Intratumoral heterogeneity of c-KIT mutations in a feline splenic mast cell tumor and their functional effects on cell proliferation.

A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.

Distal renal tubular acidosis and lethargy associated with zonisamide treatment in a dog with idiopathic epilepsy.

A 3-year-old neutered male golden retriever administered zonisamide for the treatment of seizures showed lethargy and had normal anion gap metabolic acidosis with hypokalaemia, hyperchloremia, and alkaline urine. The serum zonisamide concentration was close to the upper limit, which raised a suspicion of adverse effects of zonisamide. This is the first report showing that the fractional excretion of bicarbonate after compensation for the plasma bicarbonate concentration by a sodium bicarbonate infusion was approximately 5%, indicating distal renal tubular acidosis (RTA). The serum zonisamide concentration decreased, and adverse effects were abated by reducing the zonisamide dosage. Diagnostic therapy with bicarbonate served as a means of compensating for bicarbonate deficiency and contributed to the clinical diagnosis of the condition in zonisamide-associated RTA in dogs.

Noninvasive Thermographic Photographing as an Assessment of the State of Discomfort in a Dog Receiving Radiation Therapy.

In humans, radiation induces dilation of capillaries and inflammatory reactions to raise skin temperature. Thermography is used to detect abnormalities after radiation therapy (RT). However, in veterinary nursing, objective evaluation of the condition of dogs after RT using thermography has not been reported. We investigated the nasal irradiation temperature, behavioral changes, and post-irradiation pain scores in a dog receiving RT for intranasal tumors. The temperature of the nasal planum gradually increased after irradiation, reaching a significantly higher value at 120-240 min. The highest temperature was 42.3 °C and the average temperature increased by 4.4 °C. Behavioral analysis pre- and post-RT did not vary significantly. Post-RT pain levels evaluated by the pain scale ranged from 0 to 1 throughout. No veterinary treatment was provided. In humans, increased skin temperature after radiation causes psychological stress, i.e., pain and discomfort, but no such behavioral changes were observed in this case. Given individual differences in stress-related behaviors, such as pain and discomfort, assessing a dog's painfulness using only subjective methods, such as appearance and behavioral evaluation, is limited. We used thermography to assess changes in conditions not detectable by routine monitoring alone. This method is non-invasive, objective, and indispensable for providing appropriate care.

Effect of seasons and sex on the physical, hematological, and blood biochemical parameters of Noma horses.

This study aimed to evaluate the influence of seasons and sex on body size and hematological and biochemistry parameters of Noma horses, a native Japanese breed. Body size was larger in winter than in summer. Laboratory testing variables, including erythrocytic parameters and urea nitrogen, total cholesterol, and creatinine kinase levels, were higher in winter, while the eosinophil count was higher in summer. These seasonal differences may be related to increased energy consumption of horses due to heat stress. The higher eosinophil counts may have been related to the dermatitis observed in summer. Stallions tended to have smaller bodies compared with mares. Future studies are necessary to investigate the effect of stress in seasonal and sex-based groups.

Microparticle-associated tissue factor activity in dogs with disseminated intravascular coagulation.

Microparticle (MP)-associated tissue factor (TF) activity in plasma might play a role in human disseminated intravascular coagulation (DIC). The aim of this study was to compare MP-TF activity between non-DIC and DIC groups. Ten clinically healthy beagles and 26 diseased dogs were enrolled. The proportion of dogs with increased MP-TF activity was significantly higher in the DIC group than the non-DIC group (P=0.014). MP-TF activity in the DIC group was significantly higher than the non-DIC group (P=0.021). MP-TF activity positively correlated with plasma D-dimer concentration (r=0.42, P=0.034). Moreover, MP-TF activity was decreased by the time of recovery in some dogs with DIC. Larger prospective studies are warranted to assess its value as a diagnostic and prognostic biomarker in DIC.

Oncolytic reovirus synergizes with chemotherapeutic agents to promote cell death in canine mammary gland tumor.

The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC50) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy.

Les effets oncolytiques des reovirus dans divers cancers ont été prouvés lors de plusieurs essais cliniques en médecine humaine. La virothérapie oncolytique pour les cancers canins utilisant des reovirus est présentement en développement dans notre laboratoire. L'objectif de cette étude était d'examiner les effets synergiques anticancéreux d'une combinaison de reovirus et de faibles doses d'agents chimio-thérapeutiques variés sur les tumeurs des glandes mammaires (TGM) chez les chiens. La première partie de l'étude a démontré l'efficacité du reovirus sur des TGM in vitro et in vivo. Les reovirus utilisés seuls ont produit une quantité significative de mort cellulaire dans des lignées cellulaires canines de TGM via l'apoptose dépendante de la caspase. Une injection unique de reovirus interféra avec la croissance de TGM canines chez des souris ayant reçu une xénogreffe, mais était insuffisante pour induire une régression complète de la tumeur. La deuxième partie de cette étude a mis en évidence les effets anti-tumoraux des reovirus en combinaison avec de faibles doses de paclitaxel, de carboplatin, de gemcibatine, ou de toceranib. Une activité synergique augmentée fut observée dans la lignée cellulaire TGM traitée de manière concomitante avec du reovirus et tous les agents chimio-thérapeutique sauf le toceranib. De plus, en combinant le reovirus avec du paclitaxel ou de la gemcibatine à la moitié de la dose de la moitié de la concentration inhibitrice maximale (IC50) on augmenta la cytotoxicité en activant la caspase 3. Nos données suggèrent que la combinaison de reovirus et de faibles doses d'agents chimio-thérapeutiques fournie une option intéressante pour le traitement de cancer canin.(Traduit par Docteur Serge Messier).

