Browsing by Author "MacNeill, Amy, committee member"

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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 member
Chronic 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.
Open Access
Development of Lactobacillus acidophilus as an oral vaccine vector and effects of rice bran ingestion on the mucosal health of Malian infants
(Colorado State University. Libraries, 2020) Vilander, Allison C., author; Dean, Gregg, advisor; Abdo, Zaid, committee member; Dow, Steven, committee member; MacNeill, Amy, committee member; Ryan, Elizabeth, committee member
Most pathogens enter the body at the mucosa and induce innate and adaptive immune responses at these surfaces essential for protection against infection and disease. Induction of mucosal immune responses is best achieved locally but mucosal vaccines have been difficult to develop with few currently approved for use. Almost all are attenuated live vaccines which limits their use and efficacy in some populations. Strategies to enhance the mucosal immune response to vaccination and move away from attenuated live vaccines are needed. Prebiotics (nondigestible food ingredients that promotes growth of beneficial microorganisms) and probiotics (live microorganisms that are beneficial when ingested) are an active area of interest for improving mucosal health and increasing oral vaccine performance. Here we present the development of the probiotic Gram-positive lactic acid bacteria Lactobacillus acidophilus (LA) as a novel oral subunit vaccine. LA has many advantages as an oral vaccine vector including endogenous acid and bile resistance, heat tolerance, and numerous proteins that interact with the mucosal immune system. We show that LA can induce immune responses to weakly immunogenic neutralizing peptides from HIV-1 and rotavirus. To enhance the immune response, we developed the E. coli type I pilus protein, FimH, as a LA vaccine adjuvant. FimH increased the immune response to vaccination and increased LA trafficking by antigen presenting cells to the mesenteric lymph node, an important site of mucosal immune induction. We also evaluate the effects of ingestion of the nutrient dense prebiotic rice bran on mucosal health in a cohort of healthy Malian infants at risk for malnutrition and the subclinical condition environmental enteric dysfunction. Rice bran ingestion was found to decrease episodes of diarrhea, decrease the age to elevated fecal microbiome α-diversity, and stabilize total fecal secretory IgA concentrations over time. These results indicate that rice bran protects from diarrhea and improves the mucosal environment.
Embargo
Insights into the cell of origin, pathogenesis, and translational potential of canine peripheral T-cell lymphoma
(Colorado State University. Libraries, 2025) Owens, Eileen, author; Avery, Anne, advisor; Basaraba, Randall, committee member; Duval, Dawn, committee member; MacNeill, Amy, committee member; Regan, Daniel, committee member
Peripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms in humans and dogs with short survival times and poor treatment responses. The clinical features and immunophenotype of PTCL in dogs closely resemble PTCL, not otherwise specified (PTCL-NOS), the most common and most poorly understood subtype of PTCL in humans. This has led to interest in their potential as a preclinical model for this disease. Because these are naturally occurring tumors in dogs, they offer some unique advantages as a preclinical model compared to traditional xenograft and genetically engineered mouse models, including the ability to study these cancers in an immunocompetent host with a more comparable tumor microenvironment, the potential to investigate the genetic origins of these tumors in hosts with similar genetic diversity to humans, and the consideration of environmental risk factors, since humans and dogs largely share the same environments. One remaining barrier to this proposed canine model is our limited understanding of how the molecular features of canine PTCL compare to human PTCL-NOS. In humans, gene expression and mutational profiling studies have unveiled prognostically significant molecular subtypes of PTCL-NOS, identified enriched oncogenic signaling pathways, recognized mutations in key tumor suppressor genes and oncogenes, and discovered large structural variants such as fusion genes that contribute to the pathogenesis of PTCL-NOS and represent opportunities for novel targeted therapies. However, similar gene expression and mutational profiling studies in canine PTCL are lacking. We hypothesized that canine PTCL would exhibit a similar global gene expression profile to PTCL-NOS, would be enriched for the same oncogenic signaling pathways, and would share some of the most common gene mutations of human PTCL-NOS. We tested this hypothesis by performing bulk RNA-sequencing, differential gene expression analysis, and variant analysis on canine PTCL, normal canine nodal lymphocytes, and normal canine thymocytes. We discovered that the gene expression profile of the most common CD3+CD5+/-CD4+CD8– immunophenotype of canine PTCL closely resembles human PTCL-NOS—particularly the GATA3-expressing subtype associated with an inferior prognosis—and canine PTCL often exhibits mutations in the same genes commonly mutated in human PTCL-NOS, including DNMT3A, TP53, TET2, and PTEN. The cell of origin of human PTCL-NOS remains a subject of ongoing speculation. The