Hiroko Hiraoka

Oncolytic Reovirus in Canine Mast Cell Tumor

The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility.

Clinical observations of Babesia gibsoni infection with low parasitaemia confirmed by PCR in dogs.

2004). In preliminary experiments, this PCR was able to detect approximately 10 B gibsoni-infected red blood cells in 1 μl of peripheral blood. All 35 animals with B gibsoni infection were positive by the PCR. To confirm the PCR findings, nucleotide sequences of randomly selected products of the B gibsoni-specific PCR were determined by direct sequence analysis, using the method described by Inokuma and others (2003). All the analysed sequences showed 100 per cent homology with B gibsoni Asia-1 (GenBank accession number AF175300). The animals with IMHA showed an acute onset of signs similar to those of B gibsoni infection, such as haemolytic and regenerative anaemia, icterus, anorexia or haematuria, at presentation. All eight animals with IMHA were found to be negative for B gibsoni infection by PCR. The information obtained for the 35 babesiosis cases and eight IHMA cases included histories and the findings of physical examination, complete blood counts, serum biochemical analysis and abdominal radiography. To compare the data from the dogs with low parasitaemia with the data from those with moderate to severe parasitaemia or with IMHA cases, chisquared tests and one-way analysis of variance were performed by using StatView v 5.0 (Hulinks). When the clinical and laboratory findings of the Babesia-infected animals showing low parasitaemia were compared with those of the dogs with moderate to severe parasitaemia, there were no significant differences between the two groups (Table 1). Although the numbers of dogs that showed icterus and the mean total bilirubin level in dogs with more severe parasitaemia were slightly higher than those of the dogs with low parasitaemia, the differences were not significant. Oxidative damage to erythrocytes induced by B gibsoni infection is thought to result in severe anaemia even in the presence of low parasitaemia (Otsuka and others 2002). Overall, the degree of parasitaemia in the B gibsoni-infected dogs did not relate to the clinical and laboratory findings. The clinical and laboratory findings in the dogs with B gibsoni infection with low parasitaemia were also compared with Clinical observations of Babesia gibsoni infection with low parasitaemia confirmed by PCR in dogs

Superficial necrolytic dermatitis associated with extrapancreatic glucagonoma in a dog.

An 11-year-old Shih Tzu presented with crusting and erythema, mainly on the abdomen and the root of the tail. Based on histopathological findings, blood examinations and necropsy findings, the condition was diagnosed as superficial necrolytic dermatitis associated with a glucagon-secreting extrapancreatic neuroendocrine tumour. Gross necropsy revealed tumour invasion into the spleen, liver, adrenal glands and mesenteric lymph nodes. Immunohistochemical analysis of the neoplastic cells revealed that the tumour was a glucagonoma, consistent with earlier findings of persistent glucagonaemia and hypoaminoacidaemia.

Investigation of the cytotoxic effect of flavopiridol in canine lymphoma cell lines

The cyclin-dependent kinase (CDK) inhibitor, flavopiridol, was tested as a potential new cancer therapeutic agent to treat canine lymphoma by examining its effect on cell growth of canine lymphoma cell lines in vitro. Flavopiridol induced profound cell death in all eight lymphoma cell lines at 400 nM, and in all cases cell death was due to apoptosis. Apoptosis was inhibited by caspase inhibitor, despite the variable sensitivities between cell lines. Analysis of the mechanism of flavopiridol-induced apoptosis showed that Rb phosphorylation was inhibited, possibly due to CDK4 or CDK6 inhibition. There was also decreased expression of Rb protein and anti-apoptotic proteins, Mcl-1 and XIAP, possibly through transcriptional regulation by inhibition of CDK7 or CDK9 activation. Canine lymphoma cell line-xenotransplanted mice were then treated with flavopiridol and profound tumour shrinkage was observed. This study describes a new therapeutic approach using flavopiridol for canine lymphoma treatment.

Bimodal immunoglobulin A gammopathy in a cat with feline myeloma-related disorders.

A 10-year-old female spayed mixed breed cat with a subcutaneous mass on the right hind limb was revealed with bimodal monoclonal gammopathy composed of IgA by immunoelectrophoresis and immunofixation. Approximately 1 month after referral, the cat died due to renal failure. Postmortem immunohistopathologic evaluation of the subcutaneous mass revealed neoplastic cell proliferation of plasma cells and giant myeloma cells. Neoplastic cells were also present in the liver and spleen. These results led to the diagnosis of a rare case of feline myeloma-related disorders with extramedullary plasmacytoma infiltrating in multiple locations. This report emphasizes the necessity to accumulate cases with similar clinicopathologic findings in the future.

Molecular cloning of canine Wilms’ Tumor 1 for immunohistochemical analysis in canine tissues

Wilms’ tumor 1 (WT1) expression has been investigated in various human cancers as a target molecule for cancer immunotherapy. However, few studies have focused on WT1 expression in dogs. Firstly, cDNA of canine WT1 (cWT1) was molecularly cloned from normal canine kidney. The cross-reactivity of the anti-human WT1 monoclonal antibody (6F-H2) with cWT1 was confirmed via Western blotting using cells overexpressing cWT1. Immunohistochemical staining revealed that cWT1 expression was detected in all canine lymphoma tissues and in some normal canine tissues, including the kidney and lymph node. cWT1 is a potential immunotherapy target against canine cancers.