Caveolin-1, GATA-3, and Ki67 expressions and their correlation with pathological findings in canine bladder urothelial carcinoma.

The bladder urothelial carcinoma (UC) represents ~2% of malignant neoplasms in dogs and is a therapeutic challenge in veterinary medicine. Although it is considered the most common bladder cancer in dogs, few previous studies have investigated different markers that correlate with clinical and pathological parameters. Therefore, this study aimed to evaluate Caveolin-1, GATA-3, and Ki67 immunostaining in canine UC samples to evaluate their correlations with histopathological variables. Thirty tumor samples were obtained, and Caveolin-1, GATA-3, and Ki67 expression was assessed by immunohistochemistry and associated with pathological factors by univariate and multivariate analyses. Among the histopathological findings, lymphatic invasion was identified in 53.33% of the tumors, and the mean mitotic count (MC) was 31.82 ± 26.26. Caveolin-1 showed mild-to-high cytoplasmic expression in neoplastic cells, whereas GATA-3 showed mild-to-high nuclear expression. The Ki67 expression revealed a mean of 24.14 ± 16.88% positive cells. In the univariate analysis, no association was found between each marker and the pathological findings. On the other hand, in multivariate analysis, we identified a positive correlation between GATA-3 and MC and a negative correlation between Caveolin-1 and MC. Moreover, lymphatic invasion was positively correlated with histological type and grade, and negatively correlated with MC. In addition, the histological type was positively correlated with the histological grade. Overall, our results indicate that Caveolin-1 and GATA-3 expression could be promising markers for bladder UC aggressiveness.

Morphological and Molecular Characterization of Proliferative Inflammatory Atrophy in Canine Prostatic Samples.

Proliferative inflammatory atrophy (PIA) is an atrophic lesion of the prostate gland that occurs in men and dogs and is associated with a chronic inflammatory infiltrate. In this study, we retrospectively reviewed canine prostatic samples from intact dogs, identifying 50 normal prostates, 140 cases of prostatic hyperplasia, 171 cases of PIA, 84 with prostate cancer (PC), 14 with prostatic intraepithelial neoplasia (PIN) and 10 with bacterial prostatitis. PIA samples were then selected and classified according to the human classification. The presence of PIA lesions surrounding neoplastic areas was then evaluated to establish a morphological transition from normal to preneoplastic and neoplastic tissue. In addition, the expression of PTEN, P53, MDM2 and nuclear androgen receptor (AR) were analyzed in 20 normal samples and 20 PIA lesions by immunohistochemistry and qPCR. All PIA lesions showed variable degrees of mononuclear cell infiltration around the glands and simple atrophy was the most common histopathological feature. PIA was identified between normal glands and PC in 51 (61%) out of the 84 PC samples. PIA lesions were diffusely positive for molecular weight cytokeratin (HMWC). Decreased PTEN and AR gene and protein expression was found in PIA compared to normal samples. Overall, our results strongly suggest that PIA is a frequent lesion associated with PC. Additionally, this finding corroborates the hypothesis that in dogs, as is the case in humans, PIA is a pre neoplastic lesion that has the potential to progress into PC, indicating an alternative mechanism of prostate cancer development in dogs.

Immunoexpression and Prognostic Significance of Multidrug Resistance Markers in Feline Mammary Carcinomas.

Feline mammary carcinomas (FMCs) are commonly characterized by high clinical aggressiveness and poor prognosis. FMCs share many features with the corresponding human disease, allowing the comparative investigation of tumour biology and therapeutic strategies, including multidrug resistance (MDR) mechanisms. Although transporting/binding proteins, including permeability glycoprotein (P-gp), lung resistance protein (LRP) and metallothionein (MT), are frequently associated with tumour aggressiveness and unresponsiveness to chemotherapy in human breast cancer, they have not been analysed in FMCs. We investigated the immunoexpression of P-gp, LRP and MT in FMCs and their correlation with clinicopathological parameters and overall survival (OS) time in 46 FMCs, with a median follow-up period of 289 days. These markers were co-expressed in 85% of tumours. P-gp was expressed in 93.4% of FMCs and was positively associated with tumour grade (P = 0.049). While unequivocally observed in all FMCs, LRP immunoexpression did not correlate with any clinicopathological parameters or OS. Expression of MT was significant in triple-negative basal- and normal-like molecular subtypes of FMCs (P = 0.023). The concurrent expression of MDR proteins indicates the potential existence of chemotherapy resistance-related mechanisms in FMCs. The positive association between P-gp and MT immunoexpression and aggressive phenotypes could open new therapeutic and translational strategies for FMCs.

Prognostic value of immunohistochemical markers in canine cutaneous mast cell tumours: A systematic review and meta-analysis.