observation that subsets of human PTCL-NOS upregulate either TBX21 or GATA3—transcription factors controlling the differentiation of naïve T cells to mature T helper 1 (Th1) or T helper 2 (Th2) cells, respectively—led to suggestions that these tumors are derived from their respective normal mature T-helper cell counterparts. However, more recent studies have demonstrated that GATA3 is an oncogene upregulated by a variety of neoplasms, including immature precursor T-cell neoplasms and even non-hematopoietic neoplasms, and its expression in GATA3-PTCL is associated with promotion of general T-cell survival and proliferation rather than Th2 differentiation. Additionally, mature T cells have been shown to be highly resistant to oncogenic transformation, suggesting that even seemingly mature T-cell neoplasms may actually arise from earlier precursors. Given the similarities between human PTCL-NOS and canine CD4+ PTCL, investigations into the cell of origin in dogs may offer additional insight into the cell of origin in human PTCL-NOS. One previous study identified decreased surface expression of CD25 and MHC class II in canine PTCL, molecules normally associated with T-cell maturity and activation, suggesting that canine PTCL may arise from an earlier T-cell precursor. Recently, an RNA-sequencing study comparing the gene expression profile of canine T-zone lymphoma, a more indolent form of T-cell lymphoma in dogs, to gene signatures of human and murine naïve and activated T-cell subsets successfully identified the cell of origin for this disease as a mature, previously activated αβ T cell. Therefore, we sought to employ similar methods to test our hypothesis that the cell of origin of canine CD4+ PTCL is a thymic precursor cell. Additionally, to help overcome some of the limitations of using publicly available data in other species, we sought to develop a single-cell transcriptomic atlas of normal canine thymic and lymph node tissue to identify key changing genes throughout T-cell development and differentiation in dogs and evaluate those expression signatures in canine CD4+ PTCL. We found that canine CD4+ PTCL upregulated several markers of immaturity, including CD34, KIT, DNTT, and CCR9. Canine CD4+ PTCL was enriched for gene signatures associated with immature murine and human thymocytes compared to more mature T-cell subsets. Finally, based on data derived from our single-cell atlas of normal thymus and lymph node, canine CD4+ PTCL tended to upregulate genes expressed early in canine thymocytes and downregulate genes expressed as canine T cells progressed to more mature stages of development. Taken together, these findings support an immature thymic precursor cell of origin for canine CD4+ PTCL. For a subset of human PTCL-NOS, a type of mutation known as fusion genes play a role in tumorigenesis and offer an opportunity for targeted therapies. These mutations are characterized by the juxtaposition of two previously independent genes, which can then be transcribed and potentially translated together to oncogenic effect. In small subsets of human PTCL-NOS, the presence of a fusion gene drives malignant transformation through upregulation of specific oncogenic signaling pathways, and inhibitors of these pathways have shown promise as an alternative treatment option in these tumors. Despite their potential as actionable drivers in many hematologic cancers, fusion genes have not been previously investigated or described as a feature of canine PTCL. We hypothesized that recurrent fusion genes could be identified in canine CD4+ PTCL, and that some would correlate with enrichment for particular oncogenic pathways. To test this hypothesis, we utilized fusion calling algorithms for bulk RNA-sequencing data and long-read DNA sequencing techniques to investigate expression of fusion mRNA transcripts and the presence of large chromosomal aberrations, respectively, in canine CD4+ PTCL. We identified 13 recurrent fusion genes at the mRNA level, 3 of which were tumor-specific and not detected in dogs without lymphoma. 2 of these 3 PTCL-specific fusions, TOX2-LMO4 and PER1-EIF5A, were associated with enrichment for TNF and NF-ĸB signaling and greater enrichment for elements of the tumor microenvironment, respectively, suggesting possible mechanisms by which these fusions could contribute to CD4+ PTCL. Long-read sequencing additionally revealed 3 fusion genes in CD4+ PTCL DNA that were also predicted in bulk RNA-seq data: STAG2-SH2D1A, PLEKHA5-AEBP2, and SERPINB5-ENSCAFG00000031329. The evidence of concurrent chromosomal aberrations and fusion transcript expression for these candidates make them stronger contenders for potential driver mutations that should be explored in future studies. Taken together, the work in this dissertation supports that canine CD4+ PTCL is a naturally occurring neoplasm in dogs that arises from a thymic precursor cell of origin and closely resembles the GATA3-expressing subtype of human PTCL-NOS. Additionally, similar to human PTCL-NOS, recurrent fusion genes are a feature of canine CD4+ PTCL that may arise from a combination of structural chromosomal variants and alternative RNA splicing events, and these fusions may contribute to the pathogenesis of CD4+ PTCL through activation of certain oncogenic signaling pathways. The shared features between canine CD4+ PTCL and human PTCL-NOS represent potential opportunities to explore novel therapies in the dog that may be translated to the human disease.
