Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan. The patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated. The cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified. A new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

The lack of proofreading activity of the viral polymerase and the segmented nature of the influenza A virus (IAV) genome are responsible for the genetic diversity of IAVs and for their ability to adapt to a new host. We tried to adapt avian IAV (avIAV) to the pig by serial passages in vivo and assessed the occurrence of point mutations and their influence on viral fitness in the pig’s body. A total of 25 in vivo avIAV passages of the A/duck/Bavaria/77 strain were performed by inoculation of 50 piglets, and after predetermined numbers of passages 20 uninoculated piglets were exposed to the virus through contact with inoculated animals. Clinical signs of swine influenza were assessed daily. Nasal swabs and lung tissue were used to detect IAV RNA by real-time RT-PCR and isolates from selected passages were sequenced. Apart from a rise in rectal temperature and a sporadic cough, no typical clinical signs were observed in infected pigs. The original strain required 20 passages to improve its replication ability noticeably. A total of 29 amino-acid substitutions were identified. Eighteen of them were detected in the first sequenced isolate, of which 16 were also in all other analysed strains. Additional mutations were detected with more passages. One substitution, threonine (T) 135 to serine (S) in neuraminidase (NA), was only detected in an IAV isolate from a contact-exposed piglet. Passaging 25 times allowed us to obtain a partially swine-adapted IAV. The improvement in isolate replication ability was most likely related to S654 to glycine (G) substitution in the basic protein (PB) 1 as well as to aspartic acid (D) 701 to asparagine (N) and arginine (R) 477 to G in PB2, glutamic acid (E) 204 to D and G239E in haemagglutinin and T135S in NA.

OBJECTIVE To investigate regional differences of relative metabolite concentrations in the brain of healthy dogs with short echo time, single voxel proton magnetic resonance spectroscopy (1H MRS) at 3.0 T. ANIMALS 10 Beagles. PROCEDURES Short echo time, single voxel 1H MRS was performed at the level of the right and left basal ganglia, right and left thalamus, right and left parietal lobes, occipital lobe, and cerebellum. Data were analyzed with an automated fitting method (linear combination model). Metabolite concentrations relative to water content were obtained, including N-acetyl aspartate, total choline, creatine, myoinositol, the sum of glutamine and glutamate (glutamine-glutamate complex), and glutathione. Metabolite ratios with creatine as the reference metabolite were calculated. Concentration differences between right and left hemispheres and sexes were evaluated with a Wilcoxon signed rank test and among various regions of the brain with an independent t test and 1-way ANOVA. RESULTS No significant differences were detected between sexes and right and left hemispheres. All metabolites, except the glutamine-glutamate complex and glutathione, had regional concentrations that differed significantly. The creatine concentration was highest in the basal ganglia and cerebellum and lowest in the parietal lobes. The N-acetyl aspartate concentration was highest in the parietal lobes and lowest in the cerebellum. Total choline concentration was highest in the basal ganglia and lowest in the occipital lobe. CONCLUSIONS AND CLINICAL RELEVANCE Metabolite concentrations differed among brain parenchymal regions in healthy dogs. This study may provide reference values for clinical and research studies involving 1H MRS performed at 3.0 T.

OBJECTIVE To analytically validate a gas concentration of chromatography–mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage. SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs. PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions. RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at −80°C for ≤ 7 days. CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at −80°C for 1 week had no effect on GC-MS results.

Objective—To investigate clinical use of proton magnetic resonance spectroscopy (1H MRS) and to compare metabolic brain bioprofiles of dogs with and without hepatic encephalopathy. Animals—6 dogs with hepatic encephalopathy and 12 control dogs. Procedures—Conventional MRI and single-voxel 1H MRS were performed with a 3-T magnet. Images for routine MRI planes and sequences were obtained. Single-voxel 1H MRS was performed with a point-resolved sequence with a short echo time (35 milliseconds) and voxel of interest placement at the level of the basal ganglia. Metabolites of interest included the glutamine-glutamate complex (sum quantification of glutamate and glutamine), myoinositol, N-acetyl aspartate, total choline, and creatine. Data were analyzed with postprocessing fitting algorithm software, and metabolite concentration relative to water and ratios with creatine as the reference metabolite were calculated. Results—Compared with control dogs, dogs with hepatic encephalopathy had specific changes, which included significantly higher concentration relative to water of the glutamine-glutamate complex and significantly lower concentration of myoinositol. Choline and N-acetyl aspartate concentrations were also slightly lower in dogs with hepatic encephalopathy than in control dogs. No differences in creatine concentration were detected between groups. Conclusions and Clinical Relevance—1H MRS aided in the diagnosis of hepatic encephalopathy in dogs, and findings supported the assumption that ammonia is a neurotoxin that manifests via glutamine-glutamate complex derangements. Use of 1H MRS may provide clinically relevant information in patients with subclinical hepatic encephalopathy, equivocal results of bile acids tests, and equivocal ammonia concentrations or may be helpful in monitoring efficacy of medical management.

