Browsing by Author "Schenkel, Alan, committee member"

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Open Access
A case study of a wheat-free diet on autoimmune disease progression
(Colorado State University. Libraries, 2014) Connor, Trevor, author; Cordain, Loren, advisor; Hickey, Matthew, committee member; Ryan, Elizabeth, committee member; Schenkel, Alan, committee member
Background and Aims: Autoimmune disease encompasses a broad range of over 80 conditions for which only three have an identified environmental trigger. Gliadin is the trigger in celiac disease, a condition that has been linked to other autoimmune conditions including Crohn's disease and type I diabetes (T1D). The purpose of this study was to investigate case studies of autoimmune patients who employed a wheat-free or Paleo-style diet (WFP) to manage their conditions. Methods: A descriptive case study was performed that utilized questionnaires administered online and medical records from autoimmune disease patients who had consumed a WFP diet. Results: Fifty-seven patients were evaluated in the study (mean age 37.3 yrs., SD 10.1), including 23 males and 34 females, 24 of whom provided medical records. Thirty of the 57 volunteers demonstrated signs of disease improvement while consuming a WFP. The rates varied across conditions with eight of eight Crohn's disease patients experiencing remission, while three of four T1D patients exhibited signs of improvement. Five of 15 patients with ankylosing spondylitis, rheumatoid arthritis, or undifferentiated and multiple connective tissue disorders worsened while on the diet. Conclusions: Patients with single organ autoimmune diseases previously linked to increased intestinal permeability showed the greatest improvement after consuming a WFP. The results of these case studies warrant further controlled research examining the effects of wheat consumption on Crohn's disease and T1D.
Open Access
Biomimetic and antimicrobial surfaces for orthopedic implants
(Colorado State University. Libraries, 2021) Wigmosta, Tara, author; Kipper, Matt, advisor; Popat, Ketul, advisor; Giess, Brian, committee member; DeLong, Susan, committee member; Schenkel, Alan, committee member
The number of total knee and hip replacement surgeries is expected to continue to rise in the United States. As such, the number of revision surgeries is also expected to rise. The two most common causes of failure for these implants is aseptic loosening, caused by incomplete osseointegration, and infection. Therefore, preventing infection while increasing the osteogenic properties of the surfaces used in orthopedic implants could reduce the number of revision surgeries. It is the goal of this work to create nanostructured surfaces that both increase mineralization and antimicrobial properties of titanium surfaces commonly used in orthopedic implants. To accomplish this, chitosan/heparin polyelectrolyte multilayers (PEMs), with the addition of either bone morphogenetic protein 2 (BMP-2) or gentamicin, were adsorbed onto titania nanotubes. BMP-2 has been used in clinical applications to increase osseointegration in spinal fusions, and gentamicin is effective against the most common pathogens found in infected orthopedic implants. Both heparin and chitosan are biocompatible and have antimicrobial properties. BMP-2 has a binding site for heparin that increases BMP-2's half-life in vitro. The first chapter summarizes the motivation and previous strategies used to increase osseointegration and antimicrobial properties of nanostructured biomimetic orthopedic implant surfaces. The first chapter concludes with a shift in hypothesis testing, outlining three different hypotheses: 1) surface modification(s) increase cytocompatibility and the osteogenic properties of mammalian bone cells; 2) surface modification(s) reduce bacterial adhesion, proliferation, and infection rate, without decreasing cytocompatibility; and 3) surface modification(s) provide a favorable environment in which mammalian cells can beat bacterial cells and colonize the surface first, thus increasing the osteogenic and antimicrobial properties of the surface. The testing of these hypotheses are explored in chapters 2 through 4. The second chapter explores hypothesis 1) by testing if BMP-2 released from chitosan/heparin PEM coated titania nanotubes surfaces induce an osteogenic response from rat bone marrow cells. Chapter 3 explores hypothesis 2) by testing if iota-carrageenan/chitosan and pectin/chitosan PEMs have antimicrobial properties against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), and support rat bone marrow cell adhesion and proliferation. The last chapter explores hypothesis 3) by testing if gentamicin released from titania nanotubes coated with chitosan/heparin PEMs influences the "race to the surface" in favor of mammalian cells.
