Browsing by Author "Dow, Steven, advisor"
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Item Open Access Cationic liposome-DNA complex-based immunotherapeutic and immunization strategies for control of la Crosse virus and Leishmania major infections(Colorado State University. Libraries, 2011) Arthun, Erik Norden, author; Dow, Steven, advisor; Foy, Brian, committee member; Lappin, Michael, committee member; Callan, Robert, committee memberTo view the abstract, please see the full text of the document.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 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 Do mesenchymal stromal cells abrogate the immune response in massive cortical allograft recipients?(Colorado State University. Libraries, 2015) McNamara, Kaitlyn Louise, author; Ehrhart, Nicole, advisor; Dow, Steven, advisor; Donahue, Seth, committee member; Duncan, Colleen, committee member; Palmer, Ross, committee member; Page, Rodney, committee memberOBJECTIVE: To evaluate the humoral and cellular immune response against bone associated antigens when delivered in a vaccine, and to evaluate the immunomodulation on the aforementioned immune response with the addition of adipose-derived mesenchymal stromal cells (AD-MSCs). ANIMALS: 68 C57BL/6 mice PROCEDURES: Femur fragments harvested from Balb/C or C57BL/6 mice were resuspended in PBS and cationic liposomal DNA complexes (CLDC) to create an allograft or autograft vaccine, respectively. A positive control vaccine was created utilizing bovine alkaline phosphatase (ALP) resuspended in PBS and CLDC. Twenty C57BL/6 mice were divided into four treatment groups: non-vaccinated (n=5), ALP vaccine recipients (n=5), allograft bone vaccine recipients (n=5), or autograft bone vaccine recipients (n=5). Forty-eight C57BL/6 mice were divided into the same 4 vaccine treatment groups (n=16), and received either intravenous AD-MSCs (n=8) or a subcutaneous injection of AD-MSCs (n=8). All mice received an initial vaccine on Day 1 and a booster vaccine on Day 7, followed by euthanasia on Day 21. Blood was collected on Day 1 and Day 7 just prior to vaccination, and on Day 21 just prior to euthanasia. Serum was subjected to an antibody detection ELISA to evaluate the humoral response. Spleens were collected and flow cytometry was used to evaluate T cell proliferation as an indicator of the cellular immune response. RESULTS: The bone vaccines did no elicit a detectable humoral immune response to the total bone antigen vaccine used in this model. The addition of AD-MSCs had no effect on the lack of a detectable humoral immune response. The T cell response towards a bone antigen was dampened in mice previously vaccinated with a bone vaccine. This effect was most pronounced when looking at the T cell response towards an allograft bone antigen in mice previously vaccinated with an allograft bone vaccine, particularly with the addition of AD-MSCs. CONCLUSIONS AND CLINICAL RELEVANCE: The bone vaccine model used in this study allowed evaluation of the humoral and cellular immune response towards bone associated antigens. The model suggests that recipients of an allograft bone vaccine will dampen the T cell proliferation seen upon second exposure to the bone antigens. This model could be used in future vaccine studies looking at the effect of vaccinating mice with a bone vaccine prior to undergoing a limb-salvage procedure. If efficacious, the bone vaccine model may provide a new treatment option for decreasing the risk of transplant rejection following massive limb reconstruction.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 Evaluation of allogeneic bone marrow-derived mesenchymal stem cells for use in equine joints: in vitro to preclinical evaluation(Colorado State University. Libraries, 2019) Colbath, Aimee, author; Goodrich, Laurie, advisor; Dow, Steven, advisor; McIlwraith, Wayne, committee member; Schenkel, Alan, committee member; Nakamura, Nori, committee memberJoint disease is prominent in the equine population and horses provide a highly translational model for human joint disease. Mesenchymal stem cells (MSCs) have been investigated as a treatment of musculoskeletal disease in the horse with autologous MSCs showing promise as a treatment of desmitis, tendonitis and joint disease including meniscal injury and osteoarthritis. However, the culture expansion of autologous MSCs is both labor intensive and time consuming with an average expansion time of 2-4 weeks. Allogeneic MSCs would offer multiple potential advantages over autologous MSCs use including timing of treatment, potential for characterization, and selection of donors for desired stem cell characteristics. The safety of allogeneic MSCs must be established prior to clinical use. Allogeneic MSCs have been evaluated in vitro and in vivo, but rarely have allogeneic MSCs been directly compared with autologous MSCs. In addition, pre-clinical models must control for the large variability present in individual horses' reactions to joint injections as well as the variability in how different joints react to intra-articular treatments. Further, the safety of allogeneic MSCs must be examined in both the normal joint and inflammatory