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Browsing by Author "Webb, Craig, committee member"

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    Applications of feline immunodeficiency virus as a model to study HIV pathogenesis
    (Colorado State University. Libraries, 2018) Miller, Craig Andrew, author; VandeWoude, Susan, advisor; Hoover, Edward, committee member; Han, Sushan, committee member; Webb, Craig, committee member
    Feline immunodeficiency virus (FIV) is a naturally-occurring retrovirus that infects domestic and non-domestic feline species, and produces progressive immune depletion that eventually results in an acquired immunodeficiency syndrome (AIDS). While it is accepted that FIV is primarily transmitted by biting, few studies have evaluated FIV oral infection kinetics and transmission mechanisms over the last 20 years. Modern quantitative analyses applied to natural FIV oral infection could significantly further our understanding of lentiviral oral disease and transmission. In this Chapter 1 of this dissertation, I characterized FIV salivary viral kinetics and antibody secretions to more fully document oral viral pathogenesis. The results of this research demonstrate that (i) oral lymphoid tissues serve as a site for enhanced FIV replication, resulting in accumulation of FIV particles and FIV-infected cells in saliva, and (ii) failure to induce a virus-specific oral mucosal antibody response, and/or viral capability to overcome inhibitory components in saliva may perpetuate chronic oral cavity infection. Most importantly, these results provide a model of oral FIV pathogenesis and suggest alternative diagnostic modalities and translational approaches to study oral HIV infection. Feline immunodeficiency virus and human immunodeficiency virus (HIV) utilize parallel modes of receptor-mediated entry. The FIV surface glycoprotein (SU) is an important vaccine target for induction of virus neutralizing antibodies, and autoantibodies to the FIV binding receptor (CD134) block FIV infection ex vivo; highlighting the potential for immunotherapies which utilize anti-receptor antibodies to block viral infection. In Chapter 2 of this dissertation, I immunized cats with soluble CD134, recombinant FIV-SU protein, and/or CD134+SU complexes prior to challenge with FIV to determine if vaccination with CD134-SU complexes could induce protection against FIV infection. Immunization induced production of anti-CD134 and anti-SU antibodies in vaccinated cats, and purified anti-CD134 and anti-SU antibodies significantly inhibited FIV infection in vitro. However, no vaccine combination protected cats from FIV infection in vivo and vaccination induced high titers of antibodies directed at vaccine by-products relative to target antigens. The results of this research reinforce the need to monitor components of vaccine preparations, and emphasize that vaccination may induce proliferation of susceptible target cells and enhancement of heat-labile serum components that counteract neutralizing antibodies. Feline immunodeficiency virus induces lifelong infection in cats and may result in a spectrum of immunodeficiency-related diseases. Both prednisolone and cyclosporine A (CsA) are commonly used clinically to treat lymphoproliferative and immune-mediated diseases in cats, but the impact of these compounds on FIV infection has not been well documented, and their understanding immunomodulatory effects on FIV replication and persistence is critical to guide safe and effective use of these therapies in FIV infected cats. In Chapter 3 of this dissertation, I administered immunosuppressive doses of prednisolone or CsA to cats chronically infected with FIV and monitored alterations in hematological parameters and FIV viral/proviral loads in response to therapy. Interestingly, both treatments caused (i) acute increases in CD4+ lymphocytes, (ii) increased FIV viremia, and (iii) significant alterations in cytokine expression that favored a shift toward a Th2 response. The results of this research highlight the potential for immunosuppressive drug-induced perturbation of FIV replication and underscores the need for consideration of chronic viral infection status when prescribing immunomodulatory medications. Mucosal immune dysfunction, bacterial translocation, systemic immune activation, and chronic inflammation are well-documented features of chronic HIV infection. Despite the success of combinational antiretroviral therapy (cART) in diminishing HIV viral replication and prolonging immune function, a multitude of systemic and local manifestations of HIV infection persist, including the development of chronic inflammation (periodontitis and gingivitis). Commonly used animal models for studying HIV pathogenesis, including SIV/SHIV infections of non-human primates (NHPs) or HIV infections in humanized mice, do not reliably incite oral lesions. In contrast, gingivitis and periodontitis are primary clinical signs associated with untreated natural and experimental FIV-infection, and are principal attributes of this model that may be exploited to investigate pathogenic mechanisms involved in the perturbation of the oral immune system and microbial environment. Therefore, in Chapter 4, I outline the future directions and research goals for my career, and I present preliminary research results obtained thus far in my studies of the pathogenic mechanisms of HIV-induced oral disease. By assessing FIV-associated changes in clinical status, oral microbiota, local and systemic viral burden, and immune profile under such treatment protocols, future studies implementing the feline model of lentiviral-induced oral disease may provide a cornerstone to expand our understanding of the complex interactions between HIV infection, oral immune dysfunction, and the perturbations to the oral microbiota that occur in the context of HIV infection.
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    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 member
    Type 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.
