Expression of VCAN and its receptors in canine mammary carcinomas with or without myoepithelial proliferation.

The proteoglycan versican (VCAN) plays a complex role in cancer. The expression of this molecule has been related to invasion and progression in malignant mixed tumors, such as carcinoma in mixed tumors (CMT) of the canine mammary gland. In addition, its interaction with surface cell receptors EGFR, HER-2 and CD44 in malignant epithelial cells may be responsible for proliferation and cellular motility in early stages of cancer. We comparatively evaluated the expression of this proteoglycan and its receptors in in situ and invasive areas of simple carcinomas (SC) and CMT to investigate similarities and differences between these histological types. Immunohistochemistry was performed with anti-VCAN, anti-CD44, anti-EGFR and anti-HER-2 antibodies in 32 cases of SC or CMT. VCAN was highly expressed in stroma adjacent to invasive areas in SC and CMT. CMTs presented comparatively higher expression of VCAN in stroma adjacent to in situ and in invasive areas than in corresponding areas in SCs. In CMT, EGFR and HER-2 expressions were higher in situ compared to invasive areas. In contrast, increased CD44 and EGFR expression was found in invasive areas in SC compared to CMT. These results indicate that versican expression is similarly associated with invasiveness in SC and CMT, however higher levels were seen in CMT suggesting that the presence of myoepithelial proliferation in this tumor type participates in stromal composition and promoting an increase in the expression of versican.

Plasma biomarkers profile of female dogs with mammary carcinoma and its association with clinical and pathological features.

The immunological biomarkers profiles were evaluated using Luminex as putative measures to monitor canine mammary carcinomas (MCs). Forty female dogs were categorized into benign mixed tumour (MC-BMT = 28) and mammary carcinoma (MC=12). The ascendant biomarker signatures were used to compare the groups. For example, a higher frequency of MC-BMT animals producing IL-6, CXCL-8 and CXCL-10 was observed, whereas for the MC group IL-2 and CXCL-8 were detected. MC-BMT animals without metastasis had an increase in the levels of IL-2, CXCL-8, CXCL-10, IL-6, TNF-α, IL-15 and a decrease in IL-10 and CXCL-8. MC-BMT animals with metastasis showed only an increase in CXCL-10 and a decrease in IL-18. After comparing the ascendant signatures following the presence of metastasis in both groups, a higher frequency of dogs exhibiting IL-10 production was observed. Pearson correlation (P = 0.0273) and receiver operating characteristic (ROC) curve analysis revealed that this pattern was associated with worse outcome and lower survival rates in MC animals.

Relationship between the expression of versican and EGFR, HER-2, HER-3 and CD44 in matrix-producing tumours in the canine mammary gland.

Versican is an extracellular matrix proteoglycan that has been identified as a modulator of adhesion loss, cell motility, and tumour progression. This motility results from the interaction between versican and cell surface receptors. Studies have also demonstrated the relationship between this molecule and invasion in canine mammary tumours. Given the evidence for the participation of proteoglycans in tumour progression, this study aimed to assess versican expression and its association with cell surface receptors; human epidermal growth factor receptors 1, 2, and 3 (EGFR, HER-2, and HER-3) and CD44 through an immunohistochemical analysis of benign mixed tumours (BMTs), carcinomas in mixed tumours (CMTs), and carcinosarcomas (CSs) of the canine mammary gland. Malignant tumours were divided into low and high groups with respect to versican stromal expression. The results indicated that the BMTs showed weak stromal versican expression and correlations between the expression of stromal versican and EGFR in the epithelial membrane in benign areas (p=0.013, r=0.571). A higher stromal versican expression was observed adjacent to invasive epithelial areas compared with in situ areas in CMTs and CSs, suggesting a direct relationship between versican expression and invasiveness. Furthermore, the CSs exhibited a higher expression of HER-2, cytoplasmic HER-3, and CD44 in epithelial invasive cells in cases of higher stromal versican expression. Therefore, the cell surface receptors (HER-2, HER-3, and CD44) are more evident in CSs that overexpress versican in stroma adjacent to the invasive areas. These findings suggest that the association between these molecules may be directly related to the biological behaviour and invasiveness of these canine mammary tumours.

The Relationship Between E-Cadherin and its Transcriptional Repressors in Spontaneously Arising Canine Invasive Micropapillary Mammary Carcinoma.

