Sequence of the canine c-yes proto-oncogene

Cloning of the canine yes oncogene was attempted from a c library derived from a healthy canine spleen using a human c-yes-1 probe. The nucleotide and amino acid sequences revealed that the canine yes gene contained an open reading frame consisting of 539 amino acids. Its product had a molecular mass of 60,368 Daltons and showed 95·9 per cent and 90·4 per cent homology with human and chick p61^c-yes, respectively. Moreover, the product had a myristylation signal, src homology region (SH) 3, SH2, and tyrosine kinase domains showing 98·8 per cent and 96·0 per cent homology with those of human beings and chickens, respectively. These findings indicate that the products of the canine yes gene may have non-receptor-type tyrosine kinase activity on the cell membrane, as is the case in human and chick p61^c-yes     

Human adenovirus antibody in sera of normal and tumour-bearing dogs.

Serum specimens from 42 normal dogs and 42 with untreated malignant tumours were assayed for the presence of antibodies to human adenovirus types 5, 21 and 31 and to infectious canine hepatitis (ICH) virus. Radioimmunoassays using human adenovirus antigens showed that 71 per cent (30/42) of all dogs with tumours, but only 19 per cent (8/42) of all normal dogs, were positive for human adenovirus antibody. Canine sera reactive with antigens of one human adenovirus type in radioimmunoassays were also reactive with antigens of the other two types. Dogs bearing malignant lymphoma or squamous cell carcinoma tumours had higher levels of antibody against adenovirus type 5 antigens. Human adenovirus type 5 was neutralised by sera from four tumour-bearing and two normal dogs, while sera from two normal and five tumour-bearing dogs were positive in immunodiffusion tests with human adenovirus antigens. Levels of ICH antibody in sera of normal adult dogs and adult dogs with tumours were not markedly different when measured by radioimmunoassays. Likewise, sera from these two groups of dogs had similar ranges of ICH neutralising antibody titres. In contrast, levels of ICH antibody detected by the serological assays in sera from non-pet, non-vaccinated pups were either markedly low or absent. Possible explanations for the observed increased levels of human adenovirus antibody in sera of tumour-bearing canine pets are discussed.     

Immunohistochemical localisation of rabbit haemorrhagic disease virus VP-60 antigen in early infection of young and adult rabbits

This study evaluated the time course distribution of rabbit haemorrhagic disease virus (RHDV) structural protein VP60 in tissues from experimentally infected rabbits from three different age groups. Viral VP60 antigen could not be detected in tissue samples from animals under four weeks, and only a few hepatocytes (0.01 to 0.2 per cent) were stained in the 6-week-old animals. A 6-week-old rabbit euthanised at 72 hpi showed VP60-labelling in hepatocytes and macrophages close to areas of inflammation. Viral VP60 antigen was detected as early as 12 hpi in a few hepatocytes (0.03 per cent) from adult animals. Within this age group, the extent of hepatocyte labelling considerably increased at 18 (3.0 per cent), 24 (25.5 per cent), 36 (50 per cent) and 48 (60 per cent) hpi. Extrahepatic viral VP60 antigen was also detected at 36 and 48 hpi in spleen macrophages and lymphocytes from adult rabbits. These findings support the hypothesis that the hepatocyte is the only cell type in the liver able to support RHDV replication almost immediately after viral infection.     

