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Browsing by Author "Biller, Barbara, committee member"

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    Effects of survivin and survivin inhibition in canine models of lymphoma and osteosarcoma
    (Colorado State University. Libraries, 2014) Shoeneman, Jenette K., author; Thamm, Douglas, advisor; Gustafson, Daniel, committee member; Duval, Dawn, committee member; Biller, Barbara, committee member; Ehrhart, E. J., committee member
    Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Survivin, an IAP (inhibitor of apoptosis) family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in both dogs and humans with LSA and OS, and canine LSA and OS cell lines express high levels of survivin. Due to the strong similarities between canine and human LSA and OS, canine LSA and OS are excellent models for the human disease. In the following research, we illustrate the potential of the canine LSA and OS models as a translational tool for evaluating survivin-directed therapies, owing to the striking similarities in gross and microscopic appearance, biologic behavior, gene expression and signaling pathway alterations compared to the respective human forms of these diseases. In this research we sought to determine the effects of survivin inhibition in canine OS and LSA cell lines in vitro, and in vivo in canine OS, and to evaluate a correlation between survivin expression and outcome in canine OS patients. We hypothesized, as observed in human OS and LSA, that survivin inhibition would decrease cell proliferation and increase apoptosis and chemosensitivity in canine OS and LSA cell lines. We further hypothesized that we would observe inhibition of survivin and reduced tumor growth in murine models of canine OS treated with EZN-3042, an inhibitor of survivin. We additionally hypothesized, as observed in human OS, that increased survivin expression would correlate with a poor prognosis in canine OS patients. Survivin attenuation in canine OS cells via siRNA was confirmed by RT-PCR and western blot analysis. Cell number and viablility was assessed via manual cell counting with trypan blue. Cellular apoptosis was confirmed via caspase-3/7 and TUNEL assays. Cell cycle analysis was performed with propidium iodide staining followed by flow cytometry. Chemosensitivity to doxorubicin (DOX) was also assessed with caspase-3/7 assay. We determined that survivin inhibition via siRNA in canine OS cells inhibited cell cycle progression, and increased apoptosis, mitotic arrest and chemosensitivity. Next we inhibited survivin using EZN-3042, a locked nucleic acid oligonucleotide targeting survivin, in two canine LSA and two canine OS cell lines. Survivin inhibition was confirmed by qRT-PCR and Immunofluorescence. Percent dead and total cell number were assessed by manual cell counting with trypan blue. Growth inhibition was confirmed with a bioreductive fluorometric assay. A caspase-3/7 assay was used to determine levels of apoptosis and chemosensitivity. Survivin inhibition in vitro using EZN-3042 resulted in decreased total and viable cell numbers and increased apoptosis and chemosensitivity to DOX. In vivo, nude mice with subcutaneous and orthotopic OS xenografts were given 100 mg/kg EZN3042 intraperitoneally. Survivin inhibition was confirmed with immunohistochemistry and qRT-PCR analysis. EZN-3042 treatment in vivo in subcutaneous and orthotopic canine OS xenografts decreased tumor survivin expression. Mice treated with EZN-3042 in combination with DOX had significantly decreased tumor growth when compared to single agent treatment and control groups. Lastly, we evaluated survivin expression in archived paraffin embedded canine OS tissue samples. Survivin expression was studied via immunohistochemistry in 67 canine OS cases. Elevated survivin protein immunoreactivity in primary canine OS tissue samples correlated with increased histologic grade and mitotic index and a decreased disease free interval (DFI). These findings strongly suggest that survivin-directed therapies may be highly effective in treatment of both canine and human LSA and OS, and spontaneous canine cancer may be a valuable model for the evaluation of survivin-targeted treatment.
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    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.