joint as MSCs may react to the joint environment. The goals of the research described in this dissertation were to directly compare the immune suppressive ability of autologous and allogeneic bone marrow-derived MSCs (BMDMSCs) in vitro, and directly compare both the normal and inflamed joint response to autologous and allogeneic BMDMSCs in vivo. In the first part of this work we compared the immune suppressive properties of allogeneic and autologous BMDMSCs in vitro. No difference was detected between the ability of allogeneic versus autologous BMDMSCs to suppress lymphocytes in modified mixed lymphocyte reactions. This work also established prostaglandin E2 as an important mediator of immune suppression used by allogeneic BMDMSCs. Following in vitro studies, two preclinical, in vivo studies were performed. In the first study, allogeneic and autologous BMDMSCs were administered into clinically normal, contralateral, metacarpophalangeal joints. No difference was detected in the clinical or cytological response of the normal equine joint to allogeneic versus autologous BMDMSCs. After establishing the response of the equine tibiotarsal joint to recombinant IL-1β (rIL-1β), an additional in vivo study was conducted to determine the inflamed joint response to allogeneic versus autologous BMDMSCs. In this study, no difference was detected in synovial fluid parameters, subjective lameness, or joint effusion between the inflamed joint response to allogeneic versus autologous BMDMSCs. In addition, no decrease in joint inflammation was detected as a result of autologous or allogeneic BMDMSC administration. The work described in this dissertation has improved our understanding of the equine joint response to allogeneic and autologous BMDMSCs. Further, it supports future exploration into the use of allogeneic BMDMSCs for musculoskeletal disease in the horse. Specifically, this work should be followed with a direct comparison of the efficacy of allogeneic versus autologous BMDMSCs for joint disease in the horse.Item Open Access Immune modulatory and antimicrobial properties of canine and human mesenchymal stem cells(Colorado State University. Libraries, 2018) Chow, Lyndah, author; Dow, Steven, advisor; Koch, Peter, committee member; Zabel, Mark, committee member; Tjalkens, Ronald, committee memberTo view the abstract, please see the full text of the document.Item Open Access Impact of activated and resting mesenchymal stem cells on immune responses and gut microbiome and immune responses to gut bacteria in dogs with inflammatory bowel disease(Colorado State University. Libraries, 2019) Soontararak, Sirikul, author; Dow, Steven, advisor; Twedt, David, committee member; Lappin, Michael, committee member; Henao-Tamayo, Marcela, committee memberIdiopathic inflammatory bowel disease (IBD) is one of the common diseases that causes gastrointestinal tract (GI) disorder and encompasses a group of unknown causes of chronic gastroenteropathies found to have persistent or recurrent GI signs along with intestinal and/or gastric inflammation. Similar to IBD in humans, the pathogenesis of IBD in dogs remains undiscovered, but it is believed to involve an interaction between the abnormal host immune response against intestinal microbiota and predisposing genetic and environmental factors. IBD is mostly incurable with long-term complications despite receiving standard treatments that are typical combinations of food trial, antibiotics, anti-inflammatory and immunosuppressive drugs. However, the therapeutic outcome of medical treatment appears to be multifactorial and inconsistent therapeutic responses ranging from transient recovery to no response have been found. One of the alternative treatments that potentially accelerates therapeutic effects is the use of mesenchymal stem cell (MSC) administration. Therefore, the goal of the research presented in this dissertation was to comprehensively investigate the impact of activated and resting MSCs on immune responses, cells regeneration and gut microbiome for treatment of IBD with a specific emphasis on gaining an improved understanding of the immune responses to the gut bacteria in dogs with IBD. In the first part of the study, we needed to have better understanding of immunopathogenesis in IBD. Although it is not clear what triggers the intestinal inflammations in IBD affected dogs, we hypothesized that the disease may be mediated, in part, by an abnormal immune response directed against intestinal bacteria. We found the substantially greater percentages and overall binding of IgG and IgA with their intestinal bacteria in IBD dogs than healthy dogs, and the primary production of anti-bacterial antibodies occurs locally in the gut rather than systemically. The IgG-binding bacteria triggered an increase of phagocytosis and pro-inflammatory cytokine production by macrophages. Moreover, Actinobacteria (Collinsella genus) was the preferential target for the mucosal IgG immune response to dysbiotic bacteria. We concluded that the mucosal antibody binding to commensal gut bacteria was substantially greater in dog with IBD compared to a healthy, and that the immune response targeted particular bacteria and triggered