Open Access
Connecting structural changes to cell transformation patterns in the canine degenerative mitral valve
(Colorado State University. Libraries, 2015) Abbott, Kaitlin Marie, author; Orton, E. Christopher, advisor; Frye, Melinda, committee member; Schenkel, Alan, committee member
Degenerative mitral valve disease (DMVD) is a significant problem in the canine population and also affects humans. Recent studies have provided insight into molecular and cellular mechanisms that likely contribute to disease progression. Better understanding of the cellular processes that mediate the degenerative process could lead to treatments that prevent or slow this degeneration benefiting both canine and human patients. Structural changes to degenerative valves such as nodules, leaflet thickening, increased opacity, loss of elasticity and loss of valve architecture have been well documented. Abnormal cell transformation patterns such as the transformation of valvular interstitial cells to activated myofibroblasts have been characterized in degenerative mitral valve tissue, as well as other irregular cell behavior such as the overproduction of glycosaminoglycan and matrix remodeling factors that have become hallmarks of the disease. Despite these important discoveries, much remains unknown about cell signaling in degenerative mitral valve disease and how cell activity changes a normal valve to the diseased phenotype. An overarching hypothesis of this study is that investigating signaling mechanisms active in degenerative valves could provide insight into cellular processes mediating the disease. A specific hypothesis that emerged from initial results is that endothelial to mesenchymal transition (EndMT), a process important in valvulogenesis, could be active in degenerative mitral valves. The first goal of this study was to compare protein abundance in degenerative and normal mitral valves to determine if there exists previously unidentified signaling molecules that could be initiating or perpetuating the cellular transformations and abnormalities present in DMVD. The second goal was to investigate these proteins using immunohistochemistry to characterize their activity in the tissue matrix and show evidence of their contribution to structural changes of the valve. The first goal was accomplished by doing a targeted microarray analysis of signaling proteins comparing their relative abundance in normal and degenerative mitral valves. This analysis yielded an increased abundance of signaling proteins that have been associated with EndMT. The second goal was accomplished by immunohistochemistry to determine the spatial distribution of selected proteins from the microarray analysis with markers of endothelial cells and mesenchymal cells (activated myofibroblasts). Targeted microarray analysis of signaling proteins revealed increased abundance of 18 proteins including the growth factor HB-EGF, its partner molecule ADAM17, and the cell adhesion molecule integrin β3, all possible mediators of EndMT (Chapter 4). Immunohistochemistry studies demonstrated the presence of cells positive for the endothelial marker CD31 within the valve interstitum. These CD31 positive cells co-localized with areas of myofibroblast transformation in degenerative valves identified by positive staining for α-smooth muscle actin (αSMA). Expression of signaling proteins including HB-EGF and ADAM17 also co-localized to these areas (Chapter 5). In conclusion, these results support active EndMT in canine degenerative mitral valves. EndMT could be contributing to the formation of high cellular density myofibroblast transformation which has been postulated to mediate mitral valve degeneration.
Open Access
Development of an ultrasensitive ELISA for the detection of Mycobacterium tuberculosis antigens: an impossible challenge or a promising feat?
