Characterization of carbon nanotube (MWCNT) containing P(3HB)/bioactive glass composites for tissue engineering applications.

Poly(3-hydroxybutyrate) (P(3HB)) composites with bioactive glass particles and multiwall carbon nanotubes (MWCNTs) were prepared and used to identify whether the electrical properties of MWCNTs can be used to detect the bioactivity of P(3HB)/bioactive glass composites. The presence of MWCNTs (2-7 wt.%) increased the surface roughness of the composites. The presence of MWCNTs in low quantity enhanced MG-63 osteoblast-like cell attachment and proliferation compared to composites with higher concentration of MWCNTs. Current-voltage measurements demonstrated that the electrical resistance of the composites containing bioactive glass particles decreased over a 45-day immersion period in SBF, whereas composites without bioactive glass showed no significant change over the same period.

Late onset cerebellar cortical degeneration in a koala.

A 10-year-old male koala started to fall from the tree while sleeping. Subsequently, the koala often fell down while walking and showed a gait abnormality, abnormal nystagmus and hypersalivation. At 12 years of age, the koala became ataxic and seemed blind. At 13 years of age, the koala exhibited signs of dysstasia and was euthanased. Necropsy revealed marked symmetrical atrophy of the cerebellum. Histopathologically, a severe loss of Purkinje and granule cells was evident in the cerebellum, while the molecular layer was more cellular than normal with cells resembling small neurons, which were positively stained with parvalbumin immunohistochemistry. Reactive Bergmann glial cells (astrocytes) were present adjacent to the depleted Purkinje cell zone. The very late onset and slow progression of the cerebellar cortical degeneration in this case is particularly interesting and appears to be the first report in the koala.

Effect of bovine lactoferrin on the immune responses of captive bottlenosed dolphins (Tursiops truncatus) being transported over long distances.

Bovine lactoferrin was administered orally, in feed, to six bottlenosed dolphins (Tursiops truncatus) before they were transported for approximately six hours; their stress responses were compared with those of five untreated dolphins. During the journey the dolphins had an increased plasma concentration of cortisol, and lymphopenia, eosinopenia and mild neutrophilia, indicating a stress response. The administration of lactoferrin did not affect the function of the dolphins' polymorphonuclear leucocytes, but affected their leucogram by maintaining the number of circulating eosinophils.

Use of an incontinent end-on colostomy in a dog with annular rectal adenocarcinoma.

An 11-year-old, mixed-breed dog with dyschezia, tenesmus and haematochezia was presented. A rectal stricture caused by an adenocarcinoma was diagnosed. Following the failure of a rectal 'pull-through' procedure, which dehisced seven days later, an incontinent end-on colostomy was performed, allowing amputation of the colorectum with the anus and perineal skin. A two-piece device consisting of a flange and a drainable pouch was used for postoperative faecal evacuation and collection. Mild peristomal dermatitis was the only complication. Patient management was easily carried out by the owner at home, and the dog survived for four months with a satisfactory quality of life. Incontinent end-on colostomy may prove to be a useful treatment for canine annular colorectal tumours.

Necrotising encephalitis in the Yorkshire terrier: a case report and literature review.

A four-year-old, spayed female Yorkshire terrier was presented with a two-month history of lameness in the left forelimb, circling and falling. A magnetic resonance imaging (MRI) examination 11 days after presentation revealed dilation of the right lateral ventricle. Following euthanasia, which was performed about 10 months after the onset of clinical signs, there was gross evidence of degeneration and cavitation of the cerebrum and dilation of the lateral ventricle on the right side. Microscopically, cavitation and necrosis were observed in the white and grey matter of the right cerebrum and there was abundant gemistocytic and fibrillary astrocytosis. Haemorrhage and marked perivascular cuffing with mononuclear cells were found in the mesencephalon. Inflammatory lesions consisting of lymphocytic infiltration and glial proliferation were also present in the dorsal funiculus of the cervical spinal cord. This case was diagnosed as necrotising encephalitis in the Yorkshire terrier (NEYT) with involvement of the spinal cord. NEYT is a chronic progressive neurological disorder, resulting from widespread, destructive non-suppurative inflammation of the central nervous system of unknown cause. In the past decade, 12 cases have been documented in adult to aged Yorkshire terriers. Computed tomography and MRI can detect the characteristic multifocal cavitations and ventriculomegaly, facilitating premortem diagnosis.