the pro-inflammatory response in IBD. We noted that the more extensive studies in dogs with IBD and compared to animals with other causes for GI dysfunction may be required. Then, we focused on the use of MSCs as an alternative treatment for IBD in animals and humans. To address this question, we used a mouse model of IBD to investigate the effectiveness of using 2 types of mesenchymal stem cells (induced pluripotent MSC [iMSC] and conventional adipose-derived MSC [adMSC]) for the treatment of IBD. The impact of MSCs on immune responses, cells regeneration and the gut microbiome were evaluated. We found that iMSC and adMSC treatment effects were equivalent on the basis of significantly improving clinical abnormalities and decreasing inflammation inside the gut. Both types of MSC also stimulated a significant increase in intestinal epithelial cell proliferation and amplified intestinal angiogenesis. Furthermore, the abnormal microbiome found in mice with IBD was returned to nearly normal values in terms of complexity and composition in mice with IBD treated with adMSC or iMSC. We concluded therefore that the administration of iMSC enhanced the overall intestinal healing, suppressed inflammation, and microbiome restoration with equal effectiveness as treatment using adMSC in a mouse model of IBD. The future studies in animal model including spontaneous IBD in dog or large scale of clinical trial for long-term follow-up to determine iMSC safety and efficacy is required before clinical translation. Finally, we investigated possible ways to improve the efficacy of mouse and dog MSC treatment by preactivating the MSC with inflammatory cytokines (IFN-g or TNF-a) or TLR agonists (TLR3 or TLR9 agonists). We investigated the response of canine MSCs to the 4 activating stimuli, including measurement of cell surface phenotype and cytokine release. Contrary to previous studies in other species including mouse and man, we found that the pre-activation of dog MSC generally had little effect on either phenotype or function. Therefore, we concluded that the ex-vivo preactivation of canine MSCs by inflammatory cytokines or TLR agonists is not warranted in terms of augmenting the functionality of the cells. We further concluded that dog MSC may be hyporesponsive to preactivating stimuli compared to those of MSC from other species such as mouse and man. Further studies are required for better understanding of the biology of canine MSCs and their responses to immune activation. Overall, the work described in this dissertation has increased our understanding regarding the immunopathogenesis of the IBD in dogs. The studies have also demonstrated the equivalent activity of iMSC and conventional adMSC for treatment of IBD, and also documented a previously undescribed restorative effect of MSC on the intestinal microbiome. These studies also illustrated species specific differences in the responsiveness of MSC to common immune stimuli. These studies provide a robust foundation for further research and hopefully this work can help stimulate new investigations into alternative treatments for IBD in dogs and humans.Item Open Access Mechanisms of IFN-γ and ceftazidime interaction for synergistic killing of Burkholderia(Colorado State University. Libraries, 2014) Mosovsky, Kara, author; Dow, Steven, advisor; Schenkel, Alan, committee member; Schweizer, Herbert, committee member; Callan, Robert, committee memberBurkholderia pseudomallei is a Gram negative, facultative intracellular pathogen which infects both phagocytes and non-phagocytes and causes severe acute infections in humans and animals. Due to its inherent resistance to many classes of antibiotics, new therapies are needed which can supplement or substitute for conventional treatments in order to combat this emerging infectious disease. We have previously shown that interferon (IFN)-γ can interact with the conventionally administered antibiotic, ceftazidime, to synergistically control intracellular bacteria burden of Burkholderia infected macrophages. The goal of the studies presented here was to determine the mechanism by which IFN-γ and ceftazidime exert their synergistic effect. After investigating several potential mediators of immuno-antimicrobial synergy, we showed that IFN-γ stimulation of macrophages led to increased generation of reactive oxygen species (ROS), which led us to hypothesize that IFN-γ induced ROS may interact with ceftazidime to control intracellular bacterial burden. We next found that ROS scavenging antioxidants such as N-acetylcysteine (NAC) and reduced glutathione (GSH) were capable of reversing the IFN-γ and ceftazidime synergistic effect, while the ROS-inducing drug buthionine sulfoximine (BSO) could not only potentiate the synergy, but could completely substitute for IFN-γ to synergize with ceftazidime and control intracellular bacterial burden. These results were consistent with a ROS interaction with ceftazidime. We further showed that IFN-γ prevented vacuolar escape and actin polymerization, a finding which was recapitulated with BSO. Taken together, these results suggested that generation of IFN-γ induced ROS responses synergized with ceftazidime to enhance