Browsing by Author "Olver, Christine, advisor"
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Item Open AccessCleavage of exosomal-associated transferrin receptor in dogs, cats, and horses: progress towards a soluble transferrin receptor assay(Colorado State University. Libraries, 2018) Martinez, Caitlyn Marisa Romero, author; Olver, Christine, advisor; Santangelo, Kelly, advisor; Duval, Dawn, committee memberIron deficiency anemia and anemia of chronic disease are two complications that patients in human medicine as well as veterinary medicine often encounter. These two diseases usually occur secondary to other primary diseases and are associated with increased morbidity and a decline in prognosis. The diagnosis and differentiation of these diseases is complicated by the fact that many of the parameters used to characterize iron deficiency are also influenced by inflammatory cytokines. Consequently, the detection of iron deficiency in the presence of inflammation or the detection of combined iron deficiency anemia and anemia of chronic disease is difficult. Differentiation of these two diseases is important as treatment for each disease is different and potentially harmful if utilized on a patient who has been misdiagnosed with one disease or the other. In human medicine, soluble transferrin receptor 1 (sTfR) has shown promise as a marker, alone or in ratio with serum ferritin, that can differentiate iron deficiency anemia, anemia of chronic disease, and combined disease. sTfR is the product of cleavage of transferrin receptor 1 (TfR1) from the surface of exosomes which are released into circulation from maturing reticulocytes. Humans cleave the majority of their exosomal-associated TfR1 yielding substantial levels of circulating sTfR for detection and quantification by clinical assays. However, the level of cleavage in many of our veterinary species, including dogs, cats, and horses, remains unknown. Additionally, no currently developed sTfR clinical assays have been found to successfully detect sTfR in our veterinary species. The purpose of this study was to first confirm the presence of exosomes and exosomal-associated TfR1 in the serum of dogs, cats, and horses. Secondly, the level of cleavage of exosomal-associated TfR1 in healthy dogs, cats, and horses was explored to indirectly characterize the anticipated levels of circulating sTfR in these species. Lastly, the level of cleavage of exosomal-associated TfR1 was compared between healthy and diseased dogs and cats to investigate any potential effect of inflammation and chronic disease on the cleavage of exosomal-associated TfR1 and thus on the anticipated levels of circulating sTfR. The results of this study demonstrated significant evidence indicating the successful isolation of exosomes and identification of exosomal-associated TfR1 from the serum of dogs, cats, and horses. The level of cleavage of exosomal-associated TfR1 in dogs was found to be greater than 50% on average with significant between-individual variation. There was also no significant difference in the means of the proportion of cleavage between healthy and diseased dogs. The level of cleavage of exosomal-associated TfR1 in cats was found to be very low at about 11% without substantial variation between individuals. However, a small but significant difference between healthy and diseased cats was detected. Healthy horses do not appear to cleave exosomal-associated TfR1. These results together would suggest that development of a clinical assay for the detection and quantification of sTfR in these veterinary species may not be successful and consequently may not be worth the time, effort, and expense. Item Open AccessIron, hepcidin, and microcytosis in canine hepatocellular carcinoma(Colorado State University. Libraries, 2021) Polak, Klaudia Zofia, author; Olver, Christine, advisor; Avery, Anne, committee member; Santangelo, Kelly, committee member; Shropshire, Sarah, committee memberHepatocellular carcinoma (HCC) is the most common primary liver tumor found in dogs. There is evidence that iron dysregulation is associated with HCC pathogenesis in both humans and dogs. Anemia and thrombocytosis were common hematologic abnormalities detected in about half of dogs with massive HCC, and microcytosis was present in approximately 31% of dogs in one study. Additionally, humans with hereditary hemochromatosis have an increased risk of HCC. The liver is the major organ site for iron storage and metabolism containing numerous iron regulatory proteins which may play an important role in canine HCC. Since microcytosis is associated with iron restricted erythropoiesis, our first objective was to determine whether neoplastic hepatocytes exhibit differential expression of iron regulatory genes as well as hepatic iron stores in normocytic versus microcytic HCC cases in an initial pilot study. Next, we aimed to quantify and compare expressions of a larger set of iron regulatory and human HCC-related genes among canine HCC tumor tissue, adjacent peritumoral liver tissue, non-specific reactive hepatitis liver tissue from non-HCC dogs, and normal liver tissue, as well as to quantify and compare estimated hepatic iron stores. We hypothesized that canine HCC tumor tissue exhibits iron overloading and higher expression of hepcidin and its upstream regulators (IL-6 and BMP6), which would promote intracellular iron availability for neoplastic hepatocyte proliferation. We also hypothesized that microcytic HCC cases would exhibit higher expression levels of hepcidin in tumor tissue compared to tumors from normocytic dogs. Additionally, we explored associations between clinical parameters and RNA levels of iron regulatory genes as well as estimated hepatocellular iron stores in both HCC tumor and the adjacent, peritumoral tissues. We expected to find gene expression patterns in canine HCC tumor tissue related to abnormal regulation of iron metabolism and other pathways similar to what has been described in human malignancies. Cases were selected from a database search for canine HCC and included if complete pre-operative blood work was available and there was adequate formalin-fixed paraffin-embedded (FFPE) tissue for RNA isolation for all cases. Hematologic and clinical parameters were recorded and used for correlation studies. All liver sections were reviewed by a board-certified veterinary anatomic pathologist. RNA was isolated from FFPE blocks and NanoString nCounter platform was used to quantify RNA counts for selected genes. Sections were stained with Perls Prussian Blue stain and hepatocytic iron stores were estimated using NIS-Elements software. Contrary to our hypotheses, all canine HCC tumors had markedly decreased expression of hepcidin (HAMP) and depletion of hepatocellular iron stores. Other iron-related genes down-regulated in canine HCC tumor tissue included TfR2 (an upstream regulator of hepcidin), STEAP2, LTF, HMOX1, CYBRD1and SFXN5. Tumor tissue overexpressed TfR1, STEAP3, and LCN2. No significant differences in RNA levels or iron stores were found between tumors of microcytic and normocytic HCC cases, but the adjacent peritumoral tissue was markedly iron loaded and exhibited negative correlation between hepcidin RNA levels and mean cell volume (MCV) as well as serum iron. Microcytic HCC cases were associated with noteworthy clinical findings such as increased ALT, lower HCT and serum iron, and histologically more poorly differentiated tumors. Differential expression of genes involved in Wnt signaling and ferroptosis was observed in canine HCC tumor versus the adjacent peritumoral liver tissue.