Japan Animal Referral Medical Center

Kawasaki, Japan

Japan Animal Referral Medical Center

Kawasaki, Japan
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PubMed | Japan Animal Referral Medical Center, Bin Animal Clinic, University of Tokyo and Iwakuni Health and Welfare Center
Type: Journal Article | Journal: Veterinary pathology | Year: 2015

The authors herein describe the morphologic and immunohistochemical features of normal Merkel cells as well as the clinicopathologic findings of Merkel cell carcinoma in cats. Merkel cells were characterized as vacuolated clear cells and were individually located in the epidermal basal layer of all regions examined. Clusters of Merkel cells were often observed adjacent to the sinus hair of the face and carpus. Immunohistochemically, Merkel cells were positive for cytokeratin (CK) 20, CK18, p63, neuron-specific enolase, synaptophysin, and protein gene product 9.5. Merkel cell carcinoma was detected as a solitary cutaneous mass in 3 aged cats (13 to 16 years old). On cytology, large lymphocyte-like cells were observed in all cases. Histologic examinations of surgically resected tumors revealed nests of round cells separated by various amounts of a fibrous stroma. Tumor cells were commonly immunopositive for CK20, CK18, p63, neuron-specific enolase, and synaptophysin, representing the characteristics of normal Merkel cells.


Kitahara G.,University of Miyazaki | Nambo Y.,Hidaka Training and Research Center | El-Sheikh Ali H.,University of Miyazaki | El-Sheikh Ali H.,Mansoura University | And 4 more authors.
Journal of Reproduction and Development | Year: 2012

This study was carried out to evaluate the blood profile and tissue expression of Anti-Müllerian hormone (AMH) as a biomarker for granulosa-theca cell tumors (GTCTs) in cattle. Five cases with unilateral ovarian GTCTs (GTCT group) were investigated in comparison to other groups of Japanese Black cows, which had either cystic ovarian disease (COD group, n=5), a functional corpus luteum on Days 9 to 11 of the estrous cycle (Day 0=estrus; CL group, n=13) or received superovulation treatment (SOT group, n=13). We used transrectal ultrasonography and measured plasma AMH, estradiol-17β (E 2), progesterone (P 4) and testosterone (T) levels. Moreover, GTCT tissues were collected and examined by immunohistochemical staining (IHC) for AMH. In the GTCT group, ultrasound images of GTCTs were variable and not definitive. However, the AMH level in the GTCT group (n=3, 58.1 ± 66.3 ng/ml) was significantly higher than in the COD, CL and SOT groups (0.1 ± 0.1 ng/ml for GTCT vs. COD, P<0.05; 0.2 ± 0.1 and 0.3 ± 0.2 ng/ml, respectively for GTCT vs. CL and SOT, P<0.01). The other hormonal levels in the GTCT group had no significant differences compared with the COD or SOT group. Neoplastic granulosa cells labeled with AMH antibody clearly demonstrated a variety of tissue patterns in all cases by IHC. To the best of our knowledge, this is the first study to investigate the blood profile and IHC of AMH in bovine GTCTs. Our findings indicate that AMH may be a novel biomarker to diagnose GTCTs in cattle. © 2012 by the Society for Reproduction and Development.


PubMed | University of Miyazaki and Japan Animal Referral Medical Center
Type: Case Reports | Journal: Reproduction in domestic animals = Zuchthygiene | Year: 2015

A 14-month-old Japanese Black heifer was evaluated on Day 0 (D 0) for enlargement of the right ovary (RO). Transrectal ultrasonography (TRUS) revealed that the RO was markedly enlarged and multicystic, while the left ovary (LO) was small and inactive. The presumptive diagnosis was granulosa-theca cell tumour (GTCT), which was confirmed by markedly elevated plasma anti-Mllerian hormone (AMH) of 4.42ng/ml. Therefore, ovariectomy of the RO was the treatment of choice. The heifer was checked by TRUS and blood sampling on D 42, D 63 and immediately before ovariectomy on D 85. On D 42, TRUS did not show marked changes in either ovary in comparison with D 0. However, on D 63, the RO had transformed into a single cyst, and on D 85, the LO had resumed cyclic activity. The RO was extracted on D 85 by hand-assisted laparoscopic ovariectomy to allow better control. Unexpectedly, histopathology revealed the lesion to be a fluid-filled cystic structure, with no neoplastic proliferation of follicular epithelium that would indicate GTCT. The wall of the cystic structure consisted of collagen fibres and a few degenerated granulosa cells. The retrospective hormonal analysis revealed that the AMH concentrations had markedly dropped on D 63 and 85, which coincided with resumption of cyclicity in the LO. These findings suggest that the GTCT had self-cured and transformed into a cyst-like structure. The heifer then received an oestrous synchronization regime on D 105, was artificially inseminated on D 115 and became pregnant.