control of intracellular bacterial burden. IFN-γ induced ROS was also responsible for preventing vacuolar escape and therefore may have limited intracellular replication and spread of infection. In the second half of our study we identified and then investigated the separate and compartmentalized contributions of IFN-γ and ceftazidime to the overall synergistic effect. We determined that ceftazidime alone controlled extracellular killing in our macrophage infection model while IFN-γ alone controlled the killing of Burkholderia in the intracellular compartment. We confirmed a role for IFN-γ induced ROS responses to kill intracellular bacteria and control intracellular replication, though we also conclude that other IFN-γ-dependent and ROS-independent pathways are at play. Overall we suggest a new model to describe the dynamics of the classically used macrophage infection model. We suggest that both intracellular and extracellular control of bacteria is required for the overall synergistic effect we see with combination of IFN-γ and ceftazidime. Together our studies have implications for the use of IFN-γ, or other ROS-inducing drugs, as non-specific antibiotic potentiating agents for enhanced clearance of bacterial pathogens.Item Open Access Modulation of immune responses on mucosal surfaces through vaccination and dietary intervention(Colorado State University. Libraries, 2011) Henderson, Angela J., author; Dow, Steven, advisor; Schenkel, Alan, committee member; Biller, Barbara, committee member; Gonzalez-Juarrero, Mercedes, committee memberNumerous pathogenic organisms enter the body at the mucosal surfaces and therefore the mucosal immune response must function as the first line of defense. The ability of the body to induce protective immune responses on the mucosal surfaces is a powerful strategy for the prevention of disease. Therefore, understanding the mechanism of induction associated with protection is critical if there is to be improvement in current treatments. In these studies, the use of vaccination and diet were investigated as potential strategies for the induction of potent immune responses on the mucosal surfaces. The principle of vaccination has been used successfully for centuries. However, there is still a great need for the development of vaccines against mucosal pathogens such HIV, TB, and newly emerging pathogens. The primary way to improve mucosal vaccination is through the use of a potent vaccine adjuvant. The first part of this project focuses on the use of cationic-liposome plasmid DNA complexes (CLDC) as a mucosal vaccine adjuvant for enhancing the immune response to both particulate and soluble antigens. In these studies, intranasal vaccination using CLDC resulted in a balanced humoral and cellular immune response capable of protecting against a lethal pulmonary bacterial challenge. We found that mucosal immunization with CLDC adjuvant resulted in the increase in the pro-inflammatory cytokines IL-6 and IFN-γ. Also, cellular immune responses were shown to be dependent on MyD88 signaling. Finally, resident airway myeloid dendritic cells (DC) efficiently phagocytosed the CLDC adjuvant and efficiently trafficked the associated antigen to the draining lymph node. Therefore the effectiveness of CLDC as a mucosal vaccine adjuvant appears to depend on strong cytokine induction and efficient antigen presenting cell activation and migration. In a similar manner, dietary modulation has been shown to significantly impact the intestinal immune environment and has only recently begun to be investigated. It represents a novel approach for enhancing protective responses against pathogens and inflammatory diseases. The focus of the second part of this study is the ability of dietary rice bran to modulate the mucosal immune response as a potential mechanism to prevent disease. We found that a diet containing 10% rice bran resulted in an increase in local IgA concentrations and surface expression of IgA on mucosal B cells. Also, dietary rice bran induced a significant increase in myeloid dendritic cells residing in the lamina propria and mesenteric lymph nodes, and increased the colonization of native Lactobacillus, a beneficial gut microorganism known for its ability to positively influence the mucosal immune system. This work has increased our knowledge of the impact of vaccination and dietary modulation for the protection of the mucosal surfaces. More specifically, these findings have revealed that CLDC is a potent vaccine adjuvant and that incorporating rice bran in a balanced diet can augment the mucosal immune environment.Item Open Access PD-L1 expression by tumor macrophages: regulation and signaling(Colorado State University. Libraries, 2018) Hartley, Genevieve, author; Dow, Steven, advisor; Schenkel, Alan, committee member; Gonzalez-Juarrero, Mercedes, committee member; Biller, Barbara, committee memberTo view the abstract, please see the full text of the document.