Expression of the epidermal stem cell marker p63/CK5 in cutaneous papillomas and cutaneous squamous cell carcinomas of dogs.

Cutaneous papillomas (CPs) and cutaneous squamous cell carcinomas (CSCCs) are usual epidermal tumours in dogs. CPs and CSCCs probably arise from the neoplastic transformation of the keratinocytes within the stem cell compartment, since these cells are the only keratinocytes that would reside long enough to accumulate the number of molecular alterations to drive the progression towards a tumour cell phenotype. However, the role of these cells in common epidermal tumours in dogs is still unknown. Thus, the purpose of this study was to evaluate the immunohistochemical expression pattern of p63 together with CK5, molecular markers of epidermal stem cells, on sections of tissue microarrays constructed from canine samples of CP and CSCC to investigate the contribution of stem cells in those canine tumours. p63/CK5 coexpression was retained in most basal and some suprabasal cells in CPs and CSCCs. In addition, increased coexpression of these molecules was observed in a group of CPs and CSCCs, as a result of a higher p63 expression. These results suggest that the coexpression of p63/CK5 may mark epidermal keratinocytes that possess self-renewal capacity rather than only stem cells, and suggest that transit amplifying cells, and even differentiated keratinocytes, may also contribute to the pathogenesis of epidermal tumours in dogs.

Aberrant Expression of E-cadherin/ß-catenin During Epidermal Tumourigenesis in Dogs.

Clinically relevant epidermal tumours in dogs include cutaneous papillomas (CPs) and cutaneous squamous cell carcinomas (CSCCs). The development of CPs and CSCCs involves dysregulation in expression of E-cadherin/ß-catenin; however, knowledge about the contribution of these molecules to epidermal tumourigenesis in dogs is limited. This study examined the immunohistochemical expression pattern of E-cadherin/ß-catenin in samples of normal canine epidermis, CPs, preneoplastic epidermis and CSCCs, using tissue microarrays, in order to elucidate whether the dysregulated expression of these molecules may contribute to the pathogenesis of clinically relevant epidermal tumours in dogs. We also investigated the correlation between the immunohistochemical expression pattern of E-cadherin/ß-catenin in these tissue microarrays to further evaluate whether the disruption of the adherens junction interactions plays a relevant role in canine epidermal tumourigenesis. In samples of CP and preneoplastic epidermis, the membrane immunoreactivity of E-cadherin/ß-catenin was conserved, while in CSCC, the immunoreactivity of these molecules was significantly reduced, independently of the tumour location. There was significant correlation between the membrane expression of E-cadherin/ß-catenin in CSCC. ß-catenin also showed cytoplasmic and nuclear expression in samples of CP, preneoplastic epidermis and CSCC. These results support the hypothesis that dysregulated expression of E-cadherin/ß-catenin may play a critical role in the pathogenesis of relevant canine epidermal tumours, not only due to the disruption of the intercellular adherens junctions, but also due to the dysregulated activity of the signalling pathways in which these molecules are involved.

Dysregulated Expression of Phosphorylated Epidermal Growth Factor Receptor and Phosphatase and Tensin Homologue in Canine Cutaneous Papillomas and Squamous Cell Carcinomas.

The molecular mechanisms contributing to the development of cutaneous papillomas (CPs) and cutaneous squamous cell carcinomas (CSCCs) are still poorly understood, limiting the ability to identify molecular suitable targets for the development of novel therapies. Persistent activation of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signalling pathway is a component of epidermal carcinogenesis in dogs. The present study describes the immunohistochemical expression pattern of two key regulatory molecules involved in the PI3K/Akt/mTOR signalling pathway, phosphorylated epidermal growth factor receptor (pEGFR)Tyr1068 and phosphatase and tensin homologue (PTEN), in samples of normal canine epidermis, CP, preneoplastic epidermis and CSCC using tissue microarrays to determine whether the deregulated activity of these molecules is involved in the pathogenesis of these relevant epidermal tumours of dogs. Expression of pEGFR and PTEN was dysregulated in most samples of CP, preneoplastic epidermis and CSCC. Overexpression of pEGFR, together with decreased expression of PTEN, may facilitate the progression of some canine CPs and CSCCs by deregulation of the key cellular functions in which the PI3K/Akt/mTOR signalling pathway is involved. These findings suggest that the PI3K/Akt/mTOR signalling molecules may be potential therapeutic targets for canine patients with CP and CSCC.

