Immunohistochemical Investigation into Protein Expression Patterns of FOXO4, IRF8 and LEF1 in Canine Osteosarcoma.

Osteosarcoma (OSA) is the most common type of primary bone malignancy in people and dogs. Our previous molecular comparisons of canine OSA against healthy bone resulted in the identification of differentially expressed protein-expressing genes (forkhead box protein O4 (FOXO4), interferon regulatory factor 8 (IRF8), and lymphoid enhancer binding factor 1 (LEF1)). Immunohistochemistry (IHC) and H-scoring provided semi-quantitative assessment of nuclear and cytoplasmic staining alongside qualitative data to contextualise staining (n = 26 patients). FOXO4 was expressed predominantly in the cytoplasm with significantly lower nuclear H-scores. IRF8 H-scores ranged from 0 to 3 throughout the cohort in the nucleus and cytoplasm. LEF1 was expressed in all patients with significantly lower cytoplasmic staining compared to nuclear. No sex or anatomical location differences were observed. While reduced levels of FOXO4 might indicate malignancy, the weak or absent protein expression limits its primary use as diagnostic tumour marker. IRF8 and LEF1 have more potential for prognostic and diagnostic uses and facilitate further understanding of their roles within their respective molecular pathways, including Wnt/beta-catenin/LEF1 signalling and differential regulation of tumour suppressor genes. Deeper understanding of the mechanisms involved in OSA are essential contributions towards the development of novel diagnostic, prognostic, and treatment options in human and veterinary medicine contexts.

Immunohistochemical Characterisation of GLUT1, MMP3 and NRF2 in Osteosarcoma.

Osteosarcoma (OSA) is an aggressive bone malignancy. Unlike many other malignancies, OSA outcomes have not improved in recent decades. One challenge to the development of better diagnostic and therapeutic methods for OSA has been the lack of well characterized experimental model systems. Spontaneous OSA in dogs provides a good model for the disease seen in people and also remains an important veterinary clinical challenge. We recently used RNA sequencing and qRT-PCR to provide a detailed molecular characterization of OSA relative to non-malignant bone in dogs. We identified differential mRNA expression of the solute carrier family 2 member 1 (SLC2A1/GLUT1), matrix metallopeptidase 3 (MMP3) and nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2) genes in canine OSA tissue in comparison to paired non-tumor tissue. Our present work characterizes protein expression of GLUT1, MMP3 and NRF2 using immunohistochemistry. As these proteins affect key processes such as Wnt activation, heme biosynthesis, glucose transport, understanding their expression and the enriched pathways and gene ontologies enables us to further understand the potential molecular pathways and mechanisms involved in OSA. This study further supports spontaneous OSA in dogs as a model system to inform the development of new methods to diagnose and treat OSA in both dogs and people.

Pilot study evaluating the monitoring of canine diabetes mellitus in primary care practice.

OBJECTIVES: This study aimed to describe how canine diabetes mellitus (CDM) is monitored in primary care practice (PCP) and to report outcomes. DESIGN: Retrospective case review. SETTING: PCP. PARTICIPANTS: 40 dogs of 22 different pedigrees and five crossbreeds. Median age at diagnosis was nine years and six months (eight years six months to 10 years five months). Dogs were diagnosed with CDM between January 1, 2008 and December 30, 2012 and remained with the practice to the study end or until death. PRIMARY AND SECONDARY OUTCOME MEASURES: Stability achievement and death or euthanasia. Consultations for each dog were identified and recorded through records collected from the PCP (January 1, 2008 to December 30, 2012). RESULTS: A median of three consultations per dog occurred in the first month, subsequently falling to a median of one consultation every 19 days thereafter. After the first month postdiagnosis, weight and single blood glucose concentrations were most frequently recorded at 66.8 and 42 per cent of consultations respectively and a blood glucose curve was performed infrequently (17.4 per cent). Serum biochemistry was measured at 8 per cent of consultations and urine culture at only 0.8 per cent. Median survival time (MST) for all dogs was eight months (2-21 months). Eighteen dogs stabilised within three months of diagnosis and their MST was 20.5 months, (10.25-25.75 months), significantly longer than the 22 dogs not achieving stability within three months (MST 2.5 months, 0-5.5 months) (P<0.001). Those dogs not surviving beyond the first month had significantly fewer consultations than those still alive (P<0.005). CONCLUSIONS: This pilot study indicates dogs with CDM managed solely in PCP experience limited monitoring tests and have lower MST than reported in the literature. Recruitment of a larger cohort of CDM cases from a larger number of PCP will help determine whether these results accurately represent this demographic and verify if infrequent testing is associated with a poor outcome. Importantly, prospective evaluation of decision-making around monitoring CDM in PCP is required, to help determine the effectiveness and feasibility of more frequent monitoring strategies, such as those recommended by the American Animal Hospital Association, particularly to influence MST.