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 analysis of Burkholderia pseudomallei virulence and efficacy of potential therapeutics(Colorado State University. Libraries, 2011) Propst, Katie L., author; Schweizer, Herbert, advisor; Dow, Steven, advisor; Gentry-Weeks, Claudia, committee member; Goehring, Lutz, committee memberBurkholderia pseudomallei is the causative agent of the disease melioidosis and is classified as a category B Select Agent. There are currently many challenges associated with both the study of this pathogen and its treatment in the clinical setting. Prior to these studies, there was no attenuated B. pseudomallei strain available that was exempt of Select Agent regulations and approved for study outside of biosafety level 3 (BSL-3) containment, and consequently basic research on this pathogen was largely hindered. The first purpose of these studies was to extensively characterize the attenuation of two B. pseudomallei mutant strains using melioidosis animal models. The two mutants constructed were Bp82 and Bp190, Δ purM derivatives deficient in adenine and thiamine biosynthesis. These mutants were found to be fully attenuated in immune competent and immune deficient mouse and hamster melioidosis models. Bp82 is currently exempt of all Select Agent regulations and can be safely handled in the BSL-2 setting, greatly accelerating research on this priority pathogen. Since basic research on B. pseudomallei was not common in the Western world until its Select Agent classification, much is still unknown regarding the bacterial factors contributing to its virulence. A second purpose of this research was to determine whether resistance-nodulation-cell division (RND) efflux systems and iron acquisition siderophores impact the virulence of B. pseudomallei in a pneumonic murine melioidosis model. This was examined using a clinical isolate naturally devoid of a characterized efflux system and the gene cluster for malleobactin siderophore synthesis, and by the construction of isogenetic mutants. The two characterized B. pseudomallei efflux pumps, AmrAB-OprA and BpeAB-OprB, were both found to be completely dispensable during in vivo murine infection. The removal of one or both of these systems did not reduce lethality of the mutant strains. Unlike that observed with similar bacterial pathogens, the lethality of B. pseudomallei was also not reduced upon the removal of either the malleobactin or pyochelin siderophores. This finding indicates B. pseudomallei is likely capable of utilizing alternative systems for iron acquisition within the host. In addition to the challenges associated with the study of this pathogen, there are also many clinical challenges associated with melioidosis, providing a basis for the final two purposes of this research. One particular challenge is the high frequency of patient relapse, even after appropriate prolonged antibiotic therapy. A third purpose of this research was to determine whether traditional antibiotic therapy could be augmented by the co-administration of immunotherapy. Cationic liposome-DNA complexes (CLDC), which are potent activators of the innate immune system, were found to synergistically reduce intracellular B. pseudomallei concentrations in macrophages in vitro when combined with the antibiotic ceftazidime. In addition, this combination therapy also significantly increased mouse survival during both acute and chronic melioidosis. A similar enhancement to ceftazidime therapy was observed with recombinant IFN-γ, illustrating the potential of immunotherapy to improve clinical outcome and decrease patient relapse. The lack of an effective approved vaccine for human use is another substantial clinical challenge associated with melioidosis and its prevention. The final purpose of these studies was to develop an effective mucosal vaccine, offering both short-term protection from acute pneumonic disease and long-term protection from disseminated chronic melioidosis. CLDC was identified as a highly effective mucosal adjuvant within complexed to heat-killed B. pseudomallei, and this adjuvant offered moderate protection from acute disease when combined with Burkholderia protein subunits. The longest-term protection from lethal challenge in our murine model, lasting beyond 100 days, was elicited by the fully attenuated live Bp82 strain. Since this strain is both fully attenuated and exempt of Select Agent regulations, it has great potential clinically for high-risk persons as an effective live vaccine strain.Item Open Access Use of immune activated cellular therapy and risks with antibiotic administration in treatment of septic arthritis(Colorado State University. Libraries, 2021) Pezzanite, Lynn M., author; Dow, Steven, advisor; Goodrich, Laurie, advisor; Hendrickson, Dean, committee member; Santangelo, Kelly, committee member; Stoneback, Jason, committee memberTo view the abstract, please see the full text of the document.Item Open Access Use of liposomal bisphosphonates to deplete macrophages for cancer immunotherapy(Colorado State University. Libraries, 2011) Hafeman, Scott D., author; Dow, Steven, advisor; Gustafson, Daniel, committee member; Thamm, Douglas, committee member; Zabel, Mark, committee memberIn order for continued growth, metastasis and evasion from immune surveillance, tumor cells are dependent on a complex matrix of supportive cells and tissues. These cells make up a significant percentage of the