Cohort study of COX-1 and COX-2 expression in canine rectal and bladder tumours

OBJECTIVES: To determine the role that cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) play in malignant transformation in canine transitional cell carcinoma and rectal tumours. METHODS: Histological sections of 21 canine rectal adenocarcinomas and 18 canine transitional cell carcinomas were stained for COX-1 and COX-2. Mann-Whitney non-parametric tests were applied to determine if there was any relationship between the percentage of cells expressing COX-1 or COX-2, and between COX-1 and COX-2 staining intensity and age, breed or sex. RESULTS: For rectal adenocarcinomas, 19.0 per cent of the sections were negative for COX-1 and COX-2. A further 38.1 per cent of the sections were negative for COX-2 but positive for COX-1, and 38.1 per cent of the sections had rare or occasional single cells positive for COX-2. No significant differences were found in COX staining when compared with age, breed or sex. For transitional cell carcinomas, all of the sections were positive for COX-1 and COX-2. For COX-2 staining, 16.7 per cent had more than 30 per cent positive cells. For COX-1 staining, 38.9 per cent had more than 30 per cent positive cells. There was a significant increase in the percentage of COX-1 positive cells in small breed dogs (P = 0.0337). CLINICAL SIGNIFICANCE: The variations in COX expression reported in this study may explain the differences in the clinical response of transitional cell carcinomas and rectal adenocarcinomas following treatment with non-steroidal anti-inflammatory drugs.     

Immunohistochemical study of the expression of E-cadherin in canine mammary tumours

To investigate the possible role of E-cadherin in canine mammary tumours 20 benign and 40 malignant tumours were evaluated by immunohistochemistry in formalin-fixed paraffin wax-embedded samples. In all the benign tumours, E-cadherin was strongly expressed at the intercellular borders of epithelial cells, but it was less strongly expressed in 17 (43 per cent) of the malignant tumours. Furthermore, poorly differentiated carcinomas were less immunoreactive for E-cadherin than moderately and well differentiated carcinomas.     

Viral matrix inclusion bodies in myocardium of lymphoid leukosis virus-infected chickens

Chicken embryos and healthy adult chickens naturally infected with lymphoid leukosis virus were used to investigate viral inclusion bodies in myocardial cells by light and electron microscopies and by immunocytochemical technique. Intracytoplasmic viral matrix inclusion bodies frequently appeared in the myocardium of adult chickens, but not in that of embryos. In light microscopic preparations, inclusions were irregularly distributed, were basophilic, and contained ribonucleic acid. Ultrastructurally, inclusions in myocardial cells were in areas containing numerous interstitial C-type particles. Early inclusions were composed of clusters of ribosomes associated with sarcoplasmic tubules; spherical bodies developed among these ribosomes. Mature inclusions were composed of numerous spherical bodies (50 to 75 nm) with interspersed ribosomes and of ribosomes clustered at the periphery. Inclusions were not membrane-enclosed. Occasionally, spherical bodies were in paracrystalline arrays. Multiple budding occurred on cell membranes adjacent to matrix inclusions. The viral group-specific protein, p27, was demonstrated by the peroxidase-antiperoxidase method and by the protein A-gold method in the spherical bodies, in nucleoids of mature virus particles, and among ribosomes of inclusions. The results indicate that the matrix inclusions were the result of lymphoid leukosis virus infection and were the product of viral protein synthesis on ribosomes.     

Activins and Inhibins: Expression and Role in Normal and Pathological Canine Reproductive Organs: A review

Activins and inhibins are regulatory proteins of the reproductive function. Inhibins antagonise the activin signalling at different levels and are responsible for the negative feedback in the regulation of the release of pituitary follicle stimulating hormone (FSH), which, in turn, is promoted by locally produced activins. In the canine ovary, both peptides are expressed by developing follicles and corpora lutea. Activins may play a stimulatory role in follicular development, promoting the aromatase function; inhibins modulate these processes and suppress the hyperplasic/neoplastic stimuli. Activins are required for ovulation and corpus luteum formation, while inhibins stimulate progesterone synthesis. The exclusive production of alpha‐inhibin by granulosa cells allows the peptide to be used as marker to identify canine ovarian stromal tumours by immunohistochemistry. In the male, activins are powerful morphogenetic factors in the foetal testis. In the adult, they display a modulating action on spermatogenesis and Sertoli cell function. Inhibins, produced mainly by Leydig cells, promote testosterone secretion. Canine testicular tumours, such as Leydig, Sertoli and granulosa cell tumours (GCTs), may express inhibin subunits and produce high circulating levels of these glycoproteins. In the canine prostate, activins inhibit epithelium proliferation, antagonising androgen effects, but they are synthesised under androgenic stimulus.     