Multiple Genetic Associations with Irish Wolfhound Dilated Cardiomyopathy.

Cardiac disease is a leading cause of morbidity and mortality in dogs and humans, with dilated cardiomyopathy being a large contributor to this. The Irish Wolfhound (IWH) is one of the most commonly affected breeds and one of the few breeds with genetic loci associated with the disease. Mutations in more than 50 genes are associated with human dilated cardiomyopathy (DCM), yet very few are also associated with canine DCM. Furthermore, none of the identified canine loci explain many cases of the disease and previous work has indicated that genotypes at multiple loci may act together to influence disease development. In this study, loci previously associated with DCM in IWH were tested for associations in a new cohort both individually and in combination. We have identified loci significantly associated with the disease individually, but no genotypes individually or in pairs conferred a significantly greater risk of developing DCM than the population risk. However combining three loci together did result in the identification of a genotype which conferred a greater risk of disease than the overall population risk. This study suggests multiple rather than individual genetic factors, cooperating to influence DCM risk in IWH.

Use of a Microsoft Excel based add-in program to calculate plasma sinistrin clearance by a two-compartment model analysis in dogs.

Assessment of renal function by means of plasma clearance of a suitable marker has become standard procedure for estimation of glomerular filtration rate (GFR). Sinistrin, a polyfructan solely cleared by the kidney, is often used for this purpose. Pharmacokinetic modeling using adequate software is necessary to calculate disappearance rate and half-life of sinistrin. The purpose of this study was to describe the use of a Microsoft excel based add-in program to calculate plasma sinistrin clearance, as well as additional pharmacokinetic parameters such as transfer rates (k), half-life (t1/2) and volume of distribution (Vss) for sinistrin in dogs with varying degrees of renal function.

Long-term survival and quality of life in dogs with clinical signs associated with a congenital portosystemic shunt after surgical or medical treatment.

OBJECTIVE: To compare long-term survival and quality of life data in dogs with clinical signs associated with a congenital portosystemic shunt (CPSS) that underwent medical or surgical treatment. DESIGN: Prospective cohort study. ANIMALS: 124 client-owned dogs with CPSS. PROCEDURES: Dogs received medical or surgical treatment without regard to signalment, clinical signs, or clinicopathologic results. Survival data were analyzed with a Cox regression model. Quality of life information, obtained from owner questionnaires, included frequency of CPSS-associated clinical signs (from which a clinical score was derived), whether owners considered their dog normal, and (for surgically treated dogs) any ongoing medical treatment for CPSS. A Mann-Whitney U test was used to compare mean clinical score data between surgically and medically managed dogs during predetermined follow-up intervals. RESULTS: 97 dogs underwent surgical treatment; 27 were managed medically. Median follow-up time for all dogs was 1,936 days. Forty-five dogs (24 medically managed and 21 surgically managed) died or were euthanized during the follow-up period. Survival rate was significantly improved in dogs that underwent surgical treatment (hazard ratio, 8.11; 95% CI, 4.20 to 15.66) than in those treated medically for CPSS. Neither age at diagnosis nor shunt type affected survival rate. Frequency of clinical signs was lower in surgically versus medically managed dogs for all follow-up intervals, with a significant difference between groups at 4 to 7 years after study entry. CONCLUSIONS AND CLINICAL RELEVANCE: Surgical treatment of CPSS in dogs resulted in significantly improved survival rate and lower frequency of ongoing clinical signs, compared with medical management. Age at diagnosis did not affect survival rate and should not influence treatment choice.

Comparison of survival after surgical or medical treatment in dogs with a congenital portosystemic shunt.

OBJECTIVE: To compare survival of dogs with a congenital portosystemic shunt (CPSS) that received medical or surgical treatment. DESIGN: Prospective cohort study. ANIMALS: 126 client-owned dogs with a single CPSS. PROCEDURES: Dogs were examined at 1 of 3 referral clinics, and a single CPSS was diagnosed in each. Dogs received medical or surgical treatment without regard to signalment, clinical signs, or results of hematologic or biochemical analysis. Survival data were analyzed via a Cox regression model. RESULTS: During a median follow-up period of 579 days, 18 of 126 dogs died as a result of CPSS. Dogs treated via surgical intervention survived significantly longer than did those treated medically. Hazard ratio for medical versus surgical treatment of CPSS (for the treatment-only model) was 2.9 (95% confidence interval, 1.1 to 7.2). Age at CPSS diagnosis did not affect survival. CONCLUSIONS AND CLINICAL RELEVANCE: Both medical and surgical treatment can be used to achieve long-term survival of dogs with CPSS, although results of statistical analysis supported the widely held belief that surgery is preferable to medical treatment. However, the study population consisted of dogs at referral clinics, which suggested that efficacy of medical treatment may have been underestimated. Although surgical intervention was associated with a better chance of long-term survival, medical management provided an acceptable first-line option. Age at examination did not affect survival, which implied that early surgical intervention was not essential. Dogs with CPSS that do not achieve acceptable resolution with medical treatment can subsequently be treated surgically.

