Ryohei Nishimura

Cellular proliferative and telomerase activity in canine mammary gland tumors.

In canine mammary tumors, we examined the telomerase activity, proliferative activity by proliferative cell nuclear antigen (PCNA) immunohistochemistry, and percentage of apoptotic cells by the deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The relationship between these measures and histopathologic malignancy was also investigated. PCNA index was highest in malignant tumors (adenocarcinoma: 27.0%; malignant mixed tumor: 15.7%), followed by benign tumors (adenoma: 4.4%; benign mixed tumor: 5.3%), hyperplasia (2.1%), and normal mammary gland (0.9%). In adenoma and adenocarcinoma, papillary and solid types showing higher cellularity tended to have higher PCNA indices than did cystic and tubular types. Although the TUNEL index was <1% in all cases, the relationship between this measure and histopathologic diagnosis showed the same tendency as observed in PCNA immunostaining. Telomerase activity was detectable in all adenomas, benign mixed tumors, and adenocarcinomas examined. In contrast, all normal mammary glands, hyperplasias, and malignant mixed tumors were negative for telomerase. Relative telomerase activity (RTA) of adenocarcinoma (56.5) was significantly higher than that of adenoma (27.8) and benign mixed tumor (33.9), and a significant positive correlation (P < 0.001) was noted between RTA and PCNA index. No significant correlations were noted between either PCNA or TUNEL index and clinical features such as metastasis and tumor diameter. PCNA index and telomerase activity may be useful markers for judging malignancy of canine mammary tumors.

A Newly Developed Immunoisolated Bioartificial Pancreas with Cell Sheet Engineering

The term “immunoisolation” refers to the encapsulation of a graft in a selectively permeable membrane. Encapsulation of cellular grafts may provide a way to protect the graft from immune attack without the need for immunosuppressive agents. Although numerous types of artificial materials have been used for encapsulating membranes, their incomplete biocompatibility causes foreign body reaction against the membranes. A new technique has been developed, called cell sheet engineering using temperature-responsive culture dishes, that allows the use of living cells as an immunoisolating membrane in this study. Using this method, the cultured cells can be easily harvested in the shape of a sheet by a simple change of the temperature without the use of proteolytic enzymes. A cell sheet can be created with three-dimensional structure by making multiple cell sheet layers. In this study, a new technique of macroencapsulation (bioartificial organs) has been developed using chondrocyte sheets. Among the various candidate cells, pancreatic islet cells were selected for a bioartificial organ in this study. A chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas (CSI-BAP) was manufactured by means of cell sheet engineering. An auricular cartilage, which is a histologically elastic cartilage from dogs (beagle), was used as a source of immunoisolating membrane. CSI-BAP was made by multilayering the chondrocyte sheets, and the donors islets were located between each sheet. Islets were isolated and prepared from the dog (ALLO-model) and Brown Norway (BN) rat (XENO-model). The CSI-BAP was cultured for 83 days and the cultured medium was collected every 24 h to measure the insulin concentrations. The CSI-BAP was examined histologically using hematoxyhin and eosin (H&E), and azan dye staining. In addition, immunohistochemical staining was performed to demonstrate the insulin production of CSI-BAP. Insulin secretion of CSI-BAP on day 16 was reduced to 21.4% of the insulin secretion level of day 10, which was the start point of measurement. Although a gradual reduction was observed, insulin secretion was maintained for 3 months. The CSI-BAP was capable of secreting insulin to the culture medium during the observation period. Histological evaluations demonstrated the good viability of the islets, and immunohistochemistry showed the positive staining of insulin. This novel technology may be used for other kinds of endocrine cells or hepatocytes, which may become the models for immunoisolated bioartificial organs in the near future.

IgG-Mediated Histamine Release from Canine Mastocytoma-Derived Cells

Background: Recent data suggest that normal tissue mast cells can express functional receptors for IgG under certain conditions. However, little is known about IgG receptor expression and functional consequences in mast cell neoplasms. Methods: In this study, neoplastic mast cells were obtained from a dog with cutaneous mastocytoma (CM-MC) and from a dog with visceral mastocytoma (VI-MC). Both cell populations were characterized morphologically and functionally. Results: Most cells proliferated constantly in suspension without particular supplements. Doubling times of CM-MC and VI-MC were 52.2 and 27.5 h, respectively. Both cell types were sensitive to formalin fixation, did not contain heparin and were tryptase and chymase positive. Electron microscopy showed fine granules with electron-dense content in both cell populations. The total histamine content of CM-MC and VI-MC was 0.25 and 0.10 pg/cell, respectively. Calcium ionophore A23187 and substance P induced dose-dependent histamine release, whereas compound 48/80 had no effect. Most significantly, both cell types, when sensitized with monomeric dog IgG, released histamine upon stimulation by anti-dog IgG. Conclusions: Dog mastocytoma-derived cells may be useful to study the regulation of neoplastic mast cell growth and differentiation, as well as IgG receptor-mediated activation in neoplastic mast cells. Further research is required to clarify the pathophysiological significance of constitutive expression of IgG receptors in neoplastic (canine) mast cells.