Expression patterns of the erbB subfamily mRNA in canine benign and malignant mammary tumors.

ErbB subfamily genes, known as proto-oncogenes, encode receptor tyrosine kinases, and are expressed in relation to tumorigenesis of the mammary gland in humans. In this study, we examined the expression of erbB subfamily mRNAs in two canine normal mammary glands and 12 mammary tumor samples by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Each primer set was designed from the nucleotide sequence of the region conserved in erbB subfamily cDNA among other species. No erbB subfamily mRNAs were expressed in the normal mammary gland. In contrast, all of the subfamily mRNAs were expressed in a benign mammary tumor, and more than one type of the subfamily mRNA were observed in 11 malignant mammary tumors. The length of RT-PCR products were 380 bp for erbB1, 500 bp for erbB2, 644 bp for erbB3, and 416 bp for erbB4. These sequences were highly homologous to the cDNA sequences of other species. Therefore, these results suggest that the expression of erbB subfamily mRNAs in canine mammary tumors plays an important role in tumorigenesis of the mammary gland.

Mast cell tumor in the nasal cavity of a dog.

An 11-year-old male Shetland sheepdog displayed epistaxis and nasal discharge from the left nasal foramen. Cytological examination of a smear sample obtained by rhinotomy revealed neoplastic mast cells in the nasal cavity, a definitive diagnostic sign of mast cell tumor. The case was treated by surgery combined with radiotherapy and chemotherapy. Eighteen days after the last treatment, marked enlargement of the mandibular lymph nodes and facial edema developed, and the dog was euthanized at the owner's request. At necropsy, metastatic proliferation of mast cells was confirmed in the lymph nodes and liver, but no neoplastic mast cells were observed in the nasal cavity.

Distribution of cells immunopositive for AM-3K, a novel monoclonal antibody recognizing human macrophages, in normal and diseased tissues of dogs, cats, horses, cattle, pigs, and rabbits.

The monoclonal antibody AM-3K, which was developed using human pulmonary macrophages as the immunogen, immunocytochemically labels most human macrophages except for blood monocytes and dendritic cell populations. AM-3K also shows cross-reactivity in some animal species. To evaluate the usefulness of AM-3K, the present study investigated the detailed distribution of AM-3K-immunopositive macrophages in normal and diseased tissues of dogs, cats, horses, cattle, pigs, and rabbits. Zamboni's solution-fixed, paraffin-embedded sections were the most available for the immunocytochemistry with AM-3K. In all animal species examined, AM-3K labeled most macrophages in splenic red pulp, lymph node sinuses and thymus, and tissue macrophages in the interstitium of various organs and sites such as the kidneys, lungs, heart, pancreas, intestines, and skin. Alveolar macrophages and perivascular microglial cells were also immunoreactive for AM-3K. Interestingly, Kupffer cells of dogs, cats, and horses were labeled for AM-3K, but those of cattle, pigs, and rabbits were not. Furthermore, in tumor tissues and inflammatory lesions such as liver fibrosis and encephalomalacia that were obtained from dogs, infiltrating macrophages were stained with AM-3K, but not all infiltrating macrophages reacted to AM-3K. In addition, only 30-50% of pulmonary and peritoneal macrophages obtained from cats and dogs were reactive for AM-3K. AM-3K did not react with blood monocytes, dendritic cell populations, and osteoclasts. These observations indicate that AM-3K specifically labels most exudate and tissue macrophages in the animal species examined. However, the expression of antigens recognized by AM-3K on macrophages may be dependent on differential maturation stages or different functions evoked by some conditions. AM-3K immunoreaction products were seen on the cytoplasmic membrane of macrophages by immunoelectron microscopy. AM-3K would be useful for detection of macrophage populations in the animal species examined here.