E-cadherin downregulation is related to metastatic behaviour and a poor prognosis in cancer. It might be induced by transcriptional repression mediated by the transcription factors SNAIL, ZEB1, ZEB2 and TWIST. Here, we investigated E-cadherin expression and its relationship to those transcriptional repressors (i.e. SNAIL, ZEB1, ZEB2 and TWIST) in the progression from carcinoma 'in situ' to invasion to lymph node metastasis in spontaneously arising canine invasive micropapillary carcinoma (IMPC). E-cadherin expression decreased from carcinoma in situ to invasive progression and was likely to increase with lymph node metastasis. Expression of SNAIL decreased from carcinoma in situ to invasive areas and from invasive areas to lymph nodes. Metastatic lymph nodes had higher expression of ZEB1 than carcinoma in situ and invasive areas. ZEB2 expression was observed in 52%, 38% and 33% of carcinoma in situ areas, invasive areas and lymph node metastases, respectively. TWIST expression was observed in 52%, 38% and 33% of carcinoma in situ areas, invasive areas and lymph node metastases, respectively. In invasive areas, E-cadherin downregulation correlated significantly with SNAIL and TWIST upregulation. Additionally, in infiltrating components of IMPCs, E-cadherin(-)SNAIL(+) neoplastic epithelial cells were observed by immunofluorescence. Taken together, canine mammary IMPCs had a loss of E-cadherin from carcinoma in situ to invasive areas, which appears to be induced by the transcription factor SNAIL. In lymph node metastasis, ZEB1 appears to not exert E-cadherin transcriptional repression activity.

Histomorfometria do epitélio ruminal de cabritos 1/2 sangue Boer submetidos a dietas com tortas oriundas da produção do biodiesel / Histomorphometrics of the ruminal epithelium of 1/2 blood Bôer goats submitted to diets with pies from the production of biodiesel

Objetivou-se determinar o melhor nível de inclusão e de substituição das tortas de dendê e amendoim, respectivamente, em dietas para cabritos 1/2 sangue Boer, por meio da avaliação histomorfométrica das papilas ruminais. Foram utilizados 40 cabritos 1/2 sangue Boer, para cada período experimental, machos, não castrados, com idade aproximada de três meses e com peso inicial de 15,01±1,76kg. Os períodos experimentais constaram de 75 dias e 72 dias. As dietas consistiram de volumoso feno de Tifton-85 e de ração em mistura completa, contendo níveis de inclusão da torta de dendê nas proporções de 0,0; 7,0; 14,0; e 21,0% com base na matéria seca; e de mistura completa, contendo níveis de substituição do farelo de soja pela torta de amendoim nas proporções de 0,0; 33,0; 66,0; e 100%, constituindo-se os tratamentos. No epitélio do rúmen, foram avaliadas altura e largura das papilas, assim como densidade papilar e espessura da parede muscular do rúmen. Entre estas, a altura das papilas, no experimento com torta de amendoim, foi a única a sofrer efeito linear decrescente (P<0,0014); as demais variáveis, tanto dos animais alimentados com torta de amendoim quanto daqueles alimentados com torta de dendê, não foram afetadas pela dieta. As médias de altura encontradas foram de 2,6 e 2,3mm para as tortas de amendoim e dendê, respectivamente. Portanto, a inclusão de até 21% de torta de dendê e a substituição de 100% de torta de amendoim no concentrado de cabritos não alteraram a morfometria das papilas ruminais.

The aim of this study was to determine the best level of pies and replacement of palm oil and peanut, respectively, in diets of 1/2 blood Bôer goats from the histomorphometry of the rumen papillae. A total of 40 1/2 blood male, unneutered, aged approximately three months and initial weight of 15.01±1.76kg Bôer goats were used in each experimental period. The experimental periods consisted of 75 days and 72 days. The diets consisted of roughage hay Tifton-85 and total mixed ration containing levels of inclusion of palm kernel cake in the proportions of 0.0, 7.0, 14.0 and 21.0% based on dry matter; and the second experiment consisted of a complete mixture containing substitution levels of soybean meal by groundnut cake in the proportions of 0.0, 33.0, 66.0 and 100%. Epithelial cells were evaluated in the rumen height and width of the papillae, and density and thickness of the papillary muscle of the rumen. Among these, the height of the papillae in the experiment with peanut butter pie was the only one to suffer a negative linear effect (P<0.0014), the other variables, both in animals fed groundnut cake and palm oil, were not affected by the diet. The average heights found were 2.6 and 2.3, for pies, peanut and palm oil, respectively. Therefore, the inclusion of up to 21% palm kernel cake and replacement of 100% peanut cake in the concentrate of kids did not alter the morphology of the rumen papillae.