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    Occupational dose assessment of 64Cu-ATSM in a veterinary setting
    (Colorado State University. Libraries, 2014) Hetrick, Lucas, author; Johnson, Thomas, advisor; Kraft, Susan, committee member; Biller, Barbara, committee member
    64Cu-ATSM is an emerging radiopharmaceutical for diagnostic use in Positron Emission Tomography (PET) and has potential utility for radiation therapy but to date there are no studies that assess the occupational doses delivered to workers in either a hospital or veterinary environment. This study consisted of canine patients that were recruited at the Colorado State University James L. Voss Veterinary Teaching Hospital (VTH). The study was aimed at determining the radiation dose to veterinary workers from clinical PET/CT procedures using 64Cu-ATSM. To determine the dose to the workers, each worker was assigned two Electronic Personal Dosimeters (EPDs) to be worn on the chest and waist during the entirety of each procedure. The workers monitored during this study involved included a radiobiologist, a nuclear medicine technician, an anesthesiologist, and a veterinary surgeon. Seven canine patients were imaged over a ten month period with an average mass of 33.7 kg (a range of 20.0 - 55.1 kg) with an average injected activity of 5 MBq kg-1. The dose range for the radiobiologist was 2 -17 µSv, for the nuclear medicine technician 0 -14 µSv, for the anesthesiologist 0 - 12 µSv, and for the surgeon 0 -10 µSv. In a comparison between the results of this study and published literature on occupational exposures from human/veterinary FDG PET/CT procedures, 64Cu-ATSM veterinary PET/CT procedures, on a per patient bias, exposed workers to less radiation.
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    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.
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    TLR9 agonist produces effective mucosal immunity as a vaccine against Mycobacterium tuberculosis
    (Colorado State University. Libraries, 2016) Troy, Amber, author; Izzo, Angelo, advisor; Mclean, Jennifer, committee member; Biller, Barbara, committee member; Zabel, Mark, committee member
    The Mycobacterium tuberculosis bacillus has plagued the world for thousands of years. Researchers have found evidence of tuberculosis infection as far back as the Ice-Ages with the presence of bone lesions in mastodons. The existence of tuberculosis in humans has also been found in ancient Egyptian mummies dating as far back as 4000 years ago. Pulmonary tuberculosis is an airborne bacterial disease and the causative agent, M. tuberculosis, currently infects about 2-3 billion people globally. This deadly disease is responsible for killing an average of 5,000 people every single day, and is most prevalent in developing countries, particularly in sub-Saharan Africa where the AIDS epidemic grows yearly. TB is one of the first diseases to arise in these immune-suppressed patients, which furthers the need for a new effective disease control strategy. The incidence of drug resistance has been increasing steadily, also exemplifying the need for a prophylactic approach to combating the disease. The only currently available vaccine for M. tuberculosis in use today is Mycobacterium bovis Bacillus Calmette-Guérin (BCG), a live attenuated vaccine that has been in use since the 1930’s, and with a variable efficacy rate of 0-80% the need for a new more effective vaccine is dire. BCG is the mostly widely used vaccine in the world with some success in young children, but immunity wanes over time due to a number of reasons. In order to replace BCG or boost BCG, a better understanding of the immune response is required, allowing for better vaccine development. Animal models provide a good basis for determining immune mechanisms that are related to protective immunity, both innate and adaptive. Toll-like receptors (TLRs) are a key component of the innate immune response. TLRs are pattern recognition receptors (PRR), which recognize pathogen associated molecular patterns (PAMPs). These PAMPs are not produced by mammalian cells but are presented by invading microorganisms. Once PAMPs are detected the innate immune response is activated and begins the clearance of bacterial infection. PRRs are also required to activate antigen-presenting cells (APCs) that process bacterial antigens for presentation, in association with major histocompatibility complexes (MHC) to T-cells. One particularly stimulatory PAMP is CpG Oligodeoxynucleotide (ODN), which is unmethylated bacterial DNA. Once CpG ODN is introduced, the PRR TLR9 is activated thus inducing a potent T-helper 1 (Th1) response which plays a critical role in tuberculosis infection. Mucosally administered vaccines have been of particular interest to vaccine researches in the past twenty years. Data show the nasal mucosa is an effective route of vaccine administration because it is the first point of contact to an inhaled pathogen. Since pulmonary tuberculosis is spread via aerosolized particles, vaccines targeting the mucosa may effectively prime local antigen presenting cells like alveolar macrophages and dendritic cells. One method for vaccine delivery is the use of a carrier vehicle such as cationic liposomes. Cationic liposomes consist of a positively charged lipid bi-layer. The positive charge on these liposomes allows the addition of a negatively charged immune-stimulant such as CpG ODN. Liposomes carrying antigen are capable of inducing a robust cell mediated immune response or Th1 type response, which is crucial for mitigating M. tuberculosis infection. Since protein antigen degradation occurs quickly in mucosal sites liposomes provide increased stability of these vaccine components and thus allowing prolonged antigen presentation. Given that tuberculosis is a pulmonary infection, we hypothesize that targeting the site of infection with a TLR9 agonist; a robust mucosal immune response would be induced providing protection against infection and reducing bacterial burden. Using the mouse model of tuberculosis, we determined that intranasal inoculation of a cationic liposome carrying CpG ODN and the M. tuberculosis antigen ESAT-6 provides protection by inducing a robust Th1-type immune response capable of significantly reducing mycobacterial burden in the lungs after pulmonary infection, and also creating long-lasting immunity by stimulating the activation of memory T-cells.
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    Walleye dermal sarcoma virus Orf C: a potential oncolytic therapy
    (Colorado State University. Libraries, 2011) Magden, Elizabeth, author; Quackenbush, Sandra, advisor; VandeWoude, Sue, committee member; Biller, Barbara, committee member
    The taxonomy and nomenclature of the North American weevil genus Thecesternus Say was reviewed. Five previously described species are recognized as valid: affinis, foveolatus, hirsutus, humeralis, and maculosus. One new species, tumulosus, from Texas is described as new. The following new synonymy is proposed: longior LeConte 1856 (= affinis LeConte 1856) and albidus Pierce 1909 (= maculosus Pierce 1909). A neotype is designated for T. humeralis (Say). A key to identify the species is provided, with various illustrations of key morphological features characterizing these species. Additionally, distribution maps, species descriptions, and species differentiation for each species is provided. A cladistic hypothesis of the included species is presented. Walleye dermal sarcoma virus (WDSV) is a complex retrovirus that causes the growth of multifocal, cutaneous tumors in walleye fish (Sander vitreus vitreus). These virus-induced tumors spontaneously regress on a seasonal basis. The WDSV genome encodes three accessory proteins (rv-cyclin, Orf B, and Orf C) that are necessary for regulation of virus expression, tumor formation, and tumor regression. While rv-cyclin and B are critical for tumor development, Orf C contributes to the observed seasonal tumor regression. Previous studies have shown that Orf C targets the cell mitochondria and induces apoptosis. These studies suggest that Orf C-induced apoptosis leads to the observed tumor regression in fish infected with WDSV. To further define the mechanism(s) of apoptosis, we generated a recombinant lentivirus (Lenti Orf C) that expresses WDSV Orf C. By infecting cells with Lenti Orf C, we showed decreasing cell viability in association with increasing virus concentrations. We also demonstrated Orf C expression in mitochondrial, cytosolic, and nuclear cell fractions, with the strongest Orf C expression in cell nuclei. In addition, we identified two pro-apoptotic proteins that associate with Orf C, ANT and Bax, and identified a third protein, AIF, as a potential Orf C target. While significant progress has been made in elucidating the mechanism(s) of Orf C-induced apoptosis, further studies are necessary to determine which cellular proteins are the primary targets of Orf C. These apoptosis-inducing Orf C targets may be useful in developing future oncolytic therapies.