown that cats infected with FCoV can clear the virus, or they can become intermittent or persistent virus shedders (Marks 2016). It is thought that rapid waning of the humoral immune response predisposes cats to reinfection (Myrrha, Silva et al. 2011). A closed cat colony with circulating FCoV infection was studied longitudinally to assess mucosal immune correlates of protection. Blood and fecal samples were collected monthly and colonic biopsies were obtained at an arbitrary time 0. Virologic assessment included PCR detection of virus in feces and colonic tissue. Immunological assessment included FECV-specific serum IgG and fecal IgA. Lamina propria lymphocytes from colon biopsies were phenotyped using flow cytometry and were assessed for FCoV-specific IgA and IFNγ expression by ELISPOT. Expression of IL17 and FoxP3 was measured by qRT-PCR. Although histopathology of colonic biopsies from cats shedding virus was unremarkable, an inflammatory state was indicated by total IgA producing cells, IFNγ production, and increased IL17:FoxP3. FCoV-specific IgA was also associated with viral shedding. Taken together, results indicate mucosal and systemic antibody responses are responsible for limiting FECV infection while cell-mediated responses were not detected. Therefore, a vaccine strategy targeting antibody induction via a mucosal route may provide protection against FECV infection.Item Open Access The role of neuroinflammatory NF-KB signaling and glial crosstalk in neurodegeneration(Colorado State University. Libraries, 2013) Kirkley, Kelly Sullivan, author; Tjalkens, Ronald, advisor; Avery, Anne, committee member; Legare, Marie, committee member; Hanneman, William, committee memberNeuroinflammation or inflammatory activation of astrocytes and microglia are considered pathological hallmarks and important mechanisms in debilitating neurodegenerative diseases. However, the signaling mechanisms underlying these neuroinflammatory changes are not fully understood and there is evidence that these inflammatory responses can serve both neuroprotective and neurotoxic roles. Few studies have begun to study the complicated communications occurring between activated glia and the contributions of these cells to neuronal injury. Furthermore, the importance of these pathways in environmentally relevant animal models is currently unknown. Therefore, in order to address these knowledge gaps, I utilized three different neurodegenerative diseases models, domoic acid induced seizures in sea lions, a mouse model of Parkinson's disease (PD), and an in vitro astrocyte-microglia culture model of Manganism, to decipher the contributions of microglia and astrocytes to neuronal injury and the pathways that dictate these responses. First, for the first time, I have identified significant correlations between severity of neuronal loss and glial activation in domoic acid exposed sea lions that were associated with significant changes in glutamate metabolism. This indicates that neuroinflammation is playing a never before described role in this disease and targeting this neuroinflammation may be a new source to limit progression of seizures in these animals. Secondly, by creating a mouse with an astrocyte specific deletion of the nuclear factor kappa B (NFκB) pathway, it was determined that neuroinflammatory activation of astrocytes through NFκB is important in the initiation and progression of dopaminergic cell loss in a chemical PD model. Also, in a similar model of disease, we found that full and potent activation of astrocytes to the basal ganglia toxicant manganese (Mn) required soluble factors from Mn activated microglia. These studies indicate that glial release of inflammatory factors is not only affecting neuronal function and survival, but is important in glial crosstalk and resultant exacerbation of the inflammatory response. Targeting these glial interactions may play important roles in developing treatments to limit the neurotoxicity of neuroinflammation.Item Open Access Tracking infectious prions in the body fluids of deer infected with chronic wasting disease(Colorado State University. Libraries, 2010) Mathiason, Candace Kay, author; Hoover, Edward A., advisor; Avery, Anne, committee member; Bamburg, James R., committee member; Zabel, Mark, committee memberChronic wasting disease (CWD) is a prion disease of cervid (elk, moose and deer) with unusually high transmission efficiency. While the nidus of disease was described in a captive herd of cervid in northern Colorado/southeastern Wyoming in the late 60's, it has now been detected in both captive and free-ranging populations in 17 states and 2 Canadian provinces of North America and one Asian country. CWD is unique in being the only transmissible spongiform encephalopathy (TSE) described in a free-ranging population of animals. The etiology of CWD, like all prion diseases, is the conversion of the normal host-encoded cellular prion protein (PrPC) to an aberrantly folded protease resistant isoform (PrPRES/PrPCWD). An intriguing aspect of prion diseases is their ability to be transmitted from one organism to the next. In this dissertation work, we ask-By what means are infectious prions transmitted from one host to the next? In particular to CWD-What do infected cervids share or leave behind that contain sufficient infectious particles to initiate disease in the next cervid? We addressed this question by bioassay of