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Little is known about the anti-inflammatory activity of creatine. The aim of this study was to evaluate the anti-inflammatory effects of creatine supplements in canine chondrocytes (CnC). CnC were stimulated with IL-1β. Release of PGE2 and TNFa was measured using ELISA. Changes in oxylipin profile was assessed using HPLC/MS. Expression of COX-2 and phosphorylated NF-kB was performed using western blot. Changes in above inflammatory responses were examined following treatment with various creatine compounds including the metabolite creatinine. COX inhibitor, Rimadyl, substantially reduced PGE2 release, despite increasing both TNFa release and COX-2 expression. All creatine compounds, including creatinine, reduced PGE2, COX-2 and TNFa in stimulated CnC. In addition, all the compounds examined reduced phosphorylated NF-kB expression. The creatine compounds were also able to interfere with the production of several oxylipins in response to IL-1β. Creatine supplements may have a beneficial role in preventing inflammation within the joint and other tissues. / May 2016

Since its domestication, the dog has served in many roles, from protector, guide, hunter, and best friend, to model organism. Every role in which the dog serves is important; however, this work highlights the importance of the dog as a model organism for study of human hereditary diseases. Roughly half of the 450 hereditary diseases found in the dog have clinical presentations similar to those found in the human. Included in these are auditory-pigmentation conditions and skin diseases for which the dog is a working model. Described herein are studies of the merle coat pattern and dermatomyositis. Through research on these topics, important information can be obtained that can be used to help both the dog and the human. Merle is a pattern of coloring observed in the coat of the domestic dog and is characterized by patches of diluted pigment. Dogs heterozygous or homozygous for the merle locus exhibit a wide range of auditory and ophthalmologic abnormalities. Linkage disequilibrium was identified for a microsatellite marker with the merle phenotype in the Shetland Sheepdog. This region of the human genome contains SILV, a gene important in mammalian pigmentation. Therefore, this gene was evaluated as a candidate for merle patterning. A short interspersed element insertion at the boundary of intron 10/exon 11 was found, and this insertion segregates with the merle phenotype in multiple breeds. These data show that SILV is responsible for merle patterning and is associated with impaired function of the auditory and ophthalmologic systems. Dermatomyositis (DM) is an inflammatory disease of the skin and muscle that occurs most often in the rough collie and Shetland Sheepdog. Gene transcript profiles were generated for affected and normal skin using a canine-specific oligonucleotide array. Two-hundred and eight-five gene transcripts, many of which are involved in immune function, were found to be differentially regulated in these tissues. Also reported are western blot, immunohistochemistry, and immunofluorescence analyses. While our work suggests that canine DM is a disease that may be immune mediated, it did not detect the production of specific disease-associated autoantibodies.

Since its domestication, the dog has served in many roles, from protector, guide, hunter, and best friend, to model organism. Every role in which the dog serves is important; however, this work highlights the importance of the dog as a model organism for study of human hereditary diseases. Roughly half of the 450 hereditary diseases found in the dog have clinical presentations similar to those found in the human. Included in these are auditory-pigmentation conditions and skin diseases for which the dog is a working model. Described herein are studies of the merle coat pattern and dermatomyositis. Through research on these topics, important information can be obtained that can be used to help both the dog and the human. Merle is a pattern of coloring observed in the coat of the domestic dog and is characterized by patches of diluted pigment. Dogs heterozygous or homozygous for the merle locus exhibit a wide range of auditory and ophthalmologic abnormalities. Linkage disequilibrium was identified for a microsatellite marker with the merle phenotype in the Shetland Sheepdog. This region of the human genome contains SILV, a gene important in mammalian pigmentation. Therefore, this gene was evaluated as a candidate for merle patterning. A short interspersed element insertion at the boundary of intron 10/exon 11 was found, and this insertion segregates with the merle phenotype in multiple breeds. These data show that SILV is responsible for merle patterning and is associated with impaired function of the auditory and ophthalmologic systems. Dermatomyositis (DM) is an inflammatory disease of the skin and muscle that occurs most often in the rough collie and Shetland Sheepdog. Gene transcript profiles were generated for affected and normal skin using a canine-specific oligonucleotide array. Two-hundred and eight-five gene transcripts, many of which are involved in immune function, were found to be differentially regulated in these tissues. Also reported are western blot, immunohistochemistry, and immunofluorescence analyses. While our work suggests that canine DM is a disease that may be immune mediated, it did not detect the production of specific disease-associated autoantibodies.