Hyperthermia and radiation in the management of canine tumours

Fifty-one dogs bearing a variety of naturally occurring tumours were treated with combined radiation and hyperthermia. The treatment regime consisted of four, weekly fractions of radiation to a total dose of 3600–4000 cGys and one or two treatments with local microwave hyperthermia, heating the tumour to 44°C for 30 minutes on each occasion. Twenty-six (51 per cent) of the tumours showed a complete response to this treatment and a further eighteen (35 per cent) tumours underwent significant regression giving a total response rate of 86 per cent. Sixteen tumours later recurred at intervals of 11–50 weeks post treatment. The majority of tumours were in the oral cavity (35 cases) and in this group the survival rate compares favourably with figures for surgical treatment and radiotherapy alone. These findings support published results from other centres, indicating that hyperthermia combined with radiotherapy is a potentially useful technique for the management of certain canine malignant tumours.     

Detection of lymph node micrometastases in malignant mammary tumours in dogs by cytokeratin immunostaining

A series of 131 local and regional lymph nodes from 40 dogs with malignant mammary tumours were evaluated by staining with haematoxylin and eosin and immunohistochemically for antibodies to pancytokeratin (AE1/AE3) and cytokeratin 14. The immunohistochemical tests detected occult micrometastases in 9.2 per cent of the lymph nodes that were negative by haematoxylin and eosin staining. Under the modified TNM classification of canine mammary tumours, these results raised the clinical stage of 12.5 per cent of the affected dogs. However, if the latest TNM classification of human breast cancer had been applied, none of the animals would have been reclassified.     

Cytological features of canine ovarian tumours: a retrospective study of 19 cases

The cytological features of 19 histologically confirmed canine ovarian tumours were retrospectively examined. Seven cases were cytologically classified as papillary adenocarcinoma, eight cases as granulosa cell tumours, two cases as mature ovarian teratomas, one case as a dysgerminoma and one case as a mixed granulosa cell tumour/dysgerminoma. On cytology, papillary adenocarcinoma was characterised by a papillary glandular pattern and tight cohesiveness. Granulosa cell tumours showed monolayered clusters of loosely cohesive granulosa cells. Call-Exner-like bodies were found in five of seven cases. Granulosa cells appeared to be heterogeneous and usually contained several intracytoplasmic vacuoles. Teratoma was characterised cytologically by keratin debris (two cases) and a mixture of epithelial cells with sebaceous, basaloid, columnar/palisading or ciliated appearance (one case). The dysgerminoma contained severely atypical round cells admixed with small lymphocytes. The mixed dysgerminoma/granulosa cell tumour had a mixture of germinal and granulosa cells. Cytological diagnosis was in agreement with histopathology in 18 of 19 (94.7 per cent) cases.     

Histopathological survey of neoplasms in flat-coated retrievers, 1990 to 1998

Over the period from March 1990 to December 1998, veterinary surgeons in general practice were invited to submit tissues suspected of being neoplastic which had been removed from flat-coated retrievers. When possible, pedigree details were obtained from the owners. In addition, data were collected from flat-coated retrievers known to have suffered from a neoplastic condition and for which a histopathological report was available. A total of 1023 submissions was obtained from 782 dogs. These included 165 non-neoplastic lesions (16 per cent), 447 benign samples (44 per cent) and 411 malignant samples (40 per cent). Soft tissue sarcomas accounted for 55 per cent of the malignant samples (26 per cent of all tumour samples and 22 per cent of all submissions) with 63 per cent of them being diagnosed as undifferentiated. Carcinomas accounted for 20 per cent of malignant samples (8 per cent of all submissions). Of the benign tumours, cutaneous histiocytoma was the most common diagnosis (48 per cent of benign tumours, 25 per cent of all tumours and 21 per cent of all submissions).     