Immunohistochemical analysis of macrophages, myofibroblasts, and transforming growth factor-beta localization during rat renal interstitial fibrosis following long-term unilateral ureteral obstruction.

Renal interstitial fibrosis was induced in rats by chronic unilateral ureteral obstruction (UUO). To identify the mechanisms behind the fibrosis, macrophage influx, myofibroblast involvement, and the localization of transforming growth factor-beta (TGF-beta, a fibrogenic cytokine) were investigated immunohistochemically in rats euthanatized at 0 (controls), 3, 6, 9, 12, and 15 days after UUO. The number of alpha-smooth muscle actin-positive myofibroblasts began to increase significantly in the medulla from day 3, and the development of medullary fibrosis was confirmed from day 6 by morphometric analysis. From day 9, papillary fibrosis also developed in association with an increased number of myofibroblasts. These myofibroblasts showed a parallel orientation to the mucosal surface of the pelvis. In the medulla and papilla, from day 6 the number of ED1 (primary antibody)-positive macrophages began to increase significantly. There appeared to be a relationship between macrophage influx and myofibroblast involvement. By contrast, in the cortex there was no marked increase in myofibroblasts nor development of fibrotic tissues, regardless of increased number of macrophages from day 6. Immunohistochemically, no staining for TGF-beta was found in infiltrating macrophages or myofibroblasts. However, TGF-beta was localized on some cortical proximal renal tubules both of normal control and obstructed kidneys in the early stages on days 3, 6, and 9, suggesting that the possible origin of TGF-beta may be renal epithelia. However, the staining intensity for TGF-beta on the renal epithelia tended to be weakened in advanced obstructed kidneys on days 12 and 15. The likely contribution of TGF-beta to the advanced stages of UUO-induced renal fibrosis remains to be determined.

In vitro cytotoxicity of recombinant human-TNF-alpha and actinomycin D on canine normal and tumor cells.

The in vitro cytotoxicity of recombinant human tumor necrosis factor-alpha (rh-TNF-alpha) and actinomycin D (ACT-D) on canine normal and tumor cells was investigated. rh-TNF-alpha showed dose-dependent cytotoxic and cell-growth inhibitory effects on cultured canine kidney carcinoma cells (CKCa-1). rh-TNF-alpha alone produced little cytotoxic effect on canine normal cells. However, combined with ACT-D, it showed moderate cytotoxicity on normal canine cells from the kidney medulla, spleen, heart muscle and lung. When the effects on the spontaneous tumor cells were examined, the combination of rh-TNF-alpha and ACT-D produced substantial cytotoxic effect on 60% of the tumor cells. All mammary mixed tumors and perianal gland tumors tested were susceptible to this combination. The data indicated the combination of rh-TNF-alpha and ACT-D have in vitro cytotoxicity on certain canine tumor cells.

Cytotoxicity induced by recombinant human tumor necrosis factor-alpha dependent on the types of its receptors on canine cells.

Based on the recent findings that show how recombinant human tumor necrosis factor (rh-TNF)-alpha has potent antitumor activity on human cancer patients when it locally administrated, we have tested the cytotoxicity of rh-TNF-alpha on 3 canine cultured cells: (1) canine kidney carcinoma (CKCa-1), (2) mastocytoma and (3) Mardin Darby canine kidney cells (MDCK). The cell surface expression of TNF-alpha receptors on these canine cells was also determined with anti-human TNF RI and RII polyclonal antibodies. Our data shows that on CKCa-1 which has TNF RI receptors rh-TNF-alpha induced cytotoxicity. By contrast, it exhibited no toxicity on canine mastocytoma which has mainly RII receptors. The data also suggest actinomycin D (ACT-D), an anticancer antibiotic, enhanced the cytotoxicity of rh-TNF-alpha. Combined with ACT-D, rh-TNF-alpha showed the cytotoxicity on MDCK which possessed both TNF RI and RII receptors. The results indicate that the cytotoxicity of rh-TNF-alpha depends on the presence of TNF RI receptors on canine tumor cells.