Ito K.,Nippon Veterinary and Life Science University | Kobayashi M.,Nippon Veterinary and Life Science University | Kuroki S.,Nippon Veterinary and Life Science University | Sasaki Y.,Nippon Veterinary and Life Science University | And 12 more authors.
Veterinary Journal | Year: 2013

Canine malignant melanomas are highly aggressive and fatal neoplasms. In the present report, 21 drugs that target specific signalling pathways were screened for their growth inhibitory activity on three canine malignant melanoma cell lines. The proteasome inhibitor bortezomib inhibited the growth of these cell lines. The growth inhibitory properties of bortezomib were then examined using nine canine malignant melanoma cell lines. Bortezomib demonstrated potent growth inhibitory activity in all cell lines with calculated IC50 values of 3.5-5.6. nM.Because suppression of the NF-κB pathway by preventing proteasomic degradation of IκB is an important mechanism of the anti-tumour activity of bortezomib, the activation status of and the effect of bortezomib on the NF-κB pathway were examined using a canine malignant melanoma cell line, CMM-1. The NF-κB pathway was constitutively activated in CMM-1 cells and bortezomib efficiently suppressed this activated pathway. Using a CMM-1 xenograft mouse model, bortezomib also significantly inhibited tumour growth via suppression of tumour cell proliferation.Collectively, these findings suggest that bortezomib has growth inhibitory activity against canine malignant melanomas potentially through suppression of the constitutively activated NF-κB pathway. Targeted therapy using bortezomib could therefore be beneficial in the management of canine malignant melanomas. © 2013 Elsevier Ltd.


Kobayashi M.,Nippon Veterinary and Life Science University | Kuroki S.,Nippon Veterinary and Life Science University | Tanaka Y.,Nippon Veterinary and Life Science University | Moriya Y.,Nippon Veterinary and Life Science University | And 7 more authors.
European Journal of Haematology | Year: 2015

Although imatinib has therapeutic activity for certain subsets of patients with mastocytosis, it is not always curative. Here, molecular mechanisms that confer imatinib resistance to neoplastic mast cells were investigated using an imatinib-sensitive canine neoplastic mast cell line VI-MC carrying a KIT c.1523A>T activating mutation. Two imatinib-resistant sublines were established by culturing VI-MC cells in increasing concentrations of imatinib (1 μM resistant, rVI-MC1; 10 μM resistant, rVI-MC10). Both sublines had a second KIT mutation c.2443G>C. Recombinant KIT with the second mutation was insensitive to 1 μM but sensitive to 10 μM imatinib. The effect of imatinib on the phosphorylation of KIT and its downstream signalling proteins was then examined using these sublines. KIT and ERK were constitutively phosphorylated in both sublines, and their phosphorylation was suppressed by 10 μM imatinib in rVI-MC1 cells. However, KIT but not ERK phosphorylation was suppressed in rVI-MC10 cells. The phosphorylation of ERK in rVI-MC10 cells was also not diminished by the Src family kinase (SFK) inhibitor dasatinib. This second mutation in KIT may play an important role in imatinib resistance in neoplastic mast cells. Furthermore, KIT/SFK-independent activation of ERK would be involved in imatinib resistance when the neoplastic cells are exposed to higher concentrations of imatinib. © 2015 John Wiley & Sons A/S.


Ito K.,Nippon Veterinary and Life Science University | Kuroki S.,Nippon Veterinary and Life Science University | Kobayashi M.,Nippon Veterinary and Life Science University | Ono K.,Nippon Veterinary and Life Science University | And 3 more authors.
Veterinary Journal | Year: 2013

Canine histiocytic sarcoma (HS) is an aggressive and fatal neoplasm that has a high recurrence rate and metastatic nature. In the present report, compounds were screened for their growth inhibitory activity in two HS cell lines using a chemical library known to target specific signalling pathways. Among 171 compounds screened, dasatinib, which targets several types of kinases, clearly inhibited cell growth in one of the two HS lines. The growth inhibitory properties of dasatinib were then examined using six HS cell lines and MDCK cells. Dasatinib demonstrated potent growth inhibitory activity against four HS cell lines with calculated IC50 values of 5.4-54.5. nM, while the IC50 values in the other cell lines were in the micromolar range. In conclusion, a kinase enzyme targeted by dasatinib appears to be crucial for growth in some subsets of HS and the on-target activity of dasatinib could underlie the marked growth inhibition in HS cells. © 2012 Elsevier Ltd.