Immunohistochemical expression of selected phosphoproteins of the mTOR signalling pathway in canine cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) represents one of the most common malignant skin tumors in dogs. Research aimed at clarifying how the deregulated activity of signalling pathways contributes to cSCC progression can help to identify molecular suitable targets for the development of novel therapies. The present study describes the immunohistochemical expression pattern of two proteins (pAktSer473 and pS6Ser235/236, the latter combined with Ki-67) involved in the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signalling pathway in canine specimens of normal epidermis, pre-neoplastic epidermis, and cSCC using tissue microarrays. The results suggest that the PI3K/Akt/mTOR signalling pathway has a low expression in the normal canine epidermis, and that selected molecules involved in this signalling pathway are dysregulated during the canine epidermal carcinogenesis process. These findings provide important evidence that the persistent activation of the PI3K/Akt/mTOR signalling pathway represents one of the key events during cSCC progression in canine patients, pointing to the PI3K/Akt/mTOR pathway as a potential therapeutic target.

Lack of prognostic significance of angiogenesis in canine melanocytic tumours.

The prognostic significance of angiogenesis in some canine tumours has been investigated, but little is known about its relevance in canine melanocytic tumours (MTs). The aim of this study was to evaluate the prognostic significance of angiogenesis in canine MTs. A total of 36 cutaneous melanocytomas (benign MTs), 40 cutaneous melanomas (malignant MTs) and 43 oral melanomas were studied. Survival data were available for a subset of 59 cases. Microvessel density (MVD) and endothelial area (EA) were determined by immunolabelling using an antibody specific for von Willebrand factor (vWF). Mean MVD (expressed as the number of microvessels per mm(2)) was 129 ± 14 in melanocytomas, 191 ± 16 in cutaneous melanomas and 208 ± 16 in oral melanomas. Mean EA (expressed as the percentage of the total area) was 1.5 ± 0.14 in melanocytomas, 2.6 ± 0.2 in cutaneous melanomas and 2.4 ± 0.3 in oral melanomas. The differences in MVD and EA between melanocytomas and melanomas were significant (P = 0.001 and P = 0.003, respectively). MVD and EA were significantly correlated between cutaneous and oral MTs (r = 0.54; P <0.001 and r = 0.63; P <0.001, respectively). MVD and EA were not related to survival in cutaneous and oral MTs. In conclusion, tumour vascularization was higher in melanomas than in melanocytomas, but it seemed to have no prognostic significance in these tumours.

A simple method of infecting rabbits with Bovine herpesvirus 1 and 5.

This report describes an alternative technique to inoculate rabbits and to reproduce infection by Bovine herpesvirus 1 and 5. First, the nostrils are anaesthetized by aspersion with local anaesthetic. A few seconds later, and after proving the insensitivity of the zone, the rabbits are put on their back legs with their nostrils upwards and the inoculum is introduced slowly into each nostril by using disposable droppers. Clinical signs, viral isolation from nasal swabs, histological lesions found, positive polymerase chain reaction and antibodies production confirm the infection. This very simple and bloodless technique, where the animals are exposed to minor distress, may be useful for evaluating the virulence of BoHV-1 and BoHV-5 strains, to study the establishment of latent virus infection and to test the potential of experimental vaccines or properties of antiviral drugs. It may be also suitable for experimental infection with other respiratory viruses in this animal model.

Primary pulmonary adenocarcinoma metastatic to the uvea, brain and adrenal gland in a dog.