body secretions and excretions- ' secreta '- (saliva, blood, urine and feces) in the native white-tailed deer host and in transgenic mice expressing the normal cervid prion protein (Tg(CerPrP) mice). Cohorts of deer were exposed by oral (PO) ingestion of 'secreta', or intraperitoneal (IP)/intravenous (IV) transfusion of blood components. To replicate a more natural/realistic exposure to CWD in which a deer might travel into a contaminated area and feed for a short period of time, an additional cohort of deer was exposed to fomites (bedding, feed and water buckets) from the suites of CWD-infected deer-without direct contact with infected deer. These variously exposed deer were monitored for a minimum of 19 months post inoculation (mo pi) for CWD infection and disease by immunohistochemical (IHC) and western blot (WB) detection of PrPCWD in serial tonsil biopsies and in multiple tissues after necropsy. Parallel studies were conducted in Tg(CerPrP) mice with the addition of an intracranial (IC) inoculation group for each body fluid. We found that sufficient infectious prions were present in the saliva, whole blood, the B cell- and platelet-enriched fractions of blood, and in fomites from infected deer premises to transmit CWD. Conversely, PrPCWD was not detected in the brain or lymphoid tissues of deer or mice inoculated with urine and feces, cell-free plasma or CD14+ monocytes from CWD-infected donor deer. The results of this work: 1) suggest that the efficient transmission of CWD may be due in part to the sharing of saliva between cervids and its deposition upon surfaces frequented by cervids; 2) establish a hematogenous dissemination of infectious prions in CWD associated with the cellular fraction of blood- in particular B cells and platelets— in CWD-infected deer; 3) extend previous work localizing PrPCWD to the interface of follicular B cells and dendritic cells; 4) provide insights to PrPCWD trafficking and CWD pathogenesis; and 5) establish saliva and blood cells as viable substrates for PrPCWD antemortem detection.Item Open Access Unraveling prions: the complexities between the prion protein, complement, and B cells in diverse pathogenic settings(Colorado State University. Libraries, 2017) Kane, Sarah, author; Zabel, Mark, advisor; Bamburg, James, committee member; Tjalkens, Ronald, committee member; Avery, Anne, committee memberPrions diseases affect numerous mammalian species and may arise spontaneously, from genetic predisposition of the prion protein PrPC to misfold and aggregate, or from contacted with prion-contaminated materials. The first described prion disease, Scrapie, manifests in sheep, and records date back to the 18th century. Other mammalian species susceptible to prion diseases include humans, cats, mink, cervids (deer, elk, and moose), and cattle. The term Transmissible Spongiform Encephalopathy (TSE) arose to describe this new class of infectious diseases which exhibit spongiform degeneration in the central nervous system (CNS). TSEs are invariably fatal diseases, and only herd culling or breeding resistance mitigate disease spreading. However, chronic wasting disease (CWD) in cervids represents the first known TSE to occur in free-ranging wildlife, and the apparent facile spread demands strategies to halt its spread and prevent species eradication. Human prion disease characterization dates back to the 1920s. However, the bovine spongiform encephalopathy (BSE or mad cow disease) outbreak and subsequent transmission into a small number of humans in the 1980s and 90s pressed the need to understand the TSE agent. Many researchers since the 1960s postulated protein at least partially comprised the agent, but Stanley Prusiner provided the first scientific evidence of protein composition correlating with infectivity. Further, he coined the term proteinaceous particle, or prion. Follow-up research elegantly highlighted a host protein requisite to cause disease. Researchers now broadly accept the disease mechanism involves prions perverting the cellular prion protein to alter its conformation and join the highly stable growing prion aggregate. Upon peripheral exposure, most prion strains propagate in the lymphoreticular system prior to invading the CNS. Many elegant studies reveal the Complement system promotes initial prion trafficking and propagation in spleen and lymph nodes because mice deficient in various Complement proteins or receptors exhibit delayed or no disease. Once in the LRS, many postulate prions retrogradely infect the brain via sympathetic nerve fibers and the spinal cord. Once in the brain, prions provoke astrogliosis, neurodegeneration, and invariable death. While prion researchers made great strides in characterizing TSEs within a short few decades, many fundamental questions remain unaddressed. For example: what additional host factors foster prion pathogenesis? What is the normal function of the properly-folded, cellular prion protein? Lastly, do prion binding partners provide therapeutic targets? Data presented in this dissertation highlight crucial roles for Complement regulatory protein Factor H and Complement receptor CD21 in Scrapie pathogenesis, suggest C1q may strain-specifically impact prion disease, highlight PrPC as a crucial mediator in the adaptive immune system, and provide potential therapeutic tools and targets to combat prion disease.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.