(Colorado State University. Libraries, 2022) Early, Kala, author; Dobos, Karen, advisor; Mehaffy, Carolina, advisor; Schenkel, Alan, committee member; Henry, Charles, committee member
Tuberculosis (TB) has been classically characterized as a two-state disease with active and latent phases. Latent TB infection (LTBI) is diagnosed by either the tuberculin skin test (TST) or the Interferon Gamma Release Assay (IGRA) test. However, both diagnostic tests are unable to differentially diagnose active TB and LTBI and perform poorly in immunocompromised patients. The TST is further complicated by cross-reactivity with BCG vaccination. Therefore, further diagnostic discovery for LTBI is needed for differential diagnosis and to identify those at risk of progression to active TB for subsequent treatment. Extracellular vesicles (EVs) are nanovesicles released by eukaryotic cells. EVs from TB patients contain Mycobacterium tuberculosis (Mtb) proteins, and these protein biomarkers show promise for TB and LTBI diagnostics. Our lab previously identified 31 Mtb peptides in trypsin-treated serum EVs isolated from patients with LTBI using multiple reaction monitoring-mass spectrometry (MRM-MS) methods. MRM-MS is a highly sensitive technology but is not feasible for widespread use as a diagnostic. The goal of this study was to develop an ultrasensitive ELISA against Mtb proteins for potential use as a point-of-care diagnostic. A sandwich ELISA was initially developed against Mtb proteins DnaK, Mpt32, and GroES. Reagent development for the sandwich ELISA included polyclonal antibody production using a rabbit model, murine monoclonal antibody purification and biotinylation from an existing collection of hybridoma cell lines for each antigen, and detection using a streptavidin-HRP system with a chemiluminescent substrate for signal expansion. We observed that the sandwich ELISA was complicated by non-specific binding of the DnaK and GroES antigens to the BSA block. We hypothesized that the chaperone function of these two proteins influenced them to bind to BSA. This non-specific interaction was further characterized using SPR technology and demonstrated a concentration dependent binding of DnaK to BSA. A direct-biotinylated ELISA was subsequently developed and optimized. Limit of detection (LOD) and limit of quantification (LOQ) of the direct-biotinylated ELISA was determined for each antigen: 1) GroES had an LOD of 1.959 ng/mL and an LOQ of 6.531 ng/mL, 2) Mpt32 had an LOD of 1.884 ng/mL and an LOQ of 6.278 ng/mL, and 3) DnaK had an LOD of 6.310 ng/mL and an LOQ of 21.032 ng/mL. This direct-biotinylated ELISA platform demonstrated high sensitivity with low background for all three antigens. Thus, we successfully developed and optimized an ultrasensitive ELISA for the detection of Mtb antigens.
Open Access
Development of novel alphavirus expression and detection systems and characterization of oxidation's effect on viral replication
(Colorado State University. Libraries, 2014) Steel, J. Jordan, author; Geiss, Brian, advisor; Schenkel, Alan, committee member; Olson, Ken, committee member; Callan, Rob, committee member
Infectious diseases cause significant global suffering and death each year. Specifically, arthropod-borne viruses are emerging and re-emerging around the world and infecting millions of people. Mosquitoes that transmit these viruses are spreading to new regions of the world with naïve populations to serve as viral hosts. Climate change and human encroachment of habitat has brought mosquitoes into close proximity with humans. The viruses are evolving and are expanding their vector compatibility to include more than one species of mosquitoes. The combination of these elements results in a serious global need to develop ways to control or prevent arthropod-borne viruses. In order to discover novel antivirals and ways to inhibit these arboviruses, a better understanding of viral infection and replication is needed. This dissertation will describe a combination of projects that all aim to provide enhanced knowledge or tools to prevent, control, or treat arbovirus infection. Specifically, we improved the ability to express recombinant infectious alphaviruses, developed a novel system to detect alphavirus infection in mosquito cell culture and transgenic mosquitoes, and discovered a new role for oxidation during flavivirus replication. First, we successfully developed and established a method for transcribing infectious alphavirus RNA from a plasmid DNA platform. This approach provides an efficient way for producing high titer infectious recombinant alphavirus in multiple cell types that robustly express foreign proteins. Secondly, we optimized a system for detecting alphavirus infection in mosquito cells using the virus dependent subgenomic promoter to transcribe a reporter gene only during active infection. We demonstrated that mosquito cells can be stably transformed to transcribe an engineered viral reporter RNA that expresses a fluorescent reporter protein (mCherry) only in the presence of wild-type virus infection. The reporter protein is not detected in uninfected controls, but significant expression is readily detected during infection. Transgenic mosquitoes were also developed to transcribe the reporter RNA, which amplifies and expresses the reporter protein during infection. The transgenic mosquitoes are able to express a fluorescent reporter protein only during Sindbis virus (Alphavirus) infection, providing a novel mechanism to detect infection of wild-type virus in living mosquitoes. This transgenic reporter system is the first of its kind and demonstrated that a system based on our reporter RNAs could be optimized and used to specifically detect infected mosquitoes. Finally, I was able to study and characterize several aspects of viral RNA replication within the cell. Specifically, we identified that viral RNA replication is dependent on oxidative conditions. We determined individual residues from the flavivirus NS5 capping protein that are specifically involved in the oxidative enhancement of viral replication. Our work provided significant advances to the arbovirus field. We now have a novel method for producing recombinant alphaviruses that is more time, cost, and resource effective. We understand the ability of the subgenomic promoter to act as a virus inducible promoter to express foreign proteins only during infection to help detect or manipulate infection in mosquitoes. Finally, we have made significant discoveries on how RNA replication works on a molecular level within the cell and better understand the important role of oxidation on virus infection. The work and discoveries described in this dissertation have enhanced multiple aspects of arbovirus research and will hopefully strengthen our ability to fight and control arbovirus infections around the world.