As life expectancy in the United States increases each year, the percentage of the population that is comprised of aged individuals rises also. Researchers expect the largest increase in population to occur in the segment consisting of individuals 85 and older. Thus, investigations of the aging process, with the goals of further extending average life expectancy and improving the quality of life for aged individuals, have become increasingly important to our society. To better understand the genetics of aging, we elected to utilize another model organism, the domestic dog. The benefit to this work is that breeds exhibit extreme, natural variation in life expectancies. Here I report my contributions towards establishing the dog as another model organism for investigations of the aging process. Multiple linear regression analysis was carried out to determine the association between life spans and breed size in the dog, based upon data derived from the American pet population. A negative correlation was observed between both height and longevity and between weight and longevity with weight being the significant predictor of life span. Fifty-four genes implicated in the aging process were mapped to the canine genome. These genes were selected because of their demonstrated contribution to longevity in other organisms or based upon their proximity to a marker, D4S1564, on human chromosome 4. Four genes that are associated with dwarf mice and extended life span were analyzed in nine dog breeds of varying sizes and life expectancies. Fifty-three polymorphisms were discovered in Ghr, Ghrhr, Pit1, and Prop1. Thirteen ancestral SNPs were discovered in which both alleles were found in every breed. In Ghrhr, a transition mutation was found that changes the amino acid sequence as well as the function of the protein and is statistically significant (p=4.8 x 10-6) when large dogs are compared to medium-sized breeds, but not when they are compared to small breeds (p=0.001). This SNP warrants further investigation in additional dogs and breeds.

Cancer is essentially a genetic disease of uncontrolled cell survival and proliferation. Medical therapy has traditionally involved chemotherapy and radiation which inhibit the replication of actively dividing cells in a fairly non-selective manner. Research into the molecular pathogenesis of cancer has enabled the recent development of therapy targeted to receptors or signaling pathways crucial to the development and progression of specific cancers and revealed molecular markers that can be used to predict prognosis and treatment response. The work presented in this thesis examines the expression of two potential molecular markers of malignancy, the R1alpha regulatory subunit of cyclic AMP-dependent protein kinase A (PRKAR1A) and insulin-like growth factor 2 (IGF2) retrospectively in a population of canine osteosarcoma patients. Furthermore, it describes the derivation and preliminary characterization of canine osteosarcoma primary cell cultures and the use of these cells to assess the effects of PRKAR1A expression on sensitivity to chemotherapeutic drug treatment in vitro. Post-chemotherapy overall survival was significantly longer in canine osteosarcoma patients with tumours expressing low levels of PRKAR1A, and PRKAR1A expression did not correlate with mitotic index. IGF2 expression was generally high in the small numbers of cases examined, did not differ between axial and appendicular sites and did not correlate with either mitotic index or survival. PRKAR1A expression varied between cell cultures, but did not appear to be related to expression levels of phosphorylated cAMP response element-binding (phospho- CREB), a downstream target of cyclic AMP (cAMP)-dependent protein kinase A (PKA). In vitro chemosensitivity did not correlate with PRKAR1A expression, but faster growing cells with shorter doubling times and higher rates of BrdU incorporation tended to be more chemosensitive. In summary, this work identifies an association between low tumour PRKAR1A expression and prolonged post-chemotherapy survival in dogs with osteosarcoma and provides preliminary evidence suggesting that this survival advantage may not be associated with downstream phosphorylation of CREB or sensitivity of the tumour cells to chemotherapeutic agents.