Relationship between receptors for epidermal growth factor and steroid hormones in normal, dysplastic and neoplastic canine mammary tissues

The concentrations of receptors for epidermal growth factor (EGF-R), oestrogen (ER) and progesterone (PR) were measured in 108 samples from canine mammary tumours and 132 samples of normal mammary tissue removed surgically from 84 bitches. The history and clinical signs were also recorded. Binding sites of high affinity were detected in 70 per cent of both types of tissue and no significant variations in EGF-R concentrations or positivity were observed with the histology, location, size or number of mammary tumours or the age of the animal. A significant direct correlation (P=0002) was observed between the concentrations of ER and EGF-R only in malignant tumours. The concentrations of EGF-R were significantly correlated (P=0.04) in normal mammary tissues adjacent to and distant from the lesions, but not between normal tissue and tumour tissue. No significant differences were observed in the expression of EGF-R in normal and neoplastic tissues from the same bitches. The direct correlation between the concentrations of EGF-R and ER in malignant tumours could be related to an oestrogen-dependent expression of EGF-R or to a similar pattern of regulation of the receptors.     

Identification of hepatic stem/progenitor cells in canine hepatocellular and cholangiocellular carcinoma

Hepatic progenitor cells (HPCs) are bipotential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic or cholangiocytic lineages. HPCs are present in both hepatocellular (HCC) and cholangiocellular carcinoma (CC) in humans; and a small percentage of HCC can originate from cancer stem cells. However, its distribution in canine liver tumour has not been studied. Herein, we searched for stem/progenitor cells in 13 HCC and 7 CC archived samples by immunohistochemical analysis. We found that both liver tumours presented a higher amount of K19‐positive HPCs. Besides, 61.6% of HCC cases presented immature CD44‐positive hepatocytes. Nevertheless, only two cases presented CD133‐positive cells. As observed in humans, hepatic canine tumours presented activated HPCs, with important differentiation onto hepatocytes‐like cells and minimal role of cancer stem cells on HCC. These findings reiterate the applicability of canine model in the search for new therapies before application in humans.     

Ulex europaeus agglutinin 1 lectin histochemical staining of dog hepatocellular and bile duct carcinomas

Ulex europaeus agglutinin 1 (UEA-1) lectin-binding is a feature of canine intrahepatic bile duct epithelial cells but not of canine hepatocytes. In tumours diagnosed histologically as cholangiocarcinomas or hepatocellular carcinomas, UEA-1 staining occurred in tumour cells regardless of histological appearance. The division of primary liver carcinomas into different presumed cell types of origin is not reflected by equivalent differentiation of the tumour cells and is not of diagnostic value.     

Serum antibody titres to canine parvovirus, adenovirus and distemper virus in dogs in the UK which had not been vaccinated for at least three years

Antibody titres to canine distemper (CDV), canine parvovirus (CPV) and canine adenovirus (CAV) were measured in 144 adult dogs that had not been vaccinated for between three and 15 years. Protective antibodies to CPV were present in 95 per cent of the population, to CDV in 71.5 per cent and to CAV in 82 per cent. The prevalence of protective titres did not decrease with increasing time interval from the last vaccination for any of the three diseases studied. Booster vaccination increased the dogs CAV titres. For comparative purposes, 199 puppies were sampled at the time of their first and second vaccination. In the case of CPV and CAV a significantly higher proportion of the adult dogs were protected than of the puppies immediately after they were vaccinated. Natural CPV boosting was strongly suspected because the dogs had significantly higher titres three years after their primary vaccination than two weeks after it and three unvaccinated dogs had acquired protective antibody levels uneventfully. There was no evidence of natural exposure to CDV.     