Histological grading systems remain cornerstones in the prognosis of canine cutaneous mast cell tumours (MCTs), but the distinct biological behaviour of each tumour often necessitates the use of complementary markers. Although a plethora of immunohistochemical markers have been proposed as prognostic factors, few are presently applied in routine diagnosis. This systematic review and meta-analysis was designed to establish which immunohistochemical markers have verifiable prognostic value for cutaneous MCTs in dogs. A Boolean search of five databases identified 200 articles for screening, of which 73 were selected for full-text assessment and 24 ultimately included in the systematic review. Odds Ratio (OR) was adopted as the summary measure for subsequent meta-analysis but only 15 articles, relating to the immunomarkers Ki-67 (9), KIT (5), and BAX (2), provided either a value for OR or sufficient data to calculate this statistic. Meta-analysis verified that canine cutaneous MCTs with elevated expression of Ki-67 or BAX, as well aberrant immuno-expression of KIT, showed an increased odds of death, with respective OR values of 11.2 (95% CI 6.3-20.0; p < .01), 9.9 (95% CI 1.3-73.6; p = .03), and 4.1 (95% CI 1.1-15.3; p = .03). Despite KIT, Ki67, and BAX arise as suitable prognostic factor for canine MCTs, this study highlighted the lack of important clinical and statistical data in many published articles, rendering it impossible to complete the meta-analysis of several potentially valuable immunohistochemical markers.

Genetic variants of BRCA1 and BRCA2 genes in cats with mammary gland carcinoma.

Mammary tumours are the first and third most incident neoplasm in women and cats, respectively. Approximately 85% of feline mammary gland tumours are malignant and aggressive, especially the triple-negative and HER-2+ molecular subtypes. Triple-negative basal-like feline mammary carcinomas (FMCs) are considered suitable models due to the clinical and morphological similarities with human basal-like triple-negative breast cancer (TNBC). In women, TNBC has a poor prognosis and is often associated with mutations in the tumour suppressor genes BRCA1 and BRCA2. In light of this, the aim of the present investigation was to screen somatic and germline variants of BRCA1 and BRCA2 in nine female cats bearing FMCs. Matched whole blood and FMC samples were obtained for genetic analysis. Additional tumour samples were obtained for histopathological and immunohistochemical evaluation. Genomic DNA was isolated and 27 exonic regions of BRCA1 and BRCA2 genes were amplified and screened by next-generation sequencing. A somatic variant with high functional impact was found in exon 11 of BRCA2 at a frequency of 4.34% in one FMC-bearing cat. Four germline variants with moderate impact were detected in three of the nine FMC-bearing cats and were restricted to exon 9 of BRCA1. It is concluded that the germline genetic variants found in one-third of FMC-bearing animals might be associated with a higher risk of hereditary mammary carcinogenesis.

Use of a morcellized fragment of bone as a bone graft in a femoral fracture repair / Uso de fragmento ósseo morcelizado como enxerto ósseo no reparo de fratura femoral

As one of the most frequent reasons for presentation at the orthopedic services in veterinary practice, long bone fractures need bone continuity for consolidation to take place. This case report has demonstrates the use of a morcellized fragment of bone as a bone graft in a femoral fracture with major bone loss. A mixed-breed adult cat with a segmental femoral fracture with a large longitudinal fissure in the central bone fragment was submitted to an interlocking nail osteosynthesis. During the procedure a fracture occurred along the fissure resulting in two bone fragments with significant bone loss. Fragments were morcellized and applied over the defect to act as a bone graft. By the 15th day after the surgery, the cat had a normal gait and showed no pain response, and bone consolidation occurred after 7 months. Once harvesting of humeral, femoral and tibial bone grafts can be frustrating in cats, resulting in just small amounts of bone, the morcellation of the central fragments was vital to provide sufficient graft to cover the big defect in the femoral shaft. The authors suggest that autologous fresh morcellized cortical bone fragments can be an adjunct to the treatment of comminuted fractures in cats, as an alternative to more traditional repairs of comminuted fracture.

Fraturas de ossos longos necessitam de continuidade óssea para que a consolidação ocorra. Este relato de caso tem como objetivo demonstrar o uso de um fragmento ósseo morcelizado com a função de enxerto ósseo em uma fratura de fêmur com presença de uma grande falha óssea entre os fragmentos. Um gato adulto, sem raça definida, com uma fratura múltipla em fêmur com presença de uma fissura longitudinal no fragmento central foi submetido à osteossíntese com uso de haste bloqueada. Durante o processo, ocorreu uma fragmentação a partir da fissura, o que resultou na presença de dois fragmentos ósseos com uma grande porção de perda óssea entre os dois. Os fragmentos ósseos restantes do fragmento central foram morcelizados e aplicados no defeito para atuar com enxerto ósseo. Quinze dias após a cirurgia o paciente apresentou apoio normal e ausência de dor, e consolidação óssea ocorreu em 7 meses pós operatórios. Uma vez que a coleta de enxerto nos gatos resulta em pouca quantidade de osso, a morcelização do fragmento central foi vital para promover enxerto suficiente para cobrir o defeito ósseo. Os autores sugerem que o uso de enxerto cortical autólogo fresco morcelizado pode ser um adjuvante no tratamento de fraturas cominutivas em gatos, como um tratamento alternativo à terapia convencional.