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 member
Osteosarcoma (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.
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 member
Joint 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.
Open Access
Implications of diet-induced obesity on metabolic and immune homeostasis: the role of the mesenteric lymph nodes
(Colorado State University. Libraries, 2020) Hill, Jessica Lynn, author; Michelle, Foster T., advisor; Weir, Tiffany L., committee member; Gentile, Christopher L., committee member; Schenkel, Alan, committee member
Obesity is a major public health crisis among adolescents and adults. The development of obesity is associated with several comorbidities as a result of underlying systemic chronic inflammation, the culmination of which increases one’s risk for chronic and infectious disease. Excessive accumulation of visceral adipose tissue is shown to confer the greatest disease risk. This is primarily due to inherent depot differences, namely proximity to and a shared blood supply with the liver and gastrointestinal (GI) tract. Recent work demonstrates the considerable influence gut physiology has over both local and systemic homeostasis, as GI diseases such as inflammatory bowel disease are associated with metabolic derangements characteristic of obesity. While the mechanisms that mediate this inter-organ crosstalk continue to be elucidated, several studies suggest that inflammation originating from the gut triggers these broad metabolic and immunologic changes found in obesity. Previous work from our lab has demonstrated that high-fat diet (HFD) induced obesity results in mesenteric lymph node (MLN) fibrosis, which was associated with a localized impairment in immune function. MLNs, located within mesenteric adipose tissue (MAT) surrounding the GI tract, constitutively monitor the mesenteric adipose depot and draining sections of the small and large intestines, serving as critical inductive sites for adaptive immune responses. Subsequently, they are essential for overall tissue maintenance and protection. Hence, further study into the role of the MLNs in obesity-associated pathology is an important area of research. The goals of this dissertation research were to 1) examine the relationship between MLNs and GI inflammation on metabolic outcomes, and 2) characterize immunologic changes associated with models of chronic inflammation. To investigate the above-mentioned, we conducted four separate preclinical studies utilizing mouse models of diet-induced obesity, MLN cauterization, and dextran sulfate sodium (DSS) induced GI inflammation. In the first study (Chapter 2), we examined the contribution of the MLNs on disease pathology associated with HFD-induced obesity. We found that MLN dysfunction, either as a result of surgical manipulation or obesity-induced fibrosis, led to metabolic dysfunction. Furthermore, that functional MLNs are needed for the full restorative effects of Pirfenidone treatment. In the second study (Chapter 3), we examined the effect of chronic low-dose DSS induced GI inflammation, independent of diet and obesity, on metabolic and immune function. We found that non-obese mice treated with DSS had a modest reduction in total body weight and MAT mass yet showed substantial alterations in tissue immune cell populations and frequencies. These adaptations occurred without a concurrent change in glucose homeostasis. Finally, in the third study (Chapter 4) we characterized immunologic parameters within a normal weight and obese human population, free of disease, through the ex vivo challenge of peripheral blood mononuclear cells (PBMCs) with the T lymphocyte mitogen Concanavalin A (ConA). We found that PBMCs isolated from obese adults had a modest increase in cell proliferation and IFNγ secretion upon stimulation within ConA relative to their normal weight controls. Additionally, we found a distinct expansion of CD4+CD8+ T cells, CD16+ monocytes, and NK cells within ConA stimulated PBMCs from obese donors. Collectively, these studies provide evidence that 1) the MLNs are critical for metabolic homeostasis as their dysfunction exacerbates features of HFD-induced obesity; 2) chronic GI iv inflammation, independent of diet and obesity, can reshape the immune milieu without altering glucose homeostasis; and 3) obesity distinctly alters the PBMC response to acute ex vivo challenge as compared to that of normal weight individuals. Future studies should further elucidate mechanisms of crosstalk between the immune system, MLNs, and GI tract on metabolic homeostasis in models of obesity.