tumor mass and contribute to the hallmarks of malignancy. Of these, the tumor associated macrophage (TAM) has perhaps the most diverse role. In the majority of tumor types studied, increased percentages of these cells in the tumor correspond to a poorer prognosis for the patient. Macrophages are critical in wound healing, and as such provide a wide variety of factors that may be co-opted by the tumor to support its continued growth and metastasis. Macrophages are capable of producing a wide variety of growth factors that directly promote tumor cell growth. These factors can also be used to induce tumor cell migration and invasion, which are critical steps in metastasis. They also produce matrix metalloproteinases that actively degrade basement membranes, further aiding in invasion and metastasis. Macrophages also produce many factors that help induce angiogenesis, providing vital blood supply to the developing tumor. Through both direct and indirect mechanisms they are vital to providing new tumor blood vessels. In addition to these direct tumor aiding effects, macrophages also play a critical role aiding in both local and global immunosuppresion in tumor patients, which allows the established tumor to continue to evade the immune system. Therefore, the targeting and killing of TAMs could potentially be a promising new adjunct to traditional cancer therapies, and may increase the efficacy of traditional therapeutics. One potential drug for this purpose is liposomal clodronate. This drug is produced by encapsulating the bisphosphonate drug clodronate in a liposome. As a free drug, clodronate is very effective at inducing apoptosis of osteoclasts, a close relative to the macrophage. Encapsulation in a lipid bilayer prevents the dissemination of the drug to the bone matrix and instead allows for systemic distribution. However, only cells that phagocytize and degrade the lipsome are susceptible to killing by the enclosed clodronate. Liposomal clodronate has been used extensively to deplete macrophages in studies of autoimmune disease and more recently in tumor models. However the systemic depletion of tumor associated macrophages using liposomal clodronate (LC) has not been previously evaluated in clinical trials, and the effects of systemic LC administration on tumor growth have not been fully elucidated. Studies presented here sought to further determine the role of tumor associated macrophages in tumor growth by studying the effects of their depletion. Specifically, in vitro studies were used to determine an optimal formulation of liposome to more effectively deliver the bisphosphonate drug to macrophages. Using multiple murine macrophage cell lines and proliferation assays the most effective depleting liposome was determined. This formulation consisted of a net neutral charged phosphatidylcholine head group combined with an incorporated mannose group. These liposomes were then evaluated in vivo for their ability to deplete macrophages systemically. Once again, the modified liposome formulation was most effective. The drug was then evaluated for its ability to decrease tumor growth in a mouse fibrosarcoma model, using MCA 205 tumors subcutaneously implanted into C57BL/6 mice. The drug's ability to deplete tumor associated macrophages was also evaluated. Tumor growth rates and tumor associated macrophage numbers were significantly decreased in mice treated with liposomal clodronate as compared to untreated mice or those treated with liposomal PBS. Additional studies were undertaken to determine if liposomal clodronate could be used as an effective cancer therapeutic in a spontaneous tumor model. The tumor evaluated was malignant histiocytosis (MH). This tumor was chosen as it is a tumor derived from macrophages or dendritic cells, and LC could potentially have both primary anti-tumor effects as well as efficacy due to depletion of TAMs. In vitro studies were undertaken which showed that LC was capable of effectively killing MH cells. Based on these results, a clinical trial was conducted for dogs with MH. Dogs were treated with 0.5 mL/kg of liposomal clodronate IV every other week for six treatments. A total of 12 dogs were treated in the study. Treated dogs were evaluated for tumor response, changes in circulating blood cells, and changes in circulating cytokines. We were able to observe a 40% biologic response rate (BRR). The development of a fever was positively correlated with response. Responding dogs also had an increase in neutrophils and a decrease in monocytes while non-responding dogs did not. A significant reduction in serum Il-8 levels occurred post LC treatment. As the clinical availability of LC is currently limited to experimental use additional studies were conducted to determine if combining free bisposphonates, which are readily available, with traditional chemotherapeutics could cause synergistic killing of MH cells in vitro. The combination of clodronate with vincristine or zoledronate with doxorubicin demonstrated synergistic killing in vitro. Further evaluation of these combinations will be necessary to determine if they have a similar effect in vivo.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.