Immunohistochemical expression of calretinin in canine testicular tumours and normal canine testicular tissue

Calretinin is a calcium-binding protein expressed abundantly in the central and peripheral neural tissues. It has been demonstrated to be a valuable marker in human testicular neoplasia. The immunohistochemical expression of calretinin has been studied in 102 samples of normal (n=25) and three different neoplastic canine testicular tumours (n=77). In normal canine testis, calretinin expression was restricted to Leydig and Sertoli cells of the testis. In tumour tissues, calretinin expression was detected in all tumours investigated (interstitial cell tumours, seminoma, and Sertoli cell tumours), with a cytoplasmic and nuclear pattern of cellular distribution. The present work reports, for the first time, calretinin immunohistochemical expression in normal and neoplastic canine testis.     

Of humans and canines: Immunohistochemical analysis of PCNA, Bcl-2, p53, cytokeratin and ER in mammary tumours

Mammary tumours are the most common neoplasms in humans and canines. Human and canine mammary tumours share several important epidemiological, clinicopathological and biochemical features. Development of mammary tumours involves accumulation of mutant cells caused by excessive proliferation and insufficient apoptosis or dysregulation of cellular differentiation. The present study was therefore designed to investigate the expression of proliferation, differentiation, and apoptosis associated proteins together with expression of estrogen receptors (ER) in both human and canine mammary tumours. Thirty breast cancer patients categorized as pre- and postmenopausal, and 30 mammary gland tumours obtained from bitches were included in this study. The expression of proliferating cell nuclear antigen (PCNA), Bcl-2, p53, cytokeratin and ER in tumour tissues and adjacent tissues were investigated using immunohistochemical staining. While the expression of PCNA, Bcl-2, p53 and ER was significantly increased, expression of cytokeratin was significantly lower in both human as well as canine mammary tumours compared to corresponding adjacent tissues. The magnitude of the changes was however more pronounced in premenopausal patients compared to postmenopausal patients. The changes in proliferation, apoptosis and differentiation associated proteins in human and canine mammary tumours validate use of the canine model to understand the molecular mechanisms of mammary carcinogenesis.     

Pathology of spontaneous tumour lesions in pullets and adult chickens in commercial farms - Short communication

Twenty pullets and adult chickens, aged 100 to 403 days, from several commercial chicken farms were examined by gross and histopathology. Grossly, all chickens had white-greyish masses in the visceral organs with or without enlargement of the peripheral nerves. Histopathological examination revealed Marek's disease (MD) lymphoma, lymphoid leukosis (LL) and myeloid leukosis (ML) in 14/20, 5/20 and 1/20 of the chickens, respectively. Lesions of the sciatic nerves in chickens diagnosed as having MD lymphoma were various. No neoplastic and/or inflammatory cells were noted in the peripheral nerves of chickens diagnosed as having LL and ML. These results indicated that MD lymphoma could also develop in older chickens; thus, microscopic examination is needed to identify MD in older chickens showing lymphocyte-derived tumours.     

Outcome following removal of canine spindle cell tumours in first opinion practice: 104 cases

O bjectives : To define the outcome of a cohort of canine patients with a histological diagnosis of spindle cell tumour of soft tissue managed solely by surgery in first opinion practice.
M ethods : Clinical details of 104 spindle cell sarcomas submitted to Finn Pathologists during the year 2000 were reviewed. Questionnaires were sent to the submitting veterinarians, requesting details about the tumour, surgery performed and ultimate outcome of the patient.
R esults : The method of surgical resection was described as marginal in 45 dogs (44·2 per cent). Excision margins of 3 cm or more were described in less than 10 per cent of cases. Tumours recurred locally in 29 dogs (27·9 per cent). Eighteen dogs (21·7 per cent) died of tumour-related causes. Most deaths were unrelated to sarcoma (50 dogs, 60·2 per cent) or unknown (15 dogs, 18 per cent). The median survival time was 1013 days. Tumour size, location or degree of surgical resection were not significantly related to survival or tumour recurrence. A palpable assessment of tumour invasion into underlying tissues was significantly associated with decreased disease-free interval (P<0·0001) and survival time (P = 0·0070).
C linical S ignificance : The results of this retrospective study indicate that many spindle cell tumours managed in first opinion practice exhibit a low-grade biological behaviour and may respond well to more conservative surgery than current recommendations advise.