Takuo Shida

Determination of C‐reactive protein in serum and plasma from healthy dogs and dogs with pneumonia by ELISA and slide reversed passive latex agglutination test

The concentrations of C-reactive protein (CRP) in serum from dogs diagnosed as normal by clinical, haematological, and biochemical examination were determined by enzyme-linked immunosorbent assay (ELISA) and slide reversed passive latex agglutination (RPLA), using IgG antibody isolated from rabbit anti-canine CRP serum. The mean value of CRP in 66 normal dogs kept in private households was 8.4 +/- 4.9 micrograms/ml by ELISA and 8.5 +/- 6.3 micrograms/ml by RPLA. Thus, no significant difference was demonstrated between the values obtained by ELISA and RPLA. No significant age and sex-related differences were found in the CRP values. The mean concentration of CRP in 84 6-month-old Beagle dogs kept in kennels by breeders was 6.2 +/- 3.9 micrograms/ml by ELISA and 8.0 +/- 4.0 micrograms/ml by RPLA. Again no significant difference was found. The CRP values determined by ELISA and RPLA were closely correlated (r = 0.913). The serum and plasma concentrations of CRP measured by RPLA were also closely correlated (r = 0.994). This indicates that plasma can be used as well as serum to determine CRP concentrations by RPLA.

Late radiation response of canine mediastinal tissues

The mediastinal tissues which included heart, lung, trachea and esophagus of 70 adult beagle dogs were irradiated to a range of total radiation doses between 24 and 68 Gy given in 2, 3 and 4 Gy fractions. The purpose of the study was the calculation of alpha/beta ratios for morphologic and functional changes of the mediastinal tissues. Functional assays including echocardiography, electrocardiography, right heart hemodynamics and cardiac output were performed. Histomorphometric analyses of all tissues included in the field were done 2 years after treatment. Euthanasia was performed on 7 of 70 dogs prior to 2 years due to congestive heart failure and seven other dogs had signs of heart failure 2 years after treatment. Heart failure was thought to be caused by either pericardial effusions or constrictive pericarditis in these dogs. Heart failure occurred at doses of 62 and 68 Gy given in 2 Gy fractions, 60 Gy given in 3 Gy fractions and 52 Gy given in 4 Gy fractions. The ED50 values for pericardial fibrosis for 2, 3 and 4 Gy fractions were 46.1, 43.9 and 26.6 Gy, respectively. An alpha/beta ratio of 2.5 Gy was calculated by direct quantal response analysis. Small foci of myocytolytic lesions were detected in 11 dogs. Calculated ED50 values for myocytolysis were 70.4 Gy given in 2 Gy fractions and 50.8 Gy given in 4 Gy fractions. The estimated alpha/beta ratio was 3.2 Gy. Heart rates determined from physical examination and frequency of S-T segment changes increased with increasing dose. No other dose related changes were found in any of the other functional parameters. Functional changes were detected in the 14 dogs with clinical signs of heart failure. Focal consolidation and subpleural fibrosis were present in the irradiated lung volume. These late changes had no detectable physiologic effect in these dogs because of the small volume of lung irradiated. The ED50 values for lung consolidation were 54.3, 45.8 and 26.6 Gy after 2, 3 or 4 Gy fractions, respectively. The estimated alpha/beta ratio was 3.4 Gy. No dose-related changes could be detected in the trachea or esophagus at 2 years after treatment. These results demonstrate that lung and pericardium are the most responsive tissues in the mediastinum within the first 2 years after treatment. Myocardial lesions were present with high ED50 values, but were not found to be functionally significant at 2 years after irradiation. Human clinical data indicate that longer observation periods are needed for development of these lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of Cancer Stem Cells Derived From a Canine Lung Adenocarcinoma Cell Line

Accumulating evidence supporting the cancer stem cell (CSC) hypothesis is based on the finding that tumors contain a small population of self-renewing cells that generate differentiated progeny and thereby contribute to tumor heterogeneity. CSCs are reported to exist in several human cancers, yet only a few reports demonstrate the existence of CSCs in primary lung cancer in dogs. In this study, the authors established a cancer cell line derived from a canine primary lung adenocarcinoma and identified a side population (SP) of cells that displayed drug-resistant features. To confirm the characteristics of these SP cells, the authors investigated the tumorigenicity of the cells in vivo by using a nude mouse xenograft model. Only 100 SP cells were able to give rise to new tumors, giving a 10-fold enrichment over the main population (MP) of cells, suggesting that these cells have the cancer-initiating ability of CSCs. Further studies characterizing CSCs in canine lung adenocarcinoma might contribute to the elucidation of the mechanisms of tumorigenesis and to the establishment of novel therapeutic strategies.

