Direct comparison of four adeno-associated virus serotypes in mediating the production of antiangiogenic proteins in mouse muscle.

To determine the adeno-associated virus (AAV) serotype that most efficiently mediates muscle expression of antiangiogenic proteins, we injected four different serotype (1, 2, 7, and 8) AAV vectors encoding mouse endostatin (mEnd) or human soluble FLK-1 (hsFLK-1) into a quadriceps muscle of C57BL/6 mice. The highest plasma levels of therapeutic protein were observed in AAV8-injected mice (8 > 7 > 1 > 2). Sustained expression of mEnd was detected for 6 months, whereas concentrations of hsFLK-1 declined to the background level within 2 weeks caused by neutralizing anti-hsFLK-1 antibody. These data demonstrate that AAV8 (mEnd) serotype is the most efficient mediator for protein expression.

Imatinib elicited a favorable response in a dog with a mast cell tumor carrying a c-kit c.1523A>T mutation via suppression of constitutive KIT activation.

Target therapy using the tyrosine kinase inhibitor imatinib is one of the new therapeutic approaches for canine mast cell tumors (MCTs). In the present report, we demonstrate a clinical response to imatinib in a dog with MCT carrying a c-kit c.1523A>T mutation. Moreover, the effect of this mutation on the phosphorylation status of KIT and the inhibitory potency of imatinib on the phosphorylation of the mutant KIT were examined in vitro. A dog with a MCT tumor mass on the right forelimb sole with lymph node metastasis and mastocytemia was treated with imatinib. The MCT mass markedly shrank and mastocytemia became undetectable with 2 weeks of treatment. The lymph node enlarged by metastasis became normal in size with 5 weeks of treatment. From the sequencing analysis of c-kit in tumor cells, a substitution mutation c.1523A>T that alters the amino acid composition (p.Asn508Ile) within the extracellular domain of KIT was identified. The mutant KIT expressed on 293 cells showed ligand-independent phosphorylation and imatinib suppressed this phosphorylation in a dose-dependent manner. From these findings, imatinib was considered to elicit a clinical response in a canine case of MCT via inhibition of the constitutively activated KIT caused by a c-kit c.1523A>T mutation.

Mutations in the fifth immunoglobulin-like domain of kit are common and potentially sensitive to imatinib mesylate in feline mast cell tumours.

The purpose of the current study was to investigate the mutation status of KIT in feline mast cell tumours (MCTs) and to examine the effects of tyrosine kinase inhibition on the phosphorylation of mutant kit in vitro and in clinical cases of cats. Sequence analysis of KIT identified mutations in 42/62 MCTs (67.7%). The vast majority of the mutations were distributed in exons 8 and 9, both of which encode the fifth immunoglobulin-like domain (IgD) of kit. All five types of kit with a mutation in the fifth IgD were then expressed in 293 cells and examined for phosphorylation status. The mutant kit proteins showed ligand-independent phosphorylation. The tyrosine kinase inhibitor imatinib mesylate suppressed the phosphorylation of these mutant kit proteins in transfectant cells. In a clinical study of 10 cats with MCTs, beneficial response to imatinib mesylate was observed in 7/8 cats that had a mutation in the fifth IgD of kit in tumour cells. Mutations in the fifth IgD of kit thus appear to be common and potentially sensitive to imatinib mesylate in feline MCTs. These data provide an in vivo model for paediatric mastocytosis where mutations in the fifth IgD of kit also occur.

Immunophenotyping and gene rearrangement analysis in dogs with lymphoproliferative disorders characterized by small-cell lymphocytosis.

Lymphocytosis caused by neoplastic proliferation of small lymphocytes is occasionally difficult to distinguish by morphological examination from nonneoplastic lymphocytosis. To examine the clinical utility of gene rearrangement analysis for demonstrating neoplastic proliferation of small lymphocytes, gene rearrangement analysis was performed in comparison with immunophenotyping using peripheral lymphocytes in dogs with small lymphocytosis. Thirty-one dogs with small-cell lymphocytosis (8,100-884,300/microl) were enrolled. By immunophenotyping, lymphocytosis of all dogs was suggested to be neoplastic in nature based on the detection of marked expansion of phenotypically homogeneous lymphocytes or the presence of an aberrant antigen-expressing population of lymphocytes. In contrast, gene rearrangement analysis represented clonality in 27 dogs (detection rate of 87%). From the present study, gene rearrangement analysis was considered to be worthwhile to strengthen the evidence of neoplastic proliferation of small lymphocytes when coupled with immunophenotyping and to be a suitable diagnostic substitute if immunophenotyping is not available in clinical practice.