Diseases of the eye are common problems in dogs and can be painful, therapeutically challenging and distressing to both patient and owner. Ocular disease can result in visual impairment, vision loss or, in severe cases, enucleation. Much of the tissue damage that occurs during ocular disease results from the activity of secreted proteins that control processes such as inflammation, blood vessel growth, cellular proliferation and cellular death. These proteins are called growth factors and cytokines. The purpose of this study was to examine the expression and effects of one such cytokine, interleukin 11, in the canine eye. Interleukin 11 was found to be constitutively expressed in all layers of the canine cornea at both the protein and message level. Treatment of primary corneal cell cultures with TGF-β1 resulted in a statistically significant increase in IL-11 expression in the corneal epithelium, fibroblasts and endothelium. In order to study the biological effects of IL-11 on the canine cornea, a presumptive corneal epithelial cell line (DCE39R) was created. Such a cell line represents an important, and previously unavailable, tool to study the effects of cytokines on the corneal epithelium itself as well as create corneal constructs for research and therapeutic work. Research done using this cell line demonstrated that IL-11 has a pro-migratory effect on the corneal epithelial cells and provides a cytoprotective effect in the case of nutrient deprivation. It however, does not induce proliferation of the canine corneal epithelial cells. This study serves as an important building block for future research on the effect of IL-11 in the canine cornea, and it also provides an important tool for future research: the cell line DCE39R. / Pet Trust

This thesis is an investigation of a gentamicin impregnated collagen sponge (GICS) product implanted into an inflamed canine stifle joint. Project goals were to determine the duration for which drug concentrations remained above minimal inhibitory concentrations within the joint following sponge implantation; to determine whether there was systemic exposure to the drug following local implantation; to evaluate the impact of the sponge on local joint inflammation and lameness; and to evaluate whether sponge use resulted in any renal injury. The study design was a randomized controlled experimental trial (2 x n=9) performed with research hounds. GICS were arthroscopically implanted at a dose of 6mg/kg. Pharmacokinetic parameters were modeled using statistical moment analyses. Joint inflammation was measured by synovial fluid cell counts and cytokine concentrations, lameness was measured by force plate asymmetry indices, and renal function was measured by glomerular filtration rate (GFR) study using technetium 99 plasma clearance. The prevalence of lesions associated with aminoglycoside nephrotoxicity was assessed by renal biopsy and electronmicroscopy. Intra-articular gentamicin concentrations fell to sub-MIC for Staphylococcus sp. (4ug/ml) by 22.4hrs (95% CI=18.6-26.2) following sponge implantation. Cmax synovial was 2397ug/ml (95% CI=1161-3634 ug/ml) at 1.2 hrs (95% CI=0.5-1.8hrs). Plasma gentamicin concentrations achieved levels of Cmax plasma =8.0ug/ml (95% CI=6.1-10.0 ug/ml) at 1.5hrs (95% CI=0.8-2.1) following GICS placement and fell below target trough of 2.0ug/ml by 5.6hrs (95% CI=4.7-6.5hrs) following GICS placement. GICS implantation caused joint inflammation (p<0.01), lameness (p=0.04), and decreased GFR (p=0.04). No dog developed clinical renal failure. No difference was observed in the prevalence of renal lesions on biopsy between treatment and control group (p=0.49). Intra-articular gentamicin concentration following GICS placement at an IV-equivalent dose reached high levels and declined rapidly. The maximum plasma levels attained were approximately 1/3rd of the recommended sub-toxic target for human patients following parenteral gentamicin administration. GICS implantation in the inflamed joint caused additional inflammation and joint dysfunction that is likely to be of clinical relevance. GICS implantation affected renal function at the dose assessed. Renal effects may be exacerbated in septic patients, and care should be taken with GICS dosing in clinical patients. / Pet Trust

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