Validation of the prognostic value of histopathological grading or c-kit mutation in canine cutaneous mast cell tumours: a retrospective cohort study.

The objective of this retrospective cohort study was to validate the prognostic value of histological grading of canine cutaneous mast cell tumours (MCTs) according to the Patnaik (grades I-III) and Kiupel (low, high) grading systems, and to confirm the prognostic significance of internal tandem duplications (ITDs) within exon 11 of the c-kit gene (ITD-Exon11). The baseline characteristics and outcome data from 47 dogs diagnosed with cutaneous MCTs were collected and reviewed. Tumours were graded according to both grading systems and the nucleotide sequence of c-kit was evaluated. Results were analyzed to evaluate predictive factors for overall survival (OS) and progression-free survival (PFS). Log-rank tests indicated that dogs with Patnaik grade III MCTs had significantly reduced OS and PFS compared to those with either grade I or II tumours. However, no significant difference in OS or PFS was observed between grade I and II tumours. The dogs with Kiupel high-grade MCTs had significantly shorter OS and PFS than dogs with low-grade MCTs. The presence of ITD-Exon11 was significantly associated with shorter PFS. The result of Cox regression analysis showed that the Kiupel grading system for OS and PFS, and lymph node metastasis for OS, independently predicted prognosis. Kappa statistics confirmed a significantly higher inter-observer consistency for the Kiupel compared to the Patnaik grading system. These findings demonstrate that the Kiupel grading system is a useful prognostic tool for canine cutaneous MCTs in predicting OS and PFS, while the occurrence of ITD-Exon11 appeared to be a useful predictor for PFS.

Responses of laryngeal capsaicin-sensitive receptors to volatile anesthetics in anesthetized dogs

The responses of laryngeal capsaicin (CAPS)-sensitive receptors to halothane, enflurane, isoflurane and sevoflurane were evaluated in anesthetized spontaneously breathing dogs from the afferent activity of the internal branch of the superior laryngeal nerve. The CAPS-sensitive receptors were clearly distinguished from irritant receptors by their responsiveness to CAPS and their lack of responsiveness to water. All the CAPS-sensitive receptors were significantly stimulated by all volatile anesthetics in a concentration-related manner, and the activation by halothane, enflurane, and isoflurane was significantly greater than by sevoflurane. In contrast, responses of irritant receptors to the volatile anesthetics were divided into three types (stimulation, inhibition or non-response), and did not differ among anesthetics. In conclusion, the present study demonstrated that the CAPS-sensitive receptors were consistently stimulated by halogenated volatile anesthetics and especially by halothane, enflurane, and isoflurane, and that these responses were dissimilar to the variable responses of irritant receptors.

Comparison of injectable robenacoxib versus meloxicam for peri-operative use in cats: Results of a randomised clinical trial

The objective of this study was to evaluate the efficacy and tolerability of robenacoxib, a selective cyclooxygenase-2 inhibitor, for the treatment of post-operative pain and inflammation in cats. The study was a prospective, multi-centre, randomised, blinded, non-inferiority design clinical study to compare robenacoxib to meloxicam. Ninety-six cats undergoing surgery at eight centres in Japan were allocated randomly to receive a single s.c. injection of robenacoxib (2 mg/kg, n=67) or meloxicam (0.3 mg/kg, n=29) shortly before induction of anaesthesia. Most cats underwent soft tissue surgery (n=87), mainly ovariectomy (n=68). Post-operative pain and inflammation were assessed at 3, 8 and 22 h after recovery from anaesthesia using numerical rating scales. For the primary efficacy endpoint (total clinician score), robenacoxib had significantly better efficacy than meloxicam, the relative efficacy ratio being 1.47 (95% confidence interval 1.19-1.78, P=0.0003). For the secondary efficacy endpoints, robenacoxib was superior to meloxicam when assessed on the basis of posture, behaviour, pain on palpation and overall pain control, while meloxicam was superior with respect to wound heat. No cat in either group required rescue analgesia therapy. In tolerability assessments, pain during injection and pain and inflammation at the injection site 22 h after recovery from anaesthesia were rated significantly less with robenacoxib compared to meloxicam. Both treatments were well tolerated on the basis of clinical observations and blood tests, with no significant differences between groups. In conclusion, single pre-operative administration of robenacoxib was well tolerated and had superior efficacy to meloxicam in reducing post-operative pain in cats.