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    Coagulation abnormalities in Ehrlichia canis-infected dogs and detection and dynamics of anti-platelet antibodies in thrombocytopenic dogs
    (Colorado State University. Libraries, 2018) Shropshire, Sarah, author; Lappin, Michael, advisor; Dow, Steve, committee member; Olver, Christine, committee member; Webb, Craig, committee member; Ames, Marisa, committee member
    Vector-borne diseases affect millions of people and domestic animals worldwide resulting in significant morbidity and mortality rates. In dogs, vector-borne diseases such as Ehrlichia canis can cause a myriad of clinical signs (lethargy, weight loss, and epistaxis) and hematological abnormalities (hyperglobulinemia, thrombocytopenia, and anemia). It has been previously reported that E. canis results in systemic inflammation and vasculitis as well as the formation of immunoglobulin associated platelets or anti-platelet antibodies. It has been theorized that anti-platelet antibodies can deleteriously affect platelet function and if concurrent significant thrombocytopenia is present, signs of bleeding may manifest. However, anti-platelet antibodies and thrombocytopenia can occur in a variety of disease processes in the dog including other vector-borne diseases, neoplasia, and idiopathic primary immune syndromes. Thrombocytopenia is also one of the most common acquired hemostatic abnormality observed in dogs. Consequently, determining the underlying cause and mechanism for thrombocytopenia in dogs can represent a frequent diagnostic challenge. Additionally, inflammation is often present in dogs with thrombocytopenia due to various causes. Inflammation and immune system processes directly affect hemostasis which can lead to derangements in the coagulation system resulting in clinical signs of bleeding or thrombosis. The goals of the research described in this dissertation were to investigate the dynamic changes of anti-platelet antibodies in thrombocytopenic dogs and the changes that occur in the coagulation system during a vector-borne infection such as E. canis in dogs.
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    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 member
    Chronic 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.
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    Design and application of a droplet-digital PCR assay for detection of the STAT5BN642H mutation in feline T cell neoplasia
    (Colorado State University. Libraries, 2024) Bork, Sydney Bonnie, author; Avery, Anne, advisor; Olver, Christine, committee member; Webb, Craig, committee member
    Lymphoma is a commonly diagnosed hematopoietic neoplasm in cats. Small Cell T-cell Epitheliotropic Intestinal Lymphoma (SCL) is the most reported subtype of lymphoma in cats. Cats with SCL are presented with non-specific clinical signs such as chronic vomiting, diarrhea, and weight loss. Diagnostic work-up often includes collection of intestinal biopsies with histopathology for diagnosis. SCL is characterized by infiltration of neoplastic lymphocytes into the intestinal epithelium and lamina propria of the small intestines. Neoplastic cells are small to intermediate in size and of T-cell origin. Diagnosing SCL can be challenging for pathologists because cats also commonly develop a condition called inflammatory bowel disease (IBD), which has an almost identical clinical presentation and similar histopathologic patterns. However, in IBD, the lymphocytic infiltration is often heterogeneous (termed "lymphoplasmacytic enteritis"). When histopathology results are inconclusive, assessment of expression with immunohistochemistry markers can help further characterize the cell population. Additionally, advancements have been made with lymphocyte clonality testing by PARR (PCR for Antigen Receptor Rearrangement), a DNA-based assay that evaluates T-cell receptor (TCR) and Immunoglobulin (Ig) gene rearrangements. Cats diagnosed with SCL demonstrate a clonal TCR result, while cats with IBD demonstrate a polyclonal TCR result. Unfortunately, there are still cases where histopathology and PARR results are equivocal. Recent work in feline medicine has demonstrated that cats with SCL exhibit high expression of phosphorylated STAT5B with immunohistochemical staining on small intestinal biopsy samples compared to cats with IBD. Importantly, one group detected a STAT5BN642H mutation in cats diagnosed with SCL. In this study, 40% (17/42) of cats with intestinal lymphoma were classified as SCL by histopathology. A combination of Sanger sequencing and ARMS qPCR detected the STAT5BN642H mutation in 29.4% (5/17) of cats with SCL. This work correlates to a comparable disease entity in people, monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), which has reported the prevalence of the STAT5BN642H mutation to be 22-57%. Our group aimed to develop a droplet digital PCR (ddPCR) assay to detect wild-type and mutated STAT5B in cats. Our first aim was to design specific primers and locked-nucleic acid hydrolysis probes to detect and discriminate between wild-type and mutated STAT5B. The first step included analyzing data from control samples using wild-type DNA from cats without neoplasia and a positive control gene fragment block ("gBlock"). The second step included analyzing two cohorts of young cats (<6 years of age) without a diagnosis of lymphoid neoplasia to assess assay performance and determine if the mutated STAT5B could be considered a germ line polymorphism. The fractional abundance was calculated from the ddPCR data, estimating the percentage of mutated copies within a positive sample. The results of the first aim demonstrated that our ddPCR assay can distinguish between wild-type and mutated STAT5B with high sensitivity. Most young cats without a diagnosis of lymphoid neoplasia do not carry the STAT5BN642H mutation. Two cats with marked lymphoplasmacytic enteritis had detectable mutated STAT5BN642H. The second aim was to evaluate the prevalence of the STAT5BN642H mutation in cats with confirmed SCL. A sub-aim was to evaluate cats with CD4 T-cell leukemia to determine if this mutation could be found in other forms of T cell lymphoid neoplasia. The results from this aim demonstrate that cats with SCL frequently carry the STAT5B mutation (66.7%). We also discovered that this mutation is not exclusive to cats with SCL, as almost half of the cats with CD4 T-cell leukemia also carry this mutation (47.7%). These findings shed light on the prevalence of the STAT5BN642H mutation in cats with SCL and CD4 T-cell leukemia. This data suggests potential implications for ddPCR mutation detection to help further differentiate and diagnose cats with T cell neoplasia versus those with inflammatory conditions (such as SCL versus IBD), and investigate novel therapies (i.e., JAK/STAT inhibitors). Further research is warranted to investigate other JAK/STAT pathway mutations, particularly in cats where the STAT5BN642H mutation was not detected. Larger outcome studies should investigate the correlation of STAT5BN642H mutation status and the fractional abundance to evaluate disease risk, treatment response, and survival.
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    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 member
    Feline 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.