Evaluation of cytokine messenger RNA expression in peripheral blood mononuclear cells from dogs with canine demodicosis

Using RT-PCR and semi-quantitative PCR, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta in peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without demodicosis. mRNA expression for IFN-gamma as well as TNF-alpha in dogs with demodicosis (localized (LD) and generalized (GD)) was slightly lower than those in dogs without demodicosis (healthy controls). Expression of IL-5 mRNA in dogs with demodicosis was higher than that in control dogs, but there were no significant differences in IL-4 and IL-10 mRNA expression levels among the three groups. On the other hand, expression levels of TGF-beta mRNA in dogs with GD were higher than those in control dogs and dogs with LD. The expression levels of IL-5 and TGF-beta mRNA decreased in all three dogs with GD which showed resolution of the clinical signs. Taken together, these results suggest that the Th2-like response in PBMCs from dogs with demodicosis is up-regulated, and that subsequent increased expression of IL-5 and TGF-beta mRNA in dogs with GD is reversible after treatment. Therefore, these cytokines, particularly IL-5, might be a useful clinical index of the clinical course in demodicosis. Also, increased TGF-beta mRNA expression might be a key factor for revealing the difference in the mechanism of onset between LD and GD.     

A comparative study of the effects of glycerol and hydroxyethyl starch in canine red blood cell cryopreservation

Hydroxyethyl starch (HES) is a nonpenetrating extracellular cryoprotectant. In contrast to glycerol, it does not require labor-intensive removal from thawed red blood cells (RBCs) prior to transfusion. In this study, we compared glycerol and HES, and assessed HES as a substitute for glycerol in cryopreserved canine RBCs. The RBCs were preserved for 2 months in liquid nitrogen using a 20% (w/v) glycerol solution, and variable concentrations of HES solution. We evaluated the two cryoprotectants by the percentage of post-thaw hemolysis from the total free hemoglobin, saline stability, osmotic fragility, and by observing the erythrocyte morphology using a scanning electron microscope after thawing. The optimal concentration of HES was 12.5% (w/v) for the cryopreservation of canine RBCs. The thaw hemolysis, saline stability, and osmotic fragility index were 25.6 +/- 4.7%, 87.8 +/- 6.9%, and 0.445 +/- 0.024% NaCl respectively. These parameters resemble the results of RBCs frozen in a 20% (w/v) glycerol solution, which are 24.7 +/- 5.2%, 99.2 +/- 0.1%, and 0.485 +/- 0.023% NaCl respectively. From a morphological point of view, 12.5% (w/v) HES showed the best cryoprotection of RBCs compared to the other concentrations of HES. These results suggest that HES could be a possible substitute for glycerol for the cryopreservation of canine RBCs.     

Sesquiterpenes from the aerial part of Chloranthus japonicus and their cytotoxicities

Four new sesquiterpenes, chlorajapolides F–I (1–4), along with nine known terpenoids (5–13) were isolated from the aerial part of Chloranthus japonicus. Their structures were elucidated on the basis of spectroscopic analysis, and a lindenane sesquiterpene, named 9-hydroxy-heterogorgiolide, previously isolated from the C. japonicus, was revised as its 8-epimer (1a). Moreover, methanol extract (ME), EtOAc fraction (EF), water fraction (WF), and all isolates (1a, 1–13) were evaluated for their cytotoxicities using two human cancer cell lines.     

Detection of the anti-P53 antibodies in dogs with tumors

To detect the anti-P53 antibodies of dogs with tumors, a GST-recombinant canine (rc) P53 fusion protein was expressed and purified. Immunoblot analysis was performed using this GST-rcP53 fusion protein as an antigen and serum samples from dogs suffering from tumors as primary antibodies. Out of 16 serum samples obtained from various tumor cases, four samples showed reaction with GST-rcP53. In contrast, serum from other 12 dogs with tumors, four dogs with non-neoplastic diseases and two control healthy dogs (as controls) did not show any reaction with GST-rcP53 in immunoblotting. The p53 gene mutation and the P53 protein expression were examined, using the tumor tissues to explore the relationship between the existence of the GST-rcP53 bands, gene mutations of p53 and the accumulation of P53 protein. One case, which showed a clear GST-rcP53 band, had a point mutation of the p53 cDNA and showed nuclear accumulation of P53 protein. These results suggest that the anti-P53 antibodies are also produced in tumor dogs with p53 gene mutations.     

Application of ventriculoperitoneal shunt as a treatment for hydrocephalus in a dog with syringomyelia and Chiari I malformation

A twenty-month-old Chihuahua male dog was presented to us suffering with ataxia. Based on the physical examination, X-ray and magnetic resonance imaging (MRI) examinations, we diagnosed the dog with hydrocephalus, Chiari I malformation and syringomyelia. Treatment consisted of internal medical treatment and the placement of a ventriculoperitoneal (VP) shunt. The ventricular dilatation was relieved and the dog improved neurologically; however, the Chiari I malformation and syringomyelia remained after surgically positioning the VP shunt.     