Expression of canine Kdap in normal, hyperplastic and neoplastic epidermis.

Keratinocyte differentiation-associated protein, Kdap, is a recently identified small secretory protein that may act as a soluble regulator for the cornification and/or desquamation of keratinocytes. To clarify the role of Kdap in the terminal differentiation of keratinocytes, detailed in situ localisation of Kdap was studied using canine skin with normal, hyperplastic and neoplastic epidermis. In normal canine trunk skin, Kdap was expressed by granular keratinocytes, with polarity to the apical side of the cells, suggesting that canine Kdap is present in lamellar granules, as in humans. Expression of Kdap was widespread in the spinous layers in hyperplastic epidermis, but was undetectable in squamous cell carcinomas. These findings suggest that Kdap is closely related to the delay of terminal differentiation and/or release of cells in hyperplastic epidermis.

Induction of dendritic cell-mediated immune responses against canine malignant melanoma cells.

To establish the basis for the use of dendritic cells (DC) in the treatment of canine melanoma, dogs were vaccinated using autologous DC pulsed with canine melanoma CMM2 cell lysate in the presence of keyhole limpet haemocyanin (KLH) in vitro (CMM2-KLH-DC), and the induction of immune responses against CMM2 cells in vivo was examined using the delayed-type hypersensitivity (DTH) skin test. The DTH responses against CMM2 cells and KLH were observed in dogs vaccinated with CMM2-KLH-DC, while the responses against KLH but not CMM2 cells were detected with DC pulsed with KLH alone (KLH-DC). Recruitment of CD8 and CD4 T cells was detected in the positively responding sites, suggested that vaccination with CMM2-KLH-DC efficiently elicits T cell-mediated immunity against CMM-2 cells in vivo. These findings demonstrate the potential utility of DC-based tumour vaccination in the treatment of canine malignant melanoma.

Comparison of dendritic cell-mediated immune responses among canine malignant cells.

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.

Light-chain multiple myeloma in a cat.

A diagnosis of light-chain multiple myeloma was made in an 11-year-old male American Shorthair cat. The cat showed atypical plasma cell infiltration in the bone marrow, biclonal gammopathy caused by polymerization of myeloma protein (M-protein), and Bence-Jones proteinuria. The M-protein in the serum of the cat was analyzed by using 12% sodium dodeyl sulfate (SDS) polyacrylamide gel electrophoresis with Coomassie brilliant blue staining. An intense band with a size of 27 kDa, the size of the immunoglobulin light chain, was clearly observed, whereas the band corresponding to the immunoglobulin heavy chain (59 kDa) was undetectable. The 27-kDa band was confirmed to be an immunoglobulin light chain by Western blotting by using antibodies for feline immunoglobulin. These data suggested that the neoplastic plasma cells produce light chain only, leading to the diagnosis of light-chain multiple myeloma in the cat.

Blastic natural killer cell leukaemia in a dog--a case report.

A case of canine non-T, non-B lymphoid leukaemia was determined to be of natural killer (NK) cell lineage by detecting specific expression of canine CD56 mRNA by reverse transcriptase polymerase chain reaction analysis. Although NK cells are usually considered to be morphologically large granular lymphocytes, the malignant NK cells in this case were agranular and blast-like, resembling human blastic NK cell leukaemia. The prognosis of human NK cell leukaemia is usually poor. In this case, the dog died 10 days after initial presentation, despite chemotherapy.

Genomic organization of the T-cell receptor gamma gene and PCR detection of its clonal rearrangement in canine T-cell lymphoma/leukemia.

Because the T-cell receptor gamma (TCRgamma) gene is rearranged at an early stage of T-cell development in both TCRalphabeta and TCRgammadelta lineages, it has been preferentially targeted to detect T-cell clonality in human lymphoma/leukemia. We isolated 22 independent cDNA clones encoding canine TCRgamma and the following analysis of nucleotide sequences using the dog genome database revealed that the canine TCRgamma locus contains at least four repertories of variable genes that can be organized into two distinct subgroups and six repertories of joining genes belonging to two distinct subgroups according to the nucleotide sequence similarity. The findings allowed us to design PCR primers that were directed to the conserved or specific nucleotide sequences for each subgroup of variable and joining genes. By using four different combinations of primers, a PCR-based analysis was performed on cell samples collected from T-cell lymphoma/leukemia and B-cell lymphoma cases and hyperplastic and normal lymph nodes. All cell samples from 11 T-cell malignancy cases exhibited clonal amplification by two out of four primer combinations. This finding was considered to be valuable in PCR-based analysis for detecting T-cell clonality in canine lymphoma/leukemia.