Open Access
Inhibition of the host 5'-3' RNA decay pathway is a novel mechanism by which flaviviruses influence cellular gene expression
(Colorado State University. Libraries, 2014) Moon, Stephanie L., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor; Schenkel, Alan, committee member; Curthoys, Norman, committee member
Host gene expression is an intricate process that requires many levels of regulation to allow the cell to react properly to a given stimulus or maintain homeostasis. One mechanism by which RNA viruses perturb host gene expression and potentially favor the allocation of host cell resources for viral proliferation is through interfering with cellular post-transcriptional processes. Furthermore, because viral RNAs must persist in the host cell cytoplasm to allow translation of viral proteins and ultimately viral replication, the same post-transcriptional processes that regulate host messenger RNAs (mRNAs) likely act on viral RNAs as well. The general RNA decay machinery in the cell serves as an important regulatory step for proper gene expression at the post-transcriptional level. Many RNA viruses have evolved unique mechanisms for dealing with the cellular RNA decay machinery to preserve their transcripts and ensure a productive infection. Flaviviruses contain positive-sense, single-stranded RNA genomes that are not polyadenylated. Therefore, these viral RNAs are likely recognized by the host cell as deadenylated, incongruous mRNAs and are likely substrates for the general cellular RNA decay machinery. Remarkably, flaviviruses including the dengue viruses (DENV) and West Nile virus (WNV) produce an abundant non-coding subgenomic RNA (sfRNA) during infection that is generated through incomplete degradation of the viral genome by the host 5'-3' exoribonuclease 1 (XRN1). We demonstrate that human and mosquito XRN1 stalls on highly structured, conserved elements in the 3' untranslated region of flaviviral RNAs, resulting in sfRNA formation. Furthermore, we determined that these sfRNAs act as competitive, reversible inhibitors of XRN1. Infected cells display several signs of sfRNA-dependent XRN1 dysfunction, including the accumulation of uncapped transcripts and an overall stabilization of host mRNAs. Additionally, sfRNA acts as a weak inhibitor of the host cell RNA interference (RNAi) pathway. We propose that sfRNA likely acts as a sponge for Argonaute-2 (AGO2) and DICER, and have determined that siRNA-mediated decay is suppressed in an sfRNA-dependent fashion in flavivirus-infected human cells. This suppression of the RNAi pathway appears to alter host gene expression to a limited extent, and may be especially important for viral replication in the mosquito vector. Other flaviviruses, including hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) do not form an sfRNA from their 3' untranslated regions, but they do contain highly structured 5' untranslated regions. Herein we show that aside from acting as internal ribosome entry sites, the 5' UTRs of HCV and BVDV also stall and inhibit XRN1. Therefore, flaviviruses, pestiviruses and hepaciviruses appear to inhibit a major mRNA decay pathway by suppressing XRN1 activity via highly structured viral RNAs. Consequences of XRN1 suppression during viral infection include the stabilization and upregulation of short-lived transcripts including those encoding oncogenes, angiogenic factors, and pro-inflammatory factors. Furthermore, we present evidence that WNV sfRNA may dysregulate the coordination between mRNA stability and transcription. Therefore, the suppression of XRN1 may potentially act as an important mechanism by which diverse viruses in the Flaviviridae induce pathogenesis by dysregulating cellular gene expression.