Identification of tumor-initiating cells in a canine hepatocellular carcinoma cell line

Tumor-initiating cells (TICs) or cancer stem cells (CSCs), a small subset of tumor cells, are involved in tumor initiation, progression, recurrence and metastasis. In human hepatocellular carcinoma (HCC), TICs are enriched with cell surface markers and have the ability to self-renew and differentiate tumors at a high frequency. We established a canine HCC cell line, HCC930599, and analyzed it for stem and progenitor cell marker expression using flow cytometry. HCC930599 showed high CD44 and CD29, moderate CD90, and low CD133, CD34, CD24, CD117, and CD13 expression. CD90(+)CD44(+) and CD90(-)CD44(+) cells were characterized using the in vitro sphere assay and an in vivo transplant model. CD90(+)CD44(+) cells acquired enhanced self-renewal capacity, proliferative activity and tumourigenicity compared with CD90(-)CD44(+) cells, suggesting that TICs exist in the HCC930599 cell line and that CD90 is a marker for enriched TICs. Understanding TIC characteristics may help elucidate hepatic carcinogenesis and HCC therapy development.

Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.

IMMUNOLOGICAL DETERMINATION OF FAECAL HAEMOGLOBIN CONCENTRATIONS IN DOGS

Faecal haemoglobin (Hb) concentrations in apparently healthy experimental Beagle dogs and in dogs of various breeds kept in private households or at breeders were measured by reversed passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA) in an effort to define the physiological concentrations of faecal Hb in the dog. In 88% (53) of 60 experimental Beagle dogs (30 males and 30 females), the RPLA titres were 1:2 and 1:8 and the faecal Hb concentrations ranged from 40.0 to 431.5 (mean 184.1±92.6) μg/g faeces by ELISA. No significant difference was found in Hb levels or RPLA titres between males and females. Seven dogs (12%) had significantly greater RPLA titres and Hb concentrations by ELISA than the remaining dogs. In 84% (45) of the 53 dogs kept in private households or at breeders, the RPLA titres were >1:1 to 1:8 and the faecal Hb concentrations ranged from 7.1 to 456.7 (mean 137.5±128.7) μg/g faeces in ELISA. Eight of these dogs (15.1% of 53 dogs) had significantly greater RPLA titres and Hb concentrations by ELISA than the remaining dogs. There were no significant differences between the Beagles and dogs kept in private households or at breeders. In conclusion, in 98 (86.7% of 113) dogs the physiological concentrations of RPLA titres were >1:1 to 1:8 and the faecal Hb concentrations were 143.5–185.1 μg/g (95% confidence level). Approximately 13.3% of apparently healthy dogs had higher faecal Hb concentrations, suggesting the presence of subclinical haemorrhages. Four dogs suffering from colorectal cancer also had high faecal Hb concentrations.

A NOVEL SUPPORT DEVICE FOR HEAD IMMOBILIZATION DURING RADIATION THERAPY THAT IS APPLICABLE TO BOTH CATS AND DOGS.

Repeatable head immobilization is important for minimizing positioning error during radiation therapy for veterinary patients with head neoplasms. The purpose of this retrospective cross-sectional study was to describe a novel technique for head immobilization (Device II) and compare this technique with a previously described technique (Device I). Device II provided additional support by incorporating three teeth (vs. two teeth with Device I). Between 2011 and 2013, both devices were applied in clinically affected cats (Device I, n = 17; Device II, n = 11) and dogs (Device I, n = 85; Device II, n = 22) of various breeds and sizes. The following data were recorded for each included patient: variability in the angle of the skull (roll, yaw, and pitch), coordinates of the isocenter, and distance from the reference mark to the tumor. Devices I and II differed for skull angle variability during the treatment of dogs (roll, P = 0.0007; yaw, P = 0.0018; pitch, P = 0.0384) and for yaw of during the treatment of cats (P < 0.0001). In each case, Device II was superior to Device I. The distance from the reference mark to the center of the tumor was significantly decreased for Device II vs. Device I (dogs, P < 0.0001; cats, P = 0.0002). Device II also provided more accurate coordinates for the isocenter. Authors recommend the use of, Device II for future clinical patients.