A 10-year-old male Briard dog was presented because of listlessness, abnormal gait, fever, inappetence, and seizures. A non-pigmented growth was observed in the ventral quadrant of the left iris. Thoracic radiographs revealed multiple pulmonary metastases and the owner opted for killing. On necropsy, lung masses and nodules in left iris, right adrenal medulla, and brain were detected. Histologically the primary tumour was diagnosed as pulmonary adenocarcinoma with predominant solid pattern. Metastases to regional lymph nodes, uvea, adrenal medulla, and brain were recognized. The metastatic behaviour resembled that occurring in humans. To the authors' knowledge, this is the first report of a pulmonary adenocarcinoma with metastasis to the uvea in a dog.

Cutaneous lymphangiosarcoma in a young dog: clinical, anatomopathological and lectinhistochemical description.

Lymphangiosarcoma is a rare tumour in domestic animals arising from lymphatic endothelial cells. Occasionally, microscopic differentiation with haemangiosarcoma may be difficult. The aim of the present study was to describe a lymphangiosarcoma in a 1-year-old female Doberman Pinscher dog and to characterize its lectinhistochemical binding pattern as compared with that of haemangiosarcoma. The dog was presented because of a cutaneous painful swelling located in the left axilla. Histological diagnosis confirmed lymphangiosarcoma. The dog was killed. Necropsy revealed mediastinal lymph nodes' involvement. Twenty lectins were tested in tissue sections of this case as well as in four haemangiosarcomas from other dogs. Staining intensity was issued upon optical density determinations. Percentage of lectinhistochemical staining area was also conducted. RCA-I showed the most intense and wide distributed labelling pattern for lymphangiosarcoma. PHA-E was the counterpart for haemangiosarcoma. Should similar results be obtained in further studies, such differences could aid in the differential diagnosis between lymphangiosarcoma and haemangiosarcoma when histological pictures were not conclusive.

Peripheral neuroblastoma in a newborn piglet.

Peripheral neuroblastoma (PNB) is a rare neoplasia derived from neuroepithelial cells. PNB typically presents as a greyish mass, composed of round cells with features of neuronal differentiation. Necropsy, performed on a 1-day-old piglet, revealed a mass craniodorsally located in the abdominal cavity. Histologically, the predominant population consisted of small round to ovoid cells with scanty cytoplasm and dark round nuclei, besides, there were larger neurone-like cells. Neurone-specific enolase and S-100 protein were immunohistochemically detected, while glial fibrillary acidic protein was negative. Histological and immunohistochemical findings substantiated the diagnosis of a grade II peripheral neuroblastoma. This seems to be the first description of a PNB in a newborn piglet.

Paratuberculosis in red deer (Cervus elaphus): an immunohistochemical study.

In the present study, we compared the utility of immunohistochemistry with serological and histological results for the characterization of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) in tissues of affected red deer. Bacterial isolation was considered the standard reference. Samples were taken from seven clinically affected animals with typical macroscopic lesions. The enzyme linked immunosorbent assay (ELISA) and the gel diffusion tests (GD) were used for serological determinations. Samples from intestine and mesenteric lymph nodes were processed for bacterial isolation and histology. M. paratuberculosis was isolated from all the animals. Histologically, lymph nodes displayed necrosis and mineralization at the cortical and medullar areas. Ziehl-Neelsen stained bacteria were numerous inside macrophages and Langhans-type giant cells. Giant and epithelioid cells and lymphocytes were prominent at the ileal mucous membrane. The immunostaining of M. paratuberculosis was very clear inside epithelioid and giant cells. Image analysis was carried out to determine the immunostained area. There was total agreement among the methods employed. Immunohistochemistry can be very useful when the microorganism cannot be recovered from tissues or faeces.

Effects of plant-induced hypervitaminosis D on cutaneous structure, cell differentiation and cell proliferation in cattle.