Open Access
Investigating the use of vaccination as a tool for managing pneumonic pasteurellosis in Rocky Mountain bighorn sheep (Ovis canadensis canadensis)
(Colorado State University. Libraries, 2011) Sirochman, Michael A., author; Huyvaert, Kathryn P., advisor; Walsh, Daniel P., committee member; Schenkel, Alan, committee member; Wilson, Kenneth, committee member
To view the abstract, please see the full text of the document.
Open Access
Investigation of dietary rice bran for protection against Salmonella enterica Typhimurium infection in mice
(Colorado State University. Libraries, 2014) Kumar, Ajay, author; Ryan, Elizabeth P., advisor; Dow, Steven, committee member; Leach, Jan, committee member; Schenkel, Alan, committee member; Weir, Tiffany, committee member
Rice bran is a byproduct of rice milling for white rice. Rice bran is a rich source of nutrients such as vitamins, minerals, soluble and insoluble fibers, fatty acids, polyphenols and proteins. Research has shown the beneficial health effects of rice bran in hyperlipidemia, diabetes, immune modulation, allergies and cancer. This dissertation focuses on evaluation of rice bran for protection against Salmonella using a mouse model of oral infection. Salmonella is a food and water borne pathogen that affects a variety of hosts including plants, animals and humans. Salmonella infections are a major public health challenge around the globe. Currently, salmonellosis is treated using high doses of synthetic antimicrobials and the problem of drug resistance has increased. In this scenario, alternative and sustainable interventions are needed to control Salmonella infections. Several dietary agents have been studied for protective effects in Salmonella infection models. We tested the prophylactic effects of dietary rice bran in a Salmonella model of infection using female 129S6/SvEvTac mouse model with infection of Salmonella enterica Typhimurium 14028s strain. Feeding of 10% dietary rice bran for one week prior to infection significantly (p<0.05) reduced fecal excretion of Salmonella in orally infected mice. Salmonella-infected, rice bran fed mice also showed a significant decrease in systemic inflammatory cytokines such as TNF-α, IFN-γ and IL-12 as compared to control diet fed animals. The colonization resistance against enteric pathogens is highly influenced by composition of gut microflora. Supplementation of dietary rice bran increased the number of Lactobacillus spp. in feces of mice as compared to mice that were fed control diet. Research has shown that oral administration of some species of Lactobacillus reduces the colonization of Salmonella. We hypothesized that rice bran components also enhance mucosal protection by preventing Salmonella entry into the epithelial cells. Methanolic rice bran extracts were assessed in mouse small intestinal epithelial (MSIE) cells for blocking Salmonella entry and intracellular replication. Rice bran extract significantly reduced Salmonella entry and intracellular replication into MSIE cells. These results suggest the potential mechanisms for dietary rice bran induced improvement of colonization resistance against Salmonella. Given that rice crops have a large variation in genotype and phenotype such as in yield, disease and pest resistance, drought resistance, and nutrient quality, we hypothesized that variation in rice bran across cultivars induces differential protection against Salmonella infection due to differences in their phytochemical profile. A panel of six varieties namely IAC 600, Jasmine 85, IL 121-1-1, Wells, Red Wells and SHU 121 were tested in the in vitro and in vivo model of Salmonella infection. We found that rice bran extracts across varieties inhibited Salmonella entry into the MSIE and Caco-2 cells to different extents. IAC 600 fed animals significantly (p<0.05) reduced Salmonella fecal excretion as compared to the control diet fed animals. IAC 600 fed animals also reduced Salmonella fecal shedding significantly (p<0.05) as compared to SHU 121 diet fed animals at 2 and 6 days post Salmonella infection. Histopathological analysis revealed that IAC 600 diet fed animals had better ileal pathological scores as compared to SHU 121 and the control diet fed animals post Salmonella infection. SHU 121 and the control diet fed groups showed higher ulceration and inflammatory changes in ileum as compared to IAC 600 fed animals. Next we analyzed the fatty acid profile, mineral profile and total phenolic contents of rice bran. Stearic acid, lignoceric acid, boron and total phenol content were significantly correlated with Salmonella fecal shedding in mice across varieties. However, further studies are required to confirm the role of these nutrients from rice bran in protection against Salmonella. These results suggest that the variety of rice plays an important role in bran-induced protection against Salmonella infection and this difference in protection across the varieties could be attributed to a combination of bioactive components. Our studies suggest that dietary rice bran improves colonization resistance against Salmonella in mice. Rice bran could have important role in prevention of enteric infections in resource scarce populations and further human clinical studies are required. Rice bran may also be evaluated for supplementing diets of food animals to prevent Salmonella infections and therefore could have a potential role in food safety.