Open Access
Overactive NF-KB signaling as a druggable target and evaluation of parthenolide an NF-KB inhibitor in canine cancer
(Colorado State University. Libraries, 2022) Schlein, Lisa Janelle, author; Thamm, Douglas H., advisor; Avery, Paul, committee member; Duval, Dawn, committee member; MacNeill, Amy, committee member
This study provides a unique translational research opportunity to help both humans and dogs diagnosed with diseases that carry dismal prognoses in both species: histiocytic sarcoma (HS), hemangiosarcoma (HSA), and disseminated mastocytosis (MCT). Lymphoma is one of the most common cancer types affecting dogs and humans, and therefore, novel therapeutic approaches are always needed. For all of these cancer types, dogs and human cancers share common molecular abnormalities, consistent with a conserved pathogenesis between species. Relative to traditional murine models for human cancers, dogs are genetically diverse, large mammals with heterogeneous, spontaneous tumors. Dogs generally receive good medical care and share the environmental factors with humans, and accordingly, dogs with spontaneous tumors are an excellent model for human oncology generally. Additionally, although disseminated HS, MCT and visceral HSA are exceedingly rare diseases in humans, they are more common in some dog breeds, giving us the opportunity to study this disease in a larger population than would otherwise be available. Therapeutics evaluated in dogs with these diseases stand to benefit both canine and human patients. NF-kB proteins are a family of structurally related, eukaryotic transcription factors that have 400+ genetic targets, and are involved in many vital cellular processes, including innate immunity, inflammatory responses, development, cellular growth, and survival. Not surprisingly, overactivation of NF-kB is a feature of many chronic disease processes, including cardiac disease, neurodegenerative disease, immune-mediated disease, and cancer. While NF-kB overactivation has been documented extensively in human oncology, there is a relative paucity of data documenting the same phenomenon in veterinary medicine. As part of this study, large scale validation of NF-kB overactivation was performed in canine cancer via immunohistochemistry of 215 tumor samples (lymphoma, HS, HSA, and MCT). Antibodies were validated for use via western blot, immortalized cell pellets, and evaluation of normal canine tissues. In addition to validation of NF-kB overactivation, assays were performed to assess the therapeutic potential of parthenolide (PTL), a known, canonical NF-kB signaling inhibitor with additional mechanisms of antineoplastic activity, including alteration of cellular redox balance. Growth inhibition assays were performed with canine cell lines and primary lymphoma cells isolated from canine patients, using PTL alone or in combination with redox-perturbing standard-of-care therapeutics. Cell death was assessed using flow cytometry. Immunofluorescence was used to assess NF-kB localization, western blot was used to assess NF-kB activity with and without PTL, and canine cells were transfected with a reporter gene cassette containing the NFkB consensus sequence followed by firefly luciferase gene to study the effect of PTL on NF-kB-related luminescence. PTL's effects on glutathione and reactive oxygen species generation were assessed with a colorimetric assay and a fluorescent H2DCFDA assay, respectively. Genetic expression changes were assessed with RNA sequencing of HS cells, with and without PTL treatment. A mouse model of disseminated HS was created with NF-kB luminescent cells to study the effect of PTL on this disease in vivo. Many spontaneous canine tumor samples have nuclear p65 and p100/p52 IHC staining that is of greater magnitude than observed in comparable, normal cell populations, indicating the promise of PTL and other therapeutics that target aberrant NF-kB signaling. Canine cell lines and primary cells are sensitive to PTL and undergo dose-dependent apoptosis following exposure to drug. PTL exposure also leads to glutathione depletion, reactive oxygen species generation, and NF-kB inhibition in canine cells. Standard-of-care therapeutics broadly synergize with PTL. In two canine HS cell lines, genetic expression of NF-kB pathway signaling partners is downregulated with PTL therapy. Preliminary data suggest that PTL inhibits NF-kB activity of cells in a mouse model of disseminated HS. Overall, these data support further investigation of compounds that can antagonize canonical and alternative NF-kB pathway signaling, which are overactivated in canine lymphoma, HS, HSA, and MCT disease. PTL is one promising therapeutic that acts, in part, via canonical NF-kB antagonism in canine neoplasms. Further investigation of this compound in vivo is underway in a mouse model of disseminated HS, and if this study is successful, it will provide strong justification for clinical trials with this compound in dogs.