Lipomatosis of the canine parotid gland: case report with a literature review.

In this report, we describe a case of lipomatosis in the left parotid gland of an eight-year-old female Shetland sheepdog and review the relevant literature. Preoperative diagnosis of lipomatosis with Tru-Cut biopsy presented difficulties in this case. The postoperative diagnosis was based on the gross appearance of a non-infiltrative, circumscribed swollen mass in the parotid gland and the histological appearance of normal adipocytes that infiltrated into the parotid gland without involving surrounding structures. Additionally, flotation of the whole parotid gland in formalin solution together with well-maintained residual lobulation and a well-maintained configuration on the cut surface were identified as subsidiary characteristics of lipomatosis of the parotid gland. Lipomatosis of the parotid gland has been rarely reported in dogs and is a poorly understood condition.

Primary Lung Carcinoma with Paraneoplastic Leukocytosis in a Dog

In human lung cancers, marked leukocytosis, especially with neutrophilia and monocytosis, has been reported as a paraneoplastic syndrome. In dogs, several reports have been published on paraneoplastic leukocytosis. In this case, various examinations were conducted on a dog with extreme neutrophilic leukocytosis diagnosed via a blood test at first admission. A huge pulmonary mass was detected, which was surgically removed. The tumor was diagnosed as papillary adenocarcinoma of the lung by histopathology. The number of neutrophils recovered to normal after surgery, and the dog was diagnosed as paraneoplastic syndrome due to lung cancer. Key word:dog, leukocytosis, lung cancer, paraneoplastic syndrome 1)Sato Animal Hospital, 1-14 Miyashitachou, Ichinoseki, Iwate 021-0013, Japan 2)School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan 3)Veterinary Teaching Hospital, Azabu University, 1-17-71 Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan 4)Yamada Animal Hospital, 2-1, Koorikitanomachi, Neyagawa, Osaka 572-0080, Japan Corresponding author:Takuo Shida Telephone:042-754-7111 Facsimile:042-769-2418 Email address:shida@azabu-u.ac.jp Received:May 27, 2012 / Accepted:September 12, 2012 Note J Jpn Vet Cancer Soc, 3(2) −21− were all within normal ranges. According to these results, we surmised that the elevated BUN level was prerenal secondary to cardiac insufficiency. Based on these clinical data, the cause of the cough and the neutrophilia were considered to possibly be related to the thoracic mass. In order to improve the cough, thoracotomy was performed. The patient underwent surgery through the intercostal approach on the 15th day after first admission. The 5th rib of the patient was removed due to the large size of the mass. The mass involved the left caudal lung lobe and adhered to the pleura of the mediastinum and diaphragm. The pericardium was not involved in the mass. Blunt dissection of the mass from the pleura of the mediastinum caused some bleeding. After blunt dissection from the adhered sites, the left lung lobe was totally removed with the mass. Swelling of the tracheobronchial lymph nodes was not observed. The patient recovered from surgery without any events and showed good healthy conditions. The WBC count decreased to the normal range 2 weeks after surgery(Table 3)and was normal for 21 weeks after surgery. The patient’s cough disappeared after surgery, and the respiratory function of the patient was recovered. Furthermore, cardiac function was maintained well with the simultaneous administration of digoxin and an angiotensin converting enzyme inhibitor for cardiac insufficiency. However, cardiac function worsened, and 4 months after surgery, the patient had an increased BUN level. The patient showed collapse/syncope due to cardiac failure and died 5 months after surgery. Autopsy was not permitted by the client. Histopathological examination of the pulmonary mass revealed that the tissue consisted of multiple neoplastic foci divided by various amounts of fibrous septa. The neoplastic foci were characterized by papillary or glandular structures that were lined by cuboid to cylindroid epithelial cells. Some tumor cells showed a goblet-cell appearance, while some had cilia on their cell surface. These proliferating neoplastic cells showed moderate cellular atypia, and the mitotic figure index was 2-3 per high-power magnification(×400)field. The tumor cells invaded the lymph vessels of the surrounding tissues. Multifocal necrotic areas were also observed within the neoplastic foci. In the interstitial areas, there were moderate infiltrations of lymphocytes and macrophages, deposition of calcium, and occasional ossification. The pleurae were Fig. 1.Thoracic radiography(ventrodorsal view)revealed a large mass in the left caudal lung area that compressed and shifted the enlarged heart to the right. Fig. 2.Numerous neutrophils were observed in the blood smear specimen (×100).