Liquid chromatography-mass spectrometry for the determination of medetomidine and other anaesthetics in plasma

A liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric method is presented for the simultaneous determination of medetomidine and other anaesthetic drugs in solutions and dog plasma. The drugs examined were flumazenil, butorphanol, atropine, ketamine, xylazine, medetomidine, atipamezole and midazolam. The separation was carried out on a reversed-phase column using methanol-0.1 M ammonium acetate (3:2) as eluent.

Effects of volatile anesthetics on vagal C-fiber activities and their reflexes in anesthetized dogs

Effects of halothane, enflurane, isoflurane, and sevoflurane on vagal capsaicin (CAPS)-sensitive C-fibers were elucidated in anesthetized dogs. The CAPS-sensitive C-fibers were significantly stimulated by all volatile anesthetics with a significantly greater response to halothane than with sevoflurane. A significant increase in respiratory frequency (fR) and a significant decrease in tidal volume (VT) were observed with halothane and isoflurane, and a significant increase in fR was observed with sevoflurane. In contrast, a significant decrease in fR was induced by enflurane. The tachypnea induced by halothane, isoflurane, and sevoflurane was significantly reduced or no longer observed after perineural CAPS-treatment or bilateral vagotomy, whereas the slowing of respiration observed with enflurane was not affected by either of these treatments. These results suggest that vagal C-fibers play an important role in the reflex tachypnea that occurs with halothane, isoflurane, and sevoflurane.

CT characteristics of primary hepatic mass lesions in dogs.

Little information is available on the relationship between computed tomography (CT) imaging findings and the pathologic diagnosis of canine hepatic tumors. Our purpose was to clarify the characteristic features of CT findings in liver tumors in dogs. Data from 33 dogs with either a hepatocellular carcinoma, n = 14, hepatocellular adenoma, n = 14, or nodular hyperplasia, n = 5 were summarized from medical records. CT features for each histologic diagnosis were characterized and analyzed statistically. Common findings in hepatocellular carcinoma included central (79%, P = 0.0030) and marginal enhancement (93%, P = 0.00043) in the arterial phase, cyst-like lesions (93%), capsule formation (93%), and hypoattenuation in the portal (86%), and equilibrium phases (93%). Hepatic adenoma was characterized by a characteristic diffuse enhancement pattern during the arterial phase (57%, P = 0.013), which was also found in nodular hyperplasia (60%), but never in hepatocellular carcinoma. Nodular hyperplasia was less likely to have a capsule structure (20%, P = 0.0087). Mass size was significantly smaller in nodular hyperplasia than in hepatocellular carcinoma and hepatic adenoma (P = 0.0033 and 0.038, respectively). Hyperattenuation in the arterial and the portal phase i.e. contrast retention, was more frequent in hepatic adenoma than in the other groups (P = 0.037 and 0.037, respectively). Nodular hyperplasia was more frequently isoattenuating in the equilibrium phase (P = 0.043).

Identification of c-kit mutations-independent neoplastic cell proliferation of canine mast cells.

Gain-of-function mutations in the proto-oncogene c-kit have been considered the molecular mechanism of neoplastic proliferation of mast cells. However, the importance of c-kit gene mutations is not well evaluated in canine mast cell tumors (MCTs). In the present study, we established and characterized a mast cell line, HRMC, derived from a dog with MCT. We also examined c-kit mutations in HRMC cells and assessed an inhibitory effect of a tyrosine kinase inhibitor, STI571, on HRMC cells. HRMC cells had cytoplasmic metachromatic granules, chymase and tryptase, and expressed both KIT and FcepsilonRI on the cell surface. HRMC cells contained histamine and released beta-hexosaminidase through FcepsilonRI cross-linking and calcium ionophore stimulation. Nucleotide sequence analysis demonstrated no mutations in an open reading frame of c-kit cDNA and genomic DNA of the juxtamembrane domain of c-kit in HRMC cells. STI571 did not show any inhibitory effects on the proliferation of HRMC cells. These findings clearly demonstrated the existence of c-kit mutations-independent neoplastic canine mast cell proliferation. The growth factor-independent mast cell line established in this study might be valuable to explore novel mechanisms of c-kit mutations-independent neoplastic proliferation of mast cells in dogs.