Novel putative fragile sites observed in feline fibroblasts treated with aphidicolin and fluorodeoxyuridine.

Fragile sites are non-randomly distributed chromosomal breaks and gaps observed in the cells cultivated under certain conditions. Feline fragile sites were analyzed using skin fibroblast strains after the treatments with aphidicolin and fluorodeoxyuridine in combination with caffeine. Three aphidicolin-induced fragile sites (A1q21, C2q13 and E1p21) as well as a folate-sensitive site (B1q14) were observed in all the 3 fibroblast strains tested for each treatment group. The loci in A1q21 and B1q14 are very close to that reported previously for peripheral blood lymphocytes and lung cells. Two chromosomal break points in C2q13 and E1p21 seem to be new fragile sites. Fifteen candidates for feline fragile sites were also assigned their locations in feline chromosomes. Both the incidence and distribution of feline fragile sites in skin fibroblasts seem to be different at least in part from those in lymphocytes.

Urinary bladder rhabdomyosarcoma (sarcoma botryoides) in a young Newfoundland dog.

A 13-month-old female Newfoundland dog suffered from urinary bladder tumor. Histologically the tumor consisted of round or fusiform cells, occasionally having eosinophilic cytoplasms. Apparent mature rhabdomyoblasts possessing elongated eosinophilic cytoplasm and cross striations were infrequently observed. The tumor cells exhibited immuno-positive for anti-myoglobin, desmin and vimentin antibodies. Ultrastructurally, tumor cells have abundant myofibrils in their cytoplasm and Z bands were also detected. The present tumor was diagnosed as a urinary bladder rhabdomyosarcoma in a Newfoundland dog, which has not been frequently reported in dogs.

Lipoprotein profile in canine pancreatitis induced with oleic acid.

Lipid and lipoprotein concentrations and apolipoprotein profile were investigated in canine pancreatitis induced by infusion with oleic acid (OA) into the accessory pancreatic duct. Pancreatitis was diagnosed by physical, hematological, biochemical and pathological examinations. In OA-treated dogs, serum triglyceride (TG) concentration was increased; however, there were no changes in serum total cholesterol (TC) and phospholipid (PL) concentrations. Serum concentrations of TG, TC, PL and total lipids (TL) in beta lipoprotein and those of TC, PL and TL in pre-beta lipoprotein were increased and those of TC, PL and TL in alpha, lipoprotein were decreased. In apolipoprotein profile, the proportions of apolipoprotein B100 in low density lipoprotein fraction and apolipoprotein A-IV in high density lipoprotein fraction were increased. In addition, decreased proportion of apolipoprotein A-I and the appearance of serum amyloid A protein in high density lipoprotein fraction were observed. These results suggest that lipoprotein profiles observed in canine acute pancreatitis are attributed to the alterations in apolipoprotein compositions.

C-cell adenoma containing variously sized thyroid follicles in a horse.

A thyroid gland tumor, showing unusual histology, was identified in a 13-year-old male Andalusian horse. Microscopically, the tumor consisted of neoplastic proliferation of C-cell (parafollicular cell) with cytoplasmic fine granules, containing diffusely distributed, variously sized colloid-containing follicles. Immunohistochemically, the neoplastic C-cell were positive for calcitonin and follicleforming epithelial cells showed a positive reaction for thyroglobulin. Ultrastructurally, membrane-bound secretory granules up to 250 nm in diameter were found in the cytoplasm of the parafollicular cells, whereas the follicular epithelial cells had microvilli, junctional complex, and well-developed endoplasmic reticulum.