Histomorfometria do epitélio ruminal de cabritos 1/2 sangue Boer submetidos a dietas com tortas oriundas da produção do biodiesel / Histomorphometrics of the ruminal epithelium of 1/2 blood Bôer goats submitted to diets with pies from the production of biodiesel

Objetivou-se determinar o melhor nível de inclusão e de substituição das tortas de dendê e amendoim, respectivamente, em dietas para cabritos 1/2 sangue Boer, por meio da avaliação histomorfométrica das papilas ruminais. Foram utilizados 40 cabritos 1/2 sangue Boer, para cada período experimental, machos, não castrados, com idade aproximada de três meses e com peso inicial de 15,01±1,76kg. Os períodos experimentais constaram de 75 dias e 72 dias. As dietas consistiram de volumoso feno de Tifton-85 e de ração em mistura completa, contendo níveis de inclusão da torta de dendê nas proporções de 0,0; 7,0; 14,0; e 21,0% com base na matéria seca; e de mistura completa, contendo níveis de substituição do farelo de soja pela torta de amendoim nas proporções de 0,0; 33,0; 66,0; e 100%, constituindo-se os tratamentos. No epitélio do rúmen, foram avaliadas altura e largura das papilas, assim como densidade papilar e espessura da parede muscular do rúmen. Entre estas, a altura das papilas, no experimento com torta de amendoim, foi a única a sofrer efeito linear decrescente (P<0,0014); as demais variáveis, tanto dos animais alimentados com torta de amendoim quanto daqueles alimentados com torta de dendê, não foram afetadas pela dieta. As médias de altura encontradas foram de 2,6 e 2,3mm para as tortas de amendoim e dendê, respectivamente. Portanto, a inclusão de até 21% de torta de dendê e a substituição de 100% de torta de amendoim no concentrado de cabritos não alteraram a morfometria das papilas ruminais.(AU)

The aim of this study was to determine the best level of pies and replacement of palm oil and peanut, respectively, in diets of 1/2 blood Bôer goats from the histomorphometry of the rumen papillae. A total of 40 1/2 blood male, unneutered, aged approximately three months and initial weight of 15.01±1.76kg Bôer goats were used in each experimental period. The experimental periods consisted of 75 days and 72 days. The diets consisted of roughage hay Tifton-85 and total mixed ration containing levels of inclusion of palm kernel cake in the proportions of 0.0, 7.0, 14.0 and 21.0% based on dry matter; and the second experiment consisted of a complete mixture containing substitution levels of soybean meal by groundnut cake in the proportions of 0.0, 33.0, 66.0 and 100%. Epithelial cells were evaluated in the rumen height and width of the papillae, and density and thickness of the papillary muscle of the rumen. Among these, the height of the papillae in the experiment with peanut butter pie was the only one to suffer a negative linear effect (P<0.0014), the other variables, both in animals fed groundnut cake and palm oil, were not affected by the diet. The average heights found were 2.6 and 2.3, for pies, peanut and palm oil, respectively. Therefore, the inclusion of up to 21% palm kernel cake and replacement of 100% peanut cake in the concentrate of kids did not alter the morphology of the rumen papillae.(AU)

Double-setting alpha-tricalcium phosphate cement provided with interconection channels in rabbits after enucleation: a potential implant for the anophthalmic socket / Cimento de alfa-fosfato tricálcico de dupla pega munido de canais de interconexão em coelhos enucleados: um possível implante para cavidade anoftálmica