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 member
Burkholderia 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.
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 member
Numerous 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.
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 member
To view the abstract, please see the full text of the document.
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 member
To view the abstract, please see the full text of the document.
Open Access
Resolvin D1 modulates the pulmonary immune response to agriculture dust exposure
(Colorado State University. Libraries, 2023) Threatt, Alissa Nicole, author; Nordgren, Tara, advisor; Schaffer, Joshua, committee member; Schenkel, Alan, committee member
Occupational exposure to agriculture dust causes a variety of acute and chronic pulmonary diseases including allergies, asthma, chronic obstructive pulmonary disease (COPD) and organic dust toxic syndrome (ODTS). These diseases have high impact on the healthcare system and limited treatments with variable efficacy. In addition, workers often display low compliance with required workplace personal protective equipment (PPE), increasing their risk for developing these diseases. Therefore, the development of new pharmacological interventions is critical to alleviate the burden on the healthcare system and improve the quality of life for patients who will inevitably develop occupational-related pulmonary diseases. Interleukin-22 (IL-22) is a cytokine in the anti-inflammatory interleukin-10 (IL-10) family of cytokines that has demonstrated a protective role in murine models of acute and chronic lung injury. It has been described as being exclusively produced by lymphocytes, however methodological limitations of the primary cited study restricted the exploration of other cell types as producers of IL-22. Upregulation of this cytokine by pharmacological means could prove beneficial for delaying the progression of occupational chronic pulmonary diseases. Omega-3 fatty acids and their metabolites have well-documented anti-inflammatory and pro-resolution functions in chronic pulmonary diseases and have been implicated in the induction of IL-22. Omega-3 fatty acids have shown overwhelming evidence in being anti-inflammatory by their function as substrates for the production of specialized pro-resolving mediators (SPMs), lipid metabolites that signal immune cells to transition to a resolution and repair state following inflammation. Resolvin D1 (RvD1), a metabolite of the omega-3 fatty acid docosahexaenoic acid (DHA), has shown to have anti-inflammatory and protective functions in a murine acute lung injury model. To evaluate the source of IL-22 in the pulmonary response to agricultural dust, mouse alveolar macrophages were co-exposed to 1% hog dust extract (DE) collected from swine confinement facilities in the Midwest US and treated with either 10 nM or 100 nM RvD1. Cells were incubated for up to 24 hours, supernate was collected at the desired timepoint, and enzyme-linked immunosorbent assays (ELISAs) were performed to assess protein expression. Cells were also lysed to determine intracellular IL-22 protein concentrations. Cells exposed to DE exhibited increased pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) as well as increased IL-10 and IL-22 production, demonstrating macrophages as a source of IL-22 in the immune response to organic dust. Cells exposed to DE and treated with RvD1 demonstrated significant decreases in IL-6 and TNF-α and increases in IL-10. To determine the efficacy of RvD1 as an inducer of IL-22, and as a potential treatment for organic dust-induced lung injury, C57BL/6 (WT) and full-body IL-22 knock-out (KO) mice were intranasally instilled (IN) with 12.5% DE 5 days/week for 3 weeks and injected intraperitoneally (IP) with 250 ng RvD1 once per week. Animals were allowed to recover for 5 hours or 3 days before sacrifice where bronchoalveolar lavage fluid (BALF) was collected for cytokine and cellular infiltrate evaluation to determine the role of RvD1 in the reduction of the immune response to organic dust exposure. BALF cytokines exhibited significant increases in the production of IL-10 in KO mice exposed to DE and treated with RvD1 with a 3-day recovery. Cellular infiltrates demonstrated decreased neutrophil infiltration and increased lymphocyte recruitment in KO mice exposed to DE after a 3-day recovery and further significant decreases in mice treated with RvD1 with a 3 day recovery. The data support the production of IL-22 by alveolar macrophages and its induction by RvD1. They also demonstrate the effects of RvD1 on the pulmonary immune response to agriculture dust and as a potential therapeutic for organic dust-induced chronic pulmonary diseases.