Brain capillary function was assessed in 4- to 6-week-old calves given lead acetate (15 mg/kg of body weight) orally for 7 to 8 days. Neurologic signs of lead poisoning included CNS depression, blindness, and hyperesthesia. Brain capillaries were isolated from cerebral cortex of control and lead-treated calves and evaluated for metabolic indicators, ion transport, and prolyl hydroxylase activity. In lead-treated calves, the rate of glucose metabolism was less than half that in controls. Ion efflux of 45Ca or 36Cl from endothelial cell suspensions was not affected by lead treatment. Prolyl hydroxylase activity in endothelium and proline-to-hydroxyproline ratio in endothelial basement membranes were similar in control and lead-poisoned calves. Results indicate that lead may inhibit energy metabolism, but not ion transport or collagen biosynthesis in brain capillaries of calves and, compared with suckling rats, damage to the blood-brain barrier is less important. In calves, neuronal tissue may be the primary target for the CNS effects of lead.

Objective-To measure concentrations of glutamate, aspartate, γ-aminobutyric acid (GABA), and glycine in CSF of dogs with experimentally induced subarachnoid hemorrhage (SAH) and to assess effects of cyclosporine and simvastatin on these concentrations. Sample-CSF samples from 13 dogs. Procedures-In a previous study, SAH was induced in dogs via 2 injections of autologous blood into the cerebellomedullary cistern 24 hours apart. Dogs were untreated (control; n = 5) or received simvastatin alone (4) or simvastatin in combination with cyclosporine (4). Samples of CSF were collected before the first blood injection (baseline; time 0), before the second blood injection, and on days 3, 7, and 10. For the study reported here, neurotransmitter concentrations in CSF were analyzed via high-performance liquid chromatography. Data were analyzed with a repeated-measures model with adjustments for multiple comparisons by use of the Tukey method. Results-In control dogs, the glutamate concentration peaked on day 3 and there was a significant increase in GABA and glutamate concentrations. Glutamate concentrations were significantly lower and glycine concentrations significantly higher on day 3 after administration of simvastatin alone or simvastatin in combination with cyclosporine, compared with concentrations for the control group. No significant differences in GABA and aspartate concentrations were detected among treatment groups at any time point. Conclusions and Clinical Relevance-Glutamate concentrations were increased in the CSF of dogs with SAH. Simvastatin administration attenuated high glutamate concentrations. A combination of immunosuppression and upregulation of nitric oxide synthase may be useful in lowering high glutamate concentrations in ischemic CNS conditions.

Objective-To investigate differences in CSF concentrations of excitatory and inhibitory neurotransmitters in dogs with and without T2-weighted (T2W) MRI hyperintense areas in the limbic system. Sample-Archived CSF samples and stored brain MRI images of 5 healthy research dogs (group 1), 8 dogs with idiopathic epilepsy (IE) with no abnormal MRI findings (group 2), and 4 dogs with IE with hyperintense areas in the limbic system detected by means of T2W MRI (group 3). Procedures-Archived CSF samples and stored MRI images obtained from all dogs were evaluated. Dogs in groups 2 and 3 were matched on the basis of age and breed. High-performance liquid chromatography was used to evaluate glutamate and γ-aminobutyric acid (GABA) concentrations in CSF samples. Results-Glutamate concentrations were higher in CSF of both groups of dogs with IE than in healthy dogs. However, glutamate concentrations in CSF were not significantly higher in dogs with IE and with hyperintense areas than in dogs with IE but no abnormal MRI findings. Concentrations of GABA in CSF were higher in group 3 than in group 2 and in group 2 than in group 1. Conclusions and Clinical Relevance-No significant difference was evident between glutamate concentrations in CSF of dogs with IE and with and without hyperintense areas detected by means of T2W MRI. However, glutamate concentrations typically were higher in CSF of dogs with IE and MRI hyperintense areas. Future studies with larger sample sizes should be conducted to confirm this finding and to determine the clinical importance of high glutamate concentrations in CSF of dogs with IE.