The purpose of this study was to evaluate the macroscopy and microstructure of a double setting alpha-tricalcium phosphate bone cement sphere provided with interconnection channels (alpha-TCP-i), as well as the integration of the implant with the rabbits' orbital tissue, through macroscopic analysis and histopathology. The external and internal surfaces of the alpha-TCP-i were evaluated macroscopically and by electron microscopy. Twelve New Zealand rabbits received 12mm implants of alpha-TCP-i following enucleation of the left eye. The clinical assessment was undertaken daily during the first 15 days, followed by fortnightly assessment until the end of the study period. For the morphological analysis, exenteration was performed in 3 animals per experimental period (15, 45, 90 and 180 days). The external and internal surfaces of the implant appeared solid, smooth and compact, with six channels which interconnected centrally. The micro-architecture was characterized by the formation of columns of hexagonal crystals. No signs of infection, exposure, dehiscence of sutures or extrusion of the implant were noted in any of the animals during the entire period of the study. The morphological evaluation demonstrated the presence of a thin capsule around the implant, from whence appeared fibro-vascular projections, which penetrated it through the interconnecting channels. In the first days after the insertion of the implant, an intense inflammatory reaction was noted. At 180 days, however, there were no signs of inflammation. The alpha-tricalcium phosphate cement implant was well tolerated in this rabbit model and appeared to be relatively inert with some fibrovascular ingrowth through the large channels.

Avaliaram-se a macroscopia e a microestrutura de esferas compostas por cimento ósseo de alfa-fosfato tricálcico de dupla pega munido de canais de interconexão (alfa-TCP-i) e a integração do implante ao tecido orbital de coelhos através de análise macroscópica e histopatológica. A superfície interna e externa do alfa-TCP-i foi avaliada macroscopicamente e por microscopia eletrônica. Após enucleação do bulbo ocular esquerdo, 12 coelhos da raça Nova Zelândia receberam implantes do alfa-TCP-i com 12mm de diâmetro. A avaliação clínica foi realizada diariamente nos primeiros 15 dias e depois a cada quinze dias até o fim do período de estudo. Para análise morfológica, seguiu-se a exenteração de três animais por período experimental (15, 45, 90 e 180 dias). As superfícies externa e interna do implante mostraram-se compactas, sólidas e lisas, com seis canais que se interconectavam centralmente. A microarquitetura caracterizou-se pela formação de colunas de cristais hexagonais. Sinais de infecção, exposição, deiscência de sutura ou extrusão não foram notados em nenhum dos animais durante o período de estudo. A análise morfológica revelou a presença de fina cápsula ao redor do implante de onde surgiram projeções fibrovasculares que o penetraram através dos canais de interconexão. Nos primeiros dias após a inserção das esferas, notou-se intensa reação inflamatória que se mostrou ausente aos 180 dias. O implante do cimento de alfa-fosfato tricálcico foi bem tolerado em coelhos, mostrou-se relativamente inerte e permitiu a infiltração de tecido fibrovascular através dos canais de interconexão.

ZEB2 and ZEB1 expression in a spontaneous canine model of invasive micropapillary carcinoma of the mammary gland.

ZEB1 and ZEB2 have been recently related to cancer prognosis. We investigated their expression and its association with clinicopathological parameters and overall survival in invasive micropapillary carcinoma (IMPC), which is a metastasising neoplasm of the canine mammary gland. Immunohistochemical evaluation showed nuclear and cytoplasmic staining for ZEB2 and nuclear staining for ZEB1. 'In situ' areas presented higher positivity for cytoplasmic ZEB2 than invasive areas of IMPC did (p = 0.03). ZEB1 positivity was associated with a low histological grade (p = 0.01). A shorter overall survival rate was observed in IMPCs that were positive for cytoplasmic ZEB2 (p = 0.04). Antibodies specificity in canine species was confirmed by western blot. Our results indicated that cytoplasmic ZEB2 appears to be an important factor in the early stages of malignancy and predicts a poor overall survival rate for IMPC in this canine mammary cancer model. ZEB1 downregulation appears to be associated with the dedifferentiation process of IMPC.

Histopathological and immunohistochemical assessment of invasive micropapillary mammary carcinoma in dogs: a retrospective study.