Efficient generation of canine bone marrow-derived dendritic cells.

Because of their unsurpassed potency in presenting antigens to naive T cells, dendritic cells are considered to be an important candidate in the development of immunotherapeutic strategies. Despite the high potential of dendritic cell-based immunotherapy, as a so-called dendritic cell vaccination, few clinical approaches using dendritic cell vaccination have been performed in the dog because of very limited information regarding the generation of canine dendritic cells and their functional properties. We therefore established a protocol for the efficient generation of dendritic cells from canine bone marrow cells using recombinant feline granulocyte-macrophage colony-stimulating factor and canine interleukin-4. Dendritic cells were generated efficiently: a yield of 1-9 x 10(6) cells per approximately 0.5 ml of canine bone marrow aspiration was achieved. These dendritic cells showed features shared with mouse and human dendritic cells: dendrite morphology, expression of surface markers MHC class II and CD11c, and up-regulation of molecules related to antigen presentation (MHC class II, B7-1, and B7-2) by activation with lipopolysaccharide. Moreover, the dendritic cells demonstrated phagocytic activity, processing activity of pinocytosed proteins, and activation of allogeneic T cells far more potent than that by macrophages. Our findings suggest that the bone marrow-derived dendritic cells are functional for the capturing and processing of antigens and the initiation of T cell responses.

Identification of a c-kit exon 8 internal tandem duplication in a feline mast cell tumor case and its favorable response to the tyrosine kinase inhibitor imatinib mesylate.

The gain-of-function mutations within c-kit, a protooncogene encoding KIT, induce constitutive ligand-independent kinase activation and are important for the pathogenesis of mast cell proliferative disease in humans as well as in dogs. Despite the clinical importance of feline mast cell tumors, no mutation has been shown within the c-kit gene in cats. In the present report, we analyzed the c-kit nucleotide sequence in the case of a cat that showed systemic mastocytosis and mastocytemia. Within the c-kit cDNA prepared from the malignant mast cells, we identified an 12-bp internal tandem duplication at the region corresponding to exon 8, resulting in a four amino acid insertion between residues Thr418 and His419 within the fifth immunoglobulin-like domain of KIT. The cat underwent therapy with the kinase inhibitor imatinib mesylate (Gleevec) at a dose of 10mg/kg. The tumor masses greatly responded and were undetectable after 5 weeks of treatment. Correspondingly, the number of mast cells in the peripheral blood was markedly reduced. It is, therefore, considered that the internal tandem duplication within the domain contributes to the neoplastic transformation of mast cells in the cat by increasing KIT phosphorylation.

Neuronal expression of keratinocyte-associated transmembrane protein-4, KCT-4, in mouse brain and its up-regulation by neurite outgrowth of Neuro-2a cells.

One group of proteins that regulates neurite outgrowth and maintains neuronal networks is the immunoglobulin superfamily (IgSF). We previously identified a new member of the IgSF, keratinocyte-associated transmembrane protein-4 (KCT-4), by the signal sequence-trap method from primary cultured human keratinocytes. The KCT-4 mRNA has been found to be highly expressed in the adult human brain, although it is also distributed in various tissues. In the present study, to gain insight into the role of KCT-4 in the nervous system, we examined the expression profile and localization of KCT-4 mRNA in mouse brain. We also evaluated changes in KCT-4 mRNA expression in the differentiation of the neuroblastoma cell line Neuro-2a as the in vitro model of neurite outgrowth. KCT-4 mRNA was detected broadly in various regions of the adult mouse brain by RT-PCR. In situ hybridization revealed that it was expressed highly selectively by neurons but not by glial cells. Moreover, expression of KCT-4 mRNA was induced by neurite outgrowth of Neuro-2a. These data suggest that KCT-4 participates in the regulation of neurite outgrowth and maintenance of the neural network in the adult brain.