Solanum glaucophyllum (Sg) (synonym S. malacoxylon) is a plant toxic to cattle due to its high levels of 1,25-dihydroxyvitamin D3 as glycoside derivatives. Sg causes a disease characterized by wasting and calcification of soft tissues. The effects of vitamin D are not only important in calcium homeostasis, but also in immune regulation, cell growth and cell differentiation. Skin samples in Sg-intoxicated and control heifers were studied histologically. Cellular differentiation and proliferation were analysed by immunohistochemical expression of cytokeratins, involucrin and proliferating cell nuclear antigen (PCNA). The results were obtained by image processing and analysis and were statistically evaluated. Sg-intoxicated cattle showed atrophy of epidermis and severe involution of hair follicles and of sebaceous and sweat glands. As judged by PCNA expression, cellular proliferation was reduced, even though the reduction was not statistically significant. The analysed markers of differentiation, e.g. involucrin and cytokeratins 10 and 11, changed in relation to Sg-poisoning. The possible pathogenesis of the skin lesions is discussed.

Histochemical and lectinhistochemical studies on nasal mucosa of pigs with or without respiratory diseases.

Histochemical and lectinhistochemical examinations were carried out on nasal mucosa of pigs with or without respiratory diseases. As the results, both acid and neutral mucins coexisted in nasal mucosa of normal pigs while acid sialomucins were mainly observed in nasal mucosa of pigs infected with Bordetella bronchiseptica and/or Pasteurella multocida. Lectinhistochemistry revealed that the nasal epithelial cells of normal pigs were rich in N-acetylgalactosamine, fucose and N-acetyl-glucosamine residues which showed a tendency to disappear in porcine cytomegalovirus infection and to increase in atrophic rhinitis, respectively.

Lectin-binding sites in uterus and oviduct of normal and Campylobacter fetus subspecies venerealis-infected heifers.

This study was carried out to investigate the effect of infection with Campylobacter fetus subsp. venerealis (Cfv) on the pattern of lectin binding in the uterus and oviduct of heifers. Cfv persistence was demonstrated by bacterial isolation and immunofluorescence. Infected animals showed variations in the lectin binding pattern when compared with control animals. Cfv-infected heifers showed an increased expression of galactose and N-acetyl-galactosamine in the endometrial glands (PNA and SBA binding, respectively). The oviductal epithelium of infected heifers was strongly positive for Con A, which indicated the presence of alpha-D-mannose and alpha-D-glucose. The results of this study showed that Cfv-infection modifies the lectin binding pattern in the reproductive system of heifers. Modifications in glycoconjugates may be involved in failures of fertility and/or implantation.

Lectin histochemical study on the reproductive tract in normal and Brucella ovis-infected rams.

Histochemical studies on tissue sections showed alterations of lectin-binding reactivities in the epididymis, seminal vesicle and ampulla of Brucella ovis-infected rams. These modifications in the carbohydrate composition of organs participating in maturation, transport, and storage of spermatozoa, could be involved in the impaired fertility observed in this disease.

Intermediate filament expression and lectin histochemical features of canine transmissible venereal tumour.

Immunocharacterization of intermediate filament proteins and lectin-binding studies were carried out in canine transmissible venereal tumour (TVT), a unique neoplasia sharing some epidemiological features with Kaposi's sarcoma in humans. Neoplastic cells express vimentin, but neither cytokeratin nor desmin. Regarding lectins, TVT cells express receptors for Triticum vulgaris (WGA), Concanavalia ensiformis (Con A) and Ricinus communis I (RCA-I). They appear to be negative for Ulex europaeus-I (UEA-I), Arachis hypogaea (PNA), Glycine maximus (SBA) and Dolichos biflorus (DBA).

Double immunoenzymatic labelling of intermediate filaments in bovine urinary bladder tumours.

Double immunoenzymatic labelling made possible the simultaneous staining of two antigens with a mixture of polyclonal and monoclonal commercial antibodies. Immunocharacterization of intermediate filament proteins was found to be an accurate indicator of histogenesis in urinary bladder tumours of cattle.

Immunohistochemical methods for the visualization of Mycobacterium paratuberculosis in bovine tissues.

The peroxidase-antiperoxidase (PAP), streptavidin-biotin (SB), and avidin-biotin-complex (ABC) techniques have been evaluated for the visualization of Mycobacterium paratuberculosis (Mp) in formalin-fixed, paraffin-embedded bovine tissues. The used immunoperoxidase techniques were comparatively better than the Ziehl-Neelsen stain, specially for the demonstration of small number of mycobacteria in tissue sections.