Invasive micropapillary carcinoma (IMPC) of the mammary gland, despite its rare occurrence in humans and dogs, is an important neoplasm due to its aggressive behaviour. The aim of this study was to evaluate the clinicopathological and immunophenotypical characteristics of IMPC and to determine the overall survival of dogs with this tumour. Of the selected cases, the majority had >3 cm neoplasms (15/19, 78.95%) and lymph node metastases (16/16, 100%), but only two cases (2/9, 22.2%) had distant metastases. The IMPCs were classified as either pure (15/22, 68.18%) or mixed (7/22, 31.82%) types. There was a predominance of moderate histological grade tumours (16 grade II) and the average overall survival was 120 days. Positive immunohistochemical staining for epithelial membrane antigen and negative staining for CD-31, p63 and cytokeratin (CK) AE1AE3 in cystic formations confirmed the micropapillary nature of these neoplasms. A proportion of cases exhibited positive epithelial staining for p63 (4/20, 20%) and CK34ßE12 (20/22, 90.9%). Most cases were positive for oestrogen (19/20, 95%) and progesterone (19/20, 95%) receptors, but lacked HER-2 (16/22, 72.72%) and epidermal growth factor receptor (15/22, 68.18%) over-expression. The mean proliferation index was 14.8%. The findings demonstrate that, similar to humans, canine IMPCs behave aggressively with high rates of metastasis to regional lymph nodes and short overall survival times.

CA15.3, CEA and LDH in dogs with malignant mammary tumors.

BACKGROUND: Presence of tumor markers in serum might be connected to the number of secreting cells and with the stage of the neoplasm. However, there are few studies regarding these markers in veterinary clinical oncology. OBJECTIVES: To determine the serum concentrations of cancer antigen 15.3 (CA 15.3), carcinoembryonic antigen (CEA), and lactate dehydrogenase (LDH) in female dogs with different stages of mammary cancer. ANIMALS: Ninety female dogs, including 30 that were healthy, 40 that had nonmetastatic cancer, 12 with regional metastasis, and 8 with distant lymph node metastasis. METHODS: Prospective case-controlled observational study. Serum samples were collected to measure CA15.3, CEA, and LDH from 60 female dogs with mammary cancer during mastectomy and 30 healthy female dogs during routine check-up. CA15.3 and CEA were determined by chemiluminescent immunoassay and LDH by ultraviolet kinetic method. Western blotting analysis was performed to confirm the specificity and possible cross-reactivity of human CA15.3 and CEA antibodies with canine serum. Group data were compared by ANOVA followed by Student-Newman-Keuls and Tukey's tests. Correlations were investigated by Pearson and Spearman tests. RESULTS: CEA, CA15.3, and LDH were measurable in all groups. Higher serum concentration of CA15.3 and LDH was associated with regional and distant metastases. There was a significantly higher serum CA15.3 concentration in animals with lymph node metastasis when compared with animals without metastasis. There were no significant differences in CEA among groups. Expression of CA15.3 and CEA in canine serum was confirmed by Western blotting. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum CA15.3 can be used to distinguish nonmetastatic from metastatic carcinomas.

Tipificação genética, através da técnica de spoligotyping, de isolados de Mycobacterium bovis em animais abatidos na região metropolitana de Salvador, Bahia, Brasil / Use of spoligotyping for genetic typing of Mycobacterium bovis isolates from slaughtered animals in the metropolitan area of Salvador, Bahia, Brazil

A tuberculose é uma enfermidade infecciosa crônica, que afeta mamíferos e aves e constitui um sério problema de saúde pública e animal. Objetivando realizar um levantamento molecular da enfermidade em bovinos abatidos em matadouros frigoríficos no Estado da Bahia, Brasil, foram analisadas as lesões pulmonares e de linfonodos mediastínicos de 43 carcaças de animais abatidos em três matadouros-frigoríficos localizados na Região Metropolitana de Salvador, Bahia. Sete isolados de Mycobacterium bovis foram identificados, através da técnica do spolygotyping, e discriminados em três diferentes espoligotipos (SB1055, SB0120 e SB0268) descritos no Brasil e em diversas áreas do mundo. Os resultados indicam que o método de diagnóstico utilizado pode contribuir para a criação de uma base de dados para o estudo epidemiológico da tuberculose bovina no Estado da Bahia.

Tuberculosis is an infectious chronic disease that affects mammals and birds and constitutes a serious problem for public and animal health. Pulmonary and mediastinic lymph node lesions of 43 animals slaughtered in 3 slaughterhouses in the metropolitan region of the city of Salvador, Bahia, Brazil, were analyzed with the objective of obtaining a molecular survey of the disease in bovines slaughtered in slaughterhouses in the state. Seven isolates ofMycobacterium bovis were identified through the spoligotyping technique and classified into 3 different spoligotypes (SB1055, SB0120, SB0268), described in Brazil and in many areas worldwide. The results indicate that the diagnostic method utilized may contribute to the creation of a database for the epidemiologic study of bovine tuberculosis in the state of Bahia.