Kobayashi M.,Nippon Veterinary and Life Science University | Sugisaki O.,Nippon Veterinary and Life Science University | Ishii N.,Nippon Veterinary and Life Science University | Yamada O.,Nippon Veterinary and Life Science University | And 7 more authors.
Veterinary Journal | Year: 2012

A canine intestinal mast cell tumor with splenic metastasis was treated with imatinib. The intestinal and metastatic tumor masses markedly decreased following treatment although the clinical response was short lasting. A c-kit internal tandem duplication mutation, c.1250_1261dup, which causes an insertion of four amino acids in KIT, was identified in cDNA isolated from the tumor cells. The phosphorylation status of the mutant KIT and the effect of imatinib on its phosphorylation were examined using 293 cells transfected with c-kit carrying the c.1250_1261dup mutation. This mutation caused ligand-independent phosphorylation of KIT, which was suppressed by imatinib. Inhibition of constitutively activated mutant KIT with imatinib could underlie the tumor response in this dog. © 2011 Elsevier Ltd.


Hirose N.,University of Tokyo | Uchida K.,University of Tokyo | Matsunaga S.,Japan Animal Referral Medical Center | Chambers J.K.,University of Tokyo | Nakayama H.,University of Tokyo
Journal of Veterinary Medical Science | Year: 2015

C horoid plexus tumor (CPT) is a primary intracranial neoplasm of the choroid plexus epithelium in the central nervous system. In the current World Health Organization classification, CPT is classified into two categories; choroid plexus papilloma (CPP) and carcinoma (CPC). In the present study, we investigated immunohistochemical expressions of N-cadherin, E-cadherin and β-catenin in 5 canine CPT cases (1 disseminated CPC, 2 CPCs and 2 CPPs). One CPP case was positive for N-cadherin and β-catenin, but negative for E-cadherin. The disseminated CPC case was positive for E-cadherin and β-catenin, but negative for N-cadherin. The other cases were positive for the three molecules examined. These results suggest that loss of the N-cadherin expression might associate with the spreading of CPC cells. ©2015 The Japanese Society of Veterinary Science.


PubMed | Japan Animal Referral Medical Center
Type: Journal Article | Journal: Microbiology and immunology | Year: 2010

Recent data suggest a decreased prevalence of IFN-gamma-producing T lymphocytes (Type 1 T cells) in tumor-bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti-tumor responses.


PubMed | Japan Animal Referral Medical Center and University of Tokyo
Type: Journal Article | Journal: Veterinary pathology | Year: 2015

Granular cell tumors (GCTs) are histologically characterized by polygonal neoplastic cells with abundant eosinophilic cytoplasmic granules. In humans, these cells are considered to be derived from Schwann cells, and the cytoplasmic granules are assumed to be autophagosomes or autophagolysosomes. However, the origin and nature of the cytoplasmic granules in canine GCTs have not been well characterized. The present study examined 9 canine lingual GCTs using immunohistochemistry, transmission electron microscopy (TEM), and cell culture and xenotransplantation experiments. In some cases, the tumor cells expressed S100, CD133, and desmin. The cytoplasmic granules were positive for LC3, p62, NBR1, and ubiquitin. TEM revealed autophagosome-like structures in the cytoplasm of the granule-containing cells. The cultured GCT cells were round to spindle shaped and expressed S100, nestin, Melan-A, CD133, LC3, p62, NBR1, and ubiquitin, suggesting that they were of neural crest origin, redifferentiated into melanocytes, and exhibited upregulated autophagy. The xenotransplanted tumors consisted of spindle to polygonal cells. Only a few cells contained cytoplasmic granules, and some had melanin pigments in their cytoplasm. The xenotransplanted cells expressed S100, nestin, Melan-A, and CD133. P62 and ubiquitin were detected, regardless of the presence or absence of cytoplasmic granules, while LC3 and NBR1 were detected only in the neoplastic cells containing cytoplasmic granules. These findings suggest that some xenotransplanted cells redifferentiated into melanocytes and that autophagy was upregulated in the cytoplasmic granule-containing cells. In conclusion, canine lingual GCTs originate from the neural crest and develop cytoplasmic granules via autophagy. In addition, the microenvironment of GCT cells affects their morphology.

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