Expression of O(6)-methylguanine-DNA methyltransferase causes lomustine resistance in canine lymphoma cells.

The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT) causes resistance to nitrosoureas in various human cancers. In this study, we analyzed the correlation between canine lymphomas and MGMT in vitro. Two of five canine lymphoma cell lines required higher concentrations of lomustine to inhibit cell growth by 50%, but their sensitivity to the drug increased when they were cultured with an MGMT inhibitor. Fluorometric oligonucleotide assay and real-time polymerase chain reaction of these cell lines revealed MGMT activity and high MGMT mRNA expression, respectively. We analyzed the methylation status of the CpG islands of the canine MGMT gene by the bisulfite-sequencing method. Unlike human cells, the canine lymphoma cell lines did not show significant correlation between methylation status and MGMT suppression levels. Our results suggest that in canine lymphoma MGMT activity may influence sensitivity to nitrosoureas; thus, inhibition of MGMT activity would benefit nitrosourea-resistant patients. Additional studies are necessary to elucidate the mechanism of regulation of MGMT expression.

La protéine de réparation O6-méthylguanine-DNA méthyltransferase (MGMT) cause de la résistance aux produits nitroso-urée dans divers cancers humains. Dans la présente étude nous avons analysé in vitro la corrélation entre les lymphomes canins et le MGMT. Deux des cinq lignées cellulaires de lymphome canin ont nécessité des concentrations plus élevées de lomustine pour inhiber de 50 % la croissance cellulaire, mais leur sensibilité au médicament augmenta lorsqu'elles furent mises en culture avec un inhibiteur de MGMT. Une épreuve fluorométrique des oligonucléotides et une épreuve d'amplification en chaîne par la polymérase en temps réel sur ces lignées cellulaires ont révélé, respectivement, une activité MGMT et une expression élevée d'ARNm de MGMT. Nous avons analysé le statut de méthylation des ilots CpG du gène MGMT canin par la méthode de séquençage au bisulfite. Contrairement aux cellules humaines, les lignées cellulaires canines de lymphome n'ont pas montré de corrélation significative entre le statut de méthylation et les niveaux de suppression de MGMT. Nos résultats suggèrent que lors de lymphome canin l'activité de MGMT peut influencer la sensibilité aux produits nitroso-urée; ainsi, l'inhibition de l'activité MGMT bénéficierait les patients résistants au nitroso-urée. Des études additionnelles sont nécessaires pour élucider le mécanisme de régulation de l'expression de MGMT.(Traduit par Docteur Serge Messier).

The clinical utility of two human portable blood glucose meters in canine and feline practice.

BACKGROUND: Portable blood glucose meters (PBGMs) are useful for serial measurements of blood glucose and creation of blood glucose curves in veterinary practice. However, it is necessary to validate PBGMs designed for people for veterinary use. OBJECTIVES: Our objective was to evaluate the accuracy of 2 PBGMs designed for people for use in dogs and cats. METHODS: The blood glucose levels were determined in blood samples collected from 69 dogs and 26 cats admitted to the Kagoshima University Veterinary Teaching Hospital, using a MEDISAFE [PBGM-T] and an Antsense III [PBGM-H], and a FUJI DRI-CHEM 7000V as reference method. The correlations and agreements among the results were statistically analyzed. RESULTS: Simple regression analyses revealed a high correlation between values from both PBGMs and the reference method in both dogs and cats. However, Passing-Bablok regression and Bland-Altman analyses revealed that the data from both PBGMs did not show statistical agreement with the reference values. Concordance correlated coefficients were moderate for the PBGM-T and almost perfect for the PBGM-H for canine samples, and were poor for the PBGM-T and substantial for the PBGM-H for feline samples. Hematocrit values significantly affected the results of the PBGM-T, but not the PBGM-H. Error grid analyses revealed that all measurements from both PBGMs would lead to acceptable treatment decisions. CONCLUSIONS: Our findings suggest that both PBGMs, especially the PBGM-H, would be clinically useful in small animal practice, although there was a bias between each PBGM and the reference method.

Molecular epidemiologic survey of Bartonella, Ehrlichia, and Anaplasma infections in Japanese Iriomote and Tsushima leopard cats.

The Iriomote cat (IC; Prionailurus iriomotensis) and the Tsushima leopard cat (TLC; Prionailurus bengalensis euptilura) are endangered wild felids in Japan. As a part of ongoing conservation activities, we conducted a molecular, epidemiologic survey of Bartonella, Ehrlichia, and Anaplasma infections in wild IC and TLC populations. Blood samples (47 from 33 individual IC; 22 from 13 TLC) were collected between August 2002 and January 2011. Using PCR analysis, we confirmed the presence of Bartonella henselae in ICs and Bartonella clarridgeiae in TLCs, with prevalences of 6% and 8%, respectively. Using PCR and basic local alignment search tool analyses, we identified Ehrlichia canis in both cats and Anaplasma bovis in TLCs. The prevalence of E. canis was 12% in ICs and 8% in TLCs, and the prevalence of A. bovis was 15% in TLCs. This is the first report, to our knowledge, of B. henselae, B. clarridgeiae, E. canis, and A. bovis infections in these two endangered species. Continuous monitoring of these pathogens is needed for their conservation.