Expression of a tumor-associated antigen, RCAS1, in canine mammary tumors

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), one of novel cancer cell-surface antigens, is strongly expressed in invasive cancers. RCAS1 inhibits the in vitro growth of lymphocytes such as T cells and natural killer (NK) cells, and induces apoptotic cell death. We investigated the expression of RCAS1 in canine mammary tumor cell lines and tumor cells by immunohistochemistry, and also in situ deoxyribonucleic acid (DNA) fragmentation in tumor-infiltrating lymphocytes (TILs) by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. All canine mammary tumor cell lines expressed RCAS1 at both the messenger ribonucleic acid (mRNA) and protein level. Immunohistochemically, RCAS1 was negative in 100% of normal mammary glands, but was expressed in 100% of malignant tumors examined. In most malignant mammary tumors, RCAS1 was localized in the cytoplasm with no polarity of expression. In benign mammary tumors, it was detected on the luminal surface of the tumor cell. RCAS1 expression or localization was significantly correlated with malignancy. In situ DNA fragmentation of CD3-positive TILs was observed in RCAS1-expressing tumors. RCAS1-expressing tumors, indicating a possible induction of apoptotic cell death in TILs through RCAS1 expression. These observations suggest that RCAS1 probably plays an important role in tumor progression and escape from immune surveillance in canine mammary tumors.     

Clinical features and their course of pituitary carcinoma with distant metastasis in a dog

An 11-year-old male toy poodle with neurological symptoms was diagnosed with a macroscopic pituitary tumor, which produced adrenocorticotropic hormone. Radiation therapy with a linear accelerator was performed for the pituitary tumor, and resulted in good local tumor control. However, serum endogenous adrenocorticotropic hormone concentrations were uncontrollable even after the tumor disappeared. Abdominal computed tomography revealed splenic masses, and splenectomy was performed. Histopathological examination of the surgical specimen showed tumor cells with eosinophilic and finely granular cytoplasm suggestive of endocrine origin. Since these cells were positive for adrenocorticotropic hormone, the case was diagnosed as a pituitary carcinoma with distant metastasis. Necropsy revealed multiple metastases to the abdominal organs. This is the first case report describing canine pituitary carcinoma with distant metastasis.     

TRANSORBITAL ECHOENCEPHALOGRAPHY IN CATTLE

The purpose of this study was establishment of the criteria of transorbital echoencephalography in cattle and experimental applications to bovine practice. Quantitative investigations using magnetic resonance (MR) imaging revealed that this examination could be applied to cattle under 3 months of age. The method of transducer positioning was established in Japanese Black (J.B.) and filial (F1) cattle (turning caudally at an angle of about 16 degrees and dorsally at an angle of about 23 degrees) or in Holstein cattle (turning caudally at an angle of about 20 degrees and dorsally at an angle of about 21 degrees). Examinations in clinical calves revealed that the cerebral parenchyma and the lateral ventricle could be detected antemortem or postmortem. In this study, the diagnoses of hydrocephalus or hydranencephaly was possible using antemortem transorbital echoencephalography. Transorbital echoencephalography was especially useful as the imaging method for bovine hydranencephaly.     

In-vitro anti-proliferative effects of some anti-tumour drugs on feline mammary tumour cell lines

Six anti-tumour drugs namely; doxorubicin, mitoxantrone, vincristine, cisplatin, recombinant human tumour necrosis factor alpha (rh-TNFalpha) and recombinant feline interferon gamma (rf-IFNgamma) were singly evaluated for their anti-proliferative effects on two feline cell lines (FRM and NAC) derived from mammary adenocarcinoma and grown as monolayers. We obtained concentration response curves that enabled the determination of the concentration inhibiting growth by 50 per cent (IC50) for the chemotherapeutic agents with VCR exhibiting exponential-plateau curves. Differences in anti-proliferative effects of drugs to a given cell line and between the cell lines were also observed. NAC cells were relatively more resistant compared with FRM cells. The relative resistances for NAC cells were 4.19, 12.96, 0.05 and 2.10-fold to doxorubicin, mitoxantrone, vincristine and cisplatin, respectively. FRM cells were more resistant to VCR at lower concentrations compared with NAC cells. The cells appeared, at least in vitro, least sensitive to rh-TNFalpha and rf-IFNgamma. rh-TNFalpha and rf-IFNgamma were 23 and 29 per cent inhibitory to FRM cells and only 13 and 15 per cent inhibitory to NAC cells, respectively.     

Effect of drug-eluting bead transarterial chemoembolization loaded with cisplatin on normal dogs

Transcatheter arterial embolization (TAE) and transcatheter arterial chemoembolization (TACE) are standard treatments for advanced hepatocellular carcinoma (HCC) and particularly for unresectable tumors or liver metastases in humans. However, reports on TACE used in veterinary medicine are few. This study aimed to evaluate the feasibility and safety of drug-eluting bead transarterial chemoembolization (DEB-TACE). We performed DEB-TACE in four clinically normal dogs and pharmacokinetically compared the results against hepatic arterial infusion (HAI) of cisplatin in two dogs. Drug-eluting beads (DEB) loaded with cisplatin were injected through a microcatheter for selective embolization of the left hepatic artery. After embolization, computed tomography (CT) images and histological examination findings were obtained during a 4-week observation period. Serum platinum concentrations were measured to evaluate cisplatin after each procedure. Biochemical analysis was performed during a 12-week observation period. Embolization was successful in all dogs, and there were no clinically apparent abnormalities. Embolization was confirmed up to 4 weeks after DEB-TACE in two of the four dogs and up to 1 week in the other two dogs using postoperative CT images. Cisplatin was not detected in peripheral veins in all dogs after DEB-TACE, but it was detected in trace amounts after HAI. DEB-TACE using cisplatin was safe and well tolerated by normal dogs. DEB-TACE may be useful in terms of determining systemic toxicity and drug concentration within tumors.