Open Access
The effects of mannose capped lipoarabinomannan on dendritic cell function
(Colorado State University. Libraries, 2009) Lee, Eric John, author; Gonzalez-Juarrero, Mercedes, advisor; Schenkel, Alan, committee member; Chen, Chaoping, committee member
M. tuberculosis is one of the leading causes of death due to infectious disease in the world. While the majority of people are capable of controlling the initial infection, many progress to a latent stage of disease where the M. tuberculosis bacilli persist for long periods of time within the host. The M. tuberculosis cell wall lipoglycan mannose capped lipoarabinomannan (ManLAM) has been characterized as one of the immunomodulatory factors associated with the bacteria [3-5]. ManLAM interacts with dendritic cells (DCs) via DC-SIGN, mannose receptors and to a lesser extent TLR-2 [6-8]. Thus we set out to examine the effects that ManLAM has on DCs both in vitro and in vivo. ManLAM treatment of bone marrow derived DCs (BMDCs) prevents their phenotypic maturation reduces the expression of MHC class II and CD1d. BMDCs stimulated with ManLAM also exhibit altered phagocytic capacity and the inability to stimulate naïve CD4+ T-cell proliferation.
Open Access
Understanding Mycobacterium abscessus in cystic fibrosis mice
(Colorado State University. Libraries, 2019) Vongtongsalee, Kridakorn, author; Ordway, Diane, advisor; Schenkel, Alan, committee member; Chatterjee, Delphi, committee member; Kirby, Michael, committee member
Cystic fibrosis (CF) is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which normally encodes an ABC transporter-class ion channel protein that allows chloride and thiocyanate ions transport across epithelial cell membranes. Thus, CFTR plays an important role in airway homeostasis. Mutations of CFTR in patients with CF leads to a defect in transport of chloride and thiocyanate ions by epithelial cells, resulting in a multi-system disorder that affects the respiratory tract, gastrointestinal tract, the endocrine system, among others. The epithelial cell dysfunction in the lungs of CF patients also leads to an impaired pulmonary defense mechanism, resulting in decreased bacterial clearance and chronic inflammation. In CF patients, lung disease due to non-tuberculous mycobacteria (NTM) — an environmental organisms found in soil, water, and biofilms — is one of the most feared complications. Among the NTM, the rapidly-growing Mycobacterium abscessus is particularly notorious given its intrinsic resistance to many antibiotics. The transmission of M. abscessus to humans occurs by wound contamination, airborne transmission, or ingestion. Despite the fact that M. abscessus infection is increasing worldwide, little is known about how M. abscessus causes disease. To improve our understanding of M. abscessus in CF patients, we set out to investigate the progression of M. abscessus infection in a CF mouse model by developing a reinfection mouse model using three different strains of "CF mouse" — Beta-ENaC mice, Cftrtm1UNCTgN(FABPCFTR) mice and CFTRtm1UNC/ CFTRtm1UNC mice — to track the bacterial burden and organ pathology. Our results support the hypothesis that repeated infection with M. abscessus is more likely to result in disease progression and increased pathogenesis of the disease in CF mouse models. The high bacterial burden persisted in the lung after four infections in β-ENaC transgenic mice and CFTRtm1UNC/CFTRtm1UNC mice and maintained in the organs by day 30. Cftrtm1UNCTgN(FABPCFTR) mice tended to show slowly increasing bacterial burden in all organs. In summary, we demonstrate that reinfection of the CF mouse models with M. abscessus is more likely to result in a sustained infection in the lungs associated with increased pulmonary pathology.