Induction of fragile sites by fluorodeoxyuridine and caffeine accompanies with misincorpolation of endogenous uridine nucleotide into DNA of feline fibroblasts.

Fluorodeoxyuridine, an inhibitor of thymidylate synthetase, is known to induce chromosomal fragile sites. The drug treatment may cause deprivation of intracellular thymidine nucleotide pool followed by a serious imbalance of deoxynucleotide pool. Though the stress is probably related to the induction of folate-sensitive fragile sites, the exact mechanism is still to be investigated. The present study has been carried out to test the possibility that the fragile sites are originated, at least in part, from incorpolated uracil residues. The incorpolated uracil residue can be detected by a novel assay for abasic sites after treatment with uracil-DNA N-glycosylase (UDG). About 2.7 abasic sites per 10(4) nucleotides were detected in the DNA extracted from feline fibroblasts after the treatment with FUdR and caffeine. By digesting the DNA with UDG prior to the assay, significant increase in the number of abasic sites were observed. These results indicate that the large amount of uracil residues are present in the feline fibroblast DNA under the condition which induces chromosomal fragile sites.

Frequent expression of the c-kit proto-oncogene in canine malignant mammary tumor.

The mammary tumor is one of the popular neoplastic diseases in female dogs. In the present study, the expression of canine c-kit proto-oncogene in mammary tumor specimens was investigated to evaluate its potential usefulness as a tumor marker. By comparing the homology among the nucleotide sequences reported for human mouse, rat and feline c-kit c-DNA, a pair of primers was synthesized for the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The RT-PCR product of canine spleen total RNA was shown to have 756 bp in size and to be highly homologous to the corresponding sequences reported for the mammalian species. The expression of c-kit transcript was detected in 11 mammary tumors of different histopathology including adenocarcinomas, benign and malignant mixed tumors. The level of the transcription in adenocarcinomas was significantly higher than those in malignant mixed tumors. Fifteen canine tumor specimens originated from various tissues were also tested for their c-kit transcript. In all of the mastocytoma samples examined, high expression of the mRNA was detected. Of other 12 tumors, only low level of RT-PCR products were detected in 5 samples, whereas no apparent amplification was observed in 7 tumors. These results indicate that the high expression of c-kit transcript is helpful for the diagnosis of canine mammary tumors.

A control of a golden retriever with renal dysplasia.

A six-month-old male Golden Retriever with a three-month history of polyuria and polydipsia was examined. Hematological examinations revealed nonregenerative anemia, azotemia, high serum creatinine level, hypercalcemia, hyperphosphatemia, hypercholesterolemia, hyperamylasemia, and low level of total serum protein. Urinalysis indicated mild proteinuria, and low specific gravity. Radiographic and ultrasonographic examinations revealed bilateral small sized kidneys. Histological examination by renal biopsy confirmed the diagnosis of renal dysplasia. Treatment with a dietary protein restriction, oral adsorbents, and dried aluminum hydroxide gel have been performed in this dog, and then, azotemia, high serum creatinine level, hypercalcemia, and hyperphosphatemia were improved. During 10 months after the initiation of treatments, no significant clinical change except polydipsia and polyuria has been observed.

Partial cloning of prohibitin cDNA from canine, feline, bovine, equine, and rabbit liver mRNA by RT-PCR.

Prohibitin is the protein which has an inhibitory function in cell growth, and its gene is suggested to be one of putative tumor suppressor genes. In this report, we described a partial cloning of prohibitin cDNAs from canine, feline, bovine, equine, and rabbit liver mRNAs by RT-PCR, and their homology analysis. The sequences of these RT-PCR products were compared with each other as well as those reported for human and rat. The homology in this region of prohibitin cDNA was approximately 90%, and the amino acid sequence of each RT-PCR product shared more than 95% identity. Therefore, it is concluded that all the RT-PCR products are a part of prohibitin cDNA of each animal.