Magnetic resonance imaging of functional Schwann cell transplants labelled with magnetic microspheres.

There is increasing interest in the use of magnetic resonance imaging (MRI) methods for tracking the fate of labelled cells in vivo post-implantation. The majority of studies have employed cell labels based on nanometer-sized ultrasmall dextran-coated iron oxide particles (USPIO), which are detected through signal hypointensity in T2-weighted images. Although sensitive to MR detection, these labels can be difficult to distinguish from other sources of signal loss in vivo and can be diluted by cell division. Recently, a micron-sized cell label has been described that is much more sensitive to MR detection and which allows detection of single labels in vivo. We show here that glial cells readily take up this label in culture and that the labelled Schwann cells can be detected in vivo by MRI following their implantation into a demyelinated lesion in the rat spinal cord. Signal loss due to the label is sufficiently great that the labelled cells can easily be distinguished from surrounding haemorrhage at the lesion site. Subsequent histological analysis of the lesion area showed that the transplanted cells were remyelinating the demyelinated axons, demonstrating that the labelled cells retained their biological function and that the majority of the label had remained within the transplanted cells.

Superparamagnetic iron oxide-labeled Schwann cells and olfactory ensheathing cells can be traced in vivo by magnetic resonance imaging and retain functional properties after transplantation into the CNS.

Schwann cell (SC) and olfactory ensheathing cell (OEC) transplantation has been shown experimentally to promote CNS axonal regeneration and remyelination. To advance this technique into a clinical setting it is important to be able to follow the fates of transplanted cells by noninvasive imaging. Previous studies, using complex modification processes to enable uptake of contrast agents, have shown that cells labeled in vitro with paramagnetic contrast agents transplanted into rodent CNS can be visualized using magnetic resonance imaging (MRI). Here we show that SCs and OECs efficiently internalize dextran-coated superparamagnetic iron oxide (SPIO) from the culture medium by fluid phase pinocytosis. After transplantation into focal areas of demyelination in adult rat spinal cord both transplanted SPIO-labeled SCs and OECs produce a signal reduction using T(2)-weighted MRI in anesthetized rats that persists for up to 4 weeks. Although signal reduction was discernable after transplantation of unlabelled cells, this is nevertheless distinguishable from that produced by transplanted labeled cells. The region of signal reduction in SPIO-labeled cell recipients correlates closely with areas of remyelination. Because the retention of functional integrity by labeled cells is paramount, we also show that SPIO-labeled SCs and OECs are able to myelinate normally after transplantation into focal areas of demyelination. These studies demonstrate the feasibility of noninvasive imaging of transplanted SCs and OECs and represent a significant step toward the clinical application of promising experimental approaches.

Increasing local levels of neuregulin (glial growth factor-2) by direct infusion into areas of demyelination does not alter remyelination in the rat CNS.

Glial growth factor-2 (GGF-2) is a neuronally derived isoform of neuregulin shown in vitro to promote proliferation and survival of oligodendrocytes, the myelinating cells of the CNS. Enhanced remyelination has been demonstrated in vivo following systemic delivery of human recombinant GGF-2 (rhGGF-2) in experimental autoimmune encephalomyelitis (EAE). However, it is uncertain whether this is the result of direct effects of rhGGF-2 on cells of the oligodendrocyte lineage or due to modulation of the immune or inflammatory response. If this enhanced remyelination was due to direct effects of rhGGF-2 on cells of the oligodendrocyte lineage then one would expect rhGGF-2 to induce a similar proremyelinating response in nonimmune, gliotoxin models of demyelination. Using a gliotoxin model of demyelination we were therefore able to ascertain the in vivo effect of rhGGF-2 following local CNS delivery in a model that is not confounded by the concurrent presence of an immune-mediated process. No significant alteration in the rate or character of remyelination was evident following local delivery as compared to controls, and indeed nor following systemic delivery in the gliotoxin model. The results of this study therefore indicate that both direct infusion and systemic delivery of rhGGF-2 do not alter remyelination in a nonimmune, gliotoxin model of demyelination. This suggests that the proremyelinating effects of systemically delivered rhGGF-2 in EAE are unlikely to be due to direct effects on the oligodendrocyte lineage, but may be mediated by rhGGF-2 inducing an environment more favourable to remyelination, possibly through modulation of the immune response.