Open Access
The roles of iron, the infrapatellar fat pad, and dietary factors in the Hartley guinea pig model of spontaneous osteoarthritis
(Colorado State University. Libraries, 2018) Radakovich, Lauren, author; Santangelo, Kelly, advisor; Olver, Christine, committee member; MacNeill, Amy, committee member; Foster, Michelle, committee member; Pagliassotti, Michael, committee member
Osteoarthritis (OA) is the most prevalent musculoskeletal disorder across the world, affecting close to 300 million people. The disease manifests as degeneration and loss of articular cartilage, synovial hyperplasia, formation of osteophytes, subchondral bone remodeling, and joint space narrowing. These changes result in decreased range of motion as well as painful mobility in affected individuals. The knee joint is the most commonly afflicted joint. Osteoarthritic changes may develop secondary to a localized injury, referred to as post-traumatic OA. Degenerative changes can also develop without an inciting cause, which is referred to as spontaneous, or primary OA. Spontaneous OA is an insidious disease that is associated with aging, and, more recently, with obesity. The mechanisms contributing to disease development are not yet fully characterized, which has impeded implementation of successful treatment options. Currently, there are no treatments that are able to restore degraded cartilage. Thus, most patients with symptomatic knee OA undergo costly total knee joint replacement surgeries. The aims of this dissertation were to explore the roles that aging-associated iron accumulation, the infrapatellar fat pad, and calorie restriction with various diets may play in OA development. These studies were performed in the Hartley guinea pig, one of the only small animal models of spontaneous OA. One study was performed in Strain 13 guinea pigs, a strain that is OA-resistant. Iron is an element that acts as a double-edged sword. It is essential for oxidative phosphorylation and heme synthesis, yet its redox potential means it has the capacity to incite oxidant damage when present in excess. As there is no direct excretion mechanism for iron, it tends to accumulate slowly within tissues over time. This cellular iron accrual has been implicated in many degenerative diseases associated with aging, but its potential role in spontaneous OA has not been well-studied. In our first studies, we demonstrated that systemic iron levels have an influence on OA. We were able to induce bony and cartilage lesions in OA-resistant Strain 13 guinea pigs by systemic administration of iron dextran. Immunohistochemistry (IHC) indicated higher levels of lipid peroxidation in cartilage, menisci, the infrapatellar fat pad (IFP), and synovium in the iron overload animals. Special stains revealed that iron content was significantly higher in the IFP in these animals, which we propose serves as a local depot of oxidant damage to the knee joint. In a parallel study, we fed OA-prone Hartley guinea pigs an iron deficient diet to determine if reducing systemic iron levels may have a protective effect on the knee joint. Cartilage lesions were significantly lower in the iron deficient diet group compared to controls. Likewise, IHC for lipid peroxidation revealed less oxidant damage in the iron deficient pigs. However, no differences were noted in knee joint iron content, so the exact mechanisms for the lessened OA remain unclear. Because the iron overload study pointed to the IFP as a potential iron depot, we wanted to further characterize how this adipose tissue contributes to overall knee joint homeostasis. First, we demonstrated that quantitative iron content in the IFP was increased in aged, osteoarthritic guinea pigs compared to young, healthy animals. Gene expression data collected suggested that dysregulated iron trafficking, particularly increased expression of ZIP14 – which has been linked to pathologic iron uptake in other conditions – may be contributing to this aging-associated increase of iron in the IFP. Because of our suspicion that the IFP may be inciting local oxidant damage to the knee, we surgically removed it from a set of young Hartley guinea pigs. An identical sham procedure was performed in the contralateral limb. Four months post-surgery, animals were collected to evaluate OA in both limbs. Both cartilage and bony OA scores were markedly decreased in the IFP removal limb compared to the sham surgery limb. It is possible that removal of the IFP removed a source of local inflammatory mediators and iron, which resulted in lessened OA. As the IFP was replaced by a thick band of fibrous connective tissue, increased joint stability was also considered a contributing factor. Future studies will more closely examined contributions of biomechanical factors that may be at play. Finally, we aimed to determine how dietary manipulations may influence early OA, as previous studies suggest that calorie restriction may improve end-stage OA. Additionally, many studies have shown high fat diet (HFD)-induced obesity plays a role in OA development due to the inflammatory nature of excess adipose tissue. In our study, we demonstrated that calorie restriction with a low fat regular chow diet, but not a calorie restricted HFD, delayed onset of OA in Hartley guinea pigs. In fact, the HFD group had higher levels of systemic inflammation than the restricted regular chow group. The HFD group had similar levels of inflammation and OA scores as obese animals. Thus, we concluded that the pro-inflammatory nature of a HFD supersedes any positive effects of calorie restriction in the onset of spontaneous OA.