Use of a morcellized fragment of bone as a bone graft in a femoral fracture repair / Uso de fragmento ósseo morcelizado como enxerto ósseo no reparo de fratura femoral

As one of the most frequent reasons for presentation at the orthopedic services in veterinary practice, long bone fractures need bone continuity for consolidation to take place. This case report has demonstrates the use of a morcellized fragment of bone as a bone graft in a femoral fracture with major bone loss. A mixed-breed adult cat with a segmental femoral fracture with a large longitudinal fissure in the central bone fragment was submitted to an interlocking nail osteosynthesis. During the procedure a fracture occurred along the fissure resulting in two bone fragments with significant bone loss. Fragments were morcellized and applied over the defect to act as a bone graft. By the 15th day after the surgery, the cat had a normal gait and showed no pain response, and bone consolidation occurred after 7 months. Once harvesting of humeral, femoral and tibial bone grafts can be frustrating in cats, resulting in just small amounts of bone, the morcellation of the central fragments was vital to provide sufficient graft to cover the big defect in the femoral shaft. The authors suggest that autologous fresh morcellized cortical bone fragments can be an adjunct to the treatment of comminuted fractures in cats, as an alternative to more traditional repairs of comminuted fracture.

Fraturas de ossos longos necessitam de continuidade óssea para que a consolidação ocorra. Este relato de caso tem como objetivo demonstrar o uso de um fragmento ósseo morcelizado com a função de enxerto ósseo em uma fratura de fêmur com presença de uma grande falha óssea entre os fragmentos. Um gato adulto, sem raça definida, com uma fratura múltipla em fêmur com presença de uma fissura longitudinal no fragmento central foi submetido à osteossíntese com uso de haste bloqueada. Durante o processo, ocorreu uma fragmentação a partir da fissura, o que resultou na presença de dois fragmentos ósseos com uma grande porção de perda óssea entre os dois. Os fragmentos ósseos restantes do fragmento central foram morcelizados e aplicados no defeito para atuar com enxerto ósseo. Quinze dias após a cirurgia o paciente apresentou apoio normal e ausência de dor, e consolidação óssea ocorreu em 7 meses pós operatórios. Uma vez que a coleta de enxerto nos gatos resulta em pouca quantidade de osso, a morcelização do fragmento central foi vital para promover enxerto suficiente para cobrir o defeito ósseo. Os autores sugerem que o uso de enxerto cortical autólogo fresco morcelizado pode ser um adjuvante no tratamento de fraturas cominutivas em gatos, como um tratamento alternativo à terapia convencional.

Protective effect of genetic deletion of pannexin1 in experimental mouse models of acute and chronic liver disease.

Pannexins are transmembrane proteins that form communication channels connecting the cytosol of an individual cell with its extracellular environment. A number of studies have documented the presence of pannexin1 in liver as well as its involvement in inflammatory responses. In this study, it was investigated whether pannexin1 plays a role in acute liver failure and non-alcoholic steatohepatitis, being prototypical acute and chronic liver pathologies, respectively, both featured by liver damage, oxidative stress and inflammation. To this end, wild-type and pannexin1-/- mice were overdosed with acetaminophen for 1, 6, 24 or 48h or were fed a choline-deficient high-fat diet for 8weeks. Evaluation of the effects of genetic pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, lipid accumulation, protein adduct formation, oxidative stress and inflammation. In parallel, in order to elucidate molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. The results of this study show that pannexin1-/- diseased mice present less liver damage and oxidative stress, while inflammation was only decreased in pannexin1-/- mice in which non-alcoholic steatohepatitis was induced. A multitude of genes related to inflammation, oxidative stress and xenobiotic metabolism were differentially modulated in both liver disease models in wild-type and in pannexin1-/- mice. Overall, the results of this study suggest that pannexin1 may play a role in the pathogenesis of liver disease.

Inhibition of connexin hemichannels alleviates non-alcoholic steatohepatitis in mice.

While gap junctions mediate intercellular communication and support liver homeostasis, connexin hemichannels are preferentially opened by pathological stimuli, including inflammation and oxidative stress. The latter are essential features of non-alcoholic steatohepatitis. In this study, it was investigated whether connexin32 and connexin43 hemichannels play a role in non-alcoholic steatohepatitis. Mice were fed a choline-deficient high-fat diet or normal diet for 8 weeks. Thereafter, TAT-Gap24 or TAT-Gap19, specific inhibitors of hemichannels composed of connexin32 and connexin43, respectively, were administered for 2 weeks. Subsequently, histopathological examination was carried out and various indicators of inflammation, liver damage and oxidative stress were tested. In addition, whole transcriptome microarray analysis of liver tissue was performed. Channel specificity of TAT-Gap24 and TAT-Gap19 was examined in vitro by fluorescence recovery after photobleaching analysis and measurement of extracellular release of adenosine triphosphate. TAT-Gap24 and TAT-Gap19 were shown to be hemichannel-specific in cultured primary hepatocytes. Diet-fed animals treated with TAT-Gap24 or TAT-Gap19 displayed decreased amounts of liver lipids and inflammatory markers, and augmented levels of superoxide dismutase, which was supported by the microarray results. These findings show the involvement of connexin32 and connexin43 hemichannels in non-alcoholic steatohepatitis and, simultaneously, suggest a role as potential drug